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Sulfur is an essential macronutrient required for growth and development of plants. Plants take up sulfate from the soil environment through the function of plasma membrane-bound sulfate transporters expressed at the root surface cell layers. Plants then utilize the incorporated sulfate as the main sulfur source to synthesize sulfur-containing compounds such as cysteine and methionine. Measurement of root sulfate uptake capacity is essential for analyzing mutants showing altered levels of sulfate transporters and/or sulfur metabolic enzymes as a result of genetic modification or due to the effect of intrinsic or environmental factors modulating their gene expression. The method described in this protocol allows quantitative investigation of sulfate uptake rates and root-to-shoot sulfate distribution in Arabidopsis seedlings using [35S] sulfate as a radioactive tracer. The method is designed for parallel comparisons of multiple Arabidopsis accessions, mutants or transgenic lines at the seedling stage.

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Measurement of Uptake and Root-to-Shoot Distribution of Sulfate in Arabidopsis Seedlings
拟南芥幼苗中硫酸盐的摄取和根茎分布的测定

植物科学 > 植物生理学 > 营养
作者: Naoko Yoshimoto
Naoko YoshimotoAffiliation 1: Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan
Affiliation 2: RIKEN Plant Science Center, Japan
For correspondence: naokoy@faculty.chiba-u.jp
Bio-protocol author page: a2840
Tatsuhiko Kataoka
Tatsuhiko KataokaAffiliation: RIKEN Plant Science Center, Japan
Bio-protocol author page: a2841
Akiko Maruyama-Nakashita
Akiko Maruyama-NakashitaAffiliation 1: RIKEN Plant Science Center, Japan
Affiliation 2: Graduate School of Agricultural Science, Kyushu University, Kyushu, Japan
Bio-protocol author page: a2842
 and Hideki Takahashi
Hideki TakahashiAffiliation 1: RIKEN Plant Science Center, Japan
Affiliation 2: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, USA
For correspondence: htakaha@cns.msu.edu
Bio-protocol author page: a2843
Vol 6, Iss 1, 1/5/2016, 1759 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1700

[Abstract] Sulfur is an essential macronutrient required for growth and development of plants. Plants take up sulfate from the soil environment through the function of plasma membrane-bound sulfate transporters expressed at the root surface cell layers. Plants then utilize the incorporated sulfate as the main sulfur source to synthesize sulfur-containing compounds such as cysteine and methionine. Measurement of root sulfate uptake capacity is essential for analyzing mutants showing altered levels of sulfate transporters and/or sulfur metabolic enzymes as a result of genetic modification or due to the effect of intrinsic or environmental factors modulating their gene expression. The method described in this protocol allows quantitative investigation of sulfate uptake rates and root-to-shoot sulfate distribution in Arabidopsis seedlings using [35S] sulfate as a radioactive tracer. The method is designed for parallel comparisons of multiple Arabidopsis accessions, mutants or transgenic lines at the seedling stage.
Keywords: Arabidopsis thaliana(拟南芥), Sulfate uptake(硫酸铅), Sulfate transport(硫酸运输)

[Abstract]

Materials and Reagents

  1. Nylon mesh (300 μm mesh opening) (NBC Meshtec, model: NMG 58 or similar types)
  2. 9 cm x 9 cm square petri dishes (Simport, catalog number: D210-16 )
  3. Plastic frame [exterior dimension, 10 cm (W) x 12 cm (H) x 1.5 cm (D); window size, 7.5 cm (W) x 9 cm (H)]]
  4. Plastic container [13 cm (W) x 13 cm (H) x 3 cm (D)]-should be transparent and with no color
  5. Double-sided adhesive tape (NICHIBAN, model: NW-10 or similar types)
  6. Single-sided adhesive tape (Shamrock Scientific Specialty Systems, model: ST-12-1 or similar types)
  7. Paper towel (NIPPON PAPER INDUSTRIES CO., catalog number: 37016 or similar types)
  8. Scintillation vials (Sigma-Aldrich, catalog number: Z190527 or similar types)
  9. Seeds of Arabidopsis (Arabidopsis thaliana)
  10. Sterile deionized water
  11. Agar (Wako Pure Chemical Industries, Siyaku, catalog number: 016-11875 or similar types suitable for plant growth)
  12. Sterile agar medium for growth of Arabidopsis seedlings [General nutrient medium can be used. Nutrient source and concentrations may be modified depending on research purposes, e.g. sulfate concentrations to be adjusted to 1,500 μM or 15 μM sulfate (Maruyama-Nakashita et al., 2015)]
  13. Sterile liquid medium for labeling and post-labeling incubation (Nutrient source and concentrations to be adjusted the same as the agar medium mentioned above)
  14. Na235SO4 aqueous solution, 10 mCi/ml (370 MBq/ml) (American Radiolabeled Chemicals, catalog number: ARS0105 )
  15. Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: H1758 )
  16. Ultima GoldTM scintillation cocktail (PerkinElmer, catalog number: 6013321 )

Equipment

  1. Growth chamber (NKsystem, model: LPH-241SP or similar types. Set the growth conditions appropriately depending on research purposes, e.g. 22 °C, 16 h light/8 h dark cycles)
  2. Autoclave (TOMY SEIKO CO., model: LSX-500 or similar types)
  3. Mechanical pipettes (Gilson, model: P-200 and P-1000 ) and tips
  4. Analytical balance (readability, 0.1 mg) (Cole-Parmer Instrument Company, Mettler Toledo, model: MS204TS or similar types)
  5. Shaker (either reciprocating or rotary shaker) (TAITEC CORPORATION., model: NR-10 or similar types)
  6. Liquid scintillation counter (Hitachi Aloka Medical, model: AccuFLEX LSC-7400 or similar types)
  7. Utility knife (KOKUYO, model: HA-S200YR or similar types)
  8. Stainless steel Forceps (Sigma-Aldrich, catalog number: Z168696 or similar types)
  9. Surgical scissors (stainless steel dissecting scissors) (Sigma-Aldrich, catalog number: Z265977 or similar types)

Procedure

  1. Preparation of mesh-embedded agar medium
    1. Cut the nylon mesh (300 μm opening) into 1 cm x 8.7 cm strips. Wrap the strips of the nylon mesh tightly in aluminum foil, autoclave them and let them dry. Keep the nylon mesh in aluminum foil flattened during autoclaving and drying processes.
    2. Prepare agar medium, on which you grow Arabidopsis seedlings, in 9 cm x 9 cm square petri dishes. The agar concentrations should be 0.8% or higher to keep the roots remain on the surface of the medium.
    3. Embed the autoclaved nylon mesh vertically into a solidified agar medium. Insert the longer side of the mesh vertically against the surface of the agar medium at the position approximately 2 cm apart from one side of the petri dish (Figure 1A). Approximately one-half area of the mesh should be embedded in the agar medium. As a result, each agar medium is partitioned into 2 cm x 9 cm and 7 cm x 9 cm areas by a nylon mesh (Figure 1B).
    4. Fill in the gap between the upper side of the nylon mesh and the agar medium in the 2 cm x 9 cm area, by pouring trace amount of melted agar medium using a mechanical pipette (Figures 1C-D). Solidify the agar medium at room temperature.

  2. Vertical plate culture of Arabidopsis seedlings
    1. Sterilize the surface of Arabidopsis seeds following a standard protocol (Li, 2011) or similar methods described elsewhere. Immerse the seeds in sterile deionized water at 4 °C under dark for 3 d or more to enhance germination.
    2. Place the surface sterilized seeds on the mesh-embedded agar medium described above, approximately 2 mm apart from the nylon mesh in the 2 cm x 9 cm area. Do not sow seeds close to each side of the petri dish (Figure 1E).
    3. Set the agar plates vertically in a growth chamber and grow Arabidopsis seedlings under a controlled condition. The roots of Arabidopsis will penetrate through the nylon mesh, and elongate on the surface of the agar medium (Figure 1F). If the roots are not attached onto the surface of the medium after penetrating through the nylon mesh, dab those roots gently onto the surface of the medium using a sterile pipette tip or forceps.
    4. Following an appropriate period of incubation in a growth chamber (e.g. 10-15 d, which is generally an appropriate period to obtain the seedlings in size suitable for the analysis, for wild-type plants when grown on standard nutrient medium), you will obtain a strip of nylon mesh holding Arabidopsis seedlings to be used for the 35S-labeling experiment.


      Figure 1. Schematic illustration of mesh-embedded agar medium for vertical plate culture of Arabidopsis seedlings. A. Inserting an autoclaved mesh into an agar medium. B. Mesh-embedded agar medium. C. Filling in the gap between the upper side of the mesh and the agar medium by pouring trace amount of melted agar medium using a pipette. D. Mesh-embedded agar medium with filled gap. E. Seeding of Arabidopsis. F. Arabidopsis seedlings grown through the mesh. (Yoshimoto et al., 2007)

  3. Preparation for 35S-labeling
    1. Make a plastic frame to place the nylon mesh holding the Arabidopsis seedlings. Cut a rectangular window [7.5 cm (W) x 9 cm (H)] on the lid of a plastic tip box [10 cm (W) x 12 cm (H) x 1.5 cm (D)] using a utility knife. To support the nylon mesh in the following step (step C4), put double-sided adhesive tapes on both sides of the plastic frame (Figure 2A).
    2. Pour 50 ml of liquid medium into a 9 cm x 9 cm square petri dish, and place it under a plastic frame (Figure 2A).
    3. Pull the nylon mesh, which holds the Arabidopsis seedlings, from the agar medium using forceps (Figure 2A). Handle them carefully not to damage the roots.
    4. Carefully place two edges of the nylon mesh onto the double-sided adhesive tapes attached to the plastic frame. Then, fix the two edges of the nylon mesh to the plastic frame using single-sided adhesive tapes (Figure 2B). Usually, 4-6 strips of nylon mesh can be placed in parallel on one plastic frame (Figure 2C). After placing all the meshes on the frame, securely fix them to the frame using single-sided adhesive tapes (Figure 2D). Submerge the roots elongated under the nylon mesh into the liquid medium using forceps. This plastic frame device allows you to create approximately 1 cm of space above the surface of the liquid medium underneath the nylon mesh. Finally, place a transparent plastic container [13 cm (W) x 13 cm (H) x 3 cm (D)] bottom side up to cover the plastic frame holding the plants (Figure 2E). This prevents desiccation of aerial parts of plants. Incubate plants for >10 min.
    5. Preparation of 35S-labeling medium: Add [35S] sodium sulfate (Na235SO4) aqueous solution to the liquid medium. Usually, the appropriate amount of Na235SO4 to be added is 0.5-5% of the total sulfate contained in the liquid medium.


      Figure 2. Schematic illustration of a plastic frame device holding nylon mesh and Arabidopsis seedlings. A. Organization of a nylon mesh holding Arabidopsis seedlings, a plastic frame with double-sided adhesive tapes (yellow), and a square petri dish containing the liquid medium. B. Plastic frame holding the nylon mesh and Arabidopsis seedlings. A square petri dish is placed underneath the plastic frame. Two edges of the nylon mesh are placed onto the double-sided adhesive tapes attached to the plastic frame, and fixed with single-sided adhesive tapes (orange). Roots of Arabidopsis are submerged in the liquid medium. C. Plastic frame holding four strips of nylon mesh with Arabidopsis seedlings. D. The final setting of the plastic frame device ready for the 35S-labeling experiment. Four strips of nylon mesh are securely fixed to the plastic frame using single-sided adhesive tapes (brown). E. The final setting of the plastic frame device ready for the 35S-labeling experiment covered by a transparent plastic container which prevents desiccation of aerial parts of plants. (Yoshimoto et al., 2007)

  4. 35S-labeling of roots
    1. To start 35S-labeling, remove the transparent plastic container [13 cm (W) x 13 cm (H) x 3 cm (D)] that was covering the plants, and transfer the whole plastic frame device including the mesh and seedlings on top of a 9 cm × 9 cm square petri dish which contains 50 ml of 35S-labeling medium. Submerge the roots into the 35S-labeling medium using forceps. Place a transparent plastic container [13 cm (W) x 13 cm (H) x 3 cm (D)] back to the position to cover the plastic frame device and plants to prevent desiccation of aerial parts of plants during incubation. Then, incubate the plants for 10-60 min depending on the objectives of the experiment.
    2. To stop 35S-labeling, rinse the roots of Arabidopsis twice in 60 ml of non-labeled liquid medium in 9 cm x 9 cm square petri dishes.
    3. To wash out the radioactivities remaining in the apoplastic space of roots, submerge the roots in 75 ml of non-labeled liquid medium. Place a transparent plastic box [13 cm (W) x 13 cm (H) x 3 cm (D)] over the plastic frame device and plants to prevent desiccation of aerial parts of plants during incubation. Incubate the plants for 60 min.
    4. Harvest plant tissues from step D2 or D3. First remove the nylon mesh and the plants from the plastic frame, then cut the roots beneath the nylon mesh using surgical scissors, and detach the roots and the aerial parts of plants separately from the mesh. Remove the liquids remaining on the root surface by using a paper towel. Weigh the fresh weight of plant tissues using an analytical balance.

  5. Determination of incorporated radioactivity
    1. Place the harvested plant tissues (< 200 mg FW) in a liquid scintillation vial.
    2. In separate vials, make standard samples for calibration of radioactivity, using aliquots of the 35S-labeling medium from step C5 (e.g. 0, 1, 3, 10, 30, and 100 μl).
    3. Add 1 ml of 100 mM hydrochloric acid and gently shake the vials overnight at room temperature in the dark using a shaker, to digest the plant tissue.
    4. Add 2 ml of Ultima GoldTM scintillation cocktail and mix thoroughly.
    5. Measure the radioactivity using a liquid scintillation counter.
    6. Generate a calibration curve by plotting the amount of sulfate versus the radioactivity of the standard samples. Calculate the amount of 35S-sulfate in plant tissues using the calibration curve. Calculate the sulfate uptake rates based on the amount of 35S-sulfate incorporated into plant tissues, divided by the fresh weight of plant tissues (step D4) and the period of time for incubation in the 35S-labeling medium (step D1). The root-to-shoot sulfate distribution in plants can be estimated based on the amount of radioactivity separately detected in roots and shoots.

Notes

The composition of agar and liquid media, the concentration of [35S] sodium sulfate in the 35S-labeling medium, and the labeling period, may vary according to the research objectives (e.g., measurement of high- or low-affinity sulfate uptake rate). This protocol is adapted from Kataoka et al. (2004a), Kataoka et al. (2004b), Maruyama-Nakashita et al. (2004), Maruyama-Nakashita et al. (2006), Maruyama-Nakashita et al. (2015) and Yoshimoto et al. (2007).

Acknowledgements

N.Y. receives grant support of JSPS KAKENHI Grant Number 26460118. A.M.N. receives grant support of JSPS KAKENHI Grant Number 24380040 and 15KT0028. H.T. receives grant support from the National Science Foundation (MCB-1244300) and AgBioResearch.

References

  1. Kataoka, T., Hayashi, N., Yamaya, T. and Takahashi, H. (2004a). Root-to-shoot transport of sulfate in Arabidopsis. Evidence for the role of SULTR3;5 as a component of low-affinity sulfate transport system in the root vasculature. Plant Physiol 136(4): 4198-4204.
  2. Kataoka, T., Watanabe-Takahashi, A., Hayashi, N., Ohnishi, M., Mimura, T., Buchner, P., Hawkesford, M. J., Yamaya, T. and Takahashi, H. (2004b). Vacuolar sulfate transporters are essential determinants controlling internal distribution of sulfate in Arabidopsis. Plant Cell 16(10): 2693-2704.
  3. Li, X. (2011). Arabidopsis growing protocol-A general guide. Bio-protocol Bio101: e126.
  4. Maruyama-Nakashita, A., Nakamura, Y., Yamaya, T. and Takahashi, H. (2004). A novel regulatory pathway of sulfate uptake in Arabidopsis roots: implication of CRE1/WOL/AHK4-mediated cytokinin-dependent regulation. Plant J 38(5): 779-789.
  5. Maruyama-Nakashita, A., Nakamura, Y., Tohge, T., Saito, K. and Takahashi, H. (2006). Arabidopsis SLIM1 is a central transcriptional regulator of plant sulfur response and metabolism. Plant Cell 18(11): 3235-3251.
  6. Maruyama-Nakashita, A., Watanabe-Takahashi, A., Inoue, E., Yamaya, T., Saito, K. and Takahashi, H. (2015). Sulfur-responsive elements in the 3'-nontranscribed intergenic region are essential for the induction of SULFATE TRANSPORTER 2;1 gene expression in Arabidopsis roots under sulfur deficiency. Plant Cell 27(4): 1279-1296.
  7. Yoshimoto, N., Inoue, E., Watanabe-Takahashi, A., Saito, K. and Takahashi, H. (2007). Posttranscriptional regulation of high-affinity sulfate transporters in Arabidopsis by sulfur nutrition. Plant Physiol 145(2): 378-388.

材料和试剂

  1. 尼龙网(300μm网孔)(NBC Meshtec,型号:NMG 58或类似类型)
  2. 9cm×9cm正方形培养皿(Simport,目录号:D210-16)
  3. 塑料框架[外尺寸,10厘米(W)×12厘米(H)×1.5厘米(D);窗口尺寸,7.5cm(W)×9cm(H)]]
  4. 塑料容器[13厘米(宽)x 13厘米(H)x 3厘米(D)] - 应该是透明的,没有颜色
  5. 双面胶带(NICHIBAN,型号:NW-10或类似类型)
  6. 单面胶带(Shamrock Scientific Specialty Systems,型号:ST-12-1或类似类型)
  7. 纸巾(NIPPON PAPER INDUSTRIES CO。,目录号:37016或类似类型)
  8. 闪烁瓶(Sigma-Aldrich,目录号:Z190527或类似类型)
  9. 拟南芥( Arabidopsis thaliana )的种子
  10. 无菌去离子水
  11. 琼脂(Wako Pure Chemical Industries,Siyaku,目录号:016-11875或适于植物生长的类似类型)
  12. 用于拟南芥幼苗生长的无菌琼脂培养基[通用营养培养基可以使用。可以根据研究目的修改营养来源和浓度,例如硫酸盐浓度调整为1,500μM或15μM硫酸盐(Maruyama-Nakashita等人,2015)]
  13. 用于标记和后标记培养的无菌液体培养基(营养源和浓度与上述琼脂培养基相同)
  14. (美国放射性标记的化学品,目录号:ARS0105)的10mCi/ml(370MBq/ml)的水溶液中, br />
  15. 盐酸(HCl)(Sigma-Aldrich,目录号:H1758)
  16. Ultima Gold TM sup/TM闪烁混合物(PerkinElmer,目录号:6013321)

设备

  1. 生长室(NK系统,型号:LPH-241SP或类似类型,根据研究目的适当地设置生长条件,例如22℃,16小时光照/8小时黑暗循环)
  2. 高压灭菌器(TOMY SEIKO CO。,型号:LSX-500或类似型号)
  3. 机械移液器(Gilson,型号:P-200和P-1000)和尖端
  4. 分析天平(可读性,0.1mg)(Cole-Parmer Instrument Company,Mettler Toledo,型号:MS204TS或类似类型)
  5. 振动器(往复振动器或旋转振动器)(TAITEC CORPORATION。,型号:NR-10或类似类型)
  6. 液体闪烁计数器(Hitachi Aloka Medical,型号:AccuFLEX LSC-7400或类似类型)
  7. 公用刀(KOKUYO,型号:HA-S200YR或类似类型)
  8. 不锈钢钳(Sigma-Aldrich,目录号:Z168696或类似类型)
  9. 外科剪刀(不锈钢解剖剪)(Sigma-Aldrich,目录号:Z265977或类似类型)

程序

  1. 网状包埋的琼脂培养基的制备
    1. 将尼龙网(300μm开口)切成1 cm x 8.7 cm的条。包装 尼龙网条紧紧地放在铝箔中,高压灭菌 让它们干燥。保持尼龙网在铝箔扁平化期间 高压灭菌和干燥过程
    2. 准备琼脂培养基,其上 您在9cm×9cm正方形培养皿中生长拟南芥幼苗。的 琼脂浓度应为0.8%或更高以保持根部保持 ?介质的表面
    3. 嵌入高压灭菌的尼龙网 垂直地放入固化的琼脂培养基中。插入长边 在该位置垂直地抵靠琼脂培养基的表面 离培养皿的一侧约2cm(图1A)。 大约一半的网格应该被包埋在琼脂中 中。结果,将每种琼脂培养基分成2cm×9cm 和7cm×9cm面积的尼龙网(图1B)
    4. 填写 尼龙网的上侧与琼脂培养基之间的间隙 ?cm×9cm面积,通过使用a。注入微量的熔化的琼脂培养基 机械移液管(图1C-D)。在室温下固化琼脂培养基 温度。

  2. 拟南芥幼苗的垂直平板培养
    1. 按照标准方案杀死拟南芥种子的表面 ?(Li,2011)或其他地方描述的类似方法。浸泡种子 ?无菌去离子水中在4℃下暗处3天以上,以增强 发芽
    2. 将表面灭菌的种子放在 嵌入式琼脂培养基中,离开大约2mm ?尼龙网在2厘米×9厘米区域。不要在每个附近播种种子 侧的培养皿(图1E)
    3. 设置琼脂平板 垂直地在生长室中并在a下生长拟南芥幼苗 控制条件。 拟南芥的根将穿透 尼龙网,并在琼脂培养基的表面伸长(图 1F)。如果根没有附着在培养基的表面上 穿透尼龙网,轻轻地将这些根 表面的培养基使用无菌吸头或镊子。
    4. 在生长室中孵育适当的时间段后(例如10-15天,这通常是适当的时间以获得 幼苗的大小适合分析,适用于野生型植物 生长在标准营养培养基上),你将获得一条尼龙 用于 S标记的拟南芥苗 实验

      图1.网状嵌入琼脂的示意图 用于拟南芥幼苗的垂直平板培养的培养基。 A.插入 ?将高压灭菌网放入琼脂培养基中。 B.网状包埋的琼脂培养基。 C.填充网格的上侧和琼脂之间的间隙 通过使用移液管倾注微量的熔化的琼脂培养基。 D. ?填充间隙的网状包埋的琼脂培养基。 E.拟南芥的接种。 通过筛网生长的拟南芥幼苗。 (Yoshimoto等人, 2007)

  3. 35 S标签的准备
    1. 制作一个塑料框架,将尼龙网放在装有拟南芥苗的地方。在盖子上切一个矩形窗口[7.5厘米(宽)×9厘米(H)] 的塑料端盒[10cm(W)×12cm(H)×1.5cm(D)] 工具刀。在下面的步骤(步骤)中支撑尼龙网 C4),在塑料框架的两侧放置双面胶带 (图2A)。
    2. 将50ml液体培养基倒入9cm×9cm正方形培养皿中,并将其置于塑料框架下(图2A)。
    3. 拉出尼龙网,其中包含拟南芥幼苗 ?琼脂培养基(图2A)。小心处理他们不要 损害根。
    4. 小心地放置尼龙网的两边 到附接到塑料框架的双面胶带上。 然后,使用将尼龙网的两个边缘固定到塑料框架 单面胶带(图2B)。通常,4-6条尼龙 网可以平行放置在一个塑料框架上(图2C)。后 将所有网格放置在框架上,将它们牢固地固定在框架上 使用单面胶带(图2D)。淹没根 使用镊子在尼龙网下延伸到液体介质中。 这种塑料框架设备允许创建大约1厘米 在尼龙网下面的液体介质表面上方的空间。 最后,放置一个透明的塑料容器[13厘米(宽)×13厘米(H)×3 ?cm(D)]底面朝上以覆盖保持植物的塑料框架 (图2E)。这防止了植物地上部分的干燥。 孵育植物> 10分钟。
    5. 制备35S-标记 培养基:将[35 S]硫酸钠(Na 2 SO 4,SO 4 SO 4)水溶液加入到 液体介质。通常,添加适量的Na 2 SO 4和SO 4 SO 4, 是液体介质中包含的总硫酸盐的0.5-5%。


      图2.装有尼龙的塑料框架装置的示意图 ?网状和拟南芥幼苗。 A.组织尼龙网,保持拟南芥幼苗,具有双面胶带的塑料框架 ?(黄色)和含有液体培养基的正方形培养皿。乙。 装有尼龙网和拟南芥苗的塑料框架。一个正方形 ?培养皿放置在塑料框架下面。两边的 尼龙网放置在附接的双面胶带上 塑料框架,并用单面胶带(橙色)固定。 拟南芥的根被浸没在液体培养基中。 C.塑料 框架,其含有拟南芥幼苗的四条尼龙网。 D. 塑料框架装置的最终设置准备用于 35 S标签 ?实验。四个尼龙网带牢固地固定在塑料上 ?框架使用单面胶带(棕色)。 E.最终设置 的塑料框架装置准备好用于 S标记实验 由一个防止干燥的透明塑料容器覆盖 ?植物的地上部分。 (Yoshimoto等人,2007)

  4. 35 S标记根部
    1. 要开始 35 S标记,请取下透明塑料容器[13厘米 (W)×13cm(H)×3cm(D)],并覆盖植物 整个塑料框架装置包括顶部的网和幼苗 的9cm×9cm正方形培养皿,其含有50ml的35S-标记 中。使用镊子将根浸入35S-标记的培养基中。 放置一个透明的塑料容器[13厘米(宽)×13厘米(H)×3厘米(D)] ?返回到覆盖塑料框架装置和植物的位置 防止孵化期间植物的地上部分干燥。然后, 孵化植物10-60分钟,取决于目标 实验
    2. 为了停止 S标记,在60ml的未标记的液体培养基中在9cm×9cm正方形培养皿中冲洗拟南芥的根 菜肴
    3. 洗去剩余的放射性 根的非成形空间,将根浸没在75ml的未标记的 液体介质。放置一个透明的塑料盒[13厘米(宽)×13厘米(H)×3 ?cm(D)]在塑料框架装置和植物上以防止干燥 ?的植物地上部分。孵化植物60 ?min。
    4. 收获来自步骤D2或D3的植物组织。先删除 尼龙网和植物从塑料框架,然后切根 下面的尼龙网使用外科剪刀,并分离根和 ?植物的地上部分与网格分开。取出液体 ?通过使用纸巾保留在根表面上。称重新鲜 使用分析天平测定植物组织的重量。

  5. 结合放射性的测定
    1. 将收获的植物组织(<200mg FW)置于液体闪烁瓶中。
    2. 在单独的小瓶中,制备用于校准的标准样品 使用等分的来自步骤C5的35S-标记的培养基 (例如 0,1,3,10,30和100μl)
    3. 加入1ml的100mM 盐酸并在室温下轻轻摇动小瓶过夜 温度在黑暗中使用振动器,消化植物组织
    4. 加入2ml Ultima Gold TM 闪烁鸡尾酒,并彻底混合
    5. 使用液体闪烁计数器测量放射性。
    6. 通过绘制硫酸盐的量生成校准曲线 相对于标准样品的放射性。计算金额 使用校准曲线测定植物组织中的35 S-硫酸酯。计算 硫酸盐吸收速率基于引入的35 S-硫酸盐的量 ?到植物组织中,除以植物组织的鲜重(步骤 D4)和用于在35S标记培养基中孵育的时间段 (步骤D1)。植物中根茎间的硫酸盐分布可以是 基于在单独检测的放射性的量估计 根和芽。

笔记

琼脂和液体培养基的组成,35 S-标记培养基中的[35 S]硫酸钠的浓度和标记期可以根据研究而变化目标(例如,高或低亲和力硫酸盐摄取速率的测量)。该协议改编自Kataoka等人(2004a),Kataoka等人 (2004b),Maruyama-Nakashita (2004),Maruyama-Nakashita等人(2006),Maruyama-Nakashita等人(2015)和Yoshimoto等人/em>(2007)。

致谢

N.Y.接收JSPS KAKENHI授权号26460118的授权支持。获得JSPS KAKENHI Grant号24380040和15KT0028的资助支持。 H.T.获得国家科学基金会(MCB-1244300)和AgBioResearch的资助。

参考文献

  1. Kataoka,T.,Hayashi,N.,Yamaya,T.and Takahashi,H。(2004a)。 拟南芥中根硫酸根的转运。 证明SULTR3; 5作为低亲和力硫酸盐转运系统在根系脉管系统中的作用的作用。植物生理学136(4):4198-4204。
  2. Kataoka,T.,Watanabe-Takahashi,A.,Hayashi,N.,Ohnishi,M.,Mimura,T.,Buchner,P.,Hawkesford,M.J.,Yamaya,T.and Takahashi, 阴极硫酸盐转运蛋白是控制拟南芥中硫酸盐内部分布的必要决定因素。 植物细胞 16(10):2693-2704
  3. Li,X。(2011)。 拟南芥生长协议 - 一般指南 生物协议 Bio101:e126。
  4. Maruyama-Nakashita,A.,Nakamura,Y.,Yamaya,T.and Takahashi,H。(2004)。 拟南芥根中硫酸盐摄取的新型调节途径:CRE1的影响/WOL/AHK4介导的细胞分裂素依赖性调节。

    38(5):779-789。
  5. Maruyama-Nakashita,A.,Nakamura,Y.,Tohge,T.,Saito,K.and Takahashi,H。(2006)。 拟南芥 SLIM1是植物硫反应和代谢的中枢转录调节剂。 植物细胞 18(11):3235-3251。
  6. Maruyama-Nakashita,A.,Watanabe-Takahashi,A.,Inoue,E.,Yamaya,T.,Saito,K.and Takahashi,H。(2015)。 3'-非转录基因间区域中的硫反应元件对于诱导SULFATE TRANSPORTER 2非常重要;在硫缺乏的情况下在拟南芥根中的1个基因表达。植物细胞27(4):1279-1296。
  7. Yoshimoto,N.,Inoue,E.,Watanabe-Takahashi,A.,Saito,K.and Takahashi,H。(2007)。 硫营养对拟南芥中高亲和力硫酸转运蛋白的转录调控。 Plant Physiol 145(2):378-388。
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How to cite this protocol: Yoshimoto, N., Kataoka, T., Maruyama-Nakashita, A. and Takahashi, H. (2016). Measurement of Uptake and Root-to-Shoot Distribution of Sulfate in Arabidopsis Seedlings. Bio-protocol 6(1): e1700. DOI: 10.21769/BioProtoc.1700; Full Text



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