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