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The maize OST1 kinase homolog phosphorylates and regulates the maize SNAC1-type transcription factor.

Vilela B, Moreno-Cortés A, Rabissi A, Leung J, Pagès M, Lumbreras V - PLoS ONE (2013)

Bottom Line: Furthermore, we have identified the transcription factor, ZmSNAC1, which is directly phosphorylated by ZmOST1 with implications on its localization and protein stability.Interestingly, ZmSNAC1 binds to the ABA-box of ZmOST1, which is conserved in SnRK2s activated by ABA and is part of the contact site for the negative-regulating clade A PP2C phosphatases.Taken together, our results indicate that ZmSNAC1 is a substrate of ZmOST1 and delineate a novel osmotic stress transcriptional pathway in maize.

View Article: PubMed Central - PubMed

Affiliation: Centre for Research in Agricultural Genomics, Bellaterra, Cerdanyola del Vallés, Spain.

ABSTRACT
The Arabidopsis kinase OPEN STOMATA 1 (OST1) plays a key role in regulating drought stress signalling, particularly stomatal closure. We have identified and investigated the functions of the OST1 ortholog in Z. mays (ZmOST1). Ectopic expression of ZmOST1 in the Arabidopsis ost1 mutant restores the stomatal closure phenotype in response to drought. Furthermore, we have identified the transcription factor, ZmSNAC1, which is directly phosphorylated by ZmOST1 with implications on its localization and protein stability. Interestingly, ZmSNAC1 binds to the ABA-box of ZmOST1, which is conserved in SnRK2s activated by ABA and is part of the contact site for the negative-regulating clade A PP2C phosphatases. Taken together, our results indicate that ZmSNAC1 is a substrate of ZmOST1 and delineate a novel osmotic stress transcriptional pathway in maize.

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The interaction of ZmOST1 with SNAC1 depends on the ZmOST1 ABA-box.BiFC analysis of the interaction between ZmSNAC1 and different ZmOST1, ZmOST1 [G40R] mutant, and deletion forms as depicted on the left. Relative quantification of the BiFC interaction is shown on the right. BiFC fluorescence images analyzed by confocal microscopy are presented on the bottom. 1, YFP signals; 2, light microscope/BiFC channel. Numbers indicate ZmOST1 amino acid regions included in each construct.
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pone-0058105-g004: The interaction of ZmOST1 with SNAC1 depends on the ZmOST1 ABA-box.BiFC analysis of the interaction between ZmSNAC1 and different ZmOST1, ZmOST1 [G40R] mutant, and deletion forms as depicted on the left. Relative quantification of the BiFC interaction is shown on the right. BiFC fluorescence images analyzed by confocal microscopy are presented on the bottom. 1, YFP signals; 2, light microscope/BiFC channel. Numbers indicate ZmOST1 amino acid regions included in each construct.

Mentions: Next, we used bimolecular fluorescence complementation (BiFC) [35], [44]–[46] to determine whether and where ZmSNAC1 interacts with ZmOST1 in planta, and if so, to characterize the specific ZmOST1 domains involved in this interaction. Full-length ZmOST1 (G40R) and different deleted derivatives were fused to the C-terminal half of the YFP while the ZmSNAC1 factor was fused to the N-terminal half (Figure 4). The results showed that ZmOST1 interaction with ZmSNAC1 is mainly nuclear and the different derivatives showed a nuclear/cytosolic distribution (Figure 4). ZmOST1 interaction is independent of the kinase activity and is mediated by a site in the C-terminal regulatory domain corresponding to the region between amino acids 290–366. This domain is present in ABA-dependent SnRK2 kinases and is important for the negative regulation by the clade A PP2C phosphatases [47]–[49]. As shown in Figure 4, co-expression of the regulatory domain or the ABA-box of ZmOST1 alone, amino acids 325–366, with ZmSNAC1 reconstituted the YFP signal. No interaction was detected between YN-ZmSNAC1 and YC-ZmOST1 (1–289aa) constructs. Thus, the ABA box is necessary and sufficient for this interaction [49]. While the ABA-box has been shown to form part of the contact site for the negative regulating PP2Cs [49], [50], our results reveal that it is also important for substrate-binding. This raises the possibility that ZmSNAC1 may compete with the clade A PP2C phosphatases sharing the same docking region, highlighting the interesting perspective of substrate occupancy as a mechanism to sustain ABA signalling [48].


The maize OST1 kinase homolog phosphorylates and regulates the maize SNAC1-type transcription factor.

Vilela B, Moreno-Cortés A, Rabissi A, Leung J, Pagès M, Lumbreras V - PLoS ONE (2013)

The interaction of ZmOST1 with SNAC1 depends on the ZmOST1 ABA-box.BiFC analysis of the interaction between ZmSNAC1 and different ZmOST1, ZmOST1 [G40R] mutant, and deletion forms as depicted on the left. Relative quantification of the BiFC interaction is shown on the right. BiFC fluorescence images analyzed by confocal microscopy are presented on the bottom. 1, YFP signals; 2, light microscope/BiFC channel. Numbers indicate ZmOST1 amino acid regions included in each construct.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585266&req=5

pone-0058105-g004: The interaction of ZmOST1 with SNAC1 depends on the ZmOST1 ABA-box.BiFC analysis of the interaction between ZmSNAC1 and different ZmOST1, ZmOST1 [G40R] mutant, and deletion forms as depicted on the left. Relative quantification of the BiFC interaction is shown on the right. BiFC fluorescence images analyzed by confocal microscopy are presented on the bottom. 1, YFP signals; 2, light microscope/BiFC channel. Numbers indicate ZmOST1 amino acid regions included in each construct.
Mentions: Next, we used bimolecular fluorescence complementation (BiFC) [35], [44]–[46] to determine whether and where ZmSNAC1 interacts with ZmOST1 in planta, and if so, to characterize the specific ZmOST1 domains involved in this interaction. Full-length ZmOST1 (G40R) and different deleted derivatives were fused to the C-terminal half of the YFP while the ZmSNAC1 factor was fused to the N-terminal half (Figure 4). The results showed that ZmOST1 interaction with ZmSNAC1 is mainly nuclear and the different derivatives showed a nuclear/cytosolic distribution (Figure 4). ZmOST1 interaction is independent of the kinase activity and is mediated by a site in the C-terminal regulatory domain corresponding to the region between amino acids 290–366. This domain is present in ABA-dependent SnRK2 kinases and is important for the negative regulation by the clade A PP2C phosphatases [47]–[49]. As shown in Figure 4, co-expression of the regulatory domain or the ABA-box of ZmOST1 alone, amino acids 325–366, with ZmSNAC1 reconstituted the YFP signal. No interaction was detected between YN-ZmSNAC1 and YC-ZmOST1 (1–289aa) constructs. Thus, the ABA box is necessary and sufficient for this interaction [49]. While the ABA-box has been shown to form part of the contact site for the negative regulating PP2Cs [49], [50], our results reveal that it is also important for substrate-binding. This raises the possibility that ZmSNAC1 may compete with the clade A PP2C phosphatases sharing the same docking region, highlighting the interesting perspective of substrate occupancy as a mechanism to sustain ABA signalling [48].

Bottom Line: Furthermore, we have identified the transcription factor, ZmSNAC1, which is directly phosphorylated by ZmOST1 with implications on its localization and protein stability.Interestingly, ZmSNAC1 binds to the ABA-box of ZmOST1, which is conserved in SnRK2s activated by ABA and is part of the contact site for the negative-regulating clade A PP2C phosphatases.Taken together, our results indicate that ZmSNAC1 is a substrate of ZmOST1 and delineate a novel osmotic stress transcriptional pathway in maize.

View Article: PubMed Central - PubMed

Affiliation: Centre for Research in Agricultural Genomics, Bellaterra, Cerdanyola del Vallés, Spain.

ABSTRACT
The Arabidopsis kinase OPEN STOMATA 1 (OST1) plays a key role in regulating drought stress signalling, particularly stomatal closure. We have identified and investigated the functions of the OST1 ortholog in Z. mays (ZmOST1). Ectopic expression of ZmOST1 in the Arabidopsis ost1 mutant restores the stomatal closure phenotype in response to drought. Furthermore, we have identified the transcription factor, ZmSNAC1, which is directly phosphorylated by ZmOST1 with implications on its localization and protein stability. Interestingly, ZmSNAC1 binds to the ABA-box of ZmOST1, which is conserved in SnRK2s activated by ABA and is part of the contact site for the negative-regulating clade A PP2C phosphatases. Taken together, our results indicate that ZmSNAC1 is a substrate of ZmOST1 and delineate a novel osmotic stress transcriptional pathway in maize.

Show MeSH
Related in: MedlinePlus