Comparative transcriptome analysis to identify putative genes involved in carvacrol biosynthesis pathway in two species of Satureja, endemic medicinal herbs of Iran

Abstract

Satureja is rich in phenolic monoterpenoids, mainly carvacrol, that is of interest due to diverse biological activities including antifungal and antibacterial. However, limited information is available regarding the molecular mechanisms underlying carvacrol biosynthesis and its regulation for this wonderful medicinal herb. To identify the putative genes involved in carvacrol and other monoterpene biosynthesis pathway, we generated a reference transcriptome in two endemic Satureja species of Iran, containing different yields (Satureja khuzistanica and Satureja rechingeri). Cross-species differential expression analysis was conducted between two species of Satureja. 210 and 186 transcripts related to terpenoid backbone biosynthesis were identified for S. khuzistanica and S. rechingeri, respectively. 29 differentially expressed genes (DEGs) involved in terpenoid biosynthesis were identified, and these DEGs were significantly enriched in monoterpenoid biosynthesis, diterpenoid biosynthesis, sesquiterpenoid and triterpenoid biosynthesis, carotenoid biosynthesis and ubiquinone and other terpenoid-quinone biosynthesis pathways. Expression patterns of S. khuzistanica and S. rechingeri transcripts involved in the terpenoid biosynthetic pathway were evaluated. In addition, we identified 19 differentially expressed transcription factors (such as MYC4, bHLH, and ARF18) that may control terpenoid biosynthesis. We confirmed the altered expression levels of DEGs that encode carvacrol biosynthetic enzymes using quantitative real-time PCR (qRT-PCR). This study is the first report on de novo assembly and transcriptome data analysis in Satureja which could be useful for an understanding of the main constituents of Satureja essential oil and future research in this genus.

Fig 1. Plant materials.

A) S. khuzistanica, B) S. rechingeri, Schematic of de novo RNA-seq analysis workflow.

Fig 2. Venn diagram of four statistical methods (edgeR, NOISeq, limma, and DESeq2) of differentially expressed genes in S. khuzistanica vs S. rechingeri.

Fig 3. Gene ontology (GO) classification of annotated DEGs.

The x-axis indicates the subgroups in GO terms; y-axis shows the percentages of genes (number of a particular gene divided by total gene number).

Fig 4. The scatterplot of selected terpenoid biosynthesis view of GO category enrichment analysis of differentially expressed genes related to biological process found in S. khuzistanica vs S. rechingeri produced by REVIGO.

Circles depicted by filled color show significantly enriched GO terms with log10 p-value <0.05. The color of the bubbles shows the P-value and the size of the bubbles shows the frequency of the GO term in the underlying GOA database.

Fig 5. Top hits of KEGG metabolism pathway categories of DEGs.

Fig 6. Expression patterns of S. khuzistanica and S. rechingeri transcripts involved in the terpenoid biosynthetic pathways.

Logarithm of the FPKM value of all transcripts related to each gene was used. Ko numbers show the KEGG maps code related to each pathway. IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; GGPP, geranylgeranyl pyrophosphate; GGPS, GGPP synthase; GPP, geranyl pyrophosphate; FPP, farnesyl pyrophosphate; FPS, FPP synthase; TPS1, terpene synthase 1 (R-linalool synthase); TPS2, terpene synthase 2 ((4S)-limonene synthase), TPS3, terpene synthase 3; DH, carveol dehydrogenase; GA3, ent-kaurene oxidase; GA3ox, gibberellin 3beta-dioxygenase; GA20ox, gibberellin-44 dioxygenase; SQLE, squalene monooxygenase; FDFT1, farnesyl-diphosphate farnesyltransferase; AFS1, alpha-farnesene synthase; GERD, (-)-germacrene D synthase; LUP4; beta-amyrin synthase; ZDS, zeta-carotene desaturase; lcyB, lycopene beta-cyclase; lcyE, lycopene epsilon-cyclase; NCED, 9-cis-epoxycarotenoid dioxygenase; ABA2, xanthoxin dehydrogenase; AAO3, abscisic-aldehyde oxidase; CYP707A, (+)-abscisic acid 8’-hydroxylase; crtZ; beta-carotene 3-hydroxylase; CYP73A, trans-cinnamate 4-monooxygenase; TAT, tyrosine aminotransferase; menB, naphthoate synthase; 4CL, 4-coumarate-CoA ligase; wrbA, NAD(P)H dehydrogenase; COQ6, ubiquinone biosynthesis monooxygenase; VTE3, MPBQ/MSBQ methyltransferase.

Fig 7. Gene co-expression subnetwork of differentially expressed TFs and terpene biosynthesis-related enzyme genes.

Network was reconstructed by GeneMANIA. Connecting lines represent co-expression relationships.

Fig 8. Yield percentage of essential oil’s main components of S. khuzistanica and S. rechingeri.

Fig 9. Validation of selected genes using qRT-PCR.

(A) TPS2 (DN26728), terpene synthase 2 (B) TPS2 (DN32876), terpene synthase 2 (C) CYP71D18 (DN21230), cytochrome P450 71D18 (D) CYP71D18 (DN29590), cytochrome P450 71D18 (E) GGPS (DN26219), GGPP synthase (F) GGPS (DN32943), GGPP synthase.