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A nuclear calcium-sensing pathway is critical for gene regulation and salt stress tolerance in Arabidopsis.

Guan Q, Wu J, Yue X, Zhang Y, Zhu J - PLoS Genet. (2013)

Bottom Line: Through a forward genetic screen, we found a nuclear-localized calcium-binding protein, RSA1 (SHORT ROOT IN SALT MEDIUM 1), which is required for salt tolerance, and identified its interacting partner, RITF1, a bHLH transcription factor.We show that RSA1 and RITF1 regulate the transcription of several genes involved in the detoxification of reactive oxygen species generated by salt stress and that they also regulate the SOS1 gene that encodes a plasma membrane Na(+)/H(+) antiporter essential for salt tolerance.Together, our results suggest the existence of a novel nuclear calcium-sensing and -signaling pathway that is important for gene regulation and salt stress tolerance.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Science and Landscape Architecture, University of Maryland, College Park, Maryland, United States of America.

ABSTRACT
Salt stress is an important environmental factor that significantly limits crop productivity worldwide. Studies on responses of plants to salt stress in recent years have identified novel signaling pathways and have been at the forefront of plant stress biology and plant biology in general. Thus far, research on salt stress in plants has been focused on cytoplasmic signaling pathways. In this study, we discovered a nuclear calcium-sensing and signaling pathway that is critical for salt stress tolerance in the reference plant Arabidopsis. Through a forward genetic screen, we found a nuclear-localized calcium-binding protein, RSA1 (SHORT ROOT IN SALT MEDIUM 1), which is required for salt tolerance, and identified its interacting partner, RITF1, a bHLH transcription factor. We show that RSA1 and RITF1 regulate the transcription of several genes involved in the detoxification of reactive oxygen species generated by salt stress and that they also regulate the SOS1 gene that encodes a plasma membrane Na(+)/H(+) antiporter essential for salt tolerance. Together, our results suggest the existence of a novel nuclear calcium-sensing and -signaling pathway that is important for gene regulation and salt stress tolerance.

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RSA1 and RITF1 regulate gene expression, and RITF1 binds directly to promoters of three RSA1 target genes.(A)–(D) Expression of SOS1, At5g14130, and At2g36690 in wild-type, rsa1-1, ritf1, RITF1 overexpression, or RSA1 overexpression plants. At5g14130 and At2g36690 encode peroxidase superfamily protein, oxidoreductase (2-oxoglutarate (2OG) and Fe(II)-dependent oxygenase superfamily protein). Data in (C) and (D) are from multiple RSA1 OE or RITF1 OE plants (Figure S3F and S4C). Expression values from one representative transgenic line are presented. (E)–(H) ChIP-qPCR analysis of SOS1, At2g36690, and At5g14130 genes. Regions of amplifications in (E)–(H) are specified in Table S6A. (I) Relative luciferase activity from the dual luciferase reporter assays in tobacco leaves. RSA1(-EF), RSA1 without EF-hand motif. Error bars represent the standard deviation (n = 4 in [A]–[H], 12 in [I]). One-way ANOVA (Tukey-Kramer test) was performed, and statistically significant differences are indicated by different lowercase letters (p<0.01). These experiments were repeated at least four times with similar results, and data from one representative experiment are presented.
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pgen-1003755-g006: RSA1 and RITF1 regulate gene expression, and RITF1 binds directly to promoters of three RSA1 target genes.(A)–(D) Expression of SOS1, At5g14130, and At2g36690 in wild-type, rsa1-1, ritf1, RITF1 overexpression, or RSA1 overexpression plants. At5g14130 and At2g36690 encode peroxidase superfamily protein, oxidoreductase (2-oxoglutarate (2OG) and Fe(II)-dependent oxygenase superfamily protein). Data in (C) and (D) are from multiple RSA1 OE or RITF1 OE plants (Figure S3F and S4C). Expression values from one representative transgenic line are presented. (E)–(H) ChIP-qPCR analysis of SOS1, At2g36690, and At5g14130 genes. Regions of amplifications in (E)–(H) are specified in Table S6A. (I) Relative luciferase activity from the dual luciferase reporter assays in tobacco leaves. RSA1(-EF), RSA1 without EF-hand motif. Error bars represent the standard deviation (n = 4 in [A]–[H], 12 in [I]). One-way ANOVA (Tukey-Kramer test) was performed, and statistically significant differences are indicated by different lowercase letters (p<0.01). These experiments were repeated at least four times with similar results, and data from one representative experiment are presented.

Mentions: Because RSA1 is localized in the nucleus, we determined whether the rsa1-1 mutation affects gene expression. We performed a whole-genome microarray analysis with Arabidopsis Affymetrix ATH1 GeneChips. Compared to genes in the wild type, 41 genes displayed higher expression levels while 54 genes showed lower expression levels in rsa1-1 by at least 2-fold (p<0.01) under control conditions (Table S2). The microarray analysis also revealed that 69 genes in rsa1-1 displayed at least a 2-fold increase in transcripts levels while 76 genes in rsa1-1 showed at least a 2-fold decrease in transcripts levels relative to the wild type (p<0.01) under salt stress conditions (Table S3). Compared to genes in the wild type, 13 genes displayed higher expression levels while 27 genes showed lower expression levels in rsa1-1 by at least 2-fold (p<0.01) under both control and salt stress conditions (Table S4). The differentially expressed genes in rsa1-1 encode proteins with diverse functions, and a large portion of these proteins have predicted functions in stress responses (Table S2, S3, and S4). In addition, as indicated by comparison with publicly available expression datasets, most of the differentially expressed genes in rsa1-1 are not responsive to salt stress treatments in the wild type (Figure S5 and S6). We validated the microarray data with qRT-PCR analysis for four genes, which encode peroxidase, zinc finger protein 5, bHLH DNA-binding superfamily protein, and root hair specific 19 with putative peroxidase activity (Figure S7A–S7D). qRT-PCR analysis also revealed that the expression levels of SOS1, At2g36690, and At5g14130 are dramatically reduced in rsa1-1 under both control and salt stress conditions (Figure 6A), suggesting that RSA1 is a positive regulator of these three genes. At2g36690 and At5g14130 encode oxidoreductase and peroxidase, respectively. Therefore, RSA1 controls genes important for ROS detoxification and signal transduction. Furthermore, we observed that expression of SOS1, At2g36690, and At5g14130 is substantially reduced in the ritf1 mutant plants under both control and salt stress conditions (Figure 6B), indicating that similar to RSA1, RITF1 is a positive regulator of these three genes. Expression of SOS1, At5g14130, and At2g36690 is substantially enhanced in transgenic plants overexpressing RSA1 or RITF1 under salt stress (Figure 6C and 6D), further confirming that RSA1 and RITF1 are positive regulators for salt-induced expression of SOS1, At5g14130, and At2g36690.


A nuclear calcium-sensing pathway is critical for gene regulation and salt stress tolerance in Arabidopsis.

Guan Q, Wu J, Yue X, Zhang Y, Zhu J - PLoS Genet. (2013)

RSA1 and RITF1 regulate gene expression, and RITF1 binds directly to promoters of three RSA1 target genes.(A)–(D) Expression of SOS1, At5g14130, and At2g36690 in wild-type, rsa1-1, ritf1, RITF1 overexpression, or RSA1 overexpression plants. At5g14130 and At2g36690 encode peroxidase superfamily protein, oxidoreductase (2-oxoglutarate (2OG) and Fe(II)-dependent oxygenase superfamily protein). Data in (C) and (D) are from multiple RSA1 OE or RITF1 OE plants (Figure S3F and S4C). Expression values from one representative transgenic line are presented. (E)–(H) ChIP-qPCR analysis of SOS1, At2g36690, and At5g14130 genes. Regions of amplifications in (E)–(H) are specified in Table S6A. (I) Relative luciferase activity from the dual luciferase reporter assays in tobacco leaves. RSA1(-EF), RSA1 without EF-hand motif. Error bars represent the standard deviation (n = 4 in [A]–[H], 12 in [I]). One-way ANOVA (Tukey-Kramer test) was performed, and statistically significant differences are indicated by different lowercase letters (p<0.01). These experiments were repeated at least four times with similar results, and data from one representative experiment are presented.
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pgen-1003755-g006: RSA1 and RITF1 regulate gene expression, and RITF1 binds directly to promoters of three RSA1 target genes.(A)–(D) Expression of SOS1, At5g14130, and At2g36690 in wild-type, rsa1-1, ritf1, RITF1 overexpression, or RSA1 overexpression plants. At5g14130 and At2g36690 encode peroxidase superfamily protein, oxidoreductase (2-oxoglutarate (2OG) and Fe(II)-dependent oxygenase superfamily protein). Data in (C) and (D) are from multiple RSA1 OE or RITF1 OE plants (Figure S3F and S4C). Expression values from one representative transgenic line are presented. (E)–(H) ChIP-qPCR analysis of SOS1, At2g36690, and At5g14130 genes. Regions of amplifications in (E)–(H) are specified in Table S6A. (I) Relative luciferase activity from the dual luciferase reporter assays in tobacco leaves. RSA1(-EF), RSA1 without EF-hand motif. Error bars represent the standard deviation (n = 4 in [A]–[H], 12 in [I]). One-way ANOVA (Tukey-Kramer test) was performed, and statistically significant differences are indicated by different lowercase letters (p<0.01). These experiments were repeated at least four times with similar results, and data from one representative experiment are presented.
Mentions: Because RSA1 is localized in the nucleus, we determined whether the rsa1-1 mutation affects gene expression. We performed a whole-genome microarray analysis with Arabidopsis Affymetrix ATH1 GeneChips. Compared to genes in the wild type, 41 genes displayed higher expression levels while 54 genes showed lower expression levels in rsa1-1 by at least 2-fold (p<0.01) under control conditions (Table S2). The microarray analysis also revealed that 69 genes in rsa1-1 displayed at least a 2-fold increase in transcripts levels while 76 genes in rsa1-1 showed at least a 2-fold decrease in transcripts levels relative to the wild type (p<0.01) under salt stress conditions (Table S3). Compared to genes in the wild type, 13 genes displayed higher expression levels while 27 genes showed lower expression levels in rsa1-1 by at least 2-fold (p<0.01) under both control and salt stress conditions (Table S4). The differentially expressed genes in rsa1-1 encode proteins with diverse functions, and a large portion of these proteins have predicted functions in stress responses (Table S2, S3, and S4). In addition, as indicated by comparison with publicly available expression datasets, most of the differentially expressed genes in rsa1-1 are not responsive to salt stress treatments in the wild type (Figure S5 and S6). We validated the microarray data with qRT-PCR analysis for four genes, which encode peroxidase, zinc finger protein 5, bHLH DNA-binding superfamily protein, and root hair specific 19 with putative peroxidase activity (Figure S7A–S7D). qRT-PCR analysis also revealed that the expression levels of SOS1, At2g36690, and At5g14130 are dramatically reduced in rsa1-1 under both control and salt stress conditions (Figure 6A), suggesting that RSA1 is a positive regulator of these three genes. At2g36690 and At5g14130 encode oxidoreductase and peroxidase, respectively. Therefore, RSA1 controls genes important for ROS detoxification and signal transduction. Furthermore, we observed that expression of SOS1, At2g36690, and At5g14130 is substantially reduced in the ritf1 mutant plants under both control and salt stress conditions (Figure 6B), indicating that similar to RSA1, RITF1 is a positive regulator of these three genes. Expression of SOS1, At5g14130, and At2g36690 is substantially enhanced in transgenic plants overexpressing RSA1 or RITF1 under salt stress (Figure 6C and 6D), further confirming that RSA1 and RITF1 are positive regulators for salt-induced expression of SOS1, At5g14130, and At2g36690.

Bottom Line: Through a forward genetic screen, we found a nuclear-localized calcium-binding protein, RSA1 (SHORT ROOT IN SALT MEDIUM 1), which is required for salt tolerance, and identified its interacting partner, RITF1, a bHLH transcription factor.We show that RSA1 and RITF1 regulate the transcription of several genes involved in the detoxification of reactive oxygen species generated by salt stress and that they also regulate the SOS1 gene that encodes a plasma membrane Na(+)/H(+) antiporter essential for salt tolerance.Together, our results suggest the existence of a novel nuclear calcium-sensing and -signaling pathway that is important for gene regulation and salt stress tolerance.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Science and Landscape Architecture, University of Maryland, College Park, Maryland, United States of America.

ABSTRACT
Salt stress is an important environmental factor that significantly limits crop productivity worldwide. Studies on responses of plants to salt stress in recent years have identified novel signaling pathways and have been at the forefront of plant stress biology and plant biology in general. Thus far, research on salt stress in plants has been focused on cytoplasmic signaling pathways. In this study, we discovered a nuclear calcium-sensing and signaling pathway that is critical for salt stress tolerance in the reference plant Arabidopsis. Through a forward genetic screen, we found a nuclear-localized calcium-binding protein, RSA1 (SHORT ROOT IN SALT MEDIUM 1), which is required for salt tolerance, and identified its interacting partner, RITF1, a bHLH transcription factor. We show that RSA1 and RITF1 regulate the transcription of several genes involved in the detoxification of reactive oxygen species generated by salt stress and that they also regulate the SOS1 gene that encodes a plasma membrane Na(+)/H(+) antiporter essential for salt tolerance. Together, our results suggest the existence of a novel nuclear calcium-sensing and -signaling pathway that is important for gene regulation and salt stress tolerance.

Show MeSH