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

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