Genome-wide distribution and potential regulatory functions of AtATE, a novel family of miniature inverted-repeat transposable elements in Arabidopsis thaliana.

A study of transgenic promoter::beta-glucuronidase lines showed that the promoters of the two Arabidopsis ARGININE DECARBOXYLASE paralogues, ADC1 and ADC2, exhibited extremely different patterns of activity. One major feature of the promoter of ADC1 was the presence of a novel transposable element, which was shown to possess all of the characteristics of Miniature Inverted-repeat Transposable Elements (MITEs), and to be present in 26 full-length copies and 1617 partial copies and fragments distributed throughout the Arabidopsis genome. TRANSFAC analysis showed that this transposable element possesses a significant number of transcription-factor binding motifs. A bioinformatics approach based on a suffix-tree compilation was used to obtain an exhaustive description of exact copy numbers and positions of the element in the Arabidopsis genome. The distribution among the chromosomes was non-random, and a significant number of copies were found in regions flanking genes. Full-length copies of the transposable element were detected in the immediate vicinity of 22 genes, either upstream or downstream.

The mitochondrial isovaleryl-coenzyme a dehydrogenase of arabidopsis oxidizes intermediates of leucine and valine catabolism.

We recently identified a cDNA encoding a putative isovaleryl-coenzyme A (CoA) dehydrogenase in Arabidopsis (AtIVD). In animals, this homotetrameric enzyme is located in mitochondria and catalyzes the conversion of isovaleryl-CoA to 3-methylcrotonyl-CoA as an intermediate step in the leucine (Leu) catabolic pathway. Expression of AtIVD:smGFP4 fusion proteins in tobacco (Nicotiana tabacum) protoplasts and biochemical studies now demonstrate the in vivo import of the plant isovaleryl-CoA dehydrogenase (IVD) into mitochondria and the enzyme in the matrix of these organelles. Two-dimensional separation of mitochondrial proteins by blue native and SDS-PAGE and size determination of the native and overexpressed proteins suggest homodimers to be the dominant form of the plant IVD. Northern-blot hybridization and studies in transgenic Arabidopsis plants expressing Ativd promoter:gus constructs reveal strong expression of this gene in seedlings and young plants grown in the absence of sucrose, whereas promoter activity in almost all tissues is strongly inhibited by exogeneously added sucrose. Substrate specificity tests with AtIVD expressed in Escherichia coli indicate a strong preference toward isovaleryl-CoA but surprisingly also show considerable activity with isobutyryl-CoA. This strongly indicates a commitment of the enzyme in Leu catabolism, but the activity observed with isobutyryl-CoA also suggests a parallel involvement of the enzyme in the dehydrogenation of intermediates of the valine degradation pathway. Such a dual activity has not been observed with the animal IVD and may suggest a novel connection of the Leu and valine catabolism in plants.

Purification, characterization and cloning of isovaleryl-CoA dehydrogenase from higher plant mitochondria.

Between the different types of Acyl-CoA dehydrogenases (ACADs), those specific for branched chain acyl-CoA derivatives are involved in the catabolism of amino acids. In mammals, isovaleryl-CoA dehydrogenase (IVD), an enzyme of the leucine catabolic pathway, is a mitochondrial protein, as other acyl-CoA dehydrogenases involved in fatty acid beta-oxidation. In plants, fatty acid beta-oxidation takes place mainly in peroxisomes, and the cellular location of the enzymes involved in the catabolism of branched-chain amino acids had not been definitely assigned. Here, we describe that highly purified potato mitochondria have important IVD activity. The enzyme was partially purified and cDNAs from two different genes were obtained. The partially purified enzyme has enzymatic constant values with respect to isovaleryl-CoA comparable to those of the mammalian enzyme. It is not active towards straight-chain acyl-CoA substrates tested, but significant activity was also found with isobutyryl-CoA, implying an additional role of the enzyme in the catabolism of valine. The present study confirms recent reports that in plants IVD activity resides in mitochondria and opens the way to a more detailed study of amino-acid catabolism in plant development.

Mechanisms of primordium formation during adventitious root development from walnut cotyledon explants.

In walnut (Juglans regia L.), an otherwise difficult-to-root species, explants of cotyledons have been shown to generate complete roots in the absence of exogenous growth regulators. In the present study, this process of root formation was shown to follow a pattern of adventitious, rather than primary or lateral, ontogeny: (i) the arrangement of vascular bundles in the region of root formation was of the petiole type; (ii) a typical root primordium was formed at the side of the procambium within a meristematic ring of actively dividing cells located around each vascular bundle; (iii) the developing root apical meristem was connected in a lateral way with the vascular bundle of the petiole. This adventitious root formation occurred in three main stages of cell division, primordium formation and organization of apical meristem. These stages were characterized by expression of LATERAL ROOT PRIMORDIUM-1 and CHALCONE SYNTHASE genes, which were found to be sequentially expressed during the formation of the primordium. Activation of genes related to root cell differentiation started at the early stage of primordium formation prior to organization of the root apical meristem. The systematic development of adventitious root primordia at a precise site gave indications on the positional and biochemical cues that are necessary for adventitious root formation.

Identification, separation, and characterization of acyl-coenzyme A dehydrogenases involved in mitochondrial beta-oxidation in higher plants

The existence in higher plants of an additional beta-oxidation system in mitochondria, besides the well-characterized peroxisomal system, is often considered controversial. Unequivocal demonstration of beta-oxidation activity in mitochondria should rely on identification of the enzymes specific to mitochondrial beta-oxidation. Acyl-coenzyme A dehydrogenase (ACAD) (EC 1.3.99.2,3) activity was detected in purified mitochondria from maize (Zea mays L.) root tips and from embryonic axes of early-germinating sunflower (Helianthus annuus L.) seeds, using as the enzyme assay the reduction of 2,6-dichlorophenolindophenol, with phenazine methosulfate as the intermediate electron carrier. Subcellular fractionation showed that this ACAD activity was associated with mitochondrial fractions. Comparison of ACAD activity in mitochondria and acyl-coenzyme A oxidase activity in peroxisomes showed differences of substrate specificities. Embryonic axes of sunflower seeds were used as starting material for the purification of ACADs. Two distinct ACADs, with medium-chain and long-chain substrate specificities, respectively, were separated by their chromatographic behavior, which was similar to that of mammalian ACADs. The characterization of these ACADs is discussed in relation to the identification of expressed sequenced tags corresponding to ACADs in cDNA sequence analysis projects and with the potential roles of mitochondrial beta-oxidation in higher plants.

Higher-plant medium- and short-chain acyl-CoA oxidases: identification, purification and characterization of two novel enzymes of eukaryotic peroxisomal beta-oxidation.

Medium- and short-chain acyl-CoA oxidases were identified in and subsequently purified from dark-grown maize plantlets. The oxidase showing preference for medium-chain fatty acyl-CoAs (C10-C14) was purified to homogeneity. The oxidase showing preference for short-chain fatty acyl-CoAs (C4-C8) was purified over 150-fold. Various catalytic properties confirmed these enzymes to be true acyl-CoA oxidases. They produced trans-2-enoyl-CoA and H2O2 from the saturated acyl-CoA, as verified by various independent assay techniques. They also exhibited FAD-dependent activity; i.e. removal of loosely bound FAD by gel filtration markedly reduced activity, which could be restored upon re-addition of FAD. They showed apparent Km values between 2 and 10 microM for the acyl-CoA substrate giving maximal activity, no activity with the corresponding free fatty acid, high pH optima (8.3-8.6) and a peroxisomal subcellular location. The medium-chain acyl-CoA oxidase was determined to be a monomeric protein with a molecular mass of 62 kDa. The short-chain acyl-CoA oxidase was shown to have a native molecular mass of 60 kDa, but exhibited a labile multimeric structure, as indicated by the elution of multiple peaks of activity during several chromatographic steps, and ultimately by the purification of a subunit of molecular mass 15 kDa. The medium- and short-chain acyl-CoA oxidases were demonstrated to be distinct from the maize equivalent of the cucumber glyoxysomal long-chain acyl-CoA oxidase previously purified and characterized [Kirsch, Loffler and Kindl (1986) J. Biol. Chem. 261, 8570-8575]. The maize long-chain acyl-CoA oxidase was partially purified to permit determination of its substrate specificity; it showed activity with a broad range of acyl-CoAs of chain length greater than C8, and maximal activity with C16. The implications of the existence of multiple acyl-CoA oxidases in the regulation of plant peroxisomal beta-oxidation are discussed.

Purification and Characterization of a Novel Aminopeptidase, Plastidial Alanine-Aminopeptidase, from the Cotyledons of Etiolated Sugar Beet Seedlings.

During prolonged dark growth of sugar beet (Beta vulgaris L.) seedlings, etioplasts, rapidly after the proplastid-etioplast transition, undergo a degenerative process characterized by ultrastructural modifications, protein loss, and the decrease of carotenoid and chlorophyll accumulation upon illumination. Two plastidial aminopeptidase activities were identified as early markers of this degenerative process (A. El Amrani, I. Couee, J.-P. Carde, J.-P. Gaudillere, P. Raymond [1994] Plant Physiology 106: 1555-1565). The present study focuses on one of these markers and describes the purification to homogeneity and characterization of plastidial alanine-aminopeptidase. This novel aminopeptidase was found to be a metallo-type naphthylamidase particularly active with alanyl, arginyl, and leucyl substrates. Its plastidial location was confirmed by immunofluorescence with polyclonal antibodies against the purified enzyme. Its physico-chemical and enzymic properties are discussed with respect to other higher plant aminopeptidases and to its potential functions during prolonged dark growth.

Modifications of Etioplasts in Cotyledons during Prolonged Dark Growth of Sugar Beet Seedlings (Identification of Etiolation-Related Plastidial Aminopeptidase Activities).

We studied the effects of prolonged dark growth on proplastids and etioplasts in cotyledons of sugar beet (Beta vulgaris L.) seedlings. Differentiation of proplastids into etioplasts occurred between d 4 and d 6 after imbibition, with the typical characteristics of increased synthesis of plastidial proteins, protein and carotenoid accumulation, size increase, development of plastid membranes and of the prolamellar body, and increase of the greening capacity. However, this situation of efficient greening capacity was short-lived. The greening capacity started to decline from d 6 after imbibition. This decline was due in part to reserve depletion and glucose limitation and also to irreversible damage to plastids. Indeed, electron microscopy observations in situ showed some signs of plastidial damage, such as accumulation of plastoglobuli and membrane alterations. The biochemical characterization of purified plastids also showed a decrease of proteins per plastid. Aminopeptidase activities, and to a lesser extent, neutral endopeptidase activities, were found to increase in plastids during this degenerative process. We identified two plastidial aminopeptidases showing a sharp increase of activity at the onset of the degenerative process. One of them, an alanyl aminopeptidase, was shown to be inactivated by exposure to light or addition of exogenous glucose, thus confirming the relationship with prolonged dark growth and indicating a relationship with glucose limitation.

Effects of glucose starvation on the oxidation of fatty acids by maize root tip mitochondria and peroxisomes: evidence for mitochondrial fatty acid beta-oxidation and acyl-CoA dehydrogenase activity in a higher plant.

Fatty acid beta-oxidation was studied in organellar fractions from maize root tips by h.p.l.c. and radiometric analysis of the products of incubations with [1-14C]octanoate and [1-14C]palmitate. In crude organellar fractions containing both mitochondria and peroxisomes, octanoate and palmitate beta-oxidation, as determined by the production of acetyl-CoA, was functional and, for palmitate, was activated 4-12-fold after subjecting the root tips to 48 h of glucose starvation. The sensitivity to a 'cocktail' of respiratory-chain inhibitors containing cyanide, azide and salicylhydroxamate depended on the conditions of incubation, with no inhibition in a medium facilitating peroxisomal beta-oxidation and a significant inhibition in a medium potentially facilitating mitochondrial beta-oxidation. Indeed, preparations of highly purified mitochondria from glucose-starved root tips were able to oxidize octanoate and palmitate to give organic acids of the tricarboxylic acid cycle. This activity was inhibited 5-10-fold by the above cocktail of respiratory-chain inhibitors, with no parallel accumulation of acetyl-CoA, thus showing that the inhibition affected beta-oxidation rather than the pathway from acetyl-CoA to the organic acids. This provides the first evidence that the complete beta-oxidation pathway from fatty acids to citrate was functional in mitochondria from a higher plant. Moreover, an acyl-CoA dehydrogenase activity was shown to be present in the purified mitochondria. In contrast with the peroxisomal activity, mitochondrial beta-oxidation showed the same efficiency with octanoate and palmitate and was strictly dependent on glucose starvation.

Effects of glucose starvation on mitochondrial subpopulations in the meristematic and submeristematic regions of maize root.

Mitochondria isolated from 3-mm long maize (Zea mays L. var Dea) root tips were found to be heterogeneous on Percoll density gradients. The ultrastructure of these isolated mitochondria correlated well with that of mitochondria observed in situ and was consistent with the existence of mitochondria at different stages of maturation during cell development. The mitochondria of higher density presented an ultrastructure with many cristae and a dense matrix. These mitochondria showed classic respiratory properties, although with low ADP/O ratios. In contrast, the mitochondria of lower density showed few cristae and a clear matrix and did not seem to be fully functional because their rate of respiration was low and showed weak respiratory control. Lower- and higher- density mitochondria were shown to be differentially affected during the first stages of glucose starvation. The higher-density mitochondria from glucose-starved maize root tips retained the ultrastructure and most of the respiratory properties of nonstarved mitochondria, whereas lower- and intermediate-density mitochondria were absent in the mitochondrial preparations from glucose-starved maize root tips and were not observed in situ. Quantitatively, there was a decrease of the total mitochondrial pool when expressed as the amount of mitochondrial protein per root tip. However, this decrease affected low- and intermediate-density mitochondria, but not higher-density mitochondria. Thus, it was shown that a significant pool of functional mitochondria is maintained in maize root tips during the first stages of glucose starvation. The reasons for these apparently selective effects of glucose starvation on mitochondria are discussed in relation to effects on mitotic and differentiation processes.

Effects of anoxia on mitochondrial biogenesis in rice shoots: modification of in organello translation characteristics.

Shoots of germinating rice (Oryza sativa L.) seedlings are able to grow under anoxia and to withstand long periods of anoxic treatment. Mitochondria were purified from aerobically germinated and anaerobically treated rice shoots by differential and isopycnic centrifugation and were found to consist of two subpopulations. The mitochondrial subpopulation of higher density was used for further characterization. Ultrastructural studies showed anaerobic mitochondria to be significantly different from aerobic mitochondria, with a matrix of lower density and more developed cristae. Aerobic and anaerobic mitochondria also differed in their specific activities for fumarase and succinate dehydrogenase, which were significantly lower after the anoxic treatment. In vivo labeling of seedlings with l-[(35)S]methionine and subsequent isolation of the mitochondria indicated that anoxia induced a drastic decrease, but not a total inactivation, of the synthesis of mitochondrial proteins. In organello protein synthesis showed that anaerobic mitochondria were able to synthesize most of the polypeptides synthesized by aerobic mitochondria, although only in the presence of exogenous ATP, as would occur under anoxia. Anaerobic mitochondria, but not aerobic mitochondria, could carry out protein synthesis without a functional respiratory chain. Thus, mitochondrial protein synthesis was found to be potentially functional in the rice shoot under anoxia.

The sulphydryl groups of ox brain and liver glutamate dehydrogenase preparations and the effects of oxidation on their inhibitor sensitivities.

Glutamate dehydrogenase preparations from several sources have been shown to have suffered limited proteolysis during purification. This proteolysis has been previously shown to involve removal of the N-terminal tetrapeptide and to result in changes in the regulatory properties of the enzyme. In the present work the previously unidentified N-terminal residue of the unproteolysed enzyme from ox brain and liver is shown to be cysteine. The thiol group of this residue is masked in the native enzyme but it becomes accessible after reduction. Exposure of solutions of the unproteolysed enzyme to air oxidation causes large changes in its sensitivity to inhibition by the antipsychotic drug perphenazine, GTP and by high concentrations of NADH. No such changes occurred in the behaviour of preparations of the enzyme that had suffered proteolysis during purification under these conditions.

Inhibition of ox brain glutamate dehydrogenase by perphenazine.

Factors affecting the inhibition of ox brain glutamate dehydrogenase (GDH) by the antipsychotic drug perphenazine have been studied. Inhibition was found to be of mixed type with respect to 2-oxoglutarate and competitive towards NADH. However, the data indicate that perphenazine binds to a site distinct from the catalytic site to which NADH binds. Perphenazine also enhanced the high-substrate inhibition by these two substrates. Inhibition by perphenazine was not affected by the allosteric effector GTP but it was enhanced by increasing pH, in the range of 6.3 to 7.6, and diminished by increasing ionic strength. Low concentrations of perphenazine relieved the inhibition of GDH by phosphatidylserine and cardiolipin. However, at higher concentrations phosphatidylserine did not interfere with the inhibition by perphenazine whereas cardiolipin relieved it. The possible significance of these interactions in terms of the behaviour of this antipsychotic drug in vivo are discussed.

The inhibition of glutamate dehydrogenase by some antipsychotic drugs.

The phenothiazines chlorpromazine and perphenazine and the butyrophenone haloperidol were shown to be reversible inhibitors of glutamate dehydrogenase (GDH). Inhibition by chlorpromazine was found to be partial, whereas haloperidol and perphenazine would, apparently, give full inhibition at saturating concentrations. Double-reciprocal plots of the difference between activities in the absence and presence of the inhibitor against the inhibitor concentration were linear with chlorpromazine and perphenazine but parabolic when haloperidol was used as the inhibitor. Comparisons between the responses of commercially available preparations of ox liver GDH, which have been shown to have suffered limited proteolysis during purification, and the ox brain enzyme prepared by a procedure which does not result in such proteolysis, revealed the latter preparation to be more sensitive to inhibition because of a higher apparent affinity for these drugs. The apparent dissociation constants for enzyme-drug interactions were, however considerably higher than the concentrations that have been reported to occur in vivo following chronic administration of chlorpromazine and haloperidol. This casts doubt on earlier claims that inhibition of GDH may be involved in the antipsychotic actions of these drugs, although it might be a factor in the side effects associated with the use of such compounds.

The effects of phospholipids on the activation of glutamate dehydrogenase by L-leucine.

Glutamate dehydrogenase in disrupted mitochondrial preparations is activated by L-leucine to a much greater extent than is the purified enzyme. A factor, or factors, responsible for modulating the sensitivity of L-leucine is lost during the purification of the enzyme. Although both cardiolipin and phosphatidylserine are inhibitors of the enzyme, only the inhibition by the former phospholipid is reversed by L-leucine. The inhibition of glutamate dehydrogenase by its binding to cardiolipin in the disrupted mitochondrial preparations and its relief by L-leucine could account for the greater sensitivity of such preparations to activation by that amino acid.

Activation of glutamate dehydrogenase by L-leucine.

The activation of glutamate dehydrogenase (L-glutamate: NAD(P)+ oxidoreductase (deaminating), EC 1.4.1.3) by L-leucine has been studied. Apparently homogeneous preparations from ox liver and brain were found to respond similarly. Commercially obtained preparations of the enzyme, which had suffered limited proteolysis during the purification procedure, were shown to behave similarly to preparations which had not suffered such proteolysis when the effects of L-leucine on the oxidative deamination reaction were studied using either NAD+ or NADP+ as the coenzyme. There was also no significant difference in the responses when the reductive reaction was determined with NADPH or with 40 microM NADH. At higher concentrations of NADH (160 microM) the unproteolysed preparations were activated by L-leucine to a considerably greater extent than those which had suffered limited proteolysis. These results accord with the greater sensitivity of the former preparations to inhibition by high concentrations of NADH and the relief of such inhibition by L-leucine. This amino acid was also found to relieve the inhibition of the enzyme by GTP, resulting in an apparent increase in the activation observed in the presence of this nucleotide. In contrast, under the conditions used in this work, the apparent degree of activation by L-leucine was found to be decreased in the presence of the activators ATP or ADP. The presence of high concentrations of NADH (200 microM) potentiated the high substrate inhibition by 2-oxoglutarate, and L-leucine significantly reduced this effect. The effects of L-leucine on the activity of glutamate dehydrogenase thus appear to be composed of a direct effect on the activity of the enzyme together with a relief of high substrate inhibition. The effects of GTP and 2-oxoglutarate in potentiating inhibition by NADH can account for their effects in enhancing the apparent activation by L-leucine. The marked differences in the responses of proteolysed and unproteolysed preparations of the enzyme result from the effects of proteolysis in decreasing the sensitivity to high concentrations of NADH.