Phosphatases are involved in modulating the TCA cycle in plants

Phosphorylation is a reversible posttranslational modification that modulates the activities of numerous proteins. In plants, it is known that several mitochondrial proteins can be phosphorylated, including enzymes of the tricarboxylic acid (TCA) cycle. The kinases and phosphorylases that mediate reversible phosphorylation of TCA cycle enzymes remain largely elusive, as is the impact of protein phosphorylation on the TCA cycle. In this issue of Molecular Plant, Zhang et al., 2021Zhang Y. Giese J. Mae-Lin Kerbler S. Siemiatkowska B. Perez de Souza L. Alpers J. Medeiros D.B. Hincha D.K. Daloso D.M. Stitt M. et al.Two mitochondrial phosphatases, PP2c63 and Sal2, are required for posttranslational regulation of the TCA cycle in Arabidopsis.Mol. Plant. 2021; https://doi.org/10.1016/j.molp.2021.03.023Abstract Full Text Full Text PDF Scopus (7) Google Scholar demonstrate that two phosphatases play important roles in controlling the phosphorylation status of TCA cycle enzymes, which modulates their activities and influences carbon flux through the TCA cycle. The TCA cycle is one of the core pathways in plants and is central to aerobic respiration. It is considered an amphibolic pathway in plants, involved in the oxidative catabolism of carbohydrates, amino acids, and fatty acids, as well as providing precursors for the biosynthesis of nucleotides, fatty acids, steroids, several amino acids, and other cellular metabolites. The demand for reducing equivalents derived from catabolic reactions of the TCA cycle and for TCA cycle intermediates as precursors in anabolic reactions changes diurnally and in response to environmental conditions. Balancing ever-changing needs for energy and biosynthesis precursors requires regulation of the TCA cycle at several levels that is influenced by multiple stimuli. Operation of the TCA cycle is influenced by several factors, including enzyme expression and turnover, posttranslational modifications, and enzyme effectors. Exposing plants to various stresses causes induction of transcripts encoding enzymes of the TCA cycle as well as changes to the overall levels of TCA cycle enzymes (Lehmann et al., 2009Lehmann M. Schwarzländer M. Obata T. Sirikantaramas S. Burow M. Olsen C.E. Tohge T. Fricker M.D. Møller B.L. Fernie A.R. The metabolic response of Arabidopsis roots to oxidative stress is distinct from that of heterotrophic cells in culture and highlights a complex relationship between the levels of transcripts, metabolites, and flux.Mol. Plant. 2009; 2: 390-406Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Several potential phosphorylation, acetylation, and redox regulation sites for enzymes of the TCA cycle have been identified (Bykova et al., 2003Bykova N.V. Egsgaard H. Møller I.M. Identification of 14 new phosphoproteins involved in important plant mitochondrial processes.FEBS Lett. 2003; 540: 141-146Crossref PubMed Scopus (105) Google Scholar; König et al., 2014König A.C. Hartl M. Boersema P.J. Mann M. Finkemeier I. The mitochondrial lysine acetylome of Arabidopsis.Mitochondrion. 2014; 19: 252-260Crossref PubMed Scopus (64) Google Scholar). In Arabidopsis, thioredoxin was shown to act on redox regulatory sites on succinate dehydrogenase and fumarase, essentially fine-tuning the activities of these enzymes and modulating flux through the TCA cycle based on redox status (Daloso et al., 2015Daloso D.M. Müller K. Obata T. Florian A. Tohge T. Bottcher A. Riondet C. Bariat L. Carrari F. Nunes-Nesi A. et al.Thioredoxin, a master regulator of the tricarboxylic acid cycle in plant mitochondria.Pro. Natl. Acad. Sci. U S A. 2015; 112: 1107-1392Crossref PubMed Scopus (119) Google Scholar). Several small-molecule effectors that impact the activities of TCA cycle enzymes have been identified (Araujo et al., 2012Araujo W.L. Nunes-Nesi A. Nikoloski Z. Sweetlove L.J. Fernie A.R. Metabolic control and regulation of the tricarboxylic acid cycle in photosynthetic and heterotrophic plant tissues.Plant Cell Environ. 2012; 35: 1-21Crossref PubMed Scopus (191) Google Scholar). Pyruvate dehydrogenase complex (PDC), the enzyme that links glycolysis and the TCA cycle and whose activity strongly influences flux through the TCA cycle, is activated by pyruvate and thiamin diphosphate and inhibited by NADH, acetyl-CoA, ammonium, and glyoxylate (Tovar-Méndez et al., 2003Tovar-Méndez A. Miernyk J.A. Randall D.D. Regulation of pyruvate dehydrogenase complex activity in plant cells.Eur. J. Biochem. 2003; 270: 1043-1049Crossref PubMed Scopus (205) Google Scholar). It has recently been shown that promiscuous activities of TCA cycle enzymes can form noncanonical metabolites that are enzyme effectors; their formation and removal also influences the TCA cycle (Niehaus and Hillmann, 2020Niehaus T.D. Hillmann K.B. Enzyme promiscuity, metabolite damage, and metabolite damage control systems of the tricarboxylic acid cycle.FEBS J. 2020; 287: 1343-1358Crossref PubMed Scopus (10) Google Scholar). Although several instances of TCA cycle regulation have been reported, our understanding of the factors that control the TCA cycle are still largely fragmentary. For example, dozens of potential phosphosites have been identified in Arabidopsis TCA cycle enzymes (Zulawski et al., 2012Zulawski M. Braginets R. Schulze W.X. PhosPhAt goes kinases—searchable protein kinase target information in the plant phosphorylation site database PhosPhAt.Nucleic Acids Res. 2012; 41: D1176-D1184Crossref PubMed Scopus (86) Google Scholar). However, only a few of the enzymes involved in phosphorylation and dephosphorylation of these enzymes are known. Mitochondrial PDC is phosphorylated by PDC kinase (PDK) and dephosphorylated by phospho-PDC phosphatase, whose sequence is unknown (Tovar-Méndez et al., 2003Tovar-Méndez A. Miernyk J.A. Randall D.D. Regulation of pyruvate dehydrogenase complex activity in plant cells.Eur. J. Biochem. 2003; 270: 1043-1049Crossref PubMed Scopus (205) Google Scholar). Phosphorylation of other plant TCA cycle enzymes has not been characterized in detail. To investigate the role of phosphorylation in regulating the TCA cycle, Zhang et al., 2021Zhang Y. Giese J. Mae-Lin Kerbler S. Siemiatkowska B. Perez de Souza L. Alpers J. Medeiros D.B. Hincha D.K. Daloso D.M. Stitt M. et al.Two mitochondrial phosphatases, PP2c63 and Sal2, are required for posttranslational regulation of the TCA cycle in Arabidopsis.Mol. Plant. 2021; https://doi.org/10.1016/j.molp.2021.03.023Abstract Full Text Full Text PDF Scopus (7) Google Scholar studied two candidate phosphatases, PP2c63 and Sal2, that were previously shown to weakly interact with TCA cycle enzymes, along with the previously characterized PDK. Arabidopsis T-DNA insertion mutants were obtained for pp2c63, sal2, and pdk; pp2c63 sal2 double mutant and pp2c63 sal2 pdk triple mutant plants had markedly decreased growth rates. Quantitative phosphoproteomics indicated that PP2c63 and Sal2 cooperate in the dephosphorylation of PDC and are also likely involved in dephosphorylation of malate dehydrogenase and fumarase. Enzyme assays performed with isolated mitochondrial extracts showed that the Michaelis constant (Km) of PDC for pyruvate was lower in pdk mutant plants and higher in pp2c63 and pp2c63 sal2 mutant plants. The Km of fumarase also increased in pp2c63 and pp2c63 sal2 mutant plants. Furthermore, PDC and fumarase activities in isolated mitochondrial extracts from pp2c63 mutant cells could be altered by incubation with recombinant PP2c63 enzyme and PP2c63 was shown to physically interact with PDC in vivo. Metabolomics and heavy-isotope feeding experiments confirmed that the TCA cycle is altered in mutant plants. The data presented by Zhang et al. elegantly demonstrate that PP2c63 and Sal2 both play a role in the phosphorylation status of PDC and other TCA cycle enzymes and, by doing so, modulate their enzyme activities and impact carbon flux through the pathway (Figure 1). This is a significant finding for several reasons. The severe phenotypes of the Arabidopsis mutants indicate that regulation of the TCA cycle via enzyme phosphorylation is critical for normal growth and development. Thus, not only does this work improve our understanding of regulation of the plant TCA cycle, it justifies further studies on the enzymes described here and other kinases and phosphatases that act on TCA cycle enzymes. The phosphorylation status of several proteins not directly involved in the TCA cycle were also altered in pp2c63 and/or sal2 mutant plants, indicating that phosphoregulation of metabolism extends beyond the TCA cycle. Further experimentation is needed to understand the complex interplay of factors regulating the TCA cycle and mechanisms of crosstalk that coordinately influence a larger metabolic network. Finally, PP2c63 is a member of the D-clade of PP2c phosphatases, which have previously been shown to interact with SAUR proteins (Ren et al., 2018Ren H. Park M.Y. Spartz A.K. Wong J.H. Gray W.M. A subset of plasma membrane localized PP2C. D phosphatases negatively regulate SAUR-mediated cell expansion in Arabidopsis.PLoS Genet. 2018; 14: e1007455Crossref PubMed Scopus (43) Google Scholar). SAUR proteins are key components of hormone (e.g., auxin) signaling pathways. The association of PP2c63 with SAURS—and the observation that phosphorylation of SOUR78 and SAUR9 were impacted in this study—evokes the possibility that hormones are involved in phosphoregulation of the TCA cycle. No conflict of interest declared.