Expression analysis of TRY promoter fragments

To identify specific regulatory elements, 5' promoter fragments were generated and their regulatory function monitored by fusion to the p35S-minimal promoter and the GUS marker gene (Figure 1A and 2). Because expression of a given construct is variable between different T2 lines we present pictures of the lines with the strongest expression only (Figure 2) and provide the percentage of lines in which the basal expression as well as trichome expression and those in which only the trichome specific expression is found after 24 hours of GUS staining (Figure 1B). Assuming that promoter elements driving a weak expression yield fewer transgenic lines with a strong expression this percentage is taken as an approximation of the expression strength of the promoter fragment under consideration.

For the expression analysis we initially used a fragment starting immediately upstream of an unique putative TATA Box located 32 base pairs upstream of the possible transcription start as determined by RACE PCR [12] (Figure 1A, pTRY-A). This fragment revealed GUS expression in trichomes, but the ubiquitous expression in young leaf regions (basal expression) observed before [12] was absent (Figure 2C, D). We therefore included the fragment immediately following the A-fragment and stretching to the -4 position relative to the ATG start codon (pTRY-B). pTRY-B represents the 5' UTR identified by Schellmann et al. and includes three possible transcriptional start sides suggested by ESTs (EH866228.1, AV533156.1, AI999616.1) and two putative TATA boxes (TATTA, TATAAA) [12, 30–32]. A promoter fragment, pTRY-A, B, combining pTRY-A and pTRY-B revealed also trichome specific expression in 22,7% of the lines but in addition 61,9% of the lines (n = 35) showed the basal expression as well as the expression in trichomes. The pTRY-B fragment alone showed no expression. This indicates that the pTRY-B fragment is essential to enhance or stabilize the expression driven by the pTRY-A fragment.

A further deletion series revealed a minimal promoter region of about 620 bp (pTRY-A3, B). As found for the pTRY-A fragment the pTRY-B region is also necessary in the context of the pTRY-A3 fragment to mediate basal expression (Figure 2E, F). Further 5' deletion of about 200 bp (pTRY-A4, B) revealed trichome specific expression but only weak basal expression. Trichome-specific expression in these lines was only found in advanced stages of trichome development after branch formation (Figure 2G, H). The 200 bp fragment pTRY-A5, B revealed no basal expression and only sometimes a weak irregular expression in trichomes (Figure 2I, J).

These data do not allow to decide whether the pTRY-B fragment enhances both, the basal and trichome specific expression or whether it specifically regulates the basal expression. We therefore compared the expression pattern in pTRY-A3, B and pTRY-A3 at different time points of GUS staining procedure (data not shown). We observed that the GUS staining in young trichomes became detectable in both lines after two hours. While the basal expression in pTRY-A3, B became also detectable after two hours, no basal expression was detectable in pTRY-A3 even after 4 days of GUS staining. These data suggest, that the pTRY-B fragment specifically up-regulates the basal TRY expression in the context of the pTRY-A3 fragment.

Identification of relevant promoter regions by rescue experiments

In order to test their functionality we used various promoter fragments to express the TRY CDS in try-JC mutant plants (Figure 1A, C). In order to avoid the problem that individual transformants may show a wide range of phenotypes we did not select individual lines for analysis in the T2 but directly analyzed the phenotype of T1 plants to hold account on the full phenotypic spectrum. In these experiments both, the pTRY-A, B and the pTRY-A3, B fragments fully rescued the clustering phenotype (Figure 1C). Expression of TRY driven by pTRY-A3, however, had no significant rescue ability (Figure 1C). The smaller fragment (pTRY-A4, B) only partially rescued the try-JC clustering phenotype. Together these data indicate that the pTRY-B fragment is essential for rescue.

Regulation of the TRY promoter by TTG1, GL3, GL1 and TRY or CPC

In a next step we aimed to demonstrate the postulated activation/repression scheme of TTG1, GL3, GL1, TRY and CPC for the minimal TRY promoter fragment. The current models assume that TTG1, GL3 and GL1 can transcriptional activate the inhibitors TRY or CPC and that these in turn repress the activators and thereby also their own expression. The finding that TRY and CPC can compete with GL1 for binding to GL3 [14, 29] suggests that the inhibitors can counteract the activity of the activators at the protein level directly. In this case TRY or CPC repress their own expression without a transcriptional repression of the activators.

As TRY has been shown to be regulated by the activators in genetic experiments [27] the TRY promoter provides a tool to demonstrate that TRY/CPC can counteract the activators without a transcriptional repression of the activators. We used cotyledons for our analysis as no GUS expression was detected in this organ in pTRY-A3, B:GUS plants (Figure 3C). GL1, GL3 and TTG1 CDS under the control of the p35S promoter were used for transient transformations. In addition to these three constructs we added a p35S:GFP:YFP construct to control the bombardment efficiency. In four independent experiments analyzing each time 100 cells we found on average 68.2 ± 18.0% GUS expressing cells indicating that the simultaneous constitutive expression of TTG1, GL3 and GL1 induces the minimal TRY promoter fragment (Figure 3). Transformation with 35S:GFP:YFP alone revealed no GUS positive cells.

In a second step we tested the model whether TRY or CPC can counteract the activity of the activators. Models derived from the finding that TRY and CPC compete with GL1 for binding to GL3 in yeast three hybrid experiments suggest that differential complex formation renders the proposed activator complex inactive [14, 29]. We took advantage of the fact that in this experimental setup any indirect repression of TRY by TRY or CPC through the transcriptional repression of the activators is excluded as their expression is under the control of the 35S promoter. In four independent experiments with 100 cells in each experiment we found only 0.2 ± 0.5% or 0.2 ± 0.4% GUS-positive cells when expressing p35S:TRY or p35S:CPC respectively in addition to the three activators. This indicates that TRY and CPC can counteract TRY activation by the three activators without a transcriptional repression of the activators.

Relevance of MYB and MYC binding sites

Our finding that the activators can activate the TRY promoter in transient expression assays together with the finding that GL3 and GL1 bind to the TRY promoter in ChIP experiments [33, 34] prompted us to search for putative MYB and MYC (bHLH factor) binding sites. We found five putative MYB and two putative MYC sites in the pTRY-A3, B fragment that was the minimal promoter fragment for full rescue of the try phenotype (Table 1). Among the five putative MYB sites two seemed most promising as they were identified in the context of regulatory pathways in other plants that are also regulated by TTG1-dependent pathways [35]. In addition the MYB factor binding to these MYB binding sites are in the same clade in the phylogenetic tree as GL1 [36]. We therefore focused on these two putative binding sites. To determine the role of the two selected MYB and the two MYC sites in the regulation of the correct expression pattern we mutated each site individually and both MYB and both MYC sites together (Figure 4C). None of the mutated constructs showed a marked reduction or even absence of pTRY-A3, B:GUS expression (Figure 4B). However, we noted differences such that the MYB1 site has the most positive effect on the basal expression whereas the MYB2 and the MYC sites have a repressive role. As a pTRY-A3, B:TRY construct containing mutations in both MYC sites resulted in a complete rescue of the try mutant clustering phenotype these sites do not appear to be relevant in this context (Additional File 1)

	5'-TRY- nucleotide sequence	Position relative to the ATG (start/end)	Name of the described cis-element	Putative cis-element nucleotide sequence	Description of the putative cis-element
MYB1	GTTTGGTG	-544/-551	MYBPLANT	MACCWAMC	Binding of AmMYB305 in Antirrhinum majus to box P from gPAL2 of Phaseolus vulgaris; P box related sequences [48, 49] are identified in several promoters of phenylpropanoid biosynthesis related genes (PAL, CHS, CHI, DFR, BZ1) in different plants (Phaseolus vulgaris, Antirrhinum majus, Petunia hybrida, Petroselinum crispum, Arabidopsis thaliana, Zea mays) [50]
MYB2	CCAACC	-531/-536	MYBPZM	CCWACC	Binding in promoters of A1 and BZ1 genes of phlobaphene pigmentation and flavonoid biosynthesis in Zea mays (factors, e.g. C1, P) [51]
MYB3	TTTGTTA	-607/-613	MYBGAHV	TAACAAA	Central element of the gibberellin (GA) response complex (GARC) in the high-pI alpha-amylase gene in Hordeum vulgare, binding of GaMYB [52–54].
MYB4	CCGTT	-153/-157	MYBCOREATCYCB1	AACGG	"Myb core" found in the promoter of Arabidopsis thaliana cyclin B1:1 gene [55].
	GCCGTTCGT	-150/-158	v-MYB*	NSYAACGGN	Binding site of the v-MYB oncocgene of the avian myeloblastosis virus [56].
	GGCCGTTCGT	-150/-159	c-MYB*	NNNAACKGNC	Binding site of the c-MYB, the cellular homolog of v-MYB
MYB5	TTGAACTTGC	-404/-413	c-MYB*	NNNAACKGNC	Binding site of the c-MYB, the cellular homolog of v-MYB
MYC1	CATCTG	-399/-404	MYC CONSENSUSAT	CANNTG	Binding of AtMYC2 in pAtRD22 (dehydration responsive gene) in Arabidopsis thaliana.
MYC2	CATGTG	-243/-248	MYC CONSENSUSAT	CANNTG	Binding of AtMYC2 in pAtRD22 (dehydration responsive gene) in Arabidopsis thaliana [48, 49].
			MYCATERD1	CATGTG	Binding of AtNAC to the ERD1 gene (early responsive to dehydration) in dehydrated Arabidopsis thaliana [57, 58].
			MYCATRD22	CACATG	Binding of AtMYC2 to the RD22 gene (dehydration responsive gene) ) in Arabidopsis thaliana [49].

An additional analysis marked by "*" was done by the TRANSFAC database.

The pTRY-B region mediates the repression of the inhibitors repression

In a separate line of experiments we tested, whether reduced pTRY:GUS expression in the absence of the pTRY-B fragment or in the pTRY-A4, B lines is caused by endogenous R3MYB repressor activity. We compared the expression of the pTRY-A3, B, pTRY-A3 and pTRY-A4, B constructs in wild type and the cpc try mutant background (Figure 5). All constructs revealed a strong basal and trichome specific GUS expression. Thus the lack of basal expression in the pTRY-A3 line and the lack of basal and most of the trichome specific expression in the pTRY-A4, B line is rescued in the cpc try mutant. These data suggest that the -424 to -176 fragment (pTRY-A4) promotes the basal expression and that the pTRY-B fragment and the -623 to -424 (pTRY-A5) fragments mediate repression of the inhibitors repression.

Specific properties of the TRY protein in the regulation of cluster formation and trichome density

The fact that among the six R3 single repeat MYB inhibitor genes only mutations in TRY lead to a clustering phenotype raised the question, whether the transcriptional regulation of TRY or its protein properties constitute this difference. We therefore compared reciprocal swaps of promoters and CDS of the TRY and CPC genes for their ability to rescue the try mutant. CPC was chosen because it represents the main inhibitor for trichome density regulation and because it has a similar expression pattern including the early ubiquitous and later trichome specific expression. Here we chose 525 bp of the 5' upstream region of the CPC gene, which showed the expected CPC expression in leaves and roots (Additional File 2), and was able to rescue the cpc mutant trichome phenotype when fused to the CPC CDS (Additional File 3). Both combinations containing the CDS of TRY, pTRY:cTRY and pCPC:cTRY, completely rescued the clustering phenotype (Figure 6). By contrast, the combination of the TRY promoter with the CDS of CPC exhibited no significant rescue (Figure 6). This indicates that the specific role of TRY in preventing cluster formation is not based on its transcriptional regulation but on specific protein properties.

We also determined the trichome number in these rescued lines. In this respect try mutants have the opposite effect as all the other inhibitor mutants in showing fewer trichomes than wild type [12]. Expression of TRY under the control of the TRY promoter can significantly rescue the trichome number. By contrast, the pTRY:cCPC construct revealed weak but not significant rescue. When using the CPC promoter we found no rescue with the pCPC:cCPC construct and an overexpression phenotype leading to less or even no trichomes in pCPC:cTRY plants. Thus in summary, we recognized protein-specific properties of TRY in the context of TRY dependent trichome density regulation. The finding that the TRY CDS driven by the CPC promoter could not rescue the try density defect suggests additional relevant differences between the two promoters in this context.

Role of the pTRY-B fragment: general enhancer/suppressor or regulator of basal expression

Our promoter analyses revealed an important role of the pTRY-B fragment as it is absolutely necessary for the basal expression in the young leaf and for rescuing the clustering phenotype of the try mutant. It seems to modulate the spatial-temporal expression pattern. Several findings suggest that pTRY-B is specifically required for the basal expression of TRY. First, we never found any basal expression in the absence of the pTRY-B fragment in wild type background. Second, the expression in trichomes is similarly strong (as judged by the GUS staining time course experiments) with and without the pTRY-B fragment and the basal expression co-appears with the trichome specific expression in the presence of the pTRY-B fragment, but is not seen after 4 days without.

The absolute requirement of pTRY-B for rescue of the try clustering phenotype immediately suggests that the basal expression of TRY is relevant for patterning. This finding would be an important piece of support for the current theoretical models [2]. As the pattern is generated in a field of initially equivalent cells, the patterning system needs to start with an initially ubiquitous expression of activators and inhibitors that is necessary for the establishment of a pattern. Thus, according to this scenario the requirement of the pTRY-B fragment and therefore also of the basal expression supports this type of model.

How is the basal TRY expression regulated by the pTRY-B fragment? An answer towards this end comes from our analysis of the pTRY-A3, B:GUS and pTRY-A3:GUS constructs in try cpc mutants. The findings that the pTRY-A3 fragment can be activated by GL1 GL3 and TTG1 in the try cpc double mutants but not in wild type together with the presence of basal expression in pTRY-A3 try cpc plants indicates that the pTRY-B fragment mediates the repression of the TRY repression by TRY or CPC. Thus the apparent requirement of the pTRY-B fragment for basal expression is in fact a double negative regulatory event. The current data suggest that the -424 to -176 fragment is important for turning on the basal expression and that TRY/CPC inhibit this activation with the immediate upstream -623 to -424 and downstream -176 to -4 regions counteracting this inhibition. As the absence of the MYB2 site leads to an increased basal expression it is possible that this site is involved in this regulation loop. Similarly, the higher basal expression upon the deletion of the two MYC sites can be interpreted as a function of these sites in the double negative repression loop.

Self-repression of TRY without transcriptional regulating of the activators

Models explaining trichome formation in Arabidopsis are derived from the activator-inhibitor model formulated by Meinhardt und Gierer [37]. This theoretical model explains pattern formation with two components: an activator activates its own inhibitors and its own expression with the inhibitor being able to move faster than the activator.

When adapting this theoretical model to the biological context there are two possibilities. First, the inhibitor down-regulates its own expression indirectly through the down regulation of the activator. Second, the inhibitor represses its own expression through competitive complex formation [27, 29, 38]. While we can not exclude the first possibility, our data show that the second scenario is sufficient. We show that the minimal TRY promoter can be ectopically activated by the combined expression of GL1, GL3 and TTG1 in cotyledon cells. As the three activators are expressed under the control of the 35S promoter any transcriptional feed back loops involving these three genes are unlikely to be relevant in this experiment. The repression of the activity of the three activators by TRY or CPC provides evidence that TRY and CPC represses the TRY expression directly rather than through a transcriptional feed back loop involving the activator genes.

Specific properties of TRY protein for patterning

To further understand the molecular nature of the uniqueness of TRY among the six R3-single repeat MYB inhibitors, we used promoter swap experiments with CPC which shares all aspects of the TRY expression pattern as judged by promoter:GUS analysis. This enabled us to study the relevance of the transcriptional regulation and protein function of both genes in the try mutant cluster formation and density phenotypes. We found a different behavior of TRY and CPC proteins in these rescuing experiments such that only TRY protein could rescue the try mutant clustering and density phenotype when expressed under the TRY promoter. A similar situation was found in cpc mutant rescue experiments where the TRY protein expression under the control of the CPC promoter resulted in a stronger overexpression phenotype as compared to the CPC protein [39]. A contribution of TRY specific promoter properties was only found in the context of trichome density regulation. This is contrast to the behavior of ETC3 another homolog of TRY and CPC. The etc3 mutant could be rescued by ETC3 in the same manner by regulation through the ETC3, CPC and TRY promoter, so that the promoters were interchangeable with respect to the trichome density phenotype in the etc3 mutant but not with respect to the try mutant [14]. Thus TRY dependent regulation of trichome density is dependent on both, specific protein properties as well as specific aspects of transcriptional regulation.

The observed differences between TRY and CPC protein functions could in principle be due to various aspects including the protein stability, protein movement and their interaction with other proteins, in particular the bHLH factors. Both proteins have been shown to interact with bHLH factors [10, 11, 13, 14, 16, 40]. Their interactions, however, seem to differ as CPC binds stronger to GL3 [41] and suppresses the binding of GL1 to GL3 more efficiently than TRY [14]. Different strength in their binding to GL3 is also likely to change the intercellular movement of TRY and CPC [14]. Both proteins have been shown to move between cells and share a 79 bp N-terminal region in which W76 and M78 were shown to be necessary for movement of CPC and are conserved in the TRY protein [27, 28, 38]. However, TRY protein is lacking the first 9aa that were also be shown to be necessary for CPC movement [28] and could therefore in principle be responsible for a different movement behavior. The most obvious difference between the TRY protein and CPC, ETC1, ETC2, ETC3 and TCL1 is its c-terminal extension of unknown function [41]. While we begin to understand the functional diversification of the R3 single MYB factors in trichome development it is still elusive which properties are responsible for the differences in their requirement for clustering and density control.

Plant lines and growth conditions

Plants were grown on soil at 24°C in a 16 h light/8 h dark cycle. Plant transformations were performed by the floral dip method [43]. The transgenic pTRY-A3, B:GUS Ler and cpc-1 try-82 double mutant [12] line was generated by a genetic cross. Complementation experiments were done in try-JC and cpc-1 mutants [23, 44] and Col-0 and WS-0 respectively as a control. For the transient transformation of cotyledons pTRY-A3, B:GUS, pTRY-A3:GUS and pTRY-A4, B:GUS lines in Ler or in cpc-1 try-82 double mutant background were used. For these experiments surface sterilized seeds were grown on MS plates containing 1% sucrose and 20 μg/ml Basta for 7 days at 22°C with 16 h light/8 h dark cycle.

In silico analysis of the TRY promoter

To identify transcription factor binding sites the Plant Cis-acting Regulatory DNA Elements (PLACE, http://www.dna.affrc.go.jp/PLACE/) and the TRANSFAC (TFSEARCH: Searching Transcription Factor Binding Sites (ver 1.3), http://mbs.cbrc.jp/papia/) databases were used.