Proline Biosynthesis In Plants Research Articles

The structure of Medicago truncatula δ(1)-pyrroline-5-carboxylate reductase provides new insights into regulation of proline biosynthesis in plants.

The two pathways for proline biosynthesis in higher plants share the last step, the conversion of δ1-pyrroline-5-carboxylate (P5C) to L-proline, which is catalyzed by P5C reductase (P5CR, EC 1.5.1.2) with the use of NAD(P)H as a coenzyme. There is increasing amount of evidence to suggest a complex regulation of P5CR activity at the post-translational level, yet the molecular basis of these mechanisms is unknown. Here we report the three-dimensional structure of the P5CR enzyme from the model legume Medicago truncatula (Mt). The crystal structures of unliganded MtP5CR decamer, and its complexes with the products NAD+, NADP+, and L-proline were refined using x-ray diffraction data (at 1.7, 1.85, 1.95, and 2.1 Å resolution, respectively). Based on the presented structural data, the coenzyme preference for NADPH over NADH was explained, and NADPH is suggested to be the only coenzyme used by MtP5CR in vivo. Furthermore, the insensitivity of MtP5CR to feed-back inhibition by proline, revealed by enzymatic analysis, was correlated with structural features. Additionally, a mechanism for the modulation of enzyme activity by chloride anions is discussed, as well as the rationale for the possible development of effective enzyme inhibitors.

Overexpression of a novel feedback-desensitized Δ1-pyrroline-5-carboxylate synthetase increases proline accumulation and confers salt tolerance in transgenic Nicotiana plumbaginifolia

∆1-Pyrroline-5-carboxylate synthetase (P5CS) is a key enzyme of proline biosynthesis in plants. The Nicotiana plumbaginifolia mutant RNa was previously reported as salt tolerant, overproducing proline, and having reduced feedback inhibition of proline biosynthesis. A mutation in an RNa P5CS gene changing a conserved glutamate codon at position 155 into a glycine codon (E155G) is a possible explanation for the phenotypes in the RNa mutant. In this work, the E155G mutation was introduced in the Arabidopsis thaliana P5CS1 cDNA driven by the CaMV 35S promoter and expressed in wild type N. plumbaginifolia (P2) and in the RNa mutant. The E155G substitution decreased feedback inhibition of P5CS by proline in transgenic lines. Under normal conditions, proline content and growth were comparable in the transgenic lines and the P2 control. In contrast, when plants were exposed to salt stress, proline levels in all transgenic lines increased compared to P2. Growth rate was also less affected by salt treatment in the transgenic lines than in the controls. Semi-quantitative RT-PCR showed higher p35S-P5CS transgene derived mRNA levels in plants submitted to salt stress than in non-stressed plants. To evaluate the role of the CaMV 35S promoter during salt stress, N. plumbaginifolia lines harbouring a p35S-GUS cassette were investigated. Increased GUS activities were observed when submitting leaf discs of transformants to salt stress compared to normal conditions. This result can partly explain increased proline content in the p35S-P5CS transgenic plants when exposed to salt stress.

Two P5CS genes from common bean exhibiting different tolerance to salt stress in transgenic Arabidopsis

Many plants accumulate proline in response to salt stress. Δ-pyrroline-5-carboxylate synthetase (P5CS) is the rate-limiting enzyme in proline biosynthesis in plants. Plasmid DNA (pCHF3-PvP5CS1 and pCHF3-PvP5CS2) containing the selectable neomycin phosphotransferase gene for kanamycin resistance and Phaseolus vulgaris P5CS (PvP5CS1 and PvP5CS2) cDNA was introduced into Arabidopsis plants using Agrobacterium-mediated gene transfer. Southern blot, northern blot and RT-PCR analyses demonstrated that the foreign genes were integrated into Arabidopsis chromosomal DNA and expressed. Single-gene transformants were analysed in this study. Transgenic plants expressed higher levels of PvP5CS1 and PvP5CS2 transcripts under salt stress conditions than under normal conditions. When treated with 0, 100 and 200 mM NaCl, the average proline content in leaves of transgenic plants was significantly higher (P < 0.01) than control plants. The average relative electrical conductivity (REC) of transgenic lines was significantly lower (P < 0.01) than control plants under salt stress condition. Biomass production of transgenic lines was significantly higher (P < 0.05) than control plants under 200 mM NaCl stress treatment. These results indicated that introducing PvP5CS1 and PvP5CS2 cDNA into transgenic Arabidopsis caused proline overproduction, increasing salt tolerance. Although the expression of PvP5CS1 in L4 lines and PvP5CS2 in S4 lines was the same under salt stress condition, the S4 lines accumulated 1.6 and 1.9 times more proline than the L4 lines under 100 and 200 mM NaCl treatments, respectively. The REC of S4 plants was 0.5 (100 mM NaCl) and 0.6 times (200 mM NaCl) that of L4 plants. The biomass production of S4 plants was 1.6 times (200 mM NaCl) more than in L4 plants. Total P5CS enzyme activity of S4 was significantly higher than that of L4. These results implied that the PvP5CS2 protein had stronger capacity to catalyze proline synthesis than PvP5CS1 under salt stress condition.

Cloning, characterization and expression analysis of Δ1-pyrroline-5-carboxylate synthetase (P5CS) gene in harvested papaya (Carica papaya) fruit under temperature stress

Δ1-Pyrroline-5-carboxylate synthetase (P5CS) catalyzes the rate-limiting step in proline biosynthesis in plants. In the present study, a full-length cDNA, denominated as CpP5CS2 for Δ1-pyrroline-5-carboxylate synthetase (P5CS), was cloned from papaya using in silico cloning and 3′- or 5′-rapid amplification of cDNA ends (RACE). The full-length cDNA of CpP5CS2 was 2583bp, with a 2151bp open reading frame (ORF) encoding a 717 amino acid polypeptide. Sequence analysis showed that CpP5CS2 contained several substrate-binding and catalytic domains and had high homology to other plant P5CSs. The expression pattern of CpP5CS2 in papaya under low (7°C) and high temperature (35°C) stresses was examined using real-time quantitative PCR. The results showed that both stresses induced CpP5CS2 expression during the storage period, and the increased expression of CpP5CS2 preceded proline accumulation. In addition, the high temperature caused a more significant induction of CpP5CS2 expression and a higher level of accumulated proline than low temperature.

Modulation of intracellular proline levels affects flowering time and inflorescence architecture in Arabidopsis

We reported previously that the plant oncogene rolD anticipates and stimulates flowering in Nicotiana tabacum, and encodes ornithine cyclodeaminase, an enzyme catalysing the conversion of ornithine to proline. To investigate on the possible role of proline in flowering, we altered the expression of AtP5CS1, encoding the rate-limiting enzyme of proline biosynthesis in plants. Accordingly we characterized a mutant line containing a T-DNA insertion into AtP5CS1 and introduced in Arabidopsis thaliana AtP5CS1 under the control of the CaMV35S promoter. As expected homozygous p5cs1 mutants behaved as late flowering. In addition p5cs1 mutants exhibited a shorter size and contained lower levels of proline, compared to wild type. 35S-P5CS1 plants, manifested, early in development, overexpression of P5CS1 and accumulation of proline, leading to early flowering, both under long- and short-day conditions. Later in development, down-regulation of P5CS1 occurred in 35S-P5CS1 leaves, leading to proline reduction, and, in turn, impaired bolting and stunted growth. Salt-stress restored expression of P5CS1 and proline accumulation in P5CS1-transformed plants, as well as rescuing growth. Our data suggest that proline plays a key role in flower transition, bolting and coflorescence formation.

Response of Garden Pea to Nickel Toxicity

Hydroponically-grown Pisum sativum plants were sensitive to Ni2+ (100–1000 µmol/L). Lower Ni2+ concentration (25 µmol/L) stimulated general growth up to 20 d. Nickel (Ni)-free plants showed nodulation (average 27 per plant), but the frequency went down to 82% even in 100 µmol/L Ni2+. The Ni2+ concentration, when raised to 200 or 300 µmol/L, inhibited nodulation completely. The Ni2+ accumulation in P. sativum was concentration- and time-dependent. External Ni2+ concentration (200 or 300 µmol/L) contributed cation buildup of 34 µg g−1 dw, average. The root/shoot share of Ni2+ was invariably 60 : 40 indicating that 40% of Ni2+ accumulated gets translocated to foliar parts. Proline biosynthesis in plants was promoted by 3.36-fold. A 40:60 root/shoot share of this amino acid infers that it is synthesized primarily in foliar parts and can be adopted as a reliable metal-stress indicator. The Ni2+ load invariably lowered thiol biosynthesis in P. sativum with the exception of a 1.14-fold stimulation by the lowest Ni2+ dose (25 µmol/L) during the first 5 d. However, plants failed to sustain this stimulation any longer as the level paralleled the Ni-free control during subsequent growth. Urease activity increased to a maximum of 24.7 µmol g−1 dw during 20 d although it declined to 22.4 units at the end of the experiment (30 d). The marginal stimulation of urease by Ni2+ can be arranged as 100 > 25 > 0 > 200 > 300 µmol/L. Pisum sativum roots even at the seedling stage, had double the amount of urease as shoots. It is suggested that this may primarily be to facilitate the plant assimilation of urea from the ambient environment.

The Role of Proline in Osmoregulation in Phytophthora nicotianae

A cDNA encoding Δ1-pyrroline-5-carboxylate reductase (P5CR), the enzyme that catalyzes the final step in proline biosynthesis in plants and bacteria, has been cloned from the oomycete plant pathogen Phytophthora nicotianae. Genomic DNA blots indicated that P. nicotianae and P. cinnamomi each contain a single P5CR gene, whereas P. infestans contains one or two genes. Complementation of a strain of Escherichia coli defective in the P5CR protein by the P. nicotianae P5CR cDNA confirmed that the gene encoded a functional P5CR. RNA blots revealed that P5CR was expressed at a much higher level in P. nicotianae zoospores than in vegetative hyphae, sporulating hyphae, or germlings. Furthermore, P5CR mRNA levels increased with time in zoospores, demonstrating that transcription occurs in zoospores. mRNA encoding histidine and tryptophan biosynthetic enzymes was not highly and specifically expressed in zoospores, indicating that the developmental pattern of P5CR expression was not simply a reflection of overall amino acid biosynthesis as might be required for protein synthesis. Measurement of free proline in P. nicotianae at different stages of the life cycle revealed that proline concentration was highest in sporulating hyphae. Vegetative hyphae and germlings contained about 50% of this concentration of proline, and zoospores contained only about 1% of this level. Substantial amounts of proline were measured in the medium into which the zoospore had been released. Hypoosmotic shock of P. nicotianae hyphae led to an approximately 50% decrease in free proline concentration within 30 min of transfer to low-osmolarity medium and was accompanied by an increase in the level of P5CR mRNA. These data are discussed in terms of the possible role of proline in osmoregulation in Phytophthora.

An improved method for transformation of lettuce by Agrobacterium tumefaciens with a gene that confers freezing resistance

An efficient method for constructing transgenic lettuce cultivars by Agrobacterium tumefaciens was described by Torres et al., 1993. In the present work, an improvement of the above procedure is described and applied to transform the cultivar Grand Rapids with a mutated P5CS gene. The major modifications were concerned with turning more practical the transformation and regeneration protocols. Also we tried to improve transformation steps by increasing injured area in explants and prolonging co-cultivation with Agrobacteria (in larger concentration). A more significant selective pressure was used against non-transformed plants and bacteria. In these work we were concerned to obtain T1 and T2 seeds. The P5CS gene codes for a delta¹-pyrroline-5-carboxylate synthetase, a bifunctional enzyme that catalyzes two steps of proline biosynthesis in plants (Zhang et al., 1995; Peng et al., 1996), while the mutated gene is insensitive to feedback inhibition by proline. The potential benefit of this gene is to confer water stress resistance (drought, salt, cold) due to increased intracellular levels of proline that works like an osmoprotectant. In this work could obtain and characterize transgenic lettuce lineages which are resistant to freezing temperature.

Characterization of Δ 1-pyrroline-5-carboxylate synthetase gene promoter in transgenic Arabidopsis thaliana subjected to water stress

Many plants accumulate proline in response to osmotic stress. Δ 1-pyrroline-5-carboxylate synthetase (P5CS) is the rate limiting enzyme in proline biosynthesis in plants. The promoter of an Arabidopsis P5CS gene was isolated and analyzed using a β-glucuronidase (GUS) reporter gene in transgenic plants. NaCl stress induced GUS activity to the maximum level within 3 h while dehydration continued to enhance the GUS activity up to 24 h. The P5CS gene promoter was found to contain two transcription start sites, and dehydration stimulated the transcription predominantly from the downstream site. Abscisic acid (ABA) failed to induce the P5CS gene expression although ABA is known to increase during water stress. These observations suggest that induction of the P5CS gene is a specific response of plants to salt and water stresses.

Removal of Feedback Inhibition of Δ1-Pyrroline-5-carboxylate Synthetase, a Bifunctional Enzyme Catalyzing the First Two Steps of Proline Biosynthesis in Plants

delta 1-Pyrroline-5-carboxylate synthetase (P5CS) catalyzes the first two steps in proline biosynthesis in plants. The Vigna aconitifolia P5CS cDNA was expressed in Escherichia coli, and the enzyme was purified to homogeneity. The Vigna P5CS exhibited two activities, gamma-glutamyl kinase (gamma-GK) and glutamic acid-5-semialdehyde (GSA) dehydrogenase. The gamma-GK activity of the P5CS was detected by the hydroxamate assay and by a [14C]glutamate assay. The native molecular mass of the P5CS was 450 kDa with six identical subunits. The Vigna P5CS showed a Km of 3.6 mM for glutamate, while the Km for ATP was 2.7 mM. The gamma-GK activity of the P5CS was competitively inhibited by proline, while its GSA dehydrogenase activity was insensitive to proline. In addition, a protein inhibitor of the P5CS was observed in the plant cell. Western blot showed that the level of the P5CS was enhanced in Vigna root under salt stress. A single substitution of an alanine for a phenylalanine at amino acid residue 129 of the P5CS resulted in a significant reduction of proline feedback inhibition. The 50% inhibition values of gamma-GK activity of the wild type and the mutant P5CS were observed at 5 mM and 960 mM proline, respectively. The other properties of the mutant P5CS remained unchanged. These results may allow genetic manipulation of proline biosynthesis and overproduction of proline in plants for conferring water stress tolerance.

Allosteric regulation of proline-inhibitable glutamate kinase from winter-wheat leaves by L-proline, adenosine diphosphate and low temperatures

The determining way of regulation of proline biosynthesis in plants at normal and low temperatures has been discovered. The first enzyme of the biosynthetic pathway, allosteric glutamate kinase (GK 1), is very active at low temperatures (0°, -20°C). In the normal temperature range the biosynthesis of proline is controlled by inhibition of GK 1 through feedback by L-proline (I50 = 80μmol/l, I100 = 50mmol/l). At low temperatures the inhibition switches over into allosteric activation and the biosynthesis of proline is started. It is a new type of instantaneous change of central regulation of the enzyme with change of ecology. In a range of temperature ADP is only a partial allosteric inhibitor of GK 1. L-Proline, ADP and low temperature bring about complex heterotropic cooperatives in GK 1, which are demonstrated by changes in the relation {vo; [L-Glu]}. A hypothetical scheme of central regulation of proline-inhibitable glutamate kinase by L-proline with changes of temperature and concentration of salts has been worked out.