Assessing the Importance of Chloroplast and Mitochondrial Small Heat Shock Proteins in Arabidopsis thaliana

Abstract

Organisms have a dedicated class of proteins to safeguard their proteome – molecular chaperones. Many chaperones help proteins fold and unfold, maintain their structure, and even re‐fold denatured protein aggregates by using ATP. Small heat shock proteins (sHSPs) are ATP‐independent chaperones that are proposed to prevent irreversible aggregation of denaturing proteins and handing them off to the ATP‐dependent chaperones. sHSPs are especially diverse in plants, possibly due to the exposure of plants to environmental stress due to their sessile nature. Despite their widespread occurrence, little is known about the exact function and role of sHSPs, especially in chloroplasts and mitochondria. Both organelles are critical to plant growth, development and reproduction and are also sources of cellular stress via production of reactive oxygen species. Understanding the role of sHSPs in these organelles is vital to grasp how plants deal with stress and could allow for transferring this knowledge into crop plants to improve agricultural yield. The model plant Arabidopsis thaliana possesses four sHSPs localized to the chloroplasts and mitochondria. HSP23.5‐MI/CP and HSP23.6‐MI/CP are thought to dual‐localize to both organelles, HSP25.3‐CP localizes to chloroplasts and HSP26.5‐MII is targeted to mitochondria. These sHSPs are primarily expressed under heat stress conditions. We have developed single, double, triple and quadruple knockout mutants of these four organelle sHSPs and confirmed that they are protein nulls by immunoblotting with specific antisera. Under standard growth conditions these organelle sHSPs appear dispensable, as the knockout mutants grow similarly to wild type. Notably, the organelle sHSP mutant plants show only minimal growth defects when heat stressed, which is in stark contrast to a lethal phenotype shown by a null mutant of the ATP‐dependent chaperone HSP101. Further work is in progress to determine if chaperones or other proteins are altered in expression to compensate for absence of the sHSPs during stress. Additionally, the dual‐localization of HSPs 23.5‐MI/CP and 23.6‐MI/CP has yet to be fully established and also raises the question of crosstalk between chloroplast and mitochondrial sHSPs. To answer these questions a combination of immunoblotting and proteomics approach on isolated chloroplasts and mitochondria is being taken.