灌漿結(jié)實期弱光對水稻籽粒氮代謝酶及蛋白質(zhì)含量的影響

許光利,劉佳,梁成剛,汪燕,丁春邦,李天*(.四川農(nóng)業(yè)大學農(nóng)學院/農(nóng)業(yè)部西南作物生理生態(tài)與耕作重點實驗室,成都630;.四川農(nóng)業(yè)大學生命科學學院,四川雅安6504)

灌漿結(jié)實期弱光對水稻籽粒氮代謝酶及蛋白質(zhì)含量的影響

許光利1,劉佳1,梁成剛1,汪燕1,丁春邦2,李天1*
(1.四川農(nóng)業(yè)大學農(nóng)學院/農(nóng)業(yè)部西南作物生理生態(tài)與耕作重點實驗室,成都611130;2.四川農(nóng)業(yè)大學生命科學學院,四川雅安625014)

摘要為了探討水稻灌漿結(jié)實期弱光脅迫對籽粒氮代謝關(guān)鍵酶及蛋白質(zhì)的影響,以粳稻越光和秋田小町,秈稻IR72,以及雜交秈稻岡優(yōu)527等4個品種為材料,在灌漿結(jié)實期分別進行遮光25%(A1)和遮光50%(A2)為處理,以自然條件為對照(CK),研究了不同程度弱光對氮代謝關(guān)鍵酶谷氨酰胺合成酶(glutamine synthetase,GS)、谷氨酸-草酰乙酸轉(zhuǎn)氨酶(glutamic oxalo-acetic transaminase,GOT)和谷氨酸-丙酮酸轉(zhuǎn)氨酶(glutamate pyruvate transaminase,GPT)活性和可溶性蛋白質(zhì)、蛋白質(zhì)以及氨基酸含量的影響.結(jié)果表明,弱光下GS活性顯著降低,且隨著弱光程度加深而下降加重,而GOT、GPT、可溶性蛋白質(zhì)、蛋白質(zhì)和氨基酸含量在弱光下均顯著提高,且提高幅度A2＞A1.GS活性與可溶性蛋白質(zhì)、蛋白質(zhì)以及氨基酸含量均呈顯著負相關(guān),而GOT和GPT與可溶性蛋白質(zhì)、蛋白質(zhì)和氨基酸均呈極顯著正相關(guān).由此推測相對于GS,灌漿結(jié)實期水稻籽粒中GOT和GPT活性對蛋白質(zhì)的合成影響更加顯著.

關(guān)鍵詞水稻;灌漿結(jié)實期;弱光;氮代謝;關(guān)鍵酶;蛋白質(zhì);氨基酸

浙江大學學報(農(nóng)業(yè)與生命科學版) 42(1):53～62,2016

Journal of Zhejiang University(Agric.&Life Sci.)

http://www.journals.zju.edu.cn/agr

E-mail:zdxbnsb@zju.edu.cn

第一作者聯(lián)系方式:許光利(http://orcid.org/0000-0001-6945-7322),E-mail:guangli.xu@hotmail.com

URL:http://www.cnki.net/kcms/detail/33.1247.S.20160119.1927.006.html

Changes of nitrogen metabolism enzyme activities and protein content in response to low light during the seed filling stage in rice.Journal of Zhejiang University(Agric.&Life Sci.),2016,42(1):53-62

XU Guangli1,LIU Jia1,LIANG Chenggang1,WANG Yan1,DING Chunbang2,LI Tian1*(1.College of Agronomy,Sichuan Agricultural University/Key Laboratory of Crop Physiology,Ecology and Cultivation in Southwest,Ministry of Agriculture,Chengdu 611130,China;2.College of Life Science,Sichuan Agricultural University,Ya”an 625014,Sichuan,China)

Summary Light condition is one of the important factors affecting plant growth.During plant growth,light deficiency will result in the decrease in carbon fixation,photosynthetic efficiency,and leaf senescence acceleration,and thus decrease yield evidently.Besides yield,light also affect the rice qualities through influencing the seed compositions such as protein.Many studies have been carried out to evaluate the effect of light on protein synthesis.For example,previous studies have been certified that the protein accumulation is increased during the seed filling stage,as well as amino acids.The enzymes of glutamine synthetase(GS),glutamic oxalo-acetic transaminase (GOT),and glutamate pyruvate transaminase(GPT)are considered as the key enzymes regulating the synthesis ofprotein.Almost 95%of N was absorbed through GS/glutamate synthetase(GOGAT)cycle in plants and converted into glutamic acid(Glu)and glutamine(Gln).The Glu and Gln are then converted into other amino acids which provide the precursor materials for protein synthesis through the transamination reactions of GOT and GPT.However,researches about the effect of the activities of GS,GOT and GPT on amino acid and protein synthesis of rice are limited,especially under stress conditions such as low light.

Based on previous studies,we carried out this experiment to evaluate the effect of low light on key enzyme activities of nitrogen metabolism and protein accumulation during the seed filling stage.The aims of this study were to study the dynamic changes of GS,GOT and GPT under different low light conditions during the seed filling stage,and evaluate the relationships between the activities of enzymes and protein accumulation.

The experiment was initiated at Sichuan Agricultural University,Ya’an,China,in 2013.Four cultivars including japonica cultivars Akitakomachi and Koshihikari,indica cultivar IR72,and hybrid indica cultivar Gangyou 527 were selected as the materials.All the cultivars were managed routinely till the flowering stage,then 25%shading(A1)and 50%shading(A2),were set respectively as the treatments till maturity by using shade net,and natural condition was set as CK.Each treatment plot was repeated 3 times and the rice panicles which grown uniformly were marked for further sample collection.After 10 days of treatments,the seed samples were collected per 7-day period till maturity and stored in freezer at-80℃.After harvest,the samples were used to measure GS,GOT and GPT activities,as well as soluble protein,total protein and amino acid contents.

Results showed that the GS activities were significantly decreased by 20.01%-30.52% at A2than A1(11.61%-19.36%)compared with CK,whereas,the activities of GOT and GPT increased significantly at A2(GOT increased by 26.07%-36.82% and GPT increased by 26.07%-61.07%)than A1(GOT increased by 14.02%-23.02% GPT increased by11.81%-33.83%)compared with CK.In consistent with GOT and GPT,patterns of soluble protein,total protein,as well as amino acids were also significantly increased under low light conditions especially at A2(compared with CK,soluble protein increased by 31.19%-70.51%,total protein increased by16.02%-28.89%,and amino acids increased by16.04%-22.48% at A2).The correlation analysis showed that GS activity was negatively correlated to soluble protein,total protein and amino acids under low light conditions,however,GOT and GPT showed positive correlations with soluble protein,total protein and amino acids.

Based on the results,we speculated that the synthesis of protein of rice grain was more significantly affected by GOT and GPT which tended to increase the accumulation of both protein and amino acids,compared with GS.In addition,in consistent with previous studies,the low light conditions were beneficial for protein and amino acid synthesis of rice grain during the seed filling stage.

Key words rice;seed filling stage;low light;nitrogen metabolism;key enzyme;protein;amino acid

光照是影響植物生長發(fā)育的重要環(huán)境因子之一[1].光照不足即弱光會導(dǎo)致作物碳固定減少,光合作用降低,葉片衰老加快,因而導(dǎo)致產(chǎn)量明顯降低[2-6].除了影響產(chǎn)量,弱光脅迫通過影響稻米的組分,也對稻米品質(zhì)有著重要影響.蛋白質(zhì)作為水稻的重要組分之一,其含量以及組成直接影響著稻米品質(zhì),如何提高稻米蛋白質(zhì)含量兼顧營養(yǎng)品質(zhì)一直受到研究者的重視[7].關(guān)于弱光對稻米籽粒蛋白質(zhì)的影響,前人已做了相關(guān)研究.例如劉奇華等[8]研究指出在插秧至幼穗分化期弱光不利于蛋白質(zhì)的合成,而在抽穗至成熟期弱光則利于蛋白質(zhì)的積累.任萬軍等[9]和MO等[10]同樣研究發(fā)現(xiàn)灌漿期弱光可提高稻米蛋白質(zhì)含量.蛋白質(zhì)合成過程中,谷氨酰胺合酶(glutamine synthetase,GS)、谷氨酸-草酰乙酸轉(zhuǎn)氨酶(glutamic oxalo-acetic transaminase,GOT)和谷氨酸丙酮酸轉(zhuǎn)氨酶(glutamate pyruvate transaminase,GPT)是涉及氮代謝的關(guān)鍵酶,其活性顯著影響著蛋白質(zhì)與氨基酸含量[11-13].自然界中的無機氮主要通過谷氨酰胺合成酶/谷氨酸合成酶循環(huán)同化NH4+為谷氨酸和谷氨酰胺,該過程是整個氮代謝的中心環(huán)節(jié),進而又通過GOT和GPT等轉(zhuǎn)氨酶作用轉(zhuǎn)化為其他必需或非必需氨基酸,為蛋白質(zhì)合成提供前體物質(zhì).盡管前人關(guān)于弱光脅迫對稻米籽粒蛋白質(zhì)的影響已經(jīng)進行了大量研究,但關(guān)于弱光對氮代謝關(guān)鍵酶(GS,GOT,CPT)的影響還鮮有報道.鑒于此,本文探討了水稻灌漿結(jié)實期不同程度弱光脅迫對籽粒氮代謝關(guān)鍵酶、可溶性蛋白質(zhì)以及成熟時蛋白質(zhì)與氨基酸組分的影響,以探討影響稻米品質(zhì)的機制.

1　材料與方法

1.1試驗材料

供試水稻品種為粳稻越光和秋田小町,秈稻IR72,以及雜交秈稻岡優(yōu)527等4個品種.

1.2試驗設(shè)計

試驗于2013年4—9月在四川農(nóng)業(yè)大學教學農(nóng)場進行,供試土壤為紫色土,基礎(chǔ)肥力:有機質(zhì)20.18 g/kg,全氮1.31 g/kg,速效氮110.95 mg/kg,速效磷26.52 mg/kg,速效鉀143.43 mg/kg.試驗地的肥料用量為尿素322.5 kg/hm2、過磷酸鈣630 kg/hm2、氯化鉀240 kg/hm2,分蘗期追施尿素,基追肥施用比例為7∶3,其余采用大田常規(guī)管理,確保栽培管理措施無差異.

試驗采用對比實驗方案,設(shè)置2個處理,分別為遮光25%(A1)和遮光50%(A2),以自然條件為對照(CK).遮光處理為水稻開花后,在高約1.5 m鐵架上掛上白色紗布為A1處理,掛黑色遮陰網(wǎng)為A2處理,每小區(qū)面積16 m2,重復(fù)3次.每小區(qū)選取生長基本一致的植株掛牌標記,自遮光后第10天起每7 d選取各穗強勢籽粒取樣1次,取樣直至籽粒成熟.取樣時間在上午8:00—9:00,樣品用液氮速凍,并放入-80℃冰箱中保存,以待后期酶活測定.水稻成熟后另取樣品置于80℃烘箱中烘干后脫糙、粉碎,并過100目篩,以進行蛋白質(zhì)、氨基酸的測定.

1.3測定項目與方法

GS活性測定根據(jù)趙全志等[14]方法,取25粒水稻籽粒,加入緩沖液(p H 8.0)研磨并離心制成粗酶液.取0.7 m L粗酶液和0.7 m L ATP,并與1.6 m L反應(yīng)混合液(Tris,MgSO4.7H2O、谷氨酸鈉鹽、半胱氨酸、EDTA、鹽酸羥胺,p H 7.4)混合,于30℃下保溫30 min,加入顯色劑并離心,在540 nm測定吸光值.酶活性單位定義為30℃下30 min內(nèi)平均每1 min反應(yīng)液AODs等于0.01為1個酶活性單位.

GOT和GPT活性采用吳良歡等[15]的方法,籽粒加緩沖液(p H 7.2)研磨并離心制成粗酶液.取10 m L試管加入酶粗制劑0.1 m L和GOT(或GPT)底物液0.5 m L,37℃下水浴30 min,加入2,4-二硝基苯肼液0.5 m L終止反應(yīng),37℃水浴20 min后加入0.4 mol/L NaOH 5 m L,在500 nm下比色.GOT或GPT活性以每1 g鮮樣在30 min內(nèi)平均每1 min反應(yīng)生成的丙酮酸1μmol表示.

可溶性蛋白質(zhì)含量測定采用考馬斯亮藍法[16];蛋白質(zhì)含量測定使用瑞典Buchi B-324全自動凱氏定氮儀測定;氨基酸含量測定使用日立L-8800氨基酸分析儀測定.

1.4數(shù)據(jù)分析

以上測定至少重復(fù)3次,使用SigmaPlot 12.5 及Excel 2007制作圖表,并使用SAS Enterprise Guide 4.3進行方差分析及顯著性檢驗.

2　結(jié)果與分析

2.1弱光對水稻灌漿結(jié)實期籽粒氮代謝關(guān)鍵酶活性的影響

從圖1可以看出,水稻灌漿期GS活性呈單峰曲線變化,總的表現(xiàn)為隨灌漿進程逐漸上升,到達峰值后又逐漸下降,岡優(yōu)527和IR72均比越光和秋田小町提前7 d到達峰值.方差分析可知,GS活性品種間表現(xiàn)差異顯著,越光和秋田小町顯著高于岡優(yōu)527和IR72.弱光下GS活性顯著降低,且影響程度隨著弱光程度的增加而加深(A2＞A1).總體上,4個品種在A1和A2處理下GS活性分別比對照下降11.61%～19.36%和20.01%～30.52%,其中以岡優(yōu)527下降幅度最大.

如圖2所示,除秋田小町GOT活性在灌漿期先上升后下降外,其余3個品種GOT活性均隨著灌漿進程而下降,下降程度依次為越光＞IR72＞岡優(yōu)527.方差分析可知,弱光顯著提高了4個品種的GOT活性,且弱光程度越深GOT活性越高(A2＞A1).4個品種在A1與A2處理下GOT活性分別比對照提高14.02%～23.02%和26.07%～36.82%.

GPT趨勢總體在4個品種間表現(xiàn)較為一致(圖3),除秋田小町A2處理外,其余均隨著灌漿進程而下降,總體下降程度依次為越光＞岡優(yōu)527＞秋田小町＞IR72.與GOT表現(xiàn)相似,4個品種弱光處理均極顯著提高了GPT活性,且隨著弱光程度加深GPT提高更加明顯(A2＞A1).總體上,4個品種A1和A2處理GPT活性分別比對照提高11.81%～33.83%和26.07%～61.07%,以岡優(yōu)527影響程度最深.

2.2弱光對水稻灌漿結(jié)實期籽粒可溶性蛋白質(zhì)含量的影響

如圖4所示,4個品種籽粒的可溶性蛋白質(zhì)含量均隨著灌漿進程呈下降趨勢,下降程度依次為越光＞IR72＞秋田小町＞岡優(yōu)527.方差分析表明,弱光極顯著地提高了籽粒中的可溶性蛋白質(zhì)含量,且弱光程度越重其含量越高.在A1和A2處理下可溶性蛋白質(zhì)平均含量分別比對照提高12.06%～36.77%和31.19%～70.51%,其中以越光增加程度最大,其次是岡優(yōu)527.

2.3弱光對水稻成熟籽粒蛋白質(zhì)和氨基酸組分及含量的影響

圖5可知,弱光有利于蛋白質(zhì)的合成.其中A1處理顯著提高了蛋白質(zhì)含量,而A2處理極顯著提高了蛋白質(zhì)含量.在A1和A2處理下,蛋白質(zhì)分別比對照提高6.43%～13.38%和16.02%～28.89%.秈稻IR72和岡優(yōu)527增幅高于粳稻越光和秋田小町.

同弱光對蛋白質(zhì)影響的表現(xiàn)基本一致,弱光處理總體上顯著提高了氨基酸含量(表1),且提高程度A2＞A1.但值得一提的是岡優(yōu)527在弱光處理下的蛋氨酸含量低于對照(A1低16.41%,A2低5.13%),秋田小町和岡優(yōu)527賴氨酸在A1處理下均低于對照,其余品種在弱光處理下氨基酸各組分含量均高于對照.總體而言,必需氨基酸含量在A1和A2處理下分別比對照高3.86%～11.86%和16.37%～22.15%,非必需氨基酸分別高3.47%～22.96%和15.72%～23.64%.綜合起來,在A1和A2處理下氨基酸總含量比對照分別提高3.62%～20.25%和16.04%～22.48%.

3　討論

在水稻中,氮元素主要是通過GS/GOGAT(谷氨酰胺合成酶/谷氨酸合成酶)途徑同化為谷氨酰胺或谷氨酸,進而轉(zhuǎn)化為其他氨基酸[17 18].作為典型的喜銨植物,GS在水稻氮素營養(yǎng)過程中乃至產(chǎn)量、品質(zhì)的形成過程中起著關(guān)鍵性的作用[11].金正勛等[11]研究指出,在灌漿前期籽粒中GS活性與水稻食味值、最高黏度、崩解值呈負相關(guān),而在灌漿后期呈正相關(guān);同時,還發(fā)現(xiàn)GS在灌漿前期與蛋白質(zhì)呈正相關(guān),灌漿中后期則呈負相關(guān),據(jù)此分析GS可能主要通過影響蛋白質(zhì)的合成而間接影響水稻的蒸煮食味品質(zhì).而黃星等[19]研究指出GS與蛋白質(zhì)相關(guān)性不顯著,而與可溶性蛋白質(zhì)含量呈顯著負相關(guān).曹珍珍等[20]則指出GS與可溶性蛋白質(zhì)含量呈極顯著正相關(guān).在本實驗中,GS活性在籽粒中隨弱光程度加深而下降明顯,而可溶性蛋白質(zhì)、蛋白質(zhì)和氨基酸含量卻隨著弱光加深而顯著上升,GS與可溶性蛋白質(zhì)、蛋白質(zhì)和氨基酸均呈顯著負相關(guān).OBENDORF等[21]研究指出,谷氨酰胺主要是在葉片中生成,然后轉(zhuǎn)運到籽粒中,再在籽粒中轉(zhuǎn)化為其他氨基酸[22 23].這表明,氨同化過程可能主要發(fā)生在葉片中,籽粒中GS活性變化對蛋白質(zhì)合成影響較小[12,24],這可能與水稻籽粒在弱光脅迫下的防御代謝有關(guān),但還有待于進一步研究[19-20].除了氨同化過程,轉(zhuǎn)氨基作用同樣是植物體內(nèi)氮代謝的重要環(huán)節(jié).在水稻籽粒發(fā)育過程中,GOT可以促進L-谷氨酸與草酰乙酸合成L-天冬氨酸,進而合成限制性必需氨基酸;GPT則是負責催化谷氨酸和丙酮酸之間的轉(zhuǎn)氨作用促進生成新的酮酸和α-氨基酸;水稻籽粒通過GOT和GPT的轉(zhuǎn)氨作用最終促進蛋白質(zhì)的合成[25].前人研究表明GOT和GPT活性上升顯著提高了蛋白質(zhì)含量[12,26].同樣,在本實驗中,GOT和GPT活性均隨著弱光加深而顯著提高,相關(guān)分析表明,GOT和GPT均與蛋白質(zhì)含量呈極顯著正相關(guān).這表明,相對于GS,GOT和GPT可能顯著影響著水稻籽粒中蛋白質(zhì)合成.

前人多數(shù)研究已表明灌漿結(jié)實期弱光有利于蛋白質(zhì)的積累[8,10,27],這也可以從弱光下碳水化合物的減少可見.任萬軍等[9]研究指出,弱光下稻株全氮含量會增加并促進氮素向籽粒轉(zhuǎn)移,因而促進蛋白質(zhì)的合成.本研究同樣發(fā)現(xiàn)弱光下籽粒中蛋白質(zhì)含量均顯著增加,且隨著弱光程度的加深而增強(A2＞A1),這也同董明輝等[28]的研究結(jié)果是一致的.除了蛋白質(zhì)含量,籽?？扇苄缘鞍踪|(zhì)含量也顯著增加,且A2＞A1.可溶性蛋白質(zhì)主要在同化物代謝過程中起著重要的作用,能提高細胞可溶性物質(zhì)含量,從而增強細胞抗逆境能力[29].

稻米營養(yǎng)價值的高低,不僅取決于蛋白質(zhì)含量,還受其他品質(zhì)成分的影響,尤其是必需氨基酸含量[7].在本研究中,弱光顯著增加了籽粒的氨基酸含量,其中必需氨基酸和非必需氨基酸含量增幅較為一致,均是A2＞A1,這同周廣洽等[30]的研究結(jié)論是一致的.氨基酸含量的提高主要由于GOT和GPT等酶活性的增強,由相關(guān)性分析可知,GOT和GPT活性也同氨基酸含量呈極顯著正相關(guān).在必需氨基酸中,賴氨酸被認為是稻米蛋白質(zhì)合成的第一限制性氨基酸,蘇氨酸和蛋氨酸則分別是第二、三限制性氨基酸[31-32].在本研究中,3個氨基酸組分在品種間表現(xiàn)有所差異,其中賴氨酸含量在秋田小町和岡優(yōu)527 A1處理下甚至低于對照,其余品種在弱光處理下增幅也不高;4個品種的蘇氨酸含量在弱光處理下均顯著增加;蛋氨酸含量除岡優(yōu)527弱光下降低外,其余均顯著提高.這表明氨基酸組分除受環(huán)境因子影響外,品種間差異也不可忽視[33].

綜上所述,灌漿結(jié)實期在弱光處理下,水稻籽粒中GS活性顯著降低,而GPT、GOT、可溶性蛋白質(zhì)、蛋白質(zhì)以及氨基酸含量均明顯增加,且影響程度隨著弱光的加深而加重.說明在未來關(guān)于稻米蛋白質(zhì)研究過程中,應(yīng)更加注重GOT和GPT的活性.值得一提的是,雖然弱光下稻米的蛋白質(zhì)含量升高,但過高的蛋白質(zhì)同樣會導(dǎo)致米飯的蒸煮食味品質(zhì)下降,因此,未來研究在注意提高稻米營養(yǎng)品質(zhì)的同時,應(yīng)同時兼顧營養(yǎng)品質(zhì)與食味品質(zhì)的均衡.

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收稿日期(Received):2015-06-19;接受日期(Accepted):2015-09-13;網(wǎng)絡(luò)出版日期(Published online):2016-01-19

*通信作者(

Corresponding author):李天(http://orcid.org/0000-0003-1255-4804),E-mail:lit@sicau.edu.cn

基金項目:四川省科技廳科技支撐計劃項目(2014NZ0103).

DOI:10.3785/j.issn.1008-9209.2015.06.192

中圖分類號S 511

文獻標志碼A