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

Senescence induced alteration in the electron transport in wheat leaf chloroplasts

Aging in vivo of primary wheat leaves not only induces loss in chloroplast O2 evolution capacity but also alters the accessibility of the electron transport chain for exogenous electron acceptors and donors. The pH profile of ferricyanide Hill reaction in the presence of uncoupler methylamine shifts to the acidic side, upon leaf aging, close to the optimum pH of oxidized para-phenylenediamine-mediated photosystem-II-catalysed electron transport activity. This suggests that ferricyanide, which normally accepts electrons at more than one site in the electron transport chain, accepts electrons preferentially at a site close to that of electron acceptance by oxidized para-phenylenediamine in aged leaf chloroplasts. Leaf aging enhances the extent of inhibition by dibromothymoquinone, which suggests changes in the acceptor side of photosystem II. Leaf aging also enhances the rate of the photosystem-I-catalysed electron transport reaction supported by reduced dichlorophenol-indophenol and reduced tetramethyl-para-phenylenediamine. Furthermore, the fact that inhibition by KCN of reduced dichlorophenol-indophenol supported photosystem I activity in aged leaf chloroplasts is greater than the activity supported by reduced tetramethyl-para-phenylenediamine suggests an alteration in the site of feeding of electrons by reduced dichlorophenol-indophenol. Thus leaf aging appears to induce alterations in specific segments of the electron transport chain

Effect of temperature on photosynthetic activities of senescing detached wheat leaves

Changes in various components of photosynthetic activity during the dark induced senescence of detached wheat leaves, maintained at 25°C (control) and 35°C (mildly elevated temperature treatment), were examined. Senescence-associated decline measured up to 96 h, in photosynthetic activity was appreciably hastened at 35°C, than at 25°C as evident by the relative higher losses of chlorophyll, photosystem (PS) II and PS I catalyzed photochemical activities and ribulose-1,5-bisphosphate (RuBP) carboxylase activity. In addition, a comparatively higher rise in light scattering profile of isolated chloroplasts was noted at 35°C than at 25°C. Senescence-induced degradation of chlorophyll was faster at 35°C than at 25°C; on the other hand, the degradation of carotenoids was faster at 25°C than at 35°C. Furthermore, the ratio of carotenoids to chlorophyll increased with senescence up to 96 hours, higher ratio being obtained at 35°C than at 25°C. Both PS II and PS I activities showed a transient rise in the beginning phase of dark incubation, whereas loss in chlorophyll was continuous throughout the period of senescence. The initial rise observed in photochemical activities was attributable to the uncoupling of electron transport from photophosphorylation. Elevated temperature treatment resulted in greater inactivation of RuBP carboxylase than control. It appears that during senescence the loss in chlorophyll and RuBP carboxylase activity are triggered simultaneously

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

Kinetics of <i>p</i>BQ concentration dependent electron transport rate in thylakoids prepared from control (DMSO) and Artemisinin-treated (Artemisinin) leaves.

<p>The electron transport rate obtained from the control and treated samples (A) was further analysed for Michaelis-Menten type enzymatic reaction kinetics (B and C). The intersection of the line with the ordinate and the abscissa denotes the inverse values of V_max and K_m respectively. The values calculated for the respective K_m and V_max has been shown in the inset table. The <i>p</i>BQ titration experiments were carried out in thylakoids isolated both from spinach and rice leaves following similar <i>in vivo</i> treatments with artemisinin and the results shown here are from spinach.</p

Effect of increasing concentration of artemisinin on FeCN supported O₂ evolution activity of spinach thylakoids.

<p>The concentration of artemisinin used in µM is shown in parenthesis. The inset depicts the increase in percent inhibition of electron transfer rate relative to control with increase in artemisinin concentration. 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

Kinetics of DQ supported electron transport rate in thylakoids prepared from control (DMSO) and Artemisinin-treated (Artemisinin) leaves.

<p>The reciprocal plot of the velocity-versus-concentration is shown as inset. The intersection lines with ordinate and abscissa respectively represents the inverse of V_max and K_m.</p