B‘OCHEMCAL STUDIES OF
EMISSBN FROM ‘SOLM

Thesis for the Degree 9f M. S. ‘- ‘
MOH‘GAN STATE UNNERSWY I
‘ PETER FELKER
197-3

 

 

EDCHLOROPLASTS . _- _- I = .-

.....

 

L I B R A R Y
Mid‘ugg‘m JLdbC
University

 
       

ABSTRACT

BIOCHEMICAL STUDIES
OF THE DELAYED LIGHT EMISSION .
FROM ISOLATED CHLOROPLASTS

BY
Peter Felker

The purpose of the research reported in this thesis
was to attempt to correlate delayed light emission (DLE)
with the much better understood effects of uncouplers,
electron transport inhibitors, and ion movements on iso-
lated chlor0plast biochemistry.

Electron transPort inhibitors appear to behave in
two classes with respect to delayed light emission. It
has long been known that electron transport inhibitors,
which act between plastoquinone and photosystem II, al-
ways drastically inhibit DLE. New evidence is presented
which shows that inhibitors, which act between plasto-‘
quinone and photosystem I, actually stimulate the DLE under
some conditions.

All uncouplers of photophosphorylation except
NH4Cl markedly inhibit the DLE. Under proper conditions
NH4C1 can markedly stimulate the DLE. The conditions for

this stimulation of DLE by NH4Cl were very similar to

the conditions for light-activated NH4+ uptake by

Peter Felker

chloroplasts. Also, methylamine which uncouples phospho-
rylation in chloroplasts particles, abolishes the DLE in

chloroplasts, but not in subchloroplast particles.

Several nucleotides have also been found to stimulate
delayed light emission. ATP and ADP gave the greatest
stimulation of the DLE from chlorOplasts. This new ob-
servation of DLE stimulation by ATP and ADP appears to be
well correlated with the previously known stimulation of the
H+ uptake and inhibition of electron transport by ADP
and ATP. ‘

A scheme is presented to explain these phenomenon
in which a cation uptake creates a membrane potential which
in turn stimulates a back-reaction of holes and electrons
in the membrane to give an excited chlorOphyll molecule

and thus yield DLE.

 

BIOCHEMICAL STUDIES
OF THE DELAYED LIGHT EMISSION

FROM ISOLATED CHLOROPLASTS

BY

\0‘
Peter’Felker

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE
Department of Botany and Plant Pathology

1973

 

f0
I”)
N ACKNOWLEDGEMENTS
«e
U
I sincerely wish to thank my guidance committee
for advice and support through the course of this study.
I especially wish to thank Dr. Norman E. Good, my major
professor, for his advice and critical suggestions. I
wish to thank Dr. Seikichi Izawa for his excellent day
by day counseling on chlorOplast biochemistry and for
construction of the NH4+ selective electrodes. I wish
to thank Dr. Alfred Haug for so generously making his
phosphoroscope available to me and for his help with
controls and elctronics. Finally I wish to thank Dr.
Robert Bandurski for his encouragement and support.
This work was supported in part by a teaching
assistantship granted by the Department of Botany and
Plant Pathology, and by the United States Veterans

Administration.

ii

TABLE OF CONTENTS

LIST OF TABLES O O O O O O O 0
LIST OF FIGURES O O O O O O O O

ABBREVIATIONS . . . . . . . . . .

INTRODUCTION 0 O O O O O O O O O 0
METHODS . . . . . . . . . .
RESULTS . . . . . . . . . . . . . .

I. Electron Transport Inhibitors on Delayed

Light Emission . . . . . . . .
II. Effects of Ammonia and Organiz Amines on
Delayed Light Emission . . .

III. Effects of Nucleotides on Delayed Light
Emission . . . . . . . . . . .

DISCUSSION . . . . . . . .. . . . . .

LITERATURE CITED . . . . . . . . . . .

iii

Page

iv

vii

23

23
31
45

58

76

 

LIST OF TABLES

Table Page
I. Stimulation of Delayed Light Emission by ADP
and ATP 0 O O O O I O O O O O O 52
II. Stimulation of Delayed Light by Various
. . . . . 53

Nucleotides . . . . .

iv

 

LIST OF FIGURES

Figure Page

1. Diagram of the electrode and the apparatus
used to measure the uptake and release
of NH4+ by chloroplast suspension. . . . ll

2. Separation of the DLE and prompt fluorescence
by the instrument described . . . . . 13

3. Tests of the linearity of the photomultipliers
used for measuring DLE and fluorescence . l7

4. Waveform distortion test of the DLE apparatus 20

5. Effects of various electron transport inhibitors
on the DLE and prompt fluorescence - - - 25

6. Effects of increasing DBMIB concentrations
on the DLE and prompt fluorescence . . . 28

7. Effects of NH4Cl and other uncouplers on
the DLE decay curves with methylviologen as
electron acceptor . . . . . . . . . 32

8. Effects of amines of various pKa's on the
3.0-3.5 msec DLE . . . . . . . . . 34

‘9. Stimulation of DLE by NH4C1 at high pH and
inhibition of DLE by NH4C1 at low pH . . 37

10. A comparison of the effects of NHZOH and
NH4Cl on the DLE . . . . . . . . . 40

11. A comparison of DLE, electron transport and
NH4+ uptake as functions of pH. . . . . 43

12. A comparison of the initial rate and
extent of NH4+ uptake with the spike and
"steady state level" of the 3.0 msec DLE,
with and without high salt (50 mM LiCl). . 46

 

Figure

13.

14.

15.

16.

Page

Presence of DLE with CH3NH3C1 in sub-
chloroplast particles . . . . . . . 48

Effects of ADP, Pi, and ATP on the DLE and
fluorescence. . . . . . . . . . . 54

The effects of ADP and ATP on the DLE,'H+—
uptake and electron transport . . . . . 56

Postulated general configuration of the
thylakoid membrane discussed in this thesis 68

vi

ADP

5' AMP
ATP
Chl
Cyt
DBMIB
DCMU
DLE

E. T.
FeCy
Fluor.
GDP
HEPES

ITP

MES

msec

ABBREVIATIONS

adenosine-5'-diphosphate

adenosine-S' -monophosphate
adenosine-S'-triphosphate

chlorophyll

cytochrome

3, 6-dibromo-2-methyl-5-iSOpropylbenzoquinone
3-(3, 4-dichlorophenyl)-l, l-dimethylurea (diuron)
delayed light emission

electron transport

potassium ferricyanide

fluorescence

guanosine-5'-diphosphate
N-hydroxymethylpiperazine-N'-ethanesu1fonic acid
inosine-5'-triphosphate

Michaelis constant
N-morpholinoethanesulphonic acid

millimolar

millisecond

methylviologen

plastocyanin

orthOphosphate

photosystem I, II

photomultiplier

vii

 

PQ

TAPS

Tricine
uM

UTP

plastoquinone

reduced primary electron acceptor for photo-
system II

substrate concentration

N-tris (hydroxymethyl) methyl-3-amin0pr0pane-
sulfonic acid

tris (hydroxymethyl) methylglycine
micromolar .
uridine-5'-triphosphate

velocity of an enzyme reaction

maximal velocity of an enzyme reaction

oxidized primary electron donor for photosystem II

viii

INTRODUCTION

It has long been known that photosynthetic organisms
emit light for a short time after illumination has ceased.
The emitted light is very weak and some of it continues for
minutes. However, the highest intensity is immediately
after the light is turned off, with over half of the lumin-
escence occuring in a small fraction of a second. .The
luminescence which can be observed in green plants, in
photosynthetic bacteria, and in isolated chloroplasts, has
the emission spectrum characteristic of the type of chloro-
phyll present.

This phenomenon was first obserVed by Strehler and
Arnold (1951) while they were using the firefly luminescence
assay in an attempt to detect ATP in chloroplast preparations.
They determined that the action spectrum for the Weak red
luminescence of the chlorOplasts was the same as the action
spectrum for photosynthesis. In living algae there was a
sharp temperature Optimum at 37°C, an inhibition by ultra—
violet light, and a stimulation by C02, all of which
strikingly parallel the effects of these conditions on
photosynthesis. These workers postulated that the lumin—
escence (now called delayed light emission or DLE) involved
at least one reversible enzyme reaction.

1

Many other workers subsequently studied the phenom-
enon. Frank and Brugger (1958) studied the DLE from a
variety of plant material under N2, 02, and CO2 atmospheres
and with a variety of poisons and potential electron
acceptors. Tollin gt_§l. (1958) found the DLE measured
0.1 sec or longer after illumination was maximum at 25°C,
but at shorter times the temperature optimum was -50°C.

They also found the quantum yield to be 10-6

for Chlorella
and 10-7 for spinach chloroplasts. Bertsch and Azzie (1964)
also found that the DLE, measured 0.5 sec after illumination,
peaked fairly sharply between 15°C and 35°C, depending on
the color of the exciting light. They found the decay
kinetics of the luminescence to be affected by the color
of the exciting light. When the exciting light was 600 nm,
the decay curve was monotonic. However, when far red light
was added to the 600 nm exciting light, a shoulder appeared
in the decay curve. The decay curves after illumination
at different wavelengths actually crossed each other.
Goedheer (1964) studied the action spectrum of DLE.
He found that 660 nm stimulated DLE but longer wavelengths

decreased the DLE. The most effective wavelenghts for

decreasing DLE was 700 nm in both Chlorella and spinach

 

chloroplasts. He also showed (1962) that addition of re-
ductants such as dithionite, ascorbate, or cysteine caused
a stimulation of the DLE in chlorOplasts. The addition of
oxidents such as ferricyanide caused a drop in DLE until

the oxidant was reduced.

ChlorOplasts also emit a weak red luminescence when
they undergo a rapid acid to base transition (Mayne and
Clayton, 1966). This luminescence, which requires pre-
illumination and is therefore a special case of DLE (Mayne,
1968), is abolished by uncouplers of phosphorylation, such
as ammonia, methylamine, carbonylcyanide-3-chlorophenyl-
hydrazone (CCCP) and by inhibitors of electron transport
such as DCMU. Since Uribe and Jagendorf (1966) had shown
that similar acid-base transitions support an uncoupler-
sensitive phosphorylation reaction, it was natural to suggest
that the same ATP-generating potential was required for both
DLE and phosphorylation. Electron transport inhibitors such
as DCMU, however, inhibit only the DLE associated with acid-
base transitions, not the phosphorylation. (Therefore, it
was reasonable to postulate that DLE required both the "high
energy state" responsible for phosphorylation §§2.3t least
part of the electron transport pathway. Consistent with
this picture is the fact that not only uncouplers but also
ADP and phosphate, which use up the ATP generating potential,
decrease the DLE measured at 4 msec after illumination
(Mayne, 1967). Furthermore phlorizin, which blocks the
ATP generating reaction between ADP and phosphate, restores
the full DLE.

Miles and Jagendorf (1969) repeated the acid-base
transition luminescence studies and extended them to show

that a transition from low salt conditions to high salt

conditions also caused luminescence, even though these
salt changes did not support phosphorylation., As in the
acid-base transition and in ordinary DLE studies, the
presence of DCMU abolished the phenomenon. The nature
of the temporary trans-membrane ion gradients contributing
to DLE is not clear. Barber and Kraan (1970) reported
that a low-salt to high-salt transition with sodium salts
gave an increased luminescence if gramicidin was present.
Since gramicidin is known to increase membrance perme-
ability to a number of monovalent cations including sodium,
this implies that a sodium gradient aids DLE. On the
other hand valinomycin, which increases permeability to
potassium ions, gave increased DLE when potassium salts
were present. Therefore it seems that a potassium gradient
can also aid DLE. Barber and Kraan (1970) therefore
suggested that DLE may result from an electrical membrane
potential generated by the diffusion of any ion. Fleischman
(1971) showed that a jump in K+ concentration (a transition
from a choline chloride medium to a KCl medium) gave lumi-
nescence in bacterial chromatophores, but only if the
potassium-carrying antibiotic valinomycin was present.
Fleischman (1971) and Crofts, Wraight, and
Fleischman (1971) have suggested a model for DLE in chloro-
plasts. This model was suggested by Jackson and Croft's
demonstration (1969) of a linear correlation between the

magnitude of the artificially induced membrane potential

in Rhodopseudomonas spheroides chromatOphores and the ex-
tent of the carotenoid spectral shift, which is in turn
well correlated with the 4 msec DLE.

In their model of chlorOplast DLE, they place the
primary electron acceptor of photosystem II,Q, on the
outside of the thylakoid, in equilibrium with the plasto-
quinone pOOl- They place the primary electron donor of
photosystem II, Z, on the inside of the membrane, in equi-
librium with the water oxidation site. Since the reduction
of plastoquinone to give plastohydroquinone would Consume
H+ on the outside of the membrane and the oxidation of
water would produce H+ on the inside of the membrane, electron
transport via photosystem II should involve a H+ "pump".
This "transport" of H+ across the membrane, if unaccompanied
by a counter ion, would generate a membrane potential with
the inside being positive and the outside negative. The
forward reaction of electron tranSport in photosystem II

can therefore be depicted as

chlorophyll y + -

Z+Q+hv z+Q

 

and the back reaction which yields the DLE can be depicted

as

+ - chlorophyll

Z+Q +Z+Q+hV

 

However this back reaction is almost impossible unless

activation energy is added from another source. Now the

above mentioned membrane potential is just such an addi-
tional energy source.

Evans and Crofts (1973) have also implicated the
trans-membrane H+ gradient as a factor in causing DLE
because of the similarity of the rise and decay kinetics
of the DLE and H+ gradients.

There has been much discussion in recent years
concerning the relationship between DLE and fluorescence.
Since DLE has the same emission spectrum as fluorescence,
it's reasonable to assume that the same chlorophyll is
involved in both processes. The intensity of prompt
fluorescence is obviously a measure of the efficiency of
light emission from chlorOphyll which has been excited
by light (either by direct absorption or by energy transfer).
0n the other hand, DLE is a measure of the efficiency of
fluorescence of chlor0phyll which has been excited by some
unspecified chemical reaction. Knowing the efficiency of
prompt fluorescence, we are therefore in a position to
apply this to the DLE in order to arrive at conclusions
regarding this chemical generation of excited chlorophyll.
See Lavorel (1968), Clayton (1969), and wraight (1972).
Since our main interest has centered on those back-reactions
of photosynthesis which generate excited chlorophyll, we
have routinely measured the prompt fluorescence and the DLE
simultaneously and we have attempted to separate effects on
prompt fluorescence from effects on the chemical mechanism

responsible for DLE.

 

With the advent of mutant strains of Scendesmus

 

obliguus it has been almost conclusively shown that the DLE
originates from PSII by Bertsch §t_§l. (1967), and by Haug
32.2.1.- (1972) . ‘

Haug's group (1972) measured the entire time course
of the DLE and showed that the apparent prompt fluorescence
contained less than 1% of a DLE componenet.

There has been some recent work correlating electron
transport and DLE. Bertsch gt_al. (1971) confirmed that
DCMU markedly inhibited DLE when chloroplasts were reducing
ferricyanide. Bertsch and Lurie (1971) showed that Tris
poisoning near PS II inhibited DLE but this could be
restored by addition of artificial electron donors to PS II.
ItOh §E_al, (1971) also studied the effect of donors of
electrons to PS II on DLE and fluorescence. Butler (1971)
reported that destruction of C-550 with pancreatic lipase
markedly inhibited the DLE (C-550 is an unidentified component
detected as a light-induced absorbance change). Kessler
et_al. (1957) showed that a severely Mn-deficient

Ankistrodesmus culture gave only one—fourth as much DLE

 

as the control. Arnold (1972) has recently reported that
an alternating electric field of several hundred volts
can stimulate the DLE up to 50 fold. Zankel (1971) found
that by using single flashes of light the DLE could be
made to oscillate in a period of four in the same way as

does oxygen production (Joliot, 1969). Mar and Govindjee

 

(1971) have reported that chloroplasts subjected to a
quick temperature jump of about 15°C emit light.

It appears that DLE is primarily a function of two
major processes: first, an enzymatic catalyzed reversal
of electron flow, as originally suggested by Strehler and
Arnold (1951), and second, the involvement of a "high
energy state" and/or membrane potential, as suggested by
Mayne (1968), Fleischman (1971), and Wraight and Crofts
(1971). ‘

Electron flow therefore can be expected to affect
DLE in two ways, by generating the high energy state-
membrane potential or by influencing the size of the pools
of back-reacting endogenous electron donors and electron
acceptors.

Keeping these mechanisms in mind, we studied the
effects of various electron transport inhibitors on DLE.
In particular we investigated some new inhibitors,
Specifically dibromomethyliSOpropylbenzoquinone (DBMIB)

a plastoquinone antagonist (Trebst el_al. (1970) and KCN,

a plastocyanin inhibitor (Quitrakul and Izawa, 1973). We

also investigated correlations between DLE and membrane
gradients using various nucleotides, ions, and phosphorylation

uncouplers.

METHODS

Chloroplasts were usually prepared by the method of
Saha et_§l. (1971) from spinach (Spinacia oleracea L.) ob-
tained at a local market. The leaves were ground for 10
seconds in a Waring Blendor in a medium containing 0.3M
NaCl, 2mM MgClz, and 0.04 M N-2-hydroxyethylpiperazine-N'-
ethanesulfonic acid-NaOH (pH 7.6). The homogenate was
squeezed through several layers of cheesecloth and centri-
fuged at about 3000 x g for 4 min. The pellet was resus-
pended in a medium containing 0.2 M sucrose, 2mM MgClz,
and 0.01 M N-2-hydroxylethylpiperazine-N'-2-ethanesu1fonic
acid- NaOH (pH 7.6).. The suspension was centrifuged at
about 1200 x g for about 20 seconds to remove debris, and
then centrifuged again at 3000 x g for 4 min. The washed
chloroplasts were finally suSpended in a small volume of
the same sucrose-N-2-hydroxyethylpiperazine-N'-2-ethane-
sulfonic acid -MgCl2 medium. All operations were carried
out at as near to 0 C as possible. The chlorOphyll con—
tent of the chloroplast suspension was measured by the
method of Arnon (1949).

Crucial experiments were confirmed using chloro-
plasts prepared by the same methods from the spinach
variety "Bloomsdale Longstanding", grown in a growth chamber.
This spinach was a gift from Dr. N. E. Tolbert.

9

 

10

Under some conditions (in the presence of NH4C1)
chlorOplast preparations gave more DLE if the leaves
of commercial spinach used were illuminated overnight in
a moist chamber with their petioles in water and if the
chloroplasts stock solution was held in 0.1% bovine serum
albumin (BSA) during the experiments. These procedures
were therefore used routinely for all later experiments,
including all those reporting the effects of ammonia.

Oxygen evolution was measured using a Clark-type
oxygen electrode. The signal from the oxygen electrode
was passed to a preamplifier constructed of standard de—
sign, consisting principally of a zener diode regulated
voltage supply and a Zeltex Zel-l operational amplifier.
The signal was recorded on a sargent recorder.

Electrodes for measuring NH4+ Were constructed by
Dr. S. Izawa by using two concentric pieces of pyrex glass
tubing sealed at the lower end with 10 mu filter held
by epoxy glue. See Figure 1. The central glass tube
held a solution of 0.01 M KCl in which was inserted an
Ag/AgCl electrode. The outer tube held Orion K+ liquid
Ion Exchanger. For reference, another Ag/AgCl electrode
was used. It was immersed in a LiCl (0.03M) solution
and connected to the experimental vessel with a LiCl (0.03M)
4% agar bridge.

The potential difference between the two silver

electrodes, which is a function of the NH4+ concentration,

ll

Figure.l.—-Diagram of the electrode and the apparatus

+
used to measure the uptake and release of NH4

suspension. A 4 ml reaction volume was placed in a transparent

by chloroplast

thermostated water jacket at 20 C. The reference electrode

was connected to the reaction vessel by means of a LiCl (30 mM),
4% agar bridge in plastic surgical tubing. A copper ground wire
was connected to the power supply ground and to the reaction
vessel solution. Orion K+ "Liquid Ion Exchanger" was placed

in the outer jacket of the electrode. The 10 mu millipore

filter was sealed onto the glass with epoxy glue.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Chassis
ground
I
F
(max)
LiCl-
electrode i—agar
bfidge
#-
fls ._. __
-> — --- _.
1 - r-_
Reoctiom _‘
Mixture D, -
\
\
Thermostoted Stirring bar

water jacket

12

\
I

To

Amplifer / recorder

Pyrex gloss

 

 

 

 

' AgCI/Ag

 

 

 

 

- LiCl reservoir

Liquhi
cofion
exchanger

 

 

 

|\ I. 'll\
\

J).
m

 

 

 

 

 

 

 

____ T
\|\ n l|(

L/KCHO-IM)

 

K+electrode

13

Figure 2.-- Separation of the DLE and prompt
fluorescenceby the instrument described. Curves showing
the DLE and prompt fluorescence from a chlor0plast suspension
are above the dotted line on the left. Similar curves from
a solution of chlorophyll in acetone are given on the right.
Below the dotted line, curves for DLE and prompt fluorescence
in subchloroplast particles and chlorophyll~solution are
shown. Note that the sensitivities of the instrument were
different above and below the dotted line; below the dotted
line, the response to DLE.was about 70 times as sensitive
while the response to prompt fluorescence was actually

lowered.

 

14

Chloroplasts-l- MV Chlorophyll in Acetone Solution

DLE

 

|55ec II

Fluor. (

DLE sensitivity 70X greater than with chloroplasts

 

 

 

 

 

 

Subchloroplast particles Chlorophyll in Acetone Solution
+MV
DLE
"" lmin W‘1I" w
l—————>|
Fluor.
_J

 

15

was measured with a solid state amplifier constructed
according to the method of Brand and Rechnitz (1970). The
amplifier parts were generously loaned by Dr. Clarence
Suelter. The amplifier had very high impedance inputs

from both electrodes and consequently the reaction solution
had to be grounded. A DC power for the amplifier was
constructed with voltage regulation provided by a Silicon
General S. G. 1501 integrated circuit. A flashlight battery
was used to construct a DC offset and the output of the
amplifier was recorded on a Sargent recorder.

The phOSphorosc0pe apparatus used to measure DLE
was a modification of one previously described by Priestly
and Haug (1968). It consisted of a 10 inch diameter ro-
tating bucket, with a half inch slot subtending not quite
180 degrees cut out of it. The exciting light and the
DLE detection system were placed exactly Opposite each
other so that the exciting light could never shine on the
DLE detection system. The fluorescence detection system
was at right angles to the sample but was prevented from
seeing the exciting light by a system of complementary
filters. The exciting light from the mercury-xenon lamp
was filtered through 6 cm of saturated CuSO4 solution,
the filter being cooled with a fan. Corning 2030 red
transmission filters were used in front of the DLE and
fluorescence photomultiplier tubes to prevent scattered

exciting light from reaching them. An example of the

16

separation of fluorescence and DLE achieved by this in-
strument is given in Figure 2 which compares chlorOplasts
with an acetone solution of chlorophyll, since the latter
Obviously produces no DLE. An RCA 31000E photomultiplier was used for
DLE measurements and for best results (reduction of noise)
was cooled for 36 hours with dry ice, and typically
operated at 1800 volts. An EMI 9558B photomultiplier
used for fluorescence measurements was typically Operated
at 650 volts. Both photomultipliers were powered simul-
taneously by a Keithley 246 high voltage supply via a voltage
divider circuit. The linearity of both photomultipliers
is shown in Figure 3. The output from the fluorescence
detecting photomultiplier went directly to one channel
of a Hewlett Packard 77028 recorder. The output from the
DLE detecting photomultiplier went to a Keithley 417 high
speed picoammeter for amplification and then to a Princeton
Applied Research Boxcar Integrator cw-l for processing, and
then to the other channel of the Hewlett Packard recorder
for recording. In addition a trigger amplifier was con—
structed to raise the photocell trigger pulse to Operate
the Boxcar Integrator. The system's faithfulness in re-
production is shown in Figure 4 in which the signal in-
tegrated over 5 minutes (bottom curve) is compared with the
amplified photomultiplier output (middle curve) from
voltage pulses on a photodiode (upper curve).

A small plexiglass box was constructed to hold

sample cuvettes and about 50 mls of water to buffer

 

17

Figure 3.--Tests of the linearity of the photo-
multipliers used for measuring DLE and fluorescence. A 2
watt light bulb connected to a rheostat was placed in the
sample position in the DLE apparatus with the amplifier and
Boxcar Integrator at normal sensitivity. The voltage on the
light bulb and the iris in front of the photomultiplier were
adjusted until the signal gave a full scale deflection at
the desired photomultiplier voltage. To be on the safe side,
voltages were chosen both considerably above and considerably
below normal working voltages and the linearity was tested at
these voltage extremes. Light neutral density filters,
calibrated on a Cary 15 spectrophotometer, were placed
between the light-source and the photomultiplier. The
resulting recorder heights were then plotted versus the

percent transmission of the filter combination used.

800

PM current (I110

.000

A)

n
N
O
O
0

PM current (
3
O
O

18

 

 

I'lll‘r

Fluorescence ,
photomultiplier

603
volts

  
   

l l l I l

705 volts

 
  
 
 
  
   
 
 

I I 1 1

IO 20 so 40 so so 76 so so l00
% Transmission

 

T

 

DLE photomultiplier

2000
volts

 

L 1 l I L I

IO 20 30 4O 50 60 7O 80 90 IOO

°/o Tran smission

  
   
 
    

' l600 volts

1 l l J

d

 

 

19

temperature changes. After all pipettings were completed
the cuvette was swirled in a beaker of water at room
temperature for 30 seconds and then placed in the plexiglass
container for 30 seconds before measurements were begun.
We found that Beckman pyrex cuvettes gave almost as much
"DLE" from a chlorOphyll in acetone solution as did a
chlorOplast suspension. We were unable to measure any
"DLE" when the same chlorOphyll in acetone solution was
transferred to quartz cuvettes. Probably our pyrex cuvettes
contained phosphorescent impurities which were excited either
by excited chlorophyll or by the fluorescence given Off
by excited chlorOphyll. ‘

Cyanide treated chloroplasts were prepared by
incubating the chlorOplasts at 0°C for 90 minutes in a
30 mM KCN solution buffered at pH 7.8 as described by
Ouitrakul and Izawa (1973). Controls were incubated in
a similar medium in which KOH replaced KCN.

Thymoquinone was prepared from thymol by the
method of Gilman and Blatt (1961). Dibromothymoquinone
was prepared from thymoquinone as outlined by Carstanjen
(1871). This method consisted of adding slightly more
than two equivalents of liquid bromine to a container con-
taining the thymoquinone and water. The mono and dibromo
derivatives were separated using the fact that monobromo
compound is much more soluble in —20°C ethanol than is the

dibromo compound.

20

Figure 4.--Waveform distortion test of the DLE
apparatus. A 100 hz, 5 volt pulse from the rear of a Hewlett-
Packard counter was placed on one channel on a dual beam
oscilloscope and across a Monsanto MV-4 light emitting diode
(red), which was placed in the sample chamber facing the
photomultiplier. The output from the photomultiplier and
amplifier combination was connected to the other channel of
the dual beam oscilloscope and to the input of the Boxcar
Integrator. It can be seen that the diode-photomultiplier-
amplifier combination damps the original signal to give a
rise time of approximately 0.5 msec. The output from the
Boxcar Integrator recorded on the chart paper, if closely
examined, can also be seen to have a rise time of 0.5 msec,

with a much reduced signal to noise ratio.

21

Voltage pulse to
light emitting diode

Output from P. M.
and amplifier

 

l0msec

i 4|-

 

Output from Boxcar {—7 F1 _ m . m

Integrator recorded
at chart speed at
l inch/5minute.

 

 

 

 

 

 

 

 

.1 CJ C1 U o.

l0msec

l-——->|

22

Subchloroplast particles were-prepared from chloro-
plasts isolated in the usual manner (see above). Five
mls of chloroplast suspension having a chlorOphyll‘con-
centration of 600 ugm/ml were diluted to 20 mls with a
medium consisting of sucrose 0.2M and HEPES buffer 0.01 M
at pH 7.4 (no MgClz) to give a final chlorOphyll con-
centration of 150 ugm/ml. One ml of a 2% solution of
digitonin in ethylene glycol was added. The preparation
was shaken and incubated at 4°C for 15 minutes. It was
then centrifuged for 10 minutes at 6,000 rpm in a Sorvall
RC—2. The pellet was discarded and the supernatant was
centrifuged for 20 minutes at 16,000 rpm in the RC-2. The
pellet was resuspended in sucrose 0.2 M, HEPES 0.01 M, and
MgCl2 2mM, and was washed once and was finally resuspended

in the same medium.

RESULTS

I. Electron Transport Inhibitors
On Delayed Light Emission -

 

As was pointed out in the introduction, DLE seems
to depend on having the primary electron acceptor of PS II
in the reduced form, the primary electron donor of PS II
in the oxidized form and the thylakoid membrane in an
"energized state". Electron transport inhibitors Obviously
affect the oxidation levels of all of the electron acceptors
and electron donors in the transport chain. In this way
they presumably affect DLE. However, electron transport
also generates the "energized state" of the membrane,
whatever that may be; In an attempt to sort out these
complicated interactions we investigated the effects of a
number of very dissimilar inhibitors on DLE.

The inhibitors used were orthophenanthroline,

(3, 4~dichlorophenyl) -l, l-dimethylurea (DCMU), dibromo-
methylisoprOpylbenzoquinone (DBMIB) and KCN treatment.

At this point it is worthwhile reviewing the site of in-
hibition of these inhibitors.

DCMU is believed to inhibit between the primary
electron acceptor of PS II, Q, and plastoquinone (Duysens
and Sweers, 1963). OrthOphenanthroline seems to inhibit
at the same place (Nishmura, 1967). The cyanide treatment

23

24

has been shown to inhibit by reacting with plastocyanin
(Ouitrakul and Izawa, 1973). The newest inhibitor, DBMIB,
was first found to inhibit at the level of plastoquinone
(Trebst §E_al, 1970) and later it was postulated that
DBMIB inhibits on the PS I side of the plastoquinone pool
by Gould and Izawa (1973). These sites of inhibition are

shown in the preceding scheme.

o-phgnanthroline FOCY
DCMUG. DBMIB-9DBMIBH2 ' .MV
I 4.. A}
l H DEHHIB
A I Po /

Psrr /

 

'7OC>

 

H20\

2

 

It can be seen that these four inhibitors belong to
two different classes on the basis of their effects on DLE.
See Figure 5. OrthOphenanthroline and DCMU markedly in-
hibit the DLE at all light intensities. In contrast DBMIB
and KCN treatment only inhibit DLE slightly, and this in—
hibition was only apparent at very high light intensities.
At low and moderate light intensities these inhibitors

actually stimulated DLE.

25

.muflss oosoomonooam
may soap HOHHmEm £058 new CD UODMHOHGD mums man mGHHSmooE s0 poms muflcs mumnuflnuo one
.mHmuHHm muflmsdp Hmuusos £003 wmflnn> mums moauflmsousfl Damflq .COHDosHESHHH Osmosuom mo

sundae one OD Oouoohnsm soon was mumoamouoHso can Houmo wousmooe mm3 OOHHOQ usmfla
some mo coapocwanou map Houmo cone o.etm.m wqo mo musesoocw was .eomsu Umpwnspom mo

.80 m nmsonau Oommmm unmfla man seams .Oommovm umxosn mGHQQOSOIuanH onu SDHB .OOHSmomE mm3

muwmsousfl usmfiq .msofiuflpsoo Ham Home: Dammomm mo3 can 2 OH x m mmB somoHOH>HmnpoE

mt
Houmoooo souuooam one .zmIOH x H .0200 can “Setoa x H .osHHoucusmsmnmuo “zsnoa x m
.mHSmQ "mums mnoufinflssa Hocuo mo msOHDmuusoocoo Hmcflm .mooauoa cH confluomop

mm venomoum.ouo3 mumoamosoHso douoouptzux. .mHE N :0 HHSSQOHOHQO Ems om msflsfimucoo
momoaaonoHeo ooosaocs omen pH .2 «00.0 .maomz one 12s0.0 .Am.0 macmooztoofloane

“Ea.o .omouosm umpcsomaoo msflzoaaom men OmsHMDsoo onsuxwfi sOHDomou one .oosoomouooam

DQEOHQ can HAG can so muouflnflasw sonuooao mDOHHm> mo muoommmll.m onsmfim

 

26

000

.N .
000 00¢ 00m

 

 

_

 

3:33.82".

  

0

 

 

0

0.

ON

:50 wow no.3: stuczc.

000

203

000 00¢ 00m

 

suun Moment

 

L
Di

L

 

00

20v.
0 mime
oczocficocozauo
D 200

50:02.
3.3.5:.

 

mqo

_ _ _

 
    

 

 

0.

ON

on

0¢

suun Unmaav

27

It was noticed that at concentrations of DBMIB
forty times greater than those needed for complete in-
hibitiOn of methylviologen reduction DLE was greatly de-
creased. However it was noticed that these high concentrations
of DBMIB also completely inhibited the fluorescence. Since
Lavorel (1968) postulated that both the DLE and fluores-
cence shared common factors affecting the emmission yield,
an experiment was carried out to look at the relationship
between fluorescence and DLE as a function of DBMIB
concentration. Figure 6 presents the results of that ex-
periment. It can be immediately seen that, in three out
of four conditions, high concentrations of DBMIB inhibited
both the DLE and the fluorescence. Izawa §t_al. (1973)
have pointed out that these high concentrations of DBMIB
support electron transport from PS II; DBMIB itself acts
as an electron acceptor. It could be argued that transport
of electrons to DBMIB quenches the fluorescence. This
argument is invalid since it is shown in Figure 6 that
DCMU which inhibits all electron transport does not re-
lieve the strong quenching of fluorescence by high DBMIB.
It seems that the quenching is due to some kind of direct
effect of DBMIB on the chlorophyll itself. This is
borne out by the fact that there is a concomitant de-
crease in the quantum efficiency of electron transport, and

the fact that DBMIB at similar concentrations quenches the

fluorescence of chlorophyll solutions.

28

Figure 6.--Effects of increasing DBMIB concentrations
on the DLE and prompt fluorescence. The reaction mixture was
identical to that of figure (5) except that HEPES buffer
replaced the Tricine buffer at pH 7.5. The light intensity
was 400 Kergs/sec/cm2 of blue light. The ferricyanide when
used was 0.4 mM and the DCMU was 1.0 uM. Fluorescence
measurements with methylviologen at pH 7.5 and with DBMIB at
0.5 uM and 1.0 uM went off scale at the level indicated by
the dashed line. As in figure 5 the data presented are
recordings of the 3.5-4.0 msec DLE. The noise level could

be reduced but not without yielding "overdamped" rise curves.

29

 

 

 

 

 

DBMIB 0.5 LO 5 IO, 20 20
(FM) » +
, DCMU
DLE
FeCy' fr—
(pl-1.7.5)
Fluor.—F
. _r'—_,—————_.____,————
DLE '
FeCy l""'""'" .
(pH 8.2) "" ' "" _ —
Fluor.—F
_.| __.r"'—---.__r _ _
. 90 sec

 

30

Two pH's and two electron acceptors were used in
this experiment for reasons based on the work of Gould
and Izawa (1973). Under certain conditions DBMIB can
be reoxidized by oxygen or by ferricyanide. DBMIB can
be reoxidized at either pH 8.2 or pH 7.5, but the DBMIB
can only be reoxidized by oxygen at pH 8.2. Thus, as
shown in Figure 3, the DLE and fluorescence is very
markedly quenched when the DBMIB is oxidized i.e., at
pH 7.5 and 8.2 with ferricyanide, and at pH 8.2 with
oxygen. At pH 7.5 with methylviologen and O2 and at
DBMIB concentrations of 5 and 10 uM, the DLE and fluo-
rescence are initially low. However, during the early
stages of illumination the DBMIB becomes reduced and
cannot become reoxidized. The chlorOphyll-DBMIB in-
teraction therefore ceases and the DLE and fluorescence
rise together.

As Gould and Izawa (1973) pointed out, since DBMIB
is in the quinone form, its reduction to the hydroquinone
form must involve the uptake of 2 H+ ions as well as 2
electrons. This is how they accounted for the one pH
rise and fall in the light, which on subsequent illumi-
nations gave no more pH changes. This one and only one
pH rise, they attributed to the consumption of H+ ions
by the reduction of DBMIB. It is felt that the initial
pH 7.5 DLE and fluorescence rises are the result of DBMIB

reduction which removes the chemical quenching of the

31

chlorophyll fluorescence. The high subsequent fluorescence
is_presumed due to the remaining block of electron transport
at plastoquinone which keeps Q reduced. It is felt that the
subsequent decay of the DLE is the result of gradual loss

of the pH gradient as reported by Gould and Izawa (1973).

II. Effects of Ammonia andOrganic Amines
on Delayed Light Emission

 

It has long been dogma that the presence of un-
couplers decreased the DLE from chloroplasts (Mayne, 1967).
However this is not always the case. Ammonium ion, at
concentrations which totally abolish phosphorylation
actually increase DLE. See Figure 7. ‘The DLE data are
presented as decay curves from 3 msec to 30 msec after
light extinction. (Note that the initial steep rise and
the final steep fall are purely instrumental, representing
the mechanical opening and closing of slits and are with-
out biological signifance) . It can be seen that the pres-
ence of ammonia increases the DLE at 3 msec but that the
decay of the delayed light is much faster under these
conditions. Thus at 30 msec the DLE with ammonia is de-
creased. Valinomycin, which makes membranes permeable
to NH4+ (as well as K+), has little effect by itself but
it abolishes most of the DLE when NH4+ is present. The
uncouplers gramicidin and atebrin also abolish most of the

DLE. It is important to note that while the DLE is

32

Figure 7.--Effects of NH Cl and other uncouplers on

4

the DLE decay curves with methylviologen as electron acceptor.
Reaction mixture ingredients were as in figure 5. Concen-

trations of the added substances were, when used: NH Cl, 10 mM;

7

4
valinomycin, 5 x 10- M; gramicidin D, 4 ugm/ml; and atebrin,

1.2 x 10-5M. Data presented are decay curves from 3.0-30.0

msec after light extinction. The leading and trailing edges

1

of the traces result from the slit Opening and closing and
contains no biological information. Fmax values are the
values of the maximum prompt fluorescence observed during the

same experiment.

33

no additions valinomycin
tr"mm =l.00 (Fm =I.O7)
NH4CI NH4Cl+valinomyCin

 

.1 M

(PM sees) (Fm, =o.54)
Gramicidin Atebrin
(me =o.59) (Fm =|.00)
l l
3msec 30msec

light off

34

.0.e mm: me one omen

umooxm m mhsmflm OD HMOHDQOUH was ossuxHE COHDOMOH one .28 OH mums mmsHEm
Ham mo sowumuusoocoo HMCHE .ousuxHE soauonou men OD poops one: weep OHOMOQ
o.m mm usonm OD pepmsflpm one coausaom msoosqo CH ponmmoum one; mosHEm one
.COHDMCHESHHH Ummmoso mo msflscflmon we“ Houmm muscHE oco Umssmmofi mos mam one

.mqo ommE m.mto.m map so m.mmm mDOHHm> mo mossam mo mpomwMMIl.m ousmfim

3S

.: . o. m 0. a. o

 

 

n
_ _ _ _ _ _
... -- o...
I III! 0 3032::
I
p/ I
I
age—3:330 // l
/
I
I
I
ll
:25. .2203 I 1
Illnllllllllllllllllllllnllllulllll Illlll
3:3... IIIIO.
.5203
Box: 0

 

 

0.

ON

on

310

36

stimulated by NH4Cl, the prompt fluorescence is somewhat

inhibited. Therefore the stimulation of DLE by NH Cl

4
cannot be due to an increase in the fluorescence yield
of the chemically excited chlorophyll.

Other amines, which are equally effective un-

couplers of phosphorylation, do not have this effect of

increasing DLE. 0n the contrary they, like all other

uncouplers studied, inhibit DLE. Thus ammonia seems to be
the exception rather than the rule. See Figure 8. It is
not clear why ammonia is unique. When we first noted
that ammonia increased DLE while methylamine decreased
DLE, we considered that the marked difference in pKa
might be responsible. At pH's used in these studies
(about 8.0) an appreciable amount of ammonia is present
in the uncharged, unprotonated, membrane permeating form,
whereas very little of the methylamine is similarly
uncharge. However as Figure 8 clearly shows there is no
correlation between ammonia's pKa and its effect on DLE.
Early in the course of these experiments it be-
came apparent that the reaction mixture pH was very
critical in determing the stimulation of the DLE by NH4Cl.
At pH 8 and higher the stimulation was always present
whereas at lower pHusthe stimulation was replaced by an
inhibition. See Figure 9. At the lower pH's, for example
6.8, no concentration of NH4Cl up to 85 mM gave any

stimulation and indeed, increasing concentrations of

37

.m.m mm pm cow: was mm:

SE ow pmnu umwoxw m musmfim How Umnwuommp mm whoa mCOHuflpcoo mucuxHE cofibommm .COHp

ImcHEsHHH mo macho 30mm Hmumm ommE m.m|o.m pm UmHSmmmE Gamma was man .mm 30H um Haemz

v

»n moo wo cofluflnflsce can we boss on so mz an mum to coflnmasaflumli.m museum

38

 

 

 

.135»:

6:2 25m .032 2

m6 In

IL. IA

3:33.53...
who

65.2 25m 652 2

N6 In

39

NH4C1 simply gave lower levels of DLE. It should be
emphasized that in this Figure, in contrast to Figure 7,
the curves presented are not DLE decay curves. Rather
they represent repeated samplings of the DLE 3.0-3.5 msec
after light extinction. In other words the curves in this
Figure represent changes with time in the peak heights
depicted in Figure 7. Again the differences cannot be
correlated with fluorescence efficiencies since these did
not change very much.

Since the stimulation of the DLE by NH4C1 was
maximal at high pH, and since ammonia and other amines are
known to inhibit water oxidation at high pH, it was sus-

pected that perhaps the stimulation of DLE by NH Cl might

4
somehow be a result of electron transport inhibition. If

this were true then the electron transport inhibitor
hydroxylamine, which inhibits water oxidation much as does

high pH and NH Cl, should also stimulate DLE. Figure 10

4
gives concentration curves for the effects of hydroxylamine
and NH4C1 on the DLE. It is readily apparent that there

is little similarity between the effects of these two
compounds on the DLE.

The DLE in the presence of NH Cl and the uptake

4
of ammonium ion by chloroplasts show remarkably similar
pH Optima. Furthermore, these optima are similar to the

optima for basal electron transport and for noncyclic

phosphorylation. See Figure 11. The pH Optima for DLE

40

Figure 10.--A comparison of the effects of NHZOH

and NH4Cl on the DLE. Reaction mixture conditions were as

in figure 5 except that the pH was 8.5 for the NHZOH

treatment.

41

 

 

 

 

 

 

 

 

 

 

 

O 2 4 6 8 IO.
NHZOH (mM)
4b - I ' i . -o 4
E: ..
o 20 d
O l l l l I
O 2 4 6 8 IO

42

in the absence of NH4+ and the pH Optima for electron
transport in the presence of NH4+ are quite different.

The DLE and ammonium ion uptake were compared at
various pH's in the presence and absence of the salt
LiCl (50 mM). See Figure 12. LiCl was used instead of
NaCl or KCL since it does not interfere with the electrode

responses used to measure NH4+. It is important to note

that the DLE intensities reported in.this Figure represent
the luminescence 3.0-3.5 msec after light extinction in
each 70 msec cycle of the intermittent illumination. Thus
the insert in the upper right hand corner does not re-
present the decay of luminescence with time but rather
represents changes in the 3 msec DLE with overall time

of illumination. Clearly the DLE shown in this inset
Figure is biphasic with a fast initial spike and a subse-
quent lower steady state level. The pH curve for the
steady state component of the DLE is very similar to the
pH curve for the extent of the uptake of NH4+ in the
absence of LiCl. It should be noted particularly, however,
that the extent of the uptake of NH4+ in the presence of

LiCl showed a quite different pH Optimum. Thus the extra

+
4

seem to be correlated with the DLE at all. It should

NH uptake in the presence of the chloride salt did not
also be noted that the spike of DLE was not correlated
with the NH4+ uptake either. This spike was in any case,
very variable occuring mostly at high intensities of

actinic light (greater than 400 Kergs sec.l cm-z).

43

.28 m mw3 pH

poms was avemz coca .HE w CH Ham .HamsmonoHLO mo Em: omN msflcflmucoo mummamOMOHQO

was a: Noo.o .maumz “2 v0.0 .Hmmmsn “2 H.o..mmos05m mo pmpmflmsoo OMMpms +wmz mcflusmmme

MOM pmms mousuxflfi coflpommu wee .Umuoc mcoHuDUHqusm Hmmmsn map How umwoxw m musmflm
CH UOQHHommU mm mnt uuommcmup couuomam was man mo ucmEmssmmmfi mgp CH poms mmucuxfla
moanommh OLE .25 ow wumB mcofipmuucmocoo Hemmsn Had .o.m was .h.m .m.m mm pm poms
mmz ummmsn HalmmfiB was .m.m was o.m mm vs poms was Hmwmsn HQ: mcflOHHB .m.> was N.» mm

um pmms mm3 smmmsn Haummmmm .o.h mm Boawn pom: mm3 Hmmmsn flqnmmz .mm mo chHuoccw

v

mm OMMpQD +

mz was unommsmnu cosuomam .mqa mo somflummaoo ¢II.HH musmflm

44

 

:a
06 0.0 o.» o.m
1 u u 1 q q
u a i
4
T L
r- I
3.3.... +3.5 .3

 

 

 

 

 

to
9m O.o o.» 06
q A u q «1- q
I 4
33.30
F
r.
r
16“.. 2: .
. b - _ n —

 

 

 

OON

00¢

com

000

000.

 

 

 

 

 

45

A study of the effects of other salts and of
.mannitol on DLE was also undertaken. While the nature of
the anion present did affect the intensity of the DLE
somewhat, these effects were not striking. The same is
true Of all concentrations of mannitol employed up to 0.8M.

Since methylamine and NH4Cl do not uncouple phosphory-
lation in digitonin particles of chloroplasts (Nelson
gE_§l. 1970), DLE from these particles was investigated.
Figure 13 shows that DLE in the particles was stimulated
at pH 8.3 by NH4+ as in intact chlorOplasts. However
DLE in the particles was also stimulated by methylamine,
even though methylamine strongly inhibits the DLE in in-

tact chloroplasts. Moreover, NH Cl, which inhibits DLE

4
at pH 6.5 in intact chloroplasts, did not inhibit DLE in
the particles. As expected the DLE was obliterated by

DCMU and by the combination Of NH4C1 and valinomycin.

III. Effects of Nucleotides on Delayed
‘Light Emission

 

 

As has already been pointed out in the introduction,
DLE in chloroplasts is inhibited by the presence of ADP
and Pi (Mayne 1967). Table I confirms this observation
but adds the new information that ADP and ATP actually
increase DLE if phOsphorylation is prevented either by
omitting phosphate or by adding deoxyphlorizin. Deox-
yphlorizin was included in this experiment because Winget

et al. (1960) and Izawa et al. (1966) have shown that

46

.mcoflu

v

IwcHEumump +

m2 cuHB UmumeODCH m>mc UH503 HUM so Humz woman poops mm? HUflq COHDHGCOO
Damn moan on“ mom .HH musmflm CH Umnfluommp mm mum3 mucmEOHSmmmE mxwumz +vmz How messuxfle
cofluommu OHHSB .m mucmflm.cfl mm mum3 mucmEmHSmmmE MAD MOM mmHSDxHE coepommm .AHUHQ SE omv

namm smug “soaps; was sung .mqa omms o.m man no =Hm>ma mnmnm semmnm= mam mxflmm was

SDHB mxwums +qmz mo usmpxw can mums HMHDHGH man mo consummaoo ¢II.NH mnsmflm

47

 

 

 
 

BJIIICOZIV .25.... i

a
. , 20...:
u :03: .23 .x. 20¢:qu ..

. .ot.co
om». on ,\ o

 

  

 

 

suun Momqw

 

  
    

22.: is

_

     
           

./

5.1.3.2....

r4

  

/

20...— nudxm
«cmuxw um
Bic—ad

\

22.5.? m

 

L

l

 

 

O

O

N
HN sa|owu

{7
+

O
0
¢

anmxgw 00313081]

ooo

48

Figure 13.--Presence of DLE with CH3NH3C1 in sub-

chloroplast particles. Particles were prepared as described

in the methods. NH4C1 and CH3NH3C1 concentrations were

10 mM. The DCMU was 1 x 10-6M, and the valinomycin was

5 x 10-7M. MES-Na buffer was used at pH 6.5. All other

conditions were as in figure 5.

49

(Control) NH4CI CH3NH3cu

DLE L. K'- K.-

(pH 8.3) .. _.

Fluor. j———- r _r‘""—"'

(M! 83)

DLE NH4CI DCMU NH4CI valinomycin
Ftuor.

(pH 8.3) J J

(Contra!) NH4CI
”DLE \I N.
(pH 6.5)
Fluor.

(pH 6.5) I I

50

phlorizin and its analogs inhibit ATP synthesis and the
latent chlorOplast.ATPase. It seems, therefore, that the
stimulation of DLE by added ATP cannot be attributed to
any reaction which involves energy from the hydrolysis of
ATP.

This stimulation of DLE by nucleotides seems to
be Specific for ATP, ADP, and ITP, with ITP showing a
considerably smaller effect. This specificity is very
similar to the nucleotide specificity of the phosphoryl-
ation related activities of chloroplasts and coupling
factor (Avron, 1960). See Table II.

The actual recorder tracings which document some
of these contentions are presented in Figure 14. It can
be seen that the fluorescence change with nucleotides
present or absent are negligible in comparison with the
large DLE changes. As expected ADP and Pi inhibited DLE,
while ATP or ADP without phosphate stimulated DLE, and Pi
alone was without effect. The last tracing in Figure 14
shows the results obtained when phosphate and a limited
amount of ADP were added. This amount of ADP should have
been almost completely phosphorylated within a minute or
two. As is shown initially the ADP and Pi inhibited the
DLE, but gradually as the ADP was converted to ATP the
DLE rose in a curious two step reaction which is not
understood.

The increased DLE in the presence of the nucleo-

tides ADP and ATP seems to be related to the increased H+

51

gradient which is observed under the same conditions
(McCarty §£_al, 1971). These conditions also inhibit
the basal non-phosphorylating electron tran5port (Izawa
and Good, 1969). Figure 15 shows these phenomenon as
functions of the ATP or ADP concentration in the same
chlorOplast preparation. It is evident that the stimu-
lations and inhibitions all plateau and become maximal at
about 15 uM. The DLE data of Figure 15 shows that the
ATP stimulated the DLE more than ADP at the same con-
centration. We always found this to be true at or below
50 uM, ADP or ATP, but we also found that the difference
between ADP and ATP stimulations varied considerably
between chlorOplast preparations. Differences between
the ADP and ATP stimulated H+ uptake at the same
concentratiOn are not considered to be experimentally
significant. Nevertheless, the stimulation over the
control is quite significant and does plateau at about
15 uM. Differences between electron transport inhibition
by ATP and by ADP are considered to be experimentally
significant with ATP inhibiting slightly more than ADP.
The correlations among these three phenomenon, are not
believed to be purely coincidental as will be explained

in the discussion section.

52

TABLE I.--Stimulation of Delayed Light Emission by ADP and

 

 

 

ATP.*
Delayed Light (arbitrary units)
Additions
-deoxyphlorizin +deoxyph10rizin
Ferricyanide alone 100 95
Ferricyanide plus ADP 153 166
Ferricyanide plus Pi 113 100
Ferricyanide plus ADP and Pi 60 ~ 107
Ferricyanide plus ATP 166 166

 

*The reaction cuvette contained 2 m1. of reaction
mixture consisting of 40 mM tricine-NaOH buffer (pH 8.2),
0.1 M sucrose, 2 mM MgC12, and potassium ferricyanide 0.4
mM. Other concentrations when used were 0.5 mM ADP, 0.5
mM ATP, 5 mM Pi, and 0.5 mM deoxyphlorizin. The amount of
chloroplasts suspended in the 2.0 m1 reaction mixture was
equivalent to 30 ugm chlorophyll. Values were for steady
state delayed light taken one minute after starting the
chOpped illumination. Measurements were taken on the
segment of the decay curve from 2.5 to 3.0 msec after light
extinction.

53

TABLE II.--Stimu1ation of Delayed Light by Various
Nucleotides.*

 

Nucleotide Concentration DLE (arbitrary units)

 

(uM)
None —-- 100
ATP 50 157
ADP 50 136
5'AMP 50 100
Adenosine 500 ‘ 95
GDP 50 104
UTP 50 ‘ 104
ITP - 50 _ 123

 

*
50 uM methylviologen was used as electron acceptor.
Other conditions were as in Table I.

54

.Uonno oHomo nOHuonHEOHHH onp Houwo oomE m.muo.m nonsmoofi moz MAO one
.NEO\oom\mmHoM omH moB nanH OHnHuoo oan onn mo euHmnounH one .25 me mo3 pH pouHEHH
mo3 mod onp song .mmooxo CH mo3 mom on» cons noHuoHunoonoo mom onu moz mo 23 omH mos

med one .25 m mmz mnsoEHuomxo HHo cH Hm one .m oHDme nH mo moz ousuxHE noHuooou

one .oocmommnosam was mun men no men was .Hm .maa no muommomuu.eH mnsmHm

55

 

eels/HIE; _. a

TIIII.

SE _

 

3275: and and
C apq . 04
>2 >2 >2 >2 >2 >2

56

Figure 15.--The effects of ADP and ATP on the DLE,
H+- uptake and electron transport. The DLE experiment and
the H+ uptake were carried out on the same day with the same
chloroplast suspension. Units for the DLE are arbitrary,
units for electron transport are microequivalents/hr/mg
chlorophyll, and units for H+ uptake are micromoles/mg
chlorophyll. The reaction mixture for the DLE measurements
and the H+ uptake measurements consisted of methylviologen,

5 x lO-SM; sucrose, 0.1 M; MgCl 0.005 M; and HEPES buffer

2:
0.001 M at pH 7.8. The DLE experiments used chloroplasts
containing 30 ugm chlorophyll while the H+ uptake experi-
ments used chloroplasts cOntaining 100 ugm chlorophyll.

The reaction mixtures in both cases were 2.0 mls. Electron

transport was measured as 0 consumption using an oxygen

2

electrode. Conditions for electron transport measurements

were as in figure 5. Circles represent ATP concentrations,

triangles represent ADP concentrations, and squares represent

no additions.

57

 

 

 

 

 

 

A

- ‘1
u
\\

 

 

 

 

20

IO-

mno

ISO

50 70
ATP or ADP (UM)

30

OIO

DISCUSSION

The mechanism of DLE remains obscure.- Strehler
and Arnold (1951) postulated that a reversible enzyme re-
action was involved. However it would have to be a very
exergonic enzyme reaction since almost 40,000 calories
or l.8eV is released in the formation of an einstein of
red light. Mayne's (1967) Observation that uncouplers
greatly inhibit DLE suggests that the a "high energy
state" of the chlorOplast membrane somehow contributes to
DLE. Further, his Observation (Mayne and Clayton, 1966)
that an acid-base transition which is capable of causing
phosphorylation also elicits DLE from preilluminated
chlorOplasts adds support to this concept. However, on
considering the energetics of the situation, it becomes
clear that six or seven ATP's or ATP precursors (squiggles)
operating in a concerted fashion reaction would be re-
quired to produce an einstein Of red light. Similarly
it would take a pH difference across the membrane of 30
units at 60 mv/unit, to produce the energy for an einstein
of red light! These considerations of energy requirements
and the demonstrated requirement.for preillumination in
the acid—base transition studies make it seem very probable

58

59

that DLE somehow also involves a back-reaction of products
of the initial forward photoreactions.

There have been several proposals concerning the
ways in which these back—reactions might occur. Lavorel
(1969) postulates that DLE is the result of the re-
combination of the oxidized donor of electrons to P8 II
(2+) and the reduced acceptor Of electrons from PS II (0-).

This suggestion receives a good deal of support
from biochemical studies. The hydroxylamine - induced in-
hibition of the oxidation of water by 2+ (Joliot, 1966,
Izawa §E_al, 1969, Cheniae and Martin, 1971) results in
an inhibition Of DLE (Lavorel, 1969). ‘Furthermore,
Barbieri gt_al. (1970) have provided evidence that inter-
mediates in water oxidation (Zo to Z4 in the formalism Of
Joliot e_t__a_1. 1969) are involved in DLE. Similarly,
inhibition of electron transport by DCMU, which is reputed
to prevent the flow of electrons between Q and plasto-
quinone (Duysens, 1964), also abolishes the DLE (Sweetser
gt_al. 1961). These observations, taken together imply
not only that the back-reaction between Z+ and Q- is
required for DLE but also that the limiting factors in
determining the amount of DLE are, on the one hand, the
back-reaction of plastoquinone with Q and on the other
hand the back-reaction of some intermediate in the water

oxidation reaction with Z.

60

Lavorel's pr0posa1 should be considered from the
standpoint of energetics. Although 2+ must be more than
+0.8 volts to oxidize water and Q- is reputed to be
somewhere between +0.2 volts (Kok §E_al. 1967) and -O.2
volts (Cramer and Butler, 1969) it still does.not seem
that the recombination could provide quite all of the
requisite energy. Nor does it explain how the "high
energy state" of the membrane is involved. Nevertheless
the potentials quoted are so uncertain that one must
apply such energy computations with caution.

Arnold (1968) rationalizes the fact that there
is apparently not enough energy in the reaction of Z+ with
Q- to give DLE by abandoning the "Z scheme" of Hill and
Bandall (1960) to use 2+ with an electron from ferredoxin.
The combination of these two might then have sufficient
energy to support DLE. However, Goedheer (1962) had al-
ready shown that PS II light stimulates DLE while PS I
light markedly decreases DLE. This suggests that the
removal of electrons from the plastoquinone pool de-
creases DLE, which in turn suggests that the electrons
used in DLE come from plastoquinone and not from ferredoxin
or anything on the reducing side Of PS I.

Lavorel (1969) approached the problem of the energy
required for DLE in a different manner. He postulated
that the product of the reaction of Z+ and Q— produced

one triplet exciton. Then two of these triplet excitions

61

"collide”to form the chlorOphyll singlet which decays in
a normal manner to give the DLE. The major drawback to
this theory is that while Livingston (1960) has presented
extensive data on triplet activity in chlorophyll solutions,
the triplet state has never been detected in chloroplasts.
Further doubt is cast upon the triplet model by the
finding of Lumpkin and Hillel (1973) that a pulsed 30
kgauss magnetic field does not affect the DLE.

Fleishman (1971) and Crofts, wraight, and Fleischman
(1971) have suggested a mechanism by which the deficiency
in energy of the reaction between Q- and 2+ might be
overcome while at the same time accomodating the fact that
the high energy state of the membrane is required for
DLE. They propose that the additional energy needed
comes from an electrOchemical potential which is formed
across the membrane. They interpret Mayne and Clayton’s
(1966) observation that acid-base transitions contribute
to the DLE, and Mayne's (1967) subsequent observation
that uncouplers inhibit the DLE in terms of this model,
with the acid-base transition producing the electro-
chemical (H+) gradient across the membrane and uncouplers
abolishing this gradient, by making the membranes permeable
to H+.

Other studies have implicated trans-membrane
electrochemical gradients. Miles and Jagendorf (1969)

showed that a transition from a low salt medium to a high

62

salt medium also contributed to DLE. Several different
anions and cations were employed.. It is important to note
that such gradients, unlike H+ gradients, do not support
significant phosphorylation.. Subsequently, there were
other reports of cation gradients which stimulated DLE
but did not support phOSphorylation. For example, Barber
and Kraan (1970) showed that a low sodium chloride to
high sodium chloride transition in the presence of
gramicidin (which makes membranes permeable to Na+ but
utterly abolishes phOSphorylation) gave DLE. Similarly,
a low K+ to high K+ transition gave DLE in the presence
Of valinomycin (which makes membranes permeable to K+).
Thus DLE does indeed seem to require a membrane potential,
but this potential does not need to be of a kind which
supports phosphorylation. In fact DLE occurs under
conditions where no phOSphorylation would be possible
(in the presence of gramicidin as reported above and as
in the ammonia experiments reported herein).

These ammonia experiments show a stimulation of
DLE by NH4C1 and, under low salt conditions, the stimula-
tion seems to be correlated with NH4+ uptake. At least
both NH4+ uptake and DLE under these conditions show the
same pH Optimum. See Figure 11. This correlation disap-
pears under high salt conditions. However Izawa and Good
(1966) have shown that the chloroplast volume increases

drastically with NH + and high salt and this swelling

4

63

probably destroys any systematic relationship between
NH4+ uptake and the NH4+ concentration gradient which
might be determining the membrane potential associated
with DLE. The reason why this effect of NH4+ on DLE

is unique among amines is unclear. However, it is
probably relevant that methylamine also stimulates DLE
in subchlorOplast particles instead of inhibiting the
DLE as it does in intact chlorOplast lamellae. Perhaps
this is somehow related to the fact that ammonia and
aimes do not uncouple phosphorylation in subchloroplast
particles.

It is important to remind the reader that the
energy from an acid-base transition or a low salt to high
salt transition is not alone sufficient to create DLE,
since a recent preillumination has been shown to be re-
quired by Mayne (1968). Thus there seems to be a re-
quirement for a membrane potential AND a 2+ and 0-.
Fleischman (1971) and Crofts and co-workers (1971)
visualize the membrane potential as somehow adding to the
energy of the reaction between Z+ and Q- in such a way
as to effectively lower the activation energy of this
reaction. Even if the combined membrane potential and
the combined redox potentials of the Z+ and Q- do not
provide the 1.8 eV necessary for the DLE, there must be
a few "individuals" in the Boltzman distribution that

can reach the 1.8 eV. Furthermore the greater the membrane

64

potential due to, for instance, a H+ gradient, the greater
should be the number of "individuals" with enough energy
to make DLE, and the faster should be the rate of back-
reaction between Q- and 2+ i.e., the decay of the DLE.

See Figure 7.

The amount of DLE is very small. It has been de-
termined by Tollin gt_al. (1958) to have a quantum yield
of 10-6. Thus one would expect the activation energy
to be rather large. Indeed, this is fortunate since it
is the function of the chloroplast to get excitation
energy away from the excited chlorophyll, into the hands
Of the enzymatic machinery to make ATP and NADPH, and
not DLE.

Fleischman (1971) and Crofts gt_al. (1971) do not
limit themselves to a discussion of energetics, but also
postulate certain topographical features in the chloro-
plast membrane. We would like to develop their concepts
with new evidence. Basic features of their model are
that water splitting, and its consequent H+ production,
occurs on the inside of the thylakoid membrane " in
equilibrium" with the Z/Z+ couple, while the plasto-
quinone pool occurs on the outside of the membrane in
equilibrium with the Q/Q- couple. Then, since the H+
pump creates an electric field, positive inside and

negative outside of the membrane, the resulting membrane

potential would act to oppose the electron from Q-, going

65

to the inside of the membrane to back-react with 2+. Thus
energy would have been added to the reaction between 2+
and 0..

However, there is good evidence that the pool of
electrons used in the back reaction of DLE actually re-
sides in plastoquinone and not in the primary electron
acceptor, Q. DCMU and o-phenanthroline (Nishimura, 1967)
which probably blocks the transport of electrons from Q-
to plastoquinone abolish DLE. However, inhibitors which
block electron tranSport on the PS I side plastoquinone
actually increase the quantum efficiency of DLE, as shown
in Figure 5. Thus KCN treatment which inhibits at the
level of plastocyanin (Quitrakul and Izawa, 1973) and
DBMIB which inhibits by preventing the reoxidation of
reduced plastoquinone (Trebse et_al, 1970) cause more,
rather than less DLE at all but very high light intensities.

Furthermore, there is evidence that the back-
reaction of Q‘ with Z+ does not require much activation
energy since it is said to occur at liquid nitrogren
temperatures (Tollin et_al. 1958). Thus the "activation
energy" supplied by the membrane potential probably some-
how contributes to the transport of electrons from the
storage form in plastoquinone tO Q, and perhaps to the
transport of electrons from Z to the intermediates in the

water oxidation process.

66

With these considerations in mind we would like to
modify the Fleischman-Crofts model by suggesting that
plastoquinone and the water oxidation machinery are on
the outside and inside respectively of the membrane and
that the membrane potential "accelerates" reverse electron
flow on the way from plastoquinone to the water oxidation
site. We therefore suggest that the primary photochemical
charge separation may occur across some sort of chlorophyll
containing layer, which occupies much less than the
70-90°A width of the membrane (Kirk, 1971) and that sub-
sequent electron transport events (Q- to plastoquinone
and water oxidation to Z+) take place across the rest of
the membrane. Such a model is outlined in Figure 16.

This model would explain our data in the following
manner. First, the DBMIB and KCN blocks stimulate DLE
at low light intensities by backing up the electrons in
the plastoquinone pool which can back react to reduce
the Q. The Q reduction is very unfavorable but under
conditions where the electric field from ion gradients
make it more favroable, it can go to a small degree.

This reduction of Q by plastoquinone can be blocked by
DCMU or o-phenanthroline which then abolishes the DLE.
If Q is reduced by plastoquinone it can react with Z+ to
give an excited chlorOphyll molecule which then gives
DLE. The stimulation by NH4C1 is due to the fact that

ammonium ion uptake establishes an electrical gradient, as

67

did the H+ gradient, and this electrical field caused
by the ammonium ion gradient partially counteracts the
thermodynamically unfavorable Q reduction by plastoquinone.

It should be noted that according to our model
and to the model of Fleischman and Crofts, uncouplers
only abolish DLE if they abolish all membrane potentials.
Obviously some kinds of ion gradients and membrane poten-
tials which are incapable of supporting phosphorylation
are capable of contributing to DLE.

Probably ADP and ATP stimulate the DLE because
they stimulate the H+ uptake, presumably by interaction
with the coupling factor, and the resulting membrane
potential once again stimulates the rate of Q reduction
by plastoquinone. The fact that ADP and Pi together
inhibit the DLE is also easily explained since they
consume H+ to make ATP. This consumption of H+ reduces
the membrane potential and makes it more difficult for
electrons to go from plastoquinone to Q.

The corollary to this model is that in decreasing
the activation energy for the reduction of Q with electrons
from plastoquinone by applying an electric field favorable
for the reverse electron movement, one simultaneously
increases the activation energy for the forward reaction.
That is if one stimulates the DLE, all other conditions
being equal, one should inhibit electron transport. Figure

15 shows precisely that. ATP and ADP Stimulate the DLE

68

.mno HHannH Hon Op .MHHmHEono
Iouonm onu Eonm ouOEoH.oHOE moUHm ucosvomnsm um pun .ooumop oEom onu Op uuommcouu

sonuooHo anHnnH nOHn3 .ZUM can mHZmQ umonunoo nH .mqo pHannH AcoHuomonnnoon onu

N

nuHB ouomuoucH nOHn3V .mo mz new DEUQ pony opoz .osounfioa onp anuHB pouoUoEOoom on UHOOO

munocomEOo omonu mHnonoum .ousmHm man CH mm: nmsou euo> m CH noHMOHUCH onm HoUOE onu no

murgmflOQEOO Gnu. MO mmNHw 03H. .GUHmGH 05”.. SO OmHm N

O.Uno +m Op nouoz mo noHpmono onu Unm
.oUHmnH onu so GOHHOSUOHQ +3 unmuHEOOnoo nuHB m oEounooueo HoHnHoo nouuooHo onu en mmom
onu mo noHuoUon onu .onoHnEoE onu mo oUHmuso onu so mansooo ococHstumon mo nOHuOSUoH
onu sH +3 mo nOHumESmnoo o no oopnomoumon mH =mE:a.+m one .nomonmmnme mo unoEo>Ho>nH onu
mHmEH one s: oopmsmHmoU Honcoo mnHNHUHxO Hopos on «mom EOHH mcoupooHo mo Hommcouu onp .o.H
mam moospoum nOHnB sOHuomoH noon onp now oHnmem>m monono onu Op.©©o nsoHUmnm +m HO\Uno

Hochopom osmunfioe one .umoHUon coHuoHucoosoo o ocoHo +M no oz mo cOHmSMMHU o>Hmmom

+

OH osp Uomuono oEooon OmHo coo uH .mGOH Hounsoo an UoHsomEOocons mQOH +m mo usommnmun

on» no omsooon oomuono mofiooon eHHmanon ozonnfiofi one .Honoa mHnn CH .mHmonp mHnu nH

pommsome onoHnEoE pHoonmnu one no COHuoHsmHmcoo Hmuonom nouoHsumomll.mH onsmHm

69

2:2:

 

 

 

:2
all,
m Z\ IIIII N .
I 1 Cir: nzeflx
l 7
e o o e. o o o o o o e e
l a
.__.. (a:
40»... Anal \ Infirouzz
ob. \ mime No c_v_u_Eo._o+uz
II a ll \ on. .. c_o>Eoc__u>+v_
l0 20v.— \ . II +N %MI_O

 

(OGION
O

 

 

 

 

b NIMi O IM+GII coca.
rod m- 2200
I «ran Omroamoa
oz. o o o o o o or o o o
scum +

3.230

70

by increasing the H+ uptake while at the same time also
decreasing electron transport. When ADP and Pi were
added, H+ ions were consumed, decreasing the electrical
gradient Opposing the forward reaction and thus allowing
electron transport to proceed at a faster rate. Similarly
uncouplers, by decreasing the H+ gradient, would de-
crease the electrical gradient Opposing the forward re-
action, and thus allow electron transport to proceed
at a much faster rate.

In the course of the nucleotide stimulation of
DLE work, an interesting Observation was made, one for
which we have no good explanation. The raw data for
this Observation is presented in Figure 14. It can be
seen that Pi had little effect on the DLE, that ADP and
ATP stimulated the DLE, but that ADP and Pi inhibited the
DLE. These results were predictable. The question then
arose, if a limited amount of ADP was added, what would
happen to the DLE as the ADP was converted to ATP. Would
the DLE, which was initially very low, gradually rise
as the ADP was consumed to the level characteristic Of
the ATP and Pi reaction mixture. The last tracing in
Figure 14 shows that precisely this did happen. Follow-
ing a 15 second lag the DLE rises to its maximal height
in two steps. Since the fluorescence bore no resemblance
to the DLE curve, we assume that this phenomenon has to

do with the chemical excitation of chlorophyll. Whether

71

this could be related to successive bindings or releases
Of ADP and ATP to a protein such as the coupling factor
is purely speculative. Perhaps this phenomenon is in
the same related to the two step inhibition of phOSpho-
rylation by mercurials (Izawa and Good, 1969), Bradeen
§t_al. (1973). It is not possible to determine very
accurately the phosphorylation rate from this process

since the substrate concentration is less than the Km

(which is 1.4 X 10'4, Izawa et al. 1966) and

vm(s)

 

K
In

so that the phosphorylation rate is proportional to the
substrate concentration. Consequently it is difficult to
relate this curve of rising DLE to the decrease in the
rate of phosphorylation which must have been taking place
over the same period of time.

In these studies we have measured the intensity
Of DLE and attempted to deduce therefrom the rate of chemi-
cal excitation of the chlorophyll. However, there is a
major complicating factor which must be considered,
namely the effect of conditions on the fluorescence
efficiency of the excited chlorophyll. This factor can be
evaluated by Observing prompt fluorescence which is, of
course, a measure of the fluorescence efficiency of light

excited by chemical back-reactions with which we are

72

primarily concerned. As can be seen in Figure 6 there is
striking parallel between DLE and prompt fluorescence
when DBMIB is added at various concentrations. This
parallel is particularly noticeable when DBMIB is kept
oxidized by having ferricyanide present at the same time
(or oxygen at the higher pH's). Under these conditions
DBMIB acts as an electron acceptor (Gould and Izawa,
1973). However the suppression Of DLE and.prompt fluo—
rescence by DBMIB is in no way related to electron trans-
port since it also occurs in the presence of DCMU.' The
quenching of both kinds of fluorescence is very probably
due to a direct chemical reaction of this inhibitory
quinone with chlorophyll.. In the first place the concen-
trations of DBMIB involved greatly decrease the quantum
efficiency of electrOn tranSport (Gould and Izawa, 1973)
as must happen if there is a direct chlorophyll quenching,
.and in the second place, DBMIB quenches the fluorescence
of chlorOphyll solutions (5 mM DBMIB quenches 50% of the
fluorescence of a 5 ugm/ml solution of chlorophyll in
acetone).

At pH 7.5 with methylviologen as electron
acceptor the DBMIB cannot be reoxidized. One can see in
Figure 6 that both the DLE and the fluorescence are
initially low with 5 uM DBMIB present. However the DBMIB
becomes reduced to DBMIBH2 in the light and cannot be

reoxidized. Its reduction creates a reducing environment

73

around plastoquinone and PS II thus raising the prompt
fluorescence and the DLE simultaneously. Gould and
Izawa, (1973) measured the DBMIB reduction in chloroplasts
while looking at pH changes with a pH electrode. They
found a H+ uptake due solely to the DBMIB2 reduction.
The H+ uptake stOpped in the light as soon as the DBMIB
became reduced and then decayed in the light. The

more DBMIB present the longer the pH rise lasted. The
kinetics of Gould and Izawa's pH rise (with 5 um DBMIB
and methylviologen, pH 7.5) and the DLE presented under
the same conditions are practically identical. Thus
while the DLE and fluorescence rise identically, after
the fluorescence reaches its maximal value, the high
level is maintained, but the DLE being partially due

to the pH rise the level of DLE declines after reaching
a maximal value. At 10 um DBMIB a longer time is re-
'quired in order to reduce all of the DBMIB while the

H+ uptake is presumably going on for a longer time too.
This is undoubtedly the reason that the fluorescence and
DLE remain longer at a higher level.

Since there is considerable electron transport
and phosphorylation under all Of these conditions, we
would expect considerable DLE. The absence of DLE in
the presence of DBMIB, taken together with the fact that
prompt fluorescence is also quenched, argues strongly
that DLE shoudl be evaluated on the basis of the prompt

fluorescence efficiency as suggested above and as

74

Lavorel (1969) has pr0posed. That is to say it seems
very probable that prompt fluorescence and the chemi-
luminescence we have been concerned with here, share the
same fluorescence yield characteristics. Perhaps the
same chlorOphyll molecules are fluorescing. This does
not mean that the DLE must rise every time the prompt
fluorescence rises, since according to our model, the
mechanism of generation of the excited chlorophyll is
entirely different.

We conclude that the DLE Observed at 4 msec after
light extinction under "steady state" conditions, at
room temperature, and at fairly high light intensity:

1. Is not an Obligatory function of the

ability of chlorOplasts to phosphorylate,
though it is very often associated with
this ability.

2. Is an obligatory function of some kind

of membrane potential.

3. Absolutely requires both the reduced
form of the primary electron acceptor
for PS II Q_, and the oxidized form
of the primary electron donor for
Ps II 2+.

4. Is dependent upon the reverse flow
of electrons, from plastoquinone to

Q which is in turn causally related

75

to the above mentioned cation
generated membrane potential.
Has a final production step which is

dependent on the fluorescence yield.

LITERATURE CITED

76

LITERATURE CITED

Arnold, W., and J. R. Azzi. 1968. Chlorophyll energy
levels and electron flow in photosynthesis. Proc. Natl.
Acad. Sci. U. S. A. 61: 29-35.

Arnold, W., and J. R. Azzi. 1971. The mechanism of de-
layed light production by photosynthetic organisms and

a new effect of electric fields on chloroplasts. Photo-
chem. Photobiol. 14: 233-240.

Arnold, W. 1972. The ratio between delayed light and
fluorescence emitted by chlorOplasts. Biophys. J.
12: 793-796.

Arnon, D. I. 1949. Copper enzymes in isolated chloro-
plasts: polyphenoloxidase in Beta vulgaris. Plant. Physiol.
24: 1-15.

 

Avron, M. 1960. PhotOphOSphorylation by swiss-chard
chlorOplasts. Biochim. Biophys. Acta. 40: 257-272.

Barber, J., and G. P; B. Kraan. 1970.‘ Salt induced light
emission from chloroplasts. Biochim. Biophys. Acta.
197: 49-59.

Barbieri, G., R. Delosme, and P. Joliot. 1970. Comparai-
son entre l'emission d'oxygene et l'emission de luminescence
a la suite d'une serie d'eclairs saturants. Photochem.
Photobiol. 12: 197-206.

Bertsch, W. F., and J. R. Azzi. 1964. 'A relative maxi-
mum in the decay of long term delayed light emission
from the photosynthetic apparatus. Biochim. BiOphys.
Acta. 94: 15-26.

Bertsch, W., J. R. Azzi, and J. B. Davidson. 1967. De-
layed light studies on photosynthetic energy conversion
I Identification of the oxygen evolving photoreaction

as the delayed light emitter in mutants of Scenedesmus
Obliquus. Biochim. BiOphys. Acta. 143: 129-143.

 

Bertsch, W., J. West, and R. Hill. 1971. Delayed light
studies on photosynthetic energy conversion III Effect of
3— (3, 4-Dichlorophenyl) -l, l-Dimethyl urea on the
millisecond emission from chloroplasts performing

77

78

photoreduction of ferricyanide. Photochem. Photobiol.
14: 241-250.

Bertsch, W., and S. Lurie. 1971. Delayed light studies
on photosynthetic energy conversion IV Effect of Tris
poisoning, and its reversal by hydrogen donors, on the
millisecond emission from chloroplasts. Photochem.
Photobiol. 14: 251-260.

Bradeen, D. A., J. M. Gould, D. R. Ort, and G. D. Winget.
1973. Site-specific inhibition of photophosphorylation
in isolated chloroplasts by mercuric chloride. Submitted
Plant. Physiol.

Brand, M. J. D., and G. A. Rechnitz. 1970. Differential
potentiometry with ion-selective electrodes. Anal. Chem.
42: 616-622.

Butler, W. L. 1971. The relationship between C-550 and
delayed fluorescence. FEBS Letters. 19: 2, 125-127.

Carstanjen. 1871. Ueber chinonartige‘abkommlinge. J.
Fuer. Pract. Chemie. 3: 55-65.

Cheniae, G. M., and I. F. Martin. 1971. Effects of hydro-
xylamine on photosystem II. Plant. Physiol. 47: 568-575.

Clayton, R. K. 19691 Characteristics of prompt and de-
layed fluorescence from Spinach chloroplasts. BiOphys. J.
9: 60-76.

Cramer, W. A. and W. L. Butler. 1969. Potentiometric
titration of the fluorescence yield of spinach chlorOplasts.
Biochim. Biophys. Acta. 172: 503-510.

Crofts, A. R., C. A. Wraight, and D. E. Fleischmann. 1971.
Energy conversion in the photochemical reactions of
photosynthesis and its relation to delayed fluorescence.
FEBS Letters. 15: 2, 89-100.

Duysens, L. N. M. 1964. Photosynthesis. Prog. BiOphys.
Mol. Biol. 14: 1—31.

Duysens, L. N. M., and H. E. Sweers. 1963. Mechanism of
two photochemical reactions in algae as studied by means
of fluorescence. In Studies On Microalgae and Photo-
synthetic Bacteria. University of Tokyo Press, Tokyo.

p. 353-372.

Evans, E. H., and A. R. Crofts. 1973. The relationship
between delayed fluorescence and the H gradient in choro-
plasts. Biochim. Biophys. Acta. 292: 130-139.

79

Fleischmann, D. E. 1971. Luminescence in photosynthetic
bacteria. Photochem. Photobiol. 14: 277-286.

Franck, J., and J. E. Brugger. 1958. Experimental and
theoretical contributions to studies of the afterglow

of chorOphyll in plant materials. Arch. Biochem. Biophys.
75: 465-496.

Gilman and Blatt. 1961. Thymoquinone In Organic Synthesis,
second edition. John Wiley and Sons. vol. I, p. 511-513.

Goedheer, J. C. 1962. Afterglow of chlorophyll in vivo
and photosynthesis. Biochim. Biophys. Acta. 64: 294-308.

Gould, J. M., and S. Izawa. 1973, Studies on the energy
coupling sites of photOphosphorylation. I Separation of
site I and site II by partial reactions of the electron
transport chain. In Press. Eur. J. Biochem.

Haug, A., D. D. Jacquet, and H. C. Beall. 1972. Light
emission from the Scenedesmus Obliquus wild type, mutant
II strains, measured under steady-state conditions be-
tween 4 nanoseconds and 10 seconds. Biochim. BiOphys.
Acta. 283: 92*99.

 

Hill, R., and F. Bendall. 1960. Function of the cytochrome
components in chlorOplasts: a working hypothesis. Nature.
186: 136-137.

Itoh, S., S. Katoh, and A. Takamiya. 1971. Studies on
the delayed light emission in chloroplasts. II Participa-
tion of primary electron donor and acceptor of photo-
reaction II in producing the delayed light emission.
Biochim. Biophys. Acta. 245: 121-128.

Jackson, J. B., and A. R. Crofts. 1969. The high energy
state in chromatOphore- from Rhodopseudomonas spheroides.
FEBS Letters. 4: 3, 185-189.

 

Hess, G. P., and J. A. Rupley. 1971. Structure and func-
tion of proteins. Ann. Rev. Biochem. 40: 1,013-1,044.

Izawa, S., and N. Good. 1966. Effects of salts and
electron transport on the conformation of isolated
chlorOplasts. I Light-scattering and volume changes.
Plant. Physiol. 41: 3, 533-543.

Izawa, S., T. N. Connolly, G. D. Winget, and N. E. Good.
1966. Inhibition and uncoupling of photophosphorylation
in chloroplasts. Brookhaven Symp. Biol. 19: 169-187.

80

Izawa, S., R. L. Heath, and G. Hind. 1969. The role

of chloride in photosynthesis. III The effect of arti-
ficial electron donors upon electron transport. Biochim.
BiOphys. Acta. 180: 388-398.

Izawa, S., and N. Good. 1969. Effect of p-chloromercu-
ribenzoate and mercuric ion on chloroplast photophosphoryl-
ation. In: Prog. In Phtosyn. Research vol. III Inter-
national Union of Biological Sciences. p. 1,288-l,298.

Joliot, A. 1966. Actions du chloromethyluree et de
1'hydroxy1amine sur la reaction photochimique d'emission
d'oxygene (system II) Biochim. Biophys. Acta. 126:
587-590.

'Joliot, P., G. Barbieri, and R. Chabaud. 1969. Un
nouveau modele des centres photochimiques du systeme II.
Photochem. Photobiol. 10: 309-320.

Kirk, J. T. O. 1971. Chloroplast structure and biogenesis.
Ann. Rev. Biochemistry. 40: 161-196.

Lavorel, L. 1969. On a relation between fluorescence and
luminescence in photosynthetic systems. In: Prog. in
Photosyn. Research, Vol. III International Union of Bio-
logical Sciences. p. 883—898.

Kok, B., S. Malkin, O. Owens, and B. Forbush. 1967.
Observations on the reducing side of the O2 evolving act.
Brookhaven Symp. Biol. ‘19: 446-459.

Kressler, E., W. Arthur, and J. E. Brugger. 1957. The
influence of manganese and phosphate on delayed light
emission, fluorescence, phtoreduction and photosynthesis
in algae. Arch. Biochem. Biophys. 71: 326-335.

Livingston, R. 1960. Physciochemical aspects of some re-
cent work on photosynthesis. Quarterly Reviews, 174-198.

Lumpkin, O., and Z. Hillel. 1973. Isolation of a fast de—
cay in submillisecond Chlorella luminescence. Biochim.
Biophys. Acta. 305: 281-292.

 

Mar, T., and Govindjee. 1971. Thermoluminescence in
spinach chlorOplasts and in Chlorella. Biochim. Biophys.
Acta. 226: 200-203.

 

Mayne, B. C., and R. K. Clayton. 1966. Luminescence of
chlorophyll in spinach chloroplasts induced by acid-base
transition. Proc. Nat'l. Acad. Sci. U. S. A. 55: 494—497.

81

Mayne, B. C. 1967. The effect Of inhibitors and un-
couplers Of photosynthetic phosphorylation on the delayed
light emission Of chloroplasts. Photochem. Photobiol.

6: 187-197.

Mayne, B. C. 1968. The light requirement of acid base
transition induced luminescence of chloroplasts.
Photochem. Photobiol. 8: 107-113.

McCarty, R. B., J. S. Fuhrman, and Y. Tsuchiya. 1971.
Effects of adenine nucleotides on hydrogen-ion transport
in chloroplasts. Proc. Nat'l. Acad. Sci. U. S. A. 68: 10,
2,522-2,526.

Miles, C. D., and A. T. Jagendorf. 1969. Ionic and pH
transitions triggering chlorOplast pot-illumination
luminescence. Arch. Biochem. Biophys. 129: 711-719.

Nelson, N., Z. Drechsler, and J. Neumann. 1970. Photo—
phOSphorylation in digitonin subchloroplast particles.
J. Biol. Chem. 245: 1, 143-151.

Neumann, J., J. Barber, and P. Gregory. 1973. The re-
lation between photOphosphorylation and delayed light
emission in chlorOplasts. Plant. Physiol. 51: 1,069-l,073.

Nishmura, M. 1967. Oxidation-reduction reactions of
cytochromes in red algae. Brookhaven Symp. Biol. 19:
132-142.

Quitrakul, R., and S. Izawa. 1973. Electron transport
and photophosphorylation in chloroplasts as a function
of the electron acceptor II Acceptor-specific inhibition
by KCN. Biochim. BiOphys. Acta. 305: 105-118.

Priestly, E. B., and A. Haug. 1968. Phosphorescence
spectrum of pure crystalline naphthalene. J. Chem. Phys.
49: 622-629.

Saha, 8., R. Ouitrakul, S. Izawa, and N. E. Good. 1971.
Electron tranSport and photophosphorylation in chloro-

plasts as a function of electron acceptor. J. Biol.
Chem. 246: 10, 3,204-3,209.

Strehler, B. L., and W. Arnold. 1951. Light production
by green plants. J. Gen. Physiol. 34: 809—820.

Sweetser, P. B., C. W. Todd, and R. Titersh. 1961. Effect
of photosynthesis inhibitors on light re-emission in photo-
synthesis. Biochim. BiOphys. Acta. 51: 509-518.

82

Tollin, G., E. Fujimori, and M. Calvin. 1958. Delayed
light emission in green plant materials: Temperature de-
pendence and quantum yield. Proc. Nat'l. Acad. Sci.

U. S. A. 44: l,035-l,047.

Trebst, A., E. Harth, and W. Draber. .1970. On a new
inhibitor of photosynthetic electron transport in isolated
chloroplasts. Z. Naturforsch. 25b: l,157-l,159.

Uribe, E., and A. T. Jagendorf. 1966. ATP formation
caused by acid-base transition of spinach chloroplasts.
Proc. Nat'l. Acad. Sci. U. S. A. 55: 170-177.

Winget, G. D., S. Izawa, and N. E. Good. 1969. The in-
hibition of photophosphorylation by phlorizin and closely
related compounds. Biochemistry. 8: 2,067-2,073.

Witt, A. T., G. Doring, B. Rumberg, P. Schmidt-Mende,
U. Siggel, and H. H. Stiehl. 1966. Electron transport
in photosynthesis. Brookhaven Symp. Biol. 19: 161-168.

Wraight, C. A., and A. R. Crofts. ‘1971. Delayed fluo-
rescence and the high energy state of chloroplasts. Eur.
J. Biochem. 19: 386-397.

Zankel, K. L. 1971. Rapid delayed luminescence from
chloroplasts: Analysis of components; the relationship to
the 08 evolving system. Biochim. Biophys. Acta. 245:
373-3 5. ‘

 

lllllllllll

'
l
I
I
l