A model of the molecular mechanism of violaxanthin de-epoxidation where the reversed hexagonal structures mainly created by monogalactosyldiacylglycerol are assumed to be required for vi
Trang 1Kinetics of violaxanthin de-epoxidation by violaxanthin de-epoxidase,
a xanthophyll cycle enzyme, is regulated by membrane fluidity
in model lipid bilayers
Dariusz Latowski1, Jerzy Kruk1, Kvetoslava Burda2, Marta Skrzynecka-Jaskier1, Anna Kostecka-Gugała1 and Kazimierz Strzałka1
1
Department of Plant Physiology and Biochemistry, The Jan Zurzycki Institute of Molecular Biology and Biotechnology,
Jagiellonian University, Krako´w, Poland;2H Niewodniczanski Institute of Nuclear Physics, Krako´w, Poland
This paper describes violaxanthin de-epoxidation in model
lipid bilayers Unilamellar egg yolk phosphatidylcholine
(PtdCho) vesicles supplemented with
monogalactosyldi-acylglycerol were found to be a suitable system for studying
this reaction Such a system resembles more the native
thylakoid membrane and offers better possibilities for
studying kinetics and factors controlling de-epoxidation of
violaxanthin than a system composed only of
monogalacto-syldiacylglycerol and is commonly used in xanthophyll cycle
studies The activity of violaxanthin de-epoxidase (VDE)
strongly depended on the ratio of
monogalactosyldiacyl-glycerol to PtdCho in liposomes The mathematical model of
violaxanthin de-epoxidation was applied to calculate the
probability of violaxanthin to zeaxanthin conversion at
different phases of de-epoxidation reactions Measurements
of deepoxidation rate and EPR-spin label study at different
temperatures revealed that dynamic properties of the
membrane are important factors that might control con-version of violaxanthin to antheraxanthin A model of the molecular mechanism of violaxanthin de-epoxidation where the reversed hexagonal structures (mainly created by monogalactosyldiacylglycerol) are assumed to be required for violaxanthin conversion to zeaxanthin is proposed The presence of monogalactosyldiacylglycerol reversed hexa-gonal phase was detected in the PtdCho/monogalactosyl-diacylglycerol liposomes membrane by31P-NMR studies The availability of violaxanthin for de-epoxidation is a dif-fusion-dependent process controlled by membrane fluidity The significance of the presented results for understanding the mechanism of violaxanthin de-epoxidation in native thylakoid membranes is discussed
Keywords: xanthophyll cycle; de-epoxidation; liposomes; violaxanthin; zeaxanthin
Xanthophyll cycle is a photoprotective mechanism
wide-spread in nature operating in the thylakoid membranes of
all higher plants, ferns, mosses and several algal groups [1]
This cycle involves two reversible reactions, light-dependent
de-epoxidation of violaxanthin to zeaxanthin via
anther-axanthin as an intermediate and light-independent
epoxi-dation of zeaxanthin to anteraxanthin and violaxanthin [2]
The conversion of violaxanthin to zeaxanthin is catalysed by
violaxanthin de-epoxidase (VDE) and the reverse reaction
of violaxanthin formation from zeaxanthin is catalysed by
another enzyme, zeaxanthin epoxidase VDE has been isolated from spinach and lettuce chloroplasts and the molecular mass of the native enzyme w as estimated as
43 kDa [3–5] The gene encoding VDE has been already isolated and cloned [6] VDE is located on the lumenal side
of the thylakoid membrane, shows an optimum activity at
pH 4.8 when present in chloroplasts and at 5.2 for the isolated enzyme [7] and requires ascorbate as a reductant [8]
In the dark, when the pH in thylakoid lumen is neutral or alkaline, VDE is inactive, whereas under strong light conditions, pH in the thylakoid lumen decreases, the enzyme binds to the membrane, becomes active and converts violaxanthin to zeaxanthin [8,9] The inhibition of the enzyme activity by zeaxanthin has been reported [3] For optimal activity, VDE requires the presence of monogal-actosyldiacylglycerol, the major lipid of the thylakoid membrane [10–12] With its small head-group area and critical packing parameter value superior to one, monogal-actosyldiacylglycerol in water forms reversed hexagonal phase instead of bilayer structures [13] It is known that monogalactosyldiacylglycerol forms hexagonal phases over
a wide temperature range of)15 C to 80 C at concentra-tions higher than 50% lipid in water and this process also depends on the degree of unsaturation of the acyl chains Until now, all in vitro studies on the VDE activity have been carried out using largely undefined systems of buffered suspension of monogalactosyldiacylglycerol aggregates containing violaxanthin as substrate Here, we present a
Correspondence to K Strzalka, Department of Plant Physiology
and Biochemistry, The Jan Zurzycki Institute of Molecular Biology
and Biotechnology, Jagiellonian University, ul Gronostajowa 7,
30-387 Krako´w, Poland.
Fax: + 48 12 252 69 02, Tel.: + 48 12 252 65 09,
E-mail: strzalka@awe.mol.uj.edu.pl
Abbreviations: LHC, light harvesting complex; PSI, photosystem I;
PSII, photosystem II; VDE, violaxanthin de-epoxidase; PtdCho,
phosphatidylcholine; PtdGro, phosphatidylglycerol; VA, probability
of violaxanthin to antheraxanthin conversion; AZ, probability of
antheraxanthin to zeaxanthin conversion; VV, probability that
violaxanthin remains violaxanthin; AA, probability that
anthera-xanthin remains antheraanthera-xanthin; ZZ, probability that zeaanthera-xanthin
remains zeaxanthin; S VA , the constant rate of VA 0 decrease;
S AZ , the constant rate of AZ 0 decrease.
(Received 12 April 2002, revised 18 July 2002, accepted 5 August 2002)
Trang 2newapproach in the study of VDE activity employing
violaxanthin-containing liposomes as an experimental
sys-tem, which is a closer to the native thylakoid membrane
The use of lipid bilayers instead of
monogalactosyldiacyl-glycerol aggregates offers newpossibilities in the
investiga-tion of the kinetic parameters and mechanism of
violaxanthin de-epoxidation One of the advantages of such
system is the defined orientation of violaxanthin molecules
in the lipid bilayer, which, according to various sources [14–
16], is perpendicular to the plane of the membrane
Violaxanthin-supplemented unilamellar liposomes with
VDE present only outside the vesicles are also a good
system to study the flip-flop rate of antheraxanthin which is
probably a necessary step preceding zeaxanthin formation
in membrane Additionally, experiments carried out at
different temperatures and application of a mathematical
model of de-epoxidation for the analysis of the obtained
results provide important information on the influence of
membrane physical properties, kinetic parameters of
vio-laxanthin into zeaxanthin conversion and flip-flop rate of
antheraxanthin A possible molecular mechanism of
vio-laxanthin de-epoxidation is proposed
M A T E R I A L S A N D M E T H O D S
Preparation of unilamellar liposomes
The mixture of lipids with violaxanthin in chloroform was
evaporated under stream of nitrogen to form a thin film and
dried under vacuum for 1 h The dried lipids were dissolved
in ethanol and the solution was injected slowly with a
Hamilton syringe into 0.1Msodium citrate buffer, pH 5.1,
under continuous bubbling with nitrogen The final ethanol
concentration did not exceed 1.25% Subsequently, the
liposome suspension was extruded through a polycarbonate
membrane with a pore diameter of 100 nm [17] The final
lipid concentration in a liposome suspension was 43 lMand
violaxanthin concentration was 0.33 lM
Egg yolk phosphatidylcholine (PtdCho) was purchased
from Sigma (P2772) and plant
monogalactosyldiacylgly-cerol was obtained from Lipid Products
Electron microscopy
One drop of PtdCho/monogalactosyldiacylglycerol
lipo-somes (350 lM lipid concentration) or
monogalactosyldi-acylglycerol structures (12.9 lM lipid concentration) in
citrate buffer (pH 5.1) was placed on a Formvar coated
grid and after 30 s one drop of staining solution was added
Negative staining was performed with uranyl acetate at
room temperature [18] After 30 s, excess solution was
drained off with filter paper and the grid was allowed to dry
in the air The grids were examined in a JEM 100SX
electron microscope operated at 80 kV
Photon correlation spectroscopy (PCS) analysis
Diameter of PtdCho/monogalactosyldiacylglycerol
lipo-somes and monogalactosyldiacylglycerol reversed
hexa-gonal phase was measured by PCS analysis The 10 mW
He-Ne laser (633 nm) was used as a light source The
selected angle was 90, the viscosity was 0.890 centipoise and
refractive index 1.333 All analyses were performed at 25C
and at the equilibration time of 2 min Total lipid concen-tration in the case of PtdCho/monogalactosyldiacylgly-cerol liposomes was 43 lM (30.1 lM PtdCho, 12.9 lM monogalactosyldiacylglycerol) and 12.9 lM for monogal-actosyldiacylglycerol structures Both liposomes and monogalactosyldiacylglycerol reversed hexagonal phase were suspended in 0.1Msodium citrate buffer (pH 5.1)
Isolation of violaxanthin Violaxanthin was isolated from dark-stored leaves of lucerne (Medicago sativa) by pigment extraction with acetone, saponification of the lipid extract [19], followed
by column chromatography on Silica Gel F254 (Merck) in petroleum ether : acetone (4 : 1, v/v)
Isolation and purification of VDE VDE was isolated and purified from 7-day-old wheat leaves grown at 28C according to the method described by Hager and Holocher [9] Additionally, the enzyme was purified by gel filtration on Sephadex G100 The gel electrophoretic and ion-exchange chromatography analysis of VDE preparation showed two other minor proteins apart from VDE (data not shown) The enzyme activity was determined by dual-wavelength measurements (502–540 nm) using DW-2000 SLM Aminco spectrophotometer at 25C according to Yamamoto [20] The reaction mixture contained 0.33 lM violaxanthin, 12.9 lM monogalactosyldiacylglycerol and
30 mM sodium ascorbate in 0.1 M sodium citrate buffer (pH 5.1)
Measurement of violaxanthin de-epoxidation De-epoxidation of violaxanthin was measured at 4, 12 and
25C both in a monogalactosyldiacylglycerol reversed hexagonal phase and in liposomes The composition of the reaction mixture of the monogalactosyldiacylglycerol system w as the same as that used for the enzyme activity determination The liposomes (30.1 lM PtdCho, 12.9 lM monogalactosyldiacylglycerol, 0.33 lM violaxanthin) were prepared in 30 mMsodium ascorbate, 0.1Msodium citrate buffer (pH 5.1) In another series of experiments, liposomes with constant concentration of monogalactosyldiacylgly-cerol (12.9 lM) and violaxanthin (0.33 lM) w ere used and PtdCho was changed in order to obtain following mono-galactosyldiacylglycerol proportions: 5 mol%, 15 mol% and 30 mol%
All mixtures were placed in darkness and gently stirred The de-epoxidation reaction was initiated by addition of saturating amount of VDE, the activity of which correspon-ded to 4 nmol de-epoxidated violaxanthin per min per mL The reaction was terminated and pigments were extracted
by mixing 750 lL of the reaction medium with 750 lL of the extraction solution containing chloroform/methanol/ ammonia (1 : 2 : 0.004, v/v/v) Xanthophyll pigments were extracted by vigorous shaking and centrifugation for 10 min
at 10 000 g in Micro-Centrifuge Type-320 After centrifu-gation, the chloroform fraction (200 lL) was evaporated to dryness under stream of nitrogen Subsequently, pigments were dissolved in 50 lL tetrahydrofuran and 550 lL of the following solvent mixture, acetonitrile/methanol/water (360 : 40 : 40, v/v/v)
Trang 3Pigment separation was performed by reverse phase
HPLC using a RP-18 column, 5 lm particle size, according
to the modified method of Gilmore and Yamamoto [21] at
the flowrate of 3 mLÆmin)1 The eluted pigments were
monitored at 440 nm and quantitatively determined
Analysis of de-epoxidation kinetics
We have applied a newmathematical model [22] to
analyse the kinetics of conversion of violaxanthin to
zeaxanthin The model allowed us to follow independently
the kinetics of the two de-epoxidation steps: the
conver-sion of violaxanthin into antheraxanthin (VfiA) with a
probability VA and antheraxanthin into zeaxanthin
(AfiZ) with a probability AZ It is known from
experimental data that these two steps reach equilibrium
It means that the parameters VA and AZ must vanish In
the model w e have assumed a linear decrease of the
conversion probabilities:
VA ¼ VA0 nðSVADtÞ for VA > 0
AZ ¼ AZ0 nðSAZDtÞ for AZ > 0
where VA0and AZ0are the initial values of the conversion
probabilities, SVAand SAZare the constant rates and nÆDt is
the time of reaction (Dt ¼ a constant time interval,
n¼ number of time intervals)
Measurement of the order parameter in liposome
membrane
Temperature dependent changes of the order parameter of
lipid fatty acyl chains in liposome membranes were recorded
by EPR spin label measurements using a spin label
5-doxyl-stearic acid reporting on dynamics of membrane regions
close to the headgroup area The spin label was added to the
chloroform mixture of monogalactosyldiacylglycerol,
Ptd-Cho and V, dried under stream of nitrogen and stored under
vacuum for 1 h After this time, the dried mixture was
suspended in 0.1 M sodium citrate buffer pH 5.2 by
vortexing The final concentration of 5-doxyl-stearic acid
was 10)4M The final concentration of lipids was 10)2M
and their proportions were the same as in the section on
Preparation of unilamellar liposomes EPR spectra of the
spin label as a function of temperature were recorded using
a Bruker ESP-300E spectrometer fitted with TM110cavity
The modulation amplitude was 1 G, microwave power was
2 or 8 mW The measurements were performed within the
temperature range of 0–40C All measurements were
performed in a heating mode Temperature was stabilized
using Brucker temperature controller Spin label was
purchased from Sigma
31
P-NMR studies
31P-NMR spectra of liposomes suspended in the citrate
buffer pH (5.1), containing 10% D2O were recorded at
202.5 MHz using a Bruker AMX-500 Generally, a sweep
width of 41.7 kHz and a repetition 2.6 s using 30 radio
frequency pulses were used The exponential multiplication
of the free induction decay resulted in a 100-Hz line
broadening The number of scans was 28 000 All spectra
were recorded at 17C
R E S U L T S
Effect of monogalactosyldiacylglycerol proportion in liposomes on violaxanthin deepoxidation
Our initial attempts to use liposomes with a lipid compo-sition similar to that of the thylakoid membrane were unsuccessful because the chemical instability of violaxanthin related to the presence of phosphatidylglycerol (PtdGro) and sulphoquinovosyldiacyloglycerol, which complicates the quantitative measurements [17] Therefore, we applied PtdCho, as the lipid that most readily forms bilayers [13], supplemented with monogalactosyldiacylglycerol which was found necessary for VDE activity With the rise in monogalactosyldiacylglycerol proportion in PtdCho lipo-somes to a certain level, the percentage of transformed violaxanthin also increased (Fig 1) However, at 35 mol%
of monogalactosyldiacylglycerol the de-epoxidation rate became significantly lower due to liposome aggregation and the suspension became turbid The increase in turbidity was followed by sedimentation of the lipid aggregates formed These changes were caused probably by fusion of liposomes
or the appearance of monogalactosyldiacylglycerol aggre-gates at its high proportion to PtdCho in the lipid mixture [13,23] The liposome suspension with monogalactosyldi-acylglycerol content < 30 mol% was transparent and showed no tendency to aggregate The presence of liposomes and absence of aggregates in such a suspension was confirmed by electron microscopy and PCS (data not shown) On the other hand, 31P-NMR measurements
Fig 1 The effect of monogalactosyldiacylglycerol proportion in Ptd-Cho/monogalactosyldiacylglycerol liposomes on the level of xanthophylls after 20min of the violaxanthin de-epoxidation reaction at room tem-perature.
Trang 4revealed formation of the reversed hexagonal phase
domains existing in PtdCho/monogalactosyldiacylglycerol
liposomes (Fig 7)
Violaxanthin de-epoxidation was found to be strongly
dependent not only on the concentration of
monogalacto-syldiacylglycerol but also on the ratio of
monogalactosyl-diacylglycerol to PtdCho in liposomes, even if the absolute
amount of monogalactosyldiacylglycerol in the reaction
mixture and its proportion to violaxanthin and VDE were
constant (Fig 2) The values of transition probabilities of
the violaxanthin conversion into antheraxanthin (VA) and
antheraxanthin conversion into zeaxanthin (AZ) showthat
the varying amounts of PtdCho, which result in changes in
monogalactosyldiacylglycerol/PtdCho ratio, have much
stronger effect on conversion of violaxanthin to
anther-axanthin than on conversion of antheranther-axanthin to
zeaxan-thin (Table 1) At lowmonogalactosyldiacylglycerol
concentration (5 mol%), probability of violaxanthin to
antheraxanthin conversion is very low(VA0is 0.006 only)
However, once antheraxanthin has been formed, its
con-version to zeaxanthin occurs at relatively fast rate
(AZ0¼ 0.548) VA0and SVAparameters are very sensitive
to an increase in relative proportion of
monogalactosyldi-acylglycerol; at 30 mol% of this lipid, their values increase
43 and 76 times, respectively, while values of corresponding
parameters describing kinetics of antheraxathin to
zeaxan-thin conversion (AZ0and SAZparameters) increase only 1.5
and 2.2 times, respectively
For further studies on violaxanthin de-epoxidation,
PtdCho liposomes with 30 mol% content of
monogalacto-syldiacylglycerol were used as an optimal system
Comparison of violaxanthin de-epoxidation
in monogalactosyldiacylglycerol and liposomal systems
and the effect of temperature
The temperature dependence of de-epoxidation reaction
was measured in unilamellar
PtdCho/monogalactosyldi-acylglycerol liposomes and in the
monogalactosyldiacylglyc-erol reversed hexagonal phase system with the composition
given in Materials and methods The concentrations of
monogalactosyldiacylglycerol, violaxanthin and VDE
(saturating amount) were the same both in PtdCho/
monogalactosyldiacylglycerol liposomes and
monogalacto-syldiacylglycerol systems It was found that kinetics of
de-epoxidation reaction were different in liposomes and in
the monogalactosyldiacylglycerol system, and that
tempera-ture has a strong influence on the reaction rate in both
systems studied (Figs 3 and 4)
At the three temperatures studied (4, 12 and 25C), the
initial rate of violaxanthin de-epoxidation was always faster
in liposomes than in monogalactosyldiacylglycerol system;
this difference was most evident at 25C However, changes
Monogalctosyldiacylglycerol 5 mol%
Monogalctosyldiacylglycerol 15 mol%
Monogalctosyldiacylglycerol 30 mol%
Fig 2 Time course of violaxanthin to zeaxanthin conversion in PtdCho/ monogalactosyldiacylglycerol liposomes at 25 °C when PtdCho and monogalactosyldiacylglycerol concentrations were, respectively: 245.1 l M and 12.9 l M (5 mol% of monogalactosyldiacylglycerol); 73.1 l M and 12.9 l M (15 mol% of monogalactosyldiacylglycerol); 30.1 l M and 12.9 l M (30mol% of monogalactosyldiacylglycerol) and violaxanthin concentration was 0.33 l M
Table 1 Kinetic parameters of a de-epoxidation reaction calculated for the experimental data presented in Fig 2 by means of the mathematical model Monogalactosyldiacylglycerol
S VA
· 10)3(min)1) AZ 0
S AZ
· 10)3(min)1)
Trang 5in the temperature had different effects on the reaction
plateau reached in both systems At 25C, plateau was
achieved in 20 min in
PtdCho/cerol liposomes and in 40 min in
monogalactosyldiacylgly-cerol system At this stage, about 93% and 83% of initial
violaxanthin amount were de-epoxidated in liposomes and
monogalactosyldiacylglycerol system, respectively;
corres-ponding zeaxanthin levels amounted about to 86% and
80% of total xanthophyll pigments The plateau levels of
anteraxanthin were about 3.8% in liposomes and 2.5% in
monogalactosyldiacylglycerol reversed hexagonal phase In
our experimental systems the complete de-epoxidation of
violaxanthin was not observed A possible reason for this
may be an inhibitory effect of accumulating zeaxanthin on
VDE activity as reported previously [3] On the other hand,
it may be also connected with the presence in the
violax-anthin pool of small amount of cis isomers that cannot serve
as a substrate for VDE [24]
At 25C, the rate of antheraxanthin formation was considerably faster in liposomes than in the monogalacto-syldiacylglycerol system Its maximum level in liposomes was achieved after 2 min and amounted to about 23% of the total xanthophyll pool, whereas in monogalactosyldi-acylglycerol system the maximum level of antheraxanthin was detected after 10 min and it accounted only for about 16% of all xanthophylls
At 12C, in spite of the higher initial de-epoxidation rate
of violaxanthin in liposomes, more violaxanthin was de-epoxidated and more zeaxanthin was formed in the mono-galactosyldiacylglycerol system at the plateau stage of the reaction The same was found at 4C, although at this temperature the plateau in monogalactosyldiacylglycerol system had not been reached during 180 min reaction time The kinetic parameters of violaxanthin de-epoxidation calculated for the exeperimental data obtained from the liposome and monogalactosyldiacylglycerol systems by
Fig 4 Time course of violaxanthin to zeaxanthin conversion at different temperatures in monogalactosyldiacylglycerol systems.
Fig 3 Time course of violaxanthin to zeaxanthin conversion at different
temperatures in liposomes Monogalactosyldiacylglycerol was present
at (A) 5mol%, (B) 15mol% and (C) 30mol%.
Trang 6means of the mathematical model are compared in Table 2.
The rates of the de-epoxidation reactions are more sensitive
to temperature in the liposomal system than in the
monogalactosyldiacylglycerol system When rising the
tem-perature from 4 to 25C, zeaxanthin level at the plateau
stage increases 2.7-fold, violaxanthin level decreases
eight-fold and antheraxanthin maximal level increases twoeight-fold in
the liposomal system, whereas corresponding values for
monogalactosyldiacylglycerol system are 2.1, 3.5 and 1.7
When analysing the effect of temperature on probabilities of
VA0and AZ0transition in both systems studied (Table 2), it
is evident that these values are higher in the liposome system
than in the monogalactosyldiacylglycerol system at all
temperatures studied The increase in the temperature from
4 to 25C increases the VA0 value from 0.005 to 0.26
(52-fold) in liposomes, whereas VA0increases from 0.004 to
0.085 (21-fold) in monogalactosyldiacylglycerol system The
AZ0transition increases proportionally in both systems on
elevating the temperature from 4 to 25C and its value rises
12.5-fold in liposomal and 13.3-fold in micellar systems,
respectively On the other hand, the values SVAand SAZ
coefficients increase much more in
monogalactosyldiacyl-glycerol reversed hexagonal phase than in the liposomal
system when inceasing the temperature from 4 to 25C
Temperature-dependent changes in the value of VA0
parameter in PtdCho/monogalactosyldiacylglycerol
lipo-somes correlate well with the corresponding changes in the
value of the order parameter as found by the use of EPR
spectrometry and a spin probe 5-doxyl-stearic acid (Fig 5)
The lower value of the order parameter the higher the
violaxanthin de-epoxidation rate is observed
D I S C U S S I O N
This paper is the first work where VDE has been isolated
from a monocotyledonous plant The action and properties
of this enzyme are the same as VDE isolated previously
from dicotyledonous plants [25]
The presented results showthat VDE-mediated
conver-sion of violaxanthin via antheraxanthin into zeaxanthin can
occur in PtdCho/monogalactosyldiacylglycerol liposomes
It is worth noting that the VDE enzyme added to initiate the
reaction was present on the external and not internal side of
the liposome membrane and that similar kinetics and
decline in violaxanthin amount as in the measurements
performed with thylakoids [15] were observed For this
reason, the PtdCho/monogalactosyldiacylglycerol
unilamel-lar liposome system used in this work is a good model of the native photosynthetic membrane for studying the VDE activity
The presence of monogalactosyldiacylglycerol in Ptd-Cho-liposomes was found to be indispensable for the violaxanthin de-epoxidation reaction As we have demon-strated, the rate of violaxanthin to antheraxanthin conver-sion depends on monogalactosyldiacylglycerol/PtdCho ratio in the liposome membrane even if the absolute amount
of monogalactosyldiacylglycerol in the reaction mixture and its proportion to violaxanthin and VDE remains constant (Fig 2, Table 1) On the basis of these results, we postulate that VDE binds only to certain membrane domains that are rich in monogalactosyldiacylglycerol and the de-epoxida-tion reacde-epoxida-tions take place in these domains Violaxanthin being distributed homogeneously in the lipid bilayer has to
Table 2 Kinetic parameters of a de-epoxidation reaction calculated for the experimental data presented in Figs 3 and 4 by means of the mathematical model.
Temp.
S VA
· 10)3(min)1)
AZ 0
Liposomes
Monogalactosyldiacylglycerol system
Fig 5 PtdCho/monogalactosyldiacylglycerol liposome membrane flui-dity and percent of violaxanthin converted after 1 min de-epoxidation reaction at different temperatures.
Trang 7enter the monogalactosyldiacylglycerol-enriched domains
by lateral diffusion to be converted to antheraxanthin The
higher monogalactosyldiacylglycerol/PtdCho ratio, the
higher the amount of such domains in the liposomal
membrane This shortens the diffusion path of violaxanthin
molecules to these domains and results in higher rate of
violaxanthin de-epoxidation (see the values of VA0 in
Table 1) It is well known that nonbilayer prone lipids (e.g
monogalactosyldiacylglycerol) may form reversed
hexa-gonal phase in model lipid membranes and it has been
reported that such structures exist in biological membranes
[26–28].31P-NMR spectra shown in Fig 7 clearly
demon-strate the existence of the reversed hexagonal phase domains
in our system of PtdCho/monogalactosyldiacylglycerol
liposomes The presence of the reversed hexagonal phase
in thylakoid membranes has been also reported in our
earlier papers and by other authors using 31P-NMR
and freeze-fracturing techniques [23,29–31] These
observa-tions give a sound basis for our model of violaxanthin
de-epoxidation in liposomes and thylakoid membranes
According to the presented model, the second reaction of
de-epoxidation, i.e conversion of antheraxanthin to
zea-xanthin, also occurs in the monogalactosyldiacylglycerol
rich domains, and is greatly facilitated because
antheraxan-thin, formed in such domains, has an immediate access to
the VDE enzyme Therefore, and in contrast to the
de-epoxidation of violaxanthin to antheraxanthin, the
conversion of antheraxanthin to zeaxanthin seems to be
not limited by diffusion process The conclusion that the
conversion of violaxanthin to antheraxanthin is more
sensitive to monogalactosyldiacylglycerol concentration
than the conversion of antheraxanthin to zeaxanthin is
supported by relatively high value of the AZ0 parameter
even in conditions of very low VA0 (e.g at 5 mol% of
monogalactosyldiacylglycerol, Table 1)
The model assuming the existence of
monogalactosyldi-acylglycerol reversed hexagonal phase domains in the
membrane to which VDE binds and rather homogeneous
distribution of violaxanthin molecules explains also the
strong correlation between the rate of violaxanthin
de-epoxidation and value of the membrane lipid order
parameter It seems that decreasing value of the order
parameter permits faster lateral diffusion of violaxanthin in
the membrane and the molecules of this xanthophyll may
reach sooner the monogalactosyldiacylglycerol rich
domains where they are de-epoxidated (Fig 5, Tables 2
and 3) This model can also explain a clear temperature
effect on the level and the time of antheraxanthin
appear-ance in the liposome system The conversion of
anther-axanthin to zeanther-axanthin is less dependent on the changes in
membrane physical properties (Tables 2 and 3) for the
reasons already discussed Application of the proposed model to the results obtained shows why the conversion of violaxanthin to antheraxanthin is much slower and more sensitive to temperature than transition from the anther-axanthin to zeanther-axanthin On the basis of our results and literature data [15,22], we postulate that changes in mem-brane fluidity may play an important role in regulation of the violaxanthin de-epoxidation rate in membranes The higher rate of violaxanthin de-epoxidation to antheraxanthin and stronger temperature effect on this process in liposomes than in monogalactosyldiacylglycerol systems is probably related to the different availability of violaxanthin for VDE in both systems studied As revealed
by PCS and electron microscopy, the size of monogalacto-syldiacylglycerol aggregates differed greatly from that of PtdCho/monogalactosyldiacylglycerol liposomes The monogalactosyldiacylglycerol structures were found as large, heterogeneous aggregates with a mean diameter of
600 nm and a large standard deviation Thus, the previous assumption that monogalactosyldiacylglycerol creates small micelles with only one molecule of violaxan-thin inside [32] was not confirmed in our study The average diameter of liposomes was 110 nm (as expected) with narrowstandard deviation Apparently, the availability of violaxanthin for VDE is higher in liposomes than in the monogalactosyldiacylglycerol system where access of the enzyme to its substrate may be impeded by large scale aggregation of monogalactosyldiacylglycerol structures Neither the molecular arrangement of monogalactosyldi-acylglycerol in such aggregates nor the orientation of violaxanthin in these structures have been precisely deter-mined [23]
To convert violaxanthin into zeaxanthin, VDE has to remove two epoxy groups attached to two rings of the violaxanthin molecule In the unilamellar liposome system, where all the xanthophylls are oriented perpendicularly to the plane of the membrane and VDE is present only outside the vesicles, the formed antheraxanthin molecule, to be converted into zeaxanthin, has to reverse its orientation as a whole in such a way that the end group containing the ring with the remaining epoxy group appears on the other side of membrane Such a flip-flop of antheraxanthin molecule is a necessary step assuming that VDE cannot penetrate through the lipid bilayer and has access to the outer surface
of the liposome only Table 3 shows the maximal time in which all molecules of violaxanthin are converted to antheraxanthin and all molecules of antheraxanthin are converted to zeaxanthin at a given temperature at saturating amount of VDE and at the initial reaction rate While the time for VfiA conversion is shortened considerably on the increase of temperature from 4 to 25C, the time for AfiZ transition changes is in a much more narrowrange In the liposome system studied, the time for AfiZ transition takes 3.1 min at the temperature of 25C and 2.9 and 2.0 min at
12 and 4C, respectively This means that flip-flop of antheraxanthin in PtdCho/monogalactosyldiacylglycerol liposomes at a given temperature has to be shorter than the times specified in Table 3 Moreover, faster conversion
of antheraxanthin to zeaxanthin (AZ0values) than violax-anthin to antheraxviolax-anthin (VA0values) was observed at all temperatures studied (Tables 2 and 3) suggesting that flip-flop of antheraxanthin is not the limiting step in the transformation of violaxanthin into zeaxanthin in the
Table 3 The maximal time required for the conversion of all
violaxan-thin molecules into antheraxanviolaxan-thin (T VA ) and all molecules of
anther-axanthin into zeanther-axanthin (T AZ ) at three different temperatures in
PtdCho/monogalactosyldiacylglycerol liposomes.
Trang 8membrane system investigated Our results are in agreement
with those of Arvidsson et al [15] who suggested that in the
isolated thylakoids the flip-flop of antheraxanthin is not the
limiting factor in zeaxanthin formation
The apparently longer time necessary for antheraxanthin
to zeaxanthin conversion at higher temperatures (Table 3)
can be explained in terms of our model assuming diffusion
controlled rate of violaxanthin to antheraxanthin
conver-sion Violaxanthin and antheraxanthin compete for the
same active site of the VDE enzyme At elevated
tempera-ture, when violaxanthin lateral diffusion in the membrane is
faster, more violaxanthin molecules reach the
monogalacto-syldiacylglycerol rich domains in time unit In such a
situation, violaxanthin competes successfully with
anther-axanthin for the VDE active site; this results in enlargement
of the antheraxanthin pool As a consequence, a relatively
lower number of antheraxanthin molecules of the pool can
reach the VDE active site and become converted to
zeaxanthin This conclusion is supported by the data
presented in Fig 6, which shows that at higher temperatures
a lower proportion of total antheraxanthin pool is
conver-ted into zeaxanthin It should be also added that because
VDE was present in excess in the reaction mixture, only part
of it could be bound to the membrane, depending on the size
and number of monogalactosyldiacylglycerol-rich domains
Some conclusions drawn from our results obtained with
model lipid bilayer can be extrapolated to describe the role
of the xanthophyll cycle in the regulation of thylakoid
membrane fluidity In the darkness, due to zeaxanthin
epoxidase activity, violaxanthin accumulates in thylakoids
Illumination of plants with strong light causes acidification
of thylakoid lumen, which is a prerequisite for VDE binding
to thylakoid membrane, and also it usually increases leaf temperature, which results in the increase of the membrane dynamics A temperature-induced increase of thylakoid membranes dynamics facilitates diffusion of violaxanthin molecules into monogalactosyldiacylglycerol-VDE domains where it is converted into antheraxanthin and zeaxanthin This conclusion is supported by the results of Sarry et al [33] who found that illumination of plants at low tempera-ture results in a lower amount of zeaxanthin formed than at higher temperature There are reports that showthat zeaxanthin may act like cholesterol and play important role
in the regulation of thylakoid membrane arrangement Gruszecki and Strzalka [34] showed that light induced accumulation of zeaxanthin affects membrane fluidity Tardy and Havaux [35] found that decreased value of the thylakoid membrane order parameter was proportional to the amount of zeaxanthin present in the membrane The rigidifying effect of this xanthophyll was also found upon incorporation of exogenous zeaxanthin into isolated thyl-akoid membranes [36]
Fig 6 The percentage of total antheraxanthin pool converted into
zeaxanthin during violaxanthin de-epoxidation reaction in PtdCho/
monogalactosyldiacylglycerol liposomes at three different temperatures.
Fig 7 31P-NMR spectra of PtdCho liposomes (A) without mono-galactosyldiacylglycerol and (B) with 30mol% monogalactosyldiacyl-glycerol.
Trang 9Zeaxanthin formed in the hexagonal phase domains can
probably leave these regions and, due to its membrane
rigidifying properties, it regulates the molecular dynamics of
thylakoid membranes and protects them at elevated
temperatures resulting from intense irradiation
In conclusion, the PtdCho/monogalactosyldiacylglycerol
liposome system described in this work is more appropriate
than monogalactosyldiacylglycerol aggregates for studying
the mechanism of violaxanthin de-epoxidation catalysed by
VDE in vitro because it approaches the native
photosyn-thetic membranes The existence of de-epoxidation reactions
in liposomes opens newpossibilities in the investigation of
the xanthophyll cycle, which might contribute to a better
understanding of this process
A C K N O W L E D G E M E N T S
This work was supported by a grant no 6P04A 02819 from Committee
for Scientific Research (KBN) of Poland We wish to thank Maria
Kozlowska for electron microscopy pictures, Dr Maria Zembala for
PCS measurements and Dr F Szneler for31P- NMR analysis We are
very grateful to Dr Fabrice Franck from University of Liege, Belgium
for helpful discussion.
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