Mesophyll-specific overexpressing of SvHXK6 gene improves water use efficiency without yield loss in C4 Setaria viridis

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 C3 plants, HXKs have been found to regulate stomatal function. However, the role of HXK in C4 photosynthesis, which is inherently more water use efficient than C3 metabolism, remains poorly understood. Here, we report on the first tissue-specific modification of HXK in a C4 plant. SvHXK6 was expressed in the model C4 grass Setaria viridis under the control of the ZmPEPC promoter (ZmPEPCpro), 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 CO2 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 C4 grass, demonstrating possible widespread utility in improving water use efficiency in C4 as well as C3 species.