Osteryoung K W, Toenjes K, Hall B, Winkler V, Bennett A B
Mann Laboratory, Department of Vegetable Crops, University of California, Davis 95616.
Plant Cell. 1990 Dec;2(12):1239-48. doi: 10.1105/tpc.2.12.1239.
Tomato polygalacturonase is a cell wall enzyme secreted in large amounts during tomato fruit ripening. Polygalacturonase is synthesized as a glycoprotein precursor that undergoes numerous cotranslational and post-translational processing steps during its maturation, yielding three isozymes in tomato fruit, PG1, PG2A, and PG2B. To investigate the physiological roles of the three isozymes and the functional significance of the polygalacturonase processing domains in its intracellular transport and activity, we have examined polygalacturonase expression in transgenic tobacco plants. A full-length polygalacturonase cDNA was placed under control of the cauliflower mosaic virus 35S promoter and introduced into tobacco by way of Agrobacterium-mediated transformation. Analysis of transgenic tobacco plants indicated that (1) immunologically detectable polygalacturonase can be extracted from leaves, roots, and stems of transgenic tobacco plants; (2) only PG2A and PG2B were detectable in transgenic tobacco; (3) the polygalacturonase isozymes present in transgenic tobacco were electrophoretically indistinguishable from the tomato isozymes; (4) the N-terminal sequence, degree of N-linked glycosylation, and extent of oligosaccharide processing were similar in polygalacturonase from transgenic tobacco and tomato; (5) polygalacturonase was properly localized in cell walls of transgenic tissue; (6) the protein was enzymatically active in vitro; however, (7) accumulation of PG2A and PG2B in cell walls of transgenic tobacco did not result in pectin degradation in vivo. These results indicated that tomato polygalacturonase was properly processed and transported to the cell wall of tobacco. However, accumulation of the two polygalacturonase isozymes expressed in this heterologous host was insufficient to promote polyuronide degradation in tobacco leaf tissue.
番茄多聚半乳糖醛酸酶是一种在番茄果实成熟过程中大量分泌的细胞壁酶。多聚半乳糖醛酸酶作为一种糖蛋白前体被合成，在其成熟过程中经历了许多共翻译和翻译后加工步骤，在番茄果实中产生三种同工酶，即PG1、PG2A和PG2B。为了研究这三种同工酶的生理作用以及多聚半乳糖醛酸酶加工结构域在其细胞内运输和活性中的功能意义，我们检测了转基因烟草植株中多聚半乳糖醛酸酶的表达。将一个全长多聚半乳糖醛酸酶cDNA置于花椰菜花叶病毒35S启动子的控制下，并通过农杆菌介导的转化方法导入烟草。对转基因烟草植株的分析表明：（1）可从转基因烟草植株的叶、根和茎中提取出免疫可检测的多聚半乳糖醛酸酶；（2）在转基因烟草中仅可检测到PG2A和PG2B；（3）转基因烟草中存在的多聚半乳糖醛酸酶同工酶在电泳上与番茄同工酶无法区分；（4）转基因烟草和番茄中的多聚半乳糖醛酸酶在N端序列、N-连接糖基化程度和寡糖加工程度方面相似；（5）多聚半乳糖醛酸酶在转基因组织的细胞壁中定位正确；（6）该蛋白在体外具有酶活性；然而，（7）PG2A和PG2B在转基因烟草细胞壁中的积累并未导致体内果胶降解。这些结果表明，番茄多聚半乳糖醛酸酶得到了正确的加工并转运到烟草的细胞壁中。然而，在这种异源宿主中表达的两种多聚半乳糖醛酸酶同工酶的积累不足以促进烟草叶片组织中的多聚糖醛酸降解。
