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1
McNevin, John P., Wendy Woodward, Abdelali Hannoufa, Kenneth A. Feldmann, and Bertrand Lemieux. "Isolation and characterization of eceriferum (cer) mutants induced by T-DNA insertions in Arabidopsis thaliana." Genome 36, no. 3 (June 1, 1993): 610–18. http://dx.doi.org/10.1139/g93-082.
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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.
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Yu, I.-ching, Kevin A. Fengler, Steven J. Clough, and Andrew F. Bent. "Identification of Arabidopsis Mutants Exhibiting an Altered Hypersensitive Response in Gene-for-Gene Disease Resistance." Molecular Plant-Microbe Interactions® 13, no. 3 (March 2000): 277–86. http://dx.doi.org/10.1094/mpmi.2000.13.3.277.
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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.
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Procissi, Antonia, Solveig de Laissardière, Madina Férault, Daniel Vezon, Georges Pelletier, and Sandrine Bonhomme. "Five Gametophytic Mutations Affecting Pollen Development and Pollen Tube Growth in Arabidopsis thaliana." Genetics 158, no. 4 (August 1, 2001): 1773–83. http://dx.doi.org/10.1093/genetics/158.4.1773.
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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.
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Lolle, Susan J., Wendy Hsu, and Robert E. Pruitt. "Genetic Analysis of Organ Fusion in Arabidopsis thaliana." Genetics 149, no. 2 (June 1, 1998): 607–19. http://dx.doi.org/10.1093/genetics/149.2.607.
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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.
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Budziszewski, Gregory J., Sharon Potter Lewis, Lyn Wegrich Glover, Jennifer Reineke, Gary Jones, Lisa Schlater Ziemnik, Jennifer Lonowski, et al. "Arabidopsis Genes Essential for Seedling Viability: Isolation of Insertional Mutants and Molecular Cloning." Genetics 159, no. 4 (December 1, 2001): 1765–78. http://dx.doi.org/10.1093/genetics/159.4.1765.
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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.
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Lee, Hyoung Yool, and Kyoungwhan Back. "The phytomelatonin receptor (PMRT1) Arabidopsis Cand2 is not a bona fide G protein–coupled melatonin receptor." Melatonin Research 3, no. 2 (June 1, 2020): 177–86. http://dx.doi.org/10.32794/mr11250055.
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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.
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Shanks, Carly M., J. Hollis Rice, Yan Zubo, G. Eric Schaller, Tarek Hewezi, and Joseph J. Kieber. "The Role of Cytokinin During Infection of Arabidopsis thaliana by the Cyst Nematode Heterodera schachtii." Molecular Plant-Microbe Interactions® 29, no. 1 (January 2016): 57–68. http://dx.doi.org/10.1094/mpmi-07-15-0156-r.
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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.
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Li, Xin, Michael Lassner, and Yuelin Zhang. "Deleteagene: A Fast Neutron Deletion Mutagenesis-Based Gene Knockout System for Plants." Comparative and Functional Genomics 3, no. 2 (2002): 158–60. http://dx.doi.org/10.1002/cfg.148.
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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.
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Dawson, J., Z. A. Wilson, M. G. M. Aarts, A. F. Braithwaite, L. G. Briarty, and B. J. Mulligan. "Microspore and pollen development in six male-sterile mutants of Arabidopsis thaliana." Canadian Journal of Botany 71, no. 4 (April 1, 1993): 629–38. http://dx.doi.org/10.1139/b93-072.
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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.
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Hirschfeld, Matthew, James M. Tepperman, Ted Clack, Peter H. Quail, and Robert A. Sharrock. "Coordination of Phytochrome Levels in phyB Mutants of Arabidopsis as Revealed by Apoprotein-Specific Monoclonal Antibodies." Genetics 149, no. 2 (June 1, 1998): 523–35. http://dx.doi.org/10.1093/genetics/149.2.523.
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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.
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Koizumi, K., M. Sugiyama, and H. Fukuda. "A series of novel mutants of Arabidopsis thaliana that are defective in the formation of continuous vascular network: calling the auxin signal flow canalization hypothesis into question." Development 127, no. 15 (August 1, 2000): 3197–204. http://dx.doi.org/10.1242/dev.127.15.3197.
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For the genetic analysis of molecular mechanisms underlying temporal and spatial regulation of vascular pattern formation, we isolated mutants of Arabidopsis thaliana that are impaired in vascular patterning. Microscopic examination of the cotyledonary venation of 3,400 M(3) lines led to the identification of 12 mutant lines. Genetic analysis of 8 of these mutant lines indicated that vein pattern formation in these lines resulted from monogenic recessive mutations in 7 different genes, designated VAN1 through VAN7. Mutations in VAN1 through VAN6 genes caused fragmentation (disconnection or partial loss) of lateral veins of the cotyledon and tertiary veins of the rosette leaf whereas they were less injurious to the formation of major veins. Detailed characterization of the van3 mutant using pAthb8::GUS and pTED3::GUS, as molecular markers for the early stage of vascular tissue formation showed that the provascular tissue of the cotyledonary lateral veins was differentiated in fragments during late embryogenesis. These phenotypes of the van mutants are discussed in relation to the auxin signal flow canalization hypothesis and the diffusion-reaction prepattern hypothesis, with the fragility of the continuity in the minor vein formation favoring the latter hypothesis.
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Cho, Song Mi, Eun Young Kang, Mi Seong Kim, Seung Jin Yoo, Yang Ju Im, Young Cheol Kim, Kwang Yeol Yang, et al. "Jasmonate-dependent expression of a galactinol synthase gene is involved in priming of systemic fungal resistance in Arabidopsis thaliana." Botany 88, no. 5 (May 2010): 452–61. http://dx.doi.org/10.1139/b10-009.
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Previously, root colonization by the rhizobacterium, Pseudomonas chlororaphis O6, was shown to induce expression of galactinol synthase conferring systemic resistance against a fungal pathogen in cucumber leaves. Here, the Arabidopsis – Botrytis cinerea system is introduced to better understand signal transduction of galactinol and (or) raffinose family oligosaccharides (RFO) during O6-mediated induced systemic resistance (ISR). Among the 10 Arabidopsis galactinol synthase genes, only AtGolS1 was specifically induced upon infection with the fungal pathogen B. cinerea. AtGolS1 was primed by O6 colonization against the pathogen in Arabidopsis leaves. Arabidopsis T-DNA insertion mutants at the AtGolS1 gene site compromised O6-mediated ISR against the pathogen, thereby suggesting that AtGolS1 plays an important role in ISR. O6 colonization increased AtGolS1 transcription as well as ISR in several Arabidopsis signaling mutants, but not in the jar1-1 and coi1 mutant lines. Exogenous jasmonate treatment induced transcription of AtGolS1 in wild-type Col-0 plants, but salicylic acid and 1-aminocyclopropane-1-carboxylate did not. These studies on signaling mutants and target gene expression indicate that expression of AtGolS1 in response to O6 colonization is mediated through the jasmonate-dependent pathway, stimulating ISR in Arabidopsis against B. cinerea infection.
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O'Callaghan, Kenneth J., Richard A. Dixon, and Edward C. Cocking. "Arabidopsis thaliana: a model for studies of colonization by non-pathogenic and plant-growth-promoting rhizobacteria." Functional Plant Biology 28, no. 9 (2001): 975. http://dx.doi.org/10.1071/pp01048.
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This paper originates from an address at the 8th International Symposium on Nitrogen Fixation with Non-Legumes, Sydney, NSW, December 2000 Arabidopsis thaliana L. has many features favoring its use as a model in studies of plant-growth-promoting rhizobacteria (PGPR), such as diazotrophs. Several niches are colonized in the root system of Arabidopsis, including xylem, and intact colonized roots can be observed microscopically without sectioning of tissues. Studies of plant genes involved in colonization are facilitated by the ease with which plants are transformed and by the availability of mutant lines and other accessions obtainable from stock centers. Lines of Arabidopsis carrying reporter gene fusions are helping to reveal the pattern of expression of previously cloned plant genes induced by rhizobacteria. Studies utilizing indole-3-acetic acid (IAA)-producing PGPR and Arabidopsis that contain an auxin-responsive GUS fusion suggest that plants perceive IAA released by bacteria in the rhizosphere. The role of flavonoids in the colonization of non-legumes is being assessed using transgenic Arabidopsis with altered flavonoid metabolism and using tt mutants, which lack functional versions of specific genes for flavonoid metabolism. Studies of plant defence and of stress responses are producing molecular data on plant genes induced by inoculation of Arabidopsis roots with non-pathogens.
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Sánchez-Guerrero, Antonio, Miquel Nadal, Igor Florez-Sarasa, Miquel Ribas-Carbó, José G. Vallarino, Sabrina De Brasi-Velasco, Alisdair R. Fernie, Jaume Flexas, Ana Jiménez, and Francisca Sevilla. "Decreased Levels of Thioredoxin o1 Influences Stomatal Development and Aperture but Not Photosynthesis under Non-Stress and Saline Conditions." International Journal of Molecular Sciences 22, no. 3 (January 21, 2021): 1063. http://dx.doi.org/10.3390/ijms22031063.
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Salinity has a negative impact on plant growth, with photosynthesis being downregulated partially due to osmotic effect and enhanced cellular oxidation. Redox signaling contributes to the plant response playing thioredoxins (TRXs) a central role. In this work we explore the potential contribution of Arabidopsis TRXo1 to the photosynthetic response under salinity analyzing Arabidopsis wild-type (WT) and two Attrxo1 mutant lines in their growth under short photoperiod and higher light intensity than previous reported works. Stomatal development and apertures and the antioxidant, hormonal and metabolic acclimation are also analyzed. In control conditions mutant plants displayed less and larger developed stomata and higher pore size which could underlie their higher stomatal conductance, without being affected in other photosynthetic parameters. Under salinity, all genotypes displayed a general decrease in photosynthesis and the oxidative status in the Attrxo1 mutant lines was altered, with higher levels of H2O2 and NO but also higher ascorbate/glutathione (ASC/GSH) redox states than WT plants. Finally, sugar changes and increases in abscisic acid (ABA) and NO may be involved in the observed higher stomatal response of the TRXo1-altered plants. Therefore, the lack of AtTRXo1 affected stomata development and opening and the mutants modulate their antioxidant, metabolic and hormonal responses to optimize their adaptation to salinity.
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Makandar, Ragiba, Vamsi Nalam, Ratnesh Chaturvedi, Richard Jeannotte, Alexis A. Sparks, and Jyoti Shah. "Involvement of Salicylate and Jasmonate Signaling Pathways in Arabidopsis Interaction with Fusarium graminearum." Molecular Plant-Microbe Interactions® 23, no. 7 (July 2010): 861–70. http://dx.doi.org/10.1094/mpmi-23-7-0861.
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Fusarium graminearum is the principal causative agent of Fusarium head blight (FHB), a devastating disease of wheat and barley. This fungus can also colonize Arabidopsis thaliana. Disease resistance was enhanced in transgenic wheat and Arabidopsis plants that constitutively overexpress the NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1) gene, which regulates salicylic acid (SA) signaling and modulates the activation of jasmonic acid (JA)-dependent defenses. Here, we provide several lines of evidence that reveal an important role for SA and JA signaling in Arabidopsis defense against F. graminearum. SA level was elevated in fungus-inoculated leaves, and SA application and biologically activated systemic acquired resistance enhanced resistance. Furthermore, the disruption of SA accumulation and signaling in the sid2 mutant and NahG transgenic plant, and the npr1 and wrky18 mutants, respectively, resulted in heightened susceptibility to this fungus in leaves and inflorescence. JA signaling was activated in parallel with SA signaling in the fungus-challenged plants. However, the hyperresistance of the JA pathway mutants opr3, coi1, and jar1 indicates that this pathway contributes to susceptibility. Genetic and biochemical experiments indicate that the JA pathway promotes disease by attenuating the activation of SA signaling in fungus-inoculated plants. However, the hypersusceptibility of the jar1 npr1 double mutant compared with the npr1 mutant suggests that JAR1 also contributes to defense, signifying a dichotomous role of JA and a JAR1-dependent mechanism in this interaction.
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Jia, Qi, Paul Bundock, Paul J. J. Hooykaas, and Sylvia de Pater. "Agrobacterium tumefaciens T-DNA Integration and Gene Targeting in Arabidopsis thaliana Non-Homologous End-Joining Mutants." Journal of Botany 2012 (February 29, 2012): 1–13. http://dx.doi.org/10.1155/2012/989272.
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In order to study the role of AtKu70 and AtKu80 in Agrobacterium-mediated transformation and gene targeting, plant lines with a T-DNA insertion in AtKu80 or AtKu70 genes were functionally characterized. Such plant lines lacked both subunits, indicating that heterodimer formation between AtKu70 and AtKu80 is needed for the stability of the proteins. Homozygous mutants were phenotypically indistinguishable from wild-type plants and were fertile. However, they were hypersensitive to the genotoxic agent bleomycin, resulting in more DSBs as quantified in comet assays. They had lower end-joining efficiency, suggesting that NHEJ is a critical pathway for DSB repair in plants. Both Atku mutants and a previously isolated Atmre11 mutant were impaired in Agrobacterium T-DNA integration via floral dip transformation, indicating that AtKu70, AtKu80, and AtMre11 play an important role in T-DNA integration in Arabidopsis. The frequency of gene targeting was not significantly increased in the Atku80 and Atku70 mutants, but it was increased at least 10-fold in the Atmre11 mutant compared with the wild type.
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Jürgens, Gerd, Ulrike Mayer, Torres Ruiz Ramon A., Thomas Berleth, and Simon Miséra. "Genetic analysis of pattern formation in the Arabidopsis embryo." Development 113, Supplement_1 (January 1, 1991): 27–38. http://dx.doi.org/10.1242/dev.113.supplement_1.27.
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Virtually nothing is known about the mechanisms that generate the basic body pattern in plant embryogenesis. As a first step towards the analysis of pattern formation, we have isolated and begun to characterise putative pattern mutants in the flowering plant, Arabidopsis thaliana. A large-scale screen for morphologically abnormal seedling mutants yielded about 250 lines for further study, and genetic evidence suggests saturation of the genome for this kind of mutation. The phenotypes of putative pattern mutants fall into distinct categories, classes and groups, which may reflect specific aspects of embryonic pattern formation. Mutant seedling phenotypes result from abnormal development in the early embryo. The implications of our findings are discussed with regard to the prospects for a mechanistic understanding of pattern formation in the plant embryo.
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Howden, Ross, Soon Ki Park, James M. Moore, James Orme, Ueli Grossniklaus, and David Twell. "Selection of T-DNA-Tagged Male and Female Gametophytic Mutants by Segregation Distortion in Arabidopsis." Genetics 149, no. 2 (June 1, 1998): 621–31. http://dx.doi.org/10.1093/genetics/149.2.621.
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Abstract As a strategy for the identification of T-DNA-tagged gametophytic mutants, we have used T-DNA insertional mutagenesis based on screening for distorted segregation ratios by antibiotic selection. Screening of ~1000 transgenic Arabidopsis families led to the isolation of eight lines showing reproducible segregation ratios of ~1:1, suggesting that these lines are putative gametophytic mutants caused by T-DNA insertion at a single locus. Genetic analysis of T-DNA transmission through reciprocal backcrosses with wild type showed severe reductions in genetic transmission of the T-DNA through the male and/or female gametes. Direct evidence for mutant phenotypes in these lines was investigated by DAPI staining of mature pollen grains and by the analysis of seed set and embryo sac morphology in cleared ovules. One line, termed limpet pollen, showed a novel pollen phenotype in that the generative cell failed to migrate inward after pollen mitosis I, such that the generative or sperm cells remained against the pollen wall. Two other lines, andarta and tistrya, were defective in female transmission and showed an early arrest of embryo sac development with the viable megaspore not initiating the nuclear division cycles. These data demonstrate the efficacy of a segregation ratio distortion strategy for the identification of T-DNA-tagged gametophytic mutants in Arabidopsis.
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Veeragoni, Sravani Ram, Birgit Lange, Mario Serrano, Christiane Nawrath, Sibylle Bauer, Anton Rudolf Schäffner, Hans Thordal-Christensen, Jörg Durner, and Frank Gaupels. "Mutant Muddle: Some Arabidopsis eds5 Mutant Lines Have a Previously Unnoticed Second-Site Mutation in FAH1." Plant Physiology 182, no. 1 (November 4, 2019): 460–62. http://dx.doi.org/10.1104/pp.19.01125.
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Li, Zhilan, Yuxiao Jiang, Shuijin Hua, Yun Ren, Chiyu Jiang, Longhua Zhou, Xiaoyang Chen, and Lixi Jiang. "Characterization of seed fatty acid accumulation in DELLA mutant lines of Arabidopsis." Plant Growth Regulation 70, no. 1 (December 8, 2012): 27–37. http://dx.doi.org/10.1007/s10725-012-9775-2.
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Berná, Genoveva, Pedro Robles, and José Luis Micol. "A Mutational Analysis of Leaf Morphogenesis in Arabidopsis thaliana." Genetics 152, no. 2 (June 1, 1999): 729–42. http://dx.doi.org/10.1093/genetics/152.2.729.
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Abstract As a contribution to a better understanding of the developmental processes that are specific to plants, we have begun a genetic analysis of leaf ontogeny in the model system Arabidopsis thaliana by performing a large-scale screening for mutants with abnormal leaves. After screening 46,159 M2 individuals, arising from 5770 M1 parental seeds exposed to EMS, we isolated 1926 M2 putative leaf mutants, 853 of which yielded viable M3 inbred progeny. Mutant phenotypes were transmitted with complete penetrance and small variations in expressivity in 255 lines. Most of them were inherited as recessive monogenic traits, belonging to 94 complementation groups, which suggests that we did not reach saturation of the genome. We discuss the nature of the processes presumably perturbed in the phenotypic classes defined among our mutants.
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Richardson, Kim, Sarah Fowler, Carly Pullen, Caryl Skelton, Bret Morris, and Jo Putterill. "T-DNA tagging of a flowering-time gene and improved gene transfer by in planta transformation of Arabidopsis." Functional Plant Biology 25, no. 1 (1998): 125. http://dx.doi.org/10.1071/pp97109.
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Gene tagging with insertional mutagens greatly facilitates the isolation of novel genes. A new collection of Arabidopsis T-DNA tag insertion lines (n=2165) was generated by in planta transformation. Whole plants were vacuum-infiltrated in a suspension of Agrobacterium carrying the pGKB5 tagging vector. The efficiency of transformation increased with addition of the surfactant Silwet L-77 (0.005% v/v) to the Agrobacterium suspension. Visual screens of the T-DNA lines identified two mutants with floral defects. Allelism tests suggested that a mutation in the GIGANTEA gene was responsible for the late-flowering phenotype of one of the mutants. Linkage analysis indicated that the GIGANTEA gene was tagged in this mutant.
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Hallett, Rebecca H., Heather Ray, Jennifer Holowachuk, Juliana J. Soroka, and Margaret Y. Gruber. "Bioassay for assessing resistance of Arabidopsis thaliana L. (Heynh.) to the adult crucifer flea beetle, Phyllotreta cruciferae (Goeze) (Coleoptera: Chrysomelidae)." Canadian Journal of Plant Science 85, no. 1 (January 1, 2005): 225–35. http://dx.doi.org/10.4141/p03-122.
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A bioassay arena and a laboratory screening protocol were developed for assessing lines of Arabidopsis thaliana L. (Heynh.) for feeding damage by the adult crucifer flea beetle, Phyllotreta cruciferae (Goeze). The arena consists of a 96-well microtitre plate with a modified top to contain flea beetles and allow ventilation. Eight lines of A. thaliana, arranged in an 8 × 8 Latin square design, were screened simultaneously in each arena using 50 starved flea beetles. Two cotyledons and the first pair of true leaves per plant were rated visually under a dissecting microscope using a visual damage rating scale. The protocol was used to screen 29 wild ecotypes, eight mutant lines and a single transgenic line of A. thaliana. Discrimination between both cotyledon and leaf tissue was apparent for young beetles that were both non-reproductive or reproductive, but not for old reproductive beetles. Differences were observed between Asian and European ecotypes of A. thaliana, suggesting that geographic origin may play a role in susceptibility of Arabidopsis ecotypes to flea beetle feeding. The transparent testa regulatory gene mutants (lines 82, 111, 164) were most susceptible to flea beetle feeding, possibly indicating a role for anthocyanins and/or flavonoids in governing flea beetle susceptibility. Significant variation in damage levels indicates that expression of flea beetle resistance in the A rabidopsis genome is plastic, and that potential exists to use the wide array of publicly available Arabidopsis germplasm as tools in the transfer of resistance to agronomically important host plants. Key words: Seedling bioassay, Arabidopsis thaliana, wild ecotypes and mutants, crucifer flea beetle, Phyllotreta cruciferae, host plant resistance
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Fujisaki, Koki, Gerald B. Ravelo, Satoshi Naito, and Masayuki Ishikawa. "Involvement of THH1, an Arabidopsis thaliana homologue of the TOM1 gene, in tobamovirus multiplication." Journal of General Virology 87, no. 8 (August 1, 2006): 2397–401. http://dx.doi.org/10.1099/vir.0.81942-0.
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The TOM1 and TOM3 genes of Arabidopsis thaliana encode homologous proteins that are required for tobamovirus multiplication. Although the A. thaliana genome encodes another TOM1-like gene, THH1, the tobamovirus coat protein (CP) does not accumulate to a detectable level in the tom1 tom3 double mutant. Here, double and triple mutants of tom1, tom3 and thh1 were generated to investigate whether THH1 functions to support tobamovirus multiplication. In the tom1 thh1 double mutant, the tobamovirus CP accumulated to a level that was detectable, but lower than that in the tom1 single mutant. In tom1 tom3 double-mutant lines overexpressing THH1, the tobamovirus CP accumulated to a level similar to that observed in wild-type plants. These results suggest that THH1 supports tobamovirus multiplication, but to a lesser extent than TOM1 and TOM3. The expression level of THH1 is lower than that of TOM1 and TOM3, which might explain the smaller contribution of THH1 to tobamovirus multiplication.
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Martinelli, Laurie. "Australian Journal of Plant Physiology: Editorial report." Functional Plant Biology 25, no. 1 (1998): I. http://dx.doi.org/10.1071/ppv25n1_ed.
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Gene tagging with insertional mutagens greatly facilitates the isolation of novel genes. A new collection of Arabidopsis T-DNA tag insertion lines (n=2165) was generated by in planta transformation. Whole plants were vacuum-infiltrated in a suspension of Agrobacterium carrying the pGKB5 tagging vector. The efficiency of transformation increased with addition of the surfactant Silwet L-77 (0.005% v/v) to the Agrobacterium suspension. Visual screens of the T-DNA lines identified two mutants with floral defects. Allelism tests suggested that a mutation in the GIGANTEA gene was responsible for the late-flowering phenotype of one of the mutants. Linkage analysis indicated that the GIGANTEA gene was tagged in this mutant.
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Dzianott, Aleksandra, Joanna Sztuba-Solińska, and Jozef J. Bujarski. "Mutations in the Antiviral RNAi Defense Pathway Modify Brome mosaic virus RNA Recombinant Profiles." Molecular Plant-Microbe Interactions® 25, no. 1 (January 2012): 97–106. http://dx.doi.org/10.1094/mpmi-05-11-0137.
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RNA interference (RNAi) mechanism targets viral RNA for degradation. To test whether RNAi gene products contributed to viral RNA recombination, a series of Arabidopsis thaliana RNAi-defective mutants were infected with Brome mosaic virus (BMV) RNAs that have been engineered to support crossovers within the RNA3 segment. Single-cross RNA3-RNA1, RNA3-RNA2, and RNA3-RNA3 recombinants accumulated in both the wild-type (wt) and all knock-out lines at comparable frequencies. However, a reduced accumulation of novel 3′ mosaic RNA3 recombinants was observed in ago1, dcl2, dcl4, and rdr6 lines but not in wt Col-0 or the dcl3 line. A BMV replicase mutant accumulated a low level of RNA3-RNA1 single-cross recombinants in Col-0 plants while, in a dcl2 dcl4 double mutant, the formation of both RNA3-RNA1 and mosaic recombinants was at a low level. A control infection in the cpr5-2 mutant, a more susceptible BMV Arabidopsis host, generated similar-to-Col-0 profiles of both single-cross and mosaic recombinants, indicating that recombinant profiles were, to some extent, independent of a viral replication rate. Also, the relative growth experiments revealed similar selection pressure for recombinants among the host lines. Thus, the altered recombinant RNA profiles have originated at the level of recombinant formation rather than because of altered selection. In conclusion, the viral replicase and the host RNAi gene products contribute in distinct ways to BMV RNA recombination. Our studies reveal that the antiviral RNAi mechanisms are utilized by plant RNA viruses to increase their variability, reminiscent of phenomena previously demonstrated in fungi.
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Chinchilla, Delphine, Florian Frugier, Marcela Raices, Francisco Merchan, Veronica Giammaria, Pablo Gargantini, Silvina Gonzalez-Rizzo, Martin Crespi, and Rita Ulloa. "A mutant ankyrin protein kinase from Medicago sativa affects Arabidopsis adventitious roots." Functional Plant Biology 35, no. 1 (2008): 92. http://dx.doi.org/10.1071/fp07209.
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A family of plant kinases containing ankyrin-repeats, the Ankyrin-Protein Kinases (APKs), shows structural resemblance to mammalian Integrin-Linked Kinases (ILKs), key regulators of mammalian cell adhesion. MsAPK1 expression is induced by osmotic stress in roots of Medicago sativa (L.) plants. The Escherichia coli-purified MsAPK1 could only phosphorylate tubulin among a variety of substrates and the enzymatic activity was strictly dependent on Mn2+. MsAPK1 is highly related to two APK genes in Arabidopsis thaliana (L.), AtAPK1 and AtAPK2. Promoter-GUS fusions assays revealed that the Arabidopsis APK genes show distinct expression patterns in roots and hypocotyls. Although Medicago truncatula (L.) plants affected in MsAPK1 expression could not be obtained using in vitro regeneration, A. thaliana plants expressing MsAPK1 or a mutant MsAPK1 protein, in which the conserved aspartate 315 of the kinase catalytic domain was replaced by asparagines (DN-lines), developed normally. The DN mutant lines showed increased capacity to develop adventitious roots when compared with control or MsAPK1-expressing plants. APK-mediated signalling may therefore link perception of external abiotic signals and the microtubule cytoskeleton, and influence adventitious root development.
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Bagnall, David J., and Rod W. King. "Phytochrome, photosynthesis and flowering of Arabidopsis thaliana: photophysiological studies using mutants and transgenic lines." Functional Plant Biology 28, no. 5 (2001): 401. http://dx.doi.org/10.1071/pp99123.
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A number of phytochrome mutants have been examined for involvement in high irradiance (HIR) or red/far-red (R/FR) end-of-day (EOD) photoresponses during flowering of the long-day (LD) plant, Arabidopsis thaliana (L.) Heynh. A large component of phytochrome A (phyA) response is shown to involve an indirect effect via photosynthesis. When grown autotrophically in soil at a low irradiance (80 mol m–2 s–1), the phyA-211 mutant flowered extremely late compared with wild type and its leaf area was halved, both effects being reversed by increase in photosynthetic irradiance. Supplying sucrose via agar led to very early flowering with little indication of an additional direct phyA HIR. For light-stable phytochrome apoprotein mutants (phyB, phyD) or chromophore mutants (hy1, hy2), flowering was early and R/FR photoreversible EOD response was erased. Conversely, flowering was delayed in a transgenic line overexpressing the PHYB apoprotein. The FR EOD promotion of flowering via phyB was retained in darkness, brief night interruptions mimicking LD response. This novel finding emphasizes the importance of phyB-like phytochromes, with phyA acting indirectly. Whether phyB influences time measurement remains uncertain as we found no rhythmicity in this response to night interruptions. Overall, the role(s) of phytochromes in the regulation of flowering of Arabidopsis include EOD phyB-type response, a minor phyA photoperiodic response, and a large indirect phyA effect involving photosynthesis.
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Zou, Baohong, Zhenhua Jia, Shuangmei Tian, Xiaomeng Wang, Zhenhua Gou, Beibei Lü, and Hansong Dong. "AtMYB44 positively modulates disease resistance to Pseudomonas syringae through the salicylic acid signalling pathway in Arabidopsis." Functional Plant Biology 40, no. 3 (2013): 304. http://dx.doi.org/10.1071/fp12253.
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Plant MYB transcription factors are implicated in resistance to biotic and abiotic stresses. Here, we demonstrate that an R2-R3 MYB transcription factor, AtMYB44, plays a role in the plant defence response to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (PstDC3000). The expression of AtMYB44 was upregulated upon pathogen infection and treatments with defence-related phytohormones. Transgenic plants overexpressing AtMYB44 (35S-Ms) exhibited greater levels of PR1 gene expression, cell death, callose deposition and hydrogen peroxide (H2O2) accumulation in leaves infected with PstDC3000. Consequently, 35S-M lines displayed enhanced resistance to PstDC3000. In contrast, the atmyb44 T-DNA insertion mutant was more susceptible to PstDC3000 and exhibited decreased PR1 gene expression upon infection. Using double mutants constructed via crosses of 35S-M lines with NahG transgenic plants and nonexpressor of pathogenesis-related genes1 mutant (npr1–1), we demonstrated that the enhanced PR1 gene expression and PstDC3000 resistance in 35S-M plants occur mainly through the salicylic acid signalling pathway.
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McElver, John, Iris Tzafrir, George Aux, Rebecca Rogers, Carl Ashby, Kelsey Smith, Carla Thomas, et al. "Insertional Mutagenesis of Genes Required for Seed Development inArabidopsis thaliana." Genetics 159, no. 4 (December 1, 2001): 1751–63. http://dx.doi.org/10.1093/genetics/159.4.1751.
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AbstractThe purpose of this project was to identify large numbers of Arabidopsis genes with essential functions during seed development. More than 120,000 T-DNA insertion lines were generated following Agrobacterium-mediated transformation. Transgenic plants were screened for defective seeds and putative mutants were subjected to detailed analysis in subsequent generations. Plasmid rescue and TAIL-PCR were used to recover plant sequences flanking insertion sites in tagged mutants. More than 4200 mutants with a wide range of seed phenotypes were identified. Over 1700 of these mutants were analyzed in detail. The 350 tagged embryo-defective (emb) mutants identified to date represent a significant advance toward saturation mutagenesis of EMB genes in Arabidopsis. Plant sequences adjacent to T-DNA borders in mutants with confirmed insertion sites were used to map genome locations and establish tentative identities for 167 EMB genes with diverse biological functions. The frequency of duplicate mutant alleles recovered is consistent with a relatively small number of essential (EMB) genes with nonredundant functions during seed development. Other functions critical to seed development in Arabidopsis may be protected from deleterious mutations by extensive genome duplications.
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Fonseca, Jose Pedro, Sunhee Oh, Clarissa Boschiero, Bonnie Watson, David Huhman, and Kirankumar S. Mysore. "The Arabidopsis Iron-Sulfur (Fe-S) Cluster Gene MFDX1 Plays a Role in Host and Nonhost Disease Resistance by Accumulation of Defense-Related Metabolites." International Journal of Molecular Sciences 22, no. 13 (July 1, 2021): 7147. http://dx.doi.org/10.3390/ijms22137147.
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Until recently, genes from the iron-sulfur (Fe-S) cluster pathway were not known to have a role in plant disease resistance. The Nitrogen Fixation S (NIFS)-like 1 (NFS1) and Mitochondrial Ferredoxin-1 (MFDX1) genes are part of a set of 27 Fe-S cluster genes induced after infection with host and nonhost pathogens in Arabidopsis. A role for AtNFS1 in plant immunity was recently demonstrated. In this work, we showed that MFDX1 is also involved in plant defense. More specifically, Arabidopsis mfdx1 mutants were compromised for nonhost resistance against Pseudomonas syringae pv. tabaci, and showed increased susceptibility to the host pathogen P. syringae pv. tomato DC3000. Arabidopsis AtMFDX1 overexpression lines were less susceptible to P. syringae pv. tomato DC3000. Metabolic profiling revealed a reduction of several defense-related primary and secondary metabolites, such as asparagine and glucosinolates in the Arabidopsis mfdx1-1 mutant when compared to Col-0. A reduction of 5-oxoproline and ornithine metabolites that are involved in proline synthesis in mitochondria and affect abiotic stresses was also observed in the mfdx1-1 mutant. In contrast, an accumulation of defense-related metabolites such as glucosinolates was observed in the Arabidopsis NFS1 overexpressor when compared to wild-type Col-0. Additionally, mfdx1-1 plants displayed shorter primary root length and reduced number of lateral roots compared to the Col-0. Taken together, these results provide additional evidence for a new role of Fe-S cluster pathway in plant defense responses.
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Maleck, Klaus, Urs Neuenschwander, Rebecca M. Cade, Robert A. Dietrich, Jeffery L. Dangl, and John A. Ryals. "Isolation and Characterization of Broad-Spectrum Disease-Resistant Arabidopsis Mutants." Genetics 160, no. 4 (April 1, 2002): 1661–71. http://dx.doi.org/10.1093/genetics/160.4.1661.
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Abstract To identify Arabidopsis mutants that constitutively express systemic acquired resistance (SAR), we constructed reporter lines expressing the firefly luciferase gene under the control of the SAR-inducible PR-1 promoter (PR-1/luc). After EMS mutagenesis of a well-characterized transgenic line, we screened 250,000 M2 plants for constitutive expression of the reporter gene in vivo. From a mutant collection containing several hundred putative mutants, we concentrated on 16 mutants lacking spontaneous hypersensitive response (HR) cell death. We mapped 4 of these constitutive immunity (cim) mutants to chromosome arms. Constitutive expression of disease resistance was established by analyzing responses to virulent Peronospora parasitica and Pseudomonas syringae strains, by RNA blot analysis for endogenous marker genes, and by determination of salicylic acid levels in the mutants. The variety of the cim phenotypes allowed us to define distinct steps in both the canonical SAR signaling pathway and a separate pathway for resistance to Erysiphe cichoracearum, active in only a subset of the mutants.
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Jacques, Florian, Yingjuan Zhao, Martina Kopečná, Radka Končitíková, David Kopečný, Sonia Rippa, and Yolande Perrin. "Roles for ALDH10 enzymes in γ-butyrobetaine synthesis, seed development, germination, and salt tolerance in Arabidopsis." Journal of Experimental Botany 71, no. 22 (August 26, 2020): 7088–102. http://dx.doi.org/10.1093/jxb/eraa394.
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Abstract Plant genomes generally contain two aldehyde dehydrogenase 10 (ALDH10) genes, which encode NAD+-dependent enzymes. These oxidize various aminoaldehydes that are produced by the catabolism of amino acids and polyamines. ALDH10s are closely related to the animal and fungal trimethylaminobutyraldehyde dehydrogenases (TMABADHs) that are involved in the synthesis of γ-butyrobetaine, the precursor of carnitine. Here, we explore the ability of the Arabidopsis thaliana proteins AtALDH10A8 and AtALDH10A9 to oxidize aminoaldehydes. We demonstrate that these enzymes display high TMABADH activities in vitro. Moreover, they can complement the Candida albicans tmabadhΔ/Δ null mutant. These findings illustrate the link between AtALDH10A8 and AtALDH10A9 and γ-butyrobetaine synthesis. An analysis of single and double knockout Arabidopsis mutant lines revealed that the double mutants had reduced γ-butyrobetaine levels. However, there were no changes in the carnitine contents of these mutants. The double mutants were more sensitive to salt stress. In addition, the siliques of the double mutants had a significant proportion of seeds that failed to mature. The mature seeds contained higher amounts of triacylglycerol, facilitating accelerated germination. Taken together, these results show that ALDH10 enzymes are involved in γ-butyrobetaine synthesis. Furthermore, γ-butyrobetaine fulfils a range of physiological roles in addition to those related to carnitine biosynthesis.
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Dzikiewicz-Krawczyk, Agnieszka, Paulina Piontek, Zofia Szweykowska-Kulińska, and Artur Jarmołowski. "The nuclear cap-binding protein complex is not essential for nonsense-mediated mRNA decay (NMD) in plants." Acta Biochimica Polonica 55, no. 4 (December 16, 2008): 825–28. http://dx.doi.org/10.18388/abp.2008_3047.
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In this study we investigated whether in plants, like in mammals, components of the nuclear cap-binding protein complex (CBC) are involved in nonsense-mediated mRNA decay (NMD). We selected several genes producing at least two alternatively spliced mRNA variants: one with a premature termination codon (PTC+) and another without it (PTC-). For each gene the PTC+/PTC- ratio was calculated using RT-PCR and direct sequencing in four Arabidopsis thaliana lines: wild type, the NMD mutant atupf3-1 and two CBC mutants: cbp20 and abh1. Whereas in the NMD mutant the ratios of PTC+/PTC- splice variants were higher than in wild-type plants, the two CBC mutants investigated showed no change in the PTC+/PTC- ratios. Our results suggest that neither CBP20 nor CBP80 is involved in NMD in A. thaliana.
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Panchuk, I. I., I. M. Buzduga, and R. A. Volkov. "Total reducing capacity in Arabidopsis thaliana cat2cat3 knockout mutants under heat stress." Visnik ukrains'kogo tovaristva genetikiv i selekcioneriv 15, no. 1 (October 1, 2017): 47–51. http://dx.doi.org/10.7124/visnyk.utgis.15.1.710.
Full textAbstract:
Aim. It was investigated whether the simultaneous loss of the two catalase isoforms CAT2 and CAT3 can be compensated by the increase in content of low-molecular weight antioxidants. To clarify this question, the total reducing capacity in Arabidopsis wild type and cat2cat3 knockout mutants was evaluated under optimal growth conditions and after heat stress. Methods. Leaves of Arabidopsis thaliana wild type and cat2cat3 knockout mutants were exposed to high temperatures. The content of water-soluble low molecular weight antioxidants was evaluated by determining the total reducing capacity using iodometry. Results. In intact cat2cat3 mutants there is an 1.7 times increase in the content of low-molecular weight antioxidants compared to wild type plants. A high content of these compounds in knockout plants was also observed upon heat stress. Patterns of changes in total reducing capacity differ between wild type and knockout lines. Conclusion. The loss of activity of the catalase isoforms CAT2 and CAT3 in knock-out mutants of Arabidopsis results in activation of non-enzymatic antioxidant defenses. The increase of the content of low-molecular weight antioxidants is one of the mechanisms that provide protection of mutant plants from chronic oxidative stress, both under optimal cultivation conditions and under the influence of elevated temperatures.Keywords: multigenic family, heat shock, total reducing capacity, knockout mutants, Arabidopsis thaliana.
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Kong, Lingfei, Zeyu Li, Qin Song, Xiaohong Li, and Keming Luo. "Construction of a Full-Length cDNA Over-Expressing Library to Identify Valuable Genes from Populus tomentosa." International Journal of Molecular Sciences 22, no. 7 (March 26, 2021): 3448. http://dx.doi.org/10.3390/ijms22073448.
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Poplar wood is the main source of renewable biomass energy worldwide, and is also considered to be a model system for studying woody plants. The Full-length cDNA Over-eXpressing (FOX) gene hunting system is an effective method for generating gain-of-function mutants. Large numbers of novel genes have successfully been identified from many herbaceous plants according to the phenotype of gain-of-function mutants under normal or abiotic stress conditions using this system. However, the system has not been used for functional gene identification with high-throughput mutant screening in woody plants. In this study, we constructed a FOX library from the Chinese white poplar, Populus tomentosa. The poplar cDNA library was constructed into the plant expression vector pEarleyGate101 and further transformed into Arabidopsis thaliana (thale cress). We collected 1749 T1 transgenic plants identified by PCR. Of these, 593 single PCR bands from different transgenic lines were randomly selected for sequencing, and 402 diverse sequences of poplar genes were isolated. Most of these genes were involved in photosynthesis, environmental adaptation, and ribosome biogenesis based on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation. We characterized in detail two mutant lines carrying PtoCPCa or PtoWRKY13 cDNA insertions. Phenotypic characterization showed that overexpression of these genes in A. thaliana affected trichome development or secondary cell wall (SCW) deposition, respectively. Together, the Populus-FOX-Arabidopsis library generated in our experiments will be helpful for efficient discovery of novel genes in poplar.
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Zhai, Zhiyang, Hui Liu, and John Shanklin. "Ectopic Expression of OLEOSIN 1 and Inactivation of GBSS1 Have a Synergistic Effect on Oil Accumulation in Plant Leaves." Plants 10, no. 3 (March 9, 2021): 513. http://dx.doi.org/10.3390/plants10030513.
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During the transformation of wild-type (WT) Arabidopsis thaliana, a T-DNA containing OLEOSIN-GFP (OLE1-GFP) was inserted by happenstance within the GBSS1 gene, resulting in significant reduction in amylose and increase in leaf oil content in the transgenic line (OG). The synergistic effect on oil accumulation of combining gbss1 with the expression of OLE1-GFP was confirmed by transforming an independent gbss1 mutant (GABI_914G01) with OLE1-GFP. The resulting OLE1-GFP/gbss1 transgenic lines showed higher leaf oil content than the individual OLE1-GFP/WT or single gbss1 mutant lines. Further stacking of the lipogenic factors WRINKLED1, Diacylglycerol O-Acyltransferase (DGAT1), and Cys-OLEOSIN1 (an engineered sesame OLEOSIN1) in OG significantly elevated its oil content in mature leaves to 2.3% of dry weight, which is 15 times higher than that in WT Arabidopsis. Inducible expression of the same lipogenic factors was shown to be an effective strategy for triacylglycerol (TAG) accumulation without incurring growth, development, and yield penalties.
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Yan, Xingxing, Ying Huang, Hui Song, Feng Chen, Qingliu Geng, Min Hu, Cheng Zhang, Xi Wu, Tingting Fan, and Shuqing Cao. "A MYB4-MAN3-Mannose-MNB1 signaling cascade regulates cadmium tolerance in Arabidopsis." PLOS Genetics 17, no. 6 (June 28, 2021): e1009636. http://dx.doi.org/10.1371/journal.pgen.1009636.
Full textAbstract:
Our previous studies showed that MAN3-mediated mannose plays an important role in plant responses to cadmium (Cd) stress. However, the underlying mechanisms and signaling pathways involved are poorly understood. In this study, we showed that an Arabidopsis MYB4-MAN3-Mannose-MNB1 signaling cascade is involved in the regulation of plant Cd tolerance. Loss-of-function of MNB1 (mannose-binding-lectin 1) led to decreased Cd accumulation and tolerance, whereas overexpression of MNB1 significantly enhanced Cd accumulation and tolerance. Consistently, expression of the genes involved in the GSH-dependent phytochelatin (PC) synthesis pathway (such as GSH1, GSH2, PCS1, and PCS2) was significantly reduced in the mnb1 mutants but markedly increased in the MNB1-OE lines in the absence or presence of Cd stress, which was positively correlated with Cd-activated PC synthesis. Moreover, we found that mannose is able to bind to the GNA-related domain of MNB1, and that mannose binding to the GNA-related domain of MNB1 is required for MAN3-mediated Cd tolerance in Arabidopsis. Further analysis showed that MYB4 directly binds to the promoter of MAN3 to positively regulate the transcript of MAN3 and thus Cd tolerance via the GSH-dependent PC synthesis pathway. Consistent with these findings, overexpression of MAN3 rescued the Cd-sensitive phenotype of the myb4 mutant but not the mnb1 mutant, whereas overexpression of MNB1 rescued the Cd-sensitive phenotype of the myb4 mutant. Taken together, our results provide compelling evidence that a MYB4-MAN3-Mannose-MNB1 signaling cascade regulates cadmium tolerance in Arabidopsis through the GSH-dependent PC synthesis pathway.
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Kawamoto, Yudai, Hirotaka Toda, Hiroshi Inoue, Kappei Kobayashi, Naoto Yamaoka, Takuya Araki, and Takashi Yaeno. "Fast and Inexpensive Phenotyping and Genotyping Methods for Evaluation of Barley Mutant Population." Plants 9, no. 9 (September 6, 2020): 1153. http://dx.doi.org/10.3390/plants9091153.
Full textAbstract:
To further develop barley breeding and genetics, more information on gene functions based on the analysis of the mutants of each gene is needed. However, the mutant resources are not as well developed as the model plants, such as Arabidopsis and rice. Although genome editing techniques have been able to generate mutants, it is not yet an effective method as it can only be used to transform a limited number of cultivars. Here, we developed a mutant population using ‘Mannenboshi’, which produces good quality grains with high yields but is susceptible to disease, to establish a Targeting Induced Local Lesions IN Genomes (TILLING) system that can isolate mutants in a high-throughput manner. To evaluate the availability of the prepared 8043 M3 lines, we investigated the frequency of mutant occurrence using a rapid, visually detectable waxy phenotype as an indicator. Four mutants were isolated and single nucleotide polymorphisms (SNPs) were identified in the Waxy gene as novel alleles. It was confirmed that the mutations could be easily detected using the mismatch endonuclease CELI, revealing that a sufficient number of mutants could be rapidly isolated from our TILLING population.
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Zdanio, Malgorzata, Agnieszka Karolina Boron, Daria Balcerowicz, Sébastjen Schoenaers, Marios Nektarios Markakis, Grégory Mouille, Isabel Pintelon, et al. "The Proline-Rich Family Protein EXTENSIN33 Is Required for Etiolated Arabidopsis thaliana Hypocotyl Growth." Plant and Cell Physiology 61, no. 6 (April 25, 2020): 1191–203. http://dx.doi.org/10.1093/pcp/pcaa049.
Full textAbstract:
Abstract Growth of etiolated Arabidopsis hypocotyls is biphasic. During the first phase, cells elongate slowly and synchronously. At 48 h after imbibition, cells at the hypocotyl base accelerate their growth. Subsequently, this rapid elongation propagates through the hypocotyl from base to top. It is largely unclear what regulates the switch from slow to fast elongation. Reverse genetics-based screening for hypocotyl phenotypes identified three independent mutant lines of At1g70990, a short extensin (EXT) family protein that we named EXT33, with shorter etiolated hypocotyls during the slow elongation phase. However, at 72 h after imbibition, these dark-grown mutant hypocotyls start to elongate faster than the wild type (WT). As a result, fully mature 8-day-old dark-grown hypocotyls were significantly longer than WTs. Mutant roots showed no growth phenotype. In line with these results, analysis of native promoter-driven transcriptional fusion lines revealed that, in dark-grown hypocotyls, expression occurred in the epidermis and cortex and that it was strongest in the growing part. Confocal and spinning disk microscopy on C-terminal protein-GFP fusion lines localized the EXT33-protein to the ER and cell wall. Fourier-transform infrared microspectroscopy identified subtle changes in cell wall composition between WT and the mutant, reflecting altered cell wall biomechanics measured by constant load extensometry. Our results indicate that the EXT33 short EXT family protein is required during the first phase of dark-grown hypocotyl elongation and that it regulates the moment and extent of the growth acceleration by modulating cell wall extensibility.
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Pegler, Joseph L., Jackson M. J. Oultram, Duc Quan Nguyen, Christopher P. L. Grof, and Andrew L. Eamens. "MicroRNA-Mediated Responses to Cadmium Stress in Arabidopsis thaliana." Plants 10, no. 1 (January 10, 2021): 130. http://dx.doi.org/10.3390/plants10010130.
Full textAbstract:
In recent decades, the presence of cadmium (Cd) in the environment has increased significantly due to anthropogenic activities. Cd is taken up from the soil by plant roots for its subsequent translocation to shoots. However, Cd is a non-essential heavy metal and is therefore toxic to plants when it over-accumulates. MicroRNA (miRNA)-directed gene expression regulation is central to the response of a plant to Cd stress. Here, we document the miRNA-directed response of wild-type Arabidopsis thaliana (Arabidopsis) plants and the drb1, drb2 and drb4 mutant lines to Cd stress. Phenotypic and physiological analyses revealed the drb1 mutant to display the highest degree of tolerance to the imposed stress while the drb2 mutant was the most sensitive. RT-qPCR-based molecular profiling of miRNA abundance and miRNA target gene expression revealed DRB1 to be the primary double-stranded RNA binding (DRB) protein required for the production of six of the seven Cd-responsive miRNAs analyzed. However, DRB2, and not DRB1, was determined to be required for miR396 production. RT-qPCR further inferred that transcript cleavage was the RNA silencing mechanism directed by each assessed miRNA to control miRNA target gene expression. Taken together, the results presented here reveal the complexity of the miRNA-directed molecular response of Arabidopsis to Cd stress.
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Pegler, Joseph L., Jackson M. J. Oultram, Duc Quan Nguyen, Christopher P. L. Grof, and Andrew L. Eamens. "MicroRNA-Mediated Responses to Cadmium Stress in Arabidopsis thaliana." Plants 10, no. 1 (January 10, 2021): 130. http://dx.doi.org/10.3390/plants10010130.
Full textAbstract:
In recent decades, the presence of cadmium (Cd) in the environment has increased significantly due to anthropogenic activities. Cd is taken up from the soil by plant roots for its subsequent translocation to shoots. However, Cd is a non-essential heavy metal and is therefore toxic to plants when it over-accumulates. MicroRNA (miRNA)-directed gene expression regulation is central to the response of a plant to Cd stress. Here, we document the miRNA-directed response of wild-type Arabidopsis thaliana (Arabidopsis) plants and the drb1, drb2 and drb4 mutant lines to Cd stress. Phenotypic and physiological analyses revealed the drb1 mutant to display the highest degree of tolerance to the imposed stress while the drb2 mutant was the most sensitive. RT-qPCR-based molecular profiling of miRNA abundance and miRNA target gene expression revealed DRB1 to be the primary double-stranded RNA binding (DRB) protein required for the production of six of the seven Cd-responsive miRNAs analyzed. However, DRB2, and not DRB1, was determined to be required for miR396 production. RT-qPCR further inferred that transcript cleavage was the RNA silencing mechanism directed by each assessed miRNA to control miRNA target gene expression. Taken together, the results presented here reveal the complexity of the miRNA-directed molecular response of Arabidopsis to Cd stress.
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Jiang, C.-Z., C.-N. Yen, K. Cronin, D. Mitchell, and A. B. Britt. "UV- and Gamma-Radiation Sensitive Mutants of Arabidopsis thaliana." Genetics 147, no. 3 (November 1, 1997): 1401–9. http://dx.doi.org/10.1093/genetics/147.3.1401.
Full textAbstract:
Arabidopsis seedlings repair UV-induced DNA damage via light-dependent and -independent pathways. The mechanism of the “dark repair” pathway is still unknown. To determine the number of genes required for dark repair and to investigate the substrate-specificity of this process we isolated mutants with enhanced sensitivity to UV radiation in the absence of photoreactivating light. Seven independently derived UV sensitive mutants were isolated from EMS-mutagenized population. These fell into six complementation groups, two of which (UVR1 and UVH1) have previously been defined. Four of these mutants are defective in the dark repair of UV-induced pyrimidine [6-4]pyrimidinone dimers. These four mutant lines are sensitive to the growth-inhibitory effects of gamma radiation, suggesting that this repair pathway is also involved in the repair of some type of gamma-induced DNA damage product. The requirement for the coordinate action of several different gene products for effective repair of pyrimidine dimers, as well as the nonspecific nature of the repair activity, is consistent with nucleotide excision repair mechanisms previously described in Saccharomyces cerevisiae and nonplant higher eukaryotes and inconsistent with substrate-specific base excision repair mechanisms found in some bacteria, bacteriophage, and fungi.
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Smith, M., H. Moon, and L. Kunst. "Production of hydroxy fatty acids in the seeds of Arabidopsis thaliana." Biochemical Society Transactions 28, no. 6 (December 1, 2000): 947–50. http://dx.doi.org/10.1042/bst0280947.
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Seed-specific expression in Arabidopsis thaliana of oleate hydroxylase enzymes from castor bean and Lesquerella fendleri resulted in the accumulation of hydroxy fatty acids in the seed oil. By using various Arabidopsis mutant lines it was shown that the endoplasmic reticulum (ER) n–-3 desaturase (FAD3) and the FAE1 condensing enzyme are involved in the synthesis of polyunsaturated and very-long-chain hydroxy fatty acids, respectively. In Arabidopsis plants with an active ER Δ12-oleate desaturase the presence of hydroxy fatty acids corresponded to an increase in the levels of 18:1 and a decrease in 18:2 levels. Expression in yeast indicates that the castor hydroxylase also has a low level of desaturase activity.
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Imran, Mahnoor. "Ectopic Expression of Fiber Related Gbwri1 Complements Seed Phenotype in Arabidopsis thaliana." International Journal of Agriculture and Biology 25, no. 01 (January 1, 2021): 123–30. http://dx.doi.org/10.17957/ijab/15.1646.
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WRINKLED1 belongs to AP2/EREB family of transcription factors whose role has been well established in seed oil biosynthesis. The objective of the study was to trace the role of fiber related Gbwri1 in seed development and fatty acid biosynthesis. In this study, we isolated a transcript from elite fiber producing cotton (Gossypium barbadense), which is over-expressed in G. barbadense fibers as compared to G. hirsutum and G. arboreum. The putative protein encoded by this transcript exhibited homology in specific domains and protein structure with WRINKLED1 of Arabidopsis thaliana and was thus designated as Gbwri1. In this study, we investigated the functional homology of fiber elongation related Gbwri1 with fatty acid biosynthesis regulator Atwri1. Ectopic expression of Gbwri1 in wri1-3 mutant of A. thaliana was analyzed. In the transgenic lines of A. thaliana, Gbwri1 resumed the seed weight, seed area, and surface morphology to the wild type. Gbwri1 transformation rescued the wrinkled phenotype of wri1-3 mutants by resuming the expression of fatty acid biosynthesis genes biotin carboxyl carrier protein isoform 2 (bccp2) and keto-ACP synthase 1 (kas1). Moreover, the seedling development of transgenic lines on non-sucrose medium demonstrated that the Gbwri1 was able to regulate the supply of sucrose for normal seedling establishment. Our results showed that the transformation of Gbwri1 in A. thaliana wri1-3 mutant was able to complement wri1-3 impaired phenotype. Thus, Gbwri1 is involved in cotton fiber development and fatty acid biosynthesis in seeds. © 2021 Friends Science Publishers
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Buzduga, I. M., R. A. Volkov, and I. I. Panchuk. "Influence of sodium chloride on the dehydroascorbate reductase activity in Arabidopsis thaliana catalase 2 knokout mutant." Visnik ukrains'kogo tovaristva genetikiv i selekcioneriv 15, no. 2 (February 28, 2018): 138–44. http://dx.doi.org/10.7124/visnyk.utgis.15.2.871.
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Aim. To better understand the mechanisms of abiotic stress resistance in plants, it is important to clarify the role of individual antioxidant enzymes from the same multiproteinic family in the response to stress. It is known that the loss of some isoforms of antioxidant enzymes can be compensated by activation of other enzymes. However, the functional interaction of the ascorbate-glutathione cycle enzymes with catalase under salt stress still remains unexplored. Respectively, we determined the activity of DHAR in knock-out mutants of Arabidopsis thaliana under salt stress. Methods. The DHAR activity was determined in the knock-out line cat2 and in wild-type (WT) Arabidopsis plants after various regimes of treatment with sodium chloride. Results. After treatment with 200 mM sodium chloride in the dark, activation of DHAR was found after 8 hours in WT plants and after 4 hours in the knock-out line cat2. However stress treatment under illumination resulted in significant increase in DHAR activity after 8 hours in both studied lines. In this case, DHAR activity in cat2 was lower than in WT, whereas in non-treated plants or upon stress treatment in the dark no difference between the tested lines was detected. Conclusions. The obtained data indicate that under salt stress conditions, changes in the DHAR activity are included into functional rearrangements of the antioxidant system in cat2 line, which compensate the loss of activity of CAT2 isoenzyme.Keywords: dehydroascorbate reductase, antioxidants, reactive oxygen species (ROS), salt stress, Arabidopsis thaliana
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Pegler, Joseph L., Jackson M. J. Oultram, Shaun J. Curtin, Christopher P. L. Grof, and Andrew L. Eamens. "Further Disruption of the TAS3 Pathway via the Addition of the AGO7 Mutation to the DRB1, DRB2 or DRB4 Mutations Severely Impairs the Reproductive Competence of Arabidopsis thaliana." Agronomy 9, no. 11 (October 25, 2019): 680. http://dx.doi.org/10.3390/agronomy9110680.
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The previous assignment of functional roles for AGO7, and the DOUBLE-STRANDED RNA BINDING (DRB) proteins, DRB1, DRB2, and DRB4, in either microRNA (miRNA) or trans-acting small-interfering RNA (tasiRNA) production allowed for use of the loss-of-function mutant lines, drb1, drb2, drb4, and ago7, to further functionally characterize the TAS3 pathway in Arabidopsis thaliana (Arabidopsis). Towards achieving this goal, we also describe the developmental and molecular phenotypes expressed by three newly generated Arabidopsis lines, the drb1ago7, drb2ago7, and drb4ago7 double mutants. We show that the previously reported developmental abnormalities displayed by the drb1, drb2, drb4, and ago7 single mutants, are further exacerbated in the drb1ago7, drb2ago7, and drb4ago7 double mutants, with rosette area, silique length, and seed set all impaired to a greater degree in the double mutants. Molecular assessment of the TAS3 pathway in the floral tissues of the seven analyzed mutants revealed that DRB1 is the sole DRB required for miR390 sRNA production. However, DRB2 and DRB4 appear to play secondary roles at this stage of the TAS3 pathway to ensure that miR390 sRNA levels are tightly maintained. We further show that the expression of the TAS3-derived tasiARF target genes, AUXIN RESPONSE FACTOR2 (ARF2), ARF3, and ARF4, was altered in drb1ago7, drb2ago7, and drb4ago7 flowers. Altered ARF2, ARF3, and ARF4 expression was in turn demonstrated to lead to changes in the level of expression of KAN1, KAN3, and KAN4, three KANADI transcription factor genes known to be transcriptionally regulated by ARF2, ARF3, and ARF4. Taken together, the demonstrated relationship between altered ARF and KAN gene expression in drb1ago7, drb2ago7 and drb4ago7 flowers, could, in part, explain the more severe developmental defects displayed by the double mutants, compared to milder impact that loss of only a single piece of TAS3 pathway protein machinery was demonstrated to have on drb1, drb2, drb4 and ago7 reproductive development.
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Porco, Silvana, Aleš Pěnčík, Afaf Rashed, Ute Voß, Rubén Casanova-Sáez, Anthony Bishopp, Agata Golebiowska, et al. "Dioxygenase-encoding AtDAO1 gene controls IAA oxidation and homeostasis in Arabidopsis." Proceedings of the National Academy of Sciences 113, no. 39 (September 20, 2016): 11016–21. http://dx.doi.org/10.1073/pnas.1604375113.
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Auxin represents a key signal in plants, regulating almost every aspect of their growth and development. Major breakthroughs have been made dissecting the molecular basis of auxin transport, perception, and response. In contrast, how plants control the metabolism and homeostasis of the major form of auxin in plants, indole-3-acetic acid (IAA), remains unclear. In this paper, we initially describe the function of the Arabidopsis thaliana gene DIOXYGENASE FOR AUXIN OXIDATION 1 (AtDAO1). Transcriptional and translational reporter lines revealed that AtDAO1 encodes a highly root-expressed, cytoplasmically localized IAA oxidase. Stable isotope-labeled IAA feeding studies of loss and gain of function AtDAO1 lines showed that this oxidase represents the major regulator of auxin degradation to 2-oxoindole-3-acetic acid (oxIAA) in Arabidopsis. Surprisingly, AtDAO1 loss and gain of function lines exhibited relatively subtle auxin-related phenotypes, such as altered root hair length. Metabolite profiling of mutant lines revealed that disrupting AtDAO1 regulation resulted in major changes in steady-state levels of oxIAA and IAA conjugates but not IAA. Hence, IAA conjugation and catabolism seem to regulate auxin levels in Arabidopsis in a highly redundant manner. We observed that transcripts of AtDOA1 IAA oxidase and GH3 IAA-conjugating enzymes are auxin-inducible, providing a molecular basis for their observed functional redundancy. We conclude that the AtDAO1 gene plays a key role regulating auxin homeostasis in Arabidopsis, acting in concert with GH3 genes, to maintain auxin concentration at optimal levels for plant growth and development.
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Mordhorst, Andreas P., Keete J. Voerman, Marijke V. Hartog, Ellen A. Meijer, Jacques van Went, Maarten Koornneef, and Sacco C. de Vries. "Somatic Embryogenesis in Arabidopsis thaliana Is Facilitated by Mutations in Genes Repressing Meristematic Cell Divisions." Genetics 149, no. 2 (June 1, 1998): 549–63. http://dx.doi.org/10.1093/genetics/149.2.549.
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Abstract Embryogenesis in plants can commence from cells other than the fertilized egg cell. Embryogenesis initiated from somatic cells in vitro is an attractive system for studying early embryonic stages when they are accessible to experimental manipulation. Somatic embryogenesis in Arabidopsis offers the additional advantage that many zygotic embryo mutants can be studied under in vitro conditions. Two systems are available. The first employs immature zygotic embryos as starting material, yielding continuously growing embryogenic cultures in liquid medium. This is possible in at least 11 ecotypes. A second, more efficient and reproducible system, employing the primordia timing mutant (pt allelic to hpt, cop2, and amp1), was established. A significant advantage of the pt mutant is that intact seeds, germinated in 2,4-dichlorophenoxyacetic acid (2,4-D) containing liquid medium, give rise to stable embryonic cell cultures, circumventing tedious hand dissection of immature zygotic embryos. pt zygotic embryos are first distinguishable from wild type at early heart stage by a broader embryonic shoot apical meristem (SAM). In culture, embryogenic clusters originate from the enlarged SAMs. pt somatic embryos had all characteristic embryo pattern elements seen in zygotic embryos, but with higher and more variable numbers of cells. Embryogenic cell cultures were also established from seedling, of other mutants with enlarged SAMs, such as clavata (clv). pt clv double mutants showed additive effects on SAM size and an even higher frequency of seedlings producing embryogenic cell lines. pt clv double mutant plants had very short fasciated inflorescence stems and additive effects on the number of rosette leaves. This suggests that the PT and CLV genes act in independent pathways that control SAM size. An increased population of noncommitted SAM cells may be responsible for facilitated establishment of somatic embryogenesis in Arabidopsis.
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Negruk, Valentin, Galina Eisner, and Bertrand Lemieux. "Addition–deletion mutations in transgenic Arabidopsis thaliana generated by the seed co-cultivation method." Genome 39, no. 6 (December 1, 1996): 1117–22. http://dx.doi.org/10.1139/g96-140.
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We have characterized three mutant alleles of the CER2 gene of Arabidopsis thaliana by sequencing polymerase chain reaction products of this gene generated with template DNA isolated from lines MK1, BRL7, and BRL17. Sequence analysis of the amplification product from line BRL17 revealed a 17-bp deletion in the CER2 gene. The CER2 gene of BRL7 differs from the wild-type sequence by a 2-base substitution and 2-base insertion. As both of these lines were isolated from the transformant populations generated by Dr. K. Feldmann and his collaborators, we suggest that these small rearrangements may be caused by unsuccessful T-DNA insertions. Comparative sequence analysis of the sequence of line MK1 and the wild type revealed a single nucleotide substitution located 360 bp downstream from the intron–exon junction that changes a tryptophan triplet TGG to TGA: i.e., an opal non-sense mutation. In accordance with these observations, the cer phenotype of line MK1 was complemented by transformation with a fusion construct of the CaMV 35S promoter and the CER2 structural gene. Comparative analysis of the deduced amino acid sequences encoded by the CER2 gene from Arabidopsis and by the glossy2 gene from Zea mays revealed a significant similarity between them. This suggests that these gene products may have a similar biochemical function. Key words : epicuticular wax biosynthesis, eceriferum mutants, fatty acid elongation, glossy mutants, Zea mays.