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Strigolactones (SLs) are carotenoid-derived signaling chemicals containing two lactone moieties in their structures and induce seed germination of root parasitic plants, Striga and Orobanche spp. In the rhizosphere, SLs are essential host recognition signals not only for root parasitic plants but also for arbuscular mycorrhizal fungi. In plants, SLs play important roles as plant hormones regulating shoot and root architecture. Plants produce only trace amounts of chemically unstable SLs, which makes it difficult to determine SL contents in plant tissues. Here, we describe how to extract and quantify sorgomol and 5-deoxystrigol, major SLs produced in sorghum roots.

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Extraction and Measurement of Strigolactones in Sorghum Roots
高粱根中独脚金内酯的提取和测定

植物科学 > 植物生物化学 > 植物激素
作者: Kaori Yoneyama
Kaori YoneyamaAffiliation: Center for Bioscience Research and Education, Utsunomiya University, Utsunomiya, Japan
Bio-protocol author page: a2981
Xiaonan Xie
Xiaonan XieAffiliation: Center for Bioscience Research and Education, Utsunomiya University, Utsunomiya, Japan
Bio-protocol author page: a2982
Takahito Nomura
Takahito NomuraAffiliation: Center for Bioscience Research and Education, Utsunomiya University, Utsunomiya, Japan
Bio-protocol author page: a2983
 and Koichi Yoneyama
Koichi YoneyamaAffiliation: Center for Bioscience Research and Education, Utsunomiya University, Utsunomiya, Japan
For correspondence: yoneyama@cc.utsunomiya-u.ac.jp
Bio-protocol author page: a2984
Vol 6, Iss 6, 3/20/2016, 1692 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1763

[Abstract] Strigolactones (SLs) are carotenoid-derived signaling chemicals containing two lactone moieties in their structures and induce seed germination of root parasitic plants, Striga and Orobanche spp. In the rhizosphere, SLs are essential host recognition signals not only for root parasitic plants but also for arbuscular mycorrhizal fungi. In plants, SLs play important roles as plant hormones regulating shoot and root architecture. Plants produce only trace amounts of chemically unstable SLs, which makes it difficult to determine SL contents in plant tissues. Here, we describe how to extract and quantify sorgomol and 5-deoxystrigol, major SLs produced in sorghum roots.
Keywords: Strigolactone(独角金内酯), LC-MS/MS(LC-MS/MS), Sorgomol(sorgomol), 5-deoxystrigol(5-deoxystrigol), Quantification(量化)

[Abstract]

Materials and Reagents

  1. 50 ml Screw cap bottles (MonotaRO Co., Duran, catalog number: 371-05-20-52 )
  2. Filter paper (90 mm) (MISUMI Corporation, Toyo Roshi Kaisha Ltd, catalog number: 00011090 )
  3. pH indicator paper (Merck Millipore Corporation, catalog number: 109526 )
  4. Spin column (Merck Millipore Corporation, catalog number: UFC3 0HV 000 )
  5. Vials and caps (Chromacol, catalog number: 1030-41201 and 1030-42473 ) for LC-MS/MS analysis
  6. Sorghum (Sorghum bicolor) plants grown under P or N deficiency
    Note: Striga-tolerant sorghum cultivars may be obtained from International Crops Research Institute for the Semi-Arid Tropics (ICRISAT). We examined several sorghum “Hybrid” cultivars for their SL production and there were essentially no quantitative and qualitative differences in SL production among them.
  7. Ethyl acetate (KANTO CHEMICAL, catalog number: 14029-70 )
  8. 500 pg of d6-5-deoxystrigol (Ueno et al., 2010) added to each sample in our experiments
    Note: Internal standards if available.
  9. Anhydrous MgSO4 (KANTO CHEMICAL, catalog number: 25035-00 ) or Na2SO4 (Kanto Chemical, catalog number: 37280-00 )
  10. Acetonitrile (KANTO CHEMICAL, catalog number: 01031-2B )
  11. 0.2 M K2HPO4 (see Recipes)
  12. Acetonitrile with 0.1% acetic acid for LC-MS/MS (see Recipes)
  13. Water with 0.1% acetic acid for LC-MS/MS (see Recipes)

Equipment

  1. Funnel (45 mm diameter) (MonotaRO Co., AGJ, catalog number: 233-09-11-04 )
  2. Erlenmeyer flasks (50 ml) (Sansyo, Iwaki, catalog number: 4980FK50 )
  3. Separatory funnel (100 ml) (Iwaki, catalog number: 6402FS100R )
  4. Evaporator flasks (300 ml, 50 ml) (Sibata, catalog number: 371-13-68-34 and 371-13-68-21 )
  5. Pasteur pipette (Thermo Fisher Scientific, catalog number: 13-678-20C )
  6. Mini-benchtop centrifuge (IKA, catalog number: 969-65-03-01 )
  7. LC-MS/MS (AB Sciex, model: QTRAP 5500 )
  8. UHPLC system (Shimadzu Corporation, model: Nexera X2 )
  9. 18 column (ϕ 2.1 x 150 mm, 2.6 μm) (Phenomenex, model: Kinetex C18 )

Procedure

  1. Add 10-20 ml of ethyl acetate to 50 ml screw cap bottles and measure the weights of the bottles. Ethyl acetate volumes are adjusted to plant volumes so that all plant tissues are covered with ethyl acetate.
  2. Harvest healthy root tissues (ca. 1 g FW) from 2-4 week-old sorghum plants and immediately put them into the bottles containing ethyl acetate. It is better not to store root tissues in a freezer to avoid possible degradation of SLs. In the case of sorghum, SL production is promoted by N or P deficiency, and thus plants grown under nutrient-deficient conditions contain larger amounts of SLs. It is better to grow plants hydroponically because root tissues are easily harvested without loss. Sorghum plants grow well in hydroponic culture.
  3. Measure the weight of the bottles again to estimate the weights of collected root tissues for SL extraction.
  4. Add internal standards if available. We use d6-5-deoxystrigol as an internal standard. Amount of the internal standard should be 1/10 to 10-fold that of endogenous 5-deoxystrigol and therefore it is better to conduct a preliminary experiment to estimate levels of endogenous SL. In general 500 pg is added for 1 g FW root sample. The internal standard is dissolved in acetonitrile or ethyl acetate and added to the bottles containing ethyl acetate.
  5. Cut root tissues into small pieces (ca. 2-3 mm long) by scissors in ethyl acetate. Acetone can be used for extraction but acetone extracts need purification before LC-MS/MS analysis.
  6. Keep the bottles at 4 °C for at least 2 days. However, prolonged storage longer than a week may cause a gradual degradation of SLs.
  7. Filtrate the solution with the roots with a funnel containing the filter paper. Then, the solution is transferred into the separatory funnel. Add 10 ml of 0.2 M K2HPO4 and mix well. Check pH of aqueous (lower) phase with pH indicator paper. If aqueous phase is still acidic, repeat washing the ethyl acetate solution with 10 ml of 0.2 M K2HPO4.
  8. Collect ethyl acetate solution in an Erlenmeyer flask and add 1-2 g of anhydrous MgSO4 or Na2SO4.
  9. Transfer ethyl acetate solution by using funnel and filter paper into an evaporator flask (300 ml) and concentrate in vacuo on a rotary evaporator at below 35 °C.
  10. Dissolve the residue in a small volume of ethyl acetate (ca. 20 ml in total), transfer to a smaller evaporator flask (50 ml) and concentrate in vacuo.
  11. Dissolve the residue with 100 µl acetonitrile and transfer the sample solution to a spin column. After centrifugation at 3,000 rpm for 30 sec, transfer filtrate into vials for LC-MS/MS. These samples should be kept at or below 4 °C until use.
  12. Perform LC-MS/MS analysis (Figure 1).


    Figure 1. LC-MS/MS analysis of strigolactones in sorghum roots. Product ion scan chromatogram for sorgomol (top), 5-deoxystrigol (middle), and d6-5-deoxystrigol (bottom).

  13. LC-MS/MS analytical conditions for detection and quantification of strigolactones. Analyses of strigolactones were performed using a triple quadrapole/linear ion trap instrument (LIT) with an electrospray ionization (ESI) source and coupled to a UHPLC system.
  14. Chromatographic separation was achieved on a C18 column (ϕ 2.1 x 150 mm, 2.6 μm;) by applying acetonitrile (MeCN)-H2O contained 0.1% (v/v) acetic acid gradient to the column, starting from 35% MeCN and rising to 95% MeCN at 20.0 min. Finally, the column was equilibrated for 3 min, using this solvent composition. The column was operated at 30 °C with a flow-rate of 0.2 ml/min.
  15. MS/MS spectra were recorded in product ion scan mode using LIT. Ion source was maintained at 400 °C with curtain gas at 20 psi, collisional activated dissociation (CAD) gas at 7 psi (12 psi for LIT), ion source gas at 80 psi, and ion source gas2 at 70 psi. Ionspray voltage was set at 5,500 V in positive ion mode and -4,500 V in negative ion mode. Declustering, entrance, and collision cell exit potentials were maintained at 60, 10, and 15 V, respectively. One-fifth of the ethyl acetate extract samples dissolved in 2 μl MeCN were injected to the LC-MS/MS.
  16. The transitions of m/z 347-231, 347-233, and 347-97 were monitored for sorgomol (retention time 6.49 min); m/z 331-234, 331-216, and 331-97 for 5-deoxystrigol (retention time 12.02 min); m/z 337-240, 337-222, and 337-97 for d6-5-deoxystrigol (retention time 11.94 min) in the ESI positive mode (Xie et al., 2015).

Recipes

  1. 0.2 M K2HPO4
    34.8 g K2HPO4
    Add dH2O to 1,000 ml
    Stored at RT
  2. Acetonitrile with 0.1% acetic acid for LC-MS/MS
    Add 500 µl acetic acid to 500 ml acetonitrile
    Sonicate for a few minutes
    Stored at RT
  3. Water with 0.1% acetic acid for LC-MS/MS
    Add 500 µl acetic acid to 500 ml water
    Sonicate for a few minutes
    Stored at RT

Notes

The root content of SLs varies with nitrogen (N) and phosphorus (P) status of sorghum plant. Under N or P deficiency, root contents of sorgomol and 5-deoxystrigol would be approx. 200 and 300 pg/g root FW, respectively. These values may decrease to 1/100 when the plants are subjected to sufficient N and P. Since sorghum produces relatively large amounts of SLs, single plant (seedling) is enough for SL quantification when grown under N or P deficiency. To minimize an individual difference, it is better to perform experiments with 5 to 10 plants in triplicate.

Acknowledgements

This protocol was adapted from previously published studies, Yoneyama et al. (2013), Yoneyama et al. (2015) and Xie et al. (2015). Kaori Yoneyama was supported by a JSPS Fellowship for young scientists and JSPS Restart Postodoctoral Fellowship.

References

  1. Ueno, K., Hanada, A., Yamaguchi, S. and Asami, T. (2010). Preparation of multideuterated 5-deoxystrigol for use as an internal standard for quantitative LC/MS. J. Label Compd Radiopharm 53(13): 763-766.
  2. Xie, X., Yoneyama, K., Kisugi, T., Nomura, T., Akiyama, K., Asami, T. and Yoneyama, K. (2015). Strigolactones are transported from roots to shoots, although not through the xylem. J Pestic Sci 40(4): 214-216.
  3. Xie, X., Yoneyama, K., Kusumoto, D., Yamada, Y., Takeuchi, Y., Sugimoto, Y. and Yoneyama, K. (2008). Sorgomol, germination stimulant for root parasitic plants, produced by Sorghum bicolor. Tetrahedron Lett 49: 2066-2068.
  4. Yoneyama K., Kisugi T., Xie X., Arakawa R., Ezawa T., Nomura T. and Yoneyama K. (2015). Shoot-derived signals other than auxin are involved in systemic regulation of strigolactone production in roots. Planta 241: 687-698.
  5. Yoneyama, K., Xie, X., Kisugi, T., Nomura, T. and Yoneyama, K. (2013). Nitrogen and phosphorus fertilization negatively affects strigolactone production and exudation in sorghum. Planta 238(5): 885-894.

材料和试剂

  1. 50ml螺旋盖瓶(MonotaRO Co.,Duran,目录号:371-05-20-52)
  2. 滤纸(90mm)(MISUMI Corporation,Toyo Roshi Kaisha Ltd,目录号:00011090)
  3. pH指示剂纸(Merck Millipore Corporation,目录号:109526)
  4. 旋转柱(Merck Millipore Corporation,目录号:UFC3 0HV 000)
  5. 用于LC-MS/MS分析的小瓶和盖(Chromacol,目录号:1030-41201和1030-42473)
  6. 高粱(高粱)在P或N缺乏下生长
    注意:耐受Striga的高粱品种可从国际半干旱热带作物研究所(ICRISAT)获得。我们检查了几个高粱"杂种"品种为其SL生产,并且他们之间SL生产基本上没有定量和定性差异。
  7. 乙酸乙酯(KANTO CHEMICAL,目录号:14029-70)
  8. 在我们的实验中向每个样品中加入500pg的d 6 -5-deoxystrigol(Ueno等人,2010)
    注意:内部标准(如果有)。
  9. 无水MgSO 4(KANTO CHEMICAL,目录号:25035-00)或Na 2 SO 4(Kanto Chemical,目录号:37280-00 )
  10. 乙腈(KANTO CHEMICAL,目录号:01031-2B)

  11. 。(参见配方)。


  12. 乙腈与0.1%乙酸用于LC-MS/MS(参见配方)
  13. 含有0.1%乙酸的水用于LC-MS/MS(参见配方)

设备

  1. 漏斗(45mm直径)(MonotaRO Co.,AGJ,目录号:233-09-11-04)
  2. 锥形烧瓶(50ml)(Sansyo,Iwaki,目录号:4980FK50)
  3. 分液漏斗(100ml)(Iwaki,目录号:6402FS100R)
  4. 将蒸发烧瓶(300ml,50ml)(Sibata,目录号:371-13-68-34和371-13-68-21)
  5. 巴斯德移液管(Thermo Fisher Scientific,目录号:13-678-20C)
  6. 小台式离心机(IKA,目录号:969-65-03-01)
  7. LC-MS/MS(AB Sciex,型号:QTRAP 5500)
  8. UHPLC系统(Shimadzu Corporation,型号:Nexera X2)
  9. 18柱(φ2.1×150mm,2.6μm)(Phenomenex,型号:Kinetex C18)

程序

  1. 向50ml螺旋盖瓶中加入10-20ml乙酸乙酯,并测量瓶的重量。将乙酸乙酯体积调节至植物体积,使得所有植物组织用乙酸乙酯覆盖。
  2. 从2-4周龄的高粱植物收获健康的根组织(约1g FW),并立即将它们放入含有乙酸乙酯的瓶中。最好不要将根组织存储在冰箱中以避免SL的可能的降解。在高粱的情况下,SL生产由N或P缺乏促进,并且因此在营养缺乏条件下生长的植物含有更大量的SL。最好是水培植物,因为根组织容易收获没有损失。高粱植物在水培培养中生长良好。
  3. 再次测量瓶的重量以估计用于SL提取的收集的根组织的重量。
  4. 添加内部标准(如果可用)。我们使用d 6 -5-deoxystrigol作为内标。内标的量应该是内源性5-脱氧蓖麻油的1/10至10倍,因此最好进行初步实验以估计内源SL的水平。通常,对于1g FW根样品加入500pg。将内标溶于乙腈或乙酸乙酯中,并加入到含有乙酸乙酯的瓶中。
  5. 用剪刀在乙酸乙酯中将根组织切成小块(约2-3mm长)。丙酮可用于萃取,但丙酮萃取物需要在LC-MS/MS分析前纯化。
  6. 保持瓶子在4°C至少2天。然而,长于一周的长期储存可能导致SL的逐渐劣化。
  7. 用含有滤纸的漏斗用根过滤溶液。然后,将溶液转移到分液漏斗中。加入10ml 0.2M K 2 HPO 4并混匀。用pH指示剂纸检查水相(下)的pH。如果水相仍然是酸性的,用10ml 0.2M K 2 HPO 4水溶液重复洗涤乙酸乙酯溶液。
  8. 在锥形瓶中收集乙酸乙酯溶液,并加入1-2g无水MgSO 4或Na 2 SO 4。
  9. 通过使用漏斗和滤纸将乙酸乙酯溶液转移到蒸发烧瓶(300ml)中,并在低于35℃的旋转蒸发器上真空浓缩。
  10. 将残余物溶于少量乙酸乙酯(总共约20ml)中,转移至较小的蒸发烧瓶(50ml)中并真空浓缩。
  11. 用100μl乙腈溶解残留物,并将样品溶液转移至离心柱。在3,000rpm离心30秒后,将滤液转移到用于LC-MS/MS的小瓶中。这些样品应保持在或低于4°C,直到使用。
  12. 进行LC-MS/MS分析(图1)。


    图1.高粱根中独脚金内酯的LC-MS/MS分析。 sorgomol(顶部),5-脱氧芪醇(中间)和d 6 - 5-deoxystrigol(bottom)。

  13. 独脚金内酯的检测和定量的LC-MS/MS分析条件。独脚金内酯的分析使用具有电喷雾离子化(ESI)源并偶联至UHPLC系统的三重四极/线性离子阱仪器(LIT)进行。
  14. 在C18柱(φ2.1×150mm,2.6μm;)上通过使用含有0.1%(v/v)乙酸梯度的乙腈(MeCN)-H 2 O 2到柱上实现色谱分离,从35%MeCN开始,并在20.0分钟时升至95%MeCN。最后,使用该溶剂组合物将柱平衡3分钟。该柱在30℃下以0.2ml/min的流速操作。
  15. 使用LIT以产物离子扫描模式记录MS/MS光谱。离子源维持在400℃,窗帘气体为20psi,碰撞活化解离(CAD)气体为7psi(对于LIT为12psi),离子源气体为80psi,离子源气体2为70psi。离子溅射电压在正离子模式下设定为5500V,在负离子模式设定为-4500V。去簇,入口和碰撞单元出口电位分别维持在60,10和15V。将溶解在2μlMeCN中的1/5乙酸乙酯提取物样品注入LC-MS/MS。
  16. 监测sorgomol(保留时间6.49分钟)的m/z 347-231,347-233和347-97的跃迁; (保留时间12.02分钟)的m/z 331-234,331-216和331-97;在ESI正模式中(保留时间为11.94分钟)的d 6+ 5-脱氧立枯异丙酯(保留时间为11.94分钟)的m/z 337-240,337-222和337-97 em>等人,2015)。

食谱

  1. 0.2 M K sub 2 HPO 4
    34.8g K 2 HPO 4
    将dH <2> O添加至1,000 ml
    存储在RT
  2. 乙腈与0.1%乙酸用于LC-MS/MS
    加入500μl乙酸到500ml乙腈中 超声处理几分钟
    存储在RT
  3. 含有0.1%乙酸的水用于LC-MS/MS
    向500 ml水中加入500μl乙酸
    超声处理几分钟
    存储在RT

笔记

SL的根含量随高粱植物的氮(N)和磷(P)状态而变化。在N或P缺乏下,大豆异黄酮和5-脱氧天竺葵的根含量约为200和300pg/g根FW。当植物经受足够的N和P时,这些值可以降低到1/100。由于高粱产生相对大量的SL,当在N或P缺乏下生长时,单株植物(苗)足以用于SL定量。为了使个体差异最小化,最好一式三份用5至10株植物进行实验。

致谢

该方案改编自以前公开的研究,Yoneyama等人(2013),Yoneyama等人(2015)和Xie等人。 (2015)。 Kaori Yoneyama由JSPS青年科学家奖学金和JSPS Restart Postodoctoral Fellowship支持。

参考文献

  1. Ueno,K.,Hanada,A.,Yamaguchi,S.and Asami,T。(2010)。 制备用作定量LC/MS的内标的多氘化5-脱氧芪醇。/a> J。 Label Compd Radiopharm 53(13):763-766。
  2. Xie,X.,Yoneyama,K.,Kisugi,T.,Nomura,T.,Akiyama,K.,Asami,T.and Yoneyama,K.(2015)。 独脚金内酯从根部转移到枝条,尽管不是通过木质部。 J Pestic Sc??i 40(4):214-216
  3. Xie,X.,Yoneyama,K.,Kusumoto,D.,Yamada,Y.,Takeuchi,Y.,Sugimoto,Y.and Yoneyama,K.(2008)。 Sorgomol,根部寄生植物的萌发刺激剂,由高粱产生。 Tetrahedron Lett 49:2066-2068。
  4. Yoneyama K.,Kisugi T.,Xie X.,Arakawa R.,Ezawa T.,Nomura T.和Yoneyama K.(2015)。 除了植物生长素以外,来源于射线的信号参与根系中独脚金内酯生产的系统调节。 241:687-698。
  5. Yoneyama,K.,Xie,X.,Kisugi,T.,Nomura,T.and Yoneyama,K。(2013)。 氮磷施肥会对高粱中的独脚金内酯生产和渗出产生负面影响。 238(5):885-894。
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How to cite this protocol: Yoneyama, K., Xie, X., Nomura, T. and Yoneyama, K. (2016). Extraction and Measurement of Strigolactones in Sorghum Roots. Bio-protocol 6(6): e1763. DOI: 10.21769/BioProtoc.1763; Full Text



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