结论:辣椒果实含有两个亮氨酸氨基肽酶(LAP)基因,这些基因在成熟过程中受到一氧化氮的差异调节。LAP活性在成熟期间增加,但被硝化负调节。亮氨酸氨肽酶(LAP)是一种必需的金属酶,可从蛋白质中切割N末端亮氨酸残基,但也代谢二肽和三肽。LAP在细胞蛋白质周转中起着基本作用,并参与生理过程,例如抵抗生物和非生物胁迫的防御机制。但人们对它们与水果生理学的关系知之甚少。这项研究旨在鉴定和表征编码LAP的基因,并评估它们在辣椒(CapsicumannuumL.)果实成熟和富含一氧化氮(NO)的环境中的作用。使用辣椒植物基因组和果实转录组(RNA-seq)的数据挖掘方法,两个LAP基因,鉴定命名为CaLAP1和CaLAP2。这些基因在不同果实成熟阶段的时程表达分析显示,而CaLAP1降低,CaLAP2上调。然而,在水果的外源NO处理下,这两个基因都下调了.相反,结果表明,在果实成熟过程中,LAP活性增加了81%。在存在不同调节化合物(包括过氧亚硝酸盐(ONOO-))的情况下,LAP活性的体外测定,NO供体(S-亚硝基谷胱甘肽和亚硝基半胱氨酸),还原剂,如还原型谷胱甘肽(GSH),L-半胱氨酸(L-Cys),氰化物引发了不同的反应。因此,过氧亚硝酸盐和还原化合物引起绿色未成熟果实中LAP活性约50%的抑制,而氰化物将其上调1.5倍。据我们所知,这是辣椒果实中LAP的第一个特征,也是通过多种调节化合物对其进行调节的第一个特征。根据LAP代谢二肽和三肽的能力,可以假设LAP可能参与成熟过程中的GSH回收。
CONCLUSIONS: Pepper fruits contain two leucine
aminopeptidase (LAP) genes which are differentially modulated during ripening and by nitric oxide. The LAP activity increases during ripening but is negatively modulated by nitration. Leucine
aminopeptidase (LAP) is an essential metalloenzyme that cleaves N-terminal leucine residues from proteins but also metabolizes dipeptides and tripeptides. LAPs play a fundamental role in cell protein turnover and participate in physiological processes such as defense mechanisms against biotic and abiotic stresses, but little is known about their involvement in fruit physiology. This study aims to identify and characterize genes encoding LAP and evaluate their role during the ripening of pepper (Capsicum annuum L.) fruits and under a nitric oxide (NO)-enriched environment. Using a data-mining approach of the pepper plant genome and fruit transcriptome (RNA-seq), two LAP genes, designated CaLAP1 and CaLAP2, were identified. The time course expression analysis of these genes during different fruit ripening stages showed that whereas CaLAP1 decreased, CaLAP2 was upregulated. However, under an exogenous NO treatment of fruits, both genes were downregulated. On the contrary, it was shown that during fruit ripening LAP activity increased by 81%. An in vitro assay of the LAP activity in the presence of different modulating compounds including peroxynitrite (ONOO-), NO donors (S-nitrosoglutathione and nitrosocyteine), reducing agents such as reduced glutathione (GSH), L-cysteine (L-Cys), and cyanide triggered a differential response. Thus, peroxynitrite and reducing compounds provoked around 50% inhibition of the LAP activity in green immature fruits, whereas cyanide upregulated it 1.5 folds. To our knowledge, this is the first characterization of LAP in pepper fruits as well as of its regulation by diverse modulating compounds. Based on the capacity of LAP to metabolize dipeptides and tripeptides, it could be hypothesized that the LAP might be involved in the GSH recycling during the ripening process.