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Determination of soluble sugars is basic for the study of carbon metabolism in plants. Soluble sugar quantitation can be achieved by enzymatic methods implying different coupled reactions. Here we describe a simple method that allows rapid determination of the most abundant soluble sugars (glucose, fructose and sucrose) in Arabidopsis leaves by anion exchange chromatography. We have applied this method to study the levels of soluble sugars during the photoperiodic transition to flowering (Ortiz-Marchena et al., 2014).

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Determination of Soluble Sugars in Arabidopsis thaliana Leaves by Anion Exchange Chromatography
阴离子交换色谱法测定拟南芥中的可溶性糖

植物科学 > 植物新陈代谢 > 糖类
作者: M. Isabel Ortiz-Marchena
M. Isabel Ortiz-MarchenaAffiliation: Plant Development Unit. Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas and Universidad de Sevilla, Seville, Spain
Bio-protocol author page: a1856
M. Teresa Ruiz
M. Teresa RuizAffiliation: Plant Development Unit. Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas and Universidad de Sevilla, Seville, Spain
Bio-protocol author page: a1857
Federico Valverde
Federico ValverdeAffiliation: Plant Development Unit. Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas and Universidad de Sevilla, Seville, Spain
Bio-protocol author page: a1859
 and José M. Romero
José M. RomeroAffiliation: Plant Development Unit. Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas and Universidad de Sevilla, Seville, Spain
For correspondence: jmromero@us.es
Bio-protocol author page: a1858
Vol 4, Iss 23, 12/5/2014, 4042 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1317

[Abstract] Determination of soluble sugars is basic for the study of carbon metabolism in plants. Soluble sugar quantitation can be achieved by enzymatic methods implying different coupled reactions. Here we describe a simple method that allows rapid determination of the most abundant soluble sugars (glucose, fructose and sucrose) in Arabidopsis leaves by anion exchange chromatography. We have applied this method to study the levels of soluble sugars during the photoperiodic transition to flowering (Ortiz-Marchena et al., 2014).
Keywords: Sugar determination(糖的测定), Soluble sugars(可溶性糖), Arabidopsis(拟南芥), HPLC(高效液相色谱法)

[Abstract]

Materials and Reagents

  1. Plants grown in soil for 3 weeks
    Note: Treatment of samples is explained in the Procedure section.
  2. Liquid N2
  3. Absolute ethanol
  4. HEPES (Sigma-Aldrich, catalog number: H4034-1KG )
  5. KOH (Panreac Applichem, catalog number: 121515 )
  6. NaOH 50% (w/v) solution (AppliChem GmbH, catalog number: A3720, 1000 )
  7. Milli Q grade water
  8. 100 mM HEPES-KOH (see Recipes)
  9. Extraction buffer 1 (EB1) (see Recipes)
  10. 0.1 M NaOH (see Recipes)
  11. 21.6 mM NaOH (see Recipes)

Equipment

  1. Small mortar and pestle
  2. 1.5 ml microfuge tubes
  3. Automatic pipettes
  4. Precision scale
  5. 2 ml microcentrifuge (Eppendorf, model: 5415R )
  6. Vacufuge concentrator 5301 (Eppendorf)
  7. Nylon filters (Whatman, catalog number: UN203NPENYL )
  8. Dionex HPLC system (Dionex ICS 5000)
  9. CarboPacPA10 column (4 x 250 mm) (Thermo Fisher Scientific, catalog number: 046110 )
  10. CarboPacPA10 Pre-column (4 x 50 mm) (Thermo Fisher Scientific, catalog number: 046115 )
  11. Gold electrode (Dionex ICS 5000)
  12. Computer connected to HPLC system

Software

  1. Chromeleon software (v.7.0) (Thermo Fisher Scientific)

Procedure

  1. Collect leaf sample (or plant material) and freeze immediately in liquid N2. Stored at -80 °C until use.
  2. Grind plant tissue with mortar and pestle in the presence of liquid N2 until the sample is converted to a fine powder.
  3. Weigh 100-300 mg leaf powder into a microfuge tube and add extraction buffer 1. [The ratio of powered tissue/EB1 should be 1:1 (w/v).]
  4. Heat the samples at 80 °C for 2 h to extract soluble sugars. Allow them to cool and centrifuge at 15,000 x g (13,000 rpm in a microfuge) 10 min.
  5. Remove supernatant to a fresh microfuge tube.
  6. The supernatant is evaporated in vacuum at 45 °C for 2 h.
  7. Resuspend the dried extract in Milli Q grade water. [The ratio of water/original powdered tissue should be 0.5:1 (v/w).]
  8. Keep on ice while preparing all samples. Freeze the samples at -20 °C for long storage, when necessary.
  9. Clean soluble plant extracts by filtering through nylon filters. Suitable extract volumes (typically 50 µl) were made up to 200 µl with Milli Q grade water and filtered through Whatman Mini-UniprepTM nylon filters (0.2 µm pore size).
    1. Add 250 µl 21.6 mM NaOH to 50 µl filtered extracts and apply into a Dionex HPLC system. Set a 10 µl injection volume.
    2. Sugars are separated by isocratic elution at 20 °C with 18 mM NaOH as mobile phase at a flow of 1 ml/min [18% (v/v) 0.1 M NaOH and 82% (v/v) Milli Q grade water] through a CarboPac PA10 column and pre-column CarboPacPA10. Detection is carried out by amperometric detection with a gold electrode in nC units.
    3. The chromatograms are analysed using the Chromeleon software. Peaks are identified by their retention time in comparison with known standard sugars. The peak area is calculated by integrating the area between the start and the end of a peak (nC x min) using the Chromeleon software. A range of sugar mixtures of known concentrations (1, 0.5, 0.1, 0.05 and 0.01 mM; as an example, see the 0.5 mM standard profile in Figure 1A) is applied into the HPLC system, as described above, to generate calibration curves for each sugar, relating peak area (as determined by the software) with concentration. The amount of a certain sugar is calculated comparing its peak area (Figure 1B) with those in the calibration curves. Sugar content in samples is expressed in micromoles of sugar per gram fresh weight.


      Figure 1. Sugar elution profiles in typical chromatograms. A. Sugar standards: Mixture of trehalose (peak 1), galactose (peak 2), glucose (peak 3), fructose (peak 4) and sucrose (peak 5) at 0.5 mM each. B. Plant extract showing glucose (1), fructose (2) and sucrose (3) peaks. Retention times are indicated for each peak. nC: 10-9 C.

Representative data

  1. Between working days and during weekends, columns and pre-columns were washed with 0.2 M NaOH at 0.1 ml min-1 flow. After this treatment, they were equilibrated with 18 mM NaOH at 1 ml/min flow for at least one hour prior to sample loading. In order to monitor the reproducibility of the different analysis, aliquots of the 0.5 mM sugar standard mixture were analysed, as mentioned above, at the beginning and at the end of each sample batch.

    Table 1. Typical area values for the glucose, fructose and sucrose peaks in the 0.5 mM sugar standard aliquots (n=18)
    Area (nC x min)
    Glucose
    Fructose
    Sucrose
    Mean
    20.5
    4.3
    8.4
    SD
    1.2
    0.4
    0.9

    The peak area values from a representative plant extract (i.e. Figure 1B) were 8.3, 0.5 and 2.2 nC x min for glucose, fructose and sucrose, respectively. These values could differ among samples, as expected since they were obtained in different experimental conditions, but they always fell into the measurable range corresponding to the calibration curve.

Notes

  1. The use of Dionex equipment (or similar chromatographers containing plastic connections and tubing) is mandatory, as regular HPLC systems are not compatible with NaOH solutions.
  2. A helium stream should be used in order to degasify the mobile phase.

Recipes

  1. 100 mM HEPES-KOH (pH 7.7)
    Weigh 23.83 g HEPES
    Adjust pH to 7.7 with KOH
    Add Milli Q grade water to 1 L
    Stored at 4 °C
  2. Extraction buffer 1 (EB1)
    Mix 20 ml 100 mM HEPES-KOH (pH 7.7) with 80 ml EtOH 100% (v/v)
    Stored at RT
  3. 0.1 M and 0.2 M NaOH
    Dilute 5.25 ml or 10.5 ml (respectively) NaOH 50% (w/v) with Milli Q grade water to 1 L
    Stored at RT for no longer than 2 weeks due to carbonation
  4. 21.6 mM NaOH
    Dilute 2.16 ml 0.1 M NaOH with Milli Q grade water to 10 ml
    Stored at RT for no longer than 2 weeks due to carbonation

Acknowledgments

This work was performed with funding from projects CSD2007-00057, BIO2008-02292, and BIO2011-28847-C02-00 (Spanish Ministry of Economy and Competitiveness, MINECO) and Excellence projects P06-CVI-01450 and P08-AGR-03582 (Junta de Andalucía) partially supported by FEDER funding to F.V. and J.M.R. We also acknowledge the TRANSPLANTA consortium, Project CONSOLIDER 28317 (MINECO).

References

  1. Ortiz-Marchena, M. I., Albi, T., Lucas-Reina, E., Said, F. E., Romero-Campero, F. J., Cano, B., Ruiz, M. T., Romero, J. M. and Valverde, F. (2014). Photoperiodic control of carbon distribution during the floral transition in Arabidopsis. Plant Cell 26(2): 565-584.

材料和试剂

  1. 在土壤中生长3周的植物
    注意:在程序部分中对样品的处理进行了说明。
  2. 液体N <2>
  3. 绝对乙醇
  4. HEPES(Sigma-Aldrich,目录号:H4034-1KG)
  5. KOH(Panreac Applichem,目录号:121515)
  6. NaOH 50%(w/v)溶液(AppliChem GmbH,目录号:A3720,1000)
  7. Milli Q级水
  8. 100mM HEPES-KOH(参见配方)
  9. 提取缓冲液1(EB1)(参见配方)
  10. 0.1 M NaOH(见配方)
  11. 21.6 mM NaOH(见配方)

设备

  1. 小砂浆和杵
  2. 1.5 ml微量离心管
  3. 自动移液器
  4. 精度规模
  5. 2ml微量离心机(Eppendorf,型号:5415R)
  6. Vacufuge浓缩器5301(Eppendorf)
  7. 尼龙过滤器(Whatman,目录号:UN203NPENYL)
  8. Dionex HPLC系统(Dionex ICS 5000)
  9. CarboPacPA10柱(4×250mm)(Thermo Fisher Scientific,目录号:046110)
  10. CarboPacPA10预柱(4×50mm)(Thermo Fisher Scientific,目录号:046115)
  11. 金电极(Dionex ICS 5000)
  12. 连接到HPLC系统的计算机

软件

  1. Chromeleon软件(v.7.0)(Thermo Fisher Scientific)

程序

  1. 收集叶样品(或植物材料)并立即在液氮中冷冻。 储存于-80℃直至使用。
  2. 在液体N 2存在下用研钵和杵研磨植物组织,直到样品变成细粉。
  3. 称取100-300 mg叶粉放入离心管中,加入萃取缓冲液1. [动力组织/EB1的比例应为1:1(w/v)。]
  4. 在80℃下加热样品2小时以提取可溶性糖。 使它们冷却并在15,000×g(在微型离心机中13,000rpm)离心10分钟。
  5. 将上清液移至新鲜微量离心管中。
  6. 将上清液在45℃真空蒸发2小时
  7. 将干燥的提取物再悬浮于Milli Q级水中。 [水/原粉组织的比例应为0.5:1(v/w)。]
  8. 在准备所有样品时保持在冰上。如有必要,将样品在-20°C冷冻长期储存。
  9. 通过尼龙过滤器过滤清洁可溶性植物提取物。用Milli Q级水将合适的提取物体积(通常为50μl)补足至200μl,并通过Whatman Mini-Uniprep TM尼龙过滤器(0.2μm孔径)过滤。
    1. 加入250μl21.6 mM NaOH至50μl过滤的提取物,并应用于Dionex HPLC系统。设置10μl进样体积。
    2. 通过在18℃下用18mM NaOH在20℃下等度洗脱来分离糖 作为流动相,流速为1ml/min [18%(v/v)0.1M NaOH和82% (v/v)Milli Q级水]通过CarboPac PA10柱和预柱  CarboPacPA10。通过电流检测进行检测  金电极在nC单位。
    3. 分析色谱图 使用Chromeleon软件。峰由它们的保留来鉴定 时间与已知标准糖相比。峰面积为 通过对a的开始和结束之间的面积进行积分来计算 峰(nC x min)。一系列糖混合物  的已知浓度(1,0.5,0.1,0.05和0.01mM;作为实例, 见图1A中的0.5mM标准曲线)施加到HPLC中 系统,如上所述,以产生每个的校准曲线 糖,相关峰面积(由软件确定) 浓度。比较某糖的量来计算  峰面积(图1B)与校准曲线中的那些。糖 样品中的含量以每克新鲜的微摩尔糖表示 重量。


      图1.典型的糖洗脱曲线 色谱图。 A.糖标准品:海藻糖(峰1), 半乳糖(峰2),葡萄糖(峰3),果糖(峰4)和蔗糖 (峰5)。 B.显示葡萄糖(1),果糖的植物提取物 (2)和蔗糖(3)峰。表示每个峰的保留时间。 nC:10 -9。

代表数据

  1. 在工作日之间和周末期间,用0.2M NaOH以0.1ml min -1流速洗涤柱和预柱。 在该处理之后,在样品加载前,用18mM NaOH以1ml/min流速平衡至少1小时。 为了监测不同分析的再现性,如上所述,在每个样品批次的开始和结束时分析0.5mM糖标准混合物的等分试样。

    表1. 0.5mM糖标准等分试样(n = 18)中葡萄糖,果糖和蔗糖峰的典型面积值
    面积(nC x分钟)
    葡萄糖
    果糖
    蔗糖
    意思是
    20.5
    4.3
    8.4
    SD
    1.2
    0.4
    0.9

    来自代表性植物提取物(即图1B)的峰面积值分别为葡萄糖,果糖和蔗糖的8.3,0.5和2.2nC×min。 由于它们在不同的实验条件下获得,因此这些值可能随着样品而不同,但它们总是落入与校准曲线对应的可测量范围内。

笔记

  1. 使用Dionex设备(或包含塑料连接和管道的类似色谱仪)是强制性的,因为常规HPLC系统与NaOH溶液不兼容。
  2. 应使用氦气流以使流动相脱气

食谱

  1. 100mM HEPES-KOH(pH7.7)
    称重23.83克HEPES
    用KOH调节pH至7.7 添加Milli Q级水至1 L
    储存在4°C
  2. 提取缓冲区1(EB1)
    将20ml 100mM HEPES-KOH(pH7.7)与80ml EtOH 100%(v/v)混合 存储在RT
  3. 0.1 M和0.2 M NaOH 用Milli Q级水稀释5.25ml或10.5ml NaOH(分别)NaOH 50%(w/v)至1L
    储存在室温下不超过2周,由于碳酸化
  4. 21.6mM NaOH 用Milli Q级水稀释2.16ml 0.1M NaOH至10ml
    储存在室温下不超过2周,由于碳酸化

致谢

这项工作是用来自项目CSD2007-00057,BIO2008-02292和BIO2011-28847-C02-00(西班牙经济和竞争力部,MINECO)和卓越项目P06-CVI-01450和P08-AGR-03582(Junta deAndalucía),FEDER资金部分支持FV 和J.M.R. 我们还承认TRANSPLANTA财团,CONSOLIDER 28317项目(MINECO)。

参考文献

  1. Ortiz-Marchena,M.I.,Albi,T.,Lucas-Reina,E.,Said,F.E.,Romero-Campero,F.J.,Cano,B.,Ruiz,M.T.,Romero,J.M.and Valverde, 拟南芥花期过渡期间碳分布的光周期控制。 a> 植物细胞 26(2):565-584。
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How to cite this protocol: Ortiz-Marchena, M. I., Ruiz, M. T., Valverde, F. and Romero, J. M. (2014). Determination of Soluble Sugars in Arabidopsis thaliana Leaves by Anion Exchange Chromatography. Bio-protocol 4(23): e1317. DOI: 10.21769/BioProtoc.1317; Full Text



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