In animals, microtubules and centrosomes direct the migration of gamete
pronuclei for fertilization. By contrast, flowering plants have lost essential
components of the centrosome, raising the question of how flowering
plants control gamete nuclei migration during fertilization. Here, we use
Arabidopsis thaliana to document a novel mechanism that regulates F-actin
dynamics in the female gametes and is essential for fertilization. Live
imaging shows that F-actin structures assist the male nucleus during its
migration towards the female nucleus. We identify a female gamete-
specific Rho-GTPase that regulates F-actin dynamics, and further show that
actin-myosin interactions are also involved in male gamete nucleus
migration. Genetic analyses and imaging indicate that microtubules are
dispensable for migration and fusion of male and female gamete nuclei.
The innovation of a novel actin-based mechanism of fertilization during
plant evolution might account for the complete loss of the centrosome in
flowering plants.
Marsilea vestita
in vitro
Arabidopsis
EC1
ACTIN DN-ACTIN;
Arabidopsis ,
DN-ACTIN
DN-ACTIN
FWA
DN-ACTIN
DN-
ACTIN
cdka;1
Arabidopsis
FWA
FWA
DN-ACTIN
lac GAL4
FWA
DN-ACTIN
lac cauliflower
mosaic virus 35S
FWA
ROP8
ROP8
CA-ROP8 DN-ROP8 ROP8
CA-ROP8 DN-ROP8
DN-ROP8
DN-ROP8
CA-ROP8
DN-ROP8
Arabidopsis
PORCINO POR
por/+
por por/por
por/+
por
por
por
por
por/+
por/por
por
Arabidopsis
por
Arabidopsis
Arabidopsis
DN-ROP8
CA-ROP8
DN-ROP8 CA-ROP8
Arabidopsis
in vitro
DN-ROP8
In vitro
formin
ROP
ROP
Arabidopsis
porcino (por) erecta
pH3.10::H3.10-mRFP1 pEC1::H2B-
mRFP1 p35S::sGFP-
ROP8 pAGL80::tagRFP-TUA5
ACTIN 8 DN-
ACTIN ROP8 (CA-ROP8
DN-ROP8; ,
EN35S
por
pFWA::Lifeact-Venus, pFWA::LhGR, pOPx4::NLS-mCherry-P2A:DN-ACTIN,
pH3.10::H3.10:Clover
in vitro
pH3.10::H3.10-mRFP1
pFWA::Lifeact-Venus
f(v)
k k
k
k
Arabidopsis
p35S::sGFP-ROP8 pAGL80::tagRFP-TUA5
Development
Proceedings of the
National Academy of Sciences of the United States of America
FEBS Letters
Journal of Cell
Biology
The Plant Journal
Trends in Cell Biology
The Arabidopsis Book
Plant Physiology
Plant & Cell
Physiology
Science
Sexual Plant Reproduction
Nucleic Acids Research
BMC
Evolutionary Biology
Molecular Plant
Cell
Current Biology
Current Opinion in Plant Biology
Journal of Experimental Botany
Science
Protoplasma
Methods in Molecular Biology
Protoplasma
Planta
Sexual Plant Reproduction
Current Biology
Current Biology
Plant Physiology
The Plant Cell
Plant & Cell Physiology
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Science
PLoS One
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Experimental biology
Proceedings of the National Academy of Sciences of the United States
of America
Current Biology
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Genetics
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Science
Current Biology
Nature Cell Biology
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Science
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Nature
Cell
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Science
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Genes & Development
International Review of Cell and
Molecular Biology
Plos
Biology
Current
Biology
The
Plant Journal
International Review of Cytology
Development
Current Biology
Cell
The Plant Cell
Planta
Arabidopsis
DN-ACTIN
Figure 2. F-actin in the central cell associates with the sperm cell nucleus
during migration. A-D
A B C
D
Figure 2-figure supplement 1 Unique F-actin structures in the mature
central cell before fertilization.
Figure 2-figure supplement 2. Effects of LatA and DN-ACTIN on F-actin and
fertilization in the central cell.
Figure 3. Actin cables generate an aster-like structure around the sperm
cell nucleus during migration in the central cell. (A-H)
A-C
D-E
F-H
Figure 4. Spatio-temporal control of F-actin disruption demonstrates
importance of F-actin for fertilization. A
FWA NLS-mCherry DN-
ACTIN
lac CaMV 35S
(B DN-ACTIN
C-F DN-ACTIN
C
D DN-ACTIN
E
F
Figure 5. ROP8 is specific to the central cell and involved in fertilization. A
Arabidopsis ROP
B,C B C ROP8
D DN-ROP8
Figure 5-figure supplement 1. Ectopic expression of ROP8 in the root also
shows its association to the plasma membrane.
CaMV 35S
Figure 5-figure supplement 2. The expression of neither constitutively-
active ROP8 (CA-ROP8) nor dominant-negative ROP8 (DN-ROP8) under the
control of the ROP8 promoter affect the F-actin structure (green) in the
central cell.
Figure 6. Controls of F-actin dynamics in the central cell. A
Arabidopsis
B
C
D
E
Figure 7. Effect of microtubule disruption on cytoskeleton in the central
cell. A,B A tubulin
folding cofactors c (por)/+ B
AGL80 C,D por
D C
E por/+
FWA F
Video 1.
pH3.10::H3.10-mRFP1
pFWA::Lifeact-Venus
Video 2.
pH3.10::H3.10-mRFP1
pFWA::Lifeact-Venus
Video 3.
pFWA::Lifeact-Venus
Video 4. CA-ROP8
pFWA::Lifeact-Venus
Video 5. DN-ROP8
pFWA::Lifeact-Venus
Video 6.
pFWA::Lifeact-Venus
Video 7.
pFWA::Lifeact-Venus