ATH1 Gene Research Articles

Susceptibility to Verticillium longisporum is linked to monoterpene production by TPS23/27 in Arabidopsis.

The fungus Verticillium longisporum is a soil-borne plant pathogen of increasing economic importance, and information on plant responses to it is limited. To identify the genes and components involved in the early stages of infection, transcripts in roots of V.longisporum-challenged Arabidopsis Col-0 and the susceptible NON-RACE SPECIFIC DISEASE RESISTANCE 1 (ndr1-1) mutant were compared using ATH1 gene chips. The analysis revealed altered transcript levels of several terpene biosynthesis genes, including the monoterpene synthase TPS23/27. When transgenic 35S:TPS23/27 and TPS23/27-amiRNA plants were monitored the over-expresser line showed enhanced fungal colonization whereas the silenced genotype was indistinguishable from Col-0. Transcript analysis of terpene biosynthesis genes suggested that only the TPS23/27 pathway is affected in the two transgenic genotypes. To confirm changes in monoterpene production, emitted volatiles were determined using solid-phase microextraction and gas chromatography-mass spectrometry. Levels of all identified TPS23/27 monoterpene products were significantly altered in the transgenic plants. A stimulatory effect on conidial germination and hyphal growth of V.longisporum was also seen in co-cultivation with 35S:TPS23/27 plants and upon exposure to 1,8-cineole, the main product of TPS23/27. Methyl jasmonate treatments of myc2-1 and myc2-2 mutants and analysis of TPS23/27:uidA in the myc2-2 background suggested a dependence on jasmonic acid mediated by the transcription factor MYC2. Taken together, our results show that TPS23/27-produced monoterpenes stimulate germination and subsequent invasion of V.longisporum in Arabidopsis roots.

Enhanced freeze tolerance of baker's yeast by overexpressed trehalose-6-phosphate synthase gene (TPS1) and deleted trehalase genes in frozen dough.

Several recombinant strains with overexpressed trehalose-6-phosphate synthase gene (TPS1) and/or deleted trehalase genes were obtained to elucidate the relationships between TPS1, trehalase genes, content of intracellular trehalose and freeze tolerance of baker's yeast, as well as improve the fermentation properties of lean dough after freezing. In this study, strain TL301(TPS1) overexpressing TPS1 showed 62.92 % higher trehalose-6-phosphate synthase (Tps1) activity and enhanced the content of intracellular trehalose than the parental strain. Deleting ATH1 exerted a significant effect on trehalase activities and the degradation amount of intracellular trehalose during the first 30 min of prefermentation. This finding indicates that acid trehalase (Ath1) plays a role in intracellular trehalose degradation. NTH2 encodes a functional neutral trehalase (Nth2) that was significantly involved in intracellular trehalose degradation in the absence of the NTH1 and/or ATH1 gene. The survival ratio, freeze-tolerance ratio and relative fermentation ability of strain TL301(TPS1) were approximately twice as high as those of the parental strain (BY6-9α). The increase in freeze tolerance of strain TL301(TPS1) was accompanied by relatively low trehalase activity, high Tps1 activity and high residual content of intracellular trehalose. Our results suggest that overexpressing TPS1 and deleting trehalase genes are sufficient to improve the freeze tolerance of baker's yeast in frozen dough. The present study provides guidance for the commercial baking industry as well as the research on the intracellular trehalose mobilization and freeze tolerance of baker's yeast.

Plant stem cell maintenance involves direct transcriptional repression of differentiation program

In animal systems, master regulatory transcription factors (TFs) mediate stem cell maintenance through a direct transcriptional repression of differentiation promoting TFs. Whether similar mechanisms operate in plants is not known. In plants, shoot apical meristems serve as reservoirs of stem cells that provide cells for all above ground organs. WUSCHEL, a homeodomain TF produced in cells of the niche, migrates into adjacent cells where it specifies stem cells. Through high-resolution genomic analysis, we show that WUSCHEL represses a large number of genes that are expressed in differentiating cells including a group of differentiation promoting TFs involved in leaf development. We show that WUS directly binds to the regulatory regions of differentiation promoting TFs; KANADI1, KANADI2, ASYMMETRICLEAVES2 and YABBY3 to repress their expression. Predictions from a computational model, supported by live imaging, reveal that WUS-mediated repression prevents premature differentiation of stem cell progenitors, being part of a minimal regulatory network for meristem maintenance. Our work shows that direct transcriptional repression of differentiation promoting TFs is an evolutionarily conserved logic for stem cell regulation.

Negative regulation of defence signalling pathways by the EDR1 protein kinase

The enhanced disease resistance 1 (edr1) mutant of Arabidopsis confers enhanced resistance to bacterial and fungal pathogens. To better understand how edr1-mediated resistance occurs, we performed transcriptome analyses on wild-type and edr1 plants inoculated with the fungal pathogen Golovinomyces cichoracearum (powdery mildew). The expression of many known and putative defence-associated genes was more rapidly induced, and to higher levels, in edr1 plants relative to the wild-type. Many of the genes with elevated expression encoded WRKY transcription factors and there was enrichment for their binding sites in promoters of the genes upregulated in edr1. Confocal microscopy of transiently expressed EDR1 protein showed that a significant fraction of EDR1 was localized to the nucleus, suggesting that EDR1 could potentially interact with transcription factors in the nucleus. Analysis of gene ontology annotations revealed that genes associated with the endomembrane system, defence, reactive oxygen species (ROS) production and protein kinases were induced early in the edr1 mutant, and that elevated expression of the endomembrane system, defence and ROS-related genes was maintained for at least 4 days after infection.

Gene Expression Browser: large-scale and cross-experiment microarray data integration, management, search & visualization

BackgroundIn the last decade, a large amount of microarray gene expression data has been accumulated in public repositories. Integrating and analyzing high-throughput gene expression data have become key activities for exploring gene functions, gene networks and biological pathways. Effectively utilizing these invaluable microarray data remains challenging due to a lack of powerful tools to integrate large-scale gene-expression information across diverse experiments and to search and visualize a large number of gene-expression data points.ResultsGene Expression Browser is a microarray data integration, management and processing system with web-based search and visualization functions. An innovative method has been developed to define a treatment over a control for every microarray experiment to standardize and make microarray data from different experiments homogeneous. In the browser, data are pre-processed offline and the resulting data points are visualized online with a 2-layer dynamic web display. Users can view all treatments over control that affect the expression of a selected gene via Gene View, and view all genes that change in a selected treatment over control via treatment over control View. Users can also check the changes of expression profiles of a set of either the treatments over control or genes via Slide View. In addition, the relationships between genes and treatments over control are computed according to gene expression ratio and are shown as co-responsive genes and co-regulation treatments over control.ConclusionGene Expression Browser is composed of a set of software tools, including a data extraction tool, a microarray data-management system, a data-annotation tool, a microarray data-processing pipeline, and a data search & visualization tool. The browser is deployed as a free public web service (http://www.ExpressionBrowser.com) that integrates 301 ATH1 gene microarray experiments from public data repositories (viz. the Gene Expression Omnibus repository at the National Center for Biotechnology Information and Nottingham Arabidopsis Stock Center). The set of Gene Expression Browser software tools can be easily applied to the large-scale expression data generated by other platforms and in other species.

Within and between Whorls: Comparative Transcriptional Profiling of Aquilegia and Arabidopsis

BackgroundThe genus Aquilegia is an emerging model system in plant evolutionary biology predominantly because of its wide variation in floral traits and associated floral ecology. The anatomy of the Aquilegia flower is also very distinct. There are two whorls of petaloid organs, the outer whorl of sepals and the second whorl of petals that form nectar spurs, as well as a recently evolved fifth whorl of staminodia inserted between stamens and carpels.Methodology/Principal FindingsWe designed an oligonucleotide microarray based on EST sequences from a mixed tissue, normalized cDNA library of an A. formosa x A. pubescens F2 population representing 17,246 unigenes. We then used this array to analyze floral gene expression in late pre-anthesis stage floral organs from a natural A. formosa population. In particular, we tested for gene expression patterns specific to each floral whorl and to combinations of whorls that correspond to traditional and modified ABC model groupings. Similar analyses were performed on gene expression data of Arabidopsis thaliana whorls previously obtained using the Ath1 gene chips (data available through The Arabidopsis Information Resource).Conclusions/SignificanceOur comparative gene expression analyses suggest that 1) petaloid sepals and petals of A. formosa share gene expression patterns more than either have organ-specific patterns, 2) petals of A. formosa and A. thaliana may be independently derived, 3) staminodia express B and C genes similar to stamens but the staminodium genetic program has also converged on aspects of the carpel program and 4) staminodia have unique up-regulation of regulatory genes and genes that have been implicated with defense against microbial infection and herbivory. Our study also highlights the value of comparative gene expression profiling and the Aquilegia microarray in particular for the study of floral evolution and ecology.

Predictive network modeling of the high-resolution dynamic plant transcriptome in response to nitrate

BackgroundNitrate, acting as both a nitrogen source and a signaling molecule, controls many aspects of plant development. However, gene networks involved in plant adaptation to fluctuating nitrate environments have not yet been identified.ResultsHere we use time-series transcriptome data to decipher gene relationships and consequently to build core regulatory networks involved in Arabidopsis root adaptation to nitrate provision. The experimental approach has been to monitor genome-wide responses to nitrate at 3, 6, 9, 12, 15 and 20 minutes using Affymetrix ATH1 gene chips. This high-resolution time course analysis demonstrated that the previously known primary nitrate response is actually preceded by a very fast gene expression modulation, involving genes and functions needed to prepare plants to use or reduce nitrate. A state-space model inferred from this microarray time-series data successfully predicts gene behavior in unlearnt conditions.ConclusionsThe experiments and methods allow us to propose a temporal working model for nitrate-driven gene networks. This network model is tested both in silico and experimentally. For example, the over-expression of a predicted gene hub encoding a transcription factor induced early in the cascade indeed leads to the modification of the kinetic nitrate response of sentinel genes such as NIR, NIA2, and NRT1.1, and several other transcription factors. The potential nitrate/hormone connections implicated by this time-series data are also evaluated.

Construction of Hansenula polymorpha strains with improved thermotolerance

The methylotrophic yeast Hansenula polymorpha has the potential to be used in the process of simultaneous saccharification and fermentation (SSF) of xylan derived xylose at elevated temperatures. To improve parameters of high-temperature resistance and high-temperature fermentation of H. polymorpha, strains carrying deletion of acid trehalase gene (ATH1) and overexpressing genes coding for heat-shock proteins Hsp16p and Hsp104p were constructed. Results indicate that the corresponding recombinant strains have up to 12-fold increased tolerance to heat-shock treatment. The deletion of ATH1 gene and constitutive expression of HSP16 and HSP104 resulted in up to 5.8-fold improvement of ethanol production from xylose at 50 degrees C. Although the maximum ethanol concentration achieved from xylose was 0.9 g L(-1), our model H. polymorpha strains with elevated thermotolerance can be further modified by metabolic engineering to construct improved high-temperature ethanol producers from this pentose.

Acid trehalase is involved in intracellular trehalose mobilization during postdiauxic growth and severe saline stress inSaccharomyces cerevisiae

The role of the acid trehalase encoded by the ATH1 gene in the yeast Saccharomyces cerevisiae is still unclear. In this work, we investigated the regulation of ATH1 transcription and found a clear involvement of the protein kinase Hog1p in the induction of this gene under severe stress conditions, such as high salt. We also detected changes in the acid trehalase activity and trehalose levels, indicating a role of the acid trehalase in intracellular trehalose mobilization. Finally, the growth analysis for different mutants in neutral and acid trehalases after high salt stress implicates acid trehalase activity in saline stress resistance.

The rules of gene expression in plants: Organ identity and gene body methylation are key factors for regulation of gene expression in Arabidopsis thaliana

BackgroundMicroarray technology is a widely used approach for monitoring genome-wide gene expression. For Arabidopsis, there are over 1,800 microarray hybridizations representing many different experimental conditions on Affymetrix™ ATH1 gene chips alone. This huge amount of data offers a unique opportunity to infer the principles that govern the regulation of gene expression in plants.ResultsWe used bioinformatics methods to analyze publicly available data obtained using the ATH1 chip from Affymetrix. A total of 1887 ATH1 hybridizations were normalized and filtered to eliminate low-quality hybridizations. We classified and compared control and treatment hybridizations and determined differential gene expression. The largest differences in gene expression were observed when comparing samples obtained from different organs. On average, ten-fold more genes were differentially expressed between organs as compared to any other experimental variable. We defined "gene responsiveness" as the number of comparisons in which a gene changed its expression significantly. We defined genes with the highest and lowest responsiveness levels as hypervariable and housekeeping genes, respectively. Remarkably, housekeeping genes were best distinguished from hypervariable genes by differences in methylation status in their transcribed regions. Moreover, methylation in the transcribed region was inversely correlated (R2 = 0.8) with gene responsiveness on a genome-wide scale. We provide an example of this negative relationship using genes encoding TCA cycle enzymes, by contrasting their regulatory responsiveness to nitrate and methylation status in their transcribed regions.ConclusionOur results indicate that the Arabidopsis transcriptome is largely established during development and is comparatively stable when faced with external perturbations. We suggest a novel functional role for DNA methylation in the transcribed region as a key determinant capable of restraining the capacity of a gene to respond to internal/external cues. Our findings suggest a prominent role for epigenetic mechanisms in the regulation of gene expression in plants.

Genome-wide patterns of single-feature polymorphism in Arabidopsis thaliana

We used hybridization to the ATH1 gene expression array to interrogate genomic DNA diversity in 23 wild strains (accessions) of Arabidopsis thaliana (arabidopsis), in comparison with the reference strain Columbia (Col). At <1% false discovery rate, we detected 77,420 single-feature polymorphisms (SFPs) with distinct patterns of variation across the genome. Total and pair-wise diversity was higher near the centromeres and the heterochromatic knob region, but overall diversity was positively correlated with recombination rate (R(2) = 3.1%). The difference between total and pair-wise SFP diversity is a relative measure contrasting diversifying or frequency-dependent selection, similar to Tajima's D, and can be calibrated by the empirical genome-wide distribution. Each unique locus, centered on a gene, has a diversity and selection score that suggest a relative role in past evolutionary processes. Homologs of disease resistance (R) genes include members with especially high levels of diversity often showing frequency-dependent selection and occasionally evidence of a past selective sweep. Receptor-like and S-locus proteins also contained members with elevated levels of diversity and signatures of selection, whereas other gene families, bHLH, F-box, and RING finger proteins, showed more typical levels of diversity. SFPs identified with the gene expression array also provide an empirical hybridization polymorphism background for studies of gene expression polymorphism and are available through the genome browser http://signal.salk.edu/cgi-bin/AtSFP.

Late heading of perennial ryegrass caused by introducing an Arabidopsis homeobox gene

AbstractPerennial ryegrass (Lolium perenne L.) is the most important temperate forage grass species. Unfortunately, the nutritional value of perennial ryegrass declines as maturity progresses, mainly because of a high concentration of poorly digestible compounds in inflorescences. Therefore, the development of forage‐type ryegrass varieties with extended vegetative growth is of interest for agriculture. To delay floral transition in perennial ryegrass the Arabidopsis ATH1 gene driven by the maize ubiquitin promoter, the rice actin promoter or the rice OSH1 promoter, respectively was introduced. In ATH1‐expressing plants heading was delayed, and in a number of cases the plants never flowered at all. Such non‐ or late‐heading was accompanied by the outgrowth of normally quiescent lateral meristems into extra leaves, resulting in a leafy growth habit. When eventually heading, these plants generally produced a reduced number of inflorescences. These observations suggest that ATH1‐mediated delay of heading may be useful to improve fodder quality of perennial ryegrass.

Molecular characterization of the Aspergillus nidulans treA gene encoding an acid trehalase required for growth on trehalose.

Aspergillus nidulans conidiospores contain high levels of the non-reducing disaccharide trehalose. We show that upon induction of conidiospore germination, the trehalose pool is rapidly degraded and a glycerol pool is transiently accumulated. A trehalase with an acidic pH optimum was purified from conidiospores. Characterization of the treA gene encoding this trehalase shows that it is homologous to Saccharomyces cerevisiae vacuolar acid trehalase, the product of the ATH1 gene, and to two related proteins of unknown function identified in Mycobacterium tuberculosis and Mycobacterium leprae. A. nidulans mutants that lack acid trehalase activity were constructed by gene replacement at the treA locus. Analysis of these mutants suggests that the treA gene product is localized in the conidiospore wall, is required for growth on trehalose as a carbon source, and is not involved in the mobilization of the intracellular pool of trehalose. Therefore, it is proposed that a cytoplasmic regulatory trehalase is controlling this latter process.

Molecular Biology of Trehalose and the Trehalases in the Yeast Saccharomyces cerevisiae

The present state of knowledge of the role of trehalose and trehalose hydrolysis catalyzed by trehalase (EC 3.2.1.28) in the yeast Saccharomyces cerevisiae is reviewed. Trehalose is believed to function as a storage carbohydrate because its concentration is high during nutrient limitations and in resting cells. It is also believed to function as a stress metabolite because its concentration increases during certain adverse environmental conditions, such as heat and toxic chemicals. The exact way trehalose may perform the stress function is not understood, and conditions exist under which trehalose accumulation and tolerance to certain stress situations cannot be correlated. Three trehalases have been described in S. cerevisiae: 1) the cytosolic neutral trehalase encoded by the NTH1 gene, and regulated by cAMP-dependent phosphorylation process, nutrients, and temperature; 2) the vacuolar acid trehalase encoded by the ATH1 gene, and regulated by nutrients; and 3) a putative trehalase Nth1p encoded by the NTH2 gene (homolog of the NTH1 gene) and regulated by nutrients and temperature. The neutral trehalase is responsible for intracellular hydrolysis of trehalose, in contrast to the acid trehalase, which is responsible for utilization of extracellular trehalose. The role of the putative trehalase Nth2p in trehalose metabolism is not known. The NTH1 and NTH2 genes are required for recovery of cells after heat shock at 50 degrees C, consistent with their heat inducibility and sequence similarity. Other stressors, such as toxic chemicals, also induce the expression of these genes. We therefore propose that the NTH1 and NTH2 genes have stress-related function and the gene products may be called stress proteins. Whether the stress function of the trehalase genes is linked to trehalose is not clear, and possible mechanisms of stress protective function of the trehalases are discussed.

5587290 Stress tolerant yeast mutants : Klionsky Daniel; Holzer Helmut; Destruelle Monika Davis, CA, United States Assigned to The Regents of the University of California

The invention provides methods and compositions relating to stress tolerant yeasts; in particular, yeast mutants deficient in the expression of functional ATH1 gene product (Ath1p). Such yeast have enhanced tolerance to dehydration and freezing, are able to grow to a higher cell density over a range of fermentable carbon source concentrations, are able to produce and/or tolerate higher levels of ethanol and trehalose. Nucleic acids comprising ATH1 gene sequences are used in hybridization probes and PCR primers, in expression vectors, etc. The invention provides methods for producing a yeast mutant with improved survival ability under stress conditions which involve identifying mutations disrupting ATH1 expression using Ath1-specific reagents or ATH1 hybridization probes or primers.

Purification and biochemical characterization of the ATH1 gene product, vacuolar acid trehalase, from Saccharomyces cerevisiae

The disaccharide trehalose plays a critical role in yeast cell survival during conditions of environmental stress. The vacuole of the yeast Saccharomyces cerevisiae contains an enzyme, acid trehalose (ATH), that is capable of degrading trehalose. Recently, a gene required for ATH activity, ATH1, was cloned and sequenced [Destruelle et al., (1995) Yeast 11, 1015–1025]. The relationship between ATH1 and ATH, however, was not determined. We have purified ATH and shown that it is the ATH1 gene product; peptide sequences from the purified protein correspond to the deduced amino acid sequence of Ath1p. In addition, antiserum to Ath1p specifically recognizes purified ATH.

Deletion of the ATH1 gene in Saccharomyces cerevisiae prevents growth on trehalose

The biological function of the yeast trehalases (EC 3.2.1.28) consists of down-regulation of the concentration of trehalose via glucose formation by trehalose hydrolysis. While it is generally accepted that the cytosolic neutral trehalase (encoded by the NTH1 gene) is responsible for trehalose hydrolysis in intact cells, very little is known about a role of the vacuolar acid trehalase and the product of the recently described neutral trehalase gene YBR0106 (NTH2). We have analyzed the role of the acid trehalase in trehalose hydrolysis using the ATH1 deletion mutant (Δ athl) of Saccharomyces cerevisiae [M. Destruelle et al. (1995) Yeast 11, 1015–1025] deficient in acid trehalase activity under various nutritional conditions. In contrast to wild-type and a mutant deficient in the neutral trehalase (Δ athl ), the Aathl mutant does not grow on trehalose as a carbon source. Experiments with diploid strains heterozygous for Δ athl show a gene dosage effect for the ATH1 gene for growth on trehalose. The need for acid trehalase for growth on trehalose is supported by the finding that acid trehalase activity is induced during exponential growth of cells on trehalose while no such induction is measurable during growth on glucose. Our results show that the vacuolar acid trehalase Ath1p is necessary for the phenotype of growth on trehalose, i.e. trehalose utilization, in contrast to cytosolic neutral trehalase Nth1p which is necessary for intracellular degradation of trehalose. For explanation of the need for vacuolar acid trehalase and not cytosolic neutral trehalase for growth on trehalose, the participation of endocytosis for uptake of trehalose from medium to the vacuoles is discussed.

Disruption of the yeast ATH1 gene confers better survival after dehydration, freezing, and ethanol shock: potential commercial applications.

The accumulation of trehalose is a critical determinant of stress resistance in the yeast Saccharomyces cerevisiae. We have constructed a yeast strain in which the activity of the trehalose-hydrolyzing enzyme, acid trehalase (ATH), has been abolished. Loss of ATH activity was accomplished by disrupting the ATH1 gene, which is essential for ATH activity. The delta ath1 strain accumulated greater levels of cellular trehalose and grew to a higher cell density than the isogenic wild-type strain. In addition, the elevated levels of trehalose in the delta ath1 strain correlated with increased tolerance to dehydration, freezing, and toxic levels of ethanol. The improved resistance to stress conditions exhibited by the delta ath1 strain may make this strain useful in commercial applications, including baking and brewing.

Isolation and characterization of a novel yeast gene, ATH1, that is required for vacuolar acid trehalase activity

We have isolated a plasmid containing a gene, ATH1, that results in eight- to ten-fold higher acid trehalase activity in yeast cells when present in high copy. The screening procedure was based on overproduction-induced mislocalization of acid trehalase activity; overproduction of vacuolar enzymes that transit through the secretory pathway leads to secretion to the cell surface. A DNA fragment that confers cell surface expression of acid trehalase activity was cloned and sequenced. The deduced amino acid sequence displayed no homology to known proteins, indicating that we have identified a novel gene. A deletion in the genomic copy of the ATH1 gene eliminates vacuolar acid trehalase activity. These results suggest that ATH1 may be the structural gene encoding vacuolar acid trehalase or that the gene product may be essential regulatory component involved in control of trehalase activity.

The homeobox gene ATH1 of Arabidopsis is derepressed in the photomorphogenic mutants cop1 and det1.

A light-regulated Arabidopsis thaliana homeobox 1 gene (ATH1) was identified in a transcription factor gene collection. Primer extension analysis of this gene showed the presence of two major transcripts with unusually long 5' untranslated leader sequences. The leader sequence of the isolated cDNA clone contains several small open reading frames upstream of the initiation codon of the largest open reading frame coding for the homeodomain protein. This leader sequence was found to affect the translation efficiency negatively in an in vitro translation system. The expression of the ATH1 gene is dependent on the presence of light. After illuminating etiolated or dark-adapted Arabidopsis seedlings, the ATH1 mRNA level increased rapidly. Expression of ATH1 does not require the presence of active chloroplasts because photooxidative destruction of the chloroplast by norflurazon treatment did not influence the ATH1 mRNA level. In dark-grown seedlings of the photomorphogenic constitutive photomorphogenic 1 (cop1) and deetiolated 1 (det1) mutants, the ATH1 mRNA level was elevated. This shows that the gene products of these loci directly or indirectly repress ATH1 expression in etiolated wild-type seedlings. A correlation between the strength of the cop1 allele and the ATH1 mRNA level was found. This relationship suggests a role for the ATH1 protein in the signal transduction pathway downstream of COP1.