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

• Applicant: Professorin Dr. Veronica Maurino
• Subject Area: Plant Biochemistry and Biophysics ; Plant Physiology
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 528182088

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.

• DFG Programme: Research Grants