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Mar 2020

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Determination of Ureides Content in Plant Tissues
植物组织中酰脲含量的测定   

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Abstract

The ureides allantoin and allantoate are the main organic nitrogen compounds transported in several legumes, predominantly from N2 fixation. Moreover, recent studies point out a remarkable role for allantoin during several stress responses of plants other than legumes. The goal of this protocol is to determine ureides concentration in different plant tissues. Ureides are extracted from plant material by boiling it in phosphate buffer. The allantoin and allantoate present in the supernatants are subjected to alkaline-acidic hydrolysis to glyoxylate. The glyoxylate is converted into glycoxylic acid phenylhydrazone, that is then oxidized to red-colored 1,5-diphenylformazan. The absorbance of supernatants is measured using a spectrophotometer at 520 nm. Ureides concentration can be inferred by using a glyoxylate calibration curve. Ureide quantification of different tissues of Arabidopsis thaliana and soybean plants were carried out following this protocol.

Keywords: Allantoin (尿囊素), Allantoate (尿囊酸), Ureides (酰脲), Purine (嘌呤), Plants (植物)

Background

Determination of ureides is important for characterizing N2 fixation and assimilation in legume plants as well as stress and nutritional responses of non-ureidic plants like Arabidopsis (Brychkova et al., 2008; Watanabe et al., 2014; Irani and Todd, 2016 and 2018). The formation of allantoate is also useful for in vivo and in vitro determination of allantoinase activity (Duran and Todd, 2012). Techniques requiring expensive equipment, such as high-performance liquid chromatography (HPLC), are commonly used for ureide quantification while ethanol extraction is mostly employed for ureide recovery from plant tissues. The present protocol is based on flexible spectrometry that uses low volume of cheap reagents. The use of phosphate buffer instead of ethanol for ureide extraction and quantification greatly improved the reproducibility of the measurements, possibly by decreasing the interfering compounds that abound with the ethanol extraction.

Allantoin and allantoate present in plant extracts are converted to glyoxylate by alkaline-acidic hydrolysis (Vogels and Van der Drift, 1970). To carry out this protocol, three tubes (A, B and C) for alkaline and/or acid hydrolysis are prepared for each sample (Figure 1). The glyoxylate is converted into glycoxylic acid phenylhydrazone and is then oxidized by ferricyanide to form red-colored 1,5-diphenylformazan. The absorbance of supernatants is measured using a spectrophotometer at 520 nm. Allantoin content is obtained by subtracting the levels of glyoxylate resultant of allantoate degradation (tube B) from glyoxylate derived of allantoin alkaline-acid hydrolysis (tube C). Likewise, allantoate content can be inferred by the subtraction of basal glyoxylate levels (tube A) from glyoxylate converted from allantoate (tube B).

The described protocol was carried out for determine ureide concentration for different tissues, nutritional and stress conditions in Arabidopsis thaliana (Lescano et al., 2016 and 2020); and in roots and shoots of nodulating and non-nodulating soybean plants (Muñoz et al., 2016).


Figure 1. Alkaline-acidic hydrolysis of ureides

Materials and Reagents

  1. 1.5 ml microcentrifugue tubes (Eppendorf)
  2. 1.5 ml safe-lock microcentrifugue tubes (Eppendorf)
  3. 50 ml Falcon tubes
  4. Chemical resistant gloves
  5. Seeds (e.g., Arabidopsis)
  6. Growing medium [e.g., for Arabidopsis 0.5x MS (1% w/v agar) plates or soil: vermiculite (1:1) mix pots]
  7. Distillated water (dH2O)
  8. (Optional) Liquid air/N2
  9. Monopotassium phosphate (Cicarelli® Laboratories, catalog number: 1057211 )
  10. Dipotassium phosphate (Cicarelli® Laboratories, catalog number: 1015211 )
  11. Sodium hydroxide (NaOH) (Cicarelli® Laboratories, catalog number: 843211 )
  12. Hydrochloric acid (HCl) (Biopack, catalog number: 2000963208 )
  13. Phenylhydrazine (Merck, catalog number: 107251 ). Store at room temperature (RT) in the dark
  14. Potassium ferricyanide (Anedra, Research AG, catalog number: 7039 )
  15. Sodium glyoxylate monohydrate (Sigma-Aldrich, Merck, catalog number: G4502 )
  16. Extraction Buffer (see Recipes)
  17. Reaction Buffer (see Recipes)

Equipment

  1. Beaker
  2. (Optional) Mortar and pestle
  3. Plant growth chamber/greenhouse
  4. 60-65 °C incubator
  5. Analytical balance 0.5/0.0001 g (e.g., Mettler Toledo, model: AL204 )
  6. Centrifuge at 4°C with microliter rotor (e.g., Thermo Scientific, Sorval Biofuge Primo R)
  7. Ice bath
  8. Hot air bath at 100 °C
  9. Vortex mixer (e.g., Decalab)
  10. Fume hood
  11. VIS Spectrophotometer (e.g., Bioamerican Science, model: SP 2000 UV )
  12. Spectrophotometric plastic or glass VIS/UV-VIS semi-micro 0.75-1.5 ml cuvettes

Procedure

  1. Preparation of plant materials
    1. Grow plants in a chamber/greenhouse with the corresponding conditions.
      Note: For grow Arabidopsis thaliana, stratify seeds at 4 °C for 2-3 days. Sow seeds on 0.5x MS agar plates or on soil:vermiculite (1:1) mix, and transfer to a growth chamber under a 16 h light/8 h dark photoperiod at 22 °C and a light intensity of 100-150 μmol photons m-2s-1.
    2. Collect about 20 mg-0.5 g of plant samples in 1.5 ml safe-lock microcentrifugue tubes.
      Notes:
      1. For Arabidopsis harvest young, senescent and caulinar leaves, buds, flowers, siliques and dry seeds of adult plants (Figures 2A-2F) or collect young seedlings grown in 0.5x MS agar plates (Figure 2G).
      2. It is recommended to collect plant samples of similar weights between biological replicates.


      Figure 2. Examples of Arabidopsis thaliana tissues used for ureide quantification. (A) 8-week old plant. Details of (B) young leaves, (C) senescent leaves, (D) buds, (E) flowers and (F) seeds are shown. (G) 7 day-old seedlings grown on 0.5x agar plates. Scale bars = 2 cm (A-C, G), = 1 mm (D-F).

    3. Put microcentrifugue tubes with open lids in an oven at 60-65 °C overnight.
    4. Weight dry plant samples and put them back in the tubes. Samples can be stored at -20 °C.

  2. Ureide extraction from plant tissues
    1. Optional step: ground plant tissues to fine powder with a mortar and a pestle. Transfer the powder to microcentrifugue tubes.
      Note: Grinding plant samples to fine powder depends on the type of tissue and plant species. For example, it is not necessary for ureide extraction from Arabidopsis, but could be required for ureide extraction from hard tissues of other plant species, i.e., soybean (Muñoz et al., 2016).
    2. Add 0.5 ml of Extraction Buffer.
    3. Centrifuge at 17,758 x g for 5 min at 4 °C.
    4. Incubate in a hot air bath at 100 °C for 20 min. Ensure that all the tubes keep well closed.
    5. Transfer to ice bath for 1 min. Ensure that all the tubes keep well closed.
    6. Centrifuge at 17,758 x g for 5 min at 4 °C.
    7. Transfer the supernatant to a new microcentrifugue tube (not necessary safe-lock tube). Samples can be stored at -20 °C for several months, or at 4-8 °C prior to the next step.

  3. Alkaline/Acid Hydrolysis
    1. Prepare microcentrifugue tubes in which reactions A-C for each sample will be carried out, according the compound/s to be measured (Figure 1). Designate the tubes according which reaction take place in it: A, B and C.
      Notes:
      1. Prepare tubes according to the compound/s to be measured as indicated in Table 1. For example, to measure allantoin and allantoate concentrations in sample #1 tubes can be designated 1A, 1B and 1C.
      2. The use of duplicates for each sample is highly recommended.

      Table 1. Tubes preparation


    2. For the glyoxylate standard curve prepare a fresh serial dilution of 0, 2, 4, 6, 8 and 10 nmols sodium glyoxylate monohydrate in 100 µl Extraction Buffer and follow the steps corresponding to glyoxylate quantification (reaction A). It is not necessary to repeat this step once a proper glyoxylate standard curve is obtained.
      Note: It is recommended to use triplicates for each concentration of the glyoxylate standard curve.
      Follow the corresponding steps for each reaction as indicated in Table 2. Perform the same steps for the corresponding blank tubes, but adding 100 µl Extraction Buffer instead of the sample (Step C3a). In Steps C3d, C3e, C3h and C3i. ensure that all the tubes keep well closed.
      Samples can be stored at -20 °C for several months after Step C3j, or maintained at 4-8 °C to continue the protocol immediately.
      Note: It is recommended to perform the same reactions simultaneously for the analyzed samples. In the meantime, tubes of other reactions can be stored at 4-8 °C.

      Table 2. Ureide hydrolysis


  4. Spectrophotometric measurements
    Note: Perform HCl work in a fume hood, with the appropriate personal protective equipment.
    1. Prepare fresh 3mg/ml phenylhydrazine and 16 mg/ml potassium ferricyanide with dH2O in Falcon tubes. Mix the tubes content by inverting and shaking vigorously on a vortex. Store these solutions in the dark at RT.
    2. Add 100 µl phenylhydrazine 3 mg/ml. Mix on a vortex and incubate for 5 min at RT.
    3. Add 500 µl 37% HCl and mix by inversion.
    4. Add 100 µl 16 mg/ml potassium ferricyanide and mix by inversion. A pink to red color should be observed.
    5. Incubate for 10 min at RT.
    6. Measure the absorbance of 1,5-diphenylformazan at 520 nm (Abs520) at RT. One value of Abs520 is obtained for each reaction tube, namely Abs520A, Abs520B, Abs520C.
      Notes:
      1. If the Abs520 is above the linear range determined with the standard curve, make a dilution of the sample in 18.5% HCl and measure again immediately. 1/5 and 1/10 dilution are recommended (Dilution factors 5 and 10, respectively).
      2. Make a table to write the obtained data for each sample: Fresh weight, Dry weight, Dilution factor, Abs520A, Abs520B, Abs520C.

Data analysis

  1. Determine values of the intercept and slope of the standard curve. A typical glyoxylate standard curve is shown in Figure 3.


    Figure 3. A representative glyoxylate standard curve. The standard curve is generated by performing a serial dilution of sodium glyoxylate monohydrate. Intercept = 0.0006; Slope = 0.0214; R2 = 0.9998 (10 nmol point was not taken in account for calculation).

  2. Calculate glyoxylate content (G) in tubes A, B and C using the formula [Abs520 – intercept]/slope to obtain values GA, GB and GC.
  3. Ureide content in the sample can be calculated using the following equation:
    total ureide content (nmol) = [GC-GA] = [Allantoin content + Allantoate content]
    allantoin content (nmol) = [GC-GA]
    allantoate content (nmol) = [GB-GA]
  4. Determine ureide concentration of the sample (nmol/mg dry weight) using the formula:
    total ureide concentration = [total ureide content (nmol)] × [dilution factor]/[dry weight (mg)]
    allantoin concentration = [allantoin content (nmol)] × [dilution factor]/[dry weight (mg)]
    allantoate concentration = [total allantoate content (nmol)] × [dilution factor]/[dry weight (mg)]
  5. Report ureide concentrations of the analyzed material/s as nmol/mg dry weight (DW), e.g., allantoin and allantoate concentrations in different Arabidopsis thaliana tissues are shown in Figure 4. The described protocol was also used for determine changes in ureide concentration in shoots of Arabidopsis plants grown under different nutritional and stress conditions (Lescano et al., 2016 and 2020). Allantoin concentration was 18.2 nmol/mg in the roots and 6.3 nmol/mg in the shoots of 2 week-old Arabidopsis roots (Lescano et al., 2016, Figure 8). Allantoin and allantoate concentration from xylem sap of 6 week-old Arabidopsis were 0.27 ± 0.07 nmol/μl and 0.09 ± 0.08 nmol/μl, respectively. This protocol was also carried out for determine ureide concentration in roots and shoots of nodulating and non-nodulating soybean plants (Muñoz et al., 2016).


    Figure 4. Ureide concentration in different Arabidopsis tissues. Allantoin and allantoate content of different organs and tissues of adult plants and 7 day-old whole seedlings. DW = Dry weight. Asterisks indicates significant differences in comparison to young leaves samples (P < 0.05, Dunn’s test)

Recipes

  1. Reaction Buffer (0.4 M potassium phosphate buffer)
    1. Prepare 500 ml 0.4 M monopotassium phosphate and 500 ml 0.4 M dipotassium phosphate by dissolving the corresponding amount of salts in dH2O
    2. Put 200 ml of 0.4 M dipotassium phosphate in a beaker
    3. Bring the solution to pH 7 by adding as much as needed of 0.4 M monopotassium phosphate. The resulting solution is 0.4 M potassium phosphate buffer. Store Reaction Buffer at 4-8 °C and stock solutions at -20 °C
  2. Extraction Buffer (25 mM potassium phosphate buffer)
    1. Put 31.25 ml Reaction Buffer in a beaker
    2. Bring the solution to pH 7 with 0.5 N HCl
    3. And add dH2O to give a final volume of 500 ml

Acknowledgments

The present procedures are derived from the work of Lescano et al. (2016) and Lescano et al. (2020). This work was supported by the National Fund of Science and Technology (FONCyT, Argentina) [PICT-2009-0114] and of the Secretary of Science and Technology of the National University of Córdoba (SECyT-UNC, Argentina). The author gratefully acknowledges to Biol. María Laura Rojas for the critical revision of this protocol.

Competing interests

There are no conflict of interests.

References

  1. Brychkova, G., Alikulov, Z., Fluhr, R. and Sagi, M. (2008). A critical role for ureides in dark and senescence-induced purine remobilization is unmasked in the Atxdh1 Arabidopsis mutant. Plant J 54(3): 496-509.
  2. Duran, V. A. and Todd, C. D. (2012). Four allantoinase genes are expressed in nitrogen-fixing soybean. Plant Physiol Biochem 54: 149-155. 
  3. Irani, S. and Todd, C. D. (2016). Ureide metabolism under abiotic stress in Arabidopsis thaliana. J Plant Physiol 199: 87-95. 
  4. Irani, S. and Todd, C. D. (2018). Exogenous allantoin increases Arabidopsis seedlings tolerance to NaCl stress and regulates expression of oxidative stress response genes. J Plant Physiol 221: 43-50.
  5. Lescano, C. I., Martini, C., González, C. A. and Desimone, M. (2016). Allantoin accumulation mediated by allantoinase downregulation and transport by Ureide Permease 5 confers salt stress tolerance to Arabidopsis plants. Plant Mol Biol 91(4-5): 581-595. 
  6. Lescano, I., Bogino, M. F., Martini, C., Tessi, T. M., González, C. A., Schumacher, K. and Desimone, M. (2019). Arabidopsis thaliana Ureide Permease 5 (AtUPS5) connects cell compartments involved in Ureide metabolism. Plant Physiol: pp.01136.2019.
  7. Muñoz, N., Qi, X., Li, M. W., Xie, M., Gao, Y., Cheung, M. Y., Wong, F. L. and Lam, H. M. (2016). Improvement in nitrogen fixation capacity could be part of the domestication process in soybean. Heredity (Edinb) 117(2): 84-93. 
  8. Vogels, G. D. and Van der Drift, C. (1970). Differential analyses of glyoxylate derivatives. Anal Biochem 33(1): 143-157. 
  9. Watanabe, S., Matsumoto, M., Hakomori, Y., Takagi, H., Shimada, H. and Sakamoto, A. (2014). The purine metabolite allantoin enhances abiotic stress tolerance through synergistic activation of abscisic acid metabolism. Plant Cell Environ 37(4): 1022-1036.

简介

[摘要 ] 的酰脲尿囊素和尿囊酸是主要的有机氮化合物,在若干豆类,主要选自N 2 fixation.More,最近的研究指出期间比豆类其它植物的几个应激反应为尿囊素一个显着的作用。这样做的目的通过在磷酸盐缓冲液中煮沸从植物材料中提取尿素,然后将多糖中存在的尿囊素和尿囊酸酯进行碱酸水解以生成乙醛酸,将乙醛酸酯转化为协议,以确定不同植物组织中的尿素浓度。 酸苯腙Glycoxylic ,其随后被氧化成红色的1 ,5 -Diphenylformazan。吸光率上清测量用分光光度计在520牛米。酰脲含量可以推断使用乙醛酸校准曲线。酰脲定量不同组织的拟南芥按照该方案进行了拟南芥和大豆植株。

[背景 ] 测定尿素对于表征豆类植物中的N 2 固定和同化以及拟南芥等非尿素植物的胁迫和营养反应非常重要(Brychkova 等人,2008;Watanabe 等人,2014;Irani和Todd, 2016年和2018年)。尿囊素的形成也可用于体内和体外测定尿囊素酶的活性(Duran和Todd,2012年)。需要昂贵设备的技术,例如高效液相色谱(HPLC),通常用于尿素的定量分析,而乙醇提取主要用于从植物组织中回收尿素。本方案基于灵活的光谱分析方法,该方法使用少量廉价试剂,使用磷酸盐缓冲液代替乙醇进行尿素的提取和定量分析大大提高了可重复性。测量,PO ssibly通过减少与乙醇萃取比比皆是干扰化合物。

植物提取物中存在的尿囊素和尿囊酸酯通过碱-酸水解转化为乙醛酸酯(Vogels和Van der Drift,1970)。为执行此方案,准备了三个用于碱和/或酸水解的试管(A,B和C)。对于每个样品(图1)。该乙醛酸转化成glycoxylic酸苯腙,然后通过铁氰化物氧化形成红色1 ,5 滴的-diphenylformazan.The吸光度使用在520 nm.Allantoin内容分光光度计测定得到通过从尿囊素碱酸水解得到的乙醛酸酯中减去尿囊酸酯降解所产生的乙醛酸酯含量(试管B)(试管C),同样地,从由乙醛酸转化而来的乙醛酸酯中减去基础乙醛酸酯含量(试管A),可以推断出尿囊素的含量。尿囊酸盐(管B)。

所描述的方法进行操作用于确定用于不同的组织,在营养和胁迫条件酰脲浓度拟南芥(Lescano 等人,2016和2020);和在根和枝条的结瘤和非结节化的大豆植物(谢穆尼奥斯等。,2016)。



D:\ Reformatting \ 2020-4-7 \ 2003093--1427 CarlosLescano 861624 \ Figs jpg \ Fig。1.jpg

图1.碱性酸性水解尿素

关键字:尿囊素, 尿囊酸, 酰脲, 嘌呤, 植物

材料和试剂



 




1. 1.5毫升微量离心管(Eppendorf)      




2. 1.5毫升安全锁定微量离心管(Eppendorf)      




3. 50毫升猎鹰管      




4. 耐化学手套      




5. 种子(例如,拟南芥)。      




6. 生长培养基[ 例如,对于拟南芥0.5 x MS(1%w / v琼脂)平板或土壤:ver石(1:1)混合盆]      




7. 蒸馏水(dH 2 O)      




8. (ø Ptional)液态空气/ N 2      




9. 磷酸二氢钾(西卡雷利® 实验室,目录号:1057211)      




10. 磷酸氢二钾(西卡雷利® 实验室,目录号:1015211)   




11. 氢氧化钠(氢氧化钠)(西卡雷利® 实验室,目录号:843211)   




12. 盐酸(盐酸)(Biopack ,目录号:2000963208)   




13. 苯肼(默克公司,目录号:107251 )。在暗处室温下保存   




14. 钾的铁氰化物(Anedra ,研究AG,目录号:7039)   




15. 一水合乙醛酸钠(Sigma-Aldrich,Merck,目录号:G4502)   




16. 提取缓冲液(请参见配方)   




17. 反应缓冲液(请参见食谱)   




 




设备




 




烧杯
(Ø Ptional)杵臼
植物生长室/ 温室
60-65°C恒温箱
分析天平0.5 / 0.0001 g(例如,Mettler Toledo,型号:AL204)
用微升转子在4°C下离心(例如,Thermo Scientific,Sorval Biofuge Primo R)
冰浴
100°C的热风浴
涡流混合器(例如,Decalab )
通风柜
VIS分光光度计(例如,Bioamerican Science,型号:SP 2000 UV)
分光光度法塑料或玻璃VIS / UV-VIS半微量0.75-1.5 ml比色皿
 




程序




 




准备植物材料
在具有相应条件的温室/温室中种植植物。
注意:F 或生长拟南芥,将种子在4 °C下分层2-3 天,将种子播种在0.5 x MS琼脂平板上或土壤:ver 石(1:1)混合物中,并在16 h 下转移至生长室光照/ 8 小时黑暗光周期在22 ℃和100的光强度- 0.99 微摩尔光子米-2 小号-1 。




在1.5 ml安全锁微量离心管中收集约20 mg-0.5 g植物样品。
笔记:




对于拟南芥收获年轻,衰老和caulinar 叶,芽,花,长角果和成年植物的干种子(图小号2A- 2 F)或在收集0.5生长幼苗X 中号小号琼脂板(图2G)。
建议在生物学重复之间收集相似重量的植物样品。
 




D:\ Reformatting \ 2020-4-7 \ 2003093--1427 CarlosLescano 861624 \ Figs jpg \ Fig。2 .jpg




图2用于量化尿素的拟南芥组织示例(A)8周龄植物(B)幼叶,(C)衰老叶,(D)芽,(E)花和(F)种子的详细信息(G)7天大的幼苗在0.5 x 琼脂平板上生长,比例尺s = 2 cm(AC,G),= 1 mm(DF)。




 




将带盖的微量离心管放在60-65°C的烤箱中过夜。
将干燥的植物样品称重并放回试管中,样品可在-20°C下保存。
 




从植物组织中提取尿素
可选步骤:用研钵和研杵将植物组织研磨成细粉,然后将其转移至微量离心管中。
注意:将植物样品研磨成细粉取决于组织和植物种类的类型。例如,从拟南芥中提取尿素不是必需的,但从其他植物种类的硬组织(如大豆( Muñozet al。,2016)。




加入0.5 ml提取缓冲液。
在4°C下以17,758 xg离心5分钟。
在100°C的热空气浴中孵育20 分钟,确保所有试管保持密闭状态。
转移至冰浴中1分钟,确保所有试管保持密闭。
在4°C下以17,758 xg离心5分钟。
将补品转移至新的微量离心管中(无需使用安全锁定管),样品可在-20°C下保存数月,或在下一步之前在4-8°C下保存。








碱/酸水解
准备微量离心管,根据要测量的化合物(图1)对每个样品进行AC反应(图1),并根据其中发生反应的方式指定试管。
笔记:




根据表1中所示的待测化合物制备试管。例如,要测量样品#1中的尿囊素和尿囊酸盐浓度,可以将试管命名为1A,1B和1C。
强烈建议对每个样本使用重复项。
 




表1.管的准备




待测化合物




一个









C




尿囊素




--




X




X




尿囊酸盐




X




X




--




尿囊素和尿囊酸




X




X




X




总尿素




X




--




X




 




对于乙醛酸标准曲线,在100μl提取缓冲液中准备新的系列稀释的0、2、4、6、8和10 nmols乙醛酸钠一水合物,并按照与乙醛酸定量相对应的步骤进行(反应A)。一旦获得正确的乙醛酸酯标准曲线,就执行该步骤。
注意:对于乙醛酸标准曲线的每个浓度,建议使用一式三份。




按照相应的步骤,每个反应所表示。在表2 。执行相同的步骤为相应的空管,但增加100 Myueru提取液代替样品(步骤C3a的)。在步骤的C3d,C3E,反应3h和C3I系统。确保这所有管子保持良好密封。




样品可以在步骤C3j之后在-20°C下保存几个月,或保持在4-8°C下以立即继续操作。




注意:建议对被分析的样品同时进行相同的反应,同时,其他反应的试管可以在4-8°C下保存。












表2. 尿素水解




试剂/管




一个









C




  样品
100微升




100微升




100微升




卫生署2 Ø
400微升




300微升




200微升




  0.5 N Na(OH)
--




--




100微升




在100 °C下孵育
--




--




8分钟




  继续冰浴
1分钟




1分钟




1分钟




   0.65 N盐酸
--




--




100微升




0.15 N盐酸
--




100微升




--




在100 °C下孵育
--




4分钟




4分钟




   继续冰浴
1分钟




1分钟




1分钟




   反应缓冲液
100微升




100微升




100微升




 




分光光度法测量
注意:使用适当的个人防护设备,在通风橱中进行HCl工作。




在Falcon管中准备新鲜的3mg / ml 苯肼和16mg / ml含dH 2 O的铁氰化钾,将其倒转并在涡旋中剧烈摇晃混合,然后将其在黑暗中室温保存。
加入100 µl 3 mg / ml 苯肼,涡旋混合并在室温下孵育5分钟。
加入500 µl 37%HCl,然后颠倒混合。
加入100 µl 16 mg / ml铁氰化钾并颠倒混合,应观察到粉红色至红色。
在室温下孵育10分钟。
吸光率测量1 ,5 -Diphenylformazan在520nm吸光度(Abs 520 )在RT下,一个值的ABS 520 获得用于每个反应管,即阿布斯520 甲,ABS 520 B,阿布斯520 Ç 。
笔记:




如果Abs 520 超出标准曲线确定的线性范围,请在18.5%HCl中稀释样品并立即再次测量。建议使用1/5和1/10稀释(分别为5和10稀释因子)。
                                                                                                                                                                        制作表格以写下每个样品的获得数据:鲜重,干重,稀释系数,Abs 520 A,Abs 520 B,Abs 520C 。
 




数据分析




 




确定标准曲线的截距和斜率值。典型的乙醛酸酯标准曲线如图3 所示。
 




D:\ Reformatting \ 2020-4-7 \ 2003093--1427 CarlosLescano 861624 \ Figs jpg \图3.jpg




图3. 代表性的乙醛酸盐标准曲线通过连续稀释乙醛酸钠一水合物生成标准曲线,截距= 0.0006;斜率= 0.0214; R 2 = 0.9998(计算时未考虑10 nmol点)。




 




乙醛酸含量计算(G)在管A,B和C使用式[阿布斯520 -截] /斜率以获得值g 甲,G 乙和G Ç 。
样品中的尿素含量可以使用以下公式计算:
总脲含量(nmol)= [G C -G A ] = [ 尿囊素含量+ 尿囊酸盐含量]




尿囊素含量(nmol)= [G C -G A ]




尿囊含量(纳摩尔)= [G 乙-G 甲]




使用以下公式确定样品中的尿素浓度(nmol / mg干重):
总脲含量= [总脲含量(nmol)] × [稀释因子] / [干重(mg)]




尿囊素浓度= [尿囊素含量(nmol)] × [稀释因子] / [干重(mg)]




尿囊酸浓度= [ 尿囊酸总含量(nmol )] × [稀释系数] / [干重(mg)]




报告酰脲含量的分析材料/ S以nmol / mg干重(DW),E. G. ,尿囊素和尿囊浓度不同的拟南芥组织见图4 。所描述的协议也被用于确定修改在酰脲在不同营养和胁迫条件下生长的拟南芥植物芽中的浓度(Lescano 等人,2016和2020).2周龄拟南芥根中,尿囊素的浓度分别为18.2 nmol / mg和6.3 nmol / mg (Lescano 等人,2016 ,图8)。尿囊素和尿囊酸浓度为从木质部汁液中6周龄拟南芥是0.27±0.07 纳摩尔/ Myueru 和0.09±0.08 纳摩尔/ Myueru 分别此协议还进行了有关确定酰脲结瘤和非结瘤大豆植物的根和茎中的浓度(Muñoz 等,2016)。   
 




D:\ Reformatting \ 2020-4-7 \ 2003093--1427 CarlosLescano 861624 \ Figs jpg \ Fig.4.jpg




图Ure 4。不同拟南芥组织中的尿素浓度。成年植物和7 日龄整苗的不同器官和组织中尿囊素和尿囊素的含量。DW =干重。星号表示与幼叶样品相比有显着差异(P < 0.05 ,邓恩的测试)




 




菜谱




 




反应缓冲液(0.4 M磷酸钾缓冲液)
通过将相应量的盐溶解在dH 2 O中来制备500 ml 0.4 M磷酸一钾和500 ml 0.4 M磷酸二钾
将200 ml 0.4 M 磷酸二钾放入烧杯中
加入所需量的0.4 M磷酸二氢钾使溶液的pH值达到7,得到的溶液为0.4 M磷酸钾缓冲液。将反应缓冲液储存在4-8° C并将储备溶液储存在-20°C
提取缓冲液(25 mM磷酸钾缓冲液)
将31.25 ml反应缓冲液放入烧杯中
用0.5 N HCl将溶液调至pH 7
并添加dH 2 O,最终体积为500 ml
 




致谢




 




本程序来自Lescano 等人(2016)和Lescano 等人(2020 )的工作。这项工作得到了国家科学技术基金(FONCyT ,阿根廷)[PICT-2009-0114]和作者感谢科尔多瓦国立大学科学技术部长(阿根廷SECyT -UNC)。作者对玛丽亚·劳拉·罗哈斯(Biol。MaríaLaura Rojas)对该协议的重要修订表示感谢。












利益争夺




 




没有利益冲突。




 




参考文献




 




Brychkova ,G。,Alikulov ,Z。,Fluhr ,R。和Sagi ,M。(2008)。在Atxdh1 拟南芥突变体中,未发现脲在黑暗和衰老诱导的嘌呤迁移中的关键作用。 植物J 54(3) :496-509。
Duran,VA和Todd,CD(2012)。四个尿囊素酶基因在固氮大豆中表达,《植物生理生化》54:149-155。              
。伊拉尼,S.和Todd,CD(2016)在非生物酰脲代谢应激在拟南芥拟南芥。 Ĵ植物生理学199:87-95。              
伊朗,S.和托德,CD(2018)。外源尿囊素增加拟南芥幼苗对NaCl胁迫的耐受性并调节氧化应激反应基因的表达。植物生理学杂志221:43-50。
Lescano,CI,尼,C.,冈萨雷斯á 勒莱,CA和德西蒙,M。(2016).Allantoin积累由allantoinase下调和运输由酰脲通透5所赋予盐胁迫耐受性来介导的拟南芥植物。植物分子生物学91(4- 5):581-595。              
Lescano,I.,Bogino,MF,尼,C.,Tessi,TM,冈萨雷斯á 勒莱,CA,舒马赫,K。和德西蒙,M。(2019).Arabidopsis 拟南芥酰脲通透5连接参与(AtUPS5)细胞区室尿素代谢。植物生理学:pp.01136.2019。
Mu N Oz,N.,Qi,X.,Li,MW,Xie ,M.,Gao ,Y.,Cheung,MY,Wong,FL和Lam,HM(2016)。固氮能力的提高可能是其中的一部分大豆的驯化过程。遗传(Edinb )117(2):84-93。              
VOGELS ,GD和van der漂移,C.(1970)差分分析乙醛酸衍生物。分析生物化学33(1):143-157。              
渡边,S.,松本,M.,Hakomori ,Y。,高木,H.,岛田,H。和坂,A。(2014).The嘌呤代谢物尿囊素增强的非生物胁迫通过脱落酸代谢的协同活化的耐受性。植物细胞环境37(4):1022-1036。

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Copyright: © 2020 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. Lescano, I. (2020). Determination of Ureides Content in Plant Tissues. Bio-protocol 10(11): e3642. DOI: 10.21769/BioProtoc.3642.
  2. Lescano, I., Bogino, M. F., Martini, C., Tessi, T. M., González, C. A., Schumacher, K. and Desimone, M. (2019). Arabidopsis thaliana Ureide Permease 5 (AtUPS5) connects cell compartments involved in Ureide metabolism. Plant Physiol: pp.01136.2019.
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