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Journal of Photochemistry & Photobiology, B: Biology 166 (2017) 35–43
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Journal of Photochemistry & Photobiology, B: Biology
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Effect of phytotoxic secondary metabolites and semisyntheticcompounds from endophytic fungus Xylaria feejeensis strain SM3e-1b onspinach chloroplast photosynthesis☆
Martha Lydia Macías-Rubalcava a,⁎, Marbella Claudia García-Méndez a,Beatriz King-Díaz b, Norma Angélica Macías-Ruvalcaba c
a Instituto de Química, Departamento de Productos Naturales, Universidad Nacional Autónoma de México (UNAM), Ciudad Universitaria, Coyoacán, Ciudad de México 04510, Mexicob Facultad de Química, Departamento de Bioquímica, Universidad Nacional Autónoma de México (UNAM), Ciudad Universitaria, Coyoacán, Ciudad de México 04510, Mexicoc Facultad de Química, Departamento de Fisicoquímica, Universidad Nacional Autónoma de México (UNAM), Ciudad Universitaria, Coyoacán, Ciudad de México 04510, Mexico
☆ Taken in part from the PhD thesis of Marbella C. Garc⁎ Corresponding author.
E-mail addresses: [email protected], mam(M.L. Macías-Rubalcava).
http://dx.doi.org/10.1016/j.jphotobiol.2016.11.0021011-1344/© 2016 Elsevier B.V. All rights reserved.
a b s t r a c t
a r t i c l e i n f oArticle history:Received 28 August 2016Accepted 1 November 2016Available online 09 November 2016
We investigated themechanism of action on the photosynthesis light reactions of threemajor secondarymetab-olites produced by the endophytic fungus Xylaria feejeensis strain SM3e-1b, isolated from Sapium macrocarpum;and four novel derivatives of coriloxine, amajor compound produced by X. feejeensis. The natural phytotoxins in-clude one epoxycyclohexenone derivative, coriloxine (1), and two quinone derivatives (2–3). The semisyntheticderivatives of coriloxine are two cyclohexenone (4–6) and two quinone compounds (5–7). Cyclohexenone (4),(4R,5S,6R)-6-chloro-4,5-dihydroxy-3-methoxy-5-methylcyclohex-2-enone, inhibited ATP synthesis in freshlylysed spinach chloroplasts from water to MV; it also partly inhibited the basal and uncoupled photosyntheticelectron transport, and significantly enhanced the phosphorylating electron transport andMg2+-ATPase activity,thus demonstrating its action as an uncoupler agent. On the other hand, quinone (7), 2-((4-butylphenyl)amino)-5-methoxy-3-methylcyclohexa-2,5-diene-1,4-dione, inhibited ATP synthesis, and non-cyclic electron transportfrom water to MV in basal, phosphorylating and uncoupled conditions in a concentration-dependent manner.Hence, (7) behaves as a Hill reaction inhibitor at the PSII electron transport on the water splitting enzyme(OEC), and on the acceptor side between P680 and QA. This mechanism of action was confirmed by chlorophylla fluorescence measurements. These results indicate that coriloxine derivatives 4 and 7 could work as prototypestructures for the development of new herbicides. Contrastingly, natural products 1–3, and derivatives 5 and 6did not show a significant inhibitory effect on ATP synthesis.
© 2016 Elsevier B.V. All rights reserved.
Keywords:Xylaria feejeensisSapium macrocarpumEndophytic fungiCyclohexenone derivativeQuinone derivativeCoriloxinePhytotoxic compoundsPhotosynthetic activitiesHill reaction inhibitorsUncoupler agents
1. Introduction
The vast structural diversity found in natural products makes theman incomparable source for the discovery of new pesticides, they canbe used either as leads for the development of pest control products,or directly as pure compounds or crude preparations [1–6].
Natural products as herbicides have not been as successful as forpharmaceutical drugs or for different conventional pesticides; however,the need for new weed-management compounds is undeniable as thenumber of herbicide-resistant weeds continues to increase, and thenumber of pest control products decreases due to health concerns andregulations [7–9].
ía Méndez.
Compared to natural phytotoxins, chemically obtained herbicidesare less complex, mainly because the cost of the final product limitsthe synthetic approach [5,10–11]. On the other hand, the complex bioticinteraction between an endophytic fungi and its host often results in theproduction of secondary metabolites with biological activity on a widevariety of molecular sites, many of which are unexploited targets [2–4,10–11]. As a result of this interaction-induced evolution, metabolitesfrom these organisms aremuchmore likely to have some kind of biolog-ical activity at lower concentrations than compounds derived fromchemical synthesis [5,10–11]. Therefore, phytotoxic natural productsisolated from endophytic fungi could be used by themselves or as natu-ral prototypes of bioactive compounds, [12–20] particularly as novelherbicides.
As part of our ongoing search for novel plant growth-inhibitingagents from endophytic fungi from plants growing in areas of great bio-diversity [20–24], we recently reported the evaluation of the phytotoxicpotential of three major secondary metabolites produced by the endo-phytic fungus Xylaria feejeensis strain SM3e-1b, isolated from Sapium
36 M.L. Macías-Rubalcava et al. / Journal of Photochemistry & Photobiology, B: Biology 166 (2017) 35–43
macrocarpumMull. Arg. (Euphorbiaceae), collected at the Reserva de laBiósfera Sierra de Huautla (REBIOSH); and four novel derivatives ofcoriloxine, amajor compound present in the combined culture mediumand mycelia organic extracts from X. feejeensis. The three naturalphytotoxins and the four coriloxine derivatives are shown in Fig. 1.[24]. The phytogrowth inhibitory activity of the organic extracts, andpure and semisynthetic compounds was evaluated on the seed germi-nation, root elongation, and seedling respiration of three dicotyledon-ous species, Medicago sativa, Trifolium pretense, and Amaranthushypochondriacus; and onemonocotyledonous plant, Panicummiliaceum.All of the tested compounds show high phytotoxic activity against thefour seeds; they inhibited the three physiological processes in a concen-tration-dependentmanner. As indicated by the IC50 values, root growthis, in general, strongly inhibited by all the compounds, the effect on ger-mination and seedling respiration is weaker [24]. To deepen the under-standing of the mechanism of action of these compounds, in this workwe evaluated the effect of the combined culturemedium andmyceliumextract, and the seven pure compounds (Fig. 1) on the different phasesof the photosynthesis' light reactions, including ATP synthesis, electronflow on the uncoupled partial reactions of PSI and PSII, basal rate andphosphorylation electron transport, and Mg2+-ATPase activity; usingpolarography techniques and Chl a fluorescence analysis.
2. Materials and Methods
2.1. Tested Materials
The phytotoxic compounds evaluated include three natural com-pounds from the endophytic fungusX. feejeensis strain SM3e-1b isolatedfrom S. macrocarpum, and four semisynthetic derivatives. Additionally,the combined culture medium and mycelium extract of X. feejeensiswas tested. The three natural compounds studied were theepoxycyclohexenone derivative, (4S,5S,6S)-4-hydroxy-3-methoxy-5-methyl-5,6- epoxycyclohex-2-enone or coriloxine (1), and two quinonederivatives: 2-hydroxy-5-methoxy-3-methylcyclohexa-2,5-diene-1,4-dione (2) and 2,6-hydroxy-5-methoxy-3-methylcyclohexa-2,5-diene-
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Fig. 1. Structures of phytotoxic secondary metabolites 1, 2 and 3 from Xylaria feejeensis strain
1,4-dione or fumiquinone B (3). On the other hand, the four phytotoxicsemisynthetic derivatives were: (4R,5S,6R)-6-chloro-4,5-dihydroxy-3-methoxy-5-methylcyclohex-2-enone (4), 2-hydroxy-3-methyl-5-(methylamino)cyclohexa-2,5-diene-1,4-dione (5), (4R,5R,6R)-4,5-di-hydroxy-3-methoxy-5-methyl-6-(phenylamino)cyclohex-2-enone (6),and 2-((4-butylphenyl)amino)-5-methoxy-3-methylcyclohexa-2,5-diene-1,4-dione (7). (Fig. 1). The isolation of the natural phytotoxins,as well as the chemical preparation of the semisynthetic derivativeswas carried out as previously described by García-Méndez et al. 2016[24].
Stock solutions on dimethyl sulfoxide (DMSO, maximum finalconcentration b 0.5%) of the natural compounds, the semisynthetic de-rivatives, and the combined culture medium and mycelium extractwere prepared for fluorescence, polarography, andMg2+-ATPase assays[20].
2.2. Chloroplast Isolation and Chlorophyll Determination
Spinach leaves (Spinacea oleracea) from a localmarketwere used forthe isolation of intact chloroplasts following the procedure described byMills et al. (1980) at 4 °C under dark conditions. Once isolated, theywere re-suspended using a minimal volume of a solution with the fol-lowing composition: MgCl2 (5 mM), KCl (10 mM), sucrose (400 mM),and pH 8.0 KOH-tricine buffer (30 mM). The concentrated chloroplastssuspensions can be stored for 8 to 12 h in the darkness at 4 °C [20]. De-termination of the chlorophyll (Chl) concentration was carried out asreported by Strain and coworkers (1971) [25].
2.3. Measurement of ATP Synthesis
AnOrionmicroelectrode (Mod. 8103Ross,MA, USA) and an expand-ed-scale Corning Medical potentiometer (model 12, Acton, MA, USA)with a Gilson recorder (Kipp & Zonen, Bohemia, NY, USA) were usedfor the titrimetric determination of ATP synthesis [20,26,27]. For thisassay, intact chloroplasts (20 μg of Chl/mL) were ruptured in 3 mL ofan osmotic breaking solution: MgCl2 (5 mM), KCl (10 mM), KCN
CH3
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SM3e-1; and experimental conditions for the preparation of compounds 4, 5, 6, and 7.
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(0.5 mM), sorbitol (100 mM), and pH 8.0 KOH-tricine buffer (1 mM).The measurement was performed with adenosine diphosphate (ADP,1mM, pH 6.5), andmethylviologen (MV, 50 μM) as an artificial electronacceptor; KOH (50 mM) was used to adjust the pH to 8.0. The organicextract was evaluated in the concentration range of 50–300 μg/mL,and the natural and semisynthetic derivatives in the range of 50–400 μg/mL. The synthesized ATP was calculated as μmol of ATP/mg ofChl x h [26].
2.4. Noncyclic Electron Transport Determination
A Clark-type electrode connected to a biological oxygen monitor(Yellow Spring Instrumentmod. 5300)was used for the polarographicaldetermination of the light-induced non-cyclic electron transport fromwater toMV in 20 μg Chl/mL chloroplasts solutions. Basal electron trans-port medium was prepared with MgCl2 (5 mM), KCl (10 mM), KCN(0.5 mM), sorbitol (100mM),MV (50 μM) and pH 8.0 KOH-tricine buff-er (15mM). This same reactionmediumwas used for the phosphorylat-ing non-cyclic electron transport determinations but ADP (1 mM) andKH2PO4 (3 mM) were added for the phosphorylation step. Uncouplednon-cyclic electron transport also used the same medium than forbasal transport with the addition of NH4Cl (6 mM), an uncoupleragent [20,27–29]. Each of the measurements was carried out in 3 mLof medium at 20 °C illuminating with a projector lamp for 2 min (GAF2669). Actinic light (0.2 mW/cm2) was obtained by filtering the lightfrom the projector through a 5 cm 1% CuSO4 solution. Actinic (blue)light has a wavelength of 420 nm that corresponds to the value wherechlorophyll a absorbs during the photosynthesis. All the compounds,natural and semisynthetic, were evaluated in concentrations from 50to 300 μg/L. For each assay, the negative control consisted on the chloro-plasts in the corresponding reaction medium without the evaluatedcompound [20].
2.5. Photosystem II (PSII) and Photosystem I (PSI) Uncoupled Electron FlowDetermination
Uncoupled PSII electron flow fromwater to 2,5-dichloro-1,4-benzo-quinone (DCBQ) was determined in basal electron transport medium,with the addition of DCBQ (100 μM), 2,5-dibromo-6-isopropyl-3-meth-yl-1,4-benzoquinone (DBMIB, 1 μM) and NH4Cl (6 mM), and withoutMV, in illuminated chloroplasts (20 μg Chl/mL). DCBQ acted as an artifi-cial electron acceptor at the D1 protein site [28,30].
Electron flow from water to sodium silicomolybdate (SiMo) on PSII,was measured in 3 mL of the same medium used for water to DCBQflow, except by the addition of SiMo (50 μM) and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU, 10 μM, to inhibit PSII at QB
level), and the elimination of DBMIB, using chloroplasts containing20 μg Chl/mL [20,31–32].
Thylakoids were treated with hydroxymethyl aminomethane (Tris,0.8 M, pH 8.0) and incubated at 4 °C during 30 min for the determina-tion of uncoupled electron transport rate from diphenylcarbazide(DPC) to the oxidized 2,6-dichlorophenolindophenol (DCPIPox), the re-duction of DCPIP was measured using a Beckman DU 650 spectropho-tometer. Treatment of the thylakoids with Tris inhibits the watersplitting enzyme (OEC) [33–35].
The uncoupled PSI electron transport from the reduced DCPIP toMVwas also determined in illuminated chloroplasts (20 μg Chl/mL) using3 mL of the medium employed for the basal electron transport experi-ment but DCPIP (100 μM), ascorbate (300 μM, for DCPIP reduction),DCMU (10 μM) and NH4Cl (6 mM) were added [20,36].
Similarly to the procedure for non-cyclic electron transport determi-nation, actinic light was produced and reaction mixtures were irradiat-ed for 1 min at room temperature; the same apparatus was used tomonitor electron flow activities. Pure compounds were tested in theconcentration range of 50–300 μg/L. For each reaction, the negative
control consisted on the chloroplasts in the corresponding mediumwithout the evaluated compound.
2.6. Chlorophyll a Fluorescence
Spinach leaveswere cut into 7mmdiameter discs; 15 of these circleswere placed with 20mL of modified Krebs solution into a Petri dish, thesolution consisted of NaCl (115 mM), KCl (5.9 mM), MgCl2 (1.2 mM),KH2PO4 (1.2 mM), Na2SO4 (1.2 mM), CaCl2 (25 mM), and NaHCO3
(25 mM, pH= 7.4). After 4 h of incubation at room temperature, purecompounds were added in DMSO solutions at different concentrationsfor each Petri dish and incubated for 4 additional hours; then, thediscs were dark adapted during 30min. Finally, Chl a fluorescence tran-sients were measured using a Hansatech Handy- Plant Efficient Analyz-er (King's Lynn, Norfolk, UK) [20,37]; a three LEDs array allowed forillumination with 650 nm continuous light (gain 0.7, intensity2830 μmol photons m−2 s−1). Maximum fluorescence yield from thesample was obtained by 2 s irradiation. DMSOwas added to the controlexperiments in order to consider the solvent effect; the volume ofDMSO added was equivalent to the one used to dissolve the analyzedcompound. OJIP transients were analyzed according to the JIP testStrasser et al., 2004. Analysis of Chl a transients allowed us to determinethe following parameters: (a) F0: fluorescence intensity level when theelectron acceptor, QA, is fully oxidized, (b) FM: fluorescence intensitylevel when QA is fully reduced, (c) fluorescence intensity level at 0.1,0.3, 2 (FJ), and 30 ms, (d) the area over the curve between F0 and FM,which comprises three sections: the area over the OJ, JI and IP phases.The first area represents the reduction of the acceptor side of PSII; thearea over the JI-transients is caused by electron transfer towards PSIand a partial re-oxidation of the PQ-pool; finally, the area above theIP-phase represents the reduction of the acceptor side of PSI; thus, thetotal area measures the quantity of electron acceptors between the ac-ceptor side of PSII and the acceptor side of PSI [38] (Table 2). BiolyzerHP3 softwarewas used for the determination of the derived parameterslisted in Table 3 (http://www.unige.ch/sciences/biologie/bioen, Labora-tory University of Geneva, Prof. R. J. Strasse, software developer).
2.7. Mg2+-ATPase Assays
Approximately 35 g of spinach leaves was ground in 160 mL of a so-lution with 2-(N-morpholino)ethanesulfonic acid (MES, 20 mM,pH 6.5), sorbitol (350 mM), and ascorbic acid (5 mM) to isolate thechloroplasts. Then, they were centrifuged for 60 s at 3000g, washedwith grinding solution, and resuspended in HEPES buffer (35 mM,pH 7.6) [24]. Light-triggered Mg2+-ATPase activity was determined asdescribed by Mills and collaborators, 1980 [39]. Released inorganicphosphate (P) was measured as reported by Sumner, 1944 [40].
2.8. Statistical Analysis
Experimental results regarding the effect of the extract and theseven pure compounds on different photosynthetic activities were ana-lyzed with GraphPad Prism statistical software (ver 5.01) by Tukey andANOVA tests [41]. All the data are normally distributed and variancesare homogeneous. For each bioassay, a Probit analysis was performedto calculate the IC50 value (50% inhibitory concentration) [20,42]. Dataare represented as mean ± standard deviation (SD). Statistical signifi-cance was considered for a P value of ≤0.05.
3. Results and Discussion
The endophytic fungus X. feejeensis strain SM3e-1b was isolatedfrom symptomless healthy leaves of S. macrocarpum, collected atREBIOSH. We recently reported significant phytotoxic activity on seedgermination, root elongation, and seedling respiration of dicotyledon-ous and monocotyledonous species, by the three major secondary
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Fig. 2. Effect of increasing concentrations of organic extract (Panel A); phytotoxicsecondary metabolites: coriloxine 1 ( ), compound 2 ( ), fumiquinone B 3 ( ) (PanelB); and semisynthetic derivatives: compounds 4 ( ) and 7 ( ) (Panel C) from Xylariafeejeensis strain SM3e-1b on ATP synthesis rate on spinach thylakoids. Control averagevalue as 100% of activity was 900 ± 1.5 μM ATP/ mg Chl × h. Each point represents themean of three determinations. Each repetition was made in different batches ofchloroplasts. Vertical bars represent standard deviations. The effect of semisyntheticderivatives 4 and 7 on photophosphorylation, was statistically significant (P b 0.05).
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metabolites produced by X. feejeensis (1–3); four synthetic derivativesof coriloxine (4–7) (Fig. 1); and the combined culture medium andmy-celium extract from the fungus [24]. In order to obtain a larger amountof the three biologically active compounds, we reinvestigated the cul-ture medium and mycelium extract from X. feejeensis strain SM3e-1,grown in static conditions. The fungus was grown in liquid-substratefermentation on potato dextrose broth (PDB). The mycelium and cul-ture mediumwere extracted with CH2Cl2 and EtOAc. The combined ex-tract exhibited significant phytotoxic activity on seed germination, rootelongation, and seedling respiration of A. hypochondriacus, T. pretense,M. sativa, and P. miliaceum using a Petri dish bioassay (data notshown). Bioassay-guided fractionation of the active extract led to theisolation of three known phytotoxic compounds: coriloxine (1), andtwo quinone derivatives, compounds 2 and 3. The yields of the threesecondary metabolites produced by endophytic fungus X. feejeensis
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Fig. 3. Effect of increasing concentrations of semisynthetic derivatives from Xylariafeejeensis strain SM3e-1b. Panel A, (4R,5S,6R)-6-chloro-4,5-dihydroxy-3-methoxy-5-methylcyclohex-2-enone (4). Panel B, 2-((4-butylphenyl)amino)-5-methoxy-3-methylcyclohexa-2,5-diene-1,4-dione (7) on basal ( ), phosphorylating ( ), anduncoupled ( ) rates of electron transport. Control average value was 460, 600 and1050 μequiv e−/mg Chl × h for basal, phosphorylating and uncoupled electron flowrespectively. Control is represented as 100% of activity. Each point represents the meanof three determinations. Each repetition was made in different batches of chloroplasts.Vertical bars represent standard deviations. The inhibitory effect of semisyntheticderivative 4 on electron transport rate from water to MV on freshly lysed illuminatedspinach chloroplasts, in the concentration range 25–300 μg/mL, was statisticallysignificant (P b 0.05).
Table 1Effect of (4R,5S,6R)-6-chloro-4,5-dihydroxy-3-methoxy-5-methylcyclohex-2-enone (4)and NH4Cl on Mg2+-ATPase bound to thylakoid membranes.
Treatments μM Pi mg of Chl−1 h−1 Activity %
Compound 4(μM)0 226 ± 23 100100 389 ± 80 172200 407 ± 90 180300 425 ± 92 188NH4Cl(mM)0 226 ± 23 1001 341 ± 186 1512 418 ± 181 1853 409 ± 141 181
Each point represents themean of fivedeterminations. Each repetitionwasmade indiffer-ent batches of chloroplasts.
39M.L. Macías-Rubalcava et al. / Journal of Photochemistry & Photobiology, B: Biology 166 (2017) 35–43
strain SM3e-1b isolated from S. macrocarpumwere very similar to thosereported in our previous work [24].
Compounds 1–3 were identified by TLC, using authentic samples asreferences, with different gradient elution systems [24]. Furthermore,the spectral properties of the compounds, including 1H and 13C NMRdata, were identical to those previously described in the literature [24,43–45].
On the other hand, coriloxine derivatives, compounds 4, 5, 6, and 7(Fig. 1), were prepared as previously reported by García-Méndez, et al.(2016) [24]. Briefly, compound 4 was obtained by reacting coriloxinewith indium(III) chloride in methanol solution with a catalytic amountof SiO2. Treatment of coriloxine with an aqueous solution of methyl-amine in presence of a catalytic amount of LiClO4 produced compound5. For compounds 6 and 7, a catalytic amount of SiO2 was used to pro-mote the opening of the epoxide ring; in both cases the nucleophilic at-tack of the aromatic amine (aniline or p-butylaniline) took place at theless sterically hindered carbon atom.
Additionally, in this work we evaluated the ability of the naturalcompounds 1–3 and synthetic derivatives of coriloxine (4–7) to inhibitdifferent reactions of the photosynthetic electron transport chain andthe Mg2+-ATPase activity; also, Chl a fluorescence measurementswere performed.
3.1. Effect of the Organic Extract, Natural Compounds 1–3, and Semisyn-thetic Derivatives 4–7 on Photophosphorylation
The phytotoxic organic extract from X. feejeensis strain SM3e-1bshowed inhibitory effect in a concentration-dependent way on theATP synthesis, from water to MV in freshly lysed spinach thylakoids(Fig. 2A), reducing it by 41% at 300 μM (IC50 N 300 μg/mL). At 400 μM,coriloxine (1) and quinone derivative (2) showed a weaker effect,inhibiting ATP synthesis by only 13% and 15%, respectively (maximalconcentration tested) (Fig. 2B). Compound 3 did not show an inhibitoryeffect on ATP formation. These results suggest that the inhibitory activ-ity on ATP synthesis of the organic extract might be due to the mixtureof different organic compounds or to the presence of other minor sec-ondary metabolites in the extract. However, ATP synthesis was signifi-cantly inhibited by coriloxine derivatives 4 and 7, in a concentration-
Table 2Experimental average values and standard deviations of Chlorophyll a fluorescence obtainedbutylphenyl)amino)-5-methoxy-3-methylcyclohexa-2,5-diene-1,4-dione (7).
Compound[μM] F0 FM F(0.1 ms)
Control 221 ± 8.6 1116 ± 50.0 252 ± 12.9100 212 ± 12.8 891 ± 106 249 ± 26.8200 231 ± 30.6 820 ± 72.8 247 ± 45.0300 242 ± 34.6 933 ± 70.1 306 ± 53.9
dependent manner, causing an inhibition of 84% and 95% at 300 μM(Fig. 2 C), and with IC50 values of 258.7 and 84.1 μM respectively.Coriloxine derivatives 5 and 6 did not show a significant inhibitory ef-fect on ATP formation. Since natural compounds 1–3 and coriloxine de-rivatives 5 and 6were poorly active or inactive compared to derivatives4 and 7, they were not further evaluated; our attentionwas centered oninvestigating themechanism of action and inhibition site of compounds4 and 7.
3.2. Effect of Coriloxine Derivatives 4 and 7 on Electron Transport Rate fromWater to MV on Freshly Lysed Illuminated Spinach Chloroplasts
ATP synthesis is not only inhibited by the blockade of the phosphor-ylation reaction, it can also be affected by impeding the electron trans-port or by uncoupling ATP synthesis from the electron transport. Toelucidate the inhibition mechanism of synthetic derivatives 4 and 7,the effect on the non-cyclic electron transport fromwater toMVwas in-vestigated under basal, phosphorylating, and uncoupled conditions.
Coriloxine derivative 4 enhanced phosphorylating electron transferby 160% at 100 μM(Fig. 3 A). Contrastingly, basal and uncoupled photo-synthetic electron transport was partially inhibited at 300 μM reducingit by approximately 23%. These results indicate that coriloxine deriva-tive 4 acts as an uncoupler agent and as a weak Hill reaction inhibitor.
Coriloxine derivative 7 inhibited the electron transport under all theevaluated conditions in a concentration dependent manner (Fig. 3 B). Itwas very active inhibiting the uncoupled electron transport rate, withan IC50 value of 17.5 μM. Basal and phosphorylating electron transportrate was less affected by compound 7, with IC50 values of 50.5 and71.7 μM, respectively. Since photophosphorylation and the electrontransport rate were inhibited, these results indicate that 7 behaves asa Hill reaction inhibitor.
3.3. Coriloxine Derivative 4 on Mg2+-ATPase Activity
To corroborate the uncoupling behavior of the synthetic derivative 4,its effect on the light-activated Mg2+-ATPase bound to thylakoidmem-branes was determined for increasing concentrations of 4, from 100 to300 μM. Some classical uncoupler agents, such as NH4Cl, stimulate theactivity of the Mg2+-ATPase, so they can be used as positive controls[46]. As shown in Table 1, both 4 andNH4Cl enhanced theMg2+-ATPaseactivity. These results indicate that compound 4 affected the pH gradi-ent, thus preventing ATP synthesis (Fig. 2C), as uncoupler agents do.
3.4. Determination of Target Sites of Coriloxine Derivative 7
The effect of compound 7 on PSII and PSI and on the partial reactionswas evaluated using different combinations of artificial electron donorsand acceptors, and the appropriate inhibitors (Fig. 4) [20,36]. Thiswould allow the assessment of the action site of 7 on the thylakoid elec-tron transport chain.
Uncoupled PSI electron transport from reducedDCPIP toMVwasnotsignificantly affected, with inhibition averaging around 17% for all of theevaluated concentrations (50–300 μM). Contrastingly, the uncoupledPSII electron flow from water to DCBQ was strongly affected; and thepartial reactions, from water to SiMo, and from DPC to DCPIP, wereinhibited in a concentration-dependent manner (Fig. 5). The most
in control and in S. oleracea discs leaves at different times after treatment with 2-((4-
F(0.3 ms) F(2 ms) F(30 ms) Area
318 ± 19.1 501 ± 22.6 838 ± 45.8 49,930 ± 689314 ± 61.7 472 ± 151.4 628 ± 243.6 31,400 ± 1199401 ± 66.7 651 ± 82.9 685 ± 64.2 19,300 ± 556423 ± 73.2 680 ± 107.6 777 ± 73.9 29,520 ± 887
H20 OEC Z P
680QA
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Fig. 4. Scheme of PSII electron transportmodel. Arrows show the sites of electron donation and acceptance; broken (dashed) lines indicate sites of inhibition of commercial inhibitors. PS IImeasured from water to DCBQ (1). PSII partial reactions measured from water to SiMo (2) and from DPC to DCPIP using Tris treated chloroplasts (3).
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pronounced inhibitory effect was on the uncoupled PSII from water toDCBQ, 83% at 300 μM. (Fig. 5), also the span of electron transport fromwater to SiMo was significantly inhibited, 76% at 300 μM. SiMo acceptselectrons at the QA site, thus, it can be inferred that compound 7 inhibitsthe PSII at the span of electron transport fromwater to QA. The electronflowmeasured fromDPC (donates electrons at P680) toDCPIPox (acceptselectrons at QB site), was partially inhibited by 7 at all of the evaluatedconcentrations, electron transport from P680 to QB was reduced by 57%at 300 μM (maximum concentration evaluated) (Fig. 5).
These results suggest that semisynthetic derivative 7 interacts at thewater splitting enzyme complex (oxygen evolving complex, OEC) andthrough the electron transport chain between P680 and QA [47]. Sinceuncoupled PSI electron transport from the reduced DCPIP to MV wasnot significantly affected, the semisynthetic derivative 7 can be classi-fied as a PSII inhibitor.
0 50 100 150 200 250 300
0
20
40
60
80
100
Activ
idad
%
Compound 7 [µM]
Fig. 5. Effect of increasing concentrations of semisynthetic derivative 2-((4-butylphenyl)amino)-5-methoxy-3-methylcyclohexa-2,5-diene-1,4-dione (7) onuncoupled electron transport on PSI measured from reduced DCPIP to MV ( );uncoupled electron transport on PSII measured from H2O to DCBQ ( ); and on theuncoupled electron transport on partial reactions of PSII from H2O to SiMo ( ), andfrom DPC to DCPIP ( ). Control average value as 100% of activity was 1234, 360 and229 μequiv e−/mg Chl × h for DCPIP to MV, H2O to DCBQ and from H2O to SiMorespectively. Control average for DPC to DCPIP was 125 μM of reduced DCPIP/mgChl × h. Each point represents the mean of three determinations. Each repetition wasmade in different batches of chloroplasts. Vertical bars represent standard deviations.The inhibitory effect of semisynthetic derivative 7 on electron transport rate onuncoupled PSII on freshly lysed illuminated spinach chloroplasts, in the concentrationrange 50–300 μg/mL, was statistically significant (P b 0.05).
3.5. PSII Chlorophyll a Fluorescence Measurements in Spinach Leaf Discs
To confirm the polarography results concerning the interaction siteof the phytotoxic semisynthetic derivative 7 on PSII, fluorescence mea-surements of Chl a were carried out using fresh spinach leaves discs.DMSO was used for the control discs in order to consider solvent effect.The fluorescence transients of chlorophyll a obtained from the controland treated discs were normalized. There is an increase in intensity be-tween 2 and 4 ms (J-band, Fig. 6A); moreover, a small K-band emergednear 0.3 ms (Fig. 6B). Samples treated with DCMU show a single induc-tion phase, the FM is reached after nearly 2ms (J-step). DCMUblocks theelectron transfer between the primary and the secondary quinone ac-ceptor (QA and QB) at the D1 protein of PSII, and an accumulation ofQA− takes place. The redox state of QA
− is responsible of the fluorescenceincrease [46]. The reduction of P680+ by YZ is much faster (20 ns) than theelectron transfer (200 μs) fromQA to QB [47]; however the presence of aK-band indicates that the electron transfer from P680 to QA without thedirect participation of OEC leads to the accumulation of P680+ due to thedecreased on the reduction of YZ
+; in other words, the electron flow be-tween the donor and the acceptor side of PSII is not balanced [48].Hence, coriloxine derivative 7 behaves as a Hill reaction inhibitor,blocking the electron flow. The polarography and Chl a fluorescence re-sults confirm that this compound acts at the donor side of PSII and par-tially inhibits the acceptor site from P680 to QA.
In addition to these results, different photosynthetic parameters ofChl a fluorescence kineticswere calculated to build Fig. 7 (Table 3). Spe-cificfluxes such as: the effective antenna size (ABS/RC) and themaximalrate by which an exciton is trapped by the reaction center resulting inthe reduction of QA to QA
− (TRo/RC) increased 25 and 40% respectivelyat 300 μMof semisynthetic derivative 7. The non-photochemical de-ex-citation rate constant (Kn), the quantum yield of energy dissipation att = 0 (ϕDo), and the efficiency with which an electron moves to thePSI end electron acceptors (δRo) from the reduced intersystem electronacceptors, also increased 40, 50 and 75%, respectively. The value of thespecific activity of primary photochemistry (M0) increased N100%.However, parameters related with electron transport, such as ETo/RCand ETo/CS, the probability that a trapped exciton moves an electroninto the electron transport chain beyond QA (ψEo), and the quantumyield for electron transport at t=0 (ϕ(Eo)), decreased at all the evaluat-ed concentrations of 7; at 300 μM (highest concentration used), theseparameters decreased by 50%. The performance index (PI(ABS)) showedthe highest decrease. Other parameters, such as the quantum yield forthe reduction of end acceptors of PSI per photon absorbed (ϕ(Ro)), andthe normalized total complementary area above the O-J-I-P transient(Sm), showed a slight increase at 300 μM of 7.
These results suggest that the semisynthetic derivative 7 acts as aHill reaction inhibitor at the PSII electron transport on OEC and onthe acceptor side between P680 and QA. A similar behavior has been
A
0.01 0.1 1 10 100 1000
0.0
0.1
0.2
0.3
0.4
J-Band
Flu
orescen
ce V
ariab
le R
elative
(V
)
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B
0.5 1.0 1.5 2.0 2.5
0.000
0.025
0.050
0.075
K-Band
Relative variab
le flu
orescen
ce
(V
)
Time (ms)
Fig. 6. Panel A, appearance of a J-band and Panel B, appearance of a K-band in the normalized relative variable fluorescence of leaf discs infiltrated with 100 μM ( ), 200 μM ( ), and300 μM ( ) of semisynthetic derivative 2-((4-butylphenyl)amino)-5-methoxy-3-methylcyclohexa-2,5-diene-1,4-dione (7). For the J-band, the ordinate is the difference between theV(t) of the sample minus the V(t) of the control, where V is the relative variable fluorescence at time (t), between F0 and FM; Vt = FVt/(FM − F0) = ( Ft − F0)/(FM − F0). For the K-band, the ordinate is the difference between the W of the sample minus the W of the control, where W is the relative variable fluorescence between F0 and FJ; Wt = V0 J(t) = FVt/(FJ − F0) = (Ft − F0)/(FJ − F0). Each curve represents the average of ten replicates.
41M.L. Macías-Rubalcava et al. / Journal of Photochemistry & Photobiology, B: Biology 166 (2017) 35–43
reported for 3-hydroxy-2,5-dimethylphenyl-2,4-dihydroxy-3,6-dimethylbenzoate, and 3-hydroxy-2,5-dimethylphenyl-4-[(2,4-dihy-droxy-3,6-dimethylbenzoyl)oxy]-2-hydroxy-3,6-dimethylbenzoate,two depsides produced by Cladosporium uredinicola, an endophytic fun-gus isolated from guava fruit (Psidium guajava, Myrtaceae) [49]. Thenaphtoquinonepiroketals from endophytic fungus Edenia gomezpompaeisolated from Callicarpa acuminata, preussomerins EG1 and EG4 andpalmarumycins CP17 and CP2, also inhibited the partial reactions ofPSII electron flow in a similar way [20]. Therefore, the inhibition siteof all of these compounds, naphthoquinonepiroketals, depsides andcompound 7, is located at the PSII electron transport chain at the OEC,and on the acceptor side of PSII. This mechanism of action was support-ed by Chl a fluorescence measurements that show a J-band similarly toDiuron (DCMU), a known herbicide [50,51].
It is worth noting that X. feejeensis was isolated from symptom-less healthy leaves of S. macrocarpum; meaning there is no evidenceof damage to the plant despite 4 and 7 effects on photosynthesis. Aspart of the virulence factors implicated in the colonization process to
Fig. 7. Spider plot showing the parameters calculated from the OJIP transients obtainedfrom fluorescence experiments on the leaves discs in presence of 200 ( ) and300 μM ( ) of the semisynthetic derivative 2-((4-butylphenyl)amino)-5-methoxy-3-methylcyclohexa-2,5-diene-1,4-dione (7), compared with the control DCMU( ). Each curve represents the average of ten replicates.
the host plant, endophytes have the potential to biosynthesize a va-riety of phytotoxic mycotoxins and/or exoenzymes [52,12]. Al-though the endophyte-host plant interaction implies a continuousmutual antagonism, in part based on the secondary metabolitesthat both organisms produce, none of them is damaged becausethere is an equilibrium between the endophytic virulence factorsand the host defense response, or balanced antagonism. Disease tothe host plant or fungus death may arise only if this balanced antag-onism is broken by factors such as the host senescence or biotic andabiotic stress [52,12]. In fact, it is known that endophytic fungi play akey role increasing the uptake of nutrients and the tolerance to bioticand abiotic stress of the host plant, as well as protecting it against theattack of herbivores, insects and pathogenic microorganisms [53].Furthermore, it has been observed that plants completely devoid ofendophytes show reduced photosynthetic rates and chlorosis; there-fore, lower survival rates in nature [54].
Finally, to determine whether the secondary metabolites andsemisynthetic derivatives from endophytic fungus X. feejeensis strainSM3e-1b satisfy the requirements established for the “Lipinski's ruleof 5” Lipinski, 1997 [55] and Tice's rule Tice 2001 [56], the physico-chemical parameters of all the compounds were calculated usingthe Chemicalize software package (ChemAxon. http://www.chemicalize.org/). “Lipinski's rule of 5” is a set of empirically derivedrules that outline the molecular characteristics and the physico-chemical properties of orally bioavailable drugs. Molecules violatingmore than one of these rules exhibit limited bioavailability. Based onthe approach by Lipinski, Tice established a set of rules that can beapplied to molecules of agrochemical interest [56,57]. Calculatedphysicochemical parameters such as molecular weight, lipophilicity,number of rotatable bonds, number of hydrogen-bond donors, andnumber of hydrogen-bond acceptors, are encompassed by “Tice'srules” for screening new herbicide candidates [56,57]. As shown inTable 4, the three secondary metabolites (1–3) and four semisyn-thetic derivatives (4–7) satisfy both Tice's and Lipinski's rules, indi-cating that these compounds possess physicochemical propertiesappropriate for their use as herbicides [56].
4. Conclusions
In this study,we demonstrated that two semisynthetic derivatives ofcoriloxine are more potent as inhibitors of the photosynthetic electrontransport in isolated spinach chloroplasts than the three secondary me-tabolites from Xylaria feejeensis strain SM3e-1b isolated from S.
Table 3Derived parameters, their description and formulae, using data extracted from the Chl a fluorescence (OJIP) transient.
Fluorescence parameters derived from the extracted dataM0 = dV/dt = 4(F300 μs − F0)/(FM − F0) Approximated initial slope (in ms−1) of the fluorescence transient V = f(t)Sm = (Area)/(FM − F0) Normalized total complementary area above the O-J-I-P transient (reflecting multiple turnover QA reduction events)
Yields or flux ratiosφPo = TR0/ABS = [1 − F0/FM] Maximum quantum yield of primary photochemistry at t = 0φEo = ET0/ABS = [1 − (FJ/FM)] Quantum yield for electron transport at t = 0ψEo = ET0/TR0 = (1 − VJ) Probability (at t = 0) that a trapped exciton moves an electron into the electron transport chain beyond QA
−
φDo = 1 − φPo = (F0/FM) Quantum yield (at t = 0) of energy dissipationδR0 = RE0/ET0 = (1 − VI)/(1 − VJ) Efficiency with which an electron can move from the reduced intersystem electron acceptors to the PS I end
electron acceptors of PS IφR0 = RE0/ABS = φPo ∗ ψEo ∗ δR0 = 1 − (FI/FM) Quantum yield for the reduction of end acceptors of PSI per photon absorbedRE0/TR0 = ψEo × δR0 Efficiency with which a trapped exciton move an electron into the electron transport chain from QA
− to the PS I endelectron acceptors
Specific energy fluxes or activities (per reaction QA− reducing PS II reaction center - RC)
ABS/RC = M0(1/VJ)(1/φPo) Absorption per RCTR0/RC = M0/VJ Trapped energy flux per RC (at t = 0)ETo/RC = M0 (1/VJ)(ψEo) Electron transport flux per RC (at t = 0)
Phenomenological energy fluxes (per excited cross section – CS)ABS/CS0 Absorption flux per CSTR0/CS0 Trapped energy flux per CS (at t = 0)ET0/CS0 Electron transport flux per CS (at t = 0)
De-excitation rate constantsKp Photochemical de-excitation rate constantKn Non photochemical de-excitation rate constantSum K The sum of photochemical and non photochemical rate constants
Performance indexPI(ABS) = RC
ABS � TRoABS−TRo � ETo
TRo−EToPerformance index on absorption basis
Table 4Requirements established for “Lipinski's rule of 5” and Tice's parameters calculated for the phytotoxic secondarymetabolites 1–3 and semisynthetic derivatives 4–7 from Xylaria feejeensisstrain SM3e-1b.
Compounds Molecular weight Calculated lipophilicity (LogP) Number of rotatable bonds Number of hydrogen-bond donors Number of hydrogen-bond acceptors
1 170 −0.49 1 1 42 168 0.50 1 1 43 184 −0.02 1 2 44 206 −0.30 1 2 45 157 0.18 1 2 46 263 0.31 3 3 57 299 3.26 6 2 5Lipinski's ≤500 ≤5 ≤5 ≤10Tice 150–500 ≤5 ≤12 ≤3 2–12
42 M.L. Macías-Rubalcava et al. / Journal of Photochemistry & Photobiology, B: Biology 166 (2017) 35–43
macrocarpum. Coriloxine derivative (4R,5S,6R)-6-chloro-4,5-dihy-droxy-3-methoxy-5-methylcyclohex-2-enone (4) produced a signifi-cant enhancement on the phosphorylating electron transport rate andpartly affected basal and uncoupled photosynthetic electron transportrates. This compound also enhanced the activity of Mg2+-ATPase, cor-roborating its action as uncoupler. On the other hand, quinonederivative 2-((4-butylphenyl)amino)-5-methoxy-3-methylcyclohexa-2,5-diene-1,4-dione (7) acts as a Hill reaction inhibitor at the electrontransport on the water splitting enzyme (OEC), and on the acceptorside (between P680 and QA), similarly to the commercial herbicideDCMU. Chlorophyll a fluorescence measurements corroborated thismechanism of action.
The phytotoxic effect on photosynthesis has not been previously de-scribed for coriloxine derivatives (4) and (7). Therefore, these com-pounds could be considered as promising leads for developing newherbicides potentially useful in agriculture.
Based on the results from this investigation, it is clear that themech-anism of action associated with the phytotoxic effects of the three natu-ral products 1–3 and the semisynthetic derivatives 5 and 6 fromendophytic fungus Xylaria feejeensis strain SM3e-1b, isolated from S.macrocarpum is not on the luminous phase of the photosynthesis, so
further studies need to be carried out to better understand the mecha-nism of action of these compounds.
Acknowledgments
This work was supported by Consejo Nacional de Ciencia yTecnología grant 179194. We want to thank M. S. Elizabeth K. GalvánMiranda from Facultad de Química, UNAM, for language revision.Marbella García acknowledges the fellowship awarded by CONACyT tocarry out her graduate studies.
References
[1] O. Duke, H. Abbas, Natural products with potential use as herbicides, ACS Symp. Ser.582 (1995) 348–362.
[2] Phytotoxins of microbial origin with potential for use as herbicides, in: S.O. Duke,H.K. Abbas, T. Amagasa, T. Tanaka, L.G. Copping (Eds.), Crop Protection Agentsfrom Nature, Natural Products and Analogues, Royal Society of Chemistry, Cam-bridge, U.K. 1996, pp. 82–113.
[3] S.O. Duke, F.E. Dayan, A. Hernández, M.V. Duke, H.K. Abbas, Natural products asleads for new Herbicide Modes of action, Proceedings, Brighton Crop Prot. Conf.,Weeds, Vol. 2, 1997 579–586.
43M.L. Macías-Rubalcava et al. / Journal of Photochemistry & Photobiology, B: Biology 166 (2017) 35–43
[4] S.O. Duke, F.E. Dayan, A.M. Rimando, Natural products as tools for weed manage-ment, Proc. Jpn. Weed Sci. Soc. (1998) 1–11 (Suppl.).
[5] S.O. Duke, F.E. Dayan, J.G. Romagni, A.M. Rimando, Natural products as sources ofherbicides: current status and future trends, Weed Res. 40 (2000) 99–111.
[6] S.O. Duke, F.E. Dayan, A.M. Rimando, K.K. Schrader, G. Aliotta, A. Oliva, J.G. Romagni,Chemicals from nature for weed management, Weed Sci. 50 (2002) 138–151.
[7] F.E. Dayan, C.L.S.O. Cantrell, Duke, natural products in crop protection, Bioorg. Med.Chem. 17 (2009) 4022–4034.
[8] F.E. Dayan, D.K. Owens, S.O. Duke, Rationale for a natural products approach to her-bicide discovery, Pest Manag. Sci. 68 (2012) 519–528.
[9] C.L. Cantrell, F.E. Dayan, S.O. Duke, Natural products as sources for new pesticides, J.Nat. Prod. 75 (2012) 1231–1242.
[10] S.O. Duke, F.E. Dayan, J.G. Romagni, Natural products as sources for new mecha-nisms of herbicidal action, Crop. Prot. 19 (2000) 583–589.
[11] S.O. Duke, F.E. Dayan, A.M. Rimando, Natural products and herbicide discovery, in:A.H. Cobb, R.C. Kirkwood (Eds.), Herbicides and their Mechanisms of Action, Aca-demic Press, Sheffield, U.K. 2000, pp. 105–133.
[12] B. Schulz, C. Boyle, The endophytic continuum, Mycol. Res. 109 (2005) 661–686.[13] D.L. Hawksworth, The fungal dimension of biodiversity: magnitude, significance,
and conservation, Mycol. Res. 95 (1991) 641–655.[14] A. Aptroot, Species diversity in tropical rainforest ascomycetes: lichenized vs non-
lichenized; foliicolous vs. corticolous, Abstr. Bot. 21 (1997) 37–44.[15] M.M. Dreyfuss, I.H. Chapela, Potential of fungi in the discovery of novel, low-molec-
ular weight pharmaceuticals, Biotechnology 26 (1994) 49–80.[16] R.X. Tan, W.X. Zou, Endophytes: a rich source of functional metabolites, Nat. Prod.
Rep. 18 (2001) 448–459.[17] B. Schulz, C. Boyle, S. Draeger, A.K. Rommert, K. Krohn, Endophytic fungi: a source of
novel biologically active secondary metabolites, Mycol. Res. 106 (2002) 996–1004.[18] S. Kumar, N. Kaushik, Metabolites of endophytic fungi as novel source of
biofungicide: a review, Phytochem. Rev. 11 (2012) 507–522.[19] S. Kusari, C. Hertweck, M. Spiteller, Chemical ecology of endophytic fungi: origins of
secondary metabolites, Chem. Biol. 19 (2012) 792–798.[20] M.L. Macías-Rubalcava, M.E. Ruiz-Velasco Sobrino, C. Meléndez-González, B. King-
Díaz, B. Lotina-Hennsen, Selected phytotoxins and organic extracts from endophyticfungus Edenia gomezpompae as light reaction of photosynthesis inhibitors, J.Photochem. Photobiol. B 138 (2014) 17–26.
[21] R.E. Sánchez-Fernández, D. Diaz, G. Duarte, P. Lappe-Oliveras, S. Sánchez, M.L. Macías-Rubalcava, Antifungal volatile organic compounds from the endophyte Nodulisporiumsp. strain GS4d2II1a: a qualitative change in the intraspecific and interspecific interac-tions with Pythium aphanidermatum, Microb. Ecol. 71 (2016) 347–364.
[22] B.L. Sánchez-Ortiz, R.E. Sánchez-Fernández, G. Duarte, P. Lappe-Oliveras, M.L.Macías-Rubalcava, Antifungal, antioomycete and phytotoxic effects of volatile or-ganic compounds from the endophytic fungus Xylaria sp. strain PB3f3 isolatedfrom Haematoxylon brasiletto, J. Appl. Microbiol. 120 (2016) 1313–1325.
[23] A. Ulloa-Benítez, Y.M. Medina-Romero, E. Sánchez-Fernández, P. Lappe-Oliveras, G.Roque-Flores, G. Duarte, T. Herrera Suárez, M.L. Macías-Rubalcava, Phytotoxic andantimicrobial activity of volatile and semi-volatile organic compounds from the en-dophyte Hypoxylon anthochroum strain Blaci isolated from Bursera lancifolia(Burseraceae), J. Appl. Microbiol. 121 (2016) 380–400.
[24] M.C. García-Méndez, N.A. Macías-Ruvalcaba, P. Lappe-Oliveras, S. Hernández-Ortega, M.L. Macías-Rubalcava, Phytotoxic potential of secondary metabolites andsemisynthetic compounds from endophytic fungus Xylaria feejeensis strain SM3e-1b isolated from Sapium Macrocarpum, J. Agric. Food Chem. 64 (2016) 4255–4263.
[25] H.H. Strain, B.T. Cope, W.A. Svec, Analytical procedures for the isolation, identifica-tion, estimation and investigation of the chlorophylls, Methods Enzymol. 23(1971) 452–466.
[26] R.A. Dilley, Ion transport (H+, K+, Mg2+ exchange phenomena), Methods Enzymol.24 (1972) 68–74.
[27] B. King-Díaz, J. Granados-Pineda, M. Bah, J.F. Rivero-Cruz, B. Lotina-Hennsen, Mexi-can propolis flavonoids affect photosynthesis and seedling growth, J. Photochem.Photobiol. B 151 (2015) 213–220.
[28] F. Morales-Flores, K.S. Olivares-Palomares, M.I. Aguilar-Laurents, J.F. Rivero-Cruz, B.Lotina-Hennsen, B. King-Díaz, Flavonoids affect the light reaction of photosynthesisin vitro and in vivo as well as the growth of plants, J. Agric. Food Chem. 63 (2015)8106–8115.
[29] S. Saha, R. Ouitrakul, S. Izawa, N. Good, Electron transport and photophosphoryla-tion in chloroplasts as a function of the electron acceptor, J. Biol. Chem. 246(1971) 3204–3209.
[30] I. Yruela, G. Montoya, P.J. Alonso, R. Picorel, Identification of the pheophytin- QA-Fedomain of the reducing side of the photosystem II as the Cu(II)- inhibitory bindingsite, J. Biol. Chem. 266 (1991) 22847–22850.
[31] G. Schansker, J.J.S. Van Rensen, Characterization of the complex interaction betweenthe electron acceptor silicomolybdate and photosystem II, Photosynth. Res. 37(1993) 165–175.
[32] R.T. Giaquinta, B.R. Selman, B.J. Anderson, R.A. Dilley, Inhibition of coupling factor ac-tivity of chloroplast membrane by diazonium compounds, J. Biol. Chem. 249 (1974)2873–2878.
[33] L.P. Vernon, E.R. Shaw, Photoreduction of 2,6-dichlorophenol by diphenylcarbazide:a photosystem 2 reaction catalyzed by Tris-washed chloroplasts andsubchloroplasts fragments, Plant Physiol. 44 (1969) 1645–1649.
[34] A. Rashid, R. Popovic, Electron donation to photosystem II by diphenylcarbazide isinhibited both by the endogenous manganese complex and by exogenous manga-nese ions, Biochem. Cell Biol. 73 (1995) 241–245.
[35] C.T. Yerkes, G.T. Babcock, Photosystem II oxidation of charged electron donors: sur-face charge effects, Biochim. Biophys. Acta Bioenerg. 590 (1980) 360–372.
[36] J.F. Allen, N.G. Holmes, Electron Transport Partial Reactions, in: M.F. Hipkinns, N.R.Baker (Eds.), Photosynthesis, Energy Transduction. A Practical Approach, I.R.L.Press, Oxford, United Kingdom 1986, pp. 103–141 (Chapter 5).
[37] T.A.M. Veiga, B. King-Díaz, A.S.F. Marques, O.M. Sampaio, P.C. Vieira, M.F.G.F. da Silva,B. Lotina-Hennsen, Furoquinoline alkaloids isolated from Balfourodendronriedelianum as photosynthetic inhibitors in spinach chloroplasts, J. Photochem.Photobiol. B 120 (2013) 36–43.
[38] S.Z. Toth, G. Schansker, R.J. Strasser, A non-invasive assay of the plastoquinone poolredox state based on the OJIP-transient, Photosynth. Res. 93 (2007) 193–203.
[39] J.D. Mills, P. Mitchell, P. Schurrmann, Modulation of coupling ATPase activity in in-tact chloroplasts, FEBS Lett. 191 (1980) 144–148.
[40] J.B. Sumner, Scientific apparatus and laboratory methods. A method for the colori-metric determination of phosphorus, Science 100 (1944) 413–418.
[41] R. Mead, R.N. Curnow, A.M. Hasted, Statistical Methods in Agriculture and Experi-mental Biology, third ed. Chapman and Hall, CRC, Boca Raton, FL, 2002.
[42] J.H. Zar, Biostatistical Analysis, fifth ed. Prentice Hall, Upper Saddle River, New Jer-sey, 2010.
[43] S. Tansuwan, P. Chanaprat, T. Teerawatananond, N. Muangsin, S. Pornpakakul,(4S,5S,6S)-4-hydroxy-3-methoxy-5-methyl-5,6- epoxycyclohex-2-en-1-one, ActaCrystallogr. Sect. E Struct. Rep. Online E66 (2010) o2263.
[44] S. Tansuwan, S. Porpakakul, S. Roengsumran, A. Petsom, N. Muangsin, P. Sihanonta,N. Chaichit, Antimalarial benzoquinones from an endophytic fungus Xylaria sp. J.Nat. Prod. 70 (2007) 1620–1623.
[45] A. Hayashi, S. Fujioka, M. Nukina, T. Kawano, A. Shimada, Y. Kimura, Fumiquinones Aand B, nematicidal quinones produced by Aspergillus fumigatus, Biosci. Biotechnol.Biochem. 71 (2007) 1697–1702.
[46] L. Achnine, M. Bah, R. Pereda-Miranda, R. Mata, B. Lotina-Hennsen, Tricolorin A, apotencial natural uncoupler and inhibitor of photosystem II acceptor side of spinachchloroplasts, Physiol. Plant. 106 (1999) 240–252.
[47] H. Dau, Molecular mechanisms and quantitative models of variable Photosystem IIfluorescence, Photochem. Photobiol. 60 (1994) 1–23.
[48] B.J. Strasser, Donor side capacity of photosystem probed by chlorophyll a fluores-cence transients, Photosynth. Res. 52 (1997) 147–155.
[49] L.S. de Medeiros, O.M. Sampaio, M.F. das G.F. da Silva, E.R. Filho, T.A.M. Veiga, Eval-uation of herbicidal potential of depsides from Cladosporium uredinicola, an endo-phytic fungus found in guava fruit, J. Braz. Chem. Soc. 23 (2012) 1551–1557.
[50] S.Z. Tóth, G. Schansker, G. Garab, R.J. Strasser, Photosynthetic electron transport ac-tivity in heat-treated barley leaves: the role of internal alternative electron donorsto photosystem II, Biochim. Biophys. Acta 1767 (2007) 295–305.
[51] R.T. Giaquinta, R.A. Dilley, F.L. Crane, R. Barr, Photophosphorylation not coupled toDCMU-insensitive photosystem II oxygen evolution, Biochem. Biophys. Res.Commun. 59 (1974) 985–991.
[52] B. Schulz, A.K. Rommert, U. Dammann, H.J. Aust, D. Strack, The endophyte-host in-teraction: a balanced antagonism? Mycol. Res. 103 (1999) 1275–1283.
[53] F. Gao, C. Dai, X. Liu, Mechanisms of fungal endophytes in plant protection againstpathogens, Afr. J. Microbiol. Res. 4 (2010) 1346–1351.
[54] L.P. Partida-Martínez, M. Heil, The microbe-free plant: fact or artifact? Front. PlantSci. 2 (2011) 1–16.
[55] C.A. Lipinski, F. Lombardo, B.W. Dominy, P.J. Feeney, Experimental and computation-al approaches to estimate solubility and permeability in drug discovery and devel-opment settings, Adv. Drug Deliv. Rev. 64 (2012) 4–17.
[56] C.M. Tice, Selecting the right compounds for screening: does Lipinski's rule of 5 forpharmaceuticals apply to agrochemicals? Pest Manag. Sci. 57 (2001) 3–16.
[57] J.O.S. Varejão, L.C.A. Barbosa, G.Á. Ramos, E.V.V. Varejão, B. King-Díaz, B. Lotina-Hennsen, New rubrolide analogues as inhibitors of photosynthesis light reactions,J. Photochem. Photobiol. B 145 (2015) 11–18.