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A flavonol synthase (FLS) gene, GhFLS1, was screened out increasing salt resistance in cotton



Flavonols play important roles in antioxidation and anticancer activities, longevity, and cardiovascular protection. Flavonol synthase (FLS) is a key enzyme for flavonol synthesis.


Phenotypic, transcriptional and metabolic data were analyzed, which showed that there was a close relationship between salt stress and flavonoids, and flavonols were significantly upregulated under salt stress. Nine, seven, four, and four FLS genes were identified in Gossypium hirsutum, Gossypium barbadense, Gossypium arboreum, and Gossypium raimondii, respectively. The results of subcellular localization showed that FLS existed in the nucleus and cytoplasmic. Through phylogenetic analysis, 24 FLS genes were divided into three subfamilies. The results of the RNA sequencing showed that the expression of GhFLS genes was mainly induced by salt, drought, low temperature, and heat stress. GhFLS promoter mainly comprised plant hormone response elements and abiotic stress elements, indicating that the GhFLS gene may play a key role in abiotic stress response. The proline contents of pYL156:GhFLS1 was reduced significantly compared to pYL156 under salt stress, thereby reducing the resistance of cotton to salt stress.


This study will lay a foundation for further study on the antioxidant regulation mechanism of the FLS gene under abiotic stress.


Soil salinization is an important problem facing agriculture worldwide. Salt stress is a major environmental factor limiting plant distribution, growth, and crop production [1]. Salt accumulation in cultivated land soil mainly comes from irrigation water and seawater containing trace NaCl [2]. Plants absorb high concentrations of salt through their roots, which then causes damage to plants. High salinity stress induces osmotic stress, ionic stress, and excessive production of reactive oxygen species (ROS) [3, 4]. Therefore, plants resist salt stress mainly through antioxidation, ion antagonism, and efflux. Using exogenous substances to resist salt stress is an important way for plants to resist salt stress. In plants, antioxidant enzymes and specialized metabolites with antioxidant activity play a key role in allowing productive ROS signaling by preventing ROS from reaching damaging levels [5].

Flavonoids have antioxidant, anti-inflammatory, and anti-proliferative properties, which help protect human beings from cancer and cardiovascular disease [6, 7]. Flavonoids are also polyphenolic secondary metabolites with C6–C3–C6 carbon skeletons that are biosynthetic through the phenylpropanoid pathway [8]. Flavonoids play an important role in regulating development and stress responses [5, 9]. Flavonoids can improved salt tolerance by removing excess ROS in soybeans [10]. Flavonoids with radical scavenging activity mitigate against oxidative and drought stress in Arabidopsis thaliana [11]. Flavonols are the main flavonoid compound in plants. It is reported that flavonols have significant health-related biological activities, including antioxidation [12], anticancer [13], increased longevity [14], and cardiovascular protection properties [15]. Flavonols can be divided into three subclasses, namely, kaempferol, quercetin, and myricetin, according to the hydroxylation mode of its flavonol bring. The content of kaempferol significantly increased under salt-alkali stress [16]. Most flavonols undergo various modifications, such as glycosylation and methylation, resulting in a large number of different molecules [17]. Quercetin is an effective anti-osmotic agent that can reduce the adverse effects of mannitol-induced osmotic stress on seed germination and seed vitality [18]. Each subclass of flavonol shows different spatial and temporal distribution and accumulation patterns, and is affected by environmental factors [19]. Flavonols have a variety of physiological functions in plants, such as ultraviolet protection, regulating auxin transport, the promotion of male fertility [20, 21], and the deposition of pigment and production of anthocyanin [22]. Flavonol could reduce the level of ROS and affect the development of guard cell, roots and leaves [23,24,25]. Flavonol regulates lateral root germination through scavenging reactive oxygen species in Arabidopsis thaliana [26]. Proline has been proven to be responsible for scavenging ROS and other free radicals [27]. There was a positive correlation between ABA content and proline synthesis [28]. Abscisic acid-induced reactive oxygen species were modulated by flavonols to control stomata aperture [29].

Flavonol synthase (FLS) is a key enzyme specific to the flavonol pathway. It competes with dihydroflavonol 4-reductase (DFR) for dihydroflavonol as a substrate [30]. Therefore, the competition between FLS and DFR enzyme activities regulates different branches of the flavonoid biosynthesis pathway [31, 32]. The FLS genes convert dihydroflavonol into the corresponding flavonol by introducing a double bond between C-2 and C-3 of the C-ring [33]. Most genes of the central enzyme in flavonoid biosynthesis are encoded by single-copy genes. Only FLS1 encodes a functional FLS and thus is the major contributor to flavonol production in A. thaliana [34]. FLS is classified as a 2-hydroxyglutarate-dependent dioxygenase (2OGD), similar to flavonoid 3-hydroxylase (F3H) and anthocyanin reductase (ANS). These three enzymes showed partial amino acid sequence similarity and overlapping functions.

The first FLS cDNA was cloned from petunia (Petunia hybrida), and was functionally expressed in yeast and plants [35]. The other FLS genes have been identified and characterized in various plant species, including A. thaliana [36, 37], Citrus unshiu [38], Glycine max [39], Zea mays [40], Camellia nitidissima [41], and Litchi chinensis [42]. For example, OsFLS was expressed in both non-pigmented and pigmented rice seeds and was subject to developmental regulation during seed maturation [43]. Flavonol synthesized by nucleus FLS1 was found to play a role in Arabidopsis resistance to Pb stress [44]. The heterotopic expression of DoFLS1 in Dendrobium candidum enhanced the accumulation of flavonols in A. thaliana and the tolerance to abiotic stress [45]. FLS was involved in auxin transport and protection against environmental stresses [46].

Cotton is a crop with strong salt tolerance and an ideal pioneer crop for improving saline-alkali land [47]. However, the flavonol synthesis-related FLS gene in cotton has not been systematically analyzed and identified. The measurement of flavonoid content, analysis of existing transcriptome and metabolome data, bioinformatics analysis, subcellular localization and virus induced gene silencing (VIGS) were used to reveal the function of FLS under salt stress. The physicochemical properties, gene structure, phylogenetic evolution, and cis-acting elements were analyzed. These will provide molecular basis and reference for further exploring the relationship between the synthesis gene FLS of flavonols and salt stress in cotton.

Materials and methods

Plant materials and treatment methods

The plant material was the salt-tolerant cotton variety Zhong9807 [48]. The cultivation of cotton adopted the sand culture method. Cotton seeds were sown in 3/2 of the seedling bowls filled with sand. The seedling bowl was placed in a growth box with an alternating cycle of 28 ℃/14 h light and 25 ℃/10 h darkness. When the cotton seedlings grew to the three-leaf stage, 400 mM NaCl was applied to the bowl until it was saturated.

Determination of total flavonoid content under salt stress

The leaves were cut and sampled 0 h and 48 h after the beginning of salt stress, and then placed in an oven at 115 ℃ for 10 min. The leaves were then dried at 80℃ until the weight was constant. The flavonoid content in cotton leaves was measured according to the instructions of the Solarbio kit (BC1330) from Beijing. First, the dried leaves were ground into powder and then weighed to 0.1 g through a 50-mesh sieve. One milliliter extraction solution was added and extraction was performed with the ultrasonic extraction method. Extraction was performed for 30 min, 120,000 rpm, and 25 ℃, centrifugation was conducted for 10 min, and then the supernatant was collected and the extraction solution was added to the supernatant until it reached a volume of 1 ml for determination. The flavonoid content was calculated as follows: flavonoid content (mg/g) = x/w, where x represents the sample concentration and w represents the sample quality.

Data analysis

For the analysis of differential genes, the transcriptome data obtained in the laboratory under salt stress were used [48]. A powerful analytical method called gene set enrichment analysis (GSEA) was used to interpret the gene expression data [49]. This method derives its power from focusing on gene sets, or groups of genes that share common biological functions. GSEA was performed using the GSEA software ( The metabolic expression data analysis of 0 h and 48 h leaves was also based on the metabolic data obtained in our laboratory. A widely targeted metabolite analysis was performed for comprehensive of flavonoids in cotton leaves based on LC–MS/MS. Flavonoids are the primary classification of substances, and the secondary classification of flavonoids includes several major categories: dihydroflavonols, flavanols, flavanols, chalcones, anthocyanins, flavonoid, and flavonoid carbonoside. We compared and analyzed the metabolomic data of salt treatment for 48 h (L48) and 0 h (L0). All histograms were designed with GraphPad Prism v8.0.2.263 software. The SPSS 26.0 statistical software was used to analyze the significant differences between the control and treatments based on one-way analysis of variance at p < 0.05 (*) or p < 0.01 (**).

Database download source

The genome files and protein sequences of Gossypium arboreum (Ga) (CRI), Gossypium barbadense (Gb) (ZJU), Gossypium hirsutum (Gh) (ZJU), and Gossypium raimondii (Gr) (JGI) were downloaded from the Cotton Functional Genomics Database (CottonFGD) ( Genome data of other 10 species including Arabidopsis thaliana (At), Arabidopsis halleri (Ah), Vitis vinifera (Vv), Arabidopsis lyrata (Al), Capsella grandiflora (Cg), Theobroma cacao (Tc), Glycine max (Gm), Boechera stricta (Bs), Oryza sativa (Os), and Zea mays (Zm) were obtained from the Phytozome plant database ( The Local Blast Alignment Tool was downloaded from the National Center for Biotechnology Information (NCBI) website ( The hmmer program, which was used to search the domain, was downloaded from Hmmer (

Identification of FLS gene family members

Six Arabidopsis FLS family members have been published [50]. This study downloaded the FLS family members from the Arabidopsis website (, namely, AtFLS1, AtFLS2, AtFLS3, AtFLS4, AtFLS5, and AtFLS6. According to the protein sequence of A. thaliana, it was known that the FLS family members contained the PF03171 and PF14226 domains. The Hidden Markov Model (HMM) of FLS protein (PF03171 and PF14226 in PFAM) was downloaded from the PFAM database (, and Hmmer was used to search all possible members of the FLS gene family. A local BLAST search was used to identify family members (E-value < e−7). Then, the common genes obtained using the two methods were selected as candidate genes. To confirm these genes, these sequences were further verified using the NCBI CD Search Tool ( and the SMART database ( Finally, sequences that did not belong to the conservative binding domain and that had incomplete C and N terminals were manually deleted.

Sequence alignment and phylogenetic analysis

The full-length amino acid sequences of 14 plants, namely, G. arboreum, G. barbadense, G. hirsutum, G. raimondii, A. thaliana, A. halleri, V. vinifera, A. lyrata, C. grandiflora, T. cacao, G. max, B. stricta, O. sativa, and Z. mays were aligned using the Cluster W program with the default settings. Then, a neighborhood-joining (NJ) tree was built with 1000 bootstrap replicates using the p-distance model with default parameters in MEGA 7.0 [51]. The EvolView website ( was used to beautify the phylogenetic tree [52].

FLS chromosome positions in four species of cotton

The chromosome positions of G. arboreum, G. barbadense, G. hirsutum, and G. raimondii were mapped with TBtools software [53]. The reference genome GFF3 file was downloaded from CottonFGD.

Collinearity analysis of the FLS family in four species of cotton

To study the collinearity of four cotton FLS families and analyze their collinearity relationships, the whole genome sequence and genome annotation files of these cotton varieties were obtained using the MCScanX tool. The collinear and homologous chromosome regions of four species of cotton were visualized using the advanced circos software package. Gene duplication was evaluated using MCScanX. To visualize the duplicated regions of four species of cotton, TBtools was used to draw spectral lines between the repetitive genes in Circos [53].

Calculation of selective pressure

To study the selection pressure experienced by FLS repeat gene pairs from the four cotton species, the Ka/Ks calculator in TBtools was used to calculate synonymous (Ks) and non-synonymous (Ka) replacement rates and their ratios.

Analysis of the conserved protein motifs and gene structures

The MEME website ( was used to predict gene motifs. The parameters were as follows: the maximum number of motifs was 15, and the other parameters were set by default. The files for this domain were obtained from the hmmersearch online website ( The evolutionary relationship, gene structure, domain, and motif composition of genes were mapped using TBtools software.

Analysis of the GhFLS promoter region and different expression patterns

The 2000 bp DNA sequence of the upstream region of GhFLSs was derived from the CottonFGD database ( The predicted cis-acting elements related to abiotic stresses and plant hormones in the promotor regions of the GhFLSs were obtained from the PlantCARE website ( for further analysis. RNA Sequencing data (PRJNA490626) from the NCBI ( were used to analyze the expression level (Fragments Per Kilobase of exon model per Million mapped fragments, FPKM) of GhFLSs in 4-week-old seedlings under cold (4 °C), heat (37 °C), salt (0.4 M NaCl), and polyethylene glycol (PEG, 200 g/L) stress for 1 h, 3 h, 6 h, and 12 h, respectively [54]. RNA Sequencing data from the NCBI (PRJNA559592) [48] were analyzed to determine the FPKM of GhFLSs in different time periods under 400 mM NaCl treatment. Finally, TBtools software was used to draw an image of an evolutionary tree, cis-acting elements, and an expression level heat map for visual observation.

Verification of relative gene expression using by qRT-PCR

RNA was extracted and reversed-transcribed into cDNA as a template for qRT-PCR. The primers for the qRT-PCR of GhFLSs were designed on the GenScript website ( (Additional file 1: Table S1). According to the instructions provided by the manufacturer of TransStart Top Green qPCR Supermix reagent (TransGene Biotechnology Co., Ltd, Beijing, China), qRT-PCR was performed on an Applied Biosystem ABI7500 Fast Real-Time PCR platform, and the experiment was conducted in three independent replicates. The GhActin (AY305733) gene was used as the internal reference gene, and 2−ΔΔ Ct was used to calculate the relative expression level of GhFLS.

Interaction network analysis and subcellular localization of GhFLS1

The STRING database ( was used to analyze the GhFLS protein interaction network. The interactions between GhFLS1 and other genes in cotton were predicted based on A. thaliana homology. The Wolf-PSORT and ProtComp 9.0 websites were used to predict the subcellular location of GhFLS. A GhFLS1:121-GFP vector was constructed, and transformed into tobacco leaves.


To verify the function of FLS genes, this study selected a highly expressed gene, GhFLS1 (GH_A05G2328). VIGS fragments of 300 bp were designed using SGN-VIGS ( The fragment was connected to the pYL156 vector and the recombinant vector was transformed into Agrobacterium tumefaciens GV3101. GV3101 bacterial solution containing the control pYL156 (empty vector), pYL156: GhFLS1, pYL156: PDS (positive control), and pYL192 (auxiliary carrier) was injected into the cotyledons of cotton variety Zhong9807. After dark treatment for 24 h, cotton was grown in an incubator containing 25 °C/16 h light and 23 °C/8 h dark circulation culture. NaCl stress treatments were performed at the three-leaf stage and samples were quickly frozen with liquid nitrogen.

After 400 mM NaCl treatment, 0.1 g samples were obtained and ground with 1:9 medium homogenate. The proline content was determined using a proline content detection kit (Nanjing Jiancheng Bioengineering Research Institute, A107-1-1).


Phenotype, total flavonoid content, and expression analysis of cotton under salt stress

Cotton cotyledons showed wilting and water loss under salt stress (Fig. 1A). Analysis of the total flavonoids content in leaves revealed, significant differences between the treatment and the control (Fig. 1B). GSEA analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) enriched with the transcriptome data showed that flavonoid metabolism-related genes were significantly expressed (Fig. 1C). Analysis of the metabolomics data showed that flavonols were significantly upregulated in flavonoid-related substances (Fig. 1D). This indicated that there was a close relationship between flavonols and salt stress. FLS is a key enzyme involved in the formation of flavonols, which are a subclass of flavonoids [55]. This study speculated that the expression of the FLS gene could improve salt stress resistance, and the FLS family members were analyzed.

Fig. 1
figure 1

Phenotype, total flavonoid content, and expression analysis of cotton under salt stress. A Phenotype of cotton under salt stress. B Total flavonoid content of cotton under salt stress. C Gene set enrichment analysis (GSEA) based on RNA sequencing. D Content of flavonoid-related substances under salt stress. Flavonoids include dihydroflavonols, flavanols, flavanols, chalcones, anthocyanins, flavonoid, and flavonoid carbonoside. L01, L02, and L03 represented three replicates of the control, respectively. L48-1, L48-2, and L48-3 represented the replicates of the treatment, respectively

Identification of FLS family members

The FLS enzyme belongs to the 2OGD superfamily. A total of 24 family members have been identified from the four Gossypium species, including four in G. arboreum, seven in G. barbadense, nine in G. hirsutum, and four in G. raimondii, via tree building. Genes were renamed according to chromosome position information (Additional file 1: Table S2). The open reading frame (ORF) of 24 FLS family members of four cotton species ranged from 981 (GbFLS7) to 2270 (GrFLS1) bp. The number of amino acid-encoding protein ranged from 326 (GbFLS7) to 498 (GbFLS5). The isoelectric point (pI) ranged from 5.13 to 7.28, and the molecular weights (MWs) ranged from 37.08 to 56.62 kDa. The number of exons varied from 2 to 5.

A. thaliana, A. halleri, V. vinifera, A. lyrata, C. grandiflora, T. cacao, G. max, B. stricta, O. sativa, and Z. mays were selected to identify FLS family members that were closely related to cotton and had been studied more than cotton FLS family genes. FLS family genes also have been identified in 10 additional plants, including six in A. thaliana, six in G. max, three in O. sativa, three in Z. mays, five in V. vinifera, three in B. stricta, twelve in C. grandiflora, four in T. cacao, five in A. lyrata and nine in A. halleri. Then, FLS family members were renamed according to their position on the chromosome. The FLS family genes of two tetraploid cotton species, G. barbadense and G. hirsutum, were about twice as many as those of the two diploid cotton species, G. arboreum and G. raimondii. The FLS family members in dicotyledons were more abundant than those in monocotyledons.

Evolutionary tree analysis of FLS

To understand the evolutionary relationship of the FLS family, 80 protein sequences were used to construct the phylogenetic trees of G. arboreum, G. barbadense, G. hirsutum, G. raimondii, A. thaliana, A. halleri, V. vinifera, A. lyrata, C. grandiflora, T. cacao, G. max, B. stricta, O. sativa, and Z. mays (Fig. 2). According to sequence similarity, tree topology, gene structure characteristics, and each motif, the FLS family was divided into three branches. The results showed that the FLS branch had the largest number of group C (31), followed by group A (25), and finally group B (24). Five GhFLS genes were distributed in branch A, and group B had three GhFLS genes. AtFLS only existed in branch group C, and most of A. halleri and A. lyrata were also distributed in group C, indicating that the FLSs in branch group C were closely related. The branches of TcFLS and cotton FLS were similar, indicating that cacao was closely related to cotton and may have originated from the same ancestor. The FLS gene family members of the four cotton species always gathered together, which indicated that the four cotton species had a close evolutionary relationship.

Fig. 2
figure 2

Phylogeny tree constructed using MEGA 7 by the Neighbor-Joining (NJ) method. Phylogenetic relationship of the 80 identified FLSs from G. arboreum, G. barbadense, G. hirsutum, G. raimondii, A. thaliana, A. halleri, V. vinifera, A. lyrata, C. grandiflora, T. cacao, G. max, B. stricta, O. sativa, and Z. mays

Chromosome mapping of FLS gene family

To understand the distribution of genes on chromosomes more intuitively, this work constructed a physical map of the chromosome distribution of FLS gene family members in four cotton species (Fig. 3). Chromosome mapping analysis showed that the distribution of chromosome positions was uneven. FLSs in G. hirsutum were distributed in chromosome 5, 8, and 12 of subgenome A and in chromosome D groups 4, 5, and 8. FLS gene members of G. arboreum were distributed on chromosomes 4, 5, 8, and 12. FLS family members of G. raimondii were distributed on chromosomes 4, 9, and 12. Compared with G. hirsutum, the FLS genes were missing at the end of the A05 and D04 chromosomes in G. barbadense. It is speculated that this may be caused by gene loss or incomplete genome assembly during the evolution of G. barbadense.

Fig. 3
figure 3

The chromosome distribution of FLS gene family in four cotton Gossypium. A Chromosomal location of FLSs on chromosomes in G. hirsutum. B Chromosomal location of FLSs on chromosomes in G. barbadense. C Chromosomal location of FLSs on chromosomes in G. arboreum. D Chromosomal location of FLSs on chromosomes in G. raimondii. The scale of the genome size was given on the left

Analysis of motifs, domain structures and exon–intron structures of FLS genes

This study analyzed the evolutionary relationships, motifs, domains, exons, and introns to study the conservative structure of FLS family genes (Fig. 4). All FLS family genes had the DIOX_N domain and the 20G-Fell_Oxy domain. This domain could combine with plant hormones to achieve its catalytic function (Fig. 4C). GbFLS5 had two DIOX_N institutional domains.

Fig. 4
figure 4

Conservative motifs, domain structures, and exon–intron structures of FLS genes from G. hirsutum, G. barbadense, G. raimondii, and G. arboreum

FLSs were classified according to the topology of the evolutionary tree (Fig. 4A). The distribution patterns of exons and introns were related to their biological functions. Their arrangement could be used to analyze the evolutionary relationship between members of different gene families. Interestingly, an intron in the FLS in branch I (GbFLS5) and an intron in the FLS in branch III (GrFLS1) were significantly longer than other introns (Fig. 4D). Except for GbFLS5, the motifs of FLS family gene members were relatively consistent, which means that they have similar functions at the protein level (Fig. 4B). All FLSs contained Motif 1, Motif 2, Motif 3, Motif 4, and Motif 6, which were common conservative domains of the FLS family. Motif 9 in branch I and branch II was at the N-terminus, and Motif 9 of GaFLS4GbFLS7 in branch III was at the C-terminus. It was speculated that this may have been due to functional changes during evolution.

Gene replication and collinearity analysis

Gene replication events are considered to play an important role in the amplification of gene families. Gene replication events include whole-genome duplication (WGD), fragment replication, and tandem replication. Most plants have experienced an ancient whole-genome replication event or polyploidy. The replication region caused by WGD is usually the large-scale replication of all genes, rather than the replication of a single gene or multiple genes. Large-scale whole-genome replication and small-scale tandem replication and fragment replication can be identified from collinear fragments, which can be used as inferential data on species evolution. To explore the amplification mechanism of the FLS gene family, 95 pairs of homologous gene pairs were identified by comparing the genomes of Ga–Ga, Ga–Gb, Ga–Gh, Gb–Gb, Gb–Gr, Gr–Gr, Gr–Ga, and Gh–Gh. Genes connected by lines of the same color represent the same gene. In Fig. 5, the GhA/GhD and GbA/GbD subgenomes, as well as many chromosomes in the A and D genomes, are connected by lines of the same color, indicating that the GhA/GhD and GbA/GbD subgenomes have FLS homologs in the A and D genomes. These results indicate that these genomes/subgenomes are evolutionarily related and that most FLS genes have been retained during polyploid evolution. Eight, eight, one, and one pairs of duplicate FLS gene pairs were found in Gh–Gh, Gb–Gb, Ga–Ga, and Gr–Gr, respectively. The 18 pairs of homologous gene pairs were predicted to be fragment duplicates according to chromosome location. Seventy-seven gene pairs had undergone WGD, with 12, 12, 11, and 11 Ga–Gb, Ga–Gh, Gr–Gb, and Gh–Gr gene pairs, respectively. Based on these results, it was speculated that fragment duplication and WGD were the main reasons for the evolution of the FLS genes from diploid to tetraploid.

Fig. 5
figure 5

Collinearity relationship of FLSs duplicated genes pairs from four Gossypium (G. hirsutum, G. barbadense, G. arboreum, and G. raimondii). Chromosomal lines represented by various colors indicate the syntenic regions around the FLSs. The heatmap and line map of the outer ring represented the density of genes on chromosomes

Calculation of selective pressure (Ka/Ks) during evolution

During evolution, duplicate gene pairs may deviate from their original functions, resulting in new functionalization (the loss of original functions), sub-functionalization (a division of the original functions), and new functionalization (the acquisition of new functions). To study the driving force of FLS family genes in the evolutionary process, this study calculated the values of Ka/Ks synonymous substitution of 70 duplicate gene pairs from four cotton species. The selection pressure of duplicate gene pairs can be inferred according to the Ka/Ks ratio. It is generally believed that Ka/Ks = 1 represents neutral selection (pseudogene), Ka/Ks < 1 represents purifying or negative selection (purifying selection), and Ka/Ks > 1 represents positive selection. There were 164 duplicate gene pairs in Ga–Ga, Ga–Gb, Gb–Gb, Gb–Gr, Gh–Ga, Gh–Gb, Gh–Gh, Gh–Gr, and Gr–Gr (Fig. 6). The Ka/Ks values of four pairs of genes were greater than 1, indicating that these genes experienced positive selection during evolution (Table 1). Fifty-eight (83%) duplicate genes had a Ka/Ks ratio < 0.5, and five (7%) duplicate genes had a Ka/Ks ratio between 0.5 and 0.99. These genes were GbFLS1–GaFLS2, GhFLS1–GaFLS2, GrFLS1GbFLS6, GrFLS2–GhFLS8, and GbFLS3–GhFLS4. This showed that these FLSs evolved slowly, underwent strong purifying selection pressure, and had limited functional differences after fragment replication and WGD. In Ga–Gb, Gh–Ga, and Gh–Gr, the logarithms of genes with Ka/Ks values greater than 1 were 1, 2, and 1, respectively, indicating that these genes were actively selected during evolution and recently underwent rapid evolution. Whether it will result in harmful or beneficial characteristics remains to be studied.

Fig. 6
figure 6

Prediction of a number of duplicated gene pairs involved indifferent combinations from four Gossypium species. Gh represented G. hirsutum, Gb represented G. barbadense, Ga represented G. arboreum, Gr represented G. raimondii. Different colors represented Ka/Ks gene pairs between Ga–Ga, Ga–Gb, Ga–Gh, Gb–Gb, Gh–Gh, Gr–Gb, Gr–Gh, Gb–Gh, Ga–Gr

Table 1 Prediction of the number of duplicated gene pairs involved in different genomes of four cotton species

Promoter and expression analysis of GhFLS under salt stress

The online promoter website revealed the response of GhFLSs to hormonal and abiotic stresses, which was helpful to further analyze their regulatory networks. GhFLSs were related to plant hormones (abscisic acid, methyl jasmonate, gibberellic acid, auxin, salicylic acid) and various stresses (low temperature, drought, hypoxia, defense, and stress response). GhFLSs also had many MYB binding sites and zein metabolism regulatory elements (Fig. 7). In addition, there were light-regulated promoters.

Fig. 7
figure 7

Analysis of promoters and differentially expressed of GhFLS gene family

Transcriptome data were used to analyze the FPKM values of eight cotton tissues ((root, stem, leaf, torus, petal, stamen, pistil, and calycle tissues). The results showed that FLSs had different expression patterns in different tissues. GhFLS4 and GhFLS9 were highly expressed in all tissues, and their expression levels were significantly higher than those of other FLS family genes, which may be necessary for maintaining the normal living activities of cotton. GhFLS1 was specifically expressed in the stamen tissue, and it speculated that it may play an important role in stamen development. These results indicate that FLSs have tissue-specific expression under normal growth conditions.

The expression level of GhFLS1, GhFLS8, and GhFLS5 changed significantly at 12 h after exposure to salt stress. The expression of GhFLS1 and GhFLS8 decreased at 12 h, while the expression of GhFLS5 increased at 12 h (Additional file 1: Fig. S1). The expression of GhFLS7 was significantly decreased after exposure to cold stress, the expression GhFLS4 and GhFLS9 was significantly increased after exposure to PEG stress, and the expression of GhFLS4 and GhFLS9 was significantly decreased after exposure to heat stress. This demonstrated that different FLS family members showed different expression patterns under different abiotic stresses.

Expression patterns of GhFLSs under 400 mM NaCl stress

To study the response of GhFLSs to abiotic stress, this study examined the expression changes of the FLS genes in G. hirsutum leaves under 400 mM NaCl stress (Fig. 8). Under NaCl stress, the expression levels of GhFLS1 and GhFLS8 were significantly increased at 3 h, 12 h, 24 h, and 48 h after exposure to salt stress, and these genes continued to be overexpressed. The expression of the GhFLS2 and GhFLS4 genes increased significantly only 24 h and 48 h after exposure to stress. The expression of the GhFLS3, GhFLS5, and GhFLS7 genes did not change significantly under salt stress. The expression of GhFLS6 was significantly reduced at 12 h, 24 h, and 48 h under salt stress. The results showed that different FLS family members had different expression patterns under NaCl stress, and the expression patterns were different under different salt concentrations.

Fig. 8
figure 8

Analysis the expression level of GhFLS genes under 400 mM NaCl in different time treatment. *Represented 0.01 < p < 0.05, **represented p < 0.01

Interaction network and subcellular localization of GhFLS1 protein

To further understand the functions of GhFLS proteins, this study compared the GhFLS1 protein with Arabidopsis, obtained the Arabidopsis homologous protein AtFLS1 (AT5G08640), and used the online STRING tool to predict the interaction protein of the FLS protein (Additional file 1: Fig. S2). AtFLS1 interacts with dihydroflavonol 4-reductase (DFR), 4-coumaric acid: coenzyme A ligase (4CL), flavanone-3-hydroxylase (F3H), glycosyltransferase (UGT78D2), and cytochrome P450 (TT7) proteins. Analysis of the KEGG pathway enrichment of GhFLS1 based on transcriptome data revealed, that GhFLS1 was mainly involved in flavonoid metabolism (ko00250), and FLS was involved in the biosynthesis of flavonoids and flavonols through catalyzing the conversion of dihydroflavonols into flavonols. The 4CL3, F3H, and DFR were involved in the synthesis of flavonoids. It was speculated that FLS interacts with these proteins and responds to NaCl stress through regulating flavonol content.

According to the prediction of subcellular localization in four cotton species, nine GhFLS proteins were most located in the cytoplasmic (cyto), chloroplast (chlo) and nucleus (nucl), three were located in the chloroplast (chlo), eight were located in the cytoplasmic (cyto), and three were located in the nucleus (nucl). Due to GhFLS1 protein sequence was highly similar to AtFLS1 sequence, and only AtFLS1 was a functional gene encoding flavonol [34]. GhFLS1 protein was selected for subcellular localization verification. The results showed that GhFLS1 protein was located in the nucleus and cytoplasmic (Fig. 9).

Fig. 9
figure 9

Subcellular localization verification of GhFLS1 protein (bar = 75 μm)

VIGS of GhFLS1 in cotton

The VIGS experiment was performed to verify the role of GhFLS1 under NaCl stress (Fig. 10). PYL156: PDS showed obvious albino phenotypes (Fig. 10A). The relative expression level of GhFLS1 was detected using qRT-PCR. The results showed that the expression level of pYL156: GhFLS1 was 67% lower than pYL156 (Fig. 10B), indicating that it had a good silencing effect. Under NaCl stress, the cotton leaves wilted and the stems bent, and pYL156: GhFLS1 leaves wilted more severely than those of pYL156. The expression of pYL156: GhFLS1 was significantly lower than that of pYL156 under NaCl stress. In addition, the proline content of pYL156: GhFLS1 decreased significantly under NaCl stress (Fig. 10C).

Fig. 10
figure 10

Silencing GhFLS1 via VIGS increased sensitivity to NaCl stress. A The phenotype of cotton after GhFLS1 gene silencing under NaCl stress. pYL156: PDS as a positive control, pYL156 was an empty vector as control, and pYL156:GhFLS1 was GhFLS1 silenced lines. B The relative expression level of GhFLS1 under water and NaCl stress. C Proline content under water and NaCl stress. *Represented 0.01 < p < 0.05, **Represented p < 0.01


Flavonols respond to salt stress in cotton

As a cash crop that is widely planted around the world, upland cotton is faced with various biotic and abiotic stresses. In plants, flavonols are significantly involved in plant growth and development, and they have been identified as the most active flavonoid, as well as a regulator of polar auxin transport [56, 57]. FLS is a key enzyme for the synthesis of flavonol substances, but systematic reports on FLSs are still lacking in cotton. In this study, the transcriptome data showed significant enrichment of genes related to flavonoid synthesis under salt stress. The metabolomics data showed that flavonols were significantly upregulated under salt stress. We measured and analyzed the total flavonoid content of cotton leaves, and the results showed a significant increase in total flavonoids under salt stress. This result further verified the correctness of the metabolomics data. Due to the significant increase in the content of flavonols under salt stress, we further conducted bioinformatics analysis on the key gene for synthesizing flavonols, FLS. This study provides important reference information for further understanding the functions of FLS.

FLS genes showed evolutionary conservation in cotton

In this study, the FLS genes of G. arboreum, G. barbadense, G. hirsutum, and G. raimondii were comprehensively identified. A total of 24 FLS genes were identified in the four cotton species, and 56 FLS family members were identified in A. thaliana, A. halleri, V. vinifera, A. lyrata, C. grandiflora, T. cacao, G. max, B. stricta, O. sativa, and Z. mays.

All FLS family members had the 2OG-FeII_Oxy domain. The 2OG-FeII_Oxy oxidase domain was composed of 2-ketoglutarate and the Fe (II)-dependent oxidase superfamily. The domain of 2-ketoglutarate (2OG)/Fe(II) dependent dioxygenase (2OGDD) plays an important role in plant primary and secondary metabolism. The 2OGD gene family is extremely large. According to the sequence similarity, the members of the 2OGDD gene family could be divided into three categories: DOXA, DOXB, and DOXC. Members of the DOXA subfamily evolved from the DNA repair protein Alkb in Escherichia coli [58]. The DOXA subfamily mainly participates in primary metabolic processes. The members of the DOXB subfamily were relatively conservative, encoding prolyl 4-hydroxylases (P4Hs), which are mainly involved in the post-translational modification of polypeptide chains and are of great significance for plant signal peptide hormones and cell wall formation [59]. The members of the DOXC subfamily are complex, and their functions and numbers in different plants vary greatly, showing obvious species specificity. Members of this subfamily are mainly involved in the secondary metabolism of plants, including the biosynthesis of terpenoids, alkaloids, plant hormones, flavonoids, and phenolic acids. Most of the 2OGD genes involved in plant secondary metabolism can be classified into this subfamily [58]. This indicates that FLS family members belong to a branch of the DOXC subfamily. In addition, FLS family members were also found to contain the DIOX_N domain, a highly conserved N-terminal region of a protein with 2-ketoglutarate/Fe (II) dependent dioxygenase activity [60]. These two domains together constituted the unique gene function of FLS family members.

Replication events are one of the main driving factors for the evolution and diversification of genomes and genetic systems [61]. Environmental conditions and artificial selection affect the number of gene family members. With the occurrence of repeated WGD events, the gene sequence of cotton was doubled, and some redundant genes were selectively lost or recombined [62]. The research results showed that four, seven, nine and four FLS family members were identified in G. arboreum, G. barbadense, G. hirsutum, and G. raimondii, respectively, and the number of genes in each cotton species explained the evolutionary origin of diploid and tetraploid cotton to some extent.

The uneven distribution of genes on each chromosome indicated that there was genetic variation during evolution [63]. The chromosome location of FLSs clearly showed the physical location distribution of each FLS gene in the genome and the evolutionary relationships of some genes. There was one tandem-duplication on chromosomes A05 and D04 of G. hirsutum, 1 tandem-duplication on chromosome 12 of G. raimondii, and no tandem duplication in G. arboreum and G. barbadense, indicating that there was a special evolutionary pattern in the evolution of different cotton species. The lack of tandem duplication may be the main reason for the low number of GbFLSs and GhFLSs. The collinearity analysis showed that whole gene replication and fragment replication played important roles in the development of FLSs. The Ka/Ks values of 164 pairs of genes were calculated, among which Ka/Ks < 1 for 65 pairs of duplicate genes, indicating purifying selection. It was speculated that the cotton FLS gene family underwent strong purifying selection after fragment replication, tandem replication, and WGD, but the functional difference was limited.

Motif prediction can provide a basis for researchers to analyze the functional and structural classification of families. A motif is a short sequence of relatively conservative characteristics shared by a group of genes. This may be a recognition sequence or a functional protein [64]. In this study, it was found that most of the Motif 9 in branch I and branch II of the 24 family members was located at the N-terminus, while the Motif 9 in branch III was located at the C-terminus and the number of these 24 genes was also different, which may be the main reason why FLS had different functions.

GhFLS1 plays an important role in salt response

Under NaCl stress, the expression of GhFLS1, GhFLS2, GhFLS4, GhFLS6, GhFLS8, and GhFLS9 changed significantly. While the GhFLS6 gene was significantly downregulated, the other five genes were upregulated. After silencing the GhFLS1 in cotton, cotton seedlings were more sensitive to NaCl, and the expression of the GhFLS1 gene decreased significantly. Some studies showed that the overexpression of EkFLS significantly increased the flavonol content in guard cells, and the accumulation of flavonol reduced ABA-induced stomatal closure through inhibiting the accumulation of hydrogen peroxide in guard cells. In addition, the overexpression of EkFLS increased the content of superoxide dismutase and peroxidase enzymes in A. thaliana, thus effectively eliminating the ROS caused by drought [65]. In this study, it is speculated that GhFLS1 can resist salt stress through increasing the ROS produced by flavonol accumulation under salt stress. When salt stress is applied to plants, the FLS gene in the plant responds positively and converts dihydroflavonols into flavonols, which can cope with ROS and thus resist salt stress (Fig. 11). The structural should be changed, taking into account the circularity, the contribution of compounds and genes, and the reaction of plant and metabolism. This model needs to be further validated and improved in future experiments.

Fig. 11
figure 11

A model illustrating the role of GhFLS1 in cotton providing salinity stress tolerance. DHQ dihydroquercetin, DHK dihydrokaempferol, DHM dihydromyricetin, ROS reactive oxygen species


This study transcriptome and metabolome data showed that flavonols play an important role under salt stress. This study analyzed the phenotype and total flavonoids content of cotton leaves under salt stress. FLS was identified in cotton for the first time, and four, seven, nine, and four FLS genes were identified in G. arboreum, G. barbadense, G. hirsutum, and G. raimondii, respectively. FLSs were divided into three branches according to the composition of the phylogenetic tree, gene structure, and motifs. Fragment replication and WGD were the main evolution modes of the FLS gene family. Silencing GhFLS1 resulted in a more serious phenotype in cotton under NaCl stress, indicating that GhFLS1 was involved in the response of cotton to NaCl stress. This study provides a reference and knowledge basis for further exploring the relationship between GhFLS1 and NaCl stress.

Availability of data and materials

The original contributions presented in the study are included in the article/Additional files, further inquiries can be directed to the corresponding author.


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This work was supported by National Key Research and Development Program of China (No. 2022YFD1200300), China Agriculture Research System of MOF and MARA, Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences.

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MH: conceived and designed the experiments, writing original draft, data curation, software. RC, YC, TJ, HH, YL, XL, CR: data curation. review and editing. YF, YZ, KN, JW, SW, LS: methodology. XC, XL, DW, ZY, CC, LG and LZ: data curation. QC and WY: concept of study, supervision and revised manuscript. All authors have read and approved the final manuscript.

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Correspondence to Quanjia Chen or Wuwei Ye.

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Additional file 1:

Fig. S1. GhFLS genes displayed expression patters under cold, heat, salt and drought stress. Fig. S2. Interaction network of FLS protein. The FLS represented the protein AtFLS1 with the highest homology to GhFLS1. Table S1. Primers used in the experiment. Table S2. Basic information of 24 genes.

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Han, M., Cui, R., Cui, Y. et al. A flavonol synthase (FLS) gene, GhFLS1, was screened out increasing salt resistance in cotton. Environ Sci Eur 35, 37 (2023).

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