Icl Gene Research Articles

¹³C metabolic flux analysis identifies an unusual route for pyruvate dissimilation in mycobacteria which requires isocitrate lyase and carbon dioxide fixation.

Mycobacterium tuberculosis requires the enzyme isocitrate lyase (ICL) for growth and virulence in vivo. The demonstration that M. tuberculosis also requires ICL for survival during nutrient starvation and has a role during steady state growth in a glycerol limited chemostat indicates a function for this enzyme which extends beyond fat metabolism. As isocitrate lyase is a potential drug target elucidating the role of this enzyme is of importance; however, the role of isocitrate lyase has never been investigated at the level of in vivo fluxes. Here we show that deletion of one of the two icl genes impairs the replication of Mycobacterium bovis BCG at slow growth rate in a carbon limited chemostat. In order to further understand the role of isocitrate lyase in the central metabolism of mycobacteria the effect of growth rate on the in vivo fluxes was studied for the first time using 13C-metabolic flux analysis (MFA). Tracer experiments were performed with steady state chemostat cultures of BCG or M. tuberculosis supplied with 13C labeled glycerol or sodium bicarbonate. Through measurements of the 13C isotopomer labeling patterns in protein-derived amino acids and enzymatic activity assays we have identified the activity of a novel pathway for pyruvate dissimilation. We named this the GAS pathway because it utilizes the Glyoxylate shunt and Anapleurotic reactions for oxidation of pyruvate, and Succinyl CoA synthetase for the generation of succinyl CoA combined with a very low flux through the succinate – oxaloacetate segment of the tricarboxylic acid cycle. We confirm that M. tuberculosis can fix carbon from CO2 into biomass. As the human host is abundant in CO2 this finding requires further investigation in vivo as CO2 fixation may provide a point of vulnerability that could be targeted with novel drugs. This study also provides a platform for further studies into the metabolism of M. tuberculosis using 13C-MFA.

Characterization of a bacterial community from a Northeast Siberian seacoast permafrost sample

A combination of culture-dependent and -independent techniques was used to characterize a bacterial community, examine cold adaptation of isocitrate lyase (icl) genes, and detect genes with important ecological functions in a permafrost sample from the Bykovsky Peninsula on the Laptev Sea coast of northeast Siberia. According to the 16S rRNA gene sequence, 47 of the cultured isolates were members of the phyla Firmicutes, Proteobacteria, and Actinobacteria, with 85% of the isolates belonging to the genera Arthrobacter and Planococcus. The 16S rRNA gene clone library derived from DNA from the same permafrost sample contained sequences from the same phyla plus a few from Acidobacteria, but favored the Firmicutes at the cost of the Actinobacteria. A partial sequence of the icl gene, a proposed marker for cold adaptation, was determined for 25 isolates that grew at 0 °C. Two Psychrobacter isolates contained two of the four residues shown to be important for low-temperature activity in Colwellia maris or Colwellia psychrerythreaea. The presence in the permafrost DNA of genes with ecosystem functions was determined using geochip 2.0. The highest number of genes identified was from the categories of aromatic and natural polymer degradation genes, perhaps reflecting selection for the use of tundra vegetation-produced carbon.

Overexpression of isocitrate lyase—glyoxylate bypass influence on metabolism in Aspergillus niger

In order to improve the production of succinate and malate by the filamentous fungus Aspergillus niger the activity of the glyoxylate bypass pathway was increased by over-expression of the isocitrate lyase ( icl) gene. The hypothesis was that when isocitrate lyase was up-regulated the flux towards glyoxylate would increase, leading to excess formation of malate and succinate compared to the wild-type. However, metabolic network analysis showed that an increased icl expression did not result in an increased glyoxylate bypass flux. The analysis did show a global response with respect to gene expression, leading to an increased flux through the oxidative part of the TCA cycle. Instead of an increased production of succinate and malate, a major increase in fumarate production was observed. The effect of malonate, a competitive inhibitor of succinate dehydrogenase (SDH), on the physiological behaviour of the cells was investigated. Inhibition of SDH was expected to lead to succinate production, but this was not observed. There was an increase in citrate and oxalate production in the wild-type strain. Furthermore, in the strain with over-expression of icl the organic acid production shifted from fumarate towards malate production when malonate was added to the cultivation medium. Overall, the icl over-expression and malonate addition had a significant impact on metabolism and on organic acid production profiles. Although the expected succinate and malate formation was not observed, a distinct and interesting production of fumarate and malate was found.

Function and transcriptional regulation of the isocitrate lyase in Pseudomonas aeruginosa

Pseudomonas aeruginosa ATCC 17933 is capable of growing aerobically on ethanol as sole source of carbon and energy. This requires the glyoxylate cycle for replenishing C4-compounds to the TCA cycle. The enzyme isocitrate lyase (ICL) catalyzes the first step of this glyoxylate shunt. Its activity was induced more than 10-fold in response to the carbon sources ethanol or acetate instead of glucose or succinate. We could prove that in P. aeruginosa ICL is essential for aerobic as well as anaerobic utilization of C2-sources. Transcriptional regulation of icl gene (aceA) expression was monitored on different carbon sources by using an aceA-lacZ gene fusion. A strong correlation between promoter and ICL activity indicated regulation at the transcriptional level. But ICL was not simply induced by the mere presence of ethanol in the growth medium as was demonstrated by cultivation on mixed substrates. P. aeruginosa showed diauxic growth on media containing ethanol-succinate or ethanol-glucose mixtures and did not transcribe the aceA gene to metabolize ethanol until succinate or glucose, respectively, were exhausted. Inactivation of the chromosomal aceA gene in P. aeruginosa led to an inability to grow on ethanol and acetate. Promoter activity studies showed that all genes necessary to oxidize ethanol were downregulated in the ICL-negative mutant. But on mixed substrates like ethanol-succinate or ethanol-glucose the mutant exhibited growth and utilized ethanol as well, probably as energy source only.

Isocitrate lyase from Mycobacterium tuberculosis promotes survival of Mycobacterium smegmatis within macrophage by suppressing cell apoptosis

Isocitrate lyase (ICL) was previously demonstrated to play a pivotal role in the intracellular metabolism of Mycobacterium tuberculosis (MTB). Presently several lines of evidence suggest that ICL from MTB (MTB-ICL) may play some roles in the interaction between MTB and host macrophage. However, there has been no research on the interaction between MTB-ICL and host macrophage. MTB-icl and M. smegmatis (MS)-icl genes were amplified by polymerase chain reaction (PCR) and cloned into the E. coli-mycobacterium shuttle plasmid pUV15 to obtain recombinant shuttle plasmids pMTB-icl and pMS-icl. Following transformation into MS by electroporation, the expression of pMTB-icl and pMS-icl was verified by reverse transcriptase (RT)-PCR. The expression of recombinant plasmids derived from pUV15 when rMS was phagocytized by macrophage was also verified via fluorescence microscope. Ms 1 - 2c, rMS-pUV15, rMS-pMS-icl and rMS-pMTB-icl were used to infect RAW264.7 cells and the survival of intracellular MS was monitored by bacterial culture at 0, 24 and 48 hours after infection. The culture supernatants from macrophage infected by Ms 1 - 2c, rMS-pUV15, rMS-pMS-icl and rMS-pMTB-icl were collected and the interferon (IFN)-gamma and nitric oxide (NO) concentrations were measured by ELISA or by Griess assay, respectively. The apoptosis of macrophage was assayed by the in situ TUNEL technique. RT-PCR showed that both pMTB-icl and pMS-icl could be expressed in MS. Fluorescence microscopic observation showed that recombinant plasmids derived from pUV15 (pUV15-IG) could also be expressed in MS when MS were phagocytized by macrophage. Bacterial culture data demonstrated that rMS-pMTB-icl exhibited significantly increased intracellular survival in the murine macrophage cell line RAW264.7 compared with Ms 1 - 2c, rMS-pUV15 and rMS-pMS-icl. This increased intracellular survival was not accompanied by the upregulation of IFN-gamma and NO in host macrophage. But a lower apoptosis rate of macrophages infected with rMS-pMTB-icl was observed when compared with macrophages infected with other strains of MS. MTB-ICL could promote the intracellular survival of MS. Suppressing the apoptosis of host macrophage may be one of the important mechanisms involved in this increased intracellular survival.

Gene cloning of cold-adapted isocitrate lyase from a psychrophilic bacterium, Colwellia psychrerythraea, and analysis of amino acid residues involved in cold adaptation of this enzyme

The gene (icl) encoding cold-adapted isocitrate lyase (ICL) of a psychrophilic bacterium, Colwellia psychrerythraea, was cloned and sequenced. Open reading frame of the gene was 1,587 bp in length and corresponded to a polypeptide composed of 528 amino acids. The deduced amino acid sequence showed high homology with that of cold-adapted ICL from other psychrophilic bacterium, C. maris (88% identity), but the sequential homology with that of the Escherichia coli ICL was low (28% identity). Primer extension analysis revealed that transcriptional start site for the C. psychrerythraea icl gene was guanine, located at 87 bases upstream of translational initiation codon. The expression of this gene in the cells of an E. coli mutant defective in ICL was induced by not only low temperature but also acetate. However, cis-acting elements for cold-inducible expression known in the several other bacterial genes were absent in the promoter region of the C. psychrerythraea icl gene. The substitution of Ala214 for Ser in the C. psychrerythraea ICL introduced by point mutation resulted in the increased thermostability and lowering of the specific activity at low temperature, indicating that Ala214 is important for psychrophilic properties of this enzyme.

DNAzymes Targeting theiclGene Inhibit ICL Expression and DecreaseMycobacterium tuberculosisSurvival in Macrophages

Latent infection with Mycobacterium tuberculosis presents a big obstacle for tuberculosis therapy. In this study, we investigated the effects of sequence-specific DNAzymes targeting the mRNA of isocitrate lyase (ICL), an enzyme playing a pivotal role in the metabolism of M. tuberculosis in the latent state, on the expression of ICL and survival of M. tuberculosis. In vitro studies showed that four of five designed DNAzymes, DZ1, DZ3, DZ4, and DZ5 could cleave icl mRNA efficiently and specifically. Treatment of virulent M. tuberculosis with 5microM DZ4 plus a subinhibitory concentration of isoniazid (INH) decreased ICL expression and the survival of M. tuberculosis in macrophages but had no obvious influence on the growth of M. tuberculosis in vitro. This study demonstrates that using INH to soften the cell wall of M. tuberculosis and help the entry of biomolecules is an efficient method of improving the uptake of DNAzymes. Silencing the icl gene by DNAzyme is a promising method to combat latent infection of tuberculosis.

Soybean ( Glycine max. L.) and bacteroid glyoxylate cycle activities during nodular senescence

Soybean (Glycine max. L.) nodular senescence results in the dismantling of the peribacteroid membrane (PBM) and in an increase of soybean isocitrate lyase (ICL; EC 4.1.3.1) and malate synthase (MS; EC 4.1.3.2) mRNA and protein levels. This suggests that in senescing soybean nodular cells, the specific glyoxylate cycle enzyme activities might be induced to reallocate carbon obtained from the PBM degradation. In order to evaluate as well the carbon metabolism of the nitrogen-fixing Bradyrhizobium japonicum endosymbiotic bacteroids during nodular senescence, their glyoxylate cycle activities were also investigated. To this end, partial DNA sequences were isolated from their icl and ms genes, but the corresponding mRNAs were not detected in the microorganisms. It was also observed that the bacteroid ICL and MS activities were negligible during nodular senescence. This suggests that glyoxylate cycle activities are not reinitiated in the bacteroids under these physiological conditions. In case the microorganisms nevertheless feed on the PBM degradation products, this might occur via the citric acid cycle exclusively.

The cold-inducible icl gene encoding thermolabile isocitrate lyase of a psychrophilic bacterium, Colwellia maris.

The gene encoding isocitrate lyase (ICL; EC 4.1.3.1) of a psychrophilic bacterium, Colwellia maris, was cloned and sequenced. The ORF of the gene (icl) was 1584 bp long, and the predicted gene product consisted of 528 aa (molecular mass 58150 Da) and showed low homology with the corresponding enzymes from other organisms. The analyses of amino acid content and primary structure of the C. maris ICL suggested that it possessed many features of a cold-adapted enzyme. Primer extension and Northern blot analyses revealed that two species of the icl mRNAs with differential lengths of 5'-untranslated regions (TS1 and TS2) were present, of which the 5' end (TS1 and TS2 sites) were G and A, located at 130 and 39 bases upstream of the translation start codon, respectively. The levels of TS1 and TS2 mRNAs were increased by both acetate and low temperature. The induction of icl expression by low temperature took place in the C. maris cells grown on succinate as the carbon source but not acetate. Furthermore, a similar manner of inductions was also found in the levels of the translation and the enzyme activity in cell-free extract. These results suggest that the icl gene, encoding thermolabile isocitrate lyase, of C. maris is important for acetate utilization and cold adaptation.

Persistence of Mycobacterium tuberculosis in macrophages and mice requires the glyoxylate shunt enzyme isocitrate lyase.

Mycobacterium tuberculosis claims more human lives each year than any other bacterial pathogen. Infection is maintained in spite of acquired immunity and resists eradication by antimicrobials. Despite an urgent need for new therapies targeting persistent bacteria, our knowledge of bacterial metabolism throughout the course of infection remains rudimentary. Here we report that persistence of M. tuberculosis in mice is facilitated by isocitrate lyase (ICL), an enzyme essential for the metabolism of fatty acids. Disruption of the icl gene attenuated bacterial persistence and virulence in immune-competent mice without affecting bacterial growth during the acute phase of infection. A link between the requirement for ICL and the immune status of the host was established by the restored virulence of delta icl bacteria in interferon-gamma knockout mice. This link was apparent at the level of the infected macrophage: Activation of infected macrophages increased expression of ICL, and the delta icl mutant was markedly attenuated for survival in activated but not resting macrophages. These data suggest that the metabolism of M. tuberculosis in vivo is profoundly influenced by the host response to infection, an observation with important implications for the treatment of chronic tuberculosis.

Germination, senescence and pathogenic attack in soybean ( Glycinemax. L.): identification of the cytosolic aconitase participating in the glyoxylate cycle

During our study of the glyoxylate cycle in soybean ( Glycine max. L. var. Maple arrow), two mitochondrial and three cytosolic aconitase molecular species (EC 4.2.1.3) were detected, designated as M1, M2, C1, C2 and C3 isoforms, respectively, according to their intracellular locations and electrophoretic mobilities. Using the glyoxylate cycle marker enzymes isocitrate lyase (ICL, EC 4.1.3.1) and malate synthase (MS, EC 4.1.3.2), the activity of this pathway providing the essential link between β-oxidation and gluconeogenesis was confirmed during germination (cotyledons) and senescence (leaves). It was then established that, in both cases, the activity of the C1 aconitase isoform developed concomitantly with the transcription and translation levels of the icl and ms genes. This strongly suggests that C1 aconitase is constitutive of the glyoxylate cycle. In addition, the same isoform was found to be active during pathogenic attack as well (hypocotyls). It might be assumed that in such a case the glyoxylate cycle is reinitiated as a part of a carbon reallocation system feeding on the diseased tissue cellular components.

Three-dimensional analysis of protein aggregate body in Saccharomyces cerevisiae cells.

Modified genes of peroxisomal isocitrate lyase of Candida tropicalis (CT-ICL) were constructed and expressed in Saccharomyces cerevisiae cells. We observed subcellular localization of expressed products of the mutant CT-ICL genes by immunoelectron microscopy. An unknown structure termed a protein aggregate body (PAB) storing the expressed product was observed in cytoplasm in various mutants (Kamasawa et al. (1996) J. Electron Microsc. 45: 491-497). We chose two typical cells harbouring the mutant ICL genes delta 550 and delta 237-339 to analyse the ultrastructure and three-dimensional (3D) structure of PABs. The PABs had a homogeneous matrix with a wavy periphery in the cell image using a high-pressure freezing fixation method. Although PABs could not be separated from the cytoplasm or mitochondria under a confocal fluorescence microscope, 3D reconstruction of serial electron micrographs clearly showed the PAB was an independent structure of varying size and had the shape of an incomplete sphere. A cell was sometimes observed to have multiple PABs.

Distinct cis-acting sequences are required for the germination and sugar responses of the cucumber isocitrate lyase gene

Recent data have shown that distinct DNA sequence elements direct the germination and sugar responses of the cucumber ( Cucumis sativus, L.) malate synthase ( Ms) gene ( Sarah et al. (1996)Mol. Gen. Genet., 250, 153–161). Such information is, however, lacking for the isocitrate lyase ( Icl) gene which is coordinately regulated with Ms. Deletions from the 5′ end of the Icl promoter were therefore created specifically to address this question. Analysis of expression in seeds of transgenic Nicotiana plumbaginifolia plants showed that whereas a promoter sequence of 2.9 kilobases (kb) produced a normal germination response, deletion to −1568 base pairs (bp) dramatically reduced this response. Examination of the sugar response employed a transgenic cucumber root system. In this case, the 2900 bp and 1568 bp promoters both gave a strong sugar response, but further deletion to −1367 bp eliminated the response. Therefore, the germination and sugar responses of the Icl gene require distinct cis-acting elements, located respectively upstream and downstream of −1568 bp. This observation is consistent with distinct signal transduction systems regulating gene expression in each case.

Derepression of gene expression mediated by the 5' upstream region of the isocitrate lyase gene of Candida tropicalis is controlled by two distinct regulatory pathways in Saccharomyces cerevisiae.

The 5' upstream region of the gene encoding isocitrate lyase of Candida tropicalis (UPR-ICL) is functional as a promoter in Saccharomyces cerevisiae, and it is regulated by carbon source; the expression of the gene is repressed when cells are grown on glucose, while it increases to a higher level in acetate-grown cells. Therefore, we have investigated regions in UPR-ICL responsible for gene expression in glucose-grown and acetate-grown cells. In glucose-grown cells, a deletion of the region between -801 and -569 (region G1) significantly decreased gene expression compared with that observed with the complete UPR-ICL. The region from -421 to -379 (region G2) also repressed gene expression in glucose-grown cells. In acetate-grown cells, two regions were found to strongly enhance gene expression, one between -728 and -569 (region A1) and the other between -370 and -356 (region A2). Whereas region A2 contained a sequence motif similar to the carbon-source-responsive element (CSRE), which mediates regulation by carbon source of S. cerevisiae ICL1, region A1 did not show similarity to any reported cis-acting elements. Deletion mutants of UPR-ICL containing only one of these regions showed that each region could independently activate gene expression to a similar level when the cells were grown on acetate. The influences of null mutations in the MIG1, SNF1 and CAT8 genes on regulation of UPR-ICL-mediated gene expression were examined. Expression of the ICL gene with full-length UPR-ICL increased about tenfold in mig1 cells grown on glucose, while little difference was observed in acetate-grown cells. The effects of snf1 and cat8 mutations were different between region-A1-mediated and region-A2-mediated gene expression in acetate-grown cells. Region-A2-mediated expression decreased 95% and 86% in snf1 and cat8 cells, respectively, while region-A1-mediated expression decreased 72% in snf1 cells and was not affected by the cat8 mutation. This finding indicates that region-A1-mediated gene expression is regulated by a pathway independent of CAT8, which is necessary for derepression of CSRE-mediated gene expression in S. cerevisiae.

Expression of glyoxylate cycle genes in cucumber roots responds to sugar supply and can be activated by shading or defoliation of the shoot.

When cucumber roots are excised and incubated without a carbon source, isocitrate lyase (ICL) and malate synthase (MS) mRNAs increase significantly in amount. However, if sucrose is added to the excised roots, the mRNAs do not accumulate. Hairy roots obtained by transformation with Agrobacterium rhizogenes show the same response. Transgenic hairy roots containing the Icl and Ms gene promoters fused to the GUS reporter gene, have very low GUS activity which increases dramatically when roots are incubated in the absence of sugar, indicating regulation at the transcriptional level. Staining of sugar-deprived roots shows that GUS activity is concentrated mainly in root tips and lateral root primordia, where demand for carbohydrate is greatest. In order to determine if Icl and Ms genes are expressed in roots of whole plants under conditions which may occur in nature, cucumber plants were subjected to reduced light intensity or defoliation. In both cases increases were observed in ICL and MS mRNAs. These treatments also reduced root sugar content, consistent with the hypothesis that sugar supply could control expression of Icl and Ms genes in roots of whole plants.

The Consequences of Replacing Histidine 356 in Isocitrate Lyase fromEscherichia coli

Isocitrate lyase fromEscherichia colihas been expressed in transformedE. coliJE10 cells lacking the isocitrate lyase (icl) gene. After directed mutagenesis oficlby the restriction-site elimination method, partially purified isocitrate lyase mutants in which His 356 has been converted to Lys, Arg, Gln, Asp, or Leu have been characterized after induction of transformed, induced JE10 cells. Values ofkcatcompared to those for wild-type (wt) enzyme (100) at 37°C, pH 7.3, are 18, 1, <1, 0, and 0 for H356K, H356R, H356E, H356Q, and H356L mutant enzymes, respectively.Kmvalues for the 1:1 Mg–isocitrate complex (in millimolar units) are: 0.13, wt; 0.11, H356K; and 0.63, H356R. Further chromatographic purification of isocitrate lyase yields highly purified wt, H356K, and H356R enzymes. The pH profile of the stability of isocitrate lyase, which has never been reported, showed that the H356R enzyme was unstable in the pH range investigated; the wt and H356R variant differed but each was sufficiently stable to study the pH dependence of catalysis. The logkcat/pH profiles for highly purified wt and H356K enzymes are roughly bell-shaped and have pKaand pKbvalues for dissociation of an ionizable group on the enzyme–substrate complex of <6.3 and 8.4 for wt and 5.9 and 7.9 for H356K enzymes. Plots of pKmvs pH were different for the wt and H356K variant. Values of pKaand pKb(derived from logkcat/Kmplots vs pH) for the dissociation of an activity-related ionizable group on the variant were 5.3 and 7.6, whereas the analogous pKbvalue for the wt enzyme was 8.4. The data suggest that His 356 is an important functional residue in isocitrate lyase, perhaps in deprotonating isocitrate during catalytic cleavage.

Regulation of expression of the cucumber isocitrate lyase gene in cotyledons upon seed germination and by sucrose

A 6.5 kb cucumber genomic DNA fragment containing the icl gene was introduced into Nicotiana plumbaginifolia and shown to direct isocitrate lyase (ICL) mRNA synthesis in transgenic seedlings upon germination, in a temporally regulated manner. Two putative icl promoter fragments, of 2900 and 572 bp, were subsequently linked to the GUS reporter gene and introduced into N. plumbaginifolia. Both constructs directed GUS expression after transgenic seed germination, and although the 572 bp fragment gave only 1% of the activity of the 2900 bp fragment, it directed expression in the same cotyledon-specific and temporally regulated pattern. Seedlings were transferred to darkness after 18 days growth in the light, to induce a starvation response. The 2900 bp construct was activated by starvation and repressed by exogenous sucrose, whereas the 572 bp construct was not starvation-responsive. To localize the region of the 2900 bp promoter fragment which is responsible for regulation by sucrose, further deletions were made, linked to GUS, and assayed in a cucumber protoplast transient assay system. Constructs with promoters of 2900, 2142 and 1663 bp were activated by starvation and repressed by sucrose, but promoters of 1142 and 572 bp showed no such response. We conclude that the icl gene promoter contains at least two distinct cis-acting elements, one required for the response to sucrose and the other which participates in expression upon seed germination.

The isocitrate lyase gene of cucumber: Isolation, characterisation and expression in cotyledons following seed germination

The cucumber (Cucumis sativus L.) genome contains only a single gene encoding the glyoxylate cycle enzyme isocitrate lyase (ICL). The cucumber icl gene has been isolated and sequenced, revealing only two small introns. The predicted amino acid sequence is more than 85% identical to ICL from other higher plants, and contains the C-terminal tripeptide Ser-Arg-Met which resembles a peroxisomal targeting sequence. The icl gene is coordinately expressed with the malate synthase (ms) gene after seed germination in both the light and the dark, suggesting that these genes may contain similar DNA elements regulating transcription. The start of transcription of the icl gene was determined and the DNA sequences upstream compared with the region of the ms gene promoter known to regulate transcription. This comparison revealed a highly conserved DNA sequence at similar positions in each gene.

Molecular cloning of cucumber phosphoenolpyruvate carboxykinase and developmental regulation of gene expression.

A cDNA library from RNA of senescing cucumber cotyledons was screened for sequences also expressed in cotyledons during post-germinative growth. One clone encodes ATP-dependent phosphoenolpyruvate carboxykinase (PCK; EC 4.1.1.49), an enzyme of the gluconeogenic pathway. The sequence of a full-length cDNA predicts a polypeptide of 74,397 Da which is 43%, 49% and 57% identical to bacterial, trypanosome and yeast enzymes, respectively. The cDNA was expressed in Escherichia coli and antibodies raised against the resultant protein. The antibody recognises a single polypeptide of ca. 74 kDa, in extracts of cotyledons, leaves and roots. The cucumber genome contains a single pck gene. In the seven-day period after seed imbibition, PCK mRNA and protein steady-state levels increase in amount in cotyledons, peaking at days 2 and 3 respectively, and then decrease. Both accumulate again to a low level in senescing cotyledons. This pattern of gene expression is similar to that of isocitrate lyase (ICL) and malate synthase (MS). When green cotyledons are detached from seedlings and incubated in the dark, ICL and MS mRNAs increase rapidly in amount but PCK mRNA does not. Therefore it seems unlikely that the glyoxylate cycle serves primarily a gluconeogenic role in starved (detached) cotyledons, in contrast to post-germinative and senescing cotyledons where PCK, ICL and MS are coordinately synthesised. While exogenous sucrose greatly represses expression of icl and ms genes in dark-incubated cotyledons, it has a smaller effect on the level of PCK mRNA.