Control of plant trichome development by a cotton fiber MYB gene

Abstract

Cotton (Gossypium spp) plants produce seed trichomes (cotton fibers) that are an important commodity worldwide; however, genes controlling cotton fiber development have not been characterized. In Arabidopsis thaliana the MYB gene GLABRA1 (GL1) is a central regulator of trichome development. Here, we show that promoter of a cotton fiber gene, RD22-like1 (RDL1), contains a homeodomain binding L1 box and a MYB binding motif that confer trichome-specific expression in Arabidopsis. A cotton MYB protein GaMYB2/Fiber Factor 1 transactivated the RDL1 promoter both in yeast and in planta. Real-time PCR and in situ analysis showed that GaMYB2 is predominantly expressed early in developing cotton fibers. After transferring into Arabidopsis, GL1::GaMYB2 rescued trichome formation of a gl1 mutant, and interestingly, 35S::GaMYB2 induced seed-trichome production. We further demonstrate that the first intron of both GL1 and GaMYB2 plays a role in patterning trichomes: it acts as an enhancer in trichome and a repressor in nontrichome cells, generating a trichome-specific pattern of MYB gene expression. Disruption of a MYB motif conserved in intron 1 of GL1, WEREWOLF, and GaMYB2 genes affected trichome production. These results suggest that cotton and Arabidopsis use similar transcription factors for regulating trichomes and that GaMYB2 may be a key regulator of cotton fiber development.

Figure 1.

Expression Patterns of G. arboreum RDL1, MYB2/FIF1, and HOX3 Genes. (A) Transcript profile of GaRDL1, GaMYB2, and GaHOX3 in plants of G. arboreum. Relative transcript amounts of each gene were normalized with respect to cotton Histone3 transcript levels (100%). Mean values were obtained from three independent PCR amplifications, and the error bars indicate the standard error of the mean. A break in the scale (=) has been incorporated to show the higher amount of GaRDL1 in fibers. R, roots; S, stems; L, leaves; O-0, 0-DPA ovules; F-3, 3-DPA fibers; F-6, 6-DPA fibers; F-9, 9-DPA fibers; F-12, 12-DPA fibers; NO-6, 6-DPA naked ovules (fibers stripped off). (B) GUS staining of Arabidopsis plants expressing RDL1-P3∷GUS. Young leaf (top), mature leaf (middle), and stem (bottom) are shown.

Figure 2.

Identification of cis-Elements in the RDL1 Promoter. (A) GUS activities driven by different upstream fragments of the cotton RDL1 gene. Nucleotides are numbered from the transcription initiation site. (B) Linker scanning of RDL1-P3. The 8-bp linkers (GGAATTCC or CCTGCAGG) were used. Red and pink boxes represent linkers of SCAN-1 through -20. The L1 box and MYB motif are underlined. (C) Effects of disruption of L1 box (L1-MU) and MYB motif (MYB-MU) on promoter activity. L1-MYB-MU indicates that both L1 box and MYB motif were mutated. The L1 box was mutated from TGCATTTA to TGCcgTTA and the MYB motif from CAGTTG to CAGTgG.

Figure 3.

Transcriptional Activation of RDL1-P3 by GaMYB2 and HOX3. (A) DNA–protein interactions in yeast one-hybrid system. Yeast strain YM4271 integrating RDL1-OH (−221 to −33 bp) or RDL-OH-MYB-MU was transformed with GAL4 transcriptional activation domain (AD) (1), AD-GL1 (2), AD-WER (3), AD-GaMYB1 (4), and AD-GaMYB2 (5). (B) Effects of ectopic expression of MYB (GaMYB2 or GL1) and HOX (HOX3 or GL2) genes on the RDL1-P3∷GUS expression pattern in Arabidopsis. In GL1/GL2, the box indicates the magnified region shown at right. 35S-NOS, the 35S promoter and nopaline synthase (NOS) terminator cassette; GaMYB2, 35S∷GaMYB2/RDL1∷GUS; HOX3, 35S∷HOX3/RDL1∷GUS; GaMYB2/HOX3, 35S∷GaMYB2/35S∷HOX3/RDL1∷GUS; GL1/GL2, 35S∷GL1/35S∷GL2/RDL1∷GUS. (C) GUS activities in Arabidopsis plants expressing both RDL1-P3∷GUS and the transcription factors as indicated. MYB2, GaMYB2.

Figure 4.

GaMYB2 Regulates Arabidopsis Trichome Development. (A) The Arabidopsis wild-type and a gl1 (SALK_039478) mutant seedling. (B) and (C) Trichome phenotypes of wild-type or gl1 mutant plants transformed with various chimerical genes as indicated. For intronless cDNA, a “c” was added as a suffix.

Figure 5.

Effects of Intron 1 and the Intronic MYB Motif on Expression of Trichome-Related Genes. (A) GUS staining of Arabidopsis seedlings expressing GL1∷GUS containing the GUS intron (top) and the first intron of GL1 (middle and bottom). (B) Intron positions in GL1, WER, and GaMYB2 genes and the MYB motif in the first intron of each gene. MYB2, GaMYB2. (C) Reduced production of trichomes in gl1 plants expressing GL1∷GaMYB109, whose intron contains no MYB motif, or the MYB genes with disrupted intronic MYB motif. See also Table 1.

Figure 6.

In Situ Hybridization of Cotton Ovule and the Arabidopsis Seed Trichome. (A) In situ hybridization of GaMYB2 transcripts in the G. arboreum ovule (0 DPA) with antisense (top) and sense (middle) probes. Arrowheads indicate fiber cells; the mRNA was localized in fiber cells. (B) Trichome(s) on seed of wild-type Arabidopsis expressing 35S∷GaMYB2 observed under binoculars. Arrowheads indicate the seed-trichome. (C) Scanning electron micrographs depicting the trichome on seed of transgenic Arabidopsis expressing 35S∷GaMYB2. The box indicates the magnified region shown in the bottom panels. Arrowhead indicates the seed-trichome.