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

C4光合作用（C4 PS）在被子植物中经历了多次进化，这反映在C4综合征的结构和生化多样性上，特别是在肉质C4谱系中。然而，似乎C4谱系共享家庭基因的遗传工具箱，其中修饰的同种型被重复招募到C4代谢途径中。这些C4酶的有效功能取决于它们的调节微调，因为它们必须在正确的位置和数量表达。随着我们对C4 PS所需的遗传变化的理解的进步，现在研究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组，猪毛菜（藜科）和Sesuvium（Aizoaceae），这两个联合收割机C4 PS和肉质，虽然与年龄的C4谱系，生化亚型，解剖学和生态学的差异，其中一些显示从C3到C4 PS的发展开关。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调控基因的进化。我们将使用激光辅助显微切割（LAM）对叶肉和Kranz细胞特异性RNA进行测序，并比较密切相关的C3、C4和中间目标物种的组织特异性转录组谱。这种方法将使我们能够获得C4关键基因的调控和局部表达的新见解，并揭示共表达的已知候选基因和新的调节基因。我们假设基因调控的变化先于C4基因本身的变化。随着年龄的增长而改变光合作用的物种的发育筛选将极大地有助于识别调控候选基因，因为基因的上调或下调可以直接与一个个体内光合类型的变化相关。在该项目的后期阶段，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的C4特异性成员。我们假设，调控基因也是从一个共同的“遗传工具箱”的C3祖先和肉质和非肉质C4谱系表示在基因水平上的进化趋同反复招募。与此同时，调节和适应性遗传变化的微调将根据遗传结构（例如叶解剖学）和生态（例如沙漠植物与盐生植物）差异而有所不同。因此，我们可以期待有趣的洞察到植物中执行C4途径的基因调控的趋同进化。该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重大影响。此外，我们对C4基因的基因调节子的变化的了解的进展可能是试图生物工程C4作物的重要拼图。

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.