Purification of Starch Granules from Arabidopsis Leaves and Determination of Granule-Bound Starch Synthase Activity

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The Plant Cell
Feb 2014



Starch constitutes the most important carbon reserve in plants and is composed of branched amylopectin and linear amylose. The latter is synthesized exclusively by the Granule-Bound Starch Synthase (GBSS, EC Here we report a readily reproducible, specific and highly sensitive protocol, which includes the isolation of intact starch granules from Arabidopsis thaliana leaves and the subsequent determination of GBSS activity. We have applied this method to study GBSS activity in diurnal cycles in vegetative growth and during the photoperiodic transition to flowering in Arabidopsis (Tenorio et al., 2003; Ortiz-Marchena et al., 2014).

Keywords: Starch granule (淀粉颗粒), Starch purification (淀粉净化), GBSS activity (GBSS活性), Granule-bound starch synthase (颗粒结合型淀粉合酶)

Materials and Reagents

  1. Plant materials
    Note: Arabidopsis thaliana (A. thaliana) were grown in controlled cabinets on peat-based compost.
  2. Liquid N2
  3. HEPES (Sigma-Aldrich, catalog number: H4034 )
  4. Potassium hydroxide pellets (Panreac Applichem, catalog number: A0566 )
  5. TritonTM X-100 (Sigma-Aldrich, catalog number: X100 )
  6. Miracloth (Merck KGaA, catalog number: 475855 )
  7. Percoll® (Sigma-Aldrich, catalog number: P1644 )
  8. Glycogen (from rabbit liver Type III) (Sigma-Aldrich, catalog number: G8876 )
  9. Tricine (Sigma-Aldrich, catalog number: T0377 )
  10. Potassium acetate (Sigma-Aldrich, catalog number: P1147 )
  11. DL-Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632 )
  12. Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA) (Sigma-Aldrich, catalog number: E5134 )
  13. Sodium hydroxide pellets (Panreac Applichem, catalog number: 131687 )
  14. Maltotriose (Sigma-Aldrich, catalog number: M8378 )
  15. Adenosine 5’ diphospho, [D-glucose-14C(U)] ammonium salt (ADP [U-14C] glucose) (American Radiolabeled Chemicals, catalog number: ARC 3297 )
  16. Methanol (CARLO ERBA Reagents, catalog number: 412532 )
  17. Potassium chloride (AppliChem GmbH, catalog number: A2939 )
  18. Ecolite(+)TM liquid scintillation cocktail (MP Biomedicals, catalog number: 882475 )
  19. 1 M HEPES-KOH (see Recipes)
  20. 1 M Tricine (see Recipes)
  21. 0.25 M Potassium acetate (see Recipes)
  22. 1 M DTT (see Recipes)
  23. 0.5 M EDTA (see Recipes)
  24. 0.1 M Maltotriose (see Recipes)
  25. 3 M KCl (see Recipes)
  26. 20 mg/ml Glycogen solution (see Recipes)
  27. Extraction buffer (see Recipes)
  28. Percoll buffer (see Recipes)
  29. Washing buffer (see Recipes)
  30. Precipitation buffer (see Recipes)


  1. Small mortar and pestle
  2. 1.5 ml microfuge tubes
  3. Automatic pipettes
  4. Precision scale
  5. Centrifuge (Eppendorf, model: 5810 R )
  6. Microcentrifuge (Eppendorf, model: 5424 )
  7. Scintillation Counter (Beckman Coulter, model: LS 6000 IC )
  8. Scintillation vials (Sigma-Aldrich, catalog number: Z376817 )


  1. Starch granules isolation
    Starch granules were purified following a modification of the Percoll method described by Tenorio et al. (2003).
    1. Homogenize 600 mg (fresh weight) of Arabidopsis leaves in a mortar and pestle in the presence of liquid N2 to a fine powder and add 1 ml of extraction buffer. Leaf material was collected from fully developed rosette leaves from at least three different plants at growth stage 3.50 [according to Boyes et al. (2001)].
    2. Filter the homogenate through two layers of Miracloth on 15 ml Falcon-type tubes.
    3. Add 5 ml of Percoll buffer (4 °C) to the filtrate and mix.
    4. Centrifuge samples for 5 min, 805 x g at 4 °C (2,000 rpm, Eppendorf-5810 R centrifuge) and discard the supernatant.
    5. Wash the pellet by resuspension in 5 ml washing buffer (4 °C), centrifuge as in step A4 and discard the supernatant. Repeat three times.
    6. Air-dry the pellet (do not let it dry completely) containing the starch granules (Figure 1) and use it immediately for determination of GBSS activity as described below.

      Figure 1. Immunodetection of GBSS in starch granules isolated from Arabidopsis Col-0 leaves. Western blot employing GBSS‐specific antibodies in protein extracts from starch granules isolated from wild‐type (Col-0), GBSS mutant (gbs-1) and a recombinant line in which GBSS is fused to GFP. Molecular Mass (MM) markers are shown in kDa (left). Notice GBSS MM around 55 kDa and GBSS-GFP around 80 kDa. GBSS detection is strictly associated to starch granules fractions (three rightmost lanes) and cannot be detected in a soluble protein fraction (leftmost lane).

  2. GBSS activity assay
    The granule-bound starch synthase activity was immediately measured from freshly purified starch granules.
    1. Starch granules were resuspended in 200 μl of 100 mM Tricine (pH 8.4), 25 mM potassium acetate, 10 mM DTT, 5 mM EDTA and 10 mM ADP [U-14C] glucose (specific activity 7.4 GBq/mol).
    2. 100 µl aliquot was quickly extracted and boiled for 5 min to represent DPMtotal of the assay.
    3. The rest of the reaction was incubated at 30 °C for 20-60 min.
    4. The reaction was stopped by boiling for 5 min.
    5. Starch granules were precipitated by adding 1.875 ml of precipitation buffer and 25 µl of glycogen solution as an inert carrier to increase recovery from alcohol precipitation.
    6. Tubes were centrifuged 3 min, 2,000 x g at 4 °C (4,615 rpm, Eppendorf-5424) and the supernatant discarded.
    7. The pellet was resuspended in 0.2 ml deionized water and washed by an additional precipitation with 1.750 ml precipitation buffer without glycogen.
    8. Tubes were centrifuged as in step B5. The pellet was air-dried for a short time, resuspended in 1 ml of deionized water and transferred into an appropriate scintillation vial.
    9. 5 ml of EcoLite liquid scintillation cocktail was added to enhance 14C counting efficiency.
    10. Finally, the radioactivity incorporated into the starch granules was determined with a scintillation counter.
    11. GBSS activity (see Table 1 and Figure 2) is calculated by using the following formula:
      Activity (nmol/min/gfw)=
      DPMreac = disintegration per minute of the reaction sample minus DPM blank (Table 1)
      nmol = nmoles of ADP [U-14C]glucose (1,000 nmol according to the protocol)
      DPMtotal = total DPM of added ADP[U-14C]glucose (step B2)
      t = reaction time (min) (20-60 min according to the protocol)
      gfw = fresh weight (g) (0.3 g according to the protocol)

      Table 1. Determination of GBSS activity in starch granules from wild type (Col-0) and gbs-1 mutant
      Reaction time
      nmol ADP-gluc

      1. DPM for the blank sample. Blank sample is identical to each sample except that it was stopped at time 0, so that the reaction was not allowed to start.
      2. To simplify, a single sample measurement is shown in Table 1. We recommend calculating data from three independent experiments. Sample DPM correspond to the Disintegration Per Minute at the desired reaction time for each sample (experimental data).
      3. DPMtotal is the total amount of DPM provided by the radiolabelled substrate. It actually represents the maximum of DPM for each sample. According to the above example it consists on the total DPM given by 1,000 nmol of ADP[U-14C]glucose determined for aliquots from step B2.
      4. DPMreac = DPMsample minus DPMblank

        Figure 2. GBSS activity in starch granules from Arabidopsis thaliana leaves. GBSS activity (nmol/min/gfw) in starch granules from wild‐type (Col-0), GBSS mutant (gbs-1), GBSS fused to GFP under its own promoter (pGBSS:GBSS:GFP) in gbs-1 background and GBSS expressed under the 35S promoter (P35S:GBSS) in gbs-1 background lines. Values are the average of three independent experiments. Bars indicate the standard deviation (±SEM). Starch granules isolation and GBSS activity was carried out as described in text.

Representative data

  1. As shown in Figure 1, a GBSS activity of ca. 6 nmol/min/gfw is expected in starch granules isolated from A. thaliana ecotype col-0 leaves.
  2. Relative SD (expressed as percentage of the mean) fell in the range of 2-14 per cent for samples from three independent experiments.


  1. Use dischargeable lab ware and appropriate radioactive secure installations and equipment while manipulating ADP [U-14C] glucose.


Note: The given volumes are sufficient for 50 reactions.

  1. 1 M HEPES-KOH (pH 7.5)
    Mix 59.58 g HEPES with 175 ml deionized water
    Adjust pH to 7.5 with KOH pellets
    Add deionized water to 250 ml
    Sterilize through 0.2 µm pore diameter filters
    Stored at 4 °C
  2. 1 M Tricine (pH 8.4)
    Mix 4.48 g Tricine with 20 ml deionized water
    Adjust pH to 8.4 with KOH
    Add deionized water to 25 ml
    Stored at 4 °C
  3. 0.25 M Potassium acetate
    Mix 1.23 g Potassium acetate with 40 ml deionized water
    Sterilize through 0.2 µm pore diameter filters
    Stored at Room Temperature (RT)
  4. 1 M DTT
    Weigh 1.54 g DTT
    Add deionized water to 10 ml
    Distribute in aliquots and store at -20 °C
  5. 0.5 M EDTA (pH 8.0)
    Mix 18.61 g EDTA (disodium, dihydrate) with 80 ml deionized water
    Adjust to pH 8.0 with NaOH
    Note: It is not easy to dissolve the EDTA. It will not dissolve completely until the pH is around 8.0.
    Autoclave (15 psi, 1-2 h, 120 °C)
    Stored at RT
  6. 0.1 M Maltotriose
    Weigh 0.2655 g maltotriose
    Add deionized water to 5 ml
    Sterilize through 0.2 µm pore diameter filters
    Distribute in aliquots and store at -20 °C
  7. 3 M KCl
    Weigh 2.237 g of KCl
    Add deionized water to 10 ml
    Autoclave (15 psi, 1-2 h, 120 °C)
    Stored at RT
  8. 20 mg/ml Glycogen solution
    Weigh 25 mg of glycogen
    Add deionized water to 1.25 ml
    Stored at -20 °C
  9. Extraction buffer
    Mix 2.5 ml 1 M HEPES-KOH (pH 7.5) with 0.5 ml Triton X-100
    Add deionized water to 50 ml
    Stored at RT
  10. Percoll buffer
    Mix 12.5 ml 1 M HEPES-KOH (pH 7.5) with 125 ml Percoll
    Add deionized water to 250 ml
  11. Washing buffer
    37.5 ml of 1 M HEPES-KOH (pH 7.5)
    Add deionized water to 750 ml
  12. Precipitation buffer
    150 ml Methanol
    Add 9.4 ml 3 M KCl
    Add deionized water to 200 ml
    Stored at RT


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).


  1. Boyes, D. C., Zayed, A. M., Ascenzi, R., McCaskill, A. J., Hoffman, N. E., Davis, K. R. and Gorlach, J. (2001). Growth stage-based phenotypic analysis of Arabidopsis: a model for high throughput functional genomics in plants. Plant Cell 13(7): 1499-1510.
  2. 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.
  3. Tenorio, G., Orea, A., Romero, J. M. and Mérida, A. (2003). Oscillation of mRNA level and activity of granule-bound starch synthase I in Arabidopsis leaves during the day/night cycle. Plant Mol Biol 51(6): 949-958.


淀粉构成植物中最重要的碳储量,由支链支链淀粉和线性直链淀粉组成。 后者仅由颗粒结合的淀粉合酶(GBSS,EC合成。 在这里我们报告一个容易重现,具体和高度敏感的协议,其中包括完整的淀粉颗粒从拟南芥叶中分离和随后的GBSS活动的确定。 我们已经应用这种方法来研究营养生长的日变化周期中和在拟南芥中的光周期过渡到开花期间的GBSS活性(Tenorio等人,2003; Ortiz-Marchena& em> et al。,2014)。

关键字:淀粉颗粒, 淀粉净化, GBSS活性, 颗粒结合型淀粉合酶


  1. 植物材料
  2. 液体N <2>
  3. HEPES(Sigma-Aldrich,目录号:H4034)
  4. 氢氧化钾丸(Panreac Applichem,目录号:A0566)
  5. TritonX-100(Sigma-Aldrich,目录号:X100)
  6. Miracloth(Merck KGaA,目录号:475855)
  7. Percoll(Sigma-Aldrich,目录号:P1644)
  8. 糖原(来自兔肝III型)(Sigma-Aldrich,目录号:G8876)
  9. Tricine(Sigma-Aldrich,目录号:T0377)
  10. 乙酸钾(Sigma-Aldrich,目录号:P1147)
  11. DL-二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:D0632)
  12. 乙二胺四乙酸二钠盐二水合物(EDTA)(Sigma-Aldrich,目录号:E5134)
  13. 氢氧化钠丸(Panreac Applichem,目录号:131687)
  14. 麦芽三糖(Sigma-Aldrich,目录号:M8378)
  15. 腺苷5'二磷酸,[D-葡萄糖-14C(U)]铵盐(ADP [U-14C]葡萄糖)(American Radiolabeled Chemicals,目录号:ARC 3297)
  16. 甲醇(CARLO ERBA Reagents,目录号:412532)
  17. 氯化钾(AppliChem GmbH,目录号:A2939)
  18. Ecolite(+)TM液体闪烁鸡尾酒(MP Biomedicals,目录号:882475)
  19. 1 M HEPES-KOH(见配方)
  20. 1 M Tricine(见配方)
  21. 0.25 M醋酸钾(见配方)
  22. 1 M DTT(参见配方)
  23. 0.5 M EDTA(见配方)
  24. 0.1 M麦芽三糖(见配方)
  25. 3 M KCl(参见配方)
  26. 20 mg/ml糖原溶液(见配方)
  27. 提取缓冲液(参见配方)
  28. Percoll缓冲区(参见配方)
  29. 洗涤缓冲液(见配方)
  30. 沉淀缓冲液(参见配方)


  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. 离心机(Eppendorf,型号:5810R)
  6. 微量离心机(Eppendorf,型号:5424)
  7. 闪烁计数器(Beckman Coulter,型号:LS 6000IC)
  8. 闪烁瓶(Sigma-Aldrich,目录号:Z376817)


  1. 淀粉颗粒隔离
    1. 在研钵中均质化600mg(鲜重)的拟南芥叶子 杵在液体N 2存在下加入到细粉末中并加入1ml 提取缓冲液。 叶材料是从完全发育的 玫瑰花叶从生长阶段3.50的至少三种不同的植物叶   [根据Boyes等人(2001)]。
    2. 通过两层Miracloth在15ml Falcon型管上过滤匀浆。
    3. 向滤液中加入5ml Percoll缓冲液(4℃)并混合
    4. 在4℃(2,000rpm,Eppendorf-5810R离心机)下将样品离心5分钟,805×g,并弃去上清液。
    5. 通过重悬浮在5ml洗涤缓冲液(4℃)中洗涤沉淀, 如步骤A4中离心,并弃去上清液。 重复三 次。
    6. 风干球团(不要让它完全干燥) 含有淀粉颗粒(图1),并立即使用 测定如下所述的GBSS活性

      图1。 拟南芥中分离的淀粉颗粒中GBSS的免疫检测 Col-0叶。使用蛋白质中的GBSS特异性抗体的Western印迹   提取自分离自野生型(Col-0),GBSS的淀粉颗粒 突变体(gbs-1)和其中GBSS与GFP融合的重组品系。 分子量(MM)标记以kDa(左)显示。 注意GBSS MM 约55kDa和约80kDa的GBSS-GFP。 GBSS检测严格 与淀粉颗粒部分(三个最右侧的泳道)和 不能在可溶性蛋白质级分(最左侧泳道)中检测。

  2. GBSS活性测定
    1. 将淀粉颗粒再悬浮于200μl的100mM Tricine(pH 8.4)中, 25mM乙酸钾,10mM DTT,5mM EDTA和10mM ADP [U- 14 C] 葡萄糖(比活性7.4GBq/mol)
    2. 快速提取100μl等分试样并煮沸5分钟以代表测定的DPM总
    3. 将反应的其余部分在30℃温育20-60分钟。
    4. 通过煮沸5分钟使反应停止
    5. 通过加入1.875ml的淀粉颗粒沉淀 沉淀缓冲液和25μl作为惰性载体的糖原溶液 以增加酒精沉淀的恢复
    6. 将试管在4℃(4,615rpm,Eppendorf-5424)离心3分钟,2,000×g,弃去上清液。
    7. 将沉淀重悬浮于0.2ml去离子水中, 另外沉淀用1.750ml沉淀缓冲液无 糖原。
    8. 将管如步骤B5中离心。 沉淀是 空气干燥短时间,重悬于1ml去离子水中 转移到合适的闪烁瓶中
    9. 加入5ml的EcoLite液体闪烁混合物以增强14 C计数效率
    10. 最后,用闪烁计数器测定掺入淀粉颗粒中的放射性
    11. GBSS活性(参​​见表1和图2)通过使用下式计算:
      DPMreac =反应样品每分钟的崩解减去DPM空白(表1)
      nmol = nmol的ADP [U- 14 C]葡萄糖(根据方案为1,000nmol)
      DPM总=添加的ADP [U- 14 C]葡萄糖的总DPM(步骤B2)
      t =反应时间(min)(根据方案为20-60min)
      gfw =鲜重(g)(根据方案0.3g)

      空白 a
      示例 b
      DPMtotal c
      DPMreac d
      nmol ADP-gluc

      1. DPM用于空白样品。 空白样品与每个样品相同 除了它在时间0停止,使得反应不进行 允许启动。
      2. 为了简化,单个样本测量是 如表1所示。我们建议从三个独立的计算数据 实验。 样品DPM对应于每分钟的崩解 每个样品的期望反应时间(实验数据)。
      3. DPMtotal是由放射性标记提供的DPM的总量 基质。 它实际上代表每个样品的DPM的最大值。 根据上述例子,它包括由给定的总DPM 对来自步骤B2的等分试样测定的1,000nmol ADP [U- 14 C]葡萄糖。
      4. DPMreac = DPMsample减去DPMblank

        图2.来自拟南芥的淀粉颗粒中的GBSS活性 树叶。 来自野生型淀粉颗粒的GBSS活性(nmol/min/gfw) (Col-0),GBSS突变体(gbs-1),在其自身启动子下与GFP融合的GBSS (pGBSS:GBSS:GFP)和在 S下表达的GBSS 启动子(P35S:GBSS)在gbs-1背景系中。 值是平均值 的三次独立实验。 条表示标准偏差 (±SEM)。 淀粉颗粒分离和GBSS活性如下进行 描述在文本中。


  1. 如图1所示,GBSS活性约为 在从A中分离的淀粉颗粒中预期为6nmol/min/gfw。 thaliana 生态型col-0离开。
  2. 相对SD(表示为平均值的百分比)落在2-14%的范围内,来自三次独立实验的样品。


  1. 使用可放电的实验室器皿和适当的放射性安全装置和设备,同时操作ADP [U- 14] C葡萄糖。



  1. 1 M HEPES-KOH(pH 7.5)
    将59.58g HEPES与175ml去离子水混合
    用KOH颗粒将pH调节至7.5 加入去离子水至250 ml
    通过0.2μm孔径过滤器灭菌 储存在4°C
  2. 1 M Tricine(pH 8.4)
    将4.48g Tricine与20ml去离子水混合 用KOH调节pH至8.4 加入去离子水至25 ml
  3. 0.25 M乙酸钾
    将1.23g乙酸钾与40ml去离子水混合 通过0.2μm孔径过滤器灭菌 储存在室温(RT)
  4. 1 M DTT
    称重1.54 g DTT
  5. 0.5 M EDTA(pH 8.0)
    将18.61g EDTA(二钠,二水合物)与80ml去离子水混合 用NaOH调节至pH 8.0 注意:溶解EDTA不容易。 在pH约为8.0之前,它不会完全溶解。
    高压灭菌(15 psi,1-2 h,120℃)
  6. 0.1M麦芽三糖
    通过0.2μm孔径过滤器灭菌 以等分试样分装并在-20°C下存储
  7. 3 M KCl
    高压灭菌(15 psi,1-2 h,120℃)
  8. 20mg/ml糖原溶液 称量25mg糖原
    加入去离子水至1.25 ml
  9. 提取缓冲区
    将2.5ml 1M HEPES-KOH(pH7.5)与0.5ml Triton X-100混合 加入去离子水至50 ml
  10. Percoll缓冲区
    将12.5ml 1M HEPES-KOH(pH7.5)与125ml Percoll混合 加入去离子水至250 ml
  11. 洗涤缓冲液
    37.5ml 1M HEPES-KOH(pH7.5)
    加入去离子水至750 ml
  12. 降雨缓冲液
    加入9.4ml 3M KCl
    加入去离子水至200 ml


这项工作是用项目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资助FV和JMR部分支持我们也承认TRANSPLANTA财团项目CONSOLIDER 28317(MINECO)。


  1. Boyes,D.C.,Zayed,A.M.,Ascenzi,R.,McCaskill,A.J.,Hoffman,N.E.,Davis,K.R.and Gorlach,J。(2001)。 拟南芥的基于生长阶段的表型分析:植物中高通量功能基因组学的模型。 a> 植物细胞 13(7):1499-1510
  2. 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> Plant Cell 26(2):565-584
  3. Tenorio,G.,Orea,A.,Romero,J.M。和Mérida,A。(2003)。 拟南芥中mRNA水平和颗粒结合淀粉合成酶I的活性振荡白天/夜晚循环中的叶子。 植物分子生物学 51(6):949-958。
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引用:Albi, T., Ortiz-Marchena, M. I., Ruiz, M. T., Romero, J. M. and Valverde, F. (2014). Purification of Starch Granules from Arabidopsis Leaves and Determination of Granule-Bound Starch Synthase Activity. Bio-protocol 4(23): e1316. DOI: 10.21769/BioProtoc.1316.