The effective concentration of GA can vary markedly according to sensitivity of the variety to the hormone. For example, ‘Thompson Seedless’ requires multiple applications and large cumulative amounts of GA exceeding 100 ppm, while a single application of 10 ppm of GA can triple the size of ‘Black Finger’ berries. GA is also known to potentially delay maturity, increase pedicel thickness, and increase berry abscission depending on the application time. If applied on the whole vine on sensitive varieties, GA can also harm reproductive meristems and reduce subsequent yield. The molecular aspects of the synthesis, signal perception, and transduction of GA in grapes have been reported. The study of the effects of cytokinins on grape development focused largely on forchlorfenuron -N′-phenylurea, a synthetic cytokinin known as CPPU, that has been tested and applied to regulate fruitset, size, and shape in several fruit crops. In grapes, CPPU was also reported to delay maturation and reduce berry skin color, increase berry pedicel thickness and rigidity, increase cuticle content, and reduce weight loss of the rachis. CPPU is commercially applied at levels of 5 ppm or lower due to its potential adverse effects on maturation and post harvest quality. The time frame for application of CPPU is usually similar to that of GA and often in combination with GA at reduced concentrations. Other cytokinins such as benzyl adenine had similar effects but a concentration of 500–1000 ppm was required to increase berry size. Phenylpropanoids are a large class of plant secondary metabolites derived from aromatic amino acids, phenylalanine in most plants or tyrosine in some monocots.
The main branches of the phenylpropanoid pathway include lignans and lignins, stilbenes, coumarins, isoflavonoids, flavonoids, and PAs. The biosynthesis of PAs, anthocyanins,hydroponic pots and flavonols share common steps in the flavonoid pathway. In grape berries, the first committed steps in PA biosynthesis are catalyzed by leucoanthocyanidin reductase and anthocyanidin reductase by converting anthocyanidins to flavan-3-ols such as -catechin and -epicatechin , respectively. The resulting -epicatechin and -catechin derivatives can be oxidized to quinones, which are polymerized. However, it is not clear whether this polymerization of the soluble precursors proceeds enzymatically by laccases or non-enzymatically. The subunits of PA are derived from 2,3-cis-flflavan-3-ols -epicatechin and -epigallocatechin , as well as from 2,3-trans-flflavan-3-ols -catechin and -gallocatechin and are most commonly linked via 4 β → 8C−C bonds. The PAs are oligomeric and polymeric flflavan-3-ols that can range in size from 2 to 30 or more subunits. The regulation of the phenylpropanoid pathway was studied extensively in grapes with respect to anthocyanins, PAs, and other branches of the pathway with emphasis on the role of R2R3-MYB, bHLH, and WD40 transcription factors and their target genes. Our previous study demonstrated that CPPU causes a marked increase in tannin content of Thompson Seedless. Thompson Seedless is a major variety but it does not produce anthocyanins and is very low in volatile content. In the current study we presented the following questions: how do CPPU and GA affect tannin accumulation in a variety that is rich in anthocyanins and volatile compounds; and how are biological processes at ripening affected by the treatments. These questions were addressed at the phenological level, by the analysis of relevant metabolites from the phenylpropanoid pathway, by volatile composition, and by transcriptome analysis.Experiments were carried out on Vitis vinifera cv. Sugrasixteen that will be referred as ‘Sable’. Vines of ‘Sable’ were grafted on Richter root stock that was 7 year old and grown in Israel in the Lachish area . All viticulture practices were performed as describepreviously.
The experimental plot comprised of four replications of three vines each, arranged in a randomized block design. Clusters were manually sprayed to full wetness with the growth regulators gibberellic acid and forchlorfenuron in a concentration of 20 and 5 ppm, respectively, with 0.025% Triton-X100 as a wetting agent. A combination of GA and CPPU was also sprayed on the berry at the same concentrations. ‘Sable’ was treated on 14 May 2018 at the berry diameter of 6.0 ± 0.08 mm. Phenological data for ‘Sable’ was collected at the time of treatment , and 7, 34, 51, and 70 days post treatment . Sampling for metabolites, volatiles, and gene expression was carried out at 51 and 70 d after treatment, the time points which represent the beginning and end of commercial harvest. Sampling at all time-points was of 90 berries pooled from 20 to 30 clusters randomly collected from the four vineyard replications. For metabolite and RNA-seq analysis, a disk of 14–16 mm was removed along the longitudinal axis of each berry and was frozen in liquid nitrogen. The berry disks were homogenized in three replicates of 30 disks each using an IKA homogenizer with liquid nitrogen and were stored at −80 °C for further analysis. The remaining part of the berries was used for measurement of total soluble solids and titratable acidity .Measurements of TSS and TA in ‘Sable’ berries of GA, CPPU, and GA + CPPU treatments were carried out as previously described. The juice was obtained by using a fruit juicer from 30 berries. TSS was determined by a digital refractometer and denoted as °Brix. TA was measured by means of titration with 0.1 N NaOH to pH 8.2 with a Metrohm automatic titrator and expressed as tartaric acid equivalents. All the above analyses were performed on fresh samples at the harvest date. Informal tasting was done by three expert tasters that scored the samples in hedonic scale of 1–9 .Sample preparation and microscopic examination of ‘Sable’ berries at 33 d after GA, CPPU, and GA + CPPU treatment were performed as described by Tyagi et al.. Briefly, transverse hand sections from ‘Sable’ berries of GA, CPPU, and GA + CPPU treatment were immediately immersed in a solution containing formaldehyde, acetic acid, ethanol, and water in ratios of 10: 5: 50: 35, respectively.
After fixation, tissue sections were serially diluted by ethanol and subsequently a stepwise exchange of ethanol with Histoclear was carried out. Samples were embedded in paraffin and cut with a microtome into 12 μm thick sections. Sections were stained with Safranin O that stains nuclei, lignified suberized, or cutinized cell wall in red, and with fast green FCF that stains cellulose in green-blue and sections were examined under a light microscope .Gibberellin and cytokinin applied at fruit-set are known to increase berry size, but due to the complexity of agricultural systems, the intensity of the response can vary among seasons. The PGR treatments were performed at a fruitlet size of 6.0 ± 0.08 mm on ‘Sable Seedless’. Fruitlet diameter and weight increased significantly by application of both GA and CPPU measured 7 d after treatment . Interestingly, at 34 d post treatment, berry diameter and weight of CPPU-treated berries was smaller than the control but this trend was reversed at later time points. At 51 and 70 d after the treatments, CPPU increased berry weight and diameter significantly with respect to the control and the GA treatment. The GA treatment increased berry size by 17.5% and 13.4% at 51 and 70 d, respectively, but at a later time point, the difference was not statistically significant from the control. The diameter and weight of berries treated with the combined treatment of GA + CPPU was higher than that of CPPU alone at both 51 and 70 d. Brix of the control grapes was higher at all time-points followed by GA and CPPU . At 34 d,grow pot berries treated with GA + CPPU had higher Brix as compared to CPPU-treated berries, but at later time points there were no differences among the two treatments. CPPU had a major effect on TA that was much higher than the other treatments at 34 d after treatment . At both 34 and 51 d, GA seemed to mitigate the effect of CPPU on TA. With respect to appearance of the berries 34 d after the treatment, there was a clear delay in color development by CPPU and the treatment of GA + CPPU . Transverse sections of the berries showed that the epidermal layer was thicker either in both GA- and CPPU-treated berries or the combined treatment as compared to the untreated control . Informal tasting done by experts indicated excellent taste rated as 8 with multiple aromatic notes for the control and GA treatments, while in berries from the CPPU treatment there was some astringency that reduced the score to 7.5 or 7.0 for the combined treatment.The cytokinin CPPU was shown to increase total tannins in Thompson Seedless that bears green berries. It was therefore of interest to determine what effect CPPU has on black berries and if GA has similar effects. HPLC analysis was carried out for glucosides, acylated and coumaroylated forms of delphinidin, cyanidin, petunidin, peonidin, and malvidin . CPPU and GA + CPPU treatments reduced the levels of the majority of these compounds at both 51 and 70 d as compared to the control. Quantitatively, CPPU reduced the levels of the anthocyanin glucosides by ca. 50% at both time points. The GA + CPPU treatment reduced the levels of the anthocyanin glucosides to ca. 75% and 53% at 51 and 70 d, respectively, as compared to the control.
At 51 d post treatment, GA reduced the inhibitory effect of CPPU but this effect was not maintained at 70 d . Anthocyanin glucosides are the major form present in ‘Sable’ comprising 63–71% of the total anthocyanins and the remaining are acetylated and coumaroylated forms . Interestingly, CPPU reduced the proportion of the glucoside forms at both 51 and 70 d and this decrease was accompanied by an increase in coumaroyl glucosides at both time points and acetylated glucosides at 70 d. GA had an intermediate effect on the proportion of the anthocyanin forms. Flavonols are an important branch of the flavonoid pathway and therefore it was of interest to determine the effect of the treatments on major grape flavonols . Myrcetin glycoside was the major flavonol detected with an average of 81–89% among the compounds tested. While variation among replications was significant, two trends are worth noting. At 51 d, CPPU alone or in combination with GA reduced the levels of flavonols to ca. 60% of the control. At 70 d there was an increase in levels of the flavonols and decrease in the effect of CPPU.Flavan-3-ols are the building blocks of the PA chains that are synthesized in the early stages of berry development. Figure 2c displays the three major flavan-3-ols: -catechin, -epicatechin , and -epigallocatechin . Clearly, CPPU-treated berries contained more flavan-3-ols and their level increased during ripening. At the late sampling, GA + CPPU reduced the level of the flflavan-3-ols as compared to the CPPU treatment alone. The hydroxycinnamic acid derivatives, caffeic acid, caftaric acid, coutaric acid, and ferulic acid changed in different ways: caftaric acid was highest in the early stage in CPPU-treated berries; caffeic acid levels were reduced by CPPU at the early stage and it was absent in the late harvest; coutaric acid levels were induced by CPPU and reduced by the combined treatment relative to the control; the levels of ferulic acid were low but increased with ripening without a distinct pattern .To further investigate the effect of GA, CPPU, and GA + CPPU on PA composition, phloroglucinolysis was performed and differences were monitored using HPLC . Phloroglucinolysis hydrolyzes the polymeric PAs generating terminal units and extension units with phloroglucinol adduct . CPPU increased the levels of C, EC, EC-P, and EGC-P. GA did not affect the levels of the PA monomers while the treatment with GA + CPPU reduced the levels of C, ECG, EC-P, and EGC-P only at 70 d after the treatments . Monomeric PAs did not change with ripening with the exception of ECG that was lower at 70 d compared to the control treatment. The total PA level clearly shows the effect of CPPU and also shows that the combined treatment reduced the level of PAs at 70 d in agreement with the data on the free monomers . The treatments with GA or CPPU had minor effects on the % galloyl units but the combined treatment reduced their level. The mean degree of polymerization was significantly lower in CPPU and GA + CPPU treatments as compared to the control .