In-silico analysis of the AtMYB60promoter
In a previous study, we demonstrated that the complete 5' and 3' AtMYB60 intergenic genomic regions - cloned upstream and downstream of the β-glucoronidase (GUS) reporter gene, respectively - could specifically drive strong GUS activity in stomata of Arabidopsis seedlings and adult plants [19]. No GUS signals were detected in any other cell type or in tissues devoid of stomata [19].
To investigate the possible cis-acting elements that regulate AtMYB60 expression, we surveyed the genomic region upstream of the AtMYB60 translational start codon for the presence of known transcription factor binding sites using the PLACE software [24]. Our analysis produced a significant enrichment in the [A/T]AAAG motifs in the AtMYB60 promoter compared to the average distribution of [A/T]AAAG oligos in intergenic regions throughout the Arabidopsis genome (P < 0.01) (Figure 1). Interestingly, these [A/T]AAAG motifs, have been shown to be involved in the regulation of guard cell expression of the potato potassium channel KST1 gene [8]. Also, clusters of [A/T]AAAG motifs, required for the binding of DOF-type transcription factors [25], were over represented in different guard cells-specific promoters [6, 10, 12]. In particular, Galbiati and colleagues suggested, as guard cell-specific cis-element, a cluster of at least three [A/T]AAAG motifs located on the same strand within a region of 100 bp [10]. Using the criteria previously described by Galbiati and collaborators (2008), we found three of these guard cell-specific clusters in the 5' intergenic region of the AtMYB60 gene (Figure 1), suggesting a conserved mechanism for guard cell specific expression.
Identification of the AtMYB60minimal promoter
To gain more insights into the cis-elements that regulate the AtMYB60 expression in guard cells, we produced a set of Arabidopsis transgenic lines carrying the complete 1,307 bp 5' intergenic region upstream of the translational start codon fused to the reporter GUS (construct -1,307::GUS, Figure 2A). GUS staining analysis of 15 independent T2 lines revealed that this region contains all the cis-acting elements required for expression of the reporter in stomata (Figure 2B), while no GUS signals were detected in any other cell type or in tissues devoid of stomata (Additional file 1).
Next, we made a series of 5' deletions of the -1,307 bp genomic region to define the minimum sequence length required for the expression in guard cells (Figure 2A). These truncated promoters (fused to the GUS gene) were stably transferred to Arabidopsis and 10 to 15 independent T2 transgenic lines were analysed in detail. Deletions of the distal part of the 1,307 bp region to position -619 (construct -619::GUS), -472 (-472::GUS), or -366 (-366::GUS) from the ATG codon, did not alter expression of the reporter in guard cells located on both vegetative and floral organs (Figure 2B). Further deletions (to position -262) indicated that the 262 bp proximal region was sufficient to drive expression of the reporter in stomata (Figure 2B). However, the removal of the region between -262 bp and -205 bp (construct -205::GUS) completely abolished GUS activity in guard cell (Figure 2B). Transgenic lines carrying the -205::GUS fusion did not show GUS staining in any other cell type, even after prolonged staining (up to 48 h, Figure 2B). This finding suggests that the 57 bp region located between positions -262 and -205 contains cis-elements essential for expression in stomatal guard cells. Based on these results, we defined the -262 bp region upstream of the ATG codon as the minimal promoter of the AtMYB60 gene.
To thoroughly investigate quantitative differences in GUS expression among lines carrying different deletion:reporter constructs, we determined the relative amount of GUS transcript by quantitative RT-PCR (qRT-PCR). mRNA samples derived from two representative independent lines (A and B) were analysed for each construct (Figure 2C). Lines harbouring the 1,307 bp 5' intergenic region or the -619 deletion fused to the reporter, did not show any significant differences in their GUS transcript accumulation. Conversely, deletions to position -472 and -366 resulted in a two-fold decrease in GUS expression compared to the -1,307::GUS line, while deletion to position -262 resulted in a five-fold decrease (Figure 2C, p < 0.01). These results indicate that one or more sequences with function of enhancer are present in the genomic region between -619 bp and -472 bp and between -472 and -262 from the ATG of AtMYB60. In accordance with the results obtained from the histochemical analysis, qRT-PCR experiments did not detect significant GUS transcripts accumulation in lines carrying the -205::GUS fusion.
Site-directed mutagenesis of the AtMYB60minimal promoter
Promoter deletion experiments indicate that the AtMYB60 minimal promoter region (construct -262::GUS) contains all the cis-acting elements required to sustain expression of a reporter gene in guard cells. This region encompasses the [A/T]AAAG cluster proximal to the ATG codon, which consists of four AAAAG DOF-binding sites (Figures 1 and 3A). In addition, the PLACE software identified in this region a single W-box, corresponding to the binding site of WRKY transcription factors [26], located upstream of the [A/T]AAAG cluster (Figure 3A). To address the functional significance of the individual cis-elements present in the AtMYB60 minimal promoter, we evaluated the effects of targeted nucleotide substitutions on GUS expression (Figure 3A). Mutated versions of the minimal promoter were generated by PCR and fused to GUS and at least 30 T2 independent transgenic lines for each mutated promoter::GUS combination were visually scored and classified to reflect their relative guard-cell specific GUS staining. A representative example of each category is provided in Figure 3C.
We initially tested the role of the single W-box cis-element, by replacing the consensus sequence TTGAC, with the non-functional TTGAA motif [27]. Lines carrying the mutated W-box (mW::GUS) showed similar levels of GUS expression to the wild-type promoter, indicating that W-box does not contribute to mediate gene expression in guard cells (Figure 3B). Next, we produced mutant promoters in which single DOF motifs within the [A/T]AAAG cluster were converted to the unrelated CGCGA sequence. Inactivation of the most distal AAAAG site relative to the ATG (hereinafter referred to as DOF1) resulted in a dramatic decrease of GUS expression (mDOF1::GUS construct, Figure 4B). 30% of the lines carrying the mDOF1::GUS construct did not show GUS expression, whereas the remaining 70% only showed weak staining, thus indicating a crucial role for DOF1 in regulating AtMYB60 expression in guard cells (Figure 3B). Mutations of the second, third or fourth most proximal AAAAG site (hereinafter referred to as DOF2, DOF3 and DOF4, respectively), resulted in a reduced GUS expression, although to a lesser extent than the one in the DOF1 (Figure 4B, mDOF2::GUS, mDOF3::GUS and mDOF4::GUS plants). In particular, none of the 30 mDOF2::GUS transgenic lines displayed strong expression of the reporter, nearly 70% showed intermediate expression, 25% showed weak expression and the remaining 5% did not show any GUS staining (Figure 3B). A comparable distribution among strong, intermediate and weak lines was obtained from the analysis of the mDOF3::GUS and mDOF4::GUS plants (Figure 3B).
To establish whether DOF-binding sites could exert additive roles in mediating gene expression in stomata we produced a second series of promoters, in which two AAAAG motifs were mutated simultaneously. Mutations of DOF1 and DOF2 (mDOF(1+2)::GUS), DOF1 and DOF3 (mDOF(1+3)::GUS) or DOF1 and DOF4 (mDOF(1+4)::GUS) completely inactivated the minimal promoter, as GUS expression was abolished in all the mDOF(1+2)::GUS, mDOF(1+3)::GUS and mDOF(1+4)::GUS lines analysed (Figure 3B). Interestingly, the concurrent mutation of DOF2 and DOF3 (mDOF(2+3)::GUS) resulted in a strong, but yet not complete, inactivation of the promoter activity in guard cells, as 15% of the mDOF(2+3)::GUS lines displayed weak expression of the reporter in stomata. Likewise, concomitant inactivation of either DOF2 and DOF4, or DOF3 and DOF4 did not completely eliminate GUS expression in guard cell (Figure 3B). Taken together, these results indicate that the putative [A/T]AAAG DOF-binding sites located in the AtMYB60 promoter are necessary to mediate its expression in guard cells.
A single DOF cluster is sufficient to drive low expression in guard cell
Our deletion analysis of the AtMYB60 promoter indicates that the 57 bp region between positions -262 and -205 is essential for gene expression in stomatal guard cells (Figure 2). This region contains the DOF1 cis-element required for guard cell expression as shown by mutagenesis analysis results (Figure 3). To establish whether this 57 bp region was sufficient to activate expression in guard cells, we fused one (1x::GUS construct), two (2x::GUS) and four tandem copies (4x::GUS) of the 57 bp fragment to the minimal CaMV35S promoter [28] upstream of the GUS reporter gene (Figure 4A), effectively reconstructing an artificial DOF cluster containing one, two or four copies of the DOF1 element. However, we did not observe GUS activity in any of the 30 independent stable transformants produced for each construct, even after prolonged staining (data not shown). These data were confirmed by qRT-PCR analysis of independent lines carrying the 4x::GUS fusion (Figure 4B), indicating that the multimerisation of the DOF1 site per se is not sufficient to drive gene expression in guard cell. This might derive from an inappropriate organization and/or spatial distribution of the different DOF elements in the context of the minimal promoter. To test this hypothesis we made two 3' deletions of the AtMYB60 minimal promoter: the -148-3'::GUS and -137-3'::GUS constructs containing the first three and four DOF-binding sites respectively of the most proximal cluster fused upstream of the minimal CaMV35S promoter (Figure 4B). Our initial histochemical analysis did not reveal any GUS positive lines (data not shown). To substantiate this result we also performed a qRT-PCR analysis on fifteen independent lines for each construct. Interestingly, eight lines out of fifteen showed a low but significant GUS transcript accumulation compared to the full length minimal promoter (Figure 4B). These results suggest that the presence of the cluster containing three or four DOF-binding sites is sufficient to drive GUS activity in guard cells, even though at a very low level. This finding implies that other cis-elements present downstream of position -137 are required for the full functionality of the minimal promoter.
The guard cell-related CDF1, CDF2, CDF3 and CDF5 DOF-type transcription factors do not regulate AtMYB60expression in stomata
Target mutagenesis experiments of the AtMYB60 promoter demonstrated that [A/T]AAAG DNA consensus motifs are essential cis-acting elements in the regulation of AtMYB60 expression in guard cells. Consequently, their cognate DOF proteins represent the most likely candidates as trans-acting factors. As the Arabidopsis genome contains 36 DOF-coding genes [23], candidate DOF transcription factors involved in the regulation of AtMYB60 expression should fulfil two criteria: they should be expressed in guard cells and the loss of their gene function should abolish or significantly down-regulate the expression of AtMYB60 in this cell type.
The CYCLING DOF FACTOR 1 (CDF1, At5g62430) gene, involved in the regulation of photoperiodic flowering, has been shown to be highly expressed in the vascular tissue and guard cells [29]. We thus investigated the expression of the AtMYB60 gene in the loss-of-function cdf1-R allele. As shown in Additional file 2 we did not detect significant differences in the accumulation of AtMYB60 transcripts in homozygous cdf1-R plants compared with the wild type.
It is important to note that in photoperiodic flowering, CDF1 acts redundantly with three other DOF proteins, namely CDF2 (At5g39660), CDF3 (At3g47500) and CDF5 (At1g69570) [30], belonging to the same phylogenetic group II [31]. Similarly to CDF1, promoter::GUS analyses revealed that CDF2, CDF3 and CDF5 are strongly expressed in guard cells.
We thus analysed the expression of AtMYB60 in single, double, triple and quadruple cdf mutants to determine the possible role of these additional candidate CDF proteins. As for cdf1-R mutant, the level of expression of AtMYB60 was not significantly reduced in the cdf2-1, cdf3-1 and cdf5-1 single mutants (Additional file 2). Likewise, AtMYB60 expression was not altered in any of the double, triple or quadruple mutant combinations, indicating that, despite their expression in guard cells, these four CDF proteins are not trans-regulators of AtMYB60 expression in stomata (Additional file 2).
Identification of a promoter region that negatively responds to ABA
We previously reported that transcript accumulation of the AtMYB60 gene is rapidly down-regulated by exogenous applications of the hormone ABA, which plays a fundamental role in regulating gene expression in response to drought stress [19]. To identify the promoter region responsible for the ABA-mediated AtMYB60 down-regulation, we applied ABA to the previously described transgenic lines harbouring serial deletions of the AtMYB60 promoter (Figure 2). Quantitative RT-PCR analysis revealed a similar decrease in GUS transcript levels in transgenic lines carrying the full length as well as the -619, -472 and -366::GUS fusions (Figure 5). The kinetic of down-regulation of the GUS transcript was comparable to the one observed for the endogenous gene AtMYB60 [19], indicating that -619, -472 and -366 promoters maintain the sequences responsible for transcriptional down-regulation by ABA. Also, these results suggest that the CAAGTTG motif, present in the AtMYB60 promoter between -619 and -613 (dotted underlined in Figure 1), and recently described as overrepresented in ABA-repressed genes [16], does not play a significant role in the ABA-dependent repression of AtMYB60 expression. Rather, qRT-PCR experiments performed on different independent lines carrying the -246::GUS construct showed that the minimal promoter sequence lacks the region responsible for negative regulation by ABA, as these lines did not show changes in GUS expression in response to the hormone as shown in Figure 5.
Taken together these data indicate that, although the minimal promoter maintains the cis-elements necessary for guard cell expression, it lacks the motifs that mediate the negative regulation by ABA, becoming ABA-insensitive. We can thus conclude that the region between -366 and -262 contains elements necessary for ABA down-regulation.