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Isolation and Detection of the Chlorophyll Catabolite Hydroxylating Activity from Capsicum annuum Chromoplasts

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The Plant Cell
Oct 2016



Hydroxylation of chlorophyll catabolites at the so-called C32 position (Hauenstein et al., 2016) is commonly found in all plant species analyzed to date. Here we describe an in vitro hydroxylation assay using Capsicum annuum chromoplast membranes as a source of the hydroxylating activity, which converts the substrate epi-pFCC (epi-primary Fluorescent Chlorophyll Catabolite) (Mühlecker et al., 2000) to epi-pFCC-OH.

Keywords: TIC55 (Translocon at the inner chloroplast membrane 55 kDa) (TIC55(叶绿体内膜易位子55 kDa)), Chlorophyll breakdown (叶绿素分解), PAO/phyllobilin pathway (PAO/藻胆色素途径), Senescence (衰老), Chlorophyll catabolites (叶绿素分解代谢物), Phyllobilins (藻胆色素)


During leaf senescence and fruit ripening, light-absorbing chlorophylls are degraded to non-fluorescent catabolites to prevent oxidative damage. The chlorophyll breakdown pathway (PAO/phyllobilin pathway) consists of consecutive steps catalyzed by several enzymes and the final degradation products, called phyllobilins, are ultimately stored in the vacuole (Kräutler, 2016). epi-primary Fluorescent Chlorophyll Catabolite (epi-pFCC) is the first non-phototoxic intermediate. After its formation in the chloroplast, side-chain modifications of epi-pFCC can occur, most of which take place outside the chloroplast. One of these modifications, however, is the hydroxylation of the C32 position (Figure 1) catalyzed by the inner chloroplast envelope enzyme TIC55, a member of the family of ferredoxin (Fd)-dependent non-heme oxygenases. TIC55 contains a Rieske and a mononuclear iron-binding domain and was shown to require a Fd reducing system as well as molecular oxygen for its hydroxylating activity. Here we describe an in vitro enzyme assay for TIC55, which was used to characterize the epi-pFCC hydroxylating enzyme activity from red pepper chromoplasts.

Figure 1. Outline of the pathway of chlorophyll breakdown, highlighting the TIC55-catalyzed reaction from epi-pFCC to epi-pFCC-OH. The circle shows the C32 position, the site of hydroxylation.

Materials and Reagents

  1. Pipette tips (SARSTEDT)
  2. 2 ml SafeSeal micro tubes, PP (SARSTEDT, catalog number: 72.695.500 )
  3. Miracloth (pore size 22-25 µm) (Merck)
  4. 10 ml syringe with 0.6 mm needle
  5. Watercolor paint brush, number 10 (for example: FILA, Giotto brush art series 400)
  6. Fully ripe red-colored Capsicum annuum fruits, from local supermarket
  7. Sucrose (AppliChem, catalog number: A2211,500 0)
  8. Tris(hydroxymethyl)aminomethane (Tris) (Carl Roth, catalog number: AE15.3 )
  9. 2-(N-morpholino)ethanesulfonic acid (MES) (AppliChem, catalog number: A1074.1000 )
  10. Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA) (AppliChem, catalog number: A2937.1000 )
  11. Polyethylene glycol 4000 (PEG 4000) (Sigma-Aldrich, catalog number: 81240 )
  12. 1,4-Dithiothreitol (DTT) (Carl Roth, catalog number: 6908.3 )
  13. (+)-Sodium L-ascorbate (Vitamin C) (Sigma-Aldrich, catalog number: A4034 )
  14. Ferredoxin-NADP+ reductase (FNR) (Sigma-Aldrich, catalog number: F0628 )
  15. Ferredoxin (Fd) (Sigma-Aldrich, catalog number: F3013 )
  16. β-Nicotinamide adenine dinucleotide 2’-phosphate reduced tetrasodium salt hydrate (NADPH) (AppliChem, catalog number: A1395 )
  17. Glucose-6-phosphate dehydrogenase (GDH) (Sigma-Aldrich, catalog number: G8404 )
  18. Glucose-6-phosphate (Glc6P) (Sigma-Aldrich, catalog number: G7879 )
  19. Epi-primary fluorescent chlorophyll catabolite (epi-pFCC) (according to Mühlecker et al., 2000)
  20. Methanol, HPLC grade (Sigma-Aldrich, catalog number: 34860 )
  21. Chromoplast isolation buffer (for composition, see Recipes)
  22. Tris MES pH 8 buffer (for composition, see Recipes)


  1. Pipettes (Gilson)
  2. Fruit juicer (Vitality 4 Life, model: Oscar Vitalmax 900 ) or Sorvall mixer
  3. Microcentrifuge: Biofuge fresco (Heraeus, model: Biofuge fresco )
  4. Centrifuge: Avanti J-20 XPi (Beckman Coulter, model: Avanti® J-XN 26 ); Rotors: JLA-10.500 (Beckman Coulter, model: JLA-10.500 with 500 ml polypropylene bottles) and JA-25.50 (Beckman Coulter, model: JA-25.50 with 50 ml polypropylene tubes)
  5. Ultracentrifuge: Optima LE-80K (Beckman Coulter, model: OptimaTM LE-80K ); Rotor: SW-41Ti (Beckman Coulter, model: SW 41 Ti with 13.2 ml polyallomer tubes)
  6. -80 °C freezer (Thermo Fisher Scientific, Thermo ScientificTM, model: HERAfreezeTM HFU 586 Top )
  7. LC-MS/MS: Ultimate3000-Compact (Thermo Fisher Scientific, Thermo ScientificTM, model: UltiMate 3000 ; Bruker Daltonics, model: Compact )


  1. Chromoplast protein isolation from Capsicum annuum
    1. Collect 300 g of exocarp (with some mesocarp) from red pepper fruits (Capsicum annuum).
      For this, cut the pepper fruit in strips lengthwise (Figures 2A-2D). Now cut off most of the mesocarp tissue and collect the outer exocarp (Figure 2E). Cut the exocarp (Figure 2F) in smaller pieces to facilitate grinding.

      Figure 2. Isolation of pepper fruit mesocarp tissue. A. Entire fruit; B. Halved fruit; C. Detail of B showing exocarp (outside the dotted line) and mesocarp (inside the dotted line); D. Sliced pepper fruit; E. Separation of exocarp from mesocarp with a knife; F. Isolated exocarp.

    2. Blend the exocarp with 400 ml of cold (4 °C) chromoplast isolation buffer (see Recipes) in a fruit juicer or Sorvall mixer, with lowest setting and for three 10 sec-pulses in the Sorvall mixer, or for 30 sec in the fruit juicer. After blending, the tissue should be entirely disintegrated. Avoid warming up of the extract.
    3. Filter the extract through two layers of miracloth placed in a glass funnel into the 500 ml polypropylene bottles for centrifugation.
    4. Centrifuge for 10 min, 12,000 x g at 4 °C ( JLA-10.500 rotor).
    5. Discard supernatant by pouring it into the waste. Do this step quickly since the pellet is not very stable.
    6. Carefully resuspend the pellet in 100 ml chromoplast isolation buffer using fine strokes of a paint brush.
    7. Centrifuge for 10 min, 12,000 x g at 4 °C ( JLA-10.500 rotor).
    8. Resuspend the pellet in 8 ml of 25 mM Tris-MES (see Recipes) pH 8, this time without the paint brush, but by pipetting the solution up and down.
    9. Transfer to 50 ml polypropylene tubes by pipetting.
    10. Break chromoplasts by pressing them 10 times through a syringe with a 0.6 mm needle. For this, the solution is sucked-in and pushed-out through the 0.6 mm needle using a 10 ml syringe. Avoid sucking of air.
    11. Centrifuge for 10 min, 15,000 x g at 4 °C ( JA-25.50 rotor).
    12. Transfer the supernatant to 13.2 ml allomer tubes by pipetting.
    13. Fractionate in a soluble and membrane fraction by ultra-centrifugation for 1 h, 150,000 x g at 4 °C (SW-41Ti rotor).
    14. Resuspend the obtained membrane pellet in 8 ml of 25 mM Tris-MES pH 8 and push 10 times through a syringe with a 0.6 mm needle as above.
    15. Chromoplast membranes can now be used for hydroxylation assays or can be stored for later use at -80 °C.
    16. Freeze aliquots of 1-2 ml of the Chromoplast membranes in liquid nitrogen and store at -80 °C.

  2. Hydroxylation assay
    1. Mix all compounds on ice in a 2 ml tube except for the substrate (epi-pFCC) according to the following:

    2. Start enzyme assay by adding the substrate to the mix from above.
    3. Incubate at room temperature in darkness for up to 40 min.
    4. Terminate the reaction by adding 50 µl of methanol.
    5. Centrifuge for 2 min, 16,000 x g at 4 °C (Biofuge).
    6. Transfer supernatant to a new tube by pipetting (avoid transferring particles from the pellet).
    7. Repeat centrifugation to get rid of any particles, which could block the HPLC or LC-MS/MS tubing.
    8. Analyze supernatant of the samples by HPLC or by LC-MS/MS for formation of epi-pFCC-OH (Hauenstein et al., 2016).

Data analysis

Data was analyzed by LC-MS/MS as described (Christ et al., 2016). Figure 3 shows an example of the time-dependent formation of epi-pFCC-OH from epi-pFCC using the here described hydroxylation assay. Figure 4 shows MS/MS experiments of both epi-pFCC and epi-pFCC-OH that are characteristic for both compounds and help for their identification in LC-MS/MS experiments. Ideally, assays are performed with a minimum of three replicates to allow for statistical analysis of the results.

Figure 3. Time dependent formation of epi-pFCC-OH. The formation of epi-pFCC-OH over a time course of 60 min was measured by LC-MS. Shown are extracted ion chromatograms for the masses 629 m/z (epi-pFCC) and 645 m/z (epi-pFCC-OH).

Figure 4. MS/MS fractionation pattern of epi-pFCC (629 m/z; bottom) and its hydroxylated form (645 m/z; top). See Table 1 for MS/MS characteristics of both compounds.

Table 1. Differences between epi-pFCC and epi-pFCC-OH in mass, chemical formula and three characteristic MS/MS fragments


  1. Chromoplast isolation buffer

    Prepare 500 ml. Buffer can be prepared and then autoclaved in advance, it should be stored at 4 °C for up to a week; for longer storage freeze at -20 °C. Before use, add the Vitamin C (as powder) freshly
  2. Tris MES pH 8 buffer

    Prepare 100 ml. Buffer can be prepared and then autoclaved in advance, it should be stored at 4 °C for up to a week; for longer storage freeze at -20 °C. Before use, add the Vitamin C (as powder) freshly


This work was supported by the Swiss National Science Foundation (grant # 31003A_149389/1). This protocol was adapted from previous work (Hauenstein et al., 2016).


  1. Christ, B., Hauenstein, M. and Hortensteiner, S. (2016). A liquid chromatography-mass spectrometry platform for the analysis of phyllobilins, the major degradation products of chlorophyll in Arabidopsis thaliana. Plant J 88(3): 505-518.
  2. Hauenstein, M., Christ, B., Das, A., Aubry, S. and Hörtensteiner, S. (2016). A role for TIC55 as a hydroxylase of phyllobilins, the products of chlorophyll breakdown during plant senescence. Plant Cell 28(10): 2510-2527.
  3. Kräutler, B. (2016). Breakdown of chlorophyll in higher plants--phyllobilins as abundant, yet hardly visible signs of ripening, senescence, and cell death. Angew Chem Int Ed Engl 55(16): 4882-4907.
  4. Mühlecker, W., Kräutler, B., Moser, D., Matile, P. and Hörtensteiner, S. (2000). Breakdown of chlorophyll: a fluorescent chlorophyll catabolite from sweet pepper (Capsicum annuum). Helv Chim Acta 83: 278-286.


所谓C32位置的叶绿素分解代谢物的羟基化(Hauenstein et al。,2016)通常在迄今为止分析的所有植物物种中发现。 在这里,我们描述了使用Capsicum annuum chromoplast membrane作为羟基化活性的来源的体外羟基化测定法,其将底物epi-pFCC(外显子荧光叶绿素Catabolite)(Mühlecker等,2000)转化为epi-pFCC-OH。
【背景】在叶片衰老和果实成熟期间,吸光叶绿素被降解成非荧光分解代谢物,以防止氧化损伤。叶绿素分解途径(PAO / phyllobilin途径)由几个酶催化的连续步骤组成,最终降解产物称为叶绿素,最终储存在液泡中(Kräutler,2016)。外源荧光叶绿素Cepolite(epi-pFCC)是第一种非光毒性中间体。在叶绿体中形成后,可以发生epi-pFCC的侧链修饰,其中大部分发生在叶绿体外。然而,这些修饰之一是由内部叶绿体包膜酶TIC55(铁氧还蛋白(Fd)依赖性非血红素加氧酶家族的成员)催化的C32位置(图1)的羟基化。 TIC55含有Rieske和单核铁结合结构域,并显示其需要Fd还原系统以及分子氧作为其羟基化活性。在这里我们描述了TIC55的体外酶测定法,其用于表征红辣椒色素体的表达pFCC羟基化酶活性。
图1.叶绿素分解途径的概述,突出了从epi-pFCC到epi-pFCC-OH的TIC55催化反应。 圆圈显示C32位置,羟基化位点。

关键字:TIC55(叶绿体内膜易位子55 kDa), 叶绿素分解, PAO/藻胆色素途径, 衰老, 叶绿素分解代谢物, 藻胆色素


  1. 移液器吸头(SARSTEDT)
  2. 2 ml SafeSeal微管,PP(SARSTEDT,目录号:72.695.500)
  3. Miracloth(孔径22-25μm)(Merck)
  4. 10毫升注射器,带有0.6毫米的针头
  5. 水彩画笔,10号(例如:FILA,Giotto刷艺系列400)
  6. 来自当地超级市场的完全成熟的红色辣椒果汁,/ / />水果
  7. 蔗糖(AppliChem,目录号:A2211,5000)
  8. 三羟甲基氨基甲烷(Tris)(Carl Roth,目录号:AE15.3)
  9. 2-(N-吗啉代)乙磺酸(MES)(AppliChem,目录号:A1074.1000)
  10. 乙二胺四乙酸二钠盐二水合物(EDTA)(AppliChem,目录号:A2937.1000)
  11. 聚乙二醇4000(PEG 4000)(Sigma-Aldrich,目录号:81240)
  12. 1,4-二硫苏糖醇(DTT)(Carl Roth,目录号:6908.3)
  13. (+) - L-抗坏血酸钠(维生素C)(Sigma-Aldrich,目录号:A4034)
  14. Ferredoxin-NADP +还原酶(FNR)(Sigma-Aldrich,目录号:F0628)
  15. 铁氧还蛋白(Fd)(Sigma-Aldrich,目录号:F3013)
  16. β-烟酰胺腺嘌呤二核苷酸2'-磷酸还原四钠盐水合物(NADPH)(AppliChem,目录号:A1395)
  17. 葡萄糖-6-磷酸脱氢酶(GDH)(Sigma-Aldrich,目录号:G8404)
  18. 葡萄糖-6-磷酸(Glc6P)(Sigma-Aldrich,目录号:G7879)
  19. 荧光叶绿素分解代谢物(FCC)(根据Mühlecker等人,2000)
  20. 甲醇,HPLC级(Sigma-Aldrich,目录号:34860)
  21. 色素分离缓冲液(用于组合物,参见食谱)
  22. Tris MES pH 8缓冲液(用于组合物,参见食谱)


  1. 移液器(Gilson)
  2. 果汁榨汁机(Vitality 4 Life,型号:Oscar Vitalmax 900)或Sorvall搅拌机
  3. 微量离心机:Biofuge壁画(Heraeus,型号:Biofuge壁画)
  4. 离心机:Avanti J-20 XPi(Beckman Coulter,型号:Avanti J-XN 26);转子:JLA-10.500(Beckman Coulter,型号:JLA-10.500,500ml聚丙烯瓶)和JA-25.50(Beckman Coulter,型号:JA-25.50,用50ml聚丙烯管)
  5. 超速离心机:Optima LE-80K(Beckman Coulter,型号:Optima TM LE-80K);转子:SW-41Ti(Beckman Coulter,型号:SW 41 Ti,13.2 ml聚集管)
  6. -80°C冰箱(Thermo Fisher Scientific,Thermo Scientific TM,型号:HERAfreeze TM HFU 586 Top)
  7. LC-MS / MS:Ultimate3000-Compact(Thermo Fisher Scientific,Thermo Scientific TM,型号:UltiMate 3000; Bruker Daltonics,型号:Compact)


  1. 来自辣椒的色素淀粉蛋白分离
    1. 从红辣椒水果(辣椒)中收集300克外果皮(含一些中果皮)。

      图2.胡椒果实中果皮组织的分离。 :一种。整个水果B.一半水果C.显示外果皮(虚线外)和中果皮(虚线内)的细节;切胡椒水果;用刀将外果皮与中果皮分离; F.分离的外果皮。

    2. 在果汁榨汁机或Sorvall混合器中,将400ml冷(4℃)色素淀粉分离缓冲液(参见食谱)混合,在Sorvall混合器中以最低的设定和三个10秒脉冲混合外果皮,或在水果中混合30秒榨汁机。混合后,组织应完全分解。避免加热提取物。
    3. 将提取物通过放置在玻璃漏斗中的两层miracloth过滤到500ml聚丙烯瓶中进行离心。
    4. 在4℃(JLA-10.500转子)离心10分钟,12,000 x g 。
    5. 将上清液倒入废物中弃去。由于颗粒不稳定,请快速进行。
    6. 小心地将沉淀物重悬于100毫升染色体分离缓冲液中,使用油漆刷的细微笔触
    7. 在4℃(JLA-10.500转子)离心10分钟,12,000 x g 。
    8. 将沉淀重悬于8ml 25mM Tris-MES(参见食谱)pH 8,此时不用油漆刷,而是通过上下移动溶液。
    9. 通过移液转移到50ml聚丙烯管中
    10. 通过使用0.6毫米针头的注射器将它们压制10次来破碎色原体。为此,使用10ml注射器将溶液通过0.6mm针头吸入并推出。避免吸入空气。
    11. 在4℃(JA-25.50转子)离心10分钟,15,000 x g 。
    12. 通过移液将上清转移到13.2ml的调节管中
    13. 通过在4℃(SW-41Ti转子)超离心1小时,150,000×g /分,分离成可溶性和膜级分。
    14. 将获得的膜沉淀物重悬于8ml 25mM Tris-MES pH 8中,并用如上所述的0.6mm针头的注射器推10次。
    15. 色原膜现在可以用于羟基化测定,或者可以储存在-80°C以备将来使用。
    16. 将1-2ml的Chromoplast膜在液氮中冷冻等分,并储存在-80°C

  2. 羟基化测定
    1. 根据以下内容,将所有化合物在冰上混合在2ml管中(底物(FCC)):

    2. 通过将底物加入到上述混合物中开始酶测定
    3. 在黑暗中室温孵育长达40分钟。
    4. 加入50μl甲醇终止反应
    5. 在4℃离心2分钟,16,000 x g(Biofuge)。
    6. 通过移液将上清液转移到新管(避免从颗粒中转移颗粒)。
    7. 重复离心以除去任何可能堵塞HPLC或LC-MS / MS管道的颗粒
    8. 通过HPLC或LC-MS / MS分析样品的上清液以形成FCC-OH(Hauenstein等人,2016)。


数据通过LC-MS / MS分析,如(Christ 等人,2016)所述。图3显示了使用本文所述的羟基化测定法从epi-p FCC的时间依赖性形成FCC-OH的实例。图4显示了两种化合物特征的FCC和FCC-OH的MS / MS实验,并且有助于它们在LC-MS / MS实验。理想情况下,进行最少三次重复的分析,以便对结果进行统计分析

图3. FCC-OH的时间依赖性形式。 通过LC-MS测量60分钟的时间过程中形成的epi-p FCC-OH。所示为质量629的萃取离子色谱图(FCC)和645m / z(epi- FCC-OH)。

图4.CEM(629m / z底部)及其羟基化形式(645m / z)的MS / MS分离模式;顶部)。 两种化合物的MS / MS特性见表1.

表1.在质量,化学式和三个特征MS / MS片段中,FCC和FCC-OH之间的FCC和FCC-OH的差异强>


  1. 色素分离缓冲液

  2. Tris MES pH 8缓冲液



这项工作得到了瑞士国家科学基金会的资助(授权#31003A_149389 / 1)。该协议是从以前的工作(Hauenstein等人,2016)改编而来的。


  1. 基督,B.,Hauenstein,M.和Hortensteiner,S.(2016)。用于分析叶绿体的液相色谱 - 质谱平台,叶绿素在拟南芥中的主要降解产物。 > J 88(3):505-518。
  2. Hauenstein,M.,Christ,B.,Das,A.,Aubry,S.andHörtensteiner,S。(2016)。< a class =“ke-insertfile”href =“http://www.ncbi。 nlm.nih.gov/pubmed/27655840“target =”_ blank“> TIC55作为叶绿体的羟化酶的作用,植物衰老期间叶绿素分解的产物。植物细胞28( 10):2510-2527。
  3. Kräutler,B。(2016)。叶绿素分解更高的植物 - 叶绿素是丰富的,但几乎没有可见的成熟,衰老和细胞死亡迹象。 Angew Chem Int Ed Engl 55(16):4882-4907。
  4. Mühlecker,W.,Kräutler,B.,Moser,D.,Matile,P.andHörtensteiner,S。(2000)。叶绿素分解:荧光来自甜椒的叶绿素分解代谢物(辣椒) Helv Chim Acta 83:278-286。
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引用:Hauenstein, M. and Hörtensteiner, S. (2017). Isolation and Detection of the Chlorophyll Catabolite Hydroxylating Activity from Capsicum annuum Chromoplasts. Bio-protocol 7(18): e2561. DOI: 10.21769/BioProtoc.2561.