Studies On The Mechanisms Of Enhanced Chilling Tolerance In Potato Transformed With AtCBF3 Gene

Low temperature may cause damages which include many aspects such as the change of the membrane lipids phase, the accumulation of some secondary metabolites, the level of reactive oxygen species and so on. All these phenomena result from the damages of some enzymes which were involved in the metabolism of plants cells. Photo systems are also very sensitive to low temperature stress, under the low temperature stress Reactive Oxygen Species (ROS) accumulates a great deal, which results in photo apparatus being destroyed because of oxidation stress, Fv/Fm and other indices all deceased,at this times enzymes that catalysize photo dark reaction also reduce, at last appear significantly reducing.In the present study, the CBF3 genes of Arabidopsis thaliana are the target genes, The Arabidopsis thaliana CBF3 plant vector were introduced into potato plants by Agrobaterium tumefaciens-mediated transformation with floral dipping method under the control of CaMV35S promoter and rd29A promoter and obtained the transgenic plants. Physiological functions of these genes and molecular experiments were analyzed in the transgenic plants and wild types when they were exposed to low temperature. The main results are as follows:(1) After chilling treatment, CO₂ assimilation rate, apparent quantum yield and carboxylation efficiency of wild type potato leaves decreased greater than that of the CBF3 transgenic potato plants, rd29ACBF3 plants showed the highest resistant of CO₂ assimalation rate, apparent quamtum yield and carboxylation efficiency to chilling treatment.(2) After chilling treatment, Fv/Fm of AtCBF3 transgenic plants decreased slower than wild type plants accompanied by the slight inhibition of theirΦPSâ…¡. The increase of NPQ was faster than wild type plants, which indicated that when the carbon assimilation was inhibited at low temperature, NPQ of transgenic plants could play the role of dissipation in the excess energy and protecting the photo system. All these data show that the CBF3 gene can protect potato plants from the damages of photoinhibition at low temperature, it might be related to the over-expression of its downstream genes.(3) The analysis of chlorophyll fluorescence demonstrated that photo systemâ…¡(PSâ…¡) in transgenic plants showed higher chilling tolerance than in wild type plants, suggesting that CBF3 leads to increased tolerance to low temperature-enhanced photoinhibition. This increased tolerance was associated with an improvement of oxygen-evolving complex and the reaction center of PSâ…¡to chilling treatment.(4) Chilling stress induced a significant accumulation of H₂O₂, O₂.– in wild type and transgenic plants, but this accumulation was much less in transgenic plants. The AtCBF3 transgenic plants decreased the accumulation of ROS through maintain relatively higher antioxidant enzyme activities and antioxidants content.(5) The antioxidant enzyme activity of AtCBF3 transgenic plants were improved, which suggested that the transgenic plants could elevate the chilling tolerance.(6) Effects of chilling treatment on the relative electrical conductivity and the MDA content of the wild-type and CBF3 transgenic potato leaves. The contents of MDA and the relative electrical conductivity in controls were significantly higher than that in transgenic seedlings. The results suggest that low-temperature can cause damages to the wild type.(7) After chilling treatment, the soluble sugars and proline content of transgenic potato were higher than wide type plants. The results suggest that low-temperature can cause damages to the wild type. So, we can concluded that over-expressed constitutively CBF3 can improved the cold-resistance of potato plants indeed.(8) Compared with wild type plants,transgenic potatoes of 35S:AtCBF3 suffered serious growth inhibition, leaf shape, color and tubers weight and number are changed. In rd29A:AtCBF3 potato plants the phenomenon about growth was relieve effectively. In transgenic plants chlorophyll content increased than wild type plants. The aim of the present study was to assess the tolerance of ectoptic AtCBF3 potato plants to chilling stress. In transgenic potato plants, AtCBF3 activity also mimicked cold treatment by increasing proline and total sugar contents in the absence of cold. Relative to wild-type plants, ectopic AtCBF3 expression also induced expression of COR genes, changed the activity of antioxidant enzyme. Thus, it was of interest to determine whether AtCBF3 had an effect on the expression of predicted genes. Difference between enzyme activities and mRNA levels was observed, indicating that the control of the expression of plant antioxidant defenses is complex. In addition, the transgenic potatoes were moretolerant to the long-term chilling stress than in the wild type. These results suggest that CBF3 gene plays an important role in increasing potato freezing tolerance.