| Fruit color, as one of the most important agronomic traits for fruit trees, determines the diet and market values to a great extent in apple and other fruit industries. The red skin color of mature apple fruit is predominantly attributable to the pigment anthocyanins. The biosynthesis of anthocyanins is affected by diverse environmental factors, especially light, temperature and nutrition. In addition, the earth is warming over the past century (Abu-Asab et al., 2001). These changes appear to have had a profound effect on fruit coloration in apple. It is of emergent importance for the maintainable development of apple production to elucidate the molecular mechanism by which the cool temperature induces or warm temperature inhibits controls anthocyanin biosynthesis and fruit coloration.In our study, a low-temperature responsed bHLH transcription factor (TF) was cloned from apple fruit, which was named MdTTL1 and registered in the GeneBank as JF920725. The full length of MdTTL1 is 2687bp and encodes a protein of 710 amino acids, and this TF contains the conserved bHLH (basic Helix-Loop-Helix) domain signature. A phylogenetic tree of MdTTL1 and all of Arabidopsis bHLH proteins was constructed by means of the neighbour-joining method. This analysis showed that MdTTL1 belong to the subgroupⅢf,and it closest homologs, AtTT8, so we named our protein after AtTT8 as MdTTL1(TT8 like 1). Further analysis indicated that its closest homologs, apple MdbHLH3, grape VvMYC1, petunidin PhAN1. RT-PCR analysis from the crude extract of apple organs showed that MdTTL1 was transcripted in the fruit skin, young leaf and stem, however, in colored apple skin highest, and its expression is induced by LT. Epitope prediction based on analysis and results of biological sequence alignment software, select a protein-coding, and the synthetic polypeptide were used to prepare antibodies. Western blot analysis showed that MdTTL1 protein was increased in the UV17℃and degradated in the UV27℃.Yeast two-hybrid assays (YTH) and Bimolecular Fluorescence Complementation (BiFC) demonstrated that MdTTL1 physically interacts with MdMYB1. Serially truncated analysis showed that N-terminal 1-228 amino acids is necessary for the interaction. Both EMSA and ChIP-PCR assays confirmed the specific in vitro and in vivo binding that occurred between MdTTL1 protein and these DNA hexamers. Further transient promoter assays promoter and GUS stable expression revealed that MdTTL1 positively regulate the expression of MdUFGT, and also suggest that at low temperature MdTTL1 protein remarkably boosted GUS activity.To determine the function of MdTTL1,transgenic Gala explant and Qrin callus were created with homologic expression of MdTTL1 or MdTTL1-GFP driven by cauliflower mosaic virus 35S promoter. After treated with low temperature and UVB, transgenic plants produced increased anthocyanin, while the MdTTL1 protein did not increase at low temperature, indicating that at low temperature there might be posttranslational modification for MdTTL1 protein.Western blot suggest that low temperature rapidly induces phosphorylation of MdTTL1 protein in apple callus, and no shift in eletrophoretic mobility was detected in the transgenic callus treated with 17℃plus staurosporine (10μm). More noticeably, transgenic Gala generated much more anthocyanin in root under low temperature conditions than those under nomal conditions. Transgenic plants overexpressing MdTTL1 genes exhibit pleiotropic phenotypes including dwarfism. ChIP-PCR showed that MdTTL1 protein binds to these E-boxes of CBF promoters. To further identify the function of MdTTL1 in apple fruit coloration, transient overexpression or antisence assay was performed with a universal vector IL-60-BS using the bagged fruit just detached from apple tree. Overexpression can promote the coloring process and inhibiting its expression inhibited the coloring around the infiltration sites, which shows MdTTL1 plays a positive role in apple coloration.
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