Triticum sphaerococcum Percival) is an important landrace native to the Indian subcontinent. Several unique features, especially high protein content and resistance to drought and yellow rust, make it a very potent landrace for breeding. Our aim in this study is to identify the contrasting Indian dwarf wheat genotypes based on nitrogen use efficiency (NUE) and nitrogen deficiency tolerance (NDT) traits and the associated miRNAs differentially expressed under N deficiency in selected genotypes. Eleven Indian dwarf wheat genotypes and a high NUE bread wheat genotype (for comparison) were evaluated for NUE under control and N deficit field conditions. Based on NUE, selected genotypes were further evaluated under hydroponics and miRNome was compared by miRNAseq under control and N deficit conditions. Among the identified, differentially expressed miRNAs in control and N starved seedlings, the target gene functions were associated with N metabolism, root development, secondary metabolism and cell-cycle associated pathways. The key findings on miRNA expression, changes in root architecture, root auxin abundance and changes in N metabolism reveal new information on the N deficiency response of Indian dwarf wheat and targets for genetic improvement of NUE.The important roles of plant microRNAs (miRNAs) in adaptation to nitrogen (N) deficiency in different crop species especially cereals  have been under discussion since last decade with little focus on potential wild relatives and landraces. Indian dwarf wheat ( It is a crucial component of macromolecules like nucleic acids, proteins and secondary metabolites. Almost half of the applied N is lost from the soil for various reasons, including leaching, surface runoff, volatilization, denitrification etc. Hence farmers tend to apply more N fertilizers to the crops to mitigate the loss, resulting in environmental pollution, eutrophication, greenhouse gas emission. Besides, it is also cost-ineffective as it requires massive fossil fuels to manufacture synthetic fertilizers. Improving N use efficiency (NUE) is, therefore, the need of the hour2. Nitrogen use efficiency signifies grain dry matter per unit of N available from different sources3. It has been estimated that cereal NUE is only around 33% on average4, indicating excessive loss of N fertilizers, as discussed previously. Significant genotypic variations have been observed in terms of NUE in cereal crops earlier7. Hence the study of NUE and its related traits could be necessary selection criteria for better performing genotypes under low N and control conditions.Nitrogen (N) is one of the essential nutrients required for crop growth and improved yield9 analysed N assimilation enzymes such as nitrate reductase (NR), glutamine synthetase (GS), and glutamate dehydrogenase (GDH) activities which are good indicative of N metabolism as well as NDT. Besides, it was observed that auxin, which promotes root growth, showed a higher concentration near root meristems under N stress10. Therefore, the study of auxin concerning NDT is also imperative.Nitrogen deficiency severely affects plants, such as leaf chlorosis, stunted growth, reduction in plant height, leaf area, tiller numbers, number of spikelets, grain biomass and final yield. Relative comparisons were made previously on N deficiency tolerance (NDT) traits  under low N and control conditions and these traits were considered as selection criteria for better adaptive genotypes in rice.11. In India, the estimated production of wheat during the year 2020 was 107.59 million tonnes12. Since green revolution the yield potential of wheat has been increased, but it is not enough to feed the growing population in future. Wild wheat species possess potential traits which can be introgressed to modern cultivars for genetic improvement. Several promising results have been observed such as tolerance to drought stress31, better acquisition and utilization of nutrients15 etc. Indian dwarf wheat (T. sphaerococcum Perc.), commonly known as shot wheat, is a landrace of wheat cultivated in the Indian subcontinent since ancient times16. Moreover, it has also shown better N uptake capacity17. MicroRNAs (miRNAs) are a class of small, non-coding, approximately 22 nucleotide RNAs with regulatory roles in plants and animals18. Detailed information regarding N-responsive miRNAs and their responses to N deficiency in plants has been studied in maize and rice20. Researchers have also studied N-responsive miRNAs in bread wheat and durum wheat25. Several miRNAs associated with N deficiency response and NUE are reported in crop plants27. One of the N-regulated conserved miRNA, miR169a, targets transcription factor NFYA and overexpression of miR169a reduces N uptake due to suppression of nitrate transporters NPF6.3 and NRT2.128. Another miRNA, miR164 negatively regulates lateral root initiation through repression of its target NAC129. miR39930 is found to be upregulated in maize20 and downregulated in Arabidopsis31 roots under N deficiency. The information on N regulated changes in miRNome of Indian dwarf wheat is highly limited. In this study, we identified Indian dwarf wheat genotypes with contrasting NUE and NDT traits, and through miRNAseq, miRNAs associated with N response were delineated. The results suggest an effective and coordinated signal transduction network involving miRNAs and their target candidate genes imparting adaptation to N deficiency.Among cereals, wheat is an important crop not just for its large-scale production and area but also for its nutritional value as a food grain. The total worldwide projected wheat production during 2022–2023 is 773.43 million tonnes in an area of 221.11 million hectaresAll methods were performed in accordance with the relevant institutional, national, and international guidelines and legislation. The following traits of the studied genotypes were compared with a bread wheat genotype, BT-Schomburgk (BTS), as our previous field screening showed BTS as N responsive. Three experiments were performed, which include (1) preliminary field screening of 11 Indian dwarf wheat genotypes (S1–S11) and one bread wheat genotype (BTS) under N-sufficient (N+) and N-deficient (N−) conditions, (2) hydroponic evaluation of 3 genotypes identified from field screening and (3) miRNAseq analysis of 2 genotypes (Indian dwarf wheat and BTS) identified based on NUE, anthocyanin content and root traits from hydroponic evaluation.rabi seasons (2018 and 2019) with the recommended application of 120 kg ha−1 N fertilizer in Urea form (N120 or N+) and without the application of N fertilizer (N0 or N-) at Division of Plant Physiology, ICAR-IARI, New Delhi  field received the recommended amount of nitrogen fertiliser in three separate applications: 50% N after field preparation, 25% N during the early vegetative stage, and 25% N during the late vegetative stage. Recommended dose of P and K fertilizers (60 kg ha−1) were applied as Single Super Phosphate and as Muriate of Potash) respectively as basal dose in the N120 and N0 fields. Prior to sowing the crop in the second season, the average soil N concentration in the N120 & N0 fields was 190 kg ha−1 and 156 kg ha−1, respectively. Soil PH was approximately 6.9 in N0 and 6.95 in N120 fields while the electrical conductivity of soil solution was 3.95 dSm−1 N0 and 4.25 in N120 fields prior to planting. Before sowing, seeds of the genotypes were surface sterilized with 0.1% mercuric chloride (HgCl2) for 5 min and washed 5–6 times with double distilled water to remove the traces of HgCl2.Twelve wheat genotypes  approximately 40 days after sowing (DAS) in three replications per treatment for each variable using a Leaf area meter . Each replication consisted of three randomly selected samples. Total biomass was measured both at the vegetative stage (at 40 DAS) and at harvest in three replications per treatment in similar manner. Dry weight (DW) was measured after oven-drying the samples at 60 °C till constant weight was achieved.32. The uppermost fully expanded fresh leaves were selected and cut into small pieces, excluding the midrib. These pieces were mixed up, and 25 mg samples were taken and put into test tubes containing 5 ml of dimethyl sulphoxide (DMSO). The test tubes were kept in an oven at 65 °C for 4 h under the dark condition to facilitate the extraction of chlorophyll pigments into the solution. Using a UV–visible spectrophotometer, the absorbance was measured at 470, 645 and 663 nm . Total chlorophyll and carotenoid content were calculated according to33 and expressed as mg g−1 DW.The pigment content of plants was measured at vegetative stage  approximately 40 days after sowing (DAS) in three replications per treatment for each variable, each replication consisted of samples poled from three randomly selected plants. Chlorophyll and carotenoid contents were measured following DMSO method34. Plant leaf sheath samples were collected freshly from the uppermost second node of the stem part. Plant material weighing 20 mg was taken from each sample and put into test tubes containing a 3 ml solution of 45% methanol and 5% acetic acid. It was kept overnight at ambient temperature and covered with aluminium foil to facilitate anthocyanin extraction into the solution. The absorbance was measured at 530 nm using a UV–visible spectrophotometer , and anthocyanin content was expressed as mg g−1 FW.Anthocyanin content was measured using the protocol as described by−1.At physiological maturity, plants were harvested, and estimates of biomass and grain yield per plant (g) were made. Different yield parameters, such as the total number of tillers per plant, the number of ears per plant, the number of spikelets per ear, length of ears (cm) etc., were also measured. Nine randomly chosen plants were sampled for all measurements. Plant height was measured after harvesting using a meter scale. The measurement was taken from the topmost part of the ear to the crown region of the shoot and expressed as cm plant35. After harvesting, both shoot and grain samples were finely ground for estimation. N content was expressed in percentage (%). N utilization efficiency (here denoted as NUE) was calculated from the biomass and N content of the respective grain samples and expressed as g grain g−1 N uptake.Total N in the plant sample was measured following Kjeldahl's method2 for 5 min and rinsed thoroughly to remove the traces of HgCl2 5–6 times with double distilled water, as discussed earlier. Then the seeds were kept for germination in Petri plates on germination paper. After 4–5 days, the germinated seedlings were transplanted to plastic trays containing 10 L of N-free Hoagland solution, each with two treatments having optimum N or N+ (7.5 mM NO3−) and low N or N− (0.05 mM NO3−) and kept inside growth chambers. The seedlings were supported on Styrofoam sheets (2″ thickness), and aquarium pumps were used to keep the solutions aerated. Each tray consisted of 3 genotypes in 5 replications, and each hill consisted of 3 wheat seedlings. There were three trays per treatment, and the arrangement of genotypes in different trays was randomised  in hydroponics in a controlled environment growth chamber at National Phytotron Facility, ICAR-Indian Agricultural Research Institute, New Delhi  with two replications per treatment and data analysed in Win-RHIZO, Regent Instruments Inc. software. Several root parameters were studied, such as total root length, total root surface area, average root diameter, total root volume, primary root length (diameter > 0.5 mm), lateral root length (diameter ≤ 0.5 mm), main root surface area (diameter > 0.5 mm), lateral root surface area (diameter ≤ 0.5 mm), main root volume (diameter > 0.5 mm) and lateral root volume (diameter ≤ 0.5 mm). Photosynthetic pigments (chlorophyll and carotenoid contents) and anthocyanin content were measured spectrophotometrically, as discussed earlier. The chlorophyll content index (CCI) was measured using an MC-100 chlorophyll concentration meter .36. For enzyme extraction, freshly harvested samples were homogenized in refrigerated 0.1 M phosphate buffer (pH 7.5) containing 5 mM EDTA and 5 mM cysteine. Extracts were centrifuged (sigma 3K30) at 10,000 rpm for 15 min at 4 °C37. The supernatant was collected and kept on ice. A reaction mixture containing 0.1 M phosphate buffer (pH 7.5), 0.1 M KNO3 and 10 mM NADH was prepared for assaying enzyme activity. Enzyme extract was added in test samples only to start the reaction, whereas in the blank sample, the extract was substituted with deionized water. To stop the reaction, 1 M zinc acetate was added with 75% ethanol, followed by centrifugation at 2000 rpm for 5 min at room temperature. The supernatant was collected, and 1% (w/v) sulfanilamide solution and 0.02% (w/v) N-(1-naphthyl) ethylene diamine solution were added to it38. After 20 min of incubation, absorbance was measured at 540 nm using UV–a visible spectrophotometer . The enzyme activity was expressed as μmol nitrite formed mg−1 protein h−1.NR activity of leaf and root samples were determined by the in-vitro assay39. Extraction was done in Tris–HCl buffer containing 100 mM Tris–HCl, 100 mM sucrose, 10 mM EDTA and 10 mM MgCl2. Extracts were centrifuged (sigma 3K30) at 5000g for 10 min at 4 °C. The supernatant was collected and re-centrifuged at 12,000g for 15 min at 4 °C. The supernatant obtained after the second round of centrifugation was used to assay GS and GOGAT. The pellet was dissolved in 50 mM phosphate buffer (pH 7.5) containing 2.14 g/100 ml sucrose and was used to assay GDH.GS (Glutamine synthetase), GOGAT (Glutamine oxoglutarate aminotransferase) and GDH (Glutamate dehydrogenase) activity of leaf and root samples were determined following3 reagent was added, followed by centrifugation at 1500–2000g for 10 min. Absorbance was measured using a UV–visible spectrophotometer . The activity of GS was expressed as µmol γ-glutamyl hydroxamate formed g−1 protein h−1.A reaction mixture containing 75 mM Tris buffer, 50 mM MgSO4, 5 mM cysteine, 125 mM α-glutamate, 5 mM ATP, and 10 mM hydroxylamine was prepared for assaying GS activity. Enzyme extract was added in test samples only to start the reaction, whereas, in the blank sample, the extract is substituted with deionized water. The mixtures were incubated for 30 min at 37 °C. To stop the reaction, 0.5 ml FeCl4Cl were prepared. The rest of the steps were similar to both. Enzyme extract was added in test samples only to start the reaction, whereas, in the blank sample, the extract was substituted with deionized water. 1.5 mM NADH was added to the reaction mixture before recording the absorbance at 340 nm for 60 s. The activity of GOGAT and GDH were expressed as µmol NADH oxidized g−1 protein h−1.To assay GOGAT activity, a reaction mixture containing 75 mM Tris–HCl, 10 mM α-ketoglutaric acid, 40 mM L-glutamine, and GDH activity containing 75 mM phosphate buffer, 20 mM α-ketoglutaric acid and 300 mM NH40 to express enzyme activities in terms of the unit amount of protein. Bradford reagent (Genetix) was added to the plant extract prepared earlier, followed by vortexing. After 2 min of incubation, absorbance was measured at 595 nm using UV–a visible spectrophotometer .The soluble protein content of tissue extracts was measured by Bradford’s method41. Oven-dried samples were finely ground for estimation. An equivalent amount of sample and nitrate-free charcoal were taken in a conical flask containing 15 ml of double-distilled water and mixed thoroughly. It was then boiled for 3–4 min and filtered through Whatman filter paper-42 followed by re-extracted and refiltration and volume made up to 50 ml. After 20–30 min of incubation, absorbance was recorded at 540 nm using UV–a visible spectrophotometer .The tissue nitrate content of the leaf and root samples was measured following the hydrazine sulphate reduction method43. Fresh root and shoot samples were ground in liquid N to form a fine powder. 2.5 ml g−1 FW of 100% methanol was added and kept overnight at 4 °C in dark conditions. It was centrifuged afterwards at 16,000g for 10 min at 4 °C, and the supernatant was transferred to a new fresh tube. 2 ml of 100% methanol was added and kept in the dark condition at 4 °C for 1–2 h, followed by centrifugation under the same condition. The supernatant volume was reduced to 1/10th in a speed vacuum. 1 ml of HPLC water was added, and pH was adjusted to > 9.0 with 1 M KOH using pH strips. 100% ethyl acetate was added to double the volume and centrifuged at 16,000 g for 10 min at 4 °C. The lower aqueous phase was taken in a new tube, and pH was adjusted further to < 3.0 with concentrated acetic acid using pH strips. An equal volume of 100% ethyl acetate was added and centrifuged at 16,000 g for 2 min at 4 °C. The upper organic phase was taken and was entirely dried in a speed vac. Finally, it was dissolved in 60:40 methanol (300 μl) and filtered using 0.45 μm PVDF syringe filters. For mobile phase preparation, solvents A and B were prepared using 90% methanol with 0.3% acetic acid and 10% methanol with 0.3% acetic acid, respectively, and were filtered through a 0.45 μm PVDF membrane filter degassed by a vacuum pump. The HPLC system estimated IAA concentration in the reverse phase C18 column  (Agilent Technologies 1200 Infinity). The samples were run under standardized conditions, and peaks were detected in fluorescence detector (FLD) with an excitation wavelength of 280 and emission wavelength of 360 nm, the retention time varied from 15.5 TO 16.5 min. Standard solutions of IAA were used for the identification of peaks. The concentration of IAA was quantified from the calibration curves of the standards and expressed in μg g−1 FW.Natural auxin indole-3-acetic acid (IAA) content was estimated using the method described by3) and 35% perchloric acid (HClO4). 1–2 ml Salkowski reagent was put in watch glasses. Plant root tips were cut and dipped into it for 1 h under complete darkness by covering with black cloth. Whole plant roots were also submerged separately. The pink colour appeared where IAA concentration is more in root and was observed under EVOS XL microscope .The colourimetric assay of IAA was done using the Salkowski reagent. Salkowski reagent was prepared by mixing 0.5 M ferric chloride (FeClhttps://plants.ensembl.org/Triticum_aestivum/Info/Index). The conserved miRNA sequences were recognized using RNA central database  and mapped employing the Bowtie alignment tool44. A maximum of two mismatches was allowed for analysis. EdgeR  software was utilized to identify the differential gene expression for miRNAs.miRNAs were extracted from the leaf and root tissues of selected genotypes (S3 and BTS) based on hydroponic evaluations with the mirVana miRNA isolation kit  according to the manufacturer's protocol. RNA was quantified with a Nanodrop ND2000 spectrophotometer . For miRNA sequencing (NGS), samples were sent to Nucleome Informatics Private Ltd, Hyderabad, India. Small RNA was extracted at three-time points, the 7th day, 14th day and 21st days after treatment with control (N+) or N deficiency (N−) treatments from leaf and root tissues separately from genotypes S3 and BTS. The quality and quantity of total RNA isolated from each sample were further analysed by Bioanalyzer 2100 (Agilent Technologies). The RNA from six samples were used for small RNA library preparation using a Small RNA Sample Preparation Kit (Illumina Technologies) following the manufacturer’s instructions. The small RNA libraries were sequenced using a Hiseq Illumina 1.5. The sequence FASTQ files were analysed by FastQC ver 3 and Fastx-toolkit, ver 0.0.13. The raw sequences were then trimmed to omit adaptor/primer contamination and poly(A) tail with miR Deep adaptor filter. The unique reads having 17–23 nucleotide long sequences were retained for mapping. The reads were screened against non-coding RNA sequences found in the wheat genome database  was carried out in GraphPad Prism version 8  with variety, N treatments as treatment effects to compute adjusted P values and significance levels. Mean separation was done using Sidak's multiple comparisons test following one-way ANOVA. Graphs and heatmaps were prepared using GraphPad Prism version 8 . For yield data recorded in seasons 2018–2019 and 2019–2020, data was analysed in OPSTAT to compute statistical significance of cropping season. Statistical significance of cropping season on genotype wise yield was compared by multiple t test in GraphPad Prism version 8.2) (at 40 DAS), biomass accumulation (g) (at 40 DAS) and total biomass accumulation (g) (at harvest) of wheat genotypes were significantly different concerning N level, variety and their interaction , length of lateral root (diameter ≤ 0.5 mm), the volume of the main root, volume of the lateral root, the surface area of the main root and surface area of lateral root with N treatments and varieties Figs. , 5a. IrrThe IAA content and localization in root tips were studied in wheat seedlings grown with either N+ or N− treatment Fig. b, c. TheShoot nitrate content was significantly reduced by N deficiency, approximately 95% decline in all the genotypes. However, the highest tissue nitrate content in N+ conditions was found in S3. Under N− condition, S2 and S3 showed higher root nitrate content than BTS Fig. a,b. In sBased on miRNAseq analysis, approximately 50 miRNAs were differentially regulated in response to N deficiency in two genotypes (BTS and S3). The expression details of the miRNAs most significantly  affected by N deficiency is presented in Fig. 4. Nitrogen use efficiency is a polygenic and complex trait, and its study requires a multi-disciplinary approach. miRNA mediated regulation of gene expression in response to abiotic stresses has already been well-studied in plants46. Our objectives in this study were to evaluate Indian dwarf wheat genotypes based on NUE and miRNAseq analysis was carried out to identify miRNAs related to NUE and N deficiency. The study shows that both high and low NUE genotypes identified based on NUE, anthocyanin content and root traits have a coordinated network of the miRNA-target module in which most of the targets show similarities in terms of their functions.It is estimated that a 1% increase in cereal NUE can compensate for the cost of N fertilizers, amounting to more than 200 million US dollars on a global scale47. The reduction in photosynthesis, remobilization of N to developing parts and anthocyanin pigmentation are some of the key responses of plants to low N49. In this study, varietal mean of all the growth parameters such as leaf area, biomass accumulation, plant height and other yield-related attributes were significantly reduced under N− condition. High NUE genotype BTS showed the lowest reduction. Anthocyanin accumulation was relatively higher in Indian dwarf wheat (21%) grown in the field under N starved condition with some good contrasts found in S4 (24%), S5 (50%), S6 (67%) genotypes. Significant reduction in chlorophyll a, chlorophyll b, total chlorophyll content, chlorophyll a/b ratio and carotenoid content were also observed in low NUE genotypes compared to high NUE under limited N treatment, suggesting that chlorophyll content is a good indicator of NUE. .The previous studies have shown that N deficiency causes chlorosis of lower leaves and reduces leaf area and shoot growth in rice seedlings, with noticeable results in PB1 indicating that seedling stage screening in the field is necessary for NUE assessment50. Molecular breeding and marker-assisted selections could show promising results with regard to NUE improvement51 but NUE being a complex trait, efficient screening methods are required to accomplish this52. Here we performed preliminary field screening and found variations in NUE among genotypes. Studies showed that increasing N fertilizers application will not increase the yield beyond optima but certainly increase grain N content53. Similar results were also obtained in our study, where a significant improvement in grain N content was observed under N sufficient conditions in the field, specifically in high NUE genotypes. Grain N content can be an essential NUE attribute54. Based on NUE, the following genotypes such as BTS (High NUE), S2 (high NUE), S3 (low NUE but not affected by N level) were selected for further analysis. Screening for NUE related traits in plants is more rapid, effective, uniform and economically feasible under a hydroponic system than field screening55. Hence in this study, selected genotypes were further grown and analysed in hydroponics for change in growth, root traits, photosynthetic pigments and anthocyanin content, N assimilation enzymes activity, tissue nitrate content, soluble protein content, IAA content and localisation.In simple terms, NUE is the ratio of plant biomass or grain yield to available N in soil and supplied N56. In this study, S3 depicted robust root growth in N− treatment with clear visual evidence. Significant differences were observed in root traits like total root length, total root volume, total root surface area, primary root length, lateral root length, main root volume, lateral root volume, main root surface area and lateral root surface area for N treatments and varieties. As discussed previously, anthocyanin accumulation is a significant aspect of NSR, and our findings align with this observation. It was reported earlier that lateral root growth is governed by auxin58. Auxin promotes lateral root initiation through pericycle cell division59. In BTS, S2 and S3, the effect of N treatment on root IAA content was minimal. While IAA localisation was more visually prominent in root tips of N deficient plants of S3, IAA abundance in BTS root tips was similar under both treatments.In general, N stress induces root proliferation to a certain extent in all genotypes for better forage of nutrients61. NR activity is induced under low N supply and vice-versa63. GS and GOGAT activities also respond similarly65. Assimilatory GDH activity increased due to the application of external N66, whereas dissimilatory GDH activity showed the opposite result67. We found that N assimilatory enzyme activity was several times higher in shoots than in the roots, irrespective of treatments. Although low N induces NR activity to some extent, NR activity was found significantly lower in N− treatment than N+ , possibly due to the low availability of nitrate (substrate). We found the highest NR activity in BTS (high NUE) in leaves irrespective of treatments.N assimilation enzymes are essential for the adaptability and survival of plants68. Therefore, it can be concluded that secondary N assimilation in photorespiratory ammonia is higher under the N deficit condition by the activity of GS1. GOGAT activity was also higher in low N. There was a significant increase in GDH activity by N deficiency in leaves of wheat genotypes. We selected genotypes BTS and S3 for miRNAseq analysis based on NUE, anthocyanin content, and root traits.Similarly, GS activity increased almost twice under the N− condition. Overall, GS activity was high in low N, mainly due to higher cytosolic GS or GS1 activityNutrient deprivation, especially essential nutrients, poses severe threats to plant growth. To cope with such problems, plants usually take various means in which the miRNA-target module plays a vital role. As discussed earlier, miRNAs mediate gene regulation for low N adaptability in plants. In this study, miRNAseq analysis showed around 100 miRNAs differentially regulated under N deficiency in BTS and S3, in which 50 miRNAs were most significantly  affected by N deficiency. The expression details revealed that the target gene functions of the miRNAs are as follows: fatty acid metabolism , GTPase signaling, hormone signaling, serine/threonine-protein kinase (STK), wall-associated kinase (WAK), WPK, membrane transport , membrane vesicle traffic, methyltransferase- SAMT, N metabolism- , DUF4220, DUF668, DUF1639, DUF1645, HYPOTHETICAL protein, ROS scavenging, peroxiredoxin, secondary metabolism CYP450, PPO, terpene synthase. Dirigent protein, sugar metabolism- UGPase, transcription factor- F4, WRKY, ARF, GRAS, MYB like SPL, Tubulin, XET etc. With regard to the most differentially regulated miRNAs, the respective target genes belonged to the following four functional classes, mainly as Kinases , N metabolism , secondary metabolism  and dirigent proteins (lignin biosynthesis), transcription factor- F4, WRKY, ARF, GRAS, MYB like SPL, NAC.69. The wall-associated receptor kinases (WAKs), are principally engaged in the regulation of plant cell wall functions and control cell growth, morphogenesis, and development Anderson. Deficit of N stimulated the expression of WAK125 (Os12g0478400) and WAK37 (Os04g0365100) in rice roots. It's interesting to note that WAK125 was already identified as an early glutamate-responsive gene71. The expression of Os09g0442100 and Os06g0142650, which encode RLK homologs, was similarly quickly promoted by N deficiency, in addition to WAK125 and WAK3772. Transcriptome study of ammonium-responsive genes in rice roots revealed the Os02g0120100 gene encoding ACT domain-containing protein kinase 1 (ACTPK1), a homolog of Arabidopsis serine/threonine/tyrosine kinase 46 (STY46), that can phosphorylate and inactivate ammonium transporter AMT1;273. WPK4 is a wheat protein kinase that effectively phosphorylated the hinge 1 region of nitrate reductase and post translationally regulate NR activity74. In rice, roots the expression of os10g0576600, which encodes a tetratricopeptide repeat (TPR) protein was induced by N sufficient and N deficient conditions72.N deficit impacts all areas of plant function, including metabolism, resource allocation, growth, and development, as N is a major component of amino acids, nucleic acids, chlorophyll, ATP, coenzymes, and plant hormones76. It's interesting to note that the RING-type E3 ubiquitin-protein ligase EL5-like genes BT2 and Os05g0360400 are N-sensitive genes76. Nutrient deficiency including that of N increases ROS production in Arabidopsis and rice77. In rice roots, N deficiency activated genes encoding peroxidase, peroxidase-like proteins  and GolS2 (Os07g0687900), which encode galactinol synthase, a crucial enzyme for the manufacture of raffinose family oligosaccharide78. These findings suggest the generation of ROS and redox signalling pathways are among the earliest events connected to N deficiency. In rice, WRKY33 (Os01g0826400) Os03g0609500 (LBD38), Os03g0445700 (LBD37), Os07g0589000 (LBD37), Os05g0114400 (ZOS5-02), Os11g0184900 (NAC5), Os07g0119300 (MYB), and Os03g0764600 (MYB) and CIPK14 are among the 34 N-sensitive genes72. The heterotrimeric guanine nucleotide binding protein (G-protein), has been suggested as a key modulator of N response in rice, wheat, and Arabidopsis79. TaNBP1 has been found to encode G proteins in wheat, which increase N uptake under N deficit conditions80. Similar to this, in rice, the dep1-1 allele of the G Protein enhances N uptake, N assimilation and harvest index under moderate N supply81. Gene ontology enrichment study of rice seedling roots has showed that N-deficiency enhanced expression of two jasmonic acid biosynthesis biosynthesis genes, Os08g0508800 and Os03g0738600, that encode chloroplastic lipoxygenase 7 and linoleate 9S-lipoxygenase 2, respectively72.In Arabidopsis, it has been demonstrated that elements of the ubiquitin-mediated proteolytic degradation system modify N responses. An E3 ubiquitin-protein ligase complex (CUL3-RBX1-BTB) controls the ubiquitination and subsequent proteasomal degradation of target proteins. In rice, nitrate transporter genes and N use efficiency are negatively regulated by the rice BT2 homolog (Os01g0908200)SPX4 is involved in nitrate signalling82. Some of the targeting miRNAs of those genes in wheat include tae-miR1120a, tae-miR1120b-3p, tae-miR1120c- 5p, tae-miR1122b-3p, tae-miR1122c-3p, tae-miR1130a, tae-miR1130b-3p, tae-miR1137a, and tae-miR1137b-5p . In the present study, miRNAseq analysis showed that tae-miR1137b-5p, tae-miR1137a, tae-miR1122b-3p, tae-miR1120b-3p were differentially expressed. Apart from SPX, other targets of these miRNAs were NUE-related traits such as tae-miR1137a and tae-miR1137b-5p target NR, tae-miR1122b-3p and tae-miR1120b-3p target genes associated with secondary metabolism. It has also been observed that tae-miR156, which is conserved functionally, regulates plant morphology, and its overexpression causes excessive tillering giving a bushy appearance and defective spikelets83. In this study, expression details demonstrated that tae-csmR156-1 was differentially expressed and conserved in nature; it might also regulate plant morphological changes84. reported an inverse relationship between miR160-ARF16/17 and miR167-ARF6/8 modules stimulating lateral root growth in Arabidopsis under N-starved conditions. The differential expression of miR160 and miR167a in the current study can thus be correlated with the changes in root growth under N deficiency25. Studied N responsive miRNAs in bread wheat, and miR159, miR399 and miR408 were reported to be differentially expressed23. Also found tamiR444a regulating plant tolerance to N stress. Another report showed that the NSR-regulated miR396 targets the Growth Regulating Factor (GRF) to regulate leaf development while miR171 targets SCARECROW-like (SCL) transcription factors and regulates root growth in Arabidopsis, maize and soybean85. Similarly, our results are at par, showing altered expression of miR159b, miR171a, miR396-5p, miR444b etc. We found an approximately twofold increase in miR171 expression in BTS under the N− condition. There are four classes of miRNAs based on their conservativeness27. The most conserved group includes miR164, miR172, miR398 and miR399, whereas, highly conserved are miR156, miR167, miR395, miR319, miR408 and miR2111; miR160, miR168, miR166 and miR397 belong to moderately conserved and less conserved group consists of miR158, miR159, miR169,miR170, miR171, miR528, miR390, miR396, miR394 and miR827 y27. Some of the unique miRNAs regulated under P deficiency such as miR437, miR447, miR771, miR775, miR778, miR830, miR837, miR896, miR1122, miR1125, miR1135, miR1136, miR1211, miR1222 and miR1507 target PHO2, ARF6, ARF8 and AP2 genes. Some N-regulated unique miRNAs include miR826, miR829, miR839, miR846, miR850 and miR863; target genes are NLA, ARF6, ARF8, ARF16, ARF18, HAP2 and AP2. Our study found differential regulation of some unique miRNAs such as miR1125, miR1136 and miR1222 in N deficient wheat seedlings. PHO2 encodes a ubiquitin-conjugating E2 enzyme 24, known to play a role in maintaining phosphate homeostasis. However, the differential expression of miRNAs targeting PHO2 in the current study establishes the role of PHO2 under N deficiency. Differential expression analysis suggests that these discovered members of miRNAs are involved in reprogramming N assimilation and remobilisation machinery and developing secondary metabolites and anthocyanin in response to N deprivation. N regulated root growth and actions of different kinases  for downstream signal transduction are also reprogrammed.Like many other signalling-related genes, phosphate signalling repressor Triticum sphaerococcum Perc.) genotypes identified under low N conditions. Our findings infer that the effective and coordinated signal transduction network involving the miRNAs targeting and reprogramming N metabolism, secondary metabolism, N signal perception and signalling helps to achieve high NUE. The miRNAS targeting key genes could serve as a potential biotechnology target for enhancing nitrogen efficiency in wheat and other crops.In conclusion, the wheat homologues of N-responsive miRNAs mediate NUE-related responses through downstream signalling pathways. miRNAseq analysis revealed the expression details of miRNAs involved in signal transduction in high NUE Indian dwarf wheat (Supplementary Information 1.Supplementary Information 2.


In this paper, we aim to provide a comprehensive summary of the preparation methods employed for 11 iron-based single crystals over the past decade. Specifically, we will focus on hydrothermal, chemical vapor transport (CVT), self-flux and annealing methods. Additionally, we will discuss the quality, size, and superconductivity properties exhibited by single crystals obtained through different preparation methods. By exploring these aspects, we can gain a better understanding of the advantages and limitations associated with each technique. High-quality single crystals serve as invaluable tools for advancing both the theoretical understanding and practical utilization of high-temperature superconductivity. The 11 system in the iron-based superconducting family has become one of the most extensively studied materials in the research of high-temperature superconductivity, due to their simple structure and rich physical properties. Many exotic properties, such as multiband electronic structure, electronic nematicity, topology and antiferromagnetic order, provide strong support for the theory of high-temperature superconductivity, and have been at the forefront of condensed matter physics in the past decade. One noteworthy aspect is that a high upper critical magnetic field, large critical current density and lower toxicity give the 11 system good application prospects. However, the research on 11 iron-based superconductors faces numerous obstacles, mainly stemming from the challenges associated with producing high-quality single crystals. Since the discovery of FeSe superconductivity in 2008, researchers have made significant progress in crystal growth, overcoming the hurdles that initially impeded their studies. Consequently, they have successfully established the complete phase diagrams of 11 iron-based superconductors, including FeSe The discovery of iron-based superconductivity represents a significant breakthrough in the field of condensed matter physics, with a profound impact on the study of high-temperature superconductivity ,2. Accor4 layers stacked along the c-axis, without a charge storage layer [Ts ~ 90 K accompanied by the nematic phase [Tc) of approximately 9 K, high tunability and nematicity without magnetic order have garnered significant attention and research interest. Under high pressure, the Tc of FeSe can be elevated to approximately 38 K, and a new magnetic order emerges within a specific pressure range once the nematic phase is suppressed [Tc to over 40 K. Remarkably, monolayer FeSe films on doped SrTiO3 substrates have exhibited superconductivity with the Tc surpassing 65 K [Tc and exhibit unconventional superconducting behavior. Consequently, they have become pivotal in advancing research in the field of high-temperature superconductivity, playing a vital role similar to that of copper-based superconductors.Compared with FeAs-based superconductors, the 11 iron-based superconductors in iron-chalcogenide compounds have the advantages of a simple crystal structure and non-toxicity. FeSe consists solely of edge-sharing tetrahedral FeSege layer ,10,11. Aic phase ,13,14,15ppressed ,17,18,19ppressed ,21, ionippressed ,23,24 anppressed ,26, haveing 65 K ,28. ThesTs gradually decreases until it vanishes at x ~ 0.17, marking a nonmagnetic nematic quantum critical point (QCP) [Tc exhibits a small superconducting dome, reaching a maximum of 11 K at x ~ 0.11. Beyond the nematic regions, superconductivity is gradually suppressed, reaching a minimum at x ~ 0.45, after which the Tc slowly increases until x = 1 [The substitution of isovalent sulfur (S) in FeSe, equivalent to applying positive chemical pressure, has proven to be an effective method for tuning superconductivity and nematic order. With S doping, the nematic transition temperature nt (QCP) ,30,31,32il x = 1 . Notablyil x = 1 ,34,35. x1−Tex single crystals phase diagram, Ts linearly decreases until it disappears at x = 0.5 with Te doping [Tc initially decreases to a minimum at x ~ 0.3 and then increases to a maximum at x ~ 0.6; subsequently, the Tc is gradually suppressed and antiferromagnetic behavior emerges when x > 0.9 [Similarly, the substitution of isovalent tellurium (Te) in FeSe, equivalent to applying negative chemical pressure, is an effective method for tuning the superconductivity and various ordered states. In FeSee doping ,37,38. T x > 0.9 ,40,41,42 x > 0.9 ,45,46,47x1−Tex and FeSex1−Sx. This difficulty is also commonly encountered in the study of other iron-based superconducting families. On the one hand, the low chemical stability of FeSex1−Sx and the issue of phase separation in FeSex1−Tex (0 < x < 0.5) make it arduous to obtain single crystals or single-phase samples using traditional solid-state reactions [x1−Tex (0.5 ≤ x ≤ 1) is relatively straightforward using the self-flux method, the presence of excess Fe significantly affects the investigation of their intrinsic properties, such as the localization of charge carriers [Unfortunately, preparing high-quality single crystals is one of the challenges in the study of the 11 iron-based superconducting system, particularly FeSeeactions ,50,51,52carriers ,54,55, scarriers  and incocarriers . It is dx1−Sx single crystals using CVT for range 0 ≤ x ≤ 0.29 and the hydrothermal method for the entire region. In x1−Tex (0 ≤ x ≤ 0.5) single crystals can be directly synthesized via CVT. For the Te-high doping region (0.5 < x ≤ 1), it becomes necessary to anneal the as-grown single crystals in O2 or Te vapor. Finally, we conclude the review with a summary and outlook in To synthesize high quality single crystals across the entire doping range, different methods need to be employed. In this review, we provide an overview of the common synthesis methods for the 11 iron-based system, focusing on the optimal method for different doping regions, along with a relevant phase diagram of the entire region. Initially, we discuss the conventional methods of obtaining FeSe single crystals, namely the flux method and chemical vapor transport (CVT). In xyd, yzd, and xzd orbitals, forming well-separated electron and hole pockets [FeSe stands out as one of the most extensively studied materials within the realm of iron-based superconductors, owing to its array of unique properties. Notably, FeSe exhibits a multiband electronic structure, a nematic phase, a BCS-BEC crossover, and spin-density wave (SDW) behavior, all of which benefit from the use of high-quality single crystals. FeSe is considered a multiband compensated semimetal with a Fermi surface consisting of  pockets . Because pockets . Another pockets . Additio pockets , which w pockets . Numerou4-tetrahedra layer-by-layer, as shown schematically in 1.2Se polycrystalline with nominal stoichiometry was prepared through a traditional solid-state reaction using high purity Fe and Se powders as the raw materials. Then, the obtained Fe1.2Se polycrystal powder and NaCl/KCl mixed eutectic with mole ratio 1:1 were ground and sealed in an evacuated quartz tube. The quartz tube was slowly heated to 850 °C and kept two hours for sufficient solution of the raw materials and flux. Afterward, the temperature was gradually reduced at a rate of 3 °C/h down to 600 °C, followed by furnace cooling. FeSe single crystals were separated from the flux by dissolving the NaCl/KCl mixed eutectic in deionized water. Maw-Kuen Wu’s group reported the observation of superconductivity with zero-resistance transition temperature at 8 K in the FeSe polycrystalline bulk for the first time . The cryTc) and the small superconducting volume fraction observed suggest a low-quality superconducting tetragonal phase.7Se8 and Fe3O4 exist in all as-grown single crystals, posing a major obstacle in understanding the intrinsic properties of FeSe. The strong magnetism of these impurities often results in a prominent ferromagnetic background in the superconducting magnetization-field (M-H) loop [M-H loop is nearly absent below the Tc [Subsequently, several research groups successfully synthesized FeSe single crystals using similar methods ,64,65,66-H) loop . FeSe siw the Tc . However3 transport agent [R(T) and was confirmed to be a structural(nematic) transition from the tetragonal to orthorhombic phase [Despite the successful growth of large-sized FeSe single crystals using the flux method, the quality of the crystals and the presence of impurities hindered related research. The iodine vapor transport method did not effectively improve the crystal quality . Howeverrt agent ,68. A diic phase ,70,71,723 powders. The quartz tube was horizontally placed in a tube furnace with a double-temperature zone. The hot part of the tube containing the raw materials was heated to 390 °C while the cold part for single crystal growth was kept at 240 °C. After approximately 30 days of transport growth, a large number of single crystals with tetragonal morphology could be observed in the cold part. Similarly to the flux method, FeSe single crystals need to be separated from the flux by dissolving the KCl/AlCl3 mixed eutectic in deionized water. The schematic representation of the typical CVT growth assembly is shown in ρ-T) and magnetization (M-T), shown in The synthesis process is as follows: High-purity Fe and Se powders were sealed in an evacuated quartz tube along with KCl and AlClSince then, the preparation of FeSe single crystals using similar methods has become more prevalent, leading to a flourishing research landscape, due to the availability of high-quality single crystals. However, the quality of these single crystals is highly sensitive to the preparation conditions, primarily influenced by the complex binary Fe-Se composition–temperature phase diagram . 7Se8 phase. Additionally, the temperature conditions during growth strongly influenced the single crystal quality, with an optimal temperature gradient of 350–390 °C observed in their work. In addition, the tilt angle of the quartz tube can also have some impact on the growth. Tc), Ts and Tc. Both the Ts and Tc decrease as the RRR increases. Composition analysis using wavelength dispersive X-ray spectroscopy (WDS) indicated no correlation between the Tc and sample composition. Extrapolating the linear relation between the Ts and Tc suggests that superconductivity would be completely suppressed when the Ts reaches 64 K.A study conducted by A. E. Böhmer et al. explored the relationship between transition temperatures and residual resistivity ratio (RRR) in vapor-grown FeSe . Their fx1−Sx is significantly suppressed with S doping, which completely disappears at x = 0.17. As the S content increases, the nematic fluctuations are strongly enhanced, and the nematic susceptibility diverges as it approaches T = 0, indicating the presence of a nematic QCP at x = 0.17 [x1−Sx system proves to be an excellent platform for studying the relationship between the nematicity and superconductivity. Furthermore, the non-Fermi liquid behavior at QCP indicates that nematic critical fluctuations have a significant influence on the normal-state electronic properties [x1−Sx system.The nematicity in FeSex = 0.17 . Notablyoperties . Magnetooperties ,77,78. Tx1−Sx single crystals are typically grown by CVT from FeSe up to x ~ 0.4, using similar preparation methods as FeSe [x = 0 to 0.25. With S doping, a clear kink in resistivity gradually decreases to lower temperatures and disappears at a nonmagnetic nematic QCP, x = 0.17, as shown more clearly in ρ/dT. The discovery of QCP with nonmagnetic nematicity in the 11 system has raised the prospect of investigating the role of the relationship between nematicity and superconductivity [FeSe as FeSe ,77,79,80uctivity ,81.x1−Sx single crystals with x ≤ 0.29, the hydrothermal method has been employed to overcome this limitation. Xiaofang Lai et al. successfully synthesized tetragonal FeS through the hydrothermal reaction of Fe powder with a sulfide solution and observed bulk superconductivity for the first time at 5 K [0.8Fe1.6Sex2−Sx precursors has been widely utilized for the preparation of FeS and FeSex1−Sx single crystals [0.8Fe1.6Sex2−Sx precursors using the self-flux method, followed by the addition of Fe powder, selenourea, thiourea, and K0.8Fe1.6Sex2−Sx single crystals pieces to a solution containing dissolved NaOH in deionized water within a Teflon-linked stainless-steel autoclave (25 mL). The autoclave is then sealed and heated to 130–150 °C for 50–70 h resulting in the formation of FeSex1−Sx single crystals, as shown in While the CVT method allows the synthesis of FeSee at 5 K . Subsequcrystals ,86,87,88x1−Sx single crystals, encompassing the entire region obtained from the hydrothermal method [x ≤ 0.29) obtained from the CVT method [Ts and Tc obtained from the hydrothermal method are slightly lower than those from the CVT method, possibly due to disorder effects in the crystals [n” in the contour plot corresponds to the power law, ρ (T) = ρ0 + nAT, where ρ0 represents the residual resistivity. In the nematic phase, the resistivity exhibits a non-Fermi liquid behavior characterized by sublinear temperature dependence. Outside the nematic phase, the resistivity at low temperatures follows a prefect Fermi liquid behavior, i.e., T2 dependence. In the Fermi liquid region, the coefficient A decreases monotonically with S doping, indicating a reduction in effective mass, since A is proportional to the carrier effective mass according to the Landau Fermi liquid theory. Below the characteristic temperature T*, the resistivity displays an anomalous upturn just before the superconducting transition. The origin of this anomaly may be attributed to local magnetic impurity scattering or inelastic scattering due to crystallographic disorder.l method  and a paT method ,76,77. Tcrystals . The expx1−Tex is gradually suppressed with Te doping and disappears at x = 0.5 [x1−Tex single crystals [x > 0.1, while the superconducting dome persists, suggesting that the enhancement of superconductivity in FeSex1−Tex is not attributed to magnetism but rather to the nematic fluctuations [x1−Tex exhibits topological surface superconductivity and the presence of Majorana fermions, making it the first high-temperature topological superconductor to be discovered [x1−Tex exhibits a bi-collinear antiferromagnetism, which is distinct from the collinear antiferromagnetism observed in iron-pnictides [x1−Tex displays an excellent high upper critical field and low anisotropy, which significantly reduce the challenges associated with applications [x1−Tex are gradually being unveiled.Similar to S doping, the nematicity in FeSe x = 0.5 ,37. The crystals . The magscovered ,91. In tscovered ,42. The nictides . Additioications . Researcx1−Sx have been successfully obtained, achieving homogenous Te-doping single crystals remains challenging due to strict preparation conditions and the phase separation in the region of 0.1 ≤ x ≤ 0.4 [x1−Tex (0 ≤ x ≤ 0.41) single crystals using the flux method with a temperature gradient, including the phase separation regions, has been reported for the first time [While high-quality single crystals of FeSe x ≤ 0.4 ,51,52. Irst time . 3/KCl was placed in high-temperature zone of quartz tube. After 20–30 days, flake-like single crystals were obtained in the low-temperature zone and the residual flux was removed by dissolving it in distilled water, as shown in 0.67Te0.33 single crystal was grown using a flux method with a single-temperature zone in a box furnace [ furnace . Tc exhibits a minimum around x ~ 0.2, which is attributed to the effect of sample disorder, as indicated by the relatively small RRR value [Ts decreases linearly with increasing Te doping and disappears at approximately x ~ 0.5. The Tc exhibits a maximum around x ~ 0.6, and the Néel temperature (TN) starts to appear when x > 0.9, accompanied by the suppression of superconductivity [x1−Tex with Te doping.The results of these two works are summarized in a phase diagram, shown in RR value ,75. The uctivity . The brex1−Tex (0 ≤ x ≤ 0.5) single crystals using the CVT method, and the temperature–composition phase diagrams have been established, as shown in 3/KCl and the growth time was 1–2 weeks. The temperatures of the hot and cold sides were controlled at 420 and 250 °C for 0 ≤ x ≤ 0.25 (620 and 450 °C for 0.25 ≤ x ≤ 0.55), respectively, which play a significant role in the crystal growth process.Recently, significant progress has been made in the growth of high-quality FeSeTc and transition width ΔT. In this systematic study, a comprehensive analysis of RRR with a large number of data points, represented by ρ(200 K)/ρ(15 K), reveals a monotonous decrease with increasing Te concentration, as shown in Tc observed at x = 0.3. Additionally, when considering the temperature–pressure–composition phase diagrams of FeSex1−Tex (0 ≤ x ≤ 0.5) single crystals, it is proposed that nematic fluctuations play a role in enhancing the Tc above x = 0.3 and contribute to the formation of the observed Tc-dip. Despite the similar synthesis methods employed by different research groups, there is considerable variation in the quality of the obtained single crystals, including RRR, the superconducting transition temperature x1−Tex (0.5 < x ≤ 1) single crystals can be grown using standard melting methods, such as the Bridgeman method [FeSen method , self-fln method ,95 and on method . In the n method .x1−Tex significantly affects its intrinsic properties, such as localization of the charge carriers [0.61Se0.39 single crystals were successfully annealed in a vacuum environment for the first time at 400 °C for more than 10 days, resulting a sharp superconducting transition at around 14 K [x1−Tex (0.5 < x ≤ 1) single crystals [2 annealing can also effectively remove excess Fe [2 annealing have no effect on the excess Fe, and the observed improvement was actually due to the action of a small amount of residual O2 present during the annealing process [2 annealing, elements such as Te, Se, S, P, As, I, and Sb have been proven to effectively remove excess Fe through vapor annealing for FeSex1−Tex (0.5 < x ≤ 1) single crystals [2 annealing to remove excess Fe.The position of excess iron in the crystal structure is shown in carriers ,54,55, scarriers  and incocarriers . Annealiund 14 K . Subsequcrystals ,100. It xcess Fe . However process . Apart fcrystals ,107,108.2 [2 annealing, as-grown single crystals were cut and cleaved into thin slices with dimensions of about 2.0 × 1.0 × 0.05 mm3. These slices were then weighed and loaded into a quartz tube with an inner diameter of 10 mm. The quartz tube was carefully evacuated using a diffusion pump, and the pressure in the tube was detected using a diaphragm-type manometer with an accuracy greater than 1 mTorr. Once the gas was fully removed, the quartz tube was filled with Ar/O2 (1% Ar) mixed gas and sealed to a length of 100 mm. The pressure in the system is continuously monitored during the sealing process to prevent gas leakage and control the O2 pressure in the quartz tube. The crystals were then annealed at 400 °C for various periods of time and subsequently quenched in water.2 . To perfy1+Tex1−Sex  were established based on the magnetization, magnetic susceptibility, resistivity, and Hall effects, as shown in x (Se) < 0.05, and the spin glass state completely disappears. This confirms the effective removal of excess Fe through annealing. The superconducting state exhibits a clear bulk effect and can be easily detected by magnetic measurements. The doping–temperature phase diagram for the as-grown and annealed Fex1−Tex single crystals, with varying Te doping levels . The corresponding phase diagram is illustrated in x = 0.5. Our results also reveal that the Tc reaches its minimum at x = 0.3, which aligns with the findings of Mukasa et al. [Tc-dip phenomenon observed in FeSex1−Tex. Subsequently, the Tc increases and reaches a maximum at x = 0.6 but gradually decreases upon further Te doping, eventually leading to a transition into a non-superconducting antiferromagnetic state.In our recent work, we have successfully prepared high-quality full-range FeSea et al. , furtherTc, and strong upper critical field exhibited by FeSex1−Tex single crystals make them excellent candidates for investigating the pairing mechanism underlying high-temperature superconductivity. Consequently, the comprehensive phase diagram we have established for FeSex1−Tex provides valuable support for the ongoing exploration of the superconducting pairing mechanism in high-temperature superconductors.The high chemical stability, high x1−Tex single crystals also can be grown using the optical zone-melting technique [FeSeechnique ,109. Thi−2, and then heated at 600 °C for 20 h under a vacuum. The reacted bulk material was reground into a fine powder and loaded into a double quartz tube. The tube was loaded in an optical zone-melting furnace equipped with two 1500 W halogen lamps as infrared radiation sources, as shown in −1. After the growth, the as-grown crystals undergo an annealing process: ramping to 700–800 °C in 7 h, holding for 48 h; cool to 420 °C in 4 h, hold for 30 h; and finally shutting down the furnace and cooling to room temperature.High-purity powders of Fe, Se and Te with a nominal ratio were mixed in a ball mill for 4 h. The mixed powders were cold pressed into discs under a uniaxial pressure of 400 kg·cmR(T) curves before superconducting transition is still apparent, indicating the presence of excess Fe in the crystals [x1−Tex single crystals. Despite obtaining large-sized and well-crystallized single crystals using the optical zone-melting technique, the upwarping behavior of the crystals . Moreovex1−Sx system also exhibits superconductivity. Yoshikazu Mizuguchi et al. first reported the superconductivity in the FeTex1−Sx system and found that the Tc can reach 10 K when x is 0.2 [x1−Sx single crystals with low S doping were grown using the self-flux method, similar to FeSex1−Tex (0.5 < x ≤ 1) single crystals [x1−Sx single crystals, although the excess Fe cannot be completely removed [FeTex is 0.2 . FeTe1−xcrystals ,114,115. removed ,120,121. removed ,122. Witx1−Tex (0 ≤ x ≤ 0.15) single crystals by a hydrothermal method for the first time and provided a phase diagram of FeSx1−Tex single crystals, shown in Tc is rapidly suppressed with the Te doping for FeSx1−Tex (0 ≤ x ≤ 0.15) single crystals and finally disappears when x > 0.1. Due to the large solution limited region, only a small amount of doping can be applied at both ends of the phase diagram. The complete phase diagram needs further exploration. Caiye Zhao et al. successfully synthesized a series of FeSx1−Tex and FeSex1−Sx, through various methods. A comprehensive phase diagram has been constructed, as depicted in Tc), the onset of nematic phase (Ts), and the Néel temperature (TN) for the single crystals prepared using the optimal techniques in different intervals. In conclusion, significant progress has been made in the preparation of 11 system single crystals, including FeSex1−Sx (0 ≤ x ≤ 0.29) and FeSex1−Tex (0 ≤ x ≤ 0.55) single crystals are typically grown using CVT method with AlCl3/KCl transport agent. It is fortuitous that the range encompassing these single crystals includes the nematic phase without magnetic order. The exceptional quality of these crystals serves as an excellent platform for investigating the interplay between nematicity and superconductivity. FeSex1−Sx (0.29 ≤ x ≤ 1) single crystals, however, can only be synthesized using hydrothermal method. Although the quality of single crystals using hydrothermal is slightly inferior to those grown using CVT, they still hold great significance for studying the complete phase diagram of FeSex1−Sx. By utilizing the self-flux plus annealing technique, single crystals without excess Fe in the highly Te doping region can be obtained. In this particular region, the Tc reaches maximum of the entire phase diagram, approximately 15 K, occurring around x (Te) ~ 0.6. Furthermore, AFM state is observed within a narrow region around FeTe.High quality FeSeThe connection between the ordered states and superconductivity have not been well resolved, and the relationship between nematicity and SDW has been described as a “chicken-egg” problem . Underst


Streptomyces exfoliatus DSMZ 41693 has allowed us to identify four different lipase-encoding sequences, and one of them (SeLipC) has been successfully cloned and extracellularly expressed using Rhodococcus sp. T104 as a host. SeLipC was purified by one-step hydrophobic interaction chromatography. The enzyme is a monomeric protein of 27.6 kDa, which belongs to subfamily I.7 of lipolytic enzymes according to its phylogenetic analysis and biochemical characterization. The purified enzyme shows the highest activity at 60 °C and an optimum pH of 8.5, whereas thermal stability is significantly improved when protein concentration is increased, as confirmed by thermal deactivation kinetics, circular dichroism, and differential scanning calorimetry. Enzyme hydrolytic activity using p-nitrophenyl palmitate (pNPP) as substrate can be modulated by different water-miscible organic cosolvents, detergents, and metal ions. Likewise, kinetic parameters for pNPP are: MK = 49.6 µM, catk = 57 s−1, and cat/KMk = 1.15 × 106 s−1·M−1. SeLipC is also able to hydrolyze olive oil and degrade several polyester-type polymers such as poly(butylene succinate) (PBS), poly(butylene succinate)-co-(butylene adipate) (PBSA), and poly(ε-caprolactone) (PCL). Moreover, SeLipC can catalyze the synthesis of different sugar fatty acid esters by transesterification using vinyl laurate as an acyl donor, demonstrating its interest in different biotechnological applications.Genome mining of  Lipases  are ubiquitous enzymes that catalyze the hydrolysis of triglycerides into glycerol and fatty acids at the lipid–water interface, as well as esterification, interesterification, transesterification, acidolysis, and aminolysis reactions in aqueous and organic media . ConsequCandida antarctica (Novozyme 435), immobilized lipase from Mucor miehei (Lipozyme IM)  agar plates containing 0.1% emulsified polymers were prepared as described [A total of 0.5% PBSA, PBS, or PCL emulsions were prepared according to the procedure reported previously : 2 g polescribed . Wells wSeLipC activity were determined using pNPP as a substrate by the standard assay described above. All experiments were carried out in triplicate. The optimum pH of pure SeLipC was established by assaying the lipase activity at 37 °C using different buffer systems: 50 mM MES (pH 5.0–7.0), 50 mM HEPES (pH 7–7.5), 50 mM Tris/HCl (pH 7.5–9.0), and 50 mM glycine/NaOH (pH 9.0–10.0). To determine pH stability, 20 µL of enzyme solution containing 0.015 µg of pure lipase was incubated in 5 mM of each buffer at different pH values for 10 min at 4 °C. Afterwards, residual lipase activity was measured using the standard pNPP assay. The optimum temperature was also determined by assaying the lipase activity at several temperatures (20–80 °C) in 15 mM Tris/HCl pH 8.0 buffer. Thermal stability studies were performed by incubating purified enzyme at either 0.72 µg/mL or 19 µg/mL in 15 mM Tris/HCl pH 8.0 for 10 min at different temperatures (20 to 80 °C) in a thermostatized water bath. After 10 min in the ice bath, residual activity was measured by a standard pNPP assay in the case of the lower enzyme concentration, whereas the MPOA assay was employed for the higher enzyme concentration. Furthermore, thermal inactivation studies of SeLipC were carried out by incubating 0.72 µg/mL or 19 µg/mL enzyme solution at 45 °C at different times, and the residual activity was assessed using pNPP or MPOA as substrate, respectively. Experimental plots of residual activity versus storage time were fitted to a single exponential decay using the SigmaPlot 11.0 program. The analysis of the experimental thermal deactivation data was carried out using a first-order kinetic model that allowed the calculation of the deactivation rate constant (dk) from the slope of the semilogarithmic plot, and the half-life (1/2t = ln 2/dk) was considered as the time required to reduce 50% of the initial enzyme activity. The effect of ionic strength (I) was studied by measuring the activity in the presence of different concentrations of NaCl up to 750 mM with the standard assay. Similarly, lipase activity of pure SeLipC was assayed in the presence of CuCl2, CaCl2, BaCl2, CoCl2, ZnCl2, MgCl2, FeCl3, FeSO4, and MnCl2 at 1 mM final concentration. The effect of water-miscible organic solvents  on enzyme activity was also studied. For that purpose, pNPP was dissolved in each solvent, reaching a final concentration in the assay mixture that ranged from 10 to 40% (v/v). Likewise, the lipase activity of SeLipC was assessed in the presence of 0.2% (w/v) of several surfactants . Determination of kinetic parameters for pNPP as substrate was performed under optimal assay conditions. Standard assays with increasing concentrations of pNPP ranging from 10 to 800 µM were carried out in 50 mM Tris/HCl buffer, pH 8.0, and 37 °C. Kinetics curves were fitted to the equation: v = (maxV × S)/(MK + S), and MK and cakt were determined by nonlinear hyperbolic regression using the SigmaPlot 11.0 program . Finally, enzymatic assays were performed employing several p-nitrophenyl alkanoate esters as substrates: pNP-acetate (C2), pNP-butyrate (C4), pNP-valerate (C5), pNP-hexanoate (C6), pNP-octanoate (C8), pNP-decanoate (C10), pNP-laurate (C12), pNP-myristate (C14), pNP-palmitate (C16), and pNP-stearate (C18). All of them were dissolved in isopropanol at a final concentration of 4 mM and then employed in the same conditions as the described standard pNPP assay. Finally, the inhibition of SeLipC by phenylmethanesulfonyl fluoride (PMSF), a serine-specific modification reagent, was also addressed. For that purpose, 20 μL of enzyme solution (1.5 μg of SeLipC) were incubated with PMSF (1 mM and 10 mM in DMSO) for 30 min at 4 °C in 100 µL of final volume in Tris-HCl 15 mM, pH 7.0. The remaining enzyme activity was determined using the standard assay. In addition, to analyze protection by substrate against enzyme inactivation by PMSF, 20 μL of enzyme solution (1.5 μg of SeLipC) was preincubated with 10 μL of pNPP 4 mM for 5 min at 4 °C. Then, PMSF was added at a final concentration of 1 mM and 10 mM, and the mixture was incubated for 30 min at 4 °C in the corresponding buffer. After chemical modification, residual activity was assayed using the standard assay. Controls of the enzyme without PFMS were carried out in all the experiments.The optimal reaction conditions for SeLipC was dried by lyophilization. The THF solvent was distilled before being employed. Sugar substrates were dissolved in anhydrous THF (30 nM) in the presence of activated 3 Å molecular sieves (20 mg/mL). When the substrate was not completely soluble in THF, the mixture was sonicated for 10 min before starting the enzymatic reaction. SeLipC was added to the medium , and the reaction was started by the addition of vinyl laurate (3 equiv). The reaction was incubated at 35 °C with orbital shaking (200 rpm), and samples were withdrawn at different reaction times and then filtered through a 0.22 µm PTFE (polytetrafluoroethylene) syringe filter before HPLC analysis. Such analysis was performed using a Jasco HPLC equipped with a Mediterranean Sea 18 column (15 × 0.46 × 5 mm) , employing acetonitrile:H2O 70:30 (v/v) as the mobile phase. The flow rate was fixed at 0.7 mL/min, and an evaporative light scattering detector (Sedex LT-ELSD-80LT) was used to analyze the monoesterified products synthetized by SeLipC.Different sugars were dried in a desiccator under vacuum for 24 h before their use, while Streptomyces exfoliatus DMSZ 41693 genome. Two lipases (abbreviated as SeLipA and SeLipC) were successfully cloned and extracellularly expressed in Rhodococcus sp. T104 KACC 21099 as hosts. After its purification by hydrophobic chromatography, the SeLipC structure was studied by circular dichroism (CD), differential scanning calorimetry (DSC), and fluorescence. In addition, the lipase was kinetically characterized using p-nitrophenyl palmitate (pNPP) as a substrate. In this sense, kinetic parameters for pNPP, the effect of pH and temperature on enzyme activity and stability, and the effect of metal ions, detergents, and water-miscible organic solvents were exhaustively studied. Likewise, the effect of reducing agents on lipase activity was examined. The results suggest that those cysteine residues, suspected to be involved in disulfide bonds, are not needed for activity. In addition, the chemical modification studies of SeLipC with PMSF indicate that a serine residue is involved at the active site of the enzyme. To complete this characterization, a novel esterase assay for lipase activity determination was established using methyl phenoxyacetate (MPOA) as a substrate. Likewise, the hydrolytic activity of SeLipC towards olive oil and several polyester-type plastics such as poly(butylene succinate), poly(butylene succinate)-co-(butylene adipate), and poly(ε-caprolactone) was confirmed. Finally, SeLipC can catalyze the enzymatic synthesis of different sugar esters from Glc, Rha, Fuc, GlcNAc, and GalNAc, although further research is needed to improve reaction yields. In conclusion, the biotechnological application of SeLipC should be seriously considered.In this work, in silico searching allowed us to find four putative lipase-encoding genes in the


However, acute injection of vasotocin did not rescue the female mutant reproductive phenotype, suggesting a contribution of organizational effects of vasotocin. Together, this study provides further support for emerging roles of vasotocin in female teleost reproduction in an important teleost model species.The vertebrate nonapeptide vasotocin/vasopressin is evolutionarily highly conserved and acts as neuromodulator and endocrine/paracrine signaling molecule. Circumstantial and mechanistic evidence from pharmacological manipulations of the vasotocin system in several teleost fishes suggest sex- and species-specific reproductive roles of vasotocin. While effects of vasotocin on teleost reproductive physiology involve both courtship behaviors and the regulation of the hypothalamic-pituitary-gonadal (HPG) axes, comprehensive studies investigating behavioral and physiological reproductive consequences of genetic ablation of vasotocin in a genetically tractable fish model, such as the zebrafish, are currently lacking. Here, we report the generation of homozygous CRISPR/Cas9-based vasotocin gene knock-out zebrafish. Breeding pairs of vasotocin knock-out fish produce significantly fewer fertilized eggs per clutch compared to wildtype fish, an effect coincident with reduced female quivering courtship behavior. Crossbreeding experiments reveal that this reproductive phenotype is entirely female-dependent, as vasotocin-deficient males reproduce normally when paired with female wild-type fish. Histological analyses of vasotocin knock-out ovaries revealed an overall reduction in oocytes and differential distribution of oocyte maturation stages, demonstrating that the reproductive phenotype is linked to oocyte maturation and release. Ovarian hormone quantification and gene expression analysis in mutant fish indicated reduced synthesis of Prostaglandin F Following the identification of a female-specific impairment of reproductive function, we subsequently assessed the potential mechanistic basis of this phenotype by assessing ovarian function using histological, biochemical, and molecular experimental approaches.To address these gaps and to test the hypothesis that vasotocin contributes to reproductive success in zebrafish in either or both sexes through regulation of courtship behavior and/or regulation of the HPG axis, we here report the creation of a homozygous zebrafish vasotocin knock-out model  were attained by pairing individual male and female fish  previously separated by visual inspection of pectoral fin breeding tubercles, body shape and coloring . Wild-type (WT) zebrafish were sourced from the in-house AB strain stock at the University of Ottawa and maintained in 12 l tanks at a density of 3 fish/l in a recirculating system . The system was supplied with salt-dosed  RO water (hereafter “system water”) maintained at a pH of 7.3, a conductivity of 400 μS, and a temperature of 28°C under a 14:10 h light–dark cycle. Following transition to exogenous feeding, larval fish were fed twice daily with different-sized GEMMA zebrafish diets  according to life-stage. Adult fish were fed a mixed diet  twice daily. Embryos , excising a region of 18832 nt including all three avp exons (A current zebrafish genome annotation (GRCZ11) in Ensembl was used as reference for zebrafish gene coding and transcript information. For sgRNA design, CHOPCHOP software was employed . A proto4, 0.6 Ca(NO3)2, and 5.0 Hepes; pH 7.6). Potential founders were identified by consistent appearance of avp+/- genotypes in fin clips of a fraction of F1 offspring obtained from crosses of adult (90 dpf) F0 and WT fish. A mutant line was then established by incrossing heterozygous F1 avt+/- carrying the same deletion to generate homozygous F2 avp-/-, again assessed by fin clip genotyping (2O. The PCR reactions were run with a temperature cycle of 94°C for 3 min followed by 35 cycles of 95°C for 30 s (denaturation), 60°C for 30 s  and 72°C (extension) for 1 min. Following the last cycle, a final 72°C step was run for 10 min and samples maintained at 4°C until analysis by gel electrophoresis. Gel electrophoresis using a 1% Agarose gel containing Red Safe dye  was run for 30 min at 100 V to visualize amplicons. Using the three primers listed in avp+/- mutants, and single band (138 bp) for homozygous avp-/- mutants (F2). The PCR products containing amplicons from fin clips of fish carrying the homozygous deletion of all three avp exons were sent for Sanger sequencing , and alignment with the wild-type genomic sequence confirmed an 18832 bp deletion encompassing all three avp exons , followed by cDNA synthesis using the Quantitect reverse transcriptase kit  according to the manufacturers’ instructions. cDNA synthesized from total RNA extracted from brain and ovaries of WT and avp-/- was then used as template in PCR reactions prepared and run as described for fin clip DNA analysis with the exception that 1 μl cDNA template from brain or ovary was used in a master mix consisting of 1 μl avp FW, 1 μl avp RV primer  was run for 30 min at 100 V to visualize 121 bp avp transcript amplicons . The injection solution comprised of 40 ng/μl for each sgRNA, 20 ng/μl Cas9 protein , and 0.1% Phenol Red  suspended Danieau buffer  were combined as breeding pairs in new 2 l breeding tanks to assess reproductive success. Following the observation of decreased reproductive success in -/-avp  breeding pairs compared to wildtype breeding pairs, additional breeding pairs in which WT of one sex was paired with the avp-/- fish of the other sex were investigated to assess sex-specific contributions of vasotocin to reproductive success in zebrafish. Thus, four groups were investigated , male WT x female avt-/- (n=26) and male avt-/- x female avt-/- (n=48). While all WT and avp-/- animals used in breeding assays were derived from same-sex housed stock, each individual fish was used in a single breeding assay and thus genotype combination group. Pairs received additional feeding in the afternoon the day before the mating assay and were separated by divider overnight. The following day, the divider was removed at the onset of the light cycle (8:00 h). Breeding pairs were allowed to interact for 2 h in the Zebracube and Zebrafish Behavior platform , allowing for a controlled environment. For a subset of breeding pairs, videos of courtship behaviors were recorded using a Dragonfly2 DR2-HIBW camera with 30 frames/s . Following the 2 h breeding period, eggs, if present, were collected using a strainer and placed in a petri dish filled with methylene blue system water. The total number of fertilized eggs  were then quantified as measure of reproductive success. If present, unfertilized eggs (slightly yellow in color and with granular appearance) and/or dead eggs (white in color and often broken down) were counted separately and removed from the petri dish. Following consistent demonstration that irrespective of genotype, all analyzed courtship behaviors declined after initial interaction, the first 10 min of interaction were analyzed for each video in a subset of animals for which the interaction was recorded. Specifically, and as previously described  sampled from female holding tanks not used for courtship assays. Following terminal anesthesia of WT and -/-avp  females at the onset of lights in the morning, the fish were gently dried on paper towel, and weighed. Abdominal sections containing the ovary were then collected from WT and avp-/-, and ovaries carefully dissected and weighed. The GSI was calculated by dividing the ovarian mass by total body mass. Each section was placed in a 1.5 ml pre-weighed Eppendorf tube and the weight recorded. For n=4 WT and n=5 avp-/- ovaries, whole fish abdominal samples were placed in 4% PFA PBS solution and incubated at 4°C overnight and washed for 3 x 15 min in PBS the following day. The samples were subsequently transferred to a 0.5 M EDTA (pH= 8.0) solution to decalcify tissue for one week at 4°C. The following week, the samples were washed for 3 x 15 min in PBS, before successive dehydration steps in 10%, 20% and 30% sucrose in PBS solutions. Samples were placed in each sucrose solution until samples sunk. Finally, samples were embedded in molds with OCT (Thermo Fisher Scientific) and stored at -80°C until sectioning. Each sample was sectioned across the sagittal plane using a Cryostat  and serially cut into thirty  12 μm sections , which were then collected on Fisherbrand Superfrost Plus microscope slides (Thermo Fisher Scientific). All slides were subsequently rinsed in 1 x PBS for 3 min, then placed under a gentle stream of tap water for 3 min to wash off the OCT. For H&E staining, the slides were incubated for 30 s in Mayer’s Hematoxylin, then placed in 1 x PBS for 20 seconds and washed under a gentle stream of tap water for 1 min. The slides were then dipped in 70% and 95% EtOH for 30 s consecutively and placed in the counterstain Alcoholic Eosin for 30 s. Once removed, the slides were dehydrated through 2 x 15 seconds in 95% EtOH and 3 x 15 seconds in 99% EtOH. To ensure the tissue was cleared of any other residue, slides were immersed in xylene. 3 x for 1 min. Ovary sections were finally imaged using a dissection microscope  using a 6.7 x magnification to determine the number of eggs and largest diameter of each oocyte. Additional images of representative slides were taken using a , at 40x magnification. All images were acquired using the Infinity II microscopy camera  and images were analyzed using ImageJ (www.https://imagej.nih.gov/ij/). Briefly, the total number of oocytes per section and size-based oocyte stages (I-V) were determined for each of the eggs measured according to Li and Ge (avp-/- ovaries respectively.The gonadosomatic index (GSI) was assessed from n=25 WT and n=26 i and Ge . For all2.2.3.2avp-/- (n=18) female zebrafish (6-9 mpf) held in all female tanks not used for breeding assays were carefully extracted following terminal anesthesia and weighing of the whole animal as previously described. All animals were collected upon onset of lights in the morning. Tissues were then weighed, placed in 1.5ml microcentrifuge tubes, and stored -80°C until processing. To extract ovarian hormones, ovarian tissue samples were thawed on ice and 200 μl ELISA buffer  was added to each tube. The samples were then sonicated, ensuring the probe was cleaned with 75% EtOH and RNAse free water in between each sample. Following homogenization, 1 ml of diethyl ether was placed into each sample, vortexed and allowed to sit for 30 min. The samples were then centrifuged at 3000 g for 5 min and flash frozen at -80°C for 30 min. The liquid phase was removed and placed into new microcentrifuge tubes. The ether was evaporated under a gentle stream of nitrogen under the fume hood, and the extraction steps were then repeated two more times. After the final extraction step, 250 μl extraction buffer was added to the tubes, vortexed and placed in a heat block for 5 min at 65°C. The samples were vortexed, placed on the heat block for additional 5 min and vortexed for a final time. The extracts were then placed at -80°C until use. Extracts were used for the quantification of 17-β estradiol (E2), Progesterone (P4) and Prostaglandin F2α (PGF2α) using ELISA kits #501890, #582601 and #516011  according to manufacturer’s instructions. Assay sensitivities (defined as 80% B/B0) were 9.43 pg/ml for E2, pg/ml 7.65 pg/ml for P4 and 19.20 pg/ml PGF2α, respectively. All samples were run in duplicate and a cut-off of <20% intra-assay CV was applied for replicates. Average intra-assay CVs were 13.96% for the E2 assay, 15.75% for the P4 assay, and 13.4% for the PGF2α assay. All analyte concentrations were corrected for sample dilution factors and normalized by ovarian weight prior to analysis.Whole ovaries from adult WT (n=18) and 2.2.3.3-/-avp  mutants (n=7) were collected from all female housing tanks when lights were turned on in the morning and immediately stored at -80°C until processing as previously described. To extract total ovarian RNA, frozen tissues were homogenized in 250 μl of TRIzol reagent (Invitrogen) using a sonicator. The probe was cleaned with 75% EtOH and RNAse-free water in between each sample to avoid cross-contamination. Total RNA was extracted according to the manufacturer’s instruction and the total RNA pellet resuspended in 30 μl of DEPC water. The purity and concentration of total RNA was then assessed using a NanoDrop and was used to generate cDNA. cDNA was synthesized using the QuantiTech Reverse Transcription Kit (Qiagen) according to the manufacturer’s instructions, using 1 μg of total RNA per sample. Controls devoid of reverse transcriptase enzyme were used to monitor potential contamination during subsequent gene expression assays. All samples were stored at -20°C until used as templates in gene expression assays.Ovaries from female WT (n=7) and vasa, nanos2), the gonadotropin receptors , the lipoprotein vtg1, nuclear and membrane receptors for steroids  and genes involved in PGF2α synthesis pathway . A complete list of gene IDs, full gene names, primer sequences used in gene expression assays and annealing temperatures can be found in 2 > 0.95. Relative gene expression was subsequently normalized using the NORMA-gene method  was used to quantify relative fold-changes of 11 ovarian gene transcripts with known function in different stages of zebrafish oocyte maturation, as reviewed by Li and Ge, 2020 . Brieflye method  and rela2.2.4avp-/- zebrafish could be rescued through activational effects of vasotocin  and once deep anesthesia was confirmed, i.p. injected with either physiological saline (n=15) or 1 μg/g bw vasotocin (n=15) in physiological saline. Following ~10 min recovery from anesthesia, female fish were set up for breeding with WT male zebrafish and mating success quantified as previously described. Using the same experimental parameters, a second repeated injection rescue experiment was conducted using avp-/- zebrafish  spawned at the beginning of the experiment to release of residual eggs and subsequently injected on day 3, 6 and 9 after before quantification of reproductive success on the morning of day 10. To assess the possibility that i.p. injection itself may represent a physiological stressor sufficient to inhibit female reproductive success as measured by egg fertilization following breeding, a group of WT females was i.p. injected with physiological saline but did not exhibit different breeding success compared to un-injected WT females assessed in breeding assays as described previously (data not shown).Acute single and repeated intraperitoneal (i.p.) vasotocin  experiments were conducted in female mutant fish not previously used in breeding assays to assess whether the observed female reproductive phenotype in 2.3avp-/- knock-out to affect reproductive physiology.Given the reported role of Avp in several aspects of teleost physiology known to affect reproduction, we sought to assess the potential for altered somatic growth and basal endocrine stress axis activity in 2.3.1-/-avp  mutants (n=20) were analyzed at 30, 60 and 90 dpf. Each fish was anesthetized using 0.24 mg/ml of tricaine (Syndel Laboratories) and once deep anesthesia was confirmed, each fish was lightly dried, weighed using an analytical scale to determine body mass, and imaged using a light microscope  with camera system  to determine body length. Following recovery from anesthesia (~10 min), each fish was placed back in the housing tank. Images were scaled and measured by tracing the notochord on laterally positioned individuals on acquired images using ImageJ (www.https://imagej.nih.gov/ij/).To assess indices of somatic growth up to adulthood (3 mpf), body mass and body length (measured as fork length) in WT (n=20) and 2.3.2-/-avp  (n=3 replicates) were carefully collected in the morning within an hour and placed in 1.5 ml microcentrifuge tubes and stored at -80°C until processing. For adults randomly taken from mixed sex tanks, both WT (n=10) and -/-avp  (n=10) were terminally anesthetized as previously described and weighed. Blood was extracted by cutting of the dorsal fin and placed in 1.5ml microcentrifuge tubes stored at -80°C until processing. To extract cortisol from pooled larvae and adult blood samples, samples were thawed on ice and 200 μl extraction buffer was added to each tube. The samples were then sonicated, ensuring the probe was cleaned with 75% EtOH and RNAse free water in between each sample. Following homogenization, 1 ml of diethyl ether was placed into each sample, vortexed and allowed to sit for 30 min. The samples were then centrifuged at 3000 g for five min and flash frozen at -80°C for 30 min. The liquid phase was removed and placed into new microcentrifuge tubes. The ether was evaporated under a gentle stream of air under the fume hood and the extraction steps were then repeated two more times. After the final extraction step, 250 μl extraction buffer was added to the tubes, vortexed and placed in a heat block for five minutes at 65°C. The samples were vortexed, placed on the heat block for additional 5 min and vortexed for a final time. The extracts were then placed at -80°C until use. Extracts were used for the quantification of cortisol using ELISA kit #402710  according to the manufacturer’s instructions. In all cases, samples were run in duplicates, on a single plate, and a cut-off of <20% was applied for replicates. All samples were normalized by number of larvae in each microcentrifuge tube prior to analysis.Pooled Larvae (n=25 per pool) from WT (n=3 replicates) and 2.4post-hoc tests in cases of multiple comparison groups. In cases where data not normally distributed, Mann-Whitney U tests were used for comparisons of two groups, and Kruskal-Wallis tests for multiple groups followed by Dunn’s post-hoc test. In cases were the investigation of interaction of independent variables was warranted for non-parametric datasets, an ANOVA based on ranks followed by the Scheirer-Ray-Hare extension .For each experiment, normal distribution of data was assessed using the Shapiro-Wilk test and homoscedasticity using Levene’s test, respectively. In cases where the raw data were not parametric, standard transformations  were used to improve normality and/or homoscedasticity. Single outliers were identified in normally distributed data using Grubb’s test. For parametric and homoscedastic data, a t-test was used in case of two comparison groups and univariate ANOVAs  followed by Tukey’s xtension  was used33.1avp-/- breeding pairs, the number of successful breeding was reduced from 53% in WT to 35% in avp-/- (P=0.097). Conversely, the median number of viable eggs produced was significantly reduced , reaching a median of 40 in WT and 0 in avp-/- pairs (P=0.001), a more than 3-fold reduction was found in avp -/- breeding pairs compared to WT breeding pairs , but not male (df=1 H=0.320 P=0.572), genotype and was not dependent on the interaction of both genotypes (df=1 H=0.364 P=0.55). Regarding clutch size, a significant effect of female genotype , but not male genotype  or their interaction  was observed (avp-/- compared to WT (P<0.01).When comparing WT and 3.2P=0.118), female genotype (df=1 F=1.017 P=0.320), or their interaction (df=1 F=0.180 P=0.647). Similarly, the duration of male chasing events (P=0.051), female genotypes (df=1 F=1.032 P=0.317), or their interaction (df=1 F=0.438 P=0.513). The number of times males circled the females in breeding pairs (P=0.407) genotype, nor their interaction (df=1 H=0.012 P=0.914). The number of times the male nudged the female’s flank (P=0.166) nor female genotype (df=1 H=1.788 P=0.118) or their interaction (df=1 H=0.016 P=0.898). Conversely, quivering behavior was significantly affected by female (df=1 H=8.042 P<0.01), but not male (df=1 H=1.35 P=0.244) genotype and was significantly reduced in mating pairs with avp-/- females compared to WT females  and a significant increase in stage V oocytes (df=7 t=2.374 P=0.0493). No significant differences in the percentage of stage II , III (df=3 t=1.008 P= 0.383) and IV oocytes (df=5.345 t=1.299 P=0.964) were found. Examples of stained ovary sections used for quantification and diameter measurements of oocytes in WT and avp-/- ovaries are shown in The GSI was not significantly different between WT and 3.42 (avp-/- mutants (df=29 t=0.874 P=0.389). Similarly, ovarian tissue concentrations of P4 (avp-/- (df=32 t=0.6461 P=0.523). Conversely, PGF2α (-/-avp ovaries compared to WT ovaries (df=26 t=2.518 P=0.018).Ovarian concentrations of E3.5nanos2 (df=11 t=2.598 P=0.025), and pla2g4ab (df=12 t=3.087 P=0.009) were significantly reduced, while the ovarian transcript abundances of pgrmc1 (df=7.511 t=2.809 P=0.0244) and pgrmc2 (df=12 t=2.276 P=0.042) were significantly increased in -/-avp  mutants compared to WT (ddx4 (df=7.403 t=0.8331 P=0.43) lhcgr (df=12 t=1.770 P=0.102), fshr (df=12 t=0.9722 P=0.350) vtg1 (df=12 t=1.189 P=0.257), ar (df=12 t=1.306 P=0.216), pgr (df12 t=1.452 P=0.172) and ptgs2 (df=12 t=2.027 P=0.066) was not significantly different between avp-/- mutants and WT.The ovarian transcript abundances of 3.6avp -/- female prior to breeding assays, breeding success was marginally lower in Avp injected females compared saline injected females . No interaction effect between genotype and time was observed for body mass . An intra-assay CV of 6.68% and an inter-assay CV of 7.66% were determined for adult fish blood cortisol assays.Cortisol concentration in 44.1avp-/- breeding pairs which manifested itself by reduced egg numbers and clutch sizes, but not a significant reduction in spawning success. Backcrosses of avp-/- males and females with WT females and males, respectively, revealed that the observed reduction in these parameters was dependent on the female, but not male avp-/- genotype, as the latter reproduced similarly to WT pairs when crossed with WT females. The identification of reduced reproductive success in avp-/- breeding pairs compared to WT breeding pairs is in line with circumstantial evidence and mechanistic studies conducted in several teleost fish species reviewed in the introduction of this study, although these studies report effects on both female and male reproductive function, especially regarding courtship behavior , modulating steroidogenesis, and promoting ovulation  as percentage of total oocytes. This finding suggests that retention of mature oocytes contributed, at least in part, to the female reproductive phenotype in avp-/- mutants. Because the endocrine control of the ovulation process is well known to include PGF2α in zebrafish  from membrane phospholipids , which is further converted to mature prostaglandins including PGF2α. The physiological importance of both steps in ovarian zebrafish PGF2α synthesis has been demonstrated in detail  fishes  fishes , 62, 63 ) fishes , 64, 65,4.3avp-/- knockouts are, at least in part, dependent on Avp-dependent stimulation of ovarian PGF2α, our mechanistic studies point to additional, previously unidentified mechanisms. When considering the distribution of oocyte stages as percentage of total oocytes in avt-/- compared to WT, a significantly lower percentage of stage I oocytes are observed in avp-/- compared to WT ovarian sections. This suggests that Avp may, in addition to previously discussed roles in oocyte maturation and ovulation, play a role in germ line stem cell maintenance, proliferation and/or differentiation into stage I follicles. In zebrafish, and in contrast to mammals which possess a finite number of oocytes following embryogenesis, zebrafish can produce new oocytes throughout their lifetime due to the presence of self-renewing germline stem cells (GSCs) , which is present in all nine identified subclusters of GSC as well as stage 1A oocytes  . Indeed,ish GSCs . In a re oocytes . Such deelopment  did not ablation , it is cished in , avp, avpgrmc1, pgrmc2), which code for single transmembrane heme-binding protein containing a cytochrome b5 (Cytb5) motif and which have been proposed to function as a progesterone binding protein in vertebrates, while also interacting with various molecules such as heme, fatty acid 2-hydroxylase, cytochrome P-450 enzymes, insulin receptor, epidermal growth factor receptor (EGFR), Erbb2, and membrane progestin receptor α (mPRα), among others. Thus, its precise physiological roles difficult to dissect , a cortisol inactivating enzyme, has been described in zebrafish oocytes , critical revisIon and of the manuscript, final approval of the submitted manuscript, agreement to be accountable to all aspects of the work. All authors contributed to manuscript revision, read, and approved the submitted version.