Transgenic Medicago truncatula plants that accumulate proline display nitrogen-fixing activity with enhanced tolerance to osmotic stress

Legume root nodule nitrogen-fixing activity is severely affected by osmotic stress. Proline accumulation has been shown to induce tolerance to salt stress, and transgenic plants over-expressing Delta(1)-pyrroline-5-carboxylate synthetase (P5CS), which accumulates high levels of proline, display enhanced osmotolerance. Here, we transformed the model legume Medicago truncatula with the P5CS gene from Vigna aconitifolia, and nodule activity was evaluated under osmotic stress in transgenic plants that showed high proline accumulation levels. Nitrogen fixation was significantly less affected by salt treatment compared to wild-type (WT) plants. To our knowledge, this is the first time that transgenic legumes have been produced that display nitrogen-fixing activity with enhanced tolerance to osmotic stress. We studied the expression of M. truncatula proline-related endogenous genes M. truncatulaDelta(1)-pyrroline-5-carboxylate synthetase 1 (MtP5CS1), M. truncatulaDelta(1)-pyrroline-5-carboxylate synthetase 2 (MtP5CS2), M. truncatula ornithine delta-aminotransferase (MtOAT), M. truncatula proline dehydrogenase (MtProDH) and a proline transporter gene in both WT and transgenic plants. Our results indicate that proline metabolism is finely regulated in response to osmotic stress in an organ-specific manner. The transgenic model allowed us to analyse some of the biochemical and molecular mechanisms that are activated in the nodule in response to high salt conditions, and to ascertain the essential role of proline in the maintenance of nitrogen-fixing activity under osmotic stress.     

A novel Δ-pyrroline-5-carboxylate synthetase gene of Medicago truncatula plays a predominant role in stress-induced proline accumulation during symbiotic nitrogen fixation

Proline accumulates in environmentally stressed plant cells including those of legume roots and nodules, but how its level is regulated is poorly understood. Δ¹-Pyrroline-5-carboxylate synthetase (P5CS), the committed-step enzyme of proline biosynthesis, is encoded by two duplicated genes in many plants. Here, we isolated MtP5CS3, a third gene, from Medicago truncatula, whose predicted polypeptide sequence is highly similar to those of previously isolated MtP5CS1 and MtP5CS2 except an extra amino-terminal segment. MtP5CS3 was strongly expressed under salinity and drought in shoots and nodulating roots, while MtP5CS1 was constitutive and MtP5CS2 induced by abscisic acid. Under salinity, MtP5CS3 promoter was more active than those of MtP5CS1 and MtP5CS2, as shown by GUS fusions. Translationally fused MtP5CS1-GFP was localized in the cytoplasm, whereas significant proportions of MtP5CS2-GFP and MtP5CS3-GFP were co-localized with rubisco small subunit protein-fused RFP in transformed hairy root cells. Under salinity, RNA silencing of MtP5CS1 or MtP5CS2 strongly induced MtP5CS3 expression, while that of MtP5CS3 decreased free proline content and nodule number. Consistently, Mtp5cs3, a loss-of-function mutant, accumulated much less proline, formed fewer nodules, and fixed nitrogen significantly less efficiently than the wild type under salinity. Thus, MtP5CS3 plays a critical role in regulating stress-induced proline accumulation during symbiotic nitrogen fixation.     

Ornithine δ-aminotransferase: An enzyme implicated in salt tolerance in higher plants

This review deals with biochemical and physiological aspects of plant ornithine d-aminotransferase (OAT, EC 2.6.1.13). OAT is a mitochondrial enzyme containing pyridoxal-5′-phosphate as a cofactor, which catalyzes the conversion of L-ornithine to L-glutamate γ-semialdehyde using 2-oxoglutarate as a terminal amino group acceptor. It has been described in humans, animals, insects, plants and microorganisms. Based on the crystal structure of human OAT, both substrate binding and reaction mechanism of the enzyme are well understood. OAT shows a large structural and mechanistic similarity to other enzymes from the subgroup III of aminotransferases, which transfer an amino group from a carbon atom that does not carry a carboxyl function. In plants, the enzyme has been implicated in proline biosynthesis and accumulation (via pyrroline-5-carboxylate), which represents a way to regulate cellular osmolarity in response to osmotic stress. However, the exact metabolic pathway involving OAT remains a subject of controversy.Key words: ornithine δ-aminotransferase, osmotic stress, proline, Δ¹-pyrroline-5-carboxylate, pyridoxal-5′-phosphate, semialdehyde, transamination     

The effect of salt stress on polyamine biosynthesis and content in mung bean plants and in halophytes

The activity of L-arginine decarboxylase (EC 4.1.1.19) and L-ornithine decarboxylase (EC 4.1.1.17), polyamine content, and incorporation of arginine and ornithine into polyamines, were determined in mung bean [Vigna radiata (L.) Wilczek] plants subjected to salt (hypertonic) stress (NaCl at 0.51–2.27 MPa). Changes in enzyme activity in response to hypotonic stress were determined as well in several halophytes [Pulicaria undulata (L.), Kostei, Salsola rosmarinus (Ehr.) Solms-Laub, Mesembryanthemum forskahlei Hochst, and Atriplex halimus L.]. NaCl stress, possibly combined with other types of stress that accompanied the experimental conditions, resulted in organ-specific changes in polyamine biosynthesis and content in mung bean plants. The activity of both enzymes was inhibited in salt-stressed leaves. In roots, however, NaCl induced a 2 to 8-fold increase in ornithine decarboxylase activity. Promotion of ornithine decarboxylase in roots could be detected already 2 h after exposure of excised roots to NaCl, and iso-osmotic concentrations of NaCl and KCl resulted in similar changes in the activity of both enzymes. Putrescine level in shoots of salt-stressed mung bean plants increased considerably, but its level in roots decreased. The effect of NaCl stress on spermidine content was similar, but generally more moderate, resulting in an increased putrescine/spermidine ratio in salt-stressed plants. Exposure of plants to NaCl resulted also in organ-specific changes in the incorporation of both arginine and ornithine into putrescine: incorporation was inhibited in leaf discs but promoted in excised roots of salt-stressed mung bean plants. In contrast to mung bean (and several other glycophytes), ornithine and arginine decarboxylase activity in roots of halophytes increased when plants were exposed to tap water or grown in a pre-washed soil—i.e. a hypotonic stress with respect to their natural habitat. NaCl, when present in the enzymatic assay mixture, inhibited arginine and ornithine decarboxylase in curde extracts of mung bean roots, but did not affect the activity of enzymes extracted from roots of the halophyte Pulicaria. Although no distinct separation between NaCl stress and osmotic stress could be made in the present study, the data suggest that changes in polyamines in response to NaCl stress in mung bean plants are coordinated at the organ level: activation of biosynthetic enzymes concomitant with increased putrescine biosynthesis from its precursors in the root system, and accumulation of putrescine in leaves of salt-stressed plants. In addition, hypertonic stress applied to glycophytes and hypotonic stress applied to halophytes both resulted in an increase in the activity of polyamine biosynthetic enzymes in roots.     

Water stress-induced alterations in the proline metabolism of drought-susceptible and -tolerant cassava (Manihot esculenta) cultivars

The free proline levels and activities of ornithine aminotransferase (EC 2.6.1.13) and proline oxidase (EC 1.5.2.2), two of the enzymes involved in proline metabolism were studied during the induction of water stress in a drought susceptible (M-4) and a drought tolerant (S-1315) cultivar of cassava ( Manihot esculenta Crantz). Water stress induced by polyethylene glycol (MW 6000, osmotic potential — 1.65 MPa) caused a ca 25-fold increase in proline in young excised leaves of the susceptible cultivar (M-4) while the increase was about 9-fold in the tolerant cultivar (S-1315). The activity of ornithine aminotransferase (OAT), a key enzyme involved in the biosynthesis of proline, was found to increase 3-fold in water stressed leaves of M-4 and about 2-fold in those of S-1315. The activity of proline oxidase, which is involved in the degradation of proline to pyrroline-5-carboxylate, was reduced by 50% in M-4 and nearly 25% in S-1315 on water stress. Comparison of the kinetic properties of OAT showed that the enzyme from water-stressed leaves is more stable to heat inactivation compared to that of control. These results indicate that during water stress there are alterations in the metabolism of proline in cassava, and the extent of alteration varies between drought-susceptible and -tolerant cultivars.     

Mycorrhizal-Mediated Lower Proline Accumulation in Poncirus trifoliata under Water Deficit Derives from the Integration of Inhibition of Proline Synthesis with Increase of Proline Degradation

Proline accumulation was often correlated with drought tolerance of plants infected by arbuscular mycorrhizal fungi (AMF), whereas lower proline in some AM plants including citrus was also found under drought stress and the relevant mechanisms have not been fully elaborated. In this study proline accumulation and activity of key enzymes relative to proline biosynthesis (▵¹-pyrroline-5-carboxylate synthetase, P5CS; ornithine-δ-aminotransferase, OAT) and degradation (proline dehydrogenase, ProDH) were determined in trifoliate orange (Poncirus trifoliata, a widely used citrus rootstock) inoculated with or without Funneliformis mosseae and under well-watered (WW) or water deficit (WD). AMF colonization significantly increased plant height, stem diameter, leaf number, root volume, biomass production of both leaves and roots and leaf relative water content, irrespectively of water status. Water deficit induced more tissue proline accumulation, in company with an increase of P5CS activity, but a decrease of OAT and ProDH activity, no matter whether under AM or no-AM. Compared with no-AM treatment, AM treatment resulted in lower proline concentration and content in leaf, root, and total plant under both WW and WD. The AMF colonization significantly decreased the activity of both P5CS and OAT in leaf, root, and total plant under WW and WD, except for an insignificant difference of root OAT under WD. The AMF inoculation also generally increased tissue ProDH activity under WW and WD. Plant proline content significantly positively correlated with plant P5CS activity, negatively with plant ProDH activity, but not with plant OAT activity. These results suggest that AM plants may suffer less from WD, thereby inducing lower proline accumulation, which derives from the integration of an inhibition of proline synthesis with an enhancement of proline degradation.     

NaCl effects on proline metabolism in rice (Oryza sativa) seedlings

Salt-stress effects on osmotic adjustment, ion and proline concentrations as well as proline metabolizing enzyme activities were studied in two rice ( Oryza sativa L.) cultivars differing in salinity resistance: I Kong Pao (IKP; salt-sensitive) and Nona Bokra (salt-resistant). The salt-sensitive cultivar exposed to 50 and 100 m M NaCl in nutritive solution for 3 and 10 days accumulated higher levels of sodium and proline than the salt-resistant cultivar and displayed lower levels of osmotic adjustment. Proline accumulation was not related to proteolysis and could not be explained by stress-induced modifications in Δ¹-pyrroline-5-carboxylate reductase (P5CR; EC 1.5.1.2) or proline dehydrogenase (PDH; EC 1.5.1.2) activities recorded in vitro. The extracted ornithine Δ -aminotransferase (OAT; EC 2.6.1.13) activity was increased by salt stress in the salt-sensitive cultivar only. In both genotypes, salt stress induced an increase in the aminating activity of root glutamate dehydrogenase (GDH; EC 1.4.1.2) while deaminating activity was reduced in the leaves of the salt-sensitive cultivar. The total extracted glutamine synthetase activity (GS; EC 6.3.1.2) was reduced in response to salinity but NaCl had contrasting effects on GS1 and GS2 isoforms in salt-sensitive IKP. Salinity increased the activity of ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) extracted from leaves of both genotypes and increased the activity of NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14) in the salt-sensitive cultivar. It is suggested that proline accumulation is a symptom of salt-stress injury in rice and that its accumulation in salt-sensitive plants results from an increase in OAT activity and an increase in the endogenous pool of its precursor glutamate. The physiological significance of the recorded changes are analyzed in relation to the functions of these enzymes in plant metabolism.     

Exogenous ornithine is an effective precursor and the δ-ornithine amino transferase pathway contributes to proline accumulation under high N recycling in salt-stressed cashew leaves

The role of the δ-ornithine amino transferase (OAT) pathway in proline synthesis is still controversial and was assessed in leaves of cashew plants subjected to salinity. The activities of enzymes and the concentrations of metabolites involved in proline synthesis were examined in parallel with the capacity of exogenous ornithine and glutamate to induce proline accumulation. Proline accumulation was best correlated with OAT activity, which increased 4-fold and was paralleled by NADH oxidation coupled to the activities of OAT and Δ¹-pyrroline-5-carboxylate reductase (P5CR), demonstrating the potential of proline synthesis via OAT/P5C. Overall, the activities of GS, GOGAT and aminating GDH remained practically unchanged under salinity. The activity of P5CR did not respond to NaCl whereas Δ¹-pyrroline-5-carboxylate dehydrogenase was sharply repressed by salinity. We suggest that if the export of P5C from the mitochondria to the cytosol is possible, its subsequent conversion to proline by P5CR may be important. In a time-course experiment, proline accumulation was associated with disturbances in amino acid metabolism as indicated by large increases in the concentrations of ammonia, free amino acids, glutamine, arginine and ornithine. Conversely, glutamate concentrations increased moderately and only within the first 24 h. Exogenous feeding of ornithine as a precursor was very effective in inducing proline accumulation in intact plants and leaf discs, in which proline concentrations were several times higher than glutamate-fed or salt-treated plants. Our data suggest that proline accumulation might be a consequence of salt-induced increase in N recycling, resulting in increased levels of ornithine and other metabolites involved with proline synthesis and OAT activity. Under these metabolic circumstances the OAT pathway might contribute significantly to proline accumulation in salt-stressed cashew leaves.     

The effect of water deficit and excess copper on proline metabolism in Nicotiana benthamiana

Fluctuation in proline content is a widespread phenomenon among plants in response to heavy metal stress. To distinguish between the participation of water deficit and copper on changes in proline metabolism, potted plants and floating leaf discs of tobacco were subjected to CuSO₄ treatments. The application of copper increased the proline content in the leaves concomitantly with decreased leaf relative water content and increased abscisic acid (ABA) content in the potted plant. Excess copper increased the expression of two proline synthesis genes, pyrroline-5-carboxylate synthetase (P5CS) and ornithine aminotransferase (OAT) and suppressed proline catabolism gene, proline dehydrogenase (PDH). However, in the experiment with tobacco leaf discs floating on CuSO₄ solutions, the excess copper decreased proline content and suppressed the expression of the P5CS, OAT and PDH genes. Therefore, proline accumulation in the potted tobacco plants treated with excess Cu treatment might not be the consequence of the increased copper content in tobacco leaves but rather by the accompanied decrease in water content and/or increased ABA content.     

Influence of NaCl and mannitol on peroxidase activity and lipid peroxidation in Centaurea ragusina L. roots and shoots

Centaurea ragusina L. is a Croatian endemic plant species growing on cliffs above the Adriatic Sea, but there is no information about its physiological behavior or stress tolerance. To investigate the response of C. ragusina plants to salinity and drought, we have analysed soluble peroxidase (POD; EC 1.11.1.7) activity, anionic isoperoxidase pattern, levels of malondialdehyde (MDA) and hydrogen peroxide in C. ragusina plants exposed to these stresses. Rooted plantlets grown on MS 1/2 nutrient medium supplemented with mannitol (300 mM) and different concentrations of NaCl (150, 300, 450 or 600 mM) were harvested after 5, 10 and 15 days. Both osmotic treatments significantly increased MDA and hydrogen peroxide contents in C. ragusina shoots after 10 days of stress, while in roots these parameters showed no significant difference compared to control in overall. POD activity of salt-stressed plants changed with respect to different saline treatments and plant organs - in shoots enzymatic activity markedly increased in response to lower saline treatments, especially 300 mM NaCl; otherwise it was similar as in control plants while in roots of plants grown under 450 and 600 mM NaCl it significantly decreased. Drought increased POD activity of both shoots and roots especially after 10 days of experiment. Generally, change in the POD isoenzyme pattern of treated plants was in accordance with the activity change in time. Several POD isoforms (P3, P4 and P9) were specifically induced by salinity and drought.     

Oscillation and regulation of proline content by P5CS and ProDH gene expressions in the light/dark cycles in Arabidopsis thaliana L

The fluctuation of proline content, and protein and mRNA levels of delta1-pyrroline-5-carboxylate synthetase (P5CS) and proline dehydrogenase (ProDH), both of which are involved in proline biosynthesis and degradation, in the shoots of Arabidopsis grown in light/dark cycles were demonstrated under salt-stressed and unstressed conditions. Proline content, as well as proteins and mRNAs of these enzymes, clearly oscillated in the light/dark cycles under the stressed and unstressed conditions. A reciprocal relationship between P5CS and ProDH was observed. Protein levels of P5CS and ProDH were well synchronized with their mRNA levels, although the fluctuation of protein levels was not as significant as that of their mRNA levels. Both mRNA and protein levels of the two enzymes as well as the proline content did not oscillate under the continuous light or the dark conditions. Thus, P5CS and ProDH gene expressions seemed to be involved in light irradiation. Moreover, relative water content (RWC) in the plants oscillated in the light/dark cycles. The fluctuations of proline content in shoot reversely responded to that of RWC. It is suggested that the expression of two genes responds sensitively to a subtle change of cellular water status, and accumulated proline keeps the osmotic balance between cells and the outer environment.     

Role of Arbuscular Mycorrhizal Symbiosis in Proline Biosynthesis and Metabolism of Cicer arietinum L. (Chickpea) Genotypes Under Salt Stress

Salinity causes osmotic stress and negatively impacts plant growth and productivity. Proline is one of the most important osmoprotectants synthesized under stressed conditions. Accumulation of free proline occurs due to enhanced biosynthesis and repressed degradation, and both processes are controlled by feedback regulatory mechanisms. Arbuscular mycorrhizal (AM) fungi are considered to be bioameliorators of salinity stress due to their wide-ranging presence in contaminated soils and their role in modulation of biochemical processes. Chickpea is considered sensitive to salinity. However, reports on AM-induced osmoprotection through regulation of proline biosynthesis in chickpea genotypes are scant. The present study investigated the influence of AM symbiosis on proline metabolism in two chickpea (Cicer arietinum L.) genotypes (PBG-5 and CSG-9505) under salt stress and correlated the same with sodium (Na⁺) ion uptake. Salinity reduced plant biomass (roots and shoots), with roots being more negatively affected than shoots. Mycorrhizal colonization with Glomus mosseae was much stronger in PBG-5 and was correlated with reduced Na⁺ ion uptake and higher growth when compared with CSG-9505 under stressed and unstressed conditions. Mycorrhizal symbiosis with chickpea roots boosted proline biosynthesis by significantly increasing pyrroline-5-carboxylate synthetase (P-5-CS) and glutamate dehydrogenase (GDH) activities with a concomitant decline in proline dehydrogenase (ProDH) activity under salt stress. The enhancement of the activity of these enzymes was higher in PBG-5 than in CSG-9505 and could be directly correlated with the percent mycorrhizal colonization and Na⁺ uptake. The study indicated a strong role of AM symbiosis in enhancing stress tolerance in chickpea by significantly modulating proline metabolism and Na⁺ uptake.     

Proline Metabolism and Cross-Tolerance to Salinity and Heat Stress in Germinating Wheat Seeds

Germination/growth of wheat (Triticum aestivum L., cv. Zimai 1) seeds and changes in the levels of proline and protein as well as in activities of key enzymes involved in proline metabolism in response to salinity-, heat-stresses and their cross-stress were studied. With decreasing water potential caused by increasing concentrations of NaCl, germination percentage, fresh weight of seedlings and protein amount markedly decreased, whereas proline amount slightly increased. The activities of pyrroline-5-carboxylate synthetase (P5CS), ornithine aminotransferase (OAT), and proline dehydrogenase (PDH) peaked at ?0.2 MPa water potential. Germination percentage and amounts of proline and protein increased as germination temperature elevated to 25°C from 15°C, and decreased above 25°C; fresh weight of seedlings increased to 30°C from 15°C, and decreased above 30°C. However, the activities of P5CS, OAT and PDH gradually decreased with elevaing temperature. Seeds pretreated at 33°C or in ?0.8 MPa NaCl solution for various time length increased tolerance to subsequent salt + water stress or heat stress, as measured by germination percentage and fresh weight of seedlings 5 days after beginning of experiment. The acquisition of cross-tolerance resulting in limitation of negative stress effects does not relate directly to proline level and activities of P5CS, OAT and PDH involved in proline metabolism. Proline amount as measured four days or later after stress imposition cannot be considered a symptom of salt-, water- and heat-stress injury or an indicator of the resistance.     

Changes in foliar proline concentration of osmotically stressed barley

The amino acid proline is accumulated in plant tissues in response to a variety of stresses. The existence of two routes for its biosynthesis is well documented. However, little is known about the contribution of each pathway to the accumulation of free proline under stress conditions. In the present study young barley plants were subjected to osmotic stress by treating their roots with 25% polyethylene glycol. Prior to stress imposition roots were incubated for 24 h in nutrient solution containing proline or one of its metabolic precursors: glutamate and ornithine. Free proline quantity in the leaves was measured before and after stress. Relative water content (RWC) was used as a measure of the plant water status. Foliar proline levels showed a significant increase in ornithine- and proline-pretreated plants compared to the control. Nevertheless, no considerable changes in leaf RWC were observed. It was shown that before stress application only ornithine but not glutamate was immediately metabolized to proline. Under stress conditions, however, both precursors were converted into proline. The possible role of this amino acid in the processes of post stress recovery is discussed.     

Removal of feedback inhibition of delta(1)-pyrroline-5-carboxylate synthetase results in increased proline accumulation and protection of plants from osmotic stress

The Delta(1)-pyrroline-5-carboxylate synthetase (P5CS; EC not assigned) is the rate-limiting enzyme in proline (Pro) biosynthesis in plants and is subject to feedback inhibition by Pro. It has been suggested that the feedback regulation of P5CS is lost in plants under stress conditions. We compared Pro levels in transgenic tobacco (Nicotiana tabacum) plants expressing a wild-type form of Vigna aconitifolia P5CS and a mutated form of the enzyme (P5CSF129A) whose feedback inhibition by Pro was removed by site-directed mutagenesis. Transgenic plants expressing P5CSF129A accumulated about 2-fold more Pro than the plants expressing V. aconitifolia wild-type P5CS. This difference was further increased in plants treated with 200 mM NaCl. These results demonstrated that the feedback regulation of P5CS plays a role in controlling the level of Pro in plants under both normal and stress conditions. The elevated Pro also reduced free radical levels in response to osmotic stress, as measured by malondialdehyde production, and significantly improved the ability of the transgenic seedlings to grow in medium containing up to 200 mM NaCl. These findings shed new light on the regulation of Pro biosynthesis in plants and the role of Pro in reducing oxidative stress induced by osmotic stress, in addition to its accepted role as an osmolyte.     

Importance of ornithine-δ-aminotransferase to proline accumulation caused by water stress in detached rice leaves

Proline is synthesized either from glutamate or from ornithine in plants. Relatively little is known about the contribution of the pathway from ornithine to proline biosynthesis. In this paper we investigated the contribution of ornithine--aminotransterase (OAT), an enzyme responsible for ornithine pathway, to proline accumulation in water-stressed detached rice leaves. Although OAT activity increased with the increase of water stress duration, a pattern similar to that obtained for proline accumulation, the ornithine pathway in rice leaves seems to contribute little, if any, to proline accumulation under water stress condition. This conclusion was based on the observations that (a) gabaculine (50 M), an inhibitor of OAT, inhibited about 75% OAT activity caused by water stress but reduced only 20% of proline content and (b) cycloheximide, a protein synthesis inhibitor, had no effect on OAT activity induced by water stress but significantly reduced proline accumulation.