| Anthocyanins, which are colored products of the flavonoid biosynthetic pathway, have received particular attention in recent years. Their accumulation is induced by a range of developmental and environmental signals. Anthocyanins function as an antioxidant and photoprotective screens and play important roles in many physiological processes, including photoprotection, stress resistance, pollination and seed dispersal. In addition, anthocyanins are potentially health-protecting components in the human diet. Anthocyanin accumulation is mainly regulated at the transcriptional level by a transcriptional complex consisting of R2R3-MYB transcription factors(TF), basic helix-loop-helix(bHLH) TFs, and a WD-repeat protein(MBW complex), which activates the transcription of biosynthetic genes. SlAN2 encodes an R2R3-MYB TF involved in anthocyanin regulation in tomato.Under abiotic stresses, imbalance in metabolic processes may lead to an increased accumulation of reactive oxygen species(ROS). The excessive accumulation of ROS causes a variety of serious injuries when stress is severe. Fortunately, plants have developed enzymatic and non-enzymatic antioxidant defense systems to detoxify ROS. The non-enzymatic antioxidants, such as carotenoids, tocopherols, ascorbic acid, glutathione, and flavonoids scavenge ROS to protect plant cells. Anthocyanins may serve as antioxidants due to the hydroxyl groups present in the glycon. Tomato serves as a model for fruit ripening and SlAN2 appears unrelated to fruit ripening.In this study, the SlAN2 gene was isolated and characterized from tomato. The main results are as follows:(1) We isolated the SlAN2 gene from tomato. This gene contains an 825 bp open reading frame(ORF), and encodes 275 amino acids.(2) p35S-SlAN2-GFP fusion protein and transiently expressed in onion epidermal cells. It was observed with a fluorescence microscope that the SlAN2-GFP fusion protein was found in the nucleus.(3) qRT-PCR showed that the transcript of SlAN2 was highest in the stem, moderate in leaf, flower, and root, and lowest in fruit. The expression of SlAN2 was obviously induced by chilling and MV stresses.(4) The full-length SlAN2 cDNA was subcloned into the expression vector pBI121 to form sense and antisense constructs. The Agrobacterium tumefaciens-mediated leaf disk method was used to generate transgenic plants. The transgenic tomato plants and tobacco plants were verified by q RT-PCR. It was indicated that overexpression tomato plants, antisense tomato plants and overexpression tobacco plants were obtained. The anthocyanin level of sense transgenic tomato lines exhibited 2.2-3.2 fold increased of the WT and the transgenic tabacco lines exhibited 100-284 fold increased of the WT tobacco plants.(5) Transgenic tobaccos conferred tolerance to chilling stress by maintaining a higher chlorophyll content, net photosynthetic rate, and maximal photochemical efficiency of PSII compared to the wild type plants. Furthermore, the transgenic plants showed lower ion leakage, a lower content of reactive oxygen species, and a higher content of non-enzymatic antioxidans under the chilling stress. They also showed an enhanced resistance to the oxidative stress induced by methyl viologen based on a decreased chlorophyll content loss, lower ion leakage, and an enhanced maximal photochemical efficiency of PSII. These results indicate that overexpression of SlAN2 resulted in an increased anthocyanin accumulation and enhanced resistance to the chilling and oxidative stresses in transgenic tobacco.(6) Transgenic tomato plants showed enhanced tolerance to heat stress by maintaining higher fresh weight(FW), net photosynthetic rate(Pn) and maximal photochemical efficiency of photosystem II(PSII)(Fv/Fm) compared with wild-type(WT) plants. Furthermore, transgenic tomato plants showed higher non-enzymatic antioxidant activity, lower levels of reactive oxygen species(ROS), and higher contents of D1 protein than that in WT plants under heat stress. These results indicate that SlAN2 had an important function in heat stress resistance.(7) Fruits of transformants overexpressing SlAN2 displayed an orange colour, fast softening and elevated ethylene production. Overexpression of SlAN2 results in reduced carotenoids by an alteration of carotenoid pathway flux, elevated ethylene synthesis mainly by upregulation of ethylene biosynthetic genes and early softening of fruits by decreased the firmness of the fruits. We also found that the transcript of an important ripening-related gene, SlRIN, was up-regulated. These results suggest that SlAN2 acts as an important regulator of fruit ripening. |
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