Graduate Thesis Or Dissertation

Enhancement of cold tolerance in tomato plants by genetic engineering of glycinebetaine biosynthesis

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  • Tomato [Lycopersicon esculentum Mill.] plants are chilling sensitive and do not naturally accumulate glycinebetaine (GB), a metabolite that functions as a stress protectant in plants. While GB can increase tolerance of tomato plants to salt and drought stresses, its effect on chilling tolerance has not been examined. To evaluate whether GB improves chilling tolerance, tomato (cv. Moneymaker) plants were treated with foliar-applied GB. GB-treated plants exhibited enhanced chilling tolerance. During chilling treatment, GB-treated plants maintained lower H₂O₂ levels but a higher degree of catalase activity compared with the controls. The protective effect of GB on enhanced chilling tolerance disappeared within a week after the application. These results suggested genetic engineering of a biosynthetic GB pathway into tomato plants may provide constitutively enhanced chilling tolerance. Tomato plants (cv. Moneymaker) were transformed with a chloroplast-targeted codA gene of Arthrobacter globiformis, which encodes choline oxidase (COD) that catalyzes the conversion of choline to GB. Transgenic plants accumulated GB and their chloroplasts contain up to 86 % of total leaf GB. Over various developmental phases, transgenic plants were more tolerant of chilling stress than their wild-type (WT) counterparts. Transgenic plants also maintained lower H₂O₂ levels but a higher degree of catalase activity under chilling stress compared with the controls. Finally, GB accumulation in the chloroplasts of transgenic plants was positively correlated with their level of chilling tolerance, although GB levels in transgenic plants were very low compared to those in natural GB-accumulator plants. To increase the amount of GB in transgenic tomato plants, three codA expression cassettes were constructed to target the chloroplasts (Chl-codA), cytosol (Cyt-codA), or both locations (ChlCyt-codA). Targeting COD to the cytosol or to both locations resulted in higher GB accumulations in the Cyt-codA and ChlCyt-codA lines compared to the Chl-codA lines. Regardless of targeted location and the different amounts of GB, all three types exhibited similar degrees of enhanced tolerance to various abiotic stresses compared with the WT plants. The codA transgenic plants produced significantly enlarged flowers and fruits, which are a consequence of increased cell number and size. Fruit enlargement was caused by a pleiotropic effect of the codA gene transfer. Expression analysis revealed altered expression of genes that are involved in cell division. These results suggest that enlarged flowers and fruits are the pleiotropic effects of codA transgene expression, which may be useful for further improvement of these traits. All together, these results demonstrated that exogenous GB application enhanced chilling tolerance of non-transgenic tomato plants, and moreover introduction of a GB biosynthetic pathway into tomato confers enhanced tolerance to abiotic stresses, as well as increase in sizes of flowers and fruits.
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