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，这是一种高能耗的过程，导致释放先前固定的CO2。C4性状在生态学和农学上是重要的，因为C4植物在温暖环境中具有上级生长潜力，并且C4物种包括最高产的谷类作物。C4光合作用集中在相对较少的显花植物中，在那里它独立起源超过65次，提供了一个复杂功能性状趋同进化的例子。然而，C4光合作用可以以不同的方式进行，这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途径通过非C4祖先中存在的多个基因的共同选择而进化。在羧化步骤中，C4光合作用基因的协同选择和适应的收敛是明显的。相反，在脱羧步骤中存在分歧。在65个C4-谱系中，约43个含有使用NADP-苹果酸酶作为初级脱羧酶的物种，而NAD-苹果酸酶（NAD-ME）在20个谱系中使用，其中16个是真双子叶植物。除了NAD-ME的可及性可能较低外，C4-NAD-ME是由不同物种从不同基因谱系获得的。我们最近的工作表明，NAD-ME的C4光合途径的协同选择机制不同于所有其他C4蛋白，最有可能是因为NAD-ME主要作为异二聚体发挥作用。我们的工作还表明，NAD-ME亚基编码基因的互补选择存在可塑性。在真双子叶植物中，NAD-ME独立地被用于Carlellales和Apricales物种的C4光合作用。C4-NAD-ME基因在两个不同C4谱系的草蛉目物种中表现出相似的分子适应性：（a）NAD-ME基因α、β1和β2均受C4进化的影响;（B）β1-NAD-ME被C4代谢所选择。与此相反，在一种C4的卡氏藻目物种中，α-NAD-ME（α1）基因的额外拷贝可能是由于其在C4途径中的功能而被采用的。因此，C4-NAD-ME亚型物种的生化趋同可以通过α和β亚基的交替关联来得出。这些变化是功能性C4光合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。在这个项目中，使用互补的生化和结构分析，我们的目标是澄清是否以及在何种程度上选择不同的分子适应趋同进化产生了类似的功能，在C4-NAD-ME在不同的真双子叶植物家族。此外，我们的目标是使用A. thaliana NAD-ME来探索C3至C4生物工程方法中的代谢后果和可能的实施。

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.