Bing Wang ·Mei Luo ·Yadi Liu ·Xiaorui Guo ·Xiatong Liu ·Chong Zhang ·Zhijing Zhao·Di Liu ·Hui Li,,3·Hai Lu,,3

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 paraffi n-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·Paraffi n section·Populus tomentosa

Introduction

Raman spectroscopy has been widely applied for chemical mapping and imaging biological and biomimetic samples(Evans and Xie 2008;Roeff aers et al.2011;Sergo et al.2013;De Bleye et al.2014;Nima et al.2014).It has emerged as a powerful approach to analyze the chemical composition of the cell wall in a particular area of plants (Gorzsas 2017;Heiner et al.2018).The combination of microscopy and Raman spectroscopy has enabled descriptions of the cellular structure and composition of individual features of plant tissues in their native state.These advanced micro-Raman spectroscopy techniques include confocal Raman microspectroscopy (CRM),coherent anti-Stokes Raman scattering microspectroscopy,and stimulated Raman scattering microspectroscopy (Zeng et al.2010;Larsen and Barsberg 2010;Lelie et al.2012;D’Arco et al.2016).Micro-Raman spectroscopy can be used to quantify the chemical distribution of the cell wall in plants in situ,and has provided remarkable insight into visualizing the cell wall (Gierlinger et al.2008,2012;Pohling et al.2014).

Lignin is the major component of the secondary cell wall of the vascular system in woody plants,and has enabled plants to adapt to land from the aquatic environment (Boerjan et al.2003;Popper et al.2014;Hofte and Voxeur 2017).Lignin plays important roles in the formation of mechanical tissues,transport tissues,and protective tissues by enhancing their structural strength,water transport ability,and biotic stress resistance (Schuetz et al.2 013;Liu et al.2018).The complex and heterogeneous architecture of lignin is speciesspecific and cell type-dependent,and is closely related to cell function.Traditional chemical techniques for analyzing lignin require destroying the plant tissue but preclude in situ determination of lignin contents and distribution.In addition,it is difficult to isolate lignin from small cell wall areas or single cell wall layers of interest (Wen et al.2013;Loque et al.2015).Due to the symmetric tensile vibration of the aromatic ring at 1600 cm−1in lignin,the Raman signal attributed to 1600 cm−1was identified as the Raman characteristic peak of lignin components (Gierlinger and Schwanninger 2006).CRM provides a rapid and non-destructive method to obtain a better understanding of the natural spatial distribution of lignin.Confocal Raman microscopy can be used to visualize the intensity and distribution of lignin in situ through cytological observations.It is also possible to monitor the change in lignin components at the cellular level during lignification in woody plants (Littlejohn et al.2015;Zhao et al.2015).

The quality of a Raman image is affected by many factors,including instrument accuracy,materials,sample preparation,and algorithmic improvements in software (Gierlinger and Schwanninger 2006;Burton et al.2010).High-resolution Raman images have been obtained by improving sample preparation.Non-embedded or polyethylene glycol (PEG)embedded sampling has been the most popular approach to pretreat plant samples (Gierlinger and Schwanninger 2006;Gierlinger et al.2012).Cryosectioning has also been used for herbaceous plants (Ma et al.2014;Ji et al.2015;Marion et al.2017).In the present study,paraffi n embedding combined with microsectioning was used to obtain high quality Raman images in cells ofPopulus tomentosa.

In this study,the compositional distribution of different xylem cell types was characterized forP.tomentosausing paraffi n sections for CRM.In addition,CRM was used to compare the difference in lignin distribution and intensity in different xylem cell types betweenPtrLac4transgenic and wild-typeP.tomentosa.In short,the difference in lignin distribution and intensity in the cell walls of different cell types was effectively distinguished at the cellular level using paraffi n sections combined with confocal Raman microscopy.

Materials and methods

Plant materials

ThePtrLAC4gene ofP.tomentosawas obtained fromAtLAC4inArabidopsis thalianaby homologous cloning,and was then transferred into poplar by Agrobacteriummediated leaf disc transformation to obtain anti-sensePtr-LAC4transgenic materials.The positive transgenic materials were selected for this study and wild-type materials were designated as the control.The second internodes of the stems from 3-month-old transgenic and wild-type poplar were used for sectioning.TheP.tomentosawere cultivated at 25 ± 2 °C,under a 14 h-light/10 h-dark photoperiod,with light intensity of 1500—2000 lx.

Sample preparation for Raman imaging

Fresh stems from tissue culture material were used to prepare handmade and cryosections.The stems were fixed in 70% FAA (70% ethanol:formaldehyde:acetic acid=90:5:5)for 24 h and stored in 70% ethanol before sectioning the PEG and paraffi n embedded sections.The cryosectioned samples were embedded in OCT (Optimum Cutting Temperature Compound) embedding agent (Sakura FineTek,Torrance,CA,USA),and plunged into liquid nitrogen for 10 s.The cryosections were cut to 15 μm with a Leica CM1850 microtome cryostat machine (Leica,Zena,Germany) at − 22 °C.The following steps were used for the PEG sections as described by Ferreira et al.(2014).The samples were embedded in 90% PEG 2000 (w/v) at 60 °C overnight.The blocks were maintained at − 20 °C before sectioning.The sections were cut to 15 μm using the Leica RM2255 microtome,and PEG was dissolved in water.

The FAA fixed samples were dehydrated through an ethanol series (50%,60%,70%,80%,90%,and 100%,1 h for each step) to prepare the paraffi n sections.Then,the ethanol was gradually replaced with 50%,100%,and 100% xylene:ethanol (v:v,2 h each step).The samples were embedded in paraffi n (66.6%,100%,100% paraffi n:xylene;v:v,3 h for each step) and cut to 15 μm thickness using a Leica RM2255 microtome.These sections were dewaxed either in xylene for 1 h or in hexane for 0.5 h.An Eclipse 80i microscope (Nikon,Tokyo,Japan) was used for microscopic observations.

Raman analysis

The LabRAM HR Evolution spectrometer system (HORIBA Jobin Yvon,Paris,France) was used for Raman spectroscopy.This system is equipped with a confocal microscope(Olympus BX41,Tokyo,Japan) and a motorized scan stage.A 50-fold microscope objective lens (0.91 NA) was used for the Raman studies.A linear polarized laser excitation beam (λ=532 nm) was focused with a diff raction-limited spot size,and the Raman light was detected by a liquid nitrogen cooled charge-coupled device detector behind a grating(600 grooves mm−1) spectrometer with a spectral resolution of 0.65−1cm/pixel.The laser power on the sample was approximately 7.5 mW,focused to a 1.25 μm spot size at the tissue surface.The Raman spectra were recorded in the range 200—3500 cm−1with a 15 s integration time and a 0.5 μm step to detect the chemical composition of the different xylem cell types.Scattered Raman signals were recorded in the range of 1510 to 1700 cm−1with a 2 s integration time from a 40 × 40 μm2sample region to detect lignin intensity.Each pixel in the Raman images corresponded to a scan acquired area of 0.5 μm2.

Labspect 5 software was used for the spectral and image processing and analysis.Smoothing filtration was performed using the Savitzky-Golay algorithm by means of a five-point smoothing filter and a polynomial degree of two.The spectral background was removed by linear baseline corrections,and the baseline was subtracted from the spectrum (Ma et al.2014).Raman band intensity was calculated from 9 different areas.The boundary line of the different cells type in the regions of interest of the Raman images was selected manually to investigate lignin intensity in individual xylem cells ofP.tomentosa.Three biological replicates were used for each measurement.The spectra of 40 cells in one sample were counted using Image J.

Lignin content analysis

The modified acetyl bromide method was used to analyze lignin content from wild-type and transgenic poplar.Brief ly,poplar stems were rapidly frozen in liquid nitrogen,ground into a powder,and dried in a 100 °C oven.The precipitate was collected by centrifugation,extracted with an ethanol/toluene mixture (w/w=2:1) for 12 h,freeze dried in a vacuum,and sieved through a 100 mesh cell sieve.A 5 mg portion of the sample powder was added to a 25% (w/w) acetyl bromide/glacial acetic acid solution containing 7.5 mL glacial acetic acid,2.5 mL acetyl bromide,and 400 μL perchloric acid at 70 °C for 2 h.Then,50 mL of 2 M sodium hydroxide was added.The UV absorbance of this solution was detected at 280 nm.The extinction coefficient of lignin extracted with acetyl bromide of ε=20.09 L g−1cm−1was used to calculate the lignin content in the sample.Three biological replicates were used for each measurement.

Results

Exploring good sample preparation methods for Raman imaging

The 3 main Raman imaging steps are sample preparation,acquisition of the spectra,and algorithmic improvements in software.Sample preparation is critical,and mainly ref lects resolution of the image.Slices must be thicker than the confocal depth of the laser beam for Raman imaging and a smooth surface with intact cell walls in the slice is important(Gierlinger et al.2012).

The handmade slices did not produce high quality microscopic images due to their thickness (Fig.1 a).Thus,the frozen sections,PEG slices,and paraffi n sections were used to obtain high resolution images ofP.tomentosa.However,considering the integrity and smoothness of the sliced surface,the paraffi n sections (Fig.1 d) were better than the frozen (Fig.1 b) and PEG embedded sections (Fig.1 c).

Fig.1 Microscopic cross-sectioned images of Populus tomentosa xylem using different methods.a Image of free handmade slice;b Image of PEG embedded section;c Image of cryosection;d Image of paraffi n section.Scale bar=50 μm

Before performing Raman imaging of the paraffi n slices,the Raman spectrum of paraffi n was tested from 500 cm−1to 3500 cm−1to estimate interference from the paraffi n in the embedding agent (Faolain et al.2005).Characteristic paraffi n peaks were detected at 1063 cm−1(CC stretch),1296 cm−1(CH2 deformation),and 1441 cm−1(CH2 bending) (Fig.2 a),while the characteristic lignin peak was detected at approximately 1600 cm−1(Fig.2 b).Thus,paraffi n did not interfere with lignin in the Raman spectrum.However,the existence of paraffi n in the sections could lead to slice burning due to high intensity,long-term laser irradiation during Raman imaging (Fig.2 c).Paraffi n can be dewaxed with xylene,but the efficiency of dewaxing is low (Faolain et al.2005).Therefore,hexane was substituted for xylene during sample preparation to remove the paraffi n remnants (Fig.2 d).No section was burned when the sections were dewaxed with hexane for Raman imaging.These results indicate that the effect of dewaxing with hexane was better than that of xylene under the test conditions,as it effectively removed the paraffi n and prevented slice burning on Raman imaging.In summary,paraffi n sections combined with dewaxing using hexane was chosen for sample preparation to improve the quality of the Raman images.

Fig.2 Lignin was analyzed in paraffi n sections by CRM.a The Raman band of paraff in was measured from 500 to 3,500 cm−1 .The characteristic pure paraffi n peaks were at 1063 cm−1 (CC stretch),1296 cm−1(CH2deformation),and 1441cm−1 (CH2 bending);b The Raman band of lignin was measured from 1500 to 1700 cm−1 .The characteristic lignin peak was at 1600 cm−1,which was the stretching vibration of the aromatic ring;c Bright field image of a paraffi n section dewaxed with xylene.The red rectangular region was chosen for Raman imaging,which showed that some cells were burned during the Raman imaging process;d Bright field imaging of paraffi n sections dewaxed with hexane;the red rectangular region was chosen for Raman imaging,which showed that all cells were intact in the Raman image.Scale bar=50 μm

Detecting the cell wall components of different xylem cell types in P.tomentosa by CRM

Mature xylem consists of fiber cells,ray cells,and vessel cells (Murakami et al.1999).These cells have secondary wall deposits that do not exist in parenchyma cells;thus,the complexity and components of the cell wall are cell typedependent.The stems of the transverse paraffi n sections ofP.tomentosawere analyzed by CRM from 200 to 3500 cm−1(Fig.3).The Raman images showed that the pitch (parenchyma cells) only had a characteristic peak at the 1097 cm−1position,indicating that no lignin existed in parenchyma cells (Fig.3 a).The characteristic Raman peaks of cellulose,hemicellulose,and lignin occurred in the fiber,vessel,and ray cells,respectively (Table 1).The Raman band of the cell wall in vessel cells displayed 8 characteristic peaks at 380,1097,1122,1333,1462,1600,1660,and 2897 cm−1(Fig.3 b).The Raman characteristic peaks of the vessel and ray cell walls were almost similar (Fig.3 c).However,there were differences in the components of the cell walls between vessel cells and fiber cells,which did not exist at 380,1122,or 1462 cm−1in the Raman characteristic peaks of fiber cells(Fig.3 d).The bands at 1097 and 1122 cm−1were contributed by cellulose,glucomannan,and xylan components,while the band at 380 cm−1was contributed by cellulose alone (Gierlinger and Schwanninger 2006;Ma et al.2014).

These results indicate that different cell wall components exist in different xylem cell types ofP.tomentosa.CRM was useful to identify the cell wall components in specific cell types by detecting the characteristic Raman peaks (Fig.3).

Fig.3 Raman spectra indicate the composition of four different cell types in transverse paraffi n sections of a Populus tomentosa stems by confocal Raman spectroscopy.a Raman spectra of the pitch (parenchyma cells,purple);b Raman spectra of vessel cells (red);c Raman spectra of ray cells (blue);d Raman spectra of fiber cells (green).Scale bar=50 μm

Raman analysis of lignin intensity at the cellular level in PtrLAC4 transgenic P.tomentosa

The acetyl bromide method was used to measure total lignin content at the whole plant level,while CRM was used to examine the content and distribution of lignin at the cellular level.Laccase is involved in lignin synthesis of the secondary wall inArabidopsis,which alters lignin biosynthesis and affects lignin contents in overexpressed and downregulated transgenic plants (Zhao and Dixon 2011;Lise et al.2011).Thus,we compared the difference in lignin contents between wild-type andPtrLac4transgenicP.tomentosausing the acetyl bromide method and CRM.The results of the acetyl bromide method showed that the total amount of lignin in the stems of the wild-type,and anti-sensePtrLac4plants was 21.05% and 16.33%,respectively (Fig.4 a,Table 2).Compared with the wild-type,the lignin content of the anti-sensePtrLac4decreased significantly by 22.42%.The relative lignin content in transgenic and wild-type plants measured by confocal Raman spectroscopy was consistent with the results from the acetyl bromide method.The results of CRM showed that the total amounts of lignin in the stems of the wild type,and anti-sensePtrLac4plants were 56.38%and 33.45%,respectively (Fig.5 b,Table 2).Compared with the lignin Raman intensity in the xylem of the wild-type,the lignin Raman intensity of the anti-sensePtrLac4transgenic plants decreased by 40.67%.The fluorescence intensity profile of the lignin in the xylem of anti-sensePtrLac4transgenic plants was remarkably lower than that of the wild-type(Fig.5 b),with intensity between 20 and 320 (Fig.5 f).The intensity profile of the wild-type was between 20 and 380(Fig.5 a,e).Moreover,we examined the content and distribution of lignin at the cellular level inP.tomentosausing CRM.We estimated the lignin distribution and intensity in each cell using Image J and the Raman images to determine the detailed differences at the cellular and in situ level of the xylem region between transgenic and wild-type plants.The Raman intensity of lignin decreased by 45.78%,27.54%,and 27.21% in the vessel,ray cells,and fiber cells of antisensePtrLac4transgenic plants,compared with that of the wild-type,respectively (Table 2).In addition,lignin intensity decreased significantly in the corner of the cell compare with that in the cell wall.The lignin intensity at the cellular level obtained by CRM could not be determined using the acetyl bromide method.

Fig.5 Lignin intensity was analyzed by confocal Raman spectroscopy in the xylem of transgenic and wild-type P opulus tomentosa.a–b Confocal Raman images of paraffi n cross-sections based on the characteristic peak of the lignin band at 1600 cm− 1 in wild-type (a)and anti-sense PtrLac4 (b) transgenic P.tomentosa,respectively;c–d bright field imaging corresponding to Raman images in wild-type(c) and anti-sense PtrLac4 (d) transgenic P.tomentosa,respectively;e–f distribution of fluorescence intensity based on confocal Raman images in wild-type (e) and anti-sense PtrLac4 (f) transgenic P.tomentosa,respectively.Scale bar=200 μm

Table 2 Confocal Raman spectroscopy analysis of lignin intensity in different xylem cell types of Populus tomentosa

Fig.4 a Total lignin content measured by the acetyl bromide method in the stems of wild-type and anti-sense PtrLac4 transgenic Populus tomentosa,respectively.b Lignin intensity estimated by CRM in the stems of wild-type and anti-sense PtrLac4 transgenic P.tomentosa,respectively.Three biological replicates were used for measurement.Data presented are the means of three replicates and asterisks indicate significant difference (Mean ± SD,n=3.**,P ≤ 0.001)

Table 1 The Raman characteristic peaks of cell wall components in different cell types were analyzed in a Populus tomentosa stem(cellulose [C],glucomannan[GlcMan],and xylan [Xyl]).(Gierlingger and Schwanninger 2006)

In this study,Raman imaging was improved by preparing the samples using paraffi n.The characteristic Raman peaks of the cell wall components were determined in different xylem cell types.The difference in lignin intensity at the cellular level was determined by CRM in situ by comparing the lignin intensity of transgenicPtrLac4to the wild-type.

Discussion

CRM combines microscopy with Raman spectroscopy and improves the spatial resolution in analysis of chemical composition of the cell wall (Gierlinger et al.2012).The method of sample preparation is important to obtain high quality Raman images.Previous studies used cryosectioning and PEG embedding for most herbaceous and young woody plants,it showed that resin embedding agent gaverise to high fluorescence background in samples (Gierlinger et al.2012).In this study,by comparing four different sample preparation methods in this study,we found that ice crystals form during the cryopreservation process in stem of tissue cultures with large vacuoles,making it difficult to maintain integrity of the structure.And PEG embedding induces air into samples,making it difficult to achieve a smooth sectioned surface.However,embedding agent paraffin is suitable for CRM sample preparation in young woody plant material,because paraffin did not interfere with lignin in the Raman spectrum and paraffin can be dewaxed with hexane.In addition,paraffin sectioning has many advantages,such as high-quality microscopic detection and easy preservation.Therefore,paraffin sections combined with a hexane dewaxing process were suitable for Raman imaging analysis to measure lignin intensity in the cell wall of the xylem inP.tomentosa,particularly for young tissue culture material.

CRM collects signals based on molecular vibrations,making it suitable for in situ investigations of plant materials and for elucidating cell wall composition at the cellular level (Gierlinger et al.2012).The functional characteristic in different cell types depend on the composition of cell wall,both fiber cells and vessels undergo programmed cell death and lignification process (Meents et al.2018),it is undoubted that both fiber cells and vessels contain lignin characteristic peaks by appling CRM detection inP.tomentosa,However,our results showed that the Raman characteristic peaks of fiber cells were different from those of ray cells and vessel cells,particularly for the hemicellulose component,indicating that the function of the fiber cells were different from other cells types in the xylem ofP.tomentosa.

The acetyl bromide method to measure cell wall lignin contents is destructive,while CRM can be used to determine the detailed differences in the native state between transgenic and wild type plants.It has been shown thatLAC4contributes to the constitutive lignification of stem in Arabidopsis and that the Arabidopsislac4mutant displays decreased lignin content (Berthet et al.2011).In this study,we also found that the repression ofPtrLAC4led to decreased lignin content in transgenicP.tomentosa.Both the acetyl bromide method and CRM analysis showed similar trends in total lignin content ofPtrLac4transgenic compared to wild-typeP.tomentosa.Moreover,CRM was useful to analyze the intensity of the cell wall components in the different cell types.The lignin intensity in vessel cells displayed more changes inPtr-Lac4transgenic plants than those in fiber cells,which might be due to the difference in function ofPtrLac4in vessel cells than fiber cells during the lignin deposition process in the xylem.

Conclusions

CRM provided a rapid and non-destructive method to determine lignin intensity in plant cell walls in situ at the cellular level.The sample preparation method was improved using paraffi n-embedded sections combined with hexane dewaxing to obtain high resolution Raman images.The cell wall components of fiber cells were different from those of ray cells and vessel cells in the xylem ofP.tomentosa.CRM produced similar trends in determining total lignin content compared with the acetyl bromide method and provided detailed information on lignin intensity at the cellular level inP.tomentosa.