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Protocol to Treat Seedlings with Brassinazole and Measure Hypocotyl Length in Arabidopsis thaliana
油菜素甾醇处理拟南芥及测定下胚轴长度的方法   

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Abstract

The plant hormones brassinosteroids (BR) promote hypocotyl elongation of Arabidopsis thaliana (A. thaliana) seedlings both under light and dark (etiolated) conditions. A common assay to determine if a mutant or transgenic line is affected in BR biosynthesis or response is a sensitivity assay to brassinazole (BRZ), an inhibitor of P450 cytochromes specific to BR biosynthesis.
Here we provide a protocol to compare BRZ sensitivity of different A. thaliana genotypes in terms of hypocotyl elongation (Bernardo-García et al., 2014).

Keywords: Brassinosteroid response(brassinosteroid response), Hypocotyl elongation(下胚轴伸长), Brassinazole(brassinazole)

Materials and Reagents

  1. A. thaliana seeds (background ecotype and mutant or transgenic line to be analysed)
    Note: Seeds should be collected at the same time to avoid problems of differential germination rate.
  2. Murashige and Skoog (MS) media including vitamins and MES buffer (Duchefa M0255.0050)
  3. Sucrose
  4. Potassium hydroxide (KOH)
  5. Bacto Agar (BD Bioscience, catalog number: 214010 )
  6. Ethanol
  7. Tween-20
  8. Brassinazole (TCI America, catalog number: B2829 )
    Note: Stock solution is prepared at 2 mM in DMSO and kept at -20 °C. The appropriate volume of BRZ stock solution should be added in the fume hood to autoclaved media. The media should not be too hot (let it cool down to 55-65 °C before adding BRZ). BRZ concentration should range from 0.1 to 2 µM. We routinely use 0.1, 0.2, 0.4, 0.8 and 1.6 µM as a starting point.
  9. Growth media (see Recipes)
  10. Sterilization solution (see Recipes)

Equipment

  1. Plant growth chamber
  2. Sterile fume hood
  3. Autoclave
  4. Round (9 x 9 cm) and square (12 x 12 cm) polystyrene sterile Petri dishes

Software

  1. ImageJ software

Procedure


Figure 1. A flow chart of the steps described in our procedure

  1. Seed sterilization and sowing
    1. Seeds are surface sterilized in 1.5 ml Eppendorf tubes by shaking for 15 min at 500-1,000 rpm at room temperature with 1 ml sterilization solution, and then washed twice in the sterile fume hood with 1 ml ethanol. Ethanol should be carefully removed with a p1000 pipette. Then close the tube and shake it to stick the seeds to the tube walls and remove ethanol from the tube bottom with a p200 pipette. After carefully removing ethanol, Eppendorf tubes are left open in the fume hood until the seeds are dry (30 min-1 h). Up to 50 µl seeds can be sterilized per tube. For bigger amount of seeds, divide them in several tubes. Drying time increases with larger amounts of seeds. To verify seeds are dry close the tube and shake gently. Dry seeds do not stick to the tube.
    2. Sterile seeds are sown in round 9 cm MS plates and vernalized for 2-4 days in the dark at 4 °C. Seeds are easily sown by gently tapping in the Eppendorf tube to let them drop in the plate. If many seeds stick together, they can be separated with the help of a sterile toothpick. Up to 200 seeds can be sown per plate. Plates are sealed with parafilm or saran wrap.

  2. Germination induction and transfer to BRZ vertical plates
    1. To synchronize germination, 9 cm MS plates are placed horizontally in a culture chamber in the light for 3 h and then wrapped in aluminium foil and kept in dark for 20 h. This step is necessary because BRZ, at higher concentrations, inhibits germination and can give rise to differences in hypocotyl length due to slower germination.
    2. After germination induction, seeds are transferred to square 12 cm MS plates with increasing concentrations of BRZ keeping a control plate without BRZ (mock). Using a toothpick, seeds are transferred to square 12 cm petri dishes with appropriate concentration of BRZ. At least 20 seeds from each genotype are needed, with three replicates for each treatment. Each plate can accommodate up to 40 seeds per row. Seeds should be placed equally separated. One or two rows can be sown per plate. It can be useful to use a template (a piece of paper placed under the petri dish) (Figure 2).


      Figure 2. Template to help placing the seeds equally separated in the plate

    3. Square 12 cm petri dishes are placed in a vertical position in a growth chamber. To analyse etiolated growth, wrap them in aluminium foil and unwrap after 5 days to take pictures and measure hypocotyls. To analyse growth in the light, seedlings are grown for 7 days.
      Hypocotyl length is affected by light intensity, photoperiod and temperature. Higher light intensity, longer photoperiods and high temperature promote hypocotyl growth.
      To assess the effect of BRZ, we use the following conditions:
      Photoperiod: Short day (8 h light/16 h darkness)
      Light intensity: 10 µmol m-2 s-1
      Temperature: Constant 22 °C

  3. Hypocotyl measurement and analysis
    1. To measure hypocotyls, the plates can be photographed or scanned. Hypocotyl lengths are measured with ImageJ software (http://rsb.info.nih.gov/ij) (Abràmoff et al., 2004).
      For details on how to use ImageJ please read the following protocol (Corrales et al., 2014).
      To measure hypocotyls, choose the “segmented line” option (right-click in the highlighted button, Figure 3).


      Figure 3. Snapshot from ImageJ program showing hypocotyl measurement

    2. A graph bar is used to represent the media and standard deviation of each genotype and treatment measurement (Figure 5).
    3. Statistical analyses are carried out by one-way analysis of variance (ANOVA) followed by a Student’s t-test (P < 0.01).
    4. If compared genotypes show different hypocotyl length in control MS mock media, sensitivity to BRZ should be represented as the percentage of hypocotyl length reduction relative to control conditions.

Representative data

Representative data showing hypocotyl measurements of type Col-0 and bes1D mutant are shown in Figures 4 and 5. BES1 is a transcription factor essential for BL response. bes1D mutation renders a constitutively active form of BES1. bes1D plants display constitutive response to BL and therefore are less sensitive to BRZ.


Figure 4. 7-days old Col-0 and bes1D seedlings grown under light (A) and dark conditions (B) in the indicated BRZ concentrations


Figure 5. BRZ sensitivity of Col-0 and bes1D estimated by determining hypocotyl length reduction by increasing BRZ concentrations under light (A) and dark conditions (B). Error bars represent standard deviation. Asterisks indicate significant differences between Col-0 and bes1-D for each BRZ concentration (n = 20, p<0.01).

Recipes

  1. Growth media
    2.45 g/L Murashige and Skoog media (half strength)
    10 g/L sucrose
    Water to final volume
    Adjust pH to 5.8 with 0.5 M KOH
    Add Bacto agar to 8 g/L (horizontal plates, 9 cm round) or 11 g/L (vertical plates, 12 cm square)
    Autoclave
    Let the media cool down to 55-65 °C before adding BRZ and pour the plates.
    Pour approximately 25 ml of growth media per round 9 cm petri dishes and 50 ml of growth media per 12 cm square petri dishes.
  2. Sterilization solution
    70% ethanol
    0.01% Tween 20

Acknowledgements

We gratefully acknowledge funding through grants BIO2008-04160 and BIO2011-30546 from the Spanish Ministry of Science and Innovation.

References

  1. Abràmoff, M. D., Magalhães, P. J. and Ram, S. J. (2004). Image processing with ImageJ. Biophot Internat 11(7): 36-43.
  2. Bernardo-García, S., de Lucas, M., Martínez, C., Espinosa-Ruiz, A., Davière, J.-M. and Prat, S. (2014). BR-dependent phosphorylation modulates PIF4 transcriptional activity and shapes diurnal hypocotyl growth. Genes Dev 28(15): 1681-1694.
  3. Corrales, A. R., Carrillo, L., Nebauer, S. G., Renau-Morata, B., Sánchez-Perales, M., Fernández-Nohales, P., Marqués, J., Granell, A., Pollmann, S., Vicente-Carbajosa, J., Molina, R. V. and Medina, J. (2014). Salinity assay in Arabidopsis. Bio-protocol 4(16): e1216.

简介

植物激素油菜素类固醇(BR)在光和黑暗(etiolated)条件下促进拟南芥( a thaliana )幼苗的下胚轴延伸。 确定突变体或转基因品系是否受BR生物合成或反应影响的常用测定法是对BR生物合成特异性的P450细胞色素抑制剂 - 芸苔素(BRZ)的敏感性测定。
在这里,我们提供一个协议来比较不同 A的BRZ灵敏度。 (Bernardo-García等人,2014)。

关键字:brassinosteroid response, 下胚轴伸长, brassinazole

材料和试剂

  1. A。 拟南芥种子(背景生态型和待分析的突变体或转基因品系)
    注意:种子应同时收集,以避免发育率差异的问题。
  2. Murashige和Skoog(MS)培养基,包括维生素和MES缓冲液(Duchefa M0255.0050)
  3. 蔗糖
  4. 氢氧化钾(KOH)
  5. Bacto琼脂(BD Bioscience,目录号:214010)
  6. 乙醇
  7. 吐温-20
  8. Brassinazole(TCI America,目录号:B2829)
    注意:储备溶液以2mM在DMSO中制备并保持在-20℃。 适当体积的BRZ储备溶液应在通风橱中加入高压灭菌的培养基中。 介质不应太热(让它冷却到55-65°C,然后添加BRZ)。 BRZ浓度应在0.1至2μM范围内。 我们通常使用0.1,0.2,0.4,0.8和1.6μM作为起始点。
  9. 生长培养基(参见食谱)
  10. 灭菌溶液(见配方)

设备

  1. 植物生长室
  2. 无菌通风橱
  3. 高压灭菌器
  4. 圆形(9×9cm)和方形(12×12cm)聚苯乙烯无菌Petri培养皿

软件

  1. ImageJ软件

程序


图1.我们的过程中描述的步骤的流程图

  1. 种子灭菌和播种
    1. 种子通过振荡在1.5ml Eppendorf管中表面灭菌  在500-1,000rpm,室温下用1ml灭菌15分钟 溶液,然后在无菌通风橱中用1ml洗涤两次 乙醇。乙醇应用p1000移液管小心移除。然后 关闭管,摇动它把种子粘在管壁上 用p200移液管从管底部除去乙醇。后仔细  除去乙醇,Eppendorf管在通风橱中保持打开直到 种子干燥(30分钟-1小时)。高达50μl种子可以灭菌 管。对于更大量的种子,将其分在几个管中。烘干 时间随着更大量的种子而增加。验证种子是否干燥 关闭试管并轻轻摇动。干种子不粘在管子上。
    2. 将无菌种子播种在圆形9cm MS平板中并春化 在4℃黑暗中2-4天。种子很容易通过轻轻敲打播种  Eppendorf管让他们在板上下降。如果许多种子坚持 一起,它们可以在无菌牙签的帮助下分离。向上  可以每板播种200个种子。平板用石蜡膜密封 保鲜膜。

  2. 发芽诱导和转移到BRZ垂直板
    1. 为了同步萌发,将9cm MS板水平放置   一个培养室在光照下3小时,然后用铝包裹 并在黑暗中保持20小时。 这一步是必要的,因为BRZ,at 更高的浓度,抑制萌发并且可以产生 由于发芽速度较慢,下胚轴长度有所不同。
    2. 在发芽诱导后,将种子转移至正方形12cm MS 板具有增加浓度的BRZ,保持对照板 无BRZ(模拟)。 使用牙签,种子转移到正方形 12cm陪替氏培养皿与适当浓度的BRZ。至少20 需要来自每个基因型的种子,每个具有三个重复 治疗。每个板最多可容纳40颗种子。种子 应该分开放置。每行可以播放一行或两行 盘子。使用模板(一张纸放在下面)可能很有用  培养皿)(图2)

      图2.帮助将种子平均分布在平板中的模板

    3. 方形12厘米培养皿放置在垂直位置 生长室。为了分析钙化生长,将其包裹在铝箔中  并解开后5天拍照和测量下胚轴。至 分析光的生长,幼苗生长7天 下胚轴长度受光强度,光周期和 温度。较高的光强度,较长的光周期和高 温度促进下胚轴生长 为了评估BRZ的效果,我们使用以下条件:
      光周期:短日(8小时光照/16小时黑暗)
      光强度:10μmol/m 2 - s -1
      温度:恒定22°C

  3. 下胚轴测量和分析
    1. 为了测量下胚轴,可以对板进行拍照或扫描。 下丘脑长度用ImageJ软件(http://rsb.info.nih.gov/ij)(Abràmoff等人,2004)测量。
      有关如何使用ImageJ的详细信息,请阅读以下协议(Corrales ,,2014)。
      要测量下胚轴,请选择"分段线"选项(在突出显示的按钮中右键单击,图3)。


      图3.来自ImageJ程序的快照显示下胚轴测量

    2. 图形条用于表示每种基因型和治疗测量的介质和标准偏差(图5)。
    3. 通过单向方差分析(ANOVA),然后进行Student's t检验(P <0.01)进行统计学分析。
    4. 如果比较的基因型在对照MS模拟培养基中显示不同的下胚轴长度,则对BRZ的敏感性应表示为相对于对照条件下胚轴长度减少的百分比。

代表数据

显示Col-0和typ1D突变型的下胚轴测量的代表性数据显示于图4和图5中.BES1是BL应答必需的转录因子。 bes1D 突变呈现组成性 活性形式的BES1。 bes1D 植物显示对BL的组成性反应,因此对BRZ不太敏感

图4.在指示的BRZ浓度下在光照(A)和黑暗条件(B)下生长的7天龄的Col-0和em1 bes1D

图5.通过在光(A)和黑暗条件(B)下增加BRZ浓度确定下胚轴长度减少估计的Col-0和em1 bes1D的BRZ灵敏度。误差条表示标准偏差。星号表示对于每个BRZ浓度(n = 20,p <0.01)的Col-0和em1 bes-D之间的显着差异。

食谱

  1. 生长介质
    2.45g/L Murashige和Skoog介质(半强度)
    10g/L蔗糖 水到最终量
    用0.5 M KOH调节pH至5.8 添加Bacto琼脂至8g/L(水平板,9cm圆)或11g/L(垂直板,12cm正方形)
    高压灭菌器
    让介质冷却至55-65°C,然后加入BRZ并倒入板 每个9cm培养皿和每12cm正方形培养皿中50ml生长培养基倒入约25ml生长培养基。
  2. 灭菌溶液
    70%乙醇
    0.01%吐温20

致谢

我们衷心感谢通过拨款BIO2008-04160和BIO2011-30546从西班牙科学和创新部。

参考文献

  1. Abràmoff,M.D.,Magalhães,P.J.and Ram,S.J。(2004)。 使用ImageJ进行图像处理。 Biophot Internat 11(7):36-43。
  2. Bernardo-García,S.,de Lucas,M.,Martínez,C.,Espinosa-Ruiz,A.,Davière,J.-M. 和Prat,S。(2014)。 BR依赖性磷酸化调节PIF4转录活性并形成昼夜下胚轴生长。 em> Genes Dev 28(15):1681-1694。
  3. Corrales,AR,Carrillo,L.,Nebauer,SG,Renau-Morata,B.,Sánchez-Perales,M.,Fernández-Nohales,P.,Marqués,J.,Granell,A.,Pollmann, -Carbajosa,J.,Molina,RV和Medina,J。(2014)。 拟南芥中的盐度测定 生物协议 4(16):e1216。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Espinosa-Ruiz, A., Martínez, C. and Prat, S. (2015). Protocol to Treat Seedlings with Brassinazole and Measure Hypocotyl Length in Arabidopsis thaliana. Bio-protocol 5(16): e1568. DOI: 10.21769/BioProtoc.1568.
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