WRKY75 regulates anthocyanin accumulation in juvenile citrus tissues

Abstract

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.

Supplementary information: The online version contains supplementary material available at 10.1007/s11032-024-01490-9.

Keywords: Anthocyanin; Citrus; Juvenile stage; WRKY.

Fig. 1

Accumulation and gradual disappearance of anthocyanins in juvenile tissues of various citrus. From top to bottom are red and red fading tissues (leaf, flower, and fruit) of primitive citrus, early-diverging citrus, the three fundamental citrus species, and the hybrid sour oranges. The citrus that cannot accumulate anthocyanins in young leaves, flowers, or fruits has not been shown. The young pericarp coloration can last for 14–20 days, the coloration of young leaves and young flowers can last for 6–13 days. Numbers below the images represent anthocyanin content (A530/g). The scale bar represents 1 cm.

Fig. 2

Identifying genes that promote anthocyanin accumulation in juvenile stage. A KEGG enrichment analysis of differentially expressed genes between juvenile purple leaves and purple faded leaves in the A.buxifolia. B KEGG enrichment analysis of differentially expressed genes between purple juvenile pericarp and purple faded pericarp in the pummelo. C Expression profiles of candidate genes. The first and second columns represent the accumulation of anthocyanin in juvenile leaves of A.buxifolia and the purple faded during development, respectively. The third and fourth columns represent purple and purple faded pummelo pericarp. Numbers below the images represent anthocyanin content (A530/g). The scale bar represents 1 cm

Fig. 3

The Expression characteristics of CitWRKY75 in different tissues. A The expression level of CitWRKY75 and CitRuby1 in the blood orange pericarp during the mature stage. B The expression levels of CitWRKY75 and CitRuby1 in the blood orange pulp during the mature stage. Numbers below the images represent anthocyanin content (A530/g). C Expression of CitWRKY75 in young purple leaves and later purple fading leaves. D Expression of CitWRKY75 in young purple fruit pericarp and later purple fading pericarp. Data are mean ± standard error (n = 3 biologically independent replicates). Asterisks represent significant differences: *P < 0.05, **P < 0.01

Fig. 4

Phylogenetic relationship and Localization Analysis of CitWRKY75. A Phylogenetic tree analysis of CitWRKY75 and WRKY proteins. The comparison of amino acid sequences and phylogenetic tree (neighbor-joining method) was constructed by MEGA-X. All the amino acid sequences of WRKY proteins from Arabidopsis were obtained from the Plant Transcription Factor Database (https://planttfdb.gao-lab.org/). B CitWRKY75 is localized in the nucleus. Subcellular localization assay was carried out using leaf cells of tobacco (Nicotiana benthamiana)

Fig. 5

CitWRKY75 promotes anthocyanin synthesis. A The effect of stable overexpression of CitWRKY75 on anthocyanin accumulation in tobacco. The scale bar represents 1 cm. B Identification of positive lines of stable overexpression CitWRKY75 in tobacco, OE, overexpression line, EV, empty vector line. C Detection of anthocyanin content after stable transformation CitWRKY75 in tobacco. D Transactivation effect of CitWRKY75 on the promoters of CitRuby1 by dual-luciferase assays. E Yeast one-hybrid assay confirms that CitWRKY75 can bind to the CitRuby1 promoter. Data are mean ± standard error (n = 3 biologically independent replicates)