The biosynthesis pathway of crocins.
Classification of full-length CsWRKY amino acid sequences with different conserved domains.
Classification of 34 CsWRKY proteins. (A) Conserved motif distribution of the CsWRKYs. (B) The sequences of the 15 motifs.
Neighbor-joining Jones-Thornton-Taylor (JTT) model of the phylogenetic tree.
Multiple sequence alignment of the conserved CsWRKY domain.
Coexpression analysis of CsWRKY and crocin biosynthesis-related genes. (A) Correlation matrix heatmap. Red color indicates a positive correlation. Blue color indicates a negative correlation. The correlation coefficients were calculated using the Spearman correlation coefficient. (B) Coexpression network. The blue circle nodes represent CsWRKYs, where W1-34 refers to CsWRKY1 to CsWRKY34, respectively. The red circle nodes represent crocin biosynthesis-related genes. Solid and dashed lines (edges) represent positive and negative coexpression correlations, respectively. The width of the edges reflects the strength of the correlation.
Expression profile of key CsWRKYs in different tissues and developmental stages as measured by the quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). (A) Expression of 15 CsWRKYs in different tissues. (B) Expression of five CsWRKYs in different developmental stages.
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Crocins comprise a family of hydrophilic carotenoids with pharmacological properties that are produced in significant quantities in stigma of Crocus sativus. Although the biosynthesis pathway of crocins has been sufficiently elucidated, there is a paucity of information regarding how transcription factors (TFs) regulate crocin biosynthesis in various stigma developmental stages. WRKY TFs play a role in modulating carotenoid/apocarotenoid metabolism. To provide an overview of the WRKY family in Crocus sativus (CsWRKY) and characterize candidate CsWRKY TFs involved in the biosynthesis of crocins, CsWRKY genes were identified from RNA-sequenced stigma at different developmental stages. A phylogenetic analysis was performed to characterize their evolutionary interrelatedness. A coexpression analysis of CsWRKY genes and crocin biosynthesis-related genes was performed. A quantitative real-time polymerase chain reaction was used to corroborate the expression level of CsWRKY TFs in various tissue and at different developmental stages. A total of 34 CsWRKY TFs were identified from the stigma of C. sativus. The CsWRKY TFs, together with their orthologs from Arabidopsis, were clustered into group I, II, or III following phylogenetic analysis. A correlation analysis revealed that the expressions of the TFs CsWRKY1, CsWRKY2, CsWRKY8, CsWRKY10, CsWRKY15, and CsWRKY28 were strongly related to the expression of crocin biosynthesis-related genes CsBCH, CsCCD2L, CsALDH, and CsUGT. CsWRKY2, CsWRKY15, and CsWRKY28 exhibited identical motifs and were stratified into group IId. Transcript levels of candidate CsWRKY genes were higher in stigma than in other tissues and were proportional to the crocin content.
Crocus sativus L. (Iridaceae), commonly known as saffron, is a sterile triploid plant propagated vegetatively through corms (Husaini et al. 2022). The dried red stigma of C. sativus harnesses several compounds with medicinal properties that are beneficial to human health (Diretto et al. 2019; El Midaoui et al. 2022; Moradi et al. 2022). These include the apocarotenoids crocin, picrocin, and safranal that are produced in the crocin biosynthesis pathway and constitute major metabolites in the stigma of C. sativus (Ahrazem et al. 2022). The apocarotenoids accumulate in the stigma of the flower at specific developmental stages.
Over the past decade, the biosynthesis pathway of crocins, the characteristic apocarotenoids in the stigma of C. sativus, has been elucidated (Fig. 1) (Demurtas et al. 2018). Apocarotenoids originate from the cleavage of zeaxanthin at the 7,8:7′,8′ position in a reaction catalyzed by a specific carotenoid cleavage dioxygenase enzyme (CsCCD2L) (Frusciante et al. 2014). As result of this cleavage reaction, crocetin dialdehyde is produced and further catalyzed by CsALDH to yield crocetin, which is the key precursor of crocins (Gómez-Gómez et al. 2018). Finally, the glycosylation of crocetin by various UDP-glucosyltransferases (CsUGT) results in different crocin isoforms (López-Jimenez et al. 2021). However, the mechanism that regulates the accumulation of crocins in C. sativus and the temporal dynamics of this process have remained relatively elusive (Ashraf 2015; Bhat et al. 2021; Malik and Ashraf 2017).
Citation: J. Amer. Soc. Hort. Sci. 149, 5; 10.21273/JASHS05414-24
In our previous work, we enumerated various transcription factor (TF) families, including WRKY, bHLH, MYB, and AP2/ERF, from the stigma of C. sativus by transcriptomics (Gao et al. 2021). The WRKY TFs are among the largest families of transcriptional regulators in plants and occupy a central role in the regulation of secondary metabolites biosynthesis, defense signaling, plant growth and development, and responses to biotic and abiotic factors (Wang et al. 2023; Wani et al. 2021; Zhang et al. 2018). All known WRKY proteins are characterized by one or two WRKY domains. These domains span ∼60 conserved amino acid residues and contain the heptapeptide WRKYGQK in the N-terminal region and a C2H2 or C2HC-zinc finger motif at the C-terminal region. The WRKY proteins exhibit regulatory function by binding to cis-regulatory elements located in the 5′ upstream region of the target gene in a sequence-specific manner (Goyal et al. 2023).
In recent years, many reports of the regulatory function of WRKY TFs in the biosynthesis of various secondary metabolites such as flavonoids (Zhang et al. 2023) and alkaloids (Huang et al. 2023; Zhang et al. 2018) have been published. Increasing evidence of other species has demonstrated that WRKY TFs are involved in the regulation of carotenoid/apocarotenoid metabolism by modulating the expression of functional genes in the respective metabolic pathways (Liang and Li 2023). For instance, SlWRKY35 can regulate carotenoid biosynthesis by directly activating the expression of the SlDXS1 gene in tomato. Coexpression of SlWRKY35 and SlLCYE can promote lutein production in transgenic tomato (Yuan et al. 2022). In Osmanthus fragrans, OfWRKY3 was characterized as a positive regulator of the OfCCD4 gene (Han et al. 2016). CrWRKY42, another WRKY TF in citrus, was found to accelerate the accumulation of carotenoids by directly binding to the promoter regions of the CrBCH1, CrPDS, and CrLCYB2 genes, leading to their expression (Chen et al. 2024). Collectively, these data suggest the involvement of WRKY genes in the regulation of carotenoid/apocarotenoid biosynthesis in C. sativus.
The present study aimed to provide an exhaustive overview of the WRKY family TFs in the stigma of C. sativus and identify candidate WRKY regulatory genes involved in crocin biosynthesis. We characterized 34 CsWRKY genes from RNA-sequuencing data derived from C. sativus (Gao et al. 2021). Conserved domains and motifs were predicted. Further phylogenetic and multiple sequence alignment (MSA) analyses were performed to elucidate the functional and evolutionary aspects. To ascertain the regulatory function of CsWRKY in terms of crocin biosynthesis-related genes, a coexpression correlation analysis of these genes was performed. In addition, the expressions of candidate CsWRKYs genes in various tissues and at different developmental stages were determined by a quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). This study yielded information regarding the role of CsWRKYs in stigma of C. sativus and enumerated several candidate regulators of crocin biosynthesis.
Saffron was planted in Jiaxing, Zhejiang, China. Stigmas were collected at different developmental stages [−3 da (days after flowering), −2 da, da, +1 da, and +2 da] and sequenced as detailed previously (Gao et al. 2021). Briefly, RNA sequencing was outsourced to Shanghai Majorbio Co. (Shanghai, China) and performed using an Illumina HiSeq 2000 system. The WRKY genes in C. sativus stigmas were identified from the whole-transcriptome sequencing data. All sequences were revalidated using the Uniprot protein database (https://www.uniprot.org/) and Pfam (https://pfam.xfam.org/) and re-examined for the presence of WRKY domains using the conserved domain database [National Center for Biotechnology Information (NCBI); https://www.ncbi.nlm.nih.gov/cdd/] and through HMMScan (https://www.ebi.ac.uk/Tools/hmmer/search/hmmscan). The molecular weight, theoretical isoelectric point, instability index, aliphatic index, and grand average of hydropathicity of CsWRKY proteins were predicted using ProtParam (http://web.expasy.org/protparam/). Subcellular localization was predicted using an advanced protein subcellular localization prediction tool (WoLFPSORT; https://wolfpsort.hgc.jp/).
The conserved domains of 34 CsWRKYs were detected using the NCBI CD database. The MEME suite was used to analyze the conserved motifs of 34 CsWRKYs (http://meme-suite.org/tools/meme).
The MSA of 106 WRKY proteins was performed using 34 WRKY proteins of C. sativus and 72 from Arabidopsis thaliana (AtWRKYs). The protein sequences of Arabidopsis were downloaded from TAIR (http://www.arabidopsis.org/). The conserved regions of 34 amino acids for the WRKY proteins were searched using HMMScan and aligned using CLUSTALW for the construction of the phylogenetic tree. For the CsWRKY-based phylogenetic tree, complete protein sequences were used. The tree was constructed using MEGA 7.0 with the neighbor-joining method using the JTT substitution model and the pairwise deletion method with a 1000 bootstrap value. The conserved region of MSA of Crocus sativus with 34 amino acids was visualized using DNAMAN. The MSA included the conserved region of WRKY members that represented each group and subgroup from A. thaliana as a reference.
Eight crocin biosynthesis-related genes (CsCCD2L, CsBCH, CsUGT74AD1, CsUGT709G1, CsALDH3I1, CsALDH3898, CsALDH20158, and CsALDH54788) were obtained from previously published work (Diretto et al. 2019; Frusciante et al. 2014; Gómez-Gómez et al. 2017). The whole coding sequences (CDS) of these crocin biosynthesis-related genes (CsCCD2L, GenBank: KP887110.1; CsBCH, GenBank: AJ416711.2; CsUGT74AD1, GenBank: MF596166.1; CsUGT709G1, GenBank: KX385186.1; CsALDH3I1, GenBank: MF596165.1; CsALDH3898, GenBank: KU577905.2; CsALDH20158, GenBank: KU577906.2; CsALDH54788, GenBank: KU577904.2) were obtained from NCBI (https://www.ncbi.nlm.nih.gov/). The RNA sequencing unigenes, which were annotated as WRKY genes in the NR database, were blasted with the downloaded whole CDS. The 34 unigenes that matched with the whole CDS were used for further analyses. For the coexpression network, we used the fragments per kilobase of transcript per million mapped reads of the 34 CsWRKYs and 8 crocin biosynthesis-related genes from the stigma developmental stages. Correlation was determined using the Spearman correlation coefficient. The correlation matrix heatmap was constructed with an online tool (Majorbio Cloud Platform; https://cloud.majorbio.com/page/tools/). The coexpression network analysis was subsequently conducted using the Majorbio Cloud Platform with a Spearman correlation coefficient cutoff of ≥0.7. The interaction network was visualized using Cytoscape 3.9.1.
Total RNA of C. sativus stigmas from different developmental stages (−3 da, −2 da, da, +1 da, and +2 da) and different tissues (petal, leaf, corn, stamen, and stigma) were extracted using TRIzol reagent (Tiangen, Shanghai, China). The quality and quantity of the total RNA were determined using a NanoDrop 2000C spectrophotometer (Thermo Scientific, Waltham, MA, USA). Total RNA was used to generate complementary DNA using M-MLV Reverse Transcriptase (TaKaRa Bio, Kusatsu, Shiga, Japan) according to the manufacturer’s instructions. A qRT-PCR was performed using the SYBR Premix Ex Taq Kit (TaKaRa Bio); then, it was conducted using an ABI 7300 RT-PCR system (Applied Biosystems, Foster City, CA, USA). Each sample was assayed in triplicate. Gene-specific RT primers were designed using Primer Premier 6 according to the full-length base sequence from the RNA-seq data. The amplicon size ranged from 150 to 250 bp. The actin gene was selected as an internal reference gene. All PCRs were performed under following conditions: 95 °C for 30 s, 95 °C for 5 s, 55 °C for 30 s, and 72 °C for 30 s for a total of 40 cycles. The relative mRNA expression levels of CsWRKY genes were calculated using the 2-ΔΔCt method. The expression level of key CsWRKY genes was presented in heatmap format constructed using GraphPad Prism (GraphPad Software, San Diego, CA, USA). To compare the expression profiles of multiple experimental designs with each other, Tukey’s multiple comparisons test was performed (* P < 0.05; ** P < 0.01; *** P < 0.001).
A transcriptomics analysis of the stigma of C. sativus was performed and data pertaining to WRKY TFs were mined. Among the 63 sequences that were annotated and validated as WRKY genes, 34 CsWRKYs had complete CDS and 30 gene sequences had partial CDS (Table 1). The CsWRKYs sequences were submitted to NCBI (Table 1 and Supplemental File 1). The size of the CsWRKY proteins varied dramatically. The CDS length of the 34 CsWRKY genes ranged from 519 to 2088 bp, and the amino acid length ranged from 172 to 695 amino acids (Table 1). Of these, CsWRKY23 was the longest, and CsWRKY29 was the shortest; their molecular weight varied from 19,099.46 dalton (Da) (CsWRKY29) to 75,517.75 Da (CsWRKY23) (Table 2). The isoelectric point of 17 CsWRKYs was acidic, wherea the remaining 17 were basic proteins. All of the CsWRKYs were unstable, exhibiting a maximum instability index of 69.46 (CsWRKY17) and a minimum instability index of 41.53 (CsWRKY12) (Table 2). An instability index higher than 40.0 was considered unstable (Yang et al. 2018). Additionally, the WoLFPSORT prediction model revealed that 33 CsWRKY proteins were localized in the nucleus, suggesting that these proteins are involved in genetic regulation, whereas 11 CsWRKY had cytoplasmic subcellular localization (Table 2). Thirty-two CsWRKYs had a distinctive hepta-peptide DNA binding sequence (WRKYGQK), which is the identifying characteristic of the WRKY family, with exception of three CsWRKYs, namely CsWRKY6 and CsWRKY34 substituted with a WRKYGEK domain and CsWRKY11 substituted with a WRKYGKK domain (Table 1).
A conserved domain analysis showed that 10 CsWRKYs possessed additional domains besides the WRKY domain (Fig. 2). For example, CsWRKY1, CsWRKY25, and CsWRKY26 possessed the PHA03247 superfamily domain (170 amino acid residues) at the N-terminal, CsWRKY9 contained a lipocalin FABP super family domain (130 amino acid residues) at the N-terminal, whereas six CsWRKYs (CsWRKY2, CsWRKY3, CsWRKY15, CsWRKY20, CsWRKY28, and CsWRKY33) had a plant zinc cluster domain (36 amino acid residues) at the C-terminal (Fig. 2). The conserved motifs of CsWRKY proteins were predicted based on MEME online software. A total of 15 conserved motifs, named motif 1 to motif 15, were elucidated (Fig. 3). The number of motifs in CsWRKYs varied from one to seven, and the length of motifs ranged from 21 to 107 amino acids. CsWRKY21, CsWRKY22, CsWRKY23, and CsWRKY24, which belonged to group IC, contained the largest number of motifs, whereas CsWRKY6 and CsWRKY34 had only one motif (Fig. 3A). Motif 1 formed the main structure of the WRKY domain (WRKYGQK), which prevailed in all of the CSWRKYs proteins. Motifs 1 and 2 were present in groups IC, IIC, IIa+IIb, and III+IId. Motif 3 and motif 5 were only found in group IN. Motifs 4 and 9 were only observed in CsWRKY2 and CsWRKY20, which belonged to group III+IId, whereas motif 15 was merely detected in CsWRKY8 and CsWRKY30, which belonged to group IIC (Fig. 3A and B). Based on these results, individual groups generally contained similar motif compositions and arrangement, thus attesting to highly functional conservation.
Citation: J. Amer. Soc. Hort. Sci. 149, 5; 10.21273/JASHS05414-24
Citation: J. Amer. Soc. Hort. Sci. 149, 5; 10.21273/JASHS05414-24
To investigate the evolutionary relationships of CsWRKY genes, a phylogenetic analysis was performed using 34 CsWRKY proteins and 72 WRKYs identified from Arabidopsis thaliana (Fig. 4). The results showed that all WRKYs could be categorized into one of three groups (groups I, II, and III). Fifteen were clustered in group I, and 13 CsWRKYs were assigned to group II, which was further subdivided into five subgroups: IIa+IIb (three CsWRKYs), IIc (five CsWRKYs), and IId+IIe (five CsWRKYs). Finally, four CsWRKYs were classified into group III. The conserved region of all 34 CsWRKY proteins that contained multiple sequence alignment of 60 amino acids were clustered in eight different groups and subgroups with very high homology (>70%) (Fig. 5). Group IN displayed conserved motif 1 (DGYNWRKYGQK) and the C-X4-C-X23-HXH zinc finger pattern, thus showing conservancy with 13 CsWRKY proteins. Group IC had 13 CsWRKYs that exhibited conserved motif 2 (GPN[N/H/Y]PRSYY) and motif 3 (VRKHVERA) as well as the zinc finger pattern C-X4-C-X23-HXH. Groups IIa had two CsWRKY proteins (CsWRKY4 and CsWRKY9) that displayed motif 4 (KDG[F/Y]QWRKYGQK[V/I]TRDNP), motif 5 (VKKKVQRS), and the zinc finger motif pattern C-X5-C-X23-HNH. Group IIb had one CsWRKY (CsWRKY12) with three conserved motifs, namely motif 6 (DGCQWRKYGQK), motif 7 (PPAYYRC), motif 8 (CPVRKQVQRC), and the zinc finger motif pattern C-X5-C-X23-HNH, whereas group IIc comprised five CsWRKYs (CsWRKY8, CsWRKY11, CsWRKY17, CsWRKY18, CsWRKY30) with conserved motif 9 (C[N/G/S/T]VKK[Q/R]V[Q/E]R) and the zinc finger motif pattern C-X4-C-X23-HXH. Five CsWRKYs (CsWRKY2, CsWRKY3, CsWRKY15, CsWRKY20, and CsWRKY28) that contained conserved motif 10 (YSWRKYGQKPIKGSP[H/Y]PRGYYKCS), motif 11 (RGCPARKHVER), and zinc finger motif pattern C-X5-C-X23-HXH were categorized into group IId, whereas CsWRKY14, clustered in group IIe, showed conserved motif 12 (WRKYGQKPIK[S/G]SPYPR), motif 13 (SKGC[F/L/S]A[R/K]KQV[E/D]R), and the zinc finger motif pattern C-X5-C-X23-HXH. Three CsWRKY proteins (CsWRKY6, CsWRKY16, and CsWRKY34) were eventually clustered into group III with the zinc finger motif C-X7-C-X24-HXC and no phylogenetically conserved motif.
Citation: J. Amer. Soc. Hort. Sci. 149, 5; 10.21273/JASHS05414-24
Citation: J. Amer. Soc. Hort. Sci. 149, 5; 10.21273/JASHS05414-24
WRKY transcription factors have been identified as regulators of secondary metabolite biosynthesis (Yuan et al. 2022). To gain a better understanding of the transcriptional regulatory landscape of CsWRKY in crocin biosynthesis, we conducted a coexpression analysis of 34 CsWRKYs with eight crocin biosynthesis-related genes (CsCCD2L, CsBCH, CsUGT74AD1, CsUGT709G1, CsALDH3I1, CsALDH3898, CsALDH20158, and CsALDH54788) using an online tool (Majorbio Cloud Platform). The results are presented in the correlation matrix heatmap (Fig. 6A, Supplemental File 2). CsBCH showed a significantly strong positive correlation with CsWRKY2, CsWRKY28, and CsWRKY30 (P < 0.001); however, it showed a negative correlation with CsWRKY1, CsWRKY3, CsWRKY8, CsWRKY10, CsWRKY15, CsWRKY16, and CsWRKY19 (P < 0.001). Similar strong correlations were also found between CsUGT74AD1 and CsWRKY1, CsWRKY2, CsWRKY3, CsWRKY8, CsWRKY10, CsWRKY15, CsWRKY16, CsWRKY28, and CsWRKY30. CsUGT74AD1 was negatively correlated with CsWRKY5 (P < 0.01) and positively correlated with CsWRKY20 (P < 0.001). CsALDH54788 exhibited a positive correlation with CsWRKY7 (P < 0.001) and a negative correlation with CsWRKY11 (P < 0.01). No statistically significant correlations were found between other crocin biosynthesis-related genes and CsWRKYs.
Citation: J. Amer. Soc. Hort. Sci. 149, 5; 10.21273/JASHS05414-24
To determine the key correlation between structural genes and CsWRKYs, a Spearman correlation coefficient ≥0.7 and P ≤ 0.05 were used as cutoffs for the online analysis of Majorbio Cloud Platform, and the coexpression profile was rendered in Cytoscape 3.9.1 (Fig. 6B). A total of 47 correlations (23 nodes and 47 edges) were screened, including 18 correlations between CsWRKY and crocin biosynthesis-related genes, 28 correlations in CsWRKYs, and one correlation in crocin biosynthesis-related genes. CsBCH were correlated with CsWRKY1, CsWRKY2, CsWRKY3, CsWRKY5, CsWRKY10, CsWRKY15, CsWRKY16, CsWRKY28, and CsWRKY30. CsCCD2L was correlated with CsWRKY8, CsWRKY15, and CsWRKY32, whereas CsUGT74AD1 was correlated with CsWRKY8 and CsWRKY15. CsWRKY15 was significantly associated with CsBCH, CsCCD2L, and CsUGT74AD1 (r = −0.771, 0.714, and −0.704, respectively; P < 0.05). CsWRKY8 showed correlations with CsCCD2L (r = 0.778) and CsUGT74AD1 (r = −0.778) (P < 0.05). The strongest correlation was found between CsBCH and CsWRKY1 (r = −0.869), CsWRKY2 (r = 0.907), CsWRKY10 (r = −0.9), and CsWRKY28 (r = 0.869) (P < 0.001). CsALDH20158, CsALDH3I1, and CsALDH54788 each exhibited correlations with CsWRKY4 (r = −0.725), CsWRKY13 (r = −0.822), CsWRKY17 (r = −0.704), and CsWRKY34 (r = −0.714) based on the coexpression analysis (Supplemental File 3). Taken together, these results indicated that CsWRKYs, particularly CsWRKY15, CsWRKY8, CsWRKY1, CsWRKY2, CsWRKY10, and CsWRKY28, likely modulate the biosynthesis of crocins by regulating the expression levels of CsBCH, CsCCD2L, CsUGT74AD1, and CsALDHs.
To understand the roles of CsWRKYs in the regulation of crocin content that exhibited strong correlation with crocin biosynthesis-related genes, we studied the expression levels of 15 key CsWRKYs (r ≥ 0.7; P ≤ 0.05) in different tissues (petal, leaf, corn, stamen, and stigma) using the qRT-PCR. Six CsWRKYs (CsWRKY1, CsWRKY2, CsWRKY8, CsWRKY10, CsWRKY15, and CsWRKY28) showed relatively high expression in stigma compared with that in other tissues (Fig. 7A). In addition, the expression levels of other CsWRKYs were relatively low in the stigma. Previous evidence showed that crocins accumulate in stigma in a developmental stage-specific manner (Ashraf 2015).
Citation: J. Amer. Soc. Hort. Sci. 149, 5; 10.21273/JASHS05414-24
We further evaluated the expression pattern of highly expressed CsWRKYs (CsWRKY1, CsWRKY2, CsWRKY8, CsWRKY10, CsWRKY15, and CsWRKY28) in stigma in different developmental stages. Six CsWRKYs exhibited increasing expression from stages −3 da to da; however, it subsequently decreased from stage da to +2 da (Fig. 7B). These results indicate that the expression profile of these six key CsWRKYs are consistent with the accumulation of crocins in C. stigma, thus implicating these CsWRKY TFs in regulating crocin biosynthesis.
Secondary metabolites in plants play a prominent role in multiple biological functions throughout their life span. The biosynthesis of these metabolites is accurately tuned at the spatiotemporal level by means of transcriptional regulation of functional genes involved in these pathways. This coordinated regulation generally relies on the interplay of DNA-related mechanisms and the action of corresponding transcription factors (Colinas and Goossens 2018). The expression of genes involved in metabolic pathways has evolved to become highly correlated temporally and spatially through the process of natural selection. In C. sativus, the apocarotenoids such as crocins, picrocrocin, and safranal predictably accumulated in temporal and spatial manners (Ashraf 2015). The biosynthesis pathway of apocarotenoids has been elucidated to a considerable extent (Jain et al. 2016; López-Jimenez et al. 2021; Pu et al. 2021). However, previously, there has been a dearth of information regarding the regulatory mechanism of TFs on expression patterns of function genes (Ashraf 2015; Bhat et al. 2021; Luo et al. 2023; Malik Ashraf 2017).
In recent years, it was found that WRKY TFs act in apocarotenoids metabolism in a plethora of plant species (Duan et al. 2022; Liang and Jiang 2017; Yuan et al. 2022). We reported 34 CsWRKY TFs based on transcriptome data generated in our laboratory (Gao et al. 2021) in the stigma of C. sativus. The number of CsWRKYs obtained during this study was smaller the number observed in C. sativus (n = 40) treated with MeJA (Luo et al. 2023), which confirmed that the expression of CsWRKYs varies according to the physiological status. However, these 34 CsWRKYs are not the actual number of WRKYs in this crop because our transcriptome data were created from stigma tissue only, not the entire plant.
Based on sequence similarity and the conserved domains, the 34 CsWRKY genes were stratified into groups I, II, and III. In accordance with the classification of WRKYs in other plant species, especially Arabidopsis, Glycyrrhiza glabra, and Lonicera macranthoides (Cao et al. 2022; Goyal et al. 2020), group II was further divided into five subgroups. An evolution analysis of group II showed that subgroups IIa and IIb were closely related; the same applied to subgroups IId and IIe, revealing that these two pairs had evolved from a common ancestor separately. Moreover, subgroups IId and IIe illustrated higher divergence than subgroups IIa, IIb, and IIc in the phylogenetic analysis and were closer to group III. This result is consistent with other species previously reported, including Cynanchum thesioides (Chang et al. 2022), Caragana korshinskii (Liu et al. 2023), and Taxus chinensis (Zhang et al. 2018).
Most of the known WRKY family members are characterized by a sequence of 60 highly conserved amino acids with the WRKYGQK motif in the N-terminal. Previous studies have reported several variants, including WRKYGKK, WKKYRQK (Baranwal et al. 2016), WRKYGEK, WRKYEDK, and WKKYCEDK (Wu et al. 2017). During this study, three of 34 CsWRKY proteins had a WRKYGE/KK domain instead of the characteristic WRKYGQK domain, which also occurred in banana (Goel et al. 2016) and Arabidopsis (Hussain et al. 2018). Mutations of the conserved WRKYGQK domain may change flexibility in regard to binding to the W-box element of downstream structure genes, thereby inhibiting or disordering DNA binding activities (Goyal et al. 2023). The conserved motif distribution patterns of CsWRKY genes differ among different subgroups; however, in the same subgroup, the CsWRKYs exhibited similar conserved motif distribution patterns. The extremely conserved CsWRKY TFs may be functionally conserved in other related species exhibiting similar activities in various physiological processes.
The coexpression profile is a useful approach for identifying new metabolic regulators (Li et al. 2020; Yuan et al. 2022). Based on a correlation analysis of the expression patterns of CsCCD2L, CsBCH, CsUGT74AD1, CsUGT709G1, CsALDH3I1, CsALDH3898, CsALDH20158, and CsALDH54788, which are structure genes involved in the crocins biosynthesis pathway, and 34 CsWRKY genes, we found a strong correlation between some structure genes and CsWRKY genes. For instance, CsWRKY2, CsWRKY15, and CsWRKY28, which were all clustered in group IId, exhibited a strong correlation with the CsBCH gene, whose protein product constitutes the first enzyme in the apocarotenoids biosynthesis pathway. These three WRKY genes are orthologous of WRKY11 in Arabidopsis. The ortholog of AtWRKY11 was found to modulate the biosynthesis of secondary metabolites such as anthocyanins (Wang et al. 2022) and flavonoids (Wang et al. 2021). In addition, CsWRKY8, CsWRKY15, and CsWRKY32 were significantly correlated with CsCCD2L, which is the key enzyme that mediates crocin production. Based on the phylogenetic analysis, CsWRKY8 was evolutionarily related to AtWRKY23. WRKY23 plays a role in the accumulation of flavonols in A. thaliana (Grunewald et al. 2012). Accordingly, we proposed that CsWRKY8 may regulate the production of crocins by modulating the expression of the CsCCD2L gene during the development of stigma. In saffron, CsALDHs can oxidize crocetin dialdehyde into crocetin, which is a direct precursor of crocins.
The coexpression analysis revealed that CsWRKY4, CsWRKY13, CsWRKY17, and CsWRKY34 correlated with CsALDH, suggesting that these WRKY TFs may influence the expression of CsALDH genes. Similar to CsWRKY8, CsWRKY17 was clustered in group IIc and possessed identical motifs (motif1, motif8, and motif13), indicating that CsWRKY and CsWRKY8 execute similar functions during stigma development. Finally, there were remarkable correlations between CsWRKY8/CsWRKY15 and CsUGT74AD1, the last enzyme in the crocin biosynthesis pathways. Interestingly, CsWRKY8 and CsWRKY15 correlated with two and three structure genes, respectively, consistent with the findings of previous studies (Dong et al. 2020; Zhou et al. 2016), suggesting that these CsWRKY genes may be key regulators of crocin biosynthesis. Additionally, the expression profiles of all CsWRKY genes that were obtained from the coexpression analysis of different tissue confirmed that CsWRKY1, CsWRKY2, CsWRKY8, CsWRKY10, CsWRKY15, and CsWRKY28 were highly expressed in stigma, in contrast to other tissues. As expected, the transcript levels of these six CsWRKY genes were proportional to the crocin content during stigma development. Based on these results, we speculated that these six WRKY TFs (CsWRKY1, CsWRKy2, CsWRKY8, CsWRKY10, CsWRKY15, and CsWRKY28) are likely instrumental in crocin accumulation in stigma at specific developmental stages by regulating the expression level of structure genes in the biosynthesis pathway. However, the molecular mechanisms that underlie CsWRKY-mediated modulation of crocin biosynthesis-related genes warrant further elucidation via a series of in vitro and in vivo experiments.
In conclusion, 34 CsWRKY TFs from stigma of C. sativus were characterized by the transcriptome-wide analysis. These CsWRKY TFs were divided into groups I, II, and III by virtue of the phylogenetic tree analysis. The coexpression network between CsWRKY TFs and crocin biosynthesis-related genes revealed that CsWRRKY1, CsWRKY2, CsWRKY8, CsWRKY10, CsWRKY15, and CsWRKY28 genes are potentially involved in the regulation of crocin biosynthesis; this was validated by their expression profiles in various tissue and developmental stages. This work has enumerated six candidate CsWRKY TFs and paved the way for future research of regulation mechanisms of crocin biosynthesis.
The biosynthesis pathway of crocins.
Classification of full-length CsWRKY amino acid sequences with different conserved domains.
Classification of 34 CsWRKY proteins. (A) Conserved motif distribution of the CsWRKYs. (B) The sequences of the 15 motifs.
Neighbor-joining Jones-Thornton-Taylor (JTT) model of the phylogenetic tree.
Multiple sequence alignment of the conserved CsWRKY domain.
Coexpression analysis of CsWRKY and crocin biosynthesis-related genes. (A) Correlation matrix heatmap. Red color indicates a positive correlation. Blue color indicates a negative correlation. The correlation coefficients were calculated using the Spearman correlation coefficient. (B) Coexpression network. The blue circle nodes represent CsWRKYs, where W1-34 refers to CsWRKY1 to CsWRKY34, respectively. The red circle nodes represent crocin biosynthesis-related genes. Solid and dashed lines (edges) represent positive and negative coexpression correlations, respectively. The width of the edges reflects the strength of the correlation.
Expression profile of key CsWRKYs in different tissues and developmental stages as measured by the quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). (A) Expression of 15 CsWRKYs in different tissues. (B) Expression of five CsWRKYs in different developmental stages.
Contributor Notes
This study was funded by Jiaxing Science and Technology Planning Project (no. 2023AY11048 and 2020AY10023), the National Natural Science Foundation of China (no. 81703667), and Special Project of Jiaxing Science and Technology Commissioner (no. 2021K117).
J.L. is the corresponding author. E-mail: lijun@jxvtc.edu.cn.
The biosynthesis pathway of crocins.
Classification of full-length CsWRKY amino acid sequences with different conserved domains.
Classification of 34 CsWRKY proteins. (A) Conserved motif distribution of the CsWRKYs. (B) The sequences of the 15 motifs.
Neighbor-joining Jones-Thornton-Taylor (JTT) model of the phylogenetic tree.
Multiple sequence alignment of the conserved CsWRKY domain.
Coexpression analysis of CsWRKY and crocin biosynthesis-related genes. (A) Correlation matrix heatmap. Red color indicates a positive correlation. Blue color indicates a negative correlation. The correlation coefficients were calculated using the Spearman correlation coefficient. (B) Coexpression network. The blue circle nodes represent CsWRKYs, where W1-34 refers to CsWRKY1 to CsWRKY34, respectively. The red circle nodes represent crocin biosynthesis-related genes. Solid and dashed lines (edges) represent positive and negative coexpression correlations, respectively. The width of the edges reflects the strength of the correlation.
Expression profile of key CsWRKYs in different tissues and developmental stages as measured by the quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). (A) Expression of 15 CsWRKYs in different tissues. (B) Expression of five CsWRKYs in different developmental stages.
