Expression Of Stilbene Synthase Genes Research Articles

Identification and Expression Analysis of Stilbene Synthase Genes in Arachis hypogaea in Response to Methyl Jasmonate and Salicylic Acid Induction

Stilbene synthase is an important enzyme of the phenylpropanoid pathway, regulating the production of several biologically active stilbenoids. These compounds have antioxidant, anti-inflammatory, and anti-cancer properties. However, the detailed characterization of stilbene synthase genes in Arachis hypogaea has not yet been performed. In this study, the comprehensive characterization of stilbene synthase genes in A. hypogaea was conducted, commencing with identification, phylogenetic analysis, and study of their expression in response to exogenous hormonal treatment. We identified and isolated five AhSTSs genes and recorded their expression pattern in peanut (BARD-479) in response to methyl jasmonate (MeJA) and salicylic acid (SA) treatment. The presence of Chal_sti_synt, ACP_syn_III, and FAE1_CUT1_rppA domains in all AhSTSs indicated their role in the biosynthesis of stilbene and lipid metabolism. Cis-regulatory element analysis indicated their role in light responsiveness, defense responses, regulation of seed development, plant growth, and development. Despite close structural and functional similarities, expression and correlational analysis suggested that these genes may have a specific role in peanut, as individual AhSTS exhibited differential expression upon hormonal treatment in a genotype dependent manner. Further studies on functional characterization involving the transcriptional regulation of AhSTSs can clearly explain the differential expression of stilbene synthase genes to hormonal treatment.

Study of defense-related gene expression in grapevine infested by Colomerus vitis (Acari: Eriophyidae)

Real-time quantitative polymerase chain reaction was used to study the expression of some marker genes involved in the interaction between grape (Vitis vinifera L.) and the erineum mite Colomerus vitis Pagenstecher (Acari: Eriophyidae). Potted vines of cultivars Atabaki (resistant to C. vitis), Ghalati (susceptible to C. vitis) and Muscat Gordo (moderately resistant to C. vitis) were infested at the six-leaf stage. The expression of protease inhibitor (PIN), beta-1,3-glucanase (GLU), polygalacturonase inhibitor (PGIP), Vitis vinifera proline-rich protein 1 (PRP1), stilbene synthase (STS), and lipoxygenase (LOX) genes was assessed on young leaves collected 96, 120 and 144h after mite infestation (hami). As a control, non-infested leaves collected 24h before mite infestations were used. Differences were detected in expression of the selected genes during the C. vitis-grapevine interaction. The resistant cultivar Atabaki increased the expression of LOX, STS, GLU, PGIP and PRP1 genes during the first 120 hami. On the contrary, in the susceptible Ghalati, all selected genes showed an expression level similar or lower than non-infested leaves. Muscat Gordo increased the expression of all selected genes in comparison with non-infested leaves, but it was lower than in Atabaki. Significant transcript accumulation of PIN gene was detected for Muscat Gordo whereas it was slightly up-regulated in Ghalati and Atabaki. LOX, STS, PIN, GLU, PGIP and PRP1 genes were clearly expressed in response to C. vitis infestation. We therefore infer that expression of PGIP, PIN and PRP1 genes could represent a defense strategy against C. vitis infestations in grapevine leaves.

Stilbene content and expression of stilbene synthase genes in cell cultures of Vitis amurensis treated with cinnamic and caffeic acids

It has previously been shown that exogenous application of p-coumaric acid (CA), a precursor of phenolic compounds, improved stilbene production in cell cultures of Vitis amurensis. This study examines the effect of cinnamic (Cin) and caffeic (Caf) acids, which are also phenolic precursors, on stilbene biosynthesis in the cell cultures. Five stilbenes, t-resveratrol diglucoside, t-piceid (t-resveratrol glucoside), t-resveratrol, t-ε-viniferin, and t-δ-viniferin, were found in the treated and untreated cells. Cin acid increased the total stilbene production in the grape cell cultures 2.3-3.5 times in comparison with that in the untreated cells. Caf acid increased the total stilbene production by 1.8- to 1.9-fold, but this increase was not considerably different from stilbene production in the untreated cells. Cin acid affected the total stilbene production via a marked increase in the content of t-resveratrol diglucoside (up to 2.2 times), t-piceid (up to three times), t-resveratrol (up to 5.1 times), t-ε-viniferin (up to eight times), and t-δ-viniferin (up to 9.2 times). Transcription levels of VaSTS5, 6, 7, 8, and 10 genes considerably increased under 0.1, 0.25, and 0.5 mM Cin acid. These results indicate that Cin acid increased stilbene production in V.amurensis calli via a selective enhancement of STS gene expression.

Salicylic acid induces alterations in the methylation pattern of the VaSTS1, VaSTS2, and VaSTS10 genes in Vitis amurensis Rupr. cell cultures.

Salicylic acid (SA) treatment selectively reduced the cytosine DNA methylation of stilbene synthase ( STS ) genes and stimulated resveratrol production in cell cultures of Vitis amurensis. The effect of salicylic acid (SA) on plant growth, flowering time, and fruit number is known to correlate with the level of DNA methylation, while the potential correlation between SA-induced changes in DNA methylation and biosynthesis of secondary metabolites has not been studied. Trans-resveratrol, a naturally occurring plant phenol, has been reported to exhibit a wide range of valuable biological and pharmacological properties. In this study, cell cultures of Vitis amurensis capable of producing t-resveratrol were used as a model system to study whether the SA-induced increase in t-resveratrol production is associated with changes in DNA methylation of stilbene synthase (STS) genes. T-resveratrol is synthesized via the phenylpropanoid pathway, in which STS genes are the key enzymes. Treatment of V. amurensis callus cultures with SA significantly increased t-resveratrol production and the expression of certain STS genes (e.g., VaSTS2 and VaSTS10). A marked decrease in the methylation of the VaSTS2 and VaSTS10 genes in response to SA was demonstrated using bisulfite sequencing, while no considerable changes were detected in the methylation of VaSTS1, a constitutively and highly expressed STS gene. The obtained results show that SA treatment selectively reduced cytosine methylation of VaSTS genes. The data suggest that selective DNA demethylation of particular STS genes could be necessary for the activation of t-resveratrol biosynthesis in response to SA. This finding provides an insight into the mechanism of SA action and biosynthesis of secondary metabolites in plant cells.

Myb14, a direct activator of STS, is associated with resveratrol content variation in berry skin in two grape cultivars.

High and low resveratrol (Res) contents in two cultivars are correlated with the expression abundance of Myb14 , which could directly activate transcriptional expression of stilbene synthase gene ( STS ). Resveratrol (3,5,4'-trihydroxystilbene) is one of the natural polyphenols produced by secondary metabolism in some plants. Stilbene synthase (STS) is the key enzyme for the final step of precursor formation of resveratrol (Res) in grapevines. In this study, we found that Res contents in ripe berry skin were completely different in two grape cultivars, namely, 'Z168' (Vitis monticola × Vitis riparia) with high-Res and 'Jingzaojing' (Vitis vinifera) with low-Res. Moreover, the level of expression of STS gene was higher in the ripe berry skin of 'Z168' than in that of 'Jingzaojing'. To further investigate the underlying mechanisms, we conducted a co-expression analysis through transcriptomic data. We confirmed that Myb14, an R2R3 Myb transcription factor, is the direct regulator of STS by binding to Box-L5 motif. Moreover, the expression pattern of Myb14 is associated with the variation of Res content. To test this prediction, we conducted a number of experiments in vivo and in vitro. The expression patterns of Myb14 and STS in grapevine leaves were identical under a series of stimulus. Myb14 showed higher expression in the ripe berry skin of 'Z168' than in that of 'Jingzaojing'. Yeast one-hybrid assay indicated that grapevine Myb14 could interact with the promoter of STS in vitro, and the transient overexpression of Myb14 promoted the expression of STS. Furthermore, co-expressing 35S::Myb14 in transgenic Arabidopsis could activate GUS expression promoted by STS promoter. Thus, Myb14 is the direct activator of STS, and its expression pattern is associated with Res content variation in grapes.

VaCPK20 gene overexpression significantly increased resveratrol content and expression of stilbene synthase genes in cell cultures of Vitis amurensis Rupr

Resveratrol, a naturally occurring plant phenol, has been reported to exhibit a wide range of valuable biological and pharmacological properties. In the present investigation, we show that transformation of a Vitis amurensis Rupr. cell suspension with the gene VaCPK20 for a calcium-dependent protein kinase (CDPK) under the control of double CaMV 35S promoter increased resveratrol production in five independently transformed cell lines in 9-68 times compared with control cells. The VaCPK20-transformed calli were capable of producing 0.04-0.42 % dry wt. of resveratrol, while the control calli produced up to 0.008 % dry wt. of resveratrol Also, we characterized expression of stilbene synthase (STS) genes in the five VaCPK20-transgenic cell lines of V. amurensis. In all VaCPK20-transgenic cell lines, expression of VaSTS7 increased; while expression of VaSTS1 decreased. We suggest that transformation of V. amurensis calli with the VaCPK20 gene induced resveratrol accumulation via enhancement of expression of the VaSTS7 gene involved in resveratrol biosynthesis. The obtained data first demonstrate that overexpression of a CDPK gene resulted in increased accumulation of a stilbenoid phytoalexine in transgenic plant cells. We propose that the VaCPK20 gene could play an important role in the regulation of resveratrol biosynthesis in grape cells.

Effects of the Calmodulin Antagonist W7 on Resveratrol Biosynthesis in Vitis amurensis Rupr.

The calmodulin antagonist N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7) binds to calmodulzin and inhibits Ca2+/calmodulin-regulated enzyme activities. In plant cells, W7 inhibits the activity of calcium-dependent protein kinases (CDPKs)—the major calcium sensors in plants. In the present study, we examined the effect of W7 on increased resveratrol biosynthesis and expression of CDPK and stilbene synthase (STS) genes in a cell culture of Vitis amurensis Rupr. We used coumaric acid (CA), salicylic acid (SA), and phenylalanine (Phe) to increase the content of resveratrol in V. amurensis calli, since its content is low under standard conditions. W7 significantly decreased resveratrol production and expression of STS genes in CA-, SA-, and Phe-treated grape cells. Also, treatment of the V. amurensis calli with SA, Phe, or CA considerably increased expression of VaCDPK1a (with SA, Phe), VaCDPK1L (with SA, Phe), VaCDPK2a (with Phe) genes, and decreased expression of VaCDPK3a (with CA). Addition of W7 to CA-, SA-, and Phe-treated grape cells reversed this effect, resulting in increased VaCDPK3a expression and decreased VaCDPK1a, VaCDPK1L, and VaCDPK2a expression. The results obtained suggest that CDPK activities might play an important role in resveratrol biosynthesis.

Transcriptional expression of Stilbene synthase genes are regulated developmentally and differentially in response to powdery mildew in Norton and Cabernet Sauvignon grapevine

Stilbenic compounds are natural phytoalexins that have antimicrobial activities in plant defense against pathogens. Stilbene synthase (STS) is the key enzyme that catalyzes the biosynthesis of stilbenic compounds. Grapevine genome contains a family of preliminarily annotated 35 STS genes, the regulation of each STS gene needs to be studied to define their roles. In this study, we selected eight STS genes, STS8, STS27/31, STS16/22, STS13/17/23, and applied quantitative polymerase chain reaction (qPCR) to characterize their transcriptional expression profiles in leaf tissues upon infection by the powdery mildew fungus (PM), Erysiphe necator (Schw.) Burr. Their transcripts were also compared in young and old leaves as well as in the berry skin at five developmental stages in Vitis vinifera ‘Cabernet Sauvignon’ and Vitis aestivalis ‘Norton’. The results showed that transcripts of selected STS genes increased significantly in Cabernet Sauvignon leaves at 24 and 48h post inoculation with PM spores and remained unchanged in Norton leaves in response to the PM infection. Transcripts of STS8, STS27/31 and STS13/17/23 were more abundant in the old leaves of Norton than in Cabernet Sauvignon. STS genes showed lower expression levels in young leaves than in old leaves. Transcript levels of the eight STS genes increased drastically in the berry skin of Cabernet Sauvignon and Norton post véraison. In addition, the content of trans-resveratrol in the berry skin rapidly increased post véraison and reached the highest level at harvest. These assays demonstrated that individual STS genes are regulated differentially in response to PM infection and during development in the two grape varieties. The present study yields basic knowledge for further investigation of the regulation and function of each STS gene in grapevine and provides experimental evidences for the functional annotation of the STS gene family in the grapevine genome.

Influence of calcium influx induced by the calcium ionophore, A23187, on resveratrol content and the expression of CDPK and STS genes in the cell cultures of Vitis amurensis

The present study examines the effect of cal- cium influx induced by the calcium ionophore (CI) on the biosynthesis of resveratrol and the expression of stilbene synthase (STS) and calcium-dependent protein kinase (CDPK) genes in cell cultures of Vitis amurensis, which have different levels of resveratrol production. The present study utilized the control cell culture V2 of V. amurensis, which contains no more than 0.02 % dry weight (DW) of resveratrol, in addition to rolB transgenic cell cultures VB1 and VB2, which have increased resveratrol contents (0.1-0.8 % DW). Treatment with the CI at a 1 lM con- centration significantly increased STS gene expression (6 of 10 analyzed STS genes) and resveratrol production in the control V2 cell culture by fourfold; however, use of the CI at 10 lM significantly decreased resveratrol production by 2-4 fold in all cell cultures tested. In the control V2 grape cell culture, treatment with the CI increased expression of all of the CDPK genes except VaCDPK1a and VaCDPK3a. In the rolB transgenic VB2 grape cell culture treated with the CI, we detected alterations in expression of several CDPK genes, but these changes in gene expression were not significant. Our results indicated that treatment with 1 lM of the CI increased resveratrol content and production in control grape cells by selectively increasing the expres- sion of STS genes. Conversely, the CI treatment did not significantly increase resveratrol content and production, or the expression of CDPK or STS genes in the rolB transgenic cells. Likely, untreated VB2 cells have increased concen- trations of cytoplasmic calcium, and therefore, treatment with the CI did not significantly change CDPK expression. These results suggest that the rolB gene has an important role in the regulation of calcium-dependent transduction pathways in transformed cells.

Resveratrol Content and Expression of Phenylalanine Ammonia-lyase and Stilbene Synthase Genes in Cell Cultures of Vitis amurensis Treated with Coumaric Acid

The present study examines the effect of p-coumaric acid (CA), a precursor of stilbenes and isoflavonoids, on biosynthesis of resveratrol in cell cultures of Vitis amurensis. Earlier, we transformed V. amurensis with the rolB gene of Agrobacterium rhizogenes and showed increased level of resveratrol production in the rolB transgenic cell culture. We used control and the rolB-transgenic cell culture of V. amurensis as a model system in this study. CA was capable of increasing resveratrol production in the control and the rolB-transgenic cell cultures in 10.3 and 1.5 times, respectively. The CA-treated control and rolB transgenic calli produced up to 0.06% and 1.1% DW of resveratrol. Using quantitative real-time RT-PCR, we characterized the expression of phenylalanine ammonia-lyase (PAL) and stilbene synthase (STS) genes in the CA-treated control and rolB transgenic cell cultures. The expression of PAL genes remained essentially unchanged under 0.1 mM of CA, while expression of VaPAL1, VaPAL2, VaPAL3, and VaPAL5 genes was considerably decreased under 0.5 and 2 mM CA compared with the untreated cells. In the CA-treated calli, expression of VaSTS2 and VaSTS3 was increased, while expression of VaSTS5, VaSTS8, VaSTS9, and VaSTS10 was significantly decreased. These results indicate that CA increased resveratrol accumulation in V. amurensis calli via selective enhancement of expression of individual STS genes.

Expression pattern, genomic structure, and promoter analysis of the gene encoding stilbene synthase from Chinese wild Vitis pseudoreticulata

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.

Resveratrol content and expression patterns of stilbene synthase genes in Vitis amurensis cells treated with 5-azacytidine

DNA methylation is known to be involved in the regulation of plant development and defense mechanisms. However, there is a general lack of data on the role of methylation in plant secondary metabolism. We have investigated the effect of a cytidine analog, 5-azacytidine (azaC), which is known to block DNA methylation, on resveratrol biosynthesis and stilbene synthase (STS) gene expression in Vitis amurensis cultured cells. Resveratrol is a naturally occurring polyphenol that has been reported to exhibit a wide range of important biological and pharmacological properties. We previously obtained a control cell line of V. amurensis (VV) as well as a rolB-transgenic cell line of V. amurensis (VB2) that has a higher level of resveratrol accumulation. In our experimental setup, the azaC-treated VV and VB2 calli produced 0.092% and 0.455% dry weight (DW) resveratrol, respectively. We found that treatment with 200 μM of azaC resulted in 1.9- and 2.0-fold increases in resveratrol production in VV and VB2 calli, respectively. A quantitative real-time PCR assay for STS gene expression in the azaC-treated VV and VB2 cells revealed that there were statistically increased expression levels of VaSTS10 in VV calli and of VaSTS5, VaSTS6, and VaSTS10 in VB2 calli. These results demonstrate that azaC is able to increase resveratrol production in V. amurensis calli through a mechanism that involves the induction of STS gene expression.

Resveratrol content and expression of phenylalanine ammonia-lyase and stilbene synthase genes in rolC transgenic cell cultures of Vitis amurensis

Resveratrol, a naturally occurring polyphenol, has been reported to exhibit a wide range of valuable biological and pharmacological properties. In the present investigation, we show that transformation of Vitis amurensis Rupr. with the oncogene rolC of Agrobacterium rhizogenes increased resveratrol production in the two transformed callus cultures 3.7 and 11.9 times. The rolC-transformed calli were capable of producing 0.099% and 0.144% dry weight of resveratrol. We characterized phenylalanine ammonia-lyase (PAL) and stilbene synthase (STS) gene expression in the two rolC transgenic callus cultures of V. amurensis. In the rolC transgenic culture with higher resveratrol content, expression of VaPAL3, VaSTS3, VaSTS4, VaSTS5, VaSTS6, VaSTS8, VaSTS9, and VaSTS10 was increased; while in the rolC culture with lower resveratrol content, expression of VaPAL3 and VaSTS9 was increased. We suggest that transformation of V. amurensis calli with the rolС gene induced resveratrol accumulation via selective enhancement of expression of individual PAL and STS genes involved in resveratrol biosynthesis. We compared the data on PAL and STS gene expression in rolC transgenic calli with the previously obtained results for rolB transgenic calli of V. amurensis. We propose that the transformation of V. amurensis with the rolC and rolB genes of A. rhizogenes increased resveratrol accumulation through different regulatory pathways.

The effect of salicylic acid on phenylalanine ammonia-lyase and stilbene synthase gene expression in Vitis amurensis Cell Culture

Resveratrol is a phytoalexin with antibacterial, antiviral and cancer-preventing effects. The objective of the study was to identify PAL and STS genes of Vitis amurensis Rupr. encoding the phenylalanine ammonia-lyases (PAL) and stilbene synthases (STS), the key enzymes involved in the resveratrol biosynthesis. A V. amurensis Rupr. cell culture characterized by low resveratrol level was chosen as a model object. Salicylic acid (SA), a known secondary metabolism inducing agent, was used for enhancement of resveratrol production in this culture. PAL and STS gene expression was analyzed using the reverse transcription PCR and real-time PCR techniques. SA was originally found to specifically enhance the expression of VaPAL3, VaSTS2, VaSTS3, VaSTS4, VaSTS5, VaSTS6, and VaSTS8 of multigene families VaPAL and VaSTS. The results obtained were compared with the earlier published data on PAL and STS gene expression in the rolB transformed V. amurensis cell cultures characterized by high levels of resveratrol. The effects of SA treatment and the rolB transformation on VaPAL and VaSTS gene expression were found to be considerably different.

Phenylalanine ammonia-lyase and stilbene synthase gene expression in rolB transgenic cell cultures of Vitis amurensis

Transformation of Vitis amurensis callus culture by the plant oncogene rolB of Agrobacterium rhizogenes results in high (up to 3.15% dry wt.) levels of resveratrol in the transformed culture. The present study deals with the effect of rolB on phenylalanine ammonia-lyase (PAL) and stilbene synthase (STS) gene expression in two rolB transgenic V. amurensis callus cultures with different levels of rolB expression and resveratrol production. The total expression of PALs and STSs in rolB transgenic cultures increased 1.3-3.8 times compared with the control culture. In the rolB transgenic cultures expression of VaPAL1, VaPAL2, and six STS genes was increased, while expression of VaPAL3 and VaSTS6 was not significantly changed. These results suggest that rolB increases resveratrol production via selective enhancement of expression of individual genes from PAL and STS gene families. We propose that increase of VaPAL3, VaSTS1, and VaSTS6 transcript levels is not strongly required for high resveratrol production by rolB transgenic cell cultures.

Expression of stilbene synthase gene in transgenic tomato using salicylic acid-inducible Cre/loxP recombination system with self-excision of selectable marker

A plant transformation vector, pCLKSCLA25 (EU327498), was developed to contain eight cloning sites and the inducible self-excision system which provided an effective approach to eliminate the selectable marker gene(s) from transgenic plants. Upon induction by salicylic acid, the cre gene produced a recombinase that eliminated sequences encoding the selectable marker neomycin phosphotransferase and cre itself. The excision efficiency was 41% in transgenic tomato regenarants. The stilbene synthase gene (vst1) from Vitis vinifera L. was cloned into pCLKSCLA25. The expression of vst1 gene contributed to the accumulation of trans-reveratrol from 3.4 to 8.7 mug/g fresh wt in different marker-free transgenic tomato lines.

Effect of high CO 2 pretreatment on quality, fungal decay and molecular regulation of stilbene phytoalexin biosynthesis in stored table grapes

Table grapes ( Vitis vinifera) cv. Cardinal stored at low temperature were analysed to determine the effect of pretreatment with 20 kPa O 2 + 20 kPa CO 2 + 60 kPa N 2 for 3 days on quality and control of decay. The pattern of stilbene synthase (STS) gene expression and trans-resveratrol levels were also analyzed in grapes during low temperature storage at 0 °C and further shelf-life at 20 °C for 2 days. Our results showed that high CO 2 pretreatment was effective for improving appearance of the bunches and maintaining the quality of the berries. In CO 2-treated bunches the browning and withering index, the decline in relative water content and the weight loss were also lower than in non-treated ones. The levels of STS mRNA and the accumulation of trans-resveratrol in CO 2-treated grapes were much lower than in the non-treated grapes during low temperature storage. Moreover, the pattern of STS gene expression and trans-resveratrol content in CO 2-treated grapes was consistent with the reduction of natural total decay produced by this pretreatment. This effective non-stressing treatment avoids the induction of trans-resveratrol during low temperature storage until its synthesis is enhanced during shelf-life at 20 °C.

Molecular Basis of Ergosterol-Induced Protection of Grape Against Botrytis cinerea: Induction of Type I LTP Promoter Activity, WRKY, and Stilbene Synthase Gene Expression

Type I lipid transfer proteins (LTPs) are basic, 9-kDa cystein-rich proteins believed to be involved in plant defense mechanisms. A 2,100-bp fragment containing the coding region of Vitis vinifera lipid transfer protein 1 (VvLTP1) and 1,420-bp of its promoter region was isolated by screening a grape genomic library. In silico analysis revealed several putative, defense-related, cis-regulatory elements such as W- and MYB-boxes, involved in the binding of WRKY and MYB transcription factors, respectively. The 5'-truncated versions of the VvLTP1 promoter were generated, cloned in front of the beta-glucuronidase (GUS) reporter gene, and introduced in tobacco plants and grapevine cell suspensions using Agrobacterium spp. Single MYB- and the W-boxes identified on the 0.250-kbp fragment were sufficient to induce GUS activity in transgenic tobacco plants after transient expression of MYB and WRKY. Ergosterol, a nonspecific fungal elicitor, induced GUS activity in transgenic grapevine cell suspensions transformed with the 1,420- and 750-bp promoter containing a palindromic arrangement of two W-boxes but not the 650- or 250-bp fragment, where only one W-box was present. Moreover, ergosterol triggered WRKY, VvLTP1, and stilbene synthase gene expression in grape plantlets and enhanced protection against Botrytis cinerea. The molecular basis of ergosterol-induced protection is discussed.

Simultaneous expression of stilbene synthase genes in Japanese red pine (Pinus densiflora) seedlings

We analyzed sequences of the stilbene synthase (STS) cDNA inPinus densiflora. Three novel STS cDNA clones (pdstsl, pdsts2, pdsts3) that carry full coding sequences were isolated from a cDNA library constructed from the roots. The homologies in their coding regions were about 95%, and they have a conserved STS motif. Their phylogenetic relation was also discussed. The 3′ end of the STS cDNA fragments was amplified by the polymerase chain reaction and subcloned. The 3/t' untranslated region (3′UTR) of the fragments highlighted the structure diversity. Twelve STS cDNA clones were categorized into seven distinct subclasses according to 3′UTR sequences. We discussed the stability of the transcripts and their gene organization. The simultaneous expression of the STS members is one of the mechanisms for adaptation that pine trees have developed over time.

The molecular regulation of stilbene phytoalexin biosynthesis in Vitis vinifera during grape berry development

The molecular regulation of stilbene phytoalexin biosynthesis in developing Vitis vinifera L. grape berries was investigated using a UV induction system. Berries were collected at 1, 5, 10 and 16 weeks post-flowering from the cultivars Shiraz, Semillon, Cabernet Sauvignon and Chardonnay and the skins analysed for resveratrol production following irradiation with UV-C light. The rate and maximal level of resveratrol accumulation increased markedly in berries sampled from 1–5 weeks post-flowering and then dramatically declined in maturing berries sampled from 10–16 weeks post-flowering in all cultivars. In berries sampled at 1 and 5 weeks post-flowering, maximal levels of resveratrol accumulation were recorded at incubation periods of 24 and 48 h respectively whereas maximal resveratrol levels were not recorded in week 16 berry skins until 72 h after UV-treatment. Gene expression analysis indicated that stilbene synthase (STS) mRNA accumulated within 4–8 h of UV treatment in berries sampled at 1 and 5 weeks post-flowering, but did not increase in week 16 berries until 24–48 h following UV-irradiation. Furthermore, the overall level of STS gene expression declined in berries sampled 10–16 weeks post-flowering. The results demonstrate that inducible stilbene accumulation in ripening grape berries is highly regulated at the level of STS gene transcription. This decline in inducible STS gene expression may be a major factor contributing to the increased susceptibility of ripening grape berries to Botrytis cinerea infection.