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J Exp Bot. Oct 2008; 59(13): 3533–3542.
Published online Aug 18, 2008. doi:  10.1093/jxb/ern204
PMCID: PMC2561158

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 β-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.

Keywords: Cotton fibre, glandular, MYB, promoter, tobacco, trichome

Introduction

Trichomes are specialized epidermal appendages found in the surface of aerial organs of most land plants. There are several types of trichomes: unicellular or multicellular, branched or unbranched, and glandular or non-glandular. Trichomes contribute to many aspects of plant adaptation to biotic and abiotic stresses, such as to fence off insect herbivores, regulate surface temperature, decrease water loss through transpiration, increase tolerance to freezing, assist seed dispersal, and protect plant tissues from UV light (Eisner et al., 1998; Werker, 2000; Wagner et al., 2004). Glandular secreting trichomes (GSTs) often secrete plant secondary metabolites to constitute natural product-based resistance to herbivores and pathogens (Werker, 2000; Ranger and Hower, 2001; Wagner et al., 2004; Medeiros and Tingey, 2006). Many trichome-produced or trichome-stored compounds are of commercial value, such as those used in spice principal and pharmaceuticals production (Krings and Berger, 1998; McCaskill and Croteau, 1999; Wagner et al., 2004). For example, artemisinin, a sesquiterpene lactone that is widely used for the treatment of malaria, accumulates in glandular trichomes of Artemisia annua (Lommen et al., 2006; C Liu et al., 2006).

Different plant species may have different types of trichomes, and one plant may bear more than one type of trichomes. The annual weed Arabidopsis thaliana produces unicellular non-glandular trichomes, which are either branched or unbranched (Szymanski et al., 2000). Tobacco plants usually contain multicellular trichomes, including tall glandular secreting trichomes (GSTs) and simple glandless trichomes (Wagner et al., 2004). Recently, small procumbent glandular secreting trichomes, which accumulate antimicrobial proteins, were found in the aerial surfaces of tobacco (Shepherd et al., 2005), and sunflower (Kroumova et al., 2007). Cotton fibres are single-celled and extensively elongated seed trichomes, which provide the most important natural fibre for the textile industry (Kim and Triplett, 2001).

Cotton fibre development is a complicated and ordered process under the regulation of a vast number of genes, many of which are up-regulated or highly expressed in developing fibre cells (CH Li et al., 2002; Ruan et al., 2003; S Wang et al., 2004; Li et al., 2005; Luo et al., 2007). In recent years, comprehensive analyses of gene expression profiles have provided valuable clues to understanding cotton fibre formation (Arpat et al., 2004; Yang et al., 2006; Shi et al., 2006; Gou et al., 2007; Lee et al., 2007). To explore the molecular mechanisms regulating cotton fibre development, promoters of several cotton fibre genes have been identified. E6 was the first of such genes to be reported, and the E6 promoter has been used for engineering cotton fibre quality (John and Keller, 1996). GhRDL1, a gene highly expressed in cotton fibre cells at the elongation stage, encodes a BURP domain-containing protein (CH Li et al., 2002), and the GaRDL1 promoter exhibited a trichome-specific activity in transgenic Arabidopsis plants (S Wang et al., 2004). GhTUB1 transcripts preferentially accumulate at high levels in fibre, accordingly, the pGhTUB1::GUS fusion gene was expressed at a high level in fibre but at much lower levels in other tissues (XB Li et al., 2002). Promoters of three cotton lipid transfer protein genes, LTP3, LTP6, and FSltp4, were able to direct GUS gene expression in leaf and stem GSTs in transgenic tobacco plants (Hsu et al., 1999; Liu et al., 2000; Delaney et al., 2007), however, they did not exhibit a clear tissue-specificity. For example, in pFSltp4::GUS transgenic tobacco plants, strong GUS activity could be detected in all types of trichomes; in addition, GUS expression was also visible at the leaf margin, vascular tissue, ovules, and root tips (Delaney et al., 2007).

Previously it was reported that the cotton R2R3 MYB transcription factor GaMYB2 is a functional homologue of Arabidopsis GLABRA1 (GL1), a key regulator of Arabidopsis trichome formation. Northern blot and in situ RNA hybridization showed that GaMYB2 is expressed in cotton fibre cells at the early developmental stages (S Wang et al., 2004). In order to dissect the regulation of GaMYB2 gene expression further, the GaMYB2 promoter was isolated and its activity in cotton, Arabidopsis, and tobacco plants was analysed. It is shown that, while highly active in developing cotton fibre cells, this promoter is trichome-specific in Arabidopsis and GST head-specific in tobacco. It is further shown that a cis-element of the T/G-box, which can be recognized by bHLH transcription factors, such as Arabidopsis GL3 and cotton GhDEL65, contributes to the promoter activity in transgenic Arabidopsis.

Materials and methods

Plant materials and growth

Plants of cotton (Gossypium hirsutum cv. R15 and G. arboreum cv. Qingyangxiaozi) and tobacco (Nicotiana tabacum) were grown in a greenhouse at 28±2 °C with a natural photoperiod. Transgenic cotton and tobacco plants were at first cultured under 26 °C in a tissue culture room. Plants of Arabidopsis thaliana (Columbia-0, Col-0 ecotype) were grown indoors at 22 °C under a 16 h light period.

Genome walking

Genomic DNA was isolated from G. arboreum leaf tissue as described (XB Li et al., 2002), and genome walking was performed to isolate the GaMYB2 upstream fragment according to the Genome Walker kit (Clontech, Palo Alto, CA). The DNA was completely digested with selected restriction enzymes, and ligated to the corresponding adaptors to generate several DNA fragment libraries. The corresponding library was subjected to a first round of PCR amplification with the outer adaptor primer (AP1) and an outer gene-specific primer (GSP1), while the inner adaptor primer (AP2) and inner gene-specific primer (GSP2) were used for the second round of PCR. After two rounds of PCR, DNA fragments amplified were cloned into the pMD18-T vector (TakaRa, Japan) for sequencing.

All the primers used in this investigation are shown in Supplementary Table S1 at JXB online.

Vector construction

A 2062 bp promoter fragment of GaMYB2 was re-amplified with a pair of primers carrying an XbaI and a BamHI restriction site, respectively. Shorter promoter fragments with different lengths of 5′-terminal deletions were similarly amplified with each primer pairs. After digestion, these DNA fragments were inserted into pBI101.1 vector (Clontech), upstream of GUS gene coding region, resulting in a series of pGaMYB2::GUS binary vectors, namely P-2000 (-2062/-1, 2062 bp), P-1000 (–1000/-1, 1000 bp), P-750 (-750/-1, 750 bp), P-440 (-440/-1, 440 bp), P-360 (-360/-1, 360 bp), and P-220 (-220/-1, 220 bp). To construct the P-AB1 (-440/-1, the 20 bp fragment of -233 to -214 was deleted) and P-AB2 (-440/-1, the 87 bp fragment of -317 to -231 deleted) vectors, a PCR-based two-step DNA synthesis method was used as described (Wang and Malcolm, 2002).

The 35S promoter and NOS terminator were inserted into the SacI/EcoRI and HindIII/PstI sites of pCAMBIA1300, respectively, forming a p1300–35S-NOS intermediate vector. The Arabidopsis GLABRA3 (GL3) cDNA and the genomic sequence of GhDEL65 were amplified by PCR and were inserted into the BamHI/PstI site of the p1300–35S-NOS, respectively, generating 35S::GL3 and 35S::GhDEL65 fusion gene constructs. The vectors were transferred into Agrobacterium tumefaciens strain LBA4404 or GV3101 and used for plant transformation.

Plant transformation and GUS assay

Agrobacterium-mediated cotton transformation was performed as described (XB Li et al., 2002). The hypocotyl segments were used as explants for transformation. After the stages of callus induction, proliferation, embryogenic callus induction, embryo differentiation, and finally plantlet regeneration, the plantlets were transferred to pots in greenhouse for further growth. For tobacco transformation, a leaf disc transformation method (Horsch et al., 1985) was employed. Transformants were selected on MS medium containing 100 mg l−1 of kanamycin and 500 mg l−1 of cefotaxime. Transgenic Arabidopsis plants were generated by a floral dip method (Clough and Bent, 1998), and screened on half-strength MS agar medium containing 50 mg l−1 of kanamycin or hygromycin. Histochemical localization and fluorometric quantification of GUS activities were performed as described (Jefferson et al., 1987).

RNA analysis

Total RNAs were isolated from plant materials using a Trizol reagent (Invitrogen, Carlsbad, CA). For RT-PCR, the first strand cDNA was prepared, followed by a standard PCR protocol: 95 °C for 5 min, 27–34 cycles (according to the gene expression level) of denaturation at 95 °C for 20 s and annealing/extension at 56 °C for 30 s.

Yeast one-hybrid assay

Yeast one-hybrid assay was performed using the MATCHMAKER one-hybrid system (Clontech). The DNA fragment of four tandem copies of T/G-box [4× T/G-box (CTGCCACGTTGACAA)] was synthesized and inserted directly into the multiple cloning sites of reporter plasmids of pLacZi and pHISi-1, respectively. These two bait constructs were linearized and integrated into the genome of yeast strain YM4271, the dual reporter strain was selected and maintained on synthetic dextrose (SD)/-His/-Ura medium. For construction of the pGAD-GL3 fusion, GL3 cDNA was ligated with GAL4 activation domain in pGAD424 plasmid, and then was introduced into yeast strain with dual reporter genes, with the blank pGAD424 plasmid as control. Yeast transformants were tested on SD/-Leu/-His/-Ura medium containing different concentrations of 3-amino-1,2,4-triazole (3-AT) and 80 mg l−1 of 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) and 1× BU salt.

β-galactosidase assay was conducted as described (PT3024–1, Clontech). The ORF of GhDEL65, TT8, AtMYC2, and GL3 were in-frame fused with the GAL4 activation domain of the one-hybrid vector pGAD424, and then transferred into yeast cells containing pLacZi-4×T/G-box plasmids, respectively; the blank pGAD424 plasmid was used as control. The unit of β-Gal activity was determined by the equation of U=1000×[OD420]/(time (in min)×volume (in ml)×[OD600]. For each transformation sample, multiple independent yeast isolates were tested, each for three times.

Results

Isolation and sequence analysis of GaMYB2 promoter

Based on the cDNA sequence of GaMYB2 (S Wang et al., 2004), a 2062 bp promoter fragment upstream to the coding region was isolated from G. arboreum by genome walking (see Supplementary Table S2 at JXB online). The A of translation initiation codon (ATG) of GaMYB2 gene was defined as +1. A putative TATA box (-93 to -86) and a CAAT box (-131 to -128), which serve as basal promoter elements for the transcription of eukaryotic genes, were found in the GaMYB2 promoter. Sequence analysis using PLACE (http://dna.affrc.go.jp/PLACE) showed that a number of putative tissue-specific or stress-induced regulatory motifs corresponding to known cis-elements of plant genes were present, such as MYB recognition site, E-box, and T/G box (see Supplementary Table S3 at JXB online), implying that the GaMYB2 promoter may be under a complex regulation.

GaMYB2 promoter has a high activity in cotton fibre

Our previous investigation showed that GaMYB2 was preferentially expressed in fibre cells at the early developmental stages, and this R2R3 MYB gene was able to rescue the glabrous phenotype of the Arabidopsis gl1 mutant (S Wang et al., 2004). These experimental data suggest that GaMYB2 may play a role in controlling cotton fibre development. To dissect the GaMYB2 gene expression pattern further, its promoter activity was examined in cotton plants. The chimeric gene of P-2000::GUS, in which a β-glucuronidase (GUS) reporter gene was placed behind the promoter, was transferred into cotton (G. hirsutum) plants through Agrobacterium tumefaciens-mediated transformation. Nine T0 transgenic lines were generated, and histochemical staining of each line exhibited a similar pattern of GUS expression. Intensive GUS staining was observed in epidermis of young ovules, such as the 0-DPA ovule from which the fibre initials were emerging, and in developing fibre cells (Fig. 1A, B). To a lesser extent, GUS staining was observed in trichomes of other aerial organs, including leaves, stems, and bracts (Fig. 1C–E). Weak GUS staining was also detected in other tissues, such as roots, stamens, and petals, without a clear tissue-specificity (data not shown). Consistent with histochemical staining, the in vitro assay of protein abstracts showed the highest specific activity of GUS in cotton fibres, and a lower activity in the 0-DPA ovule. In the 9-DPA ovule from which the fibres were stripped, GUS specific activity was almost completely lost (Fig. 1F). The differences in specific activities among the organs investigated were probably a reflection of the portions of trichome proteins present in each sample, and in cotton plants the GaMYB2 promoter has a high activity in developing fibre cells and epidermal trichomes.

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 ...

GaMYB2 promoter displays trichome-specific activity in Arabidopsis

To examine the expression pattern of GaMYB2 promoter in different plant species, the P-2000::GUS gene was introduced into Arabidopsis. Arabidopsis leaf trichomes are mostly branched, but trichomes on stem and sepals are often unbranched; both branched and unbranched trichomes are unicellular (Szymanski et al., 2000). Histochemical assay of T1 P-2000::GUS plants showed that, in rosette leaves, GUS staining was located exclusively in trichomes (Fig. 2A, B). In stems, the GUS activity was also restricted to trichomes (Fig. 2C). At the flowering stage, GUS activity was again present in the unbranched trichomes of the sepals (Fig. 2D). In 1-week-old seedlings, GUS staining was observable in shoot apical meristems (SAM) and at the margins of the cotyledons, but not in roots (data not shown). Clearly, in Arabidopsis, the promoter of this cotton MYB gene drives GUS gene expression specifically in trichomes, regardless of their branching status.

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.)

GaMYB2 promoter confers specificity to glandular trichomes in tobacco

Distinguished from cotton fibres and Arabidopsis trichomes that are unicellular, tobacco plants have multicellular trichomes, including the non-glandular simple trichomes and the GSTs (Wagner et al., 2004; Shepherd et al., 2005). Most of the tobacco organs are covered with GSTs that have a head of glandular secreting cells and a long or short stalk, but on the base of the anther filaments, trichomes are mainly non-glandular. In order to test GaMYB2 promoter activities in the different types of trichomes, transgenic tobacco plants carrying P-2000::GUS were generated. GUS staining showed that in leaves, stems, and bracts, the fusion gene was expressed specifically in the GSTs. Notably, while strong GUS staining was visualized in the glandular head of GSTs, the GUS activity was undetectable in stalk cells (Fig. 3A–C, E). Occasionally faint staining appeared in the stalk cells adjacent to the glandular head, which might be a result of diffusion. No GUS staining was observed in multicellular simple trichomes of the anther filament (Fig. 3D, F). In 1-week-old seedlings which were trichomeless, no GUS staining was observed in the hypocotyl, cotyledon, and root (data not shown). These data demonstrate that, in tobacco plants, the GaMYB2 promoter drives gene expression only in glandular secreting cells, and it has no activity in other types of trichome cells.

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 ...

Promoter deletion analysis

To find regulatory regions important for trichome-specific activity of the GaMYB2 promoter, several DNA fragments of different 5′-deletions were generated by PCR and fused to the GUS gene (see Supplementary Table S4 at JXB online). These expression cassettes were then introduced into Arabidopsis and tobacco plants, respectively. Assay of transgenic Arabidopsis plants revealed that a 360 bp fragment proximal to the coding region was sufficient to drive GUS expression in Arabidopsis trichomes, with a similar expression pattern and intensity to that of P-2000 plants (Fig. 4A, B). Further deletion of the promoter decreased the gene expression level, as only about 1/3 of the P-220 plants showed weaker GUS staining under the same staining conditions used for P-360 plants (Fig. 4C), and the specific activity of GUS was decreased to about 30% of that of P-360 plants (Fig. 4E). The expression pattern and the intensity of GUS staining were similar among the five promoters ranging from P-2000 to P-360, implicating that the 360 bp fragment of GaMYB2 contained all the key cis-elements conferring trichome-specific activity.

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 ...

Similar results were obtained with transgenic tobacco plants. The 360 bp fragment directed GUS expression in secreting head cells of GSTs (Fig. 5A, B), with a similar level of specific activity in the leaf as that of P-2000 (data not shown). In the transgenic plants of P-220, the GUS signal became very weak (Fig. 5C).

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. ...

Activation of transcription by bHLH protein through binding to T/G-box

PLACE analysis revealed a T/G-box element (AACGTG) present at -226 to -221, which attracted attention. The T/G-motif has been shown to play an important regulatory role in tomato defence genes of proteinase inhibitor II and leucine aminopeptidase (LAP). JAMYC2 and JAMYC10, both encoding the basic helix-loop-helix (bHLH) domain-containing transcription factor, specifically recognize the T/G-box motif in the promoter of these two genes and transactivate their expression (Boter et al., 2004). Similar to these MYC proteins, GL3, a key regulator of Arabidopsis trichome development, also contains a conserved bHLH domain (Payne et al., 2000). A recent report showed that GL3 is able to bind to the promoter sequence and activate transcription of MYB transcription factor genes, such as CPC and ETC1, which are negative regulators of trichome development (Morohashi et al., 2007). It was then asked if GL3 could activate the GaMYB2 promoter.

First, a modified promoter, P-AB1, that lacked the T/G-box was generated by removing a 20 bp fragment (-233 to -214) from P-440. Arabidopsis plants harbouring P-AB1::GUS exhibited a strong reduction of the GUS signal in the trichome (Fig. 4D) and the specific activity of GUS in leaf proteins was dramatically decreased (Fig. 4E). A similar reduction of promoter activity was detected in P-AB1 tobacco plants, in which the GUS staining in GSTs was very faint (Fig. 5D). Another promoter, P-AB2, was then made in which a 87 bp fragment (-317 to -231) was removed from P-440, while the T/G box was intact. Analysis of Arabidopsis plants revealed that deletion of this 87 bp sequence resulted in only a marginal loss of activity (Fig. 4E). These deletion results suggest that the 17 bp region between -230 and -214 plays an important role in activating the GaMYB2 promoter, further supporting the assumption that the T/G-box present in this region may serve as a cis-acting element conferring promoter activity in trichomes.

The binding activity of GL3 to the T/G-box motif of GaMYB2 promoter was then tested by the yeast one-hybrid assay, using a 60 bp DNA fragment containing 4× T/G-box (four tandem repeats of the T/G-box element and its flanking sequence). It was found that only the yeast clones harbouring the pGAD-GL3 plasmid grew on the medium used for the assay (Fig. 6A), indicating that Arabidopsis bHLH protein GL3 is indeed able to recognize and interact with the T/G-box motif, and function as a transcriptional activator, at least in yeast.

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 ...

The Arabidopsis genome encodes more than 160 bHLH transription factors, which act as important regulatory components in diverse biological processes (Bailey et al., 2003; Toledo-Ortiz et al., 2003). Among them TT8 shares 30% amino acid sequence identity with GL3, and it plays a role in regulating the flavonoid pathway by forming a ternary complex with TT2 (a MYB) and TTG1 (a WD-repeat protein) (Baudry et al., 2004). Another bHLH transcription factor of A. thaliana, AtMYC2, is 26% identical to GL3 based on amino acid sequences. AtMYC2 is an important regulator in the jasmonic acid (JA) and abscisic acid (ABA) signalling pathways (Abe et al., 2003; Boter et al., 2004), and has been reported to bind to the MYC-site (CACATG) in the Arabidopsis RD22 gene promoter (Abe et al., 1997) and T/G-box (AACGTG) motif in the tomato LAP promoter (Boter et al., 2004). A search of the NCBI database for cotton homologues of Arabidopsis GL3 retrieved a putative bHLH protein, GhDEL65, which shares ~50% sequence identity with GL3, and is more distantly related to TT8 and AtMYC2 with sequence identities of 35% and 20%, respectively. To see if the GaMYB2 T/G-box motif was specifically recognized by a certain type of bHLH protein, the β-galactosidase activities of yeast cells expressing each bHLH proteins was compared, respectively, in a yeast one-hybrid system. It was found that while GL3 and GhDEL65 were equally active in interacting with the cis-elements, TT8 and AtMYC2 had significantly lower activities (Fig. 6B).

Recognition of the T/G-box motif by GL3 and GhDEL65 prompted the question whether both transcription factors would activate the GaMYB2 promoter in planta. The coding region of the two genes under the control of the 35S promoter was introduced into T2 plants of P-440, respectively. RT-PCR analysis of individual 35S::GL3 transformants showed that the GUS transcript level was markedly increased in plants overexpressing GL3 (Fig. 6C). In comparison with P-440 plants, GUS activities were elevated by about 2.6-fold due to 35S::GL3 expression and about 3.1-fold due to 35S::GhDEL65 expression (Fig. 6D). The trichome specificity, however, was not changed. Therefore, in transgenic Arabidopsis plants, constitutive overexpression of GL3 or GhDEL65 strongly enhanced the GaMYB2 promoter. These results suggest that cotton bHLH proteins homologous to GL3 may be involved in regulating GaMYB2 gene expression during cotton fibre development.

Discussion

It has been shown that a promoter of a cotton fibre MYB gene, GaMYB2, directs reporter gene expression specifically in trichomes of Arabidopsis and GST head cells of tobacco. Plant GSTs produce and accumulate a rich plethora of specific metabolites, particularly secondary metabolites, and are considered ideal plant cell factories for metabolic engineering (Verpoorte et al., 2000; Wagner et al., 2004; J Liu et al., 2006). Promoters of several cotton genes highly expressed in fibre cells have been reported, and those of LTP3, LTP6, FSltp4, GhGlcAT1, and GhRGP1 genes were examined using transgenic tobacco plants. Although active in GSTs as well, they are less tissue-specific (Hsu et al., 1999; Liu et al., 2000; Wu et al., 2006, 2007; Delaney et al., 2007). The high specificity of the GaMYB2 promoter makes it a valuable tool not only for engineering cotton fibre traits but also for modification of GST metabolism.

In tobacco, which has both simple and glandular secreting trichomes, activity of the GaMYB2 promoter is restricted to GST head cells. It is inactive in other types of cells, including GST stalk cells and multicellular simple trichomes. This intriguing pattern seems to suggest that unicellular trichomes of cotton and Arabidopsis share with tobacco GST head cells a conserved molecular machinery in regulating the expression of a set of genes, but this machinery is not operating in either GST stalk cells or multicellular simple trichomes of tobacco. In Arabidopsis, multimeric complexes of MYB-bHLH-WD40 play a key role in regulating trichome patterning and development (Payne et al., 2000; Ramsay and Glover, 2005; Serna and Martin, 2006), as well as anthocyanin and flavonoid biosynthesis (Hartmann et al., 2005; Koes et al., 2005). Recently, the bHLH transcription factor GL3 was shown to bind the promoter of GL2, ETC1, and CPC, a group of genes involved in the development and patterning of trichomes, and directly activate their expression (Morohashi et al., 2007). Our results that overexpression of GL3 or its cotton homologue GhDEL65 enhanced the GaMYB2 promoter activity suggest that, in cotton, bHLH transcription factor(s) are probably involved in up-regulating expression of GaMYB2 and possibly other functionally related R2R3 MYB genes.

The yeast one-hybrid assay showed that GL3 and GhDEL65 have higher binding activities to the GaMYB2 T/G-box than AtMYC2 and TT8, suggesting that the GaMYB2 promoter is prone to the recognition by GL3-type bHLH transcription factors. TT8 is similar to GL3 in working mechanisms, as both are recruited to the MYB- bHLH -WD40 activation complex. While GL3 is a trichome and non-root hair cell regulator, TT8 is involved in regulating anthocyanin and flavonoid biosynthesis (Payne et al., 2000; Zhang et al., 2003; Ramsay and Glover, 2005). The detectable binding activity of TT8 protein to the T/G motif of the GaMYB2 promoter, although comparatively low, provides a possibility that TT8 homologues in tobacco glandular trichomes, which function in secondary metabolisms, could participate in the activation of the reporter gene expression specifically in glandular cells.

Our previous analysis of the cotton GaRDL1 promoter showed that the L1-box and MYBCORE are two cis-elements conferring trichome specificity to this promoter. Mutation of either element reduced the GaRDL1 promoter activity in Arabidopsis trichomes. Furthermore, expression of MYB (GL1 or GaMYB2) and HOX (GL2 or GaHOX3) transcription factors, responsible for binding the MYBCORE and L1- box, respectively, induced a strong ectopic expression of the reporter gene in non-trichome cells (S Wang et al., 2004). The CYP71D16 promoter of tobacco was able to direct GUS gene expression in glandular cells of GSTs in transgenic tobacco plants (Wang et al., 2002), and this promoter has been used for engineering plant defence against aphid infection (E Wang et al., 2004). As the promoter deletions progressed, GUS activity decreased and the expression pattern extended. Although still trichome-specific, GUS staining was concentrated in the lowest gland cell and stalk cells, and the MYB-like sequence (CAACAG) between -56 and -51 was speculated be important for trichome specificity (Wang et al., 2002). SaPIN2b, a nightshade (Solanum americanum) proteinase inhibitor II gene, containing six MYB-binding motifs and an L1 box in its promoter region, was constitutively expressed in GSTs; similar to the CYP71D promoter, when SaPIN2b promoter deletion proceeded a small portion of the trichomes showed a shift of GUS activity to the stalk cell (J Liu et al., 2006). The Arabidopsis OASA1 promoter was reported to direct gene expression in GSTs and simple trichomes, and the MYB motifs located in the promoter and the first intron region of this gene may act as enhancer elements in trichome cells (Gutierrez-Alcala et al., 2005). AtTSG1 promoter also showed trichome-specific activity in Arabidopsis; deletion analysis of this promoter indicated that the MYB-like recognition site (AACCAAAC) was a putative element for trichome specific expression of this gene (Ni et al., 2008). In tobacco, the promoter of the T-phyllopanin gene directed reporter gene expression specifically in short procumbent trichomes, which could explain the biosynthesis of T-phylloplanin proteins only in this particular type of glandular trichome (Shepherd et al., 2005). Despite these interesting findings and speculations, cis-elements and the related transcription factors conferring glandular trichome expression await further identification.

Although the fragment of -233 to -214 containing a T/G-box was important for the activity of the GaMYB2 promoter in trichome, removal of this cis-element decreased the promoter activity greatly, but did not change the GUS staining pattern. Furthermore, ectopic expression of GL3 or GhDEL65 under the control of the 35S promoter resulted in enhanced, but not the ectopic expression of the reporter gene. It is reasonable to assume that this T/G-box serves as an enhancer, and other cis-element(s) exist that confer trichome-specificity to the GaMYB2 promoter. Identification of such cis-element(s) should help to dissect the molecular mechanisms regulating cotton fibre and tobacco GST development.

Supplementary Material

[Supplementary Material]

Acknowledgments

This work is supported by The National High-tech Research Program of China (2006AA10Z102, 2006AA10A109) and The National Key Basic Research Program of China (2007CB108800).

Glossary

Abbreviations

GUS
β-glucuronidase
4-MU
4-methylumbelliferone
3-AT
3-amino-1, 2, 4-triazole
DPA
days post-anthesis
X-Gal
5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside
X-Gluc
5-bromo-4-chloro-3-indolylglucuronide
RT-PCR
reverse transcription-PCR
GST
glandular secreting trichome
ONPG
O-nitrophenyl-β-D-galactopyranoside

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