Anthocyanins are widely applied as a marker for haploid identification after haploid induction in maize. However, the factors affecting anthocyanin biosynthesis in immature embryos and the genes regulating this process remain unclear.F3H, DFR, ANS, F3′H and the MYB transcription factor-encoding gene C1. The expression patterns of 14 selected genes were confirmed using quantitative reverse transcription-polymerase chain reaction.In this study, we analyzed the influence of genetic background of the male and female parents, embryo age and light exposure on anthocyanin accumulation in embryos. The results showed that light exposure was the most crucial factor enhancing the pigmentation of immature embryos. The identification accuracy of haploid embryos reached 96.4% after light exposure, but was only 11.0% following dark treatment. The total anthocyanin content was 7-fold higher in immature embryos cultured for 24 h under light conditions compared to embryos cultured in the dark. Transcriptome analysis revealed that the differentially expressed genes between immature embryos cultured for 24 h in dark and light chambers were significantly enriched in the pathways of flavonoid, flavone, flavonol and anthocyanin biosynthesis. Among the genes involved in anthocyanin biosynthesis, five up-regulated genes were identified: F3H, DFR, ANS, F3′H and transcription factor gene C1 were significantly up-regulated. This study provides new insight into the factors and key genes regulating anthocyanin biosynthesis in immature embryos, and supports improved efficiency of immature haploid embryo selection during doubled haploid breeding of maize.Light is the most important factor facilitating anthocyanin accumulation in immature embryos. After 24 h of exposure to light, the expression levels of the structural genes The online version contains supplementary material available at 10.1186/s12870-022-03971-5. Doubled haploid (DH) technology has dramatically improved the breeding efficiency and shortened the breeding process in maize . IdentifThe difference between haploid and diploid seeds lies mainly in the genetic constitution of their embryos. With the application of immature embryo-based chromosome doubling in breeding programs, separation of immature haploid embryos from diploid embryos is increasingly important. To date, haploid inducers such as RWS-GFP have been developed that contain fluorescent proteins expressed specifically in embryos . Neverthcolorless 1 (C1), purple leaf 1 (PL1), booster 1 (B1), red color 1 (R1) and pale aleurone color 1 (PAC1), which form the MYB transcription factor/basic helix-loop-helix (bHLH) domain protein/WD-repeat (MYB-bHLH-WD) transcription factor complex [R1-navajo  results in pigmentation of the scutellum and aleurone, and has been used for the selection of mature haploid seeds [anthocyaninless 1 (A1), anthocyaninless 2 (A2), colorless 2 (C2), bronze 1 (Bz1), and bronze 2 (Bz2), are essential for the expression of R1-nj, for which the regulatory gene C1 is also required [C1-I [C2-Idf [In1-D [Compared with fluorescent markers, the identification of haploids based on a visual anthocyanin marker is more convenient . Two cla complex . Among tid seeds . Structurequired . Anthocyrequired . Some tred [C1-I , C2-Idf  [C2-Idf  and In1-f [In1-D , and aref [In1-D , 17, 18.f [In1-D . Hence, In this study, we compared factors related to anthocyanin synthesis that affect the pigmentation of embryos, and demonstrated that light is an essential factor in the identification of immature haploid embryos. Meanwhile, transcriptome analyses revealed differentially expressed genes (DEGs) involved in anthocyanin biosynthesis, which control anthocyanin accumulation in immature embryos, under light stimulation. The results of this study provide a useful guide for further improving the efficiency of haploid identification, thereby facilitating the development of DH technology.Immature embryos derived from ZD958×CAU5 with various pigmentation area (PA) and pigmentation intensity (PI) values are shown in Fig. To observe the influence of embryo age on anthocyanin accumulation, diploid embryos derived from B73×CAU5 were isolated at 12, 15, 18 and 21 days after pollination (DAP), respectively. At 24 h after light exposure, PR was strongly correlated with embryo age, as the PR of embryos at 12 DAP was only 8.3%, while that of embryos at 21 DAP reached 96.0%  value for 32,608 total genes were obtained through transcriptome sequencing, of which 852 exhibited significant expression level differences between the dark and light treatments based on the criteria of |(log2(fold change)|> 1 and To identify the putative pathways associated with these DEGs, a Gene Ontology (GO) enrichment analysis was performed and 41 functional terms were identified. The DEGs were enriched mainly in terms associated with cellular components such as cell part, organelle and membrane part. For the biological process category, the genes were mostly enriched in the metabolic process, cellular process, biological regulation and response to stimulus terms. Within the molecular function category, the DEGs were commonly enriched in the terms catalytic activity, binding and transcription regulator activity Fig. . PreviouCHS), anthocyanidin synthase (ANS), flavanone 3′-hydroxylase (F3′H), anthocyanidin 3-O-glucosyltransferase (3GT), and flavonoid 3′-monooxygenase (Zm00001d007918), as well as one down-regulated gene, malonyl-CoA (Zm00001d052042).To further explore the biological functions of the DEGs, an enrichment analysis was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database Fig. . CompareF3′5′H) and the regulatory gene PAC1 were down-regulated. All other investigated genes, including the structural genes CHS, flavanone 3-hydroxylase (F3H), dihydroflavonol reductase (DFR), ANS, uridine diphosphate glucose flavonoid 3-glucosyltransferase (UFGT), glutathione S-transferase (GST) and F3′H, as well as the regulatory transcription factors C1/PL1 and R1/B1, were up-regulated by light exposure. Furthermore, DFR and ANS, two structural genes involved in the anthocyanin biosynthesis pathway, showed markedly increased transcript levels. In addition, a high expression level of F3H may contribute to the accumulation of anthocyanins. As noted above, the expression levels of structural genes are controlled by the MYB-bHLH-WD transcription factor complex. The expression of C1 was significantly up-regulated, whereas the up-regulation of PL1, R1 and B1 was slight and not significant (based on FPKM) and was significant based on qRT-PCR. In the terms of differential expression level, C1 was the key genes regulating anthocyanin biosynthesis in immature embryos.The expression levels of 14 genes related to the anthocyanin biosynthesis pathway, including 9 structural genes and 5 regulatory genes, were compared between the dark and light groups using RNA sequencing (RNA-seq) data  was performed Fig. C and theR1-nj, in mature haploid seeds has been studied extensively, and is significantly affected by the genetic background of the inducer  and the source germplasm  [R1-nj-based haploid inducers [Anthocyanin has long been employed in the identification of haploids. The performance of anthocyanin markers, mainly  parent) , 13, 17.inducers . Poor piR1-nj marker system. Light-induced anthocyanin accumulation has been observed not only in maize but also in pear [Arabidopsis [Nevertheless, with the development of immature embryo-based chromosome doubling methods, the chromosome doubling rate was improved significantly , the ide in pear , 23, Arabidopsis  and tomabidopsis . Aside fbidopsis , gibberebidopsis  and sucrbidopsis  improve bidopsis .CHS, F3H, DFR and ANS, are important for anthocyanin accumulation, and the expression levels of these structural genes are controlled by transcription factors [CHS and DFR is promoted by blue light, as reported in the ray florets of Gerbera hybrida [CHS, F3H, DFR, ANS and UFGT in postharvest strawberries [F3′H combined with low expression of F3′5′H led to the accumulation of cyanidin 3-glucoside anthocyanin, and F3′H is activated by C1 and R1 in the aleurone of maize seeds [C1 was significantly up-regulated after exposure to light, signifying that C1 might be a key gene in enhancing the expression of structural genes in the anthocyanin biosynthesis pathway, as reported previously [R1 is another critical gene in anthocyanin synthesis [R1 was not altered significantly by light exposure (based on FPKM), as R gene expression cannot be induced by light, in accordance with previous results [The regulatory mechanism underlying anthocyanin synthesis has been well studied in maize, but the pigmentation of diploids during haploid identification remains unclear. High expression levels of structural genes, including  factors . The exp hybrida . Similarwberries . High exze seeds . Our reseviously , 34. R1 C1 in seeds and PL1 in plant tissues contribute to the light-dependent regulation of anthocyanin accumulation, while bHLH genes such as R1, B1, scutellar node1 (Sn1), leaf color 1 (Lc1) and Hopi1 determine the tissue-specific synthesis of anthocyanins [C1/PL1 and bHLH R1/B1 gene families have been used to improve the anthocyanin contents of seeds and other tissues; for example, the pericarp of rice became dark brown when transformed with a construct of ZmC1 and ZmR-S driven by a promoter from the rice prolamin-encoding gene [ZmC1 and ZmR in wheat led to the development of anthocyanin-enriched wheat germplasms [R and C1 under the CaMV 35 S constitutive promoter, leading to accumulation of anthocyanins with no requirement for additional light-induced regulators [C1, R2, Bz1 and Bz2 [Aside from environment factors, anthocyanin synthesis can be improved through regulation of the expression of two types of transcription factors. ocyanins . The MYBing gene , and overmplasms . Furthergulators . Further and Bz2 .ZmC1 and ZmR2 in our previous study [To improve the efficiency of haploid identification using anthocyanins as a marker, the expression of genes involved in anthocyanin synthesis must be enhanced through technological and biological means. Light can activate the expression of genes involved in anthocyanin biosynthesis , 40. In us study . OverallF3H, DFR, ANS, F3′H and the transcription factor C1, which are involved in anthocyanin biosynthesis, were significantly up-regulated, suggesting roles for these genes in anthocyanin accumulation. The findings of this study provide new insight into anthocyanin accumulation in immature embryos, and will be valuable for further improving haploid embryo identification efficiency.In this study, we systematically analyzed the effects of factors including the haploid inducer line, female parent, embryo age and light exposure on the pigmentation of immature embryos. Our results demonstrate that light exposure is crucial to promoting embryo pigmentation. Transcriptome analysis revealed that DEGs are enriched in the flavonoid, flavone and flavonol and anthocyanin biosynthesis pathways. The expression levels of Three haploid inducers, CAU5, CAU6 and CHOI3, were used as the male parents in this study. We used four inbred lines, B73, Chang7-2 (C7-2), Gaoyou923 (Gy923) and Zheng58 (Z58), along with the hybrid Zhengdan958 (ZD958), to evaluate embryonic anthocyanin accumulation and the efficiency of haploid embryo identification. Our previous results showed that the tester lines C7-2, Gy923 and Z58, showed different degree of coloration and thus result in the difference of accuracy in identification of haploid seeds. All materials were grown at the experimental station of Beijing Academy of Agriculture and Forestry Sciences and China Agricultural University. Unless stated, all embryos were isolated at 15 DAP.Embryo isolation and in vitro culture were performed according to published protocols . The earWe analyzed the effects of four factors  on embryo pigmentation. To analyze the effect of the male parent on immature embryo pigmentation, we pollinated three haploid inducers, CAU5, CAU6 and CHOI3, to ZD958; the resulting embryos were isolated and cultured for 24 h under light. We used CAU5 as the male parent for crossing with three inbred lines, C7-2, Gy923 and Z58, to analyze the effect of the female parent. To study the effect of embryo age on embryo pigmentation, we isolated immature embryos from the B73×CAU5 cross at 12, 15, 18 and 21 DAP, and cultured them for 24 or 48 h under light conditions. In the light exposure experiment, we set three time widows, 0–24 h, 24–48 h and 48–72 h, with independent light exposure (+) and dark control (-) groups for each time window, constituting six treatments: 0–72 h, dark (---); 0–24 h, light and 24–72 h dark (+--); 0–48 h, dark and 48–72 h, light (--+); 0–48 h, light and 48–72 h, dark (++-); 0–24 h, dark and 24–72 h, light (-++); and 0–72 h, light (+++).We chose the incandescent lighting,, with quantity of 20,000 lx. The PR and PI of all embryos were evaluated visually, and the PR was calculated using the formula PR (%) =  × 100%. Pigmentation intensity (PI) were employed based on visual scoring of the percentage of the scutellum area that was pigmented and the degree of pigmentation of the scutellum, respectively. The immature embryos were classified into PA levels 1–3 based on the thresholds of PA < 25%, 25% < PA < 50% and PA > 50%, respectively. For PI, levels 1–3 exhibited pink, red and purple pigmentation, respectively. In addition, putative haploid embryos identified were verified based on plant characters in the field; the identification accuracy was calculated to evaluate the efficiency of haploid identification based on immature embryo pigmentation.− 1 cm− 1) and MW is the molar mass (484.84) of the selected standard [Immature embryos were ground into a fine powder under liquid nitrogen and dried at − 80 °C. To observe the total anthocyanin content of immature B73×CAU5 embryos under various culture conditions, a spectrophotometric method was used as described previously with cyanidin 3-O-glucoside (CAS: 7084-24-4) as the standard. An extraction solution was prepared by mixing 1% HCl with 90% ethanol at a volume ratio of 1:1. The operating solution was prepared at a sample-to-extraction solution volume ratio of 1:15 and subjected to ultrasonic extraction for 1 h, and then the supernatant was collected following centrifugation at 12,000 rpm for 10 min. The absorbance of a mixture of 1.0 mL of supernatant with 4.0 mL of sodium acetate buffer (pH 4.5) was measured on an ultraviolet-visible spectrophotometer at wavelengths of 520 and 700 nm. The anthocyanin concentration (mg/L) was calculated as follows: C = Af /(εL)×103×MW, where A is the absorbance difference between two pH conditions, f is the dilution factor, L is the light path of the cuvette (cm), ε is the molar absorptivity  [p < 0.05).Total RNA extraction and quality control were performed according to standard protocols. Annoroad Gene Technology Co.  constructed the cDNA library and performed Illumina sequencing on a HiSeq 4000 instrument  to yield 150-bp paired-end reads. The number of clean reads mapped to a specific gene were counted and then presented as fragments per kilobase of transcript per million mapped reads (FPKM) using Cufflinks . Differep/kegg/) . GO enrip/kegg/)  and KOBAp/kegg/) , respect(−ΔCT). Three biological and three technical replicates were performed for all RT-qPCR experiments.Total RNA was manually isolated with TRIzol reagent . cDNA was synthesized from 1 µg of RNA using a PrimeScript™ RT Kit with gDNA Eraser . qRT-PCR was performed using SYBR Premix ExTaq . The PCR cycling conditions were: 95 °C for 30 s, followed by 39 cycles of 95 °C for 10 s and 60 °C for 30 s, followed by 95 °C for 10 s, followed by melt curve 65 to 95 °C increment o.5 °C for 5 s. Expression levels were calculated relative to the reference gene (ubiquitin protein ligase) using the formula 2Additional file 1: Supplementary Table S1. Annotation of DEGs identified in light vs dark.Additional file 2: Supplementary Table S2. GO pathway analysis of DEGs under light.Additional file 3: Supplementary Table S3. KEGG pathway analysis of DEGs under light.Additional file 4: Supplementary Table S4. List of anthocyanin biosynthesis-related maize genes.Additional file 5: Supplementary Table S5. Primers used for RT-qPCR in this study.


The genes encoding the LDLr-like, Lrp1-like, Lrp1b-like, Lrp3, Lrp4-like, Lrp5-like, Lrp6, Lrp10, Lrp11, Lrp12-like, and Lr11-like proteins were found and identified by sequence and structure analysis, in addition to phylogenetic analysis. Genes encoding proteins previously implicated in follicle development and vitellogenin (Vtg) uptake in oviparous vertebrates were also identified, i.e. lr8 (including lr8 + and lr8- variants) and lrp13; their identification was reinforced by conserved synteny with orthologues in other teleost fish. Compared to other lr/lrp genes, the genes encoding Lr8 + , Lr8-, and Lrp13 were highly expressed in ovary during early development, decreasing as oocyte development advanced when induced by hypophysation. Furthermore, lr8 + , lr8-, and lrp13 were dominantly expressed in the ovary when compared with 17 other tissues. Finally, this study successfully detected the expression of both lr8 variants, which showed different expression patterns to those reported in other oviparous vertebrates and provided the first characterisation of Lrp13 in Anguilla sp. We propose that lr8 + , lr8-, and lrp13 encode putative Vtg receptors in anguillid eels.Lipoprotein receptors, including low-density lipoprotein receptor (LDLr) relatives (Lrs) and LDLr-related proteins (Lrps), belong to the LDLr supergene family and participate in diverse physiological functions. In this study, novel sequences of The online version contains supplementary material available at 10.1007/s10695-023-01169-6. FxNPxY), or for signalling  associated with Lr11, the CUB  and bone morphogenic protein-1 (Bmp1)) domain associated with the Lrp3, Lrp10, and Lrp12, or the MANEC domain associated with Lrp11.The low-density lipoprotein receptor (LDLr) family is represented by single-pass transmembrane proteins that share some distinctive common features with the first member discovered, the LDLr :  uptake. While Lr8 binds Vtg in frog  is one of the two main temperate species of anguillid eels found in New Zealand. Anguillid eels represent a group of basal teleost fish, the Elopomorpha, which correspond to one of the earliest evolved group within the teleost lineage  and the early vitellogenic stage . The database, presented in an earlier publication . For ease of reporting, the putative Vtgrs will be referred to as the lr8 gene encoding Lr8 +/- variants and lrp13/Lrp13, while the genes annotated to Lrp10 and SorLA will be referred to as lrp10/Lrp10 and lr11-like/Lr11-like, respectively. Protein sequences were further examined, and their conserved domains were identified using the CD-Search tool . Then, their protein molecular weight was predicted using the Compute pi/MW Tool from the ExPASy server  were detected with the ORF finder from NCBI (available online at G = 4.3344) to model the evolutionary rate differences among sites. Lastly, a tree was constructed with the maximum likelihood method applying 1000 bootstrap replicates. All positions with less than 95% site coverage were eliminated; thus, a total of 316 positions were kept in the final dataset. For visualisation purposes, the Lrp11 group was used as a relative outgroup, similar to Mushirobira et al. . The predicted lr8 orthologues were compared between the European eel , zebrafish , and Nile tilapia , while predicted lrp13 orthologues were compared between the European eel , zebrafish , and medaka .Due to the lack of SFE genomic data, the surrounding genomic arrangements of the predicted lr8 variants  using qPCR (“The transcript abundances of the ng qPCR “” sectionng qPCR “” sectionng qPCR “).n = 30) and a 200-L recirculating control tank (n = 5) both maintained at 30–35 ppt, 16–17 °C, 12/12 light/dark regime. Treatment eels received weekly intramuscular injections of salmon pituitary homogenate  was euthanised with an overdose of benzocaine  and dissected. Similarly, SPH-treated eels were euthanised every 2 weeks, for a maximum of 10 weeks, to detect the progression of oogenesis (n = 4–5 per sampled week). Finally, the Ringer-treated eels were euthanised after 10 weeks of treatment . During a routine water change at week 7, the water quality of the tanks was affected, resulting in deaths and reduced sample sizes for week 8 and week 10 (n = 4).The ovarian artificial maturation experiment was carried out by Damsteegt et al. (unpublished data) and Mercuriali et al. (unpublished data). Briefly, thirty-five EV stage eels (capture year 2017) were split between a 1000-L recirculating treatment tank  following the manufacturer’s instructions. Then, 5 μg of total RNA from each sample was treated with DNase TURBO DNA-free™ kit , of which 500 ng was reverse-transcribed with PrimeScript™ RT reagent kit  using Oligo(dT) and random hexamer primers. Samples were diluted with Milli-Q water (MQW) to 10 ng/μl to be used in qPCR assays “” sectionThe cDNA amplified by PCR for subsequent tenfold serial dilution, to construct qPCR standard curves “” sectionactb) and elongation factor-1α (eef1a) transcripts were validated in previous studies  following the manufacturer’s instructions. Once the identity of the PCR products was verified , they were subjected to ten-fold serial dilutions in MQW to construct qPCR standards.The PCR primers employed to generate templates to use as qPCR standards and the qPCR primers used to quantify the β-actin  was used. Initial denaturation at 95 °C for 2 min was followed by 40 cycles of denaturation at 95 °C for 30 s, annealing for 30 s (see Table lr8 variants) or 2.7 min (lrp13). Afterwards, a final extension at 72 °C for 5 min was done. The actb and eef1a PCR amplifications were done using the MangoTaq™ DNA polymerase kit . In this case, the cycling conditions involved an initial denaturation step at 95 °C for 5 min, and 35 cycles of 95 °C for 30 s, annealing for 30 s (see Table All PCR reactions were carried out on an Eppendorf Mastercycler PCR machine. To amplify ORFs encoded by lr8-), between 92.2 and 93.7% with a CV of 16.0% (lr8 +), and between 95.1 and 95.2% with a CV of 12.5% (lrp13). Assay efficiencies for the artificial maturation experiment were 95.0% (lr8-), 95.2% (lr8 +), 96.7% (lrp13), 95.3% (actb), and 95.1% (eef1a).All qPCR reactions were carried out on a QuantStudio™ 5 , and the results were analysed using QuantStudio™ Design and Analysis Software . The reactions were prepared with SYBR® Premix Ex Taq™ II  following the manufacturer’s instructions. The cycling conditions started with a hold step at 95 °C for 2 min, followed by 40 cycles of denaturation at 95 °C for 5 s, annealing for 10 s , the data were normalised over total RNA. The genes lr8- and actb transcript abundances normalised over total RNA, and lr8- transcript abundance normalised over actb) or analysed with non-parametric tests . Except for this last dataset, each variable was tested using a one-way ANOVA, followed by the Scheffé post-hoc test , the genes encoding the proteins Lr8, Lrp13, LDLr-like, Lrp1-like, Lrp1b-like, Lrp3, Lrp4-like, Lrp5-like, Lrp6, Lrp10, Lrp11, Lrp12-like, and Lr11-like were expressed in the ovary of the SFE during early development (PV and EV stages)  of this gene were detected and designated as Lr8 + and Lr8- isoforms, depending on the presence or absence, respectively, of a putative O-linked sugar domain (Online Resource lr8 + and lr8- variants display a 5′-untranslated region (UTR) of 78 bp; ORFs of 2700 bp and 2595 bp, respectively; and a 3′-UTR of 399 bp. The corresponding Lr8 + and Lr8- deduced protein sequences have a length of 899 and 864 amino acids with predicted molecular weights of 99.04 kDa and 95.36 kDa, respectively. Similarly, even though it was functionally annotated as an Lrp4 member  were found, and their nucleotide and protein sequences were further examined. Based on sequence similarity to other vertebrate Vtgrs, the gene TRINITY_DN701_c1_g1 was designated as lr8 and lrp13 genes found among the predicted sequences of the European eel  and SFE lrp13 versus European eel LOC118237195) share a high percentage of identity, supporting their inferred orthology. While SFE Lr8 + and European eel VLDLr isoform X14 share 99.4%, SFE Lr8- and European eel VLDLr isoform X19 share 99.5%. SFE Lrp13 and European eel proLrp1-like share 95.9%.The most likely orthologues of the SFE el Table  were useel Table ” sectionlr8 and lrp13 genes share genomic synteny when compared with corresponding orthologues from different teleost fish  and lrp13 (LOC118237195) genomic arrangements for European eel were compared between zebrafish and Nile tilapia and zebrafish and medaka, respectively. For both genes, similar arrangements have been found in the species compared, showing with few exceptions, a retention of direction of the reading frames and the same adjacent genes. Most importantly, the SFE lr8 and lrp13 counterparts in the European eel showed similar conserved syntenic arrangements as their orthologues in other teleost fish. In all species compared, the lr8 gene is consistently surrounded by the genes secreted phosphoprotein 1 (spp1) and SH3-domain-binding protein 2 (sh3bp2) (upstream) and potassium channel subfamily V member 2a (kcnv2a) and pumilio RNA-binding family member 3 (pum3) (downstream). The adjacent genes downstream of lrp13 are slightly more variable between the species compared, but the upstream gene ring finger and CHY zinc finger domain containing 1 (rchy1) is present in each species.Both the ish Fig. . Using tlr8 + , lr8-, and lrp13, were highly expressed in the SFE ovary during the EV stage when compared with 17 somatic tissues  . On the contrary, if the data are normalised over actb, the relative expression of lr8 + , lr8-, and lrp13 remains stable during artificial maturation, showing no significant differences in transcript abundance between the experimental groups (p > 0.05 for both one-way ANOVA and t-test) , the genes encoding Ldlr-like, Lrp1-like, Lrp1b-like, Lrp3, Lrp4-like, Lrp5-like, Lrp6, Lrp10, Lrp11, Lrp12-like, and Lr11-like were found. Since disparate levels of gene expression were detected among them, in comparison to the highly expressed putative Vtgrs , the protein components of lipoprotein complexes determining also their assembly, structure, and transport , which is orthologous to the zebrafish and human LDLrs. Instead, it was linked to the LDLr-like protein in European eel (accession number XP_035254684.1/associated to the LOC118217039 gene), sharing 96.25% identity. In addition, the gene TRINITY_DN113818_c0_g1, also annotated as LDLr, was found to share 100% identity with the predicted SFE LDLr partial sequence . Similar to rainbow trout, both transcript variants differ in 105 bp encoding 35 amino acids representing a putative O-linked sugar domain, which presents low levels of homology and glycosylation when compared to the chicken Lr8 + and the human Lr8 + , this region would be encoded by exons 25 and 26, differing from chicken and human O-linked sugar domain-coding regions that only span one exon .Two genes encoding LDLr family members involved in Vtg binding in some teleost fish, known as Morone saxatilis) and white perch (7 + 1)  (10 + 1)  (7 + 1) , spotted gar (Lepisosteus oculatus), and elephant fish , and by Wang et al.  and a unique C-terminal LDLa repeat (+ 1) , the lr8 + variant was consistently detected between 1 and 2 Ct values lower than the lr8- variant (data not shown). Notably, higher expression could imply higher protein abundance, and thus, protein abundance data should clarify the significant contribution of each receptor to the final Vtgr pool. Whether this expression pattern found in A. australis, a basal teleost, reflects the ancestral state and the predominant expression of lr8- in ovary from other teleost fish represents the derived state requires further investigation. Additionally, since the only difference between isoforms is the presence of a putative O-linked sugar domain in Lr8 + , which in mammalian receptors are thought to confer cell surface stability protecting against shedding proteases  or remain stable Below is the link to the electronic supplementary material.


Pseudophanias Raffray includes 19 species distributed in the Oriental Region. Here, the first species from continental China, P.tianmuensis Yin & Zhao sp. nov., is described and illustrated from West Tianmu Mountain. This species can be readily separated from all congeners by the coarse vestiture, along with its male features. A previous key to Pseudophanias species of East and South Asia is modified to accommodate the new species.The genus  Pseudophanias Raffray is an easily recognizable group among the Oriental genera of the ant-loving beetle tribe Tmesiphorini (Staphylinidae: Pselaphinae). After removing two species from former Chandleriella Hlaváč , Shanghai, China. The label data of the material are quoted verbatim.The type material of the new species is deposited in the Insect Collection of Shanghai Normal University  was used for image stacking. All images were modified and grouped into plates using Adobe Photoshop CC 2020.Measurements were taken as follows: total body length was measured from the anterior margin of the clypeus to the apex of the abdomen; head length was measured from the anterior margin of the clypeus to the head base, excluding the occipital constriction; head width was measured across the eyes; the length of the pronotum was measured along the midline, the width of the pronotum equals pronotum’s maximum width; the length of the elytra was measured along the suture; the width of the elytra was measured as the maximum width across both elytra; the length of the abdomen is the length of the dorsally exposed part of the abdomen along its midline, the width is abdomen’s maximum width. Since one male paratype is completely disarticulated, only the holotype and the female paratype were measured. Abdominal tergites and sternites are numbered following Taxon classificationAnimaliaColeopteraStaphylinidaeYin & Zhaosp. nov.9C307C40-F6D7-554F-BBAF-D9AFC8979F96https://zoobank.org/4476E74C-644F-4667-8AF5-45C75D21638AHolotype: China: ♂, ‘China: Zhejiang, Linan District, West Tianmu Mountain, 1100 m, 23.viii.2014, Cryptomeria leaf, sift, Tie-Xiong Zhao leg., 浙江省临安市西天目山(此路不通)’ (in SNUC). Paratypes: China: 1 ♂ [completely disarticulated specimen], China: Zhejiang, Linan District, West Tianmu Mountain, 1150 m, 2.vii.2006, decomposing log, sift, Xiao-Bin Song leg., 浙江省临安市西天目山(停车场); 1 ♀, ‘China: Zhejiang, Linan District, West Tianmu Mountain, 1180 m, 18.viii.2019, decomposing log, sift, Zi-Wei Yin leg., 浙江省临安市西天目山(消防道)’ (both in SNUC).(3 exx.). Male: antennal clubs formed by apical four enlarged, cylindrical antennomeres. Aedeagus symmetric dorso-ventrally; median lobe C-shaped laterally; parameres thin and elongate, each with two long apical and one preapical seta.Body length 2.8–2.9 mm; dorsal vestiture coarsely punctate and densely setose. Head subrounded at base, with small vertexal and frontal foveae; antennomeres moniliform. Elytra with discal and lateral longitudinal ridges, areas between ridges impressed. Tergite 1 (IV) longer than 2 (V) at middle but clearly shorter than 2 and 3 (VI) combined. Male. Body ; antennomere 1 large, subcylindrical, 2–7 moniliform, of similar shape, 8, 9 and 10 each much larger than 7, subquadrate, 11 longest, slightly narrower than 10 and as long as 9 and 10 combined.Head Fig.  subroundPronotum Fig.  approximElytra much wider than long, length 0.96 mm, width 1.24 mm, truncate at bases; each elytron Fig.  with twoMesoventrite Fig.  with rouLegs elongate; femora coarsely punctate; each tarsus Fig.  with oneAbdomen widest at lateral margins of tergite 1 (IV), length 0.86 mm, width 1.15 mm. Tergite 1 (IV) slightly less than twice as long as 2 (V), with broad, deep basal sulcus and two basolateral foveae at lateral ends of sulcus, 2 and 3 (VI) successively shorter, lacking basal sulcus nor fovea, 4 (VII), much longer than 3, narrowed posteriorly, with one pair of basolateral foveae; tergites 1–3 each with pair of quadrangular and 4 with pair of triangular paratergites, 5 (VIII) broadly rounded, with one pair of small basolateral foveae, posterior margin roundly emarginate at middle. Sternite 2 (IV) longest, with densely setose basal sulcus and pair of basolateral foveae at lateral ends of sulcus, 3 (V) and 4 (VI) each short at middle, combined approximately as long as 5 (VII), 3–5 lacking sulcus nor fovea at base, 6 (VIII) transverse, posterior margin slightly protruding at middle, 7 (IX) composed of pair of membranous lamellae.Aedeagus Fig.  0.51 mm Female. Similar to male in external morphology  leaves, while the other two individuals were found inside decomposing wood with termites. Although indicated by a set of morphological traits such as the compact body segments and moniliform antennomeres, it is still inconclusive as to whether the beetle has an obligate association with social insects, pending further observations in the field or at the laboratory.The male holotype was collected by sifting East China: Zhejiang.The specific epithet refers to West Tianmu Mountain, the type locality of this species.Pseudophanias by To accommodate the new species, the key to East and South Asian species of


Polylactic acid (PLA) coatings containing different concentrations of copper ions were prepared on Ti discs. The antibacterial activity and biocompatibility of the copper ion-incorporated Ti implants were evaluated using Staphylococcus aureus (S. aureus), bone marrow mesenchymal stem cells (BMSCs) and animal models. In vitro, the coatings produced burst release of Cu2+ in 12 h, and inhibited S. aureus growth in a dose-dependent manner. The coatings prepared from PLA solutions containing 0.5 or 1.0 mg/mL reduced the viability and osteogenic differentiation of BMSCs, but these effects were negated after the coatings were immersed in culture medium for 6 h. Four weeks after implantation, the Cu-free K-wires challenged with S. aureus had persistent infection and inferior fracture healing to the other three groups, while Cu-coated wires had no evidence of infection. Furthermore, the Cu-coated wires placed in rabbits without S. aureus challenge showed superior fracture healing to the other three groups. These results suggest that PLA coatings containing Cu2+ may be an effective design for reducing IRI without adversely affecting adjacent bone healing.Implant-related infection (IRI) is a major problem in orthopedics. Copper (Cu) is an essential trace element with strong bactericidal activity and, thus, presents potential for reducing IRI. The present study explored a straightforward strategy for releasing Cu The lea, fungi) ,3. Altho, fungi) . Coated , fungi) . Howeverw/w) Cu, and demonstrated that the Cu2+ release for >35 d could inhibit oral bacteria survival and biofilm formation [2+ release from this alloy would proceed indefinitely or terminate after a certain period. From clinical considerations, long-lasting Cu2+ release is unnecessary and even undesirable. Furthermore, many other properties of the alloy need to be well characterized before it can be considered for implant production, such as mechanical properties , corrosion rate, and forming ability. Therefore, more studies have been focused on surface modification techniques. Wan et al. implanted Cu2+ into stainless steel and Ti by ion-beam implantation [S. aureus but also inferior corrosion resistance relative to the unmodified metals. Moreover, the span of Cu2+ release  from these modified metals remains unclear. Norambuena et al. coated TiCuO thin films containing 20–80% Cu (w/w) on Ti6Al4V by magnetron sputtering, and found that in vitro Cu release peaked at 24 h and was sustained for >28 d [S. epidermidis in a Cu-dose-dependent manner; the film containing 80% Cu moderately reduced the survival of human osteoblasts at 24 h, but the cytotoxicity disappeared on day 7. However, release for 28 d is probably unnecessary, and magnetron sputtering requires special high-vacuum facilities and complex technical controls, which may adversely affect the translation of the technique to the batch manufacturing of implants. Wang et al. deposited Cu2+ on Ti substrates precoated with polydopamine [2+ in several hours in vitro, and inhibited bacterial infection and promoted osteointegration in a rat metaphyseal implantation model. However, uncertainties and concerns exist for polydopamine, such as its unclear metabolites and metabolic pathways, the formation of side products during polymerization, and unintended organocatalytic actions in vivo [Inorganic antibacterial substances represent another possibility to control IPI . As oppoormation . Howeverantation ; the impor >28 d . The fildopamine . The Cu- in vivo ,16.2+ from a biocompatible polymer coating appears a simple, straightforward approach to reducing IPIs. Because of its simplicity, it may also be technically convenient to control the dose of Cu2+ loaded and its release kinetics. Expectedly, such coatings may be applied to osteosynthetic devices , which frequently face the risk of fracture-site infection, and does not require a permanent strong fixation in the host bone. However, this simple design has not been investigated previously. Therefore, we conducted a preliminary study to evaluate the feasibility of this approach. The aims of the present study were to: (1) characterize the relation between Cu2+ dose and antibacterial activity and in vitro biocompatibility; (2) test the effect of the coating in a small animal bone fracture model.Given the successful translation of gentamicin-loaded PLA coatings to clinics , the rel2, analytical grade, Kelong Chemical, Chengdu, China) was manually ground and sieved with a 300-mesh screen. The particles passing the screen were collected.Commercially pure Ti discs  were abraded to a 1200-grit finish with silicon carbide sandpaper, and sonicated sequentially in acetone, ethanol, and distilled water (10 min in each). They were etched in a mixture of 0.5 M nitric acid and 0.3 M hydrofluoric acid for 30 s, rinsed and sonicated in ultrapure water (18.2 MΩ.cm), and air-dried. Anhydrous copper chloride , and dried in a fume hood. The process was repeated 2 times. Five groups of samples were prepared: Group A (uncoated Ti discs), Group B (Ti discs coated in PDLLA/DCM solution containing no CuCl2), and Groups C-E .Poly-D, L-lactide  was dissolved in 10 mL of dichloromethane  with the assistance of sonication in an ice bath. Then, 0, 1, 5, or 10 mg of CuCl2+ release, samples from Groups C–E were each placed in 2 mL of Dulbecco’s modified Eagle medium  and incubated  for up to 7 d. At planned intervals, 1 mL of fluid was collected for Cu2+ quantitation by inductively coupled plasma mass spectrometry  and the original medium was replenished. Contact angle was measured by placing a 5-µL water drop on the sample surface and imaging it for drop shape analysis . Surface roughness was determined with a confocal laser microscope  and expressed as Ra.Coated discs were sputter-coated with gold and observed by scanning electron microscopy (FEI Quanta 200). For in vitro CuS. aureus . Briefly, 0.5 mL of liquid bacterial suspension  was pipetted into 5 mL of presterilized (121 °C × 15 min) liquid culture medium , 5.0 g sodium chloride, 1000 mL water) and thoroughly mixed. The resulting liquid was streaked on agar medium (100 mL of liquid medium and 14.0 g agar). After incubation (37 °C) for 24 h, one colony was picked, transferred into 4 mL of sterilized liquid medium, and maintained in a shaking incubator  for 24 h. The bacterial suspension was diluted to 105–107 CFU/mL for subsequent use. Then, Ti discs were sterilized by UV irradiation for 12 h and placed in 6-well dishes; 100 μL of the bacterial suspension was pipetted on each disc. After incubation of the dish (37 °C) for 20 min, warm (45 °C) agar medium was poured into each well, gently swirled to promote mixing, and allowed to cool to room temperature and solidify. After incubation (37 °C) for 12 h, the dish was visually examined for bacterial growth.Discs were also tested for the inhibition of For observing the bacterial morphology on the Ti discs, bacterial suspension (100 μL) was pipetted on discs and incubated (37 °C) for 6 h. They were fixed with 4 mL of 2.5% glutaraldehyde , dehydrated in ethanol solutions , and dried . Finally, they were studied by scanning electron microscopy (SEM).−1). For a Group X , the bacteria survival rate was calculated by: (CFU·ml−1 for Group X/CFU·ml−1 for Group B) × 100.UV-sterilized Ti discs were placed in 6-well dishes; 100 μL of the bacterial suspension was pipetted on each disc, and dishes were incubated (37 °C) for 20 min. Then, 4 mL of the liquid culture medium was added to each well, and the dish was placed in a shaking incubator . After 0, 0.5, 2, and 6 h, the suspension was collected and diluted up to 200-fold with sterile normal saline; 100 μL of the dilution was spread on agar. After incubation , the colonies formed were counted and converted to the original bacterial concentration (in CFU·ml2 loads in ~6 h (see Results), examination under the two scenarios allowed us to better understand the biocompatibility of the coatings both during and after their Cu2+ release. Briefly, rabbit BMSCs at third passage were purchased from the Cell Bank of Institute of Zoology, Chinese Academy of Sciences . BMSCs (1 × 105 cells/well) were seeded onto UV-sterilized Ti discs cultured in DMEM containing 10% fetal bovine serum. After incubation for 1 d, 3 d or 5 d, fluid in 3 wells of each group was discarded and replaced with 200 μL of DMEM supplemented with 10% (v/v) CCK-8 cell-counting reagent . After incubation for 2 h, the absorbance at 450 nm was measured on Thermo Fisher Varioskan Flash .Biocompatibility of the coated samples was studied under two scenarios: (1) direct seeding of bone marrow mesenchymal stem cells (BMSCs) on the samples (Scenario 1) and (2) pre-elution of each sample in 4 mL of DMEM for 6 h before seeding of BMSCs (Scenario 2). As previous experiments found that all coatings released their CuCl6 BMSCs were added to each well, and the plates were incubated for 14 d with daily renewal of the medium. Subsequently, RNA was isolated using Trizol (Invitrogen) and reverse-transcribed to cDNA using first-strand cDNA kits (Invitrogen) following the manufacturer’s instructions. Then, the cDNAs were amplified by qPCR with SYBR Green  on an iQ5 multicolor real-time PCR detection system (Bio-Rad Laboratories) using corresponding primers , alkaline phosphatase (ALP), osteocalcin (OCN), and osterix (OSX)) were measured by quantitative PCR. UV-sterilized Ti discs, with or without undergoing a 6 h pre-immersion in 4 mL of DMEM, were placed in six-well culture plates ; 1 × 10 primers . The PCRS. aureus suspension (In vitro experiments observed that (see Results) Group E offered the highest antibacterial activity and acceptable cytocompatibility after pre-elution for 6 h. Thus, an in vivo model was used to evaluate its anti-infective function. All procedures were approved by the Animal Ethics Committee of North Sichuan Medical College (NSMC(A)2020(10)). Twenty-four male New Zealand rabbits  were randomized to four groups (n = 6/group) . Ti6Al4Vspension  was careMoreover, the animal was regularly checked for food/water intake and physical activity. Rectal temperature was monitored regularly. Blood sample was collected weekly and assayed for white blood cell count (WBC), C-reactive protein (CRP), procalcitonin (PCT), and copper. Four weeks after operation, the animal was killed by intravenous pentobarbital overdose. After radiographic examinations, the operated tibia was dissected and stripped of soft tissues. The wires were removed from the proximal end of the tibia under aseptic conditions and smeared with a wet cotton ball. The cotton ball was gently rolled over agar medium, incubated (37 °C) for 24 h, and examined for colony formation. Fracture healing was evaluated by radiography and callus index . For hip-value < 0.05 was considered statistically significant.Data were analyzed by analysis of variance (ANOVA) followed by Tukey test . A 2 particle agglomeration was not observed in any group. The contact angle of uncoated Ti (Group A) was 67.5 ± 5.2° (n = 3). The contact angles of Groups B–E increased to 71.9 ± 1.6°, 78.9 ± 1.8°, 72.4 ± 0.8°, and 73.9 ± 8.0°   . The slil n = 3) . This tr2+ showed a dose-dependent trend of Group E > Group D > Group C. Generally, the cumulative release of Group C reached 162 ± 4 µg at the first 12 h, while Groups D and E reached 1055 ± 60 µg and 2615 ± 115 µg at ~12 h. After 7 d, the cumulative release for Group C was 186 ± 4 µg, while the results for Groups D and E were 1183 ± 66 µg and 2814 ± 78 µg. Throughout the experiment, no direct liberation of CuCl2 particles from the coating was visually observed, confirming that Cu2+ was released via dissolution of these particles, as may be expected from the high solubility of CuCl2 (~77 g/L). Moreover, all coatings survived immersion for several days without observable detachment or fragment liberation. The SEM study on day 2  to 76.3 ± 5.2% (Group C), 42.3 ± 2.8% (Group D), and 19.2 ± 6.0 (Group E). After shaking for 6 h, the survival rates were further reduced to 4.6 ± 0.4% (Group C), 1.6 ± 0.3% (Group D), and 0.3 ± 0.0% (Group E), compared with 102.6 ± 1.3% in Group A. At any time point studied, the difference between any Cu2+-loaded group and each of the other two groups was statistically significant .The quantitative bacterial inhibition test found that , compare2-dose-dependent decrease in viable BMSCs. This indicates that the lowest CuCl2 dose used (Group C) positively affected BMSC proliferation, whereas the other two doses  impaired it. When seeded after pre-elution in DMEM for 6 h , all groups supported BMSC proliferation between days 1 and 5 .Under Scenario 1, the expression levels c of the p = 0.99, p = 0.18). One animal in Group 3 developed diarrhea and died on day 14. All other animals survived during the study.Before operation, there was no significant difference among the groups in body weight (p = 0.001). WBCs of all groups also followed an n-shaped profile (2-loaded groups) were significantly higher than the other two groups. On day 28, all these indicators were renormalized (vs. day 0).The rectal temperatures of all groups varied in an n-shaped profile b; Groups profile c, peakin profile d varied  profile e was sig profile f also vaAfter incubation, the agar inoculated with smear prepared from wires of Group 3 formed white colonies . No wirep < 0.01; p = 0.0039); the callus index of Group 4 was slightly higher than that of Group 1, but the difference was not statistically significant (p = 0.377).On postmortem radiographs a, Group Histological examinations  revealed2+ release from the simple CuCl2-loaded PLA coating produced antibacterial and pro-osteogenic effects in vitro and in vivo. Earlier studies have identified multiple mechanisms underlying the antibacterial actions of Cu2+ [E. coli, Cu2+ was reported to induce the oxidation of unsaturated fatty acids in the cell membrane, leading to membrane damage and bacterial death [2+ was believed to kill S. aureus primarily by triggering DNA breakdown, without causing membrane disintegration [In this study, Cu of Cu2+ , and difal death ,21. In cegration . Neverth2+ are not understood but, as suggested by Rodrigues et al. [2+ (5 or 50 µM Cu–histidine complex) stimulated the osteoblastic differentiation of BMSCs isolated from postmenopausal women but reduced their proliferation and ALP expression. In comparison, we observed increased osteoblastic differentiation and viability of rabbit BMSCs exposed to Cu2+-release coatings. The different trends in viability/proliferation between the studies are unclear but may, at least, include differences in experimental conditions, such as cell characteristics and the concentration and chemistry of Cu compounds .The mechanisms responsible for the pro-osteogenic effect of Cus et al. , may inv+ and Cu2+ were compared, the minimally inhibitory concentrations (MICs) of Ag+ were approximately two orders of magnitude lower than Cu2+, for both E. coli and S. aureus [E. coli and S. aureus, with minor differences. The difference between ions and metals is explained by the higher corrosion resistance  of solid Ag (vs. Cu). Additionally, Cu is an essential element for humans and thus features a relatively higher degree of safety (LD50 for the rat: 359.6 mg/kg) compared with Ag. To our knowledge, no Cu-coated orthopedic implants have been commercialized; however, copper intrauterine devices have been widely used for contraception for decades, with satisfactory safety records [A number of metallic elements have been reported to have antibacterial activities, as recently reviewed . Of thes. aureus , indicat. aureus , and obs records , suggest2+ and ~88% of this was released in vitro within 24 h. In vitro, Group E released ~91% of the CuCl2 loaded within 48 h, followed by a negligible subsequent release  and determine the optimal ones. Additionally, the CuCl2 particles were relatively homogeneously embedded in the coating, without severe agglomeration . This makes a direct comparison of results between studies difficult. For example, Wan et al. reported inhibition rates of 31–100% from Cu2+-implanted Ti after 24 h exposure to S. aureus, depending on the dose implanted [2+ adsorption after 24 h, depending on the amount of Cu2+ adsorbed [−2 Cu. The surface reduced the S. aureus number by ~1 log at 2 h, and ~2 logs at 6 h; these results are relatively consistent with our findings. Clinically, the first 6 h after the implantation operation was suggested to be a “decisive period” for the occurrence of implant-related infections, because the introduced pathogens are quiescent during this period, making it a critical opportunity to kill pathogens near the implant [The coatings killed 68.6–96% of the mplanted . Wang etadsorbed . Interesadsorbed . They ga implant ,29. Ther2+ released from the coating. In all groups, the serum Cu level peaked on day 7; however, the in vivo release profile could not be estimated because of the paucity of data between days 1 and 7. Nast et al. [2–PLA coating , a burst release similar to the in vitro pattern  that peaked on day 7 and renormalized between days 21 and 28 . Becauset et al.  evaluatet et al. . Direct t et al.  and our  pattern  can be e2 loaded. This may be achieved by controlling the CuCl2 concentration in the PLA solution and the dip–withdraw speed.The transient elevation of serum Cu resulting from the Cu-release coating is inevitable, and its impact is unknown. The normal range of serum Cu in adults is ~750–1450 µg/L. In pregnant women, plasma copper was reported to be approximately one-fold higher than in unpregnant individuals . In our PLA is a hydrophobic polymer widely reported to generate reduced cell adhesion. Thus, the significantly increased viability of BMSCs inoculated on the blank PLA coating relative to those on Ti discs  need to be clarified. Furthermore, the minimal dose that generates a clinically significant antibacterial effect without introducing observable toxicity should be established. These will be considered in future studies.2+ were prepared on Ti by dip coating in solutions containing CuCl2 particles. All coatings produced rapid Cu2+ release during the first 6 h. When BMSCs were directly seeded on the coatings, the cell viability decreased Cu2+-dose-dependently compared with the Cu2+-free coating. When the cells were seeded after pre-elution in DMEM for 6 h, the dose-dependent pattern remained. However, in this scenario, the cell viability on the coating containing the lowest Cu2+ concentration became significantly higher than those on uncoated Ti and the Cu2+-free coating; the viability on that containing the highest Cu2+ concentration became similar to the uncoated Ti, suggesting the loss of cytotoxicity. Agar diffusion and plate counting tests confirmed all coatings to be effective at inhibiting S. aureus growth. The coating containing the highest Cu2+ concentration was prepared by K-wires and tested in a tibial internal fixation model challenged with S. aureus. The coating eradicated S. aureus infection and accelerated fracture-end union and callus formation. These results suggest the coating to be a feasible technique for reducing postoperative infection associated with orthopedic implants.PLA coatings containing three concentrations of Cu


Satureja hortensis L.), an important herb used for nutritional and herbal purposes in the Middle East. Three BS treatments, including bio-fertilizers, humic acid and foliar application of amino acid (AA), were implemented. Each treatment was applied to savory plants using three irrigation regimes . Foliar application of AA increased dry matter yield, essential oil (EO) content and EO yield by 22%, 31% and 57%, respectively. The greatest EO yields resulted from the moderate (FC75) and severe water deficit stress (FC50) treatments treated with AA. Primary EO constituents included carvacrol (39–43%), gamma-terpinene (27–37%), alpha-terpinene (4–7%) and p-cymene (2–5%). Foliar application of AA enhanced carvacrol, gamma-terpinene, alpha-terpinene and p-cymene content by 6%, 19%, 46% and 18%, respectively. Physiological characteristics were increased with increasing water shortage and application of AA. Moreover, the maximum activities of superoxide dismutase (3.17 unit mg−1 min−1), peroxidase (2.60 unit mg−1 min−1) and catalase (3.08 unit mg−1 min−1) were obtained from plants subjected to severe water deficit stress (FC50) and treated with AA. We conclude that foliar application of AA under water deficit stress conditions would improve EO quantity and quality in savory.Water deficit stress exposure frequently constrains plant and agri-food production globally. Biostimulants (BSs) can be considered a new tool in mitigating water deficit stress. This study aimed to understand how BSs influence water deficit stress perceived by savory plants ( Exposure to drought (here defined as lack of plant available water for a sustained period) inhibits plant growth and development by changing plant physiological and biochemical activities, resulting in productivity and quality losses8. Physiologically, drought can reduce photosynthetic rates, enhance phenology, truncate root growth and enhance senescence11. Exposure to enduring reactive oxygen species (ROS) such as superoxide anion, hydroxyl radical, hydrogen peroxide and singlet oxygen may cause oxidative damage and cell death13. Degrading chlorophyll due to excessive ROS also decreases the photosynthetic rate and plant productivity14. To deal with stress exposure, plants have evolved various physiological and biochemical mechanisms to decrease the harmful impacts of ROS. For example, producing osmolytes or osmoprotectant compounds such as proline, glycine betaine and phenolic compounds is a well-regulated biochemical mechanism for enhancing plant tolerance against drought stress conditions15.Arable environments across much of Iran often subject plants to water deficit stress, with the country having an average annual rainfall ranging between 224 and 275 mm16. In contrast, other studies have revealed more positive impacts of BS on mitigating drought stress than synthetic chemicals17. Recent research has shown that BS applied as organic and bio-fertilizers may enhance the nutrient use efficiency in plants subjected to drought stress compared to chemical fertilizer application18. Furthermore, BSs regulate the nutrient acquisition and secretion of phytohormones such as auxin, cytokinin, and gibberellin, promote nitrogen-fixing bacteria and enhance potassium and phosphorus solubilization, potentially improving soil structure and aggregation and increase plant productivity19.Bio-stimulants (BSs), including natural substances such as proline, glycine betaine and glutamic acid, could potentially be used to alleviate water stress effects. However, their effects on plant productivity and food quality are poorly studied under water deficit. Some studies have shown that using BS decreases the negative impacts of drought stress by improving nutrient uptake efficiency and tolerance to stress through enhanced osmotic adjustment20. One such MAP is summer savory (Satureja hortensis L.), an annual species belonging to the Lamiaceae family with a pleasant aroma and high EO content. Previous work has shown that the main EO constituents of savory include carvacrol, α-thujune, p-cymene, β-myrcene, α-pinene, β-terpinene, thymol, linalool, and β-caryophyllene22. Savory has also been used in traditional medicine as a painkiller to treat ailments related to coughing, indigestion, diarrhea, nausea and loss of appetite22. Also, the relatively high vitamin C and vitamin A content of savory may contribute to long-term health by boosting the natural state of the human immune system.The global community's interest in using medicinal and aromatic plants (MAP) as natural alternatives to chemical drugs against medical disorders is growing, primarily because such plants contain essential oils (EOs), vitamins and minerals. Essential oils are natural volatile aroma compounds synthesized by aromatic plants and have been used in the medicine, cosmetics and food industries24. Across plant species, management options and geographical location, drought stress has been reported to have substantial effects on biomass production26. We propose using natural compounds as bio-fertilizers would reduce production costs and potentially maintain plant production under drought stress. Hence, the present study aimed to investigate the effects of fertilizers (organic and bio-fertilizer) and BS compounds on the productivity and phytochemical properties of savory under drought-stress conditions.For many arid and semi-arid regions in which MAP are traditionally grown, farmer financial income may be threatened by potential low yields and product quality losses. So multiple avenues have been proposed for sustainably raising farmer income under droughtTwo field experiments were conducted in 2020 and 2021 at a farm in the Higher Education Center of Shahid Bakeri Miandoab, Iran. Weather  and soil physicochemical characteristics (taken at the 0–30 cm depth) of the experimental area are shown in Tables Pantoea agglomerans + Pseudomonas putida, K-solubilizing bacteria (KSB) Pseudomonas koreensis + Pseudomonas vancouverensis, and N-fixing bacteria (NFB) Azotobacter vinelandii] and foliar application of amino acid (AA).The experiments were established in a randomized complete block design (RCBD) with a split-plot arrangement and three replicates. Each subplot was composed of 10 rows of savory with an inter-row spacing of 40 cm. There was a 25 cm distance between plants within the rows. The subplots were 2.5 × 4 m. Plots and blocks were separated by a buffer space of 2 m and 3 m, respectively. Main plots were allocated to different irrigation regimes (3 levels), including full irrigation (FC100), moderate (FC75) and severe water stress (FC50), while sub-plots were assigned to different fertilizer sources (4 levels), including no fertilization (control), humic acid (HA), bio-fertilizer (B) bacteria [mixture of phosphate-solubilizing bacteria (PSB) 8 active bacteria per g) and dried in the shade for one h before seeding. Seeds were manually sown on May 4, 2020, and May 6, 2021. There was a 25 cm distance between plants within the rows. Weeds were regularly removed by hand when needed.The savory  seeds were obtained from Urmia University, Iran. For bio-fertilizer treatments, savory seeds were soaked in biofertilizer agents . The HA treatment (Humax® 95–WSG) contained 80% humic acid, 15% folic acid, and 12% potassium. Foliar application of the AA treatment  was performed twice in each growing season, once at the beginning of the shooting stage and once four weeks later at the concentration of 2 L per 1000 L of water. Spraying was done in the afternoon near sunset. Control plants were sprayed using distilled water.Each experimental unit utilised a total application rate of 200 mg of the HA powder for the HA treatment. One-third of this amount was dissolved in water and applied at seed sowing, while the remaining two-thirds were added to the water and used at the stem elongation and later at the flowering stage. The HA treatment was added in the respective experimental units with the first irrigation  was calculated by multiplying the dry matter yield by its EO percentage. Oil constituents were analyzed using a GC–MS (Agilent 7890/5975A), as suggested by Namazi et al.27.The savory EO was extracted using the water distillation method by a Clevenger apparatus. For this, 40 g of dried plant material (leaves + flowers) was added to the balloon of the essential oil extraction machine and 500 ml of distilled water was added. The essential oil extraction operation was carried out for three hours. The extracted EO was dried with anhydrous sodium sulfate and kept at 4 °C for further chemical analysis. EO yield  and dry weight of leaves (by drying the leaves in an oven at 70 °C for 48 h), respectively.At the flower initiation stage, the leaves' relative water content (RWC) was determined according to Levitt29:In the flowering stage, 0.5 g fresh savory leaves were homogenized in 10 mL of 80% acetone and centrifuged at 12,000 rpm for 15 min. After that, the absorbance was read at 663 nm, 645 nm and 470 nm by a UV spectrophotometer . The content of chlorophyll a, b and carotenoids was calculated based on the following equationsa, b and Car represent chlorophyll a, b and carotenoid concentrations, respectively. Parameters A646.8, A663.2 and A470 represent the absorbance measured at 646.8, 663.2 and 470 nm using a spectrophotometer.In these equations, Chl 30. First, 0.5 g of fresh leaves were homogenized with 10 mL of 3% sulfosalicylic acid and centrifuged at 12,000g for 10 min at 4 °C. Then, 2 mL of each sample was boiled with 2 mL of acid-ninhydrin + 2 mL of glacial acetic acid for 40 min. The reaction mixture was extracted with 4 mL of toluene and mixed with a vortex for 20 s. Finally, the absorbance was measured spectrophotometrically at 520 nm compared to the clean toluene. The soluble sugar concentration (SSC) was determined using the phenol and sulfuric acid method. Briefly, 0.5 g of savory fresh leaves were homogenized with ethanol and mixed with 98% sulfuric acid and 5% phenol and absorbance were determined with a spectrophotometer at 485 nm31.Proline concentration was determined following the method described by Bates et al.32, Kar and Feierabend33 and Aebi34, respectively. All enzyme activity was reported in units of mg g−1 min−1.First, to assess the antioxidant enzyme activity across different irrigation levels and fertilizer sources, 0.1 g of savory leaves (frozen in liquid nitrogen) were homogenized in 2 mL sodium phosphate buffer and centrifuged at 15,000 rpm at 4 °C for 20 min. Then, the supernatant was used to determine antioxidant enzyme activity. The superoxide dismutase (SOD), peroxidase (POX) and catalase (CAT) activities were measured according to the procedures reported by Dhindsa et al.35.The 2,2-Diphenyl-1-Picrylhydrazyl (DPPH) assay was utilized to determine the plant's antioxidant activity. For this purpose, 3.8 mg of DPPH was dissolved in 25 mL MeOH. Next, 100 µL of the mixture was added into individual wells of 96-well plates and mixed with 85 µL MeOH and 15 µL of savory sample extract. Finally, the microplates were incubated in the dark and at room temperature for 30 min, and the absorbance was read at 515 nmA combined analysis of variance (ANOVA) and the comparison of the means with Duncan’s multiple range test at a 5% probability level were performed using the SAS software version 9.4 . The irrigation levels and fertilizer sources were considered fixed effects, while block, year, and interactions were considered random. Correlation coefficients among all variables under analysis were also calculated using the SAS software version 9.4 . The heatmap clusters were created in Ward’s method using heatmap.2 functions in gplots R-package.The analysis of variance in Tables The maximum plant height (68.7 cm) was obtained from FC100 treatment fertilized with AA followed by HA application (67.8 cm). However, the minimum plant height (46.8 cm) was achieved from FC50 treatment without fertilization (control). Compared with FC100, plant height decreased in FC75 and FC50 by 10% and 20%, respectively. In addition, plant height was enhanced by 10%, 7% and 14% due to HA, B and AA application  was obtained from FC100 treatment fertilized with AA. In contrast, the minimum lateral branch number (12.2) was related to FC50 treatment without fertilization (control). Lateral branch numbers decreased in FC75 and FC50 by 10% and 22%, respectively. Moreover, lateral branch numbers increased by 25%, 19% and 35% due to HA, B and AA applications  was obtained from FC100 treatment fertilized with AA, followed by HA application (40.7 cm). However, the lowest canopy diameter (20.6 cm) was achieved from FC50 without fertilization (control). Compared with FC100, the canopy diameter decreased by 20% and 34% in FC75 and FC50 water stress. In addition, fertilization with HA, B and AA increased canopy diameter by 17%, 11% and 24%, respectively  and minimum (2767 kg ha−1) DMY was achieved from FC100 treatment fertilized with AA and FC100 without fertilization. The DMY decreased by increasing water deficit stress severity. Compared to the FC100 treatment, DMY decreased in FC75 and FC50 treatments by 11% and 33%, respectively. In addition, fertilization with HA, B and AA increased canopy diameter by 14%, 11% and 22%, respectively  in the FC50 treatment treated with AA. However, the lowest EO content (0.72%) was recorded in no-stress conditions (FC100) without fertilization. Across irrigation regimes, EO content increased by 21% and 45% in moderate (FC75) and severe (FC50) water deficit stress treatments. Also, the EO content increased by 19%, 18% and 31% due to HA, B and AA applications, respectively . The highest content of carvacrol and gamma-terpinene was obtained in FC75 and FC50 treated with AA. Also, the lowest content of the above-mentioned compositions was achieved in normal irrigation conditions without fertilization (control treatment). On average, the content of carvacrol and gamma-terpinene increased by 4%, 14% due to moderate (FC75) and 4%, 19% due to severe (FC50) water stress treatments. In addition, the application of AA enhanced carvacrol, gamma-terpinene, alpha-terpinene and p-cymene content by 6%, 19%, 46% and 18%, respectively  and gamma-terpinene (26.82–37.39%), alpha-terpinene (4.01–6.88%) and a (2.3 mg g−1 fresh weight) and b (1.2 mg g−1 fresh weight) concentrations were found under no-stress conditions (FC100) fertilized with AA. The lowest values (1.02 mg g−1 fresh weight for chlorophyll a and 0.6 mg g−1 fresh weight for chlorophyll b) were recorded under severe water stress (FC50) without fertilization treatment. Compared with FC100, chlorophyll a and b were reduced by 23% and 13% in moderate (FC75) and 32% and 29% in severe water stress (FC50), respectively. In addition, fertilization with HA, B and AA enhanced chlorophyll a concentration by 23%, 21% and 35.1% and chlorophyll b concentration by 23%, 17% and 33%, respectively  was measured in FC50 treated with AA. Compared with FC100, the carotenoid concentration was enhanced by 22 and 51% in FC75 and FC50 water stress. Also, fertilization with HA, B and AA enhanced carotenoid concentration by 31%, 27% and 49%, respectively, compared with the control  and minimum (46%) RWC percentage values were related to normal irrigation conditions (FC100) treated with AA and severe water deficit (FC50) without fertilization, respectively. The RWC decreased with increasing water stress levels. Compared with FC100, RWC content was reduced by 15% and 29% in moderate (FC75) and severe water stress (FC50), respectively. Additionally, fertilization with HA, B and AA increased RWC by 11%, 10% and 16%, respectively, compared with the control Fig. B.−1 fresh weight) was obtained from FC50 fertilized with AA, followed by HA application (4.2 mg g−1 fresh weight). The SSC was enhanced with increasing water stress levels. Compared with FC100, the SSC increased by 64 and 128% in moderate (FC75) and severe water stress (FC50). Additionally, fertilization with HA, B and AA increased the SSC by 26%, 23% and 30%, respectively, compared with the control  and severe water stress (FC50). Additionally, fertilization with HA, B and AA increased proline concentration by 21%, 18% and 29%, respectively, compared with the control Fig. D.−1 min−1), POX (2.6 unit mg−1 min−1) and CAT (3.08 unit mg−1 min−1) was recorded in severe water deficit (FC50) treated with AA. Increasing water stress levels enhanced the SOD, POX, and CAT activity. Compared with FC100, the activity of antioxidant enzymes was enhanced by 65%, 24 and 45.3% in FC75 and 137.8, 47.5 and 77.7% in FC50, respectively. Moreover, fertilization with HA, B and AA increased SOD activity by 46%, 36% and 54%, POX activity by 20%, 12% and 36% and CAT activity by 26%, 22% and 40%, respectively, when compared with no fertilization  was related to severe water stress (FC50) treated with AA. Compared with FC100, the antioxidant activity was enhanced by 3.5% and 6.1% in moderate and severe water stress. Also, fertilization with HA, B and AA enhanced the antioxidant activity by 2.5%, 2.2%, and 3.8%, respectively, compared with the control Fig. D.b (r = 0.97**), RWC (r = 0.96**), canopy diameter (r = 0.96**), lateral branch (r = 0.91**), chlorophyll a (r = 0.91**), soluble sugars content (r = 0.71*), and SOD (r = 0.60*) while essential oil content showed significant and positive correlations with carotenoid content (r = 0.99**), proline concentration (r = 0.99**), antioxidant activity (r = 0.98**), soluble sugars content (r = 0.95**), and enzymes activity including CAT (r = 0.98**), SOD (r = 0.98**), and POX (r = 0.96**) , chlorophyll C r = 0.9**, canopa and b, and relative water content; (2) carotenoid and soluble sugars contents, proline, POX, SOD, and CAT activities, antioxidant activity and essential oil content. All the classified variables in the first group decreased significantly with increasing water stress. In contrast, the classified variables in the second group significantly increased by increasing water stress  essential oil yield, dry matter yield, plant height, canopy diameter, lateral branch, chlorophyll ess Fig. .Figure 5p-cymene, carvacrol, and gamma-terpinene were observed under moderate and severe water stress. The heatmap clustering shows moderate and severe water stress accompanied by amino acid and bacterial biofertilizers have the highest impact on essential oil metabolism  and bacterial (FC75 + B) biofertilizers and severe water stress along with amino acid (FC50 + AA), bacterial (FC50 + B), and humic acid (FC50 + HA) biofertilizers Fig. . In contism Fig. .Figure 636. Water limitations affect cell differentiation, division, elongation and, finally, a reduction in photosynthesis rate and dry matter production37. Daszkowska-Golec et al.38 noted that water stress conditions reduce the entry of carbon dioxide into the closed stomata due to dehydration, reducing photosynthetic materials supply and plant productivity. Similarly, Amani Machiani et al.39 noted that thyme (Thymus Vulgaris L.) dry matter yield decreased due to moderate and severe drought stress by 27% and 40%, respectively. Also, under drought stress conditions, AA application positively affected the plant yield components and dry matter yield and also decreased the negative impacts of drought stress40. The proposed mechanism for AA improving plant productivity is by improving nutrient use efficiency and increasing photosynthetic rates, improving growth41.Our results show that plant height, lateral branches, canopy diameter, and dry matter yield all decreased due to moderate (FC75) and severe (FC50) water stress. Under water deficit stress conditions, nutrient absorption decreased around the root zone due to lower mobility and rate of mineral diffusion42. Under drought stress, the massive supply of NADPH + H+ in plant cells adversely affects the photosynthesis cycle. Therefore, EOs compounds' productivity using NADPH + H + stored in plant cells balances the NADP+/NADPH+ H+ ratio and improves the photosynthesis cycle43. Among different fertilizer treatments, foliar application of AA enormously improved EO quantity and quality by increasing the main EO constituents, including carvacrol, gamma-terpinene, alpha-terpinene and p-cymene. Most EO constituents, such as terpenes composed of an isoprene structure. The isoprene productivity depends on nutrient availability and biomolecules such as ATP, NADPH, and acetyl-CoA44. The application of AA enhanced essential oil productivity and major EO constituents by increasing the availability of intermediate compounds, including ATP and NADPH. Similarly, Talaat et al.45 noted that applying amino acid bioregulators improves the essential oil quantity and quality of Khella (Ammi visnaga L.).The results showed moderate and severe water stress conditions increased the EO content, yield, and major constituents, including carvacrol and gamma-terpinene. The production of secondary metabolites such as EOs, alkaloids and flavonoids in medicinal and aromatic plants is considered one of the defense mechanisms to increase plant resistance under stressful conditions40. These conditions increase lipid peroxidation, leading to chlorophyll and photosynthesis pigment degradation46. Similar results were found by Rezaei-Chiyaneh et al.1 study, who noted that the chlorophyll concentration in black cumin (Nigella sativa L.) significantly decreased under drought stress conditions. In contrast, carotenoid content was increased under drought stress conditions due to the higher susceptibility of these compounds to oxidative stress. Therefore, an increase in carotenoid content, as a non-enzymatic antioxidant, under water deficit stress conditions plays a vital role in improving plant tolerance and decreasing the detrimental effects of oxidative stress48. In addition, chlorophyll (a and b) and carotenoid content increased because of AA foliar application. AA, as an antistress compound, enhances the activity of antioxidant enzymes and proline concentration, leading to decreased ROS harmful effects in the degradation of chlorophylls and carotenoid membranes. Noroozlo et al.49 stated that the higher chlorophyll and carotenoid content in the lettuce plants could be due to the reduction in chlorophyll degradation and the stimulating impacts of AAs on chlorophyll and carotenoid biosynthesis.In the present study, seedlings' chlorophyll content (a and b) decreased under moderate and severe water stress conditions. The reduction in the photosynthesis pigments under water deficit stress conditions may be caused by chloroplast breakdown and inhibition of its biosynthesis by enhancing the synthesis of reactive oxygen species (ROS), degradation of chlorophyll precursor, and up-regulation chlorophyllase activity50 noted that proline-based amino acid foliar application and increased proline content would increase osmoprotectants' productivity. Therefore, enhancing proline content through foliar application decreased their negative impacts by stabilizing the membranes, maintaining the osmotic balance and cell turgor, preventing electrolyte leakage, and maintaining the ROS levels in normal ranges (detoxification of ROS)51.The results showed that the maximum proline content was achieved from severe water stress (FC50) treatment with AA foliar application. Under drought-stress conditions, plants can alter water relations to maintain cellular functions. For example, plants exhibit osmotic adjustment by synthesizing and accumulating compatible solutes such as free amino acids, sugars, and proline. Also, Hanif et al.52. The results revealed that RWC was decreased under moderate and severe water stress conditions due to increased water loss from savory leaves through transpiration and decreasing water uptake from roots. In this situation, RWC decreased due to reduced leaf water potential47. In addition, the AA application strongly increased RWC content in the seedlings. Under drought stress conditions, reducing osmotic pressure by increasing osmotic adjustment such as SSC and proline in the cells helps maintain leaves’ turgor pressure. The application of AA enhanced RWC content under stressful conditions through increasing osmotic adjustment and membrane integrity53.RWC is commonly used to quantify plant water status in terms of biochemical and physiological consequences of water stress in plant cells2O2). The higher antioxidant enzyme activity reduces ROS compounds in the cells, reducing membrane damage and balancing the performance of cellular activities such as photosynthesis54. Also, increased antioxidant activity due to AA foliar application could be attributed to higher nitrogen metabolizing enzyme activity because of amino acid availability16. In a study, Shafiq et al.17 reported that a higher proline concentration under drought stress conditions protects macromolecules and enzymes from oxidative damage and increases the activities of the enzymes. Therefore, it can be concluded that AA foliar application can enhance plant protection against drought stress by enhancing organic osmolytes such as proline, SSC, and antioxidant enzyme activity.CAT, SOD and POX activity were enhanced under severe water stress treated with AA. Under water stress conditions, the antioxidant enzyme activity plays a vital role in eliminating ROS compounds such as superoxide and hydrogen peroxide (Hp-cymene. We conclude that the foliar application of amino acid could effectively increase plant tolerance against water deficit stress while improving the essential oil quantity and quality of medicinal and aromatic plants.Our study revealed that water stress negatively affects savory seedlings, reducing dry matter production by 11% and 33% under moderate and severe water deficit stress. However, foliar application of amino acid enhanced dry matter yield, essential oil content and savory yield by 22%, 31% and 57%, compared with the treatment with no fertilization. Foliar application of amino acid improved savory essential oil quality by increasing essential oil components, including carvacrol, gamma-terpinene, alpha-terpinene and


Trialkyl and triaryl phosphines are important classes of ligands in the field of catalysis and materials research. The wide usability of these low-valent phosphines has led to the design and development of new synthesis routes for a variety of phosphines. In the present work, we report the synthesis and characterization of some mixed arylalkyl tertiary phosphines via the Grignard approach. A new asymmetric phosphine is characterized extensively by multi-spectroscopic techniques. IR and UV–Vis spectra of some selected compounds are also compared and discussed. Density functional theory (DFT)-calculated results support the formation of the new compounds. Low-valent chemically functional phosphines or P(III) compounds are an important building block in organic, main group, and organometallic chemistry. They have found immense applications in the area of catalysis , as well3. Frisch and Lyons [3 or PCl5 with a corresponding Grignard reagent.Among other methods, symmetric/asymmetric alkyl and aryl phosphines are mainly obtained via the cross-coupling reaction between an organometallic compound and halogenated phosphines or phosphanes under controlled conditions . Moreovend Lyons  reportedDespite these advances, the development of efficient methods to construct asymmetric P-C remains a challenge. Therefore, application of milder methods using air-stable and economical starting materials would be most desirable. From the materials science point of view, it is desirable to construct new phosphines with balanced electronic interactions and steric properties. We report herein the synthesis and characterization of a series of mixed arylalkyl tertiary phosphines via the Grignard approach.2) was obtained by treating methyl magnesium chloride  with chlorodiphenylphosphine  in THF at −10 °C  and two-dimensional NMR spectroscopic techniques (vide infra and −1 (asym. def.) and 1346–1255 cm−1 (sym. def.), along with P-Ar stretching at around 1130–1090 cm−1, suggested the formation of the products. Moreover, the lack of strong peaks at around 1140–1320 cm−1 excluded the formation of oxidation products. Proton-decoupled 31P(1H) NMR spectra showed resonance between ~−7 ppm and −27 ppm (31P NMR spectra). In the past, one- or multi-step synthesis of compounds (2) [3) [4) [5) [7) [9) [10) [11) [2) with a 74% yield in a reaction conducted at −35 °C, while we found a yield of 66% at a moderate temperature. Compound (5) was reported using LiPPh2 and alkyl bromide, but no yield was mentioned. We also isolated compound (7) with a yield similar to that obtained in an earlier report [7) using metal-catalyzed [The molecular structure of the compounds was confirmed by FTIR, along with one-  ,17 (3) [ (2) [3) , (4) [19 [5) [7) , (9) [22[9) [10) , and (1110) [11)  has been10) [11)  reportedr report . Recentlatalyzed  of 4-bro1)  for the synthesis of asymmetric phosphine  to the solution of (8) in THF at −10 °C afforded diisopropylphenylphosphine (9) at a 52% yield. The other derivatives were prepared following a similar procedure, using 2.5 equivalents of the respective Grignard reagents with moderate yields  with a good yield. It is worth mentioning that the aryl Grignard reagents gave better conversion, as in the previous examples. However, when the alkyl Grignard reagents  were used, products in very low amounts with inseparable mixtures were obtained. Huang et al. [11) via an in-situ-generated Grignard reagent at −78 °C. Compared to the reported method (yield = 52%), our method is less time-consuming and gives a higher yield (76%).After successful investigation of the Grignard reaction on  at −10 °C, one equivalent of a given R1-MgX was added, followed by the addition of one equivalent of another R2-MgX , (7), and (12), albeit with low yields (16–33%). Too fast or too slow addition of the reagents led to the formation of mixtures in greater amounts that were not separable. However, the reasons for the low yield could be manifold; we assume that factors such as competitive reaction between the different Grignard reagents, formation of cross-products, difficult separation (due to similar polarity of the products), etc., were mainly responsible. Currently, we are investigating and optimizing the conditions to develop a transition-metal-based catalyst-free protocol for the synthesis of asymmetric phosphines in one pot.Intrigued by the abovementioned results, attempts have also been made to obtain mixed phosphines under one-pot conditions . In a ty10) by the reduction of its oxide. However, the exact source and analytical data were not given, although there was nothing remarkable about its preparation. We conducted a full structural analysis of this compound, including multidimensional NMR spectroscopy. The 1H-NMR spectrum of compound (10) in CDCl3 shows 5 aromatic protons as a multiplet between δ 7.51–7.30 ppm, a multiplet at δ 1.74–1.62 ppm for 4 P-CH2 protons, one dpd at δ 1.58 ppm for 2 CH protons, δ 1.35–1.16 ppm for 4 P-CH2-CH2 protons, and one doublet at δ 0.87 ppm for 12 protons  is clearly downfield, so the assignment is free of doubt. In 2017, Kovács et al.  reported protons . Compare1H-1H COSY results , δ 34.89–34.79 and δ 25.78–25.70 (methylene), δ 29.36–29.27 ppm (methine), and δ 22.28–22.20 ppm (methyl) were observed  and a commercially available tributylphosphine (PBu3). Due to their structural similarity, the IR spectra were found to be overlapping in most of the region, except for those characteristics relating to aryl phosphines. The peak at 1433 cm−1 in (10) and 1457 cm−1 in PBu3 can be assigned to the deformation bands present in P-CH2-R-type phosphines. In addition, compound (10) shows P-Ar stretching at 1150 cm−1, aromatic C-H stretching at 3050 cm−1, and overtones which are absent in PBu3. 5), diisopentyl(phenyl)phosphine (10), and a commercially available tributylphosphine (PBu3) were recorded in acetonitrile  shows maxima at 220 nm, along with a low-energy broad band in the region of ~260–270 nm with vibronic features. Similar, but slightly redshifted peaks can be seen in (5), owing to the presence of two phenyl groups. Note that (5) and (10) bear the features of the PBu3, methyldiphenyl phosphine, and triphenylphosphine [3 can be attributed to n → σ* transition, the absorption in the latter compounds  is due to n → π* transitions. This assignment is consistent with the theoretical calculations (vide infra) [The UV–Vis spectra of isopentyldiphenyl phosphine (onitrile . Bands chosphine . While te infra) .5), (10), and tributylphosphine (PBu3), obtained by B3LYP calculations, are shown in 5) has one while (10) has two phenyl groups, no significant differences in the structure or the highest occupied molecular orbital (HOMO)–lowest unoccupied molecular orbital (LUMO) gap were noted  is an important tool to underpin many structural features and photophysical processes ,33,34,35re noted , inset.5) and (10) and an n → σ* transition in PBu3, from HOMO to LUMO  of (5) and (10). HOMO–LUMO gaps for (5), (10), and PBu3 were found to be 3.5, 3.5, and 5.8 eV, respectively, consistent with previous works on phosphorus-based compounds [Overlaid experimentally and theoretically calculated UV spectra at the DFT level are depicted in ompounds ,37. 3. The 1H and 13C NMR spectra were referenced to solvent resonances, and the 31P NMR spectra were referenced to an external phosphoric acid standard (85% H3PO4). Splitting patterns are designated as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet. Chemical shift values are given in ppm. IR spectra were recorded using a Cary 630 FTIR spectrometer. Absorption spectra were recorded on a Varian Cary 50 UV–Visible spectrophotometer in a 1 cm quartz cuvette. Liquid chromatography/mass spectrometry (LC/MS) was performed on an Agilent LC/MS instrument (1260 Infinity II) equipped with a reverse-phase C18 column , electrospray (ESI) mass spectrometry detector, and photodiode array detector. The ground state of the phosphine compounds was calculated using the Gaussian 2009 program package [All reactions were conducted under an inert atmosphere using standard Schlenk techniques. Unless stated otherwise, all chemicals were obtained from Sigma-Aldrich and used without further purification. NMR spectra were recorded on Bruker MM-250 and WM-400 spectrometers in CDCl package . The Bec package ,40 metho package  basis se package  was perfMethod A: To the stirred solution of chlorodiphenylphosphine (1.0 equiv.) in THF (15–20 mL), a commercially available molar solution of alkyl/aryl magnesium halide (1.1 equiv.) was added dropwise at −10 °C and stirred for 12 h. The reaction was quenched by adding half-saturated NH4Cl solution (prepared using deoxygenated water), diluted with ethyl acetate (10–20 mL), and stirred for 15 min. The organic layer was separated, and the aqueous layer was washed with ethyl acetate (3 × 15 mL). The combined organic layer was dried over anhydrous Na2SO4 and concentrated under vacuum. The crude product was purified by flash column chromatography.Method B: To the stirred solution of dichlorophenylphosphine (1.0 equiv.) in THF (15 mL), a commercially available molar solution of alkyl/aryl magnesium halide (2.5 equiv.) was added dropwise at −10 °C and stirred for 12 h. The reaction was quenched by adding half-saturated NH4Cl solution (prepared using deoxygenated water), diluted with ethyl acetate (10–20 mL), and stirred for 15 min. The organic layer was separated, and the aqueous layer was washed with ethyl acetate (3 × 15 mL). The combined organic layer was dried over anhydrous Na2SO4 and concentrated under vacuum. The crude product was purified by flash column chromatography.Method C: To the stirred solution of dichlorophenylphosphine (1.0 equiv.) in THF (10 mL) at −10 °C, alkyl/aryl magnesium halide (1.0 equiv.) was added dropwise, and the mixture was stirred for 6 h. Following this, a second Grignard reagent (1.0 equiv.) was added dropwise to the reaction mixture at the same temperature, and it was stirred overnight. The reaction was quenched by adding half-saturated NH4Cl solution (prepared using deoxygenated water), diluted with ethyl acetate (10–20 mL), and stirred for 15 min. The organic layer was separated, and the aqueous layer was washed with ethyl acetate (3 × 15 mL). The combined organic layer was dried over anhydrous Na2SO4 and concentrated under vacuum. The crude product was purified by flash column chromatography.In conclusion, a variety of functionalized symmetric and asymmetric phosphines were prepared via the operationally simple phosphination of Grignard reagents in THF. However, attempts to prepare asymmetric phosphines under one-pot conditions were not very successful, and some other phosphines were obtained with yields better than those previously reported. Furthermore, we also performed extensive structural characterizations—such as multidimensional NMR—to establish the chemical structures of the products. DFT calculations also supported the spectral data of the studied compounds.