New genes and new biological roles for expansins

Expansins are extracellular proteins that loosen plant cell walls in novel ways. They are thought to function in cell enlargement, pollen tube invasion of the stigma (in grasses), wall disassembly during fruit ripening, abscission and other cell separation events. Expansins are encoded by two multigene families and each gene is often expressed in highly specific locations and cell types. Structural analysis indicates that one expansin region resembles the catalytic domain of family-45 endoglucanases but glucanase activity has not been detected. The genome projects have revealed numerous expansin-related sequences but their putative wall-loosening functions remain to be assessed.     

Analysis and expression of the alpha-expansin and beta-expansin gene families in maize

Expansins comprise a multigene family of proteins in maize (Zea mays). We isolated and characterized 13 different maize expansin cDNAs, five of which are alpha-expansins and eight of which are beta-expansins. This paper presents an analysis of these 13 expansins, as well as an expression analysis by northern blotting with materials from young and mature maize plants. Some expansins were expressed in restricted regions, such as the beta-expansins ExpB1 (specifically expressed in maize pollen) and ExpB4 (expressed principally in young husks). Other expansins such as alpha-expansin Exp1 and beta-expansin ExpB2 were expressed in several organs. The expression of yet a third group was not detected in the selected organs and tissues. An analysis of expansin sequences from the maize expressed sequence tag collection is also presented. Our results indicate that expansin genes may have general, overlapping expression in some instances, whereas in other cases the expression may be highly specific and limited to a single organ or cell type. In contrast to the situation in Arabidopsis, beta-expansins in maize seem to be more numerous and more highly expressed than are alpha-expansins. The results support the concept that beta-expansins multiplied and evolved special functions in the grasses.     

Plant cell enlargement and the action of expansins

Plant cells are caged within a distended polymeric network (the cell wall), which enlarges by a process of stress relaxation and slippage (creep) of the polysaccharides that make up the load-bearing network of the wall. Protein mediators of wall creep have recently been isolated and characterized. These proteins, called expansins, appear to disrupt the noncovalent adhesion of matrix polysaccharides to cellulose microfibrils, thereby permitting turgor-driven wall enlargement. Expansin activity is specifically expressed in the growing tissues of dicotyledons and monocotyledons. Sequence analysis of cDNAs indicates that expansins are novel proteins, without previously known functional motifs. Comparison of expansin cDNAs from cucumber, pea, Arabidopsis and rice shows that the proteins are highly conserved in size and amino acid sequence. Phylogenetic analysis of expansin sequences suggests that this multigene family diverged before the evolution of angiosperms. Speculation is presented about the role of this gene family in plant development and evolution.     

不同脱落力下蜡梅花梗CpEXP1基因的表达与扩张蛋白活性

为了研究不同脱落力下蜡梅花梗中CpEXP1基因的表达水平以及扩张蛋白活性,采用real-timePcR技术检测不同脱落力下蜡梅花梗组织CpEXP1基因的表达水平;同时采用蛋白质体外重组法以及培养基添加法对花梗组织扩张蛋白活性进行测定。结果表明：CpEXP1基因的表达与扩张蛋白活性变化趋势相同,并在蜡梅花蕾脱落过程中具有阶段特异性。CpEXPI基因在自然脱落组的表达量显著高于其他组（P〈0．05）。此时扩张蛋白活性也最强。扩张蛋白对麻点百合愈伤组织的分化无明显影响,对增殖以及生根有明显促进作用。     

ABSCISSION OF LEAVES AND LEAFLETS IN ACER NEGUNDO AND FRAXINUS AMERICANA

A comparative study of leaf and leaflet abscission in Acer negundo and Fraxinus americana was undertaken with special emphasis on leaflet abscission. Leaf fall in both species is accomplished by orderly, fragmentary abscission of leaflets followed by petiole abscission. Leaflet fall was presaged by differentiation of a separation layer at leaflet bases 10–15 days prior to leaflet fall, without an accompanying protective layer. Anatomical studies of petiole abscission revealed early differentiation of a protective layer followed by differentiation of a separation layer at petiole bases just prior to petiolar fall. Abscission at both sites was facilitated by cell division and dissolution of cell walls within separation layers.     

Three different polygalacturonases are expressed in tomato leaf and flower abscission, each with a different temporal expression pattern.

Abscission, or organ separation, is accompanied by a marked increase in hydrolases, which are responsible for the degradation of the middle lamella and the loosening of the primary cell wall surrounding cells in the separation layer. We recently reported on the cloning of a tomato (Lycopersicon esculentum) polygalacturonase (PG) cDNA, TAPG1, expressed during leaf and flower abscission. In addition to TAPG1, we have cloned two more PG cDNAs (TAPG2 and TAPG4) that are also expressed during leaf and flower abscission. The peptide sequences for the three abscission PGs are relatively similar (76-93% identity) yet different from the those of tomato fruit PG (38-41% identity). None of the three abscission PG mRNAs are expressed in fruit, stems, petioles, or anthers of fully open flowers. An RNase protection assay revealed that all three PGs are expressed in leaf and flower abscission zones and in pistils of fully open flowers. TAPG4 mRNA is detected much earlier than TAPG1 and TAPG2 mRNA during both leaf and flower abscission.     

Expression of six expansin genes in relation to extension activity in developing strawberry fruit.

Expansins are proteins which have been demonstrated to induce cell wall extension in vitro. The identification and characterization of six expansin cDNAs from strawberry fruit, termed FaExp3 to FaExp7, as well as the previously identified FaExp2 is reported here. Analysis of expansin mRNAs during fruit development and in leaves, roots and stolons revealed a unique pattern of expression for each cDNA. FaExp3 mRNA was present at much lower levels than the other expansin mRNAs and was expressed in small green fruit and in ripe fruit. FaExp4 mRNA was present throughout fruit development, but was more strongly expressed during ripening. FaExp5 was the only clone to show fruit specific expression which was up-regulated at the onset of ripening. FaExp6 and FaExp7 mRNAs were present at low levels in the fruit with highest expression in stolon tissue. During fruit development FaExp6 had the highest expression at the white, turning and orange stages whereas expression of FaExp7 was highest in white fruit. The expression profiles of FaExp2 and FaExp5 in developing fruit were similar except that FaExp2 was induced at an earlier stage. Analysis of expansin protein by Western blotting using an antibody raised against CsExp1 from cucumber hypocotyls identified two bands of 29 and 31 kDa from developing fruit. Protein extracts from developing fruit were assayed for extension activity. Considerable rates of extension were observed with extracts from ripening fruit, but no extension was observed with protein from unripe green fruit. These results demonstrate the presence of at least six expansin genes in strawberry fruit and that during ripening the fruit acquires the ability to cause extension in vitro, characteristic of expansin action.     

The Existence of Expansin and Its Properties in the Hypocotyls of Soybean Seedlings

Expansins, a newly discovered class of cell wall proteins, were the only proteins that, to date, have been shown to have the ability to restore the "acid growth" response of the heat-inactivated cell wall in an in vitro assay. In order to characterize these proteins, an automatic extensometer had been previously constructed by modification of an equal-arm mechanical balance with a linear variable differential transformer (LVDT) and with some easily available laboratory equipment. The objective of this study was to confirm and complement the work on expansin in cucumber ( Cucumis sativus L. ) seedlings carried out in the expansin-discoverers' laboratory and in addition, to further examination of the extensometer built in the authors' laboratory. It was reported that, firstly, expansin activity was maximal in cell wall from the growing region of soybean (Glycine max L. ) hypocotyls but was negligible or lacking in that from mature, basal regions and cotyledons. Corre- spondingly, walls from the growing tissue had a strong susceptibility to the action of expansin, whereas the nongrowing tissues became insensitive to the expansin action. It was concluded that the growth of soybean hypocotyl was associated with an increase in both expansin activity and wall susceptibility to the expansin action. Secondly, the heat-inactivated wall extension could be induced by cross reconstitution with crude expansin extract between soybean and cucumber species. Thirdly, once the heat-inactivated wall has been pretreated with the exogenous expansin, the reconstituted wall required no further expansin for extension indicating that exogenous expansin could specifically bind to cell wall and be enough to repeatedly exert its action without releasing from the cell wall into the external solution, i.e., a single expansin molecule could gradually break a series of load-bearing bonds one by one while moving along the cell wall, and thereby permitting the wall to extend. Fourthly, reconstitution of the wall extension activity was evidently dependent on the expansin concentration and the pH of the bathing solution, which was consistent with the catalytic characteristics of classical enzymes. Finally, endogenous and reconstituted wall extension could be significantly induced in 50 mmoL/L sodium acetate at pH 4.5 and completely inhibited in 50 mmol/L Hepes at pH 6.8, especially these phenomena could continuously be caused by switching incubation buffer from one to the other alternately, suggesting that change in pH of bathing solution could only affect the conformation of expansin (thus leading to denaturation or renaturation of it) but not the affinity of it for cell wall. In summary, these observations lend further support to the fact that expansin could mediate the acid-induced extension of the isolated wall, probably through a biochemical or enzymatic process exerting directly to the cell wall. This protein may play an essential role in the control of plant cell growth in vivo.     

水稻扩展蛋白家族的生物信息学分析

扩展蛋白是植物细胞壁的重要组成部分,具有松驰细胞壁和增加细胞壁柔韧性的作用,在植物的生长发育及抗性等方面起到重要的作用。水稻的全基因组序列统计分析显示,水稻扩展蛋白基因家族包含58个成员,分属于A(34)、B(19)、LA(4)和LB(1)4个亚家族,分布在水稻10条染色体上的58个位点,且同一亚家族成员有成簇存在的现象。扩展蛋白基因长度范围为687~1128 bp,编码蛋白质具有保守的结构域,以及保守的半胱氨酸和色氨酸残基。多数情况下,亚家族成员之间的氨基酸一致率小于35%,而同一亚家族成员之间的氨基酸一致率大于35%。在内含子、外显子组成模式上,水稻扩展蛋白呈现明显的亚家族特异性,除个别基因以外,A类基因含有1或2个内含子,B类含有3个内含子,LA和LB类含有4个内含子。密码子使用统计显示,与其他物种相比,水稻中的扩展蛋白具有更多的密码子使用偏好性,有26个高频密码子存在。研究结果展示了水稻扩展蛋白基因家族的基本信息,为深入研究扩展蛋白基因的功能、探讨物种间的进化关系奠定基础。     

Differential expression of expansin gene family members during growth and ripening of tomato fruit

cDNA clones encoding homologues of expansins, a class of cell wall proteins involved in cell wall modification, were isolated from various stages of growing and ripening fruit of tomato (Lycopersicon esculentum). cDNAs derived from five unique expansin genes were obtained, termed tomato Exp3 to Exp7, in addition to the previously described ripening-specific tomato Exp1 (Rose et al. (1997) Proc Natl Acad Sci USA 94: 5955–5960). Deduced amino acid sequences of tomato Exp1, Exp4 and Exp6 were highly related, whereas Exp3, Exp5 and Exp7 were more divergent. Each of the five expansin genes showed a different and characteristic pattern of mRNA expression. mRNA of Exp3 was present throughout fruit growth and ripening, with highest accumulation in green expanding and maturing fruit, and lower, declining levels during ripening. Exp4 mRNA was present only in green expanding fruit, whereas Exp5 mRNA was present in expanding fruit but had highest levels in full-size maturing green fruit and declined during the early stages of ripening. mRNAs from each of these genes were also detected in leaves, stems and flowers but not in roots. Exp6 and Exp7 mRNAs were present at much lower levels than mRNAs of the other expansin genes, and were detected only in expanding or mature green fruit. The results indicate the presence of a large and complex expansin gene family in tomato, and suggest that while the expression of several expansin genes may contribute to green fruit development, only Exp1 mRNA is present at high levels during fruit ripening.     

调控拟南芥花瓣脱落的果胶甲酯酶基因表达和功能分析

在基因序列和表达信息分析的基础上,重点研究初选出的两个拟南芥果胶甲酯酶基因At8g47550和At4g02330的表达和功能。半定量RT-PCR和转基因植株检测表明,At2g47550主要在花粉粒中大量表达,其次在微管组织中有明显表达。然而,At4g02330在拟南芥花和荚果整个发育时期都有不同程度的表达。主要在花的离层组织、柱头、微管组织和花粉粒中表达。结果说明,At2g47550可能参与花粉粒和花粉管的发育,而At4g02330则可能通过参与细胞壁中果胶代谢,从而达到调控细胞分离和花瓣脱落的目的。     

Further examination of abscission zone cells as ethylene target cells in higher plants

BACKGROUND AND AIMS: Two aspects of the competence of abscission zone cells as a specific class of hormone target cell are examined. The first is the competence of these target cells to respond to a remote stele-generated signal, and whether ethylene acts in concert with this signal to initiate abscission of the primary leaf in Phaseolus vulgaris. The second is to extend the concept of dual control of abscission cell competence. Can the concept of developmental memory that is retained by abscission cell of Phaseolus vulgaris post-separation in terms of the inductive/repressive control of beta-1,4-glucan endohydrolase (cellulase) activity exerted by ethylene/auxin be extended to the rachis abscission zone cells of Sambucus nigra? METHODS: Abscission assays were performed using the leaf petiole-pulvinus explants of P. vulgaris with the distal pulvinus stele removed. These (-stele) explants do not separate when treated with ethylene and require a stele-generated signal from the distal pulvinus for separation at the leaf petiole-pulvinis abscission zone. Using these explants, the role of ethylene was examined, using the ethylene action blocker, 1-methyl cyclopropene, as well as the significance of the tissue from which the stele signal originates. Further, leaf rachis abscission explants were excised from the compound leaves of S. nigra, and changes in the activity of cellulase in response to added ethylene and auxin post-separation was examined. KEY RESULTS: The use of (-stele) explants has confirmed that ethylene, with the stele-generated signal, is essential for abscission. Neither ethylene alone nor the stelar signal alone is sufficient. Further, in addition to the leaf pulvinus distal to the abscission zone, mid-rib tissue that is excised from senescent or green mid-rib tissue can also generate a competent stelar signal. Experiments with rachis abscission explants of S. nigra have shown that auxin, when added to cells post-separation can retard cellulase activity, with activity re-established with subsequent ethylene treatment. CONCLUSIONS: The triggers that initiate and regulate the separation process are complex with, in bean leaves at least, the generation of a signal (or signals) from remote tissues, in concert with ethylene, a requisite part of the process. Once evoked, abscission cells maintain a developmental memory such that the induction/repression mediated by ethylene/auxin that is observed prior to separation is also retained by the cells post-separation.