CO2 diffusion from substomatal intercellular cavities to sites of carboxylation in chloroplasts (mesophyll conductance; gm) limits photosynthetic rate and influences leaf intrinsic water-use efficiency (A/gsw). We investigated genotypic variability of gm and effects of gm on A/gsw among eleven wheat (Triticum aestivum L.) genotypes under light-saturated conditions and at either 2 or 21% O2. Significant variation in gm and A/gsw was found between genotypes at both O2 concentrations, but there was no significant effect of O2 concentration on gm. Further, gm was correlated with photosynthetic rate among the 11 genotypes, but was unrelated to stomatal conductance. The effect of leaf age differed between genotypes, with gm being lower in older leaves for one genotype but not another. This study demonstrates a high level of variation in gm between wheat genotypes; 0.5 to 1.0 μmol m−2 s−1 bar−1. Further, leaf age effects indicate that great care must be taken to choose suitable leaves in studies of genotypic variation in gm and water-use efficiency.

Mesophyll conductance (gm) is an important factor limiting photosynthesis. However, gm response to long-term growth in variable [CO2] is not well understood, particularly in crop plants. Here, we grew two cultivars of wheat (Halberd and Cranbrook), known to differ in gm under current environmental conditions, in four [CO2] treatments: glacial (206 μmol mol -1), pre-industrial (344 μmol mol -1), current ambient (489 μmol mol -1) and super-elevated (1085 μmol mol -1), and two water treatments (well-watered and moderate water limitation), to develop an evolutionary and future climate perspective on gm control of photosynthesis and water use efficiency (WUE). In the two wheat genotypes, gm increased with rising [CO2]from glacial to ambient [CO2], but declined at super-elevated [CO2]. The responses of gm to different growth [CO2]also depend on water stress; however, the specific mechanism of gm response to [CO2]remains unclear. Although gm and gm/gsc (mesophyll conductance/stomatal conductance) were strongly associated with the variability of photosynthetic rates (A) and WUE, we found that plants with higher gm may increase A without increasing gsc, which increased WUE. These results may be useful to inform plant breeding programs and cultivar selection for Australian wheat under future environmental conditions.

Summary statement: Mesophyll conductance (gm) was negatively correlated with wheat leaf age but was positively correlated with the surface area of chloroplasts exposed to intercellular airspaces (Sc). The rate of decline in photosynthetic rate and gm as leaves aged was slower for water-stressed than well-watered plants. Upon rewatering, the degree of recovery from water-stress depended on the age of the leaves, with the strongest recovery for mature leaves, rather than young or old leaves. Diffusion of CO2 from the intercellular airspaces to the site of Rubisco within C3 plant chloroplasts (gm) governs photosynthetic CO2 assimilation (A). However, variation in gm in response to environmental stress during leaf development remains poorly understood. Age-dependent changes in leaf ultrastructure and potential impacts on gm, A, and stomatal conductance to CO2 (gsc) were investigated for wheat (Triticum aestivum L.) in well-watered and water-stressed plants, and after recovery by re-watering of droughted plants. Significant reductions in A and gm were found as leaves aged. The oldest plants (15 days and 22 days) in water-stressed conditions showed higher A and gm compared to irrigated plants. The rate of decline in A and gm as leaves aged was slower for water-stressed compared to well-watered plants. When droughted plants were rewatered, the degree of recovery depended on the age of the leaves, but only for gm. The surface area of chloroplasts exposed to intercellular airspaces (Sc) and the size of individual chloroplasts declined as leaves aged, resulting in a positive correlation between gm and Sc. Leaf age significantly affected cell wall thickness (tcw), which was higher in old leaves compared to mature/young leaves. Greater knowledge of leaf anatomical traits associated with gm partially explained changes in physiology with leaf age and plant water status, which in turn should create more possibilities for improving photosynthesis using breeding/biotechnological strategies.

word count: 176 1 8 Total word count (excluding references): 4923 1 9 2 0Highlight: Mesophyll conductance increased with increasing [CO 2 ] from glacial to ambient 2 1 CO 2 levels, then declined at super-elevated CO 2 for both well-watered and water-limited 2 2 treatments. These responses of mesophyll conductance with varying [CO 2 ] have a 2 3 physiological basis.2 4 Running title: Mesophyll conductance with [CO 2 ] 2 5 2 6 2 7 2 Abstract 2 8 2 9Mesophyll conductance (g m ) is an important factor limiting photosynthesis. However, g m 3 0response to long-term growth in variable [CO 2 ] is not well understood, particularly in crop 3 1 plants. Here, we grew two cultivars of wheat (Halberd and Cranbrook), known to differ in g m 3 2under current environmental conditions, in four [CO 2 ] treatments: glacial (180 μmol mol -1 ), 3 3 pre-industrial (280 μmol mol -1 ), current ambient (450 μmol mol -1 ) and super-elevated (1000 3 4 μmol mol -1 ) in well-watered and moderate water limitation conditions, to develop an 3 5 evolutionary and future climate perspective on g m control of photosynthesis and water use 3 6 efficiency (WUE). In the two wheat genotypes, g m increased with rising [CO 2 ] from glacial to 3 7 ambient [CO 2 ], but declined at super-elevated [CO 2 ]; however, the specific mechanism of g m 3 8 response to [CO 2 ] remains unclear. Although g m and g m /g sc (mesophyll conductance/stomatal 3 9 conductance) were strongly associated with the variability of A and WUE, we found that 4 0 plants with higher g m may increase A without increasing g sc , which increased WUE. These 4 1 results may be useful to inform plant breeding programs and cultivar selection for Australian 4 2 wheat under future environmental conditions. 4 3 4 4 4 5