Background Populus trichocarpa is an important forest tree species for the generation of lignocellulosic ethanol. Understanding the genomic basis of biomass production and chemical composition of wood is fundamental in supporting genetic improvement programs. Considerable variation has been observed in this species for complex traits related to growth, phenology, ecophysiology and wood chemistry. Those traits are influenced by both polygenic control and environmental effects, and their genome architecture and regulation are only partially understood. Genome wide association studies (GWAS) represent an approach to advance that aim using thousands of single nucleotide polymorphisms (SNPs). Genotyping using exome capture methodologies represent an efficient approach to identify specific functional regions of genomes underlying phenotypic variation. Results We identified 813 K SNPs, which were utilized for genotyping 461 P. trichocarpa clones, representing 101 provenances collected from Oregon and Washington, and established in California. A GWAS performed on 20 traits, considering single SNP-marker tests identified a variable number of significant SNPs ( p -value < 6.1479E-8) in association with diameter, height, leaf carbon and nitrogen contents, and δ 15 N. The number of significant SNPs ranged from 2 to 220 per trait. Additionally, multiple-marker analyses by sliding-windows tests detected between 6 and 192 significant windows for the analyzed traits. The significant SNPs resided within genes that encode proteins belonging to different functional classes as such protein synthesis, energy/metabolism and DNA/RNA metabolism, among others. Conclusions SNP-markers within genes associated with traits of importance for biomass production were detected. They contribute to characterize the genomic architecture of P. trichocarpa biomass required to support the development and application of marker breeding technologies.

English walnut ( Juglans regia L.) is an economically important crop with > 99% of US walnuts produced in California. Changes in climate and recent drought cycles have raised concerns regarding the future of nut production and responsible water use in California agriculture. Our study used an association genetics approach to characterize ecophysiological traits such as water use efficiency as estimated by carbon isotope discrimination (Δ 13 C), and photosynthetic capacity through foliar nitrogen composition, in important individuals of the Walnut Improvement Program, located at the University of California, Davis. Stable isotope and leaf measurements of 241 mature trees, representing 60 scion genotypes in established orchards were sampled in 2015 and 2016, followed by genotyping with the Walnut Axiom 700 k SNP Array. A mean Δ 13 C of 21.7‰ (σ: 0.9‰) was calculated for all individuals, as well as a mean nitrogen/leaf area (N/area) of 3.0 gN/m 2 (σ: 0.5 gN/m 2 ). A Bayesian analysis utilizing genomic relationships revealed rankings of the most water use efficient accessions as Solano (95-011-16), 67-013 (unreleased cross), and Tulare (67-011). 126,554 SNPs were used in a two-step association genetics approach identifying four loci associated with Δ 13 C after correction for multiple testing. Investigation of identified loci revealed an annotation on the J. regia genome of protein FAR1-related sequence 5-like, related to abiotic stress response. For uncharacterized markers, homologs were identified in Arabidopsis for two loci, similarly related to drought stress.

Populus trichocarpa is a biological model and a candidate species for bioethanol production. Although intra-specific variation is recognized, knowledge about genetic variation underlying the properties of its lignocellulosic biomass is still incomplete. Genetic variation is fundamental for continuing genetic improvement. In this study, we carried out a comprehensive phenotypic characterization of this species, analyzing a suite of quantitative traits associated with growth performance and wood quality. Traits involved growth rate (height, diameter), phenology (bud flush), and ecophysiology (leaf carbon and nitrogen content and isotopic composition), along with the chemical composition (contents of sugars and lignin) and metabolome of wood. We utilized 460 clones, representing 101 provenances collected from Oregon and Washington. These genotypes were planted in California, in 2009, and sampled after three growing seasons. Trait characterization was carried out by direct measurements, determination of stable isotopes (leaf samples), and technologies based on mass spectrometry (wood samples). A significant clonal effect was observed for most of the traits, explaining up to 76.4 % of total variation. Estimates of "broad-sense heritability" were moderate to high, reaching 0.96 (for date of bud flush). Phenotypic and genetic correlations varied extensively depending on specific traits. In addition, metabolomic analyses quantified 632 metabolites. Twenty-eight of these varied significantly with experimental factors, showing low to moderate heritability and correlation estimates. The results support the presence of significant clonal variation and inheritance for the assessed traits, required for response to genetic selection.

Soil water availability represents one of the most important selective agents for plants in nature and the single greatest abiotic determinant of agricultural productivity, yet the genetic bases of drought acclimation responses remain poorly understood. Here, we developed a systems-genetic approach to characterize quantitative trait loci (QTLs), physiological traits and genes that affect responses to soil moisture deficit in the TSUxKAS mapping population of Arabidopsis thaliana. To determine the effects of candidate genes underlying QTLs, we analyzed gene expression as a covariate within the QTL model in an effort to mechanistically link markers, RNA expression, and the phenotype. This strategy produced ranked lists of candidate genes for several drought-associated traits, including water use efficiency, growth, abscisic acid concentration (ABA), and proline concentration. As a proof of concept, we recovered known causal loci for several QTLs. For other traits, including ABA, we identified novel loci not previously associated with drought. Furthermore, we documented natural variation at two key steps in proline metabolism and demonstrated that the mitochondrial genome differentially affects genomic QTLs to influence proline accumulation. These findings demonstrate that linking genome, transcriptome, and phenotype data holds great promise to extend the utility of genetic mapping, even when QTL effects are modest or complex.

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.

The importance and consequences of extreme events on the global carbon budget are inadequately understood. This includes the differential impact of extreme events on various ecosystem components, lag effects, recovery times, and compensatory processes. In the summer of 2007 in Barrow, Arctic Alaska, there were unusually high air temperatures (the fifth warmest summer over a 65-year period) and record low precipitation (the lowest over a 65-year period). These abnormal conditions were associated with substantial desiccation of the Sphagnum layer and a reduced net Sphagnum CO2 sink but did not affect net ecosystem exchange (NEE) from this wet-sedge arctic tundra ecosystem. Microbial biomass, NH4+ availability, gross primary production (GPP), and ecosystem respiration (Reco) were generally greater during this extreme summer. The cumulative ecosystem CO2 sink in 2007 was similar to the previous summers, suggesting that vascular plants were able to compensate for Sphagnum CO2 uptake, despite the impact on other functions and structure such as desiccation of the Sphagnum layer. Surprisingly, the lowest ecosystem CO2 sink over a five summer record (2005–2009) was observed during the 2008 summer (~70% lower), directly following the unusually warm and dry summer, rather than during the extreme summer. This sink reduction cannot solely be attributed to the potential damage to mosses, which typically contribute ~40% of the entire ecosystem CO2 sink. Importantly, the return to a substantial cumulative CO2 sink occurred two summers after the extreme event, which suggests a substantial resilience of this tundra ecosystem to at least an isolated extreme event. Overall, these results show a complex response of the CO2 sink and its sub-components to atypically warm and dry conditions. The impact of multiple extreme events requires further investigation.

Ecologists and physiologists have documented extensive variation in water use efficiency (WUE) in Arabidopsis thaliana, as well as association of WUE with climatic variation. Here, we demonstrate correlations of whole-plant transpiration efficiency and carbon isotope composition (δ13C) among life history classes of A. thaliana. We also use a whole-plant cuvette to examine patterns of co-variation in component traits of WUE and δ13C. We find that stomatal conductance (g s) explains more variation in WUE than does A. Overall, there was a strong genetic correlation between A and g s, consistent with selection acting on the ratio of these traits. At a more detailed level, genetic variation in A was due to underlying variation in both maximal rate of carboxylation (V cmax) and maximum electron transport rate (Jmax). We also found strong effects of leaf anatomy, where lines with lower WUE had higher leaf water content (LWC) and specific leaf area (SLA), suggesting a role for mesophyll conductance (g m) in variation of WUE. We hypothesize that this is due to an effect through g m, and test this hypothesis using the abi4 mutant. We show that mutants of ABI4 have higher SLA, LWC, and g m than wild-type, consistent with variation in leaf anatomy causing variation in g m and δ13C. These functional data also add further support to the central, integrative role of ABI4 in simultaneously altering ABA sensitivity, sugar signaling, and CO2 assimilation. Together our results highlight the need for a more holistic approach in functional studies, both for more accurate annotation of gene function and to understand co-limitations to plant growth and productivity.

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.

Flowering time and water-use efficiency (WUE) are two ecological traits that are important for plant drought response. To understand the evolutionary significance of natural genetic variation in flowering time, WUE, and WUE plasticity to drought in Arabidopsis thaliana, we addressed the following questions: (1) How are ecophysiological traits genetically correlated within and between different soil moisture environments? (2) Does terminal drought select for early flowering and drought escape? (3) Is WUE plasticity to drought adaptive and/or costly? We measured a suite of ecophysiological and reproductive traits on 234 spring flowering accessions of A. thaliana grown in well-watered and season-ending soil drying treatments, and quantified patterns of genetic variation, correlation, and selection within each treatment. WUE and flowering time were consistently positively genetically correlated. WUE was correlated with WUE plasticity, but the direction changed between treatments. Selection generally favored early flowering and low WUE, with drought favoring earlier flowering significantly more than well-watered conditions. Selection for lower WUE was marginally stronger under drought. There were no net fitness costs of WUE plasticity. WUE plasticity (per se) was globally neutral, but locally favored under drought. Strong genetic correlation between WUE and flowering time may facilitate the evolution of drought escape, or constrain independent evolution of these traits. Terminal drought favored drought escape in these spring flowering accessions of A. thaliana. WUE plasticity may be favored over completely fixed development in environments with periodic drought.

An evolutionary response to selection requires genetic variation; however, even if it exists, then the genetic details of the variation can constrain adaptation. In the simplest case, unlinked loci and uncorrelated phenotypes respond directly to multivariate selection and permit unrestricted paths to adaptive peaks. By contrast, ‘antagonistic’ pleiotropic loci may constrain adaptation by affecting variation of many traits and limiting the direction of trait correlations to vectors that are not favoured by selection. However, certain pleiotropic configurations may improve the conditions for adaptive evolution. Here, we present evidence that the Arabidopsis thaliana gene FRI ( FRIGIDA ) exhibits ‘adaptive’ pleiotropy, producing trait correlations along an axis that results in two adaptive strategies. Derived, low expression FRI alleles confer a ‘drought escape’ strategy owing to fast growth, low water use efficiency and early flowering. By contrast, a dehydration avoidance strategy is conferred by the ancestral phenotype of late flowering, slow growth and efficient water use during photosynthesis. The dehydration avoidant phenotype was recovered when genotypes with null FRI alleles were transformed with functional alleles. Our findings indicate that the well-documented effects of FRI on phenology result from differences in physiology, not only a simple developmental switch.

Fremont cottonwood seedlings are vulnerable to water stress from rapid water-table decline during river recession in spring. Water stress is usually cited as the reason for reduced establishment, but interactions of water stress with microclimate extremes are more likely the causes of mortality. We assessed photosynthetic responses of Fremont cottonwood seedlings to water, light and heat stresses, which commonly co-occur in habitats where seedlings establish. Under moderate temperature and light conditions, water stress did not affect photosynthetic function. However, stomatal closure during water stress predisposed Fremont cottonwood leaves to light and heat stress, resulting in greatly reduced photosynthesis beginning at 31 °C versus at 41 °C for well-watered plants. Ontogenetic shifts in leaf orientation from horizontal to vertical, which occur as seedlings mature, reduce heat and light stress, especially during water stress. When compared with naturally occurring microclimate extremes, seedling stress responses suggest that reduced assimilation and photoprotection are common for Fremont cottonwood seedlings on exposed point bars where they establish. These reductions in photosynthesis likely have negative impacts on growth and may predispose young (<90-day-old) seedlings to early mortality during rapid water-table declines. Interactions with heat and light stress are more important in these effects than water stress alone.

Low-nutrient adapted species have numerous mechanisms that aid in nutrient conservation. Hypothetically, species adapted to nutrient-poor soils should have tighter internal nutrient recycling, as evidenced by greater resorption. However, literature results are mixed. We suggest methodological factors may limit our understanding of this process. We hypothesized that plants adapted to serpentine soils would be more proficient in resorbing N and P than plants adapted to non-serpentine soils, although there would be differences among functional groups within each soil type. For six growing seasons, we sampled senescent leaf tissue from the dominant and co-dominant shrubs and trees found in serpentine and non-serpentine chaparral communities in the California Coast Range. Our study also explicitly included congener pairs found on both soil types. Most species were highly N proficient, but species adapted to serpentine soils were more P proficient. Surprisingly, two of the three potential N-fixing species were also highly N proficient. Evergreen Quercus congeners were more N proficient than their deciduous congener pairs, although there was no difference in P resorption proficiency. Overall, large inter-annual variation was observed among most species sampled, but at least in some years, maximum potential resorption likely was reached. However, climate (temperature and precipitation) was not strongly correlated with either N or P resorption proficiency. Our data suggest that controlling for phylogeny can aid in interpretation of resorption patterns. More importantly, our study clearly shows that resorption patterns can only be discerned through long-term datasets, of which few exist in the literature.

The regulation of gene expression is crucial for an organism's development and response to stress, and an understanding of the evolution of gene expression is of fundamental importance to basic and applied biology. To improve this understanding, we conducted expression quantitative trait locus (eQTL) mapping in the Tsu-1 (Tsushima, Japan) × Kas-1 (Kashmir, India) recombinant inbred line population of Arabidopsis thaliana across soil drying treatments. We then used genome resequencing data to evaluate whether genomic features (promoter polymorphism, recombination rate, gene length, and gene density) are associated with genes responding to the environment (E) or with genes with genetic variation (G) in gene expression in the form of eQTLs. We identified thousands of genes that responded to soil drying and hundreds of main-effect eQTLs. However, we identified very few statistically significant eQTLs that interacted with the soil drying treatment (GxE eQTL). Analysis of genome resequencing data revealed associations of several genomic features with G and E genes. In general, E genes had lower promoter diversity and local recombination rates. By contrast, genes with eQTLs (G) had significantly greater promoter diversity and were located in genomic regions with higher recombination. These results suggest that genomic architecture may play an important a role in the evolution of gene expression.

Most C3 plant species have partially open stomata during the night especially in the 3-5 h before dawn. This pre-dawn stomatal opening has been hypothesized to enhance early-morning photosynthesis (A) by reducing diffusion limitations to CO2 at dawn. We tested this hypothesis in cultivated Helianthus annuus using whole-shoot gas exchange, leaf level gas exchange and modelling approaches. One hour pre-dawn low-humidity treatments were used to reduce pre-dawn stomatal conductance (g). At the whole-shoot level, a difference of pre-dawn g (0.40 versus 0.17 mol m(-2) s(-1)) did not significantly affect A during the first hour after dawn. Shorter term effects were investigated with leaf level gas exchange measurements and a difference of pre-dawn g (0.10 versus 0.04 mol m(-2) s(-1)) affected g and A for only 5 min after dawn. The potential effects of a wider range of stomatal apertures were explored with an empirical model of the relationship between A and intercellular CO2 concentration during the half-hour after dawn. Modelling results demonstrated that even extremely low pre-dawn stomatal conductance values have only a minimal effect on early-morning A for a few minutes after dawn. Thus, we found no evidence that pre-dawn stomatal opening enhances A.

Fremont cottonwood (Populus fremontii) is an early successional foundation species found in riparian forest ecosystems in the North American Southwest. Along rivers, the upper limit of the seedling establishment zone depends on the proximity of seedling roots to the declining water table. The lower limit is a function of the maximum elevation of inundation or scone Fremont cottonwood seedlings are likely to experience short-term (1- to 5-week) inundation during their first year of growth under both natural and human-influenced hydrologic regimes. Previous studies show that inundation can account for more than 70% of seedling mortality during this time. Using controlled inundation experiments, we found that seedlings of Fremont cottonwood have high tolerance of inundation to the soil surface and a reasonable tolerance of complete shoot submergence for a duration of 1 or 2 weeks (22% and 50% mortality, respectively). Mortality increased linearly with days of complete submergence (mortality percentage = 4.6 + [2.5 x days of submergence]). Warm water temperature (25/18 degrees C day/night) during complete submergence adversely affected seedling biomass and survival, resulting in 64% mortality versus 39% with cooler water temperatures (18/11 degrees C day/night). Our results indicate that establishment of new Fremont cottonwood populations in the riparian corridor will be more successful when flows do not completely cover the shoots of seedlings for more than 2 weeks and if water temperatures during inundation are cool. From the perspective of the management of river flows for cottonwood recruitment deep, prolonged, late-season (warm water) inundations are the most detrimental.

Two instrumented test sections were constructed in summer 1999 at the Kiefer Landfill near Sacramento, California to test the hydraulic performance of two proposed alternative final covers. Both test sections simulated monolithic evapotranspiration (ET) designs that differed primarily in thickness. Both were seeded with a mix of two perennial and one annual grass species. Oleander seedlings were also planted in the thicker test section. Detailed hydrologic performance monitoring of the covers was conducted from 1999 through 2005. The thicker test section met the performance criterion (average percolation of <3 mm/y). The thinner test section transmitted considerably more percolation (average of 55 mm/y). Both test sections were decommissioned in summer 2005 to investigate changes in soil hydraulic properties, geomorphology, and vegetation and to collect data to support a revised design. Field data from hydrologic monitoring and the decommissioning study were subsequently included in a hydrologic modeling study to estimate the performance of an optimized cover system for full-scale application. The decommissioning study showed that properties of the soils changed over the monitoring period (saturated hydraulic conductivity and water holding capacity increased, density decreased) and that the perennial grasses and shrubs intended for the cover were out-competed by annual species with shallower roots and lesser capacity for water uptake. Of these changes, reduced ET from the shallow-rooted annual vegetation is believed to be the primary cause for the high percolation rate from the thinner test section. Hydrologic modeling suggests that the target hydraulic performance can be achieved using an ET cover with similar thickness to the thin test section if perennial vegetation species observed in surrounding grasslands can be established. This finding underscores the importance of establishing and maintaining the appropriate vegetation on ET covers in this climate.

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.