How to make a C4 plant: insight from comparative transcriptome analysis.

Rubisco (for ribulose-1,5-bisphosphate carboxylase/oxygenase), the enzyme that catalyzes the first major step in carbon fixation, is notoriously inefficient in this role, owing to its function as an oxygenase as well as a carboxylase. Photorespiration, the pathway followed when Rubisco catalyzes the oxygenation rather than carboxylation of the substrate ribulose-1,5-bisphosphate, can reduce the efficiency of carbon fixation in plants by up to 25% (Sharkey, 1988). C4 photosynthesis effectively increases the intracellular CO2:O2 ratio and inhibits the oxygenation reaction. Most C4 species have a characteristic leaf anatomy in which Rubisco is localized to specialized interior mesophyll cells called bundlesheath cells (reviewed in Wang et al., 2011). CO2 is initially fixed in outermesophyll cells by PEP carboxylase into a four-carbon acid, which is then shuttled into the bundle sheath cells and decarboxylated, releasing and concentrating CO2 in the vicinity of Rubisco (see figure). This protects Rubisco from exposure to O2 and inhibits photorespiration. Since these additional steps require energy, C4 photosynthesis is more efficient than the common C3 pathway only under certain conditions, namely high temperature and water limitation, when stomatal closure limits CO2 uptake and leads to high rates of photorespiration in C3 plants. Interestingly, C4 pathways have evolved multiple times in a broad range of plant families, which has led to a number of biochemical and morphological variations on the main theme. Although the majority of C4 plants are monocots, a number of species in at least 16 different dicot families also use C4 photosynthesis. C4 crop species (all of which are grasses, including maize and sugarcane) tend to be more productive and to use less water and nitrogen than C3 crops, stimulating efforts to engineer C4 photosynthesis into a major C3 crop, such as rice or soybean, to boost productivity (Sage and Zhu, 2011). Seeking further insight into the evolution of C4 photosynthesis in dicots, Gowik et al. (pages 2087–2105) used mRNA-Seq analysis to compare the transcriptomes of five closely related species in the dicot Flaveria that span a range of C3 to C4 phenotypes, including two C3 species, two C4 species, and one C3–C4 intermediate. This work follows a similar study conducted by Bräutigam et al. (2011) in the dicot Cleome. However, Gowik et al. includedmultiple C3–C4 species comparisons within the Flaveria genus to address the question of which changes in gene expression are related to the C4 syndrome rather than to the evolutionary distance of the two species. The results identified and confirmed the well-established distinct C4 enzymes and transporters in Flaveria as well as some new candidates for as yet undefined C4-related functions. The authors estimated an “upper limit” of nearly 3600 C4-related expression changes compared with C3 photosynthesis in mature leaves. In addition to genes associated with the C4 pathway, other changes were identified in processes such as photosynthetic electron transport, the C3 pathway, and nitrogen and amino acid metabolism. Intriguingly, the results point toward the evolution of a photorespiratory CO2– concentrating pump prior to the establishment of the C4 pathway, since the C3–C4 intermediate species showed a distinct increase in transcripts and metabolites related to photorespiration, compared with the true C3 or C4 species. This comprehensive study adds to our knowledge of C4 evolution and provides a database that will guide and enhance future work.