Cultivated sunflower (Helianthus annuus L.) has lower tolerance of moderate drought stress than its con-specific wild relative, but the underlying traits remain elusive.

Cultivated crops are generally expected to have less abiotic stress tolerance than their wild relatives. However, this assumption is not well supported by empirical literature and may depend on the type of stress and how it is imposed, as well as the measure of tolerance being used. Here, we investigated whether wild and cultivated accessions of Helianthus annuus differed in stress tolerance assessed as proportional decline in biomass due to drought and whether wild and cultivated accessions differed in trait responses to drought and trait associations with tolerance. In a greenhouse study, H. annuus accessions in the two domestication classes (eight cultivated and eight wild accessions) received two treatments: a well-watered control and a moderate drought implemented as a dry down followed by maintenance at a predetermined soil moisture level with automated irrigation. Treatments were imposed at the seedling stage, and plants were harvested after 2 weeks of treatment. The proportional biomass decline in response to drought was 24% for cultivated H. annuus accessions but was not significant for the wild accessions. Thus, using the metric of proportional biomass decline, the cultivated accessions had less drought tolerance. Among accessions, there was no tradeoff between drought tolerance and vigor assessed as biomass in the control treatment. In a multivariate analysis, wild and cultivated accessions did not differ from each other or in response to drought for a subset of morphological, physiological, and allocational traits. Analyzed individually, traits varied in response to drought in wild and/or cultivated accessions, including declines in specific leaf area, leaf theoretical maximum stomatal conductance (gsmax), and stomatal pore length, but there was no treatment response for stomatal density, succulence, or the ability to osmotically adjust. Focusing on traits associations with tolerance, plasticity in gsmax was the most interesting because its association with tolerance differed by domestication class (although the effects were relatively weak) and thus might contribute to lower tolerance of cultivated sunflower. Our H. annuus results support the expectation that stress tolerance is lower in crops than wild relatives under some conditions. However, determining the key traits that underpin differences in moderate drought tolerance between wild and cultivated H. annuus remains elusive.

The genomic basis of nitrogen utilization efficiency and trait plasticity to improve nutrient stress tolerance in cultivated sunflower.

Maintaining crop productivity is challenging 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 generally reduced RGR. Moreover, there was a negative correlation between vigor (RGR in control) and decline in RGR in response to stress. Given this trade-off, we focused on nutrient stress tolerance independent of vigor. This tolerance metric correlated with the change in NUtE, plasticity for a suite of morphological traits, and leaf element content. Genome-wide associations revealed regions associated with variation and plasticity in multiple traits, including two regions with seemingly additive effects on NUtE change. Our results demonstrate potential avenues for improving sunflower nutrient stress tolerance independent of vigor, and highlight specific traits and genomic regions that could play a role in enhancing tolerance.

Similar Transcriptomic Responses to Early and Late Drought Stresses Produce Divergent Phenotypes in Sunflower (Helianthus annuus L.).

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.

Genomic regions associate with major axes of variation driven by gas exchange and leaf construction traits in cultivated sunflower (Helianthus annuus L.).

Stomata 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 annuus L.). 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.

A meta-analysis of responses of C3 plants to atmospheric CO2 : dose-response curves for 85 traits ranging from the molecular to the whole-plant level.

Generalised dose-response curves are essential to understand how plants acclimate to atmospheric CO2 . We carried out a meta-analysis of 630 experiments in which C3 plants were experimentally grown at different [CO2 ] under relatively benign conditions, and derived dose-response curves for 85 phenotypic traits. These curves were characterised by form, plasticity, consistency and reliability. Considered over a range of 200-1200 µmol mol-1 CO2 , some traits more than doubled (e.g. area-based photosynthesis; intrinsic water-use efficiency), whereas others more than halved (area-based transpiration). At current atmospheric [CO2 ], 64% of the total stimulation in biomass over the 200-1200 µmol mol-1 range has already been realised. We also mapped the trait responses of plants to [CO2 ] against those we have quantified before for light intensity. For most traits, CO2 and light responses were of similar direction. However, some traits (such as reproductive effort) only responded to light, others (such as plant height) only to [CO2 ], and some traits (such as area-based transpiration) responded in opposite directions. This synthesis provides a comprehensive picture of plant responses to [CO2 ] at different integration levels and offers the quantitative dose-response curves that can be used to improve global change simulation models.

Genetic control of arbuscular mycorrhizal colonization by Rhizophagus intraradices in Helianthus annuus (L.).

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.

Plasticity and the role of mass-scaling in allocation, morphology, and anatomical trait responses to above- and belowground resource limitation in cultivated sunflower (Helianthus annuus L.).

In the face of resource limitations, plants show plasticity in multiple trait categories, including biomass allocation, morphology, and anatomy, yet inevitably also grow less. The extent to which passive mass-scaling plays a role in trait responses that contribute to increased potential for resource acquisition is poorly understood. Here, we assessed the role of mass-scaling on the direction, magnitude, and coordination of trait plasticity to light and/or nutrient limitation in cultivated sunflower (Helianthus annuus). We grew seedlings of 10 sunflower genotypes for 3 weeks in a factorial of light (50% shade) and nutrient (10% supply) limitation in the greenhouse and measured a suite of allocational, morphological, and anatomical traits for leaves, stems, fine roots, and tap roots. Under resource limitation, plants were smaller and more biomass was allocated to the organ capturing the most limiting resource, as expected. Traits varied in the magnitude of plasticity and the extent to which the observed response was passive (scaled with plant mass) and/or had an additional active component. None of the allocational responses were primarily passive. Plastic changes to specific leaf area and specific root length were primarily active, and adjusted toward more acquisitive trait values under light and nutrient limitation, respectively. For many traits, the observed response was a mixture of active and passive components, and for some traits, the active adjustment was antagonistic to the direction of passive adjustment, for example, stem height, and tap root and stem theoretical hydraulic conductance. Passive scaling with size played a major role in the coordinated response to light, but correcting for mass clarified that the active responses to both limitations were more similar in magnitude, although still resource and organ specific. Our results demonstrate that both passive plasticity and active plasticity can contribute to increased uptake capacity for limiting resources in a manner that is resource, organ, and trait specific. Indeed, passive adjustments (scaling with mass) of traits due to resource stress extend well beyond just mass allocation traits. For a full understanding of plants' response to environmental stress, both passive and active plasticity need to be taken into account.

Key Traits and Genes Associate with Salinity Tolerance Independent from Vigor in Cultivated Sunflower.

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.

Element content and distribution has limited, tolerance metric dependent, impact on salinity tolerance in cultivated sunflower (Helianthus annuus).

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.

Multiple genomic regions influence root morphology and seedling growth in cultivated sunflower (Helianthus annuus L.) under well-watered and water-limited conditions.

With climate change and an ever-increasing human population threatening food security, developing a better understanding of the genetic basis of crop performance under stressful conditions has become increasingly important. Here, we used genome-wide association studies to genetically dissect variation in seedling growth traits in cultivated sunflower (Helianthus annuus L.) under well-watered and water-limited (i.e., osmotic stress) conditions, with a particular focus on root morphology. Water limitation reduced seedling size and produced a shift toward deeper rooting. These effects varied across genotypes, and we identified 13 genomic regions that were associated with traits of interest across the two environments. These regions varied in size from a single marker to 186.2 Mbp and harbored numerous genes, some of which are known to be involved in the plant growth/development as well as the response to osmotic stress. In many cases, these associations corresponded to growth traits where the common allele outperformed the rare variant, suggesting that selection for increased vigor during the evolution of cultivated sunflower might be responsible for the relatively high frequency of these alleles. We also found evidence of pleiotropy across multiple traits, as well as numerous environmentally independent genetic effects. Overall, our results indicate the existence of genetic variation in root morphology and allocation and further suggest that the majority of alleles associated with these traits have consistent effects across environments.

Winners always win: growth of a wide range of plant species from low to future high CO2.

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.