A Potassium-Dependent Oxygen Sensing Pathway Regulates Plant Root Hydraulics.

Aerobic organisms survive low oxygen (O2) through activation of diverse molecular, metabolic, and physiological responses. In most plants, root water permeability (in other words, hydraulic conductivity, Lpr) is downregulated under O2 deficiency. Here, we used a quantitative genetics approach in Arabidopsis to clone Hydraulic Conductivity of Root 1 (HCR1), a Raf-like MAPKKK that negatively controls Lpr. HCR1 accumulates and is functional under combined O2 limitation and potassium (K(+)) sufficiency. HCR1 regulates Lpr and hypoxia responsive genes, through the control of RAP2.12, a key transcriptional regulator of the core anaerobic response. A substantial variation of HCR1 in regulating Lpr is observed at the Arabidopsis species level. Thus, by combinatorially integrating two soil signals, K(+) and O2 availability, HCR1 modulates the resilience of plants to multiple flooding scenarios.

Natural variation in response to combined water and nitrogen deficiencies in Arabidopsis.

Understanding plant responses to individual stresses does not mean that we understand real world situations, where stresses usually combine and interact. These interactions arise at different levels, from stress exposure to the molecular networks of the stress response. Here, we built an in-depth multi-omics description of plant responses to mild water (W) and nitrogen (N) limitations, either individually or combined, among five genetically different Arabidopsis (Arabidopsis thaliana) accessions. We highlight the different dynamics in stress response through integrative traits such as rosette growth and the physiological status of the plants. We also used transcriptomics and metabolomics profiling during a stage when the plant response was stabilized to determine the wide diversity in stress-induced changes among accessions, highlighting the limited reality of a 'universal' stress response. The main effect of the WxN interaction was an attenuation of the N-deficiency syndrome when combined with mild drought, but to a variable extent depending on the accession. Other traits subject to WxN interactions are often accession-specific. Multi-omics analyses identified a subset of transcript-metabolite clusters that are critical to stress responses but essentially variable according to the genotype factor. Including intra-specific diversity in our descriptions of plant stress response places our findings in perspective.

Protein kinase SnRK2.4 is a key regulator of aquaporins and root hydraulics in Arabidopsis.

Soil water uptake by roots is a key component of plant water homeostasis contributing to plant growth and survival under ever-changing environmental conditions. The water transport capacity of roots (root hydraulic conductivity; Lpr ) is mostly contributed by finely regulated Plasma membrane Intrinsic Protein (PIP) aquaporins. In this study, we used natural variation of Arabidopsis for the identification of quantitative trait loci (QTLs) contributing to Lpr . Using recombinant lines from a biparental cross (Cvi-0 x Col-0), we show that the gene encoding class 2 Sucrose-Non-Fermenting Protein kinase 2.4 (SnRK2.4) in Col-0 contributes to >30% of Lpr by enhancing aquaporin-dependent water transport. At variance with the inactive and possibly unstable Cvi-0 SnRK2.4 form, the Col-0 form interacts with and phosphorylates the prototypal PIP2;1 aquaporin at Ser121 and stimulates its water transport activity upon coexpression in Xenopus oocytes and yeast cells. Activation of PIP2;1 by Col-0 SnRK2.4 in yeast also requires its protein kinase activity and can be counteracted by clade A Protein Phosphatases 2C. SnRK2.4 shows all hallmarks to be part of core abscisic acid (ABA) signaling modules. Yet, long-term (>3 h) inhibition of Lpr by ABA possibly involves a SnRK2.4-independent inhibition of PIP2;1. SnRK2.4 also promotes stomatal aperture and ABA-induced inhibition of primary root growth. The study identifies a key component of Lpr and sheds new light on the functional overlap and specificity of SnRK2.4 with respect to other ABA-dependent or independent SnRK2s.

Genomic Basis of Adaptation to a Novel Precipitation Regime.

Energy production and metabolism are intimately linked to ecological and environmental constraints across the tree of life. In plants, which depend on sunlight to produce energy, the link between primary metabolism and the environment is especially strong. By governing CO2 uptake for photosynthesis and transpiration, leaf pores, or stomata, couple energy metabolism to the environment and determine productivity and water-use efficiency (WUE). Although evolution is known to tune physiological traits to the local environment, we lack knowledge of the specific links between molecular and evolutionary mechanisms that shape this process in nature. Here, we investigate the evolution of stomatal conductance and WUE in an Arabidopsis population that colonized an island with a montane cloud scrubland ecosystem characterized by seasonal drought and fog-based precipitation. We find that stomatal conductance increases and WUE decreases in the colonizing population relative to its closest outgroup population from temperate North Africa. Genome-wide association mapping reveals a polygenic basis of trait variation, with a substantial contribution from a nonsynonymous single-nucleotide polymorphism in MAP KINASE 12 (MPK12 G53R), which explains 35% of the phenotypic variance in WUE in the island population. We reconstruct the spatially explicit evolutionary history of MPK12 53R on the island and find that this allele increased in frequency in the population due to positive selection as Arabidopsis expanded into the harsher regions of the island. Overall, these findings show how adaptation shaped quantitative eco-physiological traits in a new precipitation regime defined by low rainfall and high humidity.

Resource allocation modeling for autonomous prediction of plant cell phenotypes.

Predicting the plant cell response in complex environmental conditions is a challenge in plant biology. Here we developed a resource allocation model of cellular and molecular scale for the leaf photosynthetic cell of Arabidopsis thaliana, based on the Resource Balance Analysis (RBA) constraint-based modeling framework. The RBA model contains the metabolic network and the major macromolecular processes involved in the plant cell growth and survival and localized in cellular compartments. We simulated the model for varying environmental conditions of temperature, irradiance, partial pressure of CO2 and O2, and compared RBA predictions to known resource distributions and quantitative phenotypic traits such as the relative growth rate, the C:N ratio, and finally to the empirical characteristics of CO2 fixation given by the well-established Farquhar model. In comparison to other standard constraint-based modeling methods like Flux Balance Analysis, the RBA model makes accurate quantitative predictions without the need for empirical constraints. Altogether, we show that RBA significantly improves the autonomous prediction of plant cell phenotypes in complex environmental conditions, and provides mechanistic links between the genotype and the phenotype of the plant cell.

The Evolutionary Dynamics of Genetic Incompatibilities Introduced by Duplicated Genes in Arabidopsis thaliana.

Although gene duplications provide genetic backup and allow genomic changes under relaxed selection, they may potentially limit gene flow. When different copies of a duplicated gene are pseudofunctionalized in different genotypes, genetic incompatibilities can arise in their hybrid offspring. Although such cases have been reported after manual crosses, it remains unclear whether they occur in nature and how they affect natural populations. Here, we identified four duplicated-gene based incompatibilities including one previously not reported within an artificial Arabidopsis intercross population. Unexpectedly, however, for each of the genetic incompatibilities we also identified the incompatible alleles in natural populations based on the genomes of 1,135 Arabidopsis accessions published by the 1001 Genomes Project. Using the presence of incompatible allele combinations as phenotypes for GWAS, we mapped genomic regions that included additional gene copies which likely rescue the genetic incompatibility. Reconstructing the geographic origins and evolutionary trajectories of the individual alleles suggested that incompatible alleles frequently coexist, even in geographically closed regions, and that their effects can be overcome by additional gene copies collectively shaping the evolutionary dynamics of duplicated genes during population history.

The complex genetic architecture of shoot growth natural variation in Arabidopsis thaliana.

One of the main outcomes of quantitative genetics approaches to natural variation is to reveal the genetic architecture underlying the phenotypic space. Complex genetic architectures are described as including numerous loci (or alleles) with small-effect and/or low-frequency in the populations, interactions with the genetic background, environment or age. Linkage or association mapping strategies will be more or less sensitive to this complexity, so that we still have an unclear picture of its extent. By combining high-throughput phenotyping under two environmental conditions with classical QTL mapping approaches in multiple Arabidopsis thaliana segregating populations as well as advanced near isogenic lines construction and survey, we have attempted to improve our understanding of quantitative phenotypic variation. Integrative traits such as those related to vegetative growth used in this work (highlighting either cumulative growth, growth rate or morphology) all showed complex and dynamic genetic architecture with respect to the segregating population and condition. The more resolutive our mapping approach, the more complexity we uncover, with several instances of QTLs visible in near isogenic lines but not detected with the initial QTL mapping, indicating that our phenotyping accuracy was less limiting than the mapping resolution with respect to the underlying genetic architecture. In an ultimate approach to resolve this complexity, we intensified our phenotyping effort to target specifically a 3Mb-region known to segregate for a major quantitative trait gene, using a series of selected lines recombined every 100kb. We discovered that at least 3 other independent QTLs had remained hidden in this region, some with trait- or condition-specific effects, or opposite allelic effects. If we were to extrapolate the figures obtained on this specific region in this particular cross to the genome- and species-scale, we would predict hundreds of causative loci of detectable phenotypic effect controlling these growth-related phenotypes.

Mild drought in the vegetative stage induces phenotypic, gene expression, and DNA methylation plasticity in Arabidopsis but no transgenerational effects.

There is renewed interest in whether environmentally induced changes in phenotypes can be heritable. In plants, heritable trait variation can occur without DNA sequence mutations through epigenetic mechanisms involving DNA methylation. However, it remains unknown whether this alternative system of inheritance responds to environmental changes and if it can provide a rapid way for plants to generate adaptive heritable phenotypic variation. To assess potential transgenerational effects induced by the environment, we subjected four natural accessions of Arabidopsis thaliana together with the reference accession Col-0 to mild drought in a multi-generational experiment. As expected, plastic responses to drought were observed in each accession, as well as a number of intergenerational effects of the parental environments. However, after an intervening generation without stress, except for a very few trait-based parental effects, descendants of stressed and non-stressed plants were phenotypically indistinguishable irrespective of whether they were grown in control conditions or under water deficit. In addition, genome-wide analysis of DNA methylation and gene expression in Col-0 demonstrated that, while mild drought induced changes in the DNA methylome of exposed plants, these variants were not inherited. We conclude that mild drought stress does not induce transgenerational epigenetic effects.

An Arabidopsis Natural Epiallele Maintained by a Feed-Forward Silencing Loop between Histone and DNA.

The extent of epigenetic variation is currently well documented, but the number of natural epialleles described so far remains very limited. Determining the relevance of epigenetic changes for natural variation is an important question of research that we investigate by isolating natural epialleles segregating in Arabidopsis recombinant populations. We previously described a genetic incompatibility among Arabidopsis strains based on the silencing of a gene involved in fitness. Here, we isolated a new epiallele resulting from the silencing of a transfer-RNA editing gene in an Arabidopsis accession from the Netherlands (Nok-1). Crosses with the reference accession Col-0 show a complete incompatibility between this epiallele and another locus localized on a different chromosome. We demonstrate that conversion of an unmethylated version of this allele occurs in hybrids, associated with modifications of small RNA populations. These epialleles can also spontaneously revert within the population. Furthermore, we bring evidence that neither METHYLTRANSFERASE 1, maintaining methylation at CGs, nor components of RNA-directed DNA methylation, are key factors for the transmission of the epiallele over generations. This depends only on the self-reinforcing loop between CHROMOMETHYLASE 3 and KRYPTONITE, involving DNA methylated in the CHG context and histone H3 lysine 9 methylation. Our findings reveal a predominant role of this loop in maintaining a natural epiallele.

AraQTL - workbench and archive for systems genetics in Arabidopsis thaliana.

Genetical genomics studies uncover genome-wide genetic interactions between genes and their transcriptional regulators. High-throughput measurement of gene expression in recombinant inbred line populations has enabled investigation of the genetic architecture of variation in gene expression. This has the potential to enrich our understanding of the molecular mechanisms affected by and underlying natural variation. Moreover, it contributes to the systems biology of natural variation, as a substantial number of experiments have resulted in a valuable amount of interconnectable phenotypic, molecular and genotypic data. A number of genetical genomics studies have been published for Arabidopsis thaliana, uncovering many expression quantitative trait loci (eQTLs). However, these complex data are not easily accessible to the plant research community, leaving most of the valuable genetic interactions unexplored as cross-analysis of these studies is a major effort. We address this problem with AraQTL (http://www.bioinformatics.nl/Ara QTL/), an easily accessible workbench and database for comparative analysis and meta-analysis of all published Arabidopsis eQTL datasets. AraQTL provides a workbench for comparing, re-using and extending upon the results of these experiments. For example, one can easily screen a physical region for specific local eQTLs that could harbour candidate genes for phenotypic QTLs, or detect gene-by-environment interactions by comparing eQTLs under different conditions.

Extensive cis-regulatory variation robust to environmental perturbation in Arabidopsis.

cis- and trans-acting factors affect gene expression and responses to environmental conditions. However, for most plant systems, we lack a comprehensive map of these factors and their interaction with environmental variation. Here, we examined allele-specific expression (ASE) in an F1 hybrid to study how alleles from two Arabidopsis thaliana accessions affect gene expression. To investigate the effect of the environment, we used drought stress and developed a variance component model to estimate the combined genetic contributions of cis- and trans-regulatory polymorphisms, environmental factors, and their interactions. We quantified ASE for 11,003 genes, identifying 3318 genes with consistent ASE in control and stress conditions, demonstrating that cis-acting genetic effects are essentially robust to changes in the environment. Moreover, we found 1618 genes with genotype x environment (GxE) interactions, mostly cis x E interactions with magnitude changes in ASE. We found fewer trans x E interactions, but these effects were relatively less robust across conditions, showing more changes in the direction of the effect between environments; this confirms that trans-regulation plays an important role in the response to environmental conditions. Our data provide a detailed map of cis- and trans-regulation and GxE interactions in A. thaliana, laying the ground for mechanistic investigations and studies in other plants and environments.

Local evolution of seed flotation in Arabidopsis.

Arabidopsis seeds rapidly release hydrophilic polysaccharides from the seed coat on imbibition. These form a heavy mucilage layer around the seed that makes it sink in water. Fourteen natural Arabidopsis variants from central Asia and Scandinavia were identified with seeds that have modified mucilage release and float. Four of these have a novel mucilage phenotype with almost none of the released mucilage adhering to the seed and the absence of cellulose microfibrils. Mucilage release was modified in the variants by ten independent causal mutations in four different loci. Seven distinct mutations affected one locus, coding the MUM2 ß-D-galactosidase, and represent a striking example of allelic heterogeneity. The modification of mucilage release has thus evolved a number of times independently in two restricted geographical zones. All the natural mutants identified still accumulated mucilage polysaccharides in seed coat epidermal cells. Using nuclear magnetic resonance (NMR) relaxometry their production and retention was shown to reduce water mobility into internal seed tissues during imbibition, which would help to maintain seed buoyancy. Surprisingly, despite released mucilage being an excellent hydrogel it did not increase the rate of water uptake by internal seed tissues and is more likely to play a role in retaining water around the seed.

Extensive natural epigenetic variation at a de novo originated gene.

Epigenetic variation, such as heritable changes of DNA methylation, can affect gene expression and thus phenotypes, but examples of natural epimutations are few and little is known about their stability and frequency in nature. Here, we report that the gene Qua-Quine Starch (QQS) of Arabidopsis thaliana, which is involved in starch metabolism and that originated de novo recently, is subject to frequent epigenetic variation in nature. Specifically, we show that expression of this gene varies considerably among natural accessions as well as within populations directly sampled from the wild, and we demonstrate that this variation correlates negatively with the DNA methylation level of repeated sequences located within the 5'end of the gene. Furthermore, we provide extensive evidence that DNA methylation and expression variants can be inherited for several generations and are not linked to DNA sequence changes. Taken together, these observations provide a first indication that de novo originated genes might be particularly prone to epigenetic variation in their initial stages of formation.

Natural variation for sulfate content in Arabidopsis thaliana is highly controlled by APR2.

Most agronomic traits of importance, whether physiological (such as nutrient use efficiency) or developmental (such as flowering time), are controlled simultaneously by multiple genes and their interactions with the environment. Here, we show that variation in sulfate content between wild Arabidopsis thaliana accessions Bay-0 and Shahdara is controlled by a major quantitative trait locus that results in a strong interaction with nitrogen availability in the soil. Combining genetic and biochemical results and using a candidate gene approach, we have cloned the underlying gene, showing how a single-amino acid substitution in a key enzyme of the assimilatory sulfate reduction pathway, adenosine 5'-phosphosulfate reductase, is responsible for a decrease in enzyme activity, leading to sulfate accumulation in the plant. This work illustrates the potential of natural variation as a source of new alleles of known genes, which can aid in the study of gene function and metabolic pathway regulation. Our new insights on sulfate assimilation may have an impact on sulfur fertilizer use and stress defense improvement.

A pair of receptor-like kinases is responsible for natural variation in shoot growth response to mannitol treatment in Arabidopsis thaliana.

Growth is a complex trait that adapts to the prevailing conditions by integrating many internal and external signals. Understanding the molecular origin of this variation remains a challenging issue. In this study, natural variation of shoot growth under mannitol-induced stress was analyzed by standard quantitative trait locus mapping methods in a recombinant inbred line population derived from a cross between the Col-0 and Cvi-0 Arabidopsis thaliana accessions. Cloning of a major QTL specific to mannitol-induced stress condition led to identification of EGM1 and EGM2, a pair of tandem-duplicated genes encoding receptor-like kinases that are potentially involved in signaling of mannitol-associated stress responses. Using various genetic approaches, we identified two non-synonymous mutations in the EGM2[Cvi] allele that are shared by at least ten accessions from various origins and are probably responsible for a specific tolerance to mannitol. We have shown that the enhanced shoot growth phenotype contributed by the Cvi allele is not linked to generic osmotic properties but instead to a specific chemical property of mannitol itself. This result raises the question of the function of such a gene in A. thaliana, a species that does not synthesize mannitol. Our findings suggest that the receptor-like kinases encoded by EGM genes may be activated by mannitol produced by pathogens such as fungi, and may contribute to plant defense responses whenever mannitol is present.

Allelic heterogeneity and trade-off shape natural variation for response to soil micronutrient.

As sessile organisms, plants have to cope with diverse environmental constraints that may vary through time and space, eventually leading to changes in the phenotype of populations through fixation of adaptive genetic variation. To fully comprehend the mechanisms of evolution and make sense of the extensive genotypic diversity currently revealed by new sequencing technologies, we are challenged with identifying the molecular basis of such adaptive variation. Here, we have identified a new variant of a molybdenum (Mo) transporter, MOT1, which is causal for fitness changes under artificial conditions of both Mo-deficiency and Mo-toxicity and in which allelic variation among West-Asian populations is strictly correlated with the concentration of available Mo in native soils. In addition, this association is accompanied at different scales with patterns of polymorphisms that are not consistent with neutral evolution and show signs of diversifying selection. Resolving such a case of allelic heterogeneity helps explain species-wide phenotypic variation for Mo homeostasis and potentially reveals trade-off effects, a finding still rarely linked to fitness.

Natural variation at the FRD3 MATE transporter locus reveals cross-talk between Fe homeostasis and Zn tolerance in Arabidopsis thaliana.

Zinc (Zn) is essential for the optimal growth of plants but is toxic if present in excess, so Zn homeostasis needs to be finely tuned. Understanding Zn homeostasis mechanisms in plants will help in the development of innovative approaches for the phytoremediation of Zn-contaminated sites. In this study, Zn tolerance quantitative trait loci (QTL) were identified by analyzing differences in the Bay-0 and Shahdara accessions of Arabidopsis thaliana. Fine-scale mapping showed that a variant of the Fe homeostasis-related FERRIC REDUCTASE DEFECTIVE3 (FRD3) gene, which encodes a multidrug and toxin efflux (MATE) transporter, is responsible for reduced Zn tolerance in A. thaliana. Allelic variation in FRD3 revealed which amino acids are necessary for FRD3 function. In addition, the results of allele-specific expression assays in F1 individuals provide evidence for the existence of at least one putative metal-responsive cis-regulatory element. Our results suggest that FRD3 works as a multimer and is involved in loading Zn into xylem. Cross-homeostasis between Fe and Zn therefore appears to be important for Zn tolerance in A. thaliana with FRD3 acting as an essential regulator.

Phenoscope: an automated large-scale phenotyping platform offering high spatial homogeneity.

Increased phenotyping accuracy and throughput are necessary to improve our understanding of quantitative variation and to be able to deconstruct complex traits such as those involved in growth responses to the environment. Still, only a few facilities are known to handle individual plants of small stature for non-destructive, real-time phenotype acquisition from plants grown in precisely adjusted and variable experimental conditions. Here, we describe Phenoscope, a high-throughput phenotyping platform that has the unique feature of continuously rotating 735 individual pots over a table. It automatically adjusts watering and is equipped with a zenithal imaging system to monitor rosette size and expansion rate during the vegetative stage, with automatic image analysis allowing manual correction. When applied to Arabidopsis thaliana, we show that rotating the pots strongly reduced micro-environmental disparity: heterogeneity in evaporation was cut by a factor of 2.5 and the number of replicates needed to detect a specific mild genotypic effect was reduced by a factor of 3. In addition, by controlling a large proportion of the micro-environmental variance, other tangible sources of variance become noticeable. Overall, Phenoscope makes it possible to perform large-scale experiments that would not be possible or reproducible by hand. When applied to a typical quantitative trait loci (QTL) mapping experiment, we show that mapping power is more limited by genetic complexity than phenotyping accuracy. This will help to draw a more general picture as to how genetic diversity shapes phenotypic variation.

Natural variation in the ATPS1 isoform of ATP sulfurylase contributes to the control of sulfate levels in Arabidopsis.

Sulfur is an essential macronutrient for all living organisms. Plants take up inorganic sulfate from the soil, reduce it, and assimilate it into bioorganic compounds, but part of this sulfate is stored in the vacuoles. In our first attempt to identify genes involved in the control of sulfate content in the leaves, we reported that a quantitative trait locus (QTL) for sulfate content in Arabidopsis (Arabidopsis thaliana) was underlain by the APR2 isoform of the key enzyme of sulfate assimilation, adenosine 5'-phosphosulfate reductase. To increase the knowledge of the control of this trait, we cloned a second QTL from the same analysis. Surprisingly, the gene underlying this QTL encodes the ATPS1 isoform of the enzyme ATP sulfurylase, which precedes adenosine 5'-phosphosulfate reductase in the sulfate assimilation pathway. Plants with the Bay allele of ATPS1 accumulate lower steady-state levels of ATPS1 transcript than those with the Sha allele, which leads to lower enzyme activity and, ultimately, the accumulation of sulfate. Our results show that the transcript variation is controlled in cis. Examination of ATPS1 sequences of Bay-0 and Shahdara identified two deletions in the first intron and immediately downstream the gene in Bay-0 shared with multiple other Arabidopsis accessions. The average ATPS1 transcript levels are lower in these accessions than in those without the deletions, while sulfate levels are significantly higher. Thus, sulfate content in Arabidopsis is controlled by two genes encoding subsequent enzymes in the sulfate assimilation pathway but using different mechanisms, variation in amino acid sequence and variation in expression levels.

Enhanced recombination empowers the detection and mapping of Quantitative Trait Loci.

Modern plant breeding, such as genomic selection and gene editing, is based on the knowledge of the genetic architecture of desired traits. Quantitative trait loci (QTL) analysis, which combines high throughput phenotyping and genotyping of segregating populations, is a powerful tool to identify these genetic determinants and to decipher the underlying mechanisms. However, meiotic recombination, which shuffles genetic information between generations, is limited: Typically only one to two exchange points, called crossovers, occur between a pair of homologous chromosomes. Here we test the effect on QTL analysis of boosting recombination, by mutating the anti-crossover factors RECQ4 and FIGL1 in Arabidopsis thaliana full hybrids and lines in which a single chromosome is hybrid. We show that increasing recombination ~6-fold empowers the detection and resolution of QTLs, reaching the gene scale with only a few hundred plants. Further, enhanced recombination unmasks some secondary QTLs undetected under normal recombination. These results show the benefits of enhanced recombination to decipher the genetic bases of traits.