Analysis of tomato polygalacturonase expression in transgenic tobacco.
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.
Osteryoung
K W
KW
Mann Laboratory, Department of Vegetable Crops, University of California, Davis 95616.
Toenjes
K
K
Hall
B
B
Winkler
V
V
Bennett
A B
AB
D000595:Amino Acid Sequence; D001692:Biological Transport; D002473:Cell Wall / Q000378:metabolism; D017343:Genes, Plant / Q000235:genetics*; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D010368:Pectins / Q000378:metabolism; D010944:Plants / Q000201:enzymology* / Q000235:genetics; D030821:Plants, Genetically Modified / Q000201:enzymology / Q000235:genetics*; D010947:Plants, Toxic; D011096:Polygalacturonase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D011499:Protein Processing, Post-Translational; D013347:Subcellular Fractions / Q000201:enzymology / Q000276:immunology; D014026:Nicotiana / Q000201:enzymology / Q000235:genetics*; D014574:Uronic Acids / Q000378:metabolism
D007527:Isoenzymes; D014574:Uronic Acids; C018402:traumacel; D010368:Pectins; D011096:Polygalacturonase