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.     

Overexpression of PhEXPA1 increases cell size, modifies cell wall polymer composition and affects the timing of axillary meristem development in Petunia hybrida

? Expansins are cell wall proteins required for cell enlargement and cell wall loosening during many developmental processes. The involvement of the Petunia hybrida expansin A1 (PhEXPA1) gene in cell expansion, the control of organ size and cell wall polysaccharide composition was investigated by overexpressing PhEXPA1 in petunia plants. ? PhEXPA1 promoter activity was evaluated using a promoter-GUS assay and the protein's subcellular localization was established by expressing a PhEXPA1-GFP fusion protein. PhEXPA1 was overexpressed in transgenic plants using the cauliflower mosaic virus (CaMV) 35S promoter. Fourier transform infrared (FTIR) and chemical analysis were used for the quantitative analysis of cell wall polymers. ? The GUS and GFP assays demonstrated that PhEXPA1 is present in the cell walls of expanding tissues. The constitutive overexpression of PhEXPA1 significantly affected expansin activity and organ size, leading to changes in the architecture of petunia plants by initiating premature axillary meristem outgrowth. Moreover, a significant change in cell wall polymer composition in the petal limbs of transgenic plants was observed. ? These results support a role for expansins in the determination of organ shape, in lateral branching, and in the variation of cell wall polymer composition, probably reflecting a complex role in cell wall metabolism.     

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.     

PcExl1 a Novel Acid Expansin-Like Protein from the Plant Pathogen Pectobacterium carotovorum,Binds Cell Walls Differently to BsEXLX1

Microbial expansins act on plant cell walls similarly to plant expansins, albeit their loosening activity levels are tenfold lesser compared to plant expansins. We report the characterization of an expansin-like gene from the plant pathogen Pectobacterium carotovorum, named exl1. PcExl1 is an acidic protein that binds cellulose (Avicel), and weakens filter paper. The acidic nature of PcExl1 confers different binding properties when compared to Bacillus subtilis BsEXLX1, which is a basic protein. PcExl1 binding to wheat cell wall increased when acidic components were depleted, reaching a similar level to the binding to Avicel, indicating that cellulose is the target of PcExl1.     

How do cell walls regulate plant growth?

The cell wall of growing plant tissues has frequently been interpreted in terms of inextensible cellulose microfibrils 'tethered' by hemicellulose polymers attached to the microfibril surface by hydrogen bonds, with growth occurring when tethers are broken or 'peeled' off the microfibril surface by expansins. This has sometimes been described as the 'sticky network' model. In this paper, a number of theoretical difficulties with this model, and discrepancies between predicted behaviour and observations by a number of researchers, are noted. (i) Predictions of cell wall moduli, based upon the sticky network model, suggest that the cell wall should be much weaker than is observed. (ii) The maximum hydrogen bond energy between tethers and microfibrils is less than the work done in expansion and therefore breakage of such hydrogen bonds is unlikely to limit growth. (iii) Composites of bacterial cellulose with xyloglucan are weaker than pellicles of pure cellulose so that it seems unlikely that hemicelluloses bind the microfibrils together. (iv) Calcium chelators promote creep of plant material in a similar way to expansins. (v) Reduced relative 'permittivities' inhibit the contraction of cell wall material when an applied stress is decreased. Revisions of the sticky network model that might address these issues are considered, as are alternatives including a model of cell wall biophysics in which cell wall polymers act as 'scaffolds' to regulate the space available for microfibril movement. Experiments that support the latter hypothesis, by demonstrating that reducing cell wall free volume decreases extensibility, are briefly described.     

Structure-function analysis of the bacterial expansin EXLX1

We made use of EXLX1, an expansin from Bacillus subtilis, to investigate protein features essential for its plant cell wall binding and wall loosening activities. We found that the two expansin domains, D1 and D2, need to be linked for wall extension activity and that D2 mediates EXLX1 binding to whole cell walls and to cellulose via distinct residues on the D2 surface. Binding to cellulose is mediated by three aromatic residues arranged linearly on the putative binding surface that spans D1 and D2. Mutation of these three residues to alanine eliminated cellulose binding and concomitantly eliminated wall loosening activity measured either by cell wall extension or by weakening of filter paper but hardly affected binding to whole cell walls, which is mediated by basic residues located on other D2 surfaces. Mutation of these basic residues to glutamine reduced cell wall binding but not wall loosening activities. We propose domain D2 as the founding member of a new carbohydrate binding module family, CBM63, but its function in expansin activity apparently goes beyond simply anchoring D1 to the wall. Several polar residues on the putative binding surface of domain D1 are also important for activity, most notably Asp82, whose mutation to alanine or asparagine completely eliminated wall loosening activity. The functional insights based on this bacterial expansin may be extrapolated to the interactions of plant expansins with cell walls.     

A novel petal up-regulated PhXTH7 promoter analysis in Petunia hybrida by using bioluminescence reporter gene

Flower opening is an important phenomenon in plant that indicates the readiness of the flower for pollination leading to petal expansion and pigmentation. This phenomenon has great impact on crop yield, which makes researches of its mechanism attractive for both plant physiology study and agriculture. Gene promoters directing the expression in petal during the petal cell wall modification and expansion when flower opens could be a convenient tool to analyze or monitor gene expression targeting this event. However, there are no reports of isolated gene promoters that can direct gene expression in petal or petal limb during the rapid cell wall dynamics when the flower opens. Xyloglucan endotransglucosylase/hydrolase 7 (XTH7), a cell wall modifying enzyme, was reported having up-regulated gene expression in the petal of Arabidopsis thaliana and Petunia hybrida. In this study, we fused a 1,904 bp length P. hybrida XTH7 promoter with a gene encoding a bright bioluminescent protein (Green enhanced Nano-lantern) to report gene expression and observed petal up-regulated bioluminescence activity by means of a consumer-grade camera. More importantly, this novel promoter demonstrated up-regulated activity in the petal limb of P. hybrida matured flower during flower opening. P. hybrida XTH7 promoter would be a useful tool for flowering study, especially for petal expansion research during flower opening.     

Cell expansion and microtubule behavior in ray floret petals of Gerbera hybrida: responses to light and gibberellic acid

It is still unclear how light and gibberellins are integrated to regulate petal size. Here, we report that light improves both the length and the width of the ray floret petals in G. hybrid, but GA(3) promotes only the petal length. It is also revealed that the control of the petal size by light and GA(3) depends on modulating the cell size, which is governed by the behavior of cortical microtubule.Light and gibberellins are important regulators of plant organ growth. However, little is known about their roles in petal size determination. Here, we report how light and gibberellic acid (GA(3)) signals are integrated to regulate the ray floret (Rf) size in Gerbera hybrida. The inflorescences of G. hybrida at stages 1.5 were cultivated in vitro for 9 d followed by the determination of the Rf petal size. Results demonstrated that the light signal significantly enhanced both the length and the width of Rf petals, but GA(3) promoted only the petal length. Moreover, GA(3) displayed a synergistic positive effect on the length but an antagonistic effect on the width with the light signal. Measurements of the petal cells revealed that the cell size, not the cell number, exhibited a dominant contribution to the petal size in response to light and GA(3) signals. Furthermore, light and GA(3) signals not only induced an obvious reorientation of cortical microtubules (MTs) into transverse arrays but also promoted the recovery of the MT lengths in petal cells following oryzalin (an MT depolymerizing agent) treatment. Importantly, disruption of the MT lengths and arrays by oryzalin could inhibit the cell expansion and the petal enlargement induced by light or/and GA(3) signals. Taken together, it is concluded that the control of the petal size by light and GA(3) signals mainly depends on modulating the cell size and, moreover, the organization of the cortical MTs plays a crucial role in the control of the cell size and hence the Rf petal growth.     

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.     

Electrostatic analysis of bacterial expansins

Expansins are a family of proteins with plant cell wall remodeling‐activity, which bind cell wall components through hydrophobic and electrostatic interactions. A shallow area on the surface of the protein serves as the polysaccharide binding site (PBS) and it is composed of conserved residues. However, electric charge differences on the opposite face of the PBS produce basic, neutral, or acidic proteins. An analysis of forty‐four bacterial expansins, homologues of BsEXLX1, revealed two main groups defined by: (a) the presence or absence of disulfide bonds; and (b) by the proteins isoelectric point (pI). We determined the location of the residues responsible for the pI on the structure of representative expansins. Our results suggest that the electric charge at the opposite site of the PBS may help in substrate differentiation among expansins from different species; in addition, electrostatic polarization between the front and the back of the molecule could affect expansin activity on cellulose. Proteins 2015; 83:215–223. © 2014 Wiley Periodicals, Inc.     

Overexpression of two cambium-abundant Chinese fir (Cunninghamia lanceolata) α-expansin genes ClEXPA1 and ClEXPA2 affect growth and development in transgenic tobacco and increase the amount of cellulose in stem cell walls

Expansins are unique plant cell wall proteins that possess the ability to induce immediately cell wall extension in vitro and cell expansion in vivo. To investigate the biological functions of expansins that are abundant in wood-forming tissues, we cloned two expansin genes from the differentiating xylem of Chinese fir (Cunninghamia lanceolata (Lamb.) Hook). Phylogenetic reconstruction indicated that they belong to α-expansin (EXPA), named ClEXPA1 and ClEXPA2. Expression pattern analysis demonstrated that they are preferentially expressed in the cambium region. Overexpression of ClEXPA1 and ClEXPA2 in tobacco plants yielded pleiotropic phenotypes of plant height, stem diameter, leaf number and seed pod. The height and diameter growth of the 35S(pro) :ClEXPA1 and 35S(pro) :ClEXPA2 transgenic plants were increased drastically, exhibiting an enlargement of pith parenchyma cell size. Isolated cell walls of ClEXPA1 and ClEXPA2 overexpressors contained 30%-50% higher cellulose contents than the wild type, accompanied by a thickening of the cell walls in the xylem region. Both ClEXPA1 and ClEXPA2 are involved in plant growth and development, with a partially functional overlap. Expansins are not only able to induce cell expansion in different tissues/organs in vivo, but they also can act as a potential activator during secondary wall formation by directly or indirectly affecting cellulose metabolism, probably in a cell type-dependent manner.     

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.     

延迟荧光分析和激光共聚焦技术在检测非洲菊发育过程中表皮细胞叶绿素变化中的应用

以非洲菊Gerberahybrida为研究对象,对其生长发育过程（P1．P6期）中外轮舌状花进行延迟荧光（delayedfluorescence,DF）分析,并利用激光共聚焦成像系统对其各期表皮细胞中叶绿素的分布和相对含量进行检测,以研究非洲菊外轮舌状花发育过程中延迟荧光改变与其叶绿素变化的关系。结果发现在非洲菊外轮舌状花从P1到P6期颜色由嫩绿色变为绿色再逐渐变为金黄色的过程中,其荧光强度变化是先升高后降低。激光共聚焦成像结果表明,其花瓣表皮细胞中叶绿素的相对含量从P1到P4期先是逐渐增多,而后从P4到P6期逐渐减少。到后期只是在保卫细胞中还能观察到叶绿素的存在。实验结果表明应用DF技术,可以有效、无损伤地快速检测非洲菊花瓣中叶绿素的相对含量,结合荧光光谱分析以及激光共聚焦扫描显微成像技术,研究花瓣在发育过程中的叶绿素降解情况。     

Measuring Plant Cell Wall Extension (Creep) Induced by Acidic pH and by Alpha-Expansin

Growing plant cell walls characteristically exhibit a property known as ''acid growth'', by which we mean they are more extensible at low pH (< 5) ¹. The plant hormone auxin rapidly stimulates cell elongation in young stems and similar tissues at least in part by an acid-growth mechanism ^{2, 3}. Auxin activates a H⁺ pump in the plasma membrane, causing acidification of the cell wall solution. Wall acidification activates expansins, which are endogenous cell wall-loosening proteins ⁴, causing the cell wall to yield to the wall tensions created by cell turgor pressure. As a result, the cell begins to enlarge rapidly. This ''acid growth'' phenomenon is readily measured in isolated (nonliving) cell wall specimens. The ability of cell walls to undergo acid-induced extension is not simply the result of the structural arrangement of the cell wall polysaccharides (e.g. pectins), but depends on the activity of expansins ⁵. Expansins do not have any known enzymatic activity and the only way to assay for expansin activity is to measure their induction of cell wall extension. This video report details the sources and preparation techniques for obtaining suitable wall materials for expansin assays and goes on to show acid-induced extension and expansin-induced extension of wall samples prepared from growing cucumber hypocotyls.To obtain suitable cell wall samples, cucumber seedlings are grown in the dark, the hypocotyls are cut and frozen at -80 °C. Frozen hypocotyls are abraded, flattened, and then clamped at constant tension in a special cuvette for extensometer measurements. To measure acid-induced extension, the walls are initially buffered at neutral pH, resulting in low activity of expansins that are components of the native cell walls. Upon buffer exchange to acidic pH, expansins are activated and the cell walls extend rapidly. We also demonstrate expansin activity in a reconstitution assay. For this part, we use a brief heat treatment to denature the native expansins in the cell wall samples. These inactivated cell walls do not extend even in acidic buffer, but addition of expansins to the cell walls rapidly restores their ability to extend.Open in a separate windowClick here to view.^{(58M, flv)}     

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.     

Disentangling loosening from softening: insights into primary cell wall structure

How cell wall elasticity, plasticity, and time‐dependent extension (creep) relate to one another, to plant cell wall structure and to cell growth remain unsettled topics. To examine these issues without the complexities of living tissues, we treated cell‐free strips of onion epidermal walls with various enzymes and other agents to assess which polysaccharides bear mechanical forces in‐plane and out‐of‐plane of the cell wall. This information is critical for integrating concepts of wall structure, wall material properties, tissue mechanics and mechanisms of cell growth. With atomic force microscopy we also monitored real‐time changes in the wall surface during treatments. Driselase, a potent cocktail of wall‐degrading enzymes, removed cellulose microfibrils in superficial lamellae sequentially, layer‐by‐layer, and softened the wall (reduced its mechanical stiffness), yet did not induce wall loosening (creep). In contrast Cel12A, a bifunctional xyloglucanase/cellulase, induced creep with only subtle changes in wall appearance. Both Driselase and Cel12A increased the tensile compliance, but differently for elastic and plastic components. Homogalacturonan solubilization by pectate lyase and calcium chelation greatly increased the indentation compliance without changing tensile compliances. Acidic buffer induced rapid cell wall creep via endogenous α‐expansins, with negligible effects on wall compliances. We conclude that these various wall properties are not tightly coupled and therefore reflect distinctive aspects of wall structure. Cross‐lamellate networks of cellulose microfibrils influenced creep and tensile stiffness whereas homogalacturonan influenced indentation mechanics. This information is crucial for constructing realistic molecular models that define how wall mechanics and growth depend on primary cell wall structure.     

Lipid transfer proteins enhance cell wall extension in tobacco

Plant cells are enclosed by a rigid cell wall that counteracts the internal osmotic pressure of the vacuole and limits the rate and direction of cell enlargement. When developmental or physiological cues induce cell extension, plant cells increase wall plasticity by a process called loosening. It was demonstrated previously that a class of proteins known as expansins are mediators of wall loosening. Here, we report a type of cell wall-loosening protein that does not share any homology with expansins but is a member of the lipid transfer proteins (LTPs). LTPs are known to bind a large range of lipid molecules to their hydrophobic cavity, and we show here that this cavity is essential for the cell wall-loosening activity of LTP. Furthermore, we show that LTP-enhanced wall extension can be described by a logarithmic time function. We hypothesize that LTP associates with hydrophobic wall compounds, causing nonhydrolytic disruption of the cell wall and subsequently facilitating wall extension.