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Tubulin perturbation leads to unexpected cell wall modifications and affects stomatal behaviour in Populus.

Swamy PS, Hu H, Pattathil S, Maloney VJ, Xiao H, Xue LJ, Chung JD, Johnson VE, Zhu Y, Peter GF, Hahn MG, Mansfield SD, Harding SA, Tsai CJ - J. Exp. Bot. (2015)

Bottom Line: The results suggest that pectin and xylan polysaccharides deposited early during cell wall biogenesis are more sensitive to subtle tubulin perturbation than cellulose and matrix polysaccharides deposited later.Pectins have been shown to confer cell wall flexibility critical for reversible stomatal movement, and results presented here are consistent with microtubule involvement in this process.Taken together, the data show the value of growth-compatible tubulin perturbations for discerning microtubule functions, and add to the growing body of evidence for microtubule involvement in non-cellulosic polysaccharide assembly during cell wall biogenesis.

View Article: PubMed Central - PubMed

Affiliation: School of Forestry and Natural Resources, University of Georgia, Athens, GA 30602, USA.

No MeSH data available.


Related in: MedlinePlus

Tubulin transcript and protein accumulation. (A, B) qRT-PCR analysis of relative tubulin transcript abundance in leaves (A) and xylem (B). Data are mean ± SD of n = 3–7 biological replicates, except for A1dEY-5 (mean ± range of n = 2) and A1dEYB15-12 (n = 1). (C, D) Western blot analysis of TUA levels in leaves (C) and xylem (D) using polyclonal antibodies raised against recombinant TUA1 (anti-TUA1) or synthetic C-terminal peptides of TUA1 (anti-dY and anti-dEY). Relative (%) signal abundance normalized to marker bands was estimated within the anti-TUA1 blot for genotypic comparison, or across the three blots (anti-TUA1, anti-dY, and anti-dEY) for isoform comparison.
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Figure 1: Tubulin transcript and protein accumulation. (A, B) qRT-PCR analysis of relative tubulin transcript abundance in leaves (A) and xylem (B). Data are mean ± SD of n = 3–7 biological replicates, except for A1dEY-5 (mean ± range of n = 2) and A1dEYB15-12 (n = 1). (C, D) Western blot analysis of TUA levels in leaves (C) and xylem (D) using polyclonal antibodies raised against recombinant TUA1 (anti-TUA1) or synthetic C-terminal peptides of TUA1 (anti-dY and anti-dEY). Relative (%) signal abundance normalized to marker bands was estimated within the anti-TUA1 blot for genotypic comparison, or across the three blots (anti-TUA1, anti-dY, and anti-dEY) for isoform comparison.

Mentions: PCR analysis of genomic DNA confirmed the presence of the TUA and TUB transgenes in the transgenic lines, except for the presumed A1dEYB15-5 and A1dEYB15-17 lines which lacked the TUB15 and NPTII PCR amplicons and were renamed A1dEY-5 and A1dEY-17 (Supplementary Fig. S2). The first cohort of transformants, including A1dYB9 (one line), A1dEYB15 (two lines), and A1dEY (one line) plants, was fully characterized. Endogenous transcript abundance was not changed in any of the transgenic lines (Fig. 1). The transcript abundance of TUA1dY or TUA1dEY in mature leaves (LPI-15) was 6–17-fold higher than endogenous TUA1 in all transgenic lines (Fig. 1A). In contrast, expression of the TUB transgenes was low, and sometimes below endogene levels (Fig. 1A). In developing xylem where endogenous tubulin transcripts were abundant, the transgenes were detected at very low levels in all viable transgenic lines we obtained. This strongly suggested that high levels of tubulin transgene expression were not tolerated in xylem during organogenesis. As in leaves, the relative transcript abundance of TUB transgenes was lower than that of TUA transgenes (Fig. 1B). The results suggest that the TUA and TUB transgenes were, much like their endogenous counterparts, differentially regulated in a gene family- and tissue-dependent manner.


Tubulin perturbation leads to unexpected cell wall modifications and affects stomatal behaviour in Populus.

Swamy PS, Hu H, Pattathil S, Maloney VJ, Xiao H, Xue LJ, Chung JD, Johnson VE, Zhu Y, Peter GF, Hahn MG, Mansfield SD, Harding SA, Tsai CJ - J. Exp. Bot. (2015)

Tubulin transcript and protein accumulation. (A, B) qRT-PCR analysis of relative tubulin transcript abundance in leaves (A) and xylem (B). Data are mean ± SD of n = 3–7 biological replicates, except for A1dEY-5 (mean ± range of n = 2) and A1dEYB15-12 (n = 1). (C, D) Western blot analysis of TUA levels in leaves (C) and xylem (D) using polyclonal antibodies raised against recombinant TUA1 (anti-TUA1) or synthetic C-terminal peptides of TUA1 (anti-dY and anti-dEY). Relative (%) signal abundance normalized to marker bands was estimated within the anti-TUA1 blot for genotypic comparison, or across the three blots (anti-TUA1, anti-dY, and anti-dEY) for isoform comparison.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4588895&req=5

Figure 1: Tubulin transcript and protein accumulation. (A, B) qRT-PCR analysis of relative tubulin transcript abundance in leaves (A) and xylem (B). Data are mean ± SD of n = 3–7 biological replicates, except for A1dEY-5 (mean ± range of n = 2) and A1dEYB15-12 (n = 1). (C, D) Western blot analysis of TUA levels in leaves (C) and xylem (D) using polyclonal antibodies raised against recombinant TUA1 (anti-TUA1) or synthetic C-terminal peptides of TUA1 (anti-dY and anti-dEY). Relative (%) signal abundance normalized to marker bands was estimated within the anti-TUA1 blot for genotypic comparison, or across the three blots (anti-TUA1, anti-dY, and anti-dEY) for isoform comparison.
Mentions: PCR analysis of genomic DNA confirmed the presence of the TUA and TUB transgenes in the transgenic lines, except for the presumed A1dEYB15-5 and A1dEYB15-17 lines which lacked the TUB15 and NPTII PCR amplicons and were renamed A1dEY-5 and A1dEY-17 (Supplementary Fig. S2). The first cohort of transformants, including A1dYB9 (one line), A1dEYB15 (two lines), and A1dEY (one line) plants, was fully characterized. Endogenous transcript abundance was not changed in any of the transgenic lines (Fig. 1). The transcript abundance of TUA1dY or TUA1dEY in mature leaves (LPI-15) was 6–17-fold higher than endogenous TUA1 in all transgenic lines (Fig. 1A). In contrast, expression of the TUB transgenes was low, and sometimes below endogene levels (Fig. 1A). In developing xylem where endogenous tubulin transcripts were abundant, the transgenes were detected at very low levels in all viable transgenic lines we obtained. This strongly suggested that high levels of tubulin transgene expression were not tolerated in xylem during organogenesis. As in leaves, the relative transcript abundance of TUB transgenes was lower than that of TUA transgenes (Fig. 1B). The results suggest that the TUA and TUB transgenes were, much like their endogenous counterparts, differentially regulated in a gene family- and tissue-dependent manner.

Bottom Line: The results suggest that pectin and xylan polysaccharides deposited early during cell wall biogenesis are more sensitive to subtle tubulin perturbation than cellulose and matrix polysaccharides deposited later.Pectins have been shown to confer cell wall flexibility critical for reversible stomatal movement, and results presented here are consistent with microtubule involvement in this process.Taken together, the data show the value of growth-compatible tubulin perturbations for discerning microtubule functions, and add to the growing body of evidence for microtubule involvement in non-cellulosic polysaccharide assembly during cell wall biogenesis.

View Article: PubMed Central - PubMed

Affiliation: School of Forestry and Natural Resources, University of Georgia, Athens, GA 30602, USA.

No MeSH data available.


Related in: MedlinePlus