Role of extensin peroxidase in tomato (Lycopersicon esculentum Mill.) seedling growth

 It is proposed that inhibition of extensin peroxidase activity leads to a less rigid cell wall and thus promotes cell expansion and plant growth. A low-molecular-weight inhibitor derived from the cell walls of suspension-cultured tomato cells was found to completely inhibit extensin peroxidase-mediated extensin cross-linking in vitro at a concentration of 260 μg/ml. The inhibitor had no effect upon guaiacol oxidation catalyzed by extensin peroxidase or horseradish peroxidase. We have demonstrated that the light-irradiated inhibition of plant growth may be partially offset by inhibition of endogenous extensin peroxidase activity. Overall plant growth was enhanced by up to 15% in the presence of inhibitor relative to control plants. Inhibitor-treated and illuminated tomato hypocotyls grew up to 15% taller than untreated controls. The inhibitor had no effect upon etiolated plants over a 15-d period, suggesting that only low levels of peroxidase-mediated cross-linking can be found in the cell walls of etiolated plants. SDS-PAGE/Western blots of ionically bound protein from both etiolated and illuminated hypocotyls identified a doublet at 57/58.5 kDa which is immuno-reactive with antibodies raised to tomato extensin peroxidase. Levels of the 58.5-kDa protein, determined by SDS-PAGE, were at least threefold higher in illuminated tomato hypocotyls than in etiolated hypocotyls. Three fold higher levels of extensin peroxidase, elevated in-vitro extensin cross-linking activity and 15% higher levels of cross-linked, non-extractable extensin were observed in illuminated tomato hypocotyls compared with etiolated tomato hypocotyls. This suggests that white-light inhibition of tomato hypocotyl growth appears to be mediated, at least partially, by deposition of cell wall extensin, a process regulated by M_r-58,500 extensin peroxidase. Our results indicate that the contribution of peroxidase-mediated extensin deposition to plant cell wall architecture may have an important role in plant growth. Received: 22 July 1999 / Accepted: 11 October 1999     

Purification of extensin from cell walls of tomato (hybrid of Lycopersicon esculentum and L. peruvianum) cells in suspension culture

Extensin, a hydroxyproline-rich glycoprotein comprising substantial amounts of -l-arabinose-hydroxyproline glycosidic linkages is believed to be insolubilized in the cell wall during host-pathogen interaction by a peroxidase/hydroperoxide-mediated cross-linking process. Both extensin precursor and extensin peroxidase were ionically eluted from intact water-washed tomato (hybrid) of Lycopersicon esculentum Mill. and L. peruvianum L. (Mill.) cells in suspension cultures and purified to homogeneity by a rapid and simple procedure under mild and non-destructive experimental conditions. The molecular weight of native extensin precursor was estimated to be greater than 240–300 kDa by Superose-12 gel-filtration chromatography. Extensin monomers have previously been designated a molecular weight of approximately 80 kDa. Our results indicate that salt-eluted extensin precursor is not monomeric. Agarose-gel electrophoresis, Superose-12-gel-filtration, extensin-peroxidase-catalysed cross-linking, Mono-S ion-exchange fast protein liquid chromatography (FPLC), and peptide-sequencing data confirmed the homogeneity of the extensin preparation. Evidence that the purified protein was extensin is attributed to the presence of the putative sequence motif — Ser (Hyp)₄ — within the N-terminal end of the protein. Treatment of extensin with trifluoroacetic acid demonstrated that arabinose was the principal carbohydrate. The amino-acid composition of the purified extensin was similar to those reported in the literature. The cross-linking of extensin in vitro upon incubation with extensin peroxidase and exogenous H₂O₂ was characteristic of other reported extensins. Furthermore, Mono-S ion-exchange FPLC of native extensin precursor resolved it into two isoforms, A (90%) and B (10%). The amino-acid compositions of extensin A and extensin B were found to be similar to each other and both extensins were cross-linked in vitro by extensin peroxidase.Abbreviations CM-cellulose carboxymethyl-cellulose - FPLC fast protein liquid chromatography - HF hydrogen fluoride - HRGP hydroxyproline-rich glycoprotein - Hyp hydroxyproline - V_c retention volume - TCA trichloroacetic acid - TFA tri-fluoroacetic acid This work was supported by a A.F.R.C. postdoctoral assistantship to Michael D. Brownleader. We thank Dr. Anthony K. Allen (Department of Biochemistry, Charing Cross and Westminster Hospital, London, UK) for performing the amino-acid analysis and Mrs. Margaret Pickering (Department of Biochemistry, Royal Holloway) for performing the peptide-sequence analysis of extensin. We also express our gratitide to Dr. A. Mort (Oklahoma State University) for performing the HF-deglycosylation of extensin.     

Antibody localization of extensin in cell walls of carrot storage roots

The accumulation and cross-linking of hydroxyproline-rich glycoproteins (HRGPs) in cell walls of dicotyledonous plants has been correlated with a number of wall-strengthening phenomena. Polyclonal antibodies raised against glycosylated extensin-1, the most abundant HRGP in carrot (Daucus carota L.) cell walls, recognize this antigen on gel and dot blots and on thin sections of epoxy-embedded carrot-root cell walls. Since wall labeling can be largely reduced by preincubating the antibodies with purified extensin-1, most labeling can be attributed to recognition of this antigen. The remaining label may be the result of recognition of extensin-2, a second carrot HRGP, or other wall components (cellulose, hemicellulose and pectin are not recognized). Extensin-1 label was distributed quite uniformly across the cell wall but was absent from the expanded middle lamella at the intersection of three or more cells and was reduced in the narrow middle lamella between two cells. This distribution is essentially the same as that of cellulose. Because of limitations of this labeling technique, it is not possible to construct a complete model of the structure of the cross-linked extensin matrix. Nonetheless, short, linear arrays of gold particles may represent small portions of the extensin matrix or of individual extensin molecules as they are exposed on the surface of sections. These and other results presented here indicate that: a) newly synthesized extensin is added to the wall by intussusception; b) extensin cannot cross the middle lamella separating the walls of adjacent cells; and c) incorporation of extensin is a late event in the development of phloem-parenchyma cell walls in carrot.Abbreviations dE-1 antibodies antibodies raised against deglycosylated extensin 1 - ELISA enzyme-linked immunosorbant assay - gE-1 antibodies antibodies raised against glycosylated extensin 1 - HRGP hydroxyproline-rich glycoprotein - PAGE polyacrylamide gel electrophoresis - RG-1 rhamnogalacturonan I - SDS sodium dodecyl sulfate     

Characterization of a hydroxyproline-rich glycoprotein in pearl millet and its differential expression in response to the downy mildew pathogen <Emphasis Type="Italic">Sclerospora graminicola</Emphasis>

A monoclonal antibody, JIM 20, derived against an extensin type of hydroxyproline-rich glycoprotein (HRGP) from pea, showed high affinity for HRGP in pearl millet [Pennisetum glaucum (L.) R. Br.]. Electrophoretic separation of Tris–SDS extracted proteins from suspension cells of pearl millet revealed a range of PM-HRGP polypeptides having a glycan epitope, which reacted with JIM 20. A high molecular mass band, probably an HRGP aggregate or polymer, and a few low molecular mass polypeptides were recognized by JIM 20 during Western blot analysis. Treatment of pearl millet suspension cells with hydrogen peroxide in the presence of an endogenous peroxidase resulted in insolubilization of HRGP polypeptides with molecular weights between 45 and 33 kDa. To investigate the gene coding for an extensin type of HRGP, a fosmid-based genomic library of pearl millet having a fourfold genome coverage was constructed. A partial sequence of 378 bp of an HRGP gene was obtained by PCR amplification of pearl millet DNA with a primer pair designed from the conserved regions of monocotyledon extensin type of HRGPs. Screening the genomic library using the homologous probe developed from the 378-bp PCR product resulted in the isolation of five fosmid clones. Restriction mapping of these fosmids resulted in an 11.8-kb region around an HRGP gene in pearl millet. The newly characterized gene, PM-HRGP, had all the characteristic features of a monocotyledon extensin type of HRGP. An intron at the 3′ untranslated region of the gene was identified by cDNA cloning. Differential expression of the PM-HRGP gene was observed during compatible and incompatible interactions of pearl millet with the downy mildew pathogen Sclerospora graminicola (Sacc) Schroet. Induced expression of the gene was observed only in case of an incompatible interaction.     

Purification and Partial Characterization of Tomato Extensin Peroxidase

Early plant defense response is characterized by elevation of activity of peroxidases and enhanced insolubilization of hydroxyproline-rich glycoproteins, such as extensin, in the cell wall. The insolubilization process (cross-linking between soluble extensin precursor molecules) is catalyzed by extensin peroxidases. We have ionically eluted extensin peroxidases from intact water-washed suspension-cultured tomato (hybrid of Lycopersicon esculentum Mill. and Lycopersicon peruvianum L. [Mill.]) cells and purified them to homogeneity by molecular sieve and cation-exchange chromatography. Four ionic forms of peroxidase (PI,PII,EPIII, and EPIV) were resolved; only the latter two cross-linked tomato soluble extensin. The molecular weight (34,000-37,000), amino acid composition, and isoelectric point (9.0) of the extensin peroxidases were determined. Substrate specificities of the enzymes were investigated: soluble extensin and potato lectin (a hydroxyproline-rich glycoprotein with a domain that strongly resembles extensin) were cross-linked by only two forms of the enzyme, whereas bovine serum albumin, aldolase, insulin, a number of other marker proteins, and proteins eluted from tomato cells (except extensin) could not be cross-linked. We have also isolated a yeast elicitor that enhances total peroxidase activity and extensin insolubilization within 1 h of challenge in cultured cells of tomato. A highly sensitive enzyme-linked immunosorbent assay technique using polyclonal antiserum raised against soluble tomato extensin was used to demonstrate extensin insolubilization in vivo. A tomato cell-wall peroxidase that cross-links extensin has been purified and may have a role in plant defense.     

Purification and Partial Characterization of a Hydroxyproline-Rich Glycoprotein in a Graminaceous Monocot, Zea mays

Graminaceous monocots generally contain low levels of hydroxyproline-rich Glycoproteins (HRGPs). As HRGPs are often at the cell surface, we used the intact cell elution technique (100 millimolar AlCl₃) to isolate soluble surface proteins from Zea mays cell suspension cultures. Further fractionation of the trichloroacetic acid-soluble eluate on the cation exchangers phospho-cellulose and BioRex-70 gave several retarded, hence presumably basic fractions, which also contained hydroxyproline (Hyp). One of these fractions yielded a pure HRGP after a final purification step involving Superose-6 gel filtration. As this HRGP was unusually rich in threonine, (25 mole%) we designated it as a threonine-hydroxyproline-rich glycoprotein (THRGP); it contained about 27% carbohydrate occurring exclusively as arabinosylated Hyp, predominantly as the monosaccharide (15%), and trisaccharide (25%) with 48% Hyp nonglycosylated—a characteristically graminaceous monocot profile. Amino acid analysis confirmed the basic character, and gave a low alanine content. Reaction with Yariv artificial antigen was negative. These characteristics show that the THRGP is not an arabinogalactan protein. On the other hand, antibodies raised against tomato extensin P1 cross-reacted significantly with the THRGP; this cross-reactivity and the above analytical data provide the best evidence to date for the presence of extensin in a graminaceous monocot.     

Isolation of pl 4.6 extensin peroxidase from tomato cell suspension cultures and identification of Val—Tyr—Lys as putative intermolecular cross-link site

Extensins and kindred hydroxyproline-rich glycoproteins occur in dicot cell walls mainly as insoluble integral components that may form an intermolecularly cross-linked network interpenetrated by other polymers. Extensins also occur in muro as a small pool of soluble monomeric precursors to network extensin. These precursors were prepared in milligram quantities by salt elution from the surface of intact cells grown as tomato suspension cultures. Based on an FPLC (Superose-6) gel filtration assay of cross-linked extensin oligomers, a pl 4.6 extensin cross-linking peroxidase isozyme was partially purified from the culture growth medium. Purification involved: volume reduction, ultracentrifugation to remove pectin and co-adsorbed cationic peroxidase, followed by chromatography of anionic extensin peroxidase (pl 4.6) on DEAE—Trisacryl and TSK-gel DEAE-5PW columns. With tomato P1 extensin as substrate and 60 µM H₂O₂ as co-substrate, at 23° pl 4.6 extensin peroxidase gave a K,m of 0.22 mM P1 and a V _max of 70 µmol P1 cross-linked min⁻¹ mg⁻¹ enzyme, at the optimum pH 5.5. Assayed with 12 different extensins from representative monocots, dicots, and gymnosperms, the pl 4.6 isozyme cross-linked highly selectively, indicating two natural groups: cross-linking or CL-extensins and non-cross-linking or NCL-extensins. CL-extensins contained the X—Hyp—Val—Tyr—Lys motif and were also highly glycosylated. However, the simplest motif common to CL-extensins but absent from NCL-extensins was Val—Tyr—Lys. Thus, peroxidative coupling of extensin monomers and resistance of the resultant oligomers to depolymerization by anhydrous HF suggests that the intermolecular cross-link involves tyrosine or lysine.     

An epitope of rice threonine- and hydroxyproline-rich glycoprotein is common to cell wall and hydrophobic plasma-membrane glycoproteins

A monoclonal antibody, LM1, has been derived that has a high affinity for an epitope of hydroxyproline-rich glycoproteins (HRGPs). In suspension-cultured rice (Oryza sativa L.) cells the epitope is carried by three major proteins with different biochemical properties. The most abundant is the 95-kDa extracellular rice extensin, a threonine- and hydroxyproline-rich glycoprotein (THRGP) occurring in the cell wall and secreted into the medium. This THRGP can be selectively oxidatively cross-linked in the presence of hydrogen peroxide and an endogenous peroxidase with the result that it does not enter a protein gel. A second polypeptide with the LM1 epitope (180 kDa), also occurring in the suspension-cultured cells and medium, is not oxidatively cross-linked. Three further polypeptides (52, 65 and 110 kDa) with the characteristics of hydrophobic proteins of the plasma-membrane also carry the LM1 epitope as determined by immuno-blotting of detergent/aqueous partitions of a plasma-membrane preparation and immuno-fluorescence studies with rice protoplasts. At the rice root apex the LM1 epitope is carried by four glycoproteins and is developmentally regulated. The major locations of the epitope are at the surface of cells associated with the developing protoxylem and metaxylem in the stele, the longitudinal radial walls of epidermal cells and a sheath-like structure at the surface of the root apex.Abbreviations AGP arabinogalactan protein - ELISA enzyme-linked immunosorbent assay - HRGP hydroxyproline-rich glycoprotein - THRGP threonine- and hydroxyproline-rich glycoprotein This work was supported by The Leverhulme Trust. We also acknowledge support from The Royal Society and thank Prof. L.A. Staehelin for the carrot extensin, N. Stacey for the rice cell culture and Dr. J. Keen for protein sequencing.     

The Arabidopsis extensin gene is developmentally regulated, is induced by wounding, methyl jasmonate, abscisic and salicylic acid, and codes for a protein with unusual motifs

A single-copy extensin gene (atExt1) has been isolated from Arabidopsis thaliana (L.) Heynh. The deduced amino acid sequence consists of 374 amino acids which are organised into highly ordered repeating blocks in which Ser(Pro)₄ and Ser(Pro)₃ motifs alternate. Two copies of the Tyr-X-Tyr-Lys motif and 13 copies of the Val-Tyr-Lys motif are present, showing that this extensin may be highly cross-linked, possessing the capacity for both intra and inter-molecular bond formation. The gene atExt1 is normally expressed in the root and is silent in the leaf; wounding reverses this pattern, turning on the gene in the leaf and repressing it in the root. The promoter contains motifs which have been found to activate plant defence genes in response to salicylic acid, abscisic acid and methyl jasmonate; when these compounds are applied to the roots, the atExt1 gene is activated in the leaf. Received: 11 September 1998 / Accepted: 20 December 1998     

Enzymic cross-linkage of monomeric extensin precursors in vitro

Rapidly growing tomato (Lycopersicon esculentum) cell suspension cultures contain transiently high levels of cell surface, salt-elutable, monomeric precursors to the covalently cross-linked extensin network of the primary cell wall. Thus, we purified a highly soluble monomeric extensin substrate from rapidly growing cells, and devised a soluble in vitro cross-linking assay based on Superose-6 fast protein liquid chromatography separation, which resolved extensin monomers from the newly formed oligomers within 25 minutes. Salt elution of slowly growing (early stationary phase) cells yielded little or no extensin monomers but did give a highly active enzymic preparation that specifically cross-linked extensin monomers in the presence of hydrogen peroxide, judging from: (a) a decrease in the extensin monomer peak on fast protein liquid chromatography gel filtration, (b) appearance of oligomeric peaks, and (c) direct electron microscopical observation of the cross-linked oligomers. The cross-linking reaction had a broad pH optimum between 5.5 and 6.5. An approach to substrate saturation of the enzyme required extensin monomer concentrations of 20 to 40 milligrams per milliliter. Preincubation with catalase completely inhibited the cross-linking reaction, which was highly dependent on hydrogen peroxide and optimal at 15 to 50 micromolar. We therefore identified the cross-linking activity as extensin peroxidase.     

Cross-linking of soluble extensin in isolated cell walls

The extensin component of primary cell walls has generally been considered to be an intrinsically insoluble cell wall glycoprotein. Recent data have established that cell wall extensin is in fact secreted in a soluble monomeric form which slowly becomes insolubilized in the cell wall probably through the oxidative formation of isodityrosine cross-links. We now show that isolated cell walls from aerated root slices of Daucus carota have the capacity to insolubilize extensin through the formation of isodityrosine. This in vitro cross-linking is specific for the extensin glycoprotein, as other wall proteins are not cross-linked by the isolated wall system. Although extensin can be cross-linked in solution by peroxidase and H₂O₂, dityrosine and not isodityrosine is the phenolic cross-link formed. Wall-catalyzed cross-linking of soluble extensin is inhibited by l-ascorbate, and both the initial rate and total extent of cross-linking are inhibited by acidic pH in the physiological range (pH 4 to 6). We suggest several mechanisms by which acid might inhibit cross-linking and propose that cytoplasmic factors (ascorbate and/or hydrogen ions) may regulate the solubility of extensin in vivo.     

Changes in onion root development induced by the inhibition of peptidyl-prolyl hydroxylase and influence of the ascorbate system on cell division and elongation

Post-translational hydroxylation of peptide-bound proline residues, catalyzed by peptidyl-prolyl-4 hydroxylase (EC 1.14.11.2) using ascorbate as co-substrate, is a key event in the maturation of a number of cell wall-associated hydroxyproline-rich glycoproteins (HRGPs), including extensins and arabinogalactan-proteins, which are involved in the processes of wall stiffening, signalling and cell proliferation. Allium cepa L. roots treated with 3,4-DL-dehydroproline (DP), a specific inhibitor of peptidyl-prolyl hydroxylase, showed a 56% decrease in the hydroxyproline content of HRGP. Administration of DP strongly affected the organization of specialized zones of root development, with a marked reduction of the post-mitotic isodiametric growth zone, early extension of cells leaving the meristematic zone and a huge increase in cell size. Electron-microscopy analysis showed dramatic alterations both to the organization of newly formed cell walls and to the adhesion of the plasma membranes to the cell walls. Moreover, DP administration inhibited cell cycle progression. Root tips grown in the presence of DP also showed an increase both in ascorbate content (+53%) and ascorbate-specific peroxidase activity in the cytosol (+72%), and a decrease in extracellular “secretory” peroxidase activity (−73%). The possible interaction between HRGPs and the ascorbate system in the regulation of both cell division and extension is discussed. Received: 14 October 1998 / Accepted: 31 May 1999     

Cross-linking patterns in salt-extractable extensin from carrot cell walls

Extensins are hydroxyproline-rich glycoproteins (HRGPs) found in the primary cell walls of dicots. Extensin monomers are secreted into the wall and covalently bound to each other, presumably by isodityrosine (IDT) cross-links, to form a rigid matrix. Expression of the extensin matrix is correlated with inhibition of cell elongation during normal development and with increased resistance to virulent pathogens. We have isolated extensin from carrot root tissue (Daucus carota L.) by published techniques and have used gel filtration chromatography to purify fractions enriched in monomers and oligomers. We refer to this protein as “extensin-1” to distinguish it from “extensin-2,” a second extensin-like HRGP from carrot which we will describe later. We prepared extensin-1 for electron microscopy by shadowing it with platinum. Monomers are highly elongated (84 nanometers) and kinked at several sites. Kinks occur at all sites on molecules with nearly equal probability, but do not appear to occur at their ends. The distribution of kinks is similar to that of tyrosine-lysine-tyrosine sequences, which have been shown to be capable of forming intramolecular IDT cross-links, so we suggest that kinks are visible manifestations of intramolecular IDTs. Oligomers likely result from IDT cross-links between monomers, and may be regarded as transient precursors of the fully cross-linked matrix. Nearly 60% of cross-links involve the ends of molecules while the rest are scattered among internal sites. We discuss how the relative positions and proportions of intra- and intermolecular cross-links in extensin-1 may affect the structure, and in turn the function, of the extensin matrix.     

Identification of a highly conserved hydroxyproline-rich glycoprotein in the cell walls of Chlamydomonas reinhardtii and two other Volvocales

The unicellular alga Chlamydomonas reinhardtii Dang, has a cell wall made entirely from hydroxyproline-rich glycoproteins (HRGPs). We recently employed a quantiative in vitro reconstitution system (Adair et al. 1987, J. Cell Biol. 105, 2373–2382) to assign outer-wall HRGPs of C. reinhardtii to specific sublayers, and describe the major interactions responsible for their assembly. Some of these interactions appear to involve relatively conserved HRGP domains, as evidenced by interspecific cell-wall reconstitution between C. reinhardtii and two multicellular Volvocales (Volvoxcarteri lyengar and Gonium pectorale Müller). In the present report we provide biochemical and immunological evidence that the outer cell-walls of V. carteri and G. pectorale both contain prominent HRGPs closely related to C. reinhardtii GP2. Identification of conserved GP2 homologues indicates a molecular basis for interspecific reconstitution and provides a useful avenue for characterization of HRGP domains mediating cell-wall formation in these algae.Abbreviations GP1, 2, 3 outer-cell wall glycoproteins 1, 2, and 3 - GP2_dg deglycosylated GP2 - HRGP hydroxyprolinerich glycoprotein - SDS-PAGE sodium docecyl sulfate polyacrylamide gel electrophoresis     

Immunolocalization of extensin and potato tuber lectin in carrot, tomato and potato

Tissue-specific expression of two members of the cell wall hydroxyproline-rich glycoprotein (HRGP) family, extensin and potato tuber lectin, was examined by immunolocalization at the light microscope level in various organs (leaves, stems, roots, fruit, tuber) of carrot ( Daucus carota cv. Thumbelina), tomato ( Lycopersicon esclentum cv. Pixie Hybrid II), and potato ( Solanum tuberosum cv. Kennebec). Extensin was prominently expressed in vascular tissue, particularly xylem and also phloem, although virtually all cells displayed some degree of staining which varied as a function of the tissue, organ, and plant under study. Antibodies against potato tuber lectin (PTL) displayed a localization pattern similar to that observed for extensin; notably PTL did not stain cambium but did stain epithelial cells lining secretory cavities. These distribution patterns are consistent with a role for extensin, and possibly PTL, in providing mechanical support in tissues subjected to compression or torsional stress imparted by vascular growth, or by similar stress brought about by transport of vascular fluids.     

Focus Issue on Plant Cell Walls: A Bioinformatics Approach to the Identification,Classification, and Analysis of Hydroxyproline-Rich Glycoproteins

Hydroxyproline-rich glycoproteins (HRGPs) are a superfamily of plant cell wall proteins that function in diverse aspects of plant growth and development. This superfamily consists of three members: hyperglycosylated arabinogalactan proteins (AGPs), moderately glycosylated extensins (EXTs), and lightly glycosylated proline-rich proteins (PRPs). Hybrid and chimeric versions of HRGP molecules also exist. In order to “mine” genomic databases for HRGPs and to facilitate and guide research in the field, the BIO OHIO software program was developed that identifies and classifies AGPs, EXTs, PRPs, hybrid HRGPs, and chimeric HRGPs from proteins predicted from DNA sequence data. This bioinformatics program is based on searching for biased amino acid compositions and for particular protein motifs associated with known HRGPs. HRGPs identified by the program are subsequently analyzed to elucidate the following: (1) repeating amino acid sequences, (2) signal peptide and glycosylphosphatidylinositol lipid anchor addition sequences, (3) similar HRGPs via Basic Local Alignment Search Tool, (4) expression patterns of their genes, (5) other HRGPs, glycosyl transferase, prolyl 4-hydroxylase, and peroxidase genes coexpressed with their genes, and (6) gene structure and whether genetic mutants exist in their genes. The program was used to identify and classify 166 HRGPs from Arabidopsis (Arabidopsis thaliana) as follows: 85 AGPs (including classical AGPs, lysine-rich AGPs, arabinogalactan peptides, fasciclin-like AGPs, plastocyanin AGPs, and other chimeric AGPs), 59 EXTs (including SP₅ EXTs, SP₅/SP₄ EXTs, SP₄ EXTs, SP₄/SP₃ EXTs, a SP₃ EXT, “short” EXTs, leucine-rich repeat-EXTs, proline-rich extensin-like receptor kinases, and other chimeric EXTs), 18 PRPs (including PRPs and chimeric PRPs), and AGP/EXT hybrid HRGPs.The genomics era has produced vast amounts of biological data that await examination. In order to “mine” such data effectively, a bioinformatics approach can be utilized to identify genes of interest, subject them to various in silico analyses, and extract relevant biological information on them from various public databases. Examination of such data produces novel insights with respect to the genes in question and can be used to facilitate and guide further research in the field. Such is the case here, where bioinformatics tools were developed to identify, classify, and analyze members of the Hyp-rich glycoprotein (HRGP) superfamily encoded by the Arabidopsis (Arabidopsis thaliana) genome.HRGPs are a superfamily of plant cell wall proteins that are subdivided into three families, arabinogalactan proteins (AGPs), extensins (EXTs), and Pro-rich proteins (PRPs), and extensively reviewed (Showalter, 1993; Kieliszewski and Lamport, 1994; Nothnagel, 1997; Cassab, 1998; José-Estanyol and Puigdomènech, 2000; Seifert and Roberts, 2007). However, it has become increasingly clear that the HRGP superfamily is perhaps better represented as a spectrum of molecules ranging from the highly glycosylated AGPs to the moderately glycosylated EXTs and finally to the lightly glycosylated PRPs. Moreover, hybrid HRGPs, composed of HRGP modules from different families, and chimeric HRGPs, composed of one or more HRGP modules within a non-HRGP protein, also can be considered part of the HRGP superfamily. Given that many HRGPs are composed of repetitive protein sequences, particularly the EXTs and PRPs, and many have low sequence similarity to one another, particularly the AGPs, BLAST searches typically identify only a few closely related family members and do not represent a particularly effective means to identify members of the HRGP superfamily in a comprehensive manner.Building upon the work of Schultz et al. (2002) that focused on the AGP family, a new bioinformatics software program, BIO OHIO, developed at Ohio University, makes it possible to search all 28,952 proteins encoded by the Arabidopsis genome and identify putative HRGP genes. Two distinct types of searches are possible with this program. First, the program can search for biased amino acid compositions in the genome-encoded protein sequences. For example, classical AGPs can be identified by their biased amino acid compositions of greater then 50% Pro (P), Ala (A), Ser (S), and Thr (T), as indicated by greater than 50% PAST. Similarly, arabinogalactan peptides (AG peptides) are identified by biased amino acid compositions of greater then 35% PAST, but the protein (i.e. peptide) must also be between 50 and 90 amino acids in length. Likewise, PRPs can be identified by a biased amino acid composition of greater then 45% PVKCYT. Second, the program can search for specific amino acid motifs that are commonly found in known HRGPs. For example, SP₄ pentapeptide and SP₃ tetrapeptide motifs are associated with EXTs, a fasciclin H1 motif is found in fasciclin-like AGPs (FLAs), and PPVX(K/T) (where X is any amino acid) and KKPCPP motifs are found in several known PRPs (Fowler et al., 1999). In addition to searching for HRGPs, the program can analyze proteins identified by a search. For example, the program checks for potential signal peptide sequences and glycosylphosphatidylinositol (GPI) plasma member anchor addition sequences, both of which are associated with HRGPs (Showalter, 1993, 2001; Youl et al., 1998; Sherrier et al., 1999; Svetek et al., 1999). Moreover, the program can identify repeated amino acid sequences within the sequence and has the ability to search for bias amino acid compositions within a sliding window of user-defined size, making it possible to identify HRGP domains within a protein sequence.Here, we report on the use of this bioinformatics program in identifying, classifying, and analyzing members of the HRGP superfamily (i.e. AGPs, EXTs, PRPs, hybrid HRGPs, and chimeric HRGPs) in the genetic model plant Arabidopsis. An overview of this bioinformatics approach is presented in Figure 1. In addition, public databases and programs were accessed and utilized to extract relevant biological information on these HRGPs in terms of their expression patterns, most similar sequences via BLAST analysis, available genetic mutants, and coexpressed HRGP, glycosyl transferase (GT), prolyl 4-hydroxylase (P4H), and peroxidase genes in Arabidopsis. This information provides new insight to the HRGP superfamily and can be used by researchers to facilitate and guide further research in the field. Moreover, the bioinformatics tools developed here can be readily applied to protein sequences from other species to analyze their HRGPs or, for that matter, any given protein family by altering the input parameters.Open in a separate windowFigure 1.Bioinformatics workflow diagram summarizing the identification, classification, and analysis of HRGPs (AGPs, EXTs, and PRPs) in Arabidopsis. Classical AGPs were defined as containing greater than 50% PAST coupled with the presence of AP, PA, SP, and TP repeats distributed throughout the protein, Lys-rich AGPs were a subgroup of classical AGPs that included a Lys-rich domain, and chimeric AGPs were defined as containing greater than 50% PAST coupled with the localized distribution of AP, PA, SP, and TP repeats. AG peptides were defined to be 50 to 90 amino acids in length and containing greater than 35% PAST coupled with the presence of AP, PA, SP, and TP repeats distributed throughout the peptide. FLAs were defined as having a fasciclin domain coupled with the localized distribution of AP, PA, SP, and TP repeats. Extensins were defined as containing two or more SP₃ or SP₄ repeats coupled with the distribution of such repeats throughout the protein; chimeric extensins were similarly identified but were distinguished from the extensins by the localized distribution of such repeats in the protein; and short extensins were defined to be less than 200 amino acids in length coupled with the extensin definition. PRPs were identified as containing greater than 45% PVKCYT or two or more KKPCPP or PVX(K/T) repeats coupled with the distribution of such repeats and/or PPV throughout the protein. Chimeric PRPs were similarly identified but were distinguished from PRPs by the localized distribution of such repeats in the protein. Hybrid HRGPs (i.e. AGP/EXT hybrids) were defined as containing two or more repeat units used to identify AGPs, extensins, or PRPs. The presence of a signal peptide was used to provide added support for the identification of an HRGP but was not used in an absolute fashion. Similarly, the presence of a GPI anchor addition sequence was used to provide added support for the identification of classical AGPs and AG peptides, which are known to contain such sequences. BLAST searches were also used to provide some support to our classification if the query sequence showed similarity to other members of an HRGP subfamily. Note that some AGPs, particularly chimeric AGPs, and PRPs were identified from an Arabidopsis database annotation search and that two chimeric extensins were identified from the primary literature as noted in the text.     

Cloning and functional analysis of a cDNA encoding a starch synthase from potato (Solanum tuberosum L.) that is predominantly expressed in leaf tissue

Three isoforms of starch synthase (SS) were shown to be present in soluble potato tuber extracts by activity staining after native gel electrophoresis. A cDNA encoding SSI from rice was used as a probe to clone a corresponding cDNA from potato. The deduced amino acid sequence identified the protein as an SS from potato with an M_r of 70.6 kDa for the immature enzyme including its transit peptide. This novel isoform was designated SSI. An analysis of the expression pattern of the gene indicated that SSI is predominantly expressed in sink and source leaves, and, to a lower extent in tubers. In several independent transgenic potato lines, where the expression of SSI was repressed using the antisense approach, the activity of a specific SS isoform was reduced to non-detectable levels as determined through activity staining after native gel electrophoresis. The reduction in the amount of this isoform of SS did not lead to any detectable changes in starch structure, probably due to the fact that this isoform only represents a minor activity in potato tubers. Received: 19 August 1998 / Accepted: 17 December 1998     

Comparative localization of three classes of cell wall proteins.

The localization of the cell wall proline-rich proteins (PRPs), and the gene expression of the cell wall glycine-rich proteins (GRPs) and the hydroxyproline-rich glycoproteins (HRGPs) were examined in several dicot species. The PRPs are accumulated in the corner walls of the cortex where several cells are joined together and in the protoxylem cell walls of 3-day-old soybean root. In 1-month-old soybean plants, the PRPs are specifically deposited in xylem vessel elements of the young stem, and they are accumulated in both phloem fibers and xylem vessel elements and fibers of the older stem. Likewise, the PRPs are localized in xylem vessel elements and fibers in tomato, petunia, potato and tobacco stems. They are also found in outer and inner phloem fiber cell walls of tomato stem and in outer phloem fiber cell walls of petunia stem. The gene expression of the HRGPs and the GRPs is developmentally regulated in tomato, petunia and tobacco stems. HRGP mRNAs are abundant in outer and inner phloem regions, while GRP mRNAs are present mostly in primary xylem and in the cambium region. Immunocytochemical localization showed that the GRPs have a localization pattern similar to that of the PRPs in tomato, petunia and tobacco stems.     

High constitutive peroxidase activity and constitutive thermotolerance in <Emphasis Type="Italic">Neurospora crassa</Emphasis>

 During the isolation of mutations in the heat-inducible hsp70-1 gene of Neurospora crassa by RIP (repeat-induced point mutations), several transformants were generated by electroporation of conidia with a plasmid harboring an incomplete copy of this gene. One isolate, designated E-45, containing ectopically integrated hsp70-1 DNA, exhibited a slow growth rate, low-temperature sensitivity, constitutive thermotolerance (without prior heat shock), and high constitutive peroxidase activity. The constitutive form of peroxidase (CP) was distinguishable from the heat-inducible form (HIP) by immunoinactivation employing polyclonal antiserum against the latter enzyme and by electrophoretic resolution in nondenaturing polyacrylamide gels. This enzyme was purified to near homogeneity and some of its properties examined. The relative molecular mass of native CP was in the range of 118–136 kDa, as estimated by gel filtration analysis on size exclusion matrices, whereas SDS-PAGE analysis yielded a size of ∼37 kDa for the polypeptide. Substrate saturation kinetics studies were conducted using ABTS [2,2′-azino-bis (3-ethylbenzthiazole-6-sulfonic acid)] and H₂O₂ as substrates: K _m, V _max, and K _cat values for H₂O₂ were ∼22 μM, ∼447 nmol mg⁻¹, and 0.33 s⁻¹, respectively, and those for ABTS were ∼55 μM, ∼453 nmol mg⁻¹, and 0.3 s⁻¹, respectively. Guaiacol was not used as a substrate by this enzyme. CP peroxidase was shown to be a heme-containing enzyme, stable at temperatures up to 58°C. Received: August 5, 2002 / Accepted: January 22, 2003 Acknowledgments This work was supported by an operating grant from the Natural Sciences and Engineering Research Council (NSERC) of Canada (to M.K.). The financial support provided to A. M. in the form of a graduate studentship award by the AHFMR (Alberta Heritage Foundation for Medical Research) and of a graduate teaching assistantship to A. S. by the Department of Biological Sciences, University of Calgary, is gratefully acknowledged. Correspondence to:M. Kapoor