# Metabolomic and Biochemical analysis of Pb accumulation and tolerance in Hirschfeldia incana

Oumaima EL KHATTABI, Youssef Lamwati, Fabrice Colin, Abdelaziz Smouni, Mouna Fahr

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Oumaima EL KHATTABI, Youssef Lamwati, Fabrice Colin, Abdelaziz Smouni, Mouna Fahr. Metabolomic and Biochemical analysis of Pb accumulation and tolerance in Hirschfeldia incana. Min-ing the Connections 2022, Apr 2022, Québec City, Canada. **hal-03833742**

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Submitted on 2 Nov 2022

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# Metabolomic and Biochemical analysis of Pb accumulation and tolerance in *Hirschfeldia incana*

Oumaima EL KHATTABI, Youssef LAMWATI, Fabrice COLIN, Abdelaziz SMOUNI and Mouna FAHR
# Mining Activities: Economic contribution

## Morocco

Rich in mineral deposits

10% of national gross domestic product

<figure><img src="image_2.png" /></figure>

Source: International council of mining and metals (LMI-AMIR)
Mining Activities: principal source of pollution

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /><figcaption>Mining Pb/ Zn slag heaps at Touissit
site - Eastern Morocco</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>Scoria of Oued El Heimer Foundry -
Eastern Morocco</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>AMD at Kettara mining site - northwest
of Marrakech Morocco</figcaption></figure>

Contamination of
water resources
Mining Activities: Principal source of pollution

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

Lead (Pb) is one of the most
toxic metals

Not biodegradable

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

Persists for long periods in the
environment (150 to 5000 years)
* Plant water status alteration

* Photosynthesis disruption

* Oxidative stress induction ....

Pb

<figure><img src="image_9.png" /></figure>

Some plant species develop
effective strategies of
protection against these
elements

<figure><img src="image_10.png" /></figure>

These natural properties are
exploited in the field of
**Phytoremediation**
Phytoremediation

* A biological Method based on
the ability of plants to extract or
block pollutants from
contaminated soil or water

- More efficient

* Less expensive

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

PHYTOSTABILISATION

<figure><img src="image_12.png" /></figure>

Little or no
transfer of
pollutants in
aerial parts

Concentration
of pollutants in
the rhizosphere
and the roots

Reduced pollutants leaching and
uptake by plants

PHYTOEXTRACTION

<figure><img src="image_13.png" /></figure>

Harvesting
biomass
enriched in
pollutants

Transfer of
pollutants
in the aerial
parts

Pollutants uptake by the
roots
Although phytoremediation
offers several advantages :

**Economic**
**Ecological**
**Aesthetic**

<figure><img src="image_14.png" /></figure>

**Phytoremediation limits**

Slow decontamination process

Accumulator plants have :

- Slow growth rate

- Low biomass production
With the aim of optimizing the use of plants in the rehabilitation of soils polluted by Pb

* *Hirschfeldia incana*

* Oued el heimer-touissit sites

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>

*Hirschfeldia incana* growing on lead slag from the site of Oued El Heimer near Oujda in Morocco
With the aim of optimizing the use of plants in the rehabilitation of soils polluted by Pb

Hirschfeldia incana

* Accumulates in its aerial parts up to 1400 mg of Pb/Kg of dry matter

- Short vegetative cycle (2 months)

* Rapid growth

- Easy to handle in the laboratory

* Its transcriptom has just been sequenced

<figure><img src="image_19.png" /></figure>

*Hirschfeldia incana* growing on lead slag from the site of Oued El Heimer near Oujda in Morocco

A good model to study Pb tolerance and accumulation mechanisms
Molecular changes
↓
Physiological changes
**Metabolites variation**

<figure><img src="image_20.png" /></figure>

**Molecular and metabolomic**

data on heavy metal
accumulating plants are
**limited**

<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /></figure>

<figure><img src="image_23.png" /></figure>
### Metabolomic analysis of Pb accumulation and tolerance in *Hirschfeldia incana*

<figure><img src="image_24.png" /></figure>

### Biochemical analysis of Pb accumulation and tolerance in *Hirschfeldia incana*

Metabolits
extraction

<figure><img src="image_25.png" /></figure>

Plants grinding in liquid nitrogen

<figure><img src="image_26.png" /></figure>

Enzymes
extraction

Transesterification

<figure><img src="image_27.png" /></figure>

<figure><img src="image_28.png" /></figure>

Analyse via
GC-MS

Metabolites implicated in Pb
tolerance and accumulation

### Molecular analysis of Pb accumulation and tolerance in *Hirschfeldia incana*

<figure><img src="image_29.png" /></figure>

Genes implicated in metabolomic
pathways

Dosage by
spectrophotometry

<figure><img src="image_30.png" /></figure>

Enzymes and molecules
implicated in Pb tolerance and
detoxification
## Metabolomic characterization

### Variation in fatty acids composition

<figure><img src="image_31.png" /></figure>

### Variation in steroids composition

<figure><img src="image_32.png" /></figure>

Plants require a period for the modulation of their
metabolism to overcome Pb stress

## Lipids after 15 days of Pb treatment

<table><thead><tr><th></th><th>metabolites</th><th>TEMOIN vs TRAITE%</th></tr></thead><tbody><tr><td rowspan="4">Acides gras insaturés</td><td>7,10,13-Hexadecatrienoic acid,</td><td>-11.87</td></tr><tr><td>9,12,15-Octadecatrienoic acid,</td><td>-18.7</td></tr><tr><td>6,9,12,15-Docosatetraenoic acid,</td><td>-68.76</td></tr><tr><td>1-(4-Fluorophenyl)-2-[(4-hydroxy-</td><td>-5.88</td></tr><tr><td rowspan="7">Acides gras saturés</td><td>Hexadecanoic acid, methyl ester</td><td>79.52</td></tr><tr><td>Methyl stearate</td><td>19.96</td></tr><tr><td>Ethyl 3-hydroxyicosanoate</td><td>+</td></tr><tr><td>Tetracosanoic acid, methyl ester</td><td>41.31</td></tr><tr><td>Hexacosanoic acid, methyl ester</td><td>58.5</td></tr><tr><td>Methyl 18-methylnonadecanoate</td><td>+</td></tr><tr><td>Octacosanoic acid, methyl ester</td><td>+</td></tr></tbody></table>

## Steroids after 15 days of Pb treatment

<table><thead><tr><th>metabolites</th><th>TEMOINvs TRAITE%</th></tr></thead><tbody><tr><td>ethyl iso-allocholate</td><td>153.81</td></tr><tr><td>campesterol</td><td>79.83</td></tr><tr><td>5-cholesten-3-ol 24-methyl-</td><td>+</td></tr><tr><td>gamma.-Sitosterol</td><td>151.54</td></tr><tr><td>Androstan-17-one, 3-ethyl-3-hydroxy-</td><td>+</td></tr></tbody></table>
## Metabolomic characterization

### Variation in organic acids composition

<figure><img src="image_33.png" /></figure>

<table><thead><tr><th></th><th>metabolites</th><th>TEMOIN vs TRAITE%</th></tr></thead><tbody><tr><td rowspan="2">Phenoles</td><td>2,4-Di-tert-butylphenol</td><td>-4.51</td></tr><tr><td>Phenol, 2,2'-methylenebis[6-(1,1-dimethylethyl)-4-methyl-</td><td>+</td></tr><tr><td rowspan="7">Acides carboxyliques</td><td>1,4-Benzenedicarboxylic acid, dimethyl ester</td><td>+</td></tr><tr><td>1,3-Benzenedicarboxylic acid, dimethyl ester</td><td>+</td></tr><tr><td>Succinic acid, tridec-2-yn-1-yl 3-methylpentyl ester</td><td>20.93</td></tr><tr><td>Succinic acid, cyclohexylmethyl 3-chlorophenyl ester</td><td>20.93</td></tr><tr><td>propanoic acid</td><td>+</td></tr></tbody></table>

### Organic acids after 15 days of Pb treatment

<figure><img src="image_34.png" /></figure>

#### Increase in organic acids concentration (succinic acid)

* Succinic acid is involved in the signaling of salicylic acid (mitigate Pb stress effects)
Biochemical characterization

Malondialdehyde (MDA) content

Effet du Pb sur la teneur en MDA chez H.incana

<figure><img src="image_35.png" /></figure>

Pb induces oxydative stress → Lipid
peroxydation → Increase in MDA

Polyphenol content

effet du Pb sur la teneur en polyphénoles totaux chez H. incana

<figure><img src="image_36.png" /></figure>

Implication of polyphenols in Pb detoxification and
Pb-induced oxydative stress attenuation
<figure><img src="image_37.png" /></figure>

## Use of the identified molecules :

* Boosting plants response to Pb

* Selection of candidate plants for phytoremediation programs

* Extraction of high value-added metal
Thank you for your attention
# Metabolomic and Biochemical analysis of Pb accumulation and tolerance in *Hirschfeldia incana*

Oumaima EL KHATABI, Youssef LAMWATI, Fabrice COLIN, Abdelaziz SMOUNI and Mouna FAHR

<figure><img src="image_38.png" /></figure>

Université Mohammed V de Rabat
Faculté des Sciences

<figure><img src="image_39.png" /></figure>

IRD
Institut de Recherche
pour le Développement
FRANCE

AMIR
Laboratoire Mixte International
Activité Minière Responsable

Laboratoire
de Physiologie
et Biotechnologie
Végétales