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

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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.