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Published on February 10, 2006; 10.1104/pp.105.074658

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Received November 23, 2005
Returned for revision December 31, 2005
Accepted January 16, 2006

Double strand break repair in plants is developmentally regulated

Alexander Boyko , Franz Zemp , Jody Filkowski , and Igor Kovalchuk *

Department of Biological Sciences, University of Lethbridge, Lethbridge, AB. T1K 3M4, Canada

* Corresponding author; email: igor.kovalchuk{at}uleth.ca.

In this paper, we analysed double strand break (DSB) repair in Arabidopsis thaliana at various developmental stages. To analyze DSB repair, we used a homologous recombination (HR) and point mutation reversion assays based on non-functional {beta}-glucuronidase reporter genes. Activation of the reporter gene through HR or point mutation reversion resulted in the appearance of blue sectors after histochemical staining. Scoring of these sectors at 3-day intervals from 2 to 31 days post germination (dpg) revealed that, although there was a 100-fold increase in the number of genomes per plant, the recombination frequency only increased 30-fold. This translates to a recombination rate at 31dpg (2.77x10-8) being only 30% of the recombination rate at 2dpg (9.14x10-8). Conversely, the mutation frequency increased nearly 180-fold, resulting in a 1.8-fold increase in mutation rate from 2 to 31dpg. Additional analysis of DSBs over the early developmental stages revealed a substantial increase in the number of strand breaks per unit of DNA. Furthermore, RNA analysis of Ku70 and Rad51, two key enzymes in two different DSB repair pathways, and further protein analysis of Ku70, revealed an increase in Ku70 levels and a decrease of Rad51 levels in the developing plants.

These data suggest that DSB repair mechanisms are developmentally regulated in Arabidopsis thaliana, whereby the proportion of breaks repaired via HR substantially decreases as the plants mature.




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