Improving sample preparation to investigate lignin intensity of xylem at the cellular level by confocal Raman microspectroscopy of Populus tomentosa

Abstract

Confocal Raman microspectroscopy (CRM) is an important tool for analyzing the compositional distribution of cell walls in situ. In this study, we improved the sample preparation method using paraffin-embedded sections combined with hexane dewaxing to obtain high resolution Raman images. We determined that the cell wall components of fiber cells were different from those of ray cells and vessel cells in the xylem of Populus tomentosa. Acetyl bromide and CRM methods produced similar trends when the difference in lignin intensity in the xylem region was compared between transgenic PtrLac4 and wild-type P. tomentosa. However, CRM proved more useful to analyze the lignin distribution in each cell type and distinguished the detailed difference in lignin intensity at the cellular level. Thus, CRM proved to be a useful in situ method to rapidly analyze the spatial variation of lignin content in the xylem of woody plants.

Keywords

Cell wall / Confocal Raman microspectroscopy / Lignin / Paraffin section / Populus tomentosa

References

[1] Berthet S, Demont-Caulet N, Pollet B, Bidzinski P, Cezard L, Le Bris P Disruption of LACCASE4 and 17 results in tissue-specific alterations to lignification of Arabidopsis thaliana stems. Plant Cell, 2011, 23: 1124-1137.

[2] Boerjan W, Ralph J, Baucher M. Lignin biosynthesis. Annu Rev Plant Biol, 2003, 54: 519-546.

[3] Burton RA, Gidley MJ, Fincher GB. Heterogeneity in the chemistry, structure and function of plant cell walls. Nat Chem Biol, 2010, 6: 724-732.

[4] Darco A, Brancati N, Ferrara MA, Indolfi M, Frucci M, Sirleto L. Subcellular chemical and morphological analysis by stimulated Raman scattering microscopy and image analysis techniques. Biomed Opt Express, 2016, 7: 1853-1864.

[5] De Bleye C, Sacre PY, Dumont E, Netchacovitch L, Chavez PF, Piel G, Lebrun P, Hubert P, Ziemons E. Development of a quantitative approach using surface-enhanced Raman chemical imaging: first step for the determination of an impurity in a pharmaceutical model. J Pharm Biomed Anal, 2014, 90: 111-118.

[6] Evans CL, Xie XS. Coherent anti-stokes Raman scattering microscopy: chemical imaging for biology and medicine. Annu Rev Anal Chem, 2008, 1: 883-909.

[7] Faolain EO, Hunter MB, Byrne JM, Kelehan P, Lambkin HA, Byrne HJ, Lyng FM. Raman spectroscopic evaluation of efficacy of current paraffin wax section dewaxing agents. J Histochem Cytochem, 2005, 53: 121-129.

[8] Ferreira BG, Teixeira CT, Isaias RM. Efficiency of the polyethylene-glycol (PEG) embedding medium for plant histochemistry. J Histochem Cytochem, 2014, 62: 577-583.

[9] Gierlinger N, Schwanninger M. Chemical imaging of poplar wood cell walls by confocal Raman microscopy. Plant Physiol, 2006, 140: 1246-1254.

[10] Gierlinger N, Sapei L, Paris O. Insights into the chemical composition of equisetum hyemale by high resolution Raman imaging. Planta, 2008, 227: 969-980.

[11] Gierlinger N, Keplinger T, Harrington M. Imaging of plant cell walls by confocal Raman microscopy. Nat Protoc, 2012, 7: 1694-1708.

[12] Gorzsas A. Chemical imaging of xylem by Raman microspectroscopy. Methods Mol Biol, 2017, 1544: 133-178.

[13] Heiner Z, Zeise I, Elbaum R, Kneipp J. Insight into plant cell wall chemistry and structure by combination of multiphoton microscopy with Raman imaging. J Biophotonics, 2018, 11: e201700164.

[14] Hofte H, Voxeur A. Plant cell walls. Curr Biol, 2017, 27: R865-R870.

[15] Ji Z, Zhang X, Ling Z, Zhou X, Ramaswamy S, Xu F. Visualization of Miscanthus x Giganteus cell wall deconstruction subjected to dilute acid pretreatment for enhanced enzymatic digestibility. Biotechnol Biofuels, 2015, 8: 103.

[16] Larsen KL, Barsberg S. Theoretical and Raman spectroscopic studies of phenolic lignin model monomers. J Phys Chem B, 2010, 114: 8009-8021.

[17] Lelie DVD, Taghavi S, Mccorkle SM, Li LL, Malfatti SA, Monteleone D, Donohoe BS, Ding SY, Adney WS, Himmel ME. The metagenome of an anaerobic microbial community decomposing poplar wood chips. PLoS ONE, 2012 7 5 e36740

[18] Lise J, Serge B, Julien M. Identification of laccases involved in lignin polymerization and strategies to deregulate their expression in order to modify lignin content in Arabidopsis and poplar. BMC Proc, 2011, 5: O39.

[19] Littlejohn GR, Mansfield JC, Parker D. In vivo chemical and structural analysis of plant cuticular waxes using stimulated Raman scattering microscopy. Plant Physiol, 2015, 168: 18-28.

[20] Liu Q, Luo L, Zheng L. Lignins: biosynthesis and biological functions in plants. Int J Mol Sci, 2018, 19: 335.

[21] Loque D, Scheller HV, Pauly M. Engineering of plant cell walls for enhanced biofuel production. Curr Opin Plant Biol, 2015, 25: 151-161.

[22] Ma J, Zhou X, Ma J, Ji Z, Zhang X, Xu F. Raman microspectroscopy imaging study on topochemical correlation between lignin and hydroxycinnamic acids in Miscanthus sinensis. Microsc Microanal, 2014, 20: 956-963.

[23] Marion J, Le Bars R, Satiat-Jeunemaitre B, Boulogne C. Optimizing CLEM protocols for plants cells: GMA embedding and cryosections as alternatives for preservation of GFP fluorescence in Arabidopsis roots. J Struct Biol, 2017, 198: 196-202.

[24] Meents MJ, Watanabe Y, Samuels AL. The cell biology of secondary cell wall biosynthesis. Ann Bot, 2018, 121: 1107-1125.

[25] Murakami Y, Funada R, Sano Y, Ohtani J. The differentiation of contact cells and isolation cells in the xylem ray parenchyma of Populus maximowiczii. Ann Bot, 1999, 84: 429-435.

[26] Nima ZA, Biswas A, Bayer IS, Hardcastle FD, Perry D, Ghosh A, Dervishi E, Biris AS. Applications of surface-enhanced Raman scattering in advanced bio-medical technologies and diagnostics. Drug Metab Rev, 2014, 46: 155-175.

[27] Pohling C, Brackmann C, Duarte A, Buckup T, Enejder A, Motzkus M. Chemical imaging of lignocellulosic biomass by CARS microscopy. J Biophotonics, 2014, 7: 126-134.

[28] Popper ZA, Ralet MC, Domozych DS. Plant and algal cell walls: diversity and functionality. Ann Bot, 2014, 114: 1043-1048.

[29] Roeffaers MB, Zhang X, Freudiger CW, Saar BG, Van Ruijven M, Van Dalen G, Xiao C, Xie XS. Label-free imaging of biomolecules in food products using stimulated Raman microscopy. J Biomed Opt, 2011, 16: 021118.

[30] Schuetz M, Smith R, Ellis B. Xylem tissue specification, patterning, and differentiation mechanisms. J Exp Bot, 2013, 64: 11-31.

[31] Sergo V, Krafft C, Codrich D, Bonifacio A, Beleites C. Raman spectroscopy and imaging: promising optical diagnostic tools in pediatrics. Curr Med Chem, 2013, 20: 2176-2187.

[32] Wen JL, Sun SL, Xue BL, Sun RC. Recent advances in characterization of lignin polymer by solution-state nuclear magnetic resonance (NMR) methodology. Materials (Basel), 2013, 6: 359-391.

[33] Zeng Y, Saar BG, Friedrich MG, Chen F, Liu YS, Dixon RA, Himmel ME, Xie XS, Ding SY. Imaging lignin-downregulated alfalfa using coherent anti-stokes Raman scattering microscopy. BioEnergy Res, 2010, 3: 272-277.

[34] Zhao Q, Dixon RA. Transcriptional networks for lignin biosynthesis: more complex than we thought?. Trends Plant Sci, 2011, 16: 227-233.

[35] Zhao Y, Lin S, Qiu Z, Cao D, Wen J, Deng X, Wang X, Lin J, Li X. MicroRNA857 is involved in the regulation of secondary growth of vascular tissues in Arabidopsis. Plant Physiol, 2015, 169: 2539-2552.