Parallelism and diversity in multiple origins of c₄ photosynthesis in the grass family

C 4 photosynthesis is thought to be an adaptation to warm environments, involving complex changes in the expression of genes governing photosynthesis, intermediary metabolism, and leaf anatomy and histology. Such complexity should be difficult to evolve, yet the pathway has arisen multiple times in the history of the flowering plants and at least four times in the grass family alone. We have used immunolocalization techniques to compare photosynthetic gene expression across all four origins, to determine which genetic changes occur in parallel and which are unique to a particular lineage. The only gene expression patterns common to all origins of the pathway are up‐regulation of PEP carboxylase and down‐regulation of RuBisCO in mesophyll cells. Both NAD‐malic enzyme and NADP‐malic enzyme are expressed in bundle sheaths. Expression patterns of light‐harvesting chlorophyll a/b binding proteins and pyruvate orthophosphate dikinase appear to be lineage specific, and may be localized to bundle sheaths or to mesophyll or expressed throughout the photosynthetic tissue of the leaf. We suggest that future studies of parallel origin of the C 4 pathway concentrate on regulation of the two carboxylases, as well as the increased density of vascular tissue, which is the only histological characteristic common to all origins of the pathway.