Upregulation of intracellular and secreted acid phosphatases (APases) is a universal response of orthophosphate-starved (-Pi) plants. APases hydrolize Pi from a broad spectrum of phosphomonoesters at an acidic pH. Plant APases belong to a relatively large multigene family whose specific functions in Pi metabolism are poorly understood. This study focuses on the identification and characterization of cell wall (CW) localized purple acid APases (PAPs) upregulated by -Pi Arabidopsis thaliana. Three glycosylated PAP isozymes secreted into the CW of -Pi Arabidopsis suspension cells were purified and identified by peptide mass fingerprinting using mass spectrometry (MALDI-TOF MS) and N-terminal microsequencing as AtPAP12 (At2g27190; subunit size 60-kDa), AtPAP25 (At4g36350; subunit size 55-kDa) and AtPAP26 (At5g34850; subunit size 55-kDa). Both AtPAP12 and AtPAP26 were previously shown to be upregulated and secreted by –Pi Arabidopsis to scavenge Pi from extracellular organic-P. However, the novel AtPAP25 has never been suggested to be involved in the plant Pi-starvation response. Biochemical characterization of AtPAP25 revealed a monomeric 55 kDa protein. Similar to other PAPs it was purple-in-solution and insensitive to tartrate. Glycoprofiling via LC MS/MS revealed highly complex NXS/T glycosylation motifs at Asn172, Asn367 and Asn424. I hypothesize that these motifs play a role in AtPAP25 targeting and function. Kinetic characterization revealed a broad pH optimum centered at 5.6 and inhibition of activity by several common APase inhibitors. AtPAP25 exhibited broad substrate selectivity, low Vmax, and a Km (phosphoenolpyruvate) value of 0.52 mM. Immunoblot and semi-quantitative RT-PCR transcript analysis indicated that AtPAP25 is exclusively synthesized under –Pi conditions. Deduced amino acid sequences were compared using multiple sequence alignment and phylogenetic analysis. Growth of atpap25 T-DNA insertion mutant knockout seedlings was completely arrested when transferred to a soluble Pi deficient organic-P containing soil mix, pointing to a potential regulatory function of AtPAP25 during nutritional Pi stress. Overall, this research is helping to shed light on the functional importance of specific PAP isozymes in facilitating plant acclimation to nutritional Pi deficiency. This is important because there is an urgent need to engineer Pi-efficient transgenic crops to minimize the huge input of expensive, non-renewable, and polluting Pi fertilizers in agriculture. / Thesis (Master, Biology) -- Queen's University, 2012-09-10 08:28:21.631
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OKQ.1974/7448 |
| Date | 10 September 2012 |
| Creators | Del Vecchio, HERNAN |
| Contributors | Queen's University (Kingston, Ont.). Theses (Queen's University (Kingston, Ont.)) |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Rights | This 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. |
| Relation | Canadian theses |
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