The ratio of plant carbon gain to water use, known as water use efficiency (WUE), has long been recognized as a key constraint on crop production and an important target for crop improvement. WUE is a physiologically and genetically complex trait that can be defined at a range of scales. Many component traits directly influence WUE, including photosynthesis, stomatal and mesophyll conductances, and canopy structure. Interactions of carbon and water relations with diverse aspects of the environment and crop development also modulate WUE. As a consequence, enhancing WUE by breeding or biotechnology has proven challenging but not impossible. This review aims to synthesize new knowledge of WUE arising from advances in phenotyping, modeling, physiology, genetics, and molecular biology in the context of classical theoretical principles. In addition, we discuss how rising atmospheric CO₂ concentration has created and will continue to create opportunities for enhancing WUE by modifying the trade-off between photosynthesis and transpiration.

Stomata allow CO₂ uptake by leaves for photosynthetic assimilation at the cost of water vapor loss to the atmosphere. The opening and closing of stomata in response to fluctuations in light intensity regulate CO₂ and water fluxes and are essential for maintaining water-use efficiency (WUE). However, a little is known about the genetic basis for natural variation in stomatal movement, especially in C₄ crops. This is partly because the stomatal response to a change in light intensity is difficult to measure at the scale required for association studies. Here, we used high-throughput thermal imaging to bypass the phenotyping bottleneck and assess 10 traits describing stomatal conductance (g_s) before, during and after a stepwise decrease in light intensity for a diversity panel of 659 sorghum (Sorghum bicolor) accessions. Results from thermal imaging significantly correlated with photosynthetic gas exchange measurements. g_s traits varied substantially across the population and were moderately heritable (h² up to 0.72). An integrated genome-wide and transcriptome-wide association study identified candidate genes putatively driving variation in stomatal conductance traits. Of the 239 unique candidate genes identified with the greatest confidence, 77 were putative orthologs of Arabidopsis (Arabidopsis thaliana) genes related to functions implicated in WUE, including stomatal opening/closing (24 genes), stomatal/epidermal cell development (35 genes), leaf/vasculature development (12 genes), or chlorophyll metabolism/photosynthesis (8 genes). These findings demonstrate an approach to finding genotype-to-phenotype relationships for a challenging trait as well as candidate genes for further investigation of the genetic basis of WUE in a model C₄ grass for bioenergy, food, and forage production.

The wild progenitors of major C₄ crops grew as individuals subjected to little shading. Today they are grown in dense stands where most leaves are shaded. Do they maintain photosynthetic efficiency in these low light conditions produced by modern cultivation? The apparent maximum quantum yield of CO₂ assimilation $(ΦCO2max,app),$ a key determinant of light-limited photosynthesis, has not been systematically studied in field stands of C₄ crops. $ΦCO2max,app$ was derived from the initial slope of the response of leaf CO₂ uptake (A) to photon flux (Q). Leaf fractional light absorptance (α) was measured to determine the absolute maximum quantum yield of CO₂ assimilation on an absorbed light basis $(ΦCO2max,abs).$ Light response curves were determined on sun and shade leaves of 49 field plants of Miscanthus × giganteus and Zea mays following canopy closure. $ΦCO2max,app$ and $ΦCO2max,abs$ declined significantly by 15–27% (P<0.05) with canopy depth. Experimentally, leaf age was shown unlikely to cause this loss. Modeling canopy CO₂ assimilation over diurnal courses suggested that the observed decline in $ΦCO2max,app$ with canopy depth costs 10% of potential carbon gain. Overcoming this limitation could substantially increase the productivity of major C₄ crops.

Chilling temperatures (0–15°C) inhibit photosynthesis in most C₄grasses, yet photosynthesis is chilling tolerant in the ‘Illinois’ clone of the C₄grassMiscanthusxgiganteus, a candidate cellulosic bioenergy crop.M.xgiganteusis a hybrid betweenMiscanthus sacchariflorusandMiscanthus sinensis; therefore chilling‐tolerant parent lines might produce hybrids superior to the current clone. Recently a collection ofM. sacchariflorusfrom Siberia, the apparent low temperature limit of natural distribution,became available, which may be a source for chilling tolerance. The collection was screened for chilling tolerance of photosynthesis by measuring dark‐adapted maximum quantum yield of PSII photochemistry (F_v/F_m) on plants in the field in cool weather. Superior accessions were selected for further phenotyping: plants were grown at 25°C, transferred to 10°C (chilling) for 15 days, and returned to 25°C for 7 days (recovery). Two experiments assessed: (a) light‐saturated net photosynthetic rate (A_sat) and operating quantum yield of PSII photochemistry (Φ_PSII), (b) response of net leaf CO₂uptake (A) to intercellular [CO₂] (c_i). Three accessions showed superior chilling tolerance: RU2012‐069 and RU2012‐114 achievedA_satup to double that ofM.xgiganteusprior to and during chilling, due to increasedc_i‐ saturated photosynthesis (V_max). RU2012‐069 and RU2012‐114 also maintained greater levels of Φ_PSIIduring chilling, indicating reduced photodamage. Additionally, accession RU2012‐112 maintained a stableA_satthroughout the 15‐day chilling period, whileA_satcontinuously declined in other accessions; this suggests RU2012‐112 could outperform others in lengthy chilling periods. Plants were returned to 25°C after the chilling period;M.xgiganteusshowed the weakest recovery after 1 day, but a strong recovery after 1 week. This study has therefore identified important genetic resources for the synthesis of improved lines ofM.xgiganteus, which could facilitate the displacement of fossil fuels by cellulosic bioenergy.

The C₄crop maize (Zea mays) is the most widely grown cereal crop worldwide and is an essential feedstock for food and bioenergy. Improving maize yield is important to achieve food security and agricultural sustainability in the 21^stcentury. One potential means to improve crop productivity is to enhance photosynthesis.ictB, a membrane protein that is highly conserved across cyanobacteria, has been shown to improve photosynthesis, and often biomass, when introduced into diverse C₃plant species. Here,ictBfromSynechococcussp. strain PCC 7942 was inserted into maize usingAgrobacterium-mediated transformation. In three controlled-environment experiments,ictBinsertion increased leaf starch and sucrose content by up to 25% relative to controls. Experimental field trials in four growing seasons, spanning the Midwestern United States (Summers 2018 & 2019) and Argentina (Winter 2018 & 2019), showed an average of 3.49% grain yield improvement, by as much as 5.4% in a given season and up to 9.4% at certain trial locations. A subset of field trial locations was used to test for modification of ear traits and ФPSII, a proxy for photosynthesis. Results suggested that yield gain in transgenics could be associated with increased ФPSII, and the production of longer, thinner ears with more kernels.ictBlocalized primarily to the microsome fraction of leaf bundle-sheath cells, but not to chloroplasts. Extramembrane domains ofictBinteractedin vitrowith proteins involved in photosynthesis and carbohydrate metabolism. To our knowledge, this is the first published evidence ofictBinsertion into a species using C₄photosynthesis and the largest-scale demonstration of grain yield enhancement fromictBinsertionin planta. Results show thatictBis a valuable yield gene in the economically important crop maize, and are an important proof of concept that transgenic manipulation of photosynthesis can be used to create economically viable crop improvement traits.