Planta (1994)193:421~429
P l a n t a
9 Springer-u
1994
ATP and N A D P H as the driving force of carbon reduction in leaves
in relation to thylakoid energization by light
Ulvi Gerst, Gerald Sch6nknecht, Ulrich Heber
Julius-von-Sachs-Institut ffir Biowissenschaften der Universit/it Wfirzburg, Mittlerer Dallenbergweg 64, D-97082 Wfirzburg, Germany
Received: 28 October 1993/Accepted: 29 November 1993
Abstract. Carbon assimilation of spinach (Spinacia oleracea L.) leaves was measured in the presence of 2000 gl.
l l CO2 and 2% 02 in the gas phase to suppress photorespiratory reactions and to reduce stomatal diffusion resistance. Simultaneously, membrane parameters such as
modulated chlorophyll fluorescence, oxidation of P7oo in
the reaction centre of photosystem I, and apparent
changes in absorbance at 535 nm were recorded. After
light-regulated enzymes were activated at a high irradiance, illumination was changed. About 3 min later (to
maintain the previous activation state of enzymes), leaves
were shock-frozen and freeze-dried. Chloroplasts were
isolated nonaqueously and analysed for ATP, ADP, inorganic phosphate, N A D P H and NADP. Observations
made under the chosen conditions differed in some important aspects from those commonly observed when
leaves are illuminated in air. (i) Not only assimilation, but
also the phosphorylation potential [ATP]/([ADP].[Pi])
increased hyperbolically with irradiance towards saturation. In contrast, the ratio of N A D P H to N A D P did not
change much as irradiances increased from low to high
photon flux densities. When ATP, the phosphorylation
potential and the assimilatory force, F A (the product of
phosphorylation potential and N A D P H / N A D P ratio),
were plotted against assimilation, ATP increased relatively less than assimilation, whereas the phosphorylation potential increased somewhat more steeply than assimilation did. A linear relationship existed between assimilation and F A at lower irradiances. The assimilatory
force F A increased more than assimilation did when irra-
Abbreviations: FA = assimilatory force; Fo = fluorescence after
long dark adaptation; Fm = maximum fluorescence level; F s =
steady-state fluorescence; PGA = 3-phosphoglycerate; PFD =
photon flux density; P7oo(PTo0+) = electron-donor pigment in the
reaction center of PSI (its oxidized form); QA = primary quinone
acceptor of PSII; qp photochemical quenching; qN = non-photochemical quenching; q)Psn = relative quantum efficiencyof energy conversation at the level of photosystem II; ~vst = relative
quantum efficiencyof photosystem II
Correspondence to: U. Heber; FAX: 49 (931) 71446
:
diances were very high. Differences from previous observations, where F A w a s under some conditions higher at
low than at high rates of carbon assimilation, are explained by differences in flux resistances caused not only
by stomatal diffusion resistance but also by differences in
the activity of light-regulated enzymes. (ii) The relationship between P700 oxidation and a fast absorption change
with a maximum close to 520 nm on one hand and carbon assimilation on the other hand was largely linear
under the specific conditions of the experiments. A similar linear relationship existed also between the quantum
efficiency of electron flow through photosystem II and
the quantum efficiency of photosystem I electron transport. (iii) Whereas the increase in non-photochemical fluorescence quenching, qN, was similar to the increase in
assimilation, the relationship between light scattering
and assimilation was distinctly sigmoidal. Light scattering appeared to be a better indicator of control of photosystem II activity under excessive irradiation than qN. (iv)
The results are discussed in relation to the relative significance of chloroplast levels of ATP and N A D P H and of
the assimilatory force F A in driving carbon assimilation.
From the observations, the proton/electron ( H + / e ) ratio
of linear electron transport is suggested to be 3 and the
H + / A T P ratio to be 4 in leaves. An H + / e - ratio of 3
implies the existence of an obligatory Q-cycle in leaves.
Key words: Assimilatory force - Chlorophyll fluorescence
- Light scattering - Photosynthesis (phosphorylation)
potential - PTo0 oxidation - Spinacia
Introduction
Light drives photosynthesis. However, how does it do
this? Several decennia of photosynthesis research have
established that, in the closed thylakoid membrane system of chloroplasts, light absorbed by chloroplast pigments initiates a series ofredox reactions which eventually lead to oxygen evolution and the reduction of water-
422
u. Gerst et al.: Role of ATP and NADPH in carbon reduction of leaves
soluble N A D P . D u r i n g c o u p l e d electron t r a n s p o r t from
water to N A D P , p r o t o n s a c c u m u l a t e inside the thylakoids. The p r o t o n m o t i v e force of the t r a n s t h y l a k o i d
p r o t o n g r a d i e n t a n d a t r a n s m e m b r a n e electrical p o t e n tial are used to synthesize A T P from A D P a n d phosphate, while p r o t o n s pass t h r o u g h the t h y l a k o i d - b o u n d
A T P synthetase from inside to outside, d e g r a d i n g the
p r o t o n g r a d i e n t which is formed d u r i n g electron transport. Both N A D P H a n d A T P are needed for, a n d cons u m e d by, the reactions of c a r b o n a s s i m i l a t i o n in p h o t o synthesis a n d c a r b o n o x i d a t i o n in p h o t o r e s p i r a t i o n . The
ratio of c o n s u m p t i o n of A T P to that of N A D P H is n o t
very different in b o t h processes. It is s o m e w h a t a b o v e 1.5.
However, it is still n o t k n o w n h o w m u c h A T P is synthesized w h e n two electrons travel from water to N A D P .
P u b l i s h e d A T P / 2 e ratios r a n g e from 1 to 2 (Hall a n d
E v a n s 1972; Hall 1976; R o b i n s o n a n d Wiskich 1976;
A r n o n 1977; D a v e n p o r t a n d M c C a r t y 1984).
T h e H +/e ratio for linear electron t r a n s p o r t from water to N A D P m a y be either 2 or 3, d e p e n d i n g o n whether
or n o t a Q-cycle operates at the level of c y t o c h r o m e b/f
c o m p l e x b e t w e e n p h o t o s y s t e m s II a n d I (Heber a n d
W a l k e r 1992). A n y value b e t w e e n 2 a n d 3 is also possible
if p r o t o n t r a n s p o r t i n t o the t h y l a k o i d s by the Q-cycle
does n o t obey stoichiometric c o u p l i n g to electron transp o r t b u t is flexible. U n c e r t a i n t y exists also c o n c e r n i n g the
H + / A T P ratio of A T P synthesis. The c o m m o n l y accepted view is that three p r o t o n s m u s t travel t h r o u g h the
A T P synthetase for the p h o s p h o r y l a t i o n of one molecule
of A D P (Junge et al. 1970; D a v e n p o r t a n d M c C a r t y
1984). This view is challenged by Grfiber et al. (1987) a n d
R u m b e r g et al. (1990) w h o claim the H + / A T P ratio to be
4.
D e p e n d i n g on the different stochiometric ratios, either
extra A T P m u s t be supplied for p h o t o s y n t h e s i s by a n
auxiliary A T P p r o d u c i n g p h o t o r e a c t i o n , or excessive
p r o t o n pressure inside the t h y l a k o i d s m u s t be released by
a m o l e c u l a r safety valve ( S t r o t m a n n et al. 1986). Flexible
c o u p l i n g w o u l d solve the p r o b l e m of conflicting stochiometries, b u t the m e c h a n i s m of a d j u s t m e n t to changing c o u p l i n g r e q u i r e m e n t s r e m a i n s obscure.
T h e r e is still the q u e s t i o n of whether a l i g h t - d e p e n d e n t
increase in the A T P / A D P ratio or rather in the ratio of
N A D P H to N A D P in the c h l o r o p l a s t s t r o m a is m o r e
i m p o r t a n t in shifting the triose p h o s p h a t e / p h o s p h o g l y c erate system from o x i d a t i o n in the d a r k to r e d u c t i o n in
the light, a n d what is the role of A T P - p r o d u c i n g reactions such as the Mehler r e a c t i o n or of cyclic electron
t r a n s p o r t in p h o t o s y n t h e s i s (see also Schreiber a n d Neub a u e r 1990; H e b e r a n d W a l k e r 1992). The experiments
r e p o r t e d in the following are i n t e n d e d to answer some of
these questions.
Materials and methods
Detached mature leaves of spinach (Spinacia oleracea L. cv. Polka,
'JuliWa', Heidelberg, Germany) grown in the greenhouse with a
10/14 h day/night cycle were used for the experiments. Part of the
lamina of a leaf was enclosed in a sandwich-type cuvette while the
petiole was kept in water. The gas flow through the cuvette was
500 ml.min ~ and the temperature was kept between 22 and 24~
Gas mixtures were prepared using mass-flow controllers (Tylan
Corporation, Eching, Germany). Carbone dioxide and water contents in the gas stream were determined using an infrared gas analyzer (BINOS; Heraeus, Hanau, Germany). The cuvette contained
windows permitting illumination of the upper surface of the leaf
with a broad band of red light (filters Calflex C from Balzers, Liechtenstein, Liechtenstein, and RG 630 from Schott, Mainz, Germany)
or far red light (filters Calflex C from Balzers and RG 9 from
Schott).
The system allowed simultaneous measurements of CO2 exchange, of changes in apparent absorbance at 535 nm (A535) of the
leaf and of modulated chlorophyll fluorescence or oxidation of Pvoo,
the electron-donor pigment in the reaction center of PSI. Light/
dark changes in A535 were used to get information on both light
scattering (Heber 1969; K6ster and Heber 1982; Bilger et al. 1988),
commonly considered to be an indicator of transthylakoid
pH gradient and the electrochromic shift (often termed P515, see
Junge 1977), which gives information on the transthylakoid electrical field. At this wavelength the faster kinetics of light-on and lightoff responses distinguish the electrochromic shift (PsJ5), which peaks
at 521 nm, but is still appreciable at 535 nm, from the slower lightscattering changes, which have a maximum at 535 nm (Heber 1969).
The detecting device, a photomultiplier, was protected against actinic light by two filters 9782 (Corning Glass Works, Corning, N.Y.,
USA) and a BG 18 filter from Schott. The measuring 535-nm light
was so weak as not to cause detectable photosynthesis.
Modulated chlorophyll fluorescence was measured with a PAM
chlorophyll fluorometer from Walz (Effeltrich, Germany). The following parameters were recorded: so-called dark fluorescence Fo
after long dark adaptation of a leaf, the maximum fluorescence level
F m produced by saturating red flashes of 1 s duration given to a
darkened leaf, steady-state fluorescence F Sin the presence of actinic
illumination, Fm' produced by saturating red flashes of I s duration
given in the presence of actinic illumination, and the so-called dark
fluorescence F o' (i.e. the level of modulated fluorescence seen immediately after actinic light was turned off and a very weak far red
beam was added in order to accelerate reoxidation of QA, the primary quinone receptor of PSII). Photochemical quenching of fluorescence, qp, non-photochemical quenching, qN, and the quantum
efficiency, q~esl~,of electron flow through PSII were calculated from
these parameters (Schreiber et al. 1986; Genty et al. 1989). The
determination of qN needs a somewhat detailed description, because
qN differed in important detail from light scattering, which is often
considered to give information equivalent to qN (Bilger et al. 1988).
In )eaves which had been predarkened for a long time, saturating
flashes raised the level of modulated fluorescence transiently from
F o to Fm. The ratio (Fm-Fo)/F m generally had values close to 0.8
(Bj6rkman and Demmig 1987). The non-photochemical fluorescence quenching qN had the value zero at Fm. When leaves were
darkened after actinic illumination,fluorescence declined rapidly to
F o' which was often lower than Fo. The difference Fm-Fo' was taken
to indicate maximally possible qN with a value of 1. Thus, qN could
have values between zero and 1.
Oxidation of P7oo was monitored close to 830 nm with a PAM
fluorometer, which was equipped with an appropriate emitter-detector unit (Schreiber et al. 1988). The observed signal is known to
have a component attributable to plastocyanin which is oxidized
together with P7o0(Klughammer and Schreiber 1991).
Experiments were performed as follows: spinach leaves were
illuminated in nitrogen containing 2000 ~tl.l ~CO2 and 2% oxygen.
After the stomata had opened and photosynthesis stabilized at a
photon flux density (PFD) of 1370 ~tmol-m 2-s ~, irradiance was
changed. After about 3 min at the lower (or higher) PFD, during
which carbon assimilation and fluorescence stabilized at a new level, metabolism of the leaf was freeze-stopped at the temperature of
liquid nitrogen using a "freeze-clamp" technique (Badger et al. 1984;
Siebke et al. 1990). In order to get sufficient leaf material for nonaqueous chloroplast extraction, and to avoid as far as possible
scattering of data due to individual differences between leaves, each
experimental point was repeated four times and two different samples were taken from one leaf. Samples were stored briefly in liquid
U. Gerst et aL: Role of ATP and N A D P H in carbon reduction of leaves
nitrogen and then freeze-dried at - 4 0 ~ Chloroplasts were isolated nonaqueously from the dry material as described in Dietz and
Heber (1984).
Inorganic phosphate (Pi), adenylates and N A D P § were determined after 30 min extraction of the chloroplasts in 6% HC104.
After centrifugation of the suspension, Pi was measured spectrophotometrically (Taussky and Shorr 1953). Another part of the supernatant was neutralized with 5 M K2CO3/0.25 M triethanolamine.
The amount of N A D P + was determined by enzymatic cycling
(Takahama et al. 1981). Since N A D P H is decomposed in strong
acid, it was oxidized by methylphenazonium methosulphate
(60 laM) in a neutral chloroplast suspension (50 mM Tris, pH 7.6)
and measured after addition of acid and subsequent neutralization
together with N A D P +. Adenylates were measured by a firefly
method (Hampp 1985; Wulff and D6ppen 1985) with a " L U M A T "
LB9501 luminometer (Berthold, Wildbad, Germany) using the procedure adopted by Dietz and Heber (1984). The sum of ATP +
ADP + A M P differed by less than 10% in all samples. We take this
as evidence that the leaf material can be considered homogeneous.
Chlorophyll (Chl) was determined as pheophytin according to Vernon (1960). The chlorophyll content of the leaves was usually close
to 40 lag. cm -2.
423
changed. After 3 min of illumination under a lower/
higher irradiance, during which CO2 exchange and fluorescence largely stabilized at a new level, leaves were
freeze-clamped. They were subsequently freeze-dried and
chloroplasts were isolated nonaqueously from the dry
material. Contents of ADP, Pi, ATP and NADP were
measured in the chloroplasts (Fig. 1). The amount of
NADPH was calculated as the difference between NADP
and the sum of NADPH and NADP+. The sum was
measured after NADPH had been oxidized. The total
pool of NADP (plus NADPH) was about 22 nmol.(mg
ChlF 1 and the total pool of adenylates about
38 nmol.(mg Chl~ 1. Levels of NADPH and ATP were
appreciable even in the dark. Whereas photosynthesis
increased with increasing irradiance in a typical hyperbolic relationship towards saturation, no corresponding
increase in NADPH levels was observed. Rather,
NADPH remained approximately constant after an initial increase, while PFDs increased by a large factor. Be-
Results
F i g u r e 1 s h o w s CO2 a s s i m i l a t i o n o f s p i n a c h l e a v e s as a
f u n c t i o n o f i r r a d i a n c e . T h e C O 2 c o n c e n t r a t i o n of t h e gas
a t m o s p h e r e w a s 2 0 0 0 gl.1 1 to p r e v e n t C O 2 f r o m b e c o m i n g r a t e - l i m i t i n g at m o s t l i g h t intensities, a n d the o x y g e n
c o n c e n t r a t i o n w a s 2 % to e n s u r e s u p p r e s s i o n o f p h o torespiration. After the stomata had opened and assimilation and chlorophyll fluorescence had reached a steady
s t a t e at a P F D o f 1 3 7 0 g m o l - m - 2 - s ~, t h e P F D w a s
100
35 'T
Z
HT:
~
80
O
O~
g
-6 60
E
x
O
O
"~ A T P
~
*~-'
30
o~
g
25 ~
e_ Tz
20 C) o
-
:3_
15 <~ ~
40
D_
D
(~
9 20
.....
NADPH
~
"
t
10 ~
cE
z
oL
5
s
....
', . . . .
500
', . . . .
1000
I ....
1500
I ....
2000
I''
0
<
z
2500
PFD, kJmol m 2 s q
Fig. 1. Carbon dioxide assimilation of spinach leaves and chloroplastic concentrations of ATP, ADP, N A D P H , N A D P and inorganic phosphate (Pi) as a function of photon flux density (PFD).
Experimental conditions: 2000 lal.1 J CO2 and 2% 02 in nitrogen,
leaf temperature 24~ average chlorophyll content of the leaves
40lag.cm -2. First assimilation was stabilized at a PFD of
1370 lamol-m 2.s ~. After gas exchange and chlorophyll fluorescence
had indicated a steady state, the PFD was changed as shown in the
figure. After 3 min of further illumination, the leaves were shockfrozen and freeze-dried, and the chloroplasts isolated non-aqueously and prepared for substrate measurements. The sum of ATP +
ADP + AMP (AMP not shown) differed by less than 10% in all
samples
i .
Light off
i:
::
~
I i 84 i
i
i . . . . .i. .84. . . .i. i
Fig. 2. Original traces showing the transient peaks of CO2 uptake in
spinach leaves after 1-s flashes of saturating white light (upper and
middle traces) and the corresponding chorophyll (Chl) fluorescence
(lower trace). Under our experimental conditions (2% 02 in nitrogen and 2000 lal'CO2, middle trace) there was a slow decline in the
height of the peaks after switching from the high-irradiance illumination (PFD 1370 lamol.m 2.s 1) to darkness, indicating slow inactivation of the light-actived enzymes. At 21% CO 2 (upper trace) there
was a faster inactivation of the enzymes participating in CO2 assimilation
424
U. Gerst et al.: Role of A T P and N A D P H in carbon reduction of leaves
cause the concentrations of ribulose bisphosphate
(RuBP), the main competitor to N A D P H for RuBP-carboxylase binding sites (Ashton 1982), have been reported
to remain constant over a wide range of light intensities
under low 02 and saturating CO2 concentrations
(Perchorowicz and Jensen 1983), we assume that binding
of N A D P H to RuBP carboxylase did not change much
as PFDs increased beyond about 200 g m o l . m 2.s ~. In
contrast to N A D P H , ATP increased with increasing
photosynthesis, while ADP and Pi decreased.
It is important to note that the experiments were performed under conditions which minimized changes in the
activity of light-regulated photosynthetic enzymes after
maximum activation had been achieved during exposure
to high-intensity illumination. After activation at a PFD
of 1370 gmol p h o t o n s . m 2.s ~, PFDs were changed and
samples were taken about 3 min after the change. Figure 2 demonstrates that during this time changes in the
activation state of light-regulated enzymes were slow
even when the oxygen concentration was 21%. In 2%
oxygen, they were much slower. In the experiments
shown, leaves were permitted to achieve the photosynthetic steady state at a high irradiance either in air or in
2% oxygen plus 2000 gl.1 ~ CO 2 before they were darkened. Staturating light flashes of 1 s duration were given
every minute. While the increase of flash-induced fluorescence indicated reversal of QN in the dark, the flashes still
produced photosynthetic carbon uptake. It was large only as long as light-regulated enzymes maintained the level
of light activation they had attained previously in the
presence of actinic illumination. Figure 2 therefore shows
that dark inactivation of the enzymes was negligible in
2% oxygen during a time span of 3 min after a transition
to darkness. It was even slower than shown in Fig. 2
when high light was replaced by low irradiances instead
of darkness (data not shown). Leegood and Walker (1982)
have reported that light-activated fructose-l,6-bisphosphatase is very slowly inactivated when irradiance is de-
100 -
"~
2,5 +
80-
2 -~
60-
+.
c~
9~
3
D
9
,,
S
E
2 <s
c
@
4@-
~:
=
1+
//"
C~
f
(~
9 20-
0-
'"
1
0,5 ~
0
NADPH/NADP+
I ....
0
500
I ....
1000
I ....
] 500
I ....
2000
I
0
2500
PFD, IJnOl ~rnol 2s ]
C a r b o n dioxide assimilation of spinach leaves as a function
of P F D , and c o r r e s p o n d i n g chloroplastic ratios of A T P to A D P
and of N A D P H to N A D P § calculated from the data in Fig. I. For
experimental conditions see legend to Fig. 1
F i g , 3,
8
d,0 [
300 'T
/
t
(ATP)
9
(ADP)(Pi)
6
_
30
250
/
.r
/-
~=
-
,-" ..- z
4
/
20
ATP /
2
200 E
/
"
10
150
J
/
~TATP/ADP
I
~/
1 ~
~
9
0 1-
0
l0
....
30
,, . . . .
50
50
I ....
70
fD_
~-~
"
, ,.~,,,'JT-,,,
-10
] O0
>.
0
90
C O 2 u p t a k e , n m o l (rag chl s)-]
Fig. 4. Plot of the chloroplastic level of ATP, of the A T P / A D P ratio,
of the chloroplast phosphorylation potential and of the assimilatory
force F A (calculated from the data shown in Fig. 1) in spinach leaves
as a function of carbon assimilation. Values of F A were calculated
according to Eq. 1 from adenylates, phosphate, and oxidized and
reduced pyridine nucleotides
creased in 2% 02. It is not inactivated when oxygen is
missing from the environment.
Figure 3 compares carbon assimilation with the redox
ratio N A D P H / N A D P and the ATP/ADP ratio as a function of irradiance. Obviously, only the adenylate ratio
follows carbon assimilation more or less closely. In Fig. 4,
assimilation is plotted against ATP, the ATP/ADP ratio,
the phosphorylation potential [ATP]/([ADP].[Pi]) and
the assimilatory force F A which is defined by
FA =
[DHAP]
K
[ATe]
[NADPH]
[PGA~ [H +] < [ADP][Pi] [NADP+] ' (Eq. 1)
where K is the overall equilibrium constant of the reactions catalyzed by the three enzymes involved in reducing
3-phosphoglycerate (PGA) to dihydroxyacetone phosphate (DHAP) (see Heber et al. 1987; Dietz and Heber
1989).
In Fig. 4, it is obvious that ATP increases relatively
less than assimilation. Considerable parallelism exists between assimilation and F A o r the phosphorylation potential at lower and intermediate PFDs. At high PFDs, the
slopes of F A and the phosphorylation potential increase
considerably. The relationship between ATP/ADP and
assimilation appears to be more complex than that between assimilation and the phosphorylation potential or
F A. Only the latter two parameters behave in relation to
assimilation as would be expected if they were to represent the driving forces in carbon reduction.
It appeared to be necessary to relate the observations
on light-dependent changes in the state of the N A D P and
the adenylate systems to changes in the chloroplast electron transport chain. In order to obtain information on
the redox state of the Q A in the reaction center of PSII
and on electron flow through PSII, modulated chlorophyll fluorescence was measured. Observed changes in
F o' , F~ and F m ' with irradiation are in agreement with
U. Gerst et al.: Role of ATP and N A D P H in carbon reduction of leaves
observations reported by Genty et al. (1989) for the photosynthetic steady state when the composition of the gas
phase was comparable. While photosynthesis increased
with increasing PFDs, the level of modulated steady-state
fluorescence, F~, remained largely constant. However, the
maximum level of modulated fluorescence, Fro', and the
so-called dark fluorescence, Fo' , declined while photosynthesis increased towards saturation (data not shown).
During the first 3 min after switching from high light to
a lower irradiation, F s and F o' adapted to the new conditions and did not change appreciably further on. Only
flash-induced F m' needed about 7 min or even more after
irradiation was changed to reach a new steady state in
darkness or under very low illumination. At higher light
intensities, stabilization was achieved within the first 3
min after a transition from the first to the second light
phase.
Fluorescence data were used to calculate the levels of
qN and qp, as described in Schreiber et al. (1986) and
Genty et al. (1989). The value of qp may be taken as an
approximate measure of oxidation of QA, if the nonlinearity introduced by energy transfer between different reaction centres is neglected. An increase in qN serves to
indicate an increase in the radiationless dissipation of
excitation energy. Figure 5 compares carbon assimilation
as a function of irradiance with oxidation of QA in the
reaction centre of PSII, as shown by qp, and with qN,
which largely followed assimilation. While assimilation
increased with increasing irradiance, oxidation of QA decreased due to increasing electron pressure on the reducing side of PSII.
The quantum efficiency of PSII electron transport is
determined by the efficiency with which an absorbed
photon can reach potentially active (open) reaction centres. It can be calculated as Ops . = (Fm F~)/F m (Genty et
al. 1989, 1990a). Measurements of P7o0 oxidation give
complementary information regarding the quantum
yield of PSI. Excitation captured by photooxidized PSI
80
.~x
1
- "~.
.~
0,8
/\'/" ~ "
0
T= 60
/
~
'
~
C02
.
0,6
.~
//
0
20
-.
:'-," \ ~
//
/
....
0
0,4 o~
""
O
o
0
I ....
500
I ....
1000
e_
f ....
1500
', . . . .
2000
', . . . .
2500
0,2
0
3000
PFD, !Jmol nn-2s -1
Fig. 5. Carbon dioxide assimilation of a spinach leaf as a function of
irradiance compared to photochemical (qp) and non-photochemical
(qN) quenching of chlorophyll fluorescence calculated from the
chlorophyll fluorescence yields as: qp = (F m' -F~)/(Fm' -Fo'
) -
(Frn' F s ) / F v ;
qN = 1 - F ~ / ( F m - F o )
425
////
/
0.8
,,'"
/
/
0.6
,/./~
0.4
0.2
0
0.2
0.4
0.6
0,8
d~PSI
Fig. 6. The relative quantum efficiency of spinach-leaf energy conversation at the level of photosystem II (a0 Psu) plotted against
reduction of P7oo as a measure of 9 psi- 9 PSU = (F~' -F~)/F,~' was
calculated according to Genty et al. (1989). The dashed line corresponds to the theoretical equal distribution of electron flows
through both photosystems
reaction centres is dissipated as heat. Thus, the in-vivo
proportion of PSI reaction centres in the nonoxidized
state constitutes a measure of the efficiency of PSI, i. e.
Ops ~(Harbinson et al. 1990). Figure 6 shows that the relationship between Opsr~ and Ops I was linear under our
experimental conditions. Simultaneous measurements of
Ops ~and Ops u have also been made by others both under
photorespiratory and non-photorespiratory conditions.
They have also shown a linear relationship between these
parameters (Harbinson et al. 1989; Genty et al. 1990b;
Peterson 1991).
Increases in qN have been reported to be related to
increases in light scattering (Bilger et al. 1988), although
the exact nature of the relationship remains to be established. Increased light scattering appears to be caused by
an intrathylakoid protonation reaction, which depends
on the formation of a large transthylakoid proton gradient (Horton 1992; Heber et al. 1993). However, it is important to note that a transthylakoid proton gradient
fully sufficient to support the A T P synthesis needed for
appreciable carbon reduction does not yet trigger a lightscattering response. Excess energization is needed for increased light scattering. Although photosynthetic carbon
assimilation which consumes ATP is appreciable already
at a photon fluence rate of 95 lamol, m 2. s 1, a light-scattering increase is seen only transiently during the induction phase of assimilation as a slow secondary hump after
a fast initial absorption increase (Fig. 7). Relaxation of
light scattering after termination of illumination, which is
characterized by a slow decrease in apparent absorbance,
is absent. A slow signal indicating relaxation of a small
light-scattering signal is seen on darkening after illumination with a P F D of 200 gmol. m 2. s 1. A large scattering
signal is produced only after a P F D of 995 gmol. m 2. s ~.
The relationship between carbon assimilation and light
scattering by the chloroplast thylakoid system is thus
sigmoidal (Fig. 8).
From traces of AAs35 such as those shown in Fig. 7, an
estimate of another energy-dependent thylakoid reaction
426
U. Gerst et al.: Role of ATP and N A D P H in carbon reduction of leaves
5 rain
I 5 t~mo! C O 2
031
"~
f
2S :
\A
_
J -qEEfi2~ P515
/ ....
~L.sc, -~ -
/
....
~
~CO2
t'
%3~
I '84
t
1
t
s
t
;
+
9
Fig. 7. Simultaneous recordings of transpiration (H20), CO 2 exchange (CO2) and apparent changes in absorbance at 535 nm of a
spinach leaf as PFDs were increased from 95 to 200 and
995 gmol.m 2.s J. The gas atmosphere contained 500 lal'l ' COa and
2% 02 in nitrogen. In A535, an electrochromic absorbance change
(termed P5,5) can be distinguished from slow scattering changes by
fast kinetics in the dark/light and light/dark transients. At a PFD of
95 gmol-m 2.s ~, a light-scattering signal is visible only transiently,
while assimilation still increases. At 200 tamol.m 2.s 1, a small scattering change persists at steady state, and at 995 lamol'm 2.s ~, the
light-scattering signal is large. As transpiration increases while
stomata open slowly, assimilation also, increases indicating that
assimilation is limited by CO2 at higher PFDs under the chosen
conditions. L.sc., light scattering
60
/
c
50
"// 9
,g
P700
03 :~>
.sc,
"~
/~"
~20
0
or-~
/
/
--030
~
/ /
,~
/
lO
~
o_
0
.-
//.
.
9
" P515
/
20
40
60
80
C 0 2 u p t a k e , nmol (mg chl sY 1
Fig. 8. Oxidation of P70o, light scattering and P5~5 (as calculated
from absorbance such as that shown in Fig. 7) versus carbon assimilation recorded 3 min after changing irradiance from a PFD
1370 pmol.m 2-s-t to higher or lower values. L.sc., light scattering
can be obtained. The fast light-off response described in
Fig. 7 as P515 is characterized by a difference spectrum
which peaks at 521 nm. A similar difference spectrum of
an absorption change caused by brief flashes has been
ascribed in the literature to the formation and decay of a
light-induced transthylakoid membrane potential (Junge
1977). However, in contrast to flash-induced P5~5 signals,
the difference spectrum of the fast absorption decrease
shown in Fig. 7 was slightly broadened toward the red
(data not shown). Probably, the signal contained also a
small scattering component. Even so, a main part of the
fast decrease in absorbance shown in Fig. 7 as Psi5 is
probably caused by the decay of a light-induced membrane potential, because Junge (1987) has shown that
P515 can also detect changes in the transthylakoid membrane potential when a transthylakoid pH gradient is
present.
Figure 8 shows that both the oxidation of P700 in the
reaction centre of PSI and the P515 signal were, over a
broad range of irradiances, linearly related to carbon assimilation. However, extrapolation of the data shows
complexity. For P7o0it shows that at low PFDs the oxidative reaction revealed by 820-nm absorption was more
than proportional to assimilation. It is known that not
only P7oo but also plastocyanin has an absorption band
at 820 nm (Klughammer and Schreiber 1991). Plastocyanin donates electrons to P700 +. It will be oxidized before oxidized P700 accumulates.
The level of light scattering which is very low or absent at low rates of assimilation (Fig. 7), increased steeply
with increasing assimilation. At very high irradiances,
light scattering reached saturation (not shown in Fig. 8).
Discussion
Chloroplast phosphorylation potentials and NADPH/
NADP ratios in relation to the state of the electron-transport chain. The experiments described above were designed to give information on the relative physiological
significance of ATP and N A D P H in carbon reduction.
Simultaneously, the relationship between the accumulation of ATP and N A D P H in the chloroplast stroma and
the state of the electron-transport chain was of interest.
Whereas it is clear that the stoichiometry of the ATP and
N A D P H requirements of carbon assimilation must be
satisfied for photosynthesis to proceed, it is not clear
which levels of ATP and N A D P H must be attained in the
chloroplast stroma to drive assimilation. It has, for instance, been reported that in air the product of phosphorylation potential and N A D P H / N A D P + ratio, which
has been termed the assimilatory force (FA) , was higher at
low rates of carbon assimilation than at high rates (Dietz
and Heber 1986; Heber et al. 1986, 1987; Siebke et al.
1990).
However, under the conditions of our measurements,
N A D P H / N A D P ratios remained largely constant over a
broad range of light intensities, whereas ATP/ADP ratios
and phosphorylation potentials increased with increasing carbon assimilation. This means that F A w a s lower at
low than at high irradiances. We were careful to avoid
appreciable changes in the activity of light-regulated enzymes during a transition from one light intensity to another. We also minimized photorespiration by maintaining the O 2 concentration of the gas atmosphere at 2%
and by increasing the CO 2 concentration to 2000 gl-1 1.
The clearly linear relationship observed between ~PS~
and q)psu implies that if some PSI-mediated cyclic elec-
U. Gerst et al.: Role of ATP and NADPH in carbon reduction of leaves
tron transport which contributes to ATP synthesis exists
under our experimental conditions, then its rate is proportional to the rate of non-cyclic electron transport (see
also Baker and Ort 1992). The slight deviation of our
measured ~psi values from the dashed line shown in
Fig. 6, which indicates the theoretical equal distribution
of electron flows through both photosystems, suggests
only a very small contribution of cyclic photophosphorylation to the ATP synthesis occurring during linear electron transport. Similar conclusions may be drawn from
the largely linear relationship between P70o oxidation or
the changes in a transmembrane electrical field indicated
by the P515 signal and carbon assimilation (Fig. 8).
By analyzing the complex relaxation kinetics of qN,
Quick and Stitt (1989) have distinquished at least three
components of qN with half-times of about 1 min, 5 min
and several hours. They ascribed the first fast component
to high-energy quenching (quenching initiated by the
transthylakoid proton gradient), the middle one to a state
transition (redistribution of excitation energy from PSII
to PSI) and the slow component to photoinhibition. The
fast component had a sigmoidal light-response curve.
Light scattering is also known to have a sigmoidal lightresponse curve (Heber 1969). In our experiments, qN
largely followed carbon assimilation (Fig. 5). Light scattering did not (Fig. 8). In contrast to light scattering
(Fig. 7) qN had a hyperbolic light-response curve. Under
many conditions, light scattering appears to be a better
indicator of the down-regulation of PSII and the dissipation of excess excitation energy than qN with its several
different components. Photosystem II catalyzes an essentially irreversible electron transfer reaction as long as
electron acceptors on its reducing side are available. In
order to prevent full reduction of the electron-transport
chain, which is known to lead to rapid photoinhibition
(Heber et al. 1987), PSII must be down-regulated at high
PFDs (Weis et al. 1987).
H +/ e and H +/A TP ratios in electron transport and photophosphorylation. It has been outlined in the Introduction that the value of the H+/e ratio for linear electron
transport from water to NADP in leaves is still not
known. Neither is it known how many protons have to
cross the thylakoid ATP synthase complex for the synthesis of one molecule of ATP. Our data may shed some
light on this unresolved question. It would be difficult to
explain our observations of largely constant NADPH/
NADP + ratios, and of ATP/ADP ratios or phosphorylation potentials which increase with photosynthesis, if
H+/e were 2 and H+/ATP either 3 or 4. Such ratios
would result in insufficient ATP formation during electron transport to NADP to cover the needs of carbon
assimilation. Additional ATP would be needed. In principle, such ATP could be synthesized during cyclic electron
transport (Katona et al. 1992) or during electron transport in the Mehler reaction and associated reactions
(Schreiber and Neubauer 1990). However, we did not see
the large increase in the NADPH/NADP ratio which
would change redox poising of the electron-transport
chain sufficiently to either allow appreciable cyclic electron transport or electron flow to oxygen. It is also possi-
427
ble to explain the observed increase in the phosphorylation potential with increasing assimilation via accompanying increases in nitrate assimilation. Our spinach
leaves contained considerable nitrate. Nitrite formed
during nitrate reduction is reduced in the chloroplasts. Its
reduction to ammonia is accompanied by ATP synthesis.
However, ATP formed during nitrite reduction is also
consumed during the reductive amination of 0~-ketoglutarate and during chloroplast protein synthesis.
The most simple interpretation of our data is that a
Q-cycle operates in the leaves. It permits the H +/e ratio
of linear electron transport to be 3. Fixed ratios of H+/e
= 3 and H+/ATP = 4, such as reported by Grfiber et al.
(1987) and Rumberg et al. (1990), would permit linear
electron transport to NADP + to produce almost all the
ATP necessary for assimilatory reactions. A small increase in the NADPH/NADP ratio not detectable in our
measurements may have been sufficient to permit the little extra ATP synthesis in cyclic or pseudocyclic phosphorylation and cause the increase of phosphorylation
potentials which accompanied the increase in carbon assimilation.
In our experiments, photorespiration was suppressed.
However, since ATP/NADPH requirements are similar
in carbon assimilation and photorespiratory carbon oxidation, conclusions which are valid for the experiments
reported here should be valid also for photosynthesis of
leaves with more or less open stomata in air when photorespiratory reactions are included.
If the H+/e ratio is 3 and H+/ATP is not 4, but 3, too
much ATP is produced for carbon assimilation. In this
case, phosphorylation potentials should be high already
at low irradiances. This is not observed. More and more
excess H + would have to be discharged when electron
flow is increased at increasing irradiances. A proton
"slip" through the ATP synthase has been documented in
experiments with isolated thylakoids (Strotmann et al.
1986; Evron and Avron 1990; Braun et al. 1991) and
intact chloroplasts (Heineke et al. 1989). However,
known properties of the slip (inhibition by submicromolar concentrations of ADP, Mg and Pi, or ATP with Mg,
see Evron and Avron 1990, or activation by low Pi, see
Heineke et al. 1989) are in conflict with our observation
of increasing phosphorylation potentials with increasing
carbon assimilation.
At a H+/ATP ratio of 3, only flexible coupling, with
H+/e ratios of linear electron transport changing between 2 and 3, would be consistent with our observations. However, our observations demand that coupling
should increase with increasing transthylakoid proton
gradients. This is very difficult to envisage.
Assimilatory force and flux resistances in photosynthesis.
An apparent discrepancy between the observations made
in this work and previously (Heber et al. 1987) concerns
the relationship between FA and carbon flux. Values of
F A can be calculated either from measurements of phosphoglycerate and dihydroxyacetone phosphate (Heber et
al. 1987) or from measurements of the phosphorylation
potential and NADPH/NADP ratios as done here. Obviously, the latter is much more difficult. A comparison of
428
U. Gerst et al.: Role of ATP and NADPH in carbon reduction of leaves
both a p p r o a c h e s was m a d e by Siebke et al. (1990). In
earlier work, we did not find a linear relationship between F A and c a r b o n assimilation (Heber et al. 1987),
whereas such a direct relationship is observed now. The
explanation of the discrepancy is simple. As for any other
flux, c a r b o n flux in photosynthesis is governed by the
general flux e q u a t i o n which, a n a l o g o u s to a well-known
specialized form, O h m ' s law, is flux = driving force/flux
resistance.
In the present work, we were as careful as possible to
minimize flux resistances or to keep them constant. In the
situation of a leaf in nature, flux resistances are altered in
m a n y ways. E n z y m e regulation m a y maintain a thermod y n a m i c gradient and control substrate interconversions
in the mesophyll cells or stomatal closure can restrict
c a r b o n flux to the chloroplasts, to mention just two imp o r t a n t flux resistances in photosynthesis. In earlier
work, no attempts were m a d e to control stomatal or enzymic resistances. If F A is considered to be related to a
driving force, it is n o t surprising that there was no direct
relationship between F A and c a r b o n flux in the earlier
work. In the present investigation, there was such a relationship, because alterations in flux resistances could be
largely avoided.
In a recent contribution, Fridlyand (1992) criticized
this concept of FA, maintaining that the idea of a driving
force which is not necessarily related to flux is not convincing. We do not believe that it is necessary to express
disagreement with a view that has a n o t h e r aim in mind.
Fridlyand p r o p o s e s that the expression
v = S'V2~• (PGA)(ATP)/(ADP)
(Eq. 2)
is better suited to describe the relationship between the
c a r b o n flux, v, in photosynthesis and the relevant
parameters determining it. In Eq. 2, S is a proportionality
coefficient and Vzmaxis the m a x i m u m rate that can be
catalyzed by g l y c e r a l d e h y d e - p h o s p h a t e dehydrogenase
which is a light-regulated enzyme. Indeed, after 3 - P G A is
formed by c a r b o x y l a t i o n or oxygenation of ribulose bisphosphate, its p h o s p h o r y l a t i o n by A T P initiates c a r b o n
reduction which is completed when N A D P H reduces the
p r o d u c t of p h o s p h o r y l a t i o n , 1,3-diphosphoglycerate. If
only the rate of c a r b o n flux is of interest and not the
potential of s u p p o r t i n g c a r b o n flux, of which F A is a
measure, then the c o n c e n t r a t i o n s of both p r i m a r y substrates A T P and P G A and of a p r o d u c t - A D P
are
i m p o r t a n t in defining flux. It is k n o w n that P G A concentrations are generally high when open s t o m a t a permit
high c a r b o n fluxes at high irradiances, whereas they are
very low when c a r b o n flux is restricted under the same
conditions by stomatal closure. The value for F A would
be lower in the first than in the second instance. In contrast to Fridlyands expression, F A describes the potential
for p r o d u c i n g a flux, not the rate of the flux. Thus, calculations of F• from measurements of the transport
metabolites P G A and d i h y d r o x y a c e t o n e p h o s p h a t e (see
Eq. 1), which are easy to carry out, and of Fridlyands
expression, which requires knowledge of chloroplast
A T P , A D P a n d P G A and, in addition, of the activation
state of g l y c e r a l d e h y d e - p h o s p h a t e dehydrogenase, ad-
dress two different questions. Values for the substrate
concentrations needed to calculate fluxes according to
Eq. 2 are difficult to obtain.
This research was supported by the Sonderforschungsbereich 251 of
the University of Wfirzburg and the Stiftung Volkswagenwerk. U.G.
is a member of the Graduate College of the Julius-von-Sachs Institut ffir Biowissenschaften, University of Wfirzburg, being on leave
from Tartu University, Tartu, Estonia. The authors are grateful to
Prof. A. Laisk, Chair of Plant Physiology, Tartu University, for
stimulating discussions.
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