Analysis of tomato polygalacturonase expression in transgenic tobacco.

Abstract

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, 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.

Activity and expression of polygalacturonase vary at different fruit ripening stages of sweet pepper cultivars

“…The results are similar to the findings of scientists who worked on the role of PG during fruit development in various kinds of fruits and found a correlation, as PG activity and softening occurs simultaneously in cherry (Barrett and Gonzalez, 1994), date (Hasegawa et al, 1969), durian (Ketsa and Daengkanit, 1999), kiwifruit (Wang et al, 2000), mango (Ketsa et al, 1998), papaya (Paull and Chen, 1983), pear (Ahmed and Labavitch, 1980), pepper (Rao and Paran, 2003;Arancibia and Motsenbocker, 2006), tobacco (Osteryoung et al, 1990), and tomato (Hobson, 1965;Crookes and Grierson, 1983;Huber, 1983;DellaPenna et al, 1987;Bird et al, 1988;Biggs and Handa, 1989;Smith et al, 1990;Fenwick et al, 1996).…”

“…It is believed that transition of fruit ripening is the result of a complex developmental program influencing metabolic processes in each compartment of the cell. Primarily, this developmental program occurs due to the degradation of pectic polymers in the cell wall, which is brought about by the action of ripening-induced enzymes, such as polygalacturonase (PG) (Themmen et al, 1982;Crookes and Grierson, 1983;Huber, 1983;Giovannoni et al, 1989;Osteryoung et al, 1990). Although the ripening mechanism has been widely studied and reviewed, it is still far from being clearly understood (Brady, 1987).…”

“…The softening phenomenon influenced by PG in fruits like apples, cherries, dates, durians, grapes, kiwis, melons, peaches, pears, pineapples, tobacco, and tomatoes during ripening has been studied during the past two decades and substantial evidence has been collected, suggesting that the cell wall enzyme PG plays a major role in the entire ripening process (Hobson, 1965;Hasegawa et al, 1969;Ahmed and Labavitch, 1980;Paull and Chen, 1983;Osteryoung et al, 1990;Barrett and Gonzalez, 1994;Ketsa et al, 1998;Ketsa and Daengkanit, 1999).…”

“…Although the ripening mechanism has been widely studied and reviewed, it is still far from being clearly understood (Brady, 1987). Some authors have reported that the fruit softening is largely associated with breakdown of the fruit cell wall (Crookes and Grierson, 1983;Osteryoung et al, 1990;Rao and Paran, 2003). The primary cell wall is composed of several polymers, including cellulose, glycans and pectins that are modified during fruit ripening (Brummell and Harpster, 2001;Giovannoni, 2001).…”

Targeted Modification of Homogalacturonan by Transgenic Expression of a Fungal Polygalacturonase Alters Plant Growth

“…Either up-and down-regulation in tomato of an endogenous gene encoding an endopolygalacturonase involved in fruit softening had no apparent effect on growth and development (Giovannoni et al, 1989;Smith et al, 1990). The expression of a tomato PG in tobacco (Nicotiana tabacum) resulted in no phenotype (Osteryoung et al, 1990), whereas the overexpression of an apple (Malus domestica) fruit PG in apple resulted in novel phenotypes involving changes in cell adhesion (Atkinson et al, 2002).…”