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Keywords:

  • Transcriptional regulation;
  • phenylalanine;
  • Myb factors;
  • ammonium assimilation;
  • conifers

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

During the life cycles of conifer trees, such as maritime pine (Pinus pinaster Ait.), large quantities of carbon skeletons are irreversibly immobilized in the wood. In energetic terms this is an expensive process, in which carbon from photosynthesis is channelled through the shikimate pathway for the biosynthesis of phenylpropanoids. This crucial metabolic pathway is finely regulated, primarily through transcriptional control, and because phenylalanine is the precursor for phenylpropanoid biosynthesis, the precise regulation of phenylalanine synthesis and use should occur simultaneously. The promoters of three genes encoding the enzymes prephenate aminotransferase (PAT), phenylalanine ammonia lyase (PAL) and glutamine synthetase (GS1b) contain AC elements involved in the transcriptional activation mediated by R2R3-Myb factors. We have examined the capacity of the R2R3-Myb transcription factors Myb1, Myb4 and Myb8 to co-regulate the expression of PAT, PAL and GS1b. Only Myb8 was able to activate the transcription of the three genes. Moreover, the expression of this transcription factor is higher in lignified tissues, in which a high demand for phenylpropanoids exits. In a gain-of-function experiment, we have shown that Myb8 can specifically bind a well-conserved eight-nucleotide-long AC-II element in the promoter regions of PAT, PAL and GS1b, thereby activating their expression. Our results show that Myb8 regulates the expression of these genes involved in phenylalanine metabolism, which is required for channelling photosynthetic carbon to promote wood formation. The co-localization of PAT, PAL, GS1b and MYB8 transcripts in vascular cells further supports this conclusion.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Wood is traditionally among the most important commercial products because of the high demand that exits for its derivatives, such as paper, fibres and building materials. Recent developments in biotechnology have inspired novel applications for this traditional raw material, such as its use for biofuel production, and these demands have greatly increased the pressure to improve forest productivity.

Wood (secondary xylem) formation represents a clear example of cell differentiation that is controlled by a wide range of internal and external factors. The regulation of the process itself involves the coordinated expression of various genes involved in cell wall synthesis, cell differentiation and programmed cell death. Owing to the diversity of gene families and the plasticity of wood formation, this complex process is difficult to study (Plomion et al., 2001).

Trees, including conifers, divert large quantities of carbon into the biosynthesis of phenylpropanoids, particularly to generate lignin, an important constituent of wood (Wagner et al., 2012). After cellulose, lignin is the most abundant organic compound on Earth, and comprises 30% of the plant biomass (Higuchi, 1990; Boerjan et al., 2003). Because lignin biosynthesis is one of the most energy demanding biosynthetic pathways in plants (Amthor, 2003), it is precisely regulated. Consequently, tree trunks act as powerful sinks in which considerable percentages of the carbon and energy derived from photosynthesis are immobilised.

Although lignin and other phenolic compounds do not contain nitrogen, phenylalanine metabolism is required to channel photosynthesis-derived carbon to phenylpropanoid biosynthesis (Figure 1). Recent genetic evidence regarding the pathway itself and the genes involved has indicated that the arogenate pathway is the predominant route for phenylalanine biosynthesis in plants (Maeda and Dudareva, 2012). Chorismate, the final product of the shikimate pathway, generates prephenate, which is subsequently aminated to arogenate in a reaction that is catalysed by prephenate aminotransferase (PAT). Until recently, the identity of the gene that encodes this enzyme was unkown. Graindorge et al. (2010) and Maeda et al. (2011) independently discovered that PAT activity was housed by a previously described prokaryotic-type plastidic aspartate aminotransferase (de la Torre et al., 2006, 2009).

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Figure 1. The channelling of photosynthetic carbon for phenylpropanoid biosynthesis and the associated nitrogen metabolism. The central role of the phenylalanine metabolism is highlighted. Critical steps in the pathway are indicated, including amination, deamination and nitrogen recycling. The enzymes PAT, PAL and GS1b are circled.

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The phenylpropane skeleton required for lignin biosynthesis is provided by the deamination of phenylalanine in the reaction catalysed by the enzyme phenylalanine ammonia-lyase (PAL), producing a molecule of cinnamic acid and a molecule of ammonium (Figure 1; Higuchi, 1997). This reaction is quantitatively important in trees because lignin biosynthesis is required for wood formation, and it releases large quantities of ammonium (Humphreys and Chapple, 2002).

The sequential action of the enzymes glutamine synthetase (GS) and glutamate synthase (GOGAT) is crucial for ammonium re-assimilation and glutamate biosynthesis (Figure 1). Nitrogen recycling can satisfy the demand for nitrogen produced during vegetative growth, and it therefore contributes to efficient nitrogen use (Cantón et al., 2005). In conifers, two genes encoding distinct GS isoforms have been described (Ávila et al., 2001). One of these genes, GS1b, is specifically expressed in vascular bundles, and is potentially involved in the recycling of ammonia derived from phenylalanine during lignin synthesis (Cánovas et al., 2007).

It is well known that the levels of lignin biosynthesis enzymes are primarily regulated at the transcriptional level, and that the presence of AC elements in the regulatory regions of these genes is necessary and sufficient to drive localized xylem expression (Lacombe et al., 2000; Wagner et al., 2012). The R2R3Myb transcription factor family is one of the largest transcription factor families in plants, with more than 120 members in Arabidopsis (Riechmann et al., 2000). Some of these genes are involved in the regulation of lignin biosynthesis genes (Stracke et al., 2001). It has been shown that R2R3Myb factors are able to bind the AC elements present in the promoter regions of genes encoding lignin biosynthesis enzymes to activate transcription (Patzlaff et al., 2003a,b). In fact, the presence of AC elements in these gene promoters is almost universal, and suggests coordinated regulation of the set of genes involved in the process (Rogers and Campbell, 2004).

An efficient and coordinated pathway for the amination of prephenate and the deamination of phenylalanine should be operative in lignifying cells to provide phenylalanine for lignin biosynthesis, and to re-assimilate ammonium because this cyclical re-assimilation maintains the efficiency of nitrogen use in these long-lived organisms (Figure 1). We hypothesized that one way to ensure efficient photosynthetic carbon channelling for lignin and other phenylpropanoid biosynthesis, together with nitrogen recycling, would be to couple both processes in time and in space by transcriptionally regulating the genes involved in phenylalanine biosynthesis and use.

The experiments described in this article attempt to test this hypothesis. To this end, we have isolated the promoter region of the three genes involved in the phenylalanine pathway in Pinus pinaster: PAL, GS1b and PAT. We have conducted both in vitro and in vivo studies using three different Myb transcription factors: PtMyb1, PtMyb4 from Pinus taeda and PpMyb8 from P. pinaster. We have studied the possible coupling in space and time of gene products for the operative co-regulation of both processes in pine trees, and have proven that Myb8 is a potential candidate to be the transcriptional regulator of phenylalanine metabolism in P. pinaster vascular cells.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Isolation of maritime pine GS1b, PAL and PAT promoters. In silico analysis of putative cis elements

The 5′ flanking regions of GS1b, PAL and PAT genes were isolated from maritime pine genomic DNA through a genome walking strategy. The full-length cDNAs for GS1b, PAL and PAT (accessions: HF548531, AY641535 and AJ628-016, respectively), genes putatively involved in the lignin pathway, were used as references for genome walking. The transcriptional start sites (TSSs) in the promoter regions (position +1) of the three genes were determined using RLM-RACE, and these data were used to define the 5′-UTR regions in all of the genes.

The isolated PpGS1b gene fragment contained 1206 bp of the sequence located upstream of the translation start codon (TSC), and the TSS was located 68 bp from the ATG (accession: HE866753). The sequence of this fragment was almost identical to the previously described PsGS1b sequence (Gómez-Maldonado et al., 2004a). The promoter region of PpPAL comprised 1722 bp of the sequence located upstream of the TSC, and the TSS was mapped at 187 bp from the ATG (accession: HE866754). The promoter region of PpPAT contained 2064 bp of the region upstream of TSC, and the TSS was positioned 199 bp from the ATG (accession: HE866755).

Bioinformatic analyses were performed to identify conserved putative cis elements that could operate in the regulation of gene expression. Figure 2 shows a diagram representing the region immediately upstream of the TSS for the three genes: PAL, GS1b and PAT. The positions and the putative classes of the AC elements are indicated. The PAL gene promoter contained five AC elements spanning the entire 1535-bp region, all of them belonging to the AC-II class. The upstream region of the GS1b gene displayed six AC elements belonging to the AC-I, AC-II and AC-III classes; most of the elements were located close to the TSS in an identical manner as previously described for the Pinus sylvestris GS1b promoter (Gómez-Maldonado et al., 2004b). The promoter region of the PAT gene contained only one canonical AC element located in the proximal region, also belonging to the AC-II class. A summary of box sequences aligned with the promoters available in other conifers is presented in Figure S1. Because only AC-II class elements were a common feature in the three promoter regions, we decided to jointly analyse the following boxes containing AC elements (Figure 2): Box 1 (from −131 to −51), Box 2 (from −207 to −187), Box 3 (from −953 to −914) and Box 4 (from −1371 to −1336) in the PAL promoter; Box 1 (from −241 to −203) and Box 2 (from −382 to −322) in the GS1b promoter; and Box 1 (from −87 to −18) in the PAT promoter.

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Figure 2. Distribution of AC elements in the PAL, GS1b and PAT promoters. Schematic representation of the Pinus pinaster promoters PAL, GS1b and PAT. The numbers indicate the distance to the relative to the transcriptional start site. The presence of different types of AC elements contained in each promoter is indicated. Only the AC elements used for further analysis were defined as boxes, and their relative positions with respect to the TSS are indicated. A detailed description of the cis elements is provided in the text.

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R2R3Myb proteins bind to the PAL, PpGS1b and PAT AC-II boxes

An in vitro analysis of protein–DNA interactions using an electrophoretic mobility shift assay (EMSA) was conducted using the individual AC boxes described in Figure 2. The EMSA assays were performed using three purified recombinant Myb proteins: PtMyb1 and PtMyb4 were previously described by Gómez-Maldonado et al. (2004b); Myb8 was previously characterized in Picea glauca (white spruce), where it is expressed in differentiating xylem and root (Bedon et al., 2007), and it is also involved in secondary cell wall biogenesis in P. taeda (Bomal et al. (2008)). In this work, the PpMyb8 cDNA was cloned (accession FN 868598), and the recombinant protein was produced and purified (Figure S2).

The EMSA analysis showed noticeable differences in the intensity of shifts, depending on the boxes and Myb proteins used (Figure 3a). Myb1, which has previously been reported as a good candidate to regulate lignin biosynthesis in the differentiating xylem of pine trees (Patzlaff et al., 2003b), was able to bind PAL Box 2 and Box 4. Myb4, the overexpression of which causes ectopic lignification in Nicotiana tabacum (tobacco; Patzlaff et al., 2003a), was able to bind PAL Box 1, Box 2 and Box 4. Interestingly, Myb8, a transcription factor that strongly accumulates in the secondary xylem, and preferentially accumulates during compression wood formation in P. glauca (white spruce; Bedon et al., 2007), was able to bind the four PAL boxes, although the binding complex in Box 3 displayed the strongest signal.

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Figure 3. Electrophoretic mobility shift assays showing the binding of Myb1, Myb4 and Myb8 to the AC-II boxes in the three promoters. (a) Protein binding to DNA targets (PAL Box 1, 2, 3 and 4; GS1b Box 1 and 2; PAT Box 1) was analysed by electrophoretic mobility shift assays. The amount of each purified recombinant protein was 0.6 μg in all of the reactions. The negative control (–) contained no protein. Oligonucleotides corresponding to the probes used in the assay are detailed in Table S1. (b) Schematic representation summarizing protein binding to the boxes in the three promoters. Circles representing the Myb proteins are placed in the relative position at which the interaction with the promoter occurred.

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An EMSA analysis using the GS1b boxes revealed that neither protein could generate a visible mobility shift with Box 2, an AC-III-containing box included as the control; however, the three Myb proteins induced mobility shifts when Box 1 was used as a target. Two bands were visible in the shift assay, indicating that Myb1 and Myb4 binding resulted in two complexes with differential mobility involving this box. This finding was also previously reported for the PsGS1b promoter (Gómez-Maldonado et al., 2004b). Myb8 generated a single band in the shift, suggesting that only one binding site was present in the target for this transcription factor.

All of the Myb proteins were able to bind the AC-II element in the PAT promoter. Myb1 was able to form two possible complexes; Myb4 and Myb8 formed a single complex, with a stronger intensity for Myb8.

Figure 3(b) summarises the EMSA assay results. Two boxes in the PAL promoter, Box 2 and Box 4, bound all of the transcription factors, Myb1, Myb4 and Myb8. Box 1 exclusively bound Myb4, and Box 3 exclusively bound Myb8. A single box in the GS1b (Box 1) and PAT (Box 1) gene promoters was able to bind the three Myb proteins included in the present study. Controls relative to the specificity of the binding complexes formed were carried out for all of the boxes and Myb proteins (Figure S3), with the exception of those relative to the interaction of the GS1b boxes and Myb1 and Myb4 that were previously performed (Gómez-Maldonado et al., 2004b).

Expression analysis of the PAL, GS1b, PAT, Myb1, Myb4 and Myb8 genes in maritime pine

To explore whether there was a correlation between the expression patterns of the PAL, GS1b and PAT genes and the transcription factors Myb1, Myb4 and Myb8 during maritime pine development, the transcript levels were analysed by quantitative PCR (qPCR) in different pine samples (Figure 4). We analysed gene expression in needles (N), stems (S) and roots (R) taken from 1-year-old trees, and cotyledons (C), hypocotyls (H) and roots (R) sampled from 3-week-old seedlings (Figure 4a). Maximum Myb1, Myb4 and Myb8 transcript levels were observed in the hypocotyls of the seedlings and the stems of young trees (Figure 4b). Importantly, Myb8 was expressed almost exclusively in the hypocotyls and stems. The PAL transcript abundance was similar in the different seedling organs; however, in the young trees, the PAL transcript was predominant in the stems. The GS1b and PAT transcripts displayed similar expression patterns in both seedlings and young trees, but higher levels were found in the non-photosynthetic organs, such as the stems and roots.

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Figure 4. Expression analysis of Myb1, Myb4, Myb8, PAL, GS1b and PAT in maritime pine. (a) Schematic representation of young trees and seedlings used for the analysis. Samples: (C) cotyledon, (H) hypocotyl and (R) root in seedlings, and (N) needle, (S) stem and (R) root in young trees. (b) Total RNA was extracted from different samples and reverse transcribed. The cDNA was amplified using specific primers for each gene described in Table S1. The transcript levels were determined by qPCR analysis. (c) Schematic representation of mechanically stressed trees used in the assay; 25-year-old trees were mechanically stressed to produce compression wood, as previously described (Villalobos et al., 2012). Samples from compression (CW) and opposite (OW) differentiating xylem were collected, and RNA was extracted as previously described. (d) Expression analysis of Myb1, Myb4, Myb8, PAL, GS1b and PAT in compression and opposite differentiating xylem from adult maritime pine trees was performed in triplicate, as before. Total RNA was reversed transcribed and transcript levels were determined by qPCR. The expression data were normalized using a geometric mean of the reference genes (ACT, 40S and EF1α). The black bars represent the levels in compression wood and the white bars correspond to opposite wood. The error bars represent the standard deviations of the replica experiments. A Student's t-test was performed to test the significance (P ≤ 0.01) of the differences observed.

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To further explore the relationships between the expression patterns of these genes, we also measured their transcript levels in samples of compression wood and opposite wood from adult trees (Figure 4c). The expression of PAL, GS1b and PAT genes was higher in compression wood than in opposite wood (Figure 4d). Increased expression levels were also observed for Myb4 and Myb8 in compression wood. In contrast, similar Myb1 transcript levels were found in opposite and compression wood as previously described in white spruce under bending stress (Bedon et al., 2007).

Myb proteins differentially activated the transcription driven by the PAL, GS1b and PAT promoters in pine cells

To determine whether the pine Myb proteins could alter gene expression by interacting with the PAL, GS1b and PAT promoters, transient expression assays were performed using protoplasts derived from pine stems. Two different construct types were used in these assays: the effector contained the coding sequences of each one of the Myb proteins, which were under the control of a tandem duplication of the cauliflower mosaic virus 35S promoter (Figure 5a), and the reporter construct contained a fusion between the corresponding promoter available (PAL 1535 bp, GS1b 1138 bp or PAT 1865 bp) and the uidA gene encoding β-glucuronidase (GUS) (Figure 5a). As shown in Figure 5(b), Myb1 and Myb4 could not enhance PAL promoter-driven GUS expression; however, Myb8 increased GUS expression by approximately twofold above the background levels observed in the absence of the transcription factor. All three Myb factors, Myb1, Myb4 and Myb8 transactivated the GS1b promoter in pine cells (Figure 5b). Finally, only Myb4 and Myb8 increased the PAT promoter-driven GUS expression (Figure 5b).

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Figure 5. Effect of Myb1, Myb4 and Myb8 on the PAL, GS1b and PAT promoter activities in pine protoplasts. (a) Schematic representations of the chimeric effector [pJIT60: Myb (1, 4 or 8)] and the reporter (pBI221: PAL, GS1b or PAT) constructs used in these experiments. (b) GUS activities in protoplasts derived from pine stems. Each value represents the average of three independent electroporation events, with the subsequent GUS quantification performed in triplicate. The differences were found to be significant, as determined by a Student's t-test (P < 0.05). The white bar represents the GUS activity of the positive control (+) corresponding to the GUS activity driven by the 35S promoter. PAL, GS1b and PAT, promoter activity without the effector; (PAL, GS1b, and PAT)/M1, (PAL, GS1b, and PAT)/M4 and (PAL, GS1b, and PAT)/M8, promoter activity with Myb1, Myb4 and Myb8, respectively. The GUS activity of the samples is expressed as a percentage of the positive control. A promoter-less derivative of plasmid pBI221 was used as a negative control, and the background level was subtracted to calculate the GUS activity. Significant changes are indicated with an asterisk.

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Role of the conserved AC-II element in the PAL, GS1b and PAT promoters

The data presented above provide evidence supporting the idea that Myb proteins interact in vitro with the PAL, GS1b and PAT promoters, and that they can regulate their promoter activity in vivo. To establish a more direct link between Myb binding activity and transcriptional regulation, we analysed conserved sequences in the putative cis elements of the three gene promoters. We focused on Myb8 because it was the only Myb that could upregulate the transcriptional activity of all three genes. Myb8 is specifically expressed, in an organ-dependent manner, in the shoots/stems, which is where lignin deposition should be quantitatively important (Bomal et al., 2008; this work). Furthermore, its expression increased in compression wood, where increased lignin biosynthesis has been well documented (Bedon et al., 2007; Villalobos et al., 2012).

We identified a consensus sequence conserved in the boxes that were used for the binding analysis (Figures 2 and 3). The consensus sequence containing the AC-II element largely overlaps with the predicted sequence in Figure 2. This sequence motif is composed of eight conserved nucleotides (Figure 6a), and is located in positions quite variable with regard to the TSS in the three genes. PAL Box 2, GS1b Box 1 and PAT Box 1 are located in the proximal portion of their respective promoters, whereas PAL Box 3 and PAL Box 4 are located in the distal portion of the promoter (Figure 2). Myb8 could always induce mobility shifts when these boxes were used as targets (Figure 3). To test our hypothesis that the identified sequence motif might act as a cis element responsible for gene regulation, we constructed a synthetic promoter containing four copies of the AC consensus sequence fused to the 35S minimal promoter (4xACmin). GUS expression driven by this promoter was analysed in pine protoplasts (Figure 6b). The basal promoter activities of the minimal promoter (min) and the 4xACmin promoter were similar, and were considerably lower than that observed with full 35S control. GUS activity with the minimal promoter and Myb8 (M8/min) was even lower than in the absence of the transcription factor; however, Myb8 drastically increased GUS expression when the four AC element copies were present in the 35S minimal promoter (M8/4xACmin).

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Figure 6. Myb responsive cis element identification in the PAL, GS1b and PAT promoters. (a) The conserved Myb 8-bp sequence present in PAL, GS1b and PAT promoters. (b) Analysis of quantitative GUS expression in pine stem protoplasts. Each value represents the average of three independent electroporation events, with the subsequent GUS quantification performed in triplicate. The error bars indicate the SE values. Reporter gene activity was significantly different in the minimal promoter construct containing four copies of the AC element, as determined by a Student's t-test analysis (P < 0.05). The white bar represents the GUS activity of the positive control (+), corresponding to the GUS activity driven by the 35S promoter. Min, minimal 35S promoter; 4xACmin, four copies of the 8-bp conserved element fused to the minimal 35S promoter; Min/M8, minimal 35S promoter in the presence of Myb8; 4xACmin/M8, four copies of the 8-bp conserved element fused to the minimal 35S promoter in the presence of Myb8. Significant changes are indicated with an asterisk.

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Pine PAL, GS1b and PAT transcripts co-localize with Myb8 in vascular cells

To establish a more direct link between Myb protein function and PAL, GS1b and PAT co-regulation, we used in situ hybridization to analyse the distribution patterns of their transcripts in developing pine seedling stems. The patterns of expression are shown in Figure 7, in which a preferential distribution was observed that was dependent on the cellular type. Previous studies in our laboratory have shown that the expression of GS1b is associated with vascular tissue throughout the plant (Ávila et al., 2001; Suárez et al., 2002). These results indicate that the four genes, GS1b, PAL, PAT and Myb8 were highly expressed in vascular cells. The presence of PAL, GS1b and PAT transcripts in the same cell types suggests a functional coordination of these genes in pine metabolism.

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Figure 7. Localization of the PAL, GS1b, PAT and Myb8 transcripts in the stems of pine seedlings. Cross sections (10-μm thick) of Pinus pinaster hypocotyls were subjected to in situ hybridization analysis using an antisense probe for PAL (a, b), GS1b (d, e), PAT (g, h), Myb8 (j, k), and a sense probe for PAL (c), GS1b (f), PAT (i) and Myb8 (l). The black arrows in (a, b, d, e, g, h, j, k) and black arrowheads in (c, f, i, l) indicate vascular cells with evident hybridization signals and vascular cells without hybridization signals, respectively. Ph, phloem; pm, parenchyma; pt, pith; x, xylem.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The understanding of the transcription regulatory network associated with key processes in conifer trees is crucial for future applications in tree improvement and sustainable forest management. In the present work, the transcriptional regulation of the phenylalanine pathway was studied in P. pinaster. We isolated the promoters of three genes involved in phenylalanine metabolism in maritime pine: PAL, GS1b and PAT (Figure 1). The structure of the PAL regulatory region in P. pinaster was found to be similar to the previously described PAL promoter in P. taeda (Osakabe et al., 2009); however, according to the primary structure of the full-length cDNA, the TSS was mapped to a location 187 bp upstream from the ATG. In contrast, the TSS in the P. taeda PAL regulatory region was positioned 22 bp upstream of the ATG. The structures of the GS1b promoters in P. pinaster and P. sylvestris (Gómez-Maldonado et al., 2004a) were almost identical, with only two single nucleotide polymorphisms.

Functional significance of AC-II elements in the PAL, GS1b and PAT promoters

The in silico analysis allowed us to identify the presence of AC elements in the promoters of the three genes under study; however, the AC-II class was the only class that was commonly present in the three genes (Figure 2). The relative positions of these AC-II elements relative to their respective TSSs were not conserved. Thus, the elements were found to be distributed along the PAL promoter, covering the proximal and distal portions, whereas they were specifically located in the proximal region of the GS1b and PAT gene promoters. AC conserved sequences are also found in the promoters of genes involved in lignin biosynthesis in angiosperms (Hauffe et al., 1993; Hatton et al., 1995; Raes et al., 2003; Zhou et al., 2009). A recent review compared the interactions between Myb proteins and their target DNA binding sites in plants and animals, and analysed the consensus sequences of the AC elements (Prouse and Campbell, 2012). Most plant Myb proteins display considerable flexibility in recognising their target sites. Thus, Myb1 and Myb4 proteins were able to bind AC-I and AC-II elements present in the PsGS1b promoter; however, the stronger shift was observed when Myb1 bound the AC-II element as the target site (Gomez-Maldonado et al., 2004b). This result was also confirmed in the present study: the Myb1, Myb4 and Myb8 proteins were able to bind PAL Box 2 and Box 4, GS1b Box 1 and PAT Box 1, but they displayed different affinities (Figure 3). However, an in vivo transient expression analysis revealed a different picture, where a particular Myb protein differentially affected the expression of each gene under study. Thus, the PAL promoter was exclusively transactivated by Myb8. In contrast Myb1, Myb4 and Myb8 activated GS1b expression, and Myb4 and Myb8 activated PAT expression (Figure 6). These data indicate that Myb8 was the only Myb transcription factor that could transactivate the PAL, GS1b and PAT genes. All of these findings suggest that besides a common function in monolignol biosynthesis, the individual gene products contribute to other metabolic pathways separately, they are likely to display differential regulatory specificities by the Myb proteins; for example, GS1b in the biosynthesis of glutamine for nitrogen transport in vascular cells (Ávila et al., 2001); and PAT in the biosynthesis of amino acids in the aspartate metabolic pathway (de la Torre et al., 2009).

The accumulation patterns of the PAL, GS1b, PAT and Myb8 transcripts support a role of these genes in the lignified tissues of maritime pine

The comparative expression profiles of genes involved in phenylalanine metabolism revealed high transcript levels of PAL, GS1b and PAT in the stems and roots of young trees, and in the compression wood of adult trees, therefore reflecting an essential role of this metabolic pathway in tissues that are undergoing lignification. Maximal transcript abundances for Myb1, Myb4 and Myb8 were also observed in pine stems, but only Myb8 was almost exclusively expressed in the stems of seedlings and young trees (Figure 4b). Enhanced gene expression levels were also observed for Myb4 and Myb8 in maritime pine compression wood (Figure 4d), which is consistent with the Myb8 expression pattern that was previously reported in white spruce compression wood (Bedon et al., 2007); however, the observed enhancement was greater for Myb8. Increased lignin deposition and the upregulation of genes encoding enzymes of the monolignol biosynthetic pathway in maritime pine compression wood have been recently reported (Villalobos et al., 2012).

Myb4 appears to be a transcriptional activator of genes involved in lignin biosynthesis in loblolly pine, and the overexpression of Myb4 resulted in ectopic lignification in tobacco (Patzlaff et al., 2003b) and Arabidopsis (Newman et al., 2004).

Previous studies have investigated the involvement of PtMyb1 and PtMyb8 in regulating secondary xylem. The authors overexpressed both Myb genes in spruce (Bomal et al., 2008), and their data clearly showed the impact of PtMyb8 in secondary cell wall biogenesis and in lignin deposition during the formation of compression wood; these results were confirmed by the current study. However, Myb1 overexpression was shown to have a lower impact in the biogenesis of secondary xylem, despite its putative role in lignin deposition (Bomal et al., 2008). These findings are consistent with the similar expression levels observed for Myb1 in the compression and opposite wood of maritime pine (Figure 4d) and white spruce (Bedon et al., 2007).

The sequence 5′-CCAACCAC/A-3′ functions as a regulatory element in the presence of Myb8

Promoter activity is generally the result of the combined effects of many cis elements. These sites mediate intercellular or intracellular stimuli, developmental signals or other functions as particular regulators of metabolic pathways. To identify cis elements mediating Myb8 transcriptional activation, a gain-of-function experiment was performed. We demonstrated that an 8-bp conserved sequence is sufficient to mediate the Myb8 response, and is necessary for the transcriptional activation of the PAL, GS1b and PAT genes. Relatively few of the possible plant R2R3Myb DNA targets have been characterized to date (Prouse and Campbell, 2012). In the current work, a consensus DNA target for Myb8 was derived from both in vitro and in vivo assays. To our knowledge, this is the only transient expression experiment conducted in woody protoplasts that demonstrates the specificity of a Myb protein for a cis element localised in three different promoters, and that demonstrates that this sequence is sufficient to activate the expression of a reporter gene under the control of a minimal promoter.

Maritime pine Myb8 co-regulates prephenate amination, phenylalanine deamination and nitrogen recycling

Considering the major contribution of PAT towards phenylalanine biosynthesis, and also the importance of this pathway in trees, we have contrasted the hypothesis of the transcriptional co-regulation of three genes involved in the process. In Figure 8, a metabolic scheme illustrates the pathways in which the three gene products are involved. Because prephenate amination occurs in the plastid, phenylalanine deamination occurs in the cytosol, and nitrogen recycling via the GS/GOGAT cycle involves enzymes that are located in the two subcellular compartments, at least the two following amino acid transporters are necessary 15 to make the route operative: i) a glutamine translocator and ii) a phenylalanine transporter. The presence of a functional glutamine/glutamate translocator in pine cells has been reported recently (Claros et al., 2010), and its specificity and activity facilitate glutamine import into the chloroplast and glutamate export to the cytosol to avoid nitrogen draining from this essential pathway in plants. It is also simultaneously necessary to translocate phenylalanine to the cytosol for phenylpropanoid biosynthesis. The mechanisms that dictate phenylalanine availability in the cytosol of lignifying cells are poorly understood. In any case, the activity of a phenylalanine transporter in the plastid membrane is necessary to satisfy the great demand of phenylalanine in the cytosol of cells undergoing lignification.

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Figure 8. Proposed model of co-regulation of PAL, GS1b and PAT by Myb8 in pine cells. The PAL, GS1b and PAT enzymes are compartmentalized in the cytosol and plastids of pine cells. The functionality of the pathway requires specific membrane transporters (black circles). The photosynthetic carbon flux is highlighted with thick arrows channelling carbon from the shikimate pathway to phenylalanine, and finally to lignin. In the process, the level of ammonium produced that is quantitatively important is also indicated with a thick arrow. The three enzymes included in this study are encircled and marked with asterisks.

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In this scenario, the simultaneous co-activation of the pathway enzymes would represent a major operational advantage by directing carbon through phenylalanine biosynthesis to promote lignin biosynthesis in different metabolic situations such as regular development or increased lignin production during wood formation.

The role of R2R3Myb transcription factors has been shown to be essential in many aspects of plant secondary metabolism, particularly in the phenylpropanoid metabolism (Stracke et al., 2001). The in vitro and functional interactions described here extend this regulatory role to a critical point at which nitrogen exchange is required to maintain the photosynthetic carbon flux for wood formation in pine trees. Our experimental approaches using different Myb proteins have revealed that despite a possible redundancy in Myb protein function, Myb8 has a prominent role in the transcriptional regulation of essential steps in phenylalanine metabolism. The co-localization studies of gene expression in pine vascular cells further support a role of Myb8 in the coordinated regulation of the phenylalanine pathway. Moreover, a recent study demonstrated that asparagine synthetase expression is downregulated by Myb proteins in maritime pine, which likely prevents the collapse of lignin biosynthesis during wood formation (Canales et al., 2012). In this metabolic context, ammonium assimilated by the GS/GOGAT cycle must be redirected towards arogenate synthesis to maintain aromatic amino acid supplies in lignifying cells.

Experimental procedures

Plant material

The maritime pine seeds (Pinus pinaster Ait.) used in all of the experiments were provided by Centro de Recursos Genéticos Forestales ‘El Serranillo’ (Ministerio de Medio Ambiente y Medio Rural y Marino, Spain). The seed germination and growth of the seedlings were performed as previously described (Cánovas et al., 1991).

For the assays, the plants were pooled, and samples were fractionated in the roots, stems and cotyledons. All of the samples were immediately frozen in liquid nitrogen and stored at −80°C until processing. Seedlings of 1.5 cm in length were used for the transient expression protoplast assays. The stems of these plants were used to produce the protoplasts.

Cloning of PpMYB8 cDNA for overexpression and purification

To clone full-length PpMyb8, RNA was extracted from roots of 60-day-old seedlings following the protocol described by Liao et al. (2004). The RNA concentration and purity were determined spectrophotometrically, and only samples with an A260/A280 nm wavelength ratio of 1.9–2.1 and A260/A230 nm wavelength ratio of more than 1.7 were used for the subsequent experiments. We designed two primers based on the P. taeda Myb8 (DQ399057.1) sequence that included ATTP sites in order to obtain GATEWAY-compatible inserts. The primers used for PCR amplification are described in Table S1 as follows: Myb8 ATTP up and Myb8ATTP down. The PCR reaction was performed with the high-fidelity enzyme AccuSure DNA Polymerase from Bioline (http://www.bioline.com), with the following programme: 94°C for 5 min (1 cycle); 94°C for 30 sec, 45°C for 30 sec and 72°C for 2 min (3 cycles); 94°C for 30 sec, 50°C for 45 sec and 72°C for 2 min (35 cycles); and 72°C for 7 min (1 cycle). The blunt-end PCR products were cloned into the pDON201 plasmid and used with the GATEWAY SYSTEM (Invitrogen, http://www.invitrogen.com).

The full-length cDNA encoding PpMyb8 (accession FN868598) was subcloned into the pDEST17 plasmid by recombination with the GATEWAY System (Invitrogen). The regions flanking the insert were sequenced before the plasmid was used for expression. The Escherichia coli strain BL21 (DE3) was used to express the recombinant protein. The bacterial growth and protein induction were performed according to the manufacturer's instructions (Novagen, now EMD Millipore, http://www.emdmillipore.com; see Figure S2a). Recombinant protein production was induced at 37°C for 2 h in the presence of 0.4 mm isopropyl-β-d-thiogalactoside (IPTG). The N-terminus 6XHis-tagged protein was purified by affinity chromatography using an Ni-NTA column (Qiagen, http://www.qiagen.com) under denaturing conditions. The purification was verified by SDS-PAGE analysis and visualization by Coomassie blue staining (Figure S2b).

Isolation of the promoter regions of the PAL, GS1b and PAT genes

The promoter regions of the P. pinaster PAL, GS1b and PAT genes were amplified by PCR walking using the protocol described by Devic et al. (1997) and following the specifications of the Universal Genome Walker kit, as indicated by Clontech (http://www.clontech.com). The transcriptional start site (+1) in the promoter regions was determined using RLM-RACE using the FirstChoice RLM-RACE Kit (Ambion, now Invitrogen, http://www.invitrogen.com). The 5′ RACE oligonucleotides were designed from the P. pinaster cDNA sequences contained in the EuroPine DB (Fernández-Pozo et al., 2011).

Electrophoretic mobility shift assay

The recombinant proteins PpMyb8 (FN868598), PtMyb1 (AY356372) and PtMyb4 (AY35-6371) were produced as previously described (Gómez-Maldonado et al., 2004b). The oligonucleotide sequence probes described in Figure 3 and Figure S3, and containing the AC elements, were generated by annealing complementary oligonucleotides designed to create 5′ overhangs that were filled-in with Klenow DNA polymerase in the presence of [α32P]dCTP for the boxes described in the PAL and GS1b promoters, and [α32P]dTTP for the box described in the PAT promoter; these are detailed in Table S1. At the end of the incubation period, the DNA–protein complexes were analysed by electrophoresis as previously described (Rueda-López et al., 2008). The binding specificity was evaluated using competition experiments with the non-specific competitor polydI-dC (NSC) or treatment with proteinase K (Gómez-Maldonado et al., 2004b).

Expression analysis

Total RNA was isolated following the protocol described by Liao et al. (2004), with minor modifications. The RNA samples were treated with RQ1 RNase-free DNase (Promega, http://www.promega.com) to eliminate any traces of genomic DNA, and cDNA synthesis was performed as described previously (Canales et al., 2012). qPCR was performed on an Mx3000P thermal cycler (Stratagene, now Agilent, http://genomics.agilent.com) with a SYBR Premix ExTaq kit (Takara, http://www.takarabioeurope.com/contact.html) under the following conditions: 95˚C for 30 sec (1 cycle), and then 95˚C for 5 sec, 60˚C for 10 sec and 72˚C for 15 sec (40 cycles). After the final cycle, a melting curve analysis was performed over a temperature range of 60−95°C in 0.5°C increments to verify the reaction specificity. Ten nanograms of reverse transcribed cDNA was used as a template for each reaction. The raw fluorescence data from each reaction were fitted with the MAK2 model, which requires no assumptions regarding the amplification efficiency of a qPCR assay (Boggy and Woolf, 2010). The initial target concentrations (D0 parameter) for each gene were deduced from the MAK2 model using the qPCR package of r environment software (Ritz and Spiess, 2008) and normalized to the geometric mean of the reference genes (actin, 40S ribosomal protein and EF-1α).

Transient expression analysis in pine protoplasts

The procedure was performed essentially as previously described by Gómez-Maldonado et al. (2004b). The full-length promoter seque-nces available were used for the reporter constructs in the initial transactivation assays. The GUS activity levels were assayed in pine stem-derived protoplasts that were co-electroporated with a combination of reporter and effector plasmids at a 1:1 molar ratio.

Construction of a reporter plasmid using a minimal promoter

To generate a reporter construct containing four copies of the consensus binding site of the AC-II element, an oligonucleotide was fused to the 35S minimal promoter into the polylinker of the GUS-encoding plasmid vector pBI221 (Jefferson et al., 1987) in frame with the GUS gene by replacing the full CaMV 35S promoter.

RNA probes for in situ hybridization

Pinus pinaster cDNA fragments of GS1b (391 bp) and PAL (487 bp) were subcloned into the pGEM®-3Zf (+) vector (Promega) and used to make riboprobes. The RNA antisense and sense probes of PAT (509-bp fragment) and the Myb8 transcription factor (618-bp fragment) mRNA transcripts were prepared by a PCR-based technique in which a T7 polymerase promoter sequence (5′-TAATACGACTCACTATAGGG-3′) was introduced at the 5′ ends of the gene-specific primers (Young et al., 1991). The PCR fragments were produced under standard PCR conditions using iProof™ High-Fidelity Master Mix (Bio-Rad, http://www.bio-rad.com), and plasmid DNA containing the cDNA of interest served as the template. The PCR fragments were gel purified using the NucleoSpin® Gel and PCR Clean-up kit (Macherey-Nagel, http://www.mn-net.com), and 500 ng was subsequently used as a DNA template for in vitro transcription by T7 RNA polymerase (Promega), incorporating dig-UTP via the DIG RNA labelling mix (Roche, http://www.roche.com). The template DNA was digested with five units of RQ1 RNAse-free DNase (Promega) in a reaction volume of 50 ml for 15 min at 37°C, and the probe was purified with the NucleoSpin® RNA Clean-Up XS kit (Macherey-Nagel). The yield of the DIG-labelled RNA probe was estimated by comparing the intensity of the sample against defined controls created with DIG-labelled control RNA (Roche).

In situ hybridization

Small pieces (approximately 5 mm3) of pine stems were fixed and paraffin-embedded, essentially as described by Cantón et al. (1999). The embedded tissue was sectioned at 10-mm thickness and affixed to poly-l-lysine-coated glass slides. The probe bound to the sections was immunologically detected using a sheep anti-digoxigenin Fab fragment that was covalently coupled to alkaline phosphatase and NBT/BCIP as chromogenic substrates (Roche). For the signal amplification, we used polyvinyl alcohol (PVA) of high molecular weight (100–120 kDa) in the BCIP/NBT detection system. An Eclipse E-800 microscope (Nikon, http://www.nikon.com) was used for sample visualization and photography.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

We are most grateful to Dr Campbell for sharing the PtMyb1 and PtMyb4 constructs used to purify both proteins that were used in the EMSA assays, and Dr Cantón for providing the RNA samples from opposite and compression wood. The authors also thank Dr Gómez-Maldonado and M Rueda-López for providing the sequences of the PpGS1b, PpPAT and PpPAL promoters, and Dr FM Suárez for providing guidance for the in situ hybrization assay. This work was supported by grants (CVI-3739) from Junta de Andalucia to C Ávila, (BIO2009-07490) from the Spanish Ministry of Science and Innovation to F.M.C., and from the SUSTAINPINE project (Plant KBBE programme).

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
  • Amthor, J.S. (2003) Efficiency of lignin biosynthesis: a quantitative analysis. Ann. Bot. 91, 673695.
  • Ávila, C., Suárez, M.F., Gómez-Maldonado, J. and Cánovas, F.M. (2001) Spatial and temporal expression of two cytosolic glutamine synthetase genes in Scots pine: functional implications on nitrogen metabolism during early stages of conifer development. Plant J. 25, 93102.
  • Bedon, F., Grima-Pettenati, J. and MacKay, J. (2007) Conifer R2R3-MYB transcription factors: sequence analysis and gene expression in wood-forming tissues of white spruce (Picea glauca). BMC Plant Biol. 7, 17.
  • Boerjan, W., Ralph, J. and Baucher, M. (2003) Lignin Biosynthesis. Ann. Rev. Plant Biol. 54, 519546.
  • Boggy, G.J. and Woolf, P.J. (2010) A mechanistic model of PCR for accurate quantification of quantitative PCR data. PLoS ONE, 5, e12355.
  • Bomal, C., Bedon, F., Caron, S. et al. (2008) Involvement of Pinus taeda MYB1 and Myb8 in phenylpropanoid metabolism and secondary cell wall biogenesis: a comparative in planta analysis. J. Exp. Bot. 59, 39253939.
  • Canales, J., Rueda-López, M., Craven-Bartle, B., Ávila, C. and Cánovas, F.M. (2012) Novel insights into regulation of asparagine synthetase in conifers. Frontiers Plant Sci. 3, 115.
  • Cánovas, F.M., Cantón, F.R., Gallardo, F., García-Gutiérrez, A. and de Vicente, A. (1991) Accumulation of glutamine synthetase during early development of maritime pine (Pinus pinaster) seedlings. Planta, 185, 372378.
  • Cánovas, F.M., Ávila, C., Cantón, F.R., Cañas, R.A. and de la Torre, F. (2007) Ammonium assimilation and amino acid metabolism in conifers. J. Exp. Bot. 58, 23072318.
  • Cantón, F.R., Suárez, M.-F., José-Estanyol, M. and Cánovas, F.M. (1999) Expression analysis of a cytosolic glutamine synthetase gene in cotyledons of Scots pine seedlings: developmental, light-dark regulation and spatial distribution of specific transcripts. Plant Mol. Biol. 40, 623634.
  • Cantón, F.R., Suárez, M.F. and Cánovas, F.M. (2005) Molecular aspects of nitrogen mobilization and recycling in trees. Photosynth. Res. 83, 265278.
  • Claros, M.G., Aguilar, M.L. and Cánovas, F.M. (2010) Evidence for an operative glutamine translocator in chloroplasts from maritime pine (Pinus pinaster Ait.). Plant Biol. 12, 717723.
  • Devic, M., Albert, S., Delseny, M. and Roscoe, T.J. (1997) Efficient PCR walking on plant genomic DNA. Plant Physiol. Biochem. 35, 331339.
  • Fernández-Pozo, N., Canales, J., Guerrero-Fernández, D. et al. (2011) EuroPineDB: a high-coverage web database for maritime pine transcriptome. BMC Genomics, 12, 366.
  • Gómez-Maldonado, J., Cánovas, F.M. and Ávila, C. (2004a) Molecular analysis of the 5′-upstream region of a gibberellin-inducible cytosolic glutamine synthetase gene (GS1b) expressed in pine vascular tissue. Planta, 218, 10361045.
  • Gómez-Maldonado, J., Ávila, C., de la Torre, F., Cañas, R., Cánovas, F.M. and Campbell, M.M. (2004b) Functional interactions between a glutamine synthetase promoter and MYB proteins. Plant J. 39, 513526.
  • Graindorge, M., Giustini, C., Jacomin, A.C., Kraut, A., Curien, G. and Matringe, M. (2010) Identification of a plant gene encoding glutamate/aspartate-prephenate aminotransferase: the last homeless enzyme of aromatic amino acids biosynthesis. FEBS Lett. 584, 43574360.
  • Hatton, D., Sablowski, R., Yung, M.H., Smith, C., Schuch, W. and Bevan, M. (1995) Two classes of cis sequences contribute to tissue-specific expression of a PAL2 promoter in transgenic tobacco. Plant J. 7, 859876.
  • Hauffe, K.D., Lee, S.P., Subramaniam, R. and Douglas, C.J. (1993) Combinatorial interactions between positive and negative cis-acting elements control spatial patterns of 4CL-1 expression in transgenic tobacco. Plant J. 4, 235253.
  • Higuchi, T. (1990) Lignin biochemistry: biosynthesis and biodegradation. Wood Sci. Technol. 24, 2363.
  • Higuchi, T. (1997) Biochemistry and Molecular Biology of Wood. New York: Springer 131181.
  • Humphreys, J.M. and Chapple, C. (2002) Rewriting the lignin roadmap. Curr. Opin. Plant Biol. 5, 224229.
  • Jefferson, R.A., Kavanagh, T.A. and Bevan, M.W. (1987) GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6, 39013907.
  • Lacombe, E., Van Doorsselaere, J., Boerjan, W., Boudet, A.M. and Grima-Pettenati, J. (2000) Characterization of cis-elements required for vascular expression of the cinnamoyl CoA reductase gene and for protein-DNA complex formation. Plant J. 23, 663676.
  • Liao, Z., Chen, M., Guo, L., Gong, Y., Tang, F., Sun, X. and Tang, K. (2004) Rapid isolation of high-quality total RNA from Taxus and Ginkgo. Prep. Biochem. Biotech. 34, 209214.
  • Maeda, H. and Dudareva, N. (2012) The shikimate pathway and aromatic amino acid biosynthesis in plants. Ann. Rev. Plant Biol. 63, 73105.
  • Maeda, H., Yoo, H. and Dudareva, N. (2011) Prephenate aminotransferase directs plant phenylalanine biosynthesis via arogenate. Nat. Chem. Biol. 7, 1921.
  • Newman, L.J., Perazza, D.E., Juda, L. and Campbell, M.M. (2004) Involvement of the R2R3-MYB, At MYB61, in the ectopic lignication and dark-photomorphogenic components of the det3 mutant phenotphype. Plant J. 37, 239250.
  • Osakabe, Y., Osakabe, K. and Chiang, V.L. (2009) Characterization of the tissue-specific expression of phenylalanine ammonia-lyase gene promoter from loblolly pine (Pinus taeda) in Nicotiana tabacum. Plant Cell Rep. 28, 13091317.
  • Patzlaff, A., McInnis, S., Courtenay, A. et al. (2003a) Characterisation of a pine MYB that regulates lignification. Plant J. 36, 743754.
  • Patzlaff, A., Newman, L.J., Dubos, C., Whetten, R.W., Smith, C., McInnis, S., Bevan, M.W., Sederoff, R.R. and Campbell, M.M. (2003b) Characterization of PtMYB1, an R2R3-MYB from pine xylem. Plant Mol. Biol. 53, 597608.
  • Plomion, C., Leprovost, G. and Stokes, A. (2001) Wood Formation in Trees. Plant Physiol. 127, 15131523.
  • Prouse, M.B. and Campbell, M.M. (2012) The interaction of MYB proteins and their target DNA binding sites. Biochim. Biophys. Acta 1819, 6777.
  • Raes, J., Rohde, A., Christensen, J.H., Van de Peer, Y. and Boerjan, W. (2003) Genome-wide characterization of the lignification toolbox in Arabidopsis. Plant Physiol. 133, 10511071.
  • Riechmann, J.L., Heard, J., Martin, G. et al. (2000) Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes. Science, 290, 21052110.
  • Ritz, C. and Spiess, A.N. (2008) qpcR: an R package for sigmoidal model selection in quantitative real-time polymerase chain reaction analysis. Bioinformatics, 24, 15491551.
  • Rogers, L.A. and Campbell, M.M. (2004) The genetic control of lignin deposition during plant growth and development. New Phytol. 164, 1730.
  • Rueda-López, M., Crespillo, R., Cánovas, F.M. and Ávila, C. (2008) Differential regulation of two glutamine synthetase genes by a single Dof transcription factor. Plant J. 56, 7385.
  • Stracke, R., Werber, M. and Weisshaar, B. (2001) The R2R3-MYB gene family in Arabidopsis thaliana. Curr. Opin. Plant Biol. 4, 447456.
  • Suárez, M.F., Ávila, C., Gallardo, F., Cantón, F.R., García-Gutiérrez, A., Claros, M.G. and Cánovas, F.M. (2002) Molecular and enzymatic analysis of ammonium assimilation in woody plants. J. Exp. Bot. 53, 891904.
  • de la Torre, F., De Santis, L., Suárez, M.F., Crespillo, R., Rodríguez-Caso, C., Cañas, R., Sánchez-Jiménez, F.M. and Cánovas, F.M. (2006) Identification and functional analysis of a prokaryotic–type aspartate aminotransferase: implications for plant amino acid metabolism. Plant J. 46, 414425.
  • de la Torre, F., De Santis, L., Suárez, M.F., Crespillo, R. and Cánovas, F.M. (2009) Molecular modelling and site directed mutagenesis reveal essential residues for catalysis in a prokaryotic-type plant aspartate aminotransferase. Plant Physiol. 149, 16481660.
  • Villalobos, D.P., Diaz-Moreno, S.M., El-Sayed, S.S., Cañas, R.A., Osuna, D., Van Kerckhoven, S.H.E, Bautista, R., Claros, M.G., Cánovas, F.M. and Cantón, F.R. (2012) Reprogramming of gene expression during compression wood formation in pine: coordinated modulation of S-adenosylmethionine, lignin and lignan related genes. BMC Plant Biol. 12, 100.
  • Wagner, A., Donaldson, L. and Ralph, J. (2012) Lignification and lignin manipulation in conifers. Adv. Bot. Res. 61, 3776.
  • Young, I.Y., Ailles, L., Deugau, K. and Kisilevsky, R. (1991) Transcription of cRNA for in situ hybridization from polymerase chain reaction-amplified DNA. Lab. Invest. 64, 709712.
  • Zhou, J., Lee, C., Zhong, R. and Zheng-Hua, Y. (2009) MYB58 and MYB63 are transcriptional activators of the lignin biosynthetic pathway during secondary cell wall formation in Arabidopsis. Plant Cell, 21, 248266.

Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
FilenameFormatSizeDescription
tpj12158-sup-0001-FigS1.tifimage/tif5836KFigure S1. Comparison of boxes containing AC elements in the PAL, GS1b and PAT promoters in conifers.
tpj12158-sup-0002-FigS2.tifimage/tif2310KFigure S2. Purification of the recombinant Myb8 protein (a) Time course induction with IPTG as described in the Experimental procedures section. (b) SDS-PAGE of the purification procedure: 1: MW markers; 2: crude extract; 3: Not attached; 4: First wash; 5: second wash; 6: First elution; 7: second elution.
tpj12158-sup-0003-FigS3.tifimage/tif17772KFigure S3. Specificity of electrophoretic mobility shift assays (a) Protein binding to DNA targets: PAL Box 2 and 4 and PAT Box 1 with Myb1. (b) PAL boxes 1, 2 and 4 and PAT Box 1 with Myb4. (c) PAL boxes 2,3 and 4 and PAT Box 1 with Myb8. (d) GS1b Box1 with Myb8. The negative control contained no protein.The positive control contained the corresponding Myb protein in each case. Nsc contained polydI-dC as non-specific competitor and in the prot K lane the corresponding Myb protein treated with proteinase K before the assay.
tpj12158-sup-0004-TableS1.docxWord document105KTable S1. Oligonucleotides used for this work.
tpj12158-sup-0005-SupplementLegends.docxWord document13K 

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