RESUMO
The R2R3-MYB family is one of the largest plant transcription factor (TF) families playing vital roles in defense, plant growth, and secondary metabolism biosynthesis. Although this gene family has been studied in many species, isoflavonoid biosynthesis-related R2R3-MYB TFs in Callerya speciosa (Champ. ex Benth.) Schot, a traditional Chinese medicinal herb, are poorly understood. Here, a total of 101 R2R3-MYB TFs were identified from C. speciosa transcriptome dataset. 25 clades divided into five functional groups were clustered based on the sequence similarity and phylogenetic tree. Conserved motifs and domain distribution, expression patterns, and coexpression networks were also employed to identify the potential R2R3-MYB TFs in the regulation of isoflavonoid biosynthesis. In silico evaluation showed that the deduced R2R3-CsMYB proteins contain highly conserved R2R3 repeat domain at the N-terminal region, that is the signature motif of R2R3-type MYB TFs. Eight potential TFs (CsMYB17, CsMYB36, CsMYB41, CsMYB44, CsMYB45, CsMYB46, CsMYB72, and CsMYB81) had high degrees of coexpression with four key isoflavonoid biosynthetic genes (CsIFS, CsCHS7, CsHID-1, and CsCHI3), in which CsMYB36 as a potential regulator possessed the highest degree. HPLC analysis showed that formononetin and maackiain contents were significantly increased during the development of tuberous roots, which might be controlled by both related R2R3-CsMYBs and structural genes involved in the isoflavonoid biosynthesis pathway. The transcriptome data were further validated by reverse transcription real-time PCR (RT-qPCR) analysis, and similar expression profiles between TFs and key structural genes were identified. This study was the first step toward the understanding of the R2R3-MYB TFs regulating isoflavonoid biosynthesis in C. speciosa. The results will provide information for further functional analysis and quality improvement through genetic manipulation of these potential R2R3-CsMYB genes in C. speciosa.
RESUMO
Callerya speciosa (Champ. ex Benth.) Schot is a traditional Chinese medicine characterized by tuberous roots as the main organ of isoflavonoid accumulation. Root thickening and isoflavonoid accumulation are two major factors for yield and quality of C. speciosa. However, the underlying mechanisms of root thickening and isoflavonoid biosynthesis have not yet been elucidated. Here, integrated morphological, hormonal and transcriptomic analyses of C. speciosa tuberous roots at four different ages (6, 12, 18, 30 months after germination) were performed. The growth cycle of C. speciosa could be divided into three stages: initiation, rapid-thickening and stable-thickening stage, which cued by the activity of vascular cambia. Endogenous changes in phytohormones were associated with developmental changes during root thickening. Jasmonic acid might be linked to the initial development of tuberous roots. Abscisic acid seemed to be essential for tuber maturation, whereas IAA, cis-zeatin and gibberellin 3 were considered essential for rapid thickening of tuberous roots. A total of 4337 differentially expressed genes (DEGs) were identified during root thickening, including 15 DEGs participated in isoflavonoid biosynthesis, and 153 DEGs involved in starch/sucrose metabolism, hormonal signaling, transcriptional regulation and cell wall metabolism. A hypothetical model of genetic regulation associated with root thickening and isoflavonoid biosynthesis in C. speciosa is proposed, which will help in understanding the underlying mechanisms of tuberous root formation and isoflavonoid biosynthesis.