Phenotypic similarity of NAD(P)-Malic Enzymes in Tomato: Unveiling Patterns of Convergent and Parallel Evolution

The Solanum lycopersicum (tomato) genome encodes seven malic enzymes (MEs): three cytosolic NADP-ME isoforms (SlNADP-ME1, -2, and -3), two plastidic NADP-ME proteins (SlNADP-ME4a and -4b), and two mitochondrial NAD-dependent subunits (SlNAD-ME1 and -2) of a heteromeric enzyme. Except for SlNADP-ME3, which is almost exclusively expressed in the root, all the genes are active in the major tissues of flowering plants (leaf, stem, flower, and root), with SlNADP-ME2 and -4b transcripts accumulating at relative high levels. During the cell expansion and ripening phases of fruit development, SlNADP-ME3 and -4b expression increased in the pericarp and seeds while SlNADP-ME2 remained at a high level. In green fruit, SlNADP-ME3 and -4b are down-regulated by ethylene, with SlNAD-ME1 and -2 being up-regulated. A correlation between SlNADP-ME4b accumulation and NADP-ME activity was observed from the early immature green to the mature green fruit stages, linking this plastidic isoform to starch and lipid biosynthesis. SlNADP-ME1 and -4a expression increase with temperature, suggesting involvement in defense mechanisms and supported by cis-elements composition in their promoters. Interesting, SlNADP-ME3 biochemical properties and its accumulation in seeds is part of an inherited and long-conserved genetic lineage in Angiosperms including Arabidopsis NADP-ME1, involved in normal seed germination. By analyzing the phylogeny and synteny of SlNAD(P)-ME genes and determining the biochemical properties of the recombinant proteins, we seek functional similarities to Arabidopsis NAD(P)-ME members. Our findings enhance the understanding of malate metabolism in tomatoes, which could inform strategies to improve fruit quality. Additionally, they prompt a re-evaluation of pre-existing notions regarding the functional orthology of enzymes based on phylogenetic relationships. SIGNIFICANCE STATEMENTEnzyme functional orthology among distant plant species cannot be inferred through phylogenetic analysis because of the large independent rearrangements of genomes during evolution. A comprehensive characterization of the tomato NAD(P)-ME family and their comparison with members from an unrelated plant species, such as Arabidopsis, allowed us to evaluate the impact of selective pressures on orthologous genes. This approach reveals that the phenotypic similarity of an entire enzyme family in different species is achieved through diverse evolutionary events such as ancestry, parallelism, and genetic convergence.