Transcriptional and Biochemical Characterization of Cytosolic Pyruvate Kinases in Arabidopsis thaliana

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.

Tonoplast-associated calcium signaling regulates manganese homeostasis in Arabidopsis

“…We found that CPK4/5/6/11 and MTP8 have similar expression patterns and are mainly expressed in whole young seedlings and in the leaves, seeds, and flowers of mature plants (Supplemental Figure 3A). In addition, previous research used GUS staining or RT-PCR to demonstrate that CPK4/5/6/11 are expressed mainly in roots, leaves, and cotyledons in the seedling stage (Zhu et al, 2007;Wulfert et al, 2020;Zhang et al, 2020). MTP8 is also mainly expressed in roots under Fe deficiency and its weak expression in cotyledons is not altered under Fe deficiency.…”

A synthetic C4 shuttle via the β-hydroxyaspartate cycle in C3 plants

“…Cytosolic phosphoenolpyruvate carboxykinase 1 (PCK1) could decarboxylate OAA to phosphoenolpyruvate [PEP ( 33 )]. PEP is then used either for gluconeogenesis or converted into pyruvate by pyruvate kinase [PK ( 34 )]. The accumulation of pyruvate strongly indicated that the cytosolic PK route is active in BHAC plants and that PEP is not channeled into gluconeogenesis, which was supported by reduced glucose and fructose levels in the same plants ( Fig.…”

MUC5AC Expression in Various Tumor Types and Nonneoplastic Tissue: A Tissue Microarray Study on 10 399 Tissue Samples

“…28 Studies have shown that the putative underlying mechanisms of MUC5AC neo-expression include promotor hypomethylation. 11 MUC5AC transcription is also directly and indirectly regulated by various cancer-associated transcription factors (eg AP-1, SP-1, NF-kB, HNF-5a-alpha) and pathways (eg MAPK, Akt/ PI3K) [39][40][41] as well as by several mediators of inflammation (IL-1-beta, IL-6, TNF-alpha, IL-13). [41][42][43][44] Tumor types that are always MUC5AC negative are of diagnostic interest, because a positive MUC5AC immunostaining will virtually exclude such entities from diagnostic considerations.…”