Gembloux Agro-Bio Tech University of Liege ML Fau auconnie ier - - PowerPoint PPT Presentation

Gembloux Agro-Bio Tech

University of Liege

ML Fau auconnie ier Karlsurhe 17/0 17/01/2019

Faculty Gembloux Agro-Bio Tech

Four masters in in Bio io-engineering

• Environmental sciences
• Forest and natural spaces
• Agronomy
• Chemistry and bio-industries

 1000 students (from 40 countries)

Research center TERRA

11 11 axis is 7 7 technical pla latforms 32 320 0 Ph D D stu tudents

Laboratory of Chemistry of Natural Molecules

Ext xtractio ion, puri rifi ficatio ion and characteriz izatio ion of f pla lant se secondary ry metaboli lites for r appli licatio ions in in agronomy or r food in industrie ies

Prof. . ML Fauconnie ier 3 post-docs 20 Ph D students 4 4 technic icia ians

Laboratory of Chemistry of Natural Molecules

Laboratory of Chemistry of Natural Molecules

Ess ssentia ial l oil ils Other VOCs Oxy xyli lipin ins Oth ther se secondary ry metaboli lites

• 1. Essential oils

Oi Oilp lprotect Proje

• ject

EOs for Protection of cereals against insects Tierry Kenne and Saskia Sergeant

• Coll. UCL- private companies

Tree Inj njectio ion Proj

• ject

EOs for Protection of trees against insects Pierre-Yves Werrie Ph D student

• Coll. UCL- private companies

EOs Os pr prod

• ductio

ion in n Sen Senegal Two senegale Ph D students Founded by WBI EOs Os slow w rele elease for

• r bi

biop

• pestic

icid ide de develo lop. Use of new polymers for encapsulation Chloé Maes Ph D student

• Coll. Reims University

Ecoe

• extractio

ion of

• f Api

Apiaceae see eed EOs Os Iness Bettaieb post-doc

• Coll. Center of Biotechnology of Borj Cedria (Tunisia)

HER HERBIOIL L pr proj

• ject

Eos as herbicides study of the action mechanisms LBMI + CGO + CA EOH OHUB pr proj

• ject

Erasmus + Knowledge alliance

• Coll. different countries

Collaboration with different universities in Morocco, Tunisia, Algeria, …

• 2. Other VOCs

VOC OCs as as ma markers for

• r mycotoxin

in con

• ntamin

inatio ion (Agr Agros

• sensor

pr proj

• ject)
• Develop. of molecular inprinted polymer sensors

Laurie Josselin PhD student- Guo Xiaodong PhD student CAAS

• Coll. Umons-Materia Nova- U Gent- private companies

VOC OCS as as ma marker of

• f abi

abiotic ic pl plant str tress (So Solindic ic pr proj

• ject)

) Bas Bastien Dur Durenne Ph D student

• Coll. CRAW

Voc

• cactio

ion pr proj

• ject

VOCs as antifungal angents in plant protection Caroline Declercq post-doc Founded by FNRS leader: phytopathology lab Fla Flavor pr profili

• filing ,

, ar aroma form

• rmulation collaboration with

different companies (Firmenich, Orval, …) VOC OCS in n tr tradit itio ional fer ermented pr prod

• ducts

VOC OCs in n bi biolog

• logic

ical com

• mmunic

icatio ions Plant-plant, Plant-insect , plant-fungi Different coll. inside and outside ULg VOC OCs and and foo

• od pr

proc

• cess
• Marinated pork Han Dong PhD student CAAS
• Peanut oil LT Hu Hui PhD student CAAS
• Fruits/vegetables Wu Xinye PhD student CAAS

• 3. Oxylipins

Fatty ac acid hydrop

• peroxid

xides ne new w el elicitor/biopesticide Strucure –function relationship Estelle Deboever PhD student Co-promotion LBMI Es Esterif ifie ied oxyl ylip ipin ins : : role

• les in

n pl plants (ar arabid idop

• psid

ide pr proj

• ject)
• Total synthesis (41 steps !)

Firmin Obonou Akong post-doc

• Coll. UNamur
• Extraction, dev. Analytical methods, localization, structure-function

relationship Manon Genva PhD student Co-promotion LBMI

• 4. Other plant secondary metabolites

Al Allelo lopathy: stru tructure-functio ion rela elatio ionship ip Simon Lebeque PhD Student Leader LBMI

Sec Secondary metabolites fr from

• m afr

fric ican pla plants s for

• r

cos

• smetic/medicin

inal ap applic ications

• Matthew Saive : valorization of Mahoran flora Ph D student
• Fatimata Nea : valorization of endemic plants from Ivory

Cost, PhD student

• Evelyne Tanoh : valorization of endemic plants from Ivory

Cost, PhD student

• Liu Jiang : valorization of secondary metabolites from sweet

potatoes, Ph D student CAAS Fabaceae roo

• ot exudates to

to improve soi soil po pollu llution bi bioremedatio ion Marie Davin PhD student Co-promotion with soil dep

Analytical platform

Vola

• latolomics
• SAFE (Solvent assisted flavor extraction)
• Supercritical CO2 extraction
• Pilot distillation
• 3 GC-FID
• 3 GC-MS including the following injection modes
• Liquid injection
• SPME
• Static Headspace
• Dynamic headspace
• MVM (multivolatile method)
• DHS-TDU
• Twister

Non

• n vola
• latile

le 4 HPLC (DAD, RI, ELSD, fluo) 1 prep HPLC (DAD, RI, ELSD, fluo) 1 LC-MS 1 IR, 1 UV-vis

LBMI

Head  LINS Laurence

l.lins@ulg.ac.be +32 81 62 25 21

http://www.gembloux.ulg.ac.be/biophysique-moleculaire-aux-interfaces/

The team

 2 permanents (FNRS)

 Non permanent researchers:

• 1 post-doc
• 7 PhD students

 Technicians:

• 0.25 FTE lab technician
• 0.1 FTE secretary

Main research topics

University of Liege (Ulg) – Gembloux Agro-Bio Tech Laboratory of Molecular Biophysics at Interfaces (LBMI)

Study of the structure-function relationships of biomolecules

• Design of new active molecules

Molecular understanding and description of mechanisms involved in biological phenomena related to interactions with biological membranes

• Perception of biomolecules by cellular membranes
• Molecular organization within a membrane

Human health Plant protection

Integrative strategy with complementary in vitro and in silico biophysical approaches

Main equipement

 Langmuir film balance  Isothermal titration calorimetry  IR spectroscopy  Fluorescence spectroscopy  Circular dichroism  Computer plateform (Linux station bi-xeon quad core)

Co-Head  DELEU Magali

magali.deleu@ulg.ac.be +32 81 62 26 38

Interaction potential with membranes Lipid specificity Membrane

• rganization

Biomolecule characte- rization

Biological membranes

Molecular recognition, Signal transduction, Enzymatic catalysis, Cellular adhesion, Membrane fusion

Biological membrane = Bilayer with a complex composition and organization

14

First contact point between molecule and cell Biomolecule/membrane interaction: a key event for the biomolecule to be active

Plant Plasma Membrane Gronnier et al., Trends Plant Sci., 23, 10 (2018)

Lipid membrane models

A B C

Lipid monolayer Supported lipid bilayer Liposome

15

Working on the lipid part = No proteins !

Bioremediation

BestBioSurf Neamine derivatives

Sautrey et al., JBC, 291, 13864-13874, (2016)

Antibiotics

Microbial NLP cytolysin

Lenarcic et al., Science, 358, 1431, (2017)

Plant toxin

Ghrelin

Drug delivery

Salade et al., Int.J.Nanomedecine, 12, 8531, (2017)

Surfactants

Sugar esters

Meline et al., Enz. Microb. Technol.,112,14(2018) Luzuriaga-Loaiza et al., Sci. Reports, 8, 8534 (2018)

Elicitors

Claereboudt et al., Sci. Reports, 8, 10845 (2018)

Tolerance

Frondoside A Remorin

Gronnier et al., eLife, 6:e26404 (2017)

Plant protein

anchor into PM

Gramine Hordenine

Allelopathy

Lebecque et al., Sci. Reports, 8, 9784 (2018) Brault et al. EMBO J, submitted

Plant membrane contact sites

C2 domains and MCTPs at plasmodesmata

Interaction with lipid membranes Structure-function relationships

Deleu et al., Colloids .Surf. B , 175 (2019)

Key intermediate

• xylipins

R e l a t i

• n

s h i p R e l a t i

• n

s h i p b e t w e e n b e t w e e n f a t t y f a t t y a c i d a c i d h y d r

• p

e r

• x

i d e s h y d r

• p

e r

• x

i d e s a n d a n d t h e i r b i

• l
• g

i c a l b i

• l
• g

i c a l acti v i t i e s as as pote n t i a l biop e s t i c i d e s

DEBOEVER Estelle

PhD Student

About the context

• (A)biotic stresses >< plants
• Complex environmental conditions
• Adaptation strategies

The LOX pathway

• Emerging pathway in plant defence
• Oxylipins
• Free fatty acids  HPO  …
• HPOs in the literature

18 Polyunsaturated C18 fatty acids

Hydroperoxides

Aldehydes Divinyl Cyclized Oxo-acids ethers compounds Hydroxides Epoxides Epoxy alcohols Hydroxides α-Hydroperoxides Aldehydes Cytochromes P450 Lipoxygenase Pathogen-induced-

• xygenase (PIOX)

½ O2 2 O2 O2

HPL DES AOS LOX Hydroperoxides

Objectives

The goal of my PhD project is to answer 3 main scientific questions:

19

• In planta, are 13-HPO antimicrobial

agents ? Are-they sufficiently powerful to eliminate totally a pathogen when applied exogenously ?

• When applied exogenously, are 13-HPO

acting as elicitors ?

• How interactions between 13-HPO and

plasma membranes (plants or pathogens) can explain those mechanisms of action ?

Multidisciplinary strategy

Use of a multidisciplinary but complementary approach

20

Complementarity

• Langmuir through
• Isothermal titration

calorimetry

• Neutron reflectometry
• SAXS/WAXS
• Cryo-TEM
• Etc.

Bioinformatic and Biophysic tools

An example of raw data obtained for the partitioning of HPO in POPE/POPG/CL liposomes Time (min) Heat flow (µcal/sec) HPOD Time (min) Heat flow (µcal/sec) HPOT

Bio-assays

Comparison

In planta, are 13-HPO antimicrobial agents ?

21

BLACK K PINK

1 2 4 3 5 6

Comparison

In planta, are 13-HPO antimicrobial agents ?

22

Comparison

How interactions between 13-HPO and plasma membranes (plants or pathogens) can explain those mechanisms of action ?

23

• 20
• 20

20 20 40 40 60 60 80 80 100 100 5 10 10 Permé méab abil ilis isat atio ion n (%) Conce cent ntra rati tion

• n (µM)

Perme meab abil ilis isat atio ion n of bacte teri rial al PM POPE/PO E/POPG/C PG/CL

13-HPOD 13-HPOD 13-HPOT 13-HPOT

• 20
• 20

20 20 40 40 60 60 80 80 100 100 5 10 10 15 15 20 20 Permé méab abil ilis isat atio ion n (%) Conce cent ntra rati tion

• n (µM)

Permeabilisat meabilisation ion of plant nt PM PLPC/Sito/Glu C/Sito/GluCer Cer

13-HPOD 13-HPOD 13-HPOT 13-HPOT

Comparison

How interactions between 13-HPO and plasma membranes (plants or pathogens) can explain those mechanisms of action ?

24

y = -0,2489x + 9,0945 R² = 0,9483 y = -0,4331x + 20,407 R² = 0,9624 5 10 15 20 25 5 10 15 20 25 30 35 40 45 50

Δπ (mN/m) πi (mN/m)

13-HPO in PLPC/Sito/GluCer monolayer

y = -0,5384x + 12,394 R² = 0,9168 y = -0,4881x + 11,352 R² = 0,9101 2 4 6 8 10 12 14 5 10 15 20 25 Δπ (mN/m) πi (mN/m)

13-HPO in POPE/POPG/CL monolayer

What next ?

• Preliminary results show that:
• HPO have a significant in vitro antimicrobial activity
• Difference between HPOD/HPOT forms: HPOT >>> HPOD
• Interactions with biomimetic plasma membrane: HPOT >>> HPOD
• Correlation between antimicrobial activity and interactions with plasma membrane
• Need to do the in planta tests :
• In planta, are 13-HPO antimicrobial agents ?
• When applied exogenously, are 13-HPO acting as elicitors ?

25

Thank You for your attention

Estelle DEBOEVER estelle.deboever@doct.uliege .be

ESTERIFIED OXYLIPINS STRUCTURE - FUNCTION

RELA TIONSHIPS

GENVA MANON

Context

28

Arabidopsis thaliana (L.) Heynh. & other plants

Stress Arabidopsides and POL production

Wounding, cold, pathogen, …

• Plant oxylipins
• Free or esterified  galactolipids, phospholipids, …
• Arabidopsides

• Arabidopsides
• Localization
• Chloroplast membranes?
• Synthesis
• Following MGDG & DGDG oxidation
• Precise

cise functions: tions: unknown wn

• Roles in
• Plant defence processes
• Developmental processes
• Direct or undirect functions

29

Context

Genva, M., Obounou Akong, F., Andersson, M.X. et al. Phytochem Rev (2018).

Part 1

• Arabidopsides

extraction and purification

• Arabidopsides

characterization

• Development and

validation of an HPLC-MS method to detect and quantify arabidopsides

30

Thesis overview

• Obtention of pure arabidopsides A, B, D & E
• Lipid extraction
• (Semi-) preparative HPLC purification
• HPLC-MS separation and detection of arabidopsides
• Utilization of purified standards for arabidopsides quantification

by HPLC-MS

• Method validation almost finished
• Will be used for arabidopside determination in other Brassicaceae

plants

31

Thesis overview

Part 2 How does arabidopside modify structure and functions of plant membranes ?

• Detection and quantification
• f arabidopsides in plant

membranes

• Preliminary assays: arabidopsides detection among

chloroplast membranes

• Detection of arabidopsides in chloroplast external

membranes and thylakoïds

• Will be repeated this year using Rpm1/col-0 plants to

induce arabidopside production

32

Thesis overview

Part 2 How does arabidopside modify structure and functions of plant membranes ?

• Detection and quantification
• f arabidopsides in plant

membranes

• Study of arabidopside

interactions with those membranes in-vitro & In-silico

• Preliminary assays: arabidopsides detection among

chloroplast membranes

• In-vitro and in-silico assays
• Interfacial properties of arabidopsides are different from

non oxidized galactolipids

• Arabidopsides A, B & D can interact favourably with

chloroplast lipid

• Arabidopsides A and B are able to permeabilize chloroplast

membranes  arabidops idopside ide prese senc nce e in chlor

• roplas

ast t membranes embranes un under er stress ess may modify dify chlor lorop

• plast

last struc uctu ture re and d fun unct ction ions

33

Thesis overview

Part 2 How does arabidopside modify structure and functions of plant membranes ?

• Detection and quantification
• f arabidopsides in plant

membranes

• Study of arabidopside

interactions with those membranes in-vitro & In-silico

• Study of arabidopsides

effect on chloroplast functions

• Preliminary assays: arabidopsides detection among

chloroplast membranes

• In-vitro and in-silico assays
• Arabidopside effects on chloroplast function: planned

for this year

• Flow cytometry analysis of arabidopsides-containing

chloroplasts shape (Rpm1/col-0 , col 0)

• Photosynthesis ability

34

Thesis overview

Part 3 Are arabidopsides used as a pool of free

• xylipins or does

arabidopsides have direct functions?

• Biological assays:

study of esterified

• xylipins formation

and remobilization under stress conditions

• Planned for next years

Infection 1

Avirulent Pseudomonas syringae

Free and esterified

• xylipins

quantification after each infection and at different times Infection 2

Virulent Pseudomonas syringae

Are e ar arabidopsides s pr produced foll

• llowin

ing the fi first t infection use used to to gen enerate fr free ee

• xy

xylip ipins du during the sec second infection? Col-0 & LOX2 mutant Correlation between oxylipin production & disease symptoms

Postdoctoral fellowship

• Arabidopside total synthesis (41 steps)

THANK YOU FOR YOUR

ATTENTION

Funding ?

FN FNRS : short term grants, post doc grant ULg ULg : co-fund post doc grants In Interreg Nor

• rth

th-West : 3 partners, companies … (March-April 2019) Belgium, Germany, Switzerland, SME of Gembloux Agro-Bio Tech, … ERA ERAnet Su SusC sCrop https://www.suscrop.eu/ ERA ERAnet Cor CoreOrganic https://www.coreorganic.org/