Genetic Engineering Of The Biosynthesis Of Glycinebetaine Leads To Increased Thermotolerance In Transgenic Tomato Plants

As sessile organisms, plants are continuously exposed to biotic and abiotic stress conditions during their whole life cycles. High temperature which affects the growth and the production of crops is a common abiotic stress during summer. Over time, plants have evolved mechanisms to overcome environmental stresses. One mechanism is the accumulation of compatible solutes such as glycinebetaine (GB). GB is one of the organic compatible solutes that can accumulate rapidly in many plants under salinity stress, drought and low temperature, and enhanced the tolerance to adverse conditions. In this study, the codA and BADH transgenic tomato were used to elucidate the effect of GB on tomato under high temperature during germination and seedling growth. Additionally, GB was exogenously applied to further investigate the role of GB in the enhancement of the tolerance to high temperature. The main results are as follows:1. After high temperature treatment during imbibition, the relative elective conductivity was greater in wild-type tomato seeds than in the codA transgenic seeds. The germination rate of codA transgenic seeds was higher than that of the wild-type seeds. During the recovery after long time high temperature treatment, the germination rate of codA transgenic seeds was higher than the wild-type seeds. The codA transgenic tomato seeds showed more thermotolerance than that of the wild type seeds.2. Exogenous application of low concentration of GB on MS medium increased the germination rate of the wild-type seeds after high temperature treatment. Imbibition in low concentration of GB also enhanced the germination rate of the wild-type seeds.3. During high temperature treatment, the transcript of gene for HSP70, HSC70 and MT-sHSP were more in the codA transgenic seeds than in the wild-type seeds. The accumulation of HSP70 was more in the codA transgenic seeds than in the wild-type seeds.4. High temperature stress decreased the CO2 assimilation. After high temperature treatment, CO2 assimilation rate, apparent quantum yield and carboxylation efficiency of wild-type tomato leaves decreased greater than that of the codA transgenic tomato leaves.The codA transgenic tomato plants showed higher thermotolerance of photosynthesis than wild type tomato plants.5. The analysis of chlorophyll fluorescence demonstrated that photosystemII (PSII) in codA transgenic plants showed more high temperature tolerance than in wild type plants, suggesting that the accumulation of GB leads to increased tolerance to high temperature-induced photo inhibition. This increased tolerance was associated with an improvement of oxygen-evolving complex and the reaction center of PSII to heat stress.6. Heat stress increased the accumulation of H2O2, O2 in the wild-type and transgenic tomato, but this accumulation was less in transgenic tomato. High temperature decreased the expression of CAT1 in the wild-type and codA transgenic tomato leaves. However the decrease was more in the wild-type plants than in the codA transgenic plants. GB decreased the accumulation of ROS through maintain relatively higher antioxidant enzyme activities.7. Exogenous application of GB also enhanced the thermotolerance of photosynthesis of wild type tomato plants.8. The gene for betaine aldehylde dehydrogenase (BADH) was successfully cloned from spinach which was supplied with 200 mM NaCl for 4 d. After the construction of eukaryote expressing vector, the A. tumefaciens mediated infection was used to infect the tomato leaf discs. After the PCR and western blot analysis of the kanamycin resistant tomato plants, the BADH transgenic seedlings were obtained.9. BADH transgenic tomato plants also showed higher thermotolerance of photosynthesis than that of wild type plants.10. High temperature induced the accumulation of HSP70 in the wild-type and codA, BADH transgenic tomato leaves. The accumulation was greater in the transgenic plants than in the wild-type plants. Exogenous application of GB in wild type tomato plants also increased the accumulation of HSP70 during high temperature stress.11. High temperature induced the degradation of D1 protein. The content of D1 protein was more in the codA, BADH transgenic tomato than that of the wild-type plants. Meanwile, the wild-type tomato plants supplied with exogenous GB also maintained greater content of D1 protein when exposed to high temperature stress.The results in this study indicated that tomato plants transformed with codA or BADH gene enhane the thermotolerance, and this resulted in the biosynthesis of glycinebetaine in transgenic plants. Under high temperature stress, the transgenic seeds and plants maintained more HSP and higher activities of antioxidant enzymes, which may associated with the improvement of the expression of some genes for heat shock protein and antioxidant enzymes by glycinebetaine during high temperature stress.