毛竹茎细胞壁半纤维素多糖的免疫细胞化学定位研究

本文以毛竹(Phyllostachys pubescens)为材料,采用免疫细胞化学标记方法对两种细胞壁半纤维素多糖成分,即木聚糖(Xylan)和(1-3)(1-4)-β-葡聚糖［(1-3)(1-4)-β-glucan］在毛竹茎中的分布进行了观察。结果表明,应用免疫细胞化学方法可以准确、有效地观察这两种半纤维素多糖成分在细胞壁中的分布;木聚糖分布在已木质化的组织细胞的细胞壁中,与细胞壁木质化有密切关系;(1-3)(1-4)-β-葡聚糖在幼竹茎基本组织中分布于短薄壁细胞细胞壁中及长薄壁细胞胞间层,而在老龄竹茎基本组织中,仅分布于短薄壁细胞细胞壁中,而长薄壁细胞细胞壁却无此成分,反映出长、短薄壁细胞细胞壁组成上的差异。     

木材形成过程中次生壁沉积和细胞程序性死亡的分子调控机制

木材的形成经历了维管形成层细胞的增殖,木质部细胞的分化和扩张,次生细胞壁的沉积和细胞程序性死亡(programmed cell death, PCD)的过程.近年来,基于遗传学和组学分析,人们已经从模式植物拟南芥和杨树中鉴定出许多调控次生壁沉积的的关键转录因子和转录抑制因子.这些转录因子调控层次分明,共同构成次生壁沉积的转录调控网络,不仅在次生壁组分木质素、纤维素、木聚糖等物质的生物合成过程中起重要作用,而且可激活下游调控细胞程序性死亡相关的水解酶,启动木质部细胞的程序化死亡过程.对这些基因的生物学功能和调控网络进行解析,为阐明木材形成的分子生物学机制奠定了理论基础.本文综述了木材形成过程中次生壁沉积的转录调控网络和细胞程序性死亡相关的酶学机制及其最新研究进展.     

毛竹竹秆基本组织发育过程中过氧化物酶的超微定位

利用电镜细胞化学技术对毛竹竹秆基本组织发育过程中过氧化物酶进行了细胞化学定位。基本组织细胞过氧化物酶活性由胞间隙处的胞间层开始逐渐向中间推进,同期过氧化物酶体、内质网等细胞器也具有酶活性,随后质膜和液泡膜出现酶反应物。次生壁形成期长细胞壁上过氧化物酶高活性主要集中在次生壁窄层中,以休眠期酶活性最高。随着年龄的增加,长细胞的过氧化物酶活性逐渐降低,九年生长、短细胞的过氧化物酶活性已很弱。短细胞的酶活性始终高于长细胞,细胞壁、质膜、运输小泡膜和纹孔也都具有较高的酶活性。短细胞伸长停止与高过氧化物酶活性有关。过氧化物酶分布和活性并不完全对应于木质素的沉积部位,短细胞的过氧化物酶可能参与了长细胞壁中木质素的合成。     

毛竹茎细胞壁半纤维素多糖的免疫细胞化学定位研究

本文以毛竹（Ｐｈｙｌｌｏｓｔａｃｈｙｓ ｐｕｂｅｓｃｅｎｓ）为材料,采用免疫细胞化学标记方法对两种细胞壁半纤维素多糖成分,即木聚糖（Ｘｙｌａｎ）和（１－３）（１－４）－β－葡聚糖「（１－３）（１－４）－β－ｇｌｕｃａｎ」在毛竹茎中的分布进行了观察。结果表明,应用免疫细胞化学方法可以准确、有效地观察这两种半纤维素多糖成分在细胞壁中的分析;木聚糖分布在已木质化的组织细胞的细胞壁中,与细胞壁木质化有密切     

参与植物细胞壁半纤维素木聚糖合成的糖基转移酶

木聚糖是双子叶植物次生细胞壁中最主要的半纤维素,含有木聚糖的次生壁是最丰富的植物生物质,广泛应用于能源、制浆、造纸和纺织业中,但其主要组分戊糖对细胞壁生物质利用具有较大影响。揭示木聚糖合成的分子机制,为遗传修饰细胞壁组成,更好地利用细胞壁生物质提供新的策略。近年来对模式植物拟南芥中多个木聚糖合成有缺陷的突变体的分析表明：GT43家族的IRX9、IRX9-L、IRX14、IRX14-L,GT47家族的FRA8、F8H、IRX10、IRX10-L,GT8家族的IRX8、PARVUS、QUA1、GUX1、GUX2等参与了木聚糖主链、还原末端序列和侧链的合成。本文主要对这些研究进展做一综述,并讨论了木聚糖合成的机制及亟待解决的问题,展望了其发展趋势。     

巴西橡胶树HbMYB52基因的克隆及其在拟南芥中的表达

为揭示Hb MYB52在巴西橡胶树(Hevea brasiliensis)木材发育过程中的功能,从其转录组中分离克隆到1个MYB转录因子G21亚组成员基因,命名为Hb MYB52,开放阅读框为726 bp,编码242个氨基酸的蛋白,在木质部中高度表达。在拟南芥(Arabidopsis thaliana)中过表达Hb MYB52,虽未改变转基因植株株型,但植株维管束间纤维细胞壁明显增厚,同时抑制了木质纤维、导管次生壁形成。转基因拟南芥株系3和株系6中纤维素和木质素含量减少,相应各组分合成的关键酶基因的表达量也不同程度下降;株系8产生了木质素异位沉积,且木质素合成关键酶基因表达活跃。因此,推测Hb MYB52参与了植物次生壁形成调控,在拟南芥次生壁形成中可能发挥了双重功能:一方面负调控维管束次生壁形成以及各组分的生物合成,另一方面具有促进束间纤维次生壁增厚的作用。     

杜仲次生木质部分化和脱分化过程中酸性磷酸酶的超微细胞化学定位

杜仲（EucommiaulmoidesOliv．）次生木质部分化过程中,在形成层刚衍生的木薄壁细胞中,酸性磷酸酶（APase）主要分布于核膜边缘和高尔基体;在分化程度较高的木薄壁细胞中,APase散布于整个核中,进而,在各种细胞器残体上聚集;在成熟的木薄壁细胞中,APase沿细胞壁内侧分布。在未成熟导管分子中,核、质膜及纹孔上明显存在APase聚集,进而,核解体;在即将分化成熟的导管分子中,APase主要集中于初生壁;在已分化成熟的导管分子中,APase集中于次生壁。脱分化过程中,只在细胞质中可见分散的APase活性,而细胞核和细胞壁上未见此酶的分布;更深层的即将分化成熟和已分化成熟的导管分子,未见有细胞分裂,其上APase的分布与剥皮前相同。通过比较分化和脱分化过程中APase的分布,推测不同的APase同工酶可能分别参与了次生木质部细胞程序性死亡过程中原生质体的解体和次生壁的建成。APase的聚集程度可能是决定细胞能否脱分化的一个重要特征。     

毛竹茎秆纤维发育过程的超微结构观察

利用透射和扫描电镜观察了毛竹（Phyllostachys edulis (Carr.) H. De Lehaie）茎秆纤维发育过程中的超微结构变化。在纤维细胞初生壁形成期,细胞质中线粒体、内质网、高尔基体等细胞器数量有明显的增加,出现大量的由内质网与高尔基体分泌形成的运输小泡,周质微管平行分布于质膜内侧,出现环状片层结构,并在细胞壁与质膜之间出现壁旁体结构。随着次生壁的逐渐形成,细胞质中细胞器逐渐地解体并出现多泡小体;纤维细胞核出现染色质凝聚并边缘化,但在8 年生的纤维中可以持续存在;在纤维次生壁形成的整个阶段都存在与周围细胞相联系的胞间连丝和运输小泡;次生壁 在前4 年加厚明显,以后加厚程度减缓,但可以持续很长一段时间,并随着加厚出现宽窄交替的多层结构。结果表明,线粒体、内质网、高尔基体和壁旁体等细胞器与周质微管一起参与了初生壁和次生壁早期的形成;纤维细胞次生壁的形成过程就是一个漫长的程序性细胞死亡（PCD）,而PCD 的产物与胞间连丝一起参与了次生壁的形成与加厚;染色质凝聚并边缘化的细胞核与胞间连丝的持续存在,证明毛竹茎秆纤维细胞是一种典型的长寿细胞。     

毛竹茎秆纤维发育过程的超微结构观察

利用透射和扫描电镜观察了毛竹(Phyllostachys edulis(Carr.)H.De Lehaie)茎秆纤维发育过程中的超微结构变化.在纤维细胞初生壁形成期,细胞质中线粒体、内质网、高尔基体等细胞器数量有明显的增加,出现大量的由内质网与高尔基体分泌形成的运输小泡,周质微管平行分布于质膜内侧,出现环状片层结构,并在细胞壁与质膜之间出现壁旁体结构.随着次生壁的逐渐形成,细胞质中细胞器逐渐地解体并出现多泡小体;纤维细胞核出现染色质凝聚并边缘化,但在8年生的纤维中可以持续存在;在纤维次生壁形成的整个阶段都存在与周围细胞相联系的胞间连丝和运输小泡;次生壁在前4年加厚明显,以后加厚程度减缓,但可以持续很长一段时间,并随着加厚出现宽窄交替的多层结构.结果表明,线粒体、内质网、高尔基体和壁旁体等细胞器与周质微管一起参与了初生壁和次生壁早期的形成;纤维细胞次生壁的形成过程就是一个漫长的程序性细胞死亡(PCD),而PCD的产物与胞间连丝一起参与了次生壁的形成与加厚;染色质凝聚并边缘化的细胞核与胞间连丝的持续存在,证明毛竹茎秆纤维细胞是一种典型的长寿细胞.     

初生纹孔场与纹孔不能混淆

细胞形成次生壁时，在一些位置上不沉积壁物质，因此形成一些间隙，这种在次生壁上的未增厚部分称为纹孔（ｐｉｔ）；细胞生长时，幼细胞的初生壁扩展，并增加表面积和厚度，但是，初生壁的某些区域较薄、明显凹陷，这个区域称为初生纹孔场（ｐｒｉｍａｒｙｐｉｔｓｆｉｅ...     

Dynamic Changes in Distribution of Lignin and Hemicelluloses in Cell Walls During Differentiation of Secondary Xylem in Eucommia ulmoides (in English)

The dynamic changes in the distribution of lignin and hemicelluloses (xylans and xyloglucans) in cell walls during the differentiation of secondary xylem in Eucommia ulmoides Oliv. were studied by means of ultraviolet light microscopy and transmission electron microscopy combined with immunogold labelling. In the cambial zone and cell expansion zone, xyloglucans were localized both in the tangential and radial walls, but no xylans or lignin were found in these regions. With the formation of secondary wall S1 layer, lignin occurred in the cell corners and middle lamella, while xylans appeared in S1 layer, and xyloglucans were localized in the primary walls and middle lamella. In pace with the formation of secondary wall S2 and S3 layer, lignification extended to S1, S2 and S3 layer in sequence, showing a patchy style of lignin deposition. Concurrently, xylans distributed in the whole secondary walls and xyloglucans, on the other hand, still localized in the primary walls and middle lamella. The results indicated that along with the formation and lignification of the secondary wall, great changes had taken place in the cell walls. Different parts of cell walls, such as cell corners, middle lamella, primary walls and various layers of secondary walls, had different kinds of hemicelluloses, which formed various cell wall architecture combined with lignin and other cell wall components.     

Formation of macromolecular lignin in ginkgo xylem cell walls as observed by field emission scanning electron microscopy

Formation of macromolecular lignin in ginkgo cell walls. In the lignifying process of xylem cell walls, macromolecular lignin is formed by polymerization of monolignols on the pectic substances, hemicellulose and cellulose microfibrils that have deposited prior to the start of lignification. Observation of lignifying secondary cell walls of ginkgo tracheids by field emission scanning electron microscopy suggested that lignin-hemicellulose complexes are formed as tubular bead-like modules surrounding the cellulose microfibrils (CMFs), and that the complexes finally fill up the space between CMFs. The size of one tubular bead-like module in the middle layer of the secondary wall (S2) was tentatively estimated to be about 16+/-2 nm in length, about 25+/-1 nm in outer diameter, with a wall thickness of 4+/-2 nm; the size of the modules in the outer layer of the secondary wall (S1) was larger and they were thicker-walled than that in the middle layer (S2). Aggregates of large globular modules were observed in the cell corner and compound middle lamella. It was suggested that the structure of non-cellulosic polysaccharides and mode of their association with CMFs may be important factors controlling the module formation and lignin concentration in the different morphological regions of the cell wall.     

Localization of cell wall polysaccharides in normal and compression wood of radiata pine: relationships with lignification and microfibril orientation

The distribution of noncellulosic polysaccharides in cell walls of tracheids and xylem parenchyma cells in normal and compression wood of Pinus radiata, was examined to determine the relationships with lignification and cellulose microfibril orientation. Using fluorescence microscopy combined with immunocytochemistry, monoclonal antibodies were used to detect xyloglucan (LM15), β(1,4)-galactan (LM5), heteroxylan (LM10 and LM11), and galactoglucomannan (LM21 and LM22). Lignin and crystalline cellulose were localized on the same sections used for immunocytochemistry by autofluorescence and polarized light microscopy, respectively. Changes in the distribution of noncellulosic polysaccharides between normal and compression wood were associated with changes in lignin distribution. Increased lignification of compression wood secondary walls was associated with novel deposition of β(1,4)-galactan and with reduced amounts of xylan and mannan in the outer S2 (S2L) region of tracheids. Xylan and mannan were detected in all lignified xylem cell types (tracheids, ray tracheids, and thick-walled ray parenchyma) but were not detected in unlignified cell types (thin-walled ray parenchyma and resin canal parenchyma). Mannan was absent from the highly lignified compound middle lamella, but xylan occurred throughout the cell walls of tracheids. Using colocalization measurements, we confirmed that polysaccharides containing galactose, mannose, and xylose have consistent correlations with lignification. Low or unsubstituted xylans were localized in cell wall layers characterized by transverse cellulose microfibril orientation in both normal and compression wood tracheids. Our results support the theory that the assembly of wood cell walls, including lignification and microfibril orientation, may be mediated by changes in the amount and distribution of noncellulosic polysaccharides.     

On the Cytochemistry of Cell Wall Formation in Poplar Trees

Abstract: The ultrastructure of cell walls and the mechanisms of cell wall formation are still not fully understood. The objective of our study was therefore to obtain additional fine structural details on the deposition of cell wall components during the differentiation of xylem cells in hybrid aspen ( Populus tremula L. × P. tremuloides Michx.) we used as a model tree. At the electron microscope level, PATAg staining revealed a successive deposition of polysaccharides with increasing distance from the cambium. Staining with potassium permanganate and UV microspectrophotometry showed that the cell walls were lignified, with some delay to the deposition of polysaccharides. Immunogold labelling of three lignin types in developing cell walls varied with progressive deposition of cell wall layers. Condensed lignin subunits were localized in corners of cells adjacent to the cambium prior to S1 formation, whereas non-condensed lignin subunits became labelled only in later stages - in secondary walls near cell corners and simultaneously with the completion of S1 formation. As S2 polysaccharide deposition progressed, the labelling extended towards the lumen. Labelling of peroxidases revealed their presence in cell corner regions of young xylem cells, still lacking a secondary wall, implying that peroxidases are incorporated into the developing cell wall at early developmental stages. A weak labelling of middle lamella regions and secondary walls could also be seen at later stages. The results are discussed in relation to current knowledge on the succession of polysaccharide and lignin deposition in woody cell walls.     

Immunohistochemical localization of hemicelluloses and pectins varies during tissue development in the bamboo culm

The distribution of hemicelluloses and pectins in bamboo internodes was studied immunocytochemistrically at various stages of development. The ultra-structures of bamboo cell walls have been reported previously at various stages. The internodes were identically classified into three developmental phases: primary wall stage (phase I), unlignified secondary wall stage (phase II) and lignified wall stage (phase III), using the same bamboo culm. (1-->3, 1-->4)-Beta-glucans were distributed in nearly all tissues in an actively elongating stage. Limited amounts of beta-glucans were deposited in primary walls and the middle lamellae, but were limited to the phloem in secondary walls. This suggests that the function of beta-glucans might be different in phloem vis-à-vis other tissues. Highly-substituted xylans were located in nearly all tissues of early phase I, but had disappeared in all tissues immediately prior to lignification. In contrast, low-branched xylan epitopes were present only in the protoxylem in phase I, but were present in all tissues immediately prior to lignification in phase II. In phase III, the epitopes were densely localized in lignified walls, suggesting that the substitution of xylans is closely related to maturation. Methyl-esterified (but not unesterified) pectins were present in all tissues of early phase I. Just before and after lignification, both types of pectins were concentrated in the phloem and protoxylem. Xyloglucans were largely distributed in the phloem and in lignified tissues, suggesting that they might be closely correlated with maturation. This represents the first account of the distribution of hemicelluloses and pectins at the tissue and ultrastructural level in bamboo internodes at various stages of development.     

Immunohistochemical Localization of Hemicelluloses and Pectins Varies During Tissue Development in the Bamboo Culm

The distribution of hemicelluloses and pectins in bamboo internodes was studied immunocytochemistrically at various stages of development. The ultra-structures of bamboo cell walls have been reported previously at various stages. The internodes were identically classified into three developmental phases: primary wall stage (phase I), unlignified secondary wall stage (phase II) and lignified wall stage (phase III), using the same bamboo culm. (1→,1→4)-β-Glucans were distributed in nearly all tissues in an actively elongating stage. Limited amounts of β-glucans were deposited in primary walls and the middle lamellae, but were limited to the phloem in secondary walls. This suggests that the function of β-glucans might be different in phloem vis-à-vis other tissues. Highly-substituted xylans were located in nearly all tissues of early phase I, but had disappeared in all tissues immediately prior to lignification. In contrast, low-branched xylan epitopes were present only in the protoxylem in phase I, but were present in all tissues immediately prior to lignification in phase II. In phase III, the epitopes were densely localized in lignified walls, suggesting that the substitution of xylans is closely related to maturation. Methyl-esterified (but not unesterified) pectins were present in all tissues of early phase I. Just before and after lignification, both types of pectins were concentrated in the phloem and protoxylem. Xyloglucans were largely distributed in the phloem and in lignified tissues, suggesting that they might be closely correlated with maturation. This represents the first account of the distribution of hemicelluloses and pectins at the tissue and ultrastructural level in bamboo internodes at various stages of development.     

Localization of dibenzodioxocin substructures in lignifying Norway spruce xylem by transmission electron microscopy–immunogold labeling

The lignification process in mature Norway spruce [Picea abies (L.) H. Karsten] xylem cell walls was studied using transmission electron microscopy (TEM)–immunogold detection with a polyclonal antibody raised against a specific lignin substructure, dibenzodioxocin. The study reveals for the first time the exact location of this abundant eight-ring structure in the cell wall layers of wood. Spruce wood samples were collected in Southern Finland at the time of active growth and lignification of the xylem cell walls. In very young tracheids where secondary cell wall layers were not yet formed, the presence of the dibenzodioxocin structure could not be shown at all. During secondary cell wall thickening, the dibenzodioxocin structure was more abundant in the secondary cell wall layers than in the middle lamella. The highest number of gold particles revealing dibenzodioxocin was in the S2+S3 layer. Statistically significant differences were found in the frequency of gold particles present in various cell wall layers. For comparison, wood sections were also cut with a cryomicrotome for light and fluorescence microscopy.     

Alfalfa Stem Tissues: Cell-wall Development and Lignification

Alfalfa stems contain a variety of tissues with different patternsof cell-wall development. Development of alfalfa cell wallswas investigated after histochemical staining and with polarizedlight using light microscopy and scanning electron microscopy.Samples of the seventh internode, from the base of stems grownon cut stems, were harvested at five defined stages of developmentfrom early internode elongation through to late maturity. Internodeseven was elongating up to the third sample harvest and internodediameter increased throughout the entire sampling period. Chlorenchyma,cambium, secondary phloem, primary xylem parenchyma and pithparenchyma stem tissues all had thin primary cell walls. Pithparenchyma underwent a small amount of cell-wall thickeningand lignification during maturation. Collenchyma and primaryphloem tissues developed partially thickened primary walls.In contrast to a recent report, the formation of a ring shaped,lignified portion of the primary wall in a number of cells inthe exterior part of the primary phloem was found to precedethe deposition of a thick, non-lignified secondary wall whichwas degradable by rumen microbes. In numerous xylem fibres fromthe fourth harvest date onwards, an additional highly degradablesecondary wall layer was deposited against a previously depositedlignified and undegradable secondary wall. The pattern of lignificationobserved in alfalfa stem tissues suggests that polymerizationof monolignols by peroxidases at the luminal border of the primarycell wall creates an impermeable zone which restricts lignificationof the middle lamella region of tissues with thick primary walls.Copyright1998 Annals of Botany Company Alfalfa,Medicago sativaL., stem tissue, cell wall, development, lignification, degradation.     

Morphology, wood structure and cell wall composition of rolC transgenic and non-transformed aspen trees

Morphology, wood structure and cell wall composition of 35S-rolC transgenic hybrid aspen (P. tremula2tremuloides) were compared with non-transformed control trees. The transgenics are characterised by stunted growth, altered physiological parameters and light green leaves of reduced size. Histometric measurements revealed thinner fibre walls as compared to the controls. UV microspectrophotometry of individual wall layers did not reveal distinctive differences in the lignification of xylem cells, but in the extremely thin-walled fibres of the transgenics the secondary walls were less lignified as revealed by KMnO₄ staining in transmission electron microscopy. In the transgenics the formation of xylem cells was delayed and the differentiation zone reduced to only a few rows. Immunocytochemical analyses revealed the deposition of lignins in less differentiated xylem cells as compared to the controls. The first labelling of condensed lignin appeared in cell corners and of non-condensed lignin in secondary walls near cell corners during the deposition of S1 polysaccharides. Because of alterations in the formation and differentiation of xylem cells, 35S-rolC transgenic aspen may be useful for studies on molecular factors controlling the differentiation continuum.