大氣汞濃度升高對水稻葉片生理效應的影響研究

陳劍，王章瑋，張曉山，秦普豐，陸海軍

1.中國科學院生態環境研究中心大氣環境科學實驗室，北京100085

2.中國科學院大學資源與環境學院，北京100049

3.湖南農業大學資源環境學院，長沙410128

大氣汞濃度升高對水稻葉片生理效應的影響研究

陳劍1,2，王章瑋1,*，張曉山1，秦普豐3，陸海軍3

1.中國科學院生態環境研究中心大氣環境科學實驗室，北京100085

2.中國科學院大學資源與環境學院，北京100049

3.湖南農業大學資源環境學院，長沙410128

采用大田開頂式氣室熏氣實驗,研究大氣汞濃度升高對水稻葉片氣體交換參數、脯氨酸、丙二醛的積累以及超氧化物歧化酶活性的影響。實驗結果顯示,水稻葉片凈光合速率(Pn)和氣孔導度(Gs)隨大氣汞濃度的升高均較對照略微下降,表明大氣汞濃度的升高對水稻光合作用和氣孔開放程度有一定影響；揚花期水稻胞間CO2濃度(Ci)隨大氣汞濃度的升高明顯降低(P＜0.05)表明Pn的略微下降屬于氣孔限制,同時蒸騰速率(Tr)顯著增加(P＜0.01)表明大氣汞對水稻的蒸騰生理功能有一定的影響。乳熟期水稻葉片氣體交換參數與大氣汞濃度無顯著差異(P＞0.05),且各指標均低于揚花期。水稻葉片脯氨酸(Pro)含量在拔節期隨大氣汞濃度的升高而顯著增加(P＜0.05),揚花期先升高后下降,在45 ng·m-3時達到最大,成熟期無顯著差異(P＞0.05)；水稻葉片丙二醛(MDA)含量在拔節期先升高后下降,45 ng·m-3時達到最大,揚花期和成熟期均無顯著差異(P＞0.05)；水稻葉片超氧化物歧化酶(SOD)活性在拔節期先升高后下降,15 ng·m-3時達到最大,揚花期無顯著差異(P＞0.05)。以上結果表明大氣汞濃度的升高可以引起水稻葉片膜脂過氧化以及脯氨酸和丙二醛含量的積累,且隨著體內Pro、MDA和SOD對大氣汞脅迫的協同反應,水稻對逆境的適應能力增強,對汞脅迫產生了耐受性。

氣態單質汞；氣體交換參數；脯氨酸；丙二醛；超氧化物歧化酶；開頂式氣室

自工業革命以來,人類活動將數千噸的汞以氣態元素汞(gaseous elemental mercury,GEM)的形式排放到大氣中[15],隨大氣傳輸擴散并通過干濕過程沉降到土壤、植物表面,進而對生物體產生毒害作用,據報道,當前大氣汞的沉降速率是工業革命前的3.4倍[16]。目前國內外大量工作關注于水培或土培實驗中Hg2+對植物根、葉及幼苗的生理生化影響研究[2,4,6,17],而大氣汞對植物生理的直接影響研究相對缺乏。大量研究表明,植物葉片中汞的含量與大氣汞濃度顯著正相關[18-21],因此本文通過大田開頂式氣室熏氣實驗,研究了大氣汞濃度升高對水稻葉片氣體交換參數、脯氨酸、丙二醛的積累以及超氧化物歧化酶活性的影響。

1 材料與方法(Materials and methods)

1.1 開頂式氣室(OTCs)熏氣實驗設計

實驗地點位于湖南農業大學農資系實驗基地(28.28°N,113.01°E),實驗田面積30 m×10 m,該地區屬于典型亞熱帶季風濕潤氣候區,季節變化明顯,年均氣溫17.2℃,供試水稻為該地區廣泛播種的中青優2號。

開頂式氣室為Heagle型[22],主要由氣室主體、GEM生成系統和布氣系統三部分組成,可以為植物提供比較接近自然的生長環境。氣室主體為長1.5 m,寬1.4 m,高1 m(地面以上部分)的長方體,頂部架設一個收縮角度為45°的平截頭體[23],總體積約為2.835 m3。氣室骨架由PVC管連接構成,四面緊密覆蓋0.08 mm厚的透明聚氯乙烯塑料薄膜。在一根上部直徑2 cm、長40 cm,下部直徑4 cm、長10 cm的玻璃管底部加入少量液態元素汞(liquid elemental mercury,LEM)沒入恒溫槽液面以下,設定恒溫槽溫度在20℃左右,為GEM均勻穩定的產生提供接近恒溫且低于環境溫度的條件。產生的GEM由一定流量的載氣(高純氮氣)通過內徑2 mm的聚四氟塑料管帶出玻璃管并引至田間,與鼓風機產生的氣流相混合,再由密布小孔的PVC管從底部通入氣室[24]。

根據城市大氣平均汞含量及實驗區近地表大氣背景汞濃度,本實驗共設4組汞濃度水平,分別為(5 ±2)ng·m-3(CK)、(15～20)ng·m-3、(45～50)ng·m-3和(90～100)ng·m-3,每個水平3個重復。為避免相互遮蔭,各氣室之間留有3 m的間距。氣室內汞濃度通過浮子流量計調節載氣流速來控制,每50 s左右氣室由離心鼓風機(690 m3·h-1)完成1次徹底換氣。從2013-08-31正式開始熏氣,到2013-11-15結束熏氣,24 h連續供氣,氣室內汞濃度由RA-915+賽曼原子吸收汞分析儀(Lumex Inc.,Russia)在線監測。

1.2 葉片生理指標的測定

1.2.1 氣體交換參數的測定

利用LI-6400便攜式光合儀測定系統(LI-6400, LICOR Inc.,USA)測定不同汞濃度水平熏氣實驗中水稻葉片凈光合速率(Pn)、氣孔導度(Gs)、胞間CO2濃度(Ci)和蒸騰速率(Tr)4個氣體交換參數[25-26]。實驗表明植物葉片Pn一般在上午9:00-11:00達到最大值[27-28],因此本研究分別選擇揚花期和乳熟期天氣狀況基本一致的2 d,在上午10:00-12:00對水稻葉片氣體交換參數進行測定,儀器CO2濃度和流速分別設定為400μmol·mol-1和500μmol·s-1,每個處理選擇4片完整無缺的劍葉進行測定。

2.3.2推進農作物秸稈資源化利用 指導長江經濟帶以縣為單元編制全量化利用實施方案，提高秸稈綜合利用的區域統籌水平。堅持農用為主、五料并舉，積極推廣深翻還田、撿拾打捆、秸稈離田多元利用等技術，指導創設秸稈還田離田利用政策機制，培育秸稈資源化利用產業化龍頭企業，推進秸稈產業化發展。

1.2.2 抗逆指標的測定

采集水稻拔節期、揚花期和成熟期的葉片鮮樣,用便攜式冰箱迅速帶回實驗室,并先后用自來水和去離子水沖洗干凈。葉片游離脯氨酸(Pro)和丙二醛(MDA)含量的測定參照朱廣廉[29]、陳建勛[30]的方法,脯氨酸含量采用磺基水楊酸提取,酸性茚三酮染色的方法進行測定；丙二醛含量采用硫代巴比妥酸(TBA)法測定；葉片總超氧化物歧化酶(T-SOD)活性采用黃嘌呤自氧化法(羥胺法)測定[31]。

2 結果(Results)

2.1 大氣汞對水稻葉片氣體交換參數的影響

實驗結果表明,在3個熏氣汞濃度下,揚花期水稻葉片凈光合速率(Pn)和氣孔導度(Gs)均比對照略低(圖1A、B),Pn在15和45 ng·m-3的大氣汞濃度下顯著低于對照(P＜0.05),但Gs在不同大氣汞濃度下無顯著差異(P＞0.05),說明在實驗處理水平下的大氣汞對水稻葉片的氣孔開放程度無明顯影響,對光合作用有一定影響；胞間CO2濃度(Ci)隨大氣汞濃度的升高有明顯降低的趨勢(圖1C,P＜0.05)；蒸騰速率(Tr)隨大氣汞濃度升高而顯著增加(圖1D,P＜0.01)。乳熟期水稻葉片Pn在45 ng·m-3的大氣汞濃度下顯著低于對照,Gs、Ci和Tr與大氣汞濃度均無顯著差異(圖1A、B、C、D,P＞0.05)；同時,除Ci與揚花期無顯著差異外,乳熟期Pn、Gs和Tr在4個大氣汞濃度水平下明顯低于揚花期,表明乳熟期水稻葉片的光合作用和蒸騰作用較揚花期弱,且大氣汞對乳熟期水稻葉片氣體交換參數均無明顯影響。

2.2 大氣汞對水稻葉片脯氨酸含量的影響

拔節期水稻葉片中脯氨酸含量隨大氣汞濃度的升高而顯著增加(圖2,P＜0.05),揚花期和成熟期水稻葉片中游離脯氨酸含量隨大氣汞濃度的升高均有增加的趨勢,表明大氣汞濃度的升高會脅迫水稻葉片產生并積累大量的游離脯氨酸。大氣汞濃度在5、15和45 ng·m-3時,揚花期葉片中脯氨酸含量明顯高于拔節期和成熟期,在90 ng·m-3的大氣汞濃度下,葉片中脯氨酸含量關系為:成熟期＞揚花期＞拔節期,表明環境濃度下的大氣汞對水稻揚花期葉片中游離脯氨酸的積累影響更大,而在高汞濃度下,汞在葉片中隨生長時期的延長而富集對脯氨酸的積累影響更大。

2.3 大氣汞對水稻葉片丙二醛含量的影響

水稻葉片中丙二醛(MDA)含量在各生長時期隨著大氣汞濃度的升高無顯著差異(圖3,P＞0.05),但在拔節期和成熟期會隨大氣汞濃度從5 ng·m-3升高到45 ng·m-3而不斷增加,在90 ng·m-3時又下降,表明大氣汞濃度的升高會脅迫葉片產生過多的丙二醛。在4個大氣汞濃度水平下,不同時期葉片中MDA含量關系均為:拔節期＞成熟期＞揚花期,拔節期明顯最高,且在15和45 ng·m-3時,葉片內MDA含量顯著高于對照(P＜0.05),表明大氣汞對水稻拔節期葉片中丙二醛含量的影響最大。

圖1 大氣汞對水稻葉片氣體交換參數的影響Fig.1 Effects of air Hg to gas exchange parameters of rice foliage

圖2 大氣汞對水稻葉片脯氨酸含量的影響Fig.2 Effects of air Hg to proline of rice foliage

圖3 大氣汞對水稻葉片丙二醛(MDA)含量的影響Fig.3 Effects of air Hg to malonaldehyde(MDA)of rice foliage

2.4 大氣汞對水稻葉片超氧化物歧化酶活性的影響

水稻葉片中總超氧化物歧化酶(T-SOD)的活性在拔節期和揚花期隨著大氣汞濃度的升高均無顯著差異(圖4,P＞0.05),但在15 ng·m-3時拔節期葉片中SOD活性顯著高于其他汞濃度下(P＜0.05)；在5、15和45 ng·m-3時,拔節期葉片中SOD活性均較揚花期高,而在90 ng·m-3時,揚花期較拔節期略高。

圖4 大氣汞對水稻葉片超氧化物歧化酶活性(SOD)的影響Fig.4 Effects of air Hg to superoxide dismutase(SOD) of rice foliage

3 討論(Discussion)

大量實驗結果表明,汞影響植物的光合作用,影響光反應和暗反應[32-33],但也有一些實驗結果表明,水培實驗中汞濃度低于10mg·L-1時,汞對植物的光合作用影響較小甚至無影響[32,34-37]。Ericksen和Gustin[34]的實驗結果表明,不同濃度的大氣汞(2.4、11和30 ng·m-3)熏蒸對楊樹葉的Pn和Gs均無顯著影響,Niu等[38]的實驗結果也表明,環境濃度的大氣汞(2、10、20和50 ng·m-3)對玉米葉片的氣體交換參數基本沒有影響。Ci的變化是分析植物氣孔與非氣孔限制的基礎,作為光合過程中CO2的中介,一方面受到作為源的外界CO2濃度和氣孔導度的影響,另一方面又受葉片光合消耗的影響[26,39],而非氣孔限制常用于解釋胞內恒定或者增加的Ci[40-41],本實驗中揚花期Ci隨著大氣汞濃度的升高明顯下降,且Pn和Gs均有一定程度的降低,說明水稻揚花期屬于氣孔限制。王孟本等[42]對河北楊和檸條的研究表明,在土壤水分充足的條件下,若樹木根系吸水力依然較強,而其蒸騰生理調控力卻大為減弱,蒸騰速率就會異常增大,本實驗中隨著大氣汞濃度的升高,水稻葉片光合作用和氣孔導度均無顯著變化,而蒸騰速率卻線性升高,說明大氣汞影響了水稻葉片的蒸騰生理功能。

脯氨酸(Pro)是植物蛋白質的組分之一,可以游離狀態廣泛存在于植物體中,在逆境(旱、鹽堿、熱、冷、凍等)脅迫條件下,許多植物體內脯氨酸含量顯著增加,在一定程度上反映了植物的抗逆性。實驗表明,重金屬脅迫下植物體內 Pro濃度會增加[11-12,43]。劉玲等[44]的砂培實驗結果表明,在低汞濃度、短時間內,玉米體內Pro含量有上升趨勢,在高汞濃度、長時間內,玉米體內Pro含量會嚴重下降,且Pro對汞的脅迫非常敏感；Niu等[38]對玉米的研究結果表明,大氣汞濃度與Pro含量之間無顯著相關性(P＞0.05),但在大氣汞濃度為20和50 ng·m-3時Pro含量顯著高于對照(P＜0.05)。本實驗中,在15和45 ng·m-3大氣汞濃度下揚花期葉片脯氨酸含量顯著較高,而在90 ng·m-3時成熟期葉片脯氨酸含量顯著較高,表明隨著大氣汞濃度的升高,水稻葉片脯氨酸含量隨著大氣汞濃度的升高均有不同程度的增加。

在植物器官衰老或在逆境條件下,細胞內會產生大量的活性自由基,并與脂質發生過氧化反應,最終產物即為丙二醛(MDA),通常利用它作為脂質過氧化指標,表示細胞膜脂過氧化程度和植物對逆境條件反應的強弱。大量實驗結果表明,重金屬脅迫下植物體內丙二醛的含量會增加[6,45-46]。Cho和Park[6]報道西紅柿葉中MDA水平隨葉汞濃度的增加而增加,Moreno-Jimeˊnez等[47]對2種野生植物的研究結果也表明葉中MDA含量與葉汞濃度顯著正相關(P＜0.05)；而Niu等[38]對玉米的研究結果表明,大氣汞濃度與MDA含量之間無顯著相關性(P＞0.05),僅在大氣汞濃度為20和50 ng·m-3時MDA含量顯著高于對照(P＜0.05)。本實驗中,高濃度的大氣汞熏氣顯著增加了丙二醛的含量,表明會引起植物葉的膜脂過氧化。

超氧化物歧化酶(SOD)是機體內天然存在的超氧自由基清除因子,是生物體內清除自由基的首要物質,與體內的過氧化氫酶(CAT)和過氧化物酶(POD)組成了一個完整的防氧化鏈條。大量實驗結果表明重金屬脅迫下植物體內SOD活性會發生變化[12,45,48-49]。馬成倉[44]用不同濃度的HgCl2溶液灌溉油菜結果顯示,油菜葉細胞內SOD活性在0.5mg·L-1的汞濃度下無明顯變化,(1～10)mg·L-1的汞濃度下逐漸升高,50mg·L-1時持續下降；陸海燕等[12]的研究結果顯示,隨著溶液中Cd2+濃度的增加,蘆葦葉片中SOD活性呈先上升后下降的趨勢；施國新等[49]的研究也表明,隨著汞濃度的升高,滿江紅葉片SOD活性逐漸增強,當濃度超過一定范圍時,SOD活性則開始降低。本實驗中,拔節期水稻葉片SOD活性隨大氣汞濃度的升高先上升后下降,15 ng·m-3時達到最大,表明水稻體內氧自由基清除能力較強,水稻耐汞性較好。

綜上分析,水稻葉片中Pro、MDA含量和SOD活性隨著大氣汞濃度的升高變化不同。在拔節期, Pro含量隨大氣汞濃度升高呈線性增加,MDA含量先急劇增加然后降低,在45 ng·m-3的汞濃度下達到最大,SOD活性先增大,在15 ng·m-3的汞濃度下達到最大后又開始下降；在揚花期,Pro含量隨大氣汞濃度的升高先增加后下降,且明顯比拔節期高, MDA含量和SOD活性無顯著變化,且較拔節期低。出現這種情況的可能原因是,汞脅迫下水稻葉片細胞膜脂出現過氧化,體內SOD活性被激發,同時Pro和MDA含量增加,Pro具有減少膜脂過氧化和穩定細胞膜結構及生物大分子的作用[50],因此對SOD這種生物蛋白大分子具有一定的保護作用,對其他膜結構也有一定的保護作用,Pro的這種雙重作用機制,使得SOD活性在迅速升高后又恢復到正常水平,并隨著Pro的不斷增加,MDA含量也開始下降；隨著生長期的延長,汞脅迫使水稻體內Pro含量升高,同時對逆境適應能力增強,對汞污染產生了耐受性,體內Pro含量開始降低。這與陸海燕等[12]在鎘污染下對蘆葦葉片丙二醛、脯氨酸及SOD保護酶反應的研究結果一致。因此,水稻葉片中Pro、MDA和SOD對大氣汞濃度升高有協同反應。

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Physiological Responses of Rice Leaves to the Elevated Gaseous Elemental Mercury in the Atmosphere

Chen Jian1,2,Wang Zhangwei1,*,Zhang Xiaoshan1,Qin Pufeng3,Lu Haijun3
1.Laboratory of Atmospheric Environmental Sciences,Research Center for Eco-Environmental Sciences,Chinese Academy of Sciences,Beijing 100085,China
2.College of Resources and Environment,University of Chinese Academy of Sciences,Beijing 100049,China
3.College of Resources&Environment,Hunan Agricultural University,Changsha 410128,China

27 January 2015 accepted 25 March 2015

The effects of elevated gaseous elemental mercury(GEM)on gas exchange parameters,accumulation of proline(Pro)and malondialdehyde(MDA),activity of superoxide dismutase(SOD)in rice foliage were studied with field open-top chambers(OTCs)fumigation experiment.The results showed that the net photosynthesis rate(Pn)and stomatal conductance(Gs)were less slightly in GEM treatment than those in the control,which indicating that elevated GEM had some effect on photosynthesis and stomatal openness of rice leaves.In flowering stage of rice,the distinct decrease(P＜0.05)of intercellular CO2concentration(Ci)with elevated GEM indicated that stomatal limitation led to the slight decrease of Pn,and the significant increase(P＜0.01)of transpiration rate(Tr)with elevated GEM showed that the physiological function of rice transpiration was effected by Hg in the atmosphere.Gas exchange parameters of rice leaves in milky stage were insignificantly difference with GEM in air(P＞0.05)and lower than that in flowering stage.Proline concentrations in rice foliage were increased obviously with elevated GEM(P＜0.05)in jointing stage,declined after increasing and reached to the maximum value at 45 ng·m-3in flowering stage,and it was no significant difference(P＞0.05)in mature stage among four treatments.The contents of MDA in rice foliage increased first and reached the highest value at 45 ng·m-3,and then decreased in jointing stage,and it was no significant difference(P＞0.05)with the increase of GEM in flowering and mature stage.The activity of SOD in rice foliage also increased first and then declined at 15 ng·m-3in jointing stage,and there was no significant difference(P＞0.05)in flowering stage.These results suggested that elevated GEM in air can cause membrane lipid peroxidation and accumulation of Pro and MDA in rice foliage,furthermore the ability of adapting to adversity and the tolerance to elevated GEM for rice were enhanced with the concerted reactions in vivo among Pro,MDA and SOD on the atmospheric mercury stress.

GEM;gas exchange parameters;proline;malondialdehyde;superoxide dismutase;open-top chamber

2015-01-27 錄用日期:2015-03-25

1673-5897(2016)1-133-08

X171.5

A

10.7524/AJE.1673-5897.20150127003

陳劍,王章瑋,張曉山,等.大氣汞濃度升高對水稻葉片生理效應的影響研究[J].生態毒理學報,2016,11(1):133-140

Chen J,Wang Z W,Zhang X S,et al.Physiological responses of rice leaves to the elevated gaseous elemental mercury in the atmosphere[J].Asian Journal of Ecotoxicology,2016,11(1):133-140(in Chinese)

國家自然科學基金項目(No.41373124,41073092)；國家重大基礎研究(973)計劃項目(2013CB430002)

陳劍(1990-),男,碩士研究生,研究方向為大氣汞循環,E-mail:chenjianev2008@126.com

),E-mail:wangzhw@rcees.ac.cn

簡介:王章瑋(1978-)，女，博士，副研究員，主要研究方向大氣汞循環。