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Plant symbiosis with arbuscular mycorrhizal (AM) fungi provides many benefits, including increased nutrient uptake, drought tolerance, and belowground pathogen resistance. To develop a better understanding of the genetic architecture of mycorrhizal symbiosis, we conducted a genome-wide association study (GWAS) of this plant-fungal interaction in cultivated sunflower. A diversity panel of cultivated sunflower (Helianthus annuus L.) was phenotyped for root colonization under inoculation with the AM fungus Rhizophagus intraradices. Using a mixed linear model approach with a high-density genetic map, we identified genomic regions that are likely associated with R. intraradices colonization in sunflower. Additionally, we used a set of twelve diverse lines to assess the effect that inoculation with R. intraradices has on dried shoot biomass and macronutrient uptake. Colonization among lines in the mapping panel ranged from 0–70% and was not correlated with mycorrhizal growth response, shoot phosphorus response, or shoot potassium response among the Core 12 lines. Association mapping yielded three single-nucleotide polymorphisms (SNPs) that were significantly associated with R. intraradices colonization. This is the first study to use GWAS to identify genomic regions associated with AM colonization in an Asterid eudicot species. Three genes of interest identified from the regions containing these SNPs are likely related to plant defense.

Maintaining crop productivity is a challenge as population growth, climate change, and increasing fertilizer costs necessitate expanding crop production to poorer lands whilst reducing inputs. Enhancing crops’ nutrient use efficiency is thus an important goal, but requires a better understanding of related traits and their genetic basis.We investigated variation in low nutrient stress tolerance in a diverse panel of cultivated sunflower genotypes grown under high and low nutrient conditions, assessing relative growth rate (RGR) as performance. We assessed variation in traits related to nitrogen utilization efficiency (NUtE), mass allocation, and leaf elemental content.Across genotypes, nutrient limitation reduced RGR. Moreover, higher vigor (higher control RGR) was associated with a greater absolute decrease under stress. Given this trade-off, we focused on nutrient stress tolerance independent from vigor. This tolerance metric correlated with the change in NUtE, plasticity for a suite of morphological traits, and leaf element content. Genome-wide association analyses revealed regions associated with variation and plasticity in multiple traits, including two key regions with ostensibly additive effects on NUtE change.Our results demonstrate potential avenues for improving sunflower nutrient stress tolerance independent from vigor and highlight specific traits and genomic regions that could play a role in enhancing tolerance.HighlightGenetic associations and trait correlations show that, in cultivated sunflower, selection for increased nitrogen utilization efficiency and plasticity in key traits is a promising avenue for increasing nutrient stress tolerance.

SummaryStomata and leaf veins play an essential role in transpiration and the movement of water throughout leaves. These traits are thus thought to play a key role in the adaptation of plants to drought and a better understanding of the genetic basis of their variation and coordination could inform efforts to improve drought tolerance. Here, we explore patterns of variation and covariation in leaf anatomical traits and analyze their genetic architecture via genome-wide association (GWA) analyses in cultivated sunflower (Helianthus annuusL.). Traits related to stomatal density and morphology as well as lower order veins were manually measured from digital images while the density of minor veins was estimated using a novel deep learning approach. Leaf, stomatal, and vein traits exhibited numerous significant correlations that generally followed expectations based on functional relationships. Correlated suites of traits could further be separated along three major principal component (PC) axes that were heavily influenced by variation in traits related to gas exchange, leaf hydraulics, and leaf construction. While there was limited evidence of colocalization when individual traits were subjected to GWA analyses, major multivariate PC axes that were most strongly influenced by several traits related to gas exchange or leaf construction did exhibit significant genomic associations. These results provide insight into the genetic basis of leaf trait covariation and showcase potential targets for future efforts aimed at modifying leaf anatomical traits in sunflower.Significance StatementUsing traditional and automated/high-throughput (using a novel deep learning approach) phenotyping methods we studied leaf anatomical variation in sunflower. Genome-wide association (GWA) analyses identified numerous genomic regions underlying individual trait variation and regions underlying major multivariate axes of phenotypic variation. These results illustrate the value of employing a multivariate approach to GWA analyses and shed light on the extent to which leaf trait (co-)variation can be genetically decoupled to explore novel phenotypic space.

Cultivated sunflower (Helianthus annuus L.) exhibits numerous phenotypic and transcriptomic responses to drought. However, the ways in which these responses vary with differences in drought timing and severity are insufficiently understood. We used phenotypic and transcriptomic data to evaluate the response of sunflower to drought scenarios of different timing and severity in a common garden experiment. Using a semi-automated outdoor high-throughput phenotyping platform, we grew six oilseed sunflower lines under control and drought conditions. Our results reveal that similar transcriptomic responses can have disparate phenotypic effects when triggered at different developmental time points. Leaf transcriptomic responses, however, share similarities despite timing and severity differences (e.g., 523 differentially expressed genes (DEGs) were shared across all treatments), though increased severity elicited greater differences in expression, particularly during vegetative growth. Across treatments, DEGs were highly enriched for genes related to photosynthesis and plastid maintenance. A co-expression analysis identified a single module (M8) enriched in all drought stress treatments. Genes related to drought, temperature, proline biosynthesis, and other stress responses were overrepresented in this module. In contrast to transcriptomic responses, phenotypic responses were largely divergent between early and late drought. Early-stressed sunflowers responded to drought with reduced overall growth, but became highly water-acquisitive during recovery irrigation, resulting in overcompensation (higher aboveground biomass and leaf area) and a greater overall shift in phenotypic correlations, whereas late-stressed sunflowers were smaller and more water use-efficient. Taken together, these results suggest that drought stress at an earlier growth stage elicits a change in development that enables greater uptake and transpiration of water during recovery, resulting in higher growth rates despite similar initial transcriptomic responses.

Generalised dose-response curves are essential to understand how plants acclimate to atmospheric CO

Plant symbiosis with arbuscular mycorrhizal (AM) fungi provides many benefits, including increased nutrient uptake, drought tolerance, and belowground pathogen resistance. To develop a better understanding of the genetic architecture of mycorrhizal symbiosis, we conducted a genome-wide association study (GWAS) of this plant-fungal interaction in cultivated sunflower. A diversity panel of cultivated sunflower (Helianthus annuus L.) was phenotyped for root colonization under inoculation with the AM fungus Rhizophagus intraradices. Using a mixed linear model approach with a high-density genetic map, we identified genomic regions that are likely associated with R. intraradices colonization in sunflower. Additionally, we used a set of twelve diverse lines to assess the effect that inoculation with R. intraradices has on dried shoot biomass and macronutrient uptake. Colonization among lines in the mapping panel ranged from 0-70% and was not correlated with mycorrhizal growth response, shoot phosphorus response, or shoot potassium response among the Core 12 lines. Association mapping yielded three single-nucleotide polymorphisms (SNPs) that were significantly associated with R. intraradices colonization. This is the first study to use GWAS to identify genomic regions associated with AM colonization in an Asterid eudicot species. Three genes of interest identified from the regions containing these SNPs are likely related to plant defense.

With rising food demands, crop production on salinized lands is increasingly necessary. Sunflower (Helianthus annuus), a moderately salt-tolerant crop, exhibits a tradeoff where more vigorous, high-performing genotypes have a greater proportional decline in biomass under salinity stress. Prior research has found deviations from this relationship across genotypes. Here, we identified the traits and genomic regions underlying variation in this expectation-deviation tolerance (the magnitude and direction of deviations from the expected effect of salinity). We grew a sunflower diversity panel under control and salt-stressed conditions and measured a suite of morphological (growth, mass allocation, plant and leaf morphology) and leaf ionomic traits. The genetic basis of variation and plasticity in these traits was investigated via genome-wide association, which also enabled the identification of genomic regions (i.e. haplotypic blocks) influencing multiple traits. We found that the magnitude and direction of plasticity in whole-root mass fraction, fine root mass fraction, and chlorophyll content, as well as leaf sodium and potassium content under saline conditions, were most strongly correlated with expectation-deviation tolerance. We identified multiple genomic regions underlying these traits as well as a single alpha-mannosidase gene directly associated with this tolerance metric. Our results show that, by taking the vigor-salinity effect tradeoff into account, we can identify unique traits and genes associated with salinity tolerance. Since these traits and genomic regions are distinct from those associated with high vigor (i.e. growth in benign conditions), they provide an avenue for increasing salinity tolerance in high-performing sunflower genotypes without compromising vigor.

Cultivated crops are expected to be less stress tolerant than their wild relatives, leading to efforts to mine wild relatives for traits to increase crop tolerance. However, empirical tests of this expectation often confound tolerance with plant vigor. We assessed whether wild and cultivated Helianthus annuus L. differed for salinity tolerance with 0 and 150 mM NaCl treatments. Salinity tolerance was assessed as the proportional reduction in biomass and as the deviation from expected performance based on vigor. Cultivated accessions had a greater proportional decline in biomass than wild accessions, but proportional decline was positively associated with vigor in both. Thus, wild and cultivated H. annuus did not differ for tolerance when variation in vigor was corrected for statistically. For traits potentially related to tolerance mechanisms, wild and cultivated accessions differed for elemental content and allocation of N, P, K, Mg, Ca, S, Na, Fe, Mn, B, Cu, and Zn for some tissues, biomass allocation, specific leaf area, and leaf succulence. However, these traits were generally unrelated to tolerance corrected for vigor. Osmotic adjustment was associated with tolerance corrected for vigor only in wild accessions where more osmotic adjustment was associated with greater tolerance. Our results for H. annuus suggest that efforts to use wild relatives to enhance crop abiotic stress tolerance will benefit from greater knowledge of traits related to plant growth responses decoupled from vigor, in order to get beyond potential growth-tolerance trade-offs.

With rising food demands, crop production on salinized lands is increasingly necessary. Sunflower, a moderately salt tolerant crop, exhibits a trade-off where more vigorous, high-performing genotypes have a greater proportional decline in biomass under salinity stress. Prior research has found deviations from this relationship across genotypes; the magnitude and direction of these deviations provides a useful metric of tolerance. Here, we identified the traits and genomic regions underlying variation in this expectation-deviation tolerance. We grew a diversity panel under control and salt-stressed conditions and measured a suite of morphological (growth, allocation, plant and leaf morphology) and leaf ionomic traits. The genetic basis of variation in these traits and their plasticity was investigated via genome-wide association studies, which also enabled the identification of genomic regions (i.e., haplotypic blocks) influencing multiple traits. We found that the magnitude of plasticity in whole root mass fraction, fine root mass fraction, and chlorophyll content, as well as leaf Na and K content under saline conditions, were the traits most strongly correlated with expectation-deviation tolerance. Additionally, we identified multiple genomic regions underlying these traits as well as a single gene directly associated with this tolerance metric. Our results show that, by taking the vigor-salinity effect trade-off into account, we can identify unique traits and genes associated with salinity tolerance. Since these traits and genomic regions are distinct from those associated with high vigor (i.e., growth in benign conditions), they provide an avenue for increasing salinity tolerance in high-performing sunflower genotypes without compromising vigor.Single sentence summaryDespite a trade-off between vigor and salinity-induced decline in biomass, distinct traits and genomic regions exist that could modulate this trade-off in cultivated sunflower.

Depending on resource availability plants exhibit a specific suite of traits. At the interspecific level these traits follow the leaf economic spectrum (LES), traits related to slow turnover when resources are poor and fast turnover when resources are plentiful. Limited data shows that within species, CO2 availability, low in the recent geologic past, high in the near future, has led to plants shifting their trait levels on the LES towards faster traits. We asked whether adjustments of physiological traits could underpin faster growth from low to high CO2 and how these responses varied among plant functional types. We analysed the trait response of seedlings of up to 28 C3 plant species grown at low (160 ppm), near-ambient (450 ppm), and high (750 ppm) CO2. We measured growth, specific leaf area (SLA), leaf gas exchange, chemical composition and stomatal traits. On average photosynthesis was reduced by 59% at low CO2 and increased by 14% at high CO2 compared to ambient CO2. Respiration decreased by 21% at low CO2 and increased by 39% at high CO2. Nitrogen content (N) per mass increased by 50% at low CO2 and decreased by 9% at high CO2. Plants drastically increased SLA at low CO2 so that despite lower carbon gain per area, carbon gain per unit mass was not reduced as much. Contrary to the responses to other resources, plant traits along the LES are adjusted towards the “fast” end of the spectrum (higher SLA, higher N) at low CO2 and towards the “slow” end (lower SLA, lower N) with increasing CO2. For a limited number of species photosynthesis per unit mass showed the same, increase at low CO2. From a resource economics perspective plants thus adjust the cost for growth towards the availability of carbon and the rate of assimilation: at lower CO2 the carbon costs decrease due to decreased respiration and lower leaf mass per area (higher SLA thinner leaves). At higher CO2 the carbon costs increase due to increased respiration and higher leaf mass per area (lower SLA thicker leaves). This suggests that CO2 increases from the past to the future are allowing plant species globally to combine faster growth with more robust, resource conservative leaves.

Disruption of ion homeostasis is a major component of salinity stress's effect on crop yield. In cultivated sunflower prior work revealed a negative relationship between vigor and salinity tolerance. Here, we determined the association of elemental content/distribution traits with salinity tolerance, both with and without taking vigor (biomass in control treatment) into account. We grew seedlings of 12 Helianthus annuus genotypes in two treatments (0, 100 mM NaCl). Plants were measured for biomass (+allocation), and element content (Na, P, K, Ca, Mg, S, Fe, B, Mn, Cu, Zn) in leaves (young and mature), stem, and roots. Genotype tolerance was assessed as both proportional decline of biomass and as expectation deviation (deviation from the observed relationship between vigor and proportional decline in biomass). Genotype rankings on these metrics were not the same. Elemental content and allocation/distribution were highly correlated both at the plant and organ level. Suggestive associations between tolerance and elemental traits were fewer and weaker than expected and differed by tolerance metric. Given the highly correlated nature of elemental content, it remains difficult to pinpoint specific traits underpinning tolerance. Results do show that taking vigor into account is important when seeking to determining traits that can be targeted to increase tolerance independent of vigor, and that the multivariate nature of associated traits should additionally be considered.

Carbon dioxide and water are crucial resources for plant growth. With anthropogenic fossil fuel emissions, CO2 availability is and has been increasing since the last glacial maximum. Simultaneously water availability is expected to decrease and the frequency and severity of drought episodes to increase in large parts of the world. How plants respond to these two changes will help in understanding plants’ responses to climate of the future. Here we sought to understand how drought affects plant traits responses to CO2 and whether there are trade-offs in responsiveness to low and elevated CO2 and drought. We grew seedlings of seven C3 annuals at past low (160 μl l−1), ambient (450 μl l−1) and elevated (750 μl l−1) CO2. At each concentration plants were subjected to well-watered conditions (100% soil water availability, SWA), 40% SWA or 20% SWA. We measured biomass allocation, relative growth rate, tissue N concentration, and gas exchange. Compared to well-watered conditions plant size was an important element in the absolute response to SWA decrease, i.e. the smaller, slow growing species were unaffected by drought at low CO2. Plants allocated less mass to root tissue at low CO2 contrasting with increased root mass fraction at lower SWA at ambient CO2. Across all traits measured, we found mostly additive effects of CO2 and water. As due to climate change regions become more drought prone these results suggest CO2 fertilization will not counteract the effects of reduced water availability.

Plant responses to carbon (C) and water availability are strongly connected. Thus, we can learn much about the responses of modern plants to rising atmospheric carbon dioxide (CO2) by studying their performance under a range of carbon and water availabilities, including very low CO2 as in past glacial periods. We hypothesized that, especially in shallow soils, the positive effects of high CO2 and the negative effects of low CO2 on growth response to drought are moderated by plant size-driven feedbacks through transpiration and soil water depletion. We grew two temperate annual C3 species, Avena sativa and Chenopodium album, in glacial (180 ppm), modern (400 ppm) and future (700 ppm) CO2 levels and five soil water regimes in climate chambers. In both species, low CO2 resulted in a much lower relative growth rate, biomass and total leaf area than at ambient CO2 with higher water availability, but this difference disappeared steadily towards severe drought conditions. Elevated CO2 increased relative growth rate, plant biomass and total leaf area of both species slightly compared with ambient CO2. These results were especially pronounced under drought. Our results support the hypothesis that, in annuals, plant size modulates the negative drought effect at low CO2. However, plant size-mediated effects of high CO2 on growth response to drought were inconclusive. Further experiments should reveal the interactive effects of CO2 and water regimes in environments closer to a field setting, both in shallow and in deep soils with unconstrained rooting, as well as in mixed communities.

Developing more stress-tolerant crops will require greater knowledge of the physiological basis of stress tolerance. Here we explore how biomass declines in response to salinity relate to leaf traits across twenty genotypes of cultivated sunflower (Helianthus annuus). Plant growth, leaf physiological traits, and leaf elemental composition were assessed after 21 days of salinity treatments (0, 50, 100, 150, or 200 mM NaCl) in a greenhouse study. There was a trade-off in performance such that vigorous genotypes, those with higher biomass at zero mM NaCl, had both a larger absolute decrease and proportional decrease in biomass due to increased salinity. More vigorous genotypes at control were less tolerant to salinity. Contrary to expectation, genotypes with a low increase in leaf Na and decrease in K:Na were not better at maintaining biomass with increasing salinity. Rather, genotypes with a greater reduction in leaf S and K content were better at maintaining biomass at increased salinity. While we found an overall trade-off between sunflower vigour and salt tolerance, some genotypes were more tolerant than expected. Further analysis of the traits and mechanisms underlying this trade-off may allow us to breed these into high vigour genotypes in order to increase their salt tolerance.

Evolutionary adaptation to variation in resource supply has resulted in plant strategies that are based on trade‐offs in functional traits. Here, we investigate, for the first time across multiple species, whether such trade‐offs are also apparent in growth and morphology responses to past low, current ambient, and future high CO 2 concentrations. We grew freshly germinated seedlings of up to 28 C3 species (16 forbs, 6 woody, and 6 grasses) in climate chambers at 160 ppm, 450 ppm, and 750 ppm CO 2. We determined biomass, allocation, SLA (specific leaf area), LAR (leaf area ratio), and RGR (relative growth rate), thereby doubling the available data on these plant responses to low CO 2. High CO 2 increased RGR by 8%; low CO 2 decreased RGR by 23%. Fast growers at ambient CO 2 had the greatest reduction in RGR at low CO 2 as they lost the benefits of a fast‐growth morphology (decoupling of RGR and LAR [leaf area ratio]). Despite these shifts species ranking on biomass and RGR was unaffected by CO 2, winners continued to win, regardless of CO 2. Unlike for other plant resources we found no trade‐offs in morphological and growth responses to CO 2 variation, changes in morphological traits were unrelated to changes in growth at low or high CO 2. Thus, changes in physiology may be more important than morphological changes in response to CO 2 variation.

A general understanding of the links between atmospheric CO2 concentration and the functioning of the terrestrial biosphere requires not only an understanding of plant trait responses to the ongoing transition to higher CO2 but also the legacy effects of past low CO2. An interesting question is whether the transition from current to higher CO2 can be thought of as a continuation of the past trajectory of low to current CO2 levels. Determining this trajectory requires quantifying the effect sizes of plant response to low CO2. We performed a meta-analysis of low CO2 growth experiments on 34 studies with 54 species. We quantified how plant traits vary at reduced CO2 levels and whether C3 versus C4 and woody versus herbaceous plant species respond differently. At low CO2, plant functioning changed drastically: on average across all species, a 50% reduction in current atmospheric CO2 reduced net photosynthesis by 38%; increased stomatal conductance by 60% and decreased intrinsic water use efficiency by 48%. Total plant dry biomass decreased by 47%, while specific leaf area increased by 17%. Plant types responded similarly: the only significant differences being no increase in SLA for C4 species and a 16% smaller decrease in biomass for woody C3 species at glacial CO2. Quantitative comparison of low CO2 effect sizes to those from high CO2 studies showed that the magnitude of response of stomatal conductance, water use efficiency and SLA to increased CO2 can be thought of as continued shifts along the same line. However, net photosynthesis and dry weight responses to low CO2 were greater in magnitude than to high CO2. Understanding the causes for this discrepancy can lead to a general understanding of the links between atmospheric CO2 and plant responses with relevance for both the past and the future.