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Trehalose is a nonreducing disaccharide. It is a common sugar in bacteria, fungi and yeast, where it functions as a carbon source and stress protectant. In contrast, plants, although encoding large trehalose biosynthesis gene families, contain only small amounts of trehalose. The intermediate compound of trehalose, trehalose-6-phosphate (T6P), is a signaling molecule in plants, regulating metabolism, growth, and development. Most plants contain only a single trehalase, the enzyme that specifically hydrolyzes trehalose into two glucose molecules. High trehalase activity has been suggested to be part of the defense mechanism in plants hosting mycorrhizal fungi, rhizobia, and the plant pathogen Plasmodiophora brassica. Recently, it was shown in Arabidopsis thaliana that high trehalase activity is associated with an increase in drought stress tolerance and that trehalase fulfills an important role in stomatal regulation. Here we describe a protocol for measuring trehalase activity in Arabidopsis tissues, optimized for 96-well plates. Dialyzed protein extracts will be incubated with trehalose, followed by the quantitation of the released glucose using glucose oxidase-peroxidase.

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Trehalase Activity in Arabidopsis thaliana Optimized for 96-well Plates
一种适用于利用96孔板测定海藻糖酶活性的方法

植物科学 > 植物生物化学 > 蛋白质 > 活性
作者: Hilde Van Houtte
Hilde Van HoutteAffiliation: VIB Department of Molecular Microbiology, KU Leuven Laboratory of Molecular Cell Biology, Leuven, Belgium
Bio-protocol author page: a925
 and Patrick Van Dijck
Patrick Van DijckAffiliation: VIB Department of Molecular Microbiology, KU Leuven Laboratory of Molecular Cell Biology, Leuven, Belgium
For correspondence: patrick.vandijck@mmbio.vib-kuleuven.be
Bio-protocol author page: a926
Vol 3, Iss 20, 10/20/2013, 3723 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.946

[Abstract] Trehalose is a nonreducing disaccharide. It is a common sugar in bacteria, fungi and yeast, where it functions as a carbon source and stress protectant. In contrast, plants, although encoding large trehalose biosynthesis gene families, contain only small amounts of trehalose. The intermediate compound of trehalose, trehalose-6-phosphate (T6P), is a signaling molecule in plants, regulating metabolism, growth, and development. Most plants contain only a single trehalase, the enzyme that specifically hydrolyzes trehalose into two glucose molecules. High trehalase activity has been suggested to be part of the defense mechanism in plants hosting mycorrhizal fungi, rhizobia, and the plant pathogen Plasmodiophora brassica. Recently, it was shown in Arabidopsis thaliana that high trehalase activity is associated with an increase in drought stress tolerance and that trehalase fulfills an important role in stomatal regulation. Here we describe a protocol for measuring trehalase activity in Arabidopsis tissues, optimized for 96-well plates. Dialyzed protein extracts will be incubated with trehalose, followed by the quantitation of the released glucose using glucose oxidase-peroxidase.
Keywords: Arabidopsis thaliana(拟南芥), Trehalose(海藻糖), Trehalase(海藻糖酶)

[Abstract]

Materials and Reagents

  1. Plant tissues
  2. Liquid nitrogen
  3. MES
  4. Phenylmethylsulfonyl fluoride (PMSF)
  5. EDTA
  6. Polyvinylpyrrolidone (PVP)
  7. Dithiothreitol (DTT)
  8. CaCl2
  9. Glucose
  10. Bovine serum albumin (BSA)
  11. Na2CO3
  12. K-Na-tartrate
  13. CuSO4.5H2O
  14. KOH
  15. NaOH
  16. Folin & Ciocalteu's phenol reagent (Sigma-Aldrich, catalog number: 47641 )
  17. Trehalose (Sigma-Aldrich, catalog number: T9531 )
  18. Glucose, GOD-PAP (DIALAB GmbH, catalog number: D95218B )
  19. Extraction buffer (see Recipes)
  20. Dialysis buffer (see Recipes)
  21. Trehalose buffer (see Recipes)
  22. Glucose standards (see Recipes)
  23. BSA standards (see Recipes)
  24. Lowry buffers (see Recipes)

Equipment

  1. Mortars and pestles
  2. Spectra/Por® 1 Dialysis Membrane (IEEE, catalog number: 132660 )
    Note: 96-well dialysis systems can be used for the dialysis of multiple samples.
  3. Transparent 96-well plates with flat bottom
  4. Rocker
  5. Refrigerated microcentrifuge
  6. Pre-chilled microcentrifuge tubes
  7. 100 ml cylinder
  8. Magnetic stir plate and magnet
  9. Cold room
  10. Spectrophotometric plate reader

Procedure

  1. Protein extraction
    Note: Work always on ice unless stated differently.
    1. Grind plant tissues in liquid nitrogen with mortar and pestle.
    2. Aliquot 100 mg tissue powder in chilled microcentrifuge tubes. Prepare at least 3 replicates per sample.
    3. Add 1 ml of ice cold extraction buffer to each sample. Homogenize samples by pipetting up and down. Centrifuge at 18,000 x g, 4 °C, 10 min.
    4. Transfer the supernatant to a new, chilled microcentrifuge tube. The obtained protein extracts can be stored at -80 °C.

  2. Dialysis
    1. Wet a piece of Spectra/Por?1 Dialysis Membrane with water and tie a knot at the bottom (Figure 1).
    2. Transfer 500 μl of the protein extract into the tubing and tie a knot at the top (Figure 1).


      Figure 1. Tying a knot at the bottom and top of a dialysis membrane

    3. Place tubing in a 100 ml cylinder filled with ice cold dialysis buffer. Dialyze the extract at 4 °C for 2-3 h under continuous stirring on a magnetic stir plate.
    4. Replace dialysis buffer and continue the dialysis at 4 °C overnight.
    5. Transfer the dialyzed extracts to new, chilled microcentrifuge tubes. The dialyzed extracts can be stored at -80 °C.

  3. Trehalase activity adapted for 96-well plates
    Note: Work always on ice unless stated differently.
    1. Prepare a water bath at 95 °C.

    For samples

  1. Transfer 10 μl of the dialysis product in a 96-well plate with flat bottom (= plate S).
  2. Transfer 10 μl of each respective glucose standard to plate S.

    For blanks

  1. Transfer 10 μl of the dialysis product in a 96-well plate with flat bottom (= plate B).
  2. Place plate B at 95 °C temperature for 5 minutes to denature the trehalase enzyme present in the blanks.
  3. Place plate B on ice for 2 min.

    For samples and blanks 

  1. Add 50 μl of trehalose buffer to plates S and B. Mix by pipetting up and down.
  2. Incubate plates S and B for 30 min on a rocker at 30 °C.
  3. Stop the reaction by boiling for 5 min at 95 °C (denaturation of the trehalase).
  4. Place plates S and B on ice for 2 min.
  5. Add 200 μl of Glucose, GOD-PAP to plates S and B for determining the glucose concentration of the samples and blanks by colorimetry. Mix by pipetting up and down.
  6. Incubate plates S and B for 15 min at 30 °C on a rocker.
  7. Measure the absorbance of plates S and B at 505 nm with a spectrophotometric plate reader (30 °C).
  8. Determine the glucose standard curve to calculate the glucose concentration (nmol) present in the samples and blanks. Subtract the glucose concentration of the blanks from the samples (see Example: Calculating the trehalase activity in excel).

    For proteins (Lowry procedure, Van Houtte et al., 2013)

  1. Transfer 10 μl of the dialysis product in a 96-well plate with flat bottom (= plate P) and add 30 μl water to these samples.
  2. Transfer 40 μl of each respective BSA standard to plate P.
  3. Add 200 μl of Reagent C (Lowry buffers) to plate P and incubate for 10 min at room temperature.
  4. Add 20 μl of Reagent D (Lowry buffers) to plate P and incubate for 30 min at 30 °C.
  5. Measure the absorbance of plate P at 546 nm with a spectrophotometric plate reader (30 °C).
  6. Use the BSA standard curve to determine the amount of protein (μg) present in the extracts (see Example: Calculating the trehalase activity in excel).
  7. Express the trehalase specific activity as nmol of glucose released per min per mg protein (see Example: Calculating the trehalase activity in excel).

Data analysis

Here we show an example how to calculate the trehalase activity from a protein extract of Arabidopsis Col-0 seedlings.

  1. Glucose standard curve
    Using excel, plot the glucose concentration of the respective glucose standards on the X axis and the corresponding absorbances (505 nm) on the Y axis (Table 1; Figure 2). Add a linear trendline to the glucose standard curve and diplay its equation (Figure 2; [1]).

    [1] y = 0.0157x + 0.0858

    Table 1. Absorbances at 505 nm (Plates S and B)
    Glucose standards
    Absorbance (505 nm)
    0 nmol glucose
    0.08
    20 nmol glucose
    0.3721
    40 nmol glucose
    0.7421
    60 nmol glucose
    1.0355
    80 nmol glucose
    1.3792
    100 nmol glucose
    1.6143
    Arabidopsis Col-0 tissues
    Absorbance (505 nm)
    Sample
    0.1483
    Blank
    0.0898


    Figure 2. Glucose standard curve

  2. Determination of the glucose concentration
    Equation [1] and the absorbances (505 nm) of the sample and blank (Table 1) can be used to calculate the glucose concentration present in the sample and blank.
    [2] Nmol glucose in sample = (0.1483-0.0858)/0.0157
      = 3.9809

    [3] Nmol glucose in blank = (0.0898-0.0858)/0.0157
      = 0.2548

    In order to know how many glucose is released per min in the extract, subtract [3] from [2], and divide by the duration of the incubation time (min).

    [4] Nmol glucose released per min = (3.9809 - 0.2548)/30
      = 0.1242


  3. BSA standard curve
    Since trehalase activity is expressed per unit of protein, we need to determine the amount of protein present in the extract. Using excel, plot the protein content of the respective BSA standards on the X axis and the corresponding absorbances (546 nm) on the Y axis (Table 2; Figure 3). Add a linear trendline to the BSA standard curve and display its equation (Figure 3; [5]).


    Figure 3. BSA standard curve

    [5] y = 0.0242x + 0.0576

  4. Determination of the protein content
    Equation [5] and the absorbance (546 nm) of the protein extract (Table 2) can be used to calculate the protein content.

    Table 2. Absorbances at 546 nm (Plate P)
    BSA standards
    Absorbance (546 nm)
    0 μg protein
    0.0488
    10 μg protein
    0.3224
    20 μg protein
    0.5603
    30 μg protein
    0.7159
    40 μg protein
    1.0637
    Arabidopsis Col-0 tissues Absorbance (505 nm)
    Protein 0.4786

    [6] μg protein in extract = (0.4786 - 0.0576)/0.0242
      = 17.3967


  5. Trehalase specific activity of Arabidopsis Col-0 seedlings
    Since the specific trehalase activity is expressed as nmol glucose produced per min per mg protein, we need to divide [4] by [6], and multiply by 1,000.

    [7] Trehalase specific activity in nmol glucose per min per mg protein = 0.1242/17.3967*1,000
      = 7.1393

Recipes

  1. Extraction buffer
    0.1 M MES-KOH, pH 6
    1 mM PMSF
    1 mM EDTA
    1% (w/v) PVP
    1 mM DTT
    Stored at 4 °C
  2. Dialysis buffer
    10 mM MES-KOH, pH 7
    50 μM CaCl2
    Stored at 4 °C
  3. Trehalose buffer
    250 mM trehalose
    62.5 mM MES-KOH, pH 7
    125 μM CaCl2
    Stored at 4 °C
  4. Glucose standards
    Make standards with 10, 8, 6, 4, 2 and 0 μl of a 10 mM glucose solution in a total V of 10 μl
    Fresh prepared or store 10 μl aliquots at -20 °C
  5. BSA standards
    Make standards with 40, 30, 20, 10 and 0 μl of a 1 mg/ml BSA solution in a total V of 40 μl
    Fresh prepared and keep on ice
  6. Lowry buffers
    Reagent A:
    2% (w/v) Na2CO3, 0.02% (w/v) K-Na-tartrate in 0.1 M NaOH
    Stored at room temperature
    Reagent B:
    1% (w/v) CuSO4.5H2O
    Stored at room temperature
    Reagent C:
    Mix solution A and B (100:1, [v/v])
    Fresh prepared
    Reagent D:
    Mix Folin & Ciocalteu's phenol reagent with MilliQ water (1:2, [v/v])
    Fresh prepared

Acknowledgments

This protocol was developed in the framework of the following paper: Van Houtte et al. (2013). It was developed based on two previous publications: Brodmann et al. (2002) and Pernambuco et al. (1996). Hilde Van Houtte was supported by the KU Leuven industrial research fund (IOF/KP/08/001). This work was supported by a grant from the FWO (G.0859.10).

References

  1. Brodmann, A., Schuller, A., Ludwig-Muller, J., Aeschbacher, R. A., Wiemken, A., Boller, T. and Wingler, A. (2002). Induction of trehalase in Arabidopsis plants infected with the trehalose-producing pathogen Plasmodiophora brassicae. Mol Plant Microbe Interact 15(7): 693-700.
  2. Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent.J Biol Chem 193(1): 265-275. 
  3. Pernambuco, M. B., Winderickx, J., Crauwels, M., Griffioen, G., Mager, W. H. and Thevelein, J. M. (1996). Glucose-triggered signaling in Saccharomyces cerevisiae: different requirements for sugar phosphorylation between cells grown on glucose and those grown on non-fermentable carbon sources. Microbiology 142(7): 1775-1782.
  4. Van Houtte, H., Vandesteene, L., Lopez-Galvis, L., Lemmens, L., Kissel, E., Carpentier, S., Feil, R., Avonce, N., Beeckman, T., Lunn, J. E. and Van Dijck, P. (2013). Overexpression of the trehalase gene AtTRE1 leads to increased drought stress tolerance in Arabidopsis and is involved in abscisic acid-induced stomatal closure. Plant Physiol 161(3): 1158-1171. 

材料和试剂

  1. 植物组织
  2. 液氮
  3. MES
  4. 苯甲基磺酰氟(PMSF)
  5. EDTA
  6. 聚乙烯吡咯烷酮(PVP)
  7. 二硫苏糖醇(DTT)
  8. CaCl <2>
  9. 葡萄糖
  10. 牛血清白蛋白(BSA)
  11. Na 2 3
  12. 酒石酸钾
  13. CuSO 4 5H sub 2 O
  14. KOH
  15. NaOH
  16. Folin& Ciocalteu的酚试剂(Sigma-Aldrich,目录号:47641)
  17. 海藻糖(Sigma-Aldrich,目录号:T9531)
  18. 葡萄糖,GOD-PAP(DIALAB GmbH,目录号:D95218B)
  19. 提取缓冲液(参见配方)
  20. 透析缓冲液(参见配方)
  21. 海藻糖缓冲液(见配方)
  22. 葡萄糖标准(见配方)
  23. BSA标准(参见配方)
  24. Lowry缓冲区(参见配方)

设备

  1. 砂浆和杵
  2. Spectra/Por ® 1 透析膜(IEEE,目录号:132660)
    注意:96孔透析系统可用于透析多个样品。
  3. 具有平底的透明96孔板
  4. 摇杆
  5. 冷藏微量离心机
  6. 预冷微量离心管
  7. 100ml气瓶
  8. 磁力搅拌板和磁铁
  9. 冷室
  10. 分光光度读数仪

程序

  1. 蛋白质提取
    注意:除非另有说明,否则始终在冰上工作。
    1. 研磨植物组织在液氮中用研钵和杵。
    2. 在冷冻的微量离心管中等分100毫克组织粉末。 每个样品至少准备3次重复。
    3. 向每个样品中加入1ml冰冷的提取缓冲液。 通过上下吹吸使样品均匀。 以18,000×g离心,4℃,10分钟。
    4. 转移上清液到一个新的,冷冻的微量离心管。 所得蛋白质提取物可在-80℃保存
  2. 透析
    1. 用水冲洗一片Spectra/Por透析膜,在底部打结(图1)。
    2. 将500μl的蛋白质提取物转移到管中,并在顶部打结(图1)。


      图1.在透析膜底部和顶部打一个结

    3. 将管放置在装有冰冷的透析缓冲液的100ml圆筒中。 在磁力搅拌板上连续搅拌下,将提取物在4℃透析2-3小时
    4. 更换透析缓冲液,并在4℃下继续透析过夜。
    5. 将透析的提取物转移到新的冷冻微量离心管中。 透析的提取物可以储存在-80℃
  3. 海藻糖酶活性适用于96孔板
    注意:除非另有说明,否则始终在冰上工作。
    1. 准备95℃的水浴。

    对于样品

  1. 转移10微升透析产品在96孔板平底(=板S)。
  2. 将10微升各葡萄糖标准品转移至平板S.

    对于空白

  1. 转移10微升透析产品在96孔板平底(=板B)。
  2. 将板B在95℃的温度下放置5分钟以使存在于空白中的海藻糖酶变性
  3. 将板B在冰上放置2分钟。

    对于样品和空白

  1. 向板S和B中加入50μl海藻糖缓冲液。通过上下吹吸混匀
  2. 孵育板S和B在30℃摇床上30分钟。
  3. 通过在95℃下煮沸5分钟(海藻糖酶的变性)停止反应
  4. 将板S和B放在冰上2分钟
  5. 添加200微升葡萄糖,GOD-PAP到板S和B,用于通过比色法测定样品和空白的葡萄糖浓度。 通过上下吹吸混合。
  6. 孵育板S和B在30°C在摇床上15分钟。
  7. 用分光光度读数板(30℃)测量板S和B在505nm处的吸光度
  8. 确定葡萄糖标准曲线以计算样品和空白中存在的葡萄糖浓度(nmol)。 从样品中减去空白的葡萄糖浓度(参见实施例:计算excel中的海藻糖酶活性)。

    对于蛋白质(Lowry过程,Van Houtte等人,2013

  1. 转移10微升的透析产品在平底96孔板(=板P),并加入30微升水到这些样品。
  2. 将40μl各自的BSA标准品转移至平板P.
  3. 加入200μl的试剂C(Lowry缓冲液)到板P,并在室温下孵育10分钟。
  4. 加入20μl试剂D(Lowry缓冲液)到板P,并在30°C孵育30分钟。
  5. 用分光光度板读数器(30℃)测量板P在546nm的吸光度
  6. 使用BSA标准曲线来确定提取物中存在的蛋白质(μg)的量(参见实施例:计算excel中的海藻糖酶活性)。
  7. 表示海藻糖酶比活性为每分钟每mg蛋白质释放的葡萄糖的nmol(参见实施例:计算excel中的海藻糖酶活性)。

数据分析

在这里我们显示如何计算来自拟南芥 Col-0幼苗的蛋白质提取物的海藻糖酶活性的实例。

  1. 葡萄糖标准曲线
    使用excel,绘制各个葡萄糖标准品的葡萄糖浓度在X轴上和相应的吸光度(505nm)在Y轴上(表1;图2)。 将线性趋势线添加到葡萄糖标准曲线和显示 其方程(图2; [1])。

    [1] y = 0.0157x + 0.0858

    表1.在505nm(板S和B)的吸光度
    葡萄糖标准
    吸光度(505nm)
    0nmol葡萄糖 0.08
    20 nmol葡萄糖 0.3721
    40nmol葡萄糖 0.7421
    60 nmol葡萄糖 1.0355
    80 nmol葡萄糖 1.3792
    100nmol葡萄糖 1.6143
    拟南芥 Col-0组织
    吸光度(505nm)
    样品
    0.1483
    空白
    0.0898


    图2.葡萄糖标准曲线

  2. 葡萄糖浓度的测定
    可以使用方程式[1]和样品和空白的吸光度(505nm)(表1)来计算样品和空白中存在的葡萄糖浓度。
    [2]样品中的Nmol葡萄糖 =(0.1483-0.0858)/0.0157
      = 3.9809

    [3] Nmol葡萄糖在空白 =(0.0898-0.0858)/0.0157
      = 0.2548

    为了知道提取物中每分钟释放多少葡萄糖,从[2]中减去[3],并除以孵育时间(min)的持续时间。

    [4]每分钟释放的Nmol葡萄糖 =(3.9809-0.2548)/30
      = 0.1242


  3. BSA标准曲线
    由于海藻糖酶活性是每单位蛋白质表达的,我们需要确定提取物中存在的蛋白质的量。 使用excel,在X轴上绘制相应BSA标准物的蛋白质含量,在Y轴上绘制相应的吸光度(546nm)(表2;图3)。 将线性趋势线添加到BSA标准曲线,并显示其方程(图3; [5])

    图3. BSA标准曲线

    [5] y = 0.0242x + 0.0576

  4. 蛋白质含量的测定
    方程[5]和蛋白质提取物的吸光度(546nm)(表2)可用于计算蛋白质含量。

    表2.在546nm(板P)的吸光度
    BSA标准
    吸光度(546 nm)
    0μg蛋白质 0.0488
    10μg蛋白质 0.3224
    20μg蛋白质 0.5603
    30μg蛋白质
    0.7159
    40μg蛋白质 1.0637
    拟南芥 Col-0组织 吸光度(505nm)
    蛋白 0.4786

    [6]提取物中的蛋白质 =(0.4786-0.0576)/0.0242
      = 17.3967


  5. 拟南芥 Col-0幼苗的海藻酸酶比活性
    由于特异性海藻糖酶活性表示为每分钟每mg蛋白质产生的nmol葡萄糖,我们需要将[4]除以[6],并乘以1,000。

    [7]海藻糖酶比活性,以nmol葡萄糖/分钟/mg蛋白质计 = 0.1242/17.3967 * 1000
      = 7.1393

食谱

  1. 提取缓冲区
    0.1M MES-KOH,pH 6
    1mM PMSF
    1mM EDTA
    1%(w/v)PVP
    1 mM DTT
    储存在4°C
  2. 透析缓冲液
    10mM MES-KOH,pH 7
    50μMCaCl 2
    储存在4°C
  3. 海藻糖缓冲液
    250mM海藻糖 62.5mM MES-KOH,pH7
    125μMCaCl 2
    储存在4°C
  4. 葡萄糖标准
    使用10,8,6,4,2和0μl的10 mM葡萄糖溶液制备标准品,总V为10μl
    新鲜配制或存储10微升等分试样在-20°C
  5. BSA标准
    使用40,30,20,10和0μl的1 mg/ml BSA溶液制备标准品,总体积为40μl
    新鲜准备,保持冰上
  6. 洛瑞缓冲区
    试剂A:
    在0.1M NaOH中的2%(w/v)Na 2 CO 3,0.02%(w/v)酒石酸钾Na / 在室温下贮存
    试剂B:
    1%(w/v)CuSO 4 sub。 5H O 在室温下贮存
    试剂C:
    混合溶液A和B(100:1,[v/v]) 新鲜准备
    试剂D:
    Mix Folin& Ciocalteu的酚试剂与MilliQ水(1:2,[v/v])
    新鲜准备

致谢

该协议在以下论文的框架中开发:Van Houtte等人(2013)。 它是基于两个以前的出版物开发的:Brodmann等人(2002)和Pernambuco等人(1996)。 Hilde Van Houtte由KU Leuven工业研究基金(IOF/KP/08/001)提供支持。 这项工作得到了FWO(G.0859.10)的资助。

参考文献

  1. Brodmann,A.,Schuller,A.,Ludwig-Muller,J.,Aeschbacher,R.A.,Wiemken,A.,Boller,T.and Wingler,A。(2002)。 在用产生海藻糖的病原体感染的拟南芥植物中诱导海藻糖酶< em> Plasmodiophora brassicae 。 Mol Plant Microbe Interact 15(7):693-700。
  2. Lowry,O.H.,Rosebrough,N.J.,Farr,A.L.and Randall,R.J。(1951)。 使用Folin酚试剂进行蛋白质测量 J Biol Chem 193(1):265-275。
  3. Pernambuco,M.B.,Winderickx,J.,Crauwels,M.,Griffioen,G.,Mager,W.H.and Thevelein,J.M。(1996)。 酿酒酵母中葡萄糖触发的信号::糖磷酸化的不同要求在葡萄糖上生长的细胞和在不可发酵的碳源上生长的细胞。 142(7):1775-1782。
  4. Van Houtte,H.,Vandesteene,L.,Lopez-Galvis,L.,Lemmens,L.,Kissel,E.,Carpentier,S.,Feil,R.,Avonce,N.,Beeckman, JE和Van Dijck,P。(2013)。 海藻糖酶基因AtTRE1的过度表达导致增加的干旱胁迫耐受性< em> Arabidopsis 并参与脱落酸诱导的气孔关闭。植物生理学 161(3):1158-1171。
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How to cite this protocol: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Houtte, H. V. and Dijck, P. V. (2013). Trehalase Activity in Arabidopsis thaliana Optimized for 96-well Plates. Bio-protocol 3(20): e946. DOI: 10.21769/BioProtoc.946; Full Text
  2. Van Houtte, H., Vandesteene, L.,Lopez-Galvis, L., Lemmens, L., Kissel, E., Carpentier, S., Feil, R., Avonce,N., Beeckman, T., Lunn, J. E. and Van Dijck, P. (2013). Overexpressionof the trehalase gene AtTRE1 leads to increased drought stress tolerancein Arabidopsis and is involved in abscisic acid-induced stomatalclosure. Plant Physiol 161(3): 1158-1171.




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