水孔蛋白在细胞延长、盐胁迫和光合作用中的作用解读

1、生物工 程学报Chin J Biotech 2009, March 25; 25(3):321-Chinese Journal of BiotechnologyISSN 1000-? 2009 Institute of Microbiology, CAS & CSM , All rights reserved综述水孔蛋白在细胞延长、盐胁迫和光合作用中的作用杨淑慎崔丽荣西北农林科技大学生命科学学院，杨凌 712100摘 要：水孔蛋白属于一个高度保守的、能够进行跨生物膜水分运输的通道蛋白MIP 家族。水孔蛋白作为膜水通道，在控制细胞和组织的水含量中扮演重要角色。本研究的重点是属于PIP 亚

2、家族的 GhPIP1;2 和属于 TIP 亚家族的 TIP1 在植物细胞延长中的作用。使用特异基因探针的Northern 杂交和实时荧光 PCR 技术证明 GhPIP1;2 和 Gh TIP1 主要在棉花纤维延长过程中显著表达，且最高表达量在开花后 5 d。在细胞延长过程中，GhPIP1;2 和 Gh TIP1 表达显著，表明 它们在促使水流迅速进入液泡这一过程中扮演重要角色。而且也研究了盐胁迫植物中钙离子对水孔蛋白的影响。分别或一起用 NaCI 或 CaCl2处理原生质体或细胞质膜。结果发现在盐胁迫条件下，水渗透率值在原生质体和质膜颗粒中都下降了，同时 PIP1 水孔蛋白的含量也下降了，表明

3、 NaCI 对水孔蛋白的功能和含量有抑制作用。同时也观察了 Ca2+的两种不同的作用。感知胁迫的胞质中游离钙离子浓度的增加可能导致水孔蛋白的关闭。而过剩的钙离子将导致水孔蛋白的上游调控。同时实验已经证明大麦的一类水孔蛋白-HvPIP2;1 有更高的水和 CO2转移率。本研究的目标是确定负责转运水和 CO2的关键水孔蛋白的类型，并且提高植物对水的利用率、抵抗胁迫的能力，促进 CO2的摄取及吸收、提高植物的产量。关键词：水孔蛋白，细胞延长，盐胁迫，光合作用The action of aquaporins in cell elongation, salt stress andphotosynthes

4、isShushe n Yang, and Lir ong CuiChinese Northwest A& F University College of Life Science Yangling 712100, ChinaAbstract: Aquaporin belongs to a highly conserved group of membrane proteins called major intrinsic proteins (MIPs) that facilitate watertransport across biological membranes. Aquapori

5、ns are membrane water channels that play critical roles in controlling the water content ofcells and tissues. We focused on GhPIP1;2 which belongs to the PIP subfamily and Gh TIP1 which belongs to theTIP group of the TIP subfamily. Northern blot analysis with gene-specific probes and real-time PCR d

6、emonstrated thatGhPIP1;2 and Gh TIP1 are predominantly expressed during cotton fiber elongation, with the highest expression levels at 5 days postanthesis. The high and preferential expression of GhPIP1;2 and Gh TIP1 suggests that they may play important roles in supporting2+the rapid influx of wate

7、r into vacuoles during cotton fiber cell expansion. Also, the effects of Ca on aquaporins in salinity-stressed plants werestudied. Researchers treated the protoplasts and plasma membrane with NaCl or CaCl2, alone or in combination. Under saline conditions,osmotic water permeability (Pf) values decre

8、ased in protoplasts and plasma membrane vesicles, and the sameReceived : December 29, 2008; Accepted : February 4, 2009 Supported by : Shaa nxi Natural Scie nee Fou ndation (No. 14210123). Corresp onding author:Liro ng Cui. E-mail: an 陕西省自然科学基金项目(No. 14210123)资助。322ISSN1000-3061CN11-1998/QChin J Bio

9、techMarch 25, 2009 Vol.25 No.3Jreduction was observed in the PIP1 aquaporin abundance, indicating inhibitory effects of NaCl on aquaporin functionality and protein2+abundance. Two different actions of Ca were observed. Increase in free cytosolic calcium concentrations associated with stress percepti

10、onmay lead to aquaporin closure, however, the extra-calcium would lead to an upregulation of aquaporins. Meanwhile, experiments havedemonstrated HvPIP2;1, one of barley aquaporins, has a higher water and CO2transport activity. The goal of our plant aquaporin researchis to determine the key aquaporin

11、 species responsible for water and CO2transport, and to improve plant water relations, stress tolerance,CO2uptake or assimilation, and plant productivity.Keywords: aquaporin, cell elongation, salt stress, photosynthesis1IntroductionWater uptake and flow across cellular membra ne is a fundame ntal re

12、quireme nt for pla nt growth and development. Ifwater uptake through the roots is reduced or blocked bywater-related stress such as drought, salt stress, or lowtemperatures, pla nt growth is seriously or lethallyinhibited.There are threedifferent components of water flow in plant tissues: symplastic

13、movement of water via plasmodesmata, transcellularmovement across cell membranes, and apoplastic flow throughthe cell walls. The relative con tributi on of each water movement pathway to flow across the composite structure of tissues andorga ns in plants varies, which provides a useful mechanism byw

14、hich thepla ntcan resp ond to cha nging1en viro nmen tal con diti ons . Moreover, we focused onthe aquapori ns that are resp on sible for the tran scellularmovement of water across the cell membrane. Although thebiophysical bases of water transport across biologicalmembranes have been enounced as ea

15、rly as in the 1950s, it isuntil 1992 that Dr. Agre discovered and reported the firstaquaporin (AQP1)2from erythrocytes . Aquaporin (AQP) belongs to a well-c onservedand an cie nt family of prote ins calledmajor in tri nsic prote ins (MIPs) that facilitate the flow of smallmolecules like water and/or

16、 glycerol across the hydrophobic interior of membra nes. The AQPsdiffere nt forms have bee n found in orga ni sms ranging frombacteria and fungi to ani mals and pla nts, and the structure,molecular biology, biophysics, and the role6ofAQPs in pla nt water tra nsport have bee n reviewed . In thisrevie

17、w, we will rather focus on the most recent findings con cerning the molecular and cellular properties of aquaporins. We willdiscuss how these findings open new perspectives forunderstanding the multiple function of aquaporins in plants.2Aquaporins structure and classificationAquaporins are small and

18、 highly hydrophobic tran smembraneprotei ns, and most of them are250-300 amino acids, in len gth with a molecular mass8between 26 kD and 34 kD . Allaquaporins have six transmembrane domains with the N- andC-termini facing the cytosol. The six transmembrane domainswere predicted to beaelices, which a

19、re connected by five loops(A-E). Loop B and loop E each contains a helical domain, HBand HE, both of which posses a con servedasparagine-proli ne-9 10ala nine (NPA) motif . The two halves of theprotein showobservesymmetry,withthehydrophobic loops containing the NPA motif overlapping in themiddle of

20、the lipid bilayer to form two hemipores that togethercreat a narrow11 12channel . Water molecules passing the channelareforcecbythqDroteinglectrostaiiiKDeipflip at the cen ter of the cha nn el.Aquaporins can be subdivided into four groups with highlycon served amino acid seque nces and intron positi

21、 onsineach group: the plasmamembra ne in tri nsic protei ns (PIPs), the ton oplast intrin sicprote ins(TIPs),the no duli n 26-likeintrinsicproteins (NIPs) and the small intrinsicproteins (SIPs) . The PIPs subfamily, thought to be localized inplasma membrane, can be further divided into two classesna

22、med PIP1 and PIP2 with the specific arrays of amino acids atthe N-and C-termini.According tothe nomenclatureby Jang14JY et al , in Arabidopsis , the PIP1 subgroup representing fivemembers of aquaporins is named PIP1;1 to PIP1;5, and PIP2subgroup consisting of eight members of aquaporins is namedPIP2

23、;1 to PIP2;8. The TIPs subfamily, thought to be prese ntedin vacuolar membra nes, can fall into several classesinciudingTlP盯IFYTIP,TlFancfcoon.Thesubcellular locationof membranes from the NIP and SIP families is still uncertain,but recent studies have shown that SIPs are localized in the ER11fracti

24、on of Arabidopsiscells .杨淑慎等：水孔蛋白在细胞延长、盐胁迫和光合作用中的作用323J3Aquaporins functionsA lot of aquaporins found in plants suggests their importaneeduring plant development and adapting to324ISSN1000-3061CN11-1998/QChin J BiotechMarch 25, 2009 Vol.25 No.3JthedifferentnvironmenrtnditiohsManysubfamrtyemberscotto

25、nhesrregiorwereIJ_II_ tIJ上IJk t J_|7processqslantaredependenrtiassiwater flowintoandoutofcellsandthereforteis reasonctblafequapeplayanimportafotnongationesponst stressand planthotosynthesisdoeseveraiellulaprocessespludinghe cellP4 Aquaporins functions in the cell elongationCotton fiber is a good mod

26、el and pro5idJPAa GhiPIJP1；2 transcripts decreasegra GhTlPI transcriptscreasecklBecaithe rapiexpansoJicottonibeasioiappefrompmeintnly)rovidebasiicnderstandin5pPAo 15DPAwethinkhaaquapoipnaytellelongatidint alscidentifidtlae important roles in cotton fiber elongation.isSABapausfr3edgltiefChanneroteinl

27、S)atof aquaporin genes preferenHowyaoboevedilaltetheimembrawaiernd elongaticeffe. Recentlypermeabiln忆espontcBenvironmefUals.界靳preferentially lanPIPandTlPsubfamiliareabundant felopmeaiieen plasmmembra3ffritonoplastndtheyplay,1g2icodingpivotarolemaintaincellturgorncwater plasrnmembrntrinspcroteira,ncG

28、lYTIP potentiaf th(I d. AfrCmtheCutvat(ipeCiC cottortGJhiFsutJm(Vacuoiei)transp6rwateicrosBembrakesexpansio agulator, of the prii clasglucanasTherapid water into thecentral vacuole is d nerateg thesolute vacuole, and this is fu llenlargement.IIitN覷瞬歸鄭懑鏈注兀需p0?t2Sf?ap6S?TS?P6SA5EP6SSIhiigHB?S?J65Ha ex

29、p翔种冲怦忡卿區isolateandcharacterizG hPlPie, codingpivotalolesnmaintainCfg|turgcfflncWaterPhylogenetic analysis showed that GhbyP1Mapl6fS9SlimportafUlnctiOinrapid tothePPgroucfthel陆ubfamilf(GTIP1 plant囲I ejongatCotton fibercell exp is toThfe group of theTIPsubfanf0i.6hfeedsturgorpfeSsali6? butalso require

30、s that the rigidi eingancp referentiapress ofrGhPIP O；nid nTIPI suggests that they may play i-pporting the rapid influx of water iicottofibecellexpansiSn).in ordetoinvesfigate the role of PIP and TIP inin su|“一FirstconstructedsUppressi6nbtractivehvbridizatCDfchoose these two aquaporin genes fo(DPA)

31、fiber, and then isolated the full-lfragmenisinj5 RACEfapjdmplificatOfnCDN-_dsturgor Pre.s alsow6wnn?n6 fife taeBwminfle6n!U(Uyj6CafndUcehangestheee9fiJCDWAIQmselVWater-chaatlfe ity, fnferreafrom themembranpermeability parameteraiseetodecreawiihincreasingedce)idoteupportatee ntryntothevacuole,惟of the

32、 aquaporin superfamily may irgearOfwatem ovementeosheonoplast ndplasma membrane during cotton fiberithdpresesitudyh etranscripsnGhPIP1;2ncGhTIPIaremaintainedhighevelirom10DPorrespontijifig9Dpeniragj ortfsoluble sugars, Knd maate All resultsshowed thathefragmenteeaquapognnepreferentiallyGh当PC2fafcure

33、x6ftalflCisreift!r6ft8hTyi79 IfviyTSlai杨淑慎等：水孔蛋白在细胞延长、盐胁迫和光合作用中的作用325J326ISSN1000-3061CN11-1998/QChin J BiotechMarch 25, 2009 Vol.25 No.3Jexpressed dur ing cott on fiber elon gati on. Some hypothesismen ti oned above can provide an expla nati on of many basicprocesses in which aquapori ns are alread

34、y implicated.5 Aquaporins function in environmental stressLand plants have evolved to cope with rapid changes inthe availability of water by regulating all aquaporins whichrepresent a major path duringwater uptake by roots.Regulation of aquaporin may alsorepresent a way to modulate membrane water pe

35、rmeability,and the factors affectingandregulati ngaquapor inbehaviors possiblyin volve2+phosphorylati on, pH, Ca , en viro nment stresses like sali nityand drought. The abu ndance andactivity ofaquaporins in the plasma membrane and tonoplast may beregulated, hence enabling the plant to tightly con t

36、rol waterfluxes into and out of cells. Un der salt stress, a n umber ofplasma membra ne-type aquapori ns were dow n-regulated,which can preve nt25contin uous dehydrati on result ing in cell death.As a step toward understandingthe aquaporinfun cti on in pla nts un der variousen viro nmen talstimuli,

37、the expressi on of the gene families en codi ngaquaporins under various abiotic stress conditions wereinvestigated. The expression of the aquaporin genesresponded significantlyand differently toen viro nmen tal stress con diti ons. One of the primary respon ses of pla nts to salt stress is the in hi

38、biti on of their rootwater uptake capacity26. Salinity has been show n todecrease the amou nts of mRNA en cod ing PIP27aquaporins in Arabidopsis . It has been observed that changes of certa in aquapor in isoforms are regulated by28salt-stress . Many laboratories have started to study themolecular an

39、d cellular mechanisms for regulation29of aquaporins under normal or stress conditions Differentstimuli can induce changes in aquaporin phosphorylation orprotonation,which playing animporta ntrole in aquapori n gati ng un der stressconditions. Experiments have shown that the waterpermeability of Arab

40、idopsis and Beta vulgaris plasma2+membrane is down-regulated by Ca , in addition to low pH30.Ca2+plays an importantrole in theup-regulationof water channelactivity. On rootwater transport, the inhibitoryeffects of HgCl2(acom mon aquapori n blocker but also a gen eral metabolismin hibitor),were only

41、see n provided thatthe pla nt n utrie nt soluti on contained Ca. Rece ntresults with pepper plants have also shown that calciumseems to be involved in plasma membrane aquapor inregulati on via a cha in of processes with in the cell but not byalteration of the stability of the plasma membrane32.To ch

42、aracterise watertra nsportin bothintracellularand plasma membranes of plant cells,osmotic water permeability values (Pf) were measured onisolated protoplasts and purified33plasma membrane vesicles . To study how NaCl affectswater tra nsport and the role of calcium un der salt stress, wetreated the i

43、solated protoplasts and plasma membrane withNaCl or CaCl2, alone or in comb in ati on.The results can be seen as follows: treatment of pla nts bycalcium alone had no sig nifica nt effect on protoplast Pf. Pfvalues were significantly decreased in NaCl-treated pla nts,but they can be restored in a2+pr

44、etreatment ofplants by Ca . Moreover, the overall Pf results obtained frommembrane vesicles of NaCl-treated plants showed the sametendencies as those obtained from protoplasts and both were34con siste ntwith the PIP1abu ndace. In allmembrane preparations except in those from NaCl-2+treated plant, an

45、 external Ca supply to the vesicle mediumcan decrease Pf. Acidic pH had similar35effects on membra ne Pf. Torn roth-Horsefield et alprovided an expla nati on of the mecha ni sms of pla ntplasma membrane aquaporin inhibitionby pH andcalcium. In summary, it has been proposed that in the closedaquapori

46、 n con formati on the sec ond cytoplasmicloop (D)caps the channel from thecytoplasm occluding the pore , whereas in the openconformationthe loop D is displaced,and thismoveme nt ope ns a hydrophobic gate block ing the35cha nnel entrance from the cytoplasm.In one word,2+exter nally supplied Cawould a

47、ct directly to blockthe aquapori n pore.The in teract ing effects of salt stress with calciumhomeostasis and signallinghas been reported. Forexample, NaCl stress can affect membrane polarity and Ca2+uptake in plant cells36. In Arabidopsis , the2+37cytosolic Cagradient can be reduced by NaCl .The con

48、 focalmicroscope studies showed that2+extended NaCl stress can reduce intracellularCa .This provides a supportion of the idea that NaCl2+affects cell Ca homeostasis which in turn wouldresult in aquaporin inhibition. Many environmental factors,such as sali ni ty, also critically in terfere with aquap

49、orinabundance. Many examples that stress in creasedthelevelof aquapori ns have bee nobserved. A recent report showed that, after NaCl treatment,the protein amounts of PIP2;1, PIP2;2 and PIP2;3 inArabidopsis suspe nsion cells in creased several fold,whereas the abu ndance of38PIP1 homologues did not

50、change . By contrast, a sig nifica ntdecrease in the abu ndance of PIP1 prote ins can beobserved in Arabidopsis pla nts after杨淑慎等：水孔蛋白在细胞延长、盐胁迫和光合作用中的作用327JsaiexposuTheimounifePIPInp，plasnrmembraaisbdecreasWdhsThiaDointeW) a rapicrespontoesaltthrough like HO and CO. Maki Kats ehapointed dynamcontrco

51、lfPlPaquap . 一“/or reg reg abi ind restorationfeppeoot To date, we have known that aquaporins；altstress. thetranspoftman9ssentjlablecule plants,328ISSN1000-3061CN11-1998/QChin J BiotechMarch 25, 2009 Vol.25 No.3JJgliggggggiglBbBEWffitneffialycofjXjlngenEyffifeRUb8llnBff bfbbilftbn鬥trquai po n wasccs

52、/ugahigher water transport activitytheoocybystemiHensm,embefPlyste mhelifferencwatqpermeabcayDeittrighitaeoelaechang insspecifaminocid quaporinsthepaannxeandNPAmotifs.Pproteiasecharacterbyadceonsutativ ulaticof ortaquaporn s watehomeostaisasaquaporjnsbmmcin chaWaitfugnifi(apnzymeSubisinhibitInternal

53、 conductance gi, conductance reofphotosynthfesionganfactoraquaporinsare probably the most important factor detof TerashimadOnoa iinthqoresenrfH glanoof mostof the aquapori杨淑慎等：水孔蛋白在细胞延长、盐胁迫和光合作用中的作用329Juooeiuisieujnop笔0i-1siuniuiuoo：(/ L ”)99乙dx3 r s6u!ipe9S -snuui|e6qdeood4aiiijBi|3Di:PUE suiiodegg

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58、6 essdg2. V禺14乙0O乙|O!sXqdiue|d suuodenbmlueid |BJ|Rzu8AV8WBM06ieaz口匸we062oo乙1puoqnaie/oojui suuodenbQ99qj|ne|/|!)|qAer9900乙1loisXqd |ue|dsieuueqo |OjeoX|6 |euepeq oiJBIILUISsoueiiwifujoosAqcl uiu!ojd。列叫。阴Jue|dAouvIE0评o炳aiqei，suossABisn9gogtzgg 0乙(i旧Qwiueuroasuodsajuauj euuseid 6uipoou8 A|iuuej eue6 e jo|eue uoissajtfeir怡/LUI?aeonclVShabS|etaJEffeof3ws3aBnna3mbaq3apeffnBntlod r ascsftsnnlBe輕waterchanne?lactiLynch JP. Effects of salinirSfArabidtitbaiSa