Characterization of the Entire Cystatin Gene Family in Barley and Their Target Cathepsin L-Like Cysteine-Proteases, Partners in the Hordein Mobilization during Seed Germination
Characterization of the Entire Cystatin Gene Family in Barley and Their Target Cathepsin L-Like Cysteine-Proteases, Partners in the Hordein Mobilization during Seed Germination
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DOI：
10.1104/pp.109.146019
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发表时间：
2009-11-01
期刊：
影响因子：
7.4
通讯作者：
Diaz, Isabel
中科院分区：
文献类型：
--
作者：
Martinez, Manuel;Cambra, Ines;Diaz, Isabel
植物半胱氨酸蛋白酶抑制剂是木瓜蛋白酶C1 A和豆荚蛋白酶C13家族的半胱氨酸蛋白酶的抑制剂。来自多个植物物种的半胱氨酸蛋白酶抑制剂数据表明，这些抑制剂作为防御蛋白质对害虫和病原体，并作为调节蛋白质周转。在这项研究中，我们的特点是整个半胱氨酸蛋白酶抑制剂基因家族从大麦（大麦），其中包含13个非冗余基因，并确定和表征其靶酶，大麦组织蛋白酶L样蛋白酶。从大肠杆菌培养物中表达和纯化胱抑素和蛋白酶。在使用特定底物的体外抑制试验中，发现每种半胱氨酸蛋白酶抑制剂对大麦半胱氨酸蛋白酶具有不同的抑制能力。实时逆转录-聚合酶链反应显示，抑制剂和酶的信使RNA表达模式存在很大差异。它们的转录本主要在发育和萌发的种子中检测到，部分在叶和根中也有表达。半胱氨酸蛋白酶抑制剂和组织蛋白酶L-样蛋白酶融合到绿色荧光蛋白的亚细胞定位证明了整个内质网和高尔基复合体的蛋白质家族的存在。蛋白酶和半胱氨酸蛋白酶抑制剂不仅共定位，但也相互作用，在植物细胞中，所揭示的双分子荧光互补。半胱氨酸蛋白酶抑制剂和组织蛋白酶L-样蛋白酶之间的功能关系推断，从他们共同的含义作为对应的动员大麦种子萌发后的存储蛋白。在赤霉素处理的糊粉中，抑制剂和酶的转录表达模式相反，允许蛋白酶特异性地降解大麦种子胚乳中储存的B、C和D大麦醇溶蛋白。
Plant cystatins are inhibitors of cysteine-proteases of the papain C1A and legumain C13 families. Cystatin data from multiple plant species have suggested that these inhibitors act as defense proteins against pests and pathogens and as regulators of protein turnover. In this study, we characterize the entire cystatin gene family from barley (Hordeum vulgare), which contain 13 nonredundant genes, and identify and characterize their target enzymes, the barley cathepsin L-like proteases. Cystatins and proteases were expressed and purified from Escherichia coli cultures. Each cystatin was found to have different inhibitory capability against barley cysteine-proteases in in vitro inhibitory assays using specific substrates. Real-time reverse transcription-polymerase chain reaction revealed that inhibitors and enzymes present a wide variation in their messenger RNA expression patterns. Their transcripts were mainly detected in developing and germinating seeds, and some of them were also expressed in leaves and roots. Subcellular localization of cystatins and cathepsin L-like proteases fused to green fluorescent protein demonstrated the presence of both protein families throughout the endoplasmic reticulum and the Golgi complex. Proteases and cystatins not only colocalized but also interacted in vivo in the plant cell, as revealed by bimolecular fluorescence complementation. The functional relationship between cystatins and cathepsin L-like proteases was inferred from their common implication as counterparts of mobilization of storage proteins upon barley seed germination. The opposite pattern of transcription expression in gibberellin-treated aleurones presented by inhibitors and enzymes allowed proteases to specifically degrade B, C, and D hordeins stored in the endosperm of barley seeds.