Limits...
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.

Show MeSH

Related in: MedlinePlus

ZmSNAC1 phosphorylation by osmotic stress is dependent on ZmOST1 activity.(A) ZmSNAC1 phosphorylation is analyzed by in gel kinase assay (KA: ZmSNAC1). Protein extracts were prepared from maize young seedlings 30 min after treatments with MS 0.5× medium (C), 100 µM ABA (ABA), 400 mM mannitol (Man) and 250 mM salt (NaCl). Sizes of the activity kinase bands obtained are shown on the right. (B) ZmSNAC1 in vitro phosphorylation by ZmOST1 (IVP). Schematic representation of ZmSNAC1 domains used in the experiment. Numbers indicate ZmSNAC1 amino acid regions included in each construct. AD marks the activation domain [35]. Phosphorylation of ZmSNAC1 and deletion forms is tested in vitro. 1, ZmSNAC1 phosphorylation by ZmOST1; 2, ZmSNAC1 alone; 3, 4, 5; ZmOST1 phosphorylation of ZmSNAC1 fragments A, B and C, respectively; 6, 7, 8 ZmSNAC1 fragments A, B and C, respectively. The expression of the different ZmSNAC1 constructs is verified by Coomassie-blue protein staining (CBB). (C) In gel kinase assay with proteins extracted from 7 day-old seedlings treated or not with 100 µM ABA (KA: ZmSNAC1; upper gel). In gel kinase assay after immunoprecipitation of the same samples with an antibody against the ZmOST1 ABA-domain (IP: OST1/KA: ZmSNAC1; centre gel). Western-blot of the immunoprecipitation experiment (IP: OST1/WB: OST1; lower gel). Lane 1, Ler wild-type seedlings; lane 2, ost1-2 mutant; lane 3, 35S::ZmOST1/ost1-2 transgenic line. The ZmSNAC1 protein was used as substrate. Sizes of the activity kinase bands obtained are shown on the right.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3585266&req=5

pone-0058105-g005: ZmSNAC1 phosphorylation by osmotic stress is dependent on ZmOST1 activity.(A) ZmSNAC1 phosphorylation is analyzed by in gel kinase assay (KA: ZmSNAC1). Protein extracts were prepared from maize young seedlings 30 min after treatments with MS 0.5× medium (C), 100 µM ABA (ABA), 400 mM mannitol (Man) and 250 mM salt (NaCl). Sizes of the activity kinase bands obtained are shown on the right. (B) ZmSNAC1 in vitro phosphorylation by ZmOST1 (IVP). Schematic representation of ZmSNAC1 domains used in the experiment. Numbers indicate ZmSNAC1 amino acid regions included in each construct. AD marks the activation domain [35]. Phosphorylation of ZmSNAC1 and deletion forms is tested in vitro. 1, ZmSNAC1 phosphorylation by ZmOST1; 2, ZmSNAC1 alone; 3, 4, 5; ZmOST1 phosphorylation of ZmSNAC1 fragments A, B and C, respectively; 6, 7, 8 ZmSNAC1 fragments A, B and C, respectively. The expression of the different ZmSNAC1 constructs is verified by Coomassie-blue protein staining (CBB). (C) In gel kinase assay with proteins extracted from 7 day-old seedlings treated or not with 100 µM ABA (KA: ZmSNAC1; upper gel). In gel kinase assay after immunoprecipitation of the same samples with an antibody against the ZmOST1 ABA-domain (IP: OST1/KA: ZmSNAC1; centre gel). Western-blot of the immunoprecipitation experiment (IP: OST1/WB: OST1; lower gel). Lane 1, Ler wild-type seedlings; lane 2, ost1-2 mutant; lane 3, 35S::ZmOST1/ost1-2 transgenic line. The ZmSNAC1 protein was used as substrate. Sizes of the activity kinase bands obtained are shown on the right.

Mentions: If ZmOST1 is activated by ABA and hyperosmotic stress, we reasoned that these treatments may lead to ZmSNAC1 phosphorylation. In fact, even though the optimal motif for OST1 phosphorylation, LXRXX(S/T) [48], is absent in the ZmSNAC1 primary sequence, we were able to predict potential phosphorylation sites using a web-based bioinformatics tool (Table S1) [51]. To test ZmSNAC1 phosphorylation we used maize extracts pre-treated or not with either ABA, mannitol or salt to detect kinase activities toward recombinant ZmSNAC1 protein. Using in-gel kinase assays our results revealed a 43–44 kDa kinase that was rapidly and strongly activated in maize seedlings by mannitol and salt. However, 30 min after ABA stimulation, this activity became barely detectable (Figure 5A) suggesting that ZmSNAC1 is phosphorylated by kinases transiently activated by hyperosmotic stress signals.


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)

ZmSNAC1 phosphorylation by osmotic stress is dependent on ZmOST1 activity.(A) ZmSNAC1 phosphorylation is analyzed by in gel kinase assay (KA: ZmSNAC1). Protein extracts were prepared from maize young seedlings 30 min after treatments with MS 0.5× medium (C), 100 µM ABA (ABA), 400 mM mannitol (Man) and 250 mM salt (NaCl). Sizes of the activity kinase bands obtained are shown on the right. (B) ZmSNAC1 in vitro phosphorylation by ZmOST1 (IVP). Schematic representation of ZmSNAC1 domains used in the experiment. Numbers indicate ZmSNAC1 amino acid regions included in each construct. AD marks the activation domain [35]. Phosphorylation of ZmSNAC1 and deletion forms is tested in vitro. 1, ZmSNAC1 phosphorylation by ZmOST1; 2, ZmSNAC1 alone; 3, 4, 5; ZmOST1 phosphorylation of ZmSNAC1 fragments A, B and C, respectively; 6, 7, 8 ZmSNAC1 fragments A, B and C, respectively. The expression of the different ZmSNAC1 constructs is verified by Coomassie-blue protein staining (CBB). (C) In gel kinase assay with proteins extracted from 7 day-old seedlings treated or not with 100 µM ABA (KA: ZmSNAC1; upper gel). In gel kinase assay after immunoprecipitation of the same samples with an antibody against the ZmOST1 ABA-domain (IP: OST1/KA: ZmSNAC1; centre gel). Western-blot of the immunoprecipitation experiment (IP: OST1/WB: OST1; lower gel). Lane 1, Ler wild-type seedlings; lane 2, ost1-2 mutant; lane 3, 35S::ZmOST1/ost1-2 transgenic line. The ZmSNAC1 protein was used as substrate. Sizes of the activity kinase bands obtained are shown on the right.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0058105-g005: ZmSNAC1 phosphorylation by osmotic stress is dependent on ZmOST1 activity.(A) ZmSNAC1 phosphorylation is analyzed by in gel kinase assay (KA: ZmSNAC1). Protein extracts were prepared from maize young seedlings 30 min after treatments with MS 0.5× medium (C), 100 µM ABA (ABA), 400 mM mannitol (Man) and 250 mM salt (NaCl). Sizes of the activity kinase bands obtained are shown on the right. (B) ZmSNAC1 in vitro phosphorylation by ZmOST1 (IVP). Schematic representation of ZmSNAC1 domains used in the experiment. Numbers indicate ZmSNAC1 amino acid regions included in each construct. AD marks the activation domain [35]. Phosphorylation of ZmSNAC1 and deletion forms is tested in vitro. 1, ZmSNAC1 phosphorylation by ZmOST1; 2, ZmSNAC1 alone; 3, 4, 5; ZmOST1 phosphorylation of ZmSNAC1 fragments A, B and C, respectively; 6, 7, 8 ZmSNAC1 fragments A, B and C, respectively. The expression of the different ZmSNAC1 constructs is verified by Coomassie-blue protein staining (CBB). (C) In gel kinase assay with proteins extracted from 7 day-old seedlings treated or not with 100 µM ABA (KA: ZmSNAC1; upper gel). In gel kinase assay after immunoprecipitation of the same samples with an antibody against the ZmOST1 ABA-domain (IP: OST1/KA: ZmSNAC1; centre gel). Western-blot of the immunoprecipitation experiment (IP: OST1/WB: OST1; lower gel). Lane 1, Ler wild-type seedlings; lane 2, ost1-2 mutant; lane 3, 35S::ZmOST1/ost1-2 transgenic line. The ZmSNAC1 protein was used as substrate. Sizes of the activity kinase bands obtained are shown on the right.
Mentions: If ZmOST1 is activated by ABA and hyperosmotic stress, we reasoned that these treatments may lead to ZmSNAC1 phosphorylation. In fact, even though the optimal motif for OST1 phosphorylation, LXRXX(S/T) [48], is absent in the ZmSNAC1 primary sequence, we were able to predict potential phosphorylation sites using a web-based bioinformatics tool (Table S1) [51]. To test ZmSNAC1 phosphorylation we used maize extracts pre-treated or not with either ABA, mannitol or salt to detect kinase activities toward recombinant ZmSNAC1 protein. Using in-gel kinase assays our results revealed a 43–44 kDa kinase that was rapidly and strongly activated in maize seedlings by mannitol and salt. However, 30 min after ABA stimulation, this activity became barely detectable (Figure 5A) suggesting that ZmSNAC1 is phosphorylated by kinases transiently activated by hyperosmotic stress signals.

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