Evolutionary Assembly of C4 Leaf Structure

二磷酸核酮糖羧化酶加氧酶（Rubisco）是含氧光合生物中主要的自养羧化酶。Rubisco的加氧酶活性导致细胞毒性磷酸乙醇酸的产生，该磷酸乙醇酸被光呼吸去除。光呼吸释放先前固定的二氧化碳，导致生长所需碳的净损失。C4光合作用将二氧化碳集中在Rubisco周围，而O2则消除了Rubisco加氧酶的活性。C4碳浓缩机制的一个重要特征是中叶基细胞中磷酸烯醇丙酮酸羧化酶对CO2的初始固定与维管组织周围束鞘（BS）细胞中Rubisco对CO2的二次再固定的空间分离。初始固定产生一种c4 -酸，这种酸被运送到BS细胞，在那里它被脱羧，使Rubisco周围的二氧化碳增加15倍。本研究的目的是验证我们的假设，即C4光合叶片结构从C3光合祖先进化源于参与组织模式、细胞和质体分裂/扩张、间连丝和VT形成以及生长素生物合成、信号传导和运输的基因的顺式和反式调控差异。我们将确定产生C4光合叶片的细胞/组织结构的时空系列事件，这对光合功能至关重要。我们将利用本实验室已经获得的RNA-seq/基因组数据，对prostrata （C3）、A. rosea （C4）及其C3xC4 F1杂交体叶片发育进行比较研究。我们将确认M和BS细胞中细胞分裂/扩增以及叶绿体/胞间连丝发育的三维模式。这些特征将通过共聚焦显微镜、连续块面扫描电镜、薄片高分辨率透射电镜以及透射电镜对蛋白质的免疫检测和活性氧H2O2的定位来获得。免疫组织化学将使用抗体检测生长素和与组织模式、细胞和质体分裂/扩增以及间连丝形成有关的蛋白质。C4光合作用已经独立进化了60多次，使其成为生物世界中进化趋同的最好例子之一。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理解还处于起步阶段。我们的研究结果将有助于鉴定C4叶片结构/功能的关键基因和这些基因的调控。为了维持全球粮食安全，到2050年，谷物产量必须增加70%。我们的研究将提供信息，帮助通过将C4途径改造成C3作物来维护全球粮食安全，就像目前在水稻中所做的那样。

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.