Title : Thylakoid rhodanase-like protein in chloroplast inner envelope forms complexes with FNR and Tic62 and its absence from thylakoids enables plants to better cope with oxidative stress
Thylakoid rhodanase-like protein (TROL) is located at the end of the photosynthetic electron transport chain, at the vicinity of photosystem I, where it facilitates the transfer of electrons from ferredoxin (Fd) to NADP+ via the dynamic binding of ferredoxin:NADPH oxydoreductase (FNR). In the absence of TROL the distribution of high-energy electrons is directed towards reactive oxygen species (ROS) scavenging pathways, rather than to the NADP+ reduction. Therefore, trol plants show increased stress resistance along with significantly elevated ROS detoxification. 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. By engineering the presequence processing site, a single amino acid exchange of Ala67 to Ile67 has been introduced to TROL, leading to inhibited processing, and resulting in protein incorporation at the single membrane location: in the inner envelope membrane. We have created Arabidopsis mutant plants, containing TROL protein just in the inner envelope (TROL-IE) to reveal its role in this compartment. TROL-IE interacts with FNR and Tic62, as shown by Western, TAP/MS, and BN-PAGE analyses. Also, TROL-IE plants seem to have increased total NADP in chloroplasts, as well as increased NADP+/NADPH ratio in physiological conditions, when compared to the WT. Like trol plants, TROL-IE mutants show a decreased amount of detected superoxide anion in comparison to the WT, in physiological conditions, indicating stronger capacity to manage oxidative stress.
What will audience learn from your presentation?
- TROL is localized both in thylakoids, where it participates in the regulation of photosynthetic electron utilization, as well as in inner envelope of chloroplasts, where its function is unknown. This work attempts to resolve this.
- By directed mutagenesis of the TROL presequence, we managed to arrest the protein in inner envelope and to subsequently engineer plants with single-localized TROL with the aim to study its function in this compartment.
- In this research, we use the variety of molecular biology and biochemical methods for protein engineering, determination of protein interactions, topology, tracking the import and localization inside the chloroplasts, as well as determining different ROS and NADP content.
- We provide new insights into photosynthetic electron distribution mechanism and the regulation of photosynthesis.
- Our results might lead to the improvement of the production of oxidative stress-resistant plants.