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Beta-carbonic anhydrases play a role in fruiting body development and ascospore germination in the filamentous fungus Sordaria macrospora.

Elleuche S, Pöggeler S - PLoS ONE (2009)

Bottom Line: No effect on vegetative growth, fruiting-body and ascospore development was seen in the single mutant strains lacking cas1 or cas3, while single mutant Deltacas2 was affected in vegetative growth, fruiting-body development and ascospore germination, and the double mutant strain Deltacas1/2 was completely sterile.Defects caused by the lack of cas2 could be partially complemented by elevated CO(2) levels or overexpression of cas1, cas3, or a non-mitochondrial cas2 variant.The results suggest that CAs are required for sexual reproduction in filamentous ascomycetes and that the multiplicity of isoforms results in redundancy of specific and non-specific functions.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology and Genetics, Department of Genetics of Eukaryotic Microorganisms, Georg-August University, Göttingen, Germany.

ABSTRACT
Carbon dioxide (CO(2)) is among the most important gases for all organisms. Its reversible interconversion to bicarbonate (HCO(3) (-)) reaches equilibrium spontaneously, but slowly, and can be accelerated by a ubiquitous group of enzymes called carbonic anhydrases (CAs). These enzymes are grouped by their distinct structural features into alpha-, beta-, gamma-, delta- and zeta-classes. While physiological functions of mammalian, prokaryotic, plant and algal CAs have been extensively studied over the past years, the role of beta-CAs in yeasts and the human pathogen Cryptococcus neoformans has been elucidated only recently, and the function of CAs in multicellular filamentous ascomycetes is mostly unknown. To assess the role of CAs in the development of filamentous ascomycetes, the function of three genes, cas1, cas2 and cas3 (carbonic anhydrase of Sordaria) encoding beta-class carbonic anhydrases was characterized in the filamentous ascomycetous fungus Sordaria macrospora. Fluorescence microscopy was used to determine the localization of GFP- and DsRED-tagged CAs. While CAS1 and CAS3 are cytoplasmic enzymes, CAS2 is localized to the mitochondria. To assess the function of the three isoenzymes, we generated knock-out strains for all three cas genes (Deltacas1, Deltacas2, and Deltacas3) as well as all combinations of double mutants. No effect on vegetative growth, fruiting-body and ascospore development was seen in the single mutant strains lacking cas1 or cas3, while single mutant Deltacas2 was affected in vegetative growth, fruiting-body development and ascospore germination, and the double mutant strain Deltacas1/2 was completely sterile. Defects caused by the lack of cas2 could be partially complemented by elevated CO(2) levels or overexpression of cas1, cas3, or a non-mitochondrial cas2 variant. The results suggest that CAs are required for sexual reproduction in filamentous ascomycetes and that the multiplicity of isoforms results in redundancy of specific and non-specific functions.

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Expression analyses of cas genes.Real time PCR was performed with total RNA isolated from S. macrospora wild type grown at 27°C in liquid BMM for three, five and seven days under ambient air conditions or 5% CO2. Comparisons are given as logarithmic values of the ambient air/5% CO2 ratios and are mean expression ratios from two independent biological replicates, each done in triplicate. For normalization, transcript levels of the SSUrRNA were calculated as described in Material and Methods. Asterisk indicate significance according to REST [70].
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pone-0005177-g002: Expression analyses of cas genes.Real time PCR was performed with total RNA isolated from S. macrospora wild type grown at 27°C in liquid BMM for three, five and seven days under ambient air conditions or 5% CO2. Comparisons are given as logarithmic values of the ambient air/5% CO2 ratios and are mean expression ratios from two independent biological replicates, each done in triplicate. For normalization, transcript levels of the SSUrRNA were calculated as described in Material and Methods. Asterisk indicate significance according to REST [70].

Mentions: The CA Nce103p of S. cerevisiae is induced by low CO2 levels, whereas C. albicans Nce103p is constitutively expressed [12], [13]. To understand the functions of CAs in the filamentous fungus S. macrospora, we investigated the transcriptional expression of cas1, cas2 and cas3 in the wild type grown in ambient air or with exposure to 5% CO2, by real time PCR. In all experiments, cas1 mRNA levels were significantly lower under ambient air conditions after three days of development and up-regulated after five days whereas cas2 transcript was not significantly regulated under the conditions investigated. Expression of cas3 was significantly down-regulated under ambient air conditions after three days of growth and not regulated by different CO2 conditions after five and seven days (Figure 2).


Beta-carbonic anhydrases play a role in fruiting body development and ascospore germination in the filamentous fungus Sordaria macrospora.

Elleuche S, Pöggeler S - PLoS ONE (2009)

Expression analyses of cas genes.Real time PCR was performed with total RNA isolated from S. macrospora wild type grown at 27°C in liquid BMM for three, five and seven days under ambient air conditions or 5% CO2. Comparisons are given as logarithmic values of the ambient air/5% CO2 ratios and are mean expression ratios from two independent biological replicates, each done in triplicate. For normalization, transcript levels of the SSUrRNA were calculated as described in Material and Methods. Asterisk indicate significance according to REST [70].
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2664464&req=5

pone-0005177-g002: Expression analyses of cas genes.Real time PCR was performed with total RNA isolated from S. macrospora wild type grown at 27°C in liquid BMM for three, five and seven days under ambient air conditions or 5% CO2. Comparisons are given as logarithmic values of the ambient air/5% CO2 ratios and are mean expression ratios from two independent biological replicates, each done in triplicate. For normalization, transcript levels of the SSUrRNA were calculated as described in Material and Methods. Asterisk indicate significance according to REST [70].
Mentions: The CA Nce103p of S. cerevisiae is induced by low CO2 levels, whereas C. albicans Nce103p is constitutively expressed [12], [13]. To understand the functions of CAs in the filamentous fungus S. macrospora, we investigated the transcriptional expression of cas1, cas2 and cas3 in the wild type grown in ambient air or with exposure to 5% CO2, by real time PCR. In all experiments, cas1 mRNA levels were significantly lower under ambient air conditions after three days of development and up-regulated after five days whereas cas2 transcript was not significantly regulated under the conditions investigated. Expression of cas3 was significantly down-regulated under ambient air conditions after three days of growth and not regulated by different CO2 conditions after five and seven days (Figure 2).

Bottom Line: No effect on vegetative growth, fruiting-body and ascospore development was seen in the single mutant strains lacking cas1 or cas3, while single mutant Deltacas2 was affected in vegetative growth, fruiting-body development and ascospore germination, and the double mutant strain Deltacas1/2 was completely sterile.Defects caused by the lack of cas2 could be partially complemented by elevated CO(2) levels or overexpression of cas1, cas3, or a non-mitochondrial cas2 variant.The results suggest that CAs are required for sexual reproduction in filamentous ascomycetes and that the multiplicity of isoforms results in redundancy of specific and non-specific functions.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology and Genetics, Department of Genetics of Eukaryotic Microorganisms, Georg-August University, Göttingen, Germany.

ABSTRACT
Carbon dioxide (CO(2)) is among the most important gases for all organisms. Its reversible interconversion to bicarbonate (HCO(3) (-)) reaches equilibrium spontaneously, but slowly, and can be accelerated by a ubiquitous group of enzymes called carbonic anhydrases (CAs). These enzymes are grouped by their distinct structural features into alpha-, beta-, gamma-, delta- and zeta-classes. While physiological functions of mammalian, prokaryotic, plant and algal CAs have been extensively studied over the past years, the role of beta-CAs in yeasts and the human pathogen Cryptococcus neoformans has been elucidated only recently, and the function of CAs in multicellular filamentous ascomycetes is mostly unknown. To assess the role of CAs in the development of filamentous ascomycetes, the function of three genes, cas1, cas2 and cas3 (carbonic anhydrase of Sordaria) encoding beta-class carbonic anhydrases was characterized in the filamentous ascomycetous fungus Sordaria macrospora. Fluorescence microscopy was used to determine the localization of GFP- and DsRED-tagged CAs. While CAS1 and CAS3 are cytoplasmic enzymes, CAS2 is localized to the mitochondria. To assess the function of the three isoenzymes, we generated knock-out strains for all three cas genes (Deltacas1, Deltacas2, and Deltacas3) as well as all combinations of double mutants. No effect on vegetative growth, fruiting-body and ascospore development was seen in the single mutant strains lacking cas1 or cas3, while single mutant Deltacas2 was affected in vegetative growth, fruiting-body development and ascospore germination, and the double mutant strain Deltacas1/2 was completely sterile. Defects caused by the lack of cas2 could be partially complemented by elevated CO(2) levels or overexpression of cas1, cas3, or a non-mitochondrial cas2 variant. The results suggest that CAs are required for sexual reproduction in filamentous ascomycetes and that the multiplicity of isoforms results in redundancy of specific and non-specific functions.

Show MeSH
Related in: MedlinePlus