Limits...
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.


Proteomic analysis of tubulin-enriched xylem extracts. (A-C) MALDI TOF-TOF spectra of cyanogen bromide-cleaved C-terminal peptide of TUA1 (A), TUA1dY (B), and TUA1dEY (C) from WT, A1dYB9, and A1dEYB15 plants, respectively. (D) Relative abundance of the three TUA1 isoforms as determined by MALDI-TOF based on the C-terminal peptides. The peptide intensity of TUA1 was set to 100% in each sample.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: Proteomic analysis of tubulin-enriched xylem extracts. (A-C) MALDI TOF-TOF spectra of cyanogen bromide-cleaved C-terminal peptide of TUA1 (A), TUA1dY (B), and TUA1dEY (C) from WT, A1dYB9, and A1dEYB15 plants, respectively. (D) Relative abundance of the three TUA1 isoforms as determined by MALDI-TOF based on the C-terminal peptides. The peptide intensity of TUA1 was set to 100% in each sample.

Mentions: The tissue-biased transgene expression was also detected at the protein level by western blot analysis. The total TUA (anti-TUA1) signal increased by as much as two-fold in transgenic leaves relative to the WT (Fig. 1C), but remained similar in xylem across genotypes (Fig. 1D). Anti-dY and anti-dEY signals were essentially absent in WT, but were readily detected in the respective transgenics. Specifically, anti-dEY signal was as strong as that of total TUA in leaves of dEYB15 and dEY plants, but accounted for only 17–27% of the total TUA signal in xylem of these plants (Fig. 1C,D). Proteomics analysis confirmed that the PTM mimics TUA1dY and/or TUA1dEY were present in transgenic xylem extracts (Fig. 2). Consistent with the western blot results, levels of the PTM isoforms were negligible in WT, but increased considerably in the transgenics, accounting for 3–6% of the TUA1 levels (Fig. 2D). In both western and proteomics analyses, the dY and dEY isoforms were only detected in the A1dYB9 and A1dEYB15-11 lines, respectively. This suggests that the enzymatic dY-to-dEY conversion reported in animal systems (Janke and Bulinski, 2011) may be absent in Populus. While the abundance estimate varied between detection platforms and cannot be directly compared, the results nevertheless provide protein-level evidence for active synthesis of TUA1 PTM mimics in the transgenics.


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)

Proteomic analysis of tubulin-enriched xylem extracts. (A-C) MALDI TOF-TOF spectra of cyanogen bromide-cleaved C-terminal peptide of TUA1 (A), TUA1dY (B), and TUA1dEY (C) from WT, A1dYB9, and A1dEYB15 plants, respectively. (D) Relative abundance of the three TUA1 isoforms as determined by MALDI-TOF based on the C-terminal peptides. The peptide intensity of TUA1 was set to 100% in each sample.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: Proteomic analysis of tubulin-enriched xylem extracts. (A-C) MALDI TOF-TOF spectra of cyanogen bromide-cleaved C-terminal peptide of TUA1 (A), TUA1dY (B), and TUA1dEY (C) from WT, A1dYB9, and A1dEYB15 plants, respectively. (D) Relative abundance of the three TUA1 isoforms as determined by MALDI-TOF based on the C-terminal peptides. The peptide intensity of TUA1 was set to 100% in each sample.
Mentions: The tissue-biased transgene expression was also detected at the protein level by western blot analysis. The total TUA (anti-TUA1) signal increased by as much as two-fold in transgenic leaves relative to the WT (Fig. 1C), but remained similar in xylem across genotypes (Fig. 1D). Anti-dY and anti-dEY signals were essentially absent in WT, but were readily detected in the respective transgenics. Specifically, anti-dEY signal was as strong as that of total TUA in leaves of dEYB15 and dEY plants, but accounted for only 17–27% of the total TUA signal in xylem of these plants (Fig. 1C,D). Proteomics analysis confirmed that the PTM mimics TUA1dY and/or TUA1dEY were present in transgenic xylem extracts (Fig. 2). Consistent with the western blot results, levels of the PTM isoforms were negligible in WT, but increased considerably in the transgenics, accounting for 3–6% of the TUA1 levels (Fig. 2D). In both western and proteomics analyses, the dY and dEY isoforms were only detected in the A1dYB9 and A1dEYB15-11 lines, respectively. This suggests that the enzymatic dY-to-dEY conversion reported in animal systems (Janke and Bulinski, 2011) may be absent in Populus. While the abundance estimate varied between detection platforms and cannot be directly compared, the results nevertheless provide protein-level evidence for active synthesis of TUA1 PTM mimics in the transgenics.

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.