Transcriptome analysis of Pueraria candollei var. mirifica for gene discovery in the biosyntheses of isoflavones and miroestrol

Abstract

Background: Pueraria candollei var. mirifica, a Thai medicinal plant used traditionally as a rejuvenating herb, is known as a rich source of phytoestrogens, including isoflavonoids and the highly estrogenic miroestrol and deoxymiroestrol. Although these active constituents in P. candollei var. mirifica have been known for some time, actual knowledge regarding their biosynthetic genes remains unknown. Results: Miroestrol biosynthesis was reconsidered and the most plausible mechanism starting from the isoflavonoid daidzein was proposed. A de novo transcriptome analysis was conducted using combined P. candollei var. mirifica tissues of young leaves, mature leaves, tuberous cortices, and cortex-excised tubers. A total of 166,923 contigs was assembled for functional annotation using protein databases and as a library for identification of genes that are potentially involved in the biosynthesis of isoflavonoids and miroestrol. Twenty-one differentially expressed genes from four separate libraries were identified as candidates involved in these biosynthetic pathways, and their respective expressions were validated by quantitative real-time reverse transcription polymerase chain reaction. Notably, isoflavonoid and miroestrol profiling generated by LC-MS/MS was positively correlated with expression levels of isoflavonoid biosynthetic genes across the four types of tissues. Moreover, we identified R2R3 MYB transcription factors that may be involved in the regulation of isoflavonoid biosynthesis in P. candollei var. mirifica. To confirm the function of a key-isoflavone biosynthetic gene, P. candollei var. mirifica isoflavone synthase identified in our library was transiently co-expressed with an Arabidopsis MYB12 transcription factor (AtMYB12) in Nicotiana benthamiana leaves. Remarkably, the combined expression of these proteins led to the production of the isoflavone genistein. Conclusions: Our results provide compelling evidence regarding the integration of transcriptome and metabolome as a powerful tool for identifying biosynthetic genes and transcription factors possibly involved in the isoflavonoid and miroestrol biosyntheses in P. candollei var. mirifica.

Identification of Isoflavonoid Biosynthesis-Related R2R3-MYB Transcription Factors in Callerya speciosa (Champ. ex Benth.) Schot Using Transcriptome-Based Gene Coexpression Analysis

“…Several MYB genes in Pueraria candollei var. Mirifica (PmMYB18, PmMYB23, PmMYB75, and PmMYB76) showed similar expression patterns with the key isoflavonoid biosynthetic genes in tuberous cortices [9]. In potato, StAN1, a R2R3-MYB activator, previously named StAN2 in some studies, was highly expressed in anthocyanin-pigmented tubers and displayed a positive correlation with the transcript levels of structural genes, as well as with anthocyanin content [38].…”

“…Then, some of the downstream key enzymes of the isoflavonoid pathway are 2hydroxyisoflavanone dehydratase (HID), isoflavone synthase (IFS), isoflavone 4′-O-methyltransferase (HI4′OMT), and vestitone reductase (VR), under the reactions of hydroxylation, dehydration, methylation, and glycosylation to form various isoflavonoid derivatives [7]. Recently, the coordinated expression of structural genes in the (iso) flavonoid biosynthetic pathway has been analyzed via the transcriptome data in several legume plant species, such as soybean, Pueraria candollei, and Pueraria lobata [6,8,9]. In C. speciosa, we have previously reported that 15 key genes might be involved in the isoflavonoid biosynthesis pathway, including IFSs, CHSs, and VRs [10].…”

“…PmMYB18 and PmMYB75 might be positive regulators of isoflavonoid biosynthesis in P. candollei var. mirifica [9]. GbMYBFL was involved in flavonoid biosynthesis, which played a critical role in flavonoids and anthocyanin accumulation in Ginkgo biloba [22].…”