C4 decarboxylases: different evolutionary innovations to feed RuBisCO

TRONCONI, M. A.; GERRARD WHEELER, M. C.; SAIGO, M.; ALVAREZ, C. E.; MAURINO, V. G.; DRINCOVICH, M. F.

Evento virtual

Workshop; Primer Workshop de la Red Argentina de Tecnología Enzimática; 2021

Red Argentina de Tecnología Enzimática

Many of the world?s most productive crops are species bearing the C4 photosynthetic metabolism, as an adaptation to high light intensities, temperatures, and dryness. C4 plants have evolved biochemical pumps to concentrate CO2 at the site of RuBisCO, which decrease the oxygenation reaction and, thus, limit the wasteful flux through photorespiration. Compared with plants using the ancestral C3 photosynthetic pathway, C4 plants show higher water and nitrogen use efficiency, which allows increased productivity in stressful habitats. The transition from C3 to C4 photosynthesis involved complex alterations of leaf anatomy and biochemistry, and occurred independently in at least 66 lineages of angiosperms being an example of convergent evolution. In C4 plants, the release of CO2 by decarboxylation of a C4 acid near RuBisCO is mainly catalyzed by the plastidic NADP-malic enzyme (ME) or the mitochondrial NAD-ME. MEs catalyze the oxidative decarboxylation of L-malate, producing pyruvate, CO2, and NADPH (or NADH) in the presence of Mg+2 (or Mn+2). The C4-MEs did not evolve de novo. Instead, they were recruited from existing housekeeping or ?nonC4?isoforms. Whilst C4-NADP-ME originated by gene duplication of a non-photosynthetic plastidic isoform and the subsequent neo-functionalization, the C4-NAD-ME followed an intricated molecular mechanism of evolution. C4-NADP-ME acquired increased catalytic efficiency, higher homo-oligomeric assembly and pH-dependent inhibition by its substrate malate. Site-directed mutagenesis and structural analyses allowed us to identify differentially substituted amino acids responsible for theses biochemical innovations. On the other hand, despite its central importance in the C4 pathway, C4-NAD-ME has not been characterized at molecular level.Higher plants possess two paralogs α- and β-NAD-ME isoforms. Both gene families were retained across angiosperms likely by a degenerative process of sub-functionalization, which resulted in a NAD-ME operating primarily as a heteromer of α- and β-subunits. Most genomes maintain a 1:1 β/α relative gene dosage, but a significantly high proportion of species with the C4-NAD-ME photosynthesis have a non-1:1 ratio. Specifically, the C4 Cleome species, Gynandropsis gynandra and Cleome angustifolia, have a 2:1 ratio due to a β-NAD-ME duplication (β1 and β2) although this was also observed in the C3-related Tarenaya hassleriana. All three genes (α, β1 and β2) were modified by C4 evolution and are expressed at higher levels than their orthologs in the C3 Cleome species. In addition, the β-NAD-MEs possess five amino acids that are identically substituted in β-NAD-ME of C4 Cleome species, two of which are positively selected.Using a combination of biochemical and proteomic analyses, we identified different NAD-ME native isoforms in C3 and C4 Cleome organs, which arise through a differential combination of α, β1 and β2 subunits. Non-photosynthetic and photosynthetic organs of T. hassleriana posses two NAD-ME isoforms, ThNAD-MEα/β1 and ThNAD-MEα/β2, with kinetic properties similar to those of A. thaliana NAD-MEs. Thus, ThNADMEα/β1 and ThNAD-MEα/β2 may represent housekeeping enzymes that participate in mitochondrial malate respiration. In the C4 species G. gynandra and C. angustifolia, this role is restricted to the NAD-MEα/β2 heteromer. NAD-MEα/β1 is exclusively present in the leaves, exhibits high catalytic efficiency and is differentially activated by the C4 intermediate aspartate, suggesting its role a photosynthetic role as C4-decarboxylase. During C4 evolution, GgNAD-MEβ1 and CaNAD-MEβ1 lost their catalytic activity; but maintained a critical role in the stabilization of associated α-subunit. This effect percolates at the active site improving the binding of the substrate and leading to the higher catalytic efficiency of GgNAD-MEα/β1 with respect to NAD-MEα/β2. Also important, our results indicate the loss of function into the active site could have led to the generation of an allosteric site for the aspartate.Finally, the association of the non-catalytic GgNAD-MEβ1 to the paralogous A, thaliana α-partner renders a NAD-ME entity with similar properties as GgNAD-MEα/β1 indicating that the interaction is largely conserved despite the evolutionary divergence between species and its different photosynthetic modes.In summary, while C4-NADP-ME originated by a standard gene duplication-neo-functionalization framework, C4-NAD-ME has evolved through the differential assembly of protein subunits that accumulated adaptive mutations during evolution. Our results will now aid in introducing C4 traits into C3 plants. Ongoing projects can now rationally design the incorporation of a NAD-ME with C4 characteristics by introducing the NAD-MEβ1 of C4 Cleome or modifying a preexistent extra NAD-MEβ copy.