Nonstructural carbohydrate dynamics' relationship to leaf development under varying environments.

Leaf-out in temperate forests is a critical transition point each spring and advancing with global change. The mechanism linking phenological variation to external cues is poorly understood. Nonstructural carbohydrate (NSC) availability may be key. Here, we use branch cuttings from northern red oak (Quercus rubra) and measure NSCs throughout bud development in branch tissue. Given genes and environment influence phenology, we placed branches in an arrayed factorial experiment (three temperatures × two photoperiods, eight genotypes) to examine their impact on variation in leaf-out timing and corresponding NSCs. Despite significant differences in leaf-out timing between treatments, NSC patterns were much more consistent, with all treatments and genotypes displaying similar NSC concentrations across phenophases. Notably, the moderate and hot temperature treatments reached the same NSC concentrations and phenophases at the same growing degree days (GDD), but 20 calendar days apart, while the cold treatment achieved only half the GDD of the other two. Our results suggest that NSCs are coordinated with leaf-out and could act as a molecular clock, signaling to cells the passage of time and triggering leaf development to begin. This link between NSCs and budburst is critical for improving predictions of phenological timing.

Patterns of pan-genome occupancy and gene coexpression under water-deficit in Brachypodium distachyon.

Natural populations are characterized by abundant genetic diversity driven by a range of different types of mutation. The tractability of sequencing complete genomes has allowed new insights into the variable composition of genomes, summarized as a species pan-genome. These analyses demonstrate that many genes are absent from the first reference genomes, whose analysis dominated the initial years of the genomic era. Our field now turns towards understanding the functional consequence of these highly variable genomes. Here, we analysed weighted gene coexpression networks from leaf transcriptome data for drought response in the purple false brome Brachypodium distachyon and the differential expression of genes putatively involved in adaptation to this stressor. We specifically asked whether genes with variable "occupancy" in the pan-genome - genes which are either present in all studied genotypes or missing in some genotypes - show different distributions among coexpression modules. Coexpression analysis united genes expressed in drought-stressed plants into nine modules covering 72 hub genes (87 hub isoforms), and genes expressed under controlled water conditions into 13 modules, covering 190 hub genes (251 hub isoforms). We find that low occupancy pan-genes are under-represented among several modules, while other modules are over-enriched for low-occupancy pan-genes. We also provide new insight into the regulation of drought response in B. distachyon, specifically identifying one module with an apparent role in primary metabolism that is strongly responsive to drought. Our work shows the power of integrating pan-genomic analysis with transcriptomic data using factorial experiments to understand the functional genomics of environmental response.

Natural Variation in 9-Cis-Epoxycartenoid Dioxygenase 3 and ABA Accumulation.

The stress hormone abscisic acid (ABA) is critical for drought resistance; however, mechanisms controlling ABA levels are unclear. At low water potential, ABA accumulation in the Arabidopsis (Arabidopsis thaliana) accession Shahdara (Sha) was less than that in Landsberg erecta (Ler) or Columbia. Analysis of a Ler × Sha recombinant inbred line population revealed a single major-effect quantitative trait locus for ABA accumulation, which included 9-cis-epoxycarotenoid dioxygenase3 (NCED3) as a candidate gene. NCED3 encodes a rate-limiting enzyme for stress-induced ABA synthesis. Complementation experiments indicated that Sha has a reduced-function NCED3 allele. Compared with Ler, Sha did not have reduced NCED3 gene expression or protein level but did have four amino acid substitutions within NCED3. Sha differed from Ler in the apparent molecular mass of NCED3, indicative of altered NCED3 proteolytic processing in the chloroplast. Site-directed mutagenesis demonstrated that substitution at amino acid 271 was critical for the altered NCED3 molecular mass pattern, while the other Sha NCED3 polymorphisms were also involved in the reduced ABA accumulation. Sha did not have a reduced level of thylakoid-bound NCED3 but did differ from Ler in the apparent molecular mass of stromal NCED3. As Sha was not impaired in known factors critical for NCED3 function in ABA synthesis (expression, chloroplast import, and thylakoid binding), the differences between Ler and Sha NCED3 may affect NCED3 activity or other factors influencing NCED3 function. These results identify functionally important sites on NCED3 and indicate a complex pattern of NCED3 posttranslational regulation in the chloroplast.

Topological features of a gene co-expression network predict patterns of natural diversity in environmental response.

Molecular interactions affect the evolution of complex traits. For instance, adaptation may be constrained by pleiotropic or epistatic effects, both of which can be reflected in the structure of molecular interaction networks. To date, empirical studies investigating the role of molecular interactions in phenotypic evolution have been idiosyncratic, offering no clear patterns. Here, we investigated the network topology of genes putatively involved in local adaptation to two abiotic stressors-drought and cold-in Arabidopsis thaliana Our findings suggest that the gene-interaction topologies for both cold and drought stress response are non-random, with genes that show genetic variation in drought expression response (eGxE) being significantly more peripheral and cold response genes being significantly more central than genes which do not show GxE. We suggest that the observed topologies reflect different constraints on the genetic pathways involved in environmental response. The approach presented here may inform predictive models linking genetic variation in molecular signalling networks with phenotypic variation, specifically traits involved in environmental response.

Interactive effects of water limitation and elevated temperature on the physiology, development and fitness of diverse accessions of Brachypodium distachyon.

An enduring question in plant physiology and evolution is how single genotypes of plants optimize performance in diverse, often highly variable, environments. We grew 35 natural accessions of the grass Brachypodium distachyon in four environments in the glasshouse, contrasting soil water deficit, elevated temperature and their interaction. We modeled treatment, genotype and interactive effects on leaf-level and whole-plant traits, including fecundity. We also assessed the relationship between glasshouse-measured traits and parameters related to climate at the place of origin. We found abundant genetic variation in both constitutive and induced traits related to plant-water relations. Most traits showed strong interaction between temperature and water availability, and we observed genotype-by-environment interaction for several traits. Notably, leaf free proline abundance showed a strong effect of genotype × temperature × water. We found strong associations between phenology, biomass and water use efficiency (WUE) with parameters describing climate of origin. Plants respond to multiple stressors in ways not directly predictable from single stressors, underscoring the complex and trait-specific mechanisms of environmental response. Climate-trait correlations support a role for WUE and phenology in local adaptation to climate in B. distachyon.

Variation in MPK12 affects water use efficiency in Arabidopsis and reveals a pleiotropic link between guard cell size and ABA response.

Plant water relations are critical for determining the distribution, persistence, and fitness of plant species. Studying the genetic basis of ecologically relevant traits, however, can be complicated by their complex genetic, physiological, and developmental basis and their interaction with the environment. Water use efficiency (WUE), the ratio of photosynthetic carbon assimilation to stomatal conductance to water, is a dynamic trait with tremendous ecological and agricultural importance whose genetic control is poorly understood. In the present study, we use a quantitative trait locus-mapping approach to locate, fine-map, clone, confirm, and characterize an allelic substitution that drives differences in WUE among natural accessions of Arabidopsis thaliana. We show that a single amino acid substitution in an abscisic acid-responsive kinase, AtMPK12, causes reduction in WUE, and we confirm its functional role using transgenics. We further demonstrate that natural alleles at AtMPK12 differ in their response to cellular and environmental cues, with the allele from the Cape Verde Islands (CVI) being less responsive to hormonal inhibition of stomatal opening and more responsive to short-term changes in vapor pressure deficit. We also show that the CVI allele results in constitutively larger stomata. Together, these differences cause higher stomatal conductance and lower WUE compared with the common allele. These physiological changes resulted in reduced whole-plant transpiration efficiency and reduced fitness under water-limited compared with well-watered conditions. Our work demonstrates how detailed analysis of naturally segregating functional variation can uncover the molecular and physiological basis of a key trait associated with plant performance in ecological and agricultural settings.

Ecological interactions and the fitness effect of water-use efficiency: Competition and drought alter the impact of natural MPK12 alleles in Arabidopsis.

The presence of substantial genetic variation for water-use efficiency (WUE) suggests that natural selection plays a role in maintaining alleles that affect WUE. Soil water deficit can reduce plant survival, and is likely to impose selection to increase WUE, whereas competition for resources may select for decreased WUE to ensure water acquisition. We tested the fitness consequences of natural allelic variation in a single gene (MPK12) that influences WUE in Arabidopsis, using transgenic lines contrasting in MPK12 alleles, under four treatments; drought/competition, drought/no competition, well-watered/competition, well-watered/no competition. Results revealed an allele × environment interaction: Low WUE plants performed better in competition, resulting from increased resource consumption. Contrastingly, high WUE individuals performed better in no competition, irrespective of water availability, presumably from enhanced water conservation and nitrogen acquisition. Our findings suggest that selection can influence MPK12 evolution, and represents the first assessment of plant fitness resulting from natural allelic variation at a single locus affecting WUE.

Deeply diverged alleles in the Arabidopsis AREB1 transcription factor drive genome-wide differences in transcriptional response to the environment.

Gene regulatory variation is an important driver of the evolution of physiological and developmental responses to the environment. The abscisic acid (ABA) signaling pathway has long been studied as a key component of the cellular response to abiotic stresses in plants. We identify two haplotypes in an Arabidopsis thaliana transcription factor, AREB1, which plays a central role in ABA-mediated response to osmotic stress. These two haplotypes show the sequence signature of long-term maintenance of genetic diversity, suggesting a role for a diversifying selection process such as balancing selection. We find that the two haplotypes, distinguished by a large number of single nucleotide polymorphisms and the presence or absence of four small insertion/deletions in AREB1 intron 1 and exon 2, are at roughly equal frequencies in Arabidopsis, and show high linkage disequilibrium and deep sequence divergence. We use a transgenic approach, along with mRNA Sequencing-based assay of genome-wide expression levels, and find considerable functional divergence between alleles representing the two haplotype groups. Specifically, we find that, under benign soil-water conditions, transgenic lines containing different AREB1 alleles differ in the expression of a large number of genes associated with pathogen response. There are relatively modest gene expression differences between the two transgenic lines under restricted soil water content. Our finding of pathogen-related activity expands the known roles of AREB1 in A. thaliana and reveals the molecular basis of gene-by-environment interaction in a putatively adaptive plant regulatory protein.

Plant hydraulics as a central hub integrating plant and ecosystem function: meeting report for 'Emerging Frontiers in Plant Hydraulics' (Washington, DC, May 2015).

Water plays a central role in plant biology and the efficiency of water transport throughout the plant affects both photosynthetic rate and growth, an influence that scales up deterministically to the productivity of terrestrial ecosystems. Moreover, hydraulic traits mediate the ways in which plants interact with their abiotic and biotic environment. At landscape to global scale, plant hydraulic traits are important in describing the function of ecological communities and ecosystems. Plant hydraulics is increasingly recognized as a central hub within a network by which plant biology is connected to palaeobiology, agronomy, climatology, forestry, community and ecosystem ecology and earth-system science. Such grand challenges as anticipating and mitigating the impacts of climate change, and improving the security and sustainability of our food supply rely on our fundamental knowledge of how water behaves in the cells, tissues, organs, bodies and diverse communities of plants. A workshop, 'Emerging Frontiers in Plant Hydraulics' supported by the National Science Foundation, was held in Washington DC, 2015 to promote open discussion of new ideas, controversies regarding measurements and analyses, and especially, the potential for expansion of up-scaled and down-scaled inter-disciplinary research, and the strengthening of connections between plant hydraulic research, allied fields and global modelling efforts.

Genome diversity in Brachypodium distachyon: deep sequencing of highly diverse inbred lines.

Brachypodium distachyon is small annual grass that has been adopted as a model for the grasses. Its small genome, high-quality reference genome, large germplasm collection, and selfing nature make it an excellent subject for studies of natural variation. We sequenced six divergent lines to identify a comprehensive set of polymorphisms and analyze their distribution and concordance with gene expression. Multiple methods and controls were utilized to identify polymorphisms and validate their quality. mRNA-Seq experiments under control and simulated drought-stress conditions, identified 300 genes with a genotype-dependent treatment response. We showed that large-scale sequence variants had extremely high concordance with altered expression of hundreds of genes, including many with genotype-dependent treatment responses. We generated a deep mRNA-Seq dataset for the most divergent line and created a de novo transcriptome assembly. This led to the discovery of >2400 previously unannotated transcripts and hundreds of genes not present in the reference genome. We built a public database for visualization and investigation of sequence variants among these widely used inbred lines.

Natural variation in abiotic stress responsive gene expression and local adaptation to climate in Arabidopsis thaliana.

Gene expression varies widely in natural populations, yet the proximate and ultimate causes of this variation are poorly known. Understanding how variation in gene expression affects abiotic stress tolerance, fitness, and adaptation is central to the field of evolutionary genetics. We tested the hypothesis that genes with natural genetic variation in their expression responses to abiotic stress are likely to be involved in local adaptation to climate in Arabidopsis thaliana. Specifically, we compared genes with consistent expression responses to environmental stress (expression stress responsive, "eSR") to genes with genetically variable responses to abiotic stress (expression genotype-by-environment interaction, "eGEI"). We found that on average genes that exhibited eGEI in response to drought or cold had greater polymorphism in promoter regions and stronger associations with climate than those of eSR genes or genomic controls. We also found that transcription factor binding sites known to respond to environmental stressors, especially abscisic acid responsive elements, showed significantly higher polymorphism in drought eGEI genes in comparison to eSR genes. By contrast, eSR genes tended to exhibit relatively greater pairwise haplotype sharing, lower promoter diversity, and fewer nonsynonymous polymorphisms, suggesting purifying selection or selective sweeps. Our results indicate that cis-regulatory evolution and genetic variation in stress responsive gene expression may be important mechanisms of local adaptation to climatic selective gradients.

Physiological genomics of response to soil drying in diverse Arabidopsis accessions.

Arabidopsis thaliana, like many species, is characterized by abundant genetic variation. This variation is rapidly being cataloged at the sequence level, but careful dissection of genetic variation in whole-organism responses to stresses encountered in the natural environment are lacking; this functional variation can be exploited as a natural mutant screen to determine gene function. Here, we document physiological and transcriptomic response to soil drying in 17 natural accessions of Arabidopsis. By imposing ecologically realistic stress conditions, we found that acclimation in Arabidopsis involved a strong signature of increased investment in photosynthesis, carbohydrate turnover, and root growth. Our results extend previous work in the Columbia accession suggesting that abscisic acid signaling pathways play an important role in drought stress response. We also identified several mechanisms, including an increase in leaf nitrogen concentration and upregulation of two-component signaling relays, that were common to most natural accessions but had not been identified in studies using only the Columbia accession. Principal component analysis reveals strong correlations between suites of genes and specific physiological responses to stress. The functional variants we identified may represent adaptive mutations in natural habitats and useful variants for agronomic improvement of crop species.

Intron-mediated alternative splicing of Arabidopsis P5CS1 and its association with natural variation in proline and climate adaptation.

Drought-induced proline accumulation is widely observed in plants but its regulation and adaptive value are not as well understood. Proline accumulation of the Arabidopsis accession Shakdara (Sha) was threefold less than that of Landsberg erecta (Ler) and quantitative trait loci mapping identified a reduced function allele of the proline synthesis enzyme Δ(1)-pyrroline-5-carboxylate synthetase1 (P5CS1) as a basis for the lower proline of Sha. Sha P5CS1 had additional TA repeats in intron 2 and a G-to-T transversion in intron 3 that were sufficient to promote alternative splicing and production of a nonfunctional transcript lacking exon 3 (exon 3-skip P5CS1). In Sha, and additional accessions with the same intron polymorphisms, the nonfunctional exon 3-skip P5CS1 splice variant constituted as much as half of the total P5CS1 transcript. In a larger panel of Arabidopsis accessions, low water potential-induced proline accumulation varied by 10-fold and variable production of exon 3-skip P5CS1 among accessions was an important, but not the sole, factor underlying variation in proline accumulation. Population genetic analyses suggest that P5CS1 may have evolved under positive selection, and more extensive correlation of exon 3-skip P5CS1 production than proline abundance with climate conditions of natural accessions also suggest a role of P5CS1 in local adaptation to the environment. These data identify a unique source of alternative splicing in plants, demonstrate a role of exon 3-skip P5CS1 in natural variation of proline metabolism, and suggest an association of P5CS1 and its alternative splicing with environmental adaptation.

Escape from adaptive conflict after duplication in an anthocyanin pathway gene.

Gene duplications have been recognized as an important source of evolutionary innovation and adaptation since at least Haldane, and their varying fates may partly explain the vast disparity in observed genome sizes. The expected fates of most gene duplications involve primarily non-adaptive substitutions leading to either non-functionalization of one duplicate copy or subfunctionalization, neither of which yields novel function. A significant evolutionary problem is thus elucidating the mechanisms of adaptive evolutionary change leading to evolutionary novelty. Currently, the most widely recognized adaptive process involving gene duplication is neo-functionalization (NEO-F), in which one copy undergoes directional selection to perform a novel function after duplication. An alternative, but understudied, adaptive fate that has been proposed is escape from adaptive conflict (EAC), in which a single-copy gene is selected to perform a novel function while maintaining its ancestral function. This gene is constrained from improving either novel or ancestral function because of detrimental pleiotropic effects on the other function. After duplication, one copy is free to improve novel function, whereas the other is selected to improve ancestral function. Here we first present two criteria that can be used to distinguish NEO-F from EAC. Using both tests for positive selection and assays of enzyme function, we then demonstrate that adaptive evolutionary change in a duplicated gene of the anthocyanin biosynthetic pathway in morning glories (Ipomoea) is best interpreted as EAC. Finally, we argue that this phenomenon likely occurs more often than has been previously believed and may thus represent an important mechanism in generating evolutionary novelty.

Arabidopsis ECERIFERUM9 involvement in cuticle formation and maintenance of plant water status.

Mutation of the ECERIFERUM9 (CER9) gene in Arabidopsis (Arabidopsis thaliana) causes elevated amounts of 18-carbon-length cutin monomers and a dramatic shift in the cuticular wax profile (especially on leaves) toward the very-long-chain free fatty acids tetracosanoic acid (C24) and hexacosanoic acid (C26). Relative to the wild type, cer9 mutants exhibit elevated cuticle membrane thickness over epidermal cells and cuticular ledges with increased occlusion of the stomatal pore. The cuticular phenotypes of cer9 are associated with delayed onset of wilting in plants experiencing water deficit, lower transpiration rates, and improved water use efficiency measured as carbon isotope discrimination. The CER9 protein thus encodes a novel determinant of plant drought tolerance-associated traits, one whose deficiency elevates cutin synthesis, redistributes wax composition, and suppresses transpiration. Map-based cloning identified CER9, and sequence analysis predicted that it encodes an E3 ubiquitin ligase homologous to yeast Doa10 (previously shown to target endoplasmic reticulum proteins for proteasomal degradation). To further elucidate CER9 function, the impact of CER9 deficiency on interactions with other genes was examined using double mutant and transcriptome analyses. For both wax and cutin, cer9 showed mostly additive effects with cer6, long-chain acyl-CoA synthetase1 (lacs1), and lacs2 and revealed its role in early steps of both wax and cutin synthetic pathways. Transcriptome analysis revealed that the cer9 mutation affected diverse cellular processes, with primary impact on genes associated with diverse stress responses. The discovery of CER9 lays new groundwork for developing novel cuticle-based strategies for improving the drought tolerance and water use efficiency of crop plants.

Characterizing genomic variation of Arabidopsis thaliana: the roles of geography and climate.

Arabidopsis thaliana inhabits diverse climates and exhibits varied phenology across its range. Although A. thaliana is an extremely well-studied model species, the relationship between geography, growing season climate and its genetic variation is poorly characterized. We used redundancy analysis (RDA) to quantify the association of genomic variation [214 051 single nucleotide polymorphisms (SNPs)] with geography and climate among 1003 accessions collected from 447 locations in Eurasia. We identified climate variables most correlated with genomic variation, which may be important selective gradients related to local adaptation across the species range. Climate variation among sites of origin explained slightly more genomic variation than geographical distance. Large-scale spatial gradients and early spring temperatures explained the most genomic variation, while growing season and summer conditions explained the most after controlling for spatial structure. SNP variation in Scandinavia showed the greatest climate structure among regions, possibly because of relatively consistent phenology and life history of populations in this region. Climate variation explained more variation among nonsynonymous SNPs than expected by chance, suggesting that much of the climatic structure of SNP correlations is due to changes in coding sequence that may underlie local adaptation.

Diversity in nonlinear responses to soil moisture shapes evolutionary constraints in Brachypodium.

Water availability is perhaps the greatest environmental determinant of plant yield and fitness. However, our understanding of plant-water relations is limited because-like many studies of organism-environment interaction-it is primarily informed by experiments considering performance at two discrete levels-wet and dry-rather than as a continuously varying environmental gradient. Here, we used experimental and statistical methods based on function-valued traits to explore genetic variation in responses to a continuous soil moisture gradient in physiological and morphological traits among 10 genotypes across two species of the model grass genus Brachypodium. We find that most traits exhibit significant genetic variation and nonlinear responses to soil moisture variability. We also observe differences in the shape of these nonlinear responses between traits and genotypes. Emergent phenomena arise from this variation including changes in trait correlations and evolutionary constraints as a function of soil moisture. Our results point to the importance of considering diversity in nonlinear organism-environment relationships to understand plastic and evolutionary responses to changing climates.

Comparative Genomics, Evolution, and Drought-Induced Expression of Dehydrin Genes in Model Brachypodium Grasses.

Dehydration proteins (dehydrins, DHNs) confer tolerance to water-stress deficit in plants. We performed a comparative genomics and evolutionary study of DHN genes in four model Brachypodium grass species. Due to limited knowledge on dehydrin expression under water deprivation stress in Brachypodium, we also performed a drought-induced gene expression analysis in 32 ecotypes of the genus' flagship species B. distachyon showing different hydric requirements. Genomic sequence analysis detected 10 types of dehydrin genes (Bdhn) across the Brachypodium species. Domain and conserved motif contents of peptides encoded by Bdhn genes revealed eight protein architectures. Bdhn genes were spread across several chromosomes. Selection analysis indicated that all the Bdhn genes were constrained by purifying selection. Three upstream cis-regulatory motifs (BES1, MYB124, ZAT) were detected in several Bdhn genes. Gene expression analysis demonstrated that only four Bdhn1-Bdhn2, Bdhn3, and Bdhn7 genes, orthologs of wheat, barley, rice, sorghum, and maize genes, were expressed in mature leaves of B. distachyon and that all of them were more highly expressed in plants under drought conditions. Brachypodium dehydrin expression was significantly correlated with drought-response phenotypic traits (plant biomass, leaf carbon and proline contents and water use efficiency increases, and leaf water and nitrogen content decreases) being more pronounced in drought-tolerant ecotypes. Our results indicate that dehydrin type and regulation could be a key factor determining the acquisition of water-stress tolerance in grasses.