Effect of phytotoxic secondary metabolites and semisynthetic compounds from endophytic fungus Xylaria feejeensis strain SM3e-1b on spinach chloroplast photosynthesis

Article abstract of DOI:10.1016/j.jphotobiol.2016.11.002

We investigated the mechanism of action on the photosynthesis light reactions of three major secondary metabolites produced by the endophytic fungus Xylaria feejeensis strain SM3e-1b, isolated from Sapium macrocarpum; and four novel derivatives of coriloxine, a major compound produced by X. feejeensis. The natural phytotoxins include one epoxycyclohexenone derivative, coriloxine (1), and two quinone derivatives (2–3). The semisynthetic derivatives 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 freshly lysed spinach chloroplasts from water to MV; it also partly inhibited the basal and uncoupled photosynthetic electron transport, and significantly enhanced the phosphorylating electron transport and Mg^2?+-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 transport from 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 Q_A. This mechanism of action was confirmed by chlorophyll a fluorescence measurements. These results indicate that coriloxine derivatives 4 and 7 could work as prototype structures for the development of new herbicides. Contrastingly, natural products 1–3, and derivatives 5 and 6 did not show a significant inhibitory effect on ATP synthesis.

Full text of DOI:10.1016/j.jphotobiol.2016.11.002

Journal of Photochemistry & Photobiology, B: Biology 166 (2017) 35–43
Contents lists available at ScienceDirect
Journal of Photochemistry & Photobiology, B: Biology
journal homepage: www.elsevier.com/locate/jphotobiol
Effect of phytotoxic secondary metabolites and semisynthetic
compounds from endophytic fungus Xylaria feejeensis strain SM3e-1b on
☆
spinach chloroplast photosynthesis
a,
a
Martha Lydia Macías-Rubalcava ⁎, Marbella Claudia García-Méndez ,
b
c
Beatriz King-Díaz , Norma Angélica Macías-Ruvalcaba
Instituto de Química, Departamento de Productos Naturales, Universidad Nacional Autónoma de México (UNAM), Ciudad Universitaria, Coyoacán, Ciudad de México 04510, Mexico
Facultad de Química, Departamento de Bioquímica, Universidad Nacional Autónoma de México (UNAM), Ciudad Universitaria, Coyoacán, Ciudad de México 04510, Mexico
Facultad de Química, Departamento de Fisicoquímica, Universidad Nacional Autónoma de México (UNAM), Ciudad Universitaria, Coyoacán, Ciudad de México 04510, Mexico
a
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
We investigated the mechanism of action on the photosynthesis light reactions of three major secondary metab-
olites produced by the endophytic fungus Xylaria feejeensis strain SM3e-1b, isolated from Sapium macrocarpum;
and four novel derivatives of coriloxine, a major compound produced by X. feejeensis. The natural phytotoxins in-
clude one epoxycyclohexenone derivative, coriloxine (1), and two quinone derivatives (2–3). The semisynthetic
derivatives of coriloxine are two cyclohexenone (4–6) and two quinone compounds (5–7). Cyclohexenone (4),
Received 28 August 2016
Accepted 1 November 2016
Available online 09 November 2016
Keywords:
Xylaria feejeensis
(4R,5S,6R)-6-chloro-4,5-dihydroxy-3-methoxy-5-methylcyclohex-2-enone, inhibited ATP synthesis in freshly
lysed spinach chloroplasts from water to MV; it also partly inhibited the basal and uncoupled photosynthetic
Sapium macrocarpum
Endophytic fungi
Cyclohexenone derivative
Quinone derivative
Coriloxine
Phytotoxic compounds
Photosynthetic activities
Hill reaction inhibitors
Uncoupler agents
2
+
electron transport, and significantly enhanced the phosphorylating electron transport and Mg -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 transport
from 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
A
(OEC), and on the acceptor side between P680 and Q . This mechanism of action was confirmed by chlorophyll
a fluorescence measurements. These results indicate that coriloxine derivatives 4 and 7 could work as prototype
structures for the development of new herbicides. Contrastingly, natural products 1–3, and derivatives 5 and 6
did not show a significant inhibitory effect on ATP synthesis.
©
2016 Elsevier B.V. All rights reserved.
1
. Introduction
Compared to natural phytotoxins, chemically obtained herbicides
are less complex, mainly because the cost of the final product limits
the synthetic approach [5,10–11]. On the other hand, the complex biotic
interaction between an endophytic fungi and its host often results in the
production of secondary metabolites with biological activity on a wide
variety of molecular sites, many of which are unexploited targets [2–4,
10–11]. As a result of this interaction-induced evolution, metabolites
from these organisms are much more likely to have some kind of biolog-
ical activity at lower concentrations than compounds derived from
chemical synthesis [5,10–11]. Therefore, phytotoxic natural products
isolated from endophytic fungi could be used by themselves or as natu-
ral prototypes of bioactive compounds, [12–20] particularly as novel
herbicides.
The vast structural diversity found in natural products makes them
an incomparable source for the discovery of new pesticides, they can
be 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 for
pharmaceutical drugs or for different conventional pesticides; however,
the need for new weed-management compounds is undeniable as the
number of herbicide-resistant weeds continues to increase, and the
number of pest control products decreases due to health concerns and
regulations [7–9].
As part of our ongoing search for novel plant growth-inhibiting
agents from endophytic fungi from plants growing in areas of great bio-
diversity [20–24], we recently reported the evaluation of the phytotoxic
potential of three major secondary metabolites produced by the endo-
phytic fungus Xylaria feejeensis strain SM3e-1b, isolated from Sapium
☆
Taken in part from the PhD thesis of Marbella C. García Méndez.
Corresponding author.
E-mail addresses: mmacias@iquimica.unam.mx, mamaciasr@gmail.com
⁎
(
M.L. Macías-Rubalcava).
http://dx.doi.org/10.1016/j.jphotobiol.2016.11.002
011-1344/© 2016 Elsevier B.V. All rights reserved.
1

36
M.L. Macías-Rubalcava et al. / Journal of Photochemistry & Photobiology, B: Biology 166 (2017) 35–43
macrocarpum Mull. Arg. (Euphorbiaceae), collected at the Reserva de la
Biósfera Sierra de Huautla (REBIOSH); and four novel derivatives of
coriloxine, a major compound present in the combined culture medium
and mycelia organic extracts from X. feejeensis. The three natural
phytotoxins and the four coriloxine derivatives are shown in Fig. 1.
1,4-dione or fumiquinone B (3). On the other hand, the four phytotoxic
semisynthetic 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 derivatives
was carried out as previously described by García-Méndez et al. 2016
[24].
[24]. The phytogrowth inhibitory activity of the organic extracts, and
pure 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 Amaranthus
hypochondriacus; and one monocotyledonous plant, Panicum miliaceum.
All of the tested compounds show high phytotoxic activity against the
four seeds; they inhibited the three physiological processes in a concen-
tration-dependent manner. As indicated by the IC50 values, root growth
is, 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 work
we evaluated the effect of the combined culture medium and mycelium
extract, and the seven pure compounds (Fig. 1) on the different phases
of the photosynthesis' light reactions, including ATP synthesis, electron
flow on the uncoupled partial reactions of PSI and PSII, basal rate and
phosphorylation electron transport, and Mg2 -ATPase activity; using
polarography techniques and Chl a fluorescence analysis.
Stock solutions on dimethyl sulfoxide (DMSO, maximum final
concentration b 0.5%) of the natural compounds, the semisynthetic de-
rivatives, and the combined culture medium and mycelium extract
2
+
were prepared for fluorescence, polarography, and Mg -ATPase assays
[20].
2.2. Chloroplast Isolation and Chlorophyll Determination
Spinach leaves (Spinacea oleracea) from a local market were used for
the isolation of intact chloroplasts following the procedure described by
Mills et al. (1980) at 4 °C under dark conditions. Once isolated, they
were re-suspended using a minimal volume of a solution with the fol-
+
2
lowing composition: MgCl (5 mM), KCl (10 mM), sucrose (400 mM),
2
. Materials and Methods
and pH 8.0 KOH-tricine buffer (30 mM). The concentrated chloroplasts
suspensions 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 as
reported by Strain and coworkers (1971) [25].
2
.1. Tested Materials
The phytotoxic compounds evaluated include three natural com-
pounds from the endophytic fungus X. feejeensis strain SM3e-1b isolated
from S. macrocarpum, and four semisynthetic derivatives. Additionally,
the combined culture medium and mycelium extract of X. feejeensis
was tested. The three natural compounds studied were the
epoxycyclohexenone derivative, (4S,5S,6S)-4-hydroxy-3-methoxy-5-
methyl-5,6- epoxycyclohex-2-enone or coriloxine (1), and two quinone
derivatives: 2-hydroxy-5-methoxy-3-methylcyclohexa-2,5-diene-1,4-
dione (2) and 2,6-hydroxy-5-methoxy-3-methylcyclohexa-2,5-diene-
2.3. Measurement of ATP Synthesis
An Orion microelectrode (Mod. 8103 Ross, 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 used
for the titrimetric determination of ATP synthesis [20,26,27]. For this
assay, intact chloroplasts (20 μg of Chl/mL) were ruptured in 3 mL of
2
an osmotic breaking solution: MgCl (5 mM), KCl (10 mM), KCN
O
O
HO
H
HO
OH
H
3
C
OCH
3
H
3
C
OCH
3
O
O
(
2)
O
(
3)
H
H
O
OCH
3
H C
3
HO
H
(1)
C
1
0
H
S
iO
1
3
N
l
3
2
H
2
InC
2
/
C
H
l
H
O
3
4
C
2
C
l
H
2
2
e
C
2
l
O
H
2
M
H
O
i
C
C
H
N
L
N
/
5
Cl
/
2
O
H
H
6
H
O
O
i
2
C
HN
H
H
S
HO
OCH
3
3
H C
HO H
H
3
C
OCH3
O
O
O
(
4)
HO
H
O
(7)
H
HN
HO
H
CH
3
H
3
C
NH
OCH3
3
H C
HO H
6)
(
5)
(
Fig. 1. Structures of phytotoxic secondary metabolites 1, 2 and 3 from Xylaria feejeensis strain SM3e-1; and experimental conditions for the preparation of compounds 4, 5, 6, and 7.

M.L. Macías-Rubalcava et al. / Journal of Photochemistry & Photobiology, B: Biology 166 (2017) 35–43
37
(
0.5 mM), sorbitol (100 mM), and pH 8.0 KOH-tricine buffer (1 mM).
The measurement was performed with adenosine diphosphate (ADP,
mM, pH 6.5), and methylviologen (MV, 50 μM) as an artificial electron
control consisted on the chloroplasts in the corresponding medium
without the evaluated compound.
1
acceptor; KOH (50 mM) was used to adjust the pH to 8.0. The organic
extract was evaluated in the concentration range of 50–300 μg/mL,
and the natural and semisynthetic derivatives in the range of 50–
2.6. Chlorophyll a Fluorescence
Spinach leaves were cut into 7 mm diameter discs; 15 of these circles
4
00 μg/mL. The synthesized ATP was calculated as μmol of ATP/mg of
were placed with 20 mL of modified Krebs solution into a Petri dish, the
Chl x h [26].
solution consisted of NaCl (115 mM), KCl (5.9 mM), MgCl
KH PO (1.2 mM), Na SO (1.2 mM), CaCl (25 mM), and NaHCO
25 mM, pH = 7.4). After 4 h of incubation at room temperature, pure
2
(1.2 mM),
2
4
2
4
2
3
(
2
.4. Noncyclic Electron Transport Determination
compounds were added in DMSO solutions at different concentrations
for each Petri dish and incubated for 4 additional hours; then, the
discs were dark adapted during 30 min. 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 for
illumination with 650 nm continuous light (gain 0.7, intensity
A Clark-type electrode connected to a biological oxygen monitor
(
Yellow Spring Instrument mod. 5300) was used for the polarographical
determination of the light-induced non-cyclic electron transport from
water to MV in 20 μg Chl/mL chloroplasts solutions. Basal electron trans-
port medium was prepared with MgCl
2
(5 mM), KCl (10 mM), KCN
−
2 −1
2
830 μmol photons m
s
). Maximum fluorescence yield from the
(
0.5 mM), sorbitol (100 mM), MV (50 μM) and pH 8.0 KOH-tricine buff-
sample was obtained by 2 s irradiation. DMSO was added to the control
experiments in order to consider the solvent effect; the volume of
DMSO added was equivalent to the one used to dissolve the analyzed
compound. OJIP transients were analyzed according to the JIP test
Strasser et al., 2004. Analysis of Chl a transients allowed us to determine
er (15 mM). This same reaction medium was used for the phosphorylat-
ing non-cyclic electron transport determinations but ADP (1 mM) and
KH
non-cyclic electron transport also used the same medium than for
basal transport with the addition of NH Cl (6 mM), an uncoupler
2 4
PO (3 mM) were added for the phosphorylation step. Uncoupled
4
the following parameters: (a) F
electron acceptor, Q , is fully oxidized, (b) F
level when Q is fully reduced, (c) fluorescence intensity level at 0.1,
M
.3, 2 (F ), and 30 ms, (d) the area over the curve between F and F ,
0
: fluorescence intensity level when the
agent [20,27–29]. Each of the measurements was carried out in 3 mL
of medium at 20 °C illuminating with a projector lamp for 2 min (GAF
A
M
: fluorescence intensity
2
A
2
669). Actinic light (0.2 mW/cm ) was obtained by filtering the light
0
J
0
4
from the projector through a 5 cm 1% CuSO solution. Actinic (blue)
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; the
area over the JI-transients is caused by electron transfer towards PSI
and a partial re-oxidation of the PQ-pool; finally, the area above the
IP-phase represents the reduction of the acceptor side of PSI; thus, the
total area measures the quantity of electron acceptors between the ac-
ceptor side of PSII and the acceptor side of PSI [38] (Table 2). Biolyzer
HP3 software was used for the determination of the derived parameters
listed in Table 3 (http://www.unige.ch/sciences/biologie/bioen, Labora-
tory University of Geneva, Prof. R. J. Strasse, software developer).
light has a wavelength of 420 nm that corresponds to the value where
chlorophyll a absorbs during the photosynthesis. All the compounds,
natural and semisynthetic, were evaluated in concentrations from 50
to 300 μg/L. For each assay, the negative control consisted on the chloro-
plasts in the corresponding reaction medium without the evaluated
compound [20].
2
.5. Photosystem II (PSII) and Photosystem I (PSI) Uncoupled Electron Flow
Determination
2
+
Uncoupled PSII electron flow from water 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-
2.7. Mg -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 the
chloroplasts. Then, they were centrifuged for 60 s at 3000g, washed
with grinding solution, and resuspended in HEPES buffer (35 mM,
pH 7.6) [24]. Light-triggered Mg2 -ATPase activity was determined as
described by Mills and collaborators, 1980 [39]. Released inorganic
phosphate (P) was measured as reported by Sumner, 1944 [40].
4
yl-1,4-benzoquinone (DBMIB, 1 μM) and NH Cl (6 mM), and without
MV, 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 DCBQ
flow, except by the addition of SiMo (50 μM) and 3-(3,4-
+
dichlorophenyl)-1,1-dimethylurea (DCMU, 10 μM, to inhibit PSII at Q
level), and the elimination of DBMIB, using chloroplasts containing
B
2
0 μg Chl/mL [20,31–32].
2.8. Statistical Analysis
Thylakoids were treated with hydroxymethyl aminomethane (Tris,
.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 water
splitting enzyme (OEC) [33–35].
The uncoupled PSI electron transport from the reduced DCPIP to MV
was also determined in illuminated chloroplasts (20 μg Chl/mL) using
0
Experimental results regarding the effect of the extract and the
seven pure compounds on different photosynthetic activities were ana-
lyzed with GraphPad Prism statistical software (ver 5.01) by Tukey and
ANOVA tests [41]. All the data are normally distributed and variances
are homogeneous. For each bioassay, a Probit analysis was performed
to calculate the IC50 value (50% inhibitory concentration) [20,42]. Data
are represented as mean ± standard deviation (SD). Statistical signifi-
cance was considered for a P value of ≤0.05.
(
3
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 NH Cl (6 mM) were added [20,36].
3. Results and Discussion
4
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 to
monitor electron flow activities. Pure compounds were tested in the
concentration range of 50–300 μg/L. For each reaction, the negative
The endophytic fungus X. feejeensis strain SM3e-1b was isolated
from symptomless healthy leaves of S. macrocarpum, collected at
REBIOSH. We recently reported significant phytotoxic activity on seed
germination, root elongation, and seedling respiration of dicotyledon-
ous and monocotyledonous species, by the three major secondary

38
M.L. Macías-Rubalcava et al. / Journal of Photochemistry & Photobiology, B: Biology 166 (2017) 35–43
metabolites produced by X. feejeensis (1–3); four synthetic derivatives
of coriloxine (4–7) (Fig. 1); and the combined culture medium and my-
celium extract from the fungus [24]. In order to obtain a larger amount
of 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-substrate
fermentation on potato dextrose broth (PDB). The mycelium and cul-
A
100
2 2
ture medium were extracted with CH Cl and EtOAc. The combined ex-
80
60
40
20
0
tract exhibited significant phytotoxic activity on seed germination, root
elongation, and seedling respiration of A. hypochondriacus, T. pretense,
M. sativa, and P. miliaceum using a Petri dish bioassay (data not
shown). Bioassay-guided fractionation of the active extract led to the
isolation of three known phytotoxic compounds: coriloxine (1), and
two quinone derivatives, compounds 2 and 3. The yields of the three
secondary metabolites produced by endophytic fungus X. feejeensis
A 180
0
50
100 150 200 250 300
Total Extract [µg/mL]
1
1
1
1
60
40
20
00
B
1
1
1
40
20
00
8
6
4
2
0
0
0
0
0
8
6
4
0
0
0
0
100
200
300
Compound 4 [µM]
B
0
100
200
300
400
100
Compounds [µM]
C
100
80
60
80
60
40
20
0
4
0
0
0
2
0
50
100 150 200 250 300
Compound 7 [ M]
µ
0
100
200
300
400
Fig. 3. Effect of increasing concentrations of semisynthetic derivatives from Xylaria
feejeensis 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 ( ), and
uncoupled ( ) rates of electron transport. Control average value was 460, 600 and
1050 μequiv e−/mg Chl × h for basal, phosphorylating and uncoupled electron flow
respectively. Control is represented as 100% of activity. Each point represents the mean
of three determinations. Each repetition was made in different batches of chloroplasts.
Vertical bars represent standard deviations. The inhibitory effect of semisynthetic
derivative 4 on electron transport rate from water to MV on freshly lysed illuminated
spinach chloroplasts, in the concentration range 25–300 μg/mL, was statistically
significant (P b 0.05).
Compounds [µM]
Fig. 2. Effect of increasing concentrations of organic extract (Panel A); phytotoxic
secondary metabolites: coriloxine 1 ( ), compound 2 ( ), fumiquinone B 3 ( ) (Panel
B); and semisynthetic derivatives: compounds 4 ( ) and 7 ( ) (Panel C) from Xylaria
feejeensis strain SM3e-1b on ATP synthesis rate on spinach thylakoids. Control average
value as 100% of activity was 900 ± 1.5 μM ATP/ mg Chl × h. Each point represents the
mean of three determinations. Each repetition was made in different batches of
chloroplasts. Vertical bars represent standard deviations. The effect of semisynthetic
derivatives 4 and 7 on photophosphorylation, was statistically significant (P b 0.05).

M.L. Macías-Rubalcava et al. / Journal of Photochemistry & Photobiology, B: Biology 166 (2017) 35–43
39
Table 1
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.
Effect of (4R,5S,6R)-6-chloro-4,5-dihydroxy-3-methoxy-5-methylcyclohex-2-enone (4)
and NH
(
2
+
4
Cl on Mg -ATPase bound to thylakoid membranes.
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 6 were poorly active or inactive compared to derivatives
4 and 7, they were not further evaluated; our attention was centered on
investigating the mechanism of action and inhibition site of compounds
μM Pi mg of Chl− h−1
1
Activity %
Treatments
Compound 4
(
μM)
0
1
2
3
226 ± 23
389 ± 80
407 ± 90
425 ± 92
100
172
180
188
00
00
00
4
and 7.
NH
4
Cl
3.2. Effect of Coriloxine Derivatives 4 and 7 on Electron Transport Rate from
Water to MV on Freshly Lysed Illuminated Spinach Chloroplasts
(mM)
0
1
2
3
226 ± 23
341 ± 186
418 ± 181
409 ± 141
100
151
185
181
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. To
elucidate the inhibition mechanism of synthetic derivatives 4 and 7,
the effect on the non-cyclic electron transport from water to MV was in-
vestigated under basal, phosphorylating, and uncoupled conditions.
Coriloxine derivative 4 enhanced phosphorylating electron transfer
by 160% at 100 μM (Fig. 3 A). Contrastingly, basal and uncoupled photo-
synthetic electron transport was partially inhibited at 300 μM reducing
it 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 the
evaluated conditions in a concentration dependent manner (Fig. 3 B). It
was very active inhibiting the uncoupled electron transport rate, with
an IC50 value of 17.5 μM. Basal and phosphorylating electron transport
rate was less affected by compound 7, with IC50 values of 50.5 and
71.7 μM, respectively. Since photophosphorylation and the electron
transport rate were inhibited, these results indicate that 7 behaves as
a Hill reaction inhibitor.
Each point represents the mean of five determinations. Each repetition was made in differ-
ent batches of chloroplasts.
strain SM3e-1b isolated from S. macrocarpum were very similar to those
reported in our previous work [24].
Compounds 1–3 were identified by TLC, using authentic samples as
references, with different gradient elution systems [24]. Furthermore,
the spectral properties of the compounds, including H and C NMR
data, were identical to those previously described in the literature [24,
3–45].
On the other hand, coriloxine derivatives, compounds 4, 5, 6, and 7
1
13
4
(
(
Fig. 1), were prepared as previously reported by García-Méndez, et al.
2016) [24]. Briefly, compound 4 was obtained by reacting coriloxine
with indium(III) chloride in methanol solution with a catalytic amount
of SiO . Treatment of coriloxine with an aqueous solution of methyl-
amine in presence of a catalytic amount of LiClO produced compound
. For compounds 6 and 7, a catalytic amount of SiO 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 the
less sterically hindered carbon atom.
Additionally, in this work we evaluated the ability of the natural
compounds 1–3 and synthetic derivatives of coriloxine (4–7) to inhibit
different reactions of the photosynthetic electron transport chain and
2
4
5
2
2
+
3.3. Coriloxine Derivative 4 on Mg -ATPase Activity
To corroborate the uncoupling behavior of the synthetic derivative 4,
its effect on the light-activated Mg2 -ATPase bound to thylakoid mem-
branes was determined for increasing concentrations of 4, from 100 to
+
300 μM. Some classical uncoupler agents, such as NH
4
Cl, stimulate the
2
+
2+
the Mg -ATPase activity; also, Chl a fluorescence measurements
were performed.
activity of the Mg -ATPase, so they can be used as positive controls
[46]. As shown in Table 1, both 4 and NH
4
Cl enhanced the Mg2 -ATPase
+
activity. These results indicate that compound 4 affected the pH gradi-
3
.1. Effect of the Organic Extract, Natural Compounds 1–3, and Semisyn-
ent, thus preventing ATP synthesis (Fig. 2C), as uncoupler agents do.
thetic Derivatives 4–7 on Photophosphorylation
3.4. Determination of Target Sites of Coriloxine Derivative 7
The phytotoxic organic extract from X. feejeensis strain SM3e-1b
showed inhibitory effect in a concentration-dependent way on the
ATP synthesis, from water to MV in freshly lysed spinach thylakoids
The effect of compound 7 on PSII and PSI and on the partial reactions
was evaluated using different combinations of artificial electron donors
and acceptors, and the appropriate inhibitors (Fig. 4) [20,36]. This
would allow the assessment of the action site of 7 on the thylakoid elec-
tron transport chain.
Uncoupled PSI electron transport from reduced DCPIP to MV was not
significantly affected, with inhibition averaging around 17% for all of the
evaluated concentrations (50–300 μM). Contrastingly, the uncoupled
PSII electron flow from water to DCBQ was strongly affected; and the
partial reactions, from water to SiMo, and from DPC to DCPIP, were
inhibited in a concentration-dependent manner (Fig. 5). The most
(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 (maximal
concentration tested) (Fig. 2B). Compound 3 did not show an inhibitory
effect on ATP formation. These results suggest that the inhibitory activ-
ity on ATP synthesis of the organic extract might be due to the mixture
of 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 2
Experimental average values and standard deviations of Chlorophyll a fluorescence obtained in control and in S. oleracea discs leaves at different times after treatment with 2-((4-
butylphenyl)amino)-5-methoxy-3-methylcyclohexa-2,5-diene-1,4-dione (7).
Compound
[
μM]
F
0
F
M
F
(0.1 ms)
F
(0.3 ms)
F
(2 ms)
F
(30 ms)
Area
Control
221 ± 8.6
212 ± 12.8
231 ± 30.6
242 ± 34.6
1116 ± 50.0
891 ± 106
820 ± 72.8
933 ± 70.1
252 ± 12.9
249 ± 26.8
247 ± 45.0
306 ± 53.9
318 ± 19.1
314 ± 61.7
401 ± 66.7
423 ± 73.2
501 ± 22.6
472 ± 151.4
651 ± 82.9
680 ± 107.6
838 ± 45.8
628 ± 243.6
685 ± 64.2
777 ± 73.9
49,930 ± 689
31,400 ± 1199
19,300 ± 556
29,520 ± 887
1
2
3
00
00
00

40
M.L. Macías-Rubalcava et al. / Journal of Photochemistry & Photobiology, B: Biology 166 (2017) 35–43
1
2
SiMo
DCBQ
H 0
OEC
Z
P680
Ph
QA
Fe
QB
PQH2
2
DCMU
3
Tris
DPC
DCPIP
Fig. 4. Scheme of PSII electron transport model. Arrows show the sites of electron donation and acceptance; broken (dashed) lines indicate sites of inhibition of commercial inhibitors. PS II
measured from water to DCBQ (1). PSII partial reactions measured from water to SiMo (2) and from DPC to DCPIP using Tris treated chloroplasts (3).
pronounced inhibitory effect was on the uncoupled PSII from water to
DCBQ, 83% at 300 μM. (Fig. 5), also the span of electron transport from
water to SiMo was significantly inhibited, 76% at 300 μM. SiMo accepts
3.5. PSII Chlorophyll a Fluorescence Measurements in Spinach Leaf Discs
To confirm the polarography results concerning the interaction site
of 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 control
and 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 emerged
near 0.3 ms (Fig. 6B). Samples treated with DCMU show a single induc-
electrons at the Q
the PSII at the span of electron transport from water to Q
flow measured from DPC (donates electrons at P680) to DCPIPox (accepts
electrons at Q site), was partially inhibited by 7 at all of the evaluated
concentrations, electron transport from P680 to Q was reduced by 57%
A
site, thus, it can be inferred that compound 7 inhibits
A
. The electron
B
B
at 300 μM (maximum concentration evaluated) (Fig. 5).
These results suggest that semisynthetic derivative 7 interacts at the
water splitting enzyme complex (oxygen evolving complex, OEC) and
tion phase, the F
electron transfer between the primary and the secondary quinone ac-
ceptor (Q and Q ) at the D1 protein of PSII, and an accumulation of
takes place. The redox state of Q
M
is reached after nearly 2 ms (J-step). DCMU blocks the
A
through the electron transport chain between P680 and Q [47]. Since
uncoupled PSI electron transport from the reduced DCPIP to MV was
not significantly affected, the semisynthetic derivative 7 can be classi-
fied as a PSII inhibitor.
A
B
−
−
Q
A
A
is responsible of the fluorescence
increase [46]. The reduction of P680 by Y is much faster (20 ns) than the
electron transfer (200 μs) from Q to Q [47]; however the presence of a
K-band indicates that the electron transfer from P680 to Q without the
direct participation of OEC leads to the accumulation of P680 due to the
Z
decreased on the reduction of Y ; in other words, the electron flow be-
+
Z
A
B
A
+
+
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-
1
00
8
6
4
2
0
0
0
0
0
A
tially inhibits the acceptor site from P680 to Q .
In addition to these results, different photosynthetic parameters of
Chl a fluorescence kinetics were calculated to build Fig. 7 (Table 3). Spe-
cific fluxes such as: the effective antenna size (ABS/RC) and the maximal
rate by which an exciton is trapped by the reaction center resulting in
−
the reduction of Q
A
to Q
A
(TRo/RC) increased 25 and 40% respectively
at 300 μM of semisynthetic derivative 7. The non-photochemical de-ex-
citation rate constant (Kn), the quantum yield of energy dissipation at
t = 0 (ϕDo), and the efficiency with which an electron moves to the
PSI end electron acceptors (δRo) from the reduced intersystem electron
acceptors, also increased 40, 50 and 75%, respectively. The value of the
0
50
100 150 200 250 300
0
specific activity of primary photochemistry (M ) increased N100%.
Compound 7 [µM]
However, parameters related with electron transport, such as ETo/RC
and ETo/CS, the probability that a trapped exciton moves an electron
A
into the electron transport chain beyond Q (ψEo), and the quantum
Fig. 5. Effect of increasing concentrations of semisynthetic derivative 2-((4-
butylphenyl)amino)-5-methoxy-3-methylcyclohexa-2,5-diene-1,4-dione (7) on
uncoupled electron transport on PSI measured from reduced DCPIP to MV ( );
yield for electron transport at t = 0 (ϕ(Eo)), decreased at all the evaluat-
ed concentrations of 7; at 300 μM (highest concentration used), these
parameters decreased by 50%. The performance index (PI(ABS)) showed
the highest decrease. Other parameters, such as the quantum yield for
the reduction of end acceptors of PSI per photon absorbed (ϕ(Ro)), and
the normalized total complementary area above the O-J-I-P transient
uncoupled electron transport on PSII measured from H
uncoupled electron transport on partial reactions of PSII from H
from DPC to DCPIP ( ). Control average value as 100% of activity was 1234, 360 and
29 μequiv e−/mg Chl × h for DCPIP to MV, H
O to DCBQ and from H O to SiMo
2
O to DCBQ ( ); and on the
2
O to SiMo ( ), and
2
2
2
respectively. Control average for DPC to DCPIP was 125 μM of reduced DCPIP/mg
Chl × h. Each point represents the mean of three determinations. Each repetition was
made in different batches of chloroplasts. Vertical bars represent standard deviations.
The inhibitory effect of semisynthetic derivative 7 on electron transport rate on
uncoupled PSII on freshly lysed illuminated spinach chloroplasts, in the concentration
range 50–300 μg/mL, was statistically significant (P b 0.05).
(
Sm), showed a slight increase at 300 μM of 7.
These results suggest that the semisynthetic derivative 7 acts as a
Hill reaction inhibitor at the PSII electron transport on OEC and on
the acceptor side between P680 and Q . A similar behavior has been
A

M.L. Macías-Rubalcava et al. / Journal of Photochemistry & Photobiology, B: Biology 166 (2017) 35–43
41
A
B
J-Band
K-Band
0
0
0
0
0
.4
.3
.2
.1
0.075
0.050
0.025
.0
0.000
0.01
0.1
1
10
100
1000
0.5
1.0
1.5
2.0
2.5
Time (ms)
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 ( ), and
00 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 the
V(t) of the sample minus the V(t) of the control, where V is the relative variable fluorescence at time (t), between F and F ; Vt = FVt/(F − F ) = ( F − F )/(F − F ). 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 F and F ; Wt = V0 J(t) = FVt/
− F ) = (F − F )/(F − F ). Each curve represents the average of ten replicates.
3
0
M
M
0
t
0
M
0
0
J
(F
J
0
t
0
J
0
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]. The
naphtoquinonepiroketals from endophytic fungus Edenia gomezpompae
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 continuous
mutual antagonism, in part based on the secondary metabolites
that both organisms produce, none of them is damaged because
there is an equilibrium between the endophytic virulence factors
and the host defense response, or balanced antagonism. Disease to
the host plant or fungus death may arise only if this balanced antag-
onism is broken by factors such as the host senescence or biotic and
abiotic stress [52,12]. In fact, it is known that endophytic fungi play a
key role increasing the uptake of nutrients and the tolerance to biotic
and abiotic stress of the host plant, as well as protecting it against the
attack of herbivores, insects and pathogenic microorganisms [53].
Furthermore, it has been observed that plants completely devoid of
endophytes show reduced photosynthetic rates and chlorosis; there-
fore, lower survival rates in nature [54].
Finally, to determine whether the secondary metabolites and
semisynthetic derivatives from endophytic fungus X. feejeensis strain
SM3e-1b satisfy the requirements established for the “Lipinski's rule
of 5” Lipinski, 1997 [55] and Tice's rule Tice 2001 [56], the physico-
chemical parameters of all the compounds were calculated using
the Chemicalize software package (ChemAxon. http://www.
chemicalize.org/). “Lipinski's rule of 5” is a set of empirically derived
rules that outline the molecular characteristics and the physico-
chemical properties of orally bioavailable drugs. Molecules violating
more than one of these rules exhibit limited bioavailability. Based on
the approach by Lipinski, Tice established a set of rules that can be
applied to molecules of agrochemical interest [56,57]. Calculated
physicochemical parameters such as molecular weight, lipophilicity,
number of rotatable bonds, number of hydrogen-bond donors, and
number of hydrogen-bond acceptors, are encompassed by “Tice's
rules” for screening new herbicide candidates [56,57]. As shown in
Table 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 properties
appropriate for their use as herbicides [56].
isolated from Callicarpa acuminata, preussomerins EG
1 4
and EG and
palmarumycins CP17 and CP , also inhibited the partial reactions of
2
PSII electron flow in a similar way [20]. Therefore, the inhibition site
of all of these compounds, naphthoquinonepiroketals, depsides and
compound 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 to
Diuron (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 evidence
of damage to the plant despite 4 and 7 effects on photosynthesis. As
part of the virulence factors implicated in the colonization process to
4. Conclusions
Fig. 7. Spider plot showing the parameters calculated from the OJIP transients obtained
In this study, we demonstrated that two semisynthetic derivatives of
from fluorescence experiments on the leaves discs in presence of 200 (
) and
coriloxine are more potent as inhibitors of the photosynthetic electron
transport in isolated spinach chloroplasts than the three secondary me-
tabolites from Xylaria feejeensis strain SM3e-1b isolated from S.
3
3
(
00 μM (
) of the semisynthetic derivative 2-((4-butylphenyl)amino)-5-methoxy-
-methylcyclohexa-2,5-diene-1,4-dione (7), compared with the control DCMU
). Each curve represents the average of ten replicates.

42
M.L. Macías-Rubalcava et al. / Journal of Photochemistry & Photobiology, B: Biology 166 (2017) 35–43
Table 3
Derived parameters, their description and formulae, using data extracted from the Chl a fluorescence (OJIP) transient.
Fluorescence parameters derived from the extracted data
Approximated initial slope (in ms− ) of the fluorescence transient V = f(t)
1
M
0
= dV/dt = 4(F300 μs − F
0
)/(F
M
0
− F )
Sm = (Area)/(F − F
M
0
)
Normalized total complementary area above the O-J-I-P transient (reflecting multiple turnover Q
A
reduction events)
Yields or flux ratios
φ
φ
ψ
φ
Po = TR
Eo = ET
Eo = ET
Do = 1 − φPo = (F
δR = RE /ET = (1 − V
0
/ABS = [1 − F
/ABS = [1 − (F
/TR = (1 − V
/F
0
/F
M
]
Maximum quantum yield of primary photochemistry at t = 0
Quantum yield for electron transport at t = 0
Probability (at t = 0) that a trapped exciton moves an electron into the electron transport chain beyond Q
Quantum yield (at t = 0) of energy dissipation
0
J
/F
M
)]
−
0
0
J
)
)
A
0
M
0
0
0
I
)/(1 − V
J
)
Efficiency with which an electron can move from the reduced intersystem electron acceptors to the PS I end
electron acceptors of PS I
φR
RE
0
= RE
/TR = ψEo × δR
0
0
/ABS = φPo ∗ ψEo ∗ δR
0
= 1 − (F
I
M
/F )
Quantum yield for the reduction of end acceptors of PSI per photon absorbed
Efficiency with which a trapped exciton move an electron into the electron transport chain from Q
−
0
0
A
to the PS I end
electron acceptors
Specific energy fluxes or activities (per reaction Q−
ABS/RC = M (1/V )(1/φPo
TR /RC = M /V
ETo/RC = M (1/V
reducing PS II reaction center - RC)
A
0
J
)
Absorption per RC
Trapped energy flux per RC (at t = 0)
Electron transport flux per RC (at t = 0)
0
0
J
0
J
)(ψEo)
Phenomenological energy fluxes (per excited cross section – CS)
ABS/CS
TR /CS
ET /CS
0
Absorption flux per CS
Trapped energy flux per CS (at t = 0)
Electron transport flux per CS (at t = 0)
0
0
0
0
De-excitation rate constants
Kp
Photochemical de-excitation rate constant
Kn
Sum K
Non photochemical de-excitation rate constant
The sum of photochemical and non photochemical rate constants
Performance index
RC
TRo
ETo
Performance index on absorption basis
PI(ABS)
=
Ã
Ã
ABS ABS−TRo TRo−ETo
Table 4
Requirements established for “Lipinski's rule of 5” and Tice's parameters calculated for the phytotoxic secondary metabolites 1–3 and semisynthetic derivatives 4–7 from Xylaria feejeensis
strain SM3e-1b.
Compounds Molecular weight Calculated lipophilicity (LogP) Number of rotatable bonds Number of hydrogen-bond donors Number of hydrogen-bond acceptors
1
2
3
4
5
6
7
170
168
184
206
157
263
299
≤500
150–500
−0.49
0.50
−0.02
−0.30
0.18
0.31
3.26
≤5
1
1
1
1
1
3
6
1
1
2
2
2
3
2
≤5
≤3
4
4
4
4
4
5
5
Lipinski's
Tice
≤10
2–12
≤5
≤12
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 and
partly affected basal and uncoupled photosynthetic electron transport
rates. This compound also enhanced the activity of Mg2 -ATPase, cor-
roborating its action as uncoupler. On the other hand, quinone
derivative 2-((4-butylphenyl)amino)-5-methoxy-3-methylcyclohexa-
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 y
Tecnología grant 179194. We want to thank M. S. Elizabeth K. Galván
Miranda from Facultad de Química, UNAM, for language revision.
Marbella García acknowledges the fellowship awarded by CONACyT to
carry out her graduate studies.
2
,5-diene-1,4-dione (7) acts as a Hill reaction inhibitor at the electron
transport on the water splitting enzyme (OEC), and on the acceptor
side (between P680 and Q ), similarly to the commercial herbicide
A
DCMU. Chlorophyll a fluorescence measurements corroborated this
mechanism 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 new
herbicides potentially useful in agriculture.
Based on the results from this investigation, it is clear that the mech-
anism of action associated with the phytotoxic effects of the three natu-
ral products 1–3 and the semisynthetic derivatives 5 and 6 from
endophytic fungus Xylaria feejeensis strain SM3e-1b, isolated from S.
macrocarpum is not on the luminous phase of the photosynthesis, so
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