Generation of Mutant Lines of Arabidopsis thaliana Chloroplast Small Heat Shock Proteins

Presenter

Alina Shkurikhina

Campus

UMass Amherst

Sponsor

Elizabeth Vierling, Department of Biochemistry and Molecular Biology, UMass Amherst

Schedule

Session 4, 2:30 PM - 3:15 PM [Schedule by Time][Poster Grid for Time/Location]

Location

Poster Board A29, Campus Center Auditorium, Row 2 (A21-A40) [Poster Location Map]

Abstract

When plants are subjected to increased temperatures, they undergo a conserved change in gene expression known as the heat shock response. One class of proteins that is upregulated during heat stress is small heat shock proteins (sHSPs), which are proposed to act as molecular chaperones to prevent irreversible denaturation and aggregation of heat-sensitive proteins. Though these proteins are vital for surviving high temperature and other stresses, much remains unclear about their mechanism of action. The focus of this project is the HSP25.3 protein, the only sHSP that is localized solely to the chloroplast. It is hypothesized that HSP25.3 assists in maintaining homeostasis in the chloroplast, but previous studies addressing its function have not shown a definitive phenotype and much is still not understood about its role and interaction. Phenotypic analysis of chloroplast sHSP mutants in Arabidopsis thaliana could shed light on the function of this highly conserved protein. To prepare for phenotypic analysis, it was required to characterize available chloroplast HSP25.3 null mutant lines and perform background research to determine potential phenotypic assays for future experiments. Two protein null mutant plant lines generated by CRISPR mutagenesis (hsp25.3-2 and hsp25.3-3) in A. thaliana were backcrossed to remove background mutations and the CRISPR machinery, reisolating the homozygous mutants in the F2 generation. A new mutant line was also developed in which the protein null line hsp25.3-1 is complemented with a wild type HSP25.3 gene containing a C-terminal affinity tag that can be used to recover HSP25.3-interacting proteins.

Keywords

plant biology, stress tolerance, molecular chaperones, CRISPR, mutant analysis

Research Area

Biological Organisms

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