Yukifumi Uesono

Osmotin, a plant antifungal protein, subverts signal transduction to enhance fungal cell susceptibility.

The plant pathogenesis-related protein osmotin is an antifungal cytotoxic agent that causes rapid cell death in the yeast S. cerevisiae. We show here that osmotin uses a signal transduction pathway to weaken defensive cell wall barriers and increase its cytotoxic efficacy. The pathway activated by osmotin includes the regulatory elements of the mating pheromone response STE4, STE18, STE20, STE5, STE11, STE7, FUS3, KSS1, and STE12. Neither the pheromone receptor nor its associated G protein alpha subunit GPA1 are required for osmotin action. However, mutation of SST2, a negative regulator of G alpha proteins, resulted in supersensitivity to osmotin. Phosphorylation of STE7 was rapidly stimulated by osmotin preceding any changes in cell vitality or morphology. These results demonstrate that osmotin subverts target cell signal transduction as part of its mechanism of action.

Ssd1p of Saccharomyces cerevisiae Associates with RNA

The SSD1 gene has been isolated as a single copy suppressor of many mutants, such as sit4,slk1/bck1, pde2, and rpc31, in the yeast Saccharomyces cerevisiae. Ssd1p has domains showing weak but significant homology with RNase II-related proteins, Cyt4p, Dss1p, VacB, and RNase II, which are involved in the modification of RNA. We found that Ssd1p had the ability to bind RNA, preferably poly(rA), as well as single-stranded DNA. Interestingly, the most conserved domain among the RNase II-related proteins was not necessary for interaction with RNA. Indirect immunofluorescence staining with anti-Ssd1p antibody revealed that Ssd1p was detected mainly in the cytoplasm. Furthermore, sucrose gradient sedimentation analysis demonstrated that Ssd1p was not cofractionated with polyribosomes, suggesting that Ssd1p is not particularly bound to a translationally active subpopulation of mRNA in the cytoplasm.

Nitrate Addition Alleviates Ammonium Toxicity Without Lessening Ammonium Accumulation, Organic Acid Depletion and Inorganic Cation Depletion in Arabidopsis thaliana Shoots

When ammonium is the sole nitrogen (N) source, plant growth is suppressed compared with the situation where nitrate is the N source. This is commonly referred to as ammonium toxicity. It is widely known that a combination of nitrate and ammonium as N source alleviates this ammonium toxicity (nitrate-dependent alleviation of ammonium toxicity), but the underlying mechanisms are still not completely understood. In plants, ammonium toxicity is often accompanied by a depletion of organic acids and inorganic cations, and by an accumulation of ammonium. All these factors have been considered as possible causes for ammonium toxicity. Thus, we hypothesized that nitrate could alleviate ammonium toxicity by lessening these symptoms. We analyzed growth, inorganic N and cation content and various primary metabolites in shoots of Arabidopsis thaliana seedlings grown on media containing various concentrations of nitrate and/or ammonium. Nitrate-dependent alleviation of ammonium toxicity was not accompanied by less depletion of organic acids and inorganic cations, and showed no reduction in ammonium accumulation. On the other hand, shoot growth was significantly correlated with the nitrate concentration in the shoots. This suggests that nitrate-dependent alleviation of ammonium toxicity is related to physiological processes that are closely linked to nitrate signaling, uptake and reduction. Based on transcript analyses of various genes related to nitrate signaling, uptake and reduction, possible underlying mechanisms for the nitrate-dependent alleviation are discussed.

The MCS1/SSD1/SRK1/SSL1 gene is involved in stable maintenance of the chromosome in yeast

A temperature-sensitive (ts) mutant of Saccharomyces cerevisiae was isolated in which mini-chromosomes were unstable at high temperature. The MCS1 gene (Mini-Chromosome Stability 1) was cloned by the ability of complementing the temperature sensitivity, and was found to be identical to SSD1/SRK1/SSL1. When MCS1/SSD1 was disrupted in a certain wild-type (wt) strain, mini-chromosomes were unstable, even at 30 degrees C, indicating that the gene is involved in chromosome stability. The Mcs1/Ssd1 protein was detected as a 170-kDa protein by immuno-blotting analysis and this 170-kDa protein could not be detected in the ts mutant and certain wt strains. Our results are consistent with the genetic data that there are two polymorphic forms of the gene, SSD1-v and ssd1-d [Sutton et al., Mol. Cell. Biol. 11 (1991) 2133-2148]. Furthermore, genetic backgrounds other than MCS1/SSD1 caused strain-specific phenotype. The protein, precipitated by specific antibodies, was phosphorylated.

A new yeast gene, HTR1, required for growth at high temperature, is needed for recovery from mating pheromone-induced G1 arrest

A new temperature-sensitive mutant of Saccharomyces cerevisiae was isolated. Arrested cells grown at the nonpermissive temperature were of dumb-bell shape and contained large vacuoles. A DNA fragment was cloned based on its ability to complement this temperature sensitivity. The HTR1 gene encodes a putative protein of 93 kDa without significant homology to any known proteins. The gene was mapped between ade5 and lys5 on the left arm of chromosome VII. The phenotype of the gene disruptant appeared to be strain-specific; disruption of the gene in strain W303 caused the cells to become temperature sensitive. The arrested phenotype here was similar to that of the original is mutant and cells in G2/M phase predominated at high temperature. Another disruptant in a strain YPH background grew slowly at high temperature due to slow progression through G2/M phase, and morphologically abnormal (elongated) cells accumulated. A single-copy suppressor that alleviated the temperature-sensitive defects in both strains was identified as MCS1/SSD1. The wild-type strains W303 and YPH are known to carry defective MCS1/SSD1 alleles; hence HTR1 may function redundantly with MCS1/SSD1 to suppress the temperature-sensitive phenotypes. In addition, based on a halo bioassay, the disruptant strains appeared to be defective in recovery from, or adaptive response to G1 arrest mediated by mating pheromone, even at the permissive temperature. Thus the gene has at least two functions and is designated HTR1 (required for high temperature growth and recovery from G1 arrest induced by mating pheromone).

Effects of AOX1a Deficiency on Plant Growth, Gene Expression of Respiratory Components and Metabolic Profile Under Low-Nitrogen Stress in Arabidopsis thaliana

Expression of alternative oxidase (AOX) and cyanide (CN)-resistant respiration are often highly enhanced in plants exposed to low-nitrogen (N) stress. Here, we examined the effects of AOX deficiency on plant growth, gene expression of respiratory components and metabolic profiles under low-N stress, using an aox1a knockout transgenic line (aox1a) of Arabidopsis thaliana. We exposed wild-type (WT) and aox1a plants to low-N stress for 7 d and analyzed their shoots and roots. In WT plants, the AOX1a mRNA levels and AOX capacity increased in proportion to low-N stress. Expression of the genes of the components for non-phosphorylating pathways and antioxidant enzymes was enhanced, but differences between WT and aox1a plants were small. Metabolome analyses revealed that AOX deficiency altered the levels of certain metabolites, such as sugars and sugar phosphates, in the shoots under low-N stress. However, the carbon (C)/N ratios and carbohydrate levels in aox1a plants were similar to those in the WT under low-N stress. Our results indicated that the N-limited stress induced AOX expression in A. thaliana plants, but the induced AOX may not play essential roles under stress due to low-N alone, and the C/N balance under low-N stress may be tightly regulated by systems other than AOX.

Effects of elevated CO2 on levels of primary metabolites and transcripts of genes encoding respiratory enzymes and their diurnal patterns in Arabidopsis thaliana: possible relationships with respiratory rates.

Elevated CO2 affects plant growth and photosynthesis, which results in changes in plant respiration. However, the mechanisms underlying the responses of plant respiration to elevated CO2 are poorly understood. In this study, we measured diurnal changes in the transcript levels of genes encoding respiratory enzymes, the maximal activities of the enzymes and primary metabolite levels in shoots of Arabidopsis thaliana grown under moderate or elevated CO2 conditions (390 or 780 parts per million by volume CO2, respectively). We examined the relationships between these changes and respiratory rates. Under elevated CO2, the transcript levels of several genes encoding respiratory enzymes increased at the end of the light period, but these increases did not result in changes in the maximal activities of the corresponding enzymes. The levels of some primary metabolites such as starch and sugar phosphates increased under elevated CO2, particularly at the end of the light period. The O2 uptake rate at the end of the dark period was higher under elevated CO2 than under moderate CO2, but higher under moderate CO2 than under elevated CO2 at the end of the light period. These results indicate that the changes in O2 uptake rates are not directly related to changes in maximal enzyme activities and primary metabolite levels. Instead, elevated CO2 may affect anabolic processes that consume respiratory ATP, thereby affecting O2 uptake rates.

Ammonium-dependent respiratory increase is dependent on the cytochrome pathway in Arabidopsis thaliana shoots.

Oxygen uptake rates are increased when concentrated ammonium instead of nitrate is used as sole N source. Several explanations for this increased respiration have been suggested, but the underlying mechanisms are still unclear. To investigate possible factors responsible for this respiratory increase, we measured the O₂ uptake rate, activity and transcript level of respiratory components, and concentration of adenylates using Arabidopsis thaliana shoots grown in media containing various N sources. The O₂ uptake rate was correlated with concentrations of ammonium and ATP in shoots, but not related to the ammonium assimilation. The capacity of the ATP-coupling cytochrome pathway (CP) and its related genes were up-regulated when concentrated ammonium was sole N source, whereas the ATP-uncoupling alternative oxidase did not influence the extent of the respiratory increase. Our results suggest that the ammonium-dependent increase of the O₂ uptake rate can be explained by the up-regulation of the CP, which may be related to the ATP consumption by the plasma-membrane H+ -ATPase.

Negative regulators of the PHO system of Saccharomyces cerevisiae: characterization of PHO80 and PHO85

SummaryBoth PHO80 and PHO85 genes are required to establish the repressed state of the PHO system of Saccharomyces cerevisiae. S1 nuclease protection analysis of the PHO85 transcript revealed that the PHO85 gene contains an intron at the 6th codon of the gene. Each of the fusion proteins, LacZ-Pho80 and LacZ-Pho85, was produced into Escherichia coli and used as an antigen to raise antibodies in a rabbit. Using the affinity-purified antibodies in Western blotting experiments, the PHO85 protein was detected as a 36 kDa and the PHO80 protein as a 34 kDa protein. The PHO80 protein was detected only in extracts prepared from an overproducing strain. The immunoprecipitate containing the PHO85 protein showed protein kinase activity suggesting that PHO85 is a protein kinase gene, which is consistent with the observation that the deduced amino acid sequence of the PHO85 protein resembles that of some protein kinases. The PHO80 protein was found to be phosphorylated in the presence of PHO85 protein.

Local Anesthetics, Antipsychotic Phenothiazines, and Cationic Surfactants Shut Down Intracellular Reactions through Membrane Perturbation in Yeast

High osmolarity and glucose deprivation cause rapid shutdowns of both actin polarization and translation initiation in yeast. Like these stresses, administration of local anesthetics and of antipsychotic phenothiazines caused similar responses. All these drugs have amphiphilic structures and formed emulsions and permeabilized the cell membrane, indicating that they have the same features as a surfactant. Consistently with this, surfactants induced responses similar to those of local anesthetics and phenothiazines. Benzethonium chloride, a cationic surfactant, showed a more potent shutdown activity than phenothiazines, whereas SDS, an anionic surfactant, transiently depolarized actin without inhibiting translation initiation, suggesting that a cationic charge in the amphiphile is important to the shutdown of both reactions. The clinical drugs and the cationic surfactants at low concentrations caused shutdown without membrane permeabilization, suggesting that these compounds and stresses activate shutdown, via perturbation rather than disruption of the cell membrane.