Effects of cadmium stress on pakchoi (Brassica chinensis L.) growth and uptake of inorganic and organic nitrogenous compounds

Accepted Manuscript Title: Effects of cadmium stress on pakchoi (Brassica chinensis L.) growth and uptake of inorganic and organic nitrogenous compounds Authors: Qingxu Ma, Xiaochuang Cao, Xiaoli Tan, Linlin Si, Lianghuan Wu PII: DOI: Reference:

S0098-8472(17)30029-1 http://dx.doi.org/doi:10.1016/j.envexpbot.2017.02.001 EEB 3180

To appear in:

Environmental and Experimental Botany

Received date: Accepted date:

18-11-2016 3-2-2017

Please cite this article as: Ma, Qingxu, Cao, Xiaochuang, Tan, Xiaoli, Si, Linlin, Wu, Lianghuan, Effects of cadmium stress on pakchoi (Brassica chinensis L.) growth and uptake of inorganic and organic nitrogenous compounds.Environmental and Experimental Botany http://dx.doi.org/10.1016/j.envexpbot.2017.02.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Effects of cadmium stress on pakchoi (Brassica chinensis L.) growth and uptake of inorganic and organic nitrogenous compounds

Qingxu Ma1,2*, Xiaochuang Cao3*, Xiaoli Tan1,2, Linlin Si1,2, Lianghuan Wu 1,2†

1Ministry

of Education Key Lab of Environmental Remediation and Ecosystem Health, College of

Environmental and Resource Sciences, Zhejiang University, Hangzhou, 310058, China 2Zhejiang

Provincial Key Laboratory of Subtropic Soil and Plant Nutrition, College of Environmental and

Resource Sciences, Zhejiang University, Hangzhou, 310058, China 3State

Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 310006, China

† Author

for correspondence:

Lianghuan Wu

Tel: (86)-571-88982922 Fax: (86)-571-88982922 Email: [email protected] * These

authors contributed equally to this work

Abstract

Exposure of cultivated plants to cadmium (Cd) can affect their growth and safety as food sources. Nitrogen (N) is important in regulating both plant growth and Cd uptake, but little research has been conducted studying the effects of Cd stress on the uptake of different forms of nitrogen. In this study, we measured the relative rates of uptake of nitrate, ammonium, and glycine by pakchoi (Brassica chinensis L.) plants under Cd stress, using substrate-specific 15N-labelling in a sterilized environment. Cd stress significantly increased the proportional contribution of nitrate in comparison with controls, while decreasing the contribution of ammonium in shoots. Overall glycine uptake decreased under Cd stress, but the proportional contribution of glycine significantly increased in roots while decreasing in shoots. The mechanism of Cd stress on glycine uptake and metabolism was detected using 15

N-labelling and

N-gas chromatography mass spectrometry (GC-MS) analysis. In shoots, Cd stress

inhibited the active uptake of 15

15

15

N-glycine, while enhancing the passive uptake of

N-glycine significantly. Additionally, the short-term total uptake of glycine in

shoots and roots was slightly affected by Cd stress. In comparison to controls, 15

N-labelled glycine was found to be significantly higher and serine lower in

Cd-stressed plants, indicating that Cd stress inhibited the conversion of glycine to serine. We posit that Cd stress affects the overall nitrogen uptake in pakchoi plants, but that an inhibition in the metabolism of glycine to serine, rather than root uptake, is the limiting step for glycine contribution.

Keywords: cadmium; pakchoi; glycine; nitrate; ammonium; uptake; metabolism

1. Introduction Cadmium (Cd) is one of the most toxic and prevalent heavy metals endangering soil resources and threatening human health. In China, more than 2.0×109 ha of agricultural land has been contaminated by heavy metals, and Cd is one of the most important pollutants in surface soils (Guo et al., 2011). More than half of soil Cd pollution is from phosphate fertilizer (Seshadri et al., 2016), and others are from application of sewage sludge mining, smelting, and burning of fossil fuels (Yong et al., 2011). Cd can be highly toxic to plants due to its high mobility and solubility in soil. Humans exposed to cadmium can suffer serious health problems, including osteoporosis, kidney damage, and cancer (Siebers et al., 2013). Therefore, reducing cadmium accumulation in cultivated food crops is an important endeavour. Several studies have documented that the application of nitrogen (N) in the soil enhances Cd uptake, including in chamomile (Kovácik et al., 2011), alpine brassica (Xie et al., 2008), wheat (LiXianglan et al., 2011), and rice (Jalloh et al., 2009). In winter wheat, an increase of 10 kg calcium nitrate (Ca(NO3)2) application over one hectare was followed by an increase of 1–3 mg/kg Cd in the harvested grain (Wangstrand et al., 2007). In addition, it is advisable to avoid over-fertilizing Cd-polluted soil due to the positive relationship between uptake of Cd and ammonium nitrate (NH4NO3) (Li et al., 2011). However, different nitrogenous sources affect Cd absorption in different ways. Cd uptake is enhanced in low-pH soils (Tsadilas et al., 2005), but because nitrate uptake increases soil pH, Tsadilas et al. (2005) suggested that Cd uptake might

ultimately be reduced under these conditions. Furthermore, several studies have suggested that Cd accumulation differs in rice under different forms of nitrogenous fertilization (Hassan et al., 2008; Jalloh et al., 2009; Yang et al., 2016). A synergistic effect is seen between nitrate (NO3-) and Cd, while an antagonistic effect is seen between ammonia (NH4+) and Cd (Hassan et al., 2008; Jalloh et al., 2009; Yang et al., 2016). Cadmium uptake, transport, and accumulation in NO3--treated rice appear faster than in NH4+ -treated rice, and NO3- appears to facilitate Cd transport from the main stem to branches and young leaves (Hu et al. 2013). In addition, Yang et al. showed that NO3- enhances Cd uptake by upregulating the expression of the iron-regulated transporter OsIRT1 in rice (Yang et al., 2016). However, an opposite result has also been demonstrated, wherein significantly lower Cd uptake occurred in a hydroponic culture under higher ratio of NO3-/NH4+ supply (Jönsson and Asp 2013). Although the effects of different nitrogenous compounds on the uptake of Cd are not clear, strategic application of these may provide a solution for reducing Cd accumulation in cultivated plants. In turn, the presence of Cd also appears to have an effect on the uptake and metabolism of nitrogenous compounds (Balestrasse et al., 2006; Yi et al., 2006). Cadmium not only limits nitrate uptake and transportation, but also inhibits nitrate assimilation by reducing nitrate reductase (NR) activity (Hernández et al., 1997). Furthermore, an increase in endogenous ammonium can be detected in Cd-treated samples, possibly from the deamination of some amino acids or other N forms (Chaffei et al., 2003). Cadmium had a deleterious effect on the activity of glutamine

synthetase (GS), a key enzyme in ammonium assimilation (Balestrasse et al., 2006). Additionally, it is also notable that glutamate dehydrogenase (GDH) activity is enhanced under Cd-stress, which may have unknown consequences for N metabolism (Astolfi et al., 2004). This suggests that Cd has differing effects on various nitrogenous compound-metabolizing enzymes. Still, the question of how Cd affects the uptake of different nitrogenous compounds such as nitrate, ammonium, and amino acids lacks detailed research. The majority of studies considering N uptake in plants have focused on inorganic forms (e.g. nitrate, NO3-, and ammonium, NH4+), but little attention has been paid to organic nitrogenous compounds. Since the initial documentation of preferential organic N uptake by a non-mycorrhizal plant (Chapin et al. 1993), many studies have shown that almost all plants possess the ability to take up organic N, even plants that live

in

subtropical

areas

(Jones

et

al.,

2005;

Näsholm

et

al.,

2009;

Paungfoo-Lonhienne et al., 2012; Warren, 2014). Organic N sources such as amino acids, peptides, and even proteins can be absorbed by plants directly, accounting for more than 50% of total N in some cold ecosystems (Ohlund and Näsholm, 2001; Warren, 2009; Warren and Adams, 2007), and account for 0.5-21% under simulated environments in the laboratory (Ge et al., 2009; Kaštovská and Šantrůčková, 2011; Reeve et al., 2009; Xiaochuang et al., 2013). Furthermore, amino acids not only provide an N source, but also play an important role in improving crop quality and resisting biotic and abiotic stress. Plants under stress accumulate proline and other amino acids, which play an important role in modulating stomatal opening,

regulating ion transport, detoxification of heavy metals, and the synthesis and activity of some enzymes and redox-homeostasis (Rai, 2002). Compared with inorganic N, amino acids show great variability in the structures, energy, and enzymes needed for their metabolism, and may play an unrecognized role in response to Cd stress. No reports have yet examined the effects of Cd stress on the relative uptake of inorganic and organic N, nor any underlying mechanisms. Pakchoi is grown across China as an important food source, but its cultivation faces risk from heavy metal pollution (Tang et al., 2016). In our study, we used

15

N

labelling to examine the following: 1) how Cd affects pakchoi growth and N uptake; 2) how Cd affects the relative uptake of nitrate, ammonium, and glycine; and 3) how Cd affects the uptake pattern and metabolism of glycine.

2. Materials and methods 2.1 Pakchoi seedling cultivation and preparation Prior to experimentation, pakchoi seedlings were cultivated in a sterile environment as described by Ma et al. (2016). Briefly, pakchoi seeds were soaked in water overnight and sterilized in 60% ethanol for 1 min, 10% H2O2 for 5 min and 0.1 M HgCl2 for 5 min, then removed to sterilized culture dishes (Wu et al., 2005). These were placed for 3 d in a sterile culture room with a day/night humidity of 60/40%, a 12 h light cycle (360 μmolm-2 s-1) and a day/night temperature of 25/20°C. After this period, each seedling was transferred to a 50 ml centrifuge tube containing 0.3% cooled agar and returned to the culture room. Each centrifuge tube was modified

with a 0.3 mm diameter hole in the cap to permit leaves to emerge. After leaf emergence, the holes were sealed with silicone rubber (Nanda 704, China).

Each

seedling and tube cap were then transferred to a fresh centrifuge tube filled with nutrient solution containing 1.4 mM MgSO4·7H2O, 4 mM CaCl2, 2 mM KH2PO4, 2 mM K2SO4, 18.3 M FeSO4·7H2O, 10 M MnCl2, 0.4 M CuSO4·5H2O, 8 M H3BO3, 1 M ZnSO4·7H2O, 5 M Na2EDTA, and 0.1 M NaMoO4·2H2O, at pH 6.2. These were covered with opaque foil to avoid the effects of light on root growth. This point marked Day 1 for each experiment. At this point, seedlings were divided into four experimental groups, as follows: 1) Group 1 was used to examine the effect of Cd stress on growth, N uptake, and N contribution of ammonium, nitrate, and glycine. 2) Group 2 was used to investigate the short-term effects of Cd stress on glycine uptake. 3) Group 3 was used to identify the effects of Cd stress on the activity of nitrogen-metabolizing enzymes. 4) Group 4 was used to examine the effect of Cd stress on the metabolism of glycine. All materials and nutrient solutions, aside from nitrogenous compound solutions, were autoclaved at 121°C for 30 min. The various N-containing solutions required for each experiment were sterilized by passing them through a 0.22-m membrane filter (Millipore, PES Membrane, Ireland) before being added to nutrient solutions. Each nutrient solution was changed every 3 d on a clean bench.

2.2

Experimental groups

2.2.1 Experiment 1: Effect of Cd stress on growth, N uptake, and N contribution of ammonium, nitrate and glycine Pakchoi seedlings were cultivated as described for 15 d with 3 mM mixed nitrogenous compounds (1 mM glycine + 1 mM nitrate + 1 mM ammonium) added to the nutrient solution for 15 d. After this period, 72 seedlings of similar size were selected. For half of these seedlings, Cd was supplied in the form of 10 mg/L CdCl2 added to the nitrogen-containing nutrient solution, after being sterilized by passing through a membrane filter. The other half of these seedlings did not receive Cd treatment. These groups were further subdivided to receive three N mixtures, each containing the same proportional composition of nitrogenous compounds, with 1 mM NO3-, 1 mM NH4+, and 1 mM glycine. Each mixture contained one specific compound labelled with

15

N at 5.0%, i.e., 5.0%

15

N-glycine, 5.0%

15

NO3-, or 5.0%

15

NH4+. For instance, under 10 mg/L Cd conditions, the three N forms were

15

NO3-:NH4+:glycine, NO3-:15NH4+:glycine, and NO3-:NH4+:15N-glycine, and the

uptake and N contribution of the different N sources in a mixed N could be separated based on this labelling scheme. These subdivisions resulted in six treatment groups (3 N sources × 2 Cd conditions), with 12 seedlings in each group. After 8 d, the shoots and roots of each plant were harvested separately. Samples from four seedlings in each group were pooled in order to reduce individual differences, yielding a total of three samples for each experimental group. Roots were washed using an ultrasonic bath for 1 min and washed in 50 mM CaCl2, then washed three times in distilled water.

Shoots and roots were freeze-dried (Labconco Freeze System, USA) and ground into powder using a ball mill (Retsch MM301, Germany). The total N content and

15

N

proportion of the powder were determined using an Elemental Analysis-Stable Isotope Mass Spectrometer (Isoprime100, UK). In addition, three extra ‘blank’ seedlings were reserved to detect the percentage of 15N atoms, which were supplied the same with the treatment but 15N-unlabelled N (Experiment 1, 2, 4).

2.2.2 Experiment 2: Effect of Cd stress on the short-term uptake of glycine Pakchoi seedlings were cultivated as described for 25 d. At the commencement of the experiment, 48 seedlings of similar size were selected for short-term testing. Each seedling’s roots and centrifuge tubes were washed with sterile distilled water. Seedlings were ‘starved’ (i.e., cultivated overnight in a nutrient solution without N) prior to the experiment. Twenty-four seedlings were cultivated with 1 mM NO3- + 1 mM NH4+ + 1 mM 98.10%-15N-glycine for 4 h with 10 mg/L CdCl2 and no Cd treatment. Simultaneously, the other twenty-four seedlings were starved and pretreated for 1 h in a 50 µM solution containing the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) (Persson and Näsholm, 2002), then cultivated with 1 mM NO3- + 1 mM NH4+ + 1 mM 98.10%-15N-glycine for 4 h with 10 mg/L CdCl2 and no Cd treatment. Afterward, as per Experiment 1, roots and shoots were harvested separately, and four seedlings pooled into a single sample, to yield 3 samples per treatment group. Root samples were washed, dried, and analysed as described in Experiment 1. The 15N values obtained in the CCCP-treated group represented the

passive uptake amounts of glycine uptake by pakchoi.

2.2.3 Experiment 3: Effect of Cd stress on the activity of N metabolic enzymes Pakchoi seedlings were cultivated for 22 d as described. Prior to experimentation, seedlings were starved overnight as described in Experiment 2. Forty-eight seedlings were cultivated for 4 d in 1 mM NO3- + 1 mM NH4+ + 1 mM glycine, 24 with 10 mg/L CdCl2 and 24 with no Cd treatment. The nutrient solution was changed every 2 d. Seedlings were harvested with roots and shoots separated, and six seedlings from each group were pooled into one sample to yield four pooled samples for each subgroup. The activities of glutamine synthetase (GS) (Horchani et al., 2010), glutamic-pyruvic transaminase (GPT), and glutamic oxaloacetic transaminase (GOT) (Lianghuan et al., 1998) in the roots and leaves were measured.

2.2.4 Experiment 4: Effect of Cd stress on the metabolism of glycine Experiment 1 showed that Cd stress inhibited the uptake of glycine (see section 3.1 of Results). However, in Experiment 2, this effect was not strongly detected over the short term (see section 3.2 of Results). Additionally, N metabolic enzymes activity under Cd stress decreased, indicating that Cd stress limits glycine metabolism rather than its uptake at the roots. In order to discern which glycine metabolism step was inhibited,

15

N labelling and GC-MS were used to measure labelled amino acids in

pakchoi roots and shoots. Pakchoi seedlings were cultivated for 25 d as described in

Experiment 1, and 96 seedlings of similar size were selected. Prior to experimentation, all seedlings’ roots were washed several times with distilled water and the seedlings were starved for 12 h as described in Experiment 2. Then, seedlings were cultivated with 1 mM 98.1% 15N-glycine + 1 mM NO3- + 1 mM NH4+ for 4 h with 360 μmolm-2 s-1 light exposure, at 60% humidity and 25°C. Half of these (48 seedlings) were also treated with 10 mg/L CdCl2 for the duration. Subsequently, the shoots and roots were harvested separately, and 8 seedlings in each treatment group were pooled to yield 6 replicates per group. The roots were washed and the shoots and roots were dried and ground as described in Experiment 1. The 15N-labelled amino acids were detected by GC-MS as described by Thornton and Robinson (2005), with minor modifications. In brief, 20 mg of each dried and milled sample was placed in 3 ml of 80% ethanol for 1 h and shaken gently every 10 min. This solution was centrifuged at 3500 g for 15 min and the supernatant reserved. The remaining pellet was then treated a second time in 80% ethanol for 1 h, with gentle shaking at 10 min intervals, and re-centrifuged. This supernatant was combined with the previously reserved supernatant and dried by a rotary evaporator (EYELA, SB-1100) at 25°C. The residue was re-suspended in 1 ml 0.1 M hydrochloric acid, then centrifuged at 12,000 g for 15 min. Each supernatant sample was added to a Dowex 50WX8-200 (2 ml bed volume, H+ form) cation exchange column. The columns were washed with 20 ml ultrapure water, and 20 ml of 4 M ammonia (NH3) solution was used to wash out the amino acids. The eluate was blown for 8 h with N2 to remove the NH3, then freeze-dried (Labconco Freezen System, U.S.A.). Amino acids in the resulting extracts were derivatised to

t-butyldimethylsilyl by 10 μl N-methyl-N-tert-butyldimethylsilyl-trifluoroacetamide. Finally, the 15N labelled amino acids in the roots and shoots were detected by GC-MS.

2.3 Statistical analyses Data are presented as the mean ± standard error (SE). We applied one-way analysis of variance (ANOVA) followed by Duncan’s multiple range method (p < 0.05) to assess differences between treatments. All statistical analyses were performed using SAS 8.2 (SAS Institute Inc., Cary, NC). Figures were created using Origin 8.1(OriginLab, Northampton, MA).

3. Results 3.1 Pakchoi biomass and long-term N uptake under Cd-stressed condition Externally-supplied Cd had a significant effect on the growth and N uptake of pakchoi (Fig. 1). Under Cd-stressed conditions, pakchoi shoot and root biomass were lower by 18.5% and 20.0%, respectively, than those of control plants not exposed to Cd (Fig. 1A). Similarly, the N uptake in shoot and root under 10 mg/L CdCl2 were decreased by 22.4% and 18.6% respectively (Fig. 1B), but the N content was not affected by Cd stress (data not shown).

3.2 Effects of Cd stress on long-term uptake of glycine, nitrate, and ammonium

Cd stress enhanced the uptake of nitrate to a greater extent than that of ammonium and glycine (Fig. 2). Because Cd stress decreased the overall biomass of pakchoi in our experiments, we used

15

N abundance per plant to demonstrate these

effects (Fig. 2A, B). This showed that Cd stress decreased the uptake of glycine in shoots, but increased the uptake of nitrate and ammonium in shoots and roots. If the difference in biomass between Cd-stressed and non-Cd-stressed plants is taken into account, it appears that the uptake of ammonium and nitrate was lower due to the decreased biomass (Fig. 2C, D). The effect of Cd stress on N contribution is inconsistent with

15

N abundance or

15

N uptake. Cd stress significantly increased the

contribution of nitrate in shoots and roots, while decreasing the contribution of ammonium in pakchoi shoots. Strikingly, the N contribution of glycine significantly increased in roots while decreasing in shoots under Cd stress (Fig. 2E, F).

3.3 Short-term uptake and transport of glycine The pattern of uptake for glycine was affected by Cd stress. In shoots, Cd stress inhibited the active uptake of

15

N-glycine, while significantly enhancing the passive

uptake of 15N-glycine (Fig. 3B). In roots, active uptake of 15N was higher by 8.4% in Cd-stressed plants than in control plants without Cd exposure (Fig. 3C). If these data from roots and shoots are merged, an inhibition of active uptake with enhancement of passive uptake under Cd stress is demonstrated, but with little effect on the total amount of

15

N-glycine (Fig. 3A). In addition, Cd stress significantly inhibited the

transport of

15

N obtained through active uptake, while significantly increasing the

passive uptake of 15N (Fig. 3D).

3.4 Activity of glycine metabolic enzymes Cd stress significantly reduced GS, GOT, and GPT enzyme activities in roots, and somewhat reduced activities in shoots. In addition, enzyme activities in roots were much higher than those in shoots (Table 1).

3.4.1 15N-labelled amino acids in shoots and roots Cd stress had a significant effect on 15N-amino acids content in roots and shoots (Fig. 4). In roots,

15

N-labelled glycine in plants under Cd stress was significantly

higher than controls, while

15

N-labelled serine, asparagine, glutamine, and

gamma-aminobutyric acid were significantly lower than in controls (Fig. 4A). In shoots,

15

N-labelled glutamic acid, glutamine, and asparagine in plants under Cd

stress were significantly lower than in controls (Fig. 4B). Additionally, the composition of roots,

where

15

N-labelled amino acids was shown to differ between shoots and

glycine

and

serine

formed

the

majority

in

roots,

but

gamma-aminobutyric acid, glutamine, and glutamic acid formed the majority in shoots.

4.0 Discussion

4.1 Effects of Cd stress on pakchoi growth and N uptake Cd stress had a considerable negative effect on pakchoi growth and N uptake. Previous studies had shown that heavy metal exposure can have an inhibiting effect on plants’ metabolic activities, with direct and indirect effects on biochemical processes causing the overall inhibition (Zhang et al., 2014; Zouari et al., 2016). For example, Cd can disrupt cellular homeostasis and lead to the overproduction of reactive oxygen species, resulting in structural damage through electrolyte leakage, lipid peroxidation, and DNA damage (Howladar, 2014). Furthermore, Cd has a negative effect on photosynthesis, and also decreases carbon assimilation by stomatal closure and changing of thylakoid membrane composition and chloroplast ultrastructure (Asgher et al., 2014; Khan et al., 2014). In addition, nutrient uptake, metabolism, transport, and relocation are affected by Cd, and it may replace essential elements that facilitate in enzymes activities (Zouari et al., 2016). In plants under Cd stress, uptake of macro-elements such as K, Mg and Ca and trace elements such as Fe, Zn, Mn, and Cu are inhibited (Ali et al., 2014; Gonçalves et al., 2009). Our results are consistent with these previous findings, and we additionally suggest that decreased biomass of Cd-stressed plants might result in a decrease in total N uptake.

4.2 Effects of Cd stress on the relative uptake of glycine, nitrate, and ammonium Several studies have shown that biotic and abiotic factors affect plants’ uptake and metabolism of nutrients. Cd not only affects micro-element and trace element

uptake as stated above, but also changes the relative uptake and contribution of different N. In order to control for the effects of difference in biomass, we examined

15

N

abundance in pakchoi (Fig. 2A, B). This showed that Cd stress decreased the uptake of glycine in shoots, but increased the uptake of nitrate and ammonium in pakchoi shoots and roots. As glycine is one of the most important amino acids in plant growth, and plants can use it directly for growth, its uptake might be expected to increase in comparison with that of ammonium and nitrate. However, the opposite was observed, wherein the uptake of nitrate was actually enhanced with respect to that of ammonium and glycine. This finding might be a result of several possible processes. First, Cd affects the uptake and metabolism of N, while N metabolism is important for plants response in counteracting Cd toxicity. Under Cd stress, plants often synthesize a set of N-containing metabolites through N metabolism, such as amino acids, phytochelatins and glutathione, which play a significant role in plants’ Cd tolerance (Sharma and Dietz, 2006). The resulting accumulation of amino acids may inhibit the glycine uptake. Second, Cd may be more toxic to roots than shoots, and may affect the metabolism of different forms of N directly. Most nitrate absorbed by roots is transported to and metabolized in shoots (Xu et al., 2012), while most amino acids and ammonium are metabolized in the roots (Warren, 2012). Roots, then, may show a higher inhibition effect in taking up amino acids and ammonium than nitrate uptake. Taking the difference in biomass into account, the uptake of all nitrogenous

forms decreased under Cd stress conditions, with the decrease in nitrate less pronounced than that of ammonium and glycine. In addition, the contribution of nitrate increased under Cd stress, while the contribution of ammonium significantly decreased in root. This suggests a synergistic effect between nitrate and Cd and an antagonistic effect between ammonium and Cd (Jalloh et al., 2009; Yang et al., 2016). We show that Cd stress increases the N contribution of nitrate, while decreasing the contribution of ammonium. Clearly, the uptake of Cd and N influence one another, and regulating Cd accumulation by controlling plant N nutrition is an important consideration. Excessive use of nitrate fertilizer increases health risk of humans by increasing Cd accumulation (Yang et al., 2016). Cd is a great risk for human health, and most of Cd uptake is from rice and vegetables (Yang et al., 2016). Furthermore, nitrate fertilization is employed in crop and vegetable production to pursue higher yield. In our test, Cd stress increased the uptake ratio of nitrate, and other studies have shown that nitrate increases Cd accumulation (Kovácik et al., 2011; Yang et al., 2016). This suggests that nitrate fertilization will increase Cd poisoning risk for humans. In addition, the increased ratio of nitrate taken up under Cd stress increases the nitrate content in vegetables, posing a further health risk. It is therefore advisable, when fertilizing cultivated food plants, to choose a nitrogen source carefully in order reduce Cd accumulation. Amino acids may be an ideal choice, as they not only act as an N source, but can also regulate plant growth. In our test, glycine-N accounts for 35.8-40.8% of total N uptake, which shown that pakchoi possesses a great ability to

take up and metabolise a large number of amino acids. In addition, amino acids and heavy metals can also become bonded, reducing the uptake and toxicity of the heavy metals. Heavy metals can conversely affect the uptake and metabolism of amino acids (Cernei et al.), and we were able to show that Cd stress did not reduce the N contribution of glycine. This indicates that amino acids are a good choice of N fertilization to reduce Cd accumulation in polluted soils.

4.3 Effects of Cd stress on the uptake and metabolism of glycine Environmental factors control plant growth by affecting N uptake, metabolism, transport, storage, and reallocation (Susanne et al., 2014). Root amino acid metabolism is influenced by several environmental factors. For example, the conversion of glycine to serine is regarded as the limiting step for glycine metabolism under high temperature (Thornton and Robinson, 2005), and the metabolism of ammonium produced from glycine is suggested to inhibit the glycine contribution for pakchoi growth under high light intensity (Ma et al., 2016). Studies of signalling pathways and bottlenecks in amino acid metabolism in plants growing in different environments could help improve the efficiency of N usage (Tegeder, 2012). Root uptake was not the limiting step for N contribution of glycine under Cd stress conditions. In the short-term uptake test, the uptake of glycine did not significantly decrease, in contrast with the long-term uptake test. This indicates that uptake did not limit the N contribution of glycine. However, active uptake decreased while passive uptake significantly increased, and the active uptake

15

N-glycine

transport from roots to shoots decreased. Cd affects the permeability of root cells, which could lead to an increase in the passive uptake of glycine. In addition, Cd inhibits biochemical processes relating to energy production (Zhang et al., 2014; Zouari et al., 2016), further reducing the active uptake of glycine. After being taken up in the roots, glycine is converted to serine and ammonium, a process catalysed by serine hydroxymethyltransferase. Then, serine is converted to other amino acids and ammonium is assimilated to glutamine catalysed by GS (Castro-Rodríguez

et

al.,

2011).

Subsequently,

glutamine

combined

with

2-oxoglutarate can be assimilated into glutamate, catalysed by glutamate synthase, which can be further converted to aspartic acid, catalysed by GOT, or converted to alanine, catalysed by GPT (Xu et al., 2012). The activities of all of these enzymes involved in N metabolism decreased under Cd stress, demonstrating that Cd can inhibit the metabolism of glycine. Furthermore, in plants under Cd stress, the 15

N-glycine in roots was found to be much higher than that in control plants, while the

content of serine, asparagine, glutamine, and gamma-aminobutyric acid were much lower. This indicates that Cd stress inhibited the conversion of glycine to serine. In addition, there was a considerable difference between composition of

15

N-labelled

amino acids in shoots and roots. In roots, 15N-glycine and serine formed the main part, while gamma-aminobutyric acid, glutamine, and glutamic acid formed the main part in shoots. According to a previous study, glycine was metabolized by deamination in roots and U-13C, and

15

N-glycine was not detected in xylem sap (Warren, 2012).

Furthermore, glutamine, asparagine, aspartic acid, and glutamic acids are the main

amino acids transported to the shoots (Xu et al., 2012). Taken together, the results of the short-term uptake test, N-metabolizing enzyme activities, and the 15N amino acids content, it appears that the metabolism of glycine to serine in roots was the limiting step for glycine uptake under Cd stress.

4.4 Conclusions Cd stress appears to have a strong inhibitory effect on pakchoi growth and N uptake. This effect was more pronounced on ammonium and amino acid uptake than on nitrate uptake. However, amino acids accounted for a large proportion of total N uptake, showing that pakchoi possesses a great ability to utilize these as a nitrogen source. In addition, root metabolism of glycine to serine, rather than root uptake, is the limiting step in N contribution of glycine under Cd stress.

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Figure 1. Effects of Cd stress on pakchoi biomass and N uptake, with shoot and root biomass (A), and N uptake (B) given under non-Cd-treated conditions (Cd0) and Cd-treated conditions (Cd10). Bars indicate mean values ± SE; n = 3. Asterisk indicates significant differences between Cd0 and Cd10 (p < 0.05 level).

Figure 2. Effects of Cd stress on 15N-glycine, nitrate, and ammonium uptake under mixed N source conditions. The 15N-abundance in shoot (A) and root (B) are presented. The uptake of glycine, nitrate, and ammonia in shoots (C) and roots (D) are shown. The N contribution of each form of N to the total N uptake (%) in shoots (E) and roots (F) are also indicated. Bars indicate mean values ± SE; n = 3. Asterisk indicates significant differences between Cd0 and Cd10 (p < 0.05 level).

Figure 3. Effects of Cd stress on the short-term uptake of g15N-glycine. 15 N-glycine in shoots (A) and roots (B), total 15N-glycine in shoots and roots (C), and the transportation rate (D) are shown. Bars indicate mean values ± SE; n = 3. Asterisk indicates significant differences between Cd0 and Cd10 (p < 0.05 level).

Figure 4. Effects of Cd stress on the content of 15N-labelled amino acids in pakchoi roots (A) and shoots (B) after 4 h uptake. Bars indicate mean values ± SE; n = 6; Asterisk indicates significant differences between Cd0 and Cd10 (p < 0.05 level).

Table 1. Effects of Cd stress on the activity of N-metabolizing enzymes in pakchoi Values represent the mean ± SE (n = 4). Different letters in each column indicate significant differences between treatments at the p < 0.05 level.

Treatment Cd0 Cd10

GPT (µmol·g-1·30min)

GOT (µmol·g-1·30min)

GS (A·mg-1 protein·h-1)

Shoot

Root

Shoot

Root

Shoot

Root

7.1 ± 0.4a 6.4 ± 0.4a

9.5 ± 0.8a 7.1 ± 0.5b

8.8 ± 0.4a 8.2 ± 0.3a

16.6 ± 0.7a 14.5 ± 0.5b

13.1 ± 0.8a 10.7 ± 0.5b

26.3 ± 1.4a 21.7 ± 1.0b