Comparative Transcriptome Analysis of the Accumulation of Anthocyanins Revealed the Underlying Metabolic and Molecular Mechanisms of Purple Pod Coloration in Okra (Abelmoschus esculentus L.)

Abstract

Color is an essential agronomic trait and the consumption of high anthocyanin containing vegetables in daily diet does provide benefits to human health, but the mechanisms on anthocyanin accumulation in tender pods of okra (Abelmoschus esculentus L.) were totally unknown. In this study, a wide characterization and quantitation of anthocyanins and flavonols in tender pods of 15 okra varieties were performed by UHPLC-Q-Orbitrap HRMS for the first time. Two major anthocyanins (delphinidin 3-O-sambubioside and cyanidin 3-O-sambubioside) and six kinds of flavonol glycosides (most are quercetin-based) were identified and quantified. The coloration of the purple okra pod mainly arises from the accumulation of both delphinidin 3-O-sambubioside and cyanidin 3-O-sambubioside in most of purple varieties (Hong Yu, Bowling Red and Burgundy), except Jing Orange. The significant differences in the compositions and contents of anthocyanins are responsible for the pod color ranging from brick-red to purplish-red among the various okra cultivars. Furthermore, four representative okra cultivars exhibiting obvious differences in anthocyanin accumulation were further analyzed with transcriptome and more than 4000 conserved differentially expressed genes were identified across the three compared groups (B vs. BR, B vs. HY and B vs. JO). Based on the comprehensive analysis of transcriptomic data, it was indicated that MBW complex consisting of AeMYB114, AeTT8, and AeTTG1 and other transcriptional factors coordinately regulate the accumulation of anthocyanins via the transcriptional regulation of structural genes. Moreover, four independent working models explaining the diversities of anthocyanin pigmentation in okra pods were also proposed. Altogether, these results improved our understanding on anthocyanin accumulation in okra pods, and provided strong supports for the development of okra pod as a functional food in the future.

Integrated Transcriptomic and Metabolomic Analyses Reveal the Mechanisms Underlying Anthocyanin Coloration and Aroma Formation in Purple Fennel

“…To reveal the molecular mechanisms of the characteristic color and aroma formation in purple fennel, a total of 273 DEGs encoding transcription factors were screened out and annotated to 16 gene families, such as MYB and bHLH ( Supplementary Tables S5 , S6 ). Expectedly, none of the identified WD40 repeat proteins show obvious expression changes between two fennel varieties, similar to the previous reports ( Figure 5A and Supplementary Table S5 ) ( 22 , 27 , 28 , 42 ). Compared with the green fennel, AfTT8, a bHLH transcription factor, exhibited significant upregulation in purple fennel leaves, indicating a critical function in the regulation of anthocyanin production.…”

“…Regardless of fennel variety, the chlorogenic acid occupies the highest proportion of the total hydroxycinnamic acid derivatives ( Table 2 ). Among the total flavonol derivatives, quercetin-3- O -ferulic acid accounts for the highest proportion, following by kaempferol 3- O -ferulic acid which is remarkably different from the flavonol glycosides identified in other species such as okra, cowpea, and eggplant ( 22 , 24 , 26 , 27 , 29 ). Therefore, several conclusions on phenylpropanoid metabolism can be made initiatively: (1) the biosynthesis of caffeic acid and sinapic acid occupies an essentially important metabolic efflux in phenylpropanoid pathway in both fennel varieties; (2) flavonol aglycones in fennel are mainly decorated by ferulic acid other than saccharide; (3) quercetin-based flavonol derivatives are preferentially produced in flavonoid pathway in fennel.…”

“…As the results shown in Table 1 , nearly all the anthocyanidins (99%) undergone the same pattern of glycosylation modification at C3 position of the C ring. Subsequently, most of these glycosylated anthocyanins are further acylated by hydroxycinnamic acids which contain aromatic ring groups, unlike the anthocyanins widely found in vegetables and fruits such as blueberry, apple, okra, and other species ( 22 , 24 – 27 , 29 ). As acylated anthocyanins show significantly higher color stability than the un-acylated, the purple fennel might be good resource for natural food colorants ( 30 ).…”

“…Total RNAs were extracted from tender leaves and tested by standard methods reported previously ( 27 ). Thence, the constructed RNA-seq libraries were sequenced by MetWare Biotechnology Co., Ltd. (Wuhan, China, https://www.metware.cn ) on NovaSeq 6000.…”

“…The unigenes were annotated using BLASTX program (E-value <1E−5) against non-redundant (nr protein sequences) in NCBI ( http://www.ncbi.nlm.nih.gov/ ), Swissprot ( http://www.expasy.ch/sprot/ ), Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway ( http://www.genome.jp/kegg/ ), Clusters of Orthologous Groups of Proteins (KOG/COG), and Pfam ( https://pfam.xfam.org/ ). Differential expression analysis was conducted as the method reported previously ( 27 ).…”