Adapting laser microirradiation and time-lapse imaging for in vivo analysis of DNA repair in plants

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00216224%3A14740%2F25%3A00143972" target="_blank" >RIV/00216224:14740/25:00143972 - isvavai.cz</a>

Výsledek na webu

<a href="https://www.czech-bioimaging.cz/activities/conference/ipl-2025/" target="_blank" >https://www.czech-bioimaging.cz/activities/conference/ipl-2025/</a>

DOI - Digital Object Identifier

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Jazyk výsledku

angličtina

Název v původním jazyce

Adapting laser microirradiation and time-lapse imaging for in vivo analysis of DNA repair in plants

Popis výsledku v původním jazyce

DNA repair is essential for genome stability, and many components of repair pathways are evolutionarily conserved from plants to humans. However, the precise timing of individual repair steps in plant cells remains poorly understood. Following DNA damage, lesions are recognized by protein complexes such as the MRE11-RAD50-NBS1 (MRN) complex, chromatin is remodeled to allow access to repair enzymes, and chromatin structure is subsequently restored. While these steps have been extensively studied using biochemical and transcriptomic methods, real-time visualization in live plant cells has remained challenging. To overcome this, we adapted laser microirradiation, which was originally developed for mammalian systems, for use in plant cells (Nespor-Dadejova et al., 2022). This technique utilizes high-intensity laser pulses from a confocal microscope to induce localized DNA lesions in living cells, enabling real-time fluorescence imaging of repair factor recruitment. Using this approach, we demonstrated that key repair proteins such as PCNA, MRE11, and PARP1 are recruited to damage sites within seconds. Fluorescence recovery after photobleaching (FRAP) further revealed dynamic accumulation patterns, highlighting the rapid kinetics of DNA repair in living plant cells. In our recent work, we extended this method to whole tissues using roots of Arabidopsis thaliana seedlings. This enabled monitoring of DNA repair and recovery in locally damaged cells over extended time frames. Initial findings show dynamic changes at damaged sites, while surrounding undamaged cells maintain their division potential. Our study establishes a platform for in vivo investigation of DNA repair in complex plant tissues, opening new possibilities for understanding how plant cells maintain genome integrity under stress.

Název v anglickém jazyce

Adapting laser microirradiation and time-lapse imaging for in vivo analysis of DNA repair in plants

Popis výsledku anglicky

DNA repair is essential for genome stability, and many components of repair pathways are evolutionarily conserved from plants to humans. However, the precise timing of individual repair steps in plant cells remains poorly understood. Following DNA damage, lesions are recognized by protein complexes such as the MRE11-RAD50-NBS1 (MRN) complex, chromatin is remodeled to allow access to repair enzymes, and chromatin structure is subsequently restored. While these steps have been extensively studied using biochemical and transcriptomic methods, real-time visualization in live plant cells has remained challenging. To overcome this, we adapted laser microirradiation, which was originally developed for mammalian systems, for use in plant cells (Nespor-Dadejova et al., 2022). This technique utilizes high-intensity laser pulses from a confocal microscope to induce localized DNA lesions in living cells, enabling real-time fluorescence imaging of repair factor recruitment. Using this approach, we demonstrated that key repair proteins such as PCNA, MRE11, and PARP1 are recruited to damage sites within seconds. Fluorescence recovery after photobleaching (FRAP) further revealed dynamic accumulation patterns, highlighting the rapid kinetics of DNA repair in living plant cells. In our recent work, we extended this method to whole tissues using roots of Arabidopsis thaliana seedlings. This enabled monitoring of DNA repair and recovery in locally damaged cells over extended time frames. Initial findings show dynamic changes at damaged sites, while surrounding undamaged cells maintain their division potential. Our study establishes a platform for in vivo investigation of DNA repair in complex plant tissues, opening new possibilities for understanding how plant cells maintain genome integrity under stress.

Druh

O - Ostatní výsledky

CEP obor

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OECD FORD obor

10611 - Plant sciences, botany

Projekt

<a href="/cs/project/EH22_008%2F0004581" target="_blank" >EH22_008/0004581: Nové poznatky pro plodiny nové generace</a><br>

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Rok uplatnění

2025

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů