Evolutionary Assembly of C4 Leaf Structure

核酮糖二磷酸羧化酶-加氧酶(Rubisco)是产氧光合作用生物中主要的自养羧基酶。Rubisco的加氧酶活性导致产生细胞毒性的磷酸乙酸酯，通过光呼吸将其去除。光呼吸作用释放先前固定的二氧化碳，导致生长所需的碳的净损失。相对于O2，C4光合作用使CO2集中在Rubisco周围，消除了Rubisco加氧酶的活性。C4碳浓缩机制的一个基本特征是中叶细胞中磷酸烯醇式丙酮酸羧基酶对CO2的初始固定与维管组织周围的束鞘(BS)细胞对CO2的二次再固定是空间分离的。最初的固定会产生一种C4酸，该酸被输送到BS细胞，在那里它被脱羧化，使二氧化碳增加约15倍。本研究的目的是验证我们的假设，即C3光合作用祖先的C4光合叶结构的进化源于组织模式、细胞和质体分裂/扩张、胞间连丝和VT形成以及生长素的生物合成、信号传递和运输相关基因的顺式和反式调节差异。我们将确定导致C4光合作用叶片细胞/组织结构的时空系列事件，这是光合作用功能所必需的。对滨藜(C3)、玫瑰滨藜(C4)及其C3xC4F1杂种的叶片发育进行了比较研究，并根据本实验室已获得的RNA-seq/基因组数据进行了分析。我们将确认细胞分裂/扩张的三维模式，以及M和BS细胞中叶绿体/胞间连丝的发育。这些特征将通过共聚焦显微镜、连续块面扫描电子显微镜、高分辨率薄片透射电子显微镜和蛋白质的免疫检测以及利用透射电子显微镜对活性氧物种H_2O_2进行定位来获得。*将使用抗体进行免疫组织化学，以检测生长素和参与组织构型、细胞和质体分裂/扩张以及胞间连丝形成的蛋白质。C4光合作用已经独立进化了60多次，使其成为生物界进化趋同的最佳例子之一。C4光合作用占陆地总初级生产力的23%，尽管它只发生在3%的植物物种中。尽管C4光合作用很重要，但我们对C4进化的细胞和分子控制的了解还处于初级阶段。我们的研究结果将有助于识别对C4叶片结构/功能至关重要的关键基因，并对这些基因进行调控。为了维护全球粮食安全，到2050年，谷物产量必须增加70%。我们的研究将提供信息，通过将C4途径转化为C3作物来帮助维持全球粮食安全，就像现在在水稻上所做的那样。

Ribulose bisphosphate carboxylase-oxygenase (Rubisco), is the primary autotrophic carboxylase in oxygenic photosynthetic organisms. Oxygenase activity of Rubisco results in production of cytotoxic phosphoglycolate that is removed by photorespiration. Photorespiration releases previously fixed CO2 resulting in a net loss of carbon for growth. C4 photosynthesis concentrates CO2 around Rubisco relative to O2 eliminating Rubisco oxygenase activity. An essential feature of the C4 carbon concentrating mechanism is the spatial separation of the initial fixation of CO2 by phosphoenolpyruvate carboxylase in mesopyhyll cells from secondary refixation of CO2 by Rubisco in bundle sheath (BS) cells surrounding vascular tissue. Initial fixation produces a C4-acid that is transported to BS cells where it is decarboxylated enhancing CO2 around Rubisco 15-fold. The purpose of this research is to test our hypothesis that evolution of C4 photosynthetic leaf structure from C3 photosynthetic ancestors arises from cis- and trans-regulatory divergence of genes involved in tissue patterning, cell and plastid division/expansion, plasmodesmata and VT formation, and auxin biosynthesis, signalling and transport. We will determine the spatial-temporal series of events giving rise to the cellular/tissue architecture of a C4 photosynthetic leaf that is essential for photosynthetic function. Comparative studies on leaf development in Atriplex prostrata (C3), A. rosea (C4) and their C3xC4 F1 hybrid will be conducted and analyzed in the context of RNA-seq/genomic data already obtained by our laboratory. We will confirm 3-D patterns of cell division/expansion, as well as chloroplast/plasmodesmata development in M and BS cells. These features will be obtained using confocal microscopy, serial block-face scanning electron microscopy, high resolution transmission electron microscopy of thin sections and immunodetection of proteins and localization of the reactive oxygen species H2O2 with transmission electron microscopy.  Immunohistochemistry will be performed using antibodies to detect auxin and proteins involved in tissue patterning, cell and plastid division/expansion, and plasmodesmata formation. C4 photosynthesis has evolved independently over 60 times making it one of the best examples of evolutionary convergence in the living world. C4 photosynthesis accounts for 23% of terrestrial gross primary productivity although it only occurs in 3% of plant species. In spite of the importance of C4 photosynthesis, our understanding of the cellular and molecular control of C4 evolution is in its infancy. Results from our study will enable identification of key genes essential for C4 leaf structure/function and regulation of those genes. To maintain global food security, cereal production will have to increase 70% by 2050. Our study will provide information to assist in maintaining global food security by engineering the C4 pathway into C3 crops, as is now being done in rice.