Tip-growing cells exhibit complex growth regimes in vitro, alternating between growing and non-growing intervals, oscillatory or more steady behavior. In Arabidopsis thaliana, pollen tube growth arrest is often accompanied by spiking behavior in intracellular ion concentrations and extracellular ion fluxes. Thus, selecting comparable growing regimes is critical for quantifying ion dynamics across cells and genotypes. Defining non-growing regimes is a fundamental step to filter out their associated data points. Here, we provide computational and statistical procedures for the quantitative phenotyping of ion fluxes associated with growth dynamics in tip-growing cells. The goal is to provide reliable estimates of ion fluxes given pairwise time-series comparisons (ion fluxes vs. growth rate), focused on growth-associated intervals and avoiding data stemming from regimes associated with growth arrest. We consider extracellular ion fluxes in growing tubes, but the analysis is applicable to other quantitative variables and tip-growing cells. After visualizing both series in a common timeframe, we extract the growth rate baseline and determine the non-growing regime threshold with a Gaussian Mixture Model, then predict the growth state at sampled flux times, filtering, and finally quantification. The protocol is presented in R but is of general use, since multiple software routines can yield similar results.
Here, we describe an in vivo dye-tracking method for measuring phloem transport velocity in seedlings, leaves, and petioles and potentially other translucent plant tissues. The method requires measurement of the fluorescent signal of a phloem-mobile dye using sensitive photo-sensors placed externally to the plant. Following dye application, velocity is determined by either following a dye pulse or using laser fluorescence bleaching. Velocity is estimated by dividing the distance traveled by the dye by the time it takes to travel. This method can be used to measure phloem transport velocity on intact plants with minimal disturbance and has the potential to be used under a variety of growth conditions. Because there are large differences among species in their anatomy, this method should be optimized for individual plants and tissue types.
Reproductive development in apomictic plants diverges from the sexual pathway at different key steps. The early steps take place in the ovule, the female organ hosting female sporogenesis and gametogenesis. Cell identities are notably more plastic in the ovule of facultative aposporous plants, where somatic cells can shift to germinal fate. This plasticity likely starts during the early morphogenesis of the ovule, concomitant with gradual differentiation of the sexual megaspore mother cell (MMC). In sexual species, 3D morphogenetic analyses have shown that ovule shape conditions MMC plasticity. However, in aposporous grasses, the morphogenetic events shaping ovule primordia are currently undescribed in 3D and at the cellular level, largely due to the inaccessibility of this organ. To fill this gap, we propose here a comprehensive workflow from ovule sampling to the extraction of 3D cellular quantitative parameters, established for the tropical apomictic grass Paspalum rufum. First, this protocol describes 3D imaging of whole-mount ovules at successive developmental stages, covering MMC differentiation, using ClearSee clearing procedure and double cell walls/nuclei staining. Second, it provides a detailed image analysis workflow in the open-source platform, MorphoGraphX. The workflow enables semiautomatic 3D cell segmentation, cell location, and annotation according to tissue layers or adjacency networks, leading to the final extraction of cellular parameters that describe geometry and topology dynamics along with ovule primordia development. This protocol applies to various species of the Paspalum genus and is potentially useful for 3D studies of large, curved, and hidden organs in multiple plant species.
Callose deposition in the phloem is an innate part of plant development and a response to biotic and abiotic stress, aiding in stress mitigation but potentially also compromising phloem functionality. Measuring callose using aniline blue staining is widely employed, but accurate quantification is hindered by image qualities such as texture and fluorescent artifacts. Here, we describe a method to quantify callose levels in the phloem of woody plants using aniline blue staining, confocal microscopy, and automated supervised machine learning-driven image analysis supported by the IlastiKlean R package. Bark peel samples from woody plants are collected from shoots, stained, and imaged to assess callose deposition. The microscopy images are preprocessed and analyzed using Fiji, Ilastik, and the IlastiKlean R package, which allows accurate quantification of the number, size, and distribution of callose deposits. This quantitative measure can be used to study, screen, and engineer plants that are better adapted to biotic or abiotic stresses, and it serves as an important tool for basic and foundational studies of callose deposition in the phloem.
Plant responses to environmental stimuli are often shaped by a history of previous interactions, forming the foundation for stress memory and adaptive plasticity. Arbuscular mycorrhizal (AM) fungi establish a mutualistic relationship with most land plants, enhancing nutrient uptake and stress resilience, and are increasingly recognized as biological agents contributing to plant stress memory. However, quantifying AM colonization, especially in large-scale or time-course experiments investigating priming or memory effects, remains a technical bottleneck. Conventional staining methods are time-consuming, destructive, and incompatible with live imaging. This chapter presents a robust, nondestructive, and quantitative protocol to assess AM colonization in Medicago truncatula roots using a visible anthocyanin pigmentation marker. The method employs a synthetic construct expressing the R2R3 MYB transcription factor MtLAP1, driven by the AM-inducible Kunitz Protease Inhibitor 106 (KPI106) promoter, enabling visualization of arbuscule-containing root cells through purple/red pigmentation. The protocol encompasses Agrobacterium rhizogenes-mediated hairy root transformation, standardized mycorrhization assays, and anthocyanin pigment extraction and quantification. Anthocyanin accumulation correlates strongly with conventional staining-based colonization estimates, and the system enables early detection, live imaging, and high-throughput screening of mutants with altered AM phenotypes. This method offers a powerful tool for dissecting the functional role of mycorrhizal symbiosis in plant stress memory and is especially suited for forward genetic screens, stress priming experiments, and live-tracking of root-fungus interactions over time.
Plants have evolved an effective defense mechanism in the phloem to prevent the spread of pathogens and minimize the loss of phloem sap following injury. Specific structural phloem proteins known as P-proteins rapidly seal affected sieve elements by plugging the sieve plates, a phenomenon defined as sieve element occlusion. This chapter describes a live cell imaging method for the analysis of P-protein responses and signal propagation in vivo without tissue sectioning or mechanical manipulation. It is based on an Arabidopsis thaliana dual-reporter line where P-proteins are labeled with fluorescent tags in a complementation background, allowing real-time visualization of the parietal protein network during sieve element occlusion. The calcium sensor Yellow Cameleon 3.6 is specifically expressed and anchored in the sieve elements, enabling the detection of calcium waves and their effects on P-protein structure over longer distances in vivo. Using this protocol, a wide range of external triggers-including chemical treatments, buffers, and wounding-can be applied with precision, allowing the analysis of P-protein functions and long-distance signaling in the phloem. The method is readily adaptable to other genetically encoded sensors and can be used to investigate P-protein-independent processes as well as the diverse signaling and structural responses of additional sieve element components.
Phloem loading of sucrose and the transport from source leaves to sink tissues is vital for plant growth and carbon allocation. Traditional methods to measure phloem loading are often time-consuming or require specialized equipment. Here, we introduce a rapid, cost-effective esculin-based fluorometric assay as a reliable proxy for sucrose loading. Esculin, a fluorescent coumarin glucoside, is specifically transported by sucrose transporters in plants with active apoplastic phloem loading. After application to source leaves, esculin fluorescence is measured in the extracted leaf sap, providing a sensitive and relatively high-throughput method for analyzing phloem loading dynamics. Validated against established techniques, the assay is accessible to nonspecialized laboratories and enables investigations into environmental and developmental regulation of phloem loading, offering insights into plant growth and stress responses.
Indole-3-acetic acid (IAA) is an important auxin phytohormone that regulates development, directional growth, and stress responses in plants. Here, we provide a detailed method and protocol for use of a corona phase molecular recognition-based IAA nanosensor that allows for direct and real-time measurement of IAA in plants. The near-infrared signal of the IAA nanosensor also allows it to be used in green tissues, as it is not affected by chlorophyll content. Furthermore, the IAA nanosensor can be easily applied across various plant species and all tissues as the sensor is not genetically encoded.
Sieve elements in the phloem transport carbon and small molecules, such as RNA and phytohormones, throughout the plant body. Understanding the physical dimensions of sieve elements and phloem tissue is thus crucial for predicting how much carbon can be moved at any given time. Quantification of sieve element diameters and areas has previously been performed using transmission electron microscopy, scanning electron microscopy, and light microscopy, but sieve element identification is difficult because the phloem is a heterogeneous tissue. The recently identified LM26 antibody labels a pectin in the sieve element cell wall, allowing the identification of sieve elements and the measurement of their properties, such as diameter, relatively quickly and the quantification of their number in cross sections using image analysis software. Here, we describe methods for immunolabelling sieve elements in fresh or fixed tissue embedded in polyethylene glycol or methacrylate. The protocol is broadly adaptable to various fixation and sectioning methods, provided they do not alter the structure of pectins in the cell wall.
Plants emit a diverse array of volatile organic compounds in response to stress. These volatiles serve as a defense mechanism against various stressors. Highly dynamic and chemically reactive, plant volatiles readily interact with other atmospheric gases, often transforming into distinct molecular forms. The precise detection of herbivore-induced plant volatiles at high purity is a fundamental prerequisite for translating scientific insights into actionable strategies. This process encompasses a series of meticulously coordinated steps, ranging from the selection of appropriate plant specimens to the quantification of their volatile emissions. While standardized protocols for the accurate estimation of plant volatiles are well established, the advancement of volatome research remains constrained by limited knowledge regarding the functional characterization of these plant volatiles. This study presents a detailed account of multiple procedures for the collection and characterization of plant volatiles, utilizing a locally engineered and specially designed volatile trapping apparatus, with particular emphasis on the critical, often subtle, procedural nuances involved.
Studying virus-infected phloem is of significant importance, as it not only enhances our understanding of viral pathogenesis but also leverages viruses as tools to expand knowledge about plant phloem physiology. The uneven distribution pattern of phloem-infecting viruses poses methodological challenges for such studies-requiring both large field of view (FOV) and high-resolution imaging. A comprehensive anatomical analysis of the phloem necessitates global visualization, while resolving viral structures demands local high-resolution observation. This chapter describes a method, the X-ray microtomography (μCT)-volume electron microscopy (vEM) correlative imaging technique, which effectively addresses these methodological requirements, where μCT provides the large FOV for identification of regions of interest, followed by vEM acquisition of high-resolution images. It is a six-step protocol, including: (1) sample preparation, (2) flaw detection, (3) overview imaging by μCT, (4) identifying viral infection regions, (5) high-resolution imaging by vEM, and (6) image processing and analysis. In this workflow, the steps of sample preparation and identification of viral infection regions are critical. This protocol was originally established for investigating Southern rice black-streaked dwarf virus (SRBSDV) infection in rice phloem, with parameters optimized for plant reoviruses. We provide advice on how to adapt the approach for studying other viral infections.
Virus-induced gene silencing (VIGS) has been applied as a functional genomics tool across diverse plant species. Integrated with the Arabidopsis sequence-tagged T-DNA homozygous mutant library, VIGS enables an efficient screening approach that combines features of both forward and reverse genetics, facilitating the identification of novel regulators in plant immunity. Plant defense against pathogens relies on a two-layered immune system, classified as pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). Dysregulation of key PTI or ETI components can lead to excessive or uncontrolled cell death. The cell death phenotype offers a unique avenue for genetic screens aimed at identifying suppressors of immune-related cell death. However, conventional genetic approaches face limitations due to seedling lethality and the consequent lack of viable seeds, restricting their efficiency. Here, we describe an Agrobacterium-mediated transient VIGS assay optimized for systematic gene silencing at seedling stages, leading to cell death phenotypes. This method enables high-throughput screening for cell death suppressors using T-DNA homozygous mutant collections. The platform provides a rapid, cost-efficient strategy for uncovering key regulators of plant immune signaling, offering new insights into mechanisms governing immune homeostasis and cell death suppression.
Histochemical staining and microscopy-based techniques have been widely used to detect, quantify, and analyze the morphology of arbuscular mycorrhizal fungi (AMF) in roots. Here, we describe a traditional standardized method for staining of AMF in colonized roots using trypan blue, along with possible modifications to adapt the protocol to specific needs, such as root type or reducing the use of toxic reagents. We also summarize common approaches for quantifying arbuscular mycorrhizal colonization. In addition, we present a simple fluorescent staining protocol, using wheat germ agglutinin-Alexa Fluor conjugates, for high-resolution imaging of fungal colonization patterns and arbuscule morphology in roots. Finally, we describe a GUS staining method for localizing the promoter activity of plant genes potentially involved in mycorrhization, using transformed mycorrhizal hairy roots carrying promoter-GUS fusions.
Sucrose is the primary transport sugar in plants, serving as an essential energy source and signaling molecule. Detection, visualization, and quantification of sucrose in various plant tissues are essential for understanding the metabolic and physiological processes that sustain plant life. Traditional metabolite-mapping techniques have struggled to visualize the quantitative distribution of sucrose at sufficient resolution to distinguish vascular bundles from surrounding tissues. Here, we present a Fourier-transform infrared (FTIR) imaging approach that can visualize sucrose in plant tissues quantitatively at a microscopic resolution (~12 µm). This IR-based, label-free method can be used with both model plants and agriculturally important crops. The assay has a detection range of 20-1000 mM and can map sucrose distribution within complex organs such as stems, leaves, and seeds. Notably, it enables the precise quantification of sucrose levels in vascular tissues. This is a trait of great interest in many current breeding and plant biotechnology approaches aimed at increasing crop yield.
Chickpea (Cicer arietinum L.), confronts substantial challenges from the emerging pathogenic fungus Macrophomina phaseolina (Tassi) Goid, causing dry root rot (DRR) disease. Chickpea plants severely affected by combined DRR and drought stress. Currently sick plot and sick pot method are utilized for germplasm screening to identify tolerant genotypes. These methods are time-consuming; therefore, we propose a novel methodology for the rapid screening of chickpea under combined DRR and osmotic stress conditions. This chapter introduces an adept high-throughput phenotyping methodology, conducted within controlled laboratory conditions, aiming to investigate the interaction between osmotic stress and DRR disease in chickpea crops. The methodology employs an innovative pouch technique for screening combined stress, providing a streamlined temporal investigation process and precise control over stress parameters. The incorporation of polyethylene glycol (PEG) enables the simultaneous imposition of osmotic stress alongside pathogen infection, making the methodology versatile for studying combined stress scenarios. This approach fills a gap in concurrent stress imposition techniques, enhancing germplasm screening by identifying genotypes with varying susceptibility and resistance levels. Thus, we suggest use of high-throughput phenotyping in combination genome-wide association study (GWAS) can take combined stress resistance breeding in chickpea at next level to combat food security and climate change.
Dry root rot (DRR) disease is a major threat to chickpea production across the world. This disease is caused by a soil-borne necrotrophic fungal pathogen, Macrophomina phaseolina. The use of disease-resistant varieties paves the way to conquer the disease spread. Though chickpea germplasm with rich genetic diversity is available around the world, its response to DRR is still unexplored. In turn, this demands screening and identification of resistant genotypes for crop protection against the disease. Here we describe an improved blotting paper technique for the large-scale screening of chickpea genotypes for DRR resistance. The method is quick, cost-effective, less labour-intensive, and thus optimized for high-throughput screening and can be efficiently used to screen a large number of chickpea genotypes for resistance against DRR.
Apomixis is an asexual reproductive mechanism that takes place deeply inside the female reproductive organs of the plant, in ovules and seeds. In gametophytic apomixis, an unreduced female gametophyte is produced by a modified meiosis of the megaspore mother cell (dipolspory) or from a somatic initial cell (apospory). The unreduced, nonrecombined egg cell develops subsequently into an embryo by parthenogenesis. The cyto-embryological study of apomixis is challenging because of the inaccessibility of these structures. Consequently, images of apomeiosis and parthenogenesis with high definition are limited to a few species. In this chapter, we show the application of a Feulgen staining protocol combined with confocal microscopy for the study of nonreductional megasporogenesis and autonomous embryo formation in diplosporous apomictic Taraxacum officinale and aposporous apomictic Pilosella piloselloides var. praealta. Using a rapid and technically simple method, performed on whole-mount ovaries, we have obtained high-resolution images of the female reproductive cells. Furthermore, we highlight the application of this protocol for the study of loss-of-diplospory and loss-of-parthenogenesis mutants in the same species.
Fluorescence microscopy is pivotal for investigating autophagy's role in plant antiviral immunity. Here, we present a standardized procedure using complementary probes, CFP-ATG8f for autophagosomal structures and monodansylcadaverine (MDC) for autophagic vacuoles, to assess autophagy during viral infection. This combined CFP-ATG8f and MDC staining system provides a powerful, reproducible method for evaluating autophagic activity in plant-virus interactions.
This protocol outlines a reproducible method for assessing the virulence of Ustilago maydis strains in Zea mays seedlings through targeted syringe inoculation of the leaf whorl. Using seedlings at the V3-V4 growth stage ensures optimal susceptibility and developmental uniformity. The method can be used with both solopathogenic strains and compatible mating-type combinations, enabling comparative analyses of infection efficiency and symptom development. Fungal cultures are prepared under controlled conditions to maintain virulence, and post-infection humidity is regulated to enhance colonization success. Symptom progression is monitored over 10-12 days and quantified using an ordinal disease scoring system. This assay provides a robust tool for evaluating effector mutants, analyzing host-pathogen interactions, and comparing strain virulence under standardized conditions.
ChickpeaRootMorphology / geometry measurementRoot system architectureStress response / tolerance
Mechanical impedance in agricultural land is a significant constraint in modern agriculture. It dramatically affects seed germination, plant growth, development, and grain yield. Soil compaction hinders root growth and the ability to access deeper nutrients and water resources, impacting climate resilience, crop productivity, and global food security. Crops display variations in root system architecture (RSA) traits when grown in compacted soils. We can better understand the mechanisms behind soil compaction by examining root-related traits and their associated genes. Our recently published study investigated RSA traits across different soil compaction levels and identified significant genomic associations in chickpeas. We developed reliable methods for creating soils with varying bulk densities (i.e., compaction levels), growing chickpea seedlings, and harvesting the roots. We also conducted high-throughput phenotyping and screening of root-related traits using winRHIZO software. By integrating these phenotypic data with available genotypic data through Genome-Wide Association Studies (GWAS), we could identify genetic loci influencing root penetration in response to increasing soil compaction. These methods will help us identify key architectural traits of roots that can be targeted in crop breeding efforts to enhance resilience and productivity in compacted soils. By improving the root system and understanding the genes involved, we aim to develop plants more responsive to root penetration.
Accurate analysis and description of plant tissues often rely on the preparation of high-quality anatomical slides, a task that becomes particularly challenging when dealing with heterogeneous tissues such as phloem, which contains both soft and rigid components. This chapter provides a comprehensive protocol outlining key techniques for the optimal preparation of phloem tissue samples for light microscopy. The protocol encompasses essential steps such as fixation, softening, embedding, sectioning, staining, and mounting, and is adaptable for examining phloem and adjacent tissues in both woody and herbaceous stems and roots. Studying phloem anatomy is crucial for understanding nutrient transport, plant development, and responses to environmental stress, offering insights into both fundamental plant biology and practical applications in agriculture and forestry.
Fusarium wilt poses a significant threat to chickpea cultivation, causing substantial yield losses. Developing resistant chickpea varieties is a crucial strategy for managing this devastating disease. Screening a large number of germplasm and breeding lines against the pathogen is necessary to achieve this goal. In this context, the seedling root dip method has emerged as an effective technique to differentiate between resistant and susceptible chickpea genotypes. This method offers the advantages of screening a large number of lines within a short time frame and limited space. Another critical aspect of breeding for disease resistance is the rapid and accurate identification of the pathogen. Traditional pathogen detection methods are labor-intensive and time-consuming. This chapter presents a detailed protocol for the seedling root dip method, enabling the screening of chickpea genotypes against Fusarium oxysporum. Additionally, a rapid approach utilizing ITS primers for identifying the pathogen is discussed, providing a precise and expedient tool for disease resistance breeding efforts.
ChickpeaChlorophyll fluorescenceSeed / grainPhysiological trait estimationGrowth / development / phenology
Seed germination is a critical physiological process that transforms a quiescent seed into a metabolically active seedling and is also a crucial factor in determining maximum crop production. This transition is influenced by various intrinsic and extrinsic factors. Interestingly, reactive oxygen species (ROS) plays an important role in breaking seed dormancy by oxidation of biomolecules, weakening of the testa and degradation of endosperm. Similarly, molecular internal oxygen is also considered vital for the transition of dormancy to seed germination. However, it is essential to establish a correlation between the internal oxygen and the generation of ROS during seed germination. This chapter details protocols for imaging internal oxygen concentrations using VisiSens and fluorescent detection of ROS using H 2 DCFDA in chickpea seeds, complemented by qPCR analysis of key ROS-related genes (RBOH, AOX 1, UCP 1, and NADH dehydrogenase). These findings from these methods help advance our understanding of the inverse relationship between molecular oxygen and ROS dynamics during seed germination.
In the context of climate change, the global rise of temperature and intense heat waves affect plant development and productivity. In order to decipher the molecular and physiological mechanism established by plants to adapt to increased temperatures, we and others have designed different high-temperature regimes to mimic as much as possible temperature variations occurring in natura. This chapter outlines these thermotolerance assays employed to assess response to high temperature in Arabidopsis thaliana. We provide detailed guidelines, including plant age considerations and timing of heat application. Moreover, we introduce new findings showing that the addition of sucrose to the growth medium can artificially enhance thermotolerance, potentially masking stress-related phenotypes. These assays, which measure both basal and acquired thermotolerance, offer a framework for assessing plant heat stress responses in a reproducible and efficient manner. To illustrate some plant responses to these different regimes, we compare the response of mutants affected in the biosynthesis of the redox buffer glutathione with wild-type plants.
Stress memory is an adaptive strategy evolved by plants that enables them to anticipate and survive stress events in a fluctuating environment. One of the most well-studied forms of stress memory in plants is thermomemory, in which exposure to moderate heat stress primes the plants, allowing them to survive subsequent, otherwise lethal, and severe temperatures. However, despite significant progress, our current understanding of heat stress memory in plants is still not complete, particularly regarding the understanding of how this priming and memory are controlled at the molecular level. We therefore provide a detailed protocol for the thermomemory assay, as well as information on how to validate the thermomemory phenotype at both the physiological and molecular levels.
OliveMicroscopyCell / cellular structureVisualization / data management
The pollen tube is widely recognized as a suitable model for investigating the structure and spatial organization of cell wall components during polarized growth. This chapter describes the application of an established immunofluorescent labeling protocol for the localization of two major cell wall components, pectins and arabinogalactan proteins, using specific monoclonal antibodies from the JIM series. JIM5 and JIM7 were employed to detect de-esterified and esterified homogalacturonan regions of pectin, respectively, while JIM8 and JIM13 were used to label distinct epitopes of arabinogalactan proteins. The protocol includes pollen germination, paraformaldehyde fixation, enzymatic digestion with cellulysin (for arabinogalactan protein detection only), and sequential antibody incubation, followed by confocal microscopy imaging using FITC filter settings. This approach enables precise visualization of the distribution patterns of pectins and arabinogalactan proteins in the pollen tube wall and provides a reliable framework for further studies on cell wall architecture in plant reproductive tissues.
Phytoalexins are plant secondary antimicrobial compounds that are rapidly and locally accumulated de novo upon pathogen attacks. They are strongly correlated with disease resistance; therefore, the timing and the location of their synthesis and accumulation have been explored transcriptionally and metabolically using various means separately. In this chapter, by focusing on the Arabidopsis camalexin (CA), we describe protocols for multimodal in situ detection of CA and elemental distribution, as well as the transcriptionally active region of its synthesis gene PHYTOALEXIN DEFICIENT 3 (PAD3) within the same leaf sample challenged with a pathogen.
Ethylene is a versatile phytohormone that is involved in the regulation of both growth and development such as senescence, and also it can act as a signaling hormone during hypoxia. Ethylene acts alone or in interaction with different phytohormones and proteins to regulate numerous cellular processes. Accumulating evidence suggest that endogenous ethylene production and emission into atmosphere are modulated by various biotic and abiotic stresses. Since it is a gaseous hormone, a precise detection, particularly under low-oxygen (hypoxic) conditions, is important for understanding its role in regulatory processes and stress signaling pathways. Currently, measurement practices such as gas chromatography, electrochemical sensing, and optical sensing are widely employed to detect ethylene. These methods are distinct from each other in terms of sensitivity, time response, selectivity, and cost. However, each method has its own advantages and limitations. Gas chromatography (GC) is one of the best techniques that is applied for the separation and measurement of ethylene due to its volatile and supersensitive nature. In this chapter, we describe a detailed GC-based procedure specifically optimized for measuring ethylene levels during hypoxic stress application in (Oryza sativa) rice plants.
Subcellular RNA localization is an underexplored regulatory layer crucial for properly adapting cells to cellular or environmental conditions. Most studies describing RNA localization have been performed by cell fractionation and subsequent RNA quantification from pools of cells, thereby missing information about cell-to-cell variability. RNA single-molecule fluorescent in situ hybridization (smFISH) is an effective technique for detecting single RNA molecules and identifying subcellular accumulation patterns. Nevertheless, obtaining quantitative results from smFISH can be challenging in tissues with high autofluorescence, like in plants. Here, we describe an automated pipeline to detect and quantify nucleocytoplasmic RNA levels from Arabidopsis root smFISH images. This pipeline utilizes free image preprocessing, segmentation, and RNA detection software. The method permits users with any programming skills to analyze batches of images. Suggestions and recommendations for image acquisition, processing, and data analysis are included. This pipeline allows quantitative differences in nucleocytoplasmic distribution at the single-cell level to be studied under different cellular, environmental, and genetic contexts.
ArabidopsisLaboratory / benchtopMicroscopyFlowerGrowth / time-series analysisGrowth / development / phenology
During the Arabidopsis reproductive process, the female whorl of the flower, known as the gynoecium, passes seven of the 20 floral stages during its development. In each of these seven stages, specific developmental events occur, ranging from gynoecium primordium establishment to complex tissue and organ differentiation. Studying gynoecium development is important for its role in fruit and seed formation. Currently, there are many Arabidopsis lines with fluorescent proteins that provide relevant information on gynoecium patterning. However, the fluorescence of some proteins is affected during the steps of histological techniques. Furthermore, the complexity of gynoecium development makes live imaging difficult in the early stages and medial tissues. To address these issues at hand, we describe a methodology that facilitates the analysis of the fluorescent signal during gynoecium development, using as an example the pMIR164c::VENUS line.
The three fungal diseases-leaf rust, stem rust, and stripe rust-are considerable challenges to wheat production, causing up to 20%, 50%, and 70% yield losses, respectively, in North America and across the world. Control strategies include regular introduction of resistant varieties as well as fungicidal applications. Stripe rust, caused by Puccinia striiformis f. sp. tritici, is best controlled by utilizing genetic resistance and regularly introducing resistant varieties to combat the rapid breakdown of host resistance by the pathogen. Genetic resistance to stripe rust can be characterized as adult plant resistance (APR) or all-stage resistance (ASR), the former being largely preferred due to the durable mechanisms governing its resistance. Breeding programs utilize large germplasm collections to screen for resistance. Field phenotyping is a critical component for breeding programs, helping in selection of resistant breeding lines to artificial epidemics of relevant pathotypes. This chapter summarizes the methods and protocols for the field phenotyping of stripe rust at the adult-plant stage, including the steps for inoculation, phenotyping, and an overview of the favorable environmental conditions for optimal results. We also summarize methods for phenotyping leaf tip necrosis (LTN), a morphological trait expressed via slow-rusting APR genes. We demonstrated the process of screening over 30,000 lines of wheat-including the organization of entries using, ideal stage for inoculum application to screen for APR, and the ideal stage for phenotyping 10-18 days postinoculation (DPI). In conclusion, field phenotyping is critical in the assessment of stripe rust resistance and development of resistant varieties and is a major component in combatting global stripe rust epidemics in a sustainable and environmentally considerate way.
Reactive oxygen species (ROS) are pivotal in regulating plant organ abscission. The buildup of ROS in the fruitlet abscission zone (FAZ) actively triggers the abscission of litchi fruitlets. In this chapter, we present a simple method to measure intracellular ROS levels in the FAZ of litchi using 2,7-dichlorodi-hydrofluorescein diacetate (DCFH-DA). Litchi FAZ samples are transverse sectioned and then incubated with a 50 μM DCFH-DA solution at room temperature for 1 h. DCF fluorescence can be visualized using a laser scanning confocal microscope, and the fluorescence intensity is then analyzed with ImageJ software.
The gaseous hormone ethylene regulates different processes in plant life. Ethylene is generally considered as a stress hormone that is stimulated by biotic and abiotic stress. To understand its role in different arrays of plant life, in-vivo quantification of ethylene is essential to understand the physiological aspects of plant metabolism. Several techniques are employed for its accurate estimation; one such popular technique is gas chromatography. Gas chromatography is commonly employed for the estimation of different types of volatile compounds. The quantification of ethylene by gas chromatography is reliable and efficient as a large number of samples can be estimated simultaneously. The measurement of ethylene is accomplished by utilizing a standard curve that is prepared from certified ethylene gas used as the standard. Here, we describe a gas chromatography-flame ionization detection (GC-FID)-based method for the quantification of ethylene from live plants.
The detached leaf assay is a valuable method for studying plant-pathogen interactions, enabling the assessment of pathogenicity, plant resistance, and treatment effects. In this protocol, we outline how to set up a Phytophthora detached leaf assay and use non-expert machine learning tools to increase the reliability and throughput of the image analysis. Utilizing ilastik for pixel classification and Python scripts for segmentation, manual correction, and temporal linking, the pipeline provides objective and quantitative data over time. The protocol covers assay setup and image segmentation and outlines key considerations, providing a comprehensive guide for setting up and analyzing detached leaf assays. The very minimal material requirements and user-friendly software make this protocol accessible for all Phytophthora researchers.
ArabidopsisLaboratory / benchtopRootMorphology / geometry measurementRoot system architecture
Understanding the root system architecture (RSA) is necessary for elucidation of plant growth patterns in response to environmental stimuli and hormonal signals. Ethylene, a gaseous phytohormone, modulates root developmental plasticity, including primary root elongation, lateral root formation, and root hair growth. We present a protocol for mapping ethylene-specific RSA traits in Arabidopsis thaliana using a hydroponic growth system. Arabidopsis seedlings grow on a polypropylene mesh supported by polycarbonate wedges in a magenta box-based setup. We treat seedlings with ethylene or its precursor, then spread root system on agar plates with an art brush. High-resolution images are recorded and analyzed with free ImageJ software. This protocol allows detailed RSA analysis under controlled ethylene treatments and can be adapted for other plant species.
The three rust diseases, yellow (stripe) rust, black (stem) rust, and brown (leaf) rust are major challenges to wheat production, causing annual global yield losses of approximately 15 million tons valued at US$ 2.9 billion. Genetic resistance, including race-specific genes (R genes) and adult plant resistance (APR), is the primary control strategy against rust diseases. Field phenotyping plays a critical role in characterizing both types of resistance, aiding in the assessment of R and APR genes for durable resistance in breeding. Field phenotyping helps breeding programs select superior resistant germplasm by evaluating wheat lines under artificial epidemics of predominant relevant pathotypes or isolates. It allows better understanding of gene effects, interactions, stability, and responses to variable pathogen races and environments. Field phenotyping ensures rust resistance evaluations align with field circumstances and high artificial epiphytotic conditions, making breeding efforts more relevant and impactful. In conclusion, field phenotyping holds paramount importance in assessing rust resistance in wheat, providing realistic, quantitative, and environment-specific data for the development of improved wheat cultivars with enhanced rust resistance and sustainable productivity. This chapter provides a comprehensive guide to leaf and stem rust of wheat, offering a step-by-step approach to understanding these diseases and conducting field evaluations and the critical role of field phenotyping in characterizing types of resistance types. The chapter equips readers with practical insights into evaluating wheat lines under artificial epidemics, enabling researchers with the knowledge and tools necessary to contribute to breeding efforts aimed at developing improved wheat varieties with enhanced rust resistance and sustainable productivity.
Fungal pathogens present a severe risk to food systems; however, complex crop-microbe interactions are challenging to study using tools developed for model species. In particular, efficient screening and rapid assessment of microbial effectors is hindered by a lack of cloned resistance (R) genes and difficulty in validating large numbers of predicted effector candidates. This chapter describes a protocol for preparing wheat protoplasts to enable positive identification of host defense induction without overexpression of a cloned R gene, increasing the available pool of host resistance genes for screening. The assay uses polyethylene glycol (PEG)-calcium-mediated transient transfection to introduce candidate effector gene constructs into wheat protoplasts, with a defense-activated reporter for inducing a positive readout with internal normalization, indicating host recognition. This protocol provides a valuable tool for the study of host-pathogen interactions in wheat, contributing to improved resources for the development of disease-resistant crops and genome-informed pathogen surveillance.
ArabidopsisCherryCell / cellular structureFlowerVisualization / data management
Petal abscission involves cell death and reactive oxygen species (ROS) accumulation in the cells at the base of petals. Visualizing changes in the properties of these cells is crucial for analyzing and understanding petal abscission, a trait with important implications, especially for ornamental flower crops. This protocol describes the guidelines, experimental setups, and conditions for visualizing cell death by trypan blue staining and ROS accumulation by 3,3'-diaminobenzidine (DAB) staining in petals. Additionally, it provides instructions for staining and sectioning the entire Arabidopsis thaliana flower to give an improved view of the cells crucial for abscission. This protocol can be used to study the mechanism of petal abscission, including temporal changes at the base of petals during abscission and comparisons with mutants. Although Arabidopsis thaliana and cherry (Prunus sp.) blossoms are used as examples here, this protocol can easily be adapted for other plant species.
The application of fungicides is a measure complementary to host genetic resistance to control the occurrence and severity of rust diseases that has been estimated to cost over $17.25 per acre annually in wheat fields on the Canadian Prairies. The most often used fungicides include the class of demethylation inhibitors (DMIs), acting on fungal sterol biosynthesis, and the class of strobilurins (quinone outside [mitochondrial respiration] inhibitor [QoI]) acting on fungal mitochondrial respiration. Fungicides are designed to target fungal pathogens but also have been reported to trigger some effects on the host plants. Therefore, an improved diagnostic protocol is developed in this chapter for evaluating the effects and efficacy of commercial fungicides: DMI and QoI on in vitro germination of rust fungus urediniospores and rust disease development of infected, detached wheat leaves as well as whole plants. The purpose is to optimize fungicide application to better control rust fungus diseases of wheat without impacting crop growth, and while mitigating fungicide applications to minimize environmental and financial costs associated with fungicide overapplication.
Field / plotLaboratory / benchtopChlorophyll fluorescencePhysiological trait estimationPhotosynthesis / fluorescence
Chlorophyll fluorescence is a rapid and noninvasive tool used for probing the activity of photosynthesis that can be used in vivo and in the field. It is highly relevant to the demands of high-throughput crop phenotyping and can be automated or manually applied. In this chapter, we describe protocols and advice for making fast timescale fluorescence measurements using handheld equipment in the laboratory or in the field in the context of phenotyping. While interpretation of some measured parameters requires caution for the purpose of identifying underlying mechanisms, we demonstrate this technique is appropriate for some applications where convenience, rapidity, and sensitivity are required.
The plant endoplasmic reticulum forms a network of tubules connected by three-way junctions or sheet-like cisternae. Although the network is three-dimensional, in many plant cells, it is constrained to thin volume sandwiched between the vacuole and plasma membrane, effectively restricting it to a 2-D planar network. The structure of the network, and the morphology of the tubules and cisternae can be automatically extracted following intensity-independent edge-enhancement and various segmentation techniques to give an initial pixel-based skeleton, which is then converted to a graph representation. ER dynamics can be determined using optical flow techniques from computer vision or persistency analysis. Collectively, this approach yields a wealth of quantitative metrics for ER structure and can be used to describe the effects of pharmacological treatments or genetic manipulation. The software is publicly available.
Infrared thermography offers a rapid, noninvasive method for measuring plant temperature, which provides a proxy for stomatal conductance and plant water status and can therefore be used as an index for plant stress. Thermal imaging can provide an efficient method for high-throughput screening of large numbers of plants. This chapter provides guidelines for using thermal imaging equipment and illustrative methodologies, coupled with essential considerations, to access plant physiological processes.
This chapter provides a methodology for evaluating plant health and leaf characteristics using spectral reflectance. It provides a step-by-step guide to using spectrometers for high-resolution point measurements of leaf spectral reflectance and multispectral imaging for capturing spatial data, emphasizing the importance of consistent measurement conditions. The chapter further explores the intricacies of multispectral imaging, including calibration, data collection, and image processing. Finally, this chapter delves into the application of various spectral indices for the quantification of key traits such as pigment content, the status of the xanthophyll cycle, water content, and how to identify spectral regions of interest for further research and development. Serving as a guide for researchers and practitioners in plant science, this chapter provides a straightforward framework for plant health assessment using spectral reflectance.
This chapter presents the application of Plantarray, a high-throughput platform commercially available for noninvasive monitoring of plant functional physiology phenotyping (FPP). The platform continuously measures water flux in the soil-plant-atmosphere for each plant in dynamic environments. To better interpret the massive phenotypic data acquired with FPP, several quantitative analysis methods were demonstrated for various types of data. Simple mathematical models were utilized to fit characteristic parameters of plant transpiration response to drought stress. Additionally, ecophysiological models were employed to quantify the sensitivity of transpiration to radiation and vapor pressure deficit (VPD) as component traits and predict more complex higher-order traits. The established protocols provide a tangible tool for integrating FPP and model analysis to address complex traits.
In this chapter, we explore the application of high-throughput crop phenotyping facilities for phenotype data acquisition and the extraction of significant information from the collected data through image processing and data mining methods. Additionally, the construction and outlook of crop phenotype databases are introduced and the need for global cooperation and data sharing is emphasized. High-throughput crop phenotyping significantly improves accuracy and efficiency compared to traditional measurements, making significant contributions to overcoming bottlenecks in the phenotyping field and advancing crop genetics.
The study of natural variations in photosynthesis in the Brassicaceae family offers the possibility of identifying mechanisms to enhance photosynthetic efficiency in crop plants. Indeed, this family, and particularly its tribe Brassiceae, has been shown to harbor species that have a higher-than-expected photosynthetic efficiency, possibly as a result of a complex evolutionary history. Over the past two decades, methods have been developed to measure photosynthetic efficiency based on chlorophyll fluorescence. Chlorophyll fluorescence measurements are performed with special cameras, such as the FluorCams, which can be included in robotic systems to create high-throughput phenotyping platforms. While these platforms have so far demonstrated high efficiency in measuring small model species like Arabidopsis thaliana, they have the drawback of limited adaptability to accommodate different plant sizes. As a result, the range of species that can be analyzed is restricted. This chapter presents our approach to analyze the photosynthetic parameters: ϕPSII and Fv/Fm for a panel of Brassicaceae species, including a high-photosynthesis species, Hirschfeldia incana, and the adaptations to the phenotyping platform that are required to accommodate this varied group of plants.
Hyperspectral imaging is a remote sensing technique that enables remote, noninvasive measurement of plant traits. Here, we outline the procedures for camera setup, scanning, and calibration, along with the acquisition of black and white reference materials, which are the key steps in collecting hyperspectral imagery. We also discuss the development of predictive models such as partial least-squares regression, using both large and small datasets, which are used to predict plant traits from hyperspectral data. To ensure practical applicability, we provide code examples that allow readers to immediately implement these techniques in real-world scenarios. We introduce these topics to beginners in an accessible and understandable manner.
Agronomists, plant breeders, and plant biologists have been promoting the need to develop high-throughput methods to measure plant traits of interest for decades. Measuring these plant traits or phenotypes is often a bottleneck since skilled personnel, resources, and ample time are required. Additionally, plant phenotypic traits from only a select number of breeding lines or varieties can be quantified because the "gold standard" measurement of a desired trait cannot be completed in a timely manner. As such, numerous approaches have been developed and implemented to better understand the biology and production of crops and ecosystems. In this chapter, we explain one of the recent approaches leveraging hyperspectral measurements to estimate different aspects of photosynthesis. Notably, we outline the use of hyperspectral radiometer and imaging to rapidly estimate two of the rate-limiting steps of photosynthesis: the maximum rate of the carboxylation of Rubisco (V cmax ) and the maximum rate of electron transfer or regeneration of RuBP (J max ).
Canopy photosynthesis (A c ), rather than leaf photosynthesis, is critical to gaining higher biomass production in the field because the daily or seasonal integrals of A c correlate with the daily or seasonal integrals of biomass production. The canopy photosynthesis and transpiration measurement system (CAPTS) was developed to enable measurement of canopy photosynthetic CO 2 uptake, transpiration, and respiration rates. CAPTS continuously records the CO 2 concentration, water vapor concentration, air temperature, air pressure, air relative humidity, and photosynthetic photon flux density (PPFD) inside the chamber, which can be used to derive CO 2 and H 2 O fluxes of a canopy covered by the chamber. This system can also be used to measure the fluxes of greenhouse gases when integrating with CH 4 and N 2 O analyzers. Here, we describe the protocol for using CAPTS to perform experiments on rice (Oryza sativa L.) in paddy field, wheat (Triticum aestivum L.) in upland field, and tobacco (Nicotiana tabacum L.) in pots.
The pace of circadian rhythms remains relatively unchanged across a physiologically relevant range of temperatures, a phenomenon known as temperature compensation. Temperature compensation is a defining characteristic of circadian rhythms, ensuring that clock-regulated processes occur at approximately the same time of day across a wide range of conditions. Despite the identification of several genes involved in the regulation of temperature compensation, the molecular mechanisms underlying this process are still not well understood. High-throughput assays of circadian period are essential for the investigation of temperature compensation. In this chapter, we present a luciferase imaging-based method that enables robust and accurate examination of temperature compensation in the plant circadian clock.
Reactive oxygen species (ROS) production is a key early defense mechanism in plants when exposed to biotic stress. Upon recognition of conserved microbe-associated molecular patterns (MAMPs) from pathogens by plant receptors, nicotinamide adenine dinucleotide phosphate (NADPH) oxidases in the plasma membrane are activated to produce hydrogen peroxide (H 2 O 2 ). This, in turn, regulates multiple signaling pathways to trigger immunity and suppress pathogen infection. Monitoring the ROS burst in plant leaves can be done within minutes of MAMPs treatment. However, there is limited research on the quantification of ROS production in plant root tissues during the activation of plant immunity. In this study, we introduce a rapid, accessible, and straightforward technique for measuring MAMPs-triggered ROS bursts in the roots of the model legume Medicago truncatula. This method will facilitate the investigation of plant root responses to biotic and abiotic stresses.
In this protocol, we present a noninvasive in planta bioimaging technique for the analysis of hydrogen peroxide (H 2 O 2 ) and glutathione redox potential in adult Arabidopsis thaliana plants. The technique is based on the use of stereo fluorescence microscopy to image A. thaliana plants expressing the two genetically encoded fluorescent sensors roGFP2-Orp1 and Grx1-roGFP2. We provide a detailed step-by-step protocol for performing low magnification imaging with mature plants grown in soil or hydroponic systems. This protocol aims to serve the scientific community by providing an accessible approach to noninvasive in planta bioimaging and data analysis.
RootMorphology / geometry measurementRoot system architectureStress response / tolerance
Due to global warming, it is important to understand how plants respond to high ambient temperature. Plant growth responses to high ambient temperature are termed thermomophogenesis and have been explored for more than a decade. However, this was mostly focused on the above-ground part of plants, the shoot. In this chapter, we describe a simple method to assess root growth phenotype to high ambient temperatures. In principle, this protocol can be applied for any other treatments to test overall seedling growth.
In this chapter, approaches to the image analysis of the choreography of the plant endoplasmic reticulum (ER) labeled with fluorescent fusion proteins ("stars," if you wish) are presented. The approaches include the analyses of those parts of the ER that are attached through membrane contact sites to moving or non-moving partners (other "stars"). Image analysis is also used to understand the nature of the tubular polygonal network, the hallmark of this organelle, and how the polygons change over time due to tubule sliding or motion. Furthermore, the remodeling polygons of the ER interact with regions of fundamentally different topologies, the ER cisternae, and image analysis can be used to separate the tubules from the cisternae. ER cisternae, like polygons and tubules, can be motile or stationary. To study which parts are attached to non-moving partners, such as domains of the ER that form membrane contact sites with the plasma membrane/cell wall, an image analysis approach called persistency mapping has been used. To study the domains of the ER that move rapidly and stream through the cell, image analysis of optic flow has been used. However, optic flow approaches confuse the movement of the ER itself with the movement of proteins within the ER. As an overall measure of ER dynamics, optic flow approaches are of value, but their limitation as to what exactly is "flowing" needs to be specified. Finally, there are important imaging approaches that directly address the movement of fluorescent proteins within the ER lumen or in the membrane of the ER. Of these, fluorescence recovery after photobleaching (FRAP), inverse FRAP (iFRAP), and single particle tracking approaches are described.
Plants generate reactive oxygen species (ROS) during different metabolic processes, which play an essential role in coordinating growth and response. ROS levels are sensitive to environmental stresses and are often used as a marker for stress in plants. While various methods can detect ROS changes, histochemical staining with nitroblue tetrazolium (NBT) and 3,3'-diaminobenzidine (DAB) is a popular method, though it has faced criticism. This staining method is advantageous as it enables both the quantification and localization of ROS and the identification of the enzymatic origin of ROS in plants, cellular compartments, or gels. In this protocol, we describe the use of NBT and DAP staining to detect ROS generation under different stresses such as nitrogen starvation, wounding, or UV-C. Additionally, we describe the use of NBT staining for detecting enzymatic generation of ROS in native and native SDS PAGE gels. Our protocol also outlines the separation and comparison of the origin of ROS generated by xanthine dehydrogenase1 (XDH1) using different substrates.
The production of stomata, the epidermal pores of plants, is influenced by diverse environmental signals including high temperature. To assess its impact on stomatal formation, researchers need to grow plants in a carefully designed regime under controlled conditions and capture clear, microscopic views of the epidermis. Here, we describe a procedure to study the effect of high temperature on stomatal formation. This method can generate high-quality epidermal images of cotyledons, leaves, and hypocotyl of young Arabidopsis seedlings, which allow the determination of the pattern, density, and index of stomata on these tissues. Besides temperature, the protocol can serve as a general approach to examine stomatal phenotype and the effect of other external signals on stomatal formation.
The recent significant progress in developmental bio-imaging of live multicellular organisms has been greatly facilitated by the development of light sheet fluorescence microscopy (LSFM). Both commercial and custom LSFM systems offer the best means for long-term rapid data collection over a wide field of view at single-cell resolution. This is thanks to the low light exposure required for imaging and consequent limited photodamage to the biological sample, and the development of custom holders and mounting techniques that allow for specimens to be imaged in near-normal physiological conditions. This method has been successfully applied to plant cell biology and is currently seen as one of the most efficient techniques for 3D time-lapse imaging for quantitative studies. LSFM allows one to capture and quantify dynamic processes across various levels, from plant subcellular compartments to whole cells, tissues, and entire plant organs. Here we present a method to carry out LSFM on Arabidopsis leaves expressing fluorescent markers targeted to the ER. We will focus on a protocol to mount the sample, test the phototoxicity of the LSFM system, set up a LSFM experiment, and monitor the dynamics of the ER during heat shock.
The endoplasmic reticulum (ER) forms an extensive network in plant cells. In leaf cells and vacuolated root cells it is mainly restricted to the cortex, whereas in the root meristem the cortical and cytoplasmic ER takes up a large volume throughout the entire cell. Only 3D electron microscopy provides sufficient resolution to understand the spatial organization of the ER in the root. Here we present two protocols for 3D EM imaging of the ER across a range of scales. For large-scale ER structure analysis, we describe selective ER staining with ZIO that allows for automated or semi-automated ER segmentation. For smaller regions of ER, we describe high-pressure freezing, which enables almost instantaneous fixation of plant tissues but without organelle specific staining. These fixation and staining techniques are suitable for a range of imaging modalities, including serial sections, array tomography, serial block face-scanning electron microscopy (SBF-SEM), or focused ion beam (FIB) SEM.
The use of nonhost, tolerant, or resistant plants, to manage plant parasitic nematodes (PPNs), is an appealing, economic, and environmentally friendly agronomic practice, which is effective when precise information on the identification of PPN species and their virulence to target host crops is available. This chapter describes suggested protocols to evaluate the reaction of the most important crops and fruit trees to infestation by the most damaging PPN with sedentary endoparasitic habits, with the aim of assessing resistance and tolerance traits, sources of resistance in progenies from breeding programs, the reaction to nematodes of newly released cultivars, and the virulence of the most noxious PPNs. These protocols consist of classical screening techniques not involving biochemical and molecular analyses. PPN species and genera considered in this chapter include (i) the most important species of root-knot nematodes Meloidogyne spp., including also M. chitwoodi, M. enterolobii, and M. graminicola, and (ii) the cyst-forming nematodes of the genera Globodera and Heterodera, such as the potato cyst nematodes (PCNs) Globodera rostochiensis and G. pallida, and also Heterodera avenae group, H. ciceri, H. glycines, and H. schachtii. Schemes are given to identify virulence groups for most of these nematodes.
One of the major plant stress level indicators is reactive oxygen species (ROS). They have been known to play a central role in regulating plant responses to various environmental stresses. This book chapter specifically covers abiotic stress induced by a drought hormone abscisic acid and biotic stress induced by Pseudomonas syringe DC3000 on single cell-type guard cells. We describe in detail the measurement of ROS production starting from sample preparation to data analysis by fluorescence intensity acquisition using ImageJ software. We discussed the problems faced while performing the experiment and addressed how to overcome them by providing specific guidelines to ensure high quality repeatable data.
Seed / grainPhysiological trait estimationGrowth / development / phenology
Seed germination of a parasitic plant Striga hermonthica is elicited by strigolactones which are exuded from roots of host plants. Here, we describe a high-throughput germination assay and a method for visualizing in vivo strigolactone receptor functions with a fluorogenic probe.
Singlet oxygen is a reactive oxygen species that causes oxidative damage to plant cells, but intriguingly it can also act as a signalling molecule to reprogram gene expression required to induce plant physiological/cellular responses. Singlet oxygen photosensitization in plants mainly occurs in chloroplasts after the molecular collision of ground-state molecular oxygen with triplet-excited-state chlorophyll. Singlet oxygen direct detection through phosphorescence emission in chloroplasts is a herculean task due to its extremely low luminescence quantum yield. Because of this, indirect alternative methods have been developed for its detection in biological systems, for example, by measuring the changes in the EPR signal or fluorescence intensity of singlet oxygen reaction-based probes. The singlet oxygen chemiluminescence (SOCL) is a chemiluminescence probe with high sensitivity and selectivity towards singlet oxygen and promising use to detect it in living cells without the inconvenience of low stability of the EPR signal of spin probes in the presence of redox compounds, spurious light scattering coming from the light source required for the excitation of fluorescence probes or the light emission of endogenous fluorescent molecules like chlorophyll in chloroplasts. The protocol presented in this chapter describes the first steps to characterizing singlet oxygen production within the biological system under study; this is accomplished through monitoring molecular oxygen consumption by SOCL using a Clark-type oxygen electrode and measuring the chemiluminescence generated by SOCL 1,2-dioxetane using a spectrofluorometer. For singlet oxygen detection within living cells, a version of SOCL with increased membrane permeability (SOCL-CPP) is described.
Reactive oxygen species (ROS) are produced by energy transfer and electron transport in plant chloroplast thylakoids at non-toxic levels under normal growth conditions, but at threatening levels under adverse or fluctuating environmental conditions. Among chloroplast ROS, singlet oxygen and superoxide anion radical, respectively, produced by photosystem II (PSII) and PSI, are known to be the major ROS under several stress conditions. Both are very unlikely to diffuse out of chloroplasts, but they are instead capable of triggering ROS-mediated chloroplast operational retrograde signalling to activate defence gene expression in concert with hormones and other molecular compounds. Therefore, their detection, identification and localization in vivo or in biological preparations is a priority for a deeper understanding of their role in (concurrent) regulation of plant growth and defence responses. Here, we present two EPR spin traps, abbreviated as TEMPD-HCl and DEPMPO, to detect and identify ROS in complex systems, such as isolated thylakoids, together with some hints and cautions to perform reliable spin trapping experiments.
ArabidopsisPeaLaboratory / benchtopRootMorphology / geometry measurementStress / disease detectionGrowth / development / phenologyRoot system architectureStress response / tolerance
The study of root growth and plasticity in situ is rendered difficult by the opacity and mechanical barrier of soil substrates. Therefore, for the analysis of developmental processes and abiotic stress and development relationships, it is essential to set up cultivation systems that overcome these hindrances in a non-invasive and non-destructive manner. For this purpose, we have developed a useful and powerful rhizobox culture system, where the roots are separated from the soil substrate by a porous membrane with a mesh of such width that allows the exchange of water and solutes without allowing the roots to penetrate the soil. This system provides direct, easy, and quick access to the roots and allows to follow root growth and development, root system architecture, and root system plasticity at different stages of plant development and under various environmental conditions. Moreover, these rhizoboxes provide clean and intact roots that can be easily harvested to perform further physiological, biochemical, and molecular analyses at different stages of development and in response to various environmental constraints. This rhizobox method was validated by assessing root response plasticity of drought-stressed Arabidopsis and pea plants grown in soil displaying water content alterations. This rhizobox system is suitable for many types of abiotic stress-development studies, including the comparison of different stress intensities or of various mutants and genotypes.
Reactive Oxygen Species (ROS) waves serve as key systemic signals within plants. Following the initial sensation of a stress, auto-propagation of ROS (the ROS wave) begins and rapidly spreads to distant, systemic tissues of the plant and invokes important physiological responses. Highly sensitive methods capable of imaging this systemic signal at the whole-plant level have long been desired for the study of ROS signaling. Here, we describe a straightforward and highly sensitive method for the detection and quantification of ROS in planta at the whole-plant level in Arabidopsis thaliana with the In Vivo Imaging System (IVIS) Lumina S5 imaging platform and the fluorescent probe 2',7'-dichlorofluorescin diacetate (H 2 DCFDA). This method can be used for high-throughput screening of the ROS Wave within Arabidopsis plants, with up to 16 plants capable of being imaged approximately every half hour.
The shoot apical meristem is the plant tissue that produces the plant aerial organs such as flowers and leaves. To better understand how the shoot apical meristem develops and adapts to the environment, imaging developing shoot meristems expressing fluorescence reporters through laser confocal microscopy is becoming increasingly important. Yet, there are not many computational pipelines enabling a systematic and high-throughput characterization of the produced microscopy images. This chapter provides a simple method to analyze 3D images obtained through laser scanning microscopy and quantitatively characterize radially or axially symmetric 3D fluorescence domains expressed in a tissue or organ by a reporter. Then, it presents different computational pipelines aiming at performing high-throughput quantitative image analysis of gene expression in plant inflorescence and floral meristems. This methodology has notably enabled the quantitative characterization of how stem cells respond to environmental perturbations in the Arabidopsis thaliana inflorescence meristem and will open new avenues in the use of quantitative analysis of gene expression in shoot apical meristems. Overall, the presented methodology provides a simple framework to analyze quantitatively gene expression domains from 3D confocal images at the tissue and organ level, which can be applied to shoot meristems and other organs and tissues.
The microtubule (MT) network is a highly dynamic subcellular structure playing an important role in the growth and development of plants, and it is able to respond to biotic and abiotic environmental signals. Recent literature shows that microtubules play a key role in the tolerance of plants to salt stress. For example, salt stress induces microtubules to undergo a process of depolymerization-repolymerization, which is necessary for Arabidopsis seedlings to survive under these conditions. However, the potential cellular and molecular mechanisms still need to be further studied. Here, we describe the protocol for salt treatment of Arabidopsis seedlings and imaging the MT array by confocal laser scanning microscopy. We also introduce the AnalyzeSkeleton (2D/3D) plugin for quantitative analysis of the microtubule array after salt stress. The application of such an image processing method can rapidly develop an appreciation of the role of microtubules in the salt stress response of plants.
Plants are constantly exposed to various environmental stresses, among which, microbial pathogens are one of the major threats. Studies have shown that the host actin cytoskeleton undergoes active rearrangement during the plant-microbe interaction. This actin remodeling is required for plant resistance to bacterial infection. In this chapter, we introduce a protocol routinely used in our laboratory to investigate actin dynamics in response to bacterial cues. We describe the bacterial inoculation methods, plant sample preparation, and imaging techniques used to monitor actin responses in different Arabidopsis cell types including epidermal cells from light-grown leaves and dark-grown hypocotyls, as well as guard cells. We further introduce a high-throughput image analysis method for quantifying cytoskeletal changes. This protocol has allowed us to dissect the host cell contribution to actin remodeling and identify actin-binding proteins as stimulus-response regulators of the cytoskeleton.
The FAIR data principle as a commitment to support long-term research data management is widely accepted in the scientific community. However, although many established infrastructures provide comprehensive and long-term stable services and platforms, a large quantity of research data is still hidden. Currently, high-throughput plant genomics and phenomics technologies are producing research data in abundance, the storage of which is not covered by established core databases. This concerns the data volume, for example, time series of images or high-resolution hyperspectral data; the quality of data formatting and annotation, e.g., with regard to structure and annotation specifications of core databases; uncovered data domains; or organizational constraints prohibiting primary data storage outside institutional boundaries. To share these potentially dark data in a FAIR way and master these challenges the ELIXIR Germany/de.NBI service Plant Genomic and Phenomics Research Data Repository (PGP) implements an on-premise approach, which allows research data to be kept in place and wrapped in FAIR-aware software infrastructure. In this chapter, the e!DAL infrastructure software and the PGP repository are presented as best practice on how to easily setup FAIR-compliant and intuitive research data services.
Chlorophyll fluorescenceCell / cellular structureVisualization / data management
The plant cell wall comprises various types of macromolecules whose abundance and spatial distribution change dynamically and are crucial for plant architecture. High-resolution live cell imaging of plant cell wall components is, therefore, a powerful tool for plant cell biology and plant developmental biology. To acquire suitable data, the experimental setup for staining and imaging of non-fixed samples must be straightforward and avoid creating stress-induced artifacts. We present a detailed sample preparation and live image acquisition protocol for fluorescence visualization of cell wall components using commercially available probes and stains.
ArabidopsisMicroscopyCell / cellular structureVisualization / data management
Labeling of the nucleolus in Arabidopsis thaliana can be achieved by incorporation of 5'-ethynyl uridine (EU) into bulk RNA. Although EU does not selectively label the nucleolus, the abundance of ribosomal transcripts results in the predominant accumulation of the signal in the nucleolus. Ethynyl uridine has the advantage of being detected via Click-iT chemistry providing a specific signal and low background. While the protocol presented here employs fluorescent dye and allows visualization of the nucleolus by microscopy, this method can also be used for other downstream applications. Though we tested nucleolar labeling only in A. thaliana, in principle it can be applied to other plant species.
Fourier transform mid-infrared spectroscopy (FTIR) is a powerful tool for compositional analysis of plant cell walls. The infrared spectrum generates a fingerprint of a sample with absorption peaks corresponding to the frequency of vibrations between the bonds of the atoms making up the material. Here we describe a method focused on the use of FTIR in combination with principal component analysis (PCA) to characterize the composition of the plant cell wall. The FTIR method described here facilitates high-throughput identification of the major compositional differences across a large set of samples in a low-cost and non-destructive manner.
ArabidopsisMicroscopyCell / cellular structureVisualization / data management
The plant cytoskeleton is instrumental in cellular processes such as cell growth, differentiation, and immune response. Microtubules, in particular, play a crucial role in morphogenesis by governing the deposition of plant cell wall polysaccharides and, in consequence, the cell wall mechanics and cell shape. Scrutinizing the microtubule dynamics is therefore integral to understanding the spatiotemporal regulation of cellular activities. In this chapter, we outline steps to acquire 3D images of microtubules in epidermal pavement cells of Arabidopsis thaliana cotyledons using a confocal microscope. We introduce the steps to assess the microtubule distribution and organization using image processing software Bitplane Imaris and ImageJ. We also demonstrate how the interpretation of image material can be facilitated by post-processing with general-purpose image enhancement software using methods trained by artificial intelligence-based algorithms.
Plants challenged with iron deficiency produce in their roots and secrete into the rhizosphere several small molecules named coumarins that derive from the phenylpropanoid pathway. Coumarins are biosynthesized in different root cell types and transported to the root epidermis prior to their secretion in the surrounding media. Taking advantage of the natural fluorescence of most coumarins glycosides when exposed to UV light, we developed a method to uncover their individual cellular localization and accumulation. This approach couples spectral imaging acquisition and linear unmixing analysis. In this protocol, we describe guidelines, experimental setup, and conditions for the analysis of coumarins localization and accumulation in Arabidopsis thaliana root seedlings grown in control and iron deficiency conditions, at both acidic and alkaline pH.
Autofluorescence of plant tissues can be used as a label-free method to detect a range of phenolic-based cell wall components including lignin, suberin, and ferulate using widefield or confocal fluorescence microscopy. Likewise, fluorescently labeled antibodies can be used to localize specific carbohydrate molecules including arabinoxylan, β-1,4 galactan, glucomannan, glucuronoxylan, pectins, and xyloglucan. When combined, these two methods allow detailed study of topochemistry in different plant tissues for phenotyping of mutant varieties and plant biology studies. This article describes the protocols for fluorescent detection and imaging of molecules in plant cell walls using autofluorescence and immunofluorescence.
Understanding the mechanisms driving plant defense responses holds the promise to provide new means to reinforce plant defense both through agrochemicals and targeted genetic improvement. The capability to quantify impacts of phytopathogens on subcellular dynamics is particularly important when elucidating the role of specific virulence mechanisms that make contributions toward infection success but do not individually alter disease outcome. Acquiring these data requires an investigator to achieve the successful handling of both plant and microbe prior to observation and an appreciation of the challenges in acquiring images under these conditions. In this chapter we describe a protocol to support the observation of cytoskeletal dynamics surrounding sites of fungal interaction, specifically the powdery mildew Blumeria graminis f.sp. hordei on the surface of Arabidopsis thaliana. Furthermore, we also describe a procedure to expose etiolated (dark-grown) hypocotyls to a molecular pattern to activate defense responses in the absence of a phytopathogen with the aim of observing localized actin-dependent trafficking.
Major advances have been made in our understanding of anther developmental processes in flowering plants through a combination of genetic studies, cell biological technologies, biochemical analyses, microarray and high-throughput sequencing-based approaches. In this chapter, we summarize widely used protocols for pollen viability staining, investigation of anther morphogenesis by scanning electron microscopy (SEM), light microscopy of semi-thin sections, ultrathin section-based transmission electron microscopy (TEM), TUNEL (terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate (dUTP) nick end labeling) assay for tapetum programmed cell death, and laser microdissection procedures to obtain specific cells or cell layers for transcriptome analysis.
Plant phenomics field has seen a great increase in scalability in the last decade mainly due to technological advances in remote sensors and phenotyping platforms. These are capable of screening thousands of plants many times throughout the day, generating massive amounts of data, which require an automated analysis to extract meaningful information. Deep learning is a branch of machine learning that has revolutionized many fields of research. Deep learning models are able to extract autonomously the underlying features within the dataset, providing a multi-level representation of the data. Our intention is to show the feasibility and effectiveness of using deep learning and low-cost technology for automated phenotyping. In this methods chapter, we describe how to train a deep neural network to segment leaf images and extract the pixels related to the disease.
High-throughput phenotyping enables the temporal detection of subtle changes in plant plasticity and adaptation to different conditions, such as nitrogen deficiency, in an accurate, nondestructive, and unbiased way. Here, we describe a protocol to assess the contribution of nitrogen addition or deprival using an image-based system to analyze plant phenotype. Thousands of images can be captured throughout the life cycle of Arabidopsis, and those images can be used to quantify parameters such as plant growth (area, caliper length, diameter, etc.), in planta chlorophyll fluorescence, and in planta relative water content.
Meiotic recombination initiates from ~100-200 s of programmed DNA double stranded breaks (DSBs) in plants. Meiotic DSBs can be repaired using homologous chromosomes to generate a crossover . Meiotic crossover is critical for chromosomal segregation and increasing genetic variation. The number of crossovers is limited to one and three per chromosome pair in most plant species. Genetic, epigenetic, and environmental factors control crossover frequency and distribution. Due to the limited number of crossovers it is challenging to measure crossover frequency along chromosomes. We adapted fluorescence-tagged lines (FTLs ) that contain quartet1 mutations and linked transgenes expressing dsRed, eYFP, and eCFP in pollen tetrads into the deep learning-based image analysis tool, DeepTetrad. DeepTetrad enables the measurement of crossover frequency and interference by classifying 12 types of tetrads from three-color FTLs in a high-throughput manner, using conventional microscope instruments and a Linux machine. Here, we provide detailed procedures for preparing tetrad samples, tetrad imaging, running DeepTetrad, and analysis of DeepTetrad outputs. DeepTetrad-based measurements of crossover frequency and interference ratio will accelerate the genetic dissection of meiotic crossover control.
Field / plotMorphology / geometry measurementArchitecture / morphology / geometry
This work provides a high-level overview of system design considerations for measuring plant architecture traits in row crops using ground-based, mobile platforms. High-throughput phenotyping technologies are commonly deployed in isolated growth chambers or greenhouses; however, there is a need for field-based systems to measure large quantities of plants exposed to natural climates throughout a growing season. High-throughput methods using ground-based mobile systems collect valuable phenotypic information at higher temporal resolutions compared to manual methods (e.g., handheld calipers and measuring sticks). Additionally, the close proximity to plants when using ground-based systems compared to aerial platforms enables plant phenotyping at the organ level. While there is no single best platform for obtaining ground-based plant measurements across crop varieties with different planting configurations, there are a wide range of off-the-shelf systems and sensors that can be integrated to accommodate varying row widths, plant spacing, plant heights, and plot sizes, in addition to emerging commercially available platforms. This chapter will provide an overview of sensor types suitable for phenotyping plant size and shape, as well as provide guidance for deployment with ground-based systems, including push carts or buggies, modified tractors, and robotic platforms.
Color patterning contributes to important plant traits that influence ecological interactions, horticultural breeding, and agricultural performance. High-throughput phenotyping of color is valuable for understanding plant biology and selecting for traits related to color during plant breeding. Here we present ColourQuant, an automated high-throughput pipeline that allows users to extract color phenotypes from images. This pipeline includes methods for color phenotyping using mean pixel values, a Gaussian density estimator of CIELAB color, and the analysis of shape-independent color patterning by circular deformation.
High-throughput phenotyping platforms for growth chamber and greenhouse-grown plants enable nondestructive, automated measurements of plant traits including shape, aboveground architecture, length, and biomass over time. However, to establish these platforms, many of these methods require expensive equipment or phenotyping expertise. Here we present a relatively inexpensive and simple phenotyping method for imaging hundreds of small- to medium-sized growth chamber or greenhouse-grown plants with a digital camera. Using this method, we image hundreds of tomato plants in 1 day.
Photosynthetic efficiency is increasingly recognized as an integration of plant responses to dynamic environments, establishing the need for data sets from both field trials and controlled environments. A robotic field scanner phenotyping platform at the University of Arizona is equipped with a high-throughput chlorophyll fluorescence imaging system capable of collecting data on field trials for genetic studies of a photosynthetic trait (Fv/Fm). A description of the fluorescence imaging system is provided in addition to methods for measurements across experimental field plots and a test to determine the impact of variable plant heights. The overall focus is on aspects of field applications of a chlorophyll fluorescence imaging system that differ from analogous systems in controlled environments.
With a rapidly increasing population, diminishing resource availability, and variation in environment, there is a need to change agricultural production to deliver long-term food security. To deliver such change, we need crops that are productive and tolerant to different stress factors. The traditional methods of obtaining data for phenotyping under field conditions, e.g., for morphological traits such as canopy structure or physiological traits such as plant stress-related traits, are laborious and time-consuming. A variety of imaging tools in the visible, spectral, and thermal infrared ranges allow data collection for quantitative studies of complex traits and crop monitoring. These tools can be used on crop phenotyping and monitoring platforms for high-throughput assessment of traits in order to better understand plant stress responses and the physiological pathways underlying yield. The applications and brief review of these imaging techniques are described and discussed in this chapter.
Plasmodesmata (PD) facilitate the exchange of nutrients and signaling molecules between neighboring plant cells, and they are therefore essential for proper growth and development. PD have been studied extensively in efforts to elucidate the ultrastructure of individual PD nanopores and the distribution of PD in a variety of cell walls. These studies often involved the use of serial ultrathin sections and manual quantification of PD by transmission electron microscopy (TEM). In recent years, a variety of techniques that offer more amenable approaches for quantifying PD distribution have been reported. Here, we describe the quantification of PD densities using the serial scanning electron microscopy technique called focused ion beam-scanning electron microscopy (FIB-SEM). For this, resin-embedded samples prepared by standard TEM methods undergo successive rounds of imaging by SEM interspersed with milling of the sample surface by a focused beam of gallium ions to reveal a new surface. In this way, the details of the sample are sequentially revealed and imaged. Over the course of a few hours, repetitive milling and imaging facilitates the automated collection of nanometer-resolution data of several μm of sample depth. FIB-SEM can be targeted to interrogate specific cell walls and cell wall junctions, and the subsequent three-dimensional renderings of the data can be used to visualize the ultrastructural details of the sample. PD densities can then be rapidly quantified by calculating the number of PD per μm 2 of cell wall observed in the renderings.
Phenotyping specific plant traits is difficult when the samples to be measured are architecturally complex. Inflorescence and root system traits are of great biological interest, but these structures present unique phenotyping challenges due to their often complicated and three-dimensional (3D) forms. We describe how a large industrial scale X-ray tomography (XRT) instrument can be used to scan architecturally complex plant structures for the goal of rapid and accurate measurement of traits that are otherwise cumbersome or not possible to capture by other means. The combination of a large imaging cabinet that can accommodate a wide range of sample size geometries and a variable microfocus reflection X-ray source allows noninvasive X-ray imaging and 3D volume generation of diverse sample types. Specific sample fixturing (mounting) and scanning conditions are presented. These techniques can be moderate to high throughput and still provide unprecedented levels of accuracy and information content in the 3D volume data they generate.
High-throughput phenotyping (HTP) allows automation of fast and precise acquisition and analysis of digital images for the detection of key traits in real time. HTP improves characterization of the growth and development of plants in controlled environments in a nondestructive fashion. Marchantia polymorpha has emerged as a very attractive model for studying the evolution of the physiological, cellular, molecular, and developmental adaptations that enabled plants to conquer their terrestrial environments. The availability of the M. polymorpha genome in combination with a full set of functional genomic tools including genetic transformation, homologous recombination, and genome editing has allowed the inspection of its genome through forward and reverse genetics approaches. The increasing number of mutants has made it possible to perform informative genome-wide analyses to study the phenotypic consequences of gene inactivation. Here we present an HTP protocol for M. polymorpha that will aid current efforts to quantify numerous morphological parameters that can potentially reveal genotype-to-phenotype relationships and relevant connections between individual traits.
Images are used frequently in plant phenotyping to capture measurements. This chapter offers a repeatable method for capturing two-dimensional measurements of plant parts in field or laboratory settings using a variety of camera styles (cellular phone, DSLR), with the addition of a printed calibration pattern. The method is based on calibrating the camera using information available from the EXIF tags from the image, as well as visual information from the pattern. Code is provided to implement the method, as well as a dataset for testing. We include steps to verify protocol correctness by imaging an artifact. The use of this protocol for two-dimensional plant phenotyping will allow data capture from different cameras and environments, with comparison on the same physical scale. We abbreviate this method as CASS, CAmera aS Scanner.
Plants live in highly dynamic surroundings and need to cope with constant environmental challenges. In order to do so, they developed quick reactions to stress that allow them to gain time while mounting a major response. This first line of defense includes the stomata, leaf epidermal pores in charge of regulating water loss and photosynthesis. Stomatal movements are controlled by the stress phytohormone abscisic acid (ABA), which induces fast closure of the stomata upon perception of stress conditions. By modulating plasma membrane ion channels, ABA leads to loss of water from the guard cells surrounding the stomatal pore and a consequent reduction of its aperture. Here, we provide a microscopy-based method to assess the plant's response to ABA through measurements of the stomatal aperture. This protocol describes a simple, quick, and unexpensive method to prepare high-quality impressions of leaves from Arabidopsis thaliana seedlings from long-lasting silicone-based casts, allowing detailed imaging and accurate determination of the aperture of stomatal pores.
ArabidopsisLaboratory / benchtopRootMorphology / geometry measurementGrowth / time-series analysisGrowth / development / phenologyRoot system architecture
The ability of roots to orient their growth relative to the vector of gravity, root gravitropism (positive gravitropism), is observed in root systems of higher plants and is an essential part of plant growth and development. While there are various methods for quantifying root gravitropism, many methods that can efficiently measure gravitropism at a reasonable throughput do not yield temporal resolution of the process, while methods that allow for high-temporal resolution are often not suitable for an efficient measurement of multiple roots. Here, we describe a method to analyze the root gravitropism activity at an increased throughput with a fine time-resolution using Arabidopsis thaliana plants.
Recently, it has been proposed to switch molecular markers to near-infrared (NIR) spectra for inferring relationships between individuals and further performing phenomic selection (PS), analogous to genomic selection (GS). The PS concept is similar to genomic-like omics-based (GLOB) selection, in which molecular markers are replaced by endophenotypes, such as metabolites or transcript levels, except that the phenomic information obtained for instance by near-infrared spectroscopy (NIRS ) has usually a much lower cost than other omics. Though NIRS has been routinely used in breeding for several decades, especially to deal with end-product quality traits, its use to predict other traits of interest and further make selections is new. Since the seminal paper on PS , several publications have advocated the use of spectral acquisition (including NIRS and hyperspectral imaging) in plant breeding towards PS , potentially providing a scope of what is possible. In the present chapter, we first come back to the concept of PS as originally proposed and provide a classification of selected papers related to the use of phenomics in breeding. We further provide a review of the selected literature concerning the type of technology used, the preprocessing of the spectra, and the statistical modeling to make predictions. We discuss the factors that likely affect the efficiency of PS and compare it to GS in terms of predictive ability. Finally, we propose several prospects for future work and application of PS in the context of plant breeding.
Field / plotLiDAR / point cloudGrowth / time-series analysisYield / yield components
The advent of plant phenomics, coupled with the wealth of genotypic data generated by next-generation sequencing technologies, provides exciting new resources for investigations into and improvement of complex traits. However, these new technologies also bring new challenges in quantitative genetics, namely, a need for the development of robust frameworks that can accommodate these high-dimensional data. In this chapter, we describe methods for the statistical analysis of high-throughput phenotyping (HTP) data with the goal of enhancing the prediction accuracy of genomic selection (GS). Following the Introduction in Sec. 1, Sec. 2 discusses field-based HTP, including the use of unoccupied aerial vehicles and light detection and ranging, as well as how we can achieve increased genetic gain by utilizing image data derived from HTP. Section 3 considers extending commonly used GS models to integrate HTP data as covariates associated with the principal trait response, such as yield. Particular focus is placed on single-trait, multi-trait, and genotype by environment interaction models. One unique aspect of HTP data is that phenomics platforms often produce large-scale data with high spatial and temporal resolution for capturing dynamic growth, development, and stress responses. Section 4 discusses the utility of a random regression model for performing longitudinal modeling. The chapter concludes with a discussion of some standing issues.
Unmanned ground vehicles can capture a sub-canopy perspective for plant phenotyping, but their design and construction can be a challenge for scientists unfamiliar with robotics. Here we describe the necessary components and provide guidelines for designing and constructing an autonomous ground robot that can be used for plant phenotyping.
Field / plotRootMorphology / geometry measurementRoot system architecture
A method has been developed to measure root intersection density (RID) on a trench-profile in field conditions. Here we describe how 2D spatial distribution mapping of RID can be processed and converted into root length density (RLD) and root distances (ARD) using a new freeware named RACINE2.2. The software also allows a simple modeling of potential root extraction ratio in the soil (PRER). The software contains models calculating RLD, ARD, and PRER from RID for several crops (maize, sorghum, sugarcane, rice, pearl millet, pineapple, eucalyptus). Models may be changed or added into RACINE2.2. RLD, ARD, and PRER are calculated for each spatial unit and can be used to generate 2D maps using RACINE2.2. Data can be exported to a spreadsheet or a surface mapping software for further analysis. It is also possible to import data into RACINE2.2 from a spreadsheet. This application thus makes studies about root-soil interactions, root growth, and root uptake easier. It opens new avenues to characterize root systems to improve root water and nutrient uptake in field conditions.
An indoor wireless fixed camera network was developed for an efficient, cost-effective method of extracting informative plant phenotypes in a controlled greenhouse environment. Deployed at the Donald Danforth Plant Science Center (DDPSC), this fixed camera platform implements rapid and automated plant phenotyping. The platform uses low-cost Raspberry Pi computers and digital cameras to monitor aboveground morphological and developmental plant phenotypes. The Raspberry Pi is a readily programmable, credit card-sized computer board with remote accessibility. A standard camera module connects to the Raspberry Pi computer board and generates eight-megapixel resolution images. With a fixed array, or "bramble," of Raspberry Pi computer boards and camera modules placed strategically in a greenhouse, we can capture automated, high-resolution images for 3D reconstructions of individual plants on timescales ranging from minutes to hours, capturing temporal changes in plant phenotypes.
Genome-wide association studies (GWAS) have benefited from the advances of sequencing methods for the generation of high-density genomic data. By bridging genotype to phenotype, several genes have been associated with traits of agricultural interest. Despite this, there is still a gap between genotyping and phenotyping due to the large difference in throughput between the two disciplines. Although cutting-edge phenomics technologies are available to the community, their costs are still prohibitive at the small lab level. Semiautomated methods of investigation provide a valid alternative to generate large-scale phenotyping data able to deeply investigate the characteristics of different plant organs. Beyond automation, phenomics data management is another major constraint to consider; while bioinformatics pipelines are well-trained for releasing high-quality genomic data, fewer efforts have been done for phenotyping information. This chapter provides a guide for generating large-scale data related to the size and shape of fruits, leaves, seeds, and roots and for downstream analysis for curation and preparation of clean datasets, through removal of outliers and performing primary statistical analysis. Different steps to be carried out in the R environment will be shown for gathering the appropriate input information to use in GWAS avoiding any possible bias.
The analysis of dynamic cellular processes such as plant cytokinesis stands and falls with live-cell time-lapse confocal imaging. Conventional approaches to time-lapse imaging of cell division in Arabidopsis root tips are tedious and have low throughput. Here, we describe a protocol for long-term time-lapse simultaneous imaging of multiple root tips on a vertical-stage confocal microscope with automated root tracking. We also provide modifications of the basic protocol to implement this imaging method in the analysis of genetic, pharmacological or laser ablation wounding-mediated experimental manipulations. Our method dramatically improves the efficiency of cell division time-lapse imaging by increasing the throughput, while reducing the person-hour requirements of such experiments.
Seed traits can easily be assessed using image processing tools to evaluate differences in crop variety performances in response to environment and stress. In this chapter, we describe a protocol to measure seed traits that can be applied to crops with small grains, including legume grains with little modification. The imaging processing tool can be applied to process a batch of images without human intervention. The method allows evaluation of geometric and color features, and currently extracts 11 seed traits that include number of seeds, seed area, major axis, minor axis, eccentricity, and mean and standard deviation of reflectance in red, green, and blue channels from seed images. Protocols or methods, including the one described in this chapter, facilitate phenotyping seed traits in a high-throughput and automated manner, which can be applied in plant breeding programs and food processing industry to evaluate seed quality.
Climate change and environmental pollution will have a great impact on food security worldwide. More than 30% of the world's irrigated areas are estimated to be perturbed by high salinity affecting the productivity of crops. Camelina sativa, also known as false flax, is a flowering plant that is mainly cultivated as an oilseed crop that has many potential economic benefits; it can be used in food products, in industrial applications, and in animal feed and converted into biofuel. However, natural disasters due to climate events have led to significant crop losses. In this work, we developed a high-throughput phenotyping protocol to analyze the effects of different concentrations of salt on C. sativa using the McGill Plant Phenomics Platform (MP3). We present an adapted protocol to be applied with phenomics facilities in a greenhouse environment and the most effective way for high-throughput phenotyping.
Quantification of plasmodesmata density on cell interfaces of plant tissues, particularly of leaves, has been a long-standing challenge. Using electron microscopy alone to quantify plasmodesmata is difficult because of the limited surface area coverage per image and hence the need to examine large numbers of sections for robust quantification. Fluorescence microscopy provides the larger surface area coverage per image but can only visualize pit fields and not individual plasmodesma. Moreover, in pigmented tissue like leaves, imaging cell interfaces beyond the epidermal layer would also require accurate sectioning. The advent of tissue clearing techniques such as PEA-CLARITY provided the opportunity to capture all pit fields within the leaf without resorting to sectioning. This paved the way toward the development of a more robust and precise plasmodesmata density quantification method by combining the three-dimensional immunolocalization fluorescence microscopy with scanning electron microscopy (SEM). Here, I describe a protocol to quantify plasmodesmata density on cell interfaces between mesophyll and bundle sheath in C 3 and C 4 monocot leaves.
Cell death in plants plays a major role during development as well as in response to certain biotic and abiotic stresses. For example, plant cell death can be triggered in a tightly regulated way during the hypersensitive response (HR) in defense against pathogens or be elicited by pathogenic toxin deployment. Monitoring cell death and its impact on plant health can aid in the quantification of plant disease symptoms and help to identify the underlying molecular pathways. Here, we describe our current protocol for monitoring plant cell death via ion leakage and Pulse-Amplitude-Modulation (PAM) fluorometry. We further provide a detailed protocol for the sample preparation, the measurement, and the data evaluation and discuss the complementary nature of ion leakage and PAM fluorometry as well as the potential of PAM fluorometry for high-throughput screenings.
Plant plasmodesmata (PD) are complex intercellular channels consisting of a thin endoplasmic reticulum (ER) tubule enveloped by the plasma membrane (PM). PD were first observed by electron microscopy about 50 years ago and, since, numerous studies in transmission and scanning electron microscopy have provided important information regarding their overall organization, revealing at the same time their diversity in terms of structure and morphology. However, and despite the fact that PD cell-cell communication is of critical importance for plant growth, development, cellular patterning, and response to biotic and abiotic stresses, linking their structural organization to their functional state has been proven difficult. This is in part due to their small size (20-50 nm in diameter) and the difficulty to resolve these structures in three dimensions at nanometer resolution to provide details of their internal organization.In this protocol, we provide in detail a complete process to produce high-resolution transmission electron tomograms of PD. We describe the preparation of the plant sample using high-pressure cryofixation and cryo-substitution. We also describe how to prepare filmed grids and how to cut and collect the sections using an ultramicrotome. We explain how to acquire a tilt series and how to reconstruct a tomogram from it using the IMOD software. We also give a few guidelines on segmentation of the reconstructed tomogram.
In many plant tissues, division plane orientation within cell files is highly predictable since all cells divide almost perpendicular to the cell file axis. Many mutations can affect division plane orientation, and the quantification of the deviation from the expected transverse orientation in various genetic backgrounds is thus an important issue.While several software tools have been proposed for the quantification of cellular morphology in plant tissues, none of them allowed investigating division plane orientation. We propose here a complete method for measuring orientation of division planes in 2D, using an open-source ImageJ plugin named "Cell File Angles." The method comprises the staining of cell wall within whole mount roots with the calcofluor dye, the acquisition of 3D Z-stacks of the stained roots, and the measurement of cell wall orientation using image processing algorithms and semi-automated analysis.
ArabidopsisPhysiological trait estimationGrowth / development / phenology
Flowering time is one of the most important developmental transitions in plants, especially in annuals such as Arabidopsis thaliana. However, flowering is also a critical agronomic trait, as it impacts the level of vegetative biomass produced (e.g., leaves) or the amount of seed (grain) generated. Therefore, uncovering flowering phenotypes would help understand the impact of any regulatory network on the overall plant life cycle, since flowering integrates multiple cues, both environmental (e.g., photoperiod, temperature) and internal (e.g., induction/repression of specific genes, phytohormone accumulation, plant age). Although the photoperiod flowering pathway has been extensively studied, and its gene circuitry characterized in great detail, specific flowering time protocols are mostly accessible to specialized laboratories in this field. In this report, we address this knowledge gap by generating a reproducible, non-expensive, and step-by-step protocol to assess flowering time under different photoperiods. We provide a comprehensive description and highlight the major pitfalls in the process. Moreover, this protocol could be expanded to include temperature changes and thus contribute to assess the impact of both environmental conditions in the plant's decision to flower.
Lourenço TF, Cordeiro AM, Frazão J, Saibo NJM, Oliveira MM.
RiceLaboratory / benchtopRoot
Unable to move, plants are physically restrained to the place where they grow. Remarkably, plants have developed a myriad of mechanisms to perceive the surrounding environment in order to maximize growth and survival. One of those mechanisms is the ability to perceive mechanical stimulus such as touch (thigmomorphogenesis), in order to adjust growth patterns (in different organs) to either attach to or surround an object. Roots are able to perceive several mechanical forces (e.g., gravity, touch). However, being the "hidden part" of a plant, it is difficult to assess their response to mechanical stimulation. In this chapter, our team presents a simple method to evaluate rice (Oryza sativa L.) root mechanosensing response that can be used to test different conditions (e.g., hormones) affecting rice root response to touch stimulus. This method is affordable to any lab and can be upgraded with a fully automated image recording system. We provide a detailed protocol with several notes for a more comprehensive application.
The development of RGB (red, green, blue) sensors has opened the way for plant phenotyping. This is relevant because plant phenotyping allows us to visualize the product of the interaction between the plant ontogeny, anatomy, physiology, and biochemistry. Better yet, this can be achieved at any stage of plant development, i.e., from seedling to maturity. Here, we describe the use of phenotyping, based on the stay-green trait, of common bean (Phaseolus vulgaris L.) plant, as a model, stressed by water deficit, to elucidate the result of that interaction. Description is based on interpretation of RGB digital images acquired using a phenomic platform and a specific software. These images allow us to obtain a data group related to the color parameters that quantify the changes and alterations in each plant growth and development.
Guédon Y, Caraglio Y, Granier C, Lauri PÉ, Muller B.
ClassificationGrowth / time-series analysisGrowth / development / phenology
Technological breakthroughs concerning both sensors and robotized plant phenotyping platforms have totally renewed the plant phenotyping paradigm in the last two decades. This has impacted both the nature and the throughput of data with the availability of data at high-throughput from the tissular to the whole plant scale. Sensor outputs often take the form of 2D or 3D images or time series of such images from which traits are extracted while organ shapes, shoot or root system architectures can be deduced. Despite this change of paradigm, many phenotyping studies often ignore the structure of the plant and therefore loose the information conveyed by the temporal and spatial patterns emerging from this structure. The developmental patterns of plants often take the form of succession of well-differentiated phases, stages or zones depending on the temporal, spatial or topological indexing of data. This entails the use of hierarchical statistical models for their identification.The objective here is to show potential approaches for analyzing structured plant phenotyping data using state-of-the-art methods combining probabilistic modeling, statistical inference and pattern recognition. This approach is illustrated using five different examples at various scales that combine temporal and topological index parameters, and development and growth variables obtained using prospective or retrospective measurements.
In plants, the hypersensitive response (HR) is a programmed cell death modality that occurs upon recognition of harmful non-self. It occurs at the site of pathogen infection, thus preventing pathogens to live off plant tissue and proliferate. Shedding light on the molecular constituents underlying this process requires robust and quantitative methods that can determine whether plants lacking functional genes are defective in HR execution compared to wild-type controls. In this chapter, we provide two quantitative protocols in which we measure cell death from Arabidopsis thaliana leaves infected with avirulent HR-causing bacterial strains. Firstly, we use trypan blue staining to quantify the stained area of leaves upon bacterial infection using a personalized macro in the Image J (Fiji) software. Alternately, we incorporate an electrolyte leakage protocol in order to measure HR caused by different avirulent bacterial strains at different bacterial titers. We encourage users to perform a combination of both methods when assessing HR in different plant genotypes.
ArabidopsisRootMorphology / geometry measurementGrowth / time-series analysisGrowth / development / phenologyRoot system architecture
Gravity is a powerful element in shaping plant development, with gravitropism, the oriented growth response of plant organs to the direction of gravity, leading to each plant's characteristic form both above and below ground. Despite being conceptually simple to follow, monitoring a plant's directional growth responses can become complex as variation arises from both internal developmental cues as well as effects of the environment. In this protocol, we discuss approaches to gravitropism assays, focusing on automated analyses of root responses. For Arabidopsis, we recommend a simple 90° rotation using seedlings that are 5-8 days old. If images are taken at regular intervals and the environmental metadata is recorded during both seedling development and gravitropic assay, these data can be used to reveal quantitative kinetic patterns at distinct stages of the assay. The use of software that analyzes root system parameters and stores this data in the RSML format opens up the possibility for a host of root parameters to be extracted to characterize growth of the primary root and a range of lateral root phenotypes.
Metabolite profiling provides insights into the metabolic signatures, which themselves are considered as phonotypes closely related to the agronomic and phenotypic traits such as yield, nutritional values, stress resistance, and nutrient use efficiency. GC-MS is a sensitive and high-throughput analytical platform and has been proved to be a vital tool for the analysis of primary metabolism to provide an overview of cellular and organismal metabolic status. The potential of GC-MS metabolite profiling as a tool for detecting metabolic changes in plants grown in a high-throughput plant phenotyping platform was explored. In this chapter, we describe an integrated workflow of semi-targeted GC-high-resolution (HR)-time-of-flight (TOF)-MS metabolomics with both the analytical and computational steps, focusing mainly on the sample preparation, GC-HR-TOF-MS analysis part, and data analysis for plant phenotyping efforts.
The phytohormone abscisic acid (ABA) regulates various aspects of plant physiology, growth, and development to maintain a balanced plant water status. Cellular ABA levels are regulated through the combined activities of biosynthesis, catabolism, and transport proteins and depend on the developmental stage, the cell-type and on environmental conditions. Genetically encoded Förster (fluorescence) Resonance Energy Transfer (FRET)-based ABA-responsive biosensors enable the direct monitoring of ABA dynamics in intact plants. Thus, ABA biosensor-based in vivo imaging can provide novel insights about the spatiotemporal patterns of biosynthesis- and transport-dependent ABA dynamics that are required for the regulation of seed dormancy and germination, root growth and hydrotropism, and stomatal closure under water limiting conditions. Here, I describe a protocol for the in vivo analysis of ABA in 5-day-old Arabidopsis seedlings (roots) expressing the FRET-based ABA biosensor ABAleonSD1-3L21.
ArabidopsisRootClassificationMorphology / geometry measurementRoot system architecture
The root system in plants plays a fundamental role in water and nutrient uptake. Lateral roots emerge from the primary root (PR) and its directional organ growth allows the plant to strategically explore the surrounding area. Compared to the main root, lateral roots initially display a distinct gravitropic set point angle, which is established shortly after emergence. Here, we describe a unifying protocol for the morphological description and classification of emerged, young lateral roots.
Array tomography (AT) is a new high-throughput imaging method for high-resolution imaging of ultrastructure and for 3-D reconstruction of cells and organelles. Here, we describe the entire procedure for obtaining a spatial image of the distribution of plasmodesmata (PD). As example, the protocol is applied here to reconstruct the number and arrangement of PD between cells undergoing differentiation during Arabidopsis somatic embryogenesis.
SoybeanRootGrowth / time-series analysisGrowth / development / phenology
Firefly luciferase is widely used as a bioluminescence reporter, which is simple, high signal-to-noise ratio and especially suitable for the long-term analysis of circadian clock-regulated gene expression. Here, we report the method of tracking circadian rhythms in Agrobacterium rhizogenes-induced soybean hairy roots via TopCount™ Microplate Scintillation Counter or Deep-Cooled CCD camera. Using transgenic soybean hairy roots, we monitored the endogenous 24-h oscillations of clock genes expression and investigated the precise parameters of circadian rhythmicity. Researchers can easily analyze the circadian phenotype in legumes and non-legumes using bioluminescence reporters carried by the hairy roots, avoiding time-consuming transgenic work.
Monitoring prompt chlorophyll fluorescence (F) by making consecutive pulse amplitude modulation (PAM) measurements is a noninvasive, nondestructive, potentially high-throughput technique for evaluating circadian rhythms in diverse plant species. The technique is also less labor-intensive than many others currently used and requires no transgenic procedures.
Plant cells are connected by cytoplasmic bridges called plasmodesmata. Plasmodesmata are lined by the plasma membrane, essentially forming tunnels that directly connect the cytoplasm of adjacent cells through which soluble molecules can move from cell to cell. This cell-to-cell mobility is underpinned by cytoplasmic advection and diffusion in a manner dependent on molecular size. This movement of molecules is regulated by the aperture of plasmodesmata. GREEN FLUORESCENT PROTEIN (GFP) is a 27 kDa soluble protein that can move passively between cells via plasmodesmata. Thus, it serves as an ideal probe to assess plasmodesmal aperture. GFP can be transgenically produced in single cells by microprojectile bombardment-mediated transformation, and its cell-to-cell mobility can be measured by live-cell imaging and counting the number of cells (or cell layers) to which it has moved. Thus, the number of cells in which GFP is visible serves as a measure of plasmodesmal aperture and functional cell-to-cell connectivity. Here we present methods for microprojectile bombardment of GFP into leaf epidermal cells and statistical analysis of resulting data.
It is essential that the scientific community develop and deploy accurate and high-throughput techniques to capture factors that influence plant phenotypes if we are to meet the projected demands for food and energy. In recognition of this fact, multiple research institutions have invested in automated high-throughput plant phenotyping (HTPP) systems designed for use in controlled environments. These systems can generate large amounts of data in relatively short periods of time, potentially allowing researchers to gain insights about phenotypic responses to environmental, biological, and management factors. Reliable inferences about these factors depends on the use of proper experimental design when planning phenotypic studies in order to avoid issues such as lack of power and confounding. In this chapter, the topic of experimental design will be discussed, from basic principles to examples specific to controlled environment plant phenotyping. Examples will be provided based on the package agricolae in the R statistical language.
Lobos GA, Estrada F, Del Pozo A, Romero-Bravo S, Astudillo CA, Mora-Poblete F.
Due to climate change and expected food shortage in the coming decades, not only will it be necessary to develop cultivars with greater tolerance to environmental stress, but it is also imperative to reduce breeding cycle time. In addition to yield evaluation, plant breeders resort to many sensory assessments and some others of intermediate complexity. However, to develop cultivars better adapted to current/future constraints, it is necessary to incorporate a new set of traits, such as morphophysiological and physicochemical attributes, information relevant to the successful selection of genotypes or parents. Unfortunately, because of the large number of genotypes to be screened, measurements with conventional equipment are unfeasible, especially under field conditions. High-throughput plant phenotyping (HTPP) facilitates collecting a significant amount of data quickly; however, it is necessary to transform all this information (e.g., plant reflectance) into helpful descriptors to the breeder. To the extent that a holistic characterization of the plant (phenomics) is performed in challenging environments, it will be possible to select the best genotypes (forward phenomics) objectively but also understand why the said individual differs from the rest (reverse phenomics). Unfortunately, several elements had prevented phenomics from developing as desired. Consequently, a new set of prediction/validation methodologies, seasonal ambient information, and the fusion of data matrices (e.g., genotypic and phenotypic information) need to be incorporated into the modeling. In this sense, for the massive implementation of phenomics in plant breeding, it will be essential to count an interdisciplinary team that responds to the urgent need to release material with greater capacity to tolerate environmental stress. Therefore, breeding programs should (i) be more efficient (e.g., early discarding of unsuitable material), (ii) have shorter breeding cycles (fewer crosses to achieve the desired cultivar), and (iii) be more productive, increasing the probability of success at the end of the breeding process (percentage of cultivars released to the number of initial crosses).
In plants, the response to stress, such as salinity, pathogen attack, drought, high concentration of metals, hyperthermia, and hypothermia, is usually accompanied by potassium ion (K + ) leakage from the cytosol to the cell wall, mediated by plasma membrane cation conductivity. Stress-induced electrolyte leakage co-occurs with accumulation of reactive oxygen species (ROS) and calcium ions (Ca 2+ ) and often results in programmed cell death (PCD). The development of X-ray and mass spectrometry (MS) based imaging techniques has enabled insight into the spatial tissue and cell-specific redistribution of major and trace elements during the stress response. In this chapter a workflow for sample preparation, imaging, and image analysis by X-ray and MS based techniques is presented.
ArabidopsisSeed / grainPhysiological trait estimationGrowth / development / phenology
Light-dependent seed germination guarantees seedling proximity to the soil surface, enabling quick photosynthetic energy supply. While seedling hypocotyl length is mainly used in phytochrome physiological assays to determine the functional impact of photoreceptor point mutations, different intracellular localizations, or the function of signal transduction components, phytochrome-controlled seed germination offers a different, very sensitive tool to test the phytochrome photoreceptor network. Photon fluences as low as 1 nmol m -2 are sufficient to elicit the phytochrome A (phyA)-dependent very low fluence response (VLFR), whereas higher fluences (> 10 μmol m -2 ) are needed to elicit the phyB-controlled and phyB-photoreversible low fluence response (LFR). Taking advantage of the different sensitivities of both phytochromes to different light qualities and quantities, a screening protocol is presented to score germination under different light conditions.
Laboratory / benchtopChlorophyll fluorescenceMicroscopyCell / cellular structureTissueVisualization / data management
The accumulation of the cell wall component callose at plasmodesmata (PD) is crucial for the regulation of symplastic intercellular transport in plants. Here we describe protocols to fluorescently image callose in sectioned plant tissue using monoclonal antibodies. This protocol achieves high-resolution images by the fixation, embedding, and sectioning of plant material to expose internal cell walls. By using this protocol in combination with high-resolution confocal microscopy, we can detect PD callose in a variety of plant tissues and species.
Activity of proteases in tissues can be influenced by various intrinsic and extrinsic factors. One of the activities that is regularly monitored in organisms ranging from prokaryotes to metazoans is the -aspase-like activity: activity of proteases, which cleave their substrates after the negatively charged amino acid residues, especially the aspartic acid. This activity is also known as the caspase-like activity, since the caspases, metazoan cysteine proteases, are one of the best characterized proteases with Asp-directed activities. Plants do not contain caspases; however, various plant proteases have been shown to exhibit caspase-like activity including saspases, phytaspases, and legumains (VPEs). The activity of these proteases can change in plants in response to stress. Here we present a simple method for monitoring of the caspase-like protease activity in roots, which have been treated with allelopathic extracts, using a set of commercially available caspase substrates. We show that activity towards some, but not all, caspase substrates is upregulated in treated but not control samples. The protocol can be used also for other plant tissues as well as for other stressors.
The role of programmed cell death (PCD) in hypersensitive response (HR)-conferred resistance depends on the type of host-pathogen interaction and therefore has to be studied for each individual pathosystem. Here we present and explain the protocol for studying the role of PCD in HR-conferred resistance in potato plants in the interaction with the viral pathogen. As an experimental system, we use genotype Rywal, where the virus spread is restricted and HR PCD develops 3 days post potato virus Y (PVY) inoculation. As a control of virus multiplication and spread, we include its transgenic counterpart impaired in salicylic acid (SA) accumulation (NahG-Rywal), in which the HR-PCD occurs but the spread of the virus is not restricted. To follow the occurrence of virus-infected cells and/or virus multiplication outside the cell death zone, we use GFP-tagged PVY (PVY-N605(123)-GFP) which can be monitored by confocal microscopy. Any other plant-pathogen system which results in PCD development could be studied using a modified version of this protocol.
Because they are highly unsaturated, plant lipids are sensitive to oxidation and constitute a primary target of reactive oxygen species. Therefore, quantification of lipid peroxidation provides a pertinent approach to evaluating oxidative stress in plants. Here, we describe a simple method to measure upstream products of the peroxidation of the major polyunsaturated fatty acids in plants, namely, linolenic acid (C18:3) and linoleic acid (C18:2). The method uses conventional HPLC with UV detection to measure hydroxy C18:3 and C18:2 after reduction of their respective hydroperoxides. The described experimental approach requires low amounts of plant material (a few hundred milligrams), monitors oxidation of both membrane and free fatty acids, and can discriminate between enzymatic and non-enzymatic lipid peroxidation.
Plant hormones can act in synergistic and antagonistic ways in response to biotic and abiotic stresses and during plant growth and development. Thus, a technique is needed to simultaneously determine the distribution and concentration of several plant hormones. A relatively new technology, mass spectrometry imaging (MSI), enables the direct mapping and imaging of biomolecules on tissue sections. MSI permits simultaneous detection of multiple analytes on a single section of plant tissue, even in the absence of target-specific markers such as antibodies. Recently, MSI has been used to localize multiple, small molecule (m/z < 500) plant hormones by the nanoparticle-assisted laser desorption/ionization (Nano-PALDI) mass spectrometry (MS) method. Here, we illustrate a technology for multiple-hormone imaging using Nano-PALDI MSI and discuss its potential in investigating the role of hormone signaling in plant development and stress responses.
Circadian rhythms affect many aspects of a plant's metabolism including, but not limited to, photosynthesis. Here, we provide a complete protocol for determining changes in the composition of photosynthetic pigments (chlorophyll and carotenoids), and we also consider its implementation within circadian experiments. We describe how to design a circadian experiment with the goal of assessing changes in pigment composition. We then perform two consecutive approaches to track changes in pigment composition: indirect noninvasive estimation of pigment composition (by reflectance or fluorescence) followed by direct pigment analysis (by chromatography or spectrophotometry). Finally, we present several considerations regarding data analyses.
TobaccoMicroscopyCell / cellular structureGrowth / time-series analysisVisualization / data management
Transgenic tobacco BY-2 cell lines stably expressing fluorescent protein-tagged marker proteins have been used to visualize the dynamic behaviors of cytoskeletons and organelles during plant cell division. Using time-lapse confocal imaging, we recently revealed that the pharmacological disruption of actin filaments results in the abnormal organization of phragmoplast microtubules during the early phase of cytokinesis in cell cycle-synchronized BY-2 cells. Additionally, disrupting the actin filaments shortens the time from cell plate emergence to the accumulation of green fluorescent protein-tagged NACK1 kinesin on the cell plate, suggesting that there are two functionally diverse types of microtubules in the phragmoplast. We herein describe a protocol for the cell cycle synchronization of BY-2 cells and the time-lapse confocal imaging of cytokinesis combined with a treatment with an actin polymerization inhibitor and the visualization of an emerging cell plate with a vital stain. This protocol is useful for examining the dynamic changes in protein localization or the intracellular architecture and the effects of actin disruption during plant cell division.
Serial block electron microscopy (SB-EM) is a technique that enables acquisition and reconstruction of 3D cellular volumes. The approach is valuable for the study of plasmodesmata (PD) as the relative positions of these structures are contained in the datasets. In this chapter, we describe how to prepare plant roots for SB-EM via fixation, embedding, and trimming steps. We also provide details and recommendations for later image acquisition and processing. The procedure is suitable to work on root vascular tissues.
Field phenotyping of crops has recently gained considerable attention leading to the development of new protocols for recording plant traits of interest. Phenotyping in field conditions can be performed by various cameras, sensors, and imaging platforms. In this chapter, practical aspects as well as advantages and disadvantages of aboveground phenotyping platforms are highlighted with a focus on drone-based imaging and relevant image analysis for field conditions. It includes useful planning tips for experimental design as well as protocols, sources, and tools for image acquisition, preprocessing, feature extraction, and machine learning highlighting the possibilities with computer vision. Several open and free resources are given to speed up data analysis for biologists.This chapter targets professionals and researchers with limited computational background performing or wishing to perform phenotyping of field crops, especially with a drone-based platform. The advice and methods described focus on potato but can mostly be used for field phenotyping of any crops.
Plant phenotyping has garnered major attention in recent years, leading to developing new strategies to measure and assess plant traits of interest. For data acquisition of large fields, devices and sensors are required that deliver detailed and reproducible temporal and spatial information on the cultivated crop. This work proposes the potential use of low-cost light drones for in-field phenotyping applications on cereal crops. The proposed method allows to obtain precise measurements of color and height of the plants for the individual plots. The method is based on a color calibration algorithm (TPS-3D interpolating function) and a 3D ortho image reconstruction. The method has been applied on an experimental field with durum and soft wheat parcels obtaining information on real color (with an error lower than 12/256) and height for each single plot.
RGB / grayscaleMultispectral / hyperspectralRootMorphology / geometry measurementPhysiological trait estimationRoot system architectureWater status / transpiration
Phenotyping root systems provide essential information for plant breeding, particularly aiming for better abiotic stress resistance. Rhizobox systems provide a field-near growth environment for in situ imaging of root systems in soil. A protocol for RGB and hyperspectral imaging of rhizobox-grown plants is presented that enables gathering of root structural (morphology, architecture) as well as functional (water content, decomposition) information. The protocol exemplifies the setup of a root phenotyping platform combining low-cost RGB with advanced short-wave infrared hyperspectral imaging. For both types of imaging approach, the essential steps of an image analysis pipeline are provided to retrieve biological information on breeding-relevant traits from the imaging datasets.
Sturtevant D, Aziz M, Romsdahl TB, Corley CD, Chapman KD.
Raman / spectroscopyTissue
Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has emerged as a major analytical platform for the determination and localization of lipid metabolites directly from tissue sections. Unlike analysis of lipid extracts, where lipid localizations are lost due to homogenization and/ or solvent extraction, MALDI-MSI analysis is capable of revealing spatial localization of metabolites while simultaneously collecting high chemical resolution mass spectra. Important considerations for obtaining high quality MALDI-MS images include tissue preservation, section preparation, MS data collection and data processing. Errors in any of these steps can lead to poor quality metabolite images and increases the chance for metabolite misidentification and/ or incorrect localization. Here, we present detailed methods and recommendations for specimen preparation, MALDI-MS instrument parameters, software analysis platforms for data processing, and practical considerations for each of these steps to ensure acquisition of high-quality chemical and spatial resolution data for reconstructing MALDI-MS images of plant tissues.
Rice is the most salt-sensitive cereal, suffering yield losses above 50% with soil salinity of 6 dS/m. Thus, understanding the mechanisms of rice salinity tolerance is key to address food security. In this chapter, we provide guidelines to assess rice salinity tolerance using a high-throughput phenotyping platform (HTP) with digital imaging at seedling/early tillering stage and suggest improved analysis methods using stress indices. The protocols described here also include computer scripts for users to improve their experimental design, run genome-wide association studies (GWAS), perform multi-testing corrections, and obtain the Manhattan plots, enabling the identification of loci associated with salinity tolerance. Notably, the computer scripts provided here can be used for any stress or GWAS experiment and independently of HTP.
Forward genetics has been extremely powerful for dissecting biological pathways in various model organisms. However, it is limited by the fact that redundant gene families and essential genes cannot be readily uncovered through such methods. Chemical genetics, on the other hand, provides a valuable complementary approach to probe biological processes and is suitable for not only genetic model organisms but also genetically less tractable species. We describe here a high-throughput chemical genetic screening method simply based on plant growth and developmental phenotypes in Arabidopsis. It was successfully utilized to study plant immunity and can be easily adapted for dissecting other plant signal transduction pathways.
Fluorescent biosensors are powerful tools for tracking analytes or cellular processes in live organisms and allowing visualization of the spatial and temporal dynamics of cellular regulators. Fluorescent protein (FP)-based biosensors are extensively employed due to their high selectivity and low invasiveness. A variety of FP-based biosensors have been engineered and applied in plant research to visualize dynamic changes in pH, redox state, concentration of molecules (ions, sugars, peptides, ATP, reactive oxygen species, and phytohormones), and activity of transporters. In this chapter, we briefly summarize reported uses of FP-based biosensors in planta and show simple methods to monitor the dynamics of intracellular Ca 2+ in Arabidopsis thaliana using a ratiometric genetically encoded Ca 2+ indicator, MatryoshCaMP6s.
Potato bacterial wilt is caused by the devastating bacterial pathogen Ralstonia solanacearum. Quantitative resistance to this disease has been and is currently introgressed from a number of wild relatives into cultivated varieties through laborious breeding programs. Here, we present two methods that we have developed to facilitate the screening for resistance to bacterial wilt in potato. The first one uses R. solanacearum reporter strains constitutively expressing the luxCDABE operon or the green fluorescent protein (gfp) to follow pathogen colonization in potato germplasm. Luminescent strains are used for nondestructive live imaging, while fluorescent ones enable precise pathogen visualization inside the plant tissues through confocal microscopy. The second method is a BIO-multiplex-PCR assay that is useful for sensitive and specific detection of viable R. solanacearum (IIB-1) cells in latently infected potato plants. This BIO-multiplex-PCR assay can specifically detect IIB-1 sequevar strains as well as strains belonging to all four R. solanacearum phylotypes and is sensitive enough to detect without DNA extraction ten bacterial cells per mL in complex samples.The described methods allow the detection of latent infections in roots and stems of asymptomatic plants and were shown to be efficient tools to assist potato breeding programs.
Since the recognition of the reactive oxygen species singlet oxygen ( 1 O 2 ) as a versatile signal that induces various stress responses, the mechanisms underlying 1 O 2 -induced signaling transduction pathways have become the subject of much current research. This in turn highlights the need for reliable detection methods for 1 O 2 . Here we describe a protocol for the detection of 1 O 2 using a commercially available fluorescent probe (Singlet Oxygen Sensor Green) and provide a simple method for direct visualization and quantification of the 1 O 2 -evolving photosensitizer protochlorophyllide in the Arabidopsis fluorescent mutant.
Late blight in potato, caused by the oomycete Phytophthora infestans, is a devastating disease that significantly impacts potato production. For a proper understanding of disease development, it is important to understand the interaction between plant and pathogen at a molecular level. Like other pathogens, P. infestans secretes effector molecules, which can be recognized by receptors in the plant and trigger immunity. In addition, effectors from P. infestans have been identified to enhance disease development. Here, we describe an assay to investigate the role of effectors in virulence of P. infestans on potato. We combine agroinfiltration to transiently express effectors in potato with detached leaf assays to monitor disease development. This protocol makes it possible to conveniently quantify the effect of individual effectors on virulence of P. infestans. The identification of effectors with an important role in late blight development can help to design better strategies to control the disease.
Understanding the biological background of strigolactone (SL) structural diversity and the SL signaling pathway at molecular level requires quantitative and sensitive tools that precisely determine SL dynamics. Such biosensors may be also very helpful in screening for SL analogs and mimics with defined biological functions.Recently, the genetically encoded, ratiometric sensor StrigoQuant was developed and allowed the quantification of the activity of a wide concentration range of SLs. StrigoQuant can be used for studies on the biosynthesis, function and signal transduction of this hormone class.Here, we provide a comprehensive protocol for establishing the use of StrigoQuant in Arabidopsis protoplasts. We first describe the generation and transformation of the protoplasts with StrigoQuant and detail the application of the synthetic SL analogue GR24. We then show the recording of the luminescence signal and how the obtained data are processed and used to assess/determine SL perception.
Light triggers changes in plant nuclear architecture to control differentiation, adaptation, and growth. A series of genetic, molecular, and imaging approaches have revealed that the nucleus forms a hub for photo-induced protein interactions and gene regulatory events. However, the mechanism and function of light-induced nuclear compartmentalization is still unclear. This chapter provides detailed experimental protocols for examining the morphology and potential functional significance of light signaling components that localize in light-induced subnuclear domains, also known as photobodies. We describe how immunolabeling of endogenous proteins and fluorescent in situ hybridization (FISH) could be combined with confocal imaging of fluorescently tagged proteins to assess co-localization in Arabidopsis nuclei. Furthermore, we employ a super-resolution imaging approach to study the morphology of photobodies at unprecedented detail.
ChickpeaLaboratory / benchtopRootMorphology / geometry measurementRoot system architecture
A semi-hydroponic phenotyping platform was constructed using inexpensive and easily obtained materials for characterizing root trait variability in a large set of chickpea (Cicer arietinum) germplasm. The system was designed to accommodate a large number of plants in a small area allowing relatively deeper root development, and thus serves as a high-throughput phenotyping tool for studying root dynamic growth. The root trait quantitative platform could provide accurate phenotyping data for parameterizing root models and for genome-wide association analyses or mapping studies of quantitative trait loci.
Quantitative assessment of freezing tolerance is essential to unravel plant adaptations to cold temperatures. Not only the survival of whole plants, but also impairment of detached leaves or small rosettes after a freeze-thaw cycle can be used to accurately quantify plant freezing tolerance in terms of LT 50 values. Here we describe two methods to determine the freezing tolerance of detached leaves or rosettes using a full or selected set of freezing temperatures and an additional method using chlorophyll fluorescence as a different physiological parameter. Firstly, we illustrate how to assess the integrity of (predominantly) the plasma membrane during freezing using an electrolyte leakage assay. Secondly, we provide a chlorophyll fluorescence imaging protocol to determine the freezing tolerance of the photosynthetic apparatus.
Determining pollen viability and other physiological parameters is of critical importance for evaluating the reproductive capacity of plants, both for fundamental and applied sciences. Flow cytometry is a powerful high-performance high-throughput tool for analyzing large populations of cells that has been in restricted use in plant cell research and in pollen-related studies, it has been minimized mostly for determination of DNA content. Recently, we developed a flow cytometry-based approach for robust and rapid evaluation of pollen viability that utilizes the reactive oxygen species (ROS) fluorescent reporter dye H 2 DCFDA (Luria et al., Plant J 98(5):942-952, 2019). This new approach revealed that pollen from Arabidopsis thaliana and Solanum lycopersicum naturally distribute into two subpopulations with different ROS levels. This method can be employed for a myriad of pollen-related studies, primarily in response to stimuli such as biotic or abiotic stress. In this chapter, we describe the protocol for H 2 DCFDA staining coupled with flow cytometry analysis providing specific guidelines. These guidelines are broadly applicable to many other types of cellular reporters to further develop this novel approach in the field of pollen biology.
Elastic properties of the cell wall play a key role in regulating plant growth and morphogenesis; however, measuring them in vivo remains a challenge. Although several new methods have recently become available, they all have substantial drawbacks. Here we describe a detailed protocol for osmotic treatments, which is based on the idea of releasing the turgor pressure within the cell and measuring the resulting deformation. When placed in hyperosmotic solution, cells lose water via osmosis and shrink. Confocal images of the tissue, taken before and after this treatment, are quantified using high-resolution surface projections in MorphoGraphX. The cell shrinkage observed can then be used to estimate cell wall elasticity. This allows qualitative comparisons of cell wall properties within organs or between genotypes and can be combined with mechanical simulations to give quantitative estimates of the cells' Young's moduli. We use the abaxial sepal of Arabidopsis thaliana as an easily accessible model system to present our approach, but it can potentially be used on many other plant organs. The main challenges of this technique are choosing the optimal concentration of the hyperosmotic solution and producing high-quality confocal images (with cell walls visualized) good enough for segmentation in MorphoGraphX.
Development and growth of plant organs is determined by a myriad of molecular processes that occur in each individual cell. As a direct consequence of these processes, cells alter in size and shape. They therefore serve as excellent parameters to thoroughly understand gene function. However, conventional single-plane analyses fail to accurately capture cell metrics. Here, we present a comprehensive illustrated guide that demonstrates how SCRI Renaissance 2200 staining of Arabidopsis thaliana embryos and roots can be combined with the open-source application MorphoGraphX to quantify cell parameters in 3D. We compare this staining method with other common staining techniques and provide examples of embryo and root tissue segmentation. With our novel approach, subtle single-cell phenotypes can be identified in their native context, providing new possibilities to dissect gene networks.
ArabidopsisMicroscopyCell / cellular structureVisualization / data managementGrowth / development / phenology
Virtually all growth, developmental, physiological, and defense responses in plants are accompanied by reorganization of subcellular structures to enable altered cellular growth, differentiation or function. Visualizing cellular reorganization is therefore critical to understand plant biology at the cellular scale. Fluorescently labeled markers for organelles, or for cellular components are widely used in combination with confocal microscopy to visualize cellular reorganization. Early during plant embryogenesis, the precursors for all major tissues of the seedling are established, and in Arabidopsis, this entails a set of nearly invariant switches in cell division orientation and directional cell expansion. Given that these cellular reorganization events are genetically regulated and coupled to formative events in plant development, they offer a good model to understand the genetic control of cellular reorganization in plant development. Until recently, it has been challenging to visualize subcellular structures in the early Arabidopsis embryo for two reasons: embryos are deeply embedded in seed coat and fruit, and in addition, no dedicated fluorescent markers, expressed in the embryo, were available. We recently established both an imaging approach and a set of markers for the early Arabidopsis embryo. Here, we describe a detailed protocol to use these new tools in imaging cellular reorganization.
The colonization of a host plant root by arbuscular mycorrhizal (AM) fungi is a progressive process, characterized by asynchronous hyphal growth in intercellular and intracellular spaces, leading to the coexistence of diverse intraradical structures, such as hyphae, coils, arbuscules, and vesicles. In addition, the relative abundance of intercellular and intracellular fungal structures is highly dependent on root anatomy and the combination of plant and fungal species. Lastly, more than one fungal species may colonize the same root, adding a further level of complexity. For all these reasons, detailed imaging of a large number of samples is often necessary to fully assess the developmental processes and functionality of AM symbiosis. To this aim, the use of rapid and efficient staining methods that can be used routinely is crucial.We herein present a simple protocol to obtain high detail images of both overall intraradical fungal colonization pattern and fine morphology, in AM root sections of Lotus japonicus. The procedure is based on tissue clearing, fluorescent staining of fungal cell walls with fluorescein isothiocyanate-conjugated wheat germ agglutinin (FITC-WGA), and the combined counterstaining of plant cell walls with propidium iodide (PI). The resulting images can be acquired using traditional or confocal fluorescence microscopes and used for qualitative and quantitative analyses of fungal colonization, of particular interest for the comparison of mycorrhizal phenotypes between different experimental conditions or genetic backgrounds.
The question of how pollen tubes orient themselves on their way to the egg cell is a major focus of plant reproduction research. The role of physical guidance through the tissues of the pistil in relation to the mechanical perception and growth adaptation of the pollen tubes has not been sufficiently investigated. In order to advance research on the mechanical perception of pollen tubes and their force application during invasive growth, we present simple methods for the observation and mechanical characterization of pollen tubes in vitro, which can be established with little effort in any biological laboratory with standard equipment. Pollen grains are germinated in a hydrogel containing agarose and their growth is recorded in 3D using brightfield microscopy. Using suitable analysis software, parameters such as growth rate and pollen tube diameter can then be determined to estimate the exerted penetration force.
Determining when a barley plant starts and finishes meiosis is not trivial as when the spikelets undergo meiosis, the spike is not visible as it is still well within the leaf sheath on the developing tiller. This is a general constraint for any experiment involving meiosis, such as cytology, RNA extractions, or abiotic stress treatments aiming to target such a developmental stage. The lack of synchronicity between barley tillers within the same plant exacerbates the difficulty to determine the overall meiotic stage of a plant at a certain time.Given the lack of a nondestructive staging system for predicting the entry into meiosis and the problems of working with large pot plant systems, a modular plant growing is proposed. This system enables the growth of a high number of plants in a small surface, each producing a single tiller. The modular tray system was used to generate a nondestructive prediction tool for meiosis by using external morphological features. As an example, the system is used here for heat treating F 1 plants in early meiosis stages to modify recombination.
Compared with small model plants like Arabidopsis containing ovules with few cell layers, embryo sac and embryo development of model crop plants such as maize and other grasses are difficult to image. Multiple layers of tissue usually surround the deeply embedded embryo sac and developing embryo. Moreover, reliable cell biological marker lines labeling, for example, nuclei, plasma membrane, cell walls, or cells of a specific identity are often not available. The introduction of markers to study mutants is difficult and time-consuming and may require several generations of backcrosses. In this chapter, we therefore present an easy protocol to image maize ovaries and developing embryo sacs before and after fertilization allowing also high-throughput mutant analysis. The laborious embedding of samples and preparation of thin sections are omitted in this fixing-Feulgen staining-clearing (FFC) method. Optical sectioning through multiple layers of tissue is possible allowing 3D reconstructions of the whole embryo sac if necessary. The advantage of staining cell nuclei using the FFC method described here compared, for example, with DAPI staining is a wide range of Schiff's type reagents available for the Feulgen reaction. Depending on the reagent of choice, various conditions such as different excitation/emission filters or even white light can be applied for imaging. Moreover, in order to better visualize cell division, nuclei polarity as well as cell extent and integrity, periodic acid staining (PAS) of cell walls can be combined with Feulgen staining.
Gene expression is tightly linked to the position of genes in the nucleus. Genomic regions associated with the nuclear envelope are usually repressed, including the heterochromatin carrying chromocenters. The shape and size of nuclei varies within tissues in plants and is dependent on proteins associated with the nuclear envelope. Here, we describe a protocol to isolate Arabidopsis thaliana nuclei and measure their size and morphology. Using this method, novel components regulating the nuclear envelope and chromatin association can be identified and analyzed.
Mechanical wounding of plant tissues triggers many different responses (Savatin DV, Gramegna G, Modesti V, Front Plant Sci 5:470, 2014). These are primarily mediated by the plant hormone Jasmonic Acid Isoleucine (JA-Ile). Recently, a fluorescent biosensor for JA-Ile showed that sample preparation (i.e., handling of samples) for fluorescent microscopy very often triggers wound response, even without apparent damage to the seedling, affecting downstream analyses (Larrieu A, Champion A, Legrand J, Nat Commun 6:6043, 2015). In this chapter, we describe how to overcome this technical limitation to monitor any fluorescent reporter or dye in response to wounding, using any type of fluorescent or confocal (inverted or upright, laser scanning or spinning disc) microscopes. Pharmacological or wound treatments can easily be performed and responses monitored over long periods of time. We further describe a simple method to extract and analyse quantitative data from confocal images using the open source software Fiji (Fiji Is Just ImageJ (Schindelin J, Arganda-Carreras I, Frise E, Nat Methods 9:676-682, 2012)) and OpenOffice.
Mutant phenotype observation is the most useful and important method to study which biological process a gene-of-interest is involved in. In flowering plants, excessive pollen grains land and germinate on the stigma, then pollen tubes grow through the transmitting tract to reach the ovules, eventually enter the micropyle to complete double fertilization. First, for mutants whose homozygotes could not be obtained due to pollen tube defects, it is difficult to observe the defect phenotype since the pollen grains of different genotypes are mixed together. Here, we provide a detailed protocol to pick out mutant pollen grains from the heterozygous mutant plants in Arabidopsis thaliana. By using this method, we could obtain sufficient mutant pollen grains for phenotypic analysis. Second, it is difficult to compare the pollen/pollen tube behavior of two different genotypes/species in vivo in a same pistil. Here, we develop a new dual staining method which combines GUS staining with aniline blue staining. By using this method, we can analyze the competence of the two different pollen tubes in the same pistil.
Low temperature is an important determinant in the configuration of natural plant communities and defines the range of distribution and growth of important crops. Some plants, including Arabidopsis thaliana, have evolved sophisticated adaptive mechanisms to tolerate freezing temperatures. Central to this adaptation is the process of cold acclimation. By means of this process, many plants from temperate regions are able to develop or increase their freezing tolerance in response to low, nonfreezing temperatures. The identification and characterization of factors involved in freezing tolerance is crucial to understand the molecular mechanisms underlying the cold acclimation response and has a potential interest to improve crop tolerance to freezing temperatures. Many genes implicated in cold acclimation have been identified in numerous plant species by using molecular approaches followed by reverse genetic analysis. Remarkably, however, direct genetic analyses have not been conveniently exploited in their capacity for identifying genes with pivotal roles in that adaptive response. In this chapter, we describe a protocol for evaluating the freezing tolerance of both nonacclimated and cold acclimated Arabidopsis plants. This protocol allows for the accurate and simple screening of mutant collections for the identification of novel factors involved in freezing tolerance and cold acclimation.
Plastids are cell organelles that, beside other functions, have the capability to store carotenoids in specialized structures, which may vary among the different plant species, tissues or according to the carotenoid complement. Fruits are an important source of carotenoids, and during ripening, chloroplasts differentiate into chromoplasts that are able to accumulate large amounts of carotenoids, rendering then the characteristic fruit coloration. Whereas lycopene or β-carotene may accumulate as crystal in the chromoplasts of some fruit, other xanthophyll-accumulating fruits differentiate plastoglobuli as a preferred system to enhance carotenoids stability and storage. Visualization of plastid ultrastructure and their transformation during ripening or in fruit of contrasting coloration are fundamental objectives within carotenoids research in fruits. Therefore, in this chapter, we describe a protocol for the visualization and analysis of plastid ultrastructure by transmission electron microscopy (TEM), specially designed and adapted to fruit tissues.
During meiosis, accurate segregation of chromosomes requires the formation of bivalents at metaphase I. In autopolyploids, there are more than two copies of each chromosome with the same chance to form chiasmata at meiosis. This leads to the formation of multivalent configurations in which chiasma quantification is rather complicated. Here, we present an improved cytological protocol, including fluorescence in situ hybridization, to obtain high quality spreads of metaphase I chromosomes from Arabidopsis thaliana autotetraploids. This method allows an accurate analysis of the different meiotic configurations and enables the assessment of the number of chiasmata formed by each tetrasome (group of four homologs).
Reverse genetics approaches for characterizing phenotypes of mutants in a gene of interest (GOI) require thorough genotyping and phenotypic analysis. However, special challenges are encountered when a GOI is expressed in reproductive tissues: a variety of assays are required to characterize the phenotype and a mutant may show sporophytic and/or gametophytic defects in male and/or female reproductive tissues, which are structurally and functionally intertwined. Here, we present a streamlined workflow to characterize mutants with reproductive defects, primarily using Arabidopsis as a model, which can also be adapted to characterize mutants in other flowering plants. Procedures described here can be used to distinguish different kinds of reproductive defects and pinpoint the defective reproductive step(s) in a mutant. Although our procedures emphasize the characterization of mutants with male reproductive defects, they can nevertheless be used to identify female reproductive defects, as those defects could manifest alongside, and sometimes require, male reproductive tissues.
Thrips are tiny, cell-content-feeding insects that are a major pest on crops and ornamentals. Besides causing direct feeding damage, thrips may also cause indirect damage by vectoring tospoviruses. Novel resistance mechanisms to thrips need to be discovered and validated. Induction of jasmonic acid-dependent defenses has been demonstrated to be essential for resistance to thrips, but underlying mechanisms still need to be discovered. For this, it is vital to use robust plant-thrips assays to analyze plant defense responses and thrips performance. In recently developed high-throughput phenotyping platforms, the feeding damage that is visible as silver spots, and the preference of thrips in a two-choice setup is assessed, using leaf discs. Here, we describe whole-plant thrips assays that are essential for (1) validation of findings obtained by the leaf disc assays, (2) assessment of longer-term effects on thrips feeding success and fecundity, (3) determination of spatial-temporal effects induced by primary thrips infestation on a secondary attack by thrips or other insects or pathogens, and (4) assessment of gene expression and metabolite changes. We present detailed methods and tips and tricks for (a) rearing and selection of thrips at different developmental stages, (b) treatment of the whole plant or an individual leaf with thrips, and (c) determination of feeding damage and visualization of thrips oviposition success in leaves.
Darrier B, Arrieta M, Mittmann SU, Sourdille P, Ramsay L, Waugh R, Colas I.
BarleyWheatLaboratory / benchtopMicroscopyCell / cellular structureVisualization / data management
Wheat and barley have large genomes of 15 Gb and 5.1 Gb, respectively, which is much larger than the human genome (3.3 Gb). The release of their respective genomes has been a tremendous advance the understanding of the genome organization and the ability for deeper functional analysis in particular meiosis. Meiosis is the cell division required during sexual reproduction. One major event of meiosis is called recombination, or the formation of crossing over, a tight link between homologous chromosomes, ensuring gene exchange and faithful chromosome segregation. Recombination is a major driver of genetic diversity but in these large genome crops, the vast majority of these events is constrained at the end of their chromosomes. It is estimated that in barley, about 30% of the genes are located within the poor recombining centromeric regions, making important traits, such as resistance to pest and disease for example, difficult to access. Increasing recombination in these crops has the potential to speed up breeding program and requires a good understand of the meiotic mechanism. However, most research on recombination in plant has been carried in Arabidopsis thaliana which despite many of the advantages it brings for plant research, has a small genome and more spread out of recombination compare to barley or wheat. Advance in microscopy and cytological procedures have emerged in the last few years, allowing to follow meiotic events in these crops. This protocol provides the steps required for cytological preparation of barley and wheat pollen mother cells for light microscopy, highlighting some of the differences between the two cereals.
ChickpeaLaboratory / benchtopRootGrowth / development / phenology
Chickpea is a major protein source in low socio-economic classes and cultivated in marginal soil without fertilizer or irrigation. As a result of its root nodule formation capacity chickpea can directly use atmospheric nitrogen. Chickpea is recalcitrant to stable transformation, particularly root regeneration efficiency of chickpea is low. The composite plant-based system with a non-transformed shoot and transformed root is particularly important for root biologist and this approach has already been used successfully for root nodule symbiosis, arbuscular mycorrhizal symbiosis, and other root-related studies. Use of fluorescent marker-based approach can accurately identify the transformed root from its non-transgenic counterpart. RNAi-based gene knockout, overexpression of genes, promoter GUS analysis to understand tissue specific expression and localization of protein can be achieved using the hairy root-based system. We have already published a hairy root-based transformation and composite plant regeneration protocol of chickpea. Here we are describing the recent modification that we have made to increase the transformation frequency and nodule morphology. Further, we have developed a pouch based artificial system, large number of plants can be scored for its nodule developmental phenotype, by using this system.
This chapter describes methods to enhanced contrast of plant material processed by high-pressure freezing and freeze substitution for improved visualization by serial block-face scanning electron microscopy (SBEM). The contrast enhancing steps are based on a protocol involving the sequential incubation of samples in heavy metals and sodium thiocarbohydrazide (OTO staining). We also describe the pipeline for imaging plant tissues in a commercial SBEM system (Gatan 3View ® ) and routines for the image analysis and three-dimensional reconstructions using open-source and commercial software packages.
Attenuated total reflectance Fourier transform mid-infrared (ATR-FTIR) spectroscopy is widely applicable for the chemical analysis of biological materials, relatively inexpensive, requires only simple sample preparation, and is of comparatively high-throughput compared to traditional wet chemical or chromatographic methods. It is particularly well suited for the nondestructive analysis of dried and finely ground plant samples for the subsequent prediction of cell wall and other compositional or processing parameters using chemometric regression models. Furthermore, analysis of mid IR spectra by nonregression methods (e.g., principal component analysis) provides a straightforward approach for multivariate comparison of the effects of experimental, processing, and environmental treatments, and genotypic and temporal differences on chemical composition including changes in cell wall composition. There is thus great potential for using ATR-FTIR in the lignocellulosic biomass industry at a number of levels. Here we describe methods for cell wall sample preparation and generation of ATR-FTIR spectra, and suggest techniques for the statistical analysis and/or chemometric pattern recognition between the analyzed samples.
Confocal microscopy is widely used to live-image plant tissue. Cell outlines can be visualized using fluorescent probes that mark the cell wall or plasma membrane, enabling the confocal microscope to be used as a 3D scanner with submicron precision. After imaging, the data needs to be analyzed by specialized software to quantify the features of interest, such as cell size and shape, growth rates and anisotropy, and gene expression. Here we present a protocol for the 3D image processing software MorphoGraphX ( www.MorphoGraphX.org ) using time-lapse images of an Arabidopsis thaliana sepal and the shoot apex of tomato.
Calcium imaging in plants requires a high-resolution microscope, able to perform volumetric acquisition in a few seconds, inducing as low photobleaching and phototoxicity as possible to the sample. Light sheet fluorescence microscopy offers these capabilities, with the further chance to mount the sample in vertical position, mimicking the plant's growth and physiological conditions.A protocol for plant preparation and mounting in a light sheet microscope is presented. First, the growth of Arabidopsis thaliana in a sample holder compatible with light sheet microscopy is described. Then, the requirements for sample alignment and image acquisition are detailed. Finally, the image processing steps to analyze calcium oscillations are discussed, with particular emphasis on ratiometric calcium imaging in Arabidopsis root hairs.
Complex geometry of plant organs and various types of organ surface deformation, including growth or hygroscopic movements, can be analyzed using sequential replica method. It enables obtaining a time-lapse series of high resolution images visualizing details of the examined surface and provides data sufficient for detailed computation of parameters characterizing surface deformation and geometry. Series of molds, made in dental polymer, representing the examined surface are used to obtain casts in epoxy resin or nail polish replicas, which are ready for microscopic examination, while the structure itself remains intact. Images obtained from the epoxy casts in scanning electron microscopy can be further used for 3D reconstruction and computation of local geometry. The sequential replica method is a universal method and can be applied to image complex shapes of a range of structures, like meristems, flowers, leaves, scarious bracts, or trichomes. Different plant species growing in various conditions can be studied.
X-ray microtomography (μCT) is a three-dimensional imaging technique, which has, over the past decade, established itself as a go-to method for nondestructive visualization of plant tissue with submicrometer resolution. μCT is closely related to medical computed tomography, in that a measurement consists of acquiring a series of radiographs from different directions around the sample. Especially with synchrotron X-ray sources, these radiographs exhibit significant phase contrast. This greatly enhances soft tissue contrast, making it well suited for plant imaging. Tomographic reconstruction techniques are then employed to convert the stack of radiographs into a 3D volumetric image. Compared with the laboratory X-ray tube-based systems, synchrotron tomography beamlines also offer high throughput, with tens of samples scanned over the course of a typical 24-h beam time.Synchrotrons are typically operated as user facilities, with a staff member assisting users in aligning the beamline and all instrumentation-related matters. From the user's point of view, success of a synchrotron μCT experiment is often dependent on secure sample mounting, choice of appropriate beam parameters, and post-processing the data, i.e., extracting scientifically meaningful results from the 3D image. In this chapter, we review the issues to consider in preparation of a μCT experiment from the point of view of a phloem researcher, emphasizing those aspects which are directly under the user's control rather than technical specifics, which vary from one beamline to another.
In vivo spectroscopy is used to directly assay phytochromes in intact plant material. The method is depending on the photoreversibility of phytochromes displaying light induced absorbance changes in response to actinic irradiation. Dual-wavelength ratio spectrophotometers (ratiospects) are the instruments successfully used for assaying phytochromes in highly scattering plant material. In the present chapter I describe the general instrument setup of an automated ratiospect and explain the measuring procedure and data calculation required to determine the total amount of photoreversible phytochromes in a sample as well as the proportion of phytochrome present in the Pfr conformation.
Rice varietal development and improvement programs are constantly seeking means to shorten the breeding cycle in order to deliver new, consumer-acceptable rice varieties to farmers and to consumers. Advances in molecular biology technologies have enabled breeders to use high-throughput genotyping to screen breeding lines. However, current phenotyping technologies, particularly for rice cooking and eating properties, have yet to match the efficiency of genotyping methodologies. A high-throughput and cost-effective phenotyping suite is essential because without phenotype, the value of genotypic information cannot be maximized. In this book chapter, we explore the application of near-infrared spectroscopy (NIRS), a high-throughput and nondestructive approach in characterizing rice grains, primarily describing method development and validation, instrument calibration, upgrading, and maintenance. We then focus on estimating protein content (PC) in brown rice as a case study because (1) PC is an attribute that contributes to the cooking behavior and the eating properties of cooked rice; and (2) proteins contain chemical bonds that can easily be detected by NIRS.
Flavescence Dorée (FD) is a contagious and incurable grapevine disease that can be perceived on leaves. In order to contain its spread, the regulations obligate winegrowers to control each plant and to remove the suspected ones. Nevertheless, this monitoring is performed during the harvest and mobilizes many people during a strategic period for viticulture. To solve this problem, we aim to develop a Multi-Spectral (MS) imaging device ensuring an automated grapevine disease detection solution. If embedded on a UAV, the tool can provide disease outbreaks locations in a geographical information system allowing localized and direct treatment of infected vines. The high-resolution MS camera aims to allow the identification of potential FD occurrence, but the procedure can, more generally, be used to detect any type of foliar diseases on any type of vegetation.Our work consists on defining the spectral bands of the multispectral camera, responsible for identifying the desired symptoms of the disease. In fact, the FD diseased samples were selected after establishing a Polymerase Chain Reaction (PCR) confirmation test and then a feature selection technique was applied to identify the best subset of wavelengths capable of detecting FD samples. An example of a preliminary version of the MS sensor was also presented along with the geometric and radiometric required corrections. An image analysis based on texture and neural networks was also detailed for an enhanced disease classification.
Panicle architecture is known to directly influence grain yield in rice, and thus is an important trait for rice varietal improvement. However, spike branching consequences trigger variation in number of superior and inferior grains and thus affect grain quality. The genetics behind the length of both primary and secondary branches were studied resulting in the identification of cloned genes. Extending this knowledge to include other physiological parameters of panicle architecture is not yet well studied, and it requires high-throughput imaging techniques that are accurate. In this chapter we put the spotlight on Panicle Trait Phenotyping Tool (P-TRAP), a freely available platform independent software to analyze the panicle architecture of rice, as one of such methods that can be used to generate a comprehensive and reproducible panicle architecture data and identify superior breeding lines. P-TRAP measures 15 panicle structure and nine spikelet traits. These quantitative traits can be used in genome-wide association studies to understand their genetic basis.
Tropic responses in plants have usually been studied by measuring changes in the deflection angle of the organ tip. However, the measurement of other geometric parameters, such as curvature along the entire length, may give us better understanding of tropic responses, particularly in shoots. Here, we describe methods for obtaining quantitative measurements of local curvature and other parameters based on digital images of bending Arabidopsis hypocotyls using the free software packages, ImageJ and R.
Here we describe an in vivo dye-tracking method for measuring phloem transport velocity in seedlings, leaves and petioles and potentially other translucent plant tissues. The method requires measurement of the fluorescent signal of a phloem-mobile fluorescent dye using sensitive photo-sensors placed external to the plant. Following dye application, velocity is determined using laser fluorescence bleaching and measuring the time it takes for the bleach front to reach a light sensor. This method can be used to measure phloem transport velocity on intact plants with minimal disturbance and has a potential to be used under a variety of growth conditions and in the field. Because there are large differences among species in their anatomy, this method should be optimized to individual plants and tissue types.
This chapter gives examples of basic procedures of quantification of plant structures with use of image analysis, which are commonly employed to describe differences among experimental treatments or phenotypes of plant material. Tasks are demonstrated with the use of ImageJ, a widely used public domain Java image processing program. Principles of sampling design based on systematic uniform random sampling for quantitative studies of anatomical parameters are given to obtain their unbiased estimations and simplified "rules of thumb" are presented. The basic procedures mentioned in the text are: (1) sampling, (2) calibration, (3) manual length measurement, (4) leaf surface area measurement, (5) estimation of particle density demonstrated on an example of stomatal density, and (6) analysis of epidermal cell shape.
The genetic improvement of field crops through plant breeding and genetic modification is highly dependent on understanding, measuring, selecting, and manipulating phenotypes. Most phenotypes result from the complex interaction of a crop's genetics with the environment and management practices in which that crop is grown. Linking gene to phenotype in field environments to create superior crop varieties can therefore be challenging, particularly for genetically complex traits that are difficult to measure. This chapter is designed to help readers overcome these difficulties by describing tools and techniques used in successful crop improvement programs. It provides methodologies that can be broadly applied across numerous situations irrespective of field crop, environment, modest financial resources, or other factors. The chapter's focus is primarily on small- and large-scale, replicated, research plot-based screening trials since these trials are crucial, ubiquitous, and costly for both public- and private-sector crop improvement programs. To ease the understanding of the protocols discussed, this chapter's materials and methods section is composed of ten subsections, with each subsection covering a critical portion of the field crop phenotyping process: regulatory, environmental, and safety considerations; trait identification and prioritization; environment characterization; field site selection; experimental design; field design, preparation, and management; crop and soil measurements; environmental monitoring; in-field data recording; and data management and analysis.
Measurements of rice grain dimensions, percent grain chalkiness, and grain elongation used to be tedious and slow due to the manual nature of measurements (e.g., use of calipers to measure grains one at a time) and the subjective nature of scoring based on visual inspection (i.e., chalkiness). Recent developments in imaging technologies have enabled more high-throughput means for measuring physical traits (i.e., grain dimensions and chalkiness) in raw grains and grain elongation by comparing ratio between raw versus cooked rice. The digital images of rice grains are captured through computer scanning and analyzed using software that can calculate area and pixel value statistics of user-defined parameters. The improvements in throughput made possible by the use of imaging technologies will allow faster quality grading of rice varieties. Market quality is usually defined based on the rice grain physical traits (grain size and shape), degree of chalkiness, and the ability of rice to elongate on cooking. In this chapter, the routine methods to measure the physical traits of rice and grain elongation using image analysis are described.
The cortical microtubule and actin meshworks play a central role in the shaping of plant cells. Transgenic plants expressing fluorescent protein markers specifically tagging the two main cytoskeletal systems are available, allowing noninvasive in vivo studies. Advanced microscopy techniques, in particular confocal laser scanning microscopy (CLSM), spinning disk confocal microscopy (SDCM), and variable angle epifluorescence microscopy (VAEM), can be nowadays used for imaging the cortical cytoskeleton of living cells with unprecedented spatial and temporal resolution. With the aid of free computing tools based on the publicly available ImageJ software package, quantitative information can be extracted from microscopic images and video sequences, providing insight into both architecture and dynamics of the cortical cytoskeleton.
UV-B phototropism in etiolated Arabidopsis seedlings has only been shown recently and needs further exploration. Here we elaborate on how to generate a customized setup with a unilateral UV-B light source, the required plant materials, different growth substrates, and a framework for data analysis.
Many aspects of light-controlled metabolism and development of plants depend on hormonal pathways. Here, a method is described to identify such hormonal dependence in light-regulated processes. A number of compounds-hormones and chemicals which interfere with hormonal pathways-are listed because of their usefulness in pharmacological treatment experiments. As an example for practical use of such compounds, elongation growth is discussed. An experimental setup is described in which plants are grown so that their structures develop predominantly in a two-dimensional plane. Time-lapse imaging is used to follow the plants in time, and image analysis reveals changes in plant morphology.
The circadian clock allows plants to synchronize their internal processes with the external environment. This synchronization occurs through daily cues, one of which is light. Phytochromes are well established as light-sensing proteins and have been identified in forming multiple signaling networks with the central circadian oscillator. However, the precise details of how these networks are formed are yet to be established. Using established promoter-luciferase lines for clock genes crossed into mutant lines, it is possible to use luciferase-based imaging technologies to determine whether specific proteins are involved in phytochrome signaling to the circadian oscillator. The methods presented here use two automated methods of luciferase imaging in Arabidopsis to allow for high-throughput measurement of circadian clock components under a range of different light conditions.
Increasing paddy yield in rice does not directly translate to enhancing food security because significant decrease in grain yield can happen during postharvest processing of the rice paddy. In parallel with enhancing paddy yield, improving the milling quality of rice is essential in ensuring food security by mitigating the impact of significant losses during the postharvest processing of rice grains. From an industrial standpoint, maximizing the milling recovery of whole grain polished rice is crucial in fetching higher revenues to rice farmers. Significant advances in rice postharvest processing technology have been achieved which are geared toward reducing the incidence of fissures and chalkiness to increase head rice yield (HRY) in rice. The genetic bases of kernel development and grain dimension are also characterized. In addition to these advancements, an integrated phenotyping suite to simultaneously characterize phenotypes related to milling quality will help in screening for breeding lines with high HRY. Toward this goal, modern imaging tools and computer algorithms are currently being developed for high-throughput characterization of rice milling quality. With the availability of more sophisticated, affordable, automated, and nondestructive phenotyping methods of milling quality, it is envisioned that significant improvement in HRY will be made possible to ensure rice food security in the future.
Phloem loading and long-distance transport of photoassimilate from source leaves to sink organs are essential physiological processes that contribute to plant growth and yield. At a minimum, three steps are involved: phloem loading in source organs, transport along the phloem path, and phloem unloading in sink organs. Each of these can have variable rates contingent on the physiological state of the plant, and thereby influence the overall transport rate. In addition to these phloem transport steps, rates of photosynthesis and photosynthate movement in the pre-phloem path, as well as photosynthate utilization in post phloem tissues of sink organs also contribute to phloem transport. The protocol described here estimates carbon allocation along the entire path from initial carbon fixation to delivery to sink organs after a labeling pulse: [ 14 C]CO 2 is photoassimilated in source leaves and loading and transport of the 14 C label to heterotrophic sink organs (roots) is quantified by scintillation counting. This method is flexible and can be adapted to quantify long-distance transport in many plant species.
Rice grain quality encompasses complex interrelated traits that cover biochemical composition, cooking, eating, nutritional, and sensory properties. Because rice endosperm is composed mainly of starch, rice grain quality is traditionally defined by characterizing starch structure and composition, which is then subsequently correlated with functional properties of the grain. The current proxy tests routinely used to describe rice grain quality preferences are rather limited to the estimation of apparent amylose content, gelatinization temperature, and gel consistency. Additional tests that characterize starch property, viscoelasticity, grain texture, and aroma are also employed in more advanced laboratories. However, these tests are not routinely applied in breeding programs to distinguish cooking quality classes to reflect evolving consumer preference and market demand. As consumer preferences in Asia and all over the world are diverse due to varied demographics and culture, defining uniform attributes to capture regional grain quality preferences becomes more challenging. Hence, novel and innovative proxy tests are needed to characterize rice grain quality to meet the demand for consumer preferences of commercially-released cultivars. In this chapter, the current methods employed in rice grain quality monitoring are succinctly reviewed. Future prospects for improvement are identified, introducing cutting edge technologies that can facilitate high-throughput screening of rice diversity panels and breeding lines. Aside from addressing the requirements for quality improvement in the traditional inbred rice breeding programs, we also tackled the need to enhance grain quality in the hybrid rice sector.
Many assays focus on determining NO content within plant tissues to assess the actual concentration that impacts on cellular processes. Diaminofluorescein fluorescent dyes (DAFs) have been very widely used by plant scientists to reveal likely sites of NO production inside and outside cells. In general, DAFs dyes react with N 2 O 3 , a byproduct of NO oxidation, resulting in fluorescence. It is initially available in the form of diacetate (DAF-2DA), which allowed the ready absorption by the cells. The diacetate group is removed by cell esterases leaving the membrane impermeable to DAF-2 and available for N 2 O 3 nitration to generate the highly fluorescent triazole (DAF-2T). Here, we describe two methods for detection of NO by fluorescence, one for NO extracellular detection by DAF-2 and the other one for NO intracellular detection, in this case using DAF-2DA.
Canopy photosynthesis (A c ), rather than leaf photosynthesis, is critical to gaining higher biomass production in the field because the daily or seasonal integrals of A c correlate with the daily or seasonal integrals of biomass production. The canopy photosynthesis and transpiration measurement system (CAPTS) was developed to enable measurement of canopy photosynthetic CO 2 uptake, transpiration, and respiration rates. CAPTS continuously records the CO 2 concentration, water vapor concentration, air temperature, air pressure, air relative humidity, and photosynthetic photon flux density (PPFD) inside the chamber, which can be used to derive CO 2 and H 2 O fluxes of a canopy covered by the chamber. Here we describe the protocol of using CAPTS to perform experiments on rice (Oryza sativa L.) in paddy field, wheat (Triticum aestivum L.) in upland field, and tobacco (Nicotiana tabacum L.) in pots.
Chlorophyll fluorescence is a rapid and non-invasive tool used for probing the activity of photosynthesis that can be used in vivo and in the field. It is highly relevant to the demands of high-throughput crop phenotyping and can be automated or manually applied. Here we describe protocols and advice for making fast timescale fluorescence measurements using handheld equipment in the laboratory or in the field. While interpretation of some measured parameters requires caution, we demonstrate that this technique is appropriate for some applications where convenience, rapidity, and sensitivity are required.
Muller L, Bennett MJ, French A, Wells DM, Swarup R.
ArabidopsisLaboratory / benchtopRootMorphology / geometry measurementSkeletonization / topologyRoot system architecture
Better understanding of root traits such as root angle and root gravitropism will be crucial for development of crops with improved resource use efficiency. This chapter describes a high-throughput, automated image analysis method to trace Arabidopsis (Arabidopsis thaliana) seedling roots grown on agar plates. The method combines a "particle-filtering algorithm with a graph-based method" to trace the center line of a root and can be adopted for the analysis of several root parameters such as length, curvature, and stimulus from original root traces.
The plant endoplasmic reticulum forms a network of tubules connected by three-way junctions or sheet-like cisternae. Although the network is three-dimensional, in many plant cells, it is constrained to a thin volume sandwiched between the vacuole and plasma membrane, effectively restricting it to a 2-D planar network. The structure of the network, and the morphology of the tubules and cisternae can be automatically extracted following intensity-independent edge-enhancement and various segmentation techniques to give an initial pixel-based skeleton, which is then converted to a graph representation. Collectively, this approach yields a wealth of quantitative metrics for ER structure and can be used to describe the effects of pharmacological treatments or genetic manipulation. The software is publicly available.
ArabidopsisMicroscopyRoot2D/3D reconstructionTrackingGrowth / development / phenology
Light sheet fluorescence microscopy (LSFM) allows sustained and repeated optical sectioning of living specimens at high spatial and temporal resolution, with minimal photodamage. Here, we describe in detail both the hardware and the software elements of a live imaging method based on LSFM and optimized for tracking and 3D scanning of Arabidopsis root tips grown vertically in physiological conditions. The system is relatively inexpensive and with minimal footprint; hence it is well suited for laboratories of any size.
High-throughput phenotyping has opened whole new perspectives for crop improvement and better understanding of quantitative traits in plants. Generation of loss-of-function and gain-of-function plant mutants requires processing and imaging a large number of plants in order to determine unknown gene functions and phenotypic changes generated by genetic modifications or selection of new traits. The use of phenomics for the evaluation of transgenic lines contributed significantly to the identification of plants more tolerant to biotic/abiotic stresses and furthermore, helped in the identification of unknown gene functions. In this chapter we describe the High-throughput phenotyping (HTP) platform working in our facility, drawing the general protocol and showing some examples of data obtainable from the platform. Tomato transgenic plants over-expressing the arginine decarboxylase 2 gene, which is involved in the polyamine biosynthetic pathway, were analyzed through our HTP facility for their tolerance to abiotic stress and significant differences in water content and ability to recover after drought stress where highlighted. This demonstrates the applicability of this methodology to the plant polyamine field.
Studying the effects of small molecules on root system development in the context of a large-scale chemical genetic screen has previously been a technical challenge. The recent development of novel seedling growth devices ("Phytostrips"), used in combination with standard 96-well microtiter plates, has made it possible to perform detailed studies of changes in root morphology and root system architecture following the application of a library of chemical compounds. Phytostrips were originally designed to allow automated robotic capture of images of roots and shoots of the model species Arabidopsis thaliana, but can also be used for manual screens that are more laborious but do not require the investment in expensive robotics.Here we describe a protocol for the use of Phytostrips to perform chemical genetic screens that rely on clearly observable changes in root morphology or root system architecture. As an example, we describe the use of polyethylene glycol to impose an abiotic stress related to reduced water potential and the application of a chemical screen for small molecules that are able to rescue Arabidopsis root development from the disruptive effect of the polyethylene glycol treatment. The protocol we describe provides a template for the application of a multiplicity of other screens for compounds that can antagonize the effects of a range of abiotic stresses on root development.
Nguyen L, Drozdzecki A, Goossens V, De Rybel B, Beeckman T, Audenaert D.
ArabidopsisWhole plant / canopy / plot / field
Phenotypic screening and subsequent target identification approaches are very valuable to identify chemical probes that can be used to explore the connection between phenotypes and biological pathways. However, assessing a phenotypic effect in plants in a high-throughput fashion is a challenging task and often requires expensive readout devices. In this chapter, we describe a cost-effective multi-parametric screening procedure that is compatible with liquid-handling systems and that enables the assessment of phenotypes in Arabidopsis thaliana seedlings in an automated way.
Reactive oxygen and nitrogen species (ROS/RNS) are signaling molecules involved in a plethora of physiological processes in plants. Especially, ROS and nitric oxide (NO) are key players that are required for programmed cell death (PCD). The PCD associated with the hypersensitive response (HR) has been well characterized and the role of H 2 O 2 and NO as key signaling molecules inducing HR has been established. Localization of ROS and NO production in plant tissues in response to pathogens can be imaged by confocal laser microscopy by using specific fluorescent probes. Deciphering the time and spatial regulation of ROS and NO is very important to establish the cellular response of plants to adverse conditions. This chapter is mainly focused on the imaging of ROS and RNS accumulation in vivo in plant tissues undergoing PCD.
Transmission electron microscopy (TEM) is used to study the fine ultrastructural organization of cells. Delicate specimen preparation is required for results to reflect the "native" ultrastructural organization of subcellular features such as the nucleus. Despite the advent of high-resolution, fluorescent imaging of chromatin components, TEM still provides a unique and complementary level of resolution capturing chromatin organization at the nanoscale level. Here, we describe the workflow, from tissue preparation, TEM image acquisition and image processing, for obtaining a quantitative description of chromatin density distribution in plant cells, informing on local fluctuations and periodicity. Comparative analyses then allow to elucidate the structural changes induced by developmental or environmental cues, or by mutations affecting specific chromatin modifiers at the nanoscale level. We argue that this approach remains affordable and merits a renewed interest by the plant chromatin community.
The hypersensitive response is one of the most powerful and complex defense reactions to survive to pathogen attacks during an incompatible plant-pathogen interaction. Local programmed cell death accompanies the hypersensitive response at the site of infection to prevent pathogen growth and spread. A precise quantitative assessment of this form of programmed cell death is essential to unravel the genetic and molecular mechanisms underlying the process. Here, we first describe the optimization of a Trypan Blue staining protocol for quantitatively measuring the HR-cell death in Arabidopsis. Furthermore, we provide an electrolyte leakage protocol based on pathogen vacuum infiltration, which allows its simultaneous application to a large number of plants as well as to Arabidopsis mutants affected by small size phenotype.
The endoplasmic reticulum (ER) forms an extensive network in plant cells. In leaf cells and vacuolated root cells it is mainly restricted to the cortex whereas in the root meristem the cortical and cytoplasmic ER takes up a large volume throughout the entire cell. Only 3D electron microscopy provides sufficient resolution to understand the spatial organization of the ER in the root. However, high contrast staining and optimally ER specific staining is essential. Here we describe a protocol for selective ER staining that allows automated or semiautomated segmentation of the organelle in 3D datasets obtained from serial sections, Array Tomography, Serial Block Face Scanning Electron Microscopy (SBFSEM), or Focused Ion Beam (FIB) SEM.
The root epidermis of Arabidopsis thaliana has been established as a model system for elucidating the mechanisms which govern the spatial patterningAbstract and morphogenesis of plant cells. Investigations into root hairs focus on various aspects of the biology of epidermal cells, using methods specifically developed to dissect the biological question under study. Despite the large number of studies related to epidermal cell differentiation, a survey of methods to analyze the phenotypic readout resulting from environmental conditions or the genetic background of the plant has not been provided so far. This protocol describes how to analyze the spatial arrangement and morphologic characteristics of cells in the root epidermis based on whole mount roots or cross sections, using confocal, scanning electron and light microscopy. This comparison of methods aids in selecting the most suitable strategy to examine the differentiation of root epidermal cells at different developmental stages.
Scanning electron microscope (SEM) is a type of electron microscope which produces detailed images of surface structures. It has been widely used in plants and animals to study cellular structures. Here, we describe a detailed protocol to prepare samples of floral abscission zones (AZs) for SEM, as well as further image analysis. We show that it is a powerful tool to detect morphologic changes at the cellular level during the course of abscission in wild-type plants and to establish the details of phenotypic alteration in abscission mutants.
Chemical biology provides an alternative way to identify genes involved in a particular biological process. It has the potential to overcome issues such as redundancy or lethality often found in genetic approaches, since the chemical compounds can simultaneously target all homologous proteins that function at the same step, and chemicals can be applied conditionally. Even with a variety of genetic approaches, the molecular mechanisms of plant hypersensitive cell death that occurs during disease resistance responses remain unclear. Therefore, application of chemical biology should provide new insights into this phenomenon. Here we describe a high-throughput chemical screening procedure to detect hypersensitive cell death quantitatively, using a suspension cell culture of Arabidopsis thaliana and a well-studied avirulent bacterial pathogen, Pseudomonas syringae pv. tomato DC3000 avrRpm1.
Cyclic guanosine-3',5'-monophosphate (cGMP) is recognized as an important second messenger in plants, mediating intracellular signal in important physiological processes, including the hypersensitive disease resistance response induced by avirulent pathogens. In this context, the analysis of cGMP levels in infected plants requires an accurate and specific detection method allowing its quantification. Here, we describe an assay based on the Alphascreen technology, developed for animal cells and further adapted and optimized for the detection of cGMP in plants. The method is applied for the measurement of cGMP in Arabidopsis thaliana plants challenged with an avirulent strain of Pseudomonas syringae pv. tomato. This protocol includes the extraction of cGMP, the assay procedure and the calculation of cGMP concentration.
In this chapter, we describe different methods for phenotyping strains or mutants of the bacterial wilt agent, Ralstonia solanacearum, on four different host plants: Arabidopsis thaliana, tomato (Solanum lycopersicum), tobacco (Nicotiana benthamiana), or Medicago truncatula. Methods for preparation of high volume or low volume inocula are first described. Then, we describe the procedures for inoculation of plants by soil drenching, stem injection or leaf infiltration, and scoring of the wilting symptoms development. Two methods for measurement of bacterial multiplication in planta are also proposed: (1) counting the bacterial colonies upon serial dilution plating and (2) determining the bacterial concentration using a qPCR approach. In this chapter, we also describe a competitive index assay to compare the fitness of two strains coinoculated in the same plant. Lastly, specific protocols describe in vitro and hydroponic inoculation procedures to follow disease development and bacterial multiplication in both the roots and aerial parts of the plant.
The plant cell wall is an important and abundant biomass with great potential for use as a modern recyclable resource. For effective utilization of this cellulosic biomass, its ability to degrade efficiently is key point. With the aim of modifying the cell wall to allow easy decomposition, we used chemical biological technology to alter its structure. As a first step toward evaluating the chemicals in the cell wall we employed a phenotype-based approach using high-throughput screening. As the plant cell wall is essential in determining cell morphology, phenotype-based screening is particularly effective in identifying compounds that bring about alterations in the cell wall. For rapid and reproducible screening, tobacco BY-2 cell is an excellent system in which to observe cell morphology. In this chapter, we provide a detailed chemical biological methodology for studying cell morphology using tobacco BY-2 cells.
Abscisic acid (ABA) is a sesquiterpenoid (15-carbon) hormone that comprehensively regulates plant stress responses, development, and senescence. Stomata are epidermal pores on plant surface used for exchanging gases such as carbon dioxide, water vapor, and oxygen. One of the mechanisms that ABA regulates leaf senescence is to control stomatal movement and thus water loss during leaf senescence. Here we describe the procedure of measuring stomatal movement in response to ABA treatments, which will provide a useful protocol to investigate ABA signaling in leaf senescence.
The study of programmed cell death (PCD) activated in a certain group of cells is complex when analyzed in the whole plant. Plant cell suspension cultures are useful when investigating PCD triggered by environmental and developmental stimuli. Due to their homogeneity and the possibility to synchronize their responses induced by external stimuli, these cultures are used for studying the signaling pathways leading to PCD. The first problem in the analysis of PCD in cell cultures is the quantification of cell viability/death over time. Cultured cells from different plant species may have specific mitotic patterns leading to calli or cell chains mixed to single cell suspensions. For this reason, not all cell cultures allow morphological parameters to be investigated using microscopy analysis, and adapted or ad hoc methods are needed to test cell viability.Here we report on some accurate methods to establish and propagate cell cultures from different plant species, including crops, as well as to determine cell viability and PCD morphological and genetic markers. In particular, we describe a protocol for extracting nucleic acids required for real-time PCR analysis which has been optimized for those cell cultures that do not allow the use of commercial kits.
In this chapter, approaches to the image analysis of the choreography of the plant endoplasmic reticulum (ER) labeled with fluorescent fusion proteins ("stars," if you wish) are presented. The approaches include the analyses of those parts of the ER that are attached through membrane contact sites to moving or nonmoving partners (other "stars"). Image analysis is also used to understand the nature of the tubular polygonal network, the hallmark of this organelle, and how the polygons change over time due to tubule sliding or motion. Furthermore, the remodeling polygons of the ER interact with regions of fundamentally different topology, the ER cisternae, and image analysis can be used to separate the tubules from the cisternae. ER cisternae, like polygons and tubules, can be motile or stationary. To study which parts are attached to nonmoving partners, such as domains of the ER that form membrane contact sites with the plasma membrane/cell wall, an image analysis approach called persistency mapping has been used. To study the domains of the ER that are moving rapidly and streaming through the cell, the image analysis of optic flow has been used. However, optic flow approaches confuse the movement of the ER itself with the movement of proteins within the ER. As an overall measure of ER dynamics, optic flow approaches are of value, but their limitation as to what exactly is "flowing" needs to be specified. Finally, there are important imaging approaches that directly address the movement of fluorescent proteins within the ER lumen or in the membrane of the ER. Of these, fluorescence recovery after photobleaching (FRAP), inverse FRAP (iFRAP), and single particle tracking approaches are described.
Plants have evolved a sophisticated innate immune system to contend with potential infection by various pathogens. Understanding and manipulation of key molecular mechanisms that plants use to defend against various pathogens are critical for developing novel strategies in plant disease control. In plants, resistance to attempted pathogen infection is often associated with hypersensitive response (HR), a form of rapid programmed cell death (PCD) at the site of attempted pathogen invasion. In this chapter, we describe a method for rapid identification of genes that are essential for plant innate immunity. It combines virus-induced gene silencing (VIGS), a tool that is suitable for studying gene function in high-throughput, with the utilization of immunity-associated PCD, particularly HR-linked PCD as the readout of changes in plant innate immunity. The chapter covers from the design of gene fragment for VIGS, the agroinfiltration of the Nicotiana benthamian plants, to the use of immunity-associated PCD induced by twelve elicitors as the indicator of activation of plant immunity.
DNA diffusion assay is a simple, sensitive and reliable technique which allows the assessment of programmed cell death (PCD) or necrosis events based on nuclear morphology. It consists in isolating nuclei from plant material, which are then embedded in agarose and subjected to lysis in alkaline buffers. Under these conditions, and due to the presence of abundant alkali-labile sites in the DNA, small pieces of DNA diffuse in the agarose gel giving a specific halo appearance when stained with fluorescent dyes like DAPI (4',6-diamidino-2-phenylindole). Here, we describe an optimized protocol for DNA diffusion assay applied to different types of plant cells/tissues, indicating all the critical steps required for a successful experimental procedure.
Bridging metabolomics with plant phenotypic responses is challenging. Multivariate analyses account for the existing dependencies among metabolites, and regression models in particular capture such dependencies in search for association with a given trait. However, special care should be undertaken with metabolomics data. Here we propose a modeling workflow that considers all caveats imposed by such large data sets.
Here we provide the instructions to build a cost-friendly rotating stage, which enables time-lapse phenotyping of seedlings, grown vertically on in vitro plates, in a medium-throughput manner.
Exposure of plants to ethylene results in drastic morphological changes. Seedlings germinated in the dark in the presence of saturating concentrations of ethylene display a characteristic phenotype known as the triple response. This phenotype is robust and easy to score. In Arabidopsis the triple response is usually evaluated at 3 days post germination in seedlings grown in the dark in rich media supplemented with 10 μM of the ethylene precursor ACC in air or in unsupplemented media in the presence of 10 ppm ethylene. The triple response in Arabidopsis consists of shortening and thickening of hypocotyls and roots and exaggeration of the curvature of apical hooks. The search for Arabidopsis mutants that fail to show this phenotype in ethylene or, vice versa, display the triple response in the absence of exogenously supplied hormone has allowed the identification of the key components of the ethylene biosynthesis and signaling pathways. Herein, we describe a simple protocol for assaying the triple response in Arabidopsis. The method can also be employed in many other dicot species, with minor modifications to account for species-specific differences in germination. We also compiled a comprehensive table of ethylene-related mutants of Arabidopsis, including many lines with auxin-related defects, as wild-type levels of auxin biosynthesis, transport, signaling, and response are necessary for the normal response of plants to ethylene.
Computational programs can be used in place of time-consuming, error-prone manual data collection. CellProfiler is a free, open source program that allows researchers to automate image analysis and collect large amounts of phenotypic data relatively easily. Here, we describe how to adapt CellProfiler to analyze cross sections of xylem tissue and use it to gather a variety of information on traits such as cell size, shape, and number. We provide step-by-step instructions to create a typical CellProfiler analysis pipeline, alongside explanations of important modules, options and parameters available to the user.
Conventional oxygen (micro-) sensors assess oxygen concentration within a particular region or across a transect of tissue, but provide no information regarding its bidimensional distribution. Here, a novel imaging technology is presented, in which an optical sensor foil (i.e., the planar optode) is attached to the surface of the sample. The sensor converts a fluorescent signal into an oxygen value. Since each single image captures an entire area of the sample surface, the system is able to deduce the distribution of oxygen at a resolution level of few micrometers. It can be deployed to dynamically monitor oxygen consumption, thereby providing a detailed respiration map at close to cellular resolution. Here, we demonstrate the application of the imaging tool to developing plant seeds; the protocol is explained step by step and some potential pitfalls are discussed.
Plants are equipped with a suite of plant pattern recognition receptors (PRRs) that must be properly trafficked to and from the plasma membrane (PM), which serves as the host-pathogen interface, for robust detection of invading pathogenic microbes. Recognition of bacterial flagellin, or the derived peptide flg22, is facilitated by the PM-localized PRR, FLAGELLIN SENSING 2 (FLS2). Upon flg22 binding, FLS2 is rapidly internalized from the PM into endosomal compartments and subsequently degraded. To understand better the integration of FLS2 endocytosis and signaling outputs, we developed methods for the quantitative analysis of FLS2 trafficking using freely available bioimage informatic tools. Emphasis was placed on robust recognition of features and ease of access for users. Using the free and open-source software Fiji (Fiji is just ImageJ) and Trainable Weka Segmentation (TWS) plug-in, we developed a workflow for the automated identification of green fluorescent protein (GFP)-tagged FLS2 in endosomal puncta. Fiji-TWS methods can be adapted with ease for the analysis of FLS2 trafficking in various genetic backgrounds as well as for the endocytic regulation of diverse plant PRRs.
ArabidopsisRootMorphology / geometry measurementRoot system architectureStress response / tolerance
The central question of genetics is how a genotype determines the phenotype of an organism. Genetic mapping approaches are a key for finding answers to this question. In particular, genome-wide association (GWA) studies have been rapidly adopted to study the architecture of complex quantitative traits. This was only possible due to the improvement of high-throughput and low-cost phenotyping methodologies. In this chapter we provide a detailed protocol for obtaining root trait data from the model species Arabidopsis thaliana using the semiautomated, high-throughput phenotyping pipeline BRAT (Busch-lab Root Analysis Toolchain) for early root growth under the stress condition of iron deficiency. Extracted root trait data can be directly used to perform GWA mapping using the freely accessible web application GWAPP to identify marker polymorphisms associated with the phenotype of interest.
A comprehensive understanding of plant growth and development requires the integration of the spatial and temporal dynamics of gene regulatory networks with changes in cellular geometry during 3D organ growth. 3DCellAtlas is an integrative computational pipeline that semi-automatically identifies cell type and position within radially symmetric plant organs, and simultaneously quantifies 3D cell anisotropy and reporter abundance at single-cell resolution. It is a powerful tool that generates digital single-cell cellular atlases of plant organs and enables 3D cell geometry and reporter abundance (gene/protein/biosensor) from multiple samples to be integrated at single-cell resolution across whole organs. Here we describe how to use 3DCellAtlas to process and analyze radially symmetric organs, and to identify cell types and extract geometric cell data within these 3D cellular datasets. We detail how to use two statistical tools in 3DCellAtlas to compare cellular geometries, and to analyze reporter abundance at single-cell resolution.
Microscope images of plant specimens showing expression of GUS markers, besides being very beautiful, provide useful information regarding various biological processes. However, the information extracted from these images is often purely qualitative, and in many publications is not subjected to quantification. Here, we describe a very simple quantification method for GUS histochemical staining that enables detection of subtle differences in gene expression at cellular, tissue, or organ level. The quantification method described is based on the freely available image analysis software ImageJ that is widely used by the scientific community. We exemplify the method by quantifying small and precise changes (at the cellular level) as well as broad changes (at the organ level) in the expression of two previously published reporter lines, such as the pPILS2::GUS and pPILS5::GUS. The method presented here represents an easy tool for converting visual information from GUS histochemical staining images into quantifiable data and is of general importance for plant biologists performing GUS activity-based evaluation of reporter genes.
Analyzing pollen quality in an efficient and reliable manner is of great importance to the industries involved in seed and fruit production, plant breeding, and plant research. Pollen quality parameters, viability and germination capacity, are analyzed by various staining methods or by in vitro germination assays, respectively. These methods are time-consuming, species-dependent, and require a lab environment. Furthermore, the obtained viability data are often poorly related to in vivo pollen germination and seed set. Here, we describe a quick, label-free method to analyze pollen using microfluidic chips inserted into an impedance flow cytometer (IFC). Using this approach, pollen quality parameters are determined by a single measurement in a species-independent manner. The advantage of this protocol is that pollen viability and germination can be analyzed quickly by a reliable and standardized method.
Observing cellular and molecular processes in living organisms is key for understanding many important biological processes. Confocal microscopy is excellently suited for this as it enables the observation of molecules and cells in tissue layers of living organisms in three dimensions over time. However, in continuously growing organs, such as plant roots, observations over extended time spans become difficult as the specimen quickly grows out the field of view. Here, we provide a protocol that allows for the acquisition of confocal microscope time-lapse images of root tips spanning many hours, as the growing root tip is tracked and the microscopy is automatized to change the position of the stage. Importantly, due to its specific setup, this protocol allows for observing the effects of chemical stimuli or for creating specific growth conditions by precisely defining the growth medium during imaging. The protocol is suitable for observing multiple fluorophores, thereby moving beyond the level of individual genes. It is also simple enough to conduct larger numbers of these assays. Here we exemplify our method by describing the observation of root growth and GFP intensity in root tips under iron depletion conditions.
Quantification of vascular morphodynamics during secondary growth has been hampered by the scale of the process. Even in the tiny model plant Arabidopsis thaliana, the xylem can include more than 2000 cells in a single cross section, rendering manual counting impractical. Moreover, due to its deep location, xylem is an inaccessible tissue, limiting live imaging. A novel method to visualize and measure secondary growth progression has been proposed: "the Quantitative Histology" approach. This method is based on a detailed anatomical atlas, and image segmentation coupled with machine learning to automatically extract cell shapes and identify cell type. Here we present a new version of this approach, with a user-friendly interface implemented in the open source software LithoGraphX.
Stem rust (SR) or black rust caused by Puccinia graminis f. sp. tritici is one of the most common diseases of wheat (Triticum aestivum L.) crops globally. Among the various control measures, the most efficient and sustainable approach is the deployment of genetically resistant cultivars. Traditionally, wheat breeding programs deployed genetic resistance in cultivars, but unknowingly this is often underpinned by a single seedling resistance gene, which is readily overcome by the pathogen. Nowadays, adult plant resistance (APR) is a widely adopted form of rust resistance because more durable mechanisms often underpin it. However, only a handful of SR APR genes are available, so breeders currently strive to combine seedling and APR genes. Phenotyping adult wheat plants for resistance to SR typically involves evaluation in the field. But establishing a rust nursery can be challenging, and screening is limited to once a year. This slows down research efforts to isolate new APR genes and breeding of genetically resistant cultivars.In this study, we report a protocol for rapid evaluation of adult wheat plants for resistance to stem rust. We demonstrate the technique by evaluating a panel of 16 wheat genotypes consisting of near isogenic lines (NILs) for known Sr genes (i.e., Sr2, Sr33, Sr45, Sr50, Sr55, Sr57, and Sr58) and three landraces carrying uncharacterized APR from the N. I. Vavilov Institute of Plant Genetic Resources (VIR). The method can be completed in just 10 weeks and involves two inoculations: first conducted at seedling stage and a second at the adult stage (using the same plants). The technique can detect APR, such as that conferred by APR gene Sr2, along with pseudo-black chaff (the morphological marker). Phenotyping can be conducted throughout the year, and is fast and resource efficient. Further, the phenotyping method can be applied to screen breeding populations or germplasm accessions using local or exotic races of SR.
RootMorphology / geometry measurementRoot system architecture
Plants are sessile organisms that can tune their body architecture to the environment. This is very pronounced in their root system. In particular, nutrient availability strongly influences the architecture of the root system; depending on the abundance of specific nutrients, root growth rates and lateral root number are modulated. The extent of these effects is important for plant adaptation and has a major impact on plant fitness. However, the assessment of quantitative effects on a scale large enough for identifying genes and variants using quantitative genetics is difficult, and well-developed methods have been largely restricted to the model species Arabidopsis thaliana. In this chapter, we present a protocol for high-throughput phenotyping of early root traits in the model legume plant Lotus japonicus. This species allows for the study of important root-associated traits that are not present in Arabidopsis, such as symbioses with nitrogen-fixing Rhizobia and arbuscular mycorrhizal fungi. The methods described in this chapter can be used in the context of reverse and forward genetics approaches to dissect the genetic basis of root growth in legumes.
The high-throughput analysis of respiratory activity has become an important component of many biological investigations. Here, a technological platform, denoted the "MultiSense tool," is described. The tool enables the parallel monitoring of respiration in 100 samples over an extended time period, by dynamically tracking the concentrations of oxygen (O 2 ) and/or carbon dioxide (CO 2 ) and/or pH within an airtight vial. Its flexible design supports the quantification of respiration based on either oxygen consumption or carbon dioxide release, thereby allowing for the determination of the physiologically significant respiratory quotient (the ratio between the quantities of CO 2 released and the O 2 consumed). It requires an LED light source to be mounted above the sample, together with a CCD camera system, adjusted to enable the capture of analyte-specific wavelengths, and fluorescent sensor spots inserted into the sample vial. Here, a demonstration is given of the use of the MultiSense tool to quantify respiration in imbibing plant seeds, for which an appropriate step-by-step protocol is provided. The technology can be easily adapted for a wide range of applications, including the monitoring of gas exchange in any kind of liquid culture system (algae, embryo and tissue culture, cell suspensions, microbial cultures).
MaizeMicroscopyCell / cellular structureVisualization / data management
The plant life cycle is characterized by the alternation of generations between genetically active diploid sporophytes and haploid gametophytes. The gametophytes of flowering plants are sexually dimorphic. While the male gametophyte consists of only three cells (two sperm and a vegetative cell) and is released by the parent sporophyte, the female gametophyte (or embryo sac) is more complex and remains imbedded within diploid sporophyte tissues. In maize, the female gametophyte is embedded in a large ovule surrounded with multiple nucellar cell layers impeding live-cell imaging approaches to study embryo sac functions. Here, we describe a simple protocol to visualize embryo sacs with hormonal fluorescent reporters by increasing accessibility of the female gametophyte. The method described is applicable for visualization of any fluorescent embryo sac reporter. The embryo sacs visualization method developed for maize could be extended to facilitate visualization of embryos sac in other important cereals like wheat, rice, and oats.
RiceMicroscopyCell / cellular structureFlower2D/3D reconstructionGrowth / development / phenology
Recent advances in fluorescence-based staining of cellular compartments coupled with confocal microscopy imaging have allowed the visualization of three-dimensional (3D) structures with cellular resolution in various intact plant tissues and species. Such approaches are of particular interest for the analysis of the reproductive lineage in plants including the meiotic precursor cells deeply embedded within the ovary of the gynoecium enclosed in the flower. Yet, their relative inaccessibility and the lack of optical clarity of plant tissues prevent robust staining and imaging across several cell layers. Several whole-mount tissue staining and clearing techniques are available. One of them specifically allows staining of cellular boundaries in thick tissue samples while providing extreme optical clarity, using an acidic treatment followed by a modified Pseudo-Schiff propidium iodide (mPS-PI) method. While commonly used for Arabidopsis tissues, its application to other species like the model crop rice required protocol adaptations for obtaining robust staining that we present here. The procedure comprises six steps: (a) Material sampling; (b) Material fixation; (c) Tissue preparation; (d) Staining; (e) Sample mounting; and (d) Microscopy imaging. Particularly, we use ethanol and acetic anhydride as fixative reagents. A modified enzymatic treatment proved essential for starch degradation influencing optical clarity hence allowing acquisition of images at high resolution. This improved protocol is efficient for analyzing the megaspore mother cells in rice (Oryza sativa) ovary but is broadly applicable to other crop tissues of complex composition, without the need for tissue sectioning.
The plant cell wall responds dynamically during interaction with various pathogens. Upon recognition of "nonself" components, plant cells deploy a variety of immune responses including cell wall fortification. Callose, a β-(1, 3)-D-glucan polymer, is a component of the material deposited at the site of infection between the plasma membrane and the preexisting cell wall that is hypothesized to serve as a physical barrier and platform for directed antimicrobial compound deposition. The defense-associated function of callose deposition is supported by its induction during pathogen-associated molecular patterns (PAMP)-triggered immunity (PTI) and its inhibition by defense suppressing virulence effectors. Thus, callose deposition is a commonly monitored read-out in plant defense. This protocol describes the use of aniline blue staining and fluorescent microscopy to measure callose deposition in bacteria-infected or elicitor-challenged Arabidopsis leaf tissues.
Histochemical assays of xylem cell death cannot take advantage of the conventional methods for detection of cell death, such as staining with propidium iodide or trypan/Evans blue or the TUNEL staining. This chapter presents two alternative histochemical methods that can be used to detect xylem cell death quickly and reliably using light microscopy. The first method is a viability stain that can be used to detect cell death of different types of xylem elements in basically any plant species. The second method reveals cell death in xylem vessel elements based on their functionality in transport of water and small water-soluble stains.
ArabidopsisRootStress / disease detectionDisease symptoms / severityRoot system architecture
Plant parasitic nematodes cause a great impact in agricultural systems. The search for effective control methods is partly based on the understanding of underlying molecular mechanisms leading to the formation of nematode feeding sites. In this respect, crosstalk of hormones such as auxins and cytokinins (IAA, CK) between the plant and the nematode seems to be crucial. Thence, the study of loss of function or overexpressing lines with altered IAA and CK functioning is entailed. Those lines frequently show developmental defects in the number, position and/or length of the lateral roots what could generate a bias in the interpretation of the nematode infection parameters. Here we present a protocol to assess differences in nematode infectivity with the lowest interference of root architecture phenotypes in the results. Thus, tailored growth conditions and normalization parameters facilitate the standardized phenotyping of nematode infection.
Respiration traits allow calculating temperature-dependent carbon use efficiency and prediction of growth rates. This protocol aims (1) to enable validation of respiration traits as non-DNA biomarkers for breeding on robust plants in support of sustainable and healthy plant production; (2) to provide an efficient, novel way to identify and predict functionality of DNA-based markers (genes, polymorphisms, edited genes, transgenes, genomes, and hologenomes), and (3) to directly help farmers select robust material appropriate for a specified region. The protocol is based on applying isothermal calorespirometry and consists of four steps: plant tissue preparation, calorespirometry measurements, data processing, and final validation through massive field-based data.The methodology can serve selection and improvement for a wide range of crops. Several of them are currently being tested in the author's lab. Among them are important cereals, such as wheat, barley, and rye, and diverse vegetables. However, it is critical that the protocol for measuring respiration traits be well adjusted to the plant species by considering deep knowledge on the specific physiology and functional cell biology behind the final target trait for production. Here, Daucus carota L. is chosen as an advanced example to demonstrate critical species-specific steps for protocol development. Carrot is an important global vegetable that is grown worldwide and in all climate regions (moderate, subtropical, and tropical). Recently, this species is also used in my lab as a model for studies on alternative oxidase (AOX) gene diversity and evolutionary dynamics in interaction with endophytes.
ArabidopsisRootMorphology / geometry measurementRoot system architecture
Genome-wide association (GWA) mapping is a powerful method for the identification of alleles that underlie quantitative traits. It enables one to understand how genetic variation translates into phenotypic variation. In particular, plant hormone signaling pathways play a key role in shaping phenotypes. This chapter presents a protocol for genome-wide association mapping of root traits of Arabidopsis thaliana in the context of hormone research. We describe a specific protocol for acquiring primary and lateral root trait data that is appropriate for GWA studies using FIJI (ImageJ), and subsequent GWA mapping using a user-friendly Internet application.
High-resolution images obtained from plant tissues processed for light microscopy, transmission electron microscopy, and immunohistochemistry have provided crucial links between plant subcellular structure and physiology during photorespiration as well as the impact of photorespiration on plant evolution and development. This chapter presents established protocols to guide researchers in the preparation of plant tissues for high-resolution imaging with a light and transmission electron microscope and detection of proteins using immunohistochemistry. Discussion of concepts and theory behind each step in the process from tissue preservation to staining of resin-embedded tissues is included to enhance the understanding of all steps in the procedure. We also include a brief protocol for quantification of cellular parameters from high-resolution images to help researchers rigorously test hypotheses.
Analytical detection of the plant hormone ethylene is an important prerequisite in physiological studies. Real-time and super sensitive detection of trace amounts of ethylene gas is possible using laser-based photo-acoustic spectroscopy. This Chapter will provide some background on the technique, compare it with conventional gas chromatography, and provide a detailed user-friendly hand-out on how to operate the machine and the software. In addition, this Chapter provides some tips and tricks for designing and performing physiological experiments suited for ethylene detection with laser-based photo-acoustic spectroscopy.
Endophytes can diversify temperature response and biomass production in plants and microalgae. Natural and inoculated endophytes that modify growth performance are increasingly considered in research and practical initiatives for sustainable agriculture. However, efficient, novel tools are required that are able to support identification of differential effects of native endophyte populations and for pre-selection of inocula.This protocol gives instructions for applying calorespirometry as a rapid means for identifying differential effects of endophytes on temperature response and predicted biomass productivity in microalgae and plant holobionts. The protocol can help discriminating hologenomes, genes, and molecular neutral or functional markers for microalgae strain and plant improvement. Here, we focus on the microalga Chlorella vulgaris and associated microorganisms as an example for highlighting the methodology for its integration in research and application.
Long-distance migration capacity, emergence of invasive lineages, and variability in adaptation to a wide range of climatic conditions make wheat rusts the most important threat to wheat production worldwide. Efficient and coordinated efforts are required for surveillance of the pathogen population at different geographical levels to enable tracking of rust pathogen populations at local, regional, continental, and ultimately worldwide scale. Here we describe a standard procedure for rust surveillance to enable comparison across various research groups for a final compilation. The procedure described would enable tracking of disease severity, field level expression of host resistance, and collection of samples for further virulence phenotyping and molecular genotyping.
Plants tightly regulate the biosynthesis of ethylene to control growth and development and respond to a wide range of biotic and abiotic stresses. To understand the molecular mechanism by which plants regulate ethylene biosynthesis as well as to identify stimuli triggering the alteration of ethylene production in plants, it is essential to have a reliable tool with which one can directly measure in vivo ethylene concentration. Gas chromatography is a routine detection technique for separation and analysis of volatile compounds with relatively high sensitivity. Gas chromatography has been widely used to measure the ethylene produced by plants, and has in turn become a valuable tool for ethylene research. Here, we describe a protocol for measuring the ethylene produced by dark-grown Arabidopsis seedlings using a gas chromatograph.
Imaging of fluorescent proteins in whole-mount tissue is a powerful tool to understand growth and developmental processes, not only in plants. With the advent of genetically encoded fluorescent reporters, which specifically label reproductive cells in Arabidopsis, deep tissue imaging has become increasingly important for the study of plant reproduction. To penetrate the surrounding layers of maternal tissue, however, the tissue has to be cleared by homogenizing the refractive index of the sample, often leading to inactivation of fluorescent proteins. 2,2'-thiodiethanol (TDE) has recently been introduced as a clearing agent that allows the imaging of fluorescent proteins in a cleared plant tissue. Here, we describe a simple protocol that combines TDE-based tissue clearing with cell wall staining to outline cells that enable deep tissue imaging in reproductive structures of Arabidopsis thaliana.
Domazakis E, Lin X, Aguilera-Galvez C, Wouters D, Bijsterbosch G, Wolters PJ, Vleeshouwers VG.
PotatoCell / cellular structure
In modern resistance breeding, effectors have emerged as tools for accelerating and improving the identification of immune receptors. Effector-assisted breeding was pioneered for identifying resistance genes (R genes) against Phytophthora infestans in potato (Solanum tuberosum). Here we show that effectoromics approaches are also well suitable for identifying pathogen recognition receptors (PRRs) that recognize apoplastic effectors. To detect genotypes that recognize apoplastic proteins of P. infestans, routine agroinfiltration and potato virus X (PVX) agroinfection methods can be applied. In addition, protein infiltrations are feasible for assessing responses to apoplastic effectors and aid in confirming results obtained from the aforementioned methods. Protocols for the effectoromics pipeline are provided, starting from phenotyping for effector responses, up to genotyping and PRR gene identification.
Subcellular targeting of vacuolar proteins depends on cellular machinery regulating vesicular trafficking. Plant-specific vacuolar trafficking routes have been reported. However, regulators mediating these processes are obscure. By combining a fluorescence imaging-based forward genetic approach and in vitro pollen germination system, we show an efficient protocol of identifying regulators of plant-specific vacuolar trafficking routes.
ArabidopsisLaboratory / benchtopMicroscopyCell / cellular structureSegmentationTrackingGrowth / development / phenology
Cells in the Arabidopsis shoot apical meristem are small and divide frequently throughout the life-time of the organism making them good candidates for studying the mechanisms of cell division in plants. But tracking these cell divisions requires multiple images to be taken of the same specimen over time which means the specimen must stay alive throughout the process. This chapter provides details on how to prepare plants for live imaging, keep them alive and growing through multiple time points, and how to process the data to extract cell boundary coordinates from three-dimensional images.
The circadian clock is a molecular timekeeper that controls a wide variety of biological processes. In plants, clock outputs range from the molecular level, with rhythmic gene expression and metabolite content, to physiological processes such as stomatal conductance or leaf movements. Any of these outputs can be used as markers to monitor the state of the circadian clock. In the model plant Arabidopsis thaliana, much of the current knowledge about the clock has been gained from time course experiments profiling expression of endogenous genes or reporter constructs regulated by the circadian clock. Since these methods require labor-intensive sample preparation or transformation, monitoring leaf movements is an interesting alternative, especially in non-model species and for natural variation studies. Technological improvements both in digital photography and image analysis allow cheap and easy monitoring of circadian leaf movements. In this chapter we present a protocol that uses an autonomous point and shoot camera and free software to monitor circadian leaf movements in tomato.
Barker R, Cox B, Silber L, Sangari A, Assadi A, Masson P.
ArabidopsisLaboratory / benchtopRGB / grayscaleRootMorphology / geometry measurementGrowth / time-series analysisGrowth / development / phenologyRoot system architecture
Arabidopsis thaliana was the first higher organism to have its genome sequenced and is now widely regarded as the model dicot. Like all plants, Arabidopsis develops distinct growth patterns in response to different environmental stimuli. This can be seen in the gravitropic response of roots. Methods to investigate this particular tropism are presented here. First, we describe a high-throughput time-lapse photographic analysis of root growth and curvature response to gravistimulation allowing the quantification of gravitropic kinetics and growth rate at high temporal resolution. Second, we present a protocol that allows a quantitative evaluation of gravitropic sensitivity using a homemade 2D clinostat. Together, these approaches allow an initial comparative analysis of the key phenomena associated with root gravitropism between different genotypes and/or accessions.
Ca(2+) is a secondary messenger involved in early signaling events triggered in response to a plethora of biotic and abiotic stimuli. In plants, environmental cues that induce cytosolic Ca(2+) elevation include touch, reactive oxygen species, cold shock, and salt or osmotic stress. Furthermore, Ca(2+) signaling has been implicated in early stages of plant-microbe interactions of both symbiotic and antagonistic nature. A long-standing hypothesis is that there is information encoded in the Ca(2+) signals (so-called Ca(2+) signatures) to enable plants to differentiate between these stimuli and to trigger the appropriate cellular response. Qualitative and quantitative measurements of Ca(2+) signals are therefore needed to dissect the responses of plants to their environment. Luminescence produced by the Ca(2+) probe aequorin upon Ca(2+) binding is a widely used method for the detection of Ca(2+) transients and other changes in Ca(2+) concentrations in cells or organelles of plant cells. In this chapter, using microbe-associated molecular patterns (MAMPs), such as the bacterial-derived flg22 or elf18 peptides as stimuli, a protocol for the quantitative measurements of Ca(2+) fluxes in apoaequorin-expressing seedlings of Arabidopsis thaliana in 96-well format is described.
ArabidopsisMicroscopyCell / cellular structureVisualization / data management
Mitosis which is a major step during plant development can also be observed in physiopathological conditions. During the compatible interaction between the root-knot nematode Meloidogyne incognita and its host Arabidopsis, the pathogen induce through repeated divisions without complete cytokinesis the formation of hypertrophied and multinucleate feeding cells, named giant cells. Due to the presence of hypertrophied plant cell material surrounding the giant cells, classical live cell imaging gave therefore very poor resolution. Here, we describe a protocol which allows the in vivo observation of the mitotic apparatus in developing giant cells using confocal imaging of vibrosliced tissues. This approach can also be used to visualize in vivo other cellular processes occurring in different steps of giant cells.
Detection of nitric oxide (NO) in plant cells is mostly undertaken using diaminofluorescein (DAF) dyes. Serious drawbacks and limitations have been identified in methods using DAF as a probe for NO detection. The present work reporting an alternative fluorescent probe for NO detection is thus proposed for varied applications in plant systems for physiological investigations. This method involves a simple, two-step synthesis, characterization, and application of MNIP-Cu {Copper derivative of [4-methoxy-2-(1H-napthol[2,3-d]imidazol-2-yl)phenol]} for specific and rapid binding with NO, leading to its detection in plant cells by epifluorescence microscopy and confocal laser scanning microscopy (CLSM). Using sunflower (Helianthus annuus L.) whole seedlings, hypocotyl segments, stigmas from capitulum, protoplasts, and isolated oil bodies, present investigations demonstrate the versatile nature of MNIP-Cu in applications for NO localization studies. MNIP-Cu can detect NO in vivo without any time lag (ex. 330-385 nm; em. 420-500 nm). It exhibits fluorescence both under anoxic and oxygen-rich conditions. This probe is specific to NO, which enhances its fluorescence due to MNIP-Cu complexing with NO and treatment with PTIO leads to quenching of fluorescence. It is relatively nontoxic when used at a concentration of up to 50 μM.
Nitric oxide (NO) plays an important role in plant signaling and in response to various stress conditions. Therefore, real-time measurements of NO production provide better insights into understanding plant processes and can help developing strategies to improve food production and postharvest quality. Using laser-based spectroscopic methods, sensitive, online, in planta measurements of plant-pathogen interactions are possible. This chapter introduces the basic principle of the optical detectors using different laser sources for accurate monitoring of fast dynamic changes of NO production. Several applications are also presented to demonstrate the suitability of these detectors for detection of NO in plants.
Smertenko A, Moschou P, Zhang L, Fahy D, Bozhkov P.
Laboratory / benchtopMicroscopyCell / cellular structureVisualization / data management
Cytokinesis is a powerful paradigm for addressing fundamental questions of plant biology including molecular mechanisms of development, cell division, cell signaling, membrane trafficking, cell wall synthesis, and cytoskeletal dynamics. Genetics was instrumental in identification of proteins regulating cytokinesis. Characterization of mutant lines generated using forward or reverse genetics includes microscopic analysis for defects in cell division. Typically, failure of cytokinesis results in appearance of multinucleate cells, formation of cell wall stubs, and isotropic cell expansion in the root elongation zone. Small fluorescent probes served as a very effective tool for the detection of cytokinetic defects. Such probes stain living or formaldehyde-fixed specimens avoiding complex preparatory steps. Although resolution of the fluorescence probes is inferior to electron microscopy, the procedure is fast, easy, and does not require expensive materials or equipment. This chapter describes techniques for staining DNA with the probes DAPI and SYTO82, for staining membranes with FM4-64, and for staining cell wall with propidium iodide.
Peroxynitrite is a highly reactive derivative of nitric oxide (NO) which is gaining attention in the plant biology community because it may play a role in NO signaling during biotic stress. Peroxynitrite can react with many different biomolecules, but its ability to nitrate the tyrosine residues of proteins is particularly important because this may regulate defense signaling in response to pathogens. The analysis of peroxynitrite levels in the context of its proposed defense role requires an accurate and specific detection method. Here, we describe a photometric assay using the fluorescent dye Hong Kong Green 2 as a specific and quantitative probe for peroxynitrite in Arabidopsis thaliana plants challenged with an avirulent strain of Pseudomonas syringae pv. tomato. This protocol includes the preparation of plant samples, the assay procedure, the measurement of peroxynitrite-specific fluorescence, and data presentation.
Synchronization of root cells through chemical treatment can generate a large number of cells blocked in specific cell cycle phases. In plants, this approach can be employed for cell suspension cultures and plant seedlings. To identify plant cells in the course of the cell cycle, especially during mitosis in meristematic tissues, chemical inhibitors can be used to block cell cycle progression. Herein, we present a simplified and easy-to-apply protocol to visualize mitotic figures, nuclei morphology, and organization in whole Arabidopsis root apexes. The procedure is based on tissue clearing, and fluorescent staining of nuclear DNA with DAPI. The protocol allows carrying out bulk analysis of nuclei and cell cycle phases in root cells and will be valuable to investigate mutants like overexpressing lines of genes disturbing the plant cell cycle.