Furano-terpene biosynthesis in Ipomoea batatas

Venus Affrignago, Amanda Fetters, Gilberto Reyes, Stephanie Stoelb, Jeanne WagesNatural Sciences Division, Bluegrass Community and Technical College, Lexington, Kentucky

email: jwages0007@kctcs.edu; stephanie.stoelb@kctcs.edu

Abstract

Terpenoids are a diverse class of plant natural products that function as plant hormones, act in plant defense responses and provide structural support to membranes. In addition, terpenoids are commercially valued as essential oils, flavors, fragrances and pharmaceutical drugs. Furano-terpenes isolated from Ipomoea batatas are produced in response to fungal infection. Because of their anti-fungal activities, these terpenes may be particularly useful as new anti-fungal treatments. We are interested in identifying genes involved in the biosynthesis of these compounds. We are currently testing different fungal elicitors to identify compounds that increase production of furano-terpenes in I. batatas. We are using GC-MS analysis to identify these compounds. In addition, we are isolating RNA to conduct RT-PCR analysis to identify candidate biosynthetic genes. Once we have identified biosynthetic genes, we will express these heterologously to confirm their functions and engineer increased production of furano-terpenes.

Discussion and Future Directions

Through GC-MS analysis, we determined that only mercuric chloride was successful in inducing production of ipomeamarone(Fig. 3). Our results indicate that methyl-jasmonate and cellulase do not induce production of ipomeamarone (Fig.4, Table 1). It could be possible that ipomeamarone was produced earlier in incubation of these samples and/or was expelled into the filter paper.

Moving forward, we would like to continue to survey alternative induction methods to identify additional treatments for ipomeamarone induction. Perhaps different fungal/viral plant pathogens could be explored and shorter or longer incubation times could be tested based on the progression of the infection. The RNA samples that were isolated from the sweet potato tissue will be used to conduct RT-PCR (reverse transcription polymerase chain reaction) to obtain complimentary DNA (cDNA) which is DNA synthesized from an RNA template. Combined with a bioinformatics approach, we can identify genes involved in ipomeamarone biosynthesis. These genes could subsequently be used to engineer production of furano-terpenes in heterologous systems.

Introduction

Sweet potato tissue can produce a variety of furano-terpenes such as ipomeamarone when injured by fungal infection or chemical treatment, such as mercuric chloride.1 Furano-terpenes are produced in the non-injured tissue adjacent to the injured region and accumulate in the injured region. The terpenes are fungitoxic and aid in defense action of the host against the parasite.1,2 Additionally, terpenes have numerous commercial uses as flavors, fragrances and pharmaceutical compounds. Paclitaxel (trade named Taxol) is a well known naturally sourced anticancer drug derived from the Pacific Yew Tree (Taxusbrevifolia) and is used to treat breast, lung and ovarian cancers as well as Kapsosi’s sarcoma.3 Parthenolide, a terpenoidcommonly found in feverfew, is currently being studied due to its cytotoxic activity and anti-inflammatory properties.4 We are interested in the synthesis of the main furano-terpene ipomeamarone as a potential anti-fungal agent. By elucidating the biosynthetic pathway, we can engineer ipomeamarone production in heterologous systems for more efficient production of the compound. We are interested in biosynthesis of ipomeamarone, a sesquiterpenoid produced by sweet potatoes infected with Ceratocystis fimbriata (Fig. 1).5

3 Day + Cellulase

3 Day - Cellulase

3 Day – Me-Ja

3 Day + Me-Ja

3 Day + Me-Ja V.

3 Day – Me-Ja V.

Results

HgCl2 Me-Ja Me-Ja V. Cellulase

3 day (+) P A A A

3 day (-) A A A A

5 day (+) P A A A

5 day (-) A A A A

7 day (+) P A A A

7 day (-) A A A A

Materials and Methods

Sample Preparation: Sweet potato samples were purchased on the same day and cut into circular slices using ~3 grams of tissue per petri plate. The plates were overlaid with white chromatography paper that had been soaked with 3 milliliters of water. For each treatment, plates were incubated at room temperature for 3 days, 5 days, and 7 days (Fig. 2).

Sample Treatment: Samples were treated with 3 different chemicals: 1. methyl-jasmonate (Me-Ja), 2. cellulase and 3. mercuric chloride (HgCl2). Comparison using 0.5mM Me-Ja solution versus Me-Ja vapor was also we performed (+/- Me-Ja and +/- Me-Ja V., respectively). Samples treated with cellulase were treated with 0.5 % cellulase from Aspergillis niger (+/-cellulase). Samples treated with HgCl2 were incubated with 0.1 % HgCl2 (+/- HgCl2). Following incubation, tissue samples were ground to a powerder in liquid nitrogen and stored at -80 °C.

RNA Isolation: We isolated RNA from the cellulase and Me-Ja 3 day samples using Tri-Reagent following the manufacturer’s instructions.9 RNA quality was visualized on a 1% agarose gel stained with Ethidium Bromide (Figure 5).

Terpene extraction: Sweet potato tissue samples were extracted with ethyl acetate with overnight at -20 °C. One microliter of extract was injected for GC-MS analysis. Each sample was spiked with 10 ng/ul of alpha-cedrene (terpene) for use as a standard. Samples were analyzed using Agilent 5977 GC-MS system; terpene presence was confirmed using MassHunter and the NIST Library.

3 Day - HgCl2

3 Day+ HgCl2

5 Day + HgCl2

5 Day - HgCl2

7 Day + HgCl2

7 Day - HgCl2

Figure 3. Trace files for +/- mercuric chloride tissue extracts (3, 5, and 7 day incubation time). Samples for all 3 incubation periods produced the signifying ipomeamarone peak at ~15 minutes when treated with mercuric chloride (alpha-cedrene standard appeared at ~12 minutes). Chemical structure as well as mass spectrum fragmentation pattern for ipomeamarone are also outlined.

Figure 4. Trace files for +/- cellulase, methyl-jasmonate, and methyl jasmonate vapor for samples incubated for 3 days. Alpha-cedrene standard present in all samples, ipomeamarone not present.

Table 1. Presence (P) or absence (A) of ipomeamarone in chemically treated sweet potato tissue with varying incubation times.

References:1. Oba, K., et al. 1976. Induction of Furano-terpene Production and Formation of the Enzyme System from Mevalonate to Isopentenyl Pyrophosphate in Sweet Potato

Root Tissue Injured by Ceratocystis fimbriata and by Toxic Chemicals. Plant Physiology, 58 (51-56).2. Oba, K and Uritani, I. 1981. Mechanism of Furano-terpene Production in Sweet Potato Root Tissue Injured by Chemical Agents. Agric. Biol. Chem., 45 (1635-1639).3. Taxol (National Cancer Institute) https://dtp.cancer.gov/timeline/flash/success_stories/S2_Taxol.htm4. Mathema, VB, et al. 2012. Parthenolide, a Sesquiterpene Lactone, Expresses Multiple Anti-cancer and Anti-inflammatory Activities. Inflammation, 35:2 (560-565).5. Dudareva, N., et al. 2013. Biosynthesis, function and metabolic engineering of plant volatile organic compounds. New Phytologist, 198:1 (16-32).6. www.forestryimages.org7. www.insectimages.org8. content.ces.ncsu.edu9. TRI Reagent Protocol, Sigma-Aldrich (www.sigmaalrdrich.com/technical-documents/protocols/bology/tri-reagent.printview.html)

Figure 5. RNA isolation of sweet potato tissue. A = cellulase treated, B = Me-Ja treated (3 day incubation). Presence of signature 28S and 18S rRNAbands. Gel was stained with Ethidium Bromide for visualization.

Figure 1. Ceratocystis fimbriata, a common fungus that attacks sweet potato tissue 6,7,8

Figure 2. Research students preparing sweet potato tissue with fungal elicitors to test for increased production of furano-terpenes.

28S rRNA18S rRNA

A B

Ipomeamarone Ipomeamarone Ipomeamarone

alpha-cedrenestandard

methyl-jasmonate

Acknowledgments

We would like to thank KY NSF EPSCoR for the continued support in funding this program (subaward 3048111570-15-087).