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First published online June 22, 2007; 10.1104/pp.107.102541 Plant Physiology 144:1936-1945 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
Phosphorylation of Phosphoenolpyruvate Carboxylase Is Not Essential for High Photosynthetic Rates in the C4 Species Flaveria bidentis1,[OA]Department of Biological Science, Hiroshima University, Higashihiroshima 739–8526, Japan (T.F); Department of Biotechnological Science, Kinki University, Wakayama 649–6493, Japan (K.I.); Molecular Plant Physiology Group, Research School of Biological Sciences, Australian National University, Canberra, Australian Capital Territory 2601, Australia (V.Q., S.v.C.); and Commonwealth Scientific and Industrial Research Organization, Division of Plant Industry, Canberra, Australian Capital Territory 2601, Australia (R.T.F.)
Phosphoenolpyruvate carboxylase (PEPC; EC4.1.1.31) plays a key role during C4 photosynthesis. The enzyme is activated by metabolites such as glucose-6-phosphate and inhibited by malate. This metabolite sensitivity is modulated by the reversible phosphorylation of a conserved serine residue near the N terminus in response to light. The phosphorylation of PEPC is modulated by a protein kinase specific to PEPC (PEPC-PK). To explore the role PEPC-PK plays in the regulation of C4 photosynthetic CO2 fixation, we have transformed Flaveria bidentis (a C4 dicot) with antisense or RNA interference constructs targeted at the mRNA of this PEPC-PK. We generated several independent transgenic lines where PEPC is not phosphorylated in the light, demonstrating that this PEPC-PK is essential for the phosphorylation of PEPC in vivo. Malate sensitivity of PEPC extracted from these transgenic lines in the light was similar to the malate sensitivity of PEPC extracted from darkened wild-type leaves but greater than the malate sensitivity observed in PEPC extracted from wild-type leaves in the light, confirming the link between PEPC phosphorylation and the degree of malate inhibition. There were, however, no differences in the CO2 and light response of CO2 assimilation rates between wild-type plants and transgenic plants with low PEPC phosphorylation, showing that phosphorylation of PEPC in the light is not essential for efficient C4 photosynthesis for plants grown under standard glasshouse conditions. This raises the intriguing question of what role this complexly regulated reversible phosphorylation of PEPC plays in C4 photosynthesis.
Phosphoenolpyruvate (PEP) carboxylase (PEPC) catalyzes the irreversible  -carboxylation of PEP in the presence of HCO3– and Mg2+ to yield oxaloacetate and inorganic phosphate (Chollet et al., 1996 ; Izui et al., 2004). PEPC is a ubiquitous cytosolic enzyme in higher plants and has an anaplerotic function replenishing the tricarboxylic acid cycle with intermediates. It is also involved in pH regulation, stomatal opening, and fruit ripening (for review, see Chollet et al., 1996). In leaves of C4 species, it is an essential component of the biochemical concentration mechanism that provides elevated CO2 partial pressure (pCO2) at the site of Rubisco carboxylation. Initially, carbonic anhydrase converts atmospheric CO2 to HCO3–, which is then fixed by PEPC to produce C4 acids, which diffuse to and are decarboxylated in the bundle sheath to supply CO2 for Rubisco. As a consequence, Rubisco operates near its Vmax, photorespiration is suppressed, and C4 photosynthesis is essentially CO2 saturated at ambient pCO2 (Hatch, 1987). There are several isoforms of PEPC in higher plants encoded by small multigene families and specific C4 isoforms have been identified (Chollet et al., 1996; Izui et al., 2004). Several of these isoforms are present in leaves of C4 plants, but the activity of the C4 isozyme predominates (Huber et al., 1994; Dever et al., 1995). In vitro, PEPC activity is modulated by a wide range of effectors, such as phosphorylated intermediates (triose- and hexose-P), amino acids, and organic acids (Doncaster and Leegood, 1987; Vidal and Chollet, 1997). For example, following earlier discoveries of the role of malate in the regulation of PEPC during Crassulacean acid metabolism (Winter, 1982), the C4 enzyme was found to be more sensitive to malate inhibition when extracted from darkened leaves than when extracted from illuminated leaves (Huber et al., 1986; Doncaster and Leegood, 1987). This change in sensitivity of the enzyme was later shown to be associated with an increase in the phosphorylation state of the enzyme in the light (Nimmo et al., 1987a, 1987b; Jiao and Chollet, 1991).
Phosphorylation of C4-PEPC at its conserved Ser residue near the N terminus is thought to be controlled primarily by the light-dependent activity of a protein kinase specific to PEPC (PEPC-PK) because the activity of the protein phosphatase 2A involved in PEPC dephosphorylation appears to be constant under various conditions (Vidal and Chollet, 1997
The efficiency of the C4 pathway and the pCO2 attained in the bundle sheath is related to both the bundle sheath resistance to CO2 diffusion and the relative biochemical capacities of the C3 and C4 cycle. Because the C4 cycle consumes energy in the form of ATP during the regeneration of PEP, leakage of CO2 out of the bundle sheath is an energy cost to the leaf and coordination between the C3 and C4 cycle is important. Biochemical interdependence of the two cycles exists at several levels (Hatch and Osmond, 1976
Characterization of Primary Transformants and T1 Progeny
We isolated a full-length PEPC-PK cDNA from the F. bidentis leaf cDNA library as described in "Materials and Methods" (accession no. AB272061). In the coding region, 843 bp, five nucleotide residues were different between F. bidentis and F. trinervia PEPC-PK genes and three of them introduced substitutions of amino acid sequences. Thus, the primary sequence of the F. bidentis PEPC-PK was almost identical to that of F. trinervia PEPC-PK and similar to other PEPC-PK sequences as described previously (Tsuchida et al., 2001
To ensure the survival of primary transformants with low PEPC-PK activity in the event of a deleterious growth phenotype in air, plants were raised in growth chambers in an atmosphere containing 15 mbar pCO2 (for details, see "Materials and Methods"). Primary transformants were initially screened with measurements of the CO2 assimilation rate at ambient pCO2 of 400 µbar pCO2 and high light. All primary transformants had CO2 assimilation rates within 20% of wild type and there were no obvious differences in the shape of the response of the CO2 assimilation rate to intercellular pCO2 (data not shown). Leaf discs were collected and stored at –70°C for later immunoblot analysis of the presence of phosphorylated PEPC; however, we found that, even in wild-type plants, PEPC was no longer phosphorylated after extended storage at –70°C. We therefore grew T1 progeny of a number of antisense and RNAi lines and screened fresh leaf material for the presence of phosphorylated PEPC in the light using the antibody that specifically recognizes phosphorylated PEPC. This antibody was raised against a synthetic peptide, mimicking the phosphorylated maize C4-PEPC. Although our previously reported antibody could not react with the PEPCs of Flaveria (Ueno et al., 2000
Characterization of T2 Progeny Some of these transgenic T1 plants with low PEPC phosphorylation in the light were grown to seed. Physiological analyses were carried out on the T2 generation of three independent transformation events (progeny of T1 plants R17#1, R111a#3, and A47b#6), which were grown in a glasshouse under natural light conditions during June and July, 2006, and then again in January and February, 2007, in Canberra, Australia. Immunoblot analysis showed that all of the progeny of antisense transformant A47b#6 and RNAi transformant R111a#3 showed a very low level of PEPC phosphorylation in comparison with wild-type leaves collected in the dark, whereas the progeny of R17#1 segregated and contained plants with various levels of phosphorylated PEPC (Fig. 2 ). We chose plants with very low phosphorylation levels from each of the three lines for further analysis.
Detection of PEPC-PK and PEPC mRNA by Reverse Transcription-PCR Detection of PEPC-PK and PEPC mRNA by reverse transcription (RT)-PCR showed that there was no difference in the amount of PpcA mRNA between leaves of the wild-type and the T2 progeny of A47b#6 and R111a#3 or wild-type leaves collected in the dark or light (Fig. 3 ). However, mRNA levels of PEPC-PK were reduced in the leaves of transformants and wild-type leaves collected in the dark compared to mRNA levels of wild-type leaves collected in the light. Because we used full-length cDNA for the construction of transgenic plasmids, it was difficult to distinguish endogenous PEPC-PK mRNA and exogenous antisense- or double-strand RNAs. For example, in line R111a#3, mRNA levels were significantly reduced, but some PEPC-PK mRNA was amplified. However, we observed significant reduction of mRNA levels in transgenic plants, which demonstrates that lack of PEPC phosphorylation is coincident with low PEPC-PK mRNA levels.
Phosphorylation of PEPC in Vitro
To check that PEPC from transformants could be phosphorylated by PEPC-PK, we incubated PEPC extracted from wild-type leaves and leaves of transformants with ATP and purified recombinant PEPC-PK from F. trinervia (Tsuchida et al., 2001
Phosphorylation of PEPC was compared to the malate sensitivity of PEPC activity in vitro (Fig. 5
). In F. bidentis, wild-type PEPC activity assayed in vitro under suboptimal conditions at pH 7.4 was less sensitive to malate if PEPC was extracted from leaves collected in the light compared to PEPC extracted from leaves collected in the dark, consistent with previous observations (Vidal and Chollet, 1997
Relationship between PEPC Phosphorylation and CO2 Assimilation Rates of Leaves Transgenic F. bidentis plants with low PEPC phosphorylation grew equally well as the wild-type plants and had no obvious visible growth phenotype. There were also no differences in the steady-state response to CO2 or light of CO2 assimilation rates (Figs. 6 and 7 ; Table I). Light induction curves of leaves that had been kept in the dark overnight also revealed no difference in the transient behavior of the CO2 assimilation rate (Fig. 8 ). The time course of phosphorylation of PEPC in the wild type shows the rate of phosphorylation of PEPC to be much slower than the induction of the CO2 assimilation rate (Fig. 8, inset).
Carbon isotope discrimination during C4 photosynthesis is, among other things, related to the extent to which Rubisco can fractionate against CO2. This, in turn, is determined by the amount of leakiness (  ), defined as the fraction of CO2 fixed by PEPC that subsequently leaks out of the bundle sheath cells. To examine whether the balance between the C4 cycle activity and the C3 cycle in the bundle sheath had been affected by the lack of PEPC phosphorylation, we compared the 13C composition of leaf dry matter of the T2 progeny with low phosphorylation levels with that of wild type, but we observed no differences (Table I).
To examine whether the lack of PEPC phosphorylation in the transformants had been compensated for by an altered balance of photosynthetic metabolites, we combined gas exchange of leaves with rapid freeze clamping of leaves. We measured a number of photosynthetic metabolites, but found no significant difference in metabolite levels between wild-type leaves and leaves of progeny of A47b#6 and R111a#3 (Table II
). Malate levels were the highest, but it has been shown previously that the photosynthetically active malate pool is only a small fraction of the total malate pool (Hatch, 1979
Antisense and RNAi Constructs Targeting mRNA of PEPC-PK Have Generated Transgenic Plants with Low Phosphorylation of PEPC
The regulatory phosphorylation of PEPC has been studied intensively in vitro (Chollet et al., 1996
It is well documented that PEPC activity is modulated in vitro by a wide range of effectors, such as phosphorylated intermediates (triose- and hexose-P), amino acids, and organic acids, such as malate, especially under suboptimal pHs that approximate cytosolic pH (Doncaster and Leegood, 1987
It has so far been difficult to test how important variation in malate sensitivity is to PEPC functioning in vivo. The mesophyll cytosolic pH is thought to be around 7.4, which forces PEPC to operate at suboptimal pH in vivo. Concentrations of PEP, triose-P, malate, and Asp have all been shown to increase with increasing pCO2, whereas Glc-6-P concentrations are relatively constant. The amounts of many of the metabolites in C4 leaves remain virtually constant in relation to irradiance, except at very low light (Leegood and von Caemmerer, 1988
Malate and Asp concentrations are highest at high light and at ambient and high pCO2. It is likely that PEPC activity does not limit the rate of CO2 assimilation under these conditions, but that the regulation of PEPC may nevertheless be important for the coordination of the C3 and C4 cycles of the pathway. We therefore combined CO2 assimilation rate measurements at high light with measurements of photosynthetic metabolite levels to see whether any changes in metabolite levels had occurred. However, there were no significant differences in metabolite levels between wild type and transformants (Table II). Unfortunately, it is not possible from these measurements to estimate photosynthetically active malate. It has been shown with 14C labeling studies that the photosynthetic malate pool is only a small fraction of leaf malate content, a large amount being presumably stored in leaf veins (Hatch, 1979
We also examined the carbon isotope composition of leaf dry matter as a sensitive tool to detect changes in coordination of C3 and C4 cycles of the pathway in transgenic plants. Carbon isotope discrimination during C4 photosynthesis is primarily dependent on the discrimination occurring during CO2 diffusion into the leaf and by the extent to which Rubisco can fractionate against CO2. This is, in turn, determined by the amount of leakiness (
Several of our transgenic F. bidentis lines with antisense or RNAi constructs targeted to PEPC-PK show low phosphorylation of PEPC in the light. This affects the malate sensitivity of the extracted PEPC, but does not appreciably affect net rates of CO2 assimilation under any condition examined. This raises the intriguing question of what physiological role this complexly regulated reversible phosphorylation of PEPC plays in C4 species.
Plasmid Construction and Plant Transformation and Regeneration
For full-length cDNA isolation, a Flaveria bidentis leaf cDNA library was constructed into
For F. bidentis transformation, antisense and RNAi plasmids were constructed using the full cDNA clone of F. bidentis PEPC-PK. For construction of the antisense plasmid, the binary vector system of pART7, pART27, was used (Gleave, 1992
F. bidentis was transformed and regenerated using the Agrobacterium method, as described by Chitty et al. (1994)
Primary transformants were grown to seed in a growth cabinet under approximately 25 mbar pCO2 and an irradiance of 400 µmol quanta m–2 s–1. Air temperature was 25°C during a 14-h day and 18°C at night. Plants were watered daily and twice weekly with a complete nutrient solution. Primary transformants were allowed to grow to seed. Subsequently, T1 generation of the primary transformants R17, R111a, R112, R113, A43a, and A47b were grown in a growth cabinet at ambient pCO2 and an irradiance of 500 µmol quanta m–2 s–1. Air temperature was 30°C during a 14-h day and 20°C at night and the relative humidity was 70%. Several plants of the T1 generations that showed low levels of PEPC phosphorylation were grown to seed. The T2 generation (progeny of T1 plants R17/1#1, R111a#3, and A47b#6) were grown in a glasshouse under natural light conditions during June and July, 2006, and during January and February, 2007, in Canberra. Glasshouse temperature was maintained at 28°C during the day and 18°C at night. Midday irradiance on clear days was approximately 800 µmol quanta m–2 s–1 in June and July, 2006, and 1,500 µmol quanta m–2 s–1 in January and February, 2007. Plants were grown in 5-L pots in a garden mix with 2.4 to 4 g of Osmocote/L soil (15/4.8/10.8/1.2 N/P/K/Mg + trace elements: B, Cu, Fe, Mn, Mo, Zn; Scotts) and watered daily.
Leaves were taken from F. bidentis wild-type and T2 generation plants grown in the glasshouse in February, 2007, at midnight and at midday. Leaf discs were ground into a fine powder with a mortar and pestle in liquid nitrogen. Total RNA was extracted by adding 1.0 mL of Tri-Reagent reagent (Sigma) to 60 mg each of the fine powder. After a 5-min incubation at room temperature, the mixture was centrifuged at 12,000g for 10 min at 4°C and the pellet was discarded. The phase separation was achieved by adding 200 µL of chloroform and the mixture was shaken for 15 s. Samples were placed at room temperature for 5 min and then centrifuged at 12,000g for 15 min at 4°C. The upper phase was collected into tubes containing 0.5 mL of isopropanol, inverted several times, and placed at room temperature for 10 min. Precipitated RNA was pelleted by centrifugation at 12,000g for 10 min at 4°C and washed twice with 75% ethanol (v/v). Samples were stored in 75% ethanol at –20°C.
One microgram of RNA was retrotranscribed with oligo(dT) primers using the SuperScript III first-strand synthesis system for RT-PCR (Invitrogen). One microliter of the total reaction mixture (20 µL) was then used as a template in the PCR reaction with gene-specific primers, PpcA (5'-atccgtacatcacaacc-3', 5'-gttctgcattccagcag-3'), PEPC-PK (5'-cgagaccgaagccgcga-3', 5'-cgagcttcaaaccccctcg-3'). PCR was performed for 18 cycles and 23 cycles, respectively.
Phosphorylated PEPC was detected in whole-leaf extracts using immunoblotting techniques. Leaf discs (approximately 1 cm2) were collected 2 to 3 h into the light period for growth cabinet-grown plants or collected at midday for glasshouse-grown plants briefly snap frozen in liquid nitrogen for transfer to the laboratory. For the dark samples, leaves were wrapped in silver foil and darkened overnight. After transfer to the laboratory, leaf discs were extracted immediately in 0.7 mL of ice-cold extraction buffer containing 50 mM HEPES-KOH, pH 7.8, 5 mM MgCl2, 2 mM EDTA, 5 mM dithiothreitol, 1% (w/v) polyvinylpolypyrrolidone, 0.1% (v/v) Triton X-100, and 4% (v/v) of protease inhibitor cocktail (Sigma), using a 2-mL glass homogenizer. Samples were centrifuged in a microcentrifuge at maximum speed for 1 min. The green pellet was discarded and the supernatant was brought to a final concentration of 2% (w/v) SDS and heated to 65°C in a water bath for 10 min. Samples were prepared for gel loading by adding 0.25 volumes of Bio-Rad XT sample buffer (Bio-Rad). Samples were loaded on an equal-leaf-area basis. Proteins were separated by electrophoresis on NuPAGE Bis-Tris precast gels (4%–12% acrylamide concentration; Novex), using the manufacturer-specified buffer system, and blotted onto nitrocellulose membranes. Blots were probed with an antiphosphorylated PEPC raised against a synthetic peptide, which corresponded to the sequence around the phosphorylation site Ser-15 of maize (Zea mays). Anti-IgG alkaline phosphatase conjugate (Bio-Rad) was used as the secondary antibody. Immunoblots were developed using the AttoPhos fluorescence substrate system (Promega). Duplicate gels were run and either stained with Coomassie Blue or blotted and probed with a polyclonal antibody to recombinant maize PEPC.
Leaves were collected at midday as described above. One leaf was then extracted in 2 mL of protein extraction buffer (50 mM Tris-HCl, pH 8.0, 0.04% Tween 20, 1 mM dithiothreitol, 15% ethylene glycol, 1 mM phenylmethylsulfonyl fluoride, 50 µM proteasome inhibitor MG132 [Peptide Institute]) and proteinase inhibitor cocktail, complete mini (Boehringer). The extract was centrifuged at 35,000g for 5 min at 4°C and 90 µL of supernatant was immediately added to 10 µL of a 10x protein kinase buffer (10 mM EGTA, 1 mM ATP, 20 mM MgCl2 with or without 0.05 µg of purified recombinant PEPC-PK from F. trinervia [Tsuchida et al., 2001
Leaf discs (approximately 1 cm2) were collected at midday for glasshouse-grown plants briefly snap frozen in liquid nitrogen for transfer to the laboratory. For the dark samples, leaves were wrapped in silver foil and darkened overnight. Leaf discs were extracted the same day in 0.7 mL of ice-cold extraction buffer containing 50 mM HEPES-NaOH, pH 7.8, 15 mM MgCl2, 1 mM EDTA, 10 mM dithiothreitol, 1% (w/v) polyvinylpolypyrrolidone, 0.01% (v/v) Triton X-100, and 4% (v/v) protease inhibitor cocktail (Sigma), using a 2-mL glass homogenizer. Samples were centrifuged in a microcentrifuge at maximum speed for 1 min and assayed immediately. The activity of PEPC was measured in 30 µL of leaf extracts at 25°C under suboptimal conditions in a 1-mL assay mixture containing 100 mM HEPES-NaOH, pH 7.4, 10 mM MgCl2, 1 mM NaHCO3, 3 mM PEP, 0.2 mM NADH, 12 units mL–1, with malate concentrations ranging from 0 to 2 mM. The reaction was started by the addition of PEP and the oxidation of NADH was monitored by A340.
Gas-exchange measurements on selected T2 plants were made in the glasshouse on young fully expanded leaves between 10 AM and 2 PM. CO2 response curves were made using the LI-COR CO2 injection system at 2,000 µmol quanta m–2 s–1 and a leaf temperature of 28°C. First measurements were made at a pCO2 of 350 µbar, then pCO2 was lowered to 50 µbar and increased in steps up to a final value of 1,000 µbar. For the light response curves, measurements were made at pCO2 of 380 µbar and a leaf temperature of 28°C and started at 2,000 µmol quanta m–2 s–1. The leaves were acclimated at this irradiance for 20 min and then irradiance was reduced stepwise at 5-min intervals.
Leaf gas exchange was measured with the LI-COR 6400 in a purpose-built leaf chamber attached to a rapid-kill apparatus (Badger et al., 1984
Frozen leaf discs were ground to a fine powder and extracted with 1 mL of frozen 1 M HCLO4 and metabolites assayed as described previously (Leegood and Furbank, 1984
Leaves similar to the one used during gas exchange were collected, oven dried at 70°C, and ground with a mortar and pestle. A subsample of ground tissue was weighted and the isotopic composition determined by combustion in a Carlo Erba elemental analyzer and the CO2 was analyzed by mass spectrometry. The Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number AB272061. Received May 17, 2007; accepted June 14, 2007; published June 22, 2007.
1 This work was supported in part by the Ministry of Education, Science, Sports and Culture of Japan (Grants-in-Aid for Scientific Research to K.I. and T.F.); by the Ministry of Agriculture, Forestry and Fisheries of Japan (grant for the Recombinant Plant Project to K.I. and T.F.); and by the New Energy and Industrial Technology Development Organization and Research Institute of Innovative Technology for the Earth (grant to T.F.).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Susanne von Caemmerer (susanne.caemmerer{at}anu.edu.au).
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.107.102541 * Corresponding author; e-mail susanne.caemmerer{at}anu.edu.au; fax 61–2–61255075.
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TOP
ABSTRACT
RESULTS
13C)
-gt10 phage as described previously (Tsuchida et al., 2001
