乙烯受體與果實(shí)成熟調控

園藝學(xué)報2004,31(4):543 ~548lcta Horticulturae Sinica乙烯受體與果實(shí)成熟調控魏紹沖’陳昆松'* 羅 云波(!浙江大學(xué)果實(shí)分子生理實(shí)驗室,農業(yè)部園藝植物生長(cháng)發(fā)育與生物技術(shù)重點(diǎn)開(kāi)放實(shí)驗室,杭州310029;2中國農業(yè)大學(xué)食品科學(xué)與營(yíng)養工程學(xué)院,北京100083 )摘要:綜述了有關(guān)乙烯受體和果實(shí)成熟衰老研究的最新進(jìn)展,主要包括擬南芥乙烯受體和信號轉導,番茄等果實(shí)乙烯受體家族基因的表達與功能研究,果實(shí)乙烯受體基因表達的調節等,對今后乙烯受體的相.關(guān)研究前景作了展望。乙烯受體轉基因等研究結果表明,乙烯的作用可以在受體水平實(shí)現高度調節。關(guān)鍵詞:果實(shí)成熟;乙烯受體;信號轉導;綜述中圖分類(lèi)號:S601 ; Q 946文獻標識碼: A文章編號: 0513-353X ( 2004 ) 04-0543-06 .Ethylene Receptor and Its Regulation in Ripening FruitsWei Shaochong' , Chen Kunsong', and Luo Yunbo2( ' Laboratory of Fruit Molecular Physiology and Biology , Zhejiang University ; The State Agriculture Ministry Laboratory of Horti-cultural Plant Growth , Development and Biotechnology , Hangzhou 310029 , China ; 2 College of Food Science and Nutritional En-gineering , China Agricultural University , Beijing 100083 , China )Abstract : The action of plant hormone ethylene is finally realized through ethylene receptor and its sig-nal transduction. The latest advances in researches on the ethylene receptor and fruit ripening is reviewed inhe present paper. The main contents include : the ethylene receptor and its signal transduction in Arabidopsis,and the expression of ethylene receptors in relation to their functions in ripening fruits. Meanwhile , the pros-pect of ethylene receptor is predicted as well.Key words : Fruit ripening ; Ethylene receptor ; Ethylene signal transduction ; Review植物激素乙烯對植物生長(cháng)發(fā)育的許多過(guò)程,如種子萌發(fā)、幼苗生長(cháng)、開(kāi)花、結實(shí)、果實(shí)成熟和衰老等有著(zhù)廣泛而深遠的影響'。乙烯也是-個(gè)植物適應環(huán)境變化的信號,它調節了機械傷和病原反應等脅迫反應(2,32。Bradford 等4認為植物激素信號可以通過(guò)內源水平及組織對其感受進(jìn)行調節,例如,果實(shí)對乙烯的敏感性可隨成熟進(jìn)程不斷在發(fā)生改變。通過(guò)控制乙烯的感受仍可使果實(shí)成熟的相關(guān)變化延緩或中斷'5。乙烯的生物合成途徑已經(jīng)于20世紀70年代末被明確, ACC合酶( ACS)和ACC氧化酶( ACO)是乙烯生物合成的兩個(gè)關(guān)鍵酶。研究表明, ACS和ACO均由多基因家族編碼,這些基因對不同發(fā)育信號和環(huán)境刺激產(chǎn)生響應,從而有效地調節植物乙烯的生成與作用06-8)。隨著(zhù)乙烯研究的不斷深入,人們開(kāi)始逐漸轉向乙烯感受和信號轉導途徑的研究。通過(guò)擬南芥乙烯反應不敏感突變體的分析,人們分離得到了一系列關(guān)鍵基因,并進(jìn)行了大量的分子遺傳學(xué)分析,建立了擬南芥的乙烯信號轉導途徑初步輪廓(9。10]。本文綜述近年來(lái)乙烯受體研究的新進(jìn)展及其在果實(shí)成熟衰老進(jìn)程中的調控途徑。1擬南芥乙烯 受體和信號轉導中國煤化工以黑暗條件下乙烯對幼苗的”三重反應” (即黑暗.MHC NMH G;在乙烯存在的條件下收稿日期: 2003 -09-20;修回日期:2003-11-06基金項目: 國家重點(diǎn)基礎研究發(fā)展規劃項目( 973項目) ( G2000046806 );國家自然科學(xué)基金項目( 30270917 );浙江省自然科學(xué)基金重點(diǎn)項目( ZD0004 )*通信陌方數據for corespondence ( E-mail : akun@ zju. edu. en)544園藝報31卷發(fā)生表型變化,包括下胚軸膨脹變短,頂端鉤狀芽彎曲加劇，莖桿偏向水平生長(cháng))作為篩選手段,人們從擬南芥中分離得到了許多乙烯合成和信號轉導發(fā)生改變的突變體"1),隨后CTR112),EIN2[13) , EIN314), ERF115) , ETR1'16)等多個(gè)乙烯信號轉導相關(guān)基因相繼被克隆。值得注意的是ETR1顯性等位基因突變體的克隆,導致了番茄中包括NR在內多個(gè)乙烯受體基因類(lèi)似物的確定[7。ETR1蛋白與原核生物中普遍存在的雙組分系統高度同源,它主要位于細胞的內質(zhì)網(wǎng)中18]。在細菌中,由感受器和反應調節器組成的雙組分系統起到調節各種發(fā)育和刺激反應的作用‘19]。遺傳和生化證據均表明ETR1編碼一個(gè)乙烯受體。ETR1 是以二聚體的形式存在,且在銅離子參與下表現出與乙烯具有高親和性20-23)。擬南芥的ETR家族由5個(gè)受體蛋白組成24-26) ,其相應的編碼基因分別為ETR1、ETR2、EIN4、 ERSI 和ERS2。擬南芥乙烯受體蛋白從功能上可分為感受器、組氨酸激酶和反應調節器3個(gè)結構域(27)。(1)感受器(Sensor)結構域:由3個(gè)疏水的跨膜節段組成。該區域高度保守,現在已知的所有引起乙烯不敏感的受體突變位點(diǎn)都處于這一區域,在乙烯不敏感的突變體(如etr1-1 )中,乙烯不能與受體蛋白結合。在這-結構域的N末端中含有形成二聚體和結合銅離子必須的氨基酸。(2)組氨酸激酶(Histidinekinase,HK)結構域:它比同源的細菌雙組分系統中的HK序列大。激酶結構域由五個(gè)被稱(chēng)為催化中心(H,N,Gl,F,G2)的亞結構域組成。在ETR1和ERS1中包含所有的五個(gè)亞結構域,而ETR2、EIN4 和ERS2三個(gè)乙烯受體中缺少一個(gè)或多個(gè)亞結構域。ETR1 在體外可檢測到HK的活性[28] ,這是目前唯-表現HK活性的乙烯受體。理論上講, HK結構域在乙烯信號轉導中起著(zhù)向下游傳遞信息的作用。但是-個(gè)明顯的問(wèn)題是,既然有的受體中不具有HK活性,那么,它們又是如何進(jìn)行信號轉導的,仍不清楚。(3)反應調節器( Response regulator)結構域:這-結構域中具有自我磷酸化的Asp殘基。與細菌相類(lèi)似,植物的乙烯受體家族的一些成員中缺乏這-結構域,如擬南芥中ERS家族成員ERS1和ERS2 ,而ETR1、ETR2 和EIN4則包含有反應調節器結構域。根據基因敲除( knock-out )對表型( phenotype )影響試驗結果得出的模式推測(29) :在無(wú)乙烯的狀態(tài)下乙烯受體應該是有活性的，下游的CTR組分也具有活性,而此時(shí)乙烯誘導的相關(guān)基因表達處于被抑制狀態(tài);當乙烯與受體結合時(shí)會(huì )使受體及CTR失活,乙烯誘導基因表達的抑制作用也相應被解除。然而,因為ERS2類(lèi)受體無(wú)HK活性,所以受體活性的精確調節目前還不清楚。2果實(shí)乙烯受體研究進(jìn)展.2.1番茄乙烯受體家族番茄是研究乙烯反應和果實(shí)成熟衰老的模式植物。目前,番茄的6個(gè)乙烯受體基因( LeETRI ~6)已分別被分離和定性‘30-37]。其中LeETR3 (即NR )基因定位在番茄的9號染色體上,序列分析發(fā)現Nr(Neverripe)突變體只是由于NR蛋白36位上單個(gè)堿基突變而使Pro變成Leu造成的(37]。Nr突變體除果實(shí)不能正常成熟外,還顯示出乙烯不敏感突變株的多效突變效應,具體表現為:不表現乙烯的三重反應,葉柄不易脫落,葉和花瓣衰老進(jìn)程明顯推遲(37]。研究表明,番茄乙烯受體( LeETR )家族的結構與擬南芥乙烯受體家族十分相似,但番茄乙烯受體的各個(gè)成員在蛋白質(zhì)水平上差別較大,在序列.上表現出50%以下的同源性,其中從LeETR4和LeETRS兩個(gè)成員的序列中推測似乎存在第4個(gè)跨膜結構域,這個(gè)結構域可能是-個(gè)信號序列或者起中國煤化工接傳遞到細胞質(zhì)的作用。其中NR受體蛋白缺少接受器結構域, LeETR4 ~6YHCNMH宅整的HK結構域。盡管各番茄乙烯受體的結構有顯著(zhù)差異,但試驗已證明除克隆較晚的LeETR6外, 其它均已被證實(shí)為乙烯受體,因為它們都已被確定有結合乙烯的能力38。2.2番茄乙烯受體基因的表達 與功能研究當考慮受體表達對調節整個(gè)乙烯反應的重要性時(shí),需注意以下幾點(diǎn)。首先,乙烯受體在信號轉導3期魏紹沖等:乙烯受體與果實(shí)成熟調控545過(guò)程中顯然是作為負調節因子起作用。在缺乏乙烯的情況下,受體會(huì )抑制乙烯誘導的相關(guān)基因表達。根據這種模式推測組織對乙烯的敏感性與受體水平之間呈負相關(guān)關(guān)系。要解除抑制,應當需更多的乙烯使受體失活,反之亦然。另一個(gè)需考慮乙烯反應的因素是受體有一個(gè)較長(cháng)的與乙烯分離的半衰期，酵母中表達的ETR1測定的半衰期大約是12 h(20]。 這意味著(zhù)受體一旦與乙烯結合,它將在較長(cháng)的一段時(shí)間內不能抑制乙烯誘導的相關(guān)基因。無(wú)論是在果實(shí)發(fā)育中或外界刺激下，番茄中的多個(gè)乙烯受體基因的表達發(fā)生明顯改變。當乙烯反應啟動(dòng)時(shí),乙烯合成的快速增加會(huì )伴隨受體合成的增加。通常當-種植物激素的合成增加或施加某種激素,植物會(huì )迅速作出反應以降低或解除這種反應,包括通過(guò)酶的合成直接消除激素的作用。例如,對擬南芥使用生長(cháng)素可以導致生長(cháng)素結合酶活性的增加59]。對于乙烯,還不知道會(huì )使哪些酶變化,因為它比較容易擴散。由于直到最近其它植物激素的受體基因才被克隆,所以還不知道其它激素的受體在激素增加時(shí)是否會(huì )出現同樣情況。2.3乙烯受體基因的表達與番茄果實(shí)成熟調節番茄乙烯受體基因在果實(shí)發(fā)育過(guò)程中表現出不同的表達模式。LeETR1 和LeETR2在不同發(fā)育時(shí)期所有組織中的表達基本保持不變,同時(shí)它們的表達不受乙烯誘導調控,這說(shuō)明它們可能與果實(shí)成熟關(guān)系不密切。相比之下,其余4個(gè)基因( LeETR3 ~6)的表達模式則是受乙烯和果實(shí)發(fā)育高度調節的38。在果實(shí)成熟過(guò)程中,盛花期子房的NR mRNA具有高水平的表達,隨后趨下降,到果實(shí)成熟期其表達水平是子房的20倍(34)。LeETR4 , LeETRS和LeETR6在生殖器官(花和果實(shí))中表達豐富,而在營(yíng)養器官中表達水平較低(38)。目前對這些受體基因在生殖器官中的顯著(zhù)高表達所產(chǎn)生的功能還不清楚。近年來(lái),人們還從番茄中分離得到了乙烯信號轉導下游CTR1 , EIN3 , EREBP ( ERF1 )等組分同源的相關(guān)基因。Lin等'40]克隆了TCTR2,它與CTR1蛋白有41%的同源性。Zegzouti等41)分離得到了ER50 ,其編碼另一個(gè)與CTR1相似的蛋白，并推斷可能有多種MAP3K參與了乙烯作用的調節。TCTR2是組成型表達的，不受外源乙烯的誘導(40);而ER50 mRNA則在果實(shí)成熟過(guò)程中正向調節( up-regulated),并受外源乙烯的誘導(41。Alexander 等(42)認為, 番茄乙烯受體可以和與Raf蛋白激酶相似的LeCTR的至少5個(gè)家族成員直接作用。此外, Gu等43)還從番茄中分離得到了EREBP的類(lèi)似物pti4。Tieman 等' 44從番茄中篩選到EIN3基因的類(lèi)似物L(fēng)eEIL1 , LeEIL2 , LeEIL3基因。結合擬南芥和番茄乙烯信號轉導的研究結果,我們對乙烯受體基因的表達與果實(shí)成熟關(guān)系有了初步認識,即乙烯與膜上受體蛋白的結合會(huì )使乙烯受體及CTR失活，下游的EIL和EREBP等組分則被激活,引起一系列成熟相關(guān)基因的表達,最終導致果實(shí)成熟;而在受體蛋白不與乙烯結合的情況下,乙烯受體和CTR是具有活性的,下游EIL和EREBP組分被抑制不表達,果實(shí)不能正常成熟。但番茄果實(shí)在成熟階段發(fā)生對乙烯的敏感性與果實(shí)中乙烯受體基因的表達模式并不是-致的,相關(guān)調節模式還需要進(jìn)-步試驗證實(shí)。2.4其它果實(shí)乙烯受體的研究盡管不同植物中乙烯受體有較大差異,但其編碼基因序列中仍存在相對保守的區域,特別是在其蛋白質(zhì)N-端與乙烯結合的結構域。除番茄外,人們已相繼從香瓜(45)、西番蓮46.47、 桃48.49)、香蕉(S0)、口果5、蘋(píng)果'52]、梨s3.54)、柑橘55、黃瓜[S0]等果實(shí)中分離得到了多個(gè)乙烯受體同源基因。目前研究結果表明,乙烯受體基因在這些植物中也可能中國煤化工在。例如，香瓜、西番蓮、桃等果實(shí)中目前均已分別分離出了兩個(gè)以上的乙MHC N M H G研究的深入，可能每一種果實(shí)中將會(huì )有更多的乙烯受體基因不斷被克隆。不同類(lèi)型果實(shí)的乙烯受體基因的表達是復雜多樣的,在西番蓮和桃果實(shí)中, ETR類(lèi)型受體基因PeETR1和Pp-ETR1在果實(shí)發(fā)育和成熟過(guò)程中是組成型表達,與番茄果實(shí)中的LeETR1和LeETR2表達相似,基本不受乙烯生成的影響;但是在香瓜和口果果實(shí)中,瓦夜葵型受體基因Cm- ETR1和METR1的表達水平則隨果實(shí)成熟進(jìn)程推進(jìn)及乙烯合成能力增546藝報31卷強而增加?？梢?jiàn),乙烯受體基因在各種果實(shí)中可能存在不同的表達和調節模式。2.5果實(shí)乙烯受體基因表達 的調節2.5.1受體基因表達的調節途徑 果實(shí)乙烯受體基因的表達受發(fā)育成熟度及果實(shí)部位、乙烯、乙烯受體抑制劑等因素調節。魏紹沖等(57)對不同成熟時(shí)期番茄果實(shí)不同部位的LeETR4表達模式研究表明,LeETR4在番茄外果皮中的表達水平明顯低于果實(shí)的輻射壁和中柱部位。乙烯對各乙烯受體基因表達的影響不一,已在前述番茄果實(shí)的基因表達部分中提及。乙烯作用抑制劑與乙烯競爭受體結合位點(diǎn)，阻止組織對乙烯響應,其對果實(shí)中各乙烯受體基因的表達影響不同。Nakatsuka 等[58)用1 -甲基環(huán)丙烯( 1-MCP )處理轉色期番茄果實(shí),發(fā)現果實(shí)中NR mRNA累積受阻,果實(shí)成熟推遲,而另一-受體基因eTAE1 ( LeETR1 )的表達基本沒(méi)有改變; 1-MCP處理對桃果實(shí)的Pp-ETR1表達沒(méi)有影響,但對Pp-ERS1表達起到抑制作用49)。2.5.2調節乙烯受體基因表達的基因工程通過(guò)調節乙烯受體基因的表達水平調控果實(shí)的成熟衰老進(jìn)程,是實(shí)現生物技術(shù)延緩果實(shí)后熟軟化的一個(gè)重要途徑。目前,番茄乙烯受體的多種轉基因植株已獲得成功,但在其它果實(shí)中未見(jiàn)成功的報道。Wilkinson 等' 59)將擬南芥乙烯受體基因ETR1-1突變序列轉入番茄中,發(fā)現能明顯推遲果實(shí)后熟及花的衰老。Ciardi等'60)通過(guò)連接組成型表達的35S啟動(dòng)子的NR eDNA獲得過(guò)量表達NR的轉基因植株對乙烯敏感性降低(61]。這些結果與擬南芥中的研究結果完全吻合,表明乙烯受體系統存在-定程度過(guò)剩。但是減少LeETR4的表達對植株影響嚴重,呈現出乙烯組成型反應,表現在葉偏上性,花瓣脫落和果實(shí)成熟提前等'61。而當這些影響發(fā)生時(shí),乙烯生成并沒(méi)有增加,說(shuō)明這些反應與乙烯受體表達水平發(fā)生改變有關(guān)。轉基因植物中受體基因表達的研究揭示了乙烯受體之間可能存在功能補償( functional compensation ),當反義控制NR的表達后, LeETR4的表達則相應提高,植株通過(guò)某種方式增加LeETR4的表達以補償NR表達的降低,因此NR反義植株整體的乙烯受體水平并未受明顯影響。Whitelaw 等62采用反義手段抑制番茄LeETR1的表達后,研究表明反義植株的葉片中NR表達水平以及果實(shí)的著(zhù)色和硬度基本未受影響,但植株的節間變短,花的衰老延遲。Hackett 等(63)反義抑制了Nr突變體植株中的NR基因表達后, 果實(shí)恢復了成熟能力,這在證明乙烯受體是負調控的同時(shí),也表明NR可能并不是果實(shí)正常成熟所必需的?？傊?，這些轉基因研究結果將有助于我們更正確地認識各個(gè)番茄乙烯受體基因的功能以及它們之間的相互關(guān)系。3展望乙烯的生理作用最終是通過(guò)乙烯受體及其信號轉導過(guò)程完成的。目前,番茄乙烯受體研究已經(jīng)較為深入,借助番茄乙烯受體的研究成果繼續開(kāi)展其它果實(shí)乙烯受體的研究將是今后乙烯作用研究的一個(gè)新熱點(diǎn)。隨著(zhù)果實(shí)成熟生理及乙烯受體研究的深入,相信從乙烯受體水平上調控果實(shí)成熟進(jìn)程的基因工程不久將會(huì )應用于生產(chǎn)實(shí)踐,并可望產(chǎn)生巨大的經(jīng)濟和社會(huì )效益。此外,乙烯的作用并不是獨立的,還需要其它激素的配合,因此要全面了解乙烯的作用,需要從分子生物學(xué)水平上對植物生長(cháng)發(fā)育過(guò)程中乙烯與其它植物激素的關(guān)系,特別是結合信號轉導開(kāi)展研究。目前許多學(xué)者已開(kāi)始致力研究ABA與乙烯信號轉導的關(guān)系(64,65)。隨著(zhù)21世紀蛋白質(zhì)組學(xué)時(shí)代的到來(lái),乙烯受體及信號轉導在果實(shí)發(fā)育等過(guò)程中蛋白水平的變化及其互作,如乙烯受體與CTR的相互作用等,將是未來(lái)研究的一個(gè)重要方向。中國煤化工參考文獻:MHCNMHG1 Abeles F B , MorganP W，Saltveit Jr M E. 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