Proline Biosynthesis Research Articles

Ethylene- and Proline-Dependent Regulation of Antioxidant Enzymes to Mitigate Heat Stress and Boost Photosynthetic Efficacy in Wheat Plants

Ethylene regulates the photosynthetic efficiency of plants grown under challenging environments by the regulation of the antioxidant system and other biomolecules, such as osmolytes (proline). The role of ethylene in modulating proline biosynthesis and subsequent changes in antioxidant system to protect wheat (Triticum aestivum L. cv. WH-711) against heat stress was studied. The effects of exogenously sourced ethylene (as 200 µL L−1 ethephon: 2-chloroethylphosphonic acid) and proline (50 mM) were studied in the protection of photosynthetic performance and heat stress tolerance by studying mechanisms of proline biosynthesis, activity and gene expression of antioxidants, and ethylene evolution. The cultivars WH-711, RAJ-3765, PBW-373, HD-2967, PBW-550, DBW-17, PBW-343, and UP-2338 were screened for their proline accumulation capacity and tolerance to heat stress. Plants of the cultivar WH-711 with higher proline accumulation and heat tolerance capacity were subjected to a temperature of 40 °C for 6 h per day over 15 days and then allowed to recover at 28 °C. These plants showed increased H2O2 and TBARS (thiobarbituric acid reactive substances), proline accumulation, and ethylene evolution, activity of antioxidant enzymes, and reduced photosynthetic characteristics. Ethephon plus proline supplementation under heat stress upregulated the antioxidant defense system, reduced oxidative stress, and upregulated psbA and psbB expression and photosynthesis. The study’s outcome may be taken to improve photosynthetic performance and heat stress tolerance through ethylene-enhanced proline accumulation and antioxidant defense system.

Foliar application of procyanidins enhanced the biosynthesis of 2-acetyl-1-pyrroline in aromatic rice (Oryza sativa L.)

BackgroundProcyanidins is a polyphenolic compound with multiple properties. However, the application of exogenous procyanidins in crops has not been reported. Aromatic rice is a high-quality rice with a special aroma and popular with consumers. The 2-acetyl-1-pyrroline (2-AP) is a key compound of aromatic rice aroma. In the current study, aromatic rice plants were sprayed with procyanidins solutions at 0.25 (Pr0.25), 0.50 (Pr0.50), 1.00 (Pr1.00), 2.00 (Pr2.00) g L−1, respectively and treatment sprayed with distilled water was taken as control (CK). The effects of exogenous procyanidins on growth and 2-AP biosynthesis of aromatic rice plants were explored.ResultsCompared with CK, Pr1.00 and Pr2.00 treatments significantly increased 2-AP content by 16.67% and 37.68%, respectively. Higher proline, 1-pyrroline-5-carboxylic acid (P5C), 1-pyrroline, methylglyoxal contents, and lower γ- aminobutyric acid (GABA) content were recorded in Pr1.00 and Pr2.00 treatments than CK. Compared with CK, Pr1.00 and Pr2.00 treatments significantly improved the activities of P5CS and OAT and diminished the activity of BADH. Furthermore, compared with CK, Pr1.00 and Pr2.00 treatments significantly up-regulated the transcript levels of P5CS2, P5CR, OAT, DAO4 and down-regulated the transcript levels of BADH2. Exogenous procyanidins had no substantial effects on plant height, stem diameter, fresh weight, and dry weight of aromatic rice plants.ConclusionsIn conclusion, our findings reported the increment of 2-AP content in aromatic rice under exogenous procyanidins. Our results indicated that the application of exogenous procyanidins enhanced 2-AP biosynthesis by improving proline biosynthesis and inhibiting GABA formation.

Crystal structure of human NADK2 reveals a dimeric organization and active site occlusion by lysine acetylation

NAD+ kinases (NADKs) are metabolite kinases that phosphorylate NAD+ molecules to make NADP+, a limiting substrate for the generation of reducing power NADPH. NADK2 sustains mitochondrial NADPH production that enables proline biosynthesis and antioxidant defense. However, its molecular architecture and mechanistic regulation remain undescribed. Here, we report the crystal structure of human NADK2, revealing a substrate-driven mode of activation. We find that NADK2 presents an unexpected dimeric organization instead of the typical tetrameric assemblage observed for other NADKs. A specific extended segment (aa 325-365) is crucial for NADK2 dimerization and activity. Moreover, we characterize numerous acetylation events, including those on Lys76 and Lys304, which reside near the active site and inhibit NADK2 activity without disrupting dimerization, thereby reducing mitochondrial NADP(H) production, proline synthesis, and cell growth. These findings reveal important molecular insight into the structure and regulation of a vital enzyme in mitochondrial NADPH and proline metabolism.

Autosomal recessive cutis laxa type IIIA: Report of a patient with severe phenotype and review of the literature

Autosomal recessive cutis laxa type IIIA is a very rare genetic condition, caused by pathogenic variants in ALDH18A1, encoding delta-1-pyrroline-5-carboxylate synthase (P5CS). This enzyme catalyzes the reduction of glutamic acid to delta1-pyrroline-5-carboxylate, playing a key role in the de novo biosynthesis of proline, ornithine, and arginine. Autosomal recessive cutis laxa type IIIA is characterized by abundant and wrinkled skin, skeletal anomalies, cataract or corneal clouding and neuro-developmental disorders of variable degree.We report on a patient with autosomal recessive cutis laxa type IIIA, due to a homozygous missense c.1273C > T; p. (Arg425Cys) pathogenic variant in ALDH18A1. The patient presented a severe phenotype with serious urological involvement, peculiar cerebro-vascular abnormalities and neurodevelopmental compromise. This description contributes to better characterize the phenotypic spectrum associated with ALDH18A1 pathogenic variants, confirming the systemic involvement as a typical feature of autosomal recessive cutis laxa type IIIA.

Autophagy loss impedes cancer-associated fibroblast activation via downregulating proline biosynthesis

ABSTRACT Cancer-associated fibroblasts (CAFs) are considered one of the most critical stromal cells that interact with pancreatic ductal adenocarcinoma (PDAC) and promote tumor growth, metastasis, and treatment resistance. Previous studies illustrated macroautophagy/autophagy contributes to CAF activation during tumor progression. Here in our study, we found that autophagy deficiency in CAFs impedes CAF activation by inhibiting proline biosynthesis and collagen production. Furthermore, we uncovered that autophagy promotes proline biosynthesis through mitophagy-mediated regulation of NADK2 (NAD kinase 2, mitochondrial), an enzyme responsible for production of mitochondrial NADP(H). Using an orthotopic mouse model of PDAC, we found that inhibiting mitophagy by targeting PRKN (parkin RBR E3 ubiquitin protein ligase) in the stroma reduced tumor weight. Thus, inhibition of CAFs mitophagy might be an attractive strategy for stroma-focused anti-cancer intervention.Abbreviations: ACTA2/α-SMA: actin alpha 2, smooth muscle, aorta; ACTB/β-actin: actin, beta; ALDH18A1/P5CS: aldehyde dehydrogenase 18 family, member A1; ATG3: autophagy related 3; ATG5: autophagy related 5; BNIP3L: BCL2/adenovirus E1B interacting protein 3-like; CAFs:cancer-associated fibroblasts; COL1A1: collagen, type I, alpha 1; DES: desmin; ECM: extracellular matrix; FABP4: fatty acid binding protein 4, adipocyte; FAP/FAPα: fibroblast activation protein; IHC: immunohistochemical staining; LAMP1: lysosomal-associated membrane protein 1; NADK2: NAD kinase 2, mitochondrial; PC1: pro-collagen 1; PDAC: pancreatic ductal adenocarcinoma; PDGFR: platelet derived growth factor receptor; PDPN: podoplanin; PRKN: parkin RBR E3 ubiquitin protein ligase; PSCs: pancreatic stellate cells; VIM: vimentin; WT: wild-type

Drought-induced proline is mainly synthesized in leaves and transported to roots in watermelon under water deficit

Proline accumulation has been shown to occur in plants in response to various environmental stresses. Although proline metabolism-related genes have been functionally characterized, the inter-organ transport of proline in stressed plants remains unclear. In this study, free proline was detected with significant accumulations in the roots, stems, and leaves of watermelon drought-tolerant germplasm M08 and drought-susceptible line Y34 under drought stress. Expression profiling and enzyme activity measurements revealed that ClP5CS1 gene, rather than its paralog ClP5CS2, mainly contributes to the proline synthesis in leaves via the Glu pathway. Additionally, over-expression of the ClP5CS genes significantly enhanced the drought tolerance of transgenic Arabidopsis lines. Furthermore, we confirmed that proline is mainly synthesized in leaves and transported to roots in watermelon under drought stress. Transcriptome and expression analyses revealed that the genes involved in proline metabolism exhibited different expression levels. Specifically, ClP5CS1 was upregulated in leaves and roots, while ClP5CS2 was downregulated under drought stress. Also, 415 and 362 differently expressed TFs were identified in roots and leaves, respectively, with the majority upregulated in the former. Ultimately, a model for proline metabolism was proposed. The findings of this study provided new insights into the biosynthesis, transport, and regulatory mechanism of drought-induced proline in plants.

Response of Potted Citrus Trees Subjected to Water Deficit Irrigation with the Application of Superabsorbent Polyacrylamide Polymers

Searching for new strategies to mitigate the effects of low water availability for citrus production, a study was carried out on potted mandarin cv. W. Murcott, with the objective of evaluating the physiological and growth response of the plants to polyacrylamide gel application in the substrate in water restriction conditions. The following treatments were evaluated, T0 (control) with 100% ETc water replenishment, T1 with 50% ETc water replenishment, and T2 with 50% ETc water replenishment plus the application of polyacrylamide polymers to the substrate. Temperature and water volumetric content (ϴ: m3 m−3) were evaluated in the substrate. Plant water-status parameters such as stem water potential (SWP), stomatal conductance (gs), and chlorophyll fluorescence (Fv/Fm), as well as biomass, nutrients levels, and proline biosynthesis were measured in the plants in response to the treatments. The results showed that the substrate moisture for T2 was kept significantly higher than T0 and T1, despite receiving the same irrigation rate as T1 and a half of T0; however, this higher moisture availability in the substrate of T2 was not reflected in the plant’s water status or growth. On the contrary, the T2 plants showed responses such as lower total biomass, lower vegetative development, and lower root biomass, as well as a higher concentration of proline in the root. According to these results, it is concluded that polymers such as polyacrylamide sodium allow the retention of water in the substrate, but do not necessarily release that water for plants, probably because that moisture is kept in the hydrogel and not released to the substrate media or the roots, or if released, in this case, this occurs with an increase in the concentration of sodium available to the plants, which could lead the citrus crop to a worse situation of water and/or osmotic stress.

Integration of Metabolomics and Transcriptomics for Investigating the Tolerance of Foxtail Millet (Setaria italica) to Atrazine Stress

Foxtail millet (Setaria italica) is a monotypic species widely planted in China. However, residual atrazine, a commonly used maize herbicide, in soil, is a major abiotic stress to millet. Here, we investigated atrazine tolerance in millet based on the field experiments, then obtained an atrazine-resistant variety (Gongai2, GA2) and an atrazine-sensitive variety (Longgu31, LG31). To examine the effects of atrazine on genes and metabolites in millet plants, we compared the transcriptomic and metabolomic profiles between GA2 and LG31 seedling leaves. The results showed that 2,208 differentially expressed genes (DEGs; 501 upregulated, 1,707 downregulated) and 192 differentially expressed metabolites (DEMs; 82 upregulated, 110 downregulate) were identified in atrazine-treated GA2, while in atrazine-treated LG31, 1,773 DEGs (761 upregulated, 1,012 downregulated) and 215 DEMs (95 upregulated, 120 downregulated) were identified. The bioinformatics analysis of DEGs and DEMs showed that many biosynthetic metabolism pathways were significantly enriched in GA2 and LG31, such as glutathione metabolism (oxiglutatione, γ-glutamylcysteine; GSTU6, GSTU1, GSTF1), amino acid biosynthesis (L-cysteine, N-acetyl-L-glutamic acid; ArgB, GS, hisC, POX1), and phenylpropanoid biosynthesis [trans-5-o-(4-coumaroyl)shikimate; HST, C3′H]. Meanwhile, the co-expression analysis indicated that GA2 plants had enhanced atrazine tolerance owing to improved glutathione metabolism and proline biosynthesis, and the enrichment of scopoletin may help LG31 plants resist atrazine stress. Herein, we screened an atrazine-resistant millet variety and generated valuable information that may deepen our understanding of the complex molecular mechanism underlying the response to atrazine stress in millet.

Metformin Induces PRODH/POX-Dependent Apoptosis in Breast Cancer Cells.

Although the antineoplastic activity of metformin (MET) is well established, the underlying mechanism of the activity is not understood. Since MET activates AMP kinase (AMPK) and proline dehydrogenase/proline oxidase (PRODH/POX) is stimulated by AMPK ligands (implicated in the regulation of cancer cell survival/apoptosis), the effect of MET on PRODH/POX-dependent apoptosis in wild-type MCF-7 cells (MCF-7WT) and POX knockdown MCF-7 cells (MCF-7crPOX cells) was studied. PRODH/POX catalyzes proline degradation generating ROS-induced apoptosis or autophagy. Availability of proline for PRODH/POX functions is regulated by the activity of prolidase (enzyme releasing proline from imidodipeptides), collagen biosynthesis (process consuming proline), and metabolism of proline, ornithine, and glutamic acid. We have found that MET is cytotoxic for MCF-7 cells (IC50∼17 mM), and to the lower extent for MCF-7crPOX cells (IC50∼28 mM). In MCF-7WT cells, the effect was accompanied by the inhibition of DNA biosynthesis, collagen biosynthesis, stimulation of ROS formation, AMPKα phosphorylation, and expression of prolidase, p53, caspase 8, caspase 9, and cleaved PARP. In MET-treated MCF-7crPOX cells, the processes were less affected than in MCF-7WT cells and the expression of caspase 9 was decreased, while cleaved caspase 8 and cleaved PARP were not detected. The effects were accompanied by an increase in the prolidase activity and proline concentration. The mechanism for MET-induced apoptosis involves the up-regulation of prolidase activity and a decrease in collagen biosynthesis contributing to an increase in the concentration of substrate (proline) for PRODH/POX-dependent ROS formation and activation of caspases −9 and −8. The data suggest that PRODH/POX participates in the MET-induced intrinsic and extrinsic apoptosis in MCF-7 cells.

The Kandelia obovata transcription factor KoWRKY40 enhances cold tolerance in transgenic Arabidopsis

BackgroundWRKY transcription factors play key roles in plant development processes and stress response. Kandelia obovata is the most cold-resistant species of mangrove plants, which are the important contributors to coastal marine environment. However, there is little known about the WRKY genes in K. obovata.ResultsIn this study, a WRKY transcription factor gene, named KoWRKY40, was identified from mangrove plant K. obovata. The full-length cDNA of KoWRKY40 gene was 1420 nucleotide bases, which encoded 318 amino acids. The KoWRKY40 protein contained a typical WRKY domain and a C2H2 zinc-finger motif, which were common signatures to group II of WRKY family. The three-dimensional (3D) model of KoWRKY40 was formed by one α-helix and five β-strands. Evolutionary analysis revealed that KoWRKY40 has the closest homology with a WRKY protein from another mangrove plant Bruguiera gymnorhiza. The KoWRKY40 protein was verified to be exclusively located in nucleus of tobacco epidermis cells. Gene expression analysis demonstrated that KoWRKY40 was induced highly in the roots and leaves, but lowly in stems in K. obovata under cold stress. Overexpression of KoWRKY40 in Arabidopsis significantly enhanced the fresh weight, root length, and lateral root number of the transgenic lines under cold stress. KoWRKY40 transgenic Arabidopsis exhibited higher proline content, SOD, POD, and CAT activities, and lower MDA content, and H2O2 content than wild-type Arabidopsis under cold stress condition. Cold stress affected the expression of genes related to proline biosynthesis, antioxidant system, and the ICE-CBF-COR signaling pathway, including AtP5CS1, AtPRODH1, AtMnSOD, AtPOD, AtCAT1, AtCBF1, AtCBF2, AtICE1, AtCOR47 in KoWRKY40 transgenic Arabidopsis plants.ConclusionThese results demonstrated that KoWRKY40 conferred cold tolerance in transgenic Arabidopsis by regulating plant growth, osmotic balance, the antioxidant system, and ICE-CBF-COR signaling pathway. The study indicates that KoWRKY40 is an important regulator involved in the cold stress response in plants.

RmZAT10, a novel Cys2/His2 zinc finger transcription factor of Rosa multiflora, functions in cold tolerance through modulation of proline biosynthesis and ROS homeostasis

The Cys2/His2-type (C2H2) zinc finger transcription factor (TF) consists of a significant gene family that has an effect on development process regulation and a wide range of stress responses, whereas the roles and regulatory pathways of the majority of C2H2-type zinc finger TFs remain unclear. Here, we characterized a C2H2-type zinc finger TF ( RmZAT10 ) from Rosa multiflora . RmZAT10 was mainly expressed in leaves and located in the nucleus. RmZAT10 was rapidly up-regulated during dehydration, cold as well as ABA treatment, with the maximal induction under cold stress. RmZAT10 overexpression facilitated the cold tolerance of tobacco, while gene silencing caused by virus-induced gene silencing (VIGS) of RmZAT10 within R.multiflora led to elevated cold sensitivity. Transgenic tobacco accumulated a larger amount of proline, consistent with elevated the expression level of P5CS (Δ -1-pyrroline-5-carboxylate synthetase ). Nevertheless, the VIGS plant achieved the opposing trend. The treatment using 24-epi-brassinolide caused transgenic tobacco plants more sensitive to cold, while exogenous proline partially restored cold tolerance of the VIGS plants. In addition, RmZAT10 could bind specifically to and activate the promoter of RmP5CS. The above results revealed the role played by RmZAT10 in cold tolerance through the modulation of proline biosynthesis and ROS homeostasis. • RmZAT10 is induced by cold, dehydration and ABA treatments, especially prominent under cold stress. • RmZAT10 overexpression enhanced the cold tolerance in tobacco, while VIGS of RmZAT10 increased cold susceptibility. • RmZAT10 played a role in cold tolerance through the modulation of proline biosynthesis and ROS homeostasis.

A Cys2His2 Zinc Finger Transcription Factor BpSZA1 Positively Modulates Salt Stress in Betula platyphylla.

Zinc finger proteins (ZFPs) are widely involved in plant growth and abiotic stress responses, however, few of these proteins have been functionally characterized in tree species. In this study, we cloned and characterized the BpSZA1 gene encoding a C2H2-type ZFP from Betula platyphylla. BpSZA1 is a transcription factor localized in the nucleus, with a transcription activation domain located at the N-terminus. BpSZA1 was predominantly expressed in stems and was induced by salt. We generated transgenic birch lines displaying overexpression (OE) or RNAi silencing (Ri) of BpSZA1 and exposed these along with wild-type birch seedlings to salinity. Phenotypic and physiological parameters such as superoxide dismutase, peroxisome, H2O2 content, proline content, water loss rate, and malondialdehyde content were examined. Overexpression of BpSZA1 in birch conferred increased salt tolerance. Chromatin immunoprecipitation-qPCR and RNA-seq showed that BpSZA1 binds to the GAGA-motif in the promoter of downstream target genes including BpAPX1, BpAPX2, BpCAT, and Bp6PGDH to activate their transcription. BpSZA1 also participates in abscisic acid (ABA) biosynthesis, proline biosynthesis, and the ABA/jasmonic acid pathway to enhance the salt stress of B. platyphylla.

Stress-induced activation of the proline biosynthetic pathway in Bacillus subtilis: a population-wide and single-cell study of the osmotically controlled proHJ promoter.

Summary Bacillus subtilis, in its natural habitat, is regularly exposed to rapid changes in the osmolarity of its surrounding. As its primary survival strategy, it accumulates large amounts of the compatible solute proline by activating the de novo proline biosynthesis pathway and exploiting the glutamate pools. This osmotically‐induced biosynthesis requires activation of a SigA‐type promoter that drives the expression of the proHJ operon. Population‐wide studies have shown that the activity of the proHJ promoter correlates with the increased osmotic pressure of the environment. Therefore, the activation of the proHJ transcription should be an adequate measure of the adaptation to osmotic stress through proline synthesis in the absence of other osmoprotectants. In this study, we investigate the kinetics of the proHJ promoter activation and the early adaptation to mild osmotic upshift at the single‐cell level. Under these conditions, we observed a switching point and heterogeneous proline biosynthesis gene expression, where the subpopulation of cells showing active proHJ transcription is able to continuously divide, and those unresponsive to osmotic stress remain dormant. Additionally, we demonstrate that bactericidal antibiotics significantly upregulate proHJ transcription in the absence of externally imposed osmotic pressure, suggesting that the osmotically‐controlled proline biosynthesis pathway is also involved in the antibiotic‐mediated stress response.

Promising role of topical caffeine mesoporous gel in collagen resynthesis and UV protection through proline assessment

BackgroundCaffeine, an alkaloid agent, has been globally used regularly in drinks, for the reduction in skin cancers and wrinkle formation. As a result of the previous, attempts have been carried out to use caffeine in cosmetology due to its antioxidant and UV ray protection effects. Our aim was to evaluate the effect of caffeine on collagen resynthesis via its effect on proline and prolidase biosynthesis on mice, orally and topically as mesoporous silica at three levels, and the influence on UV protection. In skin biopsies of orally and topically treated mice, the following was assessed using ELISA and Western blot techniques, the activity of prolidase, together with the concentrations of proline, beta integrin, insulin growth factor, protein kinases beta, and mitogen-activated protein kinase. Moreover, we loaded the caffeine on mesoporous silica and assessed the aforementioned parameters together with checkpoint kinase 1 and Rad3-related protein.ResultsCaffeine promoted collagen resynthesis in a dose-dependent manner. The mechanism of this process was found at the level of prolidase activity as caffeine significantly increased the enzyme activity. Caffeine also had a protective effect against UV exhibited by the over-expression of beta integrin, insulin growth factor together with the under-expression of protein kinases beta, mitogen-activated protein kinase, checkpoint kinase 1, and Rad3-related protein.ConclusionsOur study revealed the superiority of SYL-C12 (mesoporous silica-loaded caffeine gel), compromising the high level of the three independent factors, in terms of the measured responses in mesoporous silica with caffeine. Moreover, caffeine promoted collagen resynthesis with significant protective effect against UV apoptotic damage.

Lb1G04202, an Uncharacterized Protein from Recretohalophyte Limonium bicolor, Is Important in Salt Tolerance.

With global increases in saline soil, it has become increasingly important to decipher salt-tolerance mechanisms and identify strategies to improve salt tolerance in crops. Halophytes complete their life cycles in environments containing ≥200 mM NaCl; these remarkable plants provide a potential source of genes for improving crop salt tolerance. Recretohalophytes such as Limonium bicolor have salt glands that secrete Na+ on their leaf epidermis. Here, we identified Lb1G04202, an uncharacterized gene with no conserved domains, from L. bicolor, which was highly expressed after NaCl treatment. We confirmed its expression in the salt gland by in situ hybridization, and then heterologously expressed Lb1G04202 in Arabidopsis thaliana. The transgenic lines had a higher germination rate, greater cotyledon growth percentage, and longer roots than the wild type (WT) under NaCl treatments (50, 100 and 150 mM). At the seedling stage, the transgenic lines grew better than the WT and had lower Na+ and malonyldialdehyde accumulation, and higher K+ and proline contents. This corresponded with the high expression of the key proline biosynthesis genes AtP5CS1 and AtP5CS2 under NaCl treatment. Isotonic mannitol treatment showed that Lb1G04202 overexpression significantly relieved osmotic stress. Therefore, this novel gene provides a potential target for improving salt tolerance.

Contribution of Exogenous Proline to Abiotic Stresses Tolerance in Plants: A Review.

Abiotic stresses are the major environmental factors that play a significant role in decreasing plant yield and production potential by influencing physiological, biochemical, and molecular processes. Abiotic stresses and global population growth have prompted scientists to use beneficial strategies to ensure food security. The use of organic compounds to improve tolerance to abiotic stresses has been considered for many years. For example, the application of potential external osmotic protective compounds such as proline is one of the approaches to counteract the adverse effects of abiotic stresses on plants. Proline level increases in plants in response to environmental stress. Proline accumulation is not just a signal of tension. Rather, according to research discussed in this article, this biomolecule improves plant resistance to abiotic stress by rising photosynthesis, enzymatic and non-enzymatic antioxidant activity, regulating osmolyte concentration, and sodium and potassium homeostasis. In this review, we discuss the biosynthesis, sensing, signaling, and transport of proline and its role in the development of various plant tissues, including seeds, floral components, and vegetative tissues. Further, the impacts of exogenous proline utilization under various non-living stresses such as drought, salinity, high and low temperatures, and heavy metals have been extensively studied. Numerous various studies have shown that exogenous proline can improve plant growth, yield, and stress tolerance under adverse environmental factors.

The Investigation on the Effect Asn123Gly mutation in the Enzyme Pyrroline‐5‐Carboxylate Reductase

In this experiment, we are studying the enzyme pyrroline‐5‐carboxylate reductase from Sinorhizobium melloti. Bioinformatics proposed that the enzyme catalyzes the reduction of pyrroline‐5‐carboxylate to L‐proline using NAD(P)H, which is the final step in proline biosynthesis. The purpose of this experiment is to study the effect of the point mutation of asparagine 123 to glycine on the enzymatic activity of the enzyme. The mutation was generated using site‐directed mutagenesis to change the GGT codon to CCA in the proC gene. The mutation was confirmed via DNA sequencing and the gene was recombinantly expressed in Escherichia coli Rosetta 2 (DE3) cells for protein overproduction. The protein was purified using Ni‐NTA affinity chromatography to >90% purity (determined by SDS‐PAGE). The effect of the mutation on the function of the enzyme was determined using a continuous kinetic assay following the oxidation of the L‐proline analog L‐thiazolidine‐4‐carboxylate using NAD(P)+ and monitoring the formation of NAD(P)H at 340 nm on a UV‐visible spectrophotometer. Preliminary investigations show that the mutation results in a decreased kinetic activity. Current work is underway to identify the underlying structural reason why the Asn123Gly variant has decreased activity.

Elevated Circulatory Proline to Glutamine Ratio (PQR) in Endometriosis and Its Potential as a Diagnostic Biomarker.

Endometriosis (EM) is a hormone-dependent gynecological disease associated with chronic pelvic pain and altered immuno-inflammatory processes. It shares some cancer-like characteristics such as increased proline biosynthesis and activated glutaminolysis. Both proline and glutamine are interconvertible metabolically, and studies have shown their roles in cancer cell metabolic reprogramming, redox homeostasis, occurrence/development of endometrial carcinoma, and its further progression toward the malignant state. So based on this, we hypothesized that the circulatory proline to glutamine ratio (PQR) would be altered in EM and may serve as an indicative biomarker to improve the clinical diagnosis of EM. In present study, the circulatory-PQR levels were estimated for 39 EM patients and 48 age matched healthy female subjects using 800 MHz NMR spectroscopy. Among 39 EM patients, 15 were in the clinical stages I to II and referred to here as moderate EM (MEM) patients and 24 were in the clinical stages III to IV and referred here as severe EM (SEM) patients. The circulatory-PQR levels were significantly increased in EM patients (0.99 ± 0.13 μM in MEM; 1.39 ± 0.22 μM in SEM) compared to normal control (NC) subjects (0.52 ± 0.05 μM in NC). Further, the circulatory PQR levels exhibit the highest diagnostic potential with area under receiver operating characteristic (AUROC) curve values equal to 0.87 ± 0.04 [95%CI = 0.79–0.96] for MEM and 0.89 ± 0.04 [95% CI = 0.82–0.96] for SEM. These results suggested that circulatory-PQR has significant potential to serve as a noninvasive biomarker for diagnostic/prognostic screening of EM and further underscored the importance of these two nonessential amino acids (proline and glutamine) in cancer metabolism.

FGF21 modulates mitochondrial stress response in cardiomyocytes only under mild mitochondrial dysfunction.

The mitochondrial integrated stress response (mitoISR) has emerged as a major adaptive pathway to respiratory chain deficiency, but both the tissue specificity of its regulation, and how mitoISR adapts to different levels of mitochondrial dysfunction are largely unknown. Here, we report that diverse levels of mitochondrial cardiomyopathy activate mitoISR, including high production of FGF21, a cytokine with both paracrine and endocrine function, shown to be induced by respiratory chain dysfunction. Although being fully dispensable for the cell-autonomous and systemic responses to severe mitochondrial cardiomyopathy, in the conditions of mild-to-moderate cardiac OXPHOS dysfunction, FGF21 regulates a portion of mitoISR. In the absence of FGF21, a large part of the metabolic adaptation to mitochondrial dysfunction (one-carbon metabolism, transsulfuration, and serine and proline biosynthesis) is strongly blunted, independent of the primary mitoISR activator ATF4. Collectively, our work highlights the complexity of mitochondrial stress responses by revealing the importance of the tissue specificity and dose dependency of mitoISR.

The DnaJ-like Zinc Finger Protein ORANGE Promotes Proline Biosynthesis in Drought-Stressed Arabidopsis Seedlings.

Orange (OR) is a DnaJ-like zinc finger protein with both nuclear and plastidial localizations. OR, and its orthologs, are highly conserved in flowering plants, sharing a characteristic C-terminal tandem 4× repeats of the CxxCxxxG signature. It was reported to trigger chromoplast biogenesis, promote carotenoid accumulation in plastids of non-pigmented tissues, and repress chlorophyll biosynthesis and chloroplast biogenesis in the nucleus of de-etiolating cotyledons cells. Its ectopic overexpression was found to enhance plant resistance to abiotic stresses. Here, we report that the expression of OR in Arabidopsis thaliana was upregulated by drought treatment, and seedlings of the OR-overexpressing (OE) lines showed improved growth performance and survival rate under drought stress. Compared with the wild-type (WT) and OR-silencing (or) lines, drought-stressed OE seedlings possessed lower contents of reactive oxygen species (such as H2O2 and O2−), higher activities of both superoxide dismutase and catalase, and a higher level of proline content. Our enzymatic assay revealed a relatively higher activity of Δ1-pyrroline-5-carboxylate synthase (P5CS), a rate-limiting enzyme for proline biosynthesis, in drought-stressed OE seedlings, compared with the WT and or lines. We further demonstrated that the P5CS activity could be enhanced by supplementing exogenous OR in our in vitro assays. Taken together, our results indicated a novel contribution of OR to drought tolerance, through its impact on proline biosynthesis.