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Aims Iron (Fe) deficiency in soil is a continuing problem for soybean (Glycine max L.) production, partly as a result of continuing climate change. This study elucidates how Trichoderma harzianum strain T22 (TH) mitigates growth retardation associated with Fe-deficiency in a highly sensitive soybean cultivar. Methods and Results Soil TH supplementation led to mycelial colonization and the presence of UAOX1 gene in roots that caused substantial improvement in chlorophyll score, photosynthetic efficiency and morphological parameters, indicating a positive influence on soybean health. Although rhizosphere acidification was found to be a common feature of Fe-deficient soybean, the upregulation of Fe-reductase activity (GmFRO2) and total phenol secretion were two of the mechanisms that substantially increased the Fe availability by TH. Heat-killed TH applied to soil caused no improvement in photosynthetic attributes and Fe-reductase activity, confirming the active role of TH in mitigating Fe-deficiency. Consistent increases in tissue Fe content and increased Fe-transporter (GmIRT1, GmNRAMP2a, GmNRAMP2b and GmNRAMP7) mRNA levels in roots following TH supplementation were observed only under Fe-deprivation. Root cell death, electrolyte leakage, superoxide (O2•–) and hydrogen peroxide (H2O2) substantially declined due to TH in Fe-deprived plants. Further, the elevation of citrate and malate concentration along with the expression of citrate synthase (GmCs) and malate synthase (GmMs) caused by TH suggest improved chelation of Fe in Fe-deficient plants. Results also suggest that TH has a role in triggering antioxidant defence by increasing the activity of glutathione reductase (GR) along with elevated S-metabolites (glutathione and methionine) to stabilize redox status under Fe-deficiency. Conclusions TH increases the availability and mobilization of Fe by inducing Fe-uptake pathways, which appears to help provide resistance to oxidative stress associated with Fe-shortage in soybean. Significance and Impact of the Study These findings indicate that while Fe deficiency does not affect the rate or degree of TH hyphal association in soybean roots, the beneficial effects of TH alone may be Fe deficiency-dependent.

To understand the molecular basis underlying increased triacylglycerol (TAG) accumulation in starchless (sta) Chlamydomonas reinhardtii mutants, we undertook comparative time-course transcriptomics of strains CC-4348 (sta6 mutant), CC-4349, a cell wall-deficient (cw) strain purported to represent the parental STA6 strain, and three independent STA6 strains generated by complementation of sta6 (CC-4565/STA6-C2, CC-4566/STA6-C4, and CC-4567/STA6-C6) in the context of N deprivation. Despite N starvation-induced dramatic remodeling of the transcriptome, there were relatively few differences (5 × 10(2)) observed between sta6 and STA6, the most dramatic of which were increased abundance of transcripts encoding key regulated or rate-limiting steps in central carbon metabolism, specifically isocitrate lyase, malate synthase, transaldolase, fructose bisphosphatase and phosphoenolpyruvate carboxykinase (encoded by ICL1, MAS1, TAL1, FBP1, and PCK1 respectively), suggestive of increased carbon movement toward hexose-phosphate in sta6 by upregulation of the glyoxylate pathway and gluconeogenesis. Enzyme assays validated the increase in isocitrate lyase and malate synthase activities. Targeted metabolite analysis indicated increased succinate, malate, and Glc-6-P and decreased Fru-1,6-bisphosphate, illustrating the effect of these changes. Comparisons of independent data sets in multiple strains allowed the delineation of a sequence of events in the global N starvation response in C. reinhardtii, starting within minutes with the upregulation of alternative N assimilation routes and carbohydrate synthesis and subsequently a more gradual upregulation of genes encoding enzymes of TAG synthesis. Finally, genome resequencing analysis indicated that (1) the deletion in sta6 extends into the neighboring gene encoding respiratory burst oxidase, and (2) a commonly used STA6 strain (CC-4349) as well as the sequenced reference (CC-503) are not congenic with respect to sta6 (CC-4348), underscoring the importance of using complemented strains for more rigorous assignment of phenotype to genotype.

Debaryomyces hansenii was grown in YPD medium without or with 1.0 M NaCl or KCl. Respiration was higher with salt, but decreased if it was present during incubation. However, carbonylcyanide-3-chlorophenylhydrazone (CCCP) markedly increased respiration when salt was present during incubation. Salt also stimulated proton pumping that was partially inhibited by CCCP; this uncoupling of proton pumping may contribute to the increased respiratory rate. The ADP increase produced by CCCP in cells grown in NaCl was similar to that observed in cells incubated with or without salts. The alternative oxidase is not involved. Cells grown with salts showed increased levels of succinate and fumarate, and a decrease in isocitrate and malate. Undetectable levels of citrate and low-glutamate dehydrogenase activity were present only in NaCl cells. Both isocitrate dehydrogenase decreased, and isocitrate lyase and malate synthase increased. Glyoxylate did not increase, indicating an active metabolism of this intermediary. Higher phosphate levels were also found in the cells grown in salt. An activation of the glyoxylate cycle results from the salt stress, as well as an increased respiratory capacity, when cells are grown with salt, and a 'coupling' effect on respiration when incubated in the presence of salt.

Motivation: In recent years, several methods have been proposed for determining metabolic pathways in an automated way based on network topology. The aim of this work is to analyse these methods by tackling a concrete example relevant in biochemistry. It concerns the question whether even-chain fatty acids, being the most important constituents of lipids, can be converted into sugars at steady state. It was proved five decades ago that this conversion using the Krebs cycle is impossible unless the enzymes of the glyoxylate shunt (or alternative bypasses) are present in the system. Using this example, we can compare the various methods in pathway analysis. Results: Elementary modes analysis (EMA) of a set of enzymes corresponding to the Krebs cycle, glycolysis and gluconeogenesis supports the scientific evidence showing that there is no pathway capable of converting acetyl-CoA to glucose at steady state. This conversion is possible after the addition of isocitrate lyase and malate synthase (forming the glyoxylate shunt) to the system. Dealing with the same example, we compare EMA with two tools based on graph theory available online, PathFinding and Pathway Hunter Tool. These automated network generating tools do not succeed in predicting the conversions known from experiment. They sometimes generate unbalanced paths and reveal problems identifying side metabolites that are not responsible for the carbon net flux. This shows that, for metabolic pathway analysis, it is important to consider the topology (including bimolecular reactions) and stoichiometry of metabolic systems, as is done in EMA. Contact: ldpf@minet.uni-jena.de; schuster@minet.uni-jena.de Supplementary information: Supplementary data are available at Bioinformatics online.

Allelic variation within individuals holds information regarding the relationships of organisms, which is expected to be particularly important for reconstructing the evolutionary history of closely related taxa. However, little effort has been committed to incorporate such information for reconstructing the phylogeny of organisms. Haplotype trees represent a solution when one nonrecombinant marker is considered, but there is no satisfying method when multiple genes are to be combined. In this paper, we propose an algorithm that converts a distance matrix of alleles to a distance matrix among organisms. This algorithm allows the incorporation of allelic variation for reconstructing the phylogeny of organisms from one or more genes. The method is applied to reconstruct the phylogeny of the seven native diploid species of Rosa sect. Cinnamomeae in North America. The glyceralgehyde 3-phosphate dehydrogenase (GAPDH), the triose phosphate isomerase (TPI), and the malate synthase (MS) genes were sequenced for 40 individuals from these species. The three genes had little genetic variation, and most species showed incomplete lineage sorting, suggesting these species have a recent origin. Despite these difficulties, the networks (NeighborNet) of organisms reconstructed from the matrix obtained with the algorithm recovered groups that more closely match taxonomic boundaries than did the haplotype trees. The combined network of individuals shows that species west of the Rocky Mountains, Rosa gymnocarpa and R. pisocarpa, form exclusive groups and that together they are distinct from eastern species. In the east, three groups were found to be exclusive: R. nitida-R. palustris, R. foliolosa, and R. blanda-R. woodsii. These groups are congruent with the morphology and the ecology of species. The method is also useful for representing hybrid individuals when the relationships are reconstructed using a phylogenetic network.

During phototrophic growth on acetate and CO2Rhodospirillum rubrum 2R contained malate synthase but lacked isocitrate lyase. Acetate assimilation by R. rubrum cells was stimulated by pyruvate, propionate glyoxylate, CO2 and H2. Acetate photoassimilation by R. rubrum cells in the presence of bicarbonate was accompanied by glyoxylate secretion, which increased after addition of fluoroacetate and decreased after addition of malonate. When acetyl-CoA was incubated with pyruvate in cell-free extracts, citramalate was formed. Citramalate was also formed from propionyl-CoA and glyoxylate. The existence in R. rubrum of a CO2-dependent cyclic pathway of acetate oxidation to glyoxylate with citramalate as an intermediate is proposed. Inhibitor analysis of acetate and bicarbonate assimilation indicated that pyruvate synthase is not involved in acetate assimilation in R. rubrum. The possible anaplerotic sequences employed by R. rubrum during phototrophic growth on acetate are discussed.

In most bacteria acetate assimilation is accomplished via the glyoxylate pathway. Isocitrate lyase (ICL) and malate synthase (MS) are two key enzymes of this pathway, which results in the net generation of one molecule of succinyl-CoA from two acetyl-CoA molecules. Genetic and biochemical data have shown that genes encoding these key enzymes are present in streptomycetes, yet there has been no clear demonstration of the importance of these genes to acetate assimilation. In fact, for Streptomyces collinus an alternative butyryl-CoA pathway has been shown to be critical for growth on acetate as a sole carbon source. Crotonyl-CoA reductase (CCR) is a key enzyme in this pathway and catalyzes the last step of the conversion of 2-acetyl-CoA molecules to butyryl-CoA. In Streptomyces cinnamonensis C730.1, it has been shown that CCR and this butyryl-CoA pathway provide the majority of methylmalonyl-CoA and ethylmalonyl-CoA for monensin A biosynthesis in an oil-based fermentation medium. We have cloned a MS homologue gene from this strain. Reverse transcription and direct enzyme assays demonstrated that neither this nor other MS genes were expressed during fermentation in an oil-based fermentation of either the C730.1 or L1 strain (a ccr mutant). Similarly, no ICL activity could be detected. The C730.1 but not the L1 strain was able to grow on acetate as a sole carbon source. The Streptomyces coelicolor aceA and aceB2 genes encoding ICL and MS were cloned into a Streptomyces expression plasmid (a derivative of pSET152) to create pExIM1. Enzyme assays and transcript analyses demonstrated expression of both of these proteins in C730.1/pExIM1 and L1/pExIM1 grown in an oil-based fermentation and tryptic soy broth media. Nonetheless, L1/pExIM1, like L1, was unable to grow on acetate as a sole carbon source, and was unable to efficiently generate precursors for monensin A biosynthesis in an oil-based fermentation, indicating that the additional presence of these two enzyme activities does not permit a functional glyoxylate cycle to occur. UV mutagenesis of S. cinnamonensis L1 and L1/pExIM1 led to mutants which were able to grow efficiently on acetate despite a block in the butyryl-CoA pathway. Analysis of enzyme activity and monensin production from these mutants in an oil-based fermentation demonstrated that neither the glyoxylate cycle nor the butyryl-CoA pathway function, suggesting the possibility of alternative pathways of acetate assimilation.

We tested the hypothesis that peroxisomal citrate synthase (CSY) is required for carbon transfer from peroxisomes to mitochondria during respiration of triacylglycerol in Arabidopsis thaliana seedlings. Two genes encoding peroxisomal CSY are expressed in Arabidopsis seedlings, and seeds from plants with both CSY genes disrupted were dormant and did not metabolize triacylglycerol. Germination was achieved by removing the seed coat and supplying sucrose, but the seedlings still did not use triacylglycerol. The mutant seedlings were resistant to 2,4-dichlorophenoxybutyric acid, indicating a block in peroxisomal β-oxidation, and were unable to develop further after transfer to soil. The mutant phenotype was complemented with a cDNA encoding CSY with either its native peroxisomal targeting sequence (PTS2) or a heterologous PTS1 sequence from pumpkin (Cucurbita pepo) malate synthase. These results suggest that peroxisomal CSY in Arabidopsis is not only a key enzyme of the glyoxylate cycle but also catalyzes an essential step in the respiration of fatty acids. We conclude that citrate is exported from the peroxisome during fatty acid respiration, whereas in yeast, acetylcarnitine is exported.

The effect of culture age on intra- and extracellular metabolite levels as well as on in vitro determined specific activities of enzymes of central carbon metabolism was investigated during evolution for over 90 generations of Saccharomyces cerevisiae CEN.PK 113-7D in an aerobic glucose/ethanol-limited chemostat at a specific dilution rate of 0.052 h−1. It was found that the fluxes of consumed (O2, glucose/ethanol) and secreted compounds (CO2) did not change significantly during the entire cultivation period. However, morphological changes were observed, leading to an increased cellular surface area. During 90 generations of chemostat growth not only the residual glucose concentration decreased, also the intracellular concentrations of trehalose, glycolytic intermediates, TCA cycle intermediates and amino acids were found to have decreased with a factor 5–10. The only exception was glyoxylate which showed a fivefold increase in concentration. In addition to this the specific activities of most glycolytic enzymes also decreased by a factor 5–10 during long-term cultivation. Exceptions to this were hexokinase, phosphofructokinase, pyruvate kinase and 6-phosphogluconate dehydrogenase of which the activities remained unchanged. Furthermore, the concentrations of the adenylate nucleotides as well as the energy charge of the cells did not change in a significant manner. Surprisingly, the specific activities of glucose-6-phosphate dehydrogenase (G6PDH), malate synthase (MS) and isocitrate lyase (ICL) increased significantly during 90 generations of chemostat cultivation. These changes seem to indicate a pattern where metabolic overcapacities (for reversible reactions) and storage pools (trehalose, high levels of amino acids and excess protein in enzymes) are lost during the evolution period. The driving force is proposed to be a growth advantage in the absence of these metabolic overcapacities.

The gene, encoding malate synthase (MS), aceB, was cloned from the thermophilic bacterium Streptomyces thermovulgaris by homology-based PCR. The 1,626-bp cloned fragment encodes a protein consisting of 541 amino acids. S. thermovulgaris malate synthase (stMS) gene was over-expressed in Escherichia coli using a glutathione-S transferase (GST) fusion vector (pGEX-6P-1), purified by affinity chromatography, and subsequently cleaved from its GST fusion partner. The purified stMS was characterized and compared to a mesophilic malate synthase (scMS) from Streptomyces coelicolor. stMS exhibited higher temperature optima (40-60 degrees C) than those of scMS (28-37 degrees C). It was more thermostable and very resistant to the chemical denaturant urea. Amino acid sequence comparison of stMS with four mesophilic streptomycete MSs indicated that they share 70.9-91.4% amino acid identities, with stMS possessing slightly more charged residues (approximately 31%) than its mesophilic counterparts (approximately 28-29%). Seven charged residues (E85, R187, R209, H239, H364, R382 and K520) that were unique to stMS may be selectively and strategically placed to support its peculiar characteristics.

The tricarboxylic acid (TCA) cycle enzyme isocitrate dehydrogenase (IDH) and the glyoxylate bypass enzyme isocitrate lyase are involved in catabolism of isocitrate and play a key role in controlling the metabolic flux between the TCA cycle and the glyoxylate shunt. Two IDH genes icd1 and icd2 of Ralstonia eutropha HF39, encoding isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2), were identified and characterized. Icd1 was functionally expressed in Escherichia coli, whereas icd2 was expressed in E. coli but no activity was obtained. Interposon-mutants of icd1 (HF39Deltaicd1) and icd2 (HF39Deltaicd2) of R. eutropha HF39 were constructed and their phenotypes were investigated. HF39Deltaicd1 retained 43% of IDH activity, which was not induced by acetate, and HF39Deltaicd2 expressed 74% of acetate-induced IDH activity. Both HF39Deltaicd1and HF39Deltaicd2 kept the same growth rate on acetate as the wild-type. These data suggested that IDH1 is induced by acetate. The interposon-mutants HF39Deltaicd1 and HF39Deltaicd2 accumulated the same amount of poly(3-hydroxybutyric acid) as the wild-type.

We cloned a major aliphatic glucosinolate (GSL) gene, BoGSL-ELONG in Brassica oleracea, using the Arabidopsis sequence database. We based our work on an Arabidopsis candidate gene forming part of a gene family coding for isopropyl malate synthetase-like enzymes (IPMS). This gene is presumably responsible for synthesis of GSL possessing side chains consisting of four carbons (4C). The similarity of the Brassica homolog IPMS-Bo from broccoli to its Arabidopsis counterpart IPMS-At was on the order of 78%, both sharing the same number of exons. A nonfunctional allele of the BoGSL-ELONG gene from white cauliflower, based on the absence of 4C GSL in this crop, displayed a 30-bp deletion, which allowed us to develop a codominant marker for 4C-GSL. Gene expression analysis based on RT-PCR revealed a splicing site mutation in the white cauliflower allele. This resulted in a longer transcript containing intron 3, which failed to excise. Perfect cosegregation was observed for broccoli and cauliflower alleles at the IPMS-Bo gene and 4C-GSL content, strongly indicating that this gene indeed corresponds to BoGSL-ELONG. Cloning of two other major genes, BoGSL-ALK and BoGSL-PRO, is underway. The availability of these genes and BoGSL-ELONG is essential for the manipulation of the aliphatic GSL profile of B. oleracea.

According to different metabolic situations in various stages of Candida albicans pathogenesis the regulation of carbohydrate metabolism was investigated. We report the genetic characterization of all major C. albicans gluconeogenic and glyoxylate cycle genes (fructose-1,6-bisphosphatase, PEP carboxykinase, malate synthase and isocitrate lyase) which were isolated after functional complementation of the corresponding Saccharomyces cerevisiae deletion mutants. Remarkably, the regulation of the heterologously expressed C. albicans gluconeogenic and glyoxylate cycle genes was similar to that of the homologous S. cerevisiae genes. A C. albicans ΔCafbp1 deletion strain failed to utilize non-fermentable carbon sources but hyphal growth was not affected. Our results show that regulation of gluconeogenesis in C. albicans is similar to that of S. cerevisiae and that the current knowledge on how gluconeogenesis is regulated will facilitate the physiological understanding of C. albicans.

Malate synthase (EC 4.1.3.2), the key enzyme of the glyoxylate cycle, was purified to a homogeneous protein from the wood-rotting basidiomycete Fomitopsis palustris grown on glucose. The purified enzyme, with a molecular mass of 520 kDa, was found to consist of eight 65-kDa subunits, and to have Km of 45 and 2.2 microM for glyoxylate and acetyl-CoA, respectively. The enzyme activity was competitively inhibited by oxalate (K1, 8.5 microM) and glycolate (Ki, 17 microM), and uncompetitively by coenzyme A (Ki, 100 microM). The potent inhibition of the activity by p-chloromercuribenzoate suggests that the enzyme has a sulfhydryl group at the active center. However, the enzyme was inhibited moderately by adenine nucleotides and weakly by some of the metabolic intermediates of glycolysis and tricarboxylic acid cycle. The enzyme was completely inactive in the absence of metal ions and was maximally activated by Mg2+ (Km, 0.4 microM), which also served to significantly prevent enzyme inactivation during storage.

Cucumber fruits exposed to a 60% CO2 environment for 3 d at 20 degrees C showed a higher respiration rate, a lower respiratory quotient, and lower levels of malate, citrate + isocitrate, and glyoxylate than did control fruits. Moreover, in the CO2 treatment, fumarate and succinate increased dramatically on the first day and after the second day, respectively, while 2-oxoglutarate showed a temporary increase on the first day and a subsequent decrease. Isocitrate lyase and malate synthase activity tended to increase throughout the experimental period of the CO2 treatment. These results appear to point to a rapid operation of glyoxylate cycle due to high CO2.

Expression of a California bay lauroyl-acyl carrier protein thioesterase (MCTE) in developing seeds of transgenic oilseed rape alters the fatty acid composition of the mature seed, resulting in up to 60 mol% of laurate in triacylglycerols. In this study, we examined the metabolism of lauric acid and 14C-acetate in developing seeds of oilseed rape that express high levels of MCTE. Lauroyl-CoA oxidase activity but not palmitoyl-CoA oxidase activity was increased several-fold in developing seeds expressing MCTE. In addition, isocitrate lyase and malate synthase activities were six- and 30-fold higher, respectively, in high-laurate developing seeds. Control seeds incorporated 14C-acetate almost entirely into fatty acids, whereas in seeds expressing MCTE, only 50% of the label was recovered in lipids and the remainder was in a range of water-soluble components, including sucrose and malate. Together, these results indicate that the pathways for beta-oxidation and the glyoxylate cycle have been induced in seeds expressing high levels of MCTE. Although a substantial portion of the fatty acid produced in these seeds is recycled to acetyl-CoA and sucrose through the beta-oxidation and glyoxylate cycle pathways, total seed oil is not reduced. How is oil content maintained if lauric acid is inefficiently converted to triacylglycerol? The levels of acyl carrier protein and several enzymes of fatty acid synthesis were increased two- to threefold at midstage development in high-laurate seeds. These results indicate that a coordinate induction of the fatty acid synthesis pathway occurs, presumably to compensate for the lauric acid lost through beta-oxidation or for a shortage of long-chain fatty acids.

Nesterenkonia sp. AN1 is a polyextremophile isolated from Antarctic desert soil. Genomic analyses and genome comparisons with three mesophilic Nesterenkonia strains indicated that the unique genome fraction of Nesterenkonia sp. AN1 contains adaptive features implicated in the response to cold stress including modulation of membrane fluidity as well as response to cold-associated osmotic and oxidative stress. The core genome also encodes a number of putative cold stress response proteins. RNA-Seq-based transcriptome analyses of Nesterenkonia sp. AN1 grown at 5ºC and 21°C showed that there was significant induction of transcripts that code for antioxidants at 5ºC, demonstrated by the upregulation of sodA, bcp and bpoA2. There was also overexpression of universal stress protein genes related to uspA, along with genes encoding other characterized cold stress features. Genes encoding the two key enzymes of the glyoxylate cycle, isocitrate lyase (ICL) and malate synthase (AceB) were induced at 5ºC, suggesting possible adaptation strategies for energy metabolism in cold habitats. These genomic features may contribute to the survival of Nesterenkonia sp. AN1 in arid Antarctic soils.

Biological production in heterologous hosts is of interest for the production of the C4 alcohol (butanol) and other chemicals. However, some hurdles need to be overcome in order to achieve an economically viable process; these include avoiding the consumption of butanol and maintaining tolerance to this solvent during production. Pseudomonas putida is a potential host for solvent production; in order to further adapt P. putida to this role, we generated mini-Tn5 mutant libraries in strain BIRD-1 that do not consume butanol. We analyzed the insertion site of the mini-Tn5 in a mutant that was deficient in assimilation of butanol using arbitrary PCR followed by Sanger sequencing and found that the transposon was inserted in the malate synthase B gene. Here, we show that in a second round of mutagenesis a double mutant unable to take up butanol had an insertion in a gene coding for a multisensor hybrid histidine kinase. The genetic context of the histidine kinase sensor revealed the presence of a set of genes potentially involved in butanol assimilation; qRT-PCR analysis showed induction of this set of genes in the wild type and the malate synthase mutant but not in the double mutant.

We have previously proposed that metabolic status is important in the regulation of cucumber malate synthase (MS) and isocitrate lyase (ICL) gene expression during plant development. In this article, we used a cell culture system to demonstrate that intracellular metabolic status does influence expression of both of these genes. Starvation of cucumber cell cultures resulted in the coordinate induction of the expression of MS and ICL genes, and this effect was reversed when sucrose was returned to the culture media. The induction of gene expression was closely correlated with a drop in intracellular sucrose, glucose, and fructose below threshold concentrations, but it was not correlated with a decrease in respiration rate. Glucose, fructose, or raffinose in the culture media also resulted in repression of MS and ICL. Both 2-deoxyglucose and mannose, which are phosphorylated by hexokinase but not further metabolized, specifically repressed MS and ICL gene expression relative to a third glyoxylate cycle gene, malate dehydrogenase. However, the addition of 3-methylglucose, an analog of glucose that is not phosphorylated, did not result in repression of either MS or ICL. It is proposed that the signal giving rise to a change in gene expression originates from the intracellular concentration of hexose sugars or the flux of hexose sugars into glycolysis.

To investigate if pollen possesses glyoxysomal function, we analyzed the activities of isocitrate lyase and malate synthase genes. Because the activities of these enzymes were exceedingly low in pollen extracts, we constructed fusion genes encoding [beta]-glucuronidase (GUS) that are regulated by isocitrate lyase or malate synthase promoters from Brassica napus L. to increase the sensitivity of our assays. Expression of the fusion genes in transgenic tobacco was qualitatively similar to that of the endogenous genes; GUS activity was low in dry seeds, maximal in seedlings, and very low or undetectable in leaves, indicating that the promoters are regulated correctly. We showed that isocitrate lyase and malate synthase genes are active at specific stages of pollen development and that their activities are not enhanced during pollen germination in transgenic tobacco. We also confirmed that the endogenous genes are active by showing that the corresponding mRNAs could be detected in pollen at specific stages of development. The activation of the isocitrate lyase and malate synthase genes suggests that glyoxysomal function is induced during pollen development.

The effects of glucose starvation on the oxidation of fatty acids were studied in excised maize (Zea mays L.) root tips. After 24 hours of glucose starvation, the rate of oxidation of palmitic acid to CO(2) by the root tips was increased 2.5-fold. Different enzyme activities were tested in a crude particulate fraction from nonstarved root tips and those starved for 24 hours. The activities of the beta-oxidation enzymes crotonase, hydroxyacyl-coenzyme A (CoA) dehydrogenase, and thiolase and those of catalase, malate synthase, and peroxisomal citrate synthase were higher after starvation. However, no isocitrate lyase activity was detected, thus suggesting that the glyoxylate cycle does not operate. The overall beta-oxidation activity was assayed as the formation of [(14)C]acetyl-CoA from [(14)C]palmitic acid after high-performance liquid chromatography analysis of the CoA derivatives. An activity was detected in sugar-fed root tips, and it was increased by two-to fivefold in starved roots. Because the recovery of enzyme activities is only marginally better in starved roots compared with nonstarved roots, these results indicate that the beta-oxidation activity in the tissues is increased during sugar starvation. This increase is probably an essential part of the response to a situation in which lipids and proteins replace carbohydrates as the major respiratory substrates. These results are discussed in relation to the metabolic changes observed in senescing plant tissues.

Expression of the malate synthase (MS) gene is activated in cotyledons of cucumber seedlings during postgerminative growth and then repressed as the cotyledons become photosynthetic. MS gene expression is subsequently reactivated in the cotyledons as they senesce a few weeks later. In situ hybridization revealed that MS RNA is distributed throughout the organ during postgerminative growth and senescence, showing that the same cells express the gene at different stages of development. MS RNA also appears in senescing leaves and petals of cucumber plants. In addition, we found that MS RNA appears in mature expanded leaves and roots when they are removed from the plant and incubated in darkness for several days, thus providing a potential experimental system for the manipulation of MS gene expression. Leaves from transgenic Nicotiana plumbaginifolia containing the cucumber MS promoter fused to the [beta]-glucuronidase (GUS) reporter gene accumulated GUS activity when detached, demonstrating an activation of transcription from the MS promoter following leaf excision. These results are discussed in terms of the metabolic regulation of MS gene expression.

1. SUMMARY This paper describes ethanol metabolism in a peroxisome-deficient (PER) mutant of Hansenula polymorpha. The PER mutant was able to use ethanol as sole-carbon source but showed reduced growth rates compared to wild-type cells together with a reduced rate of ethanol utilization under ]'£max conditions. In chemostat cultures at low-dilution rates, the activities of alcohol dehydrogenase, isocitrate lyase and malate synthase were comparable in wild-type and PER cells. In PER cells the two latter enzymes, exclusively microbody-bound in wild-type cells, were active in the cytosol. The possible advantage of intact microbodies in the intermediary metabolism of ethanol in 14. polymorpha is discussed.

We have analyzed the temporal and spatial expression of genes encoding the glycoxylate cycle enzymes isocitrate lyase and malate synthase in Brassica napus L. to determine whether they are coordinately expressed. Both enzymes participate in reactions associated with lipid mobilization in oilseed plant seedlings and are sequestered in a specialized organelle, the glyoxysome. We have identified an isocitrate lyase cDNA clone containing the complete protein coding region. RNA blot and in situ hybridization studies with isocitrate lyase and malate synthase cDNA clones from B. napus showed that the genes exhibit similar expression patterns. The mRNAs begin to accumulate during late embryogeny, reach maximal levels in seedling cotyledons, are not detected at significant amounts in leaves, and are distributed similarly in cotyledons and axes of seedlings. Furthermore, transcription studies with isolated nuclei indicate that the genes are controlled primarily although not exclusively at the transcriptional level. We conclude that glyoxysome biogenesis is regulated in part through the coordinate expression of isocitrate lyase and malate synthase genes.

Developmental patterns of glyoxylate cycle and photosynthetic activities have been correlated with electrophoretic profiles of cotyledonary RNA and protein in both light- and dark-grown cucumber seedlings (Cucumis sativus L.) Cytoplasmic rRNA increases 10-fold between days 0 and 5, and the steepest increase coincides with the most rapid rise in activities of the glyoxysomal enzymes, isocitrate lyase and malate synthase. Chloroplast rRNA and ribulose bisphosphate (RuBP) carboxylase begin rising at day 3, followed about a day later by increases in glyoxylate reductase activity and chlorophyll content. Of these phototrophic indicators, only chlorophyll requires light for its initial appearance. Sodium dodecyl sulfate gel electrophoresis of total and soluble cotyledonary protein showed several developmental patterns, including: (a) progressive disappearance of storage protein present initially in particulate form; (b appearance and subsequent disappearance of a family of polypeptides identified by molecular weight, developmental profile, and density gradient centrifugation as subunits of glyoxysomal enzymes; and (c) appearance and progressive increase (in both light- and dark-grown cotyledons) of the large and small subunits of RuBP carboxylase, as well as other polypeptides presumably of chloroplast and peroxisomal origin.

Candida brumptii IFO-0731 produced a large amount of L-malic acid from n-paraffins and the acid production was not associated with cell growth. The activities of the enzymes involved in the tricarboxylic acid cycle and the glyoxylate cycle were assayed through cultivation period in order to study on the mechanism of L-malic acid production from n-paraffins. It was deduced that isocitrate lyase and malate synthase belonging to the glyoxylate cycle played an important role in L-malic acid production.

The cytochemical localizations of malate synthase (glyoxysomal marker) and glycolate oxidase (peroxisomal marker) have been examined in cotyledon segments and sucrose-gradient fractions from germinated cucumber (Cucumis sativus L.) seedlings. The seedlings were grown in the dark for 4 days, transferred to 4 hours of continuous light, then returned to the dark for 24 hours. Under these conditions, high specific activities for both glyoxysomal and peroxisomal enzymes are maintained in cotyledon homogenates and microbody-enriched fractions. Electron cytochemistry of the marker enzymes reveals that all or virtually all the microbodies observed in cotyledonary cells and sucrose-gradient fractions contain both enzymes. The staining in gradient fractions was determined from scoring a minimum of 600 photographed microbodies for each enzyme. After correcting for the number of particles stained for catalase reactivity (representing true microbodies), 94 and 97% of the microbodies were found stained for malate synthase and glycolate oxidase activity, respectively.The results from these studies provide pertinent information toward understanding the succession from glyoxysomal to peroxisomal metabolism in cotyledons of fatty seedlings. The coexistence of two separate microbody types functioning at different stages of development apparently is not the case. The localizations of both marker enzymes within one microbody type strongly suggest that the metabolic transition involves a change in enzyme complement within an ongoing population of microbodies.

The utilization of nitrate and ammonia as nitrogen sources had different effects on the metabolism of glycolate in Cholorella sorokiniana. During photolithotrophic growth with nitrate as nitrogen source, glycolate was metabolized via the glycine-serine pathway. Ammonia, produced as a result of glycolate metabolism, was reassimilated by glutamine synthetase. Two isoforms of this enzyme were present at different relative abundance in C. sorokiniana wild type and in a mutant with an increased capacity for the metabolism of glycolate (strain OR). During photolithotrophic growth in the presence of ammonia as sole nitrogen source, several lines of evidence indicated that glycolate was metabolized to malate, pyruvate, tricarboxylic acid cycle intermediates and related amino acids in C. sorokiniana wild-type cells. Malate synthase was induced and glycine decarboxylase and serine-glyoxylate aminotransferase were repressed in cells grown with ammonia. An inverse correlation was observed between aminating NADPH-glutamate dehydrogenase and the in vivo glycine decarboxylation rate.

The effects of unsaturated fatty acids on the activities of peroxisomal enzymes of Tetrahymena pyriformis were investigated. When saturated fatty acids and the corresponding unsaturated fatty acids (C18) were added to the culture medium at 0.05%, the activities of peroxisomal enzymes [fatty acyl-CoA oxidase (FAO), carnitine acetyltransferase (CAT), isocitrate lyase (ICL), and malate synthase (MS)] were significantly increased. The order of effectiveness was linoleic acid greater than oleic acid greater than stearic acid. However, alpha-linolenic acid and gamma-linolenic acid at the same concentration were lethal to the cells. The inhibitory effect on growth disappeared upon addition of an antioxidant, alpha-tocopherol. Lipid peroxides derived from unsaturated fatty acids induced marked cell lysis. In the presence of a low concentration (0.005%) of linolenic acid the production of lipid peroxide was lower and no inhibitory effect on the growth was observed, while the activities of peroxisomal enzymes participating in lipid metabolism and that of catalase were significantly increased. These results indicate that the peroxisomal enzyme systems related to the beta-oxidations of fatty acids and the glyoxylate cycle are regulated by unsaturated long-chain fatty acids, including linolenic acid, at low concentrations, as well as by saturated fatty acid in the medium.

Malate synthase (EC 4.1.3.2), termed MSH (mol. wt. 630,000), was purified from the pollen of Pinus densiflora Sieb, et Zucc. to apparent homogeneity as judged by SDS-PAGE. Part of MSH was converted to the low molecular weight form of MS, termed MSL (mol. wt. 62,000), by incubation with 5 mm ATP. Both forms of MS were maximally active at pH 7.6 in the presence of 10 mm MgCl2 and 0.01 mm EDTA. MSL was completely inactivated by heating at 50°C for 3 min, but MSH activity was retained about 65%. MSH preincubated with 5mm ATP showed unstability similar to that of MSl. MSh activity was more strongly inhibited than that of MSL by phosphoenolpyruvate or ATP. Km values of MSH for acetyl-CoA and glyoxylate were about one half of those of MSL. The Fmax of MSh was about 5 times as high as that of MSL. ATP was a noncompetitive inhibitor with respect to acetyl-CoA and Ki values of ATP for MSH and MSL were 1.0 × 10–3 m and 2.0 × 10–2 m, respectively. From these results we suggested that MSH and MSL are the active and lo...

Comparative studies on the activities of isocitrate lyase and malate synthase were performed in the cells of Candida yeasts grown on media containing glucose, n-alkane or acetate as the sole carbon source. In general, the activities of both enzymes were higher in the cells growing on n-alkane or acetate than in those growing on glucose. When the yeasts were cultivated on glucose, the level of the isocitrate lyase activity in C. tropicalis was enhanced after the middle phase of growth, while the activity in C. lipolytica was maintained at a low level. The synthesis of isocitrate lyase in C. tropicalis growing on n-alkane or acetate was repressed by the addition of glucose to the medium, and derepressed when the cells grown on glucose were transferred to n-alkane or acetate media, indicating that glyoxylate cycle has an important role in the metabolism of n-alkane as well as acetate. Cycloheximide inhibited the synthesis of isocitrate lyase in C. tropicalis growing on n-alkane or on acetate.

Exposure of dark grown resting Euglena to ethanol produced a transient increase in the specific activity of the glyoxysomal enzyme malate synthase. Enzyme specific activity increased during the first 24 hours of ethanol treatment and then declined. Light exposure or malate addition failed to increase enzyme specific activity. The increase and decrease in enzyme specific activity represented changes in the amount of active enzyme. In both wild type cells and the plastidless mutant W 3 BUL, enzyme levels were always higher in the dark than in the light. The specific activity of the peroxisomal enzyme glycolate dehydrogenase began to increase 24 hours after dark grown resting Euglena were exposed to light. Ethanol, but not malate, prevented the increase and promoted a decrease in glycolate dehydrogenase levels. Cycloheximide produced a decline in enzyme levels similar to the decline produced by ethanol addition. Glycolate dehydrogenase was present in the plastidless mutant W 3 BUL indicating that it is coded in the nucleus and synthesized on cytoplasmic ribosomes. Streptomycin, a specific inhibitor of chloroplast protein synthesis and 3-(3,4-dichlorophenyl)-1,1-dimethylurea, an inhibitor of photosynthetic CO 2 fixation, inhibited the photoinduction of glycolate dehydrogenase while having no effect on the photoinduction of NADP dependent glyceraldehyde-3-phosphate dehydrogenase, another light induced, nuclear coded, cytoplasmically synthesized enzyme. Taken together, these results suggest that microbodies are continually synthesized in resting Euglena and their enzyme complement is determined through substrate induction of glyoxysomal and peroxisomal enzymes.

When Neurospora crassa is grown with acetate as a source of carbon and energy, the metabolic pathways differ from those occurring in hyphae grown with sucrose [1-.6]. The rates of the glycolysis and of the hexose monophosphate shunt are reduced, whereas the activities of acetyl CoA synthase, isocitrate lyase, malate synthase and phosphoenolpyruvate carboxykinase are considerably enhanced, which is essential for the utilization of acetate and gluconeogenesis. Isocitrate lyase and malate synthase are located in giyoxysomes [3], but the fulfilment of the glyoxylate cycle utilizes several enzymes of the tricarboxylic acid cycle and entails a co-operation between mitochondria and glyoxysomes. Since glyoxysomes from Neurospora do not contain citrate synthase [7], these organelles would have to import citrate or isocitrate as a substrate for isocitrate lyase. Furthermore, glyoxysomes must export succinate, and possibly malate, for further oxidation. Therefore, several mitochondrial enzymes could take on an increased significance or have an altered role when Neurospora is grown with acetate, in response to the induction of isoeitrate lyase and to the increased production of malate and succinate. It has been reported before that growth with acetate results in a 3to 5-fold increase in the activity of citrate synthase, NADP-isocitrate delaydrogenase, succinate dehydrogenase, fumarase and malate dehydrogenase [2,4-7]. There is, however, no information presently available concerning the possible changes occurring in the properties of intact mitochondria, as a response to the growth of Neurospora in a medium containing acetate. The purpose of the present work was therefore to study the oxidative and phosphorylative properties of mitochondria isolated from Neurospora grown with acetate, and to compare them with mitochondria from hyphae grown with sucrose, with particular attention given to variations occurring at different stages of growth.

Comparative studies on the activities of isocitrate lyase and malate synthase were performed in the cells of Candida yeasts grown on media containing glucose, n-alkane or acetate as the sole carbon source. In general, the activities of both enzymes were higher in the cells growing on n-alkane or acetate than in those growing on glucose. When the yeasts were cultivated on glucose, the level of the isocitrate lyase activity in C. tropicalis was enhanced after the middle phase of growth, while the activity in C. lipolytica was maintained at a low level. The synthesis of isocitrate lyase in C. tropicalis growing on n-alkane or acetate was repressed by the addition of glucose to the medium, and derepressed when the cells grown on glucose were transferred to n-alkane or acetate media, indicating that glyoxylate cycle has an important role in the metabolism of n-alkane as well as acetate. Cycloheximide inhibited the synthesis of isocitrate lyase in C. tropicalis growing on n-alkane or on acetate.

Monospecific antibodies raised against four glyoxysomal enzymes (isocitrate lyase, catalase, malate synthase, and malate dehydrogenase) have been used to detect these proteins among the products of in vitro translation in a wheat germ system programmed with cotyledonary RNA from cucumber seedlings. In vitro immunoprecipitates were compared electrophoretically with the same enzymes labeled in vivo and also with the purified proteins. Isocitrate lyase yields two bands on sodium dodecyl sulfate-polyacrylamide gels, as synthesized both in vitro (61.5K and 60K products) and in vivo (63K and 61.5K polypeptides). Both the 63K and 61.5K subunits can also be demonstrated for the isolated enzyme. The two subunits are antigenically cross-reactive and yield similar electrophoretic profiles upon partial proteolytic digestion. A larger subunit is seen in vitro than in vivo for both malate dehydrogenase (38K versus 33K) and catalase (55K versus 54K); this suggests a need for processing which is often a characteristic of proteins that must be transported across or into membranes. Malate synthase has a molecular weight of 57K both in vitro and in vivo, but the isolated enzyme is a glycoprotein, containing N-acetyl glucosamine, mannose, and possibly also fucose and xylose. This indicates that the polypeptide portion of the isolated enzyme is smaller than the in vitro product and suggests processing of malate synthase also. None of the other three enzymes appears to be glycosylated. The implications of these size differences for the compartmentalization of matrix and membrane-bound glyoxysomal enzymes are discussed.

Malate synthase (EC 4.1.3.2), termed MSH (mol. wt. 630, 000), was purified from the pollen of Pinus densiflora Sieb. et Zucc. to apparent homogeneity as judged by SDS-PAGE. Part of MSH was converted to the low molecular weight form of MS, termed MSL (mol. wt. 62, 000), by incubation with 5 mM ATP. Both forms of MS were maximally active at pH 7.6 in the presence of 10 mM MgCl2 and 0.01mM EDTA. MSL was completely inactivated by heating at 50°C for 3min, but MSH activity was retained about 65%. MSH preincubated with 5mM ATP showed unstability similar to that of MSL. MSH activity was more strongly inhibited than that of MSL by phosphoenolpyruvate or ATP. Km values of MSH for acetyl-CoA and glyoxylate were about one half of those of MSL. The Vmax of MSH was about 5 times as high as that of MSL. ATP was a noncompetitive inhibitor with respect to acetyl-CoA and Ki values of ATP for MSH and MSL were 1.0 × 10-3 M and 2.0 × 10-2 M, respectively. From these results we suggested that MSH and MSL are the active and low active forms of MS, respectively.

13C Nuclear magnetic resonance spectroscopy of leaflets of Gleditsia triacanthos and Albizia julibrisin was used to determine the fate of acetate taken up during the absorption of calcium from 13C-labeled Ca-acetate solution. Small amounts of acetate accumulated temporarily in the leaf tissues, but the bulk of acetate was incorporated into malate. The initial rate of malate synthesis was very low, but increased rapidly during acetate treatment and reached its maximum after 8 hours; the enzymes involved in malate synthesis thus appear to be substrate induced. Use of acetate-2-13C yielded malate labeled in C-3, indicating that vacuolar malate accumulating during Ca-uptake might be synthesized via malate synthase from acetate and glyoxalate. However, a source of glyoxalate condensing with acetate during malate synthesis could not be identified. Glycolate produced in photorespiration is an unlikely source, because glycolate-2-13C was absorbed and metabolized by the leaf tissues into products of the glycolate pathway, but was not a major precursor in malate synthesis. Malate synthesis via the glyoxalate cycle is also unlikely, because no evidence for the recycling of a 13C-labeled 4-carbon organic acid was found. Malate synthesis in the leaflets of Gleditsia and Albizia thus appears to involve the inducible condensation of acetate with a 2-carbon compound of unidentified nature and origin.

The endosperm of 3-day germinated seedlings of Ricinus communis was homogenized in the presence or absence of Mg2+. When the Mg2+ -containing homogenate was fractionated on linear, 20 to 40% sucrose gradients, the endoplasmic reticulum (ER) reached equilibrium at a density of 1.146 grams per cubic centimeter. Absence of Mg2+ in the grinding medium resulted in displacement of the ER in the gradient from a density of 1.146 to 1.138 grams per cubic centimeter. At either density, the activities of both malate and citrate synthase were found to overlap the activity of NADH-cytochrome c reductase (an ER marker) in the gradient. Furthermore, this overlap of activities was observed whether the gradients were centrifuged for 3 or 19 hours. An analysis of sedimentation characteristics of the solubilized enzymes revealed that they exist, predominantly, as a 5.2S (s20,w × 10−13) form (malate synthase) and a 6.8S form (citrate synthase) in the glyoxysomes and cytosol. When the two enzymes were released from the ER, they appeared as aggregate forms of 70S and 55S, respectively. These results support the conclusion that the synthases are associated with the ER.

Extracts from several species and varieties of ungerminated cotton seeds plus homogenates from 18 other oilseeds (representing 11 different families) were examined for malate synthase and isocitrate lyase activity. Malate synthase activities in the various cotton seeds ranged from 35 to 129% of the units per dry seed weight found in Deltapine 16 cotton. For other oilseeds, the range was from 0.3 to 58% of Deltapine 16 cotton. Castor bean (Ricinus communis L.) had the least activity per mg dry weight (12-fold lower than the next lowest species), while Pima cotton (Gossypium barbadense L.) had the highest level (8.53 units). On a per seed basis, these values were 15 and 747 nanomoles per minute.Malate synthase activity was measurable in all seed types examined, whereas isocitrate lyase could not be detected in any of the seeds. We suggest that synthesis of malate synthase during seed development is universal among oilseeds in the absence of glyoxylate-cycle-associated isocitrate lyase activity.

Malate synthase (EC 4.1.3.2) (MS), an enzyme unique to the glyoxylate cycle, was studied in cotyledons of dark-grown cotton (Gossypium hirsutum, L.) seedlings. MS has generally been regarded as a peripheral membrane protein in glyoxysomes and believed by some to be synthesized on rough ER. Immunocyto-chemical localization of MS in both in situ and isolated cottonseed glyoxysomes, however, showed that MS was located throughout the matrix of glyoxysomes, not specifically associated with their membranes. Biochemical data also supported matrix localization. Isolated glyoxysomes were diluted in variously-buffered salt solutions (200 millimolar KCl or 100 millimolar K-phosphate) or detergents (0.1% Triton X-100, 10 millimolar deoxycholate, or 1.0% Triton X-114) and centrifuged to pellet membranes. Greater than 70% of the MS was recovered in supernatants after treatment with salt solutions, whereas generally less than 30% was released following detergent treatments. MS in pellets derived from glyoxysomes burst in low ionic strength buffer solutions was aggregated (observed on rate-zonal gradients). MS released following salt treatments was the 20S nonaggregated form indicating that salt solutions either disaggregated (or prevented aggregation of) glyoxysomal MS rather than releasing it from membranes. We confirmed reports by others that MS comigrated with ER (NADH: cytochrome c reductase) in sucrose (20-40% w/w) gradients buffered with 100 millimolar Tricine (pH 7.5) after 3 hours centrifugation. However, cottonseed MS did not comigrate with ER in gradients buffered with 10 millimolar Hepes (pH 7.0) or 20 millimolar K-phosphate (pH 7.2) after 3 hours centrifugation, or after 22 hours centrifugation in Tricine or Hepes. Collectively, our data with cotton seeds indicate that MS is not a peripheral membrane protein, and that the aggregation behavior of MS (in various buffers) very likely has led to misinterpretations of its putative associations with ER and glyoxysomal membranes.

A study was made of the enzymology of primary and intermediary pathways of C1 metabolism in three strains of non-motile obligately methylotrophic bacteria. Each uses a variant of the ribulosemonophosphate (RMP) cycle of formaldehyde fixation which involves the Entner-Doudoroff route for hexose-phosphate cleavage and transaldolase/transketolase mode of rearrangement. The organisms possess high levels of hexulose-phosphate synthase and NAD(P)-linked glucose-6-phosphate and 6-phosphogluconate dehydrogenases. In addition they contain small activities of dye-linked methanol and methylamine dehydrogenases, PMS- and NAD-linked formaldehyde and formate dehydrogenases. This indicates cyclic rather than direct oxidation of formaldehyde derived from methanol or methylamine. The tricarboxylic acid cycle is defective in 2-ketoglutarate dehydrogenase and the glyoxylate shunt is not operating because of the absence of malate synthase. Oxaloacetate is regenerated by (phosphoenol) pyruvate carboxylases. NH+4 is assimilated mainly by glutamate dehydrogenase. The results show metabolic similarities between motile and non-motile obligate methanol and methylamine utilizers.

Malate synthase (EC 4.1.3.2), an enzyme unique to the glyoxylate cycle, was purified to homogeneity from cotyledons of 72-hours, darkgrown cotton (Gossypium hirsutum L.) seedlings. Homogeneity of the enzyme was assessed by silver staining SDS-PAGE gels. Purification was accomplished by using a single buffer medium through six steps involving one ammonium sulfate fractionation and chromatography on three columns (Sephacryl S-300, DEAE Sephacel, Phenyl Sepharose). Large-scale preparation of glyoxysomes, a main step in all other published procedures, was not involved. The purified enzyme and that extracted from glyoxysomes appears to be a dodecamer with a native molecular weight of 750,000 (sedimentation coefficient of >20 Svedberg units [S] on sucrose gradients) composed of identical subunits (molecular weight approximately 63,000). The monomer (5S) occurs in the cytosol. Polyclonal antibodies raised in rabbits were judged to be monospecific for malate synthase by immunotitration, double immunodiffusion, and western blotting. Double immunodiffusion experiments revealed only partial immunological identity between the 5S (cytosolic) and 20S (glyoxysomal forms, although complete identity was observed between the 5S form in immature and germinated seeds, and the 20S form in immature and germinated seeds. Cross-reactivity of the cotton antimalate synthase serum was observed with extracts from five other oilseeds. Western blot analyses showed that malate synthase protein was not present in immature seeds prior to appearance of enzyme activity, but when present, subunit molecular weight was indistinguishable in immature, desiccated, and germinated seeds.

Seedlings of castor bean (Ricinus communis cv. Hale) were exposed to a range of concentrations of gibberellin A3 (GA3). Treatments for 20 hours with GA3 concentrations of 0.5 μM or higher resulted in increased levels of NADH-cytochrome c reductase, phosphorylcholine glyceride transferase, and malate synthase in endoplasmic reticulum (ER) isolated from endosperm on linear sucrose gradients. GA3 treatment also resulted in increased RNA associated with ER. Malate synthase and catalase in crude homogenates were enhanced by 1 to 100 μM GA3 concentrations. Isocitrate lyase, citrate synthase, malate synthase, catalase, and glycolate oxidase in isolated glyoxysomes were enhanced by 60, 20, 18, 40, and 28%, respectively, over controls. Treatment with abscisic acid led to decreased levels of glyoxysomal enzymes and reduced glyoxysomal protein. The effect of GA3 and abscisic acid on the specific activities of glyoxysomes of different densities suggests that GA3 influences enzyme levels and glyoxysome assembly.

The developmental profile of the glyoxysomes and their component enzymes catalase, malate synthase, and isocitrate lyase were compared in the scutellum of two maize (Zea mays) lines, Illinois High Oil (IHO, approximately 20% lipid content) and Illinois Low Oil (ILO, less than 0.5% lipid content). The microbodies participate in the catabolism of the seed lipids and are responsible for leading the catabolic products (acetyl-Coenzyme A) into gluconeogenesis. The aim of this study was to determine whether changes in lipid content of the seed resulted in changes in the levels of the glyoxysomal enzymes. Enzyme activity measurements, immunological measurements (in the case of catalase), cell fractionation studies, and electron microscopic observations indicated that the IHO and ILO lines contain similar populations of glyoxysomes and exhibit similar catalase and malate synthase specific activities, despite the significant difference (40-fold) in their lipid content. Only the specific activity of isocitrate lyase was higher (2-fold higher) in the IHO seeds as compared to the ILO.

The activities of isocitrate lyase and malate synthase—the key enzymes in the glyoxylate cycle—were found to be fairly high in n-alkane-, acetate-, and propionate-grown cells of Candida tropicalis compared with those in glucose-grown cells. In fact, the results of immunochemical studies showed that the increases in the enzyme levels resulted from increases in the amounts of the enzyme proteins. But the increases in these enzyme activities were not always coincident with the appearance of peroxisomes. Isocitrate lyase and malate synthase were purified from a peroxisome-containing particulate fraction of alkane-grown cells and from whole cells grown on glucose, acetate and propionate. The respective enzymes showed no significant differences in immunochemical properties, specific activities, molecular masses of active forms and subunits, on patterns of limited proteolysis with proteases, but the malate synthases of alkane- and propionate- grown cells showed higher Km values for acetyl-CoA than the enzymes of...

A cDNA clone for the glyoxysomal enzyme malate synthase was isolated from a cDNA library made with polyadenylated RNA from the cotyledons of germinating Cucumis sativus L. This cloned DNA sequence was used as a probe to characterize changes in the amounts of malate synthase gene transcripts in cotyledons of cucumber seeds grown both in the light and in the dark. Malate synthase gene transcripts increase in amount to a peak at day 3 or day 4, and thereafter decline. In the light, this rate of decline is significantly greater than in the dark. Measurement of the changes in the amounts of malate synthase by assaying enzyme activity directly, and by immunological reaction with a specific antiserum indicate that the developmentally regulated synthesis of malate synthase in germinating cucumber is brought about primarily by changes in the amount of malate synthase gene transcripts, rather than through a control of translation. Similarly, the effect of light on the amount of malate synthase correlates precisely with its effect on the abundance of malate synthase gene transcripts.

Changes in levels of isocitrate lyase, malate synthase, and catalase have been investigated during germination of flax (Linum usitatissimum L.) in the presence and absence of itaconate. Germination was accompanied by a rapid increase in these enzymes during the first 3 days. The presence of 38 millimolar itaconate inhibited the incidence of seed germination and the growth of embryo axes as well as the appearance of isocitrate lyase but did not alter the levels of malate synthase, catalase, or NADP(+)-isocitrate dehydrogenase. The specific activity for the latter enzyme was constant throughout germination. Oxalate or succinate, each at 38 millimolar, had no effect upon germination of flax seeds. Itaconate did not inhibit the activities of malate synthase, catalase, or NADP(+)-isocitrate dehydrogenase in vitro but was a potent noncompetitive inhibitor of isocitrate lyase (K(i):17 micromolar at 30 C, pH 7.6). Itaconate (at 38 millimolar) did not alter the appearance of malate synthase but reduced the incidence of germination, onset of germination, and growth of the embryo axis as well as the specific activity of isocitrate lyase in seedlings of Zea mays, Vigna glabra, Glycine hispida, Vigna sinensis, Trigonella foenumgraecum, Lens culinaris, and Medicago sativa. The incidence and onset of germination of wheat seeds were unaltered by the same concentration of itaconate but seedlings did not contain isocitrate lyase or malate synthase. The data suggest that itaconate may be isocitrate lyase-directed in inhibiting the germination of fatty seeds.

Sucrose density gradient centrifugation was employed to separate microsomes, mitochondria, and glyoxysomes from homogenates prepared from castor bean (Ricinus communis) endosperm. In the case of tissue removed from young seedlings, a significant proportion of the characteristic glyoxysomal enzyme malate synthase was recovered in the microsomal fraction. Malate synthase was purified from both isolated microsomes and glyoxysomes by a procedure involving osmotic shock, KCI solubilization, and sucrose density gradient centrifugation. All physical and catalytic properties examined were identical for the enzyme isolated from both organelle fractions. These properties include a molecular weight of 575,000, with a single subunit type of molecular weight 64,000, a pH optimum of 8, apparent K(m) for acetyl-CoA of 10 mum and glyoxylate of 2 mm. Microsomal and glyoxysomal malate synthases showed identical responses to various inhibitors. Adenine nucleotides were competitive inhibitors with respect to acetyl-CoA, and oxalate (K(i) 110 mum) and glycolate (K(i) 150 mum) were competitive inhibitors with respect to glyoxylate. Antiserum raised in rabbits against purified glyoxysomal malate synthase was used to confirm serological identity between the microsomal and glyoxysomal enzymes, and was capable of specifically precipitating (35)S-labeled malate synthase from KCI extracts of both microsomes and glyoxysomes isolated from [(35)S]methionine-labeled endosperm tissue.

The high level of lipids in seeds of some species of conifers suggested that the glyoxylate cycle might have a role in conifer seed metabolism.Six species (Pinus pinea, Pinus pinaster, Pinus canariensis, Pinus strobus, Abies alba, and Cupressus sempervirens) were investigated for their lipid content and malate synthase and isocitrate lyase level. The fatty acid composition of the triglyceride fraction was also investigated. The correlation between lipid content of germinating seed with the presence of the cycle was confirmed. The enzymes of the glyoxylate cycle were not detected in Cupressus sempervirens where the lipid content is very low.