Chaki Mounira, Álvarez de Morales Paz, Ruiz Carmelo, Begara-Morales Juan C, Barroso Juan B, Corpas Francisco J, Palma José M
Group of Antioxidants, Free Radicals and Nitric Oxide in Biotechnology, Food and Agriculture, Department of Biochemistry, Cell and Molecular Biology of Plants, Estación Experimental del Zaidín, CSIC, Apartado 419, 18008 Granada, Spain and.
Group of Biochemistry and Cell Signaling in Nitric Oxide. Department of Biochemistry and Molecular Biology, University of Jaén, 23071 Jaén, Spain.
Ann Bot. 2015 Sep;116(4):637-47. doi: 10.1093/aob/mcv016. Epub 2015 Mar 26.
Pepper (Capsicum annuum, Solanaceae) fruits are consumed worldwide and are of great economic importance. In most species ripening is characterized by important visual and metabolic changes, the latter including emission of volatile organic compounds associated with respiration, destruction of chlorophylls, synthesis of new pigments (red/yellow carotenoids plus xanthophylls and anthocyanins), formation of pectins and protein synthesis. The involvement of nitric oxide (NO) in fruit ripening has been established, but more work is needed to detail the metabolic networks involving NO and other reactive nitrogen species (RNS) in the process. It has been reported that RNS can mediate post-translational modifications of proteins, which can modulate physiological processes through mechanisms of cellular signalling. This study therefore examined the potential role of NO in nitration of tyrosine during the ripening of California sweet pepper.
The NO content of green and red pepper fruit was determined spectrofluorometrically. Fruits at the breaking point between green and red coloration were incubated in the presence of NO for 1 h and then left to ripen for 3 d. Profiles of nitrated proteins were determined using an antibody against nitro-tyrosine (NO2-Tyr), and profiles of nitrosothiols were determined by confocal laser scanning microscopy. Nitrated proteins were identified by 2-D electrophoresis and MALDI-TOF/TOF analysis.
Treatment with NO delayed the ripening of fruit. An enhancement of nitrosothiols and nitroproteins was observed in fruit during ripening, and this was reversed by the addition of exogenous NO gas. Six nitrated proteins were identified and were characterized as being involved in redox, protein, carbohydrate and oxidative metabolism, and in glutamate biosynthesis. Catalase was the most abundant nitrated protein found in both green and red fruit.
The RNS profile reported here indicates that ripening of pepper fruit is characterized by an enhancement of S-nitrosothiols and protein tyrosine nitration. The nitrated proteins identified have important functions in photosynthesis, generation of NADPH, proteolysis, amino acid biosynthesis and oxidative metabolism. The decrease of catalase in red fruit implies a lower capacity to scavenge H2O2, which would promote lipid peroxidation, as has already been reported in ripe pepper fruit.
辣椒（茄科辣椒属）果实全球皆有食用，具有重要经济价值。在大多数物种中，成熟以显著的视觉和代谢变化为特征，后者包括与呼吸相关的挥发性有机化合物的释放、叶绿素的破坏、新色素（红色/黄色类胡萝卜素以及叶黄素和花青素）的合成、果胶的形成以及蛋白质合成。一氧化氮（NO）参与果实成熟已得到证实，但仍需更多研究来详细阐述该过程中涉及NO和其他活性氮物种（RNS）的代谢网络。据报道，RNS可介导蛋白质的翻译后修饰，进而通过细胞信号传导机制调节生理过程。因此，本研究探讨了NO在加利福尼亚甜椒成熟过程中酪氨酸硝化作用的潜在作用。
采用荧光分光光度法测定青椒和红椒果实中的NO含量。将处于绿熟与红熟转变临界点的果实置于NO存在的环境中孵育1小时，然后使其再成熟3天。使用抗硝基酪氨酸（NO2-Tyr）抗体测定硝化蛋白质谱，通过共聚焦激光扫描显微镜测定亚硝基硫醇谱。通过二维电泳和基质辅助激光解吸电离飞行时间串联质谱（MALDI-TOF/TOF）分析鉴定硝化蛋白质。
NO处理延缓了果实成熟。在果实成熟过程中观察到亚硝基硫醇和硝基蛋白质增加，而添加外源NO气体可使其逆转。鉴定出六种硝化蛋白质，其特征表明它们参与氧化还原、蛋白质、碳水化合物和氧化代谢以及谷氨酸生物合成。过氧化氢酶是在青椒和红椒中发现的最丰富的硝化蛋白质。
本文报道的RNS谱表明，辣椒果实成熟的特征是亚硝基硫醇增加和蛋白质酪氨酸硝化。鉴定出的硝化蛋白质在光合作用、NADPH生成、蛋白水解、氨基酸生物合成和氧化代谢中具有重要功能。红椒中过氧化氢酶的减少意味着清除过氧化氢的能力较低，这将促进脂质过氧化，正如已在成熟辣椒果实中报道的那样。
