绪论和养分吸收

植物营养的分子生物学常春荣M:E-mail：概念Molecularbiologyofplantnutritionis“studyofstructureandfunctionofgenesregulatingand/orcontrolplantmineralnutritionatthemolecularlevel”课程主要内容1.植物养分吸收2.植物营养分子基础3.植物营养分子研究基本方法4.无机氮转运的分子基础5.低磷营养的响应机制6.钾的转运分子基础7.镁的转运分子基础8*.铁的转运分子基础9*.铜锌锰的基因表达调控10.盐胁迫调控机制课程性质与要求理论性、技术性均强的课程。要求同学在学好植物营养学和植物生理学的基础上基本掌握植物营养学在分子生物学方面的发展方向和主要的技术原理。考核：分4个小组讲微量元素部分。课程论文（至少5篇外文文章）绪论1.植物营养学科的发展从VanHelmont的柳条实验开始探讨植物生长需要的营养物质开始，植物营养学科发展已经经历了300多年的时间。对植物生长认识从定性到定量，从生长的结果到生长的过程（生理），以及实验手段的建立和完善。目前，分子生物学、分子遗传学、生物技术和生态学的发展，从细胞水平、分子水平以及生态的角度来了解植物营养的过程成为了可能。绪论1.植物营养学科的发展植物养分高效吸收利用的分子生理机制与分子遗传机制成为过去20年来植物营养学科快速发展的内容。植物营养相关的基因的克隆和表达调控的研究是植物营养学科重要的研究内容。

绪论2.分子技术与植物营养研究的关系植物营养学主要研究内容：植物营养生理学；植物根际营养；植物营养遗传学和分子生物学；植物营养生态学；肥料与施肥技术绪论2.分子技术与植物营养研究的关系植物营养学主要研究手段：田间试验；模拟试验；化学分析；数理统计；核素技术；酶学法；分子技术。绪论2.分子技术与植物营养研究的关系植物的营养过程和结果终究是由以系列的基因控制的。植物对外界环境做出的应激变化是一列基因变化的结果。分子技术成为研究植物营养的机制的重要手段。绪论3.植物营养研究新思路植物营养的研究已经从过去的化学分析、平衡施肥、生理分析发展到细胞水平、分子水平，从基因的表达和蛋白组学的角度研究植物营养的机理，从而提高植物高效营养奠定基础。Chapter1Nutrientuptake

1.1Mechanismsofnutrienttransportthroughcellmembranes

1.2Thekineticsofnutrientuptakebyplantinvivo

1.3Radicaltransportofionsacrossroots(Shorttransport)

1.4CharacteristicsofnutrientuptakebyplantsUptakefromsoilsolutionUptakefromxylemby shootcellsRemobilizationfromsenescingtissueUptakebysymplasticallyisolatedtissuesNutrientTransportoruptake1.1Mechanismsofnutrienttransportthroughcellmembranes

CELLNutrientsaccumulatedinsidethecelle.g.K+

mMRegulatecytoplasme.g.pH,Ca2+

RemovetoxicandwastesubstancesExportproductsOUTSIDEe.g.soilK+mMpHCa2+mM1.1.1MembraneFunctionanditspropertiesDiagrammaticrepresentationofcellmembraneLongchainpolyunsaturatedfattyacidsCHOPOCH2CH2N+(CH3)3O-CHO-CHOO2R1R22CH2OHORRR1R2CH2O-CH2O-CHOHOHOHHOHOHHHCH2

O-CH2O-CHOHOHOHCH2SOHOHOHHO12HPhosphatidylcholine(lecitin)磷脂酰胆碱(卵磷脂)Monogalactosyldiglyceride单半乳糖甘油二酯Sulfoquinovosyldiglyceride硫代奎诺糖甘油二酯Sometonoplasttransportproteins1.1.2ElectrochemicalequilibriumForionstherearetwomajorforcesthatareimportantfortransport:concentrationgradientelectricalgradientTogether,itistheELECTROCHEMICALthatdrivesionmovementsTransportDiffusion-free(noenergyrequired)movementofacompoundinarandomfashioncausedbythekineticenergyofthatcompound.Activetransport-movementagainstconcentrationgradientthatrequiresexpenditureofenergy.GeneralNatureofDiffusionTherateatwhichasubstancediffusesisproportionaltotheconcentrationgradientNomatterwhattypeofdiffusiontakesplace,netmovementinwardandoutwardcanonlyoccuruntilinside[]=outside[].Anythingthatmovesincanmoveout.Forelectrolytes,electricalchargecaninfluencediffusion.FacilitateddiffusionDirectionofthenetfluxdependsonrelativeconcentrationsoneithersideofthemembrane-Specificforthemolecule-exibitsaturationkineticlikeenzyme-regulatedactivityActiveandpassivetransportMovessubstancesfromlowtohighconcentrationrequiresenergy-intheformofATPhighlyselectiveexchangeoneionforanotherprimaryactivetransportH+orCa2+ATPasesecondaryactivetransportH+cotransporter2.1.3TypesofproteinthatareinvolvedinactivetransportPUMPSCHANNELSCARRIERSTRANS-PORTER(Proteinorpeptides)P-typeionpump(plasmamembrane)PlantcellshaveaH+transporterthatcontrolthecytosolicpH(H+ATPase)Ca2+ATPasealsoinplasmamembraneandendoplasmicreticulumEpithelial（上皮的）liningofthestomachH+/K+ATPasewhichsecretsasolutionofconcentratedacidintothestomachV-typeionpumpUtilizeATPenergywithoutformingaphosphorylatedproteinintermediateH+transportacrossthemembraneofcytoplasmicorganelles(细胞质体),suchaslysosomes(溶酶体),secretorygranulesandvacuolesLargecomplexmoleculeswithseveralcomponentproteins,oneburiedinmembraneAnothersimilartypeisF0F1ATPasewhichispresentinbacteria,mitochondriaandchloroplastsPUMPSINPLANTMEMBRANESPLASMAMEMBRANEH+ATPase-singleproteinbutseveraldifferenttypesCa2+ATPase-singleproteinVACUOLARMEMBRANE(TONOPLAST)H+ATPase-severalsubunitscompositionchangesH+PP-ase-singleproteinER-EndoplasmicreticulumCa2+ATPase-singleproteinIonchannelsK+channelsNandCterminaldomainsarelocatedonthecytoplasmicsideContains6membrane-spanningsegmentsTypesofcarrierComparisonofpassiveandactivetransport`NO3-NO3-NO3-=1mMpH=6.0NO3-=c5mMpH=c7.2Dy=c-100mVVacuolePMCytoplasmBathingmediumCellwallAminoacidsXylemATPADPH+H+2H+2H+NO3-NO3-=10-200mMpH=c5.5Dy=c-10mVQ1.Describethedifferenceofion

concentrationbetweenthesoilsolution

andplantcells(cytosol).

Q2.Whatisactiveandpassivetransport

throughplasmamembrane?

Q3.Whatareantiporter,uniporter,

symport?

Q4.WhatisATPase?

Q5.Whatiselectrochemicalequilibriumof

ions?2.1.4Iondrivingforce

PMF

(ProtonMotiveForce)

Electro-chemicalpotentialsofplasmamembrane

Theproductionofelectro-potentialofmembrane

1.AsaresultofworkbyATPaseinmembranes

2.AtendencyofK+diffusionfromcelltooutsideAnti-portUniportCo-transportAnti-transpot?H-ATPase+(泵)H+H++Tonoplast?PPasei(泵)H++H++ATPPPiiVacuolepH4.5~5.9~-100mVAnionsHATPase+(泵)离子通道H+PlasmamembraneH++H++ATPADP+PiiApoplasmpH~5.5CytoplasmCations+2PiiNAD(P)HNAD(P)+共运输epump离子通道H+(1~3H+)+pH7.3~7.6-120to-180mVFeIIIIIIFeIIIIH+TMredoxADP+Pii-共运输H+单向运输Cations+CationsAnionsIonchannels

OutsideR-COO-R-COO-R-COO-K+InsideMembraneK+K+K+R-COO-R-COO-R-COO-K+R-COO-K+K+MorepositiveMorenegativedμ/dx:

chemicalpotentialgradient

zFdψ/dx:

electricalpotentialgradientForaneutralsolute:

μc=μoc+RTlnC(1)

Forachargedsolute:

μ=μoc+RTlnC+zFψ(2)

whereμcischemicalpotentialofanion,μocthestandardchemicalpotential,Rgasconstant(7.95J/oC/mol),Tabsolutetemperature,Ctheconcentrationoftheion,μtheelectrochemicalpotential,zthevalanceoftheion,FFaradayconstant(92J/mV/mol),ψelectricalpotential.Whenanionisinequilibriumbetweenincellandoutcelltheelectropotentialoftheionbetweenincellandoutcellshouldbeequal:

μoc+RTlnCo+zFψo

=μoc+RTlnCi+zFψi(3)

whereCoandCirepresentanionconcentrationinoutsidesolutionandincytosol,respectively,andψoandψielectricalpotentialsofanioninoutsidesolutionandincytosol,respectively.ψi-ψo=Δψ=E=RT/zFlnCo/Ci(4)

ψi-ψoinequation(4)istheelectricalpotentialdifferenceacrossthecellmembraneandisalsocalledasNernstpotential(E)whichcanbedeterminedusingmicroeletrodemethod.SinceRT/Fisequalto25.8(assumingthatthetemperatureis20oC)andlg=2.3·ln,theequation(4)canbewrittenasthefollowingone:

E=59/zlgCo/Ci(5)

Experimentally,wecandeterminetheelectricalpotentialdifferenceacrossthecellmembraneandtheionconcentrationsincytosolandinoutsolution.AndwecanalsocalculatetheCo/CibasedonthedeterminedE.ForK+ion,forexample,ifthevalueofEdeterminedbyexperimentis–118mVthatisreplacedintoequation(5),thenlgCo/Ci=-2,whichcanalsobeexpressedaslgCo/Ci=lg10-2,or:

lgCo/Ci=lg1/100(6)Equation(6)infersthatiftheelectropotentialofcellmembraneexperimentallydeterminedis–118mVtheconcentrationofmonocation,K+forexample,canbeaccumulatedincytosolbyafactorof100relativetotheoutsolutionbypassiveuptake,alsonamelyasdownhillprocess.OnlyiftheconcentrationofK+experimentallydeterminedincytosolishigherorlowerthanthatindicatedbytheequilibriumcondition(equation(4))mustanactiveorpassiveuptakehaveoccurred,whichisalsothermodynamicallydefinedasaprocessagainstelectrochemicalgradient(uphilltransportprocess)oraprocessdownhilltheelectrochemicalgradient(SeeTable2.1.2).Activetransportofionsrequiredmoreenergythanpassiveone.

Table1-1Experimentallydeterminedandcalculatedionconcentration(mM)accordingtotheelectricalpotentialdifferencesinoatroot

IonsIonconcentrationExperimentallyCalculatedTypesofuptake

inoutsolutiondeterminedbasedonthe

electrical

potentialdifferences

determined

K+16627active

Na+1327passive

Ca2+131400passive

Cl-130.038active

NO3-1560.076active

a

Theelectropotentailofcellmembraneexperimentallydeterminedwas–84mV.BasedonHiginbothametal.

Thesizeofaglassmicroelectrodetip1.1.5Measurementofelectro-potentialofmembraneandionconcentrationincell

VSingleelectrodemeasurementscellGlassmicroelectrodeGlassmicroelectrodeinsertedintothecellmeasurestheelectricalpotentialdifferencebetweeninsideandoutsidethecell(acrosstheplasmamembrane)Methodsandmaterials

BarleygrowninHoaglandsnutrientsolutionincontrolledenvironment(pH6.0).Transferredtoelectrophysiologyrig;rootsperfusedwithsolutionscontaining5mMMES(pH6),0.5mMCaSO4and0.1mM

KClforseveralhourspriortomeasurements.Electrodesinsertedinsub-epidermalcells.InOutPetridishCover-slipmVSignalrecordedonWPIFD223coupledtodataloggersamplingat5Hz.Membranepotential(mV)10min-100-150MEMP028.DAT0.25mMNO3-0mMNO3-MembranepotentialresponsetonitratePM2H+2H+NO3-NO3-CytoplasmMediumExplanationEffectofnitrateconcentrationontheresponseofmembranepotentialtonitrateinnitrate-grownmaizeroots.Seedlingsweregrownin0.15mMCaSO4and0.05mMCa(NO3)2andimpalementsmadein0.2mMCaSO4

(FromMcClureetal.,1990).AssayingammoniumtransporteractivityinricerootsWangetal.1994PlantPhysiol.104:899Assayingnitratetransporteractivityinmutantsbymicroelectrodemeasurements.Wang&Crawford1996PNAS93:9297Ion-selectivemicroelectrodes