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In situ label-free imaging of hemicellulose in plant cell walls using stimulated Raman scattering microscopy

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ABSTRACT

Background: Plant hemicellulose (largely xylan) is an excellent feedstock for renewable energy production and second only to cellulose in abundance. Beyond a source of fermentable sugars, xylan constitutes a critical polymer in the plant cell wall, where its precise role in wall assembly, maturation, and deconstruction remains primarily hypothetical. Effective detection of xylan, particularly by in situ imaging of xylan in the presence of other biopolymers, would provide critical information for tackling the challenges of understanding the assembly and enhancing the liberation of xylan from plant materials.

Results: Raman-based imaging techniques, especially the highly sensitive stimulated Raman scattering (SRS) microscopy, have proven to be valuable tools for label-free imaging. However, due to the complex nature of plant materials, especially those same chemical groups shared between xylan and cellulose, the utility of specific Raman vibrational modes that are unique to xylan have been debated. Here, we report a novel approach based on combining spectroscopic analysis and chemical/enzymatic xylan removal from corn stover cell walls, to make progress in meeting this analytical challenge. We have identified several Raman peaks associated with xylan content in cell walls for label-free in situ imaging xylan in plant cell wall.

Conclusion: We demonstrated that xylan can be resolved from cellulose and lignin in situ using enzymatic digestion and label-free SRS microscopy in both 2D and 3D. We believe that this novel approach can be used to map xylan in plant cell walls and that this ability will enhance our understanding of the role played by xylan in cell wall biosynthesis and deconstruction.

Electronic supplementary material: The online version of this article (doi:10.1186/s13068-016-0669-9) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus

Comparison of SRS images of xylan in the native, untreated corn stover (control) and organosolv-pretreated corn stover cell walls. Two types of cell walls are compared: secondary cell walls in the vascular bundle (a–d, i–l, q–t) and parenchyma cell walls (e–h, m–p, u–x). Scale bar = 30 µm
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Fig5: Comparison of SRS images of xylan in the native, untreated corn stover (control) and organosolv-pretreated corn stover cell walls. Two types of cell walls are compared: secondary cell walls in the vascular bundle (a–d, i–l, q–t) and parenchyma cell walls (e–h, m–p, u–x). Scale bar = 30 µm

Mentions: The four xylan-specific Raman bands were also validated by SRS microscopy and tested for the possibility of in situ imaging of xylan in chemically pretreated plant cell walls. Native, untreated corn stover cell wall samples were organosolv-pretreated to remove 55–70% of the cell wall xylan (Additional file 1: Table S3, S4). Figure 5 compares the SRS images of two different types of cell walls, i.e., the vascular bundle cell wall and the parenchyma cell wall of the control and the xylan-reduced cell wall sample. When the relative cell wall xylan content was reduced from 100% to ~30 to 45% in the organosolv-pretreated cell walls, SRS signals from all four xylan frequencies also dropped. This correlation confirms that those Raman frequencies are specific to xylan content. Figure 5 also shows that the 1471 and 1219 cm−1 bands appear to have stronger SRS signals. Therefore, the SRS bands at 1471 and 1219 cm−1, especially the band at 1471 cm−1, might be more useful for imaging cell wall chemical details.Fig. 5


In situ label-free imaging of hemicellulose in plant cell walls using stimulated Raman scattering microscopy
Comparison of SRS images of xylan in the native, untreated corn stover (control) and organosolv-pretreated corn stover cell walls. Two types of cell walls are compared: secondary cell walls in the vascular bundle (a–d, i–l, q–t) and parenchyma cell walls (e–h, m–p, u–x). Scale bar = 30 µm
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5120481&req=5

Fig5: Comparison of SRS images of xylan in the native, untreated corn stover (control) and organosolv-pretreated corn stover cell walls. Two types of cell walls are compared: secondary cell walls in the vascular bundle (a–d, i–l, q–t) and parenchyma cell walls (e–h, m–p, u–x). Scale bar = 30 µm
Mentions: The four xylan-specific Raman bands were also validated by SRS microscopy and tested for the possibility of in situ imaging of xylan in chemically pretreated plant cell walls. Native, untreated corn stover cell wall samples were organosolv-pretreated to remove 55–70% of the cell wall xylan (Additional file 1: Table S3, S4). Figure 5 compares the SRS images of two different types of cell walls, i.e., the vascular bundle cell wall and the parenchyma cell wall of the control and the xylan-reduced cell wall sample. When the relative cell wall xylan content was reduced from 100% to ~30 to 45% in the organosolv-pretreated cell walls, SRS signals from all four xylan frequencies also dropped. This correlation confirms that those Raman frequencies are specific to xylan content. Figure 5 also shows that the 1471 and 1219 cm−1 bands appear to have stronger SRS signals. Therefore, the SRS bands at 1471 and 1219 cm−1, especially the band at 1471 cm−1, might be more useful for imaging cell wall chemical details.Fig. 5

View Article: PubMed Central - PubMed

ABSTRACT

Background: Plant hemicellulose (largely xylan) is an excellent feedstock for renewable energy production and second only to cellulose in abundance. Beyond a source of fermentable sugars, xylan constitutes a critical polymer in the plant cell wall, where its precise role in wall assembly, maturation, and deconstruction remains primarily hypothetical. Effective detection of xylan, particularly by in situ imaging of xylan in the presence of other biopolymers, would provide critical information for tackling the challenges of understanding the assembly and enhancing the liberation of xylan from plant materials.

Results: Raman-based imaging techniques, especially the highly sensitive stimulated Raman scattering (SRS) microscopy, have proven to be valuable tools for label-free imaging. However, due to the complex nature of plant materials, especially those same chemical groups shared between xylan and cellulose, the utility of specific Raman vibrational modes that are unique to xylan have been debated. Here, we report a novel approach based on combining spectroscopic analysis and chemical/enzymatic xylan removal from corn stover cell walls, to make progress in meeting this analytical challenge. We have identified several Raman peaks associated with xylan content in cell walls for label-free in situ imaging xylan in plant cell wall.

Conclusion: We demonstrated that xylan can be resolved from cellulose and lignin in situ using enzymatic digestion and label-free SRS microscopy in both 2D and 3D. We believe that this novel approach can be used to map xylan in plant cell walls and that this ability will enhance our understanding of the role played by xylan in cell wall biosynthesis and deconstruction.

Electronic supplementary material: The online version of this article (doi:10.1186/s13068-016-0669-9) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus