Effects of ammonium to nitrate ratio on growth, nitrogen metabolism, photosynthetic efficiency and bioactive phytochemical production of Prunella vulgaris

Abstract

Context: Prunella vulgaris L (Labiatae) is commonly used as a traditional medicinal herb in some Asian and Europe countries. To date, few studies have been conducted to determine the influence of  − N/ − N ratio on growth, physiological development, and bioactive phytochemical accumulation in hydroponically grown P. vulgaris.

Objective: The current study was conducted to evaluate the effect of five  − N/ − N ratios on growth, nitrogen metabolism, photosynthetic efficiency, and bioactive phytochemical production in P. vulgaris.

Materials and methods: Hydroponically cultivated P. vulgaris were fertilized with five  − N/ − N ratios in a greenhouse for 85 d. Dried weight of root, stem, leaf and spica, leaf area, photosynthetic efficiency, activities of nitrate reductase (NR), glutamine synthetase (GS), and the concentrations of N, soluble protein, and free amino acids in the leaves, as well as the contents of rosmarinic acid (RA), ursolic acid (UA), and oleanolic acid (OA) in the spicas were measured.

Results: Both  − N and  − N as the sole source of nitrogen had inhibitory effects on P. vulgaris growth. P. vulgaris fertilized with the 25/75 ( − N/NO₃ − N) ratio had the highest leaf area, photosynthetic rate, and chlorophyll content. The 25/75 (/) ratio increased the spica biomass by 1828%, nitrate-reductase (NR) activity by 98%, and soluble protein concentration by 29.45% compared with the 100/0 (/) treatment. Additionally, 25  − N/75 NO₃ − N resulted in the highest contents of RA and total flavonoids as well as relatively high contents of UA and OA; therefore, this ratio had the highest yield of RA, UA, OA, and total flavonoids in spicas.

Discussion and conclusion: The use of 25  − N/75  − N is recommended to improve biomass production and medicinal quality of P. vulgaris.

• Ammonium/nitrate ratios
• bioactive-component
• photosynthetic rate

Introduction

Prunella vulgaris L (Labiatae) is an herbaceous medicinal plant that is widespread in North Asia, Europe, and North Africa (Chen et al., 2011). Prunellae Spica, the dried spica of P. vulgaris, is a standard medicinal material in the Chinese Pharmacopoeia (Chinese Pharmacopoeia Commission, 2010). It is a well-known Chinese medicinal herb used to reduce fever, alleviate sore throat, and accelerate wound healing (Chen et al., 2011; Pinkas et al., 1994; Psotová et al., 2003, 2006). It is also commonly used as a traditional medicinal herb in some Asian and Europe countries, such as Korea, Japan, Turkey, the Czech Republic, Poland, and Germany (Chen et al., 2013). In addition to its pharmaceutical uses, the spicas of P. vulgaris are manufactured for tea, and the fresh leaves of P. vulgaris are consumed as a vegetable dish in southeastern China (Chen et al., 2011, 2013).

Prunella vulgaris is rich in phenolic acids, triterpenes, flavonoids, and the anionic polysaccharide prunelline (Chen et al., 2013; Cheung & Zhang, 2008; Jiang et al., 2008). Rosmarinic acid (RA), one of the phenolic acids found in P. vulgaris, suppresses lipoperoxidation, scavenges superoxide radicals, and exhibits antiinflammatory and antioxidant bioactivities (Psotová et al., 2006). Rosmarinic acid content has been used as the criterion for quality control of Prunellae Spica in the Chinese Pharmacopoeia (Chinese Pharmacopoeia Commission, 2010). Triterpenes are the dominant compounds in P. vulgaris (Cheung & Zhang, 2008). Among triterpenes, ursolic acid (UA) and oleanolic acid (OA) are especially known for their bioactivities, including hepatoprotection, antihyperglucemia, antifungal, antitumor, and antiinflammatory activities (Liu, 1995). Total flavonoids exhibit a wide range of biological effects, including antiviral, antiinflammatory, antibacterial, vasodilatory, and antiallergic actions (Chen et al., 2013; Cook & Samman, 1996; Liu et al., 2012).

Because of the medicinal and industrial importance of P. vulgaris, the demand has steadily increased in the world market. Hence, a rational agronomic practice for P. vulgaris is necessary to meet the strong market demand for uniform and high-quality raw material (Chen et al., 2011). Soilless culture is a promising cultivation method for the production of high-yield and high-quality medicinal plants using careful management of the nutrient solution and other conditions. It offers several advantages over field cultivation, such as process standardization, faster crop growth, year-round production and the production of high-quality raw material that is clean and easy to process due to minimal contamination from pollutants and micro-organisms (Letchamo et al., 2002). Moreover, secondary metabolism can be regulated in soilless culture systems via appropriate control of the nutrient solution fed to the plants (Chen et al., 2013).

Nitrogen (N) is essential for plant growth and is principally taken up by plants in the forms of nitrate () and ammonium () (Tang et al., 2011). The comparison between  − N and  − N as sources of nitrogen in uptake and growth has received considerable attention, and the most effective ratio of  − N and  − N has been of great interest in the study of N nutrition of vegetable crops (Chen et al., 2005). The form of N modifies the growth, morphology, physiological, and biochemical processes of plants, as well as the chemical composition of tissues in many plants, such as cucumber (Kotsiras et al., 2002), strawberry (Tabatabaei et al., 2006, 2008), Chinese cabbage (Chen et al., 2005), rice (Guo et al., 2007a), and tea (Ruan et al., 2007). The responses of different plants to the N form vary with species.

The ratio of  − N/ − N influences not only plant growth but also secondary metabolite formation in cell culture, hairy root culture or soilless culture of various medicinal plants (Lee & Paek, 2012; Liu et al., 2003; Naik et al., 2011; Prasad et al., 2012; Praveen & Murthy, 2013; Wang & Tan 2002). Carbon and nitrogen metabolism and their shifts in primary and secondary metabolites are largely altered by external nitrate availability. Compared with primary metabolism, the response of secondary metabolism to nitrogen form has rarely been reported and needs to be investigated in detail (Guo et al., 2012).

Although many studies have reported the pharmacological properties of P. vulgaris, agronomic practices for obtaining optimal biomass, physiological attributes, and phytochemical production of this plant remain poorly understood (Chen et al., 2012, 2013), especially with regard to the effects of N source on growth performance, physiological development, and bioactive phytochemical accumulation. In our previous study (Yu et al., 2011), we explored the response of P. vulgaris growth and photosynthetic characteristics to N source. However, the Spring-sown P. vulgaris remained in the vegetative growth phase through the experimental period, and no spicas formed. As a consequence, we could not evaluate the medicinal qualities of P. vulgaris. Hence, the present experiment using Autumn-sown P. vulgaris was conducted. The objectives of the present work were (1) to assess the effect of the  − N/ − N ratio on the growth, photosynthesis, nitrogen metabolism, and bioactive component accumulation in hydroponically grown P. vulgaris and (2) to find the optimum  − N/ − N ratio for maximal biomass yield and bioactive component production.

Materials and methods

Plant treatment and growth conditions

Hydroponic experiments were conducted in a greenhouse at the Institute of Chinese Medicinal Materials, Nanjing Agricultural University, Nanjing, China. The seeds of P. vulgaris were collected from Queshan county, Henan province in June, 2009. Seeds were germinated in propagation pots on October 2009. On 28 March 2010, after approximately 10 d of cultivation in the hydroponic solution, uniform seedlings with four fully expanded leaves were transferred to a 5 L polypropylene container with different nutrient solutions with different inorganic N compositions ( − N/ − N ratios of 100/0, 75/25, 50/50, 25/75, and 0/100). The constant N concentration, 15 mmol L⁻¹, was based on our previous work. Modified Hoagland and Arnon nutrient solution (Hoagland & Arnon, 1950) was used (Table 1). The nutrient solution also contained 23 μmol/L B, 0.16 μmol/L Cu, 4.8 μmol/L Mn, 3.8 μmol/L Zn, 25 μmol/L Fe (Fe-EDTA), and 0.07 μmol/L Mo. Throughout the experiment, the pH of each nutrient solution was adjusted to 6.2 ± 0.2 with 0.1 mol/L NaOH or 0.1 mol/L HCl. The solution was completely renewed every 7 d.

Table 1. Nutrient solution compositions (mmol/L) with a constant N concentration (15 mmol/L) and different  − N/ − N ratios.

	− N/ − N ratios
Nutrient source	100/0	75/25	50/50	25/75	0/100
Ca(NO3)2	0	1.25	0	3.75	5
KNO3	0	1.25	0	3.75	5
(NH4)2SO4	7.5	5.625	0	1.825	0
NH4NO3	0	0	7.5	0	0
CaCl2	5	3.75	5	1.25	0
KCl	5	3.75	5	1.25	0
KH2PO4	2	2	2	2	2
MgSO4	2.5	2.5	2.5	2.5	2.5

Each treatment consisted of 10 pots, and each pot contained four seedlings. The treatments were arranged in a completely randomized block design with three replications. Hydroponic culture experiments were conducted in a growth chamber with a 12 h photoperiod at 27 °C/20 °C (day/night) temperatures, 60–70% relative humidity, and 1000 µmol m⁻² s⁻¹ of photosynthetically active radiation. The seedlings were harvested on 20 June 2010.

Growth parameter determination

The harvested seedlings were separated into root, stem, leaf and spica, oven-dried at 65 °C for 72 h to a constant weight, weighed, and ground to pass through a 0.25 mm sieve. The second fully expanded leaf from each plant was collected and used to determine leaf area (LA) with a portable LI-3100 Area Meter (LI-COR, NB, Lincoln, NE).

N concentration

The concentration of N in the second fully expanded leaves was determined using the Kjeldahl method.

Physiological parameter determination

Photosynthesis and transpiration parameters were measured on the second fully expanded leaf of P. vulgaris seedlings using an LI-6400 XT portable photosynthesis system (LI-COR, NB, Lincoln, NE). Chlorophyll measurement was performed as in Arnon (1949). A 0.5-g sample of fresh leaf was incubated in 10 mL of 80% acetone in the dark for 48 h at room temperature (approximately 25 °C). The absorbance of the extract was measured at 470 nm, 645 nm, and 663 nm. Measurements were made on 6 June 2010.

On 6 June 2010, fresh samples from the second fully expanded leaves were frozen in liquid nitrogen for 1 min and subsequently stored at −80 °C for the determination of soluble protein and free amino acid (FAA) contents and for the key enzyme-activity assay. Approximately 1 g of leaves was homogenized with 10 mL of 50 mM sodium phosphate (pH 7.8) containing 2 mM EDTA and 80 mM l-ascorbic acid. After centrifugation at 15 000 × g for 20 min, the supernatants were used to determine the soluble protein content, FAA content, and key enzyme activities (Alvim et al., 2001; Cruz et al., 1970).

Nitrate-reductase (NR; EC 1.6.6.1) activity was determined as described by Baki et al. (2000). In brief, the reaction medium (a total volume of 1 mL) contained 50 mM sodium phosphate (pH 7.8), 10 µM FAD, 1 mM DTT, 5 mM KNO₃, and 20 mM EDTA. The reaction was initiated by adding 200 µL of extract and terminated after 5 min by adding 125 µL of zinc acetate solution (0.5 M). Nitrite formation was measured colorimetrically after adding 750 µL of 1% sulfanilamide in 3 M HCl and 750 µL of 0.02% N-naphthyl-ethylenediamine hydrochloride by measuring the absorption at 546 nm.

Glutamine synthetase (GS; EC 6.3.1.2) activity was determined by the synthetase reaction (Hadži-Tašković Šukalović, 1986). The volume of the reaction mixture was 2.2 mL, including 0.5 mL of enzyme extract. Hydroxamate formation was measured in the assay mixture after 15 min at 30 °C. The absorbance was measured at 540 nm.

Compound analysis

The contents of RA, UA, and OA were determined using a HPLC system consisting of an LC-20AT Liquid Chromatograph (Shimadzu, Kyoto, Japan). The methods for determining the three bioactive components have been described previously (Wang et al., 2008; Zhang et al., 2007). Determination of total flavonoid content was carried out according to the method of Liu et al. (2012). The total flavonoid content was determined with a spectrophotometric method and was calculated as rutin equivalents.

Statistical analysis

The data were subjected to a one-way analysis of variance (ANOVA) followed by Duncan's Multiple Range Test using the SPSS18.0 software (SPSS Inc., Chicago, IL). Differences among the treatments were considered significant when the p value was < 0.05.

Results

Effects of the / ratio on the growth characteristics of P. vulgaris

The / ratio showed significant effects on the leaf area of P. vulgaris (Figure 1). With the decrease of  − N in the nutrition solution, the leaf area increased initially and then decreased, and the highest values were found in the 25/75 (/) treatment. The lowest leaf area was found in the 100/0 (/) P. vulgaris plants. The leaf area in the 25/75 (/) treatment was 2.52-fold higher than the 100/0 (/) treatment.

Figure 1. Effect of / ratios on leaf area of P. vulgaris. Values followed by the same letter are not significantly different at p < 0.05.

The highest dry weight of spica, leaf, stem, and root were all obtained in the 25/75 (/) treatment (Table 2), followed by the 50/50 (/) treatment. Seedlings fed the 100/0 (/) treatment showed the lowest dry weight of spica, leaf, stem, and root. It is worth noting that 25/75 (/) ratio increased the spica biomass by 18.28-fold compared with the 100/0 (/) treatment. The corresponding increase in leaf, stem, and root dry weight were 2.72-, 5.57-, and 3.59-fold, respectively. Compared with the 25/75 (/) treatment, the 100/0 (/) treatment displayed significantly lower biomass of different organs. Specifically, the spica, leaf, stem, and root dry weight of the 100/0 (/) treatment were 24.99, 51.84, 42.24, and 77.17% lower, respectively, that those of the 25/75 (/) treatment (Table 2).

Table 2. Effect of / ratios on growth characteristic of P. vulgaris L.

	Dry weight per plant (g)
/ ratios	Spica	Leaf	Stem	Root	Total plant
100/0	0.143 e	0.561 e	0.252 e	0.103 d	1.059 d
75/25	1.125 c	0.874 d	0.471 d	0.194 c	2.664 c
50/50	1.574 b	1.591 b	1.052 b	0.379 b	4.596 b
25/75	2.757 a	2.089 a	1.655 a	0.473 a	6.973 a
0/100	0.689 d	1.083 c	0.699 c	0.365 b	2.835 c

Values followed by the same letter within a column are not significantly different at p < 0.05.

Due to the similar responses of biomass production of spica, leaf, stem, and root to the / ratios, total plant biomass followed a similar trend.

Effect of / ratio on chlorophyll content of P. vulgaris leaves

The chlorophyll a content significantly increased as the proportion decreased from 100% to 25%, and then decreased in the 100/0 (/) treatment (Table 3). The highest chlorophyll a content was found in the seedlings fed the 25/75 (/) treatment, while the lowest chlorophyll a content was observed in the 100/0 (/) treatment. The chlorophyll b content followed a similar pattern, resulting in non-significant differences in chlorophyll a/b ratio among the / ratio treatments.

Table 3. Effect of / ratio on chlorophyll content in P. vulgaris leaves.

/ratios	Chlorophyll a content (%)	Chlorophyll b content (%)	Chlorophyll a + b content (%)	Chlorophyll a/b	Carotenoid content (% )
100/0	1.341 d	0.410	1.751 d	3.299 a	0.350 c
75/25	1.477 c	0.477 ab	1.954 c	3.102 a	0.357 bc
50/50	1.602 ab	0.501 ab	2.103 ab	3.203 a	0.409 a
25/75	1.674 a	0.523 a	2.197 a	3.273 a	0.399 ab
0/100	1.565 bc	0.474 ab	2.039 bc	3.305 a	0.367 abc

Values followed by the same letter within a column are not significantly different at p < 0.05.

The carotenoid content initially increased as the proportion decreased from 100% to 50% and then decreased thereafter. The highest carotenoid content was obtained with the application of equal proportions of and (50/50), while the lowest value was found in the 100/0 (/) treatment.

Effect of / ratio on photosynthesis and transpiration parameters of P. vulgaris

The P. vulgaris plants fed the different ratios of / showed significant differences in leaf gas exchange (Table 4). The net photosynthetic rate (Pn) in P. vulgaris leaves increased as the proportion decreased from 100% to 25% and then decreased with the further decrease of proportion. The highest Pn was found in the 25/75 (/) treatment (12.026 μmol/m²·s), followed by the 100/0 (/) treatment (11.511 μmol/m²·s). The change of Tr followed a similar pattern.

Table 4. Effect of / ratio on leaf gas exchange of P. vulgaris.

/ ratios	Pn (μmol/m^2·s)	Gs (mol/m^2·s)	Ci (μmol/m^2·s)	Tr (mmol/m^2·s)
100/0	6.767 e	0.128 e	294.658 ab	1.442 c
75/25	8.205 d	0.147 d	275.803 bc	1.541 c
50/50	9.763 c	0.178 c	263.479 c	2.580 b
25/75	12.026 a	0.257 b	284.148 b	4.119 a
0/100	11.511 b	0.311 a	304.990 a	3.909 a

Values followed by the same letter within a column are not significantly different at p < 0.05.

The Gs value increased as the proportion decreased from 100% to 0%, and the Gs value in the 100/0 (/) plants was 41.16% that of the 0/100 (/) treatment. Ci decreased initially and then increased with the decrease of proportion. The lowest Ci value (263.476 μmol/m²·s) was found in the 50/50 (/) treatment, while the highest Ci value (304.990 μmol/m²·s) was observed in the 0/100 (/) treatment.

Effect of / ratio on nitrogen assimilation and recycling

Nitrate-reductase (NR) activity increased as proportion decreased from 100% to 25%, and then decreased with further decreasing proportion (Table 5). There were significant differences between any two treatments. The NR activity in the P. vulgaris plants supplied with the 25/75 (/ ratio) treatment increased by 98% in comparison with the 100/0 (/) treatment.

Table 5. Effect of / ratio on nitrate reductase (NR), glutamine synthetase (GS) and the concentrations of soluble protein and free amino acids (FAA) in the leaves of Prunella vulgaris.

/ ratios	NR (µg g^−1 FW h^−1)	GS (unit g^−1 FW h^−1)	Soluble protein (mg g^−1 FW)	FAA (mg 100 g^−1 FW)	N (mg g^−1 DW)
100/0	11.68 e	0.171 a	11.92 c	41.95 a	43.87 a
75/25	14.23 d	0.178 a	12.85 c	37.60 b	36.11 b
50/50	18.75 c	0.186 a	14.11 b	34.73 c	33.89 c
25/75	23.14 a	0.152 b	15.43 a	31.59 d	29.75 d
0/100	20.74 b	0.146 b	14.38 b	30.43 d	27.64 e

Values followed by the same letter within a column are not significantly different at p < 0.05.

There was no significant difference in GS activity when the / ratio decreased from 100% to 50%. However, the GS activity in the P. vulgaris leaves supplied with those three / ratios were higher (p<0.05) than those supplied with the 25/75 and 0/100 (/) ratios.

The soluble protein content increased as the proportion decreased from 100% to 25%, and then decreased under further decreasing proportion. The maximum soluble protein content was found in the 25/75 (/ ratio) treatment.

The FAA content decreased with the decrease of the / ratio. The FAA content in the P. vulgaris plants supplied with the 100/0 (/) treatment was 138% that of plants supplied with the 0/100 (/) treatment. The total N content of P. vulgaris leaves showed a similar tendency. The total N content in the P. vulgaris plants supplied with the 100/0 (/) treatment increased by 58.72% compared with those supplied with the 0/100 (/) treatment.

Effect of / ratio on content of medicinal components in P. vulgaris spica

The content of medicinal components in the P. vulgaris spicas was significantly influenced by the / ratio in the nutrient solution (Table 6). The RA content increased as the proportion decreased from 100% to 25% and then decreased under further decreasing proportion. The RA contents in the 100/0 (/) and 0/100 (/) plants were 15.0% and 37.7%, respectively, those of the P. vulgaris plants supplied with the 25/75 (/) treatment. The variation of the total flavonoid content followed a similar trend, except that the plants in the 100/0 (/) treatment showed significant higher total flavonoid contents than those in the 75/25 (/) treatment.

Table 6. Effect of / ratio on content of medicinal components in P. vulgaris spicas.

/ ratios	Rosmarinic acid (%)	Ursolic acid (%)	Oleanolic acid (%)	Total flavones (%)
100/0	0.129 d	0.178 a	0.044 a	4.710 d
75/25	0.133 d	0.148 ab	0.040 ab	4.503 e
50/50	0.224 c	0.135 b	0.030 ab	5.460 c
25/75	0.860 a	0.138 b	0.039 ab	8.402 a
0/100	0.324 b	0.116 b	0.026 b	5.806 b

Values followed by the same letter within a column are not significantly different at p<0.05.

The plants supplied with the 100/0 (/) treatment had significantly higher OA contents than the plants supplied with the 0/100 (/) treatment, while the combination treatment was intermediate. The UA content in the 100/0 (/) plant spicas was significantly higher than those in the 50/50, 25/75, and 0/100 (/) treatments, and the 0/100 (/) plants had the lowest UA content.

The total flavonoid, RA, UA, and OA yields were calculated by multiplying the respective levels of these factors in the spicas by the dry weight of the spicas. Although the highest contents of UA and OA were observed in the 100/0 (/) treatment, the variations in the UA and OA contents among the / treatments were markedly lower than that of the spica dry weight (Table 7). Consequently, the total yields of UA and OA increased with the decreased from 100% to 25% and peaked in the 25/75 (/) treatment. Due to a similar trend in the contents of RA, total flavonoids, and spicas, the total yields of RA and total flavonoids increased as decreased from 100% to 25%, and reached the maximum value in the 25/75 (/ ratio) treatment.

Table 7. Effect of / ratio on yield of medicinal components in P. vulgaris spicas.

/ ratios	Rosmarinic acid (mg/plant)	Ursolic acid (mg/plant)	Oleanolic acid (mg/plant)	Total flavones (mg/plant)
100/0	0.184 e	0.255 d	0.063 d	6.735 d
75/25	1.496 d	1.665 c	0.450 b	50.659 c
50/50	3.526 b	2.125 b	0.472 b	85.940 b
25/75	23.71 a	3.805 a	1.075 a	231.643 a
0/100	2.232 c	0.799 cd	0.179 c	40.003 c

Values followed by the same letter within a column are not significantly different at p<0.05.

Discussion

The effects of and nutrition on plant growth have been well studied, but the results are not consistent and are mainly dependent on plant species. Some plant species, such as maize, bean, pea, and tomato, prefer nitrate nutrition; however, some species, such as rice, pine, and tea, prefer ammonium nutrition (Chang et al., 2010; Guo et al., 2007b). The present experiment demonstrated that a high concentration of either or has an inhibitory effect on the growth of P. vulgaris. When is the sole N source, P. vulgaris plants developed symptoms of toxicity, e.g., shorter stems, margins of old leaves turning brown and withering. By comparison, P. vulgaris plant growth was relatively unaffected when was the sole N source. Hence, it is inferred that P. vulgaris prefers to , which is consistent with our preliminary work on Spring-sown P. vulgaris (Yu et al., 2011).

In many plants, is toxic and impairs plant growth when supplied at high concentrations without (Ruan et al., 2007). toxicity is considered to be the result of such effects as -induced nutrient deficiency caused by impaired uptake of cation, acidification of the root zone, alterations in the osmotic balance, modification of phytohormones, impaired N enzyme metabolism, and change of several metabolite levels, such as amino acids or organic acids (Bybordi et al., 2009; Tabatabaei et al., 2008). In this experiment, the pH of the solution was controlled to eliminate this factor as an impact on plant growth.

For most plants, mixed nitrate () and ammonium () nutrition is superior to  − N or  − N sources alone in terms of plant growth and chemical component accumulation (Bybordi et al., 2009; Chang et al., 2010; Lu et al., 2009; Tabatabaei et al., 2008). The optimal proportions of to for plant growth depend on the plant species, environmental conditions, developmental stage and the total concentration of supplied nitrogen (N) (Claussen, 2002; Lu et al., 2009). Chen et al. (2005) argued that if the / ratio was above 50/50, the yield will decrease in most of the vegetable crops as these aerobic vegetables cannot grow well in an ammonium-dominated N environment. Lu et al. (2009) proposed that the maximum growth and yield of tomatoes or peppers are obtained with an optimal concentration not exceeding 30% of total N in hydroponic cultures in a greenhouse. It was shown that a 25/75 ( − N/ − N) solution with a total N concentration of 5 mmol L⁻¹ was the optimal ratio for the growth of Chinese cabbage (Chen et al., 2005) because this ratio produced the highest photosynthesis rate and total leaf area. Similarly, Bybordi et al. (2009) found that the fresh and dry weight seed yield per plant of Canola (Brassica napus L.) was significantly higher at the 25/75 ( − N/ − N) ratio compared with other ratios.

Similar results were obtained in the present study, i.e., the biomass production of leaf, stem, spica, and root of P. vulgaris all increased as the proportion decreased from 100% to 25%, and then decreased at a higher ratio of in the solution. The highest biomass production in all organs was found in the 25/75 ( − N/ − N) treatment. In our previous work (Yu et al., 2011), the total plant biomass production was examined in the 25/75 and 0/100 ( − N/ − N) treatments, and there was no significant difference between those two treatments. The superiority of mixed N forms to either form alone is related to the maintenance of a nearly neutral pH; higher uptake of phosphorous, potassium, calcium and iron; avoidance of excessive consumption of carbohydrate upon assimilation at the expense of root growth; and cytokinin (CTK) enhancement (Lu et al., 2009).

Leaves are the main photoassimilate sources for plants, and biomass production is associated with both the leaf area and the Pn rate (Bybordi et al., 2009; Tabatabaei et al., 2008). In this study, a reduction in the Pn rate at a high ratio of either or concentration was observed. This result is in agreement with the findings of Tabatabaei et al. (2008), who reported a reduction of Pn rate in -fed strawberries. Some researchers have argued that the accumulation of in the leaves may cause uncoupling of the electron transport from photophosphorylation in chloroplasts, resulting in a lower photosynthetic rate (Tabatabaei et al., 2008). In contrast, the negative effect of nutrition on leaf expansion was explained either by reduced osmotic regulation and concomitantly reduced rates of leaf cell expansion, or by hormonal regulation between roots and shoots (Guo et al., 2007b).

In the present experiment, higher availability of carbohydrate was the result of the increased Pn rate and higher leaf area, which led to better growth of P. vulgaris plants in the 25/75 ( − N/ − N) treatment. The P. vulgaris grown in the 25/75 ( − N/ − N) treatment had the highest net photosynthesis rate, chlorophyll content, and leaf area, which may have facilitated the accumulation of more carbon in P. vulgaris. Similar results were found by Chen et al. (2005) in Chinese cabbage.

An important prerequisite of adaptation of plant species and cultivars to certain environmental conditions are enzymes that are capable of catalyzing nitrogen reduction and assimilation (Bybordi et al., 2009). The activity of NR has often been shown to decrease dramatically if plants are fed ammonium instead of nitrate nitrogen (Bybordi et al., 2009; Tabatabaei et al., 2008). In the present work, the activity of NR increased by increasing from 0 to 25% and then reduced at a higher ratio of in the solution. The highest NR activity was observed in the 25/75 ( − N/ − N) treatment. The variation of soluble protein content followed a similar trend. This result suggests that increasing the amount of ammonium in nutrient solutions will increase NR activity and nitrogen assimilation, but that it has a threshold and that increasing the ammonium above than the critical level will decrease the leaf NR activity. This interpretation is in agreement with Taghavi et al. (2004) and Bybordi et al. (2009), who showed an increase in leaf NR activity in strawberry (Fragaria x Ananassa cv. Selva) and canola (Brassica napus L.) with increasing external ammonium, respectively.

Our data suggested that P. vulgaris is able to increase leaf GS activity substantially under conditions of -sufficient solutions and the capacity to assimilate ammonium by GS might be reduced under -deficient solutions. This response to nutrition is essential to assimilate the majority of to avoid any excessive accumulation of lethal concentrations (Ruan et al., 2007). Xu et al. (2012) found no significant effect on GS activity in Cherry Tomato associated with different N forms.

In the present study, the concentration of total N in P. vulgaris leaves increased with the increasing amount in the nutrient solution and peaked in the 100/0 (/) treatment. This result is consistent with the finding of Bybordi et al. (2009), who reported that a greater concentration of total N was observed with increasing concentration in the nutrient solution. Similarly, Borgognone et al. (2013) observed that the amino acid and total protein concentrations increased in tomato leaves as the proportion increased.

Nitrogen is usually supplied in the culture medium as a combination of ammonium and nitrate salts, and the ratio of / influences secondary metabolite formation significantly in various plant cell cultures (Liu et al., 2003). Naik et al. (2011) found that adventitious shoot biomass and bacoside A content were optimum when the concentration was higher than that of and that the highest number of shoots, biomass and bacoside A content were obtained at a / ratio of 14.38 mM/37.60 mM. Similarly, the dry weight and artemisinin content of Artemisia annua were shown to be maximum when a high concentration of and a low concentration of were used (Wang & Tan, 2002). Guo et al. (2012) found that Catharanthus roseus (L.) G. Don plants in N2 (nitrate:ammonium, 1:1) nitrogen solution accumulated twofold the concentration of catharanthine and vinblastine than did the plants in N1 (nitrate:ammonium, 1:0) or N3 (nitrate:ammonium, 1:3) nitrogen solutions after long-term incubation. Our results were inconsistent with these previous works. The content and total yield of RA and total flavonoids, as well as the total yield of UA and OA, increased as decreased from 100% to 25%, and peaked in the 25/75 (/) treatment. However, the exact mechanisms by which such metabolic changes occur is not yet understood (Naik et al., 2011). The biochemical mechanisms involved in the synergistic effects of combined N regimes on growth and secondary metabolism in P. vulgaris should be investigated.

Conclusions

The present experiment demonstrated that both the 100/0 (/) and 0/100 (/) ratios have inhibitory effects on the growth of P. vulgaris. The 0/100 (/) treatment is superior to the 100/0 (/) treatment for growth and medicinal quality of P. vulgaris. The P. vulgaris plants grown under the 25/75  − N/ − N ratio had the highest photosynthetic rate and leaf area, which may facilitate the accumulation of more carbon. This finding suggests that there was a greater capacity for nitrogen assimilation and protein degradation in the plants grown in the 25/75 ( − N/ − N) solutions, as indicated by higher NR activity and higher soluble protein. In addition, the content and total yield of RA and total flavonoids, as well as the total yield of UA and OA, increased as proportion decreased from 100% to 25% and peaked in the 25/75 (/) treatment. The optimum  − N/ − N ratio for maximum growth and the highest bioactive phytochemical accumulation of P. vulgaris were 25/75 when the N concentration was 15 mmol L⁻¹. The synergistic effect of and provides a promising avenue for agronomic improvement of P. vulgaris.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

This study was funded by the programs of National Nature Science Foundation of China (Nos. 30772730 and 81072986).