ORIGINAL ARTICLE Francesco Carimi Æ Michela Zottini Æ Elide Formentin Mario Terzi Æ Fiorella Lo Schiavo Cytokinins: new apoptotic inducers in plants Received: 25 February 2002 / Accepted: 17 June 2002 / Published online: 17 September 2002 Ó Springer-Verlag 2002 Abstract High concentrations of cytokinins block cell proliferation and induce programmed cell death (PCD) in both carrot (Daucus carota L.) and Arabidopsis thaliana (L.) Heynh. cell cultures [13 and 27 lMN 6 - benzylaminopurine (BAP), respectively]. In the present work, cell death was scored by Evan’s blue staining and was also demonstrated to be programmed by various parameters, including chromatin condensation, oligo- nucleosomal DNA degradation (laddering), and release of cytochrome c from mitochondria. In carrot cells, this induction takes approximately 24 h, with proliferating cells being more sensitive than quiescent ones. Two hormones, namely abscisic acid and 2,4-dichlorophen- oxyacetic acid (2,4-D), protect cells against the cytoki- nin-induced death. PCD is not merely a consequence of the inability of the culture to proliferate, since high levels of 2,4-D block carrot cell proliferation without pro- moting PCD. Increased ethylene production was also observed in BAP-treated cultures, although this increase was not responsible for PCD because inhibitors of eth- ylene synthesis and action did not block PCD in BAP- treated cultures. Programmed cell death in the form of DNA laddering was also seen in plants treated with cytokinins. This process was accompanied by acceler- ated senescence in the form of leaf yellowing. Keywords Abscisic acid Æ Arabidopsis Æ Daucus Æ Growth regulators Æ Programmed cell death Æ Senescence Abbreviations ABA: abscisic acid Æ ACC: 1-amino- cyclopropane-1-carboxylic acid Æ AVG: aminoethoxy- vinylglycine Æ BAP: N 6 -benzylaminopurine Æ 2,4-D: 2,4-dichlorophenoxyacetic acid Æ DAPI: 4,6-diamino-2- phenylindole Æ NBD: 2,5-norbornadiene Æ PCD: programmed cell death Introduction Cytokinin, a phytohormone that controls plant growth and development, was first identified as a factor that induces cell division in the presence of auxin (Miller et al. 1955). Afterwards, Skoog and Miller (1957) discovered that cytokinins act antagonistically to auxins and de- termine cell fate by promoting shoot and root differen- tiation in callus cultures. In addition to controlling cell fate, cytokinins play a central role in the regulation of cell division (Frank and Schmu¨lling 1999) and also control the metabolism of various nutrients (Kakimoto 1998). Cytokinins have a delaying effect on leaf senes- cence (Richmond and Lang 1957; Gan and Amasino 1995) and their levels have been observed to decline in senescing leaf tissues (Sing et al. 1992). Plant development is modulated by hormonal inter- actions. In fact, ethylene and abscisic acid (ABA) will usually induce the early onset of senescence, in contrast to the delaying effect of cytokinins (Bleeker and Patt- erson 1997). Recent research continues to uncover novel interactions between these crucial regulatory compounds and in particular, recent papers describe a new kind of cross-talk between cytokinin and ethylene. Low con- centrations of cytokinin [N 6 -benzyladenine (BAP) 0.5–10 lM] elevate ethylene biosynthesis in etiolated Arabidopsis seedlings to a level that is sufficient to induce a triple response (Vogel et al. 1998a). The lack of this triple response was used in selecting for Arabidopsis mutants defective in ethylene biosynthesis (Vogel et al. 1998b). Programmed cell death (PCD) is a genetically programmed process in which a cell guides its own Planta (2003) 216: 413–421 DOI 10.1007/s00425-002-0862-x F. Carimi (&) Centro di Studio delle Biomembrane, CNR, Via G. Colombo 3, 35131 Padova, Italy E-mail: carimi@civ.bio.unipd.it Fax: +39-49-8276300 M. Zottini Æ E. Formentin Æ M. Terzi Æ F. Lo Schiavo Dipartimento di Biologia, Universita` degli Studi di Padova, Via G. Colombo 3, 35121 Padova, Italy