Jacalin domain in wheat jasmonate-regulated protein Ta-JA1 confers agglutinating activity and pathogen resistance

Ta-JA1 is a jacalin-like lectin from wheat (Triticum aestivum) plants. To date, its homologs are only observed in the Gramineae family. Our previous experiments have demonstrated that Ta-JA1 contains a modular structure consisting of an N-terminal dirigent domain and a C-terminal jacalin-related lectin domain (JRL) and this protein exhibits a mannose-specific lectin activity. The over-expression of Ta-JA1 gene provides transgenic plants a broad-spectrum resistance to diseases. Here, we report the differential activities of the dirigent and JRL domains of Ta-JA1. In vitro assay showed that the recombinant JRL domain could effectively agglutinate rabbit erythrocytes and pathogen bacteria Pseudomonas syringe pv tabaci. These hemagglutination activities could be inhibited by mannose but not by galactose. In contrast, the recombinant dirigent domain did not show agglutination activity. Corresponding to these differentiations of activities, similar to full-length of Ta-JA1, the over-expression of JRL domain in transgenic plants also increased resistance to the infection of P. syringe. Unlike JRL, the over-expression of dirigent domain in transgenic plants led to alteration of the seedling sensitivity to salts. In addition, a d_N/d_S ratio analysis of Ta-JA1 and its related proteins showed that this protein family functionally limited to a few crop plants, such as maize, rice and wheat.     

Maize beta-glucosidase-aggregating factor is a polyspecific jacalin-related chimeric lectin, and its lectin domain is responsible for beta-glucosidase aggregation

In certain maize genotypes, called "null," beta-glucosidase does not enter gels and therefore cannot be detected on zymograms after electrophoresis. Such genotypes were originally thought to be homozygous for a null allele at the glu1 gene and thus devoid of enzyme. We have shown that a beta-glucosidase-aggregating factor (BGAF) is responsible for the "null" phenotype. BGAF is a chimeric protein consisting of two distinct domains: the disease response or "dirigent" domain and the jacalin-related lectin (JRL) domain. First, it was not known whether the lectin domain in BGAF is functional. Second, it was not known which of the two BGAF domains is involved in beta-glucosidase binding and aggregation. To this end, we purified BGAF to homogeneity from a maize null inbred line called H95. The purified protein gave a single band on SDS-PAGE, and the native protein was a homodimer of 32-kDa monomers. Native and recombinant BGAF (produced in Escherichia coli) agglutinated rabbit erythrocytes, and various carbohydrates and glycoproteins inhibited their hemagglutination activity. Sugars did not have any effect on the binding of BGAF to the beta-glucosidase isozyme 1 (Glu1), and the BGAF-Glu1 complex could still bind lactosyl-agarose, indicating that the sugar-binding site of BGAF is distinct from the beta-glucosidase-binding site. Neither the dirigent nor the JRL domains alone (produced separately in E. coli) produced aggregates of Glu1 based on results from pull-down assays. However, gel shift and competitive binding assays indicated that the JRL domain binds beta-glucosidase without causing it to aggregate. These results with those from deletion mutagenesis and replacement of the JRL domain of a BGAF homolog from sorghum, which does not bind Glu1, with that from maize allowed us to conclude that the JRL domain of BGAF is responsible for its lectin and beta-glucosidase binding and aggregating activities.     

Deletion of the N-terminal dirigent domain in maize β-glucosidase aggregating factor and its homolog sorghum lectin dramatically alters the sugar-specificities of their lectin domains

Maize β-glucosidase aggregating factor (BGAF) and its homolog Sorghum Lectin (SL) are modular proteins consisting of an N-terminal dirigent domain and a C-terminal jacalin-related lectin (JRL) domain. BGAF is a polyspecific lectin with a monosaccharide preference for galactose, whereas SL displays preference for GalNAc. Here, we report that deletion of the N-terminal dirigent domain in the above lectins dramatically changes their sugar-specificities. Deletions in the N-terminal region of the dirigent domain of BGAF abolished binding to galactose/lactose, but binding to mannose was unaffected. Glucose, which was a poor inhibitor of hemagglutinating activity of BGAF, displayed higher inhibitory effect on the hemagglutinating activity of deletion mutants. Deletion of the dirigent domain in SL abolished binding to GalNAc, but binding to mannose was not affected. Surprisingly, fructose, an extremely poor inhibitor (minimum inhibitory concentration (MIC) = 125 mM) of SL hemagglutinating activity, was found to be a very potent inhibitor (MIC = 1 mM) of hemagglutinating activity of its JRL domain. These results indicate that the dirigent domain in this class of modular lectins, at least in the case of maize BGAF and SL, influences sugar specificity.     

Purification and characterisation of a jacalin-related, coleoptile specific lectin from Hordeum vulgare

A plant lectin was isolated from barley (Hordeum vulgare) coleoptiles using acidic extraction and different chromatographic methods. Sequencing of more than 50% of the protein sequence by Edman degradation confirmed a full-length cDNA clone. The subsequently identified open reading frame encodes for a 15 kDa protein which could be found in the soluble fraction of barley coleoptiles. This protein exhibited specificity towards mannose sugar and is therefore, accordingly named as Horcolin (Hordeum vulgare coleoptile lectin). Database searches performed with the Horcolin protein sequence revealed a sequence and structure homology to the lectin family of jacalin-related lectins. Together with its affinity towards mannose, Horcolin is now identified as a new member of the mannose specific subgroup of jacalin-related lectins in monocot species. Horcolin shares a high amino acid homology to the highly light-inducible protein HL#2 and, in addition to two methyl jasmonic acid-inducible proteins of 32.6 and 32.7 kDa where the jasmonic acid-inducible proteins are examples of bitopic chimerolectins containing a dirigent and jacalin-related domain. Immunoblot analysis with a cross-reactive anti-HL#2 antibody in combination with Northern blot analysis of the Horcolin cDNA revealed tissue specific expression of Horcolin in the coleoptiles. The function of Horcolin is discussed in the context of its particular expression in coleoptiles and is then compared to other lectins, which apparently share a related response to biotic or abiotic stress factors.     

Proteomic identification of differentially expressed proteins in Arabidopsis in response to methyl jasmonate

Jasmonates (JAs) are the well characterized fatty acid-derived cyclopentanone signals involved in the plant response to biotic and abiotic stresses. JAs have been shown to regulate many aspects of plant metabolism, including glucosinolate biosynthesis. Glucosinolates are natural plant products that function in defense against herbivores and pathogens. In this study, we applied a proteomic approach to gain insight into the physiological processes, including glucosinolate metabolism, in response to methyl jasmonate (MeJA). We identified 194 differentially expressed protein spots that contained proteins that participated in a wide range of physiological processes. Functional classification analysis showed that photosynthesis and carbohydrate anabolism were repressed after MeJA treatment, while carbohydrate catabolism was up-regulated. Additionally, proteins related to the JA biosynthesis pathway, stress and defense, and secondary metabolism were up-regulated. Among the differentially expressed proteins, many were involved in oxidative tolerance. The results indicate that MeJA elicited a defense response at the proteome level through a mechanism of redirecting growth-related metabolism to defense-related metabolism.     

Characterization of ZmCOLD1, novel GPCR-Type G Protein genes involved in cold stress from Zea mays L. and the evolution analysis with those from other species

Maize is one of the most vital staple crops worldwide. G proteins modulate plentiful signaling pathways, and G protein-coupled receptor-type G proteins (GPCRs) are highly conserved membrane proteins in plants. However, researches on maize G proteins and GPCRs are scarce. In this study, we identified three novel GPCR-Type G Protein (GTG) genes from chromosome 10 (Chr 10) in maize, designated as ZmCOLD1-10A, ZmCOLD1-10B and ZmCOLD1-10C. Their amino acid sequences had high similarity to TaCOLD1 from wheat and OsCOLD1 from rice. They contained the basic characteristics of GTG/COLD1 proteins, including GPCR-like topology, the conserved hydrophilic loop (HL) domain, DUF3735 (domain of unknown function 3735) domain, GTPase-activating domain, and ATP/GTP-binding domain. Subcellular localization analyses of ZmCOLD1 proteins suggested that ZmCOLD1 proteins localized on plasma membrane (PM) and endoplasmic reticulum (ER). Furthermore, amino acid sequence alignment verified the conservation of the key 187th amino acid T in maize and other wild maize-relative species. Evolutionary relationship among plants GTG/COLD1 proteins family displayed strong group-specificity. Expression analysis indicated that ZmCOLD1-10A was cold-induced and inhibited by light. Together, these results suggested that ZmCOLD1 genes had potential value to improve cold tolerance and to contribute crops growth and molecular breeding.     

Monocot chimeric jacalins: a novel subfamily of plant lectins

Abstract

Monocot chimeric jacalins are a small group of lectins (currently with nine members), each typically consisting of a dirigent domain and a jacalin-related lectin domain. This unique module structure, along with their limited taxonomic distribution and short time window in molecular evolution, makes them a novel family of lectins. Recent studies have shown that these proteins play important roles in plant stress responses and development. Our knowledge of these proteins in functional domain and evolution has also made significant progress.     

A specific beta-glucosidase-aggregating factor is responsible for the beta-glucosidase null phenotype in maize

Maize (Zea mays L.) beta-glucosidase was extracted from shoots of a wild-type (K55) and a "null" (H95) maize genotype. Enzyme activity assays and electrophoretic data showed that extracts from the null genotype had about 10% of the activity present in the normal genotype. Zymograms of the null genotype were devoid of any activity bands in the resolving gel, but had a smeared zone of activity in the stacking gel after native polyacrylamide gel electrophoresis. When extracts were made with buffers containing 0.5% to 2% sodium dodecyl sulfate, the smeared activity zone entered the resolving gel as a distinct band. These data indicated that the null genotypes have beta-glucosidase activity, but the enzyme occurs as insoluble or poorly soluble large quaternary complexes mediated by a beta-glucosidase-aggregating factor (BGAF). BGAF is a 35-kD protein and binds specifically to beta-glucosidase and renders it insoluble during extraction. BGAF also precipitates beta-glucosidase that is added exogenously to supernatant fluids of the null tissue extracts. The specific beta-glucosidase-aggregating activity of BGAF is unequivocally demonstrated. These data clearly show that the monogenic inheritance reported for the null alleles at the beta-glucosidase gene is actually for the BGAF protein, and BGAF is solely responsible for beta-glucosidase aggregation and insolubility and, thus, the apparent null phenotype.     

Molecular characterization of a new type of receptor-like kinase (wlrk) gene family in wheat

In plants, several types of receptor-like kinases (RLK) have been isolated and characterized based on the sequence of their extracellular domains. Some of these RLKs have been demonstrated to be involved in plant development or in the reaction to environmental signals. Here, we describe a RLK gene family in wheat (wlrk, wheat leaf rust kinase) with a new type of extracellular domain. A member of this new gene family has previously been shown to cosegregate with the leaf rust resistance gene Lr10. The diversity of the wlrk gene family was studied by cloning the extracellular domain of different members of the family. Sequence comparisons demonstrated that the extracellular domain consists of three very conserved regions interrupted by three variable regions. Linkage analysis indicated that the wlrk genes are specifically located on chromosome group 1 in wheat and on the corresponding chromosomes of other members of the Triticeae family. The wlrk genes are constitutively expressed in the aerial parts of the plant whereas no expression was detected in roots. Protein immunoblots demonstrated that the WLRK protein coded by the Lrk10 gene is an intrinsic plasma membrane protein. This is consistent with the hypothesis that WLRK proteins are receptor protein kinases localized to the cell surface. In addition, we present preliminary evidence that other disease resistance loci in wheat contain genes which are related to wlrk.     

Identification of a hybrid-specific expressed gene encoding novel RNA-binding protein in wheat seedling leaves using differential display of mRNA

A hybrid-specific expressed cDNA fragment, designated as AG5, has been identified in wheat seedling leaves using differential mRNA display. AG5 contains an open reading frame (ORF) encoding 183 amino acid residues. Comparison with amino acid sequences in GenBank revealed that the AG5 protein is homologous to a group of Gly-rich proteins with consensus sequence-type RNA-binding domains (CS-RBD). Structural analysis showed that AG5 protein contains five motifs, including a consensus sequence-type RNA-binding domain near its N-terminus, arginine/aspartic acid repeats and a Gly-rich region in its center, a Cys-X₂-Cys-X₄-His-X₄-Cys (CCHC) zinc finger motif in the Gly-rich region, and TrySer₂ArgAsp₂Arg repeats towards its C-terminus. Of all previously described RNA-binding proteins, only RZ-1 from tobacco has a similar structure to the AG5 protein, but RZ-1 lacks a TrySer2ArgAsp2Arg repeat motif, indicating that the two proteins may belong to a family of closely related proteins in plants. The possible role of AG5 and its relation to wheat heterosis are discussed. Received: 21 November 1999 / Accepted: 20 March 2000     

TaAbc1, a Member of Abc1-Like Family Involved in Hypersensitive Response against the Stripe Rust Fungal Pathogen in Wheat

To search for genes involved in wheat (Triticum aestivum L.) defense response to the infection of stripe rust pathogen Puccinia striiformis f. sp. tritici (Pst), we identified and cloned a new wheat gene similar to the genes in the Abc1-like gene family. The new gene, designated as TaAbc1, encodes a 717-amino acid, 80.35 kD protein. The TaAbc1 protein contains two conserved domains shared by Abc1-like proteins, two trans-membrane domains at the C-terminal, and a 36-amino acid chloroplast targeting presequence at the N-terminal. Characterization of TaAbc1 expression revealed that gene expression was tissue-specific and could be up-regulated by biotic agents (e.g., stripe rust pathogen) and/or by an abiotic stress like wounding. High-fold induction was associated with the hypersensitive response (HR) triggered only by avirulent stripe rust pathotypes, suggesting that TaAbc1 is a rust-pathotype specific HR-mediator. Down-regulating TaAbc1 reduced HR but not the overall resistance level in Suwon11 to CYR23, suggesting TaAbc1 was involved in HR against stripe rust, but overall host resistance is not HR-dependent.     

Identification and molecular characterization of ZAG1, the maize homolog of the Arabidopsis floral homeotic gene AGAMOUS.

Recent genetic and molecular studies in Arabidopsis and Antirrhinum suggest that mechanisms controlling floral development are well conserved among dicotyledonous species. To assess whether similar mechanisms also operate in more distantly related monocotyledonous species, we have begun to clone homologs of Arabidopsis floral genes from maize. Here we report the characterization of two genes, designated ZAG1 and ZAG2 (for Zea AG), that were cloned from a maize inflorescence cDNA library by low stringency hybridization with the AGAMOUS (AG) cDNA from Arabidopsis. ZAG1 encodes a putative polypeptide of 286 amino acids having 61% identity with the AGAMOUS (AG) protein. Through a stretch of 56 amino acids, constituting the MADS domain, the two proteins are identical except for two conservative amino acid substitutions. The ZAG2 protein is less similar to AG, with 49% identity overall and substantially less similarity than ZAG1 outside the well-conserved MADS domain. Like AG, ZAG1 RNA accumulates early in stamen and carpel primordia. In contrast, ZAG2 expression begins later and is restricted to developing carpels. Hybridization to genomic DNA with the full-length ZAG1 cDNA under moderately stringent conditions indicated the presence of a large family of related genes. Mapping data using maize recombinant inbreds placed ZAG1 and ZAG2 near two loci that are known to affect maize flower development, Polytypic ear (Pt) and Tassel seed4 (Ts4), respectively. The ZAG1 protein from in vitro translations binds to a consensus target site that is recognized by the AG protein. These data suggest that maize contains a homolog of the Arabidopsis floral identity gene AG and that this gene is conserved in sequence and function.     

An Arabidopsis mutant cex1 exhibits constant accumulation of jasmonate-regulated AtVSP, Thi2.1 and PDF1.2

Jasmonates (JA) act as a regulator in plant growth as well as a signal in plant defense. The Arabidopsis vegetative storage protein (AtVSP) and plant defense-related proteins thionin (Thi2.1) and defensin (PDF1.2) have previously been shown to accumulate in response to JA induction. In this report, we isolated and characterized a novel recessive mutant, cex1, conferring constitutive JA-responsive phenotypes including JA-inhibitory growth and constitutive expression of JA-regulated AtVSP, Thi2.1 and PDF1.2. The plant morphology and the gene expression pattern of the cex1 mutant could be phenocopied by treatment of wild-type plants with exogenous JA, indicating that CEX1 might be a negative regulator of the JA response pathway.     

Molecular cloning of a new wheat calreticulin gene TaCRT1 and expression analysis in plant defense responses and abiotic stress resistance

Calreticulin proteins play essential roles in regulating various metabolic processes and in molecular signal transduction in animals and plants. Using homologous PCR, we screened a cDNA library of the wheat resistance gene Yr5 from a near-isogenic line in the susceptible common wheat variety Taichung 29, which was inoculated with an incompatible race CYR32 of Puccinia striiformis. We isolated a novel full-length cDNA encoding calreticulin protein, which we named TaCRT1. Sequence analyses indicated that TaCRT1 contains an open reading frame of 1287 bp in length; it was deduced to encode 428 amino acids. Clustering analysis showed that TaCRT1 belongs to group III of the calreticulin protein family. Semi-quantitative RT-PCR was used to analyze expression profiles of the isolated gene under biotic and abiotic stresses. Expression of TaCRT1 was suppressed by exogenous application of phytohormones, such as abscisic acid and methyl jasmonate, and by dehydration; but it was induced by CYR32 infection and cold treatment. Based on the expression patterns, we propose that TaCRT1 participates in regulatory processes involved in defense responses and stress resistance in wheat.     

ZmGns,a maize class I β-1,3-glucanase,is induced by biotic stresses and possesses strong antimicrobial activity

Plant b-1,3-glucanases are members of the pathogenesis-related protein 2(PR-2) family,which is one of the 17 PR protein families and plays important roles in biotic and abiotic stress responses.One of the differentially expressed proteins(spot 842) identified in a recent proteomic comparison between five pairs of closely related maize(Zea mays L.) lines differing in aflatoxin resistance was further investigated in the present study.Here,the corresponding cDNA was cloned from maize and designated as ZmGns.ZmGns encodes a protein of338 amino acids containing a potential signal peptide.The expression of Zm Gns was detectible in all tissues studied with the highest level in silks.ZmGns was significantly induced by biotic stresses including three bacteria and the fungus Aspergillus flavus.ZmGns was also induced by most abiotic stresses tested and growth hormones including salicylic acid.In vivo,ZmGns showed a significant inhibitory activity against thebacterial pathogen Pseudomonas syringae pv.tomato DC3000 and fungal pathogen Botrytis cinerea when it overexpressed in Arabidopsis.Its high level of expression in the silk tissue and its induced expression by phytohormone treatment,as well as by bacterial and fungal infections,suggest it plays a complex role in maize growth,development,and defense.