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# Development of a multimodal approach to study plant-microorganisms interactions using mass spectrometry imaging, Raman imaging and non-targeted metabolomics

Baptiste Guillon, Justine Ferey, Léonie Chrétien, Mélia Lacaze, Cécile Pouzet, Ndeye Dieng, Laurent Debrauwer, Sylvie Fournier

► To cite this version:

Baptiste Guillon, Justine Ferey, Léonie Chrétien, Mélia Lacaze, Cécile Pouzet, et al. Development of a multimodal approach to study plant-microorganisms interactions using mass spectrometry imaging, Raman imaging and non-targeted metabolomics. 16èmes JS RFMF, Jun 2024, Saint Malo, France. hal-04838276

HAL Id: hal-04838276

https://hal.science/hal-04838276v1

Submitted on 14 Dec 2024

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**Baptiste Guillon** ¹,², **Justine Ferey** ², **Léonie Chrétien** ³, **Mélia Lacaze** ⁴, **Cécile Pouzet** ⁴, **Ndeye Dieng**, **Laurent Debrauwer** ², **Sylvie Fournier** ¹,³

1. MetaToul Agromix - Université de Toulouse, UT3 Paul Sabatier, Toulouse INP, CNRS

2. MetaToul Axiom - Université de Toulouse, ENVT, INP-Purpan, INRAe

3. Laboratoire de Recherche en Sciences Végétales - Université de Toulouse, UT3 Paul Sabatier, Toulouse INP, CNRS

4. Plateforme TRI-FRAIB - FR AIB, Université de Toulouse, UT3 Paul Sabatier, CNRS

## Introduction

450 million years ago, an essential symbiotic relationship called **arbuscular mycorrhizal symbiosis (AMS)** was formed between plants and glomeromycota fungi¹, enabling the first land plants to colonize the soil².

It begins with a molecular dialogue between partners located in the soil, leading to the development of specialized fungal structures in the roots, called **arbuscules**, facilitating the exchange of nutrients³.

Understanding this metabolic dialogue is a challenge, but innovative techniques such as LC-HRMS and MALDI* mass spectrometry imaging enable a complementary localization and quantification of metabolites in AMS. The integration of stimulated Raman microscopy enhances our understanding at the cellular level in a non-invasive way.

The first results of this study concern **Marchantia paleacea** mycorrhized by **Rhizophagus irregularis**.

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A: Marchantia paleacea

B: Thallus embedded in agarose7%

C: Transverse section (100µm thickness using vibratome) with arbuscular mycorrhizal fungi (purple area)

## Untargeted Metabolomics

<figure><img src="image_4.png" /><figcaption>Data processing</figcaption></figure>

### Data acquisition

by U-HPLC-HRMS (Thermo Scientific)

UHPLC: Vanquish with CAD and DAD detectors

MS: Q-Exactive Plus ESI +/-

Biphasic extraction following MetaToul Agromix protocol (MTBE, MeOH, H₂O).

Lipidomic analysis
(apolar compounds)

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C18 analysis
(semi-polar compounds)

Hilic analysis

(polar compounds)

<figure><img src="image_6.png" /></figure>

### Peak annotation

with dedicated bioinformatic workflow for global metabolomics

<figure><img src="image_7.png" /></figure>

Biomarker identification,

lipids involved in Arbuscular Mycorrhizal Symbiosis?

## Mass Spectrometry Imaging

<figure><img src="image_8.png" /></figure>

### Sample preparation:

* MALDI matrix: DHB

* Deposition with the **HTX TM3** sprayer (HTX technologies LLC)

Hey ! Scan me and download the file to see 3D PCA of discriminative segmentation !

**RED** = Non mycorrhized area
ions **GREEN** = Mycorrhized area
ions **BLUE** = Centre of
mycorrhize ions

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10 µm resolution
Positive mode

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⇒ **Targeting of biomarkers identified by LC-HRMS**

*Matrix-Assisted Laser Desorption-Ionization

## Conclusions

These results provide a great example on how multimodal approaches can help visualizing biological interactions between different organisms in a more constitutive way:

* Global LC-HRMS analysis helps **unravelling the metabolic segregation** occurring during AMS establishment.

* MALDI imaging helps localizing the metabolites involved at the tissue level with **high sensitivity**.

* Despite not being as resolutive as MALDI, **the high resolution** provided by Raman stimulated microscopy helps us to localize metabolites families. Allowing us to better quantify exchanges occurring during AMS formation.

## Raman Stimulated Microscopy

<figure><img src="image_11.png" /></figure>

### Data acquisition

with Raman stimulated microscopy
(STELLARIS 8 CRS - Leica)

⇒ **Identification and mapping of Raman signals characteristic of biomarkers identified by LC-HRMS** (single cell and hyphae scale) with tunable OPO laser.

Optimization of analysis conditions:

⇒ Magnification

⇒ Number of accumulations

⇒ Signal averaging

(A) BrightField snapshot of a mycorrhized *M. paleacea* cell at a x40 magnification with confocal Raman microscope.

(B) BrightField snapshot of a *R. irregularis* hyphae at a x40 magnification with confocal Raman microscope.

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(A) Raman spectrum of a non-mycorrhized cell of *M. paleacea* between 1400 and 1700cm⁻¹. (I 35 % ; SRS gain 100 ; accumulation 2)

(B) Raman spectrum from the hyphae of *R. irregularis* between 1400 and 1700cm⁻¹. (I 35 % ; SRS gain 88,5 ; accumulation 1)

(C) Raman spectrum from the inside of a mycorrhized cell of *M. paleacea* between 1400 and 1700cm⁻¹. (I 30 % ; SRS gain 100 ; accumulation 1)

⇒ Two main peaks around 1445cm⁻¹ and 1660cm⁻¹ which could be respectively attributed to the signal emitted by saturated fatty acids and unsaturated fatty acids.

⇒ The difference in intensity between non-mycorrhized and mycorrhized cells can be explained by the presence of arbuscular mycorrhizal fungi.

## References

1. Malar C M., Krüger M., Krüger C., Wang Y., Stajich J E., Keller J., Chen E C H., Yildirig G., Villeneuve-Laroche M., Roux C., Delaux P M., Corradi N., (2021) The genome of Geosiphon pyriformis reveals ancestral traits linked to the emergence of the arbuscular mycorrhizal symbiosis, *Current Biology*, 31-7; pages 1570-1577.

2. Kaur S., Suseela V. (2020) Unraveling Arbuscular Mycorrhiza-Induced Changes in Plant Primary and Secondary Metabolome, *Metabolites*, 10; 335.

3. Parniske M. (2008) Arbuscular mycorrhiza: the mother of plant root endosymbioses. *Nat Rev Microbiol* 6; pages 763-775.

4. Czamara K., Majzner K., Pilarczyk M., Kochan K., Kaczor A., and Baranska M. (2014) Raman spectroscopy of lipids: A review, *J. Raman Spectrosc.*, 46; pages 4-20.

## Acknowledgments

We thank the EVO team from LRSV for cultivating and providing the vegetal samples.

Thank you Céline Bougel from MetaToul Axiom platform for generating the QR codes.

We also thank GenoToul for funding this internship.

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