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Distribution of lipid transfer protein 1 (LTP1) epitopes associated with morphogenic events during somatic embryogenesis of Arabidopsis thaliana.

Potocka I, Baldwin TC, Kurczynska EU - Plant Cell Rep. (2012)

Bottom Line: The labelling was strongest in both the outer periclinal and anticlinal walls of the adaxial, protodermal cells of the proximal region of the cotyledon.The putative role(s) of lipid transfer proteins in the formation of lipid lamellae and in cell differentiation are discussed.Key message Occurrence of lipid transfer protein 1 epitopes in Arabidopsis explant cells accompanies changes in cell fate and may be correlated with the deposition of lipid substances in the cell walls.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biology, University of Silesia, Jagiellońska 28, 40-032, Katowice, Poland. izabela.potocka@us.edu.pl

ABSTRACT
Using immunocytochemical methods, at both the light and electron microscopic level, we have investigated the spatial and temporal distribution of lipid transfer protein 1 (LTP1) epitopes during the induction of somatic embryogenesis in explants of Arabidopsis thaliana. Immunofluorescence labelling demonstrated the presence of high levels of LTP1 epitopes within the proximal regions of the cotyledons (embryogenic regions) associated with particular morphogenetic events, including intense cell division activity, cotyledon swelling, cell loosening and callus formation. Precise analysis of the signal localization in protodermal and subprotodermal cells indicated that cells exhibiting features typical of embryogenic cells were strongly labelled, both in walls and the cytoplasm, while in the majority of meristematic-like cells no signal was observed. Staining with lipophilic dyes revealed a correlation between the distribution of LTP1 epitopes and lipid substances within the cell wall. Differences in label abundance and distribution between embryogenic and non-embryogenic regions of explants were studied in detail with the use of immunogold electron microscopy. The labelling was strongest in both the outer periclinal and anticlinal walls of the adaxial, protodermal cells of the proximal region of the cotyledon. The putative role(s) of lipid transfer proteins in the formation of lipid lamellae and in cell differentiation are discussed. Key message Occurrence of lipid transfer protein 1 epitopes in Arabidopsis explant cells accompanies changes in cell fate and may be correlated with the deposition of lipid substances in the cell walls.

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Immunofluorescence localization of LTP1 epitopes in embryogenic regions of explants at different time points of the culture. a Immature zygotic embryo at the start of culture, the most intense fluorescence visible on the embryo surface in all regions. b 1-day cultured explant, the strongest signal in the outer periclinal walls of the protoderm (empty arrowheads) and in plastids (arrow), no signal in the shoot apex. c 3-day cultured explant, the LTP1 epitopes most abundant in cell walls of the protoderm (solid arrowheads) and in external walls of subprotodermal cellular complexes (arrows), the inset shows the protoderm at higher magnification, note the labelling of the outer periclinal cell wall. d Phase contrast view of the section shown in c, clearly visible cell borders. e-g Adaxial protodermal cells of cotyledons showing intense labelling in anticlinal (arrows), outer periclinal (empty arrowheads) and inner periclinal (solid arrowheads) walls, day 6 (e), day 7 (f) and day 21 (g) of culture. h Proximal part of the cotyledon, day 11 of culture, labelling in the cell walls of the protoderm and subprotoderm and in the intercellular spaces (arrowheads). i Phase contrast view of the outlined area in h, note periclinally divided cells in the protoderm. j Negative control for h, no Cy3 labelling. k, l Sections neighbouring to the one in h, stained with Toluidine Blue O (k) and DAPI (l), dense cytoplasm, large nuclei and small vacuoles visible (arrows), arrowheads in k point to intercellular spaces between protodermal and subprotodermal cells, l corresponds to the outlined areas in h and k. m Periclinally divided protodermal cells displaying intense labelling within the walls, day 21 of culture. C cotyledon, H hypocotyl, RM root apical meristem, SM shoot apical meristem. Scale bars 100 μm (a), 10 μm (b–m)
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Fig1: Immunofluorescence localization of LTP1 epitopes in embryogenic regions of explants at different time points of the culture. a Immature zygotic embryo at the start of culture, the most intense fluorescence visible on the embryo surface in all regions. b 1-day cultured explant, the strongest signal in the outer periclinal walls of the protoderm (empty arrowheads) and in plastids (arrow), no signal in the shoot apex. c 3-day cultured explant, the LTP1 epitopes most abundant in cell walls of the protoderm (solid arrowheads) and in external walls of subprotodermal cellular complexes (arrows), the inset shows the protoderm at higher magnification, note the labelling of the outer periclinal cell wall. d Phase contrast view of the section shown in c, clearly visible cell borders. e-g Adaxial protodermal cells of cotyledons showing intense labelling in anticlinal (arrows), outer periclinal (empty arrowheads) and inner periclinal (solid arrowheads) walls, day 6 (e), day 7 (f) and day 21 (g) of culture. h Proximal part of the cotyledon, day 11 of culture, labelling in the cell walls of the protoderm and subprotoderm and in the intercellular spaces (arrowheads). i Phase contrast view of the outlined area in h, note periclinally divided cells in the protoderm. j Negative control for h, no Cy3 labelling. k, l Sections neighbouring to the one in h, stained with Toluidine Blue O (k) and DAPI (l), dense cytoplasm, large nuclei and small vacuoles visible (arrows), arrowheads in k point to intercellular spaces between protodermal and subprotodermal cells, l corresponds to the outlined areas in h and k. m Periclinally divided protodermal cells displaying intense labelling within the walls, day 21 of culture. C cotyledon, H hypocotyl, RM root apical meristem, SM shoot apical meristem. Scale bars 100 μm (a), 10 μm (b–m)

Mentions: In all explants, independent of the day of culture, LTP1 epitopes were present in the outer periclinal walls of protodermal cells (Fig. 1a–c, e–h). However, both the character and intensity of labelling changed with time. Immature zygotic embryos (Fig. 1a) as well as 1-day (Fig. 1b) and 2-day (not shown) cultured explants displayed LTP1 labelling mainly within the outer periclinal walls of protodermal cells. In some explants, at the start of the culture, a weak signal of a punctate character was observed to occur in anticlinal and inner periclinal cell walls of the protoderm, as well as in the cell walls of ground tissue (not shown). Swelling at the proximal end of the cotyledons (third day of culture onwards) appeared to be associated with an increase in signal intensity in the outer periclinal walls of the adaxial protoderm of the region, and with the occurrence of clear labelling within both anticlinal and inner periclinal walls (Fig. 1c, arrowheads). By this stage of development, the character of the signal in the outer periclinal walls of the protoderm had bifurcated into two distinct lines of label, facing the outer and inner regions of the wall, respectively (Fig. 1c, inset). From day 3 of culture onwards (in most cases), a strong signal was also detected in the external walls of subprotodermal cell complexes (Fig. 1c, arrows). Observation by the phase contrast microscopy revealed some characteristic cytological features of both protodermal and subprotodermal cells, namely central enlarged nuclei, small vacuoles and thickened cell walls (Fig. 1d).Fig. 1


Distribution of lipid transfer protein 1 (LTP1) epitopes associated with morphogenic events during somatic embryogenesis of Arabidopsis thaliana.

Potocka I, Baldwin TC, Kurczynska EU - Plant Cell Rep. (2012)

Immunofluorescence localization of LTP1 epitopes in embryogenic regions of explants at different time points of the culture. a Immature zygotic embryo at the start of culture, the most intense fluorescence visible on the embryo surface in all regions. b 1-day cultured explant, the strongest signal in the outer periclinal walls of the protoderm (empty arrowheads) and in plastids (arrow), no signal in the shoot apex. c 3-day cultured explant, the LTP1 epitopes most abundant in cell walls of the protoderm (solid arrowheads) and in external walls of subprotodermal cellular complexes (arrows), the inset shows the protoderm at higher magnification, note the labelling of the outer periclinal cell wall. d Phase contrast view of the section shown in c, clearly visible cell borders. e-g Adaxial protodermal cells of cotyledons showing intense labelling in anticlinal (arrows), outer periclinal (empty arrowheads) and inner periclinal (solid arrowheads) walls, day 6 (e), day 7 (f) and day 21 (g) of culture. h Proximal part of the cotyledon, day 11 of culture, labelling in the cell walls of the protoderm and subprotoderm and in the intercellular spaces (arrowheads). i Phase contrast view of the outlined area in h, note periclinally divided cells in the protoderm. j Negative control for h, no Cy3 labelling. k, l Sections neighbouring to the one in h, stained with Toluidine Blue O (k) and DAPI (l), dense cytoplasm, large nuclei and small vacuoles visible (arrows), arrowheads in k point to intercellular spaces between protodermal and subprotodermal cells, l corresponds to the outlined areas in h and k. m Periclinally divided protodermal cells displaying intense labelling within the walls, day 21 of culture. C cotyledon, H hypocotyl, RM root apical meristem, SM shoot apical meristem. Scale bars 100 μm (a), 10 μm (b–m)
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Fig1: Immunofluorescence localization of LTP1 epitopes in embryogenic regions of explants at different time points of the culture. a Immature zygotic embryo at the start of culture, the most intense fluorescence visible on the embryo surface in all regions. b 1-day cultured explant, the strongest signal in the outer periclinal walls of the protoderm (empty arrowheads) and in plastids (arrow), no signal in the shoot apex. c 3-day cultured explant, the LTP1 epitopes most abundant in cell walls of the protoderm (solid arrowheads) and in external walls of subprotodermal cellular complexes (arrows), the inset shows the protoderm at higher magnification, note the labelling of the outer periclinal cell wall. d Phase contrast view of the section shown in c, clearly visible cell borders. e-g Adaxial protodermal cells of cotyledons showing intense labelling in anticlinal (arrows), outer periclinal (empty arrowheads) and inner periclinal (solid arrowheads) walls, day 6 (e), day 7 (f) and day 21 (g) of culture. h Proximal part of the cotyledon, day 11 of culture, labelling in the cell walls of the protoderm and subprotoderm and in the intercellular spaces (arrowheads). i Phase contrast view of the outlined area in h, note periclinally divided cells in the protoderm. j Negative control for h, no Cy3 labelling. k, l Sections neighbouring to the one in h, stained with Toluidine Blue O (k) and DAPI (l), dense cytoplasm, large nuclei and small vacuoles visible (arrows), arrowheads in k point to intercellular spaces between protodermal and subprotodermal cells, l corresponds to the outlined areas in h and k. m Periclinally divided protodermal cells displaying intense labelling within the walls, day 21 of culture. C cotyledon, H hypocotyl, RM root apical meristem, SM shoot apical meristem. Scale bars 100 μm (a), 10 μm (b–m)
Mentions: In all explants, independent of the day of culture, LTP1 epitopes were present in the outer periclinal walls of protodermal cells (Fig. 1a–c, e–h). However, both the character and intensity of labelling changed with time. Immature zygotic embryos (Fig. 1a) as well as 1-day (Fig. 1b) and 2-day (not shown) cultured explants displayed LTP1 labelling mainly within the outer periclinal walls of protodermal cells. In some explants, at the start of the culture, a weak signal of a punctate character was observed to occur in anticlinal and inner periclinal cell walls of the protoderm, as well as in the cell walls of ground tissue (not shown). Swelling at the proximal end of the cotyledons (third day of culture onwards) appeared to be associated with an increase in signal intensity in the outer periclinal walls of the adaxial protoderm of the region, and with the occurrence of clear labelling within both anticlinal and inner periclinal walls (Fig. 1c, arrowheads). By this stage of development, the character of the signal in the outer periclinal walls of the protoderm had bifurcated into two distinct lines of label, facing the outer and inner regions of the wall, respectively (Fig. 1c, inset). From day 3 of culture onwards (in most cases), a strong signal was also detected in the external walls of subprotodermal cell complexes (Fig. 1c, arrows). Observation by the phase contrast microscopy revealed some characteristic cytological features of both protodermal and subprotodermal cells, namely central enlarged nuclei, small vacuoles and thickened cell walls (Fig. 1d).Fig. 1

Bottom Line: The labelling was strongest in both the outer periclinal and anticlinal walls of the adaxial, protodermal cells of the proximal region of the cotyledon.The putative role(s) of lipid transfer proteins in the formation of lipid lamellae and in cell differentiation are discussed.Key message Occurrence of lipid transfer protein 1 epitopes in Arabidopsis explant cells accompanies changes in cell fate and may be correlated with the deposition of lipid substances in the cell walls.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biology, University of Silesia, Jagiellońska 28, 40-032, Katowice, Poland. izabela.potocka@us.edu.pl

ABSTRACT
Using immunocytochemical methods, at both the light and electron microscopic level, we have investigated the spatial and temporal distribution of lipid transfer protein 1 (LTP1) epitopes during the induction of somatic embryogenesis in explants of Arabidopsis thaliana. Immunofluorescence labelling demonstrated the presence of high levels of LTP1 epitopes within the proximal regions of the cotyledons (embryogenic regions) associated with particular morphogenetic events, including intense cell division activity, cotyledon swelling, cell loosening and callus formation. Precise analysis of the signal localization in protodermal and subprotodermal cells indicated that cells exhibiting features typical of embryogenic cells were strongly labelled, both in walls and the cytoplasm, while in the majority of meristematic-like cells no signal was observed. Staining with lipophilic dyes revealed a correlation between the distribution of LTP1 epitopes and lipid substances within the cell wall. Differences in label abundance and distribution between embryogenic and non-embryogenic regions of explants were studied in detail with the use of immunogold electron microscopy. The labelling was strongest in both the outer periclinal and anticlinal walls of the adaxial, protodermal cells of the proximal region of the cotyledon. The putative role(s) of lipid transfer proteins in the formation of lipid lamellae and in cell differentiation are discussed. Key message Occurrence of lipid transfer protein 1 epitopes in Arabidopsis explant cells accompanies changes in cell fate and may be correlated with the deposition of lipid substances in the cell walls.

Show MeSH
Related in: MedlinePlus