Modelling Processes Determining and Limiting the Production of Secondary Metabolites during Crop Growth: the Example of the Antimalarial Artemisinin Produced in Artemisia annua

The quantitative insight in processes underlying yield and concentrations of interesting secondary metabolites in crops is still limited. Yet, this insight is essential to further improve crops and commercial production of target metabolites. Artemisia annua L. (annual or sweet wormwood, Asteraceae) was used to conceptualize a model to describe the processes determining and limiting the production of target metabolites during crop growth. A. annua is an annual herb producing the antimalarial artemisinin, a sesquiterpene lactone with an endoperoxide bridge. Artemisinin is predominantly produced in glandular trichomes present on the leaves and inflorescences. Leaves are the most important organs harvested in commercial production. The accumulation of artemisinin in the crop was analysed as the resultant of the key processes determining leaf dry weight production, accumulation of artemisinin in the leaves and losses after synthesis. A yield formation modelling approach was used to quantify artemisinin yield as a function of the individual processes and to study which processes limited production. Leaf dry weight production was limited by low total dry matter production of the crop because of poor radiation interception during early canopy expansion, and by a high proportion of dry matter allocated to stems that do not contribute to artemisinin production. Production of the target metabolite in the leaves was limited because the total production of artemisinin precursors per unit leaf dry weight was low and because the conversion of precursors to artemisinin was only partial. Possibilities to increase the values of the different yield components are discussed. A new, simple, model for explaining variation in artemisinin yield based on leaf and trichome production in combination with biosynthesis of artemisinin is proposed. INTRODUCTION Interest in plant secondary metabolites that have a protective or curative effect on human diseases has been renewed. These compounds can be taken in as drugs or as part of food and beverages. Yields and concentrations of target metabolites in crops or crop products are usually low and highly variable over studies. Also within crops, concentrations vary. The causes of these fluctuations are often unknown because commercial production methods are commonly based on applied dose-effect studies with limited attention to the crop physiology behind the effects. A great need exists for more insight into the different processes underlying the accumulation of the secondary metabolites of interest. An improved understanding can lead to more stable yield levels and concentrations, better breeding strategies, more founded selection of production environments, and agronomical and technological innovations in the production chain. To achieve a full and quantitative understanding of accumulation of secondary metabolites is difficult because mechanisms underlying their yields and concentrations in crops are more complex than those underlying yield of plant organs, yield of primary Proc. XXVII IHC Plants as Food and Medicine Eds.-in-Chief: G. Gardner and L.E. Craker Acta Hort. 765, ISHS 2008 88 metabolites, or their concentrations. The basic processes playing a role in primary metabolite formation also play a role in the synthesis of secondary metabolites, but more processes are involved: processes affecting the formation of the specialized structures in which the compounds are synthesized (such as ducts or glandular trichomes), processes affecting the production of the compounds of interest and their precursors in these structures, and processes regulating the conversion of the precursors to the target compounds. External factors quantitatively affect these underlying processes through their effects on crop development, growth rates, dry matter partitioning and partitioning of metabolites to the secondary metabolite of interest and these factors can also trigger abrupt activation of qualitative changes in secondary metabolite production. The Model Crop: Artemisia annua Artemisia annua L. (annual or sweet wormwood, Asteraceae) is an annual herb that produces the antimalarial compound artemisinin, a sesquiterpene lactone that is effective against multidrug resistant strains of Plasmodium falciparum. P. falciparum causes the severe malaria tropica resulting in more than one million deaths annually. For pharmaceutical use, artemisinin is extracted from the dry leaf mass, purified and chemically derivatised (e.g., Dhingra et al., 2000) and preferably used in combination with other antimalarials. A. annua is thus far the only economical source of artemisinin. The plant originates from South-east Asia (Ferreira et al., 1997), likely China, but is now widely distributed. The plant is cultivated mainly in Vietnam and China. Within the plant, artemisinin is found almost exclusively in leaves and inflorescences (Ferreira et al., 1995). Leaves are the most important organs for commercial production, as crops are usually harvested before full flowering. Artemisinin is phytotoxic, and production of artemisinin is limited to the very small glandular trichomes on the leaves and inflorescences (Duke and Paul, 1993; Duke et al., 1994). Recently, the biosynthetic route was elucidated for the genotypes used in our study (Bertea et al., 2005). Agronomic problems in A. annua are comparable to those in other crops grown for their secondary metabolites. Yields and concentrations of artemisinin are extremely low. Maximum yields of artemisinin are between 20 and 40 kg per ha. Maximum concentrations of artemisinin in the dry plant leaves are c. 1% (Magalhaes et al., 1996) but usually concentrations are much lower (Morales et al., 1993; Laughlin 1993, 1995). In addition, there is a high variation in yield and concentrations, and almost no insight in what is causing these variations. In our study on artemisinin production, we want to identify and quantify the processes at the level of the crop, individual organs and cells which are underlying artemisinin formation in commercial A. annua crops. The study should lead to (a) a simple model to explain artemisinin production in field crops, and (b) insight in which processes limit artemisinin production during crop growth. This paper describes the state of the art of this study. MATERIALS AND METHODS The ideas for modelling harvestable artemisinin were developed in discussions and by literature study. The experimental work was done using either an open pollinated Vietnamese selection (Bouwmeester et al., 1999; Wallaart et al., 2000; Bertea et al., 2005; Lommen et al., 2006) or the hybrid cultivar Anamed A3 (cf. Lommen et al., 2006), and was carried out at two locations (Achterberg and Wageningen) in The Netherlands; experiments included different planting densities. The experimental data presented are from one experiment using the Vietnamese selection. Plants were grown from seeds, raised in transplanting trays in the glasshouse and transplanted into the field in Wageningen, The Netherlands (N51°59'31'' E005°39'08'') on May 21, 2002, 49 days after sowing, at 75 cm betweenand 38 cm within-row distance. The plots were part of a larger experiment laid out in five blocks. Net plots were 1.5 x 1.52 m2 (2 rows x 4 plants) and were surrounded by at least 1.5 m of