孫譽(yù)育,尹春英,賀合亮,唐 波,劉 慶

1 中國科學(xué)院成都生物研究所, 中國科學(xué)院山地生態(tài)恢復(fù)與生物資源利用重點(diǎn)實(shí)驗(yàn)室, 生態(tài)恢復(fù)與生物多樣性保育四川省重點(diǎn)實(shí)驗(yàn)室, 成都 610041 2 中國科學(xué)院大學(xué), 北京 100049 3 成都理工大學(xué)材料與化學(xué)化工學(xué)院, 成都 610059

紅樺幼苗根系對水-氮耦合效應(yīng)的生理響應(yīng)

孫譽(yù)育1,2,尹春英1,*,賀合亮1,3,唐 波1,2,劉 慶1

1 中國科學(xué)院成都生物研究所, 中國科學(xué)院山地生態(tài)恢復(fù)與生物資源利用重點(diǎn)實(shí)驗(yàn)室, 生態(tài)恢復(fù)與生物多樣性保育四川省重點(diǎn)實(shí)驗(yàn)室, 成都 610041 2 中國科學(xué)院大學(xué), 北京 100049 3 成都理工大學(xué)材料與化學(xué)化工學(xué)院, 成都 610059

采用兩因素隨機(jī)區(qū)組設(shè)計(jì),設(shè)置了5個(gè)水分梯度,即40%(W1)、50%(W2)、60%(W3)、80%(W4)、100%(W5)的土壤田間持水量(FC)和3個(gè)施氮梯度,即模擬氮沉降施加0(對照,N0)、20(N1)、40(N2)gN m-2a-1的硝酸銨,研究了水-氮耦合效應(yīng)對川西亞高山主要闊葉樹種紅樺(Betulaalbosinensis)幼苗根系生理活性的影響及根系在土壤水、氮脅迫下的生理調(diào)控機(jī)制。結(jié)果表明：1)隨土壤含水量降低,根系活力和根系呼吸速率顯著降低,膜脂過氧化產(chǎn)物(丙二醛)、滲透調(diào)節(jié)物質(zhì)(脯氨酸、可溶性蛋白質(zhì)和可溶性糖)含量及抗氧化物酶(超氧化物歧化酶、過氧化物酶和過氧化氫酶)活性顯著升高。2)水-氮耦合效應(yīng)對紅樺幼苗根系生理特征影響顯著：施氮(N1和N2)在土壤水分良好(W4和W5)時(shí)使根系活力和根系呼吸速率顯著升高,而在土壤水分不足(W1和W2)時(shí)顯著降低了根系活力和根系呼吸速率；且在水分不足時(shí),施氮濃度越大根系活力、MDA含量、脯氨酸及可溶性蛋白質(zhì)含量變化越顯著；在W3條件下,只有N1對根系生理功能促進(jìn)作用顯著。3)根系活力和根系呼吸速率與丙二醛含量呈顯著負(fù)相關(guān)性。因此,一定范圍內(nèi)的氮沉降在土壤水分狀況良好時(shí)對植物根系生理特征具有顯著正效應(yīng),而在土壤水分不足時(shí)則使根系細(xì)胞膜系統(tǒng)受損,抑制根系生理活性,但根系可通過增加滲透調(diào)節(jié)物質(zhì)含量和增強(qiáng)抗氧化物酶活性來抵御一定范圍的環(huán)境脅迫。

水-氮耦合；根系活性；膜脂過氧化；滲透調(diào)節(jié)；抗氧化物酶

根系是植物重要的功能器官,它不僅擔(dān)負(fù)著吸收水分和養(yǎng)分的重要生理功能,而且通過呼吸和周轉(zhuǎn)消耗光合產(chǎn)物并向土壤輸入有機(jī)質(zhì)[1],同時(shí)也是首先感知土壤環(huán)境變化的植物器官[2- 4]。根系在受到土壤環(huán)境脅迫時(shí),細(xì)胞膜脂過氧化程度加劇[5],根系活性降低,吸收水分和養(yǎng)分的能力受到抑制[6],但植物細(xì)胞可通過主動(dòng)積累溶質(zhì)的方式,降低滲透勢和水勢,維持膨壓,進(jìn)行滲透調(diào)節(jié)；此外,環(huán)境脅迫使根系在代謝過程中通過多種途徑產(chǎn)生的活性氧及其清除系統(tǒng)的平衡遭到破壞,但輕中度的脅迫能使清除酶(抗氧化物酶)的活性增強(qiáng),根系抗氧化能力提高,緩解活性氧造成的傷害[7]。由此可見,滲透調(diào)節(jié)物質(zhì)的積累[8]和抗氧化能力的提高[9]是根系抵御環(huán)境脅迫的兩種重要生理響應(yīng)機(jī)制。

近年來,大氣氮沉降持續(xù)穩(wěn)定增長,嚴(yán)重影響著陸地及水生生態(tài)系統(tǒng)的生產(chǎn)力和穩(wěn)定性,大范圍較高濃度的氮沉降導(dǎo)致了不同程度的土壤酸化和森林退化[10-11]。同時(shí),大氣氮沉降效應(yīng)也因全球降水分布不均而異[10]。我國西南地區(qū)地處青藏高原東部,地形獨(dú)特復(fù)雜,生態(tài)環(huán)境脆弱,對氣候變化反應(yīng)十分敏感,最明顯的特點(diǎn)是干濕季明顯、冬季低溫、土壤養(yǎng)分有效性較低[12]。該區(qū)是IPCC預(yù)測的氣候變暖和氮沉降的主要區(qū)域[13],也是研究全球變化對森林生態(tài)系統(tǒng)影響的關(guān)鍵地區(qū)和重要森林類型[14],紅樺(Betualalbosinensis)是該區(qū)主要闊葉樹種,其根系生理特性與土壤水、氮有效性密切相關(guān)。因此,本試驗(yàn)著重研究不同土壤水、氮條件對該區(qū)紅樺幼苗根系活性的影響,探討幼苗根系在水、氮脅迫環(huán)境下的生理響應(yīng)機(jī)制,有助于了解該區(qū)主要植被地下生理特性及加深根系生態(tài)學(xué)的理論研究。

1 材料和方法

1.1 試驗(yàn)地基本概況

本試驗(yàn)選址于四川省阿壩州茂縣的中國科學(xué)院成都生物研究所茂縣生態(tài)定位研究站(103.90°E, 31.70°N,海拔1826 m),地處四川西部邊緣向青藏高原腹地過渡的高山峽谷區(qū),是長江的重要支流——岷江上游的重要地區(qū),該地區(qū)年平均降雨量、年平均蒸發(fā)量和年平均氣溫分別為825.2 mm、968.7 mm、9.3℃,屬暖溫帶氣候。植被屬針闊葉落葉常綠混交林,紅樺是該區(qū)主要的闊葉樹種。

1.2 試驗(yàn)材料和試驗(yàn)設(shè)計(jì)

本試驗(yàn)采用兩因素(水分和氮素)隨機(jī)區(qū)組設(shè)計(jì),根據(jù)實(shí)測的當(dāng)?shù)赝寥篮导緲O端含水量,設(shè)置5個(gè)水分梯度(結(jié)合前期紅樺對土壤水分敏感性預(yù)實(shí)驗(yàn)結(jié)果及著重研究其在干旱環(huán)境下響應(yīng)的試驗(yàn)?zāi)康?將土壤低含水量梯度設(shè)置得相對密集),即 40%(W1)、50%(W2)、60%(W3)、80%(W4)和 100%(W5)的土壤田間持水量(即實(shí)際土壤含水量分別為11.9%、14.9%、17.9%、23.9%、29.9%)；基于本區(qū)域相關(guān)研究[15],設(shè)置施氮梯度為 0(N0)、20(N1)和 40(N2)gN m-2a-1,共15個(gè)處理,每處理重復(fù)3次,5株為1個(gè)重復(fù)。

1.3 試驗(yàn)方法

于2014年8月中旬(生長季中期)進(jìn)行幼苗根系取樣,每處理隨機(jī)選取3株幼苗,用根鉆將根系最大限度帶土取出,以得到其完整根系,用清水洗凈并置于4℃冰箱保存,用于根系部分生理指標(biāo)的測定。其中,根系活力采用TTC還原法[17]測定；丙二醛(MDA)含量參照Hodges等[18]的方法測定；脯氨酸含量的測定參照Bates等[19]的方法；可溶性蛋白質(zhì)含量采用考馬斯亮藍(lán)G- 250法[20]測定；可溶性糖含量參照Shi等[21]的方法測定；超氧化物歧化酶(SOD)活性的測定參照Dhindsa等[22]的方法；過氧化物酶(POD)活性和過氧化氫酶(CAT)活性分別參照Chance和Maehly[23]的方法測定。

此外,氧氣流速(表示根系呼吸速率)參照Sun等[24]的方法,利用非損傷微測系統(tǒng)(BIO-IM-XY,Younger,USA)測定。實(shí)驗(yàn)使用的金屬微電極來自旭月(北京)科技有限公司。電極在使用前進(jìn)行校正,校正液O2濃度設(shè)置為0和21%,其他成分與測試液(0.2 mmol/L CaCl2,0.1 mmol/L KCl,0.5 g/L MES,0.1 mmol/L NH4NO3)相同,校正斜率在-9000—-2000范圍內(nèi)的電極用于檢測。測試前將根系置于裝有10—20 mL測試液的測試盒中平衡30 min后更換新的測試液開始測試,測試區(qū)域?yàn)榫嚯x根尖20 mm左右根毛分布密集的成熟區(qū)。每處理測試5個(gè)樣品,每個(gè)樣品穩(wěn)定測定10 min。

1.4 統(tǒng)計(jì)分析

數(shù)據(jù)的統(tǒng)計(jì)分析使用SPSS 17.0完成,利用兩因素方差分析(Two- way ANOVA)檢驗(yàn)水分和施氮效應(yīng),相同水分條件不同施氮梯度間的多重比較采用鄧肯式新復(fù)極差法進(jìn)行檢驗(yàn)。

2 結(jié)果和分析

2.1 水-氮耦合效應(yīng)對根系活力和根系呼吸速率的影響

紅樺幼苗根系活力和根系呼吸速率受到土壤水分、氮素供應(yīng)及其交互作用的極顯著影響(圖1)。二者均隨土壤含水量增加而增大,W4和W5時(shí)達(dá)到最大。圖1表明,當(dāng)土壤水分不足(W1和W2)時(shí),施氮(N1和N2)顯著抑制了紅樺幼苗根系活力和根系呼吸速率(W1條件下的N1除外),且施氮濃度越高,對根系活力抑制作用越顯著；在土壤水分充足(W4和W5)時(shí),N1顯著促進(jìn)了幼苗根系活力和根系呼吸速率；W3條件下,N1表現(xiàn)為顯著促進(jìn)作用,而N2表現(xiàn)為抑制效應(yīng)。

圖1 不同水、氮供應(yīng)對紅樺幼苗根系活力和根系呼吸速率的影響(平均值±標(biāo)準(zhǔn)偏差)Fig.1 Effects of different soil water and nitrogen supply on root activity, root respiration rate in Betual albosinensis seedling (mean±SD)誤差線上方不同的小寫字母表示同一水分條件下不同施氮梯度差異顯著(P<0.05);W: 水分效應(yīng)；N: 氮肥效應(yīng)；W×N: 水-氮交互作用;** P<0.05; *** P<0.005

2.2 根系MDA含量對水-氮耦合的響應(yīng)及其與根系活力和呼吸速率的關(guān)系

隨土壤含水量降低,幼苗根系MDA含量顯著升高,且不同水、氮供應(yīng)及其交互作用對MDA含量具有極顯著影響(圖2)。施氮只在土壤水分不足(W1和W2)時(shí)使MDA含量顯著增大,且施氮濃度越高,作用越顯著；而在W3和土壤水分狀況良好(W4和W5)時(shí),N1使MDA含量顯著降低。此外,根系活力和呼吸速率均與根系MDA含量呈極顯著負(fù)相關(guān)(圖2)。

圖2 紅樺幼苗根系丙二醛(MDA)含量對水、氮供應(yīng)的響應(yīng)(平均值±標(biāo)準(zhǔn)偏差)及其與根系活力和呼吸速率的相關(guān)性Fig.2 Root MDA content response to different soil water and nitrogen supply (mean±SD) and its relationship with root activity and respiration rate in Betual albosinensis seedling

2.3 根系滲透調(diào)節(jié)物質(zhì)對水-氮耦合效應(yīng)的響應(yīng)

紅樺幼苗根系滲透調(diào)節(jié)物質(zhì)(脯氨酸、可溶性蛋白質(zhì)和可溶性糖)含量也受土壤水分、氮素供應(yīng)及其交互作用的極顯著影響(圖3)。與根系MDA含量的變化相似,根系滲透調(diào)節(jié)物質(zhì)含量隨土壤含水量降低而升高。當(dāng)土壤水分不足(W1和W2)時(shí),施氮使?jié)B透調(diào)節(jié)物質(zhì)含量顯著升高,且施氮濃度越大,脯氨酸和可溶性蛋白質(zhì)含量升高越顯著；在W3和土壤水分狀況良好(W4和W5)時(shí),主要表現(xiàn)為N1顯著降低了根系滲透調(diào)節(jié)物質(zhì)含量。

圖3 紅樺幼苗根系脯氨酸含量、可溶性蛋白質(zhì)含量和可溶性糖含量(平均值±標(biāo)準(zhǔn)偏差)對不同水、氮供應(yīng)的響應(yīng)Fig.3 Responses of root proline content, soluble protein, soluble sugar in Betual albosinensis seedling (mean±SD) to different soil water and nitrogen supply

2.4 根系抗氧化物酶活性對水-氮耦合效應(yīng)的響應(yīng)

紅樺幼苗根系抗氧化物酶(SOD、POD和CAT)活性也受水-氮耦合效應(yīng)的極顯著影響(圖4),其隨土壤水分、氮素供應(yīng)的變化趨勢與MDA含量及滲透調(diào)節(jié)物質(zhì)含量一致。施氮在土壤水分脅迫(W1和W2)條件下,顯著提高了SOD活性(圖4),對POD和CAT活性無顯著影響(圖4)(W2條件下N2對POD的影響除外),在土壤水分充足(W4和W5)時(shí),則顯著降低了3種酶活性；在W3條件下,高、低濃度施氮效果不同。

圖4 紅樺幼苗根系超氧化物歧化酶(SOD)、過氧化物歧化酶(POD)和過氧化氫酶(CAT)活性(平均值±標(biāo)準(zhǔn)偏差)對不同水、氮供應(yīng)的響應(yīng)Fig.4 Responses of root SOD, POD, CAT activities in Betual albosinensis seedling (mean±SD) to different soil water and nitrogen supply

3 討論

根系活力和根系呼吸速率都是衡量根系生理活性的重要指標(biāo),在一定程度上反映了作物吸收水分和礦質(zhì)養(yǎng)分的能力,直接影響著根系的生長發(fā)育狀況,對植物的生長起著決定性作用[25]。通常,根系活力和呼吸速率越高,根系吸收水分和養(yǎng)分的能力就越強(qiáng)[24]。本研究中,根系活力和呼吸速率在土壤水分脅迫下顯著降低,而氮素的施加更是加劇了其降低(圖1),說明土壤水分脅迫不利于根系生理活性的發(fā)揮,且施氮加劇了脅迫程度,這可歸因于低土壤含水量下,氮肥使土壤水分溶質(zhì)勢降低,導(dǎo)致植物吸收水分難度增大[26],從而降低了根系生理活性。在土壤水分充足的狀況下,施氮使幼苗根系活力和呼吸速率顯著增強(qiáng),這表明充足的水分能使土壤氮素有效性增強(qiáng),促進(jìn)根系生理活性的發(fā)揮,提高根系吸收水分和養(yǎng)分的能力。本實(shí)驗(yàn)結(jié)果說明合適的水-氮配比才能使水、氮發(fā)揮其最大效應(yīng)。但也有研究表明,當(dāng)植物受到土壤環(huán)境脅迫時(shí),根系活力有所增強(qiáng)[27],這可能是由于不同植物耐受環(huán)境脅迫能力及適應(yīng)策略不同。

植物生理生化指標(biāo)的變化是植物對逆境條件的適應(yīng)性反應(yīng),可反映植物生長狀況及受傷害程度。環(huán)境脅迫可導(dǎo)致植物體內(nèi)活性氧自由基積累并引發(fā)膜脂過氧化,MDA即是細(xì)胞膜脂過氧化產(chǎn)物[28],其含量可表征膜脂過氧化程度,反映細(xì)胞膜系統(tǒng)受傷害程度[29]。大量研究表明,在受到干旱、冷凍傷害以及高溫脅迫時(shí),植物細(xì)胞內(nèi)產(chǎn)生和清除活性氧的平衡機(jī)制被破壞,從而使活性氧的產(chǎn)生和膜脂過氧化反應(yīng)加劇,導(dǎo)致MDA大量積累[30-31]。本研究中,紅樺幼苗根系MDA含量在土壤含水量不足時(shí)的變化(圖2)說明土壤水分脅迫對根系細(xì)胞膜系統(tǒng)造成了傷害,施氮更是加劇了其受傷害程度。這應(yīng)該是在較低土壤含水量的情況下,施加氮肥所導(dǎo)致的土壤滲透壓增加,而在這種緩慢滲透脅迫下根系自由基積累引起膜脂過氧化作用[32]。相反,在土壤水分充足的條件下,施氮?jiǎng)t減緩了根系細(xì)胞膜脂過氧化程度。該結(jié)果與Yin等人[33]的研究結(jié)果不完全一致,這可能與供試土壤水、氮條件以及研究物種對水、氮需求量不同有關(guān)。此外,本研究中根系MDA含量與根系活力和呼吸速率之間的顯著負(fù)相關(guān)性(圖2),說明幼苗根系膜脂過氧化程度的加劇是導(dǎo)致其生理活性降低的內(nèi)在因素之一。

在一定范圍內(nèi)的環(huán)境脅迫下,根系往往可通過增強(qiáng)其滲透調(diào)節(jié)能力[8]和抗氧化能力[9]適應(yīng)脅迫,進(jìn)而維持根系及整個(gè)植株生理過程的正常進(jìn)行[34]。本研究中,在土壤水分狀況不足時(shí),幼苗根系細(xì)胞膜系統(tǒng)受損(圖2)、生理活性降低(圖1),根系中滲透調(diào)節(jié)物質(zhì)(脯氨酸、可溶性蛋白質(zhì)和可溶性糖)含量相應(yīng)升高(圖3),這與Qayyum等[35]的研究結(jié)果相似,說明根系內(nèi)的脯氨酸等可溶性物質(zhì)能夠通過細(xì)胞內(nèi)滲透調(diào)節(jié),去除活性氧,維持膜穩(wěn)定性,從而緩解植株受到的脅迫[36-37]。但也有研究表明,干旱脅迫增加了脯氨酸含量卻降低了可溶性蛋白質(zhì)含量[27]和可溶性糖含量[38],這可能與研究物種的內(nèi)在調(diào)節(jié)機(jī)制以及相關(guān)遺傳因素[39]有關(guān)。此外,在土壤水分脅迫下,施氮?jiǎng)t進(jìn)一步提高了紅樺幼苗根系滲透調(diào)節(jié)能力,尤其是脯氨酸和可溶性蛋白質(zhì)含量隨施氮濃度的增加顯著增加(圖3),說明紅樺幼苗根系內(nèi)脯氨酸和可溶性蛋白質(zhì)對土壤環(huán)境脅迫的響應(yīng)比可溶性糖更為敏感,因?yàn)楦彼嶙鳛樗苄宰畲蟮陌被?是植物滲透調(diào)節(jié)過程中的關(guān)鍵物質(zhì)[34]。此外,根系可溶性蛋白質(zhì)和可溶性糖含量的變化與植株體內(nèi)氮含量和淀粉的積累量等也有關(guān)系[33]。

綜上,土壤水分脅迫使根系細(xì)胞膜系統(tǒng)受損,根系生理活性受到抑制,施氮更是加劇了脅迫程度,但在一定范圍內(nèi)的脅迫下,根系可通過提高滲透調(diào)節(jié)能力和抗氧化調(diào)節(jié)能力來抵御環(huán)境脅迫；此外,在土壤水分狀況良好時(shí),施氮能夠促進(jìn)紅樺幼苗根系生理活性增強(qiáng),且對于本研究的供試土壤及試驗(yàn)設(shè)置的氮素梯度而言,低濃度施氮效果尤為明顯。

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The physiological responses ofBetulaalbosinensisseedling roots to soil water-nitrogen coupling

SUN Yuyu1,2, YIN Chunying1,*, HE Heliang1,3, TANG Bo1,2, LIU Qing1

1KeyLaboratoryofMountainEcologicalRestorationandBioresourceUtilization&EcologicalRestorationBiodiversityConservationKeyLaboratoryofSichuanProvince,ChengduInstituteofBiology,ChineseAcademyofSciences,Chengdu610041,China2UniversityofChineseAcademyofSciences.Beijing100049,China3CollegeofMaterialandChemistry&ChemicalEngineering,ChengduUniversityofTechnology,Chengdu610059,China

The increasing deposition of nitrogen into the atmosphere has affected the stability and productivity of ecosystem. The effects of nitrogen deposition vary in regards to soil water content. There is uneven precipitation distribution and significant water-nitrogen coupling, which directly affects the growth and development of plants. In order to better understand the responses of plant roots to soil water-nitrogen coupling, we exposed seedlings ofBetulaalbosinensisto five different water regiments of field water holding capacity (FC) (40% [W1], 50% [W2], 60% [W3], 80% [W4], and 100% [W5]). As well as three different nitrogen (N) regiments (control, 0 [N0], 20 [N1], and 40 [N2] gN m-2a-1with NH4NO3). These experimental conditions allowed us to determine the root activity and physiology of the seedlings when exposed to various water and nitrogen supplies. The results indicated that a decrease of soil water content prompted the root vigor and root respiration rate to decrease while membrane lipid peroxide(MDA), osmotic adjustment substances (i.e. proline, soluble protein, and soluble sugar) content, and the activity of antioxidant enzymes (i.e. superoxide dismutase, peroxidase and catalase) all increased. Moreover, root physiological characteristics were significantly affected by the water-nitrogen coupling effect. When soil moisture was deficient (W1and W2), the addition of nitrogen markedly reduced root activity. However, when adequate soil water (W4and W5) was present, root activity was significantly enhanced. Additionally, the greater the concentration of nitrogen applied, the greater the effects were upon root activity and the contents of proline and soluble MDA. Only the lower concentration of nitrogen addition (N1) significantly improved root physiological functions under 60%FC. Finally, both root vigor and nitrate reductase activity had a significant negative correlation with membrane lipid peroxide level (MDA content). In conclusion, atmospheric nitrogen deposition could significantly promote root physiological characteristics ofB.albosinensisseedlings when soil moisture is plentiful. However, if soil water is deficient, the deposition of atmospheric nitrogen could cause serious damage to the cell membrane systems and consequently restrain the activity of the root system. Therefore, plant roots could defend themselves against these stresses by increasing the osmotic regulation substances content and antioxidant enzyme activity within a certain range.

water-nitrogen coupling; root activity; membrane lipid peroxidation; osmotic adjustment; antioxidant enzyme

國家自然科學(xué)基金項(xiàng)目(31370495, 31400424)

2015- 02- 15;

日期:2016- 03- 03

10.5846/stxb201502150360

*通訊作者Corresponding author.E-mail: yincy@cib.ac.cn

孫譽(yù)育,尹春英,賀合亮,唐波,劉慶.紅樺幼苗根系對水-氮耦合效應(yīng)的生理響應(yīng).生態(tài)學(xué)報(bào),2016,36(21):6758- 6765.

Sun Y Y, Yin C Y, He H L, Tang B, Liu Q.The physiological responses ofBetulaalbosinensisseedling roots to soil water-nitrogen coupling.Acta Ecologica Sinica,2016,36(21):6758- 6765.