The Role of Chloroplast-localized HSP21 in the Stress Responses of Arabidopsis Thaliana

Persistent Link:
http://hdl.handle.net/10150/146645
Title:
The Role of Chloroplast-localized HSP21 in the Stress Responses of Arabidopsis Thaliana
Author:
Samsel, Kara Ann
Issue Date:
May-2010
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
Heat shock proteins (HSPs) are a ubiquitous family of protein chaperones, which prevent irreversible protein aggregation. HSPs help confer thermotolerance to organisms and can protect against other environmental stresses, such as oxidative stress. Expression levels of HSPs increase during exposure to these stresses. Small heat shock proteins (sHSPs) bind to denaturing protein to prevent aggregation. Since sHSPs are ATP-independent, ATP-dependent chaperones are involved in actively refolding proteins after release from sHSPs. Plants, including the model organism Arabidopsis thaliana, contain multiple HSPs, which are localized to the cytosol, chloroplasts, mitochondria, the nucleus, peroxisomes, and ER. This project focuses on the function of chloroplast-localized Hsp21, which is encoded by a single nuclear gene, making it amenable to genetic analysis. One goal is to identify the phenotypic effects of elevated temperatures and other stresses on Arabidopsis plants null for Hsp21. An Hsp21 null mutant line was identified in which a point mutation in the conserved intron base at the 3' splice site prevents the accumulation of Hsp21 after heat stress. Mutants unable to synthesize Hsp21 during stress showed a mild heat-sensitive phenotype. Additional assays to study chlorophyll levels and the effects of oxidative stress will be performed. Another goal is to identify possible in vivo substrates of Hsp21. This is being approached by introducing an affinity tag (strep-tag II encoding WSHPQFEK) to the 3' end of the Hsp21 gene. The DNA encoding Hsp21-strep has been inserted into an entry vector and will be recombined with a plant transformation plasmid, which is transformed into Agrobacterium tumefaciens. This bacterium is used to insert the tagged protein gene into plants null for Hsp21. Hsp21 and any associated proteins can then be isolated by separating cell lysates from these plants via streptactin affinity chromatography. Mass spectrometry will be used to determine the identity of the in vivo substrates of Hsp21. It is hypothesized that heat-labile proteins crucial for chloroplast function are most likely to be protected by Hsp21.
Type:
text; Electronic Thesis
Degree Name:
B.S.
Degree Level:
bachelors
Degree Program:
Honors College; Biochemistry and Molecular Biophysics
Degree Grantor:
University of Arizona

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titleThe Role of Chloroplast-localized HSP21 in the Stress Responses of Arabidopsis Thalianaen_US
dc.creatorSamsel, Kara Annen_US
dc.contributor.authorSamsel, Kara Annen_US
dc.date.issued2010-05-
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractHeat shock proteins (HSPs) are a ubiquitous family of protein chaperones, which prevent irreversible protein aggregation. HSPs help confer thermotolerance to organisms and can protect against other environmental stresses, such as oxidative stress. Expression levels of HSPs increase during exposure to these stresses. Small heat shock proteins (sHSPs) bind to denaturing protein to prevent aggregation. Since sHSPs are ATP-independent, ATP-dependent chaperones are involved in actively refolding proteins after release from sHSPs. Plants, including the model organism Arabidopsis thaliana, contain multiple HSPs, which are localized to the cytosol, chloroplasts, mitochondria, the nucleus, peroxisomes, and ER. This project focuses on the function of chloroplast-localized Hsp21, which is encoded by a single nuclear gene, making it amenable to genetic analysis. One goal is to identify the phenotypic effects of elevated temperatures and other stresses on Arabidopsis plants null for Hsp21. An Hsp21 null mutant line was identified in which a point mutation in the conserved intron base at the 3' splice site prevents the accumulation of Hsp21 after heat stress. Mutants unable to synthesize Hsp21 during stress showed a mild heat-sensitive phenotype. Additional assays to study chlorophyll levels and the effects of oxidative stress will be performed. Another goal is to identify possible in vivo substrates of Hsp21. This is being approached by introducing an affinity tag (strep-tag II encoding WSHPQFEK) to the 3' end of the Hsp21 gene. The DNA encoding Hsp21-strep has been inserted into an entry vector and will be recombined with a plant transformation plasmid, which is transformed into Agrobacterium tumefaciens. This bacterium is used to insert the tagged protein gene into plants null for Hsp21. Hsp21 and any associated proteins can then be isolated by separating cell lysates from these plants via streptactin affinity chromatography. Mass spectrometry will be used to determine the identity of the in vivo substrates of Hsp21. It is hypothesized that heat-labile proteins crucial for chloroplast function are most likely to be protected by Hsp21.en_US
dc.typetexten_US
dc.typeElectronic Thesisen_US
thesis.degree.nameB.S.en_US
thesis.degree.levelbachelorsen_US
thesis.degree.disciplineHonors Collegeen_US
thesis.degree.disciplineBiochemistry and Molecular Biophysicsen_US
thesis.degree.grantorUniversity of Arizonaen_US
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