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1.
PLoS One ; 19(5): e0303145, 2024.
Article En | MEDLINE | ID: mdl-38728268

Water stress can adversely affect seed germination and plant growth. Seed osmopriming is a pre-sowing treatment in which seeds are soaked in osmotic solutions to undergo the first stage of germination prior to radicle protrusion. Seed osmopriming enhances germination performance under stressful environmental conditions, making it an effective method to improve plant resistance and yield. This study analyzed the effect of seed osmopriming with polyethylene glycol (PEG) on seed germination and physiological parameters of Coronilla varia L. Priming treatments using 10% to 30% PEG enhanced germination percentage, germination vigor, germination index, vitality index, and seedling mass and reduced the time to reach 50% germination (T50). The PEG concentration that led to better results was 10%. The content of soluble proteins (SP), proline (Pro), soluble sugars (SS), and malondialdehyde (MDA) in Coronilla varia L. seedlings increased with the severity of water stress. In addition, under water stress, electrolyte leakage rose, and peroxidase (POD) and superoxide dismutase (SOD) activities intensified, while catalase (CAT) activity increased at mild-to-moderate water stress but declined with more severe deficiency. The 10% PEG priming significantly improved germination percentage, germination vigor, germination index, vitality index, and time to 50% germination (T50) under water stress. Across the water stress gradient here tested (8 to 12% PEG), seed priming enhanced SP content, Pro content, and SOD activity in Coronilla varia L. seedlings compared to the unprimed treatments. Under 10% PEG-induced water stress, primed seedlings displayed a significantly lower MDA content and electrolyte leakage than their unprimed counterparts and exhibited significantly higher CAT and POD activities. However, under 12% PEG-induced water stress, differences in electrolyte leakage, CAT activity, and POD activity between primed and unprimed treatments were not significant. These findings suggest that PEG priming enhances the osmotic regulation and antioxidant capacity of Coronilla varia seedlings, facilitating seed germination and seedling growth and alleviating drought stress damage, albeit with reduced efficacy under severe water deficiency.


Germination , Polyethylene Glycols , Seedlings , Seeds , Polyethylene Glycols/pharmacology , Germination/drug effects , Seedlings/drug effects , Seedlings/growth & development , Seeds/drug effects , Seeds/growth & development , Dehydration , Catalase/metabolism , Malondialdehyde/metabolism , Proline/metabolism , Superoxide Dismutase/metabolism , Water/metabolism
2.
Nat Commun ; 15(1): 3888, 2024 May 08.
Article En | MEDLINE | ID: mdl-38719828

PRPF40A plays an important role in the regulation of pre-mRNA splicing by mediating protein-protein interactions in the early steps of spliceosome assembly. By binding to proteins at the 5´ and 3´ splice sites, PRPF40A promotes spliceosome assembly by bridging the recognition of the splices. The PRPF40A WW domains are expected to recognize proline-rich sequences in SF1 and SF3A1 in the early spliceosome complexes E and A, respectively. Here, we combine NMR, SAXS and ITC to determine the structure of the PRPF40A tandem WW domains in solution and characterize the binding specificity and mechanism for proline-rich motifs recognition. Our structure of the PRPF40A WW tandem in complex with a high-affinity SF1 peptide reveals contributions of both WW domains, which also enables tryptophan sandwiching by two proline residues in the ligand. Unexpectedly, a proline-rich motif in the N-terminal region of PRPF40A mediates intramolecular interactions with the WW tandem. Using NMR, ITC, mutational analysis in vitro, and immunoprecipitation experiments in cells, we show that the intramolecular interaction acts as an autoinhibitory filter for proof-reading of high-affinity proline-rich motifs in bona fide PRPF40A binding partners. We propose that similar autoinhibitory mechanisms are present in most WW tandem-containing proteins to enhance binding selectivity and regulation of WW/proline-rich peptide interaction networks.


Proline , Protein Binding , WW Domains , Humans , Amino Acid Motifs , Models, Molecular , Proline/metabolism , Proline/chemistry , RNA Splicing , RNA Splicing Factors/metabolism , RNA Splicing Factors/chemistry , RNA Splicing Factors/genetics , Scattering, Small Angle , Spliceosomes/metabolism , X-Ray Diffraction
3.
BMC Microbiol ; 24(1): 165, 2024 May 15.
Article En | MEDLINE | ID: mdl-38745279

Globally, drought stress poses a significant threat to crop productivity. Improving the drought tolerance of crops with microbial biostimulants is a sustainable strategy to meet a growing population's demands. This research aimed to elucidate microbial biostimulants' (Plant Growth Promoting Rhizobacteria) role in alleviating drought stress in oil-seed crops. In total, 15 bacterial isolates were selected for drought tolerance and screened for plant growth-promoting (PGP) attributes like phosphate solubilization and production of indole-3-acetic acid, siderophore, hydrogen cyanide, ammonia, and exopolysaccharide. This research describes two PGPR strains: Acinetobacter calcoaceticus AC06 and Bacillus amyloliquefaciens BA01. The present study demonstrated that these strains (AC06 and BA01) produced abundant osmolytes under osmotic stress, including proline (2.21 and 1.75 µg ml- 1), salicylic acid (18.59 and 14.21 µg ml- 1), trehalose (28.35 and 22.74 µg mg- 1 FW) and glycine betaine (11.35 and 7.74 mg g- 1) respectively. AC06 and BA01 strains were further evaluated for their multifunctional performance by inoculating in Arachis hypogaea L. (Groundnut) under mild and severe drought regimes (60 and 40% Field Capacity). Inoculation with microbial biostimulants displayed distinct osmotic-adjustment abilities of the groundnut, such as growth parameters, plant biomass, photosynthetic pigments, relative water content, proline, and soluble sugar in respective to control during drought. On the other hand, plant sensitivity indexes such as electrolyte leakage and malondialdehyde (MDA) contents were decreased as well as cooperatively conferred plant drought tolerance by induced alterations in stress indicators such as catalase (CAT), ascorbate peroxidase (APX), and superoxide dismutase (SOD). Thus, Acinetobacter sp. AC06 and Bacillus sp. BA01 can be considered as osmolyte producing microbial biostimulants to simultaneously induce osmotic tolerance and metabolic changes in groundnuts under drought stress.


Arachis , Droughts , Stress, Physiological , Arachis/microbiology , Arachis/growth & development , Arachis/metabolism , Arachis/physiology , Proline/metabolism , Bacillus amyloliquefaciens/metabolism , Bacillus amyloliquefaciens/physiology , Soil Microbiology , Osmotic Pressure , Betaine/metabolism , Indoleacetic Acids/metabolism , Salicylic Acid/metabolism , Acinetobacter/metabolism , Acinetobacter/growth & development , Acinetobacter/physiology , Hydrogen Cyanide/metabolism , Trehalose/metabolism
4.
Cell Mol Life Sci ; 81(1): 216, 2024 May 13.
Article En | MEDLINE | ID: mdl-38740643

p50RhoGAP is a key protein that interacts with and downregulates the small GTPase RhoA. p50RhoGAP is a multifunctional protein containing the BNIP-2 and Cdc42GAP Homology (BCH) domain that facilitates protein-protein interactions and lipid binding and the GAP domain that regulates active RhoA population. We recently solved the structure of the BCH domain from yeast p50RhoGAP (YBCH) and showed that it maintains the adjacent GAP domain in an auto-inhibited state through the ß5 strand. Our previous WT YBCH structure shows that a unique kink at position 116 thought to be made by a proline residue between alpha helices α6 and α7 is essential for the formation of intertwined dimer from asymmetric monomers. Here we sought to establish the role and impact of this Pro116. However, the kink persists in the structure of P116A mutant YBCH domain, suggesting that the scaffold is not dictated by the proline residue at this position. We further identified Tyr124 (or Tyr188 in HBCH) as a conserved residue in the crucial ß5 strand. Extending to the human ortholog, when substituted to acidic residues, Tyr188D or Tyr188E, we observed an increase in RhoA binding and self-dimerization, indicative of a loss of inhibition of the GAP domain by the BCH domain. These results point to distinct roles and impact of the non-conserved and conserved amino acid positions in regulating the structural and functional complexity of the BCH domain.


Proline , Proline/metabolism , Proline/chemistry , Proline/genetics , Tyrosine/metabolism , Tyrosine/chemistry , Tyrosine/genetics , Protein Domains , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae/genetics , Amino Acid Sequence , rhoA GTP-Binding Protein/metabolism , rhoA GTP-Binding Protein/genetics , rhoA GTP-Binding Protein/chemistry , Models, Molecular , Conserved Sequence , Humans , Protein Binding
5.
Sci Adv ; 10(19): eadl3549, 2024 May 10.
Article En | MEDLINE | ID: mdl-38718121

Metabolic reprogramming is critical in the onset of pressure overload-induced cardiac remodeling. Our study reveals that proline dehydrogenase (PRODH), the key enzyme in proline metabolism, reprograms cardiomyocyte metabolism to protect against cardiac remodeling. We induced cardiac remodeling using transverse aortic constriction (TAC) in both cardiac-specific PRODH knockout and overexpression mice. Our results indicate that PRODH expression is suppressed after TAC. Cardiac-specific PRODH knockout mice exhibited worsened cardiac dysfunction, while mice with PRODH overexpression demonstrated a protective effect. In addition, we simulated cardiomyocyte hypertrophy in vitro using neonatal rat ventricular myocytes treated with phenylephrine. Through RNA sequencing, metabolomics, and metabolic flux analysis, we elucidated that PRODH overexpression in cardiomyocytes redirects proline catabolism to replenish tricarboxylic acid cycle intermediates, enhance energy production, and restore glutathione redox balance. Our findings suggest PRODH as a modulator of cardiac bioenergetics and redox homeostasis during cardiac remodeling induced by pressure overload. This highlights the potential of PRODH as a therapeutic target for cardiac remodeling.


Mice, Knockout , Myocytes, Cardiac , Proline , Ventricular Remodeling , Animals , Proline/metabolism , Myocytes, Cardiac/metabolism , Mice , Rats , Proline Oxidase/metabolism , Proline Oxidase/genetics , Energy Metabolism , Myocardium/metabolism , Myocardium/pathology , Cardiomegaly/metabolism , Cardiomegaly/pathology , Cardiomegaly/etiology , Disease Models, Animal , Oxidation-Reduction , Male , Metabolic Reprogramming
6.
Planta ; 259(6): 142, 2024 May 04.
Article En | MEDLINE | ID: mdl-38702456

MAIN CONCLUSION: PLDα1 promoted H2S production by positively regulating the expression of LCD. Stomatal closure promoted by PLDα1 required the accumulation of H2S under drought stress. Phospholipase Dα1 (PLDα1) acting as one of the signal enzymes can respond to drought stress. It is well known that hydrogen sulfide (H2S) plays an important role in plant responding to biotic or abiotic stress. In this study, the functions and relationship between PLDα1 and H2S in drought stress resistance in Arabidopsis were explored. Our results indicated that drought stress promotes PLDα1 and H2S production by inducing the expression of PLDα1 and LCD genes. PLDα1 and LCD enhanced plant tolerance to drought by regulating membrane lipid peroxidation, proline accumulation, H2O2 content and stomatal closure. Under drought stress, the H2O2 content of PLDα1-deficient mutant (pldα1), L-cysteine desulfhydrase (LCD)-deficient mutant (lcd) was higher than that of ecotype (WT), the stomatal aperture of pldα1 and lcd was larger than that of WT. The transcriptional and translational levels of LCD were lower in pldα1 than that in WT. Exogenous application of the H2S donor NaHS or GYY reduced the stomatal aperture of WT, pldα1, PLDα1-CO, and PLDα1-OE lines, while exogenous application of the H2S scavenger hypotaurine (HT) increased the stomatal aperture. qRT-PCR analysis of stomatal movement-related genes showed that the expression of CAX1, ABCG5, SCAB1, and SLAC1 genes in pldα1 and lcd were down-regulated, while ACA1 and OST1 gene expression was significantly up-regulated. Thus, PLDα1 and LCD are required for stomatal closure to improve drought stress tolerance.


Arabidopsis Proteins , Arabidopsis , Droughts , Gene Expression Regulation, Plant , Hydrogen Sulfide , Phospholipase D , Plant Stomata , Arabidopsis/genetics , Arabidopsis/physiology , Plant Stomata/physiology , Plant Stomata/genetics , Phospholipase D/metabolism , Phospholipase D/genetics , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Hydrogen Sulfide/metabolism , Hydrogen Peroxide/metabolism , Stress, Physiological/genetics , Proline/metabolism , Cystathionine gamma-Lyase/genetics , Cystathionine gamma-Lyase/metabolism , Lipid Peroxidation
7.
J Clin Invest ; 134(9)2024 May 01.
Article En | MEDLINE | ID: mdl-38690726

Proline substitutions within the coiled-coil rod region of the ß-myosin gene (MYH7) are the predominant mutations causing Laing distal myopathy (MPD1), an autosomal dominant disorder characterized by progressive weakness of distal/proximal muscles. We report that the MDP1 mutation R1500P, studied in what we believe to be the first mouse model for the disease, adversely affected myosin motor activity despite being in the structural rod domain that directs thick filament assembly. Contractility experiments carried out on isolated mutant muscles, myofibrils, and myofibers identified muscle fatigue and weakness phenotypes, an increased rate of actin-myosin detachment, and a conformational shift of the myosin heads toward the more reactive disordered relaxed (DRX) state, causing hypercontractility and greater ATP consumption. Similarly, molecular analysis of muscle biopsies from patients with MPD1 revealed a significant increase in sarcomeric DRX content, as observed in a subset of myosin motor domain mutations causing hypertrophic cardiomyopathy. Finally, oral administration of MYK-581, a small molecule that decreases the population of heads in the DRX configuration, significantly improved the limited running capacity of the R1500P-transgenic mice and corrected the increased DRX state of the myofibrils from patients. These studies provide evidence of the molecular pathogenesis of proline rod mutations and lay the groundwork for the therapeutic advancement of myosin modulators.


Amino Acid Substitution , Distal Myopathies , Proline , Animals , Mice , Humans , Proline/genetics , Proline/metabolism , Distal Myopathies/genetics , Distal Myopathies/metabolism , Distal Myopathies/pathology , Mutation, Missense , Cardiac Myosins/genetics , Cardiac Myosins/metabolism , Myosin Heavy Chains/genetics , Myosin Heavy Chains/metabolism , Myosin Heavy Chains/chemistry , Female , Male , Mice, Transgenic , Muscle Contraction/genetics , Muscle, Skeletal/metabolism , Muscle, Skeletal/pathology
8.
Physiol Plant ; 176(3): e14319, 2024.
Article En | MEDLINE | ID: mdl-38693848

Amino acids play important roles in stress resistance, plant growth, development, and quality, with roots serving as the primary organs for drought response. We conducted biochemical and multi-omics analyses to investigate the metabolic processes of root amino acids in drought-resistant (HN44) and drought-sensitive (HN65) soybean (Glycine max) varieties. Our analysis revealed an increase in total amino acid content in both varieties, with phenylalanine, proline, and methionine accumulating in both. Additionally, several amino acids exhibited significant decreases in HN65 but slight increases in HN44. Multi-omics association analysis identified 13 amino acid-related pathways. We thoroughly examined the changes in genes and metabolites involved in various amino acid metabolism/synthesis and determined core genes and metabolites through correlation networks. The phenylalanine, tyrosine, and tryptophan metabolic pathways and proline, glutamic acid and sulfur-containing amino acid pathways were particularly important for drought resistance. Some candidate genes, such as ProDH and P4HA family genes, and metabolites, such as O-acetyl-L-serine, directly affected up- and downstream metabolism to induce drought resistance. This study provided a basis for soybean drought resistance breeding.


Amino Acids , Droughts , Glycine max , Plant Roots , Stress, Physiological , Glycine max/genetics , Glycine max/metabolism , Glycine max/physiology , Plant Roots/metabolism , Plant Roots/genetics , Plant Roots/physiology , Amino Acids/metabolism , Gene Expression Regulation, Plant , Proline/metabolism , Metabolic Reprogramming
9.
Plant Physiol Biochem ; 210: 108610, 2024 May.
Article En | MEDLINE | ID: mdl-38615447

In the face of the formidable environmental challenges precipitated by the ongoing climate change, Plant Growth-Promoting Bacteria (PGPB) are gaining widespread acknowledgement for their potential as biofertilizers, biocontrol agents, and microbial inoculants. However, a knowledge gap pertains to the ability of PGPB to improve stress tolerance in forestry species via cross-inoculation. To address this gap, the current investigation centres on PGPBs, namely, Acinetobacter johnsonii, Cronobacter muytjensii, and Priestia endophytica, selected from the phyllosphere of robust and healthy plants thriving in the face of stress-inducing conditions. These strains were selected based on their demonstrated adaptability to saline, arid, and nitrogen-deficient environments. The utilization of PGPB treatment resulted in an improvement of stomatal conductance (gs) and transpiration rate (E) in poplar plants exposed to both salt and drought stress. It also induced an increase in essential biochemical components such as proline (PRO), catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD). These reactions were accompanied by a decrease in leaf malonaldehyde (MDA) content and electrolyte leakage (EL). Furthermore, the PGPB treatment demonstrated a notable enhancement in nutrient absorption, particularly nitrogen and carbon, achieved through the solubilization of nutrients. The estimation of canopy temperature via thermal imaging proved to be an efficient method for distinguishing stress reactions in poplar than conventional temperature recording techniques. In summation, the utilization of PGPB especially Cronobacter muytjensii in this study, yielded profound improvements in the stress tolerance of poplar plants, manifesting in reduced membrane lipid peroxidation, enhanced photosynthesis, and bolstered antioxidant capacity within the leaves.


Populus , Stress, Physiological , Populus/microbiology , Populus/physiology , Endophytes/physiology , Plant Leaves/metabolism , Droughts , Proline/metabolism , Adaptation, Physiological , Acinetobacter/physiology
10.
BMC Plant Biol ; 24(1): 310, 2024 Apr 23.
Article En | MEDLINE | ID: mdl-38649811

BACKGROUND: Drought can result in yield losses, the application of plant growth regulators is an effective measure to improve drought resistance and yield. The objective of the study was to explore the application potential of mepiquat chloride (MC) in regulating soybean yield and drought resistance. METHODS: In this study, a three-year field experiment was designed and combined with drought experiments to measure the yield of popularized varieties during 2021-2022 and drought-resistant and drought-sensitive varieties were selected, and planted in the field in 2023. RESULTS: MC increased the yield of HN84 and HN87 for two consecutive years from 2021 to 2022 and improved their physiological characteristics under field conditions. Under M200 treatment, the yield of HN84 increased by 6.93% and 9.46%, and HN87 increased by 11.11% and 15.72%. Different concentrations of MC have different effects on soybeans. The maximum increase of SOD, POD and proline in HN84 under M400 treatment reached 71.92%, 63.26% and 71.54%, respectively; the maximum increase of SOD, POD and proline in HN87 under M200 treatment reached 21.96%, 93.49% and 40.45%, respectively. In 2023, the foliar application of MC improved the physiological characteristics of HN44 and HN65 under drought-stress conditions. On the eighth day of drought treatment, compared to the drought treatment, the leaf and root dry weight of HN44 under M100 treatment increased by 17.91% and 32.76%, respectively; the dry weight of leaves and roots of HN65 increased by 20.74% and 29.29% under M200 treatment, respectively. MC also reduced malondialdehyde (MDA) content, decreased antioxidant enzyme activity and proline content. In addition, different concentrations of MC increased the chlorophyll fluorescence parameters (Fs, Fv/Fm, YII, and SPAD). In the field, the plant height of the two varieties decreased significantly, the yield increased, the number of two-grain and three-grain pods increased, and the stem length at the bottom and middle decreased with MC induction. CONCLUSIONS: The application of 100-200 mg/L MC effectively improved drought resistance and increased yield. This study provided support for the rational application of MC in soybean production.


Droughts , Glycine max , Piperidines , Glycine max/drug effects , Glycine max/growth & development , Glycine max/physiology , Glycine max/metabolism , Plant Growth Regulators/pharmacology , Plant Growth Regulators/metabolism , Proline/metabolism , Drought Resistance
11.
Plant Signal Behav ; 19(1): 2334511, 2024 Dec 31.
Article En | MEDLINE | ID: mdl-38650457

Saline and alkaline stress is one of the major abiotic stresses facing agricultural production, which severely inhibits the growth and yield of plant. The application of plant growth regulators can effectively prevent crop yield reduction caused by saline and alkaline stress. Exogenous melatonin (MT) can act as a signaling molecule involved in the regulation of a variety of physiological processes in plants, has been found to play a key role in enhancing the improvement of plant tolerance to abiotic stresses. However, the effects of exogenous MT on saline and alkaline tolerance of table grape seedlings and its mechanism have not been clarified. The aim of this study was to investigate the role of exogenous MT on morphological and physiological growth of table grape seedlings (Vitis vinifera L.) under saline and alkaline stress. The results showed that saline and alkaline stress resulted in yellowing and wilting of grape leaves and a decrease in chlorophyll content, whereas the application of exogenous MT alleviated the degradation of chlorophyll in grape seedling leaves caused by saline and alkaline stress and promoted the accumulation of soluble sugars and proline content. In addition, exogenous MT increased the activity of antioxidant enzymes, which resulted in the scavenging of reactive oxygen species (ROS) generated by saline and alkaline stress. In conclusion, exogenous MT was involved in the tolerance of grape seedlings to saline and alkaline stress, and enhanced the saline and alkaline resistance of grape seedlings to promote the growth and development of the grape industry in saline and alkaline areas.


Melatonin , Plant Leaves , Seedlings , Stress, Physiological , Vitis , Vitis/drug effects , Vitis/metabolism , Vitis/physiology , Melatonin/pharmacology , Melatonin/metabolism , Seedlings/drug effects , Seedlings/metabolism , Seedlings/growth & development , Plant Leaves/drug effects , Plant Leaves/metabolism , Stress, Physiological/drug effects , Plant Senescence/drug effects , Reactive Oxygen Species/metabolism , Chlorophyll/metabolism , Alkalies , Antioxidants/metabolism , Proline/metabolism
12.
Plant Physiol Biochem ; 210: 108599, 2024 May.
Article En | MEDLINE | ID: mdl-38583313

Plant WRKY transcription factors (TFs) play important roles in abiotic stress responses. However, how WRKY facilitate physiological changes to confer salt tolerance still needs to be studied. Here, we identified a WRKY TF from birch (Betula platyphylla Suk), BpWRKY32, which is significantly (P < 0.05) induced by salt stress. BpWRKY32 binds to W-box motif and is located in the nucleus. Under salt stress conditions, fresh weights (FW) of OE lines (BpWRKY32 overexpression lines) are increased by 66.36% than that of WT, while FW of knockout of BpWRKY32 (bpwrky32) lines are reduced by 39.49% compared with WT. BpWRKY32 regulates the expression of BpRHC1, BpNRT1, and BpMYB61 to reduce stomatal, and width-length ratio of the stomatal aperture in OE lines are reduced by 46.23% and 64.72% compared with in WT and bpwrky32 lines. BpWRKY32 induces P5CS expression, but inhibits P5CDH expression, leading to the proline content in OE lines are increased by 33.41% and 97.58% compared with WT and bpwrky32 lines. Additionally, BpWRKY32 regulates genes encoding SOD and POD family members, which correspondingly increases the activities of SOD and POD. These results suggested that BpWRKY32 regulates target genes to reduce the water loss rate, enhance the osmotic potential, and reduce the ROS accumulation, leading to improved salt tolerance.


Betula , Plant Proteins , Plant Stomata , Salt Tolerance , Transcription Factors , Betula/genetics , Betula/metabolism , Gene Expression Regulation, Plant , Plant Proteins/genetics , Plant Proteins/metabolism , Plant Stomata/physiology , Plant Stomata/genetics , Plants, Genetically Modified , Proline/metabolism , Reactive Oxygen Species/metabolism , Salt Tolerance/genetics , Transcription Factors/genetics , Transcription Factors/metabolism
13.
PLoS One ; 19(4): e0289239, 2024.
Article En | MEDLINE | ID: mdl-38625918

Dipeptidyl peptidase 4 (DP4)/CD26 regulates the biological function of various peptide hormones by releasing dipeptides from their N-terminus. The enzyme is a prominent target for the treatment of type-2 diabetes and various DP4 inhibitors have been developed in recent years, but their efficacy and side effects are still an issue. Many available crystal structures of the enzyme give a static picture about enzyme-ligand interactions, but the influence of amino acids in the active centre on binding and single catalysis steps can only be judged by mutagenesis studies. In order to elucidate their contribution to inhibitor binding and substrate catalysis, especially in discriminating the P1 amino acid of substrates, the amino acids R125, N710, E205 and E206 were investigated by mutagenesis studies. Our studies demonstrated, that N710 is essential for the catalysis of dipeptide substrates. We found that R125 is not important for dipeptide binding but interacts in the P1`position of the peptide backbone. In contrast to dipeptide substrates both amino acids play an essential role in the binding and arrangement of long natural substrates, particularly if lacking proline in the P1 position. Thus, it can be assumed that the amino acids R125 and N710 are important in the DP4 catalysed substrate hydrolysis by interacting with the peptide backbone of substrates up- and downstream of the cleavage site. Furthermore, we confirmed the important role of the amino acids E205 and E206. However, NP Y, displaying proline in P1 position, is still processed without the participation of E205 or E206.


Amino Acids , Dipeptidyl Peptidase 4 , Catalytic Domain , Dipeptides/chemistry , Dipeptidyl Peptidase 4/chemistry , Dipeptidyl Peptidase 4/metabolism , Peptides , Proline/metabolism , Serine Endopeptidases/metabolism , Substrate Specificity , Humans
14.
Nat Commun ; 15(1): 3533, 2024 Apr 26.
Article En | MEDLINE | ID: mdl-38670937

Oxygen is essential for aerobic organisms, but little is known about its role in antiviral immunity. Here, we report that during responses to viral infection, hypoxic conditions repress antiviral-responsive genes independently of HIF signaling. EGLN1 is identified as a key mediator of the oxygen enhancement of antiviral innate immune responses. Under sufficient oxygen conditions, EGLN1 retains its prolyl hydroxylase activity to catalyze the hydroxylation of IRF3 at proline 10. This modification enhances IRF3 phosphorylation, dimerization and nuclear translocation, leading to subsequent IRF3 activation. Furthermore, mice and zebrafish with Egln1 deletion, treatment with the EGLN inhibitor FG4592, or mice carrying an Irf3 P10A mutation are more susceptible to viral infections. These findings not only reveal a direct link between oxygen and antiviral responses, but also provide insight into the mechanisms by which oxygen regulates innate immunity.


Hypoxia-Inducible Factor-Proline Dioxygenases , Immunity, Innate , Interferon Regulatory Factor-3 , Oxygen , Proline , Zebrafish , Animals , Hypoxia-Inducible Factor-Proline Dioxygenases/metabolism , Hypoxia-Inducible Factor-Proline Dioxygenases/genetics , Interferon Regulatory Factor-3/metabolism , Hydroxylation , Humans , Proline/metabolism , Mice , Oxygen/metabolism , HEK293 Cells , Phosphorylation , Mice, Knockout , Signal Transduction , Mice, Inbred C57BL
15.
Nat Commun ; 15(1): 3558, 2024 Apr 26.
Article En | MEDLINE | ID: mdl-38670995

The E3 ligase-degron interaction determines the specificity of the ubiquitin‒proteasome system. We recently discovered that FEM1B, a substrate receptor of Cullin 2-RING ligase (CRL2), recognizes C-degrons containing a C-terminal proline. By solving several cryo-EM structures of CRL2FEM1B bound to different C-degrons, we elucidate the dimeric assembly of the complex. Furthermore, we reveal distinct dimerization states of unmodified and neddylated CRL2FEM1B to uncover the NEDD8-mediated activation mechanism of CRL2FEM1B. Our research also indicates that, FEM1B utilizes a bipartite mechanism to recognize both the C-terminal proline and an upstream aromatic residue within the substrate. These structural findings, complemented by in vitro ubiquitination and in vivo cell-based assays, demonstrate that CRL2FEM1B-mediated polyubiquitination and subsequent protein turnover depend on both FEM1B-degron interactions and the dimerization state of the E3 ligase complex. Overall, this study deepens our molecular understanding of how Cullin-RING E3 ligase substrate selection mediates protein turnover.


Cryoelectron Microscopy , NEDD8 Protein , Receptors, Interleukin-17 , Ubiquitin-Protein Ligases , Ubiquitination , Humans , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/chemistry , NEDD8 Protein/metabolism , NEDD8 Protein/genetics , Proline/metabolism , Protein Multimerization , HEK293 Cells , Protein Binding , Substrate Specificity , Cell Cycle Proteins/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/chemistry , Models, Molecular , Cullin Proteins/metabolism , Cullin Proteins/chemistry , Cullin Proteins/genetics , Degrons
16.
BMC Plant Biol ; 24(1): 243, 2024 Apr 04.
Article En | MEDLINE | ID: mdl-38575896

BACKGROUND: Carrot is an important vegetable crop grown worldwide. The major economic problem in carrot cultivation is yellow disease caused by Bactericera trigonica, which induces biotic stress and has the greatest impact on crop productivity. Comprehensive studies on the mechanism of carrot defense response to biotic stress caused by B. trigonica infestation have yet to be conducted. METHODS: The changes in photosynthetic pigments, proline, TPC, H2O2 and MDA content, DPPH radical scavenging ability, and antioxidant enzyme activity of SOD, CAT, and POX in carrot leaves in response to insect sex (female and male), rapid response (during the first six hours), and long-term response to B. trigonica infestation were evaluated. RESULTS: The results of our study strongly suggest that B. trigonica infestation causes significant changes in primary and secondary metabolism and oxidative status of carrot leaves. Photosynthetic pigment content, TPC, and DPPH and CAT activities were significantly reduced in carrot leaves in response to insect infestation. On the other hand, proline, H2O2 content, and the activity of the antioxidant enzymes superoxide dismutase and peroxidase were increased in carrot leaves after B. trigonica infestation. The results indicate that B. trigonica attenuates and delays the oxidative stress responses of carrot, allowing long-term feeding without visible changes in the plant. Carrot responded to long-term B. trigonica infestation with an increase in SOD and POX activity, suggesting that these enzymes may play a key role in plant defense mechanisms. CONCLUSIONS: This is the first comprehensive study strongly suggesting that B. trigonica infestation causes significant changes in primary and secondary metabolism and an attenuated ROS defense response in carrot leaves that enables long-term insect feeding. The information provides new insights into the mechanisms of carrot protection against B. trigonica infestation.


Aphids , Daucus carota , Hemiptera , Phthiraptera , Animals , Daucus carota/metabolism , Aphids/physiology , Antioxidants/metabolism , Hydrogen Peroxide/metabolism , Oxidative Stress , Superoxide Dismutase/metabolism , Proline/metabolism , Phthiraptera/metabolism
17.
Sci Rep ; 14(1): 8105, 2024 04 06.
Article En | MEDLINE | ID: mdl-38582792

The response of 14 Hollyhock (Alcea rosea L.) varieties to salinity were evaluated in a field experiment over two growing seasons. Carotenoid, Chl a, Chl b, total Chl, proline and MDA content, CAT, APX and GPX activity and petal and seeds yields were determined in order to investigate the mechanism of salt tolerance exhibited by Hollyhock, and too identify salt tolerant varieties. Overall, the photosynthetic pigment content,petal and seed yields were reduced by salt stress. Whereas the proline and MDA content, and the CAT, APX and GPX activities increased as salt levels increased. However, the values of the measured traits were dependent upon the on the level of salt stress, the Varietie and the interaction between the two variables. Based upon the smallest reduction in petal yield, the Masouleh variety was shown to be the most salt tolerant, when grown under severe salt stress. However, based upon the smallest reduction in seed yield, Khorrmabad was the most tolerant variety to severe salt stress. These data suggest that the selection of more salt tolerant Hollyhock genotypes may be possible based upon the wide variation in tolerance to salinity exhibited by the varieties tested.


Malvaceae , Oxidative Stress , Oxidative Stress/physiology , Antioxidants/metabolism , Salt Tolerance/genetics , Proline/metabolism
18.
J Agric Food Chem ; 72(18): 10521-10530, 2024 May 08.
Article En | MEDLINE | ID: mdl-38656141

2-Acetyl-1-pyrroline (2-AP) is a key volatile organic compound in fragrant rice aroma. However, the effects of temperature on 2-AP biosynthesis in fragrant rice and its regulation mechanism have been rarely reported. In the present study, three fragrant rice varieties were used as plant materials, and four temperature treatments during the grain-filling stage, i.e., (T1) 22/17 °C, (T2) 27/22 °C, (T3) 32/27 °C, and (T4) 37/32 °C, were adopted. The results showed that grain contents of 2-AP, proline, and γ-aminobutyric acid (GABA) significantly (P < 0.05) increased with decreased temperature, while the lowest and highest 2-AP contents were recorded in the T4 and T1 treatments, respectively. Higher pyrroline-5-carboxylic acid (P5C) content was recorded in low-temperature treatments (T1 and T2) than in high-temperature treatments (T3 and T4). The transcript levels of genes BADH2, PRODH, and OAT significantly (P < 0.05) decreased with decreased temperature. Lower transcript levels of genes P5CR, P5CS2, DAO2, DAO4, and DAO5 were recorded in low-temperature treatments (T1 and T2) than in high-temperature treatments (T3 and T4). In conclusion, low temperature increased 2-AP content and high temperature decreased 2-AP content in fragrant rice. We deduced that temperature regulated 2-AP biosynthesis through the metabolism of proline and GABA.


Oryza , Plant Proteins , Pyrroles , Seeds , Temperature , Oryza/metabolism , Oryza/chemistry , Oryza/growth & development , Oryza/genetics , Pyrroles/metabolism , Pyrroles/analysis , Plant Proteins/metabolism , Plant Proteins/genetics , Seeds/chemistry , Seeds/metabolism , Seeds/growth & development , Seeds/genetics , Proline/metabolism , Proline/analysis , Gene Expression Regulation, Plant , gamma-Aminobutyric Acid/metabolism , gamma-Aminobutyric Acid/analysis , Volatile Organic Compounds/chemistry , Volatile Organic Compounds/metabolism
19.
Hypertension ; 81(5): 1044-1054, 2024 May.
Article En | MEDLINE | ID: mdl-38465625

BACKGROUND: Potassium (K+)-deficient diets, typical of modern processed foods, increase blood pressure (BP) and NaCl sensitivity. A K+-dependent signaling pathway in the kidney distal convoluted tubule, coined the K+ switch, that couples extracellular K+ sensing to activation of the thiazide-sensitive NaCl cotransporter (NCC) and NaCl retention has been implicated, but causality has not been established. METHODS: To test the hypothesis that small, physiological changes in plasma K+ (PK+) are translated to BP through the switch pathway, a genetic approach was used to activate the downstream switch kinase, SPAK (SPS1-related proline/alanine-rich kinase), within the distal convoluted tubule. The CA-SPAK (constitutively active SPS1-related proline/alanine-rich kinase mice) were compared with control mice over a 4-day PK+ titration (3.8-5.1 mmol) induced by changes in dietary K+. Arterial BP was monitored using radiotelemetry, and renal function measurements, NCC abundance, phosphorylation, and activity were made. RESULTS: As PK+ decreased in control mice, BP progressively increased and became sensitive to dietary NaCl and hydrochlorothiazide, coincident with increased NCC phosphorylation and urinary sodium retention. By contrast, BP in CA-SPAK mice was elevated, resistant to the PK+ titration, and sensitive to hydrochlorothiazide and salt at all PK+ levels, concomitant with sustained and elevated urinary sodium retention and NCC phosphorylation and activity. Thus, genetically locking the switch on drives NaCl sensitivity and prevents the response of BP to potassium. CONCLUSIONS: Low K+, common in modern ultraprocessed diets, presses the K+-switch pathway to turn on NCC activity, increasing sodium retention, BP, and salt sensitivity.


Potassium , Protein Serine-Threonine Kinases , Animals , Mice , Potassium/metabolism , Protein Serine-Threonine Kinases/metabolism , Potassium, Dietary/metabolism , Blood Pressure/physiology , Sodium Chloride/metabolism , Solute Carrier Family 12, Member 3/metabolism , Signal Transduction , Phosphorylation , Kidney Tubules, Distal/metabolism , Hydrochlorothiazide , Sodium/metabolism , Alanine/metabolism , Proline/metabolism
20.
Sci Rep ; 14(1): 6962, 2024 03 23.
Article En | MEDLINE | ID: mdl-38521884

Salinity is one of the most important abiotic stress factors that negatively affect plant growth and development. In contrast, fusaric acid (FA), a mycotoxin produced by Fusarium and Giberella fungal genera, has biological and metabolic effects in various plants. In this study, it was aimed to investigate the protective effect of externally applied FA (0.1 nM) against the damage caused by salt (0.15 M NaCl) stress in onion (Allium cepa L.) plant. Salt stress resulted in an increase in the chromosomal aberrations (CAs) and micronucleus (MN) frequency, a decrease in the mitotic index (MI), fresh weight, root number, germination percentage, and root length. It promoted CAs such as irregular mitosis, bilobulated nuclei, chromosome loss, bridge, unequal seperation of chromosome, vagrant chromosome and polar slip in root meristem cells. In addition, salt stress caused a enhancement in free proline (PR), catalase (CAT), superoxide dismutase (SOD) and malondialdehyde (MDA) contents in the roots of onion plant. Moreover, it revealed damage and changes that include the accumulation of some chemical substances such as proline and sugars in epidermis and cortex layer cells, epidermal cell injury, flattening of the cell nucleus, wall thickening in cortex cells, necrotic areas and indistinct transmission tissue in the anatomical structure of onion roots. On the other hand, FA application promoted bulb germination and mitotic activity, strengthened the antioxidant defense system, and reduced chromosome and anatomical structure damages. In conclusion; it has been revealed that exogenous FA application may have a positive effect on increasing the resistance of onion plants to salt stress.


Mycotoxins , Onions , Fusaric Acid/pharmacology , Sodium Chloride/pharmacology , Sodium Chloride/metabolism , Mycotoxins/metabolism , Antioxidants/pharmacology , Antioxidants/metabolism , Plant Roots/metabolism , Proline/metabolism , Cytogenetic Analysis
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