高 麗,楊海莉,王 沛,2,王鎖民
(1.蘭州大學(xué)草地農(nóng)業(yè)生態(tài)系統(tǒng)國家重點實驗室;蘭州大學(xué)草地農(nóng)業(yè)科技學(xué)院,甘肅 蘭州 730020;2.西南民族大學(xué)青藏高原研究院,四川 成都 610041)
植物在長期的進(jìn)化過程中逐漸形成了一類特殊的以脂類-酚類為基礎(chǔ)的疏水性屏障以適應(yīng)復(fù)雜的、動態(tài)的、多變的土壤及外界環(huán)境。其中包括地上部的角質(zhì)層及廣泛分布于根中的木栓質(zhì),木栓質(zhì)在根組織沉積形成的疏水性屏障不僅在控制水分和營養(yǎng)元素的運輸以及限制病原菌和毒氣入侵等方面發(fā)揮重要作用,同時在植物響應(yīng)非生物脅迫中也發(fā)揮關(guān)鍵作用。因此,近年來有關(guān)木栓質(zhì)的研究受到越來越廣泛的關(guān)注,成為植物質(zhì)外體屏障研究中的熱點。本文概述了木栓質(zhì)的結(jié)構(gòu)、化學(xué)組成、單體合成,重點綜述了木栓質(zhì)單體的合成以及近年來在其生理功能方面的研究進(jìn)展,并對目前木栓質(zhì)研究中存在的問題及未來的研究方向進(jìn)行了簡要討論和展望。
木栓質(zhì)主要分布于植物根內(nèi)皮層、外皮層,其中內(nèi)皮層木栓質(zhì)的沉積在所有高等植物中廣泛存在,且形成較早,可以隔離皮層和中柱組織,是目前研究的重點[1]。
木栓質(zhì)也廣泛存在于周皮組織,如歐洲栓皮櫟(Quercussuber)和馬鈴薯(Solanumtuberosum)塊莖的周皮含有大量的木栓質(zhì)[2-4]。此外在傷口愈合的過程中,木栓質(zhì)會在傷口邊緣處沉積以保護(hù)健康組織[1]。這些木栓化的周皮沉積于植物-環(huán)境界面,起到保護(hù)植物內(nèi)部組織的作用。
木栓質(zhì)的沉積同樣存在于組織-組織界面,使植物內(nèi)部各組織間相互隔離。例如,在柑橘屬植物的種皮的合點及珠孔區(qū)域中也發(fā)現(xiàn)有木栓質(zhì)的分布,將種子封閉起來[1]。在C4植物的維管束鞘細(xì)胞中,木栓質(zhì)也有分布,它的作用是隔離葉肉細(xì)胞與維管束鞘細(xì)胞[1]。木栓質(zhì)不僅沉積于植物體正常生長過程中的上述特定組織中,而且能響應(yīng)外界環(huán)境刺激在非木栓組織中合成[5-6]。由此可見,在任何時間,植物需要形成屏障的任何部位都可能會存在木栓質(zhì)的沉積[1,7]。
木栓質(zhì)是由甘油、脂肪酸與酚類化合物形成的高分子雜聚物[7-10]。脂肪族部分主要包括ω-羥基脂肪酸、α,ω-雙羧基脂肪酸(簡稱α,ω-二酸)、中鏈含氧脂肪酸、未被取代的脂肪酸以及伯醇[1,10]。酚類組分主要由羥基化的肉桂酸組成,通常為阿魏酸、香豆酸和單木質(zhì)醇[1]。
木栓質(zhì)單體的碳鏈長度多為C16~C26,相比角質(zhì)C16~C18更長,暗示著其疏水性可能更強(qiáng)[11-13]。木栓質(zhì)總量和單體的相對含量在植物的不同發(fā)育階段、不同組織以及不同物種間存在很大差異[14-15]。例如,擬南芥(Arabidopsisthaliana)根中的木栓質(zhì)主要為C16,C18:1和C22的單體[11],而在其種皮中則是C22和C24的單體[12]。
通過對木栓化的細(xì)胞壁切片的觀察發(fā)現(xiàn)其呈現(xiàn)明(電子半透明)暗(電子不透明)相間的薄層狀超微結(jié)構(gòu)。但關(guān)于這一明暗相間的條紋結(jié)構(gòu)中電子半透明區(qū)域(明帶)以及電子不透明區(qū)域(暗帶)的化學(xué)組成仍存在爭議。Bernards[9]以馬鈴薯周皮作為研究材料,認(rèn)為組成木栓質(zhì)的聚脂肪族域和聚芳香族域各自處在其特定的空間并以共價鍵彼此連接,且由聚脂肪族域形成了電子半透明的亮帶,由酚類物質(zhì)形成電子不透明的暗帶。但Molina等[16]的研究發(fā)現(xiàn),擬南芥ASFT/HHT敲除突變體中完全缺失酯結(jié)合態(tài)阿魏酸,但根中木栓層結(jié)構(gòu)卻沒有受到影響,這否定了Bernards的觀點。Soliday等[17]提出木栓層結(jié)構(gòu)中的亮帶是由蠟質(zhì)組成的蠟層。然而,擬南芥far1-far4-far5三突變體中,木栓質(zhì)相關(guān)的蠟質(zhì)顯著減少,木栓層的結(jié)構(gòu)同樣沒有受到影響[18]。Serra等[19]在馬鈴薯中沉默細(xì)胞色素P450脂肪酸ω-羥化酶的編碼基因CYP86A33,導(dǎo)致馬鈴薯塊莖周皮中C18:1的ω-羥基酸和α,ω-二酸單體的含量分別下降了70%和90%,且RNAi株系中木栓層的結(jié)構(gòu)扭曲變形,明暗相間的條帶消失。因此,木栓層的這一明暗相間的薄層結(jié)構(gòu)很可能與脂肪酸ω位點的羥基化有關(guān)。
木栓質(zhì)的沉積首先需要合成脂肪族、酚類以及甘油單體,然后運輸?shù)郊?xì)胞壁形成一個難溶的大分子。隨著木栓質(zhì)成分定量分析手段以及正向和反向遺傳學(xué)的發(fā)展,參與編碼木栓質(zhì)單體合成酶的基因陸續(xù)被挖掘出來。目前已知的參與木栓質(zhì)單體合成反應(yīng)的酶包括β-酮脂酰-輔酶A合成酶(KCS)、細(xì)胞色素P450單加氧酶(CYP)家族、脂肪?;€原酶(FAR)、3磷酸甘油酰基轉(zhuǎn)移酶(GPAT)家族以及羥基肉桂輔酶A轉(zhuǎn)移酶(ASFT)(表1、2)。
木栓質(zhì)單體合成途徑中涉及到的反應(yīng)主要包括脂肪酸活化為脂肪酰輔酶A硫酯、長鏈脂肪酸前體的延伸、脂肪酸的ω-羥基化以及后續(xù)的ω-羥基酸到α,ω-雙羧基酸的氧化以及脂肪酰鏈還原為脂肪醇等[7,9-10,15]。
表1 擬南芥中參與木栓質(zhì)單體生物合成途徑相關(guān)基因Table 1 Genes involved in suberin biosynthetic pathway in Arabidopsis thaliana
續(xù)表1
基因名稱GenenameAGI號AGInumber編碼蛋白Encodedprotein超表達(dá)或突變體表型Phenotypeofmutantoroverexpression參考文獻(xiàn)ReferenceGPAT7At5g06090甘油-3磷酸酰基轉(zhuǎn)移酶AcylCoA:glyc-erol-3-Phosphateacyltransferas超表達(dá)株系單酰甘油,種子和地上部角質(zhì)層蠟質(zhì)中C22:0和C24:0自由脂肪酸含量增加Overexpressiongivesrisetomonoacylglycerols,C22:0,C24:0freefattyacidsinseedsandaerialcuticularwaxes[29]ASFT/HHTAt5g41040阿魏酰-CoA轉(zhuǎn)移酶Feruloyl-CoAtransferase突變體種子和根中阿魏酸含量減少,ω-OHAs含量減少,α,ω-DCAs含量增加,改變了種子和根對鹽的透性和敏感度Mutantreducedspecificallyferulateinseedsandroots,reductioninω-hydroxyacids,increaseinα,ω-DCAs,al-teredthepermeabilityandsensitivityofseedsandrootstosaltstress[16,30]FACTAt5g63560脂肪醇:咖啡酰-CoA咖啡酰基轉(zhuǎn)移酶FattyAlcohol:Caffeoyl-CoACaffeoylTransferase根中木栓質(zhì)相關(guān)蠟質(zhì)幾乎缺失18:0-22:0的烷基咖啡酸Nearcompletelackof18:0-22:0alkylcaffeatesinrootwaxes[31]ABCG2ABCG6ABCG20At2g37360At5g13580At3g53510ATP-結(jié)合盒(ABC轉(zhuǎn)運蛋白)ATP-bindingcassette(ABC-transporter)abcg2abcg6abcg20三突變體種皮中木栓質(zhì)的裝載減少,根中木栓質(zhì)裝載增加,種皮和根透性也增加Thesuberinloadinseedcoatsoftripleabcg2abcg6ab-cg20mutantsreduced,butsuberinloadinrootsin-creased.seedcoatsandrootpermeabilityalsoincreased[32]MYB41At5g63560MYB-型轉(zhuǎn)錄因子MYB-typetranscriptionfactor超表達(dá)株系中木栓質(zhì)合成基因上調(diào)且在葉片形成木栓層類似結(jié)構(gòu)OverexpressionofMYB41resultedinupregulationofsuberinbiosyntheticgenesandtheformationofsuberin-likelamellaeinleaves[33]MYB107At3g02940MYB-型轉(zhuǎn)錄因子MYB-typetranscriptionfactor突變體種皮中C24:0ω-羥基酸和α,ω-雙羧基酸含量下降了50%,種皮透性增加Mutanthas50%reductioninω-hydroxyacidsandα,ω-DCAsinseedscoats,theseedcoatspermeabilityin-creased[34]ESB1At2g28670結(jié)構(gòu)域蛋白質(zhì)Dirigent-domainContai-ningprotein突變體根中凱氏帶缺失伴隨總木栓質(zhì)含量翻倍DefectiveCasparianstripswithtwofoldincreaseinallsuberinmonmersinroots[30-31]
通常而言,脂肪酸代謝反應(yīng)的第一步是自由脂肪酸的活化反應(yīng)。擬南芥中目前發(fā)現(xiàn)存在9個編碼長鏈?;?COA合成酶(LACS)的基因參與脂肪酸的激活反應(yīng)[40]。其中,LACS2參與角質(zhì)和角質(zhì)層蠟質(zhì)的生物合成[41]。但LACS基因在木栓質(zhì)合成過程中的作用尚未見報道,然而,在LACS2基因缺失突變體中的化學(xué)分析表明LACS2也參與木栓質(zhì)的形成[13]。MYB107參與調(diào)控擬南芥種皮中木栓質(zhì)的沉積,在myb107突變體中LACS的表達(dá)同樣出現(xiàn)了下調(diào)的現(xiàn)象[34],這也支持了LACS2參與木栓質(zhì)的沉積。此外,LACS酶也可能參與ω-羥基酸和α,ω-二酸在酯化到甘油分子上之前的激活。
脂肪酸被激活后形成的脂肪酰-COA的延長則是由定位于內(nèi)質(zhì)網(wǎng)上的脂肪酸延長酶復(fù)合體參與催化的[42]。β-酮脂酰-COA合酶(KCS)是脂肪酸延長酶復(fù)合體中的第1個酶,參與控制長鏈脂肪酰-CoA伸長的程度,同時也是這一過程中的限速酶[43]。擬南芥中有21個KCS基因,其中β-酮脂酰-COA合酶基因DAISY/AtKCS2和AtKCS20參與C20酰基鏈的木栓質(zhì)前體的延伸過程[20-21]。KCS2突變雖然并未引起根中木栓質(zhì)總量的變化,但卻導(dǎo)致C22和C24極長鏈脂肪酸衍生物減少。然而kcs2kcs20雙突變體中脂肪族木栓質(zhì)的含量相比于任一單突變體都受到了更顯著的影響,表明這兩種酶存在部分功能冗余[21]。
表2 馬鈴薯中參與木栓質(zhì)單體生物合成途徑相關(guān)基因Table 2 Genes involved in suberin biosynthetic pathway in Solanum tuberosum
NADPH依賴的細(xì)胞色素P450單加氧酶家族中的CYP86亞家族主要參與催化脂肪酰-COA ω-位點的羥基化,形成ω-羥基酸,其中一部分 ω-羥基酸又進(jìn)一步被氧化成α,ω-雙羧基酸[44-46]。Benveniste等[47]首次從擬南芥中克隆到CYP86A1基因,并在酵母中異源表達(dá),發(fā)現(xiàn)該基因參與短鏈脂肪酸的羥基化過程。擬南芥cyp86a1突變體植株根系C16和C18 ω-羥基酸和α,ω-二酸木栓質(zhì)單體含量顯著下降,且最終總脂肪族木栓質(zhì)單體含量下降了60%。同時通過RT-PCR、GUS染色及GFP定位等方法將CYP86A1定位于根內(nèi)皮層細(xì)胞的內(nèi)質(zhì)網(wǎng)上,這暗示木栓質(zhì)單體的合成是在內(nèi)質(zhì)網(wǎng)上進(jìn)行的[22]。且馬鈴薯中CYP86A1的同源蛋白StCYP86A33在馬鈴薯塊莖周皮木栓質(zhì)單體的ω-羥基化過程中同樣發(fā)揮關(guān)鍵作用[19]。CYP86B1與CYP86A1同屬一個亞家族,參與極長鏈(C22-C24)ω-羥基酸以及α,ω-雙羧基酸的形成[16,24]。CYP86B1敲除株系的根及種皮中C22和C24 ω-羥基酸和α,ω-二酸幾乎完全缺失,但脂肪族木栓質(zhì)單體C22和C24脂肪酸積累增加[16]。
脂肪酰-COA還原成伯醇的過程則是由脂肪酰還原酶(FARs)介導(dǎo)的。在擬南芥中,F(xiàn)AR家族有8個成員,各自的功能均已被鑒定[48]。其中FAR1、FAR4和FAR5這3個基因參與木栓質(zhì)相關(guān)的脂肪醇的合成[18,25]。這3個基因的T-DNA插入單突變體分別表現(xiàn)出不同鏈長伯醇的減少,其中far1突變體的根和種子中C22:0伯醇含量顯著下降,far4突變體的根和種子中C20:0伯醇含量下降,而far5突變體的根和種子中C18:0伯醇的含量有所下降[25]。因此,F(xiàn)AR1和FAR4可能參與種皮木栓質(zhì)中α,ω-二醇的合成,然而它們是否以ω-羥基酰鏈為底物仍需進(jìn)一步驗證[18]。在far1-far2-far3的三突變體中,木栓質(zhì)中總脂肪醇的含量降低了70%~80%,而其他主要單體含量沒有顯著變化,表明木栓質(zhì)的聚合過程并沒有改變,但三突變體的種皮透性卻有所增加。
?;D(zhuǎn)移反應(yīng)則是由?;?COA-依賴的甘油-3-磷酸?;D(zhuǎn)移酶(GPATs)催化生成甘油-酸酯完成的,甘油-酸酯被認(rèn)為是木栓質(zhì)大分子的基石。GPATs催化脂肪酰-COA或酰基-?;d體蛋白向甘油-3-磷酸的sn-1或sn-2位點的轉(zhuǎn)移[27]。通過這一過程,甘油被共價結(jié)合到木栓質(zhì)的脂肪族與芳香族部分[4]。目前發(fā)現(xiàn)擬南芥中至少存在20個可能的?;D(zhuǎn)移酶[13]。通過功能獲得與缺失的方法證實其中的8個GPAT基因參與角質(zhì)與木栓質(zhì)的生物合成。Yang等[28-29]的研究表明GPAT4、6和8對C16:0和C18:1 ω-位點碳氧化的酰基-COAs具有更高的親和力。而極長鏈(C20-C24)脂肪酰則作為GPAT5的底物[26]。在擬南芥gpat5突變體根和種子中C20-C24未被取代的脂肪酸、ω-羥基酸和α,ω-雙羧基酸的含量顯著減少,木栓質(zhì)的總量減少為對照的一半[26]。GPAT7基因可被創(chuàng)傷誘導(dǎo),GPAT7在葉中超表達(dá)會積累木栓質(zhì)類單體,表明其可能在創(chuàng)傷誘導(dǎo)的木栓質(zhì)單體合成中發(fā)揮作用[29]。
擬南芥和馬鈴薯中編碼阿魏酰-CoA轉(zhuǎn)移酶的基因相繼被克隆[30,36-37]。擬南芥中脂肪族木栓質(zhì)阿魏酰轉(zhuǎn)移酶(ASFT/HHT)以及馬鈴薯中的同源蛋白ω-羥基脂肪酸/脂肪醇羥基肉桂酰轉(zhuǎn)移酶(FHT)屬于BAHD?;D(zhuǎn)移酶家族,均催化阿魏酰CoA的?;颚?羥基酸和脂肪醇的轉(zhuǎn)移[28,36-37]。擬南芥asft/hht突變體根中木栓質(zhì)完全缺失阿魏酸鹽,且ω-羥基酸和α,ω-雙羧基酸的含量顯著下降[16,30]。于此類似的是,F(xiàn)HT-RNAi沉默的馬鈴薯塊莖周皮中酯結(jié)合阿魏酸大量減少,但木栓層的結(jié)構(gòu)并沒有發(fā)生變化[36]。
盡管許多植物中木栓質(zhì)生物聚酯的許多單體成分已知,但木栓質(zhì)單體合成的反應(yīng)順序依然不清楚,參與木栓質(zhì)單體與角質(zhì)單體合成反應(yīng)的酶的區(qū)別以及它們間是否存在互作等都需要進(jìn)一步研究。
各種木栓質(zhì)前體,無論是以單體還是部分形成低聚物的形式,最終都要跨膜運輸?shù)劫|(zhì)外體,然后在質(zhì)外體聚合形成木栓質(zhì)屏障。雖然木栓質(zhì)單體的合成途徑已被廣泛認(rèn)知,然而有關(guān)木栓質(zhì)單體跨膜轉(zhuǎn)運機(jī)制的研究才剛剛開始。Mcfarlane等[49]的研究發(fā)現(xiàn),高爾基體與反式高爾基體網(wǎng)絡(luò)介導(dǎo)的囊泡運輸參與表皮蠟質(zhì)向質(zhì)外體的轉(zhuǎn)運。因此也很可能參與木栓質(zhì)單體的轉(zhuǎn)運。此外,參與角質(zhì)和蠟質(zhì)向細(xì)胞壁跨膜轉(zhuǎn)運的ATP-結(jié)合盒(ABC)轉(zhuǎn)運蛋白和脂質(zhì)轉(zhuǎn)移蛋白(LTPs)也是木栓質(zhì)跨膜轉(zhuǎn)運的候選蛋白。
定位于質(zhì)膜上的ABC轉(zhuǎn)運蛋白中的G亞家族參與表皮蠟質(zhì)和孢粉素前體的轉(zhuǎn)運[50-51],因此也可能參與木栓質(zhì)的轉(zhuǎn)運。編碼ABCG亞家族中的一個子家族WBC(white-brown complex)型蛋白的基因在歐洲栓皮櫟的木栓組織中高豐度表達(dá)[52]。近年來發(fā)現(xiàn)水稻(Oryzasativa)的RCN1/OsABCG5參與水稻根周皮的木栓化[53],且Landgraf等[38]的研究表明ABCG1也參與馬鈴薯塊莖周皮木栓質(zhì)的形成。擬南芥abcg2-abcg6-abcg20的三突變體表現(xiàn)出根和種皮木栓質(zhì)結(jié)構(gòu)、組分和特性上的改變,但3個單突變體均沒有明顯的表型,表明這3個ABC轉(zhuǎn)運蛋白在木栓質(zhì)前體的跨膜運輸過程中存在功能冗余[32]。
糖基化磷脂酰肌醇(GPI)錨定的LTPs和III型LTPs分別參與表皮脂類的沉積和花粉外壁的形成[54-55]。按照分別參與角質(zhì)、木栓質(zhì)和孢粉素的合成或沉積將LTPGs分為3類[55]。然而,在擬南芥ltpg突變體中進(jìn)行深入研究卻發(fā)現(xiàn)一個對種皮透性和木栓質(zhì)組分影響極大的成員卻并不屬于木栓質(zhì)類[56-57]。最新研究表明,非特異性脂類轉(zhuǎn)移蛋白AtLtpI-4在擬南芥冠癭瘤(Crown gall)中木栓質(zhì)的形成中是必需的,其突變導(dǎo)致C18:0的木栓質(zhì)組分含量大幅下降[58]。
這些結(jié)果表明ABCG轉(zhuǎn)運蛋白和LTPs脂質(zhì)轉(zhuǎn)移蛋白參與木栓質(zhì)組分的跨膜轉(zhuǎn)運,但仍需進(jìn)一步挖掘參與木栓質(zhì)跨膜轉(zhuǎn)運的相關(guān)蛋白,進(jìn)一步闡明木栓質(zhì)單體的跨膜運輸機(jī)制。
目前對于木栓質(zhì)單體或低聚物在跨膜轉(zhuǎn)運之后在細(xì)胞壁的聚合組裝機(jī)制的理解還很有限。近年來,隨著番茄(Solanumlycopersicum)中第1個角質(zhì)合成酶CD1(cutin deficient 1)的鑒定,植物表面脂質(zhì)的組裝機(jī)制逐漸被闡明[59]。CD1定位于細(xì)胞外,屬于GDSL-motif脂酶/水解酶家族,體外實驗證實其催化sn-2-單酰甘油前體的酯基轉(zhuǎn)移作用[59]。在擬南芥中克隆得到CD1的同源基因并在體外對其功能進(jìn)行了鑒定,但是其聚合產(chǎn)物是單一的線性,表明還有其他蛋白酶參與網(wǎng)狀聚酯的最終形成[60]。但CD1是否同樣參與木栓質(zhì)在細(xì)胞壁的聚合還是未知的。一種α,β水解酶折疊蛋白BODYGUARD(BDG)被證實參與角質(zhì)的交聯(lián)過程[61]。擬南芥bdg突變體葉片中角質(zhì)含量大幅度下降,尤其是C18多元未飽和的角質(zhì)單體的含量顯著下降,且根中總木栓質(zhì)含量也有明顯的下降,GUS染色的結(jié)果也顯示BDG在根系內(nèi)皮層有定位,表明BDG還參與擬南芥根系木栓質(zhì)的聚合[61]。
木栓化的細(xì)胞壁中酚類組分的聚合被推測是由一個過氧化物酶/H2O2介導(dǎo)的過程[62]。一類陰離子過氧化物同工酶參與馬鈴薯塊莖創(chuàng)傷誘導(dǎo)的木栓化過程中酚類物質(zhì)的聚合,該酶優(yōu)先選擇阿魏酰(O-甲氧基苯酚)取代的底物,在馬鈴薯塊莖的創(chuàng)傷愈合過程中積累相應(yīng)的產(chǎn)物[63]。近年來一種NADPH依賴型的氧化酶類和過氧化物酶類被發(fā)現(xiàn)參與內(nèi)皮層凱氏帶的木質(zhì)化[64],也可能參與木栓質(zhì)的聚合組裝。
盡管木栓質(zhì)單體的跨膜轉(zhuǎn)運及組裝過程可借鑒角質(zhì)單體的,但由于兩種單體的化學(xué)組成及各自的分布部位存在差異,其跨膜轉(zhuǎn)運及組裝聚合過程可能也會存在一定差異。
木栓質(zhì)沉積于特殊的細(xì)胞類型,且被各種非生物和生物脅迫所誘導(dǎo)。木栓質(zhì)生物合成相關(guān)基因的表達(dá)模式與木栓質(zhì)的沉積位點和外界條件等密切相關(guān),木栓質(zhì)的生物合成在轉(zhuǎn)錄水平上受到嚴(yán)格的調(diào)控。隨著轉(zhuǎn)錄組學(xué)及蛋白組學(xué)的發(fā)展,參與木栓質(zhì)合成調(diào)控的因子也陸續(xù)被發(fā)現(xiàn)。最早被發(fā)現(xiàn)的參與木栓質(zhì)合成調(diào)控的轉(zhuǎn)錄因子是擬南芥AtMYB41,在擬南芥葉片持續(xù)超表達(dá)AtMYB41以及在煙草(Nicotianabenthamiana)葉片中瞬時表達(dá)AtMYB41均可誘導(dǎo)木栓質(zhì)在其葉片表皮以及葉肉細(xì)胞的細(xì)胞壁上沉積形成木栓層類似結(jié)構(gòu);同時,在超表達(dá)AtMYB41的轉(zhuǎn)基因株系中參與木栓質(zhì)合成基因的表達(dá)水平也大幅增加,此外,還發(fā)現(xiàn)AtMYB41基因在擬南芥正常發(fā)育的根中不表達(dá),但是在內(nèi)皮層響應(yīng)非生物脅迫時被特異誘導(dǎo),這表明MYB41僅調(diào)控脅迫誘導(dǎo)下的木栓質(zhì)的沉積。目前還有待于進(jìn)一步挖掘其他非脅迫誘導(dǎo)類木栓質(zhì)合成的調(diào)控因子。
蘋果(Malus×domestica)MdMYB93被發(fā)現(xiàn)參與調(diào)節(jié)黃褐色蘋果果皮中的木栓質(zhì)沉積[65]。在擬南芥、蘋果和番茄中另一項最新研究發(fā)現(xiàn),與MYB93親緣關(guān)系十分相近的兩個MYB類轉(zhuǎn)錄因子MYB9和MYB107也參與調(diào)控木栓質(zhì)在種皮和果皮中的沉積[66]。
Gou等[34]的研究進(jìn)一步證實了MYB107參與調(diào)控擬南芥種皮中木栓質(zhì)的合成。MYB107主要在長角果中表達(dá),MYB107突變導(dǎo)致種皮中聚脂肪族和聚芳香族成分均大幅度下降,導(dǎo)致種皮透性增加,木栓層結(jié)構(gòu)改變,同時,myb107突變體中參與木栓質(zhì)合成的關(guān)鍵基因FACT、CYP86A1、CYP86B1、FAR1等的表達(dá)均有所下調(diào)。但myb107突變體根系木栓質(zhì)含量以及地上部角質(zhì)的含量與野生型相比沒有明顯差異,這表明MYB107僅參與正向調(diào)控種皮中木栓質(zhì)的合成及組裝[34]。
最近,Verdaguer等[39]在馬鈴薯塊莖周皮的研究中發(fā)現(xiàn)首個負(fù)調(diào)控周皮中木栓質(zhì)及相關(guān)蠟質(zhì)生物合成的轉(zhuǎn)錄因子NAC103,StNAC103基因沉默的馬鈴薯塊莖周皮中木栓質(zhì)及相關(guān)蠟質(zhì)的裝載增加,特別是烷烴類、羥基脂肪酸、二酸、阿魏酸以及伯醇等。此外,在沉默株系中與周皮木栓質(zhì)合成及轉(zhuǎn)運有關(guān)的關(guān)鍵基因的表達(dá)均有所上調(diào)。
6.1.1木栓質(zhì)與鹽脅迫 高鹽脅迫導(dǎo)致擬南芥gpat5突變體種子的發(fā)芽率降低,且幼苗對鹽脅迫的耐受性下降[26]。木栓質(zhì)合成中編碼β-酮脂酰-COA合酶的關(guān)鍵酶DAISY/KCS2基因的表達(dá)在NaCl和滲透脅迫處理后顯著上調(diào)[22];參與木栓質(zhì)單體中脂肪醇合成的FAR1、FAR4和FAR5基因的表達(dá)也都受NaCl顯著誘導(dǎo)[29]。Krishnamurthy等[67-68]分析發(fā)現(xiàn),相比于鹽敏感品種IR20和中等耐鹽品種Jaya,強(qiáng)耐鹽型品種Pokkali的根系木栓化程度最高且地上部Na+積累最少,這3種水稻品種根系木栓化均會受鹽脅迫誘導(dǎo)加強(qiáng),且木栓質(zhì)合成基因的轉(zhuǎn)錄水平也受鹽脅迫的誘導(dǎo)。擬南芥木栓質(zhì)合成中另外一個關(guān)鍵基因CYP86A1突變之后,植株對水分和NaCl的透性都顯著增加[69];在對CYP86A在植物響應(yīng)鹽脅迫中作用的分析表明,適量NaCl誘導(dǎo)了擬南芥根的木栓化以及根中CYP86A1基因的高豐度表達(dá)[23]。鹽脅迫下,擬南芥cyp86a1突變體植株地上部生長受到顯著抑制,且地上部積累更多的Na+,而K+含量則相應(yīng)的減少,表明CYP86A1通過調(diào)控木栓質(zhì)的合成,進(jìn)而調(diào)控水分和離子轉(zhuǎn)運,在植物響應(yīng)鹽脅迫中發(fā)揮關(guān)鍵作用,同時為進(jìn)一步探究鹽生植物小花堿茅根系質(zhì)外體屏障在其適應(yīng)鹽漬土壤中的作用奠定基礎(chǔ)[23]。
這些研究均表明植物根系木栓質(zhì)受NaCl誘導(dǎo)增加,其作為質(zhì)外體屏障在植物響應(yīng)鹽脅迫過程中發(fā)揮重要作用。
6.1.2木栓質(zhì)與干旱脅迫 干旱脅迫下,木栓化的外皮層可以防止水分流失以及將溶質(zhì)留在根圍以維持根系滲透調(diào)節(jié)能力[70]。而長期干旱脅迫導(dǎo)致根系表皮和皮層細(xì)胞相繼死亡,此時,內(nèi)皮層作為根系最外層發(fā)揮功能[71-72]。干旱條件下,內(nèi)皮層木栓化程度的增加,可以保護(hù)內(nèi)部中柱鞘和維管組織免受干旱脅迫,保證其與地上部間持續(xù)的維管連接,幫助植株度過干旱期,一旦環(huán)境條件有所改善,中柱鞘/內(nèi)皮層能夠再生側(cè)根來恢復(fù)生長[6,73]。
擬南芥突變體esb1(enhancedsuberin1)植株根系中的木栓質(zhì)含量增加、日蒸騰速率下降,在營養(yǎng)生長階段其水分利用效率增加,在干旱脅迫下相比于野生型表現(xiàn)出更耐萎焉的特性,且這種木栓質(zhì)和水分運輸?shù)母淖兣c地上部Ca、Mn和Zn積累減少以及Na、S、K、As、Se和Mo等積累增加有關(guān)[74]。但esb1突變體同樣表現(xiàn)出凱氏帶形成缺陷[75],因此這些離子組模式的變化也可能與凱氏帶的缺陷有關(guān)。最近,Li等[76]通過對擬南芥低鈣敏感的lcs2-1(lowcalciumsensitive2-1)突變體和esb1突變體的研究,揭示了這兩種突變體的地上部鈣濃度的降低是由內(nèi)皮層木栓質(zhì)積累增加而非凱氏帶缺陷引起。lcs2-1突變體的根中凱氏帶缺陷,內(nèi)皮層木栓質(zhì)積累增加,無論在正常條件還是低鈣條件下,lcs2-1突變體葉片鈣濃度均下降了30%,隨后分別在lcs2-1和esb1突變體中轉(zhuǎn)入木栓質(zhì)降解酶基因CDEF1后,轉(zhuǎn)基因株系地上部鈣濃度增加,表明根系內(nèi)皮層木栓質(zhì)在離子轉(zhuǎn)運中發(fā)揮重要的作用,同時還發(fā)現(xiàn)木栓質(zhì)與側(cè)根的形成密切相關(guān)[76]。
兩個耐旱程度不同的橄欖樹(Oleaeuropaea)在持續(xù)干旱脅迫后,根系木栓化的細(xì)胞層均由內(nèi)皮層向皮層延伸,且伴隨根系水力學(xué)導(dǎo)度和氣孔導(dǎo)度下降[77]。根據(jù)橄欖樹根的形態(tài)和顏色的不同將根分為棕色根(木栓化程度高)和白色根(木栓化程度低)兩種,結(jié)果表明,兩個品種中棕色根較白色根而言,其導(dǎo)水率的下降更為明顯,氣孔導(dǎo)度呈現(xiàn)相同的下降趨勢[77]。
關(guān)于木栓質(zhì)在植物響應(yīng)干旱脅迫的作用研究多集中于其木栓質(zhì)含量的改變對植物根系透水性的改變,對于直接通過木栓質(zhì)合成關(guān)鍵基因的功能獲得或缺失直接影響植物對干旱脅迫的生理響應(yīng)的研究還相對較少。
6.1.3木栓質(zhì)與水淹脅迫 木栓質(zhì)沉積在植物響應(yīng)水淹脅迫中同樣發(fā)揮關(guān)鍵作用。植物處在水淹條件下首先會造成缺氧,有氧呼吸受到影響,進(jìn)而導(dǎo)致代謝能量不足,長期處于水淹脅迫下,植物體內(nèi)長期供能不足,且水淹土壤中有毒物質(zhì)積累導(dǎo)致植物根系死亡。木栓質(zhì)在植物根系的沉積能有效阻止水淹脅迫下植物根系徑向氧損失,且能防止?jié)碀n土壤中有毒物質(zhì)及微生物毒素進(jìn)入根系[15,78-80]。
關(guān)于木栓質(zhì)在植物適應(yīng)水淹條件下的作用研究多集中于濕地植物上,解剖學(xué)觀察、質(zhì)外體示蹤以及氧氣微電極分析表明水稻和蘆葦(Phragmitescommunis)中徑向氧損失的屏障是由木栓質(zhì)在其根系外皮層沉積形成的[81-82]。此外,De Simone等[83]對4種亞馬遜樹的下皮(外皮層)細(xì)胞壁的氧氣運輸特性及質(zhì)外體屏障進(jìn)行研究,對下皮細(xì)胞壁分離并對其進(jìn)行化學(xué)分析,并對徑向氧損失進(jìn)行測定,結(jié)果證實了木栓質(zhì)作為一種關(guān)鍵的滲透屏障在根和根際間氣體交換過程中發(fā)揮關(guān)鍵作用。水淹脅迫也會誘導(dǎo)大麥(Hordeumvulgare)根系木栓質(zhì)沉積形成徑向氧損失屏障[84-85]。
水稻RCN1/OsABCG5參與水稻根系木栓質(zhì)的沉積,水淹條件下(缺氧條件),水稻野生型根系外皮層細(xì)胞壁木栓化程度明顯增加以防止徑向氧損失[53]。RCN1/OsABCG5突變導(dǎo)致水稻根系尤其是C28-C30的脂肪酸和ω-羥基脂肪酸含量顯著下降,rcn1突變體根系外皮層木栓化缺失,導(dǎo)致其耐澇性減弱[53],且在水稻徑向氧損失屏障形成過程中,參與木栓質(zhì)合成的基因中有20%的基因表達(dá)上調(diào)[86]。與在水稻中的研究結(jié)果類似,在歐洲櫟根系徑向氧損失屏障的形成時,木栓質(zhì)合成關(guān)鍵基因CYP、KCS、GPAT等的表達(dá)也均有所上調(diào)[87]。
6.1.4木栓質(zhì)與營養(yǎng)脅迫 內(nèi)皮層的木栓化不只受到干旱和鹽脅迫的誘導(dǎo),Barberon等[88]的研究發(fā)現(xiàn)植物根系木栓化還響應(yīng)一系列由脫落酸(abscisic acid, ABA)和乙烯介導(dǎo)的營養(yǎng)脅迫。分別在Fe、Mn、Zn缺失培養(yǎng)基上生長的擬南芥幼苗根系內(nèi)皮層木栓化均有所延遲,而缺K和缺S則使擬南芥幼苗根系內(nèi)皮層木栓化增強(qiáng),這也與相應(yīng)突變體的分析結(jié)果一致,且營養(yǎng)脅迫抑制木栓化的過程由乙烯信號通路介導(dǎo),而ABA信號通路則參與營養(yǎng)脅迫誘導(dǎo)木栓化的過程,此外,ABA和乙烯可直接調(diào)控擬南芥幼苗根系木栓化。ABA引起內(nèi)皮層木栓化向皮層及更外層延伸,值得注意的是,乙烯處理卻引起內(nèi)皮層次級分化階段已沉積的木栓質(zhì)消失,這表明幼苗根系內(nèi)皮層木栓化的高度可塑性[88]。
木栓化的改變與生理適應(yīng)性相關(guān),表明了內(nèi)皮層木栓化在植物體內(nèi)營養(yǎng)穩(wěn)態(tài)維持方面的重要作用,但外皮層木栓化程度的改變是否同樣影響營養(yǎng)元素的運輸還需進(jìn)一步研究。
除了影響水分和營養(yǎng)物質(zhì)的運輸以及抵抗非生物脅迫外,木栓質(zhì)沉積在抵御病原菌入侵方面也發(fā)揮作用。馬鈴薯塊莖遭受創(chuàng)傷能誘導(dǎo)周皮的木栓化,從而對病原菌的抗性增加[5,89-90]。此外,木栓化還能提高大豆(Glycinemax)對疫霉菌(Phytophthorasojae)的抗性,從而避免根和莖的腐爛[91-92]。接種疫霉菌增加了大豆根被皮和內(nèi)皮層的木栓化,但進(jìn)一步研究發(fā)現(xiàn),木栓質(zhì)含量高的品種中菌絲的生長只是被延遲了[91]。
馬鈴薯塊莖結(jié)痂病是由鏈霉菌屬引起的,Thangavel等[93]通過細(xì)胞抗毒素篩選出具有結(jié)痂病抗性的馬鈴薯體細(xì)胞克隆,且這些馬鈴薯體細(xì)胞克隆不僅對結(jié)痂病具有抗性,對其他的馬鈴薯塊莖感染病原也具有抗性,但這些抗性機(jī)制尚不可知。在具有抗性的馬鈴薯體細(xì)胞克隆和其易感染的親本馬鈴薯塊莖的周皮組織中,對與木栓質(zhì)合成相關(guān)的基因以及與先天防御反應(yīng)相關(guān)的基因的表達(dá)模式進(jìn)行分析,結(jié)果顯示具有抗性的馬鈴薯體細(xì)胞克隆相較于易感染親本,塊莖周皮中與木栓質(zhì)相關(guān)的基因的表達(dá)量更高,且在周皮形成更多的木栓層來響應(yīng)病原菌感染,而與先天防御反應(yīng)相關(guān)的信號基因的表達(dá)在兩者間沒有差異,這一結(jié)果充分說明了周皮木栓化在馬鈴薯塊莖抵御病原菌入侵中的重要作用[93]。在其他一些植物,例如擬南芥、小麥(Triticumaestivum)以及番茄中也發(fā)現(xiàn)病原菌的侵入會誘發(fā)木栓化來響應(yīng)[11,94-95]。
綜上所述,木栓質(zhì)能通過增強(qiáng)根或塊莖周皮細(xì)胞壁的物理性屏障來部分地阻斷病菌的侵入,但尚需對控制木栓質(zhì)合成的關(guān)鍵基因在植物生物脅迫抗性中的作用進(jìn)行深入研究。
近些年,對于木栓質(zhì)生物合成的研究已經(jīng)取得了巨大進(jìn)步,但木栓質(zhì)單體合成的反應(yīng)順序、轉(zhuǎn)運及聚合組裝機(jī)制以及木栓質(zhì)合成的調(diào)控等關(guān)鍵環(huán)節(jié)依然模糊不清。可以借鑒角質(zhì)及相關(guān)蠟質(zhì)的合成,組裝以及聚合機(jī)制,進(jìn)一步完善人們對木栓質(zhì)單體的運轉(zhuǎn)、細(xì)胞外的組裝、細(xì)胞壁木栓質(zhì)聚合機(jī)制以及木栓質(zhì)生物合成的調(diào)控等過程的理解。
Naseer和Geldner[96]的研究表明,根系內(nèi)皮層質(zhì)外體屏障凱氏帶的主要組成成分是木質(zhì)素而非木栓質(zhì),因此內(nèi)皮層凱氏帶和木栓質(zhì)的沉積可能在植物根系水分和營養(yǎng)元素吸收運輸方面發(fā)揮著不同的功能。隨著一系列新技術(shù)的產(chǎn)生與發(fā)展,下一步研究可重點圍繞木栓質(zhì)與凱氏帶功能特異性與互補(bǔ)性展開,另一方面,可將木栓質(zhì)的沉積與側(cè)根的形成結(jié)合起來,最新的研究表明側(cè)根形成時,凱氏帶這一質(zhì)外體屏障被打破,但木栓質(zhì)的沉積并未受到影響,因此木栓質(zhì)可能在側(cè)根形成過程中內(nèi)皮層質(zhì)外體屏障功能的維持中發(fā)揮更為關(guān)鍵的作用[76]。這一機(jī)制的闡明將是對根系結(jié)構(gòu)適應(yīng)環(huán)境變化模型的重要補(bǔ)充。
與此同時,眾多研究表明木栓質(zhì)在植物根系外皮層以及內(nèi)皮層的沉積具有多重功能。但仍需對木栓質(zhì)生理功能進(jìn)行深入研究。木栓質(zhì)單體中的哪些成分與植物響應(yīng)生物脅迫和非生物脅迫直接相關(guān)還有待于進(jìn)一步確定,且目前關(guān)于木栓質(zhì)的生理功能研究多集中于抗逆性并不強(qiáng)的模式植物中,需要進(jìn)一步挖掘抗逆性強(qiáng)的植物中與木栓質(zhì)合成的關(guān)鍵基因,進(jìn)一步通過功能獲得或缺失的方法探究這些抗逆性強(qiáng)的植物中木栓質(zhì)與其響應(yīng)逆境間的關(guān)系。
在優(yōu)良牧草紫花苜蓿中接種土壤微生物叢枝菌根真菌(arbuscular mycorrhiza fungi,AMF)顯著提高了其抗旱性,這種土壤有益微生物與植物根系共生,其在根系周圍形成的菌絲體網(wǎng)絡(luò)可以幫助植物從更深的土層、更小的土壤縫隙中汲取更多的水分[97],但關(guān)于這些土壤有益微生物與植物根系木栓化是否存在互作來共同參與植物適應(yīng)逆境值得進(jìn)一步研究。此外,土壤環(huán)境是復(fù)雜多樣化的,其離子組成和水分含量高度不均勻,兩種對立的刺激(如缺Fe和缺K同時存在)或是多種刺激同時存在時對根系木栓化會造成何種影響、以及根系病原菌等生物因子對根系木栓化的影響同樣值得研究。
對這些深層次機(jī)理的探究對將來通過基因工程手段培育具有多重抗性的作物及牧草新品種具有十分深遠(yuǎn)的意義。
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