Diverse projected climate change scenarios affect the physiology of broccoli plants to different extents.

Climate change caused by global warming involves crucial plant growth factors such as atmospheric CO2 concentration, ambient temperature or water availability. These stressors usually co-occur, causing intricate alterations in plant physiology and development. This work focuses on how elevated atmospheric CO2 levels, together with the concomitant high temperature, would affect the physiology of a relevant crop, such as broccoli. Particular attention has been paid to those defence mechanisms that contribute to plant fitness under abiotic stress. Results show that both photosynthesis and leaf transpiration were reduced in plants grown under climate change environments compared to those grown under current climate conditions. Furthermore, an induction of carbohydrate catabolism pointed to a redistribution from primary to secondary metabolism. This result could be related to a reinforcement of cell walls, as well as to an increase in the pool of antioxidants in the leaves. Broccoli plants, a C3 crop, grown under an intermediate condition showed activation of those adaptive mechanisms, which would contribute to coping with abiotic stress, as confirmed by reduced levels of lipid peroxidation relative to current climate conditions. On the contrary, the most severe climate change scenario exceeded the adaptive capacity of broccoli plants, as shown by the inhibition of growth and reduced vigour of plants. In conclusion, only a moderate increase in atmospheric CO2 concentration and temperature would not have a negative impact on broccoli crop yields.

Virulence determines beneficial trade-offs in the response of virus-infected plants to drought via induction of salicylic acid.

It has been hypothesized that plants can get beneficial trade-offs from viral infections when grown under drought conditions. However, experimental support for a positive correlation between virus-induced drought tolerance and increased host fitness is scarce. We investigated whether increased virulence exhibited by the synergistic interaction involving Potato virus X (PVX) and Plum pox virus (PPV) improves tolerance to drought and host fitness in Nicotiana benthamiana and Arabidopsis thaliana. Infection by the pair PPV/PVX and by PPV expressing the virulence protein P25 of PVX conferred an enhanced drought-tolerant phenotype compared with single infections with either PPV or PVX. Decreased transpiration rates in virus-infected plants were correlated with drought tolerance in N. benthamiana but not in Arabidopsis. Metabolite and hormonal profiles of Arabidopsis plants infected with the different viruses showed a range of changes that positively correlated with a greater impact on drought tolerance. Virus infection enhanced drought tolerance in both species by increasing salicylic acid accumulation in an abscisic acid-independent manner. Viable offspring derived from Arabidopsis plants infected with PPV increased relative to non-infected plants, when exposed to drought. By contrast, the detrimental effect caused by the more virulent viruses overcame potential benefits associated with increased drought tolerance on host fitness.

Alterations in primary and secondary metabolism in Vitis vinifera 'Malvasía de Banyalbufar' upon infection with Grapevine leafroll-associated virus 3.

Plant defense mechanisms against pathogens result in differential regulation of various processes of primary and secondary metabolism. Imaging techniques, such as fluorescence imaging and thermography, are very valuable tools providing spatial and temporal information about these processes. In this study, effects of Grapevine leafroll-associated virus 3 (GLRaV-3) on grapevine physiology were analyzed in pot-grown asymptomatic plants of the white cultivar Malvasía de Banyalbufar. The virus triggered changes in the activity of photosynthesis and secondary metabolism. There was a decrease in the photorespiratory intermediates glycine and serine in infected plants, possibly as a defense response against the infection. The content of malate, which plays an important role in plant metabolism, also decreased. These results correlate with the increased non-photochemical quenching found in infected plants. On the other hand, the concentration of flavonols (represented by myricetin, kaempferol and quercetin derivatives) and hydroxycinnamic acids (which include derivatives of caffeic acid) increased following infection by the virus. These compounds could be responsible for the increase in multicolor fluorescence F440 (blue fluorescence) and F520 (green fluorescence) on the leaves, and changes in the fluorescence parameters F440/F680, F440/F740, F520/F680, F520/F740 and F680/F740. The combined analysis of chlorophyll fluorescence kinetics and blue-green fluorescence emitted by phenolics could constitute disease signatures allowing the discrimination between GLRaV-3 infected and non-infected plants at very early stage of infection, prior to the development of symptoms.

Picturing pathogen infection in plants.

Several imaging techniques have provided valuable tools to evaluate the impact of biotic stress on host plants. The use of these techniques enables the study of plant-pathogen interactions by analysing the spatial and temporal heterogeneity of foliar metabolism during pathogenesis. In this work we review the use of imaging techniques based on chlorophyll fluorescence, multicolour fluorescence and thermography for the study of virus, bacteria and fungi-infected plants. These studies have revealed the impact of pathogen challenge on photosynthetic performance, secondary metabolism, as well as leaf transpiration as a promising tool for field and greenhouse management of diseases. Images of standard chlorophyll fluorescence (Chl-F) parameters obtained during Chl-F induction kinetics related to photochemical processes and those involved in energy dissipation, could be good stress indicators to monitor pathogenesis. Changes on UV-induced blue (F440) and green fluorescence (F520) measured by multicolour fluorescence imaging in pathogen-challenged plants seem to be related with the up-regulation of the plant secondary metabolism and with an increase in phenolic compounds involved in plant defence, such as scopoletin, chlorogenic or ferulic acids. Thermal imaging visualizes the leaf transpiration map during pathogenesis and emphasizes the key role of stomata on innate plant immunity. Using several imaging techniques in parallel could allow obtaining disease signatures for a specific pathogen. These techniques have also turned out to be very useful for presymptomatic pathogen detection, and powerful non-destructive tools for precision agriculture. Their applicability at lab-scale, in the field by remote sensing, and in high-throughput plant phenotyping, makes them particularly useful. Thermal sensors are widely used in crop fields to detect early changes in leaf transpiration induced by both air-borne and soil-borne pathogens. The limitations of measuring photosynthesis by Chl-F at the canopy level are being solved, while the use of multispectral fluorescence imaging is very challenging due to the type of light excitation that is used.

Contribution of the non-effector members of the HrpL regulon, iaaL and matE, to the virulence of Pseudomonas syringae pv. tomato DC3000 in tomato plants.

BACKGROUND: The phytohormone indole-3-acetic acid (IAA) is widely distributed among plant-associated bacteria. Certain strains of the Pseudomonas syringae complex can further metabolize IAA into a less biologically active amino acid conjugate, 3-indole-acetyl-ε-L-lysine, through the action of the iaaL gene. In P. syringae and Pseudomonas savastanoi strains, the iaaL gene is found in synteny with an upstream gene, here called matE, encoding a putative MATE family transporter. In P. syringae pv. tomato (Pto) DC3000, a pathogen of tomato and Arabidopsis plants, the HrpL sigma factor controls the expression of a suite of virulence-associated genes via binding to hrp box promoters, including that of the iaaL gene. However, the significance of HrpL activation of the iaaL gene in the virulence of Pto DC3000 is still unclear. RESULTS: A conserved hrp box motif is found upstream of the iaaL gene in the genomes of P. syringae strains. However, although the promoter region of matE is only conserved in genomospecies 3 of this bacterial group, we showed that this gene also belongs to the Pto DC3000 HrpL regulon. We also demonstrated that the iaaL gene is transcribed both independently and as part of an operon with matE in this pathogen. Deletion of either the iaaL or the matE gene resulted in reduced fitness and virulence of Pto DC3000 in tomato plants. In addition, we used multicolor fluorescence imaging to visualize the responses of tomato plants to wild-type Pto DC3000 and to its ΔmatE and ΔiaaL mutants. Activation of secondary metabolism prior to the development of visual symptoms was observed in tomato leaves after bacterial challenges with all strains. However, the observed changes were strongest in plants challenged by the wild-type strain, indicating lower activation of secondary metabolism in plants infected with the ΔmatE or ΔiaaL mutants. CONCLUSIONS: Our results provide new evidence for the roles of non-type III effector genes belonging to the Pto DC3000 HrpL regulon in virulence.

Spatial and temporal dynamics of primary and secondary metabolism in Phaseolus vulgaris challenged by Pseudomonas syringae.

Many defense mechanisms contribute to the plant immune system against pathogens, involving the regulation of different processes of the primary and secondary metabolism. At the same time, pathogens have evolved mechanisms to hijack the plant defense in order to establish the infection and proliferate. Localization and timing of the host response are essential to understand defense mechanisms and resistance to pathogens (Rico et al. 2011). Imaging techniques, such as fluorescence imaging and thermography, are a very valuable tool providing spatial and temporal information about a series of plant processes. In this study, bean plants challenged with two pathovars of Pseudomonas syringae have been investigated. Pseudomonas syringae pv. phaseolicola 1448A and P. syringae pv. tomato DC3000 elicit a compatible and incompatible interaction in bean, respectively. Both types of host-pathogen interaction triggered different changes in the activity of photosynthesis and the secondary metabolism. We conclude that the combined analysis of leaf temperature, chlorophyll fluorescence and green fluorescence emitted by phenolics allows to discriminate compatible from incompatible P. syringae-Phaseolus vulgaris interactions in very early times of the infection, prior to the development of symptoms. These can constitute disease signatures that would allow an early identification of emerging plagues in crops.

Novel Vegetation Indices to Identify Broccoli Plants Infected With Xanthomonas campestris pv. campestris.

A rapid diagnosis of black rot in brassicas, a devastating disease caused by Xanthomonas campestris pv. campestris (Xcc), would be desirable to avoid significant crop yield losses. The main aim of this work was to develop a method of detection of Xcc infection on broccoli leaves. Such method is based on the use of imaging sensors that capture information about the optical properties of leaves and provide data that can be implemented on machine learning algorithms capable of learning patterns. Based on this knowledge, the algorithms are able to classify plants into categories (healthy and infected). To ensure the robustness of the detection method upon future alterations in climate conditions, the response of broccoli plants to Xcc infection was analyzed under a range of growing environments, taking current climate conditions as reference. Two projections for years 2081-2100 were selected, according to the Assessment Report of Intergovernmental Panel on Climate Change. Thus, the response of broccoli plants to Xcc infection and climate conditions has been monitored using leaf temperature and five conventional vegetation indices (VIs) derived from hyperspectral reflectance. In addition, three novel VIs, named diseased broccoli indices (DBI1-DBI3), were defined based on the spectral reflectance signature of broccoli leaves upon Xcc infection. Finally, the nine parameters were implemented on several classifying algorithms. The detection method offering the best performance of classification was a multilayer perceptron-based artificial neural network. This model identified infected plants with accuracies of 88.1, 76.9, and 83.3%, depending on the growing conditions. In this model, the three Vis described in this work proved to be very informative parameters for the disease detection. To our best knowledge, this is the first time that future climate conditions have been taken into account to develop a robust detection model using classifying algorithms.

An extremely low stomatal density mutant overcomes cooling limitations at supra-optimal temperature by adjusting stomatal size and leaf thickness.

The impact of global warming on transpiration and photosynthesis would compromise plant fitness, impacting on crop yields and ecosystem functioning. In this frame, we explored the performance of a set of Arabidopsis mutants carrying partial or total loss-of-function alleles of stomatal development genes and displaying distinct stomatal abundances. Using microscopy and non-invasive imaging techniques on this genotype collection, we examined anatomical leaf and stomatal traits, plant growth and development, and physiological performance at optimal (22°C) and supra-optimal (30°C) temperatures. All genotypes showed thermomorphogenetic responses but no signs of heat stress. Data analysis singled out an extremely low stomatal abundance mutant, spch-5. At 22°C, spch-5 had lower transpiration and warmer leaves than the wild type. However, at 30°C, this mutant developed larger stomata and thinner leaves, paralleled by a notable cooling capacity, similar to that of the wild type. Despite their low stomatal density (SD), spch-5 plants grown at 30°C showed no photosynthesis or growth penalties. The behavior of spch-5 at supra-optimal temperature exemplifies how the effect of very low stomatal numbers can be counteracted by a combination of larger stomata and thinner leaves. Furthermore, it provides a novel strategy for coping with high growth temperatures.

Phenotyping Plant Responses to Biotic Stress by Chlorophyll Fluorescence Imaging.

Photosynthesis is a pivotal process in plant physiology, and its regulation plays an important role in plant defense against biotic stress. Interactions with pathogens and pests often cause alterations in the metabolism of sugars and sink/source relationships. These changes can be part of the plant defense mechanisms to limit nutrient availability to the pathogens. In other cases, these alterations can be the result of pests manipulating the plant metabolism for their own benefit. The effects of biotic stress on plant physiology are typically heterogeneous, both spatially and temporarily. Chlorophyll fluorescence imaging is a powerful tool to mine the activity of photosynthesis at cellular, leaf, and whole-plant scale, allowing the phenotyping of plants. This review will recapitulate the responses of the photosynthetic machinery to biotic stress factors, from pathogens (viruses, bacteria, and fungi) to pests (herbivory) analyzed by chlorophyll fluorescence imaging both at the lab and field scale. Moreover, chlorophyll fluorescence imagers and alternative techniques to indirectly evaluate photosynthetic traits used at field scale are also revised.

Noninvasive determination of toxic stress biomarkers by high-throughput screening of photoautotrophic cell suspension cultures with multicolor fluorescence imaging.

BACKGROUND: With increasing pollution, herbicide application and interest in plant phenotyping, sensors capturing early responses to toxic stress are demanded for screening susceptible or resistant plant varieties. Standard toxicity tests on plants are laborious, demanding in terms of space and material, and the measurement of growth-inhibition based endpoints takes relatively long time. The aim of this work was to explore the potential of photoautotrophic cell suspension cultures for high-throughput early toxicity screening based on imaging techniques. The investigation of the universal potential of fluorescence imaging methods involved testing of three toxicants with different modes of action (DCMU, glyphosate and chromium). RESULTS: The increased pace of testing was achieved by using non-destructive imaging methods-multicolor fluorescence (MCF) and chlorophyll fluorescence (ChlF). These methods detected the negative effects of the toxicants earlier than it was reflected in plant growth inhibition (decrease in leaf area and final dry weight). Moreover, more subtle and transient effects not resulting in growth inhibition could be detected by fluorescence. The pace and sensitivity of stress detection was further enhanced by using photoautotrophic cell suspension cultures. These reacted sooner, more pronouncedly and to lower concentrations of the tested toxicants than the plants. Toxicant-specific stress signatures were observed as a combination of MCF and ChlF parameters and timing of the response. Principal component analysis was found to be useful for reduction of the collected multidimensional data sets to a few informative parameters allowing comparison of the toxicant signatures. CONCLUSIONS: Photoautotrophic cell suspension cultures have proved to be useful for rapid high-throughput screening of toxic stress and display a potential for employment as an alternative to tests on whole plants. The MCF and ChlF methods are capable of distinguishing early stress signatures of at least three different modes of action.

RNA-seq analysis and fluorescence imaging of melon powdery mildew disease reveal an orchestrated reprogramming of host physiology.

The cucurbit powdery mildew elicited by Podosphaera xanthii is one of the most important limiting factors in cucurbit production. Our knowledge of the genetic and molecular bases underlying the physiological processes governing this disease is very limited. We used RNA-sequencing to identify differentially expressed genes in leaves of Cucumis melo upon inoculation with P. xanthii, using RNA samples obtained at different time points during the early stages of infection and their corresponding uninfected controls. In parallel, melon plants were phenotypically characterized using imaging techniques. We found a high number of differentially expressed genes (DEGs) in infected plants, which allowed for the identification of many plant processes that were dysregulated by the infection. Among those, genes involved in photosynthesis and related processes were found to be upregulated, whereas genes involved in secondary metabolism pathways, such as phenylpropanoid biosynthesis, were downregulated. These changes in gene expression could be functionally validated by chlorophyll fluorescence imaging and blue-green fluorescence imaging analyses, which corroborated the alterations in photosynthetic activity and the suppression of phenolic compound biosynthesis. The powdery mildew disease in melon is a consequence of a complex and multifaceted process that involves the dysregulation of many plant pathways such as primary and secondary metabolism.

Use of multicolour fluorescence imaging for diagnosis of bacterial and fungal infection on zucchini by implementing machine learning.

Zucchini (Cucurbita pepo L.) is a cucurbitaceous plant ranking high in economic importance among vegetable crops worldwide. Pathogen infections cause alterations in plants primary and secondary metabolism that lead to a significant decrease in crop quality and yield. Such changes can be monitored by remote and proximal sensing, providing spatial and temporal information about the infection process. Remote sensing can also provide specific signatures of disease that could be used in phenotyping and to detect a pest, forecast its evolution and predict crop yield. In this work, metabolic changes triggered by soft rot (caused by Dickeya dadantii) and powdery mildew (caused by Podosphaera fusca) on zucchini leaves have been studied by multicolour fluorescence imaging and by thermography. The fluorescence parameter F520/F680 showed statistically significant differences between infected (with D. dadantii or P. fusca) and mock-control leaves during the whole period of study. Artificial neural networks, logistic regression analyses and support vector machines trained with a set of features characterising the histograms of F520/F680 images could be used as classifiers, discriminating between healthy and infected leaves. These results show the applicability of multicolour fluorescence imaging on plant phenotyping.

Transcriptional profiles of Arabidopsis stomataless mutants reveal developmental and physiological features of life in the absence of stomata.

Loss of function of the positive stomata development regulators SPCH or MUTE in Arabidopsis thaliana renders stomataless plants; spch-3 and mute-3 mutants are extreme dwarfs, but produce cotyledons and tiny leaves, providing a system to interrogate plant life in the absence of stomata. To this end, we compared their cotyledon transcriptomes with that of wild-type plants. K-means clustering of differentially expressed genes generated four clusters: clusters 1 and 2 grouped genes commonly regulated in the mutants, while clusters 3 and 4 contained genes distinctively regulated in mute-3. Classification in functional categories and metabolic pathways of genes in clusters 1 and 2 suggested that both mutants had depressed secondary, nitrogen and sulfur metabolisms, while only a few photosynthesis-related genes were down-regulated. In situ quenching analysis of chlorophyll fluorescence revealed limited inhibition of photosynthesis. This and other fluorescence measurements matched the mutant transcriptomic features. Differential transcriptomes of both mutants were enriched in growth-related genes, including known stomata development regulators, which paralleled their epidermal phenotypes. Analysis of cluster 3 was not informative for developmental aspects of mute-3. Cluster 4 comprised genes differentially up-regulated in mute-3, 35% of which were direct targets for SPCH and may relate to the unique cell types of mute-3. A screen of T-DNA insertion lines in genes differentially expressed in the mutants identified a gene putatively involved in stomata development. A collection of lines for conditional overexpression of transcription factors differentially expressed in the mutants rendered distinct epidermal phenotypes, suggesting that these proteins may be novel stomatal development regulators. Thus, our transcriptome analysis represents a useful source of new genes for the study of stomata development and for characterizing physiology and growth in the absence of stomata.

Imaging viral infection: studies on Nicotiana benthamiana plants infected with the pepper mild mottle tobamovirus.

We have studied by kinetic Chl-fluorescence imaging (Chl-FI) Nicotiana benthamiana plants infected with the Italian strain of the pepper mild mottle tobamovirus (PMMoV-I). We have mapped leaf photosynthesis at different points of the fluorescence induction curve as well as at different post-infection times. Images of different fluorescence parameters were obtained to investigate which one could discriminate control from infected leaves in the absence of symptoms. The non-photochemical quenching (NPQ) of excess energy in photosystem II (PSII) seems to be the most adequate chlorophyll fluorescence parameter to assess the effect of tobamoviral infection on the chloroplast. Non-symptomatic mature leaves from inoculated plants displayed a very characteristic time-varying NPQ pattern. In addition, a correlation between NPQ amplification and virus localization by tissue-print was found, suggesting that an increase in the local NPQ values is associated with the areas invaded by the pathogen. Changes in chloroplast ultrastructure in non-symptomatic leaf areas showing different NPQ levels were also investigated. A gradient of ultrastructural modifications was observed among the different areas.