搜索

Putrescine Biosynthesis Inhibition in Tomato by DFMA and DFMO Treatment
通过DFMA 和 DFMO 抑制番茄中腐胺的生物合成   

评审
匿名评审
下载 PDF 引用 收藏 提问与回复 分享您的反馈

本文章节

Abstract

This protocol can be used to inhibit the biosynthesis of polyamines, specifically putrescine, in tomato plants grown with NH4+ as a solely N source. In general, polyamines are positively charged small metabolites implicated in physiological processes, including organogenesis, embryogenesis, floral initiation and development, leaf senescence, pollen tube growth, fruit development and ripening and participate in the response to abiotic and biotic stresses (Tiburcio et al., 2014). Polyamines are synthesized from amino acids by decarboxylation of ornithine or arginine by ornithine decarboxylase (ODC) or arginine decarboxylase (ADC), respectively (Walters, 2003). Tomato plants grown with NH4+ as the sole N source presented an increase of putrescine content in leaves (Fernández-Crespo et al., 2015). To assess the importance of putrescine accumulation, DL-α-(Difluoromethyl)arginine (DFMA) and DL-α-(Difluoromethyl)ornithine (DFMO), inhibitors of putrescine synthesis, were used as irreversible inhibitors of ADC and ODC enzymes, respectively (Fallon and Phillips, 1988), with the purpose of reducing cellular putrescine accumulation induced by NH4+ nutrition.

The inhibitor solution containing 2 mM DFMA and 5 mM DFMO was applied directly to each pot during the week prior to sample collection. Putrescine content was reduced by 35.3% in tomato plants grown with NH4+.

Background

The application of the inhibitors DFMA and DFMO was normally performed in MS medium and in vitro assays (Perez-Amador et al., 2002; Stes et al., 2011). However, we needed to test effectiveness of these inhibitors in vivo with the purpose to maintain natural growth conditions. Moschou et al. (2008) demonstrated the inhibition effect of DFMA and DFMO when applied in hydroponic cultures at 0.1 mM and 1 mM respectively. In this work, we used similar approaches with some modifications: the hydroponic culture was changed by vermiculite growing medium and the concentration applied for the inhibitors was modified.

Materials and Reagents

  1. Tomato seeds (Solanum lycopersicum Mill. cv. Ailsa Craig)
  2. Vermiculite (Asfaltex SA, TERMITA®)
  3. Potassium hydroxide (KOH) (Scharlab, catalog number: PO0275 )
  4. Potassium sulfate (K2SO4) (Scharlab, catalog number: PO0365 )
  5. Ortho-Phosphoric acid (H3PO4) (Scharlab, catalog number: AC1100 )
  6. Ammonium sulfate [(NH4)2SO4] (Avantor Performance Materials, J.T.Baker, catalog number: 4628 )
  7. Calcium sulfate dihydrate (CaSO4·2H2O) (Scharlab, cataleg number: CA0285 )
  8. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Scharlab, catalog number: MA0085 )
  9. Boric acid (H3BO3) (AppliChem, catalog number: 131015 )
  10. Manganese(II) sulfate monohydrate (MnSO4) (Scharlab, catalog number: MA0131 )
  11. Zinc sulfate heptahydrate (ZnSO4·7H2O) (Scharlab, catalog number: CI0207 )
  12. Copper(II) sulfate pentahydrate (CuSO4·5H2O) (Scharlab, catalog number: CO0101 )
  13. Molybdenum trioxide (MoO3) (Panreac, Vidrafoc, catalog number: 142791 )
  14. Sequestrene (Fe 6%) (Syngenta)
  15. 2-(N-Morpholino) ethanesulfonic acid sodium salt (MES sodium salt) (Sigma-Aldrich, catalog number: M3885 )
  16. DL-α-(Difluoromethyl)arginine (DFMA) (Santa Cruz Biotechnology, catalog number: sc-211368 )
  17. DL-α-(Difluoromethyl)ornithine hydrochloride (DFMO) (Santa Cruz Biotechnology, catalog number: sc-252762 )
  18. Distilled water
  19. Nutrient solution (see Recipes)
  20. Inhibitors mix (see Recipes)

Equipment

  1. Plant growth room (A.S.L Snijders)
  2. Pots (50 ml) (Pöppelmann, model: Serie TO )
  3. pH meter (HACH LANGE SPAIN, CRISON, model: GLP21 )

Software

  1. Statgraphics-plus software of Windows V.5 (Statistical Graphics Corp., Rockville, MD, USA)

Procedure

  1. Tomato growth conditions
    1. Tomato seeds (Solanum lycopersicum Mill. cv. Ailsa Craig) are germinated in pots (50 ml) with 100% vermiculite in a growth room under the following environmental conditions: light/dark cycle of 16/8 h, temperature of 24/18 °C, light intensity of 200 μmol m-2 s-1, and relative humidity of 60%.
    2. Seeds are irrigated twice a week with approximately 50 ml/pot of distilled water during 1 week.
    3. Seedlings are irrigated twice each week for 3 weeks with approximately 50 ml/pot of nutrient solution. N is supplied in the form of [(NH4)2SO4] 0.33 g/L.
    4. The pH of the nutrient solution is adjusted to 6.0 with 1 mM KOH.

  2. Application of inhibitor mix to tomato plants
    1. 20 uniform tomato plants grown as described above are divided into two groups (10 plants are not treated and 10 plants are treated with the inhibitor solution).
    2. 21-day-old tomato plants are treated with 2 ml of the inhibitor mix by soil drench (final concentration in each pot is DMFA 80 nM and DFMO 200 nM)
    3. 23-day-old tomato plants are treated with 4 ml of the inhibitor mix by soil drench (final concentration in each pot is DMFA 160 nM and DFMO 400 nM).
    4. 25-day-old and 27-day-old tomato plants are treated with 8 ml of the inhibitor mix (final concentration in each pot is DMFA 320 nM and DFMO 800 nM) (Figure 1). Inhibitors mix treatment does not produce changes in tomato growth (Figure 2).
    5. 24 h after the last treatment, the 3rd and 4th true leaves of treated or not treated tomato plants (Figure 3) are collected and stored at -80 °C.
    6. A pool of 10 plants is crushed in liquid N2 in a mortar and 0.2 g are further used for putrescine extraction.
    7. Three biological replicated are realized.

  3. Putrescine quantification
    1. Putrescine quantification is realized following the protocol of Sanchez-Lopez et al. (2009) by ion pair LC coupled with electrospray tandem mass spectrometry.
    2. Putrescine content is expressed as the average of three independent experiments (Fernández-Crespo et al., 2015).


      Figure 1. Application of inhibitor mix to tomato plants grown in vermiculite


      Figure 2. Tomato plants treated with inhibitor mix do not display changes in growth. A. Control and treated 21-day-old tomato plants; B. The same plants after one week of inhibitors treatment.


      Figure 3. Tomato plant with five true leaves. The arrows indicate the 3rd and 4th true leaf of tomato plants collected for putrescine quantification.

Data analysis

Statistical analysis is carried out using a one-way analysis of variance in the Statgraphics-plus software of Windows V.5 (Statistical Graphics Corp., Rockville, MD, USA). The putrescine content means are calculated and expressed with standard errors and compared using a Fisher’s least-significant difference test at the 95% confidence interval. The experiment is repeated three times.

Notes

Fernández-Crespo et al. (2015) demonstrated that tomato plants grown with nutrient solution containing NH4+ 5 mM as a solely N source displayed high putrescine content compared with control plants, to which N was provided as NO3- form [(KNO3 (4 mM) and Ca(NO3)2 (5 mM)]. Putrescine reduction (35.3%) by inhibitors application was tested in tomato plants grown with (NH4)2SO4 as N source.

Recipes

  1. Nutrient solution
    0.33 g/L (NH4)2SO4
    0.35 g/L K2SO4
    0.07 ml/L H3PO4
    0.04 g/L MgSO4·7H2O
    0.54 g/L CaSO4·2H2O
    2.86 mg/L H3BO3
    2.2 mg/L ZnSO4·7H2O
    0.11 mg/L CuSO4·5H2O
    0.15 mg/L MnSO4
    0.09 mg/L MoO3
    6.7 mg/L sequestrene (6% Fe)
    217 mg/L MES sodium salt
    Adjust pH to 6.0 with 1 mM KOH
  2. Inhibitors mix
    DL-α-(Difluoromethyl)arginine DFMA (2 mM)
    DL-α-(Difluoromethyl)ornithine hydrochloride DFMO (5 mM)
    Both compounds were dissolved in distilled water.
    Note: Both solutions (Nutrient solution and Inhibitors mix) do not need to be sterilized.

Acknowledgments

The work was supported by a grant from the Spanish Ministry of Science and Innovation (AGL2013-49023-C-2-R). This protocol has been modified from Mochou et al. (2008).

References

  1. Fallon, K. M. and Phillips, R. (1988). Polyamines in relation to growth in carrot cell cultures. Plant Physiol 88(1): 224-227.
  2. Fernandez-Crespo, E., Scalschi, L., Llorens, E., Garcia-Agustin, P. and Camanes, G. (2015). NH4+ protects tomato plants against Pseudomonas syringae by activation of systemic acquired acclimation. J Exp Bot 66(21): 6777-6790.
  3. Moschou, P. N., Paschalidis, K. A., Delis, I. D., Andriopoulou, A. H., Lagiotis, G. D., Yakoumakis, D. I. and Roubelakis-Angelakis, K. A. (2008). Spermidine exodus and oxidation in the apoplast induced by abiotic stress is responsible for H2O2 signatures that direct tolerance responses in tobacco. Plant Cell 20(6): 1708-1724.
  4. Perez-Amador, M. A., Leon, J., Green, P. J. and Carbonell, J. (2002). Induction of the arginine decarboxylase ADC2 gene provides evidence for the involvement of polyamines in the wound response in Arabidopsis. Plant Physiol 130(3): 1454-1463.
  5. Sanchez-Lopez, J., Camanes, G., Flors, V., Vicent, C., Pastor, V., Vicedo, B., Cerezo, M. and Garcia-Agustin, P. (2009). Underivatized polyamine analysis in plant samples by ion pair LC coupled with electrospray tandem mass spectrometry. Plant Physiol Biochem 47(7): 592-598.
  6. Stes, E., Biondi, S., Holsters, M. and Vereecke, D. (2011). Bacterial and plant signal integration via D3-type cyclins enhances symptom development in the Arabidopsis-Rhodococcus fascians interaction. Plant Physiol 156(2): 712-725.
  7. Tiburcio, A. F., Altabella, T., Bitrian, M. and Alcazar, R. (2014). The roles of polyamines during the lifespan of plants: from development to stress. Planta 240(1): 1-18.
  8. Walters D. (2003). Resistance to plant pathogens: possible roles for free polyamines and polyamine catabolism. New Phytol 159: 109-115.

简介

该方案可用于抑制多胺,特别是腐胺在作为单独N源生长的番茄植物中的生物合成。通常,多胺是与生理过程相关的带正电荷的小代谢物,包括器官发生,胚胎发生,花起始和发育,叶衰老,花粉管生长,果实发育和成熟,并参与对非生物和生物胁迫的应答(Tiburcio等, et al 。,2014)。通过鸟氨酸脱羧酶(ODC)或精氨酸脱羧酶(ADC)将鸟氨酸或精氨酸脱羧,从氨基酸合成多胺(Walters,2003)。用NH 4+作为唯一N源生长的番茄植物在叶中表现出腐胺含量的增加(Fernández-Crespo等人,2015) )。为了评估腐胺积累的重要性,将腐胺合成抑制剂DL-α-(二氟甲基)精氨酸(DFMA)和DL-α-(二氟甲基)鸟氨酸(DFMO)分别用作ADC和ODC酶的不可逆抑制剂Fallon和Phillips,1988),其目的是减少NH 4营养物诱导的细胞腐胺积累。在样品收集之前的一周内,将含有2mM DFMA和5mM DFMO的抑制剂溶液直接施用到每个罐中。

[背景] 应用程序(应用程序) 的抑制剂DFMA和DFMO通常在MS培养基和体外试验中进行(Perez-Amador等人,2002; Stes等人, 。,2011)。然而,我们需要测试这些抑制剂在体内的有效性以维持自然生长条件的目的。 Moschou 等。 (2008)证明了当分别以0.1mM和1mM在水培培养基中应用时DFMA和DFMO的抑制效果。在这项工作中,我们使用类似的方法与一些修改:水培培养被蛭石生长培养基改变,应用于抑制剂的浓度被修改。

材料和试剂

  1. 番茄种子( Solanum lycopersicum Mill。cv。Ailsa Craig)
  2. 蛭石(Asfaltex SA,TERMITA )
  3. 氢氧化钾(KOH)(Scharlab,目录号:PO0275)
  4. 硫酸钾(K 2 SO 4)(Scharlab,目录号:PO0365)
  5. 正磷酸(H 3 PO 4)(Scharlab,目录号:AC1100)
  6. 硫酸铵[(NH 4)2 SO 4](Avantor Performance Materials,J.T.Baker,目录号:4628)
  7. 硫酸钙二水合物(CaSO 4·2H 2 O)(Scharlab,cataleg number:CA0285)
  8. 硫酸镁七水合物(MgSO 4·7H 2 O)(Scharlab,目录号:MA0085)
  9. 硼酸(H 3 BO 3)(AppliChem,目录号:131015)
  10. 硫酸锰(II)一水合物(MnSO 4)(Scharlab,目录号:MA0131)
  11. 硫酸锌七水合物(ZnSO 4·7H 2 O)(Scharlab,目录号:CI0207)
  12. 硫酸铜(II)五水合物(CuSO 4·5H 2 O)(Scharlab,目录号:CO0101)
  13. 三氧化钼(MoO 3)(Panreac,Vidrafoc,目录号:142791)
  14. Sequestrene(Fe 6%)(Syngenta)
  15. 2-(N-吗啉代)乙磺酸钠盐(MES钠盐)(Sigma-Aldrich,目录号:M3885)
  16. DL-α-(二氟甲基)精氨酸(DFMA)(Santa Cruz Biotechnology,目录号:sc-211368)
  17. DL-α-(二氟甲基)鸟氨酸盐酸盐(DFMO)(Santa Cruz Biotechnology,目录号:sc-252762)
  18. 蒸馏水
  19. 营养液(见配方)
  20. 抑制剂混合(参见配方)

设备

  1. 植物生长室(A.S.L Snijders)
  2. 盆(50ml)(Pppppmann,型号:Serie TO)
  3. pH计(HACH LANGE SPAIN,CRISON,型号:GLP21)

软件

  1. Windows V.5的Statgraphics-plus软件(Statistical Graphics Corp.,Rockville,MD,USA)

程序

  1. 番茄生长条件
    1. 在生长室中,在以下环境条件下,在具有100%蛭石的盆(50ml)中发芽番茄种子(Emanica Solanum lycopersicum Mill。cv.Ailsa Craig):16/8小时的光/,温度为24/18℃,光强度为200μmol/m 2 - s -1 s -1,相对湿度为60%。
    2. 种子每周用约50ml /罐的蒸馏水灌溉1周。
    3. 幼苗每周用约50ml /盆营养液灌溉两次,持续3周。 N以[(NH 4)2 SO 4] 0.33g/L的形式提供。
    4. 用1mM KOH将营养液的pH调节至6.0
  2. 抑制剂混合物在番茄植物上的应用
    1. 将如上所述生长的20个一致的番茄植物分成两组(10个植物未处理,10个植物用抑制剂溶液处理)。
    2. 21天的番茄植物用2ml抑制剂混合物通过土壤灌注处理(每个盆中的最终浓度为DMFA 80nM和DFMO 200nM)
    3. 通过土壤灌注用4ml抑制剂混合物处理23天的番茄植物(每个盆中的最终浓度为DMFA 160nM和DFMO 400nM)。
    4. 用8ml抑制剂混合物(每个罐中的最终浓度为DMFA 320nM和DFMO 800nM)处理25日龄和27日龄的番茄植物(图1)。抑制剂混合处理不会产生番茄生长的变化(图2)
    5. 在最后一次处理后24小时,收集处理或未处理的番茄植物的第三和第四片真叶(图3)并储存在-80℃。
    6. 将10株植物的池在液体N 2中在研钵中压碎,并将0.2g进一步用于腐胺提取。
    7. 实现三个生物复制。

  3. 腐殖质量
    1. 按照Sanchez-Lopez等人的方案实现腐胺定量。 (2009)通过离子对LC与电喷雾串联质谱联用
    2. 腐胺含量表示为三次独立实验的平均值(Fernández-Crespo等人,2015)。


      图1.抑制剂混合物在蛭石中生长的番茄植物的应用


      图2.用抑制剂混合物处理的番茄植物不显示生长的变化。A.对照和处理的21日龄番茄植株; B.在抑制剂处理一周后的相同植物

      图3.具有五片真叶的番茄植株。箭头指示为腐胺定量收集的番茄植株的第三和第四片真叶。

数据分析

使用Windows V.5(Statistical Graphics Corp.,Rockville,MD,USA)的Statgraphics-plus软件中的单向方差分析进行统计分析。计算腐胺含量,并用标准误差表示,并使用Fisher最小显着差异检验在95%置信区间进行比较。实验重复三次。

笔记

Fernández-Crespo等人。 (2015)证明了与含有NH 4+++作为单独N源的营养溶液一起生长的番茄植物与提供N的对照植物相比显示出高的腐胺含量作为NO 3 - 形式[(KNO 3)(4mM)和Ca(NO 3) - ] (NH 4)2 SO 3(5mM)]生长的番茄植物中测试通过抑制剂应用的腐胺减少(35.3%)。 4 作为N源。

食谱

  1. 营养液
    0.33g/L(NH 4)2 SO 4 4
    0.35g/L K 2 SO 4 4/s 0.07ml/L H 3 PO 4 4/
    0.04g/L MgSO 4·7H 2 O·h/v 0.54g/L CaSO 4·2H 2 O·h/v 2.86mg/L H 3 BO
    2.2mg/L ZnSO 4·7H 2 O·h/v 0.11mg/L CuSO 4·5H 2 O·h/v 0.15mg/L MnSO 4·
    0.09mg/L MoO 3
    6.7mg/L sequestrene(6%Fe)
    217mg/L MES钠盐
    用1mM KOH将pH调节至6.0
  2. 抑制剂混和
    DL-α-(二氟甲基)精氨酸DFMA(2mM) DL-α-(二氟甲基)鸟氨酸盐酸盐DFMO(5mM) 将两种化合物溶于蒸馏水中。
    注意:两种解决方案(营养液和抑制剂混合)不需要灭菌。

致谢

这项工作得到了西班牙科学和创新部(AGL2013-49023-C-2-R)的资助。此协议已修改自Mochou 等。 (2008)。

参考文献

  1. Fallon,KM和Phillips,R。(1988)。  多胺与胡萝卜细胞培养中的生长相关。植物生理学88(1):224-227。
  2. Fernandez-Crespo,E.,Scalschi,L.,Llorens,E.,Garcia-Agustin,P.和Camanes,G.(2015)。  NH + 保护番茄植物免受丁香假单胞菌通过激活系统性获得适应性。 66(21):6777-6790。
  3. Moschou,PN,Paschalidis,KA,Delis,ID,Andriopoulou,AH,Lagiotis,GD,Yakoumakis,DI和Roubelakis-Angelakis,KA(2008)。  亚麻子外排和由非生物胁迫诱导的质外体中的氧化负责H 2 O 2亚型,/sub>在烟草中指导耐受性反应的签名。植物细胞 20(6):1708-1724。
  4. Perez-Amador,MA,Leon,J.,Green,PJ和Carbonell,J.(2002)。  诱导精氨酸脱羧酶 ADC2 基因提供了多胺参与拟南芥中伤口应答的证据。 植物生理 130(3):1454-1463
  5. Sanchez-Lopez,J.,Camanes,G.,Flors,V.,Vicent,C.,Pastor,V。,Vicedo,B.,Cerezo,M.and Garcia-Agustin,P.(2009)植物样品中的非衍生多胺分析通过与电喷雾串联质谱联用的离子对LC来进行非衍生多胺分析光谱法。植物生理学(Plant Physiol Biochem)47(7):592-598。
  6. Stes,E.,Biondi,S.,Holsters,M.和Vereecke,D。(2011)。  通过D3-型细胞周期蛋白的细菌和植物信号整合增强了拟南芥 - 红球菌 fascians 相互作用中的症状发展。 > Plant Physiol 156(2):712-725
  7. Tiburcio,AF,Altabella,T.,Bitrian,M.和Alcazar,R。(2014)。  植物寿命期间聚胺的作用:从发育到胁迫。 240(1):1-18。
  8. Walters D.(2003)。  对植物病原体的抗性:游离多胺和聚胺分解代谢的可能作用。新植物 159:109-115。
  • 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: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Fernández-Crespo, E., González-Hernández, A. I., Scalschi, L., Llorens, E., García-Agustín, P. and Camañes, G. (2016). Putrescine Biosynthesis Inhibition in Tomato by DFMA and DFMO Treatment. Bio-protocol 6(21): e1987. DOI: 10.21769/BioProtoc.1987.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。