High Temperatures During the Seed-Filling Period Decrease Seed Nitrogen Amount in Pea (Pisum sativum L.): Evidence for a Sink Limitation.

Higher temperatures induced by the on-going climate change are a major cause of yield reduction in legumes. Pea (Pisum sativum L.) is an important annual legume crop grown in temperate regions for its high seed nitrogen (N) concentration. In addition to yield, seed N amount at harvest is a crucial characteristic because pea seeds are a source of protein in animal and human nutrition. However, there is little knowledge on the impacts of high temperatures on plant N partitioning determining seed N amount. Therefore, this study investigates the response of seed dry matter and N fluxes at the whole-plant level (plant N uptake, partitioning in vegetative organs, remobilization, and accumulation in seeds) to a range of air temperature (from 18.4 to 33.2°C) during the seed-filling-period. As pea is a legume crop, plants relying on two different N nutrition pathways were grown in glasshouse: N2-fixing plants or NO3 --assimilating plants. Labeled nitrate (15NO3 -) and intra-plant N budgets were used to quantify N fluxes. High temperatures decreased seed-filling duration (by 0.8 day per °C), seed dry-matter and N accumulation rates (respectively by 0.8 and 0.032 mg seed-1 day-1 per °C), and N remobilization from vegetative organs to seeds (by 0.053 mg seed-1 day-1 per °C). Plant N2-fixation decreased with temperatures, while plant NO3 - assimilation increased. However, the additional plant N uptake in NO3 --assimilating plants was never allocated to seeds and a significant quantity of N was still available at maturity in vegetative organs, whatever the plant N nutrition pathway. Thus, we concluded that seed N accumulation under high temperatures is sink limited related to a shorter seed-filling duration and a reduced seed dry-matter accumulation rate. Consequently, sustaining seed sink demand and preserving photosynthetic capacity of stressed plants during the seed-filling period should be promising strategies to promote N allocation to seeds from vegetative parts and thus to maintain crop N production under exacerbated abiotic constraints in field due to the on-going climate change.

Unexpectedly low nitrogen acquisition and absence of root architecture adaptation to nitrate supply in a Medicago truncatula highly branched root mutant.

To complement N2 fixation through symbiosis, legumes can efficiently acquire soil mineral N through adapted root architecture. However, root architecture adaptation to mineral N availability has been little studied in legumes. Therefore, this study investigated the effect of nitrate availability on root architecture in Medicago truncatula and assessed the N-uptake potential of a new highly branched root mutant, TR185. The effects of varying nitrate supply on both root architecture and N uptake were characterized in the mutant and in the wild type. Surprisingly, the root architecture of the mutant was not modified by variation in nitrate supply. Moreover, despite its highly branched root architecture, TR185 had a permanently N-starved phenotype. A transcriptome analysis was performed to identify genes differentially expressed between the two genotypes. This analysis revealed differential responses related to the nitrate acquisition pathway and confirmed that N starvation occurred in TR185. Changes in amino acid content and expression of genes involved in the phenylpropanoid pathway were associated with differences in root architecture between the mutant and the wild type.

Soil nitrogen availability and plant genotype modify the nutrition strategies of M. truncatula and the associated rhizosphere microbial communities.

Plant and soil types are usually considered as the two main drivers of the rhizosphere microbial communities. The aim of this work was to study the effect of both N availability and plant genotype on the plant associated rhizosphere microbial communities, in relation to the nutritional strategies of the plant-microbe interactions, for six contrasted Medicago truncatula genotypes. The plants were provided with two different nutrient solutions varying in their nitrate concentrations (0 mM and 10 mM). First, the influence of both nitrogen availability and Medicago truncatula genotype on the genetic structure of the soil bacterial and fungal communities was determined by DNA fingerprint using Automated Ribosomal Intergenic Spacer Analysis (ARISA). Secondly, the different nutritional strategies of the plant-microbe interactions were evaluated using an ecophysiological framework. We observed that nitrogen availability affected rhizosphere bacterial communities only in presence of the plant. Furthermore, we showed that the influence of nitrogen availability on rhizosphere bacterial communities was dependent on the different genotypes of Medicago truncatula. Finally, the nutritional strategies of the plant varied greatly in response to a modification of nitrogen availability. A new conceptual framework was thus developed to study plant-microbe interactions. This framework led to the identification of three contrasted structural and functional adaptive responses of plant-microbe interactions to nitrogen availability.

How to hierarchize the main physiological processes responsible for phenotypic differences in large-scale screening studies?

One difficulty when analyzing the determinants at the origin of plant phenotypic differences is that measured plant traits are frequently integrative: they result from the integration of a large number of physiological processes under the control of genetic and environmental factors. In a previous report, we demonstrated that dissecting integrative traits into simpler components using a simple crop physiology model was a valuable method for detecting quantitative trait loci (QTL) related to the nitrogen nutrition for a recombinant inbred lines population of Medicago truncatula. Here, using the same data set, we demonstrate the relevance of decomposing integrative traits for understanding biological differences among phenotypes, independently of QTL detection. Two examples are given to demonstrate that the dissection of integrative traits (i.e., plant leaf area and nitrogen nutrition index) into variables representing the efficiency of the plant to extract and valorize (carbon and nitrogen) resources is an effective method to determine the stream of physiological events that leads to the final phenotype.

Using a physiological framework for improving the detection of quantitative trait loci related to nitrogen nutrition in Medicago truncatula.

Medicago truncatula is used as a model plant for exploring the genetic and molecular determinants of nitrogen (N) nutrition in legumes. In this study, our aim was to detect quantitative trait loci (QTL) controlling plant N nutrition using a simple framework of carbon/N plant functioning stemming from crop physiology. This framework was based on efficiency variables which delineated the plant's efficiency to take up and process carbon and N resources. A recombinant inbred line population (LR4) was grown in a glasshouse experiment under two contrasting nitrate concentrations. At low nitrate, symbiotic N(2) fixation was the main N source for plant growth and a QTL with a large effect located on linkage group (LG) 8 affected all the traits. Significantly, efficiency variables were necessary both to precisely localize a second QTL on LG5 and to detect a third QTL involved in epistatic interactions on LG2. At high nitrate, nitrate assimilation was the main N source and a larger number of QTL with weaker effects were identified compared to low nitrate. Only two QTL were common to both nitrate treatments: a QTL of belowground biomass located at the bottom of LG3 and another one on LG6 related to three different variables (leaf area, specific N uptake and aboveground:belowground biomass ratio). Possible functions of several candidate genes underlying QTL of efficiency variables could be proposed. Altogether, our results provided new insights into the genetic control of N nutrition in M. truncatula. For instance, a novel result for M. truncatula was identification of two epistatic interactions in controlling plant N(2) fixation. As such this study showed the value of a simple conceptual framework based on efficiency variables for studying genetic determinants of complex traits and particularly epistatic interactions.

Analysis and modeling of the integrative response of Medicago truncatula to nitrogen constraints.

An integrative biology approach was conducted in Medicago truncatula for: (i) unraveling the coordinated regulation of NO3-, NH4+ and N(2) acquisition by legumes to fulfill the plant N demand; and (ii) modeling the emerging properties occurring at the whole plant level. Upon localized addition of a high level of mineral N, the three N acquisition pathways displayed similar systemic feedback repression to adjust N acquisition capacities to the plant N status. Genes associated to these responses were in contrast rather specific to the N source. Following an N deficit, NO3- fed plants maintained efficiently their N status through rapid functional and developmental up regulations while N(2) fed plants responded by long term plasticity of nodule development. Regulatory genes associated with various symbiotic stages were further identified. An ecophysiological model simulating relations between leaf area and roots N retrieval was developed and now furnishes an analysis grid to characterize a spontaneous or induced genetic variability for plant N nutrition.

Can differences of nitrogen nutrition level among Medicago truncatula genotypes be assessed non-destructively?: Probing with a recombinant inbred lines population.

The international consensus on Medicago truncatula as a model system has lead to the development of powerful approaches for dissecting the genetic and molecular bases of legume nitrogen nutrition. However, such approaches now come up against a poor knowledge of the phenotypic traits that should be used for the large-scale screening of the genotypic variability associated with nitrogen nutrition. This issue was unravelled in a previous report, in which an ecophysiological approach allowed a better understanding of the relationships between plant nitrogen nutrition and plant growth traits, for the model symbiotic association between M. truncatula cv. Jemalong and Rhizobium meliloti strain 2011. From this analysis, phenotypic traits were identified as potentially relevant for the large-scale screening of the genotypic variability. Here, by the phenotyping of a recombinant inbred lines population, we show that the proposed methodology provides a valuable support for assisting the detection of genetic variants affected for nitrogen uptake. Especially, the relative expansion rate of plant leaf area is identified as a good proxy for ranking genotypes according to their ability to uptake nitrogen in given environmental conditions. As leaf area can be measured non-destructively, such finding should pave the way for a more efficient evaluation of the genotypic variability.

Determinism of carbon and nitrogen reserve accumulation in legume seeds.

In legume plants, the determination of individual seed weight is a complex phenomenon that depends on two main factors. The first one corresponds to the number of cotyledon cells, which determines the potential seed weight as the cotyledon cell number is related to seed growth rate during seed filling. Since cell divisions take place between flowering and the beginning of seed filling, any stress occurring before the beginning of seed filling can affect individual seed growth rate (C and N reserve accumulation in seeds), and thus individual seed weights. The second factor concerns carbon and nitrogen supply to the growing seed to support reserve accumulation. Grain legume species produce protein-rich seeds involving high requirement of nitrogen. Since seed growth rate as determined by cotyledon cell number is hardly affected by photoassimilate availability during the filling period, a reduction of photosynthetic activity caused by nitrogen remobilization in leaves (e.g., remobilization of essential proteins involved in photosynthesis) can lead to shorten the duration of the filling period, and by that can provoke a limitation of individual seed weights. Accordingly, any biotic or abiotic stress during seed filling causing a decrease in photosynthetic activity should lead to a reduction of the duration of seed filling.

The model symbiotic association between Medicago truncatula cv. Jemalong and Rhizobium meliloti strain 2011 leads to N-stressed plants when symbiotic N2 fixation is the main N source for plant growth.

A better knowledge of the nitrogen nutrition of Medicago truncatula at the whole plant level and its modulation by environmental factors is a crucial step to reach a complete understanding of legume nitrogen nutrition. This study was based on the symbiotic system that is the most commonly used by the research community (M. truncatula cv. Jemalong A17 x Rhizobium meliloti strain 2011). Plant nitrogen nutrition was analysed in relation to carbon nutrition, under a range of nitrate concentrations in the nutrient solution and different light conditions. This study shows that this 'model symbiotic association' does not allow the plant to meet its nitrogen requirements, when dinitrogen fixation is the main nitrogen source for plant growth. A strong interaction between nitrogen and carbon nutrition was shown: when plant nitrogen requirements were not sustained, plant leaf area was much affected whereas photosynthesis per unit leaf area remained relatively stable. Both total nitrogen uptake and leaf area increased with increasing nitrate concentration in the nutrient solution; the magnitude of these responses varied according to the light conditions. Interestingly, the plant nitrogen nutrition level remained nearly unaffected by the light conditions. The observed nitrogen-limitation in this 'model symbiotic association' is an important finding for the research community. Based on practical recommendations regarding both the experimental conditions and the phenotypic traits to consider, a methodological framework was proposed to (i) help genomicists to assess plant nitrogen nutrition better, and (ii) assist in the detection of new genetic variants affected for nitrogen uptake in large-scale phenotyping studies.

Developmental genes have pleiotropic effects on plant morphology and source capacity, eventually impacting on seed protein content and productivity in pea.

Increasing pea (Pisum sativum) seed nutritional value and particularly seed protein content, while maintaining yield, is an important challenge for further development of this crop. Seed protein content and yield are complex and unstable traits, integrating all the processes occurring during the plant life cycle. During filling, seeds are the main sink to which assimilates are preferentially allocated at the expense of vegetative organs. Nitrogen seed demand is satisfied partly by nitrogen acquired by the roots, but also by nitrogen remobilized from vegetative organs. In this study, we evaluated the respective roles of nitrogen source capacity and sink strength in the genetic variability of seed protein content and yield. We showed in eight genotypes of diverse origins that both the maximal rate of nitrogen accumulation in the seeds and nitrogen source capacity varied among genotypes. Then, to identify the genetic factors responsible for seed protein content and yield variation, we searched for quantitative trait loci (QTL) for seed traits and for indicators of sink strength and source nitrogen capacity. We detected 261 QTL across five environments for all traits measured. Most QTL for seed and plant traits mapped in clusters, raising the possibility of common underlying processes and candidate genes. In most environments, the genes Le and Afila, which control internode length and the switch between leaflets and tendrils, respectively, determined plant nitrogen status. Depending on the environment, these genes were linked to QTL of seed protein content and yield, suggesting that source-sink adjustments depend on growing conditions.

A model-based framework for the phenotypic characterization of the flowering of Medicago truncatula.

To facilitate the phenotypic characterization of Medicago truncatula, our aim was to provide a framework of analysis of flowering in response to environmental factors. The flowering of the line A17 was analysed in different conditions of temperature, duration of vernalization and photoperiod. Flowering was characterized using three descriptors at the axis level: the position of the first reproductive node (1RN), the date of beginning of flowering (DBF) and the florochron (RFa-1) corresponding to the reciprocal of the rate of progression of flowering along each axis. As for vegetative development, it was found that flowering could be analysed as a function of thermal time using a base temperature (Tb) of 5 degrees C. Vernalization displayed a sound impact on the flowering. For all the studied axes, increasing the duration of vernalization lowered the 1RN and hastened the DBF. By contrast, for most of the studied axes, RFa-1 was only slightly affected by vernalization. For the branch B0, RFa-1 was a genotypic constant when thermal time was used. Considering B0 as a reference axis, an ecophysiological model was developed to simulate the impact of environmental factors on the three components of flowering. Concrete practical applications of the model-based framework presented herein are proposed for helping the genetic and genomic studies of M. truncatula.

Using a standard framework for the phenotypic analysis of Medicago truncatula: an effective method for characterizing the plant material used for functional genomics approaches.

A crucial step for identifying genes of interest in legume crops is to determine gene function in Medicago truncatula. To facilitate functional genomics in this species, an ecophysiological framework of analysis was developed. Our primary aim was to establish a standard terminology for identifying each organ on the plant. A standard system for the characterization of the vegetative and the reproductive developmental stages was then proposed. Using these tools, the time course of vegetative development of nitrogen-fixing A17 plants was analysed in experiments conducted under different environmental conditions. To take into account the influence of temperature on plant development timing, an original approach was used by modelling vegetative development as a function of thermal time. Interestingly, the use of thermal time highlighted genotypic constants in plant development. Thereafter, to illustrate how this methodology can be used in explaining phenotypic alterations, the phenotype of two allelic mutants was analysed. Because the tools proposed in this paper allow the following: (1) standardization of how the plant material should be characterized to be used for functional genomics; (2) prediction of plant vegetative development; and (3) a more accurate phenotyping, the use of these tools by the M. truncatula community should provide a relevant framework for facilitating the production of reproducible functional genomics data.

Genetic and genomic analysis of legume flowers and seeds.

New tools, such as ordered mutant libraries, microarrays and sequence based comparative maps, are available for genetic and genomic studies of legumes that are being used to shed light on seed production, the objective of most arable farming. The new information and understanding brought by these tools are revealing the biological processes that underpin and impact on seed production.

Dynamics of exogenous nitrogen partitioning and nitrogen remobilization from vegetative organs in pea revealed by 15N in vivo labeling throughout seed filling.

The fluxes of (1) exogenous nitrogen (N) assimilation and (2) remobilization of endogenous N from vegetative plant compartments were measured by 15N labeling during the seed-filling period in pea (Pisum sativum L. cv Cameor), to better understand the mechanism of N remobilization. While the majority (86%) of exogenous N was allocated to the vegetative organs before the beginning of seed filling, this fraction decreased to 45% at the onset of seed filling, the remainder being directed to seeds. Nitrogen remobilization from vegetative parts contributed to 71% of the total N in mature seeds borne on the first two nodes (first stratum). The contribution of remobilized N to total seed N varied, with the highest proportion at the beginning of filling; it was independent of the developmental stage of each stratum of seeds, suggesting that remobilized N forms a unique pool, managed at the whole-plant level and supplied to all filling seeds whatever their position on the plant. Once seed filling starts, N is remobilized from all vegetative organs: 30% of the total N accumulated in seeds was remobilized from leaves, 20% from pod walls, 11% from roots, and 10% from stems. The rate of N remobilization was maximal when seeds of all the different strata were filling, consistent with regulation according to the N demand of seeds. At later stages of seed filling, the rate of remobilization decreases and may become controlled by the amount of residual N in vegetative tissues.

How does temperature affect C and N allocation to the seeds during the seed-filling period in pea? Effect on seed nitrogen concentration.

The effect of moderate temperature on seed N concentration during the seed-filling period was evaluated in pea (Pisum sativum L.) kept in growth cabinets and the relation between plant assimilate availability and the variation of seed N concentration with temperature was investigated. Seed N concentration of pea was significantly lowered when temperature during the seed-filling period decreased from a day / night temperature of 25 / 20°C to 15 / 10°C. Our results demonstrate that during the seed-filling period mechanisms linked with assimilate availability can modify seed N accumulation rate and / or seed-filling duration between 25 / 20°C and 15 / 10°C. At the lower temperature (15 / 10°C), an increased C availability resulting from an enhanced carbon fixation per degree-day allowed new competing vegetative sinks to grow as pea is an indeterminate plant. Consequently N availability to filling seeds was reduced. Because the rate of seed N accumulation per degree-day mainly depends on N availability to filling seeds, the rate of seed N accumulation was lower at the low temperature of our study (15 / 10°C) than at 25 / 20°C while seed growth rate per degree-day remains unaffected, consequently seed N concentration was reduced. Concomitantly, the increased C availability at the lower temperature prolonged the duration of the seed-filling period.

Can sucrose content in the phloem sap reaching field pea seeds (Pisum sativum L.) be an accurate indicator of seed growth potential?

The composition of the translocates reaching the seeds of pea plants having various nitrogen (N) nutrition regimes was investigated under field situations. Sucrose flow in the phloem sap increased with the node number, but was not significantly different between N nutrition levels. Because N deficiency reduced the number of flowering nodes and the number of seeds per pod, the sucrose flow bleeding from cut peduncles was divided by the number of seeds to give the amount of assimilates available per seed. The sucrose concentration in phloem sap supplied to seeds at the upper nodes was higher than that at the lower nodes. The flow of sucrose delivered to the seeds during the cell division period was correlated with seed growth potential. Seeds from the more N-stressed plants had both the highest seed growth rate and received a higher sucrose flux per seed during the cell division period. As seed growth rate is highly correlated with the number of cotyledonary cells produced during the cell division period, sucrose flow in phloem sap is proposed to be an important determinant of mitotic activity in seed embryos. The carbon (C)/N ratio of the flow of translocates towards seeds was higher under conditions of N-deficiency than with optimal N nutrition, indicating that N flux towards seeds, in itself, is not the main determinant of seed growth potential.