Essay 8.1

Modulation of Phosphoenolpyruvate Carboxylase in C₄ and CAM Plants

Bob B. Buchanan, Department of Plant & Microbial Biology, University of California, Berkeley; Ricardo A. Wolosiuk, Instituto de Investigaciones Bioquímicas, University of Buenos Aires

August, 2002

The CAM and C₄ mechanisms we discussed in Chapter 8 require the separation of the initial carboxylation from the subsequent decarboxylation, to avoid a futile cycle. Therefore, three well-defined processes are necessary for the assimilation of CO₂ into organic compounds:

(1) the cytosolic fixation of CO₂ by a three-carbon compound yielding a four-carbon compound,

(2) the transport of the latter compound to the chloroplast where its decarboxylation releases CO₂ for assimilation by rubisco in the Calvin cycle, and

(3) the regeneration of the three-carbon compound in the cytosol for restarting the assimilatory cycle.

Although the two types of plants exhibit large differences in metabolites that participate in the second and third steps, they share a common mechanism for the first, i.e., the irreversible β-carboxylation of phosphoenolpyruvate (PEP) by HCO₃^- with the concurrent production of oxaloacetate and orthophosphate that is catalyzed by the enzyme phosphoenolpyruvate carboxylase (PEPCase) in the presence of Mg²⁺ (Figure 1). Experiments using ¹⁸O-labeled HCO₃^- indicate that PEPCase, a homotetramer with a 110 kDa subunit, catalyzes the efficient incorporation of two equivalents of ¹⁸O in the γ-carboxyl group of oxaloacetate and one in orthophosphate.

Figure 1 The reaction catalyzed by PEPCase. The production of oxaloacetate [OAA] and orthophosphate [P_i] requires the ordered incorporation of substrates phosphoenolpyruvate [PEP] and HCO₃^– and the cofactor Mg²⁺, to the enzyme [E] for the formation of the quaternary complex [E- Mg²⁺- PEP - HCO₃^–-]. The first (rate-limiting) step is the transfer of the phosphate for the formation of carboxyphosphate, which subsequently decomposes into E-CO₂ and orthophosphate. In the final step of the overall reaction, the enzyme-bound CO₂ combines with the enolate stabilized by Mg²⁺ and the reaction proceeds to the release of OAA and P_i. (Click image to enlarge.)

Precise initial velocity studies revealed that the reaction proceeds through an ordered mechanism in which the enzyme first binds Mg²⁺, subsequently PEP, and finally HCO₃^- (Maruyama et al. 1966; Jane et al. 1992). The very low Km for HCO₃^- (in the micromolar range) facilitates both the fixing of atmospheric CO₂ at night in CAM plants and the recapturing of photorespiratory CO₂during the day in C₄ plants. Malate is a competitive inhibitor (Ki = 0.17 mM) when PEPCase is assayed at pH 7.3 in the presence of 1 mM PEP (Chollet et al. 1996; Vidal and Chollet et al. 1997). However, the allosteric activator glucose 6-P (Ka = 1.3 mM) not only increases the affinity for the PEP but also relieves the inhibition by malate. The high dependence of these effects on pH suggests that, in vivo, negative and positive effectors act in concert with cytosolic pH in fine-tuning the activity of PEPCase.

Post-Translational Regulation of PEPCase Activity

In addition to the spatial and temporal separation exhibited by CAM and C₄ plants, respectively, a futile cycle is avoided by the regulation of PEPCase. The PEPCase is "switched on" (active) during the night in CAM plants and during the day in C₄ plants. In both plant types, PEPCase is inhibited by malate and activated by glucose-6-phosphate. In CAM plants, the upregulation at night and downregulaton during the day of PEPCase parallels the fixation of atmospheric CO₂ and the titratable acidity of the leaf (Leegood and Osmond 1990). Experiments under continuous light or darkness revealed that, not only diminished sensitivity to malate but also an endogenous circadian rhythm, is responsible for the periodic activity of PEPCase. In contrast, in C₄ plants the activating agent is light, which reversibly activates the enzyme in vivo. In both CAM and C₄ plants, the phosphorylation of a single serine residue in the N-terminal region of the 110 kDa subunit renders PEPCase less sensitive to inhibition by malate, more sensitive to activation by glucose-6-P and, as a consequence, more active in the catalytic process. Germane to this particular phosphorylation is the PEPCase-kinase (PEPCase-K, ca.33 kDa) which has high affinity for both CAM and C₄ PEPCase but lacks the capacity to phosphorylate heterologous substrates or PEPCase mutants in which functional serines of the N-terminal region are mutated (Figure 2).

Figure 2 Working model for the modulation of PEPCase in C₄ and CAM plants. First, the alkalinization of the cytosolic pH stimulates the conversion of phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] to inositol-1,4,5-trisphosphate [Ins(1,4,5)P3] and 1,2-diacylglycerol [DAG] in a reaction catalyzed by the phosphoinositide-specific phospholipase C [PI-PLC]. Subsequently, the Ins(1,4,5)P3 drives the tonoplast to enhance the cytosolic concentration of Ca²⁺ thereby activating a Ca²⁺/calmodulin-dependent protein kinase. Finally, the latter kinase directly or indirectly enhances protein synthesis with the consequent increase in expression of the PEPCase-K gene. (Click image to enlarge.)

The parallel induction of both PEPCase and PEPCase-K during the transition from C₃ to CAM metabolism elicited by salt stress in Mesembryanthenmum crystallinum provides further support to the close linkage between the enzyme and the associated kinase (Nimmo 2000). Studies with both leaves and mesophyll cell protoplasts of M. crystallinum stressed with high salt indicate that the apparent discrepancy resides in variations of a common central mechanism (Bakrim et al. 2001). Drugs that inhibit (1) the activity of phosphoinositide-specific phospholipase C; (2) the functioning of the tonoplast Ca²⁺-channel; (3) the activity of Ca²⁺/calmodulin-dependent protein kinases; and (4) cytosolic protein synthesis concurrently block the up-regulation of PEPCase-K and the phosphorylated (active) PEPCase. These collective findings suggest the functioning of a signal transduction cascade in which phosphoinositide-specific phospholipase C catalyzes the production of the second messenger inositol-1,4,5-trisphosphate [phosphatidylinositol-4,5-bisphosphate → 1,2-diacylglycerol + inositol-1,4,5-trisphosphate] for the stimulation of the Ca²⁺ efflux from the vacuole to the cytosol (see Figure 2). Upon the increase of Ca²⁺ in the cytosol, a Ca²⁺-dependent protein kinase(s) is believed to stimulate either the translation of the PEPCase-K mRNA or a putative activation factor, via phosphorylation. The most probable elicitor to trigger the operation of this signal transduction cascade is cytosolic pH because the up-regulation of PEPCase-K and the associated phosphorylation of the up-regulation of PEPCase are highly stimulated by the alkalinization of the cytosol. As these events operate under illumination in C₄ plants and in the dark in CAM plants, it becomes evident that, at the time of CO₂ fixation, different metabolic signals should trigger changes in pH. Currently, 3-phosphoglycerate—generated by photosynthesis—and malate—transported across the tonoplast—have been suggested as messengers for inducing the alkalinization of cytosolic pH, thereby up-regulating the PEPCase-K of C₄ and CAM plants, respectively.

In CAM plants, transcripts of the Ppck gene that codes for PEPCase-K accumulate mainly at night, causing the accumulation of PEPCase-K and the consequent phosphorylation of PEPCase (Figure 3). CAM plants contain further levels of regulation for the photosynthetic process. Salt- and drought-stress induce the rapid expression of both the Ppc and the Ppck genes that code for PEPCase and PEPCase-K, respectively. Moreover, a circadian oscillator enhances the cytosolic concentration of malate by increasing its efflux from the vacuole, thereby lowering Ppck gene expression, PEPCase-K activity, and PEPCase phosphorylation (low catalytic capacity PEPCase) (Borland and Taybi 2004). At night, the oscillator switches the malate flux across the tonoplast and, as a consequence, reverses these three concurrent events (high catalytic capacity PEPCase). Hence, the concerted action of the circadian oscillator and metabolites contributes to the day/night modulation of CAM PEPCase. C₄ plants do not exhibit this behavior but instead up-regulate the PEPCase-K and the associated phosphorylation of PEPCase (high catalytic capacity PEPCase) by a light-dependent transduction cascade (Giglioli-Guivarc′h et al. 1996; Coursol et al. 2000).

Figure 3 Diurnal regulation of CAM PEPCase. Phosphorylation of the serine residue (Ser-OP) yields a form of the enzyme relatively insensitive to malate that is active at night. During the day, the dephosphorylation of the specific serine (Ser-OH) by the phosphatase (PP2A) gives a form of the enzyme, which is inhibited by malate. Associated to this process environmental factors and a circadian oscillator modulate the expression of both the Ppc and the Ppck genes that code for PEPCase and PEPCase-K. (Click image to enlarge.)

References

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Borland, A. M., and Taybi, T. (2004) Synchronization of metabolic processes in plants with crassulacean acid metabolism. J. Exp. Bot. 55: 1255–1265.

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Coursol, S., Giglioli-Guivarc′h, N., Vidal, J., and Pierre J.-N. (2000) An increase in the phosphoinositide-specific phospholipase C activity precedes induction of C₄ phosphoenolpyruvate carboxylase phosphorylation in illuminated and NH₄Cl-treated protoplasts from Digitaria sanguinalis. Plant J. 23: 497–506.

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