Promoter of a cotton fibre MYB gene functional in trichomes of Arabidopsis and glandular trichomes of tobacco

Abstract

Cotton fibres are unicellular seed trichomes. Our previous study suggested that the cotton R2R3 MYB transcript factor GaMYB2 is a functional homologue of the Arabidopsis trichome regulator GLABRA1 (GL1). Here, the GaMYB2 promoter activity is reported in cotton (Gossypium hirsutum), tobacco (Nicotiana tabacum), and Arabidopsis plants. A 2062 bp promoter of GaMYB2 was isolated from G. arboreum, and fused to a beta-glucuronidase (GUS) reporter gene. In cotton, the GaMYB2 promoter exhibited activities in developing fibre cells and trichomes of other aerial organs, including leaves, stems and bracts. In Arabidopsis the promoter was specific to trichomes. Different from Arabidopsis and cotton that have unicellular non-glandular simple trichomes, tobacco plants contain more than one type of trichome, including multicellular simple and glandular secreting trichomes (GSTs). Interestingly, in tobacco plants the GaMYB2 promoter directed GUS expression exclusively in glandular cells of GSTs. A series of 5'-deletions revealed that a 360 bp fragment upstream to the translation initiation codon was sufficient to drive gene expression. A putative cis-element of the T/G-box was located at -233 to -214; a yeast one-hybrid assay showed that Arabidopsis bHLH protein GLABRA3 (GL3), also a trichome regulator, and GhDEL65, a GL3-like cotton protein, had high binding activities to the T/G-box motif. Overexpression of GL3 or GhDEL65 enhanced the GaMYB2 promoter activity in transgenic Arabidopsis plants. A comparison of GaMYB2 promoter specificities in trichomes of different plant species with different types of trichomes provides a tool for further dissection of plant trichome structure and development.

Fig. 1.

GUS expression pattern and activities in transgenic cotton (G. hirsutum) plants expressing P-2000::GUS. (A) 0-DPA ovule; (B) 9-DPA fibre (top) and ovule (bottom); (C) leaf; (D) stem; (E) bract; (F) quantitative analysis of GUS specific activities in different organs. (This figure is available in colour at JXB online.)

Fig. 2.

Histochemical staining of GUS expression pattern in transgenic Arabidopsis plants expressing P-2000::GUS. (A) 3-week-old seedling; (B) rosette leaf; (C) stem; (D) flowers showing trichomes on sepal. (This figure is available in colour at JXB online.)

Fig. 3.

Histochemical staining of GUS expression pattern in transgenic tobacco plants expressing P-2000::GUS, GUS activity was detected in glandular head cells of the multicellular glandular secreting trichome (GST). (A) GSTs on leaf; (B) GSTs on stem; (C) GSTs on flower bract; (D) multicellular simple trichomes on the base of anther filament; (E) magnified view of a leaf GST; (F) magnified view of an anther filament simple trichome. Bar = 100 μm. (This figure is available in colour at JXB online.)

Fig. 4.

Analysis of GaMYB2 promoter activities with different deletions, GUS activities in transgenic Arabidopsis plants were assayed. (A–D) GUS staining of rosette leaves of P-2000 (A), P-360 (B), P-220 (C), and P-AB1 (D) plants; (E) quantitative analysis of GUS specific activities in rosette leaves of Arabidopsis transformed with GUS gene driven by different versions of GaMYB2 promoters as indicated (see also Supplementary Table S4 at JXB online). Leaves of 3–4-week-old plants of ten different transgenic lines were used for GUS assay. **P ≤0.01 versus P-2000 plants. (This figure is available in colour at JXB online.)

Fig. 5.

GUS staining of tobacco plants transformed with the GUS gene driven by the GaMYB2 promoter with different deletions. (A–D) GSTs on leaf of the P-2000 (A), P-360 (B), P-220 (C), and P-AB1 (D) plants. All the plants assayed were at rooting stage. (This figure is available in colour at JXB online.)

Fig. 6.

Transcriptional activation of GaMYB2 promoter by bHLH protein. (A) DNA–protein interaction in a yeast one-hybrid system. pGAD-GL3 and pGAD424 plasmids were transformed into a yeast strain carrying dual report genes under the control of four-time tandem repeats of the T/G-box element. The transformants were examined for growth in the presence of 3-AT and β-galactosidase (β-Gal) activity using X-Gal as substrate. Only the yeast clones harbouring pGAD-GL3 grew on the -His-Ura-Leu synthetic dextrose (SD) base containing 20 mM 3-AT and also showed a high β-Gal activity (blue staining), demonstrating binding activity of GL3 to the T/G-box. (B) Comparison of T/G-box binding activity of four bHLH proteins. The DNA–protein interaction was determined by β-Gal activity measured through an ONPG assay. **P ≤0.01 and *P ≤0.05 versus pGAD-GL3 binding activity. (C) RT-PCR analysis of expression of GL3 and GUS genes in 35S::GL3/ P-440::GUS Arabidopsis plants. Total RNA was isolated from leaves of 4-week-old plants, Actin2 (At3g18780) was amplified as an internal control. (D) Enhanced activation of the GaMYB2 promoter by constitutive expression of GL3 and GhDEL65 in P-440 plants. Leaves of 4-week-old plants (eight transgenic lines) were used for the GUS activity assay, the mean value of GUS specific activities of P-440 plants was set as 100% and used to define the relative GUS activity of 35S::GL3/ P-440::GUS and 35S::GhDEL65/P-440::GUS plants, respectively. **P ≤0.01 versus P-440 plants. (This figure is available in colour at JXB online.)