Artemisinin Inhibits Chloroplast Electron Transport Activity: Mode of Action

Artemisinin, a secondary metabolite produced in Artemisia plant species, besides having antimalarial properties is also phytotoxic. Although, the phytotoxic activity of the compound has been long recognized, no information is available on the mechanism of action of the compound on photosynthetic activity of the plant. In this report, we have evaluated the effect of artemisinin on photoelectron transport activity of chloroplast thylakoid membrane. The inhibitory effect of the compound, under in vitro condition, was pronounced in loosely and fully coupled thylakoids; being strong in the former. The extent of inhibition was drastically reduced in the presence of uncouplers like ammonium chloride or gramicidin; a characteristic feature described for energy transfer inhibitors. The compound, on the other hand, when applied to plants (in vivo), behaved as a potent inhibitor of photosynthetic electron transport. The major site of its action was identified to be the QB; the secondary quinone moiety of photosystemII complex. Analysis of photoreduction kinetics of para-benzoquinone and duroquinone suggest that the inhibition leads to formation of low pool of plastoquinol, which becomes limiting for electron flow through photosystemI. Further it was ascertained that the in vivo inhibitory effect appeared as a consequence of the formation of an unidentified artemisinin-metabolite rather than by the interaction of the compound per se. The putative metabolite of artemisinin is highly reactive in instituting the inhibition of photosynthetic electron flow eventually reducing the plant growth

The characteristic room temperature Chl. <i>a</i> fluorescence transient of control (DMSO) and artemisinin-treated (Artemisinin) rice leaves.

<p>The leaves were washed and dark adapted for 20 min. before subjected for fluorescence measurements. The sequence of light used to evoke the various fluorescence transients are as follows. Minimal fluorescence (F<i>_O</i>) was obtained with low intensity modulated light. Saturating pulse (SP, ≤1 s duration, 18,000 µmol photons m⁻² s⁻¹) was used to give F<i>_m</i> level of fluorescence in darkness and F<i>_m′</i> in light. Actinic light (300 min duration, 615 µmol photons m⁻² s⁻¹) was applied to drive photosynthesis and gives the transient F<i>_P</i>. and F<i>_t</i> level of fluorescence. A short pulse of far–red light was used to get F<i>_O′</i> fluorescence. Each tracing is the average plot of five individual readings. The R_fd (fluorescence decline ratio from ‘<i>p</i>’ to ‘<i>t</i>’ level) value for control and treated sample was noted to be 1.98±0.04 and 0.08±0.006 respectively.</p

Oxygen exchange activity in whole chain (H₂O to FeCN, A), PSII (H₂O to <i>p</i>BQ, B) and PSI (DCIPH₂ to MV, C) catalyzed electron flow in thylakoids isolated from leaves of control (DMSO) and artemisinin-treated (Artemisinin) rice plants.

<p>The measuring cuvette contained 20 µg Chl. equivalent thylakoids suspension in 1 ml of the reaction medium. The numbers in parenthesis denote the electron transport rate, expressed as µmol O₂ evolved (A and B) or consumed (C) mg Chl.⁻¹ h⁻¹. Arrow up, light on; arrow down, light off.</p

The room temperature Chl. <i>a O-J-I-P</i> fluorescence transient of control (DMSO) and artemisinin-treated (Artemisinin) leaves (A), and the effect of DCMU (B).

<p>Leaves were floated in DCMU (20 µM) solution for 1 h in complete dark to evaluate its effect in DMSO and artemisinin sprayed leaves. The minor difference in F<i>₀</i> (<i>O</i>) and F<i>_p</i> (<i>P</i>) obtained was double normalized at F<i>_O</i> and F<i>_m</i> level using biolyzerhp3 software. Each tracing is average plot of nine individual readings. The SD for F<i>_O</i> and F<i>_m</i> in control leaves was 358±9 and 1784±13 and for treated leaves was 368±11 and 1757±19.</p

Alteration in the electron transport activity in thylakoids prepared from control (DMSO) and artemisinin-treated (Artemisinin) rice leaves under basal and uncoupled conditions.

<p>Thylakoids isolated from leaves of control and treated plants were assayed for alteration in basal and uncoupled (NH₄Cl or GS) electron transport activities with FeCN as terminal electron acceptor. Measuring cuvette contained 20 µg Chl. equivalent thylakoids suspension in 1 ml of the reaction medium. The number in parenthesis denotes the electron transport rates in µmol O₂ evolved mg Chl⁻¹ h⁻¹. Arrow up, light on; arrow down, light off. The error bars indicate ±SD of electron transport (n=3).</p

Inhibition of FeCN supported electron transport activity of spinach thylakoid incubated with the supernatant (A) and resuspended pellet (B) obtained following ultracentrifugation.

<p>Hundred µg Chl. equivalent control thylakoids suspension were incubated with both the fractions (A, supernatant; B, pelleted fraction) for two different time frames of 30 and 60 min (mentioned below the tracings). FeCN supported electron transport activity was assayed in 1 ml of reaction mixture containing thylakoid suspension equivalent to 20 µg Chl. The number in parenthesis denotes the electron transport rates in µmol O₂ evolved mg Chl⁻¹ h⁻¹. Arrow up, light on; arrow down, light off.</p