Increase in recombination rate in Arabidopsis thaliana plants sharing gaseous environment with X-ray and UVC-irradiated plants depends on production of radicals

Abstract

X-ray and UVC are the two physical agents that damage DNA directly, with both agents capable of inducing double-strand breaks. Some of our recent work has demonstrated that local exposure to UVC results in a systemic increase in recombination frequency, suggesting that information about exposure can be passed from damaged to non-damaged tissue. Indeed, we recently showed that plants sharing the same enclosed environment with UVC-irradiated plants exhibit similar increase in homologous recombination frequency as irradiated plants. Here, we further tested whether yet another DNA-damaging agent, X-ray, is capable of increasing recombination rate (RR) in neighboring plants grown in a Petri dish. To test this, we grew plants exposed to X-ray or UVC irradiation in an enclosed environment next to non-exposed plants. We found that both X-ray and UVC-irradiated plants and neighboring plants exhibited comparable increases in the levels of strand breaks and the RR. We further showed that pre-exposure of plants to radical scavenger DMSO substantially alleviates the radiation-induced increase in RR and prevents formation of bystander signal. Our results suggest that the increase in RR in bystander plants can also be triggered by X-ray and that radicals may play some role in initiation or maintenance of this signal.

Figure 1. Schematic representation of homologous recombination reporter line. GUS-based recombination substrate consists of two truncated non-functional overlapping (U region) copies of the transgene. Strand break in one of the regions of homology can be repaid using another region as a template, restoring gene function. HR, homologous recombination.

Figure 2. Increased recombination rate in X-ray- and UVC-exposed and bystander plants. (A) Experimental set-up. Shielded and non-shielded Arabidopsis plants were exposed to X-ray or UVC. Seven days post-irradiation, the plants were harvested separately as either irradiated (not covered) or bystander (covered) plants. (B) The Y-axis shows the average (with SD, calculated from three independent repeats of 25 plants each) recombination rate in control, irradiated and bystander plants grown on liquid medium. Each experiment consisted of 3–4 plates per each treatment with 25 plants per each side of the plate. The experiments were repeated three times. The statistical significance of the results was confirmed using means from all plates in each experimental round and performing a Student’s t-test. The asterisks show a statistically significant difference from control (p < 0.05).

Figure 3. The increase in recombination rate in bystander plants is triggered by gaseous signal. (A) Experimental set-up. Shielded and non-shielded Arabidopsis plants grown on divided and undivided Petri dishes were exposed to X-ray or UVC. (B) The Y-axis shows the average (with SD, calculated from three independent repeats of 25 plants each) recombination rate in control, irradiated and bystander plants grown on liquid medium. The statistical significance of the results was confirmed using means from all plates in each experimental round and performing a Student’s t-test. The asterisks show significant difference from control (p < 0.05).

Figure 4. The signal triggering genome instability in the neighboring plants is dependent on the presence of irradiated plants. (A) Experimental set-up. Shielded and non-shielded Arabidopsis plants were exposed to X-ray or UVC. The Petri dishes used contain the dividers separating the irradiated medium and non-irradiated plants. (B) The Y-axis shows the average (with SD, calculated from three independent repeats of 25 plants each) recombination rate in “media-bystander” and control plants grown on liquid medium. The statistical significance of the results was confirmed using means from all plates in each experimental round and performing a Student’s t-test. The asterisks show a statistically significant difference from control (p < 0.05).

Figure 5. X-ray irradiated and bystander plants have a higher level of strand breaks than wild type plants. Single strand breaks (SSBs) and double strand breaks (DSBs) were measured in ct, X-ray irradiated and bystander (BS) plants using ROPS. The Y-axis shows the level of strand breaks as the average (of two independent experiments, each with two technical repeats with SD) amount of ³H incorporation (dpm/µg). The statistical significance of the results was confirmed using means from all plates in each experimental round and performing a Student’s t-test. The asterisks show a statistically significant difference in strand breaks between X-ray and BS plants and ct plants (p < 0.05).

Figure 6. DMSO prevents the generation of airborne signals but does not influence the “liquid” signal. Plates containing ten-day-old Arabidopsis plants from line #11 were a subject to 20 Gy of X-ray irradiation while growing on liquid MS media in either undivided or divided Petri dishes with or without the presence of DMSO. The Y-axis shows the average (with SD, calculated from three independent repeats) recombination rate in irradiated, “bystander,” “media-bystander” and control plants grown on liquid medium. The statistical significance of the results was confirmed using means from all plates in each experimental round and performing a Student’s t-test. The asterisks show a statistically significant difference from control (p < 0.05).