Thaumatin-like Protein Research Articles

Silencing of PR-10-like proteins in Medicago truncatula results in an antagonistic induction of other PR proteins and in an increased tolerance upon infection with the oomycete Aphanomyces euteiches

Recent studies on the root proteome of Medicago truncatula (Gaertn.) showed an induction of pathogenesis-related (PR) proteins of the class 10 after infection with the oomycete pathogen Aphanomyces euteiches (Drechs.). To get insights into the function of these proteins during the parasitic root-microbe association, a gene knockdown approach using RNAi was carried out. Agrobacterium rhizogenes-mediated transformation of M. truncatula roots led to a knockdown of the Medicago PR10-1 gene in transgenic in vitro root cultures. Proteomic analyses of the MtPr10-1i root cultures showed that MtPr10-1 was efficiently knocked down in two MtPr10-1i lines. Moreover, five additional PR-10-type proteins annotated as abscisic acid responsive proteins (ABR17s) revealed also an almost complete silencing in these two lines. Inoculation of the root cultures with the oomycete root pathogen A. euteiches resulted in a clearly reduced colonization and thus in a suppressed infection development in MtPr10-1i roots as compared to that in roots of the transformation controls. In addition, MtPr10-1 silencing led to the induction of a new set of PR proteins after infection with A. euteiches including the de novo induction of two isoforms of thaumatin-like proteins (PR-5b), which were not detectable in A. euteiches-infected control roots. Thus, antagonistic induction of other PR proteins, which are normally repressed due to PR-10 expression, is supposed to cause an increased resistance of M. truncatula upon an A. euteiches in vitro infection. The results were also further confirmed by detecting increased PR-5b induction levels in 2-D gels of a previously analyzed M. truncatula line (F83.005-9) exhibiting increased A. euteiches tolerance associated with reduced PR-10 induction levels.

A thaumatin-like antifungal protein from the emperor banana

A 20-kDa protein with substantial N-terminal sequence homology to thaumatin-like proteins was isolated from ripe fruits of the emperor banana, Musa basjoo cv. ‘emperor banana’. The isolation procedure entailed (NH 4) 2SO 4 precipitation, ion exchange chromatography on DEAE-cellulose, and affinity chromatography on Affi-gel blue gel. The thaumatin-like protein inhibited mycelial growth in Fusarium oxysporum and Mycosphaerella arachidicola. However, it did not affect the mitogenic response of murine splenocytes or [methyl- 3H] thymidine incorporation by tumor cells. The activity of HIV-1 reverse transcriptase was slightly inhibited.

Severe Immediate Allergic Reactions to Grapes: Part of a Lipid Transfer Protein-Associated Clinical Syndrome

Background: Grape allergy is considered rare; grape lipid transfer protein (LTP; Vit v 1), an endochitinase and a thaumatin-like protein (TLP) have been reported as grape allergens. A considerable number of patients have referred to our department for severe reactions to grapes, and several IgE binding proteins were detected. Objectives: The aim of this study was to identify and characterise the allergens involved in severe allergic reactions to grapes and describe the population in which they occur. Methods: Patients with reported severe allergic reactions to grapes (n = 37) are described. Grape allergens were purified/fractionated by a combination of chromatographic techniques, identified by proteomic analysis and biochemically characterised. Immunoreactivity was assessed by blot (inhibitions) and RAST (inhibitions), and skin prick tests were performed with the isolated allergens. Results: All subjects were polyallergic, sensitised and reactive to several additional foods and pollen. All patients were sensitised to grape LTP. A 28-kDa expansin, a 37.5-kDa polygalacturonase-inhibiting protein, a 39-kDa β-1,3-glucanase and a 60-kDa protein were identified as minor grape allergens. Endochitinase and TLP did not play a role. Inhibition experiments revealed the possible cross-reactive role of LTP for clinical sensitivities to other LTP-containing plant foods, but also the involvement of cross-reactive carbohydrate determinants of minor allergens in IgE cross-reactivity. Conclusions: LTP is the major grape allergen, while additional minor allergens may contribute to clinical reactivity. Severe grape allergy presents in atopic patients who frequently react to other LTP-containing, plant-derived foods. The ‘LTP syndrome’ is the appropriate term to describe this condition.

Pyramiding transgenic resistance in elite indica rice cultivars against the sheath blight and bacterial blight

Elite indica rice cultivars were cotransformed with genes expressing a rice chitinase (chi11) and a thaumatin-like protein (tlp) conferring resistance to fungal pathogens and a serine-threonine kinase (Xa21) conferring bacterial blight resistance, through particle bombardment, with a view to pyramiding sheath blight and bacterial blight resistance. Molecular analyses of putative transgenic lines by polymerase chain reaction, Southern Blot hybridization, and Western Blotting revealed stable integration and expression of the transgenes in a few independent transgenic lines. Progeny analyses showed the stable inheritance of transgenes to their progeny. Coexpression of chitinase and thaumatin-like protein in the progenies of a transgenic Pusa Basmati1 line revealed an enhanced resistance to the sheath blight pathogen, Rhizoctonia solani, as compared to that in the lines expressing the individual genes. A transgenic Pusa Basmati1 line pyramided with chi11, tlp, and Xa21 showed an enhanced resistance to both sheath blight and bacterial blight.

Antimicrobial Activity of Malting Barley Grain Thaumatin-Like Protein Isoforms, S and R

Two basic proteins were isolated to homogeneity from malting barley (Hordeum vulgare L.) grain. Proteins were identified as members of a Thaumatin-Like Protein (TLP) family, by Western blot. Isoforms, assigned as TLP-S and TLP-R, have slightly different mobility at about 22 and 27 kDa in nonreducing and reducing conditions, and pI values of 9.5 and 9.4, respectively. The antifungal potency of malting barley grain TLPs isoforms was examined on Micrococcus lysodeikticus, Saccharomyces cerevisiae, Candida albicans and plant pathogen Fusarium sporotrichioides growth in vitro. It was found that that IC50 value for TLP-S was two fold higher. Antibacterial and antifungal activities of both isoforms were completely abolished by divalent (Ca2+, Mn2+, Mg2+) and monovalent (K+) cations, at concentrations approximating physiological ionic strength and higher. Glucanase activity was not observed; neither TLP-S nor TLP-R digested glucan. On the basis of these results, the importance of TLP for barley grain protection against fungal diseases has been discussed together with the mechanism of antimicrobial action.

Purification, characterization, and molecular gene cloning of an antifungal protein from Ginkgo biloba seeds

A novel basic protein with antifungal activity was isolated from the seeds of Ginkgo biloba and purified to homogeneity. The protein inhibited the growth of some fungi (Fusarium oxysporum, Trichoderma reesei, and Candida albicans) but did not exhibit antibacterial action against Escherichia coli. Furthermore, this protein showed weak inhibitory activity against the aspartic protease pepsin. To design primers for gene amplification, the NH(2)-terminal and partial internal amino acid sequences were determined using peptides obtained from a tryptic digest of the oxidized protein. The full-length cDNA of the antifungal protein was cloned and sequenced by RT-PCR and rapid amplification of cDNA ends (RACE). The cDNA contained a 402-bp open reading frame encoding a 134-aa protein with a potential signal peptide (26 residues), suggesting that this protein is synthesized as a preprotein and secreted outside the cells. The antifungal protein shows approximately 85% identity with embryo-abundant proteins from Picea abies and Picea glauca at the amino acid level; however, there is no homology between this protein and other plant antifungal proteins, such as defensin, and cyclophilin-, miraculin- and thaumatin-like proteins.

Overexpression of defense response genes in transgenic wheat enhances resistance to Fusarium head blight.

Fusarium head blight (FHB) of wheat, caused by Fusarium graminearum and other Fusarium species, is a major disease problem for wheat production worldwide. To combat this problem, large-scale breeding efforts have been established. Although progress has been made through standard breeding approaches, the level of resistance attained is insufficient to withstand epidemic conditions. Genetic engineering provides an alternative approach to enhance the level of resistance. Many defense response genes are induced in wheat during F. graminearum infection and may play a role in reducing FHB. The objectives of this study were (1) to develop transgenic wheat overexpressing the defense response genes α-1-purothionin, thaumatin-like protein 1 (tlp-1), and β-1,3-glucanase; and (2) to test the resultant transgenic wheat lines against F. graminearum infection under greenhouse and field conditions. Using the wheat cultivar Bobwhite, we developed one, two, and four lines carrying the α-1-purothionin, tlp-1, and β-1,3-glucanase transgenes, respectively, that had statistically significant reductions in FHB severity in greenhouse evaluations. We tested these seven transgenic lines under field conditions for percent FHB disease severity, deoxynivalenol (DON) mycotoxin accumulation, and percent visually scabby kernels (VSK). Six of the seven lines differed from the nontransgenic parental Bobwhite line for at least one of the disease traits. A β-1,3-glucanase transgenic line had enhanced resistance, showing lower FHB severity, DON concentration, and percent VSK compared to Bobwhite. Taken together, the results showed that overexpression of defense response genes in wheat could enhance the FHB resistance in both greenhouse and field conditions.

Co-bombardment, integration and expression of rice chitinase and thaumatin-like protein genes in barley (Hordeum vulgare cv. Conlon)

Pathogenesis-related (PR) proteins associated with degradation of structural components of pathogenic filamentous fungi were overexpressed in the two-rowed malting barley (Hordeum vulgare L.) cultivar Conlon. Transgenes were introduced by co-bombardment with two plasmids, one carrying a rice (Oryza sativa L.) chitinase gene (chi11) and another carrying a rice thaumatin-like protein gene (tlp). Each gene was under the control of the maize ubiquitin (Ubi1) promoter. Fifty-eight primary transformants from three independent transformation events were regenerated. T(1) plants with high rice chi11 and tlp protein expression levels were advanced to identify T(2) homozygotes by herbicide spray and subjected to further molecular analyses. T(3) progeny from one event (E2) had stable integration and expression of the rice chi11 and tlp while those from the other events (E1 and E3) showed stable integration only of tlp. The successful production of these lines overexpressing the antifungal chi and tlp proteins provides materials to test the effects of these genes on a variety of fungal diseases that attack barley and to serve as potential additional sources of disease resistance.

Proteomic analysis of bacterial-blight defense-responsive proteins in rice leaf blades

Plants exhibit resistance against incompatible pathogens, via localized and systemic responses as part of an integrated defense mechanism. To study the compatible and incompatible interactions between rice and bacteria, a proteomic approach was applied. Rice cv. Java 14 seedlings were inoculated with compatible (Xo7435) and incompatible (T7174) races of Xanthomonas oryzae pv. oryzae (Xoo). Cytosolic and membrane proteins were fractionated from the leaf blades and separated by 2-D PAGE. From 366 proteins analyzed, 20 were differentially expressed in response to bacterial inoculation. These proteins were categorized into classes related to energy (30%), metabolism (20%), and defense (20%). Among the 20 proteins, ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (RuBisCO LSU) was fragmented into two smaller proteins by T7174 and Xo7435 inoculation. Treatment with jasmonic acid (JA), a signaling molecule in plant defense responses, changed the level of protein accumulation for 5 of the 20 proteins. Thaumatin-like protein and probenazole-inducible protein (PBZ) were commonly up-regulated by T7174 and Xo7435 inoculation and JA treatment. These results suggest that synthesis of the defense-related thaumatin-like protein and PBZ are stimulated by JA in the defense response pathway of rice against bacterial blight.

Xanthomonas oryzae pv. oryzae infection triggers accumulation of phenolics, defense-related enzymes and thaumatin-like proteins in rice leaves

The effect of Xanthomonas oryzae pv. oryzae infection on induction of phenylalanine ammonia-lyase (PAL), peroxidase (PO), phenolics and thaumatin-like proteins (TLPs) in rice was studied. PAL activity increased significantly one day after inoculation with X. o. pv. oryzae and the maximum enzyme activity was observed two days after inoculation. The phenolic content in rice leaves increased significantly one day after inoculation and the maximum accumulation of phenols was observed two days after inoculation. Significant increase in peroxidase activity was observed in rice leaves one day after inoculation with X. o. pv. oryzae. Isozyme analysis indicated that three peroxidase isozymes (PO-1, PO-2 and PO-3) were induced after inoculation with X. o. pv. oryzae. Immunoblot analysis of protein extracts from control and pathogen inoculated rice plants revealed the induced accumulation of 16 and 24 kDa TLPs in rice leaves in response to X. o. pv. oryzae infection. TLP mRNA accumulation was induced strongly in rice leaves in response to infection by X. o. pv. oryzae.

Purification and characterization of an antifungal thaumatin-like protein from Cassia didymobotrya cell culture

A 23-kDa antifungal thaumatin-like protein was isolated and purified from Cassia didymobotrya (Fres.) cell cultures for the first time. The protein was secreted in the culture medium, but it could be also isolated after elution of whole cells with a 0.5 M CaCl 2 solution. Treatment of the cells with laminarin oligosaccharides or salicylic acid, but not with NaCl, resulted in enhancement of expression of the protein. A rapid purification protocol was used based on cationic exchange chromatography. The protein, with a highly basic character (pI 10), has an exact molecular mass of 23034 Da, as determined by MALDI-ToF mass spectrometry analysis. N-terminal sequencing of the intact polypeptide and the sequencing of two internal tryptic peptides indicated significant identity with other thaumatin-like proteins (TLP). The protein exerted antifungal activity towards some Candida species showing EC 50 values comparable to those of other antifungal TLPs. The collected data lead to classify this TLP as a new PR-5 protein.

The apoplastic antioxidant system in Prunus: response to long-term plum pox virus infection

This work describes, for the first time, the changes taking place in the antioxidative system of the leaf apoplast in response to plum pox virus (PPV) in different Prunus species showing different susceptibilities to PPV. The presence of p-hydroxymercuribenzoic acid (pHMB)-sensitive ascorbate peroxidase (APX) (class I APX) and pHMB-insensitive APX (class III APX), superoxide dismutase (SOD), peroxidase (POX), NADH-POX, and polyphenoloxidase (PPO) was described in the apoplast from both peach and apricot leaves. PPV infection produced different changes in the antioxidant system of the leaf apoplast from the Prunus species, depending on their susceptibility to the virus. In leaves of the very susceptible peach cultivar GF305, PPV brought about an increase in class I APX, POX, NADH-POX, and PPO activities. In the susceptible apricot cultivar Real Fino, PPV infection produced a decrease in apoplastic POX and SOD activities, whereas a strong increase in PPO was observed. However, in the resistant apricot cultivar Stark Early Orange, a rise in class I APX as well as a strong increase in POX and SOD activities was noticed in the apoplastic compartment. Long-term PPV infection produced an oxidative stress in the apoplastic space from apricot and peach plants, as observed by the increase in H2O2 contents in this compartment. However, this increase was much higher in the PPV-susceptible plants than in the resistant apricot cultivar. Only in the PPV-susceptible apricot and peach plants was the increase in apoplastic H2O2 levels accompanied by an increase in electrolyte leakage. No changes in the electrolyte leakage were observed in the PPV-inoculated resistant apricot leaves, although a 42% increase in the apoplastic H2O2 levels was produced. Two-dimensional electrophoresis analyses revealed that the majority of the polypeptides in the apoplastic fluid had isoelectric points in the range of pI 4-6. The identification of proteins using MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) and peptide mass fingerprinting analyses showed the induction of a thaumatin-like protein as well as the decrease of mandelonitrile lyase in peach apoplast due to PPV infection. However, most of the selected polypeptides showed no homology with known proteins. This fact emphasizes that, at least in Prunus, most of the functions of the apoplastic space remain unknown. It is concluded that long-term PPV infection produced an oxidative stress in the leaf apoplast, contributing to the deleterious effects produced by PPV infection in leaves of inoculated, susceptible Prunus plants.

CDNA sequences, MALDI-TOF analyses, and molecular modelling of barley PR-5 proteins

Barley plants are known to produce various PR-5 proteins. Transcripts encoding eight different barley PR-5 proteins (TLPs 1–8, TLP for thaumatin-like protein) were identified and cloned – seven from infected leaves and one from developing grains. Here, we describe the cDNA sequences of four of these TLP isoforms. Moreover, the TLPs from the infected leaves (TLPs 1, 2, and TLPs 4–8) were subjected to MALDI-TOF mass spectrometric measurements that resulted in protein fragments consistent with their deduced peptide sequences. Multiple sequence alignment analysis revealed that the TLPs in barley fall into two groups: long-chain proteins (TLPs 5–8) having 16 cysteine residues and short-chain proteins (TLPs 1–4) with only 10 cysteine residues. Finally, modelling experiments highlighted the effects of sequence differences between the TLP isoforms in terms of their secondary structures and their molecular electrostatic potentials. We propose that these sequence differences have implications for the target preferences of the different isomers.

Mass spectrometry in the analysis of grape and wine proteins

Wine proteins play an important role in the quality of wine, because they affect taste, clarity and stability of product. The majority of wine proteins are in the range of 20–30 kDa. Different mass spectrometry (MS) techniques have been successfully applied to study the grape and wine proteins. By liquid chromatography (LC) electrospray ionization (ESI) MS and nano-LC/MS, nine dipeptides and 80 peptides were unambiguously identified in Champagne and Sauvignon Blanc wines, respectively. Using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) and surface-enhanced laser desorption/ionization TOF, the protein and peptide fingerprints in Chardonnay, Sauvignon Blanc and Muscat of Alexandria wines were determined. MALDI-TOF identified the mesocarp proteome of six Vitis grape varieties. Proteins in different grape tissue extracts were also studied. The major grape pathogenic-related proteins are chitinases and thaumatin-like proteins, which both persist through the vinification process and cause hazes and sediments in bottled wines. ESI-MS, LC/ESI-MS and MALDI-TOF analysis of these proteins in grape and wine were also used to characterize different grape varieties.

Lentinula edodes tlg1 Encodes a Thaumatin-Like Protein That Is Involved in Lentinan Degradation and Fruiting Body Senescence

Lentinan is an antitumor product that is purified from fresh Lentinula edodes fruiting bodies. It is a cell wall component, comprising beta-1,3-glucan with beta-1,6-linked branches, which becomes degraded during postharvest preservation as a result of increased glucanase activity. In this study, we used N-terminal amino acid sequence to isolate tlg1, a gene encoding a thaumatin-like (TL) protein in L. edodes. The cDNA clone was approximately 1.0 kb whereas the genomic sequence was 2.1 kb, and comparison of the two indicated that tlg1 contains 12 introns. The tlg1 gene product (TLG1) was predicted to comprise 240 amino acids, with a molecular mass of 25 kD and isoelectric point value of 3.5. The putative amino acid sequence exhibits approximately 40% identity with plant TL proteins, and a fungal genome database search revealed that these TL proteins are conserved in many fungi including the basidiomycota and ascomycota. Transcription of tlg1 was not detected in vegetative mycelium or young and fresh mushrooms. However, transcription increased following harvest. Western-blot analysis demonstrated a rise in TLG1 levels following harvest and spore diffusion. TLG1 expressed in Escherichia coli and Aspergillus oryzae exhibited beta-1,3-glucanase activity and, when purified from the L. edodes fruiting body, demonstrated lentinan degrading activity. Thus, we suggest that TLG1 is involved in lentinan and cell wall degradation during senescence following harvest and spore diffusion.

Natural and recombinant molecules of the cherry allergen Pru av 2 show diverse structural and B cell characteristics but similar T cell reactivity

Cherry allergy is often reported in the context of allergy to other fruits of the Rosaceae family and pollinosis to trees because of cross-reactive allergens. Allergic reactions to cherry are reported by 19-29% of birch pollen-allergic patients. Pru av 2, identified as a thaumatin-like protein (TLP) from sweet cherry, was recognized by the majority of cherry-allergic patients in immunoblotting. In order to investigate the structural characteristics and the immunoglobulin (Ig)E- and T cell reactivity of cherry-derived TLP, recombinant Pru av 2 was expressed in Escherichia coli and natural Pru av 2 was purified. Parallel-His and FLAG expression vectors were used for recombinant production of Pru av 2 in the cytoplasm and the periplasm of E. coli. Natural Pru av 2 was purified from fresh cherries and verified by N-terminal sequencing. Structural characterization was performed using circular dichroism (CD) measurements, and the biologic activity was measured in a glucanase assay. Using cherry-specific sera, the IgE-binding ability of recombinant and natural Pru av 2 was investigated in IgE-ELISA and the T cell reactivity was studied in proliferation assays. Results Natural Pru av 2 revealed thaumatin-like structural features and bound IgE of 50% of cherry-allergic patients. It was demonstrated to be enzymatically active. Recombinant Pru av 2 expressed in the cytoplasm of E. coli exhibited a slightly different folding compared with the natural protein. It was not recognized by IgE from cherry-allergic subjects, but retained the ability to stimulate T lymphocytes. Periplasmic recombinant Pru av 2 was able to bind an anti-grape TLP antibody and cherry-specific IgE. We prepared two recombinant model TLPs from cherry, and compared their molecular characteristics as well as their IgE-binding activity and T cell interactions in relation to the natural counterpart. The cytoplasmic recombinant Pru av 2 can be used as a hypoallergenic variant in allergen-specific immunotherapy, whereas the periplasmic protein can be included in a component-resolved diagnosis.

Enhanced Resistance to Botrytis cinerea Mediated by the Transgenic Expression of the Chitinase Gene ch5B in Strawberry

Plants of strawberry (cultivar Pájaro) were transformed with three defense related genes: ch5B, gln2 and ap24 using Agrobacterium tumefaciens. The ch5B gene encodes for a chitinase from Phaseolus vulgaris, while gln2 and ap24 encode for a glucanase and a thaumatin-like protein, respectively, both from Nicotiana tabacum. Sixteen transgenic lines expressing one or a combination of two defense genes were obtained. Phytopathological tests showed that two transgenic lines expressing only the ch5B gene displayed high levels of resistance to gray mold disease (Botrytis cinerea). The resistance was correlated with the presence of the foreign protein CH5B and the increase of chitinolytic activity in leaves. However, resistance toward Colletotrichum acutatum, the etiological agent of the anthracnose disease, was not enhanced in the transgenic plants. These results suggest that the ch5B gene can be used to introduce transgene-mediated resistance to gray mold in strawberry, due to the lack of natural resistance to this disease in the crop.

Characterization of genes for novel thaumatin-like proteins in Cryptomeria japonica

Thaumatin-like proteins (TLPs) are induced by a variety of phytopathogens in many plants and several TLPs are allergenic. Previously, we isolated three TLP-encoding cDNAs (Cry j 3.1, Cry j 3.2 and Cry j 3.3) from a cDNA library derived from the pollen of Cryptomeria japonica D. Don. Here, we describe three new TLP cDNAs (Cry j 3.4, Cry j 3.5 and Cry j 3.6). We compared the sequences, the genetic map location and the expression patterns of the Cry j 3 genes. The amino acid sequence predicted from Cry j 3.5 exhibits only limited similarity to those predicted from the other Cry j 3 genes. Linkage analysis showed that the Cry j 3.1 to Cry j 3.4 genes are located in the same linkage group, but Cry j 3.5 is located in a different group. Organ-specificity and induction by stresses and plant hormones differed among the Cry j 3 mRNAs. In pollen grains, the Cry j 3.5 mRNA expression level was higher than that of the other Cry j 3 genes. Exposure to UV-B and salt stress induced expression of Cry j 3.1. The ethylene-releasing compound ethephon strongly induced expression of Cry j 3.4. Salt stress and salicylic acid also induced expression of Cry j 3.4. Abscisic acid weakly induced expression of Cry j 3.5. Arachidonic acid strongly induced expression of Cry j 3.4 and Cry j 3.6, and weakly induced that of Cry j 3.3, whereas expression of Cry j 3.1 and Cry j 3.5 was unaffected. These results suggest that the roles of TLPs and the cascades that regulate their expression differ among the members of the TLP family in C. japonica.

A Thaumatin-like Protein from Larvae of the Beetle Dendroides canadensis Enhances the Activity of Antifreeze Proteins

The levels of thermal hysteresis (antifreeze activity) produced by purified antifreeze proteins (DAFPs) from the larvae of the beetle Dendroides canadensis at endogenous concentrations are lower than what are present in the hemolymph of overwintering larvae. Thermal hysteresis activity of DAFPs is dependent not only on AFP concentration but also on the presence of enhancers that may be either proteins (including other hemolymph DAFPs) or low-molecular mass enhancers such as glycerol. The purpose of this study was to identify endogenous protein enhancers using yeast two-hybrid, co-immunoprecipitation, and finally the enhancement of antifreeze activity. Here we show that a thaumatin-like protein from D. canadensis, until recently known only from plants, significantly enhances the thermal hysteresis of DAFP-1 and -2. Glycerol can further this enhancement, presumably by promoting the interaction of the DAFPs and thaumatin-like protein.

Characterization of Major Stable Proteins in Chardonnay Wine

Chardonnay wine produced in the conventional manner contained 69.8 to 108mg/L soluble proteins including nondialyzable polypeptides ; with an average of 87.2mg/L. Proteins were separated by precipitation with ammonium sulfate, and fractionated into two fractions, F1 (mainly invertase and proteoglycans) and F2 (glycoproteins), by Sephadex G-100 column chromatography. The fractions were further separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) [isoelectric focusing and SDS-polyacrylamide gel electrophoresis]. More than 160 and 150 protein spots were detected on the 2-D PAGE maps of F1 and F2, respectively. The major proteins or polypeptides were determined to be fruit proteins, such as thaumatin- and osmotin-like proteins, invertase, lipid transfer protein (LTP), and their hydrolysis products. This is first report on the existence of LTP (or its hydrolysis product) and the hydrolysis products of major grape proteins (invertase, osmotin-like protein, and thaumatin-like protein) in wine.