STUDIO SUL RUOLO DELL’ACIDO Δ1-PIRROLIN-5-CARBOSSILICO NEI FENOMENI DI TOSSICITÀ INDOTTA DA PROLINA IN ARABIDOPSIS THALIANA.

A high accumulation of proline due to increased synthesis and decreased degradation under a variety of stress conditions, such as salt, drought and metals, has been documented in many plant species (Kavi Kishor et al., 2005). This accumulation (up to 80% of total free amino acids under stress, compared to 5% under normal conditions) seems to have diverse physiological roles, such as acting as a compatible osmolyte for osmotic adjustment, stabilization of proteins, membranes and subcellular structures, buffering cellular redox potential and protecting cellular functions by scavenging reactive oxygen species (ROS) (Kavi Kishor et al., 2005; Ashraf and Foolad, 2007). In plants, proline is synthesized from either glutamate or ornithine (Kavi Kishor et al.,2005; Ashraf and Foolad, 2007). The first two steps of proline biosynthesis from glutamate are catalyzed by a single bifunctional enzyme, δ1-pyrroline-5-carboxylate synthetase (P5CS), yielding glutamic-γ-semialdehyde (GSA). GSA spontaneously cyclises into pyrroline-5-carboxylate (P5C), that is reduced in turn by P5C reductase (P5CR) to proline (Zhang et al., 1995; Kavi Kishor et al., 2005). Plants also synthesize proline from ornithine, through the activity of ornithine-δ-aminotransferase (δOAT) that transaminates ornithine directly to GSA, which is subsequently converted to proline via P5C (Kavi Kishore et al., 2005). The onset of stress-induced proline accumulation is correlated with transcriptional activation of the gene encoding P5CS, which is believed to represent the key and rate limiting enzyme in this biosynthetic pathway (Stines et al., 1999; Zhang et al., 2008). During recent years a thorough understanding of the mechanisms underlying regulation of Pro biosynthesis has been achieved. However, the role of the catabolic pathway it is still unclear, consisting of two steps sequentially catalyzed by a Pro- and a P5C-dehydrogenase (DH). The oxidative pathway is induced by high intracellular levels of the imino acid; such induction is prevented under hyperosmotic stress conditions (Verbruggen et al., 1996). Besides its obvious role in Pro recycling after the re-establishment of normo-osmotic conditions, no other physiological roles have been up to now identified for the catabolic pathway. Actually, Arabidopsis thaliana knock-out seedlings for either Pro-DH1 or P5C-DH are morphologically undistinguishable from the wild type and show a normal life cycle. The main effect of the impairment of proline catabolism appears to be a hypersensitivity to the amino acid or to its analogues (Deuschle et al., 2004 ; Mani et al., 2002). Indeed, an unexpected result is that an exogenous Pro supply apparently causes phytotoxic effects. Besides necrotic area formation, the production of reactive oxygen species (ROS) and DNA laddering have been reported (Deuschle et al., 2004 ), suggesting the onset of programmed cell death (PCD). In animal systems it is well known that the PRODH gene is early induced by the tumor suppressor protein p53 (Maxwell et al., 2000), that may inhibit growth and trigger PCD; exogenously-supplied P5C may also cause apoptosis. P5CDH was also reported to represent a p53 target, but it seems able to protect cells from oxidative stress and inhibit cell death (Yoon et al., 2004). Human Pro-DH appears to catalyse ROS production directly, depending to substrate availability (Donald et al.,2001), but Pro-DH has never been purified and fully characterized to date from any plant species. On the other hand, an early induction of the gene for P5C-DH has been found in crops undergoing a fungal attack, a response typically observed in the case of compatible interactions (Roberts et al., 1995; Ayliffe et al., 2002). Moreover, Pro accumulation through activation of the biosynthetic pathway has been recently shown in Arabidopsis during incompatible plant-pathogen interactions (Fabro et al., 2004). Thus the possibility exists that Pro metabolism is involved in processes leading to PCD during the hypersensitive reaction. Proline effects have been proposed to be mediated by P5C (Phang et al.,1985; Deuschle et al.,2004 ). However, this seems inconsistent with the severe toxicity that occurs in prod1- mutants and plants expressing antisense ProDH constructs (Mani et al., 2002; Nanjo et al., 2003 ). P5C is the common metabolic intermediate in proline synthesis and catabolism. It is also produced by δOAT, which was recently demonstrated to be localized in the mitochondria (Funck et al., 2008). It is still a matter of debate whether the P5C produced by Arg/Orn catabolism enters proline biosynthesis directly, or it is metabolized to glutamate by mitochondrial P5CDH. Funck and coworkers showed experimental evidence supporting the latter hypothesis. Consistently, the occurrence of a specific P5C transporter has never been reported. This notwithstanding, some authors postulated that P5C can pass the mitochondrial membrane, and that a P5C/Pro cycle mediated by ProDH and P5CR activity can occur (Hu et al., 2007; Miller et al., 2009). Since P5C/Pro cycling is believed to cause ROS production, P5C oxidation by P5CDH would be essential for limiting cycle activity and avoiding ROS-induced damage (Miller et al., 2009). In Arabidopsis thaliana a single gene is present for both enzymes utilizing P5C as a substrate, namely P5CR and P5CDH. Until now, neither gene product has been purified in this species. Moreover, no obvious correlations between mRNA and protein levels and a RNA silencing-based regulatory mechanism were described for P5CR and P5CDH, respectively. The P5CR mRNA contains a non-coding region in the 5’ untranslated region (UTR) that at the same time mediates transcript stabilization and translation inhibition during salt and heat stress (Hua et al., 2001). Arabidopsis P5CDH has been found to be associated with another gene, which is partially overlapping on the complementary strand. The transcription of this gene is up-regulated under salt stress conditions, with a consequent production and processing of dsRNA. Short interfering RNA being able to downregulate P5CDH are generated from this process (Borsani et al., 2005). Although many physiological studies suggested that proline may be implicated in multiple stress protection mechanisms, it is also evident that proline accumulation does not represent a response conferring tolerance per se. Accordingly, several salt- and cold-hypersensitive Arabidopsis mutants accumulate proline at high levels without any apparent beneficial effect on stress tolerance (Liu and Zhu, 1997). Therefore, a proper understanding of how proline accumulation influences particular regulatory pathways in complex stress responses still requires many efforts (Maggio et al., 2002). Aiming at a better comprehension of the role of proline in plant stress response, Arabidopsis was selected as the experimental system. In this weed, P5CDH and P5CR are present as single genes, a feature that may simplify the analysis of expression patterns. Moreover, the availability of a lot of molecular data as well as the fully sequenced genome provide useful tools for the investigation. However, proper evaluations would be hampered in planta by the small size of Arabidopsis seedlings, a drawback that most likely explains why the proteins involved in proline metabolism have not been purified to date. On the other side, in differentiated plants inter-tissue transport, exclusion mechanisms and cell compartmentalization of proline do occur. As a consequence, it is difficult to know the exact conditions (i.e., ionic vs osmotic, extra- and intracellular concentrations, pressure values) to which a single cell is exposed. The availability of suitable experimental systems to quantify resistance to realistic and reproducible low water potential, or salt and freezing stress conditions is well recognized as a crucial point in genetic studies (Verslues et al., 2006). Suspension cultured cells may represent one of these systems. The possibility of using p5cdh seeds, kindly provided by Dr Dietmar Funck (Konstanz, Germany), was a further stimulus to establish cell suspension cultures from A. thaliana seedlings. Two well dispersed cultures were obtained, and used to evaluate cell responses to various experimental conditions, mainly focusing on the metabolic role of the possible effector of Pro toxicity, P5C.