Evolution of C4 gene regulation in succulent and salt-tolerant C4 lineages

在被子植物中，C4光合作用（C4 PS）发生了多次进化，这反映在C4综合征的结构和生化多样性上，特别是在多肉C4世系中。然而，似乎C4谱系共享一个家庭基因的遗传工具箱，其中修饰的同种异构体被反复招募到C4代谢途径中。这些C4酶的有效功能依赖于它们的调控微调，因为它们必须在正确的位置和数量上表达。随着近年来我们对C4 PS所需的遗传变化的理解的进步，研究C4途径起源过程中基因调控的进化变得可行。我们确定了两个有前途的研究群体，Salsola （Chenopodiaceae）和Sesuvium (Aizoaceae)，它们都结合了C4 PS和多肉质，尽管在C4谱系的年龄、生化亚型、解剖和生态方面存在差异。这两个谱系在本项目的第一阶段进行了深入研究，非常适合研究C4 PS的调控基因的进化。我们将使用激光辅助显微解剖（LAM）对叶肉和克兰兹细胞特异性RNA进行测序，并比较密切相关的C3， C4和中间靶种的组织特异性转录组图谱。这种方法将使我们对C4关键基因的调控和定位表达有新的认识，并揭示已知的共表达候选基因和新的调控基因。我们假设基因调控的变化先于C4基因本身的变化。随着年龄的增长，光合作用发生变化的物种的发育筛选将极大地有助于确定调控候选基因，因为基因的上调或下调可直接与个体光合作用类型的变化相关。在项目的后期阶段，我们还将在核苷酸和氨基酸水平上表征已确认的调控基因的基因家族，并揭示这些基因家族中c4特异性成员。我们还假设，调节基因也来自C3祖先的共同“遗传工具箱”，并被多肉和非多肉C4谱系反复招募，表明基因水平上的进化趋同。同时，调控的微调和适应性遗传变化将根据遗传结构（如叶片解剖）和生态（如沙漠植物与盐生植物）的差异而有所不同。因此，我们可以期待对执行C4途径的植物基因调控趋同进化的有趣见解。这个项目的结果将对目前的C4进化模型以及一般复杂性状的进化产生强烈的影响。此外，我们对C4基因调控基因变化的了解的进展可能是C4作物生物工程尝试的重要拼图。

C4 photosynthesis (C4 PS) evolved multiple times in angiosperms which is reflected in the structural and biochemical diversity of the C4 syndrome especially in succulent C4 lineages. However, it seems that C4 lineages share a genetic toolbox of household genes of which modified isoforms are repeatedly recruited into the C4 metabolic pathway. The efficient functioning of these C4 enzymes is dependent on their regulatory fine-tuning as they have to be expressed in the right place and amount. With the advances in our understanding of the genetic changes required for C4 PS in the recent past it becomes now feasible to study the evolution of gene regulation during the origin of the C4 pathway. We identified two promising study groups, Salsola (Chenopodiaceae) and Sesuvium (Aizoaceae), which both combine C4 PS and succulence, albeit with differences in age of the C4 lineages, biochemical subtype, anatomy and ecology, and some of which showing a developmental switch from C3 to C4 PS. These two lineages were intensively studied during the first phase of this project and are highly suitable to study the evolution of regulatory genes of the C4 PS. We will use laser-assisted microdissection (LAM) to sequence mesophyll and kranz cell specific RNA and compare tissue specific transcriptome profiles in closely related C3, C4 and intermediate target species.This approach will allow us to gain novel insights in the regulation and localized expression of C4 key genes and reveal co-expressed – known candidate and new - regulatory genes. We hypothesize that gene regulatory changes preceded changes in the C4 genes themselves. Developmental screens of species which change their photosynthesis while they age will greatly help to identify regulatory candidate genes as up- or down-regulation of genes can be directly correlated to the change of photosynthetic type within one individual. In a later stage of the project we will also characterize the gene families of confirmed regulatory genes at nucleotide and amino acid level and reveal C4-specific members in these gene families. We hypothesize that also the regulatory genes are from a common “genetic toolbox” of the C3 ancestors and repeatedly recruited by succulent and non-succulent C4 lineages denoting evolutionary convergence at gene level. At the same time the fine-tuning of the regulation and adaptive genetic changes will differ according to inherited structural (e.g. leaf anatomy) and ecological (e.g. desert plant versus halophyte) differences. Therefore, we can expect interesting insight into the convergent evolution of gene regulation in plants performing the C4 pathway. The results of this project will have strong implications for the current model of C4 evolution as well as for the evolution of complex traits in general. Furthermore, advances in our knowledge of the changes in gene regulators of C4 genes might be an important puzzle piece in the attempt to bioengineer C4 crops.