**3.1 Proline accumulation in higher plants**

Stress has been shown to induce proline accumulation in a wide range of organisms including eubacteria, protozoa, invertebrates and plants (Verbruggen & Hermans, 2008; Kostal et al., 2011) and proline accumulation is believed to be very important as part of the physiological adaptation of plants to stress. In plants a wide range of abiotic and biotic stressors have been shown to induce proline accumulation including, salt, drought, high temperatures, low temperatures, heavy metals, anaerobiosis, nutrient deficiency, organic pollutants, ultraviolet (UV) radiation and pathogen infection (Chu et al., 1978; Alia & Saradhi, 1991; Saradhi et al., 1995; Hare et al., 1999; Siripornadulsil et al., 2002). The level of proline that accumulates in plants in response to stress varies greatly and is highly dependent on the plant species, with increase of up to 100 fold compared to controls reported in the literature (Verbruggen & Hermans, 2008).

With respect to cryopreservation, numerous studies have demonstrated the importance of proline for plant cold tolerance (Swaaij, Jacobsen & Feenstra 1985, Swaaij et al. 1986; Duncan & Widholm 1987; Ait-Barka & Audran 1997; Hoffman et al., 2010; Javadian et al., 2010; Burbulis et al., 2011). Studies on plants relatively insensitive to chilling, such as barley (Chu

Fig. 2. Proposed model for proline metabolism in higher plants (adapted from Lehmann et al. 2010). Glu glutamate, Orn ornithine, P5C pyrroline-5-carboxylate, GSA glutamic- semialdehyde, KG -ketoglutarate. P5CS P5C synthetase, P5CR P5C reductase, PDH proline

Stress has been shown to induce proline accumulation in a wide range of organisms including eubacteria, protozoa, invertebrates and plants (Verbruggen & Hermans, 2008; Kostal et al., 2011) and proline accumulation is believed to be very important as part of the physiological adaptation of plants to stress. In plants a wide range of abiotic and biotic stressors have been shown to induce proline accumulation including, salt, drought, high temperatures, low temperatures, heavy metals, anaerobiosis, nutrient deficiency, organic pollutants, ultraviolet (UV) radiation and pathogen infection (Chu et al., 1978; Alia & Saradhi, 1991; Saradhi et al., 1995; Hare et al., 1999; Siripornadulsil et al., 2002). The level of proline that accumulates in plants in response to stress varies greatly and is highly dependent on the plant species, with increase of up to 100 fold compared to controls

With respect to cryopreservation, numerous studies have demonstrated the importance of proline for plant cold tolerance (Swaaij, Jacobsen & Feenstra 1985, Swaaij et al. 1986; Duncan & Widholm 1987; Ait-Barka & Audran 1997; Hoffman et al., 2010; Javadian et al., 2010; Burbulis et al., 2011). Studies on plants relatively insensitive to chilling, such as barley (Chu

dehydrogenase, P5CDH P5C dehydrogenase, OAT ornithine-daminotransferase.

Transporters and potential transporters are shown as grey circles.

reported in the literature (Verbruggen & Hermans, 2008).

**3. Proline in plant cells under stress 3.1 Proline accumulation in higher plants**  et al. 1978), rye (Koster & Lynch 1992), winter wheat (Dorffling et al. 1997), and *Arabidopsis thaliana* (Xin & Browse 1998; Nanjo et al. 1999) have demonstrated significant positive correlations between cellular proline accumulation and improved cold tolerance.

In addition, plant cells under dehydrating conditions, which are often a consequnce of cryopreservation, undergo osmotic adjustment by accumulating one or several low molecular weight organic solutes, which are often referred to as compatible osmolytes and/or osmoprotectants. These molecules play a critical role in counteracting the effect of osmotic stress in plants at the cellular level (Yoshiba et al., 1997). In plants under dehydrating conditions such as drought or high salinity, proline is one of the most common compatible osmolytes and while several amino acids are known to accumulate in response to osmotic stress, proline appears to be the preferred organic osmoticum in many plants and may have a specific protective role in the adaptation of plant cells to dehydration. For example, in a study of *Triticum aestivum* L. (durum wheat) under salinity stress, Poustini et al. (2007) found a positive correlation between proline levels and osmotic potential, and concluded that proline is an important osmolyte for osmotic adjustment in wheat under water stress. In addition, it has been demonstrated that transgenic tobacco plants with elevated levels of proline biosynthesis show increased tolerance to hyperosmotic stress (Kavi Kishot et. al., 1995), providing further evidence of a cause-and-effect relationship between proline levels and osmotic tolerance. Proline normally accumulates in the cytosol, where it contributes to the cytoplasmic osmotic adjustment in response to water loss without interfering with normal cellular processes and biochemical reactions (Ashraf & Foolad, 2007).

#### **3.2 Proline and cryopreservation**

During cryopreservation, plant cells encounter similar problems to those they encounter under freezing conditions in the field. They under go changes in the spatial organization of biological membranes, biochemical and chemical reactions can be retarded, and the status and availability of water can be altered. For these reasons proline is likely to be an effective cryoprotectant for cryopreserved plant cells and tissues.
