IMMUNOGOLD ELECTRON MICROSCOPIC OBSERVATIONSON DISTRIBUTION OF SEVERAL PHYTOHORMONES IN POLLEN TUBE GROWTH TRACK THROUGH PISTIL IN TOBACCO

Up to now,little is known about distribution and function of phytohormones in the pollen tube growth track through pistils.Distribution of tran-szeatin (t-Z),indele-3-acetic acid (IAA),gibberellins A7and A4 (GA_7/4),and(+) abscisic acid((+) ABA)in the style,micropyle and synergids of N icotiana tabacum var.macrophylla were observed with immunogold electron microscopy.Segments from middle part of the styles and ovules at various stages at and after pollination were fixed with 2 % EDC(1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide)and mixed paraformaldehyde and glutaraldehyde for acidic phytohormones (or only with the latter for t-Z),then stightly postfixed in 0.5 % O_SO₄solution for 30 min.After dehydration,samples were embedded in Epon 812 resin.Following etched in 10 % H₂ O₂ for 10 min,ultrathin sections were immunostained respectively with rabbitanti t-Z polyclonal antibody (PAb),anti-IAA methyl ester PAb,mouse anti-GA monoclonal antibody(MAb,specifically recognizes methyl esters of GA₇and GA₄),or anti(+)-ABA methyl ester MAb.Protein Acolloidal gold (Φ10 nm) was used as a marker for rabbit PAbs while sheep antimouse lg Gcolloidal gold(Φ10 nm) for mouse MAb.At pollination plenty of t-Z but a few IAA and GA_7/4 were observed in cells and intercellular matrix of the transmitting tissue.One day after pollination,when pollen tubes had penetrated the transmitting tissue,the gold granules of t-Z remarkably decreased,but those of IAA slightly increased; and those of GA_7/4 obviously increased in the same region.IAA in cells and intercellular matrix of stylar parenchyma greatly increased from just at pollination to one day after pollination.Increased IAA and GA_7/4 were mainly localized in amyloplasts,vacuoles and intercellular matrix.IAA appeared in the walls of pollen tubes passing the transmitting tissue.High level of t-Z in themicropyle and the synergids(particularly abundant t-Z in the filifrom apparatus) greatly decreased from just atpollination to 4 days after pollination.GA_7/4 were less in the synergids at pollination butincreased in both the degenerate and persistent synergids 4 days after pollination.GA_7/4 and IAA in the micropyle or IAA in the synergids maintained at lower levels-4 days after pollination.(+) ABA in the pollen tube growth track was rarely observed during the whole course.According to the results,the relationship of the distribution mode of these four phytohormones in tobacco pistil with pollen tube guidance is discussed.