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. 2007 Mar 30;7:17.
doi: 10.1186/1471-2229-7-17.

Conifer R2R3-MYB Transcription Factors: Sequence Analyses and Gene Expression in Wood-Forming Tissues of White Spruce (Picea Glauca)

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Conifer R2R3-MYB Transcription Factors: Sequence Analyses and Gene Expression in Wood-Forming Tissues of White Spruce (Picea Glauca)

Frank Bedon et al. BMC Plant Biol. .
Free PMC article

Abstract

Background: Several members of the R2R3-MYB family of transcription factors act as regulators of lignin and phenylpropanoid metabolism during wood formation in angiosperm and gymnosperm plants. The angiosperm Arabidopsis has over one hundred R2R3-MYBs genes; however, only a few members of this family have been discovered in gymnosperms.

Results: We isolated and characterised full-length cDNAs encoding R2R3-MYB genes from the gymnosperms white spruce, Picea glauca (13 sequences), and loblolly pine, Pinus taeda L. (five sequences). Sequence similarities and phylogenetic analyses placed the spruce and pine sequences in diverse subgroups of the large R2R3-MYB family, although several of the sequences clustered closely together. We searched the highly variable C-terminal region of diverse plant MYBs for conserved amino acid sequences and identified 20 motifs in the spruce MYBs, nine of which have not previously been reported and three of which are specific to conifers. The number and length of the introns in spruce MYB genes varied significantly, but their positions were well conserved relative to angiosperm MYB genes. Quantitative RTPCR of MYB genes transcript abundance in root and stem tissues revealed diverse expression patterns; three MYB genes were preferentially expressed in secondary xylem, whereas others were preferentially expressed in phloem or were ubiquitous. The MYB genes expressed in xylem, and three others, were up-regulated in the compression wood of leaning trees within 76 hours of induction.

Conclusion: Our survey of 18 conifer R2R3-MYB genes clearly showed a gene family structure similar to that of Arabidopsis. Three of the sequences are likely to play a role in lignin metabolism and/or wood formation in gymnosperm trees, including a close homolog of the loblolly pine PtMYB4, shown to regulate lignin biosynthesis in transgenic tobacco.

Figures

Figure 1
Figure 1
Alignment of predicted MYB domain protein sequences from spruce and pine. Amino acid sequence alignments of the 21 conifer MYB R2R3 domains were obtained with Clustal W (see Methods) and then separated into three groups based on their homologies to the consensus R2R3-MYB DNA-binding domain (MYBR2R3-DBD, top panel), the bHLH protein-binding motif (bHLH motif, middle panel) or the Arabidopsis calmodulin-interaction motif (AtMYB2 CaMBD, bottom panel), as indicated. Black shading indicates identical amino acid residues and grey shading the similar residues that agree with the fraction sequence of 0,4 (BoxShade 3.21) and dashes indicate gaps. The numbers on the left and right indicate the amino acid position relative to the translation start codon. The boxes and dotted line above the sequences show the predicted helix and turn structures in the R2 and R3 regions of the MYB domain. Stars show positions of conserved tryptophan residues and black arrows indicate unusual amino acid residues compared to the consensus amino acid sequence of the MYB DNA-binding domains of several plant R2R3-MYB proteins described by Avila et al. [27]. The bHLH protein-binding motif ([DE]L × 2 [RK] × 3L × 6L × 3R) identified by Zimmerman et al. [28] and the calmodulin-interaction motif [29] are shown above the middle and bottom panels, respectively (major amino acids in upper-case, bold). Ia or Ib and II indicate the positions of the first and second introns, respectively (Ib is specific to PgMYB3). Accession numbers of the newly identified spruce and pine MYBs are listed in Methods. Pg, Picea glauca; Pt, Pinus taeda; Pm, Picea mariana; At, Arabidopsis thaliana.
Figure 2
Figure 2
Phylogenetic tree of gymnosperm and angiosperm R2R3-MYB proteins. This neighbour-joining (1000 Bootstraps) tree was based on the Clustal W alignment of the complete coding sequences of 13 spruce and five pine MYB proteins identified in this study (represented by filled and empty lozenges, respectively). The bar indicates an evolutionary distance of 0.2%. Arabidopsis proteins were chosen as landmarks representing the three main groups (circles A, B and C) and subgroups (Sg next to bracket; nd, not determined) defined by Romero et al. [19] and Kranz et al. [18]. Human c-MYB [GenBank: P10242] and Mus musculus MmMYBA [GenBank: X82327] were not used as out groups but as landmarks. The accession numbers of the Arabidopsis genes are given in Methods. Other abbreviations are in Figure 1.
Figure 3
Figure 3
Transcript abundance for 13 spruce MYB genes and 4CL in various organs and tissues. Transcript abundance was determined by Q-RTPCR of six tissues from two different 33-year-old trees (number of molecules per ng of total RNA, see methods). The transcript level of an elongation factor (EF1-α) gene was used as an RNA control. N, needles; Stem tissues: P, periderm; Ph, differentiating phloem; X, differentiating xylem; Root tissues: PPh, root periderm with differentiating phloem; X, root differentiating xylem. Data are based on three technical repetitions per tree, i.e. six measurements per data point. Vertical bars represent the standard error. 4CL: 4-coumarate: CoA ligase. NS, no PCR product detected.
Figure 4
Figure 4
Transcript abundance for MYB genes and secondary cell-wall-related genes in differentiating secondary xylem and in primary growth (new flush) of spruce seedlings. Transcript abundance was determined as in Figure 4 for, a) 13 spruce MYB genes, and b) five cell-wall-related genes in differentiating secondary xylem from stem and in the elongating terminal leader (apical stem) from 3-year-old spruce seedlings. The standard error (bars) was calculated from three biological replicates and two independent technical repetitions (i.e. six independent measurements). PAL, phenylalanine ammonia lyase; 4CL, 4-coumarate: CoA ligase; CCOaOMT, caffeoyl-CoA 3-O-methyltransferase; AGP, arabinogalactan protein; CAD, cinnamyl alcohol dehydrogenase. NS, no PCR product detected.
Figure 5
Figure 5
Transcript accumulation for MYB genes and secondary cell-wall-related genes in differentiating compression wood and opposite wood. a) Compression wood and opposite wood formed in a leaning spruce seedling after 21 days of treatment, compared to the control from vertical seedling. Exposed wood (compression wood is light brown) and wood cross-sections (10 μm thick) were stained by the safranin-orange procedure [53] (magnification, ×40). Steady-state mRNA levels were determined as in Figures 4 and 5 for cell-wall-related genes (b) and for several PgMYB genes (c) in the compression wood (left panels) and opposite side wood (right panels) of spruce seedlings leaning at a 45° angle from vertical. Continuous lines indicate genes with significant variation, and standard error bars are shown three trees (biological replicates) with two independent technical repetitions). Discontinuous lines indicate examples of gene transcripts that do not fluctuate in abundance. The zero time point represents vertical control trees only. PgMYB4 (1/15) means that mRNA level is divided by 15.

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