Showing papers on "Arabidopsis published in 1989"

Alterations of Endogenous Cytokinins in Transgenic Plants Using a Chimeric Isopentenyl Transferase Gene.

Abstract: Cytokinins, a class of phytohormones, appear to play an important role in the processes of plant development. We genetically engineered the Agrobacterium tumefaciens isopentenyl transferase gene, placing it under control of a heat-inducible promoter (maize hsp70). The chimeric hsp70 isopentenyl transferase gene was transferred to tobacco and Arabidopsis plants. Heat induction of transgenic plants caused the isopentenyl transferase mRNA to accumulate and increased the level of zeatin 52-fold, zeatin riboside 23-fold, and zeatin riboside 5[prime]-monophosphate twofold. At the control temperature zeatin riboside and zeatin riboside 5[prime]-monophosphate in transgenic plants accumulated to levels 3 and 7 times, respectively, over levels in wild-type plants. This uninduced cytokinin increase affected various aspects of development. In tobacco, these effects included release of axillary buds, reduced stem and leaf area, and an underdeveloped root system. In Arabidopsis, reduction of root growth was also found. However, neither tobacco nor Arabidopsis transgenic plants showed any differences relative to wild-type plants in time of flowering. Unexpectedly, heat induction of cytokinins in transgenic plants produced no changes beyond those seen in the uninduced state. The lack of effect from heat-induced increases could be a result of the transient increases in cytokinin levels, direct or indirect induction of negating factor(s), or lack of a corresponding level of competent cellular factors. Overall, the effects of the increased levels of endogenous cytokinins in non-heat-shocked transgenic plants seemed to be confined to aspects of growth rather than differentiation. Since no alterations in the programmed differentiation pattern were found with increased cytokinin levels, this process may be controlled by components other than absolute cytokinin levels.

A dwarf mutant of Arabidopsis generated by T-DNA insertion mutagenesis

Abstract: Most plant genes that control complex traits of tissues, organs, and whole plants are uncharacterized. Plant height, structure of reproductive organs, seed development and germination, for example, are traits of great agronomic importance. However, in the absence of knowledge of the gene products, current molecular approaches to isolate these important genes are limited. Infection of germinatng seeds of Arabidopsis thaliana with Agrobacterium results in transformed lines in which the integrated T-DNA from Agrobacterium and its associated kanamycin-resistance trait cosegregate with stable, phenotypic alterations. A survey of 136 transformed lines produced plants segregating in a manner consistent with Mendelian predictions for phenotypes altered in height, flower structure, trichomes, gametogenesis, embryogenesis, and seedling development. This report is the characterization of a dwarf mutant in which the phenotype is inherited as a single recessive nuclear mutation that cosegregates with both the kanamycin-resistance trait and the T-DNA insert.

Strong cellular preference in the expression of a housekeeping gene of Arabidopsis thaliana encoding S-adenosylmethionine synthetase.

Abstract: S-Adenosylmethionine serves as a methyl group donor in numerous transmethylation reactions and plays a role in the biosynthesis of polyamines and ethylene. We have cloned and sequenced an S-adenosylmethionine synthetase gene (sam-1) of Arabidopsis thaliana. The deduced polypeptide sequence of the enzyme has extensive homology with the corresponding enzymes of Escherichia coli and yeast. Genomic hybridization indicates the presence of two adenosylmethionine synthetase genes per haploid Arabidopsis genome. RNA gel blot analysis shows that adenosylmethionine synthetase mRNA levels are high in stems and roots, correlating well with the higher enzyme activity in stems, compared with leaves. Histochemical analysis of transgenic Arabidopsis plants transformed with a chimeric beta-glucuronidase gene, under the control of 748-base pair 5' sequences of the sam-1 gene, demonstrates that the gene is expressed primarily in vascular tissues. In addition, high expression was observed in sclerenchyma and in the root cortex. A hypothesis for the strong cellular preference in the expression of the sam-1 gene is presented.

Enkephalins produced in transgenic plants using modified 2S seed storage proteins

Abstract: We have explored the possibility of producing large amounts of biologically active peptides as part of chimeric plant seed storage proteins, the 2S albumins. A portion of an Arabidopsis thaliana 2S albumin gene, encoding a region of the protein whose sequence is not highly conserved among different species, was replaced by sequences encoding the neuropeptide Leu-enkephalin, flanked by tryptic cleavage sites. Using Agrobacterium mediated transformation, the modified gene was transformed into Arabidopsis and oilseed rape and the 2S albumins isolated from the seeds of the regenerated transgenic plants. These were digested with trypsin and the enkephalin containing peptide isolated and treated to remove the extra lysine residue. Up to 200 nmol of peptide were recovered per gram of seed.

The gene family encoding the Arabidopsis thaliana translation elongation factor EF-1α: molecular cloning, characterization and expression

Abstract: The gene family encoding the Arabidopsis thaliana translation elongation factor (EF-1 alpha) was analysed. This family contains four genes (A1-A4) organized in a similar manner in different varieties of Arabidopsis. Based upon both their physical separation and a comparison of their sequences, it is suggested that the A4 gene and the A1, A2, and A3 genes constitute two distinct subfamilies within the genome. By introducing chimaeric gene constructs into Arabidopsis cells, we showed that the A1 gene promoter mediates a transient expression about twofold higher than that obtained using the CaMV 35 S promoter. This expression depends on a 348 bp DNA fragment extending from -982 to -634 bp upstream of the initiation codon. This element contains a characteristic telomeric sequence (AACCCTAA) which is also found in the promoters of the A2 and A4 genes as well as in the promoters of the Drosophila EF-1 alpha F1 gene and of several highly expressed plant genes.

Trichome Development in Arabidopsis thaliana. I. T-DNA Tagging of the GLABROUS1 Gene.

Abstract: Progeny from a transformed Arabidopsis plant (produced by the Agrobacterium-mediated seed transformation procedure) were found to be segregating for an altered trichome phenotype. The mutant plants have normal leaf trichomes but completely lack trichomes usually found on the stem. The mutation is tightly linked to a T-DNA insert. Complementation analysis with genetically characterized trichome mutants revealed that the new mutation is an allele of the GL1 locus. The new trichome mutant has been designated gl1-43. DNA gel blot analysis indicated that the insert site contains a complex array of at least four tandemly linked T-DNA units oriented as both direct and inverted repeats. A genomic library, constructed using DNA from gl1-43 plants, was used to clone DNA that flanks the left end of the T-DNA insert. The availability of DNA from the region interrupted by the insert has allowed initial characterization of the wild-type GL1 gene and will permit the eventual cloning and sequencing of this developmentally interesting gene.

Efficient Transformation of Arabidopsis-Thaliana Using Direct Gene-Transfer to Protoplasts

Abstract: Direct gene transfer has proved to be an efficient transformation method for Arabidopsis thaliana, a member of the Brassicaceae. Transgenic Arabidopsis plants resistant to hygromycin B have been regenerated from mesophyll protoplasts treated with polyethylene glycol and plasmid DNA carrying the hygromycin phosphotransferase (HPT) gene under the control of the 35 S promoter of cauliflower mosaic virus. The transformation procedure reproducibly yields transformants at frequencies of approximately 1 x 10(-4) (based on the number of protoplasts treated) or 5% (based on the number of regenerating calli). DNA from plants regenerated from hygromycin resistant colonies was analysed by Southern blot hybridization demonstrating that the foreign gene is stably integrated into the plant chromosome. Genetic analysis of several hygromycin resistant plants showed that the HPT gene is transmitted to the progeny. Transformation experiments performed with a selectable and a non-selectable gene on separate plasmids resulted in a co-transformation rate of functionally active copies in about 25% of the transformants analysed. Hence this approach can be used to introduce non-selectable genes into the Arabidopsis genome.

A Mutant of Arabidopsis Deficient in the Chloroplast 16:1/18:1 Desaturase

Abstract: Leaf tissue of a mutant of Arabidopsis thaliana contains reduced levels of both 18-carbon and 16-carbon polyunsaturated fatty acids and increased levels of the 18:1 and cis-16:1 precursors due to a single nuclear mutation at a locus designated fadC. Analysis of the fatty acid compositions of individual lipids and the kinetics of lipid labeling with [14C]acetate in vivo indicate that the mutant lacks activity of the chloroplast glycerolipid ω-6 desaturase. As a result, lipids synthesized by the prokaryotic pathway are not desaturated further than 18:1 and 16:1. Lipids derived from the eukaryotic pathway are desaturated—presumably by the endoplasmic reticulum 18:1 phosphatidylcholine desaturase. However, an increase in the level of 18:1 on all the phospholipids derived from the eukaryotic pathway in leaves of the mutant suggests that the mutation does exert an effect on the composition of extrachloroplast membranes. Synthesis of monogalactosyldiacylglycerol (MGD) by the prokaryotic pathway is reduced 30 to 35% in the mutant and there is a corresponding increase in MGD synthesis by the eukaryotic pathway. This shift in metabolism which results in a more unsaturated MGD pool, may reflect the existence of a regulatory mechanism which apportions lipid synthesis between the two pathways in response to alterations in the physical properties of the chloroplast membranes.

3-Hydroxy-3-methylglutaryl-coenzyme A reductase from Arabidopsis thaliana is structurally distinct from the yeast and animal enzymes

Abstract: We have isolated the Arabidopsis thaliana gene (HMG1) encoding 3-hydroxy-3-methylglutaryl-CoA reductase [HMG-CoA reductase; (S)-mevalonate:NAD+ oxido-reductase (CoA-acylating), EC 1.1.1.88], the catalyst of the first committed step in isoprenoid biosynthesis. cDNA copies of the plant gene were identified by hybridization with a short, highly conserved segment of yeast HMG-CoA reductase as probe. DNA sequence analysis reveals that the COOH-terminal domain of the Arabidopsis HMG-CoA reductase (containing the catalytic site of the enzyme) is highly conserved with respect to the yeast, mammalian, and Drosophila enzymes, whereas the membrane-bound amino terminus of the Arabidopsis protein is truncated and lacks the complex membrane-spanning architecture of the yeast and animal reductases. Expression of the Arabidopsis gene from the yeast GAL1 promoter in a yeast mutant lacking HMG-CoA reductase activity suppresses the growth defect of the yeast mutant. Taken together, the sequence similarity to other cloned HMG-CoA reductase genes and the suppression of the yeast hmg- mutant provide strong evidence that the novel Arabidopsis gene we have cloned encodes a functional HMG-CoA reductase enzyme.

Transgenic expression of two marker genes under the control of an Arabidopsis rbcS promoter: sequences encoding the Rubisco transit peptide increase expression levels

Abstract: Chimeric gene constructs were made in which two reporter genes, the neo and bar genes, encoding neomycin phosphotransferase II and phosphinothricin acetyl transferase, respectively, were placed under the control of the promoter of ats1A, one of four genes encoding the ribulose-1,5-bisphosphate carboxylase (Rubisco) small subunit (SSU) in Arabidopsis thaliana. In one set of constructs the fusions were made at the initiation codons, while in the second set the sequences encoding the ats1 A transit peptide were included. Significantly higher steady-state levels of RNA and protein were observed in leaves of transgenic plants varrying the latter constructions. Individual transgenic plants varied in their degree of tissue specific expression of the chimeric genes as well as in absolute levels of expression. Preliminary results suggest that the ats1 A promoter may be only weakly responsive to phytochrome.

Immunochemically detectable phytochrome is present at normal levels but is photochemically nonfunctional in the hy 1 and hy 2 long hypocotyl mutants of Arabidopsis.

Abstract: The hy 1 and hy 2 long hypocotyl mutants of Arabidopsis thaliana contain less than 20% (the detection limit) of the phytochrome in wild-type tissue as measured by in vivo difference spectroscopy. In contrast, spectral measurements for the hy 3, hy 4, and hy 5 long hypocotyl mutants indicate that they each contain levels of phytochrome equivalent to the wild-type parent. Immunoblot analysis using a monoclonal antibody directed against the chromophore-bearing region of etiolated-oat phytochrome demonstrates that extracts of all mutant and wild-type Arabidopsis tissues, prepared by extraction of proteins into hot SDS-containing buffer, have identical levels of one major immunodetectable protein (116 kDa). An assay involving controlled in vitro proteolysis, known to produce distinctive fragmentation patterns for Pr and Pfr (Vierstra RD, Quail PH, Planta 156: 158–165, 1982), indicates that the 116 kDa polypeptide from the wild-type parent represents Arabidopsis phytochrome. The 116 kDa protein from either hy 3, hy 4, or hy 5 displays the same fragmentation pattern found for the wild type. Together with the spectral data, these results indicate that the mutant phenotype of these variants does not involve lesions in the polypeptide sequence that lead to gross conformational aberrations, and suggest that the genetic lesions may affect steps in the transduction chain downstream of the photoreceptor. In contrast, this same analysis for hy 1 and hy 2 has revealed that the 116 kDa protein from either of these mutants is not degraded differently in response to the different wavelengths of irradiation given in vitro. Moreover, whereas immunoblot analysis of tissue extracts from light-grown wild-type seedlings show that the 116 kDa phytochrome protein level is greatly reduced relative to dark-grown tissue as expected, similar extracts of light-grown hy 1 and hy 2 seedlings contain the 116 kDa polypeptide in amounts equivalent to those of dark-grown tissue. Combined, these data indicate that the hy 1 and hy 2 mutants both produce normal levels of immunochemically detectable phytochrome that is photochemically nonfunctional.

Structural and functional analyses of Arabidopsis thaliana chlorophyll a/b-binding protein ( cab ) promoters

Abstract: Arabidopsis thaliana carries three functional copies of the chlorophyll a/b-binding protein (cab) gene which code for an identical mature protein. DNA sequence comparison of all three cab promoters indicated that cab2 and cab3 are more closely related compared to cab1. Although the highest degree of homology was found between the TATA box and -256 of cab3 promoter, suggesting that this region plays a major role in promoter function, this promoter regions are only 47% homologous. To study whether these promoters are regulated by identical cis-acting regulatory elements, the promoters were mutated by progressive deletions and the effects on the promoter activity were measured in either transformed plants or cultured cells. It was found that the minimum sequence necessary for the light-dependent tissue-specific promoter activity of the cab3 is the 89 bp DNA fragment (between -74 and -164) at the region of the TATA and the CCAAT boxes. However, an additional 45 bp DNA fragment (between -164 and -209) upstream of the CCAAT box was necessary for the full promoter activity in the leaves. The regulatory element in the 45 bp region appears to be a positive regulator or enhancer which is specific to photosynthetic cells, since the region did not enhance the promoter activity in cultured cells. This region contains an octamer, TGCCACGT (cab2) or TGCCACAT (cab3), which is similar to the previously identified element, TGACACGT from Arabidopsis cab1 promoter. The upstream regions of the cab promoters appear to contain additional elements which are functionally distinct in each promoter since the upstream region of cab1 activated a non-functional nos promoter whereas that of cab3 did not.

Altered regulation of β-amylase activity in mutants of Arabidopsis with lesions in starch metabolism

Abstract: Three classes of mutants of Arabidopsis thaliana (L.) Heynhold with alterations in starch metabolism were found to have higher levels of leaf amylase activity than the wild type when grown in a 12-hr photoperiod. This effect was dependent upon the developmental stage of the plants and was largely suppressed during growth in continuous light. The various amylolytic activities in crude extracts were separated by electrophoresis in nondenaturing polyacrylamide gels and visualized by activity staining. The increased amylase activity in the mutants was due to an up to 40-fold increase in the activity of an extrachloroplast β-amylase (EC 3.2.1.2). These observations indicate the existence of a regulatory mechanism that controls the amount of β-amylase activity in response to fluctuations in photosynthetic carbohydrate metabolism. It is paradoxical that β-amylase appears to be a highly regulated enzyme, but as yet no physiologically relevant function can be assigned to this enzyme due to the absence of starch in the cytoplasmic compartment of leaf cells.

Functional expression of plant acetolactate synthase genes in Escherichia coli.

Abstract: Acetolactate synthase (ALS; EC 4.1.3.18) is the first common enzyme in the biosynthetic pathways leading to leucine, isoleucine, and valine. It is the target enzyme for three classes of structurally unrelated herbicides, the sulfonylureas, the imidazolinones, and the triazolopyrimidines. A cloned ALS gene from the small cruciferous plant Arabidopsis thaliana has been fused to bacterial transcription/translation signals and the resulting plasmid has been used to transform Escherichia coli. The cloned plant gene, which includes sequences encoding the chloroplast transit peptide, is functionally expressed in the bacteria. It is able to complement genetically a strain of E. coli that lacks endogenous ALS activity. An ALS gene cloned from a line of Arabidopsis previously shown to be resistant to sulfonylurea herbicides has been similarly expressed in E. coli. The herbicide-resistance phenotype is expressed in the bacteria, as assayed by both enzyme activity and the ability to grow in the presence of herbicides. This system has been useful for purifying substantial amounts of the plant enzyme, for studying the sequence parameters involved in subcellular protein localization, and for characterizing the interactions that occur between ALS and its various inhibitors.

Isolation of a cDNA encoding mitochondrial citrate synthase from Arabidopsis thaliana.

Abstract: We isolated a cDNA clone from Arabidopsis thaliana encoding the TCA cycle enzyme, citrate synthase. The plant enzyme displays 48% and 44% amino acid residue similarity with the pig, and yeast polypeptides, respectively. Many proteins, including citrate synthase, which are destined to reside in organelles such as mitochondria and chloroplasts, are the products of the nucleocytoplasmic protein synthesizing machinery and are imported post-translationally to the site of function. We present preliminary investigations toward the establishment of an in vitro plant mitochondrial import system allowing for future studies to dissect this process in plants where the cell must differentiate between mitochondria and chloroplast and direct their polypeptides appropriately.

Isolation of Arabidopsis mutants with altered seed fatty acid composition

Abstract: By direct screening of Arabidopsis seed fatty acid methyl esters, we have isolated mutants which are deficient in the elongation of 18:1 to 20:1 and the desaturation of 18:2 to 18:3. Both the elongation and the desaturation mutants, designated MB14 and BL1 respectively, have only 10% of the wild-type levels of 20:1 and 18:3 in their seeds. The intermediate levels of 20:1 and 18:3 in F1 seeds of crosses to the wild type indicate that the level of enzyme is regulating the amount of 20:1 and 18:3 in seeds. Consistent with this observation, the mutations were found to segregate 1:2:1 in F2 seeds. We have found that the 18:2 desaturase mutation is clearly expressed in root phosphatidylcholine.

Process for obtaining intact fertile arabidopsis plants from protoplasts.

Abstract: not available for EP0378587Abstract of corresponding document: DE3802237In a regeneration process, new Arabidopsis plants are obtained from protoplasts isolated from differentiated tissues, undifferentiated tissues and various organs of Arabidopsis plants which are embedded in a polymer matrix and then cultured in the dark under constant illumination during a photoperiod. The process is used to obtain new Arabidopsis mutants and their genetic complements. The genetic material is transferred directly to the plant cells by incubating the DNA to be transformed together with Arabidopsis protoplasts in suitable incubation media.

Modifications to the Two Pathway Scheme of Lipid Metabolism Based on Studies of Arabidopsis Mutants

Abstract: In the last few years the lipid mutants of Arabidopsis have been very useful in elucidating details of the synthesis and desaturation of glycerolipids in plant leaves [1–3]. Arabidopsis is a typical 16:3-plant in which both the prokaryotic and eukaryotic pathways [4] contribute to the production of chloroplast lipids. We have investigated the pattern of lipid metabolism in wild type Arabidopsis and calculated the fluxes of carbon involved [5]. An abbreviated version of this analysis is shown in Fig. la. For every 1000 fatty acid molecules synthesized in the chloroplast 390 enter the prokaryotic pathway in the chloroplast envelope while 610 are exported as CoA esters to enter the eukaryotic pathway. Of these 340 are reimported into the chloroplast. Overall, almost equal amounts of chloroplast lipids are produced by each pathway. However, the quantities of individual lipids synthesized by the two routes are very different. All the chloroplast phosphatidylglycerol (PG) and over 70% of the monogalactosyldiacylglycerol (MGD) is derived from the prokaryotic pathway while digalactosyldiacylglycerol is synthesized mainly on the eukaryotic pathway [5].In this paper we have outlined how four of the Arabidopsis mutants have changed the way we view the operation of the two pathways involved in leaf membrane lipid synthesis. More detailedinformation on each mutant can be found elsewhere [1–3, 5,6 and in preparation].

Temperature and Light Effects on the Expression of the Arabidopsis Linolenate Mutant Phenotype in Callus Cultures

Abstract: Temperature and light have a large influence on linolenic acid levels. Low temperatures have been correlated with an increased ratio of polyunsaturated to saturated lipids in plant cell membranes, while plant tissues maintained in the dark show a significant increase in the desaturation of linoleate to linolenate upon illumination 1,2.