**1. Introduction**

414 Current Frontiers in Cryopreservation

Wilhelm E, K Hristoforoglu, S Fluch & K Burg (2005) Detection of microsatellite instability

Cryopreservation has been proven to be an effective technology for the cost-effective, longterm preservation of genetic material. A wide range of plant material including cultured cells, tissues, embryos, meristems, pollen and seeds can be effectively preserved for extended periods of time and, when thawed, can be used to rapidly produce stock plants, with good preservation of genetic and physiological characteristics. Numerous protocols including controlled rate cooling, PVS2 vitrification, encapsulation-vitrification, and encapsulation-dehydration have been developed that allow the cryopreservation of a wide range of plant germplasm (Burritt, 2008), but irrespective of the protocol used each step in a cryopreservation protocol has the potential to impose a stress on plant cells. Low temperatures that lead to freezing can impose stress on cells and tissues in two ways, by the direct effects of low temperatures on cell function and integrity or by the cellular dehydration that occurs when the cells water freezes. Several of the mechanisms by which these two forms of stress can damage plant cells are shown in Figure 1.

Numerous studies have shown that cold temperatures induce the accumulation of metabolites, including low-molecular-weight carbohydrates such as fructose, glucose, maltose and raffinose, and amino acids such as proline and glutamine (Taji et al., 2002; Cook et al., 2004). These metabolites play important protective roles in freezing tolerance in whole plants (Kaplan and Guy, 2004) and this has lead to their extensive use in the protocols developed for the cryopreservation of isolated plant cells and tissues (Burritt, 2008). In particular, the amino acid proline has been found to help confer freezing tolerance in a wide variety of both animal and plant cells, and is often added to cryoprotective solutions or is used for preconditioning plants or pretreating isolated cells or tissues prior to cryopreservation (Burritt, 2008). Despite its widespread use, little is known of the mechanisms via which proline protects cells during cryopreservation.

This chapter gives an overview of proline synthesis and metabolic regulation in plants and the changes in proline metabolism associated with desiccation and freezing tolerance, which are both of importance for the successful cryopreservation of plants cells and tissues. The

Proline and the Cryopreservation of

flowering (Mattioli et al., 2008; Mattioli et al., 2009)

**2.3 Proline biosynthesis and catabolism** 

localized in the cytosol or in plastids.

Plant Tissues: Functions and Practical Applications 417

As well as being important for primary metabolism proline appears to have numerous other functions in plants. Research has clearly demonstrated that proline levels show significant fluctuations in response to environmental stress (Bohnert et al., 1995), but the precise mode of action of proline remains largely a matter of speculation. In addition to its role in environmental stress tolerance, recent research has provided evidence that proline may also play important roles in plant development both as a metabolite and as a signal molecule (Mattioli et al., 2009). Studies have shown that proline could play important roles in embryo and seed development, stem elongation, and the transition from vegetative growth to

The proposed pathways for proline biosynthesis and catabolism in plants are outlined in Figure 2. In plants proline can be synthesized from glutamate or ornithine, however under most conditions proline is mainly synthesized from glutamate rather than from ornithine, as the enzyme ornithine-δ –aminotransferase (dOAT) is down regulated (Szabados & Savoure, 2009). Two enzymes are required for the synthesis of proline from glutamate. The first enzyme, Δ1- pyrroline-5-carboxylate synthase (P5CS) is a bifunctional enzyme that phosphorylates and reduces glutamate to glutamyl-5- semialdehyde (G5SA) that then spontaneously converts to Δ1- pyrroline-carboxylate (P5C). The second enzyme, Δ1 pyrroline-carboxylate reductase (P5CR) further reduces the P5C intermediate to proline (Delauney & Verma, 1993). P5CS has been found to be encoded by 2 genes in most plants, while P5C is encoded by only a single gene (Szekely et al. 2008; Strizhov et al., 1997) The rate-limiting step in the above pathway is the γ -glutamyl kinase activity of P5CS, which is sensitive to feedback inhibition by the presence of relatively low cellular proline levels (Zhang et al., 1995). Alternatively proline can be synthesized from ornithine by dOAT, which converts ornithine and -ketoglutarate to P5C and glutamate by transamination (Stranska et al., 2008). Funck et al., 2008, in a study of *Arabidopsis thaliana,* found that mutant plants which lacked dOAT activity could not mobilize nitrogen from arginine or ornithine, but could accumulate proline and so suggested the main role for dOAT was arginine degradation. They also suggested that as dOAT is localized in the mitochondria and that it would be unlikely that P5CR could directly utilize dOAT-generated P5C, as P5CR is

Proline degradation in plants takes place in mitochondria and so is by in large separated from the biosynthetic pathway. The first step in proline catabolism is the oxidation of proline to P5C by proline dehydrogenase (PDH), which in Arabidopsis and tobacco is encoded by two homologous genes (Mani et al., 2002; Ribarits et al., 2007; Verbruggen & Hermans 2008). The P5C generated is then converted to glutamate by pyrroline-5 carboxylate dehydrogenase (P5CDH), which is thought to be encoded by a single gene in all of the plant species analysed to date (Ayliffe et al. 2005; Mitchell et al. 2006). However, biochemical analysis P5CDH in *Nicotiana plumbaginifolia* and *Zea mays* has revealed two slightly different enzyme activities that may arise from a single gene, or a second P5CDH gene may be present (Elthon & Stewart 1982; Forlani et al. 1997). In plants under stress, the accumulation of proline is thought to be due not only to increased synthesis, but also to

inactivation of degradation pathways (Delauney & Verma, 1993)).

use of proline as a cryoprotectant or pre-growth additive for the cryopreservation of plant cells and tissues is then overviewed and the potential mechanisms via which proline can protect plant cells is critically evaluated. Future research needs are then discussed.

Fig. 1. Potential damage caused by the stresses associated with exposure of plant cells to low temperatures.
