JXB Advance Access originally published online on March 7, 2007
Journal of Experimental Botany 2007 58(6):1515-1524; doi:10.1093/jxb/erm020
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
© 2007 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER |
A truncated MYB transcription factor from Antirrhinum majus regulates epidermal cell outgrowth
1Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, UK
2Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, UK
To whom correspondence should be addressed. E-mail: bjg26{at}cam.ac.uk
Received 19 September 2006; Revised 19 December 2006 Accepted 17 January 2007
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
|---|
Plant MYB genes can be divided into subgroups on the basis of additional conserved regions of sequence. In some cases, genes within a subgroup share similarities in function, as well as sequence. The functions of three proteins in subgroup 9 have been described, with AmMYBMX regulating the differentiation of conical-papillate petal epidermal cells, PhMYB1 involved in extending the growth of these cells, and AmMYBML1 involved in differentiation of several petal epidermal cell types. Here, the isolation of a gene encoding a new member of MYB subgroup 9, AmMYBML3 (Antirrhinum majus MYB MIXTA-LIKE 3) is described, which contains the defining regions of conserved sequence but is lacking the majority of the C-terminus, including the amphipathic
-helix presumed necessary for transcriptional activation. AmMYBML3 is expressed in all aerial organs, but its expression is restricted to outgrowing epidermal cells, including trichomes, stigmatic papillae, and petal conical-papillate cells. Ectopic expression of AmMYBML3 in tobacco results in the formation of conical-papillate cells in the usually flat carpel epidermis. These data suggest that this protein is capable of altering epidermal development, thus resulting in cellular outgrowth, despite the missing C-terminus, and may act in conjunction with other transcriptional activators to enhance cellular outgrowth from the epidermis of all aerial organs. Key words: AmMYBML3, Antirrhinum, cellular differentiation, conical-papillate cell, development, epidermis, MYB, transcription factor, trichome
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
|---|
Members of the MYB family of transcription factors contain between one and four repeats of a DNA-binding domain containing about 53 amino acids (Stracke et al., 2001). Each repeat encodes three
-helices, with the second and third forming a helix–turn–helix conformation to intercalate into the major groove of the target DNA (Frampton et al., 1989; Ogata et al., 1994). Once bound to DNA, a MYB protein may function to activate transcription through its C-terminus or through interaction with another protein such as a bHLH transcription factor (Goff et al., 1992). Most plant MYB proteins contain just two of the three repeats and are thus referred to as R2R3-MYB factors (Jin and Martin, 1999). In Arabidopsis thaliana some 126 R2R3 MYB genes have been identified. The plant MYB transcription factors have been divided into 42 subgroups (plus seven outliers) based on conserved sequence motifs (Kranz et al., 2000; Stracke et al., 2001; Jiang et al., 2004). In some cases it appears that MYB proteins within a subgroup have similar functions, although insufficient proteins have been characterized to date to allow predictions of protein function based on sequence. Subgroup 9 (or G03, according to different authorities) of the plant MYB transcription factor family contains the MIXTA-LIKE MYBs, defined by a conserved sequence motif downstream of the DNA-binding domain (Kranz et al., 1998; Stracke et al., 2001; Jiang et al., 2004). The functions of only three members of this group have been characterized. AmMYBMX (or MIXTA) of Antirrhinum majus is required for conical-papillate cell formation in the petal epidermis (Noda et al., 1994; Glover et al., 1998). PhMYB1 from Petunia hybrida has also been shown to play a role in conical-papillate petal cell development (Avila et al., 1993; van Houwelingen et al., 1998). AmMYBML1 of A. majus is involved in conical-papillate petal cell development, petal trichome differentiation, and the formation of the hinge of the ventral petal (Perez-Rodriguez et al., 2005). The three known members of subgroup 9 in A. thaliana are AtMYB16, AtMYB17, and AtMYB106. To date, mutant phenotypes have not been described for loss of function in any of these Arabidopsis genes.
The first member of subgroup 9 to be fully characterized was AmMYBMX from Antirrhinum. Through analysis of a loss-of-function mutation, caused by transposon insertion in the gene, it was shown that AmMYBMX is necessary for the development of conical-papillate cells on the adaxial surface of the petals (Noda et al., 1994). The petal cells of the adaxial surface of the mixta mutant are flat compared with the wild type. The shape of the wild-type conical-papillate cells optimizes light capture by the pigments, causing the petal to appear deeper in colour (Noda et al., 1994). There is evidence to suggest that this specialized shape of the adaxial petal epidermal cells increases the attractiveness of the flower to pollinating bees (Glover and Martin, 1998; Comba et al., 2000). AmMYBMX expression in Antirrhinum is limited to a narrow window of time late in development of the petal epidermis. In situ hybridization has confirmed that the expression of AmMYBMX is confined to adaxial petal epidermal cells (Glover et al., 1998).
The ectopic expression of the AmMYBMX gene in tobacco resulted in the formation of both multicellular trichomes and conical-papillate cells (Glover et al., 1998; Payne et al., 1999). These data indicate that these two specialized cell forms use parts of a common developmental programme and suggest that a second gene may be responsible for the formation of multicellular trichomes. Two further subgroup 9 genes were isolated from Antirrhinum, using an AmMYBMX probe on a petal cDNA library. One of these genes, AmMYBML1 (Antirrhinum majus MYB MIXTA LIKE 1), has been shown to play roles in the development of three distinct specialized petal cell types. AmMYBML1 is involved in epidermal conical cell development in the ventral petal hinge region, in the elaboration of the mesophyll below this epidermis to generate the landing platform of the Antirrhinum flower, and in the development of trichomes in the throat of the flower (Perez-Rodriguez et al., 2005). The function of AmMYBML2 (Antirrhinum majus MYB MIXTA LIKE 2) is still under analysis (Martin et al., 2002). These data indicate that a family of closely related MYB genes, which are likely to specify different cell forms with different functions through expression at different developmental stages, exists in Antirrhinum.
Here, the isolation and characterization of a fourth Antirrhinum gene from MYB subgroup 9 is described. AmMYBML3 (Antirrhinum majus MYB MIXTA LIKE 3) differs from the three genes previously described in lacking a putative activation domain in its extremely reduced C-terminus. Semi-quantitative RT-PCR indicates that the gene is expressed in all aerial organs of the plant, most strongly in young flowers, while promoter–GUS analysis and in situ hybridization indicate that this expression is limited to outgrowing epidermal cells (trichomes, stigmatic papillae, and petal conical-papillate cells) in these organs. Ectopic expression of AmMYBML3 in tobacco results in the conversion of the usually flattened carpel epidermal cells to a conical-papillate form, suggesting that AmMYBML3 may play a weak but active role in the outgrowth of specialized epidermal cells of Antirrhinum.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
|---|
Gene isolation
An Antirrhinum genomic library in bacteriophage
EMBL4 was screened at very high stringency using a full-length cDNA of AmMYBML1 as a probe. Five independent plaques hybridized with the probe. One contained a genomic clone of AmMYBML1, one hybridized less strongly to the probe and was not analysed further, while three contained the same previously unknown MYB gene, which was named AmMYBML3. Following restriction digestion of phage DNA containing this unknown gene, the restriction fragments which bound AmMYBML1 probe on a Southern blot were isolated from a gel, ligated into pCR2.1, and sequenced. To obtain a cDNA clone, RNA was extracted from young Antirrhinum shoots using lithium chloride precipitation (Glover et al., 1998). First-strand cDNA was synthesized using the Amersham Pharmacia Biotech First Strand cDNA synthesis kit and a B26 primer for 3' RACE (Frohman et al., 1988). The AmMYBML3 cDNA was amplified using a gene-specific 5' primer designed to the genomic sequence and a 3' RACE primer which binds the adaptor on cDNA ends. The PCR product was cloned into pCR2.1 and sequenced.
Semi-quantitative RT-PCR
RNA was extracted from different plant organs as described above, treated with DNase I to remove contaminating genomic DNA, and cDNA pools synthesized from each sample. Ubiquitin primers were used to establish the linear phase of the PCR (25 cycles) and to ensure the use of equal amounts of cDNA from each tissue. Ubiquitin primers generate multiple bands in Antirrhinum, as they anneal to multiple repeats within the ubiquitin genes. However, the bands are consistent between samples. PCR products generated using AmMYBML3-specific primers were separated on an agarose gel.
In situ hybridization
In situ hybridizations were performed by applying digoxigenin-labelled antisense AmMYBMML3 RNA probes to 8 µm sections cut from wax-embedded samples (Hofer et al., 1997). Samples were counterstained with 0.1% (w/v) Calcofluor and viewed under epifluorescent illumination at low magnification. A sense RNA probe was used as a control.
Promoter::GUS construct
Following sequencing of bacteriophage fragments containing the AmMYBML3 genomic clone, primers were designed to amplify the complete promoter sequence (1665 bp) with BamHI and XbaI restriction sites on the end. This PCR product was ligated into a BamHI/XbaI-digested pGREEN-GUS binary vector (Hellens et al., 2000) to make construct pTRY27.
Construct for ectopic expression in tobacco
The sense construct pTRY30 for ectopic expression of AmMYBML3 in tobacco was created by excising an 813 bp XhoI fragment containing the whole AmMYBML3 cDNA from pCR2.1 and inserting it into the SalI site of a modified pBIN19 (Bevan, 1984) containing the double CaMV 35S promoter and CaMV poly(A) terminator cassette from pJIT60 (Guerineau and Mullineaux, 1993).
Plant transformation
Constructs were transformed into Agrobacterium tumefaciens LBA4404 using electroporation (Mattanovich et al., 1989). Tobacco leaf discs were transformed using the method of Horsch et al. (1985). RNA was extracted as described above and transgene expression confirmed by northern analysis, using the method of Martin et al. (1985).
GUS assays
Tissue samples were immersed in GUS staining solution (100 µl 40 mg ml–1 X-Gluc in 8 ml staining buffer [100 mM NaH2PO4, 10 mM Na2EDTA, 0.5 mM K ferrocyanide (MW 422.4), 0.5 mM K ferricyanide (MW 329.3), 0.1% Triton X-100, adjusted to pH 7.0 with NaOH)] and incubated at 37 °C for 3 h. Samples were destained with 70% ethanol and incubated at 37 °C for 30 min, twice. The samples were then transferred to a microscope slide and examined using a dissecting and light microscope.
Scanning electron microscopy
Plant tissue was examined under a CamScan mark IV scanning electron microscope with a Hexland cryostage.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
|---|
Analysis of AmMYBML3 sequence
The AmMYBML3 genomic clone was 7263 bp in length. The clone consisted of promoter sequence, three exons, two introns, and some 3' sequence. The first two exons of AmMYBML3 are identical in length to those of all other Antirrhinum and Arabidopsis MIXTA-LIKE genes. However, the sizes of the remaining exon and the two introns differ from those of other genes.
The cDNA of AmMYBML3 was isolated using RT-PCR and the sequence deposited in GenBank (accession number AY661654). Alignment of this cDNA sequence with the sequences of the other subgroup 9 genes indicated a high level of sequence conservation in the 5' region, but a highly divergent 3' sequence. Similarly, comparison of the proteins indicates a highly conserved N-terminus but a divergent C-terminus. The conservation of the N-terminus of MYB proteins is consistent with most other R2R3 MYBs and it can be seen that between previously identified MIXTA-LIKE genes and AmMYBML3 there is a very high level of conservation in the first 170 amino acids (aa) of the protein products (Fig. 1). In these first 170 aa, on the few occasions when the residue at a position differs between two members of the subgroup, it is usually the case that both amino acids are functionally similar.
|
The C-terminus, however, varies quite significantly between the proteins (Fig. 1) and it is possible that this may be indicative of different protein–protein interactions, thus resulting in different regulatory effects. AmMYBML3 is much shorter than the other subgroup 9 proteins, being only 219 aa in length compared with others which are over 300 aa. This reduction in size is due to the absence of the majority of the C-terminus of the other proteins, although the final 26 aa of AmMYBML3 do show conservation with those of AmMYBMX, AmMYBML1, AtMYB16, and AtMYB106. This unusual reduction in protein length was confirmed both by 3' RACE (which amplified the same cDNA from three separate plants) and by careful analysis of the 3' region of the genomic clone, which contained no further open reading frames suggestive of a second stop codon. Noda et al. (1994) suggested that AmMYBMX was a transcriptional activator because of the identification of a potential amphipathic
-helix, but the C-terminus of AmMYBML3 does not contain an apparent amphipathic -helix. This may have consequences for its ability to cause transactivation. Production of a phylogenetic tree of subgroup 9 proteins indicates that AmMYBML3 is most similar to AtMYB16 and AtMYB106 of Arabidopsis (Fig. 2). This suggests that at least one gene of this group existed prior to the divergence of Arabidopsis and Antirrhinum, and perhaps significantly earlier. AmMYBML2 also clusters with these proteins, and may represent an ancestral form of AmMYBML3, prior to the loss of the C-terminus.
|
Expression analysis of AmMYBML3
Semi-quantitative RT-PCR was performed on a range of different organs. AmMYBML3 transcript was found in all organs tested; floral buds, petals, sepals, stamens, carpels, inflorescence stems, leaves, hypocotyls, and the vegetative stem (Fig. 3). The lowest expression was seen in the carpel, but no difficulty was found in amplifying AmMYBML3 from any organ tested.
|
Analysis of AmMYBML3 promoter::GUS fusions in transgenic tobacco
An AmMYBML3 1665 bp promoter fragment was isolated from the genomic clone and the sequence deposited in GenBank (accession number DQ228865). This fragment was used to drive GUS expression in transgenic tobacco plants. GUS expression was found in all aerial organs of the plant, but was mainly restricted to outgrowing epidermal cells (Fig. 4). This included the glandular trichomes found over the vegetative surfaces and petals of the plant, and also the conical papillate cells of the petal and the stigmatic papillae. Stigmatic staining was particularly strong.
|
In contrast, AmMYBML3 is expressed in leaf trichomes during their active development, but the transcript disappears once the trichomes are fully mature. In the leaves, trichome development is at different stages in different places, with the youngest trichomes at the proximal end still developing while the trichomes at the distal end are fully mature (Hülskamp et al., 1994; Turner et al., 2000). GUS expression in leaf trichomes could still be visualized towards the proximal end of the leaf, but was less clear in trichomes of the distal end. This suggests that AmMYBML3 is involved in the development of the trichomes, but once they are fully developed its expression ceases.
To confirm that these results were not artefacts, the GUS vector without a promoter was transformed into tobacco but no GUS expression was detected in transformed plants. A 35S promoter–ß-galactosidase-driven construct was used to provide a positive control for GUS staining.
In situ hybridization
To confirm the results of the promoter::GUS analysis, which identified particularly strong expression in stigmatic papillae, in situ hybridization was conducted using an AmMYBML3 probe with sections of stigmatic tissue. Expression was found in the papillae themselves, but also in several cell layers beneath this surface layer (Fig. 5A). This broader distribution of AmMYBML3 expression may account for the strong staining observed with GUS. This was the only indication of AmMYBML3 expression in any non-epidermal tissue.
|
Phenotype of Nicotiana tabacum plants ectopically expressing AmMYBML3
A construct (pTRY30) containing the AmMYBML3 cDNA under the control of two copies of the CaMV 35S promoter in pBin19 was used for transformation of tobacco. Expression of AmMYBML3 in regenerated tobacco plants was confirmed by northern analysis. Detectable levels of AmMYBML3 expression in leaves were identified in 13 out of 16 lines and these 13 were used in phenotypic analysis.
The phenotype of the AmMYBML3 transformed plants was extremely similar to that of wild-type tobacco. There was no observable difference in roots, leaves, stems, sepals, petals, or stamens at any stage of development, whether in epidermal development or internal structure (determined by freeze fractures). The aerial parts of the plant appeared to have wild-type proportions of both types of trichomes, stomata, and pavement cells. The leaves appeared wild type in terms of colour, size, and trichome numbers on adaxial and abaxial surfaces, unlike the leaves of tobacco ectopically expressing AmMYBMX, which were reduced in size, paler in colour, and had rolled-in leaf margins (Glover et al., 1998). Essentially, the transgenic tobacco expressing AmMYBML3 appeared wild type for all characteristics except for the shape of the epidermal cells of the carpel tissue, surrounding the ovary, which were transformed from irregularly shaped flat epidermal cells to more regularly shaped conical-papillate cells (Fig. 6). This subtle phenotype resulting from AmMYBML3 expression was only visible using SEM. Analysis of the carpel epidermis of many wild-type tobacco plants, for comparison with a number of transgenic lines, has never resulted in the identification of such cellular outgrowths, from which it is concluded that AmMYBML3 expression is responsible for the phenotype observed here (Glover et al., 1998; Martin et al., 2002; Perez-Rodriguez et al., 2005).
|
The severity of this phenotype varied, with some plants having only a few of the epidermal cells converted to conical-papillate cells (e.g. plant Q, Fig. 6). The strongest phenotypes were shown by plants which had all carpel epidermal cells converted to the conical-papillate form (e.g. plant S, Fig. 6). No correlation was found between level of AmMYBML3 expression (as measured from transcript abundance in northern blots) and severity of phenotype. As Fig. 6 shows, some plants with very high AmMYBML3 expression levels (such as plant R) had only a gentle rounding of carpel epidermal cells. Some plants with lower expression levels, such as line Q, had outgrowth of only a few epidermal cells, while other plants with similar expression levels (such as S) had quite extreme outgrowth of all carpel epidermal cells.
Effect of ectopic AmMYBML3 expression on plants ectopically expressing AmMYBML1 or AmMYBMX
To investigate the possibility that AmMYBML3 acts as either an enhancer or repressor of the activity of related MYB transcription factors, crosses were made between the transgenic plants described above and plants ectopically expressing AmMYBMX (Glover et al., 1998) or AmMYBML1 (Perez-Rodriguez et al., 2005). Progeny containing both transgenes were selected by PCR, and expression of both transgenes confirmed by northern analysis. Ectopic expression of AmMYBMX resulted in transformation of almost all aerial epidermal cells to the conical fate, with production of excess trichomes on many organs (Glover et al., 1998). Introduction of the AmMYBML3 transgene into this background had no visible effect, either to enhance or to repress the phenotype. Similarly, ectopic expression of AmMYBML1 resulted in transformation of carpel epidermal cells to steep cones or trichomes (Perez-Rodriguez et al., 2005), but AmMYBML3 had no effect on these plants, failing either to enhance or to reduce the carpel phenotype and resulting in no novel phenotype on any other organ (Fig. 7). The plants expressing both AmMYBML3 and AmMYBML1 were indistinguishable from those expressing AmMYBML1 alone, both by eye and under the scanning electron microscope.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
|---|
The AmMYBML3 protein is a member of MYB subgroup 9 with a reduced C terminal region
The classification of A. thaliana MYB genes was based on conserved domains within the protein products. These groupings may sometimes provide information about gene function, as it has been shown that several genes that cluster together have conserved functions. These include AtMYB91 (AS1) and AmMYBPHAN, which group together but fall outside the designated subgroups. Both negatively regulate KNOX (KNOTTED) expression in organ primordia (Byrne et al., 2000). AtMYB0 (GL1) and AtMYB66 (WER) of subgroup 15 (or G07) can functionally complement each other, but display different functions due to their different expression patterns (Lee and Schiefelbein, 2001). Production of Anthocyanin Pigment 1 and 2 (PAP1/AtMYB75 and PAP2/AtMYB90) of Arabidopsis and Anthocyanin 2 (An2) of Petunia can be grouped into subgroup 6 (or N09) and have been shown to control anthocyanin biosynthesis (Quattrocchio et al., 1999; Borevitz et al., 2000).
Stracke et al. (2001) placed AtMYB16, AtMYB17, AtMYB106, and AmMYBMX into subgroup 9 based on a conserved domain (AQWESARxxAExRLxRES) in their protein products. Within these MYB proteins this domain is the next most highly conserved domain after the MYB repeats.
The genomic and cDNA sequences of AmMYBML3 confirm that it is a member of subgroup 9. The defining conserved domain is present (aa 142–172, Fig. 1). The first two exons of the genomic clone are identical in size to those of the other Antirrhinum and Arabidopsis subgroup 9 genes. Phylogenetic trees based on amino acid sequence similarity confirm that AmMYBML3 is most similar to AmMYBML2 and to AtMYB16 and AtMYB106 (Fig. 2), indicating an ancestry before the divergence of the lineages which contain Antirrhinum and Arabidopsis (the Euasterids and the Eurosids, respectively). However, the C-terminal amphipathic -helix, presumed to be necessary for transcriptional activation of target genes, is missing from the protein product. This suggests that the protein could function as a dominant negative, a suggestion used to explain the dominant negative C1-I allele of maize which negatively regulates anthocyanin production in kernels (Edwards et al., 2001; Singer et al., 1998; Paz-Ares et al., 1990). DNA-binding proteins with transcriptional activation domains usually initiate or enhance transcriptional initiation from specific promoters. However, DNA-binding proteins without transcriptional activation domains may function as competitors, binding promoter sequences without the ability to activate transcription. Thus the presence of such a dominant negative in a cell as well as a transcriptional activator may result in less mRNA of the target gene being produced than if only the transcriptional activator were present. Such systems may be used by plants to regulate the precise level of expression of transcription factor target genes. Moyano et al. (1996) showed that two MYB transcription factors of A. majus (AmMYB305 and AmMYB340) bound the promoters of the same genes encoding enzymes of flavonoid synthesis, but had differential transcriptional activation activity. As a result, variations in the expression levels of the two transcription factors with respect to each other could precisely regulate the level of target gene expression. A similar interaction has been postulated between AtMYB71 and AtMYB79, as AtMYB79 is very similar in sequence to AtMYB71, but with a truncated activation domain (Kranz et al., 1998).
While the conventional explanation for the function of a DNA-binding protein without a transcriptional activation domain is that it functions as a dominant negative, it is also possible that such proteins can enhance transcriptional activation, through interactions with other transcription factors. By analogy with proteins in other MYB subgroups with conserved functions, it is likely that AmMYBML3 plays a role, either negative or positive, in the regulation of epidermal cell outgrowth.
AmMYBML3 is expressed in outgrowing cells of all aerial organs
Semi-quantitative RT-PCR and promoter::GUS analysis both demonstrated that AmMYBML3 is expressed in all aerial tissues. These data contrast with the expression pattern of AmMYBMX, which has been shown to be expressed only in the adaxial epidermal cells of Antirrhinum petals (Glover et al., 1998). In all aerial tissues of tobacco this expression was restricted to epidermal cells, and indeed to epidermal cells which had adopted a specialized cell fate through outgrowth. This expression of AmMYBML3 in epidermal outgrowths, such as trichomes, conical-papillate petal cells, and stigmatic papillae, suggests that the protein product has a function in the development of these specialized cell types. However, this function is apparently less restricted than that of either AmMYBMX or PhMYB1, both of which are only involved in the development of petal conical-papillate cells (Noda et al., 1994; van Houwelingen et al., 1998).
Based on the reduced C-terminal domain of AmMYBML3 it is possible that the protein functions as either a negative or a positive regulator of transcription. If the protein acts as a negative regulator, AmMYBML3 expression may be triggered early in tissues which do not have many outgrowing cells, but in tissues which have many outgrowths its expression could be late, so stopping continued outgrowth. In this case the role of AmMYBML3 may be to inhibit cellular expansion or division. Alternatively, if the protein functions as a weak activator of cell expansion it could be required in many cell types to enhance their outgrowth in response to other, stronger, transcriptional activators.
Ectopic expression of AmMYBML3 causes the outgrowth of tobacco carpel epidermal cells
Ectopic expression of AmMYBML3 in tobacco caused the outgrowth of carpel epidermal cells, so that, in extreme cases, they resembled the conical-papillate cells normally found on the petal epidermis (Fig. 6). No effect was observed on any other organ. This phenotype was much more restricted than that obtained from ectopic expression of AmMYBMX or AmMYBML1 in tobacco. Glover et al. (1998) showed that ectopic AmMYBMX expression resulted in the development of conical-papillate cells on all aerial epidermal surfaces, along with an increase in multicellular trichome density in many organs. Perez-Rodriguez et al. (2005) showed that ectopic AmMYBML1 expression resulted in the development of conical-papillate cells and trichomes on carpels of transgenic tobacco. It is apparent from these data that AmMYBML3 is a less potent activator of cellular differentiation than the closely related proteins AmMYBMX and AmMYBML1. This may be a reflection of the reduction of the C-terminal domain of AmMYBML3.
Restriction of the phenotypic consequences of AmMYBML3 expression to the carpel epidermis, the same tissue which responded to AmMYBML1 expression, suggests that AmMYBML3 activity is limited by similar factors to those that limit AmMYBML1 (Perez-Rodriguez et al., 2005). Ectopic expression of AmMYBML2 from the same constitutive promoter also results in cellular outgrowth restricted to a subset of the floral organs (Cathie Martin, personal communication). The restriction of the effects of overexpression of all three MIXTA-like genes implies that carpel tissue contains some other factor which allows the activity of these proteins to result in cellular outgrowth. Many MYB transcription factors are known to interact physically with bHLH transcription factors (Ramsey and Glover, 2005). For example, the Arabidopsis MYB GLABROUS1 interacts with the bHLH protein GL3 to induce trichome development (Payne et al., 2000). However, the subgroup 9 proteins do not contain the consensus motif necessary for physical interaction with a bHLH protein (Serna and Martin, 2006), making interaction of AmMYBML3 with a bHLH protein which is present only in carpel epidermis an unlikely scenario. An explanation involving the need for a partner transcription factor, perhaps of another family, is attractive because it would also account for the apparent ability of AmMYBML3, without a transcriptional activation domain, to cause cellular outgrowth. Physical interaction with a protein with such an activation domain would allow DNA binding by AmMYBML3 to result in transcriptional activation of target genes.
It is also apparent from these data that AmMYBML3 can function as a positive regulator of epidermal cell outgrowth, and does not appear, at least in a heterologous system, to regulate the growth of trichomes or other epidermal cells negatively. No reduction in trichome density, size, or cell number was observed in the transgenic tobacco plants. This suggests that the reduced C-terminus is sufficient either to activate low levels of transcription of genes involved in cellular outgrowth, or to interact with other transcriptional activators and to enhance their activity.
Any interaction between AmMYBML3 and other transcription factors does not seem to involve AmMYBMX or AmMYBML1. Combination of ectopic expression of pairs of these genes in transgenic tobacco plants resulted in phenotypes indistinguishable from that of the parent with the strongest phenotype (Fig. 7). AmMYBML3 was unable to repress the activity of AmMYBMX or AmMYBML1, but also failed to enhance the phenotypes observed. It is possible that AmMYBML3 interacts with AmMYBML2, to which it is most similar in sequence. It is also possible that AmMYBML3 operates through interactions with less closely related proteins.
|
Conclusions |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
|---|
The truncated AmMYBML3 protein is sufficient to cause outgrowth of carpel epidermal cells in tobacco, but not to activate cellular differentiation in any other tissue. However, it is expressed in all organs of Antirrhinum, in a pattern restricted to outgrowing cells. These data suggest that AmMYBML3 functions as a very weak enhancer of the cellular differentiation induced by AmMYBMX in petal epidermal cells, and as a weak enhancer of cellular differentiation of other epidermal outgrowths regulated by other, as yet unknown, transcriptional activators. Likely candidates for the activation of trichome and stigmatic papillae outgrowth include the previously identified subgroup 9 protein AmMYBML2. Further investigation of this protein, along with analysis of the Arabidopsis members of the subgroup, should help to resolve the functions of this closely related set of transcriptional regulators.
|
Acknowledgements |
|---|
We thank Cathie Martin and Andrew Hudson for helpful discussions, and Matthew Dorling for assistance with plant care. FJ was funded by a BBSRC studentship.
|
Footnotes |
|---|
* Present address: CSIRO Plant Industry, Adelaide Laboratory, PO Box 350, Glen Osmond, SA 5064, Australia.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
|---|
Avila J, Nieto C, Canas L, Benito MJ, Paz-Ares J. (1993) Petunia hybrida genes related to maize regulatory C1 gene and to animal MYB proto-oncogenes. The Plant Journal 3 553–562.[CrossRef][Web of Science][Medline]
Bevan M. (1984) Binary Agrobacterium vectors for plant transformation. Nucleic Acids Research 12 8711–8721.
Borevitz JO, Xia Y, Blount J, Dixon RA, Lamb C. (2000) Activation tagging identifies a conserved MYB regulator of phenylpropanoid biosynthesis. The Plant Cell 12 2383–2394.
Byrne ME, Barley R, Curtis M, Arroyo JM, Dunham M, Hudson A, Martienssen RA. (2000) Asymmetric leaves 1 mediates axis leaf patterning and stem cell fate in Arabidopsis. Nature 408 967–971.[CrossRef][Medline]
Comba L, Corbet SA, Hunt H, Outram S, Parker JS, Glover BJ. (2000) The role of genes influencing the corolla in pollination of Antirrhinum majus. Plant, Cell and Environment 23 639–647.[CrossRef]
Edwards J, Stoltzfus D, Peterson PA. (2001) The C1 locus in maize (Zea mays L.): effect on gene expression. Theoretical and Applied Genetics 103 718–724.[CrossRef][Web of Science]
Frampton F, Leutz A, Gibson T, Graf T. (1989) DNA-binding domain ancestry. Nature 342 134.[CrossRef][Medline]
Frohman MA, Dush MK, Martin GR. (1988) Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proceedings of the National Academy of Sciences, USA 85 8998–9002.
Glover BJ and Martin C. (1998) The role of petal cell shape and pigmentation in pollination success in Antirrhinum majus. Heredity 80 778–784.[CrossRef][Web of Science]
Glover BJ, Perez-Rodriguez M, Martin C. (1998) Development of several epidermal cell types can be specified by the same MYB-related plant transcription factor. Development 125 3497–3508.[Abstract]
Goff SA, Cone KC, Chandler VL. (1992) Functional analysis of the transcriptional activator encoded by maize B gene: evidence for direct functional interaction between two classes of regulatory proteins. Genes and Development 6 864–875.
Guerineau F and Mullineaux P. (1993) Plant transformation and expression vectors. In Croy R (Ed.). Plant molecular biology LabFaxOxford BIOS Scientific Publishers pp. 121–148.
Hellens RP, Edwards EA, Leyland NR, Bean S, Mullineaux PM. (2000) pGreen, a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation. Plant Molecular Biology 42 819–832.[CrossRef][Web of Science][Medline]
Hofer J, Turner L, Hellens R, Ambrose M, Matthews P, Michael A, Ellis N. (1997) UNIFOLIATA regulates leaf and flower morphogenesis in pea. Current Biology 7 581–587.[CrossRef][Web of Science][Medline]
Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SG, Fraley RT. (1985) A simple and general method for transferring genes into plants. Science 227 1229–1231.
Hülskamp M, Miséra S, Jürgens G. (1994) Genetic dissection of trichome cell development in Arabidopsis. Cell 76 555–566.[CrossRef][Web of Science][Medline]
Jiang C, Gu Z, Peterson T. (2004) Identification of conserved gene structures and carboxy-terminal motifs in the Myb gene family of Arabidopsis and Oryza sative L. ssp. indica. Genome Biology 5, doi:10.1186/gb-2004–5– 7–r46.
Jin H and Martin C. (1999) Multifunctionality and diversity within the plant MYB-gene family. Plant Molecular Biology 41 577–585.[CrossRef][Web of Science][Medline]
Kranz HD, Denekamp M, Greco R, et al. (1998) Towards functional characterisation of the members of the R2R3-MYB gene family from Arabidopsis thaliana. The Plant Journal 16 263–276.[CrossRef][Web of Science][Medline]
Kranz H, Scholz K, Weisshaar B. (2000) c-MYB oncogene-like genes encoding three MYB repeats occur in all major plant lineages. The Plant Journal 21 231–235.[CrossRef][Web of Science][Medline]
Lee M and Schiefelbein J. (2001) Developmentally distinct MYB genes encode functionally equivalent proteins in Arabidopsis. Development 128 1539–1546.[Abstract]
Martin C, Bhatt K, Baumann K, Jin H, Zachgo S, Roberts K, Schwarz-Sommer Z, Glover B, Perez-Rodriguez M. (2002) The mechanics of cell fate determination in petals. Philosophical Transactions of the Royal Society B: Biological Science 357 809–813.[CrossRef]
Martin C, Carpenter R, Sommer H, Saedler H, Coen ES. (1985) Molecular analysis of instability in flower pigmentation of Antirrhinum majus following isolation of the PALLIDA locus by transposon tagging. EMBO Journal 4 1625–1630.[Web of Science][Medline]
Mattanovich D, Rüker F, Machado A, Laimer M, Regner F, Steinkellner H, Himmler G, Katinger H. (1989) Efficient transformation of Agrobacterium spp. by electroporation. Nucleic Acids Research 17 6747.
Moyano E, Martinez-Garcia JF, Martin C. (1996) Apparent redundancy in myb gene function provides gearing for the control of flavonoid biosynthesis in Antirrhinum flowers. The Plant Cell 8 1519–1532.[Abstract]
Noda K, Glover BJ, Linstead P, Martin C. (1994) Flower colour intensity depends on specialized cell shape controlled by a Myb-related transcription factor. Nature 369 661–664.[CrossRef][Medline]
Ogata K, Morikawa S, Nakamura H, Sekikawa A, Ioue T, Kanai H, Sarai A, Ishii S, Nishimura Y. (1994) Solution structure of a specific DNA complex of the MYB DNA-binding domain with cooperation helices. Cell 79 639–648.[CrossRef][Web of Science][Medline]
Payne T, Clement J, Arnold D, Lloyd AM. (1999) Heterologous myb genes distinct from GL1 enhance trichome production when overexpressed in Nicotiana tabacum. Development 126 671–682.[Abstract]
Payne CT, Zhang F, Lloyd A. (2000) GL3 encodes a bHLH protein that regulates trichome development in Arabidopsis through interaction with GL1 and TTG1. Genetics 156 1349–1362.
Paz-Ares J, Ghosal D, Saedler H. (1990) Molecular analysis of the C1-I allele from Zea mays: a dominant mutant of the regulatory C1 locus. EMBO Journal 9 315–321.[Web of Science][Medline]
Perez-Rodriguez M, Jaffe F, Butelli E, Glover BJ, Martin C. (2005) Development of three different cell types is associated with the activity of a specific MYB transcription factor in the ventral petal of Antirrhinum majus. Development 132 359–370.
Quattrocchio F, Wing J, van der Woude K, Souer E, de Vetten N, Mol J, Koes R. (1999) Molecular analysis of the anthocyanin2 gene of Petunia and its role in the evolution of flower color. The Plant Cell 11 1433–1444.
Ramsey N and Glover BJ. (2005) MYB-bHLH-WD40 protein complex and the evolution of cellular diversity. Trends in Plant Science 10 63–70.[CrossRef][Web of Science][Medline]
Serna L and Martin C. (2006) Trichomes: different regulatory networks lead to convergent structures. Trends in Plant Science 11 274–280.[CrossRef][Web of Science][Medline]
Singer T, Gierl A, Peterson PA. (1998) Three new dominant C1 suppressor alleles in Zea mays. Genetical Research 71 127–132.[CrossRef][Web of Science][Medline]
Stracke R, Werber M, Weisshaar B. (2001) The R2R3-MYB gene family in Arabidopsis thaliana. Current Opinion in Plant Biology 4 447–456.[CrossRef][Web of Science][Medline]
Turner GW, Gershenzon J, Croteau RB. (2000) Distribution of peltate glandular trichomes on developing leaves of peppermint. Plant Physiology 124 655–664.
Van Houwelingen A, Souer E, Spelt K, Kloos D, Mol J, Koes R. (1998) Analysis of flower pigmentation mutants generated by random transposon mutagensis in Petunia hybrida. The Plant Journal 13 39–50.[CrossRef][Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  S. Mintz-Oron, T. Mandel, I. Rogachev, L. Feldberg, O. Lotan, M. Yativ, Z. Wang, R. Jetter, I. Venger, A. Adato, et al. Gene Expression and Metabolism in Tomato Fruit Surface Tissues Plant Physiology, June 1, 2008; 147(2): 823 - 851. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||