Department of Stress, Development and Signaling in Plants, Group of Antioxidants, Free Radicals and Nitric Oxide in Biotechnology, Food and Agriculture, Estación Experimental del Zaidín (Spanish National Research Council, CSIC), C/Profesor Albareda, 1, Granada 18008, Spain.
Department of Stress, Development and Signaling in Plants, Group of Antioxidants, Free Radicals and Nitric Oxide in Biotechnology, Food and Agriculture, Estación Experimental del Zaidín (Spanish National Research Council, CSIC), C/Profesor Albareda, 1, Granada 18008, Spain.
Plant Sci. 2024 Dec;349:112269. doi: 10.1016/j.plantsci.2024.112269. Epub 2024 Sep 21.
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 and K 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.
NADP 依赖性异柠檬酸脱氢酶（NADP-ICDH）是细胞还原能力的主要来源之一，其形式为 NADPH。尽管人们对该酶在某些生理和应激过程中的相关性有了相当多的了解，但关于其在果实成熟过程中作用的信息却很少。本研究使用甜绿辣椒（Capsicum annuum L.）果实，通过硫酸铵沉淀富集 50-75%的蛋白，从中分离出 NADP-ICDH 并对其进行生化特性分析。结果表明，该酶具有典型的米氏动力学特性，其对异柠檬酸的 V 和 K 值分别为 97μUnits 和 78μM，对 NADP 的 V 和 K 值分别为 92μUnits 和 46μM。通过非变性聚丙烯酰胺凝胶电泳（PAGE）分离到 3 种 NADP-ICDH 同工酶，分别命名为 NADP-ICDH I、NADP-ICDH II 和 NADP-ICDH III，它们分别占总活性的 33%、24%和 43%。基于我们之前的转录组学（RNA-Seq）研究，在甜椒果实中鉴定出 3 个 CaICDH 基因（CaNADP-ICDH1、CaNADP-ICDH2 和 CaNADP-ICDH3），这些基因编码的同工酶根据与拟南芥同工酶的同源性，分别可能定位于细胞质、细胞质/线粒体和过氧化物酶体中。对这些基因在不同果实成熟阶段（包括绿熟期（G）、转色期（BP）和完熟期（R））和一氧化氮（NO）处理果实中的表达模式进行分析，发现它们具有不同的表达模式。在果实从绿熟到完熟的成熟过程中，CaNADP-ICDH1 和 CaNADP-ICDH2 的表达上调，但受到 NO 的负调控；而 CaNADP-ICDH3 的表达下调，但不受 NO 处理的影响。此外，在果实成熟过程中，NADP-ICDH 活性在完熟期增加，而 NO 气体处理则显著抑制了其活性。这些发现为非跃变型果实中 NADP-ICDH 家族的首次特征描述提供了依据，并表明 NADP-ICDH 必须在辣椒果实成熟过程中维持 NADPH 的供应中发挥重要作用，而 NO 部分调节了该 NADPH 生成系统。
