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Imaging with the fluorogenic dye Basic Fuchsin reveals subcellular patterning and ecotype variation of lignification in Brachypodium distachyon.

Kapp N, Barnes WJ, Richard TL, Anderson CT - J. Exp. Bot. (2015)

Bottom Line: It was found that the extent and intensity of Basic Fuchsin fluorescence increase over time in the Bd21-3 ecotype, that Basic Fuchsin staining is more widespread and intense in 4-week-old Bd21-3 and Adi-10 basal internodes than in Bd1-1 internodes, and that Basic Fuchsin staining reveals subcellular patterns of lignin in vascular and interfascicular fibre cell walls.Basic Fuchsin fluorescence did not correlate with lignin quantification by acetyl bromide analysis, indicating that whole-plant and subcellular lignin analyses provide distinct information about the extent and patterns of lignification in B. distachyon.Finally, it was found that flowering time correlated with a transient increase in total lignin, but did not correlate strongly with the patterning of stem lignification, suggesting that additional developmental pathways might regulate secondary wall formation in grasses.

View Article: PubMed Central - PubMed

Affiliation: Center for Lignocellulose Structure and Formation, The Pennsylvania State University, University Park, PA 16802, USA Department of Biology, The Pennsylvania State University, University Park, PA 16802, USA Department of Agricultural and Biological Engineering, The Pennsylvania State University, University Park, PA 16802, USA.

No MeSH data available.


Basic Fuchsin staining increases over the course of B. distachyon development. Basic Fuchsin fluorescence intensity μm–2 increases 2.04-fold and Wiesner staining increases in B. distachyon ecotype Bd21-3 from 4 weeks (A, A’, A’’) to 12 weeks (B, B’, B’’). Average pixel intensity above threshold μm–2 is 14 713 (AFU) for (A) and 34 389 (AFU) for (B). Basic Fuchsin fluorescence intensity of 4-week-old Bd21-3 (C) and BdPMT OX (E) plants decreases after saponification (D, F) by approximately 2-fold (G). Greyscale representations of Basic Fuchsin fluorescence (A–F), Basic Fuchsin fluorescence with a pseudo-colour look-up table applied (A’–F’) where the intensity profile shows pixel intensity values for different colours, and Wiesner staining (A’’, B’’). Scale bar=100 μm. Error bars in (G) show standard error; values with different superscript letters indicate significant differences between each ecotype pair measured by one-way ANOVA and post-hoc test, P <0.05.
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Figure 1: Basic Fuchsin staining increases over the course of B. distachyon development. Basic Fuchsin fluorescence intensity μm–2 increases 2.04-fold and Wiesner staining increases in B. distachyon ecotype Bd21-3 from 4 weeks (A, A’, A’’) to 12 weeks (B, B’, B’’). Average pixel intensity above threshold μm–2 is 14 713 (AFU) for (A) and 34 389 (AFU) for (B). Basic Fuchsin fluorescence intensity of 4-week-old Bd21-3 (C) and BdPMT OX (E) plants decreases after saponification (D, F) by approximately 2-fold (G). Greyscale representations of Basic Fuchsin fluorescence (A–F), Basic Fuchsin fluorescence with a pseudo-colour look-up table applied (A’–F’) where the intensity profile shows pixel intensity values for different colours, and Wiesner staining (A’’, B’’). Scale bar=100 μm. Error bars in (G) show standard error; values with different superscript letters indicate significant differences between each ecotype pair measured by one-way ANOVA and post-hoc test, P <0.05.

Mentions: To test whether Basic Fuchsin staining changes over time to reflect developmental increases in stem lignin content, 4-week-old and 12-week-old B. distachyon ecotype Bd21-3 basal stem sections were analysed using Basic Fuchsin staining and the observed patterns of fluorescence were compared with those observed after staining with the Wiesner reagent (Fig. 1). The basal internode was of interest in this study because it represents the oldest stem tissue and is expected to accumulate lignin throughout development. Qualitative observation of lignin staining indicated that both Basic Fuchsin fluorescence and Wiesner staining were more widespread and pronounced in 12-week-old stem sections (Fig. 1). Quantitative measurement indicated that Basic Fuchsin fluorescence intensity μm–2 was 2.34-fold higher in 12-week-old Bd21-3 stems. It was observed that this increase arose predominantly from increased staining in interfascicular regions, supporting the well-documented spatio-temporal pattern of lignification in grasses such as B. distachyon throughout maturation (Matos et al., 2013). By calculating the ratio of thresholded stem area to total stem area, it was possible to quantify the relative lignified area within a stem section. This number was 4.25-fold higher in 12-week-old Bd21-3 stems than in 4-week-old stems, suggesting that the increase in fluorescence intensity μm–2 can be attributed to an increase in Basic Fuchsin fluorescence intensity in previously lignified tissues as well as to an increase in lignified area in more mature tissue (Fig. 1). Basic Fuchsin fluorescence and Wiesner staining was also observed in the epidermis of 12-week-old Bd21-3 stems, possibly due to the presence of cutin or suberin which can also fluoresce when stained with Basic Fuchsin (Kraus et al., 1998). Overall, the similar staining pattern between Basic Fuchsin and the Wiesner reagent supports the interpretation that Basic Fuchsin fluorescence intensity correlates with the amount and location of lignin in secondary cell walls.


Imaging with the fluorogenic dye Basic Fuchsin reveals subcellular patterning and ecotype variation of lignification in Brachypodium distachyon.

Kapp N, Barnes WJ, Richard TL, Anderson CT - J. Exp. Bot. (2015)

Basic Fuchsin staining increases over the course of B. distachyon development. Basic Fuchsin fluorescence intensity μm–2 increases 2.04-fold and Wiesner staining increases in B. distachyon ecotype Bd21-3 from 4 weeks (A, A’, A’’) to 12 weeks (B, B’, B’’). Average pixel intensity above threshold μm–2 is 14 713 (AFU) for (A) and 34 389 (AFU) for (B). Basic Fuchsin fluorescence intensity of 4-week-old Bd21-3 (C) and BdPMT OX (E) plants decreases after saponification (D, F) by approximately 2-fold (G). Greyscale representations of Basic Fuchsin fluorescence (A–F), Basic Fuchsin fluorescence with a pseudo-colour look-up table applied (A’–F’) where the intensity profile shows pixel intensity values for different colours, and Wiesner staining (A’’, B’’). Scale bar=100 μm. Error bars in (G) show standard error; values with different superscript letters indicate significant differences between each ecotype pair measured by one-way ANOVA and post-hoc test, P <0.05.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 1: Basic Fuchsin staining increases over the course of B. distachyon development. Basic Fuchsin fluorescence intensity μm–2 increases 2.04-fold and Wiesner staining increases in B. distachyon ecotype Bd21-3 from 4 weeks (A, A’, A’’) to 12 weeks (B, B’, B’’). Average pixel intensity above threshold μm–2 is 14 713 (AFU) for (A) and 34 389 (AFU) for (B). Basic Fuchsin fluorescence intensity of 4-week-old Bd21-3 (C) and BdPMT OX (E) plants decreases after saponification (D, F) by approximately 2-fold (G). Greyscale representations of Basic Fuchsin fluorescence (A–F), Basic Fuchsin fluorescence with a pseudo-colour look-up table applied (A’–F’) where the intensity profile shows pixel intensity values for different colours, and Wiesner staining (A’’, B’’). Scale bar=100 μm. Error bars in (G) show standard error; values with different superscript letters indicate significant differences between each ecotype pair measured by one-way ANOVA and post-hoc test, P <0.05.
Mentions: To test whether Basic Fuchsin staining changes over time to reflect developmental increases in stem lignin content, 4-week-old and 12-week-old B. distachyon ecotype Bd21-3 basal stem sections were analysed using Basic Fuchsin staining and the observed patterns of fluorescence were compared with those observed after staining with the Wiesner reagent (Fig. 1). The basal internode was of interest in this study because it represents the oldest stem tissue and is expected to accumulate lignin throughout development. Qualitative observation of lignin staining indicated that both Basic Fuchsin fluorescence and Wiesner staining were more widespread and pronounced in 12-week-old stem sections (Fig. 1). Quantitative measurement indicated that Basic Fuchsin fluorescence intensity μm–2 was 2.34-fold higher in 12-week-old Bd21-3 stems. It was observed that this increase arose predominantly from increased staining in interfascicular regions, supporting the well-documented spatio-temporal pattern of lignification in grasses such as B. distachyon throughout maturation (Matos et al., 2013). By calculating the ratio of thresholded stem area to total stem area, it was possible to quantify the relative lignified area within a stem section. This number was 4.25-fold higher in 12-week-old Bd21-3 stems than in 4-week-old stems, suggesting that the increase in fluorescence intensity μm–2 can be attributed to an increase in Basic Fuchsin fluorescence intensity in previously lignified tissues as well as to an increase in lignified area in more mature tissue (Fig. 1). Basic Fuchsin fluorescence and Wiesner staining was also observed in the epidermis of 12-week-old Bd21-3 stems, possibly due to the presence of cutin or suberin which can also fluoresce when stained with Basic Fuchsin (Kraus et al., 1998). Overall, the similar staining pattern between Basic Fuchsin and the Wiesner reagent supports the interpretation that Basic Fuchsin fluorescence intensity correlates with the amount and location of lignin in secondary cell walls.

Bottom Line: It was found that the extent and intensity of Basic Fuchsin fluorescence increase over time in the Bd21-3 ecotype, that Basic Fuchsin staining is more widespread and intense in 4-week-old Bd21-3 and Adi-10 basal internodes than in Bd1-1 internodes, and that Basic Fuchsin staining reveals subcellular patterns of lignin in vascular and interfascicular fibre cell walls.Basic Fuchsin fluorescence did not correlate with lignin quantification by acetyl bromide analysis, indicating that whole-plant and subcellular lignin analyses provide distinct information about the extent and patterns of lignification in B. distachyon.Finally, it was found that flowering time correlated with a transient increase in total lignin, but did not correlate strongly with the patterning of stem lignification, suggesting that additional developmental pathways might regulate secondary wall formation in grasses.

View Article: PubMed Central - PubMed

Affiliation: Center for Lignocellulose Structure and Formation, The Pennsylvania State University, University Park, PA 16802, USA Department of Biology, The Pennsylvania State University, University Park, PA 16802, USA Department of Agricultural and Biological Engineering, The Pennsylvania State University, University Park, PA 16802, USA.

No MeSH data available.