Acronym

N1-0105

Contract number

N1-0105

Department:

Department of Biology

Type of project

ARIS projects

Type of project

Basic research project

Role

Lead

Duration

01.10.2019 - 30.09.2022

Total

2.49 FTE

Project manager at BF

Vogel Mikuš Katarina

Abstract

As sessile organisms, plants need to constantly adapt to environmental changes. To that end, they accumulate ions of various elements (ionome) and synthesise a plethora of biochemical compounds (metabolome). Ionome and metabolome represent dynamic and spatially distributed components, calling for the development of spatially resolved analytical techniques to relate biochemical compositions at different organisational levels (organ, tissue, cell) to the anatomical, physiological, and genetic traits of plants. Since one instrumental technique is not sufficient to provide spatially resolved information on biochemical composition of plants, the SLOEMP project aims to assemble multimodal instrumental platform and workflows for unified “single sample” imaging using accelerator-based micro-PIXE, MeV-SIMS and FTIR. The SLOEMP project will revolutionise the field of biochemical imaging by creating increasingly powerful tools leading to valuable new insights into different biomolecular processes in plants, on a long-term basis improving plant production, food quality and safety.

 

Researchers

The phases of the project and their realization 

SLOEMP is a complementary ERC CoG project (MIBPLANT) that aims to build a multimodal instrumental platform (MIP) for the correlative capture of information on the morphology of biological samples and the distribution of elements and biomolecules at the tissue level. MIP with the possibility of correlative analysis on one sample ("single sample"), would enable in-depth studies of both plant and animal samples and would represent an advanced tool for studying various issues from the impact of pollutants (metals, microplastics) on organisms to studies of various chronic diseases and cancers in humans.


The main objectives:

- Construction of a multimodal imaging instrumentation platform for comprehensive spatially resolved plant biochemical analysis at the tissue level, by combining complementary spectrometry focused-beam techniques (µPIXE, µFTIR and µMeV-SIMS).

- Novel sample-preparation workflows for “single sample” multimodal imaging aimed to directly correlate anatomical and biochemical features at the tissue level;

- Analysis and quantification of spectroscopic data to reduce spectral information of the 3D data cubes to relevant information; basic chemometric image analysis with existing software.

 

The SLOEMP project is divided into 4 sections:

DS1: Multimodal Instrumentation Platform (MIP).

We connected the following instruments in MIP: light microscope (BF), u-PIXE (IJS), MeV-SIMS, u-FTIR (Elettra, KI) and SEM (IJS).As envisaged in the project objectives, we have defined a workflow of measurements to capture information about the structure and distribution of elements and biomolecules at the tissue level. The workflow is tested on the example of a buckwheat grain tissue slice (3). An overview of the techniques is published in the chapter of monographic publication (4).

 

Task 1.1. Imaging of the histological structures

A light microscope (Carl Zeiss, Axioskop 2 MOT) was used to image the histological structures of the sample. On the basis of histological structures, it is possible to clearly define regions that serve as reference points for measurements with other techniques. We also included SEM (2,3) as a basis for the analysis of elemental and biochemical distribution images.

Task 1.2. Imaging of the distribution of biomolecules

MeV SIMS imaging of metabolite distribution was performed on buckwheat grain (1,2). We assessed the risks, which mainly included the accessibility of the instrument. Due to the COVID-19 pandemic, we did not have access to uFTIR through synchrotron institutions in 2020/21, so we contacted researchers at KI. Cooperation agreements are ongoing.

Task 1.3. Imaging of the distribution of elements

Within the framework of this task, we strengthened connections with many foreign partners in the field of mapping of the distribution of elements in plant tissues with u-PIXE and synchrotron u-XRF and published a total of 5 publications (5-9).

 

DS2: sample preparation

We have defined the main challenges in the sample preparation for MIP and tried to find appropriate solutions. The main challenge in the preparation of plant samples is to preserve the histological structure of the sample and at the same time the intact distribution of biomolecules and elements. Fresh plant tissue can be used to preserve anatomical and morphological features, but fully hydrated samples are not compatible with vacuum techniques (MeV SIMS and u-PIXE), and u-FTIR due to interference with water molecules. In addition, during each set of measurements, the tissue would decompose, which would adversely affect the biomolecular composition (activation of lytic enzymes) and the distribution of elements (plasmolysis). Another method is chemical fixation, which is inadequate because fixative residues interfere with the determination of biomolecular profiles by FTIR and SIMS, and at the same time mobile elements are washed out. The only appropriate method of preparation is therefore native cryofixation and the preparation of 20-30 um thick tissue slices on a cryotome, which we previously developed for mapping the distribution of elements and which we already routinely use (5-9). Such samples are compatible with ATR-u-FTIR mode, but not with transmission u-FTIR mode. For the latter method, slices must be 10-15 um thick (2,3,4), which is still a challenge. In 2021, we bought a vibratome (Precisionary) at BF, which enables the preparation of thin slices of fresh tissue and subsequent freezing and lyophilization. Testing of this preparation method is ongoing.

 

DS3 spectrum analysis

We prepared a workflow for processing PIXE spectra with the GEOPIXE program (10,11)


DS4 "Proof of concept"

As part of the project, we test various biological objects, as can be seen from publications (5-9)

 

Publications

1)    VOGEL-MIKUŠ K. et . Key contributions of ion beam imaging techniques to plant ionomics. V: PONGRAC, Paula (ur.). ICNMTA2020 : 17th International Conference on Nuclear Microprobe Technology and Applications, 14-15 September 2020, on-line : book of abstracts, 17th International Conference on Nuclear Microprobe Technology and Applications, 14-15 September 2020, on-line. Electronic ed. Ljubljana: Jožef Stefan Institute. 2020, str. 21. https://www.icnmta2020.org/en/. [COBISS.SI-ID 35044099]

2)    VOGEL-MIKUŠ K. et al. Challenges in the multimodal imaging of metal-stressed plants : lecture at IAEA Technical Meeting on Imaging Using Ionizing Radiation to Address Biological Challenges, 30 November - 3 December 2020, (Virtual Event). [COBISS.SI-ID 62491395]

3)    PONGRAC P. et al. Challenges in the Imaging cell-types in plants using correlative ion beam techniques MeV-SIMS and micro-PIXE at the Jožef Stefan Institute, Slovenia : lecture at IAEA Technical Meeting on Imaging Using Ionizing Radiation to Address Biological Challenges, 30 November - 3 December 2020, (Virtual Event). [COBISS.SI-ID 62489347]

4)    VOGEL-MIKUŠ K. et al. Distribution of nutritional and mineral components in important crop plants. V: UPADHYAY, Santosh Kumar (ur.). Genome engineering for crop improvement. Hoboken (NJ): Wiley. 2021, str. 22-42, doi: 10.1002/9781119672425.ch2. [COBISS.SI-ID 62668035]

5)    ALCOCK T. et al. Magnesium and calcium over-accumulate in the leaves of a schengen3 mutant of Brassica rapa. Plant physiology. IF= 6.9 [in press] 2021, 41 str. [COBISS.SI-ID 59167235]

6)    COMINELLI E. et al. Calcium redistribution contributes to the hard-to-cook phenotype and increases PHA-L lectin thermal stability in common bean low phytic acid 1 mutant seeds. Food chemistry. IF= 6.3. 15 Aug. 2020, vol. 321, 126680, str. 1-10 [COBISS.SI-ID 33286183]

7)    DETTERBECK A. et al. Temporal and spatial patterns of zinc and iron accumulation during barley (Hordeum vulgare L.) grain development. Journal of agricultural and food chemistry. IF=4.2 [in press] 2020, 12 str [COBISS.SI-ID 33245443],

8)    HÖRETH S.et al. Arabidopsis halleri shows hyperbioindicator behaviour for Pb and leaf Pb accumulation spatially separated from Zn. The new phytologist. IF=8.5 2020, vol. 226, iss. 2, str. 492-506. [COBISS.SI-ID 33005607]

9)    PONGRAC P. et al. Mineral element composition in grain of awned and awnletted wheat (Triticum aestivum L.) cultivars : tissue-specific iron speciation and phytate and non-phytate ligand ratio. Plants. . IF=2.8 2020, vol. 9, no. 1, str. 79-1-97-14. [COBISS.SI-ID 33018919]

10) PONGRAC, Paula. Image analysis of element distribution maps…. 2021. 1 spletni vir. https://repozitorij.uni-lj.si/IzpisGradiva.php?id=126923&lang=slv. [COBISS.SI-ID 62029059]

11) PONGRAC P. et al. OM DAQ - GeoPIXE for beginners. [COBISS.SI-ID 15626755]