| Trichomes are specialized structures which originate and project from the above-ground epidermal tissues of most plants. A vast array of trichome morphologies occur on the organ surface of different plants or the same plant of a given species, with diverse structure and spacing. In the field of plant molecular biology, the trichome is a useful cell type for studying regulation of plant cell differentiation. It is morphologically distinct from surrounding epidermal cells, and its genesis and development from this population are readily observed due to its accessibility to visual inspection and its relatively large size.In this study, trichome development related MYB genes were selected and constructed for over-expression and RNAi suppression in tobacco. Based on the evaluation of their impact on leaf trichome, root hair development and flower pigmentation in transgenic plants, we try to recognize the peculiar role that they played in different plant species, from which credible clues and new gene resource for engineering the MYB transcription factors in ornamental plants could be provided. The main results are as follows:1. Trichome development related MYB genes in Arabidopsis and cotton were constructed for over-expression and RNAi suppression. The sequences from petunia EST database were blasted with trichome development related MYB genes from contton and snapdragon, the ones with high scores were constructed for RNAi suppression. These plant binary expression vectors were transformed into tobacco by the mediation of agrobacterium. All transgenic plants were carefully observed and used for positive test. We found transgenic plants of one gene, named AtCPC, showed significant alterations in an ornamental property, thus this gene was selected for further functional analysis.2. In transgenic tobacco plants overexpressing AtCPC and their progeny, the pigmentation of corolla were partially or totally affected, and flowers of different lines showed different degrees of variation.3. AtCPC RNAi construct was twice transformed into transgenic tobacco plants overexpressing AtCPC with pure white flowers. The result showed the phenotype of twice tranformants was restored, from which it could be concluded that the heterologous AtCPC expression was responsible for the flower pigmentation phenotype observed here.4. Root hairs of T2 generation seedlings of transgenic tobacco overexpressing AtCPC were observed using a stereomicroscope and photographed. Leaf trichomes of T1 generation mature plants of transgenic tobacco overexpressing AtCPC were observed with a scanning electron microscope (SEM) and photographed. The numbers of both long and short trichomes were counted and analyzed, respectively. We found the ectopic expression of AtCPC also affected trichome/root hair distribution in transgenic tobacco.5. Petal epidermal cells of transgenic tobacco overexpressing AtCPC were visualized by paraffin section and SEM analysis, which indicated that varying degrees of shape variation of petal epidermal cells occurred in different lines. And it seemed that the varying degree of this phenotype has some relationship with the different degrees of flower pigmentation.6. The content of anthocyanin and flavonol in transgenic tobacco overexpressing AtCPC were measured using spectrophotometer and high performance liquid chromatography, respectively. Compared with the relative content in the wild type plants, no significant changes in flavonol content were detected while the anthocyanin concentration was remarkably decreased, and the decreasing amplitude is up to 63.03% in transgenic tobacco plants with white flowers.7. The results of AtCPC transcript accumulation analysis for transgenic tobacco plants showed that the severity of flower pigmentation phenotype correlates well with expression levels. And the transcript levels of AtCPC in twice transformed tobacco were also evaluated, which reconfirmed our conclusion that the strength of the flower pigmentation phenotype in the transgenic lines was dependent on the relative expression levels of the introduced AtCPC gene.8. The transcript levels of seven structural genes encoding the enzymes of the flavonoid pathway were measured. The results indicated that the main effect of AtCPC overexpression is the repression of the expression of three genes (NtDFR, NtANS and Nt3GT) at the lower end of the flavonid biosynthetic pathway, which were more directly related to the biosynthesis of anthocyanins.9. Several petunia genes encoding anthocynanin biosynthesis regulators were selected and examined whether they could interact with AtCPC by yeast two hybrid assays. Based on the colony growth on the selective medium and the detection ofβ-galactosidase activity, we considered that AtCPC did interact with two bHLH proteins (PhAN1 and PhJAF13).All those results demonstrated that AtCPC played a novel role in tobacco:a regulator of flavonoid secondary metabolism.
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