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

番茄果实果胶水解酶聚半乳糖醛酸酶（PG）的活性在成熟过程中急剧增加，这是由于三个密切相关的异构体PG1、PG2A和PG2B的存在。Pg2异构体由单个催化PG多肽组成，而PG1是一个异二聚体，由一个Pg2多肽与一个辅助糖蛋白PG转化器紧密结合组成。每个异构体参与果胶降解和果实成熟的其他方面仍不清楚。植物中PG异构体的水平似乎由转化蛋白的水平决定。为了增加我们对PG等细胞壁酶的生物合成、组装和生物活性的了解，我们将通过改变转基因野生型植物中转换蛋白的水平来控制PG个体同工型的水平。为此，将对该转化蛋白进行纯化、克隆，并对其在野生型果实成熟过程中的结构和表达进行研究。在转基因野生型番茄植株中，通过正义和反义方向表达转体cDNA来控制转体蛋白的水平，可能导致仅积累PG1或PG2。这些研究将增加我们对单个PG异构体对多脲醛降解和与果实成熟相关的其他生理参数的影响的认识。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发育以及果实成熟和植物防御等特殊功能至关重要。一个重要的问题是，当这些重要的蛋白质从细胞中分泌出来，而不受与细胞质酶调节相关的正常机制的控制时，它们的活性是如何受到控制的。本文描述了一种有趣的方法来研究胞外酶聚半乳糖醛酸酶的加工和功能，该酶在果实成熟过程中降解细胞壁中的多脲醛。//

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. //