Chloroplast-dependent programmed cell death is activated in Arabidopsis cell cultures after singlet oxygen production by Rose Bengal

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 E m2 s1) 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 M 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 M 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 M RB, but not with the other elicitor nor with 500 M 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 M RB and 500 M 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 M RB and 500 M 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 M 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 M 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.

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Gutiérrez J, González-Pérez S, García-García F, Daly CT, Lorenzo O, Revuelta JL, McCabe PF, Arellano JB