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H Kindl

Publications and source records attributed to H Kindl.

At least 37 records · Page 2Linked to original sources
PubMed1994

Structural organization and differential expression of three stilbene synthase genes located on a 13 kb grapevine DNA fragment.

A 13 kb DNA fragment was isolated from a grapevine (Vitis var. Optima) genomic library by hybridizing with elicitor-induced stilbene synthase cDNA as a probe. After fragmentation with Eco RI, subcloning and sequencing, two full-size stilbene synthase genes (Vst1 and Vst2) and the 3' end of a third stilbene synthase gene (Vst3) were located within the 13 kb fragment. Vst1 and Vst2, differing only slightly in the coding region, are distinguished in the intron size and in the structure of the promoter region. The 5' flanking region of gene Vst1 contains a TATAA box at nucleotide -48. The substantial structural differences found for the promoters of the two genes are paralleled by a striking difference in the expression of the two genes in elicitor-treated cells. Moreover, the accumulation upon elicitation of six different stilbene synthase mRNAs was studied and found to differ by two orders of magnitude.

Acyltransferases↗
PubMed1993

Plant dnaj homologue: molecular cloning, bacterial expression, and expression analysis in tissues of cucumber seedlings.

Proteins homologous to the bacterial dnaJ protein have been implicated as molecular chaperones in different compartments of eukaryotes. A plant equivalent is now described in various cucumber tissues. Using a cDNA library constructed from poly(A)-rich RNA of cotyledons of etiolated seedlings and dnaJ-specific probes, we isolated clones encoding full-length cDNAs: clone CSDNAJ-1 contained a 1.7-kb insert with a single open reading frame of 413 amino acid residues encoding a protein of M(r) 46, 012; pCSDNAJ-2 differed from pCSDNAJ-1 by an additional nucleotide and predicted a protein of M(r) 26, 104. Peptides of similar sizes were observed when the cDNA information was expressed by in vitro transcription and translation or expressed in vivo in bacteria. Using mRNA isolated by hybrid selection for in vitro translation we observed the formation of CSDNAJ-1 protein. Comparisons revealed that the similarity in primary structure between CSDNAJ-1 protein and dnaJ homologues in eukaryotes is most pronounced in the N-terminal region. The high degree of identity (39%) of CSDNAJ-1 protein with the yeast dnaJ homologue YDJ1 protein suggests that csdnaJ proteins assist intracellular protein transfer. The predicted CSDNAJ-1 protein is characterized by a fourfold repetition of the motif CXXCXGX(G) and by a C-terminus -QCAQQ. Analysis of gene expression at the level of mRNA showed that the putative dnaJ protein is expressed in cucumber seedlings in all tissues, but exceedingly high in hypocotyledons and roots. The level of dnaJ mRNA was transiently increased by a factor of 1.5-2.0 when heat shock was applied to the seedlings.

Amino Acid Sequence↗
PubMed1993

Disease resistance results from foreign phytoalexin expression in a novel plant.

Although phytoalexins have long been inferred to be important in the defence of plants against fungal infection, there are few reports showing that they provide resistance to infection. Several plants, including grapevine, synthesize the stilbene-type phytoalexin resveratrol when attacked by pathogens. Stilbenes with fungicidal potential are formed in several unrelated plant species, such as peanut (Arachis hypogaea), grapevine (Vitis vinifera) and pine (Pinus sylvestris). Stilbene biosynthesis only specifically requires the presence of stilbene synthase. Furthermore, the precursor molecules for the formation of hydroxy-stilbenes are malonyl-CoA and p-coumaroyl-CoA, both present in plants. To investigate the potential of stilbene biosynthetic genes in a strategy of engineering pathogen resistance, we isolated stilbene synthase genes from grapevine, where they are expressed at a high level, and transferred them into tobacco. We report here that regenerated tobacco plants containing these genes are more resistant to infection by Botrytis cinerea. This is, to our knowledge, the first report of increased disease resistance in transgenic plants based on an additional foreign phytoalexin.

Acyltransferases↗
PubMed1993

Thiolase mRNA translated in vitro yields a peptide with a putative N-terminal presequence.

Thiolase is part of the fatty acid oxidation machinery which in plants is located within glyoxysomes or peroxisomes. In cucumber cotyledons, proteolytic modification of thiolase takes place during the transfer of the cytosolic precursor into glyoxysomes prior to the intraorganellar assembly of the mature enzyme. This was shown by size comparison of the in vitro synthesized precursor and the 45 kDa subunit of the homodimeric glyoxysomal form. We isolated a full-length cDNA clone encoding the 48,539 Da precursor of thiolase. This plant protein displayed 40% and 47% identity with the precursor of fungal peroxisomal thiolase and human peroxisomal thiolase, respectively. Compared to bacterial thiolases, the precursor of the plant enzyme was distinguished by an N-terminal extension of 34 amino acid residues. This putative targeting sequence of cucumber thiolase shows similarities with the cleavable presequences of rat peroxisomal thiolase and plant peroxisomal malate dehydrogenase.

Acetyl-CoA C-Acetyltransferase↗
PubMed1993

Fatty acid degradation in plant peroxisomes: function and biosynthesis of the enzymes involved.

In plants, the fatty acid oxidation enzyme apparatus is exclusively located within glyoxysomes or peroxisomes. Following the formation of the CoA-ester, the machinery for the degradation of endogenous fatty acids consists of acyl-CoA oxidase, D-3-hydroxyacyl-CoA hydrolyase, 2,3-enoyl-CoA isomerase, isoenzymes of the multifunctional protein and thiolase. The multiple location of particular enzyme activities on different species of protein is discussed in detail. In cucumber cotyledons, the multifunctional protein exhibits a C-terminal targeting signal, -PRM like other glyoxysomal or leaf peroxisomal proteins. In contrast, proteolytic modification takes place at the N-terminus of thiolase and malate dehydrogenase. Thus, distinct mechanisms are envisaged to take place during the transfer of the cytosolic precursor into glyoxysomes prior to the intra-organellar assembly of the mature enzyme.

Acyl Coenzyme A↗
PubMed1992

Sequence analysis of cucumber cotyledon ribulosebisphosphate carboxylase/oxygenase activase cDNA.

The cDNA encoding ribulosebisphosphate carboxylase/oxygenase activase from cotyledons of dark-grown cucumber seedlings has been isolated and sequenced. The 2.0 kilobase pair cDNA encodes a polypeptide with 413 amino acid residues which shows 70% identity with the rubisco activase from green leaves of spinach. The full-length cDNA was used to prepare rubisco activase by in vitro transcription. Subsequent in vitro translation led to the formation of a 46 kDa protein which is the putative precursor of a 42 kDa plastid enzyme.

Amino Acid Sequence↗
PubMed1992

Pine stilbene synthase cDNA, a tool for probing environmental stress.

Stilbene synthase cDNAs were isolated from a pine (Pinus sylvestris) cDNA library. Poly(A)+RNA required for the preparation was obtained from young seedlings challenged with Botrytis cinerea. A full-length cDNA encoding pinosylvin-forming stilbene synthase was sequenced, and the deduced amino acid sequence was compared with sequences of resveratrol-forming stilbene synthases. The cDNA coding for the key enzyme of pinosylvin formation is a valuable tool for detecting early effects of environmental stress in pine needles.

Acyltransferases↗
PubMed1992

A lipoxygenase is the main lipid body protein in cucumber and soybean cotyledons during the stage of triglyceride mobilization.

The 90-kDa lipid body protein characterized earlier by its high expression during the stage of fat degradation was identified as a form of lipoxygenase. This organelle form was compared with lipoxygenase species purified from the cytosol. It is further shown that the antibodies raised against the lipid body membrane lipoxygenase from cucumber cotyledons cross-react with both cytosolic and lipid body lipoxygenase from soybean.

Amino Acid Sequence↗
PubMed1991

Evidence for a peroxisomal fatty acid beta-oxidation involving D-3-hydroxyacyl-CoAs. Characterization of two forms of hydro-lyase that convert D-(-)-3-hydroxyacyl-CoA into 2-trans-enoyl-CoA.

A novel D-(-)-3-hydroxyacyl-CoA hydro-lyase, forming 2-trans-enoyl-CoA and formerly designated as epimerase (EC 5.1.2.3), was extracted from fat-degrading cotyledons of cucumber seedlings. The enzyme, called D-3-hydroxyacyl-CoA hydro-lyase or D-specific 2-trans-enoyl-CoA hydratase, is shown to be required for the degradation of unsaturated fatty acids that contain double bonds extending from even-numbered C atoms. The D-3-hydroxyacyl-CoA hydro-lyase was exclusively localized within peroxisomes. A 10,000-fold purification by chromatography on a hydrophobic matrix, a cation exchanger, on hydroxyapatite and Mono S led to two proteins of apparent homogeneity, both exhibiting Mr of 65,000. The D-3-hydroxyacyl-CoA hydro-lyases are homodimers with slightly differing isoelectric points around pH = 9.0. They catalyze the conversion of 2-trans-enoyl-CoA into D-3-hydroxyacyl-CoA. The reverse reaction was observed but no reaction with 2-cis-enoyl-CoAs or L-3-hydroxyacyl-CoAs. 2-trans-Decenoyl-CoA was converted 10-times faster than 2-trans-butenoyl-CoA. The conversion of 4-cis-decenoyl-CoA into octenoyl-CoA was demonstrated in vitro with purified proteins with an assay mixture containing acyl-CoA oxidase, multifunctional protein, thiolase and the D-3-hydroxyacyl-CoA hydro-lyase. Comparisons of enzyme activities present in the cotyledons or isolated peroxisomes clearly show that the pathway via dienoyl-CoA reductase is much less effective than the sequence involving D-3-hydroxyacyl-CoA hydro-lyase.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1991

Coordinate- and elicitor-dependent expression of stilbene synthase and phenylalanine ammonia-lyase genes in Vitis cv. Optima.

The mechanisms controlling the induction of stilbene synthase and phenylalanine ammonia-lyase (PAL), two putative key regulatory enzymes of the biosynthetic pathway to stilbene phytoalexins, have been investigated. The induction was studied in cell suspension cultures of grape (Vitis cv. Optima) by treatment with fungal cell wall. Several independent cDNA clones for PAL and stilbene synthase were isolated from a cDNA library of fungal cell wall-induced grape cells and identified by sequence analysis. The stilbene synthase cDNA sequence of pSV21 predicted a protein of 392 amino acids and Mr 42,791, similar in size to that observed experimentally for immunodetected stilbene synthase. The cDNA sequences of pSV21 and pSV25 differed in 76 bp in the coding region. The sequences of grape stilbene synthase cDNAs exhibited significant homology to the sequence reported for the peanut stilbene synthase cDNA. Both PAL and stilbene synthase mRNA, measured by RNA blot hybridizations, were induced within 1 h of addition of fungal cell wall preparations to the cell cultures, rose to a maximum by the sixth hour, then declined slowly over the next 20 h. The activities of PAL and stilbene synthase were also induced in parallel, but reached their maximum at different times after fungal cell wall addition to the cell cultures. The induction patterns of stilbene synthase and PAL in grape and peanut are discussed.

Acyltransferases↗
PubMed1991

Purification and characterization of a plant peroxisomal delta 2, delta 3-enoyl-CoA isomerase acting on 3-cis-enoyl-CoA and 3-trans-enoyl-CoA.

A delta 3, delta 2-enoyl-CoA isomerase was extracted from fat-degrading cotyledons of cucumber seedlings. The enzyme is required for the degradation of cis-unsaturated fatty acids, e.g. linoleic acid being present in the storage tissue of cucumber seedlings in high amounts. The delta 3, delta 2-enoyl-CoA isomerase was exclusively localized within peroxisomes. Its purification included chromatography on a hydrophobic matrix, a cation exchanger, and on hydroxylapatite. 17,000-fold purification yielded a protein of apparent homogeneity. The isomerase is a homodimer with a Mr of 50,000 and an isoelectric point of pH approximately 8.1. Delta 3, delta 2-Enoyl-CoA isomerase reversibly catalyzes the conversion of both cis-3-enoyl-CoA and trans-3-enoyl-CoA into trans-2-enoyl-CoA. The isomerase exhibited optimal activity at pH 9 and was active with 3-enoyl-CoA species having chain lengths of C6-C12, with cis-hexanoyl-CoA being the most effective substrate. The purified enzyme did not show enoyl-CoA hydratase activity.

Carbon-Carbon Double Bond Isomerases↗
PubMed1990

Grapevine stilbene synthase cDNA only slightly differing from chalcone synthase cDNA is expressed in Escherichia coli into a catalytically active enzyme.

Stilbene synthase is responsible for the formation of resveratrol and other stilbenes which function in grapevine as phytoalexins. A full-length stilbene synthase cDNA was prepared from grapevine mRNA and sequenced. The insert in pSV25 coding for a polypeptide with 392 amino acids was inserted into the vectors pKK233-2 and pDS12/RBSII-2, respectively. Expression of the cDNA in Escherichia coli yielded an enzymatically active dimer exhibiting solely stilbene synthase activity. The protein was characterized by enzyme activity and Western blot analysis.

Acyltransferases↗
PubMed1990

Expression of a stilbene synthase gene in Nicotiana tabacum results in synthesis of the phytoalexin resveratrol.

A gene from groundnut (Arachis hypogaea) coding for stilbene synthase was transferred together with a chimaeric kanamycin resistance gene. It was found to be rapidly expressed after induction with UV light and elicitor in tobacco cells (Nicotiana tabacum). Comparative studies of stilbene synthase mRNA synthesis in groundnut and transgenic tobacco suspension cultures revealed the same kinetics of gene expression. Stilbene synthase specific mRNA was detectable 30 minutes after elicitor induction and 10 minutes after UV irradiation. The maximum of mRNA accumulation was between 2 and 8 hours post induction. 24 hours after induction stilbene synthase mRNA accumulation ceased. Furthermore, in transgenic tobacco plants, the gene was found to be inducible in sterile roots, stems and leaves. Stilbene synthase was demonstrated in crude protein extracts from transgenic tobacco cell cultures using specific antibodies. Resveratrol, the product of stilbene synthase, was identified by HPLC and antisera raised against resveratrol.

Acyltransferases↗
PubMed1990

Characterization of a cDNA Encoding Lens culinaris Glycolate Oxidase and Developmental Expression of Glycolate Oxidase mRNA in Cotyledons and Leaves.

mRNA obtained from green leaves of lentil (Lens culinaris) was used to construct a cDNA library in phage lambdagt11. The cDNA library was screened with antibodies raised against lentil glycolate oxidase and catalase. Clone CL 1 containing the full-length sequence complementary to glycolate oxidase mRNA was characterized and sequenced. In addition, a 800-base pair catalase cDNA clone was sequenced. To prove the correlation of cDNA insert in CL 1 with glycolate oxidase, the cDNA was transcribed in vitro. The mRNA was translated in vitro yielding a 43 kilodalton protein immunoprecipitable with anti-glycolate oxidase serum. Nucleotide sequences of lentil cDNA and spinach cDNA were 86% identical. Lentil glycolate oxidase was characterized by a C-terminal sequence -P-R-A-L-P-R-L. The expression of glycolate oxidase mRNA in cotyledons, leaves and roots was compared with that of catalase. In leaves, the relative amount of glycolate oxidase mRNA increased during the first 2 days of greening, but decreased later, and was hardly detectable during senescence. In cotyledons of germinating seeds, the level of glycolate oxidase mRNA was markedly lower than the catalase mRNA.

Journal Article↗
PubMed1990

Transition form of microbodies. Overlapping of two sets of marker proteins during the rearrangement of glyoxysomes into leaf peroxisomes.

Several forms of microbodies have been characterized on the basis of their biochemical functions. We have investigated cucumber cotyledons which house two different microbody forms during their development. In these cells, a shift from organelles with the enzymes of beta-oxidation and glyoxylate cycle to peroxisomes with the enzymes of the photosynthetic C2-cycle can be induced by light. The transition state and the time course of changes was studied at different levels of gene expression during the first 2 days of illumination, by quantifying the rate of de novo protein synthesis in cotyledons and by measuring the mRNA activities in vitro. Synthesis and turnover of particular proteins were determined during the transition stage by immunoprecipitation of malate synthase, isocitrate lyase, catalase, multifunctional protein, and thiolase, and quantified by fluorography. From the mRNA activities and the rate of protein synthesis, gene expression for enzymes of the glyoxylate cycle and beta-oxidation started to decrease 24-36 h after onset of continuous light. At that time the rate of synthesis of glycolate oxidase, a leaf peroxisomal marker, is already maximal. By pulse-chase experiments 0-48 h after the onset of light the speed and intensity of protein turnover were measured. Rates of proteolytic degradation of individual enzymes indicated that the different enzymes were not lost simultaneously or all at once. This excludes a destruction of the whole organelle by the lytic compartment.

Gene Expression↗
PubMed1988

Characterization of two forms of the multifunctional protein acting in fatty acid beta-oxidation.

The enzymatic apparatus of fatty acid beta-oxidation in peroxisomes and glyoxysomes includes a multifunctional protein. Two forms of this protein were detected in extracts from cotyledons of germinating cucumber seeds and separated on hydroxylapatite. The two proteins purified to apparent homogeneity possessed enoyl-CoA hydratase, 3-hydroxyacyl-CoA epimerase, and 3-hydroxyacyl-CoA dehydrogenase activity; the proteins are therefore trifunctional. Analysis of molecular structures and kinetic parameters of the two enzyme forms revealed significant differences in size and amino acid composition. The two proteins were characterized as monomers exhibiting molecular weights of 74,000 and 76,500. Likewise, the data obtained with limited proteolysis proved the occurrence of two independent proteins. Immunological comparisons were performed with antibodies raised against the 76.5-kDa protein. They indicated a weak relationship between the two proteins. From that we conclude that within one type of organelle, i.e., glyoxysome, two isoenzymes with multiple functions are located.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1988

The glyoxysomal beta-oxidation system in cucumber seedlings: identification of enzymes required for the degradation of unsaturated fatty acids.

Fat-degrading cotyledons from cucumber seedlings were investigated with respect to the enzymes metabolizing cis-unsaturated fatty acids. Isolated glyoxysomes degrade linoleic acid, the dominating fatty acid in the storage tissue of the seed. Glyoxysomes were shown to be the sole intracellular site of enzymes responsible for the degradation of unsaturated fatty acids. All three auxiliary enzyme activities discussed for the degradation of polyunsaturated fatty acids, 2,4-dienoyl-CoA reductase, enoyl-CoA isomerase, and 3-hydroxyacyl-CoA epimerase were localized within the matrix of glyoxysomes. They were not found in mitochondria. Separation of glyoxysomal matrix proteins on CM-cellulose revealed that epimerase activity was attributable to the multifunctional protein and also to another protein which apparently exhibited no other beta-oxidation activity. Furthermore, on the basis of the high epimerase activity present in glyoxysomes compared to a much lower 2,4-dienoyl-CoA reductase activity, the metabolism of unsaturated fatty acids via delta 2-cis-enoyl-CoA is considered as alternative to the reductase-dependent pathway.

Carbon-Carbon Double Bond Isomerases↗