Plant Physiol. (1989) 89, 932-940
Received for publication June 9, 1988
and in revised form August 29, 1988
0032-0889/89/89/0932/09/$01 .00/0
Photosynthetic Apparatus of Pea Thylakoid Membranes1
Response to Growth Light Intensity
Wen-Jane Lee and John Whitmarsh*
Department of Plant Biology, University of Illinois, U.S. Department of Agriculture/Agricultural Research Service,
289 Morrill Hall, 505 S. Goodwin Ave., Urbana, Illinois 61801
ABSTRACT
We investigated the effect of growth light intensity on the
photosynthetic apparatus of pea (Pisum sativum) thylakoid membranes. Plants were grown either in a growth chamber at light
intensities that ranged from 8 to 1050 microeinsteins per square
meter per second, or outside under natural sunlight. In thylakoid
membranes we determined: the amounts of active and inactive
photosystem 11, photosystem 1, cytochrome b/f, and high potential
cytochrome b559, the rate of uncoupled electron transport, and
the ratio of chlorophyll a to b. In leaves we determined: the
amounts of the photosynthetic components per leaf area, the
fresh weight per leaf area, the rate of electron transport, and the
light compensation point. To minimize factors other than growth
light intensity that may alter the photosynthetic apparatus, we
focused on peas grown above the light compensation point (2040 microeinsteins per square meter per second), and harvested
only the unshaded leaves at the top of the plant. The maximum
difference in the concentrations of the photosynthetic components was about 30% in thylakoids isolated from plants grown
over a 10-fold range in light intensity, 100 to 1050 microeinsteins
per square meter per second. Plants grown under natural sunlight
were virtually indistinguishable from plants grown in growth
chambers at the higher light intensities. On a leaf area basis,
over the same growth light regime, the maximum difference in
the concentration of the photosynthetic components was also
about 30%. For peas grown at 1050 microeinsteins per square
meter per second we found the concentrations of active photosystem 11, photosystem 1, and cytochrome b/f were about 2.1
millimoles per mol chlorophyll. There were an additional 20 to
33% of photosystem 11 complexes that were inactive. Over 90%
of the heme-containing cytochrome f detected in the thylakoid
membranes was active in linear electron transport. Based on
these data, we do not find convincing evidence that the stoichiometries of the electron transport components in the thylakoid
membrane, the size of the light-harvesting system serving the
reaction centers, or the concentration of the photosynthetic components per leaf area, are regulated in response to different
growth light intensities. The concept that emerges from this work
is of a relatively fixed photosynthetic apparatus in thylakoid
membranes of peas grown above the light compensation point.
energy into chemical free energy used for the generation of
ATP and NADPH (reviewed in Ref. 21). The antenna system,
which consists primarily of Chl a and b, absorbs photons and
converts the light energy into excitation energy. The electron
transport components include the PSII and PSI reaction
centers that convert the excitation energy into the redox free
energy that drives electrons from water to NADP, and the
intersystem electron carriers plastoquinone, the Cyt b/f 2 complex, and plastocyanin. The rate and efficiency of electron
transport depends, in part, on the intensity of the incident
light, the size of the antenna system serving the reaction
centers, and on the stoichiometries and organization of the
electron transport components. During energy conversion the
factors that limit or control the rate of electron transport are
many, and their interdependence has yet to be clearly delineated. However, no single element exhibits complete control,
and under the varied environmental conditions of plant
growth the controlling factors are likely to shift among different elements.
Our aim is to understand the role of the components of the
photosynthetic apparatus in determining the rate of electron
transport. In this study, we focus on the potential regulation
ofthe activity and concentration of the components by growth
light intensity. Specifically, we investigated whether a plant
adjusts the stoichiometries of the active electron transport
components, the size of the light-harvesting system serving
the reaction centers, or the concentration of the photosynthetic components per leaf area, in response to different
growth light intensities. This notion is not new and has been
tested in several higher plants (3, 4, 16-19, 22, 30, 31), leading
to the generally accepted conclusion that growth light intensity
regulates the stoichiometries of the components of the photosynthetic apparatus in a manner designed to balance the
tumover of each component (e.g. Refs. 17-19). For example,
Leong and Anderson (17, 18) argued that in pea plants grown
at low light the observed increase in the antenna size per
reaction center is designed to compensate for the low intensities. Despite such observations we think the case for regulation by growth light is open to question, in that: (a) the
differences reported in any single component of the photosynthetic apparatus are typically less than 50% over 10-fold
differences in growth light intensity; (b) in some studies the
2Abbreviations: Cyt b/lf plastoquinol/plastocyanin oxidoreductase
complex; DCBQ, 2,6-dichlorobenzoquinone; DMQ, 2,6-dimethylbenzoquinone; HP Cyt b559, high potential cyt b559.
The primary role of the photosynthetic apparatus of higher
plants, defined here as the antenna system and electron transport components of thylakoid vesicles, is to convert light
'This work was supported in part by the Photosynthesis Program
of the Competitive Grants Office of the U.S. Department of Agriculture (grant AG86-CRCR- 1-1987).
932
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 1989 American Society of Plant Biologists. All rights reserved.
PHOTOSYNTHETIC APPARATUS OF PEA THYLAKOID MEMBRANES
933
growth light intensities were at, or below the light compensation point (17, 18, 30, 31), which introduces additional regulatory factors beyond that of growth light intensity. In studies
in which leaves below the top of the canopy were harvested,
light quality as well as growth light intensities below the light
compensation point were introduced as potential factors in
regulation (17, 18); and (c) some assays designed to determine
concentrations of the photosynthetic components are subject
to large systematic and/or statistical errors and therefore do
not provide a reliable data base. Furthermore, assays that do
not distinguish between active and inactive components do
not answer the question of whether the observed difference
affects the rate or efficiency of energy conversion (discussed
in "Results"). Thus, despite the widespread belief that the
photosynthetic apparatus is regulated in response to growth
light intensity in order to achieve an optimum balance among
the components involved in energy conversion, we think the
phenomenon remains to be established in higher plants.
Below, we describe a study designed to characterize the
photosynthetic apparatus in pea plants and to investigate its
acclimation over a wide range in growth light intensity. One
of the key findings in this study is that pea plants construct
virtually the same photosynthetic apparatus over 80% of the
growth light intensity regime, as determined by the stoichiometries of the major components and total antenna size.
Furthermore, the concentration of the photosynthetic components on a leaf area basis was essentially constant over the
same growth light regime.
A preliminary account of some of this work was presented
earlier (27).
coln, NB). The white growth light spectrum was identical at
all intensities and is shown elsewhere (27).
Growth light intensities were measured at the top of the
pea canopy by a Quantum Photometer (LI-185B) equipped
with a Quantum Sensor (LI-190SB, LI-COR Inc., Lincoln,
NB) that detects PAR between 400 and 700 nm. At light
intensities above 100 IAE/m2 s the irradiance measured at the
top of the canopy at different times during growth varied by
10% or less, at 100 gE/m2 s the variation was 20%. The
intensity of the lowest growth light ranged from 5 to 10 AE/
s
m22S.
MATERIALS AND METHODS
Flash-Induced Proton Release
The concentration of oxygen evolving PSII complexes was
determined by measuring proton release associated with the
flash-induced oxidation of water (10, 28). The activity of PSII
complexes in thylakoids was determined in the presence of
ferricyanide and either DMQ or DCBQ using a pH electrode.
Growth Conditions
Peas (Pisum sativum L.) were grown in vermiculite from
seed (cv Progress No. 9, Ferry Morse Seed Co., Mountain
View, CA3) in controlled environment chambers (Conviron
PGW 36, Controlled Environments, Pembina, ND, and EGC
model 31-15 growth chamber, Chagrin Falls, OH). Peas were
also grown outside under natural sunlight between April and
October. In the growth chamber the conditions were: light 14
h, temperature 23 ± 2°C, relative humidity 70%; dark 10 h,
temperature 17 ± 2C, and relative humidity 90%. Pea seedlings were fertilized twice a week with a nutrient solution
consisting of approximately 4 g of Nutriculture (12-31-14,
Plant Marvel Laboratories, Chicago Heights, IL) and 1.5 g
potassium nitrate per liter of deionized water.
White growth light was provided by a combination of VHO
cool-white fluorescent lamps (F96T12/CWX/1500, General
Electric) and incandescent lamps (60W, Westinghouse). The
light intensity was adjusted by varying the distance between
the light source and the plants, and by copper screen filters.
The spectrum of the light was measured by a Spectroradiometer (model SRR, Instrumentation Specialities Co., Lin3Mention of a trademark, proprietary product, or vendor does not
constitue a guarantee or warranty of the product by the U.S. Department of Agriculture or the University of Illinois and does not imply
its approval to the exclusion of other products or vendors which may
be suitable.
Isolation of Thylakoid Membranes
Thylakoid membranes were isolated as described elsewhere
(28) from fully developed pea leaves taken from the top of
the canopy of 14 to 23 d old plants. Leaves were taken from
the growth chamber shortly after the onset of illumination to
minimize the amount of starch in the chloroplasts. The results
shown here were independent of harvest day over this growth
period. Measurements using thylakoid membranes were done
at or below 18°C to ensure that the activity of the thylakoid
membrane was stable during the assay.
Chi
The Chl concentration and Chl a/Chl b ratio was determined in 80% (v/v) acetone/H20 using specific absorption
coefficients at 664 and 647 nm for Chl a and Chl b determined
by Ziegler and Egle as described elsewhere (5).
PSII
Flash-induced Oxygen Evolution
The concentration of oxygen evolving PSII complexes was
determined by measuring oxygen evolution induced by single
turnover flashes in the presence of DMQ and ferricyanide
using a Clark-type electrode as described elsewhere (1 1).
Flash-Induced Electrochromic Shift
Electron transfer in PSII in detached pea leaves was probed
by the electrochromic shift at 518 nm which reveals charge
separation within the reaction centers. Using two short actinic
flashes separated by a variable time interval, we determined
the time required after the first flash for PSII to recover. This
technique is described in detail elsewhere (5). The measurements were done using a laboratory-built single beam spectrophotometer modified to accommodate attached and detached
leaves.
PSI
The amount of PSI was determined spectrophotometrically
by measuring the reduced minus oxidized absorption spec-
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 1989 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 89, 1989
LEE AND WHITMARSH
934
trum of P700 in thylakoid membranes. P700, reduced in the
dark, was oxidized by far-red light, and the concentration
determined using the spinach P700 reduced minus oxidized
extinction coefficient of 64 mm-' cm-' as described in elsewhere (14, 28).
Sample Size and Error
The data given in the figures and tables are the average of
measurements done on at least three different thylakoid membrane preparations. The uncertainty given is the larger of the
sample standard deviation (square root of the sample variance) or the estimated precision of the measurement.
Cyt f and HP Cyt b55s
The amount of Cyt f and HP Cyt bs59 heme present in
thylakoid membranes was determined spectrophotmetrically
by the chemically induced difference spectrum (hydrobenzoquinone reduced, ferricyanide oxidized) as described elsewhere (28). The amount of Cytfcapable of turning over in a
long actinic flash (e.g. 50 ms) was determined using a single
beam spectrophotometer. The transient light-induced oxidation of Cytfand rate of rereduction was measured at 554 nm
using a reference wavelength of 540 nm. During the measurements thylakoids were maintained at 18C. Details of the
techniques are described elsewhere (28).
Steady State Electron Transport Rate in Thylakoid
Membranes
Light-driven electron transport in thylakoid membranes
was measured using a Clark-type electrode as described elsewhere (13). Red actinic light was provided by a tungstenhalogen lamp filtered by a red blocking filter (CS 2-61, Corning Glass Works, Corning, NY) and heat filters. All measurements were done using thylakoids at 1 8°C in the presence of
the uncoupler, gramicidin (Sigma Chemicals, St. Louis, MO),
which collapsed both the electrical and chemical components
of the transmembrane potential.
Steady State Electron Transport Rate In Detached
Leaves
Oxygen evolution from whole, detached leaves was measured using a Clark-type gas phase oxygen electrode (model
LDl, Hansatech Ltd., Norfolk, U.K.) as described elsewhere
(6). A bicarbonate/carbonate buffer ensured saturating levels
of carbon dioxide (95% of 1 M NaHCO3 and 5% of 1 M
Na2CO3 mixture, corresponding to approximately 5% CO2 by
volume). Light was provided by a 300 W tungsten-halogen
lamp filtered by a heat reflecting mirror and a heat absorbing
filter. The light intensity was controlled by neutral density
filters and was measured as PAR as described above. The
system was calibrated by injecting into the chamber 1 cm3 of
air (21% 02 by volume) using a gas tight syringe. Measurements were done at 23°C.
Chi/Leaf Area and Fresh Weight/Leaf Area
Fully developed leaves were harvested from the top of the
canopy of 2 to 3 week old seedlings. After measuring the fresh
weight, the leaf outline was drawn on graph paper, cut out,
and weighed to determine the leaf area. The leaf was then
homogenized into a known volume of 80% (v/v) acetone/
H20 and the total Chl was determined as described above.
RESULTS
Effect of Growth Light Intensity on the Photosynthetic
Components in Thylakoid Membranes
PSII in thylakoid membranes has been shown to consist of
at least two distinct populations (5, 10, and refs. therein). One
fraction consists of the normally active complexes that exhibit
turnover rates of approximately 250 e/s, which we define as
active PSII. The other fraction consists of complexes that
exhibit turnover rates of 0.25 e-/s, which we define as inactive.
In spinach thylakoids the inactive fraction constitutes approximately 32 ± 4% of the total PSII. The term inactive, used to
describe the slowly turning over complexes, applies to their
contribution to energy conversion, which is negligible, and is
not intended to discount the possibility that they exhibit an
enzymatic activity that remains to be revealed. In the work
described here we measured the PSII concentration and activity using different techniques designed to quantify the concentration of active PSII, inactive PSII, and total PSII (active
+ inactive). The concentration of active PSII was determined
by measuring flash-induced proton release using thylakoid
membranes in the presence of DMQ and ferricyanide. Under
these conditions ferricyanide is the terminal electron acceptor,
and DMQ is a mediator carrying electrons from PSII to
ferricyanide. Graan and Ort (10) showed that DMQ interacts
with the active PSII fraction, but not the inactive fraction.
Presumably the binding and debinding of DMQ at the secondary quinone binding site on inactive PSII is altered in
inactive PSII complexes. The concentration of active PSII,
normalized on a Chl basis, is shown in Figure 1 for thylakoids
isolated from peas grown at different light intensities. A 10fold decrease in growth light intensity (1050 to 100 ,E/m2 s)
resulted in a 32% decrease in the ratio of active PSII to Chl.
In addition to the proton release, we measured flash-induced
oxygen evolution in the presence of DMQ and ferricyanide.
Measurements of proton release and oxygen evolution (data
not shown) gave similar results in pea thylakoids, as was
shown previously in spinach thylakoids (1 1). The concentration of total PSII was determined by measuring flash-induced
proton release as described above, except that DCBQ was
used as the mediator between PSII and ferricyanide. In the
presence of DCBQ both the active and inactive fractions of
PSII are able to transfer electrons to ferricyanide (10). In
thylakoids isolated from plants grown at 100 and 900 gE/m2
s the amount of inactive PSII was 20% (Table I).
The possibility that the inactive fraction of PSII is nonphysiological, resulting from the membrane isolation procedure,
was investigated by monitoring the recovery of the electrochromic shift in detached leaves. In dark-adapted leaves the
electrochromic shift, induced by a single turnover flash and
monitored at 518 nm, reveals the turnover of all photoactive
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 1989 American Society of Plant Biologists. All rights reserved.
PHOTOSYNTHETIC APPARATUS OF PEA THYLAKOID MEMBRANES
01
I
I
I
.
I
.
.
.
.
.
a
0
I
I
I
I
935
I
I
Z
M
-
T
E
5
2
s
0
4g3
E
E
0
2
0-
s
I~~~~
T
I
.
0<
I-,
2
E
Ln
(A
E
1
W"
U)
nf
0
500
1000
'in
2000
u A.
0
1500
2
GROWTH LIGHT INTENSITY (gE / m s)
Figure 1. Effect of growth light intensity on the ratio of active PSII/
Chi and HP Cyt b559/Chl in thylakoid membranes isolated from peas.
The concentration of active PSII (PSII*, solid line) was measured
potentiometrically by the flash-induced proton release associated with
water oxidation. The measurements were done in the presence of
DM0 and ferricyanide, conditions that reveal the active PSII centers
(see text for discussion). The concentration of HP Cyt b559 was
measured spectrophotometrically by the chemically induced difference spectrum (hydrobonzoquinone minus ferricyanide). The data
from 100 to 1050 AE/M2 s are for peas grown in a growth chamber,
and the data at 1800 ,E/m2 s (measured at midday) are for peas
grown in full sunlight. The uncertainty in the concentration of HP Cyt
b559 was ± 3%. Further details are given in the text.
Table I. Ratio of PSII to Chl in Thylakoid Membranes Isolated from
Peas Grown at Low and High Ligh Intensities.
The concentration of total PSII was determined by the flashinduced proton release due to the oxidation of water in the
presence of DCBQ and ferricyanide with a pH electrode. The
concentration of active PSII was determined by the same method,
except DM0 was used in place of DCBQ. Further details are given
in the text.
Growth Light
PSII
Total
Active
PSII
Inactive
PSII
Intensity
UE/M2 s
100
900
mmol/mol Chl mmol/mol Chl % of total PSII
1.44± 0.12
20
1.81 ± 0.11
20
2.04 ± 0.15
2.55 ± 0.22
reaction centers. A second flash, given a short time after the
first flash reveals only reaction centers that have recovered
from the first flash (see "Materials and Methods" and references cited therein for details). In detached leaves the fraction
of inactive reaction centers was 18% of the total (active PSII
+ inactive PSII + PSI) in leaves grown at 100 and 1000 ME/
m2s. This indicates that approximately 33% of the total PSII
complexes present in pea leaves are inactive, assuming 2.08
mmol PSI/mol Chl and 2.55 mmol total PSII/mol Chl (Fig.
2 and Table I). We are currently investigating why the fraction
of inactive PSII complexes measured in leaves by the electrochromic shift was greater than that measured in thylakoids by
flash-induced proton release.
Cyt b559, an integral component of PSII (20), is present at
a stoichiometry of two HP Cyt b559 hemes per active PSII,
regardless of the growth light intensity (Fig. 1). In spinach
.11
500
(GROWTH
1 000
1 500
LIGHT INTENSITY
(jiE
2000
/m
2
s)
Figure 2. Effect of growth light intensity on the ratio of PSI/Chi in
thylakoid membranes isolated from peas. The concentration of PSI
was determined spectrophotometrically by the light-induced oxidation
of P700. The filled symbols represent growth chamber light, and the
open symbol represents full sunlight measured at midday. Further
details are given in the text.
thylakoids there are also two HP Cyt b559 hemes per active
PSII (28).
The concentration of PSI was measured by the far-red lightinduced oxidation of P700. The ratio of PSI to Chl was
remarkably constant over the entire range of growth light
intensities (Fig. 2). The values ranged from 1.87 to 2.08
mmol/mol Chl. The spectrum of the absorbance change is
shown elsewhere (14).
The concentration of Cytfheme in thylakoid membranes
was determined by the chemical difference spectrum, and is
shown for plants grown at different light intensities in Figure
3. A 10-fold decrease in growth light intensity (1050 to 100
ALE/M2 s) resulted in a 32% decrease in the ratio of Cyt f to
Chl. The data show a strong correlation between the concentration of Cyt f and the concentration of active PSII (Fig. 1
and 3).
We note that ratios of Cytfto Chl reported by Leong and
Anderson (17, 18) in pea thylakoids show a larger variation
than we measure, even though the growth light conditions
were similar. We do not know the reason for the difference
between their data and ours, nor why they find large differences in the concentration of Cytfin peas that appear to have
been grown under the same conditions. For example, in pea
thylakoids grown at 840 jiE/M2 s, they report 2.1 mmol Cyt
f/mol Chl (17) and 4.7 mmol Cytf/mol Chl (18). In addition,
the Chl per reaction center ratio measured by Leong and
Anderson (see "Discussion") is significantly different from the
values we measured. In part this difference can be accounted
for by the fact that they measured the total reaction center
concentration, without differentiating between active and inactive PSII, and that different techniques were used to quantify the complexes. Another importance difference is that we
harvested leaves only from the top of the canopy, avoiding
shaded leaves grown under variable light quality and lower
light intensities.
The amount of rapidly turning over Cyt f in thylakoid
membranes isolated from peas grown at different light intensities is shown in Figure 3. The spectrum of the light-induced
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 1989 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 89, 1989
LEE AND WHITMARSH
936
5
.
.
.
I.
.
.
.
.
I
.
.
.
.
--,
4
2
.3
E2
-
I
7
n
-V
v0
'A
3
s
0
2
Q
I.-
sE
r
-
e
.
o
0
500
1000
1500
0
2000
-1
.
1000
500
2000
1500
GROWTII LIGIT IN'TENSITY (pE /m2s)
Figure 4. Effect of growth light intensity on the Chi a/Chl b ratio in
thylakoids isolated from peas. The Chi a/Chl b ratio was determined
as described in the text. The filled symbols represent growth chamber
light, and the open symbol represents full sunlight measured at
midday.
GROWTH LIGHT INTENSITY (iE / m s)
Figure 3. Effect of growth light intensity on the ratio of Cyt f/Chl in
thylakoid membranes isolated from peas. The concentration of Cyt f
was measured spectrophotometrically. The solid squares represent
the concentration of Cyt f determined by the chemically induced
difference spectrum (hydrobenzoquinone minus femcyanide). The
open circles and solid line represent the concentration of Cyt f
determined by the light-induced difference spectrum. The data from
100 to 1050 ME/M2 s are for peas grown in a growth chamber, and
the data at 1800 MAE/M2 s (measured at midday) are for peas grown
in full sunlight. The uncertainty in the amount of light-induced Cyt f
was comparable to that shown for the chemically-induced concentration. Further details are given in the text.
absorbance change was essentially the same as we find in
spinach (26, 28). Since Cytf is an integral component of the
Cyt b/f complex, present at a stoichiometry of one mol Cytf
per complex (21), we assume that the concentration of active
Cyt f is proportional to the concentration of active Cyt bif
complex. Comparison of the amount of Cyt f heme present
in the membrane with the amount turning over in the light
indicates that over 90% of the heme-containing Cytfis active
in linear electron transport, as shown previously for spinach
thylakoids (26). Based on immunochemical analysis of pea
thylakoid membrane fractions, Allred and Staehelin (1) have
concluded that there is a pool of Cytfin the membrane that
is not associated with the Cyt b/f comple. If we assume that
Cyt f can function only in the assembled Cyt b/f complex,
then these results support the suggestion that the pool of free
Cytf pool does not contain a heme prosthetic group (1).
The ratio of Chl a/Chl b was 16% lower in plants grown at
100 IAE/m2 s compared to plants grown at 1050 gE/m2 s
.
Fi
.
w
4
e.
x
5-
2
-
'
-
150
55
"
2d0 2
.
r
e
200
r
sr
0Q
50
100 B<,mr"
4
_M'-
w
un-
0
.
500
..
1 000
.. ..
..
1 500
...
20' oo
x
(gE / m s)
Figure 5. Effect of growth light intensity on the ChI/leaf area and the
leaf fresh weight/leaf area in peas. The data from 100 to 1050 uE/
mi s are for peas grown in a growth chamber, and the data at 1800
AE/M2 s (measured at midday) are for peas grown in full sunlight.
GROWTH LIGHT INTENSITY
In this study peas grown below the light compensation
point (discussed below) showed the largest differences in the
photosynthetic apparatus. For example, in plants grown at 8
ME/M2 s there was a 50% lower concentration of the active
Cytb/f complexes per Chl, a 40% lower concentration of
active PSII per Chl, and 16% fewer PSI, compared to high
light grown plants. We found that plants grown below the
light compensation point appeared spindly and unhealthy,
whereas plants grown at or above 100 uE/M2 s appeared
robust and healthy.
sponse to growth light intensity (Fig. 5). A 10-fold decrease in
growth light intensity (1050 to 100 AE/m2 s) resulted in a
24% decrease in the leaf fresh weight per leaf area.
In Table II we show the concentration of the major components of the photosynthetic apparatus on a leaf area basis.
We chose the unit leaf area to be 105 A2, which is approximately the thylakoid membrane area occupied by 500 Chl
molecules (discussed in Whitmarsh [25] and refs. therein). A
membrane area of 105 A2 contains approximately one active
PSII, one Cyt b/lf one PSI, and 500 Chl molecules (Table III).
On a leaf area basis, peas grown at high light intensity contain
approximately 430 active PSII, 430 PSI, and 390 Cytb/f per
I05 A2 leaf area. It is worth noting that a line passing vertically
through the leaf would intercept approximately 430 photosynthetic apparatuses.
Effect of Growth Light Intensity on the Photosynthetic
Components on a Leaf Area Basis
The Chl per leaf area ranged from 2.9 to 3.5 x 10-4 mol
Chl/m2, and did not exhibit a systematic difference in re-
Effect of Growth Light Intensity on Electron Transport
Rates and on the Light Compensation Point
Uncoupled electron transport rates were measured in thylakoid membranes under conditions in which water was the
(Fig. 4).
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 1989 American Society of Plant Biologists. All rights reserved.
PHOTOSYNTHETIC APPARATUS OF PEA THYLAKOID MEMBRANES
Table II. The Number of Photosynthetic Components per Leaf Area
in Peas Grown at Different Light Intensities.
The leaf area is 105 A2. PSII* and Cyt b/f* indibates active component. Further details are given in the text.
Growth Light
PSI
PSII* Cyt b/f
Chl
Intensity
1EM2 s
number of components/105 A2
100
390
302
281
2.08 x 105
200
366
311
288
1.96 x 105
350
379
320
305
1.90 x 105
550
359
315
315
1.76 x 105
700
442
425
380
2.13 x 105
850
429
429
396
2.06 x 105
1050
427
437
394
2.05 x 105
Sunlight
407
425
376
1.96 x 105
electron donor, and the terminal electron acceptor was oxymediated by methyl viologen. For growth intensities from
550 to 1050 ,E/m2 s, as well as for plants grown under natural
sunlight, the maximum rate of uncoupled electron transport
was constant within the experimental error (Fig. 6). The lowest
rate, observed for peas grown at 100 AE/m2 s, was 70% of the
average maximum rate. Comparison oflight saturation curves
for thylakoids isolated from peas grown at 100 and 1050 ,uE/
m2 s show that peas grown at lower light saturate at a 26%
lower actinic light intensity. For thylakoids the red actinic
light intensity required to give half of the maximum rate of
electron transport was 170 ME/M2 s for peas grown at 100 AtE/
m2 s, and 230 ,uE/m2 s red actinic light for peas grown at
1050 ME/M2 s.
The rate of Cytfreduction shows exactly the same dependence on growth light intensity as the steady state electron
transport rate (Fig. 6). This correlation is explained by the
observation that Cytfreduction is associated with the oxidation of plastoquinol, which is the dominant rate limitation in
light-saturated steady state electron transport (26). To test
whether the low turnover number of Cyt f in peas grown at
low light (Fig. 6) is an intrinsic characteristic of the Cyt b/f
complex, or was due to other factors, e.g. a lower concentration of plastoquinol, we measured the activity of Cytfin the
presence of duroguinol (13). In the presence of duroquinol
the rate of Cytf reduction was 52 s-', compared to the rate
gen,
937
of 43 s-' (Fig. 6) measured with water as the electron donor
in thylakoids grown at high light. This observation indicates
that the maximum turnover rate of the Cyt b/f complex is
approximately the same in peas grown at different light intensities. The lower rate of Cytfreduction in the low light grown
peas may be due to the lower concentration of active PSII,
which could result in a lower concentration of plastoquinol
in the region of the Cyt b/f complex.
The light compensation points for detached leaves grown
at 100 and 550 gE/m2 s (determined by using an oxygen
electrode to measure the C02-dependent O2 evolution at 23°C)
were 22 ± 3 and 42 ± 6 &E/m2 s, respectively. It is noteworthy
that the largest difference we observed in experiments done
using peas grown at 100 E/m2 s or above is a twofold increase
in the light compensation point.
In peas grown at 550 ,uE/m2 s the maximum rate ofelectron
transport was 360 mmol e/mol Chl s measured in leaves at
23°C in the presence of excess carbon dioxide. Based on this
rate of electron transport and the concentration of Cyt b/f
we calculate that the average turnover rate of the Cyt b/f
complex in pea leaves in the presence of saturating carbon
dioxide was 200 e-/s.
DISCUSSION
Photosynthetic Apparatus of Peas Grown at Different
Light Intensities
The photosynthetic apparatus of pea thylakoids exhibits 10
to 32% differences in the stiochiometries of the electron
transport complexes compared to the antenna system, over
the growth light regime from 100 to 1050 ,E/M2 s. The largest
difference, on a Chl basis, was a 32% decrease in the amount
of active PSII (Fig. 1) and Cyt b/f (Fig. 3), and the smallest
difference was a 10% decrease in the amount of PSI. The
results for peas grown in natural sunlight were essentially the
same as in plants grown at higher irradiances in growth
chambers. The stiochiometries of PSI:active PSII:active Cyt
b/fChl were 1:1:0.9:480 (±5%) in peas grown at high light
intensities, and were essentially the same as that found in
spinach (Table III). The maximum rate of uncoupled electron
transport measured in thylakoid membranes was constant to
within 7% over growth irradiances from 350 to 1050 ME/M2
s and natural sunlight (Fig. 6). This rate of electron transport
Table IlIl. Stoichiometries of Photosynthetic Components in Thylakoid Membranes Isolated from
Spinach and Peas Grown at Different Light Intensities.
The stoichiometries in spinach were measured as described in "Materials and Methods" using market
spinach. PSII* and Cyt b/f * indicates active component. Further details are given in the text.
Growth Light
Plant
PSI
PSII*
Chl
Cyt b/f *
Intensity
4EIM2 s
Spinach
Peas
Peas
Peas
Peas
Peas
Sunlight
Sunlight
1050
700
350
100
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.04
1.02
0.96
0.84
0.78
0.96
0.92
0.92
0.86
0.80
0.72
510
480
480
480
500
530
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 1989 American Society of Plant Biologists. All rights reserved.
LEE AND WHITMARSH
938
500 l w
*
*
I
W w * w * l 60
* -
I
O
v
400
L..
ci
el
0
A
x
I--
c
300
C6
200
6
I
z
I
40
20-i
~o
m
IE
L.
100
20
A.n
0
.
.
.
shaded leaves from the top of the plant. In addition, we
focused on data gathered at growth light intensities above the
light compensation point. In several other studies, the data
proposed to demonstrate growth light regulation include
plants that appear to have been grown near or below the light
compensation point (e.g. Refs. 17, 18, 30, and 31). It seems
likely that plants that are unable to increase their carbon
content by photosynthesis would be subject to control mechanisms beyond those of growth light intensity.
Does Growth Light Intensity Regulate the Photosynthetic
.
.
v
500
GROWTII
a
Plant Physiol. Vol. 89, 1989
1000
1500
LIGhIT INTENSITY
~~~~
Apparatus?
2000
(iE /m s)
Figure 6. Effect of growth light intensity on the rate of electron
transport and the rate of Cyt f reduction in thylakoid membranes
isolated from peas. Electron transport rates were determined polarographically as light-induced oxygen consumption in the reaction
from water to methyl viologen. The rates shown were light saturated,
and were measured in the presence of an uncoupler at 180C. The
rate of Cyt f reduction was measured spectrophotometrically. The
rate is the reciprocal of the half-time of the dark reduction observed
a 50 ms flash. The actinic light was saturating, and the
rates were measured in the presence of an uncoupler at 1 80C. The
data from 100 to 1050 gE/M2 s are for peas grown in a growth
chamber, and the data at 1800 ,iE/m2 s (measured at midday) are for
peas grown in full sunlight. Further details are given in the text.
following
corresponds to a reaction center turnover rate of approximately 160 e-/s, and a Cyt b/f turnover rate of 175 e-/s, at
1 8°C. In addition to the active PSII complexes, flash-induced
proton release measurements in thylakoids and eletrochromic
shift measurements in detached leaves each reveal a pool of
inactive PSII. Both the proton release and electrochromic
shift measurements indicate that the fraction of PSII that is
inactive is independent of growth light intensity.
In order to evaluate the contribution of electron transport
to the rate of photosynthesis we can compare the rate of Cyt
b/f turnover in uncoupled thylakoid membranes to the turnover rate in leaves under high carbon dioxide and saturating
light. The turnover rate of the Cyt b/f complex was 175 e7/s
(at 18°C) in thylakoids, compared to 200 e-/s (at 23C) in
leaves. This indicates that the maximum rate of electron
transport the photosynthetic apparatus can provide is close to
the electron transport rate required for high rates of carbon
reduction under conditions of saturating carbon dioxide. In
other words, the photosynthetic apparatus of leaves appears
to be operating near its maximum rate during carbon reduction under conditions of high carbon dioxide and saturating
light.
The aim of this study was to examine the effect of growth
irradiance on the photosynthetic apparatus ofthylakoid membranes with as little interference as possible from other factors.
The results apply to a plant that can grow well in direct
sunlight, and focus on regulation during growth, i.e. acclimation. We have investigated neither the differences between
the photosynthetic apparatuses of plants that have adapted
through evolution to low light, e.g. shade plants (2), nor the
effect of light quality. To avoid effects due to light quality and
variations in light intensity due to shading, we selected un-
To answer the question whether growth light intensity
regulates the photosynthetic apparatus, we need to establish
the criteria that the data must satisfy to demonstrate growth
light intensity regulation. The advantage to a plant of regulating the photosynthetic apparatus in response to incident light
intensity would be to get the maximum photosynthesis out of
the least number of components, thereby conserving the
energy and resources required for the synthesis and maintenance of the components. The clearest example is provided
by considering the size of the antenna system serving the
reaction centers. A reaction center exposed to high light would
require a smaller antenna system than one exposed to low
light, in order to achieve a similar turnover rate. The underlying principle is that if too much of a component is present,
then the component sits idle and does not justify its synthesis
and maintenance, whereas if too little of a component is
present, it will be rate-limiting and reduce the rate of energy
conversion.
Insight into this question is provided by considering a single
thylakoid membrane, under the assumption that regulation
is designed to maintain a fairly constant turnover rate of the
reaction centers. In this analysis we consider only the total
number of active reaction centers (PSII* + PSI), and the total
antenna system serving them. In the simplest possible model
designed to predict the ratio of antenna size to reaction center
we would expect that a three-fold increase in growth light
intensity would result in approximately a three-fold decrease
in antenna size relative to the reaction center concentration.
This model is oversimplified, ignoring the shading effect of
the antenna pigments as light penetrates the leaf, light scattering, the change in light quality as light penetrates the leaf, and
other factors, so there is no reason to expect the antenna to
reaction center ratio to be exactly inversely proportional to
growth light intensity. The problem is to extrapolate to the
level of a leaf, and then to a canopy, in order to compare the
advantages of growth light regulation to the metabolic costs
of synthesizing and maintaining a regulatory apparatus. The
other extreme would occur in the absence of regulation of the
photosynthetic apparatus, in which the antenna to reaction
center ratio would remain constant, independent of growth
light intensity. In peas we found that the antenna to reaction
center ratio to be constant over the growth light intensity
regime from 700 to 1050 ,E/m2 s and natural sunlight, and
to exhibit an increase of 28% at a growth light intensity of
100 ,uE/m2 s (Fig. 7). In our view a 28% increase in antenna
size in response to a 10-fold difference in growth light intensity
is not compelling evidence for regulation of the photosyn-
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 1989 American Society of Plant Biologists. All rights reserved.
PHOTOSYNTHETIC APPARATUS OF PEA THYLAKOID MEMBRANES
939
estimate of the actinic light intensity necessary to achieve 50%
of the maximum electron transport rate for a single thylakoid
200
100
0
0.0
..
0.5
1.0
GROWTHI LIGIIT INTENSITY (relative)
Figure 7. Effect of growth light intensity on the Chi to reaction center
(RC) ratio (Chl/(PSII + PSI)). The filled symbols represent growth
chamber light, where 1.0 is 1800 MAE/M2 s. The open symbols and
crosses represent plants grown in sunlight, where 1.0 is full sunlight.
(0) Chl/(PSII* + PSI) in pea thylakoids, this study. (+) Chl/(PSII* +
PSI) in spinach, calculated from the data of Chow and Hope (4). (A)
Chl/(total PSII + PSI) in spinach, calculated from the data of Terashima and Evans (22). (U) Chl/(total PSII + PSI) in pea thylakoids,
calculated from the data of Leong and Anderson (18).
thetic apparatus based on energy considerations. In contrast
to higher plants some algae exhibit truly large changes in their
photosynthetic apparatus in response to growth light intensity
(8, 12, 32). Wilhelm and Wild (32) found that a 5-fold
decrease in growth light intensity resulted in a 2.6-fold in-
the Chl to Cytfratio in Chlorellafusca grown above
the light compensation point. Humbeck et al. (12) observed
a similar response in Scenedesmus. In other studies of algae
similarly large differences in the photosynthetic apparatus
have been reported in response to variations in growth light
intensity (e.g. Ref. 7).
As discussed in the Introduction other studies in higher
plants including peas have been interpreted in terms of regulation of the photosynthetic apparatus by growth light irradiance (e.g. Refs. 17-19). In order to compare data from different studies in the same format we show the effect of growth
light intensity on the total Chl serving the reaction centers
using results from several different labs (Fig. 7). The data
shown are for plants grown between 50 and 1050 ,E/m2 s in
growth chambers, and in attenuated and natural sunlight. For
reasons described above we have included only data gathered
from plants grown above the light compensation point, which
we assume to be greater than 40 uE/m2 s. For spinach and
pea the differences in the Chl to reaction center ratio is not
large over several-fold changes in growth light intensity. In
these studies the average antenna size per reaction center
increased 16% (22), 18% (4), 28% (this study), and 58% (18),
while the growth light intensity decreased 6- to 10-fold. As
discussed above, we think these differences in antenna size
are too small to be accounted for by growth light regulation
in terms of energetic arguments.
In considering the influence of growth light intensity on the
photosynthetic apparatus it is useful to calculate the light
intensity necessary to saturate the turnover rate of an individual reaction center. Based on our data we can make a rough
crease in
membrane. For this calculation we assume that each reaction
center turns over 80 times per second (50% of the maximum
turnover rate at 1 8C), that the quantum yield for photochemistry is 0.8, that each reaction center is served by approximately 200 Chl molecules, that the average membrane area
occupied by a single chlorophyll molecule is 200 A2 (Whitmarsh [25] and refs. therein), that the average extinction
coefficient for Chl over the wavelength range from 400 to 700
nm is approximately 25 mM-' cm-' (33), and that the membrane is oriented normal to the incident light. Under these
assumptions uncoupled electron transport at 18°C would be
50% saturating at a sunlight intensity of approximately 90
AE/M2 s. It is important to note that at higher temperatures
we would expect the light saturation intensity to increase in
proportion to the temperature induced increase in the maximum rate of electron transport. Based on data for spinach
thylakoids (26) the light saturation intensity would increase
approximately twofold for each 10°C increase in temperature.
Since this calculation applies to a single thylakoid membrane,
it is not applicable to stacked membranes in which pigment
shading becomes a dominant factor. Based on this calculation
we expect that reaction centers in pea thylakoids would saturate below 20% of full sunlight at 18C. Experimentally we
found that uncoupled electron transport in thylakoid membranes isolated from high light intensity grown peas was 50%
saturating at a red actinic light intensity of 230 ,E/m2 s.
One of the questions we addressed in this study was whether
pea plants respond to different growth light intensities by
adjusting the amount of thylakoid membranes per leaf area,
without altering the structure or organization of the photosynthetic apparatus within the thylakoid membrane. Our data do
not support growth light regulation at this level, since neither
the Chl, nor the number of photosynthetic components per
leaf area were significantly different in peas grown at 100 to
1050 IAE/m2 s, and full sunlight (Table II).
Terashima and Inoue (23, 24) have suggested that photosynthetic components of the chloroplast adjust to different
light environments within leaves. They investigated photosynthetic components and activities at different depths within
spinach leaves. On a Chl basis they observe an decrease in
activity and the amount of Cytfin going from the top to the
bottom of the leaves. In view of our results, we do not think
the differences they report are due to a decrease in light
intensity within the leaf, in that we were unable to create a
similar response by growing peas at a low growth light intensity. The differences they observe may be due to differences
in light quality as light penetrates the leaf (9, 15, 19), or a
consequence of evolutionary adaptation.
In summary, based on the data presented here and a critical
examination of data in the literature, it is our view that the
case is not strong in support of the notion that the concentration of the photosynthetic components of the thylakoid membrane are regulated by growth light intensity in a manner
designed to balance the turnover of each element. Considering
the daily variation in light intensity that a plant is subject to,
it could be argued that regulation of photosynthetic components at the membrane level is not the most viable strategy.
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 1989 American Society of Plant Biologists. All rights reserved.
LEE AND WHITMARSH
940
For example, changing the orientation of chloroplasts within
the cells, or changing the orientation of the leaf with respect
to the incident light, may provide a more effective and timely
response to changes in growth light intensity. The concept
that emerges from this work is of a relatively fixed photosynthetic apparatus that is able to perform efflciently under a
very wide range of light intensities. Furthermore, comparison
of the photosynthetic apparatus in peas, spinach, and several
other plants (14, 27-29) leads us to suggest that evolution has
selected for a remarkably homologous photosynthetic apparatus, designed to accommodate a variety of environmental
conditions.
ACKNOWLEDGMENTS
We thank Roger Chylla for measuring the electrochromic shift in
detached leaves, and Drs. Don Ort, Mary Blackwell, and Carol
Augspurger for useful discussions.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
LITERATURE CITED
Allred DR, Staehelin LA (1987) Cytochrome f exists as two
functionally separate pools in pea thylakoid membranes. In J
Biggins, ed, Progress in Photosynthesis Research, Vol 2. Martinus Nijhoff, Dordrecht, The Netherlands, pp 293-296
Boardman NK (1977) Comparative photosynthesis of sun and
shade plants. Annu Rev Plant Physiol 28: 355-377
Boardman NK, Bjorkman 0, Anderson JM, Goodchild DJ,
Thorne SW (1975) Photosynthetic adaptation of higher plants
to light intensity: Relationship between chloroplast structure,
composition of the photosystems and photosynthetic rates. In
M Avron, ed, Proceedings of the 3rd International Congress
Photosynthesis, Vol 3. Elsevier, Amsterdam, The Netherlands,
pp 1809-1827
Chow WS, Hope AB (1987) The stoichiometries of supramolecular complexes in thylakoid membranes from spinach chloroplasts. Aust J Plant Physiol 14: 21-28
Chylla RA, Garab G, Whitmarsh J (1987) Evidence for slow
turnover in a fraction of photosystem II complexes in thylakoid
membranes. Biochim Biophys Acta 894: 562-571
Delieu T, Walker DA (1981) Polarographic measurement of
photosynthetic oxygen evolution by leaf discs. New Phytol 89:
165-178
Falkowski PG, Owens TG, Ley AC, Mauzerall DC (1981) Effects
of growth irradiance levels on the ratio of reaction centers in
two species of marine phytoplankton. Plant Physiol 68: 969973
Fleischhacker P, Senger H (1978) Adaptation of the photosynthetic apparatus of Scenedesmus obliquus to strong and weak
light conditions. II. Differences in photochemical reactions,
the photosynthetic electron transport and photosynthetic units.
Physiol Plant 43: 43-51
Glick RE, McCauley SW, Melis A (1985) Effect of light quality
on chloroplast-membrane organization and function in pea.
Planta 164: 487-494
Graan T, Ort DR (1986) Detection of oxygen-evolving photosystem II complexes inactive in plastoquinone reduction. Biochim
Biophys Acta 852: 320-330
Hangarter R, Ort DR (1985) Cooperation among electron-transfer centers in ATP synthesis in chloroplasts. Eur J Biochem
149: 503-510
Humbeck K, Hoffmann B, Senger H (1988) Influence of energy
flux and quality of light on the molecular organization of the
photosynthetic apparatus in Scenedesmus. Planta 173: 205212
Jones RW, Whitmarsh J (1988) Inhibition of electron transfer
and the electrogenic reaction in the cytochrome b/f complex
by NQNO and DBMIB. Biochim Biophys Acta 933: 258-268
Plant Physiol. Vol. 89, 1989
14. Lee WJ, Pakrasi HB, Whitmarsh J (1987) P700 spectra and
concentrations in several plants and a cyanobacterium. In J
Biggins, ed, Progress in Photosynthesis Research, Vol 2. Martinus Nijhoff Publishers, Dordrecht, The Netherlands, pp 233236
15. Leong TY, Anderson JM (1984) Effect of light quality on the
composition and function of thylakoid membranes in Atriplex
triangularis. Biochim Biophys Acta 766: 533-541
16. Leong TY, Anderson JM (1984) Adaptation of the thylakoid
membranes of pea chloroplasts to light intensities I. Study on
the distribution of chlorophyll protein complexes. Photosynth
Res5: 105-115
17. Leong TY, Anderson JM (1984) Adaptation of the thylakoid
membranes of pea chloroplasts to light intensities. II. Regulation of electron transport capacities, electron carriers, coupling
factor (CFI) activity and rates of photosynthesis. Photosynth
Res 5: 117-128
18. Leong TY, Anderson JM (1986) Light-quality and irradiance
adaptation of the composition and function of pea-thylakoid
membranes. Biochim Biophys Acta 850: 57-63
19. Melis A, Harvey GW (1981) The stoichiometries of supramolecular complexes in thylakoid membranes from spinach chloroplasts. Biochim Biophys Acta 637: 138-145
20. Nanba 0, Satoh K (1987) Isolation of a photosystem II reaction
center consisting of Dl and D2 polypeptides and cytochrome
b559. Proc Natl Acad Sci USA 84: 109-112
21. Ort DR (1986) Energy transduction in oxygenic photosynthesis:
An overview of structure and mechanism. In LA Staehelin, CJ
Arntzen, eds, Encyclopedia of Plant Physiology, Photosynthesis III, Vol 19. Springer-Verlag, Berlin, pp 143-196
22. Terashima I, Evans JR (1988) Effects of light and nitrogen
nutrition on the organization of the photosynthetic apparatus
in spinach. Plant Cell Physiol 29: 143-155
23. Terashima I, Inoue Y (1985) Palisade tissue chloroplasts and
spongy tissue chloroplasts in spinach: biochemical and ultrastructural differences. Plant Cell Physiol 26: 63-75
24. Terashima I, Inoue Y (1985) Vertical gradient in photosynthetic
properties of spinach chloroplasts dependent on intra-leaf light
environment. Plant Cell Physiol 26: 781-785
25. Whitmarsh J (1986) Mobile electron carriers in thylakoids. In
LA Staehelin, CJ Arntzen, eds, Encyclopedia of Plant Physiology, Photosynthesis III, Vol. 19. Springer-Verlag, Berlin, pp
508-527
26. Whitmarsh J, Cramer WA (1979) Cytochrome f function in
photosynthetic electron transport. Biophys J 26: 223-234
27. Whitmarsh J, Lee WJ (1986) Is the antenna to reaction center
ratio in pea chloroplasts regulated for optimum energy conversion? In G Akoyunoglou, H Senger, eds, The Regulation of
Chloroplast Differentiation. Alan R Liss, New York, pp 643652
28. Whitmarsh J, Ort DR (1984) Stoichiometries of electron transport complexes in spinach chloroplasts. Arch Biochem Biophys
231: 378-389
29. Whitmarsh J, Ort DR (1984) Quantitative determination of the
electron transport complexes in the thylakoid membranes of
spinach and several other higher plant species. Adv Photosynth
Res 3: 231-234
30. Wild A, Hopfner M, Ruhle W, Richter M (1986) Changes in the
stoichiometry of photosystem II components as an adaptive
response to high-light and low-light conditions during growth.
Z Naturforsch 41: 597-603
31. Wild A, Ke B, Shaw ER (1973) The effect of light intensity
during growth of Sinapis alba on the electron transport components. Z Pflanzenphysiol 69: 344-350
32. Wilhelm C, Wild A (1984) The variability of the photosynthetic
unit in Chlorella. II. The effect of light intensity and cell
development on the photosynthesis, P700 and cytochromefin
homocontinuous and synchronous cultures of Chlorella. J
Plant Physiol 115: 125-135
33. Zscheile FP, Comar CL (1941) Influence of preparative procedure on the purity of chlorophyll components as shown by
absorption spectra. Bot Gaz 102: 463-481
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 1989 American Society of Plant Biologists. All rights reserved.