劉建新 王金成 劉秀麗 王風(fēng)琴

(1.隴東學(xué)院生命科學(xué)與技術(shù)學(xué)院,慶陽 745000； 2.甘肅省高校隴東生物資源保護(hù)與利用省級(jí)重點(diǎn)實(shí)驗(yàn)室,慶陽 745000)

外源H2O2對(duì)混合鹽堿脅迫下燕麥幼苗葉片脯氨酸積累和代謝途徑的影響

劉建新 王金成 劉秀麗 王風(fēng)琴

(1.隴東學(xué)院生命科學(xué)與技術(shù)學(xué)院,慶陽 745000；2.甘肅省高校隴東生物資源保護(hù)與利用省級(jí)重點(diǎn)實(shí)驗(yàn)室,慶陽 745000)

為探討H2O2對(duì)鹽堿脅迫下植物脯氨酸代謝的調(diào)控機(jī)理，以燕麥新品種‘定莜6號(hào)’幼苗為材料，采用水培法研究了外源H2O2對(duì)混合鹽堿脅迫下燕麥脯氨酸積累和代謝途徑的影響。結(jié)果表明，75 mmol·L-1混合鹽堿(NaCl∶Na2SO4∶NaHCO3∶Na2CO3=12∶8∶9∶1)脅迫可促進(jìn)燕麥幼苗葉片脯氨酸的積累，提高脯氨酸合成的鳥氨酸途徑關(guān)鍵酶鳥氨酸δ-氨基轉(zhuǎn)移酶(δ-OAT)活性，抑制脯氨酸合成的谷氨酸途徑關(guān)鍵酶Δ1-吡咯啉-5-羧酸合成酶(P5CS)及脯氨酸降解限速酶脯氨酸脫氫酶(ProDH)活性。在75 mmol·L-1混合鹽堿脅迫下添加0.01～1 000 μmol·L-1H2O2可顯著提高燕麥幼苗葉片的脯氨酸含量，其中10 μmol·L-1H2O2的作用最明顯；10 μmol·L-1H2O2上調(diào)了75 mmol·L-1混合鹽堿脅迫下燕麥幼苗葉片的P5CS和δ-OAT活性，降低了ProDH活性。此外，10 μmol·L-1H2O2使75 mmol·L-1混合鹽堿脅迫下燕麥幼苗葉片內(nèi)源性H2O2含量急劇升高后迅速降低。表明外源H2O2能夠提高混合鹽堿脅迫下燕麥幼苗內(nèi)源H2O2的含量，并通過活化脯氨酸合成的谷氨酸途徑和鳥氨酸途徑，抑制脯氨酸的降解，促進(jìn)混合鹽堿脅迫下燕麥幼苗脯氨酸的積累。

燕麥;混合鹽堿脅迫;過氧化氫;脯氨酸代謝

過氧化氫(hydrogen peroxide,H2O2)是植物細(xì)胞代謝過程中產(chǎn)生的一種活性氧(reactive oxygen species,ROS)，高濃度時(shí)可損傷生物大分子，而低濃度的H2O2是一種信號(hào)分子，參與逆境脅迫應(yīng)答[7]，提高植物的耐鹽性[8]。有研究表明，外源H2O2預(yù)處理可提高脅迫蛋白的表達(dá)減輕鹽脅迫對(duì)小麥幼苗的氧化傷害[9]，誘導(dǎo)抗氧化防御機(jī)制產(chǎn)生提高水稻的耐鹽性[10]。鹽堿脅迫導(dǎo)致植物水分虧缺和碳氮代謝失調(diào)，碳氮代謝轉(zhuǎn)向滲透溶質(zhì)積累以適應(yīng)環(huán)境脅迫[11]。脯氨酸(proline,Pro)是其中一種重要的滲透調(diào)節(jié)物質(zhì)，并具有清除ROS，減輕環(huán)境脅迫對(duì)植物傷害等作用[12]。植物體內(nèi)Pro的合成有兩條途徑：一是谷氨酸(glutanate,Glu)途徑，Δ1-吡咯啉-5-羧酸合成酶(Δ1-pyrroline-5-carboxylate synthetase,P5CS)是關(guān)鍵酶；二是鳥氨酸(ornithine,Orn)途徑，Orn-δ-氨基轉(zhuǎn)移酶(ornithine-δ-aminotransferase,δ-OAT)是限速酶；Pro降解的關(guān)鍵酶是脯氨酸脫氫酶(proline dehydrogenase,ProDH)。其中P5CS定位在細(xì)胞質(zhì)中，而δ-OAT和ProDH存在于線粒體中[13]?；旌消}堿脅迫可誘導(dǎo)青山楊(Populuspseudo-cathayana×P.deltoides)Pro的積累[1]；外源H2O2能夠通過激活白刺(NitrariatangutorumBobr)Glu激酶活性和降低ProDH活性促進(jìn)Pro積累[14]；并通過提高可溶性糖和谷胱甘肽含量增強(qiáng)小麥(Triticumaestivum)幼苗對(duì)NaCl脅迫的抗性[15]。然而，外源H2O2對(duì)混合鹽堿脅迫下植物Pro積累及其代謝途徑影響的研究尚未見報(bào)道。

燕麥(Avenanuda)是一種喜陰涼、耐鹽堿的糧飼兼用型作物，其籽粒有獨(dú)特的營(yíng)養(yǎng)和保健功效[16]，被稱為鹽堿地改良的先鋒作物，在我國(guó)內(nèi)蒙古、河北、山西、甘肅等省區(qū)廣泛種植，年種植面積達(dá)55萬hm2[17]?！ㄝ?號(hào)’燕麥?zhǔn)歉拭C省定西市旱作農(nóng)業(yè)科研推廣中心選育的燕麥新品種，具有抗旱、豐產(chǎn)和品質(zhì)優(yōu)等特點(diǎn)?！ㄝ?號(hào)’幼苗對(duì)鹽脅迫的響應(yīng)及H2O2對(duì)響應(yīng)的調(diào)節(jié)已有研究報(bào)道[18]，為進(jìn)一步探討H2O2對(duì)其混合鹽堿脅迫下Pro代謝的調(diào)節(jié)機(jī)制，根據(jù)甘肅省燕麥栽培地土壤鹽分的組成，將兩種中性鹽NaCl、Na2SO4和兩種堿性鹽NaHCO3、Na2CO3按不同摩爾質(zhì)量比例混合，研究外源H2O2對(duì)混合鹽堿脅迫下‘定莜6號(hào)’幼苗Pro積累和代謝途徑的影響，以期為進(jìn)一步闡明燕麥耐鹽堿機(jī)理提供理論依據(jù)。

1 材料與方法

1.1 材料來源和混合鹽堿溶液配比

1.2 材料培養(yǎng)和實(shí)驗(yàn)設(shè)計(jì)

燕麥種子用1% CuSO4溶液表面消毒20 min，浸種6 h后播種在墊有3層濕潤(rùn)吸水紙的瓷盤中，置培養(yǎng)箱中25℃催芽3 d，挑選發(fā)芽一致的萌發(fā)種子播種在塑料盆(上口徑20 cm，高14 cm)的珍珠巖中，澆水后置日光溫室培養(yǎng)，晝/夜溫度(22～33)℃/(15～19)℃，相對(duì)濕度65%～75%，光照強(qiáng)度485～690 μmol·m-2·s-1。幼苗長(zhǎng)至2葉1心時(shí)選生長(zhǎng)一致的壯苗，經(jīng)自來水和蒸餾水沖洗后，移栽至裝有5 L Hoagland完全營(yíng)養(yǎng)液的水培池中培養(yǎng)，每池定植240株左右，營(yíng)養(yǎng)液每2 d更換1次。培養(yǎng)7 d后進(jìn)行如下處理：(1)Hoagland營(yíng)養(yǎng)液，對(duì)照(CK)；(2)含75 mmol·L-1混合鹽堿的Hoagland溶液，CSAS(complex saline-alkali stress)；(3)含75 mmol·L-1混合鹽堿和10 μmol·L-1H2O2的Hoagland溶液，CSAS+ H2O2；(4)含10 μmol·L-1H2O2的Hoagland營(yíng)養(yǎng)液，H2O2。對(duì)2葉1心期的燕麥幼苗用不同濃度的混合鹽堿處理的預(yù)備實(shí)驗(yàn)中發(fā)現(xiàn)，75 mmol·L-1濃度處理下植株生物量顯著下降，且葉色暗綠，株高明顯降低，但植株能夠承受脅迫而不會(huì)導(dǎo)致死亡。因此，選用75 mmol·L-1作為混合鹽堿脅迫的濃度。實(shí)驗(yàn)處理過程中每天更換1次溶液。

1.3 測(cè)定指標(biāo)和方法

1.3.1H2O2含量

H2O2含量測(cè)定采用Sergiev[19]的方法略有改動(dòng)：稱取0.50 g葉片，用預(yù)冷的5 mL 0.001 g·L-1三氯乙酸研磨，以每分鐘15 000轉(zhuǎn)離心20 min，取0.7 mL上清液加0.7 mL 10 mmol·L-1磷酸緩沖液(pH7)和1.4 mL 1 mol·L-1的KI，測(cè)定390 nm波長(zhǎng)的吸光值，由標(biāo)準(zhǔn)曲線計(jì)算出單位鮮重材料所含H2O2的含量。

1.3.2 Pro含量

采用李合生[20]的磺基水楊酸-酸性茚三酮顯色法測(cè)定波長(zhǎng)520 nm吸光值，以Pro梯度溶液做標(biāo)準(zhǔn)曲線計(jì)算Pro含量，結(jié)果以單位鮮重材料所含Pro的μg數(shù)表示。

1.3.3 P5CS、δ-OAT和ProDH活性

P5CS、δ-OAT和ProDH活性均按趙貴林等[21]的方法測(cè)定。以每小時(shí)ΔOD535變化0.01表示一個(gè)P5CS活性單位(U)；以每小時(shí)生成1 mmol P5C的量為一個(gè)δ-OAT活性單位(U)；以分鐘ΔOD600減少0.001為一個(gè)ProDH活性單位(U)。所有酶活性以每g鮮重材料所含的活性單位數(shù)表示。

1.4 統(tǒng)計(jì)分析

2 結(jié)果與分析

2.1H2O2實(shí)驗(yàn)濃度的篩選

為確定混合鹽堿脅迫下對(duì)Pro積累影響的適宜H2O2處理濃度，在75 mmol·L-1混合鹽堿脅迫下分別添加0、0.01、0.1、1、10、100和1 000 μmol·L-1的H2O2溶液處理燕麥幼苗6 d，并對(duì)幼苗葉片Pro含量進(jìn)行分析。結(jié)果如圖1所示，隨著H2O2濃度的遞增，混合鹽堿脅迫下燕麥幼苗葉片內(nèi)的Pro含量顯著提升。當(dāng)H2O2濃度為10 μmol·L-1時(shí)Pro含量出現(xiàn)最大值，是未經(jīng)H2O2處理的7.1倍(P<0.05)。爾后，隨H2O2濃度增加，Pro含量不斷降低，但仍高于未經(jīng)H2O2處理的幼苗。此外，不同濃度H2O2對(duì)未經(jīng)混合鹽堿脅迫的燕麥幼苗葉片中Pro含量的影響與混合鹽堿脅迫下的變化趨勢(shì)類似，只是Pro含量明顯偏低。因此，選用10 μmol·L-1H2O2作為混合鹽堿脅迫下對(duì)Pro代謝影響的實(shí)驗(yàn)濃度。

圖1 不同濃度H2O2對(duì)75 mmol·L-1混合鹽堿脅迫下燕麥幼苗葉片脯氨酸含量的影響Fig.1 Effect of different concentration of H2O2 on proline contents in leaves of oat seedlings under complex saline-alkali stress

2.2外源H2O2對(duì)混合鹽堿脅迫下燕麥葉片Pro和H2O2含量的影響

未經(jīng)混合鹽堿脅迫的燕麥幼苗葉片Pro含量在處理期間無明顯變化(P>0.05)；75 mmol·L-1混合鹽堿脅迫顯著提高了燕麥葉片的Pro含量，并隨處理時(shí)間延長(zhǎng)呈逐漸上升趨勢(shì)，脅迫6 d時(shí)的Pro含量比CK提高了5.0倍；添加10 μmol·L-1H2O2不僅能提高正常生長(zhǎng)燕麥幼苗葉片的Pro含量，更能顯著增加混合鹽堿脅迫下燕麥葉片的Pro含量(圖2)。表明外源H2O2可促進(jìn)混合鹽堿脅迫下燕麥幼苗Pro的積累。

圖2 10 μmol·L-1 H2O2對(duì)75 mmol·L-1混合鹽堿脅迫下燕麥幼苗葉片脯氨酸和內(nèi)源H2O2含量的影響CK.對(duì)照；CSAS.混合鹽堿脅迫；CSAS+H2O2.混合鹽堿脅迫+過氧化氫；H2O2.過氧化氫 圖中不同字母表示同一時(shí)間不同處理在5%水平差異顯著，下同。Fig.2 Effect of 10 μmol·L-1 H2O2 on contents of proline and endogenous H2O2 in leaves of oat seedlings under 75 mmol·L-1 complex saline-alkali stress CK.Control; CSAS.Complex saline-alkali stress; CSAS+H2O2.Complex saline-alkali stress+Hydrogen peroxide; H2O2.Hydrogen peroxide Bars superscripted with different letters are significantly different at 0.05 levels for the same time,the same as below.

圖3 10 μmol·L-1 H2O2對(duì)75 mmol·L-1混合鹽堿脅迫下燕麥幼苗葉片脯氨酸代謝關(guān)鍵酶活性的影響Fig.3 Effect of exogenous H2O2 on activities of key enzymes of proline biosynthesis in leaves of oat seedlings under complex saline-alkali stress

CK燕麥幼苗葉片H2O2含量在處理期間變化不大(P>0.05)；單獨(dú)H2O2處理使燕麥葉片H2O2含量隨處理時(shí)間延長(zhǎng)不斷提高(P<0.05)；混合鹽堿脅迫和混合鹽堿脅迫下添加H2O2處理均使燕麥葉片H2O2含量隨處理時(shí)間延長(zhǎng)呈快速升高之后急劇下降變化，處理1 d時(shí)H2O2均出現(xiàn)峰值，且處理第1和2 d的H2O2含量表現(xiàn)為混合鹽堿脅迫下添加H2O2處理顯著大于單獨(dú)混合鹽堿脅迫處理，但在處理的第3～6 d時(shí)H2O2含量變化卻相反(圖2)。

2.3外源H2O2對(duì)混合鹽堿脅迫下燕麥幼苗葉片Pro代謝關(guān)鍵酶活性的影響

P5CS和δ-OAT分別是Pro生物合成的Glu途徑和Orn途徑關(guān)鍵酶；而ProDH是Pro降解途徑的限速酶[13]。如圖3所示，與CK相比，混合鹽堿脅迫顯著抑制了燕麥幼苗葉片P5CS和ProDH活性，卻顯著增強(qiáng)了δ-OAT活性，受抑和增強(qiáng)的程度隨脅迫時(shí)間延長(zhǎng)而提高；外源H2O2雖未引起正常生長(zhǎng)燕麥幼苗葉片在處理期間δ-OAT活性的明顯改變(P>0.05)，但顯著提高了正常生長(zhǎng)燕麥幼苗葉片中P5CS的活性及混合鹽堿脅迫下燕麥葉片的P5CS和δ-OAT活性，增幅隨處理時(shí)間延長(zhǎng)而提高(P<0.05)，脅迫6 d時(shí)增幅分別為72.6%、151.1%和39.8%。外施H2O2使正常生長(zhǎng)和混合鹽堿脅迫燕麥幼苗葉片的ProDH活性隨處理時(shí)間延長(zhǎng)不斷下降，脅迫6 d時(shí)降幅分別達(dá)67.2%%和61.7%。說明外源H2O2促進(jìn)混合鹽堿脅迫下燕麥幼苗Pro的合成，抑制Pro的降解。

3 討論

Wahid等[2]研究表明，鹽脅迫可誘導(dǎo)H2O2產(chǎn)生并積累，進(jìn)而調(diào)節(jié)小麥的抗鹽反應(yīng)。本研究顯示，75 mmol·L-1混合鹽堿脅迫1 d即可引起燕麥幼苗體內(nèi)H2O2爆發(fā)式驟增，然后隨脅迫時(shí)間延長(zhǎng)逐漸下降，說明H2O2可能作為信使分子參與燕麥對(duì)混合鹽堿脅迫的響應(yīng)。添加10 μmol·L-1外源H2O2處理進(jìn)一步提高了混合鹽堿脅迫前期(1～3 d)燕麥幼苗葉片的H2O2含量，但脅迫后期(4～6 d)H2O2含量明顯下降(圖2)，這可能是外源H2O2處理提高的內(nèi)源H2O2作為信號(hào)分子刺激了抗氧化系統(tǒng)的應(yīng)答，增強(qiáng)了抗氧化酶活性和相關(guān)基因表達(dá)[9]，從而降低了混合鹽堿脅迫后期燕麥幼苗H2O2的積累。

滲透調(diào)節(jié)是植物適應(yīng)鹽堿脅迫的重要生理機(jī)制，而Pro被認(rèn)為是最重要的滲透調(diào)節(jié)物質(zhì)之一[22]。閆永慶等[1]研究表明，混合鹽堿脅迫誘導(dǎo)青山楊葉片Pro積累。外源H2O2處理能夠提高小麥幼苗的耐鹽性[15]。本研究表明，0.01～1 000 μmol·L-1的外源H2O2能夠提高75 mmol·L-1混合鹽堿脅迫6 d燕麥幼苗葉片的Pro含量，其中10 μmol·L-1H2O2促進(jìn)Pro積累的作用最明顯(圖1)，且10 μmol·L-1H2O2處理下燕麥葉片的Pro含量隨脅迫時(shí)間延長(zhǎng)不斷增加(圖2)，結(jié)合外源H2O2能夠提高混合鹽堿脅迫前期(1～3 d)燕麥幼苗葉片H2O2含量的結(jié)果，表明外源H2O2處理可能通過提高內(nèi)源H2O2含量參與鹽堿脅迫下燕麥Pro積累的調(diào)控。然而，外源H2O2調(diào)控鹽堿脅迫植物Pro積累的機(jī)制目前尚不清楚。本研究表明，混合鹽堿脅迫下，燕麥幼苗葉片Pro合成的Glu途徑關(guān)鍵酶P5CS和Pro降解限速酶ProDH活性顯著下降，而Orn途徑關(guān)鍵酶δ-OAT活性明顯增強(qiáng)，降幅和增幅均隨脅迫時(shí)間延長(zhǎng)持續(xù)提高(圖3)。說明混合鹽堿脅迫燕麥Pro的積累是以鳥氨酸途徑為主的合成增加和降解減少共同作用的結(jié)果。這與夏方山等[23]以堿地風(fēng)毛菊(Saussurearuncinata)在堿性鹽脅迫下的結(jié)果一致，而與董秋麗等[24]在芨芨草(Achnatherumsplendens)上的研究結(jié)果不同?；旌消}堿脅迫下燕麥幼苗P5CS活性下降可能是Pro積累后反饋抑制的結(jié)果，而δ-OAT活性卻不受此影響[13]。進(jìn)一步研究表明，10 μmol·L-1H2O2可顯著提高混合鹽堿脅迫下燕麥幼苗P5CS和δ-OAT活性，抑制ProDH活性，并且這種作用程度隨脅迫時(shí)間延長(zhǎng)而增強(qiáng)(圖3)。說明外源H2O2通過活化Pro合成的Glu和Orn途徑及抑制Pro的降解過程上調(diào)了混合鹽堿脅迫下燕麥幼苗Pro的積累。H2O2作為信號(hào)分子在感知脅迫信號(hào)和調(diào)控防御系統(tǒng)響應(yīng)，提高植物抗逆反應(yīng)中發(fā)揮著重要作用[25]。然而，Pro積累是涉及許多因素調(diào)控的復(fù)雜信號(hào)轉(zhuǎn)導(dǎo)過程[26]。H2O2信號(hào)調(diào)控植物Pro代謝途徑的分子機(jī)理及與其它信號(hào)的交叉對(duì)話機(jī)制還需進(jìn)一步深入探究。

4 結(jié)論

混合鹽堿脅迫下，外源H2O2可誘導(dǎo)燕麥幼苗葉片內(nèi)H2O2含量急劇增加后快速下降，Pro不斷積累；外源H2O2提高混合鹽堿脅迫下燕麥幼苗Pro代謝關(guān)鍵酶P5CS、δ-OAT活性和抑制分解代謝限速酶ProDH活性與其促進(jìn)Pro的積累密切相關(guān)。

1.閆永慶,王文杰,朱虹,等.混合鹽堿脅迫對(duì)青山楊滲透調(diào)節(jié)物質(zhì)及活性氧代謝的影響[J].應(yīng)用生態(tài)學(xué)報(bào),2009,20(9):2085-2091.

Yan Yongqing,Wang Wenjie,Zhu Hong,et al.Effects of salt-alkali stress on osmoregulation substance and active oxygen metabolism of Qingshan poplar(Populuspseudo-cathayana×P.deltoides)[J].Chinese Journal of Applied Ecology,2009,20(9):2085-2091.

2.Wahid A,Perveen M,Gelani S,et al.Pretreatment of seed with H2O2improves salt tolerance of wheat seedlings by alleviation of oxidative damage and expression of stress proteins[J].Journal of Plant Physiology,2007,164(3):283-294.

3.Tanou G,Molassiotis A,Diamantidis G.Hydrogen peroxide and nitric oxide-induced systemic antioxidant primelike activity under NaCl-stress and stress-free conditions incitrus plants[J].Journal of Plant Physiology,2009,166(17):1904 -1913.

4.劉建新,王金成,王鑫,等.外源NO對(duì)NaHCO3脅迫下黑麥草幼苗光合生理響應(yīng)的調(diào)節(jié)[J].生態(tài)學(xué)報(bào),2012,32(11):3460-3466.

Liu Jianxin,Wang Jincheng,Wang Xin,et al.Regulation of exogenous nitric oxide on photosynthetic physiological response ofLoliumperenneseedlings under NaHCO3Stress[J].Acta Ecologica Sinica,2012,32(11):3460-3466.

5.顏宏,趙偉,盛艷敏,等.堿脅迫對(duì)羊草和向日葵的影響[J].應(yīng)用生態(tài)學(xué)報(bào),2005,16(8):1497-1501.

Yan Hong,Zhao Wei,Sheng Yanmin,et al.Effects of alkali-stress onAneurolepidiumchinenseandHelianthusannuus[J].Chinese Journal of Applied Ecology,2005,16(8):1497-1501.

6.Shi D C,Sheng Y M.Effect of various salt-alkaline mixed stress conditions on sunflower seedlings and analysis of their stress factors[J].Environmental and Experimental Botany,2005,54(1):8-21.

7.馮漢青,白晶月,管冬冬,等.胞外H2O2及NADPH氧化酶參與了銅脅迫對(duì)植物細(xì)胞死亡的誘導(dǎo)[J].植物研究,2015,35(5):710-715.

Feng Hanqing,Bai Jingyue,Guan Dongdong,et al.Extracellular H2O2and NADPH Oxidase are Involved in the Copper-Induced Cell Death[J].Bulletin of Botanical Research,2015,35(5):710-715.

8.Jand-Venes Rolim Medeiros,Enéas Gomes-Filho.Hydrogen peroxide pre-treatment induces salt-stress acclimation in maize plants[J].Journal of Plant Physiology,2005,162(10):1114-1122.

9.Abdul Wahid,Mubaraka Perveena,Sadia Gelania,et al.Pretreatment of seed with H2O2improves salt tolerance of wheat seedlings by alleviation of oxidative damage and expression of stress proteins[J].Journal of Plant Physiology,2005,164(3):283-294.

10.Uchida A,Jagendorf A T,Hibino T,et al.Effects of hydrogen peroxide and nitric oxide on both salt and heat stress tolerance in rice[J].Plant Science,2002,163(3):515-523.

11.Shi D C,Wang D L.Effects of various salt-alkaline mixed stresses onAncurolepidiumchinense(Trin.) Kitag[J].Plant and Soil,2005,271(1-2):15-26.

12.Dinakar N,Nagajyothi P C,Suresh S,et al.Cadmium induced changes on proline,antioxidant enzymes,nitrate and nitrite reductases inArachishypogaeaL.[J].Journal of Environmental Biology,2009,30(2):289-294.

13.Kavi Kishor P B,Sangam S.Regulation of proline biosynthesis,degradation,uptake and transport in higher plants:Its implications in plant growth and abiotic stress tolerance[J].Current Science,2005,88(3):424-438.

14.張園園,呂秀軍,張輝,等.外源H2O2處理對(duì)唐古特白刺愈傷組織脯氨酸代謝的影響[J].植物研究,2011,31(2):213-217.

Zhang Yuanyuan,Lü Xiujun,Zhang Hui,et al.Effect of exogenous hydrogen peroxide on the proline metabolism inNitrariatangutorumBobr.Callus[J].Bulletin of Botanical Research,2011,31(2):213-217.

15.張波,張懷剛.外源H2O2對(duì)小麥幼苗耐鹽性的調(diào)節(jié)作用[J].西北植物學(xué)報(bào),2007,27(12):2491-2495.

Zhang Bo,Zhang Huaigang.Regulation of exogenous hydrogen peroxide on wheat seedling salinity tolerance[J].Acta Botanica Boreali-Occidentalia Sinica,2007,27(12):2491-2495.

16.Drzikova B,Dongowski G,Gebhardt E.Dietary fibre-rich oat-based products affect serum lipids,microbiota,formation of short-chain fatty acids and steroids in rats[J].British Journal of Nutrition,2005,94(6):1012-1025.

17.林葉春,曾昭海,任長(zhǎng)忠,等.局部根區(qū)灌溉對(duì)裸燕麥光合特征曲線及葉綠素?zé)晒馓匦缘挠绊慬J].作物學(xué)報(bào),2012,38(6):1062-1070.

Lin Yechun,Zeng Zhaohai,Ren Changzhong,et al.Effects of partial root zone irrigation on leaf photosynthetic curves and chlorophyll fluorescence parameters in naked oat[J].Acta Agronomica Sinica,2012,38(6):1062-1070.

18.劉建新,王金成,王瑞娟,等.燕麥幼苗對(duì)鹽脅迫的響應(yīng)及過氧化氫對(duì)響應(yīng)的調(diào)節(jié)[J].生態(tài)學(xué)雜志,2014,33(1):89-97.

Liu Jianxin,Wang Jincheng,Wang Ruijuan,et al.Response ofAvenanudaL. seedlings to salt stress and the modulation of hydrogen peroxide[J].Chinese Journal of Ecology,2014,33(1):89-97.

19.Sergiev I,lexieva V,karanov E.Effect of spermine,atrazine andcorabination between them on someendogenous rotective systems and stress markers in plants[J].Comptes rendus de l’Académie bulgare des Sciences,1997,51(2):122-124.

20.李合生.植物生理生化實(shí)驗(yàn)原理和技術(shù)[M].北京:高等教育出版社,2000.

Li Hesheng.Principles and techniques of plant physiological biochemical experiment [M].Beijing:Higher Education Press,2000.

21.趙貴林,陳強(qiáng),胡國(guó)霞,等.水稻脯氨酸代謝關(guān)鍵酶對(duì)水分脅迫的響應(yīng)[J].干旱地區(qū)農(nóng)業(yè)研究,2011,29(3):80-83.

Zhao Guilin,Chen Qiang,Hu Guoxia,et al.Responses of the key enzymes involved in proline metabolism in rice seedling under water stress[J].Agricultural Research in the Arid Areas,2011,29(3):80-83.

22.Szabados L,Savoure A.Proline:a multifunctional amino acid[J].Trends in Plant Science,2010,15(2):89-97.

23.夏方山,董秋麗,董寬虎.堿性鹽脅迫對(duì)堿地風(fēng)毛菊苗期脯氨酸代謝途徑的影響[J].中國(guó)草地學(xué)報(bào),2011,33(1):48-53.

Xia Fangshan,Dong Qiuli,Dong Kuanhu.Effect of alkaline salts on proline metabolism ofSaussurearuncinataat seedling stage[J].Chinese Journal of Grassland,2011,33(1):48-53.

24.董秋麗,夏方山,董寬虎.NaCl脅迫對(duì)芨芨草苗期脯氨酸代謝的影響[J].草業(yè)學(xué)報(bào),2010,19(5):71-76.

Dong Qiuli,Xia Fangshan,Dong Kuanhu.Effects of NaCl stress on proline metabolism ofAchnatherumsplendensseedling[J].Acta Prataculturae Sinica,2010,19(5):71-76.

25.Orozco-Cardenas M,Ryan C A.Hydrogen peroxide is generated systemically in plant leaves by wounding and system in via the octadecanoid pathway[J].Proceedings of the National Academy of Sciences of the United States of America,1999,96(11):6553-6557.

26.鄧?guó)P飛,楊雙龍,龔明.外源ABA對(duì)低溫脅迫下小桐子幼苗脯氨酸積累及其代謝途徑的影響[J].植物生理學(xué)報(bào),2015,51(2):221-226.

Deng Fengfei,Yang Shuanglong,Gong Ming.Effect of exogenous abscisic acid on proline accumulation and metabolic pathways inJatrophacurcasseedlings under cold stress[J].Plant Physiology Journal,2015,51(2):221-226.

The project was financially supported by the science and technology plan project of Qingyang city in Gansu province(KZ2014-19)

introduction：LIU Jian-Xin(1964—),male,professor,mainly engaged in Plant stress physiology.

date:2015-12-08

EffectofExogenousH2O2onProlineAccumulationandMetabolicPathwayinLeavesofOatSeedlingsunderComplexSaline-AlkaliStress

LIU Jian-Xin WANG Jin-Cheng LIU Xiu-Li WANG Feng-Qin

(1.College of Life Science and Technology,Longdong University,Qingyang 745000；2.University Provincial Key Laboratory for Protection and Utilization of Longdong Bio-resources in Gansu Province,Qingyang 745000)

Saline-alkali stress interferes with cell metabolism, and inhibits plant growth and development. Proline metabolism is closely related with plant salt-alkali resistance. As signaling molecules, hydrogen peroxide(H2O2) plays vital roles in the regulation of plant cell metabolism, as well as in adaptation to the environmental stress. In order to understand the regulatory mechanism of exogenous H2O2on proline metabolism in oat(Avena nuda) under salinity-alkalinity stresses, the ‘Dingyou No.6’(a new oat cultivar) seedlings with two leaves were used to investigate the effects of exogenous H2O2on proline accumulation in leaves of the seedlings under complex saline-alkali stress by the method of solution culture. The results showed that 75 mmol·L-1complex saline-alkali stress(molar ratio of NaCl∶Na2SO4∶NaHCO3∶Na2CO3=12∶8∶9∶1) led to a significant accumulation of proline in leaves, and induced a rapid increase of activities of the key enzymes ornithine-δ-aminotransferase(δ-OAT) of proline biosynthesis, and a decrease of activities of the key enzymes Δ1-pyrroline-5-carboxylate synthetase(P5CS) of proline biosynthesis, as well as the key enzyme proline dehydrogenase(ProDH) of proline degradation. Moreover, treatments with 0.01-1 000 μmol·L-1, especially, 10 μmol·L-1H2O2could enhance the accumulation of proline in oat seedling leaves under complex saline-alkali stress. H2O2of 10 μmol·L-1also increased the activities of P5CS and δ-OAT, and decreased the activity of ProDH in leaves under complex saline-alkali stress. In addition, 10 μmol·L-1H2O2treatments could rapidly increase the endogenous H2O2levels in oat seedling leaves under complex saline-alkali stress. The exogenous H2O2treatment resulted in the increase of endogenous H2O2content in oat seedlings under complex saline-alkali stress. H2O2induced proline accumulation might be a combined result of the activation of glutamate and ornithine pathways of proline biosynthesis and inhibition of proline degradation pathway.

oat;complex saline-alkali stress;hydrogen peroxide;proline metabolism

甘肅省慶陽市科技計(jì)劃項(xiàng)目(KZ2014-19)資助

劉建新(1964—)，男，教授，主要從事植物逆境生理研究。

2015-12-08

Q945.78

A

10.7525/j.issn.1673-5102.2016.03.017