Developmental and Expression Evolution in C3 and C4 Atriplex and their Hybrids

Maintaining global food security amidst climate change requires improved photosynthetic efficiency in C3 crops. Above 30°C, photosynthesis and yield are reduced 30-50% by photorespiration. The C4 photosynthetic pathway incorporates biochemical and structural features to minimize photorespiration by concentrating CO2 around Rubisco. Engineering C4 into C3 crops requires an understanding of the molecular and genetic control of C4 photosynthesis. I combined transcriptomic and developmental analyses to investigate the molecular control of leaf development in C3 Atriplex prostrata compared to A. rosea, a C4 species with Kranz anatomy. Plants with Kranz-type C4 photosynthesis have two photosynthetic cell types, mesophyll and bundle sheath. Mesophyll cells contain phosphoenolpyruvate carboxylase, the primary carbon fixation enzyme, whereas bundle sheath contains Rubisco and houses photosynthetic carbon reduction. I used C3 x C4 Atriplex F1 hybrids and allele-specific expression analysis to investigate the role of cis- and trans-regulatory effects on gene expression divergence. The majority (~80%) of expression divergence between C3 and C4 species arose from cis-only regulatory divergence, suggesting that numerous independent expression changes across the genome drove C4 photosynthesis evolution. For genes in the C4 pathway, expression divergence arose from cis-acting changes to individual genes, with little contribution from trans-changes. Genes of interest including regulators of plasmodesmata and chloroplast function did not have distinct patterns of regulatory divergence. Inheritance pattern analysis showed that misexpression was common in F1 hybrids and was supported by phenotypic evidence. I next performed comparative studies of C3 and C4 leaf development. I detected significant species*time interaction effects for a large proportion of genes, implying that evolutionary shifts in expression timing were widespread between these species. Cis-regulation was also implicated in many of these evolutionary shifts in expression timing. C4 Atriplex showed accelerated vascular tissue initiation and formation, supported by earlier expression decline of vascular tissue development and core cell cycle genes. Plasmodesmata-associated genes were differentially expressed between during secondary plasmodesmata formation. Mature leaves of F2 hybrids exhibited variation in biochemistry, leaf tissue patterning, and bundle sheath ultrastructure and would be suitable for future large-scale transcriptomic studies. Combined, these studies facilitated identification of the underlying genetics of C4 evolution and development.Ph.D