Characterization and Expression Analysis of Genes Encoding Phosphoenolpyruvate Carboxylase and Phosphoenolpyruvate Carboxylase Kinase of Lotus japonicus, a Model Legume (original) (raw)
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Archives of Biochemistry and Biophysics, 1997
vested from plants subjected to stem girdling or prolonged darkness. Furthermore, the kinase activity in Phosphoenolpyruvate carboxylase (PEPC) kinase nodules was controlled reversibly by illumination and was partially purified about 3000-fold from soybean extended darkness pretreatments of the parent plants, root nodules by a fast-protein liquid chromatography suggesting that photosynthate supply from the shoots protocol. This protein-serine kinase has an apparent is likely responsible for these striking changes in native molecular mass of about 30,000 as estimated by PEPC kinase activity observed in planta in the legume size-exclusion chromatography. Following electrophonodule. ᭧ 1997 Academic Press resis of this partially purified PEPC-kinase prepara-Key Words: PEPC kinase; phosphoenolpyruvate cartion in a denaturing gel containing dephospho maize boxylase (PEPC); protein kinase; protein phosphorylaleaf PEPC as substrate, the in situ renaturation and tion; root nodule; soybean (Glycine max). assay of protein kinase activity revealed two, PEPCdependent kinase polypeptides with molecular masses of about 32 and 37 kDa. The Ç32-kDa polypeptide was significantly more active than the Ç37-kDa catalytic Increasing evidence indicates that reversible protein subunit. The activity of this partially purified PEPC phosphorylation is an important mechanism for regukinase, and a less purified sample, was Ca 2/-insensilating plant phosphoenolpyruvate carboxylase (PEPC 3 ; tive. This protein kinase preparation was able to phos-EC 4.1.1.31) in vivo (1-4). It is well documented that phorylate purified PEPCs from soybean nodules, phosphorylation of a specific, plant-invariant serine maize leaves, and a sorghum recombinant C 4 PEPC. In residue near the N-terminus of the C 4 leaf enzyme recontrast, this PEPC kinase was unable to phosphorysults in an increased catalytic activity and a reduced late a phosphorylation-site mutant form of sorghum C 4 PEPC (S8Y), two other soybean nodule phosphopro-sensitivity to feedback inhibition by L-malate when asteins [nodulin-26 and nodulin-100 (sucrose synthase)], sayed at suboptimal but near-physiological conditions bovine serum albumin, and histone III-S. Following in (1, 2, 4). The phosphorylation state of the photosynvitro phosphorylation of purified dephospho soybean thetic PEPC isoforms in C 4 and Crassulacean acid menodule PEPC from stem-girdled plants by the partially tabolism (CAM) leaves is controlled largely by the acpurified nodule PEPC kinase, the former's activity and tivity of a Ca 2/-independent protein-Ser/Thr kinase sensitivity to L-malate inhibition increased and de-(1-3, 5, 6). In C 4 leaves, PEPC kinase and, thus, its creased, respectively. Notably, the Ca 2/-independent cytosolic target enzyme are activated by illumination PEPC kinase activity in nodules from illuminated and inactivated by darkness or inhibition of photosynplants was markedly greater than that in nodules har
Journal of Experimental Botany, 1997
A cDNA clone for phosphoenolpyruvate carboxylase (PEPC) was isolated from nodules of pea (Pisum sativum L.). The levels of activity, the PEPC polypeptide and the mRNA for PEPC in effective nodules and in ineffective nodules induced on the pea mutant E135 (sym 13) were compared to evaluate the regulation of PEPC activity in pea nodules. The cDNA was 3 222 bp long and contained an open reading frame of 2 901 bp, capable of encoding a polypeptide of 967 amino acids. The deduced amino acid sequence was similar to those of other plant PEPCs. The expression of this gene was enhanced in nitrogen-fixing effective pea nodules. The activity of PEPC in the effective nodules increased during development of the plants and the activity was maintained until senescence. This increase was associated with increased levels of the 110 kDa polypeptide and the mRNA for PEPC. By contrast, roots always had lower levels of activity, the polypeptide and the mRNA for PEPC than effective nodules. In ineffective E135 nodules, the activity of PEPC increased similarly to that in effective nodules, but then it decreased to the level in roots after 2.5 weeks post-planting. However, the levels of the 110 kDa polypeptide and the mRNA for PEPC in ineffective nodules were higher than those in roots, even though ineffective nodules had reduced levels of the polypeptide and mRNA for PEPC as compared with those in effective nodules. These findings indicate that after nodule initiation, the activity of PEPC in pea nodules may be regulated at the transcrip-tional level and after translation by a putative factor(s) that governs the effectiveness of the nodules.
In Vivo Regulatory Phosphorylation of Soybean Nodule Phosphoenolpyruvate Carboxylase
Plant Physiology, 1995
In this report we provide evidence that cytosolic phosphoenolpyruvate carboxylase (PEPC) in soybean (Clycine max 1.) root nodules is regulated in vivo by a seryl-phosphorylation cycle, as with the C4, Crassulacean acid metabolism, and C, leaf isoforms. Pretreatment of parent plants by stem girdling for 5 or 14 h caused a significant decrease in the apparent phosphorylation state of nodule PEPC, as indicated by the 50% inhibition constant (i-malate) and specific activity values assayed at suboptimal conditions, whereas short-term darkness alone was without effect. However, extended (26 h) darkness led to the formation of a relatively dephosphorylated nodule PEPC, an effect that was reversed by illuminating the darkened plants for 3 h. This reversal of the apparent phosphorylation state in the light was prevented by concomitant stem girdling. In contrast, the optimal activity of nodule PEPC and its protein leve1 showed little or no change in all pretreated plants. These results suggest that the phosphorylation state of PEPC in soybean root nodules is possibly modulated by photosynthate transported recently from the shoots. In situ [3ZPlorthophosphate labeling, immunoprecipitation, and phosphoamino acid analyses confirmed directly that PEPC in detached intact soybean nodules is phosphorylated on a serine residue(s).
PLANT PHYSIOLOGY, 2004
Phosphorylation of phosphoenolpyruvate carboxylase (PEPc; EC 4.1.1.31) plays an important role in the control of central metabolism of higher plants. This phosphorylation is controlled largely at the level of expression of PEPc kinase (PPCK) genes. We have analyzed the expression of both PPCK genes and the PEPC genes that encode PEPc in soybean (Glycine max). Soybean contains at least four PPCK genes. We report the genomic and cDNA sequences of these genes and demonstrate the function of the gene products by in vitro expression and enzyme assays. For two of these genes, GmPPCK2 and GmPPCK3, transcript abundance is highest in nodules and is markedly influenced by supply of photosynthate from the shoots. One gene, GmPPCK4, is under robust circadian control in leaves but not in roots. Its transcript abundance peaks in the latter stages of subjective day, and its promoter contains a sequence very similar to the evening element found in Arabidopsis genes expressed at this time. We report the expression patterns of five PEPC genes, including one encoding a bacterial-type PEPc lacking the phosphorylation site of the plant-type PEPcs. The PEPc expression patterns do not match those of any of the PPCK genes, arguing against the existence of specific PEPc-PPCK expression partners. The PEPC and PPCK gene families in soybean are significantly more complex than previously understood.
Cessation of photosynthesis in Lotus japonicus leaves leads to reprogramming of nodule metabolism
Journal of Experimental Botany, 2013
Symbiotic nitrogen fixation (SNF) involves global changes in gene expression and metabolite accumulation in both rhizobia and the host plant. In order to study the metabolic changes mediated by leaf-root interaction, photosynthesis was limited in leaves by exposure of plants to darkness, and subsequently gene expression was profiled by real-time reverse transcription-PCR (RT-PCR) and metabolite levels by gas chromatography-mass spectrometry in the nodules of the model legume Lotus japonicus. Photosynthetic carbon deficiency caused by prolonged darkness affected many metabolic processes in L. japonicus nodules. Most of the metabolic genes analysed were down-regulated during the extended dark period. In addition to that, the levels of most metabolites decreased or remained unaltered, although accumulation of amino acids was observed. Reduced glycolysis and carbon fixation resulted in lower organic acid levels, especially of malate, the primary source of carbon for bacteroid metabolism and SNF. The high amino acid concentrations together with a reduction in total protein concentration indicate possible protein degradation in nodules under these conditions. Interestingly, comparisons between amino acid and protein content in various organs indicated systemic changes in response to prolonged darkness between nodulated and non-nodulated plants, rendering the nodule a source organ for both C and N under these conditions.
The Plant Journal, 2003
SummaryVarious isoforms of plant phosphoenolpyruvate carboxylase (PEPC (Ppc)) are controlled post‐translationally by an intricate interaction between allosteric regulation and reversible protein phosphorylation. In leaves and root nodules of legumes, these changes in PEPC phosphorylation state are governed primarily by PEPC‐kinase (PpcK), a novel, ‘minimal but functional’ Ser/Thr kinase. To date, this plant‐specific kinase has been investigated in molecular terms exclusively in non‐leguminous plants, such as Crassulacean‐acid‐metabolism (CAM) species and Arabidopsis. As an important extension of our earlier biochemical studies on this dedicated kinase and PEPC phosphorylation in soybean (Glycine max) nodules, we now report the molecular cloning of the first legume PpcK from a soybean nodule cDNA library, which encodes a functional, 31.0 kDa PpcK polypeptide. Besides displaying organ, developmental, and spatial expression properties that are strikingly up‐regulated in mature nodules,...
European Journal of Biochemistry, 1992
The light-dependent phosphorylation of the photosynthetic phosphoennlpyruvate carboxylase (PyrPC) was shown to occur in protoplasts from Sorghum mesophyll cells. I t was accompanied by an increase in PyrPC protein-serine-kinase activity and conferred the target-specific functional properties, i. e. an increase in V,,, and apparent Ki for L-malate, as previously found with the whole leaf. The light-dependent regulatory phosphorylation of PyrPC was (a) specifically promoted by the weak bases NH4C1 and methylamine (agents which increase cytosolic pH), but not by KN03, (b) inhibited by the cytosolic protein-synthesis inhibitor, cycloheximide, thus confirming that protein turnover is a component of the signal-transduction cascade, as reported in [4], (c) found to moderately decrease in the presence of EGTA and to be strongly depressed when the Ca2+-selective ionophore A23287 was added to the incubation medium together with EGTA. Addition of Ca2+, but not of Mg2 ', to the Ca2 '-depleted protoplasts partially, but significantly, relieved the inhibition. Calcium deprivation apparently affected the in-situ light-activation of the PyrPC protein kinase. These data indicated that both Ca2+ and an increase in cytosolic pH are required for the induction of PyrPC protein kinase activitylPyrPC phosphorylation in illuminated protoplasts from Sorghum mesophyll cells. In C4 plants, the primary photosynthetic C 0 2 fixation catalysed by phosphoenolpyruvate carboxylase (PyrPC) is a key step through which the control of carbon flow is operated. Regulation of the enzyme, in the cytosol of mesophyll cells, involves both antagonistic effectors i. e. glucose-6P, triose-P (positive), L-malate (negative), [I], and a light-dependent reversible phosphorylation process [2-41. When measured at suboptimal conditions of pH and substrate PyrP (phosphoenolpyruvate) which are supposed to be physiological, the phosphorylated PyrPC displays an increased catalytic activity and Ki for L-malate. This post-translational modulation is thought to represent an additional mechanism allowing the
Biochemical and Biophysical Research Communications, 2001
In Crassulacean acid metabolism (CAM) plants, phosphoenolpyruvate carboxylase (PEPC) is subject to day-night regulatory phosphorylation of a conserved serine residue in the plant enzyme's N-terminal domain. The dark increase in PEPC-kinase (PEPC-k) activity is under control of a circadian oscillator, via the enhanced expression of the corresponding gene (1). The signaling cascade leading to PEPC-k upregulation was investigated in leaves and mesophyll cell protoplasts of the facultative, salt-inducible CAM species, Mesembryanthemum crystallinum. Mesophyll cell protoplasts had the same PEPC-k activity as leaves from which they were prepared (i.e., high at night, low during the day). However, unlike C 4 protoplasts (2), CAM protoplasts did not show marked PEPC-k up-regulation when isolated during the day and treated with a weak base such as NH 4 Cl. Investigations using various pharmacological reagents established the operation, in the darkened CAM leaf, of a PEPC-k cascade including the following components: a phosphoinositide-dependent phospholipase C (PI-PLC), inositol 1,4,5 P (IP 3)-gated tonoplast calcium channels, and a putative Ca 2؉ /calmodulin protein kinase. These results suggest that a similar signaling machinery is involved in both C 4 (2, 3) and CAM plants to regulate PEPC-k activity, the phosphorylation state of PEPC, and, thus, carbon flux through this enzyme during CAM photosynthesis.
Plant Molecular Biology, 1996
Phosphoenolpyruvate carboxylase (PEPC) genes from Corynebacterium glutamicum (cppc), Escherichia coli (eppc) or Flaveria trinervia (fppc) were transferred to Solanum tuberosum. Plant regenerants producing foreign PEPC were identified by Western blot analysis. Maximum PEPC activities measured in eppc and fppc plants grown in the greenhouse were doubled compared to control plants. For cppc a transgenic plant line could be selected which exhibited a fourfold increase in PEPC activity. In the presence of acetyl-CoA, a known activator of the procaryotic PEPC, a sixfold higher activity level was observed. In cppc plants grown in axenic culture PEPC activities were even higher. There was a 6-fold or 12-fold increase in the PEPC activities compared to the controls measured in the absence or presence of acetyl-CoA, respectively. Comparable results were obtained by transient expression in Nicotiana tabacum protoplasts. PEPC of C. glutamicum (PEPC C.g.) in S. tuberosum leaf extracts displays its characteristic Km (PEP) value. Plant growth was examined with plants showing high expression of PEPC and, moreover, with a plant cell line expressing an antisense S. tuberosum (anti-sppc) gene. In axenic culture the growth rate of a cppc plant cell line was appreciably diminished, whereas growth rates of an anti-sppc line were similar or slightly higher than in controls. Malate levels were increased in cppc plants and decreased in antisense plants. There were no significant differences in photosynthetic electron transport or steady state CO2 assimilation between control plants and transformants overexpressing PEPC C.g. or anti-sppc plants. However, a prolonged dark treatment resulted in a delayed induction of photosynthetic electron transport in plants with less PEPC. Rates of CO2 release in the dark determined after a 45 min illumination period at a high proton flux density were considerably enhanced in cppc plants and slightly diminished in anti-sppc plants. When CO2 assimilation rates were corrected for estimated rates of mitochondrial respiration in the light, the electron requirement for CO2 assimilation determined in low CO2 was slightly lower in transformants with higher PEPC, whereas transformants with decreased PEPC exhibited an appreciably elevated electron requirement. The CO2 compensation point remained unchanged in plants (cppc) with high PEPC activity, but might be increased in an antisense plant cell line. Stomatal opening was delayed in antisense plants, but was accelerated in plants overexpressing PEPC C.g. compared to the controls.