薛美昭, 儀慧蘭

(山西大學(xué)生命科學(xué)學(xué)院， 太原 030006)

砷誘導(dǎo)蠶豆氣孔保衛(wèi)細(xì)胞死亡的毒性效應(yīng)

薛美昭, 儀慧蘭*

(山西大學(xué)生命科學(xué)學(xué)院， 太原 030006)

采用蠶豆(ViciafabaL.)葉面氣孔保衛(wèi)細(xì)胞，研究砷對細(xì)胞的毒性效應(yīng)。結(jié)果表明，0.3—10 mg/L的NaAsO2能降低保衛(wèi)細(xì)胞活性，使部分細(xì)胞死亡，死亡率隨砷濃度升高而增高。死細(xì)胞中呈現(xiàn)核固縮、核崩解等典型程序性死亡特征，且泛caspase抑制劑Z-Asp-CH2-DCB能阻止NaAsO2誘發(fā)的細(xì)胞死亡。過氧化氫清除劑過氧化氫酶與NaAsO2共同作用時，細(xì)胞死亡率顯著低于砷單獨處理組，保衛(wèi)細(xì)胞內(nèi)Ca2+水平降低，具程序性死亡特征的細(xì)胞數(shù)減少；Ca2+特異性螯合劑EGTA亦能降低NaAsO2誘發(fā)的細(xì)胞死亡。研究結(jié)果表明，NaAsO2能誘發(fā)蠶豆保衛(wèi)細(xì)胞程序性死亡，該過程由脅迫引發(fā)的ROS升高引起，ROS可能通過激活質(zhì)膜Ca2+通道，使胞外Ca2+內(nèi)流，造成胞內(nèi)Ca2+濃度升高，進(jìn)而誘導(dǎo)細(xì)胞程序性死亡。

蠶豆保衛(wèi)細(xì)胞；NaAsO2；程序性細(xì)胞死亡；ROS；Ca2+

砷(As)是一種廣泛存在于自然界中的類金屬元素，通常以極低濃度存在于人類生存環(huán)境中，不會對生物造成危害。近年來，隨著砷化物在工、農(nóng)、醫(yī)藥業(yè)中的大量使用，含砷礦石的開采和冶煉，含砷廢水、廢渣的不合理排放，以及地下水的不合理開采，導(dǎo)致很多地區(qū)大氣、土壤和水體砷污染[1]。

植物葉表面的氣孔保衛(wèi)細(xì)胞是研究細(xì)胞信號轉(zhuǎn)導(dǎo)的模式系統(tǒng)，對環(huán)境變化反應(yīng)靈敏而準(zhǔn)確[9- 10]。前人采用蠶豆(ViciafabaL.)葉面氣孔保衛(wèi)細(xì)胞研究氰化物、鋁、二氧化硫的細(xì)胞毒性效應(yīng)，發(fā)現(xiàn)了它們對細(xì)胞的致死效應(yīng)及信號途徑，說明保衛(wèi)細(xì)胞對環(huán)境化學(xué)物的毒性具有靈敏的反應(yīng)[11- 13]。因此，本文以蠶豆氣孔保衛(wèi)細(xì)胞為實驗?zāi)Ｐ停鶕?jù)砷在植物體內(nèi)多以三價砷存在的事實[14]，研究NaAsO2對蠶豆氣孔保衛(wèi)細(xì)胞的致死作用，為進(jìn)一步揭示砷的毒性作用機(jī)制提供依據(jù)。

1 材料與方法

1.1 材料準(zhǔn)備

蠶豆(ViciafabaL.)種子清洗后用自來水浸泡48 h，25 ℃濕紗布包裹催芽2—3 d，然后播種于較肥沃的營養(yǎng)土中。培養(yǎng)條件：光暗周期為14 h/10 h，溫度18—25 ℃，光照強(qiáng)度240 μmol·m-2·s-1，相對濕度40%—80%。幼苗長至4周時用于實驗。

取頂端第2節(jié)完全展開的葉片，選非葉脈部位用鑷子撕取其下表皮，切成長約1 cm，寬約0.5 cm的表皮條，置于含2-(N-morpholino)ethanesulfonic acid(MES)的緩沖液中。MES緩沖液含50 mmol/L KCL，10 mmol/L MES，用三羥甲基氨基甲烷(Tris)調(diào)節(jié)pH值至7.0。

1.2 藥物處理

用MES緩沖液配制含NaAsO2濃度為0.1、0.3、1、3和10 mg/L的溶液作為砷處理液。緩解組采用一定濃度拮抗劑分別與NaAsO2同時作用。MES緩沖液作對照。每個處理用3個不同蠶豆植株上的葉片，將表皮條置于含不同藥物的處理液中，于23 ℃光照3 h。

所選拮抗劑包括：0.5 μmol/L的泛caspase 抑制劑(Z-Asp-2,6-dichlorobenzoyloxymet-hylketon，Z-Asp-CH2-DCB)，200 U/mL的CAT和1 mmol/L的Ca2+特異性螯合劑乙二醇雙四乙酸(ethylene glycol tetraacetic acid，EGTA)。實驗中，用NaAsO2處理蠶豆表皮后，檢測保衛(wèi)細(xì)胞內(nèi)ROS和Ca2+水平，與對照組比較，以分析砷脅迫誘發(fā)的細(xì)胞死亡過程中胞內(nèi)ROS和Ca2+的變化；將NaAsO2和CAT共同作用于蠶豆表皮后，檢測保衛(wèi)細(xì)胞內(nèi)Ca2+水平的改變，分析ROS水平降低時胞內(nèi)Ca2+的變化規(guī)律[12- 13]，判斷砷脅迫期間ROS對Ca2+的調(diào)節(jié)作用。

1.3 細(xì)胞活性檢測

參照Yi等實驗方法[13]，藥物處理結(jié)束后，表皮條用0.1 mg/L的二乙酸熒光素(fluorescein diacetate，F(xiàn)DA)暗染10 min，熒光顯微鏡下觀察、拍照。統(tǒng)計無綠色熒光的保衛(wèi)細(xì)胞(死細(xì)胞)數(shù)與觀察的保衛(wèi)細(xì)胞總數(shù)，計算保衛(wèi)細(xì)胞死亡率。每個處理重復(fù)3次，每次至少觀察2000個保衛(wèi)細(xì)胞。

1.4 細(xì)胞核形態(tài)觀察

藥物處理后，表皮條用卡諾氏液固定2 h，F(xiàn)eulgen法染色[15]，普通光學(xué)顯微鏡觀察。

1.5 胞內(nèi)ROS和 Ca2+水平檢測

參照González等實驗方法[16]，藥物處理后表皮條分別于10 μmol/L的活性氧熒光指示劑2′,7′-二氯熒光黃雙乙酸酯(2′,7′-dichlorofluorescein diacetate，DCFH-DA)或10 μmol/L的Ca2+熒光指示劑fluo- 3 acetomethoxyester(Fluo- 3 AM)中暗孵育60—90 min后，熒光顯微鏡(激發(fā)波長488 nm)下觀察并拍照。使用圖像分析軟件Image-Pro Plus 6.0測量每組3個表皮條中約300個細(xì)胞的熒光值，計算其平均值。將對照組熒光值計為1，各處理組的相對熒光值為處理組熒光值與對照組的比值。

1.6 數(shù)據(jù)分析

計算每組3個重復(fù)實驗的平均值和標(biāo)準(zhǔn)誤，采用SPSS17. 0對實驗結(jié)果進(jìn)行F檢驗后，采用Duncan方法進(jìn)行多重比較，分析不同處理組和對照組之間的差異顯著性(*P<0.05，差異顯著；**P<0.01，差異極顯著；不同字母間差異顯著，P<0.05)。

2 結(jié)果

2.1 砷誘發(fā)蠶豆保衛(wèi)細(xì)胞死亡

表皮條經(jīng)FDA染色后，對照組保衛(wèi)細(xì)胞發(fā)亮綠色熒光，表明細(xì)胞具有良好的活性；NaAsO2處理組保衛(wèi)細(xì)胞熒光亮度降低，說明胞內(nèi)非特異性酯酶活性降低，不能很好地將FDA水解為極性熒光素分子，其中部分細(xì)胞無綠色熒光，記為死細(xì)胞。隨著NaAsO2濃度的增高，死細(xì)胞比率增高。濃度為0.3 mg/L的NaAsO2能顯著誘導(dǎo)蠶豆氣孔保衛(wèi)細(xì)胞死亡，NaAsO2濃度為1—10 mg/L時，細(xì)胞死亡率極顯著增高(圖1)，其中10 mg/L處理組死亡率最高，達(dá)到32.2%，比對照組增加了3.4倍。

圖1 砷對蠶豆保衛(wèi)細(xì)胞活性的影響Fig.1 Effect of arsenic on guard cell viability in detached epidermis of V. faba leaves

2.2 砷誘發(fā)保衛(wèi)細(xì)胞程序性死亡

用Schiff試劑染色后，對照組保衛(wèi)細(xì)胞核質(zhì)均勻，砷處理組保衛(wèi)細(xì)胞出現(xiàn)核固縮、核崩解等典型的程序性細(xì)胞死亡(Programmed cell death, PCD)特征(圖2)，由此推測砷脅迫誘發(fā)的蠶豆保衛(wèi)細(xì)胞死亡過程中可能存在細(xì)胞程序性死亡。

Caspase是細(xì)胞程序性死亡的關(guān)鍵酶，本研究用0.5 μmol/L的泛caspase抑制劑Z-Asp-CH2-DCB分別與3 mg/L和10 mg/L的NaAsO2共同作用時，細(xì)胞死亡率顯著低于砷單獨處理組(圖3)，即caspase抑制劑抑制了NaAsO2誘發(fā)的細(xì)胞死亡。該結(jié)果說明，蠶豆保衛(wèi)細(xì)胞中的類caspase蛋白酶參與并執(zhí)行了NaAsO2誘發(fā)的細(xì)胞死亡過程，砷脅迫誘發(fā)的蠶豆保衛(wèi)細(xì)胞死亡過程中存在程序性細(xì)胞死亡。

圖2 砷誘發(fā)蠶豆氣孔保衛(wèi)細(xì)胞核形態(tài)異常(×200) Fig.2 Abnormal nucleus in V. faba guard cells exposed to arsenica: 對照; b-d: NaAsO2處理組; b: 核崩解;c: 核固縮; d: 核消失

圖3 泛caspase抑制劑對砷致蠶豆保衛(wèi)細(xì)胞死亡的緩解作用Fig.3 Antagonistic effect of caspase inhibitor on As-induced guard cell death in V. faba

2.3 胞內(nèi)ROS和Ca2+水平升高與砷誘發(fā)的細(xì)胞死亡有關(guān)

在砷處理液中加入抗氧化劑CAT或Ca2+特異性螯合劑EGTA后，細(xì)胞死亡率顯著降低。200 U/mL的CAT或1 mmol/L的EGTA與NaAsO2同時作用時，細(xì)胞死亡率顯著低于砷單獨處理組(圖4)，說明降低砷脅迫組胞內(nèi)ROS 和Ca2+水平可有效阻止砷誘發(fā)的細(xì)胞死亡，砷脅迫誘發(fā)的細(xì)胞死亡可能與脅迫引發(fā)的胞內(nèi)ROS和Ca2+升高有關(guān)。

圖4 CAT和EGTA對砷致蠶豆保衛(wèi)細(xì)胞死亡的緩解作用Fig.4 Antagonistic effects of CAT and EGTA on As-induced guard cell death in V. faba

為了證實砷脅迫中保衛(wèi)細(xì)胞內(nèi)ROS 和Ca2+水平的變化，采用特異性熒光探針標(biāo)記后檢測了保衛(wèi)細(xì)胞內(nèi)ROS和Ca2+水平。經(jīng)ROS探針DCFH-DA標(biāo)記后，NaAsO2處理組保衛(wèi)細(xì)胞ROS相對熒光值明顯高于對照組；用Ca2+探針Fluo-3AM標(biāo)記后，NaAsO2組保衛(wèi)細(xì)胞內(nèi)Ca2+相對熒光值較對照組明顯升高(圖5)。結(jié)果表明，NaAsO2處理組保衛(wèi)細(xì)胞內(nèi)ROS和Ca2+水平顯著升高。

上述結(jié)果表明，砷處理組細(xì)胞死亡與胞內(nèi)ROS和Ca2+水平升高同時發(fā)生，加入抗氧化劑降低胞內(nèi)ROS或用Ca2+特異性螯合劑減少胞外后Ca2+內(nèi)流后，保衛(wèi)細(xì)胞死亡率顯著下降，即脅迫組胞內(nèi)ROS和Ca2+水平增高參與介導(dǎo)了保衛(wèi)細(xì)胞的死亡。

加入200 U/mL的CAT后，砷處理組細(xì)胞死亡率降低(圖4)，具有程序性死亡特征的細(xì)胞數(shù)減少(圖6)，砷誘發(fā)的胞內(nèi)Ca2+升高被抑制(圖5)，表明在砷誘發(fā)蠶豆保衛(wèi)細(xì)胞死亡過程中，ROS對胞內(nèi)Ca2+水平具有正調(diào)控作用，ROS位于Ca2+的上游發(fā)揮作用。加入Ca2+特異性螯合劑EGTA 降低細(xì)胞外Ca2+濃度，阻止胞外Ca2+內(nèi)流后，可阻止砷處理誘發(fā)的細(xì)胞死亡(圖4)，說明在砷誘導(dǎo)細(xì)胞死亡的過程中，胞外Ca2+內(nèi)流對胞內(nèi)Ca2+水平升高和繼發(fā)細(xì)胞死亡發(fā)揮了重要作用。

3 討論

在砷的毒理學(xué)研究中以動物為材料的居多，砷對植物細(xì)胞的毒性效應(yīng)研究較少，相關(guān)毒理學(xué)機(jī)制的研究更是少見。本文以蠶豆氣孔保衛(wèi)細(xì)胞為實驗?zāi)Ｐ停峁┝酥参锛?xì)胞中砷的毒性作用證據(jù)。用亞砷酸鈉處理后蠶豆氣孔保衛(wèi)細(xì)胞活性降低并出現(xiàn)典型的程序性死亡特征，且泛caspase蛋白酶抑制劑Z-Asp-CH2-DCB能阻止砷誘發(fā)的細(xì)胞死亡，表明砷誘發(fā)的蠶豆保衛(wèi)細(xì)胞死亡過程存在程序性細(xì)胞死亡。通過檢測抗氧化劑CAT和鈣離子特異性螯合劑EGTA對砷毒性作用的抑制效應(yīng)，證實砷脅迫組胞內(nèi)ROS和Ca2+水平的提高是砷誘發(fā)植物細(xì)胞程序性死亡的誘因，該結(jié)果與砷誘發(fā)的多種動物細(xì)胞的凋亡途徑類似[17- 18]，表明植物細(xì)胞與動物細(xì)胞中可能存在著相似的機(jī)制。

圖5 砷對蠶豆保衛(wèi)細(xì)胞內(nèi)ROS和Ca2+水平的影響Fig.5 Changes of ROS and Ca2+ fluorescence in V. faba guard cells exposed to arsenic

圖6 CAT對砷致蠶豆保衛(wèi)細(xì)胞程序性死亡的抑制效應(yīng)Fig.6 Inhibition of CAT on PCD in V. faba guard cells exposed to arsenic

植物細(xì)胞可通過ROS介導(dǎo)的鈣信號途徑而致死，過氧化氫能激活質(zhì)膜上的鈣離子通道，使胞外Ca2+內(nèi)流，胞內(nèi)Ca2+水平升高[19]，胞內(nèi)Ca2+濃度升高可激活Ca2+依賴性核酸內(nèi)切酶，使細(xì)胞核DNA在核小體連接處被切割，從而引發(fā)細(xì)胞程序性死亡。本研究發(fā)現(xiàn)，砷處理組ROS水平對胞內(nèi)Ca2+水平具正向調(diào)節(jié)作用，減少胞外Ca2+內(nèi)流后砷處理組死亡率下降，由此推測，砷脅迫產(chǎn)生的ROS可能通過激活質(zhì)膜Ca2+通道，使Ca2+內(nèi)流導(dǎo)致胞內(nèi)Ca2+水平升高，從而介導(dǎo)了細(xì)胞死亡。研究發(fā)現(xiàn)，砷脅迫能引起酵母細(xì)胞核崩解，蠶豆和洋蔥根尖細(xì)胞氧化損傷、微核率升高，這些結(jié)果的產(chǎn)生與砷脅迫組ROS升高有關(guān)[8,20]；ROS可直接攻擊DNA、蛋白質(zhì)等生物大分子，使DNA 分子斷裂、修復(fù)酶活性降低，DNA損傷無法及時修復(fù)。DNA損傷能激活細(xì)胞死亡程序，引發(fā)細(xì)胞死亡[21- 22]，因此，砷脅迫組蠶豆保衛(wèi)細(xì)胞內(nèi)ROS升高可能通過類似途徑誘發(fā)細(xì)胞死亡。

研究表明，砷脅迫可致酵母細(xì)胞內(nèi)ROS和Ca2+水平升高，線粒體膜完整性破壞，細(xì)胞色素c釋放，類caspase蛋白酶激活，引發(fā)程序性細(xì)胞死亡[20]。因此，砷脅迫組蠶豆保衛(wèi)細(xì)胞內(nèi)ROS和Ca2+水平升高也可能通過線粒體途徑完成細(xì)胞死亡過程，通過破壞線粒體膜完整性釋放細(xì)胞色素c、凋亡誘導(dǎo)因子，并于胞質(zhì)中形成凋亡復(fù)合體，激活類caspase蛋白酶，進(jìn)而特異性切割下游底物，引發(fā)細(xì)胞凋亡。

氣孔調(diào)節(jié)著植物和外界環(huán)境間氣體與水分的交換，砷脅迫導(dǎo)致氣孔保衛(wèi)細(xì)胞活性降低或死亡會干擾氣孔正常的調(diào)節(jié)功能，影響植物的光合作用和呼吸代謝，從而干擾植株生理生化過程。但本研究觀察到砷脅迫誘發(fā)的植物細(xì)胞程序性死亡，可能在植株對環(huán)境脅迫和刺激的應(yīng)答過程中具有積極的意義，具體效應(yīng)機(jī)制有待進(jìn)一步深入研究。

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Arsenic induces guard cell death in leaf epidermis ofViciafaba

XUE Meizhao, YI Huilan*

SchoolofLifeScience,ShanxiUniversity,Taiyuan030006,China

Arsenic is a highly toxic metalloid for all forms of life including plants. Arsenic enters in plants through phosphate transporters as a phosphate analogue or through aquaglycoporins. Uptake of arsenic in plant tissues can affect plant metabolism, causing various physiological disorders, structural abnormalities and even plant death. Oxidative stress is considered to be a key mechanism of arsenic toxicity.

In this study, the effect of sodium arsenite (NaAsO2) on guard cell viability was investigated in detached epidermis ofV.fabaleaves. Epidermal strips were obtained from 4-week-old plants by peeling off the lower epidermis ofV.fabaleaves and incubated in 2-(N-morpholino) ethanesulfonic acid (MES) buffer containing some chemicals (NaAsO2with or without some antagonists) for 3 h in white light at 23 ℃ as the treatments. After treatment, the epidermal strips were stained with fluorescein diacetate (FDA) to show cell viability, or with 2′,7′-dichlorofluorescein diacetate (DCFH-DA) and fluo-3 acetomethoxyester (Fluo-3AM) respectively to indicate intracellular reactive oxygen species (ROS) and calcium ion (Ca2+) levels.

The results of our experiments showed that NaAsO2treatment significantly decreased cell viability and induced cell death in the concentration range of 0.3 to 10 mg/L. Arsenic provokes synchronous increases in cell death rate and intracellular levels of ROS and Ca2+inV.fabaguard cells. The typical nuclear morphological changes including nuclear fragmentation and nuclear condensation were observed in As-treated guard cells, while Z-Asp-2,6-dichlorobenzoyloxymethylketone (Z-Asp-CH2-DCB), a specific inhibitor of mammalian caspases, significantly blocked As-induced cell death. The occurrence of characteristic features of programmed cell death (PCD) and the inhibitory effect of caspase inhibitor Z-Asp-CH2-DCB on As-induced cell death suggest the activation of a PCD pathway evoked by arsenic exposure. Application of antioxidant catalase significantly inhibited As-induced cell death, PCD-to-total-cells ratio and intracellular Ca2+increase provoked by arsenic. A specific Ca2+chelator ethylene glycol tetraacetic acid also significantly decreased the cell death caused by arsenic. These results clearly demonstrated that arsenic induced cell death associated with obvious increases in intracellular ROS and Ca2+levels, in which ROS positively regulated intracellular Ca2+level．ROS generation and intracellular Ca2+level increase in As-treated stomatal guard cells were involved in the process of As-induced cell death. The results of the present study indicate that arsenite induced guard cell death via a PCD pathway through ROS mediating Ca2+elevation.

V.fabaguard cells; NaAsO2; programmed cell death; ROS; Ca2+

國家自然科學(xué)基金項目(30870454, 30470318); 教育部高等學(xué)校博士學(xué)科點基金項目(20070108007, 20121401110007); 山西省回國留學(xué)人員科研資助項目(2012013)

2012- 10- 17;

2013- 04- 18

10.5846/stxb201210171445

*通訊作者Corresponding author.E-mail: yihl@sxu.edu.cn

薛美昭, 儀慧蘭.砷誘導(dǎo)蠶豆氣孔保衛(wèi)細(xì)胞死亡的毒性效應(yīng).生態(tài)學(xué)報,2014,34(5):1134- 1139.

Xue M Z, Yi H L.Arsenic induces guard cell death in leaf epidermis ofViciafaba.ActaEcologicaSinica,2014,34(5):1134- 1139.