任雨敏 孟巧巧 王煜 馬丹煒
摘 要: 為探討大葉桉(Eucalyptus robusta)化感作用的細(xì)胞學(xué)機(jī)制,該研究以大葉桉揮發(fā)油及其主要成分α-蒎烯和桉油精為供體,以蠶豆(Vicia faba )的根細(xì)胞和葉保衛(wèi)細(xì)胞為靶標(biāo),運(yùn)用顯微技術(shù)、細(xì)胞化學(xué)技術(shù)和qRT-PCR技術(shù),研究了大葉桉揮發(fā)物的毒性效應(yīng)。結(jié)果表明:(1)在大葉桉揮發(fā)物作用下,蠶豆幼根生長(zhǎng)受抑制并表現(xiàn)為時(shí)間-濃度依賴效應(yīng),其化感效應(yīng)強(qiáng)弱由大到小依次為揮發(fā)油、α-蒎烯和桉油精。(2)蠶豆根邊緣細(xì)胞活性降低,分生區(qū)細(xì)胞微核率升高,有絲分裂指數(shù)下降且大部分細(xì)胞的細(xì)胞周期被阻滯在分裂前期。(3)蠶豆葉保衛(wèi)細(xì)胞內(nèi)NADPH氧化酶活性升高,活性氧(reactive oxygen species, ROS)爆發(fā),微絲聚合,氣孔開(kāi)度下降;葉保衛(wèi)細(xì)胞的核畸變率升高,細(xì)胞活性降低甚至發(fā)生caspase依賴性細(xì)胞凋亡,而Ca2+通道阻斷劑(LaCl3)、活性氧清除劑(AsA)和硝酸還原酶抑制劑(NaN3)均可顯著提高保衛(wèi)細(xì)胞存活率,說(shuō)明大葉桉揮發(fā)物改變了信號(hào)分子Ca2+、ROS和NO的信號(hào)調(diào)節(jié)。綜上表明,大葉桉揮發(fā)物的細(xì)胞毒性和遺傳毒性改變了受體細(xì)胞的信號(hào)轉(zhuǎn)導(dǎo)途徑,誘發(fā)了細(xì)胞遺傳畸變,導(dǎo)致受體根邊緣細(xì)胞保護(hù)功能障礙和氣孔運(yùn)動(dòng)障礙,從而影響受體根系生長(zhǎng)和光合作用,最終導(dǎo)致受體生長(zhǎng)受阻。該研究結(jié)果為大葉桉種植區(qū)的科學(xué)種植和管理提供了理論依據(jù)。
關(guān)鍵詞: 大葉桉, 揮發(fā)物, 化感作用, 毒性效應(yīng), 信號(hào)調(diào)節(jié)
中圖分類號(hào): Q942? 文獻(xiàn)標(biāo)識(shí)碼: A? 文章編號(hào): 1000-3142(2023)05-0947-13
Abstract: To understand the cytological mechanism of allelopathy of Eucalyptus robusta, the toxic effects of volatiles from E. robusta were studied by microscopic, cytochemical and qRT-PCR techniques, taking volatile oil from E. robusta and its main components α-pinene and eucalyptol as donors, and using root cells and leaf guard cells of Vicia faba as targets. The results were as follows: (1) The growth of radicle of V. faba were exhibited and showed a time-concentration dependent effects under the treatments of the volatiles of Eucalyptus robusta. The allelopathic effects were volatile oil, α-pinene and eucalyptol in descending order. (2) When Vicia faba roots were exposed to Eucalyptus robusta volatiles, the activity of root border cells decreased, the micronucleus rate of cells in the meristematic zone increased, mitotic index decreased, and the cell cycle of most cells was arrested in the prophase of division. (3) Under the action of the volatiles of E. robusta, the activity of NADPH oxidase increased, reactive oxygen species (ROS) burst in leaf guard cells of Vicia faba, microfilament polymerization, and stomatal aperture decreased. At the same time, the leaf epidermis strip of V. faba was treated with Eucalyptus robusta volatiles, the nuclear distortion rate of leaf guard cells increased. Moreover, the treatment of E. robusta volatiles led to the decrease of guard cell activity and caspase-dependent apoptosis in Vicia faba. However, the guard cell survival rates increased when the leaf epidermis strips of V. faba were exposed to volatiles from Eucalyptus robusta combined with different concentrations of Ca2+channel blocker (LaCl3), ROS scavenger ascorbic acid (AsA), and nitrate reductase inhibitors (NaN3), which indicated that the volatiles of E. robusta changed the signal regulation of Ca2+, ROS and NO. These results suggested that the cytotoxicity and genotoxicity of the volatiles of E. robusta altered the signal transduction pathway of the receptor cells, induced the genetic aberration of the root tip cells, then led to the dysfunction of protective function and stomatal movement of the receptor root border cells, which affected the root growth and photosynthesis of the receptor, and ultimately hindered the growth of receptor. The results provide a theoretical basis for scientific planting and management of E. robusta planting area.
Key words: Eucalyptus robusta, volatiles, allelopathy, toxic effects, signal regulation
大葉桉(Eucalyptus robusta)為桃金娘科(Myrtaceae)桉屬(Eucalyptus)喬木植物,原產(chǎn)于澳大利亞,因其適應(yīng)性強(qiáng)、生長(zhǎng)快而被稱為世界三大速生樹(shù)種之一(單體江等,2019)。大葉桉屬于藥源性植物,其葉和果實(shí)都有抗菌(Said et al., 2016)、抗氧化(Elansary et al., 2017)和抗腫瘤(Jian et al., 2017)等藥理活性,具有極其重要的社會(huì)價(jià)值、生態(tài)價(jià)值和經(jīng)濟(jì)價(jià)值。近年來(lái),因種植桉樹(shù)而引起的生物多樣性下降、生態(tài)系統(tǒng)功能和效益喪失、土壤肥力下降等問(wèn)題受到了學(xué)術(shù)界和社會(huì)的高度關(guān)注(Bayle, 2019)。桉葉揮發(fā)油及其主要成分α-蒎烯、桉油精等具有明顯的化感毒性作用,可以通過(guò)誘導(dǎo)氧化損傷抑制受體種子萌發(fā)和幼苗生長(zhǎng) (Jamil et al., 2021;Ridaoui et al., 2022)。本項(xiàng)目組前期研究發(fā)現(xiàn),大葉桉葉揮發(fā)油含α-蒎烯、β-蒎烯、桉油精、檸檬烯、松香芹醇和β-松油醇等化學(xué)成分,其中α-蒎烯和桉油精是含量較高的成分(孟巧巧等,2020)。
氣孔是CO2、水分和其他物質(zhì)進(jìn)出葉片的通道,保衛(wèi)細(xì)胞通過(guò)膨壓變化控制氣孔開(kāi)度,平衡光合作用所需的CO2和水分,從而影響植物的蒸騰和光合作用(陸雯蕓等,2016)。保衛(wèi)細(xì)胞與表皮細(xì)胞形態(tài)差異較大且對(duì)化感脅迫反應(yīng)靈敏,在鵝掌楸(Liriodendron chinense)(劉巧麗等,2021)、土荊芥(Dysphania ambrosioides)(翁可佳等,2018)和馬纓丹(Lantana camara)(Singh et al., 2014)、辣子草(Galinsoga parviflora)(周健等,2016)的化感脅迫下,受體的保衛(wèi)細(xì)胞活性降低,并發(fā)生caspase 依賴性的細(xì)胞凋亡,信號(hào)分子ROS、NO和 Ca2+調(diào)節(jié)了細(xì)胞凋亡過(guò)程(周健等,2017)。此外,植物釋放的化感物質(zhì)還能干擾受體根尖分生區(qū)細(xì)胞DNA復(fù)制、染色體分離以及紡錘體的功能(趙紅梅和王慧陽(yáng),2017;Li et al., 2018);根邊緣細(xì)胞(root border cells, RBCs)是從根冠表皮游離出來(lái)且聚集在根尖周圍的一群特殊細(xì)胞,與其胞外黏膠層在根際共同構(gòu)筑了抵御外界脅迫的第一道防線(Driouich et al., 2021)。光合作用固定的碳部分通過(guò)根系分泌物的形式釋放到土壤中(Vives-Peris et al., 2020),其中 98%的根系分泌物為根邊緣細(xì)胞及其胞外物質(zhì)(Hawes et al., 2016),在根與土壤微生物的相互作用中起主要作用(Ropitaux et al., 2020)。當(dāng)植物受到生物或非生物脅迫時(shí),根邊緣細(xì)胞基因表達(dá)改變,黏膠層厚度進(jìn)一步增大,以吸附和排斥病原體或通過(guò)螯合有毒物質(zhì),以緩解脅迫對(duì)根的傷害,從而保護(hù)根尖分生組織(Driouich et al., 2021;何勝利等,2022)。因此,進(jìn)入土壤的化感物質(zhì)只有突破根邊緣細(xì)胞及其黏膠層構(gòu)筑的防御屏障,才會(huì)傷害到根尖細(xì)胞,危及植物根系的健康生長(zhǎng)。當(dāng)揮發(fā)性化感物質(zhì)釋放到環(huán)境后,大部分揮發(fā)到空氣中作用于植物的地上部分,小部分會(huì)通過(guò)土壤吸附、淋溶、根系分泌等途徑進(jìn)入土壤。保衛(wèi)細(xì)胞構(gòu)成了物質(zhì)進(jìn)出植物體的門戶——?dú)饪祝吘壖?xì)胞則是根系和土壤之間的屏障,二者無(wú)疑是揮發(fā)性化感物質(zhì)作用的首要靶標(biāo)。受體如何響應(yīng)化感物質(zhì)是一個(gè)十分復(fù)雜的問(wèn)題,高等植物機(jī)體由多細(xì)胞構(gòu)成,每一種細(xì)胞的生命活動(dòng)均會(huì)受機(jī)體其他細(xì)胞制約。顯然,以單細(xì)胞模式系統(tǒng)作為研究化感作用的對(duì)象,可以排除其他細(xì)胞生命活動(dòng)的干擾。近年來(lái),學(xué)者們逐漸關(guān)注到根邊緣細(xì)胞(Latif et al., 2017;Ma et al., 2020;Xie et al., 2021)和保衛(wèi)細(xì)胞(周健等,2017)對(duì)化感脅迫的響應(yīng)及其機(jī)制。與以整個(gè)植株作為受試靶標(biāo)相比,保衛(wèi)細(xì)胞和根邊緣細(xì)胞不僅具有反應(yīng)靈敏、取材方便、易于控制試驗(yàn)條件、耗費(fèi)少、周期短等優(yōu)勢(shì)(周健等,2017;Wang et al., 2019),而且能夠直觀地洞察到化感作用這一自然現(xiàn)象的本質(zhì)。然而,保衛(wèi)細(xì)胞和根邊緣細(xì)胞如何響應(yīng)大葉桉揮發(fā)物的化感效應(yīng)卻鮮有報(bào)道。
大葉桉功能多樣化可應(yīng)用于醫(yī)藥(蘭宏偉等,2020)、生態(tài)(田雪晨和陳賢興,2014)、農(nóng)業(yè)(劉紅芳和邸仕忠,2018)等領(lǐng)域,而現(xiàn)有研究偏重于大葉桉揮發(fā)性成分的藥理作用,極少關(guān)注其種植對(duì)生境內(nèi)其他植物的毒害作用。為進(jìn)一步挖掘大葉桉揮發(fā)性成分的化感作用,本研究以其揮發(fā)油及主要成分α-蒎烯和桉油精為供體,以中國(guó)大葉桉種植區(qū)廣泛分布的農(nóng)作物蠶豆(Vicia faba)為受體,以葉保衛(wèi)細(xì)胞、根邊緣細(xì)胞和根尖細(xì)胞為靶標(biāo),運(yùn)用蠶豆根尖微核技術(shù)、光學(xué)顯微鏡技術(shù)、細(xì)胞化學(xué)技術(shù)和qRT-PCR技術(shù),在評(píng)估大葉桉揮發(fā)物化感效應(yīng)及其對(duì)根尖有絲分裂行為影響的基礎(chǔ)上,進(jìn)一步分析蠶豆葉保衛(wèi)細(xì)胞和根邊緣細(xì)胞對(duì)大葉桉揮發(fā)物的響應(yīng)及其信號(hào)機(jī)制,以期從細(xì)胞學(xué)角度揭示大葉桉化感作用機(jī)制,為大葉桉種植區(qū)的科學(xué)種植和管理提供理論依據(jù)。
1 材料與方法
1.1 試驗(yàn)材料
所用材料大葉桉的葉采自四川師范大學(xué)校區(qū)及其附近街道;蠶豆的種子(成胡14#)購(gòu)自成都市龍泉驛區(qū)大面鎮(zhèn)街道種子市場(chǎng);標(biāo)準(zhǔn)品α-蒎烯(≥99%)和桉油精(≥99%)購(gòu)自科賽斯特(成都)科技有限公司。
1.2 試驗(yàn)方法
A組:參照周健等(2017)的方法培養(yǎng)蠶豆幼苗,待幼苗生長(zhǎng)3~4周時(shí),取頂端第1~2對(duì)完全展開(kāi)的葉片,用蒸餾水沖洗干凈,選取非葉脈部位用鑷子撕取1 cm × 0.5 cm的葉下表皮條,浸泡于盛有MES緩沖液的EP管中。
B組:選取大小均勻且飽滿的健康蠶豆種子(趙紅梅和王慧陽(yáng),2017),在0.5% KMnO4溶液中浸泡15 min,沖洗干凈后置于(25±1)℃培養(yǎng)箱中,黑暗條件下浸種24 h后均勻置于墊有濕潤(rùn)紗布的瓷盤中,覆蓋一層浸濕的紗布,繼續(xù)培養(yǎng)至種子露白。選取長(zhǎng)勢(shì)一致的露白種子,均勻置于墊有2層濾紙的培養(yǎng)瓶(直徑6.8 cm、高度9.2 cm)中,每瓶3顆。
處理母液的制備:參照孟巧巧等(2020)的水蒸氣蒸餾法提取大葉桉揮發(fā)油,得率為0.43%,質(zhì)量濃度為836.667 mg·mL-1,采用GC-MS法確定揮發(fā)油主要成分α-蒎烯(≥99%)和桉油精(≥99%)的含量。用二甲基亞砜(DMSO)作助溶劑配制濃度為0.1 μL·μL-1的大葉桉葉揮發(fā)油母液;根據(jù)α-蒎烯和桉油精在揮發(fā)油中的含量,其處理母液濃度分別設(shè)置為0.419 1、0.076 8 μL·μL-1。
1.3 試驗(yàn)處理及指標(biāo)測(cè)定
1.3.1 遺傳毒性試驗(yàn) 分別取處理母液1、2、3、4、5 μL,均勻涂抹在B組培養(yǎng)瓶蓋中部并旋緊瓶蓋,置于(25 ± 1)℃的培養(yǎng)箱中避光培養(yǎng)24、48、72 h,以未涂抹揮發(fā)物的處理作為對(duì)照,每處理重復(fù)5次,處理結(jié)束后測(cè)定參數(shù)。
根長(zhǎng)測(cè)定:將根尖用蒸餾水洗凈,用濾紙吸干水分,用直尺測(cè)量根長(zhǎng),每個(gè)處理作15個(gè)平行測(cè)定。
有絲分裂指數(shù)和微核率測(cè)定:待處理結(jié)束后更換干凈的培養(yǎng)瓶蓋,繼續(xù)恢復(fù)培養(yǎng)24 h后,截取長(zhǎng)約0.5 cm的根尖,用卡諾固定液固定24 h、1 mol·L-1 HCl 60 ℃ 解離8 min、改良苯酚品紅染液染色,壓片,用Nikon E200攝影顯微鏡鏡檢且拍照。每個(gè)根尖計(jì)數(shù)1 000個(gè)細(xì)胞,每個(gè)處理5個(gè)根尖。計(jì)算微核率(micronucleus frequency,MCN)和有絲分裂指數(shù)(mitotic index,MI)。計(jì)算公式如下:
MCN=(微核數(shù)/觀測(cè)細(xì)胞數(shù))×1000‰;
MI=(M期細(xì)胞數(shù)/觀測(cè)細(xì)胞數(shù))×100%。
1.3.2 葉表皮條試驗(yàn) 將A組表皮條隨機(jī)分為3組:第1組,分別取2、4、6、8、10 μL處理母液,用DMSO補(bǔ)足體積為10 μL后,加入裝有表皮條的EP管中,以10 μL MES緩沖液為對(duì)照;第2組,分別加入10 μL泛caspase抑制劑Z-VAD-FMK(10、40 μmol·L-1)、Ca2+通道阻斷劑(0.1 mmol·L-1LaCl3)、活性氧清除劑抗壞血酸(0.1 mmol·L-1AsA)和硝酸還原酶抑制劑(0.1 mmol·L-1NaN3)處理5 min后,加入8 μL處理母液和2 μL DMSO,設(shè)置陰性對(duì)照(CK為MES緩沖液)和陽(yáng)性對(duì)照(處理母液8 μL);第3組,置于25 ℃、4 000 lx的光照培養(yǎng)箱2 h后,用10 μL的微絲聚合抑制劑細(xì)胞松弛素B(CB,10、20 μmol·L-1)、NADPH氧化酶抑制劑二聯(lián)苯碘(DPI,1、2 μmol·L-1)和活性氧清除劑抗壞血酸(0.1 mmol·L-1)分別處理5 min后,加入8 μL處理母液和2 μL DMSO。以不加CB、DPI和AsA的MES緩沖液為CK陰性對(duì)照,以8 μL處理液?jiǎn)为?dú)處理為陽(yáng)性對(duì)照。3組處理混勻后均置于25 ℃、4 000 lx光照培養(yǎng)箱處理30 min,待處理結(jié)束后測(cè)定氣孔開(kāi)度、細(xì)胞核形態(tài)、保衛(wèi)細(xì)胞活性、胞內(nèi)ROS、NO和Ca2+水平以及TUNEL(黃素等,2019)等指標(biāo)。
qRT-PCR分析:取第1組葉表皮條,用液氮充分研磨,按照武漢塞維爾生物科技有限公司的植物總RNA提取試劑盒操作說(shuō)明提取蠶豆葉表皮條的總RNA;用Primer Premier 5.0引物設(shè)計(jì)軟件,設(shè)計(jì)蠶豆NADPH氧化酶基因(Rboh)和內(nèi)參基因(EF-1-alpha)特異性引物,序列如下:
Rboh F:5′-GGGTATTTGCTCTGTGGATTGG-3′;
Rboh R:5′-CCTGAGCCAAGTAATGGTGTTTC-3′;
EF-1-alpha F:5′-ACGAGGCTCTCACTGAGGCTCTTCC-3′;
EF-1-alpha R:5′-CCTTGGCAGGGTCATCCTTGGAGTTG-3′。
引物均由武漢塞維爾生物科技有限公司合成。使用熒光定量PCR儀(Stepone plus,ABI)進(jìn)行qRT-PCR分析。反應(yīng)體系為25 μL,含qPCR Mix 12.5 μL、7.5 μmol·L-1基因引物2.0 μL、反轉(zhuǎn)錄產(chǎn)物2.5 μL、ddH2O 8.0 μL。PCR擴(kuò)增程序:95 ℃ 10 min,95 ℃ 15 s,60 ℃ 60 s,40個(gè)循環(huán);每處理重復(fù)3次,以MES緩沖液為CK對(duì)照;使用StepOne Software v2.3軟件分析PCR過(guò)程的CT(threshold cycle)值。
1.3.3 根邊緣細(xì)胞活性測(cè)定 將B組根培養(yǎng)至長(zhǎng)約2 cm時(shí),隨機(jī)截取 5個(gè)根尖,置于EP管中,向其中加入100 μL ddH2O,渦旋振蕩30 s,取出根尖用ddH2O沖洗2次,每次50 μL,移液槍吹打使細(xì)胞分散,得到根邊緣細(xì)胞懸液;分別取處理母液1、2、3、4、5 μL,用DMSO補(bǔ)足體積為5 μL后,分別加入200 μL細(xì)胞懸液,以DMSO為溶劑對(duì)照組、ddH2O為陰性對(duì)照組,置于(25±1)℃的培養(yǎng)箱中避光培養(yǎng)30 min,每處理重復(fù)3次;處理結(jié)束后,分別取10 μL細(xì)胞懸液,加入4 μL AO/EB染料,暗處染色2~3 s,熒光顯微鏡鏡檢,統(tǒng)計(jì)死細(xì)胞和活細(xì)胞的數(shù)量,并計(jì)算根邊緣細(xì)胞死亡率。計(jì)算公式如下:
死亡率=(死細(xì)胞/總細(xì)胞計(jì)數(shù))×100%。
1.4 數(shù)據(jù)統(tǒng)計(jì)分析
使用Microsoft Excel 2019軟件進(jìn)行數(shù)據(jù)統(tǒng)計(jì)和作圖,用SPSS 20.0軟件對(duì)數(shù)據(jù)進(jìn)行ANOVA單因素方差分析和Tukey法進(jìn)行多重比較分析。
2 結(jié)果與分析
2.1 大葉桉揮發(fā)物的化感效應(yīng)及其遺傳毒性
2.1.1 大葉桉揮發(fā)物對(duì)蠶豆的化感效應(yīng) 由圖1可知,大葉桉揮發(fā)油、α-蒎烯和桉油精對(duì)蠶豆幼根伸長(zhǎng)具有顯著抑制效應(yīng)(P<0.05),并表現(xiàn)為時(shí)間-濃度依賴效應(yīng)。用最大濃度處理72 h后,大葉桉揮發(fā)油、α-蒎烯和桉油精處理組的蠶豆根長(zhǎng)比對(duì)照組分別減少了80.62%、76.74%和75.19%。
2.1.2 大葉桉揮發(fā)物的遺傳毒性 在大葉桉揮發(fā)油、α-蒎烯和桉油精的作用下,蠶豆根尖分生區(qū)細(xì)胞的有絲分裂指數(shù)下降(圖2)。在1 μL處理母液作用下,有絲分裂指數(shù)升高,其中24 h處理組的變化最為顯著。當(dāng)處理母液大于1 μL時(shí),與對(duì)照組相比,隨著處理母液濃度增加和處理時(shí)間延長(zhǎng),有絲分裂指數(shù)顯著下降(P<0.05)。在各處理組M期(有絲分裂期)的各時(shí)相(前、中、后、末)細(xì)胞數(shù)目中大部分細(xì)胞的細(xì)胞周期被阻滯在分裂前期;當(dāng)揮發(fā)油處理母液高于4 μL時(shí),有絲分裂指數(shù)趨于平穩(wěn),表明該濃度下蠶豆根尖已嚴(yán)重受損。
各處理組的微核率均顯著高于對(duì)照組(P<0.05),呈先升高后降低的趨勢(shì)(圖3),在處理母液為3 μL時(shí)微核率達(dá)到最大值,以48 h處理組的變化最顯著,其揮發(fā)油、α-蒎烯和桉油精微核率分別為12.24%、10.89%和7.48%。大葉桉揮發(fā)物誘導(dǎo)蠶豆根尖發(fā)生染色體畸變,如染色體斷片、粘連和出現(xiàn)染色橋、微核等現(xiàn)象(圖4)。
2.2 大葉桉揮發(fā)物對(duì)蠶豆葉保衛(wèi)細(xì)胞活性的影響
由圖5可知,在大葉桉揮發(fā)油、α-蒎烯和桉油精作用下,蠶豆葉保衛(wèi)細(xì)胞活性降低,細(xì)胞核畸變率升高,并表現(xiàn)為濃度依賴效應(yīng)。其化感效應(yīng)強(qiáng)弱由大到小依次為揮發(fā)油、α-蒎烯和桉油精。在最大處理濃度下,揮發(fā)油、α-蒎烯、桉油精3個(gè)處理組的保衛(wèi)細(xì)胞活性為對(duì)照組的7.01%、29.48%、59.56%,細(xì)胞核形態(tài)異常率分別為96.96%、68.78%、24.74%。
半胱氨酸蛋白酶(caspase)是一組與細(xì)胞凋亡密切相關(guān)的蛋白水解酶,當(dāng)用泛caspase抑制劑Z-VAD-FMK與大葉桉揮發(fā)物共處理后,各處理組保衛(wèi)細(xì)胞活性均較揮發(fā)物處理組升高且與Z-VAD-FMK濃度呈正相關(guān)(圖6:B),表明大葉桉揮發(fā)物處理導(dǎo)致保衛(wèi)細(xì)胞發(fā)生凋亡。TUNEL法檢測(cè)結(jié)果發(fā)現(xiàn),揮發(fā)物處理組葉保衛(wèi)細(xì)胞的綠色熒光強(qiáng)度大于caspase抑制劑+揮發(fā)物共處理組(圖6:A),表明大葉桉揮發(fā)物誘導(dǎo)蠶豆葉保衛(wèi)細(xì)胞發(fā)生了caspase依賴性細(xì)胞凋亡。
應(yīng)用ROS、NO和Ca2+熒光定位試驗(yàn)和三者抑制劑+揮發(fā)物共處理試驗(yàn),驗(yàn)證了大葉桉揮發(fā)物作用下蠶豆葉保衛(wèi)細(xì)胞的ROS、NO和Ca2+水平的變化。由圖7可知,對(duì)照組保衛(wèi)細(xì)胞具有較弱的綠色熒光,即胞內(nèi)ROS、NO和Ca2+水平均較低;處理組保衛(wèi)細(xì)胞內(nèi)3種熒光信號(hào)均明顯高于對(duì)照組,說(shuō)明大葉桉揮發(fā)物誘導(dǎo)保衛(wèi)細(xì)胞內(nèi)ROS、NO和Ca2+水平升高。NaN3+處理液、AsA+處理液2個(gè)共處理組中,ROS、Ca2+和NO水平低于揮發(fā)物處理組,而LaCl3+處理液共處理組保衛(wèi)細(xì)胞內(nèi)的Ca2+水平低于揮發(fā)物處理組(圖8),說(shuō)明在大葉桉揮發(fā)物誘導(dǎo)的細(xì)胞死亡過(guò)程中,ROS和NO能調(diào)節(jié)胞內(nèi)Ca2+水平。
由圖9可知,與揮發(fā)物處理組相比,用Ca2+通道阻斷劑(LaCl3)、活性氧清除劑抗壞血酸(AsA)和硝酸還原酶抑制劑(NaN3)分別與處理母液共同作用時(shí),保衛(wèi)細(xì)胞存活率顯著升高(P<0.05)。這進(jìn)一步證明大葉桉揮發(fā)油、α-蒎烯和桉油精誘導(dǎo)的細(xì)胞死亡與細(xì)胞內(nèi)ROS、NO和Ca2+有關(guān)。
2.3 大葉桉揮發(fā)物對(duì)蠶豆氣孔運(yùn)動(dòng)的影響
大葉桉揮發(fā)物導(dǎo)致蠶豆葉氣孔開(kāi)度呈濃度依賴性逐漸下降(圖10),在最大濃度處理下,揮發(fā)油、α-蒎烯和桉油精3個(gè)處理組的氣孔開(kāi)度分別較對(duì)照組下降了86.65%、78.31%和75.38%;當(dāng)NADPH氧化酶抑制劑二聯(lián)苯碘(DPI)、活性氧清除劑抗壞血酸(AsA)和微絲聚合抑制劑細(xì)胞松弛素B(CB)分別與大葉桉揮發(fā)物共處理時(shí),保衛(wèi)細(xì)胞氣孔關(guān)閉現(xiàn)象顯著降低(P<0.05)(圖11),表明大葉桉揮發(fā)物誘導(dǎo)的ROS水平和NADPH氧化酶活性的升高以及微絲聚合是導(dǎo)致氣孔關(guān)閉的原因。qRT-PCR結(jié)果(圖12)顯示,在大葉桉揮發(fā)物作用下,蠶豆葉片內(nèi)NADPH氧化酶基因Rboh表達(dá)顯著上調(diào)。
2.4 大葉桉揮發(fā)物對(duì)根邊緣細(xì)胞活性的影響
由圖13可知,蠶豆根邊緣細(xì)胞活性隨大葉桉揮發(fā)物處理濃度升高而降低,死亡率顯著增加(P<0.05)。與陰性對(duì)照組(0 μL)相比,溶劑對(duì)照組(DMSO)的根邊緣細(xì)胞活性差異不顯著(P>0.05),表明助溶劑DMSO對(duì)根邊緣細(xì)胞活性沒(méi)有顯著影響。當(dāng)處理濃度達(dá)到5 μL時(shí),揮發(fā)油、α-蒎烯、桉油精處理組根邊緣細(xì)胞死亡率分別比對(duì)照組(0 μL)增加了5.65、4.13、2.12倍。
3 討論
植物釋放到環(huán)境中的化感物質(zhì)會(huì)影響其他植物的生長(zhǎng)和生態(tài)功能(Oln & Koce, 2021),如降低植物吸收水分、無(wú)機(jī)鹽、有機(jī)質(zhì)等養(yǎng)分的能力,降低對(duì)資源的利用率,從而影響該植物的生長(zhǎng)發(fā)育(馬光宗等,2022)。桉葉中含有對(duì)植物有化感作用的揮發(fā)性成分,如α-松果醇,可以抑制種子萌發(fā)和植物生長(zhǎng)(Li et al., 2020)。本研究結(jié)果表明,在大葉桉揮發(fā)物作用下,蠶豆幼根生長(zhǎng)受抑制且表現(xiàn)為時(shí)間-濃度依賴效應(yīng),其中大葉桉葉揮發(fā)油的化感效應(yīng)最強(qiáng),α-蒎烯次之,桉油精最弱。植物根的生長(zhǎng)與根尖細(xì)胞的分裂和伸長(zhǎng)密切相關(guān),當(dāng)蠶豆幼根受到大葉桉揮發(fā)物作用時(shí)分生區(qū)細(xì)胞有絲分裂指數(shù)下降,表現(xiàn)出與根長(zhǎng)相似的變化規(guī)律,表明大葉桉揮發(fā)性化感物質(zhì)作為非生物脅迫對(duì)蠶豆根的抑制效應(yīng)與其干擾根尖細(xì)胞有絲分裂行為有關(guān)。處理濃度較低時(shí),絕大多數(shù)根尖細(xì)胞有絲分裂被阻滯在前期,但隨著處理濃度增大前期細(xì)胞比例逐漸減少,推測(cè)大葉桉揮發(fā)性化感物質(zhì)可能抑制了細(xì)胞DNA的復(fù)制和蛋白質(zhì)的合成,阻礙了細(xì)胞周期的進(jìn)程,使細(xì)胞不能進(jìn)入下一次的分裂期,從而導(dǎo)致分生區(qū)有絲分裂指數(shù)下降,根生長(zhǎng)受到抑制(Vasconcelos et al., 2019)。同時(shí),蠶豆根尖細(xì)胞內(nèi)微核率上升并出現(xiàn)染色體斷片、紡錘體多極分裂、染色體粘連等多種染色體畸變現(xiàn)象,表明大葉桉樹(shù)揮發(fā)油、α-蒎烯和桉油精抑制了蠶豆根尖細(xì)胞紡錘體的形成并干擾了某些染色體運(yùn)動(dòng)而導(dǎo)致染色體斷裂和損傷,斷裂的染色體在機(jī)體免疫保護(hù)作用下形成染色體橋,無(wú)著絲粒的形成會(huì)引起微核的出現(xiàn),造成不可逆的遺傳毒性效應(yīng)。本研究結(jié)果與Aragao等(2015)研究發(fā)現(xiàn)大葉桉揮發(fā)油及其單萜烴類能夠?qū)е氯n苣(Lactuca sativa)根尖分生區(qū)細(xì)胞死亡,染色體變異這一結(jié)果相似。根邊緣細(xì)胞及其胞外黏膠層是植物根和土壤之間的保護(hù)屏障(Ropitaux et al., 2020;Driouich et al., 2021),在抵抗生物和非生物脅迫中對(duì)根尖起到一定的保護(hù)作用。何勝利等(2022)研究發(fā)現(xiàn),土荊芥揮發(fā)油及其主要化感物質(zhì)下根邊緣細(xì)胞活性下降,黏膠層厚度增加,在一定程度上抵御了揮發(fā)物化感脅迫對(duì)根尖的傷害。在大葉桉揮發(fā)物作用下,蠶豆根邊緣細(xì)胞活性呈濃度依賴性下降,由此降低且削弱其保護(hù)功能,進(jìn)而干擾根尖細(xì)胞的有絲分裂,抑制根的伸長(zhǎng)生長(zhǎng)。
氣孔是植物氣體交換、水分蒸騰和病菌入侵的主要門戶(Qi et al., 2018;Ma & Bai, 2021),氣孔開(kāi)度的調(diào)節(jié)通過(guò)保衛(wèi)細(xì)胞的運(yùn)動(dòng)來(lái)實(shí)現(xiàn)。細(xì)胞骨架、Ca2+、ROS和NO等因子通過(guò)不同的方式、信號(hào)途徑調(diào)控氣孔運(yùn)動(dòng), 使氣孔能靈活地響應(yīng)各種環(huán)境(胡子英等,2017)。ROS在植物感知脅迫、整合各種環(huán)境信號(hào)、激活脅迫響應(yīng)網(wǎng)絡(luò)中起著關(guān)鍵作用(Mittler et al., 2022),細(xì)胞內(nèi)ROS的產(chǎn)生與NADPH氧化酶和微絲動(dòng)態(tài)調(diào)控密切相關(guān)(Agurla et al., 2020)。微絲動(dòng)態(tài)的變化會(huì)引起胞內(nèi)NADPH氧化酶發(fā)生改變(Stanley et al., 2014)。本研究結(jié)果表明,在大葉桉揮發(fā)油及其主要成分作用下,蠶豆葉保衛(wèi)細(xì)胞內(nèi)NADPH氧化酶基因Rboh表達(dá)上調(diào),酶活性增加,ROS爆發(fā),保衛(wèi)細(xì)胞核畸變率升高,保衛(wèi)細(xì)胞發(fā)生caspase依賴性凋亡,氣孔開(kāi)度下降,上述現(xiàn)象具有濃度依賴效應(yīng)。這與黃素等(2019)對(duì)土荊芥揮發(fā)油作用于玉米保衛(wèi)細(xì)胞的研究結(jié)果相似。當(dāng)用微絲聚合抑制劑細(xì)胞松弛素B(CB)處理后,大葉桉揮發(fā)油及其主要成分誘導(dǎo)的氣孔關(guān)閉現(xiàn)象明顯阻滯,表明微絲可能通過(guò)調(diào)節(jié)NADPH氧化酶在保衛(wèi)細(xì)胞質(zhì)膜不同部位的分布,控制ROS在細(xì)胞壁上的產(chǎn)生部位,從而精確調(diào)控不同位置細(xì)胞壁的松弛而參與氣孔運(yùn)動(dòng)(胡子英等,2017)。當(dāng)植物受到逆境脅迫時(shí),通過(guò)NO、ROS、Ca2+等信號(hào)分子相互協(xié)調(diào),調(diào)控不同的信號(hào)途徑使保衛(wèi)細(xì)胞的膨壓變化來(lái)響應(yīng)所受的逆境脅迫(Fichman & Mittler, 2020)。在逆境脅迫下,ROS和NO之間的平衡對(duì)調(diào)控細(xì)胞凋亡至關(guān)重要(Petrov et al., 2015)。NO能夠作為一種抗氧化劑直接淬滅ROS,減少膜脂過(guò)氧化,而高濃度的NO可以導(dǎo)致ROS爆發(fā),引起細(xì)胞產(chǎn)生氧化損傷而凋亡(Hasanuzzaman et al., 2018;何勝利等,2022)。Ca2+通道能夠被ROS激活,引起胞外Ca2+內(nèi)流,胞內(nèi)Ca2+水平升高,從而導(dǎo)致DNA 在核小體的連接點(diǎn)被激活了的Ca2+依賴性核酸內(nèi)切酶切割,繼而引發(fā)細(xì)胞凋亡(Pei et al., 2000)。本研究結(jié)果發(fā)現(xiàn),在大葉桉揮發(fā)油及其主要成分α-蒎烯和桉油精誘導(dǎo)的保衛(wèi)細(xì)胞死亡過(guò)程中,保衛(wèi)細(xì)胞內(nèi)ROS、NO和Ca2+水平明顯升高,加入AsA、NaN3和LaCl3時(shí),保衛(wèi)細(xì)胞存活率顯著上升,表明在大葉桉揮發(fā)油、α-蒎烯和桉油精處理下保衛(wèi)細(xì)胞內(nèi)ROS的爆發(fā)引起胞內(nèi)Ca2+水平升高,誘發(fā)了蠶豆保衛(wèi)細(xì)胞的死亡。此外,NO 參與了大葉桉揮發(fā)油、α-蒎烯和桉油精誘導(dǎo)蠶豆保衛(wèi)細(xì)胞的死亡。ROS、NO和Ca2+信號(hào)系統(tǒng)調(diào)控程序性細(xì)胞死亡是植物抵御逆境脅迫的一種基本機(jī)制(周健等,2017)。以上結(jié)果表明,大葉桉揮發(fā)油、α-蒎烯和桉油精誘導(dǎo)的保衛(wèi)細(xì)胞死亡,可能是通過(guò)ROS 和 NO 調(diào)控保衛(wèi)細(xì)胞內(nèi) Ca2+水平的變化而引起的。
4 結(jié)論
綜上所述,大葉桉揮發(fā)性化感物質(zhì)具有細(xì)胞毒性和遺傳毒性。一方面,通過(guò)降低蠶豆根邊緣細(xì)胞活性削弱受體保護(hù)屏障,干擾分生區(qū)細(xì)胞有絲分裂行為并引起遺傳畸變,從而抑制根的生長(zhǎng);另一方面,誘導(dǎo)蠶豆葉保衛(wèi)細(xì)胞ROS爆發(fā),干擾細(xì)胞骨架功能而改變氣孔運(yùn)動(dòng),引起保衛(wèi)細(xì)胞核畸變并發(fā)生caspase依賴性凋亡,在此過(guò)程中,胞內(nèi)ROS和NO的水平上調(diào)引起胞內(nèi)Ca2+水平增加。
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(責(zé)任編輯 蔣巧媛)