Arabidopsis thaliana egy2 mutants display altered expression level of genes encoding crucial photosystem II proteins

Abstract

EGY2 is a zinc-containing, intramembrane protease located in the thylakoid membrane. It is considered to be involved in the regulated intramembrane proteolysis - a mechanism leading to activation of membrane-anchored transcription factors through proteolytic cleavage, which causes them to be released from the membrane. The physiological functions of EGY2 in chloroplasts remains poorly understood. To answer the question of what the significance is of EGY2 in chloroplast functioning, two T-DNA insertion lines devoid of EGY2 protein were obtained and the mutant phenotype and photosystem II parameters were analyzed. Chlorophyll fluorescence measurements revealed that the lack of EGY2 protease caused changes in non-photochemical quenching (NPQ) and minimum fluorescence yield (F₀) as well as a higher sensitivity of photosystem II (PSII) to photoinhibition. Further immunoblot analysis revealed significant changes in the accumulation levels of the three chloroplast-encoded PSII core apoproteins: PsbA (D1) and PsbD (D2) forming the PSII reaction center and PsbC - a protein component of CP43, a part of the inner PSII antenna. The accumulation levels of nuclear-encoded proteins,Lhcb1-3, components of the major light-harvesting complex II (LHCII) as well as proteins forming minor peripheral antennae complexes, namely Lhcb4 (CP29), Lhcb5 (CP26), and Lhcb6 (CP24) remain, however, unchanged. The lack of EGY2 led to a significant increase in the level of PsbA (D1) with a simultaneous decrease in the accumulation levels of PsbC (CP43) and PsbD (D2). To test the hypothesis that the observed changes in the abundance of chloroplast-encoded proteins are a consequence of changes in gene expression levels, real-time PCR was performed. The results obtained show that egy2 mutants display an increased expression of PSBA and a reduction in the PSBD and PSBC genes. Simultaneously pTAC10, pTAC16 and FLN1 proteins were found to accumulate in thylakoid membranes of analyzed mutant lines. These proteins interact with the core complex of plastid-encoded RNA polymerase and may be involved in the regulation of chloroplast gene expression.

Keywords: Arabidopsis thaliana; Chloroplast proteases; EGY2; Photosystem II; regulated intramembrane proteolysis.