Are high-throughput root phenotyping platforms suitable for informing root system architecture models with genotype-specific parameters? An evaluation based on the root model ArchiSimple and a small panel of wheat cultivars.

Given the difficulties in accessing plant roots in situ, high-throughput root phenotyping (HTRP) platforms under controlled conditions have been developed to meet the growing demand for characterizing root system architecture (RSA) for genetic analyses. However, a proper evaluation of their capacity to provide the same estimates for strictly identical root traits across platforms has never been achieved. In this study, we performed such an evaluation based on six major parameters of the RSA model ArchiSimple, using a diversity panel of 14 bread wheat cultivars in two HTRP platforms that had different growth media and non-destructive imaging systems together with a conventional set-up that had a solid growth medium and destructive sampling. Significant effects of the experimental set-up were found for all the parameters and no significant correlations across the diversity panel among the three set-ups could be detected. Differences in temperature, irradiance, and/or the medium in which the plants were growing might partly explain both the differences in the parameter values across the experiments as well as the genotype × set-up interactions. Furthermore, the values and the rankings across genotypes of only a subset of parameters were conserved between contrasting growth stages. As the parameters chosen for our analysis are root traits that have strong impacts on RSA and are close to parameters used in a majority of RSA models, our results highlight the need to carefully consider both developmental and environmental drivers in root phenomics studies.

Transcriptomic dissection of Bradyrhizobium sp. strain ORS285 in symbiosis with Aeschynomene spp. inducing different bacteroid morphotypes with contrasted symbiotic efficiency.

To circumvent the paucity of nitrogen sources in the soil legume plants establish a symbiotic interaction with nitrogen-fixing soil bacteria called rhizobia. During symbiosis, the plants form root organs called nodules, where bacteria are housed intracellularly and become active nitrogen fixers known as bacteroids. Depending on their host plant, bacteroids can adopt different morphotypes, being either unmodified (U), elongated (E) or spherical (S). E- and S-type bacteroids undergo a terminal differentiation leading to irreversible morphological changes and DNA endoreduplication. Previous studies suggest that differentiated bacteroids display an increased symbiotic efficiency (E > U and S > U). In this study, we used a combination of Aeschynomene species inducing E- or S-type bacteroids in symbiosis with Bradyrhizobium sp. ORS285 to show that S-type bacteroids present a better symbiotic efficiency than E-type bacteroids. We performed a transcriptomic analysis on E- and S-type bacteroids formed by Aeschynomene afraspera and Aeschynomene indica nodules and identified the bacterial functions activated in bacteroids and specific to each bacteroid type. Extending the expression analysis in E- and S-type bacteroids in other Aeschynomene species by qRT-PCR on selected genes from the transcriptome analysis narrowed down the set of bacteroid morphotype-specific genes. Functional analysis of a selected subset of 31 bacteroid-induced or morphotype-specific genes revealed no symbiotic phenotypes in the mutants. This highlights the robustness of the symbiotic program but could also indicate that the bacterial response to the plant environment is partially anticipatory or even maladaptive. Our analysis confirms the correlation between differentiation and efficiency of the bacteroids and provides a framework for the identification of bacterial functions that affect the efficiency of bacteroids.© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

Fluxomics links cellular functional analyses to whole-plant phenotyping.

Fluxes through metabolic pathways reflect the integration of genetic and metabolic regulations. While it is attractive to measure all the mRNAs (transcriptome), all the proteins (proteome), and a large number of the metabolites (metabolome) in a given cellular system, linking and integrating this information remains difficult. Measurement of metabolome-wide fluxes (termed the fluxome) provides an integrated functional output of the cell machinery and a better tool to link functional analyses to plant phenotyping. This review presents and discusses sets of methodologies that have been developed to measure the fluxome. First, the principles of metabolic flux analysis (MFA), its 'short time interval' version Inst-MFA, and of constraints-based methods, such as flux balance analysis and kinetic analysis, are briefly described. The use of these powerful methods for flux characterization at the cellular scale up to the organ (fruits, seeds) and whole-plant level is illustrated. The added value given by fluxomics methods for unravelling how the abiotic environment affects flux, the process, and key metabolic steps are also described. Challenges associated with the development of fluxomics and its integration with 'omics' for thorough plant and organ functional phenotyping are discussed. Taken together, these will ultimately provide crucial clues for identifying appropriate target plant phenotypes for breeding.

Genetic Analysis of Platform-Phenotyped Root System Architecture of Bread and Durum Wheat in Relation to Agronomic Traits.

Roots are essential for water and nutrient uptake but are rarely the direct target of breeding efforts. To characterize the genetic variability of wheat root architecture, the root and shoot traits of 200 durum and 715 bread wheat varieties were measured at a young stage on a high-throughput phenotyping platform. Heritability of platform traits ranged from 0.40 for root biomass in durum wheat to 0.82 for the number of tillers. Field phenotyping data for yield components and SNP genotyping were already available for all the genotypes. Taking differences in earliness into account, several significant correlations between root traits and field agronomic performances were found, suggesting that plants investing more resources in roots in some stressed environments favored water and nutrient uptake, with improved wheat yield. We identified 100 quantitative trait locus (QTLs) of root traits in the bread wheat panels and 34 in the durum wheat panel. Most colocalized with QTLs of traits measured in field conditions, including yield components and earliness for bread wheat, but only in a few environments. Stress and climatic indicators explained the differential effect of some platform QTLs on yield, which was positive, null, or negative depending on the environmental conditions. Modern breeding has led to deeper rooting but fewer seminal roots in bread wheat. The number of tillers has been increased in bread wheat, but decreased in durum wheat, and while the root-shoot ratio for bread wheat has remained stable, for durum wheat it has been increased. Breeding for root traits or designing ideotypes might help to maintain current yield while adapting to specific drought scenarios.

Adaptation of Medicago truncatula to nitrogen limitation is modulated via local and systemic nodule developmental responses.

Adaptation of Medicago truncatula to local nitrogen (N) limitation was investigated to provide new insights into local and systemic N signaling. The split-root technique allowed a characterization of the local and systemic responses of NO(3)(-) or N(2)-fed plants to localized N limitation. (15)N and (13)C labeling were used to monitor plant nutrition. Plants expressing pMtENOD11-GUS and the sunn-2 hypernodulating mutant were used to unravel mechanisms involved in these responses. Unlike NO(3)(-)-fed plants, N(2)-fixing plants lacked the ability to compensate rapidly for a localized N limitation by up-regulating the N(2)-fixation activity of roots supplied elsewhere with N. However they displayed a long-term response via a growth stimulation of pre-existing nodules, and the generation of new nodules, likely through a decreased abortion rate of early nodulation events. Both these responses involve systemic signaling. The latter response is abolished in the sunn mutant, but the mutation does not prevent the first response. Local but also systemic regulatory mechanisms related to plant N status regulate de novo nodule development in Mt, and SUNN is required for this systemic regulation. By contrast, the stimulation of nodule growth triggered by systemic N signaling does not involve SUNN, indicating SUNN-independent signaling.

Pea Efficiency of Post-drought Recovery Relies on the Strategy to Fine-Tune Nitrogen Nutrition.

As drought is increasingly frequent in the context of climate change it is a major constraint for crop growth and yield. The ability of plants to maintain their yield in response to drought depends not only on their ability to tolerate drought, but also on their capacity to subsequently recover. Post-stress recovery can indeed be decisive for drought resilience and yield stability. Pea (Pisum sativum), as a legume, has the capacity to fix atmospheric nitrogen through its symbiotic interaction with soil bacteria within root nodules. Biological nitrogen fixation is highly sensitive to drought which can impact plant nitrogen nutrition and growth. Our study aimed at dynamically evaluating whether the control of plant N status after drought could affect nodulated pea plant's ability to recover. Two pea genotypes, Puget and Kayanne, displaying different drought resilience abilities were compared for their capacity to tolerate to, and to recover from, a 2-weeks water-deficit period applied before flowering. Physiological processes were studied in this time-series experiment using a conceptual structure-function analysis framework focusing on whole plant carbon, nitrogen, and water fluxes combined to two 13CO2 and 15N2 labeling experiments. While Puget showed a yield decrease compared to well-watered plants, Kayanne was able to maintain its yield. During the recovery period, genotype-dependent strategies were observed. The analysis of the synchronization of carbon, nitrogen, and water related traits dynamics during the recovery period and at the whole plant level, revealed that plant growth recovery was tightly linked to N nutrition. In Puget, the initiation of new nodules after water deficit was delayed compared to control plants, and additional nodules developed, while in Kayanne the formation of nodules was both rapidly and strictly re-adjusted to plant growth needs, allowing a full recovery. Our study suggested that a rapid re-launch of N acquisition, associated with a fine-tuning of nodule formation during the post-stress period is essential for efficient drought resilience in pea leading to yield stability.

Impact of Bacterial Siderophores on Iron Status and Ionome in Pea.

Including more grain legumes in cropping systems is important for the development of agroecological practices and the diversification of protein sources for human and animal consumption. Grain legume yield and quality is impacted by abiotic stresses resulting from fluctuating availabilities in essential nutrients such as iron deficiency chlorosis (IDC). Promoting plant iron nutrition could mitigate IDC that currently impedes legume cultivation in calcareous soils, and increase the iron content of legume seeds and its bioavailability. There is growing evidence that plant microbiota contribute to plant iron nutrition and might account for variations in the sensitivity of pea cultivars to iron deficiency and in fine to seed nutritional quality. Pyoverdine (pvd) siderophores synthesized by pseudomonads have been shown to promote iron nutrition in various plant species (Arabidopsis, clover and grasses). This study aimed to investigate the impact of three distinct ferripyoverdines (Fe-pvds) on iron status and the ionome of two pea cultivars (cv.) differing in their tolerance to IDC, (cv. S) being susceptible and (cv. T) tolerant. One pvd came from a pseudomonad strain isolated from the rhizosphere of cv. T (pvd1T), one from cv. S (pvd2S), and the third from a reference strain C7R12 (pvdC7R12). The results indicated that Fe-pvds differently impacted pea iron status and ionome, and that this impact varied both according to the pvd and the cultivar. Plant iron concentration was more increased by Fe-pvds in cv. T than in cv. S. Iron allocation within the plant was impacted by Fe-pvds in cv. T. Furthermore, Fe-pvds had the greatest favorable impact on iron nutrition in the cultivar from which the producing strain originated. This study evidences the impact of bacterial siderophores on pea iron status and pea ionome composition, and shows that this impact varies with the siderophore and host-plant cultivar, thereby emphasizing the specificity of these plant-microorganisms interactions. Our results support the possible contribution of pyoverdine-producing pseudomonads to differences in tolerance to IDC between pea cultivars. Indeed, the tolerant cv. T, as compared to the susceptible cv. S, benefited from bacterial siderophores for its iron nutrition to a greater extent.

RhizoTubes as a new tool for high throughput imaging of plant root development and architecture: test, comparison with pot grown plants and validation.

BACKGROUND: In order to maintain high yields while saving water and preserving non-renewable resources and thus limiting the use of chemical fertilizer, it is crucial to select plants with more efficient root systems. This could be achieved through an optimization of both root architecture and root uptake ability and/or through the improvement of positive plant interactions with microorganisms in the rhizosphere. The development of devices suitable for high-throughput phenotyping of root structures remains a major bottleneck. RESULTS: Rhizotrons suitable for plant growth in controlled conditions and non-invasive image acquisition of plant shoot and root systems (RhizoTubes) are described. These RhizoTubes allow growing one to six plants simultaneously, having a maximum height of 1.1 m, up to 8 weeks, depending on plant species. Both shoot and root compartment can be imaged automatically and non-destructively throughout the experiment thanks to an imaging cabin (RhizoCab). RhizoCab contains robots and imaging equipment for obtaining high-resolution pictures of plant roots. Using this versatile experimental setup, we illustrate how some morphometric root traits can be determined for various species including model (Medicago truncatula), crops (Pisum sativum, Brassica napus, Vitis vinifera, Triticum aestivum) and weed (Vulpia myuros) species grown under non-limiting conditions or submitted to various abiotic and biotic constraints. The measurement of the root phenotypic traits using this system was compared to that obtained using "classic" growth conditions in pots. CONCLUSIONS: This integrated system, to include 1200 Rhizotubes, will allow high-throughput phenotyping of plant shoots and roots under various abiotic and biotic environmental conditions. Our system allows an easy visualization or extraction of roots and measurement of root traits for high-throughput or kinetic analyses. The utility of this system for studying root system architecture will greatly facilitate the identification of genetic and environmental determinants of key root traits involved in crop responses to stresses, including interactions with soil microorganisms.

(34)S and (15)N labelling to model S and N flux in plants and determine the different components of N and S use efficiency.

In order to highlight our understanding on ecosystems functioning and resource sharing/competition, either in artificial environment or agrosystems, according to changes in the climatic conditions, it is necessary to measure accurately element fluxes within plants. Stable isotopes allow tracking safely and accurately on a short time frame the behavior of elements in plants. After a short review devoted to isotopic studies of elemental flux within plants, we explain how a direct multiple labelling study might be conducted in a plant, so as to measure over short time nitrogen and sulfur acquisition, and assimilates arising from a labelled source.

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.

Systemic signaling of the plant nitrogen status triggers specific transcriptome responses depending on the nitrogen source in Medicago truncatula.

Legumes can acquire nitrogen (N) from NO(3)(-), NH(4)(+), and N(2) (through symbiosis with Rhizobium bacteria); however, the mechanisms by which uptake and assimilation of these N forms are coordinately regulated to match the N demand of the plant are currently unknown. Here, we find by use of the split-root approach in Medicago truncatula plants that NO(3)(-) uptake, NH(4)(+) uptake, and N(2) fixation are under general control by systemic signaling of plant N status. Indeed, irrespective of the nature of the N source, N acquisition by one side of the root system is repressed by high N supply to the other side. Transcriptome analysis facilitated the identification of over 3,000 genes that were regulated by systemic signaling of the plant N status. However, detailed scrutiny of the data revealed that the observation of differential gene expression was highly dependent on the N source. Localized N starvation results, in the unstarved roots of the same plant, in a strong compensatory up-regulation of NO(3)(-) uptake but not of either NH(4)(+) uptake or N(2) fixation. This indicates that the three N acquisition pathways do not always respond similarly to a change in plant N status. When taken together, these data indicate that although systemic signals of N status control root N acquisition, the regulatory gene networks targeted by these signals, as well as the functional response of the N acquisition systems, are predominantly determined by the nature of the N source.

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.