Immunolocalization of a Protein Disulfide Isomerase toArabidopsis thalianaChloroplasts and Its Association with Starch Biogenesis

Abstract

Protein disulfide isomerases (PDIs) play important roles in protein folding and redox signaling. We used antiserum generated against the chloroplast PDI, RB60, from the unicellular alga Chlamydomonas reinhardtii for the analysis of a PDI from the vascular plant Arabidopsis thaliana. Immunoanalysis was conducted on recombinant PDI and percoll gradient–purified chloroplast subfractions and via fluorescence microscopy and transmission electron microscopy of Arabidopsis leaves. An abundant immuno‐related PDI isoform of 65 kDa was detected via immunoblot analysis of total cell proteins and a 65‐kDa PDI produced by in vitro transcription/translation of a pdi cDNA. Analysis via microscopy verified that the PDI was located in chloroplasts of leaves, being mainly associated with the stromal fringes of the starch grain, in particular, the stromal‐starch interface at one end of the developing starch grain. During light‐induced chloroplast development, pdi mRNA levels increased by 50% in the light relative to etiolated seedlings. The 65‐kDa PDI protein was undetected in etiolated seedlings but increased significantly during illumination. In contrast, dark adaptation of mature green chloroplasts did not significantly affect PDI levels. We propose a new role for the redox‐regulatory PDIs in plants, that of being associated with transitory starch granule biogenesis in plant leaves.