Zinc supplementation and light intensity affect 2-acetyl-1-pyrroline (2AP) formation in fragrant rice.

BACKGROUND: Improving the yield and aroma content of fragrant rice is the focus of fragrant rice research. Light and Zinc (Zn) management generally cause regulations in the 2-acetyl-1-pyrroline (2AP) accumulation in fragrant rice. In addition, Zn promotes rice growth and improves rice yield, which has the potential to compensate for the negative impact of low light on fragrant rice yield. However, the potential of Zn to improve fragrant rice yield and 2AP content under shading conditions has not been verified. METHODS: Field experiments were conducted in the rice season (May-September) in 2019 to 2021. Two light i.e., normal light (NL) and low light (LL) and four Zn levels i.e., 0 kg Zn ha- 1 (N0), 1 kg Zn ha- 1 (Zn1), 2 kg Zn ha- 1(Zn2), and 3 kg Zn ha- 1 (Zn3), which applied at booting stage was set up. The grain yield, 2AP contents, Zn content in polished rice, photosynthesis related indicators, MDA content, antioxidant enzyme activity and the biochemical parameters related to 2AP formation were investigated. RESULTS: Shading reduced yield by 8.74% and increased 2AP content by 24.37%. In addition, shading reduced net photosynthetic rate (Pn), superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), and increased proline, γ-aminobutyric acid (GABA), and pyrroline-5-carboxylic acid (P5C), proline dehydrogenase (PDH), △1-pyrroline-5-carboxylic acid synthetase (P5CS), malondialdehyde (MDA). With increasing Zn application levels, yield, 2AP, Zn content in polished rice, Pn, proline, P5C, GABA, PDH, P5CS, SOD, CAT and POD increased, and MDA decreased. Significant Light and Zn interaction effect on 2AP content was detected, and both shading and increasing Zn application increased the 2AP content. CONCLUSION: Shading can increase the 2AP content but reduce the yield of fragrant rice. Increasing Zn application under shading conditions can further promote the biosynthesis of 2AP, but the effect of improving yield is limited.

Proline dehydrogenase in cancer: apoptosis, autophagy, nutrient dependency and cancer therapy.

L-proline catabolism is emerging as a key pathway that is critical to cellular metabolism, growth, survival, and death. Proline dehydrogenase (PRODH) enzyme, which catalyzes the first step of proline catabolism, has diverse functional roles in regulating many pathophysiological processes, including apoptosis, autophagy, cell senescence, and cancer metastasis. Notably, accumulated evidence demonstrated that PRODH plays complex role in many types of cancers. In this review, we briefly introduce the function of PRODH, then its expression in different types of cancer. We next discuss the regulation of PRODH in cancer, the downstream pathways of PRODH and the therapies that are under investigation. Finally, we propose novel insights for future perspectives on the modulation of PRODH.

Tea polyphenol epigallocatechin-3-gallate inhibits cell proliferation in a patient-derived triple-negative breast cancer xenograft mouse model via inhibition of proline-dehydrogenase-induced effects.

Triple-negative breast cancers (TNBCs) lack specific targeted therapy options and have evolved into highly chemo-resistant tumors that metastasize to multiple organs. The present study demonstrated that the proline dehydrogenase (PRODH) mRNA level in paired (tumor vs. normal) human breast tissue samples (n=234) was 6.6-fold greater than normal cells (*p=0.021). We established stable PRODH-overexpressing TNBC (HS578T) cells, and the malignant phenotypes were evaluated using soft agar colony formation and Transwell migration assays. The results demonstrated that PRODH induced epithelial-mesenchymal transition in cancer cells and increased cell proliferation. The present study found that the tea polyphenol epigallocatechin-3-gallate (EGCG) significantly inhibited PRODH and its regulated proteins, such as alpha-smooth muscle actin (alpha-SMA) expression in TNBC cells. These findings support the targeting of the PRODH signaling pathway as a potential therapeutic strategy in preventing cancer cell metastasis. The patient-derived xenograft (PDX) mouse model is highly relevant to real human tumor growth. We established a TNBC-PDX (F4, n=4 in each group)mouse model. The PDX mice were treated with EGCG (50 mg/kg), and the results indicated that EGCG significantly inhibited PDX tumor growth (*p = 0.013). These experiments provide additional evidence to evaluate the antitumor effects of EGCG-induced PRODH inhibition for clinical therapeutic application, especially in TNBC patients.

Meta-analysis on the association between genetic polymorphisms and prepulse inhibition of the acoustic startle response.

Sensorimotor gating measured by prepulse inhibition (PPI) of the acoustic startle response (ASR) has been proposed as one of the most promising electrophysiological endophenotypes of schizophrenia. During the past decade, a number of publications have reported significant associations between genetic polymorphisms and PPI in samples of schizophrenia patients and healthy volunteers. However, an overall evaluation of the robustness of these results has not been published so far. Therefore, we performed the first meta-analysis of published and unpublished associations between gene polymorphisms and PPI of ASR. Unpublished associations between genetic polymorphisms and PPI were derived from three independent samples. In total, 120 single observations from 16 independent samples with 2660 study participants and 43 polymorphisms were included. After correction for multiple testing based on false discovery rate and considering the number of analyzed polymorphisms, significant associations were shown for four variants, even though none of these associations survived a genome-wide correction (P<5∗10-8). These results imply that PPI might be modulated by four genotypes - COMT rs4680 (primarily in males), GRIK3 rs1027599, TCF4 rs9960767, and PRODH rs385440 - indicating a role of these gene variations in the development of early information processing deficits in schizophrenia. However, the overall impact of single genes on PPI is still rather small suggesting that PPI is - like the disease phenotype - highly polygenic. Future genome-wide analyses studies with large sample sizes will enhance our understanding on the genetic architecture of PPI.

Supplementation of 2-Ap, Zn and La Improves 2-Acetyl-1-Pyrroline Concentrations in Detached Aromatic Rice Panicles In Vitro.

Aromatic rice is highly prized by consumers worldwide due to its special aromatic character. 2-acetyl-1-pyrroline (2-AP) is considered to be the single most important volatile compound responsible for aroma in aromatic rice. The present study demonstrated the effects of 2-AP, zinc (Zn) and lanthanum (La) on the 2-AP concentration of detached aromatic rice panicles in vitro. Detached panicles from three well-known aromatic cultivars, Guixiangzhan, Pin14, and Pin 15, were cultured separately in basic culture medium supplemented with 2-AP, Zn and La, and 2-AP concentrations were assessed at 7 and 14 days after culture (DAC). The results show that supplementation of 2-AP, Zn and La in the basic culture medium significantly increases the accumulation of proline. 2-AP concentration and the activity of proline dehydrogenase (ProDH) were also increased in rice grains. Zn concentrations were also found to be higher when Zn was added to the basic culture medium, and La concentrations in grains were too low to be measured. Additionally, grain 2-AP concentrations were significantly and positively correlated with proline concentrations, ProDH activities in grains and 2-AP in culture medium. In summary, higher grain 2-AP concentrations might be due to Zn- and La-induced increases in proline concentrations and ProDH activities, as well as the direct uptake and transportation of 2-AP from the culture medium. Furthermore, application of both Zn and La might be helpful for improving aroma formation in rice. However, interactions of both these elements with the complex process of 2-AP formation remain to be explored.

Selenium and sulfur influence ethylene formation and alleviate cadmium-induced oxidative stress by improving proline and glutathione production in wheat.

We have studied the influence of selenium (Se) and sulfur (S) in the protection of photosynthetic capacity of wheat (Triticum aestivum) against cadmium (Cd) stress. The involvement of ethylene and its interaction with proline and antioxidant metabolism in the tolerance of plants to Cd stress was evaluated. Application of Se or S alleviated Cd-induced oxidative stress by increasing proline accumulation as a result of increased activity of glutamyl kinase (GK) and decreased activity of proline oxidase (PROX). These nutrients also induced the activity of ATP-sulfurylase and serine acetyl transferase and the content of cysteine (Cys), a precursor for the synthesis of both reduced glutathione (GSH) and ethylene. Further, application of Se and S to plants under Cd stress reduced ethylene level and increased the activity of glutathione reductase (GR) and glutathione peroxidase (GPX), reduced oxidative stress and improved photosynthesis and growth. The involvement of ethylene in Se and S-mediated alleviation of Cd stress was substantiated with the use of ethylene biosynthesis inhibitor aminoethoxyvinylglycine (AVG). The use of AVG reversed the effects of Se and S on ethylene, content of proline and GSH and photosynthesis. The results suggested that Se and S both reversed Cd-induced oxidative stress by regulating ethylene formation, proline and GSH metabolism. Thus, Se or S-induced regulatory interaction between ethylene and proline and GSH metabolism may be used for the reversal of Cd-induced oxidative stress.

New developments in diagnosis and treatment update: Schizophrenia/first episode psychosis in children and adolescents.

Childhood onset schizophrenia (COS) is diagnosed before the age of 13 years, and early onset schizophrenia (EOS) is diagnosed before the age of 18 years. EOS is considered extremely rare and its prevalence in comparison to the worldwide prevalence of schizophrenia (1%) has not adequately been studied. Patients who experience the first episode of psychosis need to be treated early and optimally to lessen the morbidity and improve the outcome of the illness. Treatment needs to be a combination of both pharmacological and non-pharmacological modalities. Pharmacological intervention is necessary for remission, improvement of positive symptoms and to aid with the efficacy of psychosocial interventions. There is a lack of efficacy and safety data of the use of antipsychotic medication in children, with most of the information available being extrapolations of adult data. An increased use of atypical antipsychotic drugs in the treatment of EOS has been accompanied by growing concern about the appropriate use and associated side effects in children and adolescents. This update highlights new developments, concepts and treatment trends in EOS.

Hyperprolinemia induces DNA, protein and lipid damage in blood of rats: antioxidant protection.

The present study investigated the effects of hyperprolinemia on oxidative damage to biomolecules (protein, lipids and DNA) and the antioxidant status in blood of rats. The influence of the antioxidants on the effects elicited by proline was also examined. Wistar rats received two daily injections of proline and/or vitamin E plus C (6th-28th day of life) and were killed 12h after the last injection. Results showed that hyperprolinemia induced a significant oxidative damage to proteins, lipids and DNA demonstrated by increased carbonyl content, malondialdehyde levels and a greater damage index in comet assay, respectively. The concomitant antioxidants administration to proline treatment completely prevented oxidative damage to proteins, but partially prevented lipids and DNA damage. We also observed that the non-enzymatic antioxidant potential was decreased by proline treatment and partially prevented by antioxidant supplementation. The plasma levels of vitamins E and C significantly increased in rats treated exogenously with these vitamins but, interestingly, when proline was administered concomitantly with vitamin E plus C, the levels of these vitamins were similar to those found in plasma of control and proline rats. Our findings suggest that hyperprolinemia promotes oxidative damage to the three major classes of macromolecules in blood of rats. These effects were accomplished by decrease in non-enzymatic antioxidant potential and decrease in vitamins administered exogenously, which significantly decreased oxidative damage to biomolecules studied. These data suggest that antioxidants may be an effective adjuvant therapeutic to limit oxidative damage caused by proline.

Vitamin D insufficiency and schizophrenia risk: evaluation of hyperprolinemia as a mediator of association.

25-Hydroxyvitamin D (25(OH)D) deficits have been associated with schizophrenia susceptibility and supplementation has been recommended for those at-risk. Although the mechanism by which a deficit confers risk is unknown, vitamin D is a potent transcriptional modulator and can regulate proline dehydrogenase (PRODH) expression. PRODH maps to chromosome 22q11, a region conferring the highest known genetic risk of schizophrenia, and encodes proline oxidase, which catalyzes proline catabolism. l-Proline is a neuromodulator at glutamatergic synapses, and peripheral hyperprolinemia has been associated with decreased IQ, cognitive impairment, schizoaffective disorder, and schizophrenia. We investigated the relationship between 25(OH)D and schizophrenia, comparing fasting plasma 25(OH)D in 64 patients and 90 matched controls. We then tested for a mediating effect of hyperprolinemia on the association between 25(OH)D and schizophrenia. 25(OH)D levels were significantly lower in patients, and 25(OH)D insufficiency associated with schizophrenia (OR 2.1, adjusted p=0.044, 95% CI: 1.02-4.46). Moreover, 25(OH)D insufficient subjects had three times greater odds of hyperprolinemia than those with optimal levels (p=0.035, 95% CI: 1.08-8.91), and formal testing established that hyperprolinemia is a significantly mediating phenotype that may explain over a third of the effect of 25(OH)D insufficiency on schizophrenia risk. This study presents a mechanism by which 25(OH)D insufficiency confers risk of schizophrenia; via proline elevation due to reduced PRODH expression, and a concomitant dysregulation of neurotransmission. Although definitive causality cannot be confirmed, these findings strongly support vitamin D supplementation in patients, particularly for those with elevated proline, who may represent a large subgroup of the schizophrenia population.

Δ(1)-pyrroline-5-carboxylate/glutamate biogenesis is required for fungal virulence and sporulation.

Proline dehydrogenase (Prodh) and Δ(1)-pyrroline-5-carboxylate dehydrogenase (P5Cdh) are two key enzymes in the cellular biogenesis of glutamate. Recombinant Prodh and P5Cdh proteins of the chestnut blight fungus Cryphonectria parasitica were investigated and showed activity in in vitro assays. Additionally, the C. parasitica Prodh and P5Cdh genes were able to complement the Saccharomyces cerevisiae put1 and put2 null mutants, respectively, to allow these proline auxotrophic yeast mutants to grow on media with proline as the sole source of nitrogen. Deletion of the Prodh gene in C. parasitica resulted in hypovirulence and a lower level of sporulation, whereas deletion of P5Cdh resulted in hypovirulence though no effect on sporulation; both Δprodh and Δp5cdh mutants were unable to grow on minimal medium with proline as the sole nitrogen source. In a wild-type strain, the intracellular level of proline and the activity of Prodh and P5Cdh increased after supplementation of exogenous proline, though the intracellular Δ(1)-pyrroline-5-carboxylate (P5C) content remained unchanged. Prodh and P5Cdh were both transcriptionally down-regulated in cells infected with hypovirus. The disruption of other genes with products involved in the conversion of arginine to ornithine, ornithine and glutamate to P5C, and P5C to proline in the cytosol did not appear to affect virulence; however, asexual sporulation was reduced in the Δpro1 and Δpro2 mutants. Taken together, our results showed that Prodh, P5Cdh and related mitochondrial functions are essential for virulence and that proline/glutamate pathway components may represent down-stream targets of hypovirus regulation in C. parasitica.

Antimicrobial activity of an Amazon medicinal plant (Chancapiedra) (Phyllanthus niruri L.) against Helicobacter pylori and lactic acid bacteria.

The potential of water extracts of the Amazon medicinal plant Chancapiedra (Phyllanthus niruri L.) from Ecuador and Peru for antimicrobial activity against Helicobacter pylori and different strains of lactic acid bacteria such as Lactobacillus acidophilus, Lactobacillus casei and Lactobacillus plantarum was investigated. H. pylori was inhibited by both water extracts in a dose dependent manner, whereas lactic acid bacterial growth was not affected. Both extracts contained ellagic acid and hydroxycinnamic acid derivatives and exhibited high free radical scavenging linked-antioxidant activities (89%). However, gallic acid was detected only in the Ecuadorian extract. Preliminary studies on the mode of action of Chancapiedra against H. pylori revealed that inhibition may not involve proline dehydrogenase-based oxidative phosphorylation inhibition associated with simple mono-phenolics and could involve ellagitannins or other non-phenolic compounds through a yet unknown mechanism. This study provides evidence about the potential of Chancapiedra for H. pylori inhibition without affecting beneficial lactic acid bacteria.

Inhibitory potential of tea polyphenolics and influence of extraction time against Helicobacter pylori and lack of inhibition of beneficial lactic acid bacteria.

Tea polyphenolics such as catechins are known to have the potential to inhibit many bacterial pathogens. Helicobacter pylori has been identified as an etiologic agent in the development of gastric ulcer, peptic ulcer, gastritis, and many other stomach-related diseases. In this study, we investigated the effect of 9 tea extracts--3 different brands representing 4 different processed types (white, green, oolong, and black)--on the inhibition of H. pylori. Extraction times of 2 and 5 minutes were compared. Most 5-minute extracts showed H. pylori inhibition, whereas 2-minute extracts only of Choice darjeeling black and Tazo white showed inhibition. No recovery was observed after the addition of 0.5 and 5 mM proline, indicating that tea polyphenols do not inhibit H. pylori by inhibition of proline oxidation via proline dehydrogenase. Extracts that showed inhibition were further evaluated for their effect on beneficial lactic acid bacteria. None of the samples showed inhibition, suggesting that tea might be able to inhibit H. pylori without affecting the beneficial lactic acid bacteria. High-performance liquid chromatography indicated the presence of gallic acid, quercetin, caffeine, and tea catechins (including catechin, epicatechin, and epigallocatechin) in all the tea samples. Our study indicates that tea can be potentially used as a low-cost dietary support to combat H. pylori-linked gastric diseases without affecting the beneficial intestinal bacteria.

Behavioral and neurochemical effects of proline.

Proline is an amino acid with an essential role for primary metabolism and physiologic functions. Hyperprolinemia results from the deficiency of specific enzymes for proline catabolism, leading to tissue accumulation of this amino acid. Hyperprolinemic patients can present neurological symptoms and brain abnormalities, whose aetiopathogenesis is poorly understood. This review addresses some of the findings obtained, mainly from animal studies, indicating that high proline levels may be associated to neuropathophysiology of some disorders. In this context, it has been suggested that energy metabolism deficit, Na(+),K(+)-ATPase, kinase creatine, oxidative stress, excitotoxicity, lipid content, as well as purinergic and cholinergic systems are involved in the effect of proline on brain damage and spatial memory deficit. The discussion focuses on the relatively low antioxidant defenses of the brain and the vulnerability of neural tissue to reactive species. This offers new perspectives for potential therapeutic strategies for this condition, which may include the early use of appropriate antioxidants as a novel adjuvant therapy, besides the usual treatment based on special diets poor in proline.

The relationship of proline content and metabolism on the productivity of maize plants.

The free proline content in maize ear-leaves, silk and pollen were analyzed in field grown plants which had matured to the pollination stage. Using maize hybrids PR34F02, PR35P12 and PR36B08 field trials were set up at two locations in eastern Croatia in two different years. Two enzymes of proline metabolism were analyzed in the same leaf samples and specific activities of synthetase (P5CS) and proline dehydrogenase (PDH). Plant productivity was evaluated at harvest by the estimation of total and fully developed grain number per ear and per plant, the mean single grain mass, and the mass of grain per plant. The year in which the plants were grown had a very significant effect on the free proline content in the leaf and pollen, as well as on the enzyme activities assayed. The differences between the plants from the two localities were very significant in all tested parameters of plant grain productivity. There was a significant genotype effect on proline content and P5CS total activity in leaf and on all the productivity parameters. Some of the correlations established suggest that the rate of proline synthesis and degradation in maize ear-leaf at pollination might contribute to the final grain production of the maize plant. Multiple regression analyses was used to further analyze the relationship between proline and grain productivity, but it is clear that future work should include other environmental conditions, plant species and organs such as roots.

Effect of exogenous γ-aminobutyric acid treatment on proline accumulation and chilling injury in peach fruit after long-term cold storage.

The effect of exogenous γ-aminobutyric acid (GABA) on chilling injury of peach fruit was investigated. Freshly harvested peaches were treated with 1, 5, or 10 mM GABA at 20 °C for 10 min and then stored at 1 °C for up to 5 weeks. The results showed that all of the GABA treatments could reduce chilling injury of peach fruit with 5 mM being the most effective concentration. GABA treatment significantly enhanced the accumulation of endogenous GABA and proline, which resulted from the increased activities of glutamate decarboxylase, Δ(1)-pyrroline-5-carboxylate synthetase, and ornithine δ-aminotransferase and decreased proline dehydrogenase activity. Our results revealed that GABA treatment may be a useful technique to alleviate chilling injury in cold-stored peach fruit, and the reduction in chilling by GABA may be due to the induction of endogenous GABA and proline accumulation. These data are the first evidence that exogenous GABA induced chilling tolerance in postharvest horticultural products.

Rhodiola-induced inhibition of adipogenesis involves antioxidant enzyme response associated with pentose phosphate pathway.

The aim of this study was to investigate whether Rhodiola crenulata extract and tyrosol, a major bioactive phenolic compound present in Rhodiola, change the activities of endogenous antioxidant enzyme response (AER) and energy pathways linked to proline-mediated pentose phosphate pathway (PPP) during adipogenesis. Treatment with Rhodiola extracts inhibited the activities of proline dehydrogenase (PDH) and glucose-6-phosphate dehydrogenase (G6PDH) as well as lipid accumulation and reactive oxygen species (ROS) production. The inhibition of PDH and G6PDH activities by Rhodiola likely prevented proline oxidation required for critical ATP generation that is coupled to AER via the PPP, leading to inhibition of adipogenesis. Rhodiola extracts dose-dependently increased superoxide dismutase (SOD) activity, resulting in a reduced ROS level during adipogenesis. Moreover, the effects of tyrosol, a major bioactive compound in Rhodiola species, were directly correlated with all observed effects by Rhodiola extracts. These results indicate that the antiadipogenic effects of Rhodiola extracts can be attributed to a phenolic tyrosol that may potentially disrupt proline-mediated energy generation and AER via PPP, resulting in the suppression of adipogenesis and lipid accumulation. This further provides a biochemical rationale to identify the roles of phenolics that modulate the cellular redox environment and therefore have relevance for obesity management.

Analysis of prepulse inhibition in mouse lines overexpressing 22q11.2 orthologues.

Animal models have been useful in elucidating the genetic basis of the cognitive and behavioural phenotypes associated with the 22q11.2 microdeletions. Loss-of-function models have implicated a number of genes as playing a role in prepulse inhibition (PPI) of the startle response. Here, we report the generation and initial analysis of bacterial artificial chromosome (BAC) transgenic (Tg) mice, overexpressing genes from within the 22q11.2 locus. We used engineered BAC constructs to generate Tg lines and quantitative RT-PCR to assess levels of gene expression in each line. We assessed PPI and open-field activity in mice from two low copy number lines. In Tg-1, a line overexpressing Prodh and Vpreb2, PPI was significantly increased at prepulse levels of 78 dB and 82 dB while no differences were found in activity measures. By contrast, no significant differences were found in PPI testing of the Tg-2 line overexpressing Zdhhc8, Ranbp1, Htf9c, T10, Arvcf and Comt. Taken together with previous loss-of-function reports, these findings suggest that Prodh has a key role in modulating the degree of sensorimotor gating in mice and possibly in humans and provide additional support for an important role of this pathway in modulating behavioural deficits associated with genomic gains or losses at 22q11.2.

Proline oxidase induces apoptosis in tumor cells, and its expression is frequently absent or reduced in renal carcinomas.

Proline oxidase is a p53-induced gene that can mediate apoptosis in lung carcinoma cells. Here, we provide evidence implicating a role for proline oxidase in renal carcinoma. We observed absent or reduced expression of proline oxidase in 8 of 12 primary renal cell carcinomas, with respect to their normal tissue counterparts. Two renal cell carcinomas, which displayed little or no expression of proline oxidase, expressed p53s that were less capable of inducing proline oxidase than p53 isolated from normal renal tissue. One of those tumor-derived p53s contained a double transition mutation at amino acid residues 125 (Ala to Thr) and 193 (Arg to His), and the other exhibited a single transition mutation at amino acid 149 (Ser to Phe). Forced up-regulation of proline oxidase induced the formation of reactive oxygen species and mediated apoptosis in the 786-0 renal cell carcinoma cell line. A proline oxidase antisense vector repressed p53-induced up-regulation of proline oxidase, release of cytochrome c from mitochondria, and apoptosis in 786-0 renal carcinoma cells. Taken together, these findings support a role for proline oxidase as a downstream effector in p53-mediated apoptosis. We hypothesize that its altered expression can contribute to the development of renal carcinomas. The presence of proline oxidase in mitochondria, a primary organelle that regulates apoptosis, places this molecule in a subcellular localization that can directly influence the apoptotic pathway and thus tumorigenesis.

Proline accumulation by mutation or disruption of the proline oxidase gene improves resistance to freezing and desiccation stresses in Saccharomyces cerevisiae.

We examined the role of intracellular proline under freezing and desiccation stress conditions in Saccharomyces cerevisiae. When cultured in liquid minimal medium, the proline-nonutilizing mutant containing the put1 mutation (proline oxidase-deficient) produced more intracellular proline, and increased the cell survival rate as compared to the wild-type strain after freezing and desiccation. We also constructed two PUT1 gene disruptants. PUT1-disrupted mutants in minimal medium supplemented with external proline at 0.1% accumulated higher proline levels than those of the control strains (17-22-fold). These disruptants also had a 2-5-fold increase in cell viability compared to the control strains after freezing and desiccation stresses. These results indicate that proline has a stress-protective function in yeast.

Proline catabolism by Pseudomonas putida: cloning, characterization, and expression of the put genes in the presence of root exudates.

Pseudomonas putida KT2442 is a root-colonizing strain which can use proline, one of the major components in root exudates, as its sole carbon and nitrogen source. A P. putida mutant unable to grow with proline as the sole carbon and nitrogen source was isolated after random mini-Tn5-Km mutagenesis. The mini-Tn5 insertion was located at the putA gene, which is adjacent to and divergent from the putP gene. The putA gene codes for a protein of 1,315 amino acid residues which is homologous to the PutA protein of Escherichia coli, Salmonella enterica serovar Typhimurium, Rhodobacter capsulatus, and several Rhizobium strains. The central part of P. putida PutA showed homology to the proline dehydrogenase of Saccharomyces cerevisiae and Drosophila melanogaster, whereas the C-terminal end was homologous to the pyrroline-5-carboxylate dehydrogenase of S. cerevisiae and a number of aldehyde dehydrogenases. This suggests that in P. putida, both enzymatic steps for proline conversion to glutamic acid are catalyzed by a single polypeptide. The putP gene was homologous to the putP genes of several prokaryotic microorganisms, and its gene product is an integral inner-membrane protein involved in the uptake of proline. The expression of both genes was induced by proline added in the culture medium and was regulated by PutA. In a P. putida putA-deficient background, expression of both putA and putP genes was maximal and proline independent. Corn root exudates collected during 7 days also strongly induced the P. putida put genes, as determined by using fusions of the put promoters to 'lacZ. The induction ratio for the putA promoter (about 20-fold) was 6-fold higher than the induction ratio for the putP promoter.