，，

(1.中國科學(xué)院 東北地理與農(nóng)業(yè)生態(tài)研究所 大豆分子設(shè)計育種重點實驗室，吉林 長春 130102；2.中國科學(xué)院大學(xué)，北京 100049)

0 引 言

血紅蛋白(Hemoglobin,Hb)廣泛存在于動物、植物及微生物體內(nèi)[1-2]，起到O2輸送與貯存以及運輸CO2的作用。植物血紅蛋白在植物根瘤固氮、抗逆脅迫、胚胎發(fā)育以及營養(yǎng)生長等方面具有重要作用[2-9]。自從1939年，Kubo從大豆根瘤中鑒定到植物血紅蛋白以來，已經(jīng)在絕大多數(shù)的物種中發(fā)現(xiàn)了植物血紅蛋白的存在，并進行了深入的研究[10-11]。在2014年XVIII 氧結(jié)合和傳感蛋白國際大會上，提出了植物血紅蛋白(Phytoglobin)新的命名體系，現(xiàn)已被廣泛采納[12]。目前植物血紅蛋白在決定細胞命運中新功能的發(fā)現(xiàn)等研究[13]已成為了一個新的研究熱點[14]，本文綜述了國際上目前對于植物血紅蛋白功能研究上的新進展。

1 植物血紅蛋白類型和命名

植物血紅蛋白最先在豆科植物的根瘤中發(fā)現(xiàn)。1938年，Pietz與Bakt發(fā)現(xiàn)蠶豆(Viciafaba)根瘤中的紅色素是共生微生物增殖和生長的必要成分，紅色素參與氧化還原反應(yīng)[15]。1939年Kubo在大豆根瘤中分離出紅色素，進一步的結(jié)晶表明紅色素是一種類似于脊椎動物馬肌紅蛋白的血紅蛋白(Haemoglobin)[16]。在最初的研究中，僅在豆科植物的根瘤中發(fā)現(xiàn)了血紅蛋白的存在，Virtanen和Laine將存在于豆科植物根瘤中的血紅蛋白命名為豆血紅蛋白或根瘤血紅蛋白(Leghemoglobin,Lb)[15]。隨后的研究中發(fā)現(xiàn)，在與植物共生的根瘤中普遍存在與Lb相似的共生血紅蛋白(Symbiotic hemoglobin,sHb)。Fukudome等根據(jù)植物系統(tǒng)進化起源和理化性質(zhì)將sHb分為Class 1、Class 2和Class 3 3種類型[7]。

后續(xù)研究發(fā)現(xiàn)，在非豆科植物中存在非共生血紅蛋白(Nonsymbiotic hemoglobin,nsHb)。Appleby等第一次在根瘤菌共生的非豆科雙子葉植物糙葉山麻黃(Parasponiaandersonii)根瘤中發(fā)現(xiàn)并提純了血紅蛋白，打破了血紅蛋白一直以來僅存在于豆科植物根瘤中的論斷，并發(fā)現(xiàn)該血紅蛋白不僅在共生的根瘤中表達，同時也在植物非共生部位表達[17]。隨后在山黃麻(Trema)、大麥(Hordeumvulgare)、玉米(Zeamays)、小麥(Triticumaestivum)、黑麥(Secalecereale)、地錢(Marchantiapolymorpha)、水稻(Oryzasativa)、棉花(Gossypiumspp.)、擬南芥(Arabidopsisthaliana)及山黃麻屬等多種單子葉和雙子葉植物中發(fā)現(xiàn)nsHb[4，6]，證明了在植物中廣泛存在非共生血紅蛋白[3]。根據(jù)與氧氣親和力的強弱可將nsHb分為nsHb-1和nsHb-2[9]。其中，nsHb-2能夠結(jié)合氧氣，這與sHb的功能類似，而nsHb-1的表達與低氧脅迫和營養(yǎng)物質(zhì)過量供給密切相關(guān)，又被稱為壓力誘導(dǎo)的植物血紅蛋白[18-19]。

進一步的研究表明，植物存在截短血紅蛋白(Truncated hemoglobin,tHb)。Watts等在擬南芥中發(fā)現(xiàn)比正常血紅蛋白少20～40氨基酸的植物血紅蛋白，并將此命名為截短血紅蛋白，它與nsHb在清除植物體內(nèi)NO功能性質(zhì)方面具有相似性，同時，tHb與其他兩類植物血紅蛋白相比具有更低的氧氣親和力[6]。另外，在棉花(Gossypiumspp.)、水稻(O.sativa)、栽培稻秈稻(O.sativavar.indica)和粳稻(O.sativavar.japonica)等植物中也發(fā)現(xiàn)了tHb[9]，證明該種血紅蛋白也廣泛存在于植物體中。

2014年的新植物血紅蛋白命名體系，為解決目前植物血紅蛋白命名混亂的現(xiàn)狀提供了一個很好的解決途徑，開始被研究者接受和使用。在此系統(tǒng)中Phytogb0代表藻類、苔蘚及裸子植物中的非共生血紅蛋白(nsHb)，Phytogb1代表被子植物中的非共生血紅蛋白nsHb-1，Phytogb2代表被子植物中的非共生血紅蛋白nsHb-2，SymPhytogb代表非豆科的共生血紅蛋白(sHb)，Lb代表豆科血紅蛋白，Phytogb3代表藻類和陸生植物中的截短血紅蛋白。各類蛋白質(zhì)的性質(zhì)詳見表1。

表1 植物(藻類、陸生植物)血紅蛋白的系統(tǒng)和特征的命名法Table 1 System and characteristics of the accepted nomenclature for plant (algae+land plants) Phytoglobins

2 共生血紅蛋白(sHb)功能

共生血紅蛋白是由植物球蛋白和來自外源根瘤菌的亞鐵血紅素輔基(Ferroheme)組成的復(fù)合蛋白，它能在根瘤中特異表達，被認為是豆科植物根瘤固氮的重要指標(biāo)[20]。植物根瘤的形成過程與植物血紅蛋白具有密切關(guān)系：在形成初期即根瘤菌侵入及感染過程中，植物根瘤中不含有植物血紅蛋白，不具有固氮功能；在根瘤發(fā)育過程中，隨血紅蛋白的形成，根瘤開始具有固氮功能；隨根瘤及根瘤菌的退化，血紅蛋白含量與固氮作用也隨之降低，根瘤中植物血紅蛋白含量與根瘤有效性呈正相關(guān)關(guān)系[21]。在豆科植物根瘤固氮過程中，植物共生血紅蛋白存在于根瘤菌固氮的場所—細菌周膜，它對O2親和力強，能夠可逆地結(jié)合O2，促進O2擴散供給固氮微生物，降低菌體內(nèi)部氧分壓，將類菌體周圍的O2環(huán)境維持在7～11 nmol·L-1，為固氮酶提供適宜氧環(huán)境，從而保護菌體內(nèi)部的固氮酶活性[22]，同時，促進游離氧的擴散[23]。

在百脈根中通過RNAi沉默血紅蛋白基因，發(fā)現(xiàn)當(dāng)根瘤中的游離氧含量增加時，類菌體內(nèi)的固氮酶受損，甚至喪失固氮能力，證實了sHb在植物根瘤共生固氮過程中具有關(guān)鍵性作用[24]。sHb通過結(jié)合O2，促進游離氧擴散，從而為類菌體提供低氧環(huán)境和ATP，并保護對O2敏感的固氮酶活性，這對特定環(huán)境下植物細胞呼吸代謝和耐澇具有重要意義。sHb的作用在植物-微生物共生中尤為重要，植物通過sHb調(diào)節(jié)NO并結(jié)合植物激素調(diào)控自身防御反應(yīng)，NO通過與生長素(Auxin)相互作用參與植物根系的發(fā)育，并為極端條件提供ATP和低氧環(huán)境，維持固氮酶活性[25-27]。

3 非共生血紅蛋白(nsHb)功能

植物非共生血紅蛋白不僅能夠運輸和存儲氧氣，促進氧氣擴散，控制微生物菌群體內(nèi)的NO水平，并利用NO控制O2的水平，從而控制氧化還原反應(yīng)信號通路[9,29-31]；還可通過控制類菌體與自由基之間的活性和反應(yīng)結(jié)合并運輸CO、硫化物和油脂等，使植物免受其害[8-9,32-33]；在結(jié)瘤植物中通過NO參與發(fā)生在土壤、微生物和植物中的不同生物過程，觸發(fā)不同的植物-微生物互作的必要途徑[27]，作為干擾激素合成、影響其活性的重要信號分子[34]，此外，nsHb還具有清除含氯化物等功能[8,35]。

注：參考[37]和[38]文獻并做修改。虛線代表潛在的或者假設(shè)的物質(zhì)轉(zhuǎn)運機制。外膜：Outer Membrane(OM)；內(nèi)膜：Inner Membrane(IM)；嵴：Cristae Junction(CJ)；基質(zhì)：Matrix(M)；線粒體膜間隙：Mitochondrial Intermembrane Space(IMS)；類菌體細胞膜：Peribacteroid Membrane(PBM)；類菌體間隙：Peribacteroid Space(PBS)；周質(zhì)：Periplasm(P)；細胞基質(zhì)：Cytosol(C)；亞硝酸鹽轉(zhuǎn)運蛋白：Nitrite Transporter(NiT)；線粒體復(fù)合體Ⅰ：Mitochondrial Complex I (Ⅰ)；輔酶Q：Ubiquinone(Q)；細胞色素bc1(復(fù)合體Ⅲ)：Cytochrome bc1(Complex III)(Cyt bc1)；細胞色素氧化酶： Cytochrome Oxidase(COX)；細胞色素C：Cytochrome c(Cyt c)；硝酸鹽還原酶：Nitrate Reductase(NR) ;植物球蛋白：Phytoglobin(Pgb)；硝酸鹽還原酶：Nitrate Reductase(Nap)；亞硝酸鹽還原酶：Nitrite Reductase(Nir)；NO還原酶：NO Reductase(Nor)；一氧化二氮還原酶：Nitrous Oxide Reductase(Nos)；細胞色素cb：Cytochrome cb(Cyt cb)；NADH-對苯二酚氧化還原酶：NADH-quinol oxidoreductase(DH)。Note:Revised[37] and [38] references.Dashed lines represent potential or bypothetical material transport mechanism.圖1 低氧條件下根瘤中線粒體Pgb-NO循環(huán)Fig.1 Schematic of mitochondrial Pgb-NO cycle pathway operation in hypoxic nodules

4 截短血紅蛋白(tHb)功能

截短血紅蛋白與非共生植物血紅蛋白在清除NO、CO和H2S等方面的功能相似，而不同的特性及分子結(jié)構(gòu)又體現(xiàn)出不同于共生、非共生植物血紅蛋白的功能[6]。截短血紅蛋白具有保守的家族特征和序列的多樣性，由于配體結(jié)構(gòu)是由截短血紅蛋白氮調(diào)控[39]，因而異常靈活，使其在植物進化中對O2有從高到低的親和力，對環(huán)境變化具有快速的功能適應(yīng)性[40-41]。截短血紅蛋白和共生血紅蛋白進化起源不同，其最早在原核生物及真核藻類中被發(fā)現(xiàn)[6]，在百脈根和弗蘭克氏菌屬中被誘導(dǎo)產(chǎn)生，通過調(diào)控自身的表達參與植物體內(nèi)NO的內(nèi)穩(wěn)態(tài)，后來在原核生物、藍細菌、細菌、古生菌、真核藻類及植物中均被發(fā)現(xiàn)[36,42-43]。

5 植物血紅蛋白調(diào)控胚胎發(fā)育及抗逆脅迫的功能

細胞程序性死亡(Programmed cell death,PCD)是影響植物體細胞胚胎發(fā)生的重要因素之一[50]。植物血紅蛋白能夠調(diào)節(jié)NO含量，而NO作為植物調(diào)控PCD的重要信號分子進一步調(diào)控植物體細胞胚胎發(fā)育。植物特定血紅蛋白的表達可減少NO含量，改變植物對逆境脅迫的反應(yīng)，終止PCD[51]。同時，體細胞胚胎發(fā)生能夠調(diào)控特定細胞血紅蛋白表達，改變胚胎發(fā)育進程的模式系統(tǒng)，影響植物激素生物合成、細胞代謝過程的PCD和胚性潛能的表達[50]。細胞內(nèi)NO含量增加會導(dǎo)致與PCD相關(guān)的Zn2+和ROS含量增加，同時降低負反饋調(diào)控IAA-的MYC2表達，影響體細胞胚胎發(fā)生[50]。在NO含量升高時ZmPgb能誘導(dǎo)玉米(Z.mays)在體細胞胚胎發(fā)生過程中發(fā)生PCD[52]；在擬南芥中，NO能激活茉莉酸生物合成通路中的關(guān)鍵酶丙二烯氧化合酶和脂氧化酶2基因的表達，促進擬南芥胚胎組織內(nèi)JA含量增加[53]，與NO共同調(diào)節(jié)體細胞內(nèi)的抑制劑MYC2和茉莉酸酯蛋白基因(JASMONATE-ZIM-PROTEIN,JAZ1)，同時，JA通過植物血紅蛋白(Class 2 Pgb)調(diào)控體細胞胚胎發(fā)生[35,47]。

同時，植物血紅蛋白在抗逆脅迫中具有重要作用。在受到低氧脅迫時，抑制玉米(Z.mays)血紅蛋白基因(ZmPgb1.1或ZmPgb1.2)表達會導(dǎo)致根部頂端分生組織異常分化，抑制根部生長；此外，Pgbs通過調(diào)節(jié)NO，間接調(diào)控乙烯含量，控制活性氧含量(Reactive Oxygen Spices,ROS)，實現(xiàn)低氧脅迫下保護根尖分生組織的功能[53]。Pgb保護細胞分生功能的作用可能是植物應(yīng)對諸如鹽分和干旱等脅迫的反應(yīng)，如紫花針茅(Stipapurpurea)中的StipurPhytogb1具有提高轉(zhuǎn)基因擬南芥在干旱和漬澇條件下的耐受能力[27]，進一步研究發(fā)現(xiàn)，這是由StipurPhytogb1通過抑制NO的積累，調(diào)節(jié)抗氧化酶活性，進而誘導(dǎo)清除活性氧的相關(guān)通路來提升上述耐受能力。谷類作物在水淹等極端條件下，過量表達Pgb基因，可增強光合進程中有關(guān)的抗氧化酶的活性，抑制抗壞血酸含量及ROS活性，減輕水淹脅迫壓力，確保植物在極端條件脅迫下具有更高的光合速率；反之，植物抗氧化酶活力降低，光合速率下降[54]。

在擬南芥中，重度PEG脅迫產(chǎn)生水分虧缺時，Class 1 Phytoglobin(AtPgb1)的表達有利于延遲其頂端分生組織的死亡和退化[55]。當(dāng)根部受到極端脅迫時，抑制AtPgb1表達導(dǎo)致細胞內(nèi)部發(fā)生PCD，這個過程可通過ROS和乙烯的調(diào)節(jié)得到緩解[56]，達到部分恢復(fù)植物根部生長的作用[57-58]。PEG脅迫致使植物根部組織細胞生長受阻是先于根尖分生組織細胞結(jié)構(gòu)變化之前發(fā)生的，包括細胞和特化組織的缺失，這可能會導(dǎo)致調(diào)節(jié)植物激素的PIN1和PIN4的改變，進而增加根部細胞極性[59-61]。抑制AtPgb1表達能延緩WOX5的相應(yīng)表達[62]，調(diào)控靜態(tài)細胞中心(Quiescent center,QCs)的功能[63]，導(dǎo)致早期營養(yǎng)生長中莖組織細胞功能差異和分生組織大小的快速減少[64-65]。同時發(fā)現(xiàn)，擬南芥根部的其它組織細胞主要基因如SCARECROW(SCR)的表達調(diào)控內(nèi)皮層和QCs的功能，通過非自主細胞信號保持根部原始細胞的分化能力[66-67]；WEREWOLF(WER)的表達會嚴(yán)重影響植物側(cè)根的生長和功能，這同樣會顯著影響AtPgb1抑制時PEG脅迫處理對根部生長及功能的影響[55,68]。AtPgb1在PEG脅迫時起到保護擬南芥根部正常功能的作用，同時確保根部組織細胞在極端脅迫下通過保留特定的細胞行使分生組織分化生長的功能[69]。

6 展 望

關(guān)于植物血紅蛋白的起源、進化及功能等方面，前人已進行了總結(jié)[5,7-8,17,20,22,46,70]，而植物中血紅蛋白基因在體細胞胚胎發(fā)生中所起的作用才剛剛起步；未來植物血紅球蛋白在植物發(fā)育中所發(fā)揮的作用將會得到進一步的揭示；植物血紅蛋白與NO信號決定細胞命運以及其在植物抗逆境脅迫上的深入研究將為人們重新認識其在植物生長發(fā)育的作用開辟一個新的視角。

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