Cloning of Tomato Fruit Polygalacturonase (PG) Converter Protein : Analysis of PG Isozyme Function

番茄果实果胶水解酶多聚半乳糖醛酸酶(PG)的活性在成熟过程中由于存在三种密切相关的同工酶PG1、PG2A和PG2B而急剧增加。Pg2亚型由单一的催化PG多肽组成，而PG1是由一个pg2多肽与辅助糖蛋白PG转换器紧密结合而成的异二聚体。每种异构体在果胶降解和果实成熟的其他方面的作用尚不清楚。植物中PG异构体的水平似乎由转换蛋白的水平决定。为了加深我们对PG等细胞壁酶的生物合成、组装和生物活性的了解，我们将在体内通过改变转基因野生型植物中转换蛋白的水平来操纵单个PG亚型的水平。为了实现这一点，将对转换蛋白进行纯化、克隆，并研究其结构和在野生型果实成熟过程中的表达。在转基因野生型番茄植株中，通过在正义和反义方向上表达转换蛋白来控制转换蛋白的水平，可能只导致PG1或PG2的积累。这些研究将增加我们对单个PG亚型对多糖醛酸解和其他与果实成熟相关的生理参数的影响的知识。几种酶和结构蛋白从植物细胞分泌到细胞壁。这些蛋白质的活性对正常生长和发育以及水果成熟和植物防御等特殊功能是必不可少的。一个重要的问题是，当这些重要蛋白质从细胞中分泌出来时，它们的活性是如何受到控制的，而不是受到与细胞质酶调节相关的正常机制的影响。这项建议描述了一种有趣的方法来研究一种胞外酶--聚半乳糖醛酸酶的加工和功能，这种酶在果实成熟期间降解细胞壁中的多糖醛酸类化合物。//

The activity of the tomato the tomato fruit pectin-hydrolyzing enzyme polygalacturonase (PG) increases dramatically during ripening due to the presence of three closely related isoforms, PG1, PG2A and PG2B. The Pg2 isoforms are composed of single catalytic PG polypeptides while PG1 is a heterodimer consisting of one PG2 polypeptide in tight association with an ancillary glycoprotein, the PG converter. The involvement of each isoform in pectin degradation and other aspects of fruit ripening remain unclear. The PG isoform levels in planta appear to be determined by the level of converter protein. In order to increase our understanding of the biosynthesis, assembly and biological activity of cell wall enzymes such as PG, the levels of individual PG isoforms will be manipulated in vivo by altering the levels of converter protein in transgenic wild-type plants. To accomplish this the converter protein will be purified, cloned and its structure and expression during wild-type fruit ripening studied. Converter protein levels will be manipulated by expressing a converter cDNA in sense and antisense orientations in transgenic wild-type tomato plants presumably resulting in accumulation of only PG1 or PG2. These studies will increase our knowledge of the effect of individual PG isoforms on polyuronide degradation and other physiological parameters associated with fruit ripening. Several enzymes and structural proteins are secreted from plant cells into the cell wall. The activities of these proteins are essential for normal growth and development as well as specialized functions such as fruit ripening and plant defense. An important question is how the activities of such important proteins is controlled when they are secreted from the cell and not subject to the normal mechanisms associated with the regulation of cytoplasmic enzymes. This proposal describes an interesting approach to studying the processing and function of an extracellular enzyme, polygalacturonase, which degrades polyuronides in the cell wall during fruit ripening. //