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Showing papers in "Plant Journal in 1998"


Journal ArticleDOI
TL;DR: The modified method should facilitate high-throughput transformation of Arabidopsis for efforts such as T-DNA gene tagging, positional cloning, or attempts at targeted gene replacement.
Abstract: Summary The Agrobacterium vacuum infiltration method has made it possible to transform Arabidopsis thaliana without plant tissue culture or regeneration. In the present study, this method was evaluated and a substantially modified transformation method was developed. The labor-intensive vacuum infiltration process was eliminated in favor of simple dipping of developing floral tissues into a solution containing Agrobacterium tumefaciens, 5% sucrose and 500 microliters per litre of surfactant Silwet L-77. Sucrose and surfactant were critical to the success of the floral dip method. Plants inoculated when numerous immature floral buds and few siliques were present produced transformed progeny at the highest rate. Plant tissue culture media, the hormone benzylamino purine and pH adjustment were unnecessary, and Agrobacterium could be applied to plants at a range of cell densities. Repeated application of Agrobacterium improved transformation rates and overall yield of transformants approximately twofold. Covering plants for 1 day to retain humidity after inoculation also raised transformation rates twofold. Multiple ecotypes were transformable by this method. The modified method should facilitate high-throughput transformation of Arabidopsis for efforts such as T-DNA

18,757 citations


Journal ArticleDOI
TL;DR: It is proposed that cold-induced expression of CRT/DRE-containing COR genes involves a low temperature-stimulated signalling cascade in which CBF gene induction is an early event and theCBF gene family is not subject to autoregulation.
Abstract: Cold-induced expression of the Arabidopsis COR (cold-regulated) genes is mediated by a DNA regulatory element termed the CRT (C-repeat)/DRE (dehydration-responsive element). Recently, we identified a transcriptional activator, CBF1, that binds to the CRT/DRE and demonstrated that its overexpression in transgenic Arabidopsis plants at non-acclimating temperatures induces COR gene expression and increases plant freezing tolerance. Here we report that CBF1 belongs to a small family of closely related proteins which includes CBF2 and CBF3. DNA sequencing of an 8.7 kb region of the Arabidopsis genome along with genetic mapping experiments indicated that the three CBF genes are organized in direct repeat on chromosome 4 at 72.8 cM, closely linked to molecular markers PG11 and m600. Like CBF1, both CBF2 and CBF3 activated expression of reporter genes in yeast that contained the CRT/DRE as an upstream activator sequence. The transcript levels for all three CBF genes increased within 15 min of transferring plants to low temperature, followed by accumulation of COR gene transcripts at about 2 h. CBF transcripts also accumulated rapidly in response to mechanical agitation. The promoter regions of the CBF genes do not contain the CRT sequence, CCGAC, and overexpression of CBF1 did not have a detectable effect on CBF3 transcript levels, suggesting that the CBF gene family is not subject to autoregulation. We propose that cold-induced expression of CRT/DRE-containing COR genes involves a low temperature-stimulated signalling cascade in which CBF gene induction is an early event.

1,090 citations


Journal ArticleDOI
TL;DR: The observations suggest that the leaf Golgi complex functions as a motile system of actin-directed stacks whose function is to pick up products from a relatively stationary ER system.
Abstract: Summary We have visualized the relationship between the endoplasmic reticulum (ER) and Golgi in leaf cells of Nicotiana clevelandii by expression of two Golgi proteins fused to green fluorescent protein (GFP). A fusion of the transmembrane domain (signal anchor sequence) of a rat sialyl transferase to GFP was targeted to the Golgi stacks. A second construct that expressed the Arabidopsis H/KDEL receptor homologue aERD2, fused to GFP, was targeted to both the Golgi apparatus and ER, allowing the relationship between these two organelles to be studied in living cells for the first time. The Golgi stacks were shown to move rapidly and extensively along the polygonal cortical ER network of leaf epidermal cells, without departing from the ER tubules. Co-localization of F-actin in the GFPexpressing cells revealed an underlying actin cytoskeleton that matched precisely the architecture of the ER network, while treatment of cells with the inhibitors cytochalasin D and N-ethylmaleimide revealed the dependency of Golgi movement on actin cables. These observations suggest that the leaf Golgi complex functions as a motile system of actin-directed stacks whose function is to pick up products from a relatively stationary ER system. Also, we demonstrate for the first time

820 citations


Journal ArticleDOI
TL;DR: A modification of this technique where mismatches in a PCR primer are used to create a polymorphism based on the target mutation to be useful for following known mutations in segregating populations and genetic mapping of isolated DNAs used for positional based cloning of new genes.
Abstract: PCR-based detection of single nucleotide polymorphisms is a powerful tool for the plant geneticist Cleaved amplified polymorphic sequence analysis is the most widely used approach for the detection of single nucleotide polymorphisms However, this technique is limited to mutations which create or disrupt a restriction enzyme recognition site This paper presents a modification of this technique where mismatches in a PCR primer are used to create a polymorphism based on the target mutation This technique is useful for following known mutations in segregating populations and genetic mapping of isolated DNAs used for positional based cloning of new genes In addition, a computer program has been developed that facilitates the design of these PCR primers

741 citations


Journal ArticleDOI
TL;DR: It is found that transient and stable expression of the talin actin-binding domain fused to the C-terminus of the green fluorescent protein (GFP-mTn) can visualize the actin cytoskeleton in different types of living plant cells without affecting cell morphology or function.
Abstract: The C-terminus of mouse talin (amino acids 2345-2541) is responsible for all of the protein's f-actin binding capacity Unlike full-length talin, the C-terminal f-actin binding domain is unable to nucleate actin polymerization We have found that transient and stable expression of the talin actin-binding domain fused to the C-terminus of the green fluorescent protein (GFP-mTn) can visualize the actin cytoskeleton in different types of living plant cells without affecting cell morphology or function Transiently expressed GFP-mTn co-localized with rhodamine-phalloidin in permeabilized tobacco BY-2 suspension cells, showing that the fusion protein can specifically label the plant actin cytoskeleton Constitutive expression of GFP-mTn in transgenic Arabidopsis thaliana plants visualized actin filaments in all examined tissues with no apparent effects on plant morphology or development at any stage during the life cycle This demonstrates that in a number of different cell types GFP-mTn can serve as a non-invasive marker for the actin cytoskeleton Confocal imaging of GFP-mTn labeled actin filaments was employed to reveal novel information on the in vivo organization of the actin cytoskeleton in transiently transformed, normally elongating tobacco pollen tubes

617 citations


Journal ArticleDOI
TL;DR: A systematic search for the function of all genes of this large family of regulatory genes in Arabidopsis thaliana revealed conserved amino acid motifs shared by subgroups of R2R3-MYB genes in addition to the characteristic DNA-binding domain.
Abstract: Transcription factors containing a conserved DNA-binding domain similar to that of the proto-oncogene c-myb have been identified in nearly all eukaryotes. MYB-related proteins from plants generally contain two related helix-turn-helix motifs, the R2 and R3 repeats. It was estimated that Arabidopsis thaliana contains more than 100 R2R3-MYB genes. The few cases where functional data are available suggest an important role of these genes in the regulation of secondary metabolism, the control of cell shape, disease resistance, and hormone responses. To determine the full regulatory potential of this large family of regulatory genes, a systematic search for the function of all genes of this family was initiated. Sequence data for more than 90 different A. thaliana R2R3-MYB genes have been obtained. Sequence comparison revealed conserved amino acid motifs shared by subgroups of R2R3-MYB genes in addition to the characteristic DNA-binding domain. No significant clustering of the genes was detected, although they are not uniformly distributed throughout the A. thaliana genome.

570 citations


Journal ArticleDOI
TL;DR: A review of transgene-induced silencing phenomena in plants and the involvement of RNA was hypothesized to explain post-transcriptional silencing in plants, fungi and nematodes.
Abstract: The recent development of gene transfer methods for almost all eukaryotes has revealed that transgenes can undergo silencing after integration in the genome. Host genes can also be silenced as a consequence of the presence of a homologous transgene, thus limiting the potential application of genetic transformation. Despite this limitation, transgene-induced gene silencing events were considered originally as anecdotal phenomena. However, as more and more similarities were found between transgene-induced gene silencing and natural epigenetic phenomena, considerable interest has been devoted to this subject (for recent reviews see Depicker and Van Montagu, 1997; Stam et al., 1997b). Epigenetics is commonly defined as ‘the study of mitotically and/or meiotically heritable changes in the function of a gene that cannot be explained by changes in its DNA sequence’ (Russo et al., 1996). For a long time, DNA was considered as the only target for epigenetic modifications. Epigenetic changes corresponding to changes in chromatin structure and affecting transcription have been reported in almost all eukaryotes: yeast, fungi, Drosophila, plants and mammals (Dorer, 1997; Foss and Selker, 1991; Rossignol and Faugeron, 1994; Ye and Signer, 1996). However, recent studies have suggested that, besides DNA, other molecules can be modified in a manner that resembles epigenetic DNA changes. First, it was shown that proteins can be converted into molecules of aberrant conformation called prions in yeast and mammals (Lacroute, 1971; Prusiner, 1982). More recently, the involvement of RNA was hypothesized to explain post-transcriptional silencing in plants, fungi and nematodes (Cogoni et al., 1996; Fire et al., 1998; Napoli et al., 1990). This review will focus on transgene-induced silencing phenomena in plants. The number of copies of a transgene that integrate into the genome of a transformed plant and

496 citations


Journal ArticleDOI
TL;DR: The hypothesis that LCOs and chitin oligosaccharides act by perturbing the auxin flow in the root during the earliest stages of nodule formation is supported, and it is shown that endogenous flavonoids could mediate this response.
Abstract: The expression of the auxin responsive reporter construct, GH3:gusA, was examined in transgenic white clover plants to assess changes in the auxin balance during the earliest stages of root nodule formation. Reporter gene expression was monitored at marked locations after the application of bacteria or signal molecules using two precise inoculation techniques: spot-inoculation and a novel method for ballistic microtargeting. Changes in GH3:gusA expression were monitored after the inoculation of Rhizobium leguminosarum biovar trifolii, non-host rhizobia, lipo-chitin oligosaccharides (LCOs), chitin oligosaccharides, a synthetic auxin transport inhibitor (naphthylphthalamic acid; NPA), auxin, the ENOD40-1 peptide or different flavonoids. The results show that clover-nodulating rhizobia induce a rapid, transient and local downregulation of GH3:gusA expression during nodule initiation followed by an upregulation of reporter gene expression at the site of nodule initiation. Microtargeting of auxin caused a local and acropetal upregulation of GH3:gusA expression, whereas NPA caused local and acropetal downregulation of expression. Both spot-inoculation and microtargeting of R. l. bv. trifolii LCOs or flavonoid aglycones induced similar changes to GH3:gusA expression as NPA. O-acetylated chitin oligosaccharides caused similar changes to GH3:gusA expression as R. l. bv. trifolii spot-inoculation, but only after delivery by microtargeting. Non-O-acetylated chitin oligosaccharides, flavonoid glucosides or the ENOD40-1 peptide failed to induce any detectable changes in GH3:gusA expression. GH3:gusA expression patterns during the later stages of nodule and lateral root development were similar. These results support the hypothesis that LCOs and chitin oligosaccharides act by perturbing the auxin flow in the root during the earliest stages of nodule formation, and that endogenous flavonoids could mediate this response.

478 citations


Journal ArticleDOI
TL;DR: It is found that mutants deficient in either auxin or ethylene response have a pronounced effect on root hair length, and studies indicate that both auxin and ethylene are required for normal root hair elongation.
Abstract: Genetic and physiological studies implicate the phytohormones auxin and ethylene in root hair development. To learn more about the role of these compounds, we have examined the root hair phenotype of a number of auxin- and ethylene-related mutants. In a previous study, Masucci and Schiefelbein (1996) showed that neither the auxin response mutations aux1 and axr1 nor the ethylene response mutations etr1 and ein2 have a significant effect on root hair initiation. In this study, we found that mutants deficient in either auxin or ethylene response have a pronounced effect on root hair length. Treatment of wild-type, axr1 and etr1 seedlings with the synthetic auxin, 2,4-D, or the ethylene precursor ACC, led to the development of longer root hairs than untreated seedlings. Furthermore, axr1 seedlings grown in the presence of ACC produce ectopic root hairs and an unusual pattern of long root hairs followed by regions that completely lack root hairs. These studies indicate that both auxin and ethylene are required for normal root hair elongation.

464 citations


Journal ArticleDOI
TL;DR: Describing ICK1 in terms of its gene structure, its interaction with both A. thaliana Cdc2a and CycD3, and its induction by the plant growth regulator, ABA suggests a molecular mechanism by which plant cell division might be inhibited by ABA.
Abstract: Cyclin-dependent kinase (CDK) inhibitor genes encode low molecular weight proteins which have important functions in cell cycle regulation, development and perhaps also in tumorigenesis. The first plant CDK inhibitor gene ICK1 was recently identified from Arabidopsis thaliana. Although the C-terminal domain of ICK1 contained an important consensus sequence with the mammalian CDK inhibitor p27Kip1, the remainder of the deduced ICK1 sequence showed little similarity to any known CDK inhibitors. In vitro assays showed that recombinant ICK1 exhibited unique kinase inhibitory properties. In the present study we characterized ICK1 in terms of its gene structure, its interaction with both A. thaliana Cdc2a and CycD3, and its induction by the plant growth regulator, abscisic acid (ABA). ICK1 was expressed at a relatively low level in the tissues surveyed. However, ICK1 was induced by ABA, and along with ICK1 induction there was a decrease in Cdc2-like histone H1 kinase activity. These results suggest a molecular mechanism by which plant cell division might be inhibited by ABA. ICK1 clones were also identified from independent yeast two-hybrid screens using the CycD3 construct. The implication that ICK1 protein could interact with both Cdc2a and CycD3 was confirmed by in vitro binding assays. Furthermore, deletion analysis indicated that different regions of ICK1 are required for the interactions with Cdc2a and CycD3. These results provide a mechanistic basis for understanding the role of CDK inhibitors in cell cycle regulation in plant cells.

446 citations


Journal ArticleDOI
TL;DR: Trans-2-hexenal induction closely mimics the group of genes induced by methyl jasmonate (MeJA), but the inductive effect seemed to be limited to C6-related volatiles, as C8-, C9- and other related volatile did not induce LOX mRNA levels.
Abstract: Summary Six-Carbon (C6-) volatiles, including the aldehydes trans-2-hexenal, hexanal and cis-3-hexenal, as well as their corresponding alcohols, are produced from damaged or wounded plant tissue as a product of the enzymatic activity of hydroperoxide lyase (HPL), a component of the lipoxygenase (LOX) pathway. Aerial treatment of Arabidopsis seedlings with 10 μM concentrations of trans-2-hexenal induces several genes known to be involved in the plant’s defense response, including phenylpropanoid-related genes as well as genes of the LOX pathway. Genes encoding the pathogenesis-related proteins PR-1 or PR-2, however, were not induced. Trans-2-hexenal induction thus closely mimics the group of genes induced by methyl jasmonate (MeJA), also a LOX-derived volatile. However, unlike MeJA, trans-2-hexenal did not induce hydroxymethylglutaryl-coenzyme A reductase (HMGR) or thionin2–1. The inductive effect seemed to be limited to C6-related volatiles, as C8-, C9- and other related volatiles did not induce LOX mRNA levels. As has been demonstrated for MeJA, trans-2-hexenal quantitatively reduced wild-type seed germination. Trans-2-hexenal also reduced the germination frequency of the MeJA resistant Arabidopsis mutant, jar1–1, supporting the notion that trans-2-hexenal and MeJA are recognized via different mechanisms. In addition, trans-2-hexenal had a moderate inhibitory effect on root length relative to similar concentrations of MeJA and was approximately 10-fold less effective than MeJA at inducing anthocyanin accumulation in Arabidopsis seedlings. These results suggest that C6-volatiles of the LOX pathway act as a wound signal in plants, but result in a moderate plant response relative to MeJA at both the physiological and molecular level.

Journal ArticleDOI
TL;DR: A putative EF-hand Ca(2+)-binding motif in the extended N-terminal region of the Atrboh proteins suggests a direct regulatory effect of Ca2+ on the activity of the NADPH oxidase in plants.
Abstract: An NADPH oxidase analogous to that in mammalian phagocytes has been hypothesized to produce reactive oxygen species (ROS) in the plant defence response. A. thaliana contains at least six gp91phox homologues, designated AtrbohA-F (A. thaliana Respiratory Burst Oxidase Homologues), which map to different positions. Transcripts of three of these genes can be detected in healthy plants by RNA gel blot analyses. The Atrboh gene products are closely related to gp91phox and the intron locations suggest a common evolutionary origin. A putative EF-hand Ca(2+)-binding motif in the extended N-terminal region of the Atrboh proteins suggests a direct regulatory effect of Ca2+ on the activity of the NADPH oxidase in plants.

Journal ArticleDOI
TL;DR: It is concluded that segregating bands from a common parent can be compared between different populations, and that AFLP bands of similar molecular size, amplified with the same primer combination in two different ecotypes, are likely to correspond to the same locus.
Abstract: An amplified fragment polymorphism (AFLP) based linkage map has been generated for a new Landsberg erecta/ Cape Verde Islands (Ler/Cvi) recombinant inbred line (RIL) population. A total of 321 molecular PCR based markers and the erecta mutation were mapped. AFLP markers were also analysed in the Landsberg erecta/Columbia (Ler/Col) RIL population (Lister and Dean, 1993) and 395 AFLP markers have been integrated into the previous Arabidopsis molecular map of 122 RFLPs, CAPSs and SSLPs. This enabled the evaluation of the efficiency and robustness of AFLP technology for linkage analyses in Arabidopsis. AFLP markers were found throughout the linkage map. The two RIL maps could be integrated through 49 common markers which all mapped at similar positions. Comparison of both maps led to the conclusion that segregating bands from a common parent can be compared between different populations, and that AFLP bands of similar molecular size, amplified with the same primer combination in two different ecotypes, are likely to correspond to the same locus. AFLPs were found clustering around the centromeric regions, and the authors have established the map position of the centromere of chromosome 3 by a quantitative analysis of AFLP bands using trisomic plants. AFLP markers were also used to estimate the polymorphism rate among the three ecotypes. The larger polymorphism rate found between Ler and Cvi compared to Ler and Col will mean that the new RIL population will provide a useful material to map DNA polymorphisms and quantitative trait loci.

Journal ArticleDOI
TL;DR: Both root growth and GCS activity of the cad2-1 mutant was less sensitive than the wild-type to inhibition by BSO, indicating that the mutation may alter the affinity of the inhibitor binding site.
Abstract: This paper reports that the glutathione (GSH)-deficient mutant, cad2-1, of Arabidopsis is deficient in the first enzyme in the pathway of GSH biosynthesis, γ-glutamylcysteine synthetase (GCS). The mutant accumulates a substrate of GCS, cysteine, and is deficient in the product, γ-glutamylcysteine. In vitro enzyme assays showed that the cad2-1 mutant has 40% of wild-type levels of GCS activity but is unchanged in the activity of the second enzyme in the pathway, GSH synthetase. The CAD2 locus maps to chromosome 4 and is tightly linked to a gene, GSHA, identified by a previously isolated cDNA. A genomic clone of GSHA complements both the phenotypic and biochemical deficiencies of the cad2-1 mutant. The nucleotide sequence of the gene has been determined and, in the mutant, this gene contains a 6 bp deletion within an exon. These data demonstrate that the CAD2 gene encodes GCS. The cad2-1 mutation is close to the conserved cysteine which is believed to bind the substrate glutamate and the specific inhibitor L-buthionine-[S,R] sulfoximine (BSO). Both root growth and GCS activity of the cad2-1 mutant was less sensitive than the wild-type to inhibition by BSO, indicating that the mutation may alter the affinity of the inhibitor binding site.

Journal ArticleDOI
TL;DR: Several inducible in vivo footprints located at or nearby these motifs demonstrate significant and highly reproducible changes in DNA accessibility following SAR induction, tightly correlated with the functionally important regions of the promoter identified by mutation analysis.
Abstract: The Arabidopsis PR-1 gene is one of a suite of genes induced co-ordinately during the onset of systemic acquired resistance (SAR), a plant defense pathway triggered by pathogen infection or exogenous application of chemicals such as salicylic acid (SA) and 2,6-dichloroisonicotinic acid (INA). We have characterized cis-acting regulatory elements in the PR-1 promoter involved in INA induction using deletion analysis, linker-scanning mutagenesis, and in vivo footprinting. Compared to promoter fragments of 815 bp or longer (which show greater than 10-fold inducibility after INA treatment), induction of a 698 bp long promoter fragment is reduced by half and promoter fragments of 621 bp or shorter have lost all inducibility. Additionally, two 10-bp linker-scanning mutations centered at 640 bp and 610 bp upstream from the transcription initiation site are each sufficient to abolish chemical inducibility of a GUS reporter fusion. The -640 linker-scanning mutation encompasses a region highly homologous to recognition sites for transcription factors of the basic leucine zipper class, while the -610 linker-scanning mutation contains a sequence similar to a consensus recognition site for the transcription factor NF-kappa B. Furthermore, several inducible in vivo footprints located at or nearby these motifs demonstrate significant and highly reproducible changes in DNA accessibility following SAR induction. This in vivo signature of protein-DNA interactions after INA induction is tightly correlated with the functionally important regions of the promoter identified by mutation analysis.

Journal ArticleDOI
TL;DR: The isolation of a P. hybrida gene, jaf13, encoding a basic helix-loop-helix protein, indicates that regulatory anthocyanin genes are conserved between species and that divergent evolution of the target gene promoters is responsible for the species-specific differences in regulatory networks.
Abstract: The regulatory anthocyanin loci, an1, an2, an4 and an11 of Petunia hybrida, and r and c1 from Zea mays, control transcription of different sets of target genes. Both an2 and c1 encode a MYB-type protein. This study reports the isolation of a P. hybrida gene, jaf13, encoding a basic helix-loop-helix protein that, on the basis of sequence homology and intron/exon structure, represents the P. hybrida orthologue of the Z. mays r genes. Ectopic expression of an2 and jaf13 is sufficient for activation of the dihydroflavonol 4-reductase-A (dfrA) promoter and enhanced pigment accumulation in P. hybrida. This indicates that an2 and jaf13 play a key role in determining the tissue-specific expression pattern of structural genes. However, because chalcone synthase (chs) and flavanone-3-hydroxylase (f3h) are not activated, the pattern of pigmentation is not fundamentally altered. Expression of an2 in Z. mays complements a mutation in pl, a c1 paralogue, indicating that an2 activates a wider set of target genes in this host. Transient expression assays in Z. mays and P. hybrida tissues showed that C1 and R or AN2 and JAF13 can activate the promoter of the c2 gene, encoding Z. mays CHS, but not the chsA promoter from P. hybrida. These results indicate that regulatory anthocyanin genes are conserved between species and that divergent evolution of the target gene promoters is responsible for the species-specific differences in regulatory networks.

Journal ArticleDOI
TL;DR: The results demonstrate that stable high-level expression of a foreign gene in Chlamydomonas is possible, and highlight a potential role of introns as modulators of gene expression in this alga.
Abstract: Summary Heterologous genes introduced into the nuclear genome of Chlamydomonas reinhardtii are often poorly expressed. To understand the molecular mechanisms underlying this effect, we examined the influence of various factors on the expression of a chimeric transgene that confers resistance to zeomycin. This marker comprises the bacterial ble gene flanked by 5′ and 3′ sequences from the Chlamydomonas RBCS2 gene. We found that the frequency with which transformants are recovered is significantly increased when ble is fused to shorter versions of the RBCS2 promoter and when Chlamydomonas introns are introduced into the coding region of ble. The latter effect is particularly evident in the case of the first intron of RBCS2, which dramatically stimulates the transformation frequency and the level of ble expression. We found that this improvement is mediated in part by an enhancer element within the intron sequence, and that this element acts in an orientation-independent manner and is effective when placed either upstream or downstream of the promoter. Our results demonstrate that stable high-level expression of a foreign gene in Chlamydomonas is possible, and highlight a potential role of introns as modulators of gene expression in this alga.

Journal ArticleDOI
TL;DR: In this article, a soil fungus, Pythium irregulare, was found to blight jar1-1, an Arabidopsis jasmonate response mutant that exhibits reduced sensitivity to methyl jamasmonate, indicating that increased susceptibility was due to the lesion in the JAR1 locus.
Abstract: Jasmonic acid has properties of a plant hormone, including the induction of specific genes associated with plant defense. We previously described jar1-1, an Arabidopsis jasmonate response mutant that exhibits reduced sensitivity to methyl jasmonate. We have further characterized this mutant and two new alleles; jar1-2 from a gamma irradiated population, and jar1-4 from a T-DNA mutant population. Seedling root growth in jar1-1 was equally insensitive to methyl jasmonate and jasmonic acid, indicating that the defect was not in the conversion of methyl jasmonate to the acid. None of the jar1 mutants showed an altered sensitivity to auxin, cytokinin, or the ethylene precursor 1-aminocyclopropane-1-carboxylic acid, indicating that the lesion does not affect the general uptake or transport of hormones. A soil fungus, Pythium irregulare, was found to blight jar1-1. Cultures of this organism caused the symptoms in all three jar1 mutants but not in wild type, indicating that increased susceptibility was due to the lesion in the JAR1 locus. A fatty acid desaturase triple mutant that is defective in the biosynthesis of jasmonic acid (J. Browse, Washington State University) was also susceptible, confirming that jasmonate is involved in resistance. The jar1-1 locus was mapped to the lower end of chromosome 2, about 11.4 cM from as1 and 1.6 cM from cer8. These results establish that jasmonate signaling plays an important role in resistance to soil micro-organisms in plants.

Journal ArticleDOI
TL;DR: Genetic analysis demonstrated that the shoot gravitropism mutants sgr1 and sgr7 are allelic to the radial pattern mutants, scr and shr, respectively, and demonstrated that SCR and SHR regulate the radial organization of the shoot axial organs in Arabidopsis.
Abstract: Shoots of higher plants exhibit negative gravitropism. However, little is known about the mechanism or site of gravity perception in shoots. We have identified two loci that are essential for normal shoot gravitropism in Arabidopsis thaliana. Genetic analysis demonstrated that the shoot gravitropism mutants sgr1 and sgr7 are allelic to the radial pattern mutants, scr and shr, respectively. Characterization of the aerial phenotype of these mutants revealed that the primary defect is the absence of a normal endodermis in hypocotyls and influorescence stems. This indicates that the endodermis is essential for shoot gravitropism and strongly suggests that this cell layer functions as the gravity-sensing cell layer in dicotyledonous plant shoots. These results also demonstrate that, in addition to their previously characterized role in root radial patterning, SCR and SHR regulate the radial organization of the shoot axial organs in Arabidopsis.

Journal ArticleDOI
TL;DR: In this paper, a sequence analysis of mutant and wild-type FUSCA3 (FUS3) alleles, as well as sequencing of fus3 cDNAs, revealed small inframe deletions at two different sites of the coding region.
Abstract: Conditionally lethal mutant alleles of the FUSCA3 (FUS3) gene of Arabidopsis thaliana are specifically defective in the gene expression program responsible for seed maturation. FUS3 was isolated by map-based cloning and expression of the FUS3 cDNA resulted in complementation of the Fus3- phenotype. In the predicted FUS3 gene product, a continuous stretch of more than 100 amino acids shows significant sequence similarity to the B3 domains of the polypeptides encoded by ABI3 (Arabidopsis) and VP1 (maize). FUS3 transcription was detected mainly in siliques and was found to be developmentally regulated during embryogenesis. Transcripts of abnormal sizes were observed in fus3 mutants due to aberrant splicing caused by point mutations at intron termini. Sequence analysis of mutant and wild-type FUS3 alleles, as well as sequencing of fus3 cDNAs, revealed small inframe deletions at two different sites of the coding region. While a deletion between B3 and the C-terminus of the predicted polypeptide was found in conjunction with normal FUS3 function, another deletion located within the conserved B3 domain (as well as truncations therein) were associated with the Fus3- phenotype. It is apparent, therefore, that an intact B3 domain is essential for the regulation of seed maturation by FUS3.

Journal ArticleDOI
TL;DR: It is shown that mechanisms in addition to regulating substrate (LA) availability and the regulation of AOS accumulation control the output of the octadecanoid pathway.
Abstract: Summary The analysis of allene oxide synthase (AOS) mRNA levels, of AOS polypeptide levels and specific enzymatic activities, as well as the quantitative determination of the levels of the octadecanoids cis-12-oxophytodienoic acid (cis-OPDA) and JA following a number of treatments, has shown that AOS is a regulatory site in octadecanoid biosynthesis in A. thaliana. AOS activity, mRNA and polypeptide levels are increased in wounded leaves locally and systemically. The methyl esters of OPDA or JA (OPDAME, JAME) and coronatine, are strong inducers of AOS mRNA, polypeptide and enzymatic activity. Ethephon also induces AOS activity. Salicylic acid (SA) was an inducer of AOS activity while abscisic acid (ABA) had no effect. At the level of the octadecanoids, the consequences of induction of AOS by the different inducers were distinctly different, depending on the nature of the inducer. Wounding led to a strong, bi-phasic accumulation of JA in wounded leaves and to a less pronounced increase in JA-levels in systemic leaves. Levels of OPDA changed very little in wounded leaves and remained constant or even declined in systemic leaves. Ethephon treatment resulted in a strong, transient increase in JA-levels kinetically coinciding with the second, more pronounced peak in wound-induced JA. In SA-treated leaves, the level of cis-OPDA increased throughout the experimental period while there was no effect on JA levels during the first 24 h following treatment and only a slight accumulation after 48 h. Clearly, mechanisms in addition to regulating substrate (LA) availability and the regulation of AOS accumulation control the output of the octadecanoid pathway.

Journal ArticleDOI
TL;DR: Arabidopsis thaliana is presented using meiotic pachytene cells in combination with fluorescence in situ hybridization and the detection of unique cosmids and YAC sequences demonstrates that detailed physical mapping of Arabidopsis chromosomes by cytogenetic techniques is feasible.
Abstract: A detailed karyotype of Arabidopsis thaliana is presented using meiotic pachytene cells in combination with fluorescence in situ hybridization. The lengths of the five pachytene bivalents varied between 50 and 80 microns, which is 20-25 times longer than mitotic metaphase chromosomes. The analysis confirms that the two longest chromosomes (1 and 5) are metacentric and the two shortest chromosomes (2 and 4) are acrocentric and carry NORs subterminally in their short arms, while chromosome 3 is submetacentric and medium sized. Detailed mapping of the centromere position further revealed that the length variation between the pachytene bivalents comes from the short arms. Individual chromosomes were unambiguously identified by their combinations of relative lengths, arm-ratios, presence of NOR knobs and FISH signals with a 5S rDNA probe and chromosome specific DNA probes. Polymorphisms were found among six ecotypes with respect to the number and map positions of 5S rDNA loci. All ecotypes contain 5S rDNA in the short arms of chromosomes 4 and 5. Three different patterns were observed regarding the presence and position of a 5S rDNA locus on chromosome 3. Repetitive DNA clones enabled us to subdivide the pericentromeric heterochromatin into a central domain, characterized by pAL1 and 106B repeats, which accommodate the functional centromere and two flanking domains, characterized by the 17 A20 repeat sequences. The upper flanking domains of chromosomes 4 and 5, and in some ecotypes also chromosome 3, contain a 5S rDNA locus. The detection of unique cosmids and YAC sequences demonstrates that detailed physical mapping of Arabidopsis chromosomes by cytogenetic techniques is feasible. Together with the presented karyotype this makes Arabidopsis a model system for detailed cytogenetic mapping.

Journal ArticleDOI
TL;DR: Insect bioassays and feeding studies showed that GNA expressed in the transgenic rice plants decreased survival and overall fecundity of the insects, retarded insect development, and had a deterrent effect on BPH feeding.
Abstract: Summary Snowdrop lectin (Galanthus nivalis agglutinin; GNA) has been shown previously to be toxic towards rice brown planthopper (Nilaparvata lugens; BPH) when administered in artificial diet. BPH feeds by phloem abstraction, and causes ‘hopper burn’, as well as being an important virus vector. To evaluate the potential of the gna gene to confer resistance towards BPH, transgenic rice (Oryza sativa L.) plants were produced, containing the gna gene in constructs where its expression was driven by a phloemspecific promoter (from the rice sucrose synthase RSs1 gene) and by a constitutive promoter (from the maize ubiquitin ubi1 gene). PCR and Southern analyses on DNA from these plants confirmed their transgenic status, and that the transgenes were transmitted to progeny after selffertilization. Western blot analyses revealed expression of GNA at levels of up to 2.0% of total protein in some of the transgenic plants. GNA expression driven by the RSs1 promoter was tissue-specific, as shown by immunohistochemical localization of the protein in the non-lignified vascular tissue of transgenic plants. Insect bioassays and feeding studies showed that GNA expressed in the transgenic rice plants decreased survival and overall fecundity (production of offspring) of the insects, retarded insect development, and had a deterrent effect on BPH feeding.

Journal ArticleDOI
Romero1, Fuertes1, Benito1, Malpica1, Leyva1, Paz‐Ares1 
TL;DR: DNA-binding studies showed that there are differences but also frequent overlaps in binding specificity among plant R2R3-MYB proteins, in line with the distinct but often related functions that are beginning to be recognized for these proteins.
Abstract: Transcription factors belonging to the R2R3-MYB family contain the related helix-turn-helix repeats R2 and R3. The authors isolated partial cDNA and/or genomic clones of 78 R2R3-MYB genes from Arabidopsis thaliana and found accessions corresponding to 31 Arabidopsis genes of this class in databanks, seven of which were not represented in the authors' collection. Therefore, there are at least 85, and probably more than 100, R2R3-MYB genes present in the Arabidopsis thaliana genome, representing the largest regulatory gene family currently known in plants. In contrast, no more than three R2R3-MYB genes have been reported in any organism from other phyla. DNA-binding studies showed that there are differences but also frequent overlaps in binding specificity among plant R2R3-MYB proteins, in line with the distinct but often related functions that are beginning to be recognized for these proteins. This large-sized gene family may contribute to the regulatory flexibility underlying the developmental and metabolic plasticity displayed by plants.

Journal ArticleDOI
TL;DR: The results indicate that the product of the MRP-like gene of A. thaliana is capable of mediating the transport of the two different classes of compounds.
Abstract: An ABC-transporter of Arabidopsis thaliana exhibiting high sequence similarity to the human (MRP1) and yeast (YCF1) glutathione-conjugate transporters has been analysed and used to complement a cadmium-sensitive yeast mutant (DTY168) that also lacks glutathione-conjugate transport activity. Comparison of the hydrophobicity plots of this A. thaliana MRP-like protein with MRP1 and YCF1 demonstrates that the transmembrane domains are conserved, even at the N-terminus where sequence identity is low. Cadmium resistance is partially restored in the complemented ycf1 mutant, and glutathione-conjugate transport activity can be observed as well. The kinetic properties of the A. thaliana MRP-like protein (AtMRP3) are very similar to those previously described for the vacuolar glutathione-conjugate transporter of barley and mung bean. Furthermore, a hitherto undescribed ATP-dependent transport activity could be correlated with the gene product, i.e. vesicles isolated from the complemented yeast, but not from DTY168 or the wild type, take up the chlorophyll catabolite Bn-NCC-1. The results indicate that the product of the MRP-like gene of A. thaliana is capable of mediating the transport of the two different classes of compounds.

Journal ArticleDOI
TL;DR: The results indicate that ozone effects are amplified in the sensitive tobacco cv.
Abstract: Summary Localized cell death is a common feature of ozone phytotoxicity and is generally thought to be initiated by the strong oxidant ozone itself as well as by ozone-derived reactive oxygen intermediates (ROIs). Here we report that ozone (150 nl l–1, 5 h) elicits cellular ROI production in the ozone-sensitive tobacco cv. Bel W3, but not in the tolerant cv. Bel B. Both cultivars exhibited a transient first maximum of apoplastic ROI accumulation followed by a comparable induction of glutathione peroxidase transcript levels. During postcultivation in pollutant-free air, a second and sustained peak of apoplastic ROI accumulation was detected only in cv. Bel W3. Histochemical staining revealed a spot-like accumulation of H2O2 and, to a lesser extent, of superoxide anion radicals in this cultivar. The H2O2 spots (‘burst initiation sites’) occurred mainly in the vicinity of leaf veins and correlated in number and distribution with discrete sites of local cell death and with visible symptoms that evolved between 15 and 72 h. The results indicate that ozone effects are amplified in the sensitive tobacco cv. Bel W3 by an oxidative burst which participates in the generation of hypersensitive cell death-like lesions.

Journal ArticleDOI
TL;DR: It is reported here that maize bm1, one of the less characterised mutants, shows severely reduced CAD activity in lignified tissues, resulting in the production of a modified lignin.
Abstract: Brown-midrib (bm) mutants of maize have modified lignin of reddish-brown colour. Although four independent bm loci are known, only one of the mutant genes has been previously identified. We report here that maize bm1, one of the less characterised mutants, shows severely reduced CAD activity in lignified tissues, resulting in the production of a modified lignin. Both the total lignin content and the structure of the polymer are altered by the mutation. We further describe the isolation and characterisation of the maize CAD cDNA and mapping of the CAD gene. CAD maps very closely to the known location of bm1 and co-segregates with the bm1 locus in two independent recombinant inbred populations. These data strongly support the premise that maize bm1 directly affects expression of the CAD gene.

Journal ArticleDOI
TL;DR: The results obtained by this study clearly demonstrate that aquaporins are important for plant water transport.
Abstract: The plant plasma membrane intrinsic protein, PIP1b, facilitates water transport. These features were characterized in Xenopus oocytes and it has asked whether aquaporins are relevant for water transport in plants. In order to elucidate this uncertainty Arabidopsis thaliana was transformed with an anti-sense construct targeted to the PIP1b gene. Molecular analysis revealed that the anti-sense lines have reduced steady-state levels of PIP1b and the highly homologous PIP1a mRNA. The cell membrane water permeability was analyzed by swelling of protoplasts, which had been transferred into hypotonic conditions. The results indicate that the reduced expression of the specific aquaporins decreases the cellular osmotic water permeability coefficient approximately three times. The morphology and development of the anti-sense lines resembles that of control plants, with the exception of the root system, which is five times as abundant as that of control plants. Xylem pressure measurement suggests that the increase of root mass compensates the reduced cellular water permeability in order to ensure a sufficient water supply to the plant. The results obtained by this study, therefore, clearly demonstrate that aquaporins are important for plant water transport.

Journal ArticleDOI
TL;DR: Results suggest that SUR2 may be involved in the control of auxin conjugation, which is a new key point in the regulation of endogenous auxin concentrations.
Abstract: A new auxin homeostasis gene in Arabidopsis called SUR2 has been identified. This gene, mapped to the bottom of chromosome 4, is defined by two recessive nuclear mutants designated superroot2 (sur2), which display several abnormalities reminiscent of auxin effects. A number of these characteristics are similar to the phenotype of the previously described auxin-overproducing mutant superroot1 (sur1); however, several lines of evidences reveal that the SUR2 gene defines a new key point in the regulation of endogenous auxin concentrations. The phenotype of the sur1 sur2 double mutant is additive. Analysis by gas chromatography coupled to mass spectrometry indicated increased levels of free indole-3-acetic acid correlated with a decreased level of bound auxin in the sur2 mutant. These results suggest that SUR2 may be involved in the control of auxin conjugation.

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TL;DR: Results provide evidence for a sucrose-specific sugar sensing and signalling system in plants and suggest a role for ATB2 in the control of processes associated with the transport or utilization of metabolites.
Abstract: Summary The Arabidopsis bZIP transcription factor gene ATB2 has been shown previously to be expressed in a light-regulated and tissue-specific way. Here we describe the precise localization of ATB2 expression, using transgenic lines containing an ATB2 promoter–GUS reporter gene construct. The observed expression pattern suggests a role for ATB2 in the control of processes associated with the transport or utilization of metabolites. Remarkably, expression of the ATB2–GUS reporter gene construct was specifically repressed by sucrose. Other sugars, such as glucose and fructose, alone or in combination, were ineffective. Repression was observed at external sucrose concentrations exceeding 25 mM. Transcript levels of both the endogenous ATB2 gene and the ATB2–GUS reporter gene were not repressed by sucrose, suggesting that sucrose affects mRNA translation. This translational regulation involves the ATB2 leader sequence because deletion of the leader resulted in loss of sucrose repression. Our results provide evidence for a sucrose-specific sugar sensing and signalling system in plants.