Academic literature on the topic 'Mutant Arabidopsis lines'

Thirteen Arabidopsis thaliana mutants with deviating epicuticular wax layers (i.e., cer mutants) were isolated by screening 13 000 transformed lines produced by the seed transformation method. After crossing the 13 mutants to some of the previously known cer mutant lines, 12 of our mutants mapped to 6 of the 21 known complementation groups (cer1 through cer4 as well as cer6 and cer10), while the other mutant corresponded to a previously unknown locus, cer21. Mutant phenotypes of 6 of the 13 mutant lines were caused by T-DNA insertions within cer genes. We also analyzed the chemical composition of the epicuticular wax layers of the cer mutants isolated in this study relative to that of Arabidopsis wild-type plants. Our results suggest that the five genes we tagged regulate different steps in wax biosynthesis, i.e., the decarbonylation of fatty aldehydes to alkanes, the elongation of hexacosanoic acid to octacosanoic acid, the reduction of fatty aldehydes to primary alcohols and the production of free aldehydes, while an insertion in the fifth gene causes an alteration in the chain length distribution of the different classes of wax compounds.Key words: epicuticular wax, glossy mutants, gas chromotography – mass spectroscopy.

A mutational study was carried out to isolate Arabidopsis thaliana plants that exhibit full or partial disruption of the RPS2-mediated hypersensitive response (HR) to Pseudomonas syringae that express avrRpt2. Five classes of mutants were identified including mutations at RPS2, dnd mutations causing a “defense, no death” loss-of-HR phenotype, a lesion-mimic mutant that also exhibited an HR¯phenotype, and a number of intermediate or partial-loss-of-HR mutants. Surprisingly, many of these mutants displayed elevated resistance to virulent P. syringae and, in some cases, to Peronospora parasitica. Constitutively elevated levels of pathogenesis-related (PR) gene expression and salicylic acid were also observed. In the lesion-mimic mutant, appearance of elevated resistance was temporally correlated with appearance of lesions. For one of the intermediate lines, resistance was shown to be dependent on elevated levels of salicylic acid. A new locus was identified and named IHR1, after the mutant phenotype of “intermediate HR.” Genetic analysis of the intermediate-HR plant lines was difficult due to uncertainties in distinguishing the partial/intermediate mutant phenotypes from wild type. Despite this difficulty, the intermediate-HR mutants remain of interest because they reveal potential new defense-related loci and because many of these lines exhibit partially elevated disease resistance without dwarfing or other apparent growth defects.

Abstract Mutant analysis represents one of the most reliable approaches to identifying genes involved in plant development. The screening of the Versailles collection of Arabidopsis thaliana T-DNA insertion transformants has allowed us to isolate different mutations affecting male gametophytic functions and viability. Among several mutated lines, five have been extensively studied at the genetic, molecular, and cytological levels. For each mutant, several generations of selfing and outcrossing have been carried out, leading to the conclusion that all these mutations are tagged and affect only the male gametophyte. However, only one out of the five mutations is completely penetrant. A variable number of T-DNA copies has integrated in the mutant lines, although all segregate at one mutated locus. Two mutants could be defined as “early mutants”: the mutated genes are presumably expressed during pollen grain maturation and their alteration leads to the production of nonfunctional pollen grains. Two other mutants could be defined as “late mutant” since their pollen is able to germinate but pollen tube growth is highly disturbed. Screening for segregation ratio distortions followed by thorough genetic analysis proved to be a powerful tool for identifying gametophytic mutations of all phases of pollen development.

Abstract Postgenital organ fusion occurs most commonly during reproductive development and is important in many angiosperms during genesis of the carpel. Although a number of mutants have been described that manifest ectopic organ fusion, little is known about the genes involved in regulating this process. In this article we describe the characterization of a collection of 29 Arabidopsis mutants showing an organ fusion phenotype. Mapping and complementation analyses revealed that the mutant alleles define nine different loci distributed throughout the Arabidopsis genome. Multiple alleles were isolated for the four complementation groups showing the strongest organ fusion phenotype while the remaining five complementation groups, all of which show only weak floral organ fusion, have a single representative allele. In addition to fusion events between aerial parts of the shoot, some mutants also show abnormal ovule morphology with adjacent ovules joined together at maturity. Many of the fusion mutants isolated have detectable differences in the rate at which chlorophyll can be extracted; however, in one case no difference could be detected between mutant and wild-type plants. In three mutant lines pollen remained unresponsive to contact with the mutant epidermis, demonstrating that organ fusion and pollen growth responses can be genetically separated from one another.

Abstract We have undertaken a large-scale genetic screen to identify genes with a seedling-lethal mutant phenotype. From screening ~38,000 insertional mutant lines, we identified >500 seedling-lethal mutants, completed cosegregation analysis of the insertion and the lethal phenotype for >200 mutants, molecularly characterized 54 mutants, and provided a detailed description for 22 of them. Most of the seedling-lethal mutants seem to affect chloroplast function because they display altered pigmentation and affect genes encoding proteins predicted to have chloroplast localization. Although a high level of functional redundancy in Arabidopsis might be expected because 65% of genes are members of gene families, we found that 41% of the essential genes found in this study are members of Arabidopsis gene families. In addition, we isolated several interesting classes of mutants and genes. We found three mutants in the recently discovered nonmevalonate isoprenoid biosynthetic pathway and mutants disrupting genes similar to Tic40 and tatC, which are likely to be involved in chloroplast protein translocation. Finally, we directly compared T-DNA and Ac/Ds transposon mutagenesis methods in Arabidopsis on a genome scale. In each population, we found only about one-third of the insertion mutations cosegregated with a mutant phenotype.

It was recently suggested that the protein Cand2 acts as a G protein–coupled receptor (GPCR) for melatonin in Arabidopsis by mediating stomatal closure via H2O2 production and Ca2+ influx. Here, we examined whether Cand2 is indeed a melatonin receptor. Contrary to previous reports, confocal microscopy analyses indicated that Cand2 protein is localized in the cytoplasm rather than the plasma membrane. The role of Cand2 was further investigated in genetic analyses using two Arabidopsis cand2 knockout mutant lines, SALK_071302 (cand2-1) and SALK_068848 (cand2-2). We found that melatonin-mediated mitogen-activated protein kinase (MAPK) activation was not abolished in the cand2 mutant lines, nor did melatonin-mediated defense gene induction (e.g., GST1) change relative to that in the wild type Col-0. Following ER stress, the two cand2 mutant lines were identical to Col-0 in terms of defense gene induction, ion leakage, and ROS levels. Two G protein mutants, gpa1 (Gα mutant) and agb1 (Gβ mutant), also exhibited no disturbance in melatonin-mediated defense gene induction or melatonin-mediated MAPK activation. Collectively, our data indicate that Cand2 is neither a phytomelatonin receptor localized in the plasma membrane nor is it involved in the melatonin-mediated defense signaling pathway via G protein components. However, it remains unclear how melatonin-mediated MAPK activation was slightly decreased in the mutant cand2-2 without affecting downstream defense gene induction. Also, it cannot rule out the possibility that Cand2 may be a melatonin binding protein and that its binding may result in a decrease of free melatonin level in plants.

Plant-parasitic cyst nematodes induce the formation of hypermetabolic feeding sites, termed syncytia, as their sole source of nutrients. The formation of the syncytium is orchestrated by the nematode, in part, by modulation of phytohormone responses, including cytokinin. In response to infection by the nematode Heterodera schachtii, cytokinin signaling is transiently induced at the site of infection and in the developing syncytium. Arabidopsis lines with reduced cytokinin sensitivity show reduced susceptibility to nematode infection, indicating that cytokinin signaling is required for optimal nematode development. Furthermore, lines with increased cytokinin sensitivity also exhibit reduced nematode susceptibility. To ascertain why cytokinin hypersensitivity reduces nematode parasitism, we examined the transcriptomes in wild type and a cytokinin-hypersensitive type-A arr Arabidopsis mutant in response to H. schachtii infection. Genes involved in the response to biotic stress and defense response were elevated in the type-A arr mutant in the absence of nematodes and were hyperinduced following H. schachtii infection, which suggests that the Arabidopsis type-A arr mutants impede nematode development because they are primed to respond to pathogen infection. These results suggest that cytokinin signaling is required for optimal H. schachtii parasitism of Arabidopsis but that elevated cytokinin signaling triggers a heightened immune response to nematode infection.

Deleteagene™(Delete-a-gene) is a deletion-based gene knockout system for plants. To obtain deletion mutants for a specific gene, random deletion libraries created by fast neutron mutagenesis are screened by polymerase chain reaction (PCR) using primers flanking the target gene. By adjusting the PCR extension time to preferentially amplify the deletion alleles, deletion mutants can be identified in pools of DNA samples with each sample representing more than a thousand mutant lines. In Arabidopsis, knockout plants for greater than 80% of targeted genes have been obtained from a population of 51 840 lines. A large number of deletion mutants have been identified and multiple deletion alleles are often recovered for targeted loci. In Arabidopsis, the method is very useful for targeting small genes and can be used to find deletion mutants mutating two or three tandem homologous genes. In addition, the method is demonstrated to be effective in rice as a deletion mutant for a rice gene was obtained with a similar approach. Because fast neutron mutagenesis is applicable to all plant genetic systems, Deleteagene™has the potential to enable reverse genetics for a wide range of plant species.

Five new recessive male-sterile mutants of Arabidopsis thaliana were isolated following seed mutagenesis by X-rays and ethyl methanesulfonate. The cytology of plants homozygous for the msY and msW mutations suggested that pollen development in these lines became abnormal at or before meiosis. The msK mutation caused faulty timing of synthesis or turnover and distribution of callose. In plants homozygous for the msZ mutation, pollen development failed at a late stage. In wild-type plants, the stamen filament elongated just prior to anther dehiscence. In contrast, in the msZ mutant stamen elongation did not occur. Pollen in msH homozygotes was fertile, but anthers failed to dehisce. The msI mutant of J.H. Van der Ween and P. Wirtz (1968. Euphytica 17: 371 – 377) was included in the present study. Pollen development in this mutant failed shortly after microspore release from tetrads. Complementation tests confirmed that the ms mutations were at different loci. Reduced transmission of certain ms genes was observed. Key words: Arabidopsis thaliana, male sterile mutants, anther dehiscence, callose, inheritance.

Abstract Accumulating evidence indicates that individual members of the phytochrome family of photoreceptors have differential but interactive roles in controlling plant responses to light. To investigate possible cross-regulation of these receptors, we have identified monoclonal antibodies that specifically detect each of the five Arabidopsis phytochromes, phyA to phyE (phytochrome A holoprotein; PHYA, phytochrome A apoprotein; PHYA, phytochrome A gene; phyA, mutant allele of phytochrome A gene), on immunoblots and have used them to analyze the effects of phyA and phyB null mutations on the levels of all five family members. In phyB mutants, but not in phyA mutants, a four- to six-fold reduction in the level of phyC is observed in tissues grown either in the dark or in the light. Coordinate expression of phyB and phyC is induced in the phyB mutant background by the presence of a complementing PHYB transgene. However, in transgenic lines that overexpress phyB 15- to 20-fold, phyC is not similarly overexpressed. In these overexpressor lines, the levels of phyA, phyC, and phyD are increased two- to four-fold over normal in light-grown but not dark-grown seedlings. These observations indicate that molecular mechanisms for coordination or cross-regulation of phytochrome levels are active in Arabidopsis and have implications for the interpretation of phytochrome mutants and overexpressor lines.

Salt, cold, and drought are major abiotic stresses that impact plants and agricultural crops. Many plants and crops are able to increase their ability to survive these and other stresses by altering gene expression. Using the Arabidopsis model plant in studying gene function and regulation is of crucial importance to plant genetics and biotechnology. Our objective was to study plant survival and stress tolerance in Arabidopsis T-DNA lines and, also chemically mutagenized and RNAi lines, for specific genes that are hypothesised or expected to have a role in at least one of the known abiotic stresses. The selection of genes was based on some previous research including QTL analysis. I included in my investigations OSMl, CHXl7, KUPl, bHLH033, CBF4, dreb2a and the NW20 and N163 lines of ERECTA. Other non-mutated genes such as COR 78, DREB2A, CBFl, CBF3, CBF4, and OSMl were used to study the specificity in gene expression in Arabidopsis leaf and epidermal tissues under a combination of cold and osmolytes. The insertional mutant analysis showed that T-DNA can be located in a different site than the one shown in database and sometimes parts of the T-DNA left-border-sequence were absent. Most of the targeted Arabidopsis mutants showed a normal Mendelian pattern of segregation with the one exception of the CBF4 insertional mutant population. RT-PCR transcript detection of bHLH and DREB2A indicated that wild-type Arabidopsis in our growth environment expressed only one splice form of each of these genes rather than the two described by the TAIR and MIPS databases. Stress results indicated that inserting T-DNA within the Arabidopsis genome for a specific gene can lead to negative or positive effects on the gene's expression and plant survival. Investigation of the role of OSMl in cold tolerance indicated that the mutant plants were more tolerant to cold as measured by plant survival and electrolyte leakage. The higher expression of OSMl gene in the mutant plants has driven me to search in the promoter region of OSMl at OSM1::T-DNA junction. The results revealed the presence of different known eis-acting elements which might have affected plant survival. Using RNAi lines for OSMl (gene expression was not tested) is also consistent with the idea that this gene has a role in cold stress. Even though the high transcripts accumulation of bHLH (lCE2) in the mutant line was discovered, there was only slight difference between homozygous and wild type plants in response to cold. DREB2A, which is well known for its dehydration involvement, was tested under cold. Using dreb2a mutant for cold stress revealed that Salt stress tolerance was obtained with osml, ehx17, and kupl mutants as measured by the high average of plant survival. Mineral content was measured in leaves and roots of CHX17 and KUP 1 mutant and wild type plants under salt conditions. The mutant plants accumulated less sodium and higher potassium especially in roots compared to wild type. Also, roots accumulated more calcium and magnesium than the wild type. These results are clearly consistent with the higher survival in kupl mutant and may indicate a correlation with the salt tolerance in ehx17. Also, the high sensitivity to salt stress was strongly linked to some phenotypic and developmental changes, such as leaf wilting, rolling, chlorosis, and desiccation. Mutant plants that had higher survival developed dark leaf colour which probably indicates anthocyanin accumulation. A higher level of expression of genes in some mutant lines than in the wild type lines, linked with higher plant survival indicated there might be some eis-acting elements in the inserted T-DNA that were influencing stress tolerance. Compared to NW20 Landsberg ereeta line, carrying the chemically produced mutant, N163 plants, which carry wild-type ERECT A gene, exhibited more plant survival under salt stress especially at an older growth stage. Abstract IV covering plants during cold acclimation increased plant's sensitivity to cold compared to uncovered plants. This suggests that the role of DREB2A in cold acclimation probably is not through water stress signalling per se but other signalling must be involved. Also, drought stress was studied and was limited to CBF4 gene. The gene expression of CBF4 in the mutant was greatly reduced. However, plant survival under drought was not affected by the mutation. Gene expression-specificity of some cold regulated genes in epidermis and leaves of Arabidopsis wild type was studied under sugar and sorbitol with or without minerals. COR 78, DREB2A, CBF1, Eli-l-o; ACT2, and ACT8 were more highly expressed under cold in epidermis than in leaves. This expression was often several-fold higher when sucrose at 90 mM with mineral salts was supplied. CBF3 also showed a higher level of transcript accumulation by cold in epidermis than in leaves but both tissues exhibited similar and higher expression with sucrose at 40mM than the control and 90mM of sucrose with minerals. It seemed that removing minerals from sugar solution reduced the gene expression but epidermal tissues conserved the preference of most genes to be expressed in higher level (relative to total RNA) than leaves. Sorbitol at 90mM with minerals highly affected the expression of CBFl and CBF3 apart from type of tissue and reduced the expression of DREB2A, CBF4, and ACT8 in both tissues. OSMl had differential gene expression by different treatments. It showed slightly higher expression in epidermis with cold treatment and its expression was completely diminished in leaves at 90mM of sorbitol with minerals. These results indicate the regulatory role of sugar in cold- induced gene expression of several genes in epidermis which reflects its importance in stress tolerance. Abstract v All in all, using T-DNA can affect plant survival and alter gene expression in ways which can be interpreted as a direct or non-direct link in the overall stress tolerance. These investigations have added new facts to understanding stress tolerance in plants but others remain to be more deeply tested. A novel observation was that the T-DNA in the promoter can increase the regulation of gene expression and stress tolerance and this may be due to stress response elements in the T-DNA. Abstract

The structure of 11S cruciferin has been solved; however, how the individual subunits contribute to its physico-chemical and functional properties are not well known. The cruciferin isoforms in Arabidopsis thaliana, CRUA, CRUB, and CRUC, were investigated with respect to their molecular structures and the relationship of structural features to the physico-chemical and functional properties of cruciferin using homology modeling and various analytical techniques. Comparison of these models revealed that hydrophobicity and electrostatic potential distribution on the surface of the CRUC homotrimer had more favorable interfacial, solubility, and thermal properties than those of CRUA or CRUB. Flavor binding and pepsin digestion were associated with hypervariable regions (HVRs) and center core regions, respectively, moreso for CRUA and CRUB homotrimers than for CRUC. Chemical imaging of a single cell area in wild type (WT) and double-knockout seeds (CRUAbc, CRUaBc, and CRUabC) using synchrotron FT-IR microscopy (amide I band, 1650 cm-1, νC=O) showed that seed storage proteins were concentrated in the cell center and protein storage vacuoles, whereas lipids were closer to the cell wall. Secondary structure components of proteins of double-knockout lines did not show major differences. Changes in protein secondary structure components of pepsin-treated CRUabC (CRUC) mutant were minimal, indicating low enzyme accessibility. A three-step chromatographic procedure allowed isolation of the hexameric form of cruciferin with high purity (>95%). Fourier transform infrared (FT-IR) and circular dichroism (CD) spectroscopic analysis of the secondary structure of these proteins revealed cruciferins were folded into higher order secondary structures; 44−50% β-sheets and 7−9% α-helices. The relative subunit ratio was approximately 1:3:6 (CRUA:CRUB:CRUC) in the WT cruciferin. The Tm values of purified cruciferin at pH 7.4 (μ = 0.0) were in the order of WT = CRUA = CRUB < CRUC. The order of surface hydrophobicity as determined by ANS (1-anilinonaphthalene-8-sulfonate) probe binding was CRUA > CRUB = WT >> CRUC. Intrinsic fluorescence studies revealed a compact molecular structure for the CRUC homohexamer compared to the CRUA and CRUB homohexamers. The order of emulsion forming abilities was CRUA = CRUB > WT > CRUC (no emulsion formation) and the order of heat-induced network structure strength was WT > CRUA = CRUB > CRUC (no gel formation). The inability of CRUC to form gels or emulsions may be attributed to its low surface hydrophobicity and molecular compactness. At pH 2.0, CRUC hexamers dissociated into trimers which allowed the formation of an O/W emulsion and heat-induced network structures. Solubility of cruciferin as a function of pH at low ionic strength gave two minima around pH 4 and 7.4 yielding a “W” shape solubility profile deviating from the typical “U” or “V” shape solubility profile of other 11S globulins. The high ionic strength (μ = 0.5) was not favorable for emulsification, heat-induced gel formation, or solubilization for all cruciferins. Furthermore, the CRUA and CRUB homohexamers exhibited rapid pepsinolysis, while the CRUC homohexamer and WT heterohexamer were digested more slowly. Although fairly well conserved regions were found in the primary structure of these three cruciferin subunits, differences were found in the hypervariable regions and extended loop regions resulting in slight differences in 3D structures and interactions that occur during association to form superstructures, such as hexamers. These differences were reflected in the physico-chemical and techno-functional properties of hexamers and trimers composed of each subunit. In silico predictions for certain functionalities were highly correlated with empirical data from laboratory experiments.