Freezing behaviours in plant tissues as visualized by NMR microscopy and their regulatory mechanisms

Introduction Since plants are immobile, they have to survive the various stresses that they encounter. Freezing is one of the severest stresses as it causes ice formation, dehydration and cell deformation. As plant tissues contain a large amount of water, the behaviour of cell water at subfreezing temperatures is a key to tissue survival. To withstand freezing, cold-hardy plants have developed various mechanisms to regulate the formation of ice in their tissues, which result in diverse but tissue-and species-specific freezing behaviours. For instance, wintering temperate woody plant species display diverse freezing behaviours: bark tissues undergo extracellular freezing and xylem ray parenchyma cells of many woody species deep supercool while flower and leaf buds of several genera undergo extra-organ freezing (Fig. 1) (Sakai and Larcher 1987). Whilst most of the tissues of cold-hardy herbaceous plants undergo extracellular freezing, t hese freezing behaviours are species-and tissue-specific. T he elucidation of freezing behaviours in cold-hardy tissues aids the development of cryopreservation techniques. Traditionally, studies on freezing behaviours in plant tissues have been done using differential thermal analysis (DTA), nuclear magnetic resonance (NMR) spectrometry, scanning electron microscopy (SEM) and visual observation. However, these methods can only provide averaged information from a bulk sample or are destructive and liable to cause artifacts (Table 1). Recently, we have developed a novel non-invasive method to visualize the localization of unfrozen water in plant tissues at subfreezing temperatures using NMR microscopy (see Price 1998). We also considered the theoretical background for interpreting the images acquired at freezing temperatures (Ishikawa et al. 1997; Price et al. 1997b). Using this method, we could successfully analyze in detail the various freezing behaviours in cold-hardy woody plant tissues (Ishikawa et al. 1997; Price et al. 1997a; Ide et al. 1998). Here, we review the use of NMR microscopy for the study of freezing behaviours in plants by introducing typical examples. We also briefly review possible regulatory mechanisms involved in the freezing behaviours. Many of the plant tissues and cultures that are used for cryopreservation are non-cold-hardy or freezing-sensitive and have not evolved innate mechanisms to regulate freezing events. The behaviour of water in these tissues is also a key to the survival at liquid nitrogen temperature. Therefore, we have to mimic innate mechanisms either by using cryoprotectants to artificially provide freezing Fundamental aspects of cryopreservation 23 (A) Extracellular freezing (B) Extra-organ freezing (C) Deep supercooling Ice Dehydration Supercool Ice R Ice Ice …