Phylogenetic analysis and transcriptional profiling of WRKY genes in sunflower (Helianthus annuus L.): Genetic diversity and their responses to different biotic and abiotic stresses
Introduction
Since the initial report of the first WRKY protein in sweet potato, WRKYs have been found to be widely distributed throughout the plant lineage (Ishiguro and Nakamura, 1994; Rushton et al., 2010; Zhang and Wang, 2005). For example, 72 and 102 WRKY genes have been identified in Arabidopsis thaliana and rice (Dong et al., 2003; Ryu et al., 2006; Wu et al., 2005). Homologs of WRKY genes have also been found in the slime mold Dictyostelium discoideum and the protist Giardia lamblia, implying that WRKYs may have an early origin prior to the evolution of plant phyla (Zhang and Wang, 2005). Generally, all WRKY transcription factors (TFs) contain at least one typical WRKY domain at the N-terminus; this domain contains approximately 60 residues, including an invariant WRKYGQK heptapeptide and a zinc finger structure (either the C2H2 or the C2HC type) (Eulgem et al., 2000; Rushton et al., 2010). Accordingly, this WRKYGQK motif can interact with a highly conserved cognate binding site (W-box, (C/T)TGAC(C/T)) within target gene promoters (Ciolkowski et al., 2008).
According to differences in the WRKY domain numbers and the type of zinc finger motif at the C-terminus (Eulgem et al., 2000; Rushton et al., 2010), WRKY proteins can be classified into three main groups: I, II and III. Generally, group I WRKY proteins have two WRKY domains and a C2H2 zinc finger motif (Eulgem et al., 2000); these WRKY proteins exist in lower plants and are believed to be the evolutionary ancestors of the other WRKYs (Wu et al., 2005; Zhang and Wang, 2005). The C-terminal domain is essential for W-box binding, whereas the N-terminal domain affects the binding affinity (de Pater et al., 1996). Group II and III WRKY proteins contain a single WRKY domain and harbor C2H2 and C2HC zinc finger motifs in their C-terminal region, respectively (Eulgem et al., 2000; Zhang and Wang, 2005). Furthermore, the members of group II can be more accurately subdivided into five subgroups (IIa-IIe) on the basis of phylogenetic relationships, and group II TF genes are not monophyletic (IIa + IIb, IIc, and IId + IIe) (Eulgem et al., 2000; Zhang and Wang, 2005). Rinerson et al. (2015) demonstrated that the evolutionary history of the WRKY family involves ancient lateral gene transfers to nonplant species and the occurrence of algal WRKY genes with no counterparts in flowering plants. The authors also proposed the following two alternative hypotheses for WRKY gene evolution: the group I hypothesis and the group IIa + b separate hypothesis (Rinerson et al., 2015).
WRKY proteins have been extensively studied for their important regulatory roles in plant defense and disease resistance, such as the response to salinity, drought, and fungi (Bai et al., 2018; Chen et al., 2012; Eulgem et al., 2000; Eulgem and Somssich, 2007; Pandey and Somssich, 2009; Rushton et al., 2010). The responses of plants to pathogens may be mediated by signaling pathways, such as the salicylic acid (SA), jasmonic acid/ethylene (JA/ET), and abscisic acid (ABA) pathways (Yang et al., 2009). For instance, sunflower WRKY10 (named HaWRKY106 in this study), which is regulated by ABA and gibberellins, can reduce carbohydrate metabolism and improve lipid metabolism in sunflower seed germination and seedling growth (Raineri et al., 2016). The expression of barley WRKY38, which is induced by the SA and ABA signaling pathways, has an antagonistic effect on GA-induced seed germination (Xie et al., 2007). WRKY3 was shown to enhance tolerance to salt and drought stress in grape and resistance to Golovinomyces cichoracearum in transgenic A. thaliana (Guo et al., 2018). Wheat WRKY93 is strongly induced by salt (NaCl) and exogenous ABA, leading to improved tolerance to salinity, drought, and low-temperature stress (Qin et al., 2015). Overexpression of VlWRKY48 in A. thaliana can enhance resistance to fungal infection and drought stress (Zhao et al., 2018). Despite the conserved functional domains and specific W-box recognition patterns of WRKY TFs, their function can vary. Several hypotheses have been proposed to explain this variation, including the partial dependence of binding site preferences on the sequence adjacent to the TTGACY core sequence (Ciolkowski et al., 2008), the temporal and spatial expression being determined by structural variations, and the effects of interacting partners (Andreasson et al., 2005; Cheng et al., 2012; Wang et al., 2010).
Sunflower (Helianthus annuus L.), one of the most important oilseed crop and ornamental plant species, can grow in regions exposed to constant biotic and abiotic stress, such as simultaneous drought, pathogen infection, and temperature stress (Badouin et al., 2017; Guo et al., 2017; Liang et al., 2017; Vangelisti et al., 2018). Giacomelli et al. (2010) identified 97 sunflower WRKY members derived from expressed sequence tag (EST) databases. More recently, Guo et al. (2019) identified 112 WRKY genes from sunflower; however, extensive studies of WRKY proteins from oil crop species and their involvement in disease resistance in domesticated sunflower are still limited. In this study, 119 HaWRKY proteins were identified from the sunflower genome by bioinformatic approaches, and 741 WRKY members from other oil crop species were retrieved from previous studies and updated (Bencke-Malato et al., 2014; He et al., 2016; Li et al., 2017, 2012; Song et al., 2016; Yang et al., 2009; Yu et al., 2016; Zou et al., 2016). Extensive phylogenetic variations among oil crop species and tissue-specific expression patterns in sunflower were systematically studied through comparative genomic analysis and RNA sequencing (RNA-seq) analysis. The responses of HaWRKY genes to various plant hormones, salinity, drought, and two different fungi provide further insights into the roles of these genes in the sunflower defense response. These results reflect a comprehensive comparative analysis of WRKY proteins and preliminary knowledge of specific WRKY proteins potentially involved in biotic and abiotic stress responses in domesticated sunflower.
Section snippets
Retrieval of WRKY genes
All sunflower protein sequences were obtained from the National Center for Biotechnology Information (NCBI) database. The HMM profile for the WRKY domain (PF03106) was downloaded from the Pfam database (http://pfam.xfam.org) (El-Gebali et al., 2019) and used to identify potential WRKY genes from the sunflower genome via HMMER 3.2.1 software (http://hmmer.janelia.org/), with an E-value threshold of 10−2 (Prakash et al., 2017). Only those proteins predicted to contain at least one WRKY domain by
Identification of sunflower WRKY genes and comparative analysis among oil crop species
In total, 119 WRKY genes were retrieved from the sunflower genome and renamed HaWRKY001 to 119 on the basis of their relative linear order on each chromosome, in accordance with widely used nomenclature (Fig. 1A) (Guo et al., 2014; Ling et al., 2011; Yu et al., 2016). Detailed information on these genes is provided in Table S3, including gene locus numbers, accession numbers, chromosome distribution (start and end sites), groups and CDS length. The gene length ranged from 896 to 24,025 bp.
Discussion
Domesticated sunflower, a widely cultivated oilseed crop species, can maintain stable yields under different environmental conditions, such as drought. Sunflower has been proposed as a model crop species for studying adaptations to climate change (Kane and Rieseberg, 2007; Škorić, 2016). Sunflower is cultivated on marginal soils and in semiarid conditions worldwide and has great economic value. Owing to its stress tolerance, sunflower serves as a valuable genetic resource for determining
Conclusion
Overall, all WRKY members from seven oil crop species, sunflower, sesame, castor bean, soybean, canola, A. duranensis, and A. ipaensis, were identified and characterized via comparative genomic analysis. The comparative analysis of OGs and the phylogenetic analysis revealed genetic diversity of WRKYs among oil crop species. Moreover, sequence features and phylogenetic analysis showed that these WRKY genes could be divided into seven well-defined (sub)groups (group I; subgroups IIa, IIb, IIc,
CRediT authorship contribution statement
Ake Liu: Conceptualization, Methodology, Software, Data curation, Writing - original draft, Software, Validation, Writing - review & editing. Chenglin Liu: Visualization, Investigation, Software, Validation. Haiying Lei: Data curation, Writing - original draft, Writing - review & editing. Zhijun Wang: Data curation, Writing - original draft. Min Zhang: Visualization, Investigation. Xinrong Yan: Visualization, Investigation. Guang Yang: Data curation, Writing - original draft. Jiahong Ren:
Declaration of Competing Interest
The authors declare that they have no conflict of interests regarding the submission of this manuscript and its probable publication.
Acknowledgments
We thank Funan He, Lanying Ning, Jianchuan Gong, and Jie Yuan for their technical support. We also thank three anonymous reviewers for their constructive comments on our manuscript revision. This study was supported by the Scientific and Technological Innovation Programs of Higher Education Institutions in Shanxi (2019L0897, 201802106), Shanxi “1331 Project” Key Innovative Research Team, and Shanxi “1331 Project” Key Subjects Construction (1331 KSC) and Shanxi Key Subjects Construction. The
References (80)
- et al.
The role of WRKY transcription factors in plant abiotic stresses
BBA-Gene Regul. Mech.
(2012) - et al.
Networks of WRKY transcription factors in defense signaling
Curr. Opin. Plant Biol.
(2007) - et al.
The WRKY superfamily of plant transcription factors
Trends Plant Sci.
(2000) - et al.
Expression analyses indicate the involvement of sunflower WRKY transcription factors in stress responses, and phylogenetic reconstructions reveal the existence of a novel clade in the Asteraceae
Plant Sci.
(2010) - et al.
Contrasting evolutionary dynamics between angiosperm and mammalian genomes
Trends Ecol. Evol. (Amst.)
(2009) - et al.
Expression divergence between duplicate genes
Trends Genet.
(2005) - et al.
Identification and expression profiles of the WRKY transcription factor family in Ricinus communis
Gene
(2012) - et al.
Genome-wide analysis of WRKY gene family in the sesame genome and identification of the WRKY genes involved in responses to abiotic stresses
BMC Plant Biol.
(2017) - et al.
Genome-wide identification of MYB genes and expression analysis under different biotic and abiotic stresses in Helianthus annuus L
Ind. Crop. Prod.
(2020) - et al.
- Sunflower diseases
Gene duplication and evolution in recurring polyploidization-diploidization cycles in plants
Genome Biol.
A wheat salinity-induced WRKY transcription factor TaWRKY93 confers multiple abiotic stress tolerance in Arabidopsis thaliana
Biochem. Biophys. Res. Commun.
WRKY transcription factors
Trends Plant Sci.
Transcription factors in plant defense and stress responses
Curr. Opin. Plant Biol.
Comparative genomic analysis of the WRKY III gene family in populus, grape, arabidopsis and rice
Biol. Direct
Identification and expression analysis of WRKY transcription factor genes in canola (Brassica napus L.) in response to fungal pathogens and hormone treatments
BMC Plant Biol.
The WRKY transcription factor superfamily: its origin in eukaryotes and expansion in plants
BMC Evol. Biol.
Differential expression analysis for sequence count data
Genome Biol.
The MAP kinase substrate MKS1 is a regulator of plant defense responses
EMBO J.
The sunflower genome provides insights into oil metabolism, flowering and Asterid evolution
Nature
The role of tomato WRKY genes in plant responses to combined abiotic and biotic stresses
Front. Plant Sci.
Genome-wide annotation of the soybean WRKY family and functional characterization of genes involved in response to Phakopsora pachyrhizi infection
BMC Plant Biol.
Expression profile matrix of Arabidopsis transcription factor genes suggests their putative functions in response to environmental stresses
Plant Cell
Structural and functional analysis of VQ motif-containing proteins in Arabidopsis as interacting proteins of WRKY transcription factors
Plant Physiol.
Comparative genomic analysis reveals extensive genetic variations of WRKYs in Solanaceae and functional variations of CaWRKYs in pepper
Front. Genet.
Studies on DNA-binding selectivity of WRKY transcription factors lend structural clues into WRKY-domain function
Plant Mol. Biol.
CAFE: a computational tool for the study of gene family evolution
Bioinformatics
Characterization of a zinc-dependent transcriptional activator from Arabidopsis
Nucleic Acids Res.
Expression profiles of the Arabidopsis WRKY gene superfamily during plant defense response
Plant Mol. Biol.
Expression pattern shifts following duplication indicative of subfunctionalization and neofunctionalization in regulatory genes of Arabidopsis
Mol. Biol. Evol.
The Pfam protein families database in 2019
Nucleic Acids Res.
Evolution and expression analysis of the grape (Vitis vinifera L.) WRKY gene family
J. Exp. Bot.
Large-scale transcriptome comparison of sunflower genes responsive to Verticillium dahliae
BMC Genomics
The grape VlWRKY3 gene promotes abiotic and biotic stress tolerance in transgenic Arabidopsis thaliana
Front. Plant Sci.
Genome-wide identification of WRKY transcription factors in the Asteranae
Plants Basel (Basel)
Genome-wide identification and expression analysis of WRKY transcription factors under multiple stresses in Brassica napus
PLoS One
GSDS 2.0: an upgraded gene feature visualization server
Bioinformatics
Characterization of a cDNA encoding a novel DNA-binding protein, SPF1, that recognizes SP8 sequences in the 5’ upstream regions of genes coding for sporamin and beta-amylase from sweet potato
Mol. Genet. Genomics
Genome-wide identification and characterization of the Populus WRKY transcription factor family and analysis of their expression in response to biotic and abiotic stresses
J. Exp. Bot.
Selective sweeps reveal candidate genes for adaptation to drought and salt tolerance in common sunflower, Helianthus annuus
Genetics
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2022, Industrial Crops and ProductsCitation Excerpt :The two tissues (root and leaf) were treated with 8 different hormones: ABA, β-indoleacetic acid (IAA), brassinosteroids (BRAS), 1-aminocyclopropane carboxylic acid (ACC), gibberellic acid 3 (GA3), kinetin, methyl-jasmonate (MeJA), and SA. All the transcriptome data obtained here were analyzed following our previous study (Liu et al., 2020). The sunflower cultivar TP3316, which has a relatively large seed size, was selected to study cadmium (Cd) stress.
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These authors contributed equally to this work.