Plant Cell Physiol. 41(2): 138–147 (2000)
2000
JSPP
Cooperation of Photosystems II and I in Leaves as Analyzed by
Simultaneous Measurements of Chlorophyll Fluorescence and Transmittance
at 800 nm
Hillar Eichelmann and Agu Laisk
Tartu Ulikooli
›
Molekulaar- ja Rakubioloogia Instituut, Riia tn 23, Tartu 51010, Estonia
energy, while between the two photosystems plastoquinone
and plastocyanin are di˙usible e carriers and e /H cotransport is accomplished by the Cyt b6/f complex. For the
maximum e‚ciency of the whole e transport chain a certain distribution of excitation between the two photosystems and fast operation of Cyt b6/f are required. The
number of PSII reaction centers is usually calculated from
measured O2 evolution from single-turnover flashes (Chow
et al. 1989) or from measured absorbance di˙erence between the states with reduced and oxidized forms of the
quinone acceptor. The number of PSI reaction centers is
determined from absorbance di˙erence between reduced
and oxidized forms of the donor pigment P700 (Melis
1989). These optical measurements are possible only on
thylakoid preparations but not on intact leaves. In vascular
plants (spinach, pea, barley, tobacco) there are in average
600 Chl molecules per PSI and 350 Chl per PSII, resulting
in PSII/PSI ratio of 1.7 (Melis 1989). However, optimal
and e‚cient electron flow between the two photosystems
requires statistically equal rates of light absorption and
utilization by the two photosystems, rather than equal
photosystem stoichiometries. Thus, the number of reaction
centers multiplied by the antenna size of PSII and PSI is
what determines the balance of excitation between the
photosystems. The antenna size of PSII can be determined
from fluorescence induction curves of DCMU-inhibited
centers. Instead of expected single exponent the transients
are slightly sigmoidal and fit with two exponents, from
which the presence of about 75% of PSIIα with a bigger
antenna (about 250 Chl) and 25% of PSIIβ with a smaller
antenna (about 115 Chl) has been detected. Since the PSIIβ
is ine‚cient in linear electron transport, it is the number
and antenna size of PSIIα that should be compared with
those of PSI. The antenna size of PSI can be determined
from an optically measured oxidation transient of P700
in KCN poisoned chloroplasts (KCN inhibits e transport
through plastocyanin) and the single-exponent transient
yields about 230 Chl/PSI. From these measurements
(Melis 1989) it can be summarized that total Chl is distributed as follows: 0.54 at PSIIα, 0.076 at PSIIβ and 0.38
at PSI. Several reasons have been proposed why more
quanta must be absorbed by PSII than by PSI: it seems
that in vivo Chl b (a component of PSII antenna) is a less
e‚cient absorber than Chl a, in the tightly appressed state
PSII absorbance is handicapped, etc. (Melis 1989). How-
Parallel measurements of CO2 assimilation, Chl fluorescence and 800 nm transmittance were carried out on
intact leaves of wild type and cytochrome b6/f deficient
transgenic tobacco grown at two di˙erent light intensities
and temperatures, with the aim to diagnose processes limiting quantum yield of photosynthesis and investigate their
adaptations to growth conditions. Relative optical crosssections of PSII and PSI antennae were calculated from
measured gas exchange rates and fluorescence-related losses at PSII and P700 oxidation-related losses at PSI. In nonstress conditions (high light grown wild type and low light
grown antisense type) optimal relative optical cross-section
of PSII (aII) was 0.48–0.51 and that of PSI (aI) was 0.38–
0.40, leaving a non-photosynthetic absorption cross-section (a0) of 0.09–0.14 for nitrite assimilation and absorption in PSIIβ and other photosynthetically inactive
pigments. Stress conditions (low light grown wild type and
high light grown antisense type, elevated growth temperatures) tend to increase a0 and decrease PSII antenna crosssection more than that of PSI antenna, but this rule is
reversed during senescence.
Key words: Chlorophyll fluorescence — P700 — Photosynthesis — Quantum yield.
In photosynthesis electrons (e ) are transported sequentially through PSII and PSI at the expense of light
Abbreviations: A, CO2 assimilation rate, µmol m 2 s 1; a,
absorption coe‚cient of leaves for PAR; aII and aI, relative optical
cross-sections of PSII and PSI antennae; a0, optical cross-section
of the leaf not supporting CO2 assimilation; Cyt b6/f, cytochrome
b6/f complex; F, steady-state fluorescence yield in the light; Fm,
pulse-saturated fluorescence yield in the light; Fmax, highest pulse-saturated fluorescence yield in the dark-adapted state; FRL,
far-red light; nI and nII, numbers of electrons transported through
PSI and PSII per CO2 fixed, respectively; NPQ, kN, nonphotochemical excitation quenching; P, Po and Pm, 800 nm signal
di˙erence from the dark level, it’s flash-oxidisable value and it’s
maximum value, respectively; P700, donor pigment of PSI; PAR,
photosynthetically active radiation; PAD and Q, photosynthetic
photon absorption density; PFD, photosynthetic photon flux
density (incident); QA, primary quinone acceptor of PSII; Y,
quantum yields of electron transport; YC, quantum yield of
CO2 fixation.
138
139
Cooperation of photosystems
ever, below we shall show that the fluorescence-related
losses, present at PSII but absent at PSI, are the most important reason why PSII must be excited more frequently
than PSI, to end up with an equal rate of electron transport
through both photosystems. In this work we propose a
simple nondestructive method for the measurement of
relative absorption cross-sections of PSII and PSI in leaves
and show how much growth conditions and leaf age can
influence these parameters. A Cyt b6/f deficient transgenic
tobacco was used to investigate the e˙ect of hindered e
transport on the antenna size of PSII.
The Cyt b6/f deficient antisense phenotype of tobacco
(Price et al. 1995) is sensitive to growth PFD, the features
indicating Rieske FeS protein deficiency is more severe at
low growth PFD. The antisense e˙ect is ameliorated and
even overcome when the plants are grown at high irradiances (Price et al. 1995). There is evidence that plant’s
overall sensitivity to photoinhibition correlates with the
redox state of QA, the primary e acceptor of PSII, and
originally it was assumed that the Cyt b6/f deficient
phenotype is more sensitive to photoinhibition. Contrary
to this, the Cyt b6/f deficient tobacco, where violaxanthine
de-epoxidation is much less than in the wild type, nonphotochemical excitation quenching is hindered and QA is
highly reduced, did not show the expected increase in susceptibility to photoinhibition (Hurry et al. 1996). Variation
in the Chl a/b ratio suggests that in these plants a smaller
PSII antenna develops in accordance with the restricted
e transport, and this may prevent photoinhibitory symptoms. We shall show that in the Cyt b6/f deficient tobacco
really a smaller PSII antenna develops under high growth
PAD than in the wild type, and that this acclimation is
absent at low growth PAD.
Quantum and electron budget in photosynthesis—The
partitioning of quanta between the two photosystems is
governed by the relative optical cross-sections of the
antennae that depend on the ratio of light-absorbing Chl
molecules at the photosystems and, to a lesser extent,
di˙erences in spectral properties of the antenna Chl. The
following Eq. 1 and 2 describe e fluxes through PSII and
PSI, driven by the partitioned quanta:
aII Fm F
nII Fm
[
] na [P P P]
I
I
o
(1)
m
and the budget of absorbed quanta
P and Pm are 800 nm signals reflecting the steady-state and
maximum oxidation of P700, and Po is the oxidizable
part of P700 corresponding to open PSI centers. Terms
in square brackets represent the e‚ciencies of excitation
use at the given photosystem, the relative photochemical
quenching of fluorescence indicating the e‚ciency of PSII
and the relative reduction state of oxidizable P700 indicating the e‚ciency of PSI. The e‚ciency of excitation
use by PSI for charge separation is considered to be close
to one (Hiyama 1985). Only these PSI centers are considered e‚cient in e transport that have reduced donor side
and oxidized acceptor side (Po P). If a fraction of the
centers have reduced acceptor side and, correspondingly,
reduced donor side (Pm Po), they would be ine‚cient even
though they have reduced P700. In the quantum budget
(Eq. 2) the term a0 accounts for quanta absorbed in photosynthetically inactive structures or pigments, such as
PSIIβ, and quanta supporting reductions not visible in
the measured process, such as Mehler type O2 reduction in
O2 and CO2 measurements and N reduction in CO2 measurements. Solving Eq. 1 and 2 for an optimal case of
(Fm F)/Fm 0.8 and (Po P)/Pm 1, nI nII 4 and a0
0.05 reveals that the maximum theoretical quantum yield
for O2 evolution is 0.105 with optimal cross-section values
of aIIO 0.53 and aIO 0.42 (O2 is added in the subscript to
indicate that these cross-sections support O2 evolution). If
Eq. 1 and 2 are solved for CO2 uptake measurements under
nonphotorespiratory conditions, then a0 is about 0.1 to
0.15, being larger due to other assimilatory processes,
particularly of nitrogen, and aI and aII will be correspondingly lower (Edwards and Baker 1993, Robinson and
Baysdorfer 1985, Robinson 1988). Solving Eq. 1 and 2 with
a0 0.15, the maximum quantum yield of CO2 assimilation
would be 0.095, with aII 0.47 and aI 0.38.
This theory predicts that for optimal distribution of
excitation PSII antenna needs to be greater than PSI antenna and the maximum quantum yield of photosynthesis
occurs when excitation is optimally distributed between
photosystems. Below we shall investigate how close to the
optimal is the actual excitation distribution in intact leaves.
From Eq. 1 we see that quantum yields of PSII and PSI are
proportional, the proportionality factor containing the ratio of optical cross-sections of the photosystems and the
ratio of e transport through photosystems:
2
[P P P ]
o
aII
aI
a0
1,
(2)
where a are relative optical cross-sections (quantum partitioning ratios) to the given photosystem (as indicated by
the subscript) and n is the number of e required to pass
through the given photosystem per unit of a given process
(e.g. per CO2 fixed or O2 evolved); F and Fm are steadystate and pulse-saturated fluorescence yields, respectively;
m
2
aIInI Fm F
aInII Fm
[
]
(3)
Eq. 3 shows that the ratio of the optical cross-sections of
the two photosystems may be found from the comparison
of fluorescence and 800 nm absorbance data, without
measuring gas exchange.
140
Cooperation of photosystems
Materials and Methods
Plant material and growth conditions—Tobacco (Nicotiana
tabaccum L.) wild type and Cyt b6/f deficient transgenic plants
(Price et al. 1995) were grown at high and low light intensities and
at elevated soil and air temperatures in a peat-soil mixture in two
homemade (0.50 0.65 m2) growth boxes. Photoperiod was 18/6
h, mean PFD in box 1 was 250 µmol m 2 s 1, measured in 9
places (range from 220 to 280), air temperature 27 to 30/18 to 21
day/night (below referred to as high temperature treatment), in
box 2 PFD was 570 in the high light treatment (from 500 to 600)
and in the low light treatment it was 180 (from 170 to 200) µmol
m 2 s 1, air temperature 22 to 25/17 to 20 day/night. To
minimize the influence of the inhomogeneity of growth illumination, plants were rearranged in the box each other day. Young
leaves, nearly full-grown leaves, and senescing leaves, attached to
the plant, were used in experiments.
T2 seeds from one individual T1 plant were used for the experiments with the transgenic tobacco. At young age the development of nonphotochemical quenching (NPQ) was measured.
Hindered NPQ is an easily detectable feature of Cyt b6/f deficiency in transgenic plants (Price et al. 1995). The value of NPQ
kN was calculated (Laisk et al. 1997) as
kN
Fmax
Fm
1
(4)
In wild type plants maximum kN was 1.8–2.1, but only those
transgenic plants were left for further growth treatments in which
kN 0.65.
Gas exchange measurements—The basic gas system (type
TREMS, Fast-Est, Tartu, Estonia) has been described before (Oja
1983, Laisk and Oja 1998). A part of a leaf was enclosed in a
sandwich-type square chamber (4.3 4.3 0.3 cm3) and exposed
to a gas flow rate of 0.8 mmol s 1. The upper side of the leaf was
sealed with starch gel to the thermostated glass window, as a result
of which leaf temperature changed from 22.8 in the dark (base
temperature in all experiments) to 24.2˚C under PFD of 3,500
µmol m 2 s 1. The actual leaf temperature was calculated from
the leaf heat budget. Gas exchange was measured through the
lower epidermis only and dissolved cell-wall CO2 concentration
was routinely calculated to consider the di˙usional limitations.
The leaf chamber was illuminated through a light guide
of plastic fibers (TREMS, Fast-Est, Tartu, Estonia). Light from
three sources—a Schott KL 1500 (H. Walz, E˙eltrich, Germany)
for white actinic light (from 0 to 3,500 µmol m 2 s 1), another KL
1500 equipped with a 720 nm interference filter for far-red light
(FRL, 70 µmol m 2 s 1) and a 1,000 W xenon arc lamp for fluorescence saturation pulses—was overlapped on the leaf area. The
fiber illumination system quaranteed homogenous illumination
over the leaf area within 5% (SD). An average PAD was used
for the calculation of the quantum yield of CO2 fixation, PAD in
the site of fluorescence measurement was 5% of the average and
in the site of 800 nm measurement it was 5% of the average.
PFD was measured with a LI-185 quantum sensor (LiCor, Lincoln, NE, U.S.A.). Leaf absorbance for PAR was measured in an
integrating sphere. Light intensity is expressed as absorbed photosynthetic photon flux density, PAD.
CO2 concentration was set by mixing pure CO2 with the help
of a capillary under stabilized pressure into the gas flow composed
of N2 and O2. Two similar gas systems (channels 1 and 2) were
mounted in one gas system, either of them could be connected
with the leaf chamber and had a CO2 analyzer (LI 6262, LiCor,
Lincon, NE, U.S.A.). Connecting the leaf chamber either with
channel 1 or channel 2 reference line of the other channel could be
checked without changing the gas flow and concentration in the
leaf chamber. This was important for correct measurements of
quantum yields of CO2 uptake at low PADs where correct measurement of low rates was dependent on frequent reference line
recording. Psychrometers (TREMS, Fast-Est, Tartu, Estonia),
one in each channel, were used for the measurement of the water
vapor pressure. If the measured light response curve displayed the
Kok e˙ect (an apparently greater quantum yield at PADs below
the light compensation point), then it was caused by decreasing
dark respiration in the light (Sharp et al. 1984) and the quantum
yield was taken from data measured at 100 and 200 µmol quanta
m 2 s 1.
Chl fluorescence and 800 nm transmission—Chl fluorescence
was recorded by PAM 101 fluorometer (H. Walz, E˙eltrich, Germany) using optical fibers placed between the illumination fibers.
This way geometric interference between light sources and fluorescence measurements was completely avoided. Fluorescence was
sensed from 2 cm2 area placed slightly away from the center of the
chamber. An additional low-pass filter cutting at 750 nm was
placed in the sensor to avoid interference with the 800 nm signal
used for P700 measurements. This decreased the fluorescence
signal for about 3 times but thanks to the geometry of fibers the
recording was still noiseless. The actinic light and saturation
flashes were filtered by heat-reflecting filters (Optical Coating
Laboratory, Inc., Santa Rosa, CA) to minimize the saturating
e˙ect of non-modulated light on the fluorescence detector. Saturation pulses were of 10,500 µmol m 2 s 1 and of about 2 s duration and Fm was read at the maximum of the induction transient.
800 nm transmission that senses the reduction state of P700
was measured by another PAM 101 equipped with a ED 800T
emitter-detector. A special bundle of fibers placed between the illumination fibers was used for guiding the detector beam to the
leaf from the physiologically upper side. Another bundle of fibers
at the lower side of the leaf collected 800 nm radiation passed
through the leaf and transferred it to the sensor diode of the ED
800T. Transmission at 800 nm was measured on the area of 1
cm2 placed in the center of the chamber separately from the
fluorescence measurement area. The pulse intensity was set to
maximum and the gain of the PAM 101 was adjusted to have the
total 800 nm transmission signal between 1.5 and 2 V, depending
on leaf. This full signal was o˙set using the zeroing function of
the PAM 101, and the o˙set signal was amplified 79 times for
subsequent recording. The percent deflections of the 800 nm signal from the dark reference line in determining P and PFRL were
calculated as follows:
P(%)
P(V)
•100
g•S(800)
(5)
where P is the deflection from the dark level (either in % or Volts,
indicated in parentheses), g is the gain factor of the amplifier
(g 79), and S(800) is the total signal at 800 nm before zeroing
(Volts). The maximum deflection under FRL in the absence of
white light, PFRL, was measured and the deflection corresponding
to completely oxidized P700 was calculated as
Pm
1.22PFRL
(6)
The correction factor accounts for the presence of PSII light in
FRL. The proportion of PSII light in FRL was found from separate experiments where a steady-state increase in CO2 uptake was
measured under FRL in sunflower with the same FRL source.
Cooperation of photosystems
Data were computer-recorded using an A/D board ME-30 (Meilhaus Electronic, Puchheim, Germany) and processed by a packet
of programs (programs Reco, RDA Synte, Fast-Est, Tartu, Estonia).
Calculation of electron transport from fluorescence and
CO2 exchange—Electron transport rate was calculated from fluorescence data using Eq. 7 (Genty et al. 1989):
JF
aIIQ
Fm F
Fm
(7)
where aII is relative optical cross-section of PSII antenna and Q is
PAD. From CO2 exchange e transport rate was calculated using
Eq. 8 (Laisk and Sumberg 1994, Laisk and Loreto 1996), which
considers the contribution of photorespiratory e transport (also
at 2% O2):
JC
4(A
Rd)
2Ks[Cw0 (rgw rmd)A] nOw0/4
2Ks[Cw0 (rgw rmd)A] Ow0
(8)
where A is the measured net assimilation rate, Rd is dark respiration rate in the light, Ks is Rubisco specificity factor (Ks 90, Laisk
and Sumberg 1994), Cw0 and Ow0 are molar CO2 and O2 concentrations in liquid phase, equilibrated with the external gaseous
CO2 and O2 concentration (Ow0 15 µM, Cw0 30 µM), rgw and
rmd are leaf gas phase and liquid phase di˙usion resistances, respectively, as calculated from leaf transpiration and photosynthesis rates, and n ( 8) is the number of e in linear e transport
required per CO2 evolved from photorespiration (see Laisk and
Loreto 1996).
Results
Standard routine—Before the measurements, a leaf
was stabilized under PAD of 1,800 µmol m 2 s 1 until stomata were maximum open (about 20–30 min). Then PAD
was increased to 3,500 µmol m 2 s 1 and decreased stepwise to the dark. A typical recording of CO2 exchange,
fluorescence and 800 nm signal during the measurement of
the light response curves is shown in Fig. 1. After the
photosynthetic rate had stabilized at a given PAD, a pulse
was given, to obtain pulse-saturated levels of fluorescence
and 800 nm signal. Fm was read at the maximum during the
flash, Po was obtained as the maximum deflection of the
800 nm signal from the dark level during the flash. Thereafter light was switched o˙ for 4 s to obtain the dark level
of the 800 nm signal, then FRL was switched on for 4 s to
oxidize interphotosystem carriers, and o˙ again, to measure dark fluorescence F0. After these procedures photosynthesis was stabilized at the next actinic PAD. The
stabilization time was about 3–5 min at each PAD (this
time is about 1 min in Fig. 1, made especially shorter for
demonstration purposes). The maximum deflection under
FRL, PFRL, was measured in the absence of white light and
the deflection corresponding to completely oxidized P700
was calculated from Eq. 6.
Light responses—In all treatments with the wild type
plants the steady-state fluorescence F remained rather
constant over the wide range of PADs (Fig. 2), which
demonstrates a good complementation between photo-
141
chemical and nonphotochemical excitation quenching, one
decreasing, the other increasing (Laisk et al. 1997). The
development of nonphotochemical excitation quenching
was clearly retarded in the antisense type tobacco in all
treatments, as a result, the steady-state F continuously increased with increasing PAD. In the transgenic plants kN
was always much lower than in the wild type, though it still
decreased at low growth PFD and in the high temperature
treatment (Fig. 2, Table 1). This indicated that the transgenic phenotype, selected at young age of the plants, was
stable throughout the di˙erent growth treatments. Despite
that the measurement routine minimized photoinhibition
(short exposure at the highest PAD and at least 15 min
darkness before Fmax was measured), some residual slowly
relaxing nonphotochemical quenching still remained in the
dark. Therefore, presented relative to the dark Fmax, the
relative F0 and F levels were apparently increased in the low
light and high temperature plants, though the absolute
values actually were not.
Quantum yield and fluorescence—Electron transport
rates were calculated from CO2 exchange rate (Eq. 8) and
the quantum yield of PSII electron transport was plotted
against (Fm F)/Fm (Fig. 3A). Relationships for all treatments are linear, there is no deflection from linearity that
could be related to the presence of an alternative e
transport or state transition in these leaves (Laisk and
Loreto 1996). Extrapolation of the relationships to the axis
of ordinate that presents the relative excitation (optical
Fig. 1 Computer-recorded traces of a segment of the measurement of light response curves. CO2 fixation rate, thick line and left
ordinate; 800 nm signal, upper thin line and right ordinate; fluorescence yield, lower thin line and right ordinate. The recording
begins at a PAD of 880 µmol m 2 s 1. At time 14 s a 2 s flash of
10,500 µmol m 2 s 1 (down in 800 nm signal, up in fluorescence
signal) followed by darkness for 4 s (up in 800 nm), FRL for 4 s
(down in 800 nm), darkness for 4 s (up in 800 nm) and the next
PAD of 670 µmol m 2 s 1. The routine is repeated at the following two PADs of 480 and 320 µmol m 2 s 1.
142
Cooperation of photosystems
Fig. 2 Light response curves of CO2 fixation rate (filled squares and left ordinate), flash-saturated fluorescence Fm (empty circles, right
ordinate, relative to dark value), steady-state fluorescence F, (empty squares, right ordinate) and dark fluorescence F0, (empty diamonds,
right ordinate). Upper panels A, C, E, wild type, lower panels B, D, F, antisense type. A and B, high light grown; C and D, low light
grown; E and F, high temperature grown plants. Maximum NPQ kN (Eq. 4) is given in the panels.
cross-section) of PSII (Laisk and Loreto 1996) varies between 0.37 and 0.51 (Table 1) and is the lowest in the high
temperature treated plants. In addition to the lower PSII
cross-section, (Fm F)/Fm did not increase beyond 0.5 in
the high temperature plants and beyond 0.6 in the high
light antisense type and low light wild type, indicating
permanent nonphotochemical quenching at low PFDs,
probably due to partial photoinhibition. No especially
pronounced photoinhibition was observed in the low light
grown antisense type plant after an exposure to PAD of
3,500 µmol m 2 s 1 during the measurement of the light
response curve.
Quantum yield and 800 nm transmission—Though
leaf transmission at 800 nm is a complex signal (Klughammer and Schreiber 1991), careful interpretation allows one
to use it for the assessment of the changes in the redox state
of P700 (Genty and Harbinson 1996, Kramer and Crofts
1996). The dark level of the signal corresponds to reduced
P700 and either FRL or saturation pulses (Klughammer
and Schreiber 1994) are used to obtain the level corresponding to oxidizable P700. The 800 nm signal behaved
very similarly in wild type and antisense type plants, continuously decreasing (more oxidation of P700) over the
whole range of increasing light (Fig. 4). However, there is a
characteristic di˙erence between plants. In high light wild
type and low light antisense type P700 stayed relatively
Table 1 Photosynthetic parameters of leaves of wild type and Cyt b6/f deficient transgenic tobacco
Rel. optical
Quantum yield Leaf absorption
Rel. optical
Nonphotochem.
cross-section of cross-section of
of CO2
for PAR
quenching
PSI aI
a
PSII aII
NPQ
assimilation YC
High light grown antisense
High light grown wild type
Low light grown antisense
Low light grown wild type
Hight temp. grown antisense
Hight temp. grown wild type
0.053
0.080
0.084
0.067
0.033
0.052
0.015
0.004
0.009
0.003
0.014
0.001
0.811
0.855
0.824
0.850
0.778
0.817
0.057
0.021
0.035
0.008
0.033
0.052
0.469
0.380
0.397
0.465
0.385
0.413
0.015
0.040
0.012
0.016
0.017
0.036
0.406
0.483
0.512
0.465
0.380
0.419
Each value is an average of 4–6 measurements carried out on expanding and fully expanded leaves.
0.060
0.050
0.010
0.018
0.036
0.060
0.65
1.76
0.41
1.27
0.16
0.80
0.16
0.26
0.12
0.09
0.06
0.20
Cooperation of photosystems
Fig. 3 Quantum yield of PSII electron transport calculated
from CO2 assimilation as a function of (Fm F)/Fm (panel A) and
quantum yield of PSI electron transport calculated from CO2 assimilation as a function of (Po P)/Pm (panel B). Squares, empty—high light wild type, filled—high light antisense type; diamonds, empty—low light wild type, filled—low light antisense
type; circles, empty—high temperature wild type, filled—high
temperature antisense type.
reduced at low PADs, a condition necessary for high PSI
e‚ciency, and it became more oxidized only as PAD approached saturating values. In low light wild type and high
light antisense type, as well as in both high temperature
treatments, the 800 nm signal deviated from the dark level
already at very low PAD, showing oxidation of P700. Such
response, when PSI looses e‚ciency by oxidizing P700 already at the lowest PADs, indicates PSI overexcitation
143
compared with PSII. As a result, at limiting PADs the
overall quantum e‚ciency of photosynthetic CO2 fixation
is smaller in these plants (Table 1).
The 800 nm data are presented by plotting Y vs.
(Po P)/Pm (Fig. 3B), a presentation of the quantum yield
of PSI which is similar to the presentation of Y vs.
(Fm F)/Fm for PSII (Fig. 3A). Typical Pm values for
plants grown under di˙erent treatments, obtained from
Eq. 6 were 1.20% for high light wild type and 0.86% for
antisense type; 0.89% for low light wild type and 0.70%
for antisense type; 0.77% for high temperature wild type
and 0.72% for high temperature antisense type. For all
treatments the relationships of Y vs. (Po P)/Pm were very
close to straight lines, which shows that e transport
through Cyt b6/f was the major bottleneck under the conditions of these experiments. These lines (dotted lines in
Fig. 3) extrapolate to Po on the abscissa, the intercept corresponding to all oxidizable P700, and to aI on the ordinate, the relative optical cross-section of PSI. In these
leaves the di˙erence between Po and Pm was not detectable,
since unoxidizable P700 (PSI with acceptor side reduced)
were practically absent. Extrapolation of the plots in
Fig. 3B to the axis of ordinates yields values of the relative
optical cross-section of PSI, aI, about 0.38 for the high
light and high temperature wild type and low light antisense type and 0.45 for low light wild type and high light
antisense plants (Table 1).
Fluorescence-related quantum losses at PSII and loss-
Fig. 4 Light response curves of CO2 fixation rate (filled squares and left ordinate), percent deflection of the 800 nm signal from the
dark reference line (Eq. 5, empty triangles, right ordinate) and the maximum deflection under FRL in the absence of white light
PFRL (empty circles, right ordinate). Upper panels A, C, E, wild type, lower panels B, D, F, antisense type. A and B, high light grown;
C and D, low light grown; E and F, high temperature grown plants.
144
Cooperation of photosystems
Fig. 5 Interdependence of the quantum e‚ciencies of PSII calculated as (Fm F)/Fm and of PSI calculated as (Po P)/Pm. Data
collected from all experiments, treatments explained at panels. Di˙erent symbols represent individual leaves. Data for senescing leaves
are shown by filled triangles in C, empty diamonds in E, empty circles in B and filled squares in F).
es by oxidation of P700 at PSI are seen from the comparison of the plots in Fig. 3A and B. Generally, in plants
which had lower aI (0.42 in the high light wild type and 0.40
in the low light antisense type) PSI was not overexcited at
low PADs (P700 was close to complete reduction). Contrary to that, in the high light antisense type and low light
wild type PSI absorbed about 0.45 of all quanta, but due to
fluorescence-related losses at PSII, PSI was forced to dissipate quanta by oxidizing P700 to about a half-level, so
that the actual quantum yield of PSI did not exceed 0.25.
The actually measured maximum quantum yield of PSII
shows the fluorescence-related losses. These were the
smallest in the high light wild type leaves (1 0.73 0.27)
and the greatest in the high temperature antisense type
plant (0.5). During senescence these losses gradually increased (e.g. to 0.85 in the senescing high temperature antisense type leaves).
Relative e‚ciencies of PSI and PSII, presented as
(Po P)/Pm and (Fm F)/Fm respectively, are compared
in Fig. 5, where data from all measurements are plotted
together. The relationships are clearly proportional, in accordance with Eq. 3. The extrapolation of the proportional
dependence to the quantum yield of unity shows which of
the two photosystems has larger antenna. For example, in
all measured leaves of the high light grown wild type plants
the quantum yield of PSI approaches unity while that of
PSII is 0.78 (Fig. 5A). This means that the ratio of aI/aII
0.78 (Eq. 3, nI nII). Very similar aI/aII was in the low light
grown antisense type leaves (Fig. 5D). In the low light and
high temperature grown wild type plants the optical crosssections of both photosystems were approximately equal
(Fig. 5C, E), but in the high light and high temperature
antisense type plants PSI had even slightly larger antenna
than PSII, aII/aI 0.9 in both cases (Fig. 5B, F). In all
treatments data for senescing leaves indicated faster
decrease of the PSI antenna compared with PSII antenna
(filled triangles in C, empty diamonds in E, empty circles in
B and filled squares in F).
Discussion
Quantum yield—The proposed theory for the quantum yield of photosynthesis considering relative optical
cross-sections of PSII and PSI antennae and the number of
e required to transport through PSII and PSI yielded the
maximum quantum yield for O2 evolution of 0.105 in C3
plants. This is equal to the experimental value obtained for
quantum yields based on net O2 evolution for 37 species
under nonphotorespiratory conditions, 0.106 (Demmig
and Björkman 1987, Lal and Edwards 1995). The coincidence of theoretical and experimental values allows us to
conclude that assumptions used in the theory are realistic:
the condition nI nII 4 is fulfilled at low PADs, meaning
that Mehler type e transport through both photosystems
and cyclic e flow around PSI are slow or absent. The slow
cyclic e flow is in good agreement with the present understanding of the energy requirements for CO2 fixation in
C3 plants, such as tobacco, where 3 ATP are needed per
Cooperation of photosystems
CO2, which can be provided by linear e flow to NADP if
the Q-cycle is active (Rich 1988, Furbank et al. 1990, Heber
et al. 1995) and 4H /ATP is the requirement of the
ATP-synthase (Rumberg et al. 1990). In this case, no cyclic
transport around PSI or Mehler reaction would be required
for steady-state photosynthesis and an equal number of
e pass through PSII and PSI per CO2 fixed. Cyclic or
Mehler type e flow through PSI are necessary to generate
initial proton gradient and to compensate proton leak
(Schreiber and Neubauer 1990), but in our experiments
these requirements were evidently so small that could not
be detected.
The calculated maximum quantum yield for CO2 fixation was 0.095. Maximum experimental quantum yields of
C3 plants under nonphotorespiratory conditions are close
to this, but variation is from 0.073 to 0.093 in di˙erent
studies (Ehleringer and Björkman 1977, Sharp et al. 1984,
Ehleringer and Pearch 1983, Long et al. 1993). In our experiments the highest values were 0.084 (low light grown
antisense) and 0.080 (high light grown wild type). As we
see, in practice, the quantum e‚ciency of photosynthesis is
frequently lower than the theoretical maximum. Eq. 1 and
2 o˙er possible explanations for this: persistent nonphotochemical excitation quenching (e.g. photoinhibition) that
decreases the e‚ciency of excitation use at PSII; an
increased fraction of optical cross-section not serving
photosynthesis, a0, e.g. increased fraction of PSIIβ;
an unoptimal ratio of excitation distribution between the
photosystems that may be caused by using measurement
light with a di˙erent spectral distribution than that prevailing on the growth site, but, also, by development of the
photosynthetic machinery under stress conditions.
Optical cross-section of photosystems and quantum
budget—The extrapolation of the graphs of the quantum
yield to (Po P)/Pm 1 and (Fm F)/Fm 1 (Fig. 3) was
used to determine the optical cross-sections of PSI and
PSII, aI and aII. Logically, the procedure finds quantum
yields of PSI and PSII in the ideal case when losses occur
neither at PSI nor at PSII under limiting PAD. The absence
of losses was nearly true for PSI in the high light grown
wild type and low light grown antisense type plants, since
P700 was almost completely reduced at low PADs and became oxidized only at higher PADs. However, fluorescence-related losses at PSII were present even at low PADs,
such that the e‚ciency of PSII did not exceed 0.64–0.66 in
these plants (Fig. 3). This indicated the presence of slowly
relaxing NPQ, but it did not alter the extrapolation rule, as
shown earlier (Laisk et al. 1997). Relative optical crosssections of PSII, aII, tended to be higher than those of PSI,
aI, in plants grown under apparently nonstress conditions,
such as high PAD for the wild type and low PAD for the
antisense type plants where, in average, aII 0.48 (0.51)
and aI 0.42 (0.40, in parentheses data for the antisense
type). The larger PSII cross-section compensated for the
145
fluorescence-related losses at this photosystem and, as a
result, the overall quantum yield of CO2 assimilation was
the highest in these plants (0.080 and 0.084 respectively).
The sum aII aI was 0.89 to 0.91 in nonstressed leaves
(high and low light grown wild type, low light grown antisense type) but decreased to a minimum of 0.72 under the
stress conditions in high temperature grown antisense type
plants. The relative proximity of the sum to unity under
nonstress conditions indicates that the quantum budget
was correctly closed in our measurements, i.e., that the
quantum yields and optical parameters were correctly
measured. The small residual of the quantum budget,
a0 0.1 accommodates absorption by nonphotosynthetic
pigments and cell structures, plus other e sinks like N
reduction and Mehler type O2 reduction, not accounted by
CO2 assimilation, but also the absorption by PSIIβ, if
present. The fraction of PSIIβ was evidently very small
under nonstress conditions, but might increase when a0
increased under stress.
The ratio aII/aI was 1.32 in the high light grown wild
type and low light grown antisense tobacco in this study,
which is equal to the ratio of 1.32 calculated on the basis of
the absorption spectra of the chlorophyll-protein complexes in high light grown pea leaves (Evans 1986) and not
far from the average ratio of 0.54/0.38 1.42 of chlorophyll associated with PSIIα and PSI (Melis 1989). Such
good agreement of our experimental results with cumbersome calculations from spectra and laborious spectroscopic measurements, and the well-closed quantum budget,
confirm that our method can be used to determine aI and
aII on intact leaves.
The relatively low total photosynthetic absorption
cross-section of 0.73 in high temperature grown antisense
type plants might partially reflect an increased fraction of
PSIIβ, which probably increased due to faster turnover of
the D1 protein under stress (Prasil et al. 1992). The faster
reaction of PSII antenna to stress conditions seems to be a
rule since a similar response has been observed in coldstressed wintering conifers (Lönneborg et al. 1985, Ottander et al. 1995, Strand and Oquist
›
1985) and in CO2
starving unicellular chlorophyte Dunaliella salina (Baroli
and Melis 1998). The deficiency of Cyt b6/f in the transgenic plant did not induce changes in the distribution of
chlorophyll between photosystems provided that the antisense type plant was grown under low light. High growth
PAD induced a pronounced stress-type decrease of the
PSII antenna, showing that adaptational responses in the
antenna size were conserved in the transgenic tobacco,
though the development of NPQ was very limited. The
conserved aII/aI ratio in low light transgenic plants, equal
to this ratio in the wild type plants, does not agree with the
observed decreased Chl a/b ratio and increased ratio of
PSII/PSI reaction centers in these plants (Price et al. 1995),
however, those plants were of extreme antisense phenotype
146
Cooperation of photosystems
and could be grown only partially heterotrophically, on
sucrose. Interestingly, a similar response towards decreasing aII/aI ratio followed when the wild type tobacco was
grown under low PAD in our experiments. One may
speculate that growth at low PAD is also a stress for such
a sun plant as tobacco and decrease in PSII antenna is a
typical reaction to it. An exception from this rule occurs
during senescence, when PSI antenna is degraded faster
than PSII antenna (Fig. 5). This situation seems to be
similar to the conditions where in transgenic plants PSI
antenna responded with a faster decrease than PSII antenna in the above cited work (Price et al. 1995).
Cooperation of photosystems—An optimal distribution of quanta between photosystems is such that ensures
equal speed of e transport through PSII and PSI considering that, as a minimum, 20% quanta will be lost at
PSII anyway. Not the factors listed in the Introduction but
inevitable fluorescence-related losses are the major reason
why the antenna of PSII has to be larger than the antenna
of PSI. The optimal PSII antenna is such that, after all
losses, provides an e‚ciency equal to the e‚ciency of PSI,
while no losses occur at PSI since P700 remains reduced at
low PAD. Our measurements show that under nonstress
conditions the antennae of PSII and PSI develop in an
optimal ratio, which is not influenced by genetic interference in the Cyt b6/f. High light grown wild type and low
light grown antisense type plants had aII/aI the closest to
the optimal, but even in these plants P700 became slightly
oxidized at low PADs (Fig. 3B, 4A, D). This was caused by
the presence of a long-lasting NPQ in these leaves that did
not allow to reach the maximum e‚ciency of PSII of 0.8.
In stressed plants the PSII antenna decreased more than
the PSI antenna, to the extent that approximately aII aI,
and there were no excitation reserves to compensate for
fluorescence-related losses at PSII. As a result, PSI su˙ered
shortage of e already at very low PADs and P700 accumulated. In these leaves the overall quantum yield of
CO2 assimilation did not exceed 0.05, due to the unoptimal
excitation distribution leading to losses at both photosystems. Contrary to the above, in senescing leaves the
antenna of PSI decreased faster than that of PSII, but due
to the very intense NPQ PSII still was rate limiting
(Fig. 5B, F). The fact that non-optimal excitation distribution can occur confirms that the optimum is not adjusted
by spillover of excitation from PSII to PSI, corresponding
to actual excitation needs under any experimental conditions, but is the result of more or less balanced development of the antennae of both photosystems during growth.
This work was supported by Estonian Science Foundation
grant No. 3907. The seeds of the Cyt b6/f deficient transgenic
tobacco were a highly appreciated gift from D. Price and M.
Badger.
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(Received July 5, 1999; Accepted November 10, 1999)