Engineering more water-use efficient crops: functional genomics of inverse stomatal control and high water use efficiency associated with Crassulacean

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Crassulacean acid metabolism (CAM) is an adaptation of photosynthesis found in a diverse range of plant species that inhabit arid and semi-arid environments. CAM plants can achieve water use efficiencies up to ten-times greater than C3 species. Our over-arching goal is to develop a systems-level view of CAM and the associated inverse stomatal control by achieving a detailed understanding of the genes, proteins and metabolites involved in these valuable adaptations. Comprehensive knowledge of the CAM and stomatal control 'parts-list' will permit forward engineering of CAM into C3 crops. In particular, we are collaborating with US scientists on a major plant synthetic biology project that aims to introduce CAM into poplar trees (http://cambiodesign.org). We have performed whole genome and transcriptome sequencing (RNA-seq) for our model CAM systems, Kalanchoë fedtschenkoi and K. laxiflora, and the CAM-performing, biomass feedstock crop, Agave sisalana. Our gene discovery work is yielding candidate genes for engineering CAM into C3 crops, but we first need to understand which are the most critical genes for CAM and stomatal control within a CAM species, before forward engineering the minimal set of CAM and stomatal control genes into a C3 species. In particular, in the last six months we have succeeded in obtaining a high quality RNA-seq dataset comparing gene regulation over the light dark cycle for both leaf epidermal peels (enriched for stomatal guard cells) and leaf mesophyll cells (where CAM photosynthesis proceeds). We are now ready to use this dataset to discover the genes that control inverse stomatal opening for CAM (stomata open in the dark and close in the light during CAM).This project will focus on the production and characterisation of transgenic Kalanchoë lines in which genes proposed to be essential for the inverse stomatal opening associated with CAM will be manipulated in a guard cell-specific manner either through gene silencing or over-expression approaches. Detailed phenotypic analysis of these transgenic lines in terms of their stomatal control, photosynthetic physiology, biochemistry and molecular biology will be undertaken thereby defining which genes are most critical for inverse stomatal opening for CAM. In the long term, this work will make a substantial contribution to the development of more drought tolerant, water use efficient bioenergy crops and new biofuel feedstock crops suitable for desert cultivation.