植物纤维素合酶基因研究进展

纤维素合酶催化合成的 β_1 ,4糖苷链构成植物细胞壁中含量最丰富的组份纤维素。植物体中存在着众多纤维素合酶 ,同时还具多种与之相关的纤维素合酶相似蛋白 ,它们组成了一个庞大的纤维素合酶超家族。纤维素合酶的催化机理尚不清楚 ,纤维素合酶相似蛋白的功能更有待于深入研究。本文综述了近年植物纤维素合酶及其相似蛋白编码基因的研究进展。     

植物纤维素合酶基因研究进展

纤维素合酶催化合成的β_1,4糖苷链构成植物细胞壁中含量最丰富的组份纤维素。植物体中存在着众多纤维素合酶,同时还具多种与之相关的纤维素合酶相似蛋白,它们组成了一个庞大的纤维素合酶超家族。纤维素合酶的催化机理尚不清楚,纤维素合酶相似蛋白的功能更有待于深入研究。本文综述了近年植物纤维素合酶及其相似蛋白编码基因的研究进展。     

高等植物纤维素合酶超家族

从1996年第一个植物纤维素合酶基因的鉴定,人们对植物体内纤维素合成的研究已经走过了10年的历程。10多年中,人们取得了很大的成果,也有很多问题有待解决。该文主要介绍拟南芥和毛果杨基因组中的纤维素合酶超家族。     

慈竹纤维素合酶BeCesA4的克隆及功能分析

非木造浆是解决国内纸浆短缺的重要手段。慈竹(Bambusa emeiensis)是我国非木造浆的主要原材料之一,提高慈竹中纤维素的含量能够有效提高竹类造浆的效率。通过前期对慈竹进行的转录组测序分析,挖掘出慈竹中一个与植物中纤维素合酶亚基A(cellulose synthase A,CesA)同源的基因,命名为BeCesA4。结果显示,克隆出的BeCesA4基因编码一个含有982个氨基酸的蛋白质,具备CesA家族的保守结构域;BeCesA4在慈竹快速生长的笋与茎中显著表达;过量表达该基因会使转基因植物出现生物量提升、纤维素含量升高和次生细胞壁加厚等现象。结果表明,BeCesA4的表达量与慈竹茎内纤维素的积累呈正相关。本研究结果为进一步揭示慈竹纤维素合成机制奠定了基础。     

高等植物细胞壁中纤维素的合成

植物细胞壁主要由纤维素、半纤维素、木质素和果胶质等构成.近年来,在细胞壁形成,如纤维素合成方面的研究取得了一系列非常令人鼓舞的进展.本文就高等植物细胞壁中纤维素合成机制的研究进展作一介绍.     

拟南芥纤维素合酶基因时空表达模式与功能预测

纤维素是细胞壁的主要组成成分, 研究纤维素合成可以从源头上解决关于高效降解纤维素的问题。该研究通过综合拟南芥(Arabidopsis thaliana)纤维素合酶基因(AtCESA)家族的进化和芯片表达分析及根据拟南芥全生育期GUS染色结果分析纤维素合酶基因的时空表达模式, 发现拟南芥纤维素合酶基因AtCESA1, 3, 6以及AtCESA4, 7, 8分别参与细胞壁初生壁和次生壁的合成并存在明显的共表达现象。其中, AtCESA1, 3, 6在全生育期表达, AtCESA4, 7, 8主要在根、茎和叶脉等次生壁细胞中表达。AtCESA5和AtCESA6、AtCESA2和AtCESA9以及AtCESA1和AtCESA10等基因对均有基因重复作用。根据AtCESA家族基因表达模式和分子演化关系可以推测, AtCESA5对AtCESA6以及AtCESA9对AtCESA2可能分别存在功能冗余。此外, AtCESA9的表达具明显的组织特异性。上述研究结果为深入认识拟南芥纤维素合酶基因的功能奠定了基础。     

内切-1,4-β-葡聚糖酶在植物细胞生长发育中的作用

内切-1,4-β-葡聚糖酶(EGases)可以催化水解具有1,4-β-葡聚糖主链的多聚糖,如纤维素和木葡聚糖分子,从而参与对细胞壁的修饰.植物细胞中存在一个EGase蛋白家族,且多为分泌蛋白;在植物细胞中还存在另一类跨膜EGase,是细胞壁纤维素生物合成所必需的,但植物EGases在体外具有降解纤维素人造底物羧甲基纤维素(CMC)的能力,而绝大多数植物EGases在活体细胞中并不能有效地降解结晶态纤维素分子和木葡聚糖分子.本文就EGases在细胞伸长、果实成熟和组织器官脱落等发育过程中的作用,以及EGases在植物纤维素合成与降解中的作用进行综述.     

苎麻纤维素合酶BnCesA1高变区蛋白的原核表达与条件优化

高等植物纤维素合酶是含有多个功能结构域的糖苷转移酶,催化合成β-1 ,4葡萄糖苷链,即高等植物细胞壁的重要成分之一纤维素。在同一物种例如在拟南芥中比对纤维素合酶家族各成员的序列信息,发现其蛋白序列中存在两个高变异区域（HVR）,其中第一个HVR靠近NH2端（NHVR）,富含酸性氨基酸。本研究克隆了苎麻纤维素合酶基因（BnCesA1） 的NHVR编码序列,并以正确的阅读框亚克隆至含有6×His接头的pQE-N1原核表达载体,构建了pQE-N1-NHVR重组表达载体。在大肠杆菌BL21（DE3）中表达的重组蛋白His-tag-NHVR经Western-blotting验证后,正交优化得到该蛋白小量表达的最优条件组合为：所挑取的2号菌落在37 ℃下,IPTG的诱导浓度为0.1 mmol/L, 诱导表达4 hr。本研究结果为纯化His-tag-NHVR融合蛋白,制备其抗体及进一步研究苎麻纤维素合酶局部功能或其组织特异性作用打下基础。     

《Molecular Plant》文章中文摘要

题目：植物细胞壁基质（matrix）多糖的生物合成（综述） 摘要：伸长中的植物细胞的细胞壁主要由纤维素微纤丝和基质多糖（半纤维素和果胶）以及少量结构蛋白和酶蛋白组成。基质多糖在高尔基体中合成,通过胞吐作用输送到细胞壁,并与纤维素微纤丝相嵌。纤维素微纤丝在细胞膜上合成并直接沉积到细胞壁。已知在生长素诱导的伸长细胞中,高尔基体中存在多糖链合成,然而直到最近才鉴定出合成多糖链酶的相关基因。在基因鉴定研究中,     

蔗糖合酶在植物生长发育中的作用研究

蔗糖合酶（SuSy）是植物蔗糖代谢的关键酶之一,在植物各组织中普遍存在。SuSy参与了植物体中许多代谢过程,包括淀粉及纤维素的合成,以及碳源的分配等。该酶还可影响植物的抗逆性、种子发育和生物固氮能力,因此,利用SUS基因改良作物品质具有良好的应用前景。对SuSy的性质、基因表达模式及其在植物生长发育中的作用进行综述。     

Cell-wall structure and anisotropy in procuste, a cellulose synthase mutant of Arabidopsis thaliana

In dark-grown hypocotyls of the Arabidopsis procuste mutant, a mutation in the CesA6 gene encoding a cellulose synthase reduces cellulose synthesis and severely inhibits elongation growth. Previous studies had left it uncertain why growth was inhibited, because cellulose synthesis was affected before, not during, the main phase of elongation. We characterised the quantity, structure and orientation of the cellulose remaining in the walls of affected cells. Solid-state NMR spectroscopy and infrared microscopy showed that the residual cellulose did not differ in structure from that of the wild type, but the cellulose content of the prc-1 cell walls was reduced by 28%. The total mass of cell-wall polymers per hypocotyl was reduced in prc-1 by about 20%. Therefore, the fourfold inhibition of elongation growth in prc-1 does not result from aberrant cellulose structure, nor from uniform reduction in the dimensions of the cell-wall network due to reduced cellulose or cell-wall mass. Cellulose orientation was quantified by two quantitative methods. First, the orientation of newly synthesised microfibrils was measured in field-emission scanning electron micrographs of the cytoplasmic face of the inner epidermal cell wall. The ordered transverse orientation of microfibrils at the inner face of the cell wall was severely disrupted in prc-1 hypocotyls, particularly in the early growth phase. Second, cellulose orientation distributions across the whole cell-wall thickness, measured by polarised infrared microscopy, were much broader. Analysis of the microfibril orientations according to the theory of composite materials showed that during the initial growth phase, their anisotropy at the plasma membrane was sufficient to explain the anisotropy of subsequent growth.     

The evolutionary origin of animal cellulose synthase

Urochordates are the only animals that produce cellulose, a polysaccharide existing primarily in the extracellular matrices of plant, algal, and bacterial cells. Here we report a Ciona intestinalis homolog of cellulose synthase, which is the core catalytic subunit of multi-enzyme complexes where cellulose biosynthesis occurs. The Ciona cellulose synthase gene, Ci-CesA, is a fusion of a cellulose synthase domain and a cellulase (cellulose-hydrolyzing enzyme) domain. Both the domains have no animal homologs in public databases. Exploiting this fusion of atypical genes, we provided evidence of a likely lateral transfer of a bacterial cellulose synthase gene into the urochordate lineage. According to fossil records, this likely lateral acquisition of the cellulose synthase gene may have occurred in the last common ancestor of extant urochordates more than 530 million years ago. Whole-mount in situ hybridization analysis revealed the expression of Ci-CesA in C. intestinalis embryos, and the expression pattern of Ci-CesA was spatiotemporally consistent with observed cellulose synthesis in vivo. We propose here that urochordates may use a laterally acquired homologous gene for an analogous process of cellulose synthesis.Electronic Supplementary Material Supplementary material is available in the online version of this article at Edited by D. Tautz     

Recent progress in cellulose biosynthesis

Cellulose comprises the major polymer of the plant cell wall. It consists of a set of parallel chains composed of glucans and these chains are highly oriented to form a structure known as a microfibril. The orientation of the microfibrils controls the extension of the direction of the plant cell. Extensive studies on the cellulose biosynthesis have been carried out for over three decades, and recently (1996) genes for cellulose biosynthesis in plants (CesA) were isolated. In the year 2002, a specific primer for cellulose biosynthesis reaction has been discovered and cellulose synthetic activity has been also confirmed by recombinant protein derived from the plant CesA gene. Furthermore, other proteins involved in cellulose biosynthesis besides CesA proteins were also proposed at the same time. One of these proteins, Korrigan cellulase, was suggested to act by removing sitosterol from the primer for biosynthesis reaction of cellulose. A membrane-bound sucrose synthase was also suggested to provide UDP-glucose as a substrate for cellulose biosynthesis. On the basis of these results, a new pathway for cellulose biosynthesis was proposed. Now, the research field of cellulose biosynthesis is facing a major turning point. Electronic Publication     

Cellulose synthase gene expression profiling of Physcomitrella patens

The cellulose synthase (CESA) gene family of seed plants comprises six clades that encode isoforms with conserved expression patterns and distinct functions in cellulose synthesis complex (CSC) formation and primary and secondary cell wall synthesis. In mosses, which have rosette CSCs like those of seed plants but lack lignified secondary cell walls, the CESA gene family diversified independently and includes no members of the six functionally distinct seed plant clades. There are seven CESA isoforms encoded in the genome of the moss Physcomitrella patens. However, only PpCESA5 has been characterised functionally, and little information is available on the expression of other PpCESA family members. We have profiled PpCESA expression through quantitative RT‐PCR, analysis of promoter‐reporter lines, and cluster analysis of public microarray data in an effort to identify expression and co‐expression patterns that could help reveal the functions of PpCESA isoforms in protein complex formation and development of specific tissues. In contrast to the tissue‐specific expression observed for seed plant CESAs, each of the PpCESAs was broadly expressed throughout most developing tissues. Although a few statistically significant differences in expression of PpCESAs were noted when some tissues and hormone treatments were compared, no strong co‐expression patterns were observed. Along with CESA phylogenies and lack of single PpCESA mutant phenotypes reported elsewhere, broad overlapping expression of the PpCESAs indicates a high degree of inter‐changeability and is consistent with a different pattern of functional specialisation in the evolution of the seed plant and moss CESA families.     

Regulation of anisotropic cell expansion in higher plants

Plant growth and development depend on anisotropic cell expansion. Cell wall yielding provides the driving force for cell expansion, and is regulated in part by the oriented deposition of cellulose microfibrils around the cell. Our current understanding of anisotropic cell expansion combines hypotheses generated by more than 50 years of research. Here, we discuss the evolving views of researchers in the field of cellulose synthesis, and highlight several unresolved questions. Recent results using live-cell imaging have illustrated novel roles for cortical microtubules in cellulose synthesis, and further research using these approaches promises to reveal exciting links between the cytoskeleton, intracellular trafficking, and anisotropic growth.     

PEG和表面活性剂对棉纤维细胞合成葡聚糖的影响

以陆地棉岱字-15号棉纤维细胞为材料,用3H-葡聚糖示踪方法测定β-1,3-葡聚糖和纤维素的合成。PEG4000促进β-1,3-葡聚糖和纤维素的合成,对刺激纤维素的合成更有效;随着非离子型表面活性剂 Trion X-100和Tween 20浓度的升高,抑制β-1,3-葡聚糖和纤维素的合成程度也增加,但抑制纤维素的合成更为强烈;而阴离子表面活性剂SDS则有所不同,在较高浓度下,又出现对β-1,3-葡聚糖合成抑制的减弱,这可能与SDS载负电荷的缘故有关。结果提示,完整的细胞膜有利于纤维素的合成,细胞膜损伤则利于β-1,3-葡聚糖的合成。     

Extraction of cellulose-synthesizing activity of Gluconacetobacter xylinus by alkylmaltoside

This study reinvestigated the synthesis of cellulose in vitro with a well-known cellulose-producing bacterium, Gluconacetobacter xylinus. Alkylmaltoside detergents, which are more frequently used in recent structural biological researches, are uniquely used in this study to solubilize cellulose-synthesizing activity from the cell membrane of G. xylinus. Activity comparable to that previously reported is obtained, while the synthesized cellulose is crystallized into a non-native polymorph of cellulose (cellulose II) as well as the previous studies. In spite of this failure to recover the native activity to synthesize cellulose I microfibril in vitro, the product is a polymer with a degree of polymerization greater than 45 as determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). It was thus concluded that the established protocol can solubilize cellulose-synthesizing activity of G. xylinus with polymerizing activity.     

利用全测序基因组数据对被子植物纤维素合成酶基因的进化分析

利用已经分离的植物纤维素合成酶基因的Cellulose_synt结构域为检索序列,从NCBI和其他数据库中调取已完成测序的物种的纤维素合成酶的氨基酸序列,共涉及10个物种的171个基因,基于以上氨基酸序列,应用MEGA4.0生成系统进化树。结果表明:CesA基因和Csl基因直向的相似度远大于平行的相似度,且它们的分化可能在单子叶和真双子叶植物分化之前,单子叶和真双子叶植物的最近共同祖先中至少存在7个CesA基因,综合已知的模式植物CesA基因的功能(初生壁或次生壁形成特异性),可为推测其他物种中该基因的功能提供帮助。     

Xylem-specific and tension stress-responsive coexpression of KORRIGAN endoglucanase and three secondary wall-associated cellulose synthase genes in aspen trees

In nature, angiosperm trees develop tension wood on the upper side of their leaning trunks and drooping branches. Development of tension wood is one of the straightening mechanisms by which trees counteract leaning or bending of stem and resume upward growth. Tension wood is characterized by the development of a highly crystalline cellulose-enriched gelatinous layer next to the lumen of the tension wood fibers. Thus experimental induction of tension wood provides a system to understand the process of cellulose biosynthesis in trees. Since KORRIGAN endoglucanases (KOR) appear to play an important role in cellulose biosynthesis in Arabidopsis, we cloned PtrKOR, a full-length KOR cDNA from aspen xylem. Using RT-PCR, in situ hybridization, and tissue-print assays, we show that PtrKOR gene expression is significantly elevated on the upper side of the bent aspen stem in response to tension stress while KOR expression is significantly suppressed on the opposite side experiencing compression stress. Moreover, three previously reported aspen cellulose synthase genes, namely, PtrCesA1, PtrCesA2, and PtrCesA3 that are closely associated with secondary cell wall development in the xylem cells exhibited similar tension stress-responsive behavior. Our results suggest that coexpression of these four proteins is important for the biosynthesis of highly crystalline cellulose typically present in tension wood fibers. Their simultaneous genetic manipulation may lead to industrially relevant improvement of cellulose in transgenic crops and trees.Suchita Bhandari and Takeshi Fujino contributed equally to this research.