陳思，范曉季，金瑜劍，李星星，宋昊，孫立偉，錢海豐,,*

1. 浙江工業(yè)大學(xué) 海洋學(xué)院，杭州 310032 2. 浙江工業(yè)大學(xué) 生物工程學(xué)院，杭州 310032 3. 浙江工業(yè)大學(xué) 環(huán)境學(xué)院，杭州 310032

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過氧化氫和一氧化氮在小球藻抗阿特拉津脅迫中的作用

陳思1，范曉季2，金瑜劍2，李星星1，宋昊1，孫立偉3，錢海豐1,3,*

1. 浙江工業(yè)大學(xué) 海洋學(xué)院，杭州 310032 2. 浙江工業(yè)大學(xué) 生物工程學(xué)院，杭州 310032 3. 浙江工業(yè)大學(xué) 環(huán)境學(xué)院，杭州 310032

過氧化氫(H2O2)和一氧化氮(NO)作為信號(hào)分子，可調(diào)節(jié)植物生長(zhǎng)、發(fā)育以及應(yīng)對(duì)外源性脅迫。利用過氧化氫酶(CAT)以及NO清除劑(PTIO)，研究了除草劑阿特拉津(atrazine，100 μg·L-1)影響小球藻生長(zhǎng)的機(jī)理，并分析內(nèi)源性H2O2和NO在小球藻抗除草劑脅迫中的作用。研究結(jié)果表明，阿特拉津在誘發(fā)小球藻細(xì)胞死亡的過程中，不同程度促發(fā)了H2O2和NO生成；外源CAT可通過清除H2O2和誘導(dǎo)NO來緩解阿特拉津?qū)π∏蛟宓纳L(zhǎng)抑制；PTIO與阿特拉津的聯(lián)合實(shí)驗(yàn)進(jìn)一步證實(shí)，小球藻體內(nèi)的NO誘導(dǎo)與H2O2的爆發(fā)無關(guān)，它們之間的合成沒有相關(guān)性。因此，除草劑阿特拉津主要通過誘導(dǎo)小球藻體內(nèi)的H2O2爆發(fā)來破壞藻細(xì)胞，抑制其生長(zhǎng)，與NO的信號(hào)傳遞無關(guān)。

阿特拉津；小球藻；CAT；一氧化氮；過氧化氫；PTIO

Received 14 April 2016 accepted 5 May 2016

過氧化氫(H2O2)是一種常見活性氧物質(zhì)，主要由線粒體、葉綠體、過氧化物酶體等產(chǎn)生[1-3]。植物的外界脅迫，如病原體、金屬化合物和除草劑暴露等，常誘導(dǎo)H2O2的產(chǎn)生，造成DNA損傷甚至細(xì)胞死亡[4-8]。不僅如此，大量研究表明，H2O2作為重要的信號(hào)分子傳遞脅迫信號(hào)[9-10]，并利用水調(diào)蛋白將脅迫信號(hào)傳遞至鄰近細(xì)胞[11-12]。另一個(gè)小分子一氧化氮(NO)由一氧化氮合成酶(nitric oxide synthase, NOS)合成。在哺乳動(dòng)物體內(nèi)，NO作為重要信號(hào)分子參與神經(jīng)調(diào)節(jié)、心血管功能和免疫系統(tǒng)調(diào)節(jié)，但在植物中的功能尚不清楚[13-14]。近年來，NO在植物中的功能也備受關(guān)注，被認(rèn)為是植物生長(zhǎng)發(fā)育過程中傳遞應(yīng)激反應(yīng)的信號(hào)分子[15-16]。研究證實(shí)，NO外源性供體硝普鈉(sodium nitroprusside, SNP)促使植物具有抗外界脅迫的功能，如鹽脅迫[17]、干旱[18]、病原體[19]、金屬化合物[20]、溫度[21]、澇災(zāi)[22]和除草劑脅迫[23]，但過量NO也會(huì)抑制植物光合作用[23-24]。這些研究表明，NO是植物體內(nèi)重要的信號(hào)分子[25]。

NO和H2O2在植物應(yīng)激反應(yīng)中的關(guān)系存在很多爭(zhēng)議。Lum等[26]發(fā)現(xiàn)，H2O2快速誘導(dǎo)綠豆細(xì)胞內(nèi)NO的積累，認(rèn)為氧化應(yīng)激反應(yīng)可誘發(fā)NO的大量產(chǎn)生。同樣，Lin等[27]證實(shí)，H2O2在水稻體內(nèi)可作為誘導(dǎo)NO的上游信號(hào)分子，并且認(rèn)為NO與H2O2是水稻葉片細(xì)胞致死的重要介質(zhì)。然而，Neill等[21]發(fā)現(xiàn)，病原體入侵植物時(shí)，植物體內(nèi)的NO和H2O2同時(shí)產(chǎn)生。這些研究說明NO和H2O2在環(huán)境脅迫中都起到信號(hào)傳遞的作用，但一些不一致的結(jié)論暗示兩者之間的復(fù)雜關(guān)系尚需進(jìn)一步解析。

阿特拉津作為全球使用最廣泛的除草劑之一，在水土中常被大量檢出[28]。阿特拉津通過阻礙光合系統(tǒng)II的電子受體，破壞植物光合作用電子傳遞；并以脂溶性方式進(jìn)入藻類葉綠體，導(dǎo)致活性氧ROS (reactive oxygen species, ROS)的爆發(fā)，對(duì)細(xì)胞造成氧化性損傷，進(jìn)而擾亂細(xì)胞內(nèi)氧化還原平衡[29-31]。過多的ROS破壞細(xì)胞膜，產(chǎn)生脂質(zhì)過氧化產(chǎn)物MDA (malondialdehyde, 丙二醛)，從而對(duì)細(xì)胞產(chǎn)生嚴(yán)重?fù)p傷[32]。本實(shí)驗(yàn)以小球藻為模式生物，研究除草劑脅迫下NO與H2O2隨時(shí)間的累積情況；清除NO后，H2O2隨時(shí)間的累積情況；以及清除H2O2后，NO隨時(shí)間的累積變化，從而闡明NO和H2O2參與植物對(duì)抗除草劑脅迫中的相互關(guān)系。

1 材料與方法 (Materials and methods)

1.1 實(shí)驗(yàn)材料

供試藻類為普通小球藻，購自中國(guó)科學(xué)院水生物研究所，水生四號(hào)培養(yǎng)液，250 mL錐形瓶中培養(yǎng)，培養(yǎng)條件為(25±0.5) °C，光照強(qiáng)度為4 500 lux左右，光暗比為14 h:10 h。接種濃度為1% (細(xì)胞濃度約為6.5×105個(gè)·mL-1)，直至培養(yǎng)到藻液在680 nm下吸光度(OD)值為0.08進(jìn)行實(shí)驗(yàn)。阿特拉津(Atr)(長(zhǎng)興化工公司，浙江，中國(guó))加入培養(yǎng)液中，單獨(dú)或與1 kU·mL-1過氧化氫酶(CAT；生物技術(shù)研究院，海門，中國(guó))或20 μmol·L-1一氧化氮清除劑(2-phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl，2-苯基-4,4,5,5-四甲基咪唑啉-3-氧代-1-氧，PTIO，西格瑪奧德里奇，圣路易斯，美國(guó))聯(lián)合使用最終濃度設(shè)置為100 μg·L-1[23]；每組設(shè)置3個(gè)平行組。

1.2 細(xì)胞生長(zhǎng)以及細(xì)胞內(nèi)NO、H2O2含量的檢測(cè)

胞內(nèi)NO含量的測(cè)定根據(jù)碧云天生物技術(shù)公司的試劑盒提供的方法：取10 mL藻液離心得沉淀，取少量石英砂研磨，使用450 mL HEPE (pH 7.2)緩沖液洗滌研磨液，11 000 r·min-1離心5 min，取200 mL上清液，具體根據(jù)試劑盒方法測(cè)定。胞內(nèi)過氧化氫含量的測(cè)定根據(jù)碧云天生物技術(shù)公司的試劑盒提供的方法。使用分光光度計(jì)測(cè)定680 nm下吸收光值，通過標(biāo)準(zhǔn)曲線換算出藻細(xì)胞密度。藻密度線性回歸方程為：

Y=162.1X+1.3463 (r2=99.34%)

公式中X為OD685值；Y為105細(xì)胞數(shù)·mL-1。

2 結(jié)果(Results)

阿特拉津處理6～48 h后小球藻細(xì)胞生長(zhǎng)變化見圖1A，對(duì)照組的小球藻細(xì)胞個(gè)數(shù)在各個(gè)時(shí)間段都明顯多于Atr處理組，說明Atr對(duì)小球藻的生長(zhǎng)有明顯的抑制作用，且隨著處理時(shí)間延續(xù)，抑制率增加，6，12，24，48 h的抑制率分別為11.8%，7.2%，20.4%和29.6%。類似現(xiàn)象在淡水藍(lán)藻、銅綠微囊藻、海洋硅藻以及三角褐指藻中也被證明[33-34]。但不同品種的藻類對(duì)阿特拉津敏感度不同，相對(duì)靈敏度依次為銅綠微囊藻>小球藻>三角褐指藻。

阿特拉津作為光合作用抑制劑，阻止電子向質(zhì)體醌的轉(zhuǎn)移，從而阻止吸收的光能轉(zhuǎn)化為電化學(xué)能[35]。多余的電子和分子氧結(jié)合，產(chǎn)生ROS來破壞細(xì)胞結(jié)構(gòu)，抑制藻的生長(zhǎng)[36]。為確認(rèn)ROS在阿特拉津脅迫下的作用，我們進(jìn)一步檢測(cè)了H2O2和NO的表達(dá)水平。結(jié)果表明，阿特拉津明顯刺激了H2O2和NO的產(chǎn)生(圖1B和C)。其中H2O2的表達(dá)量在6 h達(dá)到峰值(3.78倍)，且在48 h內(nèi)持續(xù)保持高水平(圖1B)；而NO的表達(dá)水平直到12 h處理后，才達(dá)到峰值(1.3倍)，且在24 h內(nèi)回落至正常水平(圖1C)。在阿特拉津抑制小球藻生長(zhǎng)過程中，均發(fā)現(xiàn)H2O2和NO含量在0～12 h處理期間出現(xiàn)峰值，推測(cè)這2種物質(zhì)都參與除草劑誘導(dǎo)小球藻細(xì)胞死亡過程。另外，通過觀察NO和H2O2到達(dá)峰值的時(shí)間，結(jié)合相關(guān)報(bào)道推測(cè)，NO應(yīng)該位于H2O2的下游[26,37-38]。

過氧化氫酶(CAT)可將H2O2分解成氧和水，為了確認(rèn)NO的產(chǎn)生是否依賴于H2O2，我們檢測(cè)了阿特拉津與CAT聯(lián)合暴露下小球藻的生長(zhǎng)狀況。結(jié)果顯示，1 kU·mL-1的CAT可以完全降解細(xì)胞內(nèi)的H2O2。如圖2A所示，CAT不僅能緩解阿特拉津?qū)π∏蛟宓囊种谱饔?，甚至略有促進(jìn)。在阿特拉津和CAT共同作用6，12，24，36和48 h后，細(xì)胞數(shù)量為對(duì)照組的1.4，1.86，1.73，1.82和1.59倍。有意思的是，CAT處理下，NO仍顯著增加，在12，24，36和48 h處理后NO表達(dá)量分別為對(duì)照組的1.75，2.07，3.22和1.6倍(圖2B)。

原研究表明，由于NO的抗氧化特性，能夠簇滅細(xì)胞內(nèi)ROS，對(duì)細(xì)胞起到保護(hù)作用[39]，使植物受到環(huán)境脅迫(如金屬化合物、鹽、低溫脅迫、病毒侵入)時(shí)產(chǎn)生耐受性[40-41,17]。本實(shí)驗(yàn)證實(shí)，相比于阿特拉津單獨(dú)處理，外源CAT在完全降解H2O2的同時(shí)，同時(shí)促進(jìn)NO的大量合成。該結(jié)果表明，H2O2的生成是阿特拉津抑制小球藻生長(zhǎng)的關(guān)鍵因子，而過多的H2O2可抑制NO的產(chǎn)生。盡管NO也是生物活性分子，但它在植物體內(nèi)的正效應(yīng)與其濃度相關(guān)，而過高濃度的NO對(duì)植物起到抑制效應(yīng)[22,42]。這也解釋了為什么適當(dāng)?shù)耐庠葱訬O可緩解除草劑的脅迫壓力[23]，而高濃度的外源性NO也會(huì)抑制藻細(xì)胞的生長(zhǎng)。

圖1 阿特拉津?qū)π∏蛟迳L(zhǎng)(A)，H2O2含量(B)以及NO含量(C)的影響Fig. 1 Effect of atrazine on algal growth (A), H2O2(B) and NO content (C)

圖2 阿特拉津和CAT共同暴露對(duì)小球藻生長(zhǎng)(A)，H2O2含量(B)以及NO含量(C)的影響Fig. 2 Effect of atrazine and CAT on algal growth (A), H2O2(B) and NO content (C)

圖3 阿特拉津和一氧化氮清除劑(PTIO)共同暴露對(duì)小球藻生長(zhǎng)(A)，H2O2含量(B)以及NO含量(C)的影響Fig. 3 Effect of atrazine and the NO scavenger (PTIO) on algal growth (A), H2O2(B) and NO (C) content

圖4 阿特拉津、CAT和PTIO共同暴露對(duì)小球藻生長(zhǎng)(A)，H2O2含量(B)以及NO含量(C)的影響Fig. 4 Effect of combined atrazine, CAT and PTIO treatment on (A) algal growth, the content of (B) H2O2 and (C) NO

利用PTIO能清除NO的特性，我們選取PTIO與阿特拉津共同處理，進(jìn)一步確定NO在除草劑脅迫中的作用。如圖3A所示，PTIO雖能清除NO，但并不能緩解小球藻生長(zhǎng)受抑制的現(xiàn)象。當(dāng)清除NO之后，PTIO組與阿特拉津處理組小球藻生長(zhǎng)都受到抑制，說明NO并不直接參與阿特拉津?qū)π∏蛟迳锏囊种菩?yīng)。我們同時(shí)檢測(cè)0～48 h中藻細(xì)胞內(nèi)H2O2含量變化，發(fā)現(xiàn)在清除NO后，H2O2的含量明顯高于對(duì)照組。在小球藻生長(zhǎng)受抑制的情況下，H2O2的含量顯著增加，說明H2O2是直接參與阿特拉津?qū)π∏蛟宓闹滤佬?yīng)。而在清除了NO之后，藻內(nèi)的H2O2量并沒有明顯降低，說明NO并不指導(dǎo)H2O2的合成，且NO位于H2O2信號(hào)傳導(dǎo)的下游途徑。

對(duì)于阿特拉津抑制小球藻生長(zhǎng)的2種機(jī)理推測(cè)，將H2O2清除劑(CAT)和NO特異性清除劑(PTIO)同時(shí)加入阿特拉津處理的小球藻中，我們發(fā)現(xiàn)加入CAT、PTIO和共同暴露組的細(xì)胞個(gè)數(shù)明顯多于阿特拉津單獨(dú)處理組， 說明在清除H2O2和NO的情況下，對(duì)小球藻生長(zhǎng)具顯著緩解作用。因此，我們認(rèn)為H2O2的有無，對(duì)阿特拉津的毒性有至關(guān)重要的作用，進(jìn)一步證明阿特拉津的毒性是通過H2O2的增加來實(shí)現(xiàn)的。而NO并沒有參于此脅迫過程，內(nèi)源的NO也不能緩解除草劑的脅迫。在其他外界脅迫下，H2O2和NO的關(guān)系還需要更多實(shí)驗(yàn)的驗(yàn)證。

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Effect of H2O2and NO on Atrazine Stress Resistance in Chlorella vulgaris

Chen Si1, Fan Xiaoji2, Jin Yujian2, Li Xingxing1, Song Hao1, Sun Liwei3, Qian Haifeng1,3,*

1. Ocean College, Zhejiang University of Technology, Hangzhou 310032, China 2. College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310032, China 3. College of Environment, Zhejiang University of Technology, Hangzhou 310032, China

Hydrogen peroxide (H2O2) and nitric oxide (NO) have been suggested to function as signaling molecules in plants to regulate growth, development and stress responses. In this study, we investigated the roles of endogenous H2O2and NO in herbicide stress of the algae, Chlorella vulgaris. We treated algae with the herbicide atrazine (100 μg·L-1) alone, in combination with the H2O2-degrading enzyme catalase (CAT; 1 kU·mL-1), or with the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl3-oxide (PTIO; 20 μmol·L-1) for 48 h and analyzed algal growth, H2O2and NO contents. Atrazine treatment strongly induced H2O2levels by 1.5 to 4.6 folds, mildly stimulated NO levels, and significantly increased the rate of algal cell death. CAT supplementation degraded all detectable H2O2, further increased NO levels and completely reversed the inhibitory effect of atrazine on algal growth while PTIO (the NO scavenger) had no effect on atrazine toxicity in Chlorella vulgaris. Therefore, the herbicidal effect of atrazine in Chlorella vulgaris is mediated mainly by overproduction of H2O2, and endogenous NO had no protective properties against this toxicity.

atrazine; Chlorella vulgaris; CAT; NO; H2O2; PTIO

國(guó)家自然科學(xué)基金(21577128)；浙江省大學(xué)生科技創(chuàng)新活動(dòng)計(jì)劃(新苗人才計(jì)劃)

陳思(1992-)，女，碩士，研究方向?yàn)榄h(huán)境毒理學(xué)，Email: cslock@126.com

*通訊作者(Corresponding author)， E-mail: hfqian@zjut.edu.cn

10.7524/AJE.1673-5897.20160414002

2016-04-14 錄用日期：2016-05-05

1673-5897(2016)4-102-06

X171.5

A

簡(jiǎn)介：錢海豐(1973—)，男，博士，教授，研究方向?yàn)榄h(huán)境毒理學(xué)。

陳思, 范曉季, 金瑜劍, 等. 過氧化氫和一氧化氮在小球藻抗阿特拉津脅迫中的作用[J]. 生態(tài)毒理學(xué)報(bào)，2016, 11(4): 102-107

Chen S, Fan X J, Jin Y J, et al. Effect of H2O2and NO on atrazine stress resistance in Chlorella vulgaris [J]. Asian Journal of Ecotoxicology, 2016, 11(4): 102-107 (in Chinese)