Tree Physiology 21, 899–914
© 2001 Heron Publishing—Victoria, Canada
Sensitivity of photosynthetic electron transport to photoinhibition in a
temperate deciduous forest canopy: Photosystem II center openness,
non-radiative energy dissipation and excess irradiance under field
conditions
ÜLO NIINEMETS1 and OLEVI KULL2
1
Department of Plant Physiology, Institute of Molecular and Cell Biology, University of Tartu, Riia 23, Tartu 51010, Estonia
2
Department of Ecophysiology, Institute of Ecology, Tallinn University of Educational Sciences, Riia 181, Tartu EE 51014, Estonia
Received August 18, 2000
Summary We used chlorophyll fluorescence techniques to
investigate responses of Photosystem II (PSII) quantum yield
to light availability in the short term (quantum flux density integrated over the measurement day, Q d) and in the long term
(Q d averaged over the season, Q s) in a mixed deciduous forest
comprising shade-tolerant and water-stress-sensitive Tilia
cordata Mill. in the lower canopy and shade-intolerant and water-stress-resistant Populus tremula L. in the upper canopy. In
both species, intrinsic efficiency of PSII in the dark-adapted
state (Fv /Fm) was lower during the day than during the night,
and the difference in Fv /Fm between day and night increased
with increasing Q s. Although the capacity for photosynthetic
electron transport increased with increasing Q s in both species,
maximum quantum efficiency of PSII in the light-adapted state
(α) decreased with increasing Q s. At a common Q s, α was
lower in T. cordata than in P. tremula primarily because of a
higher fraction of closed PSII centers, and to a smaller extent
because of limited, non-radiative, excitation energy dissipation in the pigment bed in T. cordata. Across both species, photochemical quenching (q P), which measures the openness of
PSII centers, varied more than fivefold, but the efficiency of
excitation energy capture by open PSII centers (Fv′/Fm′),
which is an estimate of non-radiative excitation energy dissipation in PSII antennae, varied by only 50%. Chlorophyll turnover rates increased with increasing irradiance, especially in
T. cordata, possibly because of increased photodestruction.
Diurnal measurements of PSII quantum yields (ΦPSII) indicated that, under similar environmental conditions, ΦPSII was
always lower in the afternoon than in the morning, and the fraction of daily integrated photosynthetic electron transport lost
because of diurnal declines in ΦPSII (∆) increased with increasing Qd. At a common Qd, mean daily PSII center reduction
state, the fraction of light in excess (1 – fractions of light used
in photochemistry and dissipated as heat) and ∆ were higher in
T. cordata than in P. tremula. This was attributed to greater
stomatal closure during the day, which led to a greater reduction in the requirement for assimilative electron flow in T. cordata. Across both species, ∆ scaled negatively with the fraction
of light utilized photochemically, demonstrating the leading
role of PSII center openness in maintaining high PSII efficiency. Because photosynthesis (A) at current ambient carbon
dioxide concentration is limited by CO2 availability in high
light and mainly by photosynthetic electron transport rates in
low light, overall daily down-regulation of ΦPSII primarily influences A in low light. Given that foliar water stress scales
positively with Q s in both species, we conclude that the inverse
patterns of variation in water and light availabilities in the canopy result in a greater decline in A than is predicted by decreases in stomatal conductance alone.
Keywords: chlorophyll turnover, excess light, light-use efficiency, photosynthetic acclimation, Populus tremula, Tilia
cordata, water stress.
Introduction
Photoinhibition, which embraces a plethora of phenomena
causing decreases in photosynthetic light-use efficiency because of either photodamage in thylakoids or protective downregulation of photosynthetic apparatus, or both, is an unavoidable part of the photosynthetic process (Long et al. 1994,
Osmond 1994). The rate constant for Photosystem II (PSII)
photodamage is directly proportional to absorbed quantum
flux density (Q), i.e., damage to PSII is not confined to high
light, but may occur irrespective of flux density of quanta
(Park et al. 1995a, Baroli and Melis 1996, Tyystjärvi and Aro
1996). However, recent evidence suggests that the probability
of photodamage at a common absorbed Q depends on the balance between light absorption and use in photochemistry. Increases in PSII electron transport rate allow a lower degree of
PSII reaction center closure, and reduce susceptibility to damage (Park et al. 1996a, Baroli and Melis 1998). Sustained
curbing of PSII efficiency is also a balance between damage
and repair, which take place simultaneously, and the magnitude of both determines the extent to which leaves lose efficiency for light harvesting and use (Greer et al. 1986, Schnett-
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NIINEMETS AND KULL
ger et al. 1994, Baroli and Melis 1996). Often, the capacity for
repair increases with increasing excitation pressure on PSII
(Huner et al. 1998), e.g., with increasing daily light exposure
(Tyystjärvi et al. 1992). Nevertheless, research indicates that
the fraction of active PSII centers decreases in vivo (Chylla
and Whitmarsh 1989, Park et al. 1995a, Anderson et al. 1998),
and evidence of declines in PSII overall efficiency as a result
of photodamage has been obtained from field studies (Ögren
and Sjöström 1990, Naidu and DeLucia 1997, Tognetti et al.
1997).
In addition to declines in photosynthetic efficiency resulting
from damage to the foliar photosynthetic apparatus, quantum
yields of PSII may decay because of light-induced increases in
thermal dissipation of excitation energy in pigment antennae
or in the reaction center of PSII (Krause and Weiss 1991,
Pospíšil 1997). Increases in non-radiative dissipation of excitation energy in response to high Q decrease the probability of
photodamage by relieving the excitation pressure on PSII,
thereby allowing maintenance of a greater openness of PSII
centers (Osmond 1994). Thermal dissipation of excitation energy in PSII light-harvesting antennae, which requires the
presence of the de-epoxidized xanthophyll cycle carotenoids,
zeaxanthin and antheraxanthin, as well as the trans-thylakoid
pH gradient (see Pfündel and Bilger 1994, Demmig-Adams
and Adams 1996a, Horton et al. 1996 for a review), may be the
primary route by which plants adjust the excitation pressure on
PSII under natural conditions (Demmig-Adams and Adams
1996b). There is evidence that photoinhibition observed in the
field in response to diurnal variability in Q is not related to
damage, but results from a safe down-regulation of PSII efficiency through increases in non-radiative energy dissipation
(Epron et al. 1992, Valentini et al. 1995, Krause and Winter
1996, Chaumont et al. 1997, de Mattos et al. 1997, Logan et al.
1997; for reviews see Demmig-Adams et al. 1996, Demmig-Adams and Adams 1996b). Declines in PSII efficiency
sustained over days (Demmig-Adams et al. 1998) and months
(Adams and Demmig-Adams 1994, Ottander et al. 1995, Adams and Barker 1998, Verhoeven et al. 1998) may be attributed to high rates of non-radiative energy dissipation associated with retention of a trans-thylakoid pH gradient (Verhoeven et al. 1998), and a large fraction of de-epoxidized
xanthophyll cycle carotenoids (Adams and Barker 1998,
Demmig-Adams et al. 1998, Verhoeven et al. 1998) in leaves.
In contrast, other evidence indicates that recovery of in vivo
PSII efficiencies may require significantly more time than recovery of the epoxidation state of xanthophyll cycle carotenoids (Rosenqvist et al. 1991, Leitsch et al. 1994, Thiele et al.
1998), indicating that xanthophyll cycle activity only partly
explains the decrease in PSII quantum yields in the field.
Despite the debate over the exact mechanisms by which diurnal decreases in PSII efficiency commence, the evidence indicates that changes in PSII efficiency commonly occur in the
field. Niinemets et al. (1999a) observed extreme constancy of
PSII electron transport capacities during most of the growing
season, despite large day-to-day variabilities in incident quantum flux density and temperature experienced by leaves that
were also subjected to a series of soil water deficits. This con-
stancy of foliar electron transport capacities was accompanied
by plastic adjustments in the pool size of xanthophyll cycle carotenoids, which increased with increasing daily light receipt
and decreasing daily minimum air temperature (Niinemets et
al. 1998a, 1999a). However, constancy of light-saturated values of photosynthetic electron transport does not imply constancy of light-limited rates (Ögren 1994). Despite the protection provided against excessive irradiances, elevated concentrations of zeaxanthin and antheraxanthin and the associated
decrease in PSII quantum yields may be maintained long after
relaxation of short-term light stress the leaves were subject to
earlier in the day (Björkman and Demmig-Adams 1994, Logan et al. 1997, Thiele et al. 1998), thereby hampering the efficiency of light conversion in low light during the rest of the
day. This may adversely affect cumulative daily leaf photosynthesis for most canopy leaves that photosynthesize mainly
during periods of low Q.
In the current study, we used chlorophyll fluorescence techniques to obtain quantitative estimates of reduction in PSII
electron transport rates, resulting from sustained daily decreases in PSII efficiency along a natural light gradient within
a canopy. The canopy consisted of the shade-intolerant pioneer species Populus tremula L. in the upper canopy layers,
and the late-successional shade-tolerant tree Tilia cordata
Mill. in the lower leaf layers. Reduction state of PSII centers
as well as fluorescence parameters proportional to non-radiative energy dissipation were also investigated to identify possible protective and damaging light effects leading to decreases in PSII quantum yields. Determination of the relevance of various photoinhibition mechanisms for field conditions is important, because the costs associated with recovery
from photoinhibition and with avoidance of excessive photoinhibitory damage to the leaves may differ for various pathways. Increases in xanthophyll cycle pool size, which increases the capacity for non-radiative excitation energy dissipation, may be cheap relative to rapid protein turnover, which
allows continuous replacement of damaged centers. Because
foliar chlorophyll is destroyed by excessive irradiance
(Kyparissis et al. 1995, Maxwell et al. 1995, He et al. 1996,
Bertrand and Schoefs 1999), we also analyzed the foliar pool
of chlorophyll intermediates to test the hypothesis that turnover rates of foliar chlorophyll (Chl) increase in high light to
maintain the balance between destruction and synthesis. Our
data suggest that leaves possess the capacity to adjust Chl turnover rate in response to light availability, thereby maintaining
a constant foliar Chl concentration despite its continuous destruction.
Materials and methods
Study site
The research was undertaken at Järvselja Experimental Forest
(58°22′ N, 27°20′ E, elevation 38–40 m), Estonia. The deciduous mixed tree canopy with a total leaf area index of about 6 is
dominated by P. tremula and Betula pendula Roth. in the upper layer (17–27 m), and by T. cordata in the lower layer
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PHOTOINHIBITORY DECLINE IN QUANTUM YIELDS IN NATURAL CANOPIES
(4–17 m). The woody understory primarily consists of Corylus avellana L. and a coppice of T. cordata. The soil is a fertile
gleyed pseudopodsol formed on a loamy till. Although the water storage capacity of the soil is large, soil hydraulic conductivity is low, and the trees may regularly sustain water stress,
especially in the middle and at the end of the growing season
(Niinemets et al. 1999d).
Permanent scaffolding (height 25 m) was present at the site
providing access to four trees of P. tremula with a mean height
(± SE) of 25.0 ± 1.7 m, and four trees of T. cordata (15.1 ± 0.7
m). Additional details of the study area are provided in
Niinemets et al. (1998a).
Estimation of foliar photosynthetic electron transport rates
by chlorophyll fluorescence
In July and August 1995, we measured the response of wholechain photosynthetic electron transport rate (J) to incident
quantum flux density (Q) along the canopy height profiles.
Steady-state fluorescence yield (Fs) and fluorescence yield after application of a saturating pulse of white light (Fm′) of the
light-adapted sample were estimated with a portable pulsemodulation fluorometer (PAM-2000, Heinz Walz GmbH,
Effeltrich, Germany) equipped with a leaf clip holder (Model
2030-B, see Bilger et al. 1995 for details) as outlined in
Niinemets et al. (1998a). For all fluorescence measurements,
additional experiments on separate leaves were conducted to
choose lengths and intensities of saturating pulses that were
appropriate to fully close all PSII centers, but to avoid over-reduction of the photosynthetic apparatus.
Because the effective quantum yield of PSII (ΦPSII) is equal
to (Fm′ – Fs)/Fm′, and assuming that Photosystems I and II absorb equal amounts of light, J was calculated as (Genty et al.
1989):
J = 0.5ΦPSIIΘQ,
(1)
where Θ is leaf absorptance. Quantum flux density was provided by the internal halogen lamp of the PAM-2000, and
starting from dim light (20–70 µmol m –2 s –1), Q was increased
in steps until no further increases in the steady-state values of J
were observed. Measurements of J along the canopy light gradient began at the top of the canopy, and continued from 0800
to 1100 h (morning measurements) and from 1630 to 1800 h
(afternoon measurements). In addition to J versus Q response
curves, steady-state values of J close to saturating Q
(1700 µmol m –2 s –1 for P. tremula and 600 µmol m –2 s –1 for
T. cordata; see Figure 1) were measured along the canopy
throughout the day.
To fit J versus Q response curves, we described the effects
of Q on the fluorescence parameter ΦPSII by Smith’s equation
(Smith 1937), which has often been employed to approximate
light responses of photosynthetic electron transport derived
from foliar gas exchange measurements (e.g., Tenhunen et al.
1976, Harley et al. 1992, Falge et al. 1996):
  αQ  2 
J = αQ 1 + 
 
  Jmax  
−
901
1
2
(2)
,
where α is the initial quantum yield of photosynthetic electron
transport for an incident irradiance and Jmax is the capacity for
photosynthetic electron transport. Combining Equations 1
and 2, and solving for ΦPSII we obtain:
ΦPSII
2
α   αQ  
= 2 1 + 
 
Θ   Jmax  


−
1
2
.
(3)
Although there is debate about whether estimates of photosynthetic electron transport given by Equations 1 and 2 are
equivalent (Seaton and Walker 1990, Evans et al. 1993), an
excellent relationship is generally observed between J estimated from gas exchange measurements and J estimated from
chlorophyll fluorescence analyses (Edwards and Baker 1993,
Oberhuber et al. 1993). Given that the relationship between
ΦPSII and Q is sigmoidal (Russell et al. 1995), in accordance
with the shape of the curve described by Equation 3, our formulation should fit the data at least qualitatively. Based on calculations of leaf absorptance from species-specific empirical
relationships between leaf chlorophyll concentrations and Θ
measured by an integrating sphere as described in Niinemets
et al. (1999c), the two parameters of Equation 3 were estimated by nonlinear regression. Equation 3 provided excellent
fits to the data, with r 2 values averaging 0.98 and always being
greater than 0.93 (see Figure 1).
Analysis of chlorophyll fluorescence quenching along the
canopy
In addition to the fluorescence variables measured in 1995,
minimum (Fo) and maximum (Fm) fluorescence yields of
dark-adapted, and minimum fluorescence yield (Fo′) of lightadapted leaves were assessed during August 1996. At the beginning of each measurement series, the leaf was darkened for
10 min, and Fo and Fm were determined. Thereafter, actinic
light provided by the PAM-2000 LED source was switched on
and, to allow induction of photosynthetic electron transport, Q
was increased in steps to a maximum value provided by the
LED light source of 865 µmol m –2 s –1. At that Q, a steadystate estimate of ΦPSII was taken, actinic light was switched
off, and far-red light provided by the PAM-2000 was switched
on for immediate determination of Fo′. All daytime fluorescence measurements were conducted between 1200 and
1900 h. For specific leaves, Fo and Fm were estimated between
0000 and 0100 h on the same night to determine fully relaxed
minimum and maximum fluorescence yields.
From the daytime measurements, we calculated the following fluorescence parameters (Schreiber et al. 1994): photochemical quenching coefficient (qP = (Fm′ – Fs)/(Fm′ – Fo′ )),
non-photochemical quenching coefficient (qN = 1 – (Fm′ –
Fo′ )/(Fm – Fo)), Stern-Volmer quenching coefficient (NPQ =
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902
NIINEMETS AND KULL
Figure 1. Fitting of the relationships between (A, Equation 3) incident quantum flux
density (Q) and effective
quantum yield of PSII (ΦPSII ),
and (B) illustration of the light
response curves of photosynthetic electron transport rate
calculated by Equation 2 based
on the parameters determined
in panel A for a Populus tremula (ⵧ) and a Tilia cordata
(䊉) leaf. Parameter ΦPSII was
calculated from chlorophyll fluorescence measurements as (Fm′ – Fs)/Fm′, where Fs is steady-state fluorescence yield in the light and Fm′ is
fluorescence yield after application of a saturating flash of white light. Only the steady-state values at each Q are shown. The ΦPSII value that
was apparently affected by photoinhibition at the highest Q in P. tremula was not used for data fitting.
(Fm – Fm′)/Fm′), potential maximum quantum yield of PSII,
(Fv /Fm = (Fm – Fo)/Fm), and quantum yield of open PSII centers (Fv′/Fm′ = (Fm′ – Fo′)/Fm′). From the nighttime measurements, Fv /Fm was also calculated. The Fv /Fm ratio is the
quantum yield of PSII when all PSII centers are open, and it
characterizes the intrinsic efficiency of PSII (Krause and
Weiss 1991, Schreiber et al. 1994). Effective quantum yield of
PSII (ΦPSII) depends on both qP, which is proportional to PSII
center openness, and Fv′/Fm′, which characterizes the efficiency of excitation energy capture by open PSII centers
(Genty et al. 1989):
ΦPSII = q P Fv′ / Fm′ .
(4)
As suggested by Demmig-Adams et al. (1996), we divided
the absorbed light among the fractions used in photochemistry
(PP), dissipated thermally (PD), and the fraction that remains in
excess (PE), i.e., neither used in photochemistry nor dissipated
as heat. Fraction PP is equivalent to ΦPSII (Demmig-Adams et
al. 1996). In addition to q N and NPQ, which provide an estimate of fluorescence quenching resulting from the dissipation
of excitation energy as heat, but which may also include a
slowly relaxing component resulting from photoinhibitory
damage, the quantity of 1 – Fv′/Fm′ is also positively related to
thermal energy dissipation in the antennae of PSII. Because
the non-photochemical energy dissipation process requires
conversion of the epoxidized xanthophyll violaxanthin to deepoxidized forms zeaxanthin and antheraxanthin in the xanthophyll cycle, and Fv′/Fm′ yielded the strongest correlation
with the de-epoxidation state of xanthophyll cycle carotenoids, Demmig-Adams et al. (1996) concluded that the variable
1 – Fv′/Fm′ is the best measure of PD. Finally, the fraction of
light in excess was determined as PE = 1 – PP – PD = Fv′/Fm′(1 –
qP).
Diurnal variability in chlorophyll fluorescence variables
On six representative days in August 1995, ΦPSII was measured with a PAM-2000 in 2-min steps from 0445 (before
dawn) to 2230 h (after sunset) to obtain quantitative estimates
of daily decreases in foliar PSII quantum yields and to make
inferences about possible sources of this decline. To avoid extensive shading periods during the day, Fo and Fm values were
determined for each leaf only at the beginning and end of the
day as described above. For each determination of ΦPSII, Fo′
was calculated assuming that Fo quenching results from increases in energy dissipation in PSII antennae (Oxborough
and Baker 1997):
Fo′ =
Fo
Fv /Fm + Fo / Fm′
,
(5)
and using the morning values of Fo and Fm. Use of fully relaxed predawn values of Fm in assessing the diurnal variability
in qN and NPQ is warranted because sustained decreases in Fm,
which are related to high rates of energy dissipation in the antennae of PSII, are often observed during the day (Ögren and
Sjöström 1990, Epron et al. 1992, Adams et al. 1995), leading
to underestimation of NPQ and qN (Demmig-Adams et al.
1996). All measured fluorescence parameters were corrected
for air temperature effects on the light-emitting diode of the
PAM-2000, which may result in diurnal changes in the fluorescence signal (see Epron et al. 1992, Demmig-Adams et al.
1996).
Calculating potential photosynthetic electron transport rates
during the day
Initial data analysis indicated that values of ΦPSII were lower in
the afternoon than in the morning under similar environmental
conditions. Therefore, we used the morning data (0445–
1100 h) of ΦPSII measured in relatively unstressed leaves to
parameterize Equation 3 at a certain reference temperature.
The initial estimates of α and Jmax (Figure 1) were employed to
calculate the dependence of J on light and temperature,
whereas Jmax was scaled to each leaf temperature based on the
temperature relationships of J reported in Niinemets et al.
(1999a). The relative daily photoinhibitory decrease in J was
calculated as ∆ = (JintP – JintA)/JintP, where JintP is the potential
daily integrated photosynthetic electron transport rate (mol
m –2 day –1) and JintA is the measured rate (mol m –2 day –1). The
basic assumption underlying these calculations is that there
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PHOTOINHIBITORY DECLINE IN QUANTUM YIELDS IN NATURAL CANOPIES
was no photoinhibition in early morning. Given that some sustained photodamage or xanthophyll cycle-related down-regulation of PSII efficiency may have existed in the leaves, these
estimates should be considered as lower limits to the photoinhibitory decrease in J.
Determination of integrated quantum flux densities in the
canopy
To obtain mean seasonal daily integrated quantum flux densities incident to leaves (Qs; mol m –2 day –1), we combined continuous measurements of Q with quantum sensors with estimates of fractional penetration of irradiance at sensor locations obtained from hemispherical photographs (see Niinemets et al. 1998a). Thus, for each of 18 quantum sensors, Qs
was calculated from the sensor readings taken in 1-min steps,
and the fractions of penetrating diffuse (Idif) and potential penetrating direct solar radiation of open sky (Idir) were calculated
from the hemispherical photographs taken at weekly intervals.
Thereafter, linear regression equations of Qs versus Idif and Idir
were developed. Hemispherical photographs were also taken
from the sample locations just after estimation of chlorophyll
fluorescence parameters. The Qs for each sample point was
computed either from the regression equations in the form of
Qs = aIdif + bIdir (in 1995; r 2 = 0.98, P < 0.001) or Qs = aIdif (in
1996; r 2 = 0.94, P < 0.001), where a and b are regression coefficients. Integrated values of Q used to develop the regression
equations were means between the completion of lamina expansion growth and the cessation of leaf thickness growth
(June 3–July 21 in 1995, and June 6–August 8 in 1996). In addition, daily integrated quantum flux densities averaged over
3 days preceding foliage sampling (Q3d) were computed in a
similar manner (Niinemets et al. 1998a) to characterize the
leaf light environment over a shorter term. In 1996, Q integrated during the day until the measurement of chlorophyll
fluorescence characteristics (Qd; mol m –2) was estimated analogously, developing regressions between Qd and Idif.
903
than all the carotenoids, except for β-carotene. Given the similarity in shape and spectral maxima of the absorption spectra
measured over 400–560 nm between Chls a and b and the Chl
intermediates, these compounds were identified as chlorophyllides (Chlid) a and b. Although we cannot rule out that
other Chl intermediates, e.g., protochlorophyllides (Król et al.
1995) co-eluted with Chlid a, the absorption spectra indicated
that the so-called Chlid b peak consisted of a Chl precursor in
which the methyl group on ring B was substituted by a formyl
group—a modification by which Chlid b is formed from Chlid
a (Castelfranco and Beale 1983). The calibration factors for
Chlids were obtained as the products of the calibration factors
for Chls a and b (Niinemets et al. 1998a), based on a ratio of
0.14 of Chlid to Chl calibration factors at 440 nm (Król et al.
1995).
Results
Variation in quantum yield of PSII at high and low light
In both P. tremula and T. cordata, the capacity for photosynthetic electron transport (Jmax; Equation 3) increased with increasing Q s (Figure 2A), primarily because of the positive
effects of Q s on leaf dry mass per unit area and leaf nitrogen
Foliar pigment analysis
Foliage for determination of total pigment pool sizes and pigment stoichiometry was collected in August 1994 and
July–August 1995. Leaf discs of 1.03 cm2 were punched from
the leaves between 1200–1400 h, put in labeled vials and immersed in liquid nitrogen. Pigments were solubilized in aqueous acetone, separated by HPLC with a reverse-phase column,
and detected at 440 nm as described previously (Niinemets et
al. 1998a). Data for xanthophyll cycle carotenoids (violaxanthin, antheraxanthin and zeaxanthin) as well as for total carotenoids, total chlorophyll (Chl) and Chl a/b ratio have been
reported previously (Niinemets et al. 1998a, 1999a). In addition to Chl a and b, two dephytylated chlorophyll intermediates were consistently detected in chromatograms. We deduced that these Chl intermediates were missing a phytol
residue because they were the first, i.e., the most polar pigments to resolve in the chromatograms (retention times
2.2 and 2.8 min). The intact molecules of Chl (retention times
16.2 min for Chl b and 16.6 min for Chl a) were more apolar
Figure 2. Dependencies of the capacity for photosynthetic electron
transport (Jmax; A) and initial quantum yield of photosynthetic electron transport (α; B) on the mean seasonal daily integrated quantum
flux density (Qs) in P. tremula (䊏) and T. cordata (䊊). Values of Jmax
and α were obtained from ΦPSII versus incident quantum flux density
relationships (Equation 3; see Figure 1 for sample curves). Data were
measured in the morning (0800–1130 h) during August 23–24, 1995,
and Qs was calculated as the mean for the period between June 3 and
July 21, 1995. Lines and regression statistics depicted are for linear
least squares fits.
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NIINEMETS AND KULL
content per unit area (data not shown, see Niinemets et al.
1998b, 1999a). In contrast, the initial quantum yield of photosynthetic electron transport (α), which is also directly linked
to the light-adapted maximum quantum yield of PSII according to α = 0.5ΘΦPSII at Q = 0 (Equation 3), declined with increasing Q s in both species (Figure 2B). The slope of the α
versus Q s relationship (Figure 2B) was more negative in
T. cordata than in P. tremula (separate-slope ANCOVA, P <
0.001), indicating greater down-regulation of maximum lightadapted quantum yield of PSII in T. cordata than in P. tremula.
At night, in fully relaxed leaf samples, the intrinsic efficiency of PSII (Fv /Fm) was slightly higher in P. tremula (mean
± SD = 0.825 ± 0.018) than in T. cordata (0.813 ± 0.008, P <
0.05 according to a separate sample t-test) because of moderately larger Fo values in T. cordata (P < 0.001). In contrast,
daytime Fv /Fm measured after a 10-min dark adaptation did
not differ significantly between the species (0.806 ± 0.006 in
T. cordata and 0.807 ± 0.020 in P. tremula, P > 0.8). In both
species, mean daytime Fv /Fm values were significantly lower
than mean nighttime Fv /Fm values (P < 0.001 according to
paired sample t-tests).
Because there was no evidence of sustained down-regulation of Fv /Fm with integrated irradiance at night (data not
shown), the daily decrease in Fv /Fm was standardized with respect to the nighttime measurements. The relative decline in
Fv /Fm, (Fv /Fmday – Fv /Fmnight )/Fv /Fmnight, was negatively related
to integrated irradiance (Figure 3), whereas the correlation
with Q s (Figure 3B) was significant in both species. However,
the decline in Fv /Fm with daily light receipt before leaf sampling (Figure 3A) was only significant in T. cordata.
For all data pooled, Fv′/Fm′ was strongly (P < 0.001 for linear
relationships) and negatively correlated with both qN (r 2 =
0.90) and NPQ (r 2 = 0.76), which supports the view that 1 –
Fv′/Fm′ is an estimate of thermal energy dissipation in the antennae of PSII (Demmig-Adams et al. 1996). Because qN,
NPQ and 1 – Fv′/Fm′ measure mainly the same phenomenon of
thermal energy dissipation, light relationships of qN and NPQ
mirrored those of Fv′/Fm′ (e.g., Figure 4D). The ratios of qP to
various estimates of non-photochemical fluorescence quenching (NPQ, qN, 1 – Fv′/Fm′) were positively related to Qs in both
species (data not shown), indicating that maintenance of a
greater fraction of open PSII centers was dominated by increases in foliar photosynthetic capacity rather than by enhancements in non-radiative energy dissipation capacity.
At an incident Q of 865 µmol m –2 s –1, the fraction of excess
light (PE, 1 – the fractions of light dissipated photochemically
and as heat) was lower in P. tremula (mean ± SD = 0.23 ±
0.05) than in T. cordata (0.41 ± 0.09), primarily because
high-light acclimated leaves of P. tremula had more open PSII
centers than low-light acclimated leaves of T. cordata. When
all data were pooled, qP varied more than fivefold, but Fv′/Fm′
varied by only 50%. This suggests that maintenance of high qP
through increases in photosynthetic capacity is a more important route to reducing potentially damaging excess irradiance
at a given incident Q than increases in thermal energy dissipation. This conclusion is further supported by the finding of
much stronger negative relationships between PE and qP (r 2 =
0.98 for all data pooled) than between PE and 1 – Fv′/Fm′ (r 2 =
0.72).
Photochemical and non-photochemical quenching of
chlorophyll fluorescence
The electron transport rate versus light relationship varied diurnally. When ΦPSII measured during the day was plotted
against incident quantum flux density, ΦPSII was greater at a
common Q in the morning than in the afternoon (Figures 5A
and 5B). Although there was some indication of a decline in
ΦPSII values under moderate and high incident quantum flux
densities, the differences in ΦPSII were more significant at low
Q than at light saturation (Figures 5A and 5B). Nevertheless,
comparison of a larger set of light-saturated values of J (Jsat)
measured in morning and afternoon indicated that Jsat declined
Quenching analysis of chlorophyll fluorescence indicated that
the greater yield of PSII electron transport in high light than in
low light in P. tremula (Figures 2A and 4A) was achieved by
maintenance of PSII centers in a more open state (higher q P,
Figure 4B). The second determinant of ΦPSII, Fv′/Fm′ (Figure 4C), was negatively related to Q s in T. cordata, but not in
P. tremula, indicating that maintenance of high qP relied less
on down-regulation of PSII center efficiency in P. tremula.
Diurnal variability in effective quantum yield of PSII and
photosynthetic electron transport
Figure 3. Relative difference in darkadapted quantum yield of PSII (Fv /Fm,
where Fv = Fm – Fo) between day and night
(Fv /Fmday – Fv /Fmnight)/Fv / Fmnight) in relation to incident daily quantum flux density
integrated until the measurements were
made (A) and in relation to Qs (B) in P. tremula (䊏) and T. cordata (䊊). Nighttime
measurements of Fv /Fm were used as the
reference values for leaves with a fully relaxed fast component of non-photochemical
quenching (Krause and Weiss 1991, Krause
and Winter 1996, Thiele et al. 1998). Data
were fitted by linear regressions.
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PHOTOINHIBITORY DECLINE IN QUANTUM YIELDS IN NATURAL CANOPIES
Figure 4. Influences of Qs on effective quantum yield of PSII (A),
photochemical quenching coefficient (B), quantum yield of open PSII
centers in the light (C) and on non-photochemical quenching (D) in
P. tremula (䊏) and T. cordata (䊊). Fluorescence parameters measured were: Fm′ and Fo′ (maximum and minimum fluorescence
yields, respectively, of a light-adapted sample), Fm and Fo (maximum
and minimum fluorescence yields, respectively, of a dark-adapted
sample), and ∆F = Fm′ – Fs, where Fs is the steady-state fluorescence
yield in the light. Each sample leaf was exposed to an incident quantum flux density of 865 µmol m –2 s –1 until steady-state values of
ΦPSII were observed (cf. Figure 1), and Fo′ values were measured immediately after darkening the sample. Data were fitted by linear regressions.
in the afternoon relative to the morning in P. tremula (P < 0.05
according to a common slope ANCOVA), but not in T. cordata (Figure 5C).
The photochemical quenching coefficient (qP) was lower,
905
Figure 5. Diurnal declines in initial quantum yields and capacities for
photosynthetic electron transport. (A, B) Comparison of the relationships between Q and ΦPSII measured in the morning (0445–1100 h,
䊉) and during the rest of the day (1100–2200 h, 䊊) for the same
leaves as in Figure 6. (C) Comparison of the light-saturated rates of
photosynthetic electron transport (Equation 1) versus Qs measured
during the morning (0800–1030 h in P. tremula, 䊐; 1030–1130 h in
T. cordata, 䊊) and afternoon (1630–1800 h) in P. tremula (䊏) and
T. cordata (䊉) in August 1995. In C, the steady-state rates of
photosynthetic electron transport were measured at close to saturating
Q values of 1800 µmol m –2 s –1 in P. tremula and 600 µmol m –2 s –1 in
T. cordata (Figure 1), and were standardized to 25 °C based on the relationships between Jmax and temperature given in Niinemets et al.
(1999a). Because the morning and afternoon relationships of Jmax
versus Qs (C) were not statistically different in T. cordata according
to a co-variation analysis (P > 0.6), a single linear regression line was
fitted to all data in this species.
and the non-photochemical quenching coefficients (e.g.,
NPQ) were greater at a common Q in the afternoon than in the
morning (Figures 6B and 6F). Some PSII centers remained
closed, and leaves retained a certain degree of non-photochemical quenching even after several hours of darkness in
both species (Figures 6B and 6F). Although T. cordata leaves
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Figure 6. Diurnal variability in
(A, E) incident quantum flux
density (Q) and leaf temperature (TL), and in (B–D, F–H)
chlorophyll fluorescence characteristics in a P. tremula leaf
sampled at 23 m (A–D) and in
a T. cordata leaf sampled at
16.5 m (E–H). The photochemical quenching coefficient was calculated as in Figure 4B, and NPQ as in Figure
4D. The fractions of light (C,
G) dissipated, used in photochemistry, or in excess (1 –
fractions of light dissipated
non-radiatively and used in
photochemistry) were computed as described in
Demmig-Adams et al. (1996),
and the photosynthetic electron transport rate, J, was calculated by Equation 1. The
horizontal lines in C and G denote inherent inefficiency of
excitation energy transfer for
leaves with no sustained nonphotochemical energy dissipation (Adams and Barker 1998).
This was taken equal to the
predawn value of 1 – Fv /Fm
for each leaf. Photosynthetic
electron transport rate was
modeled using ΦPSII measured
in the morning (0445–1100 h)
to parameterize Equation 3
(see Figures 5A and 5B), and
calculating J for each value of
Q by Equation 2. The inset in
panel F shows the afternoon
decreases in photosynthetic
electron transport rate at a
higher resolution. Integrated
daily quantum flux densities in
panels A and E and electron
transport rates in panels D and
H are also depicted.
were generally exposed to lower quantum flux densities than
P. tremula leaves (Figures 2, 6A and 6E), the afternoon decreases in qP and increases in NPQ were more significant in
T. cordata than in P. tremula (cf. Figures 6B and 6F). Diurnal
patterns of the different components of ΦPSII (Equation 4), qP
and Fv′/Fm′ (data not shown) were similar. However, the range
in diurnal variability in qP (15–270%; mean = 150%) for different leaves was always larger than the range in diurnal variability in Fv′/Fm′ (5–50%; mean = 30%). Accordingly, daily
differences in qP were a more important determinant of diurnal
changes in ΦPSII than down-regulation of quantum yields of
open PSII centers, Fv′/Fm′.
Although Oxborough and Baker (1997) concluded that, because Fo′ must be measured very quickly, their computed estimate of Fo′ was more accurate than direct estimations, we note
that the circumstance that Fo′ is computed (Equation 5), rather
than measured, may affect the comparisons between the
leaves. Moderate daily down-regulation of Fo′ predicted by
Equation 5 may have led to underestimation of daily variability in Fv′/Fm′ and overestimation of daily declines in qP if Fo′
did not vary or increased during the day. However, predawn Fo
values and those measured in the evening were not significantly different (data not shown). Moreover, calculation of the
chlorophyll fluorescence variables for a constant Fo′ only
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PHOTOINHIBITORY DECLINE IN QUANTUM YIELDS IN NATURAL CANOPIES
907
moderately altered the range of variation in Fv′/Fm′ and q P, indicating that the observed differences in the magnitudes of
variation in these fluorescence parameters are authentic.
Based on the chlorophyll fluorescence analysis, all leaves
intercepted light in excess (PE, Figures 6C and 6G). The excess fraction increased during the day at the expense of the
fraction of light used photochemically. The fraction of light
dissipated non-photochemically (P D) also increased during
the day, but its increase was not sufficient to prevent a rise in
PE (Figures 6C and 6G).
Relative decline in potential daily photosynthetic electron
transport
Decreases in Φ PSII at a common Q during the day brought
about lower J values than predicted from constant ΦPSII versus
Q relationships (Figures 6D and 6H). Photosynthetic electron
transport rates primarily decreased at low quantum flux densities in the afternoon, but in both species there was evidence of
decreased values of J at midday at moderate and high Q (Figures 6D and 6H).
Despite the daily decreases in J, a strong linear relationship
between daily integrated quantum flux density (Qd) and daily
integrated photosynthetic electron transport rate (Jint) was observed when the data for both species were pooled (Figure 7A). Although few data were available, the relative daily
decline in J int calculated as ∆ = (J intP – J intA)/J intP, where superscripts P and A denote potential and actual electron transport
rates, respectively, tended to increase with Q d in both species,
whereas ∆ was greater at a common Q in T. cordata than in
P. tremula (Figure 7B). This difference was accompanied by a
higher P E and lower P P in T. cordata than in P. tremula (Figure 7C). Furthermore, ∆ decreased as P P increased (Figure 8A), and increased with P E (Figure 8B). Although the
correlation between P D and ∆ was insignificant (r 2 = 0.09, P >
0.9 for both species pooled), possibly because of low variability in PD (0.191–0.255), the outlying value of P. tremula in the
∆ versus PE plot (Figure 8B) was characterized by the highest
value of PD.
Variability in the content of chlorophyll intermediates with
integrated Q
Concentrations of chlorophyll intermediates, which primarily
consisted of chlorophyllides a and b (Chlid), increased with
increasing integrated quantum flux density, whereas the correlations were somewhat stronger with Q integrated over the
3 days preceding foliar sampling (Figure 9A, mean r 2 = 0.54)
than with Q integrated over the season (mean r 2 = 0.42). All
data sampled between July 25 and August 24, 1995 fit a single
linear regression line in P. tremula, but the slopes were generally (except for August 1994 data) steeper for T. cordata than
for P. tremula (separate slope ANCOVA, P < 0.001), and also
varied with sampling date in T. cordata (Figure 9A). In
T. cordata, the slopes were steeper during both measurement
periods in 1995 than in 1994, possibly because of a greater excess Q at a common integrated Q in 1995 than in 1994. In
1994, the mean percentage of direct light with high intensities
Figure 7. Correlations of Q integrated over the measurement day with
(A) daily integrated actual (JintA; measured values) and potential
(JintP; simulated using the morning data, see Figures 6D and 6H)
photosynthetic electron transport rates, with (B) relative photoinhibitory decline in J [∆ = (JintP – JintA)/JintP], and with (C) the fractions of excess light, dissipated light and light used in photochemistry. Data for both species were fitted by the same linear regression lines in panel A (regressions with both actual and potential
electron transport rates were significant at P < 0.001); separate linear
regressions were used in other panels. Photoinhibitory decline in photosynthetic efficiency refers to decreases in measured values of J relative to modeled rates at midday and in the afternoon (Figures 5, 6D
and 6H). Daily integrated Q was measured for each leaf by the quantum sensor of the PAM-2000 in 2-min steps.
was 17% of total integrated Q, whereas 52% of light was direct
during the 3 days preceeding foliar sampling on July 19–August 2 and 46% during August 11–12, 1995. The slope was
also larger for the period (August 11–12) with severe foliar
water stress (see Niinemets et al. 1999d ) than for July 19–August 2, 1995 (separate slope ANCOVA, P < 0.05). The percentage of direct light (64%) for P. tremula was larger than
that for T. cordata, but P. tremula had lower concentrations of
Chl intermediates, possibly because of a lower fraction of ex-
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Figure 8. Relative photoinhibitory decline in J during the day (see Figure
7B) in relation to the fraction of light
used in photochemistry (A) and the
fraction in excess (B). Linear regression lines are fitted through the data for
T. cordata.
cess light at a common integrated Q (Figure 8C).
The mean Chl a/b ratio was 3.24 ± 0.31 in P. tremula and
2.82 ± 0.16 in T. cordata, whereas the Chlid a/b ratios were 6.0
± 2.4 in P. tremula and 6.3 ± 1.8 in T. cordata, indicating enrichment in Chlid a. Corelations between total Chl a + b and
Chlid were poor (r 2 = 0.01–0.21) and not significant. Relationships between integrated Q and Chlid:Chl ratios were
qualitatively similar to those with Chlid (cf. Figures 9A
and 9B), indicating that variability in Q and not in total Chl
Figure 9. Total amount of chlorophyll intermediates (A) and the ratio
of chlorophyll intermediates to total chlorophyll (B) versus daily
irradiance integrated over the 3 days preceding foliar sampling in
P. tremula (䊏) and T. cordata. All data sampled between July 25 and
August 24, 1995 are fitted by a single linear regression line in
P. tremula. In T. cordata, separate lines are used to fit the data sampled during August 10–24, 1994 (䉭), July 19–August 2, 1995 (䊊)
and August 11–17, 1995 (䊉). The pool of chlorophyll intermediates
consisted primarily of chlorophyllides a and b.
concentration (cf. Niinemets et al. 1999a) was responsible for
the observed patterns.
Discussion
Scaling of quantum yields of PSII with integrated light
Exposure of leaves to moderately elevated Q generally results
in decreased J under low and intermediate Q (Ögren and
Sjöström 1990, Demmig-Adams and Adams 1993, Ögren
1994, Russell et al. 1995; see review by Long et al. 1994) with
little effect on light-saturated values of J (Jmax). Severe light
stress may lead to decreases in Jmax (Krause et al. 1985, Rosenqvist et al. 1991, Russell et al. 1995). Although Jmax was higher
in leaves exposed to greater average seasonal integrated quantum flux densities (Qs, Figure 2A), these leaves possessed a
lower α (Equation 3, Figure 2B), indicating a greater potential
reduction in J at low and intermediate Q in leaves intercepting
more light. The variability in α with Qs was mirrored by similar daily decreases in Fv /Fm, which characterizes the intrinsic
efficiency of PSII in the dark-adapted state (Figure 3). The
greater decline in PSII efficiency in leaves growing in high Qs
than in leaves growing in low Qs corroborates other field
observations of variability in PSII quantum yields in natural irradiances (Mulkey and Pearcy 1992, Brugnoli et al. 1994, He
et al. 1996, Krause and Winter 1996, García-Plazaola et al.
1997, Valladares and Pearcy 1997).
Leaves exposed to high light in the field may exhibit sustained decreases in PSII efficiency, possibly reflecting chronic
photoinhibition resulting from non-matching rates of photodamage and repair (Greer and Laing 1988, Park et al. 1995b).
Under saturating light conditions, whole-chain linear electron
transport is limited by the intermediate steps between PSII and
PSI (Anderson 1992), so even a complete deactivation of a
certain fraction of PSII centers may not alter Jmax (e.g., Öquist
et al. 1992), because the feedback control on the remaining active PSII centers may be relieved (Foyer et al. 1990). Thus, observed decreases in α (Figure 2B) may provide evidence of
chronic photoinhibition in high light. However, declines in
PSII electron transport in low irradiance without concomitant
changes in Jmax are also compatible with regulation of PSII efficiency through increased energy dissipation in the antennae
of PSII. We did not observe any sustained decrease in Fv /Fm at
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PHOTOINHIBITORY DECLINE IN QUANTUM YIELDS IN NATURAL CANOPIES
night, so enhanced non-radiative energy dissipation at PSII
antennae may also be responsible for the negative relationship
between α and integrated irradiance.
Acclimation of leaves to high light: chlorophyll fluorescence
quenching analysis
Although the rate of PSII photodamage is roughly proportional to absorbed quantum flux density (Park et al. 1995a,
Tyystjärvi and Aro 1996, Baroli and Melis 1998), the probability of photodamage is significantly increased when the PSII
centers are closed, i.e., the primary quinone acceptor, QA, remains reduced during illumination, blocking forward electron
transport through PSII (Park et al. 1996a, Baroli and Melis
1998). Consequently, avoidance of excessive energization of
thylakoids by decreases in the reduction state of PSII centers
provides a major route for reducing the probability of photodamage (Park et al. 1996a, Baroli and Melis 1998). When estimated at a common Q, high-light acclimated leaves consistently had higher qP than low-light acclimated leaves. Thus,
acclimation to high light led to a greater fraction of open PSII
centers (Figure 4A; Demmig-Adams et al. 1996, 1998, Brugnoli et al. 1998, Špunda et al. 1998).
One way to maintain higher PSII center openness is through
increases in excitation energy dissipation as heat. In general,
light-saturated values of non-photochemical fluorescence
quenching is greater in high-light acclimated leaves than in
low-light aclimated leaves (Figures 4C and 4D; Brugnoli et al.
1994, 1998, Demmig-Adams et al. 1996, Špunda et al. 1998,
but see Demmig-Adams et al. 1998). Furthermore, pool size of
xanthophyll cycle carotenoids (violaxanthin, zeaxanthin and
antheraxanthin, VAZ) also scales positively with integrated
quantum flux density in the canopy (Thayer and Björkman
1990, Brugnoli et al. 1994, Königer et al. 1995, Logan et al.
1996), which is in accordance with the hypothesis that the
xanthophyll cycle participates in non-radiative energy dissipation. However, at the same degree of non-photochemical
quenching, sun-exposed leaves tend to possess larger values
of qP than shade-grown leaves (Špunda et al. 1998). Despite
increases of more than fourfold for VAZ to total chlorophyll
and twofold for VAZ to total carotenoids in response to integrated daily Q in the studied species (Niinemets et al. 1998a,
1999a), the ratios of qP to descriptors of non-photochemical
fluorescence quenching (NPQ, qN, 1 – Fv′/Fm′) were positively
related to Q s in both species. Thus, we conclude that enhancement of leaf photosynthetic capacity played a more important
role in maintenance of open PSII centers than increases in capacity for non-radiative energy dissipation.
We studied both photochemical and non-photochemical
fluorescence quenching at a moderately high quantum flux
density of 865 µmol m –2 s –1, which was saturating for T. cordata but not always saturating for P. tremula. Because of more
open reaction centers and a lower requirement for non-photochemical energy quenching, the highest values of NPQ are observed at irradiances higher than full sunlight in high-light
adapted leaves (Brugnoli et al. 1994, 1998, Špunda et al.
1998). In addition, non-photochemical quenching increases
909
faster with Q in shade-adapted leaves, and may exceed that in
sun leaves at lower Q (Brugnoli et al. 1994, 1998, Špunda et al.
1998). This suggests that we somewhat underestimated the capacity for non-radiative energy dissipation in P. tremula (Figures 4C and 4D). Nevertheless, diurnal measurements of chlorophyll fluorescence quenching components (Figure 6)
corroborated the conclusion that the variability in photosynthetic capacity played a primary role in the variation of the reduction state of PSII at the actual quantum flux densities encountered in the canopy.
Diurnal variability of PSII quantum yields
Because PSII electron transport becomes saturated with incoming quantum flux density, ΦPSII (Equation 1) also varies in
response to Q (Figure 1A). Daily temperature- and Q-related
variation in ΦPSII mainly resulted from variation in the openness of PSII centers, characterized by qP (Figures 6B and 6F),
rather than from changes in the quantum yield of excitation energy capture by open PSII centers, Fv′/Fm′. It is known that qP
varies diurnally (Björkman and Demmig-Adams 1994,
Valentini et al. 1995, Demmig-Adams et al. 1996, He et al.
1996, de Mattos et al. 1997, Adams and Barker 1998). That is,
leaves are not able to maintain the same degree of PSII center
openness in high light, even though Fv′/Fm′ may also decrease
at high irradiances, allowing a greater fraction of light to be
dissipated non-radiatively (Demmig-Adams et al. 1996, Adams and Barker 1998). Calculations based on chlorophyll fluorescence data show that, despite enhancement of non-radiative energy dissipation, leaves are frequently exposed to a certain fraction of potentially damaging excess light in the field
(Demmig-Adams et al. 1996, Adams and Barker 1998; Figures 6C and 6G).
Often, ΦPSII (Figure 5, Björkman and Demmig-Adams
1994, Bilger et al. 1995, Valentini et al. 1995, Chaumont et al.
1997, de Mattos et al. 1997), Fv /Fm (Ögren and Sjöström 1990,
Epron et al. 1992, Damesin and Rambal 1995, Maxwell, C. et
al. 1995, Krause and Winter 1996, Chaumont et al. 1997,
García-Plazaola et al. 1997, Thiele et al. 1998) and q P
(Björkman and Demmig-Adams 1994, de Mattos et al. 1997,
Figures 6B and 6F) are lower, and non-photochemical
quenching coefficients are higher (de Mattos et al. 1997,
García-Plazaola et al. 1997, Figures 6B and 6F) in the afternoon than in the morning under similar environmental conditions. Although few studies have examined daily variability in
qP, a strong positive correlation between qP and Fv /Fm sampled
over the day has been found (He et al. 1996), suggesting that
lower values of qP in the afternoon relative to the morning may
be general. Decreases in PSII efficiency may be interpreted in
terms of sustained non-radiative excitation energy quenching
or photodamage in PSII, or both. Because the de-epoxidation
state of the xanthophyll cycle carotenoids does not relax as
quickly as light fluctuates, high amounts of zeaxanthin and
antheraxanthin may be sustained in low light during the day
(Königer et al. 1995, Logan et al. 1997, Thiele et al. 1998), and
may provide an explanation for the observed non-photochemical quenching (Figures 6B and 6F). However, several other
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mechanisms may result in maintenance of non-photochemical
quenching, including photodamage to PSII (Krause and Weiss
1991, Ruban and Horton 1995, Pospíšil 1997).
Because the probability for PSII photodamage increases as
soon as qP decreases, negative scaling of the fraction of integrated PSII electron transport rate lost because of photoinhibition (∆) with the fraction of light used in photochemistry
(and with mean daily PSII center openness; Figure 8A), as
well as sustained reduction in qP during the day (Figures 6B
and 6F), support the idea that photodamage of PSII is involved
in the observed loss of PSII efficiency (Figures 5, 6D and 6H).
Furthermore, the data in Figure 8A are in accordance with the
concept that maintenance of high qP is quintessential in protecting leaves from photoinhibition (Ögren 1991, Ögren and
Rosenqvist 1992, Maxwell, D.P. et al. 1995, Baroli and Melis
1998, Huner et al. 1998). Although excitation energy may be
dissipated non-radiatively, the fraction of potentially damaging excess light was greater in leaves with lower qP in our
study (cf. Figures 8A and 8B). The increase in non-radiative
energy dissipation with increasing integrated Q was sufficient
to maintain the fraction of light dissipated at about 0.25 (Figure 7C) and did not avoid excessive thylakoid reduction at
high irradiance.
Species differences in sensitivity to photoinhibition: possible
interaction between water stress and light
Although Fv /Fm declined in a similar manner in both species
(Figure 3), and both species exhibited a similar intrinsic
photosynthetic capacity (Figure 4), lower Fv /Fm at night,
greater decreases in α (Figure 2B) and JintP (Figure 7B), and
larger increases in excess light (Figure 7C) with integrated
quantum flux density in T. cordata than in P. tremula indicate
that T. cordata was more sensitive to photoinhibition. Previously, Niinemets et al. (1999d) showed that stomatal conductance decreased relatively more with developing soil water
limitations in T. cordata than in P. tremula, resulting in a
lower, long-term, mean intercellular CO2 concentration (Ci) in
T. cordata (Niinemets et al. 1999b). Although moderate water
stress has no influence on dark-adapted PSII photochemistry
per se (Epron and Dreyer 1992, 1993, Lu and Zhang 1998), it
increases leaf susceptibility to photoinhibition (Lu and Zhang
1998). At a common irradiance, declines in PSII efficiency are
often disproportionately larger in water-stressed plants than in
non-stressed plants (Björkman et al. 1981, Björkman and
Powles 1984, Ludlow and Björkman 1984, Ögren and Öquist
1985, but see Flexas et al. 1998). Diurnal suppression of PSII
activity in response to daily variability in Q in the field is also
enhanced in water-limited leaves (Epron et al. 1992, Damesin
and Rambal 1995, Maxwell, C. et al. 1995, Valentini et al.
1995, García-Plazaola et al. 1997, Valladares and Pearcy
1997).
Stomatal closure is the primary response to drought stress
(Schulze 1986, Cornic 1994). When stomata close and Ci decreases, electron flow to alternative electron sinks, including
photorespiration and the Mehler peroxidase pathway, is enhanced and may protect PSII from damage (Osmond and
Grace 1995, Valentini et al. 1995, Lal et al. 1996, Lovelock
and Winter 1996, Park et al. 1996b, Flexas et al. 1998). However, the alternative electron transport processes are nonspecific, and can not fully substitute for the lack of CO2, leading to
increases in the reduction state of primary quinone acceptor
QA with decreasing Ci (Dietz et al. 1985, Sharkey et al. 1988,
Lovelock and Winter 1996, Park et al. 1996b). An increased
reduction state of PSII may predispose the leaves to damage
(Figure 8A). Thus, we conclude that the larger decline in PSII
quantum yield during the day in T. cordata than in P. tremula
(Figure 7B) is caused by an increased reduction state of PSII,
because of increased stomatal closure in T. cordata.
Significance of photoinhibition of photosynthetic electron
transport for carbon gain
Upper canopy leaves of Salix lost about 6.3–12.6% of potential carbon gain during the day because of photoinhibition,
whereas the exact percentage was dependent on photon exposure (Ögren and Sjöström 1990). A similar estimate (9%) was
reported by Long et al. (1994) for hypothetical crop canopies.
However, in both model calculations, no account was taken of
possible effects of stomatal closure on net carbon gain. Therefore, both Ögren and Sjöström (1990) and Long et al. (1994)
predicted high potential photoinhibitory loss of photosynthesis at midday. However, at current ambient [CO2],
photosynthesis is limited by Ci at light saturation rather than
by electron transport rate (see Farquhar et al. 1980), and decreases in stomatal conductance rather than in J are the primary reason for decreases in net carbon assimilation rates at
midday when Q is high (Damesin and Rambal 1995, Valentini
et al. 1995, Lal et al. 1996, Chaumont et al. 1997, Flexas et al.
1998). Although we obtained some evidence of lower Jsat in
the afternoon than in the morning (Figures 5C and 6D), we believe that this reduction would have negligible effects on carbon gain at high Q. We postulate that the observed photoinhibitory decline in J is more important for photosynthesis at
low Q, where foliar carbon uptake is less sensitive to stomatal
conductance and photosynthesis is primarily limited by light
availability. Despite increases in ∆ with increasing daily integrated Q (Figure 7B), we conclude that photoinhibition will
affect carbon gain more at low Q s, where photosynthesis is
limited by light over longer periods during the day, and efficient light harvesting and utilization is critical for survival.
We were unable to measure diurnal variability in Chl fluorescence characteristics in the uppermost leaves in P. tremula.
Nevertheless, indirect observations—variation patterns in
Fv /Fm (Figure 3), data on fluorescence quenching (Figure 4)
and the study of Chl intermediates (Figure 9)—suggest that
photoinhibitory decline in J at high irradiances would not deviate much from the line derived from measurements at intermediate irradiances (Figure 7B).
Evidence of scaling of chlorophyll turnover rates with excess
light
Because Chl intermediates are not able to transfer light to
other Chl molecules, they are potentially toxic compounds
with a high capacity to produce reactive singlet oxygen. To
avoid extensive damage to thylakoids, Chl intermediates must
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be either degraded or included in functionally active Chl–protein complexes in chloroplasts (Matile et al. 1996). We were
unable to determine whether the chlorophyllides in leaves are
Chl breakdown products or precursors of Chl synthesis, because Chlids are the intermediate products of both Chl dephytylation, which is the first step of Chl degradation (Matile et al.
1996, Bertrand and Schoefs 1999), and phytylation of Chlid,
which leads to functionally active Chl species (Castelfranco
and Beale 1983, Rudiger 1993). A higher Chlid a/b ratio than
Chl a/b ratio may be indicative of enhanced photodestruction
of Chl, because Chl b, which transfers excitation energy to
Chl a, is less sensitive to photo-oxidation (Bertrand and
Schoefs 1999). However, Chlid a (or Chl a) is also the precursor of Chlid b (Chl b; Castelfranco and Beale 1983), and consequently, this observation is also compatible with an enhanced rate of foliar Chl synthesis.
Because all Chl intermediates are prone to photodestruction, measured foliar concentrations of Chlid—up to 2.5% of
total Chl (Figure 9B)—were high. Chlorophyllide molecules
are light stable in the complex with NADPH:protochlorophyllide oxidoreductase (Franck et al. 1995, Schoefs and
Bertrand 1997). This information, plus the finding that only
Chlid a and b, but not other Chl precursors, are present in
leaves, suggest that high Chlid concentrations reflect high
rates of Chl synthesis. Because total foliar Chl concentration
per unit area was stable during the measurement period, despite large daily and seasonal differences in light and water
availabilities (Niinemets et al. 1999a), a significant concentration of Chlid may also be indicative of rapid foliar Chl turnover. Recent estimates of the half-life for foliar Chl turnover
vary from 16.4 to 108 h (see review by Bertrand and Schoefs
1999).
Higher Chlid concentration in T. cordata leaves on days
with high fractions of direct irradiance and high water stress
(Figure 9) supports the hypothesis that the Chl turnover rate is
adjusted to maintain a balance between damage and repair,
and suggests that Chl turnover may be controlled by excess
irradiance. The sensitivity of Chl to photo-oxidation does not
necessarily scale with absolute light flux, because the threshold irradiance for damage may be determined by other environmental factors (Bertrand and Schoefs 1999) that alter the
amount of excess irradiance at a common Q.
Conclusions
Although leaves acclimate to the gradient of excitation pressures on PSII along canopy light gradients by increases in
photosynthetic capacity, capacity for non-radiative energy
dissipation and possibly also capacity for repair, acclimation
is not complete in highly variable field environments. We
found that photoinhibitory decreases in foliage photosynthetic
efficiency are common in temperate forest canopies, but that
the influence on daily integrated photosynthetic electron
transport rate is moderate unless high light stress interacts with
other stress factors. Current analysis suggests that plants reduce the risk of photoinhibition primarily by maintaining an
911
appropriate level of thylakoid energization. However, because
stomatal closure, through decreases in intercellular CO2 concentration, reduces the requirement for assimilatory electron
flow, and may lead to a greater reduction state of PSII centers,
avoidance of an imbalance between excitation energy absorption and utilization may be constrained in field conditions. Because of the inherent inverse relationship between water and
light availabilities in temperate forest canopies (Niinemets et
al. 1999d ), maintenance of high openness of PSII centers may
become increasingly difficult at greater heights in the canopy,
especially in species with stomata that are highly sensitive to
water stress.
Acknowledgments
We thank Dr. Wolfgang Bilger (Department of Biology and Nature
Conservation, Agricultural University of Norway, Ås, Norway) for
the pigment analyses, and Urmas Kalla and Eve Niinemets for their
technical contributions. The study was conducted under grants from
the Estonian Science Foundation (Grants 2048, 4584), The German
Academic Exchange Service (DAAD) and from the German Federal
Minister of Research and Technology (BMBF, EST 001-98).
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