The R2R3-MYB gene CgMYB4 is involved in the regulation of cell differentiation and fiber development in the stamens of Chelone glabra L.

A Plantaginaceae flowering plant, Chelone glabra, is different from Arabidopsis thaliana and cotton (Gossypium hirsutum), as it produces fibers on the anther surface. However, the evolutionary molecular mechanism of how fiber development is controlled in the stamen is unclear. MYB genes are essential transcription factors for trichome and fiber development in plants. In this study, we isolated 29 MYB domain-containing sequences using early-stage anthers and several sets of degenerated primers conserved in the R2R3 domain of the MYB transcription factor. Among them, CgMYB4 is an R2R3-MYB gene encoding 281 amino acids. Phylogenetic analysis showed that CgMYB4 is closely related to GhMYB25L/AmMIXTA, which controls fiber initiation and development in cotton and epidermal cell differentiation in the petals of Antirrhinum. Semiquantitative RT-PCR analysis showed that CgMYB4 is strongly expressed at the stamens and carpels. Overexpression of CgMYB4 significantly enhanced root hair formation in transformed hairy roots, contrary to the root hair numbers, which were reduced in silenced CgMYB4 hairy roots. Moreover, overexpression of CgMYB4 also evidently promoted fiber development at filaments and conical cell-like epidermal cell increases at the anther wall. Our results showed that CgMYB4 is an R2R3-MYB gene and is positively involved in regulating cell division and fiber differentiation in the early stages of stamen development in C. glabra.

A rapid and stable Agrobacterium-mediated transformation method of a medicinal plant Chelone glabra L.

Transformation approach is a useful tool for the study of gene function, the mechanism of molecular regulation, and increase usefulness of components by reverse genetic approach in plants. In this study, we developed a stable and rapid method for Agrobacterium-mediated transformation of a medicinal plant Chelone glabra L. using leaf explants. Stable transformants were obtained using Agrobacterium tumefaciens strains GV2260 and GV3101 that harbored the binary vector pBI121 and contained the neomycin phosphotransferase gene (NPT II) as a selectable marker and a reporter gene ß-glucuronidase (GUS). Putative transformants were identified by kanamycin selection and a histochemical assay. PCR and Southern blot analysis confirmed the integration of the GUS gene into transformed genomes as well as detected stable expression of the ß-glucuronidase gene (GUS) by RT-PCR. Resulting transformed plants had morphologically normal phenotypes. This method requires two changes of medium and few leaf explants as well as the transformation efficiency of 2-8 % after 2-3 months of inoculation. This method can provide a quick and economical transformation method for reverse genetic approach to change the secondary metabolic pathway to increase useful components in C. glabra.

A stable and efficient Agrobacterium tumefaciens-mediated genetic transformation of the medicinal plant Digitalis purpurea L.

In this study, we developed a rapid and efficient method for in vitro propagation and Agrobacterium tumefaciens-mediated transformation of Digitalis purpurea L. (syn. foxglove), an important medicinal plant. Mature leaf explants of D. purpurea were used for 100 % adventitious shoot regeneration on Murashige and Skoog (MS) medium supplemented with 1 mg L(-1) thidiazuron (TDZ) (a cytokine) and 0.1 mg L(-1) 1-naphthaleneacetic acid (NAA) (an auxin). Transformation was achieved by inoculating leaf explants with the A. tumefaciens strains GV2260/pBI121 or GV3101/pBI121. The binary vector pBI121 contained the reporter ß-glucuronidase gene (GUS) and kanamycin selection marker nptII. Kanamycin-resistant shoots were regenerated directly on the selection medium 4-6 weeks after co-cultivation. Approximately, 52.2 and 60 % of kanamycin-resistant shoots transformed with Agrobacterium strains GV2260 and GV3101, respectively, showed strong GUS staining by histochemical assay. Furthermore, PCR and Southern blot analysis confirmed the presence of nptII and GUS on the chromosome of the transformed D. purpurea plants, and stable GUS expression was detected in the transformants by RT-PCR analysis. This efficient method of shoot regeneration and genetic transformation of D. purpurea will provide a powerful tool to increase and produce valuable components such as digitoxin, digoxin, and digoxigenin in D. purpurea through improved secondary metabolic pathways via a biotechnological approach.

The promoter structure differentiation of a MYB transcription factor RLC1 causes red leaf coloration in Empire Red Leaf Cotton under light.

The red leaf coloration of Empire Red Leaf Cotton (ERLC) (Gossypium hirsutum L.), resulted from anthocyanin accumulation in light, is a well known dominant agricultural trait. However, the underpin molecular mechanism remains elusive. To explore this, we compared the molecular biological basis of anthocyanin accumulation in both ERLC and the green leaf cotton variety CCRI 24 (Gossypium hirsutum L.). Introduction of R2R3-MYB transcription factor Rosea1, the master regulator anthocyanin biosynthesis in Antirrhinum majus, into CCRI 24 induced anthocyanin accumulation, indicating structural genes for anthocyanin biosynthesis are not defected and the leaf coloration might be caused by variation of regulatory genes expression. Expression analysis found that a transcription factor RLC1 (Red Leaf Cotton 1) which encodes the ortholog of PAP1/Rosea1 was highly expressed in leaves of ERLC but barely expressed in CCRI 24 in light. Ectopic expression of RLC1 from ERLC and CCRI 24 in hairy roots of Antirrhinum majus and CCRI 24 significantly enhanced anthocyanin accumulation. Comparison of RLC1 promoter sequences between ERLC and CCRI 24 revealed two 228-bp tandem repeats presented in ERLC with only one repeat in CCRI 24. Transient assays in cotton leave tissue evidenced that the tandem repeats in ERLC is responsible for light-induced RLC1 expression and therefore anthocyanin accumulation. Taken together, our results in this article strongly support an important step toward understanding the role of R2R3-MYB transcription factors in the regulatory menchanisms of anthocyanin accumulation in red leaf cotton under light.