Project description:Arabidopsis thaliana cell suspension cultures (ACSC) were subjected to 30-min, mild chemical treatments with three different singlet oxygen elicitors at low-medium light conditions (150 µE m–2 s–1) with the aim of getting a better understanding of singlet oxygen-mediated defence responses in plants. The three elicitors Indigo Carmine (IC), Methylene Violet (MV) and Rose Bengal (RB) at a concentration of 0.5 µM were chosen because they exhibited different abilities to permeate the plasma membrane and to accumulate in the cell soma or organelles such as chloroplasts. In addition, ACSC were treated with 500 µM H2O2 for comparison. Confocal image analysis of Arabidopsis cells revealed that IC was not retained in cells, whereas MV and RB permeated the plasma membrane and accumulated in the chloroplast envelope and inside chloroplasts, respectively. As a consequence of their different cellular location, the physiological, transcriptional and photosynthetic responses of Arabidopsis cells to singlet oxygen production varied from each other and the activation of programmed cell death (PCD) was observed in ACSC treated with 0.5 µM RB, but not with the other elicitor nor with 500 µM H2O2. The role of chloroplasts in the activation of PCD was further investigated when this physiological response was analyzed in dark-grown cell cultures containing undifferentiated plastids. Interestingly, PCD was only activated in light-grown, but not in dark-grown, Arabidopsis cell cultures, suggesting that singlet oxygen-mediated defence responses were initiated inside chloroplasts. Genome-wide transcriptional profile analyses were performed as well and the results proved that there were only statistically significant changes in the transcript expression of light-grown ACSC treated with 0.5 µM RB and 500 µM H2O2, but not with IC nor with MV. Functional enrichment analyses revealed that GO/Biological process terms associated with defence responses were common in the treatments with 0.5 µM RB and 500 µM H2O2; however, resistance response to pathogen and PCD terms were only significantly over-represented in the RB treatment. Moreover, the analysis of the up-regulated transcripts in ACSC treated with 0.5 µM RB brought out that both specific markers for singlet oxygen from the conditional fluorescence (flu) mutant of Arabidopsis and transcripts with a key role in hormone-activated PCD (i.e. ethylene and jasmonic acid) were present, although there was no evidence for the up-regulation of EDS1 encoding the ENHANCED DISEASE SUSCEPTIBILITY PROTEIN 1. Finally, a co-regulation analysis proved that ACSC treated with 0.5 µM RB exhibited higher correlation with the flu family mutants than with other singlet oxygen producer mutants of Arabidopsis or wild-type plants of Arabidopsis subjected to high light treatments, where singlet oxygen was produced in photosystem II and an acclimatory response was activated instead of PCD.

Project description:Arabidopsis thaliana cell suspension cultures (ACSC) were subjected to 30-min, mild chemical treatments with three different singlet oxygen elicitors at low-medium light conditions (150 µE m–2 s–1) with the aim of getting a better understanding of singlet oxygen-mediated defence responses in plants. The three elicitors Indigo Carmine (IC), Methylene Violet (MV) and Rose Bengal (RB) at a concentration of 0.5 µM were chosen because they exhibited different abilities to permeate the plasma membrane and to accumulate in the cell soma or organelles such as chloroplasts. In addition, ACSC were treated with 500 µM H2O2 for comparison. Confocal image analysis of Arabidopsis cells revealed that IC was not retained in cells, whereas MV and RB permeated the plasma membrane and accumulated in the chloroplast envelope and inside chloroplasts, respectively. As a consequence of their different cellular location, the physiological, transcriptional and photosynthetic responses of Arabidopsis cells to singlet oxygen production varied from each other and the activation of programmed cell death (PCD) was observed in ACSC treated with 0.5 µM RB, but not with the other elicitor nor with 500 µM H2O2. The role of chloroplasts in the activation of PCD was further investigated when this physiological response was analyzed in dark-grown cell cultures containing undifferentiated plastids. Interestingly, PCD was only activated in light-grown, but not in dark-grown, Arabidopsis cell cultures, suggesting that singlet oxygen-mediated defence responses were initiated inside chloroplasts. Genome-wide transcriptional profile analyses were performed as well and the results proved that there were only statistically significant changes in the transcript expression of light-grown ACSC treated with 0.5 µM RB and 500 µM H2O2, but not with IC nor with MV. Functional enrichment analyses revealed that GO/Biological process terms associated with defence responses were common in the treatments with 0.5 µM RB and 500 µM H2O2; however, resistance response to pathogen and PCD terms were only significantly over-represented in the RB treatment. Moreover, the analysis of the up-regulated transcripts in ACSC treated with 0.5 µM RB brought out that both specific markers for singlet oxygen from the conditional fluorescence (flu) mutant of Arabidopsis and transcripts with a key role in hormone-activated PCD (i.e. ethylene and jasmonic acid) were present, although there was no evidence for the up-regulation of EDS1 encoding the ENHANCED DISEASE SUSCEPTIBILITY PROTEIN 1. Finally, a co-regulation analysis proved that ACSC treated with 0.5 µM RB exhibited higher correlation with the flu family mutants than with other singlet oxygen producer mutants of Arabidopsis or wild-type plants of Arabidopsis subjected to high light treatments, where singlet oxygen was produced in photosystem II and an acclimatory response was activated instead of PCD. 18 Samples total. Six Samples are controls, 3 Samples each treated with Rose Bengal, Hydrogen Peroxide, Methyl Violet, and Indigo Carmine.

Project description:BACKGROUND:Thaxtomin A (TA) is a natural cellulose biosynthesis inhibitor (CBI) synthesized by the potato common scab-causing pathogen Streptomyces scabies. Inhibition of cellulose synthesis by TA compromises cell wall organization and integrity, leading to the induction of an atypical program of cell death (PCD). These processes may facilitate S. scabies entry into plant tissues. To study the mechanisms that regulate the induction of cell death in response to inhibition of cellulose synthesis, we used Arabidopsis thaliana cell suspension cultures treated with two structurally different CBIs, TA and the herbicide isoxaben (IXB). RESULTS:The induction of cell death by TA and IXB was abrogated following pretreatment with the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) and the natural auxin indole-3-acetic acid (IAA). The addition of auxin efflux inhibitors also inhibited the CBI-mediated induction of PCD. This effect may be due to intracellular accumulation of auxin. Auxin has a wide range of effects in plant cells, including a role in the control of cell wall composition and rigidity to facilitate cell elongation. Using Atomic Force Microscopy (AFM)-based force spectroscopy, we found that inhibition of cellulose synthesis by TA and IXB in suspension-cultured cells decreased cell wall stiffness to a level slightly different than that caused by auxin. However, the cell wall stiffness in cells pretreated with auxin prior to CBI treatment was equivalent to that of cells treated with auxin only. CONCLUSIONS:Addition of auxin to Arabidopsis cell suspension cultures prevented the TA- and IXB-mediated induction of cell death. Cell survival was also stimulated by inhibition of polar auxin transport during CBI-treatment. Inhibition of cellulose synthesis perturbed cell wall mechanical properties of Arabidopsis cells. Auxin treatment alone or with CBI also decreased cell wall stiffness, showing that the mechanical properties of the cell wall perturbed by CBIs were not restored by auxin. However, since auxin's effects on the cell wall stiffness apparently overrode those induced by CBIs, we suggest that auxin may limit the impact of CBIs by restoring its own transport and/or by stabilizing the plasma membrane - cell wall - cytoskeleton continuum.

Project description:BackgroundChloroplasts respond to stress and changes in the environment by producing reactive oxygen species (ROS) that have specific signaling abilities. The ROS singlet oxygen (1O2) is unique in that it can signal to initiate cellular degradation including the selective degradation of damaged chloroplasts. This chloroplast quality control pathway can be monitored in the Arabidopsis thaliana mutant plastid ferrochelatase two (fc2) that conditionally accumulates chloroplast 1O2 under diurnal light cycling conditions leading to rapid chloroplast degradation and eventual cell death. The cellular machinery involved in such degradation, however, remains unknown. Recently, it was demonstrated that whole damaged chloroplasts can be transported to the central vacuole via a process requiring autophagosomes and core components of the autophagy machinery. The relationship between this process, referred to as chlorophagy, and the degradation of 1O2-stressed chloroplasts and cells has remained unexplored.ResultsTo further understand 1O2-induced cellular degradation and determine what role autophagy may play, the expression of autophagy-related genes was monitored in 1O2-stressed fc2 seedlings and found to be induced. Although autophagosomes were present in fc2 cells, they did not associate with chloroplasts during 1O2 stress. Mutations affecting the core autophagy machinery (atg5, atg7, and atg10) were unable to suppress 1O2-induced cell death or chloroplast protrusion into the central vacuole, suggesting autophagosome formation is dispensable for such 1O2-mediated cellular degradation. However, both atg5 and atg7 led to specific defects in chloroplast ultrastructure and photosynthetic efficiencies, suggesting core autophagy machinery is involved in protecting chloroplasts from photo-oxidative damage. Finally, genes predicted to be involved in microautophagy were shown to be induced in stressed fc2 seedlings, indicating a possible role for an alternate form of autophagy in the dismantling of 1O2-damaged chloroplasts.ConclusionsOur results support the hypothesis that 1O2-dependent cell death is independent from autophagosome formation, canonical autophagy, and chlorophagy. Furthermore, autophagosome-independent microautophagy may be involved in degrading 1O2-damaged chloroplasts. At the same time, canonical autophagy may still play a role in protecting chloroplasts from 1O2-induced photo-oxidative stress. Together, this suggests chloroplast function and degradation is a complex process utilizing multiple autophagy and degradation machineries, possibly depending on the type of stress or damage incurred.

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Does singlet oxygen activate cell death in Arabidopsis cell suspension cultures?: analysis of the early transcriptional defense responses to high light stress.

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Does singlet oxygen activate cell death in Arabidopsis cell suspension cultures?: analysis of the early transcriptional defense responses to high light stress.

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