genetic evidence for essential calcium transporters in pollen growth and fertilization
DESCRIPTION
Working model for a CaM regulated calcium oscillator. Genetic evidence for essential calcium transporters in pollen growth and fertilization. Sabine Frietsch 1,3 , Shawn M. Romanowsky 1,2 , Morton Schiøtt 4 , Michael G. Palmgren 4 , Jeffrey F. Harper 1,2 - PowerPoint PPT PresentationTRANSCRIPT
Genetic evidence for essential calcium transporters in pollen growth and fertilization.
Sabine Frietsch 1,3, Shawn M. Romanowsky 1,2, Morton Schiøtt 4, Michael G. Palmgren 4, Jeffrey F. Harper 1,2
1 The Scripps Research Institute, Cell Biology, 10550 N. Torrey Pines Rd, 92037 La Jolla, CA, USA2 University of Nevada, Biochemistry, 1664 North Virginia Str, 89557 Reno, CA, USA
3 University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-01164 The Royal Veterinary and Agricultural University, Plant Biology, Thorvaldsensvej 40, DK-1871 Frederiksberg, Denmark
[email protected] and [email protected]
Introduction:
Calcium dynamics are thought to play a central role in pollen development, as evidenced by pharmaco-chemical approaches and visualization of calcium gradients and oscillations. Using Arabidopsis as a model system, we provide the first genetic evidence to support a model in which calcium signals are natural regulators of pollen tube growth and fertilization. Calcium signals are largely controlled by influx (through channels) and efflux (through pumps and antiporters). We have identified T-DNA gene disruptions in all 14 calcium pumps, and all 20 cyclic nucleotide gated channels (CNGCs). From this set of mutants, distinct pollen specific phenotypes have been found for disruptions of ACA9 (a calmodulin activated plasma membrane calcium pump) and CNGC18 (a cyclic nucleotide and calmodulin regulated putative calcium channel).
Calcium Pump Mutation aca9 Reduces Pollen Tube Growth Potential (in vivo)
cngc18 -/+ have full seed set indicating that the defect is very different than the aca9 phenotype.
Reciprocal crosses and out-crossing to the male sterile mutant, ms-1 show that cngc18 is male sterile.
CONCLUSIONS:
funiculus
funiculus
aca9 disruption results in partial male sterility
WT aca9Longest pollen tube
Aniline blue stain
WT ACA9promoter::GUS
ACA9-GFP
GFP
ACA9 appears to function as a plasma membrane pump in pollen
aca9 disruption results in >50% reduced frequency of synergidpenetration (discharge) required for fertilization
aca9 -/-
blocked
blocked
fertilized
Synergid penetration
penetration / discharge
Confocal image of GFP merged with DIC image
No synergid pentration or sperm release
Discharge + Discharge -
Discharge +:
Wild type 96%(n=178)
aca9 41%(n=247)
CNGC18 disruption results in complete male sterility
100
100
CNGC11
CNGC12
CNGC3
CNGC13
CNGC10
CNGC1
Group I
70
97
100
100
100100
100
CNGC7 CNGC8CNGC5
CNGC6CNGC9
Group II
100
100
100
CNGC19
CNGC20
Group IVA
88
CNGC4
CNGC2
Group IVB
94
10099
92CNGC18
CNGC16
CNGC17CNGC15CNGC14
Group III
100
100
CNGC11
CNGC12
CNGC3
CNGC13
CNGC10
CNGC1
Group I
70
97
100
100
100100
100
CNGC7 CNGC8CNGC5
CNGC6CNGC9
Group II
100
100
100
CNGC19
CNGC20
Group IVA
88
CNGC4
CNGC2
Group IVB
94
10099
92CNGC18
CNGC16
CNGC17CNGC15CNGC14
Group III
No homozygous cngc18 in over 400 F1 progeny of two independent gene disruption lines.
Homozygous gene disruption lines in all CNGCs but CNGC18.
~50%WT, colcngc18 (-/+)
0%cngc18 (-/+)ms-1 (-/-)
0%cngc18 (-/+)WT, col
F1 with disruption in CNGC18
♂ parent♀ parent
~50%WT, colcngc18 (-/+)
0%cngc18 (-/+)ms-1 (-/-)
0%cngc18 (-/+)WT, col
F1 with disruption in CNGC18
♂ parent♀ parent
Alexander staining for pollen viability in the quartet background (qrt) shows that cngc18 pollen is normal developed and viable.
Expression profile of CNGCs during pollen development
Modified after Maeser et al., 2001
CNGC8 and CNGC18 are highly expressed in mature pollen. Only disruption of CNGC18 results in male sterility.
100x
0
100
200
300
400
500
600
700
800
900
1000
unicellmicrospore
bicell. Pollen tricell. pollen maturepollen
av
era
ge
ex
pre
ss
ion
lev
el (
raw
va
lue
s)
CNGC7
CNGC8
CNGC9
CNGC16
CNGC18
cngc18 pollen tubes germinate, but only grow a short distance, with a “kinky-like” non-directional growth, often prematurely terminating with a bursting event.
cngc18 pollen germinate but has impaired tube growth in vitro
400x 400x
control, qrt (-/-) cngc18 (-/+), qrt (-/-)
In vitro germination assays in the quartet background (pollen doesn’t separate during development).
control, qrt (-/-) cngc18 (-/+), qrt (-/-)
Working model for a CaM regulated calcium oscillator
CNGC18
ACA9
cNMPCaM
• aca9 is the first mutant with a defect in pollen/ovule interaction.
• CNGC18 is the only CNGC essential for the plant life cycle.
• CNGC18 is the first potential calcium channel that was found to be involved in pollen tube growth.
• ACA9 and CNGC18 could be involve in a CaM regulated calcium oscillator, that is essential for pollen tube growth and fertilization.
• aca9 -/- plants display reduced pollen tube growth and >50% reduction of synergid penetration (discharge).
ACA9:
CNGC18:
Model:
• cngc18 pollen germinates with underdeveloped, kinky pollen tubes, that often burst at the tip.
• Disruption of ACA9 results in partial male sterile plants with shorter siliques and reduced seed set.
• Disruption of CNGC18 results in complete male sterility.
0
10
20
30
40
50
1 2 3 4number of germinated tubes per quartet
ger
min
atio
n f
req
uen
cy (
%)
total
all healthy
0
10
20
30
40
50
60
1 2 3 4
number of germinated tubes per quartet
ger
min
atio
n f
req
uen
cy (
%)
total
all healthy
Honys and Twell, 2003, Extracted from NASC database
WT,col
cngc18 (-/+)