Ornithine Δ-aminotransferase Research Articles

Pyrroline-5-carboxylate metabolism protein complex detected in Arabidopsis thaliana leaf mitochondria.

Proline dehydrogenase (ProDH) and pyrroline-5-carboxylate (P5C) dehydrogenase (P5CDH) catalyse the oxidation of proline into glutamate via the intermediates P5C and glutamate-semialdehyde (GSA), which spontaneously interconvert. P5C and GSA are also intermediates in the production of glutamate from ornithine and α-ketoglutarate catalysed by ornithine δ-aminotransferase (OAT). ProDH and P5CDH form a fused bifunctional PutA enzyme in Gram-negative bacteria and are associated in a bifunctional substrate-channelling complex in Thermus thermophilus; however, the physical proximity of ProDH and P5CDH in eukaryotes has not been described. Here, we report evidence of physical proximity and interactions between Arabidopsis ProDH, P5CDH, and OAT in the mitochondria of plants during dark-induced leaf senescence when all three enzymes are expressed. Pairwise interactions and localization of the three enzymes were investigated using bimolecular fluorescence complementation with confocal microscopy in tobacco and sub-mitochondrial fractionation in Arabidopsis. Evidence for a complex composed of ProDH, P5CDH, and OAT was revealed by co-migration of the proteins in native conditions upon gel electrophoresis. Co-immunoprecipitation coupled with mass spectrometry analysis confirmed the presence of the P5C metabolism complex in Arabidopsis. Pull-down assays further demonstrated a direct interaction between ProDH1 and P5CDH. P5C metabolism complexes might channel P5C among the constituent enzymes and directly provide electrons to the respiratory electron chain via ProDH.

Clinical, biochemical and molecular analysis in a cohort of individuals with gyrate atrophy

BackgroundGyrate atrophy of the choroid and retina is a rare autosomal recessive metabolic disorder caused by biallelic variants in the OAT gene, encoding the enzyme ornithine δ-aminotransferase. Impaired enzymatic activity leads to systemic hyperornithinaemia, which in turn underlies progressive chorioretinal degeneration. In this study, we describe the clinical and molecular findings in a cohort of individuals with gyrate atrophy.MethodsStudy participants were recruited through a tertiary UK clinical ophthalmic genetic service. All cases had a biochemical and molecular diagnosis of gyrate atrophy. Retrospective phenotypic and biochemical data were collected using electronic healthcare records.Results18 affected individuals from 12 families (8 male, 10 female) met the study inclusion criteria. The median age at diagnosis was 8 years (range 10 months – 33 years) and all cases had hyperornithinaemia (median: 800 micromoles/L; range: 458–1244 micromoles/L). Common features at presentation included high myopia (10/18) and nyctalopia (5/18). Ophthalmic findings were present in all study participants who were above the age of 6 years. One third of patients had co-existing macular oedema and two thirds developed pre-senile cataracts. Compliance with dietary modifications was suboptimal in most cases. A subset of participants had extraocular features including a trend towards reduced fat-free mass and developmental delay.ConclusionsOur findings highlight the importance of multidisciplinary care in families with gyrate atrophy. Secondary ophthalmic complications such as macular oedema and cataract formation are common. Management of affected individuals remains challenging due to the highly restrictive nature of the recommended diet and the limited evidence-base for current strategies.

Exogenous melatonin mitigates chilling injury in zucchini fruit by enhancing antioxidant system activity, promoting endogenous proline and GABA accumulation, and preserving cell wall stability

Zucchini fruit are sensitive to chilling injury (CI) during postharvest low temperature storage which makes their cold stored life short. In this work, zucchini fruit were treated with melatonin (200 µmol L−1) and stored at 5 ºC for 15 d. The pre-storage melatonin treatment reduced weight loss (6.51%) and suppressed symptoms of CI (1.5 Score) along with lower malondialdehyde (MDA), relative ion leakage (RIL), superoxide anion and hydrogen peroxide (H2O2) in contrast with control. The treated zucchini exhibited higher L* (51.89) and lower a* (−10.17) and b* (29.15) values of exocarp in comparison with control. Melatonin treated zucchini showed enhanced γ-aminobutyric acid (GABA) concentration due to higher glutamate content and stimulated glutamate decarboxylase (GAD) and suppressed γ-aminobutyric acid transaminase (GABA-T) activities. In addition, melatonin application increased proline accumulation owing to stimulation of Δ-1-pyrroline-5-carboxylate synthetase (P5CS) and ornithine δ-aminotransferase (OAT) enzymes and reduced proline dehydrogenase (ProDH) activity as compared with control. The activity of antioxidative such as ascorbate peroxidase (APX), peroxidase (POD), catalase (CAT) and superoxide dismutase (SOD) enzymes remained higher in melatonin treated zucchini. The fruit treated with melatonin exhibited reduced water soluble pectin (WSPN) along with higher sodium carbonate soluble pectin (SCSP), protopectin (PRPN), chelate soluble pectin (CSPN), cellulose (CLS) and hemicellulose (HLS) in comparison with control. The exogenous melatonin suppressed the activities of polygalacturonase (PG), β-galactosidase (β-Gal), cellulase (CX), and pectin methylesterase (PME) along with higher firmness. In conclusion, exogenous melatonin at 200 µmol L−1 concentration could be used for zucchini fruit CI mitigation under cold storage.

Ornithine δ-aminotransferase OsOAT is critical for male fertility and cold tolerance during rice plant development.

Cold stress is a major factor limiting the production and geographical distribution of rice (Oryza sativa) varieties. However, the molecular mechanisms underlying cold tolerance remain to be elucidated. Here, we report that ornithine δ-aminotransferase (OsOAT) contributes to cold tolerance during the vegetative and reproductive development of rice. osoat mutant was identified as a temperature-sensitive male sterile mutant with deformed floral organs and seedlings sensitive to cold stress. Comparative transcriptome analysis showed that OsOAT mutation and cold treatment of the wild-type plant led to similar changes in the global gene expression profiles in anthers. OsOAT genes in indica rice Huanghuazhan (HHZ) and japonica rice Wuyungeng (WYG) are different in gene structure and response to cold. OsOAT is cold-inducible in WYG but cold-irresponsive in HHZ. Further studies showed that indica varieties carry both WYG-type and HHZ-type OsOAT, whereas japonica varieties mostly carry WYG-type OsOAT. Cultivars carrying HHZ-type OsOAT are mainly distributed in low-latitude regions, whereas varieties carrying WYG-type OsOAT are distributed in both low- and high-latitude regions. Moreover, indica varieties carrying WYG-type OsOAT generally have higher seed-setting rates than those carrying HHZ-type OsOAT under cold stress at reproductive stage, highlighting the favorable selection for WYG-type OsOAT during domestication and breeding to cope with low temperatures.

N-α-Lauroyl-L-arginine ethyl ester hydrochloride combined with hot water treatment alleviates chilling injury of postharvest cucumber fruit

Postharvest cucumber fruit is prone to chilling injury (CI) under cold stress, leading to severe quality deterioration. In this study, the effects of N-α-Lauroyl-l-arginine ethyl ester hydrochloride (LAE), hot water (HW), and LAE combined with HW treatments on CI in cucumber fruit stored at 4 ± 1 ℃ were investigated. The response surface methodology (RSM) confirmed that the LAE concentration of 1.00 g L−1, HW temperature of 44 ℃, and immersion time of 10 min were the optimal condition for cucumber fruit preservation during cold storage. Further analysis revealed that LAE, HW, and LAE combined with HW treatments, all significantly mitigated chilling injury symptoms in cucumber fruit in comparison with the control, but the application of LAE combined with HW was the most effective method for enhancing cold tolerance in cucumber fruit. Moreover, LAE combined with HW treatment could reduce reactive oxygen species (ROS) accumulation, and enhance superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX) activities in cold-stored cucumber fruit. Meanwhile, the activities of arginine decarboxylase (ADC), ornithine decarboxylase (ODC), Δ1-pyrroline-5-carboxylate synthetase (P5CS), ornithine δ-aminotransferase (OAT), and glutamate decarboxylase (GAD) were also increased by LAE combined with HW treatment, while the activities of proline dehydrogenase (PDH) and γ-aminobutyric acid transaminase (GABA-T) were decreased by LAE combined with HW treatment, which contributed to maintaining higher polyamines, proline, and GABA contents. These findings suggested that LAE combined with HW treatment can reduce chilling injury in cucumber fruit via enhancing antioxidant enzymes activities, PA, proline, and GABA contents to maintain ROS homeostasis during cold storage.

Pyrroline-5-carboxylate dehydrogenase is an essential enzyme for proline dehydrogenase function during dark-induced senescence in Arabidopsis thaliana.

During leaf senescence, nitrogen is remobilized and carbon backbones are replenished by amino acid catabolism, with many of the key reactions occurring in mitochondria. The intermediate Δ1 -pyrroline-5-carboxylate (P5C) is common to some catabolic pathways, thus linking the metabolism of several amino acids, including proline and arginine. Specifically, mitochondrial proline catabolism involves sequential action of proline dehydrogenase (ProDH) and P5C dehydrogenase (P5CDH) to produce P5C and then glutamate. Arginine catabolism produces urea and ornithine, the latter in the presence of α-ketoglutarate being converted by ornithine δ-aminotransferase (OAT) into P5C and glutamate. Metabolic changes during dark-induced leaf senescence (DIS) were studied in Arabidopsis thaliana leaves of Col-0 and in prodh1prodh2, p5cdh, and oat mutants. Progression of DIS was followed by measuring chlorophyll and proline contents for 5 days. Metabolomic profiling of 116 compounds revealed similar profiles of Col-0 and oat metabolism, distinct from prodh1prodh2 and p5cdh metabolism. Metabolic dynamics were accelerated in p5cdh by one day. Notably, more P5C and proline accumulated in p5cdh than in prodh1prodh2. ProDH1 enzymatic activity and protein amount were significantly down-regulated in p5cdh mutant at day 4 of DIS. Mitochondrial P5C levels appeared critical in determining the flow through interconnected amino acid remobilisation pathways to sustain senescence. This article is protected by copyright. All rights reserved.

Elevated level of chilling tolerance in cucumber fruit was obtained by β-aminobutyric acid via the regulation of antioxidative response and metabolism of energy, proline and unsaturated fatty acid

• BABA treatment enhances the chilling tolerance in cucumber fruit. • BABA application improved the antioxidant capacity of harvested cucumber fruit. • BABA-treated fruit exhibited higher level of energy status. • BABA promoted proline accumulation and maintained higher level of UFA contents. This experimental study was conducted to investigate the effects of β-aminobutyric acid (BABA) on chilling injury in cold-stored cucumber fruit. Results showed that BABA decreased the development of chilling injury in cucumber fruit. BABA-treated fruit exhibited elevated antioxidant ability, indicated by high activities of superoxide dismutase (SOD) and catalase (CAT) and low levels of hydrogen peroxide and superoxide anion. BABA treatment maintained a high level of energy status by promoting the activities and genes expression of H + -ATPase, Ca 2+ -ATPase, succinate dehydrogenase (SDH) and cytochrome C oxidase (CCO). Increased enzymes activities and genes expression of Δ 1 -pyrroline-5-carboxylate synthase (PCS) and ornithine-δ-aminotransferase (OAT) and depressed enzyme activity and gene expression of proline dehydrogenase (PDH) in BABA-treated cucumber collectively promoted the accumulation of proline. Furthermore, BABA treatment delayed the decreasing trend of unsaturated fatty acid content in cucumber. Thus, BABA is effective in enhancing chilling tolerance in cucumber fruit possibly by improving the antioxidant ability and adjusting the metabolism of energy, proline and unsaturated fatty acids.

Exogenous Proline Improves Salt Tolerance of Alfalfa through Modulation of Antioxidant Capacity, Ion Homeostasis, and Proline Metabolism.

Alfalfa (Medicago sativa L.) is an important forage crop, and its productivity is severely affected by salt stress. Although proline is a compatible osmolyte that plays an important role in regulating plant abiotic stress resistance, the basic mechanism of proline requires further clarification regarding the effect of proline in mitigating the harmful effects of salinity. Here, we investigate the protective effects and regulatory mechanisms of proline on salt tolerance of alfalfa. The results show that exogenous proline obviously promotes seed germination and seedling growth of salt-stressed alfalfa. Salt stress results in stunted plant growth, while proline application alleviates this phenomenon by increasing photosynthetic capacity and antioxidant enzyme activities and decreasing cell membrane damage and reactive oxygen species (ROS) accumulation. Plants with proline treatment maintain a better K+/Na+ ratio by reducing Na+ accumulation and increasing K+ content under salt stress. Additionally, proline induces the expression of genes related to antioxidant biosynthesis (Cu/Zn-SOD and APX) and ion homeostasis (SOS1, HKT1, and NHX1) under salt stress conditions. Proline metabolism is mainly regulated by ornithine-δ-aminotransferase (OAT) and proline dehydrogenase (ProDH) activities and their transcription levels, with the proline-treated plants displaying an increase in proline content under salt stress. In addition, OAT activity in the ornithine (Orn) pathway rather than Δ1-pyrroline-5-carboxylate synthetase (P5CS) activity in the glutamate (Glu) pathway is strongly increased under salt stress, made evident by the sharp increase in the expression level of the OAT gene compared to P5CS1 and P5CS2. Our study provides new insight into how exogenous proline improves salt tolerance in plants and that it might be used as a significant practical strategy for cultivating salt-tolerant alfalfa.

Hydrogen Sulfide Treatment Alleviates Chilling Injury in Cucumber Fruit by Regulating Antioxidant Capacity, Energy Metabolism and Proline Metabolism.

Although low-temperature storage could maintain the quality of fruits and vegetables, it may also result in chilling injury (CI) in cold-sensitive produce, such as cucumbers. This can seriously affect their quality.” The antioxidant capacity, energy metabolism and proline metabolism of cucumbers treated with hydrogen sulfide (H2S) were studied in this assay. The outcomes displayed that H2S treatment effectively reduced CI and delayed the increase in electrolyte leakage (EL) and malondialdehyde (MDA) content. In addition, the H2S-treated cucumber fruit exhibited higher L* and hue angle values, as well as nutrients such as ascorbic acid (AsA). The H2S-treated fruit showed lower levels of reactive oxygen species (ROS) and higher antioxidant enzyme activities. Meanwhile, H2S treatment also increased the activities of the essential enzymes involved in energy metabolism, including cytochrome C oxidase (CCO), succinate dehydrogenase (SDH), H+-ATPase and Ca2+-ATPase, which improved the energy supply. H2S induced higher ornithine δ-aminotransferase (OAT) and Δ-1-pyrroline-5-carboxylate synthetase (P5CS) activities, and reduced proline dehydrogenase (PDH) activity, promoting the accumulation of proline. These results indicated that H2S could alleviate CI in the cucumber fruit by modulating antioxidant capacity, energy metabolism and proline metabolism, thereby extending the shelf life of postharvest cucumbers.

Physiological, biochemical and economic characteristics of transgenic winter wheat plants with gene ornitin-Δ-aminotransferases

Aim. To analyze the physiological, biochemical and economic characteristics of genetically modified plants of new promising genotypes of winter bread wheat of seed generation T2 with the heterologous gene of ornithine-δ-aminotransferase of alfalfa. Methods. Agrobacterium-mediated transformation in vitro; biochemical determination of the activity of the enzyme ornithine-δ-aminotransferase (OAT) and the content of free L-proline; morphometric indicators and elements of crop structure; mathematical statistics. Results. It was found that the presence of additional copies of the oat gene in transgenic plants leads to increased activity of the enzyme ornithine-δ-aminotransferase (on average 1.5 times compared to the original plants), but they do not differ significantly from plants of the original genotypes in free L-Proline is neither normal nor under conditions of soil drought. It has been shown that the introduction into the genome of wheat plants of a genetic construct that enhances the expression of the oat gene stimulates root growth both under normal and stressful conditions. Under conditions of insufficient moisture supply, plants of transgenic lines also exceeded untransformed plants in the number and weight of grains from the whole plant. Conclusions. Analysis of physiological and biochemical characteristics and economic characteristics of transgenic soft wheat plants containing the heterologous gene of ornithine-δ-aminotransferase of alfalfa showed their increased tolerance to soil drought compared to non-transgenic genotypes. Biotechnological plants are characterized by a more developed root system, which increased the ability of plants to grow in conditions of water scarcity.

Crystal structure of a novel type of ornithine δ-aminotransferase from the hyperthermophilic archaeon Pyrococcus horikoshii

Ornithine δ-aminotransferase (Orn-AT) activity was detected for the enzyme annotated as a γ-aminobutyrate aminotransferase encoded by PH1423 gene from Pyrococcus horikoshii OT-3. Crystal structures of this novel archaeal ω-aminotransferase were determined for the enzyme in complex with pyridoxal 5′-phosphate (PLP), in complex with PLP and l-ornithine (l-Orn), and in complex with N-(5′-phosphopyridoxyl)-l-glutamate (PLP-l-Glu). Although the sequence identity was relatively low (28%), the main-chain coordinates of P. horikoshii Orn-AT monomer showed notable similarity to those of human Orn-AT. However, the residues recognizing the α-amino group of l-Orn differ between the two enzymes. In human Orn-AT, Tyr55 and Tyr85 recognize the α-amino group, whereas the side chains of Thr92* and Asp93*, which arise from a loop in the neighboring subunit, form hydrogen bonds with the α-amino group of the substrate in P. horikoshii enzyme. Site-directed mutagenesis suggested that Asp93* plays critical roles in maintaining high affinity for the substrate. This study provides new insight into the substrate binding of a novel type of Orn-AT. Moreover, the structure of the enzyme with the reaction-intermediate analogue PLP-l-Glu bound provides the first structural evidence for the “Glu switch” mechanism in the dual substrate specificity of Orn-AT.

Postharvest 24-epibrassinolide treatment alleviates pomegranate fruit chilling injury by regulating proline metabolism and antioxidant activities

Pomegranate fruit is susceptible to chilling injury (CI) during long term cold storage. In the current work, we studied the impact of pre-storage 24-epibrassinolide (24-EBL) application for CI amelioration of pomegranate during cold storage. The fruit were treated with 0, 5, 10 and 15 µmol L−1 24-EBL and stored for 84 d at 5 ± 1 ºC. Results showed that 15 µmol L−1 24-EBL treatment markedly alleviated CI, suppressed weight loss and reduced decay. Similarly, 15 µmol L−1 24-EBL application stimulated the activity of ornithine-δ-aminotransferase (OAT) and Δ1-pyrroline-5-carboxylate synthetase (P5CS) along with suppressed increase in proline dehydrogenase (PDH) enzyme which subsequently resulted in increased proline accumulation. The 15 µmol L−1 24-EBL treated pomegranate fruit had substantially lower activity of lipoxygenase (LOX) enzyme along with reduced concentrations of malondialdehyde (MDA), electrolyte leakage (EL), hydrogen peroxide (H2O2) and superoxide anion (O2•−) contents. In addition, 15 µmol L−1 24-EBL treated pomegranate fruit showed significantly higher activity of catalase (CAT), ascorbate peroxidase (APX), peroxidase (POD) and superoxide dismutase (SOD) enzyme in contrast with control. The 15 µmol L−1 24-EBL treated pomegranate fruit showed higher phenylalanine ammonia lyase (PAL) and had lower polyphenol oxidase (PPO) activity that subsequently resulted in conserved total phenols content. In the same way, pomegranate fruit treated with 15 µmol L−1 24-EBL had markedly higher total anthocyanins content, ascorbic acid and glutathione content as compared with control. It was also observed that 15 µmol L−1 24-EBL treated pomegranate fruit showed lower juice pH, higher titratable acidity (TA) and relatively lower soluble solids content (SSC) in contrast with control. In conclusion, 15 µmol L−1 24-EBL could be considered suitable for CI alleviation and quality maintenance of pomegranate fruit under cold storage.

Cold shock treatment with oxalic acid could alleviate chilling injury in green bell pepper by enhancing antioxidant enzyme activity and regulating proline metabolism

The effects of cold shock, oxalic acid, and combined cold shock + oxalic acid treatments on chilling injury, antioxidant enzyme activity, and proline metabolism of cold-stored green bell pepper fruit were investigated. The results showed that cold shock, oxalic acid treatment, and their combination significantly reduced chilling injury, inhibited the increase of superoxide radical production, hydrogen peroxide content, malondialdehyde levels, and relative membrane permeability, and increased antioxidant enzyme activity. Furthermore, the treatments promoted the accumulation of proline associated with increase in ∆1-pyrroline-5-carboxylate synthetase and ornithine δ-aminotransferase activities, and decrease in proline dehydrogenase activity. The results indicate that cold shock, oxalic acid, and their combination alleviated chilling injury in green bell pepper fruit by increasing antioxidant enzyme activity and proline accumulation. The combined cold shock + oxalic acid treatment was the most effective method for reducing chilling injury, and therefore has broad future application prospects.

. Genes of proline metabolism in biotechnology of increasing wheat osmostability

Aim. To analyse effectiveness of introduced genes of ornithine-δ-aminotransferase (oat) Medicago truncatula and fragments of two copies of first exon and intron of the gene prolinedehydrogenase (pdh) of Arabidopsis thaliana, that form double stranded RNA suppressor of gene of the prolinedehydrogenase wheat, in the increase of her productivity for the actions ofosmotic stress. Methods. Determination of the content of free L-proline (Pro); of the activity of the enzyme ornithine-δ-aminotransferase (OAT), the activity of proline dehydrogenase (PDG), indicators of crop structure. Results. It was found that wheat plants with an additional copy of the oat gene are characterized by increased OAT activity, which is not reflected in the Pro content. Analysis of plants with integrated elements that form a double-stranded RNA suppressor of the pdh gene found a decrease in PDG activity and an increase in Pro content. It was found that T2 generation of biotechnological plants UK 322/17 with suppressor of the pdh gene and UK 95/17 with an additional copy of the oat gene, in drought conditions were characterized by higher rates for a number of crop elements compared to their original forms. Conclusions. Increased expression of the oat gene leads to improved wheat growth, which has a positive effect on plant productivity in conditions of water deficiency. Partial suppression of the proline dehydrogenase gene causes improved performance due to the increased content of free proline during drought.
 Keywords: wheat, transgenic plants, proline, ornithine-δ-aminotransferase, proline dehydrogenase, soil drought, grain productivity.

Exogenous arginine enhances the chilling tolerance in postharvest papaya fruit by regulating arginine and proline metabolism

Papaya fruit is easily affected by low temperature, causing chilling injury (CI). In this study, the exogenous arginine was applied in the storage of postharvest papaya fruit and it was found that 0.8 mM arginine treatment significantly alleviated CI of papaya fruit. Arginine treatment promoted the accumulation of polyamines (PAs), which might be ascribed to the increased activities of arginase, arginine decarboxylase (ADC), ornithine decarboxylase (ODC), and S-adenosylmethionine decarboxylase (SAMDC). A higher accumulation of proline was found in arginine-treated papaya fruit, which may be due to increased activity of ornithine-δ-aminotransferase (OAT) and Δ1-pyrroline-5-carboxylate acid synthase (P5CS) and inhibited activity of proline dehydrogenase (PDH). In addition, arginine treatment increased higher nitric oxide synthase (NOS) activity and promoted the generation of nitric oxide (NO). The results of this study indicate that exogenous arginine may improve the chilling tolerance of postharvest papaya fruit by regulating arginine and proline metabolism. Practical applications In recent years, chilling injury of tropical fruits is a significant problem, including papaya. Many studies on polyamine, proline, nitric oxide, and chilling injury of fruits, but arginine, as a common precursor of polyamine, proline, and nitric oxide, are rarely studied, especially in papaya fruit. Our findings demonstrated that the exogenous arginine can regulate endogenous arginine metabolism, induce polyamine, putrescine, and nitric oxide accumulation, and coordinate to alleviate chilling injury of papaya fruit.

Turnover and Inactivation Mechanisms for (S)-3-Amino-4,4-difluorocyclopent-1-enecarboxylic Acid, a Selective Mechanism-Based Inactivator of Human Ornithine Aminotransferase.

The inhibition of human ornithine δ-aminotransferase (hOAT) is a potential therapeutic approach to treat hepatocellular carcinoma. In this work, (S)-3-amino-4,4-difluorocyclopent-1-enecarboxylic acid (SS-1-148, 6) was identified as a potent mechanism-based inactivator of hOAT while showing excellent selectivity over other related aminotransferases (e.g., GABA-AT). An integrated mechanistic study was performed to investigate the turnover and inactivation mechanisms of 6. A monofluorinated ketone (M10) was identified as the primary metabolite of 6 in hOAT. By soaking hOAT holoenzyme crystals with 6, a precursor to M10 was successfully captured. This gem-diamine intermediate, covalently bound to Lys292, observed for the first time in hOAT/ligand crystals, validates the turnover mechanism proposed for 6. Co-crystallization yielded hOAT in complex with 6 and revealed a novel noncovalent inactivation mechanism in hOAT. Native protein mass spectrometry was utilized for the first time in a study of an aminotransferase inactivator to validate the noncovalent interactions between the ligand and the enzyme; a covalently bonded complex was also identified as a minor form observed in the denaturing intact protein mass spectrum. Spectral and stopped-flow kinetic experiments supported a lysine-assisted E2 fluoride ion elimination, which has never been observed experimentally in other studies of related aminotransferase inactivators. This elimination generated the second external aldimine directly from the initial external aldimine, rather than the typical E1cB elimination mechanism, forming a quinonoid transient state between the two external aldimines. The use of native protein mass spectrometry, X-ray crystallography employing both soaking and co-crystallization methods, and stopped-flow kinetics allowed for the detailed elucidation of unusual turnover and inactivation pathways.

High relative humidity (HRH) storage alleviates chilling injury of zucchini fruit by promoting the accumulation of proline and ABA

Abstract In order to investigate the effect of water loss on the different physiological metabolism of zucchini fruit under cold stress after harvest, two cold rooms (4 ℃) with different relative humidities (RH: 74 ± 2 % and 98 ± 2 %) were used for cold storage. The result suggested that high relative humidity (HRH) storage slowed down the increase of weight loss, chilling injury index, malondialdehyde (MDA) content and cell death rate and maintained the postharvest quality such as firmness and flesh color in zucchini fruit. HRH storage promoted the accumulation of proline and induced higher ornithine δ-aminotransferase (OAT), Δ-1-pyrroline-5-carboxylate synthetase (P5CS) enzyme activities and lower proline dehydrogenase (PDH) enzyme activity as compared with low relative humidity (LRH) storage. The content of putrescine (Put) accumulated with the progress of cold damage, which could be delayed by HRH storage due to higher activities of polyamine oxidase (PAO), diamine oxidase (DAO) and ornithine decarboxylase (ODC). Besides, chilling stress induced the biosynthesis of abscisic acid (ABA) during storage and the zucchini fruit stored in high RH with less water loss showed more accumulation of ABA and higher activity of abscisic acid aldehyde oxidase (AAO) compared to zucchini fruit stored in low RH with severe dehydration during long-term cold storage. Taken together, our results suggested that the high RH storage could reduce water loss and alleviate chilling injury in zucchini fruit after harvest by promoting the accumulation of ABA and proline.

Impact of nitric oxide on proline and putrescine biosynthesisin Chlamydomonas via transcriptional regulation

Nitric oxide plays an important role in regulating adaption of the model alga Chlamydomonas reinhardtii to various environmental stresses. One response to abiotic stresses is the accumulation of protective molecules such as proline and putrescine. The NO treatment led to a significant accumulation of proline in cells. Quantitative real-time expression analysis of proline metabolic genes in NO-treated cells showed a prolonged upregulation of the gene encoding γ-glutamyl kinase 1 (GGK1) in the glutamate biosynthetic pathway. Furthermore, truncated hemoglobin 2 (THB2)-underexpressing strains with an enhanced endogenous NO demonstrated a higher proline content and GGK1 mRNA abundances than the wild type. In contrast, transcription of the gene encoding ornithine δ-aminotransferase in the ornithine pathway of proline biosynthesis decreased after treatment with NO. This suggests the predominance of the glutamate pathway over the ornithine pathway. We also found that the expression of the proline dehydrogenase gene encoding a key enzyme in proline catabolism was downregulated in NO-treated cells. Chlamydomonas reinhardtii exposed to exogenous NO also showed an increased ornithine decarboxylase 2 mRNA and putrescine content. Our findings indicate a clear link between changes in NO application and proline and putrescine content via transcriptional regulation of respective enzymes.

Influence of Melatonin Treatment on Peel Browning of Cold-Stored “Nanguo” Pears

The effects of melatonin (MT) on chilling injury-induced peel browning of “Nanguo” pears were investigated during the fruit shelf-life at 20 °C, after storage at 0 °C for 120 days. Melatonin treatment alleviated the severity of peel browning, as evidenced by the lower incidence of peel browning and browning index of the MT-treated “Nanguo” pear, and delayed browning time compared to that observed in control fruit. In addition, MT treatment reduced lipoxygenase activity and malondialdehyde content and enhanced the activities of phenylalanine ammonia lyase, shikimate dehydrogenase, 4-coumarate:coenzyme A ligase, cinnamate-4-hydroxylase, and 6-phosphogluconate dehydrogenase but inhibited the activity of polyphenol oxidase, which catalyzes the oxidation of phenolic compounds to quinones. Furthermore, MT treatment enhanced the accumulation of total phenolic compounds, thereby improving antioxidant capacity and maintaining cell membrane integrity, and stimulated the accumulation of proline by promoting the activities of key enzymes involved in proline synthesis, namely Δ1-pyrroline-5-carboxylate synthase and ornithine δ-aminotransferase, which collectively contribute to improved chilling tolerance and delayed browning. These findings indicate that MT treatment effectively relieves peel browning of cold-stored “Nanguo” pears by regulating the metabolism of phenolic compounds and proline.

Melatonin Enhances Cold Tolerance by Regulating Energy and Proline Metabolism in Litchi Fruit.

Melatonin (MLT) is a vital signaling molecule that regulates multiple physiological processes in higher plants. In the current study, the role of MLT in regulating chilling tolerance and its possible mechanisms in litchi fruit during storage at ambient temperatures after its removal from refrigeration was investigated. The results show that the application of MLT (400 μM, dipping for 20 min) to ‘Baitangying’ litchi fruit effectively delayed the development of chilling injury (CI) while inhibiting pericarp discoloration, as indicated by higher chromacity values (L*, a*, b*) and anthocyanin levels. MLT treatment suppressed the enhancements of the relative electrical conductivity (REC) and malondialdehyde (MDA) content, which might contribute to the maintenance of membrane integrity in litchi fruit. MLT treatment slowed the decline in cellular energy level, as evidenced by higher adenosine triphosphate (ATP) content and a higher energy charge (EC), which might be ascribed to the increased activities of enzymes associated with energy metabolism including H+-ATPase, Ca2+-ATPase, succinate dehydrogenase (SDH), and cytochrome C oxidase (CCO). In addition, MLT treatment resulted in enhanced proline accumulation, which was likely a consequence of the increased activities of ornithine-δ-aminotransferase (OAT) and Δ1-pyrroline-5-carboxylate synthase (P5CS) and the suppressed activity of proline dehydrogenase (PDH). These results suggest that the enhanced chilling tolerance of litchi fruit after MLT treatment might involve the regulation of energy and proline metabolism.