A full-length Picea abies cDNA clone Pa18, encoding a protein with the characteristics of plant lipid transfer proteins, has been isolated and characterized. The size of the deduced 173 amino acid (aa) long protein is around 18 kDa. The first 100–120 aa show similarity to angiosperm lipid transfer proteins in amino acid sequence as well as in predicted secondary structure. The Pa18 gene is constitutively expressed in embryogenic cultures of Picea abies representing different stages of development as well as in non-embryogenic callus and seedlings. The Pa18 gene product has an antimicrobial activity. In situ hybridization showed that the Pa18 gene is equally expressed in all embryonic cells of proliferating embryogenic cultures but during embryo maturation the expression of the gene in maturing and mature somatic as well as in mature zygotic embryos is stronger in the outer cell layer than in other tissues. Southern blot analysis at different stringencies was consistent with a single gene with one or two copies rather than a gene family. Twenty independent transgenic sublines over- and under-expressing the Pa18 gene under the Zea mays ubiquitin promoter were established. There was a high yield of mature somatic embryos with a smooth surface only in untransformed, control cultures. Irrespective of the expression level of Pa18, the somatic embryos started to mature when given a maturation treatment. However, in the transgenic sublines, the outer cells in the maturing embryos frequently became elongated and vacuolated instead of remaining small and uniform. One explanation for this was that the expression of Pa18 was not restricted to the outer cell layer in transformed sublines. Angiosperms and gymnosperms separated about 300 million years ago and the embryo genesis is different in the two groups. The outer cell layer (protoderm), the first tissue to differentiate, is less clearly delineated in gymnosperms. For normal embryo development in angiosperms, expression of the LTP gene must be restricted to the protodermal cells. In this work we show that the expression of the Pa18 gene must be restricted to the putative protodermal cells of the gymnosperm.
Plant Molecular Biology – Springer Journals
Published: Oct 16, 2004
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