RNA sequencing on Amomum villosum Lour. induced by MeJA identifies the genes of WRKY and terpene synthases involved in terpene biosynthesis

Abstract

Amomum villosum Lour. is an important Chinese medicinal plant that has diverse medicinal functions, and mainly contains volatile terpenes. This study aims to explore the WRKY transcription factors (TFs) and terpene synthase (TPS) unigenes that might be involved in terpene biosynthesis in A. villosum, and thus providing some new information on the regulation of terpenes in plants. RNA sequencing of A. villosum induced by methyl jasmonate (MeJA) revealed that the WRKY family was the second largest TF family in the transcriptome. Thirty-six complete WRKY domain sequences were expressed in response to MeJA. Further, six WRKY unigenes were highly correlated with eight deduced TPS unigenes. Ultimately, we combined the terpene abundance with the expression of candidate WRKY TFs and TPS unigenes to presume a possible model wherein AvWRKY61, AvWRKY28, and AvWRKY40 might coordinately trans-activate the AvNeoD promoter. We propose an approach to further investigate TF unigenes that might be involved in terpenoid biosynthesis, and identified four unigenes for further analyses.

An Integrative Volatile Terpenoid Profiling and Transcriptomics Analysis for Gene Mining and Functional Characterization of AvBPPS and AvPS Involved in the Monoterpenoid Biosynthesis in Amomum villosum

“…Among 18 secondary metabolic pathways, 25.24% unigenes were annotated to be directly involved in terpenoid biosynthesis, including terpenoid backbone, monoterpenoid, diterpenoid, carotenoid (tetraterpene) biosynthesis pathways. Compared with the other transcriptome of A. villosum on the MeJA-treatment from our group ( He et al, 2018 ), all the genes involved in terpenoid backbone biosynthesis were annotated in both sets of transcriptome data. For example, DXS, DXR, MCT, CMK, MDS were annotated in the MEP pathway, and AACT, HMGS, HMGR, MVK, PMK in the MVA pathway; nevertheless more unigenes in total were annotated in this research (Supplementary Table S7 ).…”

“…The cDNA library was constructed and sequenced on the Illumina sequencing platform (IlluminaHiSeq TM 2000) using the paired-end technology by Gene Denovo Co. (Guangzhou, China). De novo assembly and annotation were performed by Gene Denovo Co. as described ( He et al, 2018 ).…”

“…In order to analyze the correlation of candidate genes and volatile terpenoid, the second transcriptome sequencing with three biological replicates for each tissue was performed by Gene Denovo Co using pericarp, seeds and creeping stems collected at different stages which were the same samples used for the volatile terpenoid profiling. The gene expression level was measured by the number of uniquely mapped reads per kilobase of exon region per million mappable reads (RPKM) ( He et al, 2018 ). The spearman correlation coefficients were calculated using gene expression and volatile terpenoid data.…”

The role of volatiles in plant communication

“…Although in these studies chemical analysis was also carried out, these data were not used to the maximum to further narrow down and/or support their candidate genes and the studies therefore remain rather descriptive. A more advanced network (co‐expression) analysis with RNA‐seq and GC‐MS results is very powerful in identifying promising candidate genes and will therefore shorten the gene discovery process substantially (He et al ., ). A next step in this process will be the use of metabolomics as exemplified for strawberry (Haugeneder et al ., ), which will allow correlation analysis also with non‐volatile precursors and/or further functionalized non‐volatile downstream products.…”

“…To allow for the identification of TFs that regulate terpene biosynthesis in the traditional Chinese medicinal plant Amomum villosum , He et al . () used a combination of RNA sequencing and volatile analysis after induction by methyl jasmonate (MeJA) (He et al ., ). Network analysis revealed correlations between putative terpene synthase Unigenes, terpene abundance and candidate WRKY TFs.…”