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.     

Expanisns

Biochemical dissection of the “acid-growth” process of plant cell walls led to the isolation of a new class of wall loosening proteins, called expansins. These proteins affect the rheology of growing walls by permitting the microfibril-matrix network to slide, thereby enabling the wall to expand. Molecular sequence analysis suggests that expansins might have a cryptic glycosyl transferase activity, but biochemical results suggest that expansins disrupt noncovalent bonding between microfibrils and the matrix. Recent discoveries of a new expansin family and gene expression in fruit, meristerms and cotton fibers have enlarged our view of the developmental functions of this group of wall loosening proteins.     

Evolutionary divergence of β–expansin structure and function in grasses parallels emergence of distinctive primary cell wall traits

Expansins are wall‐loosening proteins that promote the extension of primary cell walls without the hydrolysis of major structural components. Previously, proteins from the EXPA (α–expansin) family were found to loosen eudicot cell walls but to be less effective on grass cell walls, whereas the reverse pattern was found for EXPB (β–expansin) proteins obtained from grass pollen. To understand the evolutionary and structural bases for the selectivity of EXPB action, we assessed the extension (creep) response of cell walls from diverse monocot families to EXPA and EXPB treatments. Cell walls from Cyperaceae and Juncaceae (families closely related to grasses) displayed a typical grass response (‘β–response’). Walls from more distant monocots, including some species that share with grasses high levels of arabinoxylan, responded preferentially to α–expansins (‘α–response’), behaving in this regard like eudicots. An expansin with selective activity for grass cell walls was detected in Cyperaceae pollen, coinciding with the expression of genes from the divergent EXPB–I branch that includes grass pollen β–expansins. The evolutionary origin of this branch was located within Poales on the basis of phylogenetic analyses and its association with the ‘sigma’ whole‐genome duplication. Accelerated evolution in this branch has remodeled the protein surface in contact with the substrate, potentially for binding highly substituted arabinoxylan. We propose that the evolution of the divergent EXPB–I group made a fundamental change in the target and mechanism of wall loosening in the grass lineage possible, involving a new structural role for xylans and the expansins that target them.     

Expansins

Biochemical dissection of the "acid-growth" process of plant cell walls led to the isolation of a new class of wall loosening proteins, called expansins. These proteins affect the rheology of growing walls by permitting the microfibril matrix network to slide, thereby enabling the wall to expand. Molecular sequence analysis suggests that expansins might have a cryptic glycosyl transferase activity, but biochemical results suggest that expansins disrupt noncovalent bonding between microfibrils and the matrix. Recent discoveries of a new expansin family and gene expression in fruit meristems and cotton fibers have enlarged our view of the developmental functions of this group of wall loosening proteins.     

一种植物细胞壁松驰蛋白:膨胀素

在植物细胞的生长过程中 ,多糖和蛋白质分泌到细胞壁里层 ,并形成具有一定机械强度的网络 ,这个网络是能伸展的 ,除非细胞停止生长。在细胞的生长过程中 ,一种细胞壁蛋白—膨胀素首次被鉴定出来具有使细胞壁的多糖网络疏松的能力 ,从而使膨压驱动的细胞扩大。膨胀素由两个多基因家族即α -膨胀素和 β -膨胀素多基因家族编码 ,每种基因的表达具有部位和细胞类型的特异性 ,但最新的研究也表明拟南芥中的膨胀素可以分为三个亚家族。越来越多的膨胀素基因从各种植物中鉴定出来 ,系统分析显示它们可能从一个共同的祖先基因进化而来。膨胀素的作用机理研究的还不是很清楚 ,但因为它们具有特别的功能 ,因此展现出良好的工业化应用前景。     

Expansin gene loss is a common occurrence during adaptation to an aquatic environment

Expansins comprise a superfamily of plant cell wall loosening proteins that can be divided into four individual families (EXPA, EXPB, EXLA and EXLB). Aside from inferred roles in a variety of plant growth and developmental traits, little is known regarding the function of specific expansin clades, for which there are at least 16 in flowering plants (angiosperms); however, there is evidence to suggest that some expansins have cell‐specific functions, in root hair and pollen tube development, for example. Recently, two duckweed genomes have been sequenced (Spirodela polyrhiza strains 7498 and 9509), revealing significantly reduced superfamily sizes. We hypothesized that there would be a correlation between expansin loss and morphological reductions seen among highly adapted aquatic species. In order to provide an answer to this question, we characterized the expansin superfamilies of the greater duckweed Spirodela, the marine eelgrass Zostera marina and the bladderwort Utricularia gibba. We discovered rampant expansin gene and clade loss among the three, including a complete absence of the EXLB family and EXPA‐VII. The most convincing correlation between morphological reduction and expansin loss was seen for Utricularia and Spirodela, which both lack root hairs and the root hair expansin clade EXPA‐X. Contrary to the pattern observed in other species, four Utricularia expansins failed to branch within any clade, suggesting that they may be the result of neofunctionalization. Last, an expansin clade previously discovered only in eudicots was identified in Spirodela, allowing us to conclude that the last common ancestor of monocots and eudicots contained a minimum of 17 expansins.     

Genomic location and expression analysis of expansin gene family reveals the evolutionary and functional significance in <Emphasis Type="Italic">Triticum aestivum</Emphasis>

The plant-specific expansin proteins constitute an ancient and major gene family known to have roles in regulating diverse biological processes in plants. Although the functions of many expansin genes have been identified in wheat and other species, little is known about the evolution and genomic locations of the expansin genes in wheat (Triticum aestivum). In this study, a total of 87 expansin genes were identified in the wheat genome, including 52 EXPAs, 42 EXPBs and 4 EXLAs. The EXLB gene was not found in the wheat genome. Phylogenetic tree and comparative analysis revealed amplification of the EXPBs in rice, maize and wheat. The predicted wheat expansins were distributed across 14 of 21 chromosomes with different densities, 3 tightly co-located clusters and 15 paralogous pairs, indicating that tandem duplication and segmental duplication events also played roles in the evolution of expansins in wheat. In addition, the gene structures and conserved protein domains of wheat expansins suggest high levels of conservation within the phylogenetic subgroups. Analysis of a published microarray database showed that most wheat expansin genes exhibit different expression levels in different tissues and developmental stages. To our knowledge, this is the first report of a genome-wide analysis of the wheat expansin gene family, which should provide valuable information for further elucidating the classification and putative functions of the entire gene family.     

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.     

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.     

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.     

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.     

Use of genomic history to improve phylogeny and understanding of births and deaths in a gene family

Polyploidy events have played an important role in the evolution of angiosperm genomes. Here, we demonstrate how genomic histories can increase phylogenetic resolution in a gene family, specifically the expansin superfamily of cell wall proteins. There are 36 expansins in Arabidopsis and 58 in rice. Traditional sequence-based phylogenetic trees yield poor resolution below the family level. To improve upon these analyses, we searched for gene colinearity (microsynteny) between Arabidopsis and rice genomic segments containing expansin genes. Multiple rounds of genome duplication and extensive gene loss have obscured synteny. However, by simultaneously aligning groups of up to 10 potentially orthologous segments from the two species, we traced the history of 49 out of 63 expansin-containing segments back to the ancestor of monocots and eudicots. Our results indicate that this ancestor had 15-17 expansin genes, each ancestral to an extant clade. Some clades have strikingly different growth patterns in the rice and Arabidopsis lineages, with more than half of all rice expansins arising from two ancestral genes. Segmental duplications, most of them part of polyploidy events, account for 12 out of 21 new expansin genes in Arabidopsis and 16 out of 44 in rice. Tandem duplications explain most of the rest. We were also able to estimate a minimum of 28 gene deaths in the Arabidopsis lineage and nine in rice. This analysis greatly clarifies expansin evolution since the last common ancestor of monocots and eudicots and the method should be broadly applicable to many other gene families.     

Expansins in deepwater rice internodes.

Cell walls of deepwater rice (Oryza sativa L.) internodes undergo long-term extension (creep) when placed under tension in acidic buffers. This is indicative of the action of the cell wall-loosening protein expansin. Wall extension had a pH optimum of around 4.0 and was abolished by boiling. Acid-induced extension of boiled cell walls could be reconstituted by addition of salt-extracted rice or cucumber cell wall proteins. Cucumber expansin antibody recognized a single protein band of 24.5-kD apparent molecular mass on immunoblots of rice cell wall proteins. Expansins were partially purified by concanavalin A affinity chromatography and sulfopropyl (SP) cation-exchange chromatography. The latter yielded two peaks with extension activity (SP20 and SP29), and immunoblot analysis showed that both of these active fractions contained expansin of 24.5-kD molecular mass. The N-terminal amino acid sequence of SP20 expansin is identical to that deduced from the rice expansin cDNA Os-EXP1. The N-terminal amino acid sequence of SP29 expansin matches that deduced from the rice expansin cDNA Os-EXP2 in six of eight amino acids. Our results show that two expansins occur in the cell walls of rice internodes and that they may mediate acid-induced wall extension.     

Expansins: roles in plant growth and potential applications in crop improvement

Key message

Results from various expansin related studies have demonstrated that expansins present an opportunity to improve various crops in many different aspects ranging from yield and fruit ripening to improved stress tolerance.

Abstract

The recent advances in expansin studies were reviewed. Besides producing the strength that is needed by the plants, cell walls define cell shape, cell size and cell function. Expansins are cell wall proteins which consist of four sub families; α-expansin, β-expansin, expansin-like A and expansin-like B. These proteins mediate cell wall loosening and they are present in all plants and in some microbial organisms and other organisms like snails. Decades after their initial discovery in cucumber, it is now clear that these small proteins have diverse biological roles in plants. Through their ability to enable the local sliding of wall polymers by reducing adhesion between adjacent wall polysaccharides and the part they play in cell wall remodeling after cytokinesis, it is now clear that expansins are required in almost all plant physiological development aspects from germination to fruiting. This is shown by the various reports from different studies using various molecular biology approaches such as gene achieve these many roles through their non-enzymatic wall loosening ability. This paper reviews and summarizes some of the reported functions of expansins and outlines the potential uses of expansins in crop improvement programs.

     

Expansins in growing tomato leaves

An expansin-like protein from growing tomato leaves was identified by its ability to restore the 'acid-growth' response to heat-inactivated tomato walls and by its similarity to expansins from cucumber hypocotyls. Native walls from growing tomato leaves exhibit an endogenous acid-induced extension (creep) that resembles in various biochemical characteristics the acid-growth activity of cucumber hypocotyls. For example, the acid-growth activity is lost when the walls of tomato leaves are briefly heated and is largely restored by addition of a crude protein extract from the walls of growing leaves. Wall proteins from growing leaves enhance the stress relaxation spectrum of tomato walls in a fashion characteristic of cucumber expansins. HPLC fractionation of the crude wall protein from tomato leaves yielded an active fraction containing a major 27 kDa protein that cross-reacts with an antibody raised against cucumber expansin. The results show that tomato leafwalls possess at least one expansin that is responsible for the acid-growth property of leaves and indicate that cell wall extension in leaves shares an underlying protein mechanism common to cell wall expansion in stems.     

Detection of expansin proteins and activity during tomato fruit ontogeny

Expansins are plant proteins that have the capacity to induce extension in isolated cell walls and are thought to mediate pH-dependent cell expansion. J.K.C. Rose, H.H. Lee, and A.B. Bennett ([1997] Proc Natl Acad Sci USA 94: 5955-5960) reported the identification of an expansin gene (LeExp1) that is specifically expressed in ripening tomato (Lycopersicon esculentum) fruit where cell wall disassembly, but not cell expansion, is prominent. Expansin expression during fruit ontogeny was examined using antibodies raised to recombinant LeExp1 or a cell elongation-related expansin from cucumber (CsExp1). The LeExp1 antiserum detected expansins in extracts from ripe, but not preripe tomato fruit, in agreement with the pattern of LeExp1 mRNA accumulation. In contrast, antibodies to CsExp1 cross-reacted with expansins in early fruit development and the onset of ripening, but not at a later ripening stage. These data suggest that ripening-related and expansion-related expansin proteins have distinct antigenic epitopes despite overall high sequence identity. Expansin proteins were detected in a range of fruit species and showed considerable variation in abundance; however, appreciable levels of expansin were not present in fruit of the rin or Nr tomato mutants that exhibit delayed and reduced softening. LeExp1 protein accumulation was ethylene-regulated and matched the previously described expression of mRNA, suggesting that expression is not regulated at the level of translation. We report the first detection of expansin activity in several stages of fruit development and while characteristic creep activity was detected in young and developing tomato fruit and in ripe pear, avocado, and pepper, creep activity in ripe tomato showed qualitative differences, suggesting both hydrolytic and expansin activities.     

Expansins and coleoptile elongation in wheat

Expansins are now generally accepted to be the key regulators of wall extension during plant growth. The aim of this study was to characterize expansins in wheat coleoptiles and determine their roles in regulating cell growth. Endogenous and reconstituted wall extension activities of wheat coleoptiles were measured. The identification of beta-expansins was confirmed on the basis of expansin activity, immunoblot analysis, and beta-expansin inhibition. Expansin activities of wheat coleoptiles were shown to be sensitive to pH and a number of exogenously applied factors, and their optimum pH range was found to be 4.0 to 4.5, close to that of alpha-expansins. They were induced by dithiothreitol, K(+), and Mg(2+), but inhibited by Zn(2+), Cu(2+), Al(3+), and Ca(2+), similar to those found in cucumber hypocotyls. An expansin antibody raised against TaEXPB23, a vegetative expansin of the beta-expansin family, greatly inhibited acid-induced extension of native wheat coleoptiles and only one protein band was recognized in Western blot experiments, suggesting that beta-expansins are the main members affecting cell wall extension of wheat coleoptiles. The growth of wheat coleoptiles was closely related to the activity and expression of expansins. In conclusion, our results suggest the presence of expansins in wheat coleoptiles, and it is possible that most of them are members of the beta-expansin family, but are not group 1 grass pollen allergens. The growth of wheat coleoptiles is intimately correlated with expansin expression, in particularly that of beta-expansins.     

A genome-wide analysis of the expansin genes in Malus × Domestica

Expansins were first identified as cell wall-loosening proteins; they are involved in regulating cell expansion, fruits softening and many other physiological processes. However, our knowledge about the expansin family members and their evolutionary relationships in fruit trees, such as apple, is limited. In this study, we identified 41 members of the expansin gene family in the genome of apple (Malus × Domestica L. Borkh). Phylogenetic analysis revealed that expansin genes in apple could be divided into four subfamilies according to their gene structures and protein motifs. By phylogenetic analysis of the expansins in five plants (Arabidopsis, rice, poplar, grape and apple), the expansins were divided into 17 subgroups. Our gene duplication analysis revealed that whole-genome and chromosomal-segment duplications contributed to the expansion of Mdexpansins. The microarray and expressed sequence tag (EST) data showed that 34 Mdexpansin genes could be divided into five groups by the EST analysis; they may also play different roles during fruit development. An expression model for MdEXPA16 and MdEXPA20 showed their potential role in developing fruit. Overall, our study provides useful data and novel insights into the functions and regulatory mechanisms of the expansin genes in apple, as well as their evolution and divergence. As the first step towards genome-wide analysis of the expansin genes in apple, our results have established a solid foundation for future studies on the function of the expansin genes in fruit development.