Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is a rare medical emergency for which a correct and early diagnosis is essential. As a severe deficiency in A Disintegrin And Metalloproteinase with ThromboSpondin type 1 repeats, member 13 (ADAMTS13) is the underlying pathophysiology, diagnostic strategies require timely monitoring of ADAMTS13 parameters to differentiate TTP from alternative thrombotic microangiopathies (TMAs) and to guide initial patient management. Assays for conventional ADAMTS13 testing focus on the enzyme activity and presence of (inhibitory) anti-ADAMTS13 antibodies to discriminate immune-mediated TTP (iTTP) from congenital TTP and guide patient management. However, diagnosis of iTTP remains challenging when patients present borderline ADAMTS13 activity. Therefore, additional biomarkers would be helpful to support correct clinical judgment. Over the last few years, the evaluation of ADAMTS13 conformation has proven to be a valuable tool to confirm the diagnosis of acute iTTP when ADAMST13 activity is between 10 and 20%. Screening of ADAMTS13 conformation during long-term patient follow-up suggests it is a surrogate marker for undetectable antibodies. Moreover, some non-ADAMTS13 parameters gained notable interest in predicting disease outcome, proposing meticulous follow-up of iTTP patients. This review summarizes non-ADAMTS13 biomarkers for which inclusion in routine clinical testing could largely benefit differential diagnosis and follow-up of iTTP patients. Thrombotic thrombocytopenic purpura (TTP) is a rare disease with an incidence estimated at around four cases per million, caused by a severe deficiency of the enzyme ADAMTS13 , and is recognized as thrombotic microangiopathy (TMA) that is characterized by severe thrombocytopenia, hemolytic anemia, and ischemic organ failure . The sub9/L) and microangiopathic hemolytic anemia with highly elevated lactate dehydrogenase (LDH) and indirect bilirubin [Historically, a clinical pentad has been used for the clinical diagnosis of TTP by screening for fever, thrombocytopenia, hemolytic anemia, and renal and neurologic dysfunction . It was ilirubin ,11,12. Silirubin . Frequenilirubin . In the ilirubin . Of noteilirubin . To discilirubin . In a feilirubin . Standard iTTP therapy combines therapeutic plasma exchange (TPE) and corticosteroids and has significantly reduced mortality in acute-phase iTTP below 10–20% ,20. ReceDuring remission, treating physicians typically provide monthly follow-up for the first three months, three-monthly follow-up for the first year, and six-monthly or yearly follow-up when stable . PlateleWhen iTTP patients suffer from clinical relapse, ADAMTS13 tests reveal severely deficient ADAMTS13 activity and positive anti-ADAMTS13 IgG as laboratory findings. Persistent or recurrent deficiency of ADAMTS13 activity during follow-up of survivors of acute iTTP is an established risk factor for disease recurrence ,31. Accop < 0.005) or high anti-ADAMTS13 IgG  3 to 7 days after the initiation of TPE was associated with a higher risk for exacerbation or recurrence [As for ADAMTS13 parameters in acute phase, Sui et al. described that while plasma levels of ADAMTS13 activity, antigen, and anti-ADAMTS13 IgG on admission could not predict exacerbation or recurrence in patients with iTTP, persistently low plasma ADAMTS13 activity below 10 U/dL  and Cysteine-rich (C) domains (hereafter referred to as anti-CS antibodies) ,40,41,42As previously described, the diagnosis of iTTP is always confirmed when the laboratory parameters show an ADAMTS13 activity below 10% and the presence of anti-ADAMTS13 antibodies ,10,42. HWe showed that iTTP patient anti-ADAMTS13 antibodies induce an open ADAMTS13 conformation. Indeed, purified iTTP patient IgG and, more specifically, purified anti-CS antibodies induced an open ADAMTS13 conformation . This fiFinally, although ADAMTS13 activity was decreased in the remission patients with an activity <50%, anti-ADAMTS13 antibodies were often undetectable ,49,50. STo date, the reference assay to evaluate ADAMTS13 conformation is the 1C4 open/closed ELISA developed in our group, in which a cryptic epitope in the Spacer domain of open ADAMTS13 is specifically recognized ,43,44,47p < 0.001 by the Wald test) [Cardiac troponin-T and-I (cTnT and cTnI) are biomarkers commonly used for detecting myocardial injury and the differential diagnosis of acute coronary syndrome ,54. Patild test) . Althougld test) ,20. Acutld test) . A Japanld test) . The UK ld test) . Intriguld test) . Moreoveld test) . An incrld test) . Based old test) ,59. Howeld test) ,59. p < 0.0001) [The GCS is a clinical scale used to reliably measure a person’s level of consciousness after a brain injury. Its score is based on eye opening , verbal , and motor responses . The combined score, which ranges from 3 to 15, reflects consciousness. Generally, brain injury is classified as Severe, GCS ≤ 8; Moderate, GCS 9–12; or Minor, GCS ≥ 13 . Alwan e 0.0001) . In this 0.0001) ,61. A fuEndothelial cell (EC) activation and inflammation have been linked to the pathophysiology of iTTP ,64,65,66Although VWF antigen is increased during the acute phase upon EC activation, high VWF antigen levels were not predictive of disease outcome or relapse ,67. In cAnother protein secreted upon EC activation is big endothelin-1 (Big ET-1). Big ET-1 is a 38-amino acid polypeptide and the precursor of ET-1, a potent vasoconstrictor. The half-life of ET-1 is less than one minute, while Big ET-1 is more slowly cleared. Big ET-1 is synthesized in vascular ECs, where it has been identified in Weibel Palade bodies. It was shown that plasma levels of Big ET-1 are significantly elevated upon admission and during clinical response/remission . ElevateFinally, CECs, which are stressed endothelial cells that become detached from the endothelial membrane and indicate endothelial damage, were elevated during acute phase iTTP. This increase was linked to the presence of initial neurological symptoms and demonstrated a correlation with the patient’s clinical outcome. Whether an increase in CECs predicts relapse remains to be determined . Syndecan-1 (Sdc-1) and soluble thrombomodulin (sTM) are the main components of the endothelial glycocalyx, a layer of membrane-bound macromolecules anchored to the luminal surface of the vascular endothelium. In specific pathologic conditions, such as acute inflammation and ischemia-reperfusion injury ,71,72, lGranular, azurophilic neutrophil content, such as S100A8/A9, human neutrophil peptides 1–3 (HNP1–3), and neutrophil extracellular traps (NETs), are released upon inflammation or neutrophil activation . MoreoveMarkers of endothelial and leukocyte activation are increased in acute iTTP, and some of these are associated with neurological symptoms or patient outcomes. However, larger prospective studies are needed to prove their usefulness in predicting disease outcome and relapse.This review summarizes essential biomarkers to differentially diagnose iTTP from alternative TMAs and allow subdiagnosis of various iTTP forms . Clinica


Organic anion transporter 3 (OAT3), at the basolateral membrane of kidney proximal tubule cells, facilitates the elimination of numerous widely used drugs. Earlier investigation from our laboratory revealed that ubiquitin conjugation to OAT3 leads to OAT3 internalization from the cell surface, followed by degradation in the proteasome. In the current study, we examined the roles of chloroquine (CQ) and hydroxychloroquine (HCQ), two well-known anti-malarial drugs, in their action as proteasome inhibitors and their effects on OAT3 ubiquitination, expression, and function. We showed that in cells treated with CQ and HCQ, the ubiquitinated OAT3 was considerably enhanced, which correlated well with a decrease in 20S proteasome activity. Furthermore, in CQ- and HCQ-treated cells, OAT3 expression and OAT3-mediated transport of estrone sulfate, a prototypical substrate, were significantly increased. Such increases in OAT3 expression and transport activity were accompanied by an increase in the maximum transport velocity and a decrease in the degradation rate of the transporter. In conclusion, this study unveiled a novel role of CQ and HCQ in enhancing OAT3 expression and transport activity by preventing the degradation of ubiquitinated OAT3 in proteasomes. Numerous widely used drugs, such as antiviral drugs, anticancer therapeutics, antibiotics, antihypertensives, and anti-inflammation drugs, are removed from the body through the organic anion transporter 3 (OAT3) at the basolateral membrane of the kidney proximal tubule cells ,2,3. OATOAT3 transport activity vitally relies on its expression level at the plasma membrane, which can be changed under various diseased and pharmacological situations ,8. Our lChloroquine, a well-known anti-malarial drug, was previously reported to have an inhibitory effect on proteasome in MDAY-D2 cell (a murine leukemia cell) extracts under supra-pharmacological concentrations . HoweverHCQ differs from CQ in its chemical structure by having a hydroxyl group at the end of the N-ethyl side chain . AlthougBoth CQ and HCQ are predominantly renal eliminated with long half-lives, ranging from 20–60 days and 20–40 days ,31, resp3H]-labeled estrone sulfate from PerkinElmer . Mouse anti-myc antibody (9E10) was purchased from Roche . Mouse anti-E-cadherin antibody was purchased from Abcam . Mouse anti-ubiquitin antibody, mouse anti-β-actin antibody and normal mouse IgG were purchased from Santa Cruz Biotechnology . A 20S proteasome assay kit was purchased from Sigma-Aldrich . Normal mouse lgG, mouse monoclonal anti-ubiquitin and mouse monoclonal anti-β-actin antibodies were purchased from Santa Cruz .Parental COS-7 cells and HEK293 cells were purchased from ATCC . Hydroxychloroquine Sulfate and Chloroquine Disulfate were purchased from Sigma-Aldrich . Membrane-impermeable biotinylation reagent sulfo-NHS-SS-biotin, streptavidin agarose, protein G-agarose beads, and horseradish peroxidase-conjugated anti-mouse antibodies were obtained from Pierce Biotechnology . We purchased -estrone sulfate (300 nM) was performed, following the standard protocol previously established in our lab [3H]-estrone sulfate in the cells. Passive diffusion data were collected with mock cells .Uptake of [ our lab . A 4-min3H]-estrone sulfate was measured with an estrone sulfate concentration range of 0.3–10 µM. The same uptake procedure was applied as described in the section “Measurement of transport activity”. The kinetic values of estrone sulfate transported by OAT3 were determined using non-linear least-squares regression analysis from Michaelis–Menten equation: V = Vmax × [S]/(Km + [S]). Transport kinetic parameters were determined using the Eadie–Hofstee transformation.OAT3-expressing cells were pretreated with or without 10 µM HCQ for 4 h, and 3-min uptake of -labeled estrone sulfate was then measured. During our treatment, the fetal bovine serum was not included in the medium. Therefore, to maintain the cells in a healthy state, treatment longer than 6 h was not performed. The results (OAT3-expressing cells were treated with CQ and HCQ for various time periods (2–6 h), and OAT3-mediated uptake of [ results  demonstr3H]-estrone sulfate was then determined , without notable alteration in the substrate-binding affinity Km.To explore the mechanism underlying CQ- and HCQ-stimulated OAT3 activity, a kinetic analysis was carried out, with HCQ as the representative drug. We measured [ fitting . As showOAT3-expressing cells were treated with CQ or HCQ, and OAT3 expression both at the cell surface and in the total cell lysates was analyzed . We showWe then determined the degradation rate of cell surface OAT3 using a biotinylation approach. COS-7 Cells expressing OAT3 were biotinylated with membrane-impermeable biotinylation reagent sulfo-NHS-SS-biotin. Labeled cells were treated with or without HCQ at 10 µM for 0, 2, 4, and 6 h and then lysed. Cell surface proteins were isolated by streptavidin-agarose resin, followed by immunoblotting with an anti-myc antibody. Our results  showed tOAT3 transport activity critically relies on its expression level at the plasma membrane, which is subjected to various types of regulation ,40,41. WOAT3 recognizes a wide range of substrates . As a rePharmacokinetically, both CQ and HCQ have long mean residence times caused by their potential to distribute in aqueous cellular and intercellular compartments (~50 days for hydroxychloroquine and ~40 days for chloroquine) ,52. SignThe COS-7 cells and HEK293 cells used in the current study have several advantages: these cells are of kidney origin ,55,56. TCQ and HCQ markedly enhanced the accumulation of ubiquitinated OAT3 , which cPreviously several reports indicated that both CQ and HCQ have inhibitory effects on lysosomes ,63,64,65Our discovery of CQ and HCQ in upregulating the activity and expression of OAT3 has significant physiological implications. First, with the OAT3 function of transporting endogenous substrate, CQ and HCQ can stimulate the renal clearance of metabolites, nutrients, signaling molecules, and other OAT3 substrates, which may disturb homeostasis. Additionally, CQ and HCQ stimulate the OAT3 transport activity, leading to increased elimination of the drugs that are OAT3 substances, thereby altering their efficacy. Moreover, as proteasome inhibitors, CQ and HCQ may have potential therapeutic effects on diseases in which the proteasome activity is abnormally high such as multiple myeloma, Crohn’s disease, and ulcerative colitis ,67,68.Our current studies unveiled a novel role of both CQ and HCQ in upregulating OAT3 expression and function by inhibiting proteasome activity, thereby preventing the degradation of the transporter , which s


The Rhodanese-fold is a ubiquitous structural domain present in various protein subfamilies associated with different physiological functions or pathophysiological conditions in humans. Proteins harboring a Rhodanese domain are diverse in terms of domain architecture, with some representatives exhibiting one or several Rhodanese domains, fused or not to other structural domains. The most famous Rhodanese domains are catalytically active, thanks to an active-site loop containing an essential cysteine residue which allows for catalyzing sulfur transfer reactions involved in sulfur trafficking, hydrogen sulfide metabolism, biosynthesis of molybdenum cofactor, thio-modification of tRNAs or protein urmylation. In addition, they also catalyse phosphatase reactions linked to cell cycle regulation, and recent advances proposed a new role into tRNA hydroxylation, illustrating the catalytic versatility of Rhodanese domain. To date, no exhaustive analysis of Rhodanese containing protein equipment from humans is available. In this review, we focus on structural and biochemical properties of human-active Rhodanese-containing proteins, in order to provide a picture of their established or putative key roles in many essential biological functions. H2S is described as an ubiquitous and pleiotropic molecule with a broad spectrum of targets, including nuclear factor-kappa (NF-κB), p38 mitogen-activated kinase (p38 MAPK), nuclear factor erythroid 2-related factor 2 (Nrf2), p53 and mTOR, the target of rapamycin ..2].https://www.rcsb.org accessed on 5 September 2022) allows a direct inspection of Rhd-domain conserved structural signatures. As an example, an in silico large scale identification and analysis of Rossmann-like structural domains revealed that the Rhd fold belongs to this vast family. Its structure is composed of a three-layered α/β sandwich (a characteristic of Rossmann-like protein) in which, the central parallel β sheet is surrounded by 4 to 5 α-helices (4/5(R/T) loop motif  for thiosulfate, and 3-mercaptopyruvate sulfurtransferases (3MSTs or MPSTs) for 3-mercaptopyruvate . The twoBecause of their highly divergent nature, sequence identity search programs cannot be used to exhaustively identify Rhd domains from sequence databases. A keyword-based search of the human Rhd domains present in the UniProt library  identifiin cellulo, except for TSTD2  and three catalyze phosphatase reactions (CDC25A/B/C), whereas no typical Rhd-dependent activity is proposed for the last one (TSTD2). In terms of domain organization, TSTD1 belongs to class one, MPST and TST to class two, and CDCD25A/B/C, MOCS3 and TSTD2 to class three c. These or TSTD2 .KAT gene located on chromosome 1 (1q23.3), which is 1.6 kilobases long and consists of 4 exons and 3 introns . T. T36]. TTSTD1 transcripts accumulate in different tissues, such as the kidney, liver, skeletal muscle, heart, colon, thymus, spleen, placenta, or lung . I. ITST geKM value b,c, suggciencies .Although the TST protein is targeted to mitochondria, it does not contain a N-terminal mitochondrial targeting sequence  28,42,4,442,47. 2S produced by sulfate-reducing microbiota . A. A54]. Apyruvate a and bot2S production  and thioredoxin . Th. Th27]. 2S production in the brain, kidney, and liver at the mitochondrial level . T. T67]. Tive site ,69. The ive site .5R motif. Although Glu474 was recently proposed for CDC25B, the residue involved in acid/base catalysis for leaving group protonation and water molecule activation remains to be identified. Moreover, arsenate reductase activity was revealed for the catalytic domain of the human CDC25B and CDC25C, as these two enzymes were shown to catalyze in vitro inorganic arsenate reduction in the presence of glutathion and glutaredoxin . MOCS3 is a protein of 461 residues (49 kDa), constituted of two structural domains  and, as expected from the structure of MoeB homologs, the human Moeb-like domain is constituted of a central height strands β-sheet, surrounded by height helices , a, aMOCS3  domains b , MOCS2, or GPHN (Gephyrin) genes . Th. ThLegion a loop .E. coli, named YceA or TrhO, was recently characterized . Th. ThE. coAt the physiological level, the cellular functions of TSTD2 protein are still unknown. TSTD2’s gene expression or protein accumulation variations were reported for human traits or diseases resistance/susceptibility, but, to date, this information is too preliminary to propose physiological implications ,91,92.The increasing number of identified and characterized human Rhd domain-containing proteins supports the notion that the Rhd structural domain has evolved, as isolated protein or in combination with other domains, to fulfill different cellular functions. Although, the fine characterization of the active Rhd domains is still elusive, especially for the last one reported, the emerging picture in this catalytic promiscuity stressed the crucial role of the active-site loop, the structural domains organization and interaction networks, depending on subcellular localization. In the future, the exhaustive characterization of human Rhd-containing proteins at the biochemical and cellular level is a decisive step to better define the functions of Rhd-containing proteins, and to reveal their key contributions in human physio- and pathophysiology.


Phenylalanine and phosphorylcholine were mainly downregulated, suggesting altered neurotransmitter synthesis. N-acetylaspartate and GABA were upregulated in most regions, suggesting higher inhibitory activity in neural circuits. Other region-common metabolic pathways indicated impaired mitochondrial function and energy metabolism, while region-unique pathways indicated oxidative stress and altered immune responses. Importantly, AD caused metabolic changes in brain regions with less well-documented pathological alterations that suggest degenerative progression. The findings provide a new understanding of the biochemical mechanisms of AD and guide biomarker discovery for personalized risk prediction and diagnosis.Pathological mechanisms contributing to Alzheimer’s disease (AD) are still elusive. Here, we identified the metabolic signatures of AD in human post-mortem brains. Using  The elderly population is growing worldwide and so are the concerns over the rise in aging-associated neurodegenerative diseases, such as Alzheimer’s disease (AD) ,2. AD isThe formation of neurofibrillary tangles (NFTs) and amyloid-beta plaques (Aβ) in the brain is considered a key pathological hallmark of AD ,12 assocGenetic risk factors, such as inheritance of apolipoprotein E (APOE), presenilin-1 (PSEN1), presenilin-2 (PSEN2), and amyloid precursor protein (APP) genes, can increase the risk of neuropathological changes linked to AD ,6,12. AP1H NMR) spectroscopy demonstrated N-acetylaspartate (NAA) reduction in the grey matter of BA17 and superior temporal gyrus (BA22), among other regions  in post-mortem human tissues . The ideThis study involved post-mortem brain tissues from 11 AD patients and 11 healthy subjects (HS), including 5 females and 6 males in each group. The average age was 80 years old for AD participants and 65 years old for HS. The average neuropathology ABC score was 7.63 out of possible 9 in AD and 1.63/9 in HS subjects .AD significantly altered the metabolic profiles of all eight brain regions , BA24 (70%), PD (65%), BA17, and BA22, with 60% of metabolites being changed in AD tissues C,D. BA9 When AD-associated metabolite alterations were compared across all investigated brain regions, some common and unique metabolites were identified. Common metabolites were identified in the hallmark regions most affected by AD  and the least affected regions (DN and PB) A. One coVIAVC analysis identified common and unique metabolites that may serve as biomarkers of AD . Correspp-value < 0.05 and impact factor higher than 0.1 were considered. Notable pathways affected by AD included alanine aspartate and glutamate , phenylalanine metabolism and phenylalanine tyrosine, and tryptophan biosynthesis . Pyruvate metabolism and tricarboxylic acid (TCA) cycle was changed in BA9 and HPC and BA9, BA22, and DN, respectively. Glycerophospholipid metabolism was significantly altered in BA17, BA24, and BA40. Pathways uniquely altered in specific brain regions included D-glutamine D-glutamate in HPC, cysteine and methionine metabolism in BA22, glycolysis/gluconeogenesis, and the synthesis and degradation of ketone bodies in BA9  showed that AD altered this pathway in the frontal cortex (BA9), with upregulation in glutamate, GABA, NAA, and citrate metabolites. In this study, alanine, aspartate, and glutamate metabolisms were altered in all brain regions, except BA17 and PB. Therefore, these findings, for the first time, show changes in alanine, aspartate, and glutamate metabolisms throughout the brain, indicating that these metabolites may represent early biomarkers of AD. Alterations in these metabolites contribute not only to mitochondrial dysfunction, but also form compounds involved in the TCA cycle; glycolysis; and glycine, serine, and threonine metabolisms. Ultimately, their alteration may indicate an impaired energy metabolism, along with reduced mitochondrial efficacy, a hallmark neuropathological feature of AD ,28,41.Similar to energy metabolism alterations in AD ,19,65, tThis study provides evidence that AD is associated with brain-wide metabolic changes. Some of the same metabolites, including GABA, arginine, serine, phosphorylcholine, and phenylalanine, were previously identified in the CSF of AD patients ,16,17,57The participants were Alberta, Canada, residents who lived either at home or in a nursing home or were admitted to hospital. The basic demographics, including age, gender, post-mortem delay (PMD), and the pathological characterization of samples, are shown in Post-mortem brain tissues were obtained between 2015 and 2018. A total of 22 post-mortem human brain samples were obtained from the Calgary Brain Bank , n = 11, 5 females, 6 males, ages 65 ± 12 yrs.; AD brain tissues were collected from patients in the late stages of the disease. Brains were collected and processed according to the standardized operational protocol of human brain banking in Canada. In brief, within 18–55 h post-mortem, brains were collected, and hemispheres were dissected sagittal . At randABC score assessments were completed by a registered neuropathologist. In brief, the tissues were characterized as AD or control based on assessment for neuritic plaques and neurofibrillary tangles, as established by the Consortium to Establish a Registry for Alzheimer’s Disease (CEDAR) , amyloid2HPO4 to KH2PO4 in dH2O at a 0.625 M concentration with a final pH of 7.41 contained an antimicrobial agent of 3.75 mM NaN₃), and 150 mg of zirconium oxide beads were added to a centrifuge tube. Then, samples were homogenized in a Bullet Blender  for 1 min at intensity setting eight, and homogenate was then centrifuged for 5 min at 14,000 g. Next, 365 μL of homogenate and 135 μL of metabolomic buffer were placed in Amicon Ultra 0.5 mL 3 K centrifuge filters and centrifuged for 30 min at 14,000 g. After filtration, 360 μL of the filtrate, 120 μL of metabolomics buffer, and 120 μL of D2O with w/v 0.03% trimethylsilyl propanoic acid (TSP) were moved to a new centrifuge tube. The final dH₂O:D2O ratio was 4:1, and the final concentration was 0.5 M for the buffer salts. Finally, samples were centrifuged for 5 min at 12,000 rpm, and 550 μL of supernatant was pipetted to 5 mm NMR tubes for NMR processing.Ultrafiltration was used to extract water-soluble metabolites from each tissue sample. In brief, tissue samples were removed from −80 °C storage and kept on ice to thaw at room temperature. Approximately 150 mg of each sample, 375 μL of metabolomics buffer  equipped with a TBO-Z probe ,67,68. Ip-value < 0.05 were considered significant. Bonferroni–Holm correction was applied to correct for multiple comparisons. Supervised multivariant data analysis consisted of variable importance analysis based on random variable combination (VIAVC) [Univariate and multivariate testing was performed using MATLAB  and MetaboanalystR (v 2.0) . A Shapi (VIAVC) ,72, orth, and only metabolites formerly observed in brain tissue, cerebrospinal fluid, and blood were used. Finally, pathway topology analysis [Homo sapiens KEGG pathway library (v Oct.2019). Relative-betweenness centrality was selected for pathway topology analysis, and the hypergeometric test was used for over-representation analysis.Metabolite identification was performed for the bins that were determined to be significant via either the MW or VIAVC test using Chenomx 8.2 NMR Suite . Then, identified metabolites were confirmed using the Human Metabolite Database (HMDB) , and onlanalysis  was perfThe exact biological mechanisms and early predictive markers of AD are still incompletely understood. New biomarker discovery tools such as metabolomics enables the identification of biochemical processes and biomarkers associated with AD for better risk prediction and diagnosis. For the first time, this study uncovered the biochemical pathways associated with AD across eight brain regions. Common metabolites involved in AD pathology across brain regions include amino acids phenylalanine, phosphorylcholine, GABA, NAA, and citric acid, while unique metabolites included pyruvate, lactic acid, succinic acid, myoinositol, and arginine . The affIt remains to be confirmed if these changes in cell metabolism reflect pathological or compensatory responses. The extent of metabolic changes is consistent with the neuropathological disease progression of AD and clinical observations of cognitive impairment ,29. More


Vaccinium corymbosum). In this study, we identified 72 VcUSP genes from the Genome Database for Vaccinium. These VcUSPs could be divided into five groups based on their phylogenetic relationships. VcUSPs from groups Ⅰ, Ⅳ, and Ⅴ each possess one UspA domain; group Ⅰ proteins also contain an ATP-binding site that is not present in group Ⅳ and Ⅴ proteins. Groups Ⅱ and Ⅲ include more complex proteins possessing one to three UspA domains and UspE or UspF domains. Prediction of cis-regulatory elements in the upstream sequences of VcUSP genes indicated that their protein products are likely involved in phytohormone signaling pathways and abiotic stress responses. Analysis of RNA deep sequencing data showed that 21 and 7 VcUSP genes were differentially expressed in response to UV-B radiation and exogenous abscisic acid (ABA) treatments, respectively. VcUSP41 and VcUSP68 expressions responded to both treatments, and their encoded proteins may integrate the UV-B and ABA signaling pathways. Weighted gene co-expression network analysis revealed that VcUSP22, VcUSP26, VcUSP67, VcUSP68, and VcUSP41 were co-expressed with many transcription factor genes, most of which encode members of the MYB, WRKY, zinc finger, bHLH, and AP2 families, and may be involved in plant hormone signal transduction, circadian rhythms, the MAPK signaling pathway, and UV-B-induced flavonoid biosynthesis under UV-B and exogenous ABA treatments. Our study provides a useful reference for the further functional analysis of VcUSP genes and blueberry molecular breeding.Universal stress proteins (USPs) play essential roles in plant development, hormonal regulation, and abiotic stress responses. However, the characteristics and functional divergence of USP family members have not been studied in blueberry ( Escherichia coli; UspA was the first of these genes to be cloned and sequenced [Universal stress proteins (USPs) are members of the adenine nucleotide alpha hydrolase (AANH) superfamily (PF00582 protein family in the Pfam classification) that are widely conserved in bacteria, archaea, plants, and metazoans . There aequenced ,4,5. Theequenced ,7. The Uequenced ,10. USPsequenced .Oryza sativa). Many USPs have been characterized in a variety of plant species, including Arabidopsis thaliana, wild tomato (Solanum pennellii), wild apple , and grapevine (Vitis vinifera) [AtUSPs in Arabidopsis are induced by UV-B treatment, while most VvUSPA genes in grapevine show transcriptional responses to ABA [CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and TIMING OF CAB EXPRESSION 1 (TOC1) [Vitis yeshanensis), possibly by interacting with phytohormone signaling pathways, the ubiquitination system, ethylene-responsive element binding factors, or nuclear factors [The first plant USP was identified in rice (inifera) ,14,15,16inifera) ,18,19. Ms to ABA . In Arab1 (TOC1) . Various1 (TOC1) ; for exa1 (TOC1) . VyUSPA3Plants are subjected to a variety of abiotic stresses. UV-B radiation is an environmental signal that affects plant growth and development and controls many processes involved in physiological and biochemical acclimation. The phytohormone ABA modulates physiological processes by controlling plant responses to biotic and abiotic stress. Several studies have explored the interaction between UV-B and ABA signaling ,24,25. AVaccinium corymbosum) is an economically important small fruit crop worldwide due to the high phenolic acid and flavonoid contents of its fruits and leaves [USP genes have been identified in Arabidopsis, barley (Hordeum vulgare), grapevine, and rice, a comprehensive study of USP genes in blueberry has not yet been reported [V. corymbosum cv. Draper genome was completed, and its function was annotated in March 2019. This Genome Database offers the possibility to systematically identify and investigate the putative functions of USP family members in blueberry because of its high-quality data [Blueberry  data and examined the relationships between VcUSPs and transcription factors under UV-B and ABA treatments via weighted gene co-expression network analysis (WGCNA). Our results provide valuable information for the further functional characterization of blueberry VcUSP genes and guidance for blueberry breeding under UV-B radiation and ABA treatments.In this study, we identified 72 VcUSPs, we conducted a Hidden Markov Model (HMM) search using the USP domain (PF00582) as a query against the Genome Database for V. corymbosum cv. Draper V1.0. After removing sequences without USP or AANH domains and short and redundant sequences, 72 putative USP family genes were identified in blueberry , Arabidopsis (51 members), and grapevine (21 members). These USPs were categorized into the five blueberry USP groups . Most VcUSPs can be classified as ATP binding or non-ATP binding based on the presence or absence of ATP-binding sites ,9,10. SeWe also analyzed the UspA domains of the VcUSPs via sequence alignment . Of the VcUSP genes, we predicted the presence of 20 conserved motifs in these genes using the MEME website  found in VcUSP32, VcUSP33, and VcUSP52; VcUSP65 contained only one motif. All VcUSPs from group Ⅰ contained conserved motifs 1, 2, and 3; most VcUSPs from groups Ⅱ and Ⅲ contained conserved motifs 3, 5, 6, and 16; and most VcUSPs from groups Ⅳ and Ⅴ contained conserved motifs 2 and 4. Conserved motifs 6, 7, 13, and 14 were only observed in group Ⅰ, while motifs 10, 11, and 18 were found only in group Ⅱ. Conserved motifs 8 and 12 were detected only in group Ⅳ, while motif 15 was found in groups Ⅱ and Ⅲ. VcUSPs that clustered in the same groups shared a similar motif pattern. Conserved motifs 1, 2, 3, and 4 were present in all groups; therefore, we considered these to be the main motifs of the VcUSP family.To gain further insight into the structural characteristics of  website A. These In addition to the USP domain, we also identified six other domains in VcUSPs B. In groVcUSP genes  by predicting and visualizing the cis-regulatory elements in these regions , low temperature (LTR), drought (MBS), wounding (WUF motif), and biotic defense (TC-rich repeats). We also identified phytohormone-responsive elements, such as those responsive to auxin , GA , salicylic acid (TCA element), jasmonate (CGTCA motif and TGACG motif), and ABA (TCA element). Light-responsive elements were present in the promoters of all VcUSPs, while the ABA-responsive element was identified in the promoters of 56 VcUSPs, suggesting that most VcUSPs are involved in plant responses to light and ABA.To predict the potential functions of  regions . In addiVcUSPs in response to light stress, we downloaded RNA-seq data from blueberry calli after 0, 1, 3, 6, 12, and 24 h of UV-B treatment from the BioProject database , all six VcUSPs from group I , VcUSP22 from group Ⅱ, and VcUSP68 from group Ⅴ were downregulated, while 10 VcUSP genes from group III  and three VcUSP genes from group II  were upregulated by UV-B treatment . VcUSP5, VcUSP13, and VcUSP68 were also downregulated by UV-B treatment. In contrast, UV-B radiation promoted the expressions of VcUSP41 and VcUSP51. These results verified the accuracy and reliability of the VcUSP gene expression patterns determined based on RNA-seq data.To validate the accuracy and reliability of the analysis B. The exVcUSP genes contain cis-regulatory elements related to phytohormones, especially ABA. Therefore, we downloaded RNA-seq data of in vitro-grown blueberry seedlings subjected to ABA treatment from the BioProject database  increase in expression after 12 h of ABA treatment. These expression patterns determined using RT-qPCR were similar to those observed in the RNA-seq data, verifying the reliability of the RNA-seq data.To confirm the accuracy and reliability of the expression patterns of these seven differentially expressed  RT-qPCR B. The exVcUSPs using the WGCNA package in R and calculated their Pearson’s correlation coefficients . The WGCNA clustered all the DEGs into three modules per treatment: kMEblue, kMEbrown, and kMEturquoise for UV-B radiation and kMEblack, kMEblue, and kMEturquoise for ABA , WRKY (16.85%), bHLH (5.62%), zinc finger (21.35%), AP2 (15.73%), auxin, AUX/IAA, bZIP, TCP, and NAC families , bHLH (20.41%), MYB (18.37%), AP2 (18.37%), and other transcription factor families , families . For theor genes , includior genes . The kMEVcUSPs, we calculated Pearson’s correlation coefficients and reconstructed the co-expression networks according to the r values  pathway analysis showed that most of these transcription factors were involved in hormone signal transduction (ko04075), the circadian rhythm (ko04712), and the MAPK signaling pathway (ko04016). These results indicate that VcUSPs are mainly co-expressed with transcription factors from the MYB, AP2, zinc finger, and bHLH families and that VcUSPs may regulate the expression of downstream genes through synergistic or antagonistic effects with certain transcription factors under UV-B or ABA treatment.To screen for transcription factors whose expressions are significantly correlated with r values A–C. VcUSUSP genes have been identified in Arabidopsis, 21 in barley, 21 in grapevine, and 44 in rice [VcUSP genes in blueberry. This high number relative to Arabidopsis and other previously characterized plant species may be related to genome duplication in blueberry or to differences in genome size or evolutionary histories between the species [USPs are present in a wide variety of organisms and participate in a range of cellular responses to biotic and abiotic stress. To date, 44  in rice ,34,35,36 species ,39.Catharanthus roseus), while pkinase, pkinanse_Try, and U-box domains are found in Arabidopsis and rice USPs [We determined that VcUSPs can be structurally diverse. USPs in other species generally contain a single UspA domain or two tandem repeats of UspA domains . Most Vcice USPs ,40. In tice USPs . Most price USPs ,43,44. Tice USPs . The Tolice USPs . These dVcUSPs, we analyzed their conserved motifs and exon–intron structures. We identified 20 motifs, with motifs 1–11 found in each of the VcUSPs. Some motifs  were only present in one VcUSP group. The number of exons in VcUSPs ranged from 1 to 11 per gene  resulted in increased tolerance to freezing, salinity, osmotic stress, and methyl viologen-induced oxidative stress [VvUSPA2, VvUSPA3, VvUSPA11, VvUSPA13, and VvUSPA16 in E. coli enhanced resistance to drought stress [AtUSP (At3g53990) conferred strong tolerance to heat shock and oxidative stress in Arabidopsis [USPs participate in a broad range of cellular responses to biotic and abiotic stress, and their roles in providing stress resistance in many plants have been reported. For instance, the overexpression of e stress . The hett stress . The ovebidopsis ; howeverbidopsis . TherefoE. coli, the deletion of UspA, UspC, UspD, or UspE resulted in an enhanced sensitivity to UV-B exposure [AtUSP genes are induced by UV-B treatment [VcUSP genes that were responsive to UV-B radiation based on transcriptome data. Most of these VcUSPs belong to groups Ⅰ and Ⅲ; group I VcUSPs were downregulated and group Ⅲ VcUSPs were upregulated in response to UV-B treatment. All group Ⅰ VcUSP proteins contained one UspA domain and one ATP-binding site and shared highly similar conserved motifs, while most group Ⅲ VcUSP proteins contained not only a UspA domain but also an UspF domain and similar conserved motifs. These results indicate that proteins within the same phylogenetic clade share close evolutionary relationships, conserved structures, and similar functions. Similar results have been obtained for other proteins, including MYBs and NACs [UV-B radiation, an environmental signal, limits plant growth and development. In exposure ,48. Micrreatment . In thisand NACs ,50.SpUSP expression is markedly induced by ABA and plays important roles in drought tolerance by influencing ABA-induced stomatal movement [VyUSPA3 from Chinese wild grape improved drought tolerance in transgenic V. vinifera, likely by regulating the ABA signaling pathway [VcUSP genes responded to ABA treatment, including six upregulated and one (VcUSP4 from group Ⅰ) downregulated gene. Most of the upregulated VcUSP genes  showed a more than four-fold increase in expression compared to control conditions. Similar to other plant species, most differentially expressed VcUSPs in response to ABA were upregulated. Therefore, these genes might improve stress tolerance in blueberry by regulating the ABA signaling pathway. The large changes in expression of the seven DEGs in response to ABA treatment confirm the notion that these genes regulate ABA-related stress responses.The phytohormone ABA regulates plant responses to abiotic stress. In the wild tomato, movement . Exogeno pathway . Indeed, pathway ,22,36. IPopulus yunnanensis) [VcUSP41 and VcUSP68, with the former upregulated by both treatments and the latter upregulated by ABA but downregulated by UV-B. Therefore, it is possible that VcUSP41 and VcUSP68 act to bridge the UV-B and ABA signaling pathways for regulating stress responses or physiological and biochemical functions in blueberry.ABA mediates the core signaling network in the plant abiotic stress response ,52. Seveanensis) . Which pCatharanthus roseus, uspA-like transcripts are co-expressed with many putative ethylene-responsive bHLH or WRKY transcription factor genes [LATE ELONGATED HYPOCOTYL (LHY) of the MYB family and PSEUDO RESPONSE REGULATOR (PRR) form an early feedback loop in the circadian clock [VyUSPA3 from Chinese wild grape improved drought tolerance in transgenic V. vinifera, possibly by interacting with a phytohormone signaling pathway, an ubiquitination system, ethylene-responsive element binding factors, or nuclear factors [VcUSP26, VcUSP67, VcUSP68, VcUSP22, and VcUSP41 may be involved in the UV-B or ABA-induced MAPK signaling pathways, plant–pathogen interactions, plant hormone signal transduction, and circadian rhythms.USPs are small proteins that exist as monomers, dimers, trimers, and oligomers and regulate stress responses by interacting with various proteins ,14,16,53or genes . In the or genes ,56,57,58or genes , while Aor genes . LATE ELan clock ,62. In Aan clock . ExogenoMYB12 in response to UV-B radiation [HY5 promoter to activate its expression and interacts with BBX32 [VcUSP26, VcUSP67, and VcUSP68 were co-expressed with COP1, BBX21, BBX32, and MYB12, while VcUSP22 and VcUSP41 were co-expressed with HY5 under UV-B treatment. At the same time, VcUSP26, VcUSP67, and VcUSP68 were co-expressed with MYB114 (AtMYB114 homolog), MYBA (AtTT2 homolog), MYB11 (AtMYB114 homolog), MYB12 (AtMYB12 homolog), and MYBPA (AtMYB5 homolog) under UV-B radiation; these co-expressed genes control the biosynthesis of flavonoids, including anthocyanins, proanthocyanidins, and flavonols [Plants are typically subjected to UV-B radiation, which activates UV RESISTANCE LOCUS 8 (UVR8) to interact with the E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) ,64. ELONadiation ,66, whicadiation ,68. In tth BBX32 ,70. We flavonols ,72,73,74VcUSPs, Hidden Markov Model searches were performed in the Genome Database for the Vaccinium corymbosum cv. Draper V1.0 genome sequence (https://www.vaccinium.org/ (accessed on 1 July 2023)) using the USP domain (PF00582) from the Pfam database (http://pfam.xfam.org/ (accessed on 1 July 2023)) as a query. The candidate VcUSPs were investigated using the online programs Pfam and CDD (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi (accessed on 2 July 2023)), and genes without USP or AANH domains in their encoded proteins were removed. Finally, a list of VcUSPs with at least one USP domain was obtained by deleting redundant sequences based on sequence alignments generated using DNAMAN version 6.0.3.99 . The molecular weight, theoretical isoelectric point (pI), instability index, aliphatic index, and grand average of hydropathicity of the proteins were calculated for the VcUSP proteins using the online program ExPASy (https://web.expasy.org/protparam/ (accessed on 4 July 2023)).To identify putative A. thaliana and V. vinifera were downloaded from NCBI (https://www.ncbi.nlm.nih.gov/ (accessed on 5 July 2023)). The phylogenetic trees were constructed in MEGA X version 11.0.10 (https://www.megasoftware.net/ (accessed on 2 July 2023)) using the Maximum Likelihood method. A bootstrap analysis was carried out with 1000 replicates [The deduced amino acid sequences of the VcUSPs were aligned using DNAMAN version 6.0.3.99. The USP amino acid sequences of plicates .https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi (accessed on 10 July 2023)). Generic feature format files of the VcUSP family members were downloaded from the Genome Database for Vaccinium, including sequence information for the untranslated regions (UTRs), exons, and introns. The conserved motifs of the VcUSP amino acid sequences were uploaded to the online search tool MEME (http://meme-suite.org/tools/meme (accessed on 10 July 2023)), with the maximum number of motifs set at 20 and the order of site distribution set to zero or one occurrence per sequence. To analyze the cis-regulatory elements in the promoters of the VcUSPs, the 2000 bp upstream sequence of each gene was downloaded from the Genome Database for Vaccinium and submitted to the online program PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 20 July 2023)). The conserved domains, conserved motifs, exon–intron structures, and cis-regulatory elements in the promoters of the VcUSPs were visualized using TBtools-Ⅱ software (version 2.003) [The amino acid sequences of the VcUSPs were subjected to a BLAST search against the NCBI database to predict the conserved UspA, UspE, and UspF domains. The conserved domains used for visualization were predicted using the online program CDD (n 2.003) .VcUSP genes under 311 nm UV-B radiation  or 100 µM ABA treatment  were downloaded from the BioProject database in the NCBI repository ) [Differentially expressed t 2023)) . The DEGt 2023)) . A heatmVcUSPs and transcription factor genes from the same module were screened. Pearson’s correlation coefficient (r) analysis was performed between VcUSPs and transcription factor genes according to the FPKM values using SPSS 19.0 software . Pairs of genes with a p value ≤ 0.05 were considered to be significantly correlated. The co-expression networks were visualized based on their r values using Cytoscape version 3.9.1 [WGCNA was performed on transcriptome data obtained from plants under UV-B radiation or ABA treatment using the WGCNA package in R . The hieon 3.9.1 . The KEGon 3.9.1 , NCBI noon 3.9.1 , and Proon 3.9.1  databaseon 3.9.1  and evolon 3.9.1 , were usThe blueberry cultivar ‘Northland’ calli and in vitro–grown seedlings were used for UV-B radiation and ABA treatments, respectively. The blueberry calli and in vitro–grown seedlings were cultured on modified woody plant medium (WPM) containing Murashige and Skoog vitamins with 3.0 mg/L 2,4-dichlorophenoxyacetic acid  or 1.0 mg/L trans-Zeatin (seedlings) under a 16 h light/8 h dark photoperiod at 25 °C and subcultured every 3 weeks  and 5 weeks (seedlings). UV-B was applied by the means of narrow band lamps  positioned above the calli at the height of about 10 cm for 0, 1, 3, 6, 12, or 24 h . The seeVcUSP genes under UV-B radiation  and seven under ABA treatment  were subjected to RT-qPCR analysis using an ABI 7900HT Real-time PCR system . Glyceraldehyde-3-phosphate dehydrogenase  was used as the reference gene; the primer sequences are shown in −∆∆Ct method for RT-qPCR analysis and the fold change method for the RNA-seq data. All experiments were carried out with three independent biological replicates, and three technical replicates were performed for each biological replicate. Tukey’s test was used to identify significant differences at p value ≤ 0.05 using SPSS 19.0 software.Total RNA was extracted from each sample subjected to UV-B radiation  or 100 µM ABA treatment  using an RNA Extraction Kit , and first-strand cDNAs were synthesized using PrimeScript RT Master Mix . Six differentially expressed VcUSP genes were identified and classified into five groups, in which 21 VcUSPs responded to UV-B radiation and 7 responded to exogenous ABA, and VcUSP41 and VcUSP68 might act as bridges integrating UV-B and ABA signaling. WGCNA predicted that VcUSP22, VcUSP41, VcUSP26, VcUSP67, and VcUSP68 may be involved in plant hormone signal transduction, circadian rhythms, the MAPK signaling pathway, and UV-B-induced flavonoid biosynthesis under UV-B or ABA treatment. Our findings provide a useful reference for subsequent research investigating the biological function of VcUSP family members in blueberry.In conclusion, a total of 72 putative


LATS1) as a target of miR-497. siRNA-LATS1 increased concentrations of fatty acids, TAG, and cholesterol in cells, indicating an active role of LATS1 in milk fat metabolism. In summary, miR-497/LATS1 can regulate the biological processes associated with TAG, cholesterol, and unsaturated fatty acid synthesis in cells, providing an experimental basis for further elucidating the mechanistic regulation of lipid metabolism in BMECs.Nutrient metabolism is required to maintain energy balance in animal organisms, and fatty acids play an irreplaceable role in fat metabolism. In this study, microRNA sequencing was performed on mammary gland tissues collected from cows during early, peak, and late lactation to determine miRNA expression profiles. Differentially expressed miRNA (miR-497) was selected for functional studies of fatty acid substitution. Simulants of miR-497 impaired fat metabolism [triacylglycerol (TAG) and cholesterol], whereas knockdown of miR-497 promoted fat metabolism in bovine mammary epithelial cells (BMECs) in vitro. In addition, in vitro experiments on BMECs showed that miR-497 could down-regulate C16:1, C17:1, C18:1, and C20:1 as well as long-chain polyunsaturated fats. Thus, these data expand the discovery of a critical role for miR-497 in mediating adipocyte differentiation. Through bioinformatics analysis and further validation, we identified large tumor suppressor kinase 1 ( PPARG signaling pathway [ABCA1) and regulate the cellular transport of cholesterol and the biosynthesis of high-density lipoprotein [PPARs and reduce intracellular triglyceride synthesis [LATS2  to regulate the synthesis of milk fat [LATS1, as well as miR-497 and LATS2, are closely related to milk fat metabolism [LATS1 and miR-497 are specifically regulated by fatty acids, but whether the effects of miR-497 on LATS1 and LATS2 differ is unknown, so we further investigated their functional and regulatory relationships using molecular techniques, and biological and functional assays provided a theoretical reference for studying the genetic mechanisms of milk fat metabolism in ruminants.Milk is rich in nutrients and gradually becomes a part of people’s daily diet. Ruminant milk is the main edible dairy product worldwide, and milk is the most important source of human dairy products. Milk protein, lactose, milk fat, vitamins, and metal elements play crucial roles in human health and nutritional regulation . Milk fo pathway , the JAK pathway , and ami pathway , which a pathway ,7. MiR-3oprotein ,9, whichoprotein . Studiesoprotein , and miRynthesis ,12. Studmilk fat , and it milk fat . Previoutabolism ,16. HoweLATS1 to obtain key regulatory pathways to functionally validate the molecular mechanism of milk fat metabolism for this regulatory pair and to further study the regulatory effects of miR-497 and LATS1 in the mammary tissues and cells of dairy cows, providing a more accurate theoretical basis for molecular breeding of dairy cows.Milk fat is an important component of milk, is a high-quality natural fat, is digestible up to 98%, and contains a large number of milk fat vitamins. Fat in milk consists of 98% triglycerides, small amounts of cholesterol, and free fatty acids (saturated and unsaturated fatty acids) . The unsThe experimental cattle in this study came from the farm of Yangzhou University (China). Three cows (3 years old) of similar weights were selected for mammary gland tissue collection during early lactation (7 days after parturition), peak lactation (30 days after parturition), and late lactation (315 days after parturition). Total RNA from tissue samples was extracted using Trizol reagent  and stored at −80 °C. RNA quality and quantity were assessed with an ND-1000 spectrophotometer .Raw data were obtained by sequencing and processed for joint removal, contaminating sequences, and low-quality reads. Noncoding RNAs, such as snRNA, tRNA, and rRNA, were identified by comparison with the Rfam and Genbank databases. Clean reads were compared to the Rfam database by blastn software, and results with E-values ≤ 0.01 were extracted to annotate sequences, such as rRNA, snRNA, snoRNA, and tRNA. These comment sequences on the Rfam database were filtered and ultimately deleted. The filtered data were used for the following ratios: prediction of known miRNAs and new miRNAs .The sequencing results contained long inserted sequences, small fragments, poly A, and lower mass sequences. Then, a clean reading was generated. Small RNA is typically 18–30 nt in length. Small RNA species were grouped according to the peak length distribution. PiRNAs peaked at 28–30 nt, siRNAs peaked at 24 nt, and miRNAs peaked at 21 or 22 nt.Standardized debugging of FOSS multifunctional dairy analyzer was performed to optimize the instrument state, and then spectral information of raw milk samples was collected.The culture medium for BMEC includes 10% bovine serum and DMEM/F12. BMEC was cultured in 5% carbon dioxide, 37 ° C, and optimal humidity. When the cell density reached 80%, the biosynthetic reagents microRNA or siRNA were transferred , and theLATS1(100 nM)  using three biological replicates for each treatment. After 48 h, cells were washed with phosphate-buffered saline (PBS) , and after removal of PBS, lysis buffer (150 μL) was added. Collected cells stood at 4 °C for 15 min. The supernatant was collected by centrifugation for cholesterol, TAG detection, and bicinchoninic acid (BCA) protein quantification. The protein concentration was finally determined using BCA values [Cells  were transfected with miR-497mimic (100 nM), miR-497 inhibitor (200 nM), and siRNA-s buffer 0 μL was BMECs were cultured for 24 h and treated with miR-497 MIC, inhibitor and siRNA-LATS1, and cells were collected for fatty acid extraction after 48 h. The specific method refers to the previous research in our laboratory . The rel® QRT SuperMix (+ gDNA wiper)  kit for qPCR . The primers were designed based on the sequences of bovine PPARG, ACLY, CD36, ELOVL6, HSL, and GAPDH in NCBI  method (16). Total RNA (1 µg) was used for cDNA synthesis using a HiScript  in NCBI , and reaSDS-PAGE was used to isolate proteins from cells, and they were transferred nitrocellulose membrane . Rabbit monoclonal  and monoclonal rabbit antibodies  were used, and image collection and analysis were conducted.LATS1 is one of the target genes of miR-497 in online analysis with TargetScan  and PicTar . The results showed that LATS1 was one of the target genes of miR-497. The luciferase vector containing miR-497 binding site 3′-UTR was designed and synthesized with 3′-UTR of bovine LATS1 gene as a template  using Bowtie software. The comparison rate of nine small RNA sequencing libraries was between 85.44% and 94.27%, showing that almost all sequencing data werebovine miRNA .The characteristics of late lactation and peak lactation were compared with those of early lactation. There were 13 miRNAs with common differential expression: bta-miR-17-5p, bta-miR-25, bta-miR-101, bta-miR-151-3p, bta-miR-199a-3p, bta-miR-15b, bta-let-7c, bta-miR-125b, bta-miR-365-3p, bta-miR-495, bta-miR-497, bta-miR-423-3p, and bta-miR-1468 .p < 0.05); milk yield and lactose showed first an increase and then a decreasing trend with the progress of lactation, while milk fat and milk protein showed a continuously increasing trend  after transfection of miR-497 mimic, while the expression of LATS1 was up-regulated by 4.4 times in response to miR-497 inhibition (LATS1 were significantly down-regulated after siRNA-LATS1 treatment (LATS1 fragment containing an miR-497 target site to further demonstrate that miR-497 targets LATS1 (The expression of hibition C. The prreatment D. We dests LATS1 A. The luts LATS1 B.p < 0.01) after transfection with miR-497 mimic (p < 0.01). After transfection with mimic-miR-497, cholesterol content was decreased compared with the control, but the difference was not significant (p > 0.05) (p < 0.01). In addition, the results of oil red O staining showed that overexpression of miR-497 inhibited fat droplet formation, and transfection with miR-497-inhibitor can induce fat droplet formation  B. In conormation C. Regardand GPAM . In cont SCL27A6 .p < 0.01) compared with the control group (p < 0.01) in the siRNA-LATS1-transfected cells  and C22:6 (DHA) were significantly increased ; the resLATS1 treatment of BMECs. Meanwhile, the concentrations of C16:1, C17:1, C18:1, C20:1, C20:3, and C20:5 were significantly decreased, and the concentrations of C20:4 (EPA) and C22:6 (DHA) were significantly increased  gene [DCP1A) [SLC30A4 gene [PGC-1 and PPARA in breast cells related to lipid metabolism [LATS1 was selected in this study, and miR-497 was found to be a target by bioinformatics analysis. The results showed that LATS1 could promote TAG synthesis by bMECs. In addition, the further results indicating that LATS1 can promote the production of medium- and long-chain fatty acids and fat droplets in cells, providing a theoretical basis for improving the content and composition of beneficial fatty acids in milk, and it also helps to deeply dissect the regulatory network of milk fat metabolism involving miRNAs to regulate milk fat percentages and improve milk quality.Knowing the target genes of miRNAs is essential to further clarify the regulating signal pathways, and the target gene functions of miRNAs regulating milk fat metabolism in breast tissue mainly focus on the processes of milk fat synthesis, transport, oxidation, and secretion. There are a large number of target genes related to miRNA regulation of milk fat metabolism, of which miR-135a acts on the very low-density lipoprotein receptor (LR) gene , and miR [DCP1A) , while m [DCP1A) . Furthertabolism . It can HSL) [XDH) [ACLY) [PLIN3) [CD36) [SLC27A6) [PPARs) [The main nutrients in animal milk are milk fat and milk protein, and these nutrients play an important role in the growth and development of young animals. Milk fat is one of the most important factors for evaluating the quality of milk because of its rich fatty acid composition and large number of fat-soluble vitamins. Factors affecting milk fat percentage include genetics, feeding management, and physiological and pathological factors, of which genetic factors are the core factors determining milk fat synthesis in dairy animals . The maiHSL) , xanthinL) [XDH) , ATP cit) [ACLY) , glycero) [ACLY) , lipid d [PLIN3) , cluster) [CD36) , solute SLC27A6) , and per [PPARs) . These gGPAM. LPA is catalyzed by the AGPAT family to generate phosphatidic acid, which is then dephosphorylated by phospholipases to generate diacylglycerol, which is catalyzed by the DGAT family to generate triglycerides. GPAM is the rate-limiting enzyme in the first step of triglyceride synthesis, intracellular triglyceride levels are regulated by the GPAM enzyme [GPAM significantly reduces triglyceride synthesis and metabolism-related gene expression in bovine embryonic fibroblasts [LPIN3 belongs to the LPIN family, which can act as a transcriptional costimulator to regulate peroxisome proliferator-activated receptors (PPARγ) and PPARα and then regulate fatty acid cell differentiation and lipid metabolism, with an important impact on animal fat deposition [ACSL1 and ELOVL6, and significant up-regulation of expression of triglyceride synthesis-related genes (DGAT1 and LPIN1) and intracellular triglyceride content [CD36 and the fatty acid transporter SLC27A6 [CD36 and SLC27A6 decreased after transfection with miR-497 mimic, consistent with the function of miR-497. The fatty acid synthesis, LCFA transport, and content of C16:1, C17:1, C18:1, C18:2, and C20:1 were enhanced, which may be responsible for the expression of PPARG increasing after transfection with miR-497. It has been shown that activation of PPARG also regulates the transcription of genes in fatty acids, such as ACLY, HSL, LPIN1, and CD36 [LATS1 in fatty acids in BMECs, and these findings may help to improve milk production and develop the pasture economy.Triglycerides in BMECs are mainly synthesized in the endoplasmic reticulum, fatty acyl-CoA is produced during de novo fatty acid synthesis, and exogenous uptake of fatty acids are esterified to form a skeleton of glyceryl triphosphate, which ais then generated by the action of M enzyme ,42, and roblasts . In addiposition . It has  content , consist SLC27A6 ,39. The and CD36 ,38,46. ILATS1 and LATS2 kinases have become the focus of research due to their extensive biological activities in cell cycle regulation and differentiation and their dysregulated multiple pathological outcomes. The most significant difference between LATS1 and LATS2 occurs at the transcriptional level. The difference in transcription factors regulating LATS1 versus LATS2 may represent the need to control proliferation signaling by maintaining proficient levels of LATS under both conditions. The different expression patterns of LATS1 and LATS2 may contribute to their likelihood of encountering the same or different binding partners [LATS2 and miR-497/LATS2. First, miR-497/LATS2 was mainly involved in the regulation of five long-chain fatty acids , while miR-497/LATS1 was almost involved in the regulation of 17 fatty acids from C12:0 to DHA. Second, miR-497/LATS2 is mainly involved in the regulation of the CD36, PPARG, SCD1, ACSL1, and PPARA genes, while miR-497/LATS1 is not only involved in the regulation of CD36 and PPARG but also in the expression of the GPAM, ACLY, HSL, XDH, ELOVL6, SLC27A6, and PLIN3 genes. Finally, although both miR-497/LATS2 and miR-497/LATS1 are involved in triglyceride and cholesterol regulation, miR-497/LATS1 regulates cholesterol at a higher level than miR-497/LATS2 regulates cholesterol. Thus, many outstanding questions remain to be answered, and more meticulous studies are needed to accurately define LATS1 and LATS2′s shared versus distinct functions. Understanding advances in LATS signaling may help solve basic science mysteries. Moreover, deciphering the details of LATS-mediated tumor suppression will hopefully elucidate opportunities to improve the early detection, prognosis, and treatment of cancer.In recent years, partners . This stOur data identified for the first time that miRNA-497/LATS1 regulates PUFA metabolism in bMECs, which is a potential factor for increasing the PUFA content in milk.


Ilex paraguariensis yield. To explore the mechanisms that allow it to overcome dehydration, we investigated how the root gene expression varied between water-stressed and non-stressed plants and how the modulation of gene expression was linked to metabolite composition and physiological status. After water deprivation, 5160 differentially expressed transcripts were obtained through RNA-seq. The functional enrichment of induced transcripts revealed significant transcriptional remodelling of stress-related perception, signalling, transcription, and metabolism. Simultaneously, the induction of the enzyme 9-cis-expoxycarotenoid dioxygenase (NCED) transcripts reflected the central role of the hormone abscisic acid in this response. Consequently, the total content of amino acids and soluble sugars increased, and that of starch decreased. Likewise, osmotic adjustment and radical growth were significantly promoted to preserve cell membranes and water uptake. This study provides a valuable resource for future research to understand the molecular adaptation of I. paraguariensis plants under drought conditions and facilitates the exploration of drought-tolerant candidate genes.The appearance of water stress episodes triggers leaf abscission and decreases  Ilex paraguariensis St. Hil. (Aquifoliaceae) is an evergreen tree naturally distributed in South America between 22° and 30° south latitude and between 48° and 56° west longitude, with altitudes between 400 and 1800 m above sea level and annual rainfall between 1100 and 2300 mm [I. paraguariensis can activate several morphological, physiological, and biochemical adaptation mechanisms to cope with environmental stresses. For example, our previous studies showed that a drought-tolerant cultivar responds early to soil water shortage by promoting stomatal closure [I. paraguariensis displays other acclimation mechanisms, including root stimulation and further changes in the leaves, such as osmotic adjustment, photoprotection of the photosynthetic apparatus, and regulation of non-structural carbohydrates and amino acid metabolism [I. paraguariensis acclimation.Under these circumstances, depending on the magnitude of the strain and the stress duration,  closure . Howevertabolism . AlthougUsually, tree roots can respond to drought through various strategies that enable them to avoid and tolerate water scarcity. Responses include root biomass adjustments, anatomical alterations, and physiological acclimations . The molThe root system architecture has been the target of modern breeding programs to develop drought-tolerant varieties. The overall root system size is considered one of the main traits that maintains plant productivity, since it is related to acquiring water and nutrients from the soil and should be accompanied by a balanced leaf surface area ratio . The rooThe development of a root system that ensures an adequate supply of water in restrictive conditions is controlled by a wide range of molecular mechanisms, including the signalling of the soil water status toward the shoot and changes in the regulation of gene expression that subsequently control the molecular pathways of the plant’s drought response . UnderstIlex paraguariensis modifies its morphology to ensure the provision of resources in limiting situations. Intricate mechanisms control such plasticity, including perception, signal integration, and stress response. In the aerial part of the plant, this is related to optimising light uptake and acclimation, while root growth is promoted to ensure water and nutrient extraction. In addition, an efficient osmotic adjustment between root and leaves guarantees the water flux through the soil–atmosphere continuum.Here, we investigated (i) how the root gene expression varies under water scarcity concerning well-watered conditions, (ii) in what ways this variation is linked to the root metabolite composition and physiological status, and finally, (iii) an integrative analysis of transcriptional, metabolomic, and physiological responses of the whole plant. p < 0.05) and 54.5 ± 2.7% (p < 0.0001) when the soil water potential dropped to −1 (moderate stress) and −2 MPa (severe stress). Furthermore, this variation was correlated with a pronounced decrease in the root osmotic potential through an active solute accumulation. Consequently, a significant osmotic adjustment concerning the well-watered conditions was evident under moderate  and severe  stress.The effect of water shortage on the root relative water content and osmotic potential was analysed. The root relative water content decreased from 89.2 ± 3.2% (soil at field capacity) to 83.3 ± 2.2%  when the soil water potential reached −2 MPa. In addition, by scanning electron microscopy, we also verified a variation in the origin zone of root hair formation that contributes to water absorption. In this sense, using shoot tips collected from primary roots of similar diameter, it was observed that the distance from the root tip to the first root hair  and three non-stressed plants to construct six cDNA libraries and was sequenced on an Illumina HiSeq 1500 platform. A total of 312,265,792 paired-end reads (2 × 100 bp) were produced, with a 44% GC average content. After discarding the low-quality reads, a de novo transcriptome was assembled with 231,263,325 bases (40.92% GC) contained in 190,474 transcript contigs with an N50 length of 945 bp. Sixty-two transcripts previously obtained by the Sanger method were employed to validate the nucleotide accuracy of the assembled sequences in the de novo transcriptome using BLASTN  compared to non-stressed roots . Then, n2 = 0.934). This equivalence between RNA-seq and RT-qPCR supports an overall validity of drought-expressed transcripts (DETs) determined by bioinformatic methods from RNA-seq data , GolS , ACC oxidase (aminocyclopropanecarboxylate oxidase), TreS , and AOX  transcripts increased their expression, while PPO (polyphenol oxidase), RafS (raffinose synthase), and RSI-1 (RS-1 protein) decreased their expression in response to water deficit. The uncharacterized transcript c58016_g2_i2 maintained its expression levels without changes.The expression values obtained through both techniques were consistent , ethylene, auxins, cytokinins, jasmonic acid, brassinosteroids, and salicylic acid were identified. Results indicated an increase in ABA signalling gene expression and a general decrease in the transcription of genes related to other hormones involved in different developmental processes. In this context, the KEGG map  highlighDrought also stimulated the expression of transcripts encoding ACC oxidase (T11), which catalyses the last step in ethylene formation. In addition, positive variation was observed in the CTR1 (T12) transcript, which is considered a negative regulator of ethylene response. Consequently, the salicylic acid biosynthesis was probably altered by the upregulation of MES1 (T13) induced by ethylene.Finally, further variation in the expression of transcripts associated with auxin metabolism , signalling , and inactivation  was noticed. Likewise, the expression of several transcripts related to cytokinins , gibberellins , jasmonates , and brassinosteroids  decreased under stress .Among the mRNAs encoding other proteins involved in integrating internal and external stimuli to the cell, the overexpression of transcripts encoding the TOR (T32) protein is worth highlighting.One hundred sixty-three up-regulated and 927 down-regulated transcript factors (TFs) were identified using the PlantTFDB v4.0 database. NAC, bHLH, ERF, related to MYB, B3, C2H2, MYB, WRKY, bZIP, and C3H were the most abundant TF families . An incrhttps://string-db.org/) and the Arabidopsis thaliana database hosted on the site. Sixty-six TFs were functionally annotated, identifying four main interaction clusters organised around HSFC1 (T33), ATHB-12 (T34)/ATBH-7 (T35)/RD26 (T36), SIGE (T37), and HY5 (T38). Colour-coded transcription factors that were assigned to various GO biological processes using STRING algorithms include responses to ethylene: MYB59 (T39), MYB73 (T40), ERF10 (T41), EBP (T42), ARR2 (T43), WRKY4 (T44); ABA responses: RD26 (T36), DREB2C (T45), ATHB-12 (T34), ATHB-7 (T35), GBF3 (T46), HY5 (T38), ARF2 (T47), MYB73 (T40); auxin responses: IAA6 (T48), IAA29 (T49), HB2 (T50), ARF2 (T47), RVE1 (T51), HB40 (T52); and stress responses: WRKY33 (T53), ATHB-12 (T34), ATHB-7 (T35), SIGE (T37), and ATAF1 (T54). In addition, TFs involved in the regulation of root development, such as the A. thaliana response-regulating proteins ARR1 (T55), ARR2 (T43), ARR11 (T56), and ZAT11 (T57), are highlighted.An interaction network of stress-stimulated transcription factors was constructed  using thAn active transcription profile variation in the starch and sucrose metabolism was observed . In respThe interconversion of UDP-glucose and UDP-galactose by UDP-glucose 4-epimerase , followed by the conversion of galactose to galactinol, by galactinol synthase  , was stiMost transcripts encoding glycolytic enzymes were repressed under severe water stress, including the regulatory proteins glucokinase (T76), involved in the entry of glucose into the glycolysis pathway, 6-phosphofructokinase 1 (T77), responsible for producing fructose 1,6-bisphosphate, and pyruvate kinase (T78 and T79), which catalyses the last step to obtain pyruvate . In addiI. paraguariensis roots, drought promoted the biosynthesis of valine, leucine, and isoleucine from pyruvate , cysteine-proteases , ubiquitin-carboxy-terminal-hydrolases (T89), metalloproteases , serine-peptidases , and threonine-peptidases (T90 and T100). Similarly, an increase in the transcription of several mRNAs related to ribosome assembly was detected, including proteins such as ERAL1 , MRPL46 , and RP-S25e ; as well as those associated with pre-translational mRNA transport, processing, and stability, including UTP5 19 and 25 , adenylate kinase (T106), transportin 1 (T107), and factors involved in translation such as EIF1, EIF4A, EIF5A , and ERF3 . Consequently, the overexpression of transcripts encoding aspartyl-tRNA synthetase (T112) and valyl-tRNA synthetase (T113), related to the protein synthesis process, was observed. Lastly, the transcription of genes encoding the LEA proteins  was stimulated in the stressed roots.A. thaliana database, and 199 were annotated with the identity of at least one protein. Next, an interaction network was generated where the line width and colour intensity represented their interaction level and determined their closeness . The small 6-protein “proteolysis” cluster closely interacted with the “translation” one. Similarly, two nearby groups belonging to the GO categories “cellular wall” and “heat responses”, with ten annotated proteins, were identified. In addition, three sets of proteins with co-expressed transcripts defining the interaction of “ABA response” (green), “osmotic stress response” (yellow), and “glycolysis/glycogenolysis” (purple) clusters were apparent, containing 10, 6, and 4 annotated proteins, respectively.Concerning the protein identity assignment, the EF1Alpha protein  was differentiated in the “translation” category as an interconnection node with the “heat response”. Likewise, the proteins ELF5A-3 , SAG24 , RPS18C (T121), and other ribosomal structural proteins were identified. Linked to “proteolysis”, nodes related to polypeptide ubiquitination and proteasome degradation were confirmed, including the polyubiquitin 4 and 8 proteins , UBC11 , and SKP1 . Regarding the “cell wall” cluster, transcripts corresponding to the pectin methylesterase 58 , actin , xylematic cysteine peptidase 2 , XTR6 , peroxidases , and AIR1  proteins were observed. Among the “heat response” proteins, HSP81-3 , HSP70 , ANNAT8 , and EGY3  were annotated. For the “ABA response”, PP2CA (T2/T3), PYL4 (T1), PUB18 , GolS2 , and CPK2  proteins were defined. Among the “osmotic stress response”, MDHAR , CSD2 , RD21B , ALDH10A , MSS1 , NCED3 (T7), GRP4 , RCD1 , and RBOHF  proteins were identified. Finally, for the “glycolysis/gluconeogenesis” category, a small cluster of four protein nodes was identified, including the alcohol dehydrogenases similar to GroES, two isoforms of AT4G22110 (T147 and T148), HOT5 , and ALDH2B4 .Through the combination of different analytical tools, 55 compounds were quantified, including amino acids (20), organic acids or their anions (13), carbohydrates (13), amines (2), nucleobases (2), polyol (1), and others (4). Principal component analysis indicated differences in the metabolic profiles of roots between control and stressed plants. CP1 explained 44.24% of the total variation, allowing the separation of controls and stressed treatments, while the CP2 component explained 24.89%. The root metabolite profile of the well-watered plants was primarily correlated with those metabolites that decreased their content under stress. In contrast, the stressed root metabolic profile was mainly associated with increased metabolite levels . For exaInterestingly, due to dehydration, the total amino acid content in roots increased by two-fold. Among them, the basic amino acids asparagine and ornithine increased their concentration more than 5 and 13 times, respectively . LikewisIn response to the water shortage, the malate, glyceric acid, and gluconic acid contents increased, while the dehydroascorbate anion and 2-oxoglutaric acid dropped. On the other hand, no changes in the concentration of citric, malonic, palmitic, and succinic acids were observed . −1 dw, and the soluble sugar content increased from 26.9 ± 0.7 to 58.2 ± 3.9 mg·g−1 dw , it displays a second defense mechanism based on increased compatible osmolytes in the leaves and roots, favouring an adequate osmotic adjustment and preserving the water moving through the soil-plant-atmosphere continuum, even when the leaf relative water content falls below 65% [2 assimilation rate 48 h after rehydration [Phenotypic plasticity is the ability of an organism to alter its phenotype in response to environmental variation. It may involve changes in physiology, morphology, anatomy, development, or resource allocation resulting from a complex synergistic developmental system controlled by many genes, gene products, and the environment . Root phnd roots . As the I. paraguariensis root structure induced by water shortage.In addition, the volume of soil explored by the roots and the root surface area that interacts with the soil constitute the main parameters that determine the efficiency of a root system . In the Arabidopsis thaliana, ABA induces the expression of genes encoding auxin transporters, such as PIN2 and AUX1, which, by redistributing the hormone-free forms to cortical and epidermal cells, stimulates cell elongation [2+ signalling, and protein phosphorylation/dephosphorylation events [I. paraguariensis roots, various transcripts encoding Ca2+-dependent protein kinases , negative regulators (PP2C), and others related to G-proteins.The perception and signalling of water deficit to determine the adaptive root response constitute a complex process, where ABA plays a leading role as a system moderator . For exaongation . Likewisongation ,19 but dongation . The redongation . This van events . We idenI. paraguariensis response to water shortage [IpNCED1 (T151) and IpNCED2 (T7), encoding the key enzyme 9-cis-epoxycarotenoid dioxygenase, which, induced by drought, enhances ABA biosynthesis. The phylogenetic analysis of the deduced protein sequences indicated that IpNCED1 and IpNCED2 are strongly related to NCED1 and NCED3 proteins from other angiosperm species [A. thaliana aba3 mutants, demonstrated the existence of a new ABA-independent stress-response pathway mediated by the ABA3 enzyme. Based on their results, these authors presumed that plants develop a system that, by regulating multiple pathway metabolisms with a single enzyme (ABA3), would allow them to respond globally and cope with their constantly changing environment. However, the differential accumulation of IpNCED2 mRNA promoted by dehydration and, at the same time, the decrease in the expression of transcripts encoding ABA3 (T8) indicates that ABA mainly mediates the response to dehydration in I. paraguariensis plants.Current and previous results confirmed that ABA is the main regulator of the shortage ,5. Furth species . Additio species , workingI. paraguariensis roots enables the ABA-mediated signal transmission to the target proteins, including the AREB/ABF transcription factors, MYB/MYC [PtPYRL1 or PtPYRL5 increases tolerance to drought [PP2C gene of Populus negatively regulates this response, increasing water loss [ABA acts in plants by binding to the intracellular receptor protein Resistance 1/PYR1-like (PYR1/PYL), called PYLs, forming a complex with clade PP2Cs, resulting in the release of SnRK2 (sucrose non-fermenting 1-related protein kinase 2) from the inhibition exerted by PP2Cs ,25. Cons MYB/MYC , anion c MYB/MYC . Many co drought ; while tter loss ,31.I. paraguariensis root growth in response to drought is regulated by ABA, auxins, and ethylene crosstalk [Additionally, the overexpression of transcripts (T14) encoding SAUR32 , induced by auxins, reveals a connection point with ABA-mediated signalling at the root level  to regulrosstalk .I. paraguariensis roots. MYB, bZIP, WRKY, C2H2, and NAC were the most abundant dehydration-related families.The promoter region of ABA-inducible genes may contain multiple cis-elements, such as ABREs (PyACGTGG/TC), that are recognised by transcription factors (TFs), including ABA-responsive element (ABRE)-binding proteins, (AREB)/ABRE-binding factors (ABF), WRKY, NF-Ys, and MYB ,36. LikeI. paraguariensis-stressed roots the expression of an A. thaliana ortholog , encoding the ELONGATED HYPOCOTYL5 (HY5) transcription factor. HY5 acts as a master regulator that controls the expression of many genes in response to developmental, hormonal, and dynamically changing environments [TPS1  gene and carbohydrate transporters of the SWEET11/12 type  [I. paraguariensis roots. Furthermore, HY5 regulates the expression of several IAA/auxin proteins involved in proteolysis mediated by the ubiquitin-proteasome system, necessary for lateral root initiation [AREBs/ABF TFs, which bind to the ABRE cis-element, belong to the bZIP family, and their expression is induced by dehydration and ABA application . Water sronments . HY5 proronments , increasd 11/12) ; it is titiation . In thisA. thaliana drought tolerance response [Populus, Pinus, and Vitis [The WRKY family of transcription factors contains a conserved domain, which recognises the W-box (TAGACC/T), located in the promoter region of numerous biotic and abiotic stress response genes . We annoresponse  by regulresponse . Likewisresponse . In addind Vitis ,50,51.I. paraguariensis-stressed roots, we observed the overexpression of MYB20, MYB59, MYB73, MYB85, MIB121, and MIB305. In this context, MYB20 having ABI1 and PP2CA as target proteins would be involved in the ABA-dependent signalling [NPF7.3 expression in A. thaliana in response to the availability of K+/NO3-, coordinating its transport to the aerial part [A. thaliana xylem roots would be associated with MYB20, MYB73, and MYB85 together with the AP2-EREBP, bHLH, C2H2, C2C2-GATA, and GRAS TF families. It is worth highlighting that MYB121, a central regulator of ABA-mediated signalling [I. paraguariensis roots under stressed conditions. Likewise, ATMYB71, an ortholog of MYB305, would also be associated with the greater tolerance of sesame roots to dehydration [Populus [In some drought-sensitive ABA-dependent genes, the ABRE cis-element is substituted, specifically by MYBRS (C/TAACNA/G) and MYCRS (CANNTG) . MYB TFsgnalling . MYB59 cgnalling ,54. MYB5ial part . Meanwhiial part . Additioial part  reportedgnalling , increasydration . About 1I. paraguariensis roots, which may be related to the dehydration resilience response by regulating some heat shock proteins, with chaperone functions protecting the protein structure and folding [Zea mays [We highlight the DREB2 and NAC families of transcription factors among the ABA-independent signalling. DREB2A, DREB2B, and DREB2C belong to the petala2/ethylene-responsive factors (AP2/ERF) family, which, in turn, can act as regulators of other TFs, including the heat shock factors (HSFs) . We obse folding . In the Zea mays . I. paraguariensis roots, the transcriptional repression of the CTR1 protein kinase, whose activity is restricted by ethylene, allowing the activation of downstream proteins that leads to the expression of ethylene response TFs [AP2/ERF also can act as an interconnection point in the hormone signalling network. The ABA and ethylene signalling pathways are activated by a stressor, interconnecting with AP2/ERF through the ABI and EIN proteins . We idenonse TFs . Consequonse TFs .NCED gene [Populus spp. [Vitis vinifera [Many members of the NAC family, induced by drought or ABA, bind to the NACRS(CGTG/A) sequence in the promoter region of dehydration-responsive genes . NACs reCED gene ; NAC14 iCED gene . Numeroulus spp.  and Vitivinifera .Finally, we identified transcripts from the HD-Zip and C2H2 families whose expression is stimulated by environmental constraints, ABA, and ethylene ,74. BothTarget of rapamycin (TOR) kinase has been recognised as a key developmental regulator in plants and animals . The TORIn response to soil water shortage, we detected an 832-fold increase in the root expression of a transcript (T32) encoding serine/threonine, a target protein for rapamycin. This fact may be related to the accumulation of glucose, translocated from the leaves as sucrose, linking root development with the source/sink relation . Root glNon-structural carbohydrates (NSC) constitute an essential component of the carbon budget with which the plant must alleviate the adverse conditions generated by stress. In other words, the amount of NSC available at any given time could reflect the tree’s resilience to a drought episode. These include starch and soluble sugars consumed to produce root hairs, respiration to generate available energy, and osmotic adjustment .I. paraguariensis genotype, we determined that, in response to water deficit, the starch and sucrose content decreases in leaves due to the restriction of CO2 fixation and an increase in gene expression encoding enzymes that catalyse the hydrolysis of carbohydrates [I. paraguariensis reallocates its photosynthates (mainly sucrose) towards roots to support their hydrotropic growth.Previously, by using the same hydrates . In thisI. paraguariensis interacts with endogenous mycorrhizae [The up-regulation of genes encoding transcription factors and enzymes involved in its biosynthesis increased the trehalose content in the stressed roots. This fact could be interpreted as a resilience response since the trehalose accumulation could decrease the water potential of the root cells, allowing the entry of water. Additionally, trehalose could improve cells’ redox balance, helping preserve membrane functionality under severe dehydration conditions . Likewisorrhizae , the incorrhizae .The versatile nature of organic acids allows them to contribute in multiple ways to the biochemical processes that trigger a physiological response to a particular stressor . For exaA. thaliana, since they reflect the redox and metabolic state of the cell and can be transported between its compartments [soil = −2 MPa) still does not endanger the production of reduction equivalents necessary to maintain the metabolic processes in the root environment. Among the organic acid plants used to alleviate water stress, Krebs Cycle (KC) intermediates and other short-chain carboxylic acids are highlighted . In addiartments . In thisartments , while nLikewise, the malic acid content increased significantly in leaves and roots, correlating with the overexpression of malate synthase, an enzyme that uses acetyl-CoA and 2-oxoglutarate as substrates; therefore, the content of the latter decreased by more than half in the stressed roots. Correspondingly, the expression of transcripts encoding malate dehydrogenase, which catalyses the conversion of malic acid into oxaloacetic acid, a precursor of the amino acid aspartate and asparagine, increased by two times.I. paraguariensis roots subjected to water deficit. However, the activation of numerous genes encoding transcripts involved in protein synthesis and degradation was observed. These results suggest an apparent balance of protein metabolism, optimising the nitrogen available to the tissue.The nitrate and protein content in correspondence with the transcriptomic profile of genes involved in the uptake of inorganic nitrogen did not show variations in The total amino acid content rise was mainly attributed to increased branched-chain amino acids. In addition, a positive transcriptional variation was observed in the respective biosynthesis pathways where they doubled  or quadrupled (Ile) their endogenous levels in response to drought. Similarly, the alanine content, which can originate pyruvate by transamination with glyoxylate or 2-oxoglutarate, increased significantly in roots. The increase in the branched and related amino acid contents, such as Ala and Thr, has been associated with dehydration tolerance in numerous plant species, contributing to osmotic adjustment  and as sIlex paraguariensis St. Hil. cv SI-49 plants from rooted cuttings were grown in 3 L tube-like containers filled with lateritic red soils (Alfisols) under greenhouse conditions. One week before starting the experiment, plants were pruned and transferred to a room with controlled environmental parameters, 27 ± 1/22 ± 2 °C day/night temperatures, 50–55% relative humidity, and a 14 h photoperiod . After acclimation, pots were irrigated until the soil water potential (Ψsoil) reached field capacity  and were subsequently subjected to a continuous soil drying episode by withholding water from pots until Ψsoil at pre-dawn reached −2 MPa (severe stress). A rewatering treatment (at Ψsoil= −2 Mpa) was also included. For the control treatment, the soil water content was kept at field capacity by restoring water loss by transpiration daily. Plants from identical genotypes and ages were used in all treatments. Pots were covered with aluminium foil to prevent water loss by evaporation from the soil surface. This experiment was carried out under similar environmental conditions for 22 days. The soil water potential was determined using a psychrometer HR-33T with a PST-55 thermocouple . For root osmotic potential measurement, the roots were re-hydrated to constant fresh weight by placing them in a beaker of distilled water under controlled environmental conditions, then placed in a syringe, frozen in liquid nitrogen, and kept at −80 °C pending further analysis. Syringes were thawed until samples reached room temperature, and the osmotic potential of expressed sap was measured with a C-52 thermocouple [2 using the critical point technique, and coated with gold and palladium. A JEOL scanning electron microscope (JLV 5800) operated at 20 kv  was used to examine and photograph the root tips.Two-year-old mocouple . Root remocouple . For theThree biological replicates were used per treatment. For transcriptomic and metabolomic analyses, primary roots were harvested at midday, immediately frozen in liquid nitrogen, and kept at −80 °C until processing.Total RNA samples were obtained using the Spectrum™ Plant Total RNA Kit  with DNase I treatment, following the manufacturer’s instructions . RNA int® RNA Sample Preparation Kit  and sequenced on an Illumina HiSeq 1500 platform to produce paired-end reads (2 × 100 bp).For the qPCR reactions, cDNAs were synthesised from the RNA samples using the ImProm-II™ reverse transcription system , following the manufacturer’s instructions, with oligo(dT)20 as primers. Likewise, six cDNA libraries from both treatments were prepared as described in the TruSeqhttp://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Low-quality reads were discarded, and adapters were clipped using Trimmomatic v 0.36 [Raw-read libraries produced by the RNA-seq experiment were assessed for quality using the FastQC tool  was used to identify protein domains (Pfam database), to predict sequences of signal peptides  and transmembrane domains (TMHMM), and to obtain annotations from the EggNOG database.All assembled transcripts were annotated by sequence similarity comparisons using BLASTx against NCBI non-redundant protein sequences (nr) and the base UniProtKB/Swiss-Prot databases, with an e-value cutoff of 10software . GO  [http://www.genome.jp/kegg/tool/map_pathway1.html).Additionally, the functional enrichment of metabolic pathways was performed through diverse software and following different strategies. Using the Blast2Go PRO 5 software, the differentially expressed transcripts (DETs) were annotated as mentioned above, and subsequently, a Fisher’s exact test was performed for both the induced and the repressed sequences obtained in the differential expression analysis separately . The graphs corresponding to the different GO categories were made using the Go Graph tool. Using another approach, the MapMan software  allowed Genomes) , the funhttps://string-db.org/cgi), A. thaliana was used as a reference. In addition, to increase the sensitivity and specificity in the annotation of TFs, the induced DET sequences were also compared by BLASTx with all the protein sequences of TFs included in the published PlantTFDB 4.0 database [Finally, to generate protein–protein interaction networks enriched in transcription factors, the open reading frame (ORFs) from induced transcripts were used as input in the STRING Search Version 10.5 bioinformatics tool , and 300 nM of the corresponding primer pair. Each reaction was run in technical triplicate using a 7500 Real-Time PCR System  with an initial denaturation at 95 °C for 10 min, 40 cycles of 95 °C for 15 s, and 60 °C for 1 min. The specificity of the amplicons was assessed by a melting curve analysis after the qPCR run (by heating from 60 to 95 °C). For each primer, the real-time PCR amplification efficiency was determined by five-fold serial dilutions of the cDNAs  in a rant-test (p < 0.05). The metabolomic profile data set was subjected to a principal component analysis (PCA) to find a correlation between metabolic profiles and treatments. A biplot graph was generated using the XLSTAT plugin of Excell 2010 (Microsoft®).Metabolite extraction was performed using ground lyophilised root samples, followed by the addition of the appropriate extraction buffer as described by Gibon et al. . The sucIlex paraguariensis modifies its morphology to guarantee the uptake of resources in limited situations. Intricate mechanisms address such plasticity, including perception, signal integration, and response to the strain. In the aerial part of the plant, this is related to optimising light absorption and stress response, while root growth is required to preserve water and nutrient supplies under limiting conditions. (ii) I. paraguariensis responds early to the soil water deficit by displaying a typical isohydric behaviour based on the strict regulation of stomatal conductance modulated by rapid changes in the soil’s and roots’ hydraulic conductivity. As the stress intensifies, it deploys a second defense mechanism based on increased compatible osmolytes in leaf and root cells to maintain the water flow through the soil-atmosphere continuum. In addition, this resilience mechanism allows them to preserve cell membrane functionality and protect the photosynthetic apparatus. At the same time, morphological variations are observed that promote the differentiation and growth of adventitious roots, increasing the absorption capacity of water and nutrients. (iii) The expression of IpNCED2 (GenBank MW047100), encoding 9-cis epoxy carotenoid dioxygenase (NCED), is further induced by osmotic stress. The rise in the leaf ABA content confirmed its up-regulation. Likewise, we verified that IpNCED2 is expressed in leaves and roots. This fact, added to the isolation of several transcripts linked to the signalling process, indicates that ABA regulates the response of I. paraguariensis to drought, being the promotion of root growth, a process regulated by its interaction with ethylene and auxins. (iv) The transcriptomic variation produced changes mainly in non-structural sugar, organic acid, and amino acid content, improving I. paraguariensis resilience.From the molecular, biochemical, physiological, and morphological data that arise in leaves  and root