Abstract
BackgroundSomatic embryogenesis in spruce is a process of high importance for biotechnology, yet it comprises of orchestrated series of events whose cellular and molecular details are not well understood. In this study, we examined the role of actin cytoskeleton during somatic embryogenesis in Norway spruce line AFO 541 by means of anti-actin drugs.ResultsApplication of low doses (50-100 nM) of latrunculin B (Lat B) during the maturation of somatic embryos predominantly killed suspensor cells while leaving the cells in meristematic centres alive, indicating differential sensitivity of actin in the two cell types. The treatment resulted in faster development of more advanced embryos into mature somatic embryos and elimination of insufficiently developed ones. In searching for the cause of the differential actin sensitivity of the two cell types, we analysed the composition of actin isoforms in the culture and isolated four spruce actin genes. Analysis of their expression during embryo maturation revealed that one actin isoform was expressed constitutively in both cell types, whereas three actin isoforms were expressed predominantly in suspensor cells and their expression declined during the maturation. The expression decline was greatly enhanced by Lat B treatment. Sequence analysis revealed amino-acid substitutions in the Lat B-binding site in one of the suspensor-specific actin isoforms, which may result in a different binding affinity for Lat B.ConclusionsWe show that manipulating actin in specific cell types in somatic embryos using Lat B treatment accelerated and even synchronized the development of somatic embryos and may be of practical use in biotechnology.
Highlights
Somatic embryogenesis in spruce is a process of high importance for biotechnology, yet it comprises of orchestrated series of events whose cellular and molecular details are not well understood
Embryogenesis in Norway spruce line AFO 541 cultured on proliferation and maturation media Embryogenic suspensor mass (ESM) cultured continuously on proliferation media consisted of single early somatic embryos, free suspensor cells and polyembryogenic complexes
Polyembryogenic complexes and somatic embryos consisted of long suspensor cells connected with meristematic centres
Summary
Somatic embryogenesis in spruce is a process of high importance for biotechnology, yet it comprises of orchestrated series of events whose cellular and molecular details are not well understood. We examined the role of actin cytoskeleton during somatic embryogenesis in Norway spruce line AFO 541 by means of anti-actin drugs. Embryogenesis in multicellular organisms comprises orchestrated processes that determine the spatio-temporal cell positioning in the developing embryo. In higher plants, which lack active cell movement, the structure and position of individual tissues are determined solely by the orientation of cell division, cell growth and developmentally regulated programmed cell death (PCD). Various cytoskeletal structures have been repeatedly demonstrated to play a key role in all these processes. Actin cytoskeleton participates in the definition of cell polarity and orientation of cell division, cell elonga-. Some actins can complement each other in their biological role [10] elimination or mutation of other actin genes can lead to fatal phenotypes
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