Beyond oxidative stress: Tetrakis (4-hydroxyphenyl) porphyrin induces non-canonical programmed cell death in Tobacco BY-2 cells.

Photosensitizers are naturally occurring or synthetic light-absorbing pigments, widespread across the living kingdom, and central to biological processes such as photosynthesis (e.g., chlorophyll). Dark toxicity of photosensitizers remains largely unexplored, especially in plants. This study focuses on the neutral porphyrin tetrakis (4-hydroxyphenyl) porphyrin (THPP), tested in Tobacco Bright Yellow-2 (TBY-2) cells. THPP at 10 μM induced significant plant cell death in the dark, indicating toxicity. To understand the mechanism of this toxicity and cell death induced by THPP, THPP's self-assembly behavior in TBY-2 medium and its cellular localization were examined. After 3 h, THPP exhibited substantial stability in TBY-2 spent medium, partially explaining its dark toxicity. Incubation of cells with THPP at 4 °C limited its cellular penetration, indicating active internalization. Exposure to the THPP resulted in a significant reduction in cell viability, accompanied by decreased mitochondrial metabolic activity and dissipation of the mitochondrial membrane potential. Confocal imaging revealed a predominantly perinuclear localization of the THPP, and a diffuse cytoplasmic distribution and colocalization with mitochondria. Despite its pronounced cytotoxicity, THPP did not induce hallmarks typically associated with canonical PCD. Importantly, inhibition of autophagy with wortmannin markedly reduced THPP-induced cell death, suggesting that autophagy contributes to the THPP-induced cell death response.