Effects Of Nitrogen And Potassium On Sugar,Acid Metabolism And Anthocyanin Synthesis In Grape Berries

Soluble sugars,organic acids and anthocyanins play a vital role in the flavor and color of grape berries.Nitrogen and potassium are essential nutrients for grape growth and development,which can regulate soluble sugar,organic acid metabolism and anthocyanin synthesis.However,the research on the effects of nitrogen and potassium on the regulation of glucose and acid metabolism and anthocyanin synthesis in grape fruit is not systematic.In this study,‘Flame seedless’and‘Kyoho’grapes were used as experimental materials to investigate the effects of root application of three levels of nitrogen fertilizers,foliar application of two potassium fertilizers and nitrogen and potassium interaction on soluble sugar,organic acid and anthocyanin content of grape berries at different developmental stages（The berries of‘Flame seedless’grape in mature period is bright red;The berries of‘Kyoho’grape in mature period is purple black）.We also analyzed the expression of genes related to sugar metabolism,acid metabolism and anthocyanin synthesis in combination with transcriptome data,and preliminarily revealed effects on nitrogen and potassium sugar and acid metabolism and anthocyanin synthesis in grape berries.The main results are as follows:1.The effects of three nitrogen application levels[low nitrogen（LN）,moderate nitrogen（MN）and high nitrogen（HN）]on soluble sugar and organic acid contents and key genes of sugar and acid metabolism in‘Flame seedless’grape berries were investigated.The results showed that the contents of grape berry glucose,fructose,sucrose and total sugar gradually increased with the increase of fruit development days after full bloom in 2020 and 2021.The grapes under HN treatment for two consecutive years had the lowest sucrose and total sugar contents from the beginning of grape color transformation to the full mature stage.In 2020,except for the beginning of grape color transformation（56 days after full bloom）,the glucose content of MN treatment in the other four periods was significantly higher than that of LN and HN treatments.The contents of fructose and total sugar in MN were the highest of three treatments in five sampling periods in 2020.In2021,the glucose content of the MN treatment was the highest from the beginning of grape color transformation to the full mature stage.The fructose content under MN treatment was higher than that of LN and HN treatments at the beginning of grape color transformation and the commercial mature stage（70 d after full bloom）.From the beginning of grape color transformation to the commercial mature stage,the total sugar content of grapes was higher under MN than that of LN and HN treatments in 2021.In 2020 and 2021,the contents of tartaric acid,malic acid,citric acid,shikimic acid and total acid in grape berries of the three treatments showed a decreasing trend from the beginning of grape color transformation to the full mature stage.The tartaric and malic acid contents under HN treatment were higher than MN and LN for five periods in 2020.The total acid content of grapes was HN>MN>LN from the beginning of grape color transformation to the full mature stage in 2021.The differences in transcriptome expression of grape berries under three nitrogen application levels at70 days after full bloom in 2021 was analyzed,and KEGG enrichment analysis of differentially expressed genes（DEGs）showed that the number of DEGs in carbohydrate metabolism was higher.Further analysis of these DEGs revealed the lowest expression levels of genes for sucrose synthase（SS）,glucose-6-phosphate1-dehydrogenase（G6PDH）,hexokinase（HK）,phosphoenolpyruvate carboxylase（PEPC）,citrate synthase（CS）and malate dehydrogenase（MDH）under MN treatment.The expression levels of isocitrate dehydrogenase（IDH）and malic enzyme（ME）genes were the highest under MN treatment.2.The effects of foliar application of KH₂PO₄（T2）,K₂SO₄（T1）on the skin color and synthesis anthocyanin synthesis in‘Kyoho’grapes were investigated.The results showed that the total soluble solids（TSS）,flavonoid and total anthocyanin contents were highest under T2 treatment,followed by T1 and CK（Foliar spraying with distilled water）in the mid-ripe stage（90 days after full bloom）and the mature stage[110 days after full bloom（DAFB）].At two stages,the titratable acid content decreased and the juice p H increased under T2 treatment relative to CK.The lightness（L*）under T1 and T2 was lower than CK,and the color index of red grapes（CIRG）under T1 and T2 increased at two stages compared to CK.The differences in grape skin transcriptome expression in the three treatments at 90 and 110 DAFB showed that the flavonoid biosynthetic pathway was the most significantly enriched pathway in CK vs T2.At 90 DAFB,the expressions of phenylalanine ammonia-lyas（PAL）,cytochrome P450 CYP73A100（CYP73A）,4-coumarate:Co A ligase（4CL）,chalcone synthase（CHS）,flavanone 3-dioxygenase-like（F3H）and UDP glucose:flavonoid 3-o-glucosyl transferase（UFGT）higher under T2 than in CK and T1.PAL,CYP73A,CHS,flavonoid-3’5’-hydroxylase（F3’5’H）and ANS were down-regulated in T1 and T2 treatments at 110 days after full bloom.Foliar spraying of KH₂PO₄ improved grape skin color more significantly than foliar spraying of K₂SO₄.3.The effects of foliar spraying of KH₂PO₄and the interaction of three nitrogen levels on sugar,acid metabolism and anthocyanin synthesis in‘Flame seedless’grape were investigated.The results of two consecutive years of experiments showed that the glucose,fructose,sucrose and total sugar contents of the‘Flame seedless’grapes gradually increased with the development of the grapes in the six treatments,while the contents of tartaric acid,malic acid,citric acid,shikimic acid and total acid decreased.Foliar spraying KH₂PO₄ increased the a*,CIRG,carotenoid,flavonoid and anthocyanin contents in LN,MN and HN and decreased the tartaric acid,malic acid,citric acid,shikimic acid and total chlorophyll contents.The grape glucose and fructose contents were higher in the 6 treatments in 2020 than in those of the corresponding 6treatments in 2021.In the nitrogen and potassium interaction treatments,the glucose and fructose contents of MNK were the highest,followed by LNK,and HNK were the lowest from the beginning of grape color transformation stage to the commercial mature stage in 2020 and 2021.The tartaric acid content under MNK treatment was the lowest among nitrogen and potassium interaction treatments;followed by LNK and HNK was the highest.The malic and citric acid contents of LNK were significantly higher than those of HNK and MNK treatments from the beginning of grape color transformation stage to the full mature stage in 2020.The shikimic acid content under LNK treatment was the highest from the beginning of grape color transformation stage to the commercial mature stage in 2020.The highest tartaric,malic and citric acid contents were found in HNK treatment from the commercial stage to the full mature stage in 2021,followed by MNK and the lowest in LNK.Flavonoids,anthocyanins,a*and CIRG were extremely significantly positively correlated with glucose,fructose,sucrose and total sugar;and extremely significantly negatively correlated with tartaric acid,malic acid,citric acid,shikimic acid and total acid.The anthocyanin fractions of the grape skin from six treatments at 70 days after full bloom in 2021 revealed that MNK had the highest total anthocyanin content,followed by LNK,and HN had the least total anthocyanin content.Foliar spraying of KH₂PO₄increased the content of methylated,acetylated and coumaroylated monomeric anthocyanins in grape skin under LN,MN and HN.The grape skin transcriptome expression differences among six treatments at 70 days after full bloom in 2021 revealed that flavonoid biosynthetic pathways were the metabolic pathways that shared differences in LN vs LNK,MN vs MNK and HN vs HNK.Spraying KH₂PO₄ all up-regulated the expression levels of genes related to flavonoid biosynthetic pathways under LN,MN and HN,such as PAL,CHS,FLS,ANS and UFGT,etc.4.The effects of foliar spraying of KH₂PO₄and the interaction of three nitrogen levels on sugar,acid metabolism and anthocyanin synthesis in‘Kyoho’grape were investigated.The results showed that foliar application of KH₂PO₄ increased the grape berry firmness,TTS,p H,glucose,fructose,sucrose,a*,CIRG,flavonoids and anthocyanin contents‘Kyoho’berries under of LN,MN and HN treatments to different degrees,and decreased the contents of titratable acid,tartaric acid,malic acid,citric acid,shikimic acid,L*,b*,C*,h°,chlorophyll a,chlorophyll b and total chlorophyll.Among the nitrogen and potassium interaction treatments,the grape berry firmness and flavonoid content under LNK treatment were highest;the TTS and anthocyanin content was the highest in MNK treatment;the titratable acid,b*,h°,chlorophyll a,chlorophyll b and total chlorophyll contents under HNK were highest.At 100 and 110 days after full bloom,the contents of glucose,fructose and sucrose were the highest in MNK and the contents of tartaric acid,malic acid,citric acid and shikimic acid in LNK were the highest.Anthocyanins were extremely significantly positively correlated with glucose,fructose,sucrose and total sugar;and extremely significantly negatively correlated with malic acid,shikimic acid and total acid.In summary,nitrogen affected the soluble sugar and organic acid content of‘Flame seedless’and‘Kyoho’grapes and spraying KH₂PO₄ improved the color and anthocyanin content of the two grapes at three nitrogen application levels.The anthocyanin content of‘Flame seedless’and‘Kyoho’grape skin was found to be extremely significantly positively correlated with soluble sugar and extremely significantly negatively correlated with malic acid,shikimic acid and total acid.Nitrogen and potassium can alter the content of soluble sugars and organic acids by inducing the gene expression of such as HK,FRK,PK and CS in sugar and acid metabolism,which in turn affects the expression of anthocyanin synthesis enzymes such as PAL,CHS,F3H,ANS and UFGT,and ultimately regulates anthocyanin synthesis.