Horn Martin, Patankar Aparna G, Zavala Jorge A, Wu Jianqiang, Dolecková-Maresová Lucie, Vujtechová Milana, Mares Michael, Baldwin Ian T
Department of Protein Biochemistry, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague.
Plant Physiol. 2005 Sep;139(1):375-88. doi: 10.1104/pp.105.064006. Epub 2005 Aug 19.
Trypsin proteinase inhibitors (TPIs) of Nicotiana attenuata are major antiherbivore defenses that increase dramatically in leaves after attack or methyl jasmonate (MeJA) elicitation. To understand the elicitation process, we characterized the proteolytic fragmentation and release of TPIs from a multidomain precursor by proteases in MeJA-elicited and unelicited plants. A set of approximately 6-kD TPI peptides was purified from leaves, and their posttranslational modifications were characterized. In MeJA-elicited plants, the diversity of TPI structures was greater than the precursor gene predicted. This elicited structural heterogeneity resulted from differential fragmentation of the linker peptide (LP) that separates the seven-domain TPI functional domains. Using an in vitro fluorescence resonance energy transfer assay and synthetic substrates derived from the LP sequence, we characterized proteases involved in both the processing of the TPI precursor and its vacuolar targeting sequence. Although both a vacuolar processing enzyme and a subtilisin-like protease were found to participate in a two-step processing of LP, only the activity of the subtilisin-like protease was significantly increased by MeJA elicitation. We propose that MeJA elicitation increases TPI precursor production and saturates the proteolytic machinery, changing the processing pattern of TPIs. To test this hypothesis, we elicited a TPI-deficient N. attenuata genotype that had been transformed with a functional NaTPI gene under control of a constitutive promoter and characterized the resulting TPIs. We found no alterations in the processing pattern predicted from the sequence: a result consistent with the saturation hypothesis.
烟草（Nicotiana attenuata）中的胰蛋白酶蛋白酶抑制剂（TPI）是主要的抗食草动物防御物质，在受到攻击或茉莉酸甲酯（MeJA）诱导后，叶片中的含量会显著增加。为了了解诱导过程，我们对MeJA诱导和未诱导的植物中蛋白酶对多结构域前体中TPI的蛋白水解片段化和释放进行了表征。从叶片中纯化出一组约6-kD的TPI肽，并对其翻译后修饰进行了表征。在MeJA诱导的植物中，TPI结构的多样性大于前体基因预测的多样性。这种诱导产生的结构异质性是由连接肽（LP）的差异片段化导致的，LP将七结构域TPI功能结构域分隔开。使用体外荧光共振能量转移测定法和源自LP序列的合成底物，我们对参与TPI前体加工及其液泡靶向序列的蛋白酶进行了表征。尽管发现液泡加工酶和枯草杆菌蛋白酶样蛋白酶都参与了LP的两步加工，但只有枯草杆菌蛋白酶样蛋白酶的活性在MeJA诱导下显著增加。我们提出，MeJA诱导增加了TPI前体的产生并使蛋白水解机制饱和，从而改变了TPI的加工模式。为了验证这一假设，我们诱导了一种TPI缺陷型烟草基因型，该基因型已在组成型启动子的控制下用功能性NaTPI基因进行了转化，并对产生的TPI进行了表征。我们发现加工模式与序列预测的没有变化：这一结果与饱和假设一致。
