肖鵬程,朱端衛(wèi),羅 媛,萬(wàn)小瓊,蔡建波 (華中農(nóng)業(yè)大學(xué)植物營(yíng)養(yǎng)與生態(tài)環(huán)境研究室,湖北 武漢 430070)

環(huán)境因素對(duì)菹草根和葉細(xì)胞質(zhì)膜Ca2+-ATPase活性的影響

肖鵬程,朱端衛(wèi)*,羅 媛,萬(wàn)小瓊,蔡建波 (華中農(nóng)業(yè)大學(xué)植物營(yíng)養(yǎng)與生態(tài)環(huán)境研究室,湖北 武漢 430070)

通過(guò)改變?nèi)芤簻囟?、pH值、ATP濃度、鈣濃度和培養(yǎng)液的鈣濃度等條件,研究了菹草根和葉細(xì)胞質(zhì)膜Ca2+-ATPase的活性的變化.結(jié)果表明,根細(xì)胞質(zhì)膜Ca2+-ATPase的活性在pH 6.0時(shí)最高,其最適反應(yīng)溫度為40℃;葉細(xì)胞質(zhì)膜Ca2+-ATPase在一個(gè)較寬的pH范圍內(nèi)具有高活性,最適反應(yīng)溫度為45℃;溶液中ATP濃度分別為3mmol/L和4mmol/L時(shí)菹草根和葉細(xì)胞質(zhì)膜Ca2+-ATPase活性最大;無(wú)論是菹草根還是葉細(xì)胞質(zhì)膜 Ca2+-ATPase活性,在溶液鈣濃度為 10-4mol/L時(shí)都最高.在營(yíng)養(yǎng)液中添加不同 CaCl2濃度培養(yǎng)菹草,其根和葉細(xì)胞質(zhì)膜Ca2+-ATPase活性表現(xiàn)出差異,根細(xì)胞質(zhì)膜Ca2+-ATPase活性比葉細(xì)胞質(zhì)膜Ca2+-ATPase活性高,且隨營(yíng)養(yǎng)液中鈣濃度的增加,這種差異加大;當(dāng)營(yíng)養(yǎng)液中鈣濃度在10mg/L(2.5×10-4mol/L)以下時(shí),Ca2+-ATPase活性逐漸上升,營(yíng)養(yǎng)液中鈣濃度由10mg/L增加到15mg/L,Ca2+-ATPase活性陡然下降,這與溶液鈣濃度對(duì)Ca2+-ATPase活性的影響相呼應(yīng).

菹草；根;葉；細(xì)胞質(zhì)膜；Ca2+-ATPase

鈣作為植物細(xì)胞的第二信使,在細(xì)胞質(zhì)內(nèi)的濃度變化可以調(diào)節(jié)許多生理生化過(guò)程,在植物體的生長(zhǎng)發(fā)育以及對(duì)環(huán)境的反應(yīng)和適應(yīng)過(guò)程中發(fā)揮著舉足輕重的作用[1].激素、環(huán)境因子等信號(hào)刺激導(dǎo)致植物細(xì)胞胞質(zhì)內(nèi) Ca2+濃度大幅度增加,位于質(zhì)膜和液泡膜上的 Ca2+轉(zhuǎn)運(yùn)系統(tǒng)就會(huì)被激活,該系統(tǒng)將 Ca2+主動(dòng)運(yùn)到胞外和運(yùn)進(jìn)胞液,以維持細(xì)胞中 Ca2+濃度的正常水平[2].這種精細(xì)的 Ca2+轉(zhuǎn)運(yùn)系統(tǒng)由Ca2+-ATPase,Ca2+/nH+反向轉(zhuǎn)運(yùn)蛋白等組成[3-4].Ca2+-ATPase是一個(gè)高親和、低容量的鈣運(yùn)輸系統(tǒng),其對(duì)胞內(nèi)Ca2+平衡起微調(diào)作用[5].

另一方面,鈣是水中重要的營(yíng)養(yǎng)元素,水生植物對(duì)其吸收與釋放是湖泊生態(tài)系統(tǒng)中地球化學(xué)的重要特征[6].湖泊中可溶性磷酸鹽在鈣參與下與碳酸鈣發(fā)生共沉淀,水柱中葉綠素 a因此而減少,藻類生長(zhǎng)得到抑制[7].沉水植物對(duì)鈣的吸收與釋放將可能與水柱中碳酸鈣-磷共沉淀相聯(lián)系,但沉水植物中鈣的變化所遵循的生理生化機(jī)制及其涉及的過(guò)程目前仍缺乏論證.本研究室前期對(duì)鈣在菹草-溶液間的遷移速率研究中發(fā)現(xiàn),根室溶液中 Ca2+濃度為 10mg/L時(shí),菹草對(duì)鈣的吸收效果最好;Ca2+濃度過(guò)高,菹草根系鈣吸收速率下降.雖然根室 Ca2+濃度為 10mg/L處理的菹草根系對(duì) Ca2+的吸收量明顯小于 15mg/L的處理,但在后者中菹草莖葉分泌的鈣量反而小于在前者中的情況.這一鈣的吸收和釋放過(guò)程將可能與菹草細(xì)胞質(zhì)膜 Ca2+-ATPase活性變化有關(guān),本文將從這一假設(shè)出發(fā),探討菹草根和葉細(xì)胞質(zhì)膜Ca2+-ATPase活性受外源的影響,以闡明菹草吸收、釋放鈣的機(jī)制.

1 材料與方法

1.1 材料與處理

供試菹草(Potamogeton crispus L.)石芽采自中國(guó)科學(xué)院武漢植物園,取回清洗干凈后將其用自來(lái)水培養(yǎng)馴化7d,選取大小和重量一致并已經(jīng)萌發(fā)的石芽培養(yǎng)于 0.01×Hoagland營(yíng)養(yǎng)液里,營(yíng)養(yǎng)液pH值每天用0.1mol/L HCl或者0.1mol/L NaOH調(diào)節(jié)到6.5左右,每3d換1次營(yíng)養(yǎng)液.植物在溫室自然光條件下培養(yǎng),溫度為20～25℃.

1.2 細(xì)胞質(zhì)膜微囊的提取

菹草的離體根、葉細(xì)胞質(zhì)膜微囊的提取參照Thomson等[8]的方法稍作改動(dòng):取0.5g菹草根(或2.0g菹草葉片),按1:2(W:V)加入預(yù)冷勻漿液[蔗糖250mmol/L,Hepes-Tris (pH 值 7.5)25mmol/L, EDTA 5mmol/L,10%(W/V)甘油,1%(W/V)聚乙烯吡咯烷酮(30K),0.5%(W/V)牛血清蛋白(BSA), DTT 2mmol/L,Vc 5mmol/L]研磨成勻漿.勻漿液在9000×g、4℃下冷凍離心 15min,去除細(xì)胞核與葉綠體.將上清液于25000×g、4℃下冷凍離心60min,沉淀為膜制劑,再用少量懸浮液 [蔗糖250mmol/L, KH2PO4-K2HPO4緩 沖 液 (pH7.8) 5mmol/L, 10%(W/V)甘油]懸浮,此為粗膜提取物.提取物置于兩相系統(tǒng)[6.2%(W/W) PEG6000, 6.2% (W/W), Dextran500,蔗糖 250mmol/L, KH2PO4- K2HPO4緩沖液(pH 7.8)]中在2000 r/min、6mm振幅下振動(dòng)5s后,在1000r/min、4℃條件下冷凍離心5min,促進(jìn)分相.取上相,向所得含質(zhì)膜微囊上相中,再加入0.5mL下相,在2000r/min、6mm振幅下振動(dòng)5s后,以1000r/min、4℃冷凍離心5min促進(jìn)分相,取上相.向經(jīng)過(guò)前述兩次萃取提純的含質(zhì)膜微囊上相中加入懸浮液,在2000r/min、6mm振幅下振動(dòng)5s后,以25000×g、4℃離心60min.加入懸浮液稀釋,得到純化的質(zhì)膜微囊制劑.樣品用液氮速凍后于-80℃下保存待測(cè).

1.3 質(zhì)膜微囊Ca2+-ATPase活性的測(cè)定

提取所得細(xì)胞質(zhì)膜微囊在一定條件下測(cè)定Ca2+-ATPase活性:(1)在 ATP濃度為 1.0×10-3mol/L,溫度為37℃下設(shè)置pH值梯度:5.0,6.0,7.0, 8.0,9.0;(2)在 ATP濃度為 1.0×10-3mol/L和最適pH 值下設(shè)置溫度梯度:35℃ ,40 ℃, 45℃ , 50 ℃, 55℃;(3)在最適溫度及最適pH值條件下,設(shè)置底物ATP濃度(mol/L)梯度:2.0×10-3,3.0×10-3, 4.0× 10-3,5.0×10-3;(4)在最適溫度、最適pH值條件及根細(xì)胞 ATP 3.0×10-3mol/L或葉細(xì)胞 ATP為4.0×10-3mol/L,設(shè)置 Ca2+濃度(mol/L)梯度:1.0× 10-3,1.0×10-4,1.0×10-5,1.0×10-6,1.0×10-7.

質(zhì)膜微囊 Ca2+-ATPase活性的測(cè)定參照Chung等[9]的方法,略加改動(dòng).反應(yīng)體系含1mmol/L NaN3, 50mmol/L NaNO3, 50mmol/L KCl, 8mmol/L MgSO4, 1mmol/L EGTA, 80μmol/L的Na2MoO4, 25mmol/L Hepes-Tris.以加與不加3 mmol/L CaCl2引起的酶活之差為Ca2+-ATPase活性,反應(yīng)啟動(dòng)用0.2mL 15mmol/L的Na2-ATP,用20%(W/V)三氯乙酸(TCA)終止反應(yīng),分別按鉬藍(lán)法和考馬斯亮藍(lán)法測(cè)定無(wú)機(jī)磷和蛋白質(zhì)含量,用單位時(shí)間、單位質(zhì)量蛋白質(zhì)的無(wú)機(jī)磷量表示Ca2+-ATPase活性[μmolPi/(mgPro·min)].

1.4 環(huán)境鈣濃度對(duì)菹草細(xì)胞質(zhì)膜 Ca2+-ATPase活性的影響

將在營(yíng)養(yǎng)液中培養(yǎng) 30d后的菹草幼苗轉(zhuǎn)移至盛有不同Ca2+濃度的Hoagland完全營(yíng)養(yǎng)液的隔室中進(jìn)行培養(yǎng)(圖 1),其中,用莖葉室和根室(遮光)將菹草的莖、根分開,葉室和根室之間的菹草莖用濕棉球包裹,防止這部分莖失水而影響實(shí)驗(yàn),各室營(yíng)養(yǎng)液體積V0均為1L[10].根室鈣濃度Co依次為0,5,10,15mg/L,重復(fù)3次;莖葉用去離子水培養(yǎng).分別于40,80,240,480min取完整植株,先后進(jìn)行根和葉細(xì)胞質(zhì)膜 Ca2+-ATPase的提取和該酶在最適條件下的活性測(cè)定.

圖1 測(cè)定菹草不同部位鈣吸收/分泌的二隔室實(shí)驗(yàn)裝置Fig.1 Two-container setup for investigating uptake and exudation of calcium in different parts of P. crispus

2 結(jié)果與分析

2.1 溶液條件對(duì)菹草根和葉細(xì)胞質(zhì)膜 Ca2+-ATPase活性的影響

2.1.1 溶液溫度 由圖2可見(jiàn),菹草根、葉細(xì)胞質(zhì)膜Ca2+-ATPase的最適反應(yīng)溫度分別為40℃、45℃.就最適反應(yīng)溫度來(lái)說(shuō),菹草葉細(xì)胞質(zhì)膜Ca2+-ATPase最大活性所處溫度高于根細(xì)胞質(zhì)膜Ca2+-ATPase最大活性所對(duì)應(yīng)的溫度.圖2還表明,根細(xì)胞質(zhì)膜 Ca2+-ATPase活性隨溫度變化的趨勢(shì)較快,而葉細(xì)胞質(zhì)膜 Ca2+-ATPase活性變化稍平緩.在進(jìn)一步研究菹草根、葉細(xì)胞質(zhì)膜Ca2+-ATPase在其他條件下的活性時(shí),將根、葉細(xì)胞質(zhì)膜反應(yīng)的溫度分別控制為40℃和45℃.

2.1.2 溶液pH值 圖3表明,菹草根細(xì)胞質(zhì)膜和葉細(xì)胞質(zhì)膜中Ca2+-ATPase活性都隨溶液pH值的變化而變化,且二者差異明顯,前者的Ca2+-ATPase活性在pH值6.0時(shí)最高,后者的最適pH值為7.0.在最適pH值下,菹草根細(xì)胞質(zhì)膜Ca2+-ATPase活性是葉細(xì)胞質(zhì)膜活性的 3.76倍.此外,葉細(xì)胞質(zhì)膜Ca2+-ATPase在一個(gè)較寬的pH值范圍內(nèi)相對(duì)活性較高.根據(jù) pH值對(duì)菹草根和葉細(xì)胞質(zhì)膜 Ca2+-ATPase活性的影響,在研究底物ATP濃度和Ca2+濃度對(duì)酶活影響時(shí),根、葉細(xì)胞質(zhì)膜提取物的pH值分別控制在6.0和7.0.

圖2 溫度對(duì)菹草根和葉細(xì)胞質(zhì)膜Ca2+-ATPase活性的影響Fig.2 Effect of temperature on the activities of plasma membrane Ca2+-ATPase from P. crispus root and leaf

圖3 pH值對(duì)菹草根和葉細(xì)胞質(zhì)膜Ca2+-ATPase活性的影響Fig.3 Effect of pH on the activities of plasma membrane Ca2+-ATPase from P. crispus root and leaf

2.1.3 溶液 ATP濃度 菹草根和葉細(xì)胞質(zhì)膜Ca2+-ATPase活性與底物ATP濃度密切相關(guān)(圖4).由圖4可知,在低濃度范圍內(nèi),酶活性均隨著ATP濃度的增加而迅速上升,當(dāng)ATP濃度達(dá)到2mmol/L后,酶活性升高速度顯著降低,曲線趨于平緩.對(duì)根細(xì)胞質(zhì)膜Ca2+-ATPase而言,3mmol/L ATP可使酶活性達(dá)到最大;而對(duì)于葉細(xì)胞質(zhì)膜 Ca2+-ATPase, 4mmol/L ATP才能使酶活性達(dá)到最大.

圖4 ATP濃度對(duì)菹草根和葉細(xì)胞質(zhì)膜Ca2+-ATPase活性的影響Fig.4 Effect of ATP concentration on the activities of plasma membrane Ca2+-ATPase from P. crispus root and leaf

圖5 Ca2+濃度對(duì)菹草根和葉細(xì)胞質(zhì)膜Ca2+-ATPase活性的影響Fig.5 Effect of calcium concentration on the activities of plasma membrane Ca2+-ATPase from P. crispus root and leaf

2.1.4 溶液Ca2+濃度 由圖5可見(jiàn),在低鈣濃度范圍內(nèi),Ca2+-ATPase活性隨Ca2+濃度的負(fù)對(duì)數(shù)的減小而升高, 1.0×10-4mol/L Ca2+可使酶達(dá)到最大激活,Ca2+濃度進(jìn)一步增加反而抑制了 Ca2+-ATPase活性,使酶活性有所下降,菹草根和葉細(xì)胞質(zhì)膜Ca2+-ATPase對(duì)Ca2+濃度的響應(yīng)模式相同.

2.2 環(huán)境中鈣濃度對(duì)菹草根細(xì)胞質(zhì)膜 Ca2+-ATPase活性的影響

表1所示的是經(jīng)不同 Ca2+濃度營(yíng)養(yǎng)液培養(yǎng)的菹草根細(xì)胞質(zhì)膜 Ca2+-ATPase活性的差異,且培養(yǎng)時(shí)間不同根細(xì)胞質(zhì)膜 Ca2+-ATPase活性也不一樣.從同一時(shí)間的酶活來(lái)看,當(dāng)營(yíng)養(yǎng)液中Ca2+濃度為10mg/L時(shí),菹草根細(xì)胞質(zhì)膜Ca2+-ATPase活性最高;在 1～10mg/LCa2+范圍內(nèi),Ca2+-ATPase活性隨培養(yǎng)時(shí)間的延長(zhǎng)而增加,從能量代謝的角度說(shuō)明,菹草在這一濃度范圍內(nèi)生長(zhǎng)正常,而在相同條件下,15mg/LCa2+濃度由 240min時(shí)的0.535μmolPi/(mgPro·min)下降到 480min時(shí)的0.182μmolPi/(mgPro·min).進(jìn)一步比較可以看出,在相同時(shí)間內(nèi),10mg/LCa2+的處理時(shí),根中 Ca2+-ATPase活性最高,這一處理與其他處理相比,基本上都達(dá)到顯著性差異,而不加Ca2+的處理,根中Ca2+-ATPase活性都顯著低于其他加Ca2+處理.

表1 Ca2+濃度對(duì)菹草根細(xì)胞質(zhì)膜Ca2+-ATPase活性影響[μmolPi/(mgPro·min)]Table 1 Effect of calcium concentration on the activities of plasma membrane Ca2+-ATPase from P. crispus root [μmolPi/(mgPro·min)]

2.3 環(huán)境中鈣濃度對(duì)菹草葉細(xì)胞質(zhì)膜 Ca2+-ATPase活性的影響

由表2可知,在營(yíng)養(yǎng)液中加5mg/L和10mg/L Ca2+,培養(yǎng)的菹草葉細(xì)胞質(zhì)膜Ca2+-ATPase活性顯著高于不加鈣處理,說(shuō)明營(yíng)養(yǎng)液中添加CaCl2相應(yīng)提高了葉細(xì)胞質(zhì)膜 Ca2+-ATPase活性;隨著 Ca2+濃度進(jìn)一步升高,葉細(xì)胞質(zhì)膜Ca2+-ATPase活性反而下降,加 15mg/LCa2+培養(yǎng)的菹草葉細(xì)胞質(zhì)膜Ca2+-ATPase活性在葉中顯著低于加 5mg/L和10mg/L Ca2+培養(yǎng)的處理.從培養(yǎng)時(shí)間對(duì)菹草葉細(xì)胞質(zhì)膜 Ca2+-ATPase活性的影響來(lái)看,0～10mg/ LCa2+培養(yǎng)的菹草隨著時(shí)間的延長(zhǎng)其葉細(xì)胞質(zhì)膜Ca2+-ATPase活性在實(shí)驗(yàn)時(shí)間范圍內(nèi)逐漸加強(qiáng),這一趨勢(shì)與對(duì)應(yīng)處理的根細(xì)胞質(zhì)膜Ca2+-ATPase活性的變化一致,而 15mg/LCa2+培養(yǎng)的菹草葉細(xì)胞質(zhì)膜Ca2+-ATPase活性變化不平穩(wěn).

表2 Ca2+濃度對(duì)菹草葉細(xì)胞質(zhì)膜Ca2+-ATPase活性影響 [μmolPi/(mgPro·min)]Table 2 Effect of calcium concentration on the activities of plasma membrane Ca2+-ATPase from P. crispus leaf [μmolPi/(mgPro·min)]

3 討論

酶促反應(yīng)受多種因素影響,溫度是最重要的影響因素之一.本研究中菹草根、葉細(xì)胞質(zhì)膜Ca2+-ATPase活性分別在溫度為40℃、45℃時(shí)達(dá)到最高.就最適溫度而言,菹草葉細(xì)胞質(zhì)膜Ca2+-ATPase高于根細(xì)胞質(zhì)膜Ca2+-ATPase,這與前人在陸生植物上的研究結(jié)果相類似[2,11].菹草根細(xì)胞質(zhì)膜 Ca2+-ATPase活性隨溫度變化的趨勢(shì)較快,而葉細(xì)胞質(zhì)膜 Ca2+-ATPase活性變化稍平緩.菹草的根生長(zhǎng)在底質(zhì)中,沉積物溫度變化較小,通常在莖葉進(jìn)行光合作用的白天表層水溫度高于底層,菹草能在這種溫差條件下生長(zhǎng)[12],可能與葉細(xì)胞質(zhì)膜 Ca2+-ATPase反應(yīng)的最適溫度較高有關(guān).因此,菹草根和葉細(xì)胞質(zhì)膜 Ca2+-ATPase最適反應(yīng)溫度的差異可能是在長(zhǎng)期進(jìn)化過(guò)程中形成的.盡管離體酶活反應(yīng)的最適溫度不一定能表征植株體內(nèi) Ca2+-ATPase的準(zhǔn)確值,但卻能說(shuō)明酶的一種體外特征.另一方面,菹草根和葉細(xì)胞質(zhì)膜 Ca2+-ATPase最適反應(yīng)溫度的差異也可能反映了Ca2+在相應(yīng)部位遷移的能量差異.

菹草根和葉細(xì)胞質(zhì)膜 Ca2+-ATPase活性最適 pH值分別為 6.0和 7.0,葉細(xì)胞質(zhì)膜 Ca2+-ATPase在一個(gè)較寬的 pH值范圍內(nèi)具有較高活性.菹草生長(zhǎng)在低電導(dǎo)、高pH值的環(huán)境中[13],許多研究觀察到菹草生長(zhǎng)的水體具有高 pH值性質(zhì)

[14-16].通常認(rèn)為,菹草生長(zhǎng)過(guò)程中對(duì)CO2的吸收是菹草生長(zhǎng)導(dǎo)致水體高 pH值的原因.由此可見(jiàn),菹草根和葉細(xì)胞質(zhì)膜在不同 pH值下的 Ca2+-ATPase的活性不同,是兩種器官在相應(yīng)環(huán)境下的生化行為,這種行為為兩種器官在各自的環(huán)境中完成不同的功能提供了保證.

在對(duì)底物濃度的響應(yīng)方面,菹草根和葉細(xì)胞質(zhì)膜Ca2+-ATPase活性最大時(shí)所需的ATP濃度分別為 3,4mmol/L.根在底質(zhì)中易受缺氧脅迫,有氧呼吸經(jīng)常受到不同程度的抑制,有時(shí)ATP供應(yīng)不足,根細(xì)胞質(zhì)膜Ca2+-ATPase可在較低ATP濃度下發(fā)揮作用,使Ca2+在遷移中更易進(jìn)入根細(xì)胞.

植物體內(nèi)較低的胞質(zhì)游離 Ca2+濃度有利于Ca2+-ATPase行使第二信使功能,這種低鈣濃度的維持需要借助 Ca2+運(yùn)輸系統(tǒng),Ca2+-ATPase是植物體具有高親和力的鈣離子運(yùn)輸系統(tǒng)[5,17-19].在擬南芥中,根系細(xì)胞質(zhì)膜 Ca2+-ATPase促進(jìn)H+/Ca2+交換以加快對(duì)Ca2+的吸收[20].外界營(yíng)養(yǎng)液鈣濃度增加維持了質(zhì)膜 Ca2+-ATPase的活性,進(jìn)而促進(jìn)黃瓜根系吸收 Ca2+,并促進(jìn)鈣信號(hào)的形成和逆境信號(hào)的體內(nèi)傳遞,最終提高了黃瓜植株的低氧耐性[21].本試驗(yàn)中,營(yíng)養(yǎng)液中 Ca2+在 0～10-4mol/L之間提高了根和葉細(xì)胞質(zhì)膜Ca2+-ATPase活性,Ca2+濃度＞10-4mol/L,Ca2+-ATPase活性開始下降,這與質(zhì)膜 Ca2+濃度＞10mg/L(即＞2.5×10-4mol/L)后Ca2+-ATPase活性下降的現(xiàn)象剛好吻合.在另外的實(shí)驗(yàn)中觀察到當(dāng)培養(yǎng)液中 Ca2+濃度＞10mg/L后,菹草根吸收和莖葉釋放Ca2+的速度明顯下降,可能與細(xì)胞質(zhì)膜 Ca2+-ATPase活性在外界Ca2+濃度進(jìn)一步增加而下降有直接的關(guān)系.

相同條件下,菹草根吸收 Ca2+離子的活力保持在相當(dāng)旺盛的水平,而維持葉細(xì)胞中相當(dāng)濃度的鈣,葉細(xì)胞質(zhì)膜Ca2+-ATPase活性明顯低于根細(xì)胞質(zhì)膜 Ca2+-ATPase活性,這說(shuō)明沉積物中 Ca2+經(jīng)過(guò)菹草向上覆水遷移的動(dòng)力之一是來(lái)自根與葉細(xì)胞質(zhì)膜Ca2+-ATPase活性存在的能量差.

4 結(jié)論

4.1 菹草根、葉細(xì)胞質(zhì)膜Ca2+-ATPase的最適反應(yīng)溫度分別為40℃、45℃;菹草根細(xì)胞質(zhì)膜中Ca2+-ATPase活性在pH值6.0時(shí)最高,葉細(xì)胞質(zhì)膜中Ca2+-ATPase活性的最適pH值為7.0;在低ATP濃度范圍內(nèi),菹草根和葉細(xì)胞質(zhì)膜 Ca2+-ATPase活性均隨著ATP濃度的增加而迅速上升,根細(xì)胞質(zhì)膜Ca2+-ATPase在3mmol/L ATP活性最大,葉細(xì)胞質(zhì)膜 Ca2+-ATPase活性在 4mmol/L ATP才達(dá)到最大;在低鈣濃度范圍內(nèi),菹草根和葉細(xì)胞質(zhì)膜 Ca2+-ATPase活性隨 Ca2+濃度負(fù)對(duì)數(shù)的減小而升高,1.0×10-4mol/L Ca2+可使酶達(dá)到最大激活,Ca2+濃度進(jìn)一步增加反而抑制了 Ca2+-ATPase活性,使酶活性有所下降.

4.2 當(dāng)培養(yǎng)液中Ca2+濃度為10mg/L時(shí),菹草根細(xì)胞質(zhì)膜 Ca2+-ATPase活性最高;在 1～10mg/ LCa2+范圍內(nèi),Ca2+-ATPase活性隨培養(yǎng)時(shí)間的延長(zhǎng)而增加,而在相同條件下,15mg/LCa2+濃度培養(yǎng)一段時(shí)間,Ca2+-ATPase活性銳減.

4.3 加鈣培養(yǎng)的菹草葉細(xì)胞質(zhì)膜 Ca2+-ATPase活性顯著高于不加鈣培養(yǎng)的情況,即營(yíng)養(yǎng)液中添加鈣相應(yīng)提高了葉細(xì)胞質(zhì)膜 Ca2+-ATPase活性;在營(yíng)養(yǎng)液的培養(yǎng)下,隨著培養(yǎng)時(shí)間的延長(zhǎng),菹草葉細(xì)胞質(zhì)膜 Ca2+-ATPase活性與對(duì)應(yīng)條件下根細(xì)胞質(zhì)膜 Ca2+-ATPase活性的變化趨勢(shì)一致,從而說(shuō)明,維持環(huán)境中一定強(qiáng)度的鈣濃度是菹草生長(zhǎng)中能量代謝和離子平衡的需要.

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Impact of environmental factors on Ca2+-ATPase activity of Potamogeton crispus root and leaf plasma membrane.

XIAO Peng-cheng, ZHU Duan-wei*, LUO Yuan, WAN Xiao-qiong, CAI Jian-bo (Laboratory of Plant Nutrition and Ecological Environment Research, Huazhong Agricultural University, Wuhan 430070, China). China Environmental Science, 2012,32(6)：1123～1128

Ca2+-ATPase activity of P. crispus root and leaf plasma membrane was studied by changing the solution conditions including temperature, pH value, ATP concentration, and calcium concentration. The results indicated that the activity of root plasma membrane Ca2+-ATPase reached the maximum at pH 6.0 and 40 ℃, while the leaf enzyme highly activated at a broad range of pH at an optimum reaction temperature of 45℃. Plasma membrane Ca2+-ATPase activity in the root reached the maximum at 3 mmol/L ATP and in the leaf at 4 mmol/L ATP in the solution. P. crispus root and leaf plasma membrane Ca2+-ATPase activity increased up to the presence of 10-4mol/L calcium in the solution. The alteration of CaCl2concentration in culture solution for P. crispus cultivation resulted in different Ca2+-ATPase activity in both root and leaf plasma membrane. The root plasma membrane Ca2+-ATPase activity was higher than in the leaf, and the difference was more remarkable with increments of calcium in the culture solution. At lower concentration of calcium than 10 mg/L (2.5×10-4mol /L) in the culture solution, the Ca2+-ATPase activity was raised gradually. Between 10 mg/L to 15 mg/L, Ca2+-ATPase activity dropped sharply, which is in agreement with the impact of calcium concentration on Ca2+-ATPase activity.

Potamogeton crispus；root；leaf；plasma membrane；Ca2+-ATPase

2011-09-28

國(guó)家自然科學(xué)基金資助項(xiàng)目(40973056);博士點(diǎn)基金(20100146110020)

* 責(zé)任作者, 教授, zhudw@mail.hzau.edu.cn

X171.5

A

1000-6923(2012)06-1123-06

肖鵬程(1984-),女,湖南邵陽(yáng)人,華中農(nóng)業(yè)大學(xué)碩士研究生,主要研究方向?yàn)橹参餇I(yíng)養(yǎng)與生態(tài)環(huán)境.