**5.3 Proline as precursor for other molecules**

It has been suggested that stress-induced accumulation of amino acids like proline may not only have an osmoregulatory role, but that they could also be a mechanism to provide cells with a pool of the precursors required to synthesis other molecules known to be involved in biotic and abiotic stress responses (Sanchez et al., 2008). For example polyamines can be synthesized from arginine or ornithine and ornithine from glutamate, hence the pathways for proline and polyamine biosynthesis are interlinked, and both groups of molecules are important in plant stress responses (Groppa & Benavides 2008). Little is known about the roles of polyamine metabolism in the process of cryopreservation, but Ramon et al. (2002) reported that an increase in putrescine content was positively correlated with the survival rate after simple freezing or after vitrification of banana meristem cultures.

Proline and the Cryopreservation of

protocols.

may exist.

Plant Tissues: Functions and Practical Applications 423

Because of its chemical properties proline has a high capacity to quench singlet oxygen and hydroxyl radicals. Pyrrolidine, which forms the 5- membered ring of proline, has a low IP and so proline is able to form a charge-transfer complex, enabling it to quench singlet oxygen effectively. Proline can also react with hydroxyl radicals under hydrogen abstraction forming a stable radical (Matylsik, 2002). Therefore the accumulation of proline to high levels in plant cells under stress or plants cells treated with exogenous proline as part of a cryopreservation protocol could greatly increase the ROS scavenging capacity of said cells and reduce the potential for oxidative damage. In particular, as proline has the potential to reduce ROS levels it could help reduce oxidative damage to vital cellular macromolecules and hence stabilize proteins (Anjum, 2000),) DNA (Iakobashvil, 1999) and lipid membranes (Alia, 1991). The accumulation of proline-rich proteins and particularly proline residues in cellular proteins is thought to provide additional protection against oxidative stress (Matylsik, 2002). The increase in ROS scavenging capacity brought about by increased intracellular proline levels could be a key mechanism by which proline helps reduce the freezing and dehydration associated cellular damage associated with most cryopreservation

Fig. 5. The influence of pre-treatment with ABA (3.8 uM) and/or proline (2.15 mM) on lipid hydroperoxide levels, as determined by Mihaljevic et al. ( 1996), in post thaw *Begonia x* 

Interestingly ABA combined with proline reduced hydrogen peroxide production and oxidative damage, measured as lipid peroxidation, more effectively in post thaw *Begonia x erythrophylla* shoots than ABA or proline alone. Christianson (1998) also found that ABA and proline in combination improved the survival of moss gametophytes following cryopreservation. These results suggest a possible interaction between ABA and proline

*erythrophylla* shoots cryopreserved as described by Burritt (2008).

Stored amino acids could also be useful during the recovery process following stress. The accumulation of large cellular pools of amino acids could allow the rapid synthesis of enzymes and the repair of structural proteins, allowing a more rapid recovery of cells following cryopreservation, but this possibility has yet to investigated.

#### **5.4 Proline as an antioxidant**

Reactive oxygen species, such as the superoxide anion (O2•), hydrogen peroxide (H2O2), and the extremely reactive hydroxyl radical (•OH) are produced within cells as a consequence of normal metabolic processes, but the production of ROS often increases when cells are under stress (Smirnoff, 1993; Halliwell & Gutteridge, 1999). When ROS are produced at levels high enough to overcome the antioxidant defences that normally control cellular ROS levels, oxidation of DNA, proteins and membrane fatty acids occurs, the latter can result in lipid peroxidation and loss of membrane function (Halliwell & Gutteridge, 1999). Such damage is commonly referred to as oxidative stress (Lesser, 2006; Burritt & MacKenzie 2003; Burritt, 2008). Cryopreservation protocols comprise a number of steps, each of which has the potential to cause stress that could increase ROS production. Recent studies have shown that dehydration and freezing can both lead to increased ROS production and lead to oxidative stress (Feck et al., 2000; Roach et al., 2008). A recent study on oxidative stress and antioxidant metabolism during the cryopreservation of olive somatic embryos demonstrated the importance of oxidative stress and antioxidant metabolism for the successful cryopreservation of plant cells (Lynch et al., 2011).

As mentioned in Section 4 Burritt (2008) found that addition of ABA and proline to the pretreatment medium significantly improved the percentage of *Begonia x erythrophylla* shoots surviving freezing, this increase in percentage survival was accompanied by a decrease in levels of hydrogen peroxide (Figure 4) and oxidative damage, measured as the levels of lipid peroxides, observed in the shoots immediately following thawing (Figure 5).

Fig. 4. The influence of pre-treatment with ABA (3.8 uM) and/or proline (2.15 mM) on hydrogen peroxide levels, as determined by Chesseman ( 2006), in post thaw *Begonia x erythrophylla* shoots cryopreserved as described by Burritt (2008).

Stored amino acids could also be useful during the recovery process following stress. The accumulation of large cellular pools of amino acids could allow the rapid synthesis of enzymes and the repair of structural proteins, allowing a more rapid recovery of cells

Reactive oxygen species, such as the superoxide anion (O2•), hydrogen peroxide (H2O2), and the extremely reactive hydroxyl radical (•OH) are produced within cells as a consequence of normal metabolic processes, but the production of ROS often increases when cells are under stress (Smirnoff, 1993; Halliwell & Gutteridge, 1999). When ROS are produced at levels high enough to overcome the antioxidant defences that normally control cellular ROS levels, oxidation of DNA, proteins and membrane fatty acids occurs, the latter can result in lipid peroxidation and loss of membrane function (Halliwell & Gutteridge, 1999). Such damage is commonly referred to as oxidative stress (Lesser, 2006; Burritt & MacKenzie 2003; Burritt, 2008). Cryopreservation protocols comprise a number of steps, each of which has the potential to cause stress that could increase ROS production. Recent studies have shown that dehydration and freezing can both lead to increased ROS production and lead to oxidative stress (Feck et al., 2000; Roach et al., 2008). A recent study on oxidative stress and antioxidant metabolism during the cryopreservation of olive somatic embryos demonstrated the importance of oxidative stress and antioxidant metabolism for the successful cryopreservation

As mentioned in Section 4 Burritt (2008) found that addition of ABA and proline to the pretreatment medium significantly improved the percentage of *Begonia x erythrophylla* shoots surviving freezing, this increase in percentage survival was accompanied by a decrease in levels of hydrogen peroxide (Figure 4) and oxidative damage, measured as the levels of

lipid peroxides, observed in the shoots immediately following thawing (Figure 5).

Fig. 4. The influence of pre-treatment with ABA (3.8 uM) and/or proline (2.15 mM) on hydrogen peroxide levels, as determined by Chesseman ( 2006), in post thaw *Begonia x* 

*erythrophylla* shoots cryopreserved as described by Burritt (2008).

following cryopreservation, but this possibility has yet to investigated.

**5.4 Proline as an antioxidant** 

of plant cells (Lynch et al., 2011).

Because of its chemical properties proline has a high capacity to quench singlet oxygen and hydroxyl radicals. Pyrrolidine, which forms the 5- membered ring of proline, has a low IP and so proline is able to form a charge-transfer complex, enabling it to quench singlet oxygen effectively. Proline can also react with hydroxyl radicals under hydrogen abstraction forming a stable radical (Matylsik, 2002). Therefore the accumulation of proline to high levels in plant cells under stress or plants cells treated with exogenous proline as part of a cryopreservation protocol could greatly increase the ROS scavenging capacity of said cells and reduce the potential for oxidative damage. In particular, as proline has the potential to reduce ROS levels it could help reduce oxidative damage to vital cellular macromolecules and hence stabilize proteins (Anjum, 2000),) DNA (Iakobashvil, 1999) and lipid membranes (Alia, 1991). The accumulation of proline-rich proteins and particularly proline residues in cellular proteins is thought to provide additional protection against oxidative stress (Matylsik, 2002). The increase in ROS scavenging capacity brought about by increased intracellular proline levels could be a key mechanism by which proline helps reduce the freezing and dehydration associated cellular damage associated with most cryopreservation protocols.

Fig. 5. The influence of pre-treatment with ABA (3.8 uM) and/or proline (2.15 mM) on lipid hydroperoxide levels, as determined by Mihaljevic et al. ( 1996), in post thaw *Begonia x erythrophylla* shoots cryopreserved as described by Burritt (2008).

Interestingly ABA combined with proline reduced hydrogen peroxide production and oxidative damage, measured as lipid peroxidation, more effectively in post thaw *Begonia x erythrophylla* shoots than ABA or proline alone. Christianson (1998) also found that ABA and proline in combination improved the survival of moss gametophytes following cryopreservation. These results suggest a possible interaction between ABA and proline may exist.

Proline and the Cryopreservation of

**5.6 Other mechanisms** 

**6. Conclusions** 

different organisms.

582.

*Physiology,* Vol.138,554-558.

*enzymology,* Vol.1476,75-84.

*Journal,* Vol.47,411–414.

**6. References** 

Plant Tissues: Functions and Practical Applications 425

making them less prone to a liquid crystalline-to-gel transition (Hoekstra et al., 2001). It has also been suggested that proline molecules can directly replace missing water molecules

In addition, according to the preferential exclusion hypothesis, proline is one of a group of solutes that, when in aqueous solution, are excluded from contact with the surfaces of proteins and phospholipid bilayers (Arakawa & Timasheff, 1983). Accordingly addition of proline to a solution stabilizes the native structure of protein monomers and protects oligomeric protein complexes from denaturation and dissociation. Rudolph et al. (1986) demonstrated that the activity of the enzyme lactate dehydrogenase could be protected in part during freeze-thaw cycles by increasing the concentration of proline from 0 to 200 mM

There are several other mechanisms via which proline could contribute to over coming the stresses associated with cryopreservation. For example, the accumulation of proline could also be a mechanism to store energy as the oxidation of a single proline molecule can produce up to 30 ATP equivalents (Atkinson, 1971). Replenishment of NADP+ and redox cycling have also been sugested as potential mechanisms associated with stress tolerance,

While numerous studies have demonstrated that proline can be used to improve the survival of plant cells and organs following cryopreservation, there is little definitive evidence as to the mode of action of proline. More research is required to determine how proline protects plant cells at the cellular level and to determine how other treatments that confer cryotolerance, such as ABA pretreatments interact with proline metabolism and could hence improve the cryotolerance of plant cells. However, despite our lack of knowledge with respect to the mode of action of proline, this amino acid continues to be of great value as a cryoprotectant that can be used with a wide range of cell types from many

Ait-Barka, E.; Audran, J.C. (1997) Response of champenoise grapevine to low temperatures:

Alia; Saradhi, P.P. (1991) Proline accumulation under heavy-metal stress. *Journal of Plant* 

Anjum, F.; Rishi, V.; Ahmad, F. (2000) Compatibility of osmolytes with Gibbs energy of

Arakawa, T.; Timasheff, S.N. (1985) The stabilisation of proteins by osmolytes. *Biophysical* 

changes of shoot and bud proline concentrations in response to low temperatures and correlations with freezing tolerance. *Journal of Horticultural Science,* Vol.72,577–

stabilization of proteins. *Biochimica et biophysica acta-protein structure and molecular* 

(Hare & Cress 1999), as has a role in stress signal transdution (Hare et al., 1997).

between the phospholipids headgroups (Rudolph et al., 1986).

in the buffer in which the enzyme was solubilised.

#### **5.5 Is there an inaction between ABA and proline?**

Studies have shown a relationship between proline and ABA with respect to cold tolerance (Xi & Li, 1993; Lou & Reid, 1997). In a recent study of maize suspension-cultured cells Chen and Li (2002) showed that an ABA treatment at warm temperatures improved the tolerance of cells to subsequent chilling, and that both ABA-treated and untreated maize cells accumulated proline in response to chilling. Chen and Li also found that ABA-treated cells showed less lipid peroxidation during chilling and unlike untreated cells were able to retain the accumulated proline intracellularly.

In post thaw *Begonia x erythrophylla* shoots ABA combined with proline resulted in much higher shoot survival than pretreatment with ABA or proline alone. Interestingly ABA combined with proline resulted in far higher intracellular proline concentrations (Figure 6). The greater concentrations of proline seen in the combined treatment could be due increased endogenous synthesis of proline, induced by exposure to ABA, combined with uptake of exogenous proline during the pretreatment phase and/or to an ABA induced mechanism that helps reduce proline leakage, but further investigations are required to determine how the combined application of ABA and proline increase shoot survival after cryopreservation.

Fig. 6. The influence of pre-treatment with ABA (3.8 uM) and/or proline (2.15 mM) on proline levels, in post thaw *Begonia x erythrophylla* shoots cryopreserved as described by Burritt (2008).

#### **5.5 Proline and direct macromolecule protection**

As well as the potential protective mechanisms detailed above, proline has been shown to directly protect key cellular macromolecules, in particular lipid membranes and proteins such as enzymes (Verbruggen & Hermans, 2008). Proline molecules can intercalate between the head groups of membrane phospholipids during freeze-dehydration helping to reduce mechanical stresses in the membranes, or alter the physical properties of membranes making them less prone to a liquid crystalline-to-gel transition (Hoekstra et al., 2001). It has also been suggested that proline molecules can directly replace missing water molecules between the phospholipids headgroups (Rudolph et al., 1986).

In addition, according to the preferential exclusion hypothesis, proline is one of a group of solutes that, when in aqueous solution, are excluded from contact with the surfaces of proteins and phospholipid bilayers (Arakawa & Timasheff, 1983). Accordingly addition of proline to a solution stabilizes the native structure of protein monomers and protects oligomeric protein complexes from denaturation and dissociation. Rudolph et al. (1986) demonstrated that the activity of the enzyme lactate dehydrogenase could be protected in part during freeze-thaw cycles by increasing the concentration of proline from 0 to 200 mM in the buffer in which the enzyme was solubilised.

#### **5.6 Other mechanisms**

424 Current Frontiers in Cryopreservation

Studies have shown a relationship between proline and ABA with respect to cold tolerance (Xi & Li, 1993; Lou & Reid, 1997). In a recent study of maize suspension-cultured cells Chen and Li (2002) showed that an ABA treatment at warm temperatures improved the tolerance of cells to subsequent chilling, and that both ABA-treated and untreated maize cells accumulated proline in response to chilling. Chen and Li also found that ABA-treated cells showed less lipid peroxidation during chilling and unlike untreated cells were able to retain

In post thaw *Begonia x erythrophylla* shoots ABA combined with proline resulted in much higher shoot survival than pretreatment with ABA or proline alone. Interestingly ABA combined with proline resulted in far higher intracellular proline concentrations (Figure 6). The greater concentrations of proline seen in the combined treatment could be due increased endogenous synthesis of proline, induced by exposure to ABA, combined with uptake of exogenous proline during the pretreatment phase and/or to an ABA induced mechanism that helps reduce proline leakage, but further investigations are required to determine how the combined application of ABA and proline increase shoot survival after cryopreservation.

Fig. 6. The influence of pre-treatment with ABA (3.8 uM) and/or proline (2.15 mM) on proline levels, in post thaw *Begonia x erythrophylla* shoots cryopreserved as described by

As well as the potential protective mechanisms detailed above, proline has been shown to directly protect key cellular macromolecules, in particular lipid membranes and proteins such as enzymes (Verbruggen & Hermans, 2008). Proline molecules can intercalate between the head groups of membrane phospholipids during freeze-dehydration helping to reduce mechanical stresses in the membranes, or alter the physical properties of membranes

**5.5 Proline and direct macromolecule protection** 

**5.5 Is there an inaction between ABA and proline?** 

the accumulated proline intracellularly.

Burritt (2008).

There are several other mechanisms via which proline could contribute to over coming the stresses associated with cryopreservation. For example, the accumulation of proline could also be a mechanism to store energy as the oxidation of a single proline molecule can produce up to 30 ATP equivalents (Atkinson, 1971). Replenishment of NADP+ and redox cycling have also been sugested as potential mechanisms associated with stress tolerance, (Hare & Cress 1999), as has a role in stress signal transdution (Hare et al., 1997).
