Uptake and uptake kinetics of nitrate, ammonium and glycine by pakchoi seedlings (Brassica Campestris L. ssp. Chinensis L. Makino)

Scientia Horticulturae 186 (2015) 247–253

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Uptake and uptake kinetics of nitrate, ammonium and glycine by pakchoi seedlings (Brassica Campestris L. ssp. Chinensis L. Makino) Cao Xiaochuang a,b , Wu Lianghuan b,∗ , Yuan Ling c , Li Xiaoyan b , Zhu Yuanhong d , Jin Qianyu a a

State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou, 310006, China Ministry of Education Key Lab of Environmental Remediation and Ecosystem Health, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou, 310058, China c Department of Landscape Architecture, Wenzhou Vocational College of Science and Technology, Wenzhou, 325006, China d Department of Ecosystem Science and Mangement, The Pennsylvania State University, University Park, PA 16802, USA b

a r t i c l e

i n f o

Article history: Received 15 November 2013 Received in revised form 19 January 2015 Accepted 10 February 2015 Keywords: Glycine Inorganic N Nitrogen uptake Uptake kinetics CCCP (Protonphore Carbonyl Cyanide m-Chlorophenylhydrazone)

a b s t r a c t Plants not only absorb inorganic nitrogen (N), but also have the ability to absorb intact amino acids. Studies about pakchoi N uptake (Brassica Campestris L. ssp. Chinensis L. Makino) mainly focused on nitrate (NO− 3) uptake and its accumulation in plant edible portion. This study investigated pakchoi seedlings uptake + and uptake kinetics of NO− 3 , ammonium (NH4 ) and glycine (Gly) using a mixed equimolar concentrations of the three N forms, and examined passive and active uptake ratios of the three N forms under sterile hydroponics. Our results revealed that pakchoi N uptake was positively related to its substrate N concentration (R = 0.89–0.99, p < 0.11), while N uptake efficiency showed a negative relation with the N concentration (R = −0.64–−0.77, p < 0.36). At the N concentration from 25 to 1500 ␮mol L−1 , pakchoi took up significantly − + more NO− 3 than NH4 , but no significant difference was detected between uptakes of Gly and NO3 except −1 −1 at 250 ␮mol L . At 7500 ␮mol L , Gly uptake and uptake rate were significantly more than those for − + + NO− 3 and NH4 . Regression analysis showed uptake rates for NO3 , NH4 and Gly were well fitted to the Michaelis–Menten kinetics, and their affinity constant (Km ) was in the range of 177–2000 ␮mol L−1 and + maximal velocity (Vmax ) 18.2–46.8 ␮mol g−1 DW h−1 . Pakchoi uptake for NO− 3 , NH4 and Gly was dominated by active uptake. Gly active uptake at low concentration indicated plants have the ability to uptake amino acids that is relevant to field condition. However, passive uptake should not be overlooked in future studies, especially at high N concentration. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Traditionally, terrestrial N cycle asserted that soil organic N + must be transformed into inorganic N (NO− 3 and NH4 ) by soil microorganisms prior to becoming available to plant roots (Warren and Adams, 2007) and N mineralization has been viewed as a critical step in plant N uptake. Recent studies have demonstrated that plants have the ability to take up intact amino acids, thereby bypassing the microbial mineralization step (Näsholm et al., 2002; Persson and Näsholm, 2008; Thornton, 2001). Plant amino acid uptake was mediated by a range of different transporters, and both high- and low-affinity transporters have been identified using

∗ Corresponding author. Tel.: +86 571 88982079; fax: +86 571 88982079. E-mail address: fi[email protected] (W. Lianghuan). http://dx.doi.org/10.1016/j.scienta.2015.02.010 0304-4238/© 2015 Elsevier B.V. All rights reserved.

Arabidopsis thaliana as a model plant (Williams and Miller, 2001). In some N limited ecosystems with low temperature (or pH) and slow N mineralization rate, amino acid uptake even constituted a high proportion of plant total N economy (Jones and Kielland, 2002; Näsholm et al., 1998; Schimel and Chapin, 1996). So it has been accepted that plants are able to absorb and assimilate amino acids + as well as NO− 3 and NH4 as their main N sources. Plants possess various mechanisms that adapt N uptake to the spatial and temporal N availability changes (Hodge et al., 2003). McKane et al. (2002) reported that plant preference for a given N form was likely related to the most abundant N form in soil where plant grows. This preferential uptake could reduce N uptake competition and promote coexistence of species. Besides, some species have their particular absorbing preferences for the different and pakchoi (BrasN forms, such as rice (Oryza sativa L.) for NH+ 4 sica Campestris L. ssp. Chinensis L. Makino) for NO− 3 . Pakchoi is one

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of the main leafy vegetables grown in China and tends to accumulate high levels of NO− 3 in its edible portion (Chen et al., 2005). High − NO− 3 -N fertilizer is the main cause of high NO3 accumulation in vegetable leaves (Chen et al., 2004), which correspondingly increase the risk of nasopharyngeal and esophageal cancers (Eichholzer and Gutzwiller, 1998). In contrast, amino acid composition and their contents are important quality measures for vegetables (Yu et al., 2005). Numerous studies have demonstrated that partial replacement of NO− 3 by amino acids in the nutrient solution significantly increased pakchoi quality by reducing NO− 3 content and improving vegetable nutrition (Inal and Tarakcioglu, 2001; Wang et al., − + 2008). NO− 3 /NH4 ratios also affected NO3 concentration in cucumber (Kotsiras et al., 2002). However, of the studies to date, little is known about pakchoi uptake of amino acids and its uptake kinetics parameters. During the past 20 years, with the 13 C and 15 N double-labeled + technology, root uptake rate for amino acids, NO− 3 and NH4 have been studied using excised or intact roots in hydroponics or field experiments (Kielland et al., 2006; Persson et al., 2006; Thornton and Robinson, 2005; Warren and Adams, 2007). However, using excised roots excavated from soil was unable to differentiate the uptake and conversion of NH+ or amino acids in rhizosphere 4 microorganisms from root cell (Neumann and Römheld, 2000), because the C and N balance of root was irreversibly altered. The injection method does not ensure heterogeneous distribution of the labeled N, thus causing significant error due to the “hotspots” (Augustine and Frank, 2001; Farrar et al., 2003). In addition, the appearance of 15 N in plant might also derive from the mineralization of intact amino acids in unsterile experiment (McKane et al., 2002). These various factors have affected the reliability of the existing methodology. The concentration of individual free amino acids in bulk soil solution is in the region of 0.01–10 ␮mol L−1 , but its concentration in plant and animal cells is in the region of 1–10 mmol L−1 (Jones and Darrah, 1994; Jones et al., 2005). Therefore, it can be expected that high concentrations of amino acids will exist at least transiently in soil after cell death. However, many experiments only adopted a narrow range of amino acid concentrations (mostly below 2000 ␮mol L−1 , Warren, 2009). So we investigated the uptake and uptake kinetics of pakchoi + seedlings for NO− 3 , NH4 and Gly using solution containing equimolar of the three N forms at the concentrations of 25, 250, 1500 and + 7500 ␮mol L−1 . The equimolar of NO− 3 , NH4 and Gly were used for the purpose of investigating the preferential uptake of different N forms and their interaction effects (Persson et al., 2006). Plant seedlings, solution and cultivation environment were completely sterilized to ensure that amino acids could not be decomposed by microorganism. The objectives of this study were to (1) investigate how the N concentrations and N forms affect pakchoi preferential + uptake and uptake kinetics for NO− 3 , NH4 and Gly, (2) determine + the roles of passive and active uptakes for NO− 3 , NH4 and Gly at the different N concentrations.

2. Materials and methods 2.1. Plant material and culture The experiment was conducted in October 2012 at the Plant Organic Nutritional Laboratory, Zhejiang University. Seeds of pakchoi (B. Campestris L. ssp. Chinensis L. Makino) were obtained from the Zhejiang Provincial Academy of Agricultural Sciences, China. The cultivar was Zhebai 6. Pakchoi seeds were sterilized by the method described in Wu et al. (2005), and then placed in the sterile Petri dish covered with parafilm for germination. Three days later, the germinated seeds were transplanted to a new sterile Petri dish

with 0.5% agar (NA) substrate at a density of approximately 20 seeds per dish. When the main root length of seedlings was approximately 1.5 cm, aseptic seedlings were transplanted into centrifugal tubes containing 50 ml 0.5% (w/w) sterile agar (NA). The centrifugal tubes have internal radius 1.3 cm, total depth 11 cm. Each tube was covered with a plastic cap, which had a 0.5 cm diameter hole drilled in the center for seedling to grow out of the tube. After seedlings had completely grown out of the holes, the 0.5% agar inside the tubes was replaced with aseptic nutrient solution containing 6.25 mmol L−1 N (NO− 3 2.08, −1 , respectively), 1.0 mmol L−1 NH+ 2.08 and Gly 2.08 mmol L 4 KH2 PO4 , 1.0 mmol L−1 K2 SO4 , 0.7 mmol L−1 MgSO4 , 2.0 mmol L−1 CaCl2 , 0.00005 mmol L−1 NaMoO4 -2H2 O, 0.0002 mmol L−1 CuSO4 ·5H2 O, 0.0005 mmol L−1 ZnSO4 ·7H2 O, 0.004 mmol L−1 H3 BO3 , 0.005 mmol L−1 MnCl2 , 0.0025 mmol L−1 Na2 EDTA and 0.009 mmol L−1 FeSO4 ·7H2 O, which is one half of nutrient strength of the complete cultivation solution (1/2-nutrient solution). Then the holes in the center of the caps were sealed with Nan Da 704 silicone rubber. Five days later, the ½-nutrient solution was replaced by the complete cultivation solution, which contains double nutrient concentrations of the ½-nutrient solution. The air quality in Plant Organic Nutritional Laboratory achieved a sterility of 100-grade (USA Federal Standard, 209D) under the effects of aseptic laminar cover. The numbers of air particles (>0.5 ␮m) are less than 100 per cubic foot, which satisfies the requirement for sterile cultivation. N-free solution was sterilized by steam under high pressure at 121 ◦ C for 1 h. N nutrient solution was sterilized by passing through a 0.22 ␮m cellulose filter (Millipore, PES Membrane, Ireland). The solution pH was adjusted to 6.5 with sodium hydroxide. During cultivation, the nutrient solution was replaced every three days. To test the contamination, the replaced nutrient solution was immediately inoculated to the sterile agar (NA) substrate. If microbial growth was identified in the inoculated agar plates, the corresponding cultivation tube was discarded. The sterile seedlings were grown under mean temperature 30 ◦ C (day) and 25 ◦ C (night), relative humidity 60%, maximum photosynthetic photon flux density 300 ␮mol m−2 s−1 , and photoperiod 12 h. During the cultivation, the seedlings were watered with sterile water as needed. 2.2. Experiment design After 21 days of cultivation with unlabeled nutrient solution (seven leaves stage), the aseptic seedlings were cultivated with the 15 N labeled solution to determine pakchoi preferential uptake for + NO− 3 , NH4 and Gly under the sterile environment. Experiment 1: Pakchoi preferential uptake for the different N forms was determined using the 50.24 atom% 15 NO− 3 , 50.17 atom% 15 NH+ , 50.11 atom% 15 N-Gly, obtained from Shanghai Research 4 Institute of Chemical Industry, China. Pakchoi seedlings were first cultivated in the deionized water for 4 h to create a nutrient starvation condition. Then the pakchoi seedlings were simultaneously + supplied with the equimolar N of NO− 3 , NH4 and Gly solutions, with total N concentrations of 25, 250, 1500 and 7500 ␮mol L−1 , respectively. For each N concentration, only one N form was labeled with the 15 N resulting in 16 different treatments (3 N forms × 4 concentrations, plus four control treatments with unlabeled N sources). Cultivation solution also contained 10 mg L−1 ampicillin to control the microbial growth and 100 ␮mol L−1 Ca2+ for plant cell membrane stability (Warren, 2009). Each treatment had 12 replications. Experiment 2: The protonphore Carbonyl Cyanide mChlorophenylhydrazone (CCCP) was used to separate passive and active uptake by pakchoi seedlings (Persson and Näsholm, 2002). Since CCCP inhibits plant root active uptake, the appearance of 15 N in plant treated with CCCP is the result of passive uptake.

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Pakchoi seedlings were pre-treated with 50 ␮mol L−1 CCCP and 100 ␮mol L−1 Ca2+ for 1 h, then subsequently submerged root in centrifugal tubes containing 50 ml 25 or 1500 ␮mol L−1 mixed N + solution, which contained equimolar of NO− 3 , NH4 and Gly with 15 only one N source labeled with N as described above. Each treatment had five replications. Pakchoi seedlings in the two experiments were harvested after 15 N labeled incubation for 4 h. 15 N adsorbed on the root surface was removed by ultrasonification in sterile water, followed by rinsing the roots with 50 mmol L−1 CaCl2 and again with sterile water. Harvested plant materials were freeze-dried (Labconco Freezen System, USA), ground to fine powder with a ball mill (Retsch MM301, German). Pakchoi N content was determined by MicroKjeldahl method and titration with 0.05 mmol L−1 sulfuric acid. Then the titrated solution was condensed by a rotary evaporator (EYELA, SB-1100) until N concentration in the solution was greater than 0.5 mg N ml−1 . The 15 N enrichment of plant materials was determined using a Tracer MAT-271 (Finnigan MAT, USA). 2.3. Calculations and statistics N uptakes in different treatments were determined from the N enrichment by comparing to the control treatment with unlabeled + N forms. The amount of NO− 3 , NH4 and Gly taken up from the labeled N was calculated using the Eq. (1) (Sauheitl et al., 2009; Cao et al., 2013). Nuptake = NContent

As − Ac Af

(1)

where, Nuptake is the amount of N taken up by the different parts of pakchoi seedlings; Ncontent is the N content in different parts of pakchoi seedlings; As is the 15 N atom% in different parts of pakchoi seedlings; Ac is the average 15 N atom% in the control treatment + supplied with unlabeled NO− 3 , NH4 and Gly, which is 0.366 atom% + in this experiment; Af is the 15 N atom% of NO− 3 , NH4 and Gly used in this experiment. + N uptake efficiency is the proportion of NO− 3 , NH4 and Gly taken up by the whole pakchoi seedlings based on the amounts of 15 N + labeled NO− 3 , NH4 and Gly contained in the centrifugal tube, which was calculated from the Eq. (2). Nuptake

efficiency

=

Nuptake Nf

× 100

(2)

where, Nuptake was calculated from the Eq. (1), Nf is the N amount + of NO− 3 , NH4 or Gly contained in each centrifugal tube. All statistical analyses were performed by STATISTICA v.5.5 (StatSoft Inc., USA). Data are presented as the mean ± SE (standard error). Differences in plant total N uptake (or N uptake efficiency) at each N concentration were analyzed by one-way analysis of variance. Plant N uptake and uptake efficiency were calculated with the aboveground and belowground parts combined for the effective comparison. Two-way analysis of variance was used to test the effects of N form, N concentration and N form × N concentration on plant N uptake and N uptake efficiency. Three-way analysis of variance was used to test the effects of N form, N concentration, uptake type and their interactions on plant N uptake rate between the CCCP-treated and CCCP-untreated plants. The pakchoi uptake + rate for NO− 3 , NH4 and Gly were adapted to the Michaelis–Menten equation (Origin. 8.0). V = Vmax

C Km + C

(3)

The kinetic parameters of Km and Vmax were calculated using the non-linear regression based on the data. Vmax is the maximum uptake rate. Km is the N concentration at V = 1/2 Vmax , which repre+ sents the affinity constant of NO− 3 , NH4 and Gly with cytoplasmic

Fig. 1. Uptake of NO− , NH+ and Gly by pakchoi seedlings at the different N concen3 4 (light trations. Different lowercases indicated significant differences among NO− 3 + gray), NH4 (gray) and Gly (dark gray) (p < 0.05). Bars show mean values ±SE, n = 3.

membrane binding sites. Affinity constant was calculated as 1/Km according to Wanek and Pˇortl (2008). Significance of the differences in mean values were tested using Duncan’s multiple range method (p < 0.05). 3. Results + 3.1. Preferential uptakes of NO− 3 , NH4 and Gly

Significant amounts of the labeled N were detected in pakchoi seedlings after sterile incubation for 4 h (Fig. 1). At N concentrations of 25, 250 and 1500 ␮mol L−1 , pakchoi took up significantly + more NO− 3 than NH4 , but no significant difference was detected −1 (p < 0.05, between Gly and NO− 3 uptakes except at 250 ␮mol L Fig. 1). At the concentration of 7500 ␮mol L−1 , pakchoi seedlings + took up Gly the highest, followed by NO− 3 and NH4 , respectively. + The Gly N uptake was 104.5% and 57.0% more than NO− 3 and NH4 uptakes, respectively. Two-way analysis of variance indicated significant effects of N form, N concentration and their interactions on pakchoi seedling N uptake and uptake efficiency (p < 0.05, Table 1). Pakchoi total + N uptakes for NO− 3 , NH4 and Gly were positively related to its substrate concentrations (R = 0.89∼0.99, p < 0.11, data not shown). Contrary to the N uptake, pakchoi N uptake efficiencies for NO− 3, NH+ and Gly showed the slightly negative relationships with N 4 concentrations (R = −0.64∼−0.77, p < 0.36, data not shown). With N concentration increases, pakchoi N uptake efficiency decreased from 77.9% to 4.79%, 47.6% to 6.2% and 74.2% to 9.8% for NO− 3, Table 1 Two-way analysis of variance for the effects of N form, N concentration and N form × N concentration on pakchoi N uptake (A), and N uptake efficiency (B) after 4 h sterile cultivation. Sources of variation (A) N uptake N form N concentration N form × N concentration (B) N uptake efficiency N form N concentration N form × N concentration

Df

MS

F

P

2 3 6

3763.7 49304.6 3393.3

31.4 412.0 28.3

<0.001 <0.001 <0.05

2 3 6

510.2 7643.3 259.6

16.5 247.9 8.4

<0.05 <0.001 <0.05

250

C. Xiaochuang et al. / Scientia Horticulturae 186 (2015) 247–253 Table 2 Kinetic parameters for the uptakes of the different N forms by pakchoi seedlings. N form

Km ± SE (␮mol L−1 )

Vmax ± SE (␮mol g−1 DW h−1 )

R2

NO− 3 NH+ 4

177 ± 41 c 2000 ± 818 a 801 ± 131 b

18.2 ± 1.0 b 45.2 ± 9.3 a 46.8 ± 2.8 a

0.987 0.985 0.995

Gly

a

Note: a represents mean values ±SE, n = 3. Values were calculated on the basis of non-linear regression analysis of uptake isotherm.

Table 3 Three-way analysis of variance for the effects of N form, N concentration, uptake type and their interactions on pakchoi N uptake rate between CCCP-treated and CCCP-untreated plants after 4 h sterile cultivation. Sources of variation

, NH+ and Gly by pakchoi seedlings at the different Fig. 2. Uptake efficiency of NO− 3 4 N concentrations. Different lowercases indicated significant differences among NO− 3 + (light gray), NH4 (gray) and Gly (dark gray) (p < 0.05). Bars show mean values ±SE, n = 3.

NH+ and Gly, respectively (Fig. 2). At concentrations of 25, 250 4 and 1500 ␮mol L−1 , pakchoi NO− 3 uptake efficiencies were significantly more than NH+ (p < 0.05). At the highest N concentration 4 (7500 ␮mol L−1 ), however, Gly uptake efficiency was the highest. + 3.2. Uptake rate of NO− 3 , NH4 and Gly

Pakchoi N uptake rate was calculated as the amount of N uptake + per unit of root dry weight per hour. NO− 3 , NH4 and Gly uptake rates significantly increased with increasing N concentrations (Fig. 3). + Pakchoi Gly uptake rate was higher than NO− 3 , NH4 at concentra−1 tions of 250–7500 ␮mol L . At concentration of 25 ␮mol L−1 , NH+ 4 uptake rate was the highest, followed by Gly and then NO− 3 , but the + difference between NH4 and Gly uptake rate was not significant. From the fitted curves between N uptake rates and N concentrations, the curve slopes represented the rate of N uptake rate changes (RC) with increasing N concentrations (Fig. 3). With increasing N concentrations, pakchoi RC for NO− 3 and Gly decreased gradually and leveled off eventually. Their uptake kinetics exhibited two distinct phases, a rapid initial uptake and a slower steady state uptake. However, RC for NH+ uptake rates with increas4 ing N concentrations demonstrated a different trend, that is, the

N uptake rate N form N concentration Uptake type N form × N concentration N form × uptake type N concentration × uptake type N form × N concentration × uptake type

df

MS

F

P

2 1 1 2 2 1

15.6 384.3 103.6 15.1 5.9 89.2

37.4 923.5 248.9 36.2 14.3 214.4

<0.001 <0.001 <0.001 <0.001 0.004 <0.001

2

5.2

12.4

0.002

RC did not decrease rapidly with increasing N concentrations. The different patterns of N uptake rate were also supported by the low NO− 3 and Gly affinity constant, that is, high Km values (Table 2). The dependency of uptake rate on N concentration could be described by Michaelis–Menten kinetics equation with R2 ranging from 0.985 to 0.995. Their affinity constant (Km ) was in the range of 177–2000 ␮mol L−1 and maximal velocity (Vmax ) 18.2–46.8 ␮mol g−1 DW h−1 (Table 2). + 3.3. Passive and active uptake of NO− 3 , NH4 and Gly

Pakchoi N active uptake is the uptake difference between CCCPtreated and CCCP-untreated plants. Pakchoi uptake rates for NO− 3, NH+ and Gly in CCCP-treated plants were significantly lower than 4 that in CCCP-untreated plants (p < 0.05) (Fig. 4). Three-way analysis of variance indicated significant effects of N form, N concentration, N uptake type, and their interactions on pakchoi N uptake rate (p < 0.005, Table 3). At low N concentration (25 ␮mol L−1 ), pakchoi N passive uptake varied from 0.017 to 0.046 ␮mol g−1 DW h−1 , and passive uptake rates for NH+ and Gly were significantly 4 + greater than that for NO− (p < 0.05). Active uptake for NO− 3 3 , NH4 and Gly accounted for about 90.6%, 86.4% and 92.1% of their total N uptakes, respectively. Similarly, active uptake also dominated the pakchoi total N uptake at the high N concentration (1500 ␮mol L−1 ), with the active N uptake rate the highest for Gly −1 −1 (13.4 ␮mol g−1 DW h−1 ), followed by NO− 3 (10.0 ␮mol g DW h ) + −1 −1 and NH4 (6.42 ␮mol g DW h ). However, the proportions of pas+ sive uptake for NO− 3 , NH4 and Gly increased to 30.6%, 23.7% and 22.2%, respectively, at the high N concentration. 4. Discussion 4.1. Pakchoi Gly uptake under the sterile culture

Fig. 3. The relationship between root uptake rates (␮mol g−1 root DW h−1 ) and N , filled square; NH+ , filled triangle; Gly, filled circle). concentrations (NO− 3 4

Amino acids are excellent C and N sources for microbes, and soil microorganisms have the potential to out-compete plants for amino acids in soil (Owen and Jones, 2001). Jones et al. (2005) and Sauheitl et al. (2009) demonstrated that plants became more competitive for amino acids under high endogenous amino acid concentrations, because at the high amino acid concentration

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251

Fig. 4. Passive and active uptake rates (␮mol g−1 root DW h−1 ) of NO− , NH+ and Gly by pakchoi roots between CCCP-treated and CCCP-untreated plants (NO− , light gray; 3 3 4 , gray and Gly, dark gray) at concentration of (a): 25 ␮mol L−1 , (b): 1500 ␮mol L−1 . Bars show mean values ±SE, n = 3. CK represents CCCP-untreated and CCCP represents NH+ 4 CCCP-treated.

(>1 mmol L−1 ), the capacity of soil microbes to take up amino acids is saturated (Vinolas et al., 2001). Under the sterile culture, pakchoi seedlings had the similar uptake capacities for NO− 3 and Gly at the N concentration of 25 ␮mol L−1 (Fig. 1), indicating that pakchoi seedlings have the ability to take up amino acids at low concentration close to field conditions, which normally ranges from 1 to 10 ␮mol L−1 (Jones and Darrah, 1994). The substantial quantity of active uptake for Gly at 25 ␮mol L−1 also strengthens the point that plants are able to absorb amino acids at their low concentrations (Fig. 4). Under the sterile hydroponics, Warren (2006) speculated that amino acid uptake was the dominant way for plant N nutrition compared with the inorganic N. Our results showed that pakchoi + Gly uptake was significantly more than the NO− 3 and NH4 under the high N concentrations (7500 ␮mol L−1 ). The higher Gly uptake rate clearly showed that pakchoi seedlings have the great advantages to uptake amino acids compared with the inorganic N, especially at high N concentration. Given that NO− 3 uptake was significantly inhibited by amino acids (Wang et al., 2008), it seems that amino acids theoretically can serve as an important N sources as the inorganic N for pakchoi seedling. The result of higher Gly uptake by pakchoi seedling at the higher Gly concentrations in culture solution (Fig. 1) was consistent with the result of Warren (2009). On the other hand, the amino acid concentration and endogenous NH+ /NO− 3 level also significantly 4 affected amino acid uptake (Persson and Näsholm, 2002, 2003). Therefore, research results about amino acid uptake could vary if uptake is determined from a single N source solutions rather than from a mixture of multiple N sources (Falkengren-Grerup et al., 2000; Ge et al., 2009; Jones et al., 2005; Reeve et al., 2009). Contrary to the results of Sauheitl et al. (2009), our data revealed that amino acid uptake efficiency was inversely correlated with its concentration. These contrasting results are likely to be a consequence of the high N concentration used in our substrate solution. The concentrations of amino acids in Sauheitl et al. (2009) only varied from 2.39 ␮mol L−1 to 239 ␮mol L−1 , which is far lower than

that in our study. The amino acid transporter sites might become saturated as N concentration increases, which can also contribute to the decrease in Gly uptake efficiency. + 4.2. Effects of NO− 3 , NH4 on Gly uptake and uptake rate + Pakchoi is known to absorb NO− 3 preferable to NH4 and organic − N. When NO3 uptake rate exceeds its assimilation, NO− 3 accumulation in pakchoi tissues occurs (Chen et al., 2004). Wang et al. reported that substituting 20% or less of NO− 3 with glutamine significantly reduced NO− 3 concentration in plant tissue without significant reduction of crop yields in hydroponic culture (Wang et al., 2008). Our data showed pakchoi seedlings had the highest + affinity (lowest Km values) for NO− 3 followed by NH4 and Gly, when cultured in equimolar of the three N sources. However, uptake rate for NO− 3 was lower than that for Gly in the equimolar N culture solution, especially at the high N concentration. Previous studies have had an antagonistic effect on the reported that the presence of NH+ 4 NO− 3 uptake (Nazoa et al., 2003; Persson and Näsholm, 2002), and the external application of amino acids also inhibited both NO− 3 and NH+ uptake (Thornton, 2004). We speculated that their existed var4 + ious competitive uptakes among NO− 3 , NH4 and Gly when different N sources coexisted. It has been reported that amino acids inhibit expression of HvNRT2 transcript in root, thus blocking synthesis of mRNA encod− ing NO− 3 transporter that is directly related to NO3 uptake (Vidmar et al., 2000). Aslam et al. (2001) discovered that, in the presence of amino acids, induction of NO− 3 transporter may be normal, but the turnover of mRNA encoding NO− 3 transporter may have increased. Wang et al. (2009) demonstrated that glutamine and NH+ absorbed 4 into pakchoi plant were not converted into NO− 3 in pakchoi tissues. − So the inhibition of NO− 3 uptake rate will reduce NO3 accumulation in pakchoi tissue under the mixed N source. However, the true mechanism as to how the N form and N concentration affect root N uptake is not yet clear, due to the mutual interference to uptake

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between N forms. Plant internal selective absorption under mixed N sources may also play an important role in controlling different N uptakes.

4.3. Effects of N concentration and N form on pakchoi N uptake type Bush (1993) reported that plant roots capture amino acids and sugars from the soil using a range of H+ -ATPase driven proton cotransporters, which is active uptake. This is consistent with our finding that Gly uptakes in CCCP-treated plants were 2.5–10.7 times lower than that in CCCP-untreated plants due to the inhibition of active uptake by CCCP. Falkengren-Grerup et al. (2000) questioned the results from excised root cultivation, because the absence of root caused highly variable results relative to the presence of root. Our experiment, which used intact roots with CCCP, was able to distinguish the active uptake from passive uptake under sterile condition. Published values on affinity constants of amino acids varied from 7.0 to 590 ␮mol L−1 for Gly (Jäntgárd and Näsholm, 2008). Our Km value was considerably higher than the published values. The variation of Km may primarily to be differences in experimental conditions, especially the N concentrations, plant species and cultivation methods (sterile/non-sterile). Though pakchoi seedlings + uptake of NO− 3 , NH4 and Gly were dominated by active type, the proportions of active and passive uptake varied with the difference of N concentrations and N types. The result was consistent with the finding by Persson and Näsholm (2002) and El-Naggar et al. (2009). However, at high N concentration, passive uptake accounted for + more than 22% of total NO− 3 , NH4 and Gly uptakes. Therefore, passive uptake should not be overlooked in future study, especially at high N concentration.

5. Conclusions We have demonstrated that pakchoi uptake of Gly and inorganic N was clearly influenced by N concentration, N form and N uptake type under the sterile hydroponics. Pakchoi N uptake was positively related to the substrate N concentrations (R = 0.89–0.99, p < 0.11), but its uptake efficiency showed the opposite trend (R = −0.64–−0.77, p < 0.36). At N concentration of 25–1500 ␮mol L−1 , pakchoi NO− 3 uptake was significantly , but no significant difference was detected more than NH+ 4 −1 (p < 0.05). between Gly and NO− 3 uptake except at 250 ␮mol L At 7500 ␮mol L−1 , Gly uptake and uptake rate was significantly − + + more than NO− 3 and NH4 . Uptake rates for NO3 , NH4 and Gly were well fitted to the Michaelis–Menten kinetics equation with their affinity constants (Km ) 177, 2000 and 801 ␮mol L−1 for NO− 3, + NH+ and Gly, respectively. Pakchoi uptake for NO− 3 , NH4 and 4 Gly was dominated by active uptake. However, at high N con+ centration, more than 22% of NO− 3 , NH4 and Gly were taken up by passive type. Gly active uptake under the low N concentration indicated that plants have the ability to take up amino acids at its low concentration that is relevant to field condition.

Acknowledgments This work was funded by the National Basic Research Program of China (2015CB150502), the National Natural Science Foundation of China (no. 31172032, 31270035, 30871595) and the Special Fund for Agro-scientific Research in the Public Interest (201003016). We thank Du Xiaoning from Shanghai Research Institute of Chemical Industry, China for 15 N technical assistance.

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