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

IF 4.2 2区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Plant Science Pub Date : 2024-09-21 DOI:10.1016/j.plantsci.2024.112269

María A. Muñoz-Vargas, Salvador González-Gordo, Jorge Taboada, José M. Palma, Francisco J. Corpas

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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.

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辣椒果实成熟过程中依赖 NADP 的异柠檬酸脱氢酶（NADP-ICDH）的活性和基因表达分析及其受一氧化氮（NO）的调节。过氧化物酶同工酶的分子特征。

依赖 NADP 的异柠檬酸脱氢酶（NADP-ICDH）是以 NADPH 形式存在的细胞还原剂能力的主要来源之一。尽管人们对这种酶在某些生理和应激过程中的相关性有大量了解，但有关它参与果实成熟的现有信息却很少。利用甜青椒（Capsicum annuum L.）果实，对含有 NADP-ICDH 活性的 50-75% 富含硫酸铵的蛋白质部分进行了生化鉴定。该酶表现出典型的 Michaelis-Menten 动力学，异柠檬酸的 Vmax 和 Km 值分别为 97 μUits 和 78µM，NADP+ 的 Vmax 和 Km 值分别为 92 μUits 和 46µM。通过非变性 PAGE 鉴定出三种 NADP-ICDH 同工酶，命名为 NADP-ICDH I 至 III，分别占总活性的 33%、24% 和 43%。根据我们之前的转录组（RNA-Seq），在甜椒果实中发现了三个 CaICDH 基因（CaNADP-ICDH1、CaNADP-ICDH2 和 CaNADP-ICDH3），根据它们与拟南芥同工酶的相同百分比，这些基因编码的同工酶可能分布在细胞质、细胞质/半胱体和过氧物酶体中。对这些基因在不同果实成熟阶段（包括绿色未成熟果实（G）、破裂点果实（BP）和红色成熟果实（R））以及一氧化氮（NO）处理的果实中的时程表达分析显示出不同的表达模式。在从青果到红果的成熟过程中，CaNADP-ICDH1 和 CaNADP-ICDH2 表达上调，但受到 NO 的负面影响；然而，CaNADP-ICDH3 在成熟过程中表达下调，但不受 NO 处理的影响。此外，在成熟过程中，红熟果实中的 NADP-ICDH 活性增加，而 NO 气体处理会产生明显的抑制作用。据我们所知，这些发现首次描述了这种非成熟果实中 NADP-ICDH 家族的特征，并表明在辣椒果实成熟过程中，NADP-ICDH 必须在维持 NADPH 供应方面发挥重要作用，而 NO 可部分调节这一 NADPH 生成系统。

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来源期刊

Plant Science 生物-生化与分子生物学

CiteScore

9.10

自引率

1.90%

发文量

322

审稿时长

33 days

期刊介绍： Plant Science will publish in the minimum of time, research manuscripts as well as commissioned reviews and commentaries recommended by its referees in all areas of experimental plant biology with emphasis in the broad areas of genomics, proteomics, biochemistry (including enzymology), physiology, cell biology, development, genetics, functional plant breeding, systems biology and the interaction of plants with the environment. Manuscripts for full consideration should be written concisely and essentially as a final report. The main criterion for publication is that the manuscript must contain original and significant insights that lead to a better understanding of fundamental plant biology. Papers centering on plant cell culture should be of interest to a wide audience and methods employed result in a substantial improvement over existing established techniques and approaches. Methods papers are welcome only when the technique(s) described is novel or provides a major advancement of established protocols.