Introduction maturation and no pollen germination (Han et



angiosperms, the male gametophyte (pollen grain) plays a central role in plant
fertility and seed production.  A diploid
pollen mother cell (PMC) in the anther undergoes meiosis to produce haploid
microspores in a tetrad.  Male
gametophyte development begins from the free microspore stage.  The microspores separate, enlarge and undergo
asymmetric mitosis to generate a smaller generative cell and a larger
vegetative cell (Nonomura et al. 2007). 
The nucleus of the generative cell in the microspore further divides mitotically
to produce two sperm nuclei, resulting in mature trinucleate pollen.  At this stage, the anther dehisces to release
mature pollen grains.  The vegetative
cell occupies most of the space in the pollen and forms the pollen tube at the
time of germination.  Male gametophyte
development is completed when the sperm nuclei are released into the embryo sac
for double fertilization.  Pollen
development and maturation are complex processes requiring the coordinated
expression of several sporophytic and gametophytic genes.

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T-DNA tagging and transposon tagging have led to the
identification of a number of genes controlling anther development.  Unlike many anther-related genes studied,
only a few genes which are expressed in the pollen and play a role in male gametophyte
development and maturation have been identified by loss-of-function
gene encodes an arabinokinase-like protein and expresses preferentially in
anthers at the bicellular pollen stage. 
Heterozygous cap1 mutants
produced 50 % collapsed non-viable pollen, indicating that CAP1 is critical for
pollen development in rice (Ueda et al. 2013). 
(OsGT1) gene is highly expressed in
mature pollen grains and plays an important role in intine formation (Moon et
al. 2013).  The T-DNA insertion mutant of
OsGT1 exhibited an anamolous 1:1
segregation ratio and no homozygous progeny for the T-DNA insertion was
obtained.  The pollen grains of rice IMPORTIN ?1 mutant matured normally, but
the mutant allele could not be transmitted through the male gametophyte,
suggesting that the pollen grains containing the T-DNA insertion were
non-functional (Han et al. 2011).  RICE IMMATURE POLLEN1 (RIP1)
encodes a WD40 repeat domain protein, and its transcript was abundant in the
late stages of pollen development.  The rip1 mutant showed delayed pollen
maturation and no pollen germination (Han et al. 2006).  The
RA68 gene, which encodes a protein of unknown function, is expressed
preferentially in shoots and flowers.  RA68-silenced lines showed reduced
pollen viability, indicating its role in microspore mitosis and starch
accumulation (Li et al. 2010).  T-DNA
insertion in OsAP65, which encodes an
aspartic protease, caused a severe segregation distortion phenotype and
homozygous plants with disrupted OsAP65
were not recovered.  The mutant pollen of
OsAP65 matured normally, but did not
germinate or elongate, indicating that OsAP65
is essential for rice pollen germination and tube growth (Huang et al. 2013).  Transcriptome profiling of four stages of
anther development in rice such as pre-meiotic anther (PMA),
meiotic anther (MA), anthers at single-celled pollen (SCP) and tri-nucleate
pollen (TPA) revealed that 22,000 genes are expressed during anther development
(Deveshwar et al. 2011).  Functional analysis of these genes is important for
understanding the molecular mechanisms that control pollen development in rice.

Phospholipase A2
(PLA2) belongs to an important group of lipid hydrolysing enzymes in
plants.  PLA2 hydrolyzes the
membrane phospholipids at their sn-2
position.  Products generated in the
reaction such as free fatty acids (FFA) and lysophospholipids such as
lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) are
involved in several cellular signaling pathways (Ryu 2004).  Based on the amino acid sequence data and
biological properties, plant PLA2s are divided into two groups;
patatin like phospholipase A2 (which are homologous to animal calcium-independent
phospholipase A2, iPLA2) and low molecular weight secretory
phospholipase A2 (sPLA2) (Stahl et al. 1998; Balsinde and
Balboa 2005).  Plant sPLA2s
are characterized by the molecular weight range of 13-18 kDa and the presence
of the PA2c domain, which comprises a highly conserved Ca2+ binding
loop (YGKYCGxxxxGC) and a catalytic site domain (DACCxxHDxC).  The catalytic site domain contains well
conserved His/Asp residues (Stahl et al. 1998, 1999).  Rice genome exploration has revealed the
presence of three sPLA2
genes (?, ? and ?) (Singh et al.
2012).  The biological roles of the three
genes in rice are not known.

In this study, the rice
secretory PHOSPHOLIPASE A2? (OssPLA2?) gene was
characterized by T-DNA tagging. 
Disruption of its expression due to T-DNA integration in the transgenic rice
line TC-6 caused a severe segregation distortion phenotype and no homozygous
plant for the T-DNA insertion was recovered in the T1 generation.  Genetic and molecular analyses indicated that
loss-of-function of the OssPLA2?
gene by T-DNA insertion caused defects in the male gamete (pollen)
development.  Here, we report the key
role of OssPLA2? gene in post-meiotic
pollen development and maturation.