Tracking deuterium uptake in hydroponically grown maize roots using correlative helium ion microscopy and Raman micro-spectroscopy.

Abstract

Background Investigations into the growth and self-organization of plant roots is subject to fundamental and applied research in various areas such as botany, agriculture, and soil science. The growth activity of the plant tissue can be investigated by isotope labeling experiments with heavy water and subsequent detection of the deuterium in non-exchangeable positions incorporated into the plant biomass. Commonly used analytical methods to detect deuterium in plants are based on mass-spectrometry or neutron-scattering and they either suffer from elaborated sample preparation, destruction of the sample during analysis, or low spatial resolution. Confocal Raman micro-spectroscopy (CRM) can be considered a promising method to overcome the aforementioned challenges. The substitution of hydrogen with deuterium results in the measurable shift of the CH-related Raman bands. By employing correlative approaches with a high-resolution technique, such as helium ion microscopy (HIM), additional structural information can be added to CRM isotope maps and spatial resolution can be further increased. For that, it is necessary to develop a comprehensive workflow from sample preparation to data processing. Results A workflow to prepare and analyze roots of hydroponically grown and deuterium labeled Zea mays by correlative HIM-CRM micro-analysis was developed. The accuracy and linearity of deuterium detection by CRM were tested and confirmed with samples of deuterated glucose. A set of root samples taken from deuterated Zea mays in a time-series experiment was used to test the entire workflow. The deuterium content in the roots measured by CRM was close to the values obtained by isotope-ratio mass spectrometry. As expected, root tips being the most actively growing root zone had incorporated the highest amount of deuterium which increased with increasing time of labeling. Furthermore, correlative HIM-CRM analysis allowed for obtaining the spatial distribution pattern of deuterium and lignin in root cross-sections. Here, more active root zones with higher deuterium incorporation showed less lignification. Conclusions We demonstrated that CRM in combination with deuterium labeling can be an alternative and reliable tool for the analysis of plant growth. This approach together with the developed workflow has the potential to be extended to complex systems such as plant roots grown in soil.

Plant phenotyping relevance

植物根の成長状態を測定する相関HIM-CRMワークフローを開発し、重水素検出の精度・直線性と質量分析との一致を検証しているため、植物フェノタイピング手法が中心である。

Code and data availability

The paper's data availability statement deposits the datasets generated and analyzed (CRM/HIM phenotyping measurements of deuterium-labeled maize roots) in the UFZ Data Investigation Portal, a public repository with an explicit URL. No author analysis code with a public URL is stated.