Tsutomu Takayanagi

Characterization of thermotolerance-related genes in grapevine (Vitis vinifera)

We report the cloning and characterization of heat shock-induced genes in grapevine (Vitis vinifera L.). Using the cDNA subtraction method, four heat shock-induced genes were identified in heat shock-treated Pinot noir grapevine. The four genes were immediately induced and upregulated in leaves and berry skins by heat treatment at 45 degrees C. The recovery treatment at 26 degrees C reduced the upregulated transcription of the heat shock-induced genes to near basal levels. The predicted amino acid sequences of three genes, HSG4, HSG14, and HSG19, showed high homologies to those of known small heat shock proteins of other plants. The predicted amino acid sequence of the fourth gene, HSG1, has two conserved motifs, the IQ motif and the Bcl-2-associated athanogene (BAG) domain, suggesting that HSG1 may be a member of the BAG family in grapevine. Although no morphological changes were observed, the overexpression Arabidopsis lines of HSG1, HSG4, HSG14, or HSG19 exhibited faster growth including floral transition than the control line, suggesting that the constitutive expression of HSG1, HSG4, HSG14, or HSG19 protein resulted in increased growth rates without any detectable harm. The thermotolerance test demonstrated that all of the overexpression lines remained viable and noticeably healthy compared with the control line even after exposure to severe heat shock, suggesting that HSG1, HSG4, HSG14, or HSG19 protein might be related to the acquisition of thermotolerance in grapevine.

Nested PCR-RFLP is a high-speed method to detect fungicide-resistant Botrytis cinerea at an early growth stage of grapes.

BACKGROUNDnGrey mould caused by the fungus Botrytis cinerea Pers. ex Fr. is one of the major diseases in grapes. The use of fungicides is a simple strategy to protect grapes against B. cinerea disease. However, phenotypes exhibiting resistance to fungicides have been detected in B. cinerea populations. The variation of fungicide-resistant B. cinerea isolates renders B. cinerea disease control difficult in grapevine fields.nnnRESULTSnThe authors have developed a nested polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method to detect fungicide-resistant B. cinerea isolates at an early growth stage of grapes in grapevine fields. The nested PCR-RFLP method was carried out to detect benzimidazole-, phenylcarbamate- and/or dicarboximide-resistant B. cinerea isolates from grape berries and leaves at Eichorn-Lorenz growth stage 25 to 29. This method successfully detected fungicide-resistant B. cinerea isolates at an early growth stage of grapes. In addition, only 8 h was required from tissue sampling to phenotyping of fungicide resistance of the isolates.nnnCONCLUSIONnIt is proposed that the early diagnosis of fungicide-resistant B. cinerea isolates would contribute to further improvement of integrated pest management against B. cinerea in grapevine fields, and that the nested PCR-RFLP method is a high-speed, sensitive and reliable tool for this purpose.

Rapid method for detecting resistance to a QoI fungicide in Plasmopara viticola populations.

BACKGROUNDnThe increasing occurrence of Qo inhibitor (QoI)-fungicide-resistant Plasmopara viticola (Berk. & MA Curtis) Berl. & DeToni populations is becoming a serious problem in the control of grapevine downy mildew worldwide.nnnRESULTSnThe authors have developed a rapid method for detecting resistance to a QoI fungicide, azoxystrobin, in P. viticola populations using the nested PCR-RFLP method. With this method, a glycine-to-alanine substitution was discovered at codon 143 in the cytochrome b gene of P. viticola populations found in Japan.nnnCONCLUSIONnIt is proposed that the nested PCR-RFLP method is a high-speed, sensitive and reliable tool for detecting azoxystrobin-resistant P. viticola populations.

Cloning and characterization of VIGG, a novel virus-induced grapevine protein, correlated with fruit quality

We report here the identification and characterization of VIGG, a novel virus-induced grapevine protein. Analysis of VIGG expression in grapevine demonstrated that VIGG was constitutively expressed in leaves and stems in virus-infected grapevine, and that VIGG expression was induced by grapevine virus A (GVA) infection, but not by infection with other viruses. The virus-induced expression profile of VIGG was supported by the finding that virus-free meristem cultures prepared from virus-infected grapevines did not express VIGG. An experiment using GFP-VIGG fusion protein demonstrated that VIGG might be localized in or around the endoplasmic reticulum (ER). Treatment of grapevine cells with ER stress inducers resulted in the induction of VIGG expression. Berries from VIGG-expressing grapevines had higher organic acid and phenolic contents than those from control grapevines that did not express VIGG. Interestingly, fruit composition of a grapevine that was simultaneously infected by GVA and grapevine virus B (GVB), which did not express VIGG, was significantly different from that of GVA-infected grapevines expressing VIGG, suggesting that the effector of fruit composition alteration might be VIGG expression, but not GVA infection. Taken together, VIGG expression might suppress the decrease in organic acid content and increase phenol content in berries. Further investigation of the biological function of VIGG is expected to provide new information on the fruit quality of grapevines.

Inhibitory effect of chitinases isolated from Semillon grapes (Vitis vinifera) on growth of grapevine pathogens

Characteristics and antifungal activity of chitinases in Semillon grapes were investigated. Chitinases were isolated from the juice of Semillon grapes by chitin affinity chromatography. Native and SDS-PAGE analyses of the fraction showing chitin affinity (active fraction) demonstrated only the presence of protein bands of chitinases. Three types of class IV chitinases (chi-1a, chi-1b and chi-2) were purified from the active fraction. These chitinases actively hydrolyzed chitin under acidic conditions (pH 4.0–4.5). The isoelectric points and the molecular weights of chi-1a, chi-1b and chi-2 were 4.73, 4.60, and 7.87, and 32.1xa0kDa, 31.6xa0kDa, and 29.0xa0kDa, respectively. The active fraction was found to inhibit Botrytis cinerea mycelial growth and the inhibitory effect was due to the activity of chitinases. The active fraction inhibited twenty strains of B. cinerea collected from the experimental vineyard. The effect of chitinases was enhanced in media containing more than 20% sugar. When the active fraction was tested on Glomerella cingulata, the growth inhibitory effect observed was markedly less than that seen on B. cinerea.

Molecular characterization of Japanese indigenous grape cultivar ‘Koshu’ (Vitis vinifera) leaf and berry skin during grape development

We investigated the transcriptional profiles of Japanese indigenous grape cultivar ‘Koshu’ (Vitis vinifera) leaf and berry skin during ripening. In leaf, 64 genes were abundantly transcribed at the end of véraison (14xa0weeks post-flowering), whereas the expression of 61 genes was upregulated at the end of ripening (20xa0weeks post-flowering). In berry skin, 67 genes were abundantly transcribed at the end of véraison, whereas the expression of 86 genes was upregulated at the end of ripening. Gene expression associated with biological processes was activated in both tissues at the end of ripening. The expression of genes associated with photosynthesis, sugar synthesis, anthocyanin synthesis, cinnamic acid synthesis, and amino acid metabolism was observed in leaf and berry skin during ripening, together with the accumulation of sugars, anthocyanins, cinnamic acids, and amino acids. Transcripts of AUX/IAA family proteins that repress the activities of auxin-induced proteins were expressed in berry skin at the end of véraison. Transcripts of genes related to the ubiquitin–proteasome system that degrades AUX/IAA family proteins were abundantly expressed in berry skin at the end of ripening, suggesting that the expansion of skin cells at véraison is suppressed by AUX/IAA family proteins, and that the ubiquitin–proteasome system induces the expansion of skin cells during ripening by degrading AUX/IAA family proteins. These transcriptional profiles, which provide new information on the characteristics of ‘Koshu’ grapevine during ripening, may explain the unique characteristics of ‘Koshu’ grape in comparison with those of European grapes used for winemaking, and may contribute to the improvement of ‘Koshu’ grape quality.

Purification and properties of invertase from Muscat Bailey A grapes

Abstract A grape invertase was purified from Muscat Bailey A juice by salting out with ammonium sulfate and successive chromatographies on Sephadex G-100 and Con A-agarose to a homogeneous state as confirmed by polyacrylamide gel electrophoresis. The molecular weight of the purified enzyme was 72 kDa in gel filtration chromatography. However, SDS polyacrylamide gel electrophoresis revealed three bands with molecular weights of 56 kDa, 25 kDa and 24 kDa. Affinity staining of Western blots with lectins indicated that the enzyme was a glycoprotein. The optimum pH for the enzyme reaction was 3.5 and the optimum temperature 80°C. The enzyme was stable from pH 2.7 to 6.4, and up to 80°C. The transfructosylation reaction could not be observed. The K m value of this enzyme for sucrose was 4.4 mM at pH 4.0, but as the pH of the reaction mixture increased, the K m value decreased sharply. From the dependence of the V max and K m values on pH, the ionization constant (p K e ) of one of the two essential ionizable groups of the free enzyme was determined to be 2.7, suggesting that this essential ionizable group was a carboxyl.

In planta transformation technique for grapevines (Vitis vinifera L) using dormant buds.

Plant regeneration using the genetic transformation method often induces somatic mutation as well as epigenetic alteration. In addition, methods commonly used for grapevine (Vitis vinifera L) transformations are time-consuming due to the long reproductive cycle of the grapevine. Therefore, an improved method is needed for more efficient grapevine transformation. Five transformants (three for Chardonnay and two for Cabernet Sauvignon) were obtained by inoculation of dormant buds on cuttings with Agrobacterium tumefaciens harbouring a pBI121 binary vector. All of the transformants expressed β-glucuronidase (GUS) transcripts constitutively in all expanding leaves from the Agrobacterium-inoculated buds. Histochemical analysis showed that the transformants expressed GUS in the vascular bundle system. We demonstrate an alternative in planta transformation method for grapevines using dormant buds. Genetically modified grapevines could be obtained simply and rapidly with this method. In addition, this method avoids the tissue culture step that can cause somatic mutations, suggesting that the biological and physiological functions of transgene products could be accurately estimated in transgenic grapevines created by this new method.

Changes in Amount of Nitrogenous Compounds from Skins and Seeds of Four Grape Cultivars During Extraction Using Juice- or Fermenting Must-Like Model Solutions

Changes in the amounts of nitrogenous compounds (total and free amino acids, peptides, and proteins) were investigated during extraction under oenological winemaking conditions from seeds and skins of two Japanese cultivars (Koshu and Muscat Bailey A) and two European cultivars (Chardonnay and Cabernet Sauvignon) using juice-like model solutions and fermenting must-like model solutions. Two maceration systems, one prior to fermentation (without ethanol) and the other, during fermentation (with ethanol), were used. The amounts of nitrogenous compounds extracted increased with time. The amounts extracted varied with the cultivar. The nitrogenous compounds were gradually extracted from seeds, whereas those from skins were almost completely extracted on the first day. The presence of nitrogenous compounds, in particular free amino acids extracted from seeds and/or skins, may be relevant as nutrients for yeast growth during fermentation.