Evolutionary Assembly of C4 Leaf Structure

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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.