Anatomical drivers of stomatal conductance in sorghum lines with different leaf widths grown under different temperatures.

Sustaining crop productivity and resilience in water-limited environments and under rising temperatures are matters of concern worldwide. We investigated the leaf anatomical traits that underpin our recently identified link between leaf width (LW) and intrinsic water use efficiency (iWUE), as traits of interest in plant breeding. Ten sorghum lines with varying LW were grown under three temperatures to expand the range of variation of both LW and gas exchange rates. Leaf gas exchange, surface morphology and cross-sectional anatomy were measured and analysed using structural equations modelling. Narrower leaves had lower stomatal conductance (gs ) and higher iWUE across growth temperatures. They also had smaller intercellular airspaces, stomatal size, percentage of open stomatal aperture relative to maximum, hydraulic pathway, mesophyll thickness, and leaf mass per area. Structural modelling revealed a developmental association among leaf anatomical traits that underpinned gs variation in sorghum. Growing temperature and LW both impacted leaf gas exchange rates, but only LW directly impacted leaf anatomy. Wider leaves may be more productive under well-watered conditions, but consume more water for growth and development, which is detrimental under water stress.

Coordination of stomata and vein patterns with leaf width underpins water-use efficiency in a C4 crop.

Despite its importance for crop water use and productivity, especially in drought-affected environments, the underlying mechanisms of variation in intrinsic water-use efficiency (iWUE = net photosynthesis/stomatal conductance for water vapour, gsw ) are not well understood, especially in C4 plants. Recently, we discovered that leaf width (LW) correlated negatively with iWUE and positively with gsw across several C4 grasses. Here, we confirmed these relationships within 48 field-grown genotypes differing in LW in Sorghum bicolor, a C4 crop adapted to dry and hot conditions. We measured leaf gas exchange and modelled leaf energy balance three times a day, alongside anatomical traits as potential predictors of iWUE. LW correlated negatively with iWUE and stomatal density, but positively with gsw , interveinal distance of longitudinal veins, and the percentage of stomatal aperture relative to maximum. Energy balance modelling showed that wider leaves needed to open their stomata more to generate a more negative leaf-to-air temperature difference, especially at midday when air temperatures exceeded 40°C. These results highlight the important role that LW plays in shaping iWUE through coordination of vein and stomatal traits and by affecting stomatal aperture. Therefore, LW could be used as a predictor of higher iWUE among sorghum genotypes.

The transcriptomic responses of C4 grasses to subambient CO2 and low light are largely species specific and only refined by photosynthetic subtype.

Three subtypes of C4 photosynthesis exist (NADP-ME, NAD-ME and PEPCK), each known to be beneficial under specific environmental conditions. However, the influence of photosynthetic subtype on transcriptomic plasticity, as well as the genes underpinning this variability, remain largely unknown. Here, we comprehensively investigate the responses of six C4 grass species, spanning all three C4 subtypes, to two controlled environmental stresses: low light (200 µmol m-2  sec-1 ) and glacial CO2 (subambient; 180 ppm). We identify a susceptibility within NADP-ME species to glacial CO2 . Notably, although glacial CO2 phenotypes could be tied to C4 subtype, biochemical and transcriptomic responses to glacial CO2 were largely species specific. Nevertheless, we were able to identify subtype specific subsets of significantly differentially expressed transcripts which link resource acquisition and allocation to NADP-ME species susceptibility to glacial CO2 . Here, low light phenotypes were comparable across species with no clear subtype response, while again, transcriptomic responses to low light were largely species specific. However, numerous functional similarities were noted within the transcriptomic responses to low light, suggesting these responses are functionally relatively conserved. Additionally, PEPCK species exhibited heightened regulation of transcripts related to metabolism in response to both stresses, likely tied to their C4 metabolic pathway. These results highlight the influence that both species and subtype can have on plant responses to abiotic stress, building on our mechanistic understanding of acclimation within C4 grasses and highlighting avenues for future crop improvements.