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Multi-scale spatial heterogeneity of pectic rhamnogalacturonan I (RG-I) structural features in tobacco seed endosperm cell walls.

Lee KJ, Cornuault V, Manfield IW, Ralet MC, Knox JP - Plant J. (2013)

Bottom Line: Heterogeneous RG-I polymers are implicated in generating the mechanical properties of cell walls during cell development and plant growth, but are poorly understood in architectural, biochemical and functional terms.The analyses indicate that the features of the RG-I polymer display spatial heterogeneity at the level of the tissue and the level of single cell walls, and also heterogeneity at the biochemical level.This work has implications for understanding RG-I glycan complexity in the context of cell-wall architectures and in relation to cell-wall functions in cell and tissue development.

View Article: PubMed Central - PubMed

Affiliation: Centre for Plant Sciences, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, UK.

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Epitope detection anion-exchange chromatography of extracts of the NME and ME halves of tobacco seed endosperms. Isolated cell-wall materials were fractionated using an anion-exchange chromatography column, and epitopes were detected in the same fractions by ELISA using LM5 galactan, LM6 arabinan, RU2 RG–I backbone and LM19 homogalacturonan (HG) monoclonal antibodies. Fractions indicated by the double-line symbol indicate the presence of early-eluting material using the LM5 and LM6 epitopes ahead of the peak of RG backbone and HG elution. Galactan-containing early-eluting peaks were more abundant in the NME than in the ME, and HG was more abundant in the ME than in the NME. Open triangles indicate coincident peaks of LM5 and LM6 epitope elution. Closed triangles indicate coincident peaks of RU2 and LM19 epitope elution. Vertical dotted lines indicate peaks of HG elution. Error bars indicate the SD of three absorbance values.
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fig06: Epitope detection anion-exchange chromatography of extracts of the NME and ME halves of tobacco seed endosperms. Isolated cell-wall materials were fractionated using an anion-exchange chromatography column, and epitopes were detected in the same fractions by ELISA using LM5 galactan, LM6 arabinan, RU2 RG–I backbone and LM19 homogalacturonan (HG) monoclonal antibodies. Fractions indicated by the double-line symbol indicate the presence of early-eluting material using the LM5 and LM6 epitopes ahead of the peak of RG backbone and HG elution. Galactan-containing early-eluting peaks were more abundant in the NME than in the ME, and HG was more abundant in the ME than in the NME. Open triangles indicate coincident peaks of LM5 and LM6 epitope elution. Closed triangles indicate coincident peaks of RU2 and LM19 epitope elution. Vertical dotted lines indicate peaks of HG elution. Error bars indicate the SD of three absorbance values.

Mentions: Equivalent alkali-extracted materials (approximately five endosperm halves) were injected into a low-pressure chromatography system with a 1 ml anion-exchange column. Aliquots (100 μl) of 1 ml fractions were incubated in microtitre plate wells, and ELISAs were used to generate the detection profiles shown in Figure 6(a). The LM5 epitope was detected in fractions 18–35, and equivalent peaks of recognition in terms of elution volume (indicated by open triangles) were observed in material from both the ME and NME. However, LM5 recognition of the separated NME material had more shoulders and sub-peaks in the early-eluting fractions (less acidic) (indicated by the double line Figure 6a) than the ME material. Similar profiles were obtained for the LM6 epitope, although there was less difference between the ME and NME fractions compared with the LM5 profiles. This again reflects the occurrence of the epitopes in immunocytochemical analyses. In contrast, the peak for the RU2 epitope (which consistently produced stronger responses in the epitope detection protocols than RU1), occurred slightly later than for the LM5/LM6 peak (closed triangles) and also coincided with peaks of pectic HG elution as indicated by the LM19 pectic HG epitope. It was also noted that the RU2 and HG epitopes in the NME fraction were present slightly earlier than in the ME fraction, but both peaks occurred after the peaks of LM5/LM6 elution.


Multi-scale spatial heterogeneity of pectic rhamnogalacturonan I (RG-I) structural features in tobacco seed endosperm cell walls.

Lee KJ, Cornuault V, Manfield IW, Ralet MC, Knox JP - Plant J. (2013)

Epitope detection anion-exchange chromatography of extracts of the NME and ME halves of tobacco seed endosperms. Isolated cell-wall materials were fractionated using an anion-exchange chromatography column, and epitopes were detected in the same fractions by ELISA using LM5 galactan, LM6 arabinan, RU2 RG–I backbone and LM19 homogalacturonan (HG) monoclonal antibodies. Fractions indicated by the double-line symbol indicate the presence of early-eluting material using the LM5 and LM6 epitopes ahead of the peak of RG backbone and HG elution. Galactan-containing early-eluting peaks were more abundant in the NME than in the ME, and HG was more abundant in the ME than in the NME. Open triangles indicate coincident peaks of LM5 and LM6 epitope elution. Closed triangles indicate coincident peaks of RU2 and LM19 epitope elution. Vertical dotted lines indicate peaks of HG elution. Error bars indicate the SD of three absorbance values.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3824205&req=5

fig06: Epitope detection anion-exchange chromatography of extracts of the NME and ME halves of tobacco seed endosperms. Isolated cell-wall materials were fractionated using an anion-exchange chromatography column, and epitopes were detected in the same fractions by ELISA using LM5 galactan, LM6 arabinan, RU2 RG–I backbone and LM19 homogalacturonan (HG) monoclonal antibodies. Fractions indicated by the double-line symbol indicate the presence of early-eluting material using the LM5 and LM6 epitopes ahead of the peak of RG backbone and HG elution. Galactan-containing early-eluting peaks were more abundant in the NME than in the ME, and HG was more abundant in the ME than in the NME. Open triangles indicate coincident peaks of LM5 and LM6 epitope elution. Closed triangles indicate coincident peaks of RU2 and LM19 epitope elution. Vertical dotted lines indicate peaks of HG elution. Error bars indicate the SD of three absorbance values.
Mentions: Equivalent alkali-extracted materials (approximately five endosperm halves) were injected into a low-pressure chromatography system with a 1 ml anion-exchange column. Aliquots (100 μl) of 1 ml fractions were incubated in microtitre plate wells, and ELISAs were used to generate the detection profiles shown in Figure 6(a). The LM5 epitope was detected in fractions 18–35, and equivalent peaks of recognition in terms of elution volume (indicated by open triangles) were observed in material from both the ME and NME. However, LM5 recognition of the separated NME material had more shoulders and sub-peaks in the early-eluting fractions (less acidic) (indicated by the double line Figure 6a) than the ME material. Similar profiles were obtained for the LM6 epitope, although there was less difference between the ME and NME fractions compared with the LM5 profiles. This again reflects the occurrence of the epitopes in immunocytochemical analyses. In contrast, the peak for the RU2 epitope (which consistently produced stronger responses in the epitope detection protocols than RU1), occurred slightly later than for the LM5/LM6 peak (closed triangles) and also coincided with peaks of pectic HG elution as indicated by the LM19 pectic HG epitope. It was also noted that the RU2 and HG epitopes in the NME fraction were present slightly earlier than in the ME fraction, but both peaks occurred after the peaks of LM5/LM6 elution.

Bottom Line: Heterogeneous RG-I polymers are implicated in generating the mechanical properties of cell walls during cell development and plant growth, but are poorly understood in architectural, biochemical and functional terms.The analyses indicate that the features of the RG-I polymer display spatial heterogeneity at the level of the tissue and the level of single cell walls, and also heterogeneity at the biochemical level.This work has implications for understanding RG-I glycan complexity in the context of cell-wall architectures and in relation to cell-wall functions in cell and tissue development.

View Article: PubMed Central - PubMed

Affiliation: Centre for Plant Sciences, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, UK.

Show MeSH