Assay of Arabinofuranosidase Activity in Maize Roots
玉米根中阿拉伯呋喃糖酶的活性测定
发布: 2016年03月20日第6卷第6期 DOI: 10.21769/BioProtoc.1764 浏览次数: 9734
评审: Zhaohui LiuDušan VeličkovićAgnieszka Zienkiewicz
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参见作者原研究论文
The authors used this protocol in:
May 2015
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Abstract
Root is a perfect model for studying the mechanisms of plant cell growth. Along the root length, several zones where cells are at different stages of development can be visualized (Figure 1). The dissection of the root on these zones allows the investigation of biochemical and genetic aspects of different growth steps. Maize primary root is much more massive than the root of other Monocots and thus more convenient for such type of research. Plant cell wall, mainly consisting of polysaccharides, plays an important role in plant life. Therefore, measurement of plant carbohydrate content and glycoside-modifying enzyme activity in plant cells has become an important aspect in plant physiology. One of the well-documented changes of hemicelluloses molecules during elongation growth of monocots cells is the decrease of arabinose substitution of glucuronoarabinoxylans. This might be caused by changes in synthesis of this polysaccharide or by the action of arabinofuranosidases. Here, we describe the protocol of spectrophotometric measuring of arabinofuranosidase activity in maize root by the rate of hydrolysis of chromogenic substrate (4-nitrophenyl α-L-arabinofuranoside).
Figure 1. Scheme of plant material collection for further arabinofuranosidase assay. Four-day-old dark-grown maize seedling (left panel). Different zones of primary maize root and corresponding stages of cell development, according to Kozlova et al. (2012) (right panel).
Materials and Reagents
- Microcentrifuge tubes 1.5-2 ml
- CarboPac PA1 analytical column (4 x 250 mm) (Thermo Fisher Scientific, catalog number: 035391 )
- CarboPac PA1 guard column (4 x 50 mm) (Thermo Fisher Scientific, catalog number: 043096 )
- Maize seedlings
- 4-nitrophenyl α-L-arabinofuranoside (4NPA) (Sigma-Aldrich, catalog number: N3641 )
- 4-nitrophenol (NP) (spectrophotometric grade) (Sigma-Aldrich, catalog number: 1048 )
- DL-Dithiothreitol (Sigma-Aldrich, catalog number: D0632 )
- Quick StartTM Bradford 1x Dye (Bio-Rad Laboratories, AbD Serotec®, catalog number: 5000205 )
- Sodium Acetate Anhydrous (NaOAc) (Sigma-Aldrich, catalog number: W302406 )
- Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 306576 )
- L-(+)-ARABINOSE (Sigma-Aldrich, catalog number: W325501 )
- NaN3
- Na2CO3
- NaOCl
- Glacial acetic acid
- Ice
- MilliQ water (18 MΩ-cm)
- 5% (w/w) solution of NaOCl (see Recipes)
- 0.03% (w/w) NaN3 (see Recipes)
- 50 mM NaOAc buffer (pH 6.0) with 3 mM of dithiothreitol on 0.03% (w/w) NaN3 (see Recipes)
- 10 mM 4NPA (see Recipes)
- 1 M Na2CO3 (see Recipes)
- Calibration solutions for NP (see Recipes)
- 50% (w/w) NaOH solution (see Recipes)
- 15 mM NaOH (buffer A for HPAEC) (see Recipes)
- 1 M NaOAc in 0.1 M NaOH (buffer B for HPAEC) (see Recipes)
- Calibration solutions for arabinose (see Recipes)
Equipment
- Spectrophotometer (required wavelength is 405 nm)
- Thermostat (required temperature is 27 °C)
- Microcentrifuge (required spin rate is 10,000 x g)
- Thermoshaker (required temperature and spin rate are 37 °C and 600 rpm, correspondingly)
Note: Thermoshaker should be appropriate for incubation of microcentrifuge tubes. - Mini centrifuge/vortex
- Analytical balance with 0.1 mg readability
- Ion chromatography system DX-500 (Thermo Fisher Scientific, Dionex) equipped by electrochemical detector ED40, gradient pump GP40 and chromatography oven LC 30
- Refrigerator (required temperature is -20 °C)
- Automatic pipettes (100-1,000, 20-200 and 2-20 μl)
- Mortar and pestle
- Plastic grid and the tray of proper size
Note: The cell of grid should be approximately 1 cm2 or slightly bigger.
Software
- PeakNet software (Thermo Fisher Scientific, Dionex, model: version 5.21)
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Kozlova, L. V., Mikshina, P. V. and Gorshkova, T. A. (2016). Assay of Arabinofuranosidase Activity in Maize Roots. Bio-protocol 6(6): e1764. DOI: 10.21769/BioProtoc.1764.
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分类
植物科学 > 植物生物化学 > 糖类
生物化学 > 糖类 > 多糖
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