The Evolution of C4 Photosynthesis in Flaveria (Asteraceae): Insights from the Flaveria linearis Complex

Abstract

Flaveria is a leading model for C<inf>4</inf> plant evolution due to the presence of a dozen C<inf>3</inf>-C<inf>4</inf> intermediate species, many of which are associated with a phylogenetic complex centered around Flaveria linearis. To investigate C<inf>4</inf> evolution in Flaveria, we updated the Flaveria phylogeny and evaluated gas exchange, starch d¹³C, and activity of C<inf>4</inf> cycle enzymes in 19 Flaveria species and 28 populations within the F. linearis complex. A principal component analysis identified six functional clusters: (1) C<inf>3</inf>, (2) sub-C<inf>2</inf>, (3) full C<inf>2</inf>, (4) enriched C<inf>2</inf>, (5) sub-C<inf>4</inf>, and (6) fully C<inf>4</inf> species. The sub-C<inf>2</inf> species lacked a functional C<inf>4</inf> cycle, while a gradient was present in the C<inf>2</inf> clusters from little to modest C<inf>4</inf> cycle activity as indicated by d¹³C and enzyme activities. Three Yucatan populations of F. linearis had photosynthetic CO<inf>2</inf> compensation points equivalent to C<inf>4</inf> plants but showed little evidence for an enhanced C<inf>4</inf> cycle, indicating they have an optimized C<inf>2</inf> pathway that recaptures all photorespired CO<inf>2</inf> in the bundle sheath (BS) tissue. All C<inf>2</inf> species had enhanced aspartate aminotransferase activity relative to C<inf>3</inf> species and most had enhanced alanine aminotransferase activity. These aminotransferases form aspartate and alanine from glutamate and in doing so could help return photorespiratory nitrogen (N) from BS to mesophyll cells, preventing glutamate feedback onto photorespiratory N assimilation. Their use requires upregulation of parts of the C<inf>4</inf> metabolic cycle to generate carbon skeletons to sustain N return to the mesophyll, and thus could facilitate the evolution of the full C<inf>4</inf> photosynthetic pathway.