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PubMed2004

Nonmycorrhizal (myc-) mutants of Hebeloma cylindrosporum obtained through insertional mutagenesis.

Polyethylene glycol-mediated transformation of protoplasts was used as a method for insertional mutagenesis to obtain mutants of the ectomycorrhizal fungus Hebeloma cylindrosporum impaired in symbiotic ability. Following restriction enzyme-mediated integration or conventional plasmid insertion, a library of 1,725 hygromycin-resistant monokaryotic transformants was generated and screened for the symbiotic defect, using Pinus pinaster seedlings as host plants. A total of 51 transformants displaying a dramatically reduced mycorrhizal ability were identified. Among them, 29 were nonmycorrhizal (myc-), but only 10 of them had integrated one or several copies of the transforming plasmid in their genome. Light and scanning electron microscopy observations of pine roots inoculated with myc- mutants suggested that we selected mutants blocked at early stages of interaction between partners or at the stage of Hartig net formation. Myc- mutants with plasmid insertions were crossed with a compatible wild-type monokaryon and allowed to fruit. Monokaryotic progenies were obtained in three independent crosses and were analyzed for symbiotic activity and plasmid insertion. In all three progenies, a 1:1 myc-:myc+ segregation ratio was observed, suggesting that each myc- phenotype resulted from a single gene mutation. However, for none of the three mutants, the myc- phenotype segregated with any of the plasmid insertions. Our results support the idea that master genes, the products of which are essential for symbiosis establishment, do exist in ectomycorrhizal fungi.

Basidiomycota↗
PubMed1994

Agglutination, adherence, and root colonization by fluorescent pseudomonads.

Two fractions of agglutination activity towards fluorescent pseudomonads were detected in root washes of potato, tomato, wheat, and bean. High-molecular-mass (>10 Da) components in crude root washes agglutinated only particular saprophytic, fluorescent Pseudomonas isolates. Ion-exchange treatment of the crude root washes resulted in preparations of lower-molecular-mass (10 to 10 Da) fractions which agglutinated almost all Pseudomonas isolates examined. Also, components able to suppress agglutination reactions of pseudomonads with the lower-molecular-mass root components were detected in crude root washes of all crops studied. Pseudomonas isolates were differentially agglutinated by both types of root components. The involvement of these two types of root components in short-term adherence and in colonization was studied in potato, tomato, and grass, using Pseudomonas isolates from these crops. Short-term adherence of isolates to roots was independent of their agglutination with either type of root components. With agglutination-negative mutants, the high-molecular-mass components seemed to be involved in adherence of Pseudomonas putida Corvallis to roots of all crops studied. Short-term adherence to roots of four Pseudomonas isolates could be influenced by addition of both crude and ion-exchange-treated root washes, depending on their agglutination phenotype with these root wash preparations. Potato root colonization by 10 different isolates from this crop, over a period of 7 days, was not correlated with their agglutination phenotype. Agg mutants of P. putida Corvallis were not impaired in root colonization. It is concluded that the root agglutinins studied can be involved in short-term adherence of pseudomonads to roots but do not play a decisive role in their root colonization.

Journal Article↗
PubMed2002

Both vegetative and reproductive actin isovariants complement the stunted root hair phenotype of the Arabidopsis act2-1 mutation.

The ACT2 gene, encoding one of eight actin isovariants in Arabidopsis, is the most strongly expressed actin gene in vegetative tissues. A search was conducted for physical defects in act2-1 mutant plants to account for their reduced fitness compared with wild type in population studies. The act2-1 insertion fully disrupted expression of ACT2 RNA and significantly lowered the level of total actin protein in vegetative organs. The root hairs of the act2-1 mutants were 10% to 70% the length of wild-type root hairs, and they bulged severely at the base. The length of the mutant root hairs and degree of bulging at the base were affected by adjusting the osmolarity and gelling agent of the growth medium. The act2-1 mutant phenotypes were fully rescued by an ACT2 genomic transgene. When the act2-1 mutation was combined with another vegetative actin mutation, act7-1, the resulting double mutant exhibited extensive synergistic phenotypes ranging from developmental lethality to severe dwarfism. Transgenic overexpression of the ACT7 vegetative isovariant and ectopic expression of the ACT1 reproductive actin isovariant also rescued the root hair elongation defects of the act2-1 mutant. These results suggest normal ACT2 gene regulation is essential to proper root hair elongation and that even minor differences may cause root defects. However, differences in the actin protein isovariant are not significant to root hair elongation, in sharp contrast to recent reports on the functional nonequivalency of plant actin isovariants. Impairment of root hair functions such as nutrient mining, water uptake, and physical anchoring are the likely cause of the reduced fitness seen for act2-1 mutants in multigenerational studies.

Actins↗
PubMed2006

Identification of symbiotically defective mutants of Lotus japonicus affected in infection thread growth.

During the symbiotic interaction between legumes and rhizobia, the host cell plasma membrane and associated plant cell wall invaginate to form a tunnel-like infection thread, a structure in which bacteria divide to reach the plant root cortex. We isolated four Lotus japonicus mutants that make infection pockets in root hairs but form very few infection threads after inoculation with Mesorhizobium loti. The few infection threads that did initiate in the mutants usually did not progress further than the root hair cell. These infection-thread deficient (itd) mutants were unaffected for early symbiotic responses such as calcium spiking, root hair deformation, and curling, as well as for the induction of cortical cell division and the arbuscular mycorrhizal symbiosis. Complementation tests and genetic mapping indicate that itd2 is allelic to Ljsym7, whereas the itdl, itd3, and itd4 mutations identified novel loci. Bacterial release into host cells did occur occasionally in the itdl, itd2, and itd3 mutants suggesting that some infections may succeed after a long period and that infection of nodule cells could occur normally if the few abnormal infection threads that were formed reached the appropriate nodule cells.

Alleles↗
PubMed1999

An auxin-dependent distal organizer of pattern and polarity in the Arabidopsis root.

Root formation in plants involves the continuous interpretation of positional cues. Physiological studies have linked root formation to auxins. An auxin response element displays a maximum in the Arabidopsis root and we investigate its developmental significance. Auxin response mutants reduce the maximum or its perception, and interfere with distal root patterning. Polar auxin transport mutants affect its localization and distal pattern. Polar auxin transport inhibitors cause dramatic relocalization of the maximum, and associated changes in pattern and polarity. Auxin application and laser ablations correlate root pattern with a maximum adjacent to the vascular bundle. Our data indicate that an auxin maximum at a vascular boundary establishes a distal organizer in the root.

Arabidopsis↗
PubMed1999

ANTHOCYANINLESS2, a homeobox gene affecting anthocyanin distribution and root development in Arabidopsis.

The ANTHOCYANINLESS2 (ANL2) gene was isolated from Arabidopsis by using the maize Enhancer-Inhibitor transposon tagging system. Sequencing of the ANL2 gene showed that it encodes a homeodomain protein belonging to the HD-GLABRA2 group. As we report here, this homeobox gene is involved in the accumulation of anthocyanin and in root development. Histological observations of the anl2 mutant revealed that the accumulation of anthocyanin was greatly suppressed in subepidermal cells but only slightly reduced in epidermal cells. Furthermore, the primary roots of the anl2 mutant showed an aberrant cellular organization. We discuss a possible role of ANL2 in the accumulation of anthocyanin and cellular organization of the primary root.

Amino Acid Sequence↗
PubMed2005

Protein geranylgeranyltransferase I is involved in specific aspects of abscisic acid and auxin signaling in Arabidopsis.

Arabidopsis (Arabidopsis thaliana) mutants lacking a functional ERA1 gene, which encodes the beta-subunit of protein farnesyltransferase (PFT), exhibit pleiotropic effects that establish roles for protein prenylation in abscisic acid (ABA) signaling and meristem development. Here, we report the effects of T-DNA insertion mutations in the Arabidopsis GGB gene, which encodes the beta-subunit of protein geranylgeranyltransferase type I (PGGT I). Stomatal apertures of ggb plants were smaller than those of wild-type plants at all concentrations of ABA tested, suggesting that PGGT I negatively regulates ABA signaling in guard cells. However, germination of ggb seeds in response to ABA was similar to the wild type. Lateral root formation in response to exogenous auxin was increased in ggb seedlings compared to the wild type, but no change in auxin inhibition of primary root growth was observed, suggesting that PGGT I is specifically involved in negative regulation of auxin-induced lateral root initiation. Unlike era1 mutants, ggb mutants exhibited no obvious developmental phenotypes. However, era1 ggb double mutants exhibited more severe developmental phenotypes than era1 mutants and were indistinguishable from plp mutants lacking the shared alpha-subunit of PFT and PGGT I. Furthermore, overexpression of GGB in transgenic era1 plants partially suppressed the era1 phenotype, suggesting that the relatively weak phenotype of era1 plants is due to partial redundancy between PFT and PGGT I. These results are discussed in the context of Arabidopsis proteins that are putative substrates of PGGT I.

Abscisic Acid↗
PubMed1992

Expression and distribution of cytosolic 6-phosphogluconate dehydrogenase isozymes in maize.

Maize (Zea mays L.) cytosolic 6-phosphogluconate dehydrogenase isozymes (EC 1.1.1.44; 6-PGD) are encoded by unlinked loci Pgd1 and Pgd2. Two families from a Robertson's Mutator line were isolated which have no detectable expression of Pgd2. These Pgd2-null mutants and a Pgd1-null line were used to generate plants homozygous for null alleles at both cytosolic 6-PGD loci. The specific activity of 6-PGD in the double-null mutant was between 20 and 30% of wild-type levels in root extracts. The double-null mutant was reproductively viable in a moderate environment, suggesting that wild-type levels of cytosolic 6-PGD activity are not essential for growth. Isozyme dimer ratios in roots, leaves, and scutellum were binomial and reflected the wild-type gene copy number. 6-PGD isozymes showed tissue- and cell type-specific expression.

Cytosol↗
PubMed2005

Arabidopsis thickvein mutation affects vein thickness and organ vascularization, and resides in a provascular cell-specific spermine synthase involved in vein definition and in polar auxin transport.

Polar auxin transport has been implicated in the induction of vascular tissue and in the definition of vein positions. Leaves treated with chemical inhibitors of polar auxin transport exhibited vascular phenotypes that include increased vein thickness and vascularization. We describe a recessive mutant, thickvein (tkv), which develops thicker veins in leaves and in inflorescence stems. The increased vein thickness is attributable to an increased number of vascular cells. Mutant plants have smaller leaves and shorter inflorescence stems, and this reduction in organ size and height is accompanied by an increase in organ vascularization, which appears to be attributable to an increase in the recruitment of cells into veins. Furthermore, although floral development is normal, auxin transport in the inflorescence stem is significantly reduced in the mutant, suggesting that the defect in auxin transport is responsible for the vascular phenotypes. In the primary root, the veins appear morphologically normal, but root growth in the tkv mutant is hypersensitive to exogenous cytokinin. The tkv mutation was found to reside in the ACL5 gene, which encodes a spermine synthase and whose expression is specific to provascular cells. We propose that ACL5/TKV is involved in vein definition (defining the boundaries between veins and nonvein regions) and in polar auxin transport, and that polyamines are involved in this process.

Amino Acid Sequence↗
PubMed1989

Amyloplasts are necessary for full gravitropic sensitivity in roots of Arabidopsis thaliana.

The observation that a starchless mutant (TC7) of Arabidopsis thaliana (L.) Heynh. is gravitropic (T. Caspar and B.G. Pickard, 1989, Planta 177, 185-197) raises questions about the hypothesis that starch and amyloplasts play a role in gravity perception. We compared the kinetics of gravitropism in this starchless mutant and the wild-type (WT). Wild-type roots are more responsive to gravity than TC7 roots as judged by several parameters: (1) Vertically grown TC7 roots were not as oriented with respect to the gravity vector as WT roots. (2) In the time course of curvature after gravistimulation, curvature in TC7 roots was delayed and reduced compared to WT roots. (3) TC7 roots curved less than WT roots following a single, short (induction) period of gravistimulation, and WT, but not TC7, roots curved in response to a 1-min period of horizontal exposure. (4) Wild-type roots curved much more than TC7 roots in response to intermittent stimulation (repeated short periods of horizontal exposure); WT roots curved in response to 10 s of stimulation or less, but TC7 roots required 2 min of stimulation to produce a curvature. The growth rates were equal for both genotypes. We conclude that WT roots are more sensitive to gravity than TC7 roots. Starch is not required for gravity perception in TC7 roots, but is necessary for full sensitivity; thus it is likely that amyloplasts function as statoliths in WT Arabidopsis roots. Furthermore, since centrifugation studies using low gravitational forces indicated that starchless plastids are relatively dense and are the most movable component in TC7 columella cells, the starchless plastids may also function as statoliths.

Arabidopsis↗
PubMed2005

ABR1, an APETALA2-domain transcription factor that functions as a repressor of ABA response in Arabidopsis.

The plant hormone abscisic acid (ABA) plays an important role in plant development and stress responses. An important step of ABA action is activation or inactivation of gene expression. Although several transcription factors are identified to function as positive regulators of ABA-induced gene expression, little is known about the negative regulators of ABA-regulated gene expression. Here, we have identified an APETALA2 (AP2) domain transcription factor that serves as a repressor of ABA response during seed germination and ABA- and stress-induced gene expression in Arabidopsis (Arabidopsis thaliana). The expression of the AP2-like ABA repressor 1 (ABR1) gene itself was responsive to ABA and stress conditions including cold, high salt, and drought. Disruption of ABR1 led to hypersensitive response to ABA in seed germination and root growth assays. The mutant plants were also hypersensitive to osmotic stress conditions, such as high salt and high concentrations of mannitol. Further analyses indicated that increased stress sensitivity may result from hypersensitivity to ABA as ABA biosynthesis inhibitor rescued the stress hypersensitivity phenotype. The abr1 mutant plants accumulated significantly higher levels of ABA- and stress-inducible gene transcripts as compared to the wild-type plants, supporting the hypothesis that this AP2 domain protein serves as a repressor of ABA-regulated gene expression.

Abscisic Acid↗
PubMed2003

EXORDIUM--a gene expressed in proliferating cells and with a role in meristem function, identified by promoter trapping in Arabidopsis.

To identify new genes expressed in meristematic cells, a promoter trap insertional mutagenesis strategy was used in Arabidopsis thaliana. Transgenic line AtEM201 exhibits promoter trap GUS activity in embryos and in the regions of active cell division in the seedling, notably the apical meristems and young leaves. The tagged gene was named EXORDIUM (EXO). AtEM201 contains a single copy of the promoter trap T-DNA, located in the EXO gene promoter, resulting in a much reduced level of EXO transcription. Seedlings homozygous for the T-DNA insertion have no obvious mutant phenotype. The EXO gene, which forms part of a small gene family in Arabidopsis, is structurally related to the tobacco PHI-1 gene, which is re-activated in cultured cells following release from phosphate starvation-induced cell cycle arrest. Expression of both the EXO-GUS and the native EXO genes is downregulated by exogenous cytokinin. Expression studies using semisynchronised cells suggest that EXO mRNA is preferentially abundant during M phase of the cell cycle. Double mutant studies revealed that the exo mutation can suppress the defective root meristem phenotype of the hydra2 mutant, suggesting that EXO may be a component of a negative regulatory system for cell division.

Amino Acid Sequence↗
PubMed2002

Lineage-specific requirements of beta-catenin in neural crest development.

Beta-catenin plays a pivotal role in cadherin-mediated cell adhesion. Moreover, it is a downstream signaling component of Wnt that controls multiple developmental processes such as cell proliferation, apoptosis, and fate decisions. To study the role of beta-catenin in neural crest development, we used the Cre/loxP system to ablate beta-catenin specifically in neural crest stem cells. Although several neural crest-derived structures develop normally, mutant animals lack melanocytes and dorsal root ganglia (DRG). In vivo and in vitro analyses revealed that mutant neural crest cells emigrate but fail to generate an early wave of sensory neurogenesis that is normally marked by the transcription factor neurogenin (ngn) 2. This indicates a role of beta-catenin in premigratory or early migratory neural crest and points to heterogeneity of neural crest cells at the earliest stages of crest development. In addition, migratory neural crest cells lateral to the neural tube do not aggregate to form DRG and are unable to produce a later wave of sensory neurogenesis usually marked by the transcription factor ngn1. We propose that the requirement of beta-catenin for the specification of melanocytes and sensory neuronal lineages reflects roles of beta-catenin both in Wnt signaling and in mediating cell-cell interactions.

Animals↗
PubMed2000

Production and characterization of diverse developmental mutants of Medicago truncatula.

The diploid annual legume Medicago truncatula has been developed as a tractable genetic system for studying biological questions that are unique to, or well suited for study in legume species. An efficient mutagenesis protocol using ethyl-methyl sulfonate and a polymorphic ecotype with properties appropriate for use as a mapping parent are described. Isolation and characterization of three developmental mutants are described. The mtapetala mutation results in homeotic conversions of floral organ whorls 2 and 3 into sepals and carpelloid structures, respectively, similar to mutations in the apetala3/pistillata genes of Arabidopsis. The palmyra mutation primarily affects seedling shoot meristem initiation, and thus phenocopies meristem function mutations identified in Arabidopsis such as the zwille locus. The phenotype of the palmyra and mtapetala double mutant is additive, with seedling shoot meristems and floral organs indistinguishable from those of the single palmyra and mtapetala mutants, respectively. These results are consistent with a lack of genetic interaction between these loci. A third mutant, speckle, is characterized by spontaneous necrotic lesion formation on leaves, root, and stems, similar to necrosis mutants identified in other plant species. In addition to documenting the efficient mutagenesis of M. truncatula, the availability of developmental mutants that phenocopy characterized Arabidopsis mutants will provide a basis for establishing orthologous gene function between M. truncatula and Arabidopsis, once the genes responsible are cloned. Moreover, the male-sterile, female-fertile nature of the mtapetala mutant provides a convenient tool for genetic analyses in M. truncatula.

Amino Acid Sequence↗
PubMed2006

Characterization of a two-component high-affinity nitrate uptake system in Arabidopsis. Physiology and protein-protein interaction.

The identification of a family of NAR2-type genes in higher plants showed that there was a homolog in Arabidopsis (Arabidopsis thaliana), AtNAR2.1. These genes encode part of a two-component nitrate high-affinity transport system (HATS). As the Arabidopsis NRT2 gene family of nitrate transporters has been characterized, we tested the idea that AtNAR2.1 and AtNRT2.1 are partners in a two-component HATS. Results using the yeast split-ubiquitin system and Xenopus oocyte expression showed that the two proteins interacted to give a functional HATS. The growth and nitrogen (N) physiology of two Arabidopsis gene knockout mutants, atnrt2.1-1 and atnar2.1-1, one for each partner protein, were compared. Both types of plants had lost HATS activity at 0.2 mm nitrate, but the effect was more severe in atnar2.1-1 plants. The relationship between plant N status and nitrate transporter expression revealed a pattern that was characteristic of N deficiency that was again stronger in atnar2.1-1. Plants resulting from a cross between both mutants (atnrt2.1-1 x atnar2.1-1) showed a phenotype like that of the atnar2.1-1 mutant when grown in 0.5 mm nitrate. Lateral root assays also revealed growth differences between the two mutants, confirming that atnar2.1-1 had a stronger phenotype. To show that the impaired HATS did not result from the decreased expression of AtNRT2.1, we tested if constitutive root expression of a tobacco (Nicotiana plumbaginifolia) gene, NpNRT2.1, previously been shown to complement atnrt2.1-1, can restore HATS to the atnar2.1-1 mutant. These plants did not recover wild-type nitrate HATS. Taken together, these results show that AtNAR2.1 is essential for HATS of nitrate in Arabidopsis.

Animals↗
PubMed1997

Expression of a dominant negative mutant of interleukin-1 beta converting enzyme in transgenic mice prevents neuronal cell death induced by trophic factor withdrawal and ischemic brain injury.

To explore the role of the interleukin (IL)-1 beta converting enzyme (ICE) in neuronal apoptosis, we designed a mutant ICE gene (C285G) that acts as a dominant negative ICE inhibitor. Microinjection of the mutant ICE gene into embryonal chicken dorsal root ganglial neurons inhibits trophic factor withdrawal-induced apoptosis. Transgenic mice expressing the fused mutant ICE-lacZ gene under the control of the neuron specific enolase promoter appeared neurologically normal. These mice are deficient in processing pro-IL-1 beta, indicating that mutant ICEC285G blocks ICE function. Dorsal root ganglial neurons isolated from transgenic mice were resistant to trophic factor withdrawal-induced apoptosis. In addition, the neurons isolated from newborn ICE knockout mice are similarly resistant to trophic factor withdrawal-induced apoptosis. After permanent focal ischemia by middle cerebral artery occlusion, the mutant ICEC285G transgenic mice show significantly reduced brain injury as well as less behavioral deficits when compared to the wild-type controls. Since ICE is the only enzyme with IL-1 beta convertase activity in mice, our data indicates that the mutant ICEC285G inhibits ICE, and hence mature IL-1 beta production, and through this mechanism, at least in part, inhibits apoptosis. Our data suggest that genetic manipulation using ICE family dominant negative inhibitors can ameliorate the extent of ischemia-induced brain injury and preserve neurological function.

Animals↗
PubMed1991

Ethylene Inhibitors Restore Nodulation to sym 5 Mutants of Pisum sativum L. cv Sparkle.

The sym 5 mutants of pea, Pisum sativum L. cv Sparkle, do not differ in growth habit from their normal parent and nodulate poorly at a root temperature of 20 degrees C. If inhibitors of ethylene formation or action (Co(2+), aminoethoxyvinylglycine, or Ag(+)) are added to the substrate, nodulation of the sym 5 mutants is increased. Similar treatments of four other mutant sym lines do not restore nodulation. When Ag(+) is added to the substrate from 4 days before to 4 days after inoculation with rhizobia, nodulation of sym 5 mutants is increased. The roots of the mutant need only be exposed to Ag(+) for 4 hours to significantly increase nodule numbers. The content of free 1-aminocyclopropane-1-carboxylic acid and the production of ethylene in the lateral roots of sym 5 mutants do not differ from Sparkle.

Journal Article↗
PubMed2001

Hormonal regulation of tissue-specific ectopic expression of an Arabidopsis endoplasmic reticulum-type omega-3 fatty acid desaturase (FAD3) gene.

A common feature of the membrane lipids of higher plants is a large content of polyunsaturated fatty acids, which typically consist of dienoic and trienoic fatty acids. Two types of omega-3 fatty acid desaturase. which are present in the plastids and in the endoplasmic reticulum (ER), respectively, are responsible for the conversion of dienoic to trienoic fatty acids. To establish a system for investigating the tissue-specific, and hor-mone-regulated expression of the ER-type desaturase gene (FAD3), transgenic plants of Arabidopsis thaliana (L.) Heynh. containing the firefly luciferase gene (LUC) fused to the FAD3 promoter (FAD3::LUC) were constructed. At different times during plant development, FAD3::LUC was actively expressed at two major sites, the vegetative shoot meristem and the floral organs. Transgenic plants with LUC fused to the promoter of FAD7 (FAD7::LUC) which encodes plastid-type desaturase, were also constructed. FAD3::LUC and FAD7::LUC were expressed in the same organs during reproductive growth, but not during vegetative growth. In plants exposed to both auxin and cytokinin, FAD3::LUC expression was ectopically induced in the root tissues. However, this induction by auxin and cytokinin was inhibited when abscisic acid was also present. FAD3::LUC expression could be induced in the roots by auxin and cytokinin if the hormones were applied during vegetative growth, but not if they were applied during germination or reproductive growth. Analysis of the fatty acid composition in the roots of Arabidopsis fad mutant and wild-type plants confirmed that the response of FAD3::LUC expression to various hormones reflected the response of endogenous FAD3 gene expression. These results suggest that the expression of ER-type desaturase is regulated through synergistic and antagonistic hormonal interactions, and that such hormonal regulation and the tissue specificity of the expression of this gene are further modified in accordance with the growth phase in plant development.

Abscisic Acid↗