Gibberellic Acid (GA 3 ) Inhibits ROS Increase in Chloroplasts During Dark-Induced Senescence of Pelargonium Cuttings Shilo Rosenwasser • Eduard Belausov • Joseph Riov • Vered Holdengreber • Haya Friedman Received: 6 July 2009 / Accepted: 20 January 2010 / Published online: 14 March 2010 Ó Springer Science+Business Media, LLC 2010 Abstract The temporal and spatial changes in reactive oxygen species (ROS) during dark treatment of Pelargo- nium cuttings and the effect of gibberellic acid (GA 3 ) on ROS levels were studied. ROS-related fluorescence was detected in mitochondria and cytoplasm of epidermal cells and in chloroplasts. By monitoring dichlorofluorescein (DCF) fluorescence, an initial decrease in ROS was observed under darkness in the epidermal cell cytoplasm and the chloroplasts, which was followed by an increase on the third day. Following 3 days under darkness, the size and the structure of the chloroplasts also changed, and they became more sensitive to illumination as judged by a higher accumulation of ROS. Pretreatment of leaves with GA 3 did not prevent the structural changes in the chloro- plasts, but it inhibited the increase in ROS levels in all cell compartments, including the chloroplasts. It is suggested that the inhibition of ROS increase by GA 3 prevented complete disintegration of chloroplasts during dark- induced senescence and thereby enabled the maintenance of chlorophyll levels in the tissue. Keywords Chloroplasts Á GA 3 Á H 2 DCF-DA Á Reactive oxygen species Á Senescence Introduction Dark-induced senescence occurs during storage and ship- ment of agricultural products, including Pelargonium cut- tings (Purer and Mayak 1989). This process shares some similarities with natural senescence, which suggests that dark-induced cell death is a consequence of an active program that involves the participation of signaling mole- cules, transcription factors, and catabolic enzymes (Kleber Janke and Krupinska 1997; He and others 2001; Buchanan- Wollaston and others 2003). Reactive oxygen species (ROS) can act as toxic mole- cules, but also as signaling molecules that activate cell death programs (Thompson and others 1987; Breusegem and Dat 2006; Zentgraf and Hemleben 2008). In several cell death programs, including those activated by patho- gens, ultraviolet light (UV), and ozone, ROS were hypothesized to initiate the program (Grant and Loake 2000; Langebartels and others 2002; Mittler 2002; Mittler and others 2004; Foyer and Noctor 2005). However, in other abiotic stresses and in senescence processes, the above role of ROS has not been established. Nevertheless, the role of ROS in senescence has been investigated in Electronic supplementary material The online version of this article (doi:10.1007/s00344-010-9149-9) contains supplementary material, which is available to authorized users. S. Rosenwasser Á H. Friedman (&) Department of Postharvest Science of Fresh Produce, Agricultural Research Organization (ARO), The Volcani Center, P.O. Box 6, Bet Dagan 50250, Israel e-mail: hayafr@agri.gov.il E. Belausov Department of Plant Science, Agricultural Research Organization (ARO), The Volcani Center, P.O. Box 6, Bet Dagan 50250, Israel S. Rosenwasser Á J. Riov Institute of Plant Sciences and Genetics in Agriculture, The Robert H. Smith Faculty of Agriculture, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot, Israel V. Holdengreber Department of Plant Pathology and Weed Protection, Agricultural Research Organization (ARO), The Volcani Center, P.O. Box 6, Bet Dagan 50250, Israel 123 J Plant Growth Regul (2010) 29:375–384 DOI 10.1007/s00344-010-9149-9