Identification and characterization of victorin sensitivity in Arabidopsis thaliana

Cochliobolus victoriae is a necrotrophic fungus that produces a host-selective toxin called victorin. Victorin is considered to be host selective because it has been known to affect only certain allohexaploid oat cultivars containing the dominant Vb gene. Oat cultivars containing Vb are also the only genotypes susceptible to C. victoriae. Assays were developed to screen the "nonhost" plant of C. victoriae, Arabidopsis thaliana, for victorin sensitivity. Sensitivity to victorin was identified in six of 433 bulk populations of Arabidopsis. In crosses of Col-4 (victorin-insensitive) x victorin-sensitive Arabidopsis ecotypes, victorin sensitivity segregated as a single dominant locus, as it does in oats. This Arabidopsis locus was designated LOV, for locus orchestrating victorin effects. Allelism tests indicate that LOV loci are allelic or closely linked in all six victorin-sensitive ecotypes identified. LOV was localized to the north arm of Arabidopsis thaliana chromosome I. The victorin-sensitive Arabidopsis line LOV1 but not the victorin-insensitive line Col-4 was susceptible to C. victoriae infection. Consequently, the LOV gene appears to be a genetically dominant, disease susceptibility gene.     

Characterization of natural and induced variation in the LOV1 gene, a CC-NB-LRR gene conferring victorin sensitivity and disease susceptibility in Arabidopsis

The fungus Cochliobolus victoriae, the causal agent of Victoria blight, produces a compound called victorin that is required for pathogenicity of the fungus. Victorin alone reproduces disease symptoms on sensitive plants. Victorin sensitivity and susceptibility to C. victoriae were originally described on oats but have since been identified on Arabidopsis thaliana. Victorin sensitivity and disease susceptibility in Arabidopsis are conferred by LOV1, a coiled-coil-nucleotide-binding-leucine-rich repeat (CC-NB-LRR) protein. We sequenced the LOV1 gene from 59 victorin-insensitive mutants and found that the spectrum of mutations causing LOV1 loss of function was similar to that found to cause loss of function of RPM1, a CC-NB-LRR resistance protein. Also, many of the mutated residues in LOV1 are in conserved motifs required for resistance protein function. These data indicate that LOV1 may have a mechanism of action similar to resistance proteins. Victorin sensitivity was found to be the prevalent phenotype in a survey of 30 Arabidopsis ecotypes, and we found very little genetic variation among LOV1 alleles. As selection would not be expected to preserve a functional LOV1 gene to confer victorin sensitivity and disease susceptibility, we propose that LOV1 may function as a resistance gene to a naturally-occurring pathogen of Arabidopsis.     

Thioredoxin is required for vacuole inheritance in Saccharomyces cerevisiae

The vacuole of Saccharomyces cerevisiae projects a stream of tubules a and vesicles (a segregation structure) into the bud in early S phase. We have described an in vitro reaction, requiring physiological temperature, ATP, and cytosol, in which isolated vacuoles form segregation structures and fuse. This in vitro reaction is defective when reaction components are prepared from vac mutants that are defective in this process in vivo, Fractionation of the cytosol reveals at least three components, each of which can support the vacuole fusion reaction, and two stimulatory fractions. Purification of one low molecular weight activity (LMA1) yields a heterodimeric protein with a thioredoxin subunit. Most of the thioredoxin of yeast is in this complex rather than the well-studied monomer. A deletion of both S. cerevisiae thioredoxin genes causes a striking vacuole inheritance defect in vivo, establishing a role for thioredoxin as a novel factor in this trafficking reaction.     

The BIG gene is required for auxin-mediated organ growth in Arabidopsis

Control of organ size by cell expansion and cell proliferation is a fundamental process during development, but the importance of BIG in this process is still poorly understood. Here, we report the isolation and characterization of a new allele mutant of BIG in Arabidopsis: big-j588. The mutant displayed small aerial organs that were characterized by reduced cell size in the epidermis and short roots with decreased cell numbers. The big-j588 axr1 double and big-j588 arf7 arf19 triple mutants displayed more severe defects in leaf expansion and root elongation than their parents, implying BIG is involved in auxin-dependent organ growth. Genetic analysis suggests that BIG may act synergistically with PIN1 to affect leaf growth. The PIN1 protein level decreased in both the root cells and the tips of leaf pavement cell lobes of big-j588. Further analysis showed that the auxin maxima in the roots and the leaves of big-j588 decreased. Therefore, we concluded that the small leaves and the short roots of big-j588 were associated with reduction of auxin maxima. Overall, our study suggested that BIG is required for Arabidopsis organ growth via auxin action.     

The Arabidopsis Ethylene-Insensitive 2 gene is required for lead resistance

The Arabidopsis Ethylene-Insensitive 2 (EIN2) gene has been shown to be involved in the regulation of abiotic and biotic stresses, including ozone stress, high salt, oxidative stress and disease resistance. However, little is known about the role of EIN2 gene in lead (Pb) resistance in Arabidopsis. In this study, we showed that EIN2 gene is required for Pb(II) resistance in Arabidopsis. EIN2 gene was induced by Pb(II) treatment, and the ein2-1 mutant showed enhanced sensitivity to Pb(II). A higher Pb content was detected in ein2-1 plants than in wild-type plants when subjected to Pb(II) treatment, which was associated, at least in part, with reduction in expression of AtPDR12 gene, a pump excluding Pb(II) and/or Pb(II)-containing toxic compounds from the cytoplasm. Moreover, the ein2-1 mutation also impaired glutathione (GSH)-dependent Pb(II) resistance, which was related to constitutive reduction of express of GSH1 gene involved in GSH synthesis and consequently reduced GSH content. Taken together, all these results suggest that EIN2 gene mediates Pb(II) resistance, at least in part, through two distinct mechanisms, a GSH-dependent mechanism and a GSH-independent AtPDR12-mediated mechanism.     

Cloning and characterization of IAR1, a gene required for auxin conjugate sensitivity in Arabidopsis

Most indole-3-acetic acid (IAA) in higher plants is conjugated to amino acids, sugars, or peptides, and these conjugates are implicated in regulating the concentration of the free hormone. We identified iar1 as an Arabidopsis mutant that is resistant to the inhibitory effects of several IAA-amino acid conjugates but remains sensitive to free IAA. iar1 partially suppresses phenotypes of a mutant that overproduces IAA, suggesting that IAR1 participates in auxin metabolism or response. We used positional information to clone IAR1, which encodes a novel protein with seven predicted transmembrane domains and several His-rich regions. IAR1 has homologs in other multicellular organisms, including Drosophila, nematodes, and mammals; in addition, the mouse homolog KE4 can functionally substitute for IAR1 in vivo. IAR1 also structurally resembles and has detectable sequence similarity to a family of metal transporters. We discuss several possible roles for IAR1 in auxin homeostasis.     

Cdi gene is required for pollen germination and tube growth in Arabidopsis

Pollen germination and tube growth are of essential importance to sexual reproduction of flowering plants. Several biological processes including cell wall biosynthesis and modification are known to be involved in pollen tube growth, though the underlying molecular mechanisms remain largely to be investigated. Here we report the identification and functional characterization of the Arabidopsis gene Cdi, which encodes a putative nucleotide-diphospho-sugar transferase. Cdi is preferentially expressed in pollen grains and pollen tubes. Transient expression of Cdi:GFP fusion protein showed that CDI is localized in the cytosol. Mutation in Cdi impaired pollen germination and pollen tube growth thus leading to disrupted male transmission. These results suggest that Cdi is an essential gene required for pollen germination and tube growth.     

The dyad gene is required for progression through female meiosis in Arabidopsis

In higher plants the gametophyte consists of a gamete in association with a small number of haploid cells, specialized for sexual reproduction. The female gametophyte or embryo sac, is contained within the ovule and develops from a single cell, the megaspore which is formed by meiosis of the megaspore mother cell. The dyad mutant of Arabidopsis, described herein, represents a novel class among female sterile mutants in plants. dyad ovules contain two large cells in place of an embryo sac. The two cells represent the products of a single division of the megaspore mother cell followed by an arrest in further development of the megaspore. We addressed the question of whether the division of the megaspore mother cell in the mutant was meiotic or mitotic by examining the expression of two markers that are normally expressed in the megaspore mother cell during meiosis. Our observations indicate that in dyad, the megaspore mother cell enters but fails to complete meiosis, arresting at the end of meiosis 1 in the majority of ovules. This was corroborated by a direct observation of chromosome segregation during division of the megaspore mother cell, showing that the division is a reductional and not an equational one. In a minority of dyad ovules, the megaspore mother cell does not divide. Pollen development and male fertility in the mutant is normal, as is the rest of the ovule that surrounds the female gametophyte. The embryo sac is also shown to have an influence on the nucellus in wild type. The dyad mutation therefore specifically affects a function that is required in the female germ cell precursor for meiosis. The identification and analysis of mutants specifically affecting female meiosis is an initial step in understanding the molecular mechanisms underlying early events in the pathway of female reproductive development.     

The Arabidopsis MutS homolog AtMSH5 is required for normal meiosis

MSH5, a member of the MutS homolog DNA mismatch repair protein family, has been shown to be required for proper homologous chromosome recombination in diverse organisms such as mouse, budding yeast and Caenorhabditis elegans. In this paper, we show that a mutant Arabidopsis plant carrying the putative disrupted AtMSH5 gene exhibits defects during meiotic division, producing a proportion of nonviable pollen grains and abnormal embryo sacs, and thereby leading to a decrease in fertility. AtMSH5 expression is confined to meiotic floral buds, which is consistent with a possible role during meiosis. Cytological analysis of male meiosis revealed the presence of numerous univalents from diplotene to metaphase I, which were associated with a great reduction in chiasma frequencies. The average number of residual chiasmata in the mutant is reduced to 2.54 per meiocyte, which accounts for approximately 25% of the amount in the wild type. Here, quantitative cytogenetical analysis reveals that the residual chiasmata in Atmsh5 mutants are randomly distributed among meiocytes, suggesting that AtMSH5 has an essential role during interference-sensitive chiasma formation. Taken together, the evidence indicates that AtMSH5 promotes homologous recombination through facilitating chiasma formation during prophase I in Arabidopsis.     

The Arabidopsis FILAMENTOUS FLOWER gene is required for flower formation.

A screen for mutations affecting flower formation was carried out and several filamentous flower (fil) alleles were identified. In fil mutants, floral primordia occasionally give rise to pedicels lacking flowers at their ends. This defect is dramatically enhanced in fil rev double mutants, in which every floral primordium produces a flowerless pedicel. These data suggest that the FIL and REV genes are required for an early step of flower formation, possibly for the establishment of a flower-forming domain within the floral primordium. The FIL gene is also required for establishment of floral meristem identity and for flower development. During flower development, the FIL gene is required for floral organ formation in terms of the correct numbers and positions; correct spatial activity of the AGAMOUS, APETALA3, PISTILLATA and SUPERMAN genes; and floral organ development.     

Thioredoxin is required for deoxyribonucleotide pool maintenance during S phase

Thioredoxin was initially identified by its ability to serve as an electron donor for ribonucleotide reductase in vitro. Whether it serves a similar function in vivo is unclear. In Saccharomyces cerevisiae, it was previously shown that Deltatrx1 Deltatrx2 mutants lacking the two genes for cytosolic thioredoxin have a slower growth rate because of a longer S phase, but the basis for S phase elongation was not identified. The hypothesis that S phase protraction was due to inefficient dNTP synthesis was investigated by measuring dNTP levels in asynchronous and synchronized wild-type and Deltatrx1 Deltatrx2 yeast. In contrast to wild-type cells, Deltatrx1 Deltatrx2 cells were unable to accumulate or maintain high levels of dNTPs when alpha-factor- or cdc15-arrested cells were allowed to reenter the cell cycle. At 80 min after release, when the fraction of cells in S phase was maximal, the dNTP pools in Deltatrx1 Deltatrx2 cells were 60% that of wild-type cells. The data suggest that, in the absence of thioredoxin, cells cannot support the high rate of dNTP synthesis required for efficient DNA synthesis during S phase. The results constitute in vivo evidence for thioredoxin being a physiologically relevant electron donor for ribonucleotide reductase during DNA precursor synthesis.     

The Arabidopsis ATK1 gene is required for spindle morphogenesis in male meiosis

The spindle plays a central role in chromosome segregation during mitosis and meiosis. In particular, various kinesins are thought to play crucial roles in spindle structure and function in both mitosis and meiosis of fungi and animals. A group of putative kinesins has been previously identified in Arabidopsis, called ATK1-ATK4 (previously known as KATA-KATD), but their in vivo functions have not been tested with genetic studies. We report here the isolation and characterization of a mutant, atk1-1, which has a defective ATK1 gene. The atk1-1 mutant was identified in a collection of Ds transposon insertion lines by its reduced fertility. Reciprocal crosses between the atk1-1 mutant and wild type showed that only male fertility was reduced, not female fertility. Molecular analyses, including revertant studies, indicated that the Ds insertion in the ATK1 gene was responsible for the fertility defect. Light microscopy revealed that, in the atk1-1 mutant, male meiosis was defective, producing an abnormal number of microspores of variable sizes. Further cytological studies indicated that meiotic chromosome segregation and spindle organization were both abnormal in the mutant. Specifically, the atk1-1 mutant male meiotic cells had spindles that were broad, unfocused and multi-axial at the poles at metaphase I, unlike the typical fusiform bipolar spindle found in the wild-type metaphase I cells. Therefore, the ATK1 gene plays a crucial role in spindle morphogenesis in male Arabidopsis meiosis.     

ACAULIS5, an Arabidopsis gene required for stem elongation, encodes a spermine synthase

Polyamines have been implicated in a wide range of biological processes, including growth and development in bacteria and animals, but their function in higher plants is unclear. Here we show that the Arabidopsis: ACAULIS5 (ACL5) gene, whose inactivation causes a defect in the elongation of stem internodes by reducing cell expansion, encodes a protein that shares sequence similarity with the polyamine biosynthetic enzymes spermidine synthase and spermine synthase. Expression of the recombinant ACL5 protein in Escherichia coli showed that ACL5 possesses spermine synthase activity. Restoration of the acl5 mutant phenotype by somatic reversion of a transposon-induced allele suggests a non-cell-autonomous function for the ACL5 gene product. We also found that expression of the ACL5 cDNA under the control of a heat shock gene promoter in acl5 mutant plants restores the phenotype in a heat shock-dependent manner. The results of the experiments showed that polyamines play an essential role in promotion of internode elongation through cell expansion in Arabidopsis: We discuss the relationships to plant growth regulators such as auxin and gibberellins that have related functions.     

The Arabidopsis gene PROLIFERA is required for proper cytokinesis during seed development

The Arabidopsis thaliana (L.) Heynh. gene PROLIFERA (PRL) is a member of the MCM family of genes that are required for DNA replication during the S phase of the cell cycle. PRL is expressed in dividing cells throughout plant development. During reproductive development, PRL is expressed in both the developing megaspore mother cells and microspore mother cells, but is not expressed in the developing microgametophyte, suggesting that it does not function in the final haploid divisions leading to the production of a mature pollen grain. Disruption of PRL leads to megagametophyte and embryo lethality. prl mutant embryos arrest at a variety of stages, and often show defects in cytokinesis. Multinucleate cells and non-stereotypical cell division planes are commonly observed in developing prl mutant embryos, although mcm mutations in other organisms have not been reported to affect cytokinesis. These observations suggest that PRL may play a role in cytokinesis that is distinct from its role in regulating DNA replication. Additionally, a novel cytokinesis checkpoint that monitors cell cycle progression may exist in Arabidopsis.     

SOS4, a pyridoxal kinase gene,is required for root hair development in Arabidopsis

Root hair development in plants is controlled by many genetic, hormonal, and environmental factors. A number of genes have been shown to be important for root hair formation. Arabidopsis salt overly sensitive 4 mutants were originally identified by screening for NaCl-hypersensitive growth. The SOS4 (Salt Overly Sensitive 4) gene was recently isolated by map-based cloning and shown to encode a pyridoxal (PL) kinase involved in the production of PL-5-phosphate, which is an important cofactor for various enzymes and a ligand for certain ion transporters. The root growth of sos4 mutants is slower than that of the wild type. Microscopic observations revealed that sos4 mutants do not have root hairs in the maturation zone. The sos4 mutations block the initiation of most root hairs, and impair the tip growth of those that are initiated. The root hairless phenotype of sos4 mutants was complemented by the wild-type SOS4 gene. SOS4 promoter-beta-glucuronidase analysis showed that SOS4 is expressed in the root hair and other hair-like structures. Consistent with SOS4 function as a PL kinase, in vitro application of pyridoxine and pyridoxamine, but not PL, partially rescued the root hair defect in sos4 mutants. 1-Aminocyclopropane-1-carboxylic acid and 2,4-dichlorophenoxyacetic acid treatments promoted root hair formation in both wild-type and sos4 plants, indicating that genetically SOS4 functions upstream of ethylene and auxin in root hair development. The possible role of SOS4 in ethylene and auxin biosynthesis is discussed.