辣椒青霉色素中叶绿素的分离及含量羟化活性的测定

Mareike Hauenstein; Stefan Hörtensteiner

doi:10.21769/BioProtoc.2561

Improve Research Reproducibility A Bio-protocol resource

提交稿件
订阅
CN
- EN - English
- CN - 中文

Peer-reviewed

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

辣椒青霉色素中叶绿素的分离及含量羟化活性的测定

MH Mareike Hauenstein

SH Stefan Hörtensteiner email

发布: 2017年09月20日第7卷第18期 DOI: 10.21769/BioProtoc.2561 浏览次数: 7882

评审: Marisa RosaMohan TCWenrong He

下载 PDF

Q&A

引用

Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of The Plant Cell, featuring study using the protocol.

Oct 2016

实验方案合集

Cell Imaging - A Special Collection for Cell Bio 2023

相关实验方案

使用平板阅读器测定植物 NADP-苹果酸脱氢酶活性的半通量程序

Kevin Baudry and Emmanuelle Issakidis-Bourguet

2023年08月20日 738 阅读

探测生物分子缩合物形成的体外测定

Yu Zhang and Shen Lisha

2023年09月05日 2134 阅读

利用免疫荧光法原位检测光合生物中TOR激酶活性

Ana P. Lando [...] Giselle M. A. Martínez-Noël

2024年12月20日 894 阅读

Abstract

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)

Background

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

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

Equipment

Pipettes (Gilson)
Fruit juicer (Vitality 4 Life, model: Oscar Vitalmax 900 ) or Sorvall mixer
Microcentrifuge: Biofuge fresco (Heraeus, model: Biofuge fresco )
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)
Ultracentrifuge: Optima LE-80K (Beckman Coulter, model: Optima^TM LE-80K ); Rotor: SW-41Ti (Beckman Coulter, model: SW 41 Ti with 13.2 ml polyallomer tubes)
-80 °C freezer (Thermo Fisher Scientific, Thermo Scientific^TM, model: HERAfreeze^TMHFU 586 Top )
LC-MS/MS: Ultimate3000-Compact (Thermo Fisher Scientific, Thermo Scientific^TM, model: UltiMate 3000 ; Bruker Daltonics, model: Compact )

Procedure

English

中文翻译

文章信息

版权信息

如何引用

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.