Expansins in Plant Growth and Development: an Update on an Emerging Topic

Abstract: Expansins are a class of proteins identified by their ability to induce the extension of isolated plant cell walls. Expansins are encoded by an extensive multigene family in higher plants, several members of which have been shown to be expressed in a tissue-specific manner. Besides playing an apparently key role in wall expansion, and hence in cell growth, expansins have been implicated in an increasing number of processes during plant growth and development. These include: leaf organogenesis, fruit softening, and wall disassembly. A second class of closely related proteins (referred to as β-expansins) has been identified. Other recent advances in expansin research include the recovery of transgenic plants with altered level of expansins, and the production of recombinant expansins in het-erologous expression systems.     

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.     

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.

     

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.     

Expansins in the bryophyte Physcomitrella patens

Expansins are cell wall proteins which play a key function in basic processes of plant growth and differentiation. It has been proposed that expansins are likely to be present in all land plants and, to date, they have been reported in angiosperms, gymnosperms and pteridophytes. In this paper, we provide the first report and analysis of genes encoding expansin-like proteins in the bryophyte, Physcomitrella patens. Our analysis indicates that both - and -expansins are present as gene families in this plant and expression analysis indicates that these genes are subject to a complex regulation by both hormonal and environmental factors. In particular, the expression of many expansin genes in P. patens is upregulated by stress conditions, suggesting that they play a role in the specific cellular differentiation displayed by P. patens in response to such stress. Finally, we provide the first report on the generation and analysis of a series of knockout mutants for individual expansin genes. Abbreviations: IAA, indole-acetic acid; BAP, 6-benzylaminopurine; ABA, abscisic acid; npt, neomycin phospotransferase; KO, knockout     

The growing world of expansins

Expansins are cell wall proteins that induce pH-dependent wall extension and stress relaxation in a characteristic and unique manner. Two families of expansins are known, named alpha- and beta-expansins, and they comprise large multigene families whose members show diverse organ-, tissue- and cell-specific expression patterns. Other genes that bear distant sequence similarity to expansins are also represented in the sequence databases, but their biological and biochemical functions have not yet been uncovered. Expansin appears to weaken glucan-glucan binding, but its detailed mechanism of action is not well established. The biological roles of expansins are diverse, but can be related to the action of expansins to loosen cell walls, for example during cell enlargement, fruit softening, pollen tube and root hair growth, and abscission. Expansin-like proteins have also been identified in bacteria and fungi, where they may aid microbial invasion of the plant body.     

Plant expansins are a complex multigene family with an ancient evolutionary origin

Expansins are a group of extracellular proteins that directly modify the mechanical properties of plant cell walls, leading to turgor-driven cell extension. Within the completely sequenced Arabidopsis genome, we identified 38 expansin sequences that fall into three discrete subfamilies. Based on phylogenetic analysis and shared intron patterns, we propose a new, systematic nomenclature of Arabidopsis expansins. Further phylogenetic analysis, including expansin sequences found here in monocots, pine (Pinus radiata, Pinus taeda), fern (Regnellidium diphyllum, Marsilea quadrifolia), and moss (Physcomitrella patens) indicate that the three plant expansin subfamilies arose and began diversifying very early in, if not before, colonization of land by plants. Closely related "expansin-like" sequences were also identified in the social amoeba, Dictyostelium discoidium, suggesting that these wall-modifying proteins have a very deep evolutionary origin.     

Isolation and characterization of 18 genes encoding α- and β-expansins in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Expansins are thought to be key regulators of cell wall extension during plant growth. In this study, we isolated 18 expansin genes from wheat, nine of which encode α-expansins while the other nine code for β-expansins. The cysteine-rich and tryptophan-rich regions of the deduced amino acid sequences of all 18 expansins were highly conserved. Genomic sequences were obtained for 17 of the genes, and their intron patterns were determined. Four (A, C, D, E) of the six intron positions known in expansin genes from other species were found to be occupied in these wheat expansin genes. Five wheat expansin genes were mapped to chromosomes 1L, 2L, 5L and 6L respectively, by in silico and comparative mapping. The 18 wheat expansin genes were expressed in leaf, root and the developing seed. Moreover, it was demonstrated that four β-expansin genes were up-regulated in the internode tissue in F₁ hybrids, suggesting that changes in the regulation of these genes in hybrid might contribute to the heterosis observed in internode length and plant height. We therefore conclude that expansins are encoded by a multigene family in wheat, and could play important roles in growth and development. Z. Lin and Z. Ni contributed to this work equally     

Expansin(细胞壁松弛蛋白)的发展

Expansin是一种体外诱导分离的植物细胞壁伸展的蛋白,在修饰细胞壁基础上使细胞膨胀。Expansin的功能众多,除了促进细胞生长,还包括影响营养生长、形态发生、授粉受精、果实软化等,并表现出高度的组织、器官和细胞特异性。目前已经在多种植物及其他一些生物范围内对expansin及类expansin序列和蛋白质进行了研究,并对它们的作用机制进行了探索。     

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.     

Characterization of soybean β-expansin genes and their expression responses to symbiosis,nutrient deficiency,and hormone treatment

Expansins are plant cell wall-loosening proteins encoded by a superfamily of genes including α-expansin, β-expansin, expansin-like A, and expansin-like B proteins. They play a variety of biological roles during plant growth and development. Expansin genes have been reported in many plant species, and results primarily from graminaceous members indicate that β-expansins are more abundant in monocots than in dicots. Soybean [Glycine max (L.) Merr] is an important legume crop. This work identified nine β-expansin gene family members in soybean (GmEXPBs) that were divided into two distinct classes based on phylogeny and gene structure, with divergence between the two groups occurring more in introns than in exons. A total of 887 hormone-responsive and environmental stress-related putative cis-elements from 188 families were found in the 2-kb upstream region of GmEXPBs. Variations in number and type of cis-elements associated with each gene indicate that the function of these genes is differentially regulated by these signals. Expression analysis confirmed that the family members were ubiquitously, yet differentially expressed in soybean. Responsiveness to nutrient deficiency stresses and regulation by auxin (indole-3-acetic acid) and cytokinin (6-benzylaminopurine) varied among GmEXPBs. In addition, most β-expansin genes were associated with symbiosis of soybean inoculated with Rhizobium or abuscular mycorrhizal fungi (AMF). Taken together, these results systematically investigate the characteristics of the entire GmEXPB family in soybean and comprise the first report analyzing the relationship of GmEXPBs with rhizobial or AMF symbiosis. This information is a valuable step in the process of understanding the expansin protein functions in soybean and opens avenues for continued researches.     

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.     

小麦胚芽鞘扩展蛋白特性及对水分胁迫的响应

扩展蛋白是植物细胞壁延伸过程中的关键调节因子,在植物的生长发育以及对逆境的响应过程中起着重要作用。本文选用小麦(HF 9703)胚芽鞘为材料,采用Hepes法和SDS法分别提取小麦胚芽鞘扩展蛋白,通过改良的植物组织伸长测定仪测定其活性,并利用扩展蛋白抗体进行免疫印迹以检测其丰度,主要研究了小麦胚芽鞘扩展蛋白的特性及对水分胁迫的响应。结果表明:Hepes法提取的扩展蛋白活性较高,而SDS法的提取效率高;离体小麦胚芽鞘扩展蛋白的活性具有pH依赖性,且随缓冲液的交替更换(pH 4.5:pH 6.8)而反复逆转;扩展蛋白主要定位于细胞壁中;小麦胚芽鞘扩展蛋白和黄瓜下胚轴扩展蛋白具有交叉重组活性,但这种活性具有种属特异性。水分胁迫诱导小麦胚芽鞘扩展蛋白的活性和丰度提高,扩展蛋白活性的提高在小麦对水分胁迫的抗性方面可能具有重要作用。     

An expansin gene expressed in ripening strawberry fruit

Tissue softening accompanies the ripening of many fruit and initiates the processes of irreversible deterioration. Expansins are plant cell wall proteins proposed to disrupt hydrogen bonds within the cell wall polymer matrix. Expression of specific expansin genes has been observed in tomato (Lycopersicon esculentum) meristems, expanding tissues, and ripening fruit. It has been proposed that a tomato ripening-regulated expansin might contribute to cell wall polymer disassembly and fruit softening by increasing the accessibility of specific cell wall polymers to hydrolase action. To assess whether ripening-regulated expansins are present in all ripening fruit, we examined expansin gene expression in strawberry (Fragaria x ananassa Duch.). Strawberry differs significantly from tomato in that the fruit is derived from receptacle rather than ovary tissue and strawberry is non-climacteric. A full-length cDNA encoding a ripening-regulated expansin, FaExp2, was isolated from strawberry fruit. The deduced amino acid sequence of FaExp2 is most closely related to an expansin expressed in early tomato development and to expansins expressed in apricot fruit rather than the previously identified tomato ripening-regulated expansin, LeExp1. Nearly all previously identified ripening-regulated genes in strawberry are negatively regulated by auxin. Surprisingly, FaExp2 expression was largely unaffected by auxin. Overall, our results suggest that expansins are a common component of ripening and that non-climacteric signals other than auxin may coordinate the onset of ripening in strawberry.     

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.     

Transgenic Tobacco Plants Overexpressing a Grass PpEXP1 Gene Exhibit Enhanced Tolerance to Heat Stress

Heat stress is a detrimental abiotic stress limiting the growth of many plant species and is associated with various cellular and physiological damages. Expansins are a family of proteins which are known to play roles in regulating cell wall elongation and expansion, as well as other growth and developmental processes. The in vitro roles of expansins regulating plant heat tolerance are not well understood. The objectives of this study were to isolate and clone an expansin gene in a perennial grass species (Poa pratensis) and to determine whether over-expression of expansin may improve plant heat tolerance. Tobacco (Nicotiana tabacum) was used as the model plant for gene transformation and an expansin gene PpEXP1 from Poa pratensis was cloned. Sequence analysis showed PpEXP1 belonged to α-expansins and was closely related to two expansin genes in other perennial grass species (Festuca pratensis and Agrostis stolonifera) as well as Triticum aestivum, Oryza sativa, and Brachypodium distachyon. Transgenic tobacco plants over-expressing PpEXP1 were generated through Agrobacterium-mediated transformation. Under heat stress (42°C) in growth chambers, transgenic tobacco plants over-expressing the PpEXP1 gene exhibited a less structural damage to cells, lower electrolyte leakage, lower levels of membrane lipid peroxidation, and lower content of hydrogen peroxide, as well as higher chlorophyll content, net photosynthetic rate, relative water content, activity of antioxidant enzyme, and seed germination rates, compared to the wild-type plants. These results demonstrated the positive roles of PpEXP1 in enhancing plant tolerance to heat stress and the possibility of using expansins for genetic modification of cool-season perennial grasses in the development of heat-tolerant germplasm and cultivars.     

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。     

Cerato-platanin shows expansin-like activity on cellulosic materials

Cerato-platanin (CP) is a non-catalytic protein with a double ψβ-barrel fold located in the cell wall of the phytopathogenic fungus Ceratocystis platani. CP is released during growth and induces defence-related responses in plants. CP is also the first member of the “cerato-platanin family” (CPF) (Pfam PF07249). In the CPF, the molecular mechanism of action on plants and above all the biological role in fungal life are little-known aspects. However, an expansin-like function has recently been suggested concerning CP. Expansin-like proteins have the ability to act non-hydrolytically on cellulose. In the present work, the expansin-like activity of CP and Pop1, a CP family member, was investigated. Like expansins, CP and Pop1 were able to weaken filter paper in a concentration-dependent manner and without the production of reducing sugars. A metal-dependent polysaccharide monooxygenase-like activity was excluded. The optimum of activity was pH?5.0, 38 °C. CP was also able to cause fragmentation of the crystalline cellulose Avicel and the breakage and defibration of cotton fibres. However, the interaction did not involve a stable bond with the substrates and CP did not significantly enhance the hydrolytic activity of cellulase. On the other hand, CP and Pop1 bound quickly to chitin. We consider CP as a novel one-domain expansin-like protein. We propose a structural role for CP in the fungal cell wall due to the ability to bind chitin, and hypothesize a functional role in the interaction of the fungus with the plant for the weakening activity shown on cellulose.