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dc.contributor.authorGhahremani, Minaen
dc.date.accessioned2018-10-04T14:02:35Z
dc.date.available2018-10-04T14:02:35Z
dc.identifier.urihttp://hdl.handle.net/1974/24961
dc.description.abstractPurple acid phosphatases (PAPs) function in the acquisition and recycling of inorganic phosphate (Pi), a crucial but environmentally-limiting macronutrient for plant growth. Among 29 predicted PAPs of the model plant Arabidopsis thaliana, AtPAP26 (At5g34850) functions as the principal intra- and extracellular PAP isozyme that scavenges Pi during Pi-deprivation or leaf senescence. A pair of secreted, 55 kDa AtPAP26 ‘glycoforms’ (AtPAP26-S1 and AtPAP26-S2) were resolved during lectin-affinity chromatography of cell wall extracts from Pi-deprived Arabidopsis suspension cells and purified. High resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS) demonstrated that their glycans were comparable at Asn103, whereas Asn365 and Asn422 glycosylation sites were extensively modified in AtPAP26-S2 by the addition of up to seven terminal mannose residues (to N-acetylglucosamine) to form high mannose glycans. A 55 kDa protein that co-purified with AtPAP26-S2 was identified by LC-MS/MS as Arabidopsis GNA and apple domain-containing lectin-1 (AtGAL1). AtGAL1 (At1g78850) belongs to Arabidopsis’ Galanthus nivalis agglutinin (GNA) lectin family, whose GNA domain binds high mannose N-glycans. AtGAL1 cross-reactivity with anti-AtGAL1-IgG was markedly attenuated when the lectin was incubated in the presence of a thiol-reducing reagent (consistent with three predicted disulfide bonds in AtGAL1’s apple domain). Reciprocal far western immunodot blotting demonstrated a specific interaction between purified AtGAL1 and AtPAP26-S2, but not AtPAP26-S1. Analytical gel filtration FPLC indicated that AtGAL1 and AtPAP26-S2 associate to form a 112 kDa heterodimer. Bimolecular fluorescence complementation assays established that like AtPAP26, AtGAL1 is also targeted to lytic vacuoles of Pi-starved Arabidopsis cells, and that both proteins interact in vivo. AtGAL1 pre-incubation significantly enhanced the acid phosphatase activity and thermal stability of AtPAP26-S2, but not AtPAP26-S1. Interestingly, LC-MS/MS also revealed that purified AtGAL1 was bisphosphorylated at Tyr38 and Thr39. Secreted AtGAL1 polypeptides were upregulated to a far greater extent than AtGAL1 transcripts during Pi deprivation, indicating post-transcriptional control of AtGAL1 expression. I hypothesize that AtGAL1 plays a key role during Pi deprivation through its interaction with high mannose glycans of AtPAP26-S2, and consequent positive impact on AtPAP26-S2 phosphatase activity and stability. The current study appears to provide the first definitive evidence for involvement of glycoforms, lectins, or a phosphotyrosylated protein in plant Pi starvation responses.en
dc.language.isoengen
dc.relation.ispartofseriesCanadian thesesen
dc.rightsQueen's University's Thesis/Dissertation Non-Exclusive License for Deposit to QSpace and Library and Archives Canadaen
dc.rightsProQuest PhD and Master's Theses International Dissemination Agreementen
dc.rightsIntellectual Property Guidelines at Queen's Universityen
dc.rightsCopying and Preserving Your Thesisen
dc.rightsThis publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner.en
dc.subjectPurple acid phosphataseen
dc.subjectGlycoformsen
dc.subjectLectinen
dc.subjectCell wallen
dc.subjectPhosphate metabolismen
dc.titleA glycoform of the purple acid phosphatase AtPAP26 interacts with a GNA-apple domain lectin (AtGAL1) in cell walls of phosphate-starved Arabidopsis thalianaen
dc.typethesisen
dc.description.degreePhDen
dc.contributor.supervisorPlaxton, Williamen
dc.contributor.departmentBiologyen
dc.degree.grantorQueen's University at Kingstonen


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