**2.1 The function of proline in plants**

Essential for primary metabolism, both as a free amino acid and as a component of proteins, proline is distinctive among the proteinogenic amino acids as it contains a secondary amino group and a distinctive cyclic structure (Lehmann et al., 2010). The cyclic structure of proline causes exceptional conformational rigidity, compared to other amino acids, as proline's side chain locks its φ backbone dihedral angle at approximately -75° and this determines the arrangement of the peptide chain and can lead to the stabilization or destabilization of secondary protein structures.

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

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

Essential for primary metabolism, both as a free amino acid and as a component of proteins, proline is distinctive among the proteinogenic amino acids as it contains a secondary amino group and a distinctive cyclic structure (Lehmann et al., 2010). The cyclic structure of proline causes exceptional conformational rigidity, compared to other amino acids, as proline's side chain locks its φ backbone dihedral angle at approximately -75° and this determines the arrangement of the peptide chain and can lead to the stabilization or destabilization of

temperatures.

**2. Proline and plants** 

secondary protein structures.

**2.1 The function of proline in plants** 

protect plant cells is critically evaluated. Future research needs are then discussed.

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

### **2.3 Proline biosynthesis and catabolism**

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 localized in the cytosol or in plastids.

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)).

Proline and the Cryopreservation of

**3.2 Proline and cryopreservation** 

Plant Tissues: Functions and Practical Applications 419

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

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

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

Proline has been used for many years in numerous cryoprotection protocals for the preservation of a wide range of both animal and plant cells and tissues. For example, Li et. al. (2003) investigated the effects of addition of proline, glutamine, and glycine to the Tes-Tris-egg yolk (TTE) freezing medium used for cryopreservation of cynomolgus monkey (*Macaca fascicularis*) spermatozoa. They found that the addition of 5 mM proline, 10 mM glutamine, and 10 or 20 mM glycine to TTE significantly improved post-thaw sperm motility and membrane integrity compared to controls without an amino acid. Of the three

Proline has also been found to be useful for the cryopreservation of plant cells, meristems and embryos. Jain et al. (1996) included proline in the cryoprotectant solution as part of a protocol that was used to successfully cryopreserve embryogenic suspension cells of two commercially cultivated aromatic Indica rice varieties using a simple one-step freezing procedure that did not require a controlled-rate freezer. Brison et al. (1995 ) used a preculture medium enriched with dimethylsulfoxide and proline prior to the cropreservation of *in vitro* grown interspecific Prunus rootstock, Fereley-Jaspi (R). In a study to develop a cryoprotection protocol for highly

correlations between cellular proline accumulation and improved cold tolerance.

cellular processes and biochemical reactions (Ashraf & Foolad, 2007).

amino acids tested proline was effective at the lowest concentration.

cryoprotectant for cryopreserved plant cells and tissues.

**4. The use of proline as a cryoprotectant** 

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 dehydrogenase, P5CDH P5C dehydrogenase, OAT ornithine-daminotransferase. Transporters and potential transporters are shown as grey circles.
