UBC Theses and Dissertations
Storage protein gene expression in zygotic and somatic embryos of interior spruce Flinn, Barry Stanley
Storage proteins from interior spruce (Picea glauca/engelmanhi complex) were identified, partially characterized and used as markers to compare the developmental fidelity between zygotic and somatic embryos. The major storage proteins expressed in both embryo types had molecular weights of approximately 41, 35, 33, 24 and 22 kD. The 41 kD protein was buffer and low salt-soluble, whereas the 35-33 kD and 24-22 kD proteins were high salt-soluble and disulfide linked. All of the proteins possessed several isoelectric variants. Based on solubility and disulfide linkage characteristics, as well as cDNA sequences, these storage proteins were homologous to angiosperm vicilin-type (41 kD) and legumin-type (35-33 kD, 24-22 kD) storage proteins. Somatic embryos of different genotypes matured on 40 μM ABA accumulated significant levels of storage protein, similar to or higher than levels found in zygotic embryos. Somatic embryos on 10 μM ABA displayed initial storage protein accumulation, but the levels did not reach those found in zygotic embryos or somatic embryos matured on 40 μM ABA. Zygotic embryos and somatic embryos differentiated on 40 μM or 10 μM ABA displayed differential storage protein accumulation, with the legumin-type proteins apparent before the vicilin-type, although all showed major accumulations during cotyledon development. Zygotic embryos displayed a rapid, transient period of storage protein accumulation, with maximum storage protein levels attained at least 1 month prior to mature seed shed. In contrast, somatic embryos differentiated on 40 μM ABA displayed a more prolonged, gradual accumulation of storage proteins, which were still on the increase after 9 weeks of maturation on ABA. Somatic embryos on 10 μM ABA initally accumulated storage proteins, but these were rapidly degraded as the embryos germinated precociously. Analysis of storage protein mRNA5 indicated they were present by torpedo stage in zygotic embryos and somatic embryos matured on 40 μ M and 10 μM ABA. In all cases, the transcripts increased during development, with those of legumin reaching high levels prior to those of vicilin. Transcript levels in zygotic embryos increased during cotyledon development and then declined rapidly to very low levels at least 1 month prior to mature seed shed. Somatic embryos on 40 μM ABA displayed high transcript levels for a prolonged period, and these were still present after 9 weeks, although they had declined to 50% of maximum levels. Low levels of storage protein transcripts also appeared in somatic embryos on 10 μM ABA, but declined during precocious germination, although they were still detectable after several weeks of precocious germination. Osmotic stress, caused by the culture of somatic embryos on medium containing 15% mannitol, induced storage protein and storage protein transcript accumulation. This could be inhibited by inclusion of the ABA-biosynthetic inhibitor, fluridone, suggesting that the increase was due to osmotic stress—induced ABA biosynthesis.
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