Divergent solutions to convergent evolution: elucidating the molecular strategies for co-option of the NAD-malic enzyme for C4 biochemistry

在过去的3000万年里，光合作用的C4途径已经发展成为一种碳浓缩机制，以减少光呼吸，这是一个高能量消耗的过程，导致释放先前固定的二氧化碳。C4性状具有重要的生态和农艺学意义，因为C4植物在温暖环境中具有优越的生长潜力，C4物种是产量最高的谷类作物。C4光合作用集中在相对较少的开花植物目中，其独立起源超过65次，为复杂功能性状的趋同进化提供了一个例子。然而，C4光合作用可以以不同的方式进行，不仅表现在脱羧类型的变化上，而且表现在叶片解剖和细胞超微结构上。C4途径是通过在非C4祖先中存在的多个基因的共同选择而进化的。在羧化过程中，C4光合作用的基因选择和适应的趋同是很明显的。相反，在脱羧过程中存在分歧。在65个c4 -谱系中，有43个物种使用nadp -苹果酸酶作为初级脱羧酶，而在20个谱系中使用nadp -苹果酸酶（nadp - me），其中16个是edicots。除了可能较低的nade - me可获得性外，c4 - nade - me是由不同的物种从不同的基因谱系获得的。我们最近的工作表明，nadd - me对C4光合途径的共选择机制不同于所有其他C4蛋白，很可能是因为nadd - me主要作为异源二聚体起作用。我们的工作还表明，编码NAD-ME亚基的基因的共选择存在可塑性。在菊科植物中，颖花科和芸苔科植物的C4光合作用均独立采用nadd - me。不同C4世系的2种芸苔属植物C4- nade - me表现出相似的分子适应：(a) nade - me基因α、β1和β2均受到C4进化的影响，(b) β1- nade - me被C4代谢所吸收。相反，在石楠属植物的C4种中，α-NAD-ME （α1）基因可能因其在C4途径中的功能而被多拷贝。因此，C4-NAD-ME亚型物种的生化趋同可以通过α和β亚基的替代关联来绘制。这些变化是功能性C4光合作用途径中组分可塑性的另一个例子，支持在C3宿主中安装C4机器的可能性。在本项目中，我们利用互补的生化和结构分析，旨在阐明趋同进化对不同分子适应的选择是否以及在多大程度上产生了不同埃迪科c4 - nadd - me的类似特征。此外，我们的目标是利用拟南芥nadd - me突变系将C4- nadd - me生物工程进入BSC线粒体，以探索代谢后果和C3到C4生物工程方法的可能实现。

Over the past 30 million years, the C4 pathway of photosynthesis has evolved as a carbon concentrating mechanism to reduce photorespiration, a highly energy consuming process that leads to the release of previously fixed CO2. The C4 trait is ecologically and agronomically important as C4 plants have a superior growth potential in warm environments and C4 species comprise the most productive cereal crops. C4 photosynthesis is concentrated in relatively few orders of flowering plants, where it has originated over 65 times independently, providing an example of the convergent evolution of a complex functional trait. Nevertheless, C4 photosynthesis can be carried out in different ways, as shown not only in variations in decarboxylation types but also in leaf anatomy and cellular ultrastructure. The C4 pathway has evolved via the co-option of multiple genes present in non-C4 ancestors. Convergence in the co-option and adaptation of genes for C4 photosynthesis is apparent in the carboxylation step. In contrast, divergence exists in the decarboxylation step. About 43 of the 65 C4-lineages contain species using the NADP-malic enzyme as primary decarboxylase, while NAD-malic enzyme (NAD-ME) is used in 20 lineages, 16 of which are eudicots. Apart from likely lower accessibility for co-option of NAD-ME, C4-NAD-ME was acquired from different gene lineages by separated species. Our recent work shows that the mechanism of co-option of NAD-ME for the C4 photosynthetic pathway differs from that of all other C4 proteins, most likely because NAD-ME functions mainly as a heterodimer. Our work also shows that there exists plasticity in the co-option of genes encoding NAD-ME subunits. In the Eudicots, NAD-ME was independently adopted for C4 photosynthesis in Caryophyllales and Brassicales species. C4-NAD-ME of two Brassicales species from different C4 lineages, presented similar molecular adaptations: (a) all NAD-ME genes α, β1 and β2 were affected by C4 evolution and (b) the β1-NAD-ME was co-opted for C4 metabolism. On the contrary, in a C4 specie of the Caryophyllales, an extra copy of the α-NAD-ME (α1) gene likely was adopted for its function in the C4 pathway. Therefore, biochemical convergence in C4-NAD-ME subtype species could be drawn through an alternative association of α and β subunits. These variations are another example of the plasticity of the components in a functional C4 photosynthetic pathway, supporting the possibility of installing the C4 machinery in a C3 host. In this project, using complementary biochemical and structural analysis we aim to clarify whether and to what extent the selection of different molecular adaptations by convergent evolution has produced analogous features in C4-NAD-ME in different eudicot families. Furthermore, we aim to bioengineer a C4-NAD-ME into the BSC mitochondria using mutant lines of A. thaliana NAD-ME to explore metabolic consequences and the possible implementation in C3 to C4 bioengineering approaches.