Characterization of the WRKY Gene Family Related to Anthocyanin Biosynthesis and the Regulation Mechanism under Drought Stress and Methyl Jasmonate Treatment in Lycoris radiata

Abstract

Lycoris radiata, belonging to the Amaryllidaceae family, is a well-known Chinese traditional medicinal plant and susceptible to many stresses. WRKY proteins are one of the largest families of transcription factors (TFs) in plants and play significant functions in regulating physiological metabolisms and abiotic stress responses. The WRKY TF family has been identified and investigated in many medicinal plants, but its members and functions are not identified in L. radiata. In this study, a total of 31 L. radiata WRKY (LrWRKY) genes were identified based on the transcriptome-sequencing data. Next, the LrWRKYs were divided into three major clades (Group I–III) based on the WRKY domains. A motif analysis showed the members within same group shared a similar motif component, indicating a conservational function. Furthermore, subcellular localization analysis exhibited that most LrWRKYs were localized in the nucleus. The expression pattern of the LrWRKY genes differed across tissues and might be important for Lycoris growth and flower development. There were large differences among the LrWRKYs based on the transcriptional levels under drought stress and MeJA treatments. Moreover, a total of 18 anthocyanin components were characterized using an ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) analysis and pelargonidin-3-O-glucoside-5-O-arabinoside as well as cyanidin-3-O-sambubioside were identified as the major anthocyanin aglycones responsible for the coloration of the red petals in L. radiata. We further established a gene-to-metabolite correlation network and identified LrWRKY3 and LrWRKY27 significant association with the accumulation of pelargonidin-3-O-glucoside-5-O-arabinoside in the Lycoris red petals. These results provide an important theoretical basis for further exploring the molecular basis and regulatory mechanism of WRKY TFs in anthocyanin biosynthesis and in response to drought stress and MeJA treatment.

1. Introduction

Transcription factors (TFs) activate diverse signal transduction pathways and modulate the transcriptional mode of targeted genes, which improves plants to adapt to diverse environmental responses [1,2,3,4]. Among various TF families, WRKY TFs are one of the largest TF families in plants and play crucial roles in various biological processes, such as physiological metabolism, anthocyanin accumulation, and abiotic stress response [4,5,6]. WRKY proteins contain a highly conserved WRKY domain (WRKYGQK) at the N-terminus and C2H2 (C-X4–5-CX22–23-H-X-H) or C2HC (C-X7-C-X23-H-X-C) zinc finger motif at the C-terminus [5]. WRKY proteins can be classified into four main groups (Group I, II, III, and IV) based on the number of WRKY domains and the structure of zinc finger motifs. The Group I members contain two WRKY domains and a C2H2-type zinc finger. The Group II and Group III members have one WRKY domain; Group II members have a C2H2-type zinc finger and the Group III members consist of a C2HC type. The Group IV members contain an incomplete or partial WRKY domain and lack a zinc-finger motif, indicating that this group may have lost their function as WRKY TFs [4,7]. In addition, Group I can be divided into two subgroups, Ia and Ib, based on two WRKY domains at the N and C terminus [7,8]. The Group II WRKY can be further categorized into five subgroups (IIa, IIb, IIc, IId, and IIe) based on the sequence of the DNA-binding domain [8]. Group III is classified into two subgroups (IIIa and IIIb) based on the zinc-finger motif structure [9,10].

The WRKY gene family is widespread in many plants. For example, there are 74 WRKY genes in Arabidopsis thaliana [11], 153 WRKY genes in Brassica napus L. [12], 88 WRKY genes in Phaseolus vulgaris L. [13], 54 WRKY genes in Ananas comosus L. [14], 94 WRKY genes in Sorghum bicolor L. [15], 44 WRKY genes in Liriodendron chinense [16], 138 WRKY genes in Chrysanthemum lavandulifolium [17], and 174 WRKY genes in Arachis hypogaea L. [18]. Drought is an important abiotic stress factor that prevents plant growth and development and is a regular cause of damage. Many studies have shown that the WRKY TFs play crucial roles in the signaling and regulation of gene expression during drought stress responses [7,19,20,21]. In a recent study, GhWRKY21 was found to negatively regulate drought stress responses in cotton [22]. TaWRKY2-overexpressing transgenic wheat has been observed to be improved in drought resistance [23]. In Arabidopsis, AtWRKY11, AtWRKY17, AtWRKY28, AtWRKY30, and AtWRKY63 have been shown to play positive regulation roles in drought stress responses, while AtWRKY46, AtWRKY54, and AtWRKY70 participated in plant growth and play a negative role in drought tolerance regulation [24,25,26,27,28]. Moreover, the overexpression of GhWRKY1, IgWRKY50, IgWRKY32, MfWRKY41, TaWRKY75, OsWRKY114, ZmWRKY106, and TaWRKY133 enhances drought tolerance in transgenic Arabidopsis plants [29,30,31,32,33,34,35,36].

The WRKY genes could be up-regulated under methyl jasmonate (MeJA) treatment, suggesting that they are involved in response to abiotic stress, as well as in jasmonic acid (JA) signaling pathways in plants [37]. In Populus, most WRKY genes were induced by MeJA and SA treatments [38]. The expression level of ScWRKY3 in sugarcane was increased under salt, drought stresses, and abscisic acid (ABA) treatment but inhibited by salicylic acid (SA) treatment and MeJA treatment [39]. Differently, the expression level of ScWRKY5 was induced by salt, drought stresses, as well as SA and ABA treatments [40]. Furthermore, overexpressing HbWRKY82 of Hevea brasiliensis improved drought and salt tolerance and decreased sensitivity to ABA in Arabidopsis [41]. Recently, reports have shown that WRKY TFs have a visible correlation with the regulation of anthocyanin biosynthesis. For example, MdWRKY11 participates in anthocyanin accumulation by affecting MdMYB and MdHY5 in apples [42]. PyWRKY26 targeted the PyMYB114 promoter to regulate anthocyanin biosynthesis and transport in red-skinned pears [43]. PbWRKY75 may promote PbMYB10b expression and activate anthocyanin biosynthetic genes DFR and UFGT to promote anthocyanin accumulation [44]. Moreover, StWRKY13 could significantly activate anthocyanin biosynthetic genes StCHS, StF3H, StDFR, and StANS to promote anthocyanin biosynthesis in potato tubers [45]. In a previous study, McWRKY71 interacted with McMYB12 and directly activated McANR to participate in the regulation of proanthocyanidin biosynthesis [46]. In apples, MdWRKY75 mainly activate the promoter of MdMYB1 and induced the accumulation of anthocyanin [47].

L. radiata is a perennial herbaceous flower with good ornamental characteristics and barren-resistant, water-saving, and drought-resistant properties. Plants in the genus Lycoris have been utilized in traditional medicine preparation and more than 110 potent structurally distinct Amaryllidaceae alkaloids were isolated or identified for extensive pharmacological and phytochemical investigations [48]. For example, according to the Compendium of Materia Medica, Lycoris plants are described as potent antidotes to poison, effective agents to alleviate pain and relieve inflammation, and diuretic drugs [49]. Amaryllidaceae alkaloids from L. radiata bulbs were traditionally used for treating sore carbuncle, neurodegenerative diseases, poliomyelitis, suppurative wounds, and ulcers [50,51,52]. In addition, L. radiata is an important greening material for energy-saving medicinal construction and a genetic treasure trove for the drought resistant breeding of Lycoris and other medicinal plants. Thus, it is of great value for deeply studying the drought resistance of Lycoris. At present, amaryllidaceae alkaloid and anthocyanin biosynthesis as well as sucrose degradation have been identified in Lycoris plants through transcriptome sequencing [53,54,55,56,57,58]. In this work, 31 LrWRKY genes were identified based on our previous L. radiata transcriptome data [53] and their motif pattern and phylogenetic relationship between Arabidopsis and L. radiata were analyzed. Subcellular localization analysis revealed that these LrWRKY proteins were mainly localized in the nucleus. The transcriptome datasets and the qRT-PCR results showed that the expression levels of LrWRKY genes changed in different tissues as well as under MeJA treatment and drought stress. To better comprehend the molecular mechanism of petal color formation, we identified LrWRKYs, the key structural genes and metabolites that are involved in anthocyanin biosynthesis, combining gene expression and anthocyanin metabolome analyses. Moreover, the probable protein–protein interaction (PPI) of LrWRKYs were also predicted. Our results provide important insights into the LrWRKY genes in L. radiata and lay a foundation for the further investigation of LrWRKY genes functioning in the biological pathway.

2. Results

2.1. Identification and Characterization of LrWRKY Proteins in L. radiata

TWe used HMMER 3.0 and BLASTP to predict putative LrWRKY protein sequences in the L. radiata transcriptome database with an E-value threshold of <1 × 10⁻⁵. All the candidate sequences were confirmed in NCBI and with SMART to further identify the conserved complete WRKY domains. Thirty-one LrWRKY proteins ranged from LrWRKY1 to LrWRKY31 were identified and their basic characteristics are shown (Table S1). The coding sequence lengths of LrWRKY genes were varied from 459 bp to 1830 bp. The LrWRKY proteins ranged from 153 to 610 amino acids in size, from 17.41 to 66.53 kDa in molecular weight, and from 4.80 to 9.83 in their isoelectric points. The subcellular localization of the LrWRKY proteins was predicted by using ProtComp 9.0, Plant-mPLoc, and WOLF PSORT. It showed that most of the LrWRKY proteins were probably located in the nucleus, and few LrWRKY proteins were located in the endoplasmic reticulum and chloroplast. Sixteen LrWRKY proteins showed the highest sequence similarity with Asparagus officinalis WRKY proteins after homologous alignments. LrWRKY4 and LrWRKY24 had the highest sequence similarity with the Narcissus hybrid cultivar homologous protein. LrWRKY13 had the highest sequence identity with the Narcissus tazetta subsp. homologous protein.

2.2. Phylogenetic Analysis and Classification of LrWRKY Proteins

To investigate the evolutionary relationships of the LrWRKY and Arabidopsis WRKY (AtWRKY) proteins, a phylogenetic tree was constructed (Figure 1). The results showed that 31 LrWRKY proteins were clustered into three subfamilies consistent with the tree topology and classification of AtWRKY proteins. Meanwhile, LrWRKY proteins could be categorized into Group I, Group IIa, Group IIb, Group IIc, Group IId, Group IIe, and Group III. Ten LrWRKY proteins were classified as Group I, containing two WRKY domains and one C2H2 zinc-finger motif. A total of 18 LrWRKY proteins included one WRKY domain and one C2H2 zinc-binding motif, which were considered to be Group II. Three LrWRKY genes were categorized to Group III, consisting of a single WRKY domain and a C2CH zinc-binding motif. According to the Arabidopsis WRKY subgroup classification, the LrWRKYs in Group II were further subdivided into five subgroups, including groups IIa (1), IIb (2) IIc (6), IId (5), and IIe (4) (Figure 1). These results revealed that WRKY proteins existed in various plant species before their divergence and then independently evolved in each species.

2.3. Multiple Sequence Alignment, Conserved Motifs, and WRKY Domains of LrWRKY Proteins

To further understand the diversification and conservation of LrWRKY proteins, the conservative motifs of LrWRKYs were predicted by utilizing MEME software. We found that LrWRKY proteins contained a different number of the 10 conserved motifs (Figure 2 and Figure S1). Motif 1 and Motif 2 were the two highly conserved motifs present in most LrWRKY proteins except for LrWRKY6, LrWRKY9, LrWRKY11, LrWRKY21, LrWRKY25, and LrWRKY28. The WRKY in the same group contained similar motif structures. Motif 1 and Motif 4 were WRKY domains; Motif 1 was distributed the whole groups, whereas Motif 4 was present in Group I and IIc. Motif 10 was unique to Groups IId, whereas Motif 8 to Group I and Motif 9 to Group IIe. The clustered LrWRKY pairs, such as LrWRKY1/2, LrWRKY6/28, LrWRKY12/18, and LrWRKY22/23, displayed a highly similar motif distribution (Figure 2). In addition, the same subgroup of LrWRKY proteins has similar motifs, indicating that the protein structure of this family was conserved. The conservative motif arrangement and phylogenetic analysis of the same group proteins can be used as an important basis for categorizing protein.

In addition, conserved amino acids in the WRKY domain analysis via multiple sequence alignments of the LrWRKY proteins were performed (Figure 3). Among the 31 LrWRKY proteins, 9.55% of the proteins have homology in the “WRKYGQK” core domain, which is used for defining classification of the WRKY TFs, while only two proteins (LrWRKY1 and LrWRKY2) have “WRKYGKK” domain (Figure 3). The LrWRKY proteins also had the CX4–7-C-X22–23-H motif, forming C2HC or C2H2 zinc-finger structures.

2.4. Multiple Sequence Alignment, Conserved Motifs, and WRKY Domains of LrWRKY Proteins

To further characterize the functions of LrWRKY TFs, GO annotation and enrichment analysis were performed for all the LrWRKYs. The LrWRKY proteins were annotated to three main GO categories (biological process, cellular component, and molecular function), including 55 biological process terms, nine molecular function terms, and one cellular component terms. The top 20 GO terms of level two were visualized (Figure 4). In biological process terms, 31 LrbWRKY proteins were implicated in the regulation of transcription. Most LrWRKYs regulate a defense response to bacterium, response to chitin, defense response to fungus, metal ion binding, regulation of defense response, response to jasmonic acid and SA, as well as response to water deprivation. In cellular component terms, 31 LrWRKY proteins were components of the nucleus. In molecular function terms, 31 LrWRKY proteins were categorized as exhibiting DNA-binding transcription factor activity and sequence-specific DNA binding, respectively (Figure 4).

2.5. Multiple Sequence Alignment, Conserved Motifs, and WRKY Domains of LrWRKY Proteins

As the transcriptome analysis of different tissues (root, leaf, and bulb) has been revealed in Lycoris longituba [57], we then searched the orthologous genes of LrWRKY by using local blast within L. longituba transcriptome data; 31 orthologous genes of LrWRKY were found in L. longituba (Table S1b). As indicated in Figure S2, some LrWRKY genes exhibited differential expressions in the three tissues, whereas other LrWRKY genes showed similar expression patterns in diverse tissues. For example, two LrWRKYs, including LrWRKY14 and LrWRKY27, were relatively highly expressed in leaves, whereas LrWRKY22 and LrWRKY23 were preferentially expressed in roots. LrWRKY3, LrWRKY13, LrWRKY16, and LrWRKY30 had the highest relative expression levels in bulb. To further elucidate the biological function of LrWRKY proteins, qRT-PCR was utilized to determine the spatial specificity expression pattern of 31 LrWRKY genes in eight L. radiata organs. As indicated in Figure 5A, some LrWRKY genes exhibited differential expression in the eight tissues, whereas other LrWRKY genes showed similar expression patterns in diverse tissues, which could be attributed to the functional differentiation of LrWRKY genes during plant development. For example, three LrWRKYs (i.e., LrWRKY11, LrWRKY12, and LrWRKY17) were relatively highly expressed in leaves. LrWRKY22 and LrWRKY23 were preferentially expressed in petals. LrWRKY29 showed high expression levels in gynoecium, whereas LrWRKY30 had relatively high expression levels in bulb. In addition, five LrWRKYs (i.e., LrWRKY2, LrWRKY5, LrWRKY13, LrWRKY16, and LrWRKY19) were highly expressed in stamens tissues. In particular, LrWRKY4, LrWRKY6, LrWRKY10, LrWRKY18, LrWRKY24, and LrWRKY31 were predominantly expressed in roots. LrWRKY15 was relatively highly abundant in seeds. LrWRKY1 and LrWRKY25 had the highest relative expression levels in flower stalks. Among all the tissues, the fewest LrWRKY members were expressed the most in the roots and leaves. Conversely, some genes were not expressed specifically. We noticed that similar expression patterns for LrWRKY7, LrWRKY9, LrWRKY10, LrWRKY24, LrWRKY28, and LrWRKY31 in different tissues suggest possible redundancy. These results suggested that LrWRKYs might play important functional roles in the growth and development of L. radiata.

On the basis of tissue-specific expression, the expression of LrWRKYs was further observed at the flowering developmental stages using previously published RNA-seq data (Figure 5B). In L. radiata flowering development, FB, FL1, FL2, and R indicated the initial stage of flower—bud differentiation, partially opening flower, fully opened flower, and senescent flower stage, respectively (Figure 5B). Over half of the LrWRKY genes were expressed lower at the FB stage and then markedly increased at the R stage. In addition, the expression of LrWRKY15 and LrWRKY31 exhibited a gradual increase during flower development. Remarkably, the expression of LrWRKY16 and LrWRKY26 genes exhibited opposite trends in the flowering developmental stages, indicating they may perform diverse functions. However, the expression of LrWRKY8, LrWRKY9, LrWRKY19, and LrWRKY30 were relatively higher at the early stages of flower development.

2.6. Expression Patterns of LrWRKYs in Response to Drought Stress and MeJA Treatment

We subsequently examined the gene expression profiles of LrWRKY in the roots and leaves of one-year-old L. radiata seedlings that were subjected to drought stress for 24 h. As shown in Figure 6A, the responses of LrWRKYs were different between individuals after drought treatment. In the L. radiata leaves and roots, the majorities of LrWRKY genes were down-regulated and showed statistically significant higher expression patterns after 24 h of drought stress compared to the control (Figure 6A). Nonetheless, the expression of LrWRKY5, LrWRKY22, and LrWRKY23 reached the highest level under drought stress in L. radiata roots. Moreover, the expression levels of LrWRKY2, LrWRKY6, LrWRKY28, LrWRKY30, and LrWRKY31 genes gradually down-regulated after drought stress in L. radiata roots. In the L. radiata leaves, LrWRKY4 and LrWRKY8 were significantly up-regulated under drought treatment. Besides, the expression levels of LrWRKY1, LrWRKY9, LrWRKY16, LrWRKY19, LrWRKY20, LrWRKY21, LrWRKY25, and LrWRKY26 gradually down-regulated under drought treatment in leaves (Figure 6A). These results suggest that LrWRKY genes regulate plant physiological processes.

Previous study of Lycoris aurea transcriptome sequencing has revealed that MeJA treatment could induce the expression of LaWRKY genes [54]. Thus, the orthologous genes of LrWRKY in L. aurea transcriptome data were also searched. All the 31 LrWRKY genes were found to have orthologous transcripts in L. aurea transcriptome (Table S1). Among them, eight homologous LrWRKYs were up-regulated, while nine homologous LrWRKY genes were suppressed with MeJA treatment for 6 h (Figure S3). In addition, homologous genes of LrWRKY5 and LrWRKY17, LrWRKY15, and LrWRKY16 exhibited no expression changes after MeJA treatment. Further, the expression patterns of the LrWRKYs tested using qRT-PCR and most LrWRKYs expression levels were up-regulated by MeJA treatment and varied over time (Figure 6B). The transcription level of LrWRKYs was up-regulated after 6 h, whereas it was down-regulated at 12 h. Specifically, the expression levels of nine genes (LrWRKY6, LrWRKY11, LrWRKY12, LrWRKY13, LrWRKY15, LrWRKY18, LrWRKY24, and LrWRKY28) increased rapidly under MeJA treatment, while those of the other ten genes (LrWRKY5, LrWRKY8, LrWRKY16, LrWRKY19, LrWRKY20, LrWRKY22, LrWRKY23, LrWRKY25, LrWRKY26, and LrWRKY31) were lower under MeJA treatment than that in the control. In addition, the expression profiles of some genes changed dramatically in specific time. For instance, the expression performance of LrWRKY7 and LrWRKY9 at 24 h were lower than those in the control but were significantly unregulated at 36 h. The expression trends of LrWRKY10, LrWRKY11, LrWRKY14, and LrWRKY22 were consistent with the transcriptome data. There were certain divergences between the actual examined and transcriptome data, which can be demonstrated by the individual divergences between the material transcriptome data and the qRT-PCR analysis. In particular, the expression pattern of LrWRKY6, LrWRKY12, LrWRKY18, and LrWRKY28 up-regulated rapidly at 6 h. After 24 h of MeJA treatment, the expression pattern of LrWRKY17 and LrWRKY21 up-regulated rapidly. From these data, it can be speculated that MeJA regulates the expression pattern of the LrWRKY genes. These findings revealed that LrWRKYs displayed different expression patterns in response to MeJA hormone treatment and potentially participated in developmental processes via hormone signaling pathways in Lycoris.

2.7. Identification of the Main Anthocyanin Pigments in L. radiata Petals

To detect the metabolic mechanism of the red petal color phenotype in L. radiata, the total anthocyanins in the petals were measured [53]. To further ascertain the metabolic mechanism of the L. radiata petals, the anthocyanin metabolites were detected using UHPLC-ESI-MS/MS; 18 anthocyanin compounds were identified and quantified in the L. radiata petals (Figure 7A). We found that the pelargonidin-3-O-glucoside-5-O-arabinoside content was the highest in the L. radiata petals, followed by Cyanidin-3-O-sambubioside (Figure 7A). Given that these two compounds may play a key role in red petal color formation in L. radiata, we propose that these anthocyanins were the main compounds responsible for the red coloration of petal margins.

2.8. Integrating Related LrWRKY Genes and Metabolites in Anthocyanin Biosynthesis Pathway

WRKY TFs are important regulators of anthocyanin accumulation and they characteristically act by controlling the structural gene expression in the anthocyanin biosynthesis pathway. To enunciate the relationship between LrWRKYs and anthocyanin biosynthesis in L. radiata, we established a co-expression network of LrWRKYs and the anthocyanin metabolites (Figure 7B,C and Table S2). The PCC between the expression performance of LrWRKYs and the anthocyanin metabolites was further calculated. The results implied that eight LrWRKYs (LrWRKY1, LrWRKY3, LrWRKY6, LrWRKY7, LrWRKY13, LrWRKY15, LrWRKY17, and LrWRKY27) positively regulated the anthocyanin metabolites, whereas four LrWRKYs (LrWRKY16, LrWRKY22, LrWRKY25, and LrWRKY29) negatively regulated the anthocyanin metabolites. Furthermore, pelargonidin-3-O-glucoside-5-O-arabinoside was strongly correlated with the expression of LrWRKYs, indicating that pelargonidin-3-O-glucoside-5-O-arabinoside play a significant role in the red petal color in L. radiata. Among these LrWRKYs, the expression level of LrWRKY3 and LrWRKY27, indicated a remarkable positive correlation with the pelargonidin-3-O-glucoside-5-O-arabinoside content in the samples (PCC > 0.9, Figure 7B,C and Table S2), suggesting that LrWRKY3 and LrWRKY27 may have an essential role in the pelargonidin accumulation.

The correlation between LrWRKYs and differently expressed genes (DEGs) of anthocyanin biosynthesis in L. radiata could be divided into three main clusters (Figure 8A and Table S3). In Cluster II, several LrWRKY genes (LrWRKY1, LrWRKY3, LrWRKY5, LrWRKY7, LrWRKY13, LrWRKY15, LrWRKY17, LrWRKY24, LrWRKY26, and LrWRKY27) showed strong correlations with phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), chalcone synthase (CHS), dihydroflavonol reductase (DFR), flavonoid 3′-hydroxylase (F3′H), and leucoanthocyanidin reductase (LAR) genes in the anthocyanin pathway. In contrast, the LrWRKYs in Cluster I showed a low correlation with anthocyanin biosynthetic pathway genes in L. radiata and only correlated strongly with upstream one PAL and one 4-coumarate:CoA ligase (4CL) genes. Some LrWRKYs in Cluster III showed a strong negative correlation with the pathway genes. The co-expression analysis showed that the anthocyanin pathway genes were relevant to LrWRKYs in Cluster II and that LrWRKY3, LrWRKY7, LrWRKY26, and LrWRKY27 were selected for their modulatory potential in anthocyanin biosynthesis. In particular, we observed that the identified anthocyanin biosynthesis DEGs and key transcription factors were significantly correlated with nine anthocyanins, including Pelargonidin-3-O-glucoside-5-O-arabinoside, Malvidin-3-O-(6″-O-malonyl) glucoside, Delphinidin-3,5,3′-Tri-O-glucoside, Delphinidin-3-O-glucoside, Cyanidin-3-O-(6″-O-p-Coumaroyl) glucoside, Cyanidin-3-O-(6″-O-p-coumaroyl-2‴-O-sinapoyl)sambubioside-5-O-(6″″-O-malonyl) glucoside, Cyanidin-3-O- (6″-O-feruloyl) glucoside-5-O-glucoside, Peonidin-3-O-(6″-O-p-Coumaroyl) glucoside, and Peonidin-3-O-(6″-O-caffeoyl) glucoside (Figure 8B and Table S3). Some crucial LrWRKYs involved in anthocyanin biosynthesis, such as LrWRKY3, LrWRKY7, LrWRKY21, LrWRKY26, LrWRKY27, and LrWRKY28 were positively related to the accumulation of nine anthocyanins and anthocyanin enzyme-encoding DEGs including anthocyanidin synthetase (ANS), 4CL, CHS, C4H, chalcone isomerase (CHI), DFR, F3′H, LAR, and PAL. These results revealed that the LrWRKY genes and anthocyanin biosynthesis genes might be involved in the petal red color in L. radiata.

2.9. PPI Networks of LrWRKY Proteins

Based on the orthologs of Arabidopsis (Table S4), the STRING database predicted that numerous LrWRKY proteins associate with each other (Figure 9; Table S5), suggesting that the WRKY proteins binding activity is based on the homodimer or heterodimer formation among WRKY proteins [59]. Generally, we predicted several key interactions (Figure 9, Table S5). We identified eight high-confidence interacting proteins in the Arabidopsis WRKY family: ASC6 (aminocyclopropane-1-carboxylic acid synthase 6) and GUN5 (genomes uncoupled 5) proteins involved in abiotic stress responses; MPK1 (mitogen-activated protein kinase 1), MPK3, and MPK4 proteins involved in plant responses to pathogens and stresses; and VQ motif-containing (VQ) proteins such as MKS1 (Meckel syndrome, type 1), SIB1 (sigma factor binding protein 1), and SIB2 involved in regulating plant defense responses. In addition, the LrWRKY10 is highly homologous to Arabidopsis WRKY33, indicating that it may have potentially stronger interactions with most plant defense proteins (MPK3, MPK4, MKS1, and SIB1). Similarly, the LrWRKY13, LrWRKY21, and LrWRKY27 protein is highly homologous to Arabidopsis WRKY40, WRKY70, and WRKY6, which are presumed to have a strong interaction with the internal members of the WRKY family. These findings partially validate the anticipated interaction networks and suggest that they may have comparable roles in L. radiata.

2.10. Subcellular Localization of LrWRKY Proteins

Based on prediction analysis, most LrWRKY proteins were predicted as localized in the nucleus, whereas few of the LrWRKYs were located in the chloroplast and/or endoplasmic reticulum (Table S1). Subsequently, certain LrWRKY proteins fused to GFP were transiently expressed in N. benthamiana epidermal cells using 35S-GFP construct as a positive vector to validate their subcellular localization. The results confirmed that LrWRKY3, LrWRKY10, LrWRKY13, LrWRKY21, and LrWRKY27 proteins were localized in the nucleus (Figure 10). Thus, these fluorescent microscopy data confirm that most LrWRKYs are nucleoproteins based on their targeting and translocation to the nucleus.

3. Discussion

3.1. Characterization of the WRKY Gene Family in L. radiata

In plants, WRKY TFs are one of the largest transcription families, playing an important role in plant physiological processes, diverse biotic and abiotic stress responses, and secondary metabolite synthesis in different crop species, including Arabidopsis, Chinese cabbage, peanut, pepper, rice, and tomato [5,7,8,18,60,61,62,63,64]. WRKY TF families have been reported in many plants, but little is known concerning the functions of the WRKY TF in L. radiata. In this study, we identified 31 LrWRKY genes through homolog search and domain analysis utilizing previously published transcriptome data (Table S1; [53]). The LrWRKYs had similar properties in terms of the encoding amino acids number, isoelectric point, and motifs to WRKYs in A. thaliana and other plants [62]. In our research, we combined the phylogeny analysis with full-length protein sequences and the classification of Arabidopsis WRKY groups to categorize the WRKY groups in L. radiata [10]. Generally, highly conserved WRKYs will group together and perform similar or identical molecular functions, pertaining to the same WRKY group (Figure 1). In general, the WRKY proteins of L. radiata classified into three groups (containing Group I, Group II (II-a, II-b, II-c, II-d, II-e), and Group III) contingent on the conserved types of WRKY domains and their zinc-finger domains, which were similar to other plants [60,61,62,63,64].

Previous studies proposed four main WRKY TF lineages in flowering plants, namely Group I + IIc, Group IIa + IIb, Group IId + IIe, and Group III, which accurately reflected the evolution of the WRKY TF family members (Figure 1; [65]), which was also verified for L. radiata. For example, the Group IIa members and Group IIb members were in the same evolutionary branch and the Group IId members and Group IIe members were grouped into the same branch, suggesting that these subgroups arose indirectly from a common ancestor. Out of the 31 members, 29 were properly conserved in the “WRKYGQK” domain. The substitution of glutamine occurs instead of lysine in two WRKY heptapeptide domains (Figure 3). This “WRKYGKK” domain sequence is reported to be a major variant in many studies [60,61,62,63,64,65,66]. The mutation in the WRKYGQK conserved domain mainly occurs from “Q” to “K” amino acid. For example, LrWRKY1 and LrWRKY2 replaced “Q” by “K” as their respective domains were also reported in Camelina sativa [66]. The same kind of substitution was observed in the WRKY gene family of Chrysanthemum lavandulifolium [17]. The motif of LrWRKY genes on the same branch commonly has a similar domain component. These results can be used as an important basis for WRKY protein classification (Figure 2). These motifs are associated with transcriptional activity and protein–protein interactions of target genes [67]. Therefore, the characterizations and functions of LrWRKY TFs can be appraised by analyzing conserved motif information. All the LrWRKYs had more than two conserved motifs, coincident with other plant species reports [68,69]. Each group member had the same or similar motif constitute, suggesting that the same group WRKY TFs have same or similar protein structures and biological functions.

3.2. The WRKY Gene Expression Patterns Facilitate Their Functional Analysis

We methodically analyzed the expression patterns of LrWRKY TFs in various tissues to investigate their physiological role in L. radiata biological processes. The homologous genes with similar expression profiles are conserved due to the dosage effect, whereas the homologous genes with different expression profiles are retained by functionalization and subfunctionalization [70]. We used transcriptome data from different tissues of two Lycoris species to determine the WRKY family gene expression (Figure 5 and Figure S2). In the present study, the LrWRKYs of Lycoris were expressed differentially in the leaves, roots, flowers, and stems. The WRKY TF genes play an important role in plant development. This indicated that LrWRKYs play a critical role in the growth and development of Lycoris. The LrWRKY genes were expressed in at least one tissue and their expression patterns varied markedly among the different tissues. As shown in Figure S2, the LrWRKY genes in roots were higher than those in the leaves and bulb. This study also analyzed the differences in eight tissues of L. radiata (Figure 5A). Notably, six LrWRKY genes displayed the highest expression patterns in roots. Three LrWRKY genes were highly expressed in the leaves, whereas one LrWRKY gene was highly expressed in the gynoecium, or seed, or bulb. Five LrWRKY genes and two LrWRKYs genes were highly expressed in stamens and in flower-stalk tissues, respectively. These tissue-specific expression patterns propose that LrWRKYs may be involved in Lycoris tissue-specific developmental and signal transduction processes. As reported, the AtWRKY70 gene has significantly contributed to Arabidopsis root development. The experimental results proclaimed that the wrky70 mutant had longer roots than the wild type [28,71]. Similarly, the orthologous protein of AtWRKY70, named LrWRKY21, was specifically expressed in the roots (Figure 5A). This research indicated LrWRKY21 may be involved in regulating the L. radiata roots growth and development. Moreover, AtWRKY75 also regulate the early accumulation of anthocyanin and increase the length and number of lateral roots and root hairs [72]. The LrWRKY3 was orthologous genes of AtWRKY75, which has difference expression levels in roots and flower (Figure 5). These results displayed the significant contribution of LrWRKY3 and LrWRKY21 in the development of roots. Moreover, LrWRKY29 is significantly expressed in the gynoecium at the L. radiata flowering stage and has a close homology with Arabidopsis AtWRKY2, suggesting that LrWRKY29 play an important role in the flowering stage of Lycoris, as Arabidopsis homologs gene AtWRKY2 are the key regulators of pollen development [73].

In addition to playing a momentous role in plant development, the WRKY TFs also play critical roles in plant abiotic stress responses [21]. In the present research, AtWRKY75 gene facilitates leaf senescence and flowering in Arabidopsis [74,75]. A recent study showed that PdeWRKY75, which is orthologous with AtWRKY75, is involved in the manipulation of multiple biological processes by regulating hydrogen peroxide content in poplar [76]. In the current research, the expression level of LrWRKY3 (homolog of AtWRKY75) was increased under drought stress, suggesting that LrWRKY3 may participate in the drought stress response mechanism of Lycoris by regulating the activity of some hormone-like proteins and enhancing the Lycoris drought tolerance (Figure 6A). The overexpression of AtWRKY8, AtWRKY28, and AtWRKY17 enhance drought or salt tolerance stress in Arabidopsis [27,77]. Similarly, the expression pattern of LrWRKY11 (homolog of AtWRKY8, AtWRKY28, and AtWRKY17) can also be induced after drought treatment and interact with some regulatory factor or unknown binding protein to regulate the expression pattern of target genes, indicating that LrWRKY11 plays an important role in drought stress tolerance in L. radiata. The expression level of AtWRKY40 induces drought stress [78] and is consistent with the LrWRKYs expression dataset results, which showed that LrWRKY13 (homolog of AtWRKY40) is involved in the drought stress response. AtWRKY33 plays a critical role in broad plant stress responses in Arabidopsis [79], suggesting that drought stress induces LrWRKY10 (homolog of AtWRKY33). Therefore, LrWRKY3, LrWRKY10, LrWRKY11, and LrWRKY13 have played potential functions in the drought stress response. The results indicate that these genes potentially are involved in the drought resistance of L. radiata. Nonetheless, further experimental studies should be carried out to enunciate the accurate regulatory mechanism through which LrWRKYs respond to drought stress.

MeJA is an importantregulator in plant growth, stress response, and secondary metabolism in both angiosperms and gymnosperms [80,81]. Many studies found that anthocyanin biosynthesis and many MeJA-responsive TFs are regulated by MeJA signaling pathway [82,83,84,85]. For example, AtWRKY11 (homolog of LrWRKY6), AtWRKY17, and AtWRKY70 (homolog of LrWRKY21) are essential components of the confrontational action in JA and the SA-mediated signaling pathway [28,86]. In addition, WRKY75 (homolog of LrWRKY3) regulated the JA-mediated signaling pathway to modulate defense responses in A. thaliana. Under normal conditions, WRKY75 interacted with Jasmonate ZIM domain (JAZ) protein to inhibit the JA signaling pathway [87]. PpWRKY46 and PpWRKY53 contribute to MeJA-primed defense by regulating the energy metabolism in peaches [88]. In tomato, SlWRKY37 positively regulates jasmonic acid- and dark-induced leaf senescence [89]. This study used publicly available transcriptome data and qRT-PCR to study the LrWRKY gene expression under MeJA induction (Figure 6B and Figure S3). LrWRKY10, LrWRKY11, LrWRKY14, and LrWRKY22 showed the same expression pattern and the expression levels of LrWRKY6, LrWRKY12, LrWRKY18, and LrWRKY28 increased significantly at 6 h under MeJA treatment. In summary, our findings provide a valuable resource for select candidate LrWRKY genes, which can facilitate the further functional studies of LrWRKYs involved in various biological stresses.

3.3. LrWRKYs Are Related to L. radiata Petal Color Change

Anthocyanins are among the key metabolites of petal color formation in plants, which have antioxidant, antiaging, and anti-inflammatory functions; the anthocyanin biosynthesis pathway has been widely investigated in many plants [90,91,92]. Previous studies have demonstrated that anthocyanin regulated the plant petal color formation and influenced pigmentation [93,94]. It has been shown that six anthocyanins (cyanidin, delphinidin, malvidin, pelargonidin, peonidin, and petunidin) are common in petal color changes [95,96]. In our research, 18 differential anthocyanins were identified in the L. radiata petals and mainly contained three categories of cyanidin, peonidin, and pelargonidin (Figure 7A). Additionally, most anthocyanin compounds showed significantly high expressions in the red petals; pelargonidin (pelargonidin-3-O-glucoside-5-O-arabinoside) may especially be the main source of red petal formation. Similar results have also been acknowledged in previous academic studies [95,96,97]. Furthermore, some key structural genes (such as DFR, 4CL, CHI, ANS, CHS, and F3′H) involved in anthocyanin biosynthesis were also found based on RNA-seq profiling and were significantly up-regulated, indicating that WRKY TFs have a greater effect on anthocyanin [53].

In recent years, many studies have investigated the conservation of WRKY-based regulatory mechanisms in the anthocyanin biosynthesis pathway, such as in Arabidopsis, in which WRKY TFs combined with the MBW (MYB-bHLH-WD40) complex to regulate the anthocyanin biosynthesis pathway and control flavonoid pigment pathways [98,99]. In addition, WRKY was found to be involved in rhododendron color formation [100]. These studies illustrate that WRKY TFs play an important role in the pigment pathway. In our research, LrWRKY3 showed that significantly different expression levels in petals might be candidate regulators of anthocyanin accumulation in petal color changes in L. radiata (Figure 7 and Figure 8). The gene—metabolite correlation network showed that the metabolite pelargonidin-3-O-glucoside-5-O-arabinoside was strongly correlated with LrWRKY3 in the anthocyanin biosynthesis pathways (Figure 7). Interestingly, our results also showed that the LrWRKY3 transcription factor specifically interacted with the ANR and LAR gene, suggesting that it is involved in petal coloration in L. radiata (Figure 8). The WRKY transcription factors participate in the synthesis of anthocyanins across a diversity of different species; therefore, more research is needed to understand how WRKY has evolved throughout angiosperms and what function it serves in other lineages.

4. Materials and Methods

4.1. Plant Materials and Plant Treatments

Lycoris radiata (L′Her.) Herb. was planted in the Experimental Plantation of the Institute of Botany, Jiangsu Province and the Chinese Academy of Sciences, Nanjing, China. The seedlings with the same or similar sizes (2.8–3.2 cm) in diameter were transferred into plastic pots with a mixture of vermiculite and soil (1:1, v/v) and maintained in a plant growth chamber under the following conditions: 16 h light/8 h dark cycle at 25 °C/22 °C and 120 μmol m⁻² s⁻¹ irradiation). After one week of maintenance, the seedlings were subjected to 100 μmol L⁻¹ methyl jasmonate (MeJA) for 0 h, 6 h, 12 h, 24 h, and 36 h and drought stress (20% PEG6000) for 24 h. Each treatment was replicated three times. The tissue-specific transcription profiles of the LrWRKY genes were explored in the petals, flower-stalks, gynoeciums, stamens, leaves, seeds, roots, and bulbs of these plants. All the samples were immediately frozen in liquid nitrogen and stored at −80 °C.

4.2. Transcriptome-Wide Identification and Expression Profiling of LrWRKY Genes

The L. radiata transcriptome database during the four flower development stages with 87,584 unigenes was used for potential LrWRKY searching [53]. The AtWRKY proteins downloaded from TAIR (Arabidopsis Information Resource database, https://www.arabidopsis.org/ (accessed on 20 September 2022)) were utilized to determine the sequence homology with L. radiata transcripts from the database using basic local alignment (BLASTn). The hidden Markov model (HMM) profile of the WRKY DNA binding domain (PF03106) obtained from the Pfam protein family database (http://pfam.xfam.org (accessed on 20 September 2022)) [101] was used to search candidate LrWRKY genes. Then, we verified the WRKY domain in the predicted LrWRKY transcription factors utilizing the NCBI Batch CD-Search Tool (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi (accessed on 20 September 2022)) with default parameters. This characteristic was deemed to have a high-confidence association with the conserved domain. The sequences predicted as specific hits were retained for further analysis (Table S4). Furthermore, the PFAM and SMART (http://smart.embl-heidelberg.de/ (accessed on 20 September 2022)); [102]) databases were used for verifying the WRKY domain in all the candidate protein sequences. Finally, the ExPASy website (https://web.expasy.org/protparam (accessed on 20 September 2022)) [103]) was utilized to determine the full length of the amino acid sequences, isoelectric points (PI), molecular sizes (MW), and protein instability index.

4.3. Phylogenetic Tree and Protein Motif Analyses of LrWRKY Proteins

The phylogenetic tree of WRKYs from L. radiata and A. thaliana was constructed with MEGA7 using the neighbor-joining method (https://www.megasoftware.net/ (accessed on 20 September 2022)). The LrWRKY proteins were then classified according to their phylogenetic relationships with AtWRKY proteins. The online tool MEME (Multiple EM for Motif Elicitation, version 5.1.1) was utilized to search for the conserved motifs of LrWRKY proteins (https://meme-suite.org/meme/tools/meme; [104] (accessed on 20 September 2022)), with the motif number and a width of 21–50 for each gene. The motifs were also searched in protein databases using the SMART program (http://smart.embl.de/ (accessed on 20 September 2022)).

4.4. Gene Ontology Annotation of LrWRKYs

The Gene Ontology (GO) functional annotations were conducted using KOBAS. The top 20 functional terms for credibility in the three categories (biological processes, cellular components, and molecular function) were selected for visualization.

4.5. Expression Profiles Analysis and Real-Time Quantitative PCR Analysis of LrWRKYs Genes

The RNA-seq data for the LrWRKY genes were obtained from previous studies on gene expression in different flower developmental stages and MeJA treatment [53,54]. The LrWRKY gene expression profiles were evaluated utilizing the values of FPKM. TBtools [105] software was utilized to generate LrWRKYs expression heatmaps. The total RNA was extracted using an RNA prep Pure Plant Kit (Cat BC508, Huayueyang, Beijing, China) according to the manufacturer’s protocol. The cDNA was synthesized by using a PrimeScript™ II 1st Strand cDNA Synthesis Kit (Takara Bio, Dalian, China) and utilized for qRT-PCR assays. The relative expression levels of the genes were analyzed using qRT-PCR with SYBR^® Premix Ex Taq™ II (Takara Bio, Dalian, China) on a Bio-Rad iQ5 Real-Time PCR System (Bio-Rad, Hercules, CA, USA) in 15 μL reactions. Each reaction contained 7.5 μL 2 × TransStart^® Top Green qPCR SuperMix, 5.9 μL ddH2O, 1μL cDNA, and 0.6 μL of 10 μM forward and reverse primers, and 6.2 μL of ddH2O. The RT-qPCR protocol included the following: the PCR reaction conditions were at 95 °C for 5 min; denaturation 5 s at 95 °C; 60 °C for 30 s; and 40 cycles. The LrTIP41 gene was utilized as an endogenous control to normalize the relative expression levels based on the 2^−ΔΔCt method [106]. The specific primer sequences used in this research are listed in Supplementary Table S6.

4.6. GenesGene Cloning and Construction of Expression Vectors

The LrWRKYs were cloned based on putative ORFs of unigenes from the RNA-seq database. The primers were synthesized for ORF sequence amplification using Tks GflexTM DNA Polymerase (Takara, Dalian, China) from L. radiata petal cDNA (Table S6). The reaction conditions were: 3 min of 95 °C, 40 cycles for 30 s at 94 °C, 30 s at 58 °C, 2 min at 72 °C, and with extension at 72 °C for 10 min. The PCR products were cloned into pTOPO001 simple vectors (Genesand, Beijing, China). Afterward, those T-vectors were transferred into TOP10 competent cells (Genesand, Beijing, China) for amplification. The LrWRKYs overexpression vectors were built by linking their ORFs into a pBIN-MCS-GFP4 plant transformation vector by using the One Step Cloning Kit (Genesand, Beijing, China). Then, the 35S:LrWRKYs recombinant vectors were transformed into Agrobacterium tumefaciens GV3101 competent cells.

4.7. Anthocyanin Quantification and Data Analysis of L. radiata

The metabolite data acquisition was conducted with a UPLC-ESI-MS/MS system (Shim-pack UFLC SHIMADZU Nexera X2) (ultrafast liquid chromatography) instrument coupled to an MS/MS (Applied Biosystems 4500 QTRAP). The chromatographic separation was carried out on a Waters ACQUITY UPLC HSS T3 system (Shim-pack UFLC SHIMADZU CBM30A, see text footnote 2), which was equipped with a C18 column (1.8 mm × 2.1 mm × 100 mm), at 40 °C. The injection volume was 5 mL at a flow rate of 0.4 mL/min. The organic phase was acetonitrile (0.1% formic acid) and the mobile phase was ultrapure water (0.1% formic acid,). The elution gradient was as follows: 0 min, 95:5 water/acetonitrile (v/v); 9.0 min, 5:95 water/acetonitrile; 10.0 min, 5:95 water/acetonitrile; 11.1 min, 95:5 water/acetonitrile; and 14.0 min, 95:5 water acetonitrile. The metabolites eluted from the HPLC were monitored for each period using scheduled multiple reaction monitoring (MRM). The MRM signals were transformed and analyzed using the Analyst 1.6.3 software (Metware Biotechnology Co., Ltd., Wuhan, China). The metabolite identification and quantification were conducted following the commercially available standard Metabolites Database [107] (Metware Biotechnology Co., Ltd., Wuhan, China) and public metabolite databases [108]. The transcriptional regulatory networks were generated by combining the Pearson correlation coefficient (|PCC| > 0.8 or |PCC| > 0.9) between structural genes and transcription factors and anthocyanins metabolites. Finally, the regulatory network maps were visualized by Cytoscape (v.3.7.2, Seattle, WA, USA).

4.8. Subcellular Localization Analysis of LrWRKY Proteins

The subcellular localization of LrWRKY proteins was predicted using WolfPsort (https://wolfpsort.hgc.jp (accessed on 20 September 2022); [109]), ProtComp 9.0 (http://linux1.softberry.com (accessed on 20 September 2022)), and Plant-mPLoc (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/ (accessed on 20 September 2022)). The coding regions of each LrWRKY gene were inserted into the pBinGFP4 plant expression vector. This vector was then used to transform A. tumefaciens strain GV3101 bacteria. The recombinant A. tumefaciens strains that contained various constructs were cultivated, harvested, followed by resuspension in an invasive solution (10 mM MES, 0.2 mM Acetosyringone, and 10 mM MgCl₂) with a final OD600 value of 0.6. Forty-day-old N. benthamiana plants were used for infiltration. After infiltration, the plants were grown at 22 °C in the dark and then transferred to normal growth conditions (25 °C/16 h light and 22 °C/8 h dark cycle) for three days. The GFP fluorescent signals in the epidermal cells of N. benthamiana leaves were observed under a confocal microscope (Zeiss LSM900, Jena, Germany).

4.9. PPI Network Prediction of LrWRKY Proteins

The potential protein–protein interaction networks were predicted using the STRING online database (version 11.5) based on A. thaliana homologous proteins (https://cn.string-db.org (accessed on 20 September 2022); [110]). The protein sequences of 31 LrWRKYs (Table S4) were uploaded into the server selecting A. thaliana as the comparative organism. The LrWRKY gene interaction network was constructed after blasting with the highest bitscore.

4.10. Statistical Analysis

All the experiments were independently duplicated at least three times. The results were represented as mean ± SD of biological triplicates. The student t-test was utilized for data analysis. * p < 0.05 and ** p < 0.01 were regarded significant.

5. Conclusions

In this study, 31 LrWRKY genes were identified from the transcriptome of L. radiata and enriched by systematically analyzing the basic biochemical information, phylogenetic relationships, conserved motifs, and gene expression profiling. Most LrWRKY genes were revealed to likely play various important roles in Lycoris growth and development, especially in flower development stages. The analysis of the expression pattern of LrWRKY genes in response to drought stress and MeJA treatment suggested that the LrWRKY genes could be importantly involved in drought stress as well as in JA signaling pathway. The gene—metabolite correlation network analysis revealed a potential regulatory role of LrWRKYs in anthocyanin biosynthetic pathway. A PPI analysis of LrWRKY proteins was performed to explore the functions and regulatory mechanisms in L. radiata. In conclusion, this study provides valuable information for further research on the regulatory mechanisms of WRKY TFs in plant growth, secondary metabolism, and resistance to various stressors.