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

景天酸代谢(CAM)是一种光合作用的适应性，存在于生活在干旱和半干旱环境中的各种植物物种中。CaM植物可以实现比C3植物高出十倍的水分利用效率。我们的总体目标是通过实现对参与这些有价值的适应的基因、蛋白质和代谢物的详细了解，从系统水平上了解CAM和相关的反向气孔控制。对CAM和气孔控制‘零件清单’的全面了解将允许将CAM向前工程转化为C3作物。特别是，我们正在与美国科学家合作一个重大的植物合成生物学项目，旨在将CAM引入杨树(http://cambiodesign.org).我们已经对我们的模型CAM系统Kalancoëfedtschenkoi和K.laxiflora以及执行CAM的生物质饲料作物龙舌兰进行了全基因组和转录组测序(RNA-seq)。我们的基因发现工作正在产生将CAM工程改造成C3作物的候选基因，但在将最小的一组CAM和气孔控制基因正向工程到C3作物之前，我们首先需要了解在CAM物种中哪些基因对CAM和气孔控制最关键。特别是，在过去的六个月里，我们成功地获得了一个高质量的RNA-SEQ数据集，比较了叶片表皮(丰富的气孔保卫细胞)和叶片叶肉细胞(CAM光合作用进行的地方)在光暗周期中的基因调控。我们现在准备利用这个数据集来发现控制CAM(CAM期间气孔在黑暗中打开，在光中关闭)的基因。本项目将集中在转基因Kalancoëline的产生和特征上，其中与CAM反向气孔打开相关的基因将通过基因沉默或过度表达的方法以保卫细胞特有的方式进行操作。将从气孔控制、光合作用生理、生物化学和分子生物学等方面对这些转基因品系进行详细的表型分析，从而确定哪些基因对CAM的反向气孔开放最关键。从长远来看，这项工作将为开发更耐旱、水分利用效率更高的生物能源作物和适合沙漠种植的新型生物燃料饲料作物做出实质性贡献。

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.