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Sulfur dioxide (SO 2 ) is the most effective preservative for table grapes as it reduces the respiratory intensity of berries and inhibits mold growth. However, excessive SO 2 causes berry abscission during storage, resulting in an economic loss postharvest. In this study, grapes were exogenously treated with SO 2 , SO 2 + 1.5% chitosan, SO 2 + 1.5% eugenol, and SO 2 + eugenol-loaded chitosan nanoparticles (SN). In comparison to SO 2 treatment, SN treatment reduced the berries' abscission rate by 74% while maintaining the quality of the berries. Among the treatments, SN treatment most effectively inhibited berry abscission and maintained berry quality. RNA-sequencing (RNA-seq) revealed that SN treatment promoted the expression of genes related to cell wall metabolism. Among these genes, VlCOMT was detected as the central gene, playing a key role in mediating the effects of SN. Dual luciferase and yeast one-hybrid (Y1H) assays demonstrated that VlbZIP14 directly activated VlCOMT by binding to the G-box motif in the latter's promoter, which then participated in lignin synthesis. Our results provide key insights into the molecular mechanisms underlying the SN-mediated inhibition of berry abscission and could be used to improve the commercial value of SO 2 -treated postharvest table grapes.

Fruit set is a key stage in determining yield potential and guaranteeing quality formation and regulation. N-(2-chloro-4-pyridyl)-N'-phenylurea (CPPU) has been widely applied in grape production, the most iconic of which is the promotion of grape fruit set. However, current studies still lack the molecular mechanism of CPPU-induced grape fruit set. Here, the dynamic, high-resolution stage-specific transcriptome profiles were generated based on two different treatments and five developmental periods during fruit set in 'Kyoho' grape ( Vitis vinifera L. × V. labrusca L.). Pairwise comparison and functional category analysis showed that phytohormone action cytokinin was significantly enriched during the CPPU-induced grape fruit set, but not the natural one. Value differentially expressed gene (VDEG) was a newly proposed analysis strategy for mining genes related to the grape fruit set. Notably, the cytokinin metabolic process was significantly enriched among up-regulated VDEGs. Of importance, a key VDEG VlCKX4 related to the cytokinin metabolic process was identified as related to the grape fruit set. Overexpression of VlCKX4 gene promoted the Arabidopsis plants that produce more and heavier siliques. The transcription factor VlMYB59 directly bound to the promoter of VlCKX4 and activated its expression. Moreover, overexpression of VlMYB59 gene also promoted the Arabidopsis fruit set. Overall, VlMYB59 responded to CPPU treatment and directly activated the expression of VlCKX4 , thus promoting the fruit set. A regulatory pathway of the VlMYB59- VlCKX4 module in the fruit set was uncovered, which provides important insights into the molecular mechanisms of the fruit set and good genetic resources for high fruit set rate breeding., Competing Interests: The authors declare that they have no conflicts of interest in this work., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nanjing Agricultural University.)

Dwarfing rootstocks enhance planting density, lower tree height, and reduce both labor in peach production. Cerasus humilis is distinguished by its dwarf stature, rapid growth, and robust fruiting capabilities, presenting substantial potential for further development. In this study, Ruipan 4 was used as the scion and grafted onto Amygdalus persica and Cerasus humilis , respectively. The results indicate that compared to grafting combination R/M (Ruipan 4/ Amygdalus persica ), grafting combination R/O (Ruipan 4/ Cerasus humilis ) plants show a significant reduction in height and a significant increase in flower buds. RNA-seq indicates that genes related to gibberellin (GA) and auxin metabolism are involved in the dwarfing process of scions mediated by C. humilis . The expression levels of the GA metabolism-related gene PpGA2ox7 significantly increased in R/O and are strongly correlated with plant height, branch length, and internode length. Furthermore, GA levels were significantly reduced in R/O. The transcription factor PpGATA21 was identified through yeast one-hybrid screening of the PpGA2ox7 promoter. Yeast one-hybrid (Y1H) and dual-luciferase reporter (DLR) demonstrate that PpGATA21 can bind to the promoter of PpGA2ox7 and activate its expression. Overall, PpGATA21 activates the expression of the GA-related gene PpGA2ox7 , resulting in reduced GA levels and consequent dwarfing of plants mediated by C. humilis. This study provides new insights into the mechanisms of C. humilis and offers a scientific foundation for the dwarfing and high-density cultivation of peach trees.

Key Message: VyPUB21 plays a key role during the defense against powdery mildew in grapes. Ubiquitin-ligating enzyme (E3), a type of protein widely found in plants, plays a key role in their resistance to disease. Yet how E3 participates in the disease-resistant response of Chinese wild grapevine (Vitis yeshanensis) remains unclear. Here we isolated and identified a U-box type E3 ubiquitin ligase, VyPUB21, from V. yeshanensis. This gene's expression level rose rapidly after induction by exogenous salicylic acid (SA), jasmonic acid (JA), and ethylene (ETH) and powdery mildew. In vitro ubiquitination assay results revealed VyPUB21 could produce ubiquitination bands after co-incubation with ubiquitin, ubiquitin-activating enzyme (E1), and ubiquitin-conjugating enzyme (E2); further, mutation of the conserved amino acid site in the U-box can inhibit the ubiquitination. Transgenic VyPUB21 Arabidopsis had low susceptibility to powdery mildew, and significantly fewer conidiophores and spores on its leaves. Expression levels of disease resistance-related genes were also augmented in transgenic Arabidopsis, and its SA concentration also significantly increased. VyPUB21 interacts with VyNIMIN and targets VyNIMIN protein hydrolysis through the 26S proteasome system. Thus, the repressive effect of the NIMIN-NPR complex on the late systemic acquired resistance (SAR) gene was attenuated, resulting in enhanced resistance to powdery mildew. These results indicate that VyPUB21 encoding ubiquitin ligase U-box E3 activates the SA signaling pathway, and VyPUB21 promotes the expression of late SAR gene by degrading the important protein VyNIMIN of SA signaling pathway, thus enhancing grape resistance to powdery mildew., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Key Message: VviWOX13C plays a key regulatory role in the expansin during fruit set. Expansins as a type of non-enzymatic cell wall proteins, are responsible for the loosening and extension in cell walls leading to the enlargement of the plant cells. However, the current studies are still lacking in expansin genes associated with promoting fruit set. Here, 29 members of the expansin gene family were identified in the whole genome of grapes (Vitis vinifera L.), and the functional prediction of expansins was based on the gene annotated information. Results showed that the 29 members of grape expansin gene family could be mainly divided into four subfamilies (EXPA, EXPB, LIKE A, and LIKE B), distributed on 16 chromosomes. Replication analysis showed that there were four segmental duplications and two tandem duplications. Each expansins sequence contained two conserved domain features of grape EXPs (DPBB_1 and Expansin_C) through protein sequence analysis. The transcriptome sequencing results revealed that VviEXPA37, VviEXPA38, and VviEXPA39 were induced and upregulated by CPPU. Furthermore, transcriptional regulatory prediction network indicated that VviWOX13C targeted regulates VviEXPA37, VviEXPA38, and VviEXPA39 simultaneously. EMSA and dual luciferase assays demonstrated that VviWOX13C directly activated the expression of VviEXPA37, VviEXPA38, and VviEXPA39 by directly binding to its promoter. These results provide a basis for further studies on the function and regulatory mechanisms of expansin genes in fruit set., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Drought stress profoundly affects plant growth and development, posing a significant challenge that is extensively researched in the field. Thioredoxins (TRXs), small proteins central to redox processes, are crucial to managing both abiotic and biotic stresses. In this research, the VyTRXy gene, cloned from wild Yanshan grapes, was validated as a functional TRX through enzyme activity assays. VyTRXy was found to bolster photosynthesis, augment levels of osmotic regulators, stimulate antioxidant enzyme activities, and strengthen drought resilience in transgenic plants. These enhancements were evidenced by higher survival rates, optimized photosynthetic metrics, increased proline levels, augmented chlorophyll concentration, reduced electrolyte leakage, and decreased malondialdehyde and hydrogen peroxide (H 2 O 2 ) levels. Furthermore, there was a surge in the activities of enzymes such as catalase, ascorbate peroxidase, glutathione peroxidase, dehydroascorbate reductase, and glutathione reductase, along with an increased expression of TRX peroxidase. Notably, under drought stress, there was a marked elevation in the expression of stress-responsive genes, including the adversity stress-inducible expression gene ( NtRD29A ) and DRE-binding protein (NtDREB), in transgenic tobacco. This investigation is pivotal in the quest for drought-resistant grapevine varieties and provides significant insights into the molecular functionality of VyTRXy in enhancing grapevine drought tolerance.

Background: RING is one of the largest E3 ubiquitin ligase families and C3H2C3 type is the largest subfamily of RING, which plays an important role in plant growth and development, and growth and responses to biotic and abiotic stresses., Results: A total of 143 RING C3H2C3-type genes (RCHCs) were discovered from the grapevine genome and separated into groups (I-XI) according to their phylogenetic analysis, and these genes named according to their positions on chromosomes. Gene replication analysis showed that tandem duplications play a predominant role in the expansion of VvRCHCs family together. Structural analysis showed that most VvRCHCs (67.13 %) had no more than 2 introns, while genes clustered together based on phylogenetic trees had similar motifs and evolutionarily conserved structures. Cis-acting element analysis showed the diversity of VvRCHCs regulation. The expression profiles of eight DEGs in RNA-Seq after drought stress were like the results of qRT-PCR analysis. In vitro ubiquitin experiment showed that VyRCHC114 had E3 ubiquitin ligase activity, overexpression of VyRCHC114 in Arabidopsis improved drought tolerance. Moreover, the transgenic plant survival rate increased by 30 %, accompanied by electrolyte leakage, chlorophyll content and the activities of SOD, POD, APX and CAT were changed. The quantitative expression of AtCOR15a, AtRD29A, AtERD15 and AtP5CS1 showed that they participated in the response to drought stress may be regulated by the expression of VyRCHC114., Conclusions: This study provides valuable new information for the evolution of grapevine RCHCs and its relevance for studying the functional characteristics of grapevine VyRCHC114 genes under drought stress., (© 2021. The Author(s).)

The plant U-box E3 ubiquitin ligase-mediated ubiquitin/26S proteasome degradation system plays a key role in plant growth and development. Previously identified as a member of the grape PUB gene family, PUB38 was shown to participate in the berry-ripening progress. Here, we demonstrate that the E3 ligase VlPUB38 mediates abscisic acid (ABA) synthesis via 26S proteasome degradation and its involvement in regulating fruit-ripening processes. Strawberry-overexpressing VlPUB38 lines displayed obvious inhibition of mature phenotype, and this was rescued by exogenous ABA treatment and MG132. Post-ABA treatment, expression levels of ABA response-related genes in VlPUB38-overexpressed Arabidopsis significantly exceeded controls. Strawberry and Arabidopsis ectopic expression assays suggest that VlPUB38 negatively regulates fruit ripening in an ABA-dependent manner. Moreover, VlPUB38 has ubiquitin ligase activity, which depends on the U-box-conserved domain. VlPUB38 interacts with abscisic-aldehyde oxidase (VlAAO), targeting VlAAO proteolysis via the 26S proteasome system. These results indicate that VlPUB38 negatively regulates grape fruit ripening by mediating the degradation of key factor VlAAO in the ABA synthesis pathway., (� The Author(s) 2020. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

In recent years, plant genetic engineering has advanced agriculture in terms of crop improvement, stress and disease resistance, and pharmaceutical biosynthesis. Cells from land plants and algae contain three organelles that harbor DNA: the nucleus, plastid, and mitochondria. Although the most common approach for many plant species is the introduction of foreign DNA into the nucleus (nuclear transformation) via Agrobacterium- or biolistics-mediated delivery of transgenes, plastid transformation offers an alternative means for plant transformation. Since there are many copies of the chloroplast genome in each cell, higher levels of protein accumulation can often be achieved from transgenes inserted in the chloroplast genome compared to the nuclear genome. Chloroplasts are therefore becoming attractive hosts for the introduction of new agronomic traits, as well as for the biosynthesis of high-value pharmaceuticals, biomaterials and industrial enzymes. This review provides a comprehensive historical and biological perspective on plastid transformation, with a focus on current and emerging approaches such as the use of single-walled carbon nanotubes (SWNTs) as DNA delivery vehicles, overexpressing morphogenic regulators to enhance regeneration ability, applying genome editing techniques to accelerate double-stranded break formation, and reconsidering protoplasts as a viable material for plastid genome engineering, even in transformation-recalcitrant species.

Background: Melatonin is a ubiquitous molecule and exists across kingdoms. Studies on melatonin in plants have mainly focused on its physiological influence on growth and development, and on its biosynthesis. A number of studies have been conducted on the melatonin content and exogenous melatonin treatment of grapevine (Vitis vinifera L.). However, key genes or enzymes of the melatonin biosynthetic pathway remain unclear., Results: In this study, we cloned and identified the gene encoding serotonin N-acetyltransferase (SNAT) in grapevine (VvSNAT2). The VvSNAT2 protein was identified from a collection of 30 members of the grapevine GCN5-related N-acetyltransferase (GNAT) superfamily. Phylogenetic and protein sublocalization analyses showed that the candidate gene VvGNAT16 is VvSNAT2. Characterization of VvSNAT2 showed that its enzymatic activity is highest at a pH of 8.8 and a temperature of 45 °C. Analysis of enzyme kinetics showed the values of K m and V max of VvSNAT2 using serotonin were 392.5 μM and 836 pmol/min/mg protein, respectively. The expression of VvSNAT2 was induced by melatonin treatment and pathogen inoculation. Overexpression of VvSNAT2 in Arabidopsis resulted in greater accumulation of melatonin and chlorophyll and enhanced resistance to powdery mildew in the transgenic plants compared with the wild type (WT). Additionally, our data showed that the marker genes in the salicylic acid (SA) signaling pathway were expressed to higher levels in the transgenic plants compared with the WT., Conclusions: The VvSNAT2 gene was cloned and identified in grapevine for the first time. Our results indicate that VvSNAT2 overexpression activates the SA and JA signaling pathways; however, the SA pathway plays a central role in VvSNAT2-mediated plant defense.

Background: Resveratrol is a naturally occurring plant stilbene that exhibits a wide range of valuable biological and pharmacological properties. Although the beneficial effects of trans-resveratrol to human health and plant protection against fungal pathogens and abiotic stresses are well-established, yet little is known about the molecular mechanisms regulating stilbene biosynthesis in plant defense progress., Results: Here, we cloned and identified the Chinese wild grape (Vitis davidii) R2R3-MYB transcription factor VdMYB1, which activates defense responses against invading pathogen. VdMYB1 transcripts were significantly upregulated after inoculation with the grapevine powdery mildew fungus Erysiphe necator (Schw.) Burr. Transient expression analysis using onion epidermal cells and Arabidopsis thaliana protoplasts showed that VdMYB1 was localized in the nucleus. Yeast one-hybrid assays revealed that VdMYB1 acts as a transcriptional activator. Grapevine leaves transiently overexpressing VdMYB1 showed a lower number of fungal conidiophores compared with wild-type leaves. Overexpression of VdMYB1 in grapevine leaves did not alter the expression of genes in salicylic acid- and jasmonate-dependent pathways, but affected the expression of stilbene synthase (STS) genes, key regulators of flavonoid metabolism. Results of electrophoretic mobility shift assays and in vivo transcriptional activation assays showed that VdMYB1 binds to the MYB binding site (MYBBS) in the STS2 gene promoter, thus activating STS2 transcription. In heterologous expression assays using tobacco leaves, VdMYB1 activated STS2 gene expression and increased the accumulation of resveratrol., Conclusions: Our study showed that VdMYB1 activates STS2 gene expression to positively regulate defense responses, and increases the content of resveratrol in leaves.

Powdery mildew is a disease caused by fungal pathogens that harms grape leaves and fruits. The TIFY gene family is a plant-specific super-family involved in the process of plants' development and their biotic and abiotic stress responses. This study aimed to learn the function of the VvTIFY9 gene to investigate molecular mechanisms of grape resistance to powdery mildew. A VvTIFY9 protein encoding a conserved motif (TIF[F/Y]XG) was characterized in grape ( Vitis vinifera ). Sequence analysis confirmed that VvTIFY9 contained this conserved motif (TIF[F/Y]XG). Quantitative PCR analysis of VvTIFY9 in various grape tissues demonstrated that the expression of VvTIFY9 was higher in grape leaves. VvTIFY9 was induced by salicylic acid (SA) and methyl jasmonate (MeJA) and it also quickly responded to infection with Erysiphe necator in grape. Analysis of the subcellular localization and transcriptional activation activity of VvTIFY9 showed that VvTIFY9 located to the nucleus and had transcriptional activity. Arabidopsis that overexpressed VvTIFY9 were more resistant to Golovinomyces cichoracearum , and quantitative PCR revealed that two defense-related genes, AtPR1 and AtPDF1.2 , were up-regulated in the overexpressing lines. These results indicate that VvTIFY9 is intimately involved in SA-mediated resistance to grape powdery mildew. This study provides the basis for exploring the molecular mechanism of grape resistance to disease resistance and candidate genes for transgenic disease resistance breeding of grape plants.

Potato cold-induced sweetening (CIS) is a major drawback restricting potato process industry. Starch degradation and sucrose decomposition are considered to be the key pathways in potato CIS. Our previous study showed that the RING finger gene SbRFP1 could slow down starch degradation and the accumulation of reducing sugars (RS) through inhibiting amylase and invertase activity in cold-stored tubers. However, the regulation mechanism of SbRFP1 is not clear. In this paper, we first proved that SbRFP1 could promote starch synthesis and modify the shape of starch granules. By further yeast two hybrid, GST-pull down and inhibition of enzyme activity assays, we confirmed that SbRFP1 could slow down the transformation of starch to RS in tubers mainly through the inhibition of β-amylase StBAM1 activity. SbRFP1 was also proved to possess E3 ubiquitin ligase activity by ubiquitination assay. Thus, SbRFP1 may regulate the accumulation of RS in cold-stored tubers by ubiquitination and degradation of StBAM1. Therefore, our study reveals the regulatory mechanism of SbRFP1 in the process of CIS and provides more powerful evidence for the effect of starch degradation on potato CIS., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Ubiquitin-mediated regulation responds rapidly to specific stimuli; this rapidity is particularly important for defense responses to pathogen attack. Here, we investigated the role of the E3 ubiquitin ligase Erysiphe necator-induced RING finger protein 1 (EIRP1) in the defense response of Chinese wild grapevine Vitis pseudoreticulata. The regulatory function of E3 ubiquitin ligase EIRP1 was investigated using molecular, genetic and biochemical approaches. EIRP1 encodes a C3HC4-type Really Interesting New Gene (RING) finger protein that harbors E3 ligase activity. This activity requires the conserved RING domain, and VpWRKY11 also interacts with EIRP1 through the RING domain. VpWRKY11 localizes to the nucleus and activates W-box-dependent transcription in planta. EIRP1 targeted VpWRKY11 in vivo, resulting in VpWRKY11 degradation. The expression of EIRP1 and VpWRKY11 responds rapidly to powdery mildew in Vitis pseudoreticulata grapevine; also, overexpression of EIRP1 in Arabidopsis confers enhanced resistance to the pathogens Golovinomyces cichoracearum and Pseudomonas syringae pv tomato DC3000. Our data suggest that the EIRP1 E3 ligase positively regulates plant disease resistance by mediating proteolysis of the negative regulator VpWRKY11 via degradation by the 26S proteasome., (© 2013 College of Horticulture. New Phytologist © 2013 New Phytologist Trust.)

In both plants and animals, programmed cell death (PCD) is an indispensable process that removes redundant cells. In seedless grapes (Vitis vinifera), abnormal PCD in ovule cells and subsequent ovule abortion play key roles in stenospermocarpy. Metacaspase, a type of cysteine-dependent protease, plays an essential role in PCD. To reveal the characteristics of the metacaspase (MC) gene family and the relationship between metacaspases and the seedless trait, we identified the 6 V. vinifera metacaspases VvMC1-VvMC6, from the grape genome, using BLASTN against the 9 known Arabidopsis metacaspases. We also obtained full-length cDNAs by RT-PCR. Each of the 6 grape metacaspases contains small (p10-like) and a large (p20-like) conserved structural domains. Phylogenetic analysis of 6 grape and 9 Arabidopsis metacaspases showed that all metacaspases could be grouped into two classes: Type I and Type II. Each phylogenetic branch shares a similar exon/intron structure. Furthermore, the putative promoters of the grape metacaspases contained cis-elements that are involved in grape endosperm development. Moreover, expression analysis of metacaspases using real-time quantitative PCR demonstrated that VvMC1 and VvMC2 were able to be detected in any tissue, and VvMC3, VvMC4, VvMC5 and VvMC6 exhibited tissue-specific expression. Lastly, in cv. Thompson seedless grapes VvMC1, VvMC3, and VvMC4 were significantly up-regulated at the 35 DAF during ovule development, roughly same stage as endosperm abortion. In addition, the expression trend of VvMC2 and VvMC5 was similar between cv. Pinot Noir and cv. Thompson grape ovule development and that of VvMC6 was sustained in a relatively low level except the expression of cv. Pinot Noir significantly up-regulated in 25 DAF. Our data provided new insights into PCD by identifying the grape metacaspase gene family and provide a useful reference for further functional analysis of metacaspases in grape., (© 2013.)

RING-finger proteins (RFP) function as ubiquitin ligases and play key roles in plant responses to biotic and abiotic stresses. However, little information is available on the regulation of RFP expression. Here, we isolate and characterize the RFP promoter sequence from the disease-resistant Chinese wild grape Vitis pseudoreticulata accession Baihe-35-1. Promoter-GUS fusion assays revealed that defense signaling molecules, powdery mildew infection, and heat stress induce VpRFP1 promoter activity. By contrast, the RFP1 promoter isolated from Vitis vinifera was only slightly induced by pathogen infection and heat treatment. By promoter deletion analysis, we found that the -148 bp region of the VpRFP1 promoter was the core functional promoter region. We also found that, in Arabidopsis, VpRFP1 expressed under its own promoter activated defense-related gene expression and improved disease resistance, but the same construct using the VvRFP1 promoter slightly improve disease resistance. Our results demonstrated that the -148 bp region of the VpRFP1 promoter plays a key role in response to pathogen and heat stress, and suggested that expression differences between VpRFP1 and VvRFP1 may be key for the differing disease resistance phenotypes of the two Vitis genotypes.

RING finger proteins comprise a large family and play important roles in regulation of growth and development, hormone signalling, and responses to biotic and abiotic stresses in plants. In this study, the identification and functional characterization of a C4C4-type RING finger protein gene from the Chinese wild grapevine Vitis pseudoreticulata (designated VpRFP1) are reported. VpRFP1 was initially identified as an expressed sequence tag (EST) from a cDNA library constructed from leaves of V. pseudoreticulata inoculated with the grapevine powdery mildew Uncinula necator. Sequence analysis of the deduced VpRFP1 protein based on the full-length cDNA revealed an N-terminal nuclear localization signal (NLS) and a C-terminal C4C4-type RING finger motif with the consensus sequence Cys-X(2)-Cys-X(13)-Cys-X(1)-Cys-X(4)-Cys-X(2)-Cys-X(10)-Cys-X(2)-Cys. Upon inoculation with U. necator, expression of VpRFP1 was rapidly induced to higher levels in mildew-resistant V. pseudoreticulata plants. In contrast, expression of VpRFP1 was down-regulated in mildew-susceptible V. vinifera plants. Western blotting using an antibody raised against VpRFP1 showed that VpRFP1 was also induced to higher levels in V. pseudoreticulata plants at 12-48 hours post-inoculation (hpi). However, there was only slight increase in VpRFP in V. vinifera plants in the same time frame, even though a more significant increase was observed at 96-144 hpi in these plants. Results from transactivation assays in yeast showed that the RING finger motif of VpRFP1 exhibited some activity of transcriptional activation; however, no activity was seen with the full-length VpRFP1. Overexpression of VpRFP1 in Arabidopsis plants was found to enhance resistance to Arabidopsis powdery mildew Golovinomyces cichoracearum, which seemed to be correlated with increased transcript levels of AtPR1 and AtPR2 in the pathogen-infected tissues. In addition, the Arabidopsis transgenic lines showed enhanced resistance to a virulent bacterial pathogen Pseudomonas syringae pv. tomato DC3000. Taken together, the results suggested that VpRFP1 may be a transcriptional activator of defence-related genes in grapevines.

The gene encoding stilbene synthase (STS) plays a central role in many biochemical and physiological actions, and its metabolite resveratrol possesses broad-spectrum resistance to pathogens, as well as diverse pharmacological properties, notably an anticancer effect. Here, we report the expression analysis of the gene encoding STS and its promoter function from a powdery mildew (PM)-resistant Chinese wild Vitis pseudoreticulata, and compare it with two PM-susceptible cultivated grapevines, Vitis vinifera cvs. Carignane and Thompson Seedless. We show an unusual expression pattern of STS in V. pseudoreticulata, which differs markedly from that of the cultivated species. Sequence comparisons reveal that the genomic DNA sequences encoding STS in the three grapevines are highly conserved, but a novel residue mutation within the key motif of STS is solely present in V. pseudoreticulata. Moreover, the STS promoter in V. pseudoreticulata displays a significantly different structure from that found in the two V. vinifera. The three promoter-driven GUS differential expression patterns in transformed tobacco plants induced with Alternaria alternata, methyl jasmonate, and wounding indicated that the structurally different STS promoter of V. pseudoreticulata is responsible for its specific regulatory function. We also demonstrate that the expression of STS genes from their native promoters are functional in transformed tobacco and retain pathogen inducibility. Importantly, the genomic DNA-2 of V. pseudoreticulata under its native promoter shows good induction and the maximum level of resveratrol content. These findings further our understanding of the regulation of STS expression in a resistant grapevine and provide a new pathogen-inducible promoter system for the genetic improvement of plant disease resistance.

Stilbene synthase is a plant-specific polyketide synthase, and plays important roles in diverse metabolic processes. The genomic stilbene synthase gene was cloned from accession "Baihe-35-1" of Chinese wild Vitis pseudoreticulata, and a stilbene synthase of V. pseudoreticulata (VpSTS) transcripts expressed in the grape-powdery mildew interaction were determined by semi-quantitative RT-PCR. To monitor VpSTS expression in plant, the promoter region flanking the 5' VpSTS coding region was isolated from the genomic DNA of Chinese wild V. pseudoreticulata accession Baihe-35-1. Alignment of the VpSTS promoter sequence showed a 56.4% identity to Vitis vinifera. To identify the upstream region of the VpSTS gene required for promoter activity, a series of VpSTS promoter deletion derivatives was constructed. Each deletion construct was analyzed by Agrobacterium-mediated transient transformation in grapevine and tobacco leaves after infection by Uncinula necator and Alternaria alternata. In transiently transformed grapevine leaves, GUS activity was also determined after treatment with salicylic acid (SA) and 4 degrees C cold. Analysis of a series of 5' deletions of the VpSTS promoter in grapevine leaves indicated that the proximal 162 bp from the transcription initiation site was proved to be necessary for establishing both the constitutive and induced pattern of expression.