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.

Field-Crop Soils in Eastern France: Coldspots of Azole-Resistant Aspergillus fumigatus.

Triazole fungicides are widely used to treat fungal pathogens in field crops, but very few studies have investigated whether fields of these crops constitute hotspots of azole resistance in Aspergillus fumigatus. Soil samples were collected from 22 fields in two regions of eastern France and screened for triazole residues and azole-resistant A. fumigatus (ARAf). Real-time quantitative PCR (qPCR) was used to quantify A. fumigatus in these soil samples. All the plots contained tebuconazole at concentrations from 5.5 to 19.1 ng/g of soil, and 5 of the 22 plots also contained epoxiconazole. Only a few fungal isolates were obtained, and no ARAf was detected. A. fumigatus qPCR showed that this fungal species was, on average, 5000 times more common in soil from flowerbeds containing ARAf than in soil from field crops. Thus, field-crop soils do not appear to promote A. fumigatus development, even if treated with azole fungicides, and cannot be considered hotspots of resistance. Indeed, our results suggest that they are instead a coldspot of resistance and highlight how little is known about the ecological niche of this species.

Genome-wide DNA methylation of the liver reveals delayed effects of early-life exposure to 17-α-ethinylestradiol in the self-fertilizing mangrove rivulus.

Organisms exposed to endocrine disruptors in early life can show altered phenotype later in adulthood. Although the mechanisms underlying these long-term effects remain poorly understood, an increasing body of evidence points towards the potential role of epigenetic processes. In the present study, we exposed hatchlings of an isogenic lineage of the self-fertilizing fish mangrove rivulus for 28 days to 4 and 120 ng/L of 17-α-ethinylestradiol. After a recovery period of 140 days, reduced representation bisulphite sequencing (RRBS) was performed on the liver in order to assess the hepatic genome-wide methylation landscape. Across all treatment comparisons, a total of 146 differentially methylated fragments (DMFs) were reported, mostly for the group exposed to 4 ng/L, suggesting a non-monotonic effect of EE2 exposure. Gene ontology analysis revealed networks involved in lipid metabolism, cellular processes, connective tissue function, molecular transport and inflammation. The highest effect was reported for nipped-B-like protein B (NIPBL) promoter region after exposure to 4 ng/L EE2 (+ 21.9%), suggesting that NIPBL could be an important regulator for long-term effects of EE2. Our results also suggest a significant role of DNA methylation in intergenic regions and potentially in transposable elements. These results support the ability of early exposure to endocrine disruptors of inducing epigenetic alterations during adulthood, providing plausible mechanistic explanations for long-term phenotypic alteration. Additionally, this work demonstrates the usefulness of isogenic lineages of the self-fertilizing mangrove rivulus to better understand the biological significance of long-term alterations of DNA methylation by diminishing the confounding factor of genetic variability.

Early-life exposure to the endocrine disruptor 17-α-ethinylestradiol induces delayed effects in adult brain, liver and ovotestis proteomes of a self-fertilizing fish.

Early-life represents a critically sensitive window to endocrine disrupting chemicals, potentially leading to long-term repercussions on the phenotype later in life. The mechanisms underlying this phenomenon, referred to as the Developmental Origins of Health and Disease (DOHaD), are still poorly understood. To gain molecular understanding of these effects, we exposed mangrove rivulus (Kryptolebias marmoratus) for 28 days post hatching (dph) to 4 and 120 ng/L 17-α-ethinylestradiol, a model xenoestrogen. After 28 days, fish were raised for 140 days in clean water and we performed quantitative label-free proteomics on brain, liver and ovotestis of 168 dph adults. A total of 820, 888 and 420 proteins were robustly identified in the brain, liver and ovotestis, respectively. Effects of 17-α-ethinylestradiol were tissue and dose-dependent: a total of 31, 51 and 18 proteins were differentially abundant at 4 ng/L in the brain, liver and ovotestis, respectively, compared to 20, 25 and 39 proteins at 120 ng/L. Our results suggest that estrogen-responsive pathways, such as lipid metabolism, inflammation, and the innate immune system were affected months after the exposure. In addition, the potential perturbation of S-adenosylmethionine metabolism encourages future studies to investigate the role of DNA methylation in mediating the long-term effects of early-life exposures. SIGNIFICANCE: The Developmental Origins of Health and Disease (DOHaD) states that early life stages of humans and animals are sensitive to environmental stressors and can develop health issues later in life, even if the stress has ceased. Molecular mechanisms supporting DOHaD are still unclear. The mangrove rivulus is a new fish model species naturally reproducing by self-fertilization, making it possible to use isogenic lineages in which all individuals are highly homozygous. This species therefore permits to strongly reduce the confounding factor of genetic variability in order to investigate the effects of environmental stress on the phenotype. After characterizing the molecular phenotype of brain, liver and ovotestis, we obtained true proteomic reaction norms of these three organs in adults after early life stages have been exposed to the common endocrine disruptor 17-α-ethinylestradiol (EE2). Our study demonstrates long-term effects of early-life endocrine disruption at the proteomic level in diverse estrogen-responsive pathways 5 months after the exposure. The lowest tested and environmentally relevant concentration of 4 ng/L had the highest impact on the proteome in brain and liver, highlighting the potency of endocrine disruptors at low concentrations.

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.

DNA methylation in adults and during development of the self-fertilizing mangrove rivulus, Kryptolebias marmoratus.

In addition to genetic variation, epigenetic mechanisms such as DNA methylation might make important contributions to heritable phenotypic diversity in populations. However, it is often difficult to disentangle the contributions of genetic and epigenetic variation to phenotypic diversity. Here, we investigated global DNA methylation and mRNA expression of the methylation-associated enzymes during embryonic development and in adult tissues of one natural isogenic lineage of mangrove rivulus fish, Kryptolebias marmoratus. Being the best-known self-fertilizing hermaphroditic vertebrate affords the opportunity to work with genetically identical individuals to examine, explicitly, the phenotypic effects of epigenetic variance. Using the LUminometric Methylation Assay (LUMA), we described variable global DNA methylation at CpG sites in adult tissues, which differed significantly between hermaphrodite ovotestes and male testes (79.6% and 87.2%, respectively). After fertilization, an immediate decrease in DNA methylation occurred to 15.8% in gastrula followed by re-establishment to 70.0% by stage 26 (liver formation). Compared to zebrafish, at the same embryonic stages, this reprogramming event seems later, deeper, and longer. Furthermore, genes putatively encoding DNA methyltransferases (DNMTs), Ten-Eleven Translocation (TET), and MeCP2 proteins showed specific regulation in adult gonad and brain, and also during early embryogenesis. Their conserved domains and expression profiles suggest that these proteins play important roles during reproduction and development. This study raises questions about mangrove rivulus' peculiar reprogramming period in terms of epigenetic transmission and physiological adaptation of individuals to highly variable environments. In accordance with the general-purpose genotype model, epigenetic mechanisms might allow for the expression of diverse phenotypes among genetically identical individuals. Such phenotypes might help to overcome environmental challenges, making the mangrove rivulus a valuable vertebrate model for ecological epigenetic studies. The mangrove rivulus, Kryptolebias marmoratus, is the best-known self-fertilizing hermaphroditic vertebrate that allows to work with genetically identical individuals to examine, explicitly, the phenotypic effects of epigenetic variance. The reprogramming event is later, more dramatic and longer than in other described vertebrates. High evolutionary conservation and expression patterns of DNMT, TET, and MeCP2 proteins in K. marmoratus suggest biological roles for each member in gametogenesis and development.

Using an ecophysiological analysis to dissect genetic variability and to propose an ideotype for nitrogen nutrition in pea.

BACKGROUNDS AND AIMS: Nitrogen nutrition of legumes, which relies both on atmospheric N2 and soil mineral N, remains a major limiting factor of growth. A decade ago, breeders tried to increase N uptake through hypernodulation. Despite their high nodule biomass, hypernodulating mutants were never shown to accumulate more nitrogen than wild types; they even generally displayed depressed shoot growth. The aim of this study was to dissect genetic variability associated with N nutrition in relation to C nutrition, using an ecophysiological framework and to propose an ideotype for N nutrition in pea. METHODS: Five pea genotypes (Pisum sativum) characterized by contrasting root and nodule biomasses were grown in the field. Variability among genotypes in dry matter and N accumulation was analysed, considering both the structures involved in N acquisition in terms of root and nodule biomass and their efficiency, in terms of N accumulated through mineral N absorption or symbiotic N2 fixation per amount of root or nodule biomass, respectively. KEY RESULTS: Nodule efficiency of hypernodulating mutants was negatively correlated to nodule biomass, presumably due to the high carbon costs induced by their excessive nodule formation. Root efficiency was only negatively correlated to root biomass before the beginning of the seed-filling stage, suggesting competition for carbon between root formation and functioning during the early stages of growth. This was no longer the case after the beginning of the seed-filling stage and nitrate absorption was then positively correlated to root biomass. CONCLUSIONS: Due to the high C costs induced by nodule formation and its detrimental effect on shoot and root growth, selecting traits for the improvement of N acquisition by legumes must be engineered (a) considering inter-relationships between C and N metabolisms and (b) in terms of temporal complementarities between N2 fixation and nitrate absorption rather than through direct increase of nodule and/or root biomass.

Impacts of triclosan exposure on zebrafish early-life stage: Toxicity and acclimation mechanisms.

Triclosan (TCS) is a broad spectrum antibacterial agent widely used in personal care products and present in most aquatic ecosystems. This study investigated the occurrence of triclosan acclimation and the biological mechanisms underlying the stress response triggered in early-life stage of zebrafish. Zebrafish eggs were first exposed to four different sublethal concentrations of TCS (2, 20, 50 and 100µg/L) for 7days following fertilization and subsequently exposed to a lethal concentration of TCS (1000µg/L). During the time-to-death exposure (TTD), mortality was continuously recorded to evaluate if increased resistance occurred. Overall, larvae exposed to 50µg/L of TCS demonstrated higher sensitivity, with delayed hatching and increased mortality during the sub-lethal exposure and significant lower mean time-to-death (TTD) value compared to the other groups. Interestingly, fish exposed to the highest concentration of TCS (100µg/L) presented a similar mean TTD value as controls and a significantly better survival in comparison with embryos exposed to 50µg/L, suggesting that acclimation process has been triggered at this concentration. Proteomic and enzymatic analyses were conducted on 7days post fertilization (dpf) larvae exposed to 50µg/L and 100µg/L of TCS giving insights into the functional changes triggered at those specific concentrations. TCS seemed to affect proteins involved in cytoskeleton, stress response, eyes and neuronal development. This was endorsed by the enzymatic results, which suggest impairment in glutathione metabolism and acute neurotoxicity. A significant 2.5-fold and 3-fold increase of AChE activity was observed following TCS exposure. Moreover, GPx activity was significantly increased whereas a significant inhibition of GR activity was observed, suggesting that de novo synthesis of reduced GSH might occur in order to maintain the ratio between reduced and oxidized GSH. Proteomic results revealed possible candidate protein involved in the acclimation process of larvae exposed to 100µg/L of TCS. Our integrative analysis revealed complex non-monotonic concentration-related effects on zebrafish early-life stages with increased resistance between 50 and 100µg/L exposures. This research highlighted oxidative stress and neurotoxicity as major toxicity mechanisms of TCS during development.

Delayed impacts of developmental exposure to 17-α-ethinylestradiol in the self-fertilizing fish Kryptolebias marmoratus.

17-α-ethinylestradiol (EE2) is one of the most potent endocrine disrupting compounds found in the aquatic environments, and is known to strongly alter fish reproduction and fitness. While the effects of direct exposure to EE2 are well studied in adults, there is an increasing need to assess the impacts of exposure during early life stages. Sensitivity to pollutants during this critical window can potentially affect the phenotype later in life or in subsequent generations. This study investigated phenotypic outcome of early-life exposure to 17-α-ethinylestradiol during development and in adults of the mangrove rivulus, Kryptolebias marmoratus. Being one of the only two known self-fertilizing hermaphroditic vertebrates, this fish makes it possible to work with genetically identical individuals. Therefore, using rivulus makes it possible to examine, explicitly, the phenotypic effects of environmental variance while eliminating the effects of genetic variance. Genetically identical rivulus were exposed for the first 28days post hatching (dph) to 0, 4 or 120ng/L of EE2, and then were reared in uncontaminated water until 168dph. Growth, egg laying and steroid hormone levels (estradiol, cortisol, 11-ketotestosterone, testosterone) were measured throughout development. Exposed fish showed a reduction in standard length directly after exposure (28dph), which was more pronounced in the 120ng/L group. This was followed by compensatory growth when reared in clean water: all fish recovered a similar size as controls by 91dph. There was no difference in the age at maturity and the proportions of mature, non-mature and male individuals at 168dph. At 4ng/L, fish layed significantly fewer eggs than controls, while, surprisingly, reproduction was not affected at 120ng/L. Despite a decrease in fecundity at 4ng/L, there were no changes in hormones levels at the lower concentration. In addition, there were no significant differences among treatments immediately after exposure. However, 120ng/L exposed fish exhibited significantly higher levels of testosterone at 91 and 168dph and 11-ketotestosterone at 168dph, up to 140days after exposure. These results indicate that early-life exposure to EE2 had both immediate and delayed impacts on the adult's phenotype. While fish growth was impaired during exposure, compensatory growth, reduced fecundity and modification of the endocrine status were observed after exposure ceased.

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.

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.

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.

Genetic variability in nodulation and root growth affects nitrogen fixation and accumulation in pea.

BACKGROUND AND AIMS: Legume nitrogen is derived from two different sources, symbiotically fixed atmospheric N(2) and soil N. The effect of genetic variability of root and nodule establishment on N acquisition and seed protein yield was investigated under field conditions in pea (Pisum sativum). In addition, these parameters were related to the variability in preference for rhizobial genotypes. METHODS: Five different spring pea lines (two hypernodulating mutants and three cultivars), previously identified in artificial conditions as contrasted for both root and nodule development, were characterized under field conditions. Root and nodule establishment was examined from the four-leaf stage up to the beginning of seed filling and was related to the patterns of shoot dry matter and nitrogen accumulation. The genetic structure of rhizobial populations associated with the pea lines was obtained by analysis of nodule samples. The fraction of nitrogen derived from symbiotic fixation was estimated at the beginning of seed filling and at physiological maturity, when seed protein content and yield were determined. KEY RESULTS: The hypernodulating mutants established nodules earlier and maintained them longer than was the case for the three cultivars, whereas their root development and nitrogen accumulation were lower. The seed protein yield was higher in 'Athos' and 'Austin', the two cultivars with increased root development, consistent with their higher N absorption during seed filling. CONCLUSION: The hypernodulating mutants did not accumulate more nitrogen, probably due to the C cost for nodulation being higher than for root development. Enhancing exogenous nitrogen supply at the end of the growth cycle, by increasing the potential for root N uptake from soil, seems a good option for improving pea seed filling.

Are ABA, ethylene or their interaction involved in the response of leaf growth to soil water deficit? An analysis using naturally occurring variation or genetic transformation of ABA production in maize.

The role of abscisic acid (ABA) and its possible interaction with ethylene in mediating leaf elongation response to soil water deficit are a matter of controversy. To address this question, we used a set of maize genotypes with various levels of ABA either due to natural variability or to genetic transformation targeted on NCED/VP14, a key enzyme of ABA synthesis. The transgenic lines yielded less strong phenotypes than available mutants, making it possible to use them under normal growing conditions. We focused on leaf elongation during night periods in order to avoid the confounding effect of ABA on leaf water status. Our results suggest that over a wide range, internal ABA level (measured in both leaf extracts or xylem sap) has no clear effect on leaf elongation response to soil water deficit, except in the case of an antisense line presenting the strongest reduction in ABA accumulation that showed a slight maintenance of leaf elongation during water deficit. Leaf ethylene production rate was variable and not related to water deficit except in the ABA-deficient transgenic lines where it was increased by water deficit on average but not systematically. Moreover, variability in ethylene production rate was not linked to variability in elongation rate. Our results thus suggest that neither ABA nor ethylene seems to play a major role in the control of leaf elongation response to soil water deficit.

An efficient two-step total synthesis of the quaterpyridine nemertelline.

Regioselective and univocal Suzuki cross-coupling reactions performed on halopyridinyl boronic acids provide a flexible and versatile route to a multigram scale synthesis of 2,2'-dichloro-3,4'-bipyridine 14, which allows couplings with excess pyridin-3-yl boronic acid to give a new and efficient two-step rapid synthesis of nemertelline, the quaterpyridine neurotoxin isolated from a Hoplonemertine sea worm.