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We describe fast and reproducible sensitivity assays to quantify the response of Arabidopsis seedlings of different genotypes to a wide range of DNA damaging agents. We apply (1) γ-irradiation, which produces DNA breaks, (2) bleocin, a radiomimetic drug, (3) mitomycin C, a DNA intrastrand cross-linker, (4) hydroxyurea, an inhibitor of DNA synthesis and (5) UV-C, which causes mainly photoproducts. The “true leaf assay” and the “UV resistance assay” are based on easily determined phenotypes as readouts. Using a set of diverse damaging agents combined with different readouts allows establishing relative sensitivity/resistance compared to a reference line, e.g. wild type, determining the most effective type of induced damage and the potential repair pathway affected.

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DNA Damage Sensitivity Assays with Arabidopsis Seedlings
拟南芥幼苗的DNA损伤敏感性实验

植物科学 > 植物生理学 > 组织分析
作者: Marisa Rosa
Marisa RosaAffiliation: Gregor Mendel Institute of Molecular Plant Biology, Vienna, Austria
For correspondence: massrosa@berkeley.edu
Bio-protocol author page: a1272
 and Ortrun Mittelsten Scheid
Ortrun Mittelsten ScheidAffiliation: Gregor Mendel Institute of Molecular Plant Biology, Vienna, Austria
Bio-protocol author page: a1273
Vol 4, Iss 7, 4/5/2014, 3381 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1093

[Abstract] We describe fast and reproducible sensitivity assays to quantify the response of Arabidopsis seedlings of different genotypes to a wide range of DNA damaging agents. We apply (1) γ-irradiation, which produces DNA breaks, (2) bleocin, a radiomimetic drug, (3) mitomycin C, a DNA intrastrand cross-linker, (4) hydroxyurea, an inhibitor of DNA synthesis and (5) UV-C, which causes mainly photoproducts. The “true leaf assay” and the “UV resistance assay” are based on easily determined phenotypes as readouts. Using a set of diverse damaging agents combined with different readouts allows establishing relative sensitivity/resistance compared to a reference line, e.g. wild type, determining the most effective type of induced damage and the potential repair pathway affected.
Keywords: DNA damage(DNA损伤), Arabidopsis(拟南芥), Genotoxic stress(基因毒性应激), Sensitivity assay(敏感性分析)

[Abstract]

Materials and Reagents

  1. Arabidopsis thaliana seeds [Wild type and T-DNA insertion lines of arp6-3, swc6-1 and sensitive ku70-2 (Rosa et al., 2013) are used as examples. All mutants are in the Columbia-0 background.]
  2. Sodium hypochlorite (Sigma-Aldrich, catalog number: 425044 )
  3. Tween-80 (Sigma-Aldrich, catalog number: 4780 )
  4. Sterile H2O
  5. Solid growth medium
  6. Liquid plant growth medium (same as solid growth media but without agar)
  7. Hydroxyurea (Sigma-Aldrich, catalog number: H8627 )
  8. 70% ethanol
  9. Seed sterilization solution (see Recipes)
  10. Bleocin (commercial name for bleomycin) (EMD Millipore, catalog number: 203408 ) (see Recipes)
  11. Mitomycin C (Duchefa Biochemie BV, catalog number: M0133 ) (see Recipes)

Equipment

  1. Sterile hood, preferably a biological safety cabinet to avoid exposure to the genotoxins
  2. Bench top block shaker (e.g. Eppendorf, Thermomixer®)
  3. UV crosslinker (254-nm UV light bulbs, 15 watts each) (Stratagene, model: Stratalinker 2400 )
  4. Gamma-irradiation source (Nordion, model: Co-60- Gamma-cell 220 )
  5. Forceps
  6. Box or aluminum foil
  7. Petri dishes for plant culture with solid growth medium (see Notes) (round 200 x 15 mm and 55 x 15 mm, and square 100 x 100 x 15 mm)
  8. 1.5 ml Eppendorf tubes
  9. Plant growth facilities with 16-h-light/8-h-dark cycles, at 21 °C

Procedure

  1. Seed sterilization
    1. Wash the seeds in a 1.5 ml Eppendorf tube with 1 ml of the sterilization solution, shake in a block shaker at 300 rpm for 6 min at room temperature. Allow seeds to set in the bottom of the Eppendorf and remove supernatant. Wash with 1 ml of sterile water, 5 times for 5 min in the shaker. After the final wash, remove supernatant and dry overnight in the closed sterile hood, leaving the tubes open.
      Note: Sterilize approximately 350 seeds per genotype and treatment. All subsequent steps involving seed or plantlet manipulation should be carried in the sterile hood.

  2. True leaf assay
    1. Plate seeds in solid growth medium (≈ 75 to 100 per plate) using flame-sterilized and cooled forceps. Stratify in the dark (in a box or wrapped in aluminum foil) at 4 °C for 2 to 4 days and transfer to the growth chambers with illumination.
      Note: Any other sterile plating techniques are suitable. Each plate makes one biological replicate; for each data point at least 3 biological replicates should be analyzed.
    2. Treatments
      1. Gamma-irradiation: Treat 4-day-old-seedlings with a pulse of 100 Gray (Gy) with the Co-60- gamma source, with a dose of 27 to 34 Gy/min. Return plates to standard growth conditions to recover for 6 days until analysis.
        Note: 4-day-old = 4 days after transfer to the growth chambers.
      2. Bleocin and mitomycin C: Using flame-sterilized and cooled forceps, transfer 4-day-old seedlings to small petri dishes containing 10 ml of liquid growth medium, either without (mock) or with a drug (1 μg/ml of bleocin or 10 μg/ml of mitomycin C). After 5 days of incubation (with seedlings floating in liquid, but no shaking) in the illuminated growth chamber (9-day-old seedlings), remove the medium, wash extensively by flooding the plate 5 times with 20 ml of liquid media. Transfer seedlings to solid plates using flame-sterilized and cooled forceps. Allow seedlings to recover for 24 h before analysis.
        Note: Seedlings should be carefully picked and handled, avoiding damage by the forceps. For examples of results see Figure 1A-C.
      3. Hydroxyurea: Plate seeds directly on plates, without or with 1 mM of the chemical, and grow at standard conditions until analysis at day 10.
    3. Analysis
      At day 10, photograph the plates for reference, and score the “percentage of plants with true leaves”.
      Notes:
      1. Upon undisturbed development, 10-day-old Arabidopsis seedlings possess one pair of true leaves.
      2. DNA damage causes the cells in the apical meristem to stop dividing, so that no true leaves are developed (Figure 1A).
      3. For the quantitative assay, calculate the percentage of plants with true leaves for each treated population in relation to the number of plants with true leaves after mock treatment. This calculation separates the effect of the DNA-damaging treatment from other factors, like vigor of the mutants or environmental factors.
      4. Only paired and normally shaped leaves should be scored; single, or small and narrow unexpanded leaves are not considered as true leaves.

  3. UV damage assay
    1. Plate seeds in solid growth medium (≈ 75 to 100 per plate) using flame-sterilized and cooled forceps. Stratify in the dark (in a box or wrapped in aluminum foil) at 4 °C for 2 to 4 days and transfer to the growth chambers with illumination.
      Note: Each plate makes one biological replicate; for each data point at least 3 biological replicates should be analyzed.
    2. Treat seedlings at day 4 of growth, with 3 kJ/m2 UV-C in a Stratalinker. Grow under standard conditions until day 12 and score the percentage of resistant plants (Figure 1D). Sensitive plants have phenotypes such as reduced global size, chlorotic cotyledons and senescence, whilst resistant plants have a normal size range and remain green.
      Note: Divide the value in treated populations by the same parameter in the mock treatment, and average the biological replicates.
      Before the treatment sterilize the interior of the Stratalinker with 70% ethanol and run it for 3 min with the maximum dose of UV-C. Then place plates inside and remove the lid, keeping it inside the Stratalinker. Quickly close the door, to avoid contamination and run the desired dosage of UV-C. When the treatment is finished close the plates still inside of the Stratalinker and return them to normal growth conditions.


      Figure 1. Examples of results. (A) Phenotypes of 10-day-old bleocin-treated seedlings with (i, arrow) and without (ii) true leaves. (B) True leaf assay showing the response of wild type, arp6-3, swc6-1 and ku70-2 to different doses of bleocin. Each plotted value corresponds to the scoring of one experiment with 60-80 seedlings. (C) True leaf assay with seedlings treated with 1 μg/ml of bleocin. The percentage of 10-d-old treated plants with true leaves was calculated in relation to mock samples. Bars depict the averages between 3 to 5 experiments with 75 to 100 seedlings per treatment. (D) Phenotypes of 12-day-old UV-C-treated resistant (i) and sensitive (ii) seedlings. (E) UV resistance assay of seedlings treated with 3 kJ/m2 of UV-C. Bars depict the averages between 4 experiments with 75 to 100 seedlings per treatment. Asterisks indicate the significance between treated and non-treated samples according to P values from unpaired t tests: ***P < 0.001 and **0.001 < P < 0.01.

Notes

  1. To better characterize the response of the different genotypes to the DNA damaging agents, generate response curves with at least 3 different concentrations of the genotoxins (example in Figure 1B): 50 to 200 Gy of gamma-irradiation, 0.5 to 2 mg/ml of bleocin, 5 to 20 of mitomycin C, 0.5 to 2 of hydroxyhurea and 1.5 to 6 kJ/m2 of UV-C.
  2. Always keep the plates belonging to one experiment (mock + treatment) growing side-by-side. For example, take plates for mock and gamma-irradiation treatment out of the growth chamber at the same time.
  3. In this experiment we used Germination Media (GM) described in Rosa et al. (2013) (detailed protocol available at http://www.gmi.oeaw.ac.at/download/gm_medium_protocol_oms.pdf). We have not tested the use of regular MS plates but have no reason to believe the media will be a determining factor as long as the plates used in each experiment come from the same batch of media.

Recipes

  1. Seed sterilization solution
    5% sodium hypochlorite and 0.05% Tween-80
    Mix 500 µl of sodium hypochlorite and 5 µl of Tween-80 in 10 ml of sterile H2O
  2. Bleocin (working solution: 1 μg /ml; stock: 10 mg/ml)
    Dissolve 100 mg of Bleocin in 10 ml of H2O
    Make 0.2 ml aliquots and keep at -20 °C until use
    Avoid thawing/freezing cycles
    Dilute to the desired concentration in liquid media, using disposable 50 ml Falcon tubes
  3. Mitomycin C (working solution: 10 μg/ml; stock: 0.5 mg/ml)
    Dissolve 2 mg of Mitomycin C in 4 ml of H2O immediately before use
    Dilute to the desired concentration in liquid media, using disposable 50 ml Falcon tubes

Acknowledgments

We thank the Department of Nutritional Sciences of the University of Vienna for help with the irradiation experiments and Gudrun Böhmdorfer for helpful discussions on DNA damaging agents. Work was supported by Grants GEN-AU GZ 200.140-VI/1/2006 from the Austrian Federal Ministry of Science and Research and FWF P18986-B17 from the Austrian Science Fund. This protocol was established in Rosa et al. (2013).

References

  1. Masson, J. and Paszkowski, J. (1992). The culture response of Arabidopsis thaliana protoplasts is determined by the growth conditions of donor plants. Plant J 2(5): 829-833.
  2. Rosa, M., Von Harder, M., Cigliano, R. A., Schlögelhofer, P. and Mittelsten Scheid, O. (2013). The Arabidopsis SWR1 chromatin-remodeling complex is important for DNA repair, somatic recombination, and meiosis. Plant Cell 25(6): 1990-2001.

材料和试剂

  1. 拟南芥种子[arp6-3 ,swc6-1 和敏感 ku70-2 的野生型和T-DNA插入行/em>(Rosa et al。,2013)。 所有突变体都在哥伦比亚-0背景中。]
  2. 次氯酸钠(Sigma-Aldrich,目录号:425044)
  3. 吐温-80(Sigma-Aldrich,目录号:4780)
  4. 无菌H 2 O 2/b
  5. 固体培养基

    材料和试剂

    1. 拟南芥种子[arp6-3 ,swc6-1 和敏感 ku70-2 的野生型和T-DNA插入行/em>(Rosa et al。,2013)。 所有突变体都在哥伦比亚-0背景中。]
    2. 次氯酸钠(Sigma-Aldrich,目录号:425044)
    3. 吐温-80(Sigma-Aldrich,目录号:4780)
    4. 无菌H 2 O 2/b
    5. 固体培养基
      ...

    设备

    1. 无菌罩,优选生物安全柜,以避免暴露于基因毒素
    2. 台式块振动器(如 Eppendorf,Thermomixer ®
    3. UV交联剂(254nm UV灯泡,每种15瓦)(Stratagene,型号:Stratalinker 2400)
    4. γ照射源(Nordion,型号:Co-60-γ电池220)
    5. 镊子
    6. 箱或铝箔
    7. 用固体生长培养基(见注释)(圆形200×15mm和55×15mm,方形100×100×15mm)的植物培养用培养皿。
    8. 1.5 ml Eppendorf管
    9. 植物生长设施,在21℃下,16小时光/8小时黑暗循环

    程序

    1. 种子灭菌
      1. 在1.5ml Eppendorf管中用1ml灭菌溶液洗涤种子,在块振荡器中在室温下以300rpm振摇6分钟。使种子固定在Eppendorf的底部并除去上清液。用1ml无菌水洗涤5次,在振荡器中5分钟。最后一次洗涤后,取出上清液,在封闭的无菌罩中干燥过夜,留下管子开口 注意:每个基因型和治疗消毒约350粒种子。所有涉及种子或苗子操作的后续步骤应在无菌罩中进行。

    2. 真叶测定
      1. 使用火焰灭菌和冷却的镊子将板种子在固体生长培养基(每板约75至100个)中。在4℃下在黑暗中(在盒中或包裹在铝箔中)分层2至4天,并转移到具有照明的生长室。
        注意:任何其他无菌电镀技术都是合适的。每个板做一个生物复制;对于每个数据点,应当分析至少3个生物重复。
      2. 治疗
        1. γ辐射:用100-Gray脉冲(Gy)用Co-60-γ源以27-34Gy/min的剂量处理4日龄幼苗。将板置于标准生长条件下恢复6天直到分析。
          注意:4日龄=转移到生长室后4天。
        2. Bleocin和丝裂霉素C:使用火焰灭菌和冷却的镊子,将4天龄的幼苗转移到含有10ml液体生长培养基的小培养皿中,其不含(mock)或含有药物(1μg/ml bleocin或10 μg/ml丝裂霉素C)。在照明的生长室(9天龄的幼苗)中孵育5天(幼苗漂浮在液体中,但没有摇动)后,除去培养基,通过用20ml液体培养基浸渍板5次彻底洗涤。使用火焰灭菌和冷却镊子转移幼苗到固体板。允许幼苗恢复24 h前分析。
          注意:幼苗应仔细挑选和处理,避免镊子损坏。结果的实例参见图1A-C。
        3. 羟基脲:直接在板上种子种子,没有或含有1mM的化学物质,并在标准条件下生长直到在第10天进行分析。
      3. 分析
        在第10天,将板照片以供参考,并对"具有真叶的植物的百分比"进行评分 注意:
        1. 在未受干扰的发育中,10天龄的拟南芥幼苗具有一对真叶。
        2. DNA损伤导致顶端分生组织中的细胞停止分裂,使得没有真正的叶发育(图1A)。
        3. 对于定量测定,计算每个处理群体的具有真叶的植物的百分比与模拟处理后具有真叶的植物的数目的关系。这种计算将DNA损伤治疗与其他因素,如突变体的活力或环境因素分开。
        4. 只有成对的和正常形状的叶片才能打分;单个或小而窄的未膨胀叶片不被视为真叶。

    3. 紫外线损伤测定
      1. 使用火焰灭菌和冷却的镊子将板种子在固体生长培养基(每板约75至100个)中。在4℃下在黑暗中(在盒子中或包裹在铝箔中)分层2至4天,并用照明转移到生长室。
        注意:每个板块做一个生物复制;对于每个数据点,应当分析至少3个生物重复。
      2. 在Stratalinker中,用3kJ/m 2 UV-C处理幼苗生长的第4天。在标准条件下生长直至第12天,并对抗性植物的百分比进行评分(图1D)。敏感植物具有表型,例如减小的总体大小,氯化子叶和衰老,而抗性植物具有正常的大小范围并保持绿色。 注意:用模拟处理中的相同参数除以处理群体中的值,并平均生物重复。
        治疗前用70%乙醇消毒Stratalinker内部,运行3分钟与最大剂量的UV-C。然后将板放在里面,取下盖子,保持它在Stratalinker内。快速关闭门,以避免污染并运行所需剂量的UV-C。当治疗完成时,关闭板仍然在Stratalinker内,并恢复到正常生长条件

        图1.结果实施例(A)10天龄bleocin处理的幼苗的表型( i ,箭头) em>)true叶。 (B)真叶测定显示野生型,arp6-3 ,swc6-1 和 ku70-2 对不同剂量的bleocin 。每个绘制值对应于使用60-80个幼苗的一个实验的评分。 (C)用1μg/ml bleocin处理的幼苗的真叶测定。相对于模拟样品计算10-d-处理的具有真叶的植物的百分比。条形图描绘了3至5次实验之间的平均值,每次处理75至100个幼苗。 (D)12天龄的UV-C处理的抗性( i )和敏感( ii )幼苗的表型。 (E)用3kJ/m 2的UV-C处理的幼苗的抗UV试验。条形图描绘了4次实验之间的平均值,每次处理75至100个幼苗。星号表示根据来自未配对试验的P值的处理和未处理样品之间的显着性:*** P < 0.001和** 0.001 < P < 0.01。

    笔记

    1. 为了更好地表征不同基因型对DNA损伤剂的反应,产生具有至少3种不同浓度的基因毒素的响应曲线(图1B中的实例):50至200Gy的γ-照射,0.5至2mg/ml bleocin,5至20丝裂霉素C,0.5至2的羟基脲和1.5至6kJ/m 2的UV-C。
    2. 始终保持板属于一个实验(模拟+处理)并排生长。例如,将生长室中的模拟和γ辐照处理板同时取出
    3. 在该实验中,我们使用Rosa等人(2013)中描述的萌发培养基(GM)(详细方案可在 http://www.gmi.oeaw.ac.at/download/gm_medium_protocol_oms.pdf )。 我们没有测试过使用常规MS培养板,但没有理由相信培养基会成为决定性因素,只要每个实验中使用的培养板来自同一批培养基。

    食谱

    1. 种子灭菌溶液
      5%次氯酸钠和0.05%Tween-80 将500μl次氯酸钠和5μl吐温-80在10ml无菌H 2 O中混合
    2. Bleocin(工作溶液:1μg/ml;原液:10mg/ml) 将100mg的Bleocin溶解在10ml H 2 O中 使0.2毫升等分试样,并保持在-20°C,直到使用
      避免解冻/冻结周期
      在液体培养基中稀释至所需浓度,使用一次性50ml Falcon管
    3. 丝裂霉素C(工作溶液:10μg/ml;原液:0.5mg/ml) 在使用前立即将2mg丝裂霉素C溶于4ml H 2 O中
      在液体培养基中稀释至所需浓度,使用一次性50ml Falcon管

    致谢

    我们感谢维也纳大学营养科学系帮助辐射实验和GudrunBöhmdorfer对DNA损伤剂的有益讨论。 工作得到来自奥地利联邦科学和研究部的Grants GEN-AU GZ 200.140-VI/1/2006和来自奥地利科学基金的FWF P18986-B17的支持。 该协议在Rosa等人(2013)中建立。

    参考文献

    1. Masson,J。和Paszkowski,J。(1992)。 拟南芥的文化反应 2(5):829-833。 br />
    2. Rosa,M.,Von Harder,M.,Cigliano,R.A.,Schlögelhofer,P.and Mittelsten Scheid,O。(2013)。 拟南芥 SWR1染色质重塑 复合物对于DNA修复,体细胞重组和减数分裂是重要的。植物细胞 25(6):1990-2001。
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How to cite this protocol: Rosa, M. and Scheid, O. M. (2014). DNA Damage Sensitivity Assays with Arabidopsis Seedlings. Bio-protocol 4(7): e1093. DOI: 10.21769/BioProtoc.1093; Full Text



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Ortrun Mittelsten Scheid的其他实验方案(1)