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The decarboxylation of C4 acids in the bundle sheath cells (BSCs) is a key step in the C4 photosynthetic carbon assimilation pathway. Depending on the particular subtype of C4-species, this process can be mediated by different enzymes: NADP-malic enzyme (NADP-ME), NAD-malic enzyme (NAD-ME) and/or phosphoenolpyruvate carboxykinase (PEPCK), and each enzyme has a different subcellular compartmentalization within the BSCs. Thus, the C4 subtype cycle mediated by each decarboxylase displays distinguishing features in leaf anatomy, biochemistry and physiology. In some cases, the operation of more than one type of decarboxylating enzyme in the C4 photosynthetic process has been described. During the last few years, remarkable advances have been made in the characterization of different isoforms of each C4 decarboxylase. In most cases, non-photosynthetic isoforms of the C4-decarboxylating enzymes involved in primary and/or secondary metabolisms were characterized. These non-C4 isoforms were for sure the starting point for the evolution of the C4-specific decarboxylases through the gaining of characteristics that make them more suitable to fulfill the requirements of the photosynthetic process. For each decarboxylating enzyme, the analysis of phylogenetic relationships reveals several features of the molecular evolution of the C4 process which accomplished the same biochemical aim: the generation of CO2 in the vecinity of Rubisco in BSCs, reducing photorespiration and enhancing photosynthesis.

The Arabidopsis thaliana genome contains four genes encoding NADP-malic enzymes (NADP-ME1−4). NADP-ME4 is localized to plastids whereas the other three isoforms are cytosolic. NADP-ME2 and −4 are constitutively expressed in mature organs, while NADP-ME1 and −3 are restricted to secondary roots and to trichomes and pollen, respectively. Although the four isoforms share a high degree of identity, the recombinant NADP-ME1 to 4 show well-distinct kinetic and structural properties. NADP-ME2 exhibits the highest specific activity, while NADP-ME3 and −4 present the highest catalytic efficiency for NADP and malate, respectively. When analyzing the activity of each isoform in the presence of possible metabolic effectors, the results obtained indicate that NADP-ME2 is the most highly regulated isoform, especially by activation. The four isoforms behave differently in terms of reversibility, presenting NADP-ME4 the highest ratio between the reverse — carboxylation and reduction of pyruvate — to the forward reaction. In order to identify residues or segments of the primary structure of each NADPME isoform that could be involved in the differences in kinetic and regulatory properties among the isoforms, NADP-ME2 mutants and deletions were constructed and analysed. The results obtained show that Arg115 is involved in fumarate activation, while the regions involved in aspartate and CoA modulation are located at the amino-terminal part of the protein. On overall, these studies show that minimal structural changes are responsible for the different kinetic behaviour of each AtNADP-ME isoform.

Maize C4-NADP-malic enzyme was expressed under the control of the CaMV 35S promoter in Arabidopsis thaliana. An increase in the plastidic NADP-ME activity induced no phenotypic differences in long-day growth conditions. Analysis of metabolite levels, however, revealed a disturbed metabolic profile. Dark-induced senescence progressed more rapidly in MEm plants compared to the wild-type. A retardation of senescence in the transgenic lines was gained by exogenous supply of glucose, sucrose and malate, suggesting that the lack of a rapid energy source is likely to be the initial factor leading to the induction of senescence in these plants. A fairly complete picture of primary metabolism assessed by GC-MS and the in vitro metabolic complementation assays allow us to conclude that MEm transgenic plants entered dark induced senescence more rapidly due to an accelerated starvation. Comparison of the data obtained indicated that extremely low levels of malate and fumarate are responsible for the accelerated dark-induced senescence encountered in the MEm plants. Reinforcing previous results, our data indicate that malate and fumarate are key players in the primary metabolism of Arabidopsis thaliana.