崔玉超,陳 亮

(廈門大學(xué)生命科學(xué)學(xué)院,廈門市植物遺傳重點(diǎn)實(shí)驗(yàn)室,福建 廈門361102)

·綜 述·

擬南芥莖尖干細(xì)胞調(diào)控機(jī)制的研究進(jìn)展

崔玉超,陳 亮*

(廈門大學(xué)生命科學(xué)學(xué)院,廈門市植物遺傳重點(diǎn)實(shí)驗(yàn)室,福建 廈門361102)

植物莖尖干細(xì)胞是位于植物頂端一團(tuán)具有無限增殖能力的細(xì)胞,它是植物整個(gè)地上部分發(fā)育的源泉.干細(xì)胞分裂產(chǎn)生的子細(xì)胞一部分用來保持自我更替,另一部分形成器官原基,進(jìn)而使其處于一種動(dòng)態(tài)平衡狀態(tài),來維持植物甚至可長達(dá)千年的生長周期.現(xiàn)代分子生物學(xué)與遺傳學(xué)表明這種動(dòng)態(tài)平衡的維持受到來自干細(xì)胞微環(huán)境的各種因素的精確調(diào)控.其中起核心作用的是由WUSCHEL(WUS)基因與CLAVATA(CLV)基因之間形成的負(fù)反饋調(diào)節(jié)環(huán),而其他生物學(xué)因素如細(xì)胞分裂素、可以移動(dòng)的小RNAs和表觀遺傳作用等最終都作用于這一負(fù)反饋調(diào)節(jié)環(huán),另外與WUS-CLV信號(hào)通路相平行的SHOOTMERISTEMLESS(STM)信號(hào)通路在干細(xì)胞維持中也發(fā)揮著積極的作用.在此主要綜述了模式植物擬南芥(Arabidopsis thaliana)莖尖干細(xì)胞維持分子機(jī)制的最新研究進(jìn)展.

莖尖干細(xì)胞;WUS-CLV;細(xì)胞分裂素;小RNAs;表觀遺傳;STM

植物地上部分的胚后發(fā)育主要依賴于莖尖分生組織(shoot apical meristem,SAM).SAM通常是指最小葉原基上方的莖端區(qū)域(圖1(a)),相當(dāng)于經(jīng)典植物學(xué)中的初生分生組織或莖頂端分生組織狹義概念所涵蓋的內(nèi)容.在以擬南芥(Arabidopsis thaliana)為代表的雙子葉植物中,SAM的作用是形成葉片和花,維持莖干的生長和保持自身的位置、大小及結(jié)構(gòu).另外,在樹木的地上部分發(fā)育中,維管的環(huán)狀形成層(ringshaped cambium)和軟木形成層(phellogen cambium)分生組織對于莖的長粗具有重要作用.

SAM在細(xì)胞水平可分為頂端中央?yún)^(qū)域(central zone,CZ)、側(cè)旁圍繞CZ分布的外圍區(qū)域(peripheral zone,PZ)和CZ基部的肋狀區(qū)域(rib zone,RZ)[1-2],如圖1(b)所示.植物莖尖干細(xì)胞位于CZ,通過緩慢的細(xì)胞分裂實(shí)現(xiàn)自我更新并維持在未分化狀態(tài),它們是新器官和組織發(fā)育的來源;PZ細(xì)胞分裂較快,進(jìn)而分化成不同的器官原基;RZ細(xì)胞同PZ細(xì)胞一樣分裂也較快,它保證了植物能夠向上生長[1-2].在CZ下方有一團(tuán)細(xì)胞稱為組織中心區(qū)域(organizing center,OC),這里的細(xì)胞決定了其上方干細(xì)胞的命運(yùn)和數(shù)量,對于干細(xì)胞的維持起著決定性作用.

根據(jù)CZ干細(xì)胞的克隆特性又可將SAM分成三層,即外層的L1,表皮下的L2和內(nèi)部的L3[3](圖1 (a)).干細(xì)胞一次分裂后產(chǎn)生的兩個(gè)子細(xì)胞一個(gè)留在原位置成為新的干細(xì)胞,而另一個(gè)移向其他區(qū)域成為待分化的細(xì)胞,這種截然不同的命運(yùn)是由其所在位置決定的[4-5].在分裂形式上,L1與L2的干細(xì)胞都進(jìn)行垂周分裂,即新形成的細(xì)胞壁垂直于外表面,這樣在每一層面上干細(xì)胞分裂后產(chǎn)生的兩個(gè)子細(xì)胞中一個(gè)保持在原位置,另一個(gè)則移向PZ,進(jìn)而保證了L1與L2分別屬于不同的層次.L3細(xì)胞既可進(jìn)行垂周分裂也可以平周分裂,由此位于頂端的子細(xì)胞留在原位置而另一個(gè)子細(xì)胞移向PZ或RZ.由于干細(xì)胞具有多能性,所以每一層莖尖干細(xì)胞的子細(xì)胞都可以發(fā)展成為這一層中的各種細(xì)胞類型.最終,L1細(xì)胞發(fā)育成為表皮組織,L2發(fā)育成為皮下組織,而L3發(fā)育成為髓和維管組織[3,6-9].

在植物生長發(fā)育過程中,SAM的干細(xì)胞處于動(dòng)態(tài)平衡中,其分裂產(chǎn)生的細(xì)胞一方面進(jìn)行自我更新,另一方面為側(cè)翼器官原基的發(fā)育提供源泉.動(dòng)態(tài)平衡的維持來自于干細(xì)胞周圍不同信號(hào)因子之間的協(xié)同調(diào)控作用(圖1(b)),干細(xì)胞所處的這種環(huán)境稱為干細(xì)胞微環(huán)境.在干細(xì)胞微環(huán)境中核心信號(hào)通路是在OC表達(dá)的WUSCHEL(WUS)基因與干細(xì)胞基因CLAVATA(CLV)之間形成的負(fù)反饋調(diào)節(jié)環(huán),即WUS基因表達(dá)后決定了其頂部細(xì)胞發(fā)育成干細(xì)胞并在此處轉(zhuǎn)錄激活CLV3基因的表達(dá),CLV3基因又會(huì)反過來抑制WUS基因的表達(dá),由此形成了“走走停停”的調(diào)控模式.其他信號(hào)因子如細(xì)胞分裂素(cytokinin,CK)、可以移動(dòng)的小RNAs和表觀遺傳調(diào)控因子,最終都作用于這條信號(hào)通路來實(shí)現(xiàn)對干細(xì)胞動(dòng)態(tài)平衡的調(diào)節(jié).與WUS-CLV相平行的SHOOTMERISTEMLESS(STM)信號(hào)通路可以抑制干細(xì)胞的分化并促進(jìn)PZ細(xì)胞在形成器官原基前的分裂.

1 WUS-CLV負(fù)反饋調(diào)節(jié)環(huán)

WUS基因編碼植物中特有的同源異型轉(zhuǎn)錄因子(transcription factor,TF),它是WUSCHEL-related homeobox(WOX)基因家族的基礎(chǔ)成員[10].WUS的表達(dá)起始于胚胎的16細(xì)胞時(shí)期,表達(dá)范圍位于胚胎頂部內(nèi)層的4個(gè)細(xì)胞,并且隨著頂部細(xì)胞不斷地進(jìn)行縱向與橫向分裂,WUS的表達(dá)區(qū)域逐漸下移到達(dá)SAM的中央.苗期時(shí)WUS在OC表達(dá).花序分生組織(inflorescence meristem,IM)中WUS表達(dá)區(qū)域同苗期一樣,然而在花分生組織(floral meristem,FM)中WUS的表達(dá)會(huì)上移到L3,說明SAM與FM的組織形式存在差異[11].wus突變體在苗期時(shí)沒有類似于野生型穹頂狀的SAM,并且在干細(xì)胞區(qū)域出現(xiàn)一定程度已經(jīng)分化的細(xì)胞,在IM中也表現(xiàn)出同樣的缺陷,說明WUS可以阻止干細(xì)胞的分化[11-12].在過表達(dá)WUS時(shí)不僅導(dǎo)致SAM增大,還可以誘導(dǎo)異位干細(xì)胞產(chǎn)生,表明WUS可以決定干細(xì)胞的屬性[13-15].

CLV3基因編碼具有96個(gè)氨基酸的蛋白,其屬于32個(gè)CLV3/EMBRYO SURROUNDING REGION (CLE)蛋白家族的一員.它在N端具有18個(gè)氨基酸長度的信號(hào)肽而C端含有一個(gè)同其他CLE家族蛋白相似的14個(gè)氨基酸長度的CLE保守結(jié)構(gòu)域,此結(jié)構(gòu)域在翻譯后被加工成有活性的小分子多肽CLEp(CLE peptide)[16-18].許多其他CLE基因在CLV3啟動(dòng)子的啟動(dòng)表達(dá)下可以恢復(fù)clv3突變體的表型,這表明CLV3與這些CLE基因存在功能上的冗余[19].在植物體中CLV3前體蛋白分別在Arg70與His81或者Arg70與His82位點(diǎn)處通過細(xì)胞外水解作用而剪切成為具有12或13個(gè)氨基酸長度的CLEp[20-21].在CLEp的Pro7位點(diǎn)還需要進(jìn)一步發(fā)生羥基化和3個(gè)樹膠醛糖化修飾使其成為有功能活性的肽段[21].CLV3的表達(dá)模式在SAM頂端成楔形結(jié)構(gòu),此區(qū)域大體與干細(xì)胞一致.因?yàn)樵贚3表達(dá)CLV3的細(xì)胞數(shù)量明顯少于L1,所以在每層干細(xì)胞數(shù)量相當(dāng)時(shí),CLV3的表達(dá)區(qū)域并不完全等同于干細(xì)胞所在區(qū)域.clv3突變體的SAM和FM明顯增大,并且與野生型相比產(chǎn)生更多器官[22],這說明CLV3對干細(xì)胞的維持起負(fù)調(diào)控作用.

研究發(fā)現(xiàn)在clv3中SAM增大是由于WUS表達(dá)區(qū)域擴(kuò)大所致[15];相反地,在過表達(dá)CLV3時(shí)又抑制WUS的表達(dá),并表現(xiàn)出wus的表型[23-24].另一方面,在wus中CLV3表達(dá)消失;而在過表達(dá)WUS時(shí), CLV3表達(dá)區(qū)域擴(kuò)散[13,15].由此建立了一個(gè)反饋抑制通路,即WUS在OC表達(dá)后決定并促進(jìn)上方相鄰細(xì)胞成為干細(xì)胞,干細(xì)胞表達(dá)CLV3,而后CLV3又抑制WUS表達(dá)使其保持在適當(dāng)水平并穩(wěn)定在OC,通過這種方式維持干細(xì)胞的動(dòng)態(tài)平衡(圖1(b)).

建立在突變體研究基礎(chǔ)上的WUS-CLV反饋調(diào)節(jié)途徑只展示了基因突變后所導(dǎo)致的最終結(jié)果,并不能清楚地闡釋在干細(xì)胞動(dòng)態(tài)平衡的維持中CLV3和WUS基因的具體作用機(jī)制.由此,Reddy等[25]和Yadav等[26]分別在2005年和2010年報(bào)道了誘導(dǎo)性沉默或過表達(dá)基因后莖尖瞬時(shí)變化的結(jié)果.研究發(fā)現(xiàn)CLV3的功能是防止緊鄰CZ的PZ干細(xì)胞的子細(xì)胞脫分化重新形成干細(xì)胞,并且通過抑制PZ細(xì)胞的有絲分裂速率來維持SAM的大小[25];而WUS在CZ過表達(dá)時(shí)能促進(jìn)整個(gè)PZ細(xì)胞轉(zhuǎn)化成為干細(xì)胞,提高PZ細(xì)胞的有絲分裂速率,并控制PZ細(xì)胞參與分化和保持不分化的比例[26].由此,CLV3和WUS采用對立的機(jī)制在PZ與CZ細(xì)胞的相互轉(zhuǎn)化、PZ細(xì)胞的分裂速率和PZ細(xì)胞的分化比例這3個(gè)層面維持SAM的動(dòng)態(tài)平衡.隨后,這其中部分工作得到了分子水平的闡釋.Yadav等[27]證明大部分受WUS激活的基因在SAM的CZ表達(dá),而受WUS抑制的大部分基因在PZ表達(dá),且受WUS抑制的基因中很多是促進(jìn)細(xì)胞分化的轉(zhuǎn)錄因子,其中已經(jīng)報(bào)道的有37個(gè),包括在葉的極性建立及分化中起作用的基因KANADI1、KANADI2、ASYMMETRIC LEAVES 2(AS2)和YABBY3,以及在PZ與RZ表達(dá)而有可能對細(xì)胞命運(yùn)特化及IM干細(xì)胞分化起作用的K NAT1/BREVIPEDICELLUS (BP)、BLH5、BHLH093和ANAC083.WUS對促進(jìn)分化的轉(zhuǎn)錄因子的抑制作用是其參與維持SAM穩(wěn)定的一個(gè)重要方面.與之類似,Busch等[28]通過用不同遺傳背景下的植物(wus和誘導(dǎo)性過表達(dá)WUS的轉(zhuǎn)基因植株)進(jìn)行表達(dá)譜分析,發(fā)現(xiàn)受WUS響應(yīng)的基因有667個(gè)之多;隨后在全基因組水平進(jìn)行WUS的ChIP-chip實(shí)驗(yàn),發(fā)現(xiàn)WUS可以直接結(jié)合在118個(gè)靶基因的啟動(dòng)子上,而且這種結(jié)合至少需要每個(gè)啟動(dòng)子上2個(gè)不同的元件參與,這些靶基因主要在CK、生長素(auxin)、茉莉酸(jasmonate)幾種激素信號(hào)途徑以及發(fā)育過程和代謝過程中發(fā)揮作用.

圖1　莖尖分生組織及其維持的分子信號(hào)通路Fig.1 The shoot apical meristem and its maintenancepathway

在WUS對于干細(xì)胞的決定方向上,Yadav等[29]發(fā)現(xiàn)WUS蛋白可以移動(dòng)到干細(xì)胞區(qū)域并激活CLV3的表達(dá).在他們的研究中,IM中的eGFP-WUS融合蛋白(p WUS:eGFP-WUS)可以擴(kuò)散至L1,而其m RNA仍然在OC,并且p WUS:eGFP-WUS可以恢復(fù)wus;相比之下,2×eGFP-WUS(增加蛋白的大小)或者NLS-eGFP-WUS蛋白(將融合蛋白進(jìn)行核定位)在IM中的移動(dòng)性和對wus的恢復(fù)能力則明顯減弱,證明WUS具有移動(dòng)性并且這種移動(dòng)對SAM的維持發(fā)揮重要作用;隨后又證明了WUS移動(dòng)到CZ后會(huì)直接與CLV3啟動(dòng)子結(jié)合進(jìn)而啟動(dòng)其表達(dá)[29-30].值得注意的是,WUS蛋白在FM中的移動(dòng)性比較復(fù)雜, eGFP-WUS、2×eGFP-WUS和NLS-eGFP-WUS蛋白在FM發(fā)育早期都能在L1檢測到,而在后期2× eGFP-WUS和NLS-eGFP-WUS則定位于OC[29].對于eGFP-WUS有可能產(chǎn)生的偏差,Daum等[31]用免疫組織化學(xué)的方法進(jìn)一步確認(rèn)了內(nèi)源WUS蛋白的移動(dòng)性,并進(jìn)而證明了WUS這種細(xì)胞與細(xì)胞間的移動(dòng)需要借助于起細(xì)胞溝通橋梁作用的胞間連絲(plasmodesmata,PD),在干細(xì)胞區(qū)域誘導(dǎo)性表達(dá)胼胝質(zhì)合成酶時(shí)(胼胝質(zhì)合成酶可以在PD周圍堆積胼胝質(zhì)使細(xì)胞壁加厚從而減小PD的直徑而減弱其功能),受WUS啟動(dòng)子表達(dá)的WUS-linker-GFP融合蛋白不能移動(dòng)到L1并且干細(xì)胞出現(xiàn)分化.WUS蛋白的同源異型結(jié)構(gòu)域(homeodomain,HD)對其移動(dòng)有促進(jìn)作用,而HD與WUS-Box功能域之間的非保守區(qū)域?qū)ζ湟苿?dòng)起限制作用,這種不同區(qū)域間的相互作用保證WUS向上呈楔形移動(dòng)而不是移向SAM外圍區(qū)域進(jìn)而誘導(dǎo)形成干細(xì)胞;此外還發(fā)現(xiàn)非保守區(qū)域介導(dǎo)WUS形成二聚體,推測WUS的二聚化可能對其移動(dòng)性有影響[31].已有研究表明STM蛋白(詳見后文STM信號(hào)途徑)依賴于PD的移動(dòng)需要分子伴侶CCT8的參與[32],然而在cct8突變體中WUS的移動(dòng)并未受到影響[31],說明這些關(guān)鍵蛋白的移動(dòng)機(jī)制并不具有普遍性.

CLV3對WUS的反饋調(diào)節(jié)過程中,成熟的CLV3p從干細(xì)胞區(qū)域向下方移動(dòng),被多種富含亮氨酸重復(fù)序列(leucine-rich repeat,LRR)的受體接收從而抑制OC細(xì)胞中WUS的表達(dá)[33](圖1(b)).在這些受體中第1種就是CLV1.CLV1編碼LRR受體激酶,其主要在RZ表達(dá),也會(huì)出現(xiàn)在OC和CZ的L3[34],已有研究證明CLV3p可以直接結(jié)合在CLV1的LRR結(jié)構(gòu)域上[35],但clv1的表型要比clv3弱很多,造成這種現(xiàn)象的原因是BARELY ANY MERISTEM 1,2和3 (BAM1,2和3)基因的參與.BAM基因編碼具有LRR功能結(jié)構(gòu)域的受體激酶,然而其功能與CLV1剛好相反,bam1 bam2 bam3三突變體中SAM明顯變小[36-37].正常情況下,由于CLV1基因在RZ的表達(dá)從而抑制BAM基因在此區(qū)域的表達(dá);當(dāng)CLV1基因突變時(shí),BAM恢復(fù)在RZ的表達(dá)也進(jìn)而抑制SAM的擴(kuò)增,由此可以解釋單純clv1突變體表型較弱,而clv1 bam1 bam2 bam3四突變體表型顯著增強(qiáng)的原因[38].第2種受體是CLV2-CORYNE(CRN)復(fù)合物受體. CLV2也是類似LRR受體的跨膜蛋白但其不含細(xì)胞內(nèi)的激酶功能域;CRN具有類似于CLV1的跨膜絲氨酸/蘇氨酸激酶活性但缺少細(xì)胞外LRR功能域[39-40],其與CLV2形成異源二聚體獨(dú)立于CLV1作為CLV3p的另一受體[41].然而Nimchuk等[42]發(fā)現(xiàn)CRN不能自我磷酸化,它的功能發(fā)揮類似于動(dòng)物中的假激酶(pseudokinases),并不依靠激酶活性,而是起腳手架的作用.第3種受體是RECEPTOR-LIKE PROTEIN KINASE 2(RPK2).rpk2的表型相較于clv1與clv2要弱,但也會(huì)導(dǎo)致干細(xì)胞擴(kuò)散,并且clv1 clv2 rpk2三突變體的表型更接近于clv3,說明RPK2在CLV3主導(dǎo)的信號(hào)通路中發(fā)揮次要作用但又獨(dú)立于CLV1與CLV2-CRN受體[43].

LRR受體都是膜蛋白,在其接受到來自干細(xì)胞分泌的配體CLV3p后如何將此信號(hào)傳導(dǎo)進(jìn)入細(xì)胞內(nèi)部一直以來并不清楚.最近這一作用機(jī)制在玉米(zea mays)的相關(guān)研究中獲得了進(jìn)展[44]:玉米COMPACT PLANT 2(CT2)基因編碼異源三聚體GTP結(jié)合蛋白的α亞單位,它可以直接結(jié)合玉米的跨膜蛋白受體FASCIATED EAR(FEA,CLV2的同源蛋白).異源三聚體GTP結(jié)合蛋白是與之相聯(lián)系的膜蛋白進(jìn)行信號(hào)傳導(dǎo)的分子開關(guān),它通常是受7個(gè)跨膜蛋白接收配體后激活[45],而在玉米的研究中一個(gè)這樣的跨膜受體FEA就足以激活.玉米ct2突變體的SAM表型同擬南芥中clv突變體類似,用CLV3p處理野生型時(shí)會(huì)抑制SAM的生長而處理ct2則不會(huì).盡管如此,信號(hào)由胞外傳遞到胞內(nèi)后最終又是如何抑制WUS表達(dá)的分子機(jī)制尚待研究.

CLV3p對WUS的抑制作用存在一個(gè)平衡,因?yàn)檫^量的CLV3p或者僅在L1過表達(dá)CLV3都會(huì)完全抑制WUS的表達(dá),出現(xiàn)wus的表型[23-24].現(xiàn)在就這個(gè)問題存在2種對立的觀點(diǎn):1)CLV1受體通過直接結(jié)合屏蔽CLV3p使其不能進(jìn)入WUS的表達(dá)區(qū)域OC.這個(gè)模型建立在對CLV3-GFP融合蛋白的觀察發(fā)現(xiàn)其主要向側(cè)邊移動(dòng),而很少向正下方OC移動(dòng)[24],并且在這個(gè)過程中CLV2會(huì)輔助CLV1以提高其穩(wěn)定性,隨后的研究證實(shí)CLV3p可以直接結(jié)合CLV1也支持這一模型[21,35].2)CLV1的存在依賴于CLV3p的劑量性調(diào)控.在這種模型中,CLV1在clv3背景下于細(xì)胞質(zhì)膜中得到積累,當(dāng)CLV3p存在時(shí),CLV1被轉(zhuǎn)運(yùn)至溶酶體中被降解,CLV3p的數(shù)量決定了CLV1的量,進(jìn)而決定了信號(hào)的傳導(dǎo)[46],據(jù)此可以解釋為什么SAM承受CLV3表達(dá)水平10倍以上的變化時(shí)并不引起表型的改變和WUS表達(dá)水平先降低而后又得到恢復(fù)[47].此外,WUS本身也可以通過抑制CLV1的表達(dá)進(jìn)而降低CLV3p的抑制效應(yīng)進(jìn)行自救,通過直接結(jié)合在CLV1啟動(dòng)子上距離轉(zhuǎn)錄起始位點(diǎn)600 bp的位置而抑制CLV1的表達(dá)[28].再者,CLV信號(hào)通路下游存在POLTERGEIST(POL)和POLTERGEIST-LIKE1 (PLL1)基因,它們可以促進(jìn)WUS的轉(zhuǎn)錄,而其又受CLV信號(hào)通路抑制[48-49].POL和PLL1編碼2C型蛋白磷酸酶,它們在功能上冗余,可以通過乙?；ㄎ挥诩?xì)胞質(zhì)膜的內(nèi)表面,而CLV3的幾種受體蛋白也都定位于細(xì)胞質(zhì)膜上,它們之間或許存在著聯(lián)系進(jìn)而調(diào)控WUS-CLV信號(hào)通路的動(dòng)態(tài)平衡[50].

2 CK信號(hào)通路

在SAM中合成的CK并沒有活性,它需要CK激活酶的激活.在水稻中LONGLYGUY(LOG)[51]編碼CK激活酶,其作用于CK合成過程的最后一步,將未激活的核苷轉(zhuǎn)變成有生物活性的堿基(圖1(b)).LOG在SAM的最頂端2～3層細(xì)胞表達(dá),包含干細(xì)胞區(qū)域.水稻log在營養(yǎng)生長期間SAM體積減小,在生殖生長過程中產(chǎn)生較少的分支并且形成不完整的花,發(fā)育較早終止[51].在擬南芥中有9個(gè)LOG同源基因,其中LOG7對SAM 的維持貢獻(xiàn)最大[52],另外Chickarmane等[53]發(fā)現(xiàn)LOG4在SAM的L1表達(dá), LOG在SAM頂端表達(dá),這樣就使有活性的CK從干細(xì)胞區(qū)域向下擴(kuò)散至RZ并在此區(qū)間形成一個(gè)濃度梯度,這對于OC中干細(xì)胞的維持及其保持未分化狀態(tài)有重要作用.

在OC中,WUS的活性與CK的功能有著緊密的聯(lián)系.通過觀察與GFP相融合的CK感應(yīng)因子TCP的表達(dá),發(fā)現(xiàn)OC是SAM中CK響應(yīng)區(qū)域.CK受體ARABIDOPSIS HISTIDINE KINASE 4(AHK4)/ WOODENLEG的表達(dá)覆蓋整個(gè)OC,在這里AHK4誘導(dǎo)WUS的表達(dá)并且抑制WUS的拮抗基因CLV1的表達(dá)[54].WUS又可以直接抑制細(xì)胞內(nèi)的CK負(fù)調(diào)控因子ARR7和ARR15的表達(dá).降低ARR7和ARR15的表達(dá)水平會(huì)使SAM增大,而過表達(dá)ARR7時(shí)呈現(xiàn)wus的表型[39].同時(shí),受生長素激活的AUXIN RESPONSE FACTOR 5/MONOPTEROUS(ARF5/MP)轉(zhuǎn)錄因子也會(huì)直接抑制ARR7和ARR15的表達(dá)[55].由此在OC形成高水平的CK濃度,進(jìn)而誘導(dǎo)并穩(wěn)固了WUS的表達(dá),WUS又抑制ARR7和ARR15的表達(dá),于是構(gòu)建成一個(gè)正反饋調(diào)控途徑(圖1(b)).另外,在RZ還存在CK降解酶CYTOKININ OXIDASES 3(CKX3)來平衡CK的水平[56-57].總之,WUS介導(dǎo)了細(xì)胞內(nèi)CK的信號(hào)傳導(dǎo)途徑,OC細(xì)胞在SAM中接收并傳導(dǎo)CK信號(hào)又進(jìn)一步增強(qiáng)了WUS的表達(dá).

3 可移動(dòng)的小RNAs

HOMEODOMAIN-LEUCINE ZIPPERⅢ(HDZIPⅢ)家族基因的部分功能是調(diào)控胚胎頂部模式發(fā)生和胚胎發(fā)育時(shí)期SAM的形成[58].在擬南芥中存在5個(gè)HD-ZIPⅢ基因,分別是PHABULOSA (PHB)[59]、PHAVOLUTA(PHV)[59]、REVOLUTA (REC)、ARABIDOPSIS HOMEOBOX GENE 8 (ATHB8)和ATHB15/CORONA(CNA)[60]. HD-ZIPⅢ基因的突變會(huì)導(dǎo)致SAM終止[61-62],而提高其表達(dá)水平又可以導(dǎo)致SAM增大或者SAM異位再生[59].盡管現(xiàn)在并不清楚HD-ZIPⅢ基因家族在調(diào)控SAM方面的分子機(jī)制,但知道HD-ZIPⅢ基因本身受多種非編碼小RNAs的調(diào)控[63],在SAM中主要是micro RNA165/166的靶基因[64].

MicroRNA165/166對HD-ZIPⅢ基因的攻擊受ARGONAUTE1(AGO1)與ZWILLE(ZLL)/PINHEAD(PNH)/AGO10[65-67]的精確調(diào)控.ARGONAUTE蛋白是RNA沉默復(fù)合體中的關(guān)鍵因子. AGO1與AGO10蛋白序列相似度很高,其中包括2個(gè)重要的功能區(qū)域PAZ與MID,而且PAZ功能域可以在2個(gè)蛋白間互換,但是它們的功能卻是截然相反的[68].microRNA165/166在AGO1的介導(dǎo)下降解SAM中HD-ZIPⅢ基因的mRNA,使其表達(dá)水平下降;而micro RNA165/166與AGO10特異性結(jié)合后AGO10不會(huì)發(fā)揮其催化降解活性;生化實(shí)驗(yàn)證明AGO10與microRNA165/166的結(jié)合能力強(qiáng)于AGO1,所以AGO10的功能就是沉默體內(nèi)micro RNA165/166,使其不能與AGO1結(jié)合,從而穩(wěn)固HD-ZIPⅢ基因的表達(dá)[69].因此microRNA165/166信號(hào)因子類似于一種分子開關(guān),其通過“偏向”不同的兩側(cè)而起到截然相反的效果(圖1(b)).在zll/pnh/ ago10突變體中表現(xiàn)出胚胎發(fā)育后期SAM不能維持的表型,分子水平上microRNA165/166上升,HDZIPⅢ基因的轉(zhuǎn)錄本下降;而在SAM中增加HDZIPⅢ基因的表達(dá)水平或者降低microRNA165/166的水平時(shí)又可以部分恢復(fù)zll/pnh/ago10的異常表型[64].

擬南芥中microRNA165/166家族基因中有9個(gè)成員,他們中的5個(gè)在胚胎中的表達(dá)模式非常保守,都在胚胎基部的外圍區(qū)域表達(dá)[70].MicroRNA165/166在胚胎中具有移動(dòng)性,通過移動(dòng)降解分布于整個(gè)胚胎中的PHB m RNA最終使其只在胚胎中央的頂部表達(dá)[64].AGO10基因在圍繞SAM的原形成層處表達(dá),可能正是因?yàn)锳GO10的這種表達(dá)模式為SAM提供了一層保護(hù)屏障,通過沉默micro RNA165/166使SAM原基免受干擾;深入研究發(fā)現(xiàn)僅在OC正下方的維管組織區(qū)域表達(dá)AGO10就可以恢復(fù)其突變體的表型,說明這個(gè)區(qū)域?qū)τ赟AM的維持具有特殊的作用[69,71].

與microRNA165/166表達(dá)模式基本上相反的是microRNA394,在胚胎時(shí)期它僅在SAM的L1特異性表達(dá).MicroRNA394也具有移動(dòng)性,通過移向SAM內(nèi)部從而使干細(xì)胞維持在未分化狀態(tài)且對CLV3在CZ表達(dá)發(fā)揮重要作用(圖1(b)).MicroRNA394的靶基因是LEAF CURLING RESPONSIVENESS (LCR),其編碼的F-Box蛋白是干細(xì)胞抑制因子,可以介導(dǎo)生長素信號(hào)通路并促進(jìn)葉的發(fā)育[72].盡管microRNA394在L1表達(dá),但L1并不需要它的作用,因此Knauer等[73]認(rèn)為microRNA394的這種表達(dá)規(guī)律和作用方式正是干細(xì)胞在經(jīng)過細(xì)胞分裂和生長而始終處于SAM的原因,其作用類似于LOG.

4 表觀遺傳作用

表觀遺傳現(xiàn)象是指在DNA序列沒有改變的情況下,基因功能出現(xiàn)可逆、可遺傳的改變.在植物的生長發(fā)育過程中,細(xì)胞核基因組中等位基因尤其是有時(shí)空表達(dá)特性的基因會(huì)受某種因素影響而被選擇性地開啟或者關(guān)閉,從而引起表觀遺傳現(xiàn)象.這些因素主要包括:核小體組裝、ATP依賴型的染色質(zhì)重塑、組蛋白修飾及DNA甲基化[74].它們對SAM的維持作用最主要是通過對WUS基因的轉(zhuǎn)錄調(diào)控來實(shí)現(xiàn)的.

在參與核小體的組裝中有2個(gè)進(jìn)化上保守的蛋白CHROMATIN ASSEMBLY FACTOR 1(CAF-1)和NUCLEOSOME ASSEMBLY PROTEIN 1(NAP1),它們分別負(fù)責(zé)將組蛋白H3/H4亞基和H2A/H2B亞基包裝入核小體[74].FASCIATA 1(FAS1)和FAS2編碼CAF-1蛋白復(fù)合體的2個(gè)核心亞基,它們在SAM及葉原基中表達(dá),在fas1與fas2中SAM變得扁平肥大, WUS的表達(dá)區(qū)域明顯擴(kuò)散[75],說明CAF-1可以抑制WUS的表達(dá).BRUSHY1(BRU1)/MGOUN3(MGO3)/ TONSOKU(TSK)編碼的蛋白對DNA復(fù)制或者修復(fù)后的結(jié)構(gòu)起到穩(wěn)定作用,其突變后的表型同fas1和fas2非常相近[76-78].

在植物生長發(fā)育過程中,組蛋白與DNA之間相互作用會(huì)發(fā)生重新調(diào)整進(jìn)而改變?nèi)旧|(zhì)的結(jié)構(gòu)與活性,從而調(diào)節(jié)基因的表達(dá),即為染色質(zhì)重塑.ATP依賴型的染色質(zhì)重塑蛋白復(fù)合體在這一過程中具有重要作用,這些復(fù)合體的核心成員是 ATP酶[74]. SPLAYED(SYD)編碼一種SWI/SNF型ATP酶,它可以直接結(jié)合于WUS的啟動(dòng)子上(-435～-70 bp),并控制WUS的表達(dá)水平.在syd中,WUS的表達(dá)水平和SAM的大小全都降低[79-80].BRCA1-associated RING domain 1(BARD1)也可以抑制WUS的表達(dá),并且可以結(jié)合在SYD上,BARD1可能是通過抑制SYD的活性進(jìn)而抑制WUS的表達(dá). BARD1蛋白具有BRCA1 C-terminal(BRCT)與RING 2個(gè)前后相連的功能域,其可以直接結(jié)合于WUS的啟動(dòng)子上(-260～-206 bp).在bard1-3中, WUS m RNA表達(dá)上調(diào)238倍,其表達(dá)區(qū)域擴(kuò)散至L1～L3;而在過表達(dá)BARD1時(shí)又表現(xiàn)出wus的表型[79,81].

組蛋白修飾的方式有多種,包括甲基化、乙?；?、磷酸化和泛素化,在現(xiàn)有的研究中參與SAM調(diào)控的主要是組蛋白賴氨酸甲基化修飾(histone lysine methylation modification).組蛋白H3的N端4個(gè)賴氨酸修飾位點(diǎn)對于基因的表達(dá)至關(guān)重要,它們分別是H3K4、H3K9、H3K27和H3K36位點(diǎn),而在賴氨酸上有3個(gè)可以被甲基化修飾的化學(xué)鍵,因此又可以分為一、二和三甲基化修飾(me1,me2,me3).一般認(rèn)為H3K4和H3K36的甲基化修飾促進(jìn)基因表達(dá),而H3K9和H3K27的甲基化修飾則導(dǎo)致異染色質(zhì)化和基因沉默.賴氨酸甲基化修飾需要甲基轉(zhuǎn)移酶(lysine methyltransferase,KMT)的參與,它通常含有一個(gè)約130個(gè)氨基酸組成的SET結(jié)構(gòu)域[74](甲基轉(zhuǎn)移的催化中心).擬南芥中有47個(gè)編碼SET結(jié)構(gòu)域的蛋白,而參與對SAM維持的主要有Trithorax group(Trx G)與Polycomb group(PcG)兩類.Trx G負(fù)責(zé)H3K4me3修飾,能夠激活基因的表達(dá);而PcG的功能則與之相反,通過直接結(jié)合于DNA的可識(shí)別區(qū)域進(jìn)而誘導(dǎo)H3K27me3修飾,使基因處于沉默狀態(tài)[79].

5 STM信號(hào)途徑

在SAM的維持中存在著與WUS-CLV反饋調(diào)節(jié)通路相平行的另一條信號(hào)通路,而這條信號(hào)通路中核心成員是STM[14,82-85].STM編碼同源異型結(jié)構(gòu)域轉(zhuǎn)錄因子,屬于KNOTTED1-like homeobox(KNOX)基因家族成員[86].STM在玉米中的直系同源基因是KNOTTED1(KN1),KN1是KNOX基因家族的基礎(chǔ)成員也是在植物中發(fā)現(xiàn)的第一個(gè)能夠調(diào)控干細(xì)胞的基因[87].stm-1突變體在胚胎發(fā)育時(shí)期即沒有SAM形成,在胚胎后發(fā)育時(shí)期同樣沒有SAM并且子葉葉柄基部融合,大部分stm不會(huì)形成其他器官即老化,偶爾能夠發(fā)育的植株葉片基部也會(huì)部分融合,在花發(fā)育過程中總是以未成熟的異位器官原基融合在一起的形式終止[84].STM在胚胎發(fā)育球形期開始表達(dá),并且在胚后發(fā)育的SAM和FM中未分化的細(xì)胞中表達(dá),在分生組織鄰近的器官或者花器官原基中沒有表達(dá)[86].通過突變體表型和在生長過程中的表達(dá)變化說明STM有兩大功能:其一為防止干細(xì)胞分化,其二為促進(jìn)干細(xì)胞的子細(xì)胞在形成器官前的增殖[14,84,86,88].

STM和WUS的缺失都可以引起SAM的終止,但其機(jī)制有所不同.在stm中沒有可以識(shí)別的SAM[84],而在wus中盡管SAM終止,但其仍然存在并且變得扁平肥大,說明CZ細(xì)胞仍然存在只是沒有功能[12].隨后的研究表明STM與WUS的功能是相互獨(dú)立的.分別異位表達(dá)WUS與STM能夠誘導(dǎo)不同的下游基因表達(dá).WUS的功能主要是特化CZ的細(xì)胞成為干細(xì)胞,而STM的功能是抑制干細(xì)胞的分化和促進(jìn)干細(xì)胞子細(xì)胞的增殖[14].

像WUS一樣,STM也可以在SAM中借助于PD進(jìn)行移動(dòng),并且對于STM的可移動(dòng)性研究要比WUS更早更深入.早在1994年就發(fā)現(xiàn)KN1蛋白的表達(dá)范圍超出其m RNA的表達(dá)區(qū)域[89],隨后通過顯微注射的方式證實(shí)了KN1借助于PD移動(dòng),其可以增大PD的排阻極限(size exclusion limit,SEL),還可以介導(dǎo)KN1正義鏈m RNA借助于PD進(jìn)行移動(dòng)[90].GFPKN1可以在葉片與SAM中移動(dòng)[91],并且這種移動(dòng)有其特殊調(diào)控方式.在葉片中GFP-KN1可以從葉肉細(xì)胞向表皮細(xì)胞移動(dòng),但反方向不可以;而在SAM中, GFP-KN1與GFP-STM可以從L1向內(nèi)層移動(dòng),并且在L1特異性表達(dá)KN1可以部分恢復(fù)stm-11(強(qiáng)表型突變體)[92].KNOX家族的異型結(jié)構(gòu)域?qū)τ谄涞鞍着cmRNA的移動(dòng)是必須的[93].最近的研究發(fā)現(xiàn)KN1與STM的移動(dòng)還需要伴侶蛋白CCT8的參與(細(xì)胞質(zhì)內(nèi)二型分子伴侶復(fù)合物的亞單位),在敲除CCT8基因后表現(xiàn)出stm突變體的強(qiáng)表型.CCT8的另一個(gè)作用就是在STM移向的目的細(xì)胞中可以將STM重新折疊成有活性的蛋白[32].現(xiàn)在并不清楚STM或者KN1蛋白移動(dòng)的意義,有可能是為在SAM中形成一定的蛋白濃度梯度.

STM通過在SAM的CZ抑制器官形成基因AS1和AS2的表達(dá)進(jìn)而阻止干細(xì)胞的分化[94].AS1是金魚草(Antirrhinum majus L.)中PHAN和玉米中ROUGH SHEAT H2(RS2)的同源基因,它編碼一個(gè)具有MYB功能域的轉(zhuǎn)錄因子,而AS2編碼具有LATERAL ORGEN BOUNDARY(LOB)功能域的蛋白[95-97].在每個(gè)物種中,這些基因都參與側(cè)生器官的起始.STM及其他KNOX家族蛋白(KNAT1/BP, KNAT2和KNAT6)抑制AS1、AS2在SAM中心區(qū)域的表達(dá),而AS1、AS2反過來也會(huì)抑制K NOX基因在發(fā)育中的器官原基處的表達(dá)[95-96,98-100].STM的表達(dá)模式也反映了這種調(diào)控關(guān)系,其在SAM中的表達(dá)貫穿整個(gè)CZ,而在器官原基發(fā)育位置卻有明顯的下降[95].AS1、AS2與組蛋白分子伴侶HIRA形成轉(zhuǎn)錄抑制復(fù)合體進(jìn)而結(jié)合于KNOX家族基因的順式作用元件上來抑制K NOX的轉(zhuǎn)錄[101-102].

另一方面STM可以通過調(diào)節(jié)CK與赤霉素(gibberellin,GA)信號(hào)通路來維持SAM.CK可以通過激活細(xì)胞周期蛋白D(cyclin D)的表達(dá)進(jìn)而促進(jìn)細(xì)胞分裂[103],其本身對SAM的維持起著重要作用;而GAs包含一大類二萜類化合物,它們可以促進(jìn)器官增大與植物形態(tài)發(fā)生[104].

STM在SAM中直接激活CK合成酶基因ISOPENTENYL TRANSFERASE 7(IPT7)與CK響應(yīng)因子ARABIDOPSIS RESPONSE REGULATOR 5 (ARR5)的表達(dá),當(dāng)施用外源CK或者用STM啟動(dòng)子表達(dá)IPT7都可以部分恢復(fù)stm的表型,而在過表達(dá)STM時(shí)則可導(dǎo)致體內(nèi)CK水平上升和誘導(dǎo)異位分生組織形成[105-106].可見STM對IPT7的激活在SAM維持中起關(guān)鍵作用(圖1(b)).在過表達(dá)其他KNOX家族基因如KNAT1/BP時(shí)也會(huì)導(dǎo)致產(chǎn)生高水平的CK,這說明其他KNOX基因家族成員有同STM類似的功能[105,107].

在對GA水平的調(diào)控上,KNOX家族轉(zhuǎn)錄因子Nicotiana tabacum homeobox 15(NTH15)在CZ直接結(jié)合在GA合成酶基因GA 20-oxidase(在GA合成過程的倒數(shù)第二步發(fā)揮作用)的第一個(gè)內(nèi)含子上抑制其表達(dá)[108-109],從而阻斷了GA的生物合成,并且在表達(dá)模式上GA 20-oxidase與STM基本一致[110].然而僅通過這種方式還是不夠的,因?yàn)樵谌~原基合成的GA可以擴(kuò)散到SAM中.STM通過激活GA分解代謝基因GA 2-oxidase的表達(dá)使流入SAM的GA失活,GA 2-oxidase在SAM基部和發(fā)育中的葉原基表達(dá)除受STM調(diào)控外,CK信號(hào)對于其激活也是必要的[105].由此通過KNOX家族蛋白在SAM中建立起高水平CK活性和低水平GA活性的激素環(huán)境,這對于SAM的維持起著非常重要的作用.

6 小結(jié)與展望

近20年來對于植物SAM維持分子機(jī)制的研究取得了卓越的成績,發(fā)現(xiàn)了SAM維持中最核心的信號(hào)調(diào)節(jié)通路,并以此為基礎(chǔ)搭建了分子調(diào)控的信號(hào)網(wǎng)絡(luò).越來越多的生物學(xué)因素被發(fā)現(xiàn)參與SAM的維持,主要包括植物激素,小RNAs和表觀遺傳作用,也由此說明SAM的調(diào)控機(jī)制極其復(fù)雜.加之SAM處在一個(gè)高度聯(lián)系并且非常穩(wěn)定的調(diào)控網(wǎng)絡(luò)之中,在時(shí)間與空間上都為研究帶來諸多不便.到目前為止在WUSCLV信號(hào)途徑中還不清楚WUS決定干細(xì)胞和CLV途徑抑制WUS的具體作用機(jī)制.通過激光共聚焦技術(shù)實(shí)時(shí)觀察SAM的變化并經(jīng)過計(jì)算機(jī)建模是近幾年發(fā)展起來的解決這一科研難題的方法.

早在2007年通過在根尖中收集不同細(xì)胞類型進(jìn)行了根尖干細(xì)胞的基因表達(dá)譜分析[111-112],該方法相較于對整個(gè)根尖分生組織的轉(zhuǎn)錄本進(jìn)行檢測具有更高的靈敏度,可以檢測到一些表達(dá)豐度低或者只在根尖分生組織表達(dá)的基因.2009年Yadav等[56]對SAM也開展了同樣的工作,并且在2014年又對這種不同細(xì)胞類型的取樣進(jìn)行了優(yōu)化,細(xì)胞分類更加細(xì)化[113].通過表達(dá)譜分析發(fā)現(xiàn)有大量在SAM特異性表達(dá)的基因,然而這些基因的功能現(xiàn)在尚不清楚;另外受WUS激活與抑制又或者可以直接結(jié)合的基因也有很多,它們是否參與對SAM的維持作用也不清楚.因此SAM的分子機(jī)制研究工作任重而道遠(yuǎn).

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Research Progresses of the Regulatory Mechanism of the Shoot Apical Stem Cell in Arabidopsis

CUI Yuchao,CHEN Liang*

(Xiamen Key Laboratory for Plant Genetics,School of Life Sciences,Xiamen University,Xiamen 361102,China)

The shoot apical stem cell is a pool of pluripotent cell in the apex of the plant,which is the source of the whole parts above the ground.The stem cell divides to produce two daughter cells.One can renew itself,and the other can form the organ promordia for proliferation.At last the stem cell stablizes in homeostasis,which can maintain plant development even up to a thousand years. Modern molecular biology and genetics have indicated that the homeostasis is regulated exactly by various factors from stem cell niche.The key pathway among the network is the negative feedback loop between the WUSCHEL(WUS)gene and the CLAVATA (CLV)gene.Other biological factors,such as cytokinin,the moved small RNAs and the epigenetics effects,play a role on this loop eventually.Besides,the SHOOTMERISTEMLESS(STM)signaling pathway,which is parallel to the WUS-CLV loop,plays a positive role in stem cell maintenance.This article mainly reviewed the latest research progresses on the molecular mechanism of stem cell maintenance in model plant Arabidopsis.

stem cell maintenance;WUS-CLV;cytokinin;small RNAs;epigenetics;STM

Q 356.1

A

0438-0479(2016)06-0781-12

10.6043/j.issn.0438-0479.201604104

2016-04-13 錄用日期:2016-06-20

國家重點(diǎn)研發(fā)計(jì)劃項(xiàng)目(2016YFD0100903);國家自然科學(xué)基金(31560297)

chenlg@xmu.edu.cn

崔玉超,陳亮.擬南芥莖尖干細(xì)胞調(diào)控機(jī)制的研究進(jìn)展[J].廈門大學(xué)學(xué)報(bào)(自然科學(xué)版),2016,55(6):781-792.

CUI Y C,CHEN L.Research progresses of the regulatory mechanism of the shoot apical stem cell in Arabidopsis[J]. Journal of Xiamen University(Natural Science),2016,55(6):781-792.(in Chinese)