Embryo sac development and endogenous gibberellins in pollinated and unpollinated ovaries of walnut (Juglans regia)

Abstract

The effects of pollination on both embryo sac development and gibberellin (GA) content in walnut (Juglans regia L.) ovaries were investigated. Microscopic observations on early structural changes in the embryo sac indicated that fertilization occurred shortly after pollination (within 5 days). In unpollinated ovaries, the two polar nuclei also fused during this period, leading to a 2n endosperm which started cellularization (wall formation) about 5 days later. Zygote division in pollinated ovaries started approximately 9 days after fertilization while the endosperm at this stage was still in the free‐nuclear phase. Gas chromatography‐mass spectrometry (GC‐MS) analyses of GAs in pollinated and unpollinated ovaries demonstrated the presence of all members of the 13‐hydroxylation GA pathway (GA53, GA44, GA17, GA19, GA20, GA29, GA1, and GA8). In addition, GA4, GA9, and an unknown monohydroxy‐GA12 were also detected. Endogenous GAs were quantified by selected ion monitoring (SIM) from pollination up to 13 days later. At the end of this period, pollinated ovaries contained high levels of C20 GAs whereas C19 GAs had declined. Gibberellins A44 and A19 increased slightly shortly after fertilization, then decreased, and lastly accumulated. Gibberellin A20 followed the same step‐by‐step trend albeit in an opposite manner. Gibberellin A1 showed a transitory increase immediately after fertilization and decreased gradually subsequently. The pattern of GA change in unpollinated ovaries differed mostly as follows: (1) within 13 days after the time of pollination GA53 and GA44 decreased; (2) GA19 increased continuously; (3) GA20 decreased also continuously following the opposite pattern to GA19; and (4) the brief rise in GA1 was higher (2‐fold) than in pollinated ovaries, although 13 days after the time of pollination its levels were much lower in unpollinated ones. These results suggest that the sequence pollination/fertilization modulates GA1 levels, first reducing them immediately after fertilization, and subsequently, stimulating moderate amounts shortly thereafter at the beginning of embryogenesis. In unpollinated ovaries the lowest GAI levels coincided with ovary growth arrest, the onset of a rapid and heavy abscission (100%), and an accelerated 2n endosperm cellularization. However, repeated exogenous GA3 applications did not suppress the growth arrest and abscission.