A method for simultaneous in-situ visualizing and characterizing fine structure components of lignin in plant cell wall
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- CHINA AGRI UNIV
- Filing Date
- 2025-01-08
- Publication Date
- 2026-07-03
Smart Images
Figure CN119880551B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of analytical detection technology, and in particular to a method for synchronous in-situ visualization characterization of the fine structural components of lignin in plant cell walls. Background Technology
[0002] Lignin is an important component of the cell walls of vascular plants, mainly deposited in the cell walls of vascular tissues (such as vessels or tracheids), mechanical tissues (such as fibers and sclerenchyma), and protective tissues (such as the epidermis). It plays a crucial role in the evolution of terrestrial vascular plants and possesses vital biological functions. Lignin molecules crosslink with polysaccharide molecules such as cellulose and hemicellulose in the cell wall, increasing the mechanical strength of plant cells and tissues. Its hydrophobicity makes plant cells impermeable to water, facilitating the long-distance transport of water, minerals, and organic matter within the plant. Lignin is produced through the oxidative coupling of three p-hydroxycinnamyl alcohols (p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol) in approximately ten enzyme-mediated reactions via the phenylpropane pathway and specific pathways, forming p-hydroxyphenyl lignin (H-type), guaiacol-based lignin (G-type), and syringyl lignin (S-type). In the cell walls of grasses, hydroxycinnamic acid (HCA) couples hemicellulose and lignin via ferulic acidation, forming a ferulic acid-polysaccharide-lignin complex that cross-links the cell wall. During the growth and development of different plants, the pathways and regulatory mechanisms of lignin synthesis vary, leading to differences in lignin structure, monomer composition, and content among species, thus affecting their environmental adaptability and biomass utilization efficiency. Therefore, in-situ visualization of the distribution of fine structural components of lignin in plant cell walls is of great significance for elucidating the mechanisms of lignin monomer biosynthesis and assembly, and for exploring new pathways to improve plant stem mechanical strength, stress resistance, and efficient biomass utilization by regulating plant lignin composition.
[0003] Due to the stubbornness and complexity of lignin, there is currently no method to accurately quantify all lignin monomers and determine the chemical bond connections between them. Current research has used a series of complementary techniques to approximate the composition of lignin. For example, thioacid hydrolysis is used to degrade lignin into soluble monomers or oligomers, thereby analyzing the composition and structure of lignin; nuclear magnetic resonance (NMR) technology provides structural information about lignin molecules. However, these methods typically analyze lignin composition and structure from the perspective of the entire organ, making it difficult to obtain the distribution and differences of lignin between tissues, cell types, and cell wall layers. In the past few decades, label-free techniques such as UV absorption and fluorescence microscopy utilizing lignin autofluorescence have been widely used to study lignin polymers. Although these techniques have unique advantages in monitoring lignin, they cannot simultaneously analyze the fine structural components of lignin.
[0004] Confocal Raman microscopy combines the chemical sensitivity and multi-component simultaneous analysis advantages of molecular spectroscopy with the high spatial resolution of microscopy. It possesses high chemical specificity, can record the molecular fingerprint of a sample, and obtain information on the functional groups and chemical components of the cell wall in a single image, unlike fluorescence microscopy which requires capturing multiple images of the same sample. Significant progress has been made in characterizing lignin in wood cell walls using confocal Raman microscopy. For example, Perez-de-Lis, G., Richard, B., Quiles, F., Deveau, A., Adikurnia, IK, & Rathgeber, C. Multimodal imaging analysis in silver fir reveals coordination in cellulose and lignin deposition[J]. Plant Physiology, 2024, 195(3): 2428-2442. — Characterizes the deposition of lignin in the cell walls of Cathaya argyrophylla tracheids; Jin K, Liu X, Wang K, et al. Imaging the dynamic deposition of cell wall polymer in xylem and phloem in Populus × euramericana[J]. Planta, 2018, 248(4): 849-858. — Characterizes the difference in lignin distribution in the xylem and phloem of Populus.
[0005] In the prior art, the patent concerning in-situ characterization of cell wall lignin using confocal Raman microscopy is Chinese patent CN 102435594 A, which discloses a method for determining the degree of lignification of plant cell walls based on confocal Raman microscopy. This method utilizes lignin 1580-1660 cm⁻¹ -1 The distribution of lignin is obtained by integrating the spectrum of the range; Chinese patent CN 115266676 A discloses an in-situ synchronous quantification method for determining the lignin fiber components of crop roots and stems based on confocal Raman microscopy. This method normalizes the intensity of the processed Raman spectrum image according to the characteristic band, and generates a Raman image of the lignin distribution in crop tissue based on the single-peak imaging of the normalized relative Raman intensity.
[0006] However, the aforementioned studies failed to visualize the distribution of lignin's fine structural components. Furthermore, due to the unique structure of plant cell walls, variations in scattering volume caused by their anisotropic and heterogeneous structure, and differences in Raman intensity resulting from varying experimental conditions, the analytical results were significantly affected.
[0007] Therefore, how to develop a synchronous in-situ visualization method for the fine structural components of lignin in plant cell walls has become a technical challenge that urgently needs to be solved in this field. Summary of the Invention
[0008] To address the aforementioned technical challenges, this invention provides a method for simultaneous in-situ visualization and characterization of the fine structural components of lignin in plant cell walls, comprising:
[0009] (1) After fixing, embedding, slicing and sealing the plant cell wall, Raman imaging analysis was performed on the plant cell wall to obtain the original Raman spectrum image of the plant cell wall;
[0010] (2) Perform spectral preprocessing on the original Raman spectral images of plant cell walls;
[0011] (3) Normalize the pixel area of the preprocessed spectrum. The area normalization formula is as follows:
[0012]
[0013] In the formula, X nor（i，j） Cell wall pixels ( i , j The spectrum after area normalization. X （i，j） Cell wall pixels ( i , j The spectrum after spectral preprocessing, I （i，j，k） Cell wall pixels ( i , j After spectral preprocessing, in the first... k Raman spectral intensity at wavenumbers, p The number of Raman wavenumbers involved in the calculation;
[0014] (4) Based on the normalized Raman intensity of lignin after pixel area, characteristic peak imaging is performed to obtain the visual distribution of lignin in the cell wall.
[0015] (5) Using the characteristic peak of lignin as a reference peak, obtain the ratio of the characteristic peak intensity of the fine structure component of lignin to the characteristic peak intensity of lignin, and generate the in-situ visualization distribution image of the corresponding component; obtain the visualization distribution of the S / G ratio using the characteristic peak intensity ratio of S-type lignin and G-type lignin.
[0016] This invention achieves in-situ visualization and characterization of lignin distribution in plant cell walls based on Raman image pixel area normalization combined with feature peak imaging; furthermore, based on feature peak intensity ratio imaging, it achieves synchronous in-situ visualization and characterization of the distribution of various fine structural components of lignin.
[0017] Preferably, the spectral preprocessing in step (2) includes: selecting 3050-300 cm⁻¹ -1 For band analysis, the Savitzky-Golay smoothing algorithm was used for smoothing and noise reduction, and the concave rubberband correction method was used for baseline correction.
[0018] Preferably, the pixel area normalization in step (3) is selected as 3050-2800 cm². -1 and 1800-300 cm -1 Cell wall signal spectrum.
[0019] Preferably, the characteristic peak of lignin is 1594~1614 cm⁻¹. -1 .
[0020] Preferably, the lignin fine structure components include at least one of lignin synthesis precursors, lignin and hemicellulose linking substances, G-type lignin, S-type lignin, and H-type lignin.
[0021] Preferably, the lignin synthesis precursor is coniferyl alcohol and / or coniferyl aldehyde (CAA).
[0022] Preferably, the lignin and hemicellulose linking substance is hydroxycinnamic acid (HCA).
[0023] Preferably, the characteristic peak of the lignin synthesis precursor is 1622~1642 cm⁻¹. -1 ; and / or, the characteristic peaks of lignin and hemicellulose linkers are 1161–1181 cm⁻¹. -1 ; and / or, the characteristic peak of G-type lignin is 1257–1277 cm⁻¹. -1 ; and / or, the characteristic peak of S-type lignin is 1320–1340 cm⁻¹. -1 ; and / or, the characteristic peak of H-type lignin is 1200~1220 cm⁻¹. -1 .
[0024] Preferably, the Raman spectroscopy measurement conditions in step (1) include: an excitation wavelength of 532 nm; and / or a laser power of 25 mW; and / or a spectral resolution of 4 cm⁻¹. -1 ; and / or, the number of scans is 1 to 3; and / or, the integration time for each scan is 1000 to 2000 ms; and / or, the analysis step size is 0.5 to 1 μm.
[0025] Preferably, the specific steps of fixation, embedding, sectioning, and sealing in step (1) include: fixing the plant cell wall in formalin-acetic acid-ethanol fixative for more than 24 hours, and then embedding it with PEG; cutting a section with a thickness of 20-30 μm along the cross section direction, removing PEG, and then further sonicating to remove starch and cell contents from the cells, and then transferring the section to a glass slide, adding D2O, covering it with a coverslip and sealing it with nail polish.
[0026] Preferably, the plants include, but are not limited to, timber, wheat, corn, rice, barley, oats, rye, sorghum, millet, and forage grasses (such as alfalfa and fescue).
[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0028] This invention, based on confocal Raman microscopy combined with a characteristic peak / characteristic peak ratio method, can simultaneously obtain information on the fine structural components of lignin, including in-situ visualized distribution images of lignin, lignin synthesis precursors, lignin and hemicellulose linking substances, lignin monomers (G-type lignin, S-type lignin, H-type lignin), and S / G ratios. It has the advantages of being label-free and having high chemical specificity, and can simultaneously obtain the distribution of fine structural components of lignin. It is simple, efficient, and has broad application prospects.
[0029] Moreover, the in-situ visualization characterization method of the present invention is based on the area normalization processing of Raman image pixels and the ratio of characteristic peak intensities. It can eliminate the influence of Raman intensity differences caused by scattering volume changes due to cell structural heterogeneity and different experimental conditions on the analysis results, especially the Raman intensity fluctuations caused by the photophysical properties of the sample, such as pixel coverage, sample thickness, refractive index changes, and defocusing effects. Attached Figure Description
[0030] Figure 1 These are synchronous in-situ visualized distribution characterization images of the fine structural components of lignin in the epidermis-thick-walled tissue of the second internode at the base of the rice stem. Detailed Implementation
[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.
[0032] In the embodiments provided in this specification, unless specific techniques or conditions are specified, the techniques or conditions described in the literature in this field, or the product instructions, shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.
[0033] The rice (type species of Poaceae) used in the following examples is the Wuyungeng rice variety, grown in an outdoor paddy field environment. The samples used for testing were taken from the second internode at the base of the rice stem.
[0034] Example
[0035] This embodiment provides a method for synchronous in-situ visualization and characterization of the fine structural components of lignin in plant cell walls, the steps of which are as follows:
[0036] (1) The collected rice stem base second internode samples were divided into S1-S9 segments in the longitudinal direction. Each segment was cut into approximately 5 mm lengths and fixed in formalin-acetic acid-ethanol fixative for more than 24 h. PEG1500 was used for embedding.
[0037] (2) Cut a 30 μm thick slice along the cross section direction, soak it in deionized water for 30 min to remove PEG1500 from the slice, sonicate for 30 s to remove starch and cell contents from the cells, then transfer the slice to a glass slide, add D2O, cover with a coverslip and seal with nail polish.
[0038] (3) The sealed slices were placed under a confocal Raman microscope, and the Raman spectrum of the plant cell wall was measured at an excitation wavelength of 532 nm to obtain the original Raman spectral images of representative regions of the plant cell wall. The laser power was 25 mW and the spectral resolution was 4 cm⁻¹. -1 The number of scans was 2, the integration time for each scan was 1500 ms, and the analysis step size was 1 μm;
[0039] (4) Perform spectral preprocessing on the original Raman images of plant cell walls, including selecting 3050-300 cm⁻¹. -1 For the band analysis range, the 11-point Savitzky-Golay smoothing algorithm was used for smoothing and noise reduction, and the concave rubberband correction method was used for baseline correction. The number of iterations was 10, and the number of baseline correction points was 64.
[0040] (5) Cell wall spectra in the range of 3050-2800 cm⁻¹ -1 and 1800-300 cm -1 Pixel area normalization was performed on the main cell wall signal spectrum segments;
[0041] The area normalization formula is as follows:
[0042]
[0043] In the formula, X nor（i，j） Cell wall pixels ( i , j The spectrum after area normalization. X （i，j） Cell wall pixels ( i , j The spectrum after spectral preprocessing, I （i，j，k） Cell wall pixels ( i , j After spectral preprocessing, in the first... k Raman spectral intensity at wavenumbers, p The number of Raman spectral wavenumbers involved in the calculation; in this embodiment, p =1168;
[0044] (6) Based on the normalized Raman intensity of lignin according to the pixel area, characteristic peak imaging was performed to obtain the visual distribution of lignin in the cell wall. The characteristic peak of lignin was 1604 cm⁻¹. -1 (Aromatic skeleton stretching vibration);
[0045] (7) The characteristic peak of lignin is 1604 cm⁻¹ -1 As a reference peak, the cell wall pixel spectra at 1632 cm⁻¹ were used respectively. -1 (C=C stretching vibration of coniferyl alcohol and C=O stretching vibration of coniferyl aldehyde), 1171 cm -1 (Cinnamyl ester bond stretching vibration), 1267 cm -1 (G-type lignin structural unit C=O stretching vibration), 1330 cm -1 (Aliphatic OH bending vibrations in S-type lignin structural units) and 1210 cm -1 The characteristic peak intensity at (OCH3 stretching vibration in H-type lignin structural units) and the characteristic peak of lignin at 1604 cm⁻¹ -1 The characteristic peak intensity ratios were used to generate in-situ visualized distribution images of coniferyl alcohol / aldehyde (CAA), lignin and hemicellulose linker hydroxycinnamic acid (HCA), G-type lignin, S-type lignin, and H-type lignin.
[0046] (8) Imaging was performed using the ratio of the characteristic peak intensities of G-type lignin and S-type lignin, i.e., using the cell wall pixel spectra at 1330 cm⁻¹. -1 and 1267 cm -1 The Raman intensity ratio at a given location is used to obtain a visual distribution image of the S / G ratio.
[0047] The results are as follows: Synchronous in-situ visualization of the distribution of lignin fine structure components in the epidermis-thickness tissue of the second internode at the base of the rice stem, as shown in the image. Figure 1 As shown, lignin is continuously deposited and the degree of lignification increases with the growth and development of rice stems, mainly deposited in the outer vascular bundles and the intercellular layer of thick-walled cells. With the deposition of lignin, the lignin synthesis precursor coniferyl alcohol / aldehyde (CAA) continuously decreases; the lignin-hemicellulose linking substance hydroxycinnamic acid (HCA) continuously increases, indicating that lignin and hemicellulose are continuously deposited; G-type lignin, S-type lignin and H-type lignin are also continuously deposited and polymerized, among which H-type lignin is gradually observed in the outer vascular bundles of S5; the S / G ratio continuously increases, especially in epidermal cells.
[0048] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims
1. A method for synchronous in-situ visualization and characterization of the fine structural components of lignin in plant cell walls, characterized in that, include: (1) After fixing, embedding, slicing and sealing the plant cell wall, Raman imaging analysis was performed on the plant cell wall to obtain the original Raman spectrum image of the plant cell wall; (2) Perform spectral preprocessing on the original Raman spectral images of plant cell walls; (3) Normalize the pixel area of the preprocessed spectrum. The area normalization formula is as follows: ； In the formula, X nor（i，j） Cell wall pixels ( i , j The spectrum after area normalization. X （i，j） Cell wall pixels ( i , j The spectrum after spectral preprocessing, I （i，j，k） Cell wall pixels ( i , j After spectral preprocessing, in the first... k Raman spectral intensity at wavenumbers, p The number of Raman spectral wavenumbers involved in the calculation; the pixel area normalization in step (3) is selected as 3050-2800 cm⁻¹. -1 and 1800-300 cm -1 Cell wall signal spectrum; (4) Based on the normalized Raman intensity of lignin after pixel area, characteristic peak imaging is performed to obtain the visual distribution of lignin in the cell wall. (5) Using the characteristic peak of lignin as a reference peak, obtain the ratio of the characteristic peak intensity of the fine structure component of lignin to the characteristic peak intensity of lignin, and generate the in-situ visualization distribution image of the corresponding component; obtain the visualization distribution of the S / G ratio using the characteristic peak intensity ratio of S-type lignin and G-type lignin.
2. The method according to claim 1, characterized in that, The spectral preprocessing in step (2) includes: selecting 3050-300 cm⁻¹ -1 For band analysis, the Savitzky-Golay smoothing algorithm was used for smoothing and noise reduction, and the concave rubberband correction method was used for baseline correction.
3. The method according to claim 1, characterized in that, The characteristic peak of lignin is 1594~1614 cm⁻¹ -1 .
4. The method according to claim 1, characterized in that, The fine structure components of the lignin include at least one of lignin synthesis precursors, lignin and hemicellulose linking substances, G-type lignin, S-type lignin, and H-type lignin.
5. The method according to claim 4, characterized in that, The lignin synthesis precursor is coniferyl alcohol and / or coniferyl aldehyde.
6. The method according to claim 4, characterized in that, The lignin and hemicellulose linking substance is hydroxycinnamic acid.
7. The method according to claim 4, characterized in that, The characteristic peaks of lignin synthesis precursors are 1622–1642 cm⁻¹. -1 ; and / or, the characteristic peaks of lignin and hemicellulose linkers are 1161–1181 cm⁻¹. -1 ; and / or, the characteristic peak of G-type lignin is 1257–1277 cm⁻¹. -1 ; and / or, the characteristic peak of S-type lignin is 1320–1340 cm⁻¹. -1 ; and / or, the characteristic peak of H-type lignin is 1200~1220 cm⁻¹. -1 .
8. The method according to claim 1, characterized in that, The Raman imaging measurement conditions in step (1) include: an excitation wavelength of 532 nm; and / or a laser power of 25 mW; and / or a spectral resolution of 4 cm⁻¹. -1 ; and / or, the number of scans is 1 to 3; and / or, the integration time for each scan is 1000 to 2000 ms; and / or, the analysis step size is 0.5 to 1 μm.
9. The method according to claim 1, characterized in that, The specific steps of fixation, embedding, sectioning, and sealing in step (1) include: fixing the plant cell wall in formalin-acetic acid-ethanol fixative for more than 24 hours, and then embedding it with PEG; cutting a section with a thickness of 20-30 μm along the transverse direction, removing PEG, and then further sonicating to remove starch and cell contents from the cells, then transferring the section to a glass slide, adding D2O, covering it with a coverslip, and sealing it with nail polish.