Research article The thiol compounds glutathione and homoglutathione differentially affect cell development in alfalfa (Medicago sativa L.) Taras Pasternak a, b, * , Han Asard a , Geert Potters c, d , Marcel A.K. Jansen a, e a Department of Biology, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp, Belgium b Institute of Biology II/Molecular Plant Physiology, Albert-Ludwigs-Universität Freiburg, Schänzlestr.1, 79104 Freiburg, Germany c Department of Bioscience Engineering, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp, Belgium d Antwerp Maritime Academy, Noordkasteel Oost 6, B-2030 Antwerp, Belgium e School of Biological, Earth and Environmental Sciences, University College Cork, Distillery Field, North Mall, Cork, Ireland article info Article history: Received 20 August 2013 Accepted 22 October 2013 Available online 5 November 2013 Keywords: Glutathione Homoglutathione Medicago Alfalfa Cell differentiation Cytokinesis abstract Glutathione (GSH) is an important scavenger of Reactive Oxygen Species (ROS), precursor of metal chelating phytochelatins, xenobiotic defence compound and regulator of cell proliferation. Homo- glutathione (hGSH) is a GSH homologue that is present in several taxa in the family of Fabaceae. It is thought that hGSH performs many of the stress-defence roles typically ascribed to GSH, yet little is known about the potential involvement of hGSH in controlling cell proliferation. Here we show that hGSH/GSH ratios vary across organs and cells and that these changes in hGSH/GSH ratio occur during dedifferentiation and/or cell cycle activation events. The use of a GSH/hGSH biosynthesis inhibitor resulted in impaired cytokinesis in isolated protoplasts, showing the critical importance of these thiol- compounds for cell division. However, exposure of isolated protoplasts to exogenous GSH accelerated cytokinesis, while exogenous hGSH was found to inhibit the same process. We conclude that GSH and hGSH have distinct functional roles in cell cycle regulation in Medicago sativa L. GSH is associated with meristemic cells, and promotes cell cycle activation and induction of somatic embryogenesis, while hGSH is associated with differentiated cells and embryo proliferation. Ó 2013 Elsevier Masson SAS. All rights reserved. 1. Introduction The last decade has witnessed significant progress in our un- derstanding of the molecular mechanism of plant cell reactivation, division and differentiation. Different components of the cell cycle control machinery have been identified, their genes are sequenced, proteins purified, and antibodies have been raised (De Veylder et al., 2011). In contrast, our understanding of integration of cell cycle regulatory processes with organismal metabolism remains patchy. Best documented is the integration of the cell cycle with hormone homeostasis. Hormones such as auxin and cytokinin are capable of pushing cells towards a particular physiological fate (Dhonukshe et al., 2008; Haecker et al., 2004; Pasternak et al., 2000). The molecular connections that link cell cycle controls and organismal hormone balances are also increasingly being uncov- ered (Dudits et al., 2011; Domagalska & Leyser, 2011; Vanstraelen & Benková, 2012). In addition to, and in synergy with, hormones, oxidative stress plays a part in the regulation of cell cycle progression and plant growth (Kovtun et al., 2000; Wang et al., 2011; Zhao et al., 2011). Reichheld et al. (1999) proposed the existence of an oxidative stress-defined checkpoint in cell development. The integration of oxidative stress metabolism and the cell cycle would avoid unde- sirable expenditure of energy by balancing oxidative stress defence against division, or differentiation. Some stressors have been documented to delay or block cell cycle activity (for reviews see (Mittler et al., 2011)). Cultured, isolated leaf cells of Medicago sativa L. (alfalfa) have been extensively used to investigate the interplay between oxida- tive stress and cell development (Fehér et al., 2003). This system has been used to show how environmental parameters drive the development of alfalfa leaf-derived cells into microcalli or somatic embryos (Pasternak et al., 2002). Stressors such as menadione, Abbreviations: ASC, ascorbate; BSO, L-buthionine sulfoximine; DAPI, 4 0 ,6- diamidino-2-phenylindole; GSH, glutathione; hGSH, homoglutathione; 2,4-D, 2,4- dichlorophenoxyacetic acid; GSHS, glutathione synthetase; hGSHS, homo- glutathione synthetase; RT-PCR, reverse transcriptase polymerase chain reaction. * Corresponding author. Institute of Biology II/Molecular Plant Physiology, Albert- Ludwigs-Universität Freiburg, Schänzlestr.1, 79104 Freiburg, Germany. Tel.: þ49 (0) 761 203 2960; fax: þ49 (0)178 274 9669. E-mail addresses: taras.pasternak@biologie.uni-freiburg.de (T. Pasternak), han. asard@ua.ac.be (H. Asard), geert.potters@hzs.be (G. Potters), m.jansen@ucc.ie (M. A.K. Jansen). Contents lists available at ScienceDirect Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy 0981-9428/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.plaphy.2013.10.028 Plant Physiology and Biochemistry 74 (2014) 16e23