Effects of Water and Nitrogen Coupling on Photosynthetic Characteristics, Yield and Quality of Isatis Indigotica

In arid areas of Northwest China, Isatis indigotica is a commercial medicinal crop cultivated with higher water and nutrient in a large area, which makes rational irrigation and nitrogen application key factors for successful crop management. The objective of this study was to determine the effect of water and nitrogen coupling on photosynthetic characteristics, yield, and quality of Isatis indigotica produced in northwestern China. Field trials were conducted for two consecutive years on the irrigation test station. Data on photosynthetic parameters, yield and quality were collected from individual Isatis indigotica for each treatment during the years 2018–2019. The application of nitrogen signicantly increased photosynthetic rates and yield under the same irrigation conditions. However, the yield were reduced in the excess water treatments, W3N1, and W3N2 and in excess nitrogen treatments, W1N3, W2N3, W3N3, in contrast to the optimum W2N2 treatment. Moreover, the quality indicators of the W2N2 treatment decreased compared with CK, which was due to water stress and more photo-assimilates being available to the roots, but the effective quality index value cloud be effectively improved by greatly increasing the yield.


Introduction
In the oasis region of Hexi, China, agricultural development is limited by water shortage and excessive application of nitrogen fertilizer. The main research question is how to improve water use e ciency and reduce nitrogen application. Two essential factors for crop growth are water and fertilizer. Rational irrigation and fertilization can effectively improve crop yield. The main problem facing modern agriculture is how to promote fertilizer with water and transfer water with fertilizer [1] . Reasonable water-nitrogen coupling optimization model has been paid more and more attention by researchers. Water is the medium through which soil nutrients are effectively absorbed by plants, improving the e ciency of nitrogen utilization. However, excessive water will lead to leaching and loss of nitrogen, while excessive nitrogen application will lead to non-point source pollution [2] .
At present, more and more studies have been done in crops under the coupled conditions of water and nitrogen. For example, Liu et al [3] ., Li et al [4] ., Sui et al [5] ., Fiasconaro et al [6] ., Gholamhoseini et al [7] .,respectively studied the coupling of water and nitrogen on the yield and quality of rice, tomato, cotton, alfalfa, corn and other crops. The synergistic effect of water and nitrogen can save water, increase yield and improve crop quality effectively. The yield and water use e ciency of Isatis indigotica were not high due to ood irrigation. At the same time, in order to increase yield, the phenomenon of excessive nitrogen application is very common [8] .
Scholars have studied the effect of water on the yield and quality of Isatis indigotica from the aspects of water-saving irrigation system [9] and water stress [10] . Unfortunately, no research is currently available on the in uence of water and nitrogen fertilizer on photosynthesis, yield and quality were not discussed. Local farmers fertilized and irrigated only relying on the empirical, resulted in low e ciency of water and fertilizer utilization. The present experiment was undertaken, therefore, to study the effect of water and nitrogen coupling on photosynthesis, yields and quality of Isatis indigotica. According to the data of precipitation for years, the average annual precipitation in this area is 215 mm with little precipitation and large variation rate. The contradiction between supply and demand is prominent, and the drought is frequent. The soil is light loam with pH value of 7.22, eld water holding capacity of tillage layer soil is 24%, soil bulk density is 1.4 g·cm − 3 , groundwater level is low, and the area does not show salinization and alkalization.

Test Materials and Cultivation Methods
The full Isatis indigotica seeds were independently planted in the Department of Chinese herbal medicine in the Gansu Agricultural University, of which the seed purity was 96%, the weight per 1,000 seeds was 9.873 g, the germination rate was 87.6%, and the germination potential was 46.4%. Sowed on May 3 and harvested on October 13, the seeds have the sowing amount of 30.0 kg·hm − 2 and the planting density was 800,000 plants·hm − 2 . Before sowing, the experimental zone was ploughed for 30 cm to remove weeds manually. At the same time, 350 kg·hm − 2 calcium superphosphate (12% of P 2 O 5 , 10% of S, 16 % of Ca) and 200 kg·hm − 2 source potassium (25% of K 2 O) was applied. Each experimental plot is separated by a lm with a width of 60 cm to prevent water from seepage underground.

Experiment Design
The growth stage of Isatis indigotica is divided into four growth stages according to its growth characteristics: seedling stage, vegetative stage, eshy root growth stage and eshy root maturity. Three irrigation treatments were set in the eld experiment, W1, W2, and W3, respectively, which were 60% 7 0% of eld water capacity, 70% ~ 80% of eld water capacity, and 80% ~ 90% of eld water capacity.
Three nitrogen treatments, N1:150kg·hm − 2 , N2:200 kg·hm − 2 , N3:250 kg·hm − 2 .There are 10 water control treatments, of which CK was the control treatment. Each treatment was repeated three times, totaling 30 districts. The area of each district was 36 m 2 (9 m × 4 m). The method of irrigation is drip irrigation under mulch. The speci c experimental design is shown in Table 1. and intercellular CO 2 concentration (Ci) were measured in situ for the seventh or eighth leaf that was fully expanded [11] (counted back from the apex of new shoots). In each treatment, three to four sunlit healthy leaves for each sampled plant were randomly selected from different directions and labeled; each leaf was measured once, in triplicate for each treatment. The average value of each treatment was calculated.

Yeild and Water use e ciency (WUE)
At harvest time, the yield of plot was weighed and counted separately, and the yield of each treatment was the average of three replicates.
where WUE is water use e ciency (kg·hm − 2 ·mm), Y is yield per unit area of Isatis indigotica (kg·hm − 2 ·mm), ET a is water consumption (mm) during the whole growth period of Isatis indigotica.

Quality
Determination of indigo, indirubin, (R, S)-goitrin content: The method of Chinese Pharmacopoeia [12] was used to extract indigo, indirubin, (R, S)-goitrin, and its content was determined by high performance liquid chromatography. The content of polysaccharide in Isatis indigotica root was determined by phenolsulfuric acid colorimetry.

Statistical analysis
The data analyses were performed using SPSS software (SPSS, Chicago, IL). The signi cance of differences between treatments for the different measured parameters was evaluated Independent samples followed by Kruskal-Wallis test (P ≤ 0.05). The GraphPad Prism 5.01 was used to draw the graph. The data in each table were average values of three replicates.
3 Results And Discussion

Photosynthetic characteristics
Water and nitrogen coupling treatment had a signi cant effect on Photosynthetic characteristics (Fig. 1).
Generally, the net photosynthetic rate in the treatment order: CK, W1N1, W1N3, W3N1, W3N3, W2N1, W1N2, W3N2, W2N3 and W2N2. The treatment with low water and low nitrogen was signi cantly lower than that with W2N2. The stomatal conductance and transpiration rate showed similar changing patterns. The net photosynthetic rate showed unimodal trend with the increase of nitrogen application at the same irrigation level. Under the same nitrogen application level, the net photosynthetic rate increased rst and then decreased slowly with the increase of irrigation amount, showing the value of W2 > W3 > W1. The net photosynthetic rate was the highest and the mean value was 13.87 µmol·m − 2 ·s − 1 under treatment W2N2. The results showed that severe water stress and excessive nitrogen were not conducive to the absorption and utilization of water and nutrients by crop roots, which led to the decrease of photosynthetic rate. The effect of water and nitrogen treatment on intercellular CO 2 concentration was signi cant (Fig. 1). Under the condition of too much water or too much nitrogen, the photosynthesis of Isatis indigotica was disadvantageous, and the intercellular CO 2 concentration showed a trend opposite to the net photosynthetic rate.
Compared with the treatment of N, P and K de ciency, water-N coupling could increase the Pn of crops, which was the same as that of other fruit trees and vegetables [13] . Photoassimilates accumulated in the third internode of the upper part of the main stems, as well as in the ag leaf sheath, are mobilized in a higher proportion and can contribute to grain lling in rice plants subjected to water stress in the tillering phase [14] . The net photosynthetic rate (Pn), stomatal conductance (GS) and transpiration rate (Tr) of maize leaves at seedling stage decreased signi cantly, while the intercellular CO 2 concentration (Ci) increased signi cantly when the nitrogen application rate was low [15] .
The experiments with Isatis indigotica demonstrate that the net photosynthetic rate (Pn), stomatal conductance (GS) and transpiration rate (Tr) under the same irrigation level increased rstly and then decreased with the increase of nitrogen application rate. The net photosynthetic rate, transpiration rate and stomatal conductance of Isatis indigotica were improved by rational nitrogen application. Similar ndings have been reported for Isatis indigotica, with the decreases of N level,the net photosynthetic rate transpiration rate and stomatal conductance of leaves gradually decreased while the intercellular CO 2 concentration (Ci) increased [16] [17] .

Yeild and Water use e ciency
The Isatis indigotica yield values presented are the average of two consecutive years of water nitrogen trials (Fig. 2). The Isatis indigotica yields differed signi cantly between the water nitrogen treatments; the W2N2 and W2N3 treatments had the highest yields of 7277.5 and 6820.5 kg·hm − 2 , respectively. The lowest yield of 3264.5 kg·hm − 2 was recorded in the control treatment. The yield of all treatments was signi cantly higher than that of the control treatment. The yield of the W2N2 and W2N3 treatments was signi cantly higher than that of the W1N1 and the W3N1. With the increase of nitrogen application rate, the yield rst increased and then decreased under the same irrigation condition.
The Water use e ciency values of Isatis indigotica presented are the average of two consecutive years of water nitrogen trials (Fig. 2). The Water use e ciency of Isatis indigotica differed signi cantly between the water nitrogen treatments; the W1N2 and W2N2 treatments had the highest yields of 20.78 and 19.63 kg·mm − 1 ·hm − 2 , respectively. The lowest yield of 13.65 kg·mm − 1 ·hm − 2 was recorded in the W3N1 treatment. The Water use e ciency values of the W1N2 and W2N2 treatments was signi cantly higher than that of the W3N3, which was the treatment of excess water and nitrogen fertilizer. The Water use e ciency decreased with the increase of irrigation under the same nitrogen application condition. The Water use e ciency increased rst and then decreased with the increase of nitrogen application rate under the same irrigation conditions. TheW2N2 treatment was the highest yield and Water use e ciency. Therefore, the water-nitrogen coupling mode of medium water medium nitrogen achieved the highest yield and effectively save water.
Generally, appropriate water de cit can improve crop yield and water use e ciency [18] [19] , and rational fertilization can increase crop yield, such as fruit trees and vegetables [20][21] [22] . The yield increase in the current experiment was probably related to reasonable water stress and reasonable nitrogen application: theW2N2 treatment was the highest yield and Water use e ciency. However, too much water and too much nitrogen reduced yield and water use e ciency of the Isatis indigotica. This was consistent with recent research reports [23] [24] . Compared with the local ooding irrigation and excessive nitrogen fertilizer mode, the W2N2 treatment with middle water and nitrogen not only obtained high yield, but also signi cantly improved the water use e ciency. It can also reduce the effect of excessive application of water and fertilizer on soil productivity, which is a better water and nitrogen management mode for local Isatis indigotica production.

Quality
The Isatis indigotica quality values presented are the average of two consecutive years of water nitrogen trials (Fig. 3). It mainly included the following content indicators: Indigo, Indirubin, (R, S)-goitrin and Polysaccharide. The Isatis indigotica quality indicators differed signi cantly between the water nitrogen treatments. The CK treatments had the highest values in all quality indicators, respectively. Each quality indicator decreased gradually with the increase of water content under the same nitrogen application conditions. Each quality indicator decreased gradually with the increase of nitrogen application under the same water condition. The content (R, S)-goitrin of the W2N2 treatment decreased by 6.5% compared with CK; decreased by 3.9% compared with W1N1 treatment.
Water is the medium for improving crop quality. Generally, the crop quality was improved by suitable water de cit [25][26] [27] and reasonable fertilization [28] [29] [30] . Gradually, the quality of Isatis indigotica in the current experiment was accumulated with the decrease of water. The water de cit treatment increased the content of effective components and improved the quality of Isatis indigotica. The content of effective components in all treatments reached the pharmacopoeia standard [12] . The quality indicator values of each treatment in the current experiment were signi cantly lower than those of CK treatment, but there was little difference in the quality indicator values between each treatment. Moreover, the yield of the control treatment was much lower than other treatments. Therefore, the effective quality content of the control treatment is lower than other treatments. Too much water and nitrogen were not conducive to quality accumulation, which was not consistent with recent research reports [31] . That showed the total N applications over 280 kg ha − 1 did not increase yield or quality and N above 56 kg ha − 1 had no impact on plant biomass or tuber yield on any farm in any year of this study.

Conclusions
The current research indicates that there are some distinct bene ts of water -nitrogen coupling. The W2N2 treatment of water and nitrogen in medium could signi cantly promote net photosynthetic rates, and increase the yield and water use e ciency, but lack or excess of water and nitrogen would greatly reduce the effects. Reasonable water stress had a signi cant positive impact on the quality of Isatis indigotica under the same nitrogen application condition, but had no signi cant effect on the yield. The yield and quality of Isatis indigotica could be improved by reasonable water stress and moderate nitrogen application.