Rice phenotype of 50 % collapsed non-viable pollen.

Rice is
one of the major cereal crops.  Crop
productivity depends on successful plant reproduction, for which pollen
development is a necessary critical step. 
Deducing the pollen development at molecular level will enable us to
manipulate crop breeding and thus helps in enhancing crop productivity.  Pollen (often termed as male gametophyte or
pollen grain) development starts with diploid pollen mother cell (PMC).  Each diploid PMC generates four haploid
microspores in tetrad by meiosis.  These
tetrad microspores disintegrate into individual microspores and initiate male
gametophyte development.  These
microspores enlarge and mitotically generate a larger vegetative cell and
smaller generative cell; the generative cell further undergoes mitotic karyokinesis
to form two sperm nuclei (Nonomura et al. 2007).  The microspore at this trinucleate pollen
stage will be released upon anther dehiscence. 
Once on stigma, the pre-enlarged vegetative cell germinates to form
pollen tube, through which the nuclei are released into embryo sac to achieve
double fertilization and complete male gametophyte development.  Pollen development thus requires complex
multigene network coordination from both sporophytic and gametophytic genes.

Gene identification and characterization can be performed
by several methods.  T-DNA tagging is one
such forward genetic approach to identify the candidate gene responsible for
the observed mutant phenotype.  Several
genes with a role in anther development have been earlier identified and
characterised.  However, very few genes
with their role restricted in pollen and male gametophyte development have been
characterised by loss-of-function analysis. 
The rice cap1 mutant plant
with mutation in arabinokinase-like protein encoding COLLAPSED ABNORMAL POLLEN1 (CAP1)
gene displayed a phenotype of 50 % collapsed non-viable pollen.  Expression of the CAP1 gene was anther preferential during bicellular pollen stage (Ueda
et al. 2013).  The T-DNA insertional
mutant of rice GLYCOSYLTRANSFERASE1 (OsGT1) gene displayed anamolous 1:1
segregation ratio, and no homozygous mutant progeny was recovered.  The OsGT1
gene expression was high in the mature pollen grains and plays an important
role in intine formation (Moon et al. 2013). 
The rice IMPORTIN ?1 mutant
plants exhibited normal pollen maturation. 
However, the mutant allele did not transmit through male gametophyte, suggesting
non-functionality of the pollen that harbour the T-DNA insertion (Han et al.
2011).  The rice WD40 repeat domain protein encoding RICE IMMATURE POLLEN1 (RIP1)
showed high transcript accumulation in late stages of pollen development.  The
rip1 mutants displayed delayed pollen maturation and no pollen germination (Han
et al. 2006).  The rice RA68 gene encodes a protein of unknown
function and transcripts preferentially accumulated in male meiocytes,
developing pollen and tapetal cells as well as in shoot.  RA68
RNAi lines showed reduced pollen viability with defects in starch accumulation
in pollen.  However the meiosis was not
affected, reflecting its role in post-meiotic pollen development (Li et al.
2010).  The rice T-DNA insertional mutant
of OsAP65 gene encoding an aspartic
protease, displayed distorted segregation of 1:1, with no homozygous mutant
plant was recovered.  The hemizygous
mutant plants displayed normal pollen development, while half the pollen
population failed to germinate and elongate, suggesting the OsAP65 gene was crucial for pollen
germination and tube growth (Huang et al. 2013).  Rice transcriptome from four anther
development stages such as pre-meiotic anther (PMA),
meiotic anther (MA), anthers at single-celled pollen (SCP) and tri-nucleate
pollen (TPA), was analysed and 22,000 genes were identified to be expresses during anther development
(Deveshwar et al. 2011).  Functional validation of these genes helps us
to decipher
the molecular cues in pollen development and
to eventually apply in enhancing crop productivity.

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            Lipid
metabolism is crucial in pollen development, and its defects lead to pollen
lethality (Mariani and Wolters-Arts 2000). 
Ultrastructural changes in vacuoles, ER, and golgi during pollen
development suggest that these organelles are linked to the accumulation of
metabolites necessary for pollen development and maturation (Hesse 1991; Bedinger
1992; McCormick 1993).  Kim et al. (2011)
also demonstrated that lysophosphatidylethanolamine (LPE) is a key signal
molecule in pollen development and pollen germination.  Plant phospholipases are
glycerophospholipid hydrolyzing enzymes, with versatile activities in
development and acclimatization.  Based
on the position of the phosphodiester bond hydrolyzed, phospholipids can be
grouped into phosphoipase A1, phosphoipase A2, phosphoipase
C and phosphoipase D.  The Phosphoipase A2
(PLA2) hydrolyzes phospholipids at the sn-2 position and generates free fatty acids (FFA) and
lysophospholipids such as lysophosphatidylcholine (LPC) and
lysophosphatidylethanolamine (LPE). 
These lysophospholipids are involved in multiple signalling pathways (Ryu
2004).  The members of PLA2
group can be further divided into two sub-groups as secretory PLA2 (sPLA2)
and patatin-like PLA2 (pPLA2), and they differ with their
amino acid sequence, structure, and in turn with their biological properties (Stahl
et al. 1998; Balsinde and Balboa 2005). 
The pPLA2 sub-group member sequences show homology to animal calcium-independent
PLA2 (iPLA2).  The
sPLA2 members are of low molecular weight in nature ranging 13-18
kDa, and carry characteristic PA2c domain with embedded highly conserved Ca2+
binding loop (YGKYCGxxxxGC) and catalytic site domain (DACCxxHDxC) with conserved
His/Asp dyad (Stahl et al. 1998, 1999).

In the post-genomic era, genome-wide
mining studies identified three sPLA2
genes (sPLA2?, sPLA2? and sPLA2?) in rice (Singh et al.
2012).  The biological roles of the three
genes in rice are not known.  Singh et al. (2012) reported upregulation of OssPLA2? gene under
drought stress.  In the present study, the rice secretory PLA2? (OssPLA2?) gene was characterized by T-DNA tagging.  T-DNA insertional mutation of OssPLA2? gene in TC-6
transgenic line resulted in distorted segregation ratio with no homozygous TC-6
transgenic plant recovered.  Reverse
genetic functional characterization revealed that, the OssPLA2? gene disruption caused defect in post-meiotic
pollen development.  Here, we report the
key role of OssPLA2? gene
in post-meiotic pollen development and maturation.