Effects of nitrogen application rates on the leaf quality of 'Cabernet Gernischet' in desert areas

Abstract: 【Objective】As an essential mineral element in plant growth and development, nitrogen has irreplaceable roles in organ construction, nutritive metabolism, biochemical processes as well as fruit yield and quality. The quality and yield of grapes are greatly influenced by the amount of nitrogen applied. In recent years, the industry of China's wine grape has developed rapidly, but there are problems with unreasonable fertilization in production. The Gansu Corridor has the climatic conditions and geographical resources suitable for producing high-quality wine grapes. However, wine grapes are extensively planted on a sandy loam soil with low organic matter. In the actual production, chemical fertilizers are often applied to ensure the normal growth and development of the grapes. At the same time, watersaving agriculture has become the demand for agricultural development in this area. The aim of our study is to investigate the effects of nitrogen application rates on key enzyme activities and related gene expression during nitrogen metabolism, so as to further provide a theoretical reference for the application of nitrogen in wine grape growing in the Gansu Corridor.【Methods】The experiment was carried out in a 10-year-old'Cabernet Gernischet'vineyard of the Mo Gao located in Wuwei City from 2014 to2015. All the grapevines were planted from west to east with simple hedge-shape. The planting spacing was 0.7 m×3.0 m. The area of the plot was about 240 m2, and each plot contained 40 plants. During the test period, the irrigation was carried out according to the“Wuwei Mo Gao Brewing Grape Drip Irrigation Water Distribution Quota”, which formulated by the research team. The irrigation amount in the bud break stage was 675 m3· hm-2, the flowering stage was 900 m3· hm-2, the first berry swell period was 900 m3·hm-2, the secondary shoot growth period was 150 m3·hm-2, and the second berry swell period was 600 m3· hm-2, and overwintering water was 1 275 m3· hm-2. The total irrigation amount during the whole growth period was 4 500 m3·hm-2. Five different application amounts of nitrogen(0 kg·hm-2, 150 kg·hm-2,300 kg·hm-2, 450 kg·hm-2 and 600 kg·hm-2, characterized as CK、N1、N2、N3 and N4, respectively) were applied with a completely randomized block. Urea was used as external nitrogen and applied with water.The 750 kg·hm-2 of calcium superphosphate was applied before the first irrigation during the soil excavation. The 825 kg · hm-2 of potassium sulfate was applied in fruit color-changing period. Effects of nitrogen application rates on total nitrogen content, soluble protein content, key enzyme activities including nitrate reductase(NR), glutamine synthetase(GS), glutamate synthase(GOGAT) and glutamate dehydrogenase(GDH) as well as the related gene expression levels in leaves(VvNR1, VvGS1, VvGOGAT1 and VvGDH1) were determined at different developmental stages(5 th day before anthesis, 20 th day after anthesis, 50 th day after anthesis, 80 th day after anthesis). Ten to fifteen leaves were collected from each treatment. Fresh leaves were punched out with a punch and 1.0 g was weighed for determination of soluble protein content and enzyme activity assay with 3 replicates. The new leaves without pests and diseases on the top of the current shoots were selected to extract RNA. 2.0 g of grape leaves wrapped in tin foil were placed in a liquid nitrogen tank and stored in arefrigerator at-80℃ for RNA extraction.【Results】The results showed that nitrogen application significantly increased the net photosynthetic rate in leaves.The treatment of N2 significantly increased leaf area and the total nitrogen content in leaves in comparison with CK 5 th day before anthesis, 20 th day after anthesis and 80 th day after anthesis. The soluble protein content increased by nitrogen application in leaves on 80 th day after anthesis. The NR activity in leaves showed an increasing trend with the increase of nitrogen application in the range of 0 to 300 kg on5 th day before anthesis, 20 th day after anthesis and 80 th day after anthesis. The GS activity in leaves increased significantly by N2 treatment on 5 th day before anthesis, 50 th day after anthesis and 80 th day after anthesis, which were 1.66, 1.25 and 1.34 times more than the control, respectively. The GOGAT activity in leaves increased significantly by N2 treatment on 5 th day before anthesis and 80 th day after anthesis, 0.53 and 0.42μmol·g-1·min^-1, respectively. Compared with CK, N2 and N3 treatments could improve the GDH activity in leaves during the whole growing period. The expression level of VvNR1 in N2 treatment was significantly higher than CK and other nitrogen treatments on 5 th day before anthesis, 20 th day after anthesis and 80 th day after anthesis. VvGS1 was significantly up-regulated by N1 and N2 on5 th day before anthesis, which was up-regulated by 142.33% and 283.47% in comparison with CK, respectively. VvGDH1 was up-regulated during the whole growing period after nitrogen application. With N2 treatment, fruit soluble sugar, the ratio of sugar to acid, tannins, anthocyanin content reached the maximum, the titratable acid content was the lowest, the yield was at a medium level.【Conclusion】The results suggested that different nitrogen application rates affected the expression of VvNR1, VvGS1,VvGOGAT1 and VvGDH1 genes in leaves, thereby improving leaf nitrogen metabolism. In addition, the nitrogen application with 300 kg · hm-2(N2) among all the treatments significantly promoted the accumulation of nitrogen in leaves during the late growth period. At the same time, it promoted the increase of leaf area and net photosynthetic rate, and improved the fruit quality.