Appropriate NH4 +: NO3 − ratio improves low light tolerance of mini Chinese cabbage seedlings
© The Author(s). 2017
Received: 17 August 2016
Accepted: 12 January 2017
Published: 23 January 2017
In northwest of China, mini Chinese cabbage (Brassica pekinensis) is highly valued by consumers, and is widely cultivated during winter in solar-greenhouses where low light (LL) fluence (between 85 and 150 μmol m−2 s−1 in day) is a major abiotic stress factor limiting plant growth and crop productivity. The mechanisms with which various NH4 +: NO3 − ratios affected growth and photosynthesis of mini Chinese cabbage under normal (200 μmol m−2 s−1) and low (100 μmol m−2 s−1) light conditions was investigated. The four solutions with different ratios of NH4 +: NO3 − applied were 0:100, 10:90, 15:85 and 25:75 with the set up in a glasshouse in hydroponic culture. The most appropriate NH4 +: NO3 − ratio that improved the tolerance of mini Chinese cabbage seedlings to LL was found in our current study.
Under low light, the application of NH4 +: NO3 − (10:90) significantly stimulated growth compared to only NO3 − by increasing leaf area, canopy spread, biomass accumulation, and net photosynthetic rate. The increase in net photosynthetic rate was associated with an increase in: 1) maximum and effective quantum yield of PSII; 2) activities of Calvin cycle enzymes; and 3) levels of mRNA relative expression of several genes involved in Calvin cycle. In addition, glucose, fructose, sucrose, starch and total carbohydrate, which are the products of CO2 assimilation, accumulated most in the cabbage leaves that were supplied with NH4 +: NO3 − (10:90) under LL condition. Low light reduced the carbohydrate: nitrogen (C: N) ratio while the application of NH4 +: NO3 − (10:90) alleviated the negative effect of LL on C: N ratio mainly by increasing total carbohydrate contents.
The application of NH4 +:NO3 − (10:90) increased rbcL, rbcS, FBA, FBPase and TK expression and/or activities, enhanced photosynthesis, carbohydrate accumulation and improved the tolerance of mini Chinese cabbage seedlings to LL. The results of this study would provide theoretical basis and technical guidance for mini Chinese cabbage production. In practical production, the ratio of NH4 +:NO3 − should be adjusted with respect to light fluence for successful growing of mini Chinese cabbage.
KeywordsChlorophyll fluorescence imaging Calvin cycle Relative gene expression Low light fluence Ammonium: nitrate ratio
In northwest of China, mini Chinese cabbage (Brassica pekinensis) is highly valued by consumers, and is widely cultivated during winter in solar-greenhouses where low light (LL) fluence (between 85 and 150 μmol m−2 s−1 in day) is a major abiotic stress factor limiting plant growth and crop productivity. The exposure of most plants to LL condition affects metabolism in various ways including the alteration of enzyme activity (e.g. transketolase, superoxide dismutase, catalase) and disruption of transcription [1, 2]. Low light fluence also inhibited violaxanthin de-epoxidase, causing failure of the protective xanthophyll cycle . The alleviation role of 5-aminolevulinic acid and calcium in cucumber under LL condition was reported [4, 5]. Therefore, understanding the genetic and biochemical processes that regulate LL tolerance is a vital area of research in plant biology.
Ammonium (NH4 +) and nitrate (NO3 −) improved plants tolerance to environmental stress, including low light stress , drought , salinity , alkalinity , disease , heavy metal toxicity , higher CO2 concentration , and ultraviolet radiation . In addition, NH4 + and NO3 − have different effects on physiological and biochemical processes including photorespiration, photosynthesis, nutrient absorption and nitrogen metabolism . Cruz et al.  demonstrated that increasing NH4 + significantly increased photosynthetic acclimation to elevated CO2 during the early growth stage of cassava. However, the negative effect of NH4 + on photosynthesis was also observed at the later growth stages.
Improving photosynthesis is vital to maintaining sufficient dry biomass accumulation, especially in plants subjected to LL condition. The total CO2 assimilation rate is limited by light intensity, temperature, CO2 diffusion (stomatal conductance), enzyme activity (Rubisco), substrate availability (RuBP regeneration), and respiratory CO2 release . For increasing plant yield, identifying limiting points in photosynthetic process is the central issue. Calvin cycle contains 11 different enzymes which catalyze 13 reactions in the three phases of carboxylation, reduction and regeneration. It is initiated by the enzyme ribulose-1, 5-bisphosphate carboxylase oxygenase (Rubisco) which catalyses the carboxylation of the CO2 acceptor molecule, ribulose-1, 5-bisphosphate (RuBP). The 3-phosphoglycerate (3-PGA) formed in carboxylation stage is then used to form the 3-phosphate glyceraldehyde (GAP) and dihydroxyacetone phosphate (DHAP), through two reactions that consume ATP and NADPH. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays an important role in the second reaction. The regenerative phase of the cycle involves a series of reactions. These reactions convert GAP and DHAP into the CO2 acceptor molecule, RuBP. The fructose-1, 6-bisphosphatase (FBPase), fructose-1, 6-bisphosphate aldolase (FBA) and transketolase (TK) play the vital roles in these reactions. The GAP produced in the Calvin cycle mostly remains within the cycle to regenerate RuBP, while only few exits from the cycle and are utilized to synthesis sucrose and starch . The mechanism involved in photosynthesis with different nitrogen forms has been reviewed by Guo et al. , and includes changes in stomatal density and mesophyll conductance, alteration in photosynthetic enzyme activities, and changes in accumulation of photosynthetic outcome. As a consequence, photosynthesis is limited mainly by light intensity, light use efficiency and CO2 assimilation capacity among other factors.
As demonstrated by Lu et al. , total replacement of NO3 − by NH4 + induced a reduction of stomatal conductance and a decreased dry weight. Moreover, nitrogen in the form of NO3 − alone or NH4NO3 resulted in a greater dry weight gain in tobacco than when NH4 + was applied alone. Although there have been several studies on altered expression of genes coding for Calvin cycle enzymes after exposure to exogenous substance or under environmental stress in cucumber [2, 18], the consequences for NH4 +- induced enhancement of photosynthesis under LL condition has not been extensively explored. However, the application of moderate NH4 + was recently found to have enhanced the LL tolerance of mini Chinese cabbage seedlings in an earlier experiment . To understand the underlying mechanism of moderate NH4 +- induced promotion of net photosynthetic rate (Pn), investigations are needed to conclusively confirm that the presence of NH4 + in the nutrient solution promotes photosynthesis. In this study, we reported the mechanisms with which moderate NH4 +: NO3 − improved the tolerance of mini Chinese cabbage seedlings to LL condition in the greenhouse.
Plant growth and biomass accumulation
Effects of ammonium: nitrate on leaf area, canopy spread, leaf number, fresh weight, dry weight and total chlorophyll content in mini Chinese cabbage under normal (200 μmol m−2 s−1) and low (100 μmol m−2 s−1) light conditions
NH4 +:NO3 −
Leaf area (cm2 plant−1)
Canopy spread (cm2 plant−1)
Leaf number (# plant-1)
Fresh weight (g plant−1)
Dry weight (g plant−1)
Normal light fluence
66.86 ± 3.46 ba
94.55 ± 1.83 c
5.27 ± 0.10 a
4.409 ± 0.147 c
1.153 ± 0.014 c
21.27 ± 0.58 b
68.72 ± 4.93 b
113.06 ± 1.15 b
5.43 ± 0.09 a
6.696 ± 0.079 ab
1.638 ± 0.012 b
25.57 ± 0.33 a
89.44 ± 4.71 a
123.38 ± 1.25 a
5.47 ± 0.10 a
7.842 ± 0.308 a
1.994 ± 0.043 a
28.43 ± 0.61 a
88.71 ± 1.72 a
111.95 ± 3.29 b
5.40 ± 0.10 a
6.582 ± 0.413 b
1.604 ± 0.033 b
26.93 ± 1.26 a
Low light fluence
48.24 ± 1.43 b
57.16 ± 1.24 bc
5.00 ± 0.07 a
2.243 ± 0.078 bc
0.617 ± 0.009 bc
23.87 ± 0.50 c
58.08 ± 2.64 a
65.65 ± 1.64 a
5.13 ± 0.06 a
3.497 ± 0.080 a
0.842 ± 0.022 a
28.63 ± 0.68 a
54.95 ± 1.71 ab
63.21 ± 1.12 ab
4.93 ± 0.08 a
2.502 ± 0.065 b
0.672 ± 0.033 b
26.87 ± 0.18 ab
48.64 ± 1.13 b
55.56 ± 1.64 c
4.83 ± 0.12 a
1.991 ± 0.068 c
0.553 ± 0.013 c
25.90 ± 0.50 bc
NH4 +: NO3 − (10:90) application improved plant growth as indicated by increased total leaf area, canopy spread, fresh and dry weight of plants under LL (Table 1; Fig. 1 a, b). Moreover, application of NH4 +:NO3 − (10:90) under LL condition led to a significant increase in total chlorophyll content, almost reaching the levels of plants under normal light. In contrast, NH4 +: NO3 − (0:100 and 25:75) application under LL condition resulted in 42 and 45% reduction in fresh weight compared with NH4 +:NO3 − (10:90) application, respectively (Table 1). As shown in Table 1, all the growth parameters and chlorophyll content were almost recovered to those under normal light condition levels when NH4 +:NO3 − (10:90) was applied to the LL fluence treated plants.
Principal component analysis of tested traits of mini Chinese cabbage under four NH4 +:NO3 − ratios and normal (200 μmol m−2 s−1) and low (100 μmol m−2 s−1) light fluences
Principal component scores
NH4 +:NO3 −
Normal light fluence
Low light fluence
Gas exchange and chlorophyll fluorescence parameters
Under our growth conditions, low light fluence caused visible stress symptoms and significantly decreased the maximum quantum yield of PSII (Fv/Fm) in plants fertilized with NH4 +:NO3 − (0:100) and NH4 +:NO3 − (25:75). However, NH4 +:NO3 − (10:90) application resulted in increased Fv/Fm under LL condition (Fig. 2 a, c). In normal light plants, NH4 +:NO3 − (0:100) significantly decreased Fv/Fm, effective quantum yield of PSII (ΦPSII) and photochemical quenching (qP), whereas Fv/Fm, ΦPSII and qP did not respond to NH4 +:NO3 − ratio in the presence of NH4 + (Fig. 2 c, d, e). In LL plants, NH4 +:NO3 − (0:100) application caused reduced ΦPSII, and this reduction of ΦPSII was recovered by 10:90 or 15:85 NH4 +:NO3 − application (Fig. 2 d). Moreover, treatments with NH4 +:NO3 − (0:100, 15:85 and 25:75) significantly reduced qP, but NH4 +:NO3 − (10:90) application recovered qP to the level of qP in the plants under normal light (Fig. 2 e). ETR gradually increased and tended to be steady with the increase of photosynthetic active radiation (PAR). Low light caused significant reduction in ETR. NH4 +:NO3 − (10:90) application alleviated LL stress and enhanced the ETR. Moreover, the ETRs of plants treated with NH4 +:NO3 − (10:90) under LL fluence were significant higher than those treated with NH4 +:NO3 − (0:100) under normal light fluence (Fig. 2 b).
Activation of Calvin cycle enzymes
Relative expression of Calvin cycle genes
Carbohydrate, total nitrogen and C: N ratio
Effects of NH4 +:NO3 − on levels of glucose, fructose, sucrose and starch at 15 days after NH4 +:NO3 − treatment in mini Chinese cabbage under normal (200 μmol m−2 s−1) and low (100 μmol m−2 s−1) light conditions
NH4 +:NO3 −
Normal light fluence
4.38 ± 0.26 ba
3.53 ± 0.21 c
0.23 ± 0.03 b
2.47 ± 0.06 c
5.39 ± 0.17 ab
4.21 ± 0.39 bc
0.30 ± 0.02 b
3.28 ± 0.21 b
6.02 ± 0.46 a
6.32 ± 0.16 a
0.53 ± 0.06 a
4.34 ± 0.24 a
5.73 ± 0.25 a
5.25 ± 0.46 ab
0.54 ± 0.03 a
4.08 ± 0.19 a
Low light fluence
2.49 ± 0.14 ab
2.72 ± 0.09 bc
0.09 ± 0.02 b
1.64 ± 0.10 ab
3.35 ± 0.20 a
3.98 ± 0.12 a
0.23 ± 0.03 a
2.78 ± 0.43 a
3.05 ± 0.25 a
3.11 ± 0.14 b
0.15 ± 0.02 b
2.75 ± 0.35 a
1.76 ± 0.32 b
2.41 ± 0.18 c
0.07 ± 0.01 b
1.36 ± 0.12 b
Higher levels of nitrogen accumulated in shoot of plants treated with NH4 +:NO3 − (15:85) than in those treated with NH4 +:NO3 − (0:100) under normal light condition (Fig. 5 b). However, the highest level of total nitrogen was observed in NH4 +:NO3 − (10:90) treatment under LL condition (Fig. 5 b). Results presented in Fig. 5 c show that the C: N ratio reached the highest value in NH4 +:NO3 − (25:75) treated plants, and no significant difference was observed between NH4 +:NO3 − (15:85) applied plants. Low light decreased the C: N ratio, but the application of NH4 +:NO3 − (10:90) suppressed the negative effects of LL on C: N ratio (Fig. 5 c).
Photosynthesis is the main process by which plants produce and accumulate dry matter. The importance of water, light, nutrient and CO2 in this process cannot be over emphasized. In the present study, we found that moderate NH4 +: NO3 − application enhanced vegetative characteristics, while non-ammonium or higher ammonium application resulted in reduced growth under normal and low light conditions. This observation suggests that NH4 + is actively involved in the promotion of plant growth. Furthermore, in order to obtain the maximum biomass, less concentration of ammonium was needed in LL condition than in normal light condition. This was probably because low light reduced carbon metabolism of plants and less carbohydrate were accumulated in the tissues of the plants. Thus, less photosynthate was transported from the shoot to the root in LL condition, and thus the energy used for ammonium assimilation in plant root was low. Therefore, higher NH4 + concentration in LL condition was not completely assimilated by plant root, and then accumulated in plant cells causing ammonium toxicity. Sakakibara et al.  reported that inorganic nitrogen (NH4 + or NO3 −) was a substrate for nitrogen assimilation and also functions as a signal triggering widespread changes in gene expression that modulate metabolism and development. They also demonstrated that a large research project that focused on nitrate action in gene expression of Arabidopsis has provided the view of the extent of nitrate-dependent regulatory genes, including nitrogen metabolism, carbon metabolism and cytokinin responses. We also observed that nitrogen metabolism of plants decreased under LL condition, but the extent of the decrease of nitrogen metabolism was less than carbon metabolism, causing reduced C: N ratio. However, the application of 10% NH4 + in solutions enhanced C: N ratio mainly by increasing the total carbohydrate contents. Therefore, appropriate NH4 + concentration in solution was beneficial for plants to maintain the balance of carbon and nitrogen metabolism. These results demonstrate that the level of NH4 + is a rate-limiting factor for plant growth, and supplying appropriate NH4 + levels for plants according to the light fluence is an effective way to promote plant growth and improve vegetable yield.
Calvin cycle is the main rate-limiting factor of CO2 assimilation
Nitrogen can significantly affect the performance of the three main processes of photosynthesis: stomatal control of CO2 supply, thylakoid electron transport (light reaction), and Calvin cycle (dark reaction) [20, 21]. Our results indicate that the Pn rate of NH4 +-treated plants was higher than those of sole NO3 −-treated plants. Low light fluence caused a decrease in Pn per unit leaf area at 9 days after treatment under NH4 +:NO3 − (0:100) application. However, the negative influence of LL was not observed in plants treated with NH4 +: NO3 − (10:90). The addition of appropriate NH4 + is beneficial for improved photosynthesis in LL condition. This finding was consistent with Golvano et al. , who demonstrated that NH4 +-fed plants had higher protein content and increased activity of photosynthetic enzymes compared with NO3 −-fed plants. Frantz et al.  also reported that the inhibition of Pn was caused by ammonium toxicity. Claussen and Lenz  found that NH4 + accumulation in leaves led to uncoupling of the electron transport form photophosphorylation in chloroplasts, which consequently led to decreased photosynthetic rate. In our study, we also observed that the reduction of photosynthesis in plants fertilized with sole NO3 − was accompanied by decreased chlorophyll content, but this did not occur in plants treated with NH4 +: NO3 − (25:75). This, therefore, suggests that the reduced photosynthesis is not the result of reduced light-harvesting capacity but possibly as a consequence of decreased gas conductance. It could also be due to the activities of Calvin cycle enzymes or negative feedback regulation by accumulated carbon metabolites after sole NO3 − treatment or higher NH4 + concentration (25%) treatment. We also observed that plants fed with only NO3 − had significantly lower gs but higher Ci, thus, reduced gas conductance as a major factor for reduced CO2 assimilation was excluded. Finally, the decreased Pn in plants treated with only NO3 − was consistent with reduced contents of glucose, fructose, sucrose and starch, making it unlikely that negative feedback regulation by accumulated carbon metabolites resulted in reduced photosynthetic rates. Therefore, our results suggest that the net photosynthetic rate of plant treated by NH4 +: NO3 − and light fluence was probably limited by the Calvin cycle.
The chlorophyll fluorescence imaging is a helpful measurement to investigate several aspects of photosynthesis. This is because it reflects changes in thylakoid membrane organization and function and inhibition of photosynthesis and oxygen evolution through interactions with components of PSII [25, 26]. In the present study, the sole application of NO3 − significantly reduced ETR and Fv/Fm in normal light condition. Low light fluence obviously reduced ETR and Fv/Fm, while the addition of 10 and 15% NH4 + inhibited the negative effect of LL on ETR and Fv/Fm in different levels. As described by Krause and Weis , damage to component of thylakoid membranes, especially those of PSII, and inhibition of energy transfers from antenna molecules to reaction centers can result in lower Fv/Fm. In the chloroplast ultrastructure of mini Chinese cabbage, the degree of granal stacking increased in NH4 +:NO3 − (10:90) treated plants under LL condition . Similarly, Bi et al.  reported a decline in Fv/Fm, ΦPSII after exposure to low temperature and low light intensity in transgenic cucumber plants. In our study, in normal light plants, the addition of NH4 + increased ΦPSII and qP, but the sole NO3 − decreased the ΦPSII and qP; while ΦPSII in LL plants significantly decreased in higher NH4 + (25%) treatment. Higher ammonium concentration (25%) applied to mini Chinese cabbage seedlings under LL condition greatly degraded the grana lamella and decreased the light-captured area . The significant decrease in ΦPSII was mainly attributed to the decrease in degree of granal stacking, which is deemed as ‘down regulation’ of light energy absorbed area. In NH4 +:NO3 − (10:90) treated plants under low light, the increase in qP was attributed to the increase in the consumption rate of reductants and ATP generated from non-cyclic electron transport caused by enhanced carboxylation rate.
NH4 +:NO3 − and light fluence regulate activities of several Calvin cycle enzymes and expression of genes coding these enzymes
The photosynthesis rate is limited by the carboxylation reaction of Rubisco and the capacity of RuBP regeneration . For instance, the capacity of RuBP regeneration determines the photosynthetic rate under low irradiance or high CO2 concentration. Analysis of antisense plants with decreased activity of Rubisco showed that the control coefficient on photosynthesis varied from 0.1 to 0.9 depending on the experimental and growth conditions such as light intensity and CO2 concentration , suggesting that Rubisco activity does not always dominate the photosynthesis rate and that other rate-limiting steps exist.
Our results showed that NH4 +: NO3 − and light fluence affected light capture system and the dark reaction - Calvin cycle. Moreover, Calvin cycle is the main rate-limiting factor of CO2 assimilation. NH4 +: NO3 − regulates photosynthetic capacity, to a large extent, by affecting the capacity of Rubisco carboxylation and RuBP regeneration. The application of NH4 +: NO3 − (15:85) to the plants under normal light significantly enhanced activities of Rubisco, GAPDH, FBA, FBPase and TK, and the results was similar to the application of 10:90 (NH4 +: NO3 −) in LL plants. Raab and Terry [30, 31] demonstrated that the Rubisco content of leaf in NH4 + supplied sugar beet plants was significantly higher than those in NO3 −supplied plants, and similar results were obtained by Guo et al.  when they conducted their experiment with rice plants. In the present study, the relative expressions of rbcS, rbcL, FBA, FBPase and TK were up regulated in NH4 +-treated plants under normal light. Low light fluence inhibited the expression of these genes in different levels, but rbcL, FBA and FBPase were partially restored. The expression of rbcS and TK were above the control level in NH4 +: NO3 − (10:90) treated plants. This result is similar to what was observed in rice leaves during ontogeny [33, 34]. As described by Wingler et al. , low light intensity results in reduced expression of light-dependent genes and the disappearance of photosynthetic proteins. Several studies have illustrated that up regulation of genes involved in Calvin cycle leads to increased Pn and enhanced vegetative growth, while reduced expression of these genes results in stunted plant growth [18, 36]. We also observed that the relative expression of FBA in plants under LL fed with NH4 +: NO3 − (25:75) was higher than those fed with NH4 +: NO3 − (15:85), whereas the enzyme activity of FBA was reversed. Changes in enzyme activity and gene expression under NH4 +: NO3 − and light fluence conditions are not always positively correlated, suggesting the possibility of further regulatory mechanisms. The results were consistent with Oelze et al. , who reported that transcript abundance is poorly linked to de novo protein synthesis due to profound regulation at the level of translation. In general, effects of NH4 +: NO3 − and light fluence on transcript levels and enzymes’ activities suggest that NH4 +: NO3 − and light fluence play important role in the synthesis and activities of enzymes involved in Calvin cycle.
RuBP regeneration capacity depends on both photosynthetic electron transport chain and the enzymes downstream of Rubisco in the Calvin cycle . In this study, we observed that while the addition of appropriate NH4 + concentration promoted photosynthetic electron transport and expression of genes encoding the Calvin cycle enzymes required for RuBP regeneration, the application of sole NO3 − inhibited these. Among the examined enzymes involved in Calvin cycle, Rubisco is a vital enzyme that catalyzes the carboxylation of the CO2 acceptor molecule, RuBP, GAPDH catalysed the conversion of 1, 3 - diphosphoglyceric acid into GAP, while FBPase catalyzes a rate-limiting step in the Calvin cycle and carbohydrate metabolism . The Rubisco, GAPDH and FBPase are activated by light fluence, usually in two ways: by changing the microenvironment and by producing effector. FBA and TK are not controlled by iron - thioredoxin, but they are controlled by carbon fixation of photosynthesis to a greater extent [40, 41]. For example, a decreased in FBA level resulted in reduced RuBP content, and then inhibited photosynthesis and growth of transgenic potato . In a previous study, when TK activity decreased from 20 to 40%, it led to a significant reduction in RuBP regeneration and significantly inhibited photosynthetic rate of plants . In our current study, the activities of these enzymes in plants fed with only NO3 − or higher NH4 + decreased in 3 days after exposure to LL fluence and then gradually increased afterwards. NH4 +: NO3 − (10:90) application alleviated the negative effect of LL on the activities of these Calvin cycle enzymes. As demonstrated by Rigano et al. , ammonium assimilation required photosynthates imported through the phloem, causing a transitory decrease in the concentration of ATP, along with noticeable variations in glucose-6 -Pconcentration, a permanent decrease in free glucose concentration, an increase in respiratory oxygen consumption. The consumption of photosynthates drives the Calvin cycling process. Thus, the activities of Calvin cycling enzymes were activated. Rigano et al.  also demonstrated that less ammonium was utilized in plants under dark condition than in illuminated plants. Therefore, the addition of moderate NH4 + in LL plants is beneficial for activating Calvin cycling enzymes. The application of NH4 +: NO3 − (10:90) enhanced activity of Calvin cycle enzymes in LL fluence, and this was probably due to the fact that the absorbed NH4 +changed the microenvironment of chloroplast, or the NH4 + assimilation activated the Calvin cycle.
Our results have shown that appropriate NH4 + concentration in nutrient solution under LL condition significantly increased the Fv/Fm, the activities of Calvin cycle enzymes, the relative expression of these genes, the levels of glucose, fructose, sucrose, starch, total carbohydrate and nitrogen, and C: N ratio in mini Chinese cabbage seedlings in comparison with those from plants treated solely with NO3 − or higher amounts of NH4 +. The enhancement of photosynthesis and LL tolerance in mini Chinese cabbage seedlings supplied with NH4 +:NO3 − (10:90) is largely attributed to the increase in rbcL, rbcS, FBA, FBPase and TK expression (and/or activities).
Materials and experimental design
The seeds of Mini Chinese cabbage (Brassica pekinensis cv. “Jinwa no. 2”) obtained from Gansu Academy of Agricultural Sciences, Lanzhou, China, were germinated on moist filter paper in the dark at 25 °C for 16 h, and then sown in clean quartz sand medium and fertilized with half-strength Hoagland’s nutrient solution  once a day. The seedlings were raised in a modern climate-controlled greenhouse with a photoperiod of 12 h, temperature of 23 ± 2/13 ± 2 °C (day/night), and light fluence about 200 μmol m−2 s−1. When the second fully expanded leaves of the seedlings appeared, groups of 20 uniform seedlings were transplanted into a container (38 cm × 28 cm × 12 cm) filled with 6 L NH4 +: NO3 − treated solutions and aerated at 4 h intervals. These plants were separately placed in normal light fluence (200 μmol m−2 s−1) and low light fluence (100 μmol m−2 s−1) and supplied with one of the following NH4 +: NO3 − ratios: 0:100, 10:90, 15:85 and 25:75. The two light conditions were regulated by sunshade nets and incandescent lamps.
The composition of the nutrition solution used was as follows: 5 mmol L−1 N, using Ca(NO3)2 · 4H2O, KNO3 as NO3 −–N and using (NH4)2SO4 as NH4 +–N, 1 mmol L−1 P as KH2PO4, 3 mmol L−1 K as KNO3, K2SO4 and KH2PO4, 1.5 mmol L−1 Ca as Ca(NO3)2 · 4H2O, CaSO4 · 2H2O and CaCl2, 2 mmol L−1 Mg as MgSO4 · 7H2O, plus standard micronutrients referred to Hoagland and Arnon . All elements which plant essentially need remain the same concentration in 4 NH4 +:NO3 − treatments (Additional file 2: Table S1). Nitrification inhibitor (DCD, 7 μmol L−1) was supplied to every container. The pH of nutrient solutions in each container was adjusted to 6.5–7.0 by adding 0.1 mol L−1 HCl or NaOH solution once a day. The 6-L nutrient solution was changed once a week to avoid depletion effects. The containers, each with twenty seedlings, were arranged in a completely randomized design in the greenhouse.
Measurement of canopy spread, leaf area and biomass
Fourteen days after application of the treatments, the leaf spread of the cabbage seedlings was considered to be elliptical and therefore was computed as follows: canopy spread = π × A × B / 4, where A is the longest spread of two opposite leaves and B is the shortest spread of two opposite leaves when placed gently on a flat surface. Total leaf area was measured with leaf area analyzer (YMJ-C, Tuopu Instruments Inc. China).
When seedlings were harvested, the fresh weight of the whole plant was recorded. Subsequently, all grouped samples were kept in an oven first at 105 °C for 15 min and then at 80 °C until constant weight. The dry weight of all samples were determined and recorded using a digital balance.
Measurement of gas exchange parameters and total chlorophyll content
Net photosynthetic rate (Pn), stomatal conductance (gs), and intercellular CO2 concentration (Ci) were measured from the second young fully expanded leaves using a portable photosynthetic system (CIRAS-2, PP System, UK). The photosynthetic photon flux density (200 μmol m−2 s−1), ambient CO2 concentration (380 μmol mol−1), leaf temperature (25 °C) and relative humidity (75%) were maintained throughout the measurements.
The measured leaves were labeled and total chlorophyll content was determined with SPAD meter (leaf chlorophyll meter, SPAD-502 plus, Tuopu Instruments Inc. China). The leaf samples were then frozen in liquid nitrogen and stored at −80 °C for determining the activities and relative expression levels of the genes coding for enzymes of the Calvin cycle.
Imaging of chlorophyll fluorescence
Nine days after treatments started, chlorophyll fluorescence induction parameters were investigated using an Imaging-PAM Chlorophyll Fluorometer (Walz, Effeltrich, Germany). Before measurement, the plant leaves were kept in darkness for 30 min to allow all reaction centers to open. With the Imaging- PAM, Fs, Fo (steady chlorophyll fluorescence of light-adapted leaves, minimum fluorescence yield of the dark-adapted leaves, respectively) and Fm′, Fm (maximum fluorescence yield of the light & dark-adapted leaves, respectively) were obtained with the application of a saturation pulse. The maximum quantum yield of PSII (Fv /Fm = (Fm- Fo)/ Fm) and effective quantum yield of PSII (ФPSII) (ФPSII = (Fm’ - Fs) / Fm’) was calculated according to Genty et al. . Light- adapted minimal fluorescence (Fo’) was measured when the actinic light was turned off in the presence of far-red light. Photochemical quenching (qP) was calculated according to qP = (Fm’- Fs) / (Fm’-Fo’) . The electron transport rates (ETR) at a given actinic irradiance [Photosynthetic active radiation (PAR) = 0, 21, 56, 111, 186, 281, 396, 531, 701, 926, 1076 μmol m−2 s−1] were determined according to White and Critchley .
Measurement of Calvin cycle enzymes activity
The second leaves from the top of plants were sampled at 0, 3, 6, 9, 12 and 15 days after treatments to determine enzyme activity. The activities of Ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco, EC 188.8.131.52), Glyceraldehyde-phosphate dehydrogenase (GAPDH, EC 184.108.40.206), Fructose-1, 6-bisphosphatase (FBPase, EC 3.13.11), Fructose-1, 6-bisphosphate aldolase (FBA, EC 220.127.116.11), and Transketolase (TK, EC 18.104.22.168) were determined with ELISA kit (Shanghai Yanji Biological Technology Ltd., China) and the extraction method of these enzymes were as described by Rao and Terry  with minor modifications. The frozen leaf samples (0.5 g) were ground to a fine powder in liquid N2 with a mortar and pestle and transferred into a centrifuge tube, then extracted in pre-chilled extraction buffer (5 ml). The enzyme extraction solution was centrifuged for 15 min at 12,000× g and 4 °C. The supernatant was used for Calvin cycle enzymes activity assay. Subsequently, the activities of Calvin cycle enzymes were determined with Microplate Absorbance Reader (BioTek ELX800, USA) in absorbance at 450 nm according to the manufacturer’s instruction.
The protein concentration of each enzyme extraction solution was measured by the method of Bradford . The results were expressed as U g−1 of protein.
Total RNA extraction and gene expression analysis
Total RNA was extracted using RNAiso Plus (TaKaRa D9108A) according to the supplier’s instruction. The relative mRNA expression of Calvin cycle enzyme genes in mini Chinese cabbage plants were analyzed by real time quantitative RT-PCR using a SYBR® Green QPCR MIX QPS-201 T (TOYOBO), according to the manufacturer’s instructions. The mini Chinese cabbage actin gene (GenBank accession No. JN120480.1) was used as an internal control. The primers were designed and synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). On the basis of nucleotide, the primers for rubisco large subunit gene (rbcL), rubisco small subunit gene (rbcS) glyceraldehyde-3-phosphate dehydrogenase (GAPDH), fructose-1, 6- bisphosphatase (FBPase), fructose-1, 6-bisphosphate aldolase (FBA), transketolase (TK), and actin genes were designed and used for amplification. Gene bank accession numbers of the sequences used to design the primers were provided in the Additional file 3: Table S2.
Each real-time PCR reaction system was performed in a final volume of 20 μl on a Real-Time PCR Detection System (ABI stepone plus, USA) using the following program: 5 min at 95 °C followed by 40 cycles of 10 s at 95 °C and 30 s at 60 °C with data collection at the annealing step. After the 40 cycles, we included the dissociation /melting curve stage with 15 s at 95 °C, 60 s at 60 °C, and 15 s at 95 °C. The relative quantification of mRNA levels is based on the method of Livak and Schmittgen . The threshold cycle value (Ct) of actin was subtracted from that of the target gene to obtain a ΔCt value. The Ct value of the control sample in experiment was subtracted from the ΔCt value to obtain a ΔΔCt value. The expression level relative to the control for each sample was expressed as 2-ΔΔCt. All the samples were analyzed three times.
Carbohydrate, total nitrogen and C: N ratio analysis
The carbohydrate (glucose, fructose, sucrose and starch) levels were measured using anthrone-sulfuric method, as described by Halhoul and Kleinberg  with a little modification. The total carbohydrate content was determined by glucose, fructose, sucrose plus starch content. Total nitrogen levels in the shoot tissues were measured using the Kjeldahl method as described by Knowles and Ries  with a little modification. The carbohydrate: nitrogen (C: N) ratio was derived using the respective values of carbohydrate and nitrogen.
Tukey’s test was used for testing the significance of means difference between treatments by using the SPSS 16.0. Using the R correlation matrix method, principal component analysis of growth parameters was carried out with SPSS 16.0. All figures were created by Origin ver. 8.5 (OriginLab Institute Inc. USA).
Intercellular CO2 concentration
Electron transport rate
Fructose-1, 6-bisphosphate aldolase (EC 22.214.171.124)
Fructose-1, 6-bisphosphatase (EC 3.13.11)
Maximum quantum yield of PSII
Glyceraldehyde-phosphate dehydrogenase (EC 126.96.36.199)
Net photosynthetic rate
- rbcL :
Rubisco large subunit gene
- rbcS :
Rubisco small subunit gene
Ribulose-1, 5-bisphosphate carboxylase/oxygenase (EC 188.8.131.52)
Transketolase (EC 184.108.40.206)
Effective quantum yield of PSII
We thank Alejandro Calderón-Urrea very much for revising language usages. The authors are extremely grateful to the editors and the anonymous reviewers for their critical and valuable comments and help that have greatly improved the article.
This work was supported financially by the National Natural Science Foundation of China (No. 31260473), Special Fund for Agro-scientific Research in the Public Interest (201203001), China Agriculture Research System (CARS-25-C-07). The funds were used for the provision of laboratory chemicals and some essential materials needed for the experiment.
Availability of data and materials
The data sets supporting the conclusions of this article are included within the article and its additional files. All primers designed in this study are listed in Additional file 3: Table S2.
JY, WL and LH conceived and designed the experiment. LH and JL performed the experiment and collected the data. LH and MMD analyzed the data and wrote the paper. JY, WL, MMD and JL worked together with LH to critically revise the manuscript. All authors read and approved the manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Ali MB, Hahn EJ, Paek KY. Effects of light intensities on antioxidant enzymes and malondialdehyde content during short-term acclimatization on micropropagated Phalaenopsis plantlet. Environ Exp Bot. 2005;54:109–20.View ArticleGoogle Scholar
- Bi H, Dong X, Wu G, Wang M, Ai X. Decreased TK activity alters growth, yield and tolerance to low temperature and low light intensity in transgenic cucumber plants. Plant Cell Rep. 2015;34:345–54.View ArticlePubMedGoogle Scholar
- Bugos RC, Chang SH, Yamamoto HY. Developmental expression of violaxanthin de-epoxidase in leaves of tobacco growing under high and low light. Plant Physiol. 1999;121:207–14.View ArticlePubMedPubMed CentralGoogle Scholar
- Liang W, Wang M, Ai X. The role of calcium in regulating photosynthesis and related physiological indexes of cucumber seedlings under low light intensity and suboptimal temperature stress. Sci Hortic-Amsterdam. 2009;123:34–8.View ArticleGoogle Scholar
- Wang LJ, Jiang WB, Huang BJ. Promotion of 5-aminolevulinic acid on photosynthesis of melon (Cucumis melo) seedlings under low light and chilling stress conditions. Physiol Plant. 2004;121:258–64.View ArticlePubMedGoogle Scholar
- Hu L, Yu J, Liao W, Zhang G, Xie J, Lv J, Xiao X, Yang B, Zhou R, Bu R. Moderate ammonium: nitrate alleviates low light intensity stress in mini Chinese cabbage seedling by regulating root architecture and photosynthesis. Sci Hortic-Amsterdam. 2015;186:143–53.View ArticleGoogle Scholar
- Ding L, Gao C, Li Y, Li Y, Zhu Y, Xu G, Shen Q, Kaldenhoff R, Kai L, Guo S. The enhanced drought tolerance of rice plants under ammonium is related to aquaporin (AQP). Plant Sci. 2015;234:14–21.View ArticlePubMedGoogle Scholar
- Ali A, Tucker TC, Thompson TL, Salim M. Effects of salinity and mixed ammonium and nitrate nutrition on the growth and nitrogen utilization of barley. J Agron Crop Sci. 2001;186:223–8.View ArticleGoogle Scholar
- Roosta HR. Effect of ammonium: nitrate ratios in the response of strawberry to alkalinity in hydroponics. J Plant Nutr. 2014;37:1676–89.View ArticleGoogle Scholar
- Borrero C, Trillas MI, Delgado A, Avilés M. Effect of ammonium/ nitrate ratio in nutrient solution on control of Fusarium wilt of tomato by Trichoderma asperellum T34. Plant Pathol. 2012;61:132–9.View ArticleGoogle Scholar
- Zaccheo P, Crippa L, Pasta VDM. Ammonium nutrition as a strategy for cadmium mobilisation in the rhizosphere of sunflower. Plant Soil. 2006;283:43–56.View ArticleGoogle Scholar
- Cruz JL, Alves AAC, LeCain DR, Ellis DD, Morgan JA. Effect of elevated CO2 concentration and nitrate: ammonium ratios on gas exchange and growth of cassava (Manihot esculenta Crantz). Plant Soil. 2014;374:33–43.View ArticleGoogle Scholar
- Döhler G. Impact of UV radiation of different wavebands on pigments and assimilation of 15 N-ammonium and 15 N-nitrate by natural phytoplankton and ice algae in Antarctica. J Plant Physiol. 1997;151:550–5.View ArticleGoogle Scholar
- Guo S, Zhou Y, Shen Q, Zhang F. Effect of ammonium and nitrate nutrition on some physiological processes in higher plants-growth, photosynthesis, photorespiration, and water relations. Plant Biol. 2007;9:21–9.View ArticlePubMedGoogle Scholar
- Farquhar GD, von Caemmerer SV, Berry JA. A biochemical model of photosynthetic CO2 assimilation in leaves of C3 species. Planta. 1980;149:78–90.View ArticlePubMedGoogle Scholar
- Raines CA. The Calvin cycle revisited. Photosynthesis Res. 2003;75:1–10.View ArticleGoogle Scholar
- Lu YX, Li CJ, Zhang FS. Transpiration, potassium uptake and flow in tobacco as affected by nitrogen forms and nutrient levels. Ann Bot. 2005;95:991–8.View ArticlePubMedPubMed CentralGoogle Scholar
- Xia XJ, Huang LF, Zhou YH, Mao WH, Shi K, Wu JX. Brassinosteroids promote photosynthesis and growth by enhancing activation of Rubisco and expression of photosynthetic genes in Cucumis sativus. Planta. 2009;230:1185–96.View ArticlePubMedGoogle Scholar
- Sakakibara H, Takei K, Hirose N. Interactions between nitrogen and cytokinin in the regulation of metabolism and development. Trends Plant Sci. 2006;11:440–8.View ArticlePubMedGoogle Scholar
- Feng X, Chen Y, Qi Y, Yu C, Zheng B, Brancourt-Hulmel M, Jiang D. Nitrogen enhanced photosynthesis of Miscanthus by increasing stomatal conductance and phosphoenolpyruvate carboxylase concentration. Photosynthetica. 2012;50:577–86.View ArticleGoogle Scholar
- Miranda RDS, Mesquita RO, Freitas NS, Prisco JT, Gomes-Filho E. Nitrate: ammonium nutrition alleviates detrimental effects of salinity by enhancing photosystem II efficiency in sorghum plants. Rev Bras Eng Agric Ambient. 2014;18:8–12.View ArticleGoogle Scholar
- Golvano MP, Felipe MR, Cintas AM. Influence of nitrogen sources on chloroplast development in wheat seedlings. Physiol Plant. 1982;56:353–60.View ArticleGoogle Scholar
- Frantz TA, Peterson DM, Durbin RD. Sources of ammonium in oat leaves treated with tabtoxin or methionine sulfoximine. Plant Physiol. 1982;69:345–8.View ArticlePubMedPubMed CentralGoogle Scholar
- Claussen W, Lenz F. Effect of ammonium or nitrate nutrition on net photosynthesis, growth, and activity of the enzymes nitrate reductase and glutamine synthetase in blueberry, raspberry and strawberry. Plant Soil. 1999;208:95–102.View ArticleGoogle Scholar
- Hussain MI, Reigosa MJ. A chlorophyll fluorescence analysis of photosynthetic efficiency, quantum yield and photon energy dissipation in PSII antennae of Lactuca sativa L. leaves exposed to cinnamic acid. Plant Physiol Biochem. 2011;49:1290–8.View ArticlePubMedGoogle Scholar
- Rimando AM, Dayan FE, Czarnota MA, Weston LA, Duke SO. A new photosystem II electron transfer inhibitor from Sorghum Bicolor. J Nat Prod. 1998;61:927–30.View ArticlePubMedGoogle Scholar
- Krause GH, Weis E. Chlorophyll fluorescence as a tool in plant physiology. Photosynth Res. 1984;5:139–57.View ArticlePubMedGoogle Scholar
- Von Caemmerer SV, Farquhar GD. Some relationships between the biochemistry of photosynthesis and the gas exchange of leaves. Planta. 1981;153:376–87.View ArticleGoogle Scholar
- Stitt M, Schulze D. Does Rubisco control the rate of photosynthesis and plant growth? An exercise in molecular ecophysiology. Plant Cell Environ. 1994;17:465–87.View ArticleGoogle Scholar
- Raab TK, Terry N. Nitrogen source regulation of growth and photosynthesis in Beta vulgaris L. Plant Physiol. 1994;105:1159–66.View ArticlePubMedPubMed CentralGoogle Scholar
- Raab TK, Terry N. Carbon, nitrogen and nutrient interactions in Beta vulgaris L. as influenced by nitrogen source, NO3 − versus NH4 +. Plant Physiol. 1995;107:575–84.View ArticlePubMedPubMed CentralGoogle Scholar
- Guo S, Chen G, Zhou Y, Shen Q. Ammonium nutrition increases photosynthesis rate under water stress at early development stage of rice (Oryza sativa L.). Plant Soil. 2007;296:115–24.View ArticleGoogle Scholar
- Irving LJ, Robinson D. A dynamic model of Rubisco turnover in cereal leaves. New Phytol. 2006;169:493–504.View ArticlePubMedGoogle Scholar
- Suzuki Y, Makino A, Mae T. Changes in the turnover of Rubisco and levels of mRNAs of rbcL and rbcS in rice leaves from emergence to senescence. Plant Cell Environ. 2001;24:1353–60.View ArticleGoogle Scholar
- Wingler A, Von Schaewen A, Leegood RC, Lea PJ, Quick WP. Regulation of leaf senescence by cytokinin, sugars, and light effects on NADH-dependent hydroxypyruvate reductase. Plant Physiol. 1998;116:329–35.View ArticlePubMed CentralGoogle Scholar
- Miyagawa Y, Tamoi M, Shigeoka S. Overexpression of a cyanobacterial fructose-1, 6-/sedoheptulose-1, 7-bisphosphatase in tobacco enhances photosynthesis and growth. Nat Biotechnol. 2001;19:965–9.View ArticlePubMedGoogle Scholar
- Oelze ML, Muthuramalingam M, Vogel MO, Dietz KJ. The link between transcript regulation and de novo protein synthesis in the retrograde high light acclimation response of Arabidopsis thaliana. BMC Genomics. 2014;15:1.View ArticleGoogle Scholar
- Long SP, Zhu XG, Naidu SL, Ort DR. Can improvement in photosynthesis increase crop yields? Plant Cell Environ. 2006;29:315–30.View ArticlePubMedGoogle Scholar
- Strand Å, Zrenner R, Trevanion S, Stitt M, Gustafsson P, Gardeström P. Decreased expression of two key enzymes in the sucrose biosynthesis pathway, cytosolic fructose-1, 6-bisphosphatase and sucrose phosphate synthase, has remarkably different consequences for photosynthetic carbon metabolism in transgenic Arabidopsis thaliana. Plant J. 2000;23:759–70.View ArticlePubMedGoogle Scholar
- Haake V, Zrenner R, Sonnewald U, Stitt M. A moderate decrease of plastid aldolase activity inhibits photosynthesis, alters the levels of sugars and starch, and inhibits growth of potato plants. Plant J. 1998;14:147–57.View ArticlePubMedGoogle Scholar
- Ma W, Wei L, Wang Q, Shi D, Chen H. Increased activity of the non-regulated enzymes fructose-1, 6-bisphosphate aldolase and triosephosphate isomerase in Anabaena sp. strain PCC 7120 increases photosynthetic yield. J Appl Phycol. 2007;19:207–13.View ArticleGoogle Scholar
- Haake V, Geiger M, Walch-Liu P, Of Engels C, Zrenner R, Stitt M. Changes in aldolase activity in wild-type potato plants are important for acclimation to growth irradiance and carbon dioxide concentration, because plastid aldolase exerts control over the ambient rate of photosynthesis across a range of growth conditions. Plant J. 1999;17:479–89.View ArticleGoogle Scholar
- Henkes S, Sonnewald U, Badur R, Flachmann R, Stitt M. A small decrease of plastid transketolase activity in antisense tobacco transformants has dramatic effects on photosynthesis and phenylpropanoid metabolism. Plant Cell. 2001;13:535–51.View ArticlePubMedPubMed CentralGoogle Scholar
- Rigano C, Martino Rigano V, Vona V, Carfagna S, Carilloan P, Esposito S. Ammonium assimilation by young plants of Hordeum vulgare in light and darkness: effects on respiratory oxygen consumption by roots. New Phytol. 1996;132:375–82.View ArticlePubMedGoogle Scholar
- Hoagland DR, Arnon DI. The water-culture method for growing plants without soil. Calif Agr Exp Sta Cir. 1950;347.
- Genty B, Briantais JM, Baker NR. The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. BBA-Gen Subjects. 1989;990:87–92.View ArticleGoogle Scholar
- Kooten O, Snel JF. The use of chlorophyll fluorescence nomenclature in plant stress physiology. Photosynth Res. 1990;25:147–50.View ArticlePubMedGoogle Scholar
- White AJ, Critchley C. Rapid light curves: a new fluorescence method to assess the state of the photosynthetic apparatus. Photosynth Res. 1999;59:63–72.View ArticleGoogle Scholar
- Rao IM, Terry N. Leaf phosphate status, photosynthesis, and carbon partitioning in sugar beet I. Changes in growth, gas exchange, and Calvin Cycle enzymes. Plant Physiol. 1989;90:814–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–54.View ArticlePubMedGoogle Scholar
- Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. Methods. 2001;25:402–8.View ArticlePubMedGoogle Scholar
- Halhoul MN, Kleinberg I. Differential determination of glucose and fructose, and glucose-and fructose-yielding substances with anthrone. Anal Biochem. 1972;50:337–43.View ArticlePubMedGoogle Scholar
- Knowles NR, Ries SK. Rapid growth and apparent total nitrogen increases in rice and corn plants following applications of triacontanol. Plant Physiol. 1981;68:1279–84.View ArticlePubMedPubMed CentralGoogle Scholar