Evolutionary optimization of enzymes for their operation in the C4 photosynthetic pathway: the case of NADP-malic enzyme

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The ancestors of C4 plants evolved a biochemical pump to concentrate CO2 at the site of Rubisco, leading to greater photosynthetic efficiency. The evolutionary adaptation of cellular activities to the C4 syndrome was based on the molecular adaptation of existing enzymes, in most cases involving gene duplications followed by neo-functionalization, accompanied by changes in the regulation and biochemical properties of the gene products.A central step in C4 photosynthesis is the generation of high CO2 concentrations through the decarboxylation of a C4 acid in the bundle sheath cell (BSC) chloroplasts. Most agronomically important C4 plants, including maize, sorghum, and sugar cane, belong to the NADP-malic enzyme (ME) subtype of C4, which predominantly uses NADP-ME for this purpose. During the night, when the photosynthetic pathway is inactive, the enzymatic activity of the C4-specific isoform of NADP-ME is regulated through two processes: on the one hand, the enzyme partially loses its active quaternary oligomerization state; on the other hand, it is inhibited by malate. These regulatory mechanisms are important for the efficient concentration of CO2 in BSC chloroplasts. We recently identified specific amino acids important for the molecular adaptions of C4-NADP-ME based on strict differential conservation of amino acids, combined with solving the crystal structures of maize and sorghum C4-NADP-ME and the biochemical analysis of enzyme mutants. Several of these amino acid substitutions likely evolved to implement the necessary regulatory adaptions. In this project, we aim to elucidate the molecular mechanisms underlying the evolution of the two major regulatory properties of C4-NADP-ME, and to analyze these regulatory processes in vivo. We focus on the single C4 lineage Andropogoneae within the Panicoideae subfamily of the Poaceae, which includes the C4 grasses maize and sorghum. We will combine crystallization analyses with in silico modelling to analyze how specific amino acids present only in the C4-NADP-ME isoform influence the structural and kinetic properties of the enzyme. Through biochemical analysis and small-angle-X-ray-scattering measurements of N-terminal mutants of NADP-ME, we will determine which other amino acids are involved in the changes and stabilization of the oligomeric states. We will establish the underlying molecular mechanism of malate inhibition through the biochemical analysis of specific enzyme mutants. Furthermore, we will identify other amino acids involved in the adaptation of C4-NADP-ME in the Andropogoneae by producing and analyzing further recombinant NADP-ME mutants. Finally, to evaluate these regulatory processes in vivo, we will introduce specific mutations into the gene coding for C4-NADP-ME in maize via the CRISPR-Cas9 technology and will perform phenotypic, biochemical, and physiological analyses of the obtained plant lines.