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Aim: Nitrogen is a major element conditioning grapevine growth, yield and aromatic profiles of berries and wines. Different tools can be used in order to detect differences in N status of the plant, including direct measurements of soil, plant nitrogen status (eg. petiole; must), or indirect observations of plant nutritional status such as leaf transmittance or reflectance (eg. SPAD; NDVI). However, the relationships between these indicators of nitrogen status and the overall plant functioning over vintages remain poorly known. The present study aimed at quantifying key vegetative and reproductive responses to plant nitrogen status over two successive seasons under different nitrogen supply levels. Methods and results: Potted plants of Sauvignon Blanc grafted onto SO4 were grown outdoors in 2017 and 2018 with no water limitation. Four mineral nitrogen fertilization levels (equivalent to 0 kg of N ha-1 or 0U, 20U, 40U, 80U) and one organic nitrogen fertilization level (40U) were imposed in 2017. These treatments were doubled in 2018 to increase the degree of nitrogen supply and consequently, the range of observed effects on plant growth and yield. Plant nitrogen status (SPAD) was monitored weekly during both growing cycles. Yield components were determined over the two seasons. Lastly, plant carbon status was addressed through dynamic measurement of plant development and photosynthesis, and destructive measurement of dry matter accumulation and carbon storage in annual and perennial organs at flowering, veraison and harvest. The SPAD values progressively decreased under lower N supply (0N) during the first year (from 31 to 16) and they were more than halved between the maximum and the minimum N treatments straight after budburst in year two (40 for 160N and 19 for 0N). Then, the differences in SPAD values among treatments were maintained up to harvest (2018). The gradient of N status resulted in a gradient of berry numbers per inflorescence (from 180 to 34 berries/inflorescence for 80N and 0N, respectively in 2018) and of individual berry dry matter at harvest (from 0.13 to 0.41 g for 160N and 0N, respectively in 2018). Quantitative relationships between N status and the relative reductions (% of reduction per %SPAD decrease) in terms of C gain (leaf area, photosynthesis), C growth (shoot, berry, trunk and root dry matter) and C storage (trunk and root) were fitted at flowering, veraison and harvest. The reduction in C gain under lower N supply was mainly related to the decrease in total leaf area before flowering (-1.64%). Although the photosynthesis rate tended to decrease under N deficiency over the season, it only poorly contributed to the reduction in C gain. The whole plant C growth was inhibited when N status decreased (-1.13% at harvest), due to the inhibition of shoot dry matter before veraison (-1.81%) and to a lower extent, to the lower dry matter in berries (-0.80%), trunks (-0.42%) and roots (-0.84%) at harvest. Part of the reduction in root dry matter was related to the lower starch reserves (-0.31%) at harvest. Interestingly, starch reserves tended to be higher under organic N supply than mineral N supply. Conclusion: The present results provided a general framework of carbon gain and use over time (within and between seasons) as impacted by N supply levels and form. Such a framework will be useful when building a model of the pluri-annual dynamics of carbon balance related to yield elaboration in grapevines.

In leaves of C3 and C4 plants, stomata open during the day to favor CO2 entry for photosynthesis and close at night to prevent inefficient transpiration of water vapor. The circadian clock paces rhythmic stomatal movements throughout the diel (24–h) cycle. Leaf transitory starch is also thought to regulate the diel stomatal movements, yet the underlying mechanisms across time (key moments) and space (relevant leaf tissues) remain elusive. Here, we developed PhenoLeaks, a pipeline to analyze the diel dynamics of transpiration, and used it to screen a series of Arabidopsis (Arabidopsis thaliana) mutants impaired in starch metabolism. We detected a sinusoidal, endogenous rhythm of transpiration that overarches days and nights. We determined that a number of severe mutations in starch metabolism affect the endogenous rhythm through a phase shift, resulting in delayed stomatal movements throughout the daytime and diminished stomatal preopening during the night. Nevertheless, analysis of tissue-specific mutations revealed that neither guard-cell nor mesophyll-cell starch metabolisms are strictly required for normal diel patterns of transpiration. We propose that leaf starch influences the timing of transpiration rhythm through an interplay between the circadian clock and sugars across tissues, while the energetic effect of starch-derived sugars is usually nonlimiting for endogenous stomatal movements. Monitoring diel transpiration dynamics with the PhenoLeaks pipeline reveals that leaf starch metabolism sets the timing of endogenous stomatal rhythm. [ABSTRACT FROM AUTHOR]

Summary: The formation of Casparian strips (CS) and the deposition of suberin at the endodermis of plant roots are thought to limit the apoplastic transport of water and ions. We investigated the specific role of each of these apoplastic barriers in the control of hydro‐mineral transport by roots and the consequences on shoot growth.A collection of Arabidopsis thaliana mutants defective in suberin deposition and/or CS development was characterized under standard conditions using a hydroponic system and the Phenopsis platform.Mutants altered in suberin deposition had enhanced root hydraulic conductivity, indicating a restrictive role for this compound in water transport. In contrast, defective CS directly increased solute leakage and indirectly reduced root hydraulic conductivity. Defective CS also led to a reduction in rosette growth, which was partly dependent on the hydro‐mineral status of the plant. Ectopic suberin was shown to partially compensate for defective CS phenotypes.Altogether, our work shows that the functionality of the root apoplastic diffusion barriers greatly influences the plant physiology, and that their integrity is tightly surveyed. [ABSTRACT FROM AUTHOR]

Abstract Background Renewed interest in plant × environment interactions has risen in the post-genomic era. In this context, high-throughput phenotyping platforms have been developed to create reproducible environmental scenarios in which the phenotypic responses of multiple genotypes can be analysed in a reproducible way. These platforms benefit hugely from the development of suitable databases for storage, sharing and analysis of the large amount of data collected. In the model plant Arabidopsis thaliana, most databases available to the scientific community contain data related to genetic and molecular biology and are characterised by an inadequacy in the description of plant developmental stages and experimental metadata such as environmental conditions. Our goal was to develop a comprehensive information system for sharing of the data collected in PHENOPSIS, an automated platform for Arabidopsis thaliana phenotyping, with the scientific community. Description PHENOPSIS DB is a publicly available (URL: http://bioweb.supagro.inra.fr/phenopsis/) information system developed for storage, browsing and sharing of online data generated by the PHENOPSIS platform and offline data collected by experimenters and experimental metadata. It provides modules coupled to a Web interface for (i) the visualisation of environmental data of an experiment, (ii) the visualisation and statistical analysis of phenotypic data, and (iii) the analysis of Arabidopsis thaliana plant images. Conclusions Firstly, data stored in the PHENOPSIS DB are of interest to the Arabidopsis thaliana community, particularly in allowing phenotypic meta-analyses directly linked to environmental conditions on which publications are still scarce. Secondly, data or image analysis modules can be downloaded from the Web interface for direct usage or as the basis for modifications according to new requirements. Finally, the structure of PHENOPSIS DB provides a useful template for the development of other similar databases related to genotype × environment interactions.

Plant virus pathogenicity is expected to vary with changes in the abiotic environment that affect plant physiology. Conversely, viruses can alter the host plant response to additional stimuli from antagonism to mutualism depending on the virus, the host plant and the environment. Ecological theory, specifically the CSR framework of plant strategies developed by Grime and collaborators, states that plants cannot simultaneously optimize resistance to both water deficit and pathogens. Here, we investigated the vegetative and reproductive performance of 44 natural accessions of A. thaliana originating from the Iberian Peninsula upon simultaneous exposure to soil water deficit and viral infection by the Cauliflower mosaic virus (CaMV). Following the predictions of Grime's CSR theory, we tested the hypothesis that the ruderal character of a plant genotype is positively related to its tolerance to virus infection regardless of soil water availability. Our results showed that CaMV infection decreased plant vegetative performance and annihilated reproductive success of all accessions. In general, water deficit decreased plant performance, but, despite differences in behavior, ranking of accessions tolerance to CaMV was conserved under water deficit. Ruderality, quantified from leaf traits following a previously published procedure, varied significantly among accessions, and was positively correlated with tolerance to viral infection under both well-watered and water deficit conditions, although the latter to a lesser extent. Also, in accordance with the ruderal character of the accession and previous findings, our results suggest that accession tolerance to CaMV infection is positively correlated with early flowering. Finally, plant survival to CaMV infection increased under water deficit. The complex interactions between plant, virus and abiotic environment are discussed in terms of the variation in plant ecological strategies at the intraspecific level. Author summary: Virus pathogenicity may be influenced by changes in the abiotic environment. A common change is decrease in soil water availability, which is detrimental to plant productivity and the occurrence of which is expected to increase due to climate change, has recently been shown to interfere with plant–virus interactions. We investigated the performance of 44 natural accessions of the plant species Arabidopsis thaliana infected by Cauliflower mosaic virus under well-watered and water deficit conditions. We showed that viral infection decreased plant vegetative performance and annihilated reproductive success of all accessions, and that these pathogenic effects were increased by water deficit. Intrinsic characteristics of the accessions were related to their tolerance to the virus so that accessions with low leaf tissue density and rapid growth rate were more tolerant to viral infection regardless of watering condition. Finally, plant survival upon viral infection increased under water deficit. We discuss the role of intrinsic plant characteristics, seen as ecological strategies, in plant tolerance to viral infections under contrasting environmental conditions, and the consequences for the study of viral epidemiology. [ABSTRACT FROM AUTHOR]

Plants suffer from a broad range of abiotic and biotic stresses that do not occur in isolation but often simultaneously. Productivity of natural and agricultural systems is frequently constrained by water limitation, and the frequency and duration of drought periods will likely increase due to global climate change. In addition, phytoviruses represent highly prevalent biotic threat in wild and cultivated plant species. Several hints support a modification of epidemiological parameters of plant viruses in response to environmental changes but a clear quantification of plant-virus interactions under abiotic stresses is still lacking. Here we report the effects of a water deficit on epidemiological parameters of Cauliflower mosaic virus (CaMV), a non-circulative virus transmitted by aphid vectors, in nine natural accessions of Arabidopsis thaliana with known contrasted responses to water deficit. Plant growth-related traits and virus epidemiological parameters were evaluated in PHENOPSIS, an automated high throughput phenotyping platform. Water deficit had contrasted effects on CaMV transmission rate and viral load among A. thaliana accessions. Under well-watered conditions, transmission rate tended to increase with viral load and with CaMV virulence across accessions. Under water deficit, transmission rate and virulence were negatively correlated. Changes in the rate of transmission under water deficit were not related to changes in viral load. Our results support the idea that optimal virulence of a given virus, as hypothesized under the transmission-virulence trade-off, is highly dependent on the environment and growth traits of the host. [ABSTRACT FROM AUTHOR]

Background: Plant science uses increasing amounts of phenotypic data to unravel the complex interactions between biological systems and their variable environments. Originally, phenotyping approaches were limited by manual, often destructive operations, causing large errors. Plant imaging emerged as a viable alternative allowing non-invasive and automated data acquisition. Several procedures based on image analysis were developed to monitor leaf growth as a major phenotyping target. However, in most proposals, a time-consuming parameterization of the analysis pipeline is required to handle variable conditions between images, particularly in the field due to unstable light and interferences with soil surface or weeds. To cope with these difficulties, we developed a low-cost, 2D imaging method, hereafter called PYM. The method is based on plant leaf ability to absorb blue light while reflecting infrared wavelengths. PYM consists of a Raspberry Pi computer equipped with an infrared camera and a blue filter and is associated with scripts that compute projected leaf area. This new method was tested on diverse species placed in contrasting conditions. Application to field conditions was evaluated on lettuces grown under photovoltaic panels. The objective was to look for possible acclimation of leaf expansion under photovoltaic panels to optimise the use of solar radiation per unit soil area. Results: The new PYM device proved to be efficient and accurate for screening leaf area of various species in wide ranges of environments. In the most challenging conditions that we tested, error on plant leaf area was reduced to 5% using PYM compared to 100% when using a recently published method. A high-throughput phenotyping cart, holding 6 chained PYM devices, was designed to capture up to 2000 pictures of field-grown lettuce plants in less than 2 h. Automated analysis of image stacks of individual plants over their growth cycles revealed unexpected differences in leaf expansion rate between lettuces rows depending on their position below or between the photovoltaic panels. Conclusions: The imaging device described here has several benefits, such as affordability, low cost, reliability and flexibility for online analysis and storage. It should be easily appropriated and customized to meet the needs of various users. [ABSTRACT FROM AUTHOR]

Following the recent development of high-throughput phenotyping platforms for plant research, the number of individual plants grown together in a same experiment has raised, sometimes at the expense of pot size. However, root restriction in excessively small pots affects plant growth and carbon partitioning, andmayinteract with other stresses targeted in these experiments. In work reported here, we investigated the interactive effects of pot size and soil water deficit on multiple growth-related traits from the cellular to the whole-plant scale in oilseed rape (Brassica napus L.). The effects of pot size on responses to water deficit and allometric relationships revealed strong, multilevel interactions between pot size and watering regime. Notably, water deficit increased the root : shoot ratio in large pots, but not in small pots. At the cellular scale, water deficit decreased epidermal leaf cell area in large pots, but not in small pots. These results were consistent with changes in the level of endoreduplication factor in leaf cells. Our study illustrates the disturbing interaction of pot size with water deficit and raises the need to carefully consider this factor in the frame of the current development of high-throughput phenotyping experiments. [ABSTRACT FROM AUTHOR]

Background: Effects of abiotic and biotic stresses on plant photosynthetic performance lead to fitness and yield decrease. The maximum quantum efficiency of photosystem II (Fv/Fm) is a parameter of chlorophyll fluorescence (ChlF) classically used to track changes in photosynthetic performance. Despite recent technical and methodological advances in ChlF imaging, the spatio-temporal heterogeneity of Fv/Fm still awaits for standardized and accurate quantification. Results: We developed a method to quantify the dynamics of spatial heterogeneity of photosynthetic efficiency through the distribution-based analysis of Fv/Fm values. The method was applied to Arabidopsis thaliana grown under well-watered and severe water deficit (survival rate of 40%). First, whole-plant Fv/Fm shifted from unimodal to bimodal distributions during plant development despite a constant mean Fv/Fm under well-watered conditions. The establishment of a bimodal distribution of Fv/Fm reflects the occurrence of two types of leaf regions with contrasted photosynthetic efficiency. The distance between the two modes (called S) quantified the whole-plant photosynthetic heterogeneity. The weighted contribution of the most efficient/healthiest leaf regions to whole-plant performance (called Wmax) quantified the spatial efficiency of a photosynthetically heterogeneous plant. Plant survival to water deficit was associated to high S values, as well as with strong and fast recovery of Wmax following soil rewatering. Hence, during stress surviving plants had higher, but more efficient photosynthetic heterogeneity compared to perishing plants. Importantly, S allowed the discrimination between surviving and perishing plants four days earlier than the mean Fv/Fm. A sensitivity analysis from simulated dynamics of Fv/Fm showed that parameters indicative of plant tolerance and/or stress intensity caused identifiable changes in S and Wmax. Finally, an independent comparison of six Arabidopsis accessions grown under well-watered conditions indicated that S and Wmax are related to the genetic variability of growth. Conclusions: The distribution-based analysis of ChlF provides an efficient tool for quantifying photosynthetic heterogeneity and performance. S and Wmax are good indicators to estimate plant survival under water stress. Our results suggest that the dynamics of photosynthetic heterogeneity are key components of plant growth and tolerance to stress. [ABSTRACT FROM AUTHOR]

Mutualistic bacteria can alter plant phenotypes and confer new abilities to plants. Some plant growth-promoting rhizobacteria (PGPR) are known to improve both plant growth and tolerance to multiple stresses, including drought, but reports on their effects on plant survival under severe water deficits are scarce. We investigated the effect of Phyllobacterium brassicacearum STM196 strain, a PGPR isolated from the rhizosphere of oilseed rape, on survival, growth and physiological responses of Arabidopsis thaliana to severe water deficits combining destructive and non-destructive high-throughput phenotyping. Soil inoculation with STM196 greatly increased the survival rate of A. thaliana under several scenarios of severe water deficit. Photosystem II efficiency, assessed at the whole-plant level by high-throughput fluorescence imaging (Fv/Fm), was related to the probability of survival and revealed that STM196 delayed plant mortality. Inoculated surviving plants tolerated more damages to the photosynthetic tissues through a delayed dehydration and a better tolerance to low water status. Importantly, STM196 allowed a better recovery of plant growth after rewatering and stressed plants reached a similar biomass at flowering than non-stressed plants. Our results highlight the importance of plant-bacteria interactions in plant responses to severe drought and provide a new avenue of investigations to improve drought tolerance in agriculture. [ABSTRACT FROM AUTHOR]

Inorganic phosphate (Pi) and Zinc (Zn) are essential nutrients for normal plant growth. Interaction between these elements has been observed in many crop plants. Despite its agronomic importance, the biological significance and genetic basis of this interaction remain largely unknown. Here we examined the Pi/Zn interaction in two lettuce (Lactuca sativa) varieties, namely, "Paris Island Cos" and "Kordaat." The effects of variation in Pi and Zn supply were assessed on biomass and photosynthesis for each variety. Paris Island Cos displayed better growth and photosynthesis compared to Kordaat under all the conditions tested. Correlation analysis was performed to determine the interconnectivity between Pi and Zn intracellular contents in both varieties. Paris Island Cos showed a strong negative correlation between the accumulation levels of Pi and Zn in shoots and roots. However, no relation was observed for Kordaat. The increase of Zn concentration in the medium causes a decrease in dynamics of Pi transport in Paris Island Cos, but not in Kordaat plants. Taken together, results revealed a contrasting behavior between the two lettuce varieties in terms of the coregulation of Pi and Zn homeostasis and provided evidence in favor of a genetic basis for the interconnection of these two elements. [ABSTRACT FROM AUTHOR]

ABSTRACT Light and soil water content affect leaf surface area expansion through modifications in epidermal cell numbers and area, while effects on leaf thickness and mesophyll cell volumes are far less documented. Here, three-dimensional imaging was applied in a study of Arabidopsis thaliana leaf growth to determine leaf thickness and the cellular organization of mesophyll tissues under moderate soil water deficit and two cumulative light conditions. In contrast to surface area, thickness was highly conserved in response to water deficit under both low and high cumulative light regimes. Unlike epidermal and palisade mesophyll tissues, no reductions in cell number were observed in the spongy mesophyll; cells had rather changed in volume and shape. Furthermore, leaf features of a selection of genotypes affected in leaf functioning were analysed. The low-starch mutant pgm had very thick leaves because of unusually large palisade mesophyll cells, together with high levels of photosynthesis and stomatal conductance. By means of an open stomata mutant and a 9- cis-epoxycarotenoid dioxygenase overexpressor, it was shown that stomatal conductance does not necessarily have a major impact on leaf dimensions and cellular organization, pointing to additional mechanisms for the control of CO2 diffusion under high and low stomatal conductance, respectively. [ABSTRACT FROM AUTHOR]

Background: Renewed interest in plant x environment interactions has risen in the post-genomic era. In this context, high-throughput phenotyping platforms have been developed to create reproducible environmental scenarios in which the phenotypic responses of multiple genotypes can be analysed in a reproducible way. These platforms benefit hugely from the development of suitable databases for storage, sharing and analysis of the large amount of data collected. In the model plant Arabidopsis thaliana, most databases available to the scientific community contain data related to genetic and molecular biology and are characterised by an inadequacy in the description of plant developmental stages and experimental metadata such as environmental conditions. Our goal was to develop a comprehensive information system for sharing of the data collected in PHENOPSIS, an automated platform for Arabidopsis thaliana phenotyping, with the scientific community. Description: PHENOPSIS DB is a publicly available (URL: http://bioweb.supagro.inra.fr/phenopsis/) information system developed for storage, browsing and sharing of online data generated by the PHENOPSIS platform and offline data collected by experimenters and experimental metadata. It provides modules coupled to a Web interface for (i) the visualisation of environmental data of an experiment, (ii) the visualisation and statistical analysis of phenotypic data, and (iii) the analysis of Arabidopsis thaliana plant images. Conclusions: Firstly, data stored in the PHENOPSIS DB are of interest to the Arabidopsis thaliana community, particularly in allowing phenotypic meta-analyses directly linked to environmental conditions on which publications are still scarce. Secondly, data or image analysis modules can be downloaded from the Web interface for direct usage or as the basis for modifications according to new requirements. Finally, the structure of PHENOPSIS DB provides a useful template for the development of other similar databases related to genotype x environment interactions. [ABSTRACT FROM AUTHOR]

Variation in leaf development caused by water deficit was analysed in 120 recombinant inbred lines derived from two Arabidopsis thaliana accessions, L er and An-1. Main effect quantitative trait loci (QTLs) and QTLs in epistatic interactions were mapped for the responses of rosette area, leaf number and leaf 6 area to water deficit. An epistatic interaction between two QTLs affected the response of whole rosette area and individual leaf area but only with effects in well-watered condition. A second epistatic interaction between two QTLs controlled the response of rosette area and leaf number with specific effects in the water deficit condition. These effects were validated by generating and phenotyping new appropriate lines. Accordingly, a low reduction of rosette area was observed for lines with a specific allelic combination at the two interacting QTLs. This low reduction was accompanied by an increase in leaf number with a lengthening of the vegetative phase and a low reduction in individual leaf area with low reductions in epidermal cell area and number. Statistical analyses suggested that responses of epidermal cell area and number to water deficit in individual leaves were partly caused by delay in flowering time and reduction in leaf emergence rate, respectively. [ABSTRACT FROM AUTHOR]

Genetic variability in the plasticity of leaf area expansion in response to water deficit has been reported in Arabidopsis thaliana. Here, the objective was to identify the underlying dynamic and cellular processes involved in this variability. Twenty-five accessions were subjected to identical soil water deficit treatments. In all accessions, the plasticity of leaf production was low compared with that of individual leaf expansion. A subset of accessions was selected for further dissection of individual leaf expansion into its underlying variables: the rate and duration of leaf expansion and epidermal cell number and area. In all accessions, water deficit had opposite effects on the rate and duration of leaf expansion. The accumulation of these effects was reflected in changes in final leaf area. At the cellular level, moderate water deficits had opposite effects on cell number and cell size, but more severe ones reduced both variables. The importance of these opposing effects is highlighted by the behaviour of the accession An-1, for which the compensation between the decrease in leaf expansion rate and the increase in the duration of expansion is total. This dynamic plasticity in response to water deficit is not detectable when only final measurements are done. [ABSTRACT FROM AUTHOR]

It is clearly established that there is not a unique response to soil water deficit but that there are as many responses as soil water deficit characteristics: Drought intensity, drought duration, and drought position during plant cycle. For a same soil water deficit, responses can also differ on plant genotype within a same species. In spite of this variability, at least for leaf production and expansion processes, robust tendencies can be extracted from the literature when similar watering regimes are compared. Here, we present response curves and multi-scale dynamics analyses established on tomato plants exposed to different soil water deficit treatments. Results reinforce the trends already observed for other species: Reduction in plant leaf biomass under water stress was due to reduction in individual leaf biomass and areas whereas leaf production and specific leaf area were not affected. The dynamics of leaf expansion was modified both at the leaf and cell scales. Cell division and expansion were reduced by drought treatments as well as the endoreduplication process. Combining response curves analyses together with dynamic analyses of tomato compound leaf growth at different scales not only corroborate results on simple leaf responses to drought but also increases our knowledge on the cellular mechanisms behind leaf growth plasticity. [ABSTRACT FROM AUTHOR]

The regulation of the GORK (Guard Cell Outward Rectifying) Shaker channel mediating a massive K+ efflux in Arabidopsis guard cells by the phosphatase AtPP2CA was investigated. Unlike the gork mutant, the atpp2ca mutants displayed a phenotype of reduced transpiration. We found that AtPP2CA interacts physically with GORK and inhibits GORK activity in Xenopus oocytes. Several amino acid substitutions in the AtPP2CA active site, including the dominant interfering G145D mutation, disrupted the GORK-AtPP2CA interaction, meaning that the native conformation of the AtPP2CA active site is required for theGORKAtPP2CA interaction. Furthermore, two serines in the GORK ankyrin domain that mimic phosphorylation (Ser to Glu) or dephosphorylation (Ser to Ala) were mutated. Mutations mimicking phosphorylation led to a significant increase in GORK activity, whereas mutations mimicking dephosphorylation had no effect on GORK. In Xenopus oocytes, the interaction of AtPP2CA with "phosphorylated" or "dephosphorylated"GORK systematically led to inhibition of the channel to the same baseline level. Single-channel recordings indicated that the GORK S722E mutation increases the open probability of the channel in the absence, but not in the presence, of AtPP2CA. The dephosphorylation- independent inactivation mechanism of GORK by AtPP2CA is discussed in relation with well known conformational changes in animal Shaker-like channels that lead to channel opening and closing. In plants, PP2C activity would control the stomatal aperture by regulating both GORK and SLAC1, the two main channels required for stomatal closure. [ABSTRACT FROM AUTHOR]

High-throughput phenotyping of plant traits is a powerful tool to further our understanding of plant growth and its underlying physiological, molecular, and genetic determinisms. This protocol describes the methodology of a standard phenotyping experiment in PHENOPSIS automated platform, which was engineered in INRA-LEPSE (https://www6.montpellier.inra.fr/lepse) and custom-made by Optimalog company. The seminal method was published by Granier et al. (2006). The platform is used to explore and test various ecophysiological hypotheses (Tisné et al. , 2010; Baerenfaller et al. , 2012; Vile et al. , 2012; Bac-Molenaar et al. , 2015; Rymaszewski et al. , 2017). Here, the focus concerns the preparation and management of experiments, as well as measurements of growth-related traits ( e.g ., projected rosette area, total leaf area and growth rate), water status-related traits ( e.g ., leaf dry matter content and relative water content), and plant architecture-related traits ( e.g ., stomatal density and index and lamina/petiole ratio). Briefly, a completely randomized (block) design is set up in the growth chamber. Next, the substrate is prepared, its initial water content is measured and pots are filled. Seeds are sown onto the soil surface and germinated prior to the experiment. After germination, soil watering and image (visible, infra-red, fluorescence) acquisition are planned by the user and performed by the automaton. Destructive measurements may be performed during the experiment. Data extraction from images and estimation of growth-related trait values involves semi-automated procedures and statistical processing., (Copyright © 2018 The Authors; exclusive licensee Bio-protocol LLC.)

The regulation of the GORK (Guard Cell Outward Rectifying) Shaker channel mediating a massive K(+) efflux in Arabidopsis guard cells by the phosphatase AtPP2CA was investigated. Unlike the gork mutant, the atpp2ca mutants displayed a phenotype of reduced transpiration. We found that AtPP2CA interacts physically with GORK and inhibits GORK activity in Xenopus oocytes. Several amino acid substitutions in the AtPP2CA active site, including the dominant interfering G145D mutation, disrupted the GORK-AtPP2CA interaction, meaning that the native conformation of the AtPP2CA active site is required for the GORK-AtPP2CA interaction. Furthermore, two serines in the GORK ankyrin domain that mimic phosphorylation (Ser to Glu) or dephosphorylation (Ser to Ala) were mutated. Mutations mimicking phosphorylation led to a significant increase in GORK activity, whereas mutations mimicking dephosphorylation had no effect on GORK. In Xenopus oocytes, the interaction of AtPP2CA with "phosphorylated" or "dephosphorylated" GORK systematically led to inhibition of the channel to the same baseline level. Single-channel recordings indicated that the GORK S722E mutation increases the open probability of the channel in the absence, but not in the presence, of AtPP2CA. The dephosphorylation-independent inactivation mechanism of GORK by AtPP2CA is discussed in relation with well known conformational changes in animal Shaker-like channels that lead to channel opening and closing. In plants, PP2C activity would control the stomatal aperture by regulating both GORK and SLAC1, the two main channels required for stomatal closure., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Following the recent development of high-throughput phenotyping platforms for plant research, the number of individual plants grown together in a same experiment has raised, sometimes at the expense of pot size. However, root restriction in excessively small pots affects plant growth and carbon partitioning, and may interact with other stresses targeted in these experiments. In work reported here, we investigated the interactive effects of pot size and soil water deficit on multiple growth-related traits from the cellular to the whole-plant scale in oilseed rape (Brassica napus L.). The effects of pot size on responses to water deficit and allometric relationships revealed strong, multilevel interactions between pot size and watering regime. Notably, water deficit increased the root:shoot ratio in large pots, but not in small pots. At the cellular scale, water deficit decreased epidermal leaf cell area in large pots, but not in small pots. These results were consistent with changes in the level of endoreduplication factor in leaf cells. Our study illustrates the disturbing interaction of pot size with water deficit and raises the need to carefully consider this factor in the frame of the current development of high-throughput phenotyping experiments.

Water stresses reduce plant growth but there is no consensus on whether carbon metabolism has any role in this reduction. Sugar starvation resulting from stomatal closure is often proposed as a cause of growth impairment under long-term or severe water deficits. However, growth decreases faster than photosynthesis in response to drought, leading to increased carbohydrate stores under short-term or moderate water deficits. Here, we addressed the question of the role of carbon availability on growth under moderate water deficits using two different systems. Firstly, we monitored the day/night pattern of leaf growth in Arabidopsis plants. We show that a moderate soil water deficit promotes leaf growth at night in mutants severely disrupted in their nighttime carbohydrate availability. This suggests that soil water deficit promotes carbon satiation. Secondly, we monitored the sub-hourly growth variations of clementine fruits in response to daily, natural fluctuations in air water deficit, and at contrasting source-sink balances obtained by defoliation. We show that high carbohydrate levels prevent excessive, hydraulic shrinkage of the fruit during days with high evaporative demand, most probably through osmotic adjustment. Together, our results contribute to the view that growing organs under moderate soil or air water deficit are not carbon starved, but use soluble carbohydrate in excess to partly release a hydromechanical limitation of growth.

Leaves of flowering plants are produced from the shoot apical meristem at regular intervals and they grow according to a developmental program that is determined by both genetic and environmental factors. Detailed frameworks for multiscale dynamic analyses of leaf growth have been developed in order to identify and interpret phenotypic differences caused by either genetic or environmental variations. They revealed that leaf growth dynamics are non-linearly and nonhomogeneously distributed over the lamina, in the leaf tissues and cells. The analysis of the variability in leaf growth, and its underlying processes, has recently gained momentum with the development of automated phenotyping platforms that use various technologies to record growth at different scales and at high throughput. These modern tools are likely to accelerate the characterization of gene function and the processes that underlie the control of shoot development. Combined with powerful statistical analyses, trends have emerged that may have been overlooked in low throughput analyses. However, in many examples, the increase in throughput allowed by automated platforms has led to a decrease in the spatial and/or temporal resolution of growth analyses. Concrete examples presented here indicate that simplification of the dynamic leaf system, without consideration of its spatial and temporal context, can lead to important misinterpretations of the growth phenotype., (Copyright © 2013 Wiley Periodicals, Inc.)