Identification Of NPF2.4 And GSTZ2 And Its Role In Male And Female Gametophyte Development In Arabidopsis Thaliana

There is dire need to study angiosperm female gametophyte development and reproduction.Female gametophyte development starts early in ovule development with the formation of a diploid megaspore mother cell that go through meiosis.We identified At2gl7960(Ovule Outer Integument-OOT),NPF2.4 and GSTZ2 genes from three T-DNA insertion lines tagged with green fluorescent protein(GFP).TAIL-PCR was employed to clone these genes from T-DNA insertion lines.These genes showed distinct GFP expression pattern in root and shoot apical meristem(STM)as well as in different developmental stages of ovule and anther.At2gl7960(Ovule Outer Integument-001)was cloned from T-DNA insertion line 9204-3.OOI encodes a hypothetical protein.OOI expressed in shoot apical meristem(STM)in seedling stage while OOI also expressed FG1,FG2,FG3 and FG7 in outer integument of ovule.We confirmed its expression in Col-0 through promoter drive GFP construct.NPF2.4 was cloned from T-DNA insertion line 9204-6.NPF2.4 is member of nitrate transporter family.Here we find its role in female gametophyte development.NPF2.4 showed significant expression in root,STM and hypocotyl at seedling stage,NPF2.4 also expressed in FG1,FG2,FG3,FG6 and FG7 outer and inner integument of ovule as well as expressed in anther.Its expression was confirmed in Col-0 using promoter drive GFP vector.npf2.4 functional analysis showed its role in male and female gametophyte defects with low seed set,having40% ovule abortion,with low pollen in vitro germination of 38.01%.Ovule defects were observed through DIC,npf2.4 MMC had 25% double nucleus,while some MMC were lacking nucleus.Mature ovules of FG6 and FG7 also showed defects while these defects of npf2.4 were successfully rescued and recovered.GSTZ2 was cloned from 9333 T-DNA insertion line.GSTZ2 is member of GSTs genes family which play role in herbicide detoxification.It shows expression in hypocotyl and STM.GSTZ2 expressed in chalaza region near the nucellus in FG1 and FG2 of ovule,while later the expression was changed to ftmicullus in FG3.FG6 and FG7 showed strong GSTZ2 expression in fimicullus and chalazal end of ovule.GSTZ2 also have strong expression in anther.We confirmed its promoter expression in Col-0 using GFP and GUS expression vectors.For functional analysis we observed T-DNA mutants.gstz2 functional analysis showed 75%ovule abortion.gstz2 showed 39.6% pollen grains gennination only.DIC was carried out to find defects in ovule,gstzl had 24% double nucleus MMC and 73%FG4 showed lack of nucleus out of four nucleus in FG4.Our experiment confirmed the involvement of NPF2.4 and GSTZ2 in male and female gametophyte development and also laid a foundation for fiirther studies of gene by environment interaction.DNA binding with one finger(Dof)transcription factors(TF)femily is a class of plant specific transcription factors and has crucial roles in development of different tissues and response to stresses.Using GWAS we identified twenty six Dof genes located on 16 pineapple chromosomes.Dof family genes were distributed into five groups and contain highly conserved motif.High expression of AcoDofl,AcoDofl2,AcoDof26(root),AcoDof9,AcoDof22, AcoDof19(fruit),AcoDof23,AcoDof25(ovule),AcoDof11,AcoDof17 and AcoDof20(petal)were found in RNA-Seq analysis.RNA-Seq data also suggest that Dof genes could have important role in female gametophyte development of pineapple.Expression analysis using qRT-PCR of pineapple Dof genes family under different abiotic stress(cold,heat,salt,drought)showed a dynamic response of Dof genes.AcoDofl was up-regulated at 48 h treatment in all four abiotic stresses,while AcoDof20 was up-regulated at 24 h of treatment Moreover,AcoDof8,AcoDof12,AcoDof2 were up-regulated by salinity stress.Dof genes expression during abiotic stress reveals their vital role for pineapple growth and development which could be utilized agronomically.Present study provides a framework for fiiture studies related to the functions of Dof genes in pineapple.