CSIC-MOST Submitted Project (OSTW200003)

Hormonal elicitation of cell cultures of the medicinal plant Artemisia annua for the untargeted analysis of its volatilome and integration with its transcriptome.


Full description of Activities

Main Objective

The main aim of this project is to carry out hormonal elicitation experiments in liquid cell suspensions of A. annua and integrate their transcriptomes and metabolomes for identifying transcriptional regulators of the pathway

Detail on Specific Objectives 

  1. Elicitation of Artemisia cell cultures and metabolomic analysis of its volatilome (Activities to be conducted in Spain and Taiwan, with the visit of the Spanish Team in Taiwan) A. annua cell cultures were established by means of in vitro techniques, starting from somatic calli of A. annua that corresponds to proliferative masses of undifferentiated tissue (Figure: A. Light- and B. dark-grown calli, C. Cell suspension grown in light). The advantage of using this system is that cells can be synchronously stimulated for the production of specialized metabolites. The Spanish Team for this proposal counts on experience for the elicitation of cell cultures in different plant species (including grapevine and mulberry), together with conducting metabolite profiling and transcriptomics analysis in time-course experiments. Different elicitors will be used, such as Jasmonic acid or methyl jasmonate (MeJA), an hormone with proven effectiveness in activating secondary metabolism in plants of diverse phylogenetic origin and which has been postulated that could simultaneously stimulate artemisinin biosynthesis and promote the formation of glandular trichomes in A. annua (Caretto et al., 2011), organs that also accumulate large amounts of other terpenes. Cell suspension elicitation experiments will be carried out in Spain (samples will be lyophilized and sent to Taiwan) and also in Taiwan (fresh cell cultures) in order to see the metabolites that have been produced upon elicitation in both experiments, after which in the laboratory of Dr. Hieng-Ming Ting in the Institute of Plant Biology, National Taiwan University of Taiwan, we will analyze the volatilome of these cells by using untargeted GC/MS-based metabolomic approach. Dynamic headspace of VOCs will be collected by 200 mg Tenax TA (60/80 mesh; Markes, UK), and volatile compounds that accumulated in the suspension cells will be extracted by ethyl acetate for GC/MS analysis.
  2. Identification of the genes involved in the regulation of artemisinin and mono/sesquiterpene accumulation (Activities to be conducted in Spain, with the visit of Taiwan team in Spain). In addition to the quantifications of volatile aroma compounds, total RNA will be extracted from all time-course samples and RNA-seq experiments will be conducted to identify the elicited transcriptomes of A. annua. From these data, a differential temporal analysis of gene expression and the artemisinin and mono/sesquiterpene abundance will be carried out, in order to find co-expression modules between transcripts and metabolites. This will allow us to select those regulatory genes that have the same dynamics as different metabolites. Computationally speaking, ImpulseDE2 package will be used for an analysis of gene expression with a temporal dimension, detecting genes with a differential expression between treatment and control over time. The differentially expressed genes will be grouped into modules of similar expression patterns using the WGCNA R package which will also integrate the quantification of the metabolites of interest, thus identifying potential TF-metabolite relationships.
  3. Experimental validation of transcription factors and expression studies in A. annua plants (Activities to be conducted in Spain and Taiwan, with the visit of the Spanish Team in Taiwan). In addition to the intense bioinformatic labour of integrating volatilomes and transcriptomes we will further characterize several transcription factors identified as correlated to the identified metabolites. This characterization will be conducted using DAP-seq that interrogates all the genome regions (including gene promoters) where transcription factors bind. DAP-Seq allows studying the in vitro binding of a given TF of interest to genomic DNA (gDNA). To do this, the gDNA is extracted and fragmented into 200 base pair (bp) sequences. In a separate reaction, the TF of interest attached to an affinity tag (pHALO) is expressed and purified. Through the affinity tag, the TF is attached to an affinity column, through which the gDNA is eluted. In this way, the 200 bp fragments that have TF binding sequences will be retained on the affinity column by their interaction with TF. These fragments are subsequently amplified by PCR and sequenced. As a negative control of the technique, the affinity tag pHALO is used without any bound TF. After sequencing, the bioinformatic analysis begins by mapping the readings with Bowtie2, to then detect the TF binding sites (peaks) with GEM peak caller, which compares the TF alignments against that of pHALO. Finally, the peaks are associated with genes with the ChIPpeakAnno Bioconductor package. The expression of these TFs will be contrasted in high- and low-artemisinin/terpene producers. By the end of this Specific Objective we expect to demonstrate the binding of several TF in the promoters of artemisinin and terpenoid pathway genes.
  • List of keywords (in Spanish and English).

Spanish: Artemisia, elicitación, ómicas, VOCs, cultivos celulares

English: Artemisia, elicitation, VOCs, cellular suspensions, omics


Alignment and relevance of the application with the Sustainable Development Goals.

This project is in alignment with the Sustainable Development Goals adopted by the United Nations (https://sdgs.un.org/goals), especially with the third goal, in ensuring healthy lives and promote well-being for all at all ages, because one of the goals of the project is trying to obtain higher quantities of important molecules such as Artemisinin or health-promoting terpenoids. It is worth noting again that Artemisinin is considered the most effective treatment for malaria and would be used globally if its industrial production was possible (nowadays it’s not economically feasible). Obtaining cells that produce high quantities of artemisinin or other compounds could potentially help treat malaria, which is responsible for killing 25% of all the children that die in Africa under the age of 5 every single year. This project also follows the 15th goal in protecting, restoring and promoting sustainable use of terrestrial ecosystems, sustainably manage forests, combat desertification, and halt and reverse land degradation and halt biodiversity loss. Using suspension cell cultures could avoid using large amounts of land in order to maintain the extensive plantation of A.annua, which could intensify deforestation leading to a loss of biodiversity.


Composition of the TOMSBiolab Team

People directly involved in the Artemisia annua project

  • José Tomás Matus (PI) Ramón y Cajal Researcher. I2SysBio (JTM)
  • Jone Echeverría (PhD candidate, MSc in Research and Development in Biotechnology and Biomedicine) (JE)
  • Chen Zhang (PhD Student at UV, Program of Biodiversity and evolutive biology CSC Scholar) (CZ)

Composition of the IPB group

People directly involved in the Artemisia annua project

  • Hieng-Ming Ting (PI) Institute of Plant Biology, National Taiwan University (H-MT)
  • Yuan-Yun Zhang (PhD Student NTU) (Y-YZ)

Other team members

  • Cheng-Hsiang Kuo (MSc student NTU)
  • Yi-Ju Chen (MSc student NTU)
  • Pin-Zhe Liao  (MSc student NTU)
  • Bing-Rong Chen  (MSc student NTU)


Caretto, S., Quarta, A., Durante, M., Nisi, R., De Paolis, A., Blando, F. and Mita, G., 2010. Methyl jasmonate and miconazole differently affect arteminisin production and gene expression in Artemisia annua suspension cultures. Plant Biology, 13(1), pp.51-58.

Hisao-Hang Chung, Hieng-Ming Ting, Wei-Hsi Wang, Ya-Ting Chao, Cheng-Han Hsieh, Maria Karmella Apaya, Yi-Chang Sung, Shih-Shun Lin, Fang-Yu Hwu, Lie-Fen Shyur, Elucidation of enzymes involved in the biosynthetic pathway of bioactive polyacetylenes in Bidens pilosa using integrated omics approaches, Journal of Experimental Botany, Volume 72, Issue 2, 2 February 2021, Pages 525–541, https://doi.org/10.1093/jxb/eraa457

Matus JT, Ruggieri V, Romero F, Moretto M and Wong DCJ. 2019. Status and Prospects of Systems Biology in Grapevine Research. In: The Grape Genome (Compendium of Plant Genomes). Cantu, Walker, eds.

Orduña L, Li M, Navarro-Payá D, Zhang C, Ramšak Ž, Gruden K, Höll J, Merz P, Vannozzi A, Cantu D, Bogs J, Wong D. C. J., Huang SC, Matus JT. Orchestration of the stilbene synthase gene family and their regulators by subgroup 2 MYB genes. bioRxiv 2020.12.31.424746; doi: https://doi.org/10.1101/2020.12.31.424746

Ting, H.M., Cheah, B.H., Chen, Y.C., Yeh, P.M., Cheng, C.P., Yeo, F.K.S., Vie, A.K., Rohloff, J., Winge, P., Bones, A.M. and Kissen, R., 2020. The role of a glucosinolate-derived nitrile in plant immune responses. Frontiers in plant science, 11, p.257.

Wang, B., Kashkooli, A.B., Sallets, A., Ting, H.M., de Ruijter, N.C., Olofsson, L., Brodelius, P., Pottier, M., Boutry, M., Bouwmeester, H. and van der Krol, A.R., 2016. Transient production of artemisinin in Nicotiana benthamiana is boosted by a specific lipid transfer protein from A. annua. Metabolic engineering, 38, pp.159-169.

Wong DCJ, Matus JT. Constructing Integrated Networks for Identifying New Secondary Metabolic Pathway Regulators in Grapevine: Recent Applications and Future Opportunities. Front Plant Sci. 2017 Apr 12;8:505. doi: 10.3389/fpls.2017.00505. eCollection 2017. PubMed PMID: 28446914; PubMed Central PMCID: PMC5388765.

Wong DC, Schlechter R, Vannozzi A, Höll J, Hmmam I, Bogs J, Tornielli GB, Castellarin SD, Matus JT. A systems-oriented analysis of the grapevine R2R3-MYB transcription factor family uncovers new insights into the regulation of stilbene accumulation. DNA Res. 2016 Jul 12. pii: dsw028. [Epub ahead of print] PubMed. PMID: 27407139; PubMed Central PMCID: PMC5066171.

Zhang C, Dai ZW, Ferrier T,… Matus JT. 2021. MYB24-mediated coordination of terpene accumulation and other light-induced metabolic responses to anthocyanin depletion in the grapevine berry. Submitted to bioRxiv.