Nitrogen as an important detoxification factor to cadmium stress in poplar plants

Abstract

To verify the important role of nitrogen in detoxifying plants from heavy metals in Populus, the influence of nitrogen and cadmium on growth, chlorophyll (Chl) synthesis, and the expression of the Glutamine synthetase gene (GS₂) were studied in poplar plants. Experiments were carried out in potted plants treated with (NH₄)₂CO₃, Cd(NO₃)₂, CdCl₂ and CdCl₂ plus (NH₄)₂CO₃. After treatment, plant height, biomass, chlorophyll content, the precursors content and GS₂ were investigated. Results showed that the plants treated with cadmium showed toxicity symptoms, decrease in growth and Chl content. Cd inhibited Chl synthesis seriously by blocking the site located on the steps between UrogenIII and Coprogen III. However, the plants treated with cadmium and nitrogen grew well without any toxicity symptoms. Nitrogen supplement can alleviate Cd inhibition on chlorophyll synthesis by unblocking the pathway. The results indicated that nitrogen can effectively alleviate cadmium toxicity to poplar plants.

Keywords:

• poplar
• cadmium
• nitrogen
• growth
• chlorophyll synthesis
• Glutamine synthetase II gene

Abbreviations
cadmium	=	Cd
chlorophyll	=	Chl
nitrogen	=	N
Glutamine synthetase II gene	=	GS2
glutathione	=	GSH
Glutamine	=	Gln
Phytochelatins	=	PC
reactive oxygen species	=	ROS

Introduction

The development of urbanization and industrialization has increased heavy metal contamination which is produced and released by human activities. This increase of heavy metals may create oxidative stress on the generation of plants in the surrounding environment, which will in turn result in the ecology of the area to undergo unbalanced deterioration. Cadmium (Cd) is one of the most toxic pollutants found in the air, water and soil, which is non-essential for plants. It can be transferred through the food chain (Wagner 1993), and its accumulation in cereals represents a risk for animal and human health (Chaney et al. 1999; McLaughlin et al. 1999). Cd interacts with photosynthetic, respiratory and nitrogen (N) metabolism in plants, resulting in poor growth and low biomass accumulation (Sanitá & Gabbrielli 1999; Pereira et al. 2002). Recently, Cd pollution has been more serious, which intensifies environmental deterioration. Many farmlands have been classified as the core polluted regions in the world.

Cd harms plants through oxidative stress. It is a non-redox metal, which does not participate in redox reactions, but leads to the formation of reactive oxygen species (ROS), such as superoxide anion radicals and hydrogen peroxide (Dietz et al. 1999; Sandalio et al. 2001; Romero-Puertas et al. 2006). Therefore, a mechanism to interrupt such an autocatalytic process is required.

Although plant growth, Chl content, stomatal opening, transpiration and photosynthesis have been reported to be inhibited by Cd in nutrient solutions (Jiang & Li 1992; Baryla et al. 2001; Drążiewicz & Baszyński 2005; Sun et al. 2005), there are others’ reports that Cd treatments have no effect on photosynthesis or growth (Greger & Lindberg 1986; Haag-Kerwer et al. 1999; Li et al. 2005; Zhou & Qiu 2005). Our early study (in press) showed that after adding nitrogen to plants, the damage caused by Cd stress could be efficiently alleviated to better growth. Li et al.'s study (2007) indicates that adding the appropriate N (NO₃⁻ or NH₄⁺) in nutrient solution could enhance the development of root systems and increase the accumulation of Cd in Sedum alfredii Hance. Sedum alfredii Hance is a plant tolerant to cadmium and zinc (Yang et al. 2004)

The important physiological role of N in detoxifying plants from heavy metals has not been studied. However, the nitric oxide acting as bioactive signaling molecule in plant responses to heavy metal stress has been reported in many studies (Hassan et al. 2005; Floryszak-Wieczorek et al. 2006; Grün et al. 2006; Arasimowicz & Floryszak-Wieczorek 2007). The exogenous nitric oxide reducing the destructive action of heavy metals, ethylene and herbicides on plants was documented (Kopyra & Gwózdz 2003). Garcia-Mata and Lamattina (2001) reported that nitric oxide induces stomatal closure and enhances the adaptive plant responses against drought stress.

If nitrogen plays an important role in detoxifying plants from Cd, using tall, woody plants in combination with nitrogen fertilization to remediate contaminated sites would be an aesthetically pleasing and beneficial approach that not only promotes positive landscape effects, but reduces erosion. This study is aimed at further verifying the important physiological role of N in detoxifying plants from Cd in Populus species.

Materials and methods

Plant materials and experimental design

Two dormant, one-year-old, unrooted, 10-centimeter-long hardwood sticks of poplar clone ‘107’ (Populus deltoides×P. nigra) were set in a polyethylene pot with 5 kg of dry soil. The dimensions of the pot were 30 cm×20 cm×50 cm. The 25 pots were divided into 5 groups; each group was placed in a plant growth chamber with a 16 h photoperiod (photosynthetic active radiation about 250 µmolm⁻².s⁻¹), a 25/20 °C temperature-period, 70% air humidity, and was watered daily. A polyethylene disc was placed under each pot to avoid run-off. When the plants reached 15 cm height, approximately after a month, the following treatments were carried out: (1) a control treatment consisting of tap water without Cd or ammonium carbonate ((NH₄)₂CO₃) amendments (CK); (2) a 1.680 g Cd(NO₃)₂ treatment added to each pot every 5 days (T1); (3) a 1.303 g CdCl₂ treatment added to each pot every 5 days (T2); (4) a 1.303 g CdCl₂ and 0.682 g (NH₄)₂CO₃ treatment added to each pot every 5 days (T3); (5) 0.682 g (NH₄)₂CO₃ treatment added to each pot every 5 days (T4). The amount of Cd (Cd^{2 +}) added to each pot each time was equal to all the Cd treatments (T1, T2 and T3); similarly the amount of N added per pot each time was equal among T1, T3 and T4. The treatments were made until chlorosis was evident in T2. The duration of the experiment was 53 d. Total Cd weighing 7.988 g and N 1.989 g were added to the soil during the whole treatment.

The physicochemical properties of soil used

The experimental soil is sampled from the farmland of Sichuan agricultural university. Soil samples were collected from the surface (0–15 cm depth) of cultivated soils, and were air-dried, sieved, and analyzed in the laboratory using standard techniques. Its texture was identified by following the procedure described by Boyd (1995). The pH of the soil was obtained by a soil pH meter (USA). The organic carbon content was determined by the wet oxidation method as described by Nelson and Summers (1982). The total nitrogen and hydrolyzable nitrogen content were determined according to the methods described by Grimshaw et al. (1989). Total phosphorus and available phosphorus content were determined colourimetrically by the molybdo-phosphoric – blue method using ascorbic acid as a reducing agent (Murphy & Riley 1962). The total potassium was extracted with 1 M ammonium acetate (1 M NH₄OAc) solution buffered at pH 7.0 and was detected as described by Anderson and Ingram (1998). The physicochemical properties of soil are as followings: pH 5.8, organic matter 10.65 g·kg⁻¹, total nitrogen 0.21 g·kg⁻¹, total phosphorus 0.31 g·kg⁻¹, total potassium 2.56 g·kg⁻¹, hydrolyzable nitrogen 43.21 mg·kg⁻¹, available phosphorus 17.58 mg·kg⁻¹, available potassium, 46.17 mg·kg⁻¹.

Cadmium and nitrogen assay

Functional leaves were sampled to wash 3 times with distilled water, oven-dried for 24 h at 80 °C, and ground to a fine powder. The concentration of Cd in leaves and soil was determined with ICP-OES (OPTIMA 2000A, USA) after wet-digesting in HNO₃-H₂SO₄-HCLO₄ (Wu & Ge 1999). Nitrate-N and ammonia-N soil concentrations were measured with UV-spectrophotometer method and the phenol hypochlorite colorimetric method (Berthelot reaction) respectively.

Growth and biomass assay

Plant height and basal diameter were measured at the beginning and end of the Cd addition treatments. After the final treatment, biomass (dry weight) was determined. Samples were oven-dried at 90 °C for 15 min, kept at 70 °C for 24 h to obtain a constant dry weight and weighted for biomass.

Chlorophyll assay

Chl was extracted in 80% acetone. Absorbance was measured at 663 and 645 nm by a spectrophotometer (UV-2450, Shimadiu Corporation, Japan). Extinction coefficients and equations reported by Lichtenthaler (1987) were used to calculate the amounts of Chl a and b. Measurements were done in triplicate.

The metabolic intermediates of chlorophyll biosynthesis extraction and quantization δ-aminolevulinic acid (ALA) assay

ALA was extracted and quantified by methods of Wang et al. (1997) with some modifications. Fresh leaves (0.4 g) were ground in 4 mL of trichloroacetic acid (4%). Samples were centrifuged at 12,000 g for 10 min, and 1 mL supernatants were sampled and added with 500 µL NaAc (1 mol/L) and 50 µL Acetyl acetone. Following incubation in boiling water for 10 min, the liquid was cooled to room temperature, and then, it was centrifuged at 12,000 g for 10 min. The supernatants were sampled and added with the same volume of Ehrlich-Hg, and finally placed in a dark room for 15 min. The concentration of ALA was calculated according to the absorption at 553 nm.

Porphobilinogen (PBG) assay

PBG was measured by the methods of Bogorad (1962) with some modifications. Fresh leaves (0.2 g) were ground in liquid nitrogen and tissue was homogenized in 4 mL buffer (0.6 M Tris, 0.1 M EDTA, pH = 8.2). Samples were centrifuged at 12,000 g for 10 min. The supernatants were sampled and added with the same volumes of Ehrlich-Hg, and then placed in a dark room for 15 min. The concentration of PBG was calculated in the absorption at 553 nm.

Uroporphyrinogen III (Urogen III) and Coproporphyrinogen III (Coprogen III) assay

UrogenIII and CoprogenIII were extracted and quantified as described as Bogorad (1962) with some modifications. Fresh leaves (0.2 g) were ground in liquid nitrogen and tissue was homogenized in 4 mL of 0.067 M potassium phosphate buffer (pH 7.8). Samples were centrifuged at 12,000 g for 10 min. The supernatants were combined with 200 µL Na₂S₂O₃ (1%), shaken quickly, and then placed under the light for 20 min. The combined liquid was changed to pH 3.5 with cold glacial acetic acid and was added to a final Diethyl ether. The Urogen III concentrations were determined at 405.5 nm by a spectrophotometer (UV-2450, Shimadiu Corporation, and Japan). The final supernatants were extracted three times with 0.1 mol·L⁻¹ HCL. The concentration of Coprogen III was calculated in the absorption at 399.5 nm.

Protoporphyrin IX (ProtoIX) assay

Leaf samples (0.2 g fresh leaves) were ground in liquid nitrogen and tissue was homogenized in 6 mL of cold acetone: 0.1 M NH₄OH (90/10, v/v) and transferred into a centrifuge tube. Samples were centrifuged at 12,000 g for 10 min. The supernatants were combined and washed successively with equal volume of hexane prior to spectrophotometric analysis. The concentration of Proto IX was detected based on the method of Rebeiz et al. (1975).

Mg-protoporphyrin IX (Mg-Proto IX) and Protochlorophyllide (Pchlide) assay

Mg-Proto IX and Pchlide were extracted and measured by the method reported by Terry and Kendrick (1999) with small modifications. Leaf samples (0.2 g fresh leaves) were ground in liquid nitrogen and tissue was homogenized in 6 mL of cold acetone: 0.1 M NH₄OH (90/10, v/v) and transferred into a centrifuge tube. Samples were centrifuged at 12,000 g for 10 min. The supernatants were combined and washed successively with an equal volume and a one-third volume of hexane prior to spectrophotometric analysis. For determination of Pchlide ester, the hexane washes were pooled and analyzed directly.

Absorption spectroscopy of Mg-Proto IX samples was performed by a spectrophotometer (UV-2450, Shimadiu Corporation, and Japan). The concentration of Mg-Proto IX was determined based on a molar absorption coefficient of 16,200 M⁻¹ cm⁻¹ at 503 nm, and the Pchlide concentrations were determined by using a molar absorption coefficient of 31,100 M⁻¹ cm⁻¹ at 626 nm.

Nitrate reductase activity assay

Nitrate reductase was extracted from frozen leaf tissue stored at −80 °C. Nitrate reductase was extracted and the maximum extractable activity was measured at 4 °C (Ferrario-Mé et al., 1997; Chaffei et al., 2004).

Expression assay of Glutamine synthetase II gene

RNA isolation

Total RNA was extracted from leaves as described by Verwoerd et al. (1989). RNA integrity was verified by electrophoresis using ethidium bromide staining. First, the strand cDNA was reverse transcribed based on the standard protocol of TaKaRa prime Script^TM RT-PCR Kit. About 500 ng RNA, 1.0 µl of 10 mmol/l dNTPs, 1.0 µl of 2.5 µmol/µl OligodT primer, 1.0 µl of 20 µmol/µl random primer and 2 µl of RNase-Free H₂O were mixed and heated at 65 °C for 5 min. The mixture was immediately placed on ice for 2 min and was transitorily centrifuged. Then 4.0 µl of 5×First-Strand buffer, 0.5 µl of 40 U/µl RNase inhibitor, 0.5 µl of the Primer Script^TM RTase and 5 µl RNase-Free H₂O were added and mixed. 20 µl of reaction volume was incubated for 10 min at 30 °C, 25 min at 45 °C, 5 min at 95 °C, followed by heating at 70 °C for 15 min to stop the synthesis reaction and then held at 4 °C.

Real-time RT-PCR

The first cDNA strand was reverse transcribed from 500 ng of DNase-treated RNA as described above. Based on the homologous sequences of GS₂, gene-specific primers were designed by the Primer Express software, version 2.0 (Applied Biosystems, Courtaboeuf, France). The forward primer was 5′-TGCGTAGCAAGTCAAGGA-3′, and the reverse primer was 5′-TTCAGCCAGTAGCGAGGT-3′. Real-time quantitative PCR was performed by Bio-Rad iQ5 (Bio-Rad Company), and the results were analyzed using an optical system version 2.0 (Bio-Rad) software. The actin gene of the poplar used as the inner reference was amplified in parallel with the target gene allowing gene normalization and quantification. Based on the reaction procedure of the SYBR Premix Ex Taq^TM II Kit (TaKaRa), the reaction system was listed as follows: 10 µl of SYBR Premix Ex Taq^TM II (2×), 0.8 µl of each Primer (10 µmol/l), 1.5 µl template (cDNA solution), 6.9 µl of dH₂O. The two-step PCR reaction procedure used was as follows: 95 °C pre-denaturalized for 10 s, 95°C for 5 s and 55 °C for 20 s, the PCR reaction continued for 40 cycles, and was then kept at 72 °C for 2 min. After the PCR reaction, a melting curve was obtained with the optical version 2.0 (Bio-Rad) software and the parameters were set as the reading plate for two cycles for every increase of 0.5 °C in the range from 68 °C to 95°C. Three technical replicates of each reaction were performed. The relative changes in gene expression were quantified using the 2^−ΔΔC_T method as described by Livak and Schmittgen (2001).

Statistical analysis

Analyses of variance (ANOVA) with orthogonal contrasts and mean comparison procedures were used to detect differences among treatments. Mean separation procedures were carried out by the multiple range tests with Fisher's least significant difference (LSD) (P<0.01).

Results

Cadmium and nitrogen content in soil

Cd concentration in soil and leaves reached from 9.96 to 520 mg·kg⁻¹ respectively in the end of the experiment. The Cd in the soil of T2 was significantly higher than that of T1 and T3, while the Cd content in the leaves of T2 was lower than that of T1 and T3 (Table 1). The Cd in the soil and in the plant increased with adding exogenous Cd(NO₃)₂ or CdCl₂. The results indicate that the plants treated with Cd and N (NO₃⁻ or NH₄⁺) absorbed more Cd than the plants treated only with CdCl₂. N concentrations in the soil of T1 and T3 were substantially higher than that of CK and T2 (Table 1). Together, the results suggest that the addition of N significantly promotes one clone of populus deltoides×populus nigra's absorbtion of Cd.

Table 1. Addition of nitrogen may significantly increase cadmium absorption by poplar plants from soil.

Treatments		Cd in soil	Cd in leaves	NO3–N in soil	NH4–N in soil	NR activity (As percentage of control)
CK	Blank	20.12±0.24^A	5.13±0.17^A	12.1±0.02^A	14.6±0.06^A	100±3.76^A
T1	Cd(NO3)2	322±80^B	152.5±8.6^C	356.8±0.03^C	215.4±0.05^D	212±4.23^c
T2	CdCl2	520±40^C	91.6±5.7^B	55.6±0.06^A	13.4±0.03^A	158±2.15^B
T3	CdCl2+(NH4)2CO3	318±50^B	164.1±3.9^D	276.4±0.01^B	145.2±0.07^C	265±4.61^D
T4	(NH4)2CO3	9.96±0.31^A	3.52±0.16^A	398.7±0.04^D	123.16±0.11^B	271±4.61^D

Note: Each value is the mean±SE of six replicates, and the terms “A, B, C and D” within each column indicate significant difference between means (p < 0.01). Unit, mg·kg⁻¹.

Plant morphology and growth

No measurable differences were recorded in the treated plants at the beginning of the study. However, various phenotypes diverged in the experimental treatments. Typical treatment differences were exhibited by growth rate and leaf color. The T2 plants grew slower than the control or other treatments. The leaves of T1 and T3 gradually turned dark-green with the treatment, while leaf color in T2 slowly changed from a normal shade of green to yellow-green. At the end of the experiment, leaf chlorosis was evident in T2. CdCl₂ severely inhibited growth; a significant decrease in plant height, plant basal diameter and biomass was observed in comparison with CK, and its average plant height, basal diameter and dry weight decreased 18.6%, 16.0% and 31.0% respectively in comparison to the control. Conversely, both the T1 and T3 treatments induced a significant increase in plant growth (Table 2). Plant height, basal diameter and biomass increased 40.7%, 14.0% and 20.1% respectively under Cd(NO₃)₂ treatment in comparison to the control, and Plant height, basal diameter increased 15.8%, 3.6% and biomass decreased 3.2% compared to (NH₄)₂CO₃ only treatment respectively. Similarly, plant height, basal diameter and dry weight increased 38.3%, 24% and 32.6% under CdCl₂+(NH₄)₂CO₃ treatment compared to the control, and increased 13.8%, 12.7% and 6.9% compared to sole (NH₄)₂CO₃ treatment respectively.

Table 2. Effects of different Cd resources on plant height (cm), basal diameter (mm) and dry weight (mg plant⁻¹).

Treatments		Height (mean±SE)	Basal diameter (mean±SE)	Dry weight
CK	Blank	41.2±5.3^B	5.0±0.63^B	63.2±3.1^B
T1	Cd(NO3)2	58.0±4.5^E	5.7±0.47^CD	75.9±4.3^C
T2	CdCl2	33.5±4.8^A	4.2±0.48^A	49.3±2.7^A
T3	CdCl2+(NH4)2CO3	57.0±4.6^E	6.2±0.67^D	83.8±4.5^D
T4	(NH4)2CO3	50.1±3.2^D	5.5±0.46^C	78.4±3.9^CD

Note: Each value is the mean±SE of six replicates, and the terms “A, B, C and D” within each column indicate significant difference between means (p<0.01).

Chlorophyll content

Chl content varied widely in treatments (Table 3). Chl a, Chl b, and total Chl content in T2 were significantly lower than CK in respective measured reductions of 17.0%, 95.9% and 34.5%. The Chl a:b ratio (70.6:1) of T2 was substantially higher than the ratio of CK (3.49:1), which is indicative of the severe inhibition that Cd had on Chl b synthesis. On the other hand, Chl a and Chl b contents were increased in T1 and T3. T1 induced an increase of 77.0% and 74.1% from CK for Chl a and b. The Chl a and b contents of T3 respectively increased by 1.8 and 33.8 times CK. Interestingly, Chl a, Chl b, and total Chl content in T1 or T3 were significantly higher than those in T4. The results suggest that the addition of N can significantly increase Chl content in leaves under Cd stress.

Table 3. Effects of different Cd resources on the chlorophyll content in leaves of poplar.

Treatments		Chl a	Chl b	Chl a:b	Total Chl
CK	Blank	1.616±0.034^B	0.463±0.026^B	3.490/1	2.079±0.020^B
T1	Cd(NO3)2	2.861±0.072^D	0.806±0.026^E	3.550/1	3.666±0.097^E
T2	CdCl2	1.342±0.045^A	0.019±0.002^A	70.632/1	1.361±0.065^A
T3	CdCl2+(NH4)2CO3	2.371±0.091^C	0.642±0.041^D	3.693/1	3.012±0.132^D
T4	(NH4)2CO3	2.209±0.016^C	0.505±0.32^C	4.374/1	2.714±0.092^C

Note: Each value is the mean±SE of three replicates, and the terms “A, B, C and D” within each column indicate significant difference between means (p<0.01). Unit: mg/gFW.

Nitrate reductase (NR) activity

T2 showed an obvious increase in NR leaf activity compared to CK (Table 1). Thus, NR activity of plants is accentuated under Cd stress. When N was supplied under Cd stress (T1 and T3), NR activity was also significantly increased. The result is similar to Hecht et al.'s study. Hecht et al. (1988) reported that the addition of NO₃⁻ can up-regulate the activity of NR. NR is a key enzyme for nitrate assimilation in plants. Neill et al. (2003) showed that NR can induce the increment of nitric oxide (NO). However, nitric oxide (NO) as a bioactive signaling molecule plays an important role in plant responses to heavy metals stress (Arasimowicz & Floryszak-Wieczorek 2007). So the increase of NR activity is a help to promote plant to be tolerant to heavy metals.

The precursors of chlorophy II synthesis

The levels of seven precursors (ALA, PBG, Urogen III, Coprogen III, Proto IX, Mg-proto and Pchlide) in poplar leaves were determined by absorption spectroscope. Table 4 showed that ALA, PBG and Urogen III levels in T2 were significantly higher than CK with respective increments of 24.1%, 45.3% and 30.7%. While, Coprogen III, Proto IX, Mg-proto and Pchlide contents in T2 were obviously lower than CK with respective reduction of 44.9%, 32.9%, 52.9% and 61.3%. On the other hand, in T1 and T3, there was no remarkable difference in the seven precursors’ levels compared with CK. T2 treatment plants accumulated 1.3 fold more Urogen III and one half less Coprogen III compared to CK. These data suggested that the increased Urogen III and the reduced Coprogen III was a direct consequence of the inhibition of CoprogenIII synthesis induced by sole Cadmium stress. So, Cd seriously inhibited Chl synthesis by blocking the site located on the steps between Urogen III and Coprogen III. However, the plants treated with cadmium and nitrogen grew well without any toxicity symptoms. Nitrogen supplement can alleviate Cd inhibition on chlorophyll synthesis by unblocking the pathway.

Table 4. Analysis of seven precursors of chlorophy II synthesis in poplar leaves. They were quantified by absorption spectroscopy.

Treatments	ALA	PBG	Urogen III	Coprogen III	Proto IX	Mg-proto	Pchlide
Blank	0.079±0.002^B	0.265±0.04^B	0.326±0.05^B	0.058±0.004^B	13.752±0.12^B	6.525±0.05^B	35.452±2.53^C
Cd(NO3)2	0.082±0.005^B	0.291±0.03^B	0.342±0.02^B	0.061±0.016^B	15.256±0.17^C	6.783±0.08^B	31.885±3.11^B
CdCl2	0.098±0.002^A	0.385±0.02^A	0.426±0.01^A	0.026±0.002^A	9.232±0.12^A	3.069±0.06^A	13.712±2.05^A
CdCl2+(NH4)2CO3	0.076±0.001^B	0.234±0.03^C	0.315±0.02^B	0.054±0.008^B	14.247±0.21^B	7.012±0.07^B	32.889±3.23^B
(NH4)2CO3	0.075±0.001^B	0.196±0.01^D	0.309±0.01^B	0.075±0.007^C	16.311±0.36^C	7.095±0.03^B	36.124±1.89^C

Note: Fluorescence is given as relative units. Each value is the mean±SE of three replicates, and the terms “A, B, C and D” within each column indicate significant difference between means (p < 0.01).

Expression of GS₂ gene

Total expressed RNA was subjected to electrophoresis on 1% formaldehyde denaturation agarose gel. The total RNA bands of leaves were all trim and clear, which indicates that the extracted RNA did not degrade and could be used for quantitative PCR assay. The actin gene of the poplar used as the inner reference was amplified in parallel with the target gene allowing gene normalization and quantification. The transcript level of GS₂ varied with treatment. The expression of GS₂ showed a significant decrease in T2, but an appreciable increase in T1 and T3 (Figure 1). The transcript level of GS₂ in T1 and T3 was respectively 1.22 and 1.72 times higher than those in T2 and 1.26 and 1.52 times higher than those in T4. Therefore, Cd alone inhibited the expression of GS₂, while N significantly promoted gene expression in poplar plants under Cd stress.

Figure 1. GS₂ expression changes of stems with different cadmium treatments. Cd stress (T2) inhibited the expression of GS₂, and the combination of Cd and nitrogen, such as Cd(NO₃)₂ or CdCl₂ plus (NH₄)₂CO₃ up-regulated GS₂ expression. Therefore, nitrogen significantly promoted gene expression in poplar plants under Cd stress and can effectively alleviate cadmium toxicity to poplar plants.
Note: CK indicates control. T1, T2, T3 and T4 indicate Cd(NO₃)₂, only CdCl₂, CdCl₂ plus (NH₄)₂CO₃, only (NH₄)₂CO₃ treatments respectively.

Discussion

In general terms, Cd is inhibitory to Chl synthesis (Hammami et al. 2004; Amani 2008) and plant growth (Vassilev et al. 1995; Hammami et al. 2004; John et al. 2008). In the study, plant height, dry weight and Chl content of the plants exposed to Cd, all significantly decreased when compared to the controls. Cd seriously inhibited the Chl synthesis. The blocking site of Cd inhibition in Chl synthesis was determined to be on the steps from Urogen III to Coprogen III. Nitrogen addition could promote the growth, increase content of Chl of plants under stress of Cd, and effectively alleviate the inhibition of Cd to Chl synthesis by unblocking the pathway. The above results demonstrated that nitrogen played an important role in detoxifying heavy metals from plants. According to related research, the detoxification strategies may include: (1) Regulation of metal uptake by the root system (Hart et al. 1998; Benavides et al. 2005; Hassan et al. 2005); (2) Constraint by the cell wall (Hart et al. 1998); (3) The presence of some chelate complexes, such as phytochelatin-Cd^{2 +} or GSH-Cd^{2 +} (Mendoza-Cózatl & Moreno-Sánchez 2006; Quan et al. 2006; Gustavo et al. 2007); (4) Regulation of related gene expression (Benavides et al. 2005; Xu et al. 2006; Wu et al. 2012). Especially, sequestration with glutathione (GSH) and phytochelation (PC) is an important pathway to detoxifying Cd, which has earlier been confirmed (Singhal et al. 1987). Both GSH and PC could bind Cd^{2 +} to form a stable chelate (GSH-Cd^{2 +} or PC-Cd^{2 +}) and then transfer chelates into vacuoles. In addition, the synthesis of glutathione occurs in two ATP-dependent steps (Meister 1995). First, glutamate-cysteine ligase (GCL) catalyzes formation of γ-glutamylcysteine from Cys and Glu. This step is thought to be the rate-limiting reaction of the pathway. In the second step, glutathione synthetase (GS) adds Gly to γ-glutamylcysteine to yield glutathione. GSH also can be used for PC synthesis. So the synthesis of GSH needs the participation of glutamate (Glu). At the same time, the synthesis of Glu requires the participation of nitrogen. The above researches revealed that the important role of GSH in detoxifying Cd from plants was closely related to nitrogen. The results also demonstrated that nitrogen can effectively alleviate cadmium toxicity to poplar plants. Based on the study and other related researches on Cd toxicity, a hypothesis is proposed to explain the mechanism of detoxification of Cd in higher plants. The hypothesis includes the following main points:

1. Sequestration with GSH and PC is an important pathway to detoxify heavy metals. GSH binds Cd directly or will be used for PC synthesis and transport of sequestered Cd into the vacuole. Although GSH and PC contents were not detected in our study, others have confirmed that low molecular weight thiols, such as cysteine, GSH and PC, will significantly increase in plants under Cd stress (Cataldo et al. 1983; Strohm et al. 1995; Zhu et al. 1999; Miflin & Habash 2002; Li et al. 2006).

2. There are three metabolic pathways that utilize Gln as a resource (Figure 2): Chl synthesis pathway (Chl path); GSH synthesis pathway (GSH path); and amino acid, protein, nucleic acid synthesis pathway (Pro path). The pathways are mutually competitive; namely, when the total amount of glutamate is limited; if the consumption of glutamate increases in one of the three pathways, it will decrease in the other two. The three pathways are in balance in the absence of Cd.

3. The response of plants to Cd stress includes two phases, detoxification and toxification. Detoxification is the first response. As soon as exogenous Cd invades mesophyll cells, corroding protection is activated to preclude intercellular Cd toxicity. Typically, the genes responsible for Gln, GSH and PC synthesis would be up-regulated so that, subsequently, more GSH and PC are available for Cd chelation. No toxic symptoms will be expressed no matter how much Cd enters the cell, as long as the free Cd concentration is controlled at low levels and the amount of glutamate for biosynthesis occurs at normal levels. In the presence of exogenous N, the plant not only has enough glutamate to synthesis GSH for free Cd chelation, but also an excess amount for manifold biosyntheses, increased metabolism and plant growth.

4. The toxification phase occurs when glutamate is limited by an inadequate N supply with increasing amounts of Cd^{2 +} entering the cell. In these conditions, enhanced GSH synthesis would consume more glutamate, which would lead to glutamate deficiency and decreases in Chl, protein and nucleic acid, resulting in chlorosis and growth inhibition. Therefore, primary toxic symptoms of plants under Cd treatment are not directly caused by Cd reaction, but are induced by insufficient glutamate for the Chl-pathway and Pro-pathway in the cells. If GSH and PC are insufficient to bind the invading Cd, the ions will directly react with some proteins and enzymes, or be replaced with other metal ions, such as Mg^{2 +} and Fe^{2 +}, leading to more serious toxicity. Thus Cd toxicity includes both indirect and direct effects.

5. Because GSH synthesis needs cysteine in addition to glutamate, sulfur also plays an important role in the N-based detoxification of Cd. Namely, the detoxification depends on sufficient quantities of sulfur.

6. The detoxification and toxification of poplar to Cd may be a common mechanism of all plants and other heavy metals such as Pb, Cu, As, Hg, and so on. Our hypothesis needs verification in follow-up studies.

Figure 2. Possible mechanism of detoxification and toxification to cadmium stress in plant. There are three biosynthesis pathways: Chl synthesis pathway (Chl path); GSH synthesis pathway (GSH path); and amino acid, protein, nucleic acid synthesis pathway (Pro path), which compete for L-glutamate. Nitrogen plays a key role in the detoxification or toxification process via glutamate and GSH. The detoxification process is realized by GSH or PC binding Cd and transporting it into the vacuole. When there isn't enough glutamate to synthesis chlorophyll, protein and nucleic acid, and so on, because GSH synthesis consumes more glutamate under cadmium stress, completive inhibition would occur. With more and more Cd entering into the cell, the plant would transfer into the toxification phase and some toxic symptoms, such as chlorosis, will appear.

In the study, hybrid poplar ‘107’ (Populus deltoides×P. nigra) plants were selected as the materials and the potential species for phytoremediation because Populus ‘107’ has many excellent features, such as fast growth rates, profuse vegetative propagation, adaptability to various ecological conditions, aesthetic values and wood values. ‘107 poplar’ was widely cultivated in China and it has become one of the most economically important groups of tress. In addition, populus species, as the only woody model plants, provide the physiology and molecular basis for study of other woody plants. Using tall, woody plants in combination with nitrogen fertilization to remediate contaminated sites would be an aesthetically pleasing and beneficial approach that, not only promotes positive landscape effects, but reduces erosion and provides the valuable timber. For example, Komárek et al. (2007) used poplar plants to remediate the lead-contaminated agricultural soil. Giachetti and Sebastiani (2006) reported that poplar plants grown in industrial waste developed well, and showed high Cd accumulation. In addition, eucalyptus and populus, the short rotation woody crops, were also used to remediate phosphate mined lands in Florida USA (2006).

Conclusions

Although the toxic effects of Cd have been reported by many authors, the mechanisms are not completely understood yet. In our experiments, the poplar plants treated with very high concentrations of Cd grew well repeatedly. It is well-established that the low-molecular-weight thiol compounds, such as cysteine, GSH and PC, have an important function on Cd sequestration. Based on our results and related research, we draw the conclusion that Cd exposure does not always lead to toxic symptoms in plants. Ample N mitigates high Cd concentrations, leading to poplar plants that grew better than the controls in our experiments. Cd toxicity may be divided into indirect action and direct action; the former refers to the toxic action caused by glutamate deficiency as a result of GSH and PC's detoxification, while the direct action is caused by the interaction between Cd and protein. Both N and sulfur play an important role on the detoxification of Cd in plants. There are three biosynthesis pathways (Chl, GSH and protein), which compete for L-glutamate. N plays a key role in the detoxification or toxification process via glutamate and GSH. The detoxification process is realized by GSH- or PC-binding Cd and transporting into vacuoles. When glutamate is limited, competitive inhibition results with reductions in the biosynthesis of Chl, protein and nucleic acid, because the GSH synthesis consumes more glutamate under Cd stress. With more and more Cd^{2 +} entering the cell, the plant would transfer into the toxification phase, leading to chlorosis and growth loss. The detoxification and toxification of poplar to Cd may be a common mechanism of all plants and other heavy metals such as Pb, Cu, As, and Hg. If the reasoning is confirmed in the future, phytoremediation of contaminated soil may be enhanced with N fertilization.

Acknowledgments

The authors thank Dr Brian Stanton, Dr Chris Boswell and Dr Zhang Li for their helpful comments that improved the manuscript. This work was supported by the 12th Five Years Key Programs for forest breeding of Sichuan Province (No. 2011YZGG).