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Cotyledon Wounding of Arabidopsis Seedlings
拟南芥幼苗的子叶创伤   

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Abstract

Damage to plant organs through both biotic and abiotic injury is very common in nature. Arabidopsis thaliana 5-day-old (5-do) seedlings represent an excellent system in which to study plant responses to mechanical wounding, both at the site of the damage and in distal unharmed tissues. Seedlings of wild type, transgenic or mutant lines subjected to single or repetitive cotyledon wounding can be used to quantify morphological alterations (e.g., root length, Gasperini et al., 2015), analyze the dynamics of reporter genes in vivo (Larrieu et al., 2015; Gasperini et al., 2015), follow transcriptional changes by quantitative RT-PCR (Acosta et al., 2013; Gasperini et al., 2015) or examine additional aspects of the wound response with a plethora of downstream procedures. Here we illustrate how to rapidly and reliably wound cotyledons of young seedlings, and show the behavior of two promoters driving the expression of β-glucuronidase (GUS) in entire seedlings and in the primary root meristem, following single or repetitive cotyledon wounding respectively. We describe two procedures that can be easily adapted to specific experimental needs.

Keywords: Plant(植物), Jasmonate(茉莉), JA-Ile(JA-ILE-)

Materials and Reagents

  1. Single wounding of seedling cotyledons
    1. Sterile 6 cm x 6 cm nylon mesh, 200 μm pore size (Lanz-Anliker AG, custom made)
    2. Standard 9 cm round Petri plates
    3. 3MTM MicroporeTM Tape, 1.25 cm x 9.14 m, hypoallergenic tape, standard roll (3M, catalog number: 1530-0 )
    4. Two 25 G x 5/8” hypodermic needles, 0.5 mm x 16 mm (BD Bioscience, catalog number: 305760 )
    5. Arabidopsis thaliana sterile seeds, e.g., MYC2p-GUS reporter line (Gasperini et al., 2015)
    6. Sterile double distilled H2O (ddH2O)
    7. Murashige and Skoog (MS) medium (Duchefa Biochemie, catalog number: M0221.0050 )
    8. MES hydrate (Sigma-Aldrich, catalog number: M8250 )
    9. Agar (AppliChem GmbH, catalog number: A2111 1000 )
    10. Standard 9 cm round Petri plates filled with 30 ml of solid half-strength Murashige and Skoog (0.5x MS) medium with 0.7% agar (see Recipes)

  2. Repetitive wounding of seedling cotyledons
    1. 3MTM MicroporeTM Tape, 2.5 cm x 9.14 m, hypoallergenic tape, standard roll (3M, catalog number: 1530-1 )
    2. One 25 G x 5/8” hypodermic needle, 0.5 mm x 16 mm (BD Bioscience, catalog number: 305760 )
    3. One 36 gauge beveled NanoFil needle, 110 µM outer diameter (World Precision Instruments, catalog number: NF36BV )
    4. Standard 12 cm x 12 cm square Petri plates
    5. Racks for vertical growth (e.g., Milian, catalog number: 086680 )
    6. Arabidopsis thaliana sterile seeds, e.g., CYCB1;1p-GUS reporter line (Colón-Carmona et al., 1999)
    7. Sterile ddH2O
    8. Standard 12 cm x 12 cm square Petri plates filled with 70 ml 0.5x MS with 0.85% agar (see Recipes)

  3. GUS staining
    1. Staining dish [e.g., 12 well suspension culture plate (Greiner Bio-One GmbH, Cell Star®, catalog number: 665102 )]
    2. Ice bucket with ice
    3. Microscopy slides (e.g., Thermo Fisher Scientific, Menzel-Glaser,catalog number: AD00000112E ) and coverslips (e.g., Thermo Fisher Scientific, Menzel-Glaser, catalog number: BB024060A1 )
    4. 90% (v/v) acetone in ddH2O
    5. 50 mM sodium phosphate buffer (pH 7.0)
    6. 70% (v/v) ethanol (EtOH) in ddH2O
    7. Potassium Ferrocyanide [e.g., Potassium hexacyanoferrate(II) trihydrate (Sigma-Aldrich, catalog number: 60279 )]
    8. Potassium Ferricyanide [e.g., Potassium hexacyanoferrate(III) (Merck Millipore Corporation, catalog number: 104973 )]
    9. 5-Bromo-4-chloro-3-indoxyl-beta-D-glucuronic acid (X-Gluc) (e.g., Biosynth, catalog number: B-7300 )
    10. Chloral hydrate (e.g., Sigma-Aldrich, catalog number: 23100 )
      Safety note: acute toxicity, avoid inhalation.
    11. Glycerol (e.g., Sigma-Aldrich, catalog number: G6279 )
    12. GUS staining solution (see Recipes)
    13. Chloride hydrate solution (see Recipes)

Equipment

  1. Sterile hood (laminar flow hood)
  2. Fine forceps style 4 (e.g., Dumont, catalog number: 0508-4-PO )
  3. Micropipettes (P20, P200, P1000)
  4. Phytotron or plant growth chamber set with the following growth conditions: 21 °C, 100 μE m-2 s-1 light intensity, 14 h light/ 10 h dark photoperiod
  5. Portable dissecting stereomicroscope
  6. Vacuum pump
  7. 37 °C incubator
  8. Stereomicroscope [e.g., Leica Microsystems, model: Leica MZ16A fitted with a camera (model: DFC310FX )]
  9. Differential Interference Contrast (DIC) microscope [e.g., Leica Microsystems, model: Leica DM5500 fitted with a camera (model: DFC420 )]

Procedure

  1. Single wounding of seedling cotyledons
    Note: steps 1-3 must be performed in aseptic conditions under a sterile hood.
    1. To equidistantly sow seeds on the media, place a printed guide underneath the petri dish (File 1A).
    2. Place a sterile nylon mesh on a solid 0.5x MS medium (0.7% agar) using sterilized forceps.
      Note: The nylon mesh provides a solid support that allows seedlings to grow straight with a cotyledon surface that is easily accessible for wounding.
    3. Resuspend sterile seeds in 500 µl sterile ddH2O and equidistantly plate 120 individual seeds onto the sterile mesh with a P20 micropipette set to dispense a volume of 20 µl (Figure 1A, Video 1).
      Note: If more than one seed are ejected on the mesh in a single spot, the excess can be aspirated and repositioned.

      Video 1. Seed guide

      To play the video, you need to install a newer version of Adobe Flash Player.

      Get Adobe Flash Player

    4. Seal the plate with 1.25 cm wide micropore tape, stratify for 2 d in the dark at 4 °C (keep the plates horizontally, seeds facing upwards), transfer to the growth chamber in the morning and grow horizontally for 5 d.
      Note: Samples can be kept in the dark by wrapping them in aluminum foil.
    5. On the 5th day carefully open the plate (Figure 1B) and wound samples under a portable dissecting stereomicroscope. To minimize seedling movement and support the sample, place one hypodermic needle under the cotyledon with its hollowed face pointing downwards, and use the other hypodermic needle to pierce the center of the cotyledon with its sharper side (Figure 1C-D). With practice, it takes 3-4 min to wound 120 seedlings (1 plate).
    6. Re-seal the plate and place it back in the growth chamber. To induce the MYC2p-GUS reporter gene activity, keep seedlings in the growth chamber for 4 h prior to tissue collection. Analyze 30-60 seedlings for each treatment.
    7. Collect plant samples with forceps by gently lifting 5-10 seedlings at a time at the hypocotyl-cotyledon interface avoiding additional wounding or squeezing of tissues. Place them in staining dishes containing 1-2 ml 90% acetone and incubate for a minimum of 20 min on ice.
    8. Wash the seedlings twice with 1 ml 50 mM sodium phosphate buffer pH 7.0.
    9. Replace the rinsing buffer with 750 µl GUS staining solution.
    10. Transfer samples in a vacuum pump and vacuum-infiltrate for 5 min at room temperature, then incubate at 37 °C in the dark for 2 h.
    11. Stop the reaction by replacing the staining solution with 70% EtOH. Replace the 70% EtOH solution about twice a day until most tissues are no longer green (2-3 d). Samples can be stored in this solution for long periods at 4 °C if well sealed to prevent EtOH evaporation.
    12. Replace the 70% EtOH with chloral hydrate solution and mount samples on microscopy slides in the same chloral hydrate solution.
    13. Allow tissues to clarify for at least 24 h before photographing with a stereomicroscope.


      Figure 1. Single cotyledon wounding of horizontally grown 5-do Arabidopsis seedlings. Seeds are plated on media fitted with the nylon mesh (A, Video 1) and grown horizontally for 5 d after stratification (B). Seedlings are pierced with a hypodermic needle in the center of one of the cotyledons (C) resulting in a visible wound (D). Scale bars = 0.5 mm.

  2. Repetitive wounding of seedling cotyledons
    Note: Steps B1-4 must be performed in aseptic conditions under a sterile hood.
    1. To equidistantly sow seeds on the media, place a printed guide underneath the petri dish (File 1B).
    2. Resuspend sterile seeds in 500 µl sterile ddH2O and equidistantly plate two rows of 30 seeds each on a solid 0.5x MS medium (0.85% agar) with a P20 micropipette set to dispense a volume of 20 µl (Figure 2A).
      Note: If more than one seed are ejected on the mesh in a single spot, the excess can be aspirated and repositioned.
    3. Seal the plate with 2.5 cm wide micropore tape, stratify for 2 d in the dark at 4 °C (keep the plates horizontally, seeds facing upwards), transfer to the growth chamber in the morning and grow vertically for 3 d (Figure 2B).
      Note: Samples can be kept in the dark by wrapping them in aluminum foil.
    4. In the morning (7-8 am) of the third day, carefully open the plate in aseptic conditions, place it horizontally and wound under a portable dissecting stereomicroscope. To minimize seedling movement and support the sample, place a hypodermic needle under the cotyledon with its hollowed face pointing downwards, and use the NanoFil needle to pierce the cotyledon (Figure 2C). With practice, it takes approximately 6 min to wound 60 seedlings (1 plate).
    5. Close the plate, return to the growth chamber and keep growing vertically.
    6. Repeat the wounding procedure every 12 h on alternate cotyledons, for a total of 5 wounds per seedling (Figure 2D).
    7. After the last 5th wound keep the seedlings in growth chamber for 2 h to induce CYCB1; 1 promoter-driven GUS reporter gene activity. Collect seedlings using forceps and gently lift them at the hypocotyl-cotyledon interface avoiding additional wounding or squeezing of tissues. Analyze 30-40 seedlings for each treatment.
    8. Carefully transfer 5-10 seedlings at a time into the GUS staining solution paying attention not to damage the root tips. Incubate at 37 °C in the dark for 2 h.
    9. Stop the reaction by replacing the GUS staining solution with 1 ml 50 mM sodium phosphate buffer pH 7.0. Samples can be stored for a few h at 4 °C but they should be imaged as soon as possible.
    10. Mount roots on microscopy slides in freshly prepared chloral hydrate solution and photograph with a DIC microscope.


      Figure 2. Repetitive cotyledon wounding of vertically grown Arabidopsis seedlings. Seeds are plated on growth media (A) and grown vertically for 3 d after stratification (B). Seedlings are pierced with a NanoFil needle on alternate cotyledons every 12 h (C) resulting in 5 visible wounds (D). Scale bars = 0.5 mm.

Representative data

Expression of the MYC2p-GUS reporter in seedlings is normally confined to the roots, basal part of the hypocotyl and leaf primordia (Figure 3A; Gasperini et al., 2015) but single cotyledon wounding induces further activation in above ground tissues, including cotyledons and the upper hypocotyl (Figure 3B). Repetitive cotyledon wounding causes a reduction in root length due to decreased meristem cell number associated with reduced expression of the cell cycle gene CYCB1;1 (Gasperini et al., 2015), as visualized by a lower CYCB1;1p-GUS reporter activity in the root meristem of treated seedlings (Figure 3C-D).


Figure 3. MYC2p-GUS reporter activity in control (A) and single cotyledon wounded seedlings (B). The GUS enzyme converts X-Gluc into a blue colored product revealing sites of transcriptional activity. The orange asterisk indicates the wounding site. CYCB1; 1p-GUS reporter expression in the root meristem of the control (C) and seedling subjected to repetitive cotyledon wounding (D). Scale bars = 0.5 mm (A, B); 50 μm (C, D).

Notes

  1. The nylon mesh is sold as a large roll and should be cut in pieces of the required size. Several (8-16) mesh pieces can be wrapped in aluminum foil and sterilized together by autoclaving. The mesh can be rinsed with running tap water after use and re-sterilized multiple times.
  2. Any other sterile plating technique is suitable.
  3. Prolonged incubation of samples in chloral hydrate will improve clarifying of plant tissues that can be stored in this solution for 1-2 months. However imaging of the root tip should be done within 10-15 min after mounting in fresh chloral hydrate as meristematic cells will start to deteriorate rapidly.
  4. Repetitive wounding can be performed in older seedlings over longer periods of times (> 5 wounds) as long as there is sufficient tissue to wound.

Recipes

  1. 0.5x MS solid medium
    2.15 g/L MS
    0.5 g/L MES hydrate
    ddH2O to final volume
    Adjust pH to 5.7 with 10 M KOH
    Add agar to 7 g/L (horizontal plates, 9 cm round) or 8.5 g/L (vertical plates, 12 cm square)
    Autoclave
    Cool down to 55-65 °C and pour into plates
  2. GUS staining solution
    Note: Prepare fresh and keep in the dark.
    50 mM sodium phosphate buffer pH 7.0
    10 mM EDTA pH 8.0
    0.1% Triton X-100
    3 mM Potassium Ferrocyanide
    3 mM Potassium Ferricyanide
    0.5 mg/ml 5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc)
    ddH2O to volume
  3. Chloral hydrate solution
    8 g chloral hydrate
    2 ml glycerol
    1 ml ddH2O

Acknowledgments

The single wounding protocol was developed by Acosta et al. (2013) and the repetitive wounding protocol by Gasperini et al. (2015). This work was supported by the Swiss National Science Foundation grant 31003A-138235 to E.E.F. We thank A. Chételat, A. Kurenda and C. T. Nguyen for photography and video assistance.

References

  1. Acosta, I. F., Gasperini, D., Chetelat, A., Stolz, S., Santuari, L. and Farmer, E. E. (2013). Role of NINJA in root jasmonate signaling. Proc Natl Acad Sci U S A 110(38): 15473-15478.
  2. Colon-Carmona, A., You, R., Haimovitch-Gal, T. and Doerner, P. (1999). Technical advance: spatio-temporal analysis of mitotic activity with a labile cyclin-GUS fusion protein. Plant J 20(4): 503-508.
  3. Gasperini, D., Chetelat, A., Acosta, I. F., Goossens, J., Pauwels, L., Goossens, A., Dreos, R., Alfonso, E. and Farmer, E. E. (2015). Multilayered organization of jasmonate signalling in the regulation of root growth. PLoS Genet 11(6): e1005300.
  4. Larrieu, A., Champion, A., Legrand, J., Lavenus, J., Mast, D., Brunoud, G., Oh, J., Guyomarc'h, S., Pizot, M., Farmer, E. E., Turnbull, C., Vernoux, T., Bennett, M. J. and Laplaze, L. (2015). A fluorescent hormone biosensor reveals the dynamics of jasmonate signalling in plants. Nat Commun 6: 6043.

简介

通过生物和非生物损伤对植物器官的损害在自然界中是非常常见的。拟南芥(Arabidopsis thaliana)5天龄(5-do)幼苗代表了一种优异的系统,其中在损伤部位和远端无害组织中研究植物对机械创伤的反应。经受单次或重复子叶伤害的野生型,转基因或突变品系的幼苗可用于定量形态学改变(例如根长度,Gasperini等人,2015),分析体内报道基因的动力学(Larrieu等人,2015; Gasperini等人,2015),遵循定量的转录变化RT-PCR(Acosta等人,2013; Gasperini等人,2015)或通过大量下游程序检查伤口反应的其它方面。在这里我们说明如何快速,可靠地卷绕年轻幼苗的子叶,并显示两个启动子驱动表达的β-葡萄糖醛酸酶(GUS)在整个幼苗和主根根分生组织,单个或重复子叶伤害后的行为,分别。我们描述了可以容易地适应具体实验需要的两个程序。

关键字:植物, 茉莉, JA-ILE-

材料和试剂

  1. 苗圃子叶单发伤害
    1. 无菌6cm×6cm尼龙网,200μm孔径(Lanz-Anliker AG,定制)
    2. 标准9厘米圆形培养皿
    3. 3M TM 胶带,1.25cm×9.14m,低过敏胶带,标准卷(3M,目录号:1530-0)
    4. 两个25G×5/8"皮下注射针,0.5mm×16mm(BD Bioscience,目录号:305760)
    5. 拟南芥无菌种子, g/ 记者行(Gasperini等人,2015)
    6. 无菌双蒸水H 2 O(ddH 2 O)
    7. Murashige和Skoog(MS)培养基(Duchefa Biochemie,目录号:M0221.0050)
    8. MES水合物(Sigma-Aldrich,目录号:M8250)
    9. 琼脂(AppliChem GmbH,目录号:A2111 1000)
    10. 标准9cm圆形培养皿,装有30ml固体 半强度Murashige和Skoog(0.5x MS)培养基和0.7%琼脂 食谱)

  2. 苗木子叶的重复性伤害
    1. 3M Micropore TM 胶带,2.5cm×9.14m,低过敏胶带,标准卷(3M,目录号:1530-1)
    2. 一个25G×5/8"皮下注射针头,0.5mm×16mm(BD Bioscience,目录号:305760)
    3. 一个36号斜角NanoFil针,110μm外径(World Precision Instruments,目录号:NF36BV)
    4. 标准12cm×12cm正方形培养皿
    5. 用于垂直增长的机架(例如 Milian,目录号:086680)
    6. 拟南芥无菌种子,例如 CYCB1; 1p-GUS 报告基因线(Colón-Carmona et al。 )
    7. 无菌ddH 2 O 2 /
    8. 标准12cm×12cm正方形培养皿,填充有具有0.85%琼脂的70ml 0.5×MS(参见Recipes)

  3. GUS染色
    1. 染色皿[例如12孔悬浮培养板(Greiner Bio-One GmbH,Cell Star ,目录号:665102)]
    2. 冰桶冰
    3. 显微镜载玻片(例如 Thermo Fisher Scientific, Menzel-Glaser,目录号:AD00000112E)和盖玻片(例如 Thermo Fisher Scientific,Menzel-Glaser,目录号:BB024060A1)
    4. 90%(v/v)丙酮在ddH 2 O 2中
    5. 50mM磷酸钠缓冲液(pH7.0)
    6. 70%(v/v)乙醇(EtOH),在ddH 2 O中
    7. 亚铁氰化钾[例如三水合六氰基铁(II)酸钾(Sigma-Aldrich,目录号:60279)]
    8. 亚铁氰化钾[例如六氰基铁酸钾(III)(Merck Millipore Corporation,目录号:104973)]
    9. 5-溴-4-氯-3-吲哚氧基-β-D-葡萄糖醛酸(X-Gluc)(例如Biosynth,目录号:B-7300)
    10. 水合氯醛(例如Sigma-Aldrich,目录号:23100)
      安全注意事项:急性毒性,避免吸入。
    11. 甘油(例如Sigma-Aldrich,目录号:G6279)
    12. GUS染色溶液(参见配方)
    13. 氯化物水合物溶液(见配方)

设备

  1. 无菌罩(层流罩)
  2. 精细镊子样式4(例如 Dumont,目录号:0508-4-PO)
  3. 微量移液器(P20,P200,P1000)
  4. 植物生长室或植物生长室设置有以下生长条件:21℃,100μEm -2 s -1 s -1光强度,14小时光照/10小时黑暗光周期< br />
  5. 便携式解剖立体显微镜
  6. 真空泵
  7. 37℃孵育器
  8. 立体显微镜[例如:徕卡显微系统公司,型号:Leica MZ16A,配备相机(型号:DFC310FX)]
  9. 微分干涉对比(DIC)显微镜[例如,徕卡显微系统公司,型号:Leica DM5500,带有照相机(型号:DFC420)]

程序

  1. 苗圃子叶单发伤害
    注意:步骤1-3必须在无菌条件下在无菌罩下进行。
    1. 要在培养基上等距播种,请在培养皿下放置印刷导管( 文件1A )。
    2. 使用无菌镊子将无菌尼龙网放在固体0.5x MS培养基(0.7%琼脂)上 注意:尼龙网提供了一个固体支持,允许幼苗 生长与子叶表面是容易接近的直接 伤害。
    3. 将无菌种子重悬于500μl无菌ddH 2 O中 将120个单个种子等距地平铺在无菌网上 P20微量吸管设置以分配20μl的体积(图1A,视频 1)。
      注意:如果一个点上有多于一粒的种子在网格上喷出,可以吸出和重新定位多余的种子。

      视频1.种子指南
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    4. 用1.25cm宽的微孔胶带密封板,分层2天 ?黑暗在4℃(保持板水平,种子面向上), 在早上转移到生长室,并水平生长5 ?d。
      注意:样品可以保存在黑暗中,包装在铝箔中。
    5. 在第5天天小心地打开板(图1B)和伤口 样品在便携式解剖立体显微镜下。最小化 幼苗运动并支持样品,放置一个皮下注射针 在子叶下面,其中空的面朝下,并使用 另一个皮下注射针刺穿子叶的中心 其较尖锐的一侧(图1C-D)。与实践,需要3-4分钟的伤口 ?120株幼苗(1块)
    6. 重新密封板并将其放回 生长室。为了诱导MYC2p-GUS 报道基因活性,保持 ?幼苗在生长室中在组织收集之前4小时。 每次处理分析30-60棵幼苗。
    7. 收集植物样品 ?用镊子轻轻地提起5-10个幼苗在一次的时间 下胚轴 - 子叶界面,避免另外的伤口或挤压 的组织。将它们放在含有1-2ml 90%丙酮的染色皿中 并在冰上孵育至少20分钟
    8. 用1ml 50mM磷酸钠缓冲液pH 7.0洗涤幼苗两次
    9. 用750μlGUS染色溶液更换冲洗缓冲液。
    10. 转移样品在真空泵和真空渗透5分钟 ,然后在37℃下在黑暗中孵育2小时。
    11. 通过用70%EtOH代替染色溶液停止反应。 更换70%乙醇溶液约每天两次,直到大多数组织 不再是绿色(2-3天)。样品可以长期储存在该溶液中 ?周期在4°C,如果密封,以防止EtOH蒸发
    12. 用水合氯醛溶液代替70%EtOH,并安装样品 显微镜在同一水合氯醛溶液中滑动
    13. 允许组织澄清至少24小时,然后用立体显微镜拍照

      图1.水平生长的5-do拟南芥幼苗的单子叶伤口。将种子接种在装配有尼龙的培养基上 网格(A,视频1),并水平生长5 d后分层 (B)。在一个中心用皮下注射针刺穿幼苗 ?的子叶(C),导致可见的伤口(D)。比例尺= 0.5mm。

  2. 苗木子叶的重复性伤害
    注意:步骤B1-4必须在无菌条件下在无菌罩下进行。
    1. 要在媒体上等距播种,请在培养皿下方放置印刷的指南( File 1B )。
    2. 重悬无菌种子 在500μl无菌ddH 2 O中,并且等分铺板两行30个种子 各自在具有P20微量吸液管组的固体0.5x MS培养基(0.85%琼脂)上 以分配20μl的体积(图2A)。
      注意:如果一个点上有多于一粒的种子在网格上喷出,可以吸出和重新定位多余的种子。
    3. 用2.5cm宽的微孔胶带密封板,分层2天 黑暗在4℃(保持板水平,种子面向上), 在早晨转移到生长室,垂直生长3天 ?(图2B)。
      注意:样品可以保存在黑暗中,包装在铝箔中。
    4. 在第三天的早上(7-8 am),小心地打开盘子 在无菌条件下,将其水平放置并缠绕在便携式下 解剖立体显微镜。最小化苗移动和支持 样品,将皮下注射针放置在子叶下 空心面向下,并使用NanoFil针刺穿 子叶(图2C)。与实践,大约需要6分钟 到伤口60棵幼苗(1个平皿)
    5. 关闭板,返回生长室,并保持垂直生长。
    6. 在交替子叶上每12小时重复创伤程序,每个幼苗总共5个创伤(图2D)。
    7. 在最后5次伤口保持幼苗在生长室中2小时 ?诱导CYCB1; 1启动子驱动的GUS报告基因活性。搜集 幼苗使用镊子,轻轻地提起他们在下胚轴 - 子叶 界面,避免组织的额外伤口或挤压。分析 每次处理30-40棵幼苗。
    8. 小心转移5-10 幼苗一次进入GUS染色液注意不要 损伤根尖。在37℃在黑暗中孵育2小时。
    9. 通过用1ml 50mM替换GUS染色溶液停止反应 ?磷酸钠缓冲液pH 7.0。样品可以在4℃储存几小时 °C,但应尽快成像。
    10. 在显微镜载玻片上装载新鲜制备的水合氯醛溶液并用DIC显微镜拍照

      图2.垂直生长的拟南芥幼苗的重复子叶伤口将种子接种在生长培养基(A)上并垂直生长 分层后3天(B)。用NanoFil刺穿幼苗 针在交替子叶上每12小时(C),导致5可见 伤口(D)。比例尺= 0.5mm。

代表数据

表达 ?的MYC2p-GUS 报告基因通常限制于 根,下胚轴和叶原基的基部(图3A; Gasperini等人,2015),但单子叶伤口诱导 进一步激活上述地面组织,包括子叶和 ?上部下胚轴(图3B)。重复子叶伤口导致a 由于减少的分生组织细胞数而导致的根长度减少 与较低的CYCB1; 1p-GUS显示的细胞周期基因CYCB1; 1(Gasperini等人,2015)的表达降低有关;在处理的幼苗的根分生组织中(图3C-D)

图3.在对照(A)和单子叶受伤幼苗(B)中的MYC2p-GUS 报道基因活性。 ?GUS酶将X-Gluc转化为蓝色产物显露位点 的转录活性。橙色星号表示伤口 现场。 CYCB1; 1p-GUS 报告基因表达 对照(C)和经受重复子叶伤害的幼苗(D)。 ?比例尺= 0.5mm(A,B); 50μm(C,D)。

笔记

  1. 的 ?尼龙网作为大卷出售,应该切成片 所需尺寸。几个(8-16)网片可以用铝包裹 箔并通过高压灭菌一起灭菌。网可以用清洗 ?使用后运行自来水并重复杀菌多次
  2. 任何其他无菌电镀技术都是合适的
  3. 长期 ?孵化样品在水合氯醛中会改善澄清 植物组织可以储存在此溶液中1-2个月。 然而,根尖的成像应在10-15分钟后完成 安装在新鲜的水合氯醛中,分生细胞将开始
  4. 可以执行重复性创伤 在较长的时间段(> 5个伤口)中的较长的幼苗中 因为有足够的组织伤口

食谱

  1. 0.5x MS固体培养基
    2.15 g/L MS
    0.5g/L MES水合物
    ddH 2到最终体积
    用10M KOH调节pH至5.7 加入琼脂至7g/L(水平板,9cm圆)或8.5g/L(垂直板,12cm正方形)。
    高压灭菌器
    冷却至55-65℃,倒入板
  2. GUS染色溶液
    注意:准备新鲜,保持在黑暗中。
    50mM磷酸钠缓冲液pH7.0 10mM EDTA pH 8.0 0.1%Triton X-100 3 mM氰亚铁酸钾
    3 mM铁氰化钾
    0.5mg/ml 5-溴-4-氯-3-吲哚基葡糖苷酸(X-Gluc) ddH 2 到卷
  3. 水合氯醛溶液
    8克水合氯醛
    2ml甘油 1ml ddH 2 O 2 /

致谢

单一伤口方案由Acosta等人(2013)和Gasperini等人(2015)的重复伤口方案开发。这项工作得到了瑞士国家科学的支持 基金会赠款31003A-138235给E.E.F.我们感谢A.Chételat,A. Kurenda和C. T. Nguyen的摄影和视频援助。

参考文献

  1. Acosta,I.F.,Gasperini,D.,Chetelat,A.,Stolz,S.,Santuari,L.and Farmer,E.E。(2013)。 NINJA在根茉莉酸信号中的作用。 Proc Natl Acad Sci USA 110(38):15473-15478。
  2. Colon-Carmona,A.,You,R.,Haimovitch-Gal,T.and Doerner,P。(1999)。 技术进步:使用不稳定的细胞周期蛋白-GUS融合蛋白的有丝分裂活性的时空分析。 a> Plant J 20(4):503-508。
  3. Gasperini, ?D.,Chetelat,A.,Acosta,I.F.,Goossens,J.,Pauwels,L.,Goossens, A.,Dreos,R.,Alfonso,E.and Farmer,E.E。(2015)。 茉莉酸信号传导的多层组织在根生长调节中的作用。 PLoS Genet 11(6):e1005300。
  4. Larrieu, ?A.,Champion,A.,Legrand,J.,Lavenus,J.,Mast,D.,Brunoud,G., J.,Guyomarc'h,S.,Pizot,M.,Farmer,E.E.,Turnbull,C.,Vernoux, ?Bennett,M.J。和Laplaze,L。(2015)。 荧光激素生物传感器揭示植物中茉莉酮酸信号的动态。 Nat Commun 6:6043.
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Gasperini, D., Acosta, I. F. and Farmer, E. E. (2016). Cotyledon Wounding of Arabidopsis Seedlings. Bio-protocol 6(2): e1712. DOI: 10.21769/BioProtoc.1712.
  2. Gasperini, D., Chetelat, A., Acosta, I. F., Goossens, J., Pauwels, L., Goossens, A., Dreos, R., Alfonso, E. and Farmer, E. E. (2015). Multilayered organization of jasmonate signalling in the regulation of root growth. PLoS Genet 11(6): e1005300.
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