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Measuring the Arsenic Content and Speciation in Different Rice Tissues
水稻不同组织中砷含量的测定和形态分析   

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Abstract

Arsenic (As) plays an important role in rice production as vast soils used for rice cultivation contain As. To understand how rice plants deal with inorganic As (III) and As (V) and organic As in their tissue, it is important to obtain specific information on how much and what species of As are present in which tissue of the rice plant. The protocol presented here allows to analyse the As contents and As speciation in roots, shoots, and husks of rice plants, and thus permits direct comparison of the As contents of these rice plant tissues.

Keywords: O. sativa(水稻), Arsenate(砷酸盐), Arsenite(亚砷酸盐)

Materials and Reagents

  1. Rice tissue (roots, shoots, husks) (Muehe et al., 2014)
  2. Liquid nitrogen
  3. 65% HNO3 (Merck KGaA, catalog number: 100456 )
  4. Double-distilled or MilliQ water
  5. >30% H2O2 (Merck KGaA, catalog number: 107298 )
  6. 85% phosphoric acid (Merck KGaA, catalogue number: 100563 )
  7. 0.05 mol/L as stock solution as arsenite (Merck KGaA, catalog number: 106277 ), a 1,000 mg/L as stock solution as arsenate (Merck KGaA, catalog number: 119773 ) diluted to the appropriate concentrations
  8. NH4NO3 (Merck KGaA, catalog number: 102596 )
  9. Eluent A for LC-HPLC-ICP-MS (see Recipes)
  10. Eluent B for LC-HPLC-ICP-MS (see Recipes)

Equipment

  1. Mortar (diameter of at least 10 cm) and pestle (approximately 15 cm long)
  2. Drying oven (adjustable to 60 °C)
  3. WhatmanTM #1 filter papers (GE Healthcare, catalog number: 1440-125 )
  4. Duran® test tubes, straight rim (13 x 100 mm) (Duran Group, catalog number: 26 131 12 )
  5. Polyethylene flanged plug caps for tubes, natural (Globe Scientific, catalog number: 118240 )
  6. Polytetrafluoroethylene (PTFE)-coated spatula (Fisher Scientific, catalog number: 10081074 ) to minimize metal contamination of the plant material from the spatula
  7. Volumetric flask with glass stopper (10 ml)
  8. Tube rack
  9. Water bath
  10. Glass funnel
  11. Inductively coupled plasma atomic emission spectrometry ICP-AES (CIROS)
  12. Inductively coupled plasma mass spectroscopy ICP-MS (Agilent Technologies, model: 7700 )
  13. Autosampler vials (8 mm, brown glass, WICOM, vials are free of As) and screw caps with Teflon septum
  14. Liquid chromatography (Agilent Technologies, model: 1200 ) equipped with a Shodex RSpak NN-614 column (150 mm x 6 mm; Shodex) coupled to inductively coupled plasma mass spectroscopy HPLC-ICP-MS (Agilent Technologies, model: 7700 )

Procedure

  1. Material preparation
    Glassware (tubes, volumetric flasks), pestle, and mortar are soaked in 1 M HCl (wear gloves and safety goggles) for 24 h and washed in distilled water three times and dried before use. This is done to remove metal contaminants sorbed to the equipment.

  2. Harvesting of plant material
    1. Rice plant material is separated into different tissues (roots, shoots, husks, and seeds) using clean scissors and tweezers and wearing gloves.
    2. Plant material is dipped in distilled water for washing.
    3. (Approximately 1-5 g of fresh weight) tissue is ground in liquid nitrogen using a mortar and pestle (3 times, for 30 sec each, see Figure 1) (ground material can also be stored at -80 °C for molecular biology analysis) (Mueller et al., 2013).
    4. An approximately 1 g fresh weight aliquot of the liquid-nitrogen-ground plant material is filled into an opened falcon tube and immediately dried at 60 °C in an oven for a minimum of three days.


      Figure 1. Liquid N2-ground rice shoots

  3. Total arsenic quantification in rice material (wear gloves and goggles)
    1. 20-70 mg of the ground and dried material is weighed into Duran tubes (ensure that plant material is not aggregated).
    2. 2 ml of 65% HNO3 is added to the plant material; followed by mixing the acid and plant material by gentle shaking/swirling of the tube.
    3. Duran tubes are closed with plastic lids.
    4. Rice material is digested over night at room temperature.
    5. Tubes are placed in a rack, which is placed in a water bath.
    6. The material is boiled for 2 h at 100 °C.
    7. 2 ml of ≥30% H2O2 is added to the tubes.
    8. The rice material is boiled until brown gasses stop evolving.
    9. Samples are cooled to room temperature by incubating on the lab bench for an hour.
    10. Material is fully digested if no more tissue pieces are present in the extract.
    11. (If plant material is not fully digested (visible by small bits of see-through tissue), the extract is filtered through Whatman #1 filter papers).
    12. The extract is filled into a volumetric flask.
    13. The Duran tubes are washed with a few ml of distilled water, which is then added to the extract in the volumetric flask.
    14. The extract is filled up to 10 ml with distilled water and stored cool and dark until analysis.
    15. Total As is determined by ICP-AES (concentrations > 0.1 mg/L) or for lower concentrations by ICP-MS (Limit of detection: 0.2 µg/L).

  4. Arsenic speciation in rice material
    1. 20-50 mg of fresh plant material (roots, shoots, and husks) are cut into 1 cm long pieces and are weighed into Falcon tubes immediately after sampling.
    2. 10 ml 10 mM phosphoric acid is added to the plant material (Daus et al., 2008).
    3. Rice material is extracted for 2 h by shaking at 150 rpm and room temperature.
    4. The material is filtered through Whatman #1 filter paper.
    5. To prevent changes in As speciation, samples are immediately filled into the autosampler vials. If unavoidable, samples can be stored in the dark at low temperature (6 °C) until measurement.
    6. The redox species of arsenate and arsenite are analysed with HPLC–ICP–MS using brown glass autosampler vials.
    7. Additionally, the dry weight of the plant material is estimated by weighing an aliquot before and after drying at 60 °C.
    8. The amount of each As species in the fresh plant material is calculated per dry plant material and can be correlated to the total As content of the material.
    9. Please refer to Muehe et al. (2014) for representative amounts of As species calculated using this protocol.

Notes

  1. Seeds can also be sampled, but should be ground in a grinder beforehand.
  2. Grinding of the sample material in liquid nitrogen assures the destruction of the material and thorough mixing of the entire sample material, and thus, unbiased analysis when using small sample aliquots for further procedures.
  3. Storing the liquid N2-ground plant material at -80 °C allows for molecular analysis of the material as well. For example, in Muehe et al. (2014) we extracted RNA from these samples and performed qPCR on the cDNA product of the extracted RNA.
  4. The use of the phosphoric acid as extractant yields an almost complete recovery of the total As present in the material. The As species in these extracts are stable up to 4 weeks if they are stored cool and dark (Daus et al., 2006).
  5. More detailed information on ICP-AES and HPLC-ICP-MS can be obtained in Daus et al. (2002), Daus et al. (2006), Daus et al. (2008) and Noelte (2003).

Recipes

  1. Eluent A for LC-HPLC-ICP-MS
    5 mM HNO3
  2. Eluent B for LC-HPLC-ICP-MS
    5 mM HNO3 + 50 mM NH4NO3 [for As (III) and As (V) only 7 min isocratic 94% A]
    Flow: 1 ml/min, injection volume: 50 µl, LOD = 0.2 µg/L

Acknowledgments

This work was funded by the scholarship program of the German Federal Environmental Foundation to EMM and the Deutsche Forschungsgemeinschaft (DFG) to AK (KA 1736/14-1). The procedure was previously employed in Muehe et al. (2014).

References

  1. Daus, B., Mattusch, J., Wennrich, R. and Weiss, H. (2002). Investigation on stability and preservation of arsenic species in iron rich water samples. Talanta 58(1): 57-65.
  2. Daus, B., Mattusch, J., Wennrich, R. and Weiss, H. (2008). Analytical investigations of phenyl arsenicals in groundwater. Talanta 75(2): 376-379.
  3. Daus, B., Weiss, H., Mattusch, J. and Wennrich, R. (2006). Preservation of arsenic species in water samples using phosphoric acid--limitations and long-term stability. Talanta 69(2): 430-434.
  4. Muehe, E. M., Eisele, J. F., Daus, B., Kappler, A., Harter, K. and Chaban, C. (2014). Are rice (Oryza sativa L.) phosphate transporters regulated similarly by phosphate and arsenate? A comprehensive study. Plant Mol Biol 85(3): 301-316.
  5. Muller, K., Daus, B., Mattusch, J., Vetterlein, D., Merbach, I. and Wennrich, R. (2013). Impact of arsenic on uptake and bio-accumulation of antimony by arsenic hyperaccumulator Pteris vittata. Environ Pollut 174: 128-133.
  6. Noelte, D. (2003). ICP Emission Spectrometry, Wiley-VCH, 1st Edition.

简介

砷(As)在稻米生产中起重要作用,因为用于稻米栽培的广大土壤含有As。 要了解水稻植物如何处理无机砷(III)和As(V)和有机砷在他们的组织,重要的是获得具体的信息,有多少和什么种类的As存在于水稻植物的组织。 这里介绍的协议允许分析水稻植物的根,芽和谷物中的As含量和As形态,因此允许直接比较这些水稻植物组织的As含量。

关键字:水稻, 砷酸盐, 亚砷酸盐

材料和试剂

  1. 稻米组织(根,芽,壳)(Muehe等人,2014)
  2. 液氮
  3. 65%HNO 3(Merck KGaA,目录号:100456)
  4. 双蒸水或MilliQ水
  5. > 30%H 2 O 2(Merck KGaA,目录号:107298)< br />
  6. 85%磷酸(Merck KGaA,目录号:100563)
  7. 作为砷酸盐(Merck KGaA,目录号:106277)的储备溶液为0.05mol/L,稀释至适当浓度的作为砷酸盐的储备溶液(Merck KGaA,目录号:119773)为1,000mg /
  8. NH 4 NO 3(Merck KGaA,目录号:102596)
  9. 用于LC-HPLC-ICP-MS的洗脱液A(参见配方)
  10. 用于LC-HPLC-ICP-MS的洗脱液B(参见配方)

设备

  1. 砂浆(直径至少10厘米)和杵(约15厘米长)
  2. 烘干炉(可调至60°C)
  3. Whatman TM #1滤纸(GE Healthcare,目录号:1440-125)
  4. Duran 试管,直边(13×100mm)(Duran Group,目录号:261312)
  5. 聚乙烯法兰塞帽,管(天然)(Globe Scientific,目录号:118240)
  6. 使用涂有聚四氟乙烯(PTFE)的抹刀(Fisher Scientific,目录号:10081074),以尽量减少刮刀对植物材料的金属污染。
  7. 带玻璃塞的容量瓶(10ml)
  8. 管架
  9. 水浴
  10. 玻璃漏斗
  11. 电感耦合等离子体原子发射光谱法ICP-AES(CIROS)
  12. 电感耦合等离子体质谱ICP-MS(Agilent Technologies,型号:7700)
  13. 自动进样器样品瓶(8 mm,棕色玻璃,WICOM,样品瓶不含As)和带特氟龙隔膜的螺帽
  14. 配备有耦合到电感耦合等离子体质谱HPLC-ICP-MS(Agilent Technologies,型号:7700)的Shodex RSpak NN-614柱(150mm×6mm; Shodex)的液相色谱(Agilent Technologies,型号: />

程序

  1. 物料准备
    将玻璃器皿(试管,容量瓶),研杵和研钵在1M HCl(戴手套和安全护目镜)中浸泡24小时,并在蒸馏水中洗涤三次,并在使用前干燥。 这样做是为了去除吸附在设备上的金属污染物
  2. 植物材料的收获
    1. 将水稻植物材料分成不同的组织(根,枝条, 外壳和种子)使用干净的剪刀和镊子和佩带的手套。
    2. 植物材料浸入蒸馏水中洗涤。
    3. (大约1-5g鲜重)的组织在液体中研磨 氮气使用研钵和研杵(3次,每次30秒,参见图   1)(磨碎的材料也可以储存在-80℃用于分子生物学 分析)(Mueller等人,2013)。
    4. 大约1g新鲜 重量等分的液氮培植物材料 放入开口的Falcon管中并立即在60℃的烘箱中干燥   至少三天。


      图1.液氮 2 地下水稻

  3. 米材料(戴手套和护目镜)中的总砷量化
    1. 将20-70mg研磨和干燥的材料称重到Duran管中(确保植物材料不聚集)
    2. 将2ml的65%HNO 3加入到植物材料中; 然后混合 酸和植物材料通过轻轻摇动/涡旋管。
    3. 杜兰管用塑料盖封闭。
    4. 米材料在室温下消化过夜。
    5. 将管放置在放置在水浴中的架子中。
    6. 将该物质在100℃下煮沸2小时
    7. 向管中加入2ml≥30%H 2 O 2 2。
    8. 将米材料煮沸直到棕色气体停止生长。
    9. 通过在实验室工作台上孵育1小时将样品冷却至室温
    10. 如果提取物中没有更多的组织片,则材料完全消化
    11. (如果植物材料没有完全消化(可见的小部分) 透明组织),将提取物通过Whatman#1过滤器过滤 论文)。
    12. 将提取物装入容量瓶中
    13. Duran管用几毫升蒸馏水洗涤,然后将其加入容量瓶中的提取物中。
    14. 用蒸馏水将提取物填充至10ml并在冷却和黑暗下储存,直到分析
    15. 通过ICP-AES(浓度> 0.1mg/L)或 通过ICP-MS检测较低浓度(检测限:0.2μg/L)。

  4. 米材料中的砷形态
    1. 将20-50mg新鲜植物材料(根,芽和壳)切成   1cm长的片,并立即称重到Falcon管中 抽样
    2. 将10ml 10mM磷酸加入到植物材料中(Daus等人,2008)。
    3. 通过在150rpm和室温下摇动将稻材料提取2小时。
    4. 将材料通过Whatman#1滤纸过滤
    5. 为了防止As形态的变化,样品立即被填充 进入自动进样器样品瓶。 如果不可避免,样品可以存储在   黑暗在低温(6℃)直到测量
    6. 使用棕色玻璃自动进样器小瓶,用HPLC-ICP-MS分析砷酸盐和亚砷酸盐的氧化还原物种。
    7. 此外,植物材料的干重通过在60℃干燥前后称量等分试样来估计
    8. 新鲜植物材料中每种As种类的量 计算每个干植物材料并且可以与总As相关 材料的内容。
    9. 关于使用该方案计算的As物种的代表性量,请参考Muehe等人(2014)。

笔记

  1. 种子也可以取样,但应该事先在研磨机中研磨。
  2. 在液氮中研磨样品材料确保材料的破坏和整个样品材料的充分混合,因此,当使用小样品等分试样进行进一步处理时,可进行无偏差分析。
  3. 将液体N 2 - 离子植物材料储存在-80℃下允许对材料进行分子分析。 例如,在Muehe等人(2014)中,我们从这些样品中提取RNA并进行qPCR 对提取的RNA的cDNA产物
  4. 使用磷酸作为萃取剂产生材料中存在的总As的几乎完全回收。 如果这些提取物中的As物种储存在阴凉和黑暗中,它们可稳定达4周(Daus等人,2006)。
  5. 关于ICP-AES和HPLC-ICP-MS的更详细的信息可以在Daus等人(2002),Daus等人, (2006),Daus (2008)和Noelte(2003)。

食谱

  1. 用于LC-HPLC-ICP-MS的洗脱液A/B 5mM HNO 3
  2. 用于LC-HPLC-ICP-MS的洗脱液B / 5mM HNO 3 + 50mM NH 4 NO 3(仅对于As(III)和As(V))7分钟等度94%A ]
    流速:1ml/min,进样体积:50μl,LOD =0.2μg/L

致谢

这项工作由德国联邦环境基金会向EMM和德意志交易所(DFG)向AK(KA 1736/14-1)的奖学金计划提供资金。 该程序先前用于Muehe等人 (2014)。

参考文献

  1. Daus,B.,Mattusch,J.,Wennrich,R。和Weiss,H。(2002)。 关于富含铁的水样中砷物质的稳定性和保存的调查。 Talanta 58(1):57-65。
  2. Daus,B.,Mattusch,J.,Wennrich,R。和Weiss,H。(2008)。 地下水中苯基砷的分析研究 Talanta 75 (2):376-379。
  3. Daus,B.,Weiss,H.,Mattusch,J。和Wennrich,R。(2006)。 使用磷酸保存水样中的砷物种 - 限制和长期稳定性。 a> 69(2):430-434。
  4. Muehe,E.M.,Eisele,J.F.,Daus,B.,Kappler,A.,Harter,K.and Chaban,C。(2014)。 是水稻( Oryza sativa L.)磷酸盐转运蛋白和砷酸盐? a comprehensive study。 Plant Mol Biol 85(3):301-316。
  5. Muller,K.,Daus,B.,Mattusch,J.,Vetterlein,D.,Merbach,I.and Wennrich,R。(2013)。 砷对砷超累积剂锑的摄取和生物积累的影响 Pteris vittata < em>。 174:128-133。
  6. Noelte,D。(2003)。 ICP Emission Spectrometry,Wiley-VCH,1 版。
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引用:Muehe, E. M., Kappler, A., Chaban, C. and Daus, B. (2015). Measuring the Arsenic Content and Speciation in Different Rice Tissues. Bio-protocol 5(8): e1445. DOI: 10.21769/BioProtoc.1445.
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