The anthocyanin accumulation in juvenile tissues can enhance the ornamental value, attract pollinators, and help improve abiotic stress. Although transcriptional regulation studies of anthocyanin have been relatively extensive, there are few reports on the mechanism of anthocyanin accumulation in young tissues. This study reveals that many juvenile citrus tissues (flowers, leaves, and pericarp) undergo transient accumulation of anthocyanins, exhibiting a red coloration. Using weighted gene co-expression network analysis (WGCNA) identified CitWRKY75 as a candidate gene. After detecting the expression levels of CitWRKY75 in various citrus juvenile tissues, the expression trend of CitWRKY75 was highly consistent with the red exhibiting and fading. Overexpression of CitWRKY75 in tobacco significantly increased the anthocyanin content. LUC and yeast one-hybrid assay demonstrated that CitWRKY75 could bind to the promoter of CitRuby1(encoding the key transcription factor promoting anthocyanin accumulation) and promote its expression. Finally, comparing the expression levels of CitWRKY75 and CitRuby1 in the late development stage of blood orange found that CitWRKY75 was not the main regulatory factor for anthocyanin accumulation in the later stage. This study used reverse genetics to identify a transcription factor, CitWRKY75, upstream of CitRuby1, which promotes anthocyanin accumulation in citrus juvenile tissues.
UNASSIGNED: The online version contains supplementary material available at 10.1007/s11032-024-01490-9.

In response to biotic and abiotic stresses, the WRKY gene family plays a crucial role in plant growth and development. This study focused on Phoebe bournei and involved genome-wide identification of WRKY gene family members, clarification of their molecular evolutionary characteristics, and comprehensive mapping of their expression profiles under diverse abiotic stress conditions. A total of 60 WRKY gene family members were identified, and their phylogenetic classification revealed three distinct groups. A conserved motif analysis underscored the significant conservation of motif 1 and motif 2 among the majority of PbWRKY proteins, with proteins within the same class sharing analogous gene structures. Furthermore, an examination of cis-acting elements and protein interaction networks revealed several genes implicated in abiotic stress responses in P. bournei. Transcriptomic data were utilized to analyze the expression patterns of WRKY family members under drought and waterlogged conditions, with subsequent validation by quantitative real-time PCR (RT-qPCR) experiments. Notably, PbWRKY55 exhibited significant expression modulation under drought stress; PbWRKY36 responded prominently to waterlogging stress; and PbWRKY18, PbWRKY38, and PbWRKY57 demonstrated altered expression under both drought and waterlogging stresses. This study revealed the PbWRKY candidate genes that potentially play a pivotal role in enhancing abiotic stress resilience in P. bournei. The findings have provided valuable insights and knowledge that can guide further research aimed at understanding and addressing the impacts of abiotic stress within this species.

The WRKY gene family is ubiquitously distributed in plants, serving crucial functions in stress responses. Nevertheless, the structural organization and evolutionary dynamics of WRKY genes in cotton have not been fully elucidated. In this study, a total of 112, 119, 217, and 222 WRKY genes were identified in Gossypium arboreum, Gossypium raimondii, Gossypium hirsutum, and Gossypium barbadense, respectively. These 670 WRKY genes were categorized into seven distinct subgroups and unequally distributed across chromosomes. Examination of conserved motifs, domains, cis-acting elements, and gene architecture collectively highlighted the evolutionary conservation and divergence within the WRKY gene family in cotton. Analysis of synteny and collinearity further confirmed instances of expansion, duplication, and loss events among WRKY genes during cotton evolution. Furthermore, GhWRKY31 transgenic Arabidopsis exhibited heightened germination rates and longer root lengths under drought and salt stress. Silencing GhWRKY31 in cotton led to reduced levels of ABA, proline, POD, and SOD, along with downregulated expression of stress-responsive genes. Yeast one-hybrid and molecular docking assays confirmed the binding capacity of GhWRKY31 to the W box of GhABF1, GhDREB2, and GhRD29. The findings collectively offer a systematic and comprehensive insight into the evolutionary patterns of cotton WRKYs, proposing a suitable regulatory framework for developing cotton cultivars with enhanced resilience to drought and salinity stress.

Cadmium (Cd) is a heavy metal highly toxic to living organisms. Cd pollution of soils has become a serious problem worldwide, posing a severe threat to crop production and human health. When plants are poisoned by Cd, their growth and development are inhibited, chloroplasts are severely damaged, and respiration and photosynthesis are negatively affected. Therefore, elucidating the molecular mechanisms that underlie Cd tolerance in plants is important. Transcription factors can bind to specific plant cis-acting genes. Transcription factors are frequently reported to be involved in various signaling pathways involved in plant growth and development. Their role in the resistance to environmental stress factors, particularly Cd, should not be underestimated. The roles of several transcription factor families in the regulation of plant resistance to Cd stress have been widely demonstrated. In this review, we summarize the mechanisms of five major transcription factor families-WRKY, ERF, MYB, bHLH, and bZIP-in plant resistance to Cd stress to provide useful information for using molecular techniques to solve Cd pollution problems in the future.

BACKGROUND: Ichang papeda (Citrus ichangensis), a wild perennial plant of the Rutaceae family, is a cold-hardy plant. WRKY transcription factors are crucial regulators of plant growth and development as well as abiotic stress responses. However, the WRKY genes in C. ichangensis (CiWRKY) and their expression patterns under cold stress have not been thoroughly investigated, hindering our understanding of their role in cold tolerance.
RESULTS: In this study, a total of 52 CiWRKY genes identified in the genome of C. ichangensis were classified into three main groups and five subgroups based on phylogenetic analysis. Comprehensive analyses of motif features, conserved domains, and gene structures were performed. Segmental duplication plays a significant role in the CiWRKY gene family expansion. Cis-acting element analysis revealed the presence of various stress-responsive elements in the promoters of the majority of CiWRKYs. Gene ontology (GO) analysis and protein-protein interaction predictions indicate that the CiWRKYs exhibit crucial roles in regulation of both development and stress response. Expression profiling analysis demonstrates that 14 CiWRKYs were substantially induced under cold stress. Virus-induced gene silencing (VIGS) assay confirmed that CiWRKY31, one of the cold-induced WRKYs, functions positively in regulation of cold tolerance.
CONCLUSIONS: Sequence and protein properties of CiWRKYs were systematically analyzed. Among the 52 CiWRKY genes 14 members exhibited cold-responsive expression patterns, and CiWRKY31 was verified to be a positive regulator of cold tolerance. These findings pave way for future investigations to understand the molecular functions of CiWRKYs in cold tolerance and contribute to unravelling WRKYs that may be used for engineering cold tolerance in citrus.

The transcription factors of WRKY genes play essential roles in plant growth, stress responses, and metabolite biosynthesis. Erigeron breviscapus, a traditional Chinese herb, is abundant in flavonoids and has been used for centuries to treat cardiovascular and cerebrovascular diseases. However, the WRKY transcription factors that regulate flavonoid biosynthesis in E. breviscapus remain unknown. In this study, a total of 75 EbWRKY transcription factors were predicted through comprehensive genome-wide characterization of E. breviscapus and the chromosomal localization of each EbWRKY gene was investigated. RNA sequencing revealed transient responses of 74 predicted EbWRKY genes to exogenous abscisic acid (ABA), salicylic acid (SA), and gibberellin 3 (GA3) after 4 h of treatment. In contrast, the expression of key structural genes involved in flavonoid biosynthesis increased after 4 h in GA3 treatment. However, the content of flavonoid metabolites in leaves significantly increased at 12 h. The qRT-PCR results showed that the expression patterns of EbWRKY11, EbWRKY30, EbWRKY31, EbWRKY36, and EbWRKY44 transcription factors exhibited a high degree of similarity to the 11 structural genes involved in flavonoid biosynthesis. Protein-DNA interactions were performed between the key genes involved in scutellarin biosynthesis and candidate WRKYs. The result showed that F7GAT interacts with EbWRKY11, EbWRKY36, and EbWRKY44, while EbF6H has a self-activation function. This study provides comprehensive information on the regulatory control network of flavonoid accumulation mechanisms, offering valuable insights for breeding E. breviscapus varieties with enhanced scutellarin content.

Sweet basil (Ocimum basilicum L.) is an important aromatic plant with high edibility and economic value, widely distributed in many regions of the tropics including the south of China. In recent years, environmental problems, especially soil salinization, have seriously restricted the planting and spread of sweet basil. However, the molecular mechanism of the salt stress response in sweet basil is still largely unknown. In this study, seed germination, seedling growth, and chlorophyll synthesis in sweet basil were inhibited under salt stress conditions. Through comparative transcriptome analysis, the gene modules involved in the metabolic processes, oxidative response, phytohormone signaling, cytoskeleton, and photosynthesis were screened out. In addition, the landscape of transcription factors during salt treatment in sweet basil was displayed as well. Moreover, the overexpression of the WRKY transcription factor-encoding gene, ObWRKY16, and the phenylalanine ammonia-lyase-encoding gene, ObPAL2, enhanced the seed germination, seedling growth, and survival rate, respectively, of transgenic Arabidopsis, suggesting that they might be important candidates for the creation of salt-tolerant sweet basil cultivars. Our data enrich the study on salt responses in sweet basil and provide essential gene resources for genetic improvements in sweet basil in the future.

MYB transcription factors have been linked to anthocyanin synthesis and various color phenotypes in plants. In apple, MYB10 confers a red-flesh phenotype due to a minisatellite insertion in its R6 promoter, but R6:MYB10 genotypes exhibit various degrees of red pigmentation in the flesh, suggesting the involvement of other genetic factors. Here, it is shown that MdWRKY10, a transcription factor identified via DNA pull-down trapping, binds to the promoter of MdMYB10 and activates its transcription. MdWRKY10 specifically interacts with the WDR protein MdTTG1 to join the apple MYB-bHLH-WDR (MBW) complex, which significantly enhances its transcriptional activation activity. A 163-bp InDel detected in the promoter region of the alleles of MdWRKY10 in a hybrid population of identical heterozygous genotypes regarding R6 by structural variation analysis, contains a typical W-box element that MdWRKY10 binds to for transactivation. This leads to increased transcript levels of MdWRKY10 and MdMYB10 and enhanced anthocyanin synthesis in the flesh, largely accounting for the various degrees of flesh red pigmentation in the R6 background. These findings reveal a novel regulatory role of the WRKY-containing protein complex in the formation of red flesh apple phenotypes and provide broader insights into the molecular mechanism governing anthocyanin synthesis in plants.

UNASSIGNED: This work explored the characteristics of the WRKY transcription factor family in Rhododendron henanense subsp. lingbaoense (Rhl) and the expression patterns of these genes under abiotic stress by conducting bioinformatics and expression analyses.
UNASSIGNED: RhlWRKY genes were identified from a gene library of Rhl. Various aspects of these genes were analyzed, including genetic structures, conserved sequences, physicochemical properties, cis-acting elements, and chromosomal location. RNA-seq was employed to analyze gene expression in five different tissues of Rhl: roots, stems, leaves, flowers, and hypocotyls. Additionally, qRT-PCR was used to detect changes in the expression of five RhlWRKY genes under abiotic stress.
UNASSIGNED: A total of 65 RhlWRKY genes were identified and categorized into three subfamilies based on their structural characteristics: Groups I, II, and III. Group II was further divided into five subtribes, with shared similar genetic structures and conserved motifs among members of the same subtribe. The physicochemical properties of these proteins varied, but the proteins are generally predicted to be hydrophilic. Most proteins are predicted to be in the cell nucleus, and distributed across 12 chromosomes. A total of 84 cis-acting elements were discovered, with many related to responses to biotic stress. Among the identified RhlWRKY genes, there were eight tandem duplicates and 97 segmental duplicates. The majority of duplicate gene pairs exhibited Ka/Ks values <1, indicating purification under environmental pressure. GO annotation analysis indicated that WRKY genes regulate biological processes and participate in a variety of molecular functions. Transcriptome data revealed varying expression levels of 66.15% of WRKY family genes in all five tissue types (roots, stems, leaves, flowers, and hypocotyls). Five RhlWRKY genes were selected for further characterization and there were changes in expression levels for these genes in response to various stresses.
UNASSIGNED: The analysis identified 65 RhlWRKY genes, among which the expression of WRKY_42 and WRKY_17 were mainly modulated by the drought and MeJA, and WRKY_19 was regulated by the low-temperature and high-salinity conditions. This insight into the potential functions of certain genes contributes to understanding the growth regulatory capabilities of Rhl.

Glomerella leaf spot (GLS), caused by the fungus Colletotrichum fructicola, is considered one of the most destructive diseases affecting apples. The VQ-WRKY complex plays a crucial role in the response of plants to biotic stresses. However, our understanding of the defensive role of the VQ-WRKY complex on woody plants, particularly apples, under biotic stress, remains limited. In this study, we elucidated the molecular mechanisms underlying the defensive role of the apple MdVQ37-MdWRKY100 module in response to GLS infection. The overexpression of MdWRKY100 enhanced resistance to C. fructicola, whereas MdWRKY100 RNA interference in apple plants reduced resistance to C. fructicola by affecting salicylic acid (SA) content and the expression level of the CC-NBS-LRR resistance gene MdRPM1. DAP-seq, Y1H, EMSA, and RT-qPCR assays indicated that MdWRKY100 inhibited the expression of MdWRKY17, a positive regulatory factor gene of SA degradation, upregulated the expression of MdPAL1, a key enzyme gene of SA biosynthesis, and promoted MdRPM1 expression by directly binding to their promotors. Transient overexpression and silencing experiments showed that MdPAL1 and MdRPM1 positively regulated GLS resistance in apples. Furthermore, the overexpression of MdVQ37 increased the susceptibility to C. fructicola by reducing the SA content and expression level of MdRPM1. Additionally, MdVQ37 interacted with MdWRKY100, which repressed the transcriptional activity of MdWRKY100. In summary, these results revealed the molecular mechanism through which the apple MdVQ37-MdWRKY100 module responds to GLS infection by regulating SA content and MdRPM1 expression, providing novel insights into the involvement of the VQ-WRKY complex in plant pathogen defence responses.