Early activation of lipoxygenase in lentil (Lens culinaris) root protoplasts by oxidative stress induces programmed cell death

Abstract

Oxidative stress caused by hydrogen peroxide (H 2 O 2 ) triggers the hypersensitive response of plants to pathogens. Here, short pulses of H 2 O 2 are shown to cause death of lentil (Lens culinaris) root protoplasts. Dead cells showed DNA fragmentation and ladder formation, typical hallmarks of apoptosis (programmed cell death). DNA damage was evident 12 h after the H 2 O 2 pulse and reached a maximum 12 h later. The commitment of cells to apoptosis caused by H 2 O 2 was characterized by an early increase of lipoxygenase activity, of ultraweak luminescence and of membrane lipid peroxidation, which reached 720, 350 and 300% of controls, respectively, at 6 h after H 2 O 2 treatment. Increased lipoxygenase activity was paralleled by an increase of its protein and mRNA level. Lipoxygenase inhibitors nordihydroguaiaretic acid, eicosatetraynoic acid and plamitoyl ascorbate prevented H 2 O 2 -induced DNA fragmentation and ultraweak luminescence, only when added together with H 2 O 2 , but not when added 8 h afterwards. Inhibitory anti-lipoxygenase monoclonal antibodies, introduced into the protoplasts by electroporation, protected cells against H 2 O 2 -induced apoptosis. On the other hand, lentil lipoxygenase products 9-and 13-hydroperoxy-octadecadienoic acids and their reduced alcohol derivatives were able to force the protoplasts into apoptosis. Altogether, these findings suggest that early activation of lipoxygenase is a key element in the execution of apoptosis induced by oxidative stress in plant cells, in a way surprisingly similar to that observed in animal cells.

Lipoxygenases and their involvement in programmed cell death

“…The increase of lipoxygenase was paralleled by an enhancement of ultraweak luminescence, a marker of LOX-related membrane lipid peroxidation. 20 These findings suggest that lipoxygenase activation might contribute to membrane peroxidation during hydrogen peroxide-induced CPD, resembling previous observations on animal cells. 15 NDGA protected LRP against H 2 O 2 -induced DNA fragmentation and ultraweak luminescence.…”

“…20 Also 9-HOD and 13-HOD, the reduced forms of the LRP LOX products, were able to induce cell death and DNA fragmentation to almost the same extent as the corresponding HPODs, again matching previous observations on human cells. 20 Since similar findings were reported in H 2 O 2 -induced PCD of human cells, these observations suggest that animals and plants share a common signal transduction pathway triggering apoptosis after oxidative stress.…”

“…However, in view of the lack of specificity of this LOX inhibitor, we also used inhibitory anti-LOX monoclonal antibodies, able to block LOX activity in LRP without affecting the redox state of the cell. 20 These antibodies, too, protected LRP against H 2 O 2 -induced PCD, whilst the specific products of LRP lipoxygenase, 9-HPOD and 13-HPOD, were able per se to induce DNA fragmentation. 20 Also 9-HOD and 13-HOD, the reduced forms of the LRP LOX products, were able to induce cell death and DNA fragmentation to almost the same extent as the corresponding HPODs, again matching previous observations on human cells.…”

Plant Lipoxygenases. Physiological and Molecular Features

“…Moreover, 9-and 13-HPOT (defined in Fig. 1) cause cell death in this system (Maccarrone et al, 2000).…”

Biochemical Constituents Imparting Resistance to Sucking Pest Aphid in Cotton (Gossypium spp.)

“…(Croft et al, 1993). Increased LOX activity may also cause irreversible membrane damage, which would lead to the leakage of cellular contents (Mac Carrone et al, 2000).…”