Umeå
University
The use of fluorescence techniques
for the study of some membrane-bound
photosyntheti c properties and some
effects of copper on the thylakoid
membrane
by
Göran Samuelsson
Postal address
S-901 87
Sweden
Telephone
090-16 50 00
The use of fluorescence techniques
for the study of some membrane-bound
photosynthetic properties and some
effects of copper on the thylakoid
membrane
by
Göran Samuelsson
AKADEMISK AVHANDLING
som med tillstånd av rektorsämbetet vid
Umeå universitet för erhållande av filosofie
doktorsexamen, framlägges till offentlig
granskning fredagen den 15 maj 1981 kl. 10.00
vid Fysiologi-Botanik Hufo, seminarierum B.
Examinator:
Professor L. Eliasson, Umeå
Opponent:
Professor N. Valanne, Åbo
Title:
The use of fluorescence techniques for the study of some membrane-bound photosynthetic properties and some effects of copper on the
thylakoid membrane.
Author:
Göran Samuelsson
Address:
Department of Plant Physiology, University of Umeå, S-901 87 Umeå,
Sweden
Abstract:
Lyoph i)i zed pea chloroplasts were extracted in a stepwise manner in
an organic solvent (petroleum ether) with increasing polarity which
was obtained by addition of small amount of ETOH (0-1 %). Absorption
and low temperature fluorescence emission spectra were measured on
both the extracted thylakoids and on isolated chlorophylI-protein
complexes. Extraction of chlorophyll from the membrane increased (and
the ratio of chlorophyl l a/b decreased)with increasing polarity of
the solvent. The gel scan revealed that after extraction with petroleum
ether, CPa.. was lost from the gel and after extraction with petroleum
ether +1 % ETOH only the CPa/b was left together with SDS-free chloro­
phyll. This shows that the chlorophyll in CPa/b are situated in a less
hydrophobic environment than chlorophyll in CPa|| and CPa|. The long
wavelength absorbing and emitting chlorophyll fraction associated to
CPaj was found to be easily removed from the membrane. This caused a
blue shift in the low temperature fluorescence emission peak and in the
red absorption peak and it was also accompanied by a decrease in carotenoid absorption in isola ted CPa|. It was found in different plant material
lacking $-carotene in CPàj that a strong correlation between ß-carotene
in CPa. and the existence of the long wavelength chlorophyll in iso ­
lated cPa. existed. Based on these data, it was suggested that excited
chlorophyll can transfer energy in excess to ß-carotene by a triplet-triplet transfer.
A method based on in vivo chlorophyll £ fluorescence was developed
for studying photosynthetic capacity in unicellular algae. It was
shown that DCMU-induced fluorescence increase was a good measure of
photosynthetic capacity in four species of green algae tested.
The effect of copper chloride on photosynthetic electron transport
and chlorophyl1-protein complexes was studied in spinach chloroplasts.
Copper(11) inhibited a PS I i reaction H2O—> DPIP, a PS I r eaction
Asc/DPIP —> NADP and the overall electron transport H2O —•> NAOP to
different degrees. Chlorophyll protein complexes were only slightly
affected by copper(l l) but with both copper and ascorbate in the reac­
tion media, a rapid membrane destruction occurred. This was probably
caused by a free radical reaction catalyzed by copper(ll).
Key words:
chlorophylI-protein complexes, sequential extraction, ß-carotene
low temperature fluorescence emission, absorption, in vivo chi. £
fluorescence, copper, electron transport.
ISBN 91-717^-076-7
Umeå I98I. Distributed by the Department of Plant Physiology, University of Umeå,
S-901 87 Umeå, Sweden
CONTENTS
I
Li st of papers
II
Introduction
III
The organisation of the photosynthetic apparatus,
with special reference to the light reaction.
IV
Chlorophyl1 a fluorescence
V
Aspects of photosynthesis inhibition by copper ions.
VI
Summary
VII
Acknowledgements
V.IH-
References
1
I
LIST OF PAPERS
This thesis is based on the following papers, which will be referred
to in the text by given Roman numerals.
.v
I
Uquist, G., Samuelsson, G. (1980). Sequential extraction of
chlorophyl1 from chlorophyl1-protein complexes in lyo phi1 ized
pea thylakoids with solvents of different polarity. Physiol.
Plant. 50; 57-62.
(t
öquist, G., Samuelsson, G., Bishop, N.I. (1980). On the role of
ß-carotene in the reaction center chlorophyll £ antennae of
photosystem I . Physiol. Plant. 50: 63-70.
III
Samuelsson, G., öquist, G. (1977). A Method for studying photosynthetic capacities of unicellular algae based on in vivo
chlorophyll fluorescence. Physiol. Plant. 40; 315-319-
IV
Samuelsson, G., öquist, G. (1980). Effects of copper chloride
I
on photosynthetic electron transport and chlorophyll-protein
complexes of Spinae i a oleracea. Plant and Cell Physiol. 21(3):
kk5-k5h.
2
11
INTRODUCTION
Photosynthesis Is, undoubtedly, one of the most important biological
processes occur ing on earth. Through this series of complex reactions,
which can be found in members of the plant kingdom and in some bacte­
ria, light energy can be conserved. Thus, energy produced by nuclear
reactions occur ing in the sun can be converted Into a form able to
sustain life on earth. This is a technical solution of light energy
harvesting that no engineer has been able to reproduce. Almost every
organic molecule is a product of photosynthesis. Today, intens ified
interest has been focused towards plants and photosynthes i s in l ight
of the "food crisis", the "energy crisis", and the need for raw materials for certain industries.
Both in terre stial and in aquatic ecosystems, photosynthesis is the
bas i s for all heterotrophic existence. Therefore, accurate determina­
tion of primary production is of vital importance since it yields the
actual value of the light energy bound by a plant community or an
ecosystem. In many cases, a direct measurement is difficult to obtain.
Thus, many approximations and indi rect methods have come into use.
One such method presented as paper III.
Light is the driving force for photosynthesis. Absorption of light
i s by no means an unique process reserved for chlorophyll conta in ing
organisms. It occurs around us every second In all types of material
but photosynthes i s i s unique in th at i t involves the utilization of
absorbed light energy. This utilization is allowed by the strict
organization of pigments and electron carriers in photosynthetic
membranes. In most non-ch1orophy11 containing organisms, absorbed
light energy is rapidly converted to heat and no work is done. In
3
plants, however, absorbed light energy îs transferred
from pigment
molecule to pigment molecule with high efficiency until it finally
reaches a "trap", where a special reaction center molecule is oxi­
dized and the electron is captured by an acceptor molecule with a
lower electronic potential. The downhill transport of this electron
is coupled to the production of ATP. Many questions relating to the
organization of photosynthetic pigments in the t hylakoid membranes
still remain unanswered. Papers I and 11 de al with some aspects of
the organization and function of these pigments.
The conversion of light energy to chemical energy which is stored as
lipids, carbohydrates, and proteins also depends on a sequence of
very delicate reactions, both photochemical and enzy matic. Every pro­
cess disturbing the reaction leads to a situation of stress in the
cell. Currently, there is much interest in the effect that pollutants
such as heavy metals may have on primary production processes. The
very complex situation in whole cells makes it difficult to study the
damage or stress situation resulting
from, for example, heavy metals
binding to proteins. By isolating thylakoid membranes and studying the
different effects induced by a given metal, it is easier to give a
direct explanation of the mechanisms involved because the chloroplast
thylakoids are the most well known membranes of the pi ant-cell. Paper
IV deals with the effects of copper on some photosynthetic processes.
Abbreviations: SDS, sodium dodecyl su lphate; PS, photosystem; CP,
chlorophyll-protein; PAGE, Polyacrylamide gel electrophoresis; PE,
petroleum ether; F, fluorescence; SAN, Norflurazon; HEPES, N-2-hydroxyethyl piperazÌne-N-2-ethane-sulphonic acid; NADP, nicotinamide
adenine di nucleotide phosphate; Asc, ascorbate; DPIP, 2,6-dichlorophenol indi phenol ; DCHU, 3~(3,^-dichlorophenyl)-1,1-dimethyl urea.
/
h
III
ORGANIZATION OF THE PHOTOSYNTHETIC APPARATUS WITH
SPECIAL REFERENCE TO THE LIGHT REACTION
In all chlorophyll containing eucaryotic cells, the photosynthetic
reduction of CO2 is performed in the chloroplast. This lens-shaped,
green organelle contains pigment-bearing vesicular membranes called
thylakoids and a matrix or stromal phase which is surrounded by a
double membrane (the envelope). The stroma contain a high concentra­
tion of protein, mostly soluble enzymes required in the Calvin cycle
but also ions and intermediates formed in photosynthesis. The role
of the envelope is both to act as a barrier to keep all enzymes
needed in photosynthesis together for optimal function and as a re­
gulator of the transport out of and into the chloroplast of ions and
organic molecules. Thylakoids closely associated in stacks are cal led
grana and are in contact with those in other grana through intergranal thylakoids or stroma lamellae system, where all components neces­
sary for the photosynthetic light reaction are found. The functional
scheme proposed by Hill and Bendall (i960) for photosynthetic electron
transport is st ill in its main parts relevant. The light reaction con­
sists of two separate photochemical systems coupled in series with
an interconnecting electron transport chain. It is generally assumed
that water acts as the electron donor in a reaction where oxygen is
produced. The terminal electron acceptor is NADP. In this sequence of
reactions, photochemical energy also is transformed to chemical energy
in the form of ATP. This is only possible if light is absorbed and
efficiently channelled to reaction centers. Reaction center molecules
are beleived to be specialized chlorophyll molecules organized in
such a manner that they can take part in redo x reactions.
5
Chlorophyl1 protein complexes
It is now widely accepted that all biological membrane s can be re­
lated to the fluid mosaic model (Singer & Nicholson 1972). According
to this model, proteins can either be loosely attached to the lipid
b i layer membrane (extrinsic proteins) or more or less embedded in
the membrane (intrinsic proteins). During recent years more and
more sophisticated methods have been developed for the extraction
and separation of intrinsic membrane proteins. Solubilization of
chloroplast thylakoids with SDS and subsequent separation by SDSpolyacryl
ami degel electrophoresis has proved to be a very useful
method for studies of chlorophyll-protein complexes which, according
to Anderson (1975) may be referred to as intrinsic membrane proteins.
The use of this method for solubilization and separation of thylakoid
membrane proteins has shown the presence of three major chlorophyll
containing bands (Anderson 1975, Thornber 1975). These bands are the
chlorophyll-protein complex I (CPa^), the light harvesting chlorophyll
a/b-protein complex (CPa/b) and free chlorophyll compiexed to SDS.
Among oligomers of CPa^ and CPa/b a green band was found (Bishop and
öquist I98O) and characterized as the antennae pigments of PS II (CPa11).
In general, it appears that there is compelling evidence that the
photosynthetic pigments in higher plants and green algae are associa­
ted to proteins forming at least three major chlorop hyl1-protein com­
plexes (Markwell et al_. 1979). Two of these, namely C Pa 11 and CPa.^
are the antennae pigments directly associated to the two respective
reaction centers. The third and largest complex which binds 40-60 %
of the total chlorophyll, CPa/b, acts as a light harvesting complex
physically in close association with CPa^, but it can also transfer
absorbed excitât ion energy to PS j ( Butler
1978).
The three
different complexes should not be considered as isolated functional
units but rather as a cooperation between the pigment-protein complexes
in absorption and energy transfer to and between the two photosystems.
This has been recently discussed in a review article by Butler (1978).
The role of the immediate molecular environment in the functional pro­
perties and interactions between chlorophyll complexes has received
little attention. Is it possible that there is a chlorophyl1-proteinlipid interacti on that is importa nt for the structure and function of
chlorphyl1 in the thylakoid membrane? It was sugge sted by Anderson
(1975) that chlorophyll in the antennae forms part of the boundary
lipids of these proteins and interactions between chlorophylls and
lipids was also discussed by Katz (1979).
To date, the questions concerning interactions between chlorophylls
and lipids in the thy lakoid membrane remain unresolved. To learn more
about how Chlorophyll
is associated in the complexes a series of expe­
riments were designed in which lyophilized pea chloroplasts were extrac
ted in a stepwise manner in
solvents with increasing polarity. The sol­
vent used was petroleum ether with increasing amounts of ethanol. The
extracted membranes were sol ubi 1ized and run on a SDS-PAGE. The thylakoids and the purified chlorophyl1-protein complexes were then characte
rized by absorption and low temperature fluorescence emission spectra.
The results are presented in papers I and II.
The principle results obtained by extraction of the thylakoid membranes
with PE + 0 %EtOH can be summarized as:
a)
a decrease and a blue shift of the low temperature fluorescence
emission peak of CPaj and in the long wavelength fluorescence of
extracted thylakoid membranes
b)
a decrease in carotenoid and a blue shift in the red chlorophyl1
absorption peak of CPaj
7
c)
a lack of CPa^
the gel and of the F695 peak in low tempe­
rature fluorescence emission spectra of petroleum ether extracted
thylakoids
d)
a higher proportion of SDS-free chlorophyll in the gel
e)
only about 3 t of the chlorophyll was extracted.
With increasing polarity of the solvent the chlorophylls were extracted
in the order CPa^, CPa^ and CPa/b. Thus it may be concluded that chloro­
phyll in CPa J J is mor e hydrophobic bound than chlorophyl l in t he CPa/bcomplex. The loss of chlorophyll from the membranes is explained by:
a)
a direct effect caused by th e organic solvent and
b)
by an indirect effect due to SDS, solubilizing unextracted and
extracted thylakoids differently.
Comparison of low temperature fluorescence emission spectra of extracted
thylakoids and of chlorophyll protein complexes from similarly treated
thylakoids, extracted, solubilized and run on the gel allow us to deter­
mine if the observed differences depend upon extraction only or the SDS
treatment. Based on the presented results, it is argu ed that extractions
of thylakoid membranes by a nonpolar solvent causes a disruption of the
membrane structure that induces loss of chlorophyll mainly from the
reaction center antennae CPa j j and CPa^., respectively, during SDS-PAGE.
This is seen both in absorption and low temperature fluorescence
emission spectra. The structural disorder and resulting spectral changes
correlate well to a lowering in the carotenoid absorption.
If the lack of earotenolds (e.g. ß-carotene) is responsible for the
spectral differences obtained in absorp tion and low-temperature emission
spectra in isol ated CPaj and thylakoids then the same results should be
obtained in plan ts that for one or another reason lack ß-carotene. This
8
hypothesis was tested and the results shown in paper II. For this pur­
pose, three different types of plant material were used, lyophilized
and PE-extracted pea chloroplasts, wheat plants cultured with and with­
out the herbicide SAN 9789 and the green a1age Scenedesmus ob1iquus
mutant 6E. These plant materials were similar in that sense that is was
not possible to isolate a CPaj that contained ^-carotene.
Absorption and low temperature fluorescence emission spectra on both
isolated CPaj and on thylakoids show a strong correlation between the
lack of ß-carotene in CPaj and the lack of long wavelength absorbing
and emitting chlorophyll.
from results presented in papers I and II, it is possible to speculate
about the organization and function of ß-carotene in CPaj. As a lack
or a decreased amount of carotenoids in CPaj in b oth SAN treated wheat
plants and the algal mutant 6E resul ts in a lack of both CPaj j and
CPa/b but not of CPa^ it can be argued that a core of CPaj can be
formed without carotenoids. However, in CPaj isolate d from PE extracted
thylakoids the long wavelength absorbing and emitting chlorophyll £ is
reduced while chloroplast thylakoids still
show a dominant long
wavelength absorption and low temperature fluorescence emission peak.
One explanation may be that there exists a special long wavelength ab­
sorbing and emitting chlorophyll protein complex that is normally in
close association to CPaj. This long wavelength chlorophyll protein
complex is strictly dependent on a high degree of order between ß-caro­
tene and chlorophyll as also proposed by Junge et a]_. (1977). As a
higher degree of photobleaching is shown in carotenoid deficient mate­
rial than in the controls, it is
obvious that the proposed complex in
association with ß-carotene can function as a trap for excess energy.
This could be explained by a triplet-triplet transfer from excited
9
chlorophyll to $-carotene (Jensen and Wilbrandt 1980). By assuming a
triplet-triplet energy transfer between chlorophyll and ß-carotene and
a singlet-singlet transfer from excited ß-carotene to chlorophyll both
its role as an antennae pigment in PS I and
would be explained.
its role as photoprotectant
10
IV
CHLOROPHYLL a FLUORESCENCE
When light interacts with material one of two things can happen. It
can pass through the material with no absorption taking place or it
can be absorbed. A quantum of light is absorbed in about 10 ^ seconds
and a transition to a higher electronic state takes place. Such ground
state to singlet state transitions are responsible for the visible and
ultraviolet absorption spectra observed for molecules. If the absorbed
energy is not further dissipated by intramolecular conversion, the
electron returns to the ground electronic state with the emission of
energy. This phenomenon is called fluorescence. Because some energy is
lost in the brief period before emission occurs, the emitted energy is
of longer wavelength than the energy absorbed (see fig).
2:nd excited singlet
1 :st excited singlet
— —^
heat
flucre
internal
photochemical
ground level
Figure 1.
conversion
react ion
Different deexcitation possibilities for an excited Chlorophyll
molecule.
This is what happens is a solut ion of extracted chlorophyl 1 in an organic sol­
vent e.g. acetone. Measurements of the quantum yield of fluorescence in a
di lute chlorophyl1 a in acetone solution give values of about 25 %.
If, instead, a suspension of photosynthesizing cells (for example a
green alga) is diluted to the same chlorophyll content as in the ace­
tone solution, fluorescence is also detected but it is of a much lower
intensity than in acetone. The quantum yield of fluorescence in intact
plants is only about 3 % (Govindjee ejt al_. 1973). The lower quantum
yield for chlorophyll £ fluorescence in an intact plant is explained
by the increased possibilities for deexcitation not only through chlo­
rophyll a fluorescence and heat but also through conversion of the
11
absorbed light to chemical energy in the photosynthetic process. In
a healthy plant, about 85 % of the absorbed light energy is converted
to chemical energy through photosynthesis.
In a green plant, fluorescence can be separated into a constant and
a variable part. The constant part, often denoted "dead fluorescence"
seems to be highly independent of photosynthetic activity (Clayton
1969; Lavorei and Joliot 1972). Both constant and variable fluore­
scence have broad maxima at 685 nm at room temperature. It i is assumed
that the kinetics of variable fluorescence during the first seconds or
minutes reflects changes in the photosynthetic activity (Kautsky et al.
I960) and it has further been shown that there exists an inverse rela­
tionship between variable fluorescence yield and the photochemical
reactions during the first seconds of illumination (McAlister and
Myers 19*»0). Fluorescence studies of this type have been often used
to give information about the physiological condition of the photosyn­
thetic apparatus in b oth algae and higher plants. In healthy cells,
fluorescence is low. In such a system electrons and protons flow
smoothly from water toward the reduction of Ct^. However, if poisons
interfere with photosynthesis or if nutrient defiency occurs, the
fluorescence yield increases (Slovacek and Hannan 1977). If all elec­
tron transport is stopped by, for example, complete inhibition of
photosynthesis by 10
M DCMU in vivo, chlorophyll £ fluorescence will
reach a maximum. The difference between the DCMU-fluorescence and
fluorescence without the addition of DCMU will thus vary with physio­
logical condition. In healthy cells, it will be large and it will get
smaller as the condition for the cells get poorer. When photosynthesis
is completely inhibited it will be zero. Recent data, however indicates
that this explanation of the fluorescence phenomena may only be rele­
vant when the photosynthetic changes being observed occur mainly on
12
the reduc ing s ide of PS 11 .
Paper III describes a method based on in vivo chiorophyi 1 a_ fluore­
scence for studies of algal activity and photosynthesis in natural
waters. In contrast to the most often used methods for estimating
primary product fon in the sea, the described fluorescence method is
rapid and can easily be automated. In addition, the technique simul­
taneously gives information about the physiological condition of the
algal population as well as jn vivo chlorophyll concentration. This
method has been used both in the laboratory and in the field and has
shown good correlation to both primary production (Rey 1978) and photosynthetic rates in general (Roy and Legendre 1979)-
Low temperature fluorescence emission
At room temperature chlorophyll £ fluorescence, is mainly emitted from
PS II with a broad maximum around 680 nm. When suspensions of chloroplasts or algal cells are exposed to lower temperatures, the fluore­
scence emission at longer wavelengths tends to increase and at -196°C
(liquid nitrogen temperature) it is possibl e to see three different
peaks. These are seen at wavelengths of 685, 695 and 735 (in algae
around 720 nm) (Butler 1978). At this low temperature, deexcitation
through photosynthesis is blocked and molecular movements are decreased.
This leads to a higher fluorescence yield and a better resolution (nar­
rower band-width) of the peaks. Research in rece nt years concerning
the in vivo organization of chlorophylls has led to the development of
a model where chiorophy11, mainly in association with proteins and li­
pids, is arranged in three complexes.
The three chlorophyll protein
complexes have been separated and their fluorescence characteristics
have been determined (Bishop and b'quist 1980). These studies have shown
13
that F685 , F695 and F735 (in algae F720) results from the CPa/b, CPaj j
and CPaj, respectively. F695 which occurs under -10O°C is most likely
due to chlorophyll with no fluorescence at room temperature. The long
wavelength fluorescence, which in most plants occurs at low tempera­
tures, can either be caused by a transfer of non-fluorescent forms to
fluorescent forms at low temperatures or by a decreased deexcitation
through the long wavelength absorbing and emitting chlorophylls.
V
ASPECTS OF PHOTOSYNTHESIS INHIBITION BY COPPER IONS
Copper is required in most biological systems as a micronutrient. Some
of the proteins and enzymes in the cell need small amounts of copper
for maintaining their function. As do many other metal ions, copper
becomes toxic at high concentrations. To obtain a complete picture
of the effect that copper pol lut ion has on an ecosystem both chemistry
and biology have to be considered.
In pollutio n research many different pröblems have to be dealt with.
Speciation of the ions, complexes formed, uptake rates, excretion rates,
inhibitory effects and mechanisms for the inhibition are only examples
of the complexity of the problem. To understand the potential effect
of copper pol lution on -photosynthesising organisms, it is necessary to
examine the effect of copper at different cell organization levels. In
paper no IV, I have
studied the effect of copper on some photosynthetic
properties in isol ated spinach chloroplasts. Thus, the effect of copper
on the electron transport and pigments could be studied directly with­
out the interference of other processes taking place in whole cells
and organisms. Generally it can be concluded that copper (II) is a
very reactive ion that binds to proteins rapidly. This often leads to
the very unspecific inhibition pattern partly demonstrated
in Paper IV.
In all electron transport studies , the chloroplasts were pretreated
with different copper (II) concentrations in the dark for 5 min before
measurements were made. A PS 11 reaction H^O
—> DPIP was inhibited
by copper (I I) ions both in Hepes and phosphate buffers. Reactivation
of electron transport and oxygen evolution by Mn(ll) addition was only
possible in a phosphate buffer. This indicates that binding of copper
(II) to the membrane was different in phosphate and non phosphate con­
taining media. As oxygen evolution was restored by Mn(ll), it was
15
suggested that Mn(11) did not act as an electron donor.
It has been shown by Åkerlund and Jansson (1981) that two popypeptides
on the inside of thylakoid membrane are closely related to the function
of the water splitting system. Copper (It) might bind to one or both of
them and inhibit electron transport, but can be replaced by M n(M) and
retain its function if phosphate is present in the media. A PS
I r eac­
tion Asc/DPIP —> NADP was also inhibited by copper ions and in th at
reaction the inhibition was bigger than in the PS II reaction. The marked
inhibition of the PS I reaction was suggested not to be purely dependent
on copper but rather on an ascorbate free radical formation catalyzed by
copper. When the whole chain electron transport was tested for copper
inhibition, a slight increase was found at low copper (II) concentrations.
This was explained by an uncoupling of photophosphorylation at these
concentrations of copper. The inhibitory effect caused by copper on
photosynthetic electron transport seems to be at several sites. One site
of inhibition is between the water splitting system and the reaction
center, another is on the phosphorylating sites and a third is on PS I.
As mentioned, the inhibition of the PS I reaction could be explained by
an ascorbate radical formed that rapidly destroys the membrane . This
was tested on chloroplasts treated with copper (II) and copper (II) plus
ascorbate on pigment protein complexes and chlorophyll absorption. Poly­
acrylamide gel electrophoresis of solubi1ized chlorophyll protein complexes
showed that membrane damage had occurred in chloroplasts treated with
copper and ascorbate. In copper treated chloroplasts, however, the
damage was less marked. One would also expect destruction of membranes
only treated with copper because of 1ipid peroxidation processes cata­
lyzed by copper (II) (Sandmann and Böger, 1980). This can be seen on the
gel scan where the amount of chlorophyll in association with SDS near
the front has increased in comparison with CPaj and LHCPa/b complex.
16
VI
SUMMARY
k
Lyophilized chloroplast thylakoids were extracted in a stepwise manner
in an organic solvent with increasing polarity. Petroleum ether was
used as solvent and the polarity was increased by addition of different
amounts of ethanol (0 - 1 %). In a nonpolar solvent (PE + 0 % EtOH)
mainly chlorophyll a^was extracted, but when the polarity of the medium
was increased more and more chlorophyll wa s extracted and the chloro­
phyll a/b ratio decreasing in the extract. This indicates an increased
extraction of the CPa/b complex. The gel scan reveals that an order of
CPa jJ, CPa J and CPa/b chlorophyl l are extracted in parai lei with increasing
polarity of the solvent. Thus chlorophyll in CPa/b seems to be bound in
a much less hydrophobic environment than CPaj( and CPaj. The long wave­
length fluorescence emission peak associated with photosystem I was
blue shifted and decreased both in the isolated CPa^ and the extracted
thylakoids when extracted in a low polar solvent. Absorption spectra of
isolated CPaj also revealed that the long wavelength absorbing chloro­
phyll was lacking as was the absorption peak of carotenoids. It was
further shown that ß-carotene binding to CPaj is important for main­
taining a structural order of the long wavelength absorbing and emitting
chlorophyll a _ fraction associated to CPaj. It was suggested that excess
excitation energy can be transferred by a triplet-triplet transfer from
excited chlorophyl1 a_ to ß-earotene thereby acting as a safety valve
for photodestruction.
A method based on in vivo chlorophyll a fluorescence was used for stu­
dies of photosynthesis in some unicellular green algae. It was demon­
strated that there is a good correlation between photosynthesis measured
as
H
CO2 uptake and DCMU induced fluorescence increase in batc h cultures.
The effects of copper (II) ions on electron transport and chlorophyll-
17
protein complexes were studied in is olated spinach chloroplasts. The
inhibitory effects increased with increasing copper (II) concentration
in both a PS 11 and a PS I react ion. The effect of copper was different
in Hepe s and phosphate buffers when the PS II react ion 1^0—> OPIP was
studied. By addition of Mn(ll) it was possible to restore both electrontransport and oxygen evolution in a phosphate buffer but not in Hepe s.
The PS I reactio n Asc/DPIP—•> NADP was more inhibited by copper (II)
than the PS II r eaction. It was suggested that at least part of this
inhibition was caused by a synergistic effect between copper (II) and
ascorbate, forming a free radial reaction. This was also illustrated
on isolated chlorophyll protein complexes. At low copper (II) concentra­
tions the electron transport from 1^0 —> NADP increased. This was ex­
plained by an uncoupling of photophosphorylation in this concentration
interval.
VII
ACKNOWLEDGEMENTS
I wish to express my sincere gratitude to ali members of the Department
of Plant Physiology, University of Umeå. I am especially grateful to my
/
supervisor Dr Gunnar dquist for support and fruitful collaboration and
to Prof Lennart Eliàsson for general support. I also wish to thank Mrs
Ingali 11 Nilsson, Miss Margareta Lundberg and Miss Agneta Ehman for
preparing the manuscripts. For correcting the English I am deeply
grateful to my friend Dr Katherine Richardson.
19
VIII
REFERENCES
Anderson, J.M. (1975) Biochim. Biophys. Acta *16; 191-235.
Bishop, N.J., dquist, G. (1980) Physiol. Plant. *9; *77-*86.
Butler, W.L. (1978) Ann. Rev. Plant Physiol. 29; 3*5-378.
Clayton, R.K. (1969) Biophys. J. 9; 60-76.
Duysens, L.N.M., Sweers, H.E. (1963). In: Studies on Microalgae and
photosynthetic Bacteria. Tokyo: Univ. Tokyo pp. 353-377.
Govindjee, Papageorgiou, G., Rabinowitch, E. (1973). In: Practical
fluorescence pp. 5*3-569.
Hill, R., Bendel t, F. (I960) Nature 186; 136-137.
Jensen, N.H., Wilbrandt, R., Pagsberg, P.B. (1980) 32; 719-725.
Junge, W., Schaffernicht, H., Nelson, N. (1977) Biochim. Biophys. Acta
*62; 73-85.
Katz, J.J. (1979). In: Advances in the Biochemist ry and physiology of
plant lipids, L-Â. Appelqvist and C. Liljenberg eds. pp. 37-56.
Kautsky, H., Appel, W., Amann, N. (I960) Biochem. Z. 332; 277-292.
Kok, B. (1963) Nat. Acad. Sci. 11*5; *5-55.
Lavorel, J., Joliot, P. (1972) Biophys. J. 12; 815-831.
Lumry, R., Mayne, B., Spikes, J.D., (1959) Discussions Faraday Soc. 27; 1*9.
Markwel1, J.P., Thornber, P., Boggs, R.T. (1979) Proc. Natl. Acad. Sci.
USA. 76, 3; 1233-1235McAlister, E.D., Myers, J.(19*0) Smithsonian Inst. Misc. Col 1. 99(6); 1-37.
Rey, F. (1978). In: "Metodutveckling grundad på in vivo klorofyl1 fluorescence och luminiscence för mätning av alger och högre
växters fysiologiska tillstånd"; 35—*5Roy, S., Legendre, l.(1979) Marin Biology 55; 93-101.
Sandmann, G., Böger, P. (1980) Plant Physiol. 66, 797-800.
Singer, S.J., Nicholson, G.L. (1972) Science 175; 720—731 Slovacek, R.E., Hannan, P.J. (1977) Limnol. Oceanogr. 22(5); 919-92*.
Thornber, J.P. (1975) Ann. Rev. Plant Physiol. 26; 127-158.
Åkerlund, H.E., Jansson, C. (1981) Febs Letters 2*:2; 229-232.