Analysis of Tomato Polygalacturonase Expression Analysis of transgenic tobacco plants indicated that

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  • The Plant Cell, Vol. 2, 1239-1248, December 1990 O 1990 American Society of Plant Physiologists

    Analysis of Tomato Polygalacturonase Expression in Transgenic Tobacco

    Katherine W. Osteryoung,’ Kurt Toenjes, Bradford Hall, Vickie Winkler, and Alan 6. Bennett2 Mann Laboratory, Department of Vegetable Crops, University of California, Davis, California 9561 6

    Tomato polygalacturonase is a cell wall enzyme secreted in large amounts during tomato fruit ripening. Polygalac- turonase 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, PGPA, and PGPB. 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 PGSA and PGPB 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 enzymically active in vitro; however, (7) accumulation of PGPA and PGSB 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.


    Tomato fruit ripening is the result of a complex devel- opmental program affecting metabolic processes in every compartment of the cell. A major component of this de- velopmental program is the degradation of pectic polymers in the cell wall, which is brought about by the action of a ripening-induced enzyme, polygalacturonase (Themmen et al., 1982; Crookes and Grierson, 1983; Huber, 1983a; Giovannoni et al., 1989). Polygalacturonase accumulates to high levels in ripening tomato fruit, suggesting an im- portant role for the enzyme in the developing fruit. Although a correlation between polygalacturonase accumulation and tomato fruit softening has been documented (Huber, 1983b, and references therein), recent evidence indicates that, by itself, degradation of polyuronides by polygalac- turonase is insufficient to cause tomato fruit softening (Giovannoni et al., 1989). Thus, the physiological function of this enzyme remains unclear.

    Tomato fruit polygalacturonase comprises three iso- zymes: PGI , PGPA, and PG2B (Pressey and Avants, 1973;

    ’ Current address: Department of Biochemistry, Life Sciences South, Room 303, University of Arizona, Tucson, AZ 85721. * To whom correspondence should be addressed.

    Tucker et al., 1980; Ali and Brady, 1982). PG2A and PG2B appear to function as monomeric catalytic subunits of 45 kD and 46 kD, respectively, with the size difference arising from a difference in extent of glycosylation (DellaPenna and Bennett, 1988). PG1 is a more complex protein of approximately 1 O0 kD consisting of a single catalytic poly- galacturonase subunit (either PG2A or PGPB) in associa- tion with a second glycoprotein subunit of 41 kD (Moshrefi and Luh, 1983). This subunit is immunologically unrelated to PG2A and PG2B (T. Moore and A.B. Bennett, unpub- lished results) and may be the polygalacturonase converter that converts the PG2 isozymes to PGI in vitro (Tucker et al., 1981; Pressey, 1984).

    Most work on polygalacturonase has focused on char- acterizing the PGPA and PG2B catalytic subunits, which function both as monomers and as subunits of PG1. The biosynthesis and maturation of these monomers are them- selves complex. Both PG2A and PG2B appear to be the products of a single gene and are synthesized as a larger molecular weight precursor that undergoes a number of processing events during conversion to the mature protein. These include cotranslational removal of the hydrophobic signal sequence and asparagine-linked glycosylation in the

  • 1240 The Plant Cell

    JP Transgenic Tobacco

    2B-, 2A-

    1 Figure 1. Immunodetection of Polygalacturonase Protein in Cell Wall Extracts from Leaves of Transgenic Tobacco.

    Cell wall proteins were extracted as described in Methods, sepa- rated by SDS-PAGE, electroblotted to nitrocellulose, and probed with polygalacturonase antiserum. Lane 1, 0.5 /ig of purified tomato PG2A and PG2B; lane 2, 50 ng of cell wall protein from a control plant transformed with vector alone; lanes 3 to 7, 50 ^g of cell wall protein from five different transgenic tobacco plants. The sizes of PG2A and PG2B are 45 kD and 46 kD, respectively.

    similar analysis in transgenic rin tomato fruit expressing a chimeric polygalacturonase gene suggests that PG1 alone catalyzes polyuronide degradation in vivo (DellaPenna et al., 1990).

    To address the functional significance of processing events that contribute to the maturation, intracellular trans- port, and physiological function of the polygalacturonase isozymes, we have characterized expression of the cata- lytic polygalacturonase subunits in transgenic tobacco. The results of these experiments indicated that the protein processing apparatus, which functions in the maturation of tomato fruit polygalacturonase, is conserved in vegeta- tive tissue of tobacco. In addition, these results have provided an opportunity to assess the function of the PG2 isozymes in the absence of PG1.


    endoplasmic reticulum (DellaPenna and Bennett, 1988), as well as extensive post-translational modification of the core oligosaccharides in the Golgi (Moshrefi and Luh, 1983). Recent evidence suggests that the differences in glyco- sylation between PG2A and PG2B result from differences in cotranslational core glycosylation rather than from dif- ferential processing of the glycan side chains (DellaPenna and Bennett, 1988). Comparison of the amino acid se- quence of mature PG2A and PG2B with the amino acid sequence deduced from cDNA clones further indicates at least two additional proteolytic processing steps, one that removes 47 amino acids following the signal sequence (DellaPenna and Bennett, 1988), and another that removes 13 amino acids from the C terminus of the precursor protein (Sheehy et al., 1987). Both of these proteolytic events are presumed to occur post-translationally, al- though the subcellular sites of these cleavages and whether they occur in one or in multiple steps are not known.

    Although all three polygalacturonase isozymes are ac- tive when assayed in vitro (Pressey and Avants, 1973; Tucker et al., 1980; Ali and Brady, 1982), their distinct temporal patterns of accumulation in ripening tomato fruit have led to some controversy regarding the function of each isozyme in vivo. Pressey (1988) has suggested that PG1 is an artifact of extraction, PG2A and PG2B being the only isozymes present in vivo, whereas Knegt et al. (1988) have presented data contrary to this view and proposed that PG1 is the physiologically functional isozyme in vivo. Recent analysis of polygalacturonase isozyme accumula- tion in transgenic tomato fruit exhibiting reduced levels of polygalacturonase has led to the proposal that the PG1 and PG2 isozymes are responsible for distinct aspects of polyuronide degradation (Smith et al., 1990), whereas a

    Synthesis, Processing, and Isozyme Composition of Polygalacturonase in Transgenic Tobacco

    A full-length tomato fruit polygalacturonase cDNA (DellaPenna and Bennett, 1988) was fused to the cauli- flower mosaic virus 35S promoter and NOS 3' terminator in the binary vector pMON530 and introduced into tobacco by way of •Agrotoacter/i/m-mediated transformation. Trans- formants selected for kanamycin resistance and nopaline synthase activity were assayed for the presence of poly- galacturonase protein by immunoblotting with polygalac- turonase antiserum. Figure 1 shows an immunoblot of crude cell wall extracts from leaves of five different trans- genic tobacco plants and one control plant transformed with vector only. Levels of immunologically detectable polygalacturonase varied among transformants, as shown by the varying band intensities in Figure 1. Polygalacturon- ase was also detected in cell wall extracts from stems and roots of transgenic tobacco plants, but at somewhat lower levels than in leaves (data not shown).

    The immunoblot in Figure 1 also shows that two immu- noreactive polypeptides corresponding to purified tomato fruit PG2A and PG2B accumulated in transgenic tobacco leaves. This was true for all transgenic plants that had immunologically detectable levels of polygalacturonase. These results clearly demonstrated that PG2A and PG2B represent differentially processed forms of a single gene product because a single cDNA was transferred into to- bacco. In addition, the electrophoretic mobilities of these polypeptides on SDS gels (Figure 1) and native gels (see below) are identical to the mobilities of authentic tomato fruit PG2A and PG2B, suggesting that the cotranslational a