<i>NADP-Dependent Malic Enzyme</i> Genes in Sweet Pepper Fruits: Involvement in Ripening and Modulation by Nitric Oxide (NO)

NADPH is an indispensable cofactor in a wide range of physiological processes that is generated by a family of NADPH dehydrogenases, of which the NADP-dependent malic enzyme (NADP-ME) is a member. Pepper (<i>Capsicum annuum</i> L.) fruit is a horticultural product consumed worldwide that has great nutritional and economic relevance. Besides the phenotypical changes that pepper fruit undergoes during ripening, there are many associated modifications at transcriptomic, proteome, biochemical and metabolic levels. Nitric oxide (NO) is a recognized signal molecule with regulatory functions in diverse plant processes. To our knowledge, there is very scarce information about the number of genes encoding for NADP-ME in pepper plants and their expression during the ripening of sweet pepper fruit. Using a data mining approach to evaluate the pepper plant genome and fruit transcriptome (RNA-seq), five <i>NADP-ME</i> genes were identified, and four of them, namely <i>CaNADP</i>-<i>ME2</i> to <i>CaNADP</i>-<i>ME5</i>, were expressed in fruit. The time course expression analysis of these genes during different fruit ripening stages, including green immature (G), breaking point (BP) and red ripe (R), showed that they were differentially modulated. Thus, while <i>CaNADP</i>-<i>ME3</i> and <i>CaNADP</i>-<i>ME5</i> were upregulated, <i>CaNADP-ME2</i> and <i>CaNADP-ME4</i> were downregulated. Exogenous NO treatment of fruit triggered the downregulation of <i>CaNADP-ME4</i>. We obtained a 50–75% ammonium–sulfate-enriched protein fraction containing CaNADP-ME enzyme activity, and this was assayed via non-denaturing polyacrylamide gel electrophoresis (PAGE). The results allow us to identify four isozymes designated from CaNADP-ME I to CaNADP-ME IV. Taken together, the data provide new pieces of information on the CaNADP-ME system with the identification of five <i>CaNADP-ME</i> genes and how the four genes expressed in pepper fruits are modulated during ripening and exogenous NO gas treatment.