Rice isone of the major cereal crops. Cropproductivity depends on successful plant reproduction, for which pollendevelopment is a necessary critical step. Deducing the pollen development at molecular level will enable us tomanipulate crop breeding and thus helps in enhancing crop productivity. Pollen (often termed as male gametophyte orpollen grain) development starts with diploid pollen mother cell (PMC). Each diploid PMC generates four haploidmicrospores in tetrad by meiosis.
Thesetetrad microspores disintegrate into individual microspores and initiate malegametophyte development. Thesemicrospores enlarge and mitotically generate a larger vegetative cell andsmaller generative cell; the generative cell further undergoes mitotic karyokinesisto form two sperm nuclei (Nonomura et al. 2007). The microspore at this trinucleate pollenstage will be released upon anther dehiscence. Once on stigma, the pre-enlarged vegetative cell germinates to formpollen tube, through which the nuclei are released into embryo sac to achievedouble fertilization and complete male gametophyte development.
Pollen development thus requires complexmultigene network coordination from both sporophytic and gametophytic genes.Gene identification and characterization can be performedby several methods. T-DNA tagging is onesuch forward genetic approach to identify the candidate gene responsible forthe observed mutant phenotype. Severalgenes with a role in anther development have been earlier identified andcharacterised. However, very few geneswith their role restricted in pollen and male gametophyte development have beencharacterised by loss-of-function analysis.
The rice cap1 mutant plantwith 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 (Uedaet al. 2013). The T-DNA insertionalmutant of rice GLYCOSYLTRANSFERASE1 (OsGT1) gene displayed anamolous 1:1segregation ratio, and no homozygous mutant progeny was recovered. The OsGT1gene expression was high in the mature pollen grains and plays an importantrole in intine formation (Moon et al. 2013).
The rice IMPORTIN ?1 mutantplants exhibited normal pollen maturation. However, the mutant allele did not transmit through male gametophyte, suggestingnon-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. Therip1 mutants displayed delayed pollen maturation and no pollen germination (Hanet al. 2006). The rice RA68 gene encodes a protein of unknownfunction and transcripts preferentially accumulated in male meiocytes,developing pollen and tapetal cells as well as in shoot. RA68RNAi lines showed reduced pollen viability with defects in starch accumulationin pollen. However the meiosis was notaffected, reflecting its role in post-meiotic pollen development (Li et al.
2010). The rice T-DNA insertional mutantof OsAP65 gene encoding an asparticprotease, displayed distorted segregation of 1:1, with no homozygous mutantplant was recovered. The hemizygousmutant plants displayed normal pollen development, while half the pollenpopulation failed to germinate and elongate, suggesting the OsAP65 gene was crucial for pollengermination and tube growth (Huang et al.
2013). Rice transcriptome from four antherdevelopment stages such as pre-meiotic anther (PMA),meiotic anther (MA), anthers at single-celled pollen (SCP) and tri-nucleatepollen (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 usto decipherthe molecular cues in pollen development andto eventually apply in enhancing crop productivity.
Lipidmetabolism is crucial in pollen development, and defects failure of lipidsynthesis leads to pollen lethality (Mariani and Wolters-Arts 2000). Ultrastructural changes in vacuoles, ER, and golgiduring pollen development suggest that these organelles are linked to theaccumulation of metabolites necessary for pollen development and maturation (Hesse1991; Bedinger 1992; McCormick 1993). Kimet al.
(2011) also demonstrated that lysophosphatidylethanolamine (LPE) is akey signal molecule in pollen development and pollen germination. Plant phospholipases areglycerophospholipid hydrolyzing enzymes, with versatile activities indevelopment and acclimatization. Basedon the position of the phosphodiester bond hydrolyzed, phospholipids can begrouped into phosphoipase A1, phosphoipase A2, phosphoipaseC and phosphoipase D. The Phosphoipase A2(PLA2) hydrolyzes phospholipids at the sn-2 position and generates free fatty acids (FFA) andlysophospholipids such as lysophosphatidylcholine (LPC) andlysophosphatidylethanolamine (LPE).
These lysophospholipids are involved in multiple signalling pathways (Ryu2004). The members of PLA2group can be further divided into two sub-groups as secretory PLA2 (sPLA2)and patatin-like PLA2 (pPLA2), and they differ with theiramino acid sequence, structure, and in turn with their biological properties (Stahlet al. 1998; Balsinde and Balboa 2005). The pPLA2 sub-group member sequences show homology to animal calcium-independentPLA2 (iPLA2). ThesPLA2 members are of low molecular weight in nature ranging 13-18kDa, and carry characteristic PA2c domain with embedded highly conserved Ca2+binding loop (YGKYCGxxxxGC) and catalytic site domain (DACCxxHDxC) with conservedHis/Asp dyad (Stahl et al.
1998, 1999).In the post-genomic era, genome-widemining studies identified three sPLA2genes (sPLA2?, sPLA2? and sPLA2?) in rice (Singh et al.2012). The biological roles of the threegenes in rice are not known. Singh et al. (2012) reported upregulation of OssPLA2? gene underdrought 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-6transgenic line resulted in distorted segregation ratio with no homozygous TC-6transgenic plant recovered. Reversegenetic functional characterization revealed that, the OssPLA2? gene disruption caused defect in post-meioticpollen development. Here, we report thekey role of OssPLA2? genein post-meiotic pollen development and maturation.