Transcriptome Analysis of Pueraria Candollei Var
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Suntichaikamolkul et al. BMC Plant Biology (2019) 19:581 https://doi.org/10.1186/s12870-019-2205-0 RESEARCH ARTICLE Open Access Transcriptome analysis of Pueraria candollei var. mirifica for gene discovery in the biosyntheses of isoflavones and miroestrol Nithiwat Suntichaikamolkul1, Kittitya Tantisuwanichkul1, Pinidphon Prombutara2, Khwanlada Kobtrakul3, Julie Zumsteg4, Siriporn Wannachart5, Hubert Schaller4, Mami Yamazaki6, Kazuki Saito6, Wanchai De-eknamkul7, Sornkanok Vimolmangkang7 and Supaart Sirikantaramas1,2* 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. Keywords: Pueraria candollei var. mirifica, White Kwao Krua, Miroestrol, Isoflavones, Transcriptome * Correspondence: [email protected] 1Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok, Thailand 2Omics Sciences and Bioinformatics Center, Chulalongkorn University, Bangkok, Thailand Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Suntichaikamolkul et al. BMC Plant Biology (2019) 19:581 Page 2 of 14 Background (IFS). This pathway leads to the formation of an White Kwao Krua (Pueraria candollei var. mirifica, here- intermediate product, 2-hydroxyisoflavanone, which is after shortened to P. mirifica, shown in Additional file 1: then dehydrated to daidzein through catalysis by 2- Figure S1), has been extensively used in Thai traditional hydroxyisoflavanone dehydratase (Fig. 1)[12]. CHS medicine as a rejuvenating herb because of its numerous and IFS have been identified in P. mirifica several phytoestrogenic constituents [1]. Phytoestrogens are years ago [13, 14], however, their enzymatic functions plant-derived compounds that structurally or function- have not been elucidated. Udomsuk et al. [15]suggested ally mimic mammalian estrogen, and they have been ap- that miroestrol biosynthesis may share a common path- plied to treat different forms of cancer, heart disease, way with isoflavonoid biosynthesis due to their similar menopausal symptoms, and osteoporosis [2]. Consider- backbone structure. So far, no biosynthetic genes or en- ing the numerous effects of phytoestrogens on human zymes involved in miroestrol biosynthesis have been re- health, P. mirifica may be a promising candidate for ported, thus also the transcription factors responsible for treating various diseases and for developing novel medi- regulating the expression of these biosynthetic genes re- cinal products. main to be identified. V-myb myeloblastosis viral onco- Three major types of phytoestrogens occur in P. miri- gene homolog transcription factors (MYB TFs), which are fica: isoflavones, coumestans, and chromenes [3–5]. the largest plant transcription factor family, have been re- Isoflavones are an important type of phytoestrogens, ported to possess key functions in regulating the synthesis which are biosynthesized via the phenylpropanoid path- of phenylpropanoid-derived compounds in plants [16]. way and occur predominantly in leguminous plants [6]. These proteins have attracted substantial interest regard- Seven isoflavones that have been identified in P. mirifica ing phytoestrogen biosynthesis in plants. tubers: puerarin, daidzin, genistin, daidzein, genistein, Transcriptomes produced from high-throughput se- kwakhurin, and mirificin [3, 5]. Four coumestans that also quencing of various plants are a potential source for occur in the tuber of this plant are coumestrol, mirificou- identifying genes involved in the biosynthesis of different mestan, mirificoumestan hydrate, and mirificoumestan secondary metabolites. The transcriptome of Pueraria [7]. The chemical structure of chromenes has received lobata, a species closely related to P. mirifica, has been considerable attention because of their low toxicity and published previously [17, 18]. Although these two plants broad pharmacological application as anticancer, anti- produce similar types of isoflavones, miroestrol occurs microbial, and anti-inflammatory agents [8]. Miroestrol only in P. mirifica. In the afore-mentioned studies, P. and its precursor deoxymiroestrol are typically accumu- lobata genes that encode core isoflavone biosynthetic lated at very low levels [9], however, these compounds are enzymes were identified, and their expression levels in the predominant chromenes in the tuberous cortex of P. various tissues were examined. mirifica [10], and both compounds exhibit considerably In the current study, we high-throughput sequenced P. highest estrogenic activity [11]. Since both chromenes mirifica to produce transcriptome libraries of young have not been reported in other plant species, their bio- leaves, mature leaves, cortex-excised tubers, and tuber- synthesis is likely unique to P. mirifica. Interestingly, ous cortices, and we de novo assembled the transcrip- although miroestrol has been identified almost six decades tomes to characterize the biosynthetic pathway of ago, its biosynthetic pathway and the enzymes involved miroestrol. Additionally, MYB TFs involved in isoflavo- are still unknown. noid biosynthesis were also identified. This integrative Regarding biosynthesis of phytoestrogens, the early approach of using transcriptomics and metabolomics steps of isoflavonoid and flavonoid are generally similar, provides new insights for the prediction and identifica- starting with phenylalanine ammonia lyase removing tion of putative biosynthetic genes and transcription amides from the first substrate, phenylalanine, to produce factors that are potentially involved in P. mirifica isofla- cinnamic acid that is hydroxylated by cinnamate 4- vonoid and miroestrol biosynthetic pathways. hydroxylase to produce p-coumarate. The enzyme 4- coumarate-CoA ligase then activates p-coumarate by Results attaching a co-enzyme A (coA), and subsequently, chalcone De novo transcriptome assembly of P. mirifica synthase (CHS) binds p-coumaroyl-CoA to three molecules To obtain nucleotide sequences of expressed genes in of malonyl-CoA to form a chalcone skeleton. Chalcone can various tissues of P. mirifica, we constructed a cDNA be converted to flavanone by chalcone isomerase. Liquiriti- library from pooled tissues including, young leaves, ma- genin is a substrate for both the flavonoid and the isoflavo- ture leaves, cortex-excised tubers, and tuberous cortices noid pathway. Isoflavonoids are generally synthesized from (Fig. 2a-d). The library was processed using an Illumina common intermediates (either liquiritigenin or naringenin) Hiseq 2000 platform, yielding approximately 8.2 G base within the recognized flavonoid biosynthetic pathway by pairs and a total of 7,386,137,640 clean nucleotides (nt). aryl migration, which is catalyzed by isoflavone synthase The de novo assembly resulted in 166,923 contigs (62, Suntichaikamolkul et al. BMC Plant Biology (2019) 19:581 Page 3 of 14 Fig. 1 Proposed