Activation of caspase-like proteases and induction of apoptosis by isopentenyladenosine in tobacco BY-2 cells

Abstract

The effects of isopentenyladenosine (iPA) on tobacco (Nicotiana tabacum L.) BY-2 cells were examined. The number of BY-2 cells decreased in a time- and concentration-dependent manner after being exposed to micromolar concentrations of iPA. This decrease was mainly due to a loss of cell viability, since no substantial changes in cell cycle progression were revealed by flow-cytometric analysis. Dying cells exhibited the typical morphological and biochemical hallmarks of apoptosis, including cell shrinkage, chromatin condensation, and degradation of nuclear DNA to nucleosomal size fragments. Caspase-1-like and caspase-3-like proteases also became activated, the former being dominant. Inhibitor-sensitivity studies revealed that although synthetic caspase inhibitors failed to prevent cell death they markedly reduced cell death in tobacco BY-2 cells, Nu-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, a specific inhibitor for caspase-1, being the most effective. Our results indicate that caspase-like proteases, and particularly caspase-1-like protease, might be critically implicated in iPA-induced apoptosis of BY-2 cells. Finally, the outcome of inhibiting adenosine kinase by 4-amino-3-iodo-1(beta- D-ribofuranosyl)pyrazolo[3,4-d]-pyrimidine revealed that intracellular phosphorylation of iPA is required for its cytotoxicity to develop.

Purification and Characterization of Serine Proteases That Exhibit Caspase-Like Activity and Are Associated with Programmed Cell Death in Avena sativa

“…Other forms of PCD in N. tabacum, including those induced by bacteria (Richael et al, 2001) and treatment with a fungal elicitor (Elbaz et al, 2002), also were prevented by caspase inhibitors. Similar results were obtained with caspase inhibitors in preventing or reducing cell death after treatment with camptothecin (De Jong et al, 2000), staurosporine (Elbaz et al, 2002), and isopentenyladenosine (Mlejnek and Prochá zka, 2002). In addition, heat shock-induced PCD in N. tabacum suspension cells stimulated cleavage of PARP and activation of a caspase-3-like protease (Tian et al, 2000), whereas N. tabacum protoplast treated with menadione displayed PARP cleavage (Sun et al, 1999).…”

“…Because caspases can play such a critical role in the process and because numerous experiments have shown that caspase-specific inhibitors prevent Silver stained gel of purified SAS-1 and SAS-2; 15 mL of active fraction was loaded per lane. some forms of PCD in plants (del Pozo and Lam, 1998;Sun et al, 1999;Tian et al, 2000;Richael et al, 2001;Elbaz et al, 2002;Mlejnek and Prochá zka, 2002), it has been speculated that proteases similar to caspases are involved in plant PCD. We have purified and characterized two subtilisin-like Ser proteases that possess specificity for caspase-specific substrates and inhibitors and apparently participate in a signaling cascade involving multiple proteases, leading to the proteolysis of Rubisco.…”

High levels of the cytokinin BAP suppress programmed cell death in hybrid tobacco cells (Nicotiana suaveolens*N. tabacum) expressing hybrid lethality

“…However, leaves of cytokinindeficient engineered transgenic Arabidopsis plants show a delay of senescence, rather than an acceleration (Werner et al 2001, Werner et al 2003. On the other hand, cytokinins reportedly induce PCD in plants, including tobacco (Mlejnek and Prochazka 2002), carrot and Arabidopsis (Carimi et al 2003). Additionally, Carimi et al (2004) reported that in Arabidopsis cell suspension cultures, high levels of BAP induce features of PCD by accelerating senescence and in carrot cells, high levels of BAP and carbanilide, another cytokinin, induce some features of PCD, but kinetin and zeatin, which are also cytokinins, do not.…”

Information processing without brains – the power of intercellular regulators in plants

“…Salicylic acid, a hormone involved in the pathogen response, also modifies the auxin response . Abnormal cytokinin levels are equally monitored by plants, and unusually high concentrations of cytokinin can induce programmed cell death (Carimi et al, 2003;Mlejnek and Prochazka, 2002) and the expression of the pathogen resistance gene PR1 (Memelink et al, 1987).…”