Reciprocal interactions between plants and fluorescent pseudomonads in relation with iron in the rhizosphere

HAL Id: hal-01837545

https://hal.archives-ouvertes.fr/hal-01837545

Submitted on 3 Jun 2020

HAL is a multi-disciplinary open access

archive for the deposit and dissemination of

sci-entific research documents, whether they are

pub-lished or not. The documents may come from

teaching and research institutions in France or

abroad, or from public or private research centers.

L’archive ouverte pluridisciplinaire HAL, est

destinée au dépôt et à la diffusion de documents

scientifiques de niveau recherche, publiés ou non,

émanant des établissements d’enseignement et de

recherche français ou étrangers, des laboratoires

publics ou privés.

Reciprocal interactions between plants and fluorescent

pseudomonads in relation with iron in the rhizosphere

Laure Avoscan, Gérard Vansuyt, Jeannine Lherminier, Christine Arnould,

Geneviève Conejero, Eric Bernaud, Philippe Lemanceau

To cite this version:

Laure Avoscan, Gérard Vansuyt, Jeannine Lherminier, Christine Arnould, Geneviève Conejero, et

al.. Reciprocal interactions between plants and fluorescent pseudomonads in relation with iron in the

rhizosphere. 5. Journées Scientifiques et Techniques du Réseau des Microscopistes INRA, Nov 2014,

Dijon, France, 12-14 novembre 2014, France. 2014. �hal-01837545�

Reciprocal interactions between plants

and fluorescent pseudomonads

in relation with iron in the rhizosphere

Iron is an essential element for plants and microbes. Iron competition was demonstrated to be an important driver of the interactions between fluorescent pseudomonads and the rhizospheric microflora (Lemanceau et al., in press). To face this competition, plants and microorganisms have developed active strategies of iron uptake. In non graminaceous plants (strategy I), iron uptake relies on acidification and reduction of Fe(III) to Fe(II) which is incorporated into the roots by iron transporters (eg. IRT1). Active iron uptake by microorganisms relies on siderophores showing high affinity for iron.

We have previously shown that plants of Arabidopsis thaliana (strategy I) supplemented with Fe-pyoverdine had (i) a higher iron content than those supplemented with Fe-EDTA, (ii) iron incorporation from pyoverdine did not involve IRT1, and (iii) 15_{N-labeled pyoverdine was incorporated in planta}

(Vansuyt et al., 2007). Taken together, these observations suggest that iron from Fe-pyoverdine was incorporated in planta not through the strategy I. In the present study, we explored possible mechanisms for incorporation of iron from pyoverdine at the cellular level.

1. UMR Microbiologie du Sol et de l’Environnement, INRA/Université de Bourgogne, CMSE, BP 86510, 21065 Dijon cedex, France 2. Centre de Microscopie, INRA/Université de Bourgogne, CMSE, BP 86510, 21065 Dijon cedex, France

3. Plate-forme d'Histocytologie et Imagerie Cellulaire Végétale, UMR Biochimie et Physiologie Moleculaire des Plantes INRA CNRS UM2 SupAgro avenue Agropolis Cirad 34398 Montpellier Cedex 5

L. Avoscan

_{, G. Vansuyt}

_{, J. Lherminier}

_{, C. Arnould}

_{, G. Conejero}

_{, E. Bernaud}

_{, and P. Lemanceau}

laure.avoscan@dijon.inra.fr

RESULTS

Fe-pyoverdine localization in root Fe-EDTA Fe-Pyoverdine

Confocal sections of root labeled with pyoverdine antibody clearly indicated pyoverdine presence in

MATERIALS AND METHODS

D7 D14 Fe-PyoverdineC7R12 Fe-EDTA
50 µM
100 µM
200 µM
50 µM
100 µM
200 µM Sampling Root apex dissection Control
water • 10 plants / plate • 3-5 plates / treatment Treatments Confocal microscopy of fresh tissue Immunolocalization

• Primary antibody = pyoverdine

antibody

• Fluorescent secondary antibody

TEM of embedded tissue

Ultrastructure and immunogold labeling

• Primary antibody = pyoverdine antibody • Colloïdal gold coupled to secondary

antibody

In vitro culture of Arabidopsis plants

Plants cultured for seven days without any iron supplementation and for seven more days after having been supplemented with Fe-pyoverdine or Fe-EDTA or not supplemented

Sampling of 14-day old roots for ultrastructural studies with transmission electron microscopy (TEM) and immunolocalization of pyoverdine in roots by confocal microscopy and TEM

Ultrastructural analysis of root cells supplemented with Fe-pyoverdine, Fe-EDTA or not supplemented

INTRODUCTION

Quantification of apoplasmic vesicles in root cells pyoverdine presence in

planta.

Immunolocalization revealed the presence of pyoverdine in the root apoplasmic space.

20µm 20µm

Presence of immunogold-labeled pyoverdine in cytoplasm and apoplasm

Microscopy Conference 2009, Graz, Austria – 30 August -4 September 2009

Fe-EDTA apoplasm cytoplasm cytoplasm vacuole 500nm 50 µm apoplasm cytoplasm vacuole _{100 nm} Fe-pyoverdine 500nm apoplasmcytoplasm cytoplasm 100 nm apoplasm cytoplasm 50nm 10 µm

For each sectioned cell, apoplasmic vesicles (AVs) were classified into three classes and results are expressed in percentage of cells exhibiting 1, 2-5 and >5 AVs per cell

Fe-pyoverdine more vesicles in apoplasm

1 apoplasmic vesicle From 2 to 5 AVs > 5 AVs

Class 0 Class 1 Class 2

100 nm 100 nm 100 nm 33% 57% 10% 16% 52% 32% 13% 40% 47%

Control Fe-EDTA Fe-pyoverdine

Observations and quantification with TEM showed a more abundant presence of vesicles in the root apoplasm of plants when cultured with Fe-pyoverdine than with Fe-EDTA. However pyoverdine immunogold labeling of root sections was not sensitive enough to allow the possible detection of pyoverdine in the vesicles. Altogether, these data confirm the acquisition of iron from Fe-pyoverdine by A. thaliana and suggest that iron incorporation from Fe-pyoverdine could be related to endocytosis. Further experimental proof is required to determine if the increase of vesicles in the presence of pyoverdine mediates that process.

Incorporation of Fe-pyoverdine by endocytosis ?

Letters indicate values significantly different as measured by the Fisher’s least significant difference test (P<0.0001)

Monitoring endocytosis by uptake of the endocytosis marker FM4-64 might indicate that incorporation of Fe-pyoverdine relies on endocytosis

Total fluorescence per cell

0 100000 200000 300000 400000 500000 600000

Control FeEDTA FePVD

F lu o re s c e n c e in te n s it y ( U A ) a a b

CONCLUSION

Fe-pyoverdine Fe-EDTA Control

10 µm 10 µm

10 µm 20 µm 20 µm

References

Vansuyt G. et al., 2007. Iron acquisition from Fe-pyoverdine by Arabidopsis thaliana. Mol. Plant-Microbes Interact. 20:441-447.

Lemanceau P. et al., 2009. Iron dynamics in the rhizosphere as a case study for analysing interactions between soils, plants and microbes. Plant Soil. 321:513-535.

Lemanceau P., et al. Role of Iron in Plant–Microbe Interactions. Advances in Botanical Research, in press.

10 µm