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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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ZmOST1 complements the lack of OST1 function in drought stress signalling.(A) ZmOST1 expression in different Arabidopsis lines analyzed by RT-PCR using Ubiquitin expression as control and by western-blot using an anti-ZmOST1 antibody. Lane 1, Ler wild-type seedlings; lane 2, ost1-2 mutant; lane 3, 35S::ZmOST1/ost1-2 transgenic line (211). (B) OST1 activation by ABA in Arabidopsis protein extract analyzed by MBP in-gel kinase assay (KA: MBP). Lane 1, Ler wild-type seedlings; lane 2, ost1-2 mutant; lane 3, 35S::ZmOST1/ost1-2 transgenic line (211). Sizes of molecular markers are shown on the left. Black arrow marks AtOST1 activity. Asterisks mark two new bands of ABA-induced kinase activities resulting from ZmOST1 transgene expression. (C) Phenotypic ost1-2 complementation by ZmOST1 in drought signalling. Detached leaves from ost1-2 allele, OST1::At4g33950/ost1-2 and 35S::ZmOST1/ost1-2 transgenic lines were monitored for foliar temperature by false-colour infrared image subjected to mild drought treatment [6]. (D) Quantification of infrared images. The average leaf temperatures were 22.61±0.37 for the ost1-2 allele; 23.27±0.19 for OST1::At4g33950/ost1-2 and 23.60±0.07 for 35S::ZmOST1/ost1-2. (E) Water loss kinetics using detached leaves of wild-type (closed circles), ost1-2 mutant (open squares) and two independent 35S::ZmOST1/ost1-2 transgenic lines (line 211, closed diamonds and line 287, open circles). Water loss is expressed as the percentage of initial fresh weight. Values are means ± SD of three independent experiments.
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pone-0058105-g001: ZmOST1 complements the lack of OST1 function in drought stress signalling.(A) ZmOST1 expression in different Arabidopsis lines analyzed by RT-PCR using Ubiquitin expression as control and by western-blot using an anti-ZmOST1 antibody. Lane 1, Ler wild-type seedlings; lane 2, ost1-2 mutant; lane 3, 35S::ZmOST1/ost1-2 transgenic line (211). (B) OST1 activation by ABA in Arabidopsis protein extract analyzed by MBP in-gel kinase assay (KA: MBP). Lane 1, Ler wild-type seedlings; lane 2, ost1-2 mutant; lane 3, 35S::ZmOST1/ost1-2 transgenic line (211). Sizes of molecular markers are shown on the left. Black arrow marks AtOST1 activity. Asterisks mark two new bands of ABA-induced kinase activities resulting from ZmOST1 transgene expression. (C) Phenotypic ost1-2 complementation by ZmOST1 in drought signalling. Detached leaves from ost1-2 allele, OST1::At4g33950/ost1-2 and 35S::ZmOST1/ost1-2 transgenic lines were monitored for foliar temperature by false-colour infrared image subjected to mild drought treatment [6]. (D) Quantification of infrared images. The average leaf temperatures were 22.61±0.37 for the ost1-2 allele; 23.27±0.19 for OST1::At4g33950/ost1-2 and 23.60±0.07 for 35S::ZmOST1/ost1-2. (E) Water loss kinetics using detached leaves of wild-type (closed circles), ost1-2 mutant (open squares) and two independent 35S::ZmOST1/ost1-2 transgenic lines (line 211, closed diamonds and line 287, open circles). Water loss is expressed as the percentage of initial fresh weight. Values are means ± SD of three independent experiments.

Mentions: OST1 in Arabidopsis limits water loss in leaves through the regulation of stomatal closure [4], [6]. Since ZmOST1 shares a 83% sequence identity and a similar constitutive pattern of expression during development and stress responses with its Arabidopsis counterpart (Figures S1 and S2) we tested whether ZmOST1 is a functional OST1 ortholog by examining its ability to complement the severe ost1-2 allele carrying the point mutation (G33R) in the ATP-binding loop domain [6]. The expression levels of the ZmOST1 transgene and protein in ost1-2 transgenic plants were analyzed by RT-PCR and western-blot analyses using ZmOST1 antibody (Figure 1A). The ZmOST1 activity in the complemented Arabidopsis plants was determined by MBP in-gel kinase assays (Figure 1B). It has been reported that ABA activates OST1 but that this kinase activity is missing in the ost1-2 allele [6]. Comparing MBP phosphorylation from wild-type, ost1-2 and ZmOST1/ost1-2 seedlings we detected in the ZmOST1/ost1-2 extracts a new specific ABA-dependent kinase activity that is absent in the mutant [6]. The 42–43 kDa activity is coincidental to the expected size of ZmOST1, suggesting that the maize kinase is active in Arabidopsis (Figure 1B).


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)

ZmOST1 complements the lack of OST1 function in drought stress signalling.(A) ZmOST1 expression in different Arabidopsis lines analyzed by RT-PCR using Ubiquitin expression as control and by western-blot using an anti-ZmOST1 antibody. Lane 1, Ler wild-type seedlings; lane 2, ost1-2 mutant; lane 3, 35S::ZmOST1/ost1-2 transgenic line (211). (B) OST1 activation by ABA in Arabidopsis protein extract analyzed by MBP in-gel kinase assay (KA: MBP). Lane 1, Ler wild-type seedlings; lane 2, ost1-2 mutant; lane 3, 35S::ZmOST1/ost1-2 transgenic line (211). Sizes of molecular markers are shown on the left. Black arrow marks AtOST1 activity. Asterisks mark two new bands of ABA-induced kinase activities resulting from ZmOST1 transgene expression. (C) Phenotypic ost1-2 complementation by ZmOST1 in drought signalling. Detached leaves from ost1-2 allele, OST1::At4g33950/ost1-2 and 35S::ZmOST1/ost1-2 transgenic lines were monitored for foliar temperature by false-colour infrared image subjected to mild drought treatment [6]. (D) Quantification of infrared images. The average leaf temperatures were 22.61±0.37 for the ost1-2 allele; 23.27±0.19 for OST1::At4g33950/ost1-2 and 23.60±0.07 for 35S::ZmOST1/ost1-2. (E) Water loss kinetics using detached leaves of wild-type (closed circles), ost1-2 mutant (open squares) and two independent 35S::ZmOST1/ost1-2 transgenic lines (line 211, closed diamonds and line 287, open circles). Water loss is expressed as the percentage of initial fresh weight. Values are means ± SD of three independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585266&req=5

pone-0058105-g001: ZmOST1 complements the lack of OST1 function in drought stress signalling.(A) ZmOST1 expression in different Arabidopsis lines analyzed by RT-PCR using Ubiquitin expression as control and by western-blot using an anti-ZmOST1 antibody. Lane 1, Ler wild-type seedlings; lane 2, ost1-2 mutant; lane 3, 35S::ZmOST1/ost1-2 transgenic line (211). (B) OST1 activation by ABA in Arabidopsis protein extract analyzed by MBP in-gel kinase assay (KA: MBP). Lane 1, Ler wild-type seedlings; lane 2, ost1-2 mutant; lane 3, 35S::ZmOST1/ost1-2 transgenic line (211). Sizes of molecular markers are shown on the left. Black arrow marks AtOST1 activity. Asterisks mark two new bands of ABA-induced kinase activities resulting from ZmOST1 transgene expression. (C) Phenotypic ost1-2 complementation by ZmOST1 in drought signalling. Detached leaves from ost1-2 allele, OST1::At4g33950/ost1-2 and 35S::ZmOST1/ost1-2 transgenic lines were monitored for foliar temperature by false-colour infrared image subjected to mild drought treatment [6]. (D) Quantification of infrared images. The average leaf temperatures were 22.61±0.37 for the ost1-2 allele; 23.27±0.19 for OST1::At4g33950/ost1-2 and 23.60±0.07 for 35S::ZmOST1/ost1-2. (E) Water loss kinetics using detached leaves of wild-type (closed circles), ost1-2 mutant (open squares) and two independent 35S::ZmOST1/ost1-2 transgenic lines (line 211, closed diamonds and line 287, open circles). Water loss is expressed as the percentage of initial fresh weight. Values are means ± SD of three independent experiments.
Mentions: OST1 in Arabidopsis limits water loss in leaves through the regulation of stomatal closure [4], [6]. Since ZmOST1 shares a 83% sequence identity and a similar constitutive pattern of expression during development and stress responses with its Arabidopsis counterpart (Figures S1 and S2) we tested whether ZmOST1 is a functional OST1 ortholog by examining its ability to complement the severe ost1-2 allele carrying the point mutation (G33R) in the ATP-binding loop domain [6]. The expression levels of the ZmOST1 transgene and protein in ost1-2 transgenic plants were analyzed by RT-PCR and western-blot analyses using ZmOST1 antibody (Figure 1A). The ZmOST1 activity in the complemented Arabidopsis plants was determined by MBP in-gel kinase assays (Figure 1B). It has been reported that ABA activates OST1 but that this kinase activity is missing in the ost1-2 allele [6]. Comparing MBP phosphorylation from wild-type, ost1-2 and ZmOST1/ost1-2 seedlings we detected in the ZmOST1/ost1-2 extracts a new specific ABA-dependent kinase activity that is absent in the mutant [6]. The 42–43 kDa activity is coincidental to the expected size of ZmOST1, suggesting that the maize kinase is active in Arabidopsis (Figure 1B).

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