Download Structure and function of the filamentous proteins PP2 (Phloem

Structure and function
of the filamentous proteins PP2
(Phloem Protein 2) in Arabidopsis
Ph.D fellowship : B.V. and CEPIA departments
Supervisors : Sylvie Dinant, INRA Versailles
Denis Renard, INRA Nantes
Introduction
Phloem tissue: a superhighway
between organs
Mature leaf
SOURCE
Photosynthesis
SINK
Xylem : Transport
roots aerial parts
Phloem : Transport
source sink
The phloem sap
- Photoassimilates and others metabolites
- Macromolecules (proteins and RNA)
- Hormones, signals
Introduction
Phloem transport
Xylem : system
under tension
Phloem : system
under pressure (MPa)
Water
Mass flow
Transpiration
Water
Water
Water
saccharose
proteins
hormones
ARN
saccharose
proteins
hormones
ARN
Source
Sink
Introduction
Companion
cell
The phloem translocation pathway:
the sieve elements
P-Proteins
Sieve plate
Phloem
parenchyma
cells
Plasmodesmata
Properties of
P-proteins
• Abundant
protein bodies in
sieve elements
Mature sieve
element (SE)
• Found in all dicots
N. tabacum,
Esau, 1969
P-Proteins are found in sieve elements
of all dicotyledonous species
Introduction
P-proteins
Sieve plate
3 – 4 µm
Sieve element
200-400 nm
Longitudinal section of celery
Role of P-proteins ?
N. tabacum, fibrillar P
proteins (Esau, 1969)
Introduction
P-Proteins : hypothesis
Regulation of sieve flux
• Different aggregates
morphologies depending on
species and developmental stages
Regulation of transport of
macromolecules
A subclass of P-proteins : PP2
• Properties of fixation with :
Similar physiological role
whatever the assemblies
morphology ?
- RNA
• Sealing of sieve plate
- N-acetylglucosamine (chitine)
- Proteins
• Gel filtration
structural role in the
sieve elements?
Role in the trafficking or
retention of macromolecules ?
Introduction
Arabidopsis thaliana : a model specie for
the study of PP2
Phylogenetic analysis
Conserved domains in PP2 proteins
N-term
A
B
30 PP2 in Arabidopsis (Dinant et al., 2003)
Secondary structure prediction (GOR V)
α Helix
β Sheet
Coil
C D
Strategy, objectives
Role of PP2 in phloem functions
• Role in the trafficking or retention of macromolecules ?
• Regulation of phloem sap translocation ?
Physiological
functions ?
Structural
properties ?
PP2
Presence in
P-proteins complex ?
Conditions of
polymerisation ?
Partners ?
Strategy, objectives
Role of PP2 in phloem functions
• Role in the trafficking or retention of macromolecules ?
• Regulation of phloem sap translocation ?
Physiological
functions ?
Structural
properties ?
PP2
Presence in
P-proteins complex ?
Conditions of
polymerisation ?
Partners ?
Patterns of expression of AtPP2-A1 and AtPP2-A2
Physiological
functions ?
AtPP2-A1 and AtPP2-A2 are transcribed in the phloem
At the tissular level : expression
of genes in vascular tissues
At the cellular level : expression
in the complex sieve elements –
companion cells
PP2-A1 mRNA
Dinant et al., 2003
Expression pattern of AtPP2-A1
Spatial and temporal pattern of transcript
accumulation : lines pPP2-A1 :: GUS
pPP2-A1
GUS
Term
Blue staining
Transcription irregular as a
function of the order of the veins
More transcription in source
organs than in sink
Transcription decrease during senescence
Analysis of mutants affected in PP2 expression
At4g19840 : PP2-A1
5’
Gene
RNAi
mRNA
Protein
RNAi mutants affected in expression of PP2-A1
27 lines tested
15 retained with reduced accumulation of
PP2-A1 mRNA
Phenotype analysis (development, architecture)
Analysis of RNAi mutants
WT
RNAi
Plants are affected in early stages of development
WT
RNAi
10 days-old plants
4 week-old plants
Strong deformation of leafs
Analysis of RNAi mutants
WT
RNAi
WT
RNAi
RNAi
Problem in the formation of silique
Seeds are missing
2 month-old plants
Analysis of RNAi mutants : conclusion and
perspectives
Mutants are affected
- in architecture
- in development
- in fertility
Effect of mutation
- on the structure of vascular tissues ?
- on sieve flux ?
- on transport of molecules ?
Structure-function study of PP2
Partners ?
Do PP2 interact
together ?
Visualisation of in vivo interactions using
Bimolecular Fluorescence Complementation (BiFC)
Confocal
microscopy
observations
Visualisation of in vivo interactions by BiFC
• Cloning
• Transient expression in epidermal cells of Tobacco
• Observation by confocal microscopy
chloroplast
Interactions in the
cytoplasm
vacuole
10 µm
A1 form homopolymers
A1 and A2 form heteropolymers
Perspectives : Can A2 form homopolymers ?
cytoplasm
Structure-function study of PP2
Structural
properties ?
• Production of recombinant proteins • A1 : 28 kD
in E. coli
• A2 : 18 kD
MW T0 Tot Sol
93
66
45
30
20,1
• Purification
14,4
Antibodies production
Localisation of PP2
into P-proteins ?
Physico-chemical
analyses
Perspectives
• Structure of monomers
• in vitro polymerisation Assay
• Analyses of physico-chemical properties of PP2 polymers
- Polymerisation degree ?
- Conformation
- Conditions affecting polymerisation ( pH, Ca2+…)?
Acknowledgements
Phloem group in Versailles
Sylvie Dinant
Francoise Vilaine
Chantal Kusiak
Lionel Gissot
APR Group in Nantes
Denis Renard
Khalil Elmorjani
Cédric Gaillard
Sandrine Reze
Axelle Hermouet
Laurent Marché
Collaborations
Gary Thompson (Little Rock, USA)
INRA Colmar
Véronique Brault
INRA Grignon
Pierre Briozzo
CNRS Poitiers
Rémi Lemoine
INRA Bordeaux
Brigitte Bataille-Gélie