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Arbuscular mycorrhiza increase artemisinin accumulation in Artemisia annua by higher expression of key biosynthesis genes via enhanced jasmonic acid levels

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Abstract

It is becoming increasingly evident that the formation of arbuscular mycorrhiza (AM) enhances secondary metabolite production in shoots. Despite mounting evidence, relatively little is known about the underlying mechanisms. This study suggests that increase in artemisinin concentration in Artemisia annua colonized by Rhizophagus intraradices is due to altered trichome density as well as transcriptional patterns that are mediated via enhanced jasmonic acid (JA) levels. Mycorrhizal (M) plants had higher JA levels in leaf tissue that may be due to induction of an allene oxidase synthase gene (AOS), encoding one of the key enzymes for JA production. Non-mycorrhizal (NM) plants were exogenously supplied with a range of methyl jasmonic acid concentrations. When leaves of NM and M plants with similar levels of endogenous JA were compared, these matched closely in terms of shoot trichome density, artemisinin concentration, and transcript profile of artemisinin biosynthesis genes. Mycorrhization increased artemisinin levels by increasing glandular trichome density and transcriptional activation of artemisinin biosynthesis genes. Transcriptional analysis of some rate-limiting enzymes of mevalonate and methyl erythritol phosphate (MEP) pathways revealed that AM increases isoprenoids by induction of the MEP pathway. A decline in artemisinin concentration in shoots of NM and M plants treated with ibuprofen (an inhibitor of JA biosynthesis) further confirmed the implication of JA in the mechanism of artemisinin production.

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Acknowledgments

The research work was partially funded by the Department of Biotechnology, University Grants commission, Delhi, India. Shantanu Mandal and Shivangi Upadhyay gratefully acknowledge the Council of Scientific and Industrial Research for Research Fellowships.

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Authors and Affiliations

  1. Department of Botany, University of Delhi, Delhi, 110007, India

    Shantanu Mandal, Shivangi Upadhyay, Ved Pal Singh & Rupam Kapoor

  2. Department of Biotechnology, Centre for Transgenic Plant Development, Jamia Hamdard, New Delhi, 110062, India

    Saima Wajid & Malik Zainul Abdin

  3. Department of Green Technology, Ipca Laboratories Ltd., Ratlam, 457002, Madhya Pradesh, India

    Mauji Ram & Dharam Chand Jain

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  1. Shantanu Mandal
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Fig. S1

Induction of genes (presented in rectangular box) of mevalonate and artemisinin biosynthesis pathways in Artemisia annua plants subjected to P fertilizer, exogenous treatment of 200 μM MeJA under high and low P and mycorrhizal inoculation. Thickness of arrows indicates the significant changes in transcription levels compared to control plants of the genes coding for the enzyme cited, as determined by qRT-PCR analysis; = denotes no changes in gene expression. HMGR 3-hydroxy-3-methylglutaryl-CoA reductase; DXS 1-deoxyxylulose 5-phosphate synthase; DXR 1-deoxyxylulose 5-phosphate reductoisomerase; ADS Amorpha-4, 11-diene synthase; CYP cytochrome P 450 CYP71AV1; DBR2 double bond reductase 2; ALDH1 aldehyde dehydrogenase 1. (PPT 298 kb)

Fig. S2

Formation of arbuscules (a) and vesicles (b) in the cortical region of A. annua roots. (PPTX 219 kb)

Fig. S3

Effect of G. intraradices inoculation and exogenous application of IBU on relative frequency of arbuscules and vesicles in the cortical region of A. annua roots. (PPTX 131 kb)

Fig. S4

Scanning electron micrographs of leaves of Artemisia annua plants showing effect of G. intraradices inoculation, different exogenous concentrations of methyl jasmonate (μM MeJA) under low and high phosphorus, and exogenous application of ibuprofen (500 μM IBU) on glandular trichome density in control (a), 50 P (b), mycorrhizal (c), 100 MeJA (d), 200 MeJA (e), 300 MeJA (f), 100 MeJA + 50P (g), 200 MeJA + 50P (h), 500 IBU (i), 200 MeJA + 500 μM IBU (j), 300 MeJA + 50P (K), and M + 500 IBU (l). Arrow indicates the glandular trichome. C control, M G. intraradices inoculation, P phosphorus, MeJA methyl jasmonates, IBU ibuprofen. (PPT 3728 kb)

Fig. S5

Effect of G. intraradices inoculation, P fertilizer, and different concentrations (0, 100, 200, and 300 μM) of exogenously applied methyl jasmonate (MeJA) under low and high phosphorus on expression levels of Amorpha-4, 11-diene synthase (ADS), Cytochrome P 450 CYP71AV1 (CYP), and aldehyde dehydrogenase 1 (ALDH1) gene in leaves of Artemisia annua plants. C = NM + 0P + 0 MeJA, M = 0P + 0 MeJA. (PPT 239 kb)

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Mandal, S., Upadhyay, S., Wajid, S. et al. Arbuscular mycorrhiza increase artemisinin accumulation in Artemisia annua by higher expression of key biosynthesis genes via enhanced jasmonic acid levels. Mycorrhiza 25, 345–357 (2015). https://doi.org/10.1007/s00572-014-0614-3

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