Cotton is a very critical resource in the world, and it has a very pivotal strategic significance. It has been cultivated around the world for more than 7,000 years. Cotton can not only be used as an important raw material for textile industry, but also a nutrition resource. For every kilogram of cotton fiber produced, about 1.65kg of seeds will be produced. And cottonseed is not only rich in oil, but also contains 23% protein. But common cotton varieties contain pigment glands, which contain a toxic yellow dimeric sesquiterpene compound called gossypol, monogastric animals and humans are very sensitive to this toxicity[2, 3]. Besides, it should not be overlooked that gossypol acts as a natural plant defender to reduce the damage of pests and diseases and external abiotic stresses[4–7]. According to the characteristics of the two-sidedness of gossypol, it is of great significance to improve the economic value of cotton to cultivate cotton varieties with high content of gossypol in plants and low content of gossypol in cottonseeds.
Pigment glands, also known as gossypol glands, are unique structure on the surface of Gossypium. Cotton glands consist of a cavity and secretory cell mass formed through programmed cell death (PCD)[8, 9]. Gossypol is stored in the mature pigment glands as main component. Previous studies have shown that the number of pigment glands and gossypol content are closely related. The synthetic pathway of gossypol is relatively complete, but the mechanism of gland formation is still unclear. Previous studies have identified 6 loci involved in cotton gland formation: Gl1, Gl2, Gl3, Gl4, Gl5 and Gl6. McMichael first discovered the recessive gene gl1, which controls the trait that almost no glands in cotton bolls and stalks but a normal number of glands in cotton leaves. Subsequently, gl2gl3 was reported to be a double recessive gene that controls the absence of glands in the whole plant[12, 13]. gl4 and gl5 were found to regulate the number and density of glands, but not the complete absence of glands. gl6 is a gene with a similar but weak function to gl1. Then, a whole glandless plant was isolated from “Giza45” (G. barbadense L.). The glandless phenotype was controlled by a dominant gene Gl2e, which is an allele of Gl2[16, 17]. Since then, efforts were made to map and isolate the genes involved in gland formation. In 2016, Cheng mapped Gl2e to a 15KB region on chromosome A12, in which a transcription factor called GhMYC2-like was identified and the presence of an amino acid substitution in the conserved domain may be associated with glandless phenotype. The function of the transcription factor was verified by VIGS and it was named as Gossypium Gland Formation (GoPGF).
Gl 2 and Gl3 genes are located on chromosomes A12 and D12, respectively. gl2 and gl3 were found to be derived from premature translation termination and monomer mutation of Gl2 and Gl3. Subsequently, three CGF genes were identified by transcriptome analysis of gland and glandless cotton embryos and function verification by virus-induced gene silencing (VIGS). Gene knockout mediated by CRISPR/ Cas9 further confirmed the role of CGF2 and CGF3༈synonymous with GhMYC2-like/GoPGF༉ in gland formation.
WRKY proteins are a family of transcription factors unique to plants. WRKY transcription factors generally contain one or two conserve WRKY domains, with about 60 amino acid residues including WRKYGQK sequence and a C2H2 or C2HC zinc finger motif[21, 22]. With the cloning of the first WRKY gene SPF1 in sweet potatoes, WRKY genes have been studied and reported in more and more species, including Arabidopsis thaliana, maize, wheat, tomato, cotton, rice, castorbean, cassava, cucumber, pineapple,etc. WRKY transcription factors play important roles in a variety of complex physiological and biochemical processes, especially in the field of plant resistance to abiotic and biological stresses. BcWRKY46 could reduce the sensitivity of tobacco to freezing stress, ABA stress, salt stress and dehydration stress. SbWRKY50 can be involved in plant salt response by regulating ion homeostasis. WRKY71 were found to be involved in ethylene mediated regulation of leaf senescence. Recently, WRKY2 and WRKY10 were reported that involved in molecular mechanisms that regulate plant light signal transduction. In addition, WRKY is involved in the accumulation of secondary metabolites in a variety of plants. Maize terpenoid phytoalexins (MTPs) biosynthesis is regulated by ZmWRKY79 which is highly correlated with expression of MTPs. GaWRKY1 was found to be involved in the regulation of gossypol biosynthesis in cotton. All these indicate that WRKY is widely involved in a variety of plant biological processes, and the study of WRKY family has special production significance and application value.
The mechanism of gland development is very important for the application of cotton. GhMYC2-like has been studied as a star gene in gland development. As one of the largest transcription factor families in plants, WRKY plays an important role in various plant stress resistance processes. However, the study of the WRKY family in gland development is not clear and it is unknown whether WRKY genes participate in pigment gland development regulation.
In this study, we identified 36 WRKY group I genes and screened out one gene- GhWRKY138 from the transcriptome. GhWRKY138 expression was significantly different in glanded and glandless strains. We performed a virus-induced gene silencing assay on GhWRKY138 to obtain a phenotype with reduced number of pigment glands in cotton. Further analysis showed that the expression of GhMYC2-like decreased with the decrease of the gene expression level.
This study provides important information for improving the network between the WRKY family and gland development, and also extends the key role of WRKY in stress tolerance due to the important role of the gland in cotton stress tolerance.