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Evaluation of antidesmone alkaloid as a photosynthesis inhibitor
YPEST-03949; No of Pages 8 Pesticide Biochemistry and Physiology xxx (2016) xxx–xxx
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Pesticide Biochemistry and Physiology journal homepage: www.elsevier.com/locate/pest
Evaluation of antidesmone alkaloid as a photosynthesis inhibitor Olívia Moreira Sampaio a,⁎, Murilo Marinho de Castro Lima b, Thiago André Moura Veiga c, Beatriz King-Díaz d, Maria Fátima das Graças Fernandes da Silva b, Blas Lotina-Hennsen d a
Chemistry Department, Federal University of Mato Grosso (UFMT), Cuiabá, MT, Brazil Chemistry Department, Federal University of São Carlos (UFSCar), São Carlos, SP, Brazil Institute of Environmental, Chemistry and Pharmaceutical Sciences, Federal University of São Paulo (UNIFESP), São Paulo, SP, Brazil d Biochemistry Department, Facultad de Química, Universidad Nacional Autónoma de México, México D. F. 04510, Mexico b c
a r t i c l e
i n f o
Article history: Received 25 September 2015 Received in revised form 15 April 2016 Accepted 18 April 2016 Available online xxxx Keywords: Antidesmone Chlorophyll a Photosystem II ATP synthesis Herbicide Photosynthetic inhibitor
a b s t r a c t Antidesmone, isolated from Waltheria brachypetala Turcz., owns special structural features as two α,β-unsaturated carbonyl groups and a side alkyl chain that can compete with the quinones involved in the pool of plastoquinones at photosystem II (PSII). In this work, we showed that the alkaloid is an inhibitor of Hill reaction and its target was located at the acceptor side of PSII. Studies of chlorophyll (Chl) a fluorescence showed a J-band that indicates direct action of antidesmone in accumulation of Q− A (reduced plastoquinone A) due to the electron transport blocked at the QB (plastoquinone B) level similar to DCMU. In vivo assays indicated that antidesmone is a selective post-emergent herbicide probe at 300 μM by reducing the biomass production of Physalis ixacarpa plants. Furthermore, antidesmone also behaves as pre-emergent herbicide due to inhibit Physalis ixacarpa plant growth about 60%. Antidesmone, a natural product containing a 4(1H)-pyridones scaffold, will serve as a valuable tool in further development of a new class of herbicides. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Increasing agricultural productivity without expansion of land for food production is a necessity for a growing world population. In this sense, avoid agricultural pests is a great concern. Since weeds are one of the major pests causing a continual reduction in the quantity and quality of crops worldwide, increasing agricultural yield is highly dependent on the use of synthetic herbicides to control weeds. The continuous use of herbicides has resulted in selective pressures, leading to the replacement of sensitive weeds by herbicide-resistant biotypes [1–3]. The major goal of modern weed management research is to discover new herbicides that control the widest possible range of weed species (i.e. can be sprayed at pre- and post-emergence), and at low application rates. Furthermore, selective herbicides need to be safe to the target crop, environment and humans. In this context, natural products fit well against these objectives, and become interesting alternatives for herbicides because they are non-toxic and have demonstrated good
activity in weed control. To satisfy this demand, research in academia and industry has focused on natural products as a promising strategy for developing new herbicides. In the search of potential herbicides, several natural products, such as epifriedelinol [4], evolitrine [5] and ocotilone [6] (Fig. 1), have been studied as inhibitors of PSII with good prospects as natural inhibitors of plant growth. Waltheria brachypetala Turcz (Malvaceae) is a plant with an endemic occurrence in semi-arid areas in Brazil [7]. Previous phytochemical studies showed that W. brachypetala leaves and stems contain cyclic peptide and quinolone alkaloids, such as antidesmone [8]. Looking at the chemical structure of antidesmone, this compound has a system of two α,β-unsaturated carbonyl groups and a side alkyl chain, similarly to the quinones involved in the pool of plastoquinones at PSII (Fig. 2). Our aims in this work were to evaluate and determine the effects of antidesmone on photosynthetic apparatus, germination, roots and stems growth and dry biomass experiments. 2. Material and methods
Abbreviations: ADP, adenosine diphosphate; ATP, adenosine triphosphate; Chl, chlorophyll; DBMIB, 2,5-dibromo-6-isopropyl-3-methyl-1,4-benzoquinone; DCBQ, 2,5dichloro-1,4-benzoquinone; F0, initial fluorescence; FM, maximum fluorescence level; DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethylurea; IC50, (the half maximal inhibitory concentration); MV, methylviologen; OEC, oxygen evolving complex; PSI, photosystem I; PSII, photosystem II; SiMo, sodium silicomolybdate; Tris, (hydroxymethyl)aminoethane. ⁎ Corresponding author. E-mail address: [email protected] (O.M. Sampaio).
2.1. Antidesmone isolation The ethanol extract of W. brachypetala stems and leaves (180 g) was subjected to liquid-liquid extraction with methanol:water (1:3) and hexane to provide the hexane fraction (12.0 g). Six grams from hexane fraction was subjected to chromatographic purification using silica gel (70–230 mesh) as the stationary phase. The mobile phase was
http://dx.doi.org/10.1016/j.pestbp.2016.04.006 0048-3575/© 2016 Elsevier Inc. All rights reserved.
Please cite this article as: O.M. Sampaio, et al., Evaluation of antidesmone alkaloid as a photosynthesis inhibitor, Pesticide Biochemistry and Physiology (2016), http://dx.doi.org/10.1016/j.pestbp.2016.04.006
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O.M. Sampaio et al. / Pesticide Biochemistry and Physiology xxx (2016) xxx–xxx
2.3. Measurement of ATP synthesis ATP synthesis was determined by titration using a microelectrode (Orion model 8103; Ross, Beverly, MA, USA) connected to a Corning potentiometer model 12 (Corning Medical, Acton, MA, USA) with an expanded scale and a Gilson recorder (Kipp & Zonen, Bohemia, NY, USA) [12,13]. A non-buffered solution was employed to break the intact chloroplasts by osmotic rupture, using 100 mM sorbitol, 10 mM KCl, 5 mM MgCl2, 0.5 mM KCN, 50 μM MV, 1 mM Na+-tricine, 1 mM of ADP, with the pH adjusted to 8.0 applying 50 mM of KOH. The mixture was illuminated for 1 min and the ATP synthesis was measured as micromoles of ATP per milligram of Chl per hour. 2.4. Measurement of non-cyclic electron transport rate
Fig. 1. Natural products that are ATP synthesis inhibitors of PSII.
composed of hexane/ethyl acetate in ascending order of polarity (9:1, 8:2, 7:3, 6:4, 1:1), followed by dichloromethane:methanol (1:0, 95:5, 9:1, 8:2, 7:3, 6:4, 0:1) to obtain 49 fractions. Fraction 42 was again subjected to new chromatographic purification using silica (70–230 mesh), eluting with solvents isocratic elution (dichloromethane: methanol (95:5)) to obtain 29 new fractions. Fractions 16–18 were combined and subjected to semipreparative HPLC (Shimadzu, SCL-10 A VP) using a Phenomenex propyl-ether-diol normal bond as stationary phase (300 × 8 mm, 10 μm) with hexane:ethanol (95:5) as eluting system at 1.4 mL/min flow rate, to afford the pure alkaloid antidesmone (0.03 g). The compound was identified by spectroscopic analysis and confirmed according to thorough comparisons with the found literature data for the isolated alkaloid [9]. The obtained signal assignments are described as it follows: 1 H NMR (400 MHz, CDCl3) δ: 0.87 (t, J = 7.0 Hz, 2H, H-17), 1.25 (2H, H-17, overlapping), 1.25–1.80 (10H, H-12-16, overlapping) 1.45 (1H, H11b, overlapping), 1.75 (1H, H-11a, overlapping), 2.08 (dddd, J = 14.7, 14.0, 4.5, 4.3 Hz, 1H, H-6ax), 2.20 (dddd, J = 14.0, 5.3, 4.3, 2.4 Hz, 1H, H6eq), 2.36 (s, 3H, 2-CH3), 2.58 (ddd, J = 18.1, 4.4, 2.4 Hz, 1H, H-7eq), 2.75 (ddd, J = 18.1, 14.7, 5.3 Hz, 1H, H-7ax), 3.26 (m, 1H, H-5), 3.93 (s, 3H, OCH3), 8.73 (sl, 1H, N\\H). 13C NMR (100 MHz, CDCl3) δ: 14.0 (C-18), 14.6 (C2\\CH3), 22.6 (C-17), 24.4 (C-6), 29.2–29.6 (C-12-16), 30.3 (C-5), 30.5 (C-11), 32.2 (C-7), 59.5 (OCH3), 131.9 (C-9), 139.0 (C2), 139.1 (C-10), 147.6 (C-3), 173.3 (C-4), 194.8 (C-8). MS (ESI+) m/z 320 [M + H]. [α]25 D : +26 (c 1.50, CHCl3).
2.2. Chloroplast isolation and Chl determination Intact chloroplasts were isolated from spinach leaves (Spinacea oleracea L.) as previously described [10]. The Chl concentration was measured spectrophotometrically through a chloroplast suspension in a solution of 400 mM sucrose, 5 mM MgCl2, 10 mM KCl, 30 mM tricine-KOH and pH 8.0 [11].
Fig. 2. Structure of plastoquinona and antidesmone.
Light-induced non-cyclic electron transport activity from water to MV was determined polarographically using a Clark type electrode in the presence of 50 μM of MV [14]. Basal electron transport was measured by illuminating a solution of chloroplasts (20 μg Chl/mL) in 3 mL of 100 mM sorbitol, 10 mM KCl, 5 mM MgCl2, 0.5 mM KCN, 15 mM tricine-KOH and 50 μM MV at pH 8.0 for 1 min. The phosphorylating non-cyclic electron transport rate was measured as for the basal electron transport from water to MV, adding 1 mM of ADP and 3 mM KH2PO4. Uncoupled electron transport was evaluated in the same solution used for basal electron transport with 6 mM NH4Cl added as an uncoupler [10]. 2.5. Uncoupled PSII electron flow determination The electron flow activities were monitored with an oxygen monitor yellow spring instrument model 5300 A using a Clark type electrode. All reaction mixtures were illuminated with filtered light (filter of 5 cm of 1% CuSO4 solution) from a projector lamp (GAF 2660) at room temperature. For each reaction, a blank experiment was performed with chloroplasts in the reaction medium. The IC50 value for each activity was determined from plots of the activity versus different concentrations of antidesmone. The solution used for PSII reactions was the same medium used for basal electron transport measurements except MV. The uncoupled electron transport rate from water to DCBQ was measured by illuminating the chloroplasts for 1 min (20 μg Chl/mL) in a solution for PSII reactions, 100 μM of DCBQ, 1 μM of DBMIB and 6 mM NH4Cl [15]. To determine the uncoupled partial reaction of PSII measured from water to SiMo, a solution of 200 μM of SiMo and 10 μM of DCMU were added to the solution used for the PSII reactions (3 mL), then chloroplasts (20 μg Chl/mL) were added and illuminated for 1 min [16]. 2.6. Chl a fluorescence measurements on thylakoids Chl a fluorescence transients were measured with a Handy-PEA (Plant Efficient Analyzer, from Hansatech, King's Lynn, Norfolk, UK), as previously described [5]. The maximum fluorescence yield from the sample was generated by illumination for two seconds with continuous light (650 nm wavelength, intensity equivalent of 2830 μmol photons m−2 s−1 1 and gain of 0.7) from an array of three light-emitting diodes. The reaction medium employed was the solution used in PSII reactions. To monitor Chl a fluorescence transient, aliquots of dark-adapted thylakoids for 5 min containing 60 μg of Chl were transferred to filter paper by gravity with a dot-blot apparatus (Bio-Rad, United States) in order to ensure a homogeneous and reproducible distribution of thylakoids. The filter paper was dipped immediately in 3 mL of the medium with different concentrations of antidesmone in DMSO (150 and 300 μM) and the control was made with the medium plus DMSO. Infiltrated chloroplasts with 10 μM of DCMU and chloroplasts previously treated with 0.8 M of Tris were used as a positive control. The
Please cite this article as: O.M. Sampaio, et al., Evaluation of antidesmone alkaloid as a photosynthesis inhibitor, Pesticide Biochemistry and Physiology (2016), http://dx.doi.org/10.1016/j.pestbp.2016.04.006
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Table 1 Derived parameters, their description and formulae using data extracted from the Chl a fluorescence (OJIP) transients. Fluorescence parameters derived from the extracted data Mo = dV/dto = 4(F300 μs − F0)/(FM − F0) Sm = (Area)/(FM − F0) Yields or flux ratios φPo = TRo/ABS = [1 − F0/FM] φEo = ETo/ABS = [ − (FJ/FM)] φRo = REo/ABS = φPoψEoδRo = 1 − (FI/FM) ψo = ETo/TRo = (1 − VJ) δRo = REo/ETo = (1 − VI)/(1 − VJ) REo/TRo = ψoδRo
Approximated initial slope (in ms−1) of the fluorescence transient V = f(t) Normalized total complementary area above the O-J-I-P transient (reflecting multiple turnover QA reduction events)
Maximum quantum yield of primary photochemistry at t = 0 Quantum yield for electron transport at t = 0 Quantum yield for the reduction of end acceptors of PSI per photon absorbed Probability (at t = 0) that a trapped exciton moves an electron into the electron transport chain beyond Q− A Efficiency with which an electron can move from the reduced intersystem electron acceptors to the PSI end electron acceptors of PSI Efficiency with which a trapped exciton moves an electron into the electron transport chain from Q− A to the PSI end electron acceptors
Specific fluxes or activities per reaction center (RC) ABS/RC = Mo(1/VJ)(1/φPo) TRo/RC = Mo/VJ ETo/RC = Mo(1/VJ)ψo
Absorption per RC Trapped energy flux per RC (at t = 0) Electron transport flux per RC (at t = 0)
Phenomenological fluxes or activities per excited cross section (CS) ABS/CSo ≈ F0 TRo/CS = φPo(ABS/CSo) ETo/CS = φEo(ABS/CSo)
Absorption flux per CS, approximated by F0 Trapped energy flux per CS (at t = 0) Electron transport flux per CS (at t = 0)
De-excitation rate constants Kp Kn Sum K
Photochemical de-excitation rate constant Non-photochemical de-excitation rate constant The sum of photochemical and non-photochemical rate constants
Performance index PI = RC/(ABSRC) × φPo/(1 − φPo) × ψo/(1 − ψo)
Performance index on absorption basis
chloroplasts were incubated with slight agitation for 30 min at 4 °C with 0.8 M of Tris at pH 8.0 [17]. The O-J-I-P transient analysis was made according to the JIP test. The measured parameters were: F0, which is when the plastoquinone electron acceptor pool (QA) is fully oxidized; FM, when QA is transiently fully reduced and the area over the curve between F0 and FM, which is related to the pool size of PSII electron transport acceptors (Table 1). The other parameters were calculated employing the software Biolyzer HP3. The Chl a fluorescence transient was normalized according to the equation Vt = (Ft − F0)/(FM − F0) and the difference between the treated and control normalized transients were plotted as a function of time [11].
orbital shaker for 15 min. Then, the sodium hypochlorite solution was removed and the seeds were washed three times with distilled water. Seeds (100 units) were placed in 12 cm diameter pots containing a mixture of 50:25:25 w/w/w soil/peat-moss/agrolite. All pots were watered every third day, maintained in a greenhouse at 25–30 °C and under normal day/night illumination (12/12 h). Tomato and grass plants were selected by uniformity after 15 days of growth. Plants of similar size were separated in three groups: control, positive control (50 μM of DCMU) and experimental manually sprayed with antidesmone at 150 and 300 μM [18].
2.7. Plant material for in vivo assays A suspension of tomato (Physalis ixocarpa) and grass (Lolium perenne) seeds in a 10% sodium hypochlorite solution was kept in an
Chl a fluorescence was measured in leaves of the control plant and those sprayed with antidesmone at 150 and 300 μM. After 24, 48 and 72 h of treatment, the leaves adapted at the dark for 15 min were excited by light from an array of three light-emitting diodes delivering
Fig. 3. Effect of antidesmone concentration on photophosphorylation by freshly lysed spinach chloroplasts. Control value was 700 μM ATP/mg Chl × h.
Fig. 4. Effects of antidesmone on electron flow measured from water to MV in spinach chloroplasts in basal (■), phosphorylating (●) and uncoupled (▲) conditions. Control rate values for these conditions were 400, 480 and 1240 μequiv.e−.h−1.mg of Chl−1, respectively.
2.8. Determination of Chl a fluorescence in intact leaves in vivo
Please cite this article as: O.M. Sampaio, et al., Evaluation of antidesmone alkaloid as a photosynthesis inhibitor, Pesticide Biochemistry and Physiology (2016), http://dx.doi.org/10.1016/j.pestbp.2016.04.006
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O.M. Sampaio et al. / Pesticide Biochemistry and Physiology xxx (2016) xxx–xxx Table 2 Effect of antidesmone on uncoupled PSII electron transport from water to DCBQ. Concentration (μM)
0 25 50 75 100 200 300
PSII (H2O to DCBQ) a
b
700 ± 20.3 600 ± 17.1 500 ± 10.3 400 ± 5.8 200 ± 4.6 100 ± 3.4 0
100 85 71 57 28 14 0
a = values in μequiv. e−1. h−1. mg−1 Chl−1, b = values in percent
3000 μmol m−2 s−1 of red light (650 nm). The Chl a fluorescence induction curves were measured at room temperature with a portable Hansatech Fluorescence Handy PEA (plant efficiency analyzer) apparatus [5]. 2.9. Dry biomass determination P. ixocarpa and L. perenne plants were grown for 15 days with antidesmone (150 and 300 μM) treatment at ground level. The plants were dried in an oven at 65 °C to reach constant weight. Then, the dry biomass was measured using an analytical balance [18]. 2.10. Germination and roots and stems growth Dicotyledonous seeds of tomato (Physalis ixocarpa) and monocotyledonous seeds of grass (L. perenne) were purchased from Semillas Berentsen, S. A. de C. V. (Celaya, Guanajuato, Mexico). Germination tests were performed in triplicate with 40 seeds for each species with different concentrations of antidesmone for seven days (three days for germination and four days for growth of roots and stems). The seeds were set up in 9.0 cm Petri dishes containing 8.5 cm sheet of Whatman No. 1 filter paper and 10.0 mL of solution with antidesmone (50 μM) or DMSO (25 μL) as the control. Dishes wrapped with parafilm foil were incubated in the dark at 28 °C. The number of germinated seeds was determined according to the criterion of 1 mm extrusion of the radicle. Germination rates were counted at 72 and 120 h later for root and shoot growth measurements. The results were statistically analyzed using t-Student's test with significant p b 0.05 for two populations.
3. Results and discussion 3.1. ATP synthesis and chloroplast non-cyclic electron transport rate inhibition by antidesmone In order to investigate the potential of antidesmone as a candidate for a natural herbicide, its inhibitory effects on photosynthetic reactions were firstly evaluated through a general method employing chloroplasts isolated from spinach leaves to identify a Hill reaction inhibitor behavior [19– 21]. The light-dependent synthesis of ATP by illuminated thylakoids measured from water to MV in freshly lysed chloroplasts was inhibited by antidesmone in a concentration-dependent manner, with an IC50 39 μM (Fig. 3). This result demonstrates that antidesmone has a better ATP synthase inhibitory effect than the other natural products as the alkaloids evolitrine, kokusaginine, skimmianine and maculosidine (IC50 ˃ 50.5 μM) [22]. The light-dependent synthesis of ATP inhibition of thylakoid can occur in different ways: by blocking the electron transport, uncoupling ATP synthesis from the electron transport, and by blocking the phosphorylation reaction itself [13,17]. Thus, the effects of antidesmone on non-cyclic electron transport (in the basal, phosphorylating, and uncoupled conditions) was measured from water to MV on freshly lysed spinach chloroplasts in the absence or presence of ADP, Pi or NH4Cl, and using MV as an electron acceptor. Antidesmone inhibited electron flow in all conditions (Fig. 4). The calculated IC50 values for basal, phosphorylating and uncoupled electron flow were 45, 26 and 33 μM, respectively. Phosphorylation and uncoupled electron transport were fully inhibited at 75 μM and the basal stage at 200 μM. These results suggest that antidesmone behaves as a Hill reaction inhibitor by blocking the electron transport chain acceptor. 3.2. Localization of antidesmone interaction sites on PSI, PSII and partial reactions Artificial electron donors and acceptors were employed to localize the inhibition site of antidesmone on thylakoid electron transport chain, its effects on PSI and PSII, as well as the partial reactions were determined [23]. Antidesmone inhibited 100% of uncoupled PSII electron flow from water to DCBQ at 300 μM (Table 2). The electron transport from water to SiMo (partial reaction of donor side) was not affected by antidesmone. The polarographic measurement indicated that antidesmone inhibited the acceptor site of PSII at QB level. On the other hand, the alkaloid did not show any effect on the PSI electron transport chain (data not shown).
Fig. 5. A. Radar plot of antidesmone effects on Chl a fluorescence parameters calculated from OJIP curve of spinach thylakoids. B. Appearance of J-band about 2 ms.
Please cite this article as: O.M. Sampaio, et al., Evaluation of antidesmone alkaloid as a photosynthesis inhibitor, Pesticide Biochemistry and Physiology (2016), http://dx.doi.org/10.1016/j.pestbp.2016.04.006
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Fig. 6. A. Radar plot shows the effects on Chl a fluorescence parameters calculated from OJIP curves of antidesmone sprayed on P. ixocarpa plants at 48 h. B. Appearance of the K-band about 0.3 ms.
3.3. Chl a fluorescence measurement in spinach thylakoids To corroborate the polarographic results, Chl a fluorescence transient in the presence of antidesmone (150 and 300 μM) was measured on thylakoids obtained from freshly lysed spinach chloroplasts. The radar plot (Fig. 5A) shows antidesmone has a better activity at 300 μM than 150 μM, with the electron transport parameters: ψo, φEo, Sum K, Kp, ETo/RC and PIABS, decreased at 300 μM in 21, 24, 12, 16, 17 and 52%, respectively. The φEo parameter has a direct influence on electron transport and represents the quantum yield efficiency with which the trapped exciton can move an electron to the electron transport system. The decrease of φEo in the leaf discs of stressed plants of both species suggests that the probability for the electron transport beyond Q− A was decreased by antidesmone activity. The parameter PIABS is produced by three independent components, RC/ABS, φPo and ΨEo, and it is used to quantify the PSII behavior [11]. Any significant lowering of the PIABS values should be attributed to the change of antenna size, maximum quantum yield of primary photochemistry and the probability that an electron on reduced QA moves further into the electron transport chain [24]. The values for δRo and dV/dto parameters increased to a greater degree than others at 300 μM, 40% and 30% approximately. The flux ratios of PSI are related to δRo and φRo. The parameter δRo represents the efficiency with which an electron can move from the reduced intersystem electron acceptors to PSI end electron acceptors and φRo represents the
quantum yield of electron transport from Q− A to the PSI end electron acceptors. The increase of δRo indicates an increased capacity to reduce end acceptors beyond PSI, i.e. ferredoxine and NAP+ [25]. The Mo is the absorption of the water splitting enzyme function calculated as dV/dto, and the 30% increase in this indicates damage in the PSII. In agreement with the radar plot, the presence of a J-band between 2 and 4 ms (Fig. 5B) indicates an accumulation of Q− A , due to electron transport being blocked at the QB level, similarly to that achieved with DCMU [26]. 3.4. In vivo assays To assess the activity of antidesmone in vivo, it was sprayed at 150 and 300 μM on leaves of P. ixocarpa and L. perenne plants. After 24, 48 and 72 h of treatment, the Chl a fluorescence transients were measured and the JIP-parameters were calculated with Biolyser HP software. The results showed that the effects of antidesmone (150 and 300 μM) on P. ixocarpa plants at 24, 48 and 72 h were insignificant (data not shown). However, at 300 μM of antidesmone at 48 h, the parameter most affected was the photosynthetic index PIABS, which decreased by 30% (Fig. 6A) indicating a decrease of the photochemical efficiency of photosynthetic electron transport, and at this time a K-band appeared at the maximal concentration (Fig. 6B). The decrease of PIABS of 30% on P. ixocarpa plants represents a rapid lowering of efficiency of the redox reaction of the electron transport
Fig. 7. A. Radar plot of antidesmone effects on Chl a fluorescence parameters calculated from OJIP curve of sprayed Lolium perenne plants after 24 h. B. Appearance of K-band and I-band.
Please cite this article as: O.M. Sampaio, et al., Evaluation of antidesmone alkaloid as a photosynthesis inhibitor, Pesticide Biochemistry and Physiology (2016), http://dx.doi.org/10.1016/j.pestbp.2016.04.006
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Fig. 8. A. Radar plot of antidesmone effects on Chl a fluorescence parameters calculated from OJIP curve of sprayed Lolium perenne plants after 48 h. B. Appearance of K-band, I-band and a small G-band at 300 μM of antidesmone.
chain. A K-band (300 μs) is associated with uncoupling or inactivation of OEC. There were not significant variations in quantum yield parameters (φ). The phenomenological fluxes per CSo parameters showed pronounced variations, which caused significant effects on fluorescence transients. These results suggest that antidesmone requires time to get into P. ixocarpa plants and that these plants might have metabolized the alkaloid by 72 h after application. On L. perenne plants, antidesmone affected the parameters: φ(Ro), δRo and REo/TRo by approximately 10% (Figs. 7A, 8A and 9A), indicating a damage on PSI. The decrease of flux ratio parameters from PSI at 24 and 72 h (φ(Ro) and δRo) represents an effect on electron transport from Q− A to the PSI end electron acceptors and on the efficiency with which a move from reduced intersystem electron acceptors to the end acceptor beyond the PSI (ferredoxine and NAP+) [27]. Besides the inhibitory effects on PSI, the PIABS reduced approximately 20% over 24, 48 and 72 h, indicating effects on PSII efficiency. Furthermore, dV/dto increased by 20–30% in all experiments, demonstrating that antidesmone also acts at the enzymatic level of PSII. The increase of Sm and Kn at 150 μM and 300 μM of antidesmone, respectively, indicates that energy absorbed by the system is released as heat or transferred to other molecules, since the other parameters like quantum yield did not change (Fig. 6A). The parameters of specific fluxes or specific activities per RC, TRo/RC and ABS/RC describe the maximal rate by which an excitation is trapped by the reaction center (TRo/RC) and expresses the total number of photons absorbed by Chl molecules of all reaction centers divided by the
total number of active reaction center. These parameters showed a slight increase that identified damage on the acceptor side of PSII (Figs. 6A, 7A, 8A and 9A). These results corroborated the appearance of K and I-bands at 24, 48 and 72 h (Figs. 7B, 8B and 9B), and of the G-band solely in the 48 h experiment (Fig. 8B). The K-band (300 μs) is related to the reaction center in PSII being dissociated from the OEC, while the I-band (30 ms) is related to the reaction center of PSI being unable to provide electrons into the CO2-fixation process. On the other hand, the G-band is a small band in the I\\P region appearing near to 300 ms, which is related to plastoquinone (PQ) pool reduction, indicating that antidesmone deceases the electrons available to reduce QA, as the PQ pool forms as a consequence of non-active reaction centers [6]. Accordingly, these results indicate that antidesmone has two inhibition sites on L. perenne leaves: one at the donor site on PSII (K-band) and another one after the pool of quinones (I and G-band) [28]. 3.5. Dry-biomass Dry-biomass of P. ixocarpa and L. perenne plants were weighed after 15 days of treatment with antidesmone at 150 and 300 μM, in the absence of antidesmone (negative control) and with 10 μM of DCMU (positive control). The results showed that at low concentrations of antidesmone the growth of L. perenne plants were increased by 8% and the growth of P. ixocarpa plants decreased by 17%, a lower inhibition than DCMU. However, antidesmone at 300 μM affected the growth of
Fig. 9. A. Radar plot of antidesmone effects on Chl a fluorescence parameters calculated from OJIP curve of sprayed Lolium perenne plants after 72 h. B. Appearance of K-band and I-band.
Please cite this article as: O.M. Sampaio, et al., Evaluation of antidesmone alkaloid as a photosynthesis inhibitor, Pesticide Biochemistry and Physiology (2016), http://dx.doi.org/10.1016/j.pestbp.2016.04.006
O.M. Sampaio et al. / Pesticide Biochemistry and Physiology xxx (2016) xxx–xxx Table 3 Effect of antidesmone on dry biomass production, estimated by measuring the dry weight of P. ixocarpa and L. perenne plants. P. ixocarpa
L. perenne
Compound
Conc. (μM)
Mass (g)
%
Mass (g)
%
Control DCMU Antidesmone
0 10 150 300
0.524 ± 0.01 0.388 ± 0.04 0.433 ± 0.07 0.411 ± 0.04
100 74 83 78
0.570 ± 0.02 0.423 ± 0.04 0.613 ± 0.006 0.467 ± 0.01
100 74 108 82
7
herbicide probe. As the first natural product containing a 4(1H)pyridones scaffold with photosynthetic and plant growth activity, antidesmone has a promising potential to be explored as a tool in further development of a new class of herbicides. Acknowledgments The authors gratefully acknowledge financial support from Grants DGAPA-UNAM, IT102012 and FAPESP for the scholarship support.
Experiments were conducted with 3 replicates and data were expressed as means ± S.DE (standard errors), obtained with the program Origin 6.0.
References
P. ixocarpa and L. perenne plants by 22 and 18% respectively. DCMU inhibited growth of both plants by 18% (Table 3). These results indicated that antidesmone has a post-emergent herbicide behavior at high concentrations, which was confirmed by the fluorescence results in vivo. 3.6. Assay of germination and growth of root and stem The pre-emergent effect of antidesmone on P. ixocarpa and L. perenne seeds were evaluated through assays of germination and the growth of stems and roots in the presence of antidesmone. The results showed an increase for P. ixocarpa germination (15% at 50 μM). The effect on stem growth was insignificant at 50 μM. The major effect observed was on root growth, which was inhibited by 60% (p = 1.62 × 10−8) at 50 μM (Fig. 10). Antidesmone does not affect L. perenne germination nor the length of root and stem growth, indicating selective herbicide activity of antidesmone. 4. Conclusion This work shows for the first time that the secondary metabolite antidesmone, produced by W. brachypetala, plays a role in the inhibition of photosynthetic reactions and plant growth. The alkaloid behaves as a Hill reaction inhibitor and in vitro assays showed that it inhibits the acceptor side of PSII in the pool of QA. These data corroborated the Chl a fluorescence transient, indicating the inhibition of the PSII reactions. In vivo assays indicated a different mechanism, with antidesmone inhibiting the donor side of PSII and beyond of the PI. The effect of antidesmone on Chl a fluorescence transient of P. ixocarpa leaves was lower (effects are only present after 48 h) than for L. perenne (effects are present at 24, 48 and 72 h). However, the dry biomass decreased more in P. ixocarpa than in L. perenne, indicating that electron transport is not the only target for antidesmone in tomato plants. Although antidesmone inhibited the root growth of P. ixocarpa, it did not affect L. perenne, indicating that it is a good post-emergent
Fig. 10. Phytotoxic effect of antidesmone on P. ixocarpa germination rate, and root and stem growth. Values significant at p b 0.05 for t-Student for two populations. (G) Germination, (R) Root, (S) Stem.
[1] S.O. Duke, F.E. Dayan, J.G. Romagni, A.M. Rimando, Natural products as sources of herbicides: current status and future trends, Weed Res. 40 (1) (2000) 99–111. [2] J.R. Vyvyan, Allelochemicals as leads for new herbicides and agrochemicals, Tetrahedron 58 (9) (2002) 1631–1646. [3] F.E. Dayan, C.L. Cantrell, S.O. Duke, Natural products in crop protection, Bioorg. Med. Chem. 17 (12) (2009) 4022–4034. [4] D. Torres-Romero, B. King-Díaz, R.J. Strasser, I.A. Jiménez, B. Lotina-Hennsen, I.L. Bazzocchi, Friedelane triterpenes from Celastrus vulcanicola as photosynthetic inhibitors, J. Agric. Food Chem. 58 (20) (2010) 10847–10854. [5] T.A.M. Veiga, B. King-Díaz, A.S.F. Marques, O.M. Sampaio, P.C. Vieira, M.F.G.F. Silva, B. Lotina-Hennsen, Furoquinoline alkaloids isolated from Balfourodendron riedelianum as photosynthetic inhibitors in spinach chloroplasts, J. Photochem. Photobiol. B 120 (2013) 36–43. [6] B. King-Diaz, M.S. Soares, M.F.G.F. Silva, B. Lotina-Hennsen, T.A.M. Veiga, Triterpenes from Cabralea canjerana as in vitro inhibitors to light reactions of photosynthesis, Am. J. Plant Sci. 5 (16) (2014) 2528–2540. [7] M.M. Lima, J.A. López, J.M. David, E.P. Silva, A.M. Giulietti, L.P. De Queiroz, J.P. David, Acetylcholinesterase activity of alkaloids from the leaves of Waltheria brachypetala, Planta Med. 75 (4) (2009) 335–337. [8] K. Kubitzki, C. Bayer, Flowering plants – dicotyledons: Malvaceae, in: K. Kubitzki (Ed.), The Families and Genera of Vascular Plants, Springer-Verlag, Berlin Heidelberg, New York, NY, USA 2003, pp. 225–311. [9] G. Bringmann, J. Schlauer, H. Rischer, M. Wohlfarth, J. Muèhlbacher, A. Buske, A. Porzel, J. Schmidtb, G. Adamb, Revised structure of antidesmone, an unusual alkaloid from tropical Antidesma plants (Euphorbiaceae), Tetrahedron 56 (23) (2000) 3691–3695. [10] J.O.S. Varejão, L.C.A. Barbosa, E.V.V. Varejão, C.R.A. Maltha, B. King-Díaz, B. LotinaHennsen, Cyclopent-4-ene-1,3-diones: a new class of herbicides acting as potent photosynthesis inhibitors, J. Agric. Food Chem. 62 (25) (2014) 5772–5780. [11] Analysis of the chlorophyll a fluorescence transient, in: R.J. Strasser, A. Srivastava, M. Tsimilli-Michael, Govindjee, G.C. Papageorgiou (Eds.), Advances in Photosynthesis and Respiration, Springer, Dordrecht, The Netherlands 2004, pp. 321–362. [12] R.A. Dilley, Ion transport (H+, K+ Mg2+ exchange phenomena), Methods Enzymol. 24 (1972) 68–74. [13] M.I. Aguilar, M.G. Romero, M.I. Chávez, B. King-Dìaz, B. Lotina-Hennsen, Biflavonoids isolated from Selaginella lepidophylla inhibit photosynthesis in spinach chloroplasts, J. Agric. Food Chem. 56 (16) (2008) 6994–7000. [14] S. Izawa, G. Hind, The kinetics of the pH rise in illuminated chloroplast suspensions, Biochim. Biophys. Acta 143 (1967) 377–390. [15] I. Yruela, G. Montoya, P.J. Alonso, R. Picorel, Identification of the pheophytin-QA-Fe domain of the reducing side of the photosystem II as the Cu(II)-inhibitory binding site, J. Biol. Chem. 266 (34) (1991) 22847–22850. [16] R.T. Guiaquinta, R.A. Dilley, A partial reaction in photosystem II: reduction of silicomolybdate prior to the site of dichlorophenyldimethylurea inhibition, Biochim. Biophys. Acta 387 (2) (1975) 288–305. [17] T. Yamashida, T. Horio, Non-cyclic photophosphorylation by spinach grana treated with 0.8 M Tris buffer, Plant Cell Physiol. 9 (1968) 267–284. [18] M. Gonzalez-Ibarra, N. Farfán, C. Trejo, S. Uribe, B. Lotina-Hennsen, Selective herbicide activity of 2,5-di(benzylamine)-p-benzoquinone against the monocot weed Echinochloa crusgalli. An in vivo analysis of photosynthesis and growth, J. Agric. Food Chem. 53 (9) (2005) 3415–3420. [19] D. Torres-Romero, B. King-Díaz, I.A. Jiménez, B. Lotina-Hennsen, I.L. Bazzocchi, Sesquiterpenes from Celastrus vulcanicola as photosynthetic inhibitors, J. Nat. Prod. 71 (8) (2008) 1331–1335. [20] A.J. Demuner, L.C.A. Barbosa, T.A.M. Veiga, R.W. Barreto, B. King-Diaz, B. LotinaHennsen, Phytotoxic constituents from Nimbya alternantherae, Biochem. Syst. Ecol. 34 (11) (2006) 790–795. [21] M.L. Macías-Rubalcava, M.E.R. Sobrino, C. Meléndez-González, B. King-Díaz, B. Lotina-Hennsen, Selected phytotoxins and organic extracts from endophytic fungus Edenia gomezpompae as light reaction of photosynthesis inhibitors, J. Photochem. Photobiol. B 138 (2014) 17–26. [22] L.S. Medeiros, O.M. Sampaio, M.F.G.F. da Silva, E.R. Filho, T.A.M. Veiga, Evaluation of herbicidal potential of depsides from Cladosporium uredinicola, an endophytic fungus found in guava fruit, J. Braz. Chem. Soc. 23 (8) (2012) 1551–1557. [23] J.F. Allen, N.G. Holmes, in: M.F. Hipkins, N.R. Baker (Eds.), Photosynthesis: Energy Transduction: A Practical Approach, IRL Press, Oxford, UK 1986, pp. 103–141. [24] S. Chen, R.J. Strasser, S. Qiang, In vivo assessment of effect of phytotoxin tenuazonic acid on PSII reaction centers, Plant Physiol. Biochem. 84 (2014) 10–21. [25] E. Salvatori, L. Fusaro, S. Mereu, A. Bernardini, G. Puppi, F. Manes, Different O3 response of sensitive and resistant snap bean genotypes (Phaseolus vulgaris L.): the
Please cite this article as: O.M. Sampaio, et al., Evaluation of antidesmone alkaloid as a photosynthesis inhibitor, Pesticide Biochemistry and Physiology (2016), http://dx.doi.org/10.1016/j.pestbp.2016.04.006
8
O.M. Sampaio et al. / Pesticide Biochemistry and Physiology xxx (2016) xxx–xxx
key role of growth stage, stomatal conductance, and PSI activity, Environ. Exp. Bot. 87 (2013) 79–91. [26] S.Z. Tóth, G. Schansker, R.J. Strasser, In intact leaves, the maximum fluorescence level (FM) is independent of the redox state of the plastoquinone pool: a DCMUinhibition study, Biochim. Biophys. Acta 1708 (2) (2005) 275–282. [27] R.J. Strasser, M. Tsimilli-Michael, S. Qiang, V. Goltsev, Simultaneous in vivo recording of prompt and delayed fluorescence and 820-nm reflection changes during drying
and after rehydration of the resurrection plant Haberlea rhodopensis, Biochim. Biophys. Acta 1797 (6–7) (2010) 1313–1326. [28] M.G. Ceppi, A. Oukarroum, N. Çiçek, R.J. Strasser, G. Schansker, The IP amplitude of the fluorescence rise OJIP is sensitive to changes in the photosystem I content of leaves: a study on plants exposed to magnesium and sulfate deficiencies, drought stress and salt stress, Physiol. Plant. 144 (3) (2012) 277–288.
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Contents lists available at ScienceDirect
Pesticide Biochemistry and Physiology journal homepage: www.elsevier.com/locate/pest
Evaluation of antidesmone alkaloid as a photosynthesis inhibitor Olívia Moreira Sampaio a,⁎, Murilo Marinho de Castro Lima b, Thiago André Moura Veiga c, Beatriz King-Díaz d, Maria Fátima das Graças Fernandes da Silva b, Blas Lotina-Hennsen d a
Chemistry Department, Federal University of Mato Grosso (UFMT), Cuiabá, MT, Brazil Chemistry Department, Federal University of São Carlos (UFSCar), São Carlos, SP, Brazil Institute of Environmental, Chemistry and Pharmaceutical Sciences, Federal University of São Paulo (UNIFESP), São Paulo, SP, Brazil d Biochemistry Department, Facultad de Química, Universidad Nacional Autónoma de México, México D. F. 04510, Mexico b c
a r t i c l e
i n f o
Article history: Received 25 September 2015 Received in revised form 15 April 2016 Accepted 18 April 2016 Available online xxxx Keywords: Antidesmone Chlorophyll a Photosystem II ATP synthesis Herbicide Photosynthetic inhibitor
a b s t r a c t Antidesmone, isolated from Waltheria brachypetala Turcz., owns special structural features as two α,β-unsaturated carbonyl groups and a side alkyl chain that can compete with the quinones involved in the pool of plastoquinones at photosystem II (PSII). In this work, we showed that the alkaloid is an inhibitor of Hill reaction and its target was located at the acceptor side of PSII. Studies of chlorophyll (Chl) a fluorescence showed a J-band that indicates direct action of antidesmone in accumulation of Q− A (reduced plastoquinone A) due to the electron transport blocked at the QB (plastoquinone B) level similar to DCMU. In vivo assays indicated that antidesmone is a selective post-emergent herbicide probe at 300 μM by reducing the biomass production of Physalis ixacarpa plants. Furthermore, antidesmone also behaves as pre-emergent herbicide due to inhibit Physalis ixacarpa plant growth about 60%. Antidesmone, a natural product containing a 4(1H)-pyridones scaffold, will serve as a valuable tool in further development of a new class of herbicides. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Increasing agricultural productivity without expansion of land for food production is a necessity for a growing world population. In this sense, avoid agricultural pests is a great concern. Since weeds are one of the major pests causing a continual reduction in the quantity and quality of crops worldwide, increasing agricultural yield is highly dependent on the use of synthetic herbicides to control weeds. The continuous use of herbicides has resulted in selective pressures, leading to the replacement of sensitive weeds by herbicide-resistant biotypes [1–3]. The major goal of modern weed management research is to discover new herbicides that control the widest possible range of weed species (i.e. can be sprayed at pre- and post-emergence), and at low application rates. Furthermore, selective herbicides need to be safe to the target crop, environment and humans. In this context, natural products fit well against these objectives, and become interesting alternatives for herbicides because they are non-toxic and have demonstrated good
activity in weed control. To satisfy this demand, research in academia and industry has focused on natural products as a promising strategy for developing new herbicides. In the search of potential herbicides, several natural products, such as epifriedelinol [4], evolitrine [5] and ocotilone [6] (Fig. 1), have been studied as inhibitors of PSII with good prospects as natural inhibitors of plant growth. Waltheria brachypetala Turcz (Malvaceae) is a plant with an endemic occurrence in semi-arid areas in Brazil [7]. Previous phytochemical studies showed that W. brachypetala leaves and stems contain cyclic peptide and quinolone alkaloids, such as antidesmone [8]. Looking at the chemical structure of antidesmone, this compound has a system of two α,β-unsaturated carbonyl groups and a side alkyl chain, similarly to the quinones involved in the pool of plastoquinones at PSII (Fig. 2). Our aims in this work were to evaluate and determine the effects of antidesmone on photosynthetic apparatus, germination, roots and stems growth and dry biomass experiments. 2. Material and methods
Abbreviations: ADP, adenosine diphosphate; ATP, adenosine triphosphate; Chl, chlorophyll; DBMIB, 2,5-dibromo-6-isopropyl-3-methyl-1,4-benzoquinone; DCBQ, 2,5dichloro-1,4-benzoquinone; F0, initial fluorescence; FM, maximum fluorescence level; DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethylurea; IC50, (the half maximal inhibitory concentration); MV, methylviologen; OEC, oxygen evolving complex; PSI, photosystem I; PSII, photosystem II; SiMo, sodium silicomolybdate; Tris, (hydroxymethyl)aminoethane. ⁎ Corresponding author. E-mail address: [email protected] (O.M. Sampaio).
2.1. Antidesmone isolation The ethanol extract of W. brachypetala stems and leaves (180 g) was subjected to liquid-liquid extraction with methanol:water (1:3) and hexane to provide the hexane fraction (12.0 g). Six grams from hexane fraction was subjected to chromatographic purification using silica gel (70–230 mesh) as the stationary phase. The mobile phase was
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Please cite this article as: O.M. Sampaio, et al., Evaluation of antidesmone alkaloid as a photosynthesis inhibitor, Pesticide Biochemistry and Physiology (2016), http://dx.doi.org/10.1016/j.pestbp.2016.04.006
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2.3. Measurement of ATP synthesis ATP synthesis was determined by titration using a microelectrode (Orion model 8103; Ross, Beverly, MA, USA) connected to a Corning potentiometer model 12 (Corning Medical, Acton, MA, USA) with an expanded scale and a Gilson recorder (Kipp & Zonen, Bohemia, NY, USA) [12,13]. A non-buffered solution was employed to break the intact chloroplasts by osmotic rupture, using 100 mM sorbitol, 10 mM KCl, 5 mM MgCl2, 0.5 mM KCN, 50 μM MV, 1 mM Na+-tricine, 1 mM of ADP, with the pH adjusted to 8.0 applying 50 mM of KOH. The mixture was illuminated for 1 min and the ATP synthesis was measured as micromoles of ATP per milligram of Chl per hour. 2.4. Measurement of non-cyclic electron transport rate
Fig. 1. Natural products that are ATP synthesis inhibitors of PSII.
composed of hexane/ethyl acetate in ascending order of polarity (9:1, 8:2, 7:3, 6:4, 1:1), followed by dichloromethane:methanol (1:0, 95:5, 9:1, 8:2, 7:3, 6:4, 0:1) to obtain 49 fractions. Fraction 42 was again subjected to new chromatographic purification using silica (70–230 mesh), eluting with solvents isocratic elution (dichloromethane: methanol (95:5)) to obtain 29 new fractions. Fractions 16–18 were combined and subjected to semipreparative HPLC (Shimadzu, SCL-10 A VP) using a Phenomenex propyl-ether-diol normal bond as stationary phase (300 × 8 mm, 10 μm) with hexane:ethanol (95:5) as eluting system at 1.4 mL/min flow rate, to afford the pure alkaloid antidesmone (0.03 g). The compound was identified by spectroscopic analysis and confirmed according to thorough comparisons with the found literature data for the isolated alkaloid [9]. The obtained signal assignments are described as it follows: 1 H NMR (400 MHz, CDCl3) δ: 0.87 (t, J = 7.0 Hz, 2H, H-17), 1.25 (2H, H-17, overlapping), 1.25–1.80 (10H, H-12-16, overlapping) 1.45 (1H, H11b, overlapping), 1.75 (1H, H-11a, overlapping), 2.08 (dddd, J = 14.7, 14.0, 4.5, 4.3 Hz, 1H, H-6ax), 2.20 (dddd, J = 14.0, 5.3, 4.3, 2.4 Hz, 1H, H6eq), 2.36 (s, 3H, 2-CH3), 2.58 (ddd, J = 18.1, 4.4, 2.4 Hz, 1H, H-7eq), 2.75 (ddd, J = 18.1, 14.7, 5.3 Hz, 1H, H-7ax), 3.26 (m, 1H, H-5), 3.93 (s, 3H, OCH3), 8.73 (sl, 1H, N\\H). 13C NMR (100 MHz, CDCl3) δ: 14.0 (C-18), 14.6 (C2\\CH3), 22.6 (C-17), 24.4 (C-6), 29.2–29.6 (C-12-16), 30.3 (C-5), 30.5 (C-11), 32.2 (C-7), 59.5 (OCH3), 131.9 (C-9), 139.0 (C2), 139.1 (C-10), 147.6 (C-3), 173.3 (C-4), 194.8 (C-8). MS (ESI+) m/z 320 [M + H]. [α]25 D : +26 (c 1.50, CHCl3).
2.2. Chloroplast isolation and Chl determination Intact chloroplasts were isolated from spinach leaves (Spinacea oleracea L.) as previously described [10]. The Chl concentration was measured spectrophotometrically through a chloroplast suspension in a solution of 400 mM sucrose, 5 mM MgCl2, 10 mM KCl, 30 mM tricine-KOH and pH 8.0 [11].
Fig. 2. Structure of plastoquinona and antidesmone.
Light-induced non-cyclic electron transport activity from water to MV was determined polarographically using a Clark type electrode in the presence of 50 μM of MV [14]. Basal electron transport was measured by illuminating a solution of chloroplasts (20 μg Chl/mL) in 3 mL of 100 mM sorbitol, 10 mM KCl, 5 mM MgCl2, 0.5 mM KCN, 15 mM tricine-KOH and 50 μM MV at pH 8.0 for 1 min. The phosphorylating non-cyclic electron transport rate was measured as for the basal electron transport from water to MV, adding 1 mM of ADP and 3 mM KH2PO4. Uncoupled electron transport was evaluated in the same solution used for basal electron transport with 6 mM NH4Cl added as an uncoupler [10]. 2.5. Uncoupled PSII electron flow determination The electron flow activities were monitored with an oxygen monitor yellow spring instrument model 5300 A using a Clark type electrode. All reaction mixtures were illuminated with filtered light (filter of 5 cm of 1% CuSO4 solution) from a projector lamp (GAF 2660) at room temperature. For each reaction, a blank experiment was performed with chloroplasts in the reaction medium. The IC50 value for each activity was determined from plots of the activity versus different concentrations of antidesmone. The solution used for PSII reactions was the same medium used for basal electron transport measurements except MV. The uncoupled electron transport rate from water to DCBQ was measured by illuminating the chloroplasts for 1 min (20 μg Chl/mL) in a solution for PSII reactions, 100 μM of DCBQ, 1 μM of DBMIB and 6 mM NH4Cl [15]. To determine the uncoupled partial reaction of PSII measured from water to SiMo, a solution of 200 μM of SiMo and 10 μM of DCMU were added to the solution used for the PSII reactions (3 mL), then chloroplasts (20 μg Chl/mL) were added and illuminated for 1 min [16]. 2.6. Chl a fluorescence measurements on thylakoids Chl a fluorescence transients were measured with a Handy-PEA (Plant Efficient Analyzer, from Hansatech, King's Lynn, Norfolk, UK), as previously described [5]. The maximum fluorescence yield from the sample was generated by illumination for two seconds with continuous light (650 nm wavelength, intensity equivalent of 2830 μmol photons m−2 s−1 1 and gain of 0.7) from an array of three light-emitting diodes. The reaction medium employed was the solution used in PSII reactions. To monitor Chl a fluorescence transient, aliquots of dark-adapted thylakoids for 5 min containing 60 μg of Chl were transferred to filter paper by gravity with a dot-blot apparatus (Bio-Rad, United States) in order to ensure a homogeneous and reproducible distribution of thylakoids. The filter paper was dipped immediately in 3 mL of the medium with different concentrations of antidesmone in DMSO (150 and 300 μM) and the control was made with the medium plus DMSO. Infiltrated chloroplasts with 10 μM of DCMU and chloroplasts previously treated with 0.8 M of Tris were used as a positive control. The
Please cite this article as: O.M. Sampaio, et al., Evaluation of antidesmone alkaloid as a photosynthesis inhibitor, Pesticide Biochemistry and Physiology (2016), http://dx.doi.org/10.1016/j.pestbp.2016.04.006
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Table 1 Derived parameters, their description and formulae using data extracted from the Chl a fluorescence (OJIP) transients. Fluorescence parameters derived from the extracted data Mo = dV/dto = 4(F300 μs − F0)/(FM − F0) Sm = (Area)/(FM − F0) Yields or flux ratios φPo = TRo/ABS = [1 − F0/FM] φEo = ETo/ABS = [ − (FJ/FM)] φRo = REo/ABS = φPoψEoδRo = 1 − (FI/FM) ψo = ETo/TRo = (1 − VJ) δRo = REo/ETo = (1 − VI)/(1 − VJ) REo/TRo = ψoδRo
Approximated initial slope (in ms−1) of the fluorescence transient V = f(t) Normalized total complementary area above the O-J-I-P transient (reflecting multiple turnover QA reduction events)
Maximum quantum yield of primary photochemistry at t = 0 Quantum yield for electron transport at t = 0 Quantum yield for the reduction of end acceptors of PSI per photon absorbed Probability (at t = 0) that a trapped exciton moves an electron into the electron transport chain beyond Q− A Efficiency with which an electron can move from the reduced intersystem electron acceptors to the PSI end electron acceptors of PSI Efficiency with which a trapped exciton moves an electron into the electron transport chain from Q− A to the PSI end electron acceptors
Specific fluxes or activities per reaction center (RC) ABS/RC = Mo(1/VJ)(1/φPo) TRo/RC = Mo/VJ ETo/RC = Mo(1/VJ)ψo
Absorption per RC Trapped energy flux per RC (at t = 0) Electron transport flux per RC (at t = 0)
Phenomenological fluxes or activities per excited cross section (CS) ABS/CSo ≈ F0 TRo/CS = φPo(ABS/CSo) ETo/CS = φEo(ABS/CSo)
Absorption flux per CS, approximated by F0 Trapped energy flux per CS (at t = 0) Electron transport flux per CS (at t = 0)
De-excitation rate constants Kp Kn Sum K
Photochemical de-excitation rate constant Non-photochemical de-excitation rate constant The sum of photochemical and non-photochemical rate constants
Performance index PI = RC/(ABSRC) × φPo/(1 − φPo) × ψo/(1 − ψo)
Performance index on absorption basis
chloroplasts were incubated with slight agitation for 30 min at 4 °C with 0.8 M of Tris at pH 8.0 [17]. The O-J-I-P transient analysis was made according to the JIP test. The measured parameters were: F0, which is when the plastoquinone electron acceptor pool (QA) is fully oxidized; FM, when QA is transiently fully reduced and the area over the curve between F0 and FM, which is related to the pool size of PSII electron transport acceptors (Table 1). The other parameters were calculated employing the software Biolyzer HP3. The Chl a fluorescence transient was normalized according to the equation Vt = (Ft − F0)/(FM − F0) and the difference between the treated and control normalized transients were plotted as a function of time [11].
orbital shaker for 15 min. Then, the sodium hypochlorite solution was removed and the seeds were washed three times with distilled water. Seeds (100 units) were placed in 12 cm diameter pots containing a mixture of 50:25:25 w/w/w soil/peat-moss/agrolite. All pots were watered every third day, maintained in a greenhouse at 25–30 °C and under normal day/night illumination (12/12 h). Tomato and grass plants were selected by uniformity after 15 days of growth. Plants of similar size were separated in three groups: control, positive control (50 μM of DCMU) and experimental manually sprayed with antidesmone at 150 and 300 μM [18].
2.7. Plant material for in vivo assays A suspension of tomato (Physalis ixocarpa) and grass (Lolium perenne) seeds in a 10% sodium hypochlorite solution was kept in an
Chl a fluorescence was measured in leaves of the control plant and those sprayed with antidesmone at 150 and 300 μM. After 24, 48 and 72 h of treatment, the leaves adapted at the dark for 15 min were excited by light from an array of three light-emitting diodes delivering
Fig. 3. Effect of antidesmone concentration on photophosphorylation by freshly lysed spinach chloroplasts. Control value was 700 μM ATP/mg Chl × h.
Fig. 4. Effects of antidesmone on electron flow measured from water to MV in spinach chloroplasts in basal (■), phosphorylating (●) and uncoupled (▲) conditions. Control rate values for these conditions were 400, 480 and 1240 μequiv.e−.h−1.mg of Chl−1, respectively.
2.8. Determination of Chl a fluorescence in intact leaves in vivo
Please cite this article as: O.M. Sampaio, et al., Evaluation of antidesmone alkaloid as a photosynthesis inhibitor, Pesticide Biochemistry and Physiology (2016), http://dx.doi.org/10.1016/j.pestbp.2016.04.006
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O.M. Sampaio et al. / Pesticide Biochemistry and Physiology xxx (2016) xxx–xxx Table 2 Effect of antidesmone on uncoupled PSII electron transport from water to DCBQ. Concentration (μM)
0 25 50 75 100 200 300
PSII (H2O to DCBQ) a
b
700 ± 20.3 600 ± 17.1 500 ± 10.3 400 ± 5.8 200 ± 4.6 100 ± 3.4 0
100 85 71 57 28 14 0
a = values in μequiv. e−1. h−1. mg−1 Chl−1, b = values in percent
3000 μmol m−2 s−1 of red light (650 nm). The Chl a fluorescence induction curves were measured at room temperature with a portable Hansatech Fluorescence Handy PEA (plant efficiency analyzer) apparatus [5]. 2.9. Dry biomass determination P. ixocarpa and L. perenne plants were grown for 15 days with antidesmone (150 and 300 μM) treatment at ground level. The plants were dried in an oven at 65 °C to reach constant weight. Then, the dry biomass was measured using an analytical balance [18]. 2.10. Germination and roots and stems growth Dicotyledonous seeds of tomato (Physalis ixocarpa) and monocotyledonous seeds of grass (L. perenne) were purchased from Semillas Berentsen, S. A. de C. V. (Celaya, Guanajuato, Mexico). Germination tests were performed in triplicate with 40 seeds for each species with different concentrations of antidesmone for seven days (three days for germination and four days for growth of roots and stems). The seeds were set up in 9.0 cm Petri dishes containing 8.5 cm sheet of Whatman No. 1 filter paper and 10.0 mL of solution with antidesmone (50 μM) or DMSO (25 μL) as the control. Dishes wrapped with parafilm foil were incubated in the dark at 28 °C. The number of germinated seeds was determined according to the criterion of 1 mm extrusion of the radicle. Germination rates were counted at 72 and 120 h later for root and shoot growth measurements. The results were statistically analyzed using t-Student's test with significant p b 0.05 for two populations.
3. Results and discussion 3.1. ATP synthesis and chloroplast non-cyclic electron transport rate inhibition by antidesmone In order to investigate the potential of antidesmone as a candidate for a natural herbicide, its inhibitory effects on photosynthetic reactions were firstly evaluated through a general method employing chloroplasts isolated from spinach leaves to identify a Hill reaction inhibitor behavior [19– 21]. The light-dependent synthesis of ATP by illuminated thylakoids measured from water to MV in freshly lysed chloroplasts was inhibited by antidesmone in a concentration-dependent manner, with an IC50 39 μM (Fig. 3). This result demonstrates that antidesmone has a better ATP synthase inhibitory effect than the other natural products as the alkaloids evolitrine, kokusaginine, skimmianine and maculosidine (IC50 ˃ 50.5 μM) [22]. The light-dependent synthesis of ATP inhibition of thylakoid can occur in different ways: by blocking the electron transport, uncoupling ATP synthesis from the electron transport, and by blocking the phosphorylation reaction itself [13,17]. Thus, the effects of antidesmone on non-cyclic electron transport (in the basal, phosphorylating, and uncoupled conditions) was measured from water to MV on freshly lysed spinach chloroplasts in the absence or presence of ADP, Pi or NH4Cl, and using MV as an electron acceptor. Antidesmone inhibited electron flow in all conditions (Fig. 4). The calculated IC50 values for basal, phosphorylating and uncoupled electron flow were 45, 26 and 33 μM, respectively. Phosphorylation and uncoupled electron transport were fully inhibited at 75 μM and the basal stage at 200 μM. These results suggest that antidesmone behaves as a Hill reaction inhibitor by blocking the electron transport chain acceptor. 3.2. Localization of antidesmone interaction sites on PSI, PSII and partial reactions Artificial electron donors and acceptors were employed to localize the inhibition site of antidesmone on thylakoid electron transport chain, its effects on PSI and PSII, as well as the partial reactions were determined [23]. Antidesmone inhibited 100% of uncoupled PSII electron flow from water to DCBQ at 300 μM (Table 2). The electron transport from water to SiMo (partial reaction of donor side) was not affected by antidesmone. The polarographic measurement indicated that antidesmone inhibited the acceptor site of PSII at QB level. On the other hand, the alkaloid did not show any effect on the PSI electron transport chain (data not shown).
Fig. 5. A. Radar plot of antidesmone effects on Chl a fluorescence parameters calculated from OJIP curve of spinach thylakoids. B. Appearance of J-band about 2 ms.
Please cite this article as: O.M. Sampaio, et al., Evaluation of antidesmone alkaloid as a photosynthesis inhibitor, Pesticide Biochemistry and Physiology (2016), http://dx.doi.org/10.1016/j.pestbp.2016.04.006
O.M. Sampaio et al. / Pesticide Biochemistry and Physiology xxx (2016) xxx–xxx
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Fig. 6. A. Radar plot shows the effects on Chl a fluorescence parameters calculated from OJIP curves of antidesmone sprayed on P. ixocarpa plants at 48 h. B. Appearance of the K-band about 0.3 ms.
3.3. Chl a fluorescence measurement in spinach thylakoids To corroborate the polarographic results, Chl a fluorescence transient in the presence of antidesmone (150 and 300 μM) was measured on thylakoids obtained from freshly lysed spinach chloroplasts. The radar plot (Fig. 5A) shows antidesmone has a better activity at 300 μM than 150 μM, with the electron transport parameters: ψo, φEo, Sum K, Kp, ETo/RC and PIABS, decreased at 300 μM in 21, 24, 12, 16, 17 and 52%, respectively. The φEo parameter has a direct influence on electron transport and represents the quantum yield efficiency with which the trapped exciton can move an electron to the electron transport system. The decrease of φEo in the leaf discs of stressed plants of both species suggests that the probability for the electron transport beyond Q− A was decreased by antidesmone activity. The parameter PIABS is produced by three independent components, RC/ABS, φPo and ΨEo, and it is used to quantify the PSII behavior [11]. Any significant lowering of the PIABS values should be attributed to the change of antenna size, maximum quantum yield of primary photochemistry and the probability that an electron on reduced QA moves further into the electron transport chain [24]. The values for δRo and dV/dto parameters increased to a greater degree than others at 300 μM, 40% and 30% approximately. The flux ratios of PSI are related to δRo and φRo. The parameter δRo represents the efficiency with which an electron can move from the reduced intersystem electron acceptors to PSI end electron acceptors and φRo represents the
quantum yield of electron transport from Q− A to the PSI end electron acceptors. The increase of δRo indicates an increased capacity to reduce end acceptors beyond PSI, i.e. ferredoxine and NAP+ [25]. The Mo is the absorption of the water splitting enzyme function calculated as dV/dto, and the 30% increase in this indicates damage in the PSII. In agreement with the radar plot, the presence of a J-band between 2 and 4 ms (Fig. 5B) indicates an accumulation of Q− A , due to electron transport being blocked at the QB level, similarly to that achieved with DCMU [26]. 3.4. In vivo assays To assess the activity of antidesmone in vivo, it was sprayed at 150 and 300 μM on leaves of P. ixocarpa and L. perenne plants. After 24, 48 and 72 h of treatment, the Chl a fluorescence transients were measured and the JIP-parameters were calculated with Biolyser HP software. The results showed that the effects of antidesmone (150 and 300 μM) on P. ixocarpa plants at 24, 48 and 72 h were insignificant (data not shown). However, at 300 μM of antidesmone at 48 h, the parameter most affected was the photosynthetic index PIABS, which decreased by 30% (Fig. 6A) indicating a decrease of the photochemical efficiency of photosynthetic electron transport, and at this time a K-band appeared at the maximal concentration (Fig. 6B). The decrease of PIABS of 30% on P. ixocarpa plants represents a rapid lowering of efficiency of the redox reaction of the electron transport
Fig. 7. A. Radar plot of antidesmone effects on Chl a fluorescence parameters calculated from OJIP curve of sprayed Lolium perenne plants after 24 h. B. Appearance of K-band and I-band.
Please cite this article as: O.M. Sampaio, et al., Evaluation of antidesmone alkaloid as a photosynthesis inhibitor, Pesticide Biochemistry and Physiology (2016), http://dx.doi.org/10.1016/j.pestbp.2016.04.006
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Fig. 8. A. Radar plot of antidesmone effects on Chl a fluorescence parameters calculated from OJIP curve of sprayed Lolium perenne plants after 48 h. B. Appearance of K-band, I-band and a small G-band at 300 μM of antidesmone.
chain. A K-band (300 μs) is associated with uncoupling or inactivation of OEC. There were not significant variations in quantum yield parameters (φ). The phenomenological fluxes per CSo parameters showed pronounced variations, which caused significant effects on fluorescence transients. These results suggest that antidesmone requires time to get into P. ixocarpa plants and that these plants might have metabolized the alkaloid by 72 h after application. On L. perenne plants, antidesmone affected the parameters: φ(Ro), δRo and REo/TRo by approximately 10% (Figs. 7A, 8A and 9A), indicating a damage on PSI. The decrease of flux ratio parameters from PSI at 24 and 72 h (φ(Ro) and δRo) represents an effect on electron transport from Q− A to the PSI end electron acceptors and on the efficiency with which a move from reduced intersystem electron acceptors to the end acceptor beyond the PSI (ferredoxine and NAP+) [27]. Besides the inhibitory effects on PSI, the PIABS reduced approximately 20% over 24, 48 and 72 h, indicating effects on PSII efficiency. Furthermore, dV/dto increased by 20–30% in all experiments, demonstrating that antidesmone also acts at the enzymatic level of PSII. The increase of Sm and Kn at 150 μM and 300 μM of antidesmone, respectively, indicates that energy absorbed by the system is released as heat or transferred to other molecules, since the other parameters like quantum yield did not change (Fig. 6A). The parameters of specific fluxes or specific activities per RC, TRo/RC and ABS/RC describe the maximal rate by which an excitation is trapped by the reaction center (TRo/RC) and expresses the total number of photons absorbed by Chl molecules of all reaction centers divided by the
total number of active reaction center. These parameters showed a slight increase that identified damage on the acceptor side of PSII (Figs. 6A, 7A, 8A and 9A). These results corroborated the appearance of K and I-bands at 24, 48 and 72 h (Figs. 7B, 8B and 9B), and of the G-band solely in the 48 h experiment (Fig. 8B). The K-band (300 μs) is related to the reaction center in PSII being dissociated from the OEC, while the I-band (30 ms) is related to the reaction center of PSI being unable to provide electrons into the CO2-fixation process. On the other hand, the G-band is a small band in the I\\P region appearing near to 300 ms, which is related to plastoquinone (PQ) pool reduction, indicating that antidesmone deceases the electrons available to reduce QA, as the PQ pool forms as a consequence of non-active reaction centers [6]. Accordingly, these results indicate that antidesmone has two inhibition sites on L. perenne leaves: one at the donor site on PSII (K-band) and another one after the pool of quinones (I and G-band) [28]. 3.5. Dry-biomass Dry-biomass of P. ixocarpa and L. perenne plants were weighed after 15 days of treatment with antidesmone at 150 and 300 μM, in the absence of antidesmone (negative control) and with 10 μM of DCMU (positive control). The results showed that at low concentrations of antidesmone the growth of L. perenne plants were increased by 8% and the growth of P. ixocarpa plants decreased by 17%, a lower inhibition than DCMU. However, antidesmone at 300 μM affected the growth of
Fig. 9. A. Radar plot of antidesmone effects on Chl a fluorescence parameters calculated from OJIP curve of sprayed Lolium perenne plants after 72 h. B. Appearance of K-band and I-band.
Please cite this article as: O.M. Sampaio, et al., Evaluation of antidesmone alkaloid as a photosynthesis inhibitor, Pesticide Biochemistry and Physiology (2016), http://dx.doi.org/10.1016/j.pestbp.2016.04.006
O.M. Sampaio et al. / Pesticide Biochemistry and Physiology xxx (2016) xxx–xxx Table 3 Effect of antidesmone on dry biomass production, estimated by measuring the dry weight of P. ixocarpa and L. perenne plants. P. ixocarpa
L. perenne
Compound
Conc. (μM)
Mass (g)
%
Mass (g)
%
Control DCMU Antidesmone
0 10 150 300
0.524 ± 0.01 0.388 ± 0.04 0.433 ± 0.07 0.411 ± 0.04
100 74 83 78
0.570 ± 0.02 0.423 ± 0.04 0.613 ± 0.006 0.467 ± 0.01
100 74 108 82
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herbicide probe. As the first natural product containing a 4(1H)pyridones scaffold with photosynthetic and plant growth activity, antidesmone has a promising potential to be explored as a tool in further development of a new class of herbicides. Acknowledgments The authors gratefully acknowledge financial support from Grants DGAPA-UNAM, IT102012 and FAPESP for the scholarship support.
Experiments were conducted with 3 replicates and data were expressed as means ± S.DE (standard errors), obtained with the program Origin 6.0.
References
P. ixocarpa and L. perenne plants by 22 and 18% respectively. DCMU inhibited growth of both plants by 18% (Table 3). These results indicated that antidesmone has a post-emergent herbicide behavior at high concentrations, which was confirmed by the fluorescence results in vivo. 3.6. Assay of germination and growth of root and stem The pre-emergent effect of antidesmone on P. ixocarpa and L. perenne seeds were evaluated through assays of germination and the growth of stems and roots in the presence of antidesmone. The results showed an increase for P. ixocarpa germination (15% at 50 μM). The effect on stem growth was insignificant at 50 μM. The major effect observed was on root growth, which was inhibited by 60% (p = 1.62 × 10−8) at 50 μM (Fig. 10). Antidesmone does not affect L. perenne germination nor the length of root and stem growth, indicating selective herbicide activity of antidesmone. 4. Conclusion This work shows for the first time that the secondary metabolite antidesmone, produced by W. brachypetala, plays a role in the inhibition of photosynthetic reactions and plant growth. The alkaloid behaves as a Hill reaction inhibitor and in vitro assays showed that it inhibits the acceptor side of PSII in the pool of QA. These data corroborated the Chl a fluorescence transient, indicating the inhibition of the PSII reactions. In vivo assays indicated a different mechanism, with antidesmone inhibiting the donor side of PSII and beyond of the PI. The effect of antidesmone on Chl a fluorescence transient of P. ixocarpa leaves was lower (effects are only present after 48 h) than for L. perenne (effects are present at 24, 48 and 72 h). However, the dry biomass decreased more in P. ixocarpa than in L. perenne, indicating that electron transport is not the only target for antidesmone in tomato plants. Although antidesmone inhibited the root growth of P. ixocarpa, it did not affect L. perenne, indicating that it is a good post-emergent
Fig. 10. Phytotoxic effect of antidesmone on P. ixocarpa germination rate, and root and stem growth. Values significant at p b 0.05 for t-Student for two populations. (G) Germination, (R) Root, (S) Stem.
[1] S.O. Duke, F.E. Dayan, J.G. Romagni, A.M. Rimando, Natural products as sources of herbicides: current status and future trends, Weed Res. 40 (1) (2000) 99–111. [2] J.R. Vyvyan, Allelochemicals as leads for new herbicides and agrochemicals, Tetrahedron 58 (9) (2002) 1631–1646. [3] F.E. Dayan, C.L. Cantrell, S.O. Duke, Natural products in crop protection, Bioorg. Med. Chem. 17 (12) (2009) 4022–4034. [4] D. Torres-Romero, B. King-Díaz, R.J. Strasser, I.A. Jiménez, B. Lotina-Hennsen, I.L. Bazzocchi, Friedelane triterpenes from Celastrus vulcanicola as photosynthetic inhibitors, J. Agric. Food Chem. 58 (20) (2010) 10847–10854. [5] T.A.M. Veiga, B. King-Díaz, A.S.F. Marques, O.M. Sampaio, P.C. Vieira, M.F.G.F. Silva, B. Lotina-Hennsen, Furoquinoline alkaloids isolated from Balfourodendron riedelianum as photosynthetic inhibitors in spinach chloroplasts, J. Photochem. Photobiol. B 120 (2013) 36–43. [6] B. King-Diaz, M.S. Soares, M.F.G.F. Silva, B. Lotina-Hennsen, T.A.M. Veiga, Triterpenes from Cabralea canjerana as in vitro inhibitors to light reactions of photosynthesis, Am. J. Plant Sci. 5 (16) (2014) 2528–2540. [7] M.M. Lima, J.A. López, J.M. David, E.P. Silva, A.M. Giulietti, L.P. De Queiroz, J.P. David, Acetylcholinesterase activity of alkaloids from the leaves of Waltheria brachypetala, Planta Med. 75 (4) (2009) 335–337. [8] K. Kubitzki, C. Bayer, Flowering plants – dicotyledons: Malvaceae, in: K. Kubitzki (Ed.), The Families and Genera of Vascular Plants, Springer-Verlag, Berlin Heidelberg, New York, NY, USA 2003, pp. 225–311. [9] G. Bringmann, J. Schlauer, H. Rischer, M. Wohlfarth, J. Muèhlbacher, A. Buske, A. Porzel, J. Schmidtb, G. Adamb, Revised structure of antidesmone, an unusual alkaloid from tropical Antidesma plants (Euphorbiaceae), Tetrahedron 56 (23) (2000) 3691–3695. [10] J.O.S. Varejão, L.C.A. Barbosa, E.V.V. Varejão, C.R.A. Maltha, B. King-Díaz, B. LotinaHennsen, Cyclopent-4-ene-1,3-diones: a new class of herbicides acting as potent photosynthesis inhibitors, J. Agric. Food Chem. 62 (25) (2014) 5772–5780. [11] Analysis of the chlorophyll a fluorescence transient, in: R.J. Strasser, A. Srivastava, M. Tsimilli-Michael, Govindjee, G.C. Papageorgiou (Eds.), Advances in Photosynthesis and Respiration, Springer, Dordrecht, The Netherlands 2004, pp. 321–362. [12] R.A. Dilley, Ion transport (H+, K+ Mg2+ exchange phenomena), Methods Enzymol. 24 (1972) 68–74. [13] M.I. Aguilar, M.G. Romero, M.I. Chávez, B. King-Dìaz, B. Lotina-Hennsen, Biflavonoids isolated from Selaginella lepidophylla inhibit photosynthesis in spinach chloroplasts, J. Agric. Food Chem. 56 (16) (2008) 6994–7000. [14] S. Izawa, G. Hind, The kinetics of the pH rise in illuminated chloroplast suspensions, Biochim. Biophys. Acta 143 (1967) 377–390. [15] I. Yruela, G. Montoya, P.J. Alonso, R. Picorel, Identification of the pheophytin-QA-Fe domain of the reducing side of the photosystem II as the Cu(II)-inhibitory binding site, J. Biol. Chem. 266 (34) (1991) 22847–22850. [16] R.T. Guiaquinta, R.A. Dilley, A partial reaction in photosystem II: reduction of silicomolybdate prior to the site of dichlorophenyldimethylurea inhibition, Biochim. Biophys. Acta 387 (2) (1975) 288–305. [17] T. Yamashida, T. Horio, Non-cyclic photophosphorylation by spinach grana treated with 0.8 M Tris buffer, Plant Cell Physiol. 9 (1968) 267–284. [18] M. Gonzalez-Ibarra, N. Farfán, C. Trejo, S. Uribe, B. Lotina-Hennsen, Selective herbicide activity of 2,5-di(benzylamine)-p-benzoquinone against the monocot weed Echinochloa crusgalli. An in vivo analysis of photosynthesis and growth, J. Agric. Food Chem. 53 (9) (2005) 3415–3420. [19] D. Torres-Romero, B. King-Díaz, I.A. Jiménez, B. Lotina-Hennsen, I.L. Bazzocchi, Sesquiterpenes from Celastrus vulcanicola as photosynthetic inhibitors, J. Nat. Prod. 71 (8) (2008) 1331–1335. [20] A.J. Demuner, L.C.A. Barbosa, T.A.M. Veiga, R.W. Barreto, B. King-Diaz, B. LotinaHennsen, Phytotoxic constituents from Nimbya alternantherae, Biochem. Syst. Ecol. 34 (11) (2006) 790–795. [21] M.L. Macías-Rubalcava, M.E.R. Sobrino, C. Meléndez-González, B. King-Díaz, B. Lotina-Hennsen, Selected phytotoxins and organic extracts from endophytic fungus Edenia gomezpompae as light reaction of photosynthesis inhibitors, J. Photochem. Photobiol. B 138 (2014) 17–26. [22] L.S. Medeiros, O.M. Sampaio, M.F.G.F. da Silva, E.R. Filho, T.A.M. Veiga, Evaluation of herbicidal potential of depsides from Cladosporium uredinicola, an endophytic fungus found in guava fruit, J. Braz. Chem. Soc. 23 (8) (2012) 1551–1557. [23] J.F. Allen, N.G. Holmes, in: M.F. Hipkins, N.R. Baker (Eds.), Photosynthesis: Energy Transduction: A Practical Approach, IRL Press, Oxford, UK 1986, pp. 103–141. [24] S. Chen, R.J. Strasser, S. Qiang, In vivo assessment of effect of phytotoxin tenuazonic acid on PSII reaction centers, Plant Physiol. Biochem. 84 (2014) 10–21. [25] E. Salvatori, L. Fusaro, S. Mereu, A. Bernardini, G. Puppi, F. Manes, Different O3 response of sensitive and resistant snap bean genotypes (Phaseolus vulgaris L.): the
Please cite this article as: O.M. Sampaio, et al., Evaluation of antidesmone alkaloid as a photosynthesis inhibitor, Pesticide Biochemistry and Physiology (2016), http://dx.doi.org/10.1016/j.pestbp.2016.04.006
8
O.M. Sampaio et al. / Pesticide Biochemistry and Physiology xxx (2016) xxx–xxx
key role of growth stage, stomatal conductance, and PSI activity, Environ. Exp. Bot. 87 (2013) 79–91. [26] S.Z. Tóth, G. Schansker, R.J. Strasser, In intact leaves, the maximum fluorescence level (FM) is independent of the redox state of the plastoquinone pool: a DCMUinhibition study, Biochim. Biophys. Acta 1708 (2) (2005) 275–282. [27] R.J. Strasser, M. Tsimilli-Michael, S. Qiang, V. Goltsev, Simultaneous in vivo recording of prompt and delayed fluorescence and 820-nm reflection changes during drying
and after rehydration of the resurrection plant Haberlea rhodopensis, Biochim. Biophys. Acta 1797 (6–7) (2010) 1313–1326. [28] M.G. Ceppi, A. Oukarroum, N. Çiçek, R.J. Strasser, G. Schansker, The IP amplitude of the fluorescence rise OJIP is sensitive to changes in the photosystem I content of leaves: a study on plants exposed to magnesium and sulfate deficiencies, drought stress and salt stress, Physiol. Plant. 144 (3) (2012) 277–288.
Please cite this article as: O.M. Sampaio, et al., Evaluation of antidesmone alkaloid as a photosynthesis inhibitor, Pesticide Biochemistry and Physiology (2016), http://dx.doi.org/10.1016/j.pestbp.2016.04.006