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Caffeine-Induced Premature Chromosome Condensation Results in the Apoptosis-Like Programmed Cell Death in Root Meristems of Vicia faba.

Rybaczek D, Musiałek MW, Balcerczyk A - PLoS ONE (2015)

Bottom Line: We have demonstrated that the activation of apoptosis-like programmed cell death (AL-PCD) was a secondary result of caffeine (CF) induced premature chromosome condensation (PCC) in hydroxyurea-synchronized Vicia faba root meristem cells.In addition, the results obtained under transmission electron microscopy (TEM) further revealed apoptotic-like features at the ultrastructural level of PCC-type cells: (i) extensive vacuolization; (ii) abnormal chromatin condensation, its marginalization and concomitant degradation; (iii) formation of autophagy-like vesicles (iv) protoplast shrinkage (v) fragmentation of cell nuclei and (vi) extensive degeneration of the cells.The results obtained have been discussed with respect to the vacuolar/autolytic type of plant-specific AL-PCD.

View Article: PubMed Central - PubMed

Affiliation: Department of Cytophysiology, Institute of Experimental Biology, Faculty of Biology and Environmental Protection, University of Łódź, Łódź, Poland.

ABSTRACT
We have demonstrated that the activation of apoptosis-like programmed cell death (AL-PCD) was a secondary result of caffeine (CF) induced premature chromosome condensation (PCC) in hydroxyurea-synchronized Vicia faba root meristem cells. Initiation of the apoptotic-like cell degradation pathway seemed to be the result of DNA damage generated by treatment with hydroxyurea (HU) [double-stranded breaks (DSBs) mostly] and co-treatment with HU/CF [single-stranded breaks (SSBs) mainly]. A single chromosome comet assay was successfully used to study different types of DNA damage (neutral variant-DSBs versus alkaline-DSBs or SSBs). The immunocytochemical detection of H2AXS139Ph and PARP-2 were used as markers for DSBs and SSBs, respectively. Acridine orange and ethidium bromide (AO/EB) were applied for quantitative immunofluorescence measurements of dead, dying and living cells. Apoptotic-type DNA fragmentation and positive TUNEL reaction finally proved that CF triggers AL-PCD in stressed V. faba root meristem cells. In addition, the results obtained under transmission electron microscopy (TEM) further revealed apoptotic-like features at the ultrastructural level of PCC-type cells: (i) extensive vacuolization; (ii) abnormal chromatin condensation, its marginalization and concomitant degradation; (iii) formation of autophagy-like vesicles (iv) protoplast shrinkage (v) fragmentation of cell nuclei and (vi) extensive degeneration of the cells. The results obtained have been discussed with respect to the vacuolar/autolytic type of plant-specific AL-PCD.

No MeSH data available.


Related in: MedlinePlus

Acridine orange (AO) and ethidium bromide (EB) staining of living, dying and dead cells in Vicia faba root meristem cells in relation to (A) the localization in particular zones, and (B-D) the surface area occupied by the cells in particular zones.(A) Fluorescence picture of AO/EB stained V. faba roots in planta. (a) schematic figure presenting the outline of the roots of the control series, with marked (I) root cap, (II) zone of cell division i.e. root meristem, (III) zone of elongation, and the quiescent center. (b) the control roots, (c) the roots treated with 2.5 mM hydroxyurea (HU) for 32 h, (d) the roots treated with 2.5 mM HU for 24 h and then co-treated with 2.5 mM HU and 5 mM caffeine (CF). Scale bar = 1 mm. The schematic outline of the root from scheme (a) was placed over a root from the control series (b) on which the root outline from figure 'a' and figure 'b' are precisely overlapped, (c) a root from the series, in which seedlings were subjected to replication stress and (c) a root that was induced to premature chromosome condensation (PCC). (c-d) The continuous line marks those root fragments that in terms of size and shape were the same as the analogous areas in the roots of the control series (a-b versus c-d), while the broken line (in figures [c] and [d]) marks the root areas that indicated the appearance of aerenchymatic-like spaces that had formed in the roots that had been subjected to treatment with HU (c) or co-treated with the mixture of HU/CF (d). In places indicated by broken lines, roots of the series (c) and (d) were distinctly wider than the control (b). (B-D) quantitative presentation of surface area (%) occupied by the green, yellow-orange and red colors (that correspond to the populations of living, dying and dead cells, respectively) in the particular zones of V. faba roots. (B) quiescent center, (C) zone of cell division, i.e. root meristem, marked also as zone II, and (D) zone of elongation, marked as zone III.
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pone.0142307.g005: Acridine orange (AO) and ethidium bromide (EB) staining of living, dying and dead cells in Vicia faba root meristem cells in relation to (A) the localization in particular zones, and (B-D) the surface area occupied by the cells in particular zones.(A) Fluorescence picture of AO/EB stained V. faba roots in planta. (a) schematic figure presenting the outline of the roots of the control series, with marked (I) root cap, (II) zone of cell division i.e. root meristem, (III) zone of elongation, and the quiescent center. (b) the control roots, (c) the roots treated with 2.5 mM hydroxyurea (HU) for 32 h, (d) the roots treated with 2.5 mM HU for 24 h and then co-treated with 2.5 mM HU and 5 mM caffeine (CF). Scale bar = 1 mm. The schematic outline of the root from scheme (a) was placed over a root from the control series (b) on which the root outline from figure 'a' and figure 'b' are precisely overlapped, (c) a root from the series, in which seedlings were subjected to replication stress and (c) a root that was induced to premature chromosome condensation (PCC). (c-d) The continuous line marks those root fragments that in terms of size and shape were the same as the analogous areas in the roots of the control series (a-b versus c-d), while the broken line (in figures [c] and [d]) marks the root areas that indicated the appearance of aerenchymatic-like spaces that had formed in the roots that had been subjected to treatment with HU (c) or co-treated with the mixture of HU/CF (d). In places indicated by broken lines, roots of the series (c) and (d) were distinctly wider than the control (b). (B-D) quantitative presentation of surface area (%) occupied by the green, yellow-orange and red colors (that correspond to the populations of living, dying and dead cells, respectively) in the particular zones of V. faba roots. (B) quiescent center, (C) zone of cell division, i.e. root meristem, marked also as zone II, and (D) zone of elongation, marked as zone III.

Mentions: Double staining with acridine orange (AO) and ethidium bromide (EB) is another method used to specify the type of cell death, as well as being a useful tool in detecting and quantifying the states living, dying and dead (Fig 4, Fig 5 and S3 Fig). Fluorescence microscopy revealed images indicating the occurrence of living cells (fluoresce green), dying cells (green-yellow, yellow, yellow-orange and bright orange) and dead cells (dark orange and bright red) in the control (Fig 4A), after both HU treatment (Fig 4B) and after PCC induction (Fig 4C). The diagram presenting the color spectrum resulting from the quantitative measurements of fluorescence intensity of nuclear chromatin stained with AO/EB was made in order to determine the degree of DNA damage in the control nuclei (Fig 4A') as well as in the nuclei derived from stressed roots of V. faba (Fig 4B and 4C'). The highest number of dead cells (13.4%) was observed in HU/CF treated material (Fig 4C''). Thus, it was shown that the number of dead cells after PCC induction was over 6-fold higher compared to the control and 4.5-fold higher, compared to the HU series (Fig 4A'', 4B'' and 4C''). The fractions of living, dying and dead cells in the control, compared with the HU series were similar; we only observed an increase in the number of dead cells in the latter (1.5-fold; Fig 4A'' and 4B''). Further analysis consisted in comparing the numbers of living cells or being at various cell death stages (early-to-late) in particular zones of V. faba roots [from 'quiescent center' through the zones of cell division, i.e. root meristem (zone marked as II in Fig 5A) up to the zone of elongation (marked as III in Fig 5A)]. Our results suggest that the increase in the number of dead cells in the meristematic zone of V. faba roots after HU/CF treatment was statistically significant (p < 0.01) in relation to both the control and HU-treated series (Fig 5C). In the meristematic zone (II) the relatively high percentage (Fig 5C) populations of dying (Fig 5C, yellow-colored columns) and dead cells (i.e. 19.9%, 13.4%, and 28.8% in the control, HU-treated, and PCC-induced series, respectively) resulted from some imperfection in the method of intravital AO/EB staining, and from the fact it is not possible to omit from the calculation the population of rhizodermis cells in which the occurrence of the PCD phenomenon is typical (comp. Fig 5Aa, Fig 5Ab, Fig 5Ac, Fig 5Ad and Fig 5C; this could be the reason that no significant differences were observed herein). In the other zones of V. faba roots induced to PCC, the number of dead cells was about 1.5-fold higher than in the control and almost 2-fold higher than in the HU-treated material (Fig 5A and 5B). In turn, in the elongation zone (III, over-meristematic, Fig 5A), the highest index of dead cells was observed under the influence of HU/CF (7.7%, i.e. 1.5-fold and 3.4-fold higher than in the control and HU-series, respectively; Fig 5D). However, it seems more interesting to compare the numbers of dead cells in the meristematic zone than in the elongation zone; in the latter it was lower, where in the control, the HU and the HU/CF series, decreases of 4, 6 and 3.7 times were observed, respectively (comp. Fig 5C and 5D). S3 Fig shows longitudinal intersections of the three most representative roots stained with the intravital AO/EB method, as well as shows the proportions of living (green), dying (range: yellow-to-orange) and dead (red) cells, and their distribution in the meristem, supra-meristematic zone and in the rhizodermis. In all zones (particularly in the area of a root cap) and in all experimental series we observed red fluorescence indicating PCD processes eliminating cells, particularly from the rhizodermis, present in the external layers of the root (S3 Fig). The arrows on S3b, S3b' and S3b'' Figs show a distinct widening of the HU-treated roots forming an easily visible protuberance in which the accumulation of dead cells can be observed. These protruberances may result from the appearance of aerenchymatic-like spaces in the root cortex cells of V. faba (comp. [8]).


Caffeine-Induced Premature Chromosome Condensation Results in the Apoptosis-Like Programmed Cell Death in Root Meristems of Vicia faba.

Rybaczek D, Musiałek MW, Balcerczyk A - PLoS ONE (2015)

Acridine orange (AO) and ethidium bromide (EB) staining of living, dying and dead cells in Vicia faba root meristem cells in relation to (A) the localization in particular zones, and (B-D) the surface area occupied by the cells in particular zones.(A) Fluorescence picture of AO/EB stained V. faba roots in planta. (a) schematic figure presenting the outline of the roots of the control series, with marked (I) root cap, (II) zone of cell division i.e. root meristem, (III) zone of elongation, and the quiescent center. (b) the control roots, (c) the roots treated with 2.5 mM hydroxyurea (HU) for 32 h, (d) the roots treated with 2.5 mM HU for 24 h and then co-treated with 2.5 mM HU and 5 mM caffeine (CF). Scale bar = 1 mm. The schematic outline of the root from scheme (a) was placed over a root from the control series (b) on which the root outline from figure 'a' and figure 'b' are precisely overlapped, (c) a root from the series, in which seedlings were subjected to replication stress and (c) a root that was induced to premature chromosome condensation (PCC). (c-d) The continuous line marks those root fragments that in terms of size and shape were the same as the analogous areas in the roots of the control series (a-b versus c-d), while the broken line (in figures [c] and [d]) marks the root areas that indicated the appearance of aerenchymatic-like spaces that had formed in the roots that had been subjected to treatment with HU (c) or co-treated with the mixture of HU/CF (d). In places indicated by broken lines, roots of the series (c) and (d) were distinctly wider than the control (b). (B-D) quantitative presentation of surface area (%) occupied by the green, yellow-orange and red colors (that correspond to the populations of living, dying and dead cells, respectively) in the particular zones of V. faba roots. (B) quiescent center, (C) zone of cell division, i.e. root meristem, marked also as zone II, and (D) zone of elongation, marked as zone III.
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pone.0142307.g005: Acridine orange (AO) and ethidium bromide (EB) staining of living, dying and dead cells in Vicia faba root meristem cells in relation to (A) the localization in particular zones, and (B-D) the surface area occupied by the cells in particular zones.(A) Fluorescence picture of AO/EB stained V. faba roots in planta. (a) schematic figure presenting the outline of the roots of the control series, with marked (I) root cap, (II) zone of cell division i.e. root meristem, (III) zone of elongation, and the quiescent center. (b) the control roots, (c) the roots treated with 2.5 mM hydroxyurea (HU) for 32 h, (d) the roots treated with 2.5 mM HU for 24 h and then co-treated with 2.5 mM HU and 5 mM caffeine (CF). Scale bar = 1 mm. The schematic outline of the root from scheme (a) was placed over a root from the control series (b) on which the root outline from figure 'a' and figure 'b' are precisely overlapped, (c) a root from the series, in which seedlings were subjected to replication stress and (c) a root that was induced to premature chromosome condensation (PCC). (c-d) The continuous line marks those root fragments that in terms of size and shape were the same as the analogous areas in the roots of the control series (a-b versus c-d), while the broken line (in figures [c] and [d]) marks the root areas that indicated the appearance of aerenchymatic-like spaces that had formed in the roots that had been subjected to treatment with HU (c) or co-treated with the mixture of HU/CF (d). In places indicated by broken lines, roots of the series (c) and (d) were distinctly wider than the control (b). (B-D) quantitative presentation of surface area (%) occupied by the green, yellow-orange and red colors (that correspond to the populations of living, dying and dead cells, respectively) in the particular zones of V. faba roots. (B) quiescent center, (C) zone of cell division, i.e. root meristem, marked also as zone II, and (D) zone of elongation, marked as zone III.
Mentions: Double staining with acridine orange (AO) and ethidium bromide (EB) is another method used to specify the type of cell death, as well as being a useful tool in detecting and quantifying the states living, dying and dead (Fig 4, Fig 5 and S3 Fig). Fluorescence microscopy revealed images indicating the occurrence of living cells (fluoresce green), dying cells (green-yellow, yellow, yellow-orange and bright orange) and dead cells (dark orange and bright red) in the control (Fig 4A), after both HU treatment (Fig 4B) and after PCC induction (Fig 4C). The diagram presenting the color spectrum resulting from the quantitative measurements of fluorescence intensity of nuclear chromatin stained with AO/EB was made in order to determine the degree of DNA damage in the control nuclei (Fig 4A') as well as in the nuclei derived from stressed roots of V. faba (Fig 4B and 4C'). The highest number of dead cells (13.4%) was observed in HU/CF treated material (Fig 4C''). Thus, it was shown that the number of dead cells after PCC induction was over 6-fold higher compared to the control and 4.5-fold higher, compared to the HU series (Fig 4A'', 4B'' and 4C''). The fractions of living, dying and dead cells in the control, compared with the HU series were similar; we only observed an increase in the number of dead cells in the latter (1.5-fold; Fig 4A'' and 4B''). Further analysis consisted in comparing the numbers of living cells or being at various cell death stages (early-to-late) in particular zones of V. faba roots [from 'quiescent center' through the zones of cell division, i.e. root meristem (zone marked as II in Fig 5A) up to the zone of elongation (marked as III in Fig 5A)]. Our results suggest that the increase in the number of dead cells in the meristematic zone of V. faba roots after HU/CF treatment was statistically significant (p < 0.01) in relation to both the control and HU-treated series (Fig 5C). In the meristematic zone (II) the relatively high percentage (Fig 5C) populations of dying (Fig 5C, yellow-colored columns) and dead cells (i.e. 19.9%, 13.4%, and 28.8% in the control, HU-treated, and PCC-induced series, respectively) resulted from some imperfection in the method of intravital AO/EB staining, and from the fact it is not possible to omit from the calculation the population of rhizodermis cells in which the occurrence of the PCD phenomenon is typical (comp. Fig 5Aa, Fig 5Ab, Fig 5Ac, Fig 5Ad and Fig 5C; this could be the reason that no significant differences were observed herein). In the other zones of V. faba roots induced to PCC, the number of dead cells was about 1.5-fold higher than in the control and almost 2-fold higher than in the HU-treated material (Fig 5A and 5B). In turn, in the elongation zone (III, over-meristematic, Fig 5A), the highest index of dead cells was observed under the influence of HU/CF (7.7%, i.e. 1.5-fold and 3.4-fold higher than in the control and HU-series, respectively; Fig 5D). However, it seems more interesting to compare the numbers of dead cells in the meristematic zone than in the elongation zone; in the latter it was lower, where in the control, the HU and the HU/CF series, decreases of 4, 6 and 3.7 times were observed, respectively (comp. Fig 5C and 5D). S3 Fig shows longitudinal intersections of the three most representative roots stained with the intravital AO/EB method, as well as shows the proportions of living (green), dying (range: yellow-to-orange) and dead (red) cells, and their distribution in the meristem, supra-meristematic zone and in the rhizodermis. In all zones (particularly in the area of a root cap) and in all experimental series we observed red fluorescence indicating PCD processes eliminating cells, particularly from the rhizodermis, present in the external layers of the root (S3 Fig). The arrows on S3b, S3b' and S3b'' Figs show a distinct widening of the HU-treated roots forming an easily visible protuberance in which the accumulation of dead cells can be observed. These protruberances may result from the appearance of aerenchymatic-like spaces in the root cortex cells of V. faba (comp. [8]).

Bottom Line: We have demonstrated that the activation of apoptosis-like programmed cell death (AL-PCD) was a secondary result of caffeine (CF) induced premature chromosome condensation (PCC) in hydroxyurea-synchronized Vicia faba root meristem cells.In addition, the results obtained under transmission electron microscopy (TEM) further revealed apoptotic-like features at the ultrastructural level of PCC-type cells: (i) extensive vacuolization; (ii) abnormal chromatin condensation, its marginalization and concomitant degradation; (iii) formation of autophagy-like vesicles (iv) protoplast shrinkage (v) fragmentation of cell nuclei and (vi) extensive degeneration of the cells.The results obtained have been discussed with respect to the vacuolar/autolytic type of plant-specific AL-PCD.

View Article: PubMed Central - PubMed

Affiliation: Department of Cytophysiology, Institute of Experimental Biology, Faculty of Biology and Environmental Protection, University of Łódź, Łódź, Poland.

ABSTRACT
We have demonstrated that the activation of apoptosis-like programmed cell death (AL-PCD) was a secondary result of caffeine (CF) induced premature chromosome condensation (PCC) in hydroxyurea-synchronized Vicia faba root meristem cells. Initiation of the apoptotic-like cell degradation pathway seemed to be the result of DNA damage generated by treatment with hydroxyurea (HU) [double-stranded breaks (DSBs) mostly] and co-treatment with HU/CF [single-stranded breaks (SSBs) mainly]. A single chromosome comet assay was successfully used to study different types of DNA damage (neutral variant-DSBs versus alkaline-DSBs or SSBs). The immunocytochemical detection of H2AXS139Ph and PARP-2 were used as markers for DSBs and SSBs, respectively. Acridine orange and ethidium bromide (AO/EB) were applied for quantitative immunofluorescence measurements of dead, dying and living cells. Apoptotic-type DNA fragmentation and positive TUNEL reaction finally proved that CF triggers AL-PCD in stressed V. faba root meristem cells. In addition, the results obtained under transmission electron microscopy (TEM) further revealed apoptotic-like features at the ultrastructural level of PCC-type cells: (i) extensive vacuolization; (ii) abnormal chromatin condensation, its marginalization and concomitant degradation; (iii) formation of autophagy-like vesicles (iv) protoplast shrinkage (v) fragmentation of cell nuclei and (vi) extensive degeneration of the cells. The results obtained have been discussed with respect to the vacuolar/autolytic type of plant-specific AL-PCD.

No MeSH data available.


Related in: MedlinePlus