Genetic evidence that the endodermis is essential for shoot gravitropism in Arabidopsis thaliana

The hepatic clearance of ONO-5046 (N-[2-[4-(2,2-dimethylpropionyloxy)phenylsulphonylamino]benz oyl]aminoacetic acid), a low-molecular-weight neutrophil elastase inhibitor, has been investigated in rats and in the rat perfused liver. This ester was easily hydrolysed to its inactive metabolite EI-601 (N-[2-[(4-hydroxyphenyl)sulphonylamino]benzoyl]aminoacetic acid) in liver homogenate and in erythrocytes suspension in-vitro. On the other hand, it was stable in biological media such as plasma and whole blood, which contain plasma proteins. Scatchard plot analysis of ONO-5046 binding to bovine serum albumin (BSA) in-vitro indicated that the association constant (K) and number of binding sites (n) were 6.91 x 10(4) (M(-1)) and 4.33, respectively. Thus, ONO-5046 (100 microM) would bind to plasma proteins to an extent >99% at physiological plasma-protein concentrations. The total plasma clearance of ONO-5046 in rats was constant (approximately 9 mL min(-1) kg(-1)) under different steady-state plasma concentrations (5-50 microM) a value equivalent to the hepatic clearance. In the rat perfused liver, the hepatic extraction ratio of ONO-5046 was significantly reduced by adding BSA to the dosing solution. Thus, the relatively low hepatic clearance of ONO-5046, which has an ester linkage in its structure and is naturally susceptible to enzymatic hydrolysis, was found to be because of the extremely high protein-binding of the compound.     

A protein phosphatase 2C involved in ABA signal transduction in Arabidopsis thaliana

The plant hormone abscisic acid (ABA) mediates various responses such as stomatal closure, the maintenance of seed dormancy, and the inhibition of plant growth. All three responses are affected in the ABA-insensitive mutant abi1 of Arabidopsis thaliana, suggesting that an early step in the signaling of ABA is controlled by the ABI1 locus. The ABI1 gene was cloned by chromosome walking, and a missense mutation was identified in the structural gene of the abi1 mutant. The ABI1 gene encodes a protein with high similarity to protein serine or threonine phosphatases of type 2C with the novel feature of a putative Ca2+ binding site. Thus, the control of the phosphorylation state of cell signaling components by the ABI1 product could mediate pleiotropic hormone responses.     

The STUD gene is required for male-specific cytokinesis after telophase II of meiosis in Arabidopsis thaliana

During male meiosis in wild-type Arabidopsis the pollen mother cell (PMC) undergoes two meiotic nuclear divisions in the absence of cell division. Only after telophase II is a wall formed which partitions the PMC into four microspores. Each microspore undergoes two subsequent mitotic divisions to produce one vegetative cell and two sperm cells in the mature pollen grain. In this paper we describe the isolation and the phenotypic characterization of mutations in the STUD (STD) gene, which is specifically required for male-specific cytokinesis after telophase II of meiosis. Although the male meiotic nuclear divisions are normal in std mutant plants, no walls are formed resulting in a tetranucleate microspore. Despite the absence of cell division in the PMC, postmeiotic development in the coenocytic microspore proceeds relatively normally, resulting in the formation of large pollen grains which contain four vegetative nuclei and up to eight sperm cells. Interestingly, these enlarged pollen grains which contain multiple vegetative nuclei and extra sperm cells behave as single male gametophytes, producing only single pollen tubes and resulting in partial male fertility in std mutant plants. Characterization of the process of pollen development and pollen function in std mutants thus reveals two different types of developmental regulation. Each of the four nuclei found in a std microspore following meiosis is capable of independently undergoing the complete mitotic cell division (including cytokinesis) which the single nucleus of a wild-type microspore would normally undertake. The ability of the four meiotic products to independently continue through mitosis does not depend on their division into separate cells, but is controlled by some subcellular component found within the coenocytic microspore. By contrast, the mature std pollen grain functions as a unit and produces only a single pollen tube despite the presence of multiple nuclei within the vegetative cell, suggesting that this process is controlled at the cellular level independently of the extra subcellular components.     

A mutation in protein phosphatase 2A regulatory subunit A affects auxin transport in Arabidopsis

The phytohormone auxin controls processes such as cell elongation, root hair development and root branching. Tropisms, growth curvatures triggered by gravity, light and touch, are also auxin-mediated responses. Auxin is synthesized in the shoot apex and transported through the stem, but the molecular mechanism of auxin transport is not well understood. Naphthylphthalamic acid (NPA) and other inhibitors of auxin transport block tropic curvature responses and inhibit root and shoot elongation. We have isolated a novel Arabidopsis thaliana mutant designated roots curl in NPA (rcn1). Mutant seedlings exhibit altered responses to NPA in root curling and hypocotyl elongation. Auxin efflux in mutant seedlings displays increased sensitivity to NPA. The rcn1 mutation was transferred-DNA (T-DNA) tagged and sequences flanking the T-DNA insert were cloned. Analysis of the RCN1 cDNA reveals that the T-DNA insertion disrupts a gene for the regulatory A subunit of protein phosphatase 2A (PP2A-A). The RCN1 gene rescues the rcn1 mutant phenotype and also complements the temperature-sensitive phenotype of the Saccharomyces cerevisiae PP2A-A mutation, tpd3-1. These data implicate protein phosphatase 2A in the regulation of auxin transport in Arabidopsis.     

Arabidopsis AUX1 gene: a permease-like regulator of root gravitropism

The plant hormone auxin regulates various developmental processes including root formation, vascular development, and gravitropism. Mutations within the AUX1 gene confer an auxin-resistant root growth phenotype and abolish root gravitropic curvature. Polypeptide sequence similarity to amino acid permeases suggests that AUX1 mediates the transport of an amino acid-like signaling molecule. Indole-3-acetic acid, the major form of auxin in higher plants, is structurally similar to tryptophan and is a likely substrate for the AUX1 gene product. The cloned AUX1 gene can restore the auxin-responsiveness of transgenic aux1 roots. Spatially, AUX1 is expressed in root apical tissues that regulate root gravitropic curvature.     

NDR1, a pathogen-induced component required for Arabidopsis disease resistance

Plant disease resistance (R) genes confer an ability to resist infection by pathogens expressing specific corresponding avirulence genes. In Arabidopsis thaliana, resistance to both bacterial and fungal pathogens, mediated by several R gene products, requires the NDR1 gene. Positional cloning was used to isolate NDR1, which encodes a 660-base pair open reading frame. The predicted 219-amino acid sequence suggests that NDR1 may be associated with a membrane. NDR1 expression is induced in response to pathogen challenge and may function to integrate various pathogen recognition signals.     

Human RanGTPase-activating protein RanGAP1 is a homologue of yeast Rna1p involved in mRNA processing and transport

RanGAP1 is the GTPase activator for the nuclear Ras-related regulatory protein Ran, converting it to the putatively inactive GDP-bound state. Here, we report the amino acid sequence of RanGAP1, derived from cDNA and peptide sequences. We found it to be homologous to murine Fug1, implicated in early embryonic development, and to Rna1p from Saccharomyces cerevisiae and Schizosaccharomyces pombe. Mutations of budding yeast RNA1 are known to result in defects in RNA processing and nucleocytoplasmic mRNA transport. Concurrently, we have isolated Rna1p as the major RanGAP activity from Sc. pombe. Both this protein and recombinant Rna1p were found to stimulate RanGTPase activity to an extent almost identical to that of human RanGAP1, indicating the functional significance of the sequence homology. The Ran-specific guanine nucleotide exchange factor RCC1 and its yeast homologues are restricted to the nucleus, while Rna1p is reported to be localized to the cytoplasm. We suggest a model in which both activities, nuclear GDP-to-GTP exchange on Ran and cytoplasmic hydrolysis of Ran-bound GTP, are essential for shuttling of Ran between the two cellular compartments. Thus, a defect in either of the two antagonistic regulators of Ran would result in a shutdown of Ran-dependent transport processes, in agreement with the almost identical phenotypes described for such defects in budding yeast.     

Phosphate availability affects the thylakoid lipid composition and the expression of SQD1, a gene required for sulfolipid biosynthesis in Arabidopsis thaliana

Photosynthetic membranes of higher plants contain specific nonphosphorous lipids like the sulfolipid sulfoquinovosyl diacylglycerol in addition to the ubiquitous phospholipid phosphatidylglycerol. In bacteria, an environmental factor that drastically affects thylakoid lipid composition appears to be the availability of phosphate. Accordingly, we discovered an increase in the relative amount of sulfolipid and a concomitant decrease in phosphatidylglycerol in Arabidopsis thaliana grown on medium with reduced amounts of phosphate, as well as in the pho1 mutant of A. thaliana deficient in phosphate transport. To investigate the molecular basis of the observed change in lipid composition, we isolated a cDNA of A. thaliana, designated SQD1, that encodes a protein involved in sulfolipid biosynthesis as suggested by three lines of evidence. First, the cDNA shows high sequence similarity to bacterial sqdB genes known to be essential for sulfolipid biosynthesis; second, the SQD1 gene product is imported into chloroplasts where sulfolipid biosynthesis takes place; and third, transgenic plants expressing SQD1 in antisense orientation show a reduction in sulfolipid content. In the pho1 mutant as well as in wild-type plants grown under reduced phosphate availability, increased amounts of SQD1 mRNA and SQD1 protein are detected, suggesting that the increase in sulfolipid content under phosphate limitation is the result of an increased expression of at least one gene required for sulfolipid biosynthesis in A. thaliana. It is suggested that a certain amount of anionic thylakoid lipid is maintained by substituting sulfolipid for phosphatidylglycerol under reduced phosphate availability.     

The Arabidopsis thaliana RPM1 disease resistance gene product is a peripheral plasma membrane protein that is degraded coincident with the hypersensitive response

Disease resistance in plants is often controlled by a gene-for-gene mechanism in which avirulence (avr) gene products encoded by pathogens are specifically recognized, either directly or indirectly, by plant disease resistance (R) gene products. Members of the NBS-LRR class of R genes encode proteins containing a putative nucleotide binding site (NBS) and carboxyl-terminal leucine-rich repeats (LRRs). Generally, NBS-LRR proteins do not contain predicted transmembrane segments or signal peptides, suggesting they are soluble cytoplasmic proteins. RPM1 is an NBS-LRR protein from Arabidopsis thaliana that confers resistance to Pseudomonas syringae expressing either avrRpm1 or avrB. RPM1 protein was localized by using an epitope tag. In contrast to previous suggestions, RPM1 is a peripheral membrane protein that likely resides on the cytoplasmic face of the plasma membrane. Furthermore, RPM1 is degraded coincident with the onset of the hypersensitive response, suggesting a negative feedback loop controlling the extent of cell death and overall resistance response at the site of infection.     

The yeast SIS1 protein, a DnaJ homolog, is required for the initiation of translation

The S. cerevisiae SIS1 gene is essential and encodes a heat shock protein with similarity to the bacterial DnaJ protein. At the nonpermissive temperature, temperature-sensitive sis1 strains rapidly accumulate 80S ribosomes and have decreased amounts of polysomes. Certain alterations in 60S ribosomal subunits can suppress the temperature-sensitive phenotype of sis1 strains and prevent the accumulation of 80S ribosomes and the loss of polysomes normally seen under conditions of reduced SIS1 function. Analysis of sucrose gradients for SIS1 protein shows that a large fraction of SIS1 is associated with 40S ribosomal subunits and the smaller polysomes. These and other results indicate that SIS1 is required for the normal initiation of translation. Because DnaJ has been shown to mediate the dissociation of several protein complexes, the requirement of SIS1 in the initiation of translation might be for mediating the dissociation of a specific protein complex of the translation machinery.     

Identification and characterization of a novel cap-binding protein from Arabidopsis thaliana

Cap-binding proteins specifically bind to the 7-methyl guanosine (m7G) functional group at the 5' end of eukaryotic mRNAs. A novel Arabidopsis thaliana protein has been identified that has sequence similarity to cap-binding proteins but is clearly a different form of the protein. The most obvious primary sequence difference is the substitution of two of the eight conserved tryptophan residues with other aromatic amino acids in the novel protein. Analogous forms of this novel protein appear to be present in other higher eukaryotes but not in yeast. Analysis of the native and recombinant forms of the novel protein by retention on m7GTP-Sepharose indicate that it is a functional cap-binding protein. Measurements of the dissociation constant for this protein indicate that it binds m7GTP 5-20-fold tighter than eukaryotic initiation factor (eIF)(iso)4E. The novel protein also supports the initiation of translation of capped mRNA in vitro. Biochemical analysis and yeast two-hybrid data indicate that it interacts with eIF(iso)4G to form a complex. Based on these observations, this protein appears to be able to function as a cap-binding protein and is given the designation of novel cap-binding protein (nCBP).     

Std1, a gene involved in glucose transport in Schizosaccharomyces pombe

A wild-type strain, Sp972 h-, of Schizosaccharomyces pombe was mutagenized with ethylmethanesulfonate (EMS), and 2-deoxyglucose (2-DOG)-resistant mutants were isolated. Out of 300 independent 2-DOG-resistant mutants, 2 failed to grow on glucose and fructose (mutants 3/8 and 3/23); however, their hexokinase activity was normal. They have been characterized as defective in their sugar transport properties, and the mutations have been designated as std1-8 and std1-23 (sugar transport defective). The mutations are allelic and segregate as part of a single gene when the mutants carrying them are crossed to a wild-type strain. We confirmed the transport deficiency of these mutants by [14C]glucose uptake. They also fail to grow on other monosaccharides, such as fructose, mannose, and xylulose, as well as disaccharides, such as sucrose and maltose, unlike the wild-type strain. Lack of growth of the glucose transport-deficient mutants on maltose revealed the extracellular breakdown of maltose in S. pombe, unlike in Saccharomyces cerevisiae. Both of the mutants are unable to grow on low concentrations of glucose (10 to 20 mM), while one of them, 3/23, grows on high concentrations (50 to 100 mM) as if altered in its affinity for glucose. This mutant (3/23) shows a lag period of 12 to 18 h when grown on high concentrations of glucose. The lag disappears when the culture is transferred from the log phase of its growth on high concentrations. These mutants complement phenotypically similar sugar transport mutants (YGS4 and YGS5) reported earlier by Milbradt and Hoefer (Microbiology 140:2617-2623, 1994), and the clone complementing YGS4 and YGS5 was identified as the only glucose transporter in fission yeast having 12 transmembrane domains. These mutants also demonstrate two other defects: lack of induction and repression of shunt pathway enzymes and defective mating.     

Liz1p, a novel fission yeast membrane protein, is required for normal cell division when ribonucleotide reductase is inhibited

Ribonucleotide reductase activity is required for generating deoxyribonucleotides for DNA replication. Schizosaccharomyces pombe cells lacking ribonucleotide reductase activity arrest during S phase of the cell cycle. In a screen for hydroxyurea-sensitive mutants in S. pombe, we have identified a gene, liz1(+), which when mutated reveals an additional, previously undescribed role for ribonucleotide reductase activity during mitosis. Inactivation of ribonucleotide reductase, by either hydroxyurea or a cdc22-M45 mutation, causes liz1(-) cells in G2 to undergo an aberrant mitosis, resulting in chromosome missegregation and late mitotic arrest. liz1(+) encodes a 514-amino acid protein with strong similarity to a family of transmembrane transporters, and localizes to the plasma membrane of the cell. These results reveal an unexpected G2/M function of ribonucleotide reductase and establish that defects in a transmembrane protein can affect cell cycle progression.     

Multiple phytochromes are involved in red-light-induced enhancement of first-positive phototropism in Arabidopsis thaliana

Microsatellite instability (MSI), a symptom of defect in DNA mismatch repair function, represents a type of genomic instability frequently detected in many types of cancers. However, the involvement of MSI in non-Hodgkin's lymphomas (NHL) has not been conclusively investigated. In this study, we have tested the presence of MSI in 69 cases of B-cell NHL (B-NHL) representative of the various histologic categories of the disease and including 17 cases of acquired immunodeficiency syndrome (AIDS)-related B-NHL (AIDS-NHL). In addition, for selected B-NHL cases, consecutive samples obtained before and after clinical progression (with and without concomitant histologic transformation) were also investigated. Five distinct microsatellite repeats (2 dinucleotide, 2 trinucleotide, and 1 tetranucleotide repeats) were analyzed by polymerase chain reaction in all cases. MSI, defined by the presence of microsatellite alterations in two or more of the five microsatellite loci tested, was not found in NHL. In contrast to a previous study reporting the frequent association between MSI and AIDS-NHL, we found this abnormality in only 1 of 17 cases of AIDS-NHL representative of the major subtypes. Overall, these data indicate that defects in DNA mismatch repair do not contribute significantly to the molecular pathogenesis of B-NHL.     

Identification of a membrane protein involved in mitochondrial phosphate transport

A specific labeling by radioactive N-ethylmaleimide of a protein involved in phosphate transport was obtained by protecting one of the two-SH-groups of the transport system with low concentrations of mersalyl. Subsequently, the other free-SH groups were blocked with excess N-ethylmaleimide. Removal of mersalyl by cysteine and subsequent inbucation with labeled N-ethylmaleimide results in a "specific" binding of N-ethylmaleimide to one-SH group functionally involved in phosphate transport. The isolated inner membrane fraction of the labeled mitochondria was subjected to dodecylsulfate gel electrophoresis. The followin results were obtained. 1. The difference of the radioactivity pattern on the dodecylsulfate-polyacrylamide gel of inner membrane proteins, labeled with N-[14C]ethylmaleimide in the absence and with N-[3H-A1-ethylmaleimide in the presence of mersalyl during preincubation of mitochondria, shows only one main labeled peak. The same labeled peak is obtained from the difference of labeling after preincubation with a constant low concentration of mersalyl at 32 degrees C and at 0 degrees C. 2. The position of the labeled peak on the dodecylsulfate-polyacrylamide gel corresponds to a protein of molecular weight of 26500 +/- 800. 3. The amount of one of the two-SH groups, involved in phosphate transport, was estimated to be 30 nmol per g of mitochondrial protein.     

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.     

Fast axonal transport is required for growth cone advance

Growth cones are capable of advancing despite linkage to a stationary axonal cytoskeleton in chick and murine dorsal root ganglion neurites. Several lines of evidence point to the growth cone as the site of cytoskeletal elongation. Fast axonal transport is probably the means by which cytoskeletal elements or cofactors are rapidly moved through the axon. We report that direct, but reversible, inhibition of fast axonal transport with laser optical tweezers inhibits growth cone motility if cytoskeletal attachment to the cell body is maintained. Advancement ceases after a distance-dependent lag period which correlates with the rate of fast axonal transport. But severing the axonal cytoskeleton with the laser tweezers allows growth cones to advance considerably further. We suggest that axon elongation requires fast axonal transport but growth cone motility does not.     

Dimerization of Ste5, a mitogen-activated protein kinase cascade scaffold protein, is required for signal transduction

The mitogen-activated protein kinase cascade of the Saccharomyces cerevisiae pheromone response pathway is organized on the Ste5 protein, which binds each of the kinases of the cascade prior to signaling. In this study, a structure-function analysis of Ste5 deletion mutants uncovered new functional domains of the Ste5 protein and revealed that Ste5 dimerizes during the course of normal signal transduction. Dimerization, mediated by two regions in the N-terminal half of Ste5, was first suggested by intragenic complementation between pairs of nonfunctional Ste5 mutants and was confirmed by using the two-hybrid system. Coimmunoprecipitation of differently tagged forms of Ste5 from cells in which the pathway has been activated by Ste5 overexpression further confirmed dimerization. A precise correlation between the biological activity of various Ste5 fragments and dimerization suggests that dimerization is essential for Ste5 function.