H2S信號(hào)在擬南芥響應(yīng)SO2脅迫過程中的作用

李利紅,郭宇茹,侯俊鑫,吳麗華

H2S信號(hào)在擬南芥響應(yīng)SO2脅迫過程中的作用

李利紅1*,郭宇茹1,侯俊鑫1,吳麗華2

(1.晉中學(xué)院化學(xué)化工系,山西 榆次 030619；2.太原師范學(xué)院,山西 榆次 030619)

以擬南芥為實(shí)驗(yàn)材料,研究植物對(duì)H2S和SO2處理的轉(zhuǎn)錄響應(yīng)及其關(guān)系,并利用外源噴施H2S及其清除劑的方法,檢測(cè)SO2熏氣后植株體內(nèi)H2S的產(chǎn)生及其生理效應(yīng),探討氣體信號(hào)分子H2S在植物響應(yīng)SO2脅迫過程中的作用.高通量測(cè)序結(jié)果發(fā)現(xiàn),H2S和SO2處理誘導(dǎo)擬南芥植株多個(gè)基因轉(zhuǎn)錄水平改變,并有1220個(gè)基因在兩種處理?xiàng)l件下均差異表達(dá),其中包括多個(gè)硫代謝和谷胱甘肽代謝相關(guān)基因,表明H2S和SO2在調(diào)控硫代謝途徑中具有交互作用.SO2熏氣誘導(dǎo)擬南芥植株體內(nèi)H2S合成酶基因和轉(zhuǎn)錄水平提高,胞內(nèi)H2S含量增多.同時(shí),超氧化物歧化酶(SOD)和過氧化氫酶(CAT)活性升高,含硫抗氧化物谷胱甘肽(GSH)含量及其相關(guān)酶谷胱甘肽硫轉(zhuǎn)移酶(GST)和谷胱甘肽過氧化物酶(GPX)活性提高,活性氧H2O2含量增加,膜脂過氧化產(chǎn)物丙二醛(MDA)含量與對(duì)照相比無顯著差異.外源噴施H2S可進(jìn)一步提高SO2熏氣下擬南芥植株GSH含量及其相關(guān)防御酶的活性, H2O2含量降低.但噴施亞?；撬?HT)清除H2S后,SO2熏氣擬南芥植株GSH含量、抗氧化酶SOD、CAT和GST活性降低,MDA含量大幅增加.結(jié)果表明,SO2熏氣誘導(dǎo)產(chǎn)生的H2S可作為信號(hào)分子,提高機(jī)體抗氧化防御能力,增強(qiáng)植物對(duì)SO2脅迫的抗性.

二氧化硫；硫化氫；擬南芥；含硫化合物；抗氧化酶

H2S是繼一氧化氮、一氧化碳之后發(fā)現(xiàn)的第三種氣體信號(hào)分子.在植物體中,產(chǎn)生H2S的途徑有:直接通過葉片吸收大氣中的H2S;通過半胱氨酸脫巰基酶(CDes)催化L/D-Cys降解生成H2S,這是植物體內(nèi)源H2S形成的主要途徑;在亞硫酸鹽還原酶(SiR)的作用下, SO32–發(fā)生還原反應(yīng)生成H2S[1-2].大量研究表明, H2S參與調(diào)節(jié)植物生長發(fā)育,如促進(jìn)種子萌發(fā)、根形態(tài)建成,調(diào)節(jié)氣孔運(yùn)動(dòng),增強(qiáng)葉片的光合作用,延緩植物衰老等[3-5].此外, H2S信號(hào)還可誘導(dǎo)脅迫相關(guān)基因表達(dá)、激活體內(nèi)的抗氧化系統(tǒng)、減小氣孔孔徑,從而幫助植物抵抗干旱、鹽、溫度、重金屬等環(huán)境脅迫[6-9].

二氧化硫(SO2)是一種常見的全球性大氣污染物.高濃度SO2會(huì)引起植物葉片褪綠或壞死,抑制光合作用, 影響植株的生長發(fā)育,還可誘發(fā)細(xì)胞核固縮、DNA斷裂、染色體畸變,甚至導(dǎo)致細(xì)胞死亡[10-12].植物受到逆境脅迫時(shí),會(huì)通過調(diào)節(jié)自身生理狀態(tài)積極適應(yīng)周圍環(huán)境.研究表明, SO2熏氣激活擬南芥胞內(nèi)活性氧、植物激素等信號(hào)轉(zhuǎn)導(dǎo)途徑,誘導(dǎo)氣孔關(guān)閉、防御基因轉(zhuǎn)錄上調(diào)和抗氧化酶活性增加[13-15],還可使植物產(chǎn)生交叉抗性,提高植物對(duì)其他環(huán)境刺激的耐受性[16-18].

大氣中的SO2主要通過氣孔進(jìn)入植物體內(nèi),溶于細(xì)胞液形成HSO3-和SO32-.對(duì)植物有毒害作用的SO32-可被亞硫酸氧化酶(SO)氧化為SO42-,同時(shí)產(chǎn)生大量的活性氧[19-20],也可進(jìn)入硫同化途徑,被亞硫酸鹽還原酶(SiR)催化生成硫化物(S2-),S2-在O-乙酰絲氨酸裂解酶(OAS-TL)作用下合成半胱氨酸(Cys),半胱氨酸可作為前體進(jìn)一步合成GSH,在此過程中H2S作為副代謝產(chǎn)物釋放[21-23].早在1982年, H?llgren等[24]研究發(fā)現(xiàn)SO2熏氣后松樹針葉中釋放出H2S.近年來,有研究表明SO2熏氣誘導(dǎo)植物體內(nèi)生成的H2S可作為信號(hào)分子,介導(dǎo)植物對(duì)其他環(huán)境脅迫的抗氧化響應(yīng)[25-26].此外, SO2與H2S緩解植物衰老的作用模式相似,并且SO2和H2S之間的關(guān)系是通過硫代謝途徑建立起來的[27].這表明H2S和SO2對(duì)植物的影響存在一定的聯(lián)系,提示信號(hào)分子H2S可能參與調(diào)控植物對(duì)SO2脅迫的響應(yīng)過程.

本文以模式植物擬南芥為材料,利用高通量測(cè)序研究SO2和H2S處理后植株全基因組表達(dá)變化,并利用qRT-PCR檢測(cè)H2S合成和硫代謝相關(guān)基因的轉(zhuǎn)錄水平,探討植物對(duì)SO2和H2S處理的轉(zhuǎn)錄響應(yīng)及其關(guān)系.同時(shí),系統(tǒng)研究SO2熏氣過程中植物體內(nèi)H2S的產(chǎn)生機(jī)制及其生理效應(yīng),揭示信號(hào)分子H2S在擬南芥響應(yīng)SO2脅迫過程中的重要作用,以期為提高植物對(duì)環(huán)境脅迫的耐受性提供新思路.

1 材料與方法

1.1 植株培養(yǎng)和脅迫處理

擬南芥(L.)Columbia生態(tài)型(Col-0).4℃春化2d后播種于營養(yǎng)土中,培養(yǎng)溫度(22±1)℃,光/暗周期為16h/8h,光照強(qiáng)度140μmol/ m2/s,相對(duì)濕度約70%.

取4周齡生長一致的擬南芥植株,置于體積0.422m3的密閉箱中,適應(yīng)1d后進(jìn)行SO2熏氣和H2S處理,溫度和光照同上.前期預(yù)實(shí)驗(yàn)結(jié)果表明,0.1mmol/L NaHS處理和30mg/m3SO2熏氣誘發(fā)擬南芥植株的抗氧化防御應(yīng)答,且葉片無肉眼可見損傷.因此,本研究中選用0.1mmol/L NaHS和30mg/m3SO2,共設(shè)5個(gè)處理:

1) 對(duì)照組.

2) H2S處理: 0.1mmol/L NaHS.早、中、晚分別用NaHS處理液噴施擬南芥葉片,共處理3d.

3) SO2熏氣: 30mg/m3SO2.根據(jù)K2S2O5+2HCl→ 2KCl+H2O+2SO2的原理,定量產(chǎn)生SO2氣體,并采用甲醛吸收-副玫瑰苯胺分光光度法測(cè)定SO2濃度.前3d每天熏氣12h (08:00～20:00),第4d熏氣4h (8:00～ 12:00),共熏氣40h.

4) H2S+SO2處理: 0.1mmol/L NaHS + 30mg/ m3SO2.每晚熏氣結(jié)束后,在擬南芥葉片噴施NaHS處理液,共處理3d.

5) HT+SO2處理: 0.5mmol/L HT + 30mg/m3SO2.每晚熏氣結(jié)束后,在擬南芥葉片噴施HT處理液,共處理3d.

每處理3次重復(fù),每重復(fù)20株擬南芥.取各處理組擬南芥植株地上部分用于高通量測(cè)序、qRT-PCR和生理指標(biāo)的分析.

1.2 轉(zhuǎn)錄組高通量測(cè)序

取對(duì)照組、H2S處理組和SO2熏氣組擬南芥植株地上組織,用Trizol試劑提取總RNA,將mRNA Capture Beads置于四維旋轉(zhuǎn)儀上充分混勻,平衡30min后使用.高通量測(cè)序由北京百邁克生物科技有限公司完成.

在處理組與對(duì)照組差異表達(dá)基因檢測(cè)過程中,差異倍數(shù)(FoldChange, FC)表示兩樣品組間表達(dá)量的比值,錯(cuò)誤發(fā)現(xiàn)率(False Discovery Rate, FDR)是通過對(duì)差異顯著性P值(-value)進(jìn)行校正得到的.以處理組與對(duì)照組信號(hào)比值的log2FC值表示處理后轉(zhuǎn)錄水平的改變,并用log2FC>1且FDR<0.01作為篩選差異表達(dá)基因的標(biāo)準(zhǔn).

1.3 qRT-PCR檢測(cè)

利用qRT-PCR技術(shù)檢測(cè)H2S合成酶基因(、、)、氧乙酰絲氨酸(硫醇)裂解酶基因(、、)、亞硫酸氧化酶基因()、亞硫酸還原酶()基因表達(dá)水平.用Trizol試劑盒提取總RNA, PrimeScriptTMRT Master Mix (Takara)試劑盒合成cDNA.按照SYBR Premix Ex TaqTMII (Takara)的說明在ABI 7500Real- Time PCRSystem上進(jìn)行相關(guān)基因的qRT-PCR分析,以基因作為內(nèi)參基因,用2-??CT方法計(jì)算相對(duì)表達(dá)量.引物序列使用見表1.

表1 qRT-PCR引物序列

1.4 生理指標(biāo)檢測(cè)

H2S、谷胱甘肽(GSH)、過氧化氫(H2O2)、丙二醛(MDA)、過氧化氫酶(CAT)、超氧化物歧化酶(SOD)、谷胱甘肽硫轉(zhuǎn)移酶(GST)和谷胱甘肽過氧化物酶(GPX)測(cè)定利用南京建成生物工程研究所試劑盒.

1.5 數(shù)據(jù)分析

用SPSS 20.0進(jìn)行統(tǒng)計(jì)分析, Origin lab 2019繪圖,運(yùn)用Duncan法比較樣本間的差異顯著性(<0.05).每個(gè)實(shí)驗(yàn)獨(dú)立重復(fù)3次,數(shù)據(jù)均以3次重復(fù)的平均值和標(biāo)準(zhǔn)誤差表示.

2 結(jié)果與分析

2.1 H2S和SO2處理對(duì)擬南芥植株基因表達(dá)的影響

外源H2S處理3d后,擬南芥全基因組中差異表達(dá)基因(DEG)共有3160個(gè),其中上調(diào)表達(dá)基因1500個(gè),下調(diào)表達(dá)基因1660個(gè).在SO2熏氣40h后,差異表達(dá)基因共有1892個(gè),其中上調(diào)表達(dá)基因1034個(gè),下調(diào)表達(dá)基因858個(gè)(表2).

表2 差異表達(dá)基因數(shù)目統(tǒng)計(jì)

圖1 H2S和SO2處理共有差異表達(dá)基因的功能分類

表3 H2S和SO2處理后部分差異表達(dá)基因

通過繪制維恩圖,在H2S和SO2處理組中發(fā)現(xiàn)1220個(gè)共有差異表達(dá)基因,分別占各處理組差異表達(dá)基因的38.6%和64.5%.根據(jù)Gene Ontology (GO)分類法則對(duì)共有差異表達(dá)基因進(jìn)行初步功能分類,發(fā)現(xiàn)其功能主要包括結(jié)合、催化、轉(zhuǎn)錄因子、轉(zhuǎn)運(yùn)、分子傳感、酶調(diào)節(jié)、結(jié)構(gòu)分子、受體、電子載體、抗氧化和營養(yǎng)儲(chǔ)存(圖1),其中有多個(gè)硫代謝和谷胱甘肽代謝相關(guān)基因差異表達(dá),如、和(表3).

2.2 H2S和SO2處理后硫代謝相關(guān)基因表達(dá)水平

H2S和SO2處理后,擬南芥植株中硫代謝相關(guān)基因、、、、和表達(dá)水平發(fā)生改變. H2S處理后,H2S合成相關(guān)基因和表達(dá)量顯著降低,而表達(dá)量顯著升高.氧乙酰絲氨酸(硫醇)裂解酶基因表達(dá)量顯著提高,而和表達(dá)量顯著降低.亞硫酸氧化酶基因和亞硫酸還原酶基因表達(dá)量無明顯變化(圖2).

SO2熏氣后, H2S合成相關(guān)基因和顯著上調(diào)表達(dá),而表達(dá)量無明顯變化.氧乙酰絲氨酸(硫醇)裂解酶基因表達(dá)量顯著降低,而和表達(dá)量沒有明顯變化.亞硫酸氧化酶基因表達(dá)量增加,亞硫酸還原酶基因表達(dá)水平無明顯改變(圖2).

圖2 硫代謝相關(guān)基因表達(dá)水平

2.3 H2S對(duì)SO2脅迫下擬南芥含硫化合物水平的影響

SO2熏氣后,擬南芥植株中H2S和GSH含量增加,GST和GPX活性顯著高于對(duì)照組,說明SO2脅迫誘導(dǎo)胞內(nèi)產(chǎn)生H2S,含硫抗氧化物GSH及其相關(guān)防御酶水平提高(圖3).

圖3 H2S對(duì)SO2脅迫下擬南芥含硫化合物的影響

在SO2熏氣過程中,分別用H2S供體NaHS或H2S清除劑HT噴施擬南芥葉片.結(jié)果發(fā)現(xiàn),外源H2S處理顯著提高了SO2熏氣下擬南芥植株GSH含量、GST和GPX活性.相反,外源噴施HT后, SO2脅迫組擬南芥植株胞內(nèi)H2S和GSH含量、GST活性均降低到對(duì)照水平,說明H2S信號(hào)參與調(diào)控SO2脅迫下擬南芥體內(nèi)含硫化合物水平(圖3).

2.4 H2S對(duì)擬南芥SO2脅迫的緩解作用

SO2熏氣后,擬南芥植株中活性氧H2O2含量顯著增加,抗氧化酶SOD和CAT活性顯著升高,膜脂過氧化產(chǎn)物MDA含量與對(duì)照組相比沒有顯著差異(圖4).

在SO2熏氣過程中噴施H2S后,擬南芥植株中SOD和CAT活性維持在較高水平, H2O2含量降低,說明外源H2S可以有效緩解SO2熏氣造成的氧化脅迫.相反,外源噴施HT后, SO2熏氣擬南芥植株中H2O2含量維持在較高水平, CAT和SOD活性顯著降低, MDA含量顯著增加,說明清除H2S分子導(dǎo)致擬南芥植株發(fā)生膜脂過氧化,進(jìn)一步說明H2S參與調(diào)控植物對(duì)SO2脅迫的抗氧化應(yīng)答(圖4).

圖4 H2S對(duì)SO2脅迫下擬南芥抗氧化系統(tǒng)的影響

3 討論

SO2是一種有毒的大氣污染物.H2S是硫代謝途徑中重要的中間產(chǎn)物,也是一種新型氣體信號(hào)分子,在植物生長發(fā)育及抵抗生物和非生物脅迫過程中發(fā)揮重要作用[28-30].本研究中,外源H2S和SO2處理誘導(dǎo)擬南芥植株中多個(gè)基因轉(zhuǎn)錄水平改變,并且有1220個(gè)基因在兩種處理?xiàng)l件下均差異表達(dá),涉及轉(zhuǎn)錄調(diào)節(jié)、信號(hào)傳導(dǎo)、抗氧化應(yīng)激等多種生理過程,其中包括多個(gè)硫代謝和谷胱甘肽代謝相關(guān)基因差異表達(dá).擬南芥中多個(gè)基因既響應(yīng)SO2脅迫又受H2S調(diào)節(jié),證實(shí)SO2和H2S處理間存在一定的聯(lián)系,并為信號(hào)分子H2S調(diào)節(jié)植物SO2脅迫應(yīng)答機(jī)制研究奠定了基礎(chǔ).

SO2熏氣誘導(dǎo)擬南芥植株中亞硫酸氧化酶基因表達(dá)量增加.亞硫酸氧化酶可催化SO32-+ O2+ H2O → SO42-+ H2O2反應(yīng),緩解SO32-對(duì)植物細(xì)胞的傷害,但同時(shí)也會(huì)產(chǎn)生大量的活性氧,導(dǎo)致胞內(nèi)H2O2含量增多,造成氧化脅迫[31-32].同時(shí),SO2熏氣誘導(dǎo)胞內(nèi)抗氧化酶CAT和SOD活性上升,抗氧化物GSH含量增加, GST和GPX基因表達(dá)水平和酶活性提高,有效清除胞內(nèi)過量的活性氧,防止發(fā)生氧化損傷.

外源H2S處理降低了SO2熏氣下擬南芥植株體內(nèi)H2S含量,但是GSH含量增加,與外源H2S噴施Cr6+處理谷子中的研究結(jié)果類似[33],這可能是因?yàn)镠2S處理不僅可誘導(dǎo)轉(zhuǎn)錄水平提高,同時(shí)氧乙酰絲氨酸(硫醇)裂解酶基因表達(dá)量顯著增加,使部分H2S參與了Cys的合成,進(jìn)而生成抗氧化物質(zhì)GSH[34].因此,外源H2S對(duì)SO2熏氣下擬南芥CAT和SOD活性沒有明顯影響,但可進(jìn)一步提高植株抗氧化物GSH含量及其相關(guān)防御酶的活性,緩解SO2對(duì)植物造成的氧化脅迫.本研究表明,外源H2S處理影響植物體內(nèi)的硫代謝過程,并可通過調(diào)控含硫化合物水平來提高植物對(duì)環(huán)境的適應(yīng)能力.

對(duì)H2S合成相關(guān)基因表達(dá)水平研究發(fā)現(xiàn), SO2熏氣對(duì)擬南芥植株中亞硫酸還原酶基因轉(zhuǎn)錄水平?jīng)]有明顯影響,主要通過提高半胱氨酸脫巰基酶基因和表達(dá)水平來增加植株體內(nèi)H2S含量.同時(shí),氧乙酰絲氨酸(硫醇)裂解酶基因表達(dá)量降低,減少利用H2S合成Cys,從而使胞內(nèi)積累大量H2S.外源噴施HT清除胞內(nèi)產(chǎn)生的H2S后,擬南芥植株體內(nèi)CAT和SOD活性顯著降低, GSH含量及GST活性回落到對(duì)照水平,膜脂過氧化產(chǎn)物MDA含量大幅增加,說明SO2熏氣誘導(dǎo)胞內(nèi)產(chǎn)生H2S作為信號(hào)分子,參與調(diào)控植物體的抗氧化防御反應(yīng),有效緩解細(xì)胞氧化損傷效應(yīng).另外,有研究表明,SO2暴露誘導(dǎo)胞內(nèi)產(chǎn)生H2S,促使植株產(chǎn)生抗氧化防御應(yīng)答,提高后期干旱、Al脅迫期間植株的抗氧化酶活性,增強(qiáng)植物的逆境適應(yīng)能力[25-26].因此,H2S信號(hào)分子不僅在植物響應(yīng)SO2脅迫過程中發(fā)揮重要作用,同時(shí)還參與調(diào)控SO2暴露誘導(dǎo)植物體產(chǎn)生的對(duì)多種環(huán)境脅迫的交叉抗性.

本文不僅闡明H2S信號(hào)調(diào)節(jié)SO2脅迫應(yīng)答的機(jī)理,還揭示了H2S是SO2誘導(dǎo)植物產(chǎn)生交叉適應(yīng)性的重要基礎(chǔ),對(duì)其詳細(xì)機(jī)制的深入研究將為植物適應(yīng)復(fù)雜多變的環(huán)境條件提供一條新的有效途徑.

4 結(jié)論

4.1 高通量測(cè)序研究發(fā)現(xiàn), H2S和SO2處理組擬南芥中出現(xiàn)1220個(gè)共有差異表達(dá)基因,兩者在調(diào)控硫代謝途徑中具有交互作用.

4.2 外源噴施H2S可提高SO2脅迫下擬南芥GSH含量, GPX和GST活性增加,緩解SO2對(duì)植物造成的氧化脅迫.

4.3 SO2熏氣誘導(dǎo)擬南芥H2S合成酶基因和轉(zhuǎn)錄水平提高,胞內(nèi)H2S含量增加.H2S作為信號(hào)分子,通過提高抗氧化防御能力來調(diào)控植物對(duì)SO2脅迫的響應(yīng).

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Functions of H2S signal in response to SO2stress in.

LI Li-hong1*, GUO Yu-ru1, HOU Jun-xin1, WU Li-hua2

(1.Department of Chemistry and Chemical Engineering, Jinzhong University, Yuci 030619, China；2.Department of Biology, Taiyuan Normal University, Yuci 030619, China)., 2022,42(6)：2904～2910

Sulfur dioxide (SO2) is one of the most common and harmful air pollutant. Hydrogen sulfide (H2S), as a new gasotransmitter, is involved in the regulation of plant development and stress adaptation. To reveal the role of H2S in response to SO2stress, the relation between transcriptional response to H2S and SO2treatments, and the production and physiological effect of H2S during SO2fumigation will be studied inplants. By using high-throughput sequencing, many genes were identified to be differentially expressed inplants exposed to 30mg/m3SO2or treated with 0.1mmol/L H2S. Among these differentially expressed genes (DEGs), 122, 0genes were overlapped between SO2and H2S treatments, including some genes encoding enzymes involved in sulfur and glutathione metabolism. These results suggested a interaction between H2S and SO2in regulating sulfur metabolism pathways in plants. Moreover, two H2S-generating genes, L-cysteine desulfhydrase () and L-cysteinedesulfhydrase1 () were significantly up-regulated, and endogenous H2S content was enhanced inplants under SO2stress. Meanwhile, the activities of superoxide dismutase (SOD) and catalase (CAT), the content of glutathione (GSH) and activities of GSH-related enzymes including glutathione S-transferase (GST) and glutathione peroxidase (GPX) were significantly increased inplants exposed to SO2. And that H2O2content was markedly increased in SO2-treatedplants, while malondialdehyde (MDA) content showed no significant difference compared with the control group. Exogenous application of H2S further increased the level of GSH and GSH-related defense enzyme, and reduced the content of H2O2inplants under SO2stress. When scavenged endogenous H2S by spraying hypotaurine (HT) during SO2fumigation, the content of GSH, the activities of SOD, CAT and GST were reduced, and the content of MDA was significantly increased inplants. Together, this study indicated that H2S played an important role in response to SO2stress through improving antioxidant capacity, which would be helpful for better understanding the adaptation mechanism of plants to environmental stress.

sulfur dioxide；hydrogen sulfide；；sulfur compounds；antioxidant enzymes

X171.5

A

1000-6923(2022)06-2904-07

李利紅(1982-),女,山西呂梁人,副教授,博士,主要研究方向?yàn)榄h(huán)境污染物的生態(tài)毒理學(xué).發(fā)表論文10余篇.

2021-11-10

國家自然科學(xué)基金項(xiàng)目(21307087);山西省應(yīng)用基礎(chǔ)研究計(jì)劃項(xiàng)目(201901D111301);晉中學(xué)院“1331工程”創(chuàng)客團(tuán)隊(duì)項(xiàng)目(jzxycktd2019031)

* 責(zé)任作者, 副教授, lihongli19821129@163.com