2023 Fiscal Year Research-status Report
Functional analysis of THIOL, a novel chloroplast division protein with different roles.
| Project/Area Number |
23K14210
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| Research Institution | Nagoya University |
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Principal Investigator |
Kozgunova Elena 名古屋大学, 高等研究院(理), 特任助教 (90786120)
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| Project Period (FY) |
2023-04-01 – 2027-03-31
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| Keywords | chloroplast division / Physcomitrium patens / cell division |
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| Outline of Annual Research Achievements |
This research aims to characterize a novel chloroplast division gene THIOL, using two model plant: Physcomitrium patens and Arabidopsis thalinan. In P. patens framshift mutations in THIOL gene result in a severe phenotype with dwarf leaves and few large chloroplasts per cell. We generated frameshift mutations in the THIOL homologue in A.thaliana and observed a similar phenotype with tiny leaves and large chloroplasts. These results suggest that THIOL gene function is conserved across multiple plant species.
We also generated two fluorescent marker lines for known chloroplast division genes: FtsZ and ARC5. Chloroplasts divide by forming two contractile rings: inner ring consisting of FtsZ and outer ring, formed by ARC5. Based on detailed observation of chloroplast division, contraction or shrinkage of the chloroplast division ring is affected after THIOL mutation, preventing complete separation of dividing chloroplasts. These results provide possible explanation of THIOL frameshift phenotype.
Next, we will perform further analysis to characterize THIOL role in plant cells.
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| Current Status of Research Progress |
Current Status of Research Progress
3: Progress in research has been slightly delayed.
Reason
Funstional analysis of THIOL gene have been progressing well. We succesfully generated frameshift mutant in A. thaliana to supplement our previous results obtained from P. patens. In addition, we gained further insights into chloroplast division defects in THIOL mutant.
On the other hand, localization analysis of THIOL gene so far was not succesful. THIOL gene codes a small protein, traditional methods to observe localization such as GFP fusion did not work, as the protein was non-functional. Next, we will try split-GFP system to analyze THIOL localization.
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| Strategy for Future Research Activity |
In future, we will perform rescue and cross-rescue experiments to validate frameshift mutant phenotype. We also plan to investigate THIOL interacting partners, but performing proximity labelling experiments, such as TURBID, followed by mass-spectrometry. It will allow us to identify THIOL protein network and binding partners.
Our main focus is to show THIOL localizatio inside the cell. We will try a split-GFP system, which often works better comparing to original GFP for small proteins. If it split-GFP system does not work, we will perform immunostaining experiments using antibody against THIOL.
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| Causes of Carryover |
The underused grant budget this year primarily stems from two key factors. Firstly, we were able by purchasing consumables, such as PCR reagents, restriction enzymes, during promotional period with discounts. This resulted in the expenditure for consumables being slightly lower than initially estimated.
Secondly, the budget allocated for sequencing experiments was not fully utilized due to the ease of isolating frameshift mutants during certain experiments. As a result, fewer resources were required for sequencing than originally projected.
Given the surplus funds resulting from these factors, it has been decided to transfer the remaining amount to the next fiscal year. This decision ensures that the surplus funds are not wasted and can be utilized effectively for future research endeavors, such as additional sequencing efforts or the procurement of necessary resources for upcoming experiments.
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