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The role of actin isoforms in somatic embryogenesis in Norway spruce.

ABSTRACT: 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. 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.Application 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.We 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.

SUBMITTER: Schwarzerova K

PROVIDER: S-EPMC3095357 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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The role of actin isoforms in somatic embryogenesis in Norway spruce.

Schwarzerová Katerina K Vondráková Zuzana Z Fischer Lukás L Boríková Petra P Bellinvia Erica E Eliásová Katerina K Havelková Lenka L Fiserová Jindriska J Vágner Martin M Opatrný Zdenek Z

BMC plant biology 20100517

<h4>Background</h4>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. 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.<h4>Results</h4>Application of low doses (50-100 nM) of latrunculin B (Lat B) during the maturation of somatic embryos predominantly killed ...[more]

PMID: 20478025

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Project description:Conifer somatic embryogenesis (SE) is a process driven by exogenously supplied plant growth regulators (PGRs). Exogenous PGRs and endogenous phytohormones trigger particular ontogenetic events. Complex mechanisms involving a number of endogenous phytohormones control the differentiation of cells and tissues, as well as the establishment of structures and organs. Most of the mechanisms and hormonal functions in the SE of conifers have not yet been described. With the aim to better understand these mechanisms, we provided detailed analysis of the spectrum of endogenous phytohormones over the course of SE in Norway spruce (Picea abies). Concentrations of endogenous phytohormones including auxins, cytokinins (CKs), abscisic acid (ABA), jasmonates, and salicylic acid (SA) in somatic P. abies embryos were analyzed by HPLC-ESI-MS/MS. The results revealed that the concentrations of particular phytohormone classes varied substantially between proliferation, maturation, desiccation, and germination. Endogenous ABA showed a maximum concentration at the maturation stage, which reflected the presence of exogenous ABA in the medium and demonstrated its efficient perception by the embryos as a prerequisite for their further development. Auxins also had concentration maxima at the maturation stage, suggesting a role in embryo polarization. Endogenous jasmonates were detected in conifer somatic embryos for the first time, and reached maxima at germination. According to our knowledge, we have presented evidence for the involvement of the non-indole auxin phenylacetic acid, cis-zeatin- and dihydrozeatin-type CKs and SA in SE for the first time. The presented results represent the currently most comprehensive overview of plant hormone levels in embryos throughout the whole process of conifer SE. The differences in concentrations of various classes of phytohormones over the proliferation, maturation, desiccation, and germination in somatic P. abies embryos clearly indicate correlations between endogenous phytohormone profiles and particular developmental stages of the SE of conifers.

Project description:In Norway spruce, as in many other conifers, the germination capacity of somatic embryos is strongly influenced by the desiccation phase inserted after maturation. The intensity of drying during desiccation eminently affected the formation of emblings (i.e., seedlings developed from somatic embryos). Compared to non-desiccated embryos, the germination capacity of embryos desiccated at 100% relative humidity was about three times higher, but the reduction of relative humidity to 95 and 90% had a negative effect on the subsequent embryo development. The water loss observed in these embryos did not lead to an increase in lipid peroxidation, as shown by malondialdehyde levels. Another metabolic pathway in plants that mediates a response to abiotic stresses is directed toward the biosynthesis of polyamines (PAs). The activities of PA biosynthetic enzymes increased steadily in embryos during desiccation at 100% relative humidity, whereas they decreased at lower humidity. The total content of free PAs in the embryos gradually decreased throughout desiccation. The increase in free putrescine (Put) and perchloric acid-insoluble Put conjugates was observed in embryos desiccated at lower humidity. These changes were accompanied to some extent by the transcription of the genes for the PA biosynthesis enzymes. Desiccation at 100% relative humidity increased the activity of the cell wall-modifying enzymes β-1,3-glucanases and chitinases; the activities of these enzymes were also significantly suppressed at reduced humidity. The same pattern was observed in the transcription of some β-1,3-glucanase and chitinase genes. Desiccation treatments triggered metabolic processes that responded to water availability, suggesting an active response of the embryo to the reduction in humidity. A positive effect was demonstrated only for desiccation at high relative humidity. Some of the physiological characteristics described can be used as markers of inappropriate relative humidity during somatic embryo desiccation.

Dataset Information

The role of actin isoforms in somatic embryogenesis in Norway spruce.

Publications

The role of actin isoforms in somatic embryogenesis in Norway spruce.

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