Gamma radiation and HZE treatment of seedlings in Arabidopsis

Plants exhibit a robust transcriptional response to gamma radiation which includes the induction of transcripts required for homologous recombination and the suppression of transcripts that promote cell cycle progression. Various DNA damaging agents induce different spectra of DNA damage as well as collateral damage to other cellular components and therefore are not expected to provoke identical responses by the cell. Here we study the effects of two different types of ionizing radiation (IR) treatment, HZE (1 GeV Fe26+ high mass, high charge, and high energy relativistic particles) and gamma photons, on the transcriptome of Arabidopsis thaliana seedlings. Both types of IR induce small clusters of radicals that can result in the formation of double strand breaks (DSBs), but HZE also produces linear arrays of extremely clustered damage. We performed these experiments across a range of time points (1.5-24 h after irradiation) in both wild-type plants and in mutants defective in the DSB-sensing protein kinase ATM. The two types of IR exhibit a shared double strand break-repair-related damage response, although they differ slightly in the timing, degree, and ATM-dependence of the response. The ATM-dependent, DNA metabolism-related transcripts of the DSB response were also induced by other DNA damaging agents, but were not induced by conventional stresses. Both Gamma and HZE irradiation induced, at 24 h post-irradiation, ATM-dependent transcripts associated with a variety of conventional stresses; these were overrepresented for pathogen response, rather than DNA metabolism. In contrast, only HZE-irradiated plants, at 1.5 h after irradiation, exhibited an additional and very extensive transcriptional response, shared with plants experiencing extended night. This response was not apparent in gamma-irradiated plants. We treated 5-day-old WT and atm-1 seedlings of Arabidopsis thaliana with 100 Gy of Gamma radiation (over a span of 15 minutes) or 30 Gy of HZE (over a span of approximately 12 minutes). Gamma irradiations were completed at 8:40 am, while HZE irradiations were conducted in two runs (due to space limitations) which were completed at 1:09 and 1:28pm respectively. Gamma treated seedlings were sampled at 10:10 am, 11:40 am, 2:55 pm, 8:40 pm, and 8:40 am. HZE treated seedlings were sampled at 2:39 pm, 4:09 pm, 7:24 pm, 1:09 am, and 1:09 pm. Un-irradiated WT and atm-1 control seedlings were sampled at 10:45 am on Day #1 and 9:15 am on Day #2. There are a total of 22 experimental or control conditions, with two replicates per condition, yielding 44 samples overall.

植物对γ辐射会产生强烈的转录响应，具体包括诱导同源重组所需的转录本，同时抑制促进细胞周期进程的转录本。各类DNA损伤剂可诱导不同谱型的DNA损伤，还会对其他细胞组分造成附带损伤，因此无法引发细胞完全一致的响应。
本研究探究了两种不同类型的电离辐射（ionizing radiation, IR）处理对拟南芥幼苗转录组的影响，分别为HZE辐射（1 GeV Fe²⁶+，高原子序数、高电荷、高能量相对论粒子，high mass, high charge, and high energy relativistic particles）与γ光子辐射。两种电离辐射均可诱导少量自由基簇，进而可能引发双链断裂（double strand breaks, DSBs）；但HZE辐射还会产生高度成簇损伤的线性阵列。
我们在野生型（wild type, WT）植株以及双链断裂感知蛋白激酶ATM缺陷突变体中，设置了辐照后1.5~24小时的多个时间梯度开展实验。
两种电离辐射虽在响应时序、响应强度以及ATM依赖性上存在细微差异，但均表现出与双链断裂修复相关的共有损伤响应。
DSB响应中依赖ATM、与DNA代谢相关的转录本，同样可被其他DNA损伤剂诱导，但无法被常规胁迫诱导。
在辐照后24小时，γ辐射与HZE辐射均会诱导与多种常规胁迫相关的ATM依赖性转录本，这类转录本在病原体响应通路中富集度较高，而非DNA代谢相关通路。
与之相反，仅在辐照后1.5小时的HZE辐照植株中，会出现额外且极为广泛的转录响应，该响应与经历夜间延长处理的植株所产生的响应一致，而γ辐照植株中未观察到此响应。
我们将拟南芥5日龄的野生型（wild type, WT）与atm-1突变体幼苗分别以100 Gy的γ辐射（辐照时长15分钟）或30 Gy的HZE辐射（辐照时长约12分钟）进行处理。
γ辐照于上午8:40完成，而HZE辐照因空间限制分两次完成，分别于下午1:09与下午1:28结束。
经γ辐照的幼苗分别于上午10:10、上午11:40、下午2:55、晚上8:40以及次日上午8:40进行采样。经HZE辐照的幼苗分别于下午2:39、下午4:09、晚上7:24、次日凌晨1:09以及次日下午1:09进行采样。
未辐照的野生型（wild type, WT）与atm-1对照幼苗分别于第1日上午10:45与第2日上午9:15进行采样。
本研究共设置22种实验或对照条件，每种条件设置2个生物学重复，总计获得44个样本。