Appl Biochem Biotechnol DOI 10.1007/s12010-015-1713-0
Bridging Between Proline Structure, Functions, Metabolism, and Involvement in Organism Physiology Walid Saibi 1 & Kaouthar Feki 1 & Ines Yacoubi 1 & Faiçal Brini 1
Received: 25 February 2015 / Accepted: 9 June 2015 # Springer Science+Business Media New York 2015
Abstract Much is now known about proline multifunctionality and metabolism; some aspects of its biological functions are still unclear. Here, we discuss some cases in the proline, structure, definition, metabolism, compartmentalization, accumulation, plausible functions and also its implication in homeostasis and organism physiology. Indeed, we report the role of proline in cellular homeostasis, including redox balance and energy status and their implication as biocatalyst for aldolase activity. Proline can act as a signaling molecule to modulate mitochondrial functions, influence cell proliferation or cell death, and trigger specific gene expression, which can be essential for plant recovery from stresses. Although, the regulation and the function of proline accumulation, during abiotic stresses, are not yet completely understood. The engineering of proline metabolism could lead to new opportunities to improve plant tolerance against environmental stresses. This atypical amino acid has a potential role in the toxicity during growth of some microorganism, vegetal, and mammalian species. Furthermore, we note that the purpose through the work is to provide a rich, concise, and mostly cohesive source on proline, considered as a platform and an anchor between several disciplines and biological functions. Keywords Proline multifunctionality . Proline metabolism . Proline functions . Organism physiology . Asymmetric catalytic reaction
Proline: Definition and Structure Proline is an α-amino acid, one of the 20 DNA-encoded amino acids. This atypical residue is defined, in genetic code, by fore codons (CCU, CCC, CCA, and CCG). Because of the ability of the human body to synthesize it, proline is classified as a non-essential amino acid. It is * Walid Saibi
[email protected] 1
Biotechnology and Plant Improvement Laboratory Centre of Biotechnology of Sfax, University of Sfax, Street Sidi Mansour Km 6, P.O. Box B1177^, 3018 Sfax, Tunisia
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unique among the 20 protein-forming amino acids in that the amine nitrogen is bound to not one but two alkyl groups, thus making it a secondary amine. Proline is widely recognized as p1aying a peculiar role in the folding/unfolding transitions of the globular protein [1, 2]. Poverty or near absence of proline in the primary sequence of proteins may govern the level of disorder in the structure of the protein [3]. Among the two amino acids, proline follows an atypical Ramachandran plot. This fact is due to the ring formation connected to the β-carbon; the ψ and φ angles about the peptide bond have less allowable degrees of rotation. The repercussion related to the proline intrinsical properties is often found in Bturns^ of proteins. Indeed, the proline-free entropy is not as comparatively large to other amino acids. Hence, in a folded form vs. unfolded form, the change in entropy is less. Furthermore, proline is rarely found in α and β structures as it would reduce the stability of such structures because its side chain α-N can only form one hydrogen bond. In another hand, proline is the only amino acid that does not form a blue/purple color when developed by spraying with ninhydrin for uses in chromatography. Proline, instead, produces an orange/yellow color [4].
Proline: Physiological Functions and Importance in Homeostasis The Physiological Functions of Proline-Rich Polypeptides Proline-rich polypeptides (PRPs) are defined as extremely small chains of 10 amino acids or less; notably, proline has a very powerful effect in initiating and balancing our immune responses. PRPs enhance the ability of the thymus gland to release factors enhancing the regulation of the immune functions in the body. PRPs can induce a shift from a predominantly humeral immune response to a more protective cellular response described as a BTH2 to TH1 shift.^ Doing so may assist the immune system more effectively in fighting chronic bacterial and viral infections while simultaneously inhibiting the initiation of inappropriate inflammatory cascades associated with allergy, chemical sensitivity, and autoimmune responses. PRPs follow a great number of physiological functions that they summarized in Table 1 [5, 6].
Proline Governs the Protein Self-Organization Elastin provides extensible tissues, including arteries and skin, with the propensity for elastic recoil, whereas amyloid fibrils are associated with tissue-degenerative diseases, such as Alzheimer’s. Although both elastin-like and amyloid-like materials result from the self-organization of proteins into fibrils, the molecular basis of their differing physical properties is poorly understood. They prove that elastin-like and amyloid-like peptides are separable based on the backbone hydration and peptide-peptide hydrogen linkage. The analytical studies of diverse sequences, including those of elastin, amyloids, wheat gluten, insect resilin, and spider silks, follow a sill in proline and glycine composition above which amyloid formation is impeded and elastomeric properties become apparent. The predictive ability of this threshold is confirmed by the self-assembly of recombinant peptides in either amyloid fibrils or elastin like. These results support a unified model of protein aggregation in which hydration and conformational disorder are fundamental requirements for elastomeric function [7].
Appl Biochem Biotechnol Table 1 Some physiological functions of PRPs Some physiological functions of PRPs Modulation of the immune system Acting as molecular signaling device Undifferentiated stimulation of lymphocytes in thymus to become either helper T cells or suppressor T cells Promotion of B cells growth and differentiation Stimulate natural killer cell (NK cell) activity Stimulate the production of tumor necrosis factor-alpha and interferon-gamma Promote the proliferation of leukocytes (white blood cells) Stimulate production of cytokines by peripheral blood cells Induce differentiation and maturation of monocytes and macrophages Increase the permeability of blood vessels in the skin Produce immunity to certain viruses Inhibit viruses known to be associated with autoimmune diseases May help downregulate the Bcytokine storm^ seen in bird flu Increase T cell count in AIDS to normal or near-normal levels
Potential Role of Proline in Regulation of Pluripotent Cells The development of cell therapeutics from embryonic stem cells will request technologies which encode cell differentiation to specific somatic cell lineages, in response to defined factors. The initial step in formation of the somatic lineages from embryonic stem cells, differentiation to an intermediate, pluripotent primitive ectoderm-like cell, can be achieved in vitro by formation of early primitive ectoderm-like cells in response to a biological activity contained within the conditioned medium [8]. The fractionation of the last one has identified two activities requested for primitive ectoderm-like cell formation, an activity with a molecular mass of 100 M Lproline and some L-proline-containing peptides resulted in changes in colony morphology, cell proliferation, gene expression, and differentiation kinetics consistent with differentiation toward a primitive ectoderm-like cell. This activity appeared to be associated with L-proline since other amino acids and analogs of L-proline did not exhibit an equivalent activity. These findings represent the first report describing a role for amino acids in the regulation of pluripotency and cell differentiation and identify a novel plausible and potential role for Lproline [8].
Proline: Dynamic Molecule and Structural Implication Presence of Proline Gives Rise to Molecular Interactions In this case, we note that the proline presence in protein composition and in interaction domains with ligands would play a basic role. These findings were proved recently by
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monitoring some acoustic factors and parameters. For example, the solution behavior of proline in lecithin-ethanol mixture as a function of temperature and composition have been investigated by measuring velocity of sound in conjunction with density at the range of temperature, from 25 to 40 °C. Numerous acoustical parameters such as intermolecular free length, relative association, specific acoustic impedance, relaxation time, sound velocity number, molar volume, and internal pressure have been evaluated from velocity of sound, density, and viscosity measurements. The intermolecular free length varies linearly with the concentration of proline and increases with rise in temperature. The linear trend in intermolecular free length values with the concentration of proline has been supported by the trend obtained from relaxation time values [9].
Helix Distortions by Proline Proline residues are known to modulate the structure of helices by introducing a kink between the segment preceding and following the proline residue. It is well known that the distortion of the helical structure produced in presence of proline results from the avoided steric clash between the ring and the backbone carbonyl at position (4), as well as the elimination of helix backbone H-bonds for the carbonyls at positions (3) and (4). Both the departure from the helical pattern and the reduction in H-bond stabilization contribute to the observed flexibility of a proline-containing α-helix. Thus, the geometry of the proline kink (PK) region in an αhelix is characterized by backbone dihedral angles that deviate from α-helical values [10]. In solution, the backbone angles have been shown to vary, in agreement with a calculated broad energy minimum related to the presence of the proline in the helix. This variability indicates that in the absence of specific interactions, a PK motif produces significant flexibility that enables a number of different conformations of the helix in that region. The special local flexibility of the PK confers a dynamic behavior on the proline-containing helix that can be relevant in the conformational changes related to functional mechanisms of the protein. For example, the structural distortion of the α-helix by a conserved proline has been shown to play a key role in the voltage-dependent gating mechanism of conexin. By mediating the propagation of conformational changes from one domain in the protein to another, the proline can acquire a dynamic role in the interconversion of different protein states, as has been proposed in the activation mechanisms of G-protein-coupled receptors. Computational techniques attempting to simulate the dynamic behavior of proteins evaluate the conformational changes that can be related to such functional mechanisms of the proteins. An accurate quantification of the geometry of the distortion introduced in helices by proline, recorded both on average and for individual Bsnapshots^ along a molecular dynamics trajectory, can therefore provide a useful tool in determining and evaluating the role of proline-induced flexibility and distortions in protein function. The PK has been defined previously in terms of a bend and a twist [10].
Proline: Plausible Roles in Enzymology Proline in Catalysis: New Mechanistic Studies on the Proline-Catalyzed Aldol Reaction The mechanism of the proline-catalyzed aldol reaction has stimulated considerable debate; at least five different mechanisms have been discussed [11]. Discovered 45 years ago (since
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1970), the Hajos–Parrish–Eder–Sauer–Wiechert reaction, a proline-catalyzed intramolecular aldol reaction, represents not only the first asymmetric aldol reaction invented by chemists but also the first highly enantioselective organocatalytic transformation. Inspired by nature’s phenomenal enzymes, which catalyze direct asymmetric aldolizations of unmodified carbonyl compounds, they have recently extended to the first intermolecular variant and to several other reactions including proline-catalyzed asymmetric, α-amination, and intramolecular enolexo aldolization reactions [11]. As similarly as the aldolases, proline catalyzes direct asymmetric aldol reactions between two different carbonyl compounds to provide aldol products with excellent yields and enantioselectivities. In the enzyme classification, there are two classes of aldolase (class I and class II). Early on, it has been speculated that in addition to operating on related substrates, both class I aldolases and proline may also share a similar enamine mechanism. However, there has been some debate over several mechanistic aspects of the reaction, and a number of alternative models have been proposed. For example, Hajos suggested a mechanism that involves the Bactivation^ of one of the enantiotopic acceptor carbonyl groups as a carbinol amine. At least, the stereochemistry of this model was questioned by Jung soon after its initial proposal. An enamine mechanism was suggested by various groups already since 1970 and 1980. Nonlinearity studies have led to the proposal of a side-chain enamine mechanism that involves two proline molecules in the C–C-bond-forming transition state, one involved in enamine formation and the other as a proton transfer mediator [11].
The Increase Demand for Chiral Compounds Encodes Innovation in Catalytic Methods Development The bioactivity of great number of pharmaceutical compounds, agrochemicals, flavors, and fragrances is associated with absolute molecular configuration. Particularly, the demand is increasing in the pharmaceutical industry to make chiral drugs and it is known as Bchirotechnology.^ Under tremendous competition and intensive effort from many academic and industrial groups, new and effective catalytic reactions have been discovered at an explosive rate [11]. The most asymmetric catalysts consist of metal complexes with chiral ligands. The greatest challenge in discovering new asymmetric catalysts is conducting interdisciplinary research that combines organic, inorganic, organometallic, and biomimetic chemistry. To make an efficient transition metal catalyst, the following tasks are generally required: designing and synthesizing chiral ligands; preparing suitable substrates, catalyst precursors, and metal-ligand complexes; and searching for appropriate reaction conditions. A clear understanding of catalytic mechanisms and characteristics of metal species from an inorganic chemistry perspective can provide insights into selecting chiral ligands, substrates, and catalysts [12].
Proline Metabolism in Plant Compartmentalization of Proline Metabolism in Plants In the vegetable context and precisely in plants, this peculiar residue is anabolized mainly from glutamate, which is reduced to glutamate-semialdehyde (GSA) by the pyrroline-5-carboxylate synthetase (P5CS) and spontaneously converted to pyrroline-5-carboxylate (P5C). P5C
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reductase (P5CR) further reduces the P5C intermediate to proline. In most plant species, P5CS is encoded by two distinguished genes and P5CR is encoded by one gene [4]. Proline catabolism occurs in mitochondria via the sequential action of proline dehydrogenase 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. As an alternative pathway, proline can be synthesized from ornithine, which is transaminated first by ornithine-deltaaminotransferase (OAT) producing GSA and P5C, which is then converted to proline (Fig. 1). Intracellular proline levels are governed by the anabolism, the catabolism, and also the translocation between cells and different cellular compartments. Computer predictions suggest
Fig. 1 Proline metabolism in higher plants. Solid lines represents biosynthetic pathways while catabolic pathways are shown with dashed lines. BAC basic amino acid transporter (for arginine and ornithine exchange), Glu glutamate, G/P mitochondrial glutamate/proline antiporter, KG α-ketoglutarate, P mitochondrial proline transporter, Pi inorganic phosphate, ProT proline transporter. Question mark reprsents unknown transporters [4, 13]
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a mainly cytosolic localization of the biosynthetic enzymes (P5CS1, P5CS2, and P5CR), whereas a mitochondrial localization is predicted for the enzymes involved in proline catabolism, such as PDH1/ERD5, PDH2, P5CDH, and OAT. 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 [4]. 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. The P5CR protein and activity has been detected in the cytosol and plastid fraction of leaf, root, and nodule cells of soybean. In pea mesophyll protoplasts, P5CR activity was localized in chloroplasts, suggesting that P5CR accumulates in plastids under high osmotic conditions. Housekeeping proline biosynthesis probably occurs in the cytosol and, in Arabidopsis, it is controlled by the p5cs2 gene. During osmotic stress, proline biosynthesis is augmented in the chloroplasts, which is controlled by the stress-induced p5cs1 gene in Arabidopsis. Then, proline can be anabolized in different subcellular compartments, this fact depends on the environmental conditions [4]. PDH and P5CDH are mitochondrial enzymes that use FAD and NAD+ as electron acceptors and generate FADH2 and NADH, respectively, delivering electrons for mitochondrial respiration. In soybean nodules, PDH was detected in the cytosol, suggesting that proline oxidation provides energy for bacteroids during nitrogen fixation. Recently, a P5C–proline cycle has been described showing that P5C, produced from proline in the mitochondria, can be transported into the cytosol and reduced to proline by cytosolic P5CR. When P5CDH activity is limited, the P5C–proline cycle can transfer more electrons to the mitochondrial electron transport chain and generate reactive oxygen species (ROS). P5CDH has been found in chloroplasts by proteome analysis, suggesting that P5C is also converted to glutamate in plastids [4]. The ornithine pathway has been suggested to be more important as known (in normal physiological conditions) during the development of seedling and also in some plants for stress-induced proline accumulation. Recently, the significance of the pathway and OAT in proline anabolism has been questioned because proline levels were not affected in Arabidopsis oat knockout mutants. Instead, OAT facilitates nitrogen recycling from arginine through P5C that is converted to glutamate by P5CDH. Compartmentalization of proline metabolism implies that extensive intracellular proline transport must occur between the cytosol, chloroplasts, and mitochondria. Physiological data suggest that proline uptake into mitochondria is an active process, hinting at the existence of specific amino acid transporters. Plasma membrane proline transporters, identified in several plants, mediate proline transport between cells and organs, but are not involved in organellar transport. Two proline carriers have recently been identified in the mitochondria of durum wheat: a proline uniporter, which facilitates proline transport into the mitochondrial matrix, and a proline/glutamate antiporter, which appears to have an important role in the Pro/Glu shuttle between the mitochondrial matrix and the cytosol. Basic amino acid transporters (BAC) can deliver arginine and ornithine through the mitochondrial membrane. In the halophyte species Limonium latifolium, proline was sequestered to vacuoles in non-stressed plants, whereas in salt-stressed plants, high proline content was detected in the cytosol, suggesting the importance of de novo proline biosynthesis as well as transport for proline accumulation [13].
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Regulation of Proline Metabolism Proline was considered and will remain an important platform, which is implicated in various physiological phenomena, in microorganism, animal, and vegetal kingdoms. While proline metabolism has been studied for more than 45 years in plants, little is known about the signaling pathways embroiled in its regulation. Proline anabolism is activated and its catabolism repressed during dehydration, whereas rehydration triggers the opposite regulation. Proline anabolism is controlled by the activity of two p5cs genes in plants, encoding one housekeeping and one stress-specific p5cs isoform. Although the duplicated p5cs genes share a high level of sequence homology in coding regions, their transcriptional regulation is different. In Arabidopsis, p5cs2 is the housekeeping gene, which is active in dividing, meristematic tissues, such as the shoot and root tips, inflorescences, and cell cultures. Both p5cs genes are active in floral shoot apical meristems and supply proline for flower development. It is intriguing that, in Arabidopsis, the p5cs2 gene was identified as one of the targets of CONSTANS (CO), a transcriptional activator that promotes flowering in response to long day length. p5cs2 can be activated also by a virulent bacteria, salicylic acid (SA), and ROS signals, which trigger a hypersensitive response (HR). Arabidopsis p5cs1 is induced by osmotic and salt stresses and is activated by an abscisic acid (ABA)-dependent and ABA-insensitive 1 (ABI1)-controlled regulatory pathway and H2O2-derived signals [14]. Moreover, p5cs1 activation and proline accumulation is promoted by light and repressed by brassinosteroids. Under non-stressed conditions, phospholipase D (PLD) functions as a negative regulator of proline accumulation, whereas calcium signaling and phospholipase C (PLC) trigger p5cs transcription and proline accumulation during salt stress. In the halophyte Thellungiella halophila, PLD functions as positive regulator, whereas PLC exerts a negative control on proline accumulation. Calcium signals can be transmitted by a specific CaM4 calmodulin, which interacts with the myb2 transcription factor and upregulates p5cs1 transcription [14]. As well as transcriptional regulation, P5CS activity is submitted for metabolic control, as seen by feedback inhibition of P5CS by proline, representing the end product of the reaction. Similar allosteric inhibition of bacterial PROB has also been described. Loss of feedback inhibition of P5CS leads to elevated proline accumulation. In mammalian cells, different P5CS isoforms were generated via alternative splicing, which is function to tissue specificity, organ localization, hormonal regulation, and feedback inhibition. Conversion of P5C to proline is not a rate-limiting step in proline biosynthesis, yet the control of P5CR activity implies a complex regulation of transcription, which was shown to be under developmental and osmotic regulation. Promoter analysis of Arabidopsis p5cr identified a 69-bp promoter region that is responsible for tissue-specific expression. However, trans-acting factors that can bind to this promoter region have not yet been identified [4]. Whereas proline biosynthesis is upregulated by light and osmotic stresses, proline catabolism is activated in the dark and during stress relief and is controlled by PDH and P5CDH. pdh transcription is activated by rehydration and proline but repressed by dehydration, thus preventing proline degradation during abiotic stress. pdh1 transcription is repressed during daylight and induced in darkness; therefore, illumination has opposite effects on p5cs1 and pdh1 transcription. Promoter analysis of pdh1 identified the proline and hypo-osmolarityresponsive element (PRE) motif ACTCAT, which is necessary for the activation of the pdh gene. Basic leucine zipper protein (bZIP) transcription factors (AtbZIP-2, AtbZIP-11, AtbZIP44, AtbZIP-53) have been identified as candidates for binding to this motif. Chromatin immunoprecipitation and genetic analysis revealed combinatorial control of PDH expression
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by a network of group S bZIP transcription factors. The p5cdh gene is expressed at a low basal level in all Arabidopsis tissues, and can be upregulated by proline. A short sequence similar to the PRE motif has been identified on the promoters of p5cdh genes in Arabidopsis and cereals. The fis1 gene encodes P5CDH in flax (Linum usitatissimum) and is activated during virulent pathogen attack and wounding. Natural antisense overlapping of the 3′ UTR regions of p5cdh and the salt-induced SIMILAR TO rcd one 5 (sro5) gene generate endogenous small interfering RNA (24-nt and 21-nt siRNA) in Arabidopsis, which cleaves the p5cdh RNA and reduces transcript levels during stress. Transcriptional gene silencing is therefore important in the control of p5cdh gene activity [4].
Proline Accumulation in Plants Proline is a proteinogenic amino acid with a peculiar conformational rigidity. Numerous studies have shown that this amino acid content in higher plants increases under different abiotic stresses. Proline accumulation has been reported during conditions of drought, high salinity, high light and UV irradiation, heavy metals, and oxidative stress and in response to biotic stresses. An osmoprotective function was discovered first in bacteria, where a causal relationship between proline accumulation and salt tolerance has long been demonstrated (data in publication). Such data give birth to the assumption that proline accumulation has a protective effect in stressed plants. 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 mutants [15]. Proline content is also high in drought-tolerant rice varieties, but is not correlated with salt tolerance in barley. However, several comprehensive studies and investigations using transgenic or mutant plants demonstrate that proline metabolism has a complex effect on the development and stress responses and that proline accumulation is important for the tolerance of certain adverse environmental conditions [4].
Proline Accumulation and Stress Tolerance In this case, we noted that salt accumulation in irrigated soils is one of the main factors and causes that decreases crop productivity, since most of the plants are not halophytic. Excessive sodium inhibits growth of many salt sensitive plants, which include most crop plants [16]. The typical first response of all plants to salt stress is osmotic adjustment. The accumulation of compatible solutes in the cytoplasm is considered as a primordial mechanism contributing to salt tolerance. To counter salt stress, plants increase the osmotic potential of their cells by synthesizing and accumulating compatible osmolytes such as proline and glycine betaine, which participates in osmotic adjustment. Several plants, including halophytes, accumulate high proline levels in response to osmotic stress as a tolerance mechanism to high salinity and water deficit [16]. In plants, this atypical amino acid is anabolized from either glutamate or ornithine. However, the glutamate pathway is the primary route used under osmotic stress or nitrogen limitation conditions, whereas the ornithine pathway is prominent under high nitrogen input. Accumulation of proline in plants under stress is a result of the reciprocal regulation of two pathways: [(Δ1Δ1-pyrroline-5-carboxylate synthetase (P5CS) and P5CR] and repressed activity of proline degradation. Poline catabolism is catalyzed by pyrroline-5-carboxylate dehydrogenase and proline dehydrogenase, a mitochondrial enzyme, whose activity had been
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shown to reduce during salt stress. The first two steps of proline biosynthesis are catalyzed by P5CS by means of its γ-GK and glutamic-γ-semialdehyde dehydrogenase activities. Subsequently, the P5C formed is reduced by P5CR to proline [17]. For a long time, proline was considered as an inert compatible osmolyte that protects subcellular structures and macromolecules under osmotic stress. However, proline accumulation can influence stress tolerance in multiple ways. Proline has been shown to be as a potential and plausible effector playing molecular chaperone role and also able to protect protein integrity and enhance the activities of different enzymes. Examples of such roles include the prevention of protein aggregation and stabilization of M4 lactate dehydrogenase during extreme temperatures, protection of nitrate reductase during heavy metal and osmotic stress, and stabilization of ribonucleases and proteases upon arsenate exposure [4]. Several research findings have attributed an antioxidant feature to proline, suggesting ROS scavenging activity and proline acting as a single oxygen quencher. Proline treatment can decreases ROS levels in fungi, thus preventing programmed cell death, can protect human cells against carcinogenic oxidative stress, and can reduce lipid peroxidation in alga cells exposed to heavy metals. Proline pretreatment also alleviated Hg2+ toxicity in rice (Oryza sativa) through scavenging ROS, such as H2O2. The damaging effects of singlet oxygen and hydroxyl radicals on Photosystem II (PSII) can be reduced by proline in isolated thylakoid membranes (PSII). Free radical levels were reduced in transgenic algae and tobacco plants engineered for hyperaccumulation of proline by p5cs overexpression and acceleration of the proline biosynthetic pathway [13]. By contrast, compromised proline accumulation in p5cs1 insertion mutants led to accumulation of ROS and enhanced oxidative damage. A similar effect was observed in yeast, where low proline levels in PUT1 (proline dehydrogenase)-overexpressing lines led to enhanced ROS, whereas higher proline content in put1 mutants correlated with increased protection from oxidative damage. As an alternative to direct ROS scavenging feature, proline can protect and stabilize ROS scavenging enzymes and activate alternative detoxification pathways. In saltstressed tobacco cells, proline increased the activities of methylglyoxal detoxification enzymes, enhanced peroxidase, glutathion-S-transferase, superoxide dismutase, and catalase activities, and increased the glutathion redox state. In the desert plant Pancratium maritimum, catalase and peroxidase were found to be stabilized by proline during salt stress. The salthypersensitive p5cs1 Arabidopsis mutant shows reduced activities of key antioxidant enzymes of the glutathione-ascorbate cycle, leading to hyperaccumulation of H202, enhanced lipid peroxidation, and chlorophyll damage [18]. As well as having protective or scavenging features, it is feasible that proline metabolism can stabilize cellular homeostasis during stress conditions in a way that is still poorly understood. Accumulation of P5CS1 and P5CR in chloroplasts during salt stress suggests that, under adverse conditions, glutamate-derived proline biosynthesis increases in plastids, where photosynthesis occurs. During stress conditions, the rate of the Calvin cycle is diminished, which prevents oxidation of NADPH and restoration of NADP+ [19]. When combined with high light, electron flow in the electron transport chain is suppressed by the insufficient electron acceptor NADP+ pool, leading to singlet oxygen production in the PSI reaction center and accumulation of ROS. Proline biosynthesis is a reductive pathway, and requires NADPH for the reduction of glutamate to P5C and P5C to proline, and generates NADP+ that can be used further as electron acceptor. The phosphorylation of glutamate consumes energy (ATP) and produces ADP, which is a substrate for ATP anabolism during photosynthesis. An enhanced rate of proline biosynthesis in chloroplasts during stress can maintain the low
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NADPH/NADP+ ratio, contribute to sustaining the electron flow between photosynthetic excitation centers, stabilize the redox balance, and reduce photoinhibition and damage of the photosynthetic apparatus. In transgenic soybean plants, the inhibition of proline biosynthesis and NADPH–NADP+ conversion by antisense p5cr led to drought hypersensitivity, whereas overexpression of p5cr resulted in moderate drought tolerance, confirming that proline biosynthesis is important for maintaining NADP+ pools during stress. Such a connection between photosynthesis and proline metabolism is supported by light-dependent proline accumulation, which is regulated by the light-controlled reciprocal p5cs and pdh gene activation [20]. In mitochondria compartment, proline has distinct protective functions. Indeed, after stress, proline pools supply a reducing potential for mitochondria through the oxidation of proline by PDH and P5CDH; they provide also electrons for the respiratory chain and hence contribute to energy supply for resumed growth. The atypical amino acid was shown to protect complex II of the mitochondrial electron transport chain during salt stress and as consequence the stabilization of the mitochondrial respiration. The recently discovered P5C–proline cycle can deliver electrons to mitochondrial electron transport without producing glutamate and, under particular conditions, can generate more ROS in the mitochondria. In addition, proline catabolism is, therefore, an important regulator of cellular ROS balance and can influence numerous additional regulatory pathways [4]. Although species-specific differences in proline accumulation exist, it is an open question whether differences in proline accumulation have an adaptive value. Halophyte relatives of Arabidopsis, such as T. halophila and Lepidium crassifolium, have elevated proline levels under unstressed conditions and accumulate proline to higher levels than does Arabidopsis when exposed to high salinity. In Thellungiella, high proline accumulation results from enhanced p5cs and reduced pdh expression levels. High proline levels can improve the salt tolerance of the halophyte plant P. maritimum, by stabilizing detoxifying enzymes and protein turnover machinery and stimulating the accumulation of stress protective proteins. Other halophytes, such as Camphorosma annua or Limonium spp., do not have high proline content; instead, they accumulate carbohydrate or betain-derived osmolytes. In Silene vulgaris, constitutive proline content was higher in metal-tolerant ecotypes, whereas metal-induced proline accumulation was higher in a non-tolerant ecotype. The capacity for proline hyperaccumulation therefore accompanies the extremophile character of certain plant species and is likely that it contributes to their stress tolerance; however, it is not an absolute requirement for adaptation to extreme environmental conditions [4].
Proline Accumulation and Plausible Toxicity Proline accumulation is a common response of plants against stress. However, external supply of proline in control conditions is toxic. There is a debate whether proline or its derived product P5C seem to be the cause of toxicity [21, 22]. Application of both proline and P5C cause cell death in plants. Data of [23] support the fact that proline toxicity is mediated by GSA/P5C accumulation. Exogenously applied P5C increases ROS production, reduces growth, and induces a number of stress responsive genes. Furthermore, p5cdh overexpression decreased sensitivity to externally supplied proline while p5cdh knockout mutant was hypersensitive to proline [23]. Various studies and investigations using mutants with impaired proline catabolism support clearly that the proline toxicity is not, or not only, mediated by P5C/GSA. Moreover, proline external concentrations higher than 10 mM are toxic to Arabidopsis wild-type plants. In pdh-
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antisense plants that displayed a lower proline catabolism. Proline toxicity was observed at lower proline concentrations than in wild type [23]. pdh-sense transgenic plants, with a higher proline catabolism capacity, displayed wild-type proline sensitivity. Pdh knockout mutants were even more sensitive to proline than pdh-antisense plants, most probably because the mutation results in a more severe inhibition of proline catabolism. Transcriptomic analysis of pdh mutants treated with proline revealed the repression of genes involved in photosynthesis and genes involved in the anabolism of structural proteins (cell wall-associated proteins). This fact reinforces the belief the suggestion to account for proline toxicity. Hypersensitivity of pdhKO and of pdh-AS plants to proline is against the theory that not proline but only P5C causes toxicity [23]. In short, proline accumulation is not only a common physiological response to various stresses but is also part of the developmental program in generative tissues like pollen for example. Transgenic approaches have confirmed the beneficial effect of proline overproduction during stress. However, consensus was not achieved on the exact roles of proline accumulation. Classical gain or loss of function strategies could not bring clear answers, probably because proline also displays the essential role of being a protein component. Stresses like drought or salt stress have multiple targets and proline is also believed to play different roles. Moreover, the balance between biosynthesis and degradation of proline is also thought to be essential in the determination of the osmoprotective and developmental functions of proline.
Conclusions and Perspectives Although proline has long been considered as the most known osmolyte, recent results follow its multifunctionality in stress adaptation, recovery, and signaling. The compartmentalization of proline metabolism adds to their functional diversification complexity and may explain their importance in various fields. Stabilization of proteins in chloroplast and cytosol protects efficiently the photosynthetic apparatus and also enzymes involved in detoxification of ROS. The enhanced rate of proline anabolism in the chloroplasts can contribute to the stabilization of redox balance and to the maintenance of cellular homeostasis by dissipating the excess of reducing potential when electron transport is saturated during adverse conditions. Proline catabolism in the mitochondria is connected to oxidative respiration and administers energy to resumed growth after stress. Moreover, proline oxidation can regulate mitochondrial ROS levels and influence programmed cell death. As well as modulating responses to abiotic and biotic stresses, proline appears to function as a metabolic signal that regulates metabolite pools and redox balance controlling the expression of numerous genes and influencing plant growth and development. The enhancement of proline biosynthesis via increasing the expression of rate-limiting genes in transgenic plants is considered as the best option. Salt tolerance could be improved by the constitutive overexpression of p5cs, and further improvements have been achieved when the feedback-insensitive form of P5CS has been used. The improvement of drought or/and salt tolerance of crop plants via engineering proline metabolism is a plausible opportunity that should be explored more extensively. Eventually, the fact that proline can act as a signaling effector and influence defense pathways and regulates metabolic and developmental processes offers additional opportunities for plant improvement. Further studies are required to study the feasibility to engineer flowering time or to improve defenses against certain pathogens via the targeted engineering of proline metabolism.
Appl Biochem Biotechnol Acknowledgments This work was supported by grants from the Ministry of Higher Education, Scientific Research and Technology, Tunisia.
References 1 Michael, L. (1981). Effect of proline residues on protein folding. Journal of Molecular Biology, 145, 251– 263. 2 Grathwohl, C., & Wüthrich, K. (1976). The X-pro peptide bond as an nmr probe for conformational studies of flexible linear peptides. Biopolymers, 15(10), 2025–2041. 3 Saibi, W., Drira, M., Yacoubi, I., Feki, K., & Brini, F. (2015). Empiric, structural and in silico findings give birth to plausible explanations for the multifunctionality of the wheat dehydrin (DHN-5). Acta Physiologiae Plantarum, 37(3), 1–8. 4 Szabados, L., & Savouré, A. (2010). Proline: a multifunctional amino acid. Trends in Plant Science, 15(2), 89–97. 5 Shi, M, Hao, S, Chan, T, & Xiang, J. (2006). CD4+ T cells stimulate memory CD8+ T cell expansion via acquired pMHC I complexes and costimulatory molecules, and IL-2 secretion. Journal of Leukocyte Biology 80: 1354–1363 6 Prasad, U., bin Jalaludin, M. A., Rajadurai, P., Pizza, G., De Vinci, C., Viza, D., & Levine, P. H. (1996). Transfer factor with anti-EBV activity as an adjuvant therapy for nasopharyngeal carcinoma: a pilot study. Biotherapy, 9(1–3), 109–115. 7 Rauscher, S., Baud, S., Miao, M., Keeley, F. W., & Pomès, R. (2006). Proline and glycine control protein selforganization into elastomeric or amyloid fibrils. Structure, 14(11), 1667–1676. 8 Washington, J. M., Rathjen, J., Felquer, F., Lonic, A., Bettess, M. D., Hamra, N., Semendric, L., Tan, B. S., Lake, J. A., Keough, R. A., Morris, M. B., & Rathjen, P. D. (2010). L-Proline induces differentiation of ES cells: a novel role for an amino acid in the regulation of pluripotent cells in culture. American Journal of Physiology. Cell Physiology, 298(5), 982–992. 9 Chauhan, S., Kumar, K., & Patial, B. S. (2013). Study of acoustic parameters of proline in lecithin-ethanol mixture at varying temperature. Indian Journal of Pure and Applied Physics, 51, 531–541. 10 Visiers, I., Braunheim, B. B., & Weinstein, H. (2000). Prokink: a protocol for numerical evaluation of helix distortions by proline. Protein Engineering Design and Selection, 13(9), 603–606. 11 List, B., Hoang, L., & Martin, H. J. (2004). New mechanistic studies on the proline-catalyzed aldol reaction. PNAS, 101(16), 5839–5842. 12 Nájera, C., Sansano, J. M., & Yus, M. (2010). Metal complexes versus organocatalysts in asymmetric 1,3dipolar cycloadditions. Journal of the Brazilian Chemical Society, 21, 377–412. 13 Hayat, S., Hayat, Q., Alyemeni, M. N., Wani, A. S., Pichtel, J., & Ahmad, A. (2012). Role of proline under changing environments. Plant Signaling & Behavior, 7(11), 1456–1466. 14 Iqbala, N., Umara, S., Khanb, N. A., & Khan, M. I. R. (2014). A new perspective of phytohormones in salinity tolerance: regulation of proline metabolism. Environmental and Experimental Botany, 100, 34–42. 15 Hansom, J., Hanssen, M., Wiese, A., Hendriks, M. M. W. B., & Smeekens, S. (2008). The sucrose regulated transcription factor bZIP11 affects amino acid metabolism by regulating the expression of ASPARAGINE SYNTHETASE1 and PROLINE DEHYDROGENASE. Plant Journal, 53(6), 935–949. 16 Gobinathan, P, Murali, P.V., & Panneerselvam, R. (2009). Interactive effects of calcium chloride on salinityinduced proline metabolism in Pennisetum typoidies. Advances in Biological Research 3 (5–6): 168-173 17 Kumar Kiran Ghanti, S., Sujata, K. G., Vijay Kumar, B. M., Nataraja Karba, N., Janardhan Reddy, K., Srinath Rao, M., & Kavt Kishor, P. B. (2011). Heterologous expression of P5CS gene in chickpea enhances salt tolerance without affecting yield. Biologia Plantarum, 55(4), 634–640. 18 Ahmad, P., Ashraf, M., Younis, M., Hu, X., Kumar, A., Akram, N. A., & Al-Qurainy, F. (2012). Role of transgenic plants in agriculture and biopharming. Biotechnology Advances, 30(3), 524–540. 19 Ismail T. (2011) Plant responses to drought and salinity stress. Advances in Botanical Research 57:2–555. ISBN:978–0–12–387692-8ISSN:0065–2296 20 Foyer, C. H., & Graham, N. (2000). Oxygen processing in photosynthesis: regulation and signaling. The New Phytologist, 146, 359–388. 21 Savouré, A., Jaoua, S., Hua, X. J., Ardiles, W., Van Montagu, M., & Verbruggen, N. (1995). Isolation, characterization, and chromosomal location of a gene encoding the 1-pyrroline-5-carboxylate synthetase in Arabidopsis thaliana. FEBS Letters, 372, 13–19. 22 Verbruggen, N., & Hermans, C. (2008). Proline accumulation in plants: a review. Amino Acids, 35(4), 753–759. 23 Nicolás, M. C., Mariela, I. M., & María, E. A. (2011). Proline dehydrogenase contributes to pathogen defense in Arabidopsis. Plant Physiology, 155(4), 1947–1959.
