During 2011, two clusters of V. vinifera cv. Pinot Noir ENTAV115 were collected almost daily between 9 am and 10 am at 6–7 weeks post-flowering (wpf), corresponding to EL-33 and EL-34, at the FEM study site (San Michele all′Adige-TN Italy). Each cluster represented one biological replicate. Each cluster was divided in smaller bunches and then half of them, at random, were pressed for must analysis by means of Fourier transform infrared spectroscopy (FTIR) using the instrument WineScanTM Type 77310 (Foss Electric, Denmark) while the remaining small bunches were rapidly frozen. Frozen berries were peeled with a scalpel and the skins were ground to obtain a fine powder. Skin anthocyanin concentration was measured after methanol extraction (1 g berry skin powder in 10 mL methanol) according to the double pH differential method (Cheng and Breen, 1991). Lipid extraction and analysis were performed as described in Pilati et al. (2014). ABA detection and quantification was carried by LC-UV-MS technique. In particular we used as stationary phase a column Kinetex C18 (5 μm, 150 mm × 4.6 mm, flow 0.8 mL/min) and as mobile phase an A:B gradient elution (A = H₂O + 0.5% formic acid; B = MeOH + 0.5% formic Acid) with B changing from 40 to 55% in 10 min. 10 μL of pure ABA solution or raw skin berry extracts (solution prepared in MeOH/CHCl₃ 9:1) were injected in every chromatographic run. The retention time of ABA was 8.9 min. ABA was detected both by ion-positive mode ESI-MS analysis (characteristic ions at m/z 287, 265, 247, 229, 201, 187, and 173) and by photodiode array detector. ABA was quantified by interpolation on a working curve (absorbance vs. concentration) built on three ABA solutions at concentration 1.9, 19, and 190 ng/μL and the corresponding absorbance measured at fixed wavelength of 262 nm (R² = 0.998). During 2013, three clusters of V. vinifera cv. Pinot Noir ENTAV115 were collected between 9 and 10 am at 7 wpf, corresponding to EL-33, at the FEM study site (San Michele all′Adige-TN Italy). Each cluster represented one biological replicate. Berries were detached from each cluster by cutting the petiole, at 2–3 mm distance from the berry. Berries were washed in 0.1% Plant Preservative Mixture (PPM, Duchefa) water solution for 30 min. 20 berries per cluster were put in a 100 mL water solution containing 0.2 mM ABA (Sigma) and 0.5% methanol (used to dissolve ABA) or 0.5% methanol as control. After 20 h mild shaking, the berries were extensively rinsed and then eight berries were frozen in liquid nitrogen. The remaining berries, both treated and not treated with ABA, were plated on solid medium in Petri dishes (0.9% agar, 10% sucrose, 0.1% PPM) for 24 h, and then eight were frozen as described above. ABA and sucrose concentrations were taken from Gambetta et al. (2010).

Total RNA was extracted from the skin powder samples using Spectrum Total Plant RNA kit (Sigma) and was quantified using a Nanodrop 8000 (Thermo Scientific). The integrity was checked using Bioanalyzer 2100 (Agilent) and RNA Nano Chips. For Real-time PCR analyses, first strand cDNA was synthesized from 2 μg RNA using the SuperScript VILO cDNA Synthesis Kit (Invitrogen) according to the manufacturer’s instructions. The cDNAs were mixed with Fast SYBR Green Master Mix (Applied Biosystems) and amplified on a ViiA 7 Real Time PCR System (Applied Biosystems) using an initial heating step at 95°C for 20 min, followed by 40 cycles of 95°C for 1 min and 60°C for 20 s, using the primers reported in Supplementary Table S1. Raw fluorescence data were extracted using Viia 7 Software v1.0. Ct and reaction efficiency were calculated using LinRegPCR (Ruijter et al., 2009). Relative expression was calculated according to (Pfaffl, 2001) by centering expression values for each gene on the mean value. Three reference genes (Actin, SAND and GAPDH) were used for normalization with geNorm (Vandesompele et al., 2002). For RNA sequencing, 2 μg RNA for each sample were shipped in dry ice to Genomicx4life (Salerno, Italy).

Sequencing has been performed on Illumina Hi-seq 1500, producing 100 nt directional single-end reads. Raw reads were pre-processed for quality using fastqc v.0.11.2¹ and cleaned with cutadapt v.1.12 (Martin, 2011). The resulting reads were aligned to the grape (12x v1²) genome using the Subread aligner (Liao et al., 2013). Raw read counts were extracted from the Subread alignments using the featureCount read summarization program (Liao et al., 2014). The summarized read count data was used to identify DEGs among various treatments by using the voom method (Law et al., 2014), which estimates the mean-variance relationship of the log-counts, generating a precision weight for each observation that is fed into the limma empirical Bayes analysis pipeline (Smyth, 2004). A Volcano Plot generated using the ShinyVolcanoPlot Web App³ was used to select sets of DEGs for each comparison based on both p-value and expression fold change. In the present work, a maximum p-value of 0.05 and a minimum absolute fold change of 2 were imposed. Raw sequences were deposited at the Sequence Read Archive of the National Center for Biotechnology⁴ under BioProject accession number PRJNA369777.

Vitisnet gene annotation has been used (Grimplet et al., 2009), except for the transcription factors gene families already characterized in grapevine, for which the specific published annotation has been used (Licausi et al., 2010; Wang N. et al., 2013; Liu et al., 2014; Wang et al., 2014; Wong et al., 2016). Functional class enrichment was performed on GO (Gene Ontology) terms (annotation⁵) using the TopGO Bioconductor package (Alexa and Rahnenfuhrer, 2016) and on Vitisnet gene annotation (Grimplet et al., 2009) taking advantage of the VESPUCCI grape compendium (Moretto et al., 2016). Promoter analysis has been performed on the 1-kb promoters of the genes modulated at 20 and 44 h using DREME software (Bailey, 2011). Statistically enriched motifs were annotated using the “DAP motifs” database for Arabidopsis (O’Malley et al., 2016). The enriched motifs were searched and counted in the dataset using Patmatch software (Yan et al., 2005).

Two type of constructs were prepared for transient expression, using the Gateway system (Karimi et al., 2002). The coding sequence of ABF2 (VIT_18s0001g10450) was amplified from Pinot Noir berry cDNA using Phusion DNA polymerase (Finnzymes) and the primers ABF2Fw and ABF2rev and cloned into pENTR-D-TOPO vector (Invitrogen), sequenced and transferred into pK7WG2, under the control of 35S promoter. The 1-kb promoters of VvNAC17 (VIT_19s0014g03290), Armadillo-like (VIT_17s0000g08080), Xerico-like (VIT_01s0137g00780), VvMYB143 (VIT_00s0203g00070) and HB5 (VIT_04s0023g01330) were amplified from PN40024 genomic DNA using Phusion DNA polymerase and the pairs of primers indicated in Supplementary Table S1. These DNA fragments were cloned in pENTR-D-TOPO, sequenced and transferred into PHGWFS7, upstream of EGFP and GUS reporter genes.

Promoter activation assays were performed in 5-week-old Nicotiana benthamiana plants agroinfiltrated as described in Li (2011). Three leaves from four tobacco plants, representing four biological replicates, were co-infiltrated with the activating plasmid pK7WG2:CaMV35S:ABF2 and individual pHGWFS7:promoter:GUS target constructs. Leaves co-infiltrated with the pK7WG2 empty vector and each of the pHGWFS7:promoter:GUS plasmids were used as a control for the trans-activation assay. 48 h after the first infiltration, leaves were infiltrated with 50 μM ABA dissolved in 10 mM MgCl₂ and 0.07% EtOH, or with a mock solution (0.07% EtOH in 10 mM MgCl₂). Leaf samples were collected at 15 min, 1 and 3 h after the beginning of the ABA treatment. qPCR analysis of GUS expression was performed as previously described for the grape samples, using the primers qPCR_GUSF1 and qPCR_GUSR1 (Supplementary Table S1). GUS expression was normalized using the Elongation factor 1α (EF-1α) gene (AF120093), amplified with primers pEFfw and pEFrev (Schmidt and Delaney, 2010).

In order to identify the exact moment preceding the onset of ripening, we focused our attention on the 2 weeks preceding color break in Pinot Noir berries. Samples were collected daily during season 2011 and analyzed for biochemical and molecular parameters which are known from both literature and our experience to change dramatically at ripening inception (Pilati et al., 2014). These include biochemical profiles, such as total acidity, sugar and anthocyanins content (Figure 1A), galactolipid peroxidation state and ABA content in berry skins (Figure 1B) and gene expression profiles, such as those of lipoxygenase A (LOXA, VIT_06g0004s01510) – responsible of the enzymatic galactolipid peroxidation – and of NCED1 (VIT_19s0093g00550) – first committed enzyme in ABA biosynthesis (Figure 1C). This preliminary analysis showed that in the 48 h preceding anthocyanins accumulation (on July 14th), all these parameters undergo a transition, which marks the beginning of a distinct developmental phase, i.e., the ripening. This discontinuity implies an extensive regulation occurring within the cells to trigger all the metabolic pathways characterizing the biochemical changes of fruit ripening. To study the role of ABA as a trigger of ripening, in 2013 we collected berries at the hard green pre-véraison stage (E-L 33) and treated them with exogenous ABA. The treatment was performed on detached berries with short petioles in an aqueous medium containing 0.2 mM ABA for 20 h, which allowed for both homogenous ABA diffusion into the berry skin trough functional stomata and accurate experimental reproducibility. After 20 h, eight berries were collected, rinsed with water and frozen while the remaining were rinsed and plated for additional 24 h on solid medium containing 10% sucrose, in the absence of ABA, and then frozen (Supplementary Figure S1). Sucrose was added for avoiding osmotic stress according to (Gambetta et al., 2010) in order to mimic berry sugar content at véraison (6–7°Brix). Treatment efficacy has been verified by two independent approaches. Firstly, ABA was quantified in skin samples of control and ABA treated berries by HPLC-UV-MS showing an average accumulation of 1.62 micrograms ABA/gr FW skin powder at 20 h and 0.55 μg/gr FW at 44 h in treated samples (Figure 2A). The latter value was similar to physiological values measured at véraison in other studies, such as 300 ng/gr FW in Wheeler et al. (2009) and 200 ng/gr FW in Sun et al. (2010), whereas higher ABA level at 20 h could be explained by ABA direct uptake. The very low ABA measured in control samples is consistent with berries at the green hard pre-véraison stage (Figure 1B). Secondly, the expression of the two genes known to be up-regulated at véraison, LOXA and NCED1, has been measured by qPCR (Figure 2B). NCED1 was up-regulated 66 times by ABA treatment at 20 h and 198 times at 44 h; LOXA showed a similar behavior, as it was up-regulated 4 and 13 times at 20 and 44 h, respectively. These profiles are consistent with those observed in 2011 in the transition from E-L 33 to E-L 34.

Moreover, the induction of NCED1 can explain the intracellular ABA level measured at 44 h.

Transcriptomes of treated and control samples were analyzed by RNA-sequencing (Supplementary Table S2). Principal component analysis (PCA, Figure 3) shows that the four conditions are well-separated, while the biological replicates are grouped together. Major variance (41.5%) distinguished the two time-points (20 vs. 44 h), while treated vs. untreated berries were neatly separated along the second principal component, which explained 19.5% of the variance. It appeared that ABA treatment extensively impacted on pre-véraison berry skin transcriptome.

Treated vs. untreated samples within each time-point were statistically compared to extract the lists of significantly modulated genes and a further restriction on fold change (greater than 2) was applied. 871 genes resulted modulated at 20 h by ABA and 1512 at 44 h; 490 genes were modulated at both time-points, with a coherent trend (Figure 4; and Supplementary Table S3). Interestingly, ABA-induced transcriptional modulation increased over time, regardless of the absence of the external stimulus. This amplification in the response suggests that ABA likely acted as a primer of a broad cellular program. According to RNA-seq analysis, NCED1 was induced 32 and 64 times at 20 and 44 h, respectively, whereas LOXA was induced 3 and 12 times, thus confirming previous qPCR analysis. Genes have been functionally annotated using Vitisnet (Grimplet et al., 2009) integrated with manual curation. For functional class enrichment analyses both Vitisnet and Gene Ontology were used (Supplementary Table S4). Genes modulated exclusively at 20 h, the less abundant group, were enriched in classes related to stress, cell wall modification, photosynthesis, respiration and translation. They could represent a stress response due to the excess or sudden delivery of ABA in the treatment, which induced a high turn-over of proteins involved in basic energy metabolism and cell wall. The set of ABA-positively modulated genes at both 20 and 44 h was enriched in genes involved in cell regulation: ABA and ethylene networks were over-represented, along with transcription factors related to these hormones, such as members of the large bZIP, APETALA2 and MYB families. At 44 h post-treatment, the number of genes positively modulated equaled that of the negatively modulated and the functional categories related to the metabolic pathways typical of the ripening process were enriched. Lipid and carbohydrate metabolism, cell wall modification, and flavonoid metabolism were over-represented among the up-regulated genes, supporting the role ABA plays in regulation of sugar metabolism and accumulation, cell enlargement and softening and color development. Ethylene, Auxin and ABA-related categories remained over-represented, suggesting that these hormones regulated not only the onset but also the process of grape berry ripening. Photosynthesis was over-represented among the down-regulated genes, suggesting that ABA triggers the switching off of this basal metabolism inducing the transition to a specialized sink organ, such as the ripe berry.

In order to outline the role of ABA at ripening onset, a meta-analysis using the grapevine expression data compendium Vespucci (Moretto et al., 2016) was performed. Seven experiments in which berry ripening transcriptome had been analyzed were selected to visualize how the sets of genes modulated in the skin by ABA at the two time-points were modulated during physiological ripening. These experiments were performed in different berry tissues (seed, pulp, skin, and pericarp) in six different cultivars: Cabernet Sauvignon (GSE11406), Sauvignon Blanc (GSE34634), Corvina (GSE36128), Pinot Noir (GSE49569 and GSE31674), Muscat Hamburg (GSE41206) and Norton (GSE24561). Heatmaps representing the comparisons are shown in Figure 4, while the tables which generated the heatmaps, downloaded from Vespucci website, are available as Supplementary Table S5. Within each comparison, a percentage of genes ranging between 49 and 87% were modulated coherently by ABA and during ripening in all tissues and cultivars (Figure 4). However, the heatmaps showed also variable profiles, likely related to the tissue and/or cultivar specific modulation of these transcripts. While the smallest overlap occurred with genes up-modulated exclusively at 20 h, the sets of genes modulated at both 20 and 44 h and those modulated only after 44 h showed a more extended overlap with ripening, between 64 and 87%, higher when considering the down-regulated genes. Functional categories enrichment analyses were repeated on the restricted sets of genes modulated both by ABA and during ripening (Figure 4 and Supplementary Table S4). In general, the analysis reproduced the results described in the previous section, with small refinements, such as for genes up-modulated at 20 and 44 h in which more specific categories related to carbohydrate metabolism, such as aminosugars, galactose and glycerolipids, appeared enriched or changes, as occurred for genes up-modulated at 44 h, in which the two categories of cell wall and flavonoid biosynthesis were lost while that of fruit ripening and abscission were gained. On a broad scale, we show that green hard berries treated with ABA are not simply responding to a stimulus, rather activating genes that are typical of the ripening program.

Palumbo and collaborators highlighted that the transition from immature-to-mature stage in the berry is characterized by an extensive transcriptomic down-regulation, anti-correlated to a small group of 190 so called “switch genes” (Palumbo et al., 2014). Interestingly, we identified in our set of positively modulated genes 80 of such “switch genes” (Supplementary Table S6). This finding is consistent with ABA playing an important role in ripening regulation, partly by switching off typical vegetative pathways, such as those related to photosynthesis. Besides, 13 out of these 80 candidate “ABA-responsive switch genes” were predicted to be regulated post-transcriptionally by miRNA (Palumbo et al., 2014), suggesting that ABA modulation can occur both via direct targets activation and via post-transcriptional mechanisms.

Finally, we compared our results with the list of genes modulated in a grapevine cell culture by a 1-h treatment with 20 μM ABA (Nicolas et al., 2014). Only 55 genes were modulated by ABA in both experiments and they were mostly up-regulated (Supplementary Table S7). They are related to ABA network and cell response to abiotic stresses, such as drought, dehydration, osmotic stress and potentially represent a basal ABA signaling core conserved in any type of cell.

Abscisic acid treatment of berries at pre-véraison stage significantly modulated several genes involved in ABA metabolism, perception and signaling, which are summarized in Figure 5. All the gene families involved in ABA metabolism appeared transcriptionally affected by ABA: NCED family, involved in ABA biosynthesis, ABA 8′ hydroxylase, in its degradation, ABA glucosidase, in ABA conjugation with sugar moieties and ABC transporters of the G subfamily, involved in ABA transport. The participation of the different NCED isoforms in berry ripening has been widely reported (Lund et al., 2008; Wheeler et al., 2009; Sun et al., 2010; Young et al., 2012). Using Vespucci, we could visualize the expression of the five NCEDs present in grapevine genome in at least three cultivars during berry development and post-harvest withering (Supplementary Figure S2). By combining this information with our results, we can state that VIT_19s0093g00550, called NCED3 in Young et al. (2012) and NCED1 in Sun et al. (2010), is very rapidly induced in the skin by ABA treatment and its upregulation specifically occurs in the pulp and skin tissues at véraison, while the gene is not modulated later in ripening (Supplementary Figure S2). In our results, there are two other NCEDs which are modulated though to a lesser extent and only at 44 h: VIT_02s0087g00910, which is also modulated during post-harvest withering in Corvina, and VIT_10s0003g03750, which does not seem related to the process of berry ripening (Supplementary Figure S2). To understand their function, further investigations are needed. Instead, from Vespucci analysis, the isoform VIT_02s0087g00930 seems induced during the whole berry ripening in all tissues, slightly more in the pulp.

Concerning ABA perception and signaling, both ABA receptors of the PYL/PYR/RCAR family and PP2C phosphatases were affected by the presence of exogenous ABA. The two receptors (VIT_08s0058g00470 and VIT_02s0012g01270), previously identified as RCAR5 and 7 by Boneh et al. (2011) and as PYL3 and PYL1 by Li et al. (2012), were down-regulated in our experiment. Three PP2C phosphatases were strongly induced at 20 and 44 h: VIT_11s0016g03180 was identified as AtABI1 homolog by phylogenetic analysis (named PP2C-2 in Gambetta et al., 2010), and was characterized in leaf and root (named PP2C4) by Boneh et al. (2011). VIT_06s0004g05460 and VIT_13s0019g02200, corresponding to PP2C-6 and PP2C-3 in Gambetta et al. (2010) and PP2C9 and PP2C8 in Boneh et al. (2011) were found induced at ripening onset in developing berries, anticipated in deficit irrigation conditions and induced by ABA in the skin of in vitro cultured berries. Out of the five identified Snf1-related kinases involved in ABA response, one (VIT_02s0236g00130) corresponding to SnRK2.1 in Boneh et al. (2012) was up-regulated at 20 and 44 h. In Figure 5, other kinases belonging to the calcium dependent protein kinases family (CPK/CDPK/CDKs) or calcineurin B-like interacting protein kinases (CIPKs) and the three tiers of mitogen-activated protein kinase cascades (MAPKs, MAPKKs, and MAPKKKs) were included, based on Arabidopsis literature reporting their potential involvement in ABA signaling (reviewed in Finkelstein, 2013). In grapevine, some CPK and CDPK have been studied due to their up-regulation at véraison and relationship to ABA or drought stress (Yu et al., 2006; Cuéllar et al., 2013). Interestingly, also a LRK10 receptor kinase was found induced at 20 and 44 h and was included, as it is induced also in ABA-treated cell culture and in Arabidopsis one isoform, AtLRK10L1.2, is implicated in ABA response and drought resistance (Lim et al., 2015).

Known direct targets of SnRK2 phosphorylation are NADPH oxidases of the Respiratory Burst Oxidase family (Rboh), leading to the production of hydrogen peroxide, plasma membrane anion and K+-channels, regulating ion transport and stomata opening, and basic domain/leucine zipper (bZIP) transcription factors of the ABA responsive element binding factor subgroup (AREB/ABF), mediating ABA-responsive genes transcription (Wang P. et al., 2013). In our experiment, representatives for all these functional categories were modulated (Figure 5, top). Interestingly, only one member of the grapevine AREB/ABF predicted subgroup (Liu et al., 2014) was modulated, namely the VvAREB/ABF2 (VvbZIP045, VIT_18s0001g10450), recently characterized by Nicolas et al. (2014). VvAREB/ABF2 likely represents the isoform phosphorylated by SnRK2 in berry skin cells at ripening onset and activating down-stream genes of the ABA signaling cascade.

With the aim of identifying candidate target genes of VvABF2 activity, promoter regions of the genes modulated at 20 and 44 h were analyzed to find significantly enriched cis-acting motifs. The three most enriched motifs that were found were recognized by bZIP transcription factors, NAC and ABF subgroup of the bZIP family, respectively (Figure 6). Calculating the frequency of refined consensus motifs highlighted that the ABRE motif, “CACGTGT/GC,” was about threefold more represented in our ABA-modulated gene set compared to the whole genome set of promoters. The recurrence of ABREs within each promoter was calculated: it ranged from 0 to 4 and is represented in Figure 5. ABREs are present in genes involved in ABA network, like NCEDs, PP2Cs and PYL/RCAR receptors but also in some TFs of the NAC, MYB, HB, bZIP and ERF families. In particular, VvMYB143 and VvNAC17 were the most enriched in ABREs and were also modulated by ABA in cell cultures (Nicolas et al., 2014). VvMYB143 belongs to the subgroup S11 of R2R3-MYB TFs, whose function has not been characterized yet (Wong et al., 2016). VvNAC17 belongs to subgroup III (Wang N. et al., 2013) and has been recently characterized for VvABF2 activation in tobacco protoplasts (Nicolas et al., 2014). Other two genes, VIT_17s0000g08080 and VIT_01s0137g00780, were highly modulated by ABA in our experiment and also in grapevine cell culture (Nicolas et al., 2014) and possess 4 and 2 ABREs in their promoters, respectively. The first gene is annotated as Armadillo b-catenin, because it contains the 3D motif Armadillo, involved in binding of large molecules such as proteins or DNA but also the U-box domain, for recruiting E2-adenylated ubiquitin in the protein degradation pathway (Coates, 2003). The gene VIT_01s0137g00780 was annotated as “unknown.” RNA-seq data have been used to refine this gene prediction, revealing an incorrect splicing site (Supplementary Table S8). The improved transcript prediction based on reads mapping was used for homology search by BLAST algorithm in other species, highlighting the presence of a RING Zinc finger domain, as in Xerico (Ko et al., 2006). Interestingly, in Arabidopsis, genes containing these domains are reported to be involved in ABA response modulation and in drought resistance (Ko et al., 2006; Moody et al., 2016). These two genes will be hereafter referred to as Armadillo-like and Xerico-like, for brevity. Vespucci analysis showed that VvNAC17 and Armadillo-like were part the genes modulated by ABA at 20 and 44 h and during ripening, while VvMYB143 and Xerico-like were characterized by more variable profiles in the different cultivars and tissues and would need more specific investigation to precisely describe their modulation.

These four genes were further investigated in a transient trans-activation assay in N. benthamiana to verify their dependence on VvABF2 activity. The transcription factor HB5, activated by ABA but lacking ABREs in its promoter, was included as a negative control. Tobacco leaves were co-infiltrated with the target promoters fused to the reporter GUS along with either the VvABF2 activator or the control empty vector. The constitutive over-expression of ABF2 on its own is insufficient to induce expression of the downstream target genes, as ABF2 activity involves the ABA-dependent phosphorylation of its N-terminal domain, as demonstrated in Arabidopsis for AtABF2 (Fujita et al., 2005). Consequently, 2 days after the agro-infiltration, leaves were re-infiltrated either with 50 μM ABA or with a mock solution. GUS gene expression was analyzed by qPCR at 15 min, 1, and 3 h after the ABA treatment (Figures 7A–E). The promoters of VvNAC17 and Armadillo-like showed a remarkable activation by VvABF2 following ABA treatment, in agreement with the presence of four ABREs in their sequences (Figure 7F). Expression of VvMYB143, which contains two ABRE motifs in its promoter, was activated by ABA, irrespectively of the presence of VvABF2. Conversely, despite the presence of two ABF binding domains, the Xerico-like promoter did not show any activation in all the conditions, as observed for the negative control gene HB5.