Functional Analysis Of GhPAP1in Transgenic Cotton (Gossypium Hirsutum L.)

Anthocyanins, a diverse set of flavonoids, are water-soluble plant pigments that contribute to red, purple or blue color of flowers and fruits. In the plant kingdom, these attractive colors can draw the attention of pollinators and seed dispersers. In addition, anthocyanins can function to protect plants against UV irradiation, cold temperature, pathogen invasion, insect attack and herbivores feed.Recent studies have unraveled that the MYB transcription factors (i.e., Arabidopsis thaliana PAP1) play a major role in regulating the anthocyanin biosynthesis pathway. In our previous work, it was found that cotton PAP1homologous gene GhPAP1have the biological function to enhance anthocyanin production. In this study, we further analyzed the function of GhPAP1and the expression pattern of GhPAP1promoter in transgenic cottons.The main results are as followings.1. Red color in red-plant cotton resulted from anthocyanin accumulationIn a recombinant inbred line population, we found that the anthocyanin content and the expression levels of three anthocyanin biosynthetic structural genes(GhCHS1、 GhANS1、GhGT13) and GhPAPlwere much higher in red-plant lines than green-plant lines. Furthermore, the high levels of gene expression and anthocyanin accumulation co-segregated with red-plant trait in the population. This result showed that anthocyanin biosynthesis and accumulation determined the red color in cotton leaves.2. Overexpression of GhPAP1promoted anthocyanin accumulation and red pigmentation m cottonsGhP4P1-overexpressing vector was introduced into a green-plant cotton cultivar "Jimian14" via Agrobacterium-mediated cotton transformation. Phenotypic analysis of the GhPAP1over-expressers showed that the leaves, flowers, ovules of the transgenic plants exhibited dark purple with increased anthocyanin levels, indicating that GhPAPl overexpression might up-regulate anthocyanin biosynthesis pathway in these cotton organs. In spite of significant increased GhPAP1expression level in fibers, no color variation was observed in transgenic fibers. 3. GhPAP1activated structural genes of anthocyanin pathway both in leaves and fibers of transgenic cottonWe further examined the expression levels of the anthocyanin structural genes by quantitative RT-PCR. The expression levels of anthocyanin structural genes CHS, CHI, DFR, ANS, GT13were increased in GhPAP1-overexpressing cottons.4. Overexpression of GhPAP1inhibited fiber elongation.The fiber length of the overexpression cotton lines was shorter than that of control. Scanning electron microscopy observation indicated that fiber initiation on ovule epidermis at ODPA and1DPA delayed in GhPAP1-overexpressing cottons. Those suggested that high GhPAP1expression might suppress the development of cotton fiber.5. The expression pattern of GhPAPl promoterThe GhPAP1promoter was constructed upstream to the repoter gene GUS and introduced into cotton cultivar’Jimian14’via A. tumefaciens-mediated transformation. GUS histochemical assay showed positive staining in roots, stems, leaves, flowers and ovules of transgenic plants, demonstrating the GhPAPl promoter had activity in the related cotton tissues.In the present study, we examined the effects of GhPAPl overexpression on anthocyanin accumulation and the expression pattern of GhPAP1promoter in transgenic cottons. The results showed that GhPAP1played an important role in regulating anthocyanin biosynthesis and accumulation.