PPR蛋白響應(yīng)植物非生物脅迫的研究進(jìn)展

李程，路凱，王才林，張亞東

PPR蛋白響應(yīng)植物非生物脅迫的研究進(jìn)展

李程，路凱，王才林，張亞東

江蘇省農(nóng)業(yè)科學(xué)院糧食作物研究所/國家耐鹽堿水稻技術(shù)創(chuàng)新中心華東中心/江蘇省優(yōu)質(zhì)水稻工程技術(shù)研究中心/國家水稻改良中心南京分中心/江蘇省農(nóng)業(yè)生物學(xué)重點實驗室，南京 210014

非生物脅迫是造成全球糧食減產(chǎn)的主要因素之一。研究植物逆境相關(guān)蛋白的功能及應(yīng)答機(jī)制，對于提高作物抗逆性具有重要意義。三角狀五肽重復(fù)（PPR）蛋白屬于高等植物中最大的核編碼蛋白家族，因其包含高度特異性的PPR基序而得名。依據(jù)基序類型及其排列，PPR蛋白可分為P和PLS兩類，PLS類蛋白又可以根據(jù)其羧基末端的結(jié)構(gòu)域進(jìn)一步分為PLS、E、E+、DYW等亞類。PPR蛋白廣泛分布于陸生植物中，主要定位于葉綠體和線粒體，亦有少數(shù)定位于細(xì)胞核中。作為序列特異性RNA結(jié)合蛋白，PPR蛋白參與植物RNA加工的多個方面，包括RNA編輯、RNA剪接、RNA穩(wěn)定和RNA翻譯。PPR蛋白在植物的整個生命進(jìn)程中發(fā)揮多種重要作用，但對其在植物抗逆性中的作用機(jī)制還不清楚。本文在總結(jié)已有報道的非生物脅迫相關(guān)PPR蛋白定位和功能的基礎(chǔ)上，重點綜述了PPR蛋白參與調(diào)控植物非生物脅迫的作用機(jī)制（包括轉(zhuǎn)錄后調(diào)控和逆行信號），并對其進(jìn)行討論。轉(zhuǎn)錄后調(diào)控與PPR蛋白參與RNA轉(zhuǎn)錄后的修飾作用有關(guān)，其一般被認(rèn)為通過結(jié)合RNA并調(diào)節(jié)細(xì)胞器RNA代謝來調(diào)控逆境相關(guān)基因的表達(dá)，從而影響植物抗逆性。逆行信號方面，PPR蛋白的損傷導(dǎo)致線粒體或葉綠體功能受損，然后產(chǎn)生各類逆行信號（如ROS），進(jìn)而調(diào)控相關(guān)基因表達(dá)，抵御逆境。然而，由于質(zhì)體中的逆行信號會受到許多環(huán)境因素的影響，這些因素部分還未明確，導(dǎo)致PPR蛋白在逆行信號中的作用機(jī)制仍有很多問題有待闡明。此外，PPR蛋白存在一因多效性，部分蛋白在作用于抗逆性的同時，還會對植物的生長和生殖產(chǎn)生重要影響。最后，本文闡述了利用PPR蛋白作為RNA編輯工具的研究現(xiàn)狀，探討了目前PPR蛋白響應(yīng)植物非生物脅迫方面尚待解決的問題及研究前景，提出了未來研究仍需關(guān)注的重點和難點，為深入研究PPR蛋白的功能和作物非生物脅迫抗性育種提供參考。

PPR蛋白；植物；非生物脅迫

隨著全球人口的不斷增長，糧食安全問題日益突出[1]。由于無法移動，氣候變化所帶來的干旱、高溫、土地鹽堿化和紫外線輻射等非生物逆境對農(nóng)作物的生長、發(fā)育和結(jié)實造成了嚴(yán)重的不良影響，現(xiàn)已成為全球農(nóng)業(yè)減產(chǎn)的重要因素[2]。因此，解析植物抗逆性機(jī)制，提高作物的非生物脅迫抗性對農(nóng)業(yè)生產(chǎn)至關(guān)重要。

植物的抗逆反應(yīng)是一個復(fù)雜的調(diào)控過程，包括從脅迫信號感知到調(diào)節(jié)基因表達(dá)，再到產(chǎn)生功能性蛋白質(zhì)，最后到形態(tài)和生理生化代謝上的一系列調(diào)整[3]。脅迫應(yīng)答蛋白質(zhì)決定了植物對非生物脅迫的抗性[3]，近年來，大量與植物非生物脅迫響應(yīng)相關(guān)的蛋白已被報道。由于細(xì)胞擴(kuò)張驅(qū)動的生長與細(xì)胞壁的不斷重塑有關(guān)，當(dāng)逆境發(fā)生時，植物體內(nèi)的類受體激酶（receptor-like kinases，RLKs）先通過感應(yīng)細(xì)胞壁的變化調(diào)節(jié)逆境脅迫下的細(xì)胞生長[4]。脅迫一旦被感應(yīng)，刺激信號就會立即被第二信使（鈣離子、一氧化氮及不同類型的蛋白激酶等）傳遞和放大，以啟動復(fù)雜的特異性信號級聯(lián)反應(yīng)。比如，鈣結(jié)合蛋白可以檢測到應(yīng)激引起的細(xì)胞質(zhì)鈣離子濃度變化，并將信號傳遞給相互作用的蛋白激酶或直接與之融合的激酶，如鈣依賴性蛋白激酶（calcium-dependent protein kinases，CDPKs），以進(jìn)一步激活下游的應(yīng)答過程[5]。值得一提的是，SNF1相關(guān)蛋白激酶（SnRKs）廣泛介導(dǎo)高等植物在各種脅迫下的脅迫信號，是細(xì)胞能量穩(wěn)態(tài)的主要調(diào)節(jié)因子[6]。脅迫信號經(jīng)過感應(yīng)和轉(zhuǎn)導(dǎo)后會引起植物對脅迫的應(yīng)答。其中，bHLH、ERF/AP2、MYB和WRKY等轉(zhuǎn)錄因子在非生物脅迫下可以快速反應(yīng)，促進(jìn)脅迫信號的轉(zhuǎn)導(dǎo)并調(diào)節(jié)相關(guān)基因的表達(dá)[7]。如，的轉(zhuǎn)錄水平在非生物脅迫下顯著提高，且能夠通過直接結(jié)合的啟動子來激活其表達(dá)，正向調(diào)節(jié)水稻的耐鹽性[8]。產(chǎn)生非生物脅迫后，末端作用蛋白可以在脅迫條件下保護(hù)蛋白質(zhì)生物活性，進(jìn)而恢復(fù)細(xì)胞穩(wěn)態(tài)，減輕非生物脅迫對植物的損害，例如胚胎發(fā)育晚期豐富蛋白（late embryogenesis abundant proteins，LEA）、熱激蛋白（heat shock proteins，Hsps）和組成型光形態(tài)建成1（constitutively photomorphogenic 1，COP1）等均屬于此類[9-11]。隨著植物基因組學(xué)研究的不斷深入，越來越多新的蛋白不斷被報道和研究[12-15]。

三角狀五肽重復(fù)（pentatricopeptide repeat，PPR）蛋白因包含一種三角狀五肽重復(fù)結(jié)構(gòu)域而得名，最早在擬南芥基因組測序分析中被發(fā)現(xiàn)[16]。PPR蛋白家族成員主要由細(xì)胞核基因編碼，并以多個氨基酸螺旋重復(fù)序列簡并串聯(lián)為特征，這些重復(fù)序列堆疊在一起形成可識別RNA的延伸表面[17]。對PPR蛋白功能的單獨或系統(tǒng)研究表明，PPR蛋白主要功能是與細(xì)胞器基因組轉(zhuǎn)錄產(chǎn)物特異RNA相結(jié)合，參與轉(zhuǎn)錄后修飾，以獲得成熟的轉(zhuǎn)錄本[18-22]。PPR家族對RNA的修飾主要分為以下4種類型，包括RNA編輯：PPR蛋白特異識別編輯位點上游的順式作用元件，并招募相關(guān)編輯因子共同起作用，將胞嘧啶（C）轉(zhuǎn)化為尿嘧啶（U）或者將U轉(zhuǎn)化為C，從而改變密碼子，進(jìn)而影響氨基酸序列的構(gòu)成[23-25]；RNA剪接：DNA轉(zhuǎn)錄后的前體RNA含有豐富的內(nèi)含子，PPR蛋白從最初轉(zhuǎn)錄產(chǎn)物中去除順式和反式內(nèi)含子，并將外顯子拼接成為成熟的RNA[26-28]；RNA穩(wěn)定：PPR蛋白可以修飾mRNA前體的末端，阻礙RNA外切酶的活性，使其形成穩(wěn)定的單順反子進(jìn)行表達(dá)，以及結(jié)合在多順反子轉(zhuǎn)錄物中開放閱讀框（open reading frame，ORF）之間，處理產(chǎn)生的末端[29-31]；RNA翻譯：PPR蛋白能夠結(jié)合ORF的5′端特定非翻譯區(qū)（untranslated region，UTR），激活mRNA的翻譯調(diào)控[28, 32-33]。

本文收集了近年來與植物非生物脅迫響應(yīng)相關(guān)PPR蛋白的信息，綜述了其對植物抗逆性的影響和作用機(jī)制，并就未來PPR蛋白在作物非生物脅迫抗性育種上的研究應(yīng)用進(jìn)行了展望。

1 PPR蛋白的結(jié)構(gòu)和種類

PPR蛋白的基本結(jié)構(gòu)特征是在其氨基末端區(qū)域存在一個由35個氨基酸組成的重復(fù)序列基序——PPR基序[16]。因PPR基序具有高度特異性，根據(jù)基序類型及其排列，PPR蛋白可分為P和PLS兩類，P類蛋白僅含有35個氨基酸的典型P基序，而PLS類蛋白是由P-、L-和S-基序組成串聯(lián)重復(fù)的PLS三聯(lián)體[34]。PLS類蛋白大多具有高度保守的E、E+或DYW結(jié)構(gòu)域的羧基末端延伸，因此，PLS類蛋白可以根據(jù)其羧基末端的結(jié)構(gòu)域進(jìn)一步分為PLS、E、E+和DYW亞類[35]。此外，有人根據(jù)第一個螺旋的差異將P基序分為P1和P2；根據(jù)第二個螺旋的不同將L基序分為L1（35個氨基酸）和L2（36個氨基酸）；同樣，S基序也可以分為S1（31個氨基酸）和S2（32個氨基酸）；還有一種SS基序，其同時存在與S1和P1基序重疊的序列[18]。目前，也有一些報道稱少數(shù)P類PPR蛋白的C末端包含額外的特征序列，如小MutS相關(guān)（small MutS-related，SMR）結(jié)構(gòu)域，LAGLIDADG（His-Cys box and GIY-YIG，H-N-H）結(jié)構(gòu)域和RNA識別結(jié)構(gòu)域（RNA recognition motif，RRM）等（圖1）[36-39]。晶體結(jié)構(gòu)解析發(fā)現(xiàn)，PPR蛋白的每個重復(fù)基序通常會形成一對穩(wěn)定反向平行的α螺旋結(jié)構(gòu)，多個串聯(lián)基序還可進(jìn)一步螺旋化形成右手超螺旋結(jié)構(gòu)，從而與相關(guān)蛋白發(fā)生互作[40-41]。

圖1 PPR蛋白家族主要分類

2 PPR蛋白分布和定位

PPR蛋白主要存在于真核生物中，且在陸生植物中數(shù)量最多，原核生物中的數(shù)量很少[35]。有研究表明，PPR蛋白在陸地植物中的多數(shù)量與其調(diào)控細(xì)胞器mRNA從C到U的轉(zhuǎn)錄后編輯功能有關(guān)[42]。然而，同樣是陸生植物，低等的苔蘚中僅有103個PPR基因，而在高等的被子植物（如在擬南芥和水稻）中，PPR基因卻多達(dá)400個以上，因此，推測PPR基因的數(shù)量隨著植物從低等到高等進(jìn)化的過程中不斷增加和分化[43]。

大多數(shù)PPR蛋白的N末端具有定位信號序列[44]。在高等植物中，PPR蛋白多位于線粒體或葉綠體，也有極少數(shù)在細(xì)胞核中行使功能[45-46]。如，擬南芥MTL1蛋白定位于線粒體中，影響線粒體NADH脫氫酶亞基7（nad7）mRNA的翻譯；AtECB2定位于葉綠體的類囊體膜上，影響擬南芥葉綠體中多個基因轉(zhuǎn)錄本的編輯；細(xì)胞核定位的OsNPPR1參與了線粒體發(fā)育且影響水稻胚乳發(fā)育[47]。此外，還有少數(shù)PPR蛋白存在雙定位模式，如水稻OsPGL1蛋白（pale green leaf 1）同時定位于葉綠體和線粒體，其功能缺失會影響葉綠體和線粒體RNA編輯缺陷[48]，而擬南芥PNM1蛋白則同時定位于細(xì)胞核和線粒體，可能在線粒體和細(xì)胞核之間的基因表達(dá)調(diào)節(jié)中發(fā)揮作用[49]。因此，明確PPR蛋白的亞細(xì)胞定位將有助于揭示PPR蛋白的功能。

3 PPR蛋白對植物抗逆性的影響

葉綠體和線粒體是植物細(xì)胞中半自主性的細(xì)胞器，能夠感受逆境信號，在植物響應(yīng)內(nèi)外界環(huán)境變化的逆向信號傳導(dǎo)過程中發(fā)揮著重要的功能，因此，定位于葉綠體或線粒體中的PPR蛋白很可能與植物非生物脅迫有關(guān)。在擬南芥中，已有10個以上的PPR蛋白被證明對非生物脅迫有反應(yīng)。如，在葉綠體中，PPR蛋白RARE1負(fù)責(zé)-C794位點的編輯，其與植物耐熱性有關(guān)，人工提高C794編輯的表達(dá)能夠增強(qiáng)擬南芥的耐熱性[50]；核編碼葉綠體PPR蛋白SVR7參與擬南芥抗氧化脅迫反應(yīng)，其突變體積累更多活性氧（reactive oxygen species，ROS），并表現(xiàn)出較低的光氧化應(yīng)激耐受性[51]；GUN1亦是一種葉綠體定位的PPR蛋白，突變體對蔗糖和脫落酸（ABA）高度敏感，同時，也表現(xiàn)出對去黃化過程中引起的光氧化應(yīng)激更敏感的表型[52-55]，最新研究表明，GUN1是氧化細(xì)胞環(huán)境所必需的，通過依賴氧化還原的質(zhì)體-核通訊參與了植物基礎(chǔ)耐熱性的獲得[56-57]。此外，擬南芥中還存在相當(dāng)數(shù)量線粒體定位的PPR蛋白，如PPR40[58]、ABO5[59]、ABO8[60]、PGN[61]、AHG11[62]、SLG1[63]、SLO2[64]、PPR96[65]、POCO1[66]和LOI1/ MEF11[67]均參與多種非生物脅迫的響應(yīng)（表1）。在細(xì)胞質(zhì)-核雙定位的PPR蛋白中，SOAR1被證實是植物對非生物脅迫反應(yīng)的一個正調(diào)節(jié)因子，與ABA信號傳遞和擬南芥對干旱、鹽和冷脅迫的耐受性有關(guān)[68]。此外，PPR蛋白GEND1和PPR2都與擬南芥的耐熱性有關(guān)，其突變體植株在高溫下表現(xiàn)出高度敏感表型，但這些蛋白的準(zhǔn)確定位還未見報道[69-70]。

近年來，除了模式植物，在以水稻為代表的大田作物中，關(guān)于PPR蛋白參與非生物脅迫的研究也越來越多。Chen等[71]通過全基因組分析在水稻中共發(fā)現(xiàn)491個PPR基因，表達(dá)譜分析表明，大量PPR基因在非生物脅迫下被誘導(dǎo)，其中，鹽脅迫和干旱脅迫下分別有75和73個PPR基因表達(dá)上調(diào)，暗示這些PPR蛋白可能在水稻對非生物脅迫的反應(yīng)中發(fā)揮作用。低溫脅迫下，2種定位于葉綠體的PPR蛋白OsV4和TCD10是水稻幼苗早期葉綠體發(fā)育所必需的[72-73]。同樣是定位于葉綠體的PPR蛋白WSL，其突變體在發(fā)育早期對ABA、鹽和糖的敏感性增強(qiáng)，H2O2積累量提高[74]。此外，2種定位于線粒體的PPR蛋白PPS1和OsNBL3也被證實與水稻非生物脅迫有關(guān)，其中，的抑制導(dǎo)致水稻對高鹽度和ABA的敏感性顯著增加[75]，而的抑制卻導(dǎo)致水稻對鹽脅迫的耐受性增加[76]。最近，Luo等[77]驗證了2個定位于線粒體的PPR蛋白PPR035和PPR406在耐旱中的功能，和突變體均對干旱和鹽脅迫表現(xiàn)出較強(qiáng)的耐受性，在提高水稻抗旱性方面具有很大的應(yīng)用前景；LU等[78]在水稻中分別過表達(dá)和其同源基因，轉(zhuǎn)基因植株在幼苗生長階段的耐鹽性得到增強(qiáng)，表明SOAR1同源PPR蛋白可通過轉(zhuǎn)基因操作用于鹽脅迫條件下水稻的作物改良。Su等[79]在大豆基因組中鑒定出179個DYW亞群PPR基因，并發(fā)現(xiàn)在鹽脅迫和干旱脅迫下被誘導(dǎo)表達(dá)，其過表達(dá)轉(zhuǎn)基因植株對干旱脅迫的耐受性增強(qiáng)。

4 PPR蛋白參與植物非生物脅迫調(diào)控的作用機(jī)制

4.1 轉(zhuǎn)錄后途徑

PPR家族蛋白作為一類反式作用因子，主要參與RNA轉(zhuǎn)錄后的修飾，通過結(jié)合RNA并調(diào)節(jié)細(xì)胞器RNA代謝來調(diào)控基因的表達(dá)[42, 80-81]。一些與植物非生物脅迫響應(yīng)相關(guān)的PPR蛋白已被證明在細(xì)胞器RNA轉(zhuǎn)錄后調(diào)控中發(fā)揮作用。擬南芥的突變會導(dǎo)致多個RNA編輯缺陷，在突變體中，ABA信號通路相關(guān)基因表達(dá)下調(diào)，進(jìn)一步影響了許多與脅迫相關(guān)，特別是與干旱相關(guān)的基因表達(dá)，這與突變體的干旱敏感性增加一致[66]。另有研究表明，線粒體RNA編輯因子SLO2影響植物對ABA和非生物脅迫的敏感性，突變體中核編碼的非生物脅迫響應(yīng)基因和線粒體復(fù)合物Ⅰ基因（）及替代呼吸途徑相關(guān)基因的表達(dá)增加，進(jìn)一步支持了線粒體RNA編輯事件和應(yīng)激反應(yīng)兩者之間的聯(lián)系[64]。水稻的突變會導(dǎo)致水稻葉綠體轉(zhuǎn)錄本剪接的重大缺陷，突變體中，異常轉(zhuǎn)錄本積累及其產(chǎn)物減少，質(zhì)體編碼聚合酶依賴的質(zhì)體基因表達(dá)明顯下調(diào)，質(zhì)體rRNAs和翻譯產(chǎn)物積累到非常低的水平，這表明翻譯效率的降低可能會影響突變體對非生物脅迫的反應(yīng)[74]。最新研究發(fā)現(xiàn)，主要參與線粒體基因內(nèi)含子4的剪接，其突變會導(dǎo)致線粒體的破壞和交替呼吸途徑的增加，從而產(chǎn)生類病變表型，增強(qiáng)水稻對鹽的抗性和耐受性[76]。Xiong等[82]研究表明水稻細(xì)胞質(zhì)雄性不育系與其保持系之間的RNA編輯的差異是導(dǎo)致它們在環(huán)境脅迫下表現(xiàn)不同的原因之一，并證實了PPR基因介導(dǎo)的RNA編輯與水稻非生物脅迫耐受性的潛在關(guān)系。

表1 參與調(diào)節(jié)植物非生物脅迫反應(yīng)的部分PPR蛋白

4.2 逆行信號

一般來說，細(xì)胞器的發(fā)育和基因表達(dá)受核基因組調(diào)控，但來自葉綠體和線粒體的信號亦可“逆行”調(diào)控核基因的表達(dá)，這樣的調(diào)控信號被稱為“逆行信號”[83]。根據(jù)其功能含義，質(zhì)體逆行信號被分為與質(zhì)體發(fā)育相關(guān)的信號和與響應(yīng)環(huán)境或代謝波動的操作微調(diào)有關(guān)的信號[84]。研究表明，PPR蛋白的損傷能夠?qū)е戮€粒體或葉綠體功能受損，產(chǎn)生各類逆行信號（如ROS），從而調(diào)控抗逆相關(guān)基因表達(dá)[85]。最典型的例子是擬南芥PPR蛋白GENOMES UNCOUPLED 1（GUN1），被鑒定為葉綠體到細(xì)胞核逆行信號通路的中心整合因子[86]。GUN1的失活會在一定條件下抑制與光合作用相關(guān)的核基因（photosynthesis associated nuclear genes，）的表達(dá)，從而促進(jìn)逆行信號傳導(dǎo)。突變體植株更容易受到葉綠體干擾因素的影響，包括光、質(zhì)體翻譯抑制劑林可霉素（Linc）和類胡蘿卜素生物合成抑制劑去氟拉松（NF）處理[52-53]。一般來說，GUN1可能通過3條經(jīng)典的逆行信號通路調(diào)控的表達(dá)：四吡咯生物合成途徑（tetrapyrrole biosynthesis pathway，TPB）、氧化還原反應(yīng)和質(zhì)體基因表達(dá)（plastid gene expression，PGE）[87]。最近，Wu等[88]提出一個新模型：當(dāng)葉綠體發(fā)育過程中遭遇逆境時，GUN1通過與cpHSC70-1互作來增強(qiáng)質(zhì)體的蛋白輸入，未及時轉(zhuǎn)運(yùn)進(jìn)葉綠體的前體蛋白在細(xì)胞質(zhì)中過度積累，誘導(dǎo)細(xì)胞質(zhì)中HSP90蛋白表達(dá)上調(diào)，進(jìn)而維持表達(dá)。

除了cpHSC70-1伴侶外，還有許多其他假定的GUN1相互作用蛋白被陸續(xù)提出，包括葉綠體核糖體蛋白S1（plastid ribosomal protein S1，PRPS1）[89]、多細(xì)胞器RNA編輯因子（multiple organellar rna editing factor，MORF）[90]、核編碼RNA聚合酶（nuclear-encoded rna polymerase，NEP）[91]和各種四吡咯[92]等。研究表明，GUN1能夠在蛋白水平上控制葉綠體核糖體蛋白PRPS1的積累，并和參與葉綠體蛋白穩(wěn)態(tài)的蛋白質(zhì)相互作用，而PRPS1的功能是質(zhì)體mRNA翻譯和耐熱性所必需的[89]。MORF蛋白家族是線粒體和葉綠體中RNA編輯體系的重要組成部分，幾乎所有位點的完全編輯都需要MORF蛋白[93]。最新研究表明，GUN1通過與MORF2發(fā)生物理相互作用，直接影響葉綠體RNA中多個位點的編輯效率，并調(diào)節(jié)核編碼的葉綠體RNA聚合酶的活性，特別是在逆行信號傳遞過程中[91, 94]。然而，過表達(dá)系只有在使用NF處理時才能看到表型，而使用Linc處理時卻無法看到表型，因此，突變對質(zhì)體RNA編輯影響的潛在機(jī)制基礎(chǔ)和功能意義仍有待闡明[90]。這些相互作用支持GUN1可能作為一種支架蛋白，在各種生物環(huán)境中促進(jìn)蛋白質(zhì)復(fù)合物的形成的假設(shè)[95]。但是，由于葉綠體信號受到許多環(huán)境因素的影響，其中一些因素大多是未知的，難以控制的，導(dǎo)致不同實驗室對同一突變體得到的結(jié)果不同，因此，GUN1作為逆行通訊和細(xì)胞核信號通路的中心調(diào)節(jié)器的功能仍然有很多未解決的問題[86, 96]。部分觀點認(rèn)為，GUN1能夠通過靶向細(xì)胞核中的多種轉(zhuǎn)錄因子，包括ABSCISIC ACID INSENSITIVE4（ABI4）、GOLDEN2-LIKE 1/2（GLK1/2）和ELONGATED HYPOCOTYL 5（HY5），將信息傳遞到細(xì)胞核[97-100]，這些核轉(zhuǎn)錄調(diào)控因子被認(rèn)為是GUN1所涉及逆境信號中的一個重要的下游成分，它們的突變體在的解偶聯(lián)表達(dá)方面與突變體表現(xiàn)相似[101]。Veciana等[102-103]研究發(fā)現(xiàn)，BBX16作為GLK1的一個直接靶點，其在葉綠體損傷后通過GUN1/GLK1模塊被抑制，調(diào)節(jié)暴露在破壞性強(qiáng)光下的區(qū)域?？傊?，這些研究表明GUN1對于葉綠體RNA代謝和葉綠體-核逆行信號非常重要[104]。

近年來，亦有研究表明，其他PPR蛋白通過逆行信號調(diào)控植物抗逆性。如，擬南芥線粒體PPR蛋白LOI1參與呼吸鏈相關(guān)基因、和的RNA編輯，并調(diào)節(jié)類異戊二烯的生物合成，突變體對2種類異戊二烯合成抑制劑（真菌植物毒素洛伐他汀和除草劑氯馬松）的敏感性降低，從而引起胞質(zhì)甲戊酸（cytosolic mevalonate，MVA）途徑和質(zhì)體非甲戊酸（plastidal non-mevalonate，MEP）途徑的改變，已知此類途徑會影響防御基因的表達(dá)，以應(yīng)對損傷，揭示了從線粒體到細(xì)胞質(zhì)逆行信號的間接作用[105-106]；PPR蛋白PGN的失活會引起擬南芥線粒體編碼轉(zhuǎn)錄物差異表達(dá)，最顯著的是和表達(dá)水平的升高，它們在線粒體功能改變或抑制誘導(dǎo)的逆行信號傳導(dǎo)中發(fā)揮作用，突變體幼苗內(nèi)源ABA和鹽脅迫下的ROS積累增加，因此，PGN可能通過轉(zhuǎn)錄編輯協(xié)調(diào)影響整體線粒體基因表達(dá)，從而有助于植物防御和維持細(xì)胞氧化還原平衡[61]；PPR蛋白AHG11和SLG1分別參與線粒體和的編輯，和是線粒體中電子傳遞鏈復(fù)合體Ⅰ的2個亞基，和的突變致使線粒體功能的部分損傷誘導(dǎo)氧化還原失衡，突變體植株表現(xiàn)出對干旱等非生物脅迫敏感性增強(qiáng)[62-63]；另有研究發(fā)現(xiàn)，ABA缺失抑制位點HAS2編碼參與線粒體RNA編輯的PPR蛋白LOI1/ MEF11，證明了ABA在線粒體逆行信號調(diào)節(jié)中起著重要作用[67]。

5 PPR蛋白的多效性

目前，鑒定到的很多PPR基因都具有一因多效性，通常在影響植物適應(yīng)非生物脅迫的同時，對植物的生長和生殖也會產(chǎn)生重要影響。例如，水稻突變體在三葉期表現(xiàn)為白化表型和葉綠體異常，這種現(xiàn)象與葉綠素含量和葉綠體發(fā)育變化有關(guān)，且受溫度影響[72]；同樣，與低溫相關(guān)的PPR蛋白基因突變體亦表現(xiàn)出白化和葉綠體畸形[73]；擬南芥突變體不僅表現(xiàn)出光氧化應(yīng)激耐受性降低，而且其幼期葉綠素含量也降低，呈現(xiàn)淡綠色表型[51]，這些PPR蛋白在葉綠體早期發(fā)育過程中起重要的作用，其功能的缺失會影響葉綠體的發(fā)育，從而影響葉片生長。此外，影響線粒體功能的PPR蛋白亦被證明具有一因多效性。例如，ABO8不僅和擬南芥對ABA的敏感性有關(guān)，還能夠通過ABA介導(dǎo)的線粒體ROS調(diào)控擬南芥根的分生組織活性[60]；突變體植株不僅根系變短，分生組織大小和細(xì)胞數(shù)量減少，還表現(xiàn)出對高溫的敏感性增強(qiáng)，功能分析表明，GEND1能夠結(jié)合并編輯線粒體mRNA，其突變會導(dǎo)致擬南芥細(xì)胞色素c水平降低[69]；的突變會使植株產(chǎn)生類病變表型（自發(fā)的細(xì)胞死亡反應(yīng)和H2O2積累，對真菌和細(xì)菌病原體稻瘟病菌和水稻黃單胞菌的抗性增強(qiáng)），同時增強(qiáng)了水稻對鹽脅迫的耐受性[76]。POCO1被證明能夠影響擬南芥的開花時間，在長、短日照條件下，突變體均表現(xiàn)出早花表型，后續(xù)研究表明，POCO1還參與了擬南芥的抗旱，但二者之間的聯(lián)系還未見報道[107]。

6 展望

以往研究綜述大多針對PPR蛋白的起源、分類、定位，以及在植物生長發(fā)育中的功能。本文綜述了近年來PPR蛋白在植物非生物脅迫中的功能研究進(jìn)展，并總結(jié)分析了PPR蛋白參與植物非生物脅迫調(diào)控的分子機(jī)制，以期為作物非生物脅迫分子育種提供參考。盡管已經(jīng)鑒定出多種植物PPR蛋白，但與數(shù)目眾多的PPR成員相比，還有很多發(fā)揮重要功能的PPR蛋白未被研究，它們是否和植物非生物脅迫抗性有關(guān)？此外，由于PPR蛋白基因普遍存在的一因多效性，當(dāng)利用PPR蛋白改良作物抗逆性時，需要注意其對作物其他生理功能的影響，這也是應(yīng)用PPR蛋白育種時的重點和難點之一。

因為PPR蛋白在植物線粒體和葉綠體中負(fù)責(zé)C-to-U和U-to-C的RNA編輯，已有研究將其作為RNA編輯工具來利用[25]。如，Oldenkott等[108]通過表達(dá)來自腎葉白頭翁（）的含有單個DYW結(jié)構(gòu)域的PPR蛋白，在大腸桿菌中構(gòu)建了C-to-U RNA編輯；Ichinose等[25]成功開發(fā)了一種基于DYW:KP蛋白的U-to-C RNA編輯因子，該因子在細(xì)菌和人類細(xì)胞中起作用。在臨床治療中，相比于現(xiàn)在流行的以CRISPR-Cas9為代表的DNA編輯而言，RNA編輯更靈活、更安全，因為RNA編輯其通常只在特定的細(xì)胞類型中或在特定的時間表達(dá)，預(yù)期脫靶導(dǎo)致的編輯副作用更少，而且由于基因組序列不受影響，錯誤的RNA編輯也不會影響胎兒發(fā)育，停止治療后，突變的RNA會迅速降解[109]。這些研究為未來的基因治療和作物改良提供了參考。

近年來，雖然PPR蛋白的研究取得了重要的進(jìn)展，但關(guān)于PPR蛋白參與植物非生物脅迫響應(yīng)中的分子機(jī)制還不十分清楚，需要進(jìn)一步深入探究。首先，PPR蛋白在調(diào)節(jié)植物非生物脅迫過程中是否存在時空性和組織特異性？其次，PPR蛋白之間如何相互作用或與其他蛋白相互作用以實現(xiàn)其最終功能，這些相互作用又是如何在細(xì)胞器RNA轉(zhuǎn)錄后加工中影響PPR活性的？第三，PPR蛋白是如何特異識別并與RNA結(jié)合的？最后，PPR蛋白在陸生植物核-細(xì)胞質(zhì)相互作用的逆行信號調(diào)控網(wǎng)絡(luò)中的具體角色是什么？也許大量的進(jìn)化分析和更多其他PPR蛋白的功能鑒定，以及對共表達(dá)PPR基因的進(jìn)一步分析將揭示這些問題的答案。然而，為了更詳細(xì)地闡明PPR蛋白在植物非生物脅迫抗性中的具體機(jī)制，有必要對PPR蛋白的RNA靶點進(jìn)行鑒定，并對蛋白質(zhì)-RNA復(fù)合物的晶體結(jié)構(gòu)進(jìn)行分析，同時，還應(yīng)加強(qiáng)對MORF等直接影響PPR蛋白作用的相互作用蛋白給予關(guān)注。這些研究將有助于更好地闡明PPR蛋白在植物非生物脅迫下的調(diào)控網(wǎng)絡(luò)和特異性，有助于了解植物細(xì)胞器RNA加工的細(xì)節(jié)，從而為作物育種改良提供支撐。值得期待的是，人工PPR蛋白可以被定制并在體內(nèi)結(jié)合特定的內(nèi)源性RNA，這為開發(fā)用于分子設(shè)計育種的RNA結(jié)合蛋白（RNA binding protein，RBPs）提供了廣闊的研究前景[110]。

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Research progress of PPR protein in plant abiotic stress response

LI Cheng, LU Kai, WANG CaiLin, ZHANG YaDong

Institute of Food Crops, Jiangsu Academy of Agricultural Sciences/East China Branch of National Center of Technology Innovation for Saline-Alkali Tolerant Rice/Jiangsu High Quality Rice R&D Center/Nanjing Branch of China National Center for Rice Improvement/Key laboratory of Jiangsu Province for Agrobiology, Nanjing 210014

Abiotic stress is one of the main factors causing global grain yield reduction. It is of great significance to study the function and response mechanisms of plant stress-related proteins to improve crop stress resistance. Pentatricopeptide repeat (PPR) proteins, belong to the largest family of nuclear coding proteins in higher plants and are named because they contain highly specific PPR motifs. Depending on motif type and arrangement, PPR proteins can be classified as P and PLS, and PLS proteins can be further classified as PLS, E, E+, DYW, and other subclasses based on their carboxyl-terminal domains. PPR proteins are widely distributed in terrestrial plants, mainly in chloroplasts and mitochondria, and a few in the nucleus. As sequence-specific RNA binding proteins, PPR proteins are involved in multiple aspects of plant RNA processing, including RNA editing, splicing, stabilization, and translation. PPR protein plays a variety of important roles in the whole life process of plants, but the mechanism of its action in plant stress resistance is not well understood. Based on the localization and function of PPR proteins related to abiotic stress reported, the mechanism of PPR proteins involved in regulation of abiotic stress, including post-transcriptional regulation and retrograde signaling, was reviewed and discussed in this paper. Post-transcriptional regulation is related to the role of PPR proteins in the modification of RNA after transcription. It is generally believed that PPR affects stress resistance in plants by regulating the expression of stress-related genes via binding RNA and by regulating the metabolism of organelle RNA. In terms of retrograde signaling, damage to PPR proteins can lead to impaired mitochondrial or chloroplast function, and then produce various retrograde signals (such as ROS), thereby regulating the expression of related genes and resisting adversity. However, since plastid signaling is affected by many environmental factors, some of which are still unclear, the mechanism of the PPR protein in retrograde signaling remains to be clarified. In addition, PPR proteins are pleiotropic and some have important effects on plant growth and reproduction while acting on stress resistance. Finally, this paper further analyzed the current research status of PPR protein as an RNA editing tool, discussed the remaining problems and research prospects of PPR protein in the direction of abiotic stress, and pointed out the key points and difficulties that need to be paid attention to in future research, to provide references for further research on PPR protein and crop abiotic stress resistance breeding.

PPR protein; plant; abiotic stress

10.3864/j.issn.0578-1752.2023.24.001

2023-05-25；

2023-07-24

江蘇省種業(yè)振興揭榜掛帥項目（JBGS[2021]001）

李程，E-mail：cli1024shine@163.com。通信作者張亞東，E-mail：zhangyd@jaas.ac.cn

（責(zé)任編輯 李莉）