Construction of the Arabidopsis isogenic lines containing dually localized protein TROL only in the inner chloroplast envelope membrane

The thylakoid rhodanese-like protein (TROL) is located at the end of the photosynthetic electron transport chain, at the vicinity of photosystem I, where it dynamically interacts with the ferredoxin:NADP+ oxidoreductase (FNR) and is postulated to facilitate the transfer of electrons from reduced ferredoxin (Fd) to NADP+. TROL is one of the few so far known dually localized chloroplast proteins. Besides being localized in the thylakoid membranes as the 66 kDa mature form, it has also been found in the inner envelope membrane of chloroplasts as the 70 kDa precursor. In thylakoids, the interaction between TROL and FNR acts like a switch, prioritizing the photosynthetic electron destination sinks according to the organellar needs. The role of TROL in the chloroplast inner envelope membrane is, however, presently unknown. By engineering the presequence protease processing site, a single amino acid exchange of Ala67 to Ile67 has been introduced to TROL, leading to inhibited cleavage of the presequence and resulting in protein incorporation at the inner envelope membrane. In this work, we engineered the Arabidopsis mutant plants that contain TROL almost exclusively in the inner envelope membrane (TROL-IE). To facilitate studying the role of this protein in this chloroplast compartment, we also produced the antiserum specific for the IE form of the TROL.