Identification of an Arabidopsis thaliana mutation (vsm1) that restricts systemic movement of tobamoviruses

Following inoculation, many plant viruses spread locally from cell to cell until they reach the vascular system, through which they then move to other parts of the plant, resulting in systemic infection. To isolate host genes involved in systemic transport of plant viruses, ethyl methanesulfonate-mutagenized Arabidopsis thaliana plants were screened for significant delays in the systemic movement of turnip vein clearing virus (TCVC). One such mutant, designated vsm1 (virus systemic movement), was identified. Unlike the wild-type plants, vsm1 did not develop viral disease and did not allow the systemic spread of the virus. The local viral movement within the inoculated vsm1 leaves, however, was not affected. TVCV systemic movement within the vsm1 plants was likely blocked at the step of viral entry into the host plant vasculature from the infected leaf tissue. vsm1 plants also restricted the systemic movement of another tobamovirus but not of an unrelated carmovirus.     

Suppressors of the arabidopsis lsd5 cell death mutation identify genes involved in regulating disease resistance responses

Cell death is associated with the development of the plant disease resistance hypersensitive reaction (HR). Arabidopsis lsd mutants that spontaneously exhibit cell death reminiscent of the HR were identified previously. To study further the regulatory context in which cell death acts during disease resistance, one of these mutants, lsd5, was used to isolate new mutations that suppress its cell death phenotype. Using a simple lethal screen, nine lsd5 cell death suppressors, designated phx (for the mythological bird Phoenix that rises from its ashes), were isolated. These mutants were characterized with respect to their response to a bacterial pathogen and oomycete parasite. The strongest suppressors-phx2, 3, 6, and 11-1-showed complex, differential patterns of disease resistance modifications. These suppressors attenuated disease resistance to avirulent isolates of the biotrophic Peronospora parasitica pathogen, but only phx2 and phx3 altered disease resistance to avirulent strains of Pseudomonas syringae pv tomato. Therefore, some of these phx mutants define common regulators of cell death and disease resistance. In addition, phx2 and phx3 exhibited enhanced disease susceptibility to different virulent pathogens, confirming probable links between the disease resistance and susceptibility pathways.     

The late elongated hypocotyl mutation of Arabidopsis disrupts circadian rhythms and the photoperiodic control of flowering

The dominant late elongated hypocotyl (lhy) mutation of Arabidopsis disrupted circadian clock regulation of gene expression and leaf movements and caused flowering to occur independently of photoperiod. LHY was shown to encode a MYB DNA-binding protein. In wild-type plants, the LHY mRNA showed a circadian pattern of expression with a peak around dawn but in the mutant was expressed constantly at high levels. Increased LHY expression from a transgene caused the endogenous gene to be expressed at a constant level, suggesting that LHY was part of a feedback circuit that regulated its own expression. Thus, constant expression of LHY disrupts several distinct circadian rhythms in Arabidopsis, and LHY may be closely associated with the central oscillator of the circadian clock.     

Phytochrome controls the number of endoreduplication cycles in the Arabidopsis thaliana hypocotyl

In contrast to mammals, all teleost fish examined thus far exhibit an enormous potential to regenerate not only neuronal processes (axonal regeneration), but even whole neurons (neuronal regeneration) after injuries in the central nervous system. By application of lesions to one subdivision of the cerebellum, the corpus cerebelli, the role of apoptosis in neuronal regeneration was examined in the gymnotiform fish, Apteronotus leptorhynchus. Apoptotic cells were identified by examination of cryosections with the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) reaction, an in situ technique employed for detection of nuclear DNA fragmentation. Additional evidence for the apoptotic nature of dying cells was obtained through analysis of morphologies displayed at both the light microscopic and the ultrastructural level. The first TUNEL-positive cells at the site of the lesion appeared as rapidly as 5 min following mechanical damage of the tissue. Thirty minutes after stab wound lesion, their number reached maximum levels. Starting with 2 days of postlesioning survival time, a gradual decline in the number of TUNEL-positive cells was evident, until this process reached background levels 20 days after the lesion. We hypothesize that apoptosis is used in A. leptorhynchus as an efficient mechanism for the removal of cells damaged through injury in the central nervous system. Since apoptosis is not accompanied by the side-effects known from necrosis (which is predominant after injuries in the mammalian central nervous system), this "clean" type of cell death may, at least partially, explain the tremendous regenerative capability of teleosts.     

Receptor-like protein kinase genes of Arabidopsis thaliana

The isolation of a maize cDNA clone that encodes a membrane spanning protein kinase related to the self-incompatibility glycoproteins (SLG) of Brassica and structurally similar to the growth factor receptor tyrosine kinases has recently been reported. Three distinct receptor-like protein kinase (RLK) cDNA clones from Arabidopsis thaliana have now been identified. Two of the Arabidopsis RLK genes encode SLG-related protein kinases but have different patterns of expression: one is expressed predominantly in rosettes while the other is expressed primarily in roots. The third RLK gene contains an extracellular domain that consists of 21 leucine-rich repeats that are analogous to the leucine-rich repeats found in proteins from humans, flies and yeast. The Arabidopsis leucine-rich gene is expressed at equivalent levels in roots and rosettes. These results show that there are several genes in higher plants that encode members of the receptor protein kinase superfamily. The structural diversity and differential expression of these genes suggest that each plays a distinct and possibly important role in cellular signaling in plants.     

Chromosomal mapping of genes in the RBCS family in Arabidopsis thaliana

We evaluated the usefulness of magnetic resonance angiography (MRA) for showing the topography of paraclinoid carotid artery aneurysms in 27 patients with 30 paraclinoid aneurysms undergoing conventional angiography, three-dimensional time-of-flight MRA and surgery. The anatomy shown on the axial MRA source images was consistent with that found at surgery. The neck of the aneurysm could always be identified on the source images, while it could not be analysed exactly on conventional angiography in 3 cases (10%). The optic nerves, including those displaced by the aneurysm, were recognised in all patients. The anterior clinoid process was shown as a low-intensity rim or area contiguous with the cortical bone. The source images were of great value in understanding the topography of paraclinoid carotid artery aneurysms.     

bor1-1, an Arabidopsis thaliana mutant that requires a high level of boron

Catenins are proteins associated with the cytoplasmic domain of cadherins, a family of transmembrane cell adhesion molecules. The cadherin-catenin adhesion system is involved in morphogenesis during development and in the maintenance of the integrity of different tissue types. Using a gene trap strategy, we have isolated a mouse mutation for the gene encoding the alpha-E-catenin. This form of the alpha-catenin appears frequently coexpressed with E-cadherin in epithelial cell types. The mutation obtained eliminates the carboxyl-terminal third of the protein but nevertheless provokes a complete loss-of-function phenotype. Homozygous mutants show disruption of the trophoblast epithelium (the first differentiated embryonic tissue), and development is consequently blocked at the blastocyst stage. This phenotype parallels the defects observed in E-cadherin mutant embryos. Our results show the requirement of the alpha-E-catenin carboxy terminus for its function and represent evidence of the role of the alpha-E-catenin in vivo, identifying this molecule as the natural partner of the E-cadherin in trophoblast epithelium.     

Suppressors of ectopic UNC-5 growth cone steering identify eight genes involved in axon guidance in Caenorhabditis elegans

The UNC-5 guidance receptor, in response to the UNC-6/netrin path cue, orients growing axons in a dorsal direction along the epidermis of Caenorhabditis elegans. When ectopically expressed in the touch neurons, which normally extend ventrally or longitudinally, UNC-5 is able to reorient their axons toward the dorsal side in an UNC-6-dependent manner. This forms the basis of a genetic screen to identify other mutations that, like unc-6 mutations, suppress unc-5-induced growth cone guidance. These mutations may identify new components required for pioneer axon guidance by unc-5. In this paper, we describe eight genes that are required for ectopic unc-5-induced growth cone steering. Mutations in four of these identify the previously known axon guidance genes unc-6, unc-40, unc-34, and unc-44 and mutations in four others identify the novel genes unc-129, seu-1, seu-2, and seu-3. Several of these mutations cause axon guidance defects similar to those found in unc-5 mutants. We propose that some or all of these genes may function in a developmentally important unc-5 signaling pathway.     

Characterization of an Arabidopsis thaliana gene that defines a new class of putative plant receptor kinases with an extracellular lectin-like domain

We have characterized an Arabidopsis receptor-like serine/threonine kinase gene, Ath.lecRK1 (Arabidopsis thaliana lectin-receptor kinase), defining a new and putatively important class of plant receptor kinases. Structural features of the predicted polypeptide include an amino-terminal membrane-targeting signal sequence, a legume lectin-like extracellular domain, a single membrane-spanning domain, and a characteristic serine/threonine protein kinase domain. A recombinant protein containing the kinase domain can be autophosphorylated on a serine residue. Ath.lecRK1 is a member of a gene family of at least two closely related genes. Northern blot analysis indicates that the Ath.lecRK1 gene is weakly expressed in a variety of organs and is regulated in Arabidopsis cell suspension cultures according to the growth phase of cells. The role this new class of plant receptor kinase could play is discussed with regard to the transduction of oligosaccharide and plant hormone signals.     

Suppressors of cdc25p overexpression identify two pathways that influence the G2/M checkpoint in fission yeast

Checkpoints maintain the order of cell-cycle events. At G2/M, a checkpoint blocks mitosis in response to damaged or unreplicated DNA. There are significant differences in the checkpoint responses to damaged DNA and unreplicated DNA, although many of the same genes are involved in both responses. To identify new genes that function specifically in the DNA replication checkpoint pathway, we searched for high-copy suppressors of overproducer of Cdc25p (OPcdc25(+)), which lacks a DNA replication checkpoint. Two classes of suppressors were isolated. One class includes a new gene encoding a putative DEAD box helicase, suppressor of uncontrolled mitosis (sum3(+)). This gene negatively regulates the cell-cycle response to stress when overexpressed and restores the checkpoint response by a mechanism that is independent of Cdc2p tyrosine phosphorylation. The second class includes chk1(+) and the two Schizosaccharomyces pombe 14-3-3 genes, rad24(+) and rad25(+), which appear to suppress the checkpoint defect by inhibiting Cdc25p. We show that rad24Delta mutants are defective in the checkpoint response to the DNA replication inhibitor hydroxyurea at 37 degrees and that cds1Delta rad24Delta mutants, like cds1Delta chk1Delta mutants, are entirely checkpoint deficient at 29 degrees. These results suggest that chk1(+) and rad24(+) may function redundantly with cds1(+) in the checkpoint response to unreplicated DNA.     

The ROTUNDIFOLIA3 gene of Arabidopsis thaliana encodes a new member of the cytochrome P-450 family that is required for the regulated polar elongation of leaf cells

The polarized processes of cell elongation play a crucial role in morphogenesis of higher plants. We reported previously that the rotundifolia3 (rot3) mutant of Arabidopsis has a defect in the polar elongation of leaf cells. In the present study, we isolated two additional alleles with mutations in the ROT3 gene. The ROT3 gene was cloned by a T-DNA-tagging method and isolation of the gene was confirmed by a molecular analysis of three rot3 mutant alleles obtained from different mutagenesis. The ROT3 gene encodes a cytochrome P-450 (CYP90C1) with domains homologous to regions of steroid hydroxylases of animals and plants. Expression of the ROT3 gene was detected in all major plant organs. Especially, higher expression was detected in the tissues that had high activity of cell division. We confirmed that the ROT3 gene controls polar elongation specifically in leaf cells by an analysis of three rot3 mutants obtained from different mutagenesis experiments. Our results imply that the ROT3 protein is a member of a new class of cytochrome P-450 encoding putative steroid hydroxylases, which is required for the regulated polar elongation of cells in leaves of Arabidopsis.     

UV- and gamma-radiation sensitive mutants of Arabidopsis thaliana

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 an 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.     

Characterization of an Arabidopsis thaliana mutant that has a defect in ABA accumulation: ABA-dependent and ABA-independent accumulation of free amino acids during dehydration

In an attempt to elucidate the physiological role of ABA in seed dormancy and the adaptive response to dehydration, we isolated an ABA-deficient mutant of Arabidopsis thaliana (L.) Heynh. which germinated in the presence of a gibberellin biosynthetic inhibitor. Genetic analysis showed this mutation is a new allele of a recently reported locus aba2, and therefore has been designated aba2-2. The levels of endogenous ABA in fresh and dehydrated tissues of the aba2-2 mutant were highly reduced compared to those of wild-type plants. As a consequence, aba2-2 plants wilt and produce seeds with reduced dormancy. Dark germinated seedlings of the aba2-2 mutant showed true leaves, which were not observed in those of the wild type, indicating that aba2-2 embryos grew precociously during seed maturation. In the dehydrated tissues of the wild-type plants, the levels of free proline, isoleucine and leucine were elevated to a content approximately 100-fold higher than those in fresh tissues. In contrast to the wild-type plants, dehydration-induced accumulation of proline was highly suppressed in the aba2-2 mutant plants while that of leucine and isoleucine accumulated. Furthermore, exogenous application of ABA to wild-type plants promoted accumulation of free proline, but not leucine nor isoleucine. These results suggest that dehydration-induced accumulation of free leucine and isoleucine is achieved independent of ABA.     

The Arabidopsis thaliana gametophytic mutation gemini pollen1 disrupts microspore polarity, division asymmetry and pollen cell fate

Pollen development and male gametogenesis are critically dependent upon cell polarization leading to a highly asymmetric cell division termed pollen mitosis I. A mutational approach was adopted in Arabidopsis thaliana to identify genes involved these processes. Four independent gemini pollen mutants were isolated which produce divided or twin-celled pollen. The gemini pollen1 mutant was characterized in detail and shown to act gametophytically resulting in reduced transmission through both sexes. gemini pollen1 showed an incompletely penetrant phenotype resulting in equal, unequal and partial divisions at pollen mitosis I. The division planes in gemini pollen1 were shown to be aligned with the polar axis (as in wild type) and evidence was obtained for incomplete nuclear migration, which could account for altered division symmetry. gemini pollen1 also showed division phenotypes consistent with spatial uncoupling of karyokinesis and cytokinesis suggesting that GEMINI POLLEN1 may be required for the localization of phragmoplast activity. Cell fate studies showed that in both equal and unequal divisions a vegetative cell marker gene was activated in both daughter cells. Daughter cells with a range of intermediate or hybrid vegetative/generative cell fates suggests that cell fate is quantitatively related to cell size. The potential mode of action of GEMINI POLLEN1 and its effects on cell fate are discussed in relation to proposed models of microspore polarity and cell fate determination.     

Genetic control of male fertility in Arabidopsis thaliana: structural analyses of postmeiotic developmental mutants

Seven new male-sterile mutants (ms7-ms13) of Arabidopsis thaliana (L.) Heynh. (ecotype columbia) are described that show a postmeiotic defect of microspore development. In ms9 mutants, microspores recently released from the tetrad appear irregular in shape and are often without exines. The earliest evidence of abnormality in ms12 mutants is degeneration of microspores that lack normal exine sculpturing, suggesting that the MS12 product is important in the formation of pollen exine. Teratomes (abnormally enlarged microsporocytes) are also occasionally present and each has a poorly developed exine. In ms7 mutant plants, the tapetal cytoplasm disintegrates at the late vacuolate microspore stage, apparently causing the degeneration of microspores and pollen grains. With ms8 mutants, the exine of the microspores appears similar to that of the wild type. However, intine development appears impaired and pollen grains rupture prior to maturity. In ms11 mutants, the first detectable abnormality appears at the mid to late vacuolate stage. The absence of fluorescence in the microspores and tapetal cells after staining with 4',6-diamidino-2-phenylindole (DAPI) and the occasional presence of teratomes indicate degradation of DNA. Viable pollen from ms10 mutant plants is dehisced from anthers but appears to have surface abnormalities affecting interaction with the stigma. Pollen only germinates in high-humidity conditions or during in-vitro germination experiments. Mutant plants also have bright-green stems, suggesting that ms10 belongs to the eceriferum (cer) class of mutants. However, ms10 and cer6 are non-allelic. The ms13 mutant has a similar phenotype to ms10, suggesting is also an eceriferum mutation. Each of these seven mutants had a greater number of flowers than congenic male-fertile plants. The non-allelic nature of these mutants and their different developmental end-points indicate that seven different genes important for the later stages of pollen development have been identified.     

Floral organ-specific and constitutive expression of an Arabidopsis thaliana heat-shock HSP18.2::GUS fusion gene is retained even after homeotic conversion of flowers by mutation

Organ-specific and constitutive expression of the Arabidopsis HSP18.2 gene under normal growth conditions (22 degrees C) was observed in transgenic A. thaliana, which carried a fusion gene composed of the promoter region of HSP18.2, one of the genes for low molecular weight heat-shock proteins in Arabidopsis, and the gene for beta-glucuronidase (GUS) from Escherichia coli. In order to clarify the organ-specific nature of promoter expression, various mutations that affect flower morphology were introduced into the transgenic Arabidopsis line, AHS9. The results show that the pattern of expression observed in sepals, filaments, and styles is regulated in a structure-dependent manner, and suggest that the HSP18.2 gene might have an important role in the process of differentiation of flower buds, as do several other stress-related genes.     

A novel blue light- and abscisic acid-inducible gene of Arabidopsis thaliana encoding an intrinsic membrane protein

Continuous irradiation with blue light (400-500 nm) induces flower formation in plantlets of Arabidopsis thaliana (C24) while red light (600-700 nm) is ineffective. This observation started a search for genes that are activated by blue light and initiate the morphogenic programme leading to flower formation. Several genes were identified via their cDNAs. From these clone AthH2, with an open reading frame for a hydrophobic 30.5 kDa polypeptide, was selected for further characterization of the corresponding gene. From a genomic library a DNA fragment of about 6.4 kb was isolated, comprising the coding region as well as 5'-upstream and 3'-downstream flanking segments. The coding region is composed of four exons, which specify a polypeptide of 286 amino acids. Several potential regulatory elements were found between position -670 and -1140 including GA and ABA sequence motifs. The latter could account for the observed induction of the AthH2 gene by ABA. Southern blot analysis of Arabidopsis genomic DNA suggests that the AthH2 gene is encoded by a single-copy gene. Hydropathy plots and secondary structure analysis of the putative polypeptide predict six membrane-spanning domains implicating a function as transmembrane channel protein. It displays significant homology with the proteins TR7a of pea (82%) and RD 28 of A. thaliana (68%).     

The intron of Arabidopsis thaliana polyubiquitin genes is conserved in location and is a quantitative determinant of chimeric gene expression

We have isolated and determined DNA sequence for the 5'-flanking regions of three Arabidopsis thaliana polyubiquitin genes, UBQ3, UBQ10, and UBQ11. Comparison to cDNA sequences revealed the presence of an intron in the 5'-untranslated region at the same position immediately upstream of the initiator methionine codon in each of the three genes. An intron at this position is also present in two sunflower and two maize polyubiquitin genes. An intron is also found in the 5'-untranslated regions of several animal polyubiquitin genes, although the exact intron position is not conserved among them, and none are in the same position as those in the higher plant polyubiquitin genes. Chimeric genes containing the 5'-flanking regions of UBQ3, UBQ10, and UBQ11 in front of the coding regions for the reporter enzyme Escherichia coli beta-glucuronidase (GUS) were constructed. When introduced transiently into Arabidopsis leaves via microprojectile bombardment, all resulted in readily detectable levels of GUS activity that were quantitatively similar. The introns of UBQ3 and UBQ10 in the corresponding promoter fragments were removed by replacement with flanking cDNA sequences and chimeric genes constructed. These constructs resulted in 2.5- to 3-fold lower levels of marker enzyme activity after transient introduction into Arabidopsis leaves. The UBQ10 promoter without the 5' intron placed upstream of firefly luciferase (LUX) resulted in an average of 3-fold lower LUX activity than from an equivalent construct with the UBQ10 intron. A UBQ3 promoter cassette was constructed for the constitutive expression of open reading frames in dicot plants and it produced readily detectable levels of GUS activity in transient assays.