Author
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Smith, David |
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Gross, Kenneth |
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Submitted to: Plant Physiology
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 3/26/2000 Publication Date: N/A Citation: N/A Interpretive Summary: Because of the critical relationship changes in fruit texture have to quality and shelf-life, we are studying mechanisms involved in the loss of firmness that occurs during tomato ripening. A critical feature of fruit softening is the disassembly of the cell wall (the sugar envelope surrounding plant cells) that occurs during ripening. If this disassembly and loss of integrity can be slowed down, we should be able to create fruit that can be picked vine-ripe and still withstand current commercial handling practices. Previous research showed a dramatic loss of galactose (a sugar cell wall component) from the wall during ripening, and we propose this loss may play a significant role in softening. In the present research, several enzymes (proteins that accelerate specific transformation of materials) were identified in tomatoes which can remove galactose from the cell wall. These enzymes are called beta-galactosidases; each is encoded for by a single corresponding gene. We identified seven beta-galactosidase genes/enzymes that exist and are active during tomato fruit development. By cloning the DNA for these genes, we were able to study when and where they are expressed in tomato fruit. Based on this information the genes most likely to be involved in the removal of galactose from the cell wall during ripening were determined. The cloned genes and knowledge of their regulation in tomato fruit now provide us with the tools necessary to use molecular genetic techniques to turn off each of the seven genes, individually or in groups, and to determine what direct effect they have on fruit softening. Technical Abstract: At least seven beta-galactosidase (EC 3.2.1.23) genes are expressed during tomato (Lycopersicon esculentum Mill.) fruit development. During our search for a cDNA encoding beta-galactosidase II, an enzyme known to have exo-galactanase activity and be present during fruit ripening, five full-length and two partial-length tomato beta-galactosidase (TBG) clones were identified. All of the TBG clones contain the putative active site-containing consensus sequence pattern G-G-P-[LIVM]-x-Q-x-E- N-E-[FY] belonging to glycosyl hydrolase family 35 [3, E1]. Six of the seven single-copy genes were mapped. Six of the genes were expressed during fruit ripening stages, whereas TBG6 expression ceased before the onset of ripening. Five of the seven genes were expressed in all fruit tissues. Only TBG2 mRNA appeared to have a fruit-specific expression pattern and mRNA for the other TBGs was detected in either root, stem, leaf or flower tissues, mRNA abundance was essentially the same in wild- type and rin, nor and Nr mutant fruit for five of the seven genes, however TBG4 mRNA level was reduced and TBG6 mRNA persisted longer than normal in the three mutants. The TBG4 encoded protein was expressed in yeast and exo-galactanase activity was confirmed via a quantified release of galactosyl residues from cell wall fractions containing (1,4)-D-galactan purified from tomato fruit. |
