Inducible Repression of Multiple Expansin Genes Leads to Growth Suppression during Leaf Development

Expansins are cell wall proteins implicated in the control of plant growth via loosening of the extracellular matrix. They are encoded by a large gene family, and data linked to loss of single gene function to support a role of expansins in leaf growth remain limited. Here, we provide a quantitative growth analysis of transgenics containing an inducible artificial microRNA construct designed to down-regulate the expression of a number of expansin genes that an expression analysis indicated are expressed during the development of Arabidopsis (Arabidopsis thaliana) leaf 6. The results support the hypothesis that expansins are required for leaf growth and show that decreased expansin gene expression leads to a more marked repression of growth during the later stage of leaf development. In addition, a histological analysis of leaves in which expansin gene expression was suppressed indicates that, despite smaller leaves, mean cell size was increased. These data provide functional evidence for a role of expansins in leaf growth, indicate the importance of tissue/organ developmental context for the outcome of altered expansin gene expression, and highlight the separation of the outcome of expansin gene expression at the cellular and organ levels.     

Distribution of expansins in graviresponding maize roots

To test if expansins, wall loosening proteins that disrupt binding between microfibrils and cell wall matrix, participate in the differential elongation of graviresponding roots, Zea mays L. cv. Merit roots were gravistimulated and used for immunolocalization with anti-expansin. Western blots showed cross-reaction with two proteins of maize, one of the same mass as cucumber expansin (29 kDa), the second slightly larger (32 kDa). Maize roots contained mainly the larger protein, but both were found in coleoptiles. The expansin distribution in cucumber roots and hypocotyls was similar to the distribution in maize. Roots showed stronger expansin signals on the expanding convex side than the concave flank as early as 30 min after gravistimulation. Treatment with brefeldin A, a vesicle transport inhibitor, or the auxin transport inhibitor, naphthylphthalamic acid, showed delayed graviresponse and the appearance of differential staining. Our results indicate that expansins may be transported and secreted to cell walls via vesicles and function in wall expansion.     

Expression of expansin genes is correlated with growth in deepwater rice.

Expansins are a family of proteins that catalyze long-term extension of isolated cell walls. Previously, two expansin proteins have been isolated from internodes of deepwater rice, and three rice expansin genes, Os-EXP1, Os-EXP2, and Os-EXP3, have been identified. We report here on the identification of a fourth rice expansin gene, Os-EXP4, and on the expression pattern of the rice expansin gene family in deepwater rice. Rice expansin genes show organ-specific differential expression in the coleoptile, root, leaf, and internode. In these organs, there is increased expression of Os-EXP1, Os-EXP3, and Os-EXP4 in developmental regions where elongation occurs. This pattern of gene expression is also correlated with acid-induced in vitro cell wall extensibility. Submergence and treatment with gibberellin, both of which promote rapid internodal elongation, induced accumulation of Os-EXP4 mRNA before the rate of growth started to increase. Our results indicate that the expression of expansin genes in deepwater rice is differentially regulated by developmental, hormonal, and environmental signals and is correlated with cell elongation.     

扩展蛋白与植物抗逆性关系研究进展

扩展蛋白是一种细胞壁蛋白,可调节细胞壁的松弛和伸展。目前研究表明,扩展蛋白几乎参与调节植物生长发育的整个进程。扩展蛋白还与植物的多种抗性反应有关,在植物对干旱、高盐以及病虫害等生物胁迫和非生物胁迫响应方面起着重要的调节作用。干旱胁迫下扩展蛋白基因的表达与植物的抗旱性有一定的关系;植物的耐盐性受到扩展蛋白基因表达的影响;淹水促进植物的伸长生长与扩展蛋白的表达密切相关;扩展蛋白调节细胞壁松弛为植物抗病性研究提供了新的思路。     

PhEXPA1, a Petunia hybrida expansin,is involved in cell wall metabolism and in plant architecture specification

Expansins are wall-loosening proteins that induce wall stress relaxation and irreversible wall extension in a pH-dependent manner. Despite a substantial body of work has been performed on the characterization of many expansins genes in different plant species, the knowledge about their precise biological roles during plant development remains scarce. To yield insights into the expansion process in Petunia hybrida, PhEXPA1, an expansin gene preferentially expressed in petal limb, has been characterized. The constitutive overexpression of PhEXPA1 significantly increased expansin activity, cells size and organ dimensions. Moreover, 35S::PhEXPA1 transgenic plants exhibited an altered cell wall polymer composition and a precocious timing of axillary meristem development compared with wild-type plants. These findings supported a previous hypothesis that expansins are not merely structural proteins involved in plant cell wall metabolism but they also take part in many plant development processes. Here, to support this expansins dual role, we discuss about differential cell wall-related genes expressed in PhEXPA1 expression mutants and gradients of altered petunia branching pattern.     

Ectomycorrhizas of two species of <Emphasis Type="Italic">Tuber</Emphasis> (clade Puberulum) in the Mexican subtropical cloud forest

Expansins are non-enzymatic cell wall proteins that mediate plant growth by catalyzing loosening of cell walls without lysing the wall polymers. Advances in the field of bioinformatics have facilitated the prediction of the members of expansin gene family across several model plants. Expansins constitutes into four sub-families; α-expansin, β-expansin, expansin-like A and expansin-like B. Biological functions of expansin gene family include diverse aspects of plant growth and development, shoot and root elongation, leaf morphogenesis, flower and fruit development, embryogenesis, pollen tube growth, stress tolerance, etc. Recent studies have demonstrated the role of expansins in plant-symbiotic interactions. The present review reveals the factors that govern plant-arbuscular mycorrhizal fungi (AMF) and legume-rhizobia symbioses; and the genes that participate in these diverse symbiont interactions. Further, we focus on the expression profiles and the functions of expansins during plant-AMF and legume-rhizobia interactions. The key roles of expansin proteins during AMF invasion, arbuscule formation, rhizobial infection and nodule organogenesis were uncovered during symbioses. This review summarizes discoveries that support the key and versatile roles of various expansin members in the plant-mycorrhizal and legume-rhizobial symbioses.     

Expansin mode of action on cell walls. Analysis of wall hydrolysis, stress relaxation, and binding

The biochemical mechanisms underlying cell wall expansion in plants have long been a matter of conjecture. Previous work in our laboratory identified two proteins (named "expansins") that catalyze the acid-induced extension of isolated cucumber cell walls. Here we examine the mechanism of expansin action with three approaches. First, we report that expansins did not alter the molecular mass distribution or the viscosity of solutions of matrix polysaccharides. We conclude that expansins do not hydrolyze the major pectins or hemicelluloses of the cucumber wall. Second, we investigated the effects of expansins on stress relaxation of isolated walls. These studies show that expansins account for the pH-sensitive and heat-labile components of wall stress relaxation. In addition, these experiments show that expansins do not cause a progressive weakening of the walls, as might be expected from the action of a hydrolase. Third, we studied the binding of expansins to the cell wall and its components. The binding characteristics are consistent with this being the site of expansin action. We found that expansins bind weakly to crystalline cellulose but that this binding is greatly increased upon coating the cellulose with various hemicelluloses. Xyloglucan, either solubilized or as a coating on cellulose microfibrils, was not very effective as a binding substrate. Expansins were present in growing cell walls in low quantities (approximately 1 part in 5000 on a dry weight basis), suggesting that they function catalytically. We conclude that expansins bind at the interface between cellulose microfibrils and matrix polysaccharides in the wall and induce extension by reversibly disrupting noncovalent bonds within this polymeric network. Our results suggest that a minor structural component of the matrix, other than pectin and xyloglucan, plays an important role in expansin binding to the wall and, presumably, in expansin action.     

Changes in expansin activity and cell wall susceptibility to expansin action during cessation of internodal elongation in floating rice

We investigated the involvement of expansin action in determining the growth rate of internodes of floating rice (Oryza sativa L.). Floating rice stem segments in which rapid internodal elongation had been induced by submergence for 2 days were exposed to air or kept in submergence for 2 more days. Both treatments reduced the elongation rate of the internodes, and the degree of reduction was much greater in air-exposed stem segments than in continually submerged segments. These rates of internodal elongation were correlated with acid-induced cell wall extensibility and cell wall susceptibility to expansins in the cell elongation zone of the internodes, but not with extractable expansin activity. These results suggest that the reduced growth rate of internodes must be due, at least in part, to the decrease in acid-induced cell wall extensibility, which can be modulated through changes in the cell wall susceptibility to expansins rather than through expansin activity. Analysis of the cell wall composition of the internodes showed that the cellulosic and noncellulosic polysaccharide contents increased in response to exposure to air, but they remained almost constant under continued submergence although the cell wall susceptibility to expansins gradually declined even under continued submergence. The content of xylose in noncellulosic neutral sugars in the cell walls of internodes was closely and negatively correlated with changes in the susceptibility of the walls to expansins. These results suggest that the deposition of xylose-rich polysaccharides into the cell walls may be related to a decrease in acid-induced cell wall extensibility in floating rice internodes through the modulation of cell wall susceptibility to expansins.     

Impaired growth in transgenic plants over-expressing an expansin isoform

Expansins are cell wall proteins characterised by their ability to stimulate wall loosening during cell expansion. The expression of some expansin isoforms is clearly correlated with growth and the external application of expansins can stimulate cell expansion in vivo in several systems. We report here the expression of a heterologous expansin coding sequence in transgenic tomato plants (Lycopersicon esculentum Mill.) under the control of a constitutive promoter. In some transgenic lines with high levels of expansin activity extractable from cell walls, we observed alterations of growth: mature plants were stunted, with shorter leaves and internodes, and dark-grown seedlings had shorter and wider hypocotyls than their wild-type counterparts. Examination of hypocotyl sections revealed similar differences at the cellular level: cortical and epidermal cells were shorter and wider than those from wild-type seedlings. The observed stimulation of radial expansion did not compensate for the decreased elongation, and overall growth was reduced in the transgenics. As this observation can seem paradoxical given the known effect of expansins on isolated cell walls, we examined the mechanical behaviour of transgenic tissue. We measured a decrease in hypocotyl elongation in response to acidic pH in the transformants. This result may account for the alterations in cell expansion, and could itself be explained by a reduced susceptibility of transgenic cell walls to expansin action.     

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.     

Localized upregulation of a new expansin gene predicts the site of leaf formation in the tomato meristem.

Expansins are extracellular proteins that increase plant cell wall extensibility in vitro and are thought to be involved in cell expansion. We showed in a previous study that administration of an exogenous expansin protein can trigger the initiation of leaflike structures on the shoot apical meristem of tomato. Here, we studied the expression patterns of two tomato expansin genes, LeExp2 and LeExp18. LeExp2 is preferentially expressed in expanding tissues, whereas LeExp18 is expressed preferentially in tissues with meristematic activity. In situ hybridization experiments showed that LeExp18 expression is elevated in a group of cells, called I1, which is the site of incipient leaf primordium initiation. Thus, LeExp18 expression is a molecular marker for leaf initiation, predicting the site of primordium formation at a time before histological changes can be detected. We propose a model for the regulation of phyllotaxis that postulates a crucial role for expansin in leaf primordium initiation.     

Evidence for Variation in the Optimal Translation Initiation Complex: Plant eIF4B,eIF4F,and eIF(iso)4F Differentially Promote Translation of mRNAs

Expansins are cell wall proteins associated with the process of plant growth. However, investigations in which expansin gene expression has been manipulated throughout the plant have often led to inconclusive results. In this article, we report on a series of experiments in which overexpression of expansin was targeted to specific phases of leaf growth using an inducible promoter system. The data indicate that there is a restricted window of sensitivity when increased expansin gene expression leads to increased endogenous expansin activity and an increase in leaf growth. This phase of maximum expansin efficacy corresponds to the mid phase of leaf growth. We propose that the effectiveness of expansin action depends on the presence of other modulating factors in the leaf and we suggest that it is the control of expression of these factors (in conjunction with expansin gene expression) that defines the extent of leaf growth. These data help to explain some of the previously observed variation in growth response following manipulation of expansin gene expression and highlight a potential linkage of the expression of modifiers of expansin activity with the process of exit from cell division.Expansins were initially identified as cell wall proteins that had the ability to promote the extension of plant tissue in vitro (McQueen-Mason et al., 1992). Further work on these proteins and the genes encoding them has revealed a picture in which, although a general correlation with growth has often been substantiated, it is clear that control of growth is a much more complex process than the control of expression of a single protein type (for review, see Cosgrove, 2000; Lee et al., 2001; Li et al., 2003). In addition, although it is clear that expansins play a role in many growth processes, there are a number of open questions about exactly how expansins contribute to these processes. First, we still have a very limited understanding of the molecular mechanism of expansin action. Efforts to identify classical enzymatic activities associated with expansins have proven fruitless (McQueen-Mason and Cosgrove, 1995; Li and Cosgrove, 2001) and the remaining, somewhat speculative, interpretation is that expansins intercalate within carbohydrate matrices in the cell wall, leading to transient loosening of noncovalent interactions and, thus, the ability of these matrices to move relative to each other (McQueen-Mason and Cosgrove., 1994). In addition, by unlocking aspects of the molecular architecture of the cell wall, expansins may allow access of other cell wall proteins/enzymes to particular substrates. Depending on the nature of these other proteins/enzymes, expansin activity could thus be associated not only with growth processes, but also with cell wall modifications linked with differentiation. Such a mechanism would help to explain observations (described below) that the effectiveness of expansin action appears to be context dependent and is not only associated with changes in plant growth but also with differentiation.Various analyses have revealed that expansins are present in a wide range of plants, including bryophytes, ferns, angiosperms, and conifers (Hutchison et al., 1999; Kim et al., 2000; Schipper et al., 2002). Moreover, they are generally encoded by relatively large gene families whose members often show distinct patterns of gene expression (Kende et al., 2004). Some of these expression patterns correlate with growth processes, such as root growth (Wu et al., 1996), internode growth (Cho and Kende, 1997), leaf growth (Muller et al., 2007), and cotton (Gossypium hirsutum) fiber growth (Ruan et al., 2001), whereas others correlate with events of differentiation, such as fruit ripening (Rose et al., 1997; Brummell et al., 1999b), grass tiller formation (Reidy et al., 2001), and endosperm breakdown (Chen and Bradford, 2000). In addition, some novel nonplant expansin activities have been identified that suggest that pathogens may induce altered cell wall structure via an expansin-mediated mechanism (Qin et al., 2004). Since in vitro assays have suggested that the activities of expansins extracted from different sources tend to be similar (Cosgrove, 2000), it has been proposed that this tissue, organ, and environmental specificity of expression pattern reflects a specialized role for expansins in specific contexts rather than any major difference in activity of the protein. As stated above, this specific function may depend on the presence (or absence) of tissue-specific cofactors, the nature of which is as yet unclear.In addition to biochemical approaches to understanding expansin function, numerous groups have undertaken transgenic experiments to alter expansin gene expression in plants to observe the outcome on plant phenotype. Although some successes with antisense strategies have been reported (Brummell et al., 1999a; Cho and Cosgrove, 2000), the encoding of expansin by large gene families means that genetic redundancy poses a significant problem for such approaches (e.g. Schipper et al., 2002). Simple overexpression strategies to alter expansin activity may also be difficult to interpret. For example, when expansins were constitutively overexpressed throughout Arabidopsis (Arabidopsis thaliana), tomato (Solanum lycopersicum), and rice (Oryza sativa) plants, the outcomes tended to be pleiotropic, including a decrease in overall plant growth (Cho and Cosgrove, 2000; Rochange et al., 2001; Choi et al., 2003). However, when altered expansin expression was targeted more specifically to a particular tissue or organ, then more easily interpretable results were obtained. For example, when altered expansin expression was directed to the developing leaf petiole in Arabidopsis, altered leaf growth was observed (Cho and Cosgrove, 2000), consistent with the idea that expansins promote growth, and when inducible expression of expansin was targeted throughout rice plants, quantitative changes in growth were observed (Choi et al., 2003). The results of these experiments indicate that expansin gene expression can be used as a tool to modulate growth, but that the timing and spatial extent of expression can have a significant influence on the phenotype observed. Again, these data support the hypothesis that the effectiveness of expansin in promoting specific growth or differentiation events is dependent on the presence of particular tissue- or developmental-specific cofactors. So far, little progress has been made on the identification and characterization of these cofactors.In previous work, we reported on the characterization of transgenic lines of tobacco (Nicotiana tabacum) in which a cucumber (Cucumis sativus) expansin (CsEXP1) could be induced by application of a chemical inducer (anhydrotetracycline [Ahtet]). In these experiments, we targeted expansin overexpression to localized regions of either the shoot apical meristem or very young leaf primordia, which led to localized promotion of growth (Pien et al., 2001), consistent with the idea that expansins play a role in the endogenous mechanism of leaf initiation (Reinhardt et al., 1998). However, when inductions were performed throughout the plant the resulting phenotypes were variable and difficult to interpret (S. Pien and A. Fleming, unpublished data), in line with other reports (Rochange et al., 2001). To investigate the possibility that this variable response reflected a differential sensitivity to expansin in different tissues at different stages of development, we performed a series of experiments (reported here) in which overexpression of expansin was targeted to specific stages of leaf growth. Our data indicate that the efficacy of expansin action depends on the presence of other factors that are present in a developmentally controlled fashion, so that increased expansin gene expression is only effective in promoting leaf growth during a specific developmental period of leaf growth. This period corresponds to the inflection point of relative growth rate (RGR) and, thus, to the phase of maximum leaf growth rate. An intriguing article by Cookson et al. (2005) reported on potential correlations between various parameters of leaf growth and final leaf size. They found that the best predictor of final leaf size was the maximum value of absolute leaf growth rate. Thus, the experiments reported here identify a novel, developmental control of expansin efficacy in the regulation of leaf growth, investigate the reported correlation between maximal leaf expansion rate and leaf size, and provide an insight into potential means of controlling leaf growth.     

Growth maintenance of the maize primary root at low water potentials involves increases in cell-wall extension properties, expansin activity, and wall susceptibility to expansins

Previous work on the growth biophysics of maize (Zea mays L.) primary roots suggested that cell walls in the apical 5 mm of the elongation zone increased their yielding ability as an adaptive response to low turgor and water potential (psi w). To test this hypothesis more directly, we measured the acid-induced extension of isolated walls from roots grown at high (-0.03 MPa) or low (-1.6 MPa) psi w using an extensometer. Acid-induced extension was greatly increased in the apical 5 mm and was largely eliminated in the 5- to 10-mm region of roots grown at low psi w. This pattern is consistent with the maintenance of elongation toward the apex and the shortening of the elongation zone in these roots. Wall proteins extracted from the elongation zone possessed expansin activity, which increased substantially in roots grown at low psi w. Western blots likewise indicated higher expansin abundance in the roots at low psi w. Additionally, the susceptibility of walls to expansin action was higher in the apical 5 mm of roots at low psi w than in roots at high psi w. The basal region of the elongation zone (5-10 mm) did not extend in response to expansins, indicating that loss of susceptibility to expansins was associated with growth cessation in this region. Our results indicate that both the increase in expansin activity and the increase in cell-wall susceptibility to expansins play a role in enhancing cell-wall yielding and, therefore, in maintaining elongation in the apical region of maize primary roots at low psi w.