-
Increased Thermostability of Thylakoid Membranesin
Isoprene-Emitting Leaves Probed with ThreeBiophysical
Techniques1[W][OA]
Violeta Velikova, Zsuzsanna Várkonyi, Milán Szabó, Liliana
Maslenkova, Isabel Nogues, László Kovács,Violeta Peeva, Mira
Busheva, Győző Garab, Thomas D. Sharkey, and Francesco
Loreto*
Institute of Plant Physiology and Genetics (V.V., L.M., V.P.)
and Institute of Biophysics and BiomedicalEngineering (M.B.),
Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria; Institute of
Plant Biology, BiologicalResearch Center, Hungarian Academy of
Sciences, 6726 Szeged, Hungary (Z.V., M.S., L.K., G.G.); Institute
ofAgroenvironmental and Forest Biology, National Research Council,
00015 Monterotondo, Rome, Italy (I.N.);Department of Biochemistry
and Molecular Biology, Michigan State University, East Lansing,
Michigan 48824(T.D.S.); and Institute for Plant Protection,
National Research Council, 50019 Sesto Fiorentino, Florence,
Italy(F.L.)
Three biophysical approaches were used to get insight into
increased thermostability of thylakoid membranes in
isopre-ne-emittingplants.Arabidopsis (Arabidopsis thaliana) plants
genetically modified to make isoprene and Platanus orientalis
leaves,in which isoprene emission was chemically inhibited, were
used. First, in the circular dichroism spectrum the
transitiontemperature of the main band at 694 nm was higher in the
presence of isoprene, indicating that the heat stability of
chiralmacrodomains of chloroplast membranes, and specifically the
stability of ordered arrays of light-harvesting complex
II-photosystem II in the stacked region of the thylakoid grana, was
improved in the presence of isoprene. Second, the decay
ofelectrochromic absorbance changes resulting from the electric
field component of the proton motive force (DA515) wasevaluated
following single-turnover saturating flashes. The decay of DA515
was faster in the absence of isoprene when leaves ofArabidopsis and
Platanus were exposed to high temperature, indicating that isoprene
protects the thylakoid membranesagainst leakiness at elevated
temperature. Finally, thermoluminescence measurements revealed that
S2QB
2 charge recombi-nation was shifted to higher temperature in
Arabidopsis and Platanus plants in the presence of isoprene,
indicating higheractivation energy for S2QB
2 redox pair, which enables isoprene-emitting plants to perform
efficient primary photochemistry ofphotosystem II even at higher
temperatures. The data provide biophysical evidence that isoprene
improves the integrity andfunctionality of the thylakoid membranes
at high temperature. These results contribute to our understanding
of isoprenemechanism of action in plant protection against
environmental stresses.
Vegetation is a source of large quantities of biogenicvolatile
organic compounds emitted into the atmo-sphere, of which isoprene
is the most abundant(Guenther et al., 2006). Owing to the high
reactivityand fast oxidation by hydroxyl radicals in the
atmo-sphere, isoprene can increase the formation of atmo-
spheric ozone (Monson and Holland, 2001; Kleindienstet al.,
2007), organic nitrates (O’Brien et al., 1995),
andperoxyacylnitrates (Sun and Huang, 1995). Isoprene isalso a
significant factor in secondary aerosol forma-tion, with direct and
indirect effects on the global ra-diation balance of the atmosphere
(Claeys et al., 2004;Matsunaga et al., 2005; Kroll et al., 2006;
Ervens et al.,2008; Paulot et al., 2009).
Besides having significant influences on atmo-spheric chemistry,
many biogenic volatile organiccompounds play an important role in
plant biology.Experimental evidence shows that isoprene
protectsphotosynthesis under thermal and oxidative stressconditions
(for review, see Vickers et al., 2009a; Loretoand Schnitzler,
2010). It was demonstrated that leavesin which isoprene
biosynthesis was blocked by themethyl erythritol pathway inhibitor
fosmidomycin,were more sensitive to high temperature and
ozoneexposure, and developed stronger oxidative damage,compared to
isoprene-emitting leaves (Loreto andVelikova, 2001; Sharkey et al.,
2001; Velikova andLoreto, 2005). Plants fumigated with isoprene
suffer
1 This work was supported by bilateral projects in the
frameworkof Bulgarian Academy of Sciences, Italian National
Research Coun-cil, and Hungarian Academy of Sciences agreements, by
the Euro-pean Science Foundation-Eurocores project
EuroVOL-MOMEVIP(to F.L.), by the grant OTKA/NKTH-CNK (grant no.
80345 to G.G.),and by the National Science Foundation (grant no.
IOS–0950574 toT.D.S.).
* Corresponding author; e-mail [email protected]
author responsible for distribution of materials integral to
the
findings presented in this article in accordance with the
policydescribed in the Instructions for Authors
(www.plantphysiol.org) is:Francesco Loreto
([email protected]).
[W] The online version of this article contains Web-only
data.[OA] Open Access articles can be viewed online without a
sub-
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less damage when exposed to oxidative stresses (Loretoet al.,
2001) and recover more rapidly from heat stressthan untreated
controls (Singsaas et al., 1997; Sharkeyet al., 2001; Velikova et
al., 2006). Genetic approaches todevelop isoprene-emitting species
from nonemittingwild types (Sharkey et al., 2005; Loivamäki et
al.,2007; Sasaki et al., 2007; Vickers et al., 2009b) or tosuppress
isoprene synthesis in strong emitters (Behnkeet al., 2007) have
also been used to clarify the role ofisoprene in plant protection.
Studies with geneticallymodified plants mostly confirmed improved
toleranceassociated with the capacity to form and emit iso-prene.
Recently important compensatory responses,such as the activation of
alternative defensive bio-chemical pathways, e.g. phenolics
biosynthesis, to copewith stressful conditions, were also
highlighted (Fareset al., 2010), especially when isoprene
biosynthesis isknocked out (Behnke et al., 2009).
Several hypotheses have been put forward toexplain the
physiological mechanism(s) by which iso-prene protects the
photosynthetic apparatus (Loretoand Schnitzler, 2010). The oldest
and most widelyaccepted idea is that isoprene stabilizes
chloroplastmembranes (Sharkey and Singsaas, 1995).
Thylakoidmembranes become leaky at high temperature (Pastenesand
Horton, 1996; Bukhov et al., 1999; Schrader et al.,2004; Zhang et
al., 2009). It was suggested that thepositive effect of isoprene
might be due to the hydro-phobic nature of the molecule, the
localization ofisoprene synthase enzyme near the thylakoid
mem-branes (Wildermuth and Fall, 1998; Schnitzler et al.,2005), and
the high octanol/water partitioning coeffi-cient (Copolovici and
Niinemets, 2005). Lipophilicisoprene partitioned into membranes
might preventthe formation of water channels responsible for
themembrane leakiness at high temperature, or may pre-vent the
formation of nonlamellar aggregates, or helpstabilize the
photosynthetic complexes embedded inthylakoid membranes (Singsaas
et al., 1997; Sharkeyet al., 2001). Isoprene could also enhance
hydrophobicinteractions within thylakoids and thereby
stabilizeinteractions between lipids and/or membrane proteinsduring
episodes of heat shock or high-temperaturestress conditions
(Sharkey and Yeh, 2001). Based onmolecular dynamics simulations of
phospholipid bi-layers with and without isoprene, Siwko et al.
(2007)suggested that isoprene enhances the packing of lipidtails.
The authors suggested that the role of isopreneas a membrane
stabilizer can be related to the factthat it fits well into the
available pockets of the freevolume inside the membrane, and adds
cohesiveness,amplifying membrane packing, while not affectingthe
dynamics of phospholipid bilayers (Siwko et al.,2007). However,
Logan and Monson (1999) workingwith reconstituted liposomes were
not able to provethat isoprene improves the thermal stability of
mem-branes.
Another hypothesis to explain the generally positiverole of
isoprene in plant metabolism is based on thehigh reactivity of
volatile isoprenoids with radicals
and other reactive compounds. Loreto et al. (2001)
firstsuggested that isoprene might operate as a volatilemolecule,
scavenging reactive oxygen species in theintercellular spaces of
the leaf mesophyll. More re-cently, it was proven that isoprene
also removes reac-tive nitrogen species from the mesophyll
(Velikovaet al., 2008). Vickers et al. (2009a) reviewed the
possiblemechanisms of isoprene function and suggested thatthe
molecule may have a general antioxidant role.
A demonstration of effects of isoprene on biophys-ical
measurements of thylakoid function at high tem-perature could help
in working toward a resolution ofthe primary mechanism of isoprene
action. Here threewell-known techniques in biophysical studies of
thy-lakoid membrane function were tested for the effect ofisoprene
at high temperature. Two plant systems wereused. Arabidopsis
(Arabidopsis thaliana), which doesnot normally make isoprene, was
engineered with anisoprene synthase gene from kudzu (Pueraria
lobata) sothat wild-type plants (nonemitting) could be com-pared to
the transformed plants that do make iso-prene. Leaves of Platanus
(plane tree) normally domake isoprene but this was inhibited by
fosmido-mycin so that emitting (water-fed) and
nonemitting(fosmidomycin-fed) leaves could be compared. Thethermal
stability of the thylakoid membranes was char-acterized with
biophysical approaches not previouslyused in isoprene studies,
namely circular dichroism(CD) spectrosocopy, electrochromic
absorbance tran-sients (DA515), and thermoluminescence (TL).
Thesemeasurements revealed that in the presence of iso-prene, the
macroorganization of the pigment-proteincomplexes in the membranes
were more stable toelevated temperature, the membranes were better
ableto maintain a light-induced transmembrane electricfield at
elevated temperatures, and the recombinationof the PSII donor and
acceptor side charges occurred upto higher temperature.
RESULTS
Suitability of Plant Material
Genetic manipulation of Arabidopsis plants didnot affect their
photosynthetic performance. IspS andwild-type plants were
characterized by similar photo-synthesis (Fig. 1; absolute values
7.0 6 0.3 mmolm22 s21, n = 14). Inhibition of isoprene emission
inPlatanus leaves by the chemical inhibitor also did notinfluence
photosynthesis. Absolute values of photo-synthesis in Platanus
leaves were 5.3 6 0.4 mmolm22 s21, n = 14. Isoprene emission was
not detect-able in wild-type plants of Arabidopsis, whereas
infosmidomycin-fed Platanus leaves isoprene emissionwas inhibited
to about 10% of the original level (Fig. 1).
CD Spectroscopy
CD spectra in wild-type and IspSArabidopsis plantsshowed
considerable differences at 20�C. In particular,
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it was found that the amplitude of the main CD band(at [+]694
nm) was lower in wild-type than in IspSplants (compare Fig. 2, A
and B, solid lines), sug-gesting that constitutive presence of
isoprene mightdetermine structural changes in the thylakoid
mem-branes.To investigate the possible role of isoprene in the
conformational stability of chloroplast membranessubjected to
high temperatures, measurements of CDspectra were performed in
wild-type and IspS Arabi-dopsis leaves after 10 min incubations at
20�C, 30�C,40�C, 45�C, 50�C, 55�C, and 60�C. At 40�C the ampli-tude
of the main band at (+)694 nm was considerablylower in wild-type
than in IspS leaves (compare Fig. 2,A and B, gray lines), while the
weaker, excitonic bandsat (+)440 and (2)650 nm and the excitonic
band pair(+)482/(2)470 were not affected by isoprene. Thetransition
temperature of the band at (+)694 nm, wasshifted to higher
temperature in IspS leaves; the tran-
sition temperatures were 40.1�C and 49.4�C in wild-type and IspS
leaves, respectively (Fig. 2C).
Measurements of CD spectra were also performedon
isoprene-emitting and isoprene-inhibited Platanusleaves (Fig. 3).
The CD spectra of Platanus leaves at25�Cwere not affected by the
fosmidomycin treatmentsuppressing isoprene biosynthesis (compare
Fig. 3, Aand B, black lines). At elevated temperatures,
theamplitudes of the (2)675 and (+)694 nm CD bandswere higher in
isoprene-emitting (Fig. 3A, gray line)than in isoprene-inhibited
leaves (Fig. 3B, gray line).The CD band at (+)694 nm was completely
missingin isoprene-inhibited leaves already at 55�C and
inisoprene-emitting leaves over 60�C (data not shown).The
transition temperature was shifted to lower value(46.4�C) in
isoprene-inhibited leaves, compared toisoprene-emitting leaves
(55.3�C; Fig. 3C).
Flash-Induced Electrochromic Shift at 515 nm
To test the membrane integrity, the electrochromicabsorbance
changes at 515 nm (DA515) induced bysingle-turnover, saturating
flashes was recorded. Thehalf-times of the decay of DA515
transients character-izing the membrane permeability are shown in
Table I.We found that the decay times on IspS and
wild-typeArabidopsis leaves were essentially identical at
20�C,suggesting that the thylakoid membrane of theseplants possess
similar permeability; however, after a5-min-long 40�C treatment,
the decay of DA515 becamefaster in the wild type, while remained
unaffected inIspS (Table I).
In Platanus leaves that were maintained at 25�C thedecay
half-times of DA515 were also similar in isopre-ne-emitting and
isoprene-inhibited leaves (Table I). Theheat treatment at 45�C (4 h
in weak light) resulted instronger decrease of the half-time of
DA515 decay inisoprene-inhibited than in isoprene-emitting leaves.
Tworepresentative traces registered after high-temperaturetreatment
(40�C for Arabidopsis and 45�C for Platanus)are shown in
Supplemental Figure S1 to exemplify thechange driven by the
high-temperature treatment.
Flash-Induced TL from Arabidopsis and Platanus Leaves
TL measurements were performed to assess thepossibility of
isoprene-induced alterations to PSII pri-mary photochemistry for
direct estimation and com-parison of redox properties of PSII. Upon
illuminationby a single flash at 1�C, the wild-type
Arabidopsisleaves showed a main TL B band (S2QB
2) peaking at23.0�C 6 1.3�C (Fig. 4A). In IspS leaves, the
peakposition of the B band was up shifted by about 10�C, to32.6�C6
1.0�C (Fig. 4C), showing a significant increasein the activation
energy for S2QB
2 charge recombina-tion. Similarly, higher emission temperatures
wererecorded when two (compare with Fig. 4, A and C)and more (data
not shown) flashes were given, con-firming the role of S3 oxidation
state in charge stabi-lization upon multiple turnovers. The higher
emission
Figure 1. Photosynthesis and isoprene emission in Arabidopsis
(A) andP. orientalis (B) leaves. Values represent means 6 SE (n =
7) and areexpressed as percent of photosynthesis and isoprene
emission of wild-type Arabidopsis and isoprene-emitting Platanus.
Measurements wereperformed at growth conditions: 22�C and 150 mmol
photons m22 s21
for Arabidopsis, and 25�C and 350 mmol photons m22 s21 for
Platanus.In Platanus isoprene emission was inhibited by adding
fosmidomycin(5 mM) to the water. Asterisks indicate significant
differences (P, 0.01).n.d. identifies emissions that were not
detectable (,0.05 nmol m22 s21).
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temperature of TL B band in IspS leaves is indicativefor more
stably stored S2(3)QB
2 charge pairs.When both wild-type and IspS Arabidopsis
plants
were illuminated by a flash series, the intensity of TL Bband
exhibited a period four-oscillation pattern withmaximum on the
second flash (data not shown),typical for active PSII. TL
oscillations are related todark distribution of the S states of
oxygen-evolvingcomplex and QB/QB
2 ratio (Rutherford et al., 1984),thus suggesting no differences
in these parametersbetween the wild-type and IspS plants.
In Platanus leaves, the TL traces could not berecorded in frozen
material and therefore samplesthat were not previously frozen were
used. The inhi-bition of isoprene emission by fosmidomycin did
notcause significant changes in the main B-band temper-ature (Fig.
4, B and D). The B band induced by a sin-gle flash in
isoprene-inhibited and isoprene-emittingleaves peaked at 33.1�C 6
2.0�C and 35.8�C 6 2.5�C,respectively. These values were similar to
the B-bandtemperature observed in IspS Arabidopsis plants(Fig.
4C).
Figure 2. CD spectra measured in Arabidopsis wild type, which do
not emit isoprene (A), and IspS isoprene-emitting (B) plants.CD was
recorded in leaves at 20�C (black line) and 40�C (gray line).
Temperature dependences of the intensity of the (+)694 nmband for
the wild type (white circles) and IspS (black circles) are shown in
C. All measurements were done on 35-d-old plants.Measurements were
repeated on seven leaves from different plants; representative
spectra are shown in A and B. All spectra werecorrected for a flat
baseline obtained by setting the CD values at 400 and 750 nm to
zero. In C, the temperature at which theintensity of the CD band is
50% of its value at 20�C (transition temperature, Tt) is shown.
Statistical significance of differencesbetween Tt in wild-type and
IspS leaves (P , 0.01) was determined by Student’s t test. Means 6
SE are given (n = 7), for thereplicates at 40�C. rel. u., Relative
units.
Figure 3. CD spectra measured in isoprene-emitting (A) and
isoprene-inhibited (B) Platanus leaves. CDwas recorded in leaves
at25�C (black line) and 50�C (gray line). Temperature dependences
of the intensity of the (+)694 nm band for the
isoprene-emitting(black circles) and isoprene-inhibited,
fosmidomycin-treated leaves (white circles) are shown in C.
Measurements were repeatedon seven leaves from different plants;
representative spectra are shown in A and B. All spectra were
corrected for a flat baselineobtained by setting the CD values at
400 and 750 nm to zero. In C, the temperature at which the
intensity of the CD band is 50%of its value at 20�C (transition
temperature, Tt) is shown. Statistical significance of differences
between Tt in wild-type and IspSleaves (P, 0.01) was determined by
Student’s t test. Means6 SE are given (n = 7), for the replicates
at 50�C. rel. u., Relative units.
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TL measurements were compared on leaf discs thatwere maintained
at 25�C and on leaf discs incubatedat 40�C for 5 min. The effect of
the heat treatment onB-band peak temperature and intensity is shown
inFigure 5. The heat treatment of wild-type Arabidopsisand
isoprene-inhibited Platanus leaves reduced theB-band peak emission
temperature (Fig. 5, A and B)and intensity (Fig. 5, C and D). In
isoprene-emittingleaves (IspS and isoprene-emitting Platanus
leaves) theB-band peak emission temperature was unchanged(Fig. 5, A
and B); the intensity of the B-band peak wasreduced by the heat
treatment, but significantly lessthan in isoprene-inhibited leaves
of the two plants(Fig. 5, C and D).
DISCUSSION
CD
CD measures the difference in the extinction of left-handed
versus right-handed circularly polarized light.CD spectroscopy is
used to study chiral moleculesand their assemblies of all types and
sizes, and it isparticularly important in the study of
hierarchicallyorganized biological samples. A primary use is
inanalyzing the secondary structure or conformation
ofmacromolecules, particularly proteins, and becausesecondary
structure is sensitive to its environment,e.g. temperature or pH.
In plants, CD spectroscopyin the visible range originates from
pigment-pigmentinteractions and hence is a valuable tool for
probing themolecular architecture of the photosynthetic
complexesand supercomplexes and their macroorganization inthe
membrane system (Garab, 1996; Garab and vanAmerongen, 2009). CD can
originate from differentlevels of complexity in the sample.
Short-range (exci-tonic) interactions are observed for
pigment-pigmentinteractions within one pigment-protein complex orin
supercomplexes between molecules on adjacentcomplexes. In leaves,
characteristic excitonic CD bandsare typically observed around
(+)440 and (2)650 nm,originating from PSI and light-harvesting
complex II(LHCII), respectively. Additionally the band pair
(+)482/(2)470 nm has been shown to correlate with thetrimeric
organization of LHCII (Garab et al., 2002).
Long-range (polymer- or salt-induced or C-type) in-teractions
are due to pigment interactions in denselypacked chirally organized
macroaggregates with di-mensions of hundreds of nanometers,
membranedomains, also called chiral macrodomains. C-Typeaggregates
are abundant in biological systems; e.g.nuclei, chromosomes,
viruses, and numerous otherhighly organized systems. In these
biological systems,C-type CD signals are determined by the size,
hand-edness, and strength of long-range coupling of the
Table I. Half-times (t1/2, ms) of the decay kinetics of the
flash-induced electrochromic absorbance changes at 515 nm in
detached Arabidopsis andP. orientalis leaves
In Arabidopsis, the measurements were performed at 20�C after
preincubating the wild-type (nonemitting) and IspS
(isoprene-emitting) leaves for 5min at 20�C or 40�C in the dark. In
Platanus, the preincubation of the control (isoprene-emitting) and
fosmidomycin-treated (isoprene-inhibited)leaves was done at 25�C or
45�C for 4 h in weak white light (40 mmol photons m22 s21). All
measurements were then performed at 25�C. Means6 SEare shown; n = 6
for both Arabidopsis and Platanus leaves. The kinetic traces were
obtained by averaging 64 transients with a repetition rate of 1
s21.The half-times were obtained by fitting the decay phase with
single exponential decay functions. Asterisks (*P , 0.05; **P ,
0.01) indicatesignificant differences within isoprene-emitting and
nonemitting leaves of the two plant species when comparing the
temperature treatments.
Arabidopsis P. orientalis
IspS Wild Type Isoprene Emitting Isoprene Inhibited
20�C 40�C 20�C 40�C 25�C 45�C 25�C 45�C
57.8 6 6.5 52.0 6 6.0 61.2 6 2.8 37.1 6 3.8* 83.3 6 5.4 65.2 6
2.4 74.5 6 4.4 45.0 6 1.1**
Figure 4. TL curves of wild-type (A) and IspS (C) Arabidopsis
leaves andof isoprene-inhibited (B) and isoprene-emitting (D)
Platanus leaves afterillumination with one or two single-turnover
flashes of white saturatinglight at 1�C. TL was measured during
heating of the samples to 70�C indarkness with a ramp of
temperature at constant rate of 0.6�C s21.Curves are representative
of measurements on 10 leaves per treatmentfrom different plants.
a.u., Arbitary units.
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chromophores (Keller and Bustamante, 1986). In thy-lakoids the
CD bands at (2)675 and (+)694 nm thatposses such C-type features
are usually correlatedwith the macroorganization of LHCII (Garab
andMustárdy, 1999) and with the presence of orderedarrays of
LHCII-PSII supercomplexes, as shown alsoby advanced electron
microscopy (Kovács et al.,2006).
CD can be used to observe how secondary structureof proteins
changes with environmental conditions oron interaction with other
molecules. In fact, our CDresults show that the heat stability of
the chiral macro-domains in the grana, i.e. of the ordered arrays
ofLHCII-PSII in the stacked regions, was positivelyaffected by the
presence of isoprene. Probably, in theIspS leaves, isoprene
significantly modifies the or-ganization of the pigment-protein
complexes in thethylakoid membranes. At physiological
temperatures(20�C), however, the C-type CD at (+)694 nm
wasincreased in IspS leaves. This last C-type CD featuremight
indicate the presence of better-ordered arraysof LHCII-PSII
supercomplexes or of LHCII macro-domains. This interesting
observation requires furtherinvestigations since it might indicate
a modified two-dimensional organization of the LHCII-PSII
super-complexes in the stacked membranes of plants that
aretransformed to emit isoprene (compare with Kovácset al., 2006).
In contrast, Platanus leaves in which iso-prene emission was
chemically inhibited did notshow changes of (+)694 nm CD band with
respect
to isoprene-emitting leaves at physiological tempera-tures
(25�C). This indicates that the inhibition ofisoprene biosynthesis
does not affect the macroorga-nization of the photosynthetic
complexes that havebeen formed when isoprene was actively
synthesizedduring the ontogeny of leaves, or that the smallamount
of isoprene still being made by chemicallyinhibited Platanus leaves
was enough to stabilize theLHCII-PSII supercomplexes revealed by
the (+)694 nmband.
CD spectroscopy is also a useful tool for probing thethermal
stability of the macrodomains formed by thephotosynthetic complexes
in the membranes (Garabet al., 2002; Dobrikova et al., 2003). Our
data demon-strated that modified membrane structure in IspSleaves
is related to the increased thermostability ofthe supercomplexes in
isoprene-transformed Arabi-dopsis plants, and that thermal
stability of the chiralmacrodomains in the thylakoid membranes is
higherin isoprene-emitting Platanus leaves compared
toisoprene-inhibited leaves. Under increasing temper-atures, the
transition temperature that revealed thedisassembly of the protein
macrodomain constitutedby the LHCII-PSII supramolecular
organization (asmonitored by the [+]694 nm CD band), was about8�C
higher in IspS Arabidopsis and isoprene-emittingPlatanus leaves
compared to the plants that do not emitisoprene. In general, heat-
or light-induced reorganiza-tions occur mostly at the
supramolecular level of struc-tural complexity, rather than at the
level of the building
Figure 5. TL B-band (S2QB2) peak tem-
perature (A and B) and TL maximumintensity (C andD) inwild-type
and IspSArabidopsis (A and C) and in isoprene-emitting and
isoprene-inhibited Plata-nus leaves (B and D).The measurementswere
done after a 5-min incubation at25�C (white bars) or 40�C (gray
bars).Values are means of five different leavesexposed to each
treatment 6 SE Thesignificant differences between meansat different
temperatures were deter-mined by Student’s t test. Means
signif-icantly different at P , 0.05 and 0.01confidence levels are
labeled by one ortwo asterisks, respectively. a.u.,
Arbitaryunits.
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blocks of macroassemblies. For example, the stability ofthe
single constituents of pigment-protein complexeshas been shown to
be considerably higher than thestability of their macroassemblies
(Cseh et al., 2000,2005; Dobrikova et al., 2003). The experiment
using CDspectroscopy therefore confirms that a higher
thermalstability is conferred by isoprene to thylakoid mem-branes,
and suggests that this might be due to a sus-tained stability of
the ordered arrays of supercomplexesin the thylakoid membranes,
rather than to a specifi-cally induced resistance in a single
photosystem com-ponent.It was shown that the structural flexibility
of LHCII
macrodomains depends largely on the lipid environ-ment of the
complexes (Simidjiev et al., 1998). Thepositive stabilizing effect
of isoprene on membranescould therefore be related to changes in
the lipid phaseand/or alterations induced by isoprene in the
phasebehavior of lipids. The mixed lipid phases can havesignificant
effects on the organization of thylakoidmembranes (Williams and
Quinn, 1987). Lateral or-ganization of membranes into domains can
dependcritically on temperature and small molecules havebeen found
to significantly affect large-scale phaseseparations. Molecules
that affect the miscibility of dif-ferent phases will significantly
alter the stability of dif-ferent membrane domains under heat
stress (Veatch,2007). As under physiological conditions the CD
spec-trum at (+)694 nm is similar in isoprene-emitting
andisoprene-inhibited Platanus leaves, we infer that thechemical
inhibition of isoprene does not alter the mac-romolecular structure
of the membranes. If isoprenesimply alters the lipid phase of
membranes, then theprotective effect may be more rapidly lost after
iso-prene inhibition. This would explain why the protec-tive effect
of isoprene is seen on both Arabidopsis IspSplants and in
isoprene-emitting Platanus under heatstress, independently of the
stability of the LHCII-PSIIsupramolecular organization and of the
way isopreneemission is manipulated. Recent data have shown thatthe
dgd1 Arabidopsis mutant, which is deficient in thebilayer lipid
digalatosyldiacyl glycerol and enriched inthe nonbilayer
monogalactosyldiacyl glycerol, displayconsiderably lower thermal
stability of the chiralmacrodomains, measured with the temperature
de-pendence of the main C-type CD bands (Krumovaet al.,
2010).Inwhole leaves and intact granal chloroplasts, usually
the C-type bands are dominating the spectra and themuch weaker
excitonic bands become visible only upondisintegration of the
macrodomains, e.g. at hypotoniclow-salt medium (Garab et al., 1991)
or at elevatedtemperatures (Cseh et al., 2000). Interestingly, the
ca-pacity to emit isoprene does not seem to affect signifi-cantly
the excitonic (non-C-type) bands arising fromsupercomplexes as also
monitored by CD spectroscopy.Namely, the trimer-to-monomer
transition temperaturesof LHCII, between 55�C and 60�C, as tested
with theexcitonic band pair (+)482/(2)470 (Garab et al., 2002)was
unchanged (data not shown), indicating that the
trimeric LHCII is resistant to heat independently ofisoprene
presence, in both tested species.
Electrochromic Shift and Membrane Conductance
Electrochromic absorbance changes at 515 nm(DA515) are used to
monitor ion permeability of mem-branes (Junge, 1977; Witt, 1979;
Peters et al., 1984)because of the effect on the electrical
potential acrossthe thylakoid membranes. In particular, the
decaykinetics of the absorbance changes are proportionalto the ion
flux across the thylakoid membranes and aresensitive indicators of
thylakoid membrane intactness(Peters et al., 1984), i.e. of the
ability of membranes tomaintain the light-induced transmembrane
electricfield. In dark-adapted leaves the ATP synthase isturned off
and so membrane effects are more prom-inent than in light-adapted
leaves. The decay is slowerin dark-adapted leaves than
light-adapted leaves butis variable among species. Therefore, the
absolute de-cay rate cannot be compared among species but thedecay
for one species can be compared under differ-ent conditions such as
moderate or high temperatureand with or without isoprene
present.
Increased permeability has been found to occurparticularly at
high temperatures, concurrently withdenaturation of membrane lipids
(Bukhov et al., 1999;Schrader et al., 2004; Zhang and Sharkey,
2009; Zhanget al., 2009). The increased membrane permeability
ofheat-treated samples can be due to conformationalchanges of
membrane lipids, opening of ion channels,or changes in lipid-lipid
interactions (Santarius, 1980).At moderate (20�C–25�C) temperature,
the thylakoidmembranes exhibit similar electrochromic
absorbancechanges at 515 nm (DA515), in spite of the presence
(IspSArabidopsis, and control water-fed Platanus leaves)or absence
(wild-type Arabidopsis, and fosmidomy-cin-fed Platanus leaves) of
isoprene. Thus, the mem-brane permeability to ions is low and is
not affectedby isoprene presence at growth temperature. How-ever,
at high temperature (40�C–45�C) wild-type Arab-idopsis membranes,
and isoprene-inhibited Platanusmembranes became more permeable to
ions, whereasisoprene-emitting Platanus membranes were
lessaffected, and IspS membranes remained unaffected.Thus, isoprene
presence is associatedwith better mem-brane integrity under
heat-stress conditions, as shownby lower ion leakage at high
temperatures.
TL
TL measurements also support the thermotolerancehypothesis,
demonstrating the protective role of iso-prene in increasing the
membrane thermostability.TL is a sensitive and reliable tool for
monitoring thefunctionality of PSII donor and acceptor side
compo-nents (Sane and Rutherford, 1986; Vass and Inoue,1992;
Ducruet, 2003; Sane, 2004). Illumination of dark-adapted samples
with single-turnover flashes gener-ates charge pairs within PSII
reaction centers that are
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energetically stabilized on the primary and secondaryquinone
acceptors of the chloroplast’s electron trans-port chain. The
samples are rapidly cooled down totrap separated charges that, on
heating, yield a TLlight emission at characteristic temperatures
(for de-tails, see Sane, 2004). Two major TL bands, B and Q,appear
in TL curves as a result of the recombination ofthe trapped
electrons and stabilized positive holes onthe reduced quinone
acceptors (QA or QB) and on theS2 (or S3) oxidation state of the
water-splitting enzyme.The B band is generated by the recombination
of QB
2
with S2 state of the water-splitting complex. Evensmall changes
in the redox properties of the radicalpairs affect the intensity
and the peak position of TLbands. This complexity of information of
TL emissioncurves is widely used for detection of structuralchanges
in both the donor and acceptor side of PSII.
We tested with TL measurements the impact ofisoprene presence in
leaves that were exposed tophysiological or high temperatures
before runningthe assay. In our experiment with Arabidopsis
plantsexposed to physiological temperatures, the peak posi-tion of
the B band was up shifted by about 10�C in IspSleaves, compared to
wild-type leaves. Alterations inthe peak positions of a TL band
indicates changes inthe stability, redox potential, or activation
energy ofthe recombining charge pairs, while the peak ampli-tude is
proportional to the concentration of the corre-sponding charge
pairs (Rutherford et al., 1984). Theobserved stabilization of the
charge separation statebetween the redox components on PSII donor
andacceptor side in IspS Arabidopsis clearly indicates
anisoprene-induced modification that may enable plantsto perform
efficient primary photochemistry of PSII athigher temperatures. In
Platanus leaves exposed tophysiological temperatures, however, the
B band wasnot shifted to lower temperatures when isoprene
waschemically inhibited, as we would have expected. Asin the case
of CD spectroscopy, we attribute this resultto the possibility that
the chemical inhibition of iso-prene does not interfere with the
molecular structureof membrane architecture that was assembled
whenisoprene was still available. Specifically, our resultsmight
indicate that the isoprene-related membranemodification affecting
PSII reaction centers, and gen-erating the shift in the energy
levels of the S2(3)QB
2, is along-term, structural effect that cannot be altered
byfast removal of isoprene. Alternatively, however, thelow rate of
isoprene synthesis in the fosmidomycin-fedPlatanus leaves might be
sufficient to stabilize chargeseparation. In any case, a change in
TL peak position isnot directly associated with the presence of
isopreneunder physiological conditions.
When TL measurements were repeated in samplespreviously exposed
to heat stress, the B band clearlypeaked at a lower temperature in
plants that do notemit isoprene, whereas the B-band peak position
wasnot affected in isoprene-emitting leaves of both Arab-idopsis
and Platanus. Absence of isoprene caused astrong reduction of the
intensity of the B band in both
plant species that were subjected to the heat treat-ment. It has
been shown that the peak temperature ofB band increases in
stress-tolerant photosyntheticorganisms; for example in
thermophilic cyanobacte-ria or desiccation-tolerant species
(Govindjee et al.,1985; Sass et al., 1996; Maslenkova and
Homann,2000; Peeva and Maslenkova, 2004). It has also beenshown
that stresses do not affect the energetic levelsof the stabilized
S2QB
2 charge pairs in stress-tolerantplants (Sass et al., 1996;
Georgieva et al., 2005).
It is of interest that the heat stress also affected
TLparameters in isoprene-inhibited Platanus leaves thatdid not show
TL changes at physiological tempera-tures. However, the TL
intensity of the B band inisoprene-inhibited Platanus leaves was
less affectedafter heat treatment, than in wild-type
Arabidopsisthat constitutively do not emit isoprene. We
interpretthis result as showing that a continuous biosynthesisof
isoprene is needed to maintain membrane stabilityunder heat stress,
whereas the residual isoprene thatis likely stabilizing membranes
under physiologicaltemperatures in isoprene-inhibited Platanus (see
above)may only partially fulfill this role under elevated
tem-peratures.
Since the production of isoprene is strongly stimu-lated at
elevated temperatures in the range of 30�C to40�C (Monson et al.,
1992; Singsaas and Sharkey, 2000),we suggest that isoprene emission
represents an adap-tive mechanism of membrane stabilization under
hightemperatures. Isoprene is highly lipophilic so that itmay
partition into chloroplast membranes and there-fore it could act
within the thylakoid lipid phase byrendering membranes more viscous
and changing thefeatures of secondary electron acceptor QB. It
wasshown that this plastoquinone molecule is the mostsensitive to
the lipid environment (Gombos et al.,2002). However, this does not
rule out other volatileisoprenoids (e.g. monoterpenes,
sesquiterpenes), zea-xanthin synthesis, accumulation of soluble
thermo-protectants such as sugars or free amino acids, orchanges in
membrane lipid composition involvementin membrane stabilization
(Ducruet et al., 2007).
CONCLUSION
There currently is a controversy about the mecha-nism of action
of isoprene (Loreto and Schnitzler, 2010).Vickers et al. (2009b)
suggests that the mechanismcould be interaction/quenching of
reactive oxygenspecies that allows plants to tolerate heat and
oxidativestress. Sharkey et al. (2008) suggest that isoprene
mayaffect membrane organization and function and thatthis allows
plants to tolerate heat and reduces theformation of reactive
oxygen. The molecular dynamicsmodeling of Siwko et al. (2007) and
the octanol/waterpartitioning (Copolovici and Niinemets, 2005)
showthat isoprene will accumulate inside membranes butuntil now
there were no data showing that this couldaffect biophysical
signals of membrane function.
Velikova et al.
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Three biophysical techniques, which are often usedto study
thylakoid membrane function, show thatisoprene can extend thylakoid
membrane functionalityto higher temperature while having little or
no effect atmoderate temperature. While the three
techniquesspecifically investigated different aspects of the
pho-tochemical apparatus, they converge in indicating thatisoprene
improves the integrity of the photochemistryof photosynthesis under
heat-stress conditions. TheCD spectra in particular show that
isoprene can affectlarge-scale thylakoid membrane organization
while notaffecting the short-range interactions that arise
frompigment-protein complexes and their supercomplexes.As more is
learned about how heat affects photo-
synthetic function and what mechanisms improvethermotolerance it
will be possible to interpret thesebiophysical measures of isoprene
effects in terms ofimproved thermotolerance and ultimately in terms
ofthe evolutionary pressure that favors isoprene emis-sion from
plants in some conditions. These experi-ments show that there are
biophysical mechanisms bywhich the integrity and functionality of
the thylakoidmembranes are better preserved at high temperaturein
the presence of isoprene. This strongly supports theidea that
isoprene improves thermotolerance of plants(Sharkey and Singsaas,
1995; Loreto and Schnitzler,2010) and favors effects on membrane
function as thereason. This suggests that isoprene-emitting
plantswill cope better with temperature extremes that maybecomemore
frequent in the near future due to currentclimate change (e.g. De
Boeck et al., 2011).
MATERIALS AND METHODS
Plant Material and Growth Conditions
Wild-type Arabidopsis (Arabidopsis thaliana; ecotype
Wassilewskija), which
does not naturally emit isoprene (wild type), and genetically
modified
Arabidopsis plants (IspS) producing isoprene as a natural
metabolite were
used in this study. Arabidopsis IspS plants were transformed
inserting the
kudzu (Pueraria lobata) ISPS genomic sequence under the control
of a consti-
tutive promoter (Sharkey et al., 2005). The T4 generation was
used for the
experiments. Plants were grown in a climate chamber (Percival
Scientific)
under a photoperiod of 8 h light at 22�C and 16 h dark at 20�C.
Light intensitywas 150 mmol photons m22 s21 and relative humidity
was 50% to 60%. Fully
grown rosettes were used for the experiments.
Three-year-old Platanus (Platanus orientalis) plants were grown
in 5-L pots
with sand and peat-based commercial soil (1:1) in a greenhouse
under con-
trolled environmental factors. In particular the greenhouse was
thermostated
with air conditioning modules and the light intensity was
supplemented,
if needed, with artificial lamps characterized by a solar
spectrum (Osram
PowerStar HQI-TS 150W). Growth temperature was 25�C/22�C
(day/night),daily light intensity was 350 mmol photons m22 s21,
with a 12-h photoperiod,
and relative humidity was maintained around 65%. The experiments
were
performed on fully expanded leaves.
Plants were regularly watered to pot water capacity and
fertilized once a
week with full-strength Hoagland solution.
Fosmidomycin and High-Temperature Treatments of
Platanus Leaves
To inhibit isoprene emission from Platanus leaves, fosmidomycin,
a specific
inhibitor of 2-deoxyxylulose 5-phosphate reductoisomerase
(Zeidler et al.,
1998), was used. Platanus leaves were cut under water and
maintained with
the petiole in a glass vial with 15 mL of distilled water for 1
h. Fosmidomycin
(5 mM) was then added to the water and taken up by the leaf
through
transpiration stream. After 1 h of incubation isoprene emission
was inhibited
to approximately 10% of the original level, as monitored by gas
exchange and
gas chromatography (Loreto and Velikova, 2001). Cut leaves with
the petiole
immersed in a vial with distilled water only were used as a
control (isoprene-
emitting leaves). Isoprene-emitting and isoprene-inhibited
leaves were used
for all measurements.
Measurements
Gas-Exchange and Isoprene Emission Measurements
CO2 and water exchange were measured by using a LI-7000 infrared
gas
analyzer (LI-COR). Two entire Arabidopsis plants were placed in
a 1.7-L glass
cuvette. Plants were exposed to synthetic air made by mixing O2,
N2, and CO2from cylinders deprived of contaminants. The absence of
contamination by
trace gases that could interfere with isoprene measurements was
tested every
time a cylinder was changed, using the spectrometric analysis
outlined below.
Atmospheric concentrations of the three gases (20%, 80%, and 390
mmol
mol21, respectively) were set with mass flow controllers. The
air flow was set
at 2 L min21. During gas-exchange measurements leaf temperature,
light
intensity, and relative humidity were maintained at constant
levels close to
growth conditions (22�C, 150 mmol m22 s21, and 56%,
respectively).Steady-state photosynthesis, stomatal conductance,
and transpiration in
Platanus leaves were measured by a portable gas-exchange system
(LI-6400)
equipped with a 6-cm2 cuvette. Measurements were made in ambient
CO2concentration (390 mmol mol21) on individual leaves enclosed
into a leaf
cuvette under a rate of 0.44 L min21 air flow, relative humidity
within the
cuvette at 50% to 55%, a leaf temperature of 25�C, and 500 mmol
photons m22
s21 light intensity.
Isoprene emission was measured on both Arabidopsis and Platanus
plants
online, by diverting the air at the exit of the gas-exchange
cuvettes into a
proton transfer reaction-mass spectrometer (Ionicon). Details on
isoprene
analysis by proton transfer reaction-mass spectrometer can be
found in Tholl
et al. (2006).
Data represent means6 SE of measurements on seven different
plants. Thesignificant differences between means were determined by
Student’s t test.
Means significantly different at P, 0.05 and 0.01 confidence
levels are labeledby one or two asterisks, respectively.
CD Spectroscopy
CD was measured in a JobinYvon CD6 dichrograph (JobinYvon
ISA)
equipped with a thermostated sample holder on Arabidopsis
leaves, and with
Jasco 815 dichrograph equippedwith Peltier sample holder on
Platanus leaves.
The spectra were recorded between 400 and 750 nm in 1-nm steps
with an
integration time of 0.3 s and a band pass of 2 nm. For the
measurements of
thermal stability, Arabidopsis or Platanus leaves were placed in
the sample
holder and were dark adapted for 10 min at 20�C. Then the
temperature wasgradually increased up to 80�C (60�C in Arabidopsis
due to higher thermalsensitivity of this plant) in 5�C steps. The
leaves were incubated for 10 minbefore measurements at each
temperature. The transition temperature (Tt) is
the temperature at which the intensity of the CD band is
decreased by 50% of
its value at 25�C (Cseh et al., 2000). The transition
temperatures for the mainC-type CD band at around (+)694 nm were
calculated from the inflection
point of the fitted curve of the temperature dependence of the
CD ampli-
tude. The experiment was repeated on seven leaves of different
plants per
treatment, on each one of the two species. The mean transition
temper-
atures of isoprene-emitting and nonemitting leaves were
separated by
Student’s t test and positively tested against a P , 0.01.The
spectra werecorrected for a flat baseline obtained by setting the
CD values at 400 and
750 nm to zero.
Electrochromic Absorbance Changes (DA515)
Electrochromic absorbance changes (DA515; also named
electrochromic
shift) induced by saturating single-turnover flashes were
measured at 515 nm
on detached leaves in a setup described earlier (Horváth et
al., 1979; Barabás
et al., 1985; Büchel and Garab, 1995). Briefly, the single-beam
kinetic spectro-
photometer consists of a tungsten light source, a grating
monochromator, and
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a photomultiplier with an optical filter for the blue spectral
region, and is
equipped with two xenon lamps (Stroboslave, General Electric)
providing red
flashes of 3 ms half-duration. For recording the signal and data
acquisition, a
differential amplifier and a digital averaging storage scope
(Tektronix AM502
and 2224, respectively) are used in combination with a
home-built timer unit
and a personal computer. The Arabidopsis plants used for these
measure-
ments were dark adapted at 20�C for 20 min. Then, detached
wild-type andIspS leaves were incubated for 5 min either at 20�C or
40�C in the dark, andthereafter measured at 20�C. In Platanus,
control (isoprene-emitting) andfosmidomycin-treated
(isoprene-inhibited) leaves were exposed to 25�C or45�C for 4 h in
weak white light (40 mmol photons m22 s21). The treatmentwith
Platanus leaves was much longer than in the case of Arabidopsis and
was
carried out in dim light to allow complete infiltration of
fosmidomycin in the
leaves. In the last 20 min of the heat treatment, the light was
turned off to
ensure the dark-adapted state of the leaves. Kinetic traces (n =
64) were
collected with a repetition rate of 1 s21, and averaged; the
time constant of the
measurements was adjusted to 100 ms. The measurements were
repeated at
least six times on different leaves. The half-times of the decay
were obtained
by fitting the curves with single exponential decay
functions.
TL
TL was detected with a photomultiplier tube (Hamamatsu
R943-02,
Hamamatsu Photonics) linked to an amplifier in a home-built
apparatus. A
detailed description of the block diagram of the TL equipment,
handling,
temperature regulation, personal computer-based TL data
acquisition, and
graphical simulation is in Zeinalov and Maslenkova (1996). After
a 30-min
dark incubation at room temperature leaf segments were placed on
a sample
holder and covered with a thin plastic plate. TL was excited by
one or two
saturating (4J) single-turnover flashes (approximately 10 ms
half-band with
1-Hz frequency) given at 1�C. After the flash exposure, the
sample fromArabidopsis leaves was quickly cooled to 250�C with the
aid of a metal blockcooled by liquid nitrogen before being warmed
to 70�C with 0.6�C/s heatingrate. TL emission from Platanus leaves
was recorded similarly, but samples
were not frozen before warming from 0�C to 70�C. This way the
distortion ofthe TL signal during freezing that was observed in
Platanus as well as in some
other species (Homann, 1999), was avoided. The nomenclature of
Vass and
Govindjee (1996) was used for characterization of the main TL
curve peaks. TL
measurements were repeated on 10 leaves from different plants
per treatment,
on each plant species. TL curves shown in Figure 4 are
representative of 10
measurements on leaves from different plants.
In another experiment, Arabidopsis and Platanus leaves were
incubated for
5 min at 25�C or 40�C before TL measurements. TL emission was
thenrecorded following the same procedure as outlined above. Means
and SEs for
TL B-peak parameters (temperature positions and amplitude; Fig.
5) were
calculated from five replicates of each nontreated and
high-temperature-
treated leaf samples from different plants. The significant
differences between
means at different temperatures were determined by Student’s t
test. Means
significantly different at P, 0.05 and 0.01 confidence levels
are labeled by oneor two asterisks, respectively. Analysis of the
TL curves was carried out using
a Microcal Origin v.6.0 software package (Microcal
Software).
Supplemental Data
The following materials are available in the online version of
this article.
Supplemental Figure S1. Representative traces of electrochromic
absor-
bance changes measured at 515 nm in isoprene-emitting and
nonemit-
ting leaves.
Received June 27, 2011; accepted July 28, 2011; published August
1, 2011.
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