The Roles of the NANA and LEPIDA Genes in Regulating the Stem Growth in Arabidopsis thaliana

Genetic, physiological, and morphological studies of dwarf mutants of Arabidopsis thaliana (L.) Heynh. from the collection of the Department of Genetics and Breeding, Moscow State University, showed that the NA and LE genes are involved in regulating elongation of internode cells and sensitivity to various hormones. The na mutation suppressed stem growth only in the presence of the active LE gene. The absence of the LE activity (in the lele homozygote) restored stem growth of the na mutant to the level characteristic of thele-2 mutant, and a decrease inLE activity (in LE/le heterozygote) almost completely suppressed the na phenotype. Phenotypic analysis of homozygous double mutants and heterozygotes obtained by crossing the na and le-2 mutants showed that the recessive le-2 allele has an epistatic effect on the semidominant na allele and that the genes possibly control consecutive steps of one biochemical pathway or one morphogenetic process. A hypothetical scheme was proposed for the interaction of the NA and LE gene products.     

The pea gene NA encodes ent-kaurenoic acid oxidase

The gibberellin (GA)-deficient dwarf na mutant in pea (Pisum sativum) has severely reduced internode elongation, reduced root growth, and decreased leaflet size. However, the seeds develop normally. Two genes, PsKAO1 and PsKAO2, encoding cytochrome P450 monooxygenases of the subfamily CYP88A were isolated. Both PsKAO1 and PsKAO2 had ent-kaurenoic acid oxidase (KAO) activity, catalyzing the three steps of the GA biosynthetic pathway from ent-kaurenoic acid to GA(12) when expressed in yeast (Saccharomyces cerevisiae). In addition to the intermediates ent-7alpha-hydroxykaurenoic acid and GA(12)-aldehyde, some additional products of the pea KAO activity were detected, including ent-6alpha,7alpha-dihydroxykaurenoic acid and 7beta-hydroxykaurenolide. The NA gene encodes PsKAO1, because in two independent mutant alleles, na-1 and na-2, PsKAO1 had altered sequences and the five-base deletion in PsKAO1 associated with the na-1 allele cosegregated with the dwarf na phenotype. PsKAO1 was expressed in the stem, apical bud, leaf, pod, and root, organs in which GA levels have previously been shown to be reduced in na plants. PsKAO2 was expressed only in seeds and this may explain the normal seed development and normal GA biosynthesis in seeds of na plants.     

Gibberellin biosynthesis mutations and root development in pea

Dwarf mutants of pea (Pisum sativum), with impaired gibberellin (GA) biosynthesis in the shoot, were studied to determine whether the roots of these genotypes had altered elongation and GA levels. Mutations na, lh-2, and ls-1 reduced GA levels in root tips and taproot elongation, although in lh-2 and ls-1 roots the reduction in elongation was small (less than 15%). The na mutation reduced taproot length by about 50%. The roots of na plants elongated in response to applied GA(1) and recombining na with mutation sln (which blocks GA catabolism) increased GA(1) levels in root tips and completely restored normal root development. In shoots, Mendel's le-1 mutation impairs the 3beta-hydroxylation of GA(20) to the bioactive GA(1), resulting in dwarfism. However, GA(1) and GA(20) levels were normal in le-1 roots, as was root development. The null mutation le-2 also did not reduce root GA levels or elongation. The results support the theory that GAs are important for normal root elongation in pea, and indicate that a 3beta-hydroxylase gene other than LE operates in pea roots.     

Mutant alleles for hypoxanthine phosphoriboxyltransferase: codominant expression, complementation, and segregation in hybrid Chinese hamster cells.

Chinese hamster ovary cell mutants resistant to the purine analogs 6-thioguanine or 8-azaguanine have been isolated following mutagenesis with ethyl methane sulfonate. The activities of hypoxanthine phosphoribosyltransferase (HPRT) in three such mutants have been found to exhibit an increased Km for the substrate 5-phosphoribosyl-1-pyrophosphate. The isoelectric point of the mutant enzyme activity has also changed in two mutants. Hybrid cells containing one mutant and one wild-type allele express both genes. Segregants that have lost only the wild-type allele can be selected on the basis of drug resistance. Two mutants exhibiting different alterations in HPRT activity can complement in a hybrid cell to yield a wild-type growth pattern and enzyme activity with intermediate electrophoretic and kinetic properties. The results suggest intracistronic complementation between structural gene mutants of HPRT.     

Identification of opsA,a Gene Involved in Solute Stress Mitigation and Survival in Soil,in the Polycyclic Aromatic Hydrocarbon-Degrading Bacterium Novosphingobium sp. Strain LH128

The aim of this study was to identify genes involved in solute and matric stress mitigation in the polycyclic aromatic hydrocarbon (PAH)-degrading Novosphingobium sp. strain LH128. The genes were identified using plasposon mutagenesis and by selection of mutants that showed impaired growth in a medium containing 450 mM NaCl as a solute stress or 10% (wt/vol) polyethylene glycol (PEG) 6000 as a matric stress. Eleven and 14 mutants showed growth impairment when exposed to solute and matric stresses, respectively. The disrupted sequences were mapped on a draft genome sequence of strain LH128, and the corresponding gene functions were predicted. None of them were shared between solute and matric stress-impacted mutants. One NaCl-affected mutant (i.e., NA7E1) with a disruption in a gene encoding a putative outer membrane protein (OpsA) was susceptible to lower NaCl concentrations than the other mutants. The growth of NA7E1 was impacted by other ions and nonionic solutes and by sodium dodecyl sulfate (SDS), suggesting that opsA is involved in osmotic stress mitigation and/or outer membrane stability in strain LH128. NA7E1 was also the only mutant that showed reduced growth and less-efficient phenanthrene degradation in soil compared to the wild type. Moreover, the survival of NA7E1 in soil decreased significantly when the moisture content was decreased but was unaffected when soluble solutes from sandy soil were removed by washing. opsA appears to be important for the survival of strain LH128 in soil, especially in the case of reduced moisture content, probably by mitigating the effects of solute stress and retaining membrane stability.     

Lrg1p functions as a putative GTPase-activating protein in the Pkc1p-mediated cell integrity pathway in Saccharomyces cerevisiae

In Saccharomyces cerevisiae the ROM2 gene encodes a GDP/GTP exchange factor for the small G-protein Rho1p, a known activator of protein kinase C. In a screen designed to isolate suppressors of a rom2 mutant allele, we identified a mutant defective in the gene coding for the putative GTPase-activating protein Lrg1p. This protein was previously suggested to be involved in sporulation and mating. Here we provide evidence for its role in Pkc1p-mediated signal transduction based on the following results. (1) Deletion of LRG1 suppresses the growth phenotypes associated with mutations in SLG1 (which codes for a putative sensor of cell wall damage). (2) Using two-hybrid assays an interaction between the GAP domain of Lrg1p and Rho1p was demonstrated. (3) The lrg1 mutant shows enhanced activity of the Pkc1p pathway. (4) Overexpression of LRG1 leads to a cell lysis defect that can be suppressed by the addition of osmotic stabilizers. Phenotypic comparison of lrg1 mutants with mutants defective in other GTPase-activating proteins (Sac7p, Bem2p, Bag7p) presumed to act on Rho1p revealed that deletion of SAC7, but not BEM2 or BAG7, suppresses the phenotype of rom2 mutants. Pairwise combination of mutations in all these genes showed that the simultaneous deletion of SAC7 and LRG1 is synthetically lethal. We therefore suggest that Lrg1p acts as a negative regulator of the Pkc1p pathway in conjunction with its known homologue Sac7p.     

Accumulation of pre-tRNA splicing ''2/3'' intermediates in a Saccharomyces cerevisiae mutant.

In an effort to identify genes involved in the excision of tRNA introns in Saccharomyces cerevisiae, temperature-sensitive mutants were screened for intracellular accumulation of intron-containing tRNA precursors by RNA hybridization analysis. In one mutant, tRNA splicing intermediates consisting of the 5' exon covalently joined to the intron ('2/3' pre-tRNA molecules) were detected in addition to unspliced precursors. The mutant cleaves pre-tRNA(Phe) in vitro at the 3' exon/intron splice site, generating the 3' half molecule and 2/3 intermediate. The 5' half molecule and intron are not produced, indicating that cleavage at the 5' splice site is suppressed. This partial splicing activity co-purifies with tRNA endonuclease throughout several chromatographic steps. Surprisingly, the splicing defect does not appreciably affect cell growth at normal or elevated temperatures, but does confer a pseudo cold-sensitive phenotype of retarded growth at 15 degrees C. The mutant falls into the complementation group SEN2 previously defined by the isolation of mutants defective for tRNA splicing in vitro [Winey, M. and Culbertson, M.R. (1988) Genetics, 118, 609-617], although its phenotypes are distinct from those of the previous sen2 isolates. The distinguishing genetic and biochemical properties of this new allele, designated sen2-3, suggests the direct participation of the SEN2 gene product in tRNA endonuclease function.     

Identification and characterization of yeast mutants that overcome an experimentally introduced block to splicing at the 3' splice site.

An experimentally introduced secondary structure in exon 2 adjacent to the 3' splice site of a yeast ACT-Escherichia coli lacZ fusion gene abolishes splicing in vivo and inhibits beta-galactosidase production. We have devised a genetic screen to isolate both cis and trans-acting mutants that restore beta-galactosidase activity. Two cis-acting mutants potentially destabilize the stem in the region close to the 3' splice site. One trans-acting mutant, designated rss1-1, partially restores beta-galactosidase activity by both increasing the splicing efficiency and stabilizing the precursor and lariat intermediate. The trans-acting suppression activity of rss1-1 is specific for a particular structure because another artificially introduced secondary structure, which also blocks splicing, is not suppressed by this mutant allele. We have cloned the gene encoding the trans-acting mutant protein. The RSS1 gene is located on Saccharomyces cerevisiae chromosome V and is a single copy, essential gene. The predicted RSS1 protein has marked similarity to members of the putative ATP-dependent RNA helicase family. At the nonpermissive temperature, the rss1-1 mutant allele decreases the steady-state levels of several endogenous messenger RNAs and increases the ratio of pre-mRNA to mRNA of specific messages. RSS1 is likely to play an interesting role in RNA processing.     

Catalytic and framework mutations in the neuraminidase active site of influenza viruses that are resistant to 4-guanidino-Neu5Ac2en.

Here we report the isolation of influenza virus A/turkey/Minnesota/833/80 (H4N2) with a mutation at the catalytic residue of the neuraminidase (NA) active site, rendering it resistant to the novel NA inhibitor 4-guanidino-Neu5Ac2en (GG167). The resistance of the mutant stems from replacement of one of three invariant arginines (Arg 292-->Lys) that are conserved among all viral and bacterial NAs and participate in the conformational change of sialic acid moiety necessary for substrate catalysis. The Lys292 mutant was selected in vitro after 15 passages at increasing concentrations of GG167 (from 0.1 to 1,000 microM), conditions that earlier gave rise to GG167-resistant mutants with a substitution at the framework residue Glu119. Both types of mutants showed similar degrees of resistance in plaque reduction assays, but the Lys292 mutant was more sensitive to the inhibitor in NA inhibition tests than were mutants bearing a substitution at framework residue 119 (Asp, Ala, or Gly). Cross-resistance to other NA inhibitors (4-amino-Neu5Ac2en and Neu5Ac2en) varied among mutants resistant to GG167, being lowest for Lys292 and highest for Asp119. All GG167-resistant mutants demonstrated markedly reduced NA activity, only 3 to 50% of the parental level, depending on the particular amino acid substitution. The catalytic mutant (Lys292) showed a significant change in pH optimum of NA activity, from 5.9 to 5.3. All of the mutant NAs were less stable than the parental enzyme at low pH. Despite their impaired NA activity, the GG167-resistant mutants grew as well as parental virus in Madin-Darby canine kidney cells or in embryonated chicken eggs. However, the infectivity in mice was 500-fold lower for Lys292 than for the parental virus. These findings demonstrate that amino acid substitution in the NA active site at the catalytic or framework residues, followed by multiple passages in vitro, in the presence of increasing concentrations of the NA inhibitor GG167, generates GG167-resistant viruses with reduced NA activity and decreased infectivity in animals.     

New genes involved in carbon catabolite repression and derepression in the yeast Saccharomyces cerevisiae.

A mutation causing resistance to carbon catabolite repression in gene HEX2, mutant allele hex2-3, causes an extreme sensitivity to maltose when in combination with the genes necessary for maltose metabolism. This provided a convenient system for the selective isolation of mutations in genes specifically required for maltose metabolism and other genes involved in general carbon catabolite repression. In addition to reversion of the hex2-3 allele, mutations in three other genes were detected. These genes were called CAT1, CAT3, and MUR1 and in a mutated form abolished maltose inhibition caused by mutant allele hex2-3. Mutant alleles cat1 and cat3 also restored normal repression in the presence of the hex2-3 allele. Segregants having only mutant alleles cat1 or cat3 were obtained by tetrad analysis. These segregants could not grow on nonfermentable carbon sources. Mutant alleles of gene CAT1 were allelic to a mutant allele cat1-1 previously isolated (Zimmermann et al., Mol. Gen. Genet. 151:95-103). Such mutants prevented derepression not only of the maltose catabolizing system, the selected property, but also of glyoxylate shunt and gluconeogenic enzymes. However, respiratory activities and invertase formation were not affected under derepressing conditions. cat3 mutants had the same phenotypic properties as cat1 mutants. This showed that carbon metabolism in yeast cells is under a very complex and ramified control of repressing and derepressing genes, which are interdependent.     

Identification and characterization of a novel gene, hos2+, the function of which is necessary for growth under high osmotic stress in fission yeast

hos2 mutants of the fission yeast Schizosaccharomyces pombe showed the phenotype of high osmolarity sensitivity for growth. An S. pombe strain carrying the hos2-M10 allele cannot form colonies on agar plates containing 2 M glucose, but the parental strain can do so very well, as demonstrated previously. In this study, the hos2+ gene was identified as one that encodes a small protein of 94 amino acids, which shows no sequence similarity to any other proteins in the current databases. The hos2-M10 mutation resulted in Gln-62 to TAG-termination codon. A Hos2-defective (hos2delta) strain, which we then constructed, showed the phenotype of high osmolarity sensitivity, as in the case of the original hos2-M10 mutant. For this hos2delta mutant, three multicopy suppressor genes were isolated and one of which was identified as the pgk1+ gene, encoding a phosphoglycerate kinase.     

A carbon catabolite repression mutant of Saccharomyces cerevisiae with elevated hexokinase activity: Evidence for regulatory control of hexokinase PII synthesis

Summary Mutants were investigated that had elevated hexokinase activity and had been isolated previously as resistant to carbon catabolite repression (Zimmermann and Scheel 1977). They were allele tested with mutant strains of Lobo and Maitra (1977), which had defects in one or more of the genes coding for glucokinase and unspecific hexokinases. It was shown, that the mutation abolishing carbon catabolite repression had occured in a gene that was not allelic to any of the structural genes coding for hexokinases. This indicated that a regulatory defect was responsible for elevated hexokinase activity. This agreed with observations that hexokinase activities were like wild-type during growth on non-fermentable carbon sources in hex2 mutants. Recombination between the mutant allele hex2 and mutant alleles hxk1 and hxk2, coding for hexokinase PI and PII respectively, clearly demonstrated that only hexokinase PII was elevated in hex2 mutants. When hex2 mutant cells grown on YEP ethanol were shifted to YEP glucose media, hexokinase activity increased after 30min. This increase depended on de novo protein synthesis. hex2 mutants provide evidence, that carbon catabolite repression and synthesis of hexokinase PII are under common regulatory control.     

The BLADE-ON-PETIOLE 1 gene controls leaf pattern formation through the modulation of meristematic activity in Arabidopsis

The plant leaf provides an ideal system to study the mechanisms of organ formation and morphogenesis. The key factors that control leaf morphogenesis include the timing, location and extent of meristematic activity during cell division and differentiation. We identified an Arabidopsis mutant in which the regulation of meristematic activities in leaves was aberrant. The recessive mutant allele blade-on-petiole1-1 (bop1-1) produced ectopic, lobed blades along the adaxial side of petioles of the cotyledon and rosette leaves. The ectopic organ, which has some of the characteristics of rosette leaf blades with formation of trichomes in a dorsoventrally dependent manner, was generated by prolonged and clustered cell division in the mutant petioles. Ectopic, lobed blades were also formed on the proximal part of cauline leaves that lacked a petiole. Thus, BOP1 regulates the meristematic activity of leaf cells in a proximodistally dependent manner. Manifestation of the phenotypes in the mutant leaves was dependent on the leaf position. Thus, BOP1 controls leaf morphogenesis through control of the ectopic meristematic activity but within the context of the leaf proximodistality, dorsoventrality and heteroblasty. BOP1 appears to regulate meristematic activity in organs other than leaves, since the mutation also causes some ectopic outgrowths on stem surfaces and at the base of floral organs. Three class I knox genes, i.e., KNAT1, KNAT2 and KNAT6, were expressed aberrantly in the leaves of the bop1-1 mutant. Furthermore, the bop1-1 mutation showed some synergistic effect in double mutants with as1-1 or as2-2 mutation that is known to be defective in the regulation of meristematic activity and class I knox gene expression in leaves. The bop1-1 mutation also showed a synergistic effect with the stm-1 mutation, a strong mutant allele of a class I knox gene, STM. We, thus, suggest that BOP1 promotes or maintains a developmentally determinate state in leaf cells through the regulation of class I knox genes.     

BIN2, a new brassinosteroid-insensitive locus in Arabidopsis

Brassinosteroids (BRs) play important roles throughout plant development. Although many genes have been identified that are involved in BR biosynthesis, genetic approaches in Arabidopsis have led to the identification of only one gene, BRI1, that encodes a membrane receptor for BRs. To expand our knowledge of the molecular mechanism(s) of plant steroid signaling, we analyzed many dwarf and semidwarf mutants collected from our previous genetic screens and identified a semidwarf mutant that showed little response to exogenous BR treatments. Genetic analysis of the bin2 (BR-INSENSITIVE 2) mutant indicated that the BR-insensitive dwarf phenotype was due to a semidominant mutation in the BIN2 gene that mapped to the middle of chromosome IV between the markers CH42 and AG. A direct screening for similar semidwarf mutants resulted in the identification of a second allele of the BIN2 gene. Despite some novel phenotypes observed with the bin2/+ mutants, the homozygous bin2 mutants were almost identical to the well-characterized bri1 mutants that are defective in BR perception. In addition to the BR-insensitive dwarf phenotype, bin2 mutants exhibited BR insensitivity when assayed for root growth inhibition and feedback inhibition of CPD gene expression. Furthermore, bin2 mutants displayed an abscisic acid-hypersensitive phenotype that is shared by the bri1 and BR-deficient mutants. A gene dosage experiment using triploid plants suggested that the bin2 phenotypes were likely caused by either neomorphic or hypermorphic gain-of-function mutations in the BIN2 gene. Thus, the two bin2 mutations define a novel genetic locus whose gene product might play a role in BR signaling.     

The maize low-phytic acid mutant lpa2 is caused by mutation in an inositol phosphate kinase gene

Reduced phytic acid content in seeds is a desired goal for genetic improvement in several crops. Low-phytic acid mutants have been used in genetic breeding, but it is not known what genes are responsible for the low-phytic acid phenotype. Using a reverse genetics approach, we found that the maize (Zea mays) low-phytic acid lpa2 mutant is caused by mutation in an inositol phosphate kinase gene. The maize inositol phosphate kinase (ZmIpk) gene was identified through sequence comparison with human and Arabidopsis Ins(1,3,4)P(3) 5/6-kinase genes. The purified recombinant ZmIpk protein has kinase activity on several inositol polyphosphates, including Ins(1,3,4)P(3), Ins(3,5,6)P(3), Ins(3,4,5,6)P(4), and Ins(1,2,5,6)P(4). The ZmIpk mRNA is expressed in the embryo, the organ where phytic acid accumulates in maize seeds. The ZmIpk Mutator insertion mutants were identified from a Mutator F(2) family. In the ZmIpk Mu insertion mutants, seed phytic acid content is reduced approximately 30%, and inorganic phosphate is increased about 3-fold. The mutants also accumulate myo-inositol and inositol phosphates as in the lpa2 mutant. Allelic tests showed that the ZmIpk Mu insertion mutants are allelic to the lpa2. Southern-blot analysis, cloning, and sequencing of the ZmIpk gene from lpa2 revealed that the lpa2-1 allele is caused by the genomic sequence rearrangement in the ZmIpk locus and the lpa2-2 allele has a nucleotide mutation that generated a stop codon in the N-terminal region of the ZmIpk open reading frame. These results provide evidence that ZmIpk is one of the kinases responsible for phytic acid biosynthesis in developing maize seeds.     

Genetic and biochemical evidence for hexokinase PII as a key enzyme involved in carbon catabolite repression in yeast

Summary Mutants with reduced hexokinase activity previously isolated as resistant to carbon catabolite repression of invertase and maltase (Zimmermann and Scheel, 1977) were allele tested with mutant strains of Lobo and Maitra (1977) which had defects in one or several of the genes coding for glucokinase and the two unspecific hexokinases. It could be demonstrated, that the mutation abolishing carbon catabolite repression had occurred in a gene allelic to the structural gene of hexokinase PII. Moreover, the defective mutant allele for hexokinase PII isolated by Lobo and Maitra (1977) was also defective in carbon catabolite repression. Neither glucokinase nor hexokinase PI showed any effect on this regulatory system. Biochemical analysis in crude extracts also showed altered kinetic properties of hexokinases in the hex1 mutants. The results directly support the hypothesis previously put forward, that one of the hexokinases is not only active as a catalytic, but also as a regulatory protein.     

Nana gene--a regulator of cell division and elongation of stem cells in Arabidopsis thaliana (L.) Heynh

The dominant nana (na) mutation localized to the upper arm of Arabidopsis thaliana chromosome 1 blocks cell proliferation in apical meristem (AM) of the flower-bearing stem at its early development and suppresses the subsequent elongation of its internode cells. The na mutation reduces the sensitivity of cells of the flower-bearing stem to gibberellin (GA) and paclobutrazole (PBZ) and prevents resting and immature seeds from restoring the germinating ability in response to exogenous GA. On the other hand, exogenous GA and PBZ affects the onset of flowering, hypocotyl length, and leaf color; i.e., the na mutant displays a distortion of only several, rather than all, GA-dependent processes. Based on the results obtained, the product of the NA gene was assumed to play a role in the negative regulation of GA signaling and to act later than the products of the known GAI and SPY genes.