NITRIC OXIDE BINDS TO AND MODULATES THE ACTIVITY OF A POLLEN SPECIFIC ARABIDOPSIS DIACYLGLYCEROL KINASE

Handle URI:
http://hdl.handle.net/10754/320295
Title:
NITRIC OXIDE BINDS TO AND MODULATES THE ACTIVITY OF A POLLEN SPECIFIC ARABIDOPSIS DIACYLGLYCEROL KINASE
Authors:
Wong, Aloysius Tze ( 0000-0002-9105-5845 )
Abstract:
Nitric oxide (NO) is an important signaling molecule in plants. In the pollen of Arabidopsis thaliana, NO causes re-orientation of the growing tube and this response is mediated by 3′,5′-cyclic guanosine monophosphate (cGMP). However, in plants, NO-sensors have remained somewhat elusive. Here, the findings of an NO-binding candidate, Arabidopsis thaliana DIACYLGLYCEROL KINASE 4 (ATDGK4; AT5G57690) is presented. In addition to the annotated diacylglycerol kinase domain, this molecule also harbors a predicted heme-NO/oxygen (H-NOX) binding site and a guanylyl cyclase (GC) catalytic domain which have been identified based on the alignment of functionally conserved amino acid residues across species. A 3D model of the molecule was constructed, and from which the locations of the kinase catalytic center, the ATP-binding site, the GC and H-NOX domains were estimated. Docking of ATP to the kinase catalytic center was also modeled. The recombinant ATDGK4 demonstrated kinase activity in vitro, catalyzing the ATP-dependent conversion of sn-1,2-diacylglycerol (DAG) to phosphatidic acid (PA). This activity was inhibited by the mammalian DAG kinase inhibitor R59949 and importantly also by the NO donors diethylamine NONOate (DEA NONOate) and sodium nitroprusside (SNP). Recombinant ATDGK4 also has GC activity in vitro, catalyzing the conversion of guanosine-5'-triphosphate (GTP) to cGMP. The catalytic domains of ATDGK4 kinase and GC may be independently regulated since the kinase but not the GC, was inhibited by NO while Ca2+ only stimulates the GC. It is likely that the DAG kinase product, PA, causes the release of Ca2+ from the intracellular stores and Ca2+ in turn activates the GC domain of ATDGK4 through a feedback mechanism. Analysis of publicly available microarray data has revealed that ATDGK4 is highly expressed in the pollen. Here, the pollen tubes of mis-expressing atdgk4 recorded slower growth rates than the wild-type (Col-0) and importantly, they showed altered NO responses. Specifically, the mis-expressing atdgk4 pollen tubes have growth rates that were less affected by NO and showed reduced bending angles when challenged by an NO source. Further works on atdgk4 knockout/knockdown mutants will reveal the biological functions of ATDGK4 in NO and/or cGMP signaling in the pollen, and in the broader fertilization process.
Advisors:
Gehring, Christoph A ( 0000-0003-4355-4591 )
Committee Member:
Aranda, Manuel ( 0000-0001-6673-016X ) ; Pain, Arnab ( 0000-0002-1755-2819 ) ; Pasqualini, Stefania
KAUST Department:
Biological and Environmental Sciences and Engineering (BESE) Division
Program:
Bioscience
Issue Date:
Jun-2014
Type:
Dissertation
Appears in Collections:
Bioscience Program; Dissertations; Biological and Environmental Sciences and Engineering (BESE) Division

Full metadata record

DC FieldValue Language
dc.contributor.advisorGehring, Christoph Aen
dc.contributor.authorWong, Aloysius Tzeen
dc.date.accessioned2014-06-10T12:43:02Z-
dc.date.available2014-06-10T12:43:02Z-
dc.date.issued2014-06en
dc.identifier.urihttp://hdl.handle.net/10754/320295en
dc.description.abstractNitric oxide (NO) is an important signaling molecule in plants. In the pollen of Arabidopsis thaliana, NO causes re-orientation of the growing tube and this response is mediated by 3′,5′-cyclic guanosine monophosphate (cGMP). However, in plants, NO-sensors have remained somewhat elusive. Here, the findings of an NO-binding candidate, Arabidopsis thaliana DIACYLGLYCEROL KINASE 4 (ATDGK4; AT5G57690) is presented. In addition to the annotated diacylglycerol kinase domain, this molecule also harbors a predicted heme-NO/oxygen (H-NOX) binding site and a guanylyl cyclase (GC) catalytic domain which have been identified based on the alignment of functionally conserved amino acid residues across species. A 3D model of the molecule was constructed, and from which the locations of the kinase catalytic center, the ATP-binding site, the GC and H-NOX domains were estimated. Docking of ATP to the kinase catalytic center was also modeled. The recombinant ATDGK4 demonstrated kinase activity in vitro, catalyzing the ATP-dependent conversion of sn-1,2-diacylglycerol (DAG) to phosphatidic acid (PA). This activity was inhibited by the mammalian DAG kinase inhibitor R59949 and importantly also by the NO donors diethylamine NONOate (DEA NONOate) and sodium nitroprusside (SNP). Recombinant ATDGK4 also has GC activity in vitro, catalyzing the conversion of guanosine-5'-triphosphate (GTP) to cGMP. The catalytic domains of ATDGK4 kinase and GC may be independently regulated since the kinase but not the GC, was inhibited by NO while Ca2+ only stimulates the GC. It is likely that the DAG kinase product, PA, causes the release of Ca2+ from the intracellular stores and Ca2+ in turn activates the GC domain of ATDGK4 through a feedback mechanism. Analysis of publicly available microarray data has revealed that ATDGK4 is highly expressed in the pollen. Here, the pollen tubes of mis-expressing atdgk4 recorded slower growth rates than the wild-type (Col-0) and importantly, they showed altered NO responses. Specifically, the mis-expressing atdgk4 pollen tubes have growth rates that were less affected by NO and showed reduced bending angles when challenged by an NO source. Further works on atdgk4 knockout/knockdown mutants will reveal the biological functions of ATDGK4 in NO and/or cGMP signaling in the pollen, and in the broader fertilization process.en
dc.language.isoenen
dc.subjectArabidopsis Thalianaen
dc.subjectNitric Oxideen
dc.subjectPollen/Pollen Tubeen
dc.subjectCyclic Nucleotides (cGMP, cAMP)en
dc.subjectHomology modeling and dockingen
dc.subjectGyanylyl Cyclaseen
dc.titleNITRIC OXIDE BINDS TO AND MODULATES THE ACTIVITY OF A POLLEN SPECIFIC ARABIDOPSIS DIACYLGLYCEROL KINASEen
dc.typeDissertationen
dc.contributor.departmentBiological and Environmental Sciences and Engineering (BESE) Divisionen
thesis.degree.grantorKing Abdullah University of Science and Technologyen_GB
dc.contributor.committeememberAranda, Manuelen
dc.contributor.committeememberPain, Arnaben
dc.contributor.committeememberPasqualini, Stefaniaen
thesis.degree.disciplineBioscienceen
thesis.degree.nameDoctor of Philosophyen
dc.person.id118497en
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