ISSN 10214437, Russian Journal of Plant Physiology, 2015, Vol. 62, No. 3, pp. 349–359. © Pleiades Publishing, Ltd., 2015.

RESEARCH PAPERS

Exogenous Nitric OxideMediated GSH–PC Synthesis Pathway in Tomato under Copper Stress1 J. Wang, S. X. Yu, M. Zhang, and X. M. Cui College of Resources and Environment, Shandong Agricultural University, Tai’an 271018, China; fax number: 86+05388242250; email: [email protected] Received May 12, 2014

Abstract—Nitric oxide (NO) is a bioactive molecule that is extensively used at various biotic and abiotic stresses. This study investigated the law governing the variation of related enzymatic activity and metabolites in exogenous NOmediated GSH–PC synthesis pathway in tomato solution culture subjected to copper stress. Results demonstrated that relative to control copper stress was more effective in the activation of γECS and GS in tomato. Moreover, sharp increases in root GSH and PCs were observed, which keep upward as the process continued. Moreover, adding exogenous SNP (NO donor) can further improve γECS and GS activities in tomato roots and facilitate the synthesis of GSH and PCs, thereby enhancing its peroxide removal ability, chelating excessive Cu2+, and reducing its biotoxicity. The GSH–PC metabolism in the tomato leaves lagged behind that in the roots to a certain extent. Although exogenous GSH synthesis inhibitor BSO inhib ited γECS activity in tomato roots, as well as GSH and PC syntheses, adding SNP can counteract this effect by lessening the influence to the PCs in leaves. Under copper stress, exogenous NO may stimulate a signaling mechanism and reduce the biotoxicity and oxidative damage caused by excessive Cu2+ through activating or enhancing the enzymatic and nonenzymatic systems in the GSH–PC synthesis pathway. Keywords: Solanum lycopersicum, copper stress, glutathione, nitric oxide, phytochelatins DOI: 10.1134/S1021443715030188 1

INTRODUCTION

Phytochelatins (PCs) comprise a type of small pep tide molecules with heavy metal complexation. PCs are composed of cysteine (Cys), glutamic acid (Glu), and glycine (Gly). PCs in plants are unique sulfhydryl proteins that are rich in –SH, which are enzymatic products synthesized from Glu and Cys rather than direct products of structural genes [1]. After entering the plant, the heavy metal ions will combine with PCs through the –SH on Cys to form compounds with the low molecular weight. These compounds will combine with molecular PCs as they enter the vacuole in to lower the activity of metal ions and form macromolec ular compounds that are characterized by small heavy metal toxicity to plant tissues [2]. Copper is a microelement essential for plant growth. As an activator or prosthetic group of enzymes, copper 1

This text was submitted by the authors in English. Abbreviations: ASA—Ascorbic acid; BSO—Lbuthionine(S,R) sulfoximine; DTNB—5,5dithiobis(2nitrobenzoic acid); DTPA—diethylenetriaminepentaacetic acid; GS—glutathione synthetase; GSH—reduced glutathione; GSSG—oxidized glu tathione; Hb—bovine hemoglobin; H2O2—hydrogen perox ide; NEM—Nethylmaleimide; OPT—ophthalaldehyde; PCs—phytochelatins; ROS—reactive oxygen species; SNP— sodium nitroprusside dihydrate; TAST—total acidsoluble thiols; TG—total glutathione [GSH + GSSG]; γECs—γglutamylcys teine synthetase.

facilitates various important physiological and bio chemical activities, such as photosynthesis, respiration, and signal transduction [3]. As a heavy metal element, excessive copper in plants will result in copper toxicity and accumulation of free superoxide anion radical, H2O2, and reactive oxygen species (ROS) similar to per oxide in the plant, thereby destroying the components of cells (such as the DNA, proteins, and membrane lip ids)[4, 5]. As an important micromolecular signaling sub stance, nitric oxide (NO) is extensively involved in various physiological and biochemical processes in plants [6, 7]. Under heavy metal stress, NO can remove the ROS produced under stress and offset the adverse effect of excessive heavy metal ions on the plant [8, 9]. Under copper stress, exogenous NO can offset the growth inhibition by Cu stress and facilitate the absorption and accumulation of excess copper by the plant. Undoubtedly, some mechanisms result in the reduction of the toxicity of the excessive absorbed copper in plants [10, 11]. This paper studied the effects of exogenous NO on the activities of γglutamylcysteinesynthetase (γECS) and glutathione synthetase (GS), as well as metabo lites, in commonly cultivated tomato seedlings in the solution culture that were under copper stress, with the aim of discussing the exogenous NOmediated GSH– PC synthesis pathway. Compared with heavy metal

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hyperaccumulators, tomato has the large biomass and the high growth rate. Therefore, investigating the exogenous substanceinduced copper tolerance mechanism of tomato can provide for novel ideas for bioremediation and agricultural production in cop perpolluted areas. MATERIALS AND METHODS Plant material. The testing of the tomato Solanum lycopersicum L. involved cv. Improved Maofen 802F1. The improved Hoagland nutrient solution was com posed of Ca(NO3)2 ⋅ 4H2O, KNO3, NH4NO3, KH2PO4, MgSO4, and micronutrients. All the reagents were ana lytically pure and prepared with distilled water. Donors and appropriate concentrations of NO and Cu2+ were determined through a preliminary test. Cu2+ was sup plied as CuCl2, whereas NO was supplied by (Na2Fe(CN)5)NO (SNP, bought from Sigma, United States). First, these reagents were used to prepare 200 μM stock solutions with distilled water, which was kept in a dark place under 4°C and diluted to achieve desired concentrations. Bovine hemoglobin (Hb, Sigma) was used as the scavenger of NO, and Lbuthioninesulfoximine (BSO, Sigma) was used as the GSH synthesis inhibitor. Test design. The test was conducted in the green house of Shandong Agricultural University. Seeds were first disinfected through hot water treatment (55°C) for 15 min and then underwent to accelerated germination on wet absorbent paper under 28°C. When buds appeared, these seedlings were sown into clean fine sand. After the seedlings emerged, 0.25strength Hoagland nutrient solution was given to them. When the seedlings had three to four true leaves, seedlings that had the same growth rate were selected and transplanted into 5L plastic basins cov ered with 3cmthick round foam plastic board after the fine sands were cleaned from the roots. Each plastic basin was planted with five seedlings cultured in 0.5strength Hoagland nutrient solution. One week later, full nutrient solution was introduced. The nutrient solution was then replaced every 3 days. Air was supplied 24 h a day through an electric pump during the entire cultivation period. Stress treatment was applied when the tomato seedlings developed five to six true leaves. The test set comprised six groups (CK, Cu, Cu+S, Cu+S+H, Cu + B, and Cu + B + S): (1) control group: complete Hoagland nutrient solution; (2) 50 μM CuCl2; (3) 50 μM CuCl2 + 100 μM SNP; (4) 50 μM CuCl2 + 100 SNP + 0.1% Hb; (5) 50 μM CuCl2 + 100 μM BSO; and (6) 50 μM CuCl2 + 100 μM BSO + 100 μM SNP. Each group was tested three times and randomly ranked in the greenhouse. During stress treatment, the nutrient solution was changed every 3 days. The pH of the nutri ent solution was adjusted to 5.0 ± 0.2 through lowcon centration KOH or HCl. The greenhouse enjoyed about 12 h of sunlight (light intensity: 320 μmol/(m2 s)) and

the temperature ranged from 32°C (day) to 15°C (night). Roots (washed off) and leaves were collected at 1, 3, 6, 24, and 48 h of the stress treatment. Fresh samples were used for enzyme activity test, whereas several samples were frozen in liquid nitrogen and stored at a temperature of –80°C. Major test items and methods. PCs were assayed according to De Vos et al. [12] with minor modifica tions: PCs (μg/g fr wt) = TAST (μg/g fr wt) – TG (μg/g fr wt). For TAST test: extraction was carried out by grinding 1 g of freezed fresh plant material (using a mortar and pestle and quartz sand) in 2 mL of 5% (w/v) sulfosalicylic acid with 6.3 mM (DTPA) (pH < 1) at 0°C. After centrifugation at 10000 g for 15 min (4°C), the supernatants were immediately assayed. The con centration of TAST was determined using Ellman’s reagent [13]. The supernatant (300 μL) was mixed with 630 μL of 0.5 M K2HPO4, and the absorbance was mea sured after 2 min at 412 nm (30°C). After the addition of 25 μL of DTNB solution (10 mM DTNB; 0.143 K2HPO4; 6.3 mM DTPA, pH 7.5), the A412 was remeasured after 2 min. The increase in absorbance was corrected for the absorbance of DTNB. Values were calculated using the molar extinction coefficient of 13600/(M cm). For GSH and GSSG test: extraction was carried out by grinding 1 g of freezed dry plant material in 1 mL of 25% H3PO4 and 3.5 mL of phos phate buffer (PH 8.0). After centrifugation at 5000 g for 30 min, the supernatants were immediately assayed for GSH and GSSG. GSH test: the supernatant (0.5 mL) was mixed with 2.5 mL of phosphate buffer (pH 8.0), 200 μL of the mixture 200 μL OPT and 3.6 mL of phos phate buffer was added; and the reaction was lasted for 15 min at room temperature; then test the fluorescence intensity was assessed (excitation wavelength of 350 nm; emission wavelength of 420 nm). GSSG test: the super natant (0.5 mL) was added to 200 μL of NEM (25– 30 min) and 200 μL of the mixture 200 μL OPT and 3.6 mL 0.1 M NaOH was added; the reaction was lasted for 15 min at room temperature; then test the fluores cence intensity was measured (excitation wavelength of 350 nm; emission wavelength of 420 nm)[14]. γECS activity was tested using a kit from Nanjing Jiancheng (Nanjing Jiancheng Chemical Industrial Co. Ltd, China). GS activity was tested using a kit from Jianglai Biology Company (China). Finally, the copper content was tested through HNO3−HClO4 heating digestion method [15] and using an atomic absorption spectro photometer (AA370MC). Method for protein content measurement. Fresh root and leaves (1 g) were triturated and kept at 20– 25°C for 1 h; then centrifuged at 4000 rpm; the super natant fraction was used for protein assay. The supernatant (0.1 mL) was mixed with 5 mL of protein reagent (0.01% (w/v) Coomassie Brilliant Blue G250 (Fluka Chemicals, United Kingdom); 4.7% (w/v) ethanol; 8.5% (w/v) phosphoric acid) [16]. After 2 min, the absorbance was measured at 595 nm. The BSA was used as a standard protein.

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Fig. 1. Effect of exogenous NO on copper content in (a) tomato roots and (b) leaves under copper stress. (1) Control, CK; (2) Cu; (3) Cu + SNP; (4) Cu + SNP + Hb; (5) Cu + BSO;(6) Cu + BSO + SNP.

Data processing. Microsoft Excel was used for data processing and drawing. DPS software was used for multiple comparisons of mean values using the Dun can range method.

absorption by SNP. Compared with Cu, Cu + BSO decreased copper content in tomato roots by 46.3% by the end of the treatment. In contrast to Cu + BSO, Cu + BSO + SNP weakened the inhibition of BSO and increased copper content in roots.

RESULTS

The change in the copper content in the leaves was different from that in the roots. Compared with Cu, Cu + SNP decreased copper content in leaves under copper stress. This decreasing trend was offset to some extent by adding 0.1% Hb. However, Cu + BSO increased copper content in leaves by 45.8% by the end of the treatment. The copper content in leaves in Cu + BSO + SNP was 26.7% lower compared with that in Cu +BSO. In sum, under copper stress, exoge nous SNP increased copper content in roots but decreased copper content in leaves, whereas exoge nous BSO decreased copper content in roots but increased copper content in leaves. The 0.1% Hb weakened the effect of SNP.

Influence of Exogenous NO on Copper Content in Tomato under Copper Stress Figure 1 shows that compared with CK Cu stress sig nificantly increased the copper content in roots and leaves, especially in roots. This increase continued dur ing the course of the treatment. By the end of the treat ment, copper contents in the roots and leaves were 89.2% and 63% higher compared with those of CK, respectively. Compared with Cu, Cu + SNP showed continuous increase of copper content in roots. How ever, Cu + SNP + Hb demonstrated a sharp decrease of copper content in roots, indicating the weakening Cu2+ RUSSIAN JOURNAL OF PLANT PHYSIOLOGY

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Fig. 2. Effect of exogenous NO on PCs in (a) tomato roots and (b) leaves under copper stress. (1) Control, CK; (2) Cu; (3) Cu + SNP; (4) Cu + SNP + Hb; (5) Cu + BSO;(6) Cu + BSO + SNP.

Influence of Exogenous NO on PCs in Tomato under Copper Stress

Influence of Exogenous NO on GSH in Tomato under Copper Stress

In Figure 2 shows a continuous growth of PCs in tomato roots and leaves with time, in contrast to CK. Compared with Cu, Cu + SNP increased the PC con tent in tomato roots and leaves. This increasing trend continued during the course of the treatment. However, Cu + BSO decreased PC content in tomato roots and leaves continuously during the course of the treatment. Cu + BSO + SNP indicated a slow growth of PCs in roots until 6 h after the treatment, during which the inductive effect of SNP on PC synthesis had weakened.

As shown in Fig. 3, Cu showed significantly higher GSH content in roots than CK during the entire course of the treatment. As treatment continued, the GSH content in roots in Cu treatment gradually increased, becoming by 46% higher than that in CK at 48 h. Compared with Cu, Cu + SNP showed the quicker growth of GSH content in roots during the treatment, exceeding that of Cu alone at 3 h. Exoge nous Hb can weaken the inductive effect of SNP. However, Cu + BSO continuously reduced the GSH content in roots and leaves, decreasing by 78% and 31%, respectively, at 48 h. Compared with Cu + BSO, Cu + BSO + SNP continuously increased the GSH content in roots until 24 h after the treatment. The change in the GSH content in leaves differed from that in roots. Unlike CK, Cu, Cu + SNP, Cu + SNP + Hb, Cu + BSO, and Cu + BSO + SNP decreased the GSH content in leaves continuously under Cu stress. The GSH content in Cu was by 56% lower than that in CK at 48 h. Compared with Cu, Cu + SNP increased the GSH content in leaves. Hb

Cu + SNP + Hb decreased PC content in leaves during the course of the treatment and became stable in 24 h after the treatment. This observation indicates that under copper stress and exogenous SNP can increase PC content in tomato roots and leaves, whereas exoge nous 0.1% Hb can weaken the positive effect of SNP on PC growth. Both Cu + BSO and Cu + BSO + SNP had lower PC content in roots and leaves compared with Cu alone. However, to a certain extent, SNP can still regu late the transition of GSH–PCs under copper stress.

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Fig. 3. Influence of exogenous NO on GSH content in (a) tomato roots and (b) leaves under copper stress. (1) Control, CK; (2) Cu; (3) Cu + SNP; (4) Cu + SNP + Hb; (5) Cu + BSO;(6) Cu + BSO + SNP.

can significantly weaken the inductive effect of SNP. Cu + BSO continuously decreased GSH content in leaves by 24% at 48 h. However, Cu + BSO + SNP increased the GSH content in tomato leaves. This observation indicated that under copper stress, exoge nous SNP can continuously increase the GSH content in tomato roots but continuously decrease GSH con tent in leaves. Exogenous BSO can significantly inhibit GSH synthesis. Influence of Exogenous NO on GSSG in Tomato under Copper Stress Figure 4 shows that unlike CK, Cu, Cu + SNP, Cu + SNP + Hb, Cu + BSO, and Cu + BSO + SNP continuously increased the GSSG content in roots of tomato seedlings during the course of the treatment, showing the higher GSSG content in roots than in CK. Compared with Cu, Cu + SNP facilitated the production of GSSG in tomato roots, whereas 0.1% Hb weakened SNP positive effect on GSSG produc tion. Compared with Cu, Cu + BSO had the lower RUSSIAN JOURNAL OF PLANT PHYSIOLOGY

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GSSG content in the roots of tomato seedlings. How ever, Cu + BSO + SNP had significantly higher GSSG content in roots, increased by 11% at 48 h. Compared with CK, Cu, Cu + SNP, and Cu + SNP + Hb increased the GSSG content in the tomato leaves. The GSSG content in the leaves was higher than that in CK. Compared with Cu, Cu +SNP had the higher GSSG content in leaves, whereas exoge nous 0.1% Hb hindered SNPinduced GSSG produc tion. However, Cu + BSO showed continuous decline in GSSG content in leaves, which was lower than that in CK. Compared with Cu + BSO, Cu + BSO + SNP increased GSSG content in leaves of tomato seed lings, almost reaching the CK level at 48 h. Therefore, BSO can lower GSSG content in both tomato roots and leaves under copper stress. Influence of Exogenous NO on GSH/GSSG Ratio in Tomato under Copper Stress As shown in Fig. 5, although the GSH/GSSG ratio in roots with Cu was lower than that in CK within the No. 3

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Fig. 4. Effect of exogenous NO on GSSG content in (a) tomato roots and (b) leaves under copper stress. 1⎯control, CK; 2⎯Cu; 3⎯Cu + SNP; 4⎯Cu + SNP + Hb; 5⎯Cu + BSO; 6⎯Cu + BSO + SNP.

6 h of treatment, the ratio continuously increased and exceeded the CK level at 6 h. Exogenous SNP can increase the GSH/GSSG ratio in tomato roots under copper stress. GSH/GSSG ratio in tomato roots con tinuously increased throughout the treatment. Com pared with Cu + SNP, Cu + SNP + Hb had the lower GSH/GSSG ratio with slight changes throughout the treatment process. Compared with Cu, Cu + BSO presented the lower GSH/GSSG ratio in roots, con tinuously decreased during the whole treatment. Cu + BSO + SNP showed the higher GSH/GSSG ratio than Cu + BSO. Cu, Cu + SNP, Cu + SNP + Hb, Cu + BSO, and Cu + BSO + SNP had the lower GSH/GSSG ratio in tomato leaves compared with CK and exhibited con tinuous decrease in the GSH/GSSG ratio in tomato leaves during the course of the treatment. Compared with Cu, Cu + SNP showed the higher GSH/GSSG ratio in tomato leaves under copper stress. However, 0.1% Hb can slow down the increase trend. At 6 h, exogenous BSO contributed to the peak of the GSH/GSSG ratio in tomato leaves, which was fol

lowed by a continuous decline. Under copper stress, Cu + BSO failed to show significant changes in the GSH/GSSG ratio in tomato leaves, although Cu + BSO + SNP significantly increased the GSH/GSSG ratio. This observation showed that under copper stress, exogenous SNP can increase the GSH/GSSG ratio in tomato roots and leaves, whereas exogenous 0.1% Hb can weaken SNP positive effect on the increase in the GSH/GSSG ratio. Influence of Exogenous NO on γ–ECS Activity in Tomato under Copper Stress As shown in Fig. 6, compared with CK, Cu contin uously increased the γ–ECS activity in tomato roots and leaves throughout the duration of treatment. Cu + SNP exhibited significantly higher γECS activity in roots and leaves than Cu alone. The γ–ECS activity presented a sharp increase in roots in 6 h after treat ment, whereas that in leaves increased slowly, with 44.4% and 13.6% increase at the end of the treatment, respectively. Exogenous 0.1% Hb worked against the

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Fig. 5. Effect of exogenous NO on GSH/GSSG ratio in (a) tomato roots and (b) leaves under copper stress. 1⎯control, CK; 2⎯Cu; 3⎯Cu + SNP; 4⎯Cu + SNP + Hb; 5⎯Cu + BSO; 6⎯Cu + BSO + SNP.

inductive effect of SNP to γ–ECS activity in roots and leaves. Under copper stress, exogenous BSO contrib uted significant continuous reduction of γECS activ ity in tomato roots and leaves during the entire treat ment, decreasing by 83% and 31.6%, respectively, compared with the activity in Cu. Exogenous SNP can reduce copper inhibition of γECS activity and facili tate γECS activity in roots and leaves at 48 h to recover by 50% and 66.6%, respectively. Overall, cop per stress increased γECS activity in roots and leaves, which can be further increased by SNP. However, BSO inhibited γECS activity, which can be relieved through exogenous SNP under copper stress. Influence of Exogenous NO on GS Activity in Tomato under Copper Stress As shown in Fig. 7, Cu showed a “Λshaped” vari ation of GS activity in roots. The GS activity in roots increased by 22.5% compared with the activity in CK. In contrast to Cu, Cu + SNP presented a continuous increase of GS activity in roots throughout the treat RUSSIAN JOURNAL OF PLANT PHYSIOLOGY

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ment procedure, with an activity that was by 46.6% higher than in Cu by the end of the treatment. Exoge nous 0.1% Hb weakened the inductive effect of SNP. Compared with Cu, Cu + BSO showed no significant change in GS activity in the roots, indicating the insig nificance of BSO on GS activity. Cu + BSO + SNP continuously increased the GS activity in roots throughout the treatment procedure, with a result that was by 25% higher than that in Cu + BSO by the end of the treatment. The GS activity in leaves continuously increased throughout the entire Cu treatment, with an activity that was by 80.1% higher than that in CK by the end of the treatment. Compared with Cu, Cu + SNP showed a dramatic increase in GS activity. Exogenous 0.1% Hb weakened the inductive effect of SNP to GS activity. Exogenous BSO had no significant effect on GS activ ity. However, Cu + BSO + SNP demonstrated a “Vshaped” variation of GS activity at 3 h. No. 3

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Fig. 6. Effect of exogenous NO on γECS activity in (a) tomato roots and (b) leaves under copper stress. 1⎯control, CK; 2⎯Cu; 3⎯Cu + SNP; 4⎯Cu + SNP + Hb; 5⎯Cu + BSO; 6⎯Cu + BSO + SNP.

DISCUSSION Several studies have reported that exogenous NO can significantly relieve copper stress in tomato [17]. Although NO can facilitate the absorption or accumu lation of copper in roots under copper stress [18], plants showed no intensifying stress, but almost recov ered to the CK level. This result further prove that by the dynamic change of Cu2+ after stress treatment and the tomato growth test performed in this paper. Li et al. [19] found that under copper stress, exog enous NO mediated the GSH–ASA pathway to improve the oxidative stress resistance of tomato. In addition to being an important ROS scavenger in plants, GSH can likewise protect plants from environ mental stress, oxidative stress, and toxic effect of heavy metals [20]. Glutathione includes GSH and GSSG. GSH can provide electrons, whereas GSSG can only eliminate H2O2 after being restored to GSH. Plants growing normally demonstrate balanced ratios of GSH/GSSG. When plants suffer from harmful stress,

oxygen radicals produced under the stress will oxidize GSH into GSSG [21]. Such change in the GSH/GSSG ratio can provide electrons for eliminat ing metabolites (such as ROS, hydroperoxide, and lipid peroxide) to maintain the redox equilibrium in cells and mitigate the suffering of plants from oxidative stress [22]. In this test, exogenous SNP increased GSH content in tomato roots and leaves under copper stress, adjusting the GSH/GSSG ratio, and mitigating the tomato suffering from the produced ROS. The variation of GSH content in the roots disproves the study by Li et al. [19], which may be ascribed to the stress treatment time. This paper adopted dynamic continuous sampling. Aside from eliminating oxygen radicals in plants, the synthesized GSH likewise contributed to the syn thesis of PCs. Under heavy metal stress, ligands with sulfhydryl groups (such as GSH, PCs, and metal lothionein) in plant cells can chelate and passivate metal ions in cells to form lowmolecularweight compounds. These lowmolecularweight compounds

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Fig. 7. Effect of exogenous NO on GS activity in (a) tomato roots and (b) leaves under copper stress. 1⎯control, CK; 2⎯Cu; 3⎯Cu + SNP; 4⎯Cu + SNP + Hb; 5⎯Cu + BSO; 6⎯Cu + BSO + SNP.

will thus combine with molecular PCs after entering the vacuole to form highmolecularweight com pounds with low biotoxicity. As much as possible, Cu was retained in roots to reduce its interference in pho tosynthesis and other physiological and biochemical processes [23]. When tomato plants in soil suffer from copper stress, roots that are in direct contact with the soil will promptly respond to the stress [24], increase GSH synthesis in the roots, and synthesize more PCs to combine excessive Cu2+ in the roots to lower Cu2+ activity and transport to the aboveground part. With out external biotic or abiotic stresses, plants maintain the lower GSH content. The GSH content is higher in the above ground than in the underground parts. How ever, under external biotic or abiotic stresses, GSH content in plants will rapidly increase, not only to eliminate produced ROS and peroxide effectively, but also to provide more substrates for PC synthesis [25]. In this test, Cu + SNP had significantly higher GSH and PCs in roots compared with Cu. Significant posi tive correlation was observed in the variation trends of RUSSIAN JOURNAL OF PLANT PHYSIOLOGY

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GSH and PCs (R = 0.9778), whereas PC concentra tion was consistently higher than GSH, indicating that under copper stress, NO facilitated GSH–PC metab olism to produce more PCs–copper complex to reduce the biotoxicity of Cu2+. However, the variation of GSH in leaves was negatively correlated with that of PCs (R = –0.995). On one hand, this result may have been caused by the exogenous SNPinduced acceler ated GSH transport to the underground parts under copper stress [26]. On the other hand, this result may be related to the strong GSH–PC metabolism of leaves, thus enabling the produced PCs–Cu complex to quickly enter the vacuole and ensure that appropri ate Cu2+ in the protoplasm is engaged in normal metabolism activities [27, 28]. Under copper stress, NO released from exogenous SNP can cause the GSH–PC metabolism to produce more PCs, which may be beneficial for mitigating or reducing the damage caused by excessive copper to the tomato. With respect to Cu, Cu + SNP, and Cu + SNP + Hb, Cu2+ content change in roots and leaves No. 3

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demonstrated good agreement with PC variation. Among them, Cu + SNP + Hb demonstrated the highest correlation coefficient in roots and leaves (R = 0.9999 and R = 0.9950, respectively), showing the sig nificant function of PCs under copper stress. γECS and GS are essential enzymes for GSH syn thesis. Glu and Cys in plants are catalyzed by γ–ECS into γglutamylcysteine, which will be further cata lyzed by GS into GSH. In this test, γECS and GS in the tomato roots and leaves remained highly activated under copper stress. This phenomenon may have been caused by the plant’s need to continuously synthesize ample GSH to eliminate the peroxide resulting from the excess copper content, complexing metal ions, and lowering ion activity [29]. Under copper stress, exogenous SNP can further activate γECS and GS in tomato roots and leaves, thereby facilitating GSH syn thesis in tomato roots and leaves. This effect may be related to the increasing gene expressions of γ–ECS and GS, which resulted from the NO released by exog enous SNP under copper stress [30]. As the precursor of PC synthesis, GSH growth will facilitate PC syn thesis in tomato plants. As a result, the GSH and PC content in tomato roots and leaves will be higher in Cu + SNP (Figs. 2, 3), although the GSH in Cu + SNP + Hb will be lower. BSO is the γ–ECS activity inhibitor. In this paper, the activity of γ–ECS in tomato roots and leaves under copper stress was inhib ited by the addition of BSO, thus resulting in a signif icant decline in PC content [22, 31]. However, the activity of GS in leaves only decreased significantly at 6 h. Therefore, PC synthesis in leaves significantly lagged behind that in roots under copper stress, which explains the significantly lower PC content in leaves in Cu + BSO and Cu + BSO + SNP compared with that in Cu (Fig. 2b). Cu + BSO + SNP had significantly higher GSH and PC content in roots compared with Cu + BSO. This result may be ascribed to the NO released by SNP, which first initiated some signal net work in roots and offset BSO inhibition to γ–ECS. Li et al. [19] demonstrated that additional exogenous SNP under 250 μM BSO can further activate GR in tomato roots under copper stress, as well as induce the restoration of GSSG to GSH, thereby enhancing PC synthesis. This result may be a positive response path way of GSH–PC metabolism to exogenous NO sig nals under copper stress. ACKNOWLEDGMENTS This work was supported by National Natural Sci ence Foundation of China (no. 31201619). REFERENCES 1. Cobbett, C. and Goldsbrough, P., Phytochelatins and metallothioneins: roles in heavy metal detoxification and homeostasis, Annu. Rev. Plant Biol., 2002, vol. 53, pp. 159–182.

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