周萬海, 馮瑞章, 師尚禮, 寇江濤

1 宜賓學(xué)院生命科學(xué)與食品工程學(xué)院 西南特色經(jīng)濟(jì)植物實(shí)驗(yàn)室, 宜賓 644000 2 甘肅農(nóng)業(yè)大學(xué)草業(yè)學(xué)院 草業(yè)生態(tài)系統(tǒng)教育部重點(diǎn)實(shí)驗(yàn)室, 蘭州 730070

NO對鹽脅迫下苜蓿根系生長抑制及氧化損傷的緩解效應(yīng)

周萬海1,2, 馮瑞章1, 師尚禮2,*, 寇江濤2

1 宜賓學(xué)院生命科學(xué)與食品工程學(xué)院 西南特色經(jīng)濟(jì)植物實(shí)驗(yàn)室, 宜賓 644000 2 甘肅農(nóng)業(yè)大學(xué)草業(yè)學(xué)院 草業(yè)生態(tài)系統(tǒng)教育部重點(diǎn)實(shí)驗(yàn)室, 蘭州 730070

一氧化氮; 苜蓿; 鹽脅迫; 抗氧化系統(tǒng); 根系

根系是植物與土壤環(huán)境接觸的主要界面，不僅在植物水分、養(yǎng)分吸收、土壤固著及內(nèi)源激素合成中起關(guān)鍵作用，也是土壤鹽漬危害中植物最直接的受害部位，因此改善和提高植物根系的生長對植物適應(yīng)環(huán)境有重要意義[1- 2]。一氧化氮(NO)是一種重要的信號分子，在植物生長發(fā)育和抗逆反應(yīng)中起關(guān)鍵作用[3]，研究表明，一定濃度的NO對植物側(cè)根、不定根和根毛的發(fā)育有促進(jìn)作用, 但對主根的伸長具抑制效應(yīng)[4- 5]。外源NO能提高鹽脅迫下根組織的滲透調(diào)節(jié)能力、降低自由基積累，緩解根系氧化損傷，改善根系對離子的選擇性吸收、促進(jìn)根系生長，從而提高植株耐鹽性[6- 8]。然而，植物能通過一氧化氮合酶(NOS)或硝酸還原酶(NR)催化的酶學(xué)途徑或非酶學(xué)途徑形成內(nèi)源NO，且內(nèi)源NO的產(chǎn)生受植物種、組織和細(xì)胞類型及環(huán)境條件的影響[9]，使得NO在植物體中的來源及在抗逆中的作用有較大爭議。因此，研究NO對根系耐鹽性的調(diào)控效應(yīng)及作用機(jī)制具有重要的理論意義和實(shí)踐價(jià)值。

苜蓿(MedicagosativaL.)是我國西北地區(qū)廣泛種植的一種優(yōu)質(zhì)豆科牧草，鹽漬已成為限制苜蓿生產(chǎn)的主要土壤環(huán)境因子之一，雖然苜蓿耐鹽性研究已有一些報(bào)道，但有關(guān)NO對苜蓿根系耐鹽性的作用機(jī)制仍然模糊不清，且苜蓿根系中內(nèi)源NO的來源和對根系抗逆性的影響亦無相關(guān)報(bào)道，為進(jìn)一步闡明NO在這一過程中的作用，本試驗(yàn)以苜蓿為材料，研究外源NO對鹽脅迫下苜蓿根系生長和氧化損傷的緩解效應(yīng)；同時(shí)還研究了苜蓿根系中內(nèi)源NO的來源及其在根系耐鹽中的作用，為進(jìn)一步理解鹽脅迫對豆科雙子葉植物根系生長抑制的作用機(jī)制及調(diào)控方法提供依據(jù)。

1 材料和方法

1.1 材料的培養(yǎng)和處理

供試苜蓿品種為甘農(nóng)4號(MedicagosativaL. cv. Gannong No.4)。選取飽滿均勻的苜蓿種子，用0.1% HgCl2溶液消毒5 min, 去離子水洗凈，播種于滅菌的蛭石培養(yǎng)缽中，種子萌發(fā)后每盆定植10株，轉(zhuǎn)移至光照培養(yǎng)室(相對濕度60%，光照時(shí)間為14 h, 光通量密度400 μmol m-2s-1，晝/夜溫度為25 ℃/20 ℃)，每3 d澆灌1次1/2 Hoagland營養(yǎng)液，生長35 d后，挑選整齊一致的幼苗洗凈后移栽到盛有1/2 Hoagland營養(yǎng)液的塑料缽中預(yù)培養(yǎng)，預(yù)培養(yǎng)3 d后開始處理。試驗(yàn)中以亞硝基鐵氰化鈉(sodium nitroprusside,SNP)為NO供體，亞鐵氰化鈉(sodium ferrocyanide，SF)為SNP類似物(不產(chǎn)生NO)，2- 4-羧苯基四甲基咪唑烷- 1-氧- 3-氧化物(2-(4-carboxyphenyl)- 4,4,5,5-tetramethylimidazoline- 1-oxyl- 3-oxide,c-PTIO)為NO特異清除劑，鎢酸鹽(tungstate，縮寫為T)為硝酸還原酶活性抑制劑，N-硝基-L-精氨酸甲脂(NG-nitro-L-Arg-methyl ester,L-NAME)為一氧化氮合酶抑制劑，以上化學(xué)試劑均購于Sigma公司。試驗(yàn)設(shè)11種處理：1)CK，2)SNP(100 μmol/L SNP)，3)NaCl(150 mmol/L NaCl)，4)NaCl+SNP(150 mmol/L NaCl+100 μmol/L SNP)，5)NaCl+SF(150 mmol/L NaCl+100 μmol/L 亞鐵氰化鈉)，6)NaCl+c-PTIO(150 mmol/L NaCl+100 μmol/L c-PTIO)，7)NaCl+c-PTIO+SNP(150 mmol/L NaCl+100 μmol/L c-PTIO+100 μmol/L SNP)，8)NaCl+T(150 mmol/L NaCl+100 μmol/L Tungstate)，9)NaCl+SNP+T(150 mmol/L NaCl+100 μmol/L Tungstate+100 μmol/L SNP)，10)NaCl+L-NAME(150 mmol/L NaCl+100 μmol/L L-NAME)，11)NaCl+SNP+L-NAME(150 mmol/L NaCl+100 μmol/L L-NAME+100 μmol/L SNP)；所用溶液均用1/2 Hoagland營養(yǎng)液溶解配制，試驗(yàn)采用隨機(jī)區(qū)組設(shè)計(jì)，每處理10盆，重復(fù)3次。為保證處理濃度穩(wěn)定，每隔2 d更換1次處理液，用空氣壓縮泵給營養(yǎng)液間歇通入空氣。處理第8天取根樣測定各項(xiàng)指標(biāo)，每處理取3個(gè)重復(fù)。

1.2 測定項(xiàng)目與方法

1.2.1 根系干重測定

處理結(jié)束后，將植株的地上部和地下部分開，取苜蓿幼苗根系，用去離子水沖洗干凈，擦干水分，105 ℃殺青15 min，70 ℃烘干至恒重，稱干質(zhì)量。

1.2.2 生理生化指標(biāo)及NO產(chǎn)生量測定

根系活力用氯化三苯基四氮唑(triphenyltetrazolium chloride，TTC)法測定[10]，游離脯氨酸含量用水合茚三酮法測定[10]，用蒽酮法測定可溶性糖含量[10]，考馬斯亮藍(lán)G- 250法測定可溶性蛋白含量[10]，硫代巴比妥酸法測定丙二醛(malondialdehyde，MDA)含量[10]，羥自由基含量測定參考Halliwell的方法[11]，過氧化氫(H2O2)含量測定采用Velikova的方法[12]，超氧陰離子產(chǎn)生速率測定參考王愛國的方法[13]；粗酶液的制備參考Azevedo-Neto的方法[14]，超氧化物歧化酶(superoxide dismutase，SOD)活性測定參考Giannopolitis和Ries的方法[15]，過氧化氫酶(catalase，CAT)活性測定參考Beers和Sizer的方法[16]，抗壞血酸過氧化物酶(ascorbate peroxidase，APX)活性測定參考Nakano and Asada(1981)的方法[17]，愈創(chuàng)木酚過氧化物酶(guaiacol peroxidase，GPX)活性測定參考Urbanek的方法[18]，谷胱甘肽還原酶(glutathione reductase，GR)活性測定參考Foyer的方法[19]；還原型抗壞血酸(reduced ascorbic acid，AsA)含量測定參考Law的方法[20]，還原型谷胱甘肽(reduced glutathion，GSH)含量測定參考Griffith的方法[21]；NO產(chǎn)生量測定參考Zhou的方法[22]。

1.3 數(shù)據(jù)處理

所有數(shù)據(jù)均為3個(gè)重復(fù)的平均值±標(biāo)準(zhǔn)誤(±SE)，SPSS(16.0)軟件進(jìn)行單因素方差(ANOVA)統(tǒng)計(jì)分析，Duncan 法多重比較，差異顯著性定義為P<0.05，Excel 2003制作相應(yīng)圖表。

2 結(jié)果與分析

2.1 SNP對苜蓿根系生長和根系活力的影響

鹽脅迫抑制了苜蓿幼苗根系生長和根系活力(圖1)，與CK相比，根系干重和活力分別降低15.9%和58.7%(P<0.05)；SNP緩解了鹽脅迫對根系生長和活力的抑制，干重和根系活力分別比CK降低8.1%和16.7%(P<0.05)。NO的特異清除劑c-PTIO、硝酸還原酶活性抑制劑鎢酸鹽和一氧化氮合酶活性抑制劑L-NAME分別與NaCl共處理進(jìn)一步抑制了苜蓿根系干重和根系活力(P<0.05)，與CK相比根系干重分別降低26.7%、24.9%和32.7%，根系活力降低63.3%、62.5%和68.9%；c-PTIO、鎢酸鹽和L-NAME對根系干重和活力的抑制效應(yīng)能被SNP緩解，各處理根系干重和活力與NaCl處理無差異；鹽脅迫對苜蓿幼苗根系生長和根系活力的抑制不能被SNP類似物亞鐵氰化鈉緩解(P>0.05)。

二是要有閑。時(shí)間是保證自駕游的前提。這次去西北，我們兩口子是退休，小弟是休年假，還帶了正放暑假的侄女。時(shí)間不受限制。自駕游的特點(diǎn)是寬松自由，一個(gè)好的景點(diǎn)甚至一個(gè)好的畫面，想看了，可以隨時(shí)停車欣賞，時(shí)間長一點(diǎn)短一點(diǎn)，自己掌握，無關(guān)緊要。

圖1 SNP對鹽脅迫下苜蓿根系干重和根系活力的影響

2.2 SNP對鹽脅迫下苜蓿根系滲透調(diào)節(jié)物質(zhì)含量的影響

鹽脅迫下苜蓿根系中游離脯氨酸和可溶性糖含量分別比CK增加3.4倍和2.1倍，可溶性蛋白含量降低43.7%，表明鹽脅迫引起苜蓿根系的滲透脅迫。添加SNP使可溶性蛋白含量升高，游離脯氨酸和可溶性糖含量降低。c-PTIO、鎢酸鹽和L-NAME與NaCl共處理進(jìn)一步提高了游離脯氨酸和可溶性糖含量，降低了可溶性蛋白含量(P<0.05)，與CK相比，游離脯氨酸增加3.8—4.1倍，可溶性糖含量提高2.3—2.6倍，可溶性蛋白分別降低56.1%、52.3%和58.7%，添加SNP能逆轉(zhuǎn)c-PTIO、鎢酸鹽和L-NAME處理對苜蓿根系游離脯氨酸、可溶性糖、可溶性蛋白的影響。亞鐵氰化鈉處理使苜蓿根系中游離脯氨酸、可溶性糖和可溶性蛋白含量與NaCl處理無差異(P>0.05)(表1)。

2.3 SNP對鹽脅迫下苜蓿根系自由基和MDA含量的影響

表1 SNP對鹽脅迫下苜蓿根系可溶性蛋白、游離脯氨酸和可溶性糖含量的影響

圖2 SNP對鹽脅迫下苜蓿根系MDA、H2O2、OH·含量和產(chǎn)生速率的影響

2.4 SNP對鹽脅迫下苜蓿根系抗氧化系統(tǒng)的影響

與CK相比，正常條件下添加SNP能在一定程度上提高苜蓿根系抗氧化酶活性和抗氧化物含量(表2)；鹽脅迫使苜蓿根系SOD、CAT、GPX和APX活性比CK降低48.5%、50.2%、23.9%和57.4%， GR活性升高34.4%(P<0.05)，AsA和GSH含量分別提高1.17和1.28倍。SNP提高根系抗氧化系統(tǒng)活性，SOD、CAT、GPX、APX和GR活性分別比NaCl處理提高54.2%、83.3%、125.9%、78.3%和25.6%，AsA和GSH含量也進(jìn)一步提高(P<0.05)(表2)。c-PTIO、鎢酸鹽和L-NAME處理能抑制SOD、GPX、CAT、APX和GR活性，降低AsA和GSH含量(P<0.05)。添加SNP能緩解c-PTIO、鎢酸鹽和L-NAME處理對抗氧化系統(tǒng)活性的抑制；亞鐵氰化鈉處理對苜蓿幼苗根系抗氧化系統(tǒng)的影響與NaCl處理類似(P>0.05)(表2)。

表2 SNP對鹽脅迫下苜蓿根系抗氧化系統(tǒng)活性和NO含量的影響

2.5 SNP處理對鹽脅迫下苜蓿根系NO含量的影響

3 結(jié)論與討論

根系生長受抑制是鹽脅迫下植物最早和最明顯的癥狀[23]，本試驗(yàn)中NaCl脅迫明顯抑制苜蓿幼苗根系生長，用100 μmol/L SNP處理苜蓿幼苗，根系干重顯著增加，NO清除劑c-PTIO處理則進(jìn)一步降低了苜蓿幼苗根系干重，添加SNP能逆轉(zhuǎn)c-PTIO對根生長的抑制，由于SNP被廣泛用做NO供體，且SNP類似物亞鐵氰化鈉處理對苜蓿根的生長無促進(jìn)效應(yīng)，表明NO能夠緩解鹽脅迫對苜蓿幼苗根生長的抑制效應(yīng)。

根系活力是判斷植物對逆境適應(yīng)能力的優(yōu)良指標(biāo)。本研究中，鹽脅迫導(dǎo)致苜蓿幼苗根系活力顯著降低，說明鹽脅迫破壞了苜蓿根系的功能。NO作為生長素下游的一個(gè)信號分子，能替代IAA誘導(dǎo)植物側(cè)根發(fā)育并促進(jìn)根系對水分和養(yǎng)分的吸收[4,24]；此外，NO還能調(diào)控生長素在根器官中的轉(zhuǎn)運(yùn)和平衡[25]，抑制IAA氧化酶的活性，提高膜的轉(zhuǎn)運(yùn)活性和根系吸收功能[26]，從而促進(jìn)細(xì)胞的分裂和生長[27]，增強(qiáng)根系活力和植株對逆境的適應(yīng)能力，本研究也得到類似結(jié)果。

植物能通過積累可溶性蛋白、氨基酸和可溶性糖等一些小分子有機(jī)物緩解鹽脅迫的不利影響[28]。其中可溶性蛋白除參與滲透調(diào)節(jié)外[29]，還在一定程度上代表植物器官功能的變化[30]；脯氨酸含量的增加也能調(diào)控植物滲透勢, 提高植株耐性[31]。本試驗(yàn)中鹽脅迫顯著降低苜蓿幼苗根系中可溶性蛋白含量，提高了游離脯氨酸含量，說明鹽脅迫破壞了根系功能，但可溶性蛋白的分解會(huì)產(chǎn)生大量的游離氨基酸，這有利于根系進(jìn)行滲透調(diào)節(jié)。因此，鹽脅迫下根系中可溶性蛋白的分解可能是游離脯氨酸含量升高的原因之一。SNP處理顯著降低了根系中游離脯氨酸含量，但提高了可溶性蛋白含量，表明NO促進(jìn)了植株蛋白的合成，這有助于維持根系滲透壓和恢復(fù)根系功能。鹽脅迫也提高了苜蓿根系中可溶性糖含量，但SNP處理使其含量顯著降低，由于可溶性糖在植物體內(nèi)具有滲透保護(hù)、碳儲(chǔ)存和清除自由基的作用[32]，這種變化可能與NO對碳水化合物的代謝調(diào)控有關(guān)，需做進(jìn)一步研究。因此，NO通過滲透調(diào)節(jié)來維持根系滲透壓和功能是促進(jìn)鹽脅迫下苜蓿根系生長的原因之一。

NOS和NR是植物體內(nèi)源NO的最主要來源，但仍存在爭議[9]。為明確苜蓿根系內(nèi)源NO的來源和其對苜蓿根系耐鹽性的影響，本試驗(yàn)用NO特異清除劑c-PTIO、NOS活性抑制劑和NR活性抑制劑處理苜蓿幼苗，結(jié)果表明，c-PTIO處理進(jìn)一步提高根系中自由基含量，加劇膜脂過氧化作用，添加SNP能逆轉(zhuǎn)c-PTIO的效應(yīng)，SNP類似物處理對苜蓿根系的膜脂過氧化和ROS代謝無緩解效應(yīng)，表明NO能緩解鹽脅迫對苜蓿幼苗的氧化脅迫，且內(nèi)源NO對根系耐鹽也有一定作用。與c-PTIO處理類似，NOS抑制劑 L-NAME降低了根組織中NO的產(chǎn)生量和根系活力，加劇根系中ROS的積累和膜脂過氧化作用，抑制了根系的抗氧化系統(tǒng)活性和根系生長。但添加SNP則能提高根系中NO含量，緩解L-NAME的處理效應(yīng)，表明根系中內(nèi)源NO的合成可能依賴于NOS途徑；用NR活性抑制劑鎢酸鹽處理，也得到與L-NAME類似的結(jié)果，然而，需要注意的是，鎢酸鹽不僅抑制NR活性，還能抑制植物根系生長、影響皮層微管的形成和誘導(dǎo)細(xì)胞的程序性死亡[41]，且通過NR途徑來源的NO也受培養(yǎng)液中氮源的影響，因此，本研究中，鎢酸鹽處理下根系傷害的加劇和內(nèi)源NO來源及代謝變化的原因還需被進(jìn)一步研究和闡明。

逆境脅迫可以改變內(nèi)源NO的代謝[42]，本試驗(yàn)中鹽脅迫降低了苜蓿根系中NO的積累，這與一年生蒺藜苜蓿[26]上的研究結(jié)果一致，但與擬南芥[43]上的研究結(jié)果相反，這可能與研究中使用的材料類型，脅迫因子及處理時(shí)間有關(guān)。然而，內(nèi)源NO必須積累達(dá)到一定濃度才能啟動(dòng)根的生長[44]。因此，內(nèi)源NO濃度降低也可能是鹽脅迫下苜蓿根系生長降低的原因，而添加外源SNP后，促進(jìn)了內(nèi)源NO的積累，達(dá)到啟動(dòng)根系生長所需的濃度閾值，從而促進(jìn)苜蓿植株根系的生長，但相關(guān)機(jī)制還應(yīng)做進(jìn)一步研究。

綜上所述，SNP處理能明顯促進(jìn)根系中NO的積累，提高苜蓿幼苗根系可溶性蛋白含量、根系活力和根系中抗氧化系統(tǒng)活性，降低自由基含量和膜脂過氧化作用，緩解鹽脅迫對苜蓿幼苗根系的氧化傷害和生長抑制。NO特異清除劑c-PTIO、NOS活性抑制劑和NR活性抑制劑處理鹽脅迫下苜蓿幼苗，進(jìn)一步降低了根系活力和根系中NO產(chǎn)量，抑制了根系抗氧化系統(tǒng)活性，提高了根系中自由基含量和膜脂過氧化作用，加劇了鹽漬對苜蓿根系生長的抑制，表明依賴于NOS活性和/或NR活性產(chǎn)生的內(nèi)源NO也參與鹽脅迫下苜蓿幼苗根系耐鹽性的調(diào)節(jié)。

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Nitric oxide protection of alfalfa seedling roots against salt-induced inhibition of growth and oxidative damage

ZHOU Wanhai1,2, FENG Ruizhang1, SHI Shangli2,*, KOU Jiangtao2

1LaboratoryofSouthwesternEconomicPlants,CollegeofLifeScience&FoodEngineering,YibinUniversity,Yibin644000,China2KeyEcosystemLaboratoryoftheMinistryofEducation，CollegeofGrasslandScience,GansuAgriculturalUniversity,Lanzhou730070,China

Roots play important roles in anchoring plants in the soil and absorbing water and nutrients from the soil. Soil salinity is one of the major abiotic stresses that adversely affect root growth and development. The exogenous application of some bioactive substances is a simple and effective approach to improve plant stress tolerance in agricultural production. Nitric oxide (NO) is a bioactive, gaseous, multifunctional molecule which plays a central role and mediates a variety of physiological processes and responses to biotic and abiotic stresses, including salt. However, information on the effects of exogenous SNP on alfalfa root systems under salt stress conditions and its physiological mechanisms is lacking. In this study,MedicagosativaL. cv. Gannong No.4 was used as the experimental material and NO-donor sodium nitroprusside (SNP), NO-scavenger 2-(4-carboxyphenyl)- 4,4,5,5-tetramethylimidazoline- 1-oxyl- 3-oxide (c-PTIO), tungstate, the nitrate reductase (NR) inhibitor, nitric oxide synthase (NOS) inhibitor NG-nitro-L-Arg-methyl ester (L-NAME) and sodium ferrocyanide (SNP analogue, does not release NO) were used to investigate the effects of NO on growth, root vigor, osmoregulatory molecules, membrane lipid peroxidation, reactive oxygen species (ROS) accumulation and the activity of antioxidant enzyme in alfalfa roots under NaCl stress. Results showed that when 100 μmol/L SNP was applied in the nutrient solution under NaCl stress, the growth of alfalfa roots was significantly improved for eight days, the root vigor value and the content of free proline were increased and the content of soluble protein was decreased throughout the treatment period. SNP also caused an increase in the activities of ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR), guaiacol peroxidase (GPX), superoxide dismutase (SOD), and the contents of reduced ascorbic acid (AsA) and reduced glutathion (GSH) under salt stress. Whereas, the content of H2O2, OH·, production rate of O·-2and MDA level were decreased in alfalfa roots. Meanwhile, SNP pretreatment improved the endogenous NO accumulation. Sodium ferrocyanide, the NO-donor SNP analogue, has no noticeable effect on the damage caused by NaCl stress for alfalfa roots. When applied with the NO-scavenger c-PTIO, under NaCl stress, the NR inhibitor tungstate and NOS inhibitor L-NAME both reduced the accumulation of endogenous NO in roots, further inhibited the activity of the antioxidant system, aggravated membrane lipid peroxidation and root growth, however, their harmful effects on growth, antioxidant system and membrane lipid peroxidation could be reversed by the addition of SNP. Thus, the results showed that application of SNP significantly increased root vigor, activated the antioxidant system, alleviated the oxidative root damage induced by salt stress and promoted root growth under NaCl stress. Endogenous NO may also take part in the regulation of salt-tolerance of alfalfa roots under NaCl stress, and the release of NO according to the NOS and NR pathways may play an important role in alleviating the inhibitive effect in alfalfa root growth. These results may provide a theoretical basis for the mechanisms of salt resistance, help research into NO in the chemical regulation of alfalfa salt tolerance, the breeding of salt tolerant alfalfa cultivars, and the utilization of saline land in the future.

Nitric oxide; alfalfa; salt stress; antioxidant systems; roots

牧草種質(zhì)資源保護(hù)利用(NB2130135); 國家牧草產(chǎn)業(yè)技術(shù)體系專項(xiàng)(CARS- 35); 宜賓學(xué)院重點(diǎn)科研項(xiàng)目(2013QD06)

2013- 10- 14;

2014- 08- 22

10.5846/stxb201310142472

*通訊作者Corresponding author.E-mail: shishl@gsau.edu.cn

周萬海, 馮瑞章, 師尚禮, 寇江濤.NO對鹽脅迫下苜蓿根系生長抑制及氧化損傷的緩解效應(yīng).生態(tài)學(xué)報(bào),2015,35(11):3606- 3614.

Zhou W H, Feng R Z, Shi S L, Kou J T.Nitric oxide protection of alfalfa seedling roots against salt-induced inhibition of growth and oxidative damage.Acta Ecologica Sinica,2015,35(11):3606- 3614.