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[14C] Glucose Cell Wall Incorporation Assay for the Estimation of Cellulose Biosynthesis
采用14C 葡萄糖细胞壁掺入实验估测纤维素的生物合成   

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Abstract

Cellulose is synthesized by Cellulose Synthase A proteins at the plasma membrane using the substrate UDP glucose. Herein, we provide a detailed method for measuring the incorporation of radiolabeled glucose into the cellulose fraction of the cell wall. In this method Arabidopsis seedlings are treated for 2 h with a cellulose biosynthesis inhibitor in the presence of radiolabeled glucose, and are subsequently boiled in acetic-nitric acid to solubilize non-cellulosic material. The radiolabeled glucose detected in the insoluble fraction indicates the amount of cellulose synthesized during the experimental timeframe. The short-term nature of this method is a useful tool in determining if inhibition of cellulose biosynthesis is the herbicides primary mode of action.

Materials and Reagents

  1. Arabidopsis seed (Arabidopsis thaliana L.)
  2. Dextrose (Sigma-Aldrich, catalog number: D9434 )
  3. Radiolabeled glucose [glucose D-14C(U)] (American Radiolabeled Chemicals, catalog number: ARC 0122D )
  4. Acetic Acid, Glacial (Certified ACS) (Fisher Scientific, catalog number: A38-212 )
  5. Nitric Acid, Fuming (Certified ACS) (Fisher Scientific, catalog number: A202-212 )
  6. Counting cocktail (Biodegradable Scintillation Cocktails) (Biosafe II, catalog number: 111195 )
  7. Deionized sterile water (autoclaved at 120 °C for 30 min)
  8. Murashige & Skoog (MS) Basal Salt Mixture (PhytoTechnology Laboratories®, catalog number: M524 )
  9. MES monohydrate (Sigma-Aldrich, catalog number: 69889 )
  10. Model A307 Sample Oxidizer (PerkinElmer, catalog number: A030700 )
  11. Aluminum foil
  12. Eppendorf tubes (1.5 ml) (autoclaved at 120 °C for 30 min)
  13. MS media (see Recipes)
  14. Acetic-nitric acid reagent (see Recipes)

Equipment

  1. Eppendorf tube holders
  2. Pasteur pipets and tips
  3. 1 L Erlenmeyer flask (autoclaved at 120 °C for 30 min)
  4. Microscale
  5. Orbital shaker
  6. Microcentrifuge
  7. Glass scintillation vials (20 ml)
  8. Scintillation Counter (Packard Tri-Carb Liquid Scintillation Counter)
  9. Autoclave

Procedure

  1. Setup (Notes 1 and 2)
    1. In a 1.5 ml Eppendorf tube, add Arabidopsis seeds (~100 mg of seed = ~ 2 g of seedling weight wet) and surface sterilize. Briefly, in a laminar flow hood, add 1 ml of 30% household bleach solution supplemented with 20 µl of 5% v/v sodium dodecyl sulfate (SDS) and shake for 15 min. Remove the bleach supernatant with the seed settling at the bottom of the tube, then sequentially wash three times with 1 ml of autoclaved deionized water. Remove the final wash solution and aliquot 500 µl of sterilized water into the Eppendorf tube. Transfer the seed to a 4 °C refrigerator and store in darkness for 2 days to promote even germination.
    2. Prepare two bottles of liquid MS media (a 250 ml) with and without glucose (2% w/v), respectively. Autoclave in a liquid cycle at 120 °C for 30 min.
    3. Add the seed and the cooled and sterilized MS media supplemented with 2% glucose into a pre-sterilized 1 L Erlenmeyer flask.
    4. Place and secure the flask on an orbital shaker and agitate (180 rpm) the mixture for 2 h in light to promote germination. Afterwards, wrap the flask with aluminum foil to prevent light penetration. Place the flask back on the shaker and agitate for 72 h. (Note 3).

  2. Experimental Procedure
    Note: Seedlings need to be kept in the dark from steps B5-8.
    1. After 72 h, remove the glucose supplemented media and wash seedlings to remove all traces of glucose (Note 4). A depiction of how the seedlings should look is in Figure 1.


      Figure 1. A representation of dark-grown etiolated Arabidopsis seedlings that were grown in liquid MS media for 72 h. A square is equal to 1 cm.

    2. Add 20 mg (wet weight) worth of etiolated seedling to an Eppendorf tube. Wrap each tube in aluminum foil to prevent light penetration (Notes 5, 6, and 7).
      Note: All the following steps should be conducted in an area designed for radioactive experiments. Wear and use the appropriate personal protective equipment. Waste material must be disposed in a proper manner.
    3. Make a master mix of glucose free-MS media that is supplement with radiolabeled glucose (1 µCi ml-1 per tube) and the respective drug concentration for each treatment (Notes 8 and 9). For example, say I want to know the inhibitory effects of isoxaben at 100 nM. For 3 replications, I would create a 4x master mix. The master mix would contain 2 ml of glucose free-MS media, 2 µl of isoxaben at 100 µM, and 2 µl of 14C-glucose from the 1 mCi ml-1 stock.
    4. Transfer 0.5 ml of the master-mix to their respective Eppendorf tube and place in an Eppendorf holder. Cover the holder with aluminum foil and allow seedlings to sit in tubes for 2 h (Note 10). Shake holder every 30 min or agitate if possible.
    5. After treatment, remove unincorporated radiolabeled glucose by centrifuging for 10 min at max speed and decant supernatant. Then add 1 ml of water, centrifuged for 10 min at max speed, and decant supernatant (Note 11). Repeat twice.
    6. Bring a water bath to boil.
    7. Add 0.5 ml of acetic-nitric acid regent in each tube.
    8. Submerge closed tubes in boiling water bath for 30 min.
    9. Let samples cool, then centrifuge for 15 min at max speed. Carefully remove 400 µl of supernatant and place into a scintillation vial. This is a representative sample of the soluble cell wall fraction. While removing supernatant make sure not to disturb the pelleted insoluble fraction (Note 12).
    10. Next, wash the remaining sample to remove radioactivity that was not incorporated into the insoluble fraction. Wash each sample with 1 ml of water, centrifuged for 10 min at max speed, and carefully discard supernatant. Repeat 2 more times.
    11. Re-suspend pelleted material with 1 ml of water and transfer it to a scintillation vial. The insoluble fraction is considered cellulose (Updegraff, 1960) (Note 13).
    12. Add 10 and 5 ml of scintillation cocktail to the vials containing the insoluble and soluble material, respectively. Place vials in a scintillation counter and choose appropriate program for 14C to determine disintegrations per minute (dpm). Include a blank containing only the scintillation cocktail to determine the background radioactivity count (Note 14).
    13. Analyze the results.

Notes

  1. It is important that steps 1 through 4 are done in a sterile environment and all growth media kept sterile.
  2. It is extremely important to keep seedlings in the dark until step B11.
  3. This timing is important because the hypocotyls are rapidly elongating and require large amounts of glucose.
  4. Etiolated seedlings were placed in a seed-cleaning sieve and subsequently washed with deionized water.
  5. The amount of seedling-wet weight can change if needed; however a significant quantity of seedlings is needed to take up a statistically relevant amount of radioactivity.
  6. It is extremely important to keep your weight similar among all tubes (replication). If not, this will have a significant effect on the amount of radioactivity uptake. If you are so inclined you can present data as dpm gram-1. Each tube is an independent replication within a trial. We suggest at least using 3 analytic replications per treatment and repeat the experiment in time.
  7. The majority of excess water was drained and a subset of etiolated seedlings were placed in a petri dish. Three tubes were filled at a time then a new subset of seedlings was used. During this step we relied on the light source from the scale display as are only light source.
  8. Greater than 1 µCi ml-1 of radiolabeled glucose is required to detect radioactivity in the insoluble fraction over background noise in the scintillation counter (> 400 dpm).
  9. At minimum a 2X rate should be used, however we suggest a saturating 20X rate. X is equal to the rate that reduces hypocotyl or root growth by 50% (GR50) and should be predetermined in a dose response experiment. For example the GR50 for isoxaben is ~5 nM and thus a 100 nM concentration would be used. The compound carrier and control, DMSO, should not exceed more than 0.01% of the solution. DMSO at higher concentrations can be detrimental to seedling growth.
    The 2-hour time point is important in determining if inhibition of cellulose biosynthesis is a direct effect of the drugs mode of action.
  10. Important to use water to remove excess radioactivity, as MS media or sulfuric acid does not mix with the counting cocktail solution.
  11. 400 µl was chosen as a representative sample of the soluble fraction because the leftover solution is hard to remove without removing the potential insoluble fraction (cellulose).
  12. Transfer can be aided by cutting the tip off of a 1 ml tip and pipette with it.
  13. Important that the cocktail be >75% of the mixture otherwise the solution will become cloudy and counting will not be possible. If this happens dilute into another vial and add the count sums.
  14. This method is only applicable to 72-hour-old Arabidopsis seedlings. To measure the insoluble fraction in thicker tissue, for example grass roots, tissue should be first burned in an oxidizer. The created 14CO2 is captured and rinsed into a vial to be counted in a scintillation counter.

Recipes

  1. MS media
    1. Half MS (Murashige and Skoog) basal salt media (2.32 g/L)
    2. MES monohydrate (0.6 g/L)
    3. Adjust pH to 5.7 with 5M KOH
  2. Acetic-nitric acid reagent [acetic acid:nitric acid:water (8:1:2)]
    Add 15 ml of concentrated nitric acid to 150 ml of 80% acetic acid

Acknowledgments

The aforementioned methodology was adapted from the protocol of Heim et al. (1990) and used by Brabham et al. (2014). This work was supported by the U. S. Department of Energy (grant no. DOE–FOA 10–0000368 to S. D. and C. B.), the U. S. Department of Agriculture (Hatch Act grant to S. D. and J. S.)

References

  1. Brabham, C., Lei, L., Gu, Y., Stork, J., Barrett, M. and DeBolt, S. (2014). Indaziflam herbicidal action: a potent cellulose biosynthesis inhibitor. Plant Physiol 166(3): 1177-1185.
  2. Heim, D. R., Skomp, J. R., Tschabold, E. E. and Larrinua, I. M. (1990). Isoxaben inhibits the aynthesis of acid insoluble cell wall materials in Arabidopsis thaliana. Plant Physiol 93(2): 695-700.
  3. Updegraff, D. M. (1969). Semimicro determination of cellulose in biological materials. Anal Biochem 32(3): 420-424.

简介

纤维素由纤维素合酶A蛋白质在质膜上使用底物UDP葡萄糖合成。 本文中,我们提供了测量放射性标记的葡萄糖到细胞壁的纤维素部分中的掺入的详细方法。 在该方法中,将拟南芥幼苗在放射性标记的葡萄糖存在下用纤维素生物合成抑制剂处理2小时,随后在乙酸 - 硝酸中煮沸以溶解非纤维素材料。 在不溶性级分中检测的放射性标记的葡萄糖表示在实验时间段期间合成的纤维素的量。 该方法的短期性质是确定纤维素生物合成的抑制是否是除草剂主要作用模式的有用工具。

材料和试剂

  1. 拟南芥种子( Arabidopsis thaliana L。)
  2. 葡萄糖(Sigma-Aldrich,目录号:D9434)
  3. 放射性标记的葡萄糖[葡萄糖D-葡萄糖(U)](American Radiolabeled Chemicals,目录号:ARC 0122D)
  4. 乙酸,冰川(Certified ACS)(Fisher Scientific,目录号:A38-212)
  5. 硝酸,Fuming(Certified ACS)(Fisher Scientific,目录号:A202-212)
  6. 计数鸡尾酒(Biodegradable Scintillation Cocktails)(Biosafe II,目录号:111195)
  7. 去离子无菌水(在120℃高压灭菌30分钟)
  8. Murashige& Skoog(MS)基础盐混合物( Phyto Technology Laboratories ,目录号:M524)
  9. MES一水合物(Sigma-Aldrich,目录号:69889)
  10. 型号A307样品氧化剂(PerkinElmer,目录号:A030700)
  11. 铝箔
  12. Eppendorf管(1.5ml)(在120℃高压灭菌30分钟)
  13. MS介质(参见配方)
  14. 乙酸硝酸试剂(见配方)

设备

  1. Eppendorf管支架
  2. 巴斯德吸管和提示
  3. 1L锥形烧瓶(在120℃高压灭菌30分钟)
  4. 微观
  5. 轨道振动器
  6. 微量离心机
  7. 玻璃闪烁管(20ml)
  8. 闪烁计数器(Packard Tri-Carb液体闪烁计数器)
  9. 高压灭菌器

程序

  1. 设置(注1和2)
    1. 在1.5ml Eppendorf管中,加入拟南芥种子(〜100mg种子=〜2   g的幼苗湿重)和表面灭菌。 简而言之,在层流中 流动罩,加1ml 30%家用漂白液补充 20μl的5%v/v十二烷基硫酸钠(SDS)并摇动15分钟。 去除漂白剂上清液,种子沉降在底部 然后依次用1ml高压灭菌器洗涤三次 去离子水。 取出最终的洗涤溶液,并等分500微升 灭菌水倒入Eppendorf管中。 将种子转移至4℃ 冰箱和商店在黑暗中2天促进均匀 发芽。
    2. 准备两瓶液体MS培养基(250ml) 有和没有葡萄糖(2%w/v)。 在液体中高压灭菌 在120℃下循环30分钟。
    3. 添加种子和冷却和 灭菌的MS培养基补充2%葡萄糖到预灭菌的1   L锥形烧瓶。
    4. 放置并将烧瓶固定在轨道上 摇动并搅拌(180rpm)混合物2小时,以促进 发芽。 然后,用铝箔包裹烧瓶以防止 光穿透。 将烧瓶放回振荡器并搅拌72   H。 (注3)。

  2. 实验程序
    注意:幼苗需要保持在黑暗中从步骤B5-8。
    1. 72小时后,取出葡萄糖补充培养基并洗涤幼苗 去除所有痕量的葡萄糖(注4)。 描述幼苗如何 应该如图1所示。


      图1.黑暗生长的表示   etiolated的拟南芥幼苗,其在液体MS培养基中生长 72小时。 正方形等于1厘米。


  3. 实验程序
    注意:幼苗需要保持在黑暗中从步骤B5-8。
    1. 72小时后,取出葡萄糖补充培养基并洗涤幼苗 去除所有痕量的葡萄糖(注4)。 描述幼苗如何 应该如图1所示。


      图1.黑暗生长的表示   etiolated的拟南芥幼苗,其在液体MS培养基中生长 72小时。 正方形等于1厘米。

    2. ... Transfer 0.5 ml of the master-mix to their respective Eppendorf tube and place in an Eppendorf holder. Cover the holder with aluminum foil and allow seedlings to sit in tubes for 2 h (Note 10). Shake holder every 30 min or agitate if possible.
    3. After treatment, remove unincorporated radiolabeled glucose by centrifuging for 10 min at max speed and decant supernatant. Then add 1 ml of water, centrifuged for 10 min at max speed, and decant supernatant (Note 11). Repeat twice.
    4. Bring a water bath to boil.
    5. Add 0.5 ml of acetic-nitric acid regent in each tube.
    6. Submerge closed tubes in boiling water bath for 30 min.
    7. Let samples cool, then centrifuge for 15 min at max speed. Carefully remove 400 µl of supernatant and place into a scintillation vial. This is a representative sample of the soluble cell wall fraction. While removing supernatant make sure not to disturb the pelleted insoluble fraction (Note 12).
    8. Next, wash the remaining sample to remove radioactivity that was not incorporated into the insoluble fraction. Wash each sample with 1 ml of water, centrifuged for 10 min at max speed, and carefully discard supernatant. Repeat 2 more times.
    9. Re-suspend pelleted material with 1 ml of water and transfer it to a scintillation vial. The insoluble fraction is considered cellulose (Updegraff, 1960) (Note 13).
    10. 加入10和5毫升闪烁 混合物到含有不溶性和可溶性材料的小瓶中, 分别。 将小瓶放在闪烁计数器中,然后选择 用于 14的适当程序以确定每分钟的崩解 (dpm)。 包括只含有闪烁鸡尾酒的空白 确定背景放射性计数(注14)。
    11. 分析结果。

笔记

  1. 重要的是,步骤1至4在无菌环境中进行,并且所有生长培养基保持无菌
  2. 在步骤B11之前将幼苗保持在黑暗中是非常重要的
  3. 这个时间是重要的,因为下胚轴迅速伸长并需要大量的葡萄糖
  4. 将olated化的幼苗放置在种子清洗筛中,随后用去离子水洗涤
  5. 如果需要,幼苗 - 湿重量可以改变;然而,需要大量的幼苗来吸收统计学上相关的放射性量
  6. 保持你的重量在所有管之间相似(复制)是非常重要的。如果不是,这将对放射性摄取量具有显着影响。如果你这样倾向,你可以将数据显示为dpm gram -1 。每个试管是试验中的独立复制。我们建议至少每次治疗使用3个分析重复,并及时重复实验
  7. 排出大部分过量的水,并将一小部分的乙醇幼苗放置在培养皿中。一次填充三个管,然后使用新的子集的子集。在这个步骤中,我们依靠来自标尺显示的光源作为只有光源
  8. 需要大于1μCiml -1 sup -1的放射性标记葡萄糖来检测不溶性级分中的放射性而不是闪烁计数器中的背景噪声(> 400dpm)。
  9. 至少应使用2倍速率,但我们建议饱和20倍速率。 X等于将下胚轴或根生长减少50%(GR 50)的速率,并且应当在剂量响应实验中预先确定。例如,异恶唑的GR 50为〜5nM,因此使用100nM浓度。化合物载体和对照,DMSO,不应超过溶液的0.01%。在较高浓度的DMSO可能对幼苗生长有害。
    2小时时间点对于确定纤维素生物合成的抑制是否是药物作用模式的直接作用是重要的。
  10. 使用水去除过量的放射性,重要的是MS培养基或硫酸不与计数鸡尾酒溶液混合。
  11. 选择400μl作为可溶性级分的代表性样品,因为剩余的溶液难以除去而不除去潜在的不溶性级分(纤维素)。
  12. 转移可以通过切除1毫升的提示和移液器的尖端帮助
  13. 重要的是混合物大于混合物的75%,否则溶液将变得混浊并且计数将是不可能的。 如果发生这种情况,稀释到另一个小瓶中,并添加计数总和
  14. 该方法仅适用于72小时的拟南芥幼苗。 为了测量较厚组织(例如草根)中的不溶性级分,应首先在氧化剂中燃烧组织。 捕获所产生的CO 2 CO 2并将其冲洗到小瓶中以在闪烁计数器中计数。

食谱

  1. MS媒体
    1. 半MS(Murashige和Skoog)基础盐培养基(2.32g/L)
    2. MES一水合物(0.6g/L)
    3. 用5M KOH调节pH至5.7
  2. 乙酸 - 硝酸试剂[乙酸:硝酸:水(8:1:2)]
    将15ml浓硝酸加入到150ml 80%乙酸中

致谢

上述方法改编自Heim等人(1990)的方案,并由Brabham等人(2014)使用。这项工作得到了美国能源部的支持(批准号为DOE-FOA 10-0000368,S.D.和C.B.),美国农业部(Hatch Act grant to S.D.and J.S.)

参考文献

  1. Brabham,C.,Lei,L.,Gu,Y.,Stork,J.,Barrett,M。和DeBolt,S。(2014)。 Indaziflam除草作用:有效的纤维素生物合成抑制剂 植物生理 em> 166(3):1177-1185。
  2. Heim,D.R.,Skomp,J.R.,Tschabold,E.E。和Larrinua,I.M。(1990)。 Isoxaben抑制拟南芥中酸不溶性细胞壁物质的合成。 Plant Physiol 93(2):695-700。
  3. Updegraff,D.M。(1969)。 半定量测定生物材料中的纤维素。 Anal Biochem 32(3):420-424。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2015 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. Brabham, C., Stork, J. and Debolt, S. (2015). [14C] Glucose Cell Wall Incorporation Assay for the Estimation of Cellulose Biosynthesis. Bio-protocol 5(18): e1589. DOI: 10.21769/BioProtoc.1589.
  2. Brabham, C., Lei, L., Gu, Y., Stork, J., Barrett, M. and DeBolt, S. (2014). Indaziflam herbicidal action: a potent cellulose biosynthesis inhibitor. Plant Physiol 166(3): 1177-1185.
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