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 Km [PEP] coupled with elevated I50 [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.

Figure 1.

Time course of proteoid root development for –Pi plants supplemented with 1 µm Pi. A, Each developmental stage is labeled according to the number of days following rootlet emergence observed at day 1 until senescence at day 20. Scale bar = 1 cm. B and C, Rootlets from immature stage I (B) and mature stage III (C) proteoid roots were stained for lignified tissues, cleared, and imaged as described in “Materials and Methods.” Scale bar = 0.35 mm.

Figure 2.

Influence of proteoid root development on PEPC activity, subunit composition, and PTMs. A, All values represent the mean (±se) PEPC-specific activity of duplicate determinations of desalted extracts from n = four biological replicates determined under optimal (pH 8.2, 2.5 mm PEP) or suboptimal (pH 7.2, 0.2 mm PEP, 0.125 mm malate) conditions. All differences in specific activity and suboptimal:optimal activity ratio between stage I and III roots were statistically significant (P < 0.05). B, Hakea extracts were subjected to SDS-PAGE (10% gels; 0.5 μg protein lane⁻¹), electroblotted onto a poly(vinylidene difluoride) membrane, and probed with anti-PEPC. Relative band intensity (±se) was quantified on n = five biological replicates from scanned immunoblots using ImageJ. C, Immunoblots were probed with anti-pSer11 in the presence of 10 μg mL⁻¹ of the corresponding dephosphopeptide or phosphopeptide (20 μg of protein lane⁻¹ for hakea extracts). D, Desalted extracts of stage I roots were incubated with and without 15 μΜ USP-2 and then immunoblotted with anti-PEPC (2 μg protein lane⁻¹). E, In-gel PEPC activity staining followed nondenaturing PAGE of proteoid root extracts (7.5 μg protein lane⁻¹) on 7% gels. The lanes labeled “Castor PEPC” (B and D) contained 50 and 25 ng, respectively, of purified monoubiquitinated Class-1 PEPC (RcPPC3) from germinating COS (Uhrig et al., 2008). The lanes labeled “At PEPC” (C and E) contained 250 and 500 ng, respectively, of purified phosphorylated Class-1 PEPC (AtPPC1) from –Pi Arabidopsis suspension cells (Gregory et al., 2009).

Figure 3.

Kinetic constants of PEPC in desalted extracts prepared from stage I versus stage III proteoid roots. A and C, All assays were performed at pH 7.2. I₅₀ (malate and Asp) and K_a (Glc-6-P and Gly-3-P) values were determined using subsaturating PEP (0.2 mm). A, Values in parentheses represent mean (±se) fold activation of PEPC by saturating Glc-6-P or Gly-3-P. B, Cellular concentration of malate and Glc-6-P in stage I versus stage III roots. C, Influence of combined presence of 0.25 mm malate and 2.5 mm Asp on Glc-6-P activation of PEPC from stage III roots. All values represent the mean (±se) of duplicate determinations on n = four biological replicates. The asterisks denote statistically significant differences (P < 0.05).

Figure 4.

Coelution of PEPC activity with p110 and p107 during Superdex 200 FPLC of PEPC from stage I and II proteoid roots. Aliquots (1 μL) from various fractions along with purified PEPC (50 ng) from germinating COS (“Castor PEPC”) were subjected to immunoblotting with anti-PEPC. V_o denotes the void volume.

Figure 5.

MALDI Q-TOF MS spectra of p110 (A) and p107 (B) tryptic peptides derived from purified PEPC of stage I and II proteoid roots. C, MALDI Q-TOF MS/MS analysis of four peptides common to both subunits (boldface in A and B) yielded identical amino acid sequences.

Figure 6.

Influence of in vitro dephosphorylation by PP2A (A and B) or deubiquitination by USP-2 (C and D) on activity of PEPC purified from stage I and II proteoid roots. A, Immunoblot analysis was performed using anti-PEPC or anti-pSer11 (75 ng or 1 μg of hakea PEPC lane⁻¹, respectively. “Castor PEPC” denotes 50 ng of monoubiquitinated PEPC (RcPPC3) purified from germinating COS (Uhrig et al., 2008), whereas “At PEPC” denotes 500 ng of phosphorylated-PEPC (AtPPC1) purified from –Pi Arabidopsis (Gregory et al., 2009). B, Time course for PP2A-dependent inhibition of hakea PEPC activity (±50 nm microcystin-LR). C, Immunoblot analysis was performed using anti-PEPC, anti-pSer11, or antiubiquitin (75 ng, 1 μg, or 4 μg of hakea PEPC lane⁻¹, respectively; reference lanes contained 50 ng of germinating COS PEPC, 0.5 μg of –Pi Arabidopsis PEPC, or 1 μg of germinating COS PEPC, respectively). D, Influence of USP-2 mediated deubiquitination, followed by bovine heart protein kinase-A mediated phosphorylation after 30 min, on activity of hakea PEPC. PEPC was assayed using suboptimal conditions (pH 7.2, 0.2 mm PEP, 0.125 mm malate). All values represent the mean (±se) of n = three independent experiments.