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Nov 2021

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A Quick Method to Quantify Iron in Arabidopsis Seedlings
一种快速定量拟南芥幼苗中铁的方法   

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Abstract

Iron (Fe) is an indispensable micronutrient for plant growth and development. Since both deficiency, as well as a surplus of Fe, can be detrimental to plant health, plants need to constantly tune uptake rates to maintain an optimum level of Fe. Quantification of Fe serves as an important parameter for analyzing the fitness of plants from different accessions, or mutants and transgenic lines with altered expression of specific genes. To quantify metals in plant samples, methods based on inductively coupled plasma-optical emission spectrometry (ICP-OES) or inductively coupled plasma-mass spectrometry (ICP-MS) have been widely employed. Although these methods are highly accurate, these methodologies rely on sophisticated equipment which is not always available. Moreover, ICP-OES and ICP-MS allow for surveying several metals in the same sample, which may not be necessary if only the Fe status is to be determined. Here, we outline a simple and cost-efficient protocol to quantify Fe concentrations in roots and shoots of Arabidopsis seedlings, by using a spectroscopy-based assay to quantify Fe2+-BPDS3 complexes against a set of standards. This protocol provides a fast and reproducible method to determine Fe levels in plant samples with high precision and low costs, which does not depend on expensive equipment and expertise to operate such equipment.

Keywords: Arabidopsis (拟南芥), Iron quantification (铁量化), Spectrophotometry (分光光度法), BPDS (BPDS), Plant Nutrition (植物营养)

Background

Iron (Fe) is an essential micronutrient, which is involved in numerous biochemical and physiological processes in plants. A deficiency or an excess of Fe in plants limit plant growth and cause severe losses in crop yield and quality. Imbalances of Fe levels in plants also affect the homeostasis of other nutrients (Schmidt et al., 2020). Therefore, the determination of Fe concentrations in plant tissues is mandatory for the assessment of the nutritional status of the plant and serves as an important parameter for studying Fe-related genes. Currently, inductively coupled plasma (ICP) spectrometry-based methods are considered the gold standard for determining Fe levels. Although these methods are highly sensitive and reliable, their accessibility is rather limited due to costly equipment, specialized training required for the operation of such equipment, and the requirement of generally large amounts of material, which may render the analysis difficult in cases where sample size is limited. Therefore, a simple, reliable, and easily accessible method to measure Fe levels at high accuracy is a valuable alternative to ICP-based methods.


Here, we describe a method for quantifying total Fe in roots and shoots of plant seedlings. The method is based on a protocol established for the analysis of Fe concentration in Plantago lanceolata L. leaves (Schmidt, 1996), which we have adapted and optimized for convenient and routine analysis of the more commonly used model plant Arabidopsis thaliana. The method involves wet acid digestion of dried plant samples with nitric acid and hydrogen peroxide, to solubilize Fe and reduction of Fe(III) complexes by hydroxylammonium chloride, followed by a colorimetric assay based on the ability of the chelating agent bathophenanthroline disulfonate (BPDS) to form red-colored complexes with ferrous Fe. The resulting Fe2+(BPDS)3 complex has a distinct absorbance band at 535nm under acid conditions, allowing for the quantification of Fe concentrations by interpolation from a standard curve. BPDS has been widely used for the determination of Fe levels in a variety of samples (Pré and Benlatrèche, 1977; Tangerås, 1983; Hirayama and Nagasawa, 2017; Freinbichler et al., 2020). The present protocol was optimized for Arabidopsis thaliana samples, and has been used for quantifying Fe concentrations as low as 50 μg/g dry weight sample or less (Tsai et al., 2018; Gautam et al., 2021), but can be modified and applied to determine Fe levels in samples from other plant species.

Materials and Reagents

  1. Butter paper

  2. Tissue paper/Kimwipes

  3. 15 mL screw cap conical tubes (Sarstedt, catalog number: NC1377856)

  4. 96-well microplate (Greiner CELLSTAR®, catalog number: 655180)

  5. Nitric acid (65%) (Merck, catalog number: 1.00456.1000, store at room temperature)

  6. Hydrogen peroxide solution (30%) (Merck, catalog number: 1.07209.1000, store at room temperature)

  7. ddH2O

  8. Bathophenanthrolinedisulfonic acid disodium salt hydrate (Sigma, catalog number: B1375-5G, store at room temperature)

  9. Sodium acetate (Sigma, catalog number: S8750-500G, store at room temperature)

  10. Hydroxylamine hydrochloride (Alfa Aesar, catalog number: A15398.36, store at room temperature)

  11. Iron (III) chloride hexahydrate (Merck, catalog number: 1.03943.0250, store at room temperature inside a dry cabinet)

  12. Assay solution (see Recipes)

  13. Fe standard solutions (see Recipes)

Equipment

  1. Vannas scissors

  2. Drying oven (Hot air oven, PREMA®)

  3. Vortex mixer (Vortex-Genie 2, Scientific Industries)

  4. Heat block (Elite Dry Bath Incubator, Major Science)

  5. Microplate spectrophotometer (Power Wave XS2, BioTek Instruments, Agilent Technologies)

Software

  1. Gen5TM BioTek Instruments (Agilent Technologies)

  2. GraphPad Prism version 9

Procedure

  1. Sampling

    1. Sow seeds on Estelle and Somerville (ES) nutrient media (Estelle and Somerville, 1987), and stratify for at least two days in the dark at 4°C, before transferring to a growth chamber. Grow seedlings at 21–22°C under continuous illumination (50 μmol m2 s−1) for two weeks.

    2. To separate seedlings into shoots and roots for sample harvesting, use Vannas scissors to cut seedlings on the media at hypocotyl junctions.

    3. For shoot samples, collect 25–30 shoots of two-week-old seedlings in butter paper.

    4. For root samples, collect 30–36 roots of two-week-old seedlings in butter paper. Before placing roots into butter paper, make sure that the roots don’t contain any residual media, which also contains Fe. Remove any media from the roots with tissue paper/Kimwipes, followed by washing twice with ddH2O. Then gently dry roots with tissue paper/Kimwipes.

    5. Dry samples in an oven for at least two days at 60–65°C.

    6. Determine the dry weight of each sample.

    7. After weighing, transfer the samples into 15 mL conical tubes.


  2. Sample digestion

    1. Add 225 µL of nitric acid (65%) into each tube and screw the caps tightly.

    2. Incubate for 6 h at 95°C on a heat block, vortexing the tubes every 1–2 h. Make sure that samples do not stick to the walls of the tubes and no precipitates are present, as these could affect measurements.

    3. Carefully unscrew the caps of the tubes, add 150 µL of H2O2 (30%) into each tube, and screw the caps back tightly.

    4. Incubate for 2 h at 56°C on a heat block, and vortex every 30–60 min to make sure no precipitates are formed, as this could also affect measurements.

    5. Add 225 µL of ddH2O. Fully digested samples should be pale yellow in color, and ideally should have no white precipitates.

    6. For the ease of pipetting in the following steps, carefully transfer the digested samples into 1.5 mL microcentrifuge tubes. At this point, samples are ready to be immediately used for the Fe quantification in procedure C, or can be stored at 4°C in the dark for up to 1 month for later analysis.


  3. Standard and sample preparation, and spectroscopic reading

    1. Prepare the assay solution (see Recipe 1) and Fe standard solutions (see Recipe 2).

    2. Add 5 µL of standards/samples in microplate wells. Run three technical replicates for each standard/sample.

    3. Add 245 µL of assay solution to each well with standard/sample.

    4. Cover the microplate and keep it in the dark to incubate at room temperature for 5 min before spectrophotometric measurement. Following incubation, the standards will turn pink/red, with increasing color saturation as the Fe level increases (Figure 1A), and the samples will turn light yellowish-pink/orange-red depending on the Fe content.

    5. Measure absorbance at 535 nm. Use the 0 Fe standards as blanks to zero the plate reader.

Data analysis

  1. Create a standard curve in an Excel sheet by plotting the average blank-corrected 535 nm absorbance values vs. the concentration of standards. Make sure that the coefficient of determination (R2) value is ≥0.990.

  2. Use the equation derived from the standard curve to calculate the Fe concentrations in the samples. Table 1 shows the calculation of Fe concentrations in root samples.

  3. Divide the Fe concentrations of the samples by their respective dry weight (DW), to obtain the final Fe concentration per g DW (Table 1).

  4. Plot the final graph in Excel or any suitable software. The representative plot shown in Figure 1B was generated using GraphPad Prism 9. The sample data shown only represents one biological replicate, with three technical replicates. A complete experiment should have at least three biological replicates.


    Table 1. Calculations to quantify Fe in root samples.

    Spectrophotometric readings of the standards were used to plot a calibration curve. The Fe concentrations in the samples were calculated using the equation derived from the slope. Wild-type (Col-0) plants, a coumarin-deficient mutant (f6’h1-1; Schmid et al., 2014), and an Fe over-accumulating genotype (IMA2ox; Gautam et al., 2021) grown on nutrient media containing 50 µM Fe-EDTA are used to represent Fe quantification. The data represent the standard deviation of three technical replicates from the spectrophotometer reading. The microplate layout used to make spectrophotometric readings is shown at the top. STD, standard; S, sample; Abs., absorbance; DW, dry weight; Avg., average; SD, standard deviation.




    Figure 1. Fe content-dependent color pattern of Fe2+(BPDS)3 complexes and their spectrophotometric quantification.

    A. Representative figure showing the change in color of Fe2+(BPDS)3 complex across an increasing Fe content gradient. Equal volume of assay solution was added in a 96 well plate microplate containing different amounts of Fe (0, 2.5, 5, 10, 20, and 40 µg) in three technical repeats. The microplate was kept in the dark to incubate at room temperature for 5 min, a photo was captured immediately afterwards. B. Graph showing the Fe concentrations in different samples (genotypes). Wild-type (Col-0) plants, a coumarin-deficient mutant (f6’h1-1; Schmid et al., 2014), and an Fe over-accumulating genotype (IMA2ox) (Gautam et al., 2021) grown on nutrient media containing 50 µM of Fe-EDTA are used to represent the Fe quantification. The data represent standard deviation of three technical replicates from the spectrophotometer reading. DW, dry weight.

Notes

  1. This method can be used to quantify Fe in samples grown on both soil and nutrient media.

  2. Steps B to C need to be carried out inside a fume hood with proper protection, including corrosion resistant gloves, safety glasses, lab coat, and shoes.

  3. The sample volume should be reduced or the reagent volumes should be adjusted for complete digestion of samples.

  4. Use only high-grade acid-resistant falcon, PTFE, or TeflonTM tubes for acid digestion. Make sure to tightly cap the tubes during the wet acid digestion steps.

  5. Although the volume of the digested sample is only 600 µL, a 15 mL Falcon tube is used for safety reasons, and to avoid damaging the tubes due to pressure from gas build-up during the heat assisted digestion steps.

Recipes

  1. Assay solution (for 100 mL)

    No.
    Reagent
    Working concentration
    Amount to add

    1.
    Bathophenanthrolinedisulfonic acid disodium salt hydrate (MW: 590.53
    g/mol)
    1 mM
    0.06 g
    2.
    Sodium acetate (MW: 82.03 g/mol)
    0.6 M
    4.92 g

    3.
    Hydroxylamine
    hydrochloride (MW: 69.49 g/mol)
    0.48 M
    3.34 g
    4.
    ddH2O
    -
    adjust to 100 mL
    Note: Prepare fresh assay solution in a light-shield bottle and store at 4°C until use.

  2. Fe standard solutions

    Prepare a 50 mM FeCl3 stock solution (50 mM FeCl3 contains 2.79 µg/µL of Fe). With this stock solution, prepare Fe standards in 1.5 mL microcentrifuge tubes as follows:

    Standards
    Blank
    STD1
    STD2
    STD3
    STD4
    STD5
    STD6
    Fe content (µg)
    0
    1.25
    2.5
    5.0
    10.0
    20.0
    40.0
    Amount of stock to add (µL)
    0
    0.45
    0.90
    1.79
    3.58
    7.17
    14.34
    Nitric acid (65%)
    225 µL into each tube
    H2O(30%)
    150 µL into each tube
    ddH2O
    225 µL into each tube

Acknowledgments

This work was supported by a grant from the Ministry of Science and Technology to W.S. (grant No.: 108-2311-B-001-033-MY3). The data used to create the representative graph and table has been extracted from previously published work (Gautam et al., 2021).

Competing interests

The authors declare no conflict of interest.

References

  1. Freinbichler, W., Misini, B., Colivicchi, M. A., Linert, W., Tipton, K. F. and Della Corte, L. (2020). The application of bathophenanthroline for the determination of free iron in parallel with hROS in microdialysis samples. J Neurosci Methods 331: 108530.
  2. Gautam, C. K., Tsai, H. H. and Schmidt, W. (2021). IRONMAN tunes responses to iron deficiency in concert with environmental pH. Plant Physiol 187(3): 1728-1745.
  3. Hirayama, T. and Nagasawa, H. (2017). Chemical tools for detecting Fe ions. J Clin Biochem Nutr 60(1): 39-48.
  4. Pré, J. and Benlatrèche, C. (1977). Rapid determination of serum iron concentration using bathophenanthroline sulfonate in a formate buffered system. Pathol Biol(Paris) 25(3): 203-205.
  5. Estelle, M. A. and Somerville, C. (1987). Auxin-resistant mutants of Arabidopsis thaliana with an altered morphology. MGG Mol Gen Genet 206: 200-206.
  6. Schmid, N. B., Giehl, R. F., Doll, S., Mock, H. P., Strehmel, N., Scheel, D., Kong, X., Hider, R. C. and von Wiren, N. (2014). Feruloyl-CoA 6'-Hydroxylase1-dependent coumarins mediate iron acquisition from alkaline substrates in Arabidopsis. Plant Physiol 164(1): 160-172.
  7. Schmidt, W., (1996). Influence of chromium(lll) on root-associated Fe(lll) reductase in Plantago lanceolata L. J Exp Bot 47: 805-810.
  8. Schmidt, W., Thomine, S. and Buckhout, T. J. (2020). Editorial: Iron Nutrition and Interactions in Plants. Front Plant Sci 10: 1670.
  9. Tangerås, A., (1983). Iron content and degree of lipid peroxidation in liver mitochondria isolated from iron-loaded rats. Biochim Biophys Acta 757(1): 59-68.
  10. Tsai, H. H., Rodriguez-Celma, J., Lan, P., Wu, Y. C., Velez-Bermudez, I. C. and Schmidt, W. (2018). Scopoletin 8-hydroxylase-mediated fraxetin production is crucial for iron mobilization. Plant Physiol 177(1): 194-207.

简介

[摘要]铁(Fe)是植物生长发育不可缺少的微量营养素。由于铁的缺乏和过量都可能对植物健康有害,因此植物需要不断调整吸收率以保持最佳的铁水平。 Fe 的定量是分析来自不同种质的植物、或具有特定基因表达改变的突变体和转基因系的适应性的重要参数。为了量化植物样品中的金属,基于电感耦合等离子体发射光谱法 (ICP-OES) 或电感耦合等离子体质谱法 (ICP-MS) 的方法已被广泛采用。尽管这些方法非常准确,但这些方法依赖于并不总是可用的复杂设备。此外,ICP-OES 和 ICP-MS 允许测量同一样品中的多种金属,如果仅确定 Fe 状态,则可能没有必要。在这里,我们概述了一个简单且具有成本效益的协议,通过使用基于光谱的测定法根据一组标准量化 Fe 2+ -BPDS 3配合物来量化拟南芥幼苗根和芽中的 Fe 浓度。该协议提供了一种快速且可重复的方法,以高精度和低成本确定植物样品中的 Fe 水平,这不依赖于昂贵的设备和操作此类设备的专业知识。


[背景] 铁 (Fe) 是一种必需的微量营养素,参与植物的许多生化和生理过程。植物中铁的缺乏或过量会限制植物的生长并导致作物产量和质量的严重损失。植物中铁含量的不平衡也会影响其他营养物质的稳态(Schmidt等,2020) 。因此,测定植物组织中的铁浓度对于评估植物的营养状况是必不可少的,并且是研究铁相关基因的重要参数。目前,基于电感耦合等离子体 (ICP) 光谱的方法被认为是确定 Fe 水平的金标准。尽管这些方法具有高度的敏感性和可靠性,但由于设备昂贵、操作此类设备需要专门培训以及通常需要大量材料,因此在样本量大的情况下可能难以进行分析。是有限的。因此,一种简单、可靠且易于访问的高精度测量 Fe 水平的方法是基于 ICP 方法的有价值的替代方法。
在这里,我们描述了一种量化植物幼苗根和芽中总铁的方法。该方法基于为分析车前子叶中铁浓度而建立的协议(Schmidt, 1996) ,我们已经对其进行了调整和优化,以便对更常用的模式植物拟南芥进行方便和常规的分析。该方法包括用硝酸和过氧化氢对干燥的植物样品进行湿酸消化,以溶解 Fe 并通过羟氯化铵还原Fe( III) 配合物,然后基于螯合剂红菲咯啉二磺酸盐(BPDS)的能力进行比色测定与亚铁形成红色络合物。生成的 Fe 2+ (BPDS) 3复合物在酸性条件下在 535 nm 处具有明显的吸收带,允许通过从标准曲线插值来量化 Fe 浓度。 BPDS 已广泛用于测定各种样品中的铁含量(Pré 和 Benlatrèche,1977;Tangerås,1983;Hirayama 和 Nagasawa,2017;Freinbichler等,2020) 。本协议针对拟南芥样品进行了优化,并已用于量化低至 50 的 Fe 浓度 μg /g 干重样品或更少(Tsai等人,2018 年;Gautam等人,2021 年),但可以修改并应用于测定其他植物物种样品中的铁含量。

关键字:拟南芥, 铁量化, 分光光度法, BPDS, 植物营养



材料和试剂


1.黄油纸
2.卫生纸/ Kimwipes
3.15 mL螺帽锥形管( Sarstedt ,目录号:NC1377856)
4.96孔微孔板(Greiner CELLSTAR ® ,目录号:655180)
5.硝酸(65%)(Merck,目录号:1.00456.1000,室温储存)
6.过氧化氢溶液(30%)(Merck,目录号:1.07209.1000,室温储存)
7.ddH 2 O
8.Bathophenanthrolinedisulfonic acid disodium salt hydrate(Sigma,目录号: B1375-5G,室温储存)
9.乙酸钠(Sigma,目录号: S8750-500G,室温储存)
10.盐酸羟胺(Alfa Aesar ,目录号: A15398.36,室温储存)
11.氯化铁(III)六水合物(Merck,目录号:1.03943.0250 ,室温下储存在干燥柜内)
12.检测溶液(见配方)
13.Fe 标准溶液(见配方)




设备


1.万纳斯剪刀
2.烘箱(热风烘箱,PREMA ® )
3.涡流混合器(Vortex-Genie 2,科学工业)
4.热块(精英干浴培养箱,主要科学)
5.微孔板分光光度计(Power Wave XS2, BioTek仪器,安捷伦科技)


软件 


1.第 5代 BioTek仪器(安捷伦科技) 
2.GraphPad 棱镜版本 9


程序


A.采样
1.Estelle 和 Somerville (ES)播种 营养培养基( Estelle 和 Somerville, 1987 年) ,并在 4°C 的黑暗中分层至少两天,然后转移到生长室。在 21 – 22°C 下连续光照 (50 μmol m 2 s -1 ) 培养幼苗两周。
2.要将幼苗分离成芽和根以进行样品采集,请使用Vannas剪刀在下胚轴连接处的介质上切割幼苗。
3.对于芽样,在黄油纸中收集 25 - 30 个两周龄幼苗的芽。
4.对于根部样本,在黄油纸中收集 30 – 36 根两周大的幼苗。在将根放入黄油纸之前,请确保根不包含任何残留介质,其中也包含 Fe。用纸巾/ Kimwipes从根部去除任何培养基,然后用 ddH 2 O 清洗两次。然后用纸巾/ Kimwipes轻轻擦干根部。
5.– 65°C的烘箱中干燥样品至少两天。
6.确定每个样品的干重。
7.称重后,将样品转移到 15 mL 锥形管中。


B.样品消解
1.在每个管中加入 225 μL 的硝酸(65%),并拧紧瓶盖。
2.在 95°C 的加热块上孵育 6 小时,每1-2 小时涡旋一次试管。确保样品不会粘在管壁上并且不存在沉淀物,因为这些可能会影响测量。
3.小心拧开管盖,将 150 μL 的 H 2 O 2 (30%) 添加到每个管中,然后将盖子拧紧。
4.在 56°C 的加热块上孵育 2 小时,每 30-60 分钟涡旋一次,以确保没有形成沉淀物,因为这也会影响测量。
5.加入 225 µL 的 ddH 2 O。完全消化的样品应呈淡黄色,理想情况下不应有白色沉淀。
6.为了便于在以下步骤中移液,请小心地将消化的样品转移到 1.5 mL 微量离心管中。此时,样品已准备好立即用于程序 C 中的 Fe 定量,或者可以在 4°C 的黑暗中储存长达 1 个月以供以后分析。


C.标准品和样品制备,以及光谱读数
1.准备检测溶液(见配方 1)和 Fe 标准溶液(见配方 2)。
2.在微孔板中添加 5 μL 的标准/样品。为每个标准/样品运行三个技术复制。
3.将 245 μL 的测定溶液添加到每个带有标准/样品的孔中。
4.在分光光度测量之前,盖上微孔板并在黑暗中在室温下孵育 5 分钟。孵育后,标准品将变为粉红色/红色,随着Fe 含量的增加,颜色饱和度增加(图 1A) ,样品将根据 Fe 含量变为淡黄粉红色/橙红色。
5.在 535 nm 处测量吸光度。使用 0 Fe 标准品作为空白,将读板器归零。


数据分析


1.通过绘制平均空白校正 535 nm 吸光度值与标准浓度的关系,在 Excel 表中创建标准曲线。确保决定系数 (R 2 ) 值≥ 0.990。
2.使用从标准曲线导出的方程来计算样品中的 Fe 浓度。表 1显示了根样品中 Fe 浓度的计算。
3.将样品的 Fe 浓度除以它们各自的干重 (DW), 以获得每 g DW 的最终 Fe 浓度 (表 1 )。
4.在 Excel 或任何合适的软件中绘制最终图形。图 1B所示的代表性图是使用 GraphPad Prism 9 生成的。显示的样本数据仅代表一个生物复制品,具有三个技术复制品。一个完整的实验应该至少有三个生物学重复。


桌子。 1 根样品中 Fe 的量化计算。
标准品的分光光度读数用于绘制校准曲线。使用从斜率导出的方程计算样品中的 Fe 浓度。野生型 (Col-0) 植物、香豆素缺乏突变体 ( f6'h1-1 ; Schmid等人, 2014)和铁过度积累基因型 ( IMA2ox ; Gautam等人, 2021)生长在养分上含有 50 µM Fe-EDTA 的介质用于表示 Fe 量化。数据代表分光光度计读数的三个技术重复的标准偏差。 用于进行分光光度读数的微孔板布局显示在顶部。 STD,标准; S,样品;绝对值,吸光度; DW,干重;平均,平均; SD,标准偏差。
 




 
数字。 1 Fe 2+ (BPDS) 3配合物的Fe含量依赖性颜色模式及其分光光度定量。
A.代表图显示了 Fe 2+ (BPDS) 3配合物随着 Fe 含量梯度的增加而发生的颜色变化。 将等体积的测定溶液添加到 96 孔板微孔板中,其中包含不同量的 Fe(0、2.5、5、10、20 和 40 µg),重复三次。将微孔板置于暗处室温孵育 5 分钟,然后立即拍照。 B.图表显示不同样品(基因型)中的 Fe 浓度。 野生型 (Col-0) 植物、香豆素缺乏突变体 ( f6'h1-1; Schmid et al. , 2014)和 Fe 过度积累基因型 ( IMA2ox ) (Gautam et al. , 2021)含有 50 μM Fe-EDTA 的营养培养基用于表示 Fe 量化。数据代表分光光度计读数的三个技术重复的标准偏差。 DW,干重。


笔记


1.该方法可用于量化在土壤和营养培养基上生长的样品中的 Fe。
2.步骤 B 到 C 需要在具有适当保护的通风柜内进行,包括防腐蚀手套、安全眼镜、实验室外套和鞋子。
3.应减少样品体积或调整试剂体积以完全消化样品。
4.仅使用高级耐酸 falcon、PTFE 或 Teflon TM管进行酸消解。确保在湿酸消化步骤期间盖紧管子。
5.虽然消化样品的体积仅为 600 µL,但出于安全原因使用 15 mL Falcon 管,并避免在热辅助消化步骤期间由于气体积聚的压力而损坏管。


食谱


1.检测溶液(100 mL)
不。试剂工作浓度添加金额
1.Bathophenanthrolinedisulfonic acid disodium salt hydrate (MW: 590.53 g/mol)1毫米0.06 克
2.乙酸钠(分子量:82.03 g/mol)0.6M4.92 克
3.盐酸羟胺(分子量:69.49 g/mol)0.48 万3.34 克
4.ddH 2 O-调整至 100 毫升
注意:在遮光瓶中制备新鲜的检测溶液,并在 4 ° C 下储存直至使用。


2.铁标准溶液
准备 50 mM FeCl 3库存溶液(50 mM FeCl 3含有 2.79 µg/µL 的 Fe)。使用此库存溶液,在 1.5 mL 微量离心管中制备 Fe 标准品,如下所示:
标准空白的性病1性病2性病3性病4性病5性病6
铁含量 (µg)01.252.55.010.020.040.0
要添加的库存量 (µL)00.450.901.793.587.1714.34
硝酸 (65%)每管 225 µL
H 2 O 2 (30%)每管 150 µL
ddH 2 O每管 225 µL


致谢


这项工作得到了科技部对 WS 的资助(资助号:108-2311-B-001-033-MY3)。用于创建代表性图形和表格的数据是从先前发表的作品中提取的(Gautam等人,2021 年) 。


利益争夺


作者宣称没有利益冲突。


参考


1.Freinbichler, W.、Misini, B.、Colivicchi, MA、Linert, W.、Tipton, KF 和 Della Corte, L. (2020)。红菲咯啉在微透析样品中与 hROS 平行测定游离铁的应用。 J神经科学方法331:108530。
2.Gautam, CK, Tsai, HH 和施密特, W. (2021)。 IRONMAN 可根据环境 pH 值调节对缺铁的反应。 植物生理学187(3):1728-1745。
3.Hirayama, T. 和 Nagasawa, H. (2017)。用于检测 Fe 离子的化学工具。 J Clin Biochem Nutr 60(1):39-48。
4.Pré , J. 和 Benlatrèche, C. (1977)。在甲酸盐缓冲系统中使用红菲咯啉磺酸盐快速测定血清铁浓度。 Pathol Biol(巴黎) 25(3):203-205。
5.Estelle, MA 和 Somerville, C. (1987 年)。具有改变形态的拟南芥生长素抗性突变体。 MGG 分子基因基因 206 :200-206。
6.Schmid, NB, Giehl, RF, Doll, S., Mock, HP, Strehmel, N., Scheel, D., Kong, X., Hider, RC 和 von Wiren, N. (2014)。 Feruloyl-CoA 6'-Hydroxylase1-dependent coumarins 介导拟南芥碱性底物的铁获取。 植物生理学164(1):160-172。
7.施密特,W. (1996)。铬(III)对车前子根相关Fe(III)还原酶的影响。 J Exp Bot 47:805-810。
8.Schmidt, W.、Thomine, S. 和 Buckhout, TJ (2020)。社论:植物中的铁营养和相互作用。 前植物科学10:1670。
9.Tangerås ,A . (1983 年)。从载铁大鼠分离的肝线粒体中铁含量和脂质过氧化程度。 Biochim Biophys Acta 757(1):59-68。
10.Tsai, HH, Rodriguez-Celma, J., Lan, P., Wu, YC, Velez-Bermudez, IC 和 Schmidt, W. (2018)。东莨菪碱8-羟基化酶介导的fraxetin生产对于铁的流动至关重要。 植物生理学177(1):194-207。




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Copyright: © 2022 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Gautam, C. K., Tsai, H. H. and Schmidt, W. (2022). A Quick Method to Quantify Iron in Arabidopsis Seedlings. Bio-protocol 12(5): e4342. DOI: 10.21769/BioProtoc.4342.
  2. Gautam, C. K., Tsai, H. H. and Schmidt, W. (2021). IRONMAN tunes responses to iron deficiency in concert with environmental pH. Plant Physiol 187(3): 1728-1745.
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