PYM: a new, affordable, image-based method using a Raspberry Pi to phenotype plant leaf area in a wide diversity of environments.

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.

Abscisic Acid Down-Regulates Hydraulic Conductance of Grapevine Leaves in Isohydric Genotypes Only.

Plants evolved different strategies to cope with water stress. While isohydric species maintain their midday leaf water potential (ΨM) under soil water deficit by closing their stomata, anisohydric species maintain higher stomatal aperture and exhibit substantial reductions in ΨM It was hypothesized that isohydry is related to a locally higher sensitivity of stomata to the drought-hormone abscisic acid (ABA). Interestingly, recent lines of evidence in Arabidopsis (Arabidopsis thaliana) suggested that stomatal responsiveness is also controlled by an ABA action on leaf water supply upstream from stomata. Here, we tested the possibility in grapevine (Vitis vinifera) that different genotypes ranging from near isohydric to more anisohydric may have different sensitivities in these ABA responses. Measurements on whole plants in drought conditions were combined with assays on detached leaves fed with ABA. Two different methods consistently showed that leaf hydraulic conductance (Kleaf) was down-regulated by exogenous ABA, with strong variations depending on the genotype. Importantly, variation between isohydry and anisohydry correlated with Kleaf sensitivity to ABA, with Kleaf in the most anisohydric genotypes being unresponsive to the hormone. We propose that the observed response of Kleaf to ABA may be part of the overall ABA regulation of leaf water status.

Reduced nighttime transpiration is a relevant breeding target for high water-use efficiency in grapevine.

Increasing water scarcity challenges crop sustainability in many regions. As a consequence, the enhancement of transpiration efficiency (TE)-that is, the biomass produced per unit of water transpired-has become crucial in breeding programs. This could be achieved by reducing plant transpiration through a better closure of the stomatal pores at the leaf surface. However, this strategy generally also lowers growth, as stomatal opening is necessary for the capture of atmospheric CO2 that feeds daytime photosynthesis. Here, we considered the reduction in transpiration rate at night (En) as a possible strategy to limit water use without altering growth. For this purpose, we carried out a genetic analysis for En and TE in grapevine, a major crop in drought-prone areas. Using recently developed phenotyping facilities, potted plants of a cross between Syrah and Grenache cultivars were screened for 2 y under well-watered and moderate soil water deficit scenarios. High genetic variability was found for En under both scenarios and was primarily associated with residual diffusion through the stomata. Five quantitative trait loci (QTLs) were detected that underlay genetic variability in En Interestingly, four of them colocalized with QTLs for TE. Moreover, genotypes with favorable alleles on these common QTLs exhibited reduced En without altered growth. These results demonstrate the interest of breeding grapevine for lower water loss at night and pave the way to breeding other crops with this underexploited trait for higher TE.

Phenotypic plasticity toward water regime: response of leaf growth and underlying candidate genes in Populus.

Phenotypic plasticity is considered as an important mechanism for plants to cope with environmental challenges. Leaf growth is one of the first macroscopic processes to be impacted by modification of soil water availability. In this study, we intended to analyze and compare plasticity at different scales. We examined the differential effect of water regime (optimal, moderate water deprivation and recovery) on growth and on the expression of candidate genes in leaves of different growth stages. Candidates were selected to assess components of growth response: abscisic acid signaling, water transport, cell wall modification and stomatal development signaling network. At the tree scale, the four studied poplar hybrids responded similarly to water regime. Meanwhile, leaf growth response was under genotype × environment interaction. Patterns of candidate gene expression enriched our knowledge about their functionality in poplars. For most candidates, transcript levels were strongly structured according to leaf growth performance while response to water regime was clearly dependent on genotype. The use of an index of plasticity revealed that the magnitude of the response was higher for gene expression than for macroscopic traits. In addition, the ranking of poplar genotypes for macroscopic traits well paralleled the one for gene expression.

Relationships between biomass allocation, axis organogenesis and organ expansion under shading and water deficit conditions in grapevine.

The relationships between whole-plant growth and morphogenetic processes under abiotic stresses are still partly unknown. Whole-plant biomass growth can be decreased by many abiotic stresses, including water deficit and shading. Two experiments were performed on potted plants of one grapevine cultivar (Vitis vinifera L. cv. Syrah) subjected to watering and shading treatments. Under water stress, plants reduced their primary and secondary axis leaf production rate, whereas secondary axis budburst was relatively unaffected. Individual leaf area was reduced and a strong decrease in leaf expansion rate was observed. Under shading, primary axis organogenesis was maintained, both secondary axis budburst rate and phytomer appearance rate were decreased, and individual leaf area slightly increased. Specific leaf area did not change under soil water deficit, whereas it increased under shading. These results confirm the existence of dynamic changes in organ sink strength and biomass allocation patterns to favour plant leaf area growth under shading, and to reduce plant leaf area and water losses by transpiration under water stress. From a modelling point of view, this study shows that functional structural models based on a C balance are not fully relevant for simulating plant growth under abiotic constraints if they do not include non-trophic relationships (hormonal signalling or plant hydraulic properties) that modify organ sink strength according to abiotic constraints.

Genetic variation in a grapevine progeny (Vitis vinifera L. cvs Grenache×Syrah) reveals inconsistencies between maintenance of daytime leaf water potential and response of transpiration rate under drought.

In the face of water stress, plants evolved with different abilities to limit the decrease in leaf water potential, notably in the daytime (ΨM). So-called isohydric species efficiently maintain high ΨM, whereas anisohydric species cannot prevent ΨM from dropping as soil water deficit develops. The genetic and physiological origins of these differences in (an)isohydric behaviours remain to be clarified. This is of particular interest within species such as Vitis vinifera L. where continuous variation in the level of isohydry has been observed among cultivars. With this objective, a 2 year experiment was conducted on the pseudo-F1 progeny from a cross between the two widespread cultivars Syrah and Grenache using a phenotyping platform coupled to a controlled-environment chamber. Potted plants of all the progeny were analysed for ΨM, transpiration rate, and soil-to-leaf hydraulic conductance, under both well-watered and water deficit conditions. A high genetic variability was found for all the above traits. Four quantitative trait loci (QTLs) were detected for ΨM under water deficit conditions, and 28 other QTLs were detected for the different traits in either condition. Genetic variation in ΨM maintenance under water deficit weakly correlated with drought-induced reduction in transpiration rate in the progeny, and QTLs for both traits did not completely co-localize. This indicates that genetic variation in the control of ΨM under water deficit was not due simply to variation in transpiration sensitivity to soil drying. Possible origins of the diversity in (an)isohydric behaviours in grapevine are discussed on the basis of concurrent variations in soil-to-leaf hydraulic conductance and stomatal control of transpiration.

Nitrogen supply controls vegetative growth, biomass and nitrogen allocation for grapevine (cv. Shiraz) grown in pots.

Maintaining grapevine productivity with limited inputs is crucial in Mediterranean areas. Apart from water, nitrogen (N) is also an important limiting factor in grape growing. The effects of N deficiency on grapevine growth were investigated in this study. Two-year-old Vitis vinifera L.cv. Shiraz plants grafted on 110 R were grown in pots placed outside and exposed to various N supplies (0, 0.6, 1.2, 2.4 and 12g plant-1) under well-watered conditions. At veraison, plants were harvested and organs separately dried, weighed and analysed for N. During plant growth, the length of the primary and secondary axes and the number of leaves on them were recorded. The N content of leaves was also analysed at three phenological stages (flowering, bunch closure and veraison). All growth processes were inhibited by N deficiency in an intensity-dependent manner. Quantitative relationships with N supply were established. Vegetative growth responded negatively to N stress when comparing control N supply with no N supply: primary axis elongation (-61%), leaf emergence on the primary axis (-47%), leaf emergence on the secondary axis (-94%) and lamina area expansion (-45%). Significant differences on the plant N status were observed from flowering onwards which might be useful for managing fertilisation.

Use of water extraction variability to screen for sunflower genotypes well adapted to soil water limitation.

In conditions of water deficit, plant yield depends mostly on the ability of the plant to explore soil profile and its water uptake capacity per unit volume of soil. In this study, the value of soil water extraction properties for use in sunflower breeding was evaluated. Five experiments were carried out in pots, in greenhouses, from 2005 to 2009, in Montpellier, France. Elite sunflower cultivars and experimental hybrids obtained from a factorial cross between five female and five male inbred lines were grown. The soil water extraction performance of the plants was characterised by the soil water content at minimal stomatal conductance (SWCgs=0) and the index of water extraction (IEgen), which was calculated as the relative value of SWCgs=0 to the performance of the cultivar NKMelody. Heritability (H2) was estimated for the experimental hybrids. Phenotypic variability of the SWCgs=0 was observed with a significant effect of the environment and the genotype. The latest released cultivars were observed as the best performing one in water extraction with an IEgen under 0.85. This trait was found to be suitable for use in comparisons of the soil water extraction performances of different genotypes. The high H2 value for SWCgs=0 (0.77 and 0.81) and the significant correlation (r2=0.70, P<0.001) between the values obtained for the experimental hybrids and the mean values of the general combining ability (GCA) for the parental lines showed that this trait is heritable and could be used in plant breeding programs. Phenotyping methods and the usefulness of this trait in crop modelling are discussed.

Arabidopsis plants acclimate to water deficit at low cost through changes of carbon usage: an integrated perspective using growth, metabolite, enzyme, and gene expression analysis.

Growth and carbon (C) fluxes are severely altered in plants exposed to soil water deficit. Correspondingly, it has been suggested that plants under water deficit suffer from C shortage. In this study, we test this hypothesis in Arabidopsis (Arabidopsis thaliana) by providing an overview of the responses of growth, C balance, metabolites, enzymes of the central metabolism, and a set of sugar-responsive genes to a sustained soil water deficit. The results show that under drought, rosette relative expansion rate is decreased more than photosynthesis, leading to a more positive C balance, while root growth is promoted. Several soluble metabolites accumulate in response to soil water deficit, with K(+) and organic acids as the main contributors to osmotic adjustment. Osmotic adjustment costs only a small percentage of the daily photosynthetic C fixation. All C metabolites measured (not only starch and sugars but also organic acids and amino acids) show a diurnal turnover that often increased under water deficit, suggesting that these metabolites are readily available for being metabolized in situ or exported to roots. On the basis of 30 enzyme activities, no in-depth reprogramming of C metabolism was observed. Water deficit induces a shift of the expression level of a set of sugar-responsive genes that is indicative of increased, rather than decreased, C availability. These results converge to show that the differential impact of soil water deficit on photosynthesis and rosette expansion results in an increased availability of C for the roots, an increased turnover of C metabolites, and a low-cost C-based osmotic adjustment, and these responses are performed without major reformatting of the primary metabolism machinery.

Using a mathematical model to evaluate the trophic and non-trophic determinants of axis development in grapevine.

The grapevine (Vitis vinifera L.) shoot is a complex modular branching system, with one primary axis and many secondary axes organised into a repetitive structure of three successive phytomers (P0-P1-P2). P1-P2 phytomers bear one tendril or cluster, whereas P0 phytomers bear no tendrils or clusters. Axis development displays a high variability, due, partly, to trophic competition. The aim of this study was to estimate changes in trophic competition within the shoot, and to relate plasticity in axis development to changes in trophic competition. 'Grenache N.' and 'Syrah' cultivars were grown with two contrasting levels of cluster load. Organogenesis and organ mass were measured during shoot development. Changes in trophic competition were estimated, using the solver functions of the GreenLab model. Internodes and clusters were strong sinks. They affected the shoot development to the same extent, but the internodes showed an earlier effect. The cessation of development of the secondary axis was affected by trophic competition, but the primary axis continued to develop, regardless of trophic competition. Secondary axes differed in sensitivity to trophic competition as a function of two criteria: their type and their size. The most highly developed axes were less affected than the smaller axes, and secondary axes arising from a P0 phytomer were also less affected than secondary axes arising from a P1 or P2 phytomer.

Influence of intra-shoot trophic competition on shoot development in two grapevine cultivars (Vitis vinifera).

The effect of trophic competition between vegetative sources and reproductive sinks on grapevine (Vitis vinifera L.) shoot development was analyzed. Two international cultivars (Grenache N and Syrah) grown in pots, which were well watered, were studied. A large range of trophic competition levels was obtained by modifying the cluster loads per plant. An analytical breakdown of the branching system was used to analyze the effects of trophic competition. Phytomer production on the primary axis and the probability and timing of axillary budburst were not affected by trophic competition. However, the duration of development and leaf production rate for secondary axes were both significantly affected. The impact of trophic competition differed within the P0-P1-P2 architectural module, locally within the shoot and between cultivars. Trophic competition reduced the organogenesis of secondary axes most strongly close to clusters, on P1-P2 phytomers and in Grenache N. Based on these results, a modeling approach simulating sink strength variation and the local effects of sink proximity would be more relevant than a model considering only development as a function of thermal time or the global distribution of available biomass.

A model-based analysis of the dynamics of carbon balance at the whole-plant level in Arabidopsis thaliana.

Arabidopsis thaliana (L.) Heynh. is used as a model plant in many research projects. However, few models simulate its growth at the whole-plant scale. The present study describes the first model of Arabidopsis growth integrating organogenesis, morphogenesis and carbon-partitioning processes for aerial and subterranean parts of the plant throughout its development. The objective was to analyse competition among sinks as they emerge from patterns of plant structural development. The model was adapted from the GreenLab model and was used to estimate organ sink strengths by optimisation against biomass measurements. Dry biomass production was calculated by a radiation use efficiency-based approach. Organogenesis processes were parameterised based on experimental data. The potential of this model for growth analysis was assessed using the Columbia ecotype, which was grown in standard environmental conditions. Three phases were observed in the overall time course of trophic competition within the plant. In the vegetative phase, no competition was observed. In the reproductive phase, competition increased with a strong increase when lateral inflorescences developed. Roots and internodes and structures bearing siliques were strong sinks and had a similar impact on competition. The application of the GreenLab model to the growth analysis of A. thaliana provides new insights into source-sink relationships as functions of phenology and morphogenesis.

Quantitative contributions of blue light and PAR to the photocontrol of plant morphogenesis in Trifolium repens (L.).

Shade-avoidance is a major adaptive response of plants, and is usually considered to be controlled by phytochromes through the perception of changes in the red:far red light ratio. However, few studies on the effects of blue light (BL) and of light intensity [photosynthetically active radiation (PAR)] on light-grown plants have been conducted, especially concerning changes in PAR at constant BL. The objective here was to quantify the photocontrol of aerial morphogenesis by BL and PAR. Experiments were conducted varying BL and PAR independently, with three BL levels (4, 38, and 83 micromol m(-2) s(-1)) at constant PAR (300 micromol m(-2) s(-1)) and three PAR levels (338, 705, and 163 micromol m(-2) s(-1)) at constant BL (36 micromol m(-2) s(-1)). Effects on morphogenetic processes were analysed as quantitative modulations of ontogenic trends and response curves were produced. White clover (Trifolium repens L.) was used, as it is a typical shade-avoider displaying the whole syndrome of shade-avoidance in a purely vegetative stage. Morphological responses were strongly controlled by both BL and PAR changes, through antagonist effects on leaf appearance rate and additive effects on petiole elongation. All the other responses appeared to be the indirect consequences of changes in the leaf appearance rates. BL acted as a light signal for plant morphogenesis. However, the PAR control probably implicates two distinct mechanisms, such as a trophic effect and a signal. Both PAR and BL actions involved organ-specific differences, which are central in the control of the shade-avoidance responses.