Effects of Phosphorus Level on Quality Traits and Quality-Related Enzymatic Activity in Lettuce

In this experiment, we studied the effects of the application of various amounts phosphorus on the quality traits and quality-related enzyme activity in lettuce. When we supplied lettuce with 0.3 mmol/L or 0.4 mmol/L phosphorus, the yield was higher than that of the control. In addition, the quality traits were better, the nitrate content was lower and the nitrate reductase, catalase and ascorbic acid oxidase activities were higher in these plants than in the control. In addition, the chlorophyll content was higher, and the root activity was stronger, in plants supplied with 0.3 or 0.4 mM/L phosphorus than in the control. These results suggest that the application of 0.3 or 0.4 mM/L phosphorus to lettuce is optimal for plant growth.


Introduction
Phosphorus is one of the most important elements that are required for crop growth. Phosphorus is required for many physiological and biochemical processes in plants, including photosynthesis and carbohydrate synthesis, transformation, nitrogen metabolism and adipose synthesis, and it has an important effect on the growth, yield and quality vegetables 1,2,3 . However, due to the long-term emphasis on the application of nitrogen and potassium to crops, fertilizers have a relative lack of phosphorus, which is the biggest obstacle to optimal vegetable production. If plants lack phosphorus, protein synthesis is blocked. This seriously impacts plant growth, nitrate absorption and assimilation 4 .
Lactuca sativa L., commonly known as lettuce, belongs to asteraceae, genus Lactuca. Lettuce, an annual or biennial herb, is one of the four vegetables that are grown by soilless cultivation in worldwide. The cultivation area of lettuce continues to expand, as lettuce has been greatly enjoyed by consumers in recent years. To achieve high production quality, chemical fertilizers are often utilized in vegetable cultivation 5 . The incorrect application of fertilizer is one of the most important factors that affect the yield and quality of vegetables. Recent studies have focused on the effects of the level of nitrogen application, nitrogen form and ratio and the amount of potassium on the growth and development of leafy vegetables, as well as nitrate accumulation 6,7,8 , but studies on the effects of phosphorus on leafy vegetable growth and development have been limited to the study of nitrate, most of which is single factors 9,10 . Therefore, it is important to conduct an indepth, systematic study on the effects of phosphorus on lettuce.
In this study, we grew lettuce plants in soil with xed levels of nitrogen and potassium and varied amounts of phosphorus to study the effects of phosphorus levels on plant physiological indicators, quality-related enzyme activity, production and quality. The aim of this study was to determine an economically feasible and effective level of phosphorus for the production of high-yielding, high-quality lettuce to enhance the e cient production of this crop.

Results
Effects of different phosphate levels on nitrate content and nitrate reductase activity in lettuce The effects of different phosphate levels on nitrate content and nitrate reductase activity of lettuce are shown in Figure 1.
As the phosphorus concentration increased, the nitrate levels generally decreased; only treatment 6 increased the nitrate levels slightly. These results show that phosphorus levels are closely related to nitrate absorption and nitrogen metabolism. As the phosphorus concentration increased, nitrate reductase activity increased, followed by a decline. The nitrate reductase activity was signi cantly higher in treatment 4 plants than in the other treatments, while nitrate reductase activity was the lowest in treatment 1 plants. These results indicate that nitrate reductase activity is closely related to nitrate content.
Effects of different phosphate levels on catalase and ascorbate oxidase activity in lettuce The effects of different phosphate levels on the activity of catalase and ascorbate in lettuce are shown in Figure 2. As phosphorus levels increased, the hydrogen peroxide enzyme activity generally increased; only treatment 6 produced a relative decline in enzyme activity. These results illustrate that catalase activity is closely related to plant metabolism. With increasing levels of phosphorus, ascorbic acid oxidase activity rst increased, followed by a decline. There was no signi cant difference between the ascorbic acid oxidase activity levels in treatments 4 and 5, but these levels were signi cantly higher than in other treatments, and treatment 1 produced the lowest ascorbic acid oxidase activity level; ascorbic acid oxidase activity is closely related to plant stress resistance.
Effects of different phosphate levels on chlorophyll content and root activity in lettuce The effects of different phosphate levels on chlorophyll content and root activity in lettuce are shown in Figure 3. As the phosphorus content increased, the chlorophyll content increased, but treatment 6 led to signi cantly decreased chlorophyll content. These results indicate that chlorophyll content is closely related to nutrition and metabolism; with increasing phosphorus content, root activity increased, followed by a decrease. The root activity in treatment 5 plants was signi cantly higher than that of other treatments. Treatment 1 plants had the lowest root activity, but there was no signi cant difference in root activity between treatments 6 and 1. Root activity is an important factor that re ects the strength of crop metabolic activity.

Effects of different phosphate levels on lettuce yield and nutritional quality
The effects of different phosphate levels on lettuce yield and nutritional quality are shown in Table 1. With increasing phosphorus content, the yield per plant of lettuce increased initially. When a certain level of phosphorus was applied, the lettuce yield per plant began to decline. In addition, the vitamin C content rst increased and then decreased; the vitamin C content of treatment 5 plants was signi cantly higher than that of the other treatments, and treatment 1 plants had the lowest vitamin C content. With increasing phosphorus application, the protein level decreased, followed by an increase. There was no signi cant difference in protein level between treatments 4 and 5, but these levels were signi cantly higher in treatment 4 and 5 than in the other treatments; the vitamin C content in treatment 1 was the lowest. The soluble sugar content rst increased gradually, and then slowly declined, in response to increasing phosphorus levels. The soluble sugar content was signi cantly higher in treatment 4 plants than in the other treatments, while the soluble sugar content of treatment 6 plants was the lowest. The soluble solids content increased, followed by a decrease, in response to increasing phosphorus levels. The soluble solid content in treatment 5 plants was signi cantly higher than that in the other treatments, while treatment 6 plants had the lowest soluble solids content.

Discussion
Food safety in an important issue of widespread concern. Vegetables are some of the most widely consumed foods. With the increasing application of chemical fertilizers, there has been a sharp increase in nitrite and nitrate accumulation in vegetables. Green leafy vegetables are subjected to high levels of fertilization, so these vegetables accumulate high levels of nitrate in vivo, which lowers the nutritional value of the crop 11 .
Nitrate accumulation and metabolism in leafy vegetables is a complex process; exogenous application of nitrogen is the dominant factor that promotes an increase in nitrate content. With increasing levels of nitrogen application, the accumulation of nitrate content increases with no threshold. Therefore, the primary way to reduce nitrate content is to control the rate of nitrogen application 12 . Phosphorus is a nutrient that is required for plant growth and development.
Phosphorus promotes nitrogen absorption and assimilation, and phosphorus is also an important part of nitrate reductase (NR) and nitrite reductase (NiR), which participate in NO 3 restoration and assimilation 13 . In this experiment, with increasing phosphorus content, the nitrate content generally decreased. Treatment 6 (0.5 mmol/L phosphorus) improved the nitrate content, but the nitrate contents in all of the treatments were below the maximum safe levels for leafy vegetables (432 mg/kg) 14 , so all of the treatments produced safe, high quality vegetables.
Nitrate reductase (NR) is a key enzyme in the process of plant nitrogen metabolism; NR is the rate-limiting enzyme in this process. Increased NR activity (NRA) can improve the utilization of nitrogen and reduce the nitrate content of vegetables 15 .
Studies have shown that under soilless cultivation conditions, 7-day-old cucumber seedlings exhibit some tolerance to osmotic stress; the plant growth potential of the plant is not affected by moderate levels of osmotic stress. However, the application of sodium sulfate or PEG causes a 20% decline in both the fresh weight and dry mass of the roots, accompanied by slower cotyledon growth. Salt stress leads to a signi cant reduction in the nitrate contents of roots and cotyledons, while nitrate reductase activity in roots does not change signi cantly, and the enzyme activity in cotyledons decreases slightly 16 . In the current experiment, the nitrate reductase activity in treatment 4 (0.3 mmol/L) plants was signi cantly higher than that in the other treatments. In addition, as the phosphorus content increased, the nitrate reductase activity declined, which indicates that high phosphorus levels stress the plants, thereby affecting nitrate reductase activity.
Catalase (CAT), which is ubiquitous in plants, plays a major role in the decomposition of hydrogen peroxide produced in the process of respiration, which helps eliminate damage caused by the accumulation of peroxide 17  Chlorophyll content is an important indicator of crop nutrition and growth conditions. Chlorophyll content is closely related to photosynthetic rate, especially during the accumulation of photosynthetic assimilates; chlorophyll content and the photosynthetic intensity of leaves are positively correlated 20 . At the same time, root activity is an objective physiological indicator 21 . Root absorption vitality re ects the overall performance of root weight, quantity, root water uptake and water delivery; high root absorption vitality indicates that the weight, quantity, water uptake and water delivery of roots are good, which lays the foundation for high yield. In this experiment, the appropriate level of phosphate treatment led to a signi cant increase in chlorophyll content and root activity, which increased the yield of the lettuce.
In this experiment, the yield per lettuce plant was higher in treatment 4 (0.3 mmol/L) and treatment 5 (0.4 mmol/L) plants than in the other treatments. This trend was also observed for vitamin C content, protein content, soluble sugar content and soluble solids content. However, high levels of phosphorus treatment led to a decrease in the levels of these indicators.
These results are similar to the results of Chesti M H et al. 22 and Ahmad M et al. 23 .
In this experiment, we found that treatment 4 (0.3 mmol/L phosphorus) and treatment 5 (0.4 mmol/L phosphorus) led to the production of lettuce plants with lower nitrate content, higher nitrate reductase, catalase and ascorbic acid oxidase activity, higher chlorophyll content, stronger root activity, higher yields and better quality than the other treatments. These phosphate treatment levels are therefore suitable for lettuce production.

Materials And Methods
The lettuce (Solanum tuberosum L) seeds, Grand Rapid, were purchased from Horticultural Branch of Heilongjiang Academy of Agriculture Sciences (Harbin, Heilongjiang, China, 150069). The compositions of the major elements of the nutrient solutions are shown in Table 2, and the trace element composition of the nutrient solution is shown in Table 3. The seeds were disinfected with 0.1% HgCl 2 solution prior to planting. The seeds were sown in plastic pots on May 16, and the seedlings emerged after 3 days. The pots were watered with tap water during for rst 5 days of the experiment, after which they were watered with nutrient solution (containing the proper concentration of phosphorus) once per day for 5 days and 1-2 times per day thereafter. The plants were watered at a rate of 1.5 L per pot (to maintain appropriate moisture levels). The plants were harvested 40 days after emergence and subjected to analysis.
All the experiments are conducted in the accordance with the relevant institutional, national, and international guidelines /legislation.

Plant Analysis
Thirty plants of uniform appearance per treatment were randomly selected, washed with distilled water, dried with absorbent paper and weighed using an electronic balance.
The nitrate content of the lettuce was determined spectrophotometrically at 540 nm, and the mg/kg fresh weight was calculated as described by Bian et al 24 . The nitrate reductase activity (NRA) of the plant material was spectrophotometrically determined 25 . Details about the NRA test are also presented in Arslan and Güleryüz 26 . Total CAT activity was spectrophotometrically measured using the method of Kato M and Shimizu 27 . Ascorbic acid oxidase extraction and activity analysis were performed using the method of Pignocchi et al 28 .
The extraction and measurement of chlorophyll were performed as follows: 0.1 g of green leaf tissue was placed in a sample vial containing 25 ml of acetone, which was then sealed; each extraction was performed in triplicate. The chlorophyll extracts were stored in a dark environment for 12 h under ambient conditions or at 4°C. The absorption spectrum of chlorophyll was recorded between 350 and 750 nm at a scan speed of 400 nm/min using an ultraviolet-visible (UV-VIS) spectrophotometer. The chlorophyll content was then calculated using the extinction coe cients and equations of Gratani 29 . Measurements of root vigor were performed using the triphenyltetrazolium chloride (TTC) method 30 .
The extraction and measurement of ascorbate (vitamin C) was performed using the standard 2,6 dichlorophenolindophenol dye method 31 . Soluble proteins were measured by Coomassie brilliant blue method 32 . The content of soluble sugars was measured by the method of sulfuric acid anthrone 33 . Soluble sugars were extracted in 5 mL of boiling 80% (v/v) ethanol for an hour, followed by centrifugation at 10,000 × g at 4°C for 10 min. The process was repeated for complete extraction. The amounts of total soluble sugar were determined using anthrone reagent, with glucose as the standard. The soluble solid contents were determined using Abel refractometer direct determination 34 .

Statistical Analysis
The results obtained in this experiment were subjected to statistical analysis using the SPSS 13.0 statistics program. Multiway analysis of variance and Duncan's test (P ≤ 0.05) were applied for the determination of the signi cance of differences between means. All experiments were performed with three technological replications from two years of investigation, and the results were presented as the mean of all data combined.

Conclusion
Yield and quality have always been the core factors of lettuce production. It is of great signi cance to study the regulation of nutritional conditions on lettuce production. As an essential nutrient element for crops, phosphorus plays an important role in the growth and development, yield and quality formation of vegetables. The results showed that appropriate phosphorus could increase chlorophyll content and root activity, improve nitrate reductase activity, catalase activity and ascorbic acid oxidase activity, increase quality traits including vitamin C, protein, soluble sugar and soluble solid content, reduce nitrate content and increase yield. These ndings provide a better guidance for the production practice of lettuce with high yield, high quality and safety.