Abstract
The aim of this work was to investigate the effect of decreased cytosolic pyruvate kinase (PKc) on potato (Solanum tuberosum) tuber metabolism. Transgenic potato plants with strongly reduced levels of PKc were generated by RNA interference gene silencing under the control of a tuber-specific promoter. Metabolite profiling showed that decreased PKc activity led to a decrease in the levels of pyruvate and some other organic acids involved in the tricarboxylic acid cycle. Flux analysis showed that this was accompanied by changes in carbon partitioning, with carbon flux being diverted from glycolysis toward starch synthesis. However, this metabolic shift was relatively small and hence did not result in enhanced starch levels in the tubers. Although total respiration rates and the ATP to ADP ratio were largely unchanged, transgenic tubers showed a strong decrease in the levels of alternative oxidase (AOX) protein and a corresponding decrease in the capacity of the alternative pathway of respiration. External feeding of pyruvate to tuber tissue or isolated mitochondria resulted in activation of the AOX pathway, both in the wild type and the PKc transgenic lines, providing direct evidence for the regulation of AOX by changes in pyruvate levels. Overall, these results provide evidence for a crucial role of PKc in the regulation of pyruvate levels as well as the level of the AOX in heterotrophic plant tissue, and furthermore reveal that these parameters are interlinked in vivo.
Highlights
Pyruvatke inase(PK;EC 2.7.1.40c) atalyzesthefinal fromhexose-phosphatteos pyruvateowingto PEP's reactionof theglycolytipcathwayc,onvertinAg DP pronouncedfeedbackallosteriicnhibitioonfATP-and andphosphoenoZpyruv(PatEeP)toATPandpyruvate, inorganicpyrophosphate-dependepnhtosphofructowhichis subsequentlyimportedintomitochondria kinases(Dennisand Greyson1,987).PK inplantshas iarnrdeuvseerdsaisbalreseuacbtsitornaintfevorirveosipnircaatsitoonPrK.becaanta(lRyizceisannus been foundto be subjectto a numberofregulatory mechanismsi,ncludingallostericregulationby Glu communiGs'e,igenbergeertal.,1993)andis probablya andAsp (Turneretal.,2005),activatiobnychangesin primaryregulationsiteofglycolysiasnd respiration, cytosolicpH (Podestaand Plaxton1991,1992,1994), as indicatedbyvariousbiochemicasltudiesin mammalianand bacterialsystems(Pilkisand Granner, inhibitionby ATP (Podestaand Plaxton,1991,1992, 1993),and phosphorylatioannd ubiquitinatio(nTang
Internal oxygen concentrationswere measured with a needle-type, 230-/im-diameterm, inimallyinvasive oxygen microoptode, which was directlyinserted into growing tubers of similar size
Consistent with the lack of changesin respirationratesin discs,reductionofPKc did not lead to significantchanges in internaloxygen levels of growing tubers
Summary
A setoftobacco(Nicotiantaabacump)lantslackingPKc alternativpeathwayof respirationin heterotrophic in leaves was previouslyanalyzedwithrespectto potatotubers. Growthp,hotosynthesains,drespirationan,da number ofconditionaglrowthdefectws ereidentifiedm, ainly as a consequenceofalteredregulationofsink-source metabolismdue toa lossofPKc,specificalliynleaves
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