Expansion of the STS family in grapevine
To date, STS genes have been cloned from several plant species including peanut, sorghum, pine and grapevine . In peanut and pine STS genes are organised in multigenic families composed of 2–5 members, although in the absence of a whole genome sequence for these species an accurate estimate of the number of family members is difficult. Grapevine and sorghum represent the only two species which possess stilbene biosynthetic genes for which the genomes have been completely sequenced. Screening of the sorghum genome sequence revealed the presence of a single, unique STS gene [10; 49]. In this study, a search for STS genes in the most update version of the genome assembly of the grape PN40024 genotype referred to as 12X V1, led to the identification of 48 members, designated VvSTS1 to VvSTS48 and included at least 33 full-length coding genes, 8 pseudogenes and 7 sequences that remain to be resolved (Table 1).
The striking size of the grapevine STS gene family, compared to other stilbene-producing plant species, is not surprising given that analysis of the grape genome sequence has already indicated an expansion in the size of other gene families related to secondary metabolism in grapevine [50, 51]. For example, it is estimated that there may be up to 35 terpene synthase (TPS) genes in grapevine based on the genome assembly of the PN ENTAV 115 genotype . The phenylalanine ammonia-lyase (PAL) gene, which encodes for the key enzyme of the phenylpropanoid pathway, has 13 members in grapevine, whereas only 4–8 genes are present in Arabidopsis, rice, and poplar . More recently, Falginella et al.  reported on the expansion and subfunctionalization of the grapevine flavonoid 3’,5’-hydroxylase (F3’5’H) gene family, responsible for the biosynthesis of precursors of blue anthocyanins. Large-scale (segmental or whole) genome duplication has been recurring during angiosperm evolution and is one of the driving forces in the evolution of genomes and genetic systems [56, 57]. Subsequent gene loss and gene rearrangements further affect gene copy number and fractionate ancestral gene lineages across multiple chromosomes. The expansion of the F3’5’H family, which is composed of 16 members, appears to be the result of multiple events of segmental and tandem duplications that occurred in the Vitaceae lineage, after the separation from other dicots . Of the 16 copies of F3'5'Hs present in the PN40024 genome, 15 reside in a tandem array within a 650 Kb region on chr 6 with an isolated copy on chr 8. Although a detailed study of the VvSTS evolution was not the major aim of this study, the model proposed for the F3’5’H family could also be applied to the VvSTS gene family. The majority of VvSTS members (VvSTS7
VvSTS48) are located in a 500 Kb region on chr16, which shows numerous paralogous zones, not only at the level of coding regions, but also in non-coding regions (data not shown) suggesting multiple events of tandem and segmental duplication. Something similar could have happened for members VvSTS1
VvSTS6 located within an 80 Kb region of chr 10. A recent analysis of the genome architecture of the PN40024 line and its high-identity duplication content by , identified that 85 Mb out of the 487 Mb comprising the grapevine genome is duplicated. Furthermore, they found that chr 16, which contains the majority of VvSTS family members, has the highest percentage (25.08%) of segmental duplication among the assembled non-random chromosomes.
It is noteworthy that duplicate genes involved in secondary metabolism or involved in the response to exogenous stimuli, appear to be more frequently maintained than duplicate genes belonging to other categories [59–61]. Moreover it’s generally assumed that the maintenance of duplicate genes provides a foundation for consolidation and refinement of established functions, particularly in secondary metabolism, by preserving extra copies that guarantee a gene reservoir for adaptive evolution [62–64]. What is particularly interesting in the case of the STS gene family is that the majority of plants don’t even possess a single STS gene, whilst grapevine has evolved such a large STS gene-reservoir. The fact that a single STS gene is present in the monocot Sorghum [10, 49] suggests that the evolution of STS from CHS, the common ancestor of PKSs, occurred before the monocot-dicot separation. Nevertheless, it’s difficult to explain the lack of stilbene-producing genes in the majority of plant species and the conservation and retention of many duplicated STS genes in a restricted group of unrelated species. It could be argued that the production of stilbenes did not confer an evolutionary advantage in those species that lost their biosynthetic genes or, on the other hand, that the majority of species were not able to cope with the production of compounds such as resveratrol, that, although related to benefits at low range of concentrations, are phytotoxic to plant cells at higher concentrations .
Structure/Function of VvSTS proteins
All full-length VvSTS coding genes were found to encode proteins of 392 amino acids in length and contain the conserved CHS/STS active site (Additional file 2). In some cases (Table 1), it was not possible, based on currently available sequence information, to determine with certainty whether the genes encode for a complete or truncated ORF. This includes the genes VvSTS1 and VvSTS4, which are particularly interesting as they possess the conserved CHS/STS active site within the truncated allele (Additional file 6).
In a previous study, which compared the enzymological properties of three STS proteins (PdSTS1, PdSTS2 and PdSTS3) and one CHS protein (PdCHSX) from Japanese red pine, it was observed that PdSTS3, which has a frame-shift mutation leading to a premature stop codon, presents a functional divergence compared to the other full-length STS/CHS proteins . In particular, the PdSTS3 protein showed poor solubility compared to PdSTS2, but despite being truncated, still demonstrated a high potential for pinosylvin production. Furthermore, neither pinosylvin nor pinocembrin inhibited the PdSTS3 activity in vitro, whereas these metabolites effectively inhibited the activity of both PdSTS2 and PdCHSX. Thus, although the truncated ORFs of VvSTS1 and VvSTS4 are shorter than that observed for PdSTS3 (Additional file 6) we cannot rule out the possibility that these truncated alleles may still contribute to stilbene synthesis biosynthesis in grape cells.
Together with the CHSs, STSs represent the most studied enzymes of the plant type III PKS proteins and for this reason this group is often referred as the CHS/STS type III PKS family. The two enzymes compete for the same substrates, share very close amino acid sequences, and possess very similar crystallographic structures . Previous phylogenetic analyses of the STS and CHS families indicated that STSs of Scots pine, peanut and grapevine do not form a separate cluster, but instead cluster with the CHSs proteins from the same or related plants . This observation, reinforced by the observation that only three amino acids exchanges were required within the N-terminal 107 aa of CHS to shift the activity to a STS-type function, suggests that STS may have evolved from CHS several times during the course of evolution . In this study, the three CHS genes identified in the PN40024 genotype, based on clones previously isolated in Cabernet Sauvignon , were included in the phylogenetic analysis performed on the STS family to investigate whether any of the predicted VvSTS proteins cluster more closely to the VvCHS clade. Sequence alignment and phylogenetic tree analyses revealed the existence of 3 VvSTS clades or groups, designated as A, B and C (Figure 2). Group A is composed of genes located on chr10, whereas groups B and C are composed of members located on chr16. However, neighbour-joining analysis indicated that all predicted VvSTS proteins cluster separately from the three VvCHSs, suggesting a conservation of function amongst all VvSTS members. This observation is in agreement with a recent functional study in which 10 different VvSTS genes (including members of each group) were transiently expressed in tobacco and all led to an accumulation of resveratrol and stilbenes, with no evidence for the production of any other products (Parage et al, in preparation).
Temporal and spatial patterns of STS gene expression in grapevine
Using an expression atlas of V. vinifera cv. Corvina (Fasoli et al., in preparation), it was possible to investigate patterns of expression of all of the predicted coding members of the VvSTS and VvCHS gene families in different grapevine tissues and at different developmental stages (Figure 3).
Expression of the majority of VvSTS genes was found to be very low in most non-stressed grapevine tissues analysed. The two exceptions to this were in vitro roots and the berry rachis. The high level of VvSTS expression in in vitro roots is in agreement with the detection of high levels of oligostilbenes in this organ . Moreover, the propagation of this organ in vitro is an artificial procedure that could represent a stress for the plant, leading to the stress-induced transcription of VvSTS genes as observed in Figure 4. The elevated levels of VvSTSs expression in the berry rachis, however, are more surprising. What is particularly striking is the dramatic increase in transcription of group B and C VvSTS genes in the rachis during maturation of the Corvina berries while there is no detectable induction of VvSTS genes in the berries themselves (Figure 3). As discussed in more detail below, VvSTS expression in grape tissues such as leaves and berries appears to be strongly associated with senescence. Thus, the results shown in Figure 3 may reflect the fact that the rachis on Corvina berries undergoes maturation and senescence during berry ripening. This is also supported by the fact that rachis are generally brown, dehydrated and lignified by the time berries reach full maturity.
Interestingly, the microarray results did not show any significant increase in VvSTS expression in Corvina berries during both véraison and ripening. This is in contrast with previously reported studies, which indicate that healthy grape berries synthesise stilbene compounds under natural environmental conditions [14, 68–70]. However, stilbene production during berry ripening has been shown to be genotype dependent with “high” producers such as Pinot noir producing up to 20 ug resveratrol per g berry fresh wt at maturity  compared to a low producer like Corvina which was found to synthesize only 1.5 μg g-1 at harvest . It would appear, therefore, that the microarray technique was not sufficiently sensitive to detect the low level changes in VvSTS expression during ripening of the Corvina berries.
In general, VvSTS expression was low in young grape leaves except for two VvSTS gene members of group A. However, as observed for the rachis, grapevine leaves also show a dramatic increase in VvSTS transcription as they reach maturity and begin to senesce (Figure 3). This was true of gene members of each VvSTS group with individual genes increasing by as much as 2 (VvSTS5-6) to 130 fold (VvSTS9) in senescing leaves compared to young leaves. Leaf senescence is an active and highly regulated process that involves an integrated response of leaf cells to age information and other internal and environmental signals . It is accompanied by a decreased expression of genes related to photosynthesis and protein synthesis and an increase in the expression of hundreds of senescence-associated genes . Many of these genes are associated with the remobilization of nutrients to other developing organs . However, it is not immediately clear as to what role stilbene biosynthesis would play in such a process. The observation that a number of pathogenesis-related (PR) genes are induced during leaf senescence has lead to the suggestion that the senescence program might have incorporated features of the pathogen-defense response to protect the senescing leaf against opportunistic pathogens . Alternatively, the induction of STS genes in senescing leaves may simply be a consequence of changes in the levels of various phytohormones including abscissic acid (ABA), salicylic acid (SA), jasmonates (JA) and ethylene which are known to play an important role in regulating leaf senescence and which have also been shown to be involved in the induction of stilbene biosynthesis. For example, treatment of Cabernet Sauvignon cuttings with Ethephon, an ethylene-releasing compound, resulted in an enhancement of both PAL and STS gene induction leading to an increase in phytoalexins biosynthesis by . Similarly, JA, another key hormone in the senescence response, has been shown to induce high levels of STS transcription in cell cultures of V. vinifera cv. Cabernet Sauvignon . Therefore, it is likely that the increased expression of STS genes during leaf senescence is related to an accumulation of hormones such as ethylene and jasmonates, which are well known to be involved in these particular plants developmental stages.
Stress-induced VvSTS gene expression in grapevine tissues
The majority of previous studies on the accumulation of stilbene compounds and their biosynthetic genes performed on peanut and grapevine tissues, indicated that these genes are highly inducible in response to a number of biotic and abiotic stresses including mechanical damage [24, 25], UV-C light irradiation [26, 27], treatments with chemicals such as aluminium ions, cyclodextrins and ozone [28–30] and infection, including powdery mildew, downy mildew and gray mold [35–40]. Although these studies have made important contributions to our general understanding of the behaviour of stilbene biosynthetic genes, in light of the information we now have regarding the size of the VvSTS gene family and the strong sequence conservation amongst its members, the interpretation of some of this data needs to be reconsidered. To this end, we investigated the transcriptional response of all of the predicted coding members of the VvSTS and VvCHS gene families to three abiotic stress treatments (post-harvest drying, wounding and exposure to UV-C radiation) and one biotic treatment (downy mildew infection) using either grape berries or grape leaves.
The process of post-harvest berry drying (berry withering) involves harvesting of ripe grapes and allowing them to dry over a period of three months in a naturally ventilated room. Its primary purpose is to alter berry quality characteristics and increase the concentration of simple sugars in the production of dessert and fortified wines typical of the Valpolicella region in Italy. However, the drying of harvested grapes in this way results in a loss of over 30% of their weight through evaporation during this post-harvest treatment  and, as such, imposes a significant water stress on the berries. It also results in a dramatic induction of the majority of VvSTS genes (Figure 3) demonstrating that drying berries are still capable of undergoing a significant stress response. Versari et al.  previously observed an increase in the resveratrol content of skins sampled from Corvina berries which had undergone an artificial berry withering treatment. A later study by Zamboni et al.  showed that berry withering was associated with an increase in the transcription of a range of genes involved in hexose metabolism and transport, cell wall composition, and secondary metabolism including a number of VvSTS genes. Our data extends these original observations to show that nearly all of the VvSTS gene members are markedly induced by the dehydration stress. Furthermore the increase in VvSTS expression was detected predominately within skin of the drying grape berry (Additional files 3 and 4). This is in agreement with the immuno-detection of STS proteins performed on berry extracts by Fornara et al.  who showed that STS protein is located mainly in berry exocarp during the véraison phase and is detected only occasionally within the mesocarp.
In order to obtain more control over the stress treatments imposed, the second set of experiments employed young rapidly expanding leaves harvested from glasshouse-grown V. vinifera cv. Pinot noir plants and utilised whole transcriptome mRNA-seq analysis to investigate the expression patterns of all of the predicted coding members of the VvSTS and VvCHS gene families in response to mechanical wounding, UV-C exposure and downy mildew (P. viticola) infection. In agreement with data obtained from the Corvina expression atlas (Figure 3), there appears to be a much higher level of constitutive expression of the group A VvSTS gene family members (VvSTS5 and VvSTS6) than VvSTS gene members belonging to groups B and C raising the question as to the role of group A VvSTS proteins in young leaves. In terms of stress-induced expression, the results indicate that among the three stress treatments examined, UV-C exposure resulted in the highest VvSTS induction, followed by downy mildew infection and wounding (Figure 4), confirming previous observations . The much larger increase in VvSTS induction in response to UV-C exposure may reflect the much larger number of cells within the leaf disc that are subjected to UV-C exposure compared to the wounding and downy mildew treatments which are only affecting a subset of cells. The data also indicates that members within the same VvSTS groups are not only related through protein homology (Figure 2) but also appear to show similar transcriptional responses (Figure 4). Thus, members of group B showed the highest response to all stress treatments, whereas group C members showed a reduced response, while the two group A genes showed little or no transcriptional response to the three stress treatments imposed.
In an attempt to validate the different stress-induced transcriptional responses within the VvSTS gene family, a more detailed analysis of individual members of group A (VvSTS6), group B (VvSTS48) and group C (VvSTS16) was undertaken using qPCR (Figure 5). The qPCR analysis confirmed the significant differences in the quantitative response of these different group members to the different abiotic and biotic stress treatments observed using the mRNA-seq analysis (Figure 4). At the peak of induction, the transcript copy number of VvSTS48 was found to be 15–50 fold higher than the levels of VvSTS16 and VvSTS6. If one assumes there are no major differences in translational efficiency between these different transcripts, this means that the bulk of the observed increase in the biosynthetic capacity of the stilbene pathway under stress conditions would appear to be contributed by the group B VvSTS family members.
Not only did qPCR analysis of stress-induced VvSTS induction in grape leaves confirm the quantitative differences in the transcriptional response of the different group members, it also demonstrated clear differences in the pattern and timing of the response to the different abiotic and biotic stress treatments. The transcriptional response of VvSTS6 and VvSTS16 to both UV-C treatment and downy mildew infection appears to be similar and more rapid than the response of VvSTS48 (Figures 5 & 7) leading one to speculate that the genes within the VvSTS groups A and C may be responding to different transcriptional signals to those in group B. The differential timing in the stress-response of VvSTS genes from the different groups provides an explanation for previous observations that total STS transcription in grape cells, as detected with Northern blot assays or PCR using generic primers, following stress or elicitor treatment, is often observed to be biphasic [27, 76, 77]. Indeed, Wiese et al.  previously suggested that the biphasic nature of the VvSTS response indicated that the VvSTS gene family may be divided into two groups: some expressed early with rapid degradation of the mRNA and others which are expressed later, providing more stable mRNA.
The different patterns of transcriptional response between the VvSTS groups further suggest that these genes may be responding to different signalling pathways. Both the JA and ethylene signalling pathways have previously been shown to have a role in STS transcription [31–33, 74, 78, 79]. Faurie et al.  were able to show that co-treatment of Cabernet sauvignon suspension cells with methyl-jasmonate (MeJ) + Ethephon (ethylene) not only led to both a higher level of total stilbenes and VvSTS transcription compared to treatment with either elicitor alone, but also resulted in a biphasic pattern of transcription which was not observed in cells treated with MeJ or Ethephon only. These observations lend support to the hypothesis that VvSTS genes within the different groups respond to different stress/defense signalling pathways.
Transcriptional subfunctionalization has also been reported between the 15 members of the F3’5’H family , where the development of structural variation in the promoter regions of recently duplicated gene copies has led to differences in member-specific patterns of accumulation across organs, developmental stages and cultivars. Indeed, in the absence of transcriptional subfunctionalization, it would be hard to explain the retention of so many functionally identical VvSTS gene family members.
One question yet to be resolved is the identity of the transcription factor(s) which regulate VvSTS transcription. The expression of phenylpropanoid pathway genes is regulated by the binding of R3R3-type MYB transcription factors (TFs) to highly conserved cis-elements in their promoters [81, 82]. Over the last few years a number of R2R3-type MYB TFs have been identified which regulate flavonol pathway genes in grapevine [83–87], however, to date, no transcription factor responsible for the regulation of VvSTS transcription has been reported. We have undertaken a PTM (Pavlidis Template matching) analysis of the whole mRNA-seq dataset for all 26,346 genes annotated in the 12X V1 PN40024 assembly to identify TF genes that show co-expression with VvSTS under the different stress conditions applied. This has resulted in the identification of two R3R3-MYB candidates which we believe have a role in the transcriptional regulation of the stilbene biosynthetic pathway (Vannozzi et al., in preparation).
Differential regulation of VvSTS and VvCHS genes in grapevine during development and in response to stress
Although there appears to have been little divergence in sequence since the evolution of STS from CHS, there has been sufficient mutation to lead to changes in the products synthesised. These products clearly fulfill very different roles in plant growth and development. Chalcone synthase catalyses the first committed step of the flavonoid biosynthetic pathway, which leads to the synthesis of anthocyanins, tannins and flavonols. Stilbene synthase, on the other hand, appears to function primarily as a stress-response protein, and has been implicated to have a role in defence against pathogens including powdery mildew, downy mildew and Botrytis cinerea[88, 89]. As these two proteins represent branch points in the same pathway, the diversion of carbon skeletons into either secondary metabolism via CHS or stilbenic defence compounds via STS would be expected to be under tight control.
Evidence for the existence of crosstalk between these two pathways in grapevine cells is clearly evident from the analysis of gene expression data in Corvina tissues at various developmental stages (Figure 3). Tissues in which VvSTS expression levels are generally low i.e. stem, bud, young leaves, rachis at fruit set and developing berries are characterised by high constitutive expression of at least one of the three different VvCHS genes (Figure 3). Conversely, expression of all three VvCHS genes is suppressed in tissues in which VvSTS transcription is strongly induced i.e. roots, senescing leaves, maturing rachi and berries undergoing withering treatment. A similar pattern of inverse expression patterns between the members of the VvSTS and VvCHS gene families is also evident in grape leaves exposed to UV-C or inoculated with downy mildew (Figure 7). While both stress treatments resulted in dramatic increase in VvSTS transcription, the expression of all three VvCHS genes was strongly suppressed relative to the untreated leaf discs.
While a number of previous studies have shown that the expression of CHS can be induced by UV-A and UV-B light and pathogen infection (reviewed in ), this is the first study, to our knowledge, that has investigated the effect of UV-C light on CHS transcription. The other major difference between our study and previous investigations is that our research has been carried out on grapevine which has a highly evolved stilbene biosynthetic pathway which is strongly induced by both UV-C and downy mildew infection. As such, one might expect there to be an enhanced level of cross-talk between the flavonoid and stilbene biosynthetic pathways in grapevine.
It has been well documented that the triggering of defence pathways in plants causes a suppression of genes associated with photosynthesis and basic metabolism leading to the suggestion that there is a diversion of metabolic resources from general metabolism to defense-related metabolism, during pathogen attack. This is particularly true for the flavonoid pathway, which has been shown to be suppressed in a number of different plant species following exposure to fungal pathogens or fungal elicitors [91–94]. Recently Schenke et al.  demonstrated that the induction of biosynthetic pathways, in Arabidopsis, responsible for the synthesis of lignin and the phytoalexin scopoletin, by the bacterial elicitor flg22, was associated with a strong suppression of flavonol biosynthesis genes including CHS. They concluded that as flavonols, lignin and scopoletin are all derived from phenylalanine, that under stress conditions, the plant appears to refocuses it’s metabolism on the production of scopoletin and lignin, at the expense of flavonol. We propose that a similar antagonistic relationship exists between flavonol biosynthesis and stilbene biosynthesis in grapevine and that during periods of abiotic or biotic stress, stilbene biosynthesis takes precedence over flavonol biosynthesis.
How might this antagonistic relationship be regulated? In Arabidopsis, it appears that the antagonistic relationship between the flavonol and stress/defense biosynthetic pathways involves the action of at least two opposing MYB TFs: MYB12 (positive regulator) and MYB4 (negative regulator), which compete for binding to MYB-recognition elements within the promoters of the flavonol biosynthetic pathway genes. We are currently investigating whether R2R3-MYB candidates in grapevine might also repress the transcription of the VvCHS genes during the induction of the stilbene biosynthesis pathway.