# 2003 Kluwer Academic Publishers. Printed in the Netherlands. Effects of salicylic acid and cold on freezing tolerance in winter wheat leaves Esen Tas° gı ´n 1 ,O ¨ kkes° Atı ´cı ´ 2 and Barbaros Nalbantog ˘lu 1, * 1 Department of Chemistry and 2 Department of Biology, Faculty of Science and Arts, Atat€ urk University, Erzurum, Turkey; *Author for correspondence (e-mail: barbarosn@yahoo.com; phone: +442 23 14 440; fax: +442 23 60 948) Received 7 January 2003; accepted in revised form 18 July 2003 Key words: Apoplastic protein, Cold, Freezing injury, Ice nucleation activity, Salicylic acid Abstract The effects of salicylic acid (SA) (0.01, 0.1 and 1 mM) and cold on freezing tolerance (freezing injury and ice nucleation activity) were investigated in winter wheat (Triticum aestivum cv. Dogu-88) grown under control (20/18  C for 15, 30 and 45-day) and cold (15/10  C for 15-day, 10/5  C for 30-day and 5/3  C for 45-day) conditions. Cold acclimatisation caused a decrease of injury to leaf segments removed from the plants and subjected to freezing conditions. Exogenous SA also decreased freezing injury in the leaves grown under cold (15/10  C) and control (15 and 30-day) conditions. Cold conditions (10/5 and 5/3  C) caused an increase in ice nucleation activity by apoplastic proteins, which were isolated from the leaves. For the first time, it was shown that exogenous SA caused an increase in ice nucleation activity under cold (15/10 and 10/5  C) and control conditions. These results show that salicylic acid can increase freezing tolerance in winter wheat leaves by affecting apoplastic proteins. Introduction The stresses imposed by temperature have impor- tant implications for agriculture. Plants differ in their resistance to chilling and freezing tempera- ture. Freezing is lethal to most cellular organisms. Dehydration of the intracellular environment and physical damage by ice crystals are major causes for freezing injury and death (Lewitt 1980). Freezing tolerance (lower freezing injury in leaves and higher ice nucleation activity of apoplastic proteins in leaves) is due to the capacity to avoid intracellular ice formation and to withstand extra- cellular ice formation. Freezing-tolerant plants exhibit injury only at temperatures lower than the temperature at which extracellular ice formation begins (Antikainen 1996). Plants produce several compounds to protect cells against fatal intracellu- lar and intercellular ice formation. Many overwin- tering plants accumulate sugars, amino acids and antifreeze compounds including antifreeze proteins in the apoplastic region (Griffith et al. 1997; Atı ´cı ´ and Nalbantog ˘lu 1999a, b; Ewart et al. 1999; Hoshino et al. 1999; Yu et al. 2001). Salicylic acid (SA) is synthesised endogenously, playing an essential role in thermogenesis and in the activation of certain plant defense responses including pathogenesis-related gene expression (Gaffney et al. 1993; Klessing and Malamy 1994). A role for SA in plant growth and development, flowering, ion uptake, stomatal regulation and photosythesis has been investigated (Pancheva et al. 1996; Popova et al. 1997; Uzunova and Popova 2000). Only a few recent studies have reported the effects of SA on freezing injury. These studies demonstrated that SA and methyl salicylate (MeSA) treatments increased resistance to chilling injury in maize plants (Janda et al. 1999), tomato fruit (Ding et al. 2002) and banana seed- lings (Kang et al. 2003). 231 Plant Growth Regulation 231–236, 2003. 41: