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# Influence of cell wall pectic fraction modification on Cadmium stress capacities

Jonathan Robilliard<fnref n="1" />,<fnref n="3" />, Sophie Bouton<fnref n="1" />, Karine Pageau<fnref n="1" />, Romain Roulard<fnref n="1" />, Stéphanie Guénin<fnref n="2" />,
Laurent Gutierrez<fnref n="2" />, Pierre Delaplace<fnref n="3" />, Olivier Van Wuytswinkel<fnref n="1" />

## Abstract

Pectin MethylEsterases (PME) catalyze the hydrolysis of the C6 Galacturonic Acid (GalA) methylester group present in newly synthesized HomoGalacturonans (HG) [1]. Analysis of recent literature suggests a potential role for pectin demethylesterification in the plant's response to nutritional stresses such as nitrogen and phosphorus [2, 3, 4] or metal stresses such as cadmium and aluminum [5, 6, 7]. This is evidenced by changes in PME activity under these conditions. These observations raise the question of whether pectin methylesterification rate is involved in the apoplastic transport and translocation of ions in both root and aerial plant tissues.

## Objective

Assess how modifications to the cell wall pectic fraction impact the absorption and translocation of cadmium within *Arabidopsis thaliana*

## Results

### 1 Impact of modifications on the absorption and transport of Cadmium

**Pre-culture on MS ½**
(WT + mutants):

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**Hydroponic culture:**

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21°C

Short day

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→ Cell wall analysis + measurement of different ions quantity in plant tissues (sugar composition of cell wall, quantification of metal ions)

### 3 Accumulation of cadmium in leaves

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➤ Significant ↘ of Cadmium in cgr2cgr3 mutant compared to Col 0

➤ Significant ↗ of Copper and Iron in cgr2cgr3 mutant compared to Col 0

➤ No significant difference between Col 0 and gaut10gaut11

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*: 2,5µM of Cadmium has not been tested on cgr2cgr3 line

Significant differences (letters) were determined according to one-way ANOVA followed by a multiple comparison with Tukey's test

### 2 Characterization of *A. thaliana* cell wall mutants

**Double mutant gaut10gaut11**

(GalactUronosyl Transferase → synthesis of HG)

➤ Ø visible phenotypic difference between gaut10gaut11 and Col 0

➤ Lower amount of GalA in cell wall (obtained by F. Sénéchal and P. Trezel : BioEcoAgro)

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Cell wall analysis (Leaves at 0µM of Cd<fnref n="2" />⁺)

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**Double mutant cgr2cgr3**

(HG MethylTransferase → HG methylesterification)

➤ Growth ↘ (Dwarf phenotype)

➤ Early appearance of inflorescence

➤ Defect in silique formation

➤ Lower methylesterification rate (Kim et al. 2015)

Obtained thanks to F. Brandizzi

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Student t-test (*p<0,05 ; **p<0,01 ; ***p<0,001)

### 4 Effect of Cadmium on cell wall composition

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Cell wall analysis (Leaves: Galacturonic A.)

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Galacturonic A.

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➤ Significant ↘ of Gal A on WT as Cd concentration ↗ : in roots only

➤ Other cell wall components (roots or leaves) remain unchanged between cadmium conditions

Cell wall analysis (Roots: Galacturonic A.)

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Galacturonic A.

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Cell wall analysis (Roots)

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Significant differences (letters) were determined according to one-way ANOVA followed by a multiple comparison with Tukey's test

Col 0

## Conclusions

➤ *gaut10gaut11* mutant did show a significant *decrease of GalA* in addition to a *decrease of Rhamnose*

➤ Cadmium stress induces a *decrease in GalA* content in roots cell wall

➤ Metallic ions accumulation in *cgr2cgr3* leaves, whatever condition, suggests that *lower methylesterification level results in cation accumulation*

➤ Upon cadmium stress, accumulation of cadmium in *cgr2cgr3* mutants is slightly decreased. Which can be explained by an *overload of cell wall* by other ions as shown by copper and iron hyperaccumulation

## Perspectives

➤ Plants overexpressing a *fungal PME* [8] in different tissues will be tested to confirm importance of methylesterification rate under cadmium stress conditions

➤ Cadmium accumulation *kinetics* and the effect of cadmium on cell wall modifications will be tested

➤ Influence of cadmium on *root architecture* will be tested on different mutants

➤ Capacity of cell wall to *bind cadmium* will be tested using extracted cell wall from different mutants

➤ PME and PMEi genes expression upon cadmium stress will be measured using RT-qPCR

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<footnote n="1">¹Université de Picardie Jules Verne - UMRT INRAE 1158 BioEcoAgro - Biologie des Plantes et Innovation (BioPI)</footnote>

<footnote n="2">² Université de Picardie Jules Verne – Centre de Ressources Régionales en Biologie Moléculaire (CRRBM)</footnote>

<footnote n="3">³ Université de Liège - Gembloux Agro-Bio Tech (GxABT) - TERRA - Plant Sciences</footnote>