Molecular cloning and expression characterization of flavonol synthase genes in peanut (Arachis hypogaea)

Flavonol is an important functional bioactive substance in peanut seeds, and plays important roles responding to abiotic stress. The flavonol content is closely related to the activity and regulation of gene expression patterns of flavonol synthase (FLS). In this study, eight FLS genes, AhFLSs were cloned and their expression characterization in different peanut organ and seedling under different abiotic stress were conducted. The results showed that the expressions levels of AhFLSs were differed in all assayed peanut organs and seedlings under abiotic stress treatments. Expression levels of AhFLS2, AhFLS3, AhFLS4, and AhFLS6 were higher than those of other AhFLSs. The flavonol contents of peanut organs and seedlings under different abiotic stress were also determined using high performance liquid chromatography (HPLC). Dried mature peanut seeds were the organ tissue with the highest flavonol content, and flavonol content increased with seed development. Under abiotic stress treatments, the types of flavonols induced differed among stress treatments. Correlation analysis results suggested that eight AhFLS genes may have different functions in peanut. Moreover, changes in the expression of the eight genes appear to has substrate preference. These results can lay the foundation for the study of improving nutritional value of peanut seed and resistance of peanut plant.

In this study, the identification and characteristic of AhFLS genes under different organ and abiotic stress treatments were carried out. The contents of three flavonol components in peanut organs, seeds at different developmental stages, and seedlings under drought, salt stress, low temperature, high temperature, UV, heavy metals, and MeJA treatments were also conducted. The expression patterns of AhFLS in the regulation of peanut flavonol content was suggested, which provides a theoretical basis for the improvement of peanut quality through enhancements of peanut flavonol content and for future research on the functional mechanisms by which flavonols enhance abiotic stress resistance.

Results
Identification and characteristic of AhFLS genes. Based on conserved FLS sequences, such as those in Arabidopsis thaliana, soybean, and alfalfa, and Peanutbase data (https ://www.peanu tbase .org/), eight cDNAs were identified and cloned. There was a highly similar sequence on the A02 chromosome of the AA genome (Arachis duranensis V14167). There were four and three highly similar sequences in the A05 and A10 clusters, respectively, as well. There were two highly similar sequences on the corresponding B10 chromosomes of the BB genome (Arachis ipaensis K30076) ( Table 1). The results of the sequence alignment indicated that the above eight AhFLS coding genes had two sequences with high similarity within the 150-300 base pair (bp) and 600-800 bp fragments. In this research, the aforementioned AhFLS genes were named AhFLS1 through AhFLS8.
The peptide chains encoded by AhFLS1 through AhFLS8 are between 333 amino acids (aa) and 361 aa in length (Table S1). The secondary structure of each protein contains α-helix, β-sheet, and random coil regions. Among them, AhFLS4 has the most α-helix regions, while AhFLS1 and AhFLS6 have the most β-sheets (Fig. S1). AhFLS3 and AhFLS4 contain transmembrane region signals (Fig. S2), but none of them are transmembrane proteins. Analysis of protein acidity and alkalinity showed that AhFLS3 is a typical basic protein (Table S1). In addition, each AhFLS protein has a distinct hydrophilic region, while the hydrophobic region is not obvious (Fig. S3). Signal P online analysis showed that the inferred AhFLS proteins did not contain significant signal peptides, but contained two conserved domains of FLS, including N-terminal DIOX_N (located at 96-133 aa) and C-terminal 2OG-FeII_Oxy (located at 93-101 aa) (Fig. 1). These structural features indicate that the tested AhFLS proteins participate in the biochemical metabolism of flavonols in peanut plants.
The gene tree of FLS sequences from Arachis hypogaea, Arabidopsis thaliana, Medicago truncatula, Zea mays, and Glycine max (Fig. 2) revealed that AhFLS2, AhFLS4 and AhFLS5 had the highest homology with other plant species' FLS proteins. The sequences identity with MtrFLS4 was 88%. The homology of AhFLS6 to MtrFLS3 was 100%. AhFLS8 was also highly homologous to the MtrFLS1 and MtrFLS2 sequences (100% identity). Studies have shown that there is high sequence divergence between AhFLS3 and the other seven AhFLS proteins, indicating that the gene family members have been under divergent evolutionary pressures.

Expression pattern of AhFLS genes among different peanut organs. The expression level of
AhFLS genes in each organ of the peanut plant are shown in Fig. 3. Among AhFLS genes, AhFLS2 expression was highest in roots, stems, leaves, and flowers. During the reproductive growth period from R5 to R8, the expression of AhFLS4 was higher than that of the other AhFLS genes, with the highest expression occurring when seed www.nature.com/scientificreports/ coat color was formed during the R7 stage (Fig. 4). The expression of this gene decreased rapidly and decreased throughout the maturation and drying processes. The expression of AhFLS6 at the seed development stage was higher than that during the drying period.
AhFLS expression patterns under abiotic stress. The varying abundance of AhFLSs transcripts in seedlings under different abiotic stresses is shown in Fig. 3. Compared with the test genes, the expression levels of AhFLS2, AhFLS3, AhFLS4, and AhFLS6 were higher than those of the other AhFLS genes under various treatments. AhFLSs expression levels decreased over time under the 50-μM MeJA and UV treatments. AhFLS2 expression increased after 3 h under the CdCl 2 treatment and then decreased thereafter. Under the 42 °C, 20% PEG6000, and NaCl treatments, AhFLS2 expression was highest at 6, 12, and 24 h after treatment, respectively. AhFLS4 expression was higher than that of AhFLS2 after 24 h under the CdCl 2 and 20% PEG6000 treatments.
AhFLS6 was up-regulated by the high temperature treatment at 42 °C, reaching its highest expression at 12 h.
Flavonol content in various peanut organs. Peanut plants are rich in flavonols. The combined of quercetin, myricetin, and kaempferol in stems is 46.69 μg/g fresh weight (FW), which is the lowest amount among peanut organs. The flavonol content in the reproductive organs is higher than that in the vegetative organs, especially in the seeds. The flavonol content of the seeds increased from the R5 stage, at 78.04 μg/g FW, during which the peanut seeds began to expand, to the mature D4 stage after drying, at 137.44 μg/g FW (Figs. 4,5).
Quercetin is the main flavonol substance in various peanut organs, especially in seeds, accounting for 79% of the total flavonol content. The distribution of quercetin in organs corresponded to the total flavonol content. There were differences among organs in the percentage of the three flavonol compounds comprised of quercetin, myricetin, and kaempferol. In the vegetative organs, kaempferol content was higher than myricetin content. In root, stem and leaf of peanut plant, kaempferol contents were 12.58, 9.38, and 25.98 μg/g FW, myricetin contents were 10.41, 8.56 and 15.28 μg/g FW. In the reproductive organs, the myricetin content was higher than kaempferol content (Fig. 5).
Effects of abiotic stress on flavonol content in peanut seedlings. The amounts of myricetin, quercetin, and kaempferol in leaves of 0-, 3-, 6-, 12-, 24-h seedlings after different abiotic stress treatments are shown in Fig. 5. Under these stress treatments, the quercetin content was the highest among the three flavonols assayed. Under the high temperature, low temperature, and NaCl treatments, flavonol content decreased first and then increased. Under PEG and MeJA treatments, the myricetin content changed significantly. Myricetin content increased to 35.21 μg/g FW and 33.63 μg/g FW at 3 h after MeJA and PEG treatments, then decreased to 18.68 μg/g FW at 12 h after MeJA treatments and 18.17 μg/g FW at 6 h after PEG treatments. Similar to myricetin content, the quercetin and kaempferol contents were also significantly regulated by PEG treatments. In addition, quercetin also showed a significant response to temperature and salt stress, and kaempferol showed a significant response to the heavy metal stress.

Relationship between flavonol content and expression level of AhFLSs. The relationships
between expression level of AhFLS genes and flavonol content are shown in Table S2 and Fig. 6. AhFLS4 expression levels were highly correlated with quercetin content (r = 0.6577) in roots, stems, leaves, and flowers. But at the seed development stage and drying processes, the expression abundance of AhFLS4 was highly negatively correlated with quercetin content (r = − 0.6203) and myricetin content (r = − 0.6396) and positively correlated with kaempferol content (r = 0.6120). It is likely that AhFLS4 changes in substrate specificity across plant development. Studies in Arabidopsis indicate that AtFLS1 is more prone to using dihydrokaempferol as a substrate and produces kaempferol 9,16 . In Scutellaria baicalensis, SbFLS expression is associated with kaempferol synthesis 17 . Park 18 isolated two AcFLS genes in onion, which favors the catalysis of dihydroquercetin, which in turn synthesizes quercetin.
Correlation analysis (Table S2) showed that under heavy metal stress, the expression of AhFLS2 was highly negatively correlated with myricetin content (r = − 0.8506), and AhFLS4 expression was highly negatively correlated with the total content of the three flavonols (r = − 0.6385). Under high temperature stress, the expression of AhFLS2 was highly negatively correlated with quercetin content (r = − 0.6094). Under UV irradiation, the expression of AhFLS4 was highly positively correlated with the total content of the three flavonols (r = 0.6155). Under low temperature stress, AhFLS3 expression was highly negatively correlated with quercetin content (r = − 0.6604). These results indicate that abiotic stress treatments have important effects on the metabolic synthesis of flavonol compounds in peanut through the regulation of different AhFLSs expression levels.

Discussion
Variation in flavonol composition among plant organs. The contents and proportions of the three flavonol compounds examined-myricetin, quercetin, and kaempferol-vary among the different plant species and tissues that have been examined. It was found that the leaves and flowers of S. baicalensis contained more myricetin, while kaempferol content was highest in roots, but with quercetin as the main flavonol in stems 17 . The quercetin content in leaves of Allium fistulosum was higher than that of pseudostem tissue 19 . Wang et al. 3 showed that quercetin is the main component among five flavonoids in peanut seeds, namely daidzein, genistein, myricetin, quercetin, and kaempferol. In this study, quercetin was identified as the main flavonol in peanut plants. The quercetin content in seeds gradually increased throughout seed development. The contents of the flavonols Scientific Reports | (2020) 10:17717 | https://doi.org/10.1038/s41598-020-74763-w www.nature.com/scientificreports/ myricetin, quercetin, and kaempferol in peanut seeds after drying were higher than those of the fresh seeds, which improves the functional food value of peanut seeds.
Variation in flavonol components in plant seedlings under abiotic stress. Flavonoids scavenge free radicals and thus play an important role in protecting cells and tissues from oxidative stress 8 . Many studies have confirmed that plant tolerance to abiotic stresses such as drought, salt, and high temperatures is related to flavonoid content. For example, when Triticum turgidum (L.) subsp. turgidum (L.) convar. durum (Desf.) is subjected to a high-temperature treatment during seed development, its anthocyanin synthesis is promoted, thereby improving the antioxidant capacity of the plant 20 . Similarly, the flavonoid content of grape seedlings and fruits was significantly increased by UV-B treatment 21 . MeJA treatment improved the salt tolerance of various plant species, including petunia 10 , Medicago sativa 11 , and soybean 12,22 , by promoting flavonoid anabolism. A low-temperature treatment at 4 °C can effectively increase the quercetin content of tobacco plants 23 . These studies show that the response of plants to abiotic stresses and specific hormones is closely related to the anabolism and content of flavonoids. In this study, the flavonol content of peanut seedlings was induced by various abiotic stresses. However, there was a difference in the degree of change in the flavonol components among myricetin, quercetin, and kaempferol. Biochemical mechanisms associated with specific stresses on the anabolic response of the aforementioned components require further investigation.
Characteristics of AhFLS proteins. The sequences and lengths of FLS proteins are highly conserved across plant species. The approximately 330-aa-long peptide sequences contain a DIOX_N conserved domain at the N-terminus and a 2OG-FeII_Oxy conserved domain at the C-terminus. FLS proteins localize to the endoplasmic reticulum membrane and cytoplasm, and they lack signal peptides and transmembrane domains 17 . The observed characteristics of the eight AhFLS proteins in cultivated peanuts were consistent with those reported by previous studies 17 , indicating that these genes are members of the FLS family. The phylogenetic analysis of the eight AhFLS proteins showed that the sequence similarity between AhFLS3 and the other AhFLS proteins was low. However, this protein also contains DIXO_N and 2OG-FeII_Oxy conserved domains, as well as the eight aa residues conserved in the C-terminal conserved domain, which are involved in ferrous ions (His221, Asp223, and His277) binding to ketoglutarate (Arg287) and affecting protein folding (G1y68, His75, Pro207, and G1y261) 24 . The study also showed that the eight conserved amino acid residues of the tested AhFLS pro-  23 showed that tobacco exhibits a significant change in the expression of specific FLS family members after chilling, high temperature, salt, and H 2 O 2 stress. The present study similarly showed that AhFLS expression was down-regulated in peanut seedlings after UV irradiation. In addition, AhFLS expression patterns among different organs and various abiotic stresses revealed that AhFLS2, AhFLS3, AhFLS4, and AhFLS6 were dominantly expressed genes in various tissues and organs and under different stresses. Some structural genes involved in flavonoid biosynthesis are members of larger gene families. Specific gene member expression patterns differ among growth stages, organs, and types of stress, perhaps in relation to the different regulatory elements contained by their promoter regions. Previous studies have confirmed that GmFNSII-1 expression in soybean seedling leaves treated with MeJA decreased gradually throughout treatment, which was lower than that of GmFNSII-2. However, GmFNSII-1 expression in roots and stems was higher than that of GmFNSII-2 12 . MtFN-SII-1 and MtFNSII-2 are two FNSII members strongly expressed in M. truncatula. MtFNSII-2 is highly expressed in roots under stress conditions 25 . Previous studies on the transcriptional regulation of flavonoid metabolism genes, such as Sm4CL2 26 , PgD1 27 , VER2 28 , and GmFNSII-1 12 , revealed that MeJA regulatory elements are present in the promoter regions of these genes 12 . Further identification of enhancers, especially cis-acting elements associated with organ-and stress-specific expression that are contained in the promoter regions of AhFLS2, AhFLS3, AhFLS4, and AhFLS6, is important for revealing the transcriptional regulation mechanism of the AhFLS genes.   13,29 . High FLS expression may increase the content of rutin, a downstream product, while increasing the stress tolerance of plants. The accumulation of flavonols in roots of drought-treated Arabidopsis seedlings is associated with increased AtFLS1 expression in this organ 9,14 . However, there were no significant differences in stress responses between a AtFLS1-overexpression mutant and wild-type seedlings 9 . This indicated that AtFLS1 is a member of the AtFLS gene family that affects the flavonol content of Arabidopsis seedlings but that it is not closely related to the ability of plants to resist stress. In addition, compared to other 2-OOD enzymes in the flavonoid biosynthetic pathway, such as F3H, and ANS, FLS has a wider range of substrates. Some FLS members can convert both flavanones into dihydroflavonols and dihydroflavonols into flavonols and are thus considered to be bifunctional enzymes with both F3H and FLS activity 30 . The present study also found that although AhFLS2, AhFLS3, AhFLS4, and AhFLS6 have higher expression levels under stress treatments, the expression levels of these genes were less correlated with flavonol content in vivo. This indicates that flavonol anabolism in plants under abiotic stress is not only related to the transcription of FLS, but is also to a large extent regulated at protein translation and post-translational levels. In this study, the expression of AhFLS1, AhFLS5, AhFLS7, and AhFLS8 remained low in various organs and under stress treatments. The biological functions of the above genes in peanut tissues, organs, and the mediation of abiotic stress responses of plants merits further exploration.

Methods
Plant materials and treatments. Peanut seeds (cultivar 'Yinduzhaiye') were planted in the experimental station of College of Agronomy, Hebei Agricultural University. The roots, stems, leaves, and flowers of the plants were collected at the flowering stage and stored at − 80 °C after being quickly frozen with liquid nitrogen. The flowering stage is the coexistence period of the various organs of peanut, and the vigorous growth period of the plant, which is suitable for comparing the flavonol content of various organs. Seeds were collected at different developmental stages, which were categorized according to the pod development criteria used by Boote 31 and Gupta et al. 32 , at R5 (28 days after flowering), R6 (40 days after flowering), R7 (55 days after flowering), and R8 (86 days after flowering) stages. Tissues were stored at − 80 °C after being quickly frozen with liquid nitrogen. The pods were harvested at 90 days after flowering and dried in an oven at 35 °C. Seeds were dried for 2, 4, 6, and 8 days and were stored at − 80 °C after being quickly frozen with liquid nitrogen. The aforementioned peanut organs and seeds were used for the determination of flavonol content and RNA extraction. The purpose of this study was to elucidate the accumulation dynamics of flavonols and the expression characteristics of AhFLS genes throughout peanut seed development.
Seedlings were maintained at 25 °C under a light intensity of 700 μmol photons m -2 s -1 , and a photoperiod of 14 h in light and 10 h in dark. During seedling growth, Murashige and Skoog nutrient solution (MS) was provided Gene cloning and sequencing. Highly conserved FLS sequence motifs were identified from DNA sequences of Arabidopsis thaliana, Medicago truncatula, and Glycine max in NCBI's GenBank database. The peanut sequencing database (https ://www.peanu tbase .org) was searched for AhFLS coding gene sequences with the conserved FLS sequence. Primers used to clone AhFLS members were designed according to the obtained peanut FLS sequences (Table 2). Peanut RNA was extracted using the RNAiso Plus kit (Takara Biotechnology (Dalian) Co., Ltd, Dalian, China). The RNA was reverse transcribed into cDNA using the One-Step RT-PCR Kit (Takara Biotechnology (Dalian) Co., Ltd). PCR-amplified products of expected sizes were gel-purified using a SanPrep column DNA gel recovery kit (Sangon Biotech, Shanghai, China), cloned into a pMD19-T vector (Takara Biotechnology (Dalian) Co., Ltd), and transformed into Escherichia coli DH5α (TIANGEN Biotech (Beijing) Co., Ltd, Beijing, China). The amplicons were then sequenced at the Beijing Genomics Institute (Beijing, China).
Multiple sequence alignments and phylogenetic and gene structure analyses. DNAStar software (DNASTAR, Madison, WI, USA) was used to predict the encoded protein sequences of the cloned AhFLS genes, and the AhFLS protein sequence alignment and phylogenetic analysis were performed using MEGA5.0. Physical and chemical properties and protein hydrophilicity were predicted using the ExPASy server (https :// web.expas y.org). Swiss model (https ://swiss model .expas y.org/inter activ e), NCBI CDD (https ://www.ncbi.nlm. nih.gov/cdd), Signal P4.1(https ://www.cbs.dtu.dk/servi ces/Signa lP/), and TMPred (https ://www.ch.embne t.org/ softw are/TMPRE D _form.html) online tools were used to predict three-dimensional structure model, conserved domains, signal peptides, and transmembrane domain features of AhFLS proteins. Schematic diagrams of conserved domains of AhFLS proteins were drawn by Illustrator for Biological Sequences (IBS) 1.0 software.
Gene expression analysis using quantitative real-time PCR. Real-time quantitative methods were used to analyze the expression patterns of AhFLS genes across different stages of seed development and under various abiotic stresses. Fluorescent primers and standard curve primers of each of the tested AhFLS genes were amplified as shown in Table 2. The real-time PCR instrument used was the Bio-Rad CHROM4 platform (Bio-Rad Laboratories, Hercules, CA, USA), and the amplification product fluorescence data were obtained using Determination of quercetin, kaempferol, and myricetin contents by HPLC. The myricetin, quercetin, and kaempferol contents were determined by the HPLC method used by Wang et al. 3 , which is briefly described as follows. Ground tissues were mixed with 80% methanol containing 25% hydrochloric acid. The mixture was incubated at 80 °C for 1.5 h and then ultrasonically extracted for 30 min at 25 kHz. After extraction, the samples were centrifuged for 10 min at 10,000×g. The supernatant containing flavonols was filtered through a syringe with a 0.2-mm filter prior to injection into an HPLC system. Flavonol separation was performed on the Agilent 1100 LC-MSD-Trap-XCT HPLC system (Agilent Technologies, Santa Clara, CA, USA) using a C18 column. The flavonol standards were purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). The mobile phase consisted of A (acetonitrile) and B (water with 0.1% formic acid) solutions. The injection volume was 10 μL. A gradient elution was carried out at a flow rate 1 mL/min with a column temperature of 30 °C. The detection wave length was 360 nm. The gradient profile was programmed at 14% A solution from 0 to 8 min, 14 AhFLS1   Clone  GAA GTT TAT TAA TTT ACC G  CTT AAC TAC AAG GCA GCT AG   RT-qPCR  TGT TGC ATA GAG CAC TGG TA  ATG TGA AAT TTG TGT ACT TGGGA   Standard curve  TGT TGC ATA GAG CAC TGG TA  ATG TGA AAT TTG TGT ACT TGGGA   AhFLS2   Clone  GTT GGT TAG TAC CCA CTG AAC  CCA GCC TTA TTA AGG TGC TA   RT-qPCR  GTC TTC AAG TTA AGC GAC GAA  CAC CTG CAT TAT GTC ACC A   Standard curve  TCT TCA AGT TAA GCG ACG AA  CTT ATT AAG GTG CTA TCC