Getting the most out of natural variation in [C.sub.4] photosynthesis

[C.sub.4] photosynthesis is a complex trait that has a high degree of natural variation, involving anatomical and biochemical changes relative to the ancestral [C.sub.3] state. It has evolved at least 66 times across a variety of lineages and the evolutionary route from [C.sub.3] to [C.sub.4] is likely conserved but not necessarily genetically identical. As such, a variety of [C.sub.4] species are needed to identify what is fundamental to the [C.sub.4] evolutionary process in a global context. In order to identify the genetic components of [C.sub.4] form and function, a number of species are used as genetic models. These include Zea mays (maize), Sorghum bicolor (sorghum), Setaria viridis (Setaria), Flaveria bidentis, and Cleome gynandra. Each of these species has different benefits and challenges associated with its use as a model organism. Here, we propose that RNA profiling of a large sampling of [C.sub.4], [C.sub.3]-[C.sub.4], and [C.sub.3] species, from as many lineages as possible, will allow identification of candidate genes necessary and sufficient to confer [C.sub.4] anatomy and/or biochemistry. Furthermore, [C.sub.4] model species will play a critical role in the functional characterization of these candidate genes and identification of their regulatory elements, by providing a platform for transformation and through the use of gene expression profiles in mesophyll and bundle sheath cells and along the leaf developmental gradient. Efforts should be made to sequence the genomes of F. bidentis and C. gynandra and to develop congeneric [C.sub.3] species as genetic models for comparative studies. In combination, such resources would facilitate discovery of common and unique [C.sub.4] regulatory mechanisms across genera.