Trichome Dynamics and Artemisinin Accumulation during Development and Senescence of Artemisia annua Leaves

Abstract

Artemisinin is a sesquiterpene lactone endoperoxide and an important antimalarial drug produced in Artemisia annua. To unravel the diverse processes determining artemisinin yield in A. annua crops, artemisinin accumulation during the development of individual leaves was studied in two field experiments. During the life cycle of a leaf, artemisinin was always present. Quantities were low at leaf appearance and increased steadily. In leaves studied until after senescence, maximum quantities and concentrations were achieved after the leaf had turned brown. The total quantity of possible artemisinin precursors per leaf (dihydroartemisinic acid and other upstream precursors) was highest early in the leaf cycle when the leaf was still expanding. Dihydroartemisinic acid was more abundant than the other compounds and its quantity declined during leaf development whereas that of artemisinin increased. Dihydroartemisinic acid was not converted directly into artemisinin, because on a per leaf basis the decline in molar quantity of precursors in the earliest formed leaves was not compensated for by a simultaneous increase in artemisinin. Our results suggest that a (putative) intermediate such as dihydroartemisinic acid hydroperoxide temporarily may have accumulated in considerable quantities. The number of mature, capitate trichomes on the adaxial leaf side increased after leaf appearance until the end of leaf expansion, and then decreased, probably due to collapse of trichomes. Artemisinin production thus (also) occurred when trichomes were collapsing. Later formed leaves achieved higher concentrations of artemisinin than earlier formed leaves, because of a higher trichome density and a higher capacity per trichome.

Key words

Artemisia annua - artemisinin formation - leaf position - leaf ontogenesis - glandular trichomes

References

• 1 Dhingra V, Rao K V, Narasu M L. Current status of artemisinin and its derivatives as antimalarial drugs.  Life Sci. 2000;  66 279-300
• 2 Ferreira J FS, Simon J E, Janick J. Developmental studies of Artemisia annua: Flowering and artemisinin production under greenhouse and field conditions.  Planta Med. 1995;  61 167-70
• 3 Ferreira J FS, Janick J. Distribution of artemisinin in Artemisia annua .  In: Janick J, editor. Progress in new crops.  Arlington; ASHS Press; 1996: p. 579-84
• 4 Gupta S K, Singh P, Bajpai P, Ram G, Singh D, Gupta M M. et al . Morphogenetic variation for artemisinin and volatile oil in Artemisia annua .  Ind Crop Prod. 2002;  16 217-24
• 5 Duke S O, Paul R N. Development and fine structure of the glandular trichomes of Artemisia annua L.  Int J Plant Sci. 1993;  154 107-18
• 6 Duke M V, Paul R N, Elsohly H N, Sturtz G, Duke S O. Localization of artemisinin and artemisitene in foliar tissues of glanded and glandless biotypes of Artemisia annua L.  Int J Plant Sci. 1994;  155 365-72
• 7 Bouwmeester H J, Wallaart T E, Janssen M HA, van Loo B, Jansen B JM, Posthumus M A. et al . Amorpha-4,11-diene synthase catalyses the first probable step in artemisinin biosynthesis.  Phytochemistry. 1999;  52 843-54
• 8 Bertea C M, Freije J R, van der Woude H, Verstappen F WA, Perk L, Marquez V. et al . Identification of intermediates and enzymes involved in the early steps of artemisinin biosynthesis in Artemisia annua L.  Planta Med. 2005;  71 40-7
• 9 Wallaart T E, van Uden W, Lubberink H GM, Woerdenbag H J, Pras N, Quax W J. Isolation and identification of dihydroartemisinic acid from Artemisia annua and its possible role in the biosynthesis of artemisinin.  J Nat Prod. 1999;  62 430-3
• 10 Sy L K, Brown G D. The mechanism of the spontaneous autoxidation of dihydroartemisinic acid.  Tetrahedron. 2002;  58 897-908
• 11 Brown G D, Sy L K. In vivo transformations of dihydroartemisinic acid in Artemisia annua plants.  Tetrahedron. 2004;  60 1139-59
• 12 Wallaart T E, Pras N, Beekman A C, Quax W J. Seasonal variation of artemisinin and its biosynthetic precursors in plants of Artemisia annua of different geographical origin: Proof for the existence of chemotypes.  Planta Med. 2000;  66 57-62
• 13 Laughlin J C. Effect of agronomic practices on plant yield and anti-malarial constituents of Artemisia annua L.  Acta Hortic. 1993;  331 53-61
• 14 Laughlin J C. The influence of distribution of antimalarial constituents in Artemisia annua L. on time and method of harvest.  Acta Hortic. 1995;  390 67-73
• 15 Woerdenbag H J, Pras N, Nguyen Gia C han, Bui Thi B ang, Bos R, van Uden W. et al . Artemisinin, related sesquiterpenes, and essential oil in Artemisia annua during a vegetation period in Vietnam.  Planta Med. 1994;  60 272-5
• 16 Liersch R, Soicke H, Stehr C, Tüllner H U. Formation of artemisinin in Artemisia annua during one vegetation period.  Planta Med. 1986;  52 387-90
• 17 Morales M M, Charles D J, Simon J E. Seasonal accumulation of artemisinin in Artemisia annua L.  Acta Hortic. 1993;  344 416-20
• 18 Munsell . Munsell Color Charts for Plant Tissues,. 2nd edition. Baltimore; Munsell Color Division, Kollmorgen Corporation; 1972: p 19
• 19 Wallaart T E, Pras N, Quax W J. Seasonal variations of artemisinin and its biosynthetic precursors in tetraploid Artemisia annua plants compared with the diploid wild-type.  Planta Med. 1999;  65 723-8
• 20 Laughlin J C. Post-harvest drying treatment effects on antimalarial constituents of Artemisia annua L.  Acta Hortic. 2002;  576 315-20
• 21 Ascensão L, Pais M SS. Glandular trichomes of Artemisia campestris (ssp. maritima) ontogeny and histochemistry of the secretory product.  Bot Gaz. 1987;  148 221-7
• 22 Shanker S, Ajayakumar P V. Essential oil gland number and ultrastructure during Mentha arvensis leaf ontogeny.  Biol Plantarum. 1999;  42 379-87
• 23 Wallaart T E, Pras N, Quax W J. Isolation and identification of dihydroartemisinic acid hydroperoxide from Artemisia annua: a novel biosynthetic precursor of artemisinin.  J Nat Prod. 1999;  62 1160-2
• 24 Woerdenbag H J, Lugt C B, Pras N. Artemisia annua L.: a source of novel antimalarial drugs.  Pharmaceutisch Weekblad, Sci Ed. 1990;  12 169-81