Reciprocal Control of Anaplerotic Phosphoenolpyruvate Carboxylase by in Vivo Monoubiquitination and Phosphorylation in Developing Proteoid Roots of Phosphate-Deficient Harsh Hakea    

Abstract

Accumulating evidence indicates important functions for phosphoenolpyruvate (PEP) carboxylase (PEPC) in inorganic phosphate (Pi)-starved plants. This includes controlling the production of organic acid anions (malate, citrate) that are excreted in copious amounts by proteoid roots of nonmycorrhizal species such as harsh hakea (Hakea prostrata). This, in turn, enhances the bioavailability of mineral-bound Pi by solubilizing Al 3+ , Fe 3+ , and Ca 2+ phosphates in the rhizosphere. Harsh hakea thrives in the nutrient-impoverished, ancient soils of southwestern Australia. Proteoid roots from Pi-starved harsh hakea were analyzed over 20 d of development to correlate changes in malate and citrate exudation with PEPC activity, posttranslational modifications (inhibitory monoubiquitination versus activatory phosphorylation), and kinetic/allosteric properties. Immature proteoid roots contained an equivalent ratio of monoubiquitinated 110-kD and phosphorylated 107-kD PEPC polypeptides (p110 and p107, respectively). PEPC purification, immunoblotting, and mass spectrometry indicated that p110 and p107 are subunits of a 430-kD heterotetramer and that they both originate from the same plant-type PEPC gene. Incubation with a deubiquitinating enzyme converted the p110:p107 PEPC heterotetramer of immature proteoid roots into a p107 homotetramer while significantly increasing the enzyme's activity under suboptimal but physiologically relevant assay conditions. Proteoid root maturation was paralleled by PEPC activation (e.g. reduced K m [PEP] coupled with elevated I 50 [malate and Asp] values) via in vivo deubiquitination of p110 to p107, and subsequent phosphorylation of the deubiquitinated subunits. This novel mechanism of posttranslational control is hypothesized to contribute to the massive synthesis and excretion of organic acid anions that dominates the carbon metabolism of the mature proteoid roots.

Light-dependent activation of phosphoenolpyruvate carboxylase by reversible phosphorylation in cluster roots of white lupin plants: diurnal control in response to photosynthate supply

“…2B indicating that PEPC's phosphorylation status was not markedly altered as the CRs matured. These findings agree with our study of PEPC in immature CRs of harsh hakea showing that only p107 (and not the monoubiquitinated p110 subunit) is phosphorylated (Shane et al, 2013). Non-denaturing PAGE followed by in-gel activity staining yielded a single PEPC activity band at each stage of CR development that comigrated with homogeneous 440-kDa PEPC purified from ÀPi arabidopsis suspension cells (Fig.…”

Plant adaptations to severely phosphorus-impoverished soils

“…Enzymes involved in malate and citrate metabolism, such as PEP carboxylase [54,55] and aconitase [30], show greater and lower activities, respectively, in cluster roots of white lupin. PEP carboxylase is activated by deubiquitination followed by phosphorylation [56]. Activities of enzymes of carbohydrate metabolism, such as sucrose synthase, are also increased during cluster-root development [57].…”

Evolution of the Phosphoenolpyruvate Carboxylase Protein Kinase Family in C3 and C4 Flaveria spp.

“…Monoubiquitination of PEPC has been identified only recently; it was found to be tissue specific and to influence the feedback regulation of PEPC by various metabolites (Agetsuma et al, 2005;Uhrig et al, 2008;O'Leary et al, 2011;Shane et al, 2013).…”