| 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 6th 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 2nd 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.
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