Effect of Exogenous NaCl On Anthocyanin Accumulation and Biosynthesis of Cabernet Sauvignon Grape Berries


 Backgroud:

The coloring mechanism of grape (Vitis vinifera L.) berry in response to salinity during maturation is poorly understood. To determine the effect of salinity on fruit quality, especially anthocyanin accumulation, the grapevine cultivar Cabernet Sauvignon were sprayed with different concentrations of sodium chloride. Dynamic changes in anthocyanin accumulation and eight biosynthetic enzyme activities during maturation were also investigated to clarify the anthocyanin biosynthesis regulation.
Results

The analysis showed that the fruit’s fresh weight was decreased by NaCl spray. But the grape quality including reducing sugar, soluble sugars, total phenols and tannins increased significantly on harvest time. Ten individual anthocyanins were detected from the skin of Cabernet Sauvignon by ultra performance liquid chromatography (UPLC). And there was a significantly greater accumulation of total anthocyanin contents under salt treatment. The NaCl spray provoked metabolic responses in grapes and enhanced biosynthetic enzyme activities during riping. Correlation analysis showed that anthocyanin accumulation was closely related to the key enzyme LDOX both in the control and 100 mM NaCl treatment berries.
Conclusion

The application of NaCl to grape foliage effectively increase the quality of the Cabernet Sauvignon grape, improving soluble sugar, organic acid, phenolics and tannin contents, and the total anthocyanin contents in grape skins after varasion. These findings provide novel insight into the crucial factors that directly modulate anthocyanin biosynthesis and consequently control grape coloration.

The experiment was carried out in a Vitis vinifera L. cv Cabernet Sauvignon vineyard of the Great Wall Wine Co., Ltd in Penglai, Shandong Province, China (37°25′∼37°50′ N, 120°35′∼121°09′E) during the 2015 vintage. The vines were planted in 2005, trained on a vertical trellis system and pruned using unilateral Royat cordon system. Six representative and consecutive rows were selected for the analysis. The grape leaves were sprayed with three different concentrations of NaCl solution (40,60, 100 mM) every two weeks starting at 7 days after anthesis (DAA) until berry maturation respectively. At 90, 105 and 120 days after anthesis, 300 berries were taken randomly from three different positions in the sample clusters: the shoulder, middle and bottom. The berry size and fresh weight of the collected grapes were analyzed within 1 h after harvest . Berries collected for enzyme and biochemical analyses were immediately frozen in liquid nitrogen and kept at −80 °C until analysis.

Measurement of grape berry indexes
The total soluble solids (TSS) of collected berries was measured using a digital refractometer (TD-45, TOP Instrument, Zhejiang, China). And the titratable acidity was titrated with 0.1 M NaOH to an end-point at pH 8. 3. Reducing sugar of the supernatant was analyzed by the dinitrosalicylic acid (DNS) method (L., 1959). Quanti cation of total phenolics was performed by the Folin-Ciocalteu's method (Minussi et al., 2003), using gallic acid as standard. While tannin quanti cation used Folin-Denis reagent, using catechinic acid as standard.

Soluble sugar and organic acid determination
The pulp was ground into powder with liquid nitrogen. And 3 g of powder was extracted three times in 6 ml of 80% (v/v) methanol (phosphoric acid for organic acid) and ultrasonic extraction for 20 min. The extract was concentrated with a rotary evaporator and lted with a 0.45 mm membrane lter. Then the ltrate was analysed using a HPLC System (SHIMADZU LC-20AT, Kyoto, Japan) to determine the soluble sugar and organic acid content, respectively.

Determination of anthocyanin pro le
The extraction of anthocyanins was performed according to the method previously reported (Mattivi et al., 2006 Mass spectrometry was performed using an electrospray source in positive ion mode, with a selected mass range of 20−1200 m/z. The ionization parameters were the following: 3.2 kV capillary voltage, source temperature at 100 °C, and desolvation gas temperature at 400°C. Malvidin-3-Oglucoside (Sigma-Aldrich, St. Louis, USA) was used as a common external standard, and other anthocyanin components were quanti ed by it.

Enzyme assay
The activities of key enzymes involved in anthocyanin biosynthesis were assayed by ELISA kit (Bangyi, Shanghai, China).

Statistical Analysis
All data were expressed as the mean ± SD of three replicates. Statistical difference between control and treated groups was analyzed by the LSD't-test at P < 0.05 using SPSS software (version 19.0, SPSS Inc., Chicago, IL, USA).

Results
General quality parameters of mature grape The average fresh weight, transverse and vertical diameter of the berries decreased gradually with increasing salt concentrations. Berries sprayed with 40 mM NaCl had the lowest vertical diameter and fresh weight (Table 1). Both the reducing sugar and TSS content elevated under moderate salinity but declined under high salinity. The grapes under 40 mM NaCl treatment produced the highest levels of reducing sugar and TSS than in the control and other salt-treated berries (Table 1). However, the titratable acidity had the opposite trend (Table 1). Moreover, the salt treatment enhanced the total phenols and tannins of the berries signi cantly (Table 1).

Soluble sugars and organic acids
The grape berries mostly accumulate soluble sugars (fructose and glucose) during riping (Hilbert et al., 2011). In the study, the salt-treated berries showed higher fructose and glucose concentrations than the control. And the most effective treatment was 40 mM NaCl ( Table 2).
The concentrations of the four organic acids were also tested ( Table 2). Tartaric and malic acid accounted for 46.6% and 48.1% in the control. While citric and oxalic acid just accounted for 2.84% and 2.52%. A signi cant increase of tartaric acid and oxalic acid was found under high salinity (100 mM NaCl) . And malic acid did not have a signi cant variation after NaCl spray. The percentage of tartaric acid increased whereas malic acid decreased under salt treatment., However, citric acid did not signi cantly changed in both content and percentage. In short, the sum of the four organic acids increased under 100 mM NaCl, which was mainly due to increased tartaric acid.
Total anthocyanin content (TAC) of grape skin extracts during ripening were shown in Fig. 1. The TAC during maturation were higher under NaCl treatments than that in control (Fig 1). According to the position of substituents on B-ring of avonoid skeleton, ve non-acylated individual anthocyanins can be divided into 3'-substituted anthocyanins (Cy, Pn) and 3'5'-substituted anthocyanins (Mv, Dp and Pt). During grape maturation, the account for the 3'5'-substituted anthocyanins (Mv, Dp and Pt) of the TAC increased from 87.17% to 88.31% in the control. After varasion, although NaCl treatment increased the content of Mv, Dp and Pt. The account for the 3'5'-substituted anthocyanins was lower than that in control. And the composition of 3'-substituted anthocyanins increased under salt treatment than control during maturation (Fig 1, Table 3).

Effects of NaCl treatment on enzyme activities
In the avonoid pathway, the action of many related enzymes may contribute to the anthocyanin accumulation in grape skins (Duan et al., 2019). In order to better understand the impact of NaCl on anthocyanin biosynthesis, the activities of eight pivotal enzymes, CHS, CHI, DFR, F3H, F3'5'H, F3'H, LDOX and UFGT were investigated during grape ripening. The original step of avonoid pathway is catalyzed by CHS to produce chalcone. During grape maturation, compared with the control, the 60 mM NaCl treated samples rst had lower CHS activity levels at 90 DAA, then it signi cantly enhanced during ripening (Fig.2). At 120 DAA, the CHS activity of NaCl treated samples was signi cantly higher than the control group. CHI is one of the key enzymes in anthocyanin synthesis. It can catalyze isomerization of tetrahydroxychalcone. The CHI activity of all salt treatments increased rapidly after varasion. In the control group, the CHI decreased from 90 DAA to 120 DAA. Only 40 mM NaCl treated samples had higher CHI activity than control at 120 DAA (Fig.2). After CHS, F3H hydroxylates avanones to form dihydro avonols. Subsequently, DFR catalyses the conversion of dihydro avonols to leucoanthocyanidins, followed by the production of anthocyanidin from leucoanthocyanidins, which involves LDOX (Boss et al., 1996). DFR is the key enzyme committed to anthocyanin and proanthocyanidin biosynthesis in the avonoid biosynthetic pathway (Katsu et al., 2017). The DFR activity of the control group peaked at 105 DAA and then decreased. The NaCl treatments signi cantly increased the DFR activity in the ripen grape (Fig.2). F3H is part of the 2-oxoglutarate-dependent dioxygenases family and is fundamental for avonol synthesis in Vitis. It is a crucial enzyme in regulating cyanidin derivatives (Kumar and Yadav, 2013). The F3H activity in the control increased rapidly after varasion. Only 100 mM NaCl treatment had higher F3H activity than control. F3'H promotes cyanidin and peonidin anthocyanin accumulations, while F3'5'H catalyzes the production of delphinidin and its derivatives petunidin and malvidin. These two enzymes competitively control diand trihydroxylated anthocyanin synthesis (Falginella et al., 2010). The activities of F3'H and F3'5'H exhibited similar dynamic changes in the control groups. They decreased from 105 DAA to 120 DAA. However, the F3'5'H activity of each salt treatment was higher than that of the control. The 60mM NaCl treated samples rst had lower F3'H activity levels, then it signi cantly increased throughout ripening. In mature grapes, the activity of F3'H under NaCl treatments was signi cantly greater than that of control (Fig.2).
LDOX can catalyze the transformation of colorless cyanidin and delphinidin into coloured anthocyanins. The LDOX activity in each salt treatment was signi cantly higher than that of the control. LDOX activity gradually increased with the salt concentration and peaked under 60 mM NaCl. But the 100 mM NaCl decreased LDOX activity from 105 DAA to 120 DAA during grape ripening (Fig.2). The enzyme UFGT catalyzes the nal step of anthocyanin biosynthesis (Davies et al., 1997). The UFGT activity peaked at 105 DAA and then decreased during grape ripening. The 40 mM NaCl treated grapes got highest UFGT activity at 120 DAA, while UFGT of other salt treatments was lower than the control (Fig.2).

Correlation analysis between anthocyanin contents and key enzymes in anthocyanin biosynthesis
The correlation between the activity of eight enzymes and the total content of anthocyanins in grape skins during maturation was analyzed. The results showed that in the control group, only LDOX activity was positively correlated with total anthocyanin content (p< 0.05) ( Table 4). After treatment with different concentrations of NaCl, only 100 mM treatment showed a signi cant positive correlation between LDOX activity and total anthocyanins (P < 0.05). And there was no signi cant correlation between the eight enzyme activities and total anthocyanins under other NaCl concentration (Table 4).
During anthocyanin synthesis, the enzymes F3'H and F3'5'H are responsible for the hydroxylation of the B-ring of avonoids at position 3' or 5' (Falginella et al., 2010). In the test, the total trihydroxylated anthocyanins in the control group and NaCl treatment groups were correlated with F3'5'H, UFGT, IDOX activity and 100 mM NaCl treatment group, the total amount of trihydroxylated and dihydroxylated anthocyanins were positively correlated with LDOX activity (Table   5). However they were not signi cantly correlated with other enzyme activities (Table 5).

Discussion
Salinity can induce complex effects on grapevines. Moderate salt stress can improve the quality of plants. Low salt stress increased the content of total sugar and ascorbic acid in 'Moldova' grape fruit which improved the quality of grape (Gong et al., 2013). The organoleptic quality and avours of table grapes are mostly determined by the content and composition of sugars and organic acids. Therefore, the ratio of TSS to TA is ordinarily used to assess fruit avor.
Tomatoes (Sato et al., 2006) and strawberries (Anna ) under moderate salinity have increased TSS, TA and stable TSS/TA amounts. Long-term NaCl treatment on Kyoho grape berry increased the average grain weight and cross diameter of grape fruit. Moderate salinity treatment raised the soluble sugars glucose, fructose and sucrose (Li et al., 2013). Our experiment indicates that different concentrations of NaCl decreased the average grain weight, the vertical and transverse diameter of grapes. And NaCl solution signi cantly increased the content of reducing sugar and TSS in mature grapes (Table 1). Glucose and fructose are the predominant sugars accumulated during berry ripening (Li et al., 2013). During grape ripening, the content of glucose and fructose in the fruits of different NaCl concentrations were signi cantly higher than that of the control ( Table 2) (Table 2) in grape fruit not only improve the sweetness and avour under salinity, but also improve the potential alcohol of wine, which is conducive to the production of high-quality wine. Sugars can provide a framework for the biosynthesis of many secondary metabolites, such as phenols, terpenes and organic acids (Pan et al., 2009). The type and concentration of organic acids in grape is an important aspect of quality control. Among the organic acids, tartaric acid and malic acid account for over 90% of the total acids. And tartaric acid is more sour than malic acid (Amerine et al., 1965;Liu et al., 2006). The altered percentage in the organic acid composition suggested a change in sourness under salinity. Although salt treatment reduced the yield of grape, it improved the fruit quality.
Flavonoids are reported to be responsive to various abiotic stresses (Treutter, 2007). Anthocyanins represent a group of natural avonoid compounds in plants and are responsible for the coloration of berrys. The anthocyanin content of Kyoho grape berry increased after 20 mM and 60 mM NaCl treatment (Li et al., 2013). Salt stress increased the antioxidant capacity and anthocyanins in two strawberry cultivars (Keutgen and Pawelzik, 2008). Similar to the previous study, in our present work, NaCl treatment could enhance anthocyanin content at different development stages of grapes. When plants are under salt stress, they will spontaneously carry out a series of biochemical reactions to improve the adaptability to salt environment. The synthesis of compatible substances is one of the important strategies. The substances includes sugars, polyols, proline and avonoids (Parida et al., 2002). Anthocyanins, as the main component of avonoids, have the function of osmoregulation: the content of total anthocyanins in hypocotyls and cotyledons of tomato and purple cabbage seedlings cultivating with nutrient solutions of different salt concentrations is signi cantly higher than that of the control (Eryılmaz, 2006;Tahkokorpi et al., 2012). The nutrient solution containing 40 or 80 mmol/L NaCl enhanced the anthocyanin content of the salt resistant strawberry Korona. But the anthocyanin of the salt sensitive variety Elsanta decreased signi cantly (Keutgen and Pawelzik, 2007). Plants produce reactive oxygen and malondialdehyde (MDA) to cause cell damage under salt stress., As a natural non-enzyme antioxidant, anthocyanin will react with superoxide dismutase (SOD) in plants to eliminate excessive active oxygen radicals and reduce the damage of MDA to cell membrane, which will improve the adaptability of plants to salt environment (Keutgen and Pawelzik, 2007). Therefore, one reason of the elevated content of anthocyanin could be attributed to its salt resistance in plants.
Anthocyanins are secondary metabolites, primarily synthesized via phenylpropanoid and avonoid pathways. These pathways consist of a number of enzymatic steps, each of which catalyzed by a consecutive reaction for anthocyanin biosynthesis (Zorenc et al., 2017). In the study, we explored the activity of key regulatory enzymes in the avonoid pathway of anthocyanin metabolism in response to NaCl treatment. The activity of eight enzymes during grape riping were shown in Fig. 2. Only LDOX activity was positively correlated with total anthocyanin content (p < 0.05) in the control and 100 mM NaCl treated group. And there was no signi cant correlation between the eight enzyme activities and total anthocyanins under other NaCl concentration (Fig. 2). We also detected no signi cant correlation between di-and trihydroxylated anthocyanin with F3'H and F3'5'H activity. Flavonoids mainly include avanone, anthocyanin and proanthocyanidins. They share a part of synthesis path in grape skins (Matus et al., 2009). The nonsigni cant difference of correlation may be because salt stress also affects the activity of enzymes related to the non anthocyanin avonoids synthesis, or the expression level of enzyme genes. Therefore anthocyanin precursors are more used in the synthesis of other avonoids.
The regulatory effect of MYB transcription factor on avonoids has been con rmed in many plants (Feng et al., 2010;Niu et al., 2010;Umemura et al., 2013). MYB regulating avonoid synthesis has been shown to be differentially affected by abiotic stimuli in grapevines (Matus et al., 2010). MYB1 is speci cally expressed in grape skin and induce anthocyanidin synthesis. MYB2 can activate the promoter of UFGT e ciently (Guohui et al., 2013). Therefore, the anthocyanin content in the test is not only the result of the co regulation of regulatory genes and structural genes, but also closely related to the activity of related enzymes or the expression of coding genes in the synthesis of other avonoids.
The synthesis and decomposition of anthocyanins keep a dynamic balance in grape skins, so the total anthocyanins in the experiment are net content. Various physical and chemical factors will destroy anthocyanin's stability and make it degrade in vitro. The degradation of anthocyanin in vitro has been widely studied, but there are few studies on degradation in vivo. Anthocyanins are secondary metabolites of plants in response to environmental changes. Salt stress not only affect the biosynthesis genes and enzymes, but also affect the enzymes or genes in the pathway of anthocyanin catabolism. Both anabolism and catabolism determine the change of anthocyanin content and composition in plants. Further studies are needed in catabolic enzymes and genes on anthocyanin synthesis.

Conclusion
In summary, the application of NaCl to grape foliage effectively increased the quality of the Cabernet Sauvignon grape, improving soluble sugar, organic acid, phenolics and tannin contents. Salt treatment signi cantly increased the total anthocyanin contents in grape skins after varasion. And 40mm NaCl has the greatest impact on the anthocyanin content. However, because the anthocyanin synthesis of grapes are in uenced by many environmental factors in eld, such as light, temperature, rainfall and biotic stress. Furthermore, more researches are needed into the molecular mechanisms of exogenous NaCl increasing the anthocyanin content and synthesis of grape skins.