To improve sustainability of agriculture, high yielding crop varieties with improved water use efficiency (WUE) are needed. Despite the feasibility of assessing WUE using different measurement techniques, breeding for WUE and high yield is a major challenge. Factors influencing the trait under field conditions are complex, including different scenarios of water availability. Plants with C₃ photosynthesis are able to moderately increase WUE by restricting transpiration, resulting in higher intrinsic WUE (iWUE) at the leaf level. However, reduced CO₂ uptake negatively influences photosynthesis and possibly growth and yield as well. The negative correlation of growth and WUE could be partly disconnected in model plant species with implications for crops. In this paper, we discuss recent insights obtained for Arabidopsis thaliana (L.) and the potential to translate the findings to C₃ and C₄ crops. Our data on Zea mays (L.) lines subjected to progressive drought show that there is potential for improvements in WUE of the maize line B73 at the whole plant level (WUE_plant). However, changes in iWUE of B73 and Arabidopsis reduced the assimilation rate relatively more in maize. The trade-off observed in the C₄ crop possibly limits the effectiveness of approaches aimed at improving iWUE but not necessarily efforts to improve WUE_plant.

A genomic segment on maize chromosome 7 influences carbon isotope composition, water use efficiency, and leaf growth sensitivity to drought, possibly by affecting stomatal properties.

Key message: Carbon isotope discrimination is a promising trait for indirect screening for improved water use efficiency of C₄crops. In the context of a changing climate, drought is one of the major factors limiting plant growth and yield. Hence, breeding efforts are directed toward improving water use efficiency (WUE) as a key factor in climate resilience and sustainability of crop production. As WUE is a complex trait and its evaluation is rather resource consuming, proxy traits, which are easier to screen and reliably reflect variation in WUE, are needed. In C₃crops, a trait established to be indicative for WUE is the carbon isotopic composition (δ¹³C) of plant material, which reflects the preferential assimilation of the lighter carbon isotope¹²C over¹³C during photosynthesis. In C₄crops, carbon fixation is more complex and δ¹³C thus depends on many more factors than in C₃crops. Recent physiological and genetic studies indicate a correlation between δ¹³C and WUE also in C₄crops, as well as a colocalization of quantitative trait loci for the two traits. Moreover, significant intraspecific variation as well as a medium to high heritability of δ¹³C has been shown in some of the main C₄crops, such as maize, sorghum and sugarcane, indicating its potential for indirect selection and breeding. Further research on physiological, genetic and environmental components influencing δ¹³C is needed to support its application in improving WUE and making C₄crops resilient to climate change.

Altering plant water use efficiency (WUE) is a promising approach for achieving sustainable crop production in changing climate scenarios. Here, we show that WUE can be tuned by alleles of a single gene discovered in elite maize (Zea mays) breeding material. Genetic dissection of a genomic region affecting WUE led to the identification of the gene ZmAbh4 as causative for the effect. CRISPR/Cas9-mediated ZmAbh4 inactivation increased WUE without growth reductions in well-watered conditions. ZmAbh4 encodes an enzyme that hydroxylates the phytohormone abscisic acid (ABA) and initiates its catabolism. Stomatal conductance is regulated by ABA and emerged as a major link between variation in WUE and discrimination against the heavy carbon isotope (Δ¹³C) during photosynthesis in the C₄ crop maize. Changes in Δ¹³C persisted in kernel material, which offers an easy-to-screen proxy for WUE. Our results establish a direct physiological and genetic link between WUE and Δ¹³C through a single gene with potential applications in maize breeding.

Stomata play a crucial role in balancing carbon dioxide uptake and water vapor loss, thereby regulating plant water use efficiency (WUE). Enhancing WUE is important for sustainable agriculture and food security, particularly for crops such as maize (Zea mays L.), as climate change and growing global food demand exacerbate limitations on water availability. Genetic factors controlling stomatal density and levels of the plant hormone abscisic acid (ABA) in leaves, which affect stomatal aperture, are key determinants of stomatal conductance (g_s) and intrinsic WUE (iWUE). In this study, we demonstrate that stomatal density and stomatal aperture have a combined effect on g_s and iWUE in maize. Using near-isogenic lines (NILs) and CRISPR/Cas9 mutants, we show that combining reduced stomatal density and reduced stomatal aperture can improve iWUE without compromising photosynthesis. This effect is pronounced at both, optimal and high temperatures. These findings highlight the potential of targeting multiple stomatal traits through genetic stacking to enhance WUE, offering a promising strategy for crop adaptation to water-limited environments.