Abstract
Glycolysis is a central catabolic pathway in every living organism with an essential role in carbohydrate breakdown and ATP synthesis, thereby providing pyruvate to the tricarboxylic acid cycle (TCA cycle). The cytosolic pyruvate kinase (cPK) represents a key glycolytic enzyme by catalyzing phosphate transfer from phosphoenolpyruvate (PEP) to ADP for the synthesis of ATP. Besides its important functions in cellular energy homeostasis, the activity of cytosolic pyruvate kinase underlies tight regulation, for instance by allosteric effectors, that impact stability of its quaternary structure. We determined five cytosol-localized pyruvate kinases, out of the fourteen putative pyruvate kinase genes encoded by the Arabidopsis thaliana genome, by investigation of phylogeny and localization of yellow fluorescent protein (YFP) fusion proteins. Analysis of promoter β-glucuronidase (GUS) reporter lines revealed an isoform-specific expression pattern for the five enzymes, subject to plant tissue and developmental stage. Investigation of the heterologously expressed and purified cytosolic pyruvate kinases revealed that these enzymes are differentially regulated by metabolites, such as citrate, fructose-1,6-bisphosphate (FBP) and ATP. In addition, measured in vitro enzyme activities suggest that pyruvate kinase subunit complexes consisting of cPK2/3 and cPK4/5 isoforms, respectively, bear regulatory properties. In summary, our study indicates that the five identified cytosolic pyruvate kinase isoforms adjust the carbohydrate flux through the glycolytic pathway in Arabidopsis thaliana, by distinct regulatory qualities, such as individual expression pattern as well as dissimilar responsiveness to allosteric effectors and enzyme subgroup association.
Highlights
During plant development and adaptation to environmental changes, the glycolytic network provides an enormous metabolic flexibility
The Arabidopsis thaliana genome encodes several putative cytosolic and plastidial pyruvate kinase (PK), and glycolytic metabolites can be exchanged between the cytosol and plastids [1] since both compartments are connected through diverse transporters located in the inner plastid envelope membrane [2,3,4]
PK5 by heterologous expression of yellow fluorescent protein (YFP) fusion constructs in Nicotiana benthamiana, we aimed to identify the different roles of cytosolic PK enzymes in dependence of changing developmental and environmental conditions
Summary
During plant development and adaptation to environmental changes, the glycolytic network provides an enormous metabolic flexibility. Thereby, flux regulation is achieved by the fine control of key regulatory enzymes, including pyruvate kinase (PK). On the other hand, reduced pyruvate kinase activity will lead to a backlog of PEP and other glycolytic intermediates, thereby increasing the flux rate of carbon skeletons into branching biosynthetic pathways. Despite the assumable key regulatory function of pyruvate kinase so far, only plastidial isoforms have been described [5]. The high number of isoenzymes with potential redundant physiological roles, as well as the compartmentalized system with glycolytic intermediates equilibrating through plastid membrane transporters, may hamper their investigation
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