Alphabetized listing of articles related to expansins, cell walls and acid growth. Hot-links to pdf files are provided, when available. These files are provided for non-profit educational use only and copyright remains with the copyright owner.  Authors are invited to send me [dcosgrove(at)psu.edu] relevant pdf's to include in this listing.

Aleman L, Kitamura J, Abdel-mageed H, Lee J, Sun Y, Nakajima M, Ueguchi-Tanaka M, Matsuoka M, Allen RD. 2008. Functional analysis of cotton orthologs of GA signal transduction factors GID1 and SLR1. Plant Mol Biol. 68(1-2):1-16.
ABSTRACT: Gibberellic acid (GA) is both necessary and sufficient to promote fiber elongation in cultured fertilized ovules of the upland cotton variety Coker 312. This is likely due to the temporal and spatial regulation of GA biosynthesis, perception, and subsequent signal transduction that leads to alterations in gene expression and morphology. Our results indicate that the initiation of fiber elongation by the application of GA to cultured ovules corresponds with increased expression of genes that encode xyloglucan endotransglycosylase/hydrolase (XTH) and expansin (EXP) that are involved in promoting cell elongation. To gain a better understanding of the GA signaling components in cotton, that lead to such changes in gene expression, two GA receptor genes (GhGID1a and GhGID1b) and two DELLA protein genes (GhSLR1a and GhSLR1b) that are orthologous to the rice GA receptor (GID1) and the rice DELLA gene (SLR1), respectively, were characterized. Similar to the GA biosynthetic genes, expression of GhGID1a and GhGID1b is under the negative regulation by GA while GA positively regulates GhSLR1a. Recombinant GST-GhGID1s showed GA-binding activity in vitro that was augmented in the presence of GhSLR1a, GhSLR1b, or rice SLR1, indicating complex formation between the receptors and repressor proteins. This was further supported by the GA-dependent interaction of these proteins in yeast cells. Ectopic expression of the GhGID1a in the rice gid1-3 mutant plants rescued the GA-insensitive dwarf phenotype, which demonstrates that it is a functional GA receptor. Furthermore, ectopic expression of GhSLR1b in wild type Arabidopsis led to reduced growth and upregulated expression of DELLA-responsive genes. Download pdf (C) Springer

An C, Saha S, Jenkins JN, Scheffler BE, Wilkins TA, Stelly DM. 2007.  Transcriptome profiling, sequence characterization, and SNP-based chromosomal assignment of the EXPANSIN genes in cotton. Mol Genet Genomics. 278(5):539-53
ABSTRACT: The knowledge of biological significance associated with DNA markers is very limited in cotton. SNPs are potential functional marker to tag genes of biological importance. Plant expansins are a group of extracellular proteins that directly modify the mechanical properties of cell walls, enable turgor-driven cell extension, and likely affect length and quality of cotton fibers. Here, we report the expression profiles of EXPANSIN transcripts during fiber elongation and the discovery of SNP markers, assess the SNP characteristics, and localize six EXPANSIN A genes to chromosomes. Transcriptome profiling of cotton fiber oligonucleotide microarrays revealed that seven EXPANSIN transcripts were differentially expressed when there was parallel polar elongation during morphogenesis at early stage of fiber development, suggesting that major and minor isoforms perform discrete functions during polar elongation and lateral expansion. Ancestral and homoeologous relationships of the six EXPANSIN A genes were revealed by phylogenetic grouping and comparison to extant A- and D-genome relatives of contemporary AD-genome cottons. The average rate of SNP per nucleotide was 2.35% (one SNP per 43 bp), with 1.74 and 3.99% occurring in coding and noncoding regions, respectively, in the selected genotypes. An unequal evolutionary rate of the EXPANSIN A genes at the subgenome level of tetraploid cotton was recorded. Chromosomal locations for each of the six EXPANSIN A genes were established by gene-specific SNP markers. Results revealed a strategy for discovering SNP markers in a polyploidy species like cotton. These markers could be useful to associate candidate genes with the complex fiber traits in MAS. Download pdf (C) Springer

Anand A, Vaghchhipawala Z, Ryu CM, Kang L, Wang K, del-Pozo O, Martin GB, Mysore KS. 2007.  Identification and characterization of plant genes involved in Agrobacterium-mediated plant transformation by virus-induced gene silencing. Mol Plant Microbe Interact. 20(1):41-52.
ABSTRACT: Genetic transformation of plant cells by Agrobacterium tumefaciens represents a unique case of trans-kingdom sex requiring the involvement of both bacterial virulence proteins and plant-encoded proteins. We have developed in planta and leaf-disk assays in Nicotiana benthamiana for identifying plant genes involved in Agrobacterium-mediated plant transformation using virus-induced gene silencing (VIGS) as a genomics tool. VIGS was used to validate the role of several genes that are either known or speculated to be involved in Agrobacterium-mediated plant transformation. We showed the involvement of a nodulin-like protein and an alpha-expansin protein (alpha-Exp) during Agrobacterium infection. Our data suggest that alpha-Exp is involved during early events of Agrobacterium-mediated transformation but not required for attaching A. tumefaciens. By employing the combination of the VIGS-mediated forward genetics approach and an in planta tumorigenesis assay, we identified 21 ACG (altered crown gall) genes that, when silenced, produced altered crown gall phenotypes upon infection with a tumorigenic strain of A. tumefaciens. One of the plant genes identified from the screening, Histone H3 (H3), was further characterized for its biological role in Agrobacterium-mediated plant transformation. We provide evidence for the role of H3 in transfer DNA integration. The data presented here suggest that the VIGS-based approach to identify and characterize plant genes involved in genetic transformation of plant cells by A. tumefaciens is simple, rapid, and robust and complements other currently used approaches.  Download pdf

Anjanasree,K.N. and Bansal,K.C. (2003) Isolation and characterization of ripening-related expansin cDNA from tomato. Journal of Plant Biochemistry and Biotechnology 12:31-35.
Abstract: Among different ripening-related genes, expansins are unique due to their non-enzymatic nature and their ability to stimulate cell wall disassembly by reversible disruption of hydrogen bonds at cellulose-hemicellulose interface. In the present study, LeExp1 cDNA from an Indian tomato cv Pusa Sheetal has been cloned and sequenced. The LeExp1 gene expression was analyzed in vegetative tissues, flowers and fruit tissues at different stages of ripening. The gene was found to express only in fruit tissues from breaker stage onwards. The transcript level progressively increased beyond the breaker stage and achieved a maximum level at the pink and red ripe stages of fruit development. The nucleotide and predicted amino acid sequences of the LeExp1 cDNA were compared with ripening-related expansins from others species and revealed high degree of conservation.

Arana MV, de Miguel LC, Sanchez RA. (2006) A phytochrome-dependent embryonic factor modulates gibberellin responses in the embryo and micropylar endosperm of Datura ferox seeds. Planta. 223(4):847-57
Abstract: The promotion of germination by phytochrome is associated with extensive changes both in the embryo and in the micropylar region of the endosperm (ME) of Datura ferox seeds. These changes require de novo gibberellins (GAs) biosynthesis in the embryo, the site where the light stimulus is perceived. GAs stimulate embryo growth potential and move to ME, promoting the expression of genes related with weakening. We report here that, in addition, phytochrome stimulates the sensitivity of the seeds to gibberellic acid (GA). The phytochrome-induced signal is produced in the embryo and enhances the stimulus by GA of embryo growth potential (EGP) and the promotion of the expression of proteins thought to participate in ME weakening: endo-beta-mannanase (EC 3.2.1.78), endo-beta-mannosidase (EC 3.2.1.25) and expansin. Our results suggest that the cytokinins may be a component of the embryonic signal. Phytochrome also modulates DfPHOR and DfMYB transcript levels in ME. These genes show a high identity with components of GAs signaling identified in other species. Expression of DfPHOR in the ME is apparently regulated by phytochrome through the supply of GAs from the embryo to ME, whereas DfMYB expression is regulated by an embryonic factor with some of the characteristics of the one that modulates seed sensitivity to GAs.

Avesani L, Marconi G, Morandini F, Albertini E, Bruschetta M, Bortesi L, Pezzotti M, Porceddu A. 2007.  Stability of Potato virus X expression vectors is related to insert size: implications for replication models and risk assessment. Transgenic Res. 16(5):587-97.
ABSTRACT: We investigated the stability of expression constructs based on Potato virus X (PVX) as a function of insert length. Five different inserts ranging in length from 261 to 1,758 bp (human proinsulin, murine interleukin-10, HIV-1 nef, petunia expansin-1 and human gad65) were expressed using a PVX vector in Nicotiana benthamiana plants for three sequential passages. Using a competitive RT-PCR approach we demonstrated that insert-deletion could occur in the first infection cycle for all inserts, but that this was much more likely to be the case for longer ones. This suggested a negative correlation between insert length and vector stability. Sequence analysis of the deleted constructs suggested that recombination usually occurred at sites close to the duplicated sub-genomic promoter, but in a smaller number of cases the foreign gene itself was probably involved, resulting in partially deleted constructs containing transgene fragments. The implications of these results in the context of recombinant protein expression and its risks are discussed. Download pdf  

Bacic A, Harris PJ, Stone BA (1988) Structure and function of plant cell walls. In J Preiss, ed, The Biochemistry of Plants. New York, Academic, pp 297-371

Baluska F, Salaj J, Mathur J, Braun M, Jasper F, Samaj J, Chua NH, Barlow PW, Volkmann D (2000)  Root Hair Formation: F-Actin-Dependent Tip Growth Is Initiated by Local Assembly of Profilin-Supported F-Actin Meshworks Accumulated within Expansin-Enriched Bulges. Dev. Biol. 227:618-632
Abstract: Plant root hair formation is initiated when specialized elongating root epidermis cells (trichoblasts) assemble distinct domains at the plasma membrane/cell wall cell periphery complexes facing the root surface. These localities show accumulation of expansin and progressively transform into tip-growing root hair apices. Experimentation showed that trichoblasts made devoid of microtubules (MTs) were unaffected in root hair formation, whereas those depleted of F-actin by the G-actin sequestering agent latrunculin B had their root hair formation blocked after the bulge formation stage. In accordance with this, MTs are naturally depleted from early outgrowing bulges in which dense F-actin meshworks accumulate. These F-actin caps remain associated with tips of emerging and growing root hairs. Constitutive expression of the GFP- mouse talin fusion protein in transgenic Arabidopsis, which visualizes all classes of F-actin in a noninvasive mode, allowed in vivo confirmation of the presence of distinct F-actin meshworks within outgrowing bulges and at tips of young root hairs. Profilin accumulates, at both the protein and the mRNA levels, within F-actin- enriched bulges and at tips of emerging hairs. ER-based calreticulin and HDEL proteins also accumulate within outgrowing bulges and remain enriched at tips of emerging hairs. All this suggests that installation of the actin-based tip growth machinery takes place only after expansin- associated bulge formation and requires assembly of profilin-supported dynamic F-actin meshworks.  Download pdf file (768 KB)  Copyright 2000 Academic Press.

Barre,A. and Rouge,P. (2002) Homology modeling of the cellulose-binding domain of a pollen allergen from rye grass: structural basis for the cellulose recognition and associated allergenic properties. Biochemical and Biophysical Research Communications 296:1346-1351.
Abstract: A three-dimensional model of the cellulose-binding domain of the rye-grass pollen allergen Lol pI built by homology modeling is proposed as a structural scaffold for expansins and other expansin-related proteins. A groove and an extended strip of aromatic and polar residues presumably account for the cellulose-binding properties of the protein domain. Two of the four predicted T-cell epitopes readily exposed on the surface of the cellulose-binding domain match with previously reported IgE-binding regions. A close structural relationship occurs between the cellulose-binding and allergenic properties. (C) 2002 Elsevier Science (USA).

Beemster,G.T.S., De Vusser,K., De Tavernier,E., De Bock,K., and Inze,D. (2002) Variation in growth rate between Arabidopsis ecotypes is correlated with cell division and A-type cyclin-dependent kinase activity. Plant Physiology 129:854-864.
Abstract: We used a kinematic analysis to investigate the growth processes responsible for variation in primary root growth between 18 ecotypes of Arabidopsis. Root elongation rate differed 4-fold between the slowest (Landsberg erecta, 71 mum h(-1)) and fastest growing line (Wassilewskija [Ws]; 338 Am h(-1)). This difference was contributed almost equally by variations in mature cortical cell length (84 mum [Landsberg erecta] to 237 mum [Ws]) and rate of cell production (0.63 cell h(-1) [NW108] to 1.83 cell h(-1) [Ws]). Cell production, in turn, was determined by variation in cell cycle duration (19 h [Tsu] to 48 h [NW108]) and, to a lesser extent, by differences in the number of dividing cells (32 [Weiningen] to 61 [Ws]). We found no correlation between mature cell size and endoreduplication, refuting the hypothesis that the two are linked. However, there was a strong correlation between cell production rates and the activity of the cyclin-dependent kinase (CDKA). The level of the protein could explain 32% of the variation in CDKA. Therefore, it is likely that regulators of CDKA, such as cyclins and inhibitors, are also involved. These data provide a functional link between cell cycle regulation and whole-plant growth rate as affected by genetic differences.

Belfield EJ, Ruperti B, Roberts JA, McQueen-Mason S. 2005.  Changes in expansin activity and gene expression during ethylene-promoted leaflet abscission in Sambucus nigra. J.Exp.Bot. 56:817-823, 2005.
Abstract:  During ethylene-promoted leaflet abscission in Sambucus nigra a 7-fold increase in expansin activity was detected specifically in tissues undergoing cell separation, whilst only low levels of activity were seen in adjacent non-abscising tissues. An RT-PCR strategy was used to amplify expansin fragments from a cDNA library generated from mRNA extracted from ethylene-treated leaflet abscission-zone tissue. Two different full-length expansin-encoding cDNAs were isolated with sizes of 1190 bp and 1169 bp and named SniExp2 and SniExp4, respectively. The deduced protein sequences of SniExp2 and SniExp4 exhibited 67% homology and comprised 256 and 249 amino acids, respectively. Both putative proteins contained signal sequences at their N terminal ends, suggesting that they were likely to encode secreted or transmembrane proteins. Northern analyses with probes specific for each gene revealed that transcripts for both of these cDNAs accumulated specifically in abscission zone tissues in response to ethylene treatment, with no expression being apparent in either freshly excised material or non-separating tissues. These data support a role for expansins in ethylene-stimulated abscission and provide evidence that specific family members contribute to the cell separation process that takes place during organ shedding. Download pdf

Bennett,A.B. (2002) Biochemical and genetic determinants of cell wall disassembly in ripening fruit: A general model. Hortscience 37:447-450.

Ding X, Cao Y, Huang L, Zhao J, Xu C, Li X, Wang S. 2008.  Activation of the indole-3-acetic acid-amido synthetase GH3-8 suppresses expansin expression and promotes salicylate- and jasmonate-independent basal immunity in rice. Plant Cell. 20(1):228-40
ABSTRACT: New evidence suggests a role for the plant growth hormone auxin in pathogenesis and disease resistance. Bacterial infection induces the accumulation of indole-3-acetic acid (IAA), the major type of auxin, in rice (Oryza sativa). IAA induces the expression of expansins, proteins that loosen the cell wall. Loosening the cell wall is key for plant growth but may also make the plant vulnerable to biotic intruders. Here, we report that rice GH3-8, an auxin-responsive gene functioning in auxin-dependent development, activates disease resistance in a salicylic acid signaling- and jasmonic acid signaling-independent pathway. GH3-8 encodes an IAA-amino synthetase that prevents free IAA accumulation. Overexpression of GH3-8 results in enhanced disease resistance to the rice pathogen Xanthomonas oryzae pv oryzae. This resistance is independent of jasmonic acid and salicylic acid signaling. Overexpression of GH3-8 also causes abnormal plant morphology and retarded growth and development. Both enhanced resistance and abnormal development may be caused by inhibition of the expression of expansins via suppressed auxin signaling. Download pdf (C) ASPB

Bishop-Hurley,S.L., Gardner,R.C., and Walter,C. (2003) Isolation and molecular characterization of genes expressed during somatic embryo development in Pinus radiata. Plant Cell Tissue and Organ Culture 74:267-281.
Abstract: Pinus radiata somatic embryogenic tissue offers many advantages over seed material for plantation forestry. Although much progress has been made in embryogenesis during the past few years, more research is needed to understand mechanisms underlying the difference in response by genotypes and the conversion of immature to mature embryos. As an initial genetic characterization of early embryo development in P. radiata, a cDNA library was constructed using mRNA from cultured tissue that was forming stage 1 embryos. This library was differentially screened against non-embryogenic tissues ( roots, shoots and needles) and six gene families preferentially expressed during conifer embryogenesis were identified. These genes showed high mRNA transcript levels in embryogenic tissue and little or no expression in non-embryogenic ( roots, shoots and needles) or callus tissue ( needle and fibre culture callus). These gene families included four putative extracellular proteins ( germin, beta-expansin, 21 kDa protein precursor and cellulase), a cytochrome P450 enzyme and a gene with unknown function (PRE87). We report data on the copy number of these genes and examine their expression in both P. radiata and Picea abies embryogenic tissues

Bogoslavsky L, Neumann PM.  (1998) Rapid regulation by acid pH of cell wall adjustment and leaf growth in maize plants responding to reversal of water stress. Plant Physiol. 118 (2):701-709

Boudart,G., Charpentier,M., Lafitte,C., Martinez,Y., Jauneau,A., Gaulin,E., Esquerre-Tugaye,M.T., and Dumas,B. (2003) Elicitor activity of a fungal endopolygalacturonase in tobacco requires a functional catalytic site and cell wall localization. Plant Physiology 131:93-101.
Abstract: CLPG1, an endopolygalacturonase (endoPG) gene of Colletotrichum lindemuthianum, was transferred to tobacco (Nicotiana tabacum) leaves by using the Agrobacterium tumefaciens transient delivery system. The following four constructs were prepared: CLPG1 with or without its signal peptide (SP; PG1, PG1DeltaSP); CLPG1 with the tobacco expansin1 SP instead of its own SP (Exp::PG1DeltaSP); and a mutated version of the latter on two amino acids potentially involved in the catalytic site of CLPG1 (D202N/D203N). Chlorotic and necrotic lesions appeared 5 to 7 d postinfiltration, exclusively in response to CLPG1 fused to the expansin SP. The lesions were correlated to the production of an active enzyme. Necrosis-inducing activity, as well as endoPG activity, were completely abolished by site-directed mutagenesis. Ultrastructural immunocytolocalization experiments indicated that the expansin SP addressed CLPG1 to the cell wall. Staining of parenchyma cells revealed the progressive degradation of pectic material in junction zones and middle lamella as a function of time after infiltration, ultimately leading to cell separation. A 30% decrease in the GalUA content of the cell walls was simultaneously recorded, thereby confirming the hydrolytic effect of CLPG1 on pectic polysaccharides, in planta. The elicitor activity of CLPG1 was further illustrated by the induction of defense responses comprising active oxygen species and beta-1,3-glucanase activity, before leaf necrosis. Altogether, the data demonstrate that an appropriate SP and a functional catalytic site are required for the proper expression and elicitor activity of the fungal endoPG CLPG1 in tobacco.

Bouzarelou D, Billini M, Roumelioti K, Sophianopoulou V. 2008. EglD, a putative endoglucanase, with an expansin like domain is localized in the conidial cell wall of Aspergillus nidulans. Fungal Genet Biol. 45(6):839-50.
ABSTRACT: Although the process of conidial germination in filamentous fungi has been extensively studied, many aspects remain to be elucidated since the asexual spore or conidium is vital in their life cycle. Breakage and reformation of cell wall polymer bonds along with the maintenance of cell wall plasticity during conidia germination depend upon a range of hydrolytic enzymes whose activity is analogous to that of expansins, a highly conserved group of plant cell wall proteins with characteristic wall loosening activity. In the current study, we identified and characterized the eglD gene in Aspergillus nidulans, an expansin-like gene the product of which shows strong similarities with bacterial and fungal endo-beta1,4-glucanases. However, we failed to show such activity in vitro. The eglD gene is constitutively expressed in all developmental stages and compartments of A. nidulans asexual life cycle. However, the EglD protein is exclusively present in conidial cell walls. The role of the EglD protein in morphogenesis, growth and germination rate of conidia was investigated. Our results show that EglD is a conidial cell wall localized expansin-like protein, which could be involved in cell wall remodeling during germination.  Download pdf (C) Elsevier

Brotman Y, Briff E, Viterbo A, Chet I. 2008. Abstract Role of swollenin, an expansin-like protein from Trichoderma, in plant root colonization. Plant Physiol. 147(2):779-89.
ABSTRACT: Swollenin, a protein first characterized in the saprophytic fungus Trichoderma reesei, contains an N-terminal carbohydrate-binding module family 1 domain (CBD) with cellulose-binding function and a C-terminal expansin-like domain. This protein was identified by liquid chromatography-mass spectrometry among many other cellulolytic proteins secreted in the coculture hydroponics medium of cucumber (Cucumis sativus) seedlings and Trichoderma asperellum, a well-known biocontrol agent and inducer of plant defense responses. The swollenin gene was isolated and its coding region was overexpressed in the same strain under the control of the constitutive pki1 promoter. Trichoderma transformants showed a remarkably increased ability to colonize cucumber roots within 6 h after inoculation. On the other hand, overexpressors of a truncated swollenin sequence bearing a 36-amino acid deletion of the CBD did not differ from the wild type, showing in vivo that this domain is necessary for full protein activity. Root colonization rates were reduced in transformants silenced in swollenin gene expression. A synthetic 36-mer swollenin CBD peptide was shown to be capable of stimulating local defense responses in cucumber roots and leaves and to afford local protection toward Botrytis cinerea and Pseudomonas syringae pv lachrymans infection. This indicates that the CBD domain might be recognized by the plant as a microbe-associated molecular pattern in the Trichoderma-plant interaction. Download pdf (C) ASPB

Brummell DA, Harpster MH (2001) Cell wall metabolism in fruit softening and quality and its manipulation in transgenic plants.  Plant Mol Biol 47:311-40.
ABSTRACT: Excessive softening is the main factor limiting fruit shelf life and storage. Transgenic plants modified in the expression of cell wall modifying proteins have been used to investigate the role of particular activities in fruit softening during ripening, and in the manufacture of processed fruit products. Transgenic experiments show that polygalacturonase (PG) activity is largely responsible for pectin depolymerization and solubilization, but that PG-mediated pectin depolymerization requires pectin to be de-methyl-esterified by pectin methylesterase (PME), and that the PG beta-subunit protein plays a role in limiting pectin solubilization. Suppression of PG activity only slightly reduces fruit softening (but extends fruit shelf life), suppression of PME activity does not affect firmness during normal ripening, and suppression of beta-subunit protein accumulation increases softening. All these pectin-modifying proteins affect the integrity of the middle lamella, which controls cell-to-cell adhesion and thus influences fruit texture. Diminished accumulation of either PG or PME activity considerably increases the viscosity of tomato juice or paste, which is correlated with reduced polyuronide depolymerization during processing. In contrast, suppression of beta-galactosidase activity early in ripening significantly reduces fruit softening, suggesting that the removal of pectic galactan side-chains is an important factor in the cell wall changes leading to ripening-related firmness loss. Suppression or overexpression of endo-(1-->4)beta-D-glucanase activity has no detectable effect on fruit softening or the depolymerization of matrix glycans, and neither the substrate nor the function for this enzyme has been determined. The role of xyloglucan endotransglycosylase activity in softening is also obscure, and the activity responsible for xyloglucan depolymerization during ripening, a major contributor to softening, has not yet been identified. However, ripening-related expansin protein abundance is directly correlated with fruit softening and has additional indirect effects on pectin depolymerization, showing that this protein is intimately involved in the softening process. Transgenic work has shown that the cell wall changes leading to fruit softening and textural changes are complex, and involve the coordinated and interdependent activities of a range of cell wall-modifying proteins. It is suggested that the cell wall changes caused early in ripening by the activities of some enzymes, notably beta-galactosidase and ripening-related expansin, may restrict or control the activities of other ripening-related enzymes necessary for the fruit softening process.

Brummell, DA; Harpster, MH; Dunsmuir, P  (1999)  Differential expression of expansin gene family members during growth and ripening of tomato fruit.  Plant Mol. Biol. 39:161-169.  
ABSTRACT: cDNA clones encoding homologues of expansins, a class of cell wall proteins involved in cell wall modification, were isolated from various stages of growing and ripening fruit of tomato (Lycopersicon esculentum). cDNAs derived from five unique expansin genes were obtained, termed tomato Exp3 to Exp7, in addition to the previously described ripening-specific tomato Exp1 (Rose et al. (1997) Proc Natl Acad Sci USA 94: 5955-5960). Deduced amino acid sequences of tomato Exp1, Exp4 and Exp6 were highly related, whereas Exp3, Exp5 and Exp7 were more divergent. Each of the five expansin genes showed a different and characteristic pattern of mRNA expression. mRNA of Exp3 was present throughout fruit growth and ripening, with highest accumulation in green expanding and maturing fruit, and lower, declining levels during ripening. Exp4 mRNA was present only in green expanding fruit, whereas Exp5 mRNA was present in expanding fruit but had highest levels in full-size maturing green fruit and declined during the early stages of ripening. mRNAs from each of these genes were also detected in leaves, stems and flowers but not in roots. Exp6 and Exp7 mRNAs were present at much lower levels than mRNAs of the other expansin genes, and were detected only in expanding or mature green fruit. The results indicate the presence of a large and complex expansin gene family in tomato, and suggest that while the expression of several expansin genes may contribute to green fruit development, only Exp1 mRNA is present at high levels during fruit ripening.

Brummell DA, Harpster MH, Civello PM, Palys JM, Bennett AB, and Dunsmuir P.  (1999) Modification of expansin protein abundance in tomato fruit alters softening and cell wall polymer metabolism during ripening. Plant Cell 11 (11):2203-2216.  
ABSTRACT:  The role of the ripening-specific expansin Exp1 protein in fruit softening and cell wall metabolism was investigated by suppression and overexpression of Exp1 in transgenic tomato plants. Fruit in which Exp1 protein accumulation was suppressed to 3% that of wild-type levels were firmer than controls throughout ripening. Suppression of Exp1 protein also substantially inhibited polyuronide depolymerization late in ripening but did not prevent the breakdown of structurally important hemicelluloses, a major contributor to softening. In contrast, fruit overexpressing high levels of recombinant Exp1 protein were much softer than controls, even in mature green fruit before ripening commenced. This softening was correlated with the precocious and extensive depolymerization of structural hemicelluloses, whereas polyuronide depolymerization was not altered. These data are consistent with there being at least three components to fruit softening and textural changes. One component is a relaxation of the wall directly mediated by Exp1, which indirectly limits part of a second component due to polyuronide depolymerization late in ripening, perhaps by controlling access of a pectinase to its substrate. The third component is caused by depolymerization of hemicelluloses, which occurs independently of or requires only very small amounts of Exp1 protein. Download PFD file (371 KB) (C) ASPP

Brummell,D.A., Howie,W.J., Ma,C., and Dunsmuir,P. (2002) Postharvest fruit quality of transgenic tomatoes suppressed in expression of a ripening-related expansin. Postharvest Biology and Technology 25:209-220.
Abstract: Expansins are proteins that cause cell wall loosening, and are involved in many aspects of cell wall modification during development. In tomato, the expansin gene LeExp1 shows ripening-related accumulation of mRNA and protein, and transgenic silencing of the expression of this gene results in tomato fruit that are significantly firmer than corresponding azygous controls throughout ripening. Examination of postharvest quality characteristics of fruit suppressed in accumulation of LeExp1 protein found that increased firmness resulted in significantly improved fruit integrity during storage at 13 degreesC. Based upon the first appearance of noticeable deterioration, fruit shelf life was extended by 5-10 days, depending upon the packaging. However, the increased firmness of LeExp1-suppressed fruit did not result in increased resistance to the necrophytic pathogens Botrytis cinerea and Alternaria alternata. Juice prepared from LeExp1-suppressed fruit following a microwave break had a soluble solids content (degreesBrix), insoluble solids content (precipitate weight ratio) and serum viscosity similar to controls. Resuspension of the insoluble pelleted particulate material in 15% of the serum produced a thick paste, which allowed estimation of gross viscosity in a Bostwick consistometer. The viscosity of paste from LeExp1-suppressed fruit was 19% greater than that from corresponding azygous controls, presumably due to changes in the insoluble particulate components affecting flow characteristics. No significant effects of the LeExp1 transgene on fruit size or fruit number per plant were noted. The data suggest that fruit suppressed in expression of LeExp1 have improved shelf life and processing properties. 2002 Elsevier Science B.V.

Caderas D, Muster M, Vogler H, Mandel T, Rose JKC, McQueen-Manson S, Kuhlemeier C (2000) Limited correlation between expansin gene expression and elongation growth rate. Plant Physiology 123: 1399-1414.
ABSTRACT:  The aim of this work was to study the role of the cell wall protein expansin in elongation growth. Expansins increase cell wall extensibility in vitro and are thought to be involved in cell elongation. Here, we studied the regulation of two tomato (Lycopersicon esculentum cv Moneymaker) expansin genes, LeExp2 and LeExp18, in rapidly expanding tissues. LeExp2 was strongly expressed in the elongation zone of hypocotyls and in the faster growing stem part during gravitropic stimulation. LeExp18 expression did not correlate with elongation growth. Exogenous application of hormones showed a substantial auxin-stimulation of LeExp2 mRNA in etiolated hypocotyls and a weaker auxin-stimulation of LeExp18 mRNA in stem tissue. Analysis of transcript accumulation revealed higher levels of LeExp2 and LeExp18 in light-treated, slow-growing tissue than in dark-treated, rapidly elongating tissue. Expansin protein levels and cell wall extension activities were similar in light- and dark-grown hypocotyl extracts. The results show a strong correlation between expansin gene expression and growth rate, but this correlation is not absolute. We conclude that elongation growth is likely to be controlled by expansin acting in concert with other factors that may limit growth under some physiological conditions. Download pdf file (1146K) (C) ASPP

D. Cantu, A. R. Vicente, L. C. Greve, F. M. Dewey, A. B. Bennett, J. M. Labavitch, and A. L. T. Powell (2008), The intersection between cell wall disassembly, ripening, and fruit susceptibility to Botrytis cinerea.  PNAS 105: 859-864.
ABSTRACT: Fruit ripening is characterized by processes that modify texture and flavor but also by a dramatic increase in susceptibility to necrotrophic pathogens, such as Botrytis cinerea. Disassembly of the major structural polysaccharides of the cell wall (CW) is a significant process associated with ripening and contributes to fruit softening. In tomato, polygalacturonase (PG) and expansin (Exp) are among the CW proteins that cooperatively participate in ripening-associated CW disassembly. To determine whether endogenous CW disassembly influences the ripening-regulated increase in necrotropic pathogen susceptibility, B. cinerea susceptibility was assessed in transgenic fruit with suppressed polygalacturonase (LePG) and expansin (LeExp1) expression. Suppression of either LePG or LeExp1 alone did not reduce susceptibility but simultaneous suppression of both dramatically reduced the susceptibility of ripening fruit to B. cinerea, as measured by fungal biomass accumulation and by macerating lesion development. These results demonstrate that altering endogenous plant CW disassembly during ripening influences the course of infection by B. cinerea, perhaps by changing the structure or the accessibility of CW substrates to pathogen CW-degrading enzymes. Recognition of the role of ripening-associated CW metabolism in postharvest pathogen susceptibility may be useful in the design and development of strategies to limit pathogen losses during fruit storage, handling, and distribution.  Download pdf (C)PNAS

Carey RE, Cosgrove DJ. 2007 Portrait of the expansin superfamily in Physcomitrella patens: comparisons with angiosperm expansins. Ann Bot (Lond). 99(6):1131-41
ABSTRACT:  BACKGROUND AND AIMS: Expansins are plant cell wall loosening proteins important in a variety of physiological processes. They comprise a large superfamily of genes consisting of four families (EXPA, EXPB, EXLA and EXLB) whose evolutionary relationships have been well characterized in angiosperms, but not in basal land plants. This work attempts to connect the expansin superfamily in bryophytes with the evolutionary history of this superfamily in angiosperms. METHODS: The expansin superfamily in Physcomitrella patens has been assembled from the Physcomitrella sequencing project data generated by the Joint Genome Institute and compared with angiosperm expansin superfamilies. Phylogenetic, motif, intron and distance analyses have been used for this purpose. KEY RESULTS: A gene superfamily is revealed that contains similar numbers of genes as found in arabidopsis, but lacking EXLA or EXLB genes. This similarity in gene numbers exists even though expansin evolution in Physcomitrella diverged from the angiosperm line approx. 400 million years ago. Phylogenetic analyses suggest that there were a minimum of two EXPA genes and one EXPB gene in the last common ancestor of angiosperms and Physcomitrella. Motif analysis seems to suggest that EXPA protein function is similar in bryophytes and angiosperms, but that EXPB function may be altered. CONCLUSIONS: The EXPA genes of Physcomitrella are likely to have maintained the same biochemical function as angiosperm expansins despite their independent evolutionary history. Changes seen at normally conserved residues in the Physcomitrella EXPB family suggest a possible change in function as one mode of evolution in this family.  Download pdf (C) Oxford

Carpita NC (1996) Structure and biogenesis of the cell walls of grasses. Annu. Rev. Plant Physiol. Plant Mol. Biol. 47: 445-476

Carpita NC, Gibeaut DM (1993) Structural models of primary cell walls in flowering plants: consistency of molecular structure with the physical properties of the walls during growth. Plant J. 3: 1-30

Catala C, Rose JK, Bennett AB (2000) Auxin-regulated genes encoding cell wall-modifying proteins are expressed during early tomato fruit growth. Plant Physiol 122 (2):527-534
ABSTRACT: An expansin gene, LeExp2, was isolated from auxin-treated, etiolated tomato (Lycopersicon esculentum cv T5) hypocotyls. LeExp2 mRNA expression was restricted to the growing regions of the tomato hypocotyl and was up-regulated during incubation of hypocotyl segments with auxin. The pattern of expression of LeExp2 was also studied during tomato fruit growth, a developmental process involving rapid cell enlargement. The expression of genes encoding a xyloglucan endotransglycosylase (LeEXT1) and an endo-1,4-beta-glucanase (Cel7), which, like LeExp2, are auxin-regulated in etiolated hypocotyls (C. Catala, J.K.C. Rose, A.B. Bennett [1997] Plant J 12: 417-426), was also studied to examine the potential for synergistic action with expansins. LeExp2 and LeEXT1 genes were coordinately regulated, with their mRNA accumulation peaking during the stages of highest growth, while Cel7 mRNA abundance increased and remained constant during later stages of fruit growth. The expression of LeExp2, LeEXT1, and Cel7 was undetectable or negligible at the onset of and during fruit ripening, which is consistent with a specific role of these genes in regulating cell wall loosening during fruit growth, not in ripening-associated cell wall disassembly. Download PDF file.

Caturla,M., Chaparro,C., Schroeyers,K., and Holsters,M. (2002) Suppression subtractive hybridization to enrich low-abundance and submergence-enhanced transcripts of adventitious root primordia of Sesbania rostrata. Plant Science 162:915-921.
Abstract: Upon waterlogging, the tropical legume Sesbania rostrata activates dormant meristems of stem-located root primordia, leading to adventitious root outgrowth. By suppression subtractive hybridization (SSH), cDNA clones were collected that were enriched for low-abundant and water-induced transcripts. From. 192 sequenced SSH clones, ranging from 64 to 1064 by in size, 66 unigenes were obtained. Complex cDNA probes were generated to screen cDNA macro-arrays. Sequence homologies of putative up-regulated unigenes and of non-induced unigenes will be briefly discussed. (C) 2002 Elsevier Science Ireland Ltd.

Ceccardi, TL; Barthe, GA; Derrick, KS  (1998)  A novel protein associated with citrus blight has sequence similarities to expansin.  Plant Mol. Biol. 38: 775-783 
ABSTRACT:   A protein associated with citrus blight (CB), a disease of unknown cause, was partially characterized. The 12 kDa protein, designated p12, is diagnostic of CB and is present in leaves and xylem fluid from roots and stems of CB-affected trees. The protein, and up to six other CB-specific proteins, are readily detected by SDS-PAGE of xylem fluid from CB-affected trees. The partial N-terminal amino acid sequence of p12 was found to be unique based on database searches. A cDNA library from CB-affected root cambium was screened with a 60 bp fragment, obtained by PCR amplification of cDNA with degenerate primers designed using the amino acid sequence of p12, and two clones were selected. These clones were sequenced revealing a 674 nucleotide cDNA with a 393 nt ORF which included sequence predicted by the N-terminal amino acid sequence of p12. The amino acid sequence based on the p12 BRF was found to be up to 49% similar and 31% identical to expansins. Bacterial expression of the cloned ORE which encodes an 11.8 kDa protein plus an N-terminal hydrophobic signal peptide, produced an immunoreactive protein of the expected size. By northern blot analysis, it was determined that p12 transcripts are present in root and stem cambium, but not in leaves of CB-affected trees, suggesting transport of the protein to leaves. Southern hybridization analysis of citrus genomic DNA indicated that p12 is a citrus encoded protein.

E. Chanliaud, J. de Silva, B. Strongitharm, G. Jeronimidis, and M. J. Gidley. Mechanical effects of plant cell wall enzymes on cellulose/xyloglucan composites. Plant J. 38 (1):27-37, 2004.
Abstract:  Xyloglucan-acting enzymes are believed to have effects on type I primary plant cell wall mechanical properties. In order to get a better understanding of these effects, a range of enzymes with different in vitro modes of action were tested against cell wall analogues (bio-composite materials based on Acetobacter xylinus cellulose and xyloglucan). Tomato pericarp xyloglucan endo transglycosylase (tXET) and nasturtium seed xyloglucanase (nXGase) were produced heterologously in Pichia pastoris. Their action against the cell wall analogues was compared with that of a commercial preparation of Trichoderma endo-glucanase (EndoGase). Both 'hydrolytic' enzymes (nXGase and EndoGase) were able to depolymerise not only the cross-link xyloglucan fraction but also the surface-bound fraction. Consequent major changes in cellulose fibril architecture were observed. In mechanical terms, removal of xyloglucan cross-links from composites resulted in increased stiffness (at high strain) and decreased visco-elasticity with similar extensibility. On the other hand, true transglycosylase activity (tXET) did not affect the cellulose/xyloglucan ratio. No change in composite stiffness or extensibility resulted, but a significant increase in creep behaviour was observed in the presence of active tXET. These results provide direct in vitro evidence for the involvement of cell wall xyloglucan-specific enzymes in mechanical changes underlying plant cell wall re-modelling and growth processes. Mechanical consequences of tXET action are shown to be complimentary to those of cucumber expansin

Chen F, Dahal P, Bradford KJ (2001)  Two Tomato Expansin Genes Show Divergent Expression and Localization in Embryos during Seed Development and Germination. Plant Physiology 127: 928-36 
ABSTRACT:   Expansins are plant proteins that can induce extension of isolated cell walls and are proposed to mediate cell expansion. Three expansin genes were expressed in germinating tomato (Lycopersicon esculentum Mill.) seeds, one of which (LeEXP4) was expressed specifically in the endosperm cap tissue enclosing the radicle tip. The other two genes (LeEXP8 and LeEXP10) were expressed in the embryo and are further characterized here. LeEXP8 mRNA was not detected in developing or mature seeds but accumulated specifically in the radicle cortex during and after germination. In contrast, LeEXP10 mRNA was abundant at an early stage of seed development corresponding to the period of rapid embryo expansion; it then decreased during seed maturation and increased again during germination. When gibberellin-deficient (gib-1) mutant seeds were imbibed in water, LeEXP8 mRNA was not detected, but a low level of LeEXP10 mRNA was present. Expression of both genes increased when gib-1 seeds were imbibed in gibberellin. Abscisic acid did not prevent the initial expression of LeEXP8 and LeEXP10, but mRNA abundance of both genes subsequently decreased during extended incubation. The initial increase in LeEXP8, but not LeEXP10, mRNA accumulation was blocked by low water potential, but LeEXP10 mRNA amounts fell after longer incubation. When seeds were transferred from abscisic acid or low water potential solutions to water, abundance of both LeEXP8 and LeEXP10 mRNAs increased in association with germination. The tissue localization and expression patterns of both LeEXP8 and LeEXP10 suggest developmentally specific roles during embryo and seedling growth. Download PDF file.

Chen F, Bradford KJ (2000)  Expression of an expansin is associated with endosperm weakening during tomato seed germination. Plant Physiol. 124: 1265-1274
ABSTRACT: Expansins are extracellular proteins that facilitate cell wall extension, possibly by disrupting hydrogen bonding between hemicellulosic wall components and cellulose microfibrils. In addition, some expansins are expressed in non-growing tissues such as ripening fruits, where they may contribute to cell wall disassembly associated with tissue softening. We have identified at least three expansin genes that are expressed in tomato (Lycopersicon esculentum Mill.) seeds during germination. Among these, LeEXP4 mRNA is specifically localized to the micropylar endosperm cap region, suggesting that the protein might contribute to tissue weakening that is required for radicle emergence. In gibberellin (GA)-deficient (gib-1) mutant seeds, which germinate only in the presence of exogenous GA, GA induces the expression of LeEXP4 within 12 hours of imbibition. When gib-1 seeds were imbibed in GA solution combined with 100 muM abscisic acid, the expression of LeEXP4 was not reduced, although radicle emergence was inhibited. In wild-type seeds, LeEXP4 mRNA accumulation was blocked by far-red light and decreased by low water potential but was not affected by abscisic acid. The presence of LeEXP4 mRNA during seed germination parallels endosperm cap weakening determined by puncture force analysis. We hypothesize that LeEXP4 is involved in the regulation of seed germination by contributing to cell wall disassembly associated with endosperm cap weakening. Download pdf file (429 KB) (c) ASPP

Cho HT,  Cosgrove DJ  (2000)  Altered expression of expansin modulates leaf growth and pedicel abscission in Arabidopsis thaliana. Proc. Nat'l Acad. Sci. 97: 9783-9788
ABSTRACT:  The spatial distribution pattern of expansin transcripts and proteins in tissues and organs of deepwater rice were characterized using in situ mRNA hybridization and immunohisto-chemical analysis. Expansin transcripts and proteins were present at high levels in the growing internodal epidermis, which has thick cell walls and acts, therefore, as a growth-limiting cell layer. Expansins were also concentrated in differentiating vascular bundles of internodes. In the primary root, expansins were predominantly expressed in the tip region, particularly in the epidermis, in the differentiating vascular cylinder and around the pericyle. Developing adventitious roots and lateral root primordia also contained high levels of expansin mRNA. In the shoot apex, expansin transcripts were abundant in the emerging leaf primordia. Results indicate that expansins play an important role in the expansion and differentiation of plant tissues and organs.   Download pdf file (275 kB)  (c) PNAS.

Cho,H.T. and Cosgrove,D.J. (2002) Regulation of root hair initiation and expansin gene expression in Arabidopsis. Plant Cell 14:3237-3253.
Abstract: The expression of two Arabidopsis expansin genes (AtEXP7 and AtEXP18) is tightly linked to root hair initiation; thus, the regulation of these genes was studied to elucidate how developmental, hormonal, and environmental factors orchestrate root hair formation. Exogenous ethylene and auxin, as well as separation of the root from the medium, stimulated root hair formation and the expression of these expansin genes. The effects of exogenous auxin and root separation on root hair formation required the ethylene signaling pathway. By contrast, blocking the endogenous ethylene pathway, either by genetic mutations or by a chemical inhibitor, did not affect normal root hair formation and expansin gene expression. These results indicate that the normal developmental pathway for root hair formation (i.e., not induced by external stimuli) is independent of the ethylene pathway. Promoter analyses of the expansin genes show that the same promoter elements that determine cell specificity also determine inducibility by ethylene, auxin, and root separation. Our study suggests that two distinctive signaling pathways, one developmental and the other environmental/hormonal, converge to modulate the initiation of the root hair and the expression of its specific expansin gene set.  Download pdf

H. T. Cho and D. J. Cosgrove. Expansins as agents of hormone action. In: Plant hormones: biosynthesis, signal transduction, action!, edited by P. J. Davies,   p. 262-281.     Dordrecht:Kluwer, 2004
Abstract: A review of the expansin/hormone action field.   Download pdf

Cho HT, Kende H (1997) Expansins and internodal growth of deepwater rice. Plant Physiol. 113:1145-1151
ABSTRACT:  The distribution and activity of the cell wall-loosening protein expansin was correlated with internodal growth in deepwater rice (Oryza sativa) cv. Pin Gaew 56. Acid-induced extension of native cell walls and reconstituted extension of boiled cell walls were confined to the growing region of the internode, i.e. to the intercalary meristem (IM) and the elongation zone. Immunolocalization by tissue printing and immunoblot analysis, using an antibody against cucumber expansin 29 as a probe, confirmed that rice expansin occurred primarily in the IM and elongation zone. Rice expansin was localized mainly around the vascular bundles at the base of the IM and along the inner epidermal cell layer surrounding the internodal cavity. Submergence greatly promoted the growth of rice internodes, and cell walls of submerged internodes extended much more in response to acidification than did the cell walls of air-grown internodes. Susceptibility of cell walls to added expansin was also increased in submerged internodes, and analysis by immunoblotting showed that cell walls of submerged internodes contained more expansin than did cell walls of air-grown internodes. It is proposed that expansin is involved in mediating rapid internodal elongation in submerged deepwater rice internodes.

Cho HT, Kende H (1997) Expansins in deepwater rice internodes.    Plant Physiol. 113: 1137-1143
ABSTRACT:   Expansins are cell-wall-loosening proteins that induce stress relaxation and extension of plant cell walls. To evaluate their hypothesized role in cell growth, we genetically manipulated expansin gene expression in Arabidopsis thaliana and assessed the consequent changes in growth and cell-wall properties. Various combinations of promoters were used to drive antisense and sense sequences of AtEXP10, which is maximally expressed in the growing leaf and at the base of the pedicel. Compared with controls, antisense lines had smaller rosettes because of shorter petioles and leaf blades and often acquired a twisted leaf morphology. Petiole cells from antisense plants were smaller than controls and their cell walls were significantly less extensible in vitro. Sense plants had slightly longer petioles, larger leaf blades, and larger cells than controls. Abscission at the base of the pedicel, where AtEXP10 is endogenously expressed, was enhanced in sense plants but reduced in antisense lines. These results support the concept that expansins function endogenously as cell-wall-loosening agents and indicate that expansins have versatile developmental roles that include control of organ size, morphology, and abscission. 

Cho HT, Kende H (1997c) Expression of expansin genes is correlated with growth in deepwater rice. Plant Cell 9: 1661-1671
ABSTRACT:  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.

Cho HT,  Kende H  (1998)  Tissue localization of expansins in deepwater rice. Plant J. 1998. 15: 6, 805-812 
ABSTRACT:  The spatial distribution pattern of expansin transcripts and proteins in tissues and organs of deepwater rice were characterized using in situ mRNA hybridization and immunohisto-chemical analysis. Expansin transcripts and proteins were present at high levels in the growing internodal epidermis, which has thick cell walls and acts, therefore, as a growth-limiting cell layer. Expansins were also concentrated in differentiating vascular bundles of internodes. In the primary root, expansins were predominantly expressed in the tip region, particularly in the epidermis, in the differentiating vascular cylinder and around the pericyle. Developing adventitious roots and lateral root primordia also contained high levels of expansin mRNA. In the shoot apex, expansin transcripts were abundant in the emerging leaf primordia. Results indicate that expansins play an important role in the expansion and differentiation of plant tissues and organs.

Choi,D.S., Lee,Y., Cho,H.T., and Kende,H. (2003) Regulation of expansin gene expression affects growth and development in transgenic rice plants. Plant Cell 15:1386-1398.
Abstract: To investigate the in vivo functions of expansins, we generated transgenic rice plants that express sense and antisense constructs of the expansin gene OsEXP4. In adult plants with constitutive OsEXP4 expression, 12% of overexpressors were taller and 88% were shorter than the average control plants, and most overexpressors; developed at least two additional leaves. Antisense plants were shorter and flowered earlier than the average control plants. In transgenic plants with inducible OsEXP4 expression, we observed a close correlation between OsEXP4 protein levels and seedling growth. Coleoptile and mesocotyl length increased by up to 31 and 97%, respectively, in overexpressors, whereas in antisense seedlings, they decreased by up to 28 and 43%, respectively. The change in seedling growth resulted from corresponding changes in cell size, which in turn appeared to be a function of altered cell wall extensibility. Our results support the hypothesis that expansins are involved in enhancing growth by mediating cell wall loosening.

Civello PM, Powell ALT, Sabehat A, and Bennett AB.  (1999) An expansin gene expressed in ripening strawberry fruit. Plant Physiol. 121 (4):1273-1279.    
ABSTRACT: 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 × 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.  (C) ASPP

Cleland RE (1992) Auxin-induced growth of Avena coleoptiles involves two mechanisms with different pH optima. Plant Physiol. 99: 1556-1561

T. D. Colmer, A. J. Peeters, C. A. Wagemaker, W. H. Vriezen, A. Ammerlaan, and L. A. Voesenek. Expression of alpha-expansin genes during root acclimations to O2 deficiency in Rumex palustris. Plant Mol.Biol. 56 (3):423-437, 2004.
Abstract:  Thirteen alpha-expansin genes were isolated from Rumex palustris , adding to the six already documented for this species. Five alpha-expansin genes were selected for expression studies in various organs/tissues of R. palustris , with a focus on roots exposed to aerated or O2)-deficient conditions, using real-time RT-PCR. Several cases of differential expression of alpha-expansin genes in the various root types of R. palustris were documented, and the identity of the dominant transcript differed between root types (i.e., tap root vs. lateral roots vs. adventitious roots). Several genes were expressed differentially in response to low O2. In situ hybridizations showed expansin mRNA expression in the oldest region of the tap root was localized to cells near the vascular cambium; this being the first report of expansin expression associated with secondary growth in roots. In situ hybridization also showed abundant expression of expansin mRNA in the most apical 1 mm of adventitious roots. Such early expression of expansin mRNA in cells soon after being produced by the root apex presumably enables cell wall loosening in the elongation zone of roots. In addition, expression of some expansin mRNAs increased in 'mature zones' of roots; these expansins might be involved in root hair formation or in formation of lateral root primordia. The present findings support the notion that large gene families of alpha-expansins enable flexibility in expression for various organs and tissues as a normal part of plant development, as well as in response to abiotic stress.

Cosgrove DJ (1989) Characterization of long-term extension of isolated cell walls from growing cucumber hypocotyls. Planta 177: 121-130
ABSTRACT:  Walls from frozen-thawed hypocotyls are known to extend for many hours when placed in tension under acidic conditions. It was found that chemical denaturants inhibited such wall 'creep', some acting reversibly and others irreversibly. Brief (15 s) boiling in water irreversibly inhibited creep, as did pre-incubation with proteases. Creep exhibited a high Q10 (3.8) between 20 and 30 deg C, with slow inactivation at higher temperatures, whereas the viscous flow of pectin solutions exhibited a much lower Q10 (1.35). On the basis of its temperature sensitivity, involvement of pectic gel-sol transitions appeared to be of little importance in creep. Pre-incubation of walls at neutral pH irreversibly inactivated their ability to creep, with a half-time of about 40 minutes. At 1 mM concentration, Cu, Hg and Al were strongly inhibitory whereas most other cations, including Ca, had little effect. Sulphhydryl-reducing agents strongly stimulated creep, apparently by stabilizing wall enzyme(s). The physical effects of these treatments on polymer interactions were examined by Instron and stress-relaxation analyses. Some treatments, such as pH and Cu, had significant effects on wall viscoelasticity, but others had little or no apparent effect, thus implicating an enzymatic mechanism for creep. The results indicate that creep depends on relatively rugged enzymes that are firmly attached to or entangled in the wall. The sensitivity of creep to SH-reducing agents indicates that thiol reduction of wall enzymes might provide a control mechanism for endogenous cell growth.. © Springer-Verlag. Download pdf file:   Long version (3.6 MB)      Short version (363 KB, some errors). 

Cosgrove DJ (1993) Wall extensibility: its nature, measurement, and relationship to plant cell growth. New Phytologist. 124: 1-23 
ABSTRACT: Expansive growth of plant cells is controlled principally by processes that loosen the wall and enable it to expand irreversibly. The central role of wall relaxation for cell expansion is reviewed. The most common methods for assessing the extension properties of plant cell walls ('wall extensibility') are described, categorized and assessed critically. What emerges are three fundamentally different approaches which test growing cells for their ability (a) to enlarge at different values of turgor, (b) to induce wall relaxation, and (c) to deform elastically or plastically in response to an applied tensile force. Analogous methods with isolated walls are similarly reviewed. The results of these different assays are related to the nature of plant cell growth and pertinent biophysical theory. I argue that the 'extensibilities' measured by these assays are fundamentally different from one another and that some are more pertinent to growth than others. Download pdf

Cosgrove DJ  (1996) BioEssays 18: 533-540.  Plant cell enlargement and the action of expansins
ABSTRACT:  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. © ICSU Press

Cosgrove DJ (1997) Relaxation in a high-stress environment: the molecular bases of extensible cell walls and cell enlargement. Plant Cell 9: 1031-1041.
ABSTRACT: This article reviews current physical and molecular concepts of the mechanism of cell wall expansion. Headings include the following: Introduction. Special rheological properties of growing cell walls: stress relaxation and polymer creep. Major structural polymers of the cell wall. Potential wall loosening mechanisms. Expansins and acid growth . The expansin gene family. A second family of expansins. Glucanases and Xyloglucan endotransglycosylases. Cell wall synthesis: another brick in the wall? Rigidifying the cell wall. Summary and perspective. Download pdf © ASPP

Cosgrove DJ (1997)  Assembly and enlargement of the primary cell wall in plants. Annual Review of Cell and Developmental Biology 13: 171-201.
ABSTRACT:  Growing plant cells are shaped by an extensible wall that is a complex amalgam of cellulose microfibrils bonded noncovalently to a matrix of hemicelluloses, pectins, and structural proteins. Cellulose is synthesized by complexes in the plasma membrane and is extruded as a self-assembling microfibril, whereas the matrix polymers are secreted by the Golgi apparatus and become integrated into the wall network by poorly understood mechanisms. The growing wall is under high tensile stress from cell turgor and is able to enlarge by a combination of stress relaxation and polymer creep. A pH-dependent mechanism of wall loosening, known as acid growth, is characteristic of growing walls and is mediated by a group of unusual wall proteins called expansins. Expansins appear to disrupt the noncovalent bonding of matrix hemicelluloses to the microfibril, thereby allowing the wall to yield to the mechanical forces generated by cell turgor. Other wall enzymes, such as (1-->4) beta-glucanases and pectinases, may make the wall more responsive to expansin-mediated wall creep whereas pectin methylesterases and peroxidases may alter the wall so as to make it resistant to expansin-mediated creep. Download pdf file. (c) Annual Reviews Inc.

Cosgrove DJ (1997) Creeping walls, softening fruit, and penetrating pollen tubes: the growing roles of expansins.  Proc. Nat'l Acad. Sci. 94: 5504-5505
ABSTRACT:  This commentary describes new results that imply a broadening biological role for expansins.  Download the full article in Acrobat pdf format (206 kB file). © PNAS

Cosgrove DJ (1998)  Update on cell growth: Wall loosening by expansins. Plant Physiol. 118: 333-339
ABSTRACT:  This minireview discusses expansins in the context of plant growth and describes emerging concepts about its roles in plant development. Section headings include the following: Growing cell walls are pliant and extend at acidic pH. Expansins mediate pH-dependent creep of cell walls. Expansins are novel proteins comprising a large superfamily. Homology with grass pollen allergens reveals the b-expansin family. Why so many expansin genes? The precise molecular mechanism of expansin-induced wall creep is still enigmatic. A model of wall enlargement and its control. New roles for expansins. Conclusions and prospectus.  Download the full article in Acrobat pdf format (296 kB file). © ASPP

Cosgrove DJ (1999)  Enzymes and other agents that enhance cell wall extensibility. Annual Review of Plant Physiology and Plant Molecular Biology 50: 391-417
ABSTRACT: Polysaccharides and proteins are secreted to the inner surface of the growing cell wall, where they assemble into a network that is mechanically strong, yet remains extensible until the cells cease growth. This review focuses on the agents that directly or indirectly enhance the extensibility properties of growing walls. The properties of expansins, endoglucanases, and xyloglucan transglycosylases are reviewed and their postulated roles in modulating wall extensibility are evaluated. A summary model for wall extension is presented, in which expansin is a primary agent of wall extension, whereas endoglucanases, xyloglucan endotransglycosylase, and other enzymes that alter wall structure act secondarily to modulate expansin action.  Download the full article in Acrobat pdf format (296 KB).  © Annual Reviews Inc.

Cosgrove DJ (2000)  New genes and new biological roles for expansins. Current Opinion in Plant Biology 3: 73-78
ABSTRACT: Expansins are extracellular proteins that loosen plant cell walls in novel ways. They are thought to function in cell enlargement, pollen tube invasion of the stigma (in grasses), wall disassembly during fruit ripening, abscission and other cell separation events. Expansins are encoded by two multigene families and each gene is often expressed in highly specific locations and cell types. Structural analysis indicates that one expansin region resembles the catalytic domain of family-45 endoglucanases but glucanase activity has not been detected. The genome projects have revealed numerous expansin-related sequences but their putative wall-loosening functions remain to be assessed..  Download the full article in Acrobat pdf format (276 KB).  © Current Opinions

Cosgrove DJ (2000)  Expansive growth of plant cell walls. Plant Physiol. Biochem. 38: 109-124.
ABSTRACT:  The enlargement of plant cell walls is a key determinant of plant morphogenesis. Current models of the cell wall are reviewed with respect to their ability to account for the mechanism of cell wall enlargement. The concept of primary and secondary wall loosening agents is presented, and the possible roles of expansins, xyloglucan endotransglycosylase, endo-1,4-beta-D-glucanase, and wall synthesis in the process of cell wall enlargement are reviewed and critically evaluated. Experimental results indicate that cell wall enlargement may be regulated at many levels. (C) 2000 Editions scientifiques et medicales Elsevier SAS. Download full article in Acrobat pdf format  (Long version 2.3 MB;  short version, with minor format errors, 302 KB).  

Cosgrove, DJ  (2000)  Loosening of plant cell walls by expansins. Nature  407: 321-326
ABSTRACT:  Plant cell walls are the starting materials for many commercial products, from lumber, paper and textiles to thickeners, films and explosives. The cell wall is secreted by each cell in the plant body, forming a thin fibreglass-like network with remarkable strength and flexibility. During growth, plant cells secrete a protein called expansin, which unlocks the network of wall polysaccharides, permitting turgor-driven cell enlargement. Germinating grass pollen also secretes an unusual expansin that loosens maternal cell walls to aid penetration of the stigma by the pollen tube. Expansin's action has puzzling implications for plant cell-wall structure. The recent explosion of gene sequences and expression data has given new hints of additional biological functions for expansins.   Download the full article in Acrobat pdf format (529 kB file). © Nature

Cosgrove DJ (2001) Wall structure and wall loosening. a look backwards and forwards.   Plant Physiol 125:131-134
ABSTRACT: a short review on our concepts of wall structure and how wall loosening and extension occur. Download pdf file (366 KB)  (c) ASPB

Cosgrove DJ. Growth of the plant cell wall. Nat.Rev.Mol.Cell Biol. 6 (11):850-861, 2005.
Abstract: Plant cells encase themselves within a complex polysaccharide wall, which constitutes the raw material that is used to manufacture textiles, paper, lumber, films, thickeners and other products. The plant cell wall is also the primary source of cellulose, the most abundant and useful biopolymer on the Earth. The cell wall not only strengthens the plant body, but also has key roles in plant growth, cell differentiation, intercellular communication, water movement and defense. Recent discoveries have uncovered how plant cells synthesize wall polysaccharides, assemble them into a strong fibrous network and regulate wall expansion during cell growth.  Download pdf

Cosgrove, DJ,  Bedinger P, and  Durachko DM (1997)  Group I allergens of grass pollen as cell wall-loosening agents.  Proc. Natl. Acad. Sci. 94: 6559-6564
ABSTRACT:  Group I allergens are the major allergens of grass pollen, but their biological function is unknown. These proteins are shown here to be structurally related to expansins, which are able to induce extension (creep) of plant cell walls. Extracts of maize pollen possess potent expansin-like activity, as measured in wall extension and wall stress- relaxation assays. This activity is selective for grass cell walls and is, at least partly, due to the action of maize group I allergens. We propose that group I allergens facilitate invasion of the pollen tube into the maternal tissues by loosening the cell walls of the grass stigma and style. Additionally, the presence of related mRNAs in vegetative tissues of rice, Arabidopsis, and soybean implies that allergen homologs may function to loosen walls in growing vegetative tissues as well.   Download the full article in Acrobat pdf format (407 kB file). © PNAS

Cosgrove DJ, Durachko DM (1994) Autolysis and extension of isolated walls from growing cucumber hypocotyls.  J. Exp. Bot. 45: 1711-1719
ABSTRACT: Cell walls isolated from cucumber hypocotyls retain autolytic activities and the ability to extend when placed under the appropriate conditions. To test whether autolysis and extension are related, hypocotyl cell walls from cv. Burpee Pickler were treated in various ways to enhance or inhibit long-term wall extension ('creep') and autolysis was measured as release of various saccharides from the wall. Except for some non-specific inhibitors of enzymatic activity, no correlation was found between wall extension and wall autolysis. Most notably, autolysis and extension differed strongly in their pH dependence. Exogenous cellulases and pectinases enhanced extension in native walls, but when applied to walls previously inactivated with heat or protease these enzymes caused breakage without sustained extension. In contrast, pretreatment of walls with pectinase or cellulase, followed by boiling in methanol to inactivate the enzymes, resulted in walls with much stronger expansin-mediated extension responses. Crude protein preparations from the digestive tracts of snails enhanced extension of both native and inactivated walls, and these preparations contained expansin-like proteins (assessed by Western blotting). The results indicate that the extension of isolated cucumber walls does not depend directly on the activity of endogenous wall-bound autolytic enzymes. The results with exogenous enzymes suggest that the hydrolysis of matrix polysaccharides may not induce wall creep by itself, but may act synergistically with expansins to enhance wall extension.

Cosgrove, D.J., Li, L.-C., Cho, H.T., Hoffmann-Benning, S. Moore, R.C., Blecker, D. 2002. The growing world of expansins. Plant & Cell Physiology 43: 1236-1244
ABSTRACT:  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 - and [1]-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. Download pdf.

Cosgrove DJ, Li Z-C (1993) Role of expansin in developmental and light control of growth and wall extension in oat coleoptiles. Plant Physiol. 103: 1321-1328
Abstract: Expansins are wall proteins that mediate a type of acid-induced extension in isolated plant cell walls (S. McQueen-Mason, D.M. Durachko, 0.1. Cosgrove [1992] Plant Cell4: 1425-1433). To assess the role of these proteins in the process of cell enlargement in living tissues, we compared the spatial and temporal growth patterns of oat (Avena sativa 1.) coleoptiles with four wall properties related to expansin action. These properties were (a) the ability of isolated walls and living segments to extend in acidic buffer, (b) the ability of heat-inadivated walls to extend upon application of expansins, (c) the amount of immunologically detedable expansin in wall protein extrads, and (d) the extradable expansin adivity of walls. Crowth rate was maximal in the apical half of dark-grown coleoptiles and negligible in the basal region. This growth pattern correlated with properties a and b; in contrast, the amount and adivity of extradable expansin (properties c and d) were reduced only in the most basal region. Upon exposure to white light, coleoptiles abruptly ceased elongation at 8 to 10 h after start of irradiation, and this cessation correlated with reductions in properties a to E. The growth cessation at 8 to 10 h also coincided with the loss of growth response to exogenous auxin and fusicoccin in excised coleoptile segments. These results lend correlative support to the hypothesis that expansin adion is important for growth responses of living oat coleoptiles (e.g. responses to acidic buffers, auxin, fusicoccin, aging, and light). Our results suggest that changes in the susceptibility of the wall to expansin adion, rather than changes in expansin adivity, may be a key determinant of the growth patterns in oat coleoptiles. Download pdf

M. C. Cox, J. J. Benschop, R. A. Vreeburg, C. A. Wagemaker, T. Moritz, A. J. Peeters, and L. A. Voesenek. The roles of ethylene, auxin, bbscisic acid, and gibberellin in the hyponastic growth of submerged Rumex palustris petioles. Plant Physiol, 2004.
Abstract: Rumex palustris responds to complete submergence with upward movement of the younger petioles. This so-called hyponastic response, in combination with stimulated petiole elongation, brings the leaf blade above the water surface and restores contact with the atmosphere. We made a detailed study of this differential growth process, encompassing the complete range of the known signal transduction pathway: from the cellular localization of differential growth, to the hormonal regulation, and the possible involvement of a cell wall loosening protein (expansin) as a downstream target. We show that hyponastic growth is caused by differential cell elongation across the petiole base, with cells on the abaxial (lower) surface elongating faster than cells on the adaxial (upper) surface. Pharmacological studies and endogenous hormone measurements revealed that ethylene, auxin, abscisic acid (ABA), and gibberellin regulate different and sometimes overlapping stages of hyponastic growth. Initiation of hyponastic growth and (maintenance of) the maximum petiole angle are regulated by ethylene, ABA, and auxin, whereas the speed of the response is influenced by ethylene, ABA, and gibberellin. We found that a submergence-induced differential redistribution of endogenous indole-3-acetic acid in the petiole base could play a role in maintenance of the response, but not in the onset of hyponastic growth. Since submergence does not induce a differential expression of expansins across the petiole base, it is unlikely that this cell wall loosening protein is the downstream target for the hormones that regulate the differential cell elongation leading to submergence-induced hyponastic growth in R. palustris.

Crowell DN (1994) Cytokinin regulation of a soybean pollen allergen gene. Plant Mol. Biol. 25: 829-835

Darley CP, Forrester AM, McQueen-Mason SJ (2001)  The molecular basis of plant cell wall extension. Plant Mol Biol 47:179-95
ABSTRACT: In all terrestrial and aquatic plant species the primary cell wall is a dynamic structure, adjusted to fulfil a diversity of functions. However a universal property is its considerable mechanical and tensile strength, whilst being flexible enough to accommodate turgor and allow for cell elongation. The wall is a composite material consisting of a framework of cellulose microfibrils embedded in a matrix of non-cellulosic polysaccharides, interlaced with structural proteins and pectic polymers. The assembly and modification of these polymers within the growing cell wall has, until recently, been poorly understood. Advances in cytological and genetic techniques have thrown light on these processes and have led to the discovery of a number of wall-modifying enzymes which, either directly or indirectly, play a role in the molecular basis of cell wall expansion.

Darley,C.P., Li,Y., Schaap,P., and McQueen-Mason,S.J. (2003) Expression of a family of expansin-like proteins during the development of Dictyostelium discoideum. FEBS Lett. 546:416-418.

Dotto MC, Martinez GA, Civello PM. (2006) Expression of expansin genes in strawberry varieties with contrasting fruit firmness.  Plant Physiol Biochem. 44(5-6):301-7
Abstract: Fruit softening is associated with cell wall disassembly mediated by the action of a complex set of enzymes and proteins. Expansins, a group of proteins with unknown enzymatic activity, are proposed to be involved in this process. In order to study the involvement of expansins in strawberry fruit softening we have analyzed the expression level of five expansin mRNAs (FaEXP1, FaEXP2, FaEXP4, FaEXP5 and FaEXP6) in the cultivars "Selva", "Camarosa" and "Toyonaka", which differ in fruit firmness during ripening. We have found a correlation between mRNA expression levels and fruit firmness for FaEXP1, FaEXP2 and FaEXP5. For these three mRNAs we have observed higher expression levels in the softest cultivar (Toyonaka) than in the other two firmer cultivars (Selva and Camarosa) at the beginning of ripening. This correlation was not found in the case of FaEXP4 and FaEXP6, although both genes displayed a different expression pattern in the three cultivars analyzed. Western-blot analysis revealed that the accumulation of expansin proteins begins earlier in the softest cultivar during ripening. Download pdf.

Downes, BP; Crowell, DN (1998)   Cytokinin regulates the expression of a soybean beta-expansin gene by a post-transcriptional mechanism.  Plant Mol.  Biol., 37:  437-444 
ABSTRACT: The cytokinin-inducible soybean mRNA Cim1 accumulates 20-60-fold upon cytokinin addition to cytokinin-starved soybean suspension cultures. In this report, we demonstrate that cytokinin-induced stability of the Cim1 mRNA plays an important role in the accumulation of the message. We also present evidence that cytokinin-induced Cim1 stability is blocked by the addition of the protein phosphatase inhibitor okadaic acid. Thus, we suggest that protein phosphatase activity is required for the cytokinin-induced stability and subsequent accumulation of Cim1 in soybean cells. The deduced amino acid sequence of the Cim1 protein product is similar to the group I pollen allergens from various plants, which constitute a subfamily of expansin proteins. The relatedness between Cim1 and the expansins supports our hypothesis that the protein product of Cim1 is localized to the cell wall and suggests a role for Cim1 in cytokinin-regulated cell wall expansion. Thus, post-transcriptional regulation of Cim1 by cytokinin may represent a molecular link between cytokinin and changes in cell shape and size.

Downes,B.P.; Steinbaker,C.R.; Crowell,D.N. (2001) Expression and Processing of a Hormonally Regulated beta-Expansin from Soybean. Plant Physiology 126: 244-252
ABSTRACT. Expansin proteins are essential components of acid-induced cell wall loosening in plants. beta-Expansins, which constitute a subfamily of related expansin proteins, include the group I grass pollen allergens. To provide a better description of beta-expansin expression, we have characterized a cytokinin-inducible beta-expansin from soybean (Glycine max cv Mandarin) called Cim1. Our results demonstrate that the hormones cytokinin and auxin act synergistically to induce the accumulation and proteolytic processing of Cim1. Carboxyl terminal truncation of a 35-kD form of Cim1 is predicted to remove the putative cellulose binding domain from the amino terminal cysteine-rich domain, resulting in a 20- kD form of the protein. Furthermore, the identical amino termini of the 35- and 20-kD forms of Cim1 correspond to a position 11 amino acids downstream of the predicted signal sequence cleavage site, suggesting proteolysis of a short amino terminal propeptide after removal of the signal peptide. This propeptide fragment contains a consensus site for N-glycosylation and our data suggest that it is glycosylated by a tunicamycin-sensitive mechanism in cultured soybean cells. The onset of Cim1 expression correlates with increased growth of soybean cultures. Ultimately, Cim1 is rapidly and specifically proteolyzed as soybean cultures reach stationary phase. These findings are consistent with the hypothesis that beta-expansin proteins are extensively modified by post- translational N-glycosylation and proteolysis. (c)ASPP Download Acrobat PDF file (526kb)

Esmon CA, Tinsley AG, Ljung K, Sandberg G, Hearne LB, Liscum E.  A gradient of auxin and auxin-dependent transcription precedes tropic growth responses. Proc Natl Acad Sci U S A. 2006 Jan 3; 103(1): 236-241.
ABSTRACT: Plants, although sessile, can reorient growth axes in response to changing environmental conditions. Phototropism and gravitropism represent adaptive growth responses induced by changes in light direction and growth axis orientation relative to gravitational direction, respectively. The nearly 80-year-old Cholodny–Went theory [Went, F. W. & Thimann, K. V. (1937) Phytohormones (Macmillan, New York)] predicts that formation of a gradient of the plant morphogen auxin is central to the establishment of tropic curvature. Loss of tropic responses in seedling stems of Arabidopsis thaliana mutants lacking the auxin-regulated transcriptional activator NPH4/ARF7 has further suggested that a gradient of gene expression represents an essential output from the auxin gradient. Yet the molecular identities of such output components, which are likely to encode proteins directly involved in growth control, have remained elusive. Here we report the discovery of a suite of tropic stimulus-induced genes in Brassica oleracea that are responsive to an auxin gradient and exhibit morphologically graded expression concomitant with, or before, observable curvature responses. These results provide compelling molecular support for the Cholodny–Went theory and suggest that morphologically graded transcription represents an important mechanism for interpreting tropically stimulated gradients of auxin. Intriguingly, two of the tropic stimulus-induced genes, EXPA1 and EXPA8, encode enzymes involved in cell wall extension, a response prerequisite for differential growth leading to curvatures, and are up-regulated before curvature in the flank that will elongate. This observation suggests that morphologically graded transcription likely leads to the graded expression of proteins whose activities can directly regulate the establishment and modulation of tropic curvatures. Download PDF file (500K)

A. Ferrante, D. A. Hunter, and M. S. Reid. Towards a molecular strategy for improving harvesting of olives (Olea europaea L.). Postharvest Biology and Technology 31 (2):111-117, 2004.
Abstract: Mature olive fruit that produced barely detectable levels of ethylene produced much greater quantities when they were briefly dipped in a solution containing 2 mM 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene. A single 10 s dip resulted in a transient increase in ethylene production that peaked I day after the application. When the fruits were dipped daily, their ethylene production remained elevated and their attachment to their pedicels weakened after 3 days. As a first step towards producing mature olive fruit with elevated levels of ACC leading to weakened abscission zone tissue, we examined the expression of genes whose promoters might be potential candidates for driving maturation-specific expression of an ACC synthase construct in the olive. We examined the accumulation of three distinct transcripts (chalcone synthase, OE-CHS1; anthocyanidin synthase, OE-ANS1; expansin, OE-EXP1) at five stages of olive fruit development. Northern analysis showed that all three gene transcripts accumulated during ripening. Transcript abundance was lowest in green fruit, higher as the pericarp began to darken and highest at the stage where the exocarp was completely purple and the mesocarp beginning to color. None of the transcripts were detected in either young or aged leaves suggesting that they may be specific to the fruit and useful candidates for promoter isolation.

Fleming AJ, McQueen-Mason S, Mandel T, Kuhlemeier C (1997) Induction of leaf primordia by the cell wall protein expansin. Science 276: 1415-1418
ABSTRACT:

Fleming, AJ; Caderas, D; Wehrli, E, McQueen-Mason, S; Kuhlemeier, C (1999) Analysis of expansin-induced morphogenesis on the apical meristem of tomato.   Planta  208: 166-174 
ABSTRACT: Our previous work has shown that localised activity of the cell-wall-loosening protein expansin is sufficient to induce primordia on the apical meristem of tomato, consistent with the hypothesis that tissue expansion plays a key role in leaf initiation. In this paper we describe the earliest morphogenic events visible on the surface of the apical meristem of tomato (Lycopersicon esculentum Mill.) following treatment with expansin and report on the spectrum of final structures formed. Our observations are consistent with a proposed primary function of expansin effecting morphogenesis via altered biophysical stress patterns in the meristem. The primordia induced by expansin do not complete the full program of leaf development. We present data indicating that one reason for this might be the inability of exogenous expansin to mimic the endogenous pattern of expansin activity in the meristem. These data provide the first detailed analysis at the cellular level of expansin action on living tissue, the first description of the spectrum of structures induced by expansin on the apical meristem, and give an insight into a potentially fundamental mechanism in plant development.

Fonseca S., L. Monteiro, M. G. Barreiro, and M. S. Pais. 2005. Expression of genes encoding cell wall modifying enzymes is induced by cold storage and reflects changes in pear fruit texture. J.Exp.Bot. 56 (418):2029-2036.
Abstract: Preclimacteric 'Rocha' pears stored under chilling conditions, had a larger increase of ACO (1-aminocyclopropane-1-carboxylate oxidase) activity and softened faster than those treated with ethylene. Non-treated fruit did not ripen or soften, acquired a rubbery texture, and showed barely detectable levels of ACO activity. The transcript accumulation of seven genes encoding cell wall modifying enzymes was followed during fruit growth, ripening, and senescence, and in fruit that failed to ripen, by quantitative real-time PCR. Transcripts from 'Rocha' pear polygalacturonase1 and 2 (PcPG1, PcPG2), beta-galactosidase (Pc beta GAL) and beta-xylosidase (PcXYL) genes accumulated up to 1000-fold at the climacteric onset, while low transcript levels were detected in growing fruit. In fruit that did not ripen, this transcript accumulation was lower compared with fruits that ripened normally. Transcripts for expansin1 and 2 (PcEXPA1, PcEXPA2) accumulated in growing fruit, but about 10-fold more in fruit after rewarming. Xyloglucan endotransglucosylase/hydrolase (PcXTH) had the highest basal expression levels in all samples, showing only a small increase during fruit growth and ripening. PcEXPA2 and PcXTH transcripts accumulated in untreated fruit, 21 d after harvest, to levels similar to those of fruit that ripened normally. Since in untreated fruit ACO activity was barely detectable, it is likely that the activation of these genes might occur at very low ethylene levels. Results suggest that PcXTH and PcEXPA2 gene induction might be associated with cell wall maintenance during 'Rocha' pear development and ripening, while PcEXPA1, PcPG1, PcPG2, Pc beta GAL, and PcXYL expression is likely to be related to cell wall disassembly and loosening.

Fudali S, Janakowski S, Sobczak M, Griesser M, Grundler FM, Golinowski W. 2008.  Two tomato alpha-expansins show distinct spatial and temporal expression patterns during development of nematode-induced syncytia. Physiol Plant. 132(3):370-83.
ABSTRACT: Cyst nematodes induce specific syncytial feeding structures within the root which develop from an initial cell by successive incorporation of neighbouring cells through local cell wall dissolutions followed by hypertrophy of included cells. Expansins are known to induce cell wall relaxation and extension in acidic pH, and they are involved in many processes requiring wall modification from cell expansion to cell wall disassembly. We studied the expression pattern of tomato (Lycopersicon esculentum L., cv. Money Maker) expansins during development of syncytia induced by the potato cyst nematode (Globodera rostochiensis Woll.). Based on semi-quantitative reverse transcription-polymerase chain reaction, two expansin genes, LeEXPA4 and LeEXPA5, were selected for detailed examinations because their expression was either elevated in infected roots (LeEXPA4) or specifically induced in the root upon nematode infection (LeEXPA5). Both genes have distinct spatial and temporal expression patterns that may reflect their different roles in syncytium development. LeEXPA4 transcripts were localized predominantly in parenchymatous vascular cylinder cells surrounding syncytia. This finding suggests that LeEXPA4 might be involved in cell wall disassembly or relaxation, mediating syncytium expansion and/or development of conductive tissues. By contrast, LeEXPA5 transcripts were localized in enlarging syncytial elements. Similarly, in immunogold localization experiments, polyclonal antibodies localized the LeEXPA5 protein in cell walls of syncytial elements. This expression pattern suggests that LeEXPA5 gene is specifically involved in enlargement of cells incorporated into syncytium. Download pdf (C) Blackwell

Gao Q, Guo QF, Xing SC, Zhao MR, Li F, Wang W. 2007.  The characteristics of expansins in wheat coleoptiles and their responses to water stress. Zhi Wu Sheng Li Yu Fen Zi Sheng Wu Xue Xue Bao 33(5):402-10
ABSTRACT: As the key regulators of cell wall extension during plant growth, expansins play an important role in regulating the development and response of plants to adverse environment. The characteristics of expansins in wheat coleoptiles and their responses to water stress were studied. Expansin proteins were extracted from wheat coleoptiles by the methods of Hepes or SDS. The activities of expansins were measured with an improved extensometer and the amount of expansins was measured by immunoblot analysis with the expansin antibody. The results showed that in coleoptiles, the extension of native cell walls depended on acidic pH, and the expansins were found to be located at cell walls by location analysis. Expansins from wheat coleoptiles could induce cell wall extension both of cucumber hypocotyls and coleoptiles, and vice versa, albeit with differences noted in extension activity. The changes in activity and abundance of expansins in wheat coleoptiles in response to water stress suggest that expansins may play a significant role in the tolerance of wheat plants to water stress. 

Gao Q, Zhao M, Li F, Guo Q, Xing S, Wang W. 2008. Expansins and coleoptile elongation in wheat. Protoplasma. 2008 DOI 10.1007/s00709-008-0303-1
ABSTRACT: 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.  Download pdf (C) Springer

Gal TZ, Aussenberg ER, Burdman S, Kapulnik Y, Koltai H.Expression of a plant expansin is involved in the establishment of root knot nematode parasitism in tomato. Planta:1-8, 2006.
Abstract: A group of plant proteins, expansins, have been identified as wall-loosening factors and as facilitators of cell expansion in vivo. The root knot nematode Meloidogyne javanica establishes a permanent feeding site composed of giant cells surrounded by gall tissue. We used quantitative PCR and in situ localization to demonstrate the induction of a tomato (Lycopersicon esculentum cv. VF36) expansin (LeEXPA5) expression in gall cells adjacent to the nematode feeding cells. To further characterize the biological role of LeEXPA5 we have generated LeEXPA5-antisense transgenic roots. The ability of the nematode to establish a feeding site and complete its life cycle, the average root cell size and the rate of root elongation were determined for the transgenic roots, as well as the level of LeEXPA5 expression in non-infected and nematode-infected roots. Our results demonstrated that a decrease of LeEXPA5 expression reduces the ability of the nematode to complete its life cycle in transgenic roots. We suggest that a plant-originated expansin is necessary for a successful parasitic nematode-plant interaction. Download pdf

Gehring,C.A. and Irving,H.R. (2003) Natriuretic peptides - a class of heterologous molecules in plants. International Journal of Biochemistry & Cell Biology 35:1318-1322.
Abstract: Immunological and physiological evidence suggests the presence of biologically active natriuretic peptide hormones (NPs) in plants. Evidence includes specific binding of rat atrial NP, [rANP (99-126)] to plant membranes and the promotion of cyclic guanosine-3',5'-monophosphate (cGMP) mediated stomatal responses. Furthermore, anti-ANP affinity purifies biologically active plant immunoreactants (irPNPs) and a biologically active Arabidopsis thaliana irPNP (AtPNP-A) has been identified. AtPNP-A belongs to a novel class of molecules that share some similarity with the cell wall loosening expansins but do not contain the carbohydrate-binding wall anchor, thus suggesting that irPNPs and ANP are heterologues. We hypothesise that irPNP-like molecules have evolved from primitive glucanase-like molecules that have been recruited to become systemically mobile modulators of homeostasis acting via the plasma membrane. Such a function is compatible with localisation in the conductive tissue and the physiological and cellular modes of action of irPNPs reported to-date. (C) 2003 Elsevier Science Ltd.

Geshi N, Rechinger K, Brandt A. (1998) A Full Length cDNA Clone from Brassica napus Encoding Expansin (Accession No. AJ000885). (PGR98-068). Plant Physiol. 116: 1605

Giordano W, Hirsch AM. (2004) The expression of MaEXP1, a Melilotus alba expansin gene, is upregulated during the sweetclover-Sinorhizobium meliloti interaction. Mol Plant Microbe Interact. 17:613-22.
Abstract:  Expansins are a highly conserved group of cell wall-localized proteins that appear to mediate changes in cell wall plasticity during cell expansion or differentiation. The accumulation of expansin protein or the mRNA for specific expansin gene family members has been correlated with the growth of various plant organs. Because expansin proteins are closely associated with plant cell wall expansion, and as part of a larger study to determine the role of different gene products in the legume-Rhizobium spp. symbiosis, we investigated whether a Melilotus alba (white sweetclover) expansin gene is expressed during nodule development. A cDNA fragment encoding an expansin gene (EXP) was isolated from Sinorhizobium meliloti-inoculated sweetclover root RNA by reverse-transcriptase polymerase chain reaction using degenerate primers, and a full-length sweetclover expansin sequence (MaEXP1) was obtained using 5' and 3' rapid amplification of cDNA end cloning. The predicted amino acid of the sweetclover expansin is highly conserved with the various alpha-expansins in the GenBank database. MaEXP1 contains a series of eight cysteines and four tryptophans that are conserved in the alpha-expansin protein family. Northern analysis and whole-mount in situ hybridization analyses indicate that MaEXP1 mRNA expression is enhanced in roots within hours after inoculation with S. meliloti and in nodules. Western and immunolocalization studies using a cucumber expansin antibody demonstrated that a cross-reacting protein accumulated in the expanding cells of the nodule.

Gookin,T.E., Hunter,D.A., and Reid,M.S. (2003) Temporal analysis of alpha and beta-expansin expression during floral opening and senescence. Plant Science 164:769-781.
Abstract: We have identified a family of expansin transcripts that include seven alpha-expansins (MjExp1 through MjExp7) and three beta-expansins (MjExpB1 through MjExpB3) from Mirabilis jalapa (Nyctaginaceae) that show dramatic changes in transcript abundance during the rapid expansion and subsequent senescence of the ephemeral flowers. In general, alpha-expansin expression was low in small buds, high during maximal elongation of the floral tube, reduced during floral display, and upregulated during calyx infolding and collapse. Transcripts encoding auxin responsive proteins (Aux/IAA) showed a similar pattern of expression. Northern analysis using a set of overlapping probes designed to the MjExp2 transcript demonstrated a gradient of sequence conservation along its length (high to low, from the 5' to the 3' end of the coding region), and identified the presence of floral senescence-specific expansins. Beta expansin transcripts were found to be preferentially expressed during early floral development and sharply downregulated coincident with rapid growth. All three beta-expansin transcripts are highly related, and psiMjExpB2 is an intronless pseudogene derived from MjExpB1. MjExpB3 appears to have derived from MjExpB1 in a separate gene duplication event, and is predicted to encode a truncated protein. (C) 2003 Elsevier Science Ireland Ltd.

Gray-Mitsumune M, Blomquist K, McQueen-Mason S, Teeri TT, Sundberg B, Mellerowicz EJ. 2008. Ectopic expression of a wood-abundant expansin PttEXPA1 promotes cell expansion in primary and secondary tissues in aspen. Plant Biotechnol J. 6(1):62-72.
ABSTRACT: Expansins are primary agents inducing cell wall extension, and are therefore obvious targets in biotechnological applications aimed at the modification of cell size in plants. In trees, increased fibre length is a goal of both breeding and genetic engineering programmes. We used an alpha-expansin PttEXPA1 that is highly abundant in the wood-forming tissues of hybrid aspen (Populus tremula L. x P. tremuloides Michx.) to evaluate its role in fibre elongation and wood cell development. PttEXPA1 belongs to Subfamily A of alpha-expansins that have conserved motifs at the N- and C-termini of the mature protein. When PttEXPA1 was over-expressed in aspen, an extract of the cell wall-bound proteins of the transgenic plants exhibited an increased expansin activity on cellulose-xyloglucan composites in vitro, indicating that PttEXPA1 is an active expansin. The transgenic lines exhibited increased stem internode elongation and leaf expansion, and larger cell sizes in the leaf epidermis, indicating that PttEXPA1 protein is capable of increasing the growth of these organs by enhancing cell wall expansion in planta. Wood cell development was also modified in the transgenic lines, but the effects were different for vessel elements and fibres, the two main cell types of aspen wood. PttEXPA1 stimulated fibre, but not vessel element, diameter growth, and marginally increased vessel element length, but did not affect fibre length. The observed differences in responsiveness to expansin of these cell types are discussed in the light of differences in their growth strategies and cell wall composition.  Downlaod pdf  (C) Blackwell

Gray-Mitsumune M, Mellerowicz EJ, Abe H, Schrader J, Winzell A, Sterky F, Blomqvist K, McQueen-Mason S, Teeri TT, Sundberg B. (2004) Expansins Abundant in Secondary Xylem Belong to Subgroup A of the {alpha}-Expansin Gene Family. Plant Physiology
Abstract: Differentiation of xylem cells in dicotyledonous plants involves expansion of the radial primary cell walls and intrusive tip growth of cambial derivative cells prior to the deposition of a thick secondary wall essential for xylem function. Expansins are cell wall-residing proteins that have an ability to plasticize the cellulose-hemicellulose network of primary walls. We found expansin activity in proteins extracted from the cambial region of mature stems in a model tree species hybrid aspen (Populus tremula x Populus tremuloides Michx). We identified three alpha-expansin genes (PttEXP1, PttEXP2, and PttEXP8) and one beta-expansin gene (PttEXPB1) in a cambial region expressed sequence tag library, among which PttEXP1 was most abundantly represented. Northern-blot analyses in aspen vegetative organs and tissues showed that PttEXP1 was specifically expressed in mature stems exhibiting secondary growth, where it was present in the cambium and in the radial expansion zone. By contrast, PttEXP2 was mostly expressed in developing leaves. In situ reverse transcription-PCR provided evidence for accumulation of mRNA of PttEXP1 along with ribosomal rRNA at the tips of intrusively growing xylem fibers, suggesting that PttEXP1 protein has a role in intrusive tip growth. An examination of tension wood and leaf cDNA libraries identified another expansin, PttEXP5, very similar to PttEXP1, as the major expansin in developing tension wood, while PttEXP3 was the major expansin expressed in developing leaves. Comparative analysis of expansins expressed in woody stems in aspen, Arabidopsis, and pine showed that the most abundantly expressed expansins share sequence similarities, belonging to the subfamily A of alpha-expansins and having two conserved motifs at the beginning and end of the mature protein, RIPVG and KNFRV, respectively. This conservation suggests that these genes may share a specialized, not yet identified function. (c) ASPB  PDF file

Green PB (1997) Expansin and morphology: a role for biophysics. Trends.Plant Sci. 2: 365-366

Grobe,K., Poppelmann,M., Becker,W.M., and Petersen,A. (2002) Properties of group I allergens from grass pollen and their relation to cathepsin B, a member of the C1 family of cysteine proteinases. European Journal of Biochemistry 269:2083-2092.
Abstract: Expansins are a family of proteins that catalyze pH-dependent long-term extension of isolated plant cell walls. They are divided into two groups, alpha and beta, the latter consisting of the grass group 1 pollen allergens and their vegetative homologs. Expansins are suggested to mediate plant cell growth by interfering with either structural proteins or the polysaccharide network in the cell wall. Our group reported papain-like properties of beta-expansin of Timothy grass (Phleum pratense) pollen, Phl p 1, and suggested that cleavage of cell wall structural proteins may be the underlying mechanism of expansin-mediated wall extension. Here, we report additional data showing that beta-expansins resemble ancient and modern cathepsin B, which is a member of the papain (C1) family of cysteine proteinases. Using the Pichia pastoris expression system, we show that cleavage of inhibitory prosequences from the recombinant allergen is facilitated by its N-glycosylation and that the truncated, activated allergen shows proteolytic activity, resulting in very low stability of the protein. We also show that deglycosylated, full-length allergen is not activated efficiently and therefore is relatively stable. Motif and homology search tools detected significant similarity between beta-expansins and cathepsins of modern animals as well as the archezoa Giardia lamblia, confirming the presence of inhibitory prosequences, active site and other functional amino-acid residues, as well as a conserved location of these features within these molecules. Lastly, we demonstrate by site-directed mutagenesis that the conserved His 104 residue is involved in the catalytic activity of beta-expansins. These results indicate a common origin of cathepsin B and beta-expansins, especially if taken together with their previously known biochemical properties (editor's note: these conclusions are not supported by the crystal structure of expansin (Yennawar et al. 2006) or other activity analyses (Li & Cosgrove 2001) ).

Harmer,S.E., Orford,S.J., and Timmis,J.N. (2002) Characterisation of six alpha-expansin genes in Gossypium hirsutum (upland cotton). Molecular Genetics and Genomics 268:1-9.
Abstract: A genomic library screen and PCR-based strategies were employed to isolate six genes with sequence similarity to a cotton fibre-specific mRNA encoding an alpha-expansin. alpha-Expansins are cell wall proteins that facilitate cell wall extension by disruption of non-covalent bonds between wall components. The characterisation and expression analysis of these six novel genes (GhExp1-GhExp6) is described. Four of them (GhExp3-GhExp6) are expressed within multiple tissues of the cotton plant and two (GhExp1 and GhExp2) give rise to transcripts that are specific to the developing cotton fibre. GhExp1 transcripts are highly abundant in the fibre, while transcripts for GhExp2 are detected at a low level. Cotton fibres are highly elongated cells of the ovule epidermis, and we envisage that GhExp1 may play an important role in cell wall extension during development

Hayama,H., Ito,A., Moriguchi,T., and Kashimura,Y. (2003) Identification of a new expansin gene closely associated with peach fruit softening. Postharvest Biology and Technology 29:1-10.
Abstract: Expansins are proteins that have been shown to contribute to fruit softening in tomato. However, expansins that have been correlated with loss of fruit firmness have not yet been identified in peach (Prunus persica (L.) Batsch). Along with the previously isolated PpExp1, two new expansin cDNAs, termed PpExp2 and PpExp3, were isolated from ripe peach fruit, and their mRNA expression patterns were characterized during fruit development and in other tissues, including the flower bud, leaf, and stem. All three expansins were detected in the fruit and not in the other tissues, but each showed differential patterns of mRNA accumulation during fruit development. The PpExp2 mRNA was constitutively expressed throughout fruit development but was abundant in Stage III, when the fruit expands exponentially and then matures. The PpExp1 and PpExp3 mRNAs were up-regulated at the onset of ripening, but PpExp1 was induced at an earlier stage. In order to identify the expansins whose expression correlates with the loss of peach fruit firmness, the mRNA expression levels of the three expansins were compared in the ripe fruit of the 'Akatsuki' and 'Manami' cultivars during postharvest storage. During storage, the ripe fruit of 'Akatsuki' rapidly softened as the level of ethylene increased significantly, while 'Manami' fruit remained firm and exhibited very low levels of ethylene production. The PpExp1 and PpExp2 mRNAs were constitutively detectable during the 8-day storage of both cultivars, whereas PpExp3 mRNA was detectable in 'Akatsuki' but hardly detectable in 'Manami,' suggesting that PpExp3 expression may be related to the changes in fruit firmness. To address the detailed role of PpExp3 in the loss of fruit firmness, the fruit of 'Manami' was treated by ethylene to artificially induce softening. The PpExp3 mRNA accumulation in the ethylene-treated 'Manami' was detectable and similar to that observed in 'Akatsuki.' These results show that, while several expansins show a general increase in expression levels during the later stages of fruit development, some isoforms show a greater association with softening than others. In this regard, PpExp3 is more likely to play a role in peach fruit softening than PpExp1 or PpExp2. (C) 2002 Elsevier Science B.V.

Hayama,H., Shimada,T., Ito,A., Yoshioka,H., and Kashimura,Y. (2001) Changes in the levels of mRNAs for putative cell wall-related genes during peach fruit development. Scientia Horticulturae 91:239-250.
Abstract: Cell wall changes are important factors for understanding fruit development. Four cDNA clones putatively encoding xyloglucan endotransglycosylase. (pTpXet1), expansin (pfPpExp2), sucrose synthase (pfPpSS1), and cellulose synthase (pfPpCesA1), which are all assumed to relate to cell wall modification, were isolated from developing fruit of peach (Prunus persica cv. Akatsuki), and the relationships between their mRNA accumulations and fruit development were investigated. Each of these four clones showed a different and characteristic pattern of mRNA expression. For example, the accumulation pattern of pfPpExp2 mRNA was in accordance with peach fruit development; i.e., it is abundant in the fruit when the fruit is growing and hardly detectable in the fruit when the fruit growth slows. The pfPpXet1 mRNA was detected in fruit in the earlier stages of development only. The result suggests that cell wall-related genes are differentially regulated during fruit development in peach. (C) 2001 Elsevier Science B.V. All rights reserved

Hiwasa,K., Rose,J.K.C., Nakano,R., Inaba,A., and Kubo,Y. (2003) Differential expression of seven alpha-expansin genes during growth and ripening of pear fruit. Physiologia Plantarum 117:564-572.
Abstract: Seven cDNAs, designated PcExp1 to PcExp7 , encoding expansin homologues, were isolated from mature pear fruit and their expression profiles were investigated in ripening fruit and other tissues, and in response to ethylene. Accumulation of PcExp2 , -3, -5 and -6 mRNA increased markedly with fruit softening and then declined at the over-ripe stage. Treatment of fruit at an early ripening stage with 1-methylcyclopropene (MCP), an inhibitor of ethylene action, suppressed ethylene biosynthesis, fruit softening and the accumulation of the expansin mRNAs. Conversely, propylene treatment at the preclimacteric stage induced accumulation of the same four expansin genes, as well as ethylene production and fruit softening. The expression patterns correlated with alteration in the rate and extent of fruit softening. The abundance of PcExp1 mRNA increased at the late expanding phase of fruit development and further increased during ripening, whereas PcExp4 mRNA levels were constant throughout fruit growth and ripening. The MCP and propylene treatments had little effect on PcExp1 and PcExp4 expression. PcExp7 was expressed in young but not mature fruit. PcExp4 and PcExp6 mRNA was also detected in flowers. The accumulation of PcExp4, -5, -6 and -7 mRNA was more abundant in young growing tissues, but not in fully expanded tissues, suggesting roles for these genes in cell expansion. These results demonstrate that characteristically, multiple expansin genes show differential expression and hormonal regulation during pear fruit development and at least six expansins show overlapping expression during ripening

Hoeberichts,F.A., Van der Plas,L.H.W., and Woltering,E.J. (2002) Ethylene perception is required for the expression of tomato ripening-related genes and associated physiological changes even at advanced stages of ripening. Postharvest Biology and Technology 26:125-133.
Abstract: Treatment of tomato fruit (Lycopersicon esculentum L. cv Prisca) with 1-metbyleyelopropene (1-MCP), a potent inhibitor of ethylene action, delayed colour development, softening, and ethylene production in tomato fruit harvested at the mature green breaker, and orange stages. 1-MCP treatment also decreased the mRNA abundance of phytoene synthase 1 (PSY1), expansin 1 (EXP1), and 1-aminocyclopropane-l-carboxylic acid (ACC) oxidase 1 (ACO1), three ripening-related tomato genes, in mature green, breaker, orange, and red ripe fruit. These results demonstrate that the ripening process can be inhibited both on a physiological and molecular level, even at very advanced stages of ripening. The effects of 1-MCP on ripening lasted 5-7 days and could be prolonged by renewed exposure. (C) 2002 Elsevier Science B.V.

Huang J, Takano T, Akita S.  (2000)  Expression of alpha-expansin genes in young seedlings of rice (Oryza sativa L.).  Planta  211: 467-473
ABSTRACT: Rice is the only cereal in which germination and coleoptile elongation occur in hypoxia or anoxia. Little is known of the molecular basis directly underlying coleoptile cell extension. In this paper, we describe the expression of alpha-expansin genes in embryos during seed development and young seedlings grown under various oxygen concentrations. The genes Os-EXP2 and Os-EXP1 were predominantly expressed in the developing seeds, mainly in newly developed leaves, coleoptiles, and seminal roots. These expansins expressed in the developing seeds may give cells the potential to expand after seed imbibition begins. In coleoptiles, Os-EXP4 and Os-EXP2 mRNAs were greatly induced by submergence, while they were weakly detected in aerobic or anoxic conditions. Under submerged soil conditions, the signals hybridized with probes Os-EXP4 and Os-EXP2 in coleoptiles were strongest when coleoptiles elongated in the water layer. These data show that expansin gene expression is highly correlated with coleoptile elongation in response to oxygen concentrations. The Os-EXP4 gene was also expressed in leaves, mesocotyls, and coleorhizas of young seedlings. The growth of these tissues was also correlated with the presence of expansins. Therefore, the evidence derived from this study clearly demonstrates that expansins are indispensable for the growing tissues of rice seedlings Download pdf file (284 kB) (c) Springer-Verlag

Hutchison, KW; Singer, PB; McInnis, S; Diaz-Salaz, C; Greenwood, MS   (1999)  Expansins are conserved in conifers and expressed in hypocotyls in response to exogenous auxin.  Plant Physiol. 120: 827-831
ABSTRACT: Differential display reverse transcription-polymerase chain reaction was used to detect the induction of gene expression during adventitious root formation in loblolly pine (Pinus taeda) after treatment with the exogenous auxin indole-3-butyric acid. A BLAST search of the GenBank database using one of the clones obtained revealed very strong similarity to the alpha-expansin gene family in angiosperms. A near-full-length loblolly pine alpha-expansin sequence was obtained using 5'- and 3'-rapid amplification of cDNA end cloning, and the deduced amino acid sequence was highly conserved relative to those of angiosperm expansins. Northern analysis indicates that alpha-expansin mRNA expression increases 50- to 100-fold in the base of hypocotyl stem cuttings from loblolly pine seedlings in response to indole-3-butyric acid, with peak expression occurring 24 to 48 h after induction. Download pdf file (284 kB) (c) ASPP

Im, H-K, Cosgrove, DJ, Jones AM.  (2000)  Subcellular localization of expansin mRNA in xylem cells.  Plant Physiol. 123:463-470
ABSTRACT:  Terminal differentiation of many vascular cells involves cell wall changes. Cells first elongate their primary wall, then lay down a lignified secondary wall, which is often followed by digestion of the primary wall. Expansins are wall proteins that regulate wall changes, but little is known about the specific functions of the many individual expansin isoforms. An in vitro cell culture of synchronously differentiating tracheary elements was used to identify three new expansins and to compare their expression kinetics with the timing of wall changes. The genes encoding these expansins from zinnia (Zinnia elegans), designated ZeExp1, ZeExp2, ZeExp3, are expressed during cell elongation. ZeExp1 and ZeExp2 mRNA decrease at the early stage of secondary wall formation, whereas ZeExp3 does not. In planta, all three ZeExp mRNAs are found predominantly in a single flank of cells adjacent to protoxylem and metaxylem vessels and in cells roughly at the radial position of the fasicular and interfasicular cambium. Furthermore, within these cells, Exp mRNA is localized exclusively either to the apical or basipetal end of cells depending on the expansin gene and organ, providing the first evidence for polar localization of mRNA in plant cells. ZeExp1 and ZeExp3 mRNA are localized at the apical tip, whereas ZeExp2 mRNA is found in the basal tip. These observations indicate that these three expansins are xylem cell specific and possibly involved in the intrusive growth of the primary walls of differentiating xylem cells.  Download pdf file (1,290 KB) (c) ASPP

Imin N, Kerim T, Rolfe BG, Weinman JJ. 2004.  Effect of early cold stress on the maturation of rice anthers. Proteomics. 4(7):1873-82.
ABSTRACT: Male reproductive development in rice (Oryza sativa Linnaeus is very sensitive to various forms of environmental stresses including low temperature. Here, we present our findings on the proteomic analysis of the later developmental consequences of low temperature treatment on rice anthers. Anther proteins at the trinucleate stage, with or without cold treatment for four days at 12 degrees C at the young microspore stage, were extracted, separated by two-dimensional gel electrophoresis (2-DE) and compared. More than 3000 rice anther proteins of cold-sensitive cultivar Doongara plants at the trinucleate stage were resolved on 2-DE gels over a pH range of 4-7 and detected by silver-staining. Seventy protein spots were differentially displayed after four days of cold treatment at the young microspore stage. Of these, 12 protein spots were newly-induced, 47 were up-regulated, and 11 were down-regulated by cold treatment at the early microspore stage. We identified 18 by matrix-assisted laser desorption/ionization mass spectrometry time of flight (MALDI-TOF) analysis. Of the identified proteins, seven were observed as breakdown (cleavage) products by a combination of 2-DE and MALDI-TOF analysis, thus demonstrating for the first time that cold temperature stress at the young microspore stage enhances and induces partial degradation of proteins in the rice anthers at the trinucleate stage.  Download pdf

Ishimaru M, Smith DL, Gross KC, Kobayashi S. 2007.  Expression of three expansin genes during development and maturation of Kyoho grape berries. J Plant Physiol. 164(12):1675-82.
ABSTRACT: Expansins are cell-wall-localized proteins that induce loosening of isolated plant cell walls in vitro in a pH-dependent manner, but exhibit no detectable hydrolase or transglycosylase activity. Three putative expansin cDNAs, Vlexp1, Vlexp2, and Vlexp3 were isolated from a cDNA library made from mature berries of the Kyoho grape. Expression profiles of the 3 genes were analyzed throughout berry development. Accumulation of the Vlexp3 transcript was closely correlated with berry softening, and expression of this gene was detected before véraison and markedly increased at véraison (onset of berry softening). Expression of Vlexp3 was berry-specific. Vlexp1 and Vlexp2 mRNA accumulation began during the expansion stage of berry development and expression increased for both genes during ripening. Vlexp1 and Vlexp2 mRNA was detected in leaf, tendril and flower tissues and Vlexp2 mRNA was additionally detected in root and seed tissues. These findings suggest that the three expansin genes are associated with cell division or expansion and berry ripening. Vlexp3, in particular, is most likely to play a role in grape berry softening at véraison. Download pdf 

Ji,S.J., Lu,Y.C., Feng,J.X., Wei,G., Li,J., Shi,Y.H., Fu,Q., Liu,D., Luo,J.C., and Zhu,Y.X. (2003) Isolation and analyses of genes preferentially expressed during early cotton fiber development by subtractive PCR and cDNA array. Nucleic Acids Research 31:2534-2543.
Abstract: Cotton fibers are differentiated epidermal cells originating from the outer integuments of the ovule. To identify genes involved in cotton fiber elongation, we performed subtractive PCR using cDNA prepared from 10 days post anthesis (d.p.a.) wild-type cotton fiber as tester and cDNA from a fuzzless-lintless (fl) mutant as driver. We recovered 280 independent cDNA fragments including most of the previously published cotton fiber-related genes. cDNA macroarrays showed that 172 genes were significantly up-regulated in elongating cotton fibers as confirmed by in situ hybridization in representative cases. Twenty-nine cDNAs, including a putative vacuolar (H+)-ATPase catalytic subunit, a kinesin-like calmodulin binding protein, several arabinogalactan proteins and key enzymes involved in long chain fatty acid biosynthesis, accumulated to greater than 50-fold in 10 d.p.a. fiber cells when compared to that in 0 d.p.a. ovules. Various upstream pathways, such as auxin signal transduction, the MAPK pathway and profilin- and expansin-induced cell wall loosening, were also activated during the fast fiber elongation period. This report constitutes the first systematic analysis of genes involved in cotton fiber development. Our results suggest that a concerted mechanism involving multiple cellular pathways is responsible for cotton fiber elongation.

Jin Y, Tashpulatov AS, Katholnigg H, Heberle-Bors E, Touraev A. (2006)  Isolation and characterisation of two wheat beta-expansin genes expressed during male gametophyte development. Protoplasma. 228:13-9
Abstract: Two novel beta-expansin genes, TaEXPB1 and TaEXPB2, were isolated from wheat microspores by suppression subtractive hybridisation. Northern blot and reverse transcription PCR analyses showed that the expression of both genes was restricted to early stages of male gametophyte development (from microspores to immature pollen). A homology search showed high similarity of the newly discovered genes to generative beta-expansins in grass pollen (group 1 pollen allergens). Southern hybridisation revealed that the isolated genes belong to a distinct group within the subfamily of beta-expansin genes in the wheat genome. A comparison of full-length cDNAs with the corresponding genomic sequences showed that there are two introns in the TaEXPB1 gene, whereas TaEXPB2 has three introns. Both genes were predicted to encode highly similar basic proteins (pI 9.0) with molecular masses of approximately 29 kDa consisting of a signal peptide, catalytic, and polysaccharide binding domains, which include conserved cysteines and tryptophans and motifs characteristic for beta-expansins.

Jones L, McQueen-Mason S.  (2004) A role for expansins in dehydration and rehydration of the resurrection plant Craterostigma plantagineum. FEBS Lett. 2004 Feb 13;559(1-3):61-5.
Abstract:  Craterostigma plantagineum is one of the few higher plants capable of surviving desiccation throughout its vegetative tissues. Water loss results in cell shrinkage and a commensurate folding of the cell wall indicating an unusual degree of wall flexibility. We show that wall extensibility undergoes a marked increase during dehydration and rehydration. Similar increases were observed in the activity of expansins in cell walls during these processes suggesting a role for these proteins in increasing wall flexibility. Three alpha-expansin cDNAs were cloned from dehydrating leaves and transcript levels for one correlated closely with the observed changes in expansin activity during the dehydration and rehydration of leaves.  pdf file

Kalamaki MS, Harpster MH, Palys JM, Labavitch JM, Reid DS, Brummell DA. (2003) Simultaneous Transgenic Suppression of LePG and LeExp1 Influences Rheological Properties of Juice and Concentrates from a Processing Tomato Variety. J Agric Food Chem. Dec 3;51(25):7456-7464.
Abstract:  Processing tomato lines suppressed in the accumulation of ripening-related polygalacturonase or expansin were generated by introduction of transgenes to silence expression of the LePG and LeExp1 genes, respectively. The rheological properties of juice and juice reconstituted from paste produced from lines suppressed in one of these genes, or in both, were compared with azygous controls. When assayed by measuring Bostwick consistency, paste produced from either suppressed LePG or suppressed LeExp1 lines and diluted to 5 degrees Brix was approximately 18% more viscous than that produced from controls. Simultaneous suppression of LePG and LeExp1 produced a small additional increase in viscosity of 4%. Rheometric flow analysis at 5 or 10 degrees Brix also showed substantial increases in the consistency index due to suppression of either LePG or LeExp1 alone, and a small additional increase when both genes were suppressed in the same transgenic line. Measurements by laser diffraction and [(1)H]NMR showed that suppression of LePG or LeExp1 accumulation altered the size distribution of insoluble particles and modified their surface properties. The data are consistent with suppression of LePG increasing serum viscosity, and suppression of either LePG or LeExp1 altering the properties of the insoluble particles and improving some aspect of particle-particle or particle-serum interaction, or both. However, relative to that caused by suppression of either gene alone, the additional increase in viscosity caused by simultaneous suppression of LePG and LeExp1 together was slight.

Kalamaki MS, Powell AL, Struijs K, Labavitch JM, Reid DS, Bennett AB. )2003) Transgenic Overexpression of Expansin Influences Particle Size Distribution and Improves Abstract:  Viscosity of Tomato Juice and Paste. J Agric Food Chem.  Dec 3;51(25):7465-7471.
Suppression of the expression of a ripening-related expansin gene, LeExp1, in tomato enhanced fruit firmness and overexpression of LeExp1 resulted in increased fruit softening. Because of the incompletely understood relationship between fresh fruit texture and the consistency of processed products, we examined the effects of LeExp1 overexpression on the processing characteristics of tomato fruit. As determined by Bostwick consistency and by controlled strain rheometry, juices and pastes prepared from transgenic tomatoes with suppressed LeExp1 expression had a higher viscosity than preparations from control fruits. However, the viscosity of juice and paste prepared from fruit overexpressing LeExp1 was significantly greater than products from controls or lines with reduced LeExp1. Bostwick consistency increased by 9% (juice) and 6% (paste) in lines with suppressed LeExp1 expression but increased by 27.5% (juice) and 19.5% (paste) in lines overexpressing LeExp1, relative to controls. Determined by laser diffraction, the particles in juice and paste prepared from transgenic fruits with reduced LeExp1 expression were smaller, and preparations from fruits overexpressing LeExp1 had a size distribution indicating more large particles. Analysis of cell wall polysaccharides size indicated that LeExp1 overexpression enhanced depolymerization of water soluble pectins as well as tightly bound matrix glycans. LeExp1 overexpression may allow increased cell wall hydration, resulting in expanded particle size and increased viscosity of products. Because either LeExp1 suppression or overexpression leads to improved consistency, the interactions that contribute to optimal product rheological properties are complex. Download pdf file (c) ACS

M. J. Kam, H. S. Yun, P. B. Kaufman, S. C. Chang, and S. K. Kim. Two expansins, EXP1 and EXPB2, are correlated with the growth and development of maize roots. Journal of Plant Biology 48:304-310, 2005.
Abstract: Expansins are proteins that can confer extensibility in plant cells by modifying the cross-links between cellulose microfibrils and pollysacchrides. Because they are present as multi-gene families, their various patterns of differential expression suggest that each expansin plays a specific role in growth and development. Here, we describe the expression of expansin genes in maize roots in response to stage of growth, hormone treatment, or environmental stimuli. ExpB2 was the most strongly detected, with its transcript level being much higher than any other expansin in the regions undergoing cell division and elongation. Indole-3-acetic acid, which generally inhibits root elongation, induced expression of Ex l, but repressed that of ExpB2. This auxin-induced alteration was negated by treatment with 1-aminoethoxyvinylglysin (AVG), indicating that transcript levels may be modified by auxin-induced ethylene biosynthesis. ExpB2 expression was also induced by wounding and gravistimulus treatments. Therefore, our results imply that ExpB2 plays a role in the elongation of maize roots, and may be also involved in plant responses to environmental stimuli.

Keller E, Cosgrove DJ (1995) Expansins in growing tomato leaves.  Plant J. 8:795-802
ABSTRACT: An expansin-like protein from growing tomato cv. Caruso leaves was identified by its ability to restore the 'acid-growth' response to heat-inactivated tomato cell walls and by its similarity to expansins from cucumber cv. Burpee Pickler hypocotyls. Native walls from growing tomato leaves exhibited an endogenous acid-induced extension (creep) that resembled in various biochemical characteristics the acid-growth activity of cucumber hypocotyls. For example, the acid-growth activity was lost when the walls of tomato leaves were briefly heated and was largely restored by addition of a crude protein extract from the walls of growing leaves. Wall proteins from growing leaves enhanced 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-reacted with an antibody raised against cucumber expansin. The results show that tomato leaf walls 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.

Kende H, Bradford K, Brummell D, Cho HT , Cosgrove DJ, Fleming A, Gehring C, Lee Y,  McQueen-Mason SM, Rose J., and  Voesenek LA.  (2004) Nomenclature for members of the expansin superfamily of genes and proteins. Plant Mol.Biol. 55 (3):311-314.
This article by the expansin research community defines expansins and recommends naming conventions. Download pdf file.

Kerff F, Amoroso A, Herman R, Sauvage E, Petrella S, Filée P, Charlier P, Joris B, Tabuchi A, Nikolaidis N, Cosgrove DJ.  2008.  Crystal structure and activity of Bacillus subtilis YoaJ (EXLX1), a bacterial expansin that promotes root colonization. Proc Natl Acad Sci U S A.  105(44):16876-81.
Abstract:  We solved the crystal structure of a secreted protein, EXLX1, encoded by the yoaJ gene of Bacillus subtilis. Its structure is remarkably similar to that of plant beta-expansins (group 1 grass pollen allergens), consisting of 2 tightly packed domains (D1, D2) with a potential polysaccharide-binding surface spanning the 2 domains. Domain D1 has a double-psi beta-barrel fold with partial conservation of the catalytic site found in family 45 glycosyl hydrolases and in the MltA family of lytic transglycosylases. Domain D2 has an Ig-like fold similar to group 2/3 grass pollen allergens, with structural features similar to a type A carbohydrate-binding domain. EXLX1 bound to plant cell walls, cellulose, and peptidoglycan, but it lacked lytic activity against a variety of plant cell wall polysaccharides and peptidoglycan. EXLX1 promoted plant cell wall extension similar to, but 10 times weaker than, plant beta-expansins, which synergistically enhanced EXLX1 activity. Deletion of the gene encoding EXLX1 did not affect growth or peptidoglycan composition of B. subtilis in liquid medium, but slowed lysis upon osmotic shock and greatly reduced the ability of the bacterium to colonize maize roots. The presence of EXLX1 homologs in a small but diverse set of plant pathogens further supports a role in plant-bacterial interactions. Because plant expansins have proved difficult to express in active form in heterologous systems, the discovery of a bacterial homolog opens the door for detailed structural studies of expansin function. Download_pdf    Suppl. data

Kerim,T., Imin,N., Weinman,J.J., and Rolfe,B.G. (2003) Proteome analysis of male gametophyte development in rice anthers. Proteomics 3:738-751
Abstract: We used proteomic analysis to investigate the changing patterns of protein synthesis during pollen development in anthers from rice plants grown under strictly controlled growth conditions. Cytological analysis and external growth measurements such as anther length, auricle distances and days before flowering were used to determine pollen developmental stages. This allowed the collection of synchronous anther materials representing six discrete pollen developmental stages. Proteins were extracted from the anther samples and separated by two-dimensional gel electrophoresis to produce proteome maps. The anther proteome maps of different developmental stages were compared and 150 protein spots, which were changed consistently during development, were analysed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry to produce peptide mass fingerprint (PMF) data. Database searches using these PMF data revealed the identities of 40 of the protein spots analyzed. These 40 proteins represent 33 unique gene products. Four protein spots that could not be identified by PMF analysis were analysed by N-terminal microsequencing. Multiple charge-isoforms of vacuolar acid invertase, fructokinase, beta-expansin and profilin were identified. These proteins are closely associated with sugar metabolism, cell elongation and cell expansion, all of which are cell activities that are essential to pollen germination. The existence of multiple isoforms of the same proteins suggests that during the process of pollen development some kind of post-translational modification of these proteins occurs.

Dong Wook Kim, Sang Ho Lee, Sang-Bong Choi, Su-Kyung Won, Yoon-Kyung Heo, Misuk Cho, Youn-Il Park, and Hyung-Taeg Cho.  (2006)  Functional Conservation of a Root Hair Cell-Specific cis-Element in Angiosperms with Different Root Hair Distribution Patterns.  Plant Cell. 2006 November; 18(11): 2958–2970
ABSTRACT:  Vascular plants develop distinctive root hair distribution patterns in the root epidermis, depending on the taxon. The three patterns, random (Type 1), asymmetrical cell division (Type 2), and positionally cued (Type 3), are controlled by different upstream fate-determining factors that mediate expression of root hair cell-specific genes for hair morphogenesis. Here, we address whether these root hair genes possess a common transcriptional regulatory module (cis-element) determining cell-type specificity despite differences in the final root hair pattern. We identified Arabidopsis thaliana expansinA7 (At EXPA7) orthologous (and paralogous) genes from diverse angiosperm species with different hair distribution patterns. The promoters of these genes contain conserved root hair–specific cis-elements (RHEs) that were functionally verified in the Type-3 Arabidopsis root. The promoter of At EXPA7 (Type-3 pattern) also showed hair cell–specific expression in the Type 2 rice (Oryza sativa) root. Root hair–specific genes other than EXPAs also carry functionally homologous RHEs in their promoters. The RHE core consensus was established by a multiple alignment of functionally characterized RHEs from different species and by high-resolution analysis of At EXPA7 RHE1. Our results suggest that this regulatory module of root hair–specific genes has been conserved across angiosperms despite the divergence of upstream fate-determining machinery.  Download pdf (C)ASPB

Kim JH, Cho HT, Kende H   (2000) Alpha-expansins in the semiaquatic ferns Marsilea quadrifolia and Regnellidium diphyllum: evolutionary aspects and physiological role in rachis elongation. Planta 212:85-92
ABSTRACT: To investigate the evolutionary history of expansins and their role in cell elongation in early land plants, we isolated two alpha-expansin genes, Mq-EXP1 and Rd-EXP1, respectively, from the semiaquatic ferns Marsilea quacdrifolia L. and Regnellidium diphyllum Lindm. The deduced amino acid sequences of the fern expansins exhibit a high degree of identity to those of seed plants, showing that expansin genes were conserved during the evolution of vascular plants. Gel-blot analysis of M. quadrifolia and R. diphyllum genomic DNA indicated that, in both ferns, alpha-expansins are encoded by multigene families. Expression of alpha-expansin genes probed with Mq-EXP1 was confined to the elongating region of the Marsilea rachis. Cell-wall proteins of M. quadrifolia induced in-vitro extension of acidified cucumber cell walls. In R. diphyllum, expression of Rd-EXP1 increased when elongation of the rachis was enhanced by submergence or ethylene. These results indicate that alpha-expansins act as wall-loosening proteins in ferns, as has been proposed for angiosperms. In addition, Rd-EXP1 may play a role in mediating elongation of the rachis in submerged plants. Download the full article in Acrobat pdf format (230 kB file). © Springer

Knox B, Suphioglu C (1996) Environmental and molecular biology of pollen allergens. Trends. Plant Sci. 1: 156-164

Kudla U, Qin L, Milac A, Kielak A, Maissen C, Overmars H, Popeijus H, Roze E, Petrescu A, Smant G, Bakker J, Helder J.  2005. Origin, distribution and 3D-modeling of Gr-EXPB1, an expansin from the potato cyst nematode Globodera rostochiensis. FEBS Lett. 579:2451-2457 2005.
Abstract: Southern analysis showed that Gr-EXPB1, a functional expansin from the potato cyst nematode Globodera rostochiensis, is member of a multigene family, and EST data suggest expansins to be present in other plant parasitic nematodes as well. Homology modeling predicted that Gr-EXPB1 domain 1 (D1) has a flat beta-barrel structure with surface-exposed aromatic rings, whereas the 3D structure of Gr-EXPB1-D2 was remarkably similar to plant expansins. Gr-EXPB1 shows highest sequence similarity to two extracellular proteins from saprophytic soil-inhabiting Actinobacteria, and includes a bacterial type II carbohydrate-binding module. These results support the hypothesis that a number of pathogenicity factors of cyst nematodes is of procaryotic origin and were acquired by horizontal gene transfer.  Download pdf

Kwasniewski M, Szarejko I. (2006) Molecular cloning and characterization of beta-expansin gene related to root hair formation in barley. Plant Physiol. 141:1149-58
Abstract: Root hairs are specialized epidermal cells that play a role in the uptake of water and nutrients from the rhizosphere and serve as a site of interaction with soil microorganisms. The process of root hair formation is well characterized in Arabidopsis (Arabidopsis thaliana); however, there is a very little information about the genetic and molecular basis of root hair development in monocots. Here, we report on isolation and cloning of the beta-expansin (EXPB) gene HvEXPB1, tightly related to root hair initiation in barley (Hordeum vulgare). Using root transcriptome differentiation in the wild-type/root-hairless mutant system, a cDNA fragment present in roots of wild-type plants only was identified. After cloning of full-length cDNA and genomic sequences flanking the identified fragment, the subsequent bioinformatics analyses revealed homology of the protein coded by the identified gene to the EXPB family. Reverse transcription-PCR showed that expression of HvEXPB1 cosegregated with the root hair phenotype in F2 progeny of the cross between the hairless mutant rhl1.a and the wild-type Karat parent variety. Expression of the HvEXPB1 gene was root specific; it was expressed in roots of wild-type forms, but not in coleoptiles, leaves, tillers, and spikes. The identified gene was active in roots of two other analyzed root hair mutants: rhp1.a developing root hair primordia only and rhs1.a with very short root hairs. Contrary to this, a complete lack of HvEXPB1 expression was observed in roots of the spontaneous root-hairless mutant bald root barley. All these observations suggest a role of the HvEXPB1 gene in the process of root hair formation in barley. Download pdf.

Kwon YR, Lee HJ, Kim KH, Hong SW, Lee SJ, Lee H. 2008.  Ectopic expression of Expansin3 or Expansinbeta1 causes enhanced hormone and salt stress sensitivity in Arabidopsis. Biotechnol Lett. 30(7):1281-8.
ABSTRACT: Expansins are cell wall loosening proteins that appear to permit the microfibril matrix network to slide in growing plant cell walls, thereby enabling the wall to expand. To scrutinize possible impacts on plant growth and development when expansins are over-expressed, we characterized phenotypic alterations of the transgenic plants that constitutively expressed AtEXP3 or AtEXP-beta1 under control of 35S-CaMV promoter. Our results suggest that both AtEXP3-OX and AtXPbeta1-OX are very sensitive to salt stress. However, the mechanisms underlying their enhanced salt sensitivity appear to be different. Download pdf  (C) Springer

Lasanthi-Kudahettige R, Magneschi L, Loreti E, Gonzali S, Licausi F, Novi G, Beretta O, Vitulli F, Alpi A, Perata P. 2007.  Transcript profiling of the anoxic rice coleoptile. Plant Physiol. 144(1):218-31.
ABSTRACT: Rice (Oryza sativa) seeds can germinate in the complete absence of oxygen. Under anoxia, the rice coleoptile elongates, reaching a length greater than that of the aerobic one. In this article, we compared and investigated the transcriptome of rice coleoptiles grown under aerobic and anaerobic conditions. The results allow drawing a detailed picture of the modulation of the transcripts involved in anaerobic carbohydrate metabolism, suggesting up-regulation of the steps required to produce and metabolize pyruvate and its derivatives. Sugars appear to play a signaling role under anoxia, with several genes indirectly up-regulated by anoxia-driven sugar starvation. Analysis of the effects of anoxia on the expansin gene families revealed that EXPA7 and EXPB12 are likely to be involved in rice coleoptile elongation under anoxia. Genes coding for ethylene response factors and heat shock proteins are among the genes modulated by anoxia in both rice and Arabidopsis (Arabidopsis thaliana). Identification of anoxia-induced ethylene response factors is suggestive because genes belonging to this gene family play a crucial role in rice tolerance to submergence, a process closely related to, but independent from, the ability to germinate under anoxia. Genes coding for some enzymes requiring oxygen for their activity are dramatically down-regulated under anoxia, suggesting the existence of an energy-saving strategy in the regulation of gene expression. Download pdf (C) ASPB

Laine, MJ,  M Haapalainen,  T Wahlroos,  K Kankare,  R Nissinen,  S Kassuwi,  M C Metzler (2000).  The cellulase encoded by the native plasmid of Clavibacter michiganensis ssp. sepedonicus plays a role in virulence and contains an expansin-like domain. Physiological and Molecular Plant Pathology Vol. 57: 221-233. 

Lamport DT (2001)  Life behind cell walls: paradigm lost, paradigm regained. Cell Mol Life Sci 58:1363-85
ABSTRACT: This review of the living cell wall and its protein components is in two parts. The first is anecdotal. A personal account spanning over 40 years research may perhaps be an antidote to one stereotypical view of scientists as detached and humorless. The second part deals with the meaning of function, particularly as it applies to hydroxyproline-rich glycoproteins. Function is a difficult word to define objectively. However, with help from such luminaries as Humpty Dumpty: "A word means what I want it to mean, neither more nor less," and Wittgenstein: "Giving examples of usage ... is the only way to talk about meaning," it is possible to construct a ziggurat representing increasingly complex levels of organization from molecular structure to ecology. Forty years ago I suggested that hydroxyproline-rich structural proteins played a key role in cell wall functioning. But because the bulk of the wall is carbohydrate, there has been an understandable resistance to paradigm change. Expansins, paradoxically, contribute greatly to this resistance because their modus operandi as cell-wall-loosening proteins is based on the idea that they break hydrogen bonds between polysaccharide chains allowing slippage. However, this view is not consistent with the recent discovery [Grobe et al. (1999) Eur. J. Biochem 263: 33-40] that beta-expansins may be proteases, as it implies that the extensin network is not a straightjacket but a substrate for expansin in muro. Such a direct role for extensins in both negative and positive regulation of cell expansion and elongation may constitute a major morphogenetic mechanism operating at all levels of plant growth and development. Editor's note:  the hypothesis that b-expansins are proteases was refuted by Li and Cosgrove 2001 Eur. J. Biochem. 268: 4217.

Lee DJ, Park JY, Ku SJ, Ha YM, Kim S, Kim MD, Oh MH, Kim J. 2007.  Genome-wide expression profiling of ARABIDOPSIS RESPONSE REGULATOR 7(ARR7) overexpression in cytokinin response. Mol Genet Genomics. 277(2):115-37.
ABSTRACT: The type-A ARRs of cytokinin two-component signaling system act as negative regulators for cytokinin signaling except for ARR4, but the molecular mechanism by which the A-type ARRs regulate cytokinin signaling remain elusive. To get insights into the molecular function of A-type ARR in cytokinin response, we sought to find the components that function downstream of A-type ARR protein by investigating the effects of ARR7 overexpression on cytokinin-regulated gene expression with the Affymetrix full genome array. To examine early cytokinin response, plants were treated with cytokinin for 30 min or 2 h, followed by GeneChip analysis. The hierarchical clustering analysis of our GeneChip data showed that ARR7 overexpression had distinctively repressive impacts on various groups of the cytokinin-regulated genes. In particular, the induction of all A-type ARRs except for ARR22, and AHK(ARABIDOPSIS HISTIDINE KINASE)1 and AHK4 was suppressed by ARR7. Cytokinin-induced expression of most of 12 expansin genes were repressed by ARR7, indicating potential involvement of ARR7 in cell expansion and plant development. Up-regulation of five cytokinin oxidase genes by cytokinins was negatively affected by ARR7. Our GeneChip analysis suggest that ARR7 mainly acts as a transcriptional repressor for a variety of early cytokinin-regulated genes encoding transcription factors, signal transmitters, plant development, and cellular metabolism, which may be responsible for reduced sensitivity of Arabidopsis transgenic plants overexpressing ARR7 to exogenous cytokinins. Download pdf

Lee,D.K., Ahn,J.H., Song,S.K., Choi,Y.D., and Lee,J.S. (2003) Expression of an expansin gene is correlated with root elongation in soybean. Plant Physiol 131:985-997.
Abstract: Expansin is a family of proteins that catalyze long-term expansion of cell walls and has been considered a principal protein that affects cell expansion in plants. We have identified the first root-specific expansin gene in soybean (Glycine max), GmEXP1, which may be responsible for root elongation. Expression levels of GmEXP1 were very high in the roots of 1- to 5-d-old seedlings, in which rapid root elongation takes place. Furthermore, GmEXP1 mRNA was most abundant in the root tip region, where cell elongation occurs, but scarce in the region of maturation, where cell elongation ceases, implying that its expression is closely related to root development processes. In situ hybridization showed that GmEXP1 transcripts were preferentially present in the epidermal cells and underlying cell layers in the root tip of the primary and secondary roots. Ectopic expression of GmEXP1 accelerated the root growth of transgenic tobacco (Nicotiana tabacum) seedlings, and the roots showed insensitivity to obstacle-touching stress. These results imply that the GmEXP1 gene plays an important role in root development in soybean, especially in the elongation and/or initiation of the primary and secondary roots.

Y. Lee and D. Choi. Biochemical properties and localization of the beta-expansin OsEXPB3 in rice (Oryza sativa L.). Mol.Cells 20 (1):119-126, 2005.
Abstract: Alpha-expansins are bound to the cell wall of plants and can be solubilized with an extraction buffer containing 1 M NaCl. Localization of alpha-expansins in the cell wall was confirmed by immunogold labeling and electron microscopy. The subcellular localization of vegetative beta-expansins has not yet been studied. Using antibodies specific for OsEXPB3, a vegetative beta-expansin of rice (Oryza sativa L.), we found that OsEXPB3 is tightly bound to the cell wall and, unlike alpha-expansins, cannot be solubilized with extraction buffer containing 1 M NaCl. OsEXPB3 protein could only be extracted with buffer containing SDS. The subcellular localization of the OsEXPB3 protein was confirmed by immunogold labeling and electron microscopy. Gold particles were mainly distributed over the primary cell walls. Immunohistochemistry showed that OsEXPB3 is present in all regions of the coleoptile and root tissues tested. pdf file.

Lee Y,  Kende H  (2001)  Expression of b-Expansins Is Correlated with Internodal Elongation in Deepwater Rice. Plant Physiology 127: 645-54
ABSTRACT:  Fourteen putative rice (Oryza sativa) beta-expansin genes, Os-EXPB1 through Os-EXPB14, were identified in the expressed sequence tag and genomic databases. The DNA and deduced amino acid sequences are highly conserved in all 14 beta-expansins. They have a series of conserved C (cysteine) residues in the N-terminal half of the protein, an HFD (histidine-phenylalanine-aspartate) motif in the central region, and a series of W (tryptophan) residues near the carboxyl terminus. Five beta- expansin genes are expressed in deepwater rice internodes, with especially high transcript levels in the growing region. Expression of four beta-expansin genes in the internode was induced by treatment with gibberellin and by wounding. The wound response resulted from excising stem sections or from piercing pinholes into the stem of intact plants. The level of wound-induced beta-expansin transcripts declined rapidly 5 h after cutting of stem sections. We conclude that the expression of beta-expansin genes is correlated with rapid elongation of deepwater rice internodes, it is induced by gibberellin and wounding, and wound- induced beta-expansin mRNA appears to turn over rapidly. Download PDF file. (c) ASPB

Lee Y, Choi D, Kende H (2001) Expansins: ever-expanding numbers and functions. Curr Opin Plant Biol 2001 4:527-32
ABSTRACT:  Expansins were first identified as cell-wall-loosening proteins that, at least in part, mediate pH-dependent extension of the plant cell wall and growth of the cell. More recently, it has been realized that expansins belong to two protein families, the alpha-and beta-expansins, and that they appear to be involved in regulating, besides cell expansion, a variety of plant processes, including morphogenesis, softening of fruits, and growth of the pollen tube of grasses through the stigma and the style. The Arabidopsis genome contains 26 alpha-expansin genes and the rice genome at least 26. There are more beta-expansin genes in monocots than in dicots, at least 14 in rice and five in Arabidopsis. Expansin genes are differentially regulated by environmental and hormonal signals, and hormonal regulatory elements have been found in their promoter regions. An analysis of exon/intron structure led to the hypothesis that alpha-and beta-expansins evolved from a common ancestral gene. Download PDF file.

Lee,Y. and Kende,H. (2002) Expression of alpha-expansin and expansin-like genes in deepwater rice. Plant Physiology 130:1396-1405.
Abstract: Previously, we have studied the expression and regulation of four alpha- and 14 beta-expansin genes in deepwater rice (Oryza sativa). We now report on the structure, expression, and regulation of 22 additional a-expansin (Os-EXP) genes, four expansin-like (Os-EXPL) genes, and one expansin-related (Os-EXPR) gene, which have recently been identified in the expressed sequence tag and genomic databases of rice. alpha-Expansins are characterized by a series of conserved Cys residues in the N-terminal half of the protein, a histidine-phenylalanine-aspartate (HFD) motif in the central region, and a series of tryptophan residues near the carboxyl terminus. Of the 22 additional alpha-expansin genes, five are expressed in internodes and leaves, three in coleoptiles, and nine in roots, with high transcript levels in the growing regions of these organs. Transcripts of five alpha-expansin genes were found in roots only. Expression of five alpha-expansin genes was induced in the internode by treatment with gibberellin (GA) and by wounding. The wound response resulted from excising stem sections or from piercing pinholes into the stem of intact plants. EXPL proteins lack the HFD motif and have two additional Cys residues in their C- and N-terminal regions. The positions of conserved tryptophan residues at the C-terminal region are different from, those of alpha- and beta-expansins. Expression of the Os-EXPL3 gene is correlated with elongation and slightly induced by applied GA. However, the expression of the Os-EXPL1 and Os-EXPL2 genes showed limited correlation with cell elongation and was not induced by GA. We found no expression of the Os-EXPR1 gene in the organs examined.  Download PDF

Li,L.C., Bedinger,P.A., Volk,C., Jones,A.D., and Cosgrove,D.J. (2003) Purification and characterization of four beta-expansins (Zea m 1 isoforms) from maize pollen. Plant Physiology 132:2073-2085.
Abstract: Four proteins with wall extension activity on grass cell walls were purified from maize (Zea mays) pollen by conventional column chromatography and high-performance liquid chromatography. Each is a basic glycoprotein (isoelectric point = 9.1-9.5) of approximately 28 kD and was identified by immunoblot analysis as an isoform of Zea m 1, the major group 1 allergen of maize pollen and member of the beta-expansin family. Four distinctive cDNAs for Zea m 1 were identified by cDNA library screening and by GenBank analysis. One pair (GenBank accession nos. AY104999 and AY104125) was much closer in sequence to weR-characterized allergens such as Lol p1 and Phl p1 from ryegrass (Lolium perenne) and Phleum pretense, whereas a second pair was much more divergent. The N-terminal sequence and mass spectrometry fingerprint of the most abundant isoform. (Zea m 1d) matched that predicted for AY197353, whereas N-terminal sequences of the other isoforms matched or nearly matched AY104999 and AY104125. Highly purified Zea m 1d induced extension of a variety of grass walls but not dicot walls. Wall extension activity of Zea m 1d was biphasic with respect to protein concentration, had a broad pH optimum between 5 and 6, required more than 50 mug mL(-1) for high activity, and led to cell wall breakage after only approximately 10% extension. These characteristics differ from those of alpha-expansins. Some of the distinctive properties of Zea m 1 may not be typical of beta-expansins as a class but may relate to the specialized function of this beta-expansin in pollen function. Download PDF

Link BM, Cosgrove  (1998) Acid-Growth Response and a-Expansins in Suspension Cultures of Bright Yellow 2 Tobacco Plant Physiology 118: 907-916
ABSTRACT: The possibility that Bright Yellow 2 (BY2) tobacco (Nicotiana tabacum L.) suspension-cultured cells possess an expansin-mediated acid-growth mechanism was examined by multiple approaches. BY2 cells grew three times faster upon treatment with fusicoccin, which induces an acidification of the cell wall. Exogenous expansins likewise stimulated BY2 cell growth 3-fold. Protein extracted from BY2 cell walls possessed the expansin-like ability to induce extension of isolated walls. In western-blot analysis of BY2 wall protein, one band of 29 kD was recognized by anti-expansin antibody. Six different classes of a-expansin mRNA were identified in a BY2 cDNA library. Northern-blot analysis indicated moderate to low abundance of multiple a-expansin mRNAs in BY2 cells. From these results we conclude that BY2 suspension-cultured cells have the necessary components for expansin-mediated cell wall enlargement.   Download the full article in Acrobat pdf format (290 kB file). © ASPP

Link BM, Wagner ER, Cosgrove DJ. 2001.  The effect of a microgravity (space) environment on the expression of expansins from the peg and root tissues of Cucumis sativus. Physiol Plant 113:292-300
ABSTRACT: In young cucumber seedlings, the peg is a polar outgrowth of tissue that functions by snagging the seed coat, thereby freeing the cotyledons. The development of the peg is thought to be gravity-dependent and has become a model system for plant-gravity response. Peg development requires rapid cell expansion, a process thought to be catalyzed by alpha-expansins, and thus was a good system to identify expansins that were regulated by gravity. This study identified 7 new alpha-expansin cDNAs from cucumber seedlings (Cucumis sativus L. cv Burpee Hybrid II) and examined their expression patterns. Two alpha-expansins (CsExp3 and CsExp4) were more highly expressed in the peg and the root. Earlier reports stated that pegs tend not to form in the absence of gravity, so the expression levels were compared in the pegs of seedlings grown in space (STS-95), on a clinostat, and on earth (1 g). Pegs were observed to form at high frequency on clinostat and space-grown seedlings, yet on clinostats there was more than a 4-fold reduction in the expression of CsExp3 in the pegs of seedlings grown on clinostats vs. those grown at 1 g, while the CsExp4 gene appeared to be turned off (below detection limits). There were no detectable differences in expansin gene expression levels for the pegs of seedlings grown in space or in the orbiter environmental simulator (OES) (1 g) at NASA. The microgravity environment did not affect the expression of CsExp3 or CsExp4, and the clinostat did not simulate the microgravity environment well. Download pdf file

Liu Y, Liu D, Zhang H, Gao H, Guo X, Wang D, Zhang X, Zhang A. 2007.  The alpha- and beta-expansin and xyloglucan endotransglucosylase/hydrolase gene families of wheat: molecular cloning, gene expression, and EST data mining. Genomics. 90(4):516-29
ABSTRACT: Expansins and xyloglucan endotransglucosylase/hydrolases (XTHs) are families of extracellular proteins with members that have been shown to play an important role in cell wall growth. In this study, three, six, and five members of the wheat alpha-expansin (TaEXPA1 to TaEXPA3), beta-expansin (TaEXPB1 to TaEXPB6), and XTH (TaXTH1 to TaXTH5) gene families, respectively, were isolated from a dwarf wheat line. The mRNA expression analysis by real-time RT-PCR indicates that these genes display different transcription levels in different stages/organs/treatments, possibly suggesting their functional roles in the cell wall expansion process. Moreover, the comparison of the expression levels reveals that most of the expansins show lower expression than the XTHs. Finally, we present the analysis of wheat alpha- and beta-expansins and XTH families by expressed sequence tag data mining.  Download pdf (C) Elsevier

Ludidi,N.N., Heazlewood,J.L., Seoighe,C., Irving,H.R., and Gehring,C.A. (2002) Expansin-like molecules: Novel functions derived from common domains. Journal of Molecular Evolution 54:587-594.
Abstract: An Arabidopsis thaliana transcript (AtPNP-A) encoding an immunoreactant plant natriuretic peptide (irPNP) analog was identified and isolated. The encoded protein shows similarity to CjBAp12, a functionally undefined protein from citrus that is induced in response to blight infection. CjBAp12 shows significant sequence identity to domains found in the cell wall loosening expansins but has tested negative for cell wall loosening activity. We have thus undertaken to establish the evolutionary and functional relationships of irPNP-like molecules within the super-family of expansins, pollen allergens, and distantly related molecules such as endoglucanases. We show that irPNP-like molecules are related to expansins and fall in two groups; one includes CjBAp12 and the other AtPNP-A. Members of both groups share distinct sequence motifs (K[VI]VD and [LM]SxxAFxxI) but do not contain the tryptophan and tyrosine rich C-terminal putative polysaccharide-binding domain typical of expansins or bacterial cellulases and hemicellulases. We argue that both irPNP-like molecules and expansin have evolved from primitive/ancestral glucanase-like molecules that hydrolysed the cell wall. Importantly, we have previously demonstrated that irPNPs act on protoplasts, that is plant cells without cell walls as well as microsomes, indicating that these novel proteins specifically interact with the plasma membrane. It follows that the cell wall cannot be an obligatory substrate for irPNPs. Thus, both irPNP function and domain structure point to these molecules having a systemic role in H2O and solute homeostasis.

F. Marga, M. Grandbois, D. J. Cosgrove, and T. I. Baskin. Cell wall extension results in the coordinate separation of parallel microfibrils: evidence from scanning electron microscopy and atomic force microscopy. Plant J 43 (2):181-190, 2005.
Abstract: Enlargement of the cell wall requires separation of cellulose microfibrils, mediated by proteins such as expansin; according to the multi-net growth hypothesis, enlargement passively reorients microfibrils. However, at the molecular scale, little is known about the specific movement of microfibrils. To find out, we examined directly changes in microfibril orientation when walls were extended slowly in vitro under constant load (creep). Frozen-thawed cucumber hypocotyl segments were strained by 20-30% by incubation in pH 4.5 buffer or by incubation of heat-inactivated segments in alpha-expansin or a fungal endoglucanase (Cel12A). Subsequently, the innermost layer of the cell wall was imaged, with neither extraction nor homogenization, by field-emission scanning electron microscopy (FESEM) and atomic force microscopy (AFM). AFM images revealed that sample preparation for FESEM did not appreciably alter cell wall ultrastructure. In both FESEM and AFM, images from extended and non-extended samples appeared indistinguishable. To quantify orientational order, we used a novel algorithm to characterize the fast Fourier transform of the image as a function of spatial frequency. For both FESEM and AFM images, the transforms of non-extended samples were indistinguishable from those of samples extended by alpha-expansin or Cel12A, as were AFM images of samples extended by acidic buffer. We conclude that cell walls in vitro can extend slowly by a creep mechanism without passive reorientation of innermost microfibrils, implying that wall loosening agents act selectively on the cross-linking polymers between parallel microfibrils, rather than more generally on the wall matrix.  pdf download

Mbeguie,A.M., Gouble,B., Gomez,R.M., Audergon,J.M., Albagnac,G., and Fils-Lycaon,B. (2002) Two expansin cDNAs from Prunus armeniaca expressed during fruit ripening are differently regulated by ethylene. Plant Physiology and Biochemistry 40:445-452.
Abstract: Little is known about gene expression during fruit ripening of apricot (Prunes armeniaca L. cv. Bergeron), especially for enzymes involved in cell wall modifications. A partial cDNA clone encoding a protein homologous to expansin was isolated from a ripe apricot fruit cDNA library. This clone was used to isolate two full-length expansin cDNAs, Pa-Exp1 (accession no. U93157) and Pa-Exp2 (accession no. AF038815) from the same cDNA library. The predicted polypeptides encoded by these two cDNAs are different and belong to the a-expansin family; Pa-Exp1 and Pa-Exp2 are two different members of a multigene family. These two clones are mostly expressed in fruit, during its ripening. Pa-Exp1 mRNA accumulated abundantly at the half-ripe stage of fruit development and decreased thereafter. Pa-Exp2 mRNA level increased from the immature-green stage to the half-ripe stage where it peaked before declining. During the ripening process, Pa-Exp1 and Pa-Exp2 gene expression appeared to be positively correlated with fruit size. Post-harvest treatments by air, ethylene, and 1-methyl cyclopropene led us to conclude that Pa-Exp1 appears to be developmentally down-regulated by ethylene while Pa-Exp2 is not affected. The relationship between Pa-Exp1, Pa-Exp2 and the softening process is also discussed. (C) 2002 Editions scientifiques et medicales Elsevier SAS.

McQueen-Mason S (1995) Expansins and cell wall expansion. J. Exp. Bot. 46: 1639-1650

McQueen-Mason, SJ; Rochange, F  (1999)  Expansins in plant growth and development: an update on an emerging topic.  Plant Biology 1:19-25 
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 beta-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 heterologous expression systems.

McQueen-Mason S, Cosgrove DJ (1994) Disruption of hydrogen bonding between wall polymers by proteins that induce plant wall extension. Proc. Natl. Acad. Sci. USA 91: 6574-6578
ABSTRACT: Plant cell enlargement is controlled by the ability of the constraining cell wall to expand. This ability has been postulated to be under the control of polysaccharide hydrolases or transferases that weaken or rearrange the loadbearing polymeric networks in the wall. We recently identified a family of wall proteins, called expansins, that catalyze the extension of isolated plant cell walls. Here we report that these proteins mechanically weaken pure cellulose paper in extension assays and stress relaxation assays, without detectable cellulase activity (exo- or endo- type). Because paper derives its mechanical strength from hydrogen bonding between cellulose microfibrils, we conclude that expansins can disrupt hydrogen bonding between cellulose fibers. This conclusion is further supported by experiments in which expansin-mediated wall extension (i) was increased by 2 M urea (which should weaken hydrogen bonding between wall polymers) and (ii) was decreased by replacement of water with deuterated water, which has a stronger hydrogen bond. The temperature sensitivity of expansin-mediated wall extension suggests that units of 3 or 4 hydrogen bonds are broken by the action of expansins. In the growing cell wall, expansin action is likely to catalyze slippage between cellulose microfibrils and the polysaccharide matrix, and thereby catalyze wall stress relaxation, followed by wall surface expansion and plant cell enlargement. Download PDF file.

McQueen-Mason S, Cosgrove DJ (1995)  Expansin mode of action on cell walls: Analysis of wall hydrolysis, stress relaxation, and binding. Plant Physiol. 107: 87-100
ABSTRACT:  Previous work identified 2 proteins (named "expansins") that catalyse the acid-induced extension of isolated cucumber cell walls [see Plant Cell (1992) 4, 1425-1433]. Here the mechanism of expansin action was examined by 3 approaches using cell walls from cucumber cv. Burpee Pickler hypocotyls. Firstly, expansins did not alter the molecular mass distribution or the viscosity of solutions of matrix polysaccharides. It was concluded from this that expansins do not hydrolyze the major pectins or hemicelluloses of the cucumber wall. Second, the effects of expansins on stress relaxation of isolated walls were studied. These studies showed that expansins account for the pH-sensitive and heat-labile components of wall stress relaxation, and also that expansins do not cause a progressive weakening of the walls as might be expected from the action of a hydrolase. Thirdly, the binding of expansins to the cell wall and its components was studied. The binding characteristics were consistent with this being the site of expansin action. Expansins bound weakly to crystalline cellulose but this binding was 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 (approx. 1 part in 5000 on a DW basis), suggesting that they function catalytically. It is concluded that expansins bind at the interface between cellulose microfibrils and matrix polysaccharides in the wall and induce extension by reversibly disrupting non-covalent bonds within this polymeric network. The 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. Download PDF (1,500 kB)

McQueen-Mason S, Durachko DM, Cosgrove DJ (1992) Two endogenous proteins that induce cell wall expansion in plants. Plant Cell 4: 1425-1433
ABSTRACT: Plant cell enlargement is regulated by wall relaxation and yielding, which is thought to be catalysed by elusive "wall-loosening" enzymes. By employing a reconstitution approach, a crude protein extract from the cell walls of growing cucumber seedlings was found to possess the ability to induce the extension of isolated cell walls. This activity was restricted to the growing region of the stem and could induce the extension of isolated cell walls from stems of various dicotyledons (pea, radish, tomato and cucumber) and the leaves of 2 monocotyledons (onion and Zephyranthes candida), but was less effective on coleoptile walls of graminaceous monocotyledons (maize and barley). Endogenous and reconstituted wall extension activities showed similar sensitivities to pH, metal ions, thiol reducing agents, proteases, and boiling in methanol or water. Sequential HPLC fractionation of the active wall extract revealed 2 proteins with molecular masses of 29 and 30 kDa associated with the activity. Each protein, by itself, could induce wall extension without detectable hydrolytic breakdown of the wall. These proteins appear to mediate "acid growth" responses of isolated walls and may catalyse plant cell wall extension by a novel biochemical mechanism.  Download the full article in Acrobat pdf format (1,556 kB file). © ASPP

McQueen-Mason S, Fry SC, Durachko DM, Cosgrove DJ (1993) The relationship between xyloglucan endotransglycosylase and in vitro cell wall extension in cucumber hypocotyls. Planta 190: 327-331

Meir S, Hunter DA, Chen JC, Halaly V, Reid MS. (2006) Molecular changes occurring during acquisition of abscission competence following auxin depletion in Mirabilis jalapa. Plant Physiol. 141:1604-16
Abstract: To understand how auxin regulates sensitivity of abscission zone (AZ) tissues to ethylene, we used a polymerase chain reaction-based subtractive approach to identify gene transcripts in Mirabilis jalapa AZs that changed in abundance during the time the zones became competent to abscise in response to exogenous ethylene. Transcript expression was then examined in leaf and stem AZs over the period they became ethylene competent following indole-3-acetic acid (IAA) depletion either by leaf deblading, treatment with the IAA transport inhibitor naphthylphthalamic acid, or cutting the stem above a node (decapitation). Transcripts down-regulated by deblading/decapitation included Mj-Aux/IAA1 and Mj-Aux/IAA2, encoding Aux/IAA proteins, and three other transcripts showing highest identity to a polygalacturonase inhibitor protein, a beta-expansin, and a beta-tubulin. Application of IAA to the cut end of petioles or stumps inhibited abscission, and prevented the decline in the levels of transcripts in both AZs. Transcripts up-regulated in the AZ following deblading/decapitation or treatment with naphthylphthalamic acid were isolated from plants pretreated with 1-methylcyclopropene before deblading to help select against ethylene-induced genes. Some of the up-regulated transcripts showed identity to proteins associated with ethylene or stress responses, while others did not show homology to known sequences. Sucrose infiltration of stem stumps enhanced abscission following ethylene treatment and also enhanced the induction of some of the up-regulated genes. Our results demonstrate a correlation between acquisition of competence to respond to ethylene in both leaf and stem AZs, and decline in abundance of auxin regulatory gene transcripts.

Muller B, Bourdais G, Reidy B, Bencivenni C, Massonneau A, Condamine P, Rolland G, Conéjéro G, Rogowsky P, Tardieu F. 2007.  Association of specific expansins with growth in maize leaves is maintained under environmental, genetic, and developmental sources of variation. Plant Physiol. 143(1):278-90.
ABSTRACT: We aimed to evaluate whether changes in maize (Zea mays) leaf expansion rate in response to environmental stimuli or developmental gradients are mediated by common or specific expansins, a class of proteins known to enhance cell wall extensibility. Among the 33 maize expansin or putative expansin genes analyzed, 19 were preferentially expressed at some point of the leaf elongation zone and these expansins could be organized into three clusters related to cell division, maximal leaf expansion, and cell wall differentiation. Further analysis of the spatial distribution of expression was carried out for three expansins in leaves displaying a large range of expansion rates due to water deficit, genotype, and leaf developmental stage. With most sources of variation, the three genes showed similar changes in expression and consistent association with changes in leaf expansion. Moreover, our analysis also suggested preferential association of each expansin with elongation, widening, or both of these processes. Finally, using in situ hybridization, expression of two of these genes was increased in load-bearing tissues such as the epidermis and differentiating xylem. Together, these results suggest that some expansins may be preferentially related to elongation and widening after integrating several spatial, environmental, genetic, and developmental cues.  Download pdf (C)ASPB\

Nishiyama K, Guis M, Rose JK, Kubo Y, Bennett KA, Wangjin L, Kato K, Ushijima K, Nakano R, Inaba A, Bouzayen M, Latche A, Pech JC, Bennett AB. 2007. Ethylene regulation of fruit softening and cell wall disassembly in Charentais melon. J Exp Bot. 58(6):1281-90.
ABSTRACT: Cell wall disassembly in ripening fruit is highly complex, involving the dismantling of multiple polysaccharide networks by diverse families of wall-modifying proteins. While it has been reported in several species that multiple members of each such family are expressed in the same fruit tissue, it is not clear whether this reflects functional redundancy, with protein isozymes from a single enzyme class performing similar roles and contributing equally to wall degradation, or whether they have discrete functions, with some isoforms playing a predominant role. Experiments reported here sought to distinguish between cell wall-related processes in ripening melon that were softening-associated and softening-independent. Cell wall polysaccharide depolymerization and the expression of wall metabolism-related genes were examined in transgenic melon (Cucumis melo var. cantalupensis Naud.) fruit with suppressed expression of the 1-aminocyclopropane-1-carboxylate oxidase (ACO) gene and fruits treated with ethylene and 1-methylcyclopropene (1-MCP). Softening was completely inhibited in the transgenic fruit but was restored by treatment with exogenous ethylene. Moreover, post-harvest application of 1-MCP after the onset of ripening completely halted subsequent softening, suggesting that melon fruit softening is ethylene-dependent. Size exclusion chromatography of cell wall polysaccharides, from the transgenic fruits, with or without exogenous ethylene, indicated that the depolymerization of both pectins and xyloglucans was also ethylene dependent. However, northern analyses of a diverse range of cell wall-related genes, including those for polygalacturonases, xyloglucan endotransglucosylase/hydrolases, expansin, and beta-galactosidases, identified specific genes within single families that could be categorized as ethylene-dependent, ethylene-independent, or partially ethylene-dependent. These results support the hypothesis that while individual cell wall-modifying proteins from each family contribute to cell wall disassembly that accompanies fruit softening, other closely related family members are regulated in an ethylene-independent manner and apparently do not directly participate in fruit softening.  Download pdf (C) Oxford

Obenland,D.M., Crisosto,C.H., and Rose,J.K.C. (2003) Expansin protein levels decline with the development of mealiness in peaches. Postharvest Biology and Technology 29:11-18.
Abstract: Expansin mRNA and protein expression in peaches was examined to investigate the possibility that expansins may be involved in the development of mealy flesh texture. Immunoblot analysis, using an expansin antibody, detected a 27-kDa protein corresponding to the predicted molecular mass of expansins in the later stages of ripening but not in full-size green fruit and indicated that expansin is associated with the progression of ripening in peaches. Peach cultivars 'O' Henry' and 'Summer Lady' were stored at 5 degreesC to induce the development of mealiness and individual fruit samples collected periodically to measure mealiness (free water) and provide samples for expansin quantification. Initially, stored fruit were juicy (50-60% free water), but as storage progressed they became visibly mealy at free water percentages of 30% for 'O' Henry' and 46% for 'Summer Lady'. In both cultivars expansin protein abundance decreased as the fruit became mealy. Development of mealiness within individual fruit was often not uniform and lead to the existence of distinct juicy and mealy regions. Immunoblot analysis indicated that mealy regions contained substantially less expansin than juicy regions. Analysis of expansin mRNA abundance using a ripening-related expansin cDNA probe from peach indicated that expansin mRNA expression was also markedly reduced in mealy tissues. The relationship observed in this study between expansin expression and mealiness suggests a possible role for expansin in the development of the disorder. Published by Elsevier Science B.V

Oka M, Tasaka Y, Iwabuchi M, Mino M. Elevated sensitivity to gibberellin by vernalization in the vegetative rosette plants of Eustoma grandiflorum and Arabidopsis thaliana. Plant Sci 160(6):1237-1245
ABSTRACT: Changes in the sensitivity to gibberellin (GA) after vernalization were studied in the vegetative rosette of Eustoma grandiflorum and late flowering Arabidopsis thaliana mutant, fca-1. The sensitivity to GA after vernalization was monitored using the bolting rate of plants that were grown on a medium containing GA(3) or ancymidol. The bolting rates were higher in vernalized plants than non-vernalized plants when the same GA(3) concentration was used. There was a positive relationship between the duration of vernalization and the bolting rate in E. grandiflorum. In contrast, a negative relationship between the duration of treatment and bolting rate was found in the non-vernalized plants. In fca-1, the flowering time of vernalized plants was significantly reduced compared with the non-vernalized plants under various concentrations of GA(3) treatment. To elucidate whether this elevated sensitivity relates to the efficiency of GA signal transduction, we measured the transcript amounts of Expansin (Exp), which is up-regulated by GA, and GA20-oxidase (GA20ox) and GA3 beta-hydroxylase (GA3betahy), which are down-regulated by GA. The transcript amounts were estimated using the Taq-Man PCR system based on combinations of primers and probes that specifically detect the genes, and normalized by the transcript amount of ubiquitin gene measured as an internal standard. For each concentration of GA treatments examined, Expansin of both E. grandiflorum and A. thaliana was induced at a higher rate in the vernalized plants than in the non-vernalized plants. The expression of GA20ox and GA3betahy of E. grandiflorum decreased faster in the vernalized plants than the non-vernalized plants. We conclude that vernalization is a critical environmental cue not only for initiating GA biosyntheses in vegetative rosette, but also for elevating the GA sensitivity of the plants via a GA signal transduction pathway.

R. Ookawara, S. Satoh, T. Yoshioka, and K. Ishizawa. Expression of alpha-expansin and xyloglucan endotransglucosylase/hydrolase genes associated with shoot elongation enhanced by anoxia, ethylene and carbon dioxide in arrowhead (Sagittaria pygmaea Miq.) tubers. Ann.Bot.(Lond) 96 (4):693-702, 2005.
Abstract:  BACKGROUND AND AIMS: Shoot elongation of arrowhead tubers (Sagittaria pygmaea Miq.) is stimulated by anoxia, ethylene and CO2. The aim of this study was to characterize anoxic elongation by comparison with elongation stimulated by ethylene and CO2. METHODS: The effects of the inhibitors aminoethoxyvinylglycine (AVG) as an ethylene biosynthesis inhibitor, 1-methylcyclopropene (1-MCP) as a potent inhibitor of ethylene action, and pyrazol, an inhibitor of alcohol dehydrogenase, on shoot elongation were examined. Moreover, the effects of these gaseous factors on expression of genes possibly involved in modification of cell wall architecture were examined by polymerase chain reaction (PCR) methods. KEY RESULTS AND CONCLUSIONS: In air, promotion by 5% CO2 and 5 microL L-1 ethylene of shoot elongation occurred. At 1% O2, ethylene also stimulated shoot elongation but CO2 did not. Pyrazol inhibited shoot elongation in hypoxia but not in normoxia, suggesting that alcohol fermentation contributes to elongation enhanced by hypoxia. AVG and 1-MCP partially prevented shoot elongation both in normoxia and in hypoxia, but they did not have significant effects in anoxia, suggesting that endogenous ethylene acts as a stimulator of shoot elongation in normoxia and in hypoxia but not in anoxia. Ethylene is not involved in anoxia-enhanced elongation. We cloned four cDNAs (SpEXPA1, 2, 3 and 4) encoding alpha-expansin (EXPA) and five cDNAs (SpXTH1, 2, 3, 4 and 5) encoding xyloglucan endotransglucosylase/hydrolase (XTH) from shoots of arrowhead tubers. The transcript levels of SpEXPA1 and 2 were increased by anoxia and those of SpEXPA2 were increased by 5% CO2. Ethylene slightly elevated the level of SpEXPA4 transcripts. Anoxia enhanced the transcript levels of SpXTH1 and 4; neither ethylene nor CO2 had any effect. CO2 enhanced transcript levels of SpXTH3 and depressed those of SpXTH5. Ethylene decreased transcript levels of SpXTH5. These results suggest that four SpEXPA genes and five SpXTH genes are differently responsive to anoxia, CO2 and ethylene. Enhancement of SpEXPA1 and 2, and SpXTH1 and 4 transcript levels suggests that these gene products are involved in anoxic shoot elongation through modification of cell wall architecture

Orford SJ, Timmis JN   (1998)  Specific expression of an expansin gene during elongation of cotton fibres. Biochim Biophys Acta 1998 Jul 9;1398(3):342-6       
ABSTRACT: A differential screening experiment resulted in the isolation of a full-length cDNA clone encoding an expansin from cotton fibres. Nucleotide and derived amino acid sequence data showed that pGhEX1 encodes an expansin of 258 amino acids, with an N-terminal signal peptide. Northern blot analysis showed that the corresponding transcript is abundant in cotton fibre cells but absent in all other tissues tested, and that the gene is developmentally regulated during fibre elongation.

Osumi M. The ultrastructure of yeast: cell wall structure and formation. Micron. 1998 Apr-Jun;29(2-3):207-33
Abstract: Yeasts are unicellular eukaryotes, and are used widely as a model system in basic and applied fields of life science, medicine, and biotechnology. The ultrastructure of yeast cells was first studied in 1957 and the techniques used have advanced greatly in the 40 years since then; an overview of these methods is first presented in this review. The ultrastructure of budding and dimorphic yeast cells observed with a scanning electron microscope (SEM) and a transmission electron microscope (TEM) after thin sectioning and freeze-etching are then described, followed by discussion of the regeneration of the cell wall of Candida albicans protoplasts detected by cryosectioning. C. albicans protoplasts are regenerated to synthesize microfibrils on their surface. They are aggregated into thicker bundles which are intermeshed, forming a wide-meshed network of long fibrils. These microfibrillar structures are chains of beta-1,3-glucan which are broken down after treatment with beta-1,3-glucanase. Morphologically identical microfibrils are synthesized in vitro by a cell-free system in which the active cell membrane fraction as a source of beta-1,3-glucan synthetase and UDP glucose as the sole substrate are used. The diameter of an elemental fibril of beta-glucan is estimated to be 2.8 nm from the pattern of autocorrelation of the image obtained by computer processing. In contrast, in the presence of aculeacin A the formation of normal fibrillar nets or bundles is significantly inhibited, resulting in the occurrence of short fibrils. These electron microscopic data suggest that aculeacin A inhibits not only the synthesis of beta-1,3-glucan but the aggregation of microfibrils of this polysaccharide, allowing formation of the crystalline structure. On the basis of the cumulative data obtained from the electron microscopic studies, we are led to the assumption that de novo synthesized beta-glucan chains might initially form fine particles which are then transformed into thin fibrils with single to multiple strands which appear to be oriented parallel to each other so that they develop into fibrillar structures. This process of assembly of beta-glucan molecules leads to the development of a fibrous network within the regenerating Candida cell wall. Third, the mechanism of cell wall formation is shown by low-voltage (LV) SEM and TEM, using various techniques and computer graphics, of the regeneration system of Schizosaccharomyces pombe protoplasts: after 10 min of regeneration, the protoplasts begin to grow fibrillar substances of a beta-glucan nature, and a fibrillar network covers the surface of all protoplasts. The network is originally formed as fine particles on the protoplast surface and these are subsequently lengthened to microfibrils 2 nm thick. The microfibrils twist around each other and develop into 8 nm thick fibrils forming flat bundles 16 nm thick. Interfibrillar spaces are gradually filled with amorphous particles of an alpha-galactomannan nature and, finally, the complete cell wall is formed after 12 h. Treatment of reverting protoplasts with RuO4 provided clear TEM images of glucan fibrils with high electron density. The relationship between cell wall regeneration and intracellular organelles was examined by using serial thin sections stained with PATAg and computer-aided three-dimensional reconstruction. The secretory vesicles in a protoplast had increased markedly by 1.4, 3.4, and 5.8 times at 1.5, 3.0, and 5 h, respectively. Three-dimensional analysis indicates that Golgi apparatuses are located close together in the nucleus of the protoplast and are dispersed into the cytoplasm during the progress of cell wall formation.  download_pdf (12 MB)

Robert E. Paull, Beth Irikura, Pingfang Wu, Helen Turano, Nancy Jung Chen, Andrea Blas, John K. Fellman, Andrea R. Gschwend, Ching Man Wai, Qingyi Yu, Gernot Presting, Maqsudul Alam, Ray Ming. 2008. Fruit Development, Ripening and Quality Related Genes in the Papaya Genome. Tropical Plant Biology 1: 246-277.
Abstract Papaya (Carica papaya L.) is the first fleshy fruit with a climacteric ripening pattern to be sequenced. As a member of the Rosids superorder in the order Brassicales, papaya apparently lacks the genome duplication that occurred twice in Arabidopsis. The predicted papaya genes that are homologous to those potentially involved in fruit growth, development, and ripening were investigated. Genes homologous to those involved in tomato fruit size and shape were found. Fewer predicted papaya expansin genes were found and no Expansin Like-B genes were predicted. Compared to Arabidopsis and tomato, fewer genes that may impact sugar accumulation in papaya, ethylene synthesis and response, respiration, chlorophyll degradation and carotenoid synthesis were predicted. Similar or fewer genes were found in papaya for the enzymes leading to volatile production than so far determined for tomato. The presence of fewer papaya genes in most fruit development and ripening categories suggests less subfunctionalization of gene action. The lack of whole genome duplication and reductions in most gene families and biosynthetic pathways make papaya a valuable and unique tool to study the evolution of fruit ripening and the complex regulatory networks active in fruit ripening.

Rochange SF, Wenzel CL, McQueen-Mason SJ. (2001) Impaired growth in transgenic plants over-expressing an expansin isoform. Plant Mol Biol 46(5):581-9
ABSTRACT: 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.  PDF file not available. 

 Zhang N, Hasenstein KH (2000)  Distribution of expansins in graviresponding maize roots. Plant Cell Physiol. 41: 1305-1312
ABSTRACT:  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. Download pdf file (2041 K). (C) Plant & Cell Physiology.

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