Mesophyll-specific overexpressing ofSvHXK6gene improves water use efficiency without yield loss in C₄Setaria viridis

Abstract Hexokinases (HXK) were the first sugar signalling proteins identified in plants and are well known for their feedback regulation of photosynthetic gene expression. In some C 3 plants, HXKs have been found to regulate stomatal function. However, the role of HXK in C 4 photosynthesis, which is inherently more water use efficient than C 3 metabolism, remains poorly understood. Here, we report on the first tissue-specific modification of HXK in a C 4 plant. SvHXK6 was expressed in the model C 4 grass Setaria viridis under the control of the ZmPEPC promoter ( ZmPEPC pro ), which directs expression in the leaf mesophyll tissue. Three S. viridis transgenic lines with increased abundance of SvHXK6 transcripts in the leaf tissue showed significant reduction in stomatal conductance with minimal effects on leaf CO 2 assimilation rate. Consequently, the transgenic lines had higher leaf-level water use efficiency relative to the control (wild-type and null) plants. Overexpression of SvHXK6 had no effect on shoot biomass or seed yield of the S. viridis plants. Our study shows conserved function of HXK in regulating stomatal conductance in a C 4 grass, demonstrating possible widespread utility in improving water use efficiency in C 4 as well as C 3 species.