植物细胞壁同聚半乳糖醛酸的代谢与功能

果胶是细胞壁多糖的重要组成成分,对植物正常的生长发育十分重要。作为初生细胞壁中果胶的一种主要组成成分,同聚半乳糖醛酸（homogalacturonan,HG）是由α-D-半乳糖醛酸单体经α-（1,4）-糖苷键连接起来的一种长链大分子物质。HG的合成和降解参与了细胞壁中的多糖代谢,影响了细胞壁的结构和功能。同时,HG精确的去甲酯化以及HG所参与的细胞壁关联激酶（WAKs）和促分裂原活化蛋白激酶（MAPKs）相关的信号转导途径,在植物生长发育中也发挥着重要作用。该文主要从HG的合成、降解和循环利用以及HG的作用等方面对植物细胞壁中HG的研究进展进行了阐述。     

裂果易发性不同的荔枝品种果皮中细胞壁代谢酶活性的比较

“糯米糍”荔枝裂果率极显著高于“淮枝”,前者果皮中的果胶酶、纤维素酶和果胶甲酯酶的活性高于后者,其中以果胶酶活性差异最明显,其次是纤维素酶,果胶甲酯酶差异最小;“糯米糍”细胞壁结合型的过氧化物酶(POD)和多酚氧化酶(PP0)活性明显高于“淮枝”,而水溶性POD和PP0的活性则两个品种间无明显差异。据此认为,果皮细胞壁水解酶活性以及细胞壁结合型的POD和PPO的活性高的荔枝品种,其裂果率也高。文章对细胞壁代谢相关酶类在果皮抗裂性形成中的作用进行了讨论。     

植物果胶的生物合成与功能

果胶作为植物细胞壁多糖之一,其结构和功能非常复杂。果胶主要由同型半乳糖醛酸聚糖(HG)、鼠李半乳糖醛酸聚糖I (RGI)和鼠李半乳糖醛酸聚糖II (RGII)组成。果胶类成分在维持细胞壁结构的完整性以及细胞间黏附和信号转导等方面发挥重要作用。研究果胶类成分的结构、分布和功能,对理解细胞壁高级结构的构建和功能具有重要意义...     

具有免疫调节活性的果胶类物质

果胶类物质是广泛存在于植物细胞壁中的一类复杂多糖,对于植物生长与病害防御等起着重要的调节作用,随着糖类分离纯化和结构研究技术的进步,尤其随着糖类体内外活性测试方法的发展,果胶类物质的结构与活性研究得到了普遍重视。     

蚜虫取食中的细胞壁修饰与免疫功能

蚜虫是世界性害虫,它通过独特的口针结构和丰富的唾液组分破坏植物细胞壁,穿过表皮细胞和叶肉细胞间隙,克服多重植物抗性,到达韧皮部取食为害。已有报道蚜虫唾液中含有多种细胞壁修饰酶能够降解修饰细胞壁,帮助蚜虫在细胞间刺探,更为有效的定位韧皮部。而细胞壁作为保护植物细胞的重要屏障,能感知和传递细胞壁损伤信号,通过调控细胞壁修饰酶的表达水平启动胞内诱导抗性,从而影响蚜虫的刺探、取食和定殖。此外,蚜虫唾液中的一些效应因子还能抑制细胞壁免疫和胞内抗性。可见,细胞壁免疫在蚜虫持续取食和成功定殖中发挥重要功能。为深入理解细胞壁免疫在蚜虫刺探与取食过程中的作用机制,本文概述了蚜虫唾液关键组分对细胞壁修饰与免疫的调控作用,从植物细胞壁多糖结构修饰、损伤信号传导和胞内抗性等方面重点论述对蚜虫取食行为的影响,结合病原菌与细胞壁免疫互作机制,进一步揭示蚜虫与细胞壁免疫互作新机制,为基于阻断蚜虫韧皮部取食的分子抗虫育种提供新思路。     

植物果胶甲酯酶及其花粉特异基因的分离

果胶甲酯酶与植物的多种重要生长发育过程有关,是目前植物生物学研究中的一个热点。根据相关文献,对植物果胶甲酯酶的结构模型、作用方式以及花粉特异的果胶甲酯酶基因的分离进行了综述。     

香蕉叶片发育过程中不同甲酯化程度果胶的变化

采用免疫荧光标记技术,利用5种识别不同甲酯化程度聚半乳糖醛酸(HGs)果胶及香蕉果胶甲酯酶(PME)的单克隆抗体,对不同株龄香蕉叶片中PME及不同甲酯化程度的HGs定位、相对含量以及PME活性的变化进行分析,为探讨HGs和PME在香蕉生长发育及抵抗逆境过程中的功能和生理机制奠定基础。结果显示:(1)PME主要在组培苗的叶肉和保卫细胞中表达,其表达量及其酶的活性均随着香蕉株龄的增长而呈现下降趋势。(2)叶肉细胞也是各类不同甲酯化程度HGs的主要分布区域,其含量随着香蕉株龄的增长有不同程度的下降,但不同HGs在叶肉细胞中的含量以及在表皮、保卫细胞及叶脉中的分布模式不尽相同,保卫细胞中HGs的甲酯化程度较高。研究表明,香蕉的叶肉细胞是PME及这5种不同甲酯化程度HGs的主要分布场所,且这些HGs在香蕉发育过程中的含量变化趋势与PME相似。     

水分胁迫对柑橘果皮细胞壁结构与代谢的影响

研究水分胁迫下,盆栽'暗柳橙(Citrus Sinensis Osbeck cv. Anliu)'的果实成熟期果皮细胞壁超微结构、细胞壁物质成分、细胞壁代谢相关酶活性的变化规律及其之间关系.结果表明,在果实发育成熟期,果皮细胞壁代谢相关水解酶果胶酶、纤维素酶、果胶甲酯酶的活性随着水分胁迫的加强而增加,多酚氧化酶活性与果胶酶活性变化趋势相反,果皮细胞壁代谢相关成分离子结合型果胶、共价结合型果胶、半纤维素、纤维素的含量随着水分胁迫的加强而降低,水溶性果胶含量随着水分胁迫的加强而增加,果皮细胞壁超微结构随着水分胁迫的加强而加速解体.     

组蛋白修饰调控植物对胁迫的记忆与警备抗性的研究进展

植物扎根土壤,面对不利的环境胁迫无法逃避。然而,植物已经进化出对环境胁迫的记忆(stress memory)与警备抗性(或防御警备defense priming)等机制适应环境。环境胁迫在短时间内无法改变植物的DNA碱基序列,因此表观遗传被认为是植物对环境胁迫产生记忆和产生防御警备的主要机制,而组蛋白修饰被认为是最重要的机制,为胁迫记忆提供了可能。本文综述了非生物和生物胁迫下植物分别以胁迫记忆和防御警备机制为主导的组蛋白修饰参与抵御不良环境的最新进展,并提出该研究领域存在的问题和今后研究的重点与方向。深入探究组蛋白修饰与植物适应环境胁迫的关系,可为提高植物抗性、植物表型塑造、器官再生和作物改良等方面提供理论和技术指导。     

边缘细胞对荞麦根尖铝毒的防护效应和对细胞壁多糖的影响

以2个荞麦(Fygopyrum esculentum Moench)基因型‘江西荞麦’(耐性)和‘内蒙荞麦’(敏感)为材料,采用悬空培养(保持边缘细胞附着于根尖和去除根尖边缘细胞),研究边缘细胞对根尖铝毒的防护效应以及对细胞壁多糖组分的影响。结果表明,铝毒抑制荞麦根系伸长,导致根尖Al积累。去除边缘细胞的根伸长抑制率和根尖Al含量高于保留边缘细胞的根。去除边缘细胞使江西荞麦和内蒙荞麦根尖的酸性磷酸酶(APA)活性显著升高,前者在铝毒下增幅更大。同时,铝毒胁迫下去除边缘细胞的根尖果胶甲酯酶(PME)活性和细胞壁果胶、半纤维素1、半纤维素2含量显著高于保留边缘细胞的酶活性和细胞壁多糖含量。表明边缘细胞对荞麦根尖的防护效应,与其阻止Al的吸收,降低根尖细胞壁多糖含量及提高酸性磷酸酶活性有关,以此缓解Al对根伸长的抑制。     

High‐throughput screening of Erwinia chrysanthemi pectin methylesterase variants using carbohydrate microarrays

Pectin methylesterases (PMEs) catalyse the removal of methyl esters from the homogalacturonan (HG) backbone domain of pectin, a ubiquitous polysaccharide in plant cell walls. The degree of methyl esterification (DE) impacts upon the functional properties of HG within cell walls and plants produce numerous PMEs that act upon HG in muro. Many microbial plant pathogens also produce PMEs, the activity of which renders HG more susceptible to cleavage by pectin lyase and polygalacturonase enzymes and hence aids cell wall degradation. We have developed a novel microarray‐based approach to investigate the activity of a series of variant enzymes based on the PME from the important pathogen Erwinia chrysanthemi. A library of 99 E. chrysanthemi PME mutants was created in which seven amino acids were altered by various different substitutions. Each mutant PME was incubated with a highly methyl esterified lime pectin substrate and, after digestion the enzyme/substrate mixtures were printed as microarrays. The loss of activity that resulted from certain mutations was detected by probing arrays with a mAb (JIM7) that preferentially binds to HG with a relatively high DE. Active PMEs therefore resulted in diminished JIM7 binding to the lime pectin substrate, whereas inactive PMEs did not. Our findings demonstrate the feasibility of our approach for rapidly testing the effects on PME activity of substituting a wide variety of amino acids at different positions.     

Phenylephrine,a small molecule,inhibits pectin methylesterases

Pectin methylesterases (PMEs) catalyze pectin demethylation and facilitate the determination of the degree of methyl esterification of cell wall in higher plants. The regulation of PME activity through endogenous proteinaceous PME inhibitors (PMEIs) alters the status of pectin methylation and influences plant growth and development. In this study, we performed a PMEI screening assay using a chemical library and identified a strong inhibitor, phenylephrine (PE). PE, a small molecule, competitively inhibited plant PMEs, including orange PME and Arabidopsis PME. Physiologically, cultivation of Brassica campestris seedlings in the presence of PE showed root growth inhibition. Microscopic observation revealed that PE inhibits elongation and development of root hairs. Molecular studies demonstrated that Root Hair Specific 12 (RHS12) encoding a PME, which plays a role in root hair development, was inhibited by PE with a Ki value of 44.1?μM. The biochemical mechanism of PE-mediated PME inhibition as well as a molecular docking model between PE and RHS12 revealed that PE interacts within the catalytic cleft of RHS12 and interferes with PME catalytic activity. Taken together, these findings suggest that PE is a novel and non-proteinaceous PME inhibitor. Furthermore, PE could be a lead compound for developing a potent plant growth regulator in agriculture.     

A coupled spectrophotometric enzyme assay for the determination of pectin methylesterase activity and its inhibition by proteinaceous inhibitors

Pectin methylesterase (PME; EC 3.1.1.11) activities are widespread in bacteria, fungi, and plants. PME-mediated changes in cell wall pectin structure play important roles in plant development. Genome sequencing projects have revealed the existence of large PME multigene families in higher plants. Additional complexity for PME regulation arises from the presence of specific PME inhibitor proteins (PMEI) in plant cells. Several assay procedures for the determination of PME activity have been reported. However, previous protocols suffered from various limitations. Here we report a protocol for a coupled enzyme assay based on methanol oxidation via alcohol oxidase (AO; EC 1.1.3.13) and subsequent oxidation of formaldehyde by formaldehyde dehydrogenase (FDH; EC 1.2.1.3). This simple and robust assay allows the continuous monitoring of PME activity in the neutral pH range. Furthermore, as plant PMEIs do not interfer with AO and FDH activities, this assay is suitable for the characterization of the inhibition kinetics of PMEI.     

The ectopic expression of a pectin methyl esterase inhibitor increases pectin methyl esterification and limits fungal diseases in wheat

Cell wall pectin methyl esterification can influence plant resistance because highly methyl-esterified pectin can be less susceptible to the hydrolysis by pectic enzymes such as fungal endopolygalacturonases (PG). Pectin is secreted into the cell wall in a highly methyl-esterified form and, here, is de-methyl esterified by pectin methyl esterase (PME). The activity of PME is controlled by specific protein inhibitors called PMEI; consequently, an increased inhibition of PME by PMEI might modify the pectin methyl esterification. In order to test the possibility of improving wheat resistance by modifying the methyl esterification of pectin cell wall, we have produced durum wheat transgenic lines expressing the PMEI from Actinidia chinensis (AcPMEI). The expression of AcPMEI endows wheat with a reduced endogenous PME activity, and transgenic lines expressing a high level of the inhibitor showed a significant increase in the degree of methyl esterification. These lines showed a significant reduction of disease symptoms caused by the fungal pathogens Bipolaris sorokiniana or Fusarium graminearum. This increased resistance was related to the impaired ability of these fungal pathogens to grow on methyl-esterified pectin and to a reduced activity of the fungal PG to hydrolyze methyl-esterified pectin. In addition to their importance for wheat improvement, these results highlight the primary role of pectin despite its low content in the wheat cell wall.     

Arabidopsis PME17 Activity can be Controlled by Pectin Methylesterase Inhibitor4

The degree of methylesterification (DM) of homogalacturonans (HGs), the main constituent of pectins in Arabidopsis thaliana, can be modified by pectin methylesterases (PMEs). Regulation of PME activity occurs through interaction with PME inhibitors (PMEIs) and subtilases (SBTs). Considering the size of the gene families encoding PMEs, PMEIs and SBTs, it is highly likely that specific pairs mediate localized changes in pectin structure with consequences on cell wall rheology and plant development. We previously reported that PME17, a group 2 PME expressed in root, could be processed by SBT3.5, a co-expressed subtilisin-like serine protease, to mediate changes in pectin properties and root growth. Here, we further report that a PMEI, PMEI4, is co-expressed with PME17 and is likely to regulate its activity. This sheds new light on the possible interplay of specific PMEs, PMEIs and SBTs in the fine-tuning of pectin structure.     

Modulation of the degree and pattern of methyl-esterification of pectic homogalacturonan in plant cell walls. Implications for pectin methyl esterase action, matrix properties, and cell adhesion

Homogalacturonan (HG) is a multifunctional pectic polysaccharide of the primary cell wall matrix of all land plants. HG is thought to be deposited in cell walls in a highly methyl-esterified form but can be subsequently de-esterified by wall-based pectin methyl esterases (PMEs) that have the capacity to remove methyl ester groups from HG. Plant PMEs typically occur in multigene families/isoforms, but the precise details of the functions of PMEs are far from clear. Most are thought to act in a processive or blockwise fashion resulting in domains of contiguous de-esterified galacturonic acid residues. Such de-esterified blocks of HG can be cross-linked by calcium resulting in gel formation and can contribute to intercellular adhesion. We demonstrate that, in addition to blockwise de-esterification, HG with a non-blockwise distribution of methyl esters is also an abundant feature of HG in primary plant cell walls. A partially methyl-esterified epitope of HG that is generated in greatest abundance by non-blockwise de-esterification is spatially regulated within the cell wall matrix and occurs at points of cell separation at intercellular spaces in parenchymatous tissues of pea and other angiosperms. Analysis of the properties of calcium-mediated gels formed from pectins containing HG domains with differing degrees and patterns of methyl-esterification indicated that HG with a non-blockwise pattern of methyl ester group distribution is likely to contribute distinct mechanical and porosity properties to the cell wall matrix. These findings have important implications for our understanding of both the action of pectin methyl esterases on matrix properties and mechanisms of intercellular adhesion and its loss in plants.     

Tuning of Pectin Methylesterification: PECTIN METHYLESTERASE INHIBITOR 7 MODULATES THE PROCESSIVE ACTIVITY OF CO-EXPRESSED PECTIN METHYLESTERASE 3 IN A pH-DEPENDENT MANNER*

Pectin methylesterases (PMEs) catalyze the demethylesterification of homogalacturonan domains of pectin in plant cell walls and are regulated by endogenous pectin methylesterase inhibitors (PMEIs). In Arabidopsis dark-grown hypocotyls, one PME (AtPME3) and one PMEI (AtPMEI7) were identified as potential interacting proteins. Using RT-quantitative PCR analysis and gene promoter::GUS fusions, we first showed that AtPME3 and AtPMEI7 genes had overlapping patterns of expression in etiolated hypocotyls. The two proteins were identified in hypocotyl cell wall extracts by proteomics. To investigate the potential interaction between AtPME3 and AtPMEI7, both proteins were expressed in a heterologous system and purified by affinity chromatography. The activity of recombinant AtPME3 was characterized on homogalacturonans (HGs) with distinct degrees/patterns of methylesterification. AtPME3 showed the highest activity at pH 7.5 on HG substrates with a degree of methylesterification between 60 and 80% and a random distribution of methyl esters. On the best HG substrate, AtPME3 generates long non-methylesterified stretches and leaves short highly methylesterified zones, indicating that it acts as a processive enzyme. The recombinant AtPMEI7 and AtPME3 interaction reduces the level of demethylesterification of the HG substrate but does not inhibit the processivity of the enzyme. These data suggest that the AtPME3·AtPMEI7 complex is not covalently linked and could, depending on the pH, be alternately formed and dissociated. Docking analysis indicated that the inhibition of AtPME3 could occur via the interaction of AtPMEI7 with a PME ligand-binding cleft structure. All of these data indicate that AtPME3 and AtPMEI7 could be partners involved in the fine tuning of HG methylesterification during plant development.