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

C4植物的祖先进化出一种生化泵，在Rubisco处浓缩二氧化碳，导致更高的光合作用效率。细胞活动对C4综合征的进化适应是建立在现有酶的分子适应的基础上的，大多数情况下涉及基因复制和新功能化，伴随着基因产物的调节和生化性质的变化。C4光合作用的核心步骤是通过束鞘细胞(BSC)叶绿体中的C4酸脱羧基产生高浓度的CO2。大多数农学上重要的C4植物，包括玉米、高粱和甘蔗，都属于C4的NADP-苹果酸酶(ME)亚型，主要利用NADP-ME来实现这一目的。在夜间，当光合作用途径不活跃时，C4特异性NADP-ME亚型的酶活性通过两个过程进行调节：一方面，该酶部分失去其活性的四元齐聚状态；另一方面，它被苹果酸抑制。这些调节机制对于BSC叶绿体中有效的CO2浓度是重要的。最近，我们基于氨基酸的严格微分保守，结合玉米和高粱C4-NADP-ME的晶体结构和酶突变体的生化分析，发现了对C4-NADP-ME分子适应重要的特定氨基酸。这些氨基酸替换中的几个很可能进化为实现必要的调节适应。在本项目中，我们旨在阐明C4-NADP-ME两个主要调控特性演变的分子机制，并在体内分析这些调控过程。我们关注的是禾本科Panicoideae亚科中的单一C4系雄配子，包括C4禾草玉米和高粱。我们将结合结晶分析和计算机模拟来分析只存在于C4-NADP-ME亚型中的特定氨基酸如何影响酶的结构和动力学性质。通过对NADP-ME N端突变体的生化分析和小角X射线散射测量，我们将确定还有哪些氨基酸参与了寡聚体状态的变化和稳定。我们将通过对特定酶突变体的生化分析来建立苹果酸抑制的潜在分子机制。此外，我们将通过产生和分析进一步的重组NADP-ME突变体来鉴定参与C4-NADP-ME在雄配子体中适应的其他氨基酸。最后，为了在体内评估这些调控过程，我们将通过CRISPR-Cas9技术将特定的突变引入玉米C4-NADP-ME基因编码中，并对获得的植株系进行表型、生化和生理分析。

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.