Early male-female communication in flowering plants : When mechanical properties of a cell affect its neighboring cell behavior 2018-Mecacell project
Early male-female communication in flowering plants : When mechanical properties of a cell affect its
neighboring cell behavior
2018-Mecacell project
Isabelle Fobis-Loisy, CR CNRS Lucie Riglet, PhD Thierry Gaude, DR CNRS
Catherine Quilliet, MdC UGA Karin John, CR CNRS
Mecacell
Univ. Grenoble Alpes ENS de LYON
UNIVERSITE de LYON
Mecacell Consortium
Denis Bartolo, PR ENS
2018 project
Our scientific interest
Cell-cell interaction How cells communicate with each others?
How mechanical forces serve as cues for cellular behavior?
Model to address mechanical questions
A plant model: Arabidopsis thaliana
Interaction between male and female reproductive partners
10 mm
Reproduction in flowering plants
Pollen grains contain male gametes
50 mm
SEM
A. thaliana flower
Tube
50 mm
Elongated epidermal cells = stigmatic cells
ovules
SEM
Stigma
50 mm
Pollen tube : transports male gametes towards the ovules
Pollen tube growth at the stigma surface
Pollen grains
ovules
10 mm
Stigma
Our biological question
Pollen grains
ovules Early interaction at the stigma surface
The stigmatic cell structure
The plant cell wall
Cell Wall
TEM
A stigmatic cell 2 µm
Bordeaux Image Center
Cytoplasm
Vacuole
Plasma Membrane
Cuticle
Pollen tube “invades” stigma cell wall
The pollen tube digests locally the cuticle + cell wall and grows within the wall
Inner CW layer
Outer CW layer
Tube Stigmatic cell
2 µm
Bordeaux Image Center
TEM
Mechanical tensions
CMT cytoskeleton in stigmatic cell?
The plant cell cytoskeleton = sensor of mechanical pressure
microneedle
Cortical Microtubules (CMT) sense modification of tensions
(Hamant et al., 2008; Hardham et al., 2008)
Cytoskeleton of stigmatic cell
In stigmatic cells, CMT are oriented perpendicular to the longitudinal axis :
A. thaliana stigmatic cells
10 mm
CMT
WT plant mutant plant Katanin (ktn1)
A genetic approach to study role of CMT
CMT are disorganized in the ktn1 plant
5 µm
L. Riglet
50 µm
Ktn1 mutant : pollen tube coiled phenotype SEM
Stigma ktn1 pollen WT
Stigma WT pollen WT
50 µm
Ktn1 mutant : pollen tube coiled phenotype SEM
Stigma ktn1 pollen WT
Stigma WT pollen WT
50 µm
Ktn1 mutant : pollen tube coiled phenotype SEM
Stigma ktn1 pollen WT
Stigma WT pollen WT
Starting point of the Mecacell project
How the properties of one cell (stigmatic cell) can influence the behavior of a neighboring cell (pollen tube) ?
Ktn1
Where le pollen tube is growing in ktn1? kt
n1
x W
T
Pollen tube is growing inside the ktn1 cell wall
WT
x W
T
WT Pollen tube
WT Pollen tube
2 µm 10 µm
Mechanical properties of the stigma cell wall: stiffness
Atomic Force Microscopy
60nN force Point and Shoot mode
Lucie Riglet, Arezki Boudaoud, RDP
ktn1 cell wall is softer
WT ktn1
Young’s
mo
du
lus
(Mp
a)
= el
asti
city
20
40
0
***
Ktn1
CMT disorganized
Cell Wall softer
Pollen tube directionality affected
Deciphering the coiled phenotype ?
Ktn1 mutant
Golgi
PLASMA
MEMBRANE
Microtubules
Cellulose synthase
complex
Cellulose microfibrils
Vesicles
CELL WALL
CYTOPLASM
Hemicellulose
Pectins
CSI1
Cell wall proteins
20
Link between CMT and Cell Wall
Cellulose microfibrils
Hemicellulose fibers
Pectin matrix
Cellulose synthase complex
Cell wall
Plasma membrane
cytoplasm
CMT
Golgi
PLASMA
MEMBRANE
Microtubules
Cellulose synthase
complex
Cellulose microfibrils
Vesicles
CELL WALL
CYTOPLASM
Hemicellulose
Pectins
CSI1
Cell wall proteins
Cellulose microfibrils
Hemicellulose fibers
Pectin matrix
Cellulose synthase complex
CMT
Pollen tube path on cell wall mutant stigmas
xxt1 xxt2 xyl1
qua2
kor1 Prc1 any1
ktn1
Pollen tube growth not affected on CW mutant stigmas
Col-0 xxt1 xxt2
Young’s
mo
du
lus
(Mp
a)
20
40
0
***
n.s
xxt1xxt2 stigmatic cells are softer
Mechanical properties of xxt1xxt2 stigmatic cells
Alteration of the cell wall stiffness is not sufficient to induced the pollen tube coiled phenotype
ktn1-5 xxt1 xxt2
10 µm
ktn1-5
AFM: Stiffness
Mechanical properties of stigmatic cell wall : Resistance
Pollen tube speed:
Resistance
0
10
20
30
40
50
60
70
1 2 3 4 5 6 7 8 9 10 11
WT
xxt1 xxt2
Time after germination (min)
Dis
tan
ce (
µm
)
ktn1
1min
2min
3min
Despite similar stiffness, pollen tubes behave differently on ktn1 and xxt1xxt2 stigmas
WT and xxt1xxt2 ktn1
Conclusions/perspectives
Our 2019 project aims at characterizing the mechanical properties of the stigmatic cell wall, focusing on cellulose organization
How can we explain the
coiled phenotype ?
K. John C. Quilliet
The pollen tube coiled phenotype : answer from modeling???
A first mathematic model based on:
Stigmatic cell shape
Modelling pollen tube growth
Creation of a stigmatic cell profile :
The stigmatic cell profile
a kt n 1 -5 x W T W T x W T
10 µm
ktn1 WT
B
D
L
z
B
D
L
z
- 4 distances experimentally measured (B/D/z/L)
c
k
c
k
- 2 adjustable parameters (c/k)
Pollen tube growth parameters
Starting pollen tube angle
f
Initial pollen position
z/L
L
z
L
z
Tip growth Minimal local curvature of the tube
Keep contact with the stigma surface
Star
tin
g an
gle
(deg
ree
)
90°
60°
30°
120°
150°
180°
Starting position (z/L)
0.1 0.3 0.5 0.7 0.9
Pilot model
Pollen tube path on ktn1 stigma : Phase diagram
z/L: 0.15 = 170° 3 coils
Some parameters are missing to reproduce the observed coiled phenotype !
1
2
>3
Nu
mb
er o
f co
il
Conclusions/perspectives
2019 project: refining our mathematical model, adding mechanical surface tensions
Tensions imposed by the stigmatic Cell Wall
2019 Mecacell project
Mechanics, mediated by CMT organization, is a key player in early pollen tube guidance at the stigma surface
Improving our model
+ mechanical anisotropy of the stigma cell wall
long axis
transversal axis
Alignment of the pollen tube growth direction with a preferred axis
WT ktn1
Mechanical properties of the stigmatic Cell Wall
Cell Wall organization and structure
Organization of Cellulose Microfibrils
Staining confocal microscopy
TEM
Acknowledgements
Mission pour les Initiatives Transverses et Interdisciplinaires
Isabelle Fobis-Loisy, CR CNRS Lucie Riglet, PhD Thierry Gaude, DR CNRS
Catherine Quilliet, MdC UGA Karin John, CR CNRS
Mecacell
Univ. Grenoble Alpes ENS de LYON
UNIVERSITE de LYON
Denis Bartolo, PR ENS