Activity and gene expression analysis of the NADP-dependent isocitrate dehydrogenase (NADP-ICDH) through pepper fruit ripening and its modulation by nitric oxide (NO). Molecular characterization of the peroxisomal isozyme

Abstract

NADP-dependent isocitrate dehydrogenase (NADP-ICDH) is one of the main sources of cellular reductant capacity in the form of NADPH. Although there is significant knowledge about the relevance of this enzyme during some physiological and stress processes, the available information about its involvement in fruit ripening is scarce. Using sweet green pepper (Capsicum annuum L.) fruits, a 50–75 % ammonium-sulfate-enriched protein fraction containing the NADP-ICDH activity allowed its biochemical characterization. The enzyme displayed a typical Michaelis-Menten kinetics and exhibited V_max and K_m values of 97 μUnits and 78 µM for isocitrate, and 92 μUnits and 46 µM for NADP⁺. Three NADP-ICDH isozymes were identified by non-denaturing PAGE designated as NADP-ICDH I to III, each representing 33 %, 24 %, and 43 %, respectively, of the total activity. Based on our previous transcriptome (RNA-Seq), three CaICDH genes (CaNADP-ICDH1, CaNADP-ICDH2, and CaNADP-ICDH3) were identified in sweet pepper fruits encoding isozymes potentially distributed in the cytosol, cytosol/mitochondrion, and peroxisome, according to their percentage of identity with the Arabidopsis isozymes. The time-course expression analysis of these genes during different fruit ripening stages including green immature (G), breaking point (BP), and red ripe (R), and in fruits subjected to nitric oxide (NO) treatments, showed dissimilar expression patterns. During ripening from green to red fruits, CaNADP-ICDH1 and CaNADP-ICDH2 were upregulated but were negatively affected by NO; however, CaNADP-ICDH3 was downregulated during ripening but unaffected by NO treatment. Furthermore, during ripening, the NADP-ICDH activity increased in red ripe fruits whereas the NO gas treatment produced a significant inhibition. These findings provide, to our knowledge, the first characterization of the NADP-ICDH family in this non-climacteric fruit and suggest that NADP-ICDH must play an important role in maintaining the supply of NADPH during pepper fruit ripening and that NO partially modulates this NADPH-generating system.