Abstract
Cytosolic NADPH can be directly oxidized by a calcium-dependent NADPH dehydrogenase, NDB1, present in the plant mitochondrial electron transport chain. However, little is known regarding the impact of modified cytosolic NADPH reduction levels on growth and metabolism. Nicotiana sylvestris plants overexpressing potato (Solanum tuberosum) NDB1 displayed early bolting, whereas sense suppression of the same gene led to delayed bolting, with consequential changes in flowering time. The phenotype was dependent on light irradiance but not linked to any change in biomass accumulation. Whereas the leaf NADPH/NADP(+) ratio was unaffected, the stem NADPH/NADP(+) ratio was altered following the genetic modification and strongly correlated with the bolting phenotype. Metabolic profiling of the stem showed that the NADP(H) change affected relatively few, albeit central, metabolites, including 2-oxoglutarate, glutamate, ascorbate, sugars, and hexose-phosphates. Consistent with the phenotype, the modified NDB1 level also affected the expression of putative floral meristem identity genes of the SQUAMOSA and LEAFY types. Further evidence for involvement of the NADPH redox in stem development was seen in the distinct decrease in the stem apex NADPH/NADP(+) ratio during bolting. Additionally, the potato NDB1 protein was specifically detected in mitochondria, and a survey of its abundance in major organs revealed that the highest levels are found in green stems. These results thus strongly suggest that NDB1 in the mitochondrial electron transport chain can, by modifying cell redox levels, specifically affect developmental processes.
Highlights
The typeII NAD(P)H dehydrogenasesare locatedon theinternaland externalsides of the innermitochondrialmembraneand oxidize NADH or NADPH from [[wOA]jY]O^peeonnlAincecvesesrsairotnicolfetshcainsarbteicvlieecwonedtaionnslWineebw-oitnhlyoduattaa.subscription
Consistentlyt,he E. coliexpressedAtNDBl was shown to be specificfor NADPH and tobindcalciumvia itsEF-handdomain as a prerequisitfeoritsactivity(Geisleretal, 2007)
ExternalNADPH-specifictypeII dehydrogenases have to date been reportedin the ETC of plants (Michaleckaet al.,2004)and thefilamentoufsungus Neurosporcarassa(Melo et al.,2001)butareabsentin
Summary
The typeII NAD(P)H dehydrogenasesare locatedon theinternaland externalsides of the innermitochondrialmembraneand oxidize NADH or NADPH from [[wOA]jY]O^peeonnlAincecvesesrsairotnicolfetshcainsarbteicvlieecwonedtaionnslWineebw-oitnhlyoduattaa.subscription. AmongthetypeII NAD(P)H dehydrogenases, NDB proteinosfpotato(Solariumtuberosuman)dArabidopsis(Arabidopstihs alianaa)re presenton theexternal surfaceof the innermitochondriaml embrane (Rasmussonetal, 1999;Elhafezetal, 2006).TheNDB proteincsharacteristicaplloyssessan inserteddomain carryingmoreor less conservedEF-handmotifsfor calciumbinding(Michaleckeatal.,2003).Uponanalyses ofcytoplasmimc embraneosfEscherichcioaliproducingArabidopsiNs DB fusionproteinsA,tNDB2and AtNDB4oxidizedNADH butnotNADPH, and calciumionsboundto and stimulatedAtNDB2butnot AtNDB4(Geisleretal, 2007).Thiswas consistenwtith thegenerallyobservedpartialcalciumdependenceof externaNl ADH oxidation(Rasmussonetal, 2004).In contrastp,otatoStNDBl expressedin Nicotiansaylvestris catalyzedcalcium-dependenNtADPH oxidation (Michaleckaet al., 2004). ColiexpressedAtNDBl was shown to be specificfor NADPH and tobindcalciumvia itsEF-handdomain as a prerequisitfeoritsactivity(Geisleretal, 2007). Iventhatit is notdirectlylinkedto energyconservatioNn,DB1,likeothertypeII NAD(P)H dehydrogenasesm, ay be able to modulatecellular NADP(H) redoxlevels,irrespectivoefthecellularATP phosphorylatiosntatusI.nN. Sylvestroivserexpressing StNDBland thushavingelevatedlevelsofmitochondrialNADPH dehydrogenasae,decreaseinNADPH/ NADP+ratioindependenotftheNADH/NAD+ratio was observedinleavesduringtheday.Thisprovided a physiologicadl emonstratioinn whichthe NDB1 enzymewas activeand abletospecificallmy odifythe cellularNADP(H) pools (Liu et al, 2008).unnotdyerptewhaesgorbowsetrhvceodn,adnidtioitnisus sheidt,hneortvoniosutaklnpohwenwhatthephysiologicailmportancoef mitochondrial NADPH oxidationis. Here,we reportthatwhengrownunderhigherlight intensitieNs,. sylvestrpilsantsoverexpressinSgtNDBl exhibitan earliertransitionfromrosettestage to bolting,whereasa line suppressingboth StNDBl and NsNDBl is delayedin thisparameterT.he phenotypewas stronglycorrelatedwith stem-specific changesin NADPH reductionlevels.Thesechanges werein turncorrelatedto thelevelsof a relatively smallsetofmetaboliteasnd affectetdheexpressionof gcdheunarneinsgagesbssionolcttihianetwgeadiwsldiw-ttheyflllpoaaesrpaiapcl ahhlaNisgAehtDarPbaHnuns/idNtaiAonnDcSsePp.o+efrcatithfiieoc StNDBl proteinin potatostemmitochondrpiarovide furthesrupportforan importanrtoleofmitochondrial NADPH oxidationinstemdevelopment
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