Kranz and single-cell forms of C(4) plants in the subfamily Suaedoideae show kinetic C(4) convergence for PEPC and Rubisco with divergent amino acid substitutions

The two carboxylation reactions performed by phosphoenolpyruvate carboxylase (PEPC) and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) are vital in the fixation of inorganic carbon for C(4) plants. The abundance of PEPC is substantially elevated in C(4) leaves, while the location of Rubisco is restricted to one of two chloroplast types. These differences compared with C(3) leaves have been shown to result in convergent enzyme optimization in some C(4) species. Investigation into the kinetic properties of PEPC and Rubisco from Kranz C(4), single cell C(4), and C(3) species in Chenopodiaceae s. s. subfamily Suaedoideae showed that these major carboxylases in C(4) Suaedoideae species lack the same mutations found in other C(4) systems which have been examined; but still have similar convergent kinetic properties. Positive selection analysis on the N-terminus of PEPC identified residues 364 and 368 to be under positive selection with a posterior probability >0.99 using Bayes empirical Bayes. Compared with previous analyses on other C(4) species, PEPC from C(4) Suaedoideae species have different convergent amino acids that result in a higher K (m) for PEP and malate tolerance compared with C(3) species. Kinetic analysis of Rubisco showed that C(4) species have a higher catalytic efficiency of Rubisco (k (catc) in mol CO(2) mol(–1) Rubisco active sites s(–1)), despite lacking convergent substitutions in the rbcL gene. The importance of kinetic changes to the two-carboxylation reactions in C(4) leaves related to amino acid selection is discussed.