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Conifer R2R3-MYB transcription factors: sequence analyses and gene expression in wood-forming tissues of white spruce (Picea glauca).

Bedon F, Grima-Pettenati J, Mackay J - BMC Plant Biol. (2007)

Bottom Line: The number and length of the introns in spruce MYB genes varied significantly, but their positions were well conserved relative to angiosperm MYB genes.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre d'étude de la Forêt, Université Laval, Pavillon Charles-Eugène Marchand, Sainte Foy G1K7P4, Québec, Canada. frank.bedon@rsvs.ulaval.ca

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.

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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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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.

Mentions: We followed the expression of the 13 spruce MYB genes and five cell-wall-related genes during the early phases of compression wood formation, in order to explore further the potential involvement of MYBs in wood formation and lignin biosynthesis. Gymnosperm trees form a type of reaction wood (known as compression wood) on the lower side of a bent or leaning stem, or in branches. Compression wood is enriched in lignin and contains lignins that are more condensed. Therefore compression wood formation requires the modulation of lignin biosynthesis, which we hypothesized to involve such gene sequences as R2R3-MYBs. We induced the formation of compression wood in actively growing 3-year-old spruces by maintaining at a 45° angle (relative to vertical) (Fig. 5). After 21 days of growth in this leaning position, characteristic compression wood was well developed on the lower side of the stems (Fig. 5a). We chose to monitor transcript abundance over a 76-hour period immediately after induction, and found that several transcripts accumulated between 28 and 76 hours (Fig. 5c). The transcripts of PgMYB2, 4 and 8 clearly increased in the xylem forming compression wood compared to the opposite wood and compared to the vertical trees (0 hour time point). The transcripts of PgMYB9, 11 and 13 RNA were slightly increased and the seven others did not fluctuate significantly. By contrast, no significant variation in spruce MYB RNA abundance was observed in the opposite wood, which is found on the upper side of the stem (opposite to the the compression wood). Transcripts for PAL, 4CL, CCoAOMT and CAD lignin biosynthesis enzymes as well as the AGP also increased within the same time-frame as the MYB transcripts (Fig. 5b). In the opposite wood, only CCoAOMT RNA transcripts decreased. No significant variation in transcript abundance was observed for the spruce MYB or lignin genes in the terminal shoots of the same seedlings (data not shown).


Conifer R2R3-MYB transcription factors: sequence analyses and gene expression in wood-forming tissues of white spruce (Picea glauca).

Bedon F, Grima-Pettenati J, Mackay J - BMC Plant Biol. (2007)

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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Related In: Results  -  Collection

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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.
Mentions: We followed the expression of the 13 spruce MYB genes and five cell-wall-related genes during the early phases of compression wood formation, in order to explore further the potential involvement of MYBs in wood formation and lignin biosynthesis. Gymnosperm trees form a type of reaction wood (known as compression wood) on the lower side of a bent or leaning stem, or in branches. Compression wood is enriched in lignin and contains lignins that are more condensed. Therefore compression wood formation requires the modulation of lignin biosynthesis, which we hypothesized to involve such gene sequences as R2R3-MYBs. We induced the formation of compression wood in actively growing 3-year-old spruces by maintaining at a 45° angle (relative to vertical) (Fig. 5). After 21 days of growth in this leaning position, characteristic compression wood was well developed on the lower side of the stems (Fig. 5a). We chose to monitor transcript abundance over a 76-hour period immediately after induction, and found that several transcripts accumulated between 28 and 76 hours (Fig. 5c). The transcripts of PgMYB2, 4 and 8 clearly increased in the xylem forming compression wood compared to the opposite wood and compared to the vertical trees (0 hour time point). The transcripts of PgMYB9, 11 and 13 RNA were slightly increased and the seven others did not fluctuate significantly. By contrast, no significant variation in spruce MYB RNA abundance was observed in the opposite wood, which is found on the upper side of the stem (opposite to the the compression wood). Transcripts for PAL, 4CL, CCoAOMT and CAD lignin biosynthesis enzymes as well as the AGP also increased within the same time-frame as the MYB transcripts (Fig. 5b). In the opposite wood, only CCoAOMT RNA transcripts decreased. No significant variation in transcript abundance was observed for the spruce MYB or lignin genes in the terminal shoots of the same seedlings (data not shown).

Bottom Line: The number and length of the introns in spruce MYB genes varied significantly, but their positions were well conserved relative to angiosperm MYB genes.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre d'étude de la Forêt, Université Laval, Pavillon Charles-Eugène Marchand, Sainte Foy G1K7P4, Québec, Canada. frank.bedon@rsvs.ulaval.ca

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.

Show MeSH