植物叶片原生质体分离的可能机制

分析了植物叶片在分离液环境中形成原生质体的过程,文中提出,分离液配方中的酸性物质使植物叶片处于酸性环境中并导致植物正常细胞首先发生细胞壁酸性降解,随后出现原生质体脱离细胞壁进入分离液,继而又进一步发生质膜的酸性降解,使细胞核和细胞器进入分离液中,最终分离液中的细胞器以细胞核为中心进行细胞器重组,最后产生外貌形态一致的新的原生质体。植物细胞壁和质膜是植物细胞的包被系统。植物细胞包被系统的酸性降解使植物细胞器重组并产生新的原生质体成为可能。     

植物叶片愈伤组织形成的可能机制

分析了植物叶片在组培条件下形成愈伤组织的过程.文中提出,培养基配方中的酸性物质使植物叶片处于酸性环境中并导致植物正常细胞首先发生细胞壁酸性降解,随后出现原生质体脱离细胞壁,进一步发生细胞器重组或细胞重建,人工培养基的酸性环境使细胞壁强制性地降解后,植物原生质体失去细胞壁的包被后直接处于较酸性的环境中,可能会促使原生体出现酸性快速分裂.因此,植物细胞壁是控制植物细胞完成正常细胞周期的信号载体.     

组织培养条件下喜树叶片细胞壁酸性降解的pH值观察

喜树叶片外植体经组织培养第13 d和23 d后进行解剖学观察,同时比较正常叶片的解剖学特征,发现在pH值为5.8的酸性培养基中,喜树叶片外植体中的海绵组织和栅栏组织等薄壁细胞相继发生明显的细胞壁降解,而表皮细胞发生较微弱的细胞壁降解现象;进行组织培养前,正常叶片的各类细胞未观察到细胞壁降解现象发生。应用BCECF-AM pH荧光探标记并采用激光共聚焦在480 nm波长下进行pH值测定发现,喜树叶片外植体经组织培养第13 d和第23 d后的海绵组织和栅栏组织等薄壁细胞部位的pH值均为5.2,但表皮细胞部位的pH值则为5.7~5.8,而正常叶片各类细胞的pH值平均为5.7。这说明, pH值为5.8的酸性培养基和喜树叶片薄壁细胞内的酸性成分自泌可能共同诱导了其细胞壁的酸性降解。     

植物原生质体培养

六十年代初英国植物生理学家 Cocking首先用酶解方法降解细胞壁,获得了蕃茄根尖细胞的原生质体。由于酶解方法能获得遗传性状和生理性状较一致的原生质体群体,为从植物细胞获得大量原生质体开辟了新的途径,并已为国内外科研工作者广泛采用。现用酶解法     

一种特殊的长寿细胞:毛竹茎秆纤维细胞

利用显微和细胞化学方法,对毛竹(Phyllostachys edulis)茎秆纤维次生壁形成过程中超微结构变化以及ATP酶、Ca2 -ATPase和酸性磷酸酶的超微细胞化学定位进行了研究.研究发现,次生壁形成早期,细胞核具有双层核膜,染色质凝聚,可见大量的线粒体、粗面内质网和高尔基体等细胞器存在于纤维细胞中;随后,双层核膜消失,细胞器将逐渐解体,多泡体开始出现在纤维细胞的细胞质;随着年龄的增加,纤维细胞壁逐渐增厚,并出现多层结构现象,而运输小泡、细胞膜、胞间连丝和凝聚的染色质将持续存在.在次生壁形成的整个过程中,ATP酶、Ca2 -ATPase和酸性磷酸酶在运输小泡、细胞膜、质膜内陷、胞间连丝和凝聚的染色质中将持续存在.结果表明,毛竹茎秆纤维细胞是一种不同于木本双子叶植物的长寿细胞,纤维原生质体中ATP酶和酸性磷酸酶的持续存在与次生壁的持续增厚密切相关.     

第八讲 植物原生质体培养

一、前言植物原生质体就是除去细胞壁以后的裸露细胞。英国植物生理学家Cocking(1960)首先用酶解的方法降解番茄根尖的细胞壁,获得大量而完整的原生质体。植物原生质体可直接从植物各种器官、如根、茎、叶、花、果实的细胞中获得,也可以从培养细胞中获得。一般认为由叶肉组织分离的原生质体遗传性状较一致。而一般     

超声破碎拟南芥原生质体后的再生培养初报

拟南芥原生质体在超声破碎过滤后,对其叶绿体、细胞核等细胞器进行再生培养.观察到超声破碎对叶绿体有一定破坏作用导致叶绿体在蓝色荧光下不发荧光,在培养115 h后叶绿体有聚集现象;细胞核明显增多;出现内质网的明亮绿色荧光点.细胞核和内质网都是形成新原生质体的必要细胞器,通过对其再生培养可为细胞器重组乃至细胞重建提供启示.     

小麦叶片细胞周质微管的研究

采用铜网粘附-负染色法,并结合超薄切片,对小麦幼叶和成熟叶片细胞内的周质微管进行了研究,结果如下: (1) 粘附于铜网支持膜上的质膜片段,往往包含一个组织中心的微管体系。微管组织中心具有电子致密度很高的浓密物质。微管从组织中心呈辐射状或扇形分布。微管之间,有单个或数根成束排列, 有的相互平行,有的则相互交叉形成网状结构。微管的外径为24—24.76毫微米,最大长度为12微米。(2) 周质微管与质膜之间有密切联系,两者之间有连丝结构(“桥”)相连接。微管-桥-质膜三者结合形成一个稳定的体系。(3) 不仅质膜能粘附于铜网的福尔马支持膜上,分离原生质体残留的细胞壁纤维素微丝也能粘附于其上。被粘附的网状排列的纤维素微丝与幼叶细胞中周质微管的网状排列相一致,说明周质微管与纤维素微丝排列方向的密切关系。(4) 正在迅速生长的幼叶细胞比成熟叶片具有更多的周质微管和小泡结构(Vesicles),显示这两种细胞器的数量与细胞生长及细胞壁增生加厚的活动强度成正相关。     

植物遗传工程转化

将大分子导入完植物细胞的简易方法 美国弗古尼亚州立大学的F.-S.Wu、A.B.Cahoon及M.Shulleeta的研究证实:向完整植物细胞直接转移基因或其它大分子的困难不是由于其细胞壁本身的存在,而是由于细胞壁与质膜之间的紧密接触(限制了大分子通过细胞壁而进入质膜)引起的。他们发现,当利用渗透压变化使细胞壁与质膜之间产生空隙后,大分子就可通过细胞壁,利用直接转移技术(目前需用原生质体)就很容易把大分子导入细胞。 研究还报道,当洋葱表皮细胞或烟草茎细胞进行质壁分离时,蛋白质和DNA就能够穿透进入细胞壁,     

梭梭原生质体快速制备方法的建立及其在亚细胞定位中的应用

梭梭是中国西北荒漠半荒漠地区防沙治沙首选建群树种。目前梭梭蛋白亚细胞定位采用洋葱表层细胞以及鹰嘴豆和拟南芥原生质体等进行,但采用不同体系进行亚细胞定位,可能会出现不同结果,这与同源或异源表达的植物细胞特性有关。因此,梭梭蛋白采用其原生质体进行亚细胞定位结果更真实可信。通过对酶种类及配比、质壁分离时间、酶解液pH、酶解时间等的优化,本研究拟建立梭梭原生质体快速制备方法,并探讨其在亚细胞定位中的应用。结果表明:以2～3 cm长的幼嫩同化枝,置于pH值5.6的CPW酶解液(10%甘露醇, 0.1%MES, 1.0%纤维素酶, 0.4%离析酶)中,黑暗条件下27℃恒温水浴45 r/min酶解10～11 h,为梭梭原生质体快速制备最佳条件,原生质体产量可达0.41×10~7个/g·FW,存活率为70.32%;所得原生质体应用于亚细胞定位研究效果良好。本制备方法可应用于梭梭原生质体培养、细胞研究、基因瞬时表达等领域。     

Inhibition of phenylpropanoid biosynthesis increases cell wall digestibility, protoplast isolation, and facilitates sustained cell division in American elm (Ulmus americana)

ABSTRACT: BACKGROUND: Protoplast technologies offer unique opportunities for fundamental research and to develop novel germplasm through somatic hybridization, organelle transfer, protoclonal variation, and direct insertion of DNA. Applying protoplast technologies to develop Dutch elm disease resistant American elms (Ulmus americana L.) was proposed over 30 years ago, but has not been achieved. A primary factor restricting protoplast technology to American elm is the resistance of the cell walls to enzymatic degradation and a long lag phase prior to cell wall resynthesis and cell division. RESULTS: This study suggests that resistance to enzymatic degradation in American elm was due to water soluble phenylpropanoids. Incubating tobacco (Nicotiana tabacum L.) leaf tissue, an easily digestible species, in aqueous elm extract inhibits cell wall digestion in a dose dependent manner. This can be mimicked by p-coumaric or ferulic acid, phenylpropanoids known to re-enforce cell walls. Culturing American elm tissue in the presence of 2-aminoindane-2-phosphonic acid (AIP; 10-150 uM), an inhibitor of phenylalanine ammonia lyase (PAL), reduced flavonoid content, decreased tissue browning, and increased isolation rates significantly from 11.8% (+/-3.27) in controls to 65.3% (+/-4.60). Protoplasts isolated from callus grown in 100 uM AIP developed cell walls by day 2, had a division rate of 28.5% (+/-3.59) by day 6, and proliferated into callus by day 14. Heterokaryons were successfully produced using electrofusion and fused protoplasts remained viable when embedded in agarose. CONCLUSIONS: This study describes a novel approach of modifying phenylpropanoid biosynthesis to facilitate efficient protoplast isolation which has historically been problematic for American elm. This isolation system has facilitated recovery of viable protoplasts capable of rapid cell wall re-synthesis and sustained cell division to form callus. Further, isolated propotoplasts survived electrofusion and viable heterokaryons were produced. Together, these results provide the first evidence of sustained cell division, callus regeneration, and potential application of somatic cell fusion in American elm, suggesting that this source of protoplasts may be ideal for genetic manipulation of this species. The technological advance made with American elm in this study has potential implications in other woody species for fundamental and applied research which require availability of viable protoplasts.     

Two endogenous proteins that induce cell wall extension in plants.

Plant cell enlargement is regulated by wall relaxation and yielding, which is thought to be catalyzed by elusive "wall-loosening" enzymes. By employing a reconstitution approach, we found that a crude protein extract from the cell walls of growing cucumber seedlings possessed the ability to induce the extension of isolated cell walls. This activity was restricted to the growing region of the stem and could induce the extension of isolated cell walls from various dicot stems and the leaves of amaryllidaceous monocots, but was less effective on grass coleoptile walls. Endogenous and reconstituted wall extension activities showed similar sensitivities to pH, metal ions, thiol reducing agents, proteases, and boiling in methanol or water. Sequential HPLC fractionation of the active wall extract revealed two proteins with molecular masses of 29 and 30 kD associated with the activity. Each protein, by itself, could induce wall extension without detectable hydrolytic breakdown of the wall. These proteins appear to mediate "acid growth" responses of isolated walls and may catalyze plant cell wall extension by a novel biochemical mechanism.     

酸性培养基对喜树叶片细胞壁降解的影响

在组织培养过程中对喜树叶片外植体进行解剖学观察。发现在组织培养条件下,喜树叶片在培养基的酸性环境中细胞壁呈现模糊微弱降解、明显降解和完全降解直至消失的解剖学特征。在同样的组培条件和相同的时间内,同一喜树叶片不同部位出现细胞壁程度不同的降解和消失现象,可能是喜树叶片因上表皮凸凹不平进而导致其不同部位与酸性培养基接触的程度不同,因而使培养基中的酸性物质对喜树叶片上表皮的不同部位影响出现异质化。本文对培养基中的酸性成分对喜树叶片细胞壁降解的影响有了进一步的认识和理解。     

Building bridges: formin1 of Arabidopsis forms a connection between the cell wall and the actin cytoskeleton

Actin microfilament (MF) organization and remodelling is critical to cell function. The formin family of actin binding proteins are involved in nucleating MFs in Arabidopsis thaliana. They all contain formin homology domains in the intracellular, C‐terminal half of the protein that interacts with MFs. Formins in class I are usually targeted to the plasma membrane and this is true of Formin1 (AtFH1) of A. thaliana. In this study, we have investigated the extracellular domain of AtFH1 and we demonstrate that AtFH1 forms a bridge from the actin cytoskeleton, across the plasma membrane and is anchored within the cell wall. AtFH1 has a large, extracellular domain that is maintained by purifying selection and that contains four conserved regions, one of which is responsible for immobilising the protein. Protein anchoring within the cell wall is reduced in constructs that express truncations of the extracellular domain and in experiments in protoplasts without primary cell walls. The 18 amino acid proline‐rich extracellular domain that is responsible for AtFH1 anchoring has homology with cell‐wall extensins. We also have shown that anchoring of AtFH1 in the cell wall promotes actin bundling within the cell and that overexpression of AtFH1 has an inhibitory effect on organelle actin‐dependant dynamics. Thus, the AtFH1 bridge provides stable anchor points for the actin cytoskeleton and is probably a crucial component of the signalling response and actin‐remodelling mechanisms.     

Genetic transformation with cell wall-associated deoxyribonucleic acid in Bacillus subtilis

Cell walls from bacillus subtilis 168 were prepared by conventional methods and found to contain deoxyribonucleic acid (DNA). In transformation assays, after autolysis, it was found that two major regions of the chromosome were selectively enriched in the wall preparations. One region clustered around the replication origin and is represented by the markers purA16, ts8132, thiC5, sacA321, and hisA1. The other region included the replication terminus with representative loci metB10, citK5, gltA292, and pyrA1. All other (internal) loci which were examined showed no statistical enrichment. The two areas of enrichment were similar to but more extensive than those reported for membrane-DNA complexes. The wall preparations also contained protein and lipid, indicating a possible membrane involvement. Analyses of the cell walls revealed that the fatty acid composition of the membrane component was not typical of the for B. subtilis protoplast membranes or for lipoteichoic acids. In addition, radioiodination of cell wall autolysates, followed by gel electrophoresis and autoradiography, demonstrated the presence of proteins not readily detectable in bulk protoplast membranes or on the surfaces of intact cells. These data suggest that a unique component of the membrane and regions of the B. subtilis genome involved in DNA replication events are tightly associated with cell walls. The binding of DNA-membrane complexes to the "rigid" cell wall and the replication of the wall could be a mechanism by which the segregation of growing chromosomes occurs.     

Force extension analysis of Avena coleoptile cell walls

Summary A method is described for measuring the cell wall mechanical properties of Avena coleoptiles in the absence of turgor stress or influences of a living protoplast. Forceextension curves obtained with a constant-rate-of-extension instrument and standard fiber-testing techniques demonstrate the permanence of cell wall loosening effects of prior indoleacetic acid (IAA) treatment of living tissue and provide evidence that these changes involve interactions between cell wall polymers. By this method various chemical and enzymatic modifications of cell walls can be evaluated in terms of altered mechanical properties. Thus, it was possible to remove over 97% of the cell nitrogen (including some hydroxyproline-containing protein) by hot methanol followed by enzymatic treatment and not change the extensibility properties of the tissue. In contrast, coleoptile mechanical properties were markedly influenced by chemical acetylation procedures or cellulase treatment.With 3 Figures in the Text     

Invertase activity associated with the walls of Solanum tuberosum tubers

Three fractions with invertase activity (beta-D-fructofuranoside fructohydrolase, EC 3.2.1.26) were isolated from mature Solanum tuberosum tubers: acid soluble invertase, invertase I and invertase II. The first two invertases were purified until electrophoretic homogeneity. They are made by two subunits with an apparent M(r) value of 35,000 and their optimal pH is 4.5. Invertase I was eluted from cell walls with ionic strength while invertase II remained tightly bound to cell walls after this treatment. This invertase was solubilized by enzymatic cell wall degradation (solubilized invertase II). Their K(m)s are 28, 20, 133 and 128 mM for acid soluble invertase, invertase I, invertase II and solubilized invertase II, respectively. Glucose is a non-competitive inhibitor of invertase activities and fructose produces a two site competitive inhibition with interaction between the sites. Bovine serum albumin produces activation of the acid soluble invertase and invertase I while a similar inhibition by lectins and endogenous proteinaceous inhibitor from mature S. tuberosum tubers was found. Invertase II (tightly bound to the cell walls) shows a different inhibition pattern. The test for reassociation of the acid soluble invertase or invertase I on cell wall, free of invertase activity, caused the reappearance of all invertase forms with their respective solubilization characteristics and molecular and kinetic properties. The invertase elution pattern, the recovery of cell wall firmly bound invertase and the coincidence in the immunological recognition, suggest that all three invertases may be originated from the same enzyme. The difference in some properties of invertase II and solubilized invertase II from the other two enzymes would be a consequence of the enzyme microenvironment in the cell wall or the result of its wall binding.     

Biochemical and immunohistochemical analysis of pectic polysaccharides in the cell walls of Arabidopsis mutant QUASIMODO 1 suspension-cultured cells: implications for cell adhesion

Mutation in the Arabidopsis thaliana QUASIMODO 1 gene (QUA1), which encodes a putative glycosyltransferase, reduces cell wall pectin content and cell adhesion. Suspension-cultured calli were generated from roots of wild-type (wt) and qua1-1 A. thaliana plants. The altered cell adhesion phenotype of the qua1-1 plant was also found with its suspension-cultured calli. Cell walls of both wt and qua1-1 calli were analysed by chemical, enzymatic and immunohistochemical techniques in order to assess the role of pectic polysaccharides in the mutant phenotype. Compared with the wt, qua1-1 calli cell walls contained more arabinose (23.6 versus 21.6 mol%), rhamnose (3.1 versus 2.7 mol%), and fucose (1.4 versus 1.2 mol%) and less uronic acid (24.2 versus 27.6 mol%), and they were less methyl-esterified (DM: 22.9% versus 30.3%). When sequential pectin extraction of calli cell walls was performed, qua1-1 water-soluble and chelator-soluble extracts contained more arabinose and less uronic acid than wt. Water-soluble pectins were less methyl-esterified in qua1-1 than in wt. Chelator-soluble pectins were more acetyl-esterified in qua1-1. Differences in the cell wall chemistry of wt and mutant calli were supported by a reduction in JIM7 labelling (methyl-esterified homogalacturonan) of the whole wall in small cells and particularly by a reduced labelling with 2F4 (calcium-associated homogalacturonan) in the middle lamella at tricellular junctions of large qua1-1 cells. Differences in the oligosaccharide profile obtained after endopolygalacturonase degradation of alkali extracts from qua1-1 and wt calli indicated variations in the structure of covalently bonded homogalacturonan. About 29% more extracellular polymers rich in pectins were recovered from the calli culture medium of qua1-1 compared with wt. These results show that perturbation of QUASIMODO 1-1 gene expression in calli resulted in alterations of homogalacturonan content and cell wall location. The consequences of these structural variations are discussed with regard to plant cell adhesion.