沈國明

(菏澤學(xué)院植物生物學(xué)重點(diǎn)實(shí)驗(yàn)室，菏澤學(xué)院生命科學(xué)系，山東菏澤274015)

Cd2+誘導(dǎo)的鎘敏感水稻突變體cadB-1葉片抗壞血酸循環(huán)的變化

沈國明

(菏澤學(xué)院植物生物學(xué)重點(diǎn)實(shí)驗(yàn)室，菏澤學(xué)院生命科學(xué)系，山東菏澤274015)

【目的】鎘離子 (Cd2+) 為非必需的微量元素，植物易從土壤中吸收并積累Cd2+，通過食物鏈進(jìn)入人體內(nèi)，對人類的健康造成重大威脅。為了闡明Cd2+誘導(dǎo)氧化脅制和抑制生長的機(jī)制，對 Cd2+敏感水稻突變體 (cadB-1) 進(jìn)行了水培試驗(yàn)。【方法】植物材料為水稻粳稻中花11(OryzasativaL. sspjaponicavariety, Zhonghua 11)，經(jīng)農(nóng)桿菌(Agrobacteriumtumefaciens)介導(dǎo)轉(zhuǎn)入T-DNA/Ds的突變體庫(M1代)。將M1代種子用1%稀硝酸清洗后，30℃浸種2 d，于墊有2層濾紙的培養(yǎng)皿中加7 mL滅菌水，28℃催芽4 d，種子露白后播于含1/2水稻培養(yǎng)液的水稻育苗盤中，待苗長到三葉期時(shí)移至含8 L培養(yǎng)液的直徑25 cm塑料桶中，桶外壁涂黑，每桶種8穴，每穴2株，用塑料板分隔各穴，海綿固定使水稻垂直生長。置于人工氣候箱(MC1000 system, Snijders)中，溫度周期32℃/27℃ (日溫/夜溫) ，相對濕度65%， 12 h光周期光照強(qiáng)度為500 μmol/(m2·s)，每隔5 d換一次營養(yǎng)液，直到結(jié)出M2代種子。將中花11野生型與M2代突變體種子用以上同樣方法培養(yǎng)，長到五葉期。以不加Cd2+作為對照，分別加入0.1、 0.25、 0.5和0.75 mmol/L Cd2+進(jìn)行篩選，每種處理平行培養(yǎng)3桶，作為重復(fù)，共6001桶，每天定時(shí)觀察。12 d后，發(fā)現(xiàn)0.5 mmol/L Cd2+中的中花11野生型沒有死亡，而M2代突變體出現(xiàn)部分死亡。按所在位置，選取表型最明顯的株系命名為cadB-1。取cadB-1 種子按上述方法萌發(fā)，然后均勻發(fā)芽的幼苗與上述相同條件培養(yǎng)，至七葉期，水稻幼苗包括野生型 (WT)和cadB-1 用 0.5 mmol/L CdCl2處理2、4、6、8和 12 d?！窘Y(jié)果】1)葉片中Cd和過氧化氫(H2O2)積累量cadB-1高于野生型； 2)葉片中還原型谷胱甘肽(GSH)和氧化型谷胱甘肽、抗壞血酸和脫氫抗壞血酸及還原型煙酰胺腺嘌呤二核苷酸磷酸和氧型煙酰胺腺嘌呤二核苷酸磷酸的比值都是cadB-1低于野生型； 3)葉片中抗壞血酸氧化酶 (ascorbate peroxidase, APX, EC 1.11.1.11), 還原型谷胱甘肽酶(glutathione reductase, GR, EC 1.6.4.2), 脫氫抗壞血酸還原酶(dehydroascorbate reductase, DHAR, EC 1.8.5.1) 和單脫氫抗壞血酸還原酶(monodehydroascorbate reductase，MDHAR, EC 1.6.5.4) 活性都是cadB-1低于野生型?！窘Y(jié)論】cadB-1具有低水平的抗氧化劑和抗氧化酶活性。此外，cadB-1比 WT 積累更多的 Cd 從而產(chǎn)生更多的活性氧 (reactive oxygen species, ROS)。也就是說，與野生型相比，cadB-1 更缺乏防御力來清除更多的活性氧，從而導(dǎo)致較低的生長勢和對Cd的敏感。

抗壞血酸-谷胱甘肽循環(huán)； 鎘敏感突變體； 生長抑制； 過氧化氫； 水稻

Cadmium can be readily taken up by roots and often accumulates to a large number in plant system[1], the presence of Cd can induce the generation of reactive oxygen species (ROS). Plants have developed antioxidant mechanisms to alleviate hazardous effects imposed by oxide stress. These mechanisms include antioxidative enzymatic and antioxidative non-enzymatic systems. The ascorbate-glutathione (ASC-GSH) cycle is the keystone of the nonenzymatic antioxidative defense system and has been suggested as the source for H2O2remova1 into organelles[2-3]. ASC and GSH, two low molecular weight antioxidants are of great importance in preserving a wide range of metabolic processes[4]. They can both react directly with ROS as well as participating in ROS detoxification through the ASC-GSH cycle[2, 5-7]. Moreover, ASC and GSH are also associated with the cellular redox balance and the ratios of ASC ∶DHA and GSH ∶GSSG may function as signals for the regulation of antioxidant mechanisms[8].

Previously, we screened a rice cadmium sensitive and hyper-accumulation mutant byAgrobacteriumtumefacienssystem, investigated the enzymatic defense system, root morphology and cadmium uptake kinetics[9-10]. In present research, we mainly compared the differences in the ASC-GSH metabolism betweencadB-1 and WT seedling leaves after increasing exposure periods of Cd. Although recently we reported some results of ASC-GSH metabolism[11-12], we also want more evidences to confirm that higher ASC, GSH, or NADPH are more able to resist Cd toxicity.

1 Materials and methods

1.1 Plant materials and culture conditions

Stable inheritance rice (OrizasativaL.) cadmium sensitive mutant (cadB-1) and the same rice variety wild-type (WT) were used in this experiment. The seeds were surface sterilized in 0.5% sodium hypochlorite for 20 min, rinsed, and germinated in the dark on moistened filter paper at 30℃ for 2 d, and then moved to a plastic screen floating on distilled water at 28℃ for 4 d. Then uniformly germinated seedlings were transferred to black polyethylene barrels which contained 6 L of rice culture solution. Seedlings were grown in a growth chamber with a photo flux density of 500 μmol/(m2·s), relative humidity of approximately 65% and day/night temperatures of 32℃/27℃ (14 h/10 h). During the growth period, the solution was renewed every 5 d. At the seven leaf stage, rice seedlings include wild-type (WT) andcadB-1 exposed to 0.5 mmol/L CdCl2for 0 (as control), 2, 4, 6, 8，or 12 d.

1.2 Cd and H2O2content analysis

The Cd contents in seedling leaves, stems and roots were determined according to the method of Shah and Dubey[13]. H2O2content was determined according to the method described by Jana and Choudhuri[14].

1.3 Ratios of ASC/DHA, GSH/GSSG and NADPH/NADP+analysis

ASC and DHA content were determined according to the method of Lawetal.[15].GSH and GSSG content was determined according to the method of Andersonetal.[16]. NADPH and NADP+ content was determined according to the method of Nisselbaum and Gree[17].

1.4 Enzyme assays

Frozen materials (400 mg fresh weight) were homogenized in 4 mL of 50 mmol/L potassium phosphate buffer, pH 7.8, containing 0.1% Triton X-100. The homogenate was centrifuged at 15,000×g for 20 min at 4℃ and the supernatant was used for enzyme assays. APX and GR activities were determined according to the method of Nakanoetal.[18]. DHAR activity was determined according to the method of Daltonetal.[19]. NDHAR activity was determined according to the method of Arrigonietal.[20].

1.5 Statistical Analysis

Data were analyzed with the statistical package SPSS15 for Windows on the website (www.nbs.ntu.edu.sg/userguide/SPSS/SPSS15/). Significance levels 0.05 and 0.01 were used in presenting the results. The experiments were repeated in triplicate, and the data presented are the mean values±standard error (SE). The difference was considered significant at P levels lower than 0.05 (P<0.05) and this significance is denoted in the figures by an asterisk (*) while significant at P levels lower than 0.01 (P<0.01) denoted in the figures by double asterisk (**).

2 Results

2.1 Cadmium accumulation and effect on rice seedling growth

At the seven-leaf stage, CdCl2was added to the nutrient solution to achieve the final Cd2+concentration is 0.5 mmol/L. After 12 d exposure to Cd2+, the leaves ofcadB-1 faded seriously and the roots were more exiguous than WT. The fresh weight of shoots, and roots of wild type seedlings declined by 45.97%, and 46.99%, respectively, while the percent decrease ofcadB-1 are 63.56% and 51.28%, compared to the control-cultivated seedlings (Table 1).

表1 侵染于0.5 mmol/L Cd2+12天后野生型(WT)和Cd-敏感型(cadB-1)水稻秧苗不同部位鮮重(g, FW)

The Cd contents increased in all organs of both WT andcadB-1 with 0.5 mmol/L Cd2+exposure for 12 d (Table 2). A larger increase was seen in all organs ofcadB-1 compared to WT seedlings. However, the increase was not statistically significant.

2.2 H2O2accumulation in rice seedlings

H2O2contents increased in the leaves of WT andcadB-1 rice seedlings during Cd2+exposure period (Fig.1). In general,cadB-1 rice seedlings accumulated more H2O2than WT rice seedlings, except the 2nd day. Significant difference of H2O2contents between WT andcadB-1 rice seedling leaves was only detected the 12th exposure day.

表2 侵染于0.5 mmol/L Cd2+12天后野生型(WT)和Cd-敏感型(cadB-1)

圖1 野生型(WT)和Cd-敏感型(cadB-1)水稻秧苗葉片中H2O2含量Fig.1 H2O2 contents in leaves of WT and cadB-1 rice seedlings[注(Note)：External Cd2+ concentration is 0.5 mmol/L 外源Cd2+濃度為0.5 mmol/L. Error bars represent standard error (n=3)誤差線代表標(biāo)準(zhǔn)差(n=3)].

2.3 Cd effect on ratios of ASC/DHA, GSH/GSSG and NADPH/NADP+in rice seedlings

During exposure period, leaf contents of ASC decreased both in WT andcadB-1 (Fig.2-a), however the opposite effect was observed with DHA contents in leaves (Fig. 2-b). Significant differences were seen in ASC content between WT andcadB-1 rice seedling leaves at the 8th and 12th exposure day. Therefore, the ratio of ASC/DHA was reduced with prolonging exposure time (Fig.2-c). Furthermore, in WT andcadB-1 rice seedlings the ratio varied concomitantly with prolonging time at 0.5 mmol/L Cd2+. After 12 d exposure to 0.5 mmol/L Cd2+, the ratio of ASC/DHA at the 2nd, 4th, 6th, 8th and 12th exposure day compared to the control declined by 9.76%, 20.10%, 38.00%, 53.00% and 66.55%, respectively in the leaves of WT rice seedlings, while incadB-1 rice seedlings the percentage of decrease was 16.56%, 32.92%, 48.57%, 65.86% and 85.06%, respectively. Overall, the ratio of ASC:DHA declined more in leaves ofcadB-1 rice seedlings than in WT rice seedlings.

The GSH contents decrease and GSSG increase in leaves occurred not only in WT but also incadB-1 during the exposure (Fig. 2-d, e). Significant differences took place in GSH contents but not in GSSG between WT andcadB-1 rice seedling leaves during exposure period. Changes in GSH and GSSG led to changes in GSH/GSSG ratio (Fig. 2-f), significant differences could be found in the GSH/GSSG ratio between WT andcadB-1 rice seedling leaves at the 4th, 8th and 12th exposure day. During exposure period, the GSH/GSSG ratio in leaves ofcadB-1 rice seedlings declined by 35.80%, 61.09%, 70.65%, 82.31% and 85.91%, respectively, while ratios for leaves of the WT rice seedlings were increased by 26.37%, 37.52%, 59.15%, 63.00% and 68.09%, respectively compared to the control. GSH/GSSG ratios decreased more incadB-1 rice seedlings than in WT rice seedlings.

Oxidized nicotinamide adenine dinucleotide phosphate (NADP+) contents increased during exposure period (Fig. 2-h), while the NADPH contents meet the opposite to NADP+in leaves. NADPH:NADP+ratios were reduced (Fig. 2-i), similar to GSH/GSSG and ASC/DHA ratios. The leaves ofcadB-1 rice seedlings showed a decrease in the NADPH/NADP+ratios of 7.77%, 19.34%, 35.76%, 55.03%, and 67.94%, respectively. In the WT seedlings, the NADPH/NADP+ratios compared to the control were decreased by 3.81%, 18.30%, 28.76%, 45.09%, and 58.37%. The decrease in the NADPH/NADP+ratios was more pronounced incadB-1 rice seedlings than in WT rice seedlings.

圖2 野生型(WT)和Cd-敏感型(cadB-1)水稻秧苗葉片中ASC、DHA、GSH、GSSG、NADPH、NADP+值和ASC/DHA, GSH/GSSG and NADPH/NADP+比率Fig.2 The contesnt ASC, DHA, GSH, GSSG, NADPH, NADP+ and the ratio of ASC/DHA, GSH/GSSG and NADPH/NADP+ in leaves of WT and cadB-1 rice seedlings[注(Note): 外源Cd濃度為0.5 mmol/L External Cd concentration is 0.5 mmol/L; *P<0.05 and **P<0.01. 誤差線代表標(biāo)準(zhǔn)差(n=3) Error bars represent standard error (n=3).]

2.4 Cd effect on APX, GR, DHAR and MDHAR activities in rice seedlings

APX activities increased and then decreased both in WT and incadB-1 rice seedlings during exposure (Fig.3-a). APX activity reached its highest level in WT and incadB-1 rice seedlings at the 6thexposure day, and the APX activities showed significant differences between WT andcadB-1 rice seedling. APX activities decreased more in the leaves ofcadB-1 seedlings than in WT seedlings during exposure.

GR activities increased first and then decreased in both WT andcadB-1 rice seedlings with the prolongation of exposure (Fig.3-b). GR activities were highest in both WT andcadB-1 rice seedlings at the 4th exposure day, GR activities were observed higher in WT than incadB-1 rice seedlings. to 0.5 mmol/L Cd2+, on the 4th day of exprosure, comparing to the control, GR activities increased by 64.85% incadB-1 seedlings and 101% in the WT rice seedlings. At the 8th day, significant differences in GR activities were observed betweencadB-1 and the WT rice seedling.

In this experiment, DHAR activities were lower incadB-1 than in WT seedlings with Cd2+exposure (Fig.3-c). DHAR activities in the WT increased and then decreased,cadB-1 varied concomitantly with WT. In thecadB-1 rice seedlings, DHAR activities reached maximum levels at the 4th exposure day, while those in WT seedlings reached maximum levels at the 8th day. Differences in DHAR activities betweencadB-1 and WT plants were significant at the 6th, 8th and 12th exposure day.

MDHAR activities increased and then decreased both in thecadB-1 and WT rice seedlings (Fig. 3-d). MDHAR activities were maximum at the 4th day incadB-1, and at the 6th day in WT seedlings. MDHAR activities decreased more in thecadB-1 than the WT rice seedlings. At the 12th exposure day, MDHAR activities decreased by 56.82% in the MT, and 47.79% in thecadB-1. MDHAR activity differences were significant betweencadB-1 and WT rice seedlings at the 6th exposure day.

圖3 野生型(WT)和Cd-敏感型(cadB-1)水稻秧苗根系中APX, GR, DHAR and MDHAR活性Fig.3 APX, GR, DHAR and MDHAR activities in roots of WT and cadH-5 rice seedlings[注(Note): 外源Cd濃度為0.5 mmol/L External Cd concentration is 0.5 mmol/L; *—P<0.05； **—P< 0.01. 誤差線代表標(biāo)準(zhǔn)差 (n=3) Error bars represent standard error (n=3).]

3 Discussion

H2O2is induced inArabidopistreated with Cd2+[21], moreover, H2O2considered, as a signaling molecule in stress, is well documented[22], it defense and provide acclimation during various abiotic and biotic stresses. In general, we found the H2O2contents increased with prolongation of Cd2+exposure in bothcadB-1 and WT plants, and accumulated more incadB-1 than in WT (Fig.1).

ASC behaves as an electron donor for APX scavenging of H2O2, and would be oxidated to DHA. On the other hand, DHA could be regenerated to ASC by DHAR, using GSH as an electron donor. The balance of ASC and DHA is crucial for the enzymatic systems that scavenge H2O2. In the present study, we found that prolonged Cd2+exposure time produced a decrease in the ASC content while increased in the DHA content both incadB-1 and WT. These changes resulted in decreases in the ASC:HA ratio. The decrease in ASC content might be due to an inhibition of the DHAR activity, or more excessive use of ASC in metal detoxification, or because the activity of GSH, an electron donor, is lower. The ASC content and the ASC:DHA ratio were lower in the mutant than in the wild type. This could be the result of lower DHAR activities incadB-1 than in the wild type (Fig.3). From this, we inferred that plants resistant to Cd2+toxicity maintain a high ASC content and require a high ratio of ASC/DHA.

H2O2scavenging by APX is the first step in the ASC-GSH cycle[26]. As demonstrated in the choroplasts of pea (Pisumsativum) and spinach (Spinaceaoleracea), GR, DHAR, and MDHAR also participate in this cycle[26]. In this study, we found at the short time of Cd2+exposure can induce APX activity, and may indicate the H2O2content is increasing; after long time of Cd2+exposure the APX activities decreased, which not suggesting the H2O2content is decreasing but showing Cd2+inhibition of APX activities. With the prolongation of exposure, APX activity reductions were more pronounced in the mutant than in the wild type, showing that Cd2+strongly inhibits APX activity in the mutant. GR activities vary concomitantly with APX activities; short time Cd2+exposure can induce GR activities, which catalyze GSSG to synthesize GSH by consuming NADPH as an electron donor. DHAR and MDHAR take part in the regeneration of ASC. At first can induce DHAR and MDHAR activity, possibly as the result of increased APX activities, hence DHAR and MDHAR caught regenerate enough ASC as an electron donor for APX scavenging for H2O2.With long time of Cd2+, DHAR and MDHAR activities are inhibited, and DHAR and MDHAR activities decrease more in the mutant than in the wild type, showing that Cd2+inhibited both DHAR and MDHAR activities in the mutant acutely. At the short time, ROS is effectively scavenged; at the long time the capability is overridden and they cannot scavenge ROS effectively. From this we inferred the plant is irreversibly damaged and inhibited by ROS at the long time to Cd2+exposure.

4 Conclusion

The higher ASC, GSH and NADPH levels and the higher ratios of ASC/DHA, GSH/GSSG and NADPH/NADP+, as well as the higher antioxidative enzymatic activities in plants will be more effective to resist Cd2+toxicity. Compare to WT, the mutantcadB-1 has lower level of antioxidants as well as lower activity of antioxidant enzymes;A little more Cd accumulated incadB-1 means a little more reactive oxygen species production (ROS). Videlicet,cadB-1 is deficient of the defense power against increased level of ROS which leads to a lower growth potential and sensitive to Cd.

Acknowledgements:

The authors are very grateful to Dr Wang Jiang-Xin (Shenzhen University) read the manuscript carefully, and proposed many revisions. This work supported by Heze University fund to PhD (XY13BS01).

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Cd2+induced changes of ascorbate-glutathione cycle in Cd sensitive rice mutantcadB-1 leaves

SHEN Guo-ming

(KeyLaboratoryofPlantBiology/DepartmentofLifeSciences,HezeUniversity,Heze,Shandong274015,China)

【Objectives】 Cd2+is easily absorbed from the soil by plants and accumulation in plants which health threat to humans through human food chain. To investigate the mechanism of cadmium (Cd2+) induced oxidative stress and inhibit growth in a Cd sensitive rice mutant (cadB-1), a hydroponic experiment was conducted. 【Methods】 Ajaponicarice (Oryzasativa) variety Zhonghua 11 and the mutant rice seedlings obtained from the same rice variety as that formerly constructed with T-DNA/Ds insertion mediated byAgrobacterium. The transgenetic rice generations have stable hereditability and were used in this experiment. The seeds were surface sterilized in 0.5% sodium hypochlorite for 20 min, rinsed, and germinated in the dark on moistened filter paper at 30℃ for 2 d, and then moved to a plastic screen floating on distilled water at 28℃ for 4 d. Then uniformly germinated seedlings were transferred to black polyethylene barrels which contained 6 L of rice culture solution. Seedlings were grown in a growth chamber with a photo flux density of 500 μmol/(m2·s), relative humidity of approximately 65% and day/night temperatures of 32℃/27℃ (14 h/10 h). During the growth period, the solution was renewed every 5 d. At the seven leaf stage, rice seedlings include wild-type (WT) andcadB-1 exposed to 0.5 mmol/L CdCl2for 0 (as control), 2, 4, 6, 8，or 12 d.【Results】 1) Cd and hydrogen peroxide (H2O2) accumulation were higher incadB-1 than in wild one； 2) The ratios of reduced glutathione (GSH) and oxidized glutathione (GSSG), ascorbate (ASC) and dehydroascorbate (DHA), or reduced nicotinamide adenine dinucleotide phosphate (NADPH) and oxidized nicotinamide adenine dinucleotide phosphate (NADP+) were lower incadB-1 than in WT； 3) Ascorbate peroxidase (APX, EC 1.11.1.11), glutathione reductase (GR, EC 1.6.4.2), dehydroascorbate reductase (DHAR, EC 1.8.5.1) and monodehydroascorbate reductase (MDHAR, EC 1.6.5.4) activities were lower incadB-1 than in WT in leaves during CdCl2exposure periods.【Conclusion】cadB-1 has lower level of antioxidants as well as lower activity of antioxidant enzymes. In addition,cadB-1 accumulates more Cd means that it can produce more reactive oxygen species (ROS). Videlicet,cadB-1 is deficient of the defense power against increased level of ROS which leads to a lower growth potential and sensitive to Cd.Key words: ascorbate-glutathione cycle; cadmium sensitive mutant; growth inhibit; hydrogen peroxide; rice

2014-08-18 接受日期： 2015-01-22

菏澤學(xué)院博士基金項(xiàng)目(XY13BS01)資助。

沈國明(1975—)， 男， 浙江紹興人， 博士，講師，主要從事植物逆境分子生理和農(nóng)產(chǎn)品安全生產(chǎn)研究。 E-mail: gmshen@tzc.edu.cn

S511.01

A

1008-505X(2015)02-0346-08