土荊芥揮發(fā)性化感物質(zhì)誘導(dǎo)蠶豆保衛(wèi)細(xì)胞死亡及信號調(diào)節(jié)

周 健,王亞男,馬丹煒,黃 素,辛文媛,張 紅

四川師范大學(xué)生命科學(xué)學(xué)院, 成都 610101

土荊芥揮發(fā)性化感物質(zhì)誘導(dǎo)蠶豆保衛(wèi)細(xì)胞死亡及信號調(diào)節(jié)

周 健,王亞男,馬丹煒*,黃 素,辛文媛,張 紅

四川師范大學(xué)生命科學(xué)學(xué)院, 成都 610101

為了探討入侵植物土荊芥的化感作用機(jī)制,以其入侵地廣泛種植的農(nóng)作物蠶豆葉片下表皮為受試材料,通過對保衛(wèi)細(xì)胞的活性分析,研究了土荊芥揮發(fā)油及其兩種主要成分α-萜品烯和對傘花素誘導(dǎo)保衛(wèi)細(xì)胞死亡及其信號調(diào)節(jié)的機(jī)制。結(jié)果表明：土荊芥揮發(fā)油、α-萜品烯、對傘花素具有顯著的細(xì)胞毒性,隨著處理劑量增加,保衛(wèi)細(xì)胞存活率顯著下降,細(xì)胞核出現(xiàn)了畸形、碎裂和降解等程序性細(xì)胞死亡的典型特征；活性氧(Reactive oxygen species, ROS)、一氧化氮合酶(Nitric oxide synthetase, NOS)和Ca2+的組織化學(xué)定位顯示,在土芥揮發(fā)油、α-萜品烯和對傘花素作用下,保衛(wèi)細(xì)胞內(nèi)ROS、NOS和Ca2+的水平明顯高于對照組；活性氧清除劑(AsA)、Ca2+螯合劑(EGTA)和硝酸還原酶抑制劑(NaN3)均可有效緩解土荊芥揮發(fā)油、α-萜品烯和對傘花素的細(xì)胞毒性,顯著提高了保衛(wèi)細(xì)胞的存活率(P<0.05)。上述結(jié)果表明,ROS、NO和Ca2+參與了土荊芥揮發(fā)油、α-萜品烯和對傘花素誘導(dǎo)蠶豆保衛(wèi)細(xì)胞死亡的信號調(diào)節(jié)過程。土荊芥揮發(fā)油、α-萜品烯和對傘花素誘導(dǎo)的保衛(wèi)細(xì)胞死亡,可能是通過ROS和NO調(diào)控保衛(wèi)細(xì)胞內(nèi)Ca2+水平的變化而引起的。

土荊芥；化感脅迫；保衛(wèi)細(xì)胞；程序性細(xì)胞死亡；信號調(diào)節(jié)

Abstract： Allelopathy is a direct or indirect effect of one plant on another and occurs by releasing allelochemicals into the surrounding environment. When allelopathy inhibits the growth and development of receptor plants, it is considered a biotic stress called “allelochemical stress.” Allelopathy is one of the successful mechanisms of alien plant invasions. Allelochemicals can enhance the production of reactive oxygen species (ROS) and increase membrane lipid peroxidation. Allelochemicals can also show obvious cytotoxicity and genotoxicity on receptor plants by inducing enzyme inactivation, DNA damage, and programmed cell death (PCD).ChenopodiumambrosioidesL. (Mexican tea) is one of the aromatic herbaceous species of theChenopodiumgenus, native to tropical Central and South America. It has recently become a major invasive plant in China, and has strong allelopathic effects on surrounding plants. This plant is threatening the ecological security of China in more than 20 provinces and cities by crowding out and suppressing native species. The guard cells are highly specialized cells, which are sensitive to the change of environment. These specialized cells could receive and integrate a great number of external and internal stimuli to accurately respond to various physiological requirements of the plant. Therefore, stomata are considered an optimal material for studying environmental stress. This study aimed to explore the mechanism of apoptosis and its signal regulation in guard cells induced by volatile oil fromC.ambrosioidesand by α-terpinene and cymene.ViciafabaL. (broad bean), which is widely planted in the areas invaded byC.ambrosioides, was chosen as the receptor plant. Epidermal strips of leaves were incubated in 2-(N-morpholino) ethanesulfonic acid (MES) buffer containing volatile oil, α-terpinene and cymene for 30 minutes in light at 25℃ in an illumination incubator. After treatment, the epidermal strips were stained with acridine orange/ethidium bromide (AO/EB) to detect cell viability, and were also stained with histochemical localization to indicate intracellular reactive oxygen species (ROS), nitric oxide synthase (NOS) and calcium ion (Ca2+) levels. The results showed that volatile oil, α-terpinene, and cymene treatments resulted in a decrease in guard cell viability of broad bean leaves, and these effects were dose-dependent. Apoptosis features including nuclear malformation, karyorrhexis, and degradation were observed in guard cells under the volatile oil, α- terpinene, and cymene treatments. ROS, NOS and Ca2+histochemical localization showed that the coloration of ROS, NOS and Ca2+in guard cells deepened considerably under the treatments of volatile oil, α-terpinene and cymene. Meanwhile, the guard cell survival rates increased when epidermal strips were exposed to volatile oil, α-terpinene and cymene combined with different concentrations of ROS scavenger ascorbic acid (AsA), nitrite reductase inhibitor sodium azide (NaN3), or Ca2+chelator ethylene glycol tetraacetic acid (EGTA). All results suggested that ROS,NO, and Ca2+participate in apoptosis signal pathways induced by volatile oil, α-terpinene, and cymene inV.favastomata guard cells. Changes in Ca2+levels regulated by ROS and NO may mediate programmed cell death induced by volatile oil, α-terpinene, and cymene.

KeyWords:ChenopodiumambrosioidesL.; Allelochemical stress; guard cells; programmed cell death; signal regulation

入侵植物土荊芥(ChenopodiumambrosioidesL.)為藜科藜屬1年生或多年生芳香性草本植物,原產(chǎn)熱帶美洲,現(xiàn)廣泛分布于我國20多個地區(qū),嚴(yán)重破壞了我國生態(tài)系統(tǒng)的結(jié)構(gòu)與功能[1]。土荊芥全株富含揮發(fā)油,當(dāng)這些揮發(fā)油釋放到周圍環(huán)境后,對受體植物具有強(qiáng)烈的化感作用,可誘導(dǎo)受體植物根細(xì)胞內(nèi)活性氧(Reactive oxygen species, ROS)爆發(fā),干擾細(xì)胞有絲分裂和基因表達(dá),導(dǎo)致微核率和畸變率增加,引起氧化損傷和細(xì)胞死亡[2-5],表現(xiàn)出較強(qiáng)的遺傳毒性和細(xì)胞毒性。植物釋放到環(huán)境中的揮發(fā)性化感物質(zhì)除了一小部分通過淋溶[6]、根系分泌[7]或腐解[8]進(jìn)入土壤外,很大一部分會揮發(fā)進(jìn)入大氣作用于植物的地上器官,尤其是葉片。氣孔是存在于植物莖和葉表面由一對保衛(wèi)細(xì)胞圍成的小孔,是大氣中物質(zhì)進(jìn)入植物體的主要門戶,控制著外界環(huán)境和植物內(nèi)部的氣體交換[9]。氣孔保衛(wèi)細(xì)胞能夠接受和整合一系列內(nèi)外刺激并做出準(zhǔn)確的響應(yīng)[10],以此減輕環(huán)境脅迫程度和提高植物的抗性[11],是研究環(huán)境脅迫的良好材料,也是研究單個細(xì)胞內(nèi)信號轉(zhuǎn)導(dǎo)的模式系統(tǒng)[12]。研究表明,氣孔保衛(wèi)細(xì)胞對植物的化感脅迫十分敏感,在馬纓丹(Lantanacamara)[13]和狗牙根(Cynodondactylon)[14]的化感脅迫下,受體植物的氣孔導(dǎo)度顯著下降。本研究室前期研究發(fā)現(xiàn),土荊芥揮發(fā)油及其兩個主要成分α-萜品烯和對傘花素處理導(dǎo)致蠶豆(ViciafabaL.)氣孔保衛(wèi)細(xì)胞發(fā)生Caspase依賴性的細(xì)胞凋亡[15]。信號分子NO、ROS和 Ca2+在脅迫誘導(dǎo)的植物程序性細(xì)胞死亡中起著重要的調(diào)節(jié)作用,如參與了SO2誘導(dǎo)氣孔保衛(wèi)細(xì)胞凋亡的信號轉(zhuǎn)導(dǎo)過程[16-17],但是否參與土荊芥化感物質(zhì)誘導(dǎo)保衛(wèi)細(xì)胞凋亡的信號轉(zhuǎn)導(dǎo)過程卻知之甚少。本研究模擬土荊芥揮發(fā)途徑產(chǎn)生的化感作用,使土荊芥揮發(fā)油及其主要成分α-萜品烯和對傘花素與蠶豆葉片表皮條充分接觸,通過分析葉片保衛(wèi)細(xì)胞活性并運(yùn)用ROS、一氧化氮合酶(Nitric oxide synthetase, NOS)和Ca2+的組織化學(xué)定位法,進(jìn)一步研究了土荊芥揮發(fā)途徑化感脅迫誘導(dǎo)蠶豆保衛(wèi)細(xì)胞死亡相關(guān)的信號調(diào)控機(jī)制,為深入揭示土荊芥化感作用機(jī)制提供理論依據(jù)。

1 材料與方法

1.1 供試材料

供試植物土荊芥采自四川省成都市包江橋。采用水蒸氣蒸餾法[18]提取揮發(fā)油,用無水 Na2SO4除去水分,得到淡黃色具有強(qiáng)烈芳香味的揮發(fā)油,測得密度為843 mg/mL。4 ℃保存?zhèn)溆?。?jīng)四川大學(xué)測試中心分析鑒定,該揮發(fā)油中α-萜品烯和對傘花素的含量分別為151 mg/mL和156 mg/mL；試驗用α-萜品烯和對傘花素的標(biāo)準(zhǔn)品購自成都市銳可思生化試劑公司。

受體植物蠶豆種子(成胡14#)購于成都市五塊石種子市場。

1.2 試驗方法

1.2.1 材料培養(yǎng)和準(zhǔn)備

挑選大小均一、飽滿的蠶豆種子,用0.5%KMnO4浸泡15 min,浸種24 h,避光 25 ℃催芽2—3 d,出芽后播種于盛有石英砂的花盆(直徑10 cm,高度6 cm)中,置于光暗周期 14 h/10 h(光周期25 ℃,暗周期18 ℃)的條件下培養(yǎng)。培養(yǎng)期間盆內(nèi)Hoagland營養(yǎng)液(CaNO3·4H2O 945 mg/mL, KNO3506 mg/mL, NH4NO380 mg/mL, KH2PO4136 mg/mL, MgSO4·7H2O 493 mg/mL,鐵鹽溶液2.5 mL,微量元素液5 mL,pH 6.0)保持在0.2%。待蠶豆生長4—5周時取頂端完全展開的葉片,撕取1 cm×0.5 cm的葉下表皮,置于含有5 mL MES緩沖液[50 mmol/L KCl, 0.1 mmol/L, 0.1 mol/L Tris, 10 mmol/L 2-(N-morpholino) ethanesulfonic acid(MES), pH 7.0]的EP管中。

1.2.2 試驗處理

用二甲基亞砜(DMSO)配制揮發(fā)油、α-萜品烯和對傘花素處理母液。將10 μL揮發(fā)油用DMSO稀釋至100 μL得到0.1 μL/μL的揮發(fā)油處理母液。根據(jù)10 μL揮發(fā)油中α-萜品烯和對傘花素的含量,配制二者的處理母液,其終濃度分別為0.0169 μL/μL和0.0186 μL/μL。

分別取處理母液2,4,6,8,10 μL(分別記為梯度1、2、3、4、5),用DMSO補(bǔ)充至10 μL,分別加入1.2.1的EP管中處理表皮條,其中MES緩沖液和DMSO作為對照；緩解組在1.2.1的EP管中分別加入0.5 mmol/L和2.5 mmol/L的活性氧清除劑抗壞血酸(AsA)、Ca2+螯合劑乙二醇雙四乙酸(EGTA)和硝酸還原酶抑制劑NaN310 μL,10 min后加入梯度4的處理液母液,以MES緩沖液和梯度4處理液為對照。所有處理均置于25 ℃光照30 min。每處理重復(fù)3次。

1.2.3 細(xì)胞活性檢測和細(xì)胞核形態(tài)觀察

細(xì)胞活性檢測采用馬丹煒等[19]的方法略有改進(jìn)。處理結(jié)束后將表皮條用MES緩沖液清洗后,平鋪在載玻片上,滴加少許吖啶橙/溴乙錠(AO/EB)染液,避光染色3 min,用 LEICA DM300熒光顯微鏡觀察并拍照。具有綠色熒光的為活細(xì)胞,具有橘紅色熒光的為死細(xì)胞。每個處理組觀察1000個細(xì)胞,統(tǒng)計保衛(wèi)細(xì)胞的存活率。

細(xì)胞核形態(tài)檢測采用Feulgen染色法[19]。用 Schiff試劑對處理后的表皮條避光染色30 min,光學(xué)顯微鏡下觀察細(xì)胞核形態(tài)并拍照。

1.2.4保衛(wèi)細(xì)胞內(nèi)ROS、NOS和Ca2+組織化學(xué)定位

Ca2+定位參照Li等[21]的方法,用0.01 mmol/L Ca2+熒光指示劑(Fluo- 3/AM)暗孵育60 min。

由于NO半衰期短,對其定位較難,因此本研究在脅迫處理后,通過對NOS染色來推測保衛(wèi)細(xì)胞內(nèi)NO的生成。參照曹冰等[22]的方法略有改進(jìn),用含有0.05%β-NADPH、0.01%NBT、0.3% tritonx- 100的100 mmol/L磷酸緩沖液(pH 7.4)孵育60 min,用 100 mmol/L的磷酸緩沖液清洗。

上述定位試驗均用LEICA DM300熒光顯微鏡觀察拍照。

1.3 數(shù)據(jù)統(tǒng)計與分析

采用SPSS17.0對數(shù)據(jù)進(jìn)行ANOVA方差分析和Duncan法進(jìn)行多重比較,Microsoft Excel 2007作圖。

2 結(jié)果與分析

2.1 土荊芥揮發(fā)油及其兩種主要成分對蠶豆保衛(wèi)細(xì)胞活性的影響

經(jīng)AO/EB染色后,對照組中保衛(wèi)細(xì)胞細(xì)胞核呈均勻的亮綠色熒光(圖1d),土荊芥揮發(fā)油、α-萜品烯和對傘花素3個處理組中保衛(wèi)細(xì)胞細(xì)胞核出現(xiàn)橘紅色熒光(圖1e, f),表明保衛(wèi)細(xì)胞活性喪失。隨著處理劑量的增加,呈橘紅色熒光的細(xì)胞核數(shù)目也隨之增加(圖1a—c),且細(xì)胞核出現(xiàn)明顯的畸變特征,如碎裂(圖2b, f)、畸形(圖2c, g)和降解(圖2d, h)等,由此推測保衛(wèi)細(xì)胞可能發(fā)生了細(xì)胞程序性死亡。

圖1 土荊芥揮發(fā)油、α-萜品烯和對傘花素對蠶豆保衛(wèi)細(xì)胞活性的影響Fig.1 Effects of volatile oil from Chenopodium ambrosioides L., cymene and α-terpinene on guard cell viabilities in Vicia faba L.a: 對照組；b:低劑量藥物處理；c:高劑量藥物處理；d: 對照組； e:氣孔單保衛(wèi)細(xì)胞活性喪失；f: 氣孔雙保衛(wèi)細(xì)胞活性喪失

圖2 土荊芥揮發(fā)油、α-萜品烯和對傘花素對蠶豆保衛(wèi)細(xì)胞核形態(tài)的影響Fig.2 Effects of volatile oil from Chenopodium ambrosioides L., cymene and α-terpinene on guard cell nucleus morphology in Vicia faba L.a: 對照組；b: 核碎裂；c: 核畸形；d: 核降解(a—d AO/EB 染色)e: 對照組；f: 核碎裂； g: 核畸形；h: 核降解(e—h Schiff試劑染色)

從圖3可以看出,DMSO處理組保衛(wèi)細(xì)胞存活率與對照組相比無顯著差異,而土荊芥揮發(fā)油、α-萜品烯和對傘花素處理組中,保衛(wèi)細(xì)胞存活率不同程度的降低。揮發(fā)油處理組中,細(xì)胞存活率隨著揮發(fā)油處理劑量的增加呈顯著下降的趨勢,在最高劑量處理下細(xì)胞存活率為5.0%,僅為對照組的5.5%；α-萜品烯和傘花素處理處理組中,隨處理劑量的增加保衛(wèi)細(xì)胞存活率與揮發(fā)油處理組相似均呈顯著下降的趨勢,在最高劑量處理下,α-萜品烯處理組中細(xì)胞存活率為18.0%,對傘花素處理組中細(xì)胞存活率為34.8%,分別為對照組的19.7%和38.1%。由此可見,土荊芥揮發(fā)油、α-萜品烯和對傘花素對蠶豆保衛(wèi)細(xì)胞產(chǎn)生了明顯的細(xì)胞毒性,且具有劑量依耐性,其中土荊芥揮發(fā)油的毒性最大,α-萜品烯次之,對傘花素的毒性最小。

圖3 土荊芥揮發(fā)油、α-萜品烯和對傘花素對蠶豆保衛(wèi)細(xì)胞活性的影響Fig.3 Effects of volatile oil from Chenopodium ambrosioides L., α-terpinene and cymene on guard cell viabilities in Vicia faba L.

2.2 ROS參與土荊芥揮發(fā)油及其兩種主要成分誘導(dǎo)的蠶豆保衛(wèi)細(xì)胞死亡

DAB溶液染色結(jié)果顯示(圖4),土荊芥揮發(fā)油、α-萜品烯和對傘花素處理后保衛(wèi)細(xì)胞中生成的棕色化合物明顯多于對照組,表明土荊芥揮發(fā)油及其兩種主要成分誘發(fā)了蠶豆保衛(wèi)細(xì)胞中大量生成H2O2。其中,揮發(fā)油處理組中棕色化合物分布最多,α-萜品烯和對傘花素處理組中差異不明顯。

圖4 土荊芥揮發(fā)油、α-萜品烯和對傘花素處理后保衛(wèi)細(xì)胞內(nèi)H2O2的定位Fig.4 Location of H2O2 in guard cell under treatment by volatile oil from Chenopodium ambrosioides L., α-terpinene and cymene

圖5土荊芥揮發(fā)油、α-萜品烯和對傘花素處理后保衛(wèi)細(xì)胞內(nèi)的定位Fig.5 Location of in guard cell under treatment by volatile oil from Chenopodium ambrosioides L., α-terpinene and cymene

圖6 AsA對土荊芥揮發(fā)油、α-萜品烯和對傘花素誘導(dǎo)的蠶豆保衛(wèi)細(xì)胞死亡的緩解效應(yīng)Fig.6 The alleviated effects of AsA on volatile oil from Chenopodium ambrosioides L., α-terpinene and cymene induced guard cell death in Vicia faba L.

2.3Ca2+參與土荊芥揮發(fā)油及其兩種主要成分誘導(dǎo)的蠶豆保衛(wèi)細(xì)胞死亡

Ca2+定位顯示(圖7),對照組中保衛(wèi)細(xì)胞呈現(xiàn)較弱的綠色熒光,揮發(fā)油處理組綠色熒光最強(qiáng),而α-萜品烯和對傘花素處理組的熒光強(qiáng)度介于對照組和揮發(fā)油處理組之間。表明在土荊芥揮發(fā)油、α-萜品烯和對傘花素脅迫下蠶豆保衛(wèi)細(xì)胞內(nèi)Ca2+水平升高。由圖8可以看出,Ca2+螯合劑 EGTA減少了土荊芥揮發(fā)油、α-萜品烯和對傘花素誘導(dǎo)的保衛(wèi)細(xì)胞死亡。其中,0.5 mmol/L的EGTA有效地提高了揮發(fā)油和α-萜品烯脅迫下保衛(wèi)細(xì)胞的存活率(P<0.05),但對傘花素處理組細(xì)胞存活率變化不明顯；2.5 mmol/L的EGTA對3個處理組細(xì)胞死亡均具有抑制效應(yīng)。由此可見,土荊芥揮發(fā)油、α-萜品烯和對傘花素誘導(dǎo)的細(xì)胞死亡與細(xì)胞內(nèi)Ca2+水平的升高有關(guān)。

圖7 土荊芥揮發(fā)油、α-萜品烯和對傘花素處理后保衛(wèi)細(xì)胞內(nèi)Ca2+的定位Fig.7 Location of Ca2+ in guard cell under treatment by volatile oil from Chenopodium ambrosioides L., α-terpinene and cymene

圖8 EGTA對土荊芥揮發(fā)油、α-萜品烯和對傘花素誘導(dǎo)的蠶豆保衛(wèi)細(xì)胞死亡的緩解效應(yīng)Fig.8 The alleviated effects of EGTA on volatile oil from Chenopodium ambrosioides L., α-terpinene and cymene induced guard cell death in Vicia faba L.

2.4 NO參與土荊芥揮發(fā)油及其兩種主要成分誘導(dǎo)的蠶豆保衛(wèi)細(xì)胞死亡

由圖9可以看出,處理組中藍(lán)色顆粒要明顯多于對照組,說明保衛(wèi)細(xì)胞內(nèi)NOS活性升高。因此,土荊芥揮發(fā)油、α-萜品烯和對傘花素可能誘發(fā)了蠶豆保衛(wèi)細(xì)胞中NO的大量生成。其中揮發(fā)油處理組藍(lán)色顆粒最多,α-萜品烯處理組次之,對傘花素處理組最少。

NaN3可減少土荊芥揮發(fā)油、α-萜品烯和對傘花素誘導(dǎo)的保衛(wèi)細(xì)胞死亡(圖10)。低濃度的NaN3有效地緩解了揮發(fā)油和α-萜品烯誘導(dǎo)的細(xì)胞死亡(P<0.05),而對對傘花素誘導(dǎo)細(xì)胞死亡的緩解效果并不顯著；高濃度的NaN3處理顯著提高了3個處理組的細(xì)胞存活率。由此可見,土荊芥揮發(fā)油、α-萜品烯和對傘花素誘導(dǎo)的細(xì)胞死亡與細(xì)胞內(nèi)NO水平的升高有關(guān)。

3 討論

3.1土荊芥揮發(fā)油及其兩種主要成分對蠶豆保衛(wèi)細(xì)胞的毒性效應(yīng)

3.2ROS、NO和Ca2+參與土荊芥揮發(fā)油及其兩種主要成分誘導(dǎo)的保衛(wèi)細(xì)胞死亡

植物體受到各種生物或非生物脅迫時細(xì)胞內(nèi)平衡的自我調(diào)節(jié)就會被擾亂,同時伴隨著ROS的大量生成。當(dāng)細(xì)胞內(nèi) ROS的水平超出機(jī)體所承受的范圍時,細(xì)胞就處于氧化脅迫狀態(tài),并引發(fā)脂質(zhì)過氧化、酶失活、核酸損傷,甚至是激活細(xì)胞的程序性死亡[31-32]。酚酸類化感物質(zhì)肉桂酸和咖啡酸導(dǎo)致萵苣(Lactucasativa)體內(nèi)大量產(chǎn)生和積累ROS,使其根尖細(xì)胞的死亡數(shù)明顯增加[33]。ROS能激活質(zhì)膜上的Ca2+通道,使胞外Ca2+內(nèi)流,引起胞內(nèi)Ca2+水平升高,從而激活了Ca2+依賴性的核酸內(nèi)切酶,切割DNA在核小體的連接點(diǎn),致使細(xì)胞發(fā)生程序性死亡[34-35]。本研究結(jié)果表明,土荊芥揮發(fā)油、α-萜品烯和對傘花素處理組中蠶豆保衛(wèi)細(xì)胞內(nèi)ROS和Ca2+水平明顯升高,同時細(xì)胞存活率顯著降低,當(dāng)加入外源性ROS 清除劑AsA和Ca2+螯合劑 EGTA時,保衛(wèi)細(xì)胞的死亡得到了有效遏制,細(xì)胞存活率顯著上升。這些現(xiàn)象說明,在土荊芥揮發(fā)油、α-萜品烯和對傘花素的脅迫下保衛(wèi)細(xì)胞內(nèi)ROS的爆發(fā)引起了胞內(nèi)Ca2+水平升高,誘發(fā)了蠶豆保衛(wèi)細(xì)胞的死亡。

圖9 土荊芥揮發(fā)油、α-萜品烯和對傘花素處理后保衛(wèi)細(xì)胞內(nèi)NOS的定位Fig.9 Location of NOS in guard cell under treatment by volatile oil from Chenopodium ambrosioides L., α-terpinene and cymene

圖10 NaN3對土荊芥揮發(fā)油、α-萜品烯和對傘花素誘導(dǎo)的蠶豆保衛(wèi)細(xì)胞死亡的緩解效應(yīng)Fig.10 The alleviated effects of NaN3 on volatile oil from Chenopodium ambrosioides L., α-terpinene and cymene induced guard cell death in Vicia faba L.

NO是一個氣體型的信號分子,可修飾ROS信號。當(dāng)細(xì)胞受到外界脅迫時隨著ROS瞬間爆發(fā),NOS活性隨之增強(qiáng)而使NO合成到達(dá)高峰,因此大多情況下ROS和NO幾乎是在細(xì)胞的同一個區(qū)室內(nèi)同時爆發(fā)的[36-37]。NO和ROS及抗氧化劑之間的化學(xué)反應(yīng)會形成大量的活性氮(reactive nitrogen species, RNS),當(dāng)細(xì)胞內(nèi)平衡失控,ROS和RNS信號不受控制的自我放大可能會激起氮化應(yīng)激,最終導(dǎo)致程序性細(xì)胞死亡[35-37]。本研究結(jié)果表明,土荊芥揮發(fā)油、α-萜品烯和對傘花素處理組中蠶豆保衛(wèi)細(xì)胞內(nèi)NOS的活性明顯高于對照組,表明NO的合成量增加,當(dāng)加入外源性的硝酸還原酶抑制劑NaN3時,保衛(wèi)細(xì)胞死亡得到了有效遏制,細(xì)胞存活率顯著上升,表明NO參與了土荊芥揮發(fā)油、α-萜品烯和對傘花素誘導(dǎo)蠶豆保衛(wèi)細(xì)胞的死亡。

植物體通過復(fù)雜的信號調(diào)控網(wǎng)絡(luò)控制著細(xì)胞內(nèi)的各種生理過程,其中ROS和NO通過Ca2+信號系統(tǒng)調(diào)控程序性細(xì)胞死亡是植物體抵御環(huán)境中非生物或生物脅迫的一種基本機(jī)制[37]。由此可見,土荊芥揮發(fā)油、α-萜品烯和對傘花素誘導(dǎo)的保衛(wèi)細(xì)胞死亡,可能是通過ROS和NO調(diào)控保衛(wèi)細(xì)胞內(nèi)Ca2+水平的變化而引起的。

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VolatileallelochemicalsfromChenopodiumambrosioidesL.inducedguardcellsapoptosisanditssignalregulationinViciafabaL.

ZHOU Jian, WANG Ya′nan, MA Danwei*, HUANG Su, XIN Wenyuan, ZHANG Hong

CollegeofLifeScience,SichuanNormalUniversity,Chengdu610101,China

國家自然基金項目(31370549); 四川省應(yīng)用基礎(chǔ)研究重點(diǎn)項目(2017JY0017);四川省教育廳重點(diǎn)項目(16ZA0056); 四川省教育廳一般項目(16ZB0058); 四川省高校重點(diǎn)實(shí)驗室開放項目(SCYZ201410)

2016- 06- 01; < class="emphasis_bold">網(wǎng)絡(luò)出版日期

日期:2017- 04- 24

10.5846/stxb201606011058

*通訊作者Corresponding author.E-mail: danwei10ma@163.com

周健,王亞男,馬丹煒,黃素,辛文媛,張紅.土荊芥揮發(fā)性化感物質(zhì)誘導(dǎo)蠶豆保衛(wèi)細(xì)胞死亡及信號調(diào)節(jié).生態(tài)學(xué)報,2017,37(17):5713- 5721.

Zhou J, Wang Y N, Ma D W, Huang S, Xin W Y, Zhang H.Volatile allelochemicals fromChenopodiumambrosioidesL. induced guard cells apoptosis and its signal regulation inViciafabaL..Acta Ecologica Sinica,2017,37(17):5713- 5721.