Thaumatin-like Protein Research Articles

Changes in the activity, dissociation, aggregation, and the secondary and tertiary structures of a thaumatin-like protein with a high polyphenol oxidase activity induced by high pressure CO2

Abstract Effects of high pressure CO 2 (HPCD) on the activity, dissociation and aggregation, as well as secondary and tertiary structures of a thaumatin-like protein (TLP) with polyphenol oxidase (PPO) activity from apples ( Malus domestica , Fuji) were investigated. The PPO activity of TLP subjected to HPCD was initially increased and then decreased after increasing pressure, temperature, time and cycles. The secondary structure was changed by HPCD. The λ max of fluorescence spectra was red-shifted with intensity increasing or decreasing, depending on the treatment parameters. HPCD induced dissociation and aggregation of the native TLP aggregates. More intense HPCD treatment caused the dissociation of larger aggregates and further aggregation in the smaller aggregates. Activation of TLP may be attributed to the dissociation of its aggregates and the structural changes at the catalytic centre; inactivation was due to the aggregation of TLP and collapse of the catalytic centre. Industrial relevance High pressure carbon dioxide (HPCD) is one merging non-thermal technique for inactivating microorganisms and enzymes in food products. Polyphenol oxidase (PPO) can cause quality deterioration in many food products. This study analyzed the effects of HPCD on the activity, dissociation, aggregation, and the secondary and tertiary structure of a thaumatin-like protein (TLP) with a high PPO activity. Available data provided in this study will benefit the food industry.

Effect of carboxymethyl cellulose on tartrate salt, protein and colour stability of red wine

Background and Aims Recent studies have confirmed a long-term effect of carboxymethyl cellulose (CMC) for tartrate salt stabilisation in white wine. It has been argued that CMC is not only less effective in red wine but also interacts with proteins and polyphenols generating turbidity and change in colour. In order to explain these effects, we studied in detail the impact of CMC on haze formation and colour stability of red wine. Methods and Results The influence of CMC concentration was tested with ten samples of red wine produced from several grape cultivars. The haze-forming material was analysed by sodium dodecyl-sulfate polyacrylamide electrophoresis and the protein composition by high-performance liquid chromatography-mass spectrometry. Colour alteration was documented by Vis-spectroscopy. Three samples of wine that were tested developed significant turbidity when treated with oenological doses of CMC. The haze formation coincided with a high-colour density and protein instability of the wine. The insoluble fraction contained pathogenesis-related wine proteins (thaumatin-like proteins, lipid transfer protein) and was associated with colouring matter. Conclusions Carboxymethyl cellulose is of value for tartrate salt stabilisation in red wine. Occasionally, it promotes development of protein haze and colour loss. At a first glance, this behaviour appears to limit the oenological suitability of CMC, but it might also be considered as a new tool to remove unstable wine proteins. Significance of the Study The results indicate the conditions under which red wine turbidity is triggered by CMC. Thus, they are important for the elaboration of recommendations for the optimal use of the polymer.

Sequential allergen microarray testing during the follow-up of allergic patients

Results Median age was 6 years (10 months to 17 years) when ISAC was first performed, and 8 years (22 months to 18 years) when the latest ISAC was performed. With respect to the number and intensity of IgE reactivities, ISAC follow-up results entered one of the following categories: progression (16), stability (12), attenuation (8). Two patients displayed complex alterations of their molecular profile over time. In most cases, ISAC followup provided aid for the management of food exclusion or reintroduction regimens. Storage protein reactivity was the most frequent setting, but transition from one molecular profile to another, which were not clinically distinguishable, such as lipid tranfer proteins versus thaumatin-like proteins, was als noted. Complex cross-reactivity patterns or allergy to components which are unavailable for individual testing (sesame Ses i 1, thaumatin-like Act d 2, 7S vicillins other than those from Fabaceae, wheat Tri a 14) gained better insight from repeated ISAC assays. Finally, sequential “allergen landscape views” improved the understanding of the patient’s biological and clinical evolution.

Induction of extracellular defense-related proteins in suspension cultured-cells of Daucus carota elicited with cyclodextrins and methyl jasmonate

Suspension cultured-cells (SCC) of Daucus carota were used to evaluate the effect of methyl jasmonate and cyclodextrins, separately or in combination, on the induction of defense responses, particularly the accumulation of pathogenesis-related proteins. A comparative study of the extracellular proteome (secretome) between control and elicited carrot SCC pointed to the presence of amino acid sequences homologous to glycoproteins which have inhibitory activity against the cell-wall-degrading enzymes secreted by pathogens and/or are induced when carrot cells are exposed to a pathogen elicitor. Other amino acid sequences were homologous to Leucine-Rich Repeat domain-containing proteins, which play an essential role in defense against pathogens, as well as in the recognition of microorganisms, making them important players in the innate immunity of this plant. Also, some tryptic peptides were shown to be homologous to a thaumatin-like protein, showing high specificity to abiotic stress and to different reticuline oxidase-like proteins that displayed high levels of antifungal activity, suggesting that methyl jasmonate and cyclodextrins could play a role in mediating defense-related gene product expression in SCC of D. carota. Apart from these elicitor-inducible proteins, we observed the presence of PR-proteins in both control and elicited carrot SCC, suggesting that their expression is mainly constitutive. These PR-proteins are putative class IV chitinases, which also have inhibitory activity against pathogen growth and the class III peroxidases that participate in response to environmental stress (e.g. pathogen attack and oxidative), meaning that they are involved in defense responses triggered by both biotic and abiotic factors.

Application of airlift bioreactors to accelerate genetic transformation in American chestnut

Airlift bioreactors (ALBs) were constructed and tested for their potential to enhance chestnut embryogenic tissue proliferation for genetic transformation and mass propagation. Multiple genotypes of American chestnut (Castanea dentata) and backcross hybrids of American chestnut and Chinese chestnut (C. dentata × Castanea mollissima) were cultured in ALBs. Medium for tissue proliferation in ALBs was woody plant medium supplemented with 3 g l−1 sucrose, 0.5 g l−1l-glutamine and 2 mg l−1 2,4-dichlorophenoxyacetic acid. Optimum culture conditions for 1-l ALBs included 2 % (w/v) tissue inoculation density for culture initiation, 200 ml min−1 airflow rate, and weekly feeding with fresh medium (80–85 %, v/v). Compared with flasks, ALBs generated higher yields of tissue mass and larger fractions of small cell clumps (<1 mm in diameter) that were optimum target material for transformation. Bioreactor-produced tissue demonstrated high amenability to transformation with reporter genes and chestnut blight resistance candidate genes (CGs) via Agrobacterium co-cultivation. The thaumatin-like protein CG was transformed effectively into ALB-produced target material of four genotypes, resulting in thousands of G418-resistant cell clumps, or transgenic events, which reached up to 70 % of the total target cell clumps in liquid selection medium for 3–6 weeks. Substantial acceleration of chestnut transformation was achieved with ALB technology, by which larger quantities of high quality target tissue was produced at lower labor and operating expense than previously used approaches.

Characterization of a pathogen induced thaumatin-like protein gene AdTLP from Arachis diogoi, a wild peanut.

Peanut (Arachis hypogaea L) is one of the widely cultivated and leading oilseed crops of the world and its yields are greatly affected by various biotic and abiotic stresses. Arachis diogoi, a wild relative of peanut, is an important source of genes for resistance against various stresses that affect peanut. In our previous study a thaumatin-like protein gene was found to be upregulated in a differential expression reverse transcription PCR (DDRT-PCR) study using the conidial spray of the late leaf spot pathogen, Phaeoisariopsis personata. In the present study, the corresponding full length cDNA was cloned using RACE-PCR and has been designated as AdTLP. It carried an open reading frame of 726 bp potentially capable of encoding a polypeptide of 241 amino acids with 16 conserved cysteine residues. The semi-quantitative RT-PCR analysis showed that the transcript level of AdTLP increased upon treatment with the late leaf spot pathogen of peanut, P. personata and various hormone treatments indicating its involvement in both, biotic and abiotic stresses. The antifungal activity of the purified recombinant protein was checked against different fungal pathogens, which showed enhanced anti-fungal activity compared to many other reported TLP proteins. The recombinant AdTLP-GFP fusion protein was found to be predominantly localized to extracellular spaces. Transgenic tobacco plants ectopically expressing AdTLP showed enhanced resistance to fungal pathogen, Rhizoctonia solani. The seedling assays showed enhanced tolerance of AdTLP transgenic plants against salt and oxidative stress. The transcript analysis of various defense related genes highlighted constitutively higher level expression of PR1a, PI-I and PI-II genes in transgenic plants. These results suggest that the AdTLP is a good candidate gene for enhancing stress resistance in crop plants.

Identification and characterization of a basic thaumatin‐like protein (TLP 2) as an allergen in sapodilla plum (Manilkara zapota)

Cases of oral allergy syndrome following the ingestion of sapodilla plum (Manilkara zapota) have been reported rarely. As the causative allergens are not known, the main objective of this study was to identify and characterize the important allergens in sapodilla. Allergy to sapodilla was diagnosed by case history, skin prick test, and serum allergen-specific IgE. The allergen was detected by IgE immunoblotting, purified on SP-Sepharose and characterized by native/SDS-PAGE, IEF, MS, and amino acid composition. Several cases of allergy to sapodilla fruit were identified; majority of the sapodilla-allergic subjects (6/7) experienced typical oral allergy syndrome symptoms, and allergen-specific IgE to the purified protein was positive. The allergen has a pI ≥9.5 and high contents of arginine, threonine, glycine, and cysteine. Circular dichroism revealed a secondary structure rich in beta sheets/turns. Based on its N-terminal sequence of A-T-F-D-I-Q-N-N-C-X-Y-, the allergen (21 578 Da) was identified as a thaumatin-like protein by homology. The causative allergen in sapodilla plum has been identified and characterized as a highly basic thaumatin-like protein belonging to the pathogenesis-related protein (PR-5) family, which has been recognized as a new family of conserved, cross-reactive plant allergens.

INHIBITING WILDFIRE AND INDUCING DEFENSE-RELATED GENE EXPRESSION BY LED TREATMENT ON NICOTIANA BENTHAMIANA

Light plays an important role in the induction of resistance responses in various plants against diseases. In this study, the inhibition was investigated of wildfire disease caused by Pseudomonas syringae pv. tabaci in Nicotiana benthamiana in response to irradiation with light emitting diodes (LEDs) To identify the effects of various wavelengths on the induction of defense responses, N. benthamiana plants were irradiated for three days with various LEDs (380, 440, 470, and 660 nm) before and after inoculation. Symptom development showed greater suppression in response to irradiation with blue (440 nm) and red (660 nm) light than fluorescent lights. Additionally, fewer bacterial cells were recovered from the lesions of LEDtreated leaves than those of fluorescent light-treated leaves. Simple LED irradiation induced the expression of defense-related genes including catalase (CAT), chalcone synthase (CHS), gluthatione-S-transferase (GST), pathogenesis-related protein (PR Q), proteinase inhibitor II (PinII), and thaumatin-like protein (TLP). Taken together, these data suggest that irradiation by LEDs could inhibit wildfire development by inducing defense responses in N. benthamiana plants.

063 Functions of insect antifreeze proteins and glycolipids

Thermal hysteresis (TH), indicative of antifreeze proteins (AFPs) or antifreeze glycolipids (AFGLs), has been identified in numerous species of insects. The AFPs or AFGLs responsible for the TH in most of these have not been described, and, given the diversity of insects, there are numerous new TH-producing substances yet to be discovered. While it is assumed that AFPs and AFGLs function to prevent freezing, details of their function(s) in vivo are sparse. Freeze avoiding larvae of the beetle Dendroides canadensis produce a family of over 30 AFPs (DAFPs) that are structurally similar to those of other beetles such as Tenebrio molitor. There is tissue specific expression with certain DAFPs present in hemolymph, mid- and hind-gut, primary urine, and epidermis, suggesting variation in function specific to the site. Some DAFPs exhibit self-enhancement whereby activity is increased by certain other DAFPs, as well as by endogenous glycerol (∼0.5M in hemolymph), a thaumatin-like protein, or AFGL. Hemolymph and epidermal DAFPs inhibit inoculative freezing across the cuticle when larvae are in contact with ice. Hemolymph and gut DAFPs inhibit ice nucleator proteins and bacteria, thereby promoting supercooling. Likewise, DAFPs in the primary urine inhibit ice nucleating crystals present in insect urine. Similar AFPs in freeze avoiding larvae of the beetle Cucujus clavipes assist (along with high glycerol and cryoprotective dehydration) in the extreme supercooling ability of the Alaskan subspecies whereby when in a mid-winter deep supercooling state they do not freeze even at −150 °C, but vitrify at ∼−60 to −70 °C. While low hemolymph TH is present in several freeze tolerant insects, AFPs have not been sequenced from these sources. However, a xylomannan-AFGL with TH activity similar to insect AFPs was purified from freeze tolerant Alaskan beetles, Upis ceramboides and a few other freeze tolerant insects, plants, and frogs. As AFPs and the AFGL inhibit recrystallization, this is likely to be a function in freeze tolerant species. However, most of the AFGL is present on cell membranes where it could also inhibit inoculation of the cytoplasm by extracellular ice. There may be additional functions of AFPs unrelated to antifreeze. Several insects have low TH in summer, and D. canadensis larvae synthesize certain DAFPs, but not AFGL, through the summer. TH was first identified by Ramsay in his classic studies of the water reabsorption ability of the cryptonephridial rectal complex of Tenebrio molitor. TH was present in hemolymph, but was especially high in the perirectal space and Ramsay speculated that TH-proteins were involved in water reabsorption. Later we demonstrated that T. molitor held at low relative humidity increase hemolymph TH, and any acclimation that increased hemolymph TH (low temperature, short photoperiod or low humidity) greatly extended their ability to survive 15% RH at 22 °C. DAFP and/or AFGLs may be involved (perhaps by membrane stabilization) in the higher upper lethal temperatures exhibited by D. canadensis in winter (∼41 °C) relative to summer (∼35 °C), even though lower lethal temperatures are −20 to −25 °C in winter but only ∼4 °C in summer. Source of funding: National Science Foundation IOS0618342 and 1025929. Conflict of interest: None declared. duman.1@nd.edu

Osmotic stress-induced polyamine oxidation mediates defence responses and reduces stress-enhanced grapevine susceptibility to Botrytis cinerea

Abiotic factors inducing osmotic stress can influence the plant immune response and resistance to pathogen infections. In this study, the effect of polyethylene glycol (PEG)- and sucrose-induced osmotic stress on polyamine (PA) homeostasis and the basal immune response in grapevine plantlets before and after Botrytis cinerea infection was determined. Pharmacological approaches were also addressed to assess the contribution of osmotic stress-induced PA oxidation to the regulation of defence responses and the susceptibility of grapevine to B. cinerea. Following osmotic stress or pathogen infection, PA homeostasis was linked to enhanced activity of diamine oxidases (CuAO) and PA oxidases (PAO) and the production of 1,3-diaminopropane. These responses paralleled the accumulation of the main stilbenic phytoalexins, resveratrol and ε-viniferin and upregulation of gene transcripts including STS (a stilbene synthase), PR-2 (a β-1,3-glucanase), PR3-4c (acidic chitinase IV), and PR-5 (a thaumatin-like protein), as well as NCED2 involved in abscisic acid biosynthesis. It was also demonstrated that leaves pre-exposed to osmotic stress and later inoculated with B. cinerea showed enhanced PA accumulation and attenuation of CuAO and PAO activities. This was consistent with the impaired production of phytoalexins and transcript levels of defence- and stress-related genes following infection, and the enhanced susceptibility to B. cinerea. Pharmacological experiments revealed that, under osmotic stress conditions, CuAO and PAO were involved in PA homeostasis and in the regulation of defence responses. Specific inhibition of CuAO and PAO in osmotically stressed leaves strongly attenuated the induction of defence responses triggered by B. cinerea infection and enhanced susceptibility to the pathogen. Taken together, this study reveals a contribution of PA catabolism to the resistance state through modulation of immune response in grapevine following osmotic stress and/or after B. cinerea infection.

In silico analyses of structural and allergenicity features of sapodilla (Manilkara zapota) acidic thaumatin-like protein in comparison with allergenic plant TLPs

Thaumatin-like proteins (TLPs) belong to the pathogenesis-related family (PR-5) of plant defense proteins. TLPs from only 32 plant genera have been identified as pollen or food allergens. IgE epitopes on allergens play a central role in food allergy by initiating cross-linking of specific IgE on basophils/mast cells. A comparative analysis of pollen- and food-allergenic TLPs is lacking. The main objective of this investigation was to study the structural and allergenicity features of sapodilla (Manilkara zapota) acidic TLP (TLP 1) by in silico methods. The allergenicity prediction of composite sequence of sapodilla TLP 1 (NCBI B3EWX8.1, G5DC91.1) was performed using FARRP, Allermatch and Evaller web tools. A homology model of the protein was generated using banana TLP template (1Z3Q) by HHPRED-MODELLER. B-cell linear epitope prediction was performed using BCpreds and BepiPred. Sapodilla TLP 1 matched significantly with allergenic TLPs from olive, kiwi, bell pepper and banana. IgE epitope prediction as performed using AlgPred indicated the presence of 2 epitopes (epitope 1: residues 36–48; epitope 2: residues 51–63), and a comprehensive analysis of all allergenic TLPs displayed up to 3 additional epitopes on other TLPs. It can be inferred from these analyses that plant allergenic TLPs generally carry 2–3 IgE epitopes. ClustalX alignments of allergenic TLPs indicate that IgE epitopes 1 and 2 are common in food allergenic TLPs, and IgE epitopes 2 and 3 are common in pollen allergenic TLPs; IgE epitope 2 overlaps with a portion of the thaumatin family signature. The secondary structural elements of TLPs vary markedly in regions 1 and 2 which harbor all the predicted IgE epitopes in all food and pollen TLPs in either of the region. Further, based on the number of IgE epitopes, food TLPs are grouped into rosid and non-rosid clades. The number and distribution of the predicted IgE epitopes among the allergenic TLPs may explain the specificity of food or pollen allergy as well as the varied degree of cross-reactivity among plant foods and/or pollens.

Identification and characterisation of main allergic proteins in Vitis vinifera vitis

Background. Vitis vinifera vitis (grape) cultivar Sultana is a causative agent of oral allergy syndrome (OAS) in Iran. In this study, the IgE-reactive proteins of this cultivar were characterised. Methods. IgE-reactivity of sera from patients with history of OAS to grape and positive skin prick test (SPT) were evaluated. Grape immunoreactive components were also purified and characterised. Results. The purified 10 kDa allergen showed the highest immunoreactivity in component-resolved ELISA and the results were in correlation with SPT results. The patients' sera were mostly reactive with 10, 28 and 60 kDa allergens; meanwhile, a 16 kDa protein can be considered as a novel marker for OAS to grape. The 24, 28 and 30 kDa allergens were identified as thaumatin-like protein, class IV chitinase and beta-1,3-glucanase, respectively. Conclusion. The Sultana cultivar showed a rather similar pattern of IgE reactivity with other cultivars. Several allergens play role in OAS to grapes, however, the 10 kD protein is the main allergen.

Identification of thaumatin-like protein and aspartyl protease as new major allergens in lettuce (Lactuca sativa).

Today, about 2-8% of the population of Western countries exhibits some type of food allergy whose impact ranges from localized symptoms confined to the oral mucosa to severe anaphylactic reactions. Consumed worldwide, lettuce is a Compositae family vegetable that can elicit allergic reactions. To date, however, only one lipid transfer protein has been described in allergic reaction to lettuce. The aim of this study was to identify potential new allergens involved in lettuce allergy. Sera from 42 Spanish lettuce-allergic patients were obtained from patients recruited at the outpatient clinic. IgE-binding proteins were detected by SDS-PAGE and immunoblotting. Molecular characterization of IgE-binding bands was performed by MS. Thaumatin was purified using the Agilent 3100 OFFGEL system. The IgE-binding bands recognized in the sera of more than 50% of patients were identified as lipid transfer protein (9 kDa), a thaumatin-like protein (26 kDa), and an aspartyl protease (35 and 45 kDa). ELISA inhibition studies were performed to confirm the IgE reactivity of the purified allergen. Two new major lettuce allergens-a thaumatin-like protein and an aspartyl protease-have been identified and characterized. These allergens may be used to improve both diagnosis and treatment of lettuce-allergic patients.

White Wine Proteins: How Does the pH Affect Their Conformation at Room Temperature?

Our studies focused on the determination of aggregation mechanisms of proteins occurring in wine at room temperature. Even if the wine pH range is narrow (2.8 to 3.7), some proteins are affected by this parameter. At low pH, the formation of aggregates and the development of a haze due to proteins sometimes occur. The objective of this work was to determine if the pH impacted the conformational stability of wine proteins. Different techniques were used: circular dichroism and fluorescence spectroscopy to investigate the modification of their secondary and tertiary structure and also SAXS to determine their global shape. Four pure proteins were used, two considered to be stable (invertase and thaumatin-like proteins) and two considered to be unstable (two chitinase isoforms). Two pH values were tested to emphasize their behavior (pH 2.5 and 4.0). The present work highlighted the fact that the conformational stability of some wine proteins (chitinases) was impacted by partial modifications, related to the exposure of some hydrophobic sites. These modifications were enough to destabilize the native state of the protein. These modifications were not observed on wine proteins determined to be stable (invertase and thaumatin-like proteins).

Prevalence of Sensitization to Cannabis sativa. Lipid-Transfer and Thaumatin-Like Proteins Are Relevant Allergens

Background: Although allergy to Cannabis sativa was first reported over 40 years ago, the allergenicity has scarcely been studied. The objectives of this study were to investigate the frequency of sensitization to this plant, to analyze the clinical characteristics and allergenic profile of sensitized individuals and to identify the allergens involved. Methods: Five hundred and forty-five individuals in Spain attending allergy clinics with respiratory or cutaneous symptoms underwent a skin-prick test (SPT) with C. sativa leaf extract. The extract was characterized by SDS-PAGE and 2-dimensional electrophoresis. Specific IgE to C. sativa was measured in positive SPT individuals. The clinical and allergenic profiles of sensitized individuals were investigated and the most-recognized allergens sequenced and characterized by liquid chromatography-mass spectrometry/mass spectrometry. Results: Of this preselected population, 44 individuals had positive SPT to C. sativa (prevalence 8.1%). Prevalence was higher in individuals who were C. sativa smokers (14.6%). Two individuals reported mild symptoms with C. sativa. Twenty-one individuals from 32 available sera (65.6%) had positive specific IgE to C. sativa. Twelve sera recognized at least 6 different bands in a molecular-weight range of between 10 and 60 kDa. Six of them recognized a 10-kDa band, identified as a lipid transfer protein (LTP) and 8 recognized a 38-kDa band, identified as a thaumatin-like protein. Conclusions: There is a high prevalence of sensitization to C. sativa leaves. The clinical symptoms directly attributed to C. sativa were uncommon and mild. The sensitization profile observed suggests that C. sativa sensitization may be mediated by two mechanisms, i.e. cross-reactivity, mainly with LTP and thaumatin-like protein, and exposure-related ‘de novo' sensitization.

The Anti-Cancer Property of Proteins Extracted from Gynura procumbens (Lour.) Merr

Gynura procumbens (Lour.) Merr. belongs to the Asteraceae Family. The plant is a well-known traditional herb in South East Asia and it is widely used to treat inflammation, kidney discomfort, high cholesterol level, diabetic, cancer and high blood pressure. Our earlier study showed the presence of valuable plant defense proteins, such as peroxidase, thaumatin-like proteins and miraculin in the leaf of G. procumbens. However, the effects of these defense proteins on cancers have never been determined previously. In the present study, we investigated the bioactivity of gel filtration fractionated proteins of G. procumbens leaf extract. The active protein fraction, SN-F11/12, was found to inhibit the growth of a breast cancer cell line, MDA-MB-231, at an EC50 value of 3.8 µg/mL. The mRNA expressions of proliferation markers, Ki67 and PCNA, were reduced significantly in the MDA-MB-23 cells treated with SN-F11/12. The expression of invasion marker, CCL2, was also found reduced in the treated MDA-MB-231 cells. All these findings highlight the anti-cancer property of SN-F11/12, therefore, the proteins in this fraction can be a potential chemotherapeutic agent for breast cancer treatment.

Screening Differential Expressions of Defense-related Responses in Cold-treated 'Kyoho' and 'Campbell Early' Grapevines

Low temperature is one of the major environmental factors that affect productivity including reduced growth and budding of vines, and changes of metabolic processes in grape (Vitis spp.). To screen the specific expression of abiotic stress-related genes against cold treatment in 'Kyoho' and 'Campbell Early' grapevines, expression of various defense-related genes was investigated by RT-PCR and real-time PCR. Among the 67 genes analyzed by RT-PCR and real-time PCR, 17 and 16 types of cDNA were up-regulated, while 5 and 6 types were down-regulated in cold-treated 'Kyoho' and 'Campbell Early' grapevines, respectively. Genes encoding carotene (Cart3564 and Cart4472), chalcone isomerase (CHI), cytochrome P450 (CYP), flavonol synthase (FLS), endo-<TEX>${\beta}$</TEX>-glucanase precursor (Glu), glutathione peroxidase (GPX), glutathione-S-transferase (GST), leucine-rich repeats (LRR), manganese superoxide dismutase (Mn-SOD), phenylalanine ammonia lyase (PAL), polygalacturonase-inhibiting protein (PGIP), proline rich protein 2 (PRP2), small heat shock protein (sHSP), temperature induced lipocalin (TIL), and thaumatin-like protein (TLP) were up-regulated, while those encoding CBF like transcription factor (CBF1), chitinase-like protein (CLP), cold induced protein (CIP), glycerol-3-phosphate acyltransferase (GPAT), and mitogen-activated protein kinase (MAPK) were down-regulated by low temperature treatment in both in 'Kyoho' and 'Campbell Early'.

Cloning and characterization of a thaumatin-like protein gene PeTLP in Populus deltoides × P. euramericana cv. ‘Nanlin895’

Thaumatin-like protein (TLP) genes constitute a large, highly complex gene family involved in host defense against abiotic stresses, as well as other physiological processes. However, their functions in the abiotic stress-induced defense response in poplar remain largely unknown. In this study, one full-length cDNA from a hybrid poplar (Populusdeltoides × P. euramericana cv. ‘Nanlin895’) that encodes a TLP was isolated by RACE and designated PeTLP. The deduced amino acid sequence of PeTLP encodes a protein of a predicted molecular mass 24.5 kDa and a pI of 8.19. Transcript of PeTLP accumulated in stems, but the gene showed low transcriptional levels in roots and leaves. PeTLP was upregulated in leaves after abiotic treatments, including 200 mM NaCl, 10 % PEG6000, 100 μM gibberellin, 1 mM salicylic acid and 100 μM abscisic acid. Subcellular localization analysis showed that GFP-tagged PeTLP predominantly localizes as discrete points in the cytoplasm and the periphery of the plasma membrane. Recombinant PeTLP obtained using Escherichiacoli expression produced antifungal activities to Rhizopus sp., Aspergillusniger and Penicilliumcitrinum. These results suggest that PeTLP is an abiotic stresses-induced protein and plays roles in physiological processes of poplar development, and is involved in the defense of poplar against abiotic stresses. Manipulation of PeTLP in poplar or other trees should improve their performance under various abiotic stress conditions.

Comparative proteomic profiling in compatible and incompatible interactions between hop roots and Verticillium albo-atrum

Verticillium wilt, caused by the soil borne fungal pathogen Verticillium albo-atrum, is a serious threat to hop (Humulus lupulus L.) production in several hop-growing regions. A proteomic approach was applied to analyse the response of root tissue in compatible and incompatible interactions between hop and V.albo-atrum at 10, 20 and 30 days after inoculation, using two-dimensional difference gel electrophoresis (2D-DIGE) coupled with de novo sequencing of derivatized peptides. Approximately 1200 reproducible spots were detected on the gels, of which 102 were identified. In the compatible interaction, 252 spots showed infection-specific changes in spot abundance and an accumulation of defence-related proteins, such as chitinase, β-glucanase, thaumatin-like protein, peroxidase and germin-like protein, was observed. However, no significant infection-specific changes were detected in the incompatible interaction. The results indicate that resistance in this pathosystem may be conferred by constitutive rather than induced defence mechanisms. The identification and high abundance of two mannose/glucose-specific lectin isoforms present only in the roots of the resistant cultivar suggests function of lectins in hop resistance against V.albo-atrum.

Identification and expression analysis of candidate genes related to endodormancy induction and breaking in Pyrus pyrifolia

To identify genes related to endodormancy in Pyrus pyrifolia, 124 clones exhibiting differential expression at three stages of bud endodormancy in the Japanese pear (Pyrus pyrifolia Nakai) ‘Gold Nijisseiki’ were isolated using the GeneFishing PCR method. Among the clones, 30, 54, and 19 clones showed high expression during the induction period (IP), deepest period (DP), and breaking period (BP) of endodormancy, respectively. In addition, 21 clones were overexpressed during IP and BP. The basic local alignment search tool searches revealed that 13 of these highly expressed genes have been previously identified. Based on the results of real-time PCR analysis, we focused on 8 genes as potential candidate genes related to bud endodormancy. Expression of 2 genes decreased from IP to DP during endodormancy in ‘Gold Nijisseiki,’ but increased in ‘Hengshanli,’ a plant that enters endodormancy at a different period. These genes were highly homologous to those encoding auxin-responsive family protein and E3 ubiquitin ligase PUB14. Expression of tonoplast intrinsic protein 1 increased briefly before BP as well as after 24h of hydrogen cyanamide (HC) treatment. During BP, genes highly homologous to those encoding thaumatin-like protein and auxin influx carrier component exhibited decreased and increased expression, respectively. This was also observed following HC treatment. Genes identified in this study may play a role in regulating the induction or breaking of endodormancy in the vegetative buds of P. pyrifolia.