RESEARCH ARTICLE 2221
Development 139, 2221-2233 (2012) doi:10.1242/dev.081224
© 2012. Published by The Company of Biologists Ltd
Inositol polyphosphate 5-phosphatase-controlled
Ins(1,4,5)P3/Ca2+ is crucial for maintaining pollen dormancy
and regulating early germination of pollen
Yuan Wang*, Yu-Jia Chu* and Hong-Wei Xue‡
SUMMARY
Appropriate pollen germination is crucial for plant reproduction. Previous studies have revealed the importance of dehydration in
maintaining pollen dormancy; here, we show that phosphatidylinositol pathway-controlled Ins(1,4,5)P3/Ca2+ levels are crucial for
maintaining pollen dormancy in Arabidopsis thaliana. An interesting phenotype, precocious pollen germination within anthers,
results from a disruption of inositol polyphosphate 5-phosphatase 12 (5PT12). The knockout mutant 5pt12 has normal early
pollen development and pollen dehydration, and exhibits hypersensitive ABA responses, indicating that precocious pollen
germination is not caused either by abnormal dehydration or by suppressed ABA signaling. Deficiency of 5PT13 (a close paralog
of 5PT12) synergistically enhances precocious pollen germination. Both basal Ins(1,4,5)P3 levels and endogenous Ca2+ levels are
elevated in pollen from 5pt12 mutants, and 5pt12 5pt13 double mutants show an even higher precocious germination rate along
with much higher levels of Ins(1,4,5)P3/Ca2+. Strikingly, exogenous Ca2+ stimulates the germination of wild-type pollen at floral
stage 12, even in very low humidity, both in vitro and in vivo, and treatment with BAPTA, a [Ca2+]cyt inhibitor, reduces the
precocious pollen germination rates of 5pt12, 5pt13 and 5pt12 5pt13 mutants. These results indicate that the increase in the
levels of Ins(1,4,5)P3/Ca2+ caused by deficiency of inositol polyphosphate 5-phosphatases is sufficient to break pollen dormancy
and to trigger early germination. The study reveals that independent of dehydration, the control of Ins(1,4,5)P3/Ca2+ levels by
Inositol polyphosphate 5-phosphatases is crucial for maintaining pollen dormancy.
INTRODUCTION
Pollen dormancy and germination are crucial for reproductive
growth of flowering plants. In Arabidopsis, mature pollen grains
are released from anthers in a partially dehydrated and dormant
state. Once they fall onto stigma, rehydration of desiccated pollen
grains breaks dormancy and turns on pollen germination
(McCormick, 2004). Inappropriate pollen germination will
inevitably affect plant fertility, and thus studies on pollen dormancy
and germination are of great significance to agricultural production.
Precocious pollen germination in anthers was first observed in the
Arabidopsis gametophytic mutant raring-to-go (rtg), in which
pollen grain development is affected as early as the bicellular stage
and affected pollen grains are unable to desiccate completely
(Johnson and McCormick, 2001). In addition, rtg pollen tubes were
dramatically elongated within anthers under high humidity
conditions. These findings indicate the importance of
dehydration/rehydration in pollen dormancy and germination; to
date, dehydration is considered to be the main factor in maintaining
pollen dormancy and preventing precocious germination.
Abscisic acid (ABA) promotes and maintains seed dormancy
(Finkelstein et al., 2008). Deficiencies in ABA synthesis or signaling
frequently result in reduced dormancy, seed precocious germination
or vivipary (Koornneef and Karssen, 1994; McCarty, 1995; Bewley,
National Key Laboratory of Plant Molecular Genetics, Shanghai Institute of Plant
Physiology and Ecology, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai
200032, China.
*These authors contribute equally to this work
‡
Author for correspondence ([email protected])
Accepted 26 March 2012
1997). Supportive evidence comes from the studies of maize vp
(viviparous) (Neill et al., 1986), tomato sitiens (Hilhorst and Downie,
1996), rice phs (pre-harvest sprouting) (Fang et al., 2008), and
Arabidopsis ABA-deficient (aba) and ABA-insensitive (abi) mutants
(Koornneef et al., 1989). Moreover, recent studies show that the
ABA content in the lily pollen is increased before anthesis, which
correlates with the pollen dehydration stage (Hsu et al., 2010). A
unique desiccation-associated ABA signaling transduction has been
reported, through which a Rop (Rho GTPase of plants) gene is
regulated during the stage of pollen dehydration (Hsu et al., 2010).
These results indicate the importance of ABA-mediated dehydration
for pollen dormancy.
Both hydration/humidity and Ca2+ are essential for pollen
germination. In Arabidopsis, pollen germination starts with the
rehydration of pollen grains on the stigma. During this process,
Ca2+ flows into the pollen grains and triggers cytoplasmic
reorganization. Subsequently, a cytoplasmic gradient of Ca2+ is
formed with the highest [Ca2+] at the germination site, which is
crucial for polar tip growth of prospective pollen tubes (HeslopHarrison and Heslop-Harrison, 1992a; Heslop-Harrison and
Heslop-Harrison, 1992b; Franklin-Tong, 1999). Dynamic analysis
of cytosolic [Ca2+] ([Ca2+]cyt) in Arabidopsis pollen grains reveals
an increase in [Ca2+]cyt at the potential germination site soon after
rehydration; this high Ca2+ concentration is maintained until the
onset of pollen germination (Iwano et al., 2004). In hydrated pollen
of Nicotiana tabacum L., a high level of calmodulin (CaM) is
consistently present in the region of the germination apertures and
is associated with the plasma membrane of the germination bubble
and with the onset of germination (Tirlapur et al., 1994). High
environmental humidity and exogenous Ca2+ are also requisites for
pollen germination in vitro (Taylor et al., 1997).
DEVELOPMENT
KEY WORDS: Inositol polyphosphate 5-phosphatase (5PT), Cytosolic Ca2+, Pollen dormancy, Inositol 1,4,5-trisphosphate [Ins(1,4,5)P3],
Arabidopsis thaliana
2222 RESEARCH ARTICLE
MATERIALS AND METHODS
Plant materials and growth conditions
The Arabidopsis Columbia ecotype was used. The 5pt13 mutant has been
described by Lin et al. (Lin et al., 2005). Seedlings were grown in a
phytotron at 22°C with a 16-hour light/8-hour dark photoperiod.
Identification of 5pt12 and 5pt14 knockout mutant
The 5pt12 mutant was obtained from the Arabidopsis Biological Resource
Center (SALK_065920, http://www.arabidopsis.org/). 5pt12 mutant seeds
were screened on MS medium containing 30 mg/l kanamycin for
segregation ratio analysis and a PCR-based approach was employed to
confirm the T-DNA insertion and to identify the homozygous mutant.
Genotyping primers 5PT12-F (5⬘-CACAGCCTCGACGACATTC-3⬘),
5PT12-R (5⬘-TTGCCAAGCTAGACCTTCC-3⬘) and LBa1 (5⬘TGGTTCACGTAGTGGG CCATCG-3⬘) were used.
The 5pt14 mutant was obtained from the Arabidopsis Biological
Resource Center (GK-309D03, http://www.arabidopsis.org/). Genotyping
primers 5PT14-F (5⬘-GGTGGTGTAAGAGGATGGT-3⬘), 5PT14-R (5⬘CGGCACTTCCTTCAACCC-3⬘)
and
GK-LB
(5⬘-ATATTGACCATCATACTCATTGC-3⬘) were used to confirm the T-DNA insertion
and to identify the homozygous mutant.
Quantitative real-time RT-PCR (qRT-PCR) analysis
qRT-PCR was performed to test the expression patterns of 5PT12, 5PT13
and 5PT14 in various tissues, the transcription levels of 5PT12 and 5PT14
in mutant or wild-type plants, and the expression of ABA-responsive genes
in seedlings or flower tissues.
For analyzing the expression pattern of 5PT12, total RNAs were
extracted from 4-day-old seedlings, roots, 4-week-old rosette leaves,
flowers and stems. For analyzing the expression pattern of 5PT12, 5PT13
and 5PT14 at different floral stages, total RNAs were extracted from
flowers at stages 12, 13 and 14. For testing the transcriptional levels of
5PT12 and 5PT14 in mutant or wild-type plants, total RNAs were extracted
from flowers of 4-week-old plants. For examining the expressions of ABAresponsive genes in flower tissues or seedlings, flowers of 4-week-old
plants at floral stages 12-14 or an earlier stage were collected and
submerged in ABA (100 M) solution for 0, 1 or 3 hours, and 8-day-old
wild-type and 5pt12 seedlings were submerged in ABA (100 M) solution
for 0, 0.5, 1 or 2 hours.
Total RNAs were extracted using Trizol reagent (Invitrogen) and then
reversely transcribed according to the manufacturer’s instructions
(ReverTra Ace kit, Toyobo). RNA samples were incubated for 30 minutes
at 37°C with RNase-free DNase I (Takara), and then precipitated for further
analysis.
RotorGene RG-3000A (Corbett Research) and a SYBR green detection
kit (SYBR Green Realtime PCR Master Mix kit; TOYOBO) were used,
with a program of denaturation (5 minutes, 95°C), 40 cycles of
denaturation (95°C, 10 seconds), annealing (55°C, 15 seconds) and
elongation (72°C, 20 seconds). Fluorescence is monitored during each
annealing and denaturation phase. The product amounts were determined
by the method of comparative Delta-Delta Ct (Fleige et al., 2006). Primers
used were as follows: 5PT12 (5⬘-AGAATCGGTAGGAGGAAACG-3⬘ and
5⬘-TTCTCAGATTCTTCCTCACC-3⬘),
5PT13
(5⬘-CCGAGGACAAAATCTAAGTAACG-3⬘ and 5⬘-CAGCGCCGGTGCTTGGAATAG3⬘),
5PT14
(5⬘-GGTGGTGTAAGAGGATGGT-3⬘
and
5⬘CGGCACTTCCTTCAACCC-3⬘), ABA-responsive genes Rd22 (5⬘TTACCAAACACTCCCATTCC-3⬘
and
5⬘-CGTACACCTCCCTTTCCAAC-3⬘), COR47 (5⬘-GAACAAGCCTAGTGTCATCG-3⬘ and 5⬘TCTTGTCGTGGTGTCCTGG-3⬘),
KIN2
(5⬘-ACCAACAAGAATGCCTTCCA-3⬘ and 5⬘-ACTGCCGCATCCGATATACT-3⬘) and
COR15
(5⬘-CTCAGTTCGTCGTCGTTTC-3⬘
and
5⬘-CATCTGCTAATGCCTCTT T-3⬘). Arabidopsis ACTIN gene (At5g09810) was
amplified with primers (5⬘-CCGGTATTGTGCTCGATTCTG-3⬘ and 5⬘TTCCCGTTCTGCGGTAGTGG-3⬘) and used as positive internal control.
Primers of 5PT13 have been previously described (Chen et al., 2008). All
the experiments were repeated three times.
Promoter-reporter gene fusion studies
The genomic DNA region in front of the translation initiation site ATG was
PCR
amplified
with
primers
5PT12p-1
(5⬘CCCAAGCTTTAAGGCAGTGTCTGTCGAGG-3⬘, added HindIII site
underlined)
and
5PT12p-2
(5⬘-CGGGATCCGGAAGAGAGAATGAGAGAAATG-3⬘, added BamHI site underlined). The amplified
1216 bp DNA fragment was confirmed by sequencing and then subcloned
into modified pCAMBIA1301 (Liu et al., 2003), resulting in the construct
containing 5PT12 promoter-GUS (-glucuronidase) fusion. The resultant
construct was transferred into Agrobacterium tumefaciens strain GV3101
and then transformed into Arabidopsis using the floral dip method. T1
transgenic seedlings were screened on MS medium supplemented with 30
mg/l hygromycin and the T-DNA integration was confirmed by PCR
analysis using primers annealing to the hygromycin resistance gene (hygs, 5⬘-GCTTCTGCGGGCGATTTGTGT-3⬘ and hyg-a, 5⬘-GGTCGCGGAGGCTATGGATGC-3⬘). Seedlings of T3 homozygous plants were
analyzed for GUS activities and observed using a SMZ 800 stereoscope
(Nikon).
The transgenic seedling harboring a 5PT13 promoter-GUS reporter gene
construct has been described previously (Lin et al., 2005).
Complementary expression of 5PT12 in 5pt12 mutant
A 7.4 kb fragment of genomic DNA containing the full-length 5PT12 gene
and the promoter region was cut from a BAC clone F6E13 (from ABRC,
http://www.arabidopsis.org) and subcloned into modified pCAMBIA1301
vector (Liu et al., 2003). The resultant construct was transferred into the
5pt12 mutant by Agrobacterium-mediated transformation through the floral
dipping method. Complemented expression of 5PT12 was confirmed
through qRT-PCR using primers (5⬘-AGTACTTCTCCCACCACTTG-3⬘
and 5⬘-TCTGAACATGACCACAAATCG-3⬘). T2 homozygous plants
were used for complementation studies.
DEVELOPMENT
Inositol polyphosphate 5-phosphatase (5PT), a key enzyme of
the phosphatidylinositol (PI) signaling pathway, functions in
growth, development and responses to stress in plant, yeast and
mammals (Stevenson et al., 2000; Astle et al., 2006; Xue et al.,
2007; Xue et al., 2009). 5PT removes the phosphate at the 5position of the inositol ring of specifically phosphorylated (5position) phosphoinositides [PtdIns(4,5)P2, PtdIns(3,4,5)P3 and/or
PtdIns(3,5)P2], and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] or
inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4].
There are 15 5PTs in Arabidopsis (Berdy et al., 2001; Ercetin
and Gillaspy, 2004; Zhong and Ye, 2004) that have diverse roles in
plant growth and development, and phytohormone signaling.
5PT13 modulates cotyledon vein development through the
regulation of auxin homeostasis (Lin et al., 2005). FRAGILE
FIBER 3 (FRA3) and COTYLEDON VASCULAR PATTERN 2
(CVP2) are two 5PTs containing the 5PTase domain. FRA3 is
required for secondary wall synthesis and actin organization in
fiber cells (Zhong et al., 2004), and CVP2-mediated Ins(1,4,5)P3
signal transduction is essential for closed venation patterns of
Arabidopsis foliar organs (Carland and Nelson, 2004). In addition,
5PTs participate in seedling growth (Gunesekera et al., 2007;
Ercetin et al., 2008) and blue-light signaling via the regulation of
Ins(1,4,5)P3/[Ca2+]cyt (Chen et al., 2008), and are important
regulators of Ins(1,4,5)P3-mediated ABA signaling (Sanchez et al.,
2001; Burnette et al., 2003; Lin et al., 2005; Gunesekera et al.,
2007). However, no 5PTs have been reported to function in the
regulation of reproductive development.
Here, through the functional characterization of Arabidopsis
5PT12 and 5PT13, we show that the increase of 5PT-controlled
Ins(1,4,5)P3/Ca2+ is sufficient for breaking pollen dormancy and
triggering early germination, and further confirm that,
independently of dehydration, 5PT-controlled Ins(1,4,5)P3/Ca2+ is
crucial for pollen dormancy.
Development 139 (12)
IP3/Ca2+ in pollen dormancy
To generate 5pt12 5pt13 double mutant by genetic cross, 5pt13
homozygotes (carrying Basta-resistant gene) were used as female parent,
and 5pt12 homozygotes (carrying kanamycin-resistant gene) were used as
male parent. The cross was carried out by removing the petals, sepals and
androecia from large green buds in 5pt13 plants, followed by artificial
fecundation with the 5pt12 pollen grains on the next day. F1 cross
progenies were selected on kanamycin-containing MS plates and positive
plants were used for harvesting F2 seeds. F2 generation plants were used
for PCR analysis to confirm the T-DNA insertions in 5PT12 gene loci and
5PT13 gene loci. Identified homozygotes were grown for one additional
generation and harvested seeds were used for further experiments.
Seed germination assay
Surface-sterilized seeds of wild-type or 5pt12 plants were sown onto MS
medium containing different concentrations of ABA (0, 0.1, 0.3 and 0.5
M). Following stratification in 4°C for 2 days, seeds were grown at 22°C
for 7 days and germination rates were calculated. One-hundred seeds were
used for each line, and all the assays were performed in triplicate. Assays
were repeated at least twice.
Scanning electron microscope analysis
Dehiscent anthers of wild-type and 5pt12 plants were harvested and fixed
by FAA, followed by dehydration with a graded ethanol series. Specimens
were dried at crucial points and coated with gold before examination with
a Scanning Electron Microscope (JSM-6360LV, Tokyo, Japan).
Observation and measurement of dehydrated pollen grains
Dehydrated pollen grains were stripped out from dehiscent anthers of wildtype and 5pt12 plants and covered with type N immersion oil to retain their
original shapes. Image J software was used to measure major and minor
axes of dehydrated pollen grains, and the ratio of minor axes/major axes
was calculated. More than 50 pollen grains of each genotype were
analyzed, and the assays were repeated twice.
Measurement of pollen viability and DAPI staining
Flower buds prior to opening were harvested, and dispersed pollen grains
were treated with FDA (fluorescein diacetate, diluted with acetone to a
concentration of 100 mg/l) for 30 minutes, and then observed in ultraviolet
light with a microscope (Leica).
Pollen harvested from open flowers was treated with DAPI solution
(final concentration of 2 mg/l dissolved in 7% sucrose, w/v) for 5 minutes
and imaged with a confocal laser scanning microscope (FITC488, Zeiss
LSM500).
Pollen germination in vitro and in vivo
Open flowers from wild-type and 5pt12 mutant plants were dehydrated in
a desiccator at room temperature for at least 2 hours. Pollen germination
was performed as described by Li et al. (Li et al., 1999) and 2 mM Ca2+
(with an equal molar ratio of CaCl2 and Ca[NO3]2) was added into basal
medium to obtain the optimal pollen germination rate. Soaked filter paper
was used to provide environment humidity. After incubation for 6 hours at
25°C, germinated pollen were observed and photographed.
To examine the effect of extracellular Ca2+ on pollen germination, pollen
was cultured in basal agar medium supplemented with different
concentrations of Ca2+ [with an equal molar ratio of CaCl2 and Ca(NO3)2].
For the humidity assay, dry filter paper was used to provide a coarse
gradient of environmental humidity conditions by adding different volumes
of distilled water onto filter papers. 0, 2.3, 4.6 or 7 ml of distilled water
was added to five pieces of dry filter paper to provide very low (~0-10%),
low (~25-35%), middle (~55-65%) or saturation (high humidity, ~85-95%).
Pollen grains (>200) collected from more than three individual plants were
used for each genotype, and each assay was performed in triplicate. Assays
were repeated at least twice.
Different concentrations of Ca2+ [with an equal molar ratio of CaCl2 and
Ca(NO3)2] were applied to anthers at floral stage 11 to examine the pollen
germination in anthers. BAPTA (2 mM, Sigma-Aldrich) was used to block
the cytosolic Ca2+ in anthers of 5pt12, 5pt13 and 5pt12 5pt13 mutants. At
late floral stage 12, Ca2+- or BAPTA-treated anthers were harvested for
pollen tube staining by DAB assay.
Pollen tube staining with Aniline Blue
Dehiscent anthers or dispersed pollen grains from wild-type and 5pt12,
5pt13, 5pt12 5pt13, 5pt14 and 5pt12 plants with complementary expression
of 5PT12 were treated with 0.1% decolorized Aniline Blue (DAB) for 5
minutes followed by observation in ultraviolet light with a microscope
(Leica).
Quantification of ABA content
Anthers at floral stage 12 or anthers at earlier stage (pollen were too small
to collect) were collected for ABA measurement using liquid
chromatography-mass spectrometry (LC-MS) (Welsch et al., 2008).
Measurement of Ins(1,4,5)P3 content
Stamens at floral stages 12-14 from wild-type, 5pt12, 5pt13, 5pt12 5pt13
and 5pt12 plants with complementary expression of 5PT12 were harvested
for measuring Ins(1,4,5)P3 content. Extraction of Ins(1,4,5)P3 was
performed according to a previously published method (Burnette et al.,
2003). Ins(1,4,5)P3 content was measured using the D-myo-inositol-1,4,5trisphosphate [3H] assay kit (GE healthcare) and determined from a
standard curve generated with known amounts of Ins(1,4,5)P3.
For inositol phosphates (IPs) control assay, flower tissues at floral stages
12-14 of wild-type, 5pt12, 5pt13, and 5pt12 5pt13 plants were collected
and used for the assay. A mixture of unlabelled PtdIns(4,5)P2, Ins(1,3,4)P3
and InsP6 were added into assays. As carried out previously (Perera et al.,
2008), PtdIns(4,5)P2, InsP6 and Ins(1,3,4)P3 were added, respectively, at
18⫻, 10⫻ and 1⫻ the concentration of Ins(1,4,5)P3 found in wild-type
stamen tissues. Assays were performed in triplicate and experiments were
repeated twice.
Measurement of calcium content using ICP-MS
Anthers at floral stages 11-13 from wild-type, 5pt12, 5pt13 and 5pt12
5pt13 plants were harvested, following addition of 1 ml 100% HNO3 and
kept overnight. The samples were kept at 99°C for 1 hour and diluted to
14 ml with ultra-pure water. Samples were analyzed using an ELAN DRCe ICP-MS system (PerkinElmer) and Multi-Element calibration Standard2A (Agilent) samples were used as standards. Assays were performed in
triplicate.
Indo-1 staining and examination of [Ca2+]cyt in microspores, pollen
grains and root hair cells
Microspores or pollen grains at different floral stages from wild-type,
5pt12, 5pt13, and 5pt12 5pt13 plants were stained with 20 M Indo-1
(57180, Sigma) on slides in the dark. Two hours after loading and washing
of the dye, fluorescence was observed with a confocal SMZ510 under UV
light excitation (364-nm excision, and 400- to 435-nm and 480 nm
emission) and imaged. [Ca2+]cyt was pseudocolored according to the scale.
Pseudocolor ratio images of the [Ca2+]cyt distribution was calculated with
Olympus Fluoview Ver. 1.7a Viewer tool (Olympus) to measure the
fluorescence intensity. White indicates approximately twice the [Ca2+]cyt of
that indicated by the blue and 1.5 times the [Ca2+]cyt indicated by the
yellow. For the root hair plasmolysis, 7-day-old seedlings of wild-type,
5pt12, 5pt13, and 5pt12 5pt13 plants were stained with Indo-1, followed
by treatment with 500 mM mannitol for 10 minutes.
A pollen protoplast staining assay was performed as described
previously (Wu et al., 2011). Pollen grains at different floral stages from
wild-type, 5pt12, 5pt13, and 5pt12 5pt13 plants were harvested for the
assay.
Accession numbers
Sequence data from this article can be found in the GenBank/EMBL data
libraries under the following accession numbers: 5PT12 (At2g43900),
5PT13 (At1g05630), 5PT14 (At2g31830), KIN2 (At5g15970), COR15
(At2g42540), RD22 (At5g25610) and COR47 (At1g20440).
DEVELOPMENT
Genetic cross and characterization of 5pt12 5pt13 double mutant
RESEARCH ARTICLE 2223
Development 139 (12)
RESULTS
The Arabidopsis 5PT12 gene is preferentially
expressed in pollen grains at floral stages 12-14
Our previous studies have shown that inositol polyphosphate 5phosphatase 13 (5PT13) plays crucial roles in auxin-regulated
cotyledon vein development (Lin et al., 2005), blue light-mediated
photomorphogenesis (Chen et al., 2008) and root gravitropism
(Wang et al., 2009). To further characterize the 5PT functions in
Arabidopsis, we focused on 5PT12, a close paralog of 5PT13
(because the mutant of the closest paralog, 5PT14, was not
available when we started). Based on domain prediction, 5PT12
has a conserved 5-phosphatase domain (5PTase domain), which is
required for 5-phosphatase activity towards inositol phosphates and
phosphoinositides, and three WD40 repeats (supplementary
material Fig. S1A). Phylogenetic analysis of 15 Arabidopsis 5PTs
and representative yeast and mammalian 5PTs showed that 5PT12
is most closely related to 5PT13, 5PT14 and FRA3 (supplementary
material Fig. S1B).
The gene expression pattern of 5PT12 in various tissues was
analyzed using quantitative real-time RT-PCR (qRT-PCR) and the
results showed that 5PT12 was mostly expressed in leaves and
flowers, whereas it was only weakly expressed in roots, stem and
young seedlings (Fig. 1A). To investigate the detailed expression
pattern, a construct containing the 1.2 kb 5PT12 promoter region
fused to the Escherichia coli -glucuronidase (GUS)-coding gene
was generated and transformed into Arabidopsis. Consistent results
from five independent transgenic lines showed that 5PT12 is
transcribed in cotyledon tips and the hydathode of leaves (Fig.
1Ba,b).
Interestingly, 5PT12 is preferentially expressed in pollen grains
at floral stages (Smyth et al., 1990; Sanders et al., 1999) 12 to 14
(Fig. 1Bc-f). At stage 13, the filaments have elongated to the height
of the stigma, mature pollen and pollen tubes (Fig. 1Bg,h),
suggesting a potential role for 5PT12 in pollen development and
germination. qRT-PCR analysis indicated the gradually increased
expression of 5PT12 as floral development proceeded (from floral
stage 12 to 14, Fig. 1C), which is consistent with the microarray
data (https://www.genevestigator.ethz.ch/) showing that the 5PT12
gene is largely expressed in anthers, with high transcript abundance
in tri-cellular pollen and mature pollen, and low abundance in unicellular microspore and bi-cellular pollen. Furthermore, 5PT12,
5PT13, 5PT14 and 5PT5 are also highly expressed in pollen grains,
based on the microarray data (supplementary material Table S1;
qRT-PCR analysis confirmed relatively less expression of 5PT13
in comparison with 5PT12, Fig. 1C). As the 5pt14 mutant was not
available and 5PT5 is not a close paralog to 5PT12, 5PT13 and
5PT14, we first focused on the function of 5PT12 in pollen
development.
plants by PCR amplification of NPTII (selectable marker located
in T-DNA for resistance to kanamycin) showed that 30 out of 41
progenies carried NPTII (supplementary material Fig. S2B). In
addition, scoring for kanamycin (Kan) resistance showed that 212
F2 seedlings were Kan resistant and 72 were Kan sensitive. A chisquare test for both assays confirmed the 3:1 segregation ratio.
These results validated that the two T-DNA insertions are closely
linked without segregation. Transcription analysis by qRT-PCR
showed that the 5PT12 gene was completely disrupted (Fig. 1F),
indicating that 5pt12 is a knockout mutant.
Owing to the predominant expression of 5PT12 in pollen
grains at floral stages 12-14, in mature pollen and in pollen
tubes, the function of 5PT12 in pollen development and
germination was specifically examined. Phenotypic observation
showed that, unlike wild-type dehiscent anthers, in which very
few pollen grains remained (floral stage 13), many more pollen
grains were left in the 5pt12 dehiscent anthers, and they had
tube-like structures (Fig. 1G). Pollen grains were washed off and
tube-like structures could be easily distinguished (Fig. 1H).
Further staining with decolored Aniline Blue (DAB), a widely
used dye for pollen tube observations (Escobar-Restrepo et al.,
2007), confirmed that the tube-like structures are indeed pollen
tubes (Fig. 1I).
The precocious pollen germination rates of 5PT12/5pt12
heterozygotes were 20-30% and those of 5pt12 homozygotes
reached 40-70% in comparison with the rate of 1-3% in wild-type
anthers (Fig. 1J), indicating that 5PT12 deficiency resulted in
precocious pollen germination within the anther. This is a very
interesting finding, because, in Arabidopsis, only two mutants
(raring-to-go and callose synthase 9) and one transgenic line
overexpressing callose synthase 5 have been reported so far to
show a similar phenotype with unknown mechanism (Johnson and
McCormick, 2001; Xie et al., 2010).
The link between T-DNA insertion in 5PT12 gene and the
precocious germination phenotype was further tested using
randomly selected 40 progenies of one single 5pt12 plant. PCR
analysis using the genotyping primers confirmed that all the 40
progenies were homozygotes (supplementary material Fig. S2C)
and all exhibited precocious pollen germination at a rate of 4070%. Complementation studies were performed by transferring
a 7.4 kb fragment of genomic DNA containing the entire 5PT12
gene and promoter region into the 5pt12 mutant (Fig. 1F). The
results showed that the Aniline Blue fluorescence was barely
detectable in complementation lines (Fig. 1I) and the precocious
pollen germination rates of complementation lines (T2
homozygous lines) were also similar to those in wild type (Fig.
1J), confirming that 5PT12 deficiency is responsible for
precocious pollen germination.
Deficiency of 5PT12 results in precocious pollen
germination within anthers
A putative T-DNA insertion mutant of 5PT12 was obtained from
SALK, and PCR analysis confirmed the T-DNA insertion at the
first exon of the 5PT12 (Fig. 1E). The homozygote was
characterized and designated as a 5pt12 mutant. Both forward and
reverse gene-specific genotyping primers could amplify the PCR
product when combined with T-DNA border primer LBa1 (Fig.
1E), indicating that two tandem T-DNA insertions are located backto-back in 5PT12. This was further confirmed by the corresponding
T-DNA flanking sequences. Back-cross between 5pt12 and wildtype plants was performed (supplementary material Fig. S2A) and
segregation analysis of F2 progenies of a single 5PT12/5pt12 F1
Normal pollen development and unaffected
fertility in 5pt12 mutant
To investigate whether there are developmental defects in 5pt12
that cause precocious pollen germination, pollen development of
5pt12 was examined. The observations showed the normal
development of 5pt12 pollen, and the DAPI staining assay revealed
that precociously germinated pollen grains within 5pt12 anthers
have regular tri-cellular structures, similar to wild-type pollen
grains (Fig. 2A, left panel). Further analysis of pollen viability with
FDA staining showed that 5pt12 pollen grains were
indistinguishable from wild type (Fig. 2A, right panel). Besides,
mature pollen grains of 5pt12 that did not germinate precociously
within the anther could germinate well in vitro (Fig. 2B), and the
DEVELOPMENT
2224 RESEARCH ARTICLE
RESEARCH ARTICLE 2225
Fig. 1. 5PT12 is preferentially expressed in leaf and mature pollen grains, and knockout expression of 5PT12 results in precocious
germinated pollen grains within anthers. (A)Quantitative real-time RT-PCR (qRT-PCR) assay shows that 5PT12 is expressed in different tissues,
preferentially in the leaves and flowers. (B)Temporal and spatial expression analysis of 5PT12 by promoter-GUS fusion studies reveals that 5PT12 is
transcribed in seedlings (a), leaves (b), pollen in anthers at different floral stages (c-f, images d-f represent floral stages 12-14), mature pollen (g),
and germinating pollen and pollen tubes (h). Scale bars: 1 mm in a-c; 200m in d-f; 50m in g; 20m in h. (C)qRT-PCR assay shows that 5PT12
and 5PT13 are expressed in flowers at different floral stages. (D)Promoter-GUS fusion studies reveal the expression of Arabidopsis 5PT13 in flowers
(a), pollen at different floral stages (b-e, representing floral stages 12, 13, 14, and later than 14, respectively), germinating pollen (f) and pollen
tubes (g). Scale bars: 1 mm in a; 200m in b-e; 20m in g,h. (E)Schematic structure of 5PT12 (At2g43900, boxes represent exons). The position of
the T-DNA insertion and primers used for genotyping are indicated (top panel). Analysis of heterozygous (5PT12/5pt12), homozygous (5pt12) and
wild-type plants is shown in the bottom panel. (F)qRT-PCR assay revealed that expression of 5PT12 is deficient in the 5pt12 mutant, which is
recovered by complementary expression of 5PT12 driven by its native promoter. RNA extracted from flowers was used for analysis, and wild-type
expression of 5PT12 was set as 1.0. (G)Scanning electron microscope (SEM) analyses of flowers at floral stage 13 of wild-type and 5pt12 plants
show that pollen grains in 5pt12 anthers have tube-shaped structures (arrows, a representative image is shown in the right panel). (H)Tube-shaped
structures of 5pt12 pollen grains at floral stage 12. Pollen grains were washed off the anthers of flowers at floral stage 12 of both wild-type and
5pt12 plants. Scale bar: 20m. (I)The tube-shaped structures in 5p12 anthers are precociously germinated pollen tubes. Anthers of flowers at floral
stage 13 are stained with decolored Aniline Blue solution. Complementary expression of 5PT12 rescues normal pollen germination. Fluorescence is
detected under UV light. Scale bars: 50m. (J)Measurement of precocious germination incidence confirms the precocious pollen germination of
homozygous 5pt12 and heterozygous 5PT12/5pt12 mutants, whereas complementary expression of 5PT12 rescues normal pollen germination.
Experiments are repeated three times, and more than 150 pollen grains are observed and measured. Data are presented as average±s.d.
DEVELOPMENT
IP3/Ca2+ in pollen dormancy
2226 RESEARCH ARTICLE
Development 139 (12)
seed set rate of 5pt12 was not affected, despite the precocious
pollen germination (at the rate of 40-70%, Fig. 2C). All these
results confirm the normal development of 5pt12 pollen grains.
Unaltered dehydration of 5PT12-deficient pollen
grains
Previous studies have demonstrated the significance of
dehydration, which is probably mediated by ABA signaling, in
pollen dormancy and germination (Hsu et al., 2010). Our
observations showed that precociously germinated pollen grains
in 5pt12 anthers keep their dehydrated shape (Fig. 3A). The axis
lengths of pollen grains from dehiscent anthers were further
measured and there was no statistical difference in the ratio of
minor axis:major axis between wild-type and 5pt12 pollen,
suggesting that the mature pollen grains of 5pt12, including the
precocious germinated ones, undergo normal dehydration
process.
Deficiencies in ABA synthesis or signaling lead to seed
precocious germination or vivipary, and pollen dehydration is
probably mediated by ABA signaling. As disruption or
overexpression of several characterized Arabidopsis 5PTs has
been reported to alter ABA responses, we therefore tested
whether 5pt12 has altered ABA content or ABA signaling. Wild-
Fig. 3. Normal dehydration of precocious germinated pollen grains
and hypersensitive ABA responses of 5p12 mutant. (A)Precociously
germinated pollen grains from dehiscent anthers of 5pt12 mutant have
similar shapes to those of wild-type plants (left panel). Statistical analysis
of the ratio of minor axis to major axis of pollen grains from dehiscent
anthers further confirms the similar dehydration of 5pt12 mutants and
wild-type pollen grains (right panel). More than 50 pollen grains of wild
type or 5pt12 mutants are analyzed, and the assays are repeated three
times. Arrowheads indicate the pollen tubes. Scale bars: 20m. (B)LCMS analysis of anthers of wild type and 5pt12 mutant revealed no
changes of the ABA level in 5pt12 mutants at floral stage 12 or earlier
stages. (C)qRT-PCR analysis of the ABA signaling-related genes KIN2,
COR15, RD22 and COR47 indicate the enhanced ABA responses of
5pt12 seedlings. Eight-day-old seedlings are used for ABA treatments (0,
0.5, 1 or 2 hours). (D)Seed germination of the 5pt12 mutant was more
sensitive to ABA treatment, further confirming the enhanced ABA
response under 5PT12 deficiency. Seed germination percentage was
calculated after 7 days ABA treatment (0, 0.1, 0.3 or 0.5M) of wild-type
and 5pt12 seeds. Data are presented as average±s.d. (E)Pollen
germination percentages under different relative humidity conditions.
Wild-type pollen grains at floral stage 12 are used for in vitro germination
assay on germination media without additional Ca2+. Data are presented
as average±s.d.
DEVELOPMENT
Fig. 2. The 5pt12 mutant exhibits normal early pollen
development and fertility. (A)Mature pollen grains of the 5pt12
mutant have regular tri-cellular structures similar to those of wild-type
plants, as revealed by DAPI staining (left panel). In addition, viability
analysis by FDA indicates normal viability of 5pt12 pollen grains.
Mature pollen grains at floral stage 14 are used for analyses. Scale bars:
10m (left); 100m (right). (B)Pollen grains that do not germinate in
anthers germinate and grow normally in vitro. Scale bars: 20m.
(C)Fertility is not altered under 5PT12 deficiency. Well-developed seeds
in mature siliques of both wild-type and 5pt12 plants are calculated,
and the data are presented as the average±s.d. (more than 20
independent plants of each line are analyzed).
type or 5pt12 anthers at different floral stages were collected for
ABA measurements. There was no significant difference in ABA
content between wild type and 5pt12 either at floral stage 12 or
at earlier stages (Fig. 3B). Further analysis of the ABA-induced
expression of ABA-responsive genes (KIN2, COR15, RD22 and
COR47) in seedlings showed a much enhanced induction of
these genes under 5PT12 deficiency (Fig. 3C), indicating
stimulated ABA signaling in 5pt12 mutant. Consistently, the
5pt12 mutant was more sensitive to exogenous ABA in seed
germination assays (Fig. 3D). The expression patterns of ABAresponsive genes in floral tissues were also tested. However,
ABA treatment could not induce uniform and consistent
expression of all the tested ABA-responsive genes, although
some expression was induced at specific floral stages
(supplementary material Fig. S3). The reason might be that ABA
cannot effectively penetrate into the pollen grains owing to thick
anther walls (no similar ABA treatment has been reported
previously). Taken together, these results indicate unaffected
ABA synthesis and stimulated ABA signaling in the 5pt12
mutant; thus, precocious pollen germination seen in 5pt12
mutants is not due to altered ABA responses.
We also examined the germination rate of pollen (at early floral
stage 12) in response to different humidity conditions. The
germination rate of wild-type stage 12 pollen grains increased by
2.8-fold (from 18% to 52%) as the environmental humidity rose,
whereas that of 5pt12 early stage 12 pollen grains (at this time the
pollen grains are not germinated) showed a moderate increase of
1.6-fold (from 46% to 76%, Fig. 3E). Compared with wild-type
pollen grains, 5pt12 pollen grains showed a higher germination rate
under low humidity conditions and a significantly higher
germination percentage at high environmental humidity (76%
versus 45% of wild type). This ruled out the possibility that the
altered environmental humidity caused the precocious pollen
germination in 5pt12 mutant.
Taken together, these results strongly suggested that the
precocious pollen germination in the 5pt12 mutant is not caused by
alterations in dehydration/rehydration or by the ABA response, and
indicated the existence of another key regulator in pollen dormancy
and germination, which is possibly triggered in the 5pt12 mutant.
Ca2+ is the potential candidate, based on the fact that Ca2+ is crucial
for pollen development and germination, and that Ins(1,4,5)P3
stimulates the cytosolic Ca2+.
5PT13 deficiency synergistically enhances
precocious pollen germination, and Ins(1,4,5)P3
basal level/[Ca2+]cyt is increased in 5pt12 and 5pt12
5pt13 mutants
Based on microarray data (https://www.genevestigator.ethz.ch/),
5PT13, a close paralog of 5PT12, is weakly expressed in pollen
grains. qPT-PCR analysis confirmed the expression of 5PT13 at
floral stages 12 to 14 (Fig. 1C), with a similar pattern to that of
5PT12, and a detailed expression analysis by promoter-reporter
gene fusion studies shows that the 5PT13 gene is largely
expressed in pollen grains at floral stages 13 and 14, as well as
in mature pollen and pollen tubes (Fig. 1D). Although the
expression level of 5PT13 is lower than 5PT12 at floral stage 12
to 14, especially at floral stage 12, the 5PT13 knockout mutant
(5pt13) also exhibits precocious pollen germination in dehiscent
anthers (Fig. 4A,B) at a low incidence. Double mutant 5pt12
5pt13 has a much higher incidence of precocious pollen
germination (~60-70%) than 5pt12 or 5pt13 single mutants (Fig.
4C). In addition, abnormal pollen tubes were occasionally
RESEARCH ARTICLE 2227
observed in precociously germinated pollen grains, including
more than one pollen tube formed in one pollen grain or aberrant
pollen tube structures (Fig. 4D).
Previous studies have shown that in vitro, both 5PT12 and
5PT13 exhibit phosphatase activity towards only Ins(1,4,5)P3,
rather than towards inositol lipids (Zhong and Ye, 2004). Thus,
Ins(1,4,5)P3 is suggested to be involved in the regulation of pollen
dormancy and germination. Ins(1,4,5)P3 receptor binding protein
was employed specifically to determine the intrinsic Ins(1,4,5)P3
content by using the D-myo-inositol-1,4,5-trisphosphate [3H] assay
kit. The basal level of Ins(1,4,5)P3 was significantly increased in
5pt12 anthers and was even more increased in 5pt12 5pt13 double
mutants in comparison with wild type, while the complementary
expression of 5PT12 restored Ins(1,4,5)P3 levels (Fig. 5A). Basal
Ins(1,4,5)P3 levels corresponded to the precocious germination
incidences in each genotype.
It has been reported that the basal level of InsP2 or InsP6 is
higher than Ins(1,4,5)P3 in Arabidopsis (Perera et al., 2008). To
exclude the potential influence caused by non-specific binding,
unlabelled PtdIns(4,5)P2, InsP6 and Ins(1,3,4)P3 [Ins(1,4,5)P3
stereoisomer] were added into the samples and the results showed
that the addition of these inositol phosphates and phospholipids did
not affect the measurement (Fig. 5B), consistent with the specificity
described by the manufacturer (the cross-reactivity for other
inositol
phosphates
is
lower
than
1%,
http://www.gehealthcare.com/lifesciences). These results, thus,
indicate a close correlation between Ins(1,4,5)P3 content and
precocious pollen germination, and indicate that Ins(1,4,5)P3 is
involved in controlling pollen grain dormancy in anthers.
As Ca2+ is crucial for pollen germination on stigma or in vitro,
total Ca2+ contents in anthers of wild type, 5pt12, 5pt13 and 5pt12
5pt13 double mutant were measured using ICP-MS (inductively
coupled plasma mass spectrometric analysis) (Chen et al., 2003).
The results showed that the Ca2+content in 5pt12 mutant anthers
(stage 13) was higher than that in wild-type anthers, and the 5pt12
5pt13 double mutant had an even higher Ca2+ content in stage 12
and 13 anthers (Fig. 5C). No significant difference in Ca2+ content
was detected between the mutants and wild-type stage 11 anthers,
where 5PT12 and 5PT13 have very low expression.
Although the Ins(1,4,5)P3 receptor has not been identified in
higher plants, it is assumed that Ins(1,4,5)P3 can stimulate the Ca2+
release from the internal Ca2+ store. The concentration and
distribution of [Ca2+]cyt in 5pt12, 5pt13 or 5pt12 5pt13 pollen were
further detected by Indo-1 (an indicator of [Ca2+]cyt) staining.
[Ca2+]cyt was gradually increased along with the pollen
development, and reached its highest level in mature tricellular
pollen grains at floral stage 12 (Fig. 5D). At floral stage 9, no
differences in Indo-1-indicated [Ca2+]cyt were detected between
wild type and 5pt mutants, whereas, at stage 12, significantly
higher [Ca2+]cyt was detected in 5pt12 pollen grains, consistent with
the expression pattern of 5PT12. Furthermore, the distribution of
[Ca2+]cyt showed a diffuse pattern rather than accumulation in
germination furrows (high concentration of [Ca2+]cyt is
accumulated in germination furrows of wild-type pollen grains for
subsequent germination of pollen tubes, Fig. 5D). The alterations
of [Ca2+]cyt were much more significant in the 5pt12 5pt13 double
mutant (Fig. 5D).
To rule out the possible effect of pollen coat on Indo-1 staining,
pollen protoplast was used for detecting Indo-1-indicated [Ca2+]cyt
and similar results were obtained (Fig. 5E). Observation of
plasmolyzed root hair cells stained with Indo-1 further indicated
that Indo-1 fluorescence came from cytoplasm rather than the cell
DEVELOPMENT
IP3/Ca2+ in pollen dormancy
2228 RESEARCH ARTICLE
Development 139 (12)
wall (supplementary material Fig. S4), confirming that Ca2+
associated with the pollen coat or cell wall is not labeled by
Indo-1.
These results indicate that the Ins(1,4,5)P3-mediated [Ca2+]cyt
increase is responsible for the precocious pollen germination in
5pt12 and 5pt12 5pt13 mutants, and suggest that 5PTs and
Ins(1,4,5)P3/Ca2+ are crucial for maintaining pollen dormancy.
[Ca2+]cyt breaks pollen dormancy and stimulates
precocious pollen germination
Although Ca2+ is known to be required for pollen germination and
pollen tube growth, the effect of Ca2+ on pollen dormancy has not
been reported before. We therefore tested the influence of
exogenous Ca2+ on pollen grains at floral late stage 11. [Ca2+]cyt in
5pt12, 5pt13 and 5pt12 5pt13 pollen at floral late stage 11 was only
slightly higher than that in wild-type pollen (Fig. 5D), and all these
mutants did not show the precocious pollen germination within
anthers at this stage. However, in vitro pollen germination assays
showed that under 100% humidity, wild-type pollen at floral late
stage 11 could germinate in a low percentage (~26%, Fig. 6A),
indicating the low germination potential of pollen at late floral
stage 11. As exogenous Ca2+ concentration rose, the precocious
germination rate of wild-type pollen increased, especially at
concentrations of 1 or 1.5 mM (Fig. 6A), indicating that exogenous
Ca2+ can break the dormancy and promote precocious germination
of wild-type pollen at late floral stage 11 in a dose-dependent
manner. As expected, 5pt12 and 5pt12 5pt13 mutants showed
higher precocious pollen germination rates than wild type when
supplemented with 1 or 1.5 mM Ca2+ (Fig. 6A), consistent with the
slightly increased [Ca2+]cyt in pollen of these mutants at floral late
stage 11. There was no difference in precocious germination rate
under high concentrations of exogenous Ca2+ (2 mM,
supplementary material Fig. S5).
At floral stage 12, pollen grains are in enclosed anthers. As
pollen dehydration is supposed to occur at late stage 12, the inside
of the anther should have very low environmental humidity.
Therefore, we further tested the effect of Ca2+ on pollen
germination in very low relative humidity (0-10%). In the absence
of additional Ca2+, wild-type pollen grains (floral stage 12)
germinated at a very low rate, whereas supplementation with
external Ca2+ significantly stimulated the germination (4 mM Ca2+
produces a 44% germination rate, Fig. 6B). This indicated that
DEVELOPMENT
Fig. 4. Both 5pt13 and 5pt12 5pt13
had precocious germination of
pollen grains within anthers.
(A)Scanning electron microscopy (SEM)
analyses of flowers at floral stage 13 of
wild-type, 5pt12, 5pt13 and 5pt12
5pt13 mutant plants showed that pollen
grains in 5pt13 and 5pt12 5pt13
mutant anthers have tube-shaped
structures (outlined region is enlarged in
the bottom panel). Scale bars: Top row,
left to right, 20, 50, 20, 20 m; bottom
row, 10 m. (B)The tube-shaped
structures in 5pt12, 5pt13 and 5pt12
5pt13 anthers are precociously
germinated pollen tubes (outlined
regions are enlarged in the bottom
panel). Anthers of flowers at floral stage
13 are stained with decolored Aniline
Blue solution and fluorescence is
detected under UV light. Scale bars:
100m (top panel); 30m (bottom
panel). (C)Analysis of precocious
germination incidence confirms the
precocious pollen germination in 5pt13
mutants and the 5pt12 5pt13 double
mutant has a much higher incidence of
precocious germination in comparison
with 5pt12 or 5pt13 single mutant.
More than 300 pollen grains are
observed and calculated. Data are
presented as average±s.d. (D)The
precociously germinated pollen grains in
5pt12 5pt13 mutant anthers
occasionally have distorted pollen tubes
or more than one pollen tube on a
single pollen grain (right panel).
Arrowhead indicates the abnormal
pollen tube. Scale bars: 20m.
IP3/Ca2+ in pollen dormancy
RESEARCH ARTICLE 2229
[Ca2+]cyt, independently of hydration/humidity, can break pollen
dormancy and stimulate precocious pollen germination, which
mimicked the precocious pollen germination of 5PTs-deficient
mutants to some extent.
To further demonstrate that Ca2+ is crucial for precocious pollen
germination in vivo, exogenous Ca2+ was applied to wild-type
anthers and was found to stimulate precocious pollen germination,
which could reach to 35% with application of 6 mM Ca2+ (Fig.
6C). Moreover, the precocious germination rates in anthers of
5pt12, 5pt13 and 5pt12 5pt13 mutant were decreased when
applying the [Ca2+]cyt inhibitor BAPTA (Fig. 6D), confirming the
role of [Ca2+]cyt in pollen dormancy and germination. These results
indicate that the increase of Ins(1,4,5)P3/Ca2+ in 5PTs-deficient
pollen grains is responsible for precocious pollen germination and
confirm that 5PT-controlled Ins(1,4,5)P3/Ca2+ is crucial for
maintaining pollen dormancy.
DEVELOPMENT
Fig. 5. Increased basal Ins(1,4,5)P3 levels, and increased concentration and altered distribution of Ca2+ in 5pt12, 5pt13 and 5pt12 5pt13
mutants. (A)Basal Ins(1,4,5)P3 levels in anthers of wild-type, 5pt12, 5pt13, 5pt12 5pt13 and 5pt12 mutant lines with complementary expression of
5PT12. (B)Basal Ins(1,4,5)P3 levels in flower tissues of wild-type, 5pt12, 5pt13 and 5pt12 5pt13 mutant plants. ‘–IPs’ and ‘+IPs’ indicate the assays
without or with a mixture of unlabelled PtdIns(4,5)P2, InsP6 and Ins(1,3,4)P3 [18-, 10- and 1-fold of Ins(1,4,5)P3]. (C)ICP-MS analysis of Ca2+
concentration in anthers at floral stages 11, 12 and 13 of wild-type, 5pt12, 5pt13 and 5pt12 5pt13 mutants. For A-C, assays were performed in
triplicate and data are presented as average±s.d. Statistical analysis by a one-tailed Student’s t-test indicates significant differences (*P<0.05;
**P<0.01). (D)Distribution of [Ca2+]cyt in pollen grains at floral stages 9, 11 and 12 of wild-type, 5pt12, 5pt13 and 5pt12 5pt13 mutant plants, as
detected with Indo-1 staining. Scale bars: 20m. (E)Distribution of [Ca2+]cyt in pollen protoplast at floral stages 9, 11 and 12 of wild-type, 5pt12,
5pt13 and 5pt12 5pt13 mutant plants, detected by Indo-1 staining. Pollen grains at floral stage 12 treated with mock solution (DMGA) were used as
controls. Scale bars: 10m. All images in D and E show pseudocolor representation of Indo-1 fluorescence.
Development 139 (12)
Fig. 6. [Ca2+]cyt breaks pollen dormancy and stimulates precocious pollen germination. (A)Under the 100% humidity condition, exogenous
Ca2+ (0.5-1.5 mM) promotes in vitro precocious germination of pollen at floral late stage 11, especially those of 5pt12 and 5pt12 5pt13 mutants.
Data are presented as average±s.d. (n150-300). (B)Under very low relative humidity, exogenous Ca2+ significantly promotes in vitro precocious
germination of pollen at floral stage 12. Data are presented as average±s.d. (n>200). Representative images highlight germinated pollen grains
when supplemented with exogenous Ca2+ (right panel). Scale bars: 50m. (C)Observation (left panels) and statistical analysis (right panel) showed
that exogenous Ca2+ significantly promotes in vivo precocious germination of pollen at floral stage 12. More than 300 pollen grains are observed
and calculated. Data are presented as average±s.d. Scale bars: 100m. (D)Observation (left panels) and statistical analysis (right panel) confirm that
BAPTA treatment blocks the in vivo precocious germination of pollen at floral stage 12 of 5pt12, 5pt13 and 5pt12 5pt13 mutant plants (left panel).
More than 300 pollen grains are observed and calculated. Data are presented as average±s.d. Scale bar: 100m. (E)Model of the role for 5PTcontrolled Ins(1,4,5)P3/[Ca2+]cyt in maintaining pollen dormancy. In wild-type plants, 5PT12, 5PT13 and 5PT14 are synergistically highly expressed in
tri-cellular pollen at floral stage 12, to restrain the level of Ins(1,4,5)P3/[Ca2+]cyt in tri-cellular pollen and maintain the pollen in dormant state.
[Ca2+]cyt is mainly controlled by intrinsic Ins(1,4,5)P3 levels to prevent pollen grains from germinating. This prevents the precocious pollen
germination within anthers. The dehydration process at late stage 12 further maintains this state. Once the dehydrated pollen grains fall on pistils
and rehydrate, Ca2+ levels will increase and [Ca2+]cyt aggregates in the germination hole, resulting in the broken dormancy and pollen germination.
Deficiency of 5PT12 or 5PT13 results in increased intrinsic Ins(1,4,5)P3/[Ca2+]cyt in tri-cellular pollen at floral stage 12 or earlier stages, preventing the
maintenance of dormancy and resulting in the precocious germination of pollen grains within anthers.
DEVELOPMENT
2230 RESEARCH ARTICLE
DISCUSSION
5PT-controlled Ins(1,4,5)P3/Ca2+ is crucial for
maintaining pollen dormancy
After maturation, pollen grains undergo dehydration and are then
released from anthers in a partially dehydrated and dormant state.
Pollen germination on the stigma starts with the rehydration that is
subject to stringently spatiotemporal control (Heslop-Harrison,
1979). Inappropriate dehydration/rehydration results in premature
or earlier germination within the anther, or germination on the
wrong surface (Lolle and Cheung, 1993; Lolle et al., 1998).
Therefore, pollen dehydration is considered to be the major
determinant that controls pollen dormancy. Here, our study first
reveal that, in addition to dehydration, 5PTs and Ins(1,4,5)P3/Ca2+
play crucial roles in pollen dormancy and uncover a novel
mechanism for controlling it.
Deficiency of 5PT12, 5PT13 leads to precocious pollen
germination within anthers, suggesting the essential roles of these
5PTs in pollen dormancy. However, the precociously germinated
pollen grains in 5pt12 anthers could dehydrate normally and
pollens of the 5pt12 mutant is dehydrated before release from the
anthers (Fig. 3A), suggesting that humidity or altered sensitivity to
environmental humidity are not responsible for precocious
germination, As the 5pt12 mutant exhibits hypersensitive ABA
responses, further excluding the influence of dehydration/hydration
or ABA signaling in altered pollen dormancy and germination of
the 5pt12 mutant.
As speculated, the basal levels of Ins(1,4,5)P3, the common
substrate of 5PT12 and 5PT13, are elevated significantly in the
5pt12 or 5pt12 5pt13 mutant, and an increased [Ca2+]cyt
concentration is detected in pollen grains of the 5pt12 mutant or
5pt12 5pt13 double mutant (more significantly) at floral stage 12.
Previous studies have shown that a 1.6-fold increase in Ins(1,4,5)P3
did affect the seedling growth (Ercetin et al., 2008) and here an
approximate doubling of Ins(1,4,5)P3 in the 5pt12 5pt13 double
mutant is sufficient to lead to precocious pollen germination. In
addition, preventing the increase in basal Ins(1,4,5)P3 levels in the
5pt12 mutant by complementary expression of 5PT12 resulted in
the normal pollen germination rate, further confirming the role of
increased Ins(1,4,5)P3 levels in precocious pollen germination. No
significantly increase in Ins(1,4,5)P3 or total Ca2+ content was
detected in 5pt13 mutant anthers, which may be because whole
anthers were used as the material for Ins(1,4,5)P3 and total Ca2+
measurement (because pollen grains are too small to obtain enough
material for measurement), and the minor alterations in 5pt13
mutant pollen grains might be covered up. However, the increased
Ca2+ in the 5pt13 mutant pollen grains can be clearly observed by
the Indo-1 staining assay, which is consistent with precocious
pollen germination in the 5pt13 mutant. These findings indicate
that precocious pollen germination, a higher basal level of
Ins(1,4,5)P3 and increased [Ca2+]cyt in pollen grains are closely
correlated.
A more diffuse [Ca2+]cyt distribution pattern was observed in the
pollen of the 5pt12 5pt13 double mutant (distinguished from the
aggregated pattern in wild-type pollen grains prior to germination
on stigma) (Fig. 5D,E), which may explain the emergence of more
than one pollen tube. The concentration and distribution of [Ca2+]cyt
is supposed to be stringently controlled during pollen dormancy
and 5PTs/Ins(1,4,5)P3 may be the regulators.
In vitro pollen germination assays showed that exogenous Ca2+
could break dormancy and stimulate the early germination of wildtype pollen grains at floral late stage 11, which confirms the crucial
roles of Ca2+ in pollen dormancy. In addition, exogenous Ca2+ can
RESEARCH ARTICLE 2231
break dormancy and trigger early germination of wild-type pollen
grains at floral stage 12 under very low humidity (Fig. 6B).
BAPTA treatment resulted in the decreased pollen germination
rates in anther of 5pt12, 5pt13 and 5pt12 5pt13 mutants,
confirming that the precocious pollen germination in 5PT mutants
is caused by increased [Ca2+]cyt (Fig. 6D). In addition, 5PT14, a
close paralog of 5PT12 and 5PT13, is also expressed at floral stage
12 to 14, and the expression level of 5PT14 is higher than 5PT12
and 5PT13 at these stages (Fig. 1C, supplementary material Fig.
S6A). Recently, we identified a T-DNA insertion mutant of 5PT14,
which also exhibited precocious pollen germination in dehiscent
anthers (~15-30%) (supplementary material Fig. S6B,C,D).
Although all 5PT12, 5PT13 and 5PT14 are expressed in pollen
grain, each single mutant exhibits the phenotype of precocious
pollen germination and the 5pt12 5pt13 double mutant with much
higher percentage of the of precocious pollen germination,
suggesting the dose effect of 5PTs in regulating the pollen
germination. A further study with mutation of all 5PT12, 5PT13
and 5PT14 is on the way.
Take together, these findings indicate that 5PT-controlled
Ins(1,4,5)P3/Ca2+ has a crucial role in maintaining pollen dormancy
and the increase of 5PT-controlled Ins(1,4,5)P3/Ca2+ is sufficient to
break pollen dormancy and trigger early germination, independent
of hydration/humidity. This study sheds light on the mechanism
that controls pollen dormancy and germination (Fig. 6E). In wildtype plants, 5PT12, 5PT13 and 5PT14 are highly expressed in tricellular pollen at floral stage 12 in order to restrain the level of
Ins(1,4,5)P3/[Ca2+]cyt in tri-cellular pollen. The pollen at stage 12
is then maintained in a dormant state, and prevented from
precocious germinating. At late stage 12, tri-cellular pollen
undergoes dehydration to further maintain the dormant state. Once
the dehydrated pollen grains fall on pistils and rehydrate, Ca2+ will
flow into pollen grains and aggregates in the germination pore,
breaking dormancy and stimulating pollen early germination.
Deficiency of 5PT genes will result in the increased intrinsic
Ins(1,4,5)P3/[Ca2+]cyt in tri-cellular pollen at floral stage 12, which
sufficiently breaks the pollen dormancy and results in the
precocious germination of pollen grains in anthers.
Ins(1,4,5)P3 can be converted into InsP6 (inositol
hexakisphosphate) and InsP6 was reported to mobilize intracellular
Ca2+ stores (Lemtiri-Chlieh et al., 2003) in guard cells. Although
there is no evidence that InsP6 is involved in breaking pollen
dormancy, it is possible that the InsP6 content in 5PT mutants is
also increased and, to some extent, contributes to the alteration of
[Ca2+]cyt in 5PT mutants.
Biological significance and engineering prospect
of precocious pollen germination
Here, we report a striking phenotype in Arabidopsis: precocious
pollen germination within anthers at an incidence of 40-70%,
caused by 5PT12 deficiency. Pollen grains in wild-type
Arabidopsis do not germinate until falling onto stigma. In fact,
pollen grains in almost all the flowering plants will not germinate
prior to pollination. The only exceptions are some cleistogamous
species, in which pollen germinates inside the anther, and the
pollen tubes extend via the anther stomium to reach to the stigma
for fertilization (Lord, 1981; Trent, 1942), e.g. Cardamine
chenopodifolia (Lord, 1981). It is noteworthy that C.
chenopodifolia is a Cruciferae plant, similar to Arabidopsis. In
species in which the anther sac fails to open, germinated pollen
tubes go through the anther walls to the stigma (Hanson, 1943;
Lord, 1981). However, little is known about the genetic
DEVELOPMENT
IP3/Ca2+ in pollen dormancy
mechanism. Although a similar phenotype has been reported in the
raring-to-go (rtg) mutant, the pollen grains of which do not
desiccate completely and thus germinate precociously (Johnson and
McCormick, 2001), the responsible gene and genetic mechanism
are still unknown. In our study, 5pt12 and 5pt12 5pt13 mutants
showed precocious pollen germination within anthers, similar to
these cleistogamous plants, providing convincing evidence of the
crucial roles of 5PTs and Ins(1,4,5)P3/[Ca2+]cyt in pollen dormancy,
and revealing a novel mechanism for keeping pollen dormancy.
These results provide insights into the mechanisms of pollen
germination in such cleistogamous species. It is speculated that
Ins(1,4,5)P3/[Ca2+]cyt might also function in breaking dormancy
and stimulating pollen germination in these cleistogamous plants,
and 5PTs or other enzymes controlling Ins(1,4,5)P3 metabolism in
the pollen of these species are expressed at low levels or are
deficient, which promotes pollen germination in anthers.
Increased Ins(1,4,5)P3/Ca2+ is sufficient to break pollen
dormancy and trigger early germination, suggesting an engineering
prospect of 5PTs in altering dormancy. The mechanism controlling
seed dormancy is very likely to be similar to pollen dormancy and
thereby 5PTs may be applied to alter seed dormancy. Recently, two
lines with altered expression of callose synthases were reported to
show precocious pollen germination, but at a very low incidence
(4-8%) (Xie et al., 2010). However, in contrast to the 5pt12 mutant,
all the reported lines, including rtg mutant, have abnormal pollen
development as early as the bi-cellular stage. Thus, 5PT12 may be
a better candidate for genetic engineering in crops for improving
fertility and sterility, especially under environmental stress. For
cereal crops, seed dormancy before harvest is a desired trait. In
some cultivars, mature grains germinate on maternal plants
following high temperature or rains, causing large economic losses.
This phenomenon is characterized as pre-harvest sprouting or
vivipary. It is worth testing whether the vivipary phenotype could
be somehow rescued by overexpression of 5PT12. On the contrary,
some crops, such as barley, exhibit excessive dormancy at harvest
that prevent grains from rapid and uniform germination, which is
required in the malting process. Using a transgenic strategy (using
antisense fragment of 5PT12 under the control of seed-specific
promoter) to specifically silence 5PT orthologs in mature seeds of
these crops might inhibit seed dormancy. Thus, our research
provides possibilities of genetic engineering for improving crop
traits and agricultural production.
Funding
The study was supported by the National Science Foundation of China
[31130060, 30740006] and Shanghai Institutes for Biological Sciences
[SIBS2008004].
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.081224/-/DC1
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