Study on brassinosteroid-mediated regulation of stem cell regeneration in roots

2022 Fiscal Year Research-status Report

• Project/Area Number: 22K15143
• Research Institution: Nara Institute of Science and Technology
• Principal Investigator: ZHANG YE 奈良先端科学技術大学院大学, 先端科学技術研究科, 博士研究員 (10899470)
• Project Period (FY): 2022-04-01 – 2024-03-31
• Keywords: stem cell / DNA damage / cell division / regeneration

Outline of Annual Research Achievements

I have experimentally tested the effects of brassinosteriod (brassinolide) application and the effects of inhibiting brassinosteroid biosynthesis (by brassinozole treatment) on DNA damage induced stem cell death and the replenishing cell division afterwards. The results showed that brassinosteroid plays a positive role both in promoting DNA damage-induced stem cell death and the replenishing cell division afterwards.
I have examined the regeneration capacity of transgenic plants in which the feedback activation on BRL3 expression is defective. Quantification analysis revealed that the feedback defective lines showed less replenishing cell division than brl3 mutant but not comparable to the wildtype, indicating that the feedback effect BRL3 contributes to activate replenishing cell division.
Using translational fusion of fluorescent proteins, I have examined the expression pattern of BRL3 in the root tip during genotoxic stress exposure (before and after stem cell death) and during the afterward stem cell regeneration phase. The result revealed a positive correlation between the expression level of BRL3 and the activation of replenishing cell division. At the time point prior to the activation of replenishing cell division, the expression level of the mutant version of BRL3, on which the feedback activation is defective, was significantly lower than that of the wild type BRL3.

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

In this year, I have completed the phenotypical analysis of the transgenic lines in which the positive feedback onto BRL3 expression is abolished. I have also completed the preliminary experiment on the effects of brassinosteroid on DNA damage response and stem cell regeneration.
However, the timelapse live imaging of the fluorescent protein reporters for hormone signaling levels and key gene expressions in the root tip was lagging behind the plan because of the time-consuming process of plant crossing and the difficult nature of timelapse imaging over long period of time.

Strategy for Future Research Activity

In the second year, I will continue with live imaging to reveal the spatiotemporal dynamics of hormone signaling levels and key gene expressions. I will optimize the plant growth and treatment conditions and the microscope settings.
To further dissect the BRL3-centered regulatory network for stem cell regeneration, I will generate and examine the regeneration capacities of the following transgenic plant lines. Firstly, mutating the DNA damage inducible cis-element in the gene BRL3's promoter may demonstrate the importance of the rapid input from DNA damage. Secondly, an internalization-deficient version of BRL3 in the brl3 mutant background will serve to test whether internalization of BRL3-BR plays a physiologically relevant role in the transient activation of replenishing cell division that enables proper cellular organization in the regenerated tissues.

Causes of Carryover

Due to the adjustments in the priorities of the planned experiments, the article cost and personnel expenditure of the first year was lower than planned. The postponed experiments and research activities will be nevertheless carried out in the next year. Therefore I would like to request to use the incurring amount in the next fiscal year.