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A Cyclic Nucleotide-Gated Channel (CNGC16)
in Pollen Is Critical for Stress Tolerance in Pollen
Reproductive Development1[W][OA]
Meral Tunc-Ozdemir, Chong Tang, Maryam Rahmati Ishka, Elizabeth Brown, Norman R. Groves,
Candace T. Myers2, Claudia Rato3, Lisbeth R. Poulsen, Stephen McDowell, Gad Miller,
Ron Mittler, and Jeffrey F. Harper*
Department of Biochemistry, University of Nevada, Reno, Nevada 89557 (M.T.-O., C.T., M.R.I., E.B., C.T.M.,
L.R.P., S.M., J.F.H.); Department of Molecular Genetics, The Ohio State University, Columbus, Ohio 43210
(N.R.G.); Universidade de Lisboa, Faculdade de Ciências de Lisboa, BioFIG, 1749–016 Lisboa, Portugal (C.R.);
Department of Plant Biology and Biotechnology, Centre for Membrane Pumps in Cells and Disease
(PUMPKIN), University of Copenhagen, Danish National Research Foundation, 2000 Frederiksberg, Denmark
(L.R.P.); The Mina and Everard Goodman Faculty of Life Sciences Bar Ilan University, Ramat-Gan 52900,
Israel (G.M.); and Department of Biological Sciences, University of North Texas, Denton, Texas 76203 (R.M.)
Cyclic nucleotide-gated channels (CNGCs) have been implicated in diverse aspects of plant growth and development, including
responses to biotic and abiotic stress, as well as pollen tube growth and fertility. Here, genetic evidence identifies CNGC16 in
Arabidopsis (Arabidopsis thaliana) as critical for pollen fertility under conditions of heat stress and drought. Two independent
transfer DNA disruptions of cngc16 resulted in a greater than 10-fold stress-dependent reduction in pollen fitness and seed set.
This phenotype was fully rescued through pollen expression of a CNGC16 transgene, indicating that cngc16-1 and 16-2 were both
loss-of-function null alleles. The most stress-sensitive period for cngc16 pollen was during germination and the initiation of
pollen tube tip growth. Pollen viability assays indicate that mutant pollen are also hypersensitive to external calcium chloride,
a phenomenon analogous to calcium chloride hypersensitivities observed in other cngc mutants. A heat stress was found to
increase concentrations of 39,59-cyclic guanyl monophosphate in both pollen and leaves, as detected using an antibody-binding
assay. A quantitative PCR analysis indicates that cngc16 mutant pollen have attenuated expression of several heat-stress
response genes, including two heat shock transcription factor genes, HsfA2 and HsfB1. Together, these results provide
evidence for a heat stress response pathway in pollen that connects a cyclic nucleotide signal, a Ca2+-permeable ion channel,
and a signaling network that activates a downstream transcriptional heat shock response.
The reproductive phase in flowering plants can be
highly sensitive to hot or cold temperature stresses. Even
a single hot day or cold night can sometimes be fatal to
reproductive success. Pollen development and fertilization
are often the most temperature-sensitive part of
reproductive development (Zinn et al., 2010). A heat
stress response in pollen, like vegetative tissues, involves
changes in gene expression, including increased
mRNA levels for heat shock transcription factors (e.g.
HsfA2) and heat shock proteins (e.g. Heat Shock
Protein17-CII and Thermosensitive Male Sterile1; Frank
et al., 2009; Yang et al., 2009; Giorno et al., 2010).
Nevertheless, the signaling pathways underlying these
responses remain poorly understood, especially in
pollen development.
Cyclic nucleotide monophosphates (cNMPs) 39,59-
cyclic guanyl monophosphate (cGMP) and cAMP play
key roles in the regulation of diverse cellular processes
in eukaryotes and prokaryotes (Jammes et al., 2011),
including biotic and abiotic stresses. In plants, cNMPs
have been implicated in pathogen responses and salt
and osmotic stresses (Jammes et al., 2011; Li et al.,
2011; Ma and Berkowitz, 2011; Moeder et al., 2011).
1 This work was supported by grants from the National Science
Foundation (DBI–0420033 to J.F.H. and R.M.) for stress-dependent
phenotype screens, and from the National Institutes of Health
(1RO1 GM070813–01 to J.F.H.) for studies on forming calcium signals
in pollen and for studies on membrane biogenesis and function (DE–
FG03–94ER20152 to J.F.H.). Bioinformatics was made possible by the
IDeA Network of Biomedical Research Excellence Program of the
National Center for Research Resources (National Institutes of Health
grant no. P20 RR–016464). Confocal microscopy was made possible
by support from National Institutes of Health Center of Biomedical
Research Excellence grant no. RR024210.
2 Present address: Department of Cellular and Molecular Medicine,
Molecular Cardiovascular Research Program, University of
Arizona, Tucson, AZ 85724.
3 Present address: European Molecular Biology Laboratory, European
Bioinformatics Institute, Wellcome Trust Genome Campus,
Hinxton CB10 1SD, UK.
* Corresponding author; e-mail jfharper@unr.edu.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described
in the Instructions for Authors (www.plantphysiol.org) is:
Jeffrey F. Harper (jfharper@unr.edu).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription.
www.plantphysiol.org/cgi/doi/10.1104/pp.112.206888
1010 Plant Physiology, February 2013, Vol. 161, pp. 1010–1020, www.plantphysiol.org 2012 American Society of Plant Biologists. All Rights Reserved.
Recently, cNMPs have also been linked to heat stress
responses in vegetative tissues from Arabidopsis (Arabidopsis
thaliana) and the moss Physcomitrella patens (Finka
et al., 2012; Gao et al., 2012). In these cases, genetic and
electrophysiological evidence suggest that cNMPs activate
cyclic nucleotide-gated ion channels (CNGCs).
CNGCs are Ca2+-permeable cation transport channels
that are activated by cNMPs and deactivated by
binding Ca2+/calmodulin (Cukkemane et al., 2011; Ma
and Berkowitz, 2011; Spalding and Harper, 2011).
They have been identified in both plant and animal
systems (Schuurink et al., 1998; Köhler and Neuhaus,
2000; Becchetti et al., 2009; Zelman et al., 2012). In
plants, CNGCs have a binding site for calmodulin that
overlaps with a site for cNMP (Köhler et al., 1999).
Thus, CNGCs have the potential for integrating signals
from cyclic nucleotide and Ca2+ signaling pathways
(Newton and Smith, 2004).
Arabidopsis contains 20 CNGC family members (Mäser
et al., 2001) that are differentially expressed in all tissues
(Talke et al., 2003). CNGC18 has been shown to provide
an essential function in pollen tube tip growth (Frietsch
et al., 2007). This is consistent with pharmacological evidence
that cyclic nucleotide signals in pollen can trigger
growth-altering Ca2+ signals (Moutinho et al., 2001; Rato
et al., 2004; Wu et al., 2011). Here, we show that CNGC16,
another pollen expressing CNGC, is critical for heat stress
tolerance, providing a link between a stress-triggered
cNMP signal and a downstream transcriptional heat
shock response.
RESULTS
cngc16 Disruptions Show a Stress-Dependent
Segregation Distortion
To identify genes involved in stress tolerance, we initiated
a screen for transfer DNA (T-DNA) mutations that
showed a stress-dependent segregation distortion under
conditions of hot days and cold nights (Fig. 1B). For in
vivo pollen tube growth in Arabidopsis, approximately
2 h is required for the first pollen tubes to grow through
the stigma and enter the ovary and about 4 to 5 h for
tubes to traverse 50% of the distance to the bottom of
the ovary (Schiøtt et al., 2004; Crawford et al., 2007). The
stress conditions chosen here were designed to provide
only a small window of optimal growth conditions to
force pollen to cope with a stress condition in order to
complete a fertilization event. The daily cycling between
hot and cold conditions allowed plants to be grown under
the same stress regime for their entire life cycle,
whereas a continuous exposure to either the hot or cold
stress would have been lethal. The stress cycle used was
found to reduce seed set in wild-type siliques by nearly 2-
fold (Fig. 1C; Supplemental Fig. S1), providing an optimal
stress condition in which to screen for mutants that either
increased or decreased reproductive stress tolerance.
A stress-dependent decrease in transmission was
observed (Table I) for two independent T-DNA insertions
in a gene encoding cyclic nucleotide-gated
channel16 (CNGC16; Fig. 1A).
Both insertions were associated with a T-DNA harboring
a Basta resistance marker (Sessions et al., 2002).
When plants heterozygous for cngc16-1(2/+) and 16-2
(2/+) were allowed to self-fertilize under conditions
of hot days and cold nights, the Basta marker showed
an approximately 50% transmission instead of an expected
75% (n = 720, P , 0.001; Table I). This distortion
was confirmed in a subset of progeny by PCR genotyping
of cngc16-1 and cngc16-2 T-DNA insertion site
borders (n = 61, P , 0.01). In contrast, plants grown
under standard nonstress conditions showed segregation
very close to the expected 75% (e.g. 69%–73%,
n = 536).
To determine if a heat stress alone was sufficient to
reduce transmission of the cngc16 T-DNA insertions,
heterozygous mutants were transferred into a heat
stress chamber (32°C or 37°C) for 4 to 6 d while
flowering and then returned to normal conditions for
seed maturation. Flower meristems were pruned to
Figure 1. Knockout mutations for cngc16 and corresponding stressdependent
seed set phenotype. A, Schematic diagram of CNGC16
gene model (AT3G48010) and T-DNA insertion sites in cngc16-1 and
cngc16-2. Positions are shown for T-DNA insertions (triangles), exons
(rectangles), introns (lines), and primers (arrows). Black regions represent
transmembranes (S1–S6) and pore (P) domains in the corresponding
protein. Gray shading represents the cyclic nucleotide
binding domain (CNBD). B, Schematic diagram of the hot day and
cold night stress cycling from 21°C to 40°C and forcing the period of
pollen tube growth and fertilization to overlap with suboptimal temperatures.
C, Seed set analysis of cngc16 shows a near-sterile phenotype
under the hot/cold stress conditions diagrammed in B. n, Number
of siliques counted. Student’s t test was performed to detect significant
differences between cngc16 mutants and the wild type (wt) under hot
and cold stress. **Student’s t test significant at P , 0.01.
Plant Physiol. Vol. 161, 2013 1011
CNGC16 Is Critical for Stress Tolerance in Pollen
leave only siliques that resulted from self-fertilization
events that occurred during the stress period. Seed
from these siliques were then harvested and used to
determine the transmission frequency of T-DNA mutations
to F1 progeny conceived only during the limited
stress period. The heat stress alone resulted in
an equivalent segregation distortion to that observed
under a hot day/cold night stress regime (Table I).
To determine if a drought stress could also result
in a segregation distortion, heterozygous plants were
subjected to a period of severe drought and then rescued
through restored watering. As the drought stress
became more severe, the resulting siliques became
shorter and contained fewer seeds. To focus only on
F1 progeny whose reproductive origins were actually
impacted by the period of drought stress, seeds were
selectively harvested from the shortest siliques, which
contained progeny conceived during the most severe
stages of the drought stress. These progeny showed a
segregation distortion equivalent to that observed with
a hot day/cold night stress regime (Table I).
cngc16 Mutations Result in a Stress-Dependent
Pollen Defect
To determine whether the observed stress-dependent
segregation distortion in self-fertilized plants was due
to a defect in either the male or female gametophytes,
we conducted reciprocal crosses between cngc16(+/2)
mutants and plants that were either wild type or male
sterile (ms1-1). When cngc16 heterozygotes were used
as females, a normal 50% transmission frequency was
observed with or without stress conditions (Table II,
groups A and E). In contrast, cngc16 pollen transmission
under stress conditions was reduced to 1% to
2.4% (Table II, groups B and D). This indicated that
cngc16 pollen alone had a stress-dependent defect in
transmission.
To determine which aspects of cngc16 pollen are
stress sensitive, plants were exposed to stress either
before and/or after a cross. When pollen were allowed
to develop under a stress of hot days and cold nights
and then outcrossed and allowed to fertilize under
nonstress conditions, transmission efficiency dropped
approximately 1.5-fold from 31% observed for control
conditions down to 21% (Table II, group C). For these
crosses the expected transmission efficiency was considered
to be 31%, based on our nonstress control
conditions. This slight decrease in transmission for the
nonstress control (i.e. from 50% down to 31%) is likely
due to manual pollination resulting in multiple stresses,
such as wounding from brushing pollen onto the stigma,
in addition to a low humidity stress that is common in
dry climates when a growth chamber door is opened to
perform a cross.
The largest relative decrease in cngc16 transmission
efficiency occurred when pollen from a heterozygous
cngc16 mutant (grown without stress) was used to
pollinate an ms1-1 female, and the outcrossed plant
moved into the stress chamber within 30 min. Under
these conditions, the cngc16 transmission efficiency
was reduced more than 10-fold, down to 1.2% to 2.4%
(31% expected; Table II, group B). However, if the
stress exposure was delayed by 1 to 2 h, the transmission
efficiency was less severe, with only a 1.5-fold
decrease similar to that observed when pollen were
stressed only during maturation (Table II, group G).
This bracketing indicates that while mutant pollen
were still sensitive to stress during grain maturation or
tube growth, they were most sensitive during the first
1 to 2 h postpollination, corresponding to germination
and early tube growth into the stigma (Crawford et al.,
2007).
Homozygous cngc16 Mutants Have Reduced Seed Set
under Conditions of a Hot/Cold Stress
Two possible types of pollen defects could explain a
stress-dependent segregation distortion: (1) A reduction
Table I. Segregation analysis showing non-Mendelian transmission of the T-DNA in cngc16 mutants
under various stresses
cngc16-1(+/2) and cncg 16-2(+/2) were self-pollinated under no stress, hot/cold (hot days and cold
nights; Fig. 1B), 32°C, 37°C, or drought stresses. Statistical significance was determined by Pearson’s x2
test.
Allele (2/+) Selfing Condition F1Total
Segregation of Basta Resistance Marker
Expected Observed P Value
%
cngc16-1 No Stress 282 75 73 ,0.700
cngc16-2 No Stress 254 75 69 ,0.300
cngc16-1 Hot/Cold 380 75 49 ,0.001
cngc16-2 Hot/Cold 340 75 52 ,0.001
cngc16-1 32°C 323 75 53 ,0.001
cngc16-2 32°C 945 75 52 ,0.001
cngc16-1 37°C 369 75 52 ,0.001
cngc16-2 37°C 213 75 48 ,0.001
cngc16-1 Drought 104 75 48 ,0.001
cngc16-2 Drought 390 75 48 ,0.001
1012 Plant Physiol. Vol. 161, 2013
Tunc-Ozdemir et al.
in competitiveness compared with the wild type, for
example, from a slightly slower rate of pollen grain
germination or tube growth; or (2) complete sterility, for
example, from death or the inability to grow or discharge
sperm cells. To help distinguish between these
two alternatives, we grew homozygous mutants under
conditions of hot days and cold nights and quantified
the number of seeds per silique (Fig. 1C; Supplemental
Fig. S1). For both cngc16-1(2/2) and cngc16-2(2/2)
mutants, we observed a stress-dependent 10-fold decrease
in seed set. This indicates that there is stressdependent
transmission defect even in the absence of
competition with wild-type pollen. This is consistent
with a model in which the mutant pollen show a stressdependent
lethality, as opposed to slightly slower rate of
pollen grain germination or tube growth.
Expression of GFP-CNGC16 Rescues the cngc16
Stress-Dependent Phenotype
To determine if pollen expression of a GFP-tagged
CNGC16 could rescue the stress dependent phenotypes,
a transgene encoding a GFP-CNGC16 was stably
transformed into homozygous cngc16-1 and
cngc16-2 backgrounds. The CNGC18 promoter was
used for relatively weak levels of pollen expression
(Frietsch et al., 2007), whereas an ACA9 promoter was
used for strong expression (Schiøtt et al., 2004). The
ACA9 promoter was previously used with a parallel
construct to drive the expression of a GFP-CNGC18,
which provided sufficiently high expression levels to
allow the GFP-GNCG18 to be visualized in pollen
(Frietsch et al., 2007). However, for both GFP-CNGC16
rescue constructs, we were unable to detect a GFP
signal, suggesting that mRNA or protein expression
levels were kept very low, despite the use of a relatively
strong ACA9 promoter. Nevertheless, both rescue
constructs were observed to restore seed set to
levels equivalent to the wild type (approximately 35
seeds) grown under parallel stress conditions (Fig. 2A).
In addition to rescuing the seed set phenotype, the
CNGC16 transgene also rescued the stress-dependent
segregation distortion phenotype. This was observed
in a competition assay in which 50% of the mutant
pollen harbored a rescue construct (i.e. cngc162/2
with a hemizygous transgene). The transmission frequency
of the rescue construct was assayed using the
associated hygromycin marker. In the absence of
stress, a normal 50% transmission efficiency was observed
for a pollen outcross, indicating that under
control conditions, all pollen were equally competitive
(Fig. 2B). In contrast, under conditions of a hot/cold
Table II. Segregation analysis showing a stress-dependent defect in cngc16 pollen transmission
Transmission efficiencies are shown for reciprocal crosses between cngc16-1(–/+), cngc16-2(–/+), and wild-type (WT) plants or ms1-1 plants under
various conditions. All outcrosses in which pollinated plants were moved to a stress chamber were done between 2 and 5 PM (10°C) on the stress
cycle shown in Figure 1. “Pre” and “Post” refer to application of stress to the female recipient or pollen before or after the manual cross. All post-cross
stress treatments occurred within 30 min of the cross unless otherwise indicated. Statistical significance was determined by Pearson’s x2 test.
Group
Hot/Cold Stress
Regime Female 3 Male F1 Total
Segregation of Basta Resistance Marker
Pre Post Expected Observed P Value
%
A
Female 2 2 cngc16-1(–/+) 3 WT 285 50 48 ,0.7
cngc16-2(–/+) 3 WT 156 50 50 1
Pollen 2 2 WT 3 cngc16-1(–/+) 490 50 33 ,0.001
WT 3 cngc16-2(–/+) 792 50 31 ,0.001
B
Female 2 + ms1-1 3 cngc16-1(–/+) 602 50 2.4 ,0.001
Pollen 2 + ms1-1 3 cngc16-2(–/+) 162 50 1.2 ,0.001
C
Female 2 2 WT 3 cngc16-1(–/+) 170 50 21 ,0.001
Pollen + 2 WT 3 cngc16-2(–/+) 40 50 20 ,0.01
D
Female 2 + ms1-1 3 cngc16-2(–/+) 96 50 1 ,0.001
Pollen + +
E
Female + + cngc 16-1(–/+) 3 WT 97 50 48.4 ,0.95
Pollen 2 +
F
Female + + ms1-1 3 cngc16-2(–/+) 97 50 2.1 ,0.001
Pollen + +
G
Female 2 +a ms1-1 3 cngc16-1(–/+) 458 50 20.6 ,0.001
Pollen 2 +a ms1-1 3 cngc16-2(–/+) 427 50 22.6 ,0.001
aIndicates a 1-h delay before crossed plants were transferred to the stress chamber.
Plant Physiol. Vol. 161, 2013 1013
CNGC16 Is Critical for Stress Tolerance in Pollen
stress, the only pollen transmission observed was for
mutant pollen harboring GFP-CNGC16 (n = 108, P ,
0.001). Together, the observation that the stress-dependent
segregation distortion and reduced seed set phenotypes
could be rescued using a pollen-expressed GFP-CNGC16
corroborates that cngc16-1 and cngc16-2 represent loss-offunction
null mutations.
The Viability of cngc16 Pollen Can Be Reduced by
Temperature Stress or Elevated CaCl2
Alexander staining was used as an assay to evaluate
pollen viability in response to environmental conditions.
In pollen isolated from plants grown under a
stress of hot days and cold nights, pollen from homozygous
cngc16 mutants were twice as likely to be dead
compared with wild-type controls (Fig. 3; Supplemental
Fig. S2).
In an attempt to use an in vitro pollen growth assay
to study the cngc16 phenotype, we observed that less
than 1% of cngc16 mutant pollen grew tubes longer
than 200 mm. Instead, more than 80% of the pollen
grains either failed to germinate, arrested as short
pollen tubes (i.e. ,30 mm), or ruptured (Fig. 4).
To determine if a specific component of the pollen
germination medium was responsible for the observed
developmental block, we examined the effects of
varying concentrations of CaCl2 from 0 to 10 mM in a
standard liquid germination medium. After 3 h, the
viability of pollen grains was assayed using Alexander’s
stain (Fig. 5). At 10 mM CaCl2, cngc16 pollen
showed a more than 2-fold increase in pollen death
compared with the wild type. This increase in lethality
also occurred with 10 mM CaCl2 in a Tris-MES buffer,
suggesting that additional nutrient components normally
present in our standard germination medium
were not necessary for the hypersensitivity to CaCl2.
The Induction of Key Thermotolerance Genes Is Impaired
in cngc16 Pollen
To elucidate the underlying cause of the cngc16
stress sensitivity, we performed real-time quantitative
reverse transcription (RT)-PCR analyses on a subset of
genes previously identified as stress markers (Fig. 6).
As an example of a nonresponsive marker, Zat12 failed
to show any temperature-dependent changes in mutants
or the wild type. In contrast, stress-induced changes
were observed for two HSF genes, HsfA2 and HsfB1.
While both HSFs still showed a weak stress-induced
increase in cngc16-1 and cngc16-2 backgrounds, their
induction was 2- to 4-fold lower compared with wildtype
controls. Similarly, a downstream gene under the
control of HsfA2 was also assayed (BCL-2-associated
athanogene6 [Bag6], At2g46240; Nishizawa-Yokoi et al.,
2009) and found to have a 2- to 7-fold lower induction
Figure 2. Seed set and segregation analysis showing the rescue of the male sterile phenotype. A, Seed set analyses of cngc16
knockouts rescued with a CNGC18p(i)-GFP-CNGC16 construct [seed stock nos. 1646 and 1647 for cngc16-1(2/2) and
cngc16-2(2/2) backgrounds, respectively] showing seed set levels equivalent to the wild type (wt) under a hot and cold stress
regime (siliques counted = 5). B, Outcrosses with representative rescue lines seed stock numbers 1646 and 1647 [for cngc16-1
(2/2) and cngc16-2(2/2) backgrounds, respectively]. The rescue construct CNGC18p(i)-GFP-CNGC16 was hemizygous in all
crosses. A rescue experiment was repeated with similar results using two additional lines harboring the same GFP-CNGC16
under the control of the ACA9 pollen promoter (seed stock nos. 1648 and 1649; data not shown). For crosses done with a hot/
cold stress, the female had a ms1-1 phenotype. After a manual fertilization, the plants were moved to a hot day/cold night stress
chamber, with the entry time at approximately 3 PM and temperature at 10°C (see Figure 1B). Statistical significance was determined
by Pearson’s x2 test.
Figure 3. Viability staining showing cngc16 mutants are hypersensitive
to stress. Viability was assayed using Alexander’s reagent. Pollen
were harvested from plants growing under conditions of hot days and
cold nights (see Figure 1B). Values represent means 6 SD of three independent
experiments (n = 50–100 pollen grains for each experiment).
Student’s t test was done to compare the pollen viability of
cngc16 mutants with wild-type (wt) plants grown under a hot and cold
stress regime. **Student’s t test significant at P , 0.01.
1014 Plant Physiol. Vol. 161, 2013
Tunc-Ozdemir et al.
compared with the wild type. It is noteworthy that the
BAG6 induction was more impaired in the cngc16-1 mutant
background than cngc16-2, which might indicate that
lines harboring cngc16-1 and cngc16-2 have functional
differences. At a minimum, the cngc16-1 line includes an
additional quartet mutation that was included in subset
of lines generated for the Syngenta Arabidopsis Insertion
Library T-DNA knockout collection (Alonso et al., 2003).
The quartet mutant has a defect that alters the pollen cell
wall and keeps the four meiotic products physically
linked together in a tetrad (Rhee and Somerville, 1998).
Regardless of potential modifiers in cngc16-1, both mutants
show an impaired stress-dependent transcriptional
response.
Heat Stress Increases cGMP Levels
To determine if a heat stress can trigger a rise or fall
in the level of a cyclic nucleotide, we attempted to
quantify levels of cAMP and cGMP in pollen with and
without a heat stress. To detect cNMPs, we used antibodies
that could distinguish between cAMP and
cGMP. Unfortunately, we were unable to detect cAMP
above background levels, leaving us to estimate that
its concentration in pollen grains and leaves is less
than 74 pmol g–1 dry weight. However, we were able
to detect cGMP, at 13.65 pmol g–1 dry weight in leaves
and 0.019 pmol g–1 dry weight in pollen and observed a
small but statistically significant heat stress-dependent
increase in both leaves and pollen (Fig. 7).
DISCUSSION
Genetic evidence presented here identifies CNGC16
in Arabidopsis as critical to reproductive success
under conditions of heat stress or drought (Table I).
Two independent T-DNA disruptions of cngc16 (Fig.
1A) resulted in stress-dependent reductions in pollen
fitness (Table II) and seed set (Fig. 1C). This phenotype
was fully rescued through pollen expression of a CNGC16
transgene (Fig. 2), indicating that cngc16-1 and cngc16-2
were both loss-of-function null alleles. CNGC16 is
expressed primarily in pollen (Fig. 8), which is consistent
with a stress-dependent phenotype associated
with pollen transmission. In contrast, there was no
detectable transmission deficiency through the female
gametophyte (Table II, group E).
Germination of cngc16 Pollen Grains Is Highly Sensitive to
Environmental Conditions
The most stress-sensitive period for cngc16 pollen
was observed as a 10-fold decrease in transmission
efficiency when the stress environment was introduced
just after a manual pollination (Table II). When the
stress exposure was limited to prepollination, there
was only a 1.5- to 2.5-fold decrease in transmission
efficiency. This is consistent with a viability assay
showing that when pollen grains developed under
Figure 4. cngc16 pollen show poor growth and bursting when germinated
in vitro. Pollen grains were harvested from plants grown under
normal conditions. Germination and growth were allowed to proceed
for approximately 12 h on a standard in vitro agar-based growth medium
containing 1 mM CaCl2. Values represent means 6 SD of three to
five independent experiments, each with approximately 200 pollen
grains. wt, Wild type.
Figure 5. Alexander viability staining demonstrates that cngc16 pollen
are hypersensitive to elevated CaCl2 concentrations. Pollen grains were
harvested into a water suspension from plants grown under normal
conditions. Aliquots were modified as indicated and incubated for 3 h
at 20°C in parallel. Incubations were done in solutions corresponding
to water only, standard liquid in vitro germination medium, pH 7.5
(GM), or Tris-MES buffer (pH 7.5). Solutions were amended as indicated
with Ca2+ using CaCl2. Alexander staining was done after 3 h by
pelleting pollen and resuspending pellets in 1 mL of Alexander stain
for 30 min. Within the relatively short post hydration time frame
assayed, wild-type (wt) controls for each solution showed less than
0.5% pollen grain germination. Viability counts were done with a
digital camera mounted on a Leica DM IRE2 microscope. Values
represent means 6 SD of three independent experiments, each with
approximately 50 pollen grains. Student’s t test was done to compare
the pollen viability of cngc16 mutants to wild-type plants incubated at
the same condition. *Student’s t test significant at P , 0.05. **Student’s
t test significant at P , 0.01.
Plant Physiol. Vol. 161, 2013 1015
CNGC16 Is Critical for Stress Tolerance in Pollen
stress conditions, the cngc16 pollen were twice as likely
to die compared with the wild type (Fig. 3). When the
stress environment was applied 2 h postpollination,
a similar 1.5- to 2.5-fold decrease in transmission efficiency
was observed. This bracketing suggests that
cngc16 mutant pollen are most sensitive to stress conditions
at the time of germination and/or growth into
the stigma surface.
In vitro pollen growth assays corroborate that
cngc16 pollen grains are highly sensitive to environmental
conditions. In comparison with wild-type pollen
germinated under a standard in vitro growth
condition (with 1 mM CaCl2), less than 1% of the pollen
grains produced tubes and more than 80% arrested as
short tubes (,30 mm) or ruptured. This phenotype
shows similarities to that observed for knockouts of
cngc18 (Frietsch et al., 2007) and double knockouts
of cngc7 and cngc8 (J.F. Harper, unpublished data).
However, a major difference between cngc18, cngc7,
cngc8, and cngc16 was that at least some of cngc16
pollen grains produced long tubes. In addition, cngc16
mutant pollen showed normal Mendelian transmission
under nonstress conditions, whereas cngc18, cngc7,
and cngc8 are male sterile.
The sensitivity of cngc16 pollen to in vitro growth
conditions was at least partially due to an increased
sensitivity to external CaCl2 (Fig. 5). Using a viability
assay based on Alexander staining, nearly 50% of
cngc16 pollen were found to be dead after a 3-h incubation
in a buffered solution of 10 mM CaCl2. This level
of lethality is 2- to 3-fold higher than a wild-type control.
An analogous CaCl2 hypersensitivity has been reported
for two other cngc mutant phenotypes (Chaiwongsar
et al., 2009; Urquhart et al., 2011). For example, the
growth reduction phenotype associated with a knockout
of cngc2 in Arabidopsis was further decreased by approximately
2-fold by supplementing soil with 10 mM
CaCl2.

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