Enzyme In Biosynthesis Research Articles

Combining multi-omics analysis to identify host-targeted targets for the control of Brucella infection.

Human infections caused by Brucella (called brucellosis) are among the most common zoonoses worldwide with an estimated 500,000 cases each year. Since chronic Brucella infections are extremely difficult to treat, there is an urgent need for more effective therapeutics. As a facultative intracellular bacterium, Brucella is strictly parasitic in the host cell. Here, we performed proteomic and transcriptomic and metabolomic analyses on Brucella infected patients, mice and cells that provided an extensive "map" of physiological changes in brucellosis patients and characterized the metabolic pathways essential to the response to infection, as well as the associated cellular response and molecular mechanisms. This is the first report utilizing multi-omics analysis to investigate the global response of proteins and metabolites associated with Brucella infection, and the data can provide a comprehensive insight to understand the mechanism of Brucella infection. We demonstrated that Brucella increased nucleotide synthesis in the host, consistent with increased biomass requirement. We also identified IMPDH2, a key regulatory complex that controls nucleotide synthesis during Brucella infection. Pharmacological targeting of IMPDH2, the rate-limiting enzyme in guanine nucleotide biosynthesis, efficiently inhibits B. abortus growth both in vitro and in vivo. Through screening a library of natural products, we identified oxymatrine, an alkaloid obtained primarily from Sophora roots, is a novel and selective IMPDH2 inhibitor. In further in vitro bacterial inhibition assays, oxymatrine effectively inhibited the growth of B. abortus, which was impaired by exogenous supplementation of guanosine, a salvage pathway of purine nucleotides. This moderately potent, structurally novel compound may provide clues for further design and development of efficient IMPDH2 inhibitors and also demonstrates the potential of natural compounds from plants against Brucella.

Wax Ester and Triacylglycerol Production in Acinetobacter baumannii: Role in Osmostress Protection, Reactive Oxygen Species, and Antibiotic Sensitivity.

Wax esters (WEs) are neutral lipids that are produced by many different bacteria as potential carbon and energy storage compounds. Comparatively little is known about the role of WE in pathogenic bacteria. The opportunistic pathogen Acinetobacter baumannii is a major cause of hospital-acquired infections worldwide. Salt and desiccation resistance foster A. baumannii infections such as urinary tract infections and allow for reinfection when bacteria are taken up from dry surfaces in the hospital environment. Here we report on WE and triacylglycerol (TAG) production in A. baumannii as a response to nitrogen limitation and high salt stress. Fatty acids and fatty alcohols with chain lengths of C16 and C18 were identified as the most prominent WE constituents. We identified the terminal key enzyme of WE biosynthesis, the bifunctional wax ester synthase/acylCoA:diacylglycerol acyltransferase (WS/DGAT) encoded by the wax/dgat gene, and demonstrated that transcription of wax/dgat and production of WS/DGAT are independent of the nitrogen concentration. A Δwax/dgat mutant was impaired in growth in the presence of high salt concentration and was more sensitive to imipenem and reactive oxygen species.

Plant Responses of Maize to Two formae speciales of Sporisorium reilianum Support Recent Fungal Host Jump.

Host jumps are a major factor for the emergence of new fungal pathogens. In the evolution of smut fungi, a putative host jump occurred in Sporisorium reilianum that today exists in two host-adapted formae speciales, the sorghum-pathogenic S. reilianum f. sp. reilianum and maize-pathogenic S. reilianum f. sp. zeae. To understand the molecular host-specific adaptation to maize, we compared the transcriptomes of maize leaves colonized by both formae speciales. We found that both varieties induce many common defense response-associated genes, indicating that both are recognized by the plant as pathogens. S. reilianum f. sp. reilianum additionally induced genes involved in systemic acquired resistance. In contrast, only S. reilianum f. sp. zeae induced expression of chorismate mutases that function in reducing the level of precursors for generation of the defense compound salicylic acid (SA), as well as oxylipin biosynthesis enzymes necessary for generation of the SA antagonist jasmonic acid (JA). In accordance, we found reduced SA levels as well as elevated JA and JA-Ile levels in maize leaves inoculated with the maize-adapted variety. These findings support a model of the emergence of the maize-pathogenic variety from a sorghum-specific ancestor following a recent host jump.

High Plant Protein Diet Ameliorated Hepatic Lipid Accumulation Through the Modulation of Gut Microbiota.

Substituting plant protein for animal protein has emerged as a promising strategy for managing atherogenic lipids. However, the impact of long-term intake of a high plant protein diet (HPD) on hepatic lipid disorder remains unclear. Eight-week-old apolipoprotein E deficient (apoE-/- ) mice are fed with either a normal protein diet (NCD) or HPD for 12 weeks. HPD intervention results in decreased body weight accompanied by increased energy expenditure, with no significant effect on glycemic control. Long-term intake of HPD improves the serum and hepatic lipid and cholesterol accumulation by suppressing hepatic squalene epoxidase (SQLE) expression, a key enzyme in cholesterol biosynthesis. Integrated analysis of 16S rDNA sequencing and metabolomics profiling reveals that HPD intervention increases the abundance of the Lachnospiraece family and serum levels of 12,13-DiHOME. Furthermore, in vivo studies demonstrate that 12,13-DiHOME significantly inhibits lipid accumulation, as well as SQLE expression induced by oleic acid in HepG2 cells. Diet rich in plant protein diet alleviates hyperlipidemia via increased microbial production of 12,13-DiHOME.

Tyrosinase from Citreicella sp. as an organophilic enzyme for catechol biosynthesis

Tyrosinases can directly catalyze the biosynthesis of catechol compounds without additional cofactors under mild conditions. However, the low stability of tyrosinases in organic media has limited their use. Herein, a tyrosinase (csTyr) was identified from Citreicella sp. SE45 that possesses a characteristic feature of halophilic proteins and can be functionally expressed in Escherichia coli. Remarkably, the enzymatic activity of csTyr was effectively induced by primary alcohols in cosolvent media, whereas csTyr did not have enzymatic activity in aqueous media, minimizing unwanted reactions during tyrosinase preparation. In particular, the catalytic efficiency and stability of csTyr in a 50 % (v/v) ethanol-water cosolvent were comparable to those of other tyrosinases in water. Using csTyr as a catalyst for ortho-hydroxylation reactions of acetaminophen and resveratrol, the high catalytic activity and stability in 50 % (v/v) ethanol efficiently enabled the production of 3-hydroxy-acetaminophen and piceatannol. Therefore, csTyr, an organophilic tyrosinase, can be greatly useful in the biosynthesis of catechol compounds by addressing the issue of poor enzymatic stability in organic media.

Proteolitic Activity of Marine Strain Bacillus sp. 051

The main interest in the study of marine microorganisms is due to their ability to produce a wide range of unique enzymes, including peptidases with different specificities. In recent years, interest has increased in peptidases that are able to cleave elastin as a specific substrate. Streptomyces fradiae and Bacillus thermoproteolyticus elastases are among the most potent elastolytic proteinases discovered to date because they are 4-8-fold more effective than pancreatic elastases. The disadvantages of these producers include the fact that most of them are pathogenic for humans, and the elastase enzyme secreted from them is directly involved in the initiation of the pathogenetic process. All this significantly limits the scope of their practical application. Therefore, the search for new, more effective, safe for humans’ producers continues to be an urgent question, taking into account the fact that there are no highly active elastase producers in Ukraine. Previously we found elastase activity in only 4 of the 10 studied isolates of bacteria from the Black Sea. Since among them, the elastase activity of the Bacillus sp. 051 was the highest, the aim of this work was to study the physicochemical properties and substrate specificity of the enzyme. Methods. We used methods of determining proteolytic (caseinolytic, elastolytic, fibrinolytic, fibrinogenolytic) activity. Protein concentration was determined by the Lowry method. The study of the effect of temperature on the enzymatic activity was carried out in the range from 4 to 70 °C and pH values from 2.0 to 12.0, created using 0.01 M phosphate-citrate buffer. Results. It has been shown that the growing temperature of 12°C is the most optimal for biosynthesis of enzyme by the culture of Bacillus sp. 051. The complex enzyme preparation capable of hydrolyzing elastin, casein and fibrinogen. The enzyme showed maximum activity in relation to elastin (3.65 U/mg). The optimum pH of the enzyme action is 8.0, the thermal optimum is 40°C. The rate of casein hydrolysis compared to elastin was 2.7 times lower and amounted to 1.35 U/mg. The complex enzyme preparation also hydrolyzed fibrinogen (1.16 U/mg). Conclusions. According to its physicochemical and catalytic properties, the representative of the Black Sea, Bacillus sp. 051 is promising for further research as an enzyme producer with elastolytic activity.

A Systematic Review on the Impact of Cholesterol Levels on Diabetes Mellitus

Diabetes mellitus (DM) is a common metabolic disorder characterized by defects in insulin secretion and action, or both, resulting in chronic hyperglycemia and altered metabolism of carbohydrates, fats, and proteins. In the absence of the major risk factors, diabetes is associated with increased morbidity and mortality from cardiovascular disease. The prevalence of diabetes is 10.1 crores, as per the Indian Council of Medical Research – India Diabetes (ICMR INDIAB) study published in 2023. Serum lipid abnormalities (dyslipidemia) are commonly seen in diabetic populations, irrespective of insulin deficiency or insulin resistance. When insulin activity is very low, it severely inhibits lipoprotein lipase production, which significantly impairs the digestion of triglyceride-rich lipoproteins. As a result, there is an increase in triglyceride-rich lipoproteins and a delay in the clearance of chylomicrons and VLDL. Additionally, insulinopenia leads to a substantial increase in lipolysis in adipose tissue, causing the release of free fatty acids into the bloodstream. This higher supply of fatty acids to the liver improves triglyceride synthesis in the liver, ultimately resulting in increased production and secretion of VLDL. By lowering serum cholesterol with statins, which inhibit 3-hydroxy-3-methylglutaryl-coenzyme A reductase, the rate-limiting enzyme in cholesterol biosynthesis, decreases the risk of CHD.

Sterol Biosynthesis Contributes to Brefeldin-A-Induced Endoplasmic Reticulum Stress Resistance inChlamydomonas reinhardtii.

The endoplasmic reticulum (ER) stress response is an evolutionarily conserved mechanism in most eukaryotes. In this response, sterols in the phospholipid bilayer play a crucial role in controlling membrane fluidity and homeostasis. Despite the significance of both the ER stress response and sterols in maintaining ER homeostasis, their relationship remains poorly explored. Our investigation focused on Chlamydomonas strain CC-4533 and revealed that free sterol biosynthesis increased in response to ER stress, except in mutants of the ER stress sensor Inositol-requiring enzyme 1 (IRE1). Transcript analysis of Chlamydomonas experiencing ER stress unveiled the regulatory role of the IRE1/basic leucine zipper 1 pathway in inducing the expression of ERG5, which encodes C-22 sterol desaturase. Through the isolation of three erg5 mutant alleles, we observed a defect in the synthesis of Chlamydomonas' sterol end products, ergosterol and 7-dehydroporiferasterol. Furthermore, these erg5 mutants also exhibited increased sensitivity to ER stress induced by brefeldin A (BFA, an inhibitor of ER-Golgi trafficking), whereas tunicamycin (an inhibitor of N-glycosylation) and dithiothreitol (an inhibitor of disulfide-bond formation) had no such effect. Intriguingly, the sterol biosynthesis inhibitors fenpropimorph and fenhexamid, which impede steps upstream of the ERG5 enzyme in sterol biosynthesis, rescued BFA hypersensitivity in CC-4533 cells. Collectively, our findings support the conclusion that the accumulation of intermediates in the sterol biosynthetic pathway influences ER stress in a complex manner. This study highlights the significance and complexity of regulating sterol biosynthesis during the ER stress response in microalgae.

Transcriptomics of Temporal- versus Substrate-Specific Wood Decay in the Brown-Rot Fungus Fibroporia radiculosa.

Brown-rot fungi lack many enzymes associated with complete wood degradation, such as lignin-attacking peroxidases, and have developed alternative mechanisms for rapid wood breakdown. To identify the effects of culture conditions and wood substrates on gene expression, we grew Fibroporia radiculosa in submerged cultures containing Wiley milled wood (5 days) and solid wood wafers (30 days), using aspen, pine, and spruce as a substrate. The comparative analysis revealed that wood species had a limited effect on the transcriptome: <3% of genes were differentially expressed between different wood species substrates. The comparison between gene expression during growth on milled wood and wood wafer conditions, however, indicated that the genes encoding plant cell wall-degrading enzymes, such as glycoside hydrolases and peptidases, were activated during growth on wood wafers, confirming previous reports. On the other hand, it was shown for the first time that the genes encoding Fenton chemistry enzymes, such as hydroquinone biosynthesis enzymes and oxidoreductases, were activated during submerged growth on ground wood. This illustrates the diversity of wood-decay reactions encoded in fungi and activated at different stages of this process.

Serine 31 Phosphorylation-Driven Regulation of AGPase Activity: Potential Implications for Enhanced Starch Yields in Crops.

ADP-Glc pyrophosphorylase (AGPase), which catalyzes the transformation of ATP and glucose-1-phosphate (Glc-1-P) into adenosine diphosphate glucose (ADP-Glc), acts as a rate-limiting enzyme in crop starch biosynthesis. Prior research has hinted at the regulation of AGPase by phosphorylation in maize. However, the identification and functional implications of these sites remain to be elucidated. In this study, we identified the phosphorylation site (serine at the 31st position of the linear amino acid sequence) of the AGPase large subunit (Sh2) using iTRAQTM. Subsequently, to ascertain the impact of Sh2 phosphorylation on AGPase, we carried out site-directed mutations creating Sh2-S31A (serine residue replaced with alanine) to mimic dephosphorylation and Sh2-S31D (serine residue replaced with aspartic acid) or Sh2-S31E (serine residue replaced with glutamic acid) to mimic phosphorylation. Preliminary investigations were performed to determine Sh2 subcellular localization, its interaction with Bt2, and the resultant AGPase enzymatic activity. Our findings indicate that phosphorylation exerts no impact on the stability or localization of Sh2. Furthermore, none of these mutations at the S31 site of Sh2 seem to affect its interaction with Bt2 (smaller subunit). Intriguingly, all S31 mutations in Sh2 appear to enhance AGPase activity when co-transfected with Bt2, with Sh2-S31E demonstrating a substantial five-fold increase in AGPase activity compared to Sh2. These novel insights lay a foundational groundwork for targeted improvements in AGPase activity, thus potentially accelerating the production of ADP-Glc (the primary substrate for starch synthesis), promising implications for improved starch biosynthesis, and holding the potential to significantly impact agricultural practices.

Targeting LRRK2 mRNA stability in Parkinson’s disease

In a recent study, Liu and colleagues demonstrated a role for the purine biosynthesis enzyme ATIC and its substrate in regulating the protein levels of the Parkinson’s disease kinase LRRK2, which rescues neurodegeneration and neuroinflammation in distinct animal models. This work highlights a novel avenue to target LRRK2 protein levels as a strategy to prevent neurodegeneration in Parkinson’s disease.

CRYAB plays a role in terminating the presence of pro-inflammatory macrophages in the older, injured mouse peripheral nervous system

Evidence indicates that dysfunction of older Schwann cells and macrophages contributes to poor regeneration of more mature peripheral nervous system (PNS) neurons after damage. Since the underlying molecular factors are largely unknown, we investigated if CRYAB, a small heat shock protein that is expressed by Schwann cells and axons and whose expression declines with age, impacts prominent deficits in the injured, older PNS including down-regulation of cholesterol biosynthesis enzyme genes, Schwann cell dysfunction, and macrophage persistence. Following sciatic nerve transection injury in 3- and 12-month-old wildtype and CRYAB knockout mice, we found by bulk RNA sequencing and RT-PCR, that while gene expression of cholesterol biosynthesis enzymes is markedly dysregulated in the aging, injured PNS, CRYAB is not involved. However, immunohistochemical staining of crushed sciatic nerves revealed that more macrophages of the pro-inflammatory but not immunosuppressive phenotype persisted in damaged 12-month-old knockout nerves. These pro-inflammatory macrophages were more efficient at engulfing myelin debris. CRYAB thus appears to play a role in resolving pro-inflammatory macrophage responses after damage to the older PNS.

Elevated high-mannose N-glycans hamper endometrial decidualization

Decidualization of endometrial stromal cells is a hallmark of endometrial receptivity for embryo implantation, and dysfunctional decidualization is associated with pregnancy failure. Protein glycosylation is an important posttranslational modification that affects the structure and function of glycoproteins. Our results showed that high-mannose epitopes were elevated in the decidual tissues of miscarriage patients compared with early pregnant women by Lectin microarray. Furthermore, the level of mannosyl-oligosaccharide α-1,2 mannosidase IA (MAN1A1), a key enzyme for high-mannose glycan biosynthesis, was decreased in the decidual tissues of miscarriage patients. Screening of lncRNAs showed that lncNEAT1 level was increased in the serum and decidua of miscarriage patients, and negatively correlated with MAN1A1 expression. The results also revealed that specific binding of lncNEAT1 with nucleophosmin (NPM1)-SP1 transcription complex inhibited MAN1A1 expression and hampered endometrial decidualization and embryo implantation potential. The study suggests the new insights into the function of high-mannose glycans/MAN1A1 modification during endometrial decidualization.

Low acrosin activity is associated with decreased Spam1/acrosin expression and GSH deficiency-caused premature acrosome release of human sperm cells.

Low acrosin activity (LAA) is associated with sperm function anomaly and poor outcomes of in vitro fertilization. In this study, we confirm that 993 semen samples with LAA had a reduced sperm motility and low in vitro fertilization rate in comparison with 1332 normal controls (NC). Proteomic comparison between 11 LAA and 11 NC sperm samples identified 35 upregulated and 99 downregulated proteins in the LAA group. Indeed, proteomic data showed that acrosome enzymes Spam1 and Acrosin were among the downregulated proteins in the LAA group, which was validated by quantitative PCR and immunefluorescent staining of sperm cells. The KEEG pathway analysis revealed a deficiency of GSH and Gln biosynthesis in LAA sperm cells. Immunofluorescent staining of sperms and quantitative PCR verified downregulation of GLUL and GCLC, the key enzymes for GSH and Gln biosynthesis. Moreover, the results of ELISA assay confirmed low levels of GSH and Gln in LAA sperm cells. Mechanistic studies showed that addition of 10mM H2O2 to semen samples led to a significant reduction of acrosin activity and sperm motility, most possibly by triggering premature acrosome release. In contrast, the presence of 20mM GSH blocked the oxidative effects of H2O2. Since GSH counteracts the oxidative stress and Gln participates in TCA cycling, their deficiency may affect the redox balance as well as energy production of sperm cells. These findings shed new light on the pathological mechanisms of infertility associated with LAA. Male infertility patients could benefit from GSH supplement by improvement of acrosin activity and other sperm functions.

Phenylalanyl-tRNA synthetase deficiency caused by biallelic variants in FARSA gene and literature review

BackgroundAminoacyl-tRNA synthetases (ARSs) are indispensable enzymes for protein biosynthesis in cells. The phenylalanyl-tRNA synthetase (FARS1) located in cytoplasm which consists of two FARS alpha subunits (FARSA) and two FARS beta subunits (FARSB). Autosomal recessive inheritance of pathogenic variants of FARSA or FARSB can result in defective FARS1 which are characterized by interstitial lung disease, liver disease, brain abnormalities, facial dysmorphism and growth restriction.MethodsExome sequencing was used to detect the candidate variants. The in silico prediction and expressional level analysis were performed to evaluate the pathogenicity of the variations. Additionally, we presented the patient’s detailed clinical information and compared the clinical feature with other previously reported patients with FARSA-deficiency.ResultsWe identified compound heterozygous rare missense variants (c.1172 T > C/ p.Leu391Pro and c.1211G > A/ p.Arg404His) in FARSA gene in a Chinese male patient. The protein structure prediction and the analysis of levels of FARSA and FARSB subunits indicated both variants pathogenic. Clinical feature review indicated inflammatory symptoms in young infants may be an additional key feature. Thyroid dysfunction should be considered as a phenotype with variable penetrance.ConclusionsOur results expanded the current phenotypic and genetic spectrum of FARSA-deficiency.

Stearoyl-CoA desaturase 1 inhibition impairs triacylglycerol accumulation and lipid droplet formation in colorectal cancer cells.

Increases in fatty acid (FA) biosynthesis meet the higher lipid demand by intensely proliferating cancer cells and promoting their progression. Stearoyl-CoA desaturase 1 (SCD1) is the key enzyme in FA biosynthesis, converting saturated FA (SFA) into monounsaturated FA (MUFA). Increases in the MUFA/SFA ratio and SCD1 expression have been observed in cancers of various origins and correlate with their aggressiveness. However, much is still unknown about the SCD1-dependent molecular mechanisms that promote specific changes in metabolic pathways of cancer cells. The present study investigated the involvement of SCD1 in shaping glucose and lipid metabolism in colorectal cancer (CRC) cells. Excess FAs that derive from de novo lipogenesis are stored in organelles, called lipid droplets (LDs), mainly in the form of triacylglycerol (TAG) and cholesteryl esters. LD accumulation is associated with key features of cancer development and progression. Consistent with our findings, the pharmacological inhibition of SCD1 activity affects CRC cell viability and impairs TAG accumulation and LD formation in these cells through the activation of lipolytic and lipophagic pathways. We showed that SCD1 suppression affects crucial lipogenic processes that promote lipid accumulation in CRC cells but in a sterol regulatory element-binding protein 1-independent manner. We propose that adenosine monophosphate-activated protein kinase contributes to these changes through the activation of lipolysis and inhibition of TAG synthesis. We also provide evidence of the involvement of SCD1 in the regulation of glucose uptake and utilization in CRC cells. These findings underscore the importance of SCD1 in regulating cellular processes that promote cancer development and progression.

A first-in-class dimethyl 2-acetamido terephthalate inhibitor targeting Conyza canadensis SHMT1 with a novel herbicidal mode-of-action

IntroductionHerbicide application is a highly efficiency method of weed control that boots agricultural output and assures food security. The development of novel herbicides focuses on improved bioactivity and new modes of action. The amino acid biosynthesis was validated as a promising novel mode of action for herbicidal compounds. However, the amino acid biosynthesis enzyme remains largely unexplored for herbicidal targets. ObjectivesSerine hydroxymethyl transferase (SHMT) is an essentialenzyme in the photorespiratory cycle. The study aims to explore Conyza canadensis SHMT1 (CcSHMT1) as a promising target for herbicide discovery. MethodsStructure determination of CcSHMT1 was resolved by X-ray crystallography. Virtual screening docking experiments were performed with Glide version 5.5. Novel derivatives of dimethyl 2-acetamido terephthalate were further designed, synthesized, and bioassay. The druggability of the inhibitor was evidenced by ultrastructural changes in mitochondria, in vivo and vitro enzyme activity assays, and genetics analysis. ResultsCcSHMT1 has a typical PLP-dependent enzyme 3D structure. The dimethyl 2-acetamido terephthalate-containing compounds had herbicidal activity. Dimethyl 2-(2-(4-(2-(4-bromo-2-chlorophenoxy) acetyl)piperazin-1-yl)acetamido) terephthalate (Compound 9ay, EC50 = 193.8 g a.i./ ha) exhibited the highest herbicidal activity on tested weed among the synthesized compounds. Compound 9ay had no obvious adverse effect on the growth of maize and honeybees. Compound 9ay was verified to target CcSHMT1 as an herbicide candidate. ConclusionA first-in-class CcSHMT1 inhibitor that could be developed as a potent herbicide with a new mode of action and provide an avenue for discovering novel inhibitors of pyridoxal-5-phosphate-dependent enzymes.

CmNAC25 targets CmMYB6 to positively regulate anthocyanin biosynthesis during the post-flowering stage in chrysanthemum

BackgroundAnthocyanin is a class of important secondary metabolites that determines colorful petals in chrysanthemum, a famous cut flower. ‘Arctic Queen’ is a white chrysanthemum cultivar that does not accumulate anthocyanin during the flowering stage. During the post-flowering stage, the petals of ‘Arctic Queen’ accumulate anthocyanin and turn red. However, the molecular mechanism underlying this flower color change remains unclear.ResultsIn this study, by using transcriptome analysis, we identified CmNAC25 as a candidate gene promoting anthocyanin accumulation in the post-flowering stage of ‘Arctic Queen’. CmNAC25 is directly bound to the promoter of CmMYB6, a core member of the MBW protein complex that promotes anthocyanin biosynthesis in chrysanthemum, to activate its expression. CmNAC25 also directly activates the promoter of CmDFR, which encodes the key enzyme in anthocyanin biosynthesis. CmNAC25 was highly expressed during the post-flowering stage, while the expression level of CmMYB#7, a known R3 MYB transcription factor interfering with the formation of the CmMYB6–CmbHLH2 complex, significantly decreased. Genetic transformation of both chrysanthemum and Nicotiana tabacum verified that CmNAC25 was a positive regulator of anthocyanin biosynthesis. Another two cultivars that turned red during the post-flowering stages also demonstrated a similar mechanism.ConclusionsAltogether, our data revealed that CmNAC25 positively regulates anthocyanin biosynthesis in chrysanthemum petals during the post-flowering stages by directly activating CmMYB6 and CmDFR. Our results thus revealed a crucial role of CmNAC25 in regulating flower color change during petal senescence and provided a target gene for molecular design breeding of flower color in chrysanthemum.

Endomicrobiome of in vitro and natural plants deciphering the endophytes-associated secondary metabolite biosynthesis in Picrorhiza kurrooa, a Himalayan medicinal herb.

Picrorhiza kurrooa an endangered high-altitude Himalayan medicinal herb, is used as a potent hepatoprotective due to the presence of various secondary metabolites, with picrosides being the most bioactive. In-vitro propagation is used as a sustainable strategy for its conservation and cultivation. The in-vitro propagation produces P. kurrooa plants (Tc) in mass, but these plants have reduced secondary metabolites (especially picrosides) compared to plants growing in their natural habitats (Wt). Therefore, considering the well-established role of microbes in secondary metabolite biosynthesis, in this study, endomicrobiome of P. kurroa plants (Tc and Wt) was explored. Using high-throughput DNA-sequencing, the endophytic communities associated with leaves, roots, and rhizomes of Wt and Tc plants were characterized. Diversity analysis revealed a loss of diversity during in-vitro propagation, and the abundant phyla were Proteobacteria, Bacteroidetes, Parcubacteria (OD1), Firmicutes, and Verrucomicrobia. Besides, the presence of distinct genera specific to different parts of Wt plants was also revealed. Quantification of secondary metabolites demonstrated the reduced accumulation of picrosides and intermediates of picroside biosynthesis in the Tc plants compared to Wt plants. Host-secondary metabolite production was positively correlated to microbial community abundance, suggesting a dynamic interplay of host-endomicrobiota interaction. Predictive functional analysis revealed the abundance of enzymes of secondary metabolite biosynthesis (especially MVA/MEP and phenylpropanoid/shikimate pathway involved in picrosides biosynthesis) in the associated-endophytic community with predominance in roots and rhizomes of Wt plants. This investigation provides novel insight into the change in the endomicrobiome of Wt and Tc plants and their correlation to the biosynthesis of secondary metabolites, and that needs to be considered for cultivation practices.IMPORTANCEPicrorhiza kurrooa is a major source of picrosides, potent hepatoprotective molecules. Due to the ever-increasing demands, overexploitation has caused an extensive decline in its population in the wild and placed it in the endangered plants' category. At present plant in-vitro systems are widely used for the sustainable generation of P. kurrooa plants, and also for the conservation of other commercially important, rare, endangered, and threatened plant species. Furthermore, the in-vitro-generated plants had reduced content of therapeutic secondary metabolites compared to their wild counterparts, and the reason behind, not well-explored. Here, we revealed the loss of plant-associated endophytic communities during in-vitro propagation of P. kurrooa plants which also correlated to in-planta secondary metabolite biosynthesis. Therefore, this study emphasized to consider the essential role of plant-associated endophytic communities in in-vitro practices which may be the possible reason for reduced secondary metabolites in in-vitro plants.

Interplay of Cholesterol and Actin in Neurotransmitter GPCR Signaling: Insights from Chronic Cholesterol Depletion Using Statin.

Serotonin1A receptors are important neurotransmitter receptors in the G protein-coupled receptor (GPCR) family and modulate a variety of neurological, behavioral, and cognitive functions. We recently showed that chronic cholesterol depletion by statins, potent inhibitors of HMG-CoA reductase (the rate-limiting enzyme in cholesterol biosynthesis), leads to polymerization of the actin cytoskeleton that alters lateral diffusion of serotonin1A receptors. However, cellular signaling by the serotonin1A receptor under chronic cholesterol depletion remains unexplored. In this work, we explored signaling by the serotonin1A receptor under statin-treated condition. We show that cAMP signaling by the receptor is reduced upon lovastatin treatment due to reduction in cholesterol as well as polymerization of the actin cytoskeleton. To the best of our knowledge, these results constitute the first report describing the effect of chronic cholesterol depletion on the signaling of a G protein-coupled neuronal receptor. An important message arising from these results is that it is prudent to include the contribution of actin polymerization while analyzing changes in membrane protein function due to chronic cholesterol depletion by statins. Notably, our results show that whereas actin polymerization acts as a negative regulator of cAMP signaling, cholesterol could act as a positive modulator. These results assume significance in view of reports highlighting symptoms of anxiety and depression in humans upon statin administration and the role of serotonin1A receptors in anxiety and depression. Overall, these results reveal a novel role of actin polymerization induced by chronic cholesterol depletion in modulating GPCR signaling, which could act as a potential therapeutic target.