Summary

元素成像保护湿地根和岩石圈的方法

Published: February 15, 2021
doi:

Summary

我们描述了一个协议,以水稻(Oryza水稻 L.)为模型物种,从湿地环境中取样、保存和分割完整的根部和周围的岩石圈土壤。保存后,可以使用元素成像技术(如同步加速器 X 射线荧光 (XRF) 化学光谱成像技术分析样品。

Abstract

根与土壤环境广泛相互作用,但可视化根与周围岩石圈之间的这种相互作用是具有挑战性的。湿地植物的岩石圈化学由于从根部到散装土壤的陡峭氧梯度,在捕获方面尤其具有挑战性。这里描述了一个协议,通过猛击冷冻和冷冻干燥,有效地保留了湿地植物的根系结构和岩石圈化学。大满贯冷冻,样品被冻结在铜块之间预先冷却液氮,尽量减少根部损伤和样品失真,可能发生闪光冻结,同时仍然尽量减少化学规格的变化。虽然样品失真仍有可能,但快速且成本最低的多个样品的能力增加了获得满意样品和优化成像时间的潜力。资料显示,该方法成功地保存了与铁斑块相关的稻根和生虫的减少砷物种。这种方法可用于研究各种湿地环境中的植物-土壤关系,这些环境跨越了从微量元素循环到植物化应用等各种浓度范围。

Introduction

根及其生殖球是动态的,异质的,对于理解植物如何获得矿物养分和污染物1,2,3至关重要。根系是营养物质(例如磷)和污染物(例如砷)从土壤转移到植物的主要途径,因此了解这个过程对食物数量和质量、生态系统功能和植物灭菌有影响。然而,根是动态的空间和时间增长,以满足营养获取的需求,他们往往在功能,直径和结构(例如,横向根,冒险根,根毛)2。根系的异质性可以在从细胞到生态系统的空间尺度上以及从每小时到十进制的时间尺度上进行研究。因此,根及其周围土壤(或日光圈)的动态和异质性对捕捉日光圈化学提出了挑战。尽管面临这一挑战,但必须研究土壤环境中的根源,以描述这种关键的植物与土壤关系。

湿地植物的岩石圈化学研究尤其具有挑战性,因为从散装土壤到根部存在陡峭的氧梯度,在空间和时间上都发生了变化。由于根部需要氧气来恢复,湿地植物已经适应了湿地土壤的低氧条件,创造了4,5。Aerenchyma 是空心皮质组织,从芽延伸到根部,允许空气通过植物扩散到根部。然而,其中一些空气泄漏到岩石圈在较低的亚化部分的根,特别是附近的横向根结,不太成熟的根尖和拉长区6,7,8,9。这种径向缺氧在湿地植物的日生圈中形成了一个氧化区,影响生化层(生物-地理)化学,与减少的散装土壤10、11、12有区别。要了解湿地生殖圈和根部营养物质和污染物的命运和运输,必须保存化学减少的散装土壤、氧化的生殖圈和湿地植物的根部进行分析。然而,由于散装土壤中含有对氧敏感的土壤成分减少,根部和土壤保护方法必须保留根部结构并尽量减少对氧敏感的反应。

存在修复植物组织和保存超结构成像的方法,但这些方法不能应用于化学保存生长在湿地土壤中的根部。对于只需要植物细胞内元素分布的调查,植物通常以水力方式生长,根部可以很容易地从溶液中去除,在高压冻结和冷冻替代下固定,并分割成各种成像应用,包括高分辨率二次离子质谱(nanoSIMS)、电子显微镜和同步加速器X射线荧光 (S-XRF) 分析13, 1415.为了研究湿地根部外的Fe斑块,这些水培研究必须在溶液16中人工诱导Fe斑块的形成,不能准确地代表Fe斑块形成的分布和矿物成分的异质性以及原位17、18、19、20等相关元素。有保存湿地土壤和与冻土有关的微生物的方法,但很难用这种技术获得根基。目前可视化土壤中生长的根及其生殖层化学的方法包括两种主要测量类型:元素通量和总元素浓度(和规格)。前者通常使用薄膜 (DGT)22、23、24中的扩散梯度进行测量,其中土壤被放置在 rhizobox 中,以支持实验室环境中的植物生长,土壤中的阴唇元素通过凝胶扩散到结合层中。然后,可以成像此绑定层,以量化感兴趣的阴唇元素。这项技术可以成功地说明根与日光圈24、25、26、27之间的关系,但根界的人工制品可能通过在rhizots中生长植物而存在,而且没有用DGT捕获有关根内部的信息。后者涉及根部和岩石圈的采样,保存样本,并直接分析样本部分的元素分布。对于湿地植物根部及其周围生虫圈的环境采样,需要仔细的样品处理,以避免样品制备中的人工制品。

这里描述了一个协议,通过猛击冷冻和冷冻干燥,有效地保护了湿地植物的根系结构和岩石圈化学。闪光冻结可以大大减缓对氧敏感溶质的转化,但可能会损坏根部,当样品干涸时,可能会引起动员。然而,在用液氮预冷的铜块之间冷冻样品的重度冻结,可最大限度地减少根部损伤和样品失真28。然后,保存下来的样品被嵌入环氧树脂中,保存为20、29,并可以切割和抛光,以成像其岩石圈土壤中的根部。本报告中的样本在薄截面后由 S-XRF 化学规格成像分析。但是,还可以使用其他成像技术,包括激光消融感应耦合等离子质谱 (LA-ICP-MS)、粒子诱导 X 射线发射 (PIXE)、二次离子质谱 (SIMS) 和激光诱导故障光谱 (LIBS) 成像。

Protocol

1. 准备防冻设备 将两个铜块(+5 厘米 x 5 厘米 x 15 厘米)水平放置在能够容纳液氮的清洁冷却器内,并倒入足够的液氮以淹没方块。一旦冒泡消退,将两个隔垫放在每端一个铜块的顶部。注:垫片高度决定要冻结的样品的高度:此示例使用 2 厘米隔间创建约 3 厘米 x 3 厘米 x 2 厘米的立方体。液氮的体积取决于冷却器的大小。此示例对大约 5 个立方体系列使用大约 1 L。注意:使用适?…

Representative Results

这种方法允许在湿地植物的根和生土层中保存根部和化学物种,并进入散装土壤。在这项工作中,该方法用于评估稻米(Oryza水稻 L.)中与Fe和Mn氧化物以及植物养分的规格和共定位。水稻是在特拉华大学的水稻设施中种植的,在那里,30个稻田间皮(每株2米x2米,49株)用于在各种水土管理条件下种植水稻,目的是降低水稻谷物的吸收率和CD。这个实验提供了1470种单个植物,在整个生长季节…

Discussion

本文描述了一种利用可用于元素成像和/或化学规格映射的重度冷冻技术获取湿地植物根部保存的散装土壤和生肖球的协议。

与现有方法比较,此方法有几个优点。首先,这种方法允许同时调查根部和周围的生虫圈。目前存在的方法是,通过冲走土壤和保存根31、32或在人工环境中种植植物(如rhizotbox),以及使用DGT方法检查根-土壤…

Disclosures

The authors have nothing to disclose.

Acknowledgements

作者承认向塞弗斯和塔佩罗提供联合种子赠款,以支持特拉华大学和布鲁克黑文国家实验室之间的合作。这项研究的部分内容使用了国家同步光源II的XFM(4-BM)光束线,这是美国能源部(DOE)科学用户设施办公室,由布鲁克黑文国家实验室根据合同号为能源部科学办公室运营。DE-SC0012704。

Materials

Copper blocks McMaster Carr 89275K42
Diamond blade Buehler 15 LC, 102 mm x 0.3 mm operation speed: 225 rpm
Epoxy forms Struers 40300085 FixiForm
Epoxy Epotek 301-2FL
Superglue Loctite 404
Thin sectioning machine Buehler PetroThin
Wet saw Buehler IsoMet 1000

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Cite This Article
Seyfferth, A. L., Limmer, M. A., Tappero, R. A Method to Preserve Wetland Roots and Rhizospheres for Elemental Imaging. J. Vis. Exp. (168), e62227, doi:10.3791/62227 (2021).

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