Molecular evolution of starch degradation in the guard cells of CAM plants

CAM光合作用的保水特性已经将该途径确定为合成生物学的靶点。CAM通过将净CO2吸收转移到夜间来保存水，此时叶蒸散率与白天相比降低。CAM提高了水分利用效率（每单位水分损失固定的CO2），比具有其他类型光合作用的植物高出约5至10倍。因此，将CAM生物工程转化为非CAM作物提供了维持植物生产力的潜力，用于食品，饲料，纤维和生物燃料生产，同时减少水投入（Borland等人，2014）。CAM生物工程的基本原理是植根于这样的知识，即由于CAM在整个进化历史中经常出现在C3光合作用中，CAM所需的所有酶都是C3物种中发现的祖先形式的同源物。因此，了解CAM所需的关键基因的分子进化是CAM工程的重要组成部分。 将CAM工程化到C3植物中需要气孔运动的日/夜重新安排，使得气孔（叶孔）在夜间打开并且在白天关闭。Borland实验室最近未发表的工作表明CAM和C3植物之间的保卫细胞代谢存在根本差异，值得进一步研究。具体而言，淀粉分解气孔保卫细胞，增强保卫细胞膨压和加速白天气孔开放C3植物，CAM保卫细胞受到抑制。C3保卫细胞中的淀粉降解需要一种与叶肉不同的酶促途径，最近在拟南芥中鉴定了C3保卫细胞淀粉周转所需的关键酶。我们对CAM保卫细胞的观察提供了两个假设：1）CAM保卫细胞缺乏淀粉降解所需的关键酶; 2）CAM和C3植物保卫细胞淀粉降解受到相反的调控模式。该项目将使用一系列湿和干实验室方法来测试这些假设，包括：a）分析CAM模式植物Kalamë fedtschenkoi的富含保卫细胞的蛋白质组，以及它如何与已发表的拟南芥保卫细胞蛋白质组进行比较，特别关注与淀粉和渗透调节物质周转有关的蛋白质; B）对CAM保卫细胞淀粉代谢中涉及的所选候选基因进行遗传操作，和c）对已发表的C3和CAM基因组进行生物信息学研究，以建立与保卫细胞淀粉降解有关的候选基因的分子遗传学，并测试这些基因在CAM和C3植物中进化不同。

The water conserving properties of CAM photosynthesis have identified the pathway as a target for synthetic biology. CAM conserves water by shifting net CO2 uptake to the night when rates of leaf evapotranspiration are reduced compared to the day. CAM improves water-use efficiency (CO2 fixed per unit water lost) some 5 to 10 fold over that in plants with other types of photosynthesis. Thus, bioengineering CAM into non-CAM crops offers the potential to sustain plant productivity for food, feed, fibre, and biofuel production whilst curtailing water inputs (Borland et al, 2014). The rationale for CAM-bioengineering is rooted in the knowledge that since CAM emerged frequently from C3 photosynthesis throughout evolutionary history, all of the enzymes required for CAM are homologues of ancestral forms found in C3 species. Thus, understanding the molecular evolution of key genes required for CAM is a crucial component for informing CAM engineering. Engineering CAM into C3 plants requires a day/night rescheduling of stomatal movement so that stomata (leaf pores) open at night and close during the day. Recent unpublished work in the Borland lab has suggested a fundamental difference in guard cell metabolism between CAM and C3 plants which merits further study. Specifically, starch breakdown in stomatal guard cells, which enhances guard cell turgor and accelerates day-time stomatal opening in C3 plants, is suppressed in CAM guard cells. Starch degradation in C3 guard cells requires a different enzymatic route to that in the leaf mesophyll and key enzymes required for C3 guard cell starch turnover have recently been identified in Arabidopsis. Our observations for CAM guard cells provide two hypotheses: 1) CAM guard cells lack key enzymes required for starch degradation; 2) Guard cell starch degradation is subject to contrasting modes of regulation in CAM and C3 plants. This project will test these hypotheses using a range of wet and dry lab approaches that include; a) analysis of the guard cell-enriched proteome of the model CAM plant Kalanchoë fedtschenkoi and how it compares to published guard cell proteomes of Arabidopsis, with particular focus on proteins implicated in starch and osmolyte turnover; b) genetic manipulation of selected candidate genes implicated in CAM guard cell starch metabolism and c) bioinformatics interrogation of published C3 and CAM genomes to establish molecular phylogenies of candidate genes implicated in guard cell starch degradation and to test if these genes have evolved differently in CAM and C3 plants.