Functional divergence of chloroplast Cpn60α subunits during Arabidopsis embryo development

Abstract

Chaperonins are a class of molecular chaperones that assist in the folding and assembly of a wide range of substrates. In plants, chloroplast chaperonins are composed of two different types of subunits, Cpn60α and Cpn60β, and duplication of Cpn60α and Cpn60β genes occurs in a high proportion of plants. However, the importance of multiple Cpn60α and Cpn60β genes in plants is poorly understood. In this study, we found that loss-of-function of CPNA2 ( AtCpn60α2), a gene encoding the minor Cpn60α subunit in Arabidopsis thaliana, resulted in arrested embryo development at the globular stage, whereas the other AtCpn60α gene encoding the dominant Cpn60α subunit, CPNA1 ( AtCpn60α1), mainly affected embryonic cotyledon development at the torpedo stage and thereafter. Further studies demonstrated that CPNA2 can form a functional chaperonin with CPNB2 (AtCpn60β2) and CPNB3 (AtCpn60β3), while the functional partners of CPNA1 are CPNB1 (AtCpn60β1) and CPNB2. We also revealed that the functional chaperonin containing CPNA2 could assist the folding of a specific substrate, KASI (β-ketoacyl-[acyl carrier protein] synthase I), and that the KASI protein level was remarkably reduced due to loss-of-function of CPNA2. Furthermore, the reduction in the KASI protein level was shown to be the possible cause for the arrest of cpna2 embryos. Our findings indicate that the two Cpn60α subunits in Arabidopsis play different roles during embryo development through forming distinct chaperonins with specific AtCpn60β to assist the folding of particular substrates, thus providing novel insights into functional divergence of Cpn60α subunits in plants.

Author summary

Chaperonins are large oligomeric complexes that are involved in the folding and assembly of numerous proteins in various species. In contrast to other types of chaperonins, chloroplast chaperonins are characterized by the hetero-oligomeric structure composed of two unique types of subunits, Cpn60α and Cpn60β, each of which is present in two or more paralogous forms in most of higher plants. However, the functional significance underlying the wide array of subunit types and complex oligomeric arrangement remains largely unknown. Here, we investigated the role of the minor Cpn60α subunit AtCpn60α2 in Arabidopsis embryo development, and found that AtCpn60α2 is important for the transition of globular embryos to heart-shaped embryos, whereas loss of the dominant Cpn60α subunit AtCpn60α1 affects embryonic cotyledon development. Further studies demonstrated that AtCpn60α2 could form functional chaperonins with AtCpn60β2 and AtCpn60β3 to specifically assist in folding of the substrate KASI, which is important for the formation of heart-shaped embryos. Our results suggest that duplication of Cpn60α genes in higher plants can increase the potential number of chloroplast chaperonin substrates and provide chloroplast chaperonins with more roles in plant growth and development, thus revealing the relationship between duplication and functional specialization of chaperonin genes.