Proline: a multifunctional amino acid La ´ szlo ´ Szabados 1 and Arnould Savoure ´ 2 1 Institute of Plant Biology, Biological Research Center, Temesva ´ ri krt. 62., H-6726 Szeged, Hungary 2 University Pierre and Marie Curie, UR5 EAC7180 CNRS, Faculty of Life Sciences, 4 Place Jussieu, case 156, 75005 Paris, France Proline accumulates in many plant species in response to environmental stress. Although much is now known about proline metabolism, some aspects of its bio- logical functions are still unclear. Here, we discuss the compartmentalization of proline biosynthesis, accumu- lation and degradation in the cytosol, chloroplast and mitochondria. We also describe the role of proline in cellular homeostasis, including redox balance and energy status. Proline can act as a signaling molecule to modulate mitochondrial functions, influence cell pro- liferation or cell death and trigger specific gene expres- sion, which can be essential for plant recovery from stress. Although the regulation and function of proline accumulation are not yet completely understood, the engineering of proline metabolism could lead to new opportunities to improve plant tolerance of environ- mental stresses. Proline accumulation in plants Proline is a proteinogenic amino acid with an exceptional conformational rigidity, and is essential for primary metabolism. Since the first report on proline accumulation in wilting perennial rye grass (Lolium perenne) [1], numer- ous studies have shown that the proline content in higher plants increases under different environmental stresses. Proline accumulation has been reported during conditions of drought [2] high salinity [3] high light and UV irradia- tion [4], heavy metals [5], oxidative stress [6] and in response to biotic stresses [7,8]. An osmoprotective func- tion of proline was discovered first in bacteria, where a causal relationship between proline accumulation and salt tolerance has long been demonstrated [9,10]. Such data led to the assumption that proline accumulation in stressed plants has a protective function, which has been emphasized in numerous reviews [11–13]. However, the correlation between proline accumulation and abiotic stress tolerance in plants is not always apparent. For example, high proline levels can be characteristic of salt- and cold-hypersensitive Arabidopsis (Arabidopsis thali- ana) mutants [14,15]. Proline content is also high in drought-tolerant rice varieties [2], but is not correlated with salt tolerance in barley (Hordeum vulgare) [16,17]. Nevertheless, several comprehensive studies using trans- genic plants or mutants demonstrate that proline metab- olism has a complex effect on development and stress responses, and that proline accumulation is important for the tolerance of certain adverse environmental con- ditions [18–21]. Compartmentalization of proline metabolism in plants In plants, proline is synthesized mainly from glutamate, which is reduced to glutamate-semialdehyde (GSA) by the pyrroline-5-carboxylate synthetase (P5CS) enzyme, and spontaneously converted to pyrroline-5-carboxylate (P5C) [22,23] (Figure 1). P5C reductase (P5CR) further reduces the P5C intermediate to proline [24,25]. In most plant species, P5CS is encoded by two genes and P5CR is encoded by one [25–27]. Proline catabolism occurs in mito- chondria via the sequential action of proline dehydrogen- ase or proline oxidase (PDH or POX) producing P5C from proline, and P5C dehydrogenase (P5CDH), which converts P5C to glutamate. PDH is encoded by two genes, whereas a single P5CDH gene has been identified in Arabidopsis and tobacco (Nicotiana tabacum) [28–31]. As an alternative pathway, proline can be synthesized from ornithine, which is transaminated first by ornithine-delta-aminotransfer- ase (OAT) producing GSA and P5C, which is then con- verted to proline [32,33]. Intracellular proline levels are determined by biosyn- thesis, catabolism and transport between cells and differ- ent cellular compartments. Computer predictions suggest a mainly cytosolic localization of the biosynthetic enzymes (P5CS1, P5CS2 and P5CR), whereas a mitochondrial local- ization is predicted for the enzymes involved in proline catabolism, such as PDH1/ERD5, PDH2, P5CDH and OAT (Table 1). Although signal peptides could not be identified within the primary structure of P5CS1, P5CS2 and P5CR enzymes, the PDH1, P5CDH and OAT proteins have well recognizable mitochondrial targeting signals. P5CS1-GFP is normally localized in the cytosol of leaf mesophyll cells, but in embryonic cells and roots it is associated with organelles that are similar to fusiform bodies. When cells are exposed to salt or osmotic stress, P5CS1-GFP, but not P5CS2-GFP, accumulates in the chloroplasts. GFP-labeled Arabidopsis P5CS2 has been shown to be predominantly localized in the cytosol [20]. The P5CR protein and activity has been detected in the cytosol and plastid fraction of leaf, root and nodule cells of soybean (Glycine max) [24,34]. In pea (Pisum sativum) mesophyll protoplasts, P5CR activity was localized in chloroplasts, suggesting that P5CR accumulates in plas- tids under high osmotic conditions [35]. Housekeeping proline biosynthesis probably occurs in the cytosol and, in Arabidopsis, it is controlled by the P5CS2 gene [20]. During osmotic stress, proline biosynthesis is augmented in the chloroplasts, which is controlled by the stress- induced P5CS1 gene in Arabidopsis [20,23,26]. Therefore, proline can be synthesized in different subcellular com- partments, depending on the environmental conditions (Figure 1). Review Corresponding author: Szabados, L. (szabados@brc.hu). 1360-1385/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.tplants.2009.11.009 Available online 23 December 2009 89