王華華+楊麗丹+黃俊駿

摘要:以大豆為試驗(yàn)材料,研究了干旱脅迫下NO對(duì)抗氧化酶活性的調(diào)節(jié)作用。結(jié)果顯示,干旱脅迫處理顯著地增強(qiáng)了大豆根中NO產(chǎn)生和抗氧化酶SOD、CAT和POD活性;外源NO處理進(jìn)一步增加了抗氧化酶活性,而NO清除劑處理則抑制了NO產(chǎn)生和抗氧化酶活性;NO合成酶(NOS)抑制劑和硝酸還原酶(NR)抑制劑處理均明顯抑制了干旱脅迫下NO產(chǎn)生和抗氧化酶活性,且NOS抑制劑影響程度更大。此外,干旱脅迫增強(qiáng)了NOS和NR活性,而且NOS活性增幅比NR大。這些結(jié)果表明,干旱脅迫下NOS和NR途徑介導(dǎo)的NO產(chǎn)生參與了大豆根中抗氧化酶活性的調(diào)節(jié),而且NOS途徑起著主要作用。

關(guān)鍵詞:大豆;干旱脅迫;一氧化氮(NO);抗氧化酶

中圖分類(lèi)號(hào):S565.1;Q945.78文獻(xiàn)標(biāo)識(shí)碼:A文章編號(hào):0439-8114(2014)11-2493-04

Effects of NO on the Activity of Antioxidative Enzymes in Soybean Seedlings

under Drought Stress

WANG Hua-hua, YANG Li-dan, HUANG Jun-jun

(College of Life Science, Henan Normal University, Xinxiang 453007, Henan, China)

Abstract: Soybean was used to investigate the regulatory role of NO on the activity of antioxidative enzymes in soybean seedlings under drought stress. The results showed that NO production and activities of SOD, CAT and POD were significantly increased in the soybean roots exposed to drought stress. Application of exogenous NO to soybean seedlings further increased the activity of antioxidative enzymes, while NO scavenger inhibited NO production and the activity of antioxidative enzymes. Both inhibitors of NO synthase (NOS) and nitrate reductase (NR) significantly inhibited NO production and the activity of antioxidative enzymes under drought stress, and NOS inhibitor had greater effect than NR inhibitor had.The activities of NOS and NR were increased under drought stress, and NOS activity was more than NR activity. The results indicated that both NOS and NR mediated NO production was involved in regulating the activity of antioxidative enzymes under drought stress. NOS pathway played a dominant role in this process.

Key words: soybean; drought sress; nitric oxide(NO); antioxidative enzymes

基金項(xiàng)目:國(guó)家自然科學(xué)基金項(xiàng)目(U1204305);河南省教育廳科學(xué)技術(shù)研究重點(diǎn)項(xiàng)目(13A180515);河南省基礎(chǔ)與前沿技術(shù)研究項(xiàng)目(132300410455)

干旱是影響世界上許多地區(qū)農(nóng)業(yè)生產(chǎn)的最主要環(huán)境因子之一。研究表明,全世界由于干旱造成的作物減產(chǎn)超過(guò)了其他逆境因子危害的總和[1]。干旱脅迫可以導(dǎo)致活性氧的過(guò)量積累,使得細(xì)胞內(nèi)自由基的代謝平衡遭到破壞,引發(fā)或加劇膜脂過(guò)氧化作用,造成細(xì)胞膜系統(tǒng)的損傷。植物在長(zhǎng)期的適應(yīng)過(guò)程中,形成了一整套清除活性氧的抗氧化酶防御體系,包括超氧化物歧化酶(SOD)、過(guò)氧化氫酶(CAT)、過(guò)氧化物酶(POD)等,這些酶已廣泛用于植物抗逆境反應(yīng)機(jī)理的研究[2]。目前在小麥[3]、大豆[4]、花生[5]等方面的研究工作均證明抗氧化酶活性與植物抗旱性有著密切的關(guān)系。然而,對(duì)于干旱脅迫下抗氧化酶活性的調(diào)節(jié)機(jī)制報(bào)道很少。

一氧化氮(NO)是生物體中重要的氧化還原信號(hào)分子,在植物體內(nèi)的酶促途徑主要是通過(guò)硝酸還原酶(NR)和一氧化氮合成酶(NOS)催化合成[6]。大量的研究表明,NO參與了植物生長(zhǎng)發(fā)育和環(huán)境脅迫響應(yīng)過(guò)程,如種子萌發(fā)、根生長(zhǎng)、細(xì)胞凋亡、防御相關(guān)基因的表達(dá)以及植物的耐逆反應(yīng)等[7,8]。近些年來(lái),NO對(duì)植物抗逆性調(diào)節(jié)作用的研究已經(jīng)受到了廣泛的重視。在小麥[3]、刺槐[9]、枳[10]等植物中的證據(jù)表明,施用外源NO可緩解干旱脅迫對(duì)植物造成的傷害,其原因與NO增強(qiáng)的抗氧化酶活性密切相關(guān)。但是,對(duì)于干旱脅迫下內(nèi)源NO是否參與了植物抗氧化酶活性的調(diào)節(jié)目前還不清楚。

目前對(duì)于干旱脅迫下NO與抗氧化酶關(guān)系的研究多集中于外源NO對(duì)抗氧化酶系活性的影響,很少探討干旱脅迫下內(nèi)源NO對(duì)調(diào)節(jié)抗氧化酶系的作用。本研究以大豆為試驗(yàn)材料,利用NO清除劑、NO產(chǎn)生途徑相關(guān)抑制劑處理,探討了干旱脅迫下內(nèi)源NO在調(diào)節(jié)抗氧化酶中的作用以及干旱脅迫下NO的產(chǎn)生來(lái)源,以期為大豆抗旱機(jī)理的研究和選育抗旱品種提供理論依據(jù)。

1材料與方法

1.1供試材料

供試大豆(Glycine max)品種為河南省大面積種植的豫豆19(種子由河南省農(nóng)業(yè)科學(xué)院提供)。

1.2材料培養(yǎng)

挑選大小一致的大豆種子,用5%次氯酸鈉消毒15 min后,用自來(lái)水反復(fù)沖洗干凈,放入水中浸泡3 h使種子充分吸脹,然后將種子于恒溫箱內(nèi)25 ℃黑暗條件下萌發(fā)2 d。挑選萌發(fā)一致的大豆種子,將其種在放有蛭石的托盤(pán)里,并用1/4 Hoagland溶液澆灌。培養(yǎng)條件:25 ℃,14 h光周期,相對(duì)濕度控制在70%。

1.3材料處理

以聚乙二醇(PEG)6000模擬干旱脅迫處理。將生長(zhǎng)3 d的幼苗取出洗凈后轉(zhuǎn)移到盛有10% PEG、 100 μmol/L 硝普鈉(SNP)、200 μmol/L N-硝基-L-精氨酸(L-NNA)、20 μmol/L疊氮化鈉(NaN3)和200 μmol/L 2-苯基-4,4,5,5-四甲基咪唑-1-氧-3-氧化物(PTIO)溶液的塑料容器中進(jìn)行不同處理。處理24 h后收集大豆主根用于各項(xiàng)指標(biāo)的測(cè)定。

1.4測(cè)定方法

相對(duì)電導(dǎo)率采用DDS-307A型電導(dǎo)率儀測(cè)定,參照Wang等[11]的方法;丙二醛(MDA)含量測(cè)定采用硫代巴比妥酸法[11];超氧化物歧化酶(SOD)活性測(cè)定采用氮藍(lán)四唑(NBT)光氧化還原法[12],SOD活性以抑制NBT光化還原的50%為一個(gè)酶活性單位;過(guò)氧化氫酶(CAT)活性測(cè)定參照Wang等[12]的方法,CAT活性以消耗1 μmol(H2O2)/min為一個(gè)酶活性單位;過(guò)氧化物酶(POD)活性測(cè)定采用愈創(chuàng)木酚法[12],以每分鐘A470 nm變化0.01為1個(gè)酶活性單位。NO含量通過(guò)氧合血紅蛋白向高鐵血紅蛋白轉(zhuǎn)化的量來(lái)計(jì)算,參照Murphy等[13]的方法;NOS和NR活性測(cè)定參照Tian等[14]的方法。所測(cè)指標(biāo)均選用大豆的主根為試驗(yàn)材料,3次重復(fù),取平均值。

2結(jié)果與分析

2.1干旱脅迫對(duì)大豆幼苗相對(duì)電導(dǎo)率和MDA含量的影響

相對(duì)電導(dǎo)率和MDA含量是反映植物細(xì)胞遭受傷害程度的常用指標(biāo)。由圖1可知,5% PEG處理大豆1 d,根中相對(duì)電導(dǎo)率和MDA含量略微增加;隨著PEG用量的增加,相對(duì)電導(dǎo)率和MDA含量表現(xiàn)出顯著增加的趨勢(shì)。從形態(tài)上看,15% PEG處理時(shí),植株表現(xiàn)出明顯的萎蔫現(xiàn)象;20%PEG處理時(shí)則表現(xiàn)出非常嚴(yán)重的萎蔫。表明輕度脅迫(5% PEG)對(duì)大豆相對(duì)電導(dǎo)率和MDA含量影響較小,重度脅迫(>15% PEG)對(duì)其影響則較大。根據(jù)以上試驗(yàn)結(jié)果,后續(xù)試驗(yàn)選定中等脅迫程度(10% PEG)進(jìn)行干旱脅迫處理。

2.2干旱脅迫下NO對(duì)大豆幼苗抗氧化酶活性的影響

如表1所示,PEG處理顯著增加了大豆根中SOD、CAT和POD的活性,比對(duì)照分別增加了59.7%、91.5%和75.9%。SNP(NO供體)處理則進(jìn)一步增加了干旱脅迫下大豆根中SOD、CAT和POD活性,分別比PEG處理增加了15.2%、27.9%和33.3%。結(jié)果表明,外源NO可增加干旱脅迫誘導(dǎo)的抗氧化酶活性。PTIO(NO清除劑)處理則抑制了干旱脅迫誘導(dǎo)的抗氧化酶活性,表現(xiàn)出與對(duì)照相近的水平,表明內(nèi)源NO參與了干旱脅迫誘導(dǎo)的抗氧化酶活性的調(diào)節(jié)。L-NNA(NOS抑制劑)處理顯著地抑制了干旱脅迫下大豆根中SOD、CAT和POD活性;NaN3(NR抑制劑)處理也抑制了干旱脅迫下大豆根中SOD、CAT和POD活性。這些結(jié)果表明,NOS和NR介導(dǎo)的NO產(chǎn)生途徑均參與了干旱脅迫下抗氧化酶活性的調(diào)節(jié),并且NOS途徑起著主導(dǎo)作用。

2.3干旱脅迫對(duì)大豆幼苗NO產(chǎn)生的影響

為了進(jìn)一步證實(shí)NO參與調(diào)節(jié)了干旱脅迫下抗氧化酶活性的誘導(dǎo),本試驗(yàn)檢測(cè)了干旱脅迫下NO的產(chǎn)生情況。如圖2所示,干旱脅迫下大豆根中NO產(chǎn)生情況與抗氧化酶活性變化趨勢(shì)一致。PEG處理增加了NO的產(chǎn)生,與對(duì)照相比增加了89.3%;PTIO處理則完全抑制了干旱脅迫誘導(dǎo)的NO產(chǎn)生;L-NNA和NaN3處理均抑制了干旱脅迫下NO產(chǎn)生,與PEG處理相比均顯著降低。這些結(jié)果進(jìn)一步表明,干旱脅迫下NO參與了抗氧化酶活性的調(diào)節(jié)。

2.4干旱脅迫對(duì)大豆幼苗NOS和NR活性的影響

為了進(jìn)一步弄清干旱脅迫下NO產(chǎn)生的來(lái)源,本試驗(yàn)檢測(cè)了干旱脅迫下NOS和NR活性的變化。如圖3所示,干旱脅迫下大豆根中NOS和NR活性變化趨勢(shì)與NO產(chǎn)生趨勢(shì)一致。PEG處理顯著增加了NOS和NR活性,且NOS比NR增加的幅度大。結(jié)果表明,干旱脅迫下NO來(lái)源于NOS和NR兩條途徑,但是主要來(lái)源于NOS途徑。

3討論

逆境脅迫下,植物體內(nèi)會(huì)產(chǎn)生大量的活性氧,對(duì)植物造成氧化傷害[10,15]。然而,植物在遭受逆境時(shí),其體內(nèi)也會(huì)產(chǎn)生相應(yīng)的活性氧清除機(jī)制。研究表明,SOD、CAT和POD是幾種重要的抗氧化酶,在清除活性氧保護(hù)植物避免遭受活性氧誘導(dǎo)的氧化傷害中發(fā)揮著重要的作用[16]。現(xiàn)已在小麥[3]、大豆[4]、刺槐[9]、枳[10]等多種植物中發(fā)現(xiàn)干旱脅迫誘導(dǎo)的抗氧化酶活性的增加與植物的抗旱性有關(guān)。但有關(guān)抗氧化酶系統(tǒng)與植物抗旱性關(guān)系的研究結(jié)果也不盡相同[2,17],這可能與植物的種類(lèi)、脅迫程度、抗氧化酶系統(tǒng)活性大小等因子的不同都有關(guān)系。本試驗(yàn)結(jié)果表明,干旱脅迫處理增強(qiáng)了大豆根中SOD、CAT和POD活性,說(shuō)明大豆根中也存在著類(lèi)似的活性氧清除的機(jī)制,這與莫紅等[2]在干旱脅迫下大豆葉片抗氧化酶活性的研究中得到的結(jié)論是一致的。

然而,目前對(duì)于干旱脅迫下抗氧化酶活性的調(diào)節(jié)機(jī)制仍不清楚。大量證據(jù)表明,NO參與了植物多種非生物脅迫響應(yīng),包括鹽脅迫、干旱脅迫、低溫脅迫、UV-B輻射等,并能增強(qiáng)植物對(duì)逆境的耐受性[10,18-20]。有證據(jù)表明,干旱脅迫下施用外源NO可增強(qiáng)抗氧化酶活性并緩解干旱脅迫對(duì)植物造成的傷害[3,10]。本試驗(yàn)結(jié)果也進(jìn)一步證實(shí)了外源NO可增強(qiáng)大豆根中抗氧化酶活性。這些研究結(jié)果表明NO可能參與了干旱脅迫條件下大豆中抗氧化酶活性的調(diào)節(jié),但是以前的研究都是集中在探討外源NO對(duì)抗氧化酶活性的影響,很少有探討干旱脅迫下內(nèi)源NO是否參與了大豆抗氧化酶活性調(diào)節(jié)的報(bào)道。

本試驗(yàn)結(jié)果表明,干旱脅迫處理增加了大豆根中NO的產(chǎn)生,當(dāng)干旱脅迫誘導(dǎo)的NO被抑制或被清除時(shí),干旱脅迫誘導(dǎo)的抗氧化酶活性也被抑制,表現(xiàn)出與NO產(chǎn)生呈正相關(guān)的關(guān)系,表明NO參與了干旱脅迫下抗氧化酶活性的調(diào)節(jié)。為了弄清干旱脅迫下內(nèi)源NO產(chǎn)生途徑,本試驗(yàn)中運(yùn)用了NOS和NR抑制劑。結(jié)果表明,NOS和NR抑制劑處理均抑制了干旱脅迫下NO的產(chǎn)生,并且NOS抑制劑處理比NR抑制劑處理抑制程度更大,表明干旱脅迫下NOS途徑和NR途徑干旱脅迫下均能產(chǎn)生NO,而且NOS途徑對(duì)NO產(chǎn)生的貢獻(xiàn)更大。為了進(jìn)一步證實(shí)這個(gè)結(jié)論,本試驗(yàn)檢測(cè)了干旱脅迫下NOS和NR活性。結(jié)果表明,干旱脅迫增加了NOS和NR活性,并且NOS活性的增幅比NR活性的增幅大,表明干旱脅迫下NOS途徑對(duì)NO的產(chǎn)生確實(shí)起著主要作用。以前有研究報(bào)道,NOS介導(dǎo)的NO產(chǎn)生參與了擬南芥中鹽脅迫響應(yīng)[21],而在紅蕓豆中發(fā)現(xiàn)NR介導(dǎo)的NO參與了鹽脅迫響應(yīng)[19]。此外,干旱脅迫條件下NOS介導(dǎo)的NO產(chǎn)生參與了枳中干旱脅迫響應(yīng)[10]。這些研究結(jié)果表明,植物中NOS途徑或NR途徑對(duì)NO產(chǎn)生貢獻(xiàn)的大小可能取決于植物種類(lèi)和環(huán)境脅迫的種類(lèi)。

綜上所述,本試驗(yàn)結(jié)果表明,干旱脅迫刺激了大豆根中NOS和NR活性的增加,從而增強(qiáng)了NO產(chǎn)生;增強(qiáng)的NO使干旱脅迫下抗氧化酶活性增強(qiáng),使得植物體內(nèi)避免活性氧的過(guò)量累積而對(duì)植物細(xì)胞造成氧化傷害,從而增強(qiáng)植物的抗旱性。但對(duì)于干旱脅迫下NO對(duì)抗氧化酶活性的調(diào)節(jié)機(jī)制還需要進(jìn)一步研究。

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[5] 陳由強(qiáng),葉冰瑩,朱錦懋,等.滲透協(xié)迫對(duì)花生葉活性氧傷害和膜脂過(guò)氧化作用的影響[J].中國(guó)油料作物學(xué)報(bào),2000,22(1):53-56.

[6] 李早霞,趙軍.一氧化氮對(duì)植物抗旱和抗病性的調(diào)控[J].中國(guó)農(nóng)業(yè)科技導(dǎo)報(bào),2008,10(S1):7-11.

[7] WENDEHENNE D, PUGIN A, KLESSIG D F, et al. Nitric oxide: Comparative synthesis and signaling in animal and plant cells[J]. Trends Plant Science,2001,6(4):177-183.

[8] NEILL S J, DESIKAN R, HANCOCK J T. Nitric oxide signaling in plants [J]. New Phytologist,2003,159(1):11-35.

[9] 李建新,任永信.外源NO對(duì)干旱下刺槐幼苗抗氧化特性的影響[J].河南農(nóng)業(yè)科學(xué),2012,41(11):129-131.

[10] FAN Q J, LIU J H. Nitric oxide is involved in dehydration/drought tolerance in Poncirus trifoliata seedlings through regulation of antioxidant systems and stomatal response[J].Plant Cell Reports,2012,31(1):145-154.

[11] WANG H H,HUANG J J, LIANG X L, et al. Involvement of hydrogen peroxide, calcium, and ethylene in the induction of the alternative pathway in chilling-stressed Arabidopsis callus[J]. Planta,2012,235(1):53-67.

[12] WANG H H, HUANG J J, BI Y R. Nitrate reductase-dependent nitric oxide production is involved in aluminum tolerance in red kidney bean roots[J]. Plant Science,2010,179(3):281-288.

[13] MURPHY M E,NOACK E.Nitric oxide assay using hemoglobin method[J].Methods Enzymol,1994,233:240-250.

[14] TIAN Q Y, SUN D H, ZHAO M G,et al. Inhibition of nitric oxide synthase(NOS) underlies aluminum-induced inhibition of root elongation in Hibiscus moscheutos[J].New Phytologist,2007,174(2):322-331.

[15] WANG H H, LIANG X L, HUANG J J, et al. Involvement of ethylene and hydrogen peroxide in induction of alternative respiratory pathway in salt-treated Arabidopsis calluses[J]. Plant and Cell Physiology, 2010,51(10):1754-1765.

[16] MILLER G, SUZUKI N, CIFTCI-YILMAZ S, et al. Reactive oxygen species homeostasis and signaling during drought and salinity stresses[J].Plant Cell Environ,2010,33(4):453-467.

[17] 王啟明,徐心誠(chéng),馬原松,等.干旱脅迫下大豆開(kāi)花期的生理生化變化與抗旱性的關(guān)系[J].干旱地區(qū)農(nóng)業(yè)研究,2004,23(4):98-102.

[18] SHI S Y, WANG G, WANG L G, et al. Protective effect of nitric oxide against oxidative stress under ultraviolet-B radiation [J].Nitric Oxide, 2005,13(1):1-9.

[19] LIU Y G, WU R R, WAN Q, et al. Glucose-6-phosphate dehydrogenase plays a pivotal role in nitric oxide-involved defense against oxidative stress under salt stress in red kidney bean roots[J].Plant and Cell Physiology,2007,48(3):511-522.

[20] ZHAO M G, CHEN L, ZHANG L L, et al. Nitric reductase-dependent nitric oxide production is involved in cold acclimation and freezing tolerance in Arabidopsis[J].Plant Physiology, 2009,151(2):755-767.

[21] ZHAO M G, TIAN Q Y, ZHANG W H. Nitrate oxide synthase-dependent nitric oxide production is associated with salt tolerance in Arabidopsis[J].Plant Physiology,2007,144(1):206-217.

[8] NEILL S J, DESIKAN R, HANCOCK J T. Nitric oxide signaling in plants [J]. New Phytologist,2003,159(1):11-35.

[9] 李建新,任永信.外源NO對(duì)干旱下刺槐幼苗抗氧化特性的影響[J].河南農(nóng)業(yè)科學(xué),2012,41(11):129-131.

[10] FAN Q J, LIU J H. Nitric oxide is involved in dehydration/drought tolerance in Poncirus trifoliata seedlings through regulation of antioxidant systems and stomatal response[J].Plant Cell Reports,2012,31(1):145-154.

[11] WANG H H,HUANG J J, LIANG X L, et al. Involvement of hydrogen peroxide, calcium, and ethylene in the induction of the alternative pathway in chilling-stressed Arabidopsis callus[J]. Planta,2012,235(1):53-67.

[12] WANG H H, HUANG J J, BI Y R. Nitrate reductase-dependent nitric oxide production is involved in aluminum tolerance in red kidney bean roots[J]. Plant Science,2010,179(3):281-288.

[13] MURPHY M E,NOACK E.Nitric oxide assay using hemoglobin method[J].Methods Enzymol,1994,233:240-250.

[14] TIAN Q Y, SUN D H, ZHAO M G,et al. Inhibition of nitric oxide synthase(NOS) underlies aluminum-induced inhibition of root elongation in Hibiscus moscheutos[J].New Phytologist,2007,174(2):322-331.

[15] WANG H H, LIANG X L, HUANG J J, et al. Involvement of ethylene and hydrogen peroxide in induction of alternative respiratory pathway in salt-treated Arabidopsis calluses[J]. Plant and Cell Physiology, 2010,51(10):1754-1765.

[16] MILLER G, SUZUKI N, CIFTCI-YILMAZ S, et al. Reactive oxygen species homeostasis and signaling during drought and salinity stresses[J].Plant Cell Environ,2010,33(4):453-467.

[17] 王啟明,徐心誠(chéng),馬原松,等.干旱脅迫下大豆開(kāi)花期的生理生化變化與抗旱性的關(guān)系[J].干旱地區(qū)農(nóng)業(yè)研究,2004,23(4):98-102.

[18] SHI S Y, WANG G, WANG L G, et al. Protective effect of nitric oxide against oxidative stress under ultraviolet-B radiation [J].Nitric Oxide, 2005,13(1):1-9.

[19] LIU Y G, WU R R, WAN Q, et al. Glucose-6-phosphate dehydrogenase plays a pivotal role in nitric oxide-involved defense against oxidative stress under salt stress in red kidney bean roots[J].Plant and Cell Physiology,2007,48(3):511-522.

[20] ZHAO M G, CHEN L, ZHANG L L, et al. Nitric reductase-dependent nitric oxide production is involved in cold acclimation and freezing tolerance in Arabidopsis[J].Plant Physiology, 2009,151(2):755-767.

[21] ZHAO M G, TIAN Q Y, ZHANG W H. Nitrate oxide synthase-dependent nitric oxide production is associated with salt tolerance in Arabidopsis[J].Plant Physiology,2007,144(1):206-217.

[8] NEILL S J, DESIKAN R, HANCOCK J T. Nitric oxide signaling in plants [J]. New Phytologist,2003,159(1):11-35.

[9] 李建新,任永信.外源NO對(duì)干旱下刺槐幼苗抗氧化特性的影響[J].河南農(nóng)業(yè)科學(xué),2012,41(11):129-131.

[10] FAN Q J, LIU J H. Nitric oxide is involved in dehydration/drought tolerance in Poncirus trifoliata seedlings through regulation of antioxidant systems and stomatal response[J].Plant Cell Reports,2012,31(1):145-154.

[11] WANG H H,HUANG J J, LIANG X L, et al. Involvement of hydrogen peroxide, calcium, and ethylene in the induction of the alternative pathway in chilling-stressed Arabidopsis callus[J]. Planta,2012,235(1):53-67.

[12] WANG H H, HUANG J J, BI Y R. Nitrate reductase-dependent nitric oxide production is involved in aluminum tolerance in red kidney bean roots[J]. Plant Science,2010,179(3):281-288.

[13] MURPHY M E,NOACK E.Nitric oxide assay using hemoglobin method[J].Methods Enzymol,1994,233:240-250.

[14] TIAN Q Y, SUN D H, ZHAO M G,et al. Inhibition of nitric oxide synthase(NOS) underlies aluminum-induced inhibition of root elongation in Hibiscus moscheutos[J].New Phytologist,2007,174(2):322-331.

[15] WANG H H, LIANG X L, HUANG J J, et al. Involvement of ethylene and hydrogen peroxide in induction of alternative respiratory pathway in salt-treated Arabidopsis calluses[J]. Plant and Cell Physiology, 2010,51(10):1754-1765.

[16] MILLER G, SUZUKI N, CIFTCI-YILMAZ S, et al. Reactive oxygen species homeostasis and signaling during drought and salinity stresses[J].Plant Cell Environ,2010,33(4):453-467.

[17] 王啟明,徐心誠(chéng),馬原松,等.干旱脅迫下大豆開(kāi)花期的生理生化變化與抗旱性的關(guān)系[J].干旱地區(qū)農(nóng)業(yè)研究,2004,23(4):98-102.

[18] SHI S Y, WANG G, WANG L G, et al. Protective effect of nitric oxide against oxidative stress under ultraviolet-B radiation [J].Nitric Oxide, 2005,13(1):1-9.

[19] LIU Y G, WU R R, WAN Q, et al. Glucose-6-phosphate dehydrogenase plays a pivotal role in nitric oxide-involved defense against oxidative stress under salt stress in red kidney bean roots[J].Plant and Cell Physiology,2007,48(3):511-522.

[20] ZHAO M G, CHEN L, ZHANG L L, et al. Nitric reductase-dependent nitric oxide production is involved in cold acclimation and freezing tolerance in Arabidopsis[J].Plant Physiology, 2009,151(2):755-767.

[21] ZHAO M G, TIAN Q Y, ZHANG W H. Nitrate oxide synthase-dependent nitric oxide production is associated with salt tolerance in Arabidopsis[J].Plant Physiology,2007,144(1):206-217.