Comprehensive RNA sequencing and co-expression network analysis to complete the biosynthetic pathway of coumestrol, a phytoestrogen

Abstract

Coumestrol (CMS), a coumestan isoflavone, plays key roles in nodulation through communication with rhizobia, and has been used as phytoestrogens for hormone replacement therapy in humans. Because CMs content is controlled by multiple genetic factors, the genetic basis of CMs biosynthesis has remained unclear. We identified soybean genotypes with consistently high (Daewonkong) or low (SS0903-2B-21-1-2) CMS content over 2 years. We performed RNA sequencing of leaf samples from both genotypes at developmental stage R7, when CMS levels are highest. Within the phenylpropanoid biosynthetic pathway, 41 genes were tightly connected in a functional co-expression gene network; seven of these genes were differentially expressed between two genotypes. We identified 14 candidate genes involved in CMs biosynthesis. Among them, seven were annotated as encoding oxidoreductases that may catalyze the transfer of electrons from daidzein, a precursor of CMs. two of the other genes, annotated as encoding a MYB domain protein and a MLp-like protein, may increase CMs accumulation in response to stress conditions. our results will help to complete our understanding of the CMs biosynthetic pathway, and should facilitate development of soybean cultivars with high CMs content that could be used to promote the fitness of plants and human beings. Plants synthesize secondary metabolites to promote their own survival, and some of these compounds have antioxidant or antibiotic effects 1,2. Several lines of evidence suggest that plant secondary metabolites, especially isoflavones, can improve the fitness of both humans and plants. Accordingly, a great deal of research has been conducted on isoflavones. Legume species, including the economically important crop plant soybean, are rich in isoflavones with estrogenic and antioxidant functions 3,4. Within plants themselves, isoflavones play crucial roles in nodulation and nitrogen fixation 5 and defense against environmental stresses 6. In the context of human health, isoflavones, as phytoestrogens, can decrease the risk of menopausal symptoms, breast cancer, osteoporosis, dementia, and cardiovascular disease 7-13. Declining estrogen levels in postmenopausal women are associated with a variety of cutaneous changes, including dryness, wrinkling, poor healing, and hot flashes, many of which can be improved by estrogen supplementation 14. However, the estrogens used in hormone replacement therapy can promote the initiation and progression of breast cancer 15-17. The effects of estrogen are mediated by two estrogen receptors (ERs), ERα and ERβ, which are distributed differently in each tissue 18,19. ERα mediates the breast cancer-promoting effects of estrogens, whereas ERβ inhibits breast cancer cell proliferation and tumor formation 20. Therefore, it has been proposed that dietary or synthetic ERβ-selective estrogens would lack the breast cancer-promoting properties of the estrogens used in hormone replacement regimens 20 .

Candidate Domestication-Related Genes Revealed by Expression Quantitative Trait Loci Mapping of Narrow-Leafed Lupin (Lupinus angustifolius L.)

“…Indeed, Lup028431 was selected in another study as potential L. angustifolius quinolizidine alkaloid biosynthetic gene because it revealed similar expression pattern to the LDC gene [63]. Lup015923 has been annotated as MAJOR LATEX PROTEIN 423 ( MLP423 , AT1G24020), which is hypothesized to be involved in stress responsive activation of biosynthetic pathway of coumestrol, a coumestan isoflavone in soybean [64]. Lup015923 was recently highlighted as one of candidate quinolizidine alkaloid biosynthesis genes in L. angustifolius due to highly elevated expression in bitter P27255 accession [58].…”

Is Chickpea a Potential Substitute for Soybean? Phenolic Bioactives and Potential Health Benefits

“…Recently, an MYB transcription factor was found to regulate isoflavone contents in soybean hairy roots, the GmMYB29 activated the promoters IFS2 and CHS8 , while its overexpression or silencing by RNAi had positive and negative effects on isoflavone content [89]. Nonetheless, further studies to verify and confirm biosynthetic pathways using RNA-sequencing and co-expression network analysis can lead to the identification of specialized varieties, for example, producing coumestrol [90].…”

The Induction of the Isoflavone Biosynthesis Pathway Is Associated with Resistance to Common Bacterial Blight in Phaseolus vulgaris L.

“…Genes for early biosynthetic steps (e.g., PvPAL2 ) included those that were previously determined to be phylogenetically similar to known phenylpropanoid genes for other legume organisms, such as barrel clover ( Medicago truncatula Gaertn.) and soybean [ 13 , 26 ]. In addition, increased transcript levels were apparent for a C4H , a 4CL1 , a CHI , two PAL s (i.e., Phvul.001G177700 and Phvul.008G289500 ), several CHS s including a transcript that mapped to three genes (i.e., Phvul.002G039100 , Phvul.002G039166 , Phvul.002G039232 ), a transcript mapping to six genes (i.e., Phvul.002G038600 , Phvul.002G038700 , Phvul.002G038800 , Phvul.002G038900 , Phvul.002G039000 , Phvul.002G039300 ), a transcript aligning to many CHR genes, and a transcript mapping to both PvCHR2 and PvCHR3 ( Figure 7 ; Supplementary Information, Tables S5 and S41 ).…”

“…In fact, several isoflavones, coumestrol, and phaseollinisoflavan accumulate in seedlings of a related species Phaseolus coccineus L. treated with the fungus Rhizopus oryzae [ 25 ]. Coumestrol is proposed to be derived from daidzein by way of the oxidation products of pterocarpans [ 26 ]. A soybean VR converts the IFR product 2′-hydroxyisoflavanone to a 3,9-dihydroxypterocarpan [ 27 ], with the latter compound proposed as an intermediate for coumestrol biosynthesis by a hitherto unknown enzyme [ 26 ].…”

“…Coumestrol is proposed to be derived from daidzein by way of the oxidation products of pterocarpans [ 26 ]. A soybean VR converts the IFR product 2′-hydroxyisoflavanone to a 3,9-dihydroxypterocarpan [ 27 ], with the latter compound proposed as an intermediate for coumestrol biosynthesis by a hitherto unknown enzyme [ 26 ]. By comparison, an alfalfa ( Medicago sativa L.) VR converts vestitone to 7,2′-dihydroxy-4′-isoflavanol, which is subsequently dehydrated to medicarpin ( Figure 1 ) [ 28 ].…”