【作者】 刘波；

【导师】 姜笑梅；

【作者基本信息】 中国林业科学研究院 ， 木材科学与技术， 2008， 博士

【摘要】 与木材相比,竹材细胞壁的研究工作仍处于起步阶段,对竹茎杆细胞的发育过程,尤其是细胞壁形成和分化过程的研究远远不够。细胞壁是构成竹材细胞的实体物质,是竹类植物遗传改良和定向培育的重要目标,是竹材各种性质的成因,也是竹材加工和利用的主要部分。因此,针对竹类植物开展细胞壁的生物形成过程的研究,具有非常重要的理论和现实意义。本研究是以我国种植面积最广、利用价值最大的毛竹（Phyllostachys pubescens MaxelExh.De Lehaie）为研究对象,紧紧围绕竹材生物形成机理与加工利用相互关系这一主题,利用多学科的技术手段和方法以及先进的实验仪器,从竹材发育过程中的显微结构变化、细胞壁超微结构变化和细胞壁力学性能及其与细胞壁显微和超微结构关系三个方面,系统研究了各类细胞的分化和细胞壁的形成过程。在竹材发育过程中显微结构变化的研究中,采用定量解剖学和显微图像分析技术,得到了毛竹各类组织和细胞的解剖形态在发育过程中的变化;并采用X-射线衍射仪获得了毛竹在发育过程中微纤丝角（MFA）和结晶度（CrI）的变化。在竹材发育过程中细胞壁超微结构变化的研究中,通过透射电子显微镜（TEM）观察了毛竹各类细胞在发育各阶段细胞壁和原生质体的变化特征,确定了细胞的分化方式,讨论了细胞程序化死亡（PCD）过程中细胞壁形成与原生质体之间的相互关系。采用场发射扫描电子显微镜（FE-SEM）、TEM结合直接碳复型（DCR）技术,首次系统地观察到了毛竹细胞壁纤维素微纤丝（CMfs）从初生壁到次生壁多层上的排列和沉积过程,提出了毛竹纤维细胞壁的构成模型。在国内首次将免疫细胞化学技术结合激光共聚焦显微镜（CLSM）的方法,运用到竹材骨架物质微管的研究中,得到了各类型细胞分化过程中周质微管（CMTs）的排列方式,并讨论了CMTs的排列与细胞壁上CMfs沉积方式的相互关系,研究结果为竹类植物细胞分化过程中CMTs排列方式控制细胞壁上CMfs的沉积提供了新的证据。通过可见光显微分光光度计结合组织化学染色方法,首次系统地观察到了毛竹各类细胞分化过程中细胞壁上木质素的沉积过程和微区分布特点,半定量分析了木质素组成单元的分布和含量变化。在竹材发育过程中细胞壁力学性能及其与结构相互关系的研究中,通过纳米压痕技术获得了毛竹纤维细胞的纵向弹性模量和硬度性能指标,并分析了MFA、CrI、细胞壁木质素含量和分布以及细胞壁超微结构对细胞壁力学性能的影响。本文旨在通过以上所涉及的三个方面的研究内容和结果为纽带,针对细胞壁研究比较薄弱的竹类植物,揭示细胞壁发育的生物形成过程,建立竹材解剖学研究与现代生物技术改良研究和竹材加工利用研究之间的紧密联系,为揭示竹子生长发育规律、结构与功能的关系以及合理利用提供科学的基础数据,为竹材分子生物学研究提供有价值的细胞学依据。以上研究内容的主要结果如下:1.定量解剖学研究结果表明,随着细胞分化和发育的进行,维管束分布密度、维管束径向长度和弦向长度、纤维长度和宽度、导管分子长度、微纤丝角、结晶度随竹龄、径向位置和纵向高度变化趋势各不相同。其中,维管束分布密度在7天～1年内不断减小;在1～8年内保持稳定,一般为11～18个/5mm²;在8～9年内逐渐增大。纤维长度在7～17天内纤维长度迅速增大;17天～8年内基本保持稳定,竹青位置平均2295μm,竹黄位置平均2205μm;8～9年内纤维长度略有下降。X-射线衍射研究结果显示,MFA随竹龄的变化趋势为:竹青位置在1～2年内小幅上升;2～8年内小幅回落后稳定在平均值9.98°左右;8～9年内大幅增加至平均值11.41°。竹黄位置各节间在1～8年内保持稳定在平均值9.16°左右;8～9年内大幅增加至平均11.35°,与竹青处相差不多。结晶度随竹龄的变化趋势为,竹青位置在1～2年内逐渐减小;随后2～8年内迅速回升,然后稳定平均值46.78%左右;8～9年间又有所减小。竹黄位置处结晶度在1～9年内基本围绕在平均40.79%值左右波动变化。2.TEM观察结果表明纤维、基本薄壁组织细胞、导管分子的分化过程均是典型的衰退过程,它是由次生壁的沉积和原生质体的衰退这两个密切相关的过程组成。纤维、基本薄壁组织细胞、导管分子的终端分化就是PCD;并且PCD过程是一个具有较长周期的过程,周期长短比较为基本薄壁组织细胞＞纤维＞导管分子;三类细胞最后的凋亡决定于细胞内原生质体完全降解的时刻。在细胞PCD过程中,原生质体内的各种细胞器对次生壁形成起到了不同的推进作用,并且实现了各类型细胞向功能定位的转变——纤维细胞组织起机械支持作用,基本薄壁组织细胞起贮藏作用,导管起输导作用。3.场发射和复型技术揭示纤维、基本薄壁组织细胞和导管的细胞壁上CMfs的沉积方式。纤维的细胞壁为典型的薄厚层相间的同心圆多层结构,初生壁CMfs排列方向与细胞轴向垂直（90°）;次生壁中厚层的CMfs均以接近平行的小角度（0～5°）沉积;薄层一般以大角度沉积,且薄层的CMfs角度由壁外侧向细胞腔里逐渐增加,直到完全垂直。纤维单纹孔由初生壁CMfs在初生纹孔场区域凹陷形成,各层次生壁CMfs围绕纹孔排列形成椭圆形,纹孔长轴随着次生壁每层CMfs排列的主方向变化。将纤维素微纤丝排列变化与细胞壁各层厚度的研究结果相结合,本文提出了毛竹纤维细胞壁的构成模型。基本薄壁组织细胞的细胞壁为多层结构,无明显的薄厚层变化规律。初生壁CMfs排列方向与细胞轴向垂直;次生壁奇数层CMfs排列与细胞轴向近平行或呈小角度,偶数层CMfs排列与细胞轴向接近垂直或呈大角度。其单纹孔由初生壁CMfs在初生纹孔场周围盘绕而成,次生壁各层的CMfs均绕开纹孔膜排列,椭圆形纹孔的长轴总与各壁层CMfs排列方向相同。导管的细胞壁为多层结构,每层次生壁的CMfs排列均与细胞轴向垂直。其具缘纹孔由初生壁CMfs在初生纹孔场区域网状交织、在初生纹孔场周围加高隆起而形成。多层次生壁在纹孔周围不断沉积,使具缘纹孔形成了长型椭圆形,其长轴方向始终与细胞轴向垂直。4.免疫细胞化学研究分别得到了纤维、基本薄壁组织细胞和导管的CMTs在分化中的动态变化。在各类细胞发育之初,纤维细胞中最早形成CMTs;在细胞发育间期,纤维和导管中只存在CMTs,而基本薄壁组织细胞中还存在胞质微管（CYMTs）,随着基本薄壁组织细胞伸长期的结束或CMTs的大量增加,CYMTs逐渐消失。纤维和基本薄壁组织细胞中的CMTs束经历由细变粗,数量由少到多,排列由随机到定向,分布由稀疏到密集的变化过程。在基本薄壁组织细胞中还观察到CMTs的微管组织中心（MTOC）的存在,它是由微管、不规则的小泡和浓密基质构成的复合物。在纤维和基本薄壁组织细胞增厚中期,CMTs均存在解聚现象,但其特征有所不同。结合场发射和透射电镜的研究结果表明,无论初生壁还是次生壁发育阶段,CMTs排列方式的变化与CMfs沉积方向的变化具有明显的一致性。5.木质素在纤维、基本薄壁组织细胞、导管分子和石细胞的细胞壁上均有沉积,但沉积先后和周期不同。沉积先后顺序为:原生木质部导管＞纤维＞后生木质部导管=石细胞＞基本薄壁组织细胞;沉积周期长短顺序为:基本薄壁组织细胞＞后生木质部导管＞石细胞＞纤维。各类型细胞的木质素总含量随细胞壁的增厚而不断增加。可见光吸收光谱表明,纤维、基本薄壁组织细胞、后生木质部导管的木质素中具有愈创木基单元（G）和紫丁香单元（S）。三类细胞胞壁各微区的木质素总含量和木质素组成单元含量随竹龄、径向位置的变化规律各不相同,纤维的木质素含量随纤维在维管束纤维帽中位置的变化而不同。木质素的沉积和细胞次生壁的发育过程紧密相关。6.纤维、基本薄壁组织细胞和导管细胞壁层数在发育过程中不断增加,发育快慢程度为:基本薄壁组织细胞＞纤维＞导管。三类细胞壁层发育程度在径向上的比较为:竹青位置的细胞始终快于同时期同类型竹黄位置的细胞。纤维和基本薄壁组织细胞的壁层数在6年时达到最大值,分别形成了6～11层和7～13层次生壁;导管的壁层始终处于不断增加的状态,在9年时达到5～6层。三类细胞各壁层厚度变化为:纤维在发育的各个时期,细胞壁的最厚层均为次生壁S₂层;基本薄壁组织细胞和导管分子各壁层的厚度变化规律不一致,最厚壁层在发育的不同时期出现于不同的次生壁层。维管束内纤维壁层和壁厚,是近维管束中心的先发育、程度高,远维管束中心的后发育、程度低,发育程度是以维管束中心为原点,呈辐射状向四个纤维帽外侧过渡。7.纳米压痕技术原位测定结果显示,随着竹龄的增加,纤维细胞壁的纵向弹性模量和硬度的变化不同。次生壁（SW）S₂层的纵向弹性模量和硬度范围分别在13.2～24.7GPa和0.46～0.71GPa;细胞角隅（CC）处分别为9.0～16.6GPa和0.38～0.66GPa;细胞胞间层（ML）分别为8.9～13.6GPa和0.27～0.66GPa。细胞壁三个不同微区的纵向弹性模量和硬度在径向上的变化趋势也不同。同竹龄同径向位置的细胞壁三个不同微区上纵向弹性模量大小为SW＞ML＞CC,硬度大小为ML＞CC＞SW。在纤维细胞的发育过程中,细胞壁力学性能与细胞壁木质素含量和分布呈正相关,尤其是硬度性能与木质素含量密切相关。同时,细胞壁力学性能与细胞壁纤维素结晶度呈正相关,与纤维素微纤丝角度关系密切,呈负相关。竹材的高强度、高硬度等力学性能,主要由竹材细胞的细胞壁结构特性决定。更多还原

【Abstract】 In comparison with wood,Research of bamboo cell wall is still at the early stage.The study on development and differentiation of cells in bamboo culm is very limited,especially on formation and development of cell wall.Cell wall is the main substance of bamboo cell structure.Basically,the bamboo properties is determinated by the cell wall of bamboo,which is the main part for bamboo processing and utilization,and the key aim of genetic modifications and tree treeding of bamboo plant as well.So it is very important and essential to conduct the study on the developmental formation of cell wall in bamboo.The objective of this paper is to obtain more details of the cell wall formation during developmental process of bamboo.The bamboo species Phyllostachys pubescens Maxel Exh. De Lehaie at different ages were selected for the wide planting area and great potential use in China.Histological and cytological studies were implemented respectively on microstructural features,ultrastructural features,and mechanical properties of cell wall,as well as the relationship between the structural features with the mechanical properties of cell wall.A series of testing techniques and methods and advanced instruments from different research fields were adopted in this study.In the study on developmental changes of microstructural features,quantitative anatomy and image analyzing methods were used to get anatomic properties of main kinds of tissues and cells in bamboo.X-ray diffracometer was applied to obtain the microfibril angle（MFA） and crystallinity index（CrI） of cell wall of bamboo at different ages.In the research of cell wall ultrastructural features,the changes of cell wall and protoplast of different kinds of cells at different developmental phases were observed by transmission Electron Microscope（TEM）.The general type of cell differentiation was determined,and the relationship of cell wall formation and protoplast changes in programmed cell death（PCD） process was discussed.Meanwhile,Field Emission Scanning Electron Microscope（FE-SEM）, TEM and direct carbon replica technique（DCR） were used in the systemic observations of cellulose microfibrils（CMfs） deposition process and arrangement from primary wall to each secondary wall.The new evidence of the relationship between cellulose-synthesizing complexes and CMfs deposition was obtained.A model of fiber wall structure of bamboo Phyllostachys pubescens was proposed on the basis of observation.Consequently,the method of indirect immunofluorescence staining ofα-tubulin combined with confocal laser scanning microscope（CLSM） was adopted at the first time to investigate cortical microtubules（CMTs） array during cell differentiating process of fiber,parenchyma cell and vessel element of bamboo in China.The relationship between CMfs deposition and CMTs arrangement was then discussed to provided a new evidence for CMfs deposition controlled by CMTs arrangement. Furthermore,by means of visible-light microspectrophotometry（VLMS） accompanying with histochemistry staining technique,the lignification progress and distribution characteristics in micro-morphological regions were systemically observed during bamboo cells differentiation process.The distribution of lignin components and variety of lignin content were also measured in semi-quantitative level.In the investigation of relationship between structural features with mechanical properties of cell wall,longitudinal elasticity modulus and hardness of fiber were determinated by means of nanoindentation technique.Meanwhile,the effects of MFA,CrI,lignin content and distribution in cell wall and ultrastructural features of cell wall on mechenical of cell wall were also analyzed.The results from above studies were summarized as follows:1.The data of quantitative anatomy indicated that the variety trends of vascular bundle distributing density,length and width of vascular bundle,fiber length and width,vessel element length,MFA and CrI were different with bamboo age,radial location or longitudinal location. Distributing density of vascular bundles decreased in 7～17 days,then preserved at 11～18/5mm² in 1～8 years,and gradually increased in 8～9 years.Fiber length rapidly increased in 7～17 days,then preserved at 2295μm on outer part of bamboo and 2205μm on inner part of bamboo,but slightly decreased in 8～9 years.X-ray diffracometer results suggested that MFA increased slightly in 1～2 years and then kept 9.98°in 2～8 years on outer part of bamboo, however it increased greatly to 11.41°in 8～9 years.On inner part of bamboo,MFA kept stable at 9.16°in 1～8 years and increased greatly to 11.35°in 8～9 years.On outer part of bamboo, CrI decreased gradually in 1-2years and returned rapidly and kept at 46.78%in 2～8 years, however it decreased slightly in 8～9 years.2.TEM observation suggested that the differentiation of fiber,parenchyma cell and vessel was a typical deterioration process.It consists of two tight corresponding parts,the secondary wall deposition and the protoplast deterioration.The terminal differentiation of fiber, parenchyma cell and vessel was PCD,which was a long periodic process.The comparison in the period length of three types of cells was parenchyma cell＞fiber＞vessel.The terminal cell apoptosis depended on the time of complete degradation of protoplast in cell.During PCD process,all kinds of cell organelles in protoplast promoted secondary wall deposition at different degree.Furthermore,the changes of cell organelles realized different cell functions, which fiber for mechanical support,parenchyma cell for preservation and vessel for transportation.3.FE-SEM and DCR technique revealed the arrangements of CMfs on cell wall of fiber, parenchyma cell and vessel.Firstly,fiber cell formed multilamellate concentric structure with thin and thick lamellae alternate.CMfs orientation on fiber primary wall was perpendicular to cell axis（90°）.CMfs orientation on thick fiber secondary wall was parallel to cell axis or presented small angle to cell axis（0～5°）.However,CMfs orientation on thin fiber secondary wall usually presented large angle to cell axis,and the angle increased from outside to inside of secondary wall,until perpendicular to cell axis.Simple pit on fiber cell wall formed by CMfs of primary wall sunk on the area of primary pits field.CMfs of each secondary wall surrounded Simple pit to an oval.And the long axis of simple pit Varied along with CMfs orientation of each secondary wall.This paper proposed a structure model of Phyllostachys pubescens cell wall,based on the results of CMfs deposition and thickness of each secondary wall.Secondly, it was a multilamellate structure of parenchyma cell wall,but its thin and thick lamellate was not evident.CMfs orientation on primary wall of parenchyma cell was perpendicular to cell axis.CMfs orientation on odd secondary wall was nearly parallel to cell axis or presented small angle to cell axis.CMfs orientation on even secondary wall was nearly perpendicular to cell axis or presented large angle to cell axis.The simple pit of parenchyma cell formed by CMfs of primary wall circled primary pits field and the long axis of pit were always same to the CMfs arrangement of each secondary wall.Thirdly,vessel cell wall was a multilamellate structure, which CMfs orientation of each wall lamellate was perpendicular to cell axis.Bordered pit of vessel formed by CMfs of primary wall intercrossed on primary pits field and protruded around primary pits field.Bordered pits became long ellipse because of the sustaining deposition of secondary wall CMfs around the pits,moreover,the long axis of bordered pit was always perpendicular to cell axis.4.Indirect immunofluorescence staining ofα-tubulin showed clearly the dynamic changes in the array of CMTs in dfferentiating fiber,parenchyma cell and vessel.At early stages of cells differentiation,the CMTs array was present firstly in fiber.During cell interphase,there was only CMTs in fiber and vessel,but there was another microtubule called cytoplasmic microtubules（CYMTs） in parenchyma cell.The number of CYMTs greatly increased accompanying with the end of elongation stage of parenchyma cell,but would be disappeared gradually.CMTs bundles in fiber and parenchyma cells experienced the changes from slender to thick,from scarce to abundant,from random arrangement to oriented arrangement,and from sparse to dense.A kind of microtubule organizing center（MTOC） was also observed in parenchyma cells,which was a complex composed of microtubule,vesicles and dense cytoplasm.In the middle stage of wall increasing of fiber and parenchyma cell, CMTs showed disassembly phenomenon,and the characteristics of them were different.The present results from the observations by FE-SEM and TEM have provided evident consistency between CMTs and CMfs arrangement during the development of primary wall or secondary wall.5.Lignin deposited were occurred in cell walls of fiber,parenchyma cell,vessel and stone cell,However,the deposition time and period were different.The time order of lignin deposition was:protoxylem vessel＞fiber＞metaxylem vessel=stone cell＞ground tissue parenchyma cell.The period length of lignin deposition was:fiber＜stone cell＜metaxylem vessel＜ground tissue parenchyma cell.The iignin content of each kind of cell wall increased constantly accompanying with the thickness increasing of cell wall.Visible-light absorbance spectrum showed,there were Guaiacyl lignin and Syringyl lignin in cell wall of fiber,ground tissue parenchyma cell and vessel.However,the total lignin content and content of each lignin component varied respectively by bamboo age,radial location and longitudinal location.In the meantime,the lignin content of fiber varied with fiber location in vascular bundle.There was a close relationship between lignin deposition and development of secondary wall.6.The layer number of cell wall of fiber,parenchyma cell and vessel increased continuously during bamboo developmental process.The developmental order of three cells was:parenchyma cell＞fiber＞vessel.The radial variety of wall layers of three cells was that the development of cell on outer part of bamboo was always earlier than the cell on inner part of bamboo.The maximum number of cell wall layers appeared at the 6^th year in fiber and parenchyma cell.There were 6～11 layers and 7～13 layers respectively in fiber and parenchyma cell.In addition,the layer number of vessel wall gradually increased from shoot to the 9th year, and reached 5～6 layers.The thickness variety of three cells was as following.The thickest layer was the 2^nd layer of secondary wall（S₂） during the whole developmental stages in fiber. The changes of wall thickness of parenchyma cell and vessel were disorder,and the thickest layer of secondary wall was different during different developmental stages.The developmental variety of thickness and layer number of fiber cell wall was that,the fiber near the center of vascular bundle was earlier than the fiber far from the center of vascular bundle in developmental speed,as well as the developmental degree.So the developmental degree presented radiate from the center of vascular bundle to outside of four fiber caps,as the center of vascular bundle for the origin. 7.Nanoindentation technique results showed that the varieties of longitudinal elasticity modulus and hardness of cell wall was different with bamboo age.The change ranges of them on S₂ was 13.2～24.7GPa and 0.46～0.71GPa respectively.The change ranges of them on cell comer（CC） was 9.0～16.6GPa and 0.38～0.66GPa respectively.The change ranges of them on middle lamella（ML） was 8.9～13.6GPa and 0.27～0.66GPa respectively.The radial varieties of them on three micro-morphological regions of cell wall were different too.About the longitudinal elasticity modulus and hardness on three micro-morphological regions-regions of the same bamboo age and radial location,the compare results were SW＞ML＞CC,and ML＞CC＞SW respectively.In fiber developmental progress,it was positive correlation between lignin content and distribution of cell wall and mechanical strength of cell wall,especially lignin content had a close relationship with hardness.Furthermore,it was the positive correlation between cell wall mechanics and CrI,and negative correlation between mechanical strength of cell wall and MFA.The high strength and hardness of bamboo were mainy determined by the structural characteristics of cell wall.更多还原