Reproductive development modulates gene expression and metabolite levels with possible feedback inhibition of artemisinin in Artemisia annua

Abstract

The relationship between the transition to budding and flowering in Artemisia annua and the production of the antimalarial sesquiterpene, artemisinin (AN), the dynamics of artemisinic metabolite changes, AN-related transcriptional changes, and plant and trichome developmental changes were measured. Maximum production of AN occurs during full flower stage within floral tissues, but that changes in the leafy bracts and nonbolt leaves as the plant shifts from budding to full flower. Expression levels of early pathway genes known to be involved in isopentenyl diphosphate and farnesyl diphosphate biosynthesis leading to AN were not immediately positively correlated with either AN or its precursors. However, we found that the later AN pathway genes, amorpha-4,11-diene synthase (ADS) and the cytochrome P450, CYP71AV1 (CYP), were more highly correlated with AN's immediate precursor, dihydroartemisinic acid, within all leaf tissues tested. In addition, leaf trichome formation throughout the developmental phases of the plant also appears to be more complex than originally thought. Trichome changes correlated closely with the levels of AN but not its precursors. Differences were observed in trichome densities that are dependent both on developmental stage (vegetative, budding, and flowering) and on position (upper and lower leaf tissue). AN levels declined significantly as plants matured, as did ADS and CYP transcripts. Spraying leaves with AN or artemisinic acid inhibited CYP transcription; artemisinic acid also inhibited ADS transcription. These data allow us to present a novel model for the differential control of AN biosynthesis as it relates to developmental stage and trichome maturation and collapse.

Figure 1.

Isopentenyl diphosphate and AN biosynthetic pathway. MEP, Nonmevalonate pathway (plastid pathway).

Figure 2.

A. annua during vegetative and reproductive growth. A, Vegetative growth. B, Floral bolt developed. C, Full flower. Arrows indicate the leafy bracts. D to F, Schematic of locations of leaves of different ages on vegetative plants (D), on budding plants (F), and on plants in full flower (F). [See online article for color version of this figure.]

Figure 3.

Artemisinic metabolite response to budding and flowering. Metabolites were extracted from fresh plant material and assayed by liquid chromatography-mass spectrometry. Letters show statistical significance for each metabolite; values are means ± sd (n = 3). FW, Fresh weight.

Figure 4.

Responses of artemisinic metabolite-specific genes (DXS, DXR, HMGR, FPS, ADS, and CYP71AV1) to budding and flowering. Values displayed are mean fold change as calculated by the 2^−ΔΔCT method ± sd (n = 5). * P < 0.05, relative to vegetative plants. [See online article for color version of this figure.]

Figure 5.

AN levels correlate with trichome densities regardless of leaf type. Bars show AN content in various leaf types. Points represent total glandular trichome (adaxial + abaxial) populations. Error bars represent sd. FW, Fresh weight. [See online article for color version of this figure.]

Figure 6.

Changes in gene expression with spraying of 50 μg mL⁻¹ AN in 70% ethanol relative to spraying with 70% ethanol controls. Asterisks show significant differences at P ≤ 0.05 (n = 4).

Figure 7.

Summation of trichome populations, transcript levels, and artemisinic metabolites throughout the development of A. annua from vegetative growth through full flowering. Numbers within each box are average values obtained for each leaf type analyzed for the indicated factor. Shading of the boxes indicates the relative level of response. Fold change is against VEG leaves. FW, Fresh weight. [See online article for color version of this figure.]