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An integrated genetic, genomic and systems approach defines gene networks regulated by the interaction of light and carbon signaling pathways in Arabidopsis.

Thum KE, Shin MJ, GutiƩrrez RA, Mukherjee I, Katari MS, Nero D, Shasha D, Coruzzi GM - BMC Syst Biol (2008)

Bottom Line: One transcription factor, HAT22 appears to be a regulatory "hub" in the cli186 network as it shows regulatory connections linking a metabolic network of genes involved in "amino acid metabolism", "C-compound/carbohydrate metabolism" and "glycolysis/gluconeogenesis".The global misregulation of gene networks controlled by light and carbon signaling in cli186 indicates that it represents one of the first Arabidopsis mutants isolated that is specifically disrupted in the integration of both carbon and light signals to control the regulation of metabolic, developmental and regulatory genes.The network analysis of misregulated genes suggests that CLI186 acts to integrate light and carbon signaling interactions and is a master regulator connecting the regulation of a host of downstream metabolic and regulatory processes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, New York University, New York, NY, 10003, USA. karen.thum@gmail.com

ABSTRACT

Background: Light and carbon are two important interacting signals affecting plant growth and development. The mechanism(s) and/or genes involved in sensing and/or mediating the signaling pathways involving these interactions are unknown. This study integrates genetic, genomic and systems approaches to identify a genetically perturbed gene network that is regulated by the interaction of carbon and light signaling in Arabidopsis.

Results: Carbon and light insensitive (cli) mutants were isolated. Microarray data from cli186 is analyzed to identify the genes, biological processes and gene networks affected by the integration of light and carbon pathways. Analysis of this data reveals 966 genes regulated by light and/or carbon signaling in wild-type. In cli186, 216 of these light/carbon regulated genes are misregulated in response to light and/or carbon treatments where 78% are misregulated in response to light and carbon interactions. Analysis of the gene lists show that genes in the biological processes "energy" and "metabolism" are over-represented among the 966 genes regulated by carbon and/or light in wild-type, and the 216 misregulated genes in cli186. To understand connections among carbon and/or light regulated genes in wild-type and the misregulated genes in cli186, the microarray data is interpreted in the context of metabolic and regulatory networks. The network created from the 966 light/carbon regulated genes in wild-type, reveals that cli186 is affected in the light and/or carbon regulation of a network of 60 connected genes, including six transcription factors. One transcription factor, HAT22 appears to be a regulatory "hub" in the cli186 network as it shows regulatory connections linking a metabolic network of genes involved in "amino acid metabolism", "C-compound/carbohydrate metabolism" and "glycolysis/gluconeogenesis".

Conclusion: The global misregulation of gene networks controlled by light and carbon signaling in cli186 indicates that it represents one of the first Arabidopsis mutants isolated that is specifically disrupted in the integration of both carbon and light signals to control the regulation of metabolic, developmental and regulatory genes. The network analysis of misregulated genes suggests that CLI186 acts to integrate light and carbon signaling interactions and is a master regulator connecting the regulation of a host of downstream metabolic and regulatory processes.

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Reporter construct used for mutant screen and verification of ASN1 gene expression in cli186. (a) Schematic representation showing regulation of the ASN1-HPT2 reporter construct used to select for carbon and light insensitive (cli) plants. A 148-bp region of the ASN1 promoter from pea was placed upstream of the hygromycin phosphotransferase gene, HPT2. ASN1 is transcriptionally repressed by sucrose and by light independently, where sucrose and light together have a synergistic repressive effect. (b) Three mutagenized lines, cli186, cli12-2-1 and cli16-1 that exhibit hygromycin-resistance when screened on 0.5% sucrose in L/D cycling conditions. Controls consist of a 'wild-type' (WT) unmutagenized line containing the ASN1-HPT2 transgene and a transgenic line (NOS) containing the HPT2 transgene driven by a NOS promoter, allowing for constitutive expression of the HPT2 gene. (c) Fold-repression as determined via Q-PCR of ASN1 in WT and cli186 plants. Seven day old etiolated seedlings were subject to four treatments: -C-L, +C-L, -C+L and +C+L. Fold-repression of ASN1 was determined by comparing all treatments against their respective backgrounds of -C-L. Asterisks indicate a significant difference between WT and cli186 in expression based on a t-test, p > 0.05.
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Figure 1: Reporter construct used for mutant screen and verification of ASN1 gene expression in cli186. (a) Schematic representation showing regulation of the ASN1-HPT2 reporter construct used to select for carbon and light insensitive (cli) plants. A 148-bp region of the ASN1 promoter from pea was placed upstream of the hygromycin phosphotransferase gene, HPT2. ASN1 is transcriptionally repressed by sucrose and by light independently, where sucrose and light together have a synergistic repressive effect. (b) Three mutagenized lines, cli186, cli12-2-1 and cli16-1 that exhibit hygromycin-resistance when screened on 0.5% sucrose in L/D cycling conditions. Controls consist of a 'wild-type' (WT) unmutagenized line containing the ASN1-HPT2 transgene and a transgenic line (NOS) containing the HPT2 transgene driven by a NOS promoter, allowing for constitutive expression of the HPT2 gene. (c) Fold-repression as determined via Q-PCR of ASN1 in WT and cli186 plants. Seven day old etiolated seedlings were subject to four treatments: -C-L, +C-L, -C+L and +C+L. Fold-repression of ASN1 was determined by comparing all treatments against their respective backgrounds of -C-L. Asterisks indicate a significant difference between WT and cli186 in expression based on a t-test, p > 0.05.

Mentions: In previous studies, it has been shown that the asparagine synthetase (ASN1) gene in plants is transcriptionally repressed by transient treatments with sucrose and/or light, where both light and sucrose together have a synergistic repressive effect [24,29,30]. To identify genetic components involved in the integration of light and carbon signaling, the ASN1 promoter was used in a positive genetic selection, designed to identify mutants defective in the transcriptional repression of the ASN1 gene by both light and carbon. An Arabidopsis line was created that contained a transgene in which a 148-bp region of the ASN1 promoter from pea was placed upstream of the hygromycin phosphotransferase gene (HPT2) [30,31] (Figure 1a). As light and carbon treatments repress expression of ASN1 in the light on carbon-containing media, the ASN1::HPT2 lines whose growth was suppressed under these conditions were selected for mutagenesis. A similar positive genetic selection scheme using the reporter gene construct, CAB3::HPT2 was used in the isolation of the dark overexpression of cab mutants (doc), which identified genes controlling expression of the CAB3 gene [32].


An integrated genetic, genomic and systems approach defines gene networks regulated by the interaction of light and carbon signaling pathways in Arabidopsis.

Thum KE, Shin MJ, GutiƩrrez RA, Mukherjee I, Katari MS, Nero D, Shasha D, Coruzzi GM - BMC Syst Biol (2008)

Reporter construct used for mutant screen and verification of ASN1 gene expression in cli186. (a) Schematic representation showing regulation of the ASN1-HPT2 reporter construct used to select for carbon and light insensitive (cli) plants. A 148-bp region of the ASN1 promoter from pea was placed upstream of the hygromycin phosphotransferase gene, HPT2. ASN1 is transcriptionally repressed by sucrose and by light independently, where sucrose and light together have a synergistic repressive effect. (b) Three mutagenized lines, cli186, cli12-2-1 and cli16-1 that exhibit hygromycin-resistance when screened on 0.5% sucrose in L/D cycling conditions. Controls consist of a 'wild-type' (WT) unmutagenized line containing the ASN1-HPT2 transgene and a transgenic line (NOS) containing the HPT2 transgene driven by a NOS promoter, allowing for constitutive expression of the HPT2 gene. (c) Fold-repression as determined via Q-PCR of ASN1 in WT and cli186 plants. Seven day old etiolated seedlings were subject to four treatments: -C-L, +C-L, -C+L and +C+L. Fold-repression of ASN1 was determined by comparing all treatments against their respective backgrounds of -C-L. Asterisks indicate a significant difference between WT and cli186 in expression based on a t-test, p > 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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Figure 1: Reporter construct used for mutant screen and verification of ASN1 gene expression in cli186. (a) Schematic representation showing regulation of the ASN1-HPT2 reporter construct used to select for carbon and light insensitive (cli) plants. A 148-bp region of the ASN1 promoter from pea was placed upstream of the hygromycin phosphotransferase gene, HPT2. ASN1 is transcriptionally repressed by sucrose and by light independently, where sucrose and light together have a synergistic repressive effect. (b) Three mutagenized lines, cli186, cli12-2-1 and cli16-1 that exhibit hygromycin-resistance when screened on 0.5% sucrose in L/D cycling conditions. Controls consist of a 'wild-type' (WT) unmutagenized line containing the ASN1-HPT2 transgene and a transgenic line (NOS) containing the HPT2 transgene driven by a NOS promoter, allowing for constitutive expression of the HPT2 gene. (c) Fold-repression as determined via Q-PCR of ASN1 in WT and cli186 plants. Seven day old etiolated seedlings were subject to four treatments: -C-L, +C-L, -C+L and +C+L. Fold-repression of ASN1 was determined by comparing all treatments against their respective backgrounds of -C-L. Asterisks indicate a significant difference between WT and cli186 in expression based on a t-test, p > 0.05.
Mentions: In previous studies, it has been shown that the asparagine synthetase (ASN1) gene in plants is transcriptionally repressed by transient treatments with sucrose and/or light, where both light and sucrose together have a synergistic repressive effect [24,29,30]. To identify genetic components involved in the integration of light and carbon signaling, the ASN1 promoter was used in a positive genetic selection, designed to identify mutants defective in the transcriptional repression of the ASN1 gene by both light and carbon. An Arabidopsis line was created that contained a transgene in which a 148-bp region of the ASN1 promoter from pea was placed upstream of the hygromycin phosphotransferase gene (HPT2) [30,31] (Figure 1a). As light and carbon treatments repress expression of ASN1 in the light on carbon-containing media, the ASN1::HPT2 lines whose growth was suppressed under these conditions were selected for mutagenesis. A similar positive genetic selection scheme using the reporter gene construct, CAB3::HPT2 was used in the isolation of the dark overexpression of cab mutants (doc), which identified genes controlling expression of the CAB3 gene [32].

Bottom Line: One transcription factor, HAT22 appears to be a regulatory "hub" in the cli186 network as it shows regulatory connections linking a metabolic network of genes involved in "amino acid metabolism", "C-compound/carbohydrate metabolism" and "glycolysis/gluconeogenesis".The global misregulation of gene networks controlled by light and carbon signaling in cli186 indicates that it represents one of the first Arabidopsis mutants isolated that is specifically disrupted in the integration of both carbon and light signals to control the regulation of metabolic, developmental and regulatory genes.The network analysis of misregulated genes suggests that CLI186 acts to integrate light and carbon signaling interactions and is a master regulator connecting the regulation of a host of downstream metabolic and regulatory processes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, New York University, New York, NY, 10003, USA. karen.thum@gmail.com

ABSTRACT

Background: Light and carbon are two important interacting signals affecting plant growth and development. The mechanism(s) and/or genes involved in sensing and/or mediating the signaling pathways involving these interactions are unknown. This study integrates genetic, genomic and systems approaches to identify a genetically perturbed gene network that is regulated by the interaction of carbon and light signaling in Arabidopsis.

Results: Carbon and light insensitive (cli) mutants were isolated. Microarray data from cli186 is analyzed to identify the genes, biological processes and gene networks affected by the integration of light and carbon pathways. Analysis of this data reveals 966 genes regulated by light and/or carbon signaling in wild-type. In cli186, 216 of these light/carbon regulated genes are misregulated in response to light and/or carbon treatments where 78% are misregulated in response to light and carbon interactions. Analysis of the gene lists show that genes in the biological processes "energy" and "metabolism" are over-represented among the 966 genes regulated by carbon and/or light in wild-type, and the 216 misregulated genes in cli186. To understand connections among carbon and/or light regulated genes in wild-type and the misregulated genes in cli186, the microarray data is interpreted in the context of metabolic and regulatory networks. The network created from the 966 light/carbon regulated genes in wild-type, reveals that cli186 is affected in the light and/or carbon regulation of a network of 60 connected genes, including six transcription factors. One transcription factor, HAT22 appears to be a regulatory "hub" in the cli186 network as it shows regulatory connections linking a metabolic network of genes involved in "amino acid metabolism", "C-compound/carbohydrate metabolism" and "glycolysis/gluconeogenesis".

Conclusion: The global misregulation of gene networks controlled by light and carbon signaling in cli186 indicates that it represents one of the first Arabidopsis mutants isolated that is specifically disrupted in the integration of both carbon and light signals to control the regulation of metabolic, developmental and regulatory genes. The network analysis of misregulated genes suggests that CLI186 acts to integrate light and carbon signaling interactions and is a master regulator connecting the regulation of a host of downstream metabolic and regulatory processes.

Show MeSH