Effect of nitrogen regulation on berry quality and flavonoids during veraison stage

Abstract Nitrogen regulation can effectively promote the improvement of berry components and the formation of flavor compounds in wine grapes. In order to understand the effects of foliar nitrogen spraying on grape quality and flavonoid substance, took Cabernet Sauvignon as the test subject, grape leaves were sprayed by ammonium sulfate, calcium ammonium nitrate, urea, phenylalanine, and glutamate during veraison, and clear water was used as the control. The results showed that spraying ammonium sulfate could improve the contents of soluble solids, anthocyanins, and total phenols of grape berries; spraying phenylalanine significantly increased the content of titratable acid and tannin and decreased the ratio of sugar to acid in grape berries; compared with the control group, spraying glutamate could significantly upregulate some flavonol monomers; spraying calcium ammonium nitrate can adjust the monomer content of some flavanols; urea spraying significantly increased the contents of most anthocyanins, flavanols, and flavonol and increased the contents of total anthocyanins, total flavanols, and total flavonol in grape skins, laying a foundation for the improvement of the nutritional value of grapes and wine in the future.


| INTRODUC TI ON
Nitrogen is an essential nutrient element in plant growth and development (Zhao et al., 2017), and rational utilization of nitrogen affects the absorption of mineral nutrients, photosynthate, and hormone levels of plants, and further affects the growth and development of fruit trees and fruit yield (Chen et al., 2018;Miquel et al., 2016;Zhao et al., 2018). Appropriate application of nitrogen fertilizer could significantly increase leaf nitrogen content, dry matter weight, and yield (Zhang et al., 2007), and excessive application of nitrogen fertilizer will lead to problems such as overgrowth of tree body nutrition, decreased fruit setting rate, and yield (Zhang et al., 2017). Restricting nitrogen application in vineyards can prevent excessive vegetative growth of grape and improve grape quality (Bell & Henschke, 2005). However, excessive nitrogen control will lead to the decrease in nitrogen content in grapes, and the failure to meet the normal nitrogen metabolism requirements of yeast in the process of alcohol fermentation, leading to fermentation stop (Bell & Henschke, 2005;Garde-Cerdán & Ancín-Azpilicueta, 2008;Gutiérrez-Gamboa et al., 2017). Leaf nitrogen spraying did not affect the nutritional growth of wine grapes, but could promote the nitrogen content in the fruit, and provide sufficient yeast assimilable nitrogen for the later grape alcohol fermentation (Gutiérrez-Gamboa et al., 2017;Hannam et al., 2016Hannam et al., ). al., 2019, and it also plays an important role in the quality parameters of red wine Ma et al., 2020). These compounds are produced by the flavonoid metabolic pathway (Wei et al., 2020); the most important precursor of this pathway is phenylalanine ; moreover, spraying phenylalanine on the leaves could promote the synthesis of phenolic compounds in grapes . Flavonoids synthesis in grapefruits was most active in the late stage of color turning. Flavonoids in grape and wine could be effectively improved by rapid nitrogen supplementation on leaf surface (Cheng et al., 2020). Carina et al., (2019) found that nitrogen fertilizer could significantly affect the aroma and sensory characteristics of grapes and wine, and the levels of 33 metabolites in leaves and 55 metabolites in wine were significantly different due to the application of fertilizers with different nitrogen forms. Javier et al., (2015) found in the study of the effects of foliar spraying of phenylalanine and urea on grape flavonoid substances that 0.9 kg N/ha urea could increase the monomer content of several anthocyanins and flavonols. Some studies have also shown that the application of nitrogen fertilizer before the turning period has no significant effect on the flavonoids and volatile compounds in grape wine and wine due to the influence of special environmental factors, such as water shortage and sunlight Javier et al., 2017;Martínez-Lüscher et al., 2017).
There are many studies on the effects of soil nitrogen application and nitrogen application amount on the fermentation, quality, and yeast assimilated nitrogen of wine. However, the effects of nitrogen regulation at the veraison stage on grape quality and flavonoid substances are less studied. The aim of this study was to understand the effects of nitrogen regulation on grape berry composition and flavonoid compounds in Cabernet Sauvignon vineyards during the color turning period, so as to provide support for grape quality improvement and wine fermentation.

| Test design
The experimental site was located in Lilan Chateau (105°58′20″ E, 38°16′38″ N) in Yongning County, Ningxia. The soil type is gravelly light lime soil, and the soil texture was gravelly sand soil. The experimental grapes were 8 years old Cabernet Sauvignon, the planting direction was north-south, the tree shape was "Inclined upper frame shape," the plant row spacing was 0.6 m × 3.5 m, and the irrigation method was drip irrigation.
There were six treatments in the experiment, which were spraying ammonium sulfate (AS), calcium ammonium nitrate (CAN), urea (Ur), phenylalanine (Phe), glutamate (Glu), and clear water (control), and the amount of nitrogen fertilizer in each treatment was converted to 1.5‰ (Table 1). A single-factor random block design was adopted in the experiment. Each treatment had 5 replicates, and there were 30 plots in total, with a total area of 1,890 m 2 . Spraying was carried out three times (July 15, July 31, and August 13) during the grape veraison stage, and irrigation, pruning, pest control, and other production management measures were consistent.

| Determination of grapefruit quality
Soluble solids were measured with a handheld sugar meter, and the titratable acid content was determined by the standard 0.1 mol·L -1 NaOH method (Jin et al., 2016); reducing sugar was determined by anthrone reagent method (Sohrab et al., 2016); the total phenol was determined by Foling-Shocka method. Tannins were determined by Flynn-Dennis method, and anthocyanins were determined by the pH differential method (Yang, 2016).

| Preparation and extraction of flavonoids from grapes
The sample was placed in a freeze-drying machine (SCIENTZ-100F) for vacuum freeze-drying, and the grinding machine (MM400, Retsch) was used to grind the sample at 30 Hz for 1.5 min to powder form. 100 mg of the freeze-dried powder was dissolved in 1.2 ml 70% methanol solution for vortex and then placed in a refrigerator at 4℃ after vortex. The sample was centrifuged at 12,000 rpm for 10 min and then filtered with a microporous membrane (pore size was 0.22 μm) for later use. and remained at 95% for 1 min. In 10-11.1 min, the proportion of phase B was reduced to 5%, and the balance was 5% to 14 min. The flow rate was set at 0.35 ml/min. The temperature of the column box is set to 40℃; the amount of injection was 4 μl.

| Chromatographic analysis of flavonoids in grapes
The LIT and triple quadrupole (QQQ) scans were obtained on triple quadrupole linear ion trap mass spectrometer (Q TRAP), AB4500 Q TRAP UPLC/MS/MS system, and the system is equipped with ESI Turbo Ion-Spray in 29 interface, which has two negative ion modes, and is controlled by Analyst 1.6.3 software (AB SCIEX). The instrument was tuned and calibrated with 10 and 100 μmol/L polypropylene glycol solutions in QQQ and LIT modes, respectively. QQQ scan was obtained through MRM experiment, and the collision gas (nitrogen) was set to medium.
Through further DP and CE optimization, DP and CE for a single MRM transition were completed. A specific set of MRM ion pairs was monitored in each period based on the metabolites eluted in each period.

| Statistical analysis
Microsoft Excel 2016 and SPSS 24.0 were used to process and analyze the data, and origin 2018 was used to plot the data, and the significant level was (p < .05, n = 5).

| Effects of nitrogen regulation on grape and berry components during veraison stage
AS treatment significantly improved the contents of TSS, TA, and TP in grape berries, while Phe treatment increased the contents of TAC and TN in grape berries and decreased the contents of TSS/ TAC ( AS treatment also significantly increased the TP content of grape berries.

| Effects of nitrogen regulation on anthocyanins in grape skins during veraison stage
GAN treatment significantly improved Pt and cMvcoum, Phe treatment significantly improved Mvacet, and Ur treatment significantly improved the content of total anthocyanins in grape skins (Table 3). There were 22 anthocyanins in grape skins by UPLC-MS analysis, and they are mallow malvidin class (5), cyanidin class (5), peonidin class (4), petunidin class (4) Under Phe treatment, the content of Mvacet in grape peel was the highest, which increased by 18.35% compared with control; except for Cy, Pngluc, Pt, and cMvcoum, almost all single anthocyanins in Ur treatment were significantly higher than those in other treatments; In terms of total anthocyanins, the content of total anthocyanin in grape skins under Ur treatment was the highest, which increased by 12.73%, 28.56%, 23.21%, 17.18%, and 16.50%, respectively, compared with control, AS, GAN, Phe, and Glu. Note: All the parameters are given with their standard deviation (n = 5). TSS = total soluble solids (%). TAC: titratable acid content (expressed in gram equivalent tartaric acid L −1 ). RS: reducing sugar (expressed in gram equivalent glucose L −1 ). TN, tannins (mg tannin/100 g fresh weight); TA, anthocyanins (mg anthocyanin/100 g fresh weight); TP, total phenols (mg gallic acid/100 g fresh weight); Different lowercase letters indicate significant differences between treatments as calculated by Tukey's HSD test (p < .05).

| Effects of nitrogen regulation on flavanols and flavonols in grape skins during veraison stage
The main flavanols in grape skins are catechin, followed by epicatechin. Among the 19 flavanols, catechin and its derivatives and epicatechin and its derivatives account for 63.16% of the total (Table 4).

| Principal component analysis of flavonoids under nitrogen regulation at veraison stage
Principal component analysis (PCA) was used to represent the differences between different treatments (Figure 1). It can be seen from

| Effects of nitrogen regulation on grape and berry components during veraison stage
Nitrogen can promote the nutritional growth of wine grapes and then affect the quality of wine grapes (Carina et al., 2019) Note: Different lowercase letters indicate significant differences between treatments as calculated by Tukey's HSD test (p < .05).

| Effects of nitrogen regulation on anthocyanins in grape skins during veraison stage
Anthocyanin is a natural colorant existing in the skins of red grapes, which is the fundamental cause of the red appearance of grapes. The proportion and amount of each anthocyanin are greatly affected by varieties and cultivation conditions (Mattivi et al., 2006;Stéphane et al., 2004). In this experiment, it was found that nitrogen regula-

| Effects of nitrogen regulation on flavanols and flavonols in grape skins during veraison stage
Flavanols and flavonols are subgroups of flavonoids and are synthesized mainly in grape skins (González-Manzano et al., 2019). In this study, flavanols were mainly catechins, followed by epicatechins, which was also observed by Sergio et al., (2007). Nitrogen regulation at the veraison stage would increase the contents of Meepi, Epgal, and Cagal, but the contents of other flavanols did not significantly increase compared with the control. Schreiner et al., (2014) also found that the supply of different kinds of nitrogen fertilizer did not change the contents of catechin, epicatechin, or epicatechin-3gallate. Only the content of total flavanols in low-concentration urea treatment was significantly higher than that in the control group (Javier et al., 2015), and this study also concluded that urea treatment could improve the content of total flavanols in grape skins. In summary, urea can increase the total content of flavanols and then improve the quality and taste of wine and grapes.
Flavonols are important pigments that help to stabilize red anthocyanins (Boulton, 2001). Javier et al., (2015) found that the increase in flavonol content in grapes may improve the quality of wine, because flavonol, as an adjuvant, indirectly affects the formation of wine color. In addition, Ritchey and Waterhouse (1999) found that high-quality wines contain higher levels of flavonol compounds, indicating that the flavonol content in grapes affects the quality of wine. Javier et al., (2017) believed that myricetin was the main compound of flavonol, followed by quercetin. Due to the influence of nitrogen application rate, grape growing soil, and climate conditions, different metabolites will be generated. In this experiment, it was found that quercetin and its derivatives are the main flavonols, followed by myricetin, kaempferol, and its derivatives, which is contrary to previous conclusions. Javier et al., (2017) showed that there was control treatment and foliar application of nitrogen fertilizer. In this study, it was also found that except urea, other nitrogen did not increase the flavonol content, or even decreased the flavonol content.

| CON CLUS ION
In this study, we determined the effects of nitrogen regulation at the veraison stage on the composition of grape berries and the content of flavonoids in grape skins. The contents of soluble solids, anthocyanins, and total phenols in grape berries were increased by spraying ammonium sulfate on leaf surface. Spraying phenylalanine could increase the content of titratable acid and tannin and decrease the ratio of sugar to acid. Compared with the control, foliar spraying of nitrogen fertilizer will increase the content of some flavonoid monomers. And the treatment of spraying urea can significantly increase the content of total anthocyanins, total flavanols, and total flavonols in wine grape skins. These results have important oenological significance for grape quality.

ACK N OWLED G M ENTS
This work was supported by Ningxia Natural Science Foundation

(2020AAC03281) and National Key Research and Development
Project (2019YFD1002500). We thank our colleagues for their comments regarding this paper and the journal's editors and anonymous reviewers for their critical reviews and comments regarding this manuscript.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.