Characterization of waldmeister, a novel developmental mutant in Arabidopsis thaliana

A novel Arabidopsis thaliana (L.) Heynh. developmental mutant,waldmeister (wam), is described. This mutant was found in theprogeny arising from an Ac-Ds tagging experiment, but does notappear to be tagged by an introduced transposon. This recessivenuclear mutation maps between GAPB and ap1 on chromosome 1 andshows extreme morphological and physiological changes in bothfloral and vegetative tissues. Changes to the vegetative phenotypeinclude altered leaf morphology, multiple rosettes, stem fasciation,retarded senescence and disturbed geotropic growth. Changesto the floral phenotype include delayed flowering, increasednumber of inflorescences, determinate inflorescences, alterednumber and morphology of floral organs, chimeric floral organs,and ectopic ovules . wam was crossed to a number of previouslydescribed floral mutants: apetela 2, apetela 3, pistillata,agamous, and leafy. The phenotype of the double mutant was ineach case additive. In the case of agamous, however, the indeterminaterepetitive floral structure of agamous was lacking, emphasizingthe determinate inflorescence growth of wam. The extreme phenotypeof the wam mutant is suggestive of a disturbance to a gene ofglobal importance in the regulation of plant growth and development. Key words: Arabidopsis thaliana, waldmeister, developmental mutant, flower mutant     

Characterization of AtNST-KT1, a novel UDP-galactose transporter from Arabidopsis thaliana

Nucleotide sugar transporters (NST) mediate the transfer of nucleotide sugars from the cytosol into the lumen of the endoplasmatic reticulum and the Golgi apparatus. Because the NSTs show similarities with the plastidic phosphate translocators (pPTs), these proteins were grouped into the TPT/NST superfamily. In this study, a member of the NST-KT family, AtNST-KT1, was functionally characterized by expression of the corresponding cDNA in yeast cells and subsequent transport experiments. The histidine-tagged protein was purified by affinity chromatography and reconstituted into proteoliposomes. The substrate specificity of AtNST-KT1 was determined by measuring the import of radiolabelled nucleotide mono phosphates into liposomes preloaded with various unlabelled nucleotide sugars. This approach has the advantage that only one substrate has to be used in a radioactively labelled form while all the nucleotide sugars can be provided unlabelled. It turned out that AtNST-KT1 represents a monospecific NST transporting UMP in counterexchange with UDP-Gal but did not transport other nucleotide sugars. The AtNST-KT1 gene is ubiquitously expressed in all tissues. AtNST-KT1 is localized to Golgi membranes. Thus, AtNST-KT1 is most probably involved in the synthesis of galactose-containing glyco-conjugates in plants.     

Characterization of a chlorate-hypersensitive,high nitrate reductase Arabidopsis thaliana mutant

Summary A population of A. thaliana, produced by self-fertilization of ethylmethane sulfonate treated plants, was exposed to chlorate in the watering solution, and plants showing early susceptibility symptoms were rescued. Among the progeny lines of these plants five were shown to be repeatably chlorate-hypersusceptible. One of these lines (designated C-4) possessed elevated activity of nitrate reductase (NR). The NR activity of mutant C-4 was higher than that of normal plants throughout the life cycle. Nitrite reductase and glutamine synthetase activities of C-4 were normal, as were chlorate uptake rate and tissue nitrate content. The elevated NR activity apparently was responsible for the chlorate hypersusceptibility of C-4. Inheritance studies of NR indicated that the elevated activity of C-4 was probably controlled by a single recessive allele.     

Characterization of ISRgn1, a novel insertion sequence of the IS3 family isolated from a bacteriocin-negative mutant of Ruminococcus gnavus E1

ISRgn1, an insertion sequence of the IS3 family, has been identified in the genome of a bacteriocin-negative mutant of Ruminococcus gnavus E1. The copy number of ISRgn1 in R. gnavus E1, as well as its distribution among phylogenetically E1-related strains, has been determined. Results obtained suggest that ISRgn1 is not indigenous to the R. gnavus phylogenetic group but that it can transpose in this bacterium.     

Characterization of a novel developmentally retarded mutant (drm1) associated with the autonomous flowering pathway in Arabidopsis

INTRODUCTIONThe transition from vegetative growth to reproductionis one of the most important developmental events in flow-ering plants since it is related to the competence and sur-vivability of a particular species living in a particularenvironment. The…     

Characterization of a cytokinesis defective (cyd1) mutant of Arabidopsis.

Although several mutations and genes affecting plant cytokinesis have been identified, mutant screens are not yet saturated and knowledge about gene function is still limited. A novel Arabidopsis mutation, cytokinesis defective1 (cyd1), was identified by partial or missing cell walls in stomata. Stomata with incomplete or no cytokinesis still differentiate and some contain swellings of the outer wall not found in the wild type. The incomplete walls are correctly placed opposite stomatal wall thickenings suggesting that the mutation interferes with the execution of cytokinesis rather than with the placement of the division site. Cytokinesis defects are also detectable in other cell types throughout the plant, defects which include cell wall protrusions, two or more nuclei in one cell, and reduced cell number. The extent of cytokinetic partitioning correlates with nuclear number in abnormal stomata. Many cyd1 epidermal cells, stomata and pollen are larger, and trichomes have more branches. cyd1 is partially lethal with poor seed set and some defective ovules, but many plants are fertile despite abnormalities in vegetative and reproductive development such as missing, reduced, fused or misshapen leaves and floral organs. cyd1 appears to be the only cytokinesis mutant described where defects are known to occur in both mature vegetative and reproductive organs. Thus, the CYD1 gene product appears to be necessary for the execution of cytokinesis throughout the shoot. The examination of stomata by microscopy may be a useful screen for the directed isolation of additional cytokinesis mutations that are not embryo or seedling lethal     

Characterization of tt15, a novel transparent testa mutant of Arabidopsis thaliana (L.) Heynh.

The Arabidopsis thaliana seed coat typically has a brown color due to the accumulation of flavonoid pigments in the testa. Mutants of A. thaliana with defects in pigment biosynthesis often produce seeds that are olive brown or even yellow in appearence, and the responsible genetic loci are referred to as TRANSPARENT TESTA (TT). Large-scale screening for mutants affected in seed development and complementation analysis of a candidate mutant line with all published A. thalianatt mutants identified a new tt locus designated tt15. The tt15 mutation maps to the lower part of chromosome 1. Mutant plants produced pale greenish-brown seeds whose dormancy was slightly reduced. The phenotype was consistent with the maternal origin of the testa. Analysis of pigment accumulation and the study of expression patterns of genes involved in flavonoid biosynthesis in tt15 plants and seeds indicated a seed-specific phenotype. Most notable was a reduction of the cyanidin and quercetin content of tt15 seeds. Received: 2 October 1998 / Accepted: 10 October 1998     

A novel ethanol-hypersensitive mutant of Arabidopsis

A novel ethanol-hypersensitive mutant, geko1 (gek1), was isolated from Arabidopsis thaliana. The gek1 mutant displays an enhanced sensitivity (10-100 times greater than the wild type) to ethanol in growth medium, while it grows normally in the absence of ethanol, and responds normally to other alcohols and to environmental stresses such as heat shock and high salinity. The ethanol-hypersensitive phenotype of gek1 requires alcohol dehydrogenase activity, indicating that gek1 is sensitive not to ethanol itself but to the metabolites of ethanol. Consistent with this, gek1 shows enhanced sensitivity to acetaldehyde in the medium. The endogenous acetaldehyde levels were not different between gek1-2 and wild-type seedlings treated with ethanol. These results indicate that the ethanol hypersensitivity of gek1 is due to an enhanced sensitivity to acetaldehyde toxicity, instead of abnormally elevated accumulation of toxic acetaldehyde, which has been thought to be the major cause of ethanol toxicity in mammal cells.     

Characterization of grape Gibberellin Insensitive1 mutant alleles in transgenic Arabidopsis

We generated 12 different mutations in the grape Gibberellin Insensitive1 (VvGAI1) sequences, transformed them into Arabidopsis under the control of 35S, Arabidopsis GAI or grape GAI1 promoter, and evaluated the impact of these mutant alleles on plant growth and development. These VvGAI1 sequence variants included some mimics of the known GAI-like mutant alleles discovered in grape, wheat, barley, corn, Brassica, and Arabidopsis. In general, plant height and related traits such as length of internodes and inflorescences were significantly reduced for most of the mutant alleles studied, regardless of which promoter was used. Interestingly, the numbers of rosette leaves and lateral branches were generally reduced when a 35S promoter was used to express the mutant alleles, but increased when an Arabidopsis or grape GAI promoter was used. Furthermore, the 35S plants often displayed curly and small leaves. In contrast, the leaves of the plants carrying mutant alleles controlled by a GAI promoter were of variable size, dark green and rarely curly. In addition, when certain VvGAI1 mutant alleles were under the control of the grape GAI1 promoter, the number of pods on inflorescences was significantly increased, but some of the pods produced few seeds due to partial sterility. On the basis of the systematic evaluation of various VvGAI1 mutant alleles in Arabidopsis, we concluded that the VvGAI1 mutant alleles mimicking the GAI or GAI-like mutant variants discovered in wheat, barley and Brassica could potentially be useful for the improvement of grapevine plant architecture.     

Characterization of a new Arabidopsis mutant exhibiting enhanced disease resistance

In many plant-pathogen interactions, resistance is associated with the synthesis and accumulation of salicylic acid (SA) and pathogenesis-related (PR) proteins. At least two general classes of mutants with altered resistance to pathogen attack have been identified in Arabidopsis. One class exhibits increased susceptibility to pathogen infection; the other class exhibits enhanced resistance to pathogens. In an attempt to identify mutations in resistance-associated loci, we screened a population of T-DNA tagged Arabidopsis thaliana ecotype Wassilewskija (Ws) for mutants showing constitutive expression of the PR-1 gene (cep). A mutant was isolated and shown to constitutively express PR-1, PR-2, and PR-5 genes. This constitutive phenotype segregated as a single recessive trait in the Ws genetic background. The mutant also had elevated levels of SA, which are responsible for the cep phenotype. The cep mutant spontaneously formed hypersensitive response (HR)-like lesions on the leaves and cotyledons and also exhibited enhanced resistance to virulent bacterial and fungal pathogens. Genetic analyses of segregating progeny from outcrosses to other ecotypes unexpectedly revealed that alterations in more than one gene condition the constitutive expression of PR genes in the original mutant. One of the mutations, designated cpr20, maps to the lower arm of chromosome 4 and is required for the cep phenotype. Another mutation, which has been termed cpr21, maps to chromosome 1 and is often, but not always, associated with this phenotype. The recessive nature of the cep trait suggests that the CPR20 and CPR21 proteins may act as negative regulators in the disease resistance signal transduction pathway.     

Characterization of Arabidopsis AtAMT2, a novel ammonium transporter in plants

We have cloned and characterized the first member of a novel family of ammonium transporters in plants: AtAMT2 from Arabidopsis thaliana. AtAMT2 is more closely related to bacterial ammonium transporters than to plant transporters of the AMT1 family. The protein was expressed and functionally characterized in yeast. AtAMT2 transported ammonium in an energy-dependent manner. In contrast to transporters of the AMT1 family, however, AtAMT2 did not transport the ammonium analogue, methylammonium. AtAMT2 was expressed more highly in shoots than roots and was subject to nitrogen regulation.     

Transcriptional and biochemical regulation of a novel Arabidopsis thaliana bifunctional aspartate kinase-homoserine dehydrogenase gene isolated by functional complementation of a yeast hom6 mutant

An aspartate kinase-homoserine dehydrogenase (AK-HSDH) cDNA of Arabidopsis thaliana has been cloned by functional complementation of a Saccharomyces cerevisiae strain mutated in its homoserine dehydrogenase (HSDH) gene (hom6). Two of the three isolated clones were also able to complement a mutant yeast aspartate kinase (AK) gene (hom3). Sequence analysis showed that the identified gene (akthr2), located on chromosome 4, is different from the previously cloned A. thaliana AK-HSDH gene (akthr1), and corresponds to a novel bifunctional AK-HSDH gene. Expression of the isolated akthr2 cDNA in a HSDH-less hom6 yeast mutant conferred threonine and methionine prototrophy to the cells. Cell-free extracts contained a threonine-sensitive HSDH activity with feedback properties of higher plant type. Correspondingly, cDNA expression in an AK-deficient hom3 yeast mutant resulted in threonine and methionine prototrophy and a threonine-sensitive AK activity was observed in cell-free extracts. These results confirm that akthr2 encodes a threonine-sensitive bifunctional enzyme. Transgenic Arabidopsis thaliana plants (containing a construct with the promoter region of akthr2 in front of the gus reporter gene) were generated to compare the expression pattern of the akthr2 gene with the pattern of akthr1 earlier described in tobacco. The two genes are simultaneously expressed in meristematic cells, leaves and stamens. The main differences between the two genes concern the time-restricted or absent expression of the akthr2 gene in the stem, the gynoecium and during seed formation, while akthr1 is less expressed in roots.     

Characterization of a new simian virus 40 mutant, tsA3900, isolated from deletion mutant tsA1499.

The simian virus 40 (SV40) mutant tsA1499 contains an 81-base-pair deletion in the region of A gene encoding the C-terminal portion of the large T antigen. This mutant is particularly interesting, since it is a temperature-sensitive mutant that is apparently able to separate the lytic growth and transforming functions of the SV40 large T antigen at 38.5 degrees C. We report the isolation of a tsA1499 revertant (tsA1499-Rev) which is no longer temperature sensitive for lytic growth but still contains the 81-base-pair deletion of tsA1499. Marker rescue experiments with tsA1499-Rev or wild-type strain 830 (wt830) DNAs revealed that the original tsA1499 mutant contained a second mutation within the HindIII-Fnu4HI restriction fragment between 0.425 and 0.484 map units. Sequencing of this DNA fragment from the tsA1499, tsA1499-Rev, and wt830 viruses revealed that tsA1499 contained a single-base transversion (C to G) at 0.455 map units (nucleotide 4261). This transversion resulted in the creation of a new RsaI cleavage site in the tsA1499 DNA and predicts an arginine-to-threonine substitution at amino acid position 186 in the mutant large T antigen. The DNA sequence of the tsA1499-Rev HindIII-Fnu4HI fragment was identical to that of wt830. To determine whether tsA1499 was temperature sensitive for lytic growth solely as a result of the newly discovered point mutation or because of a combination of the point and deletion mutations, a series of viruses were constructed which contained the point mutation, the deletion mutation, both mutations, or neither. This was done by ligating the PstI A and B DNA fragments from either tsA1499 or wt830 and transfecting the ligated DNA into BSC-1H monkey kidney cells. This experiment revealed that all viruses containing the point mutation (the tsA1499 PstI A DNA fragment) were temperature sensitive for lytic growth, regardless of the presence of the 81-base-pair deletion (the tsA1499 PstI B DNA fragment). This newly discovered point mutation, at nucleotide 4261, is therefore unique, since to our knowledge it is the first tsA mutation to be described in the 0.455-map-unit region of the SV40 genome. We then tested the effect of this unique mutation on the ability of the SV40 virus to transform cultured rat cells to anchorage independence.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization and fine mapping of a novel rice albino mutant low temperature albino 1

Albino mutants are useful genetic resource for studying chlorophyll biosynthesis and chloroplast development and cloning genes involved in these processes in plants.Here we report a novel rice mutant low temperature albino 1(lta1) that showed albino leaves before 4-leaf stage when grown under temperature lower than 20℃,but developed normal green leaves under temperature higher than 24℃or similar morphological phenotypes in dark as did the wild-type(WT).Our analysis showed that the contents of chlorophylls and chlorophyll precursors were remarkably decreased in the ltal mutant under low temperature compared to WT.Transmission electron microscope observation revealed that chloroplasts were defectively developed in the albino lta1 leaves,which lacked of well-stacked granum and contained less stroma lamellae.These results suggested that the lta1 mutation may delay the light-induced thylakoid assembly under low temperature.Genetic analysis indicated that the albino phenotype was controlled by a single recessive locus.Through map-based approach,we finally located the Lta1 gene to a region of 40.3 kb on the short arm of chromosome 11.There are 8 predicted open reading frames(ORFs) in this region and two of them were deleted in lta1 genome compared with the WT genome.The further characterization of the Ltal gene would provide a good approach to uncover the novel molecular mechanisms involved in chloroplast development under low temperature stress.     

Characterization of an Arabidopsis thaliana DNA hypomethylation mutant.

We have recently isolated two Arabidopsis thaliana DNA hypomethylation mutations, identifying the DDM1 locus, that cause a 70% reduction in genomic 5-methylcytosine levels [1]. Here we describe further phenotypic and biochemical characterization of the ddm1 mutants. ddm1/ddm1 homozygotes exhibited altered leaf shape, increased cauline leaf number, and a delay in the onset of flowering when compared to non-mutant siblings in a segregating population. Our biochemical characterization investigated two possible mechanisms for DNA hypomethylation. In order to see if ddm1 mutations affect DNA methyltransferase function, we compared DNA methyltransferase activities in extracts from wild-type and ddm1 mutant tissues. The ddm1 mutant extracts had as much DNA methyltransferase activity as that of the wild-type for both the CpI and CpNpG substrates suggesting that the DDM1 locus does not encode a DNA methyltransferase. Moreover, the ddm1 mutations did not affect the intracellular level of S-adenosylmethionine, the methyl group donor for DNA methylation. The possibility that the DDM1 gene product functions as a modifier of DNA methylation is discussed.