Tree Physiology 28, 1863–1871

© 2008 Heron Publishing—Victoria, Canada

Aluminum-induced effects on Photosystem II photochemistry in Citrus

leaves assessed by the chlorophyll a fluorescence transient
HUAN-XIN JIANG,1,2 LI-SONG CHEN,1,3,4 JIN-GUI ZHENG,5 SHUANG HAN,1,3 NING
TANG1,3 and BRANDON R. SMITH6
1

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Institute of Horticultural Plant Physiology, Biochemistry and Molecular Biology, Fujian Agriculture and Forestry University, Fuzhou 350002,
P.R. China
2
College of Life Science, Fujian Agriculture and Forestry University, Fuzhou 350002, P.R China
3
College of Horticulture, Fujian Agriculture and Forestry University, Fuzhou, 350002, P.R. China
4
Corresponding author (lisongchen2002@hotmail.com)
5
Biotechnology Center, Fujian Agriculture and Forestry University, Fuzhou, 350002, P.R. China
6
Organic and Alternative Crop Production, Department of Plant Sciences, University of Tennessee, 2431 Joe Johnson Drive, Knoxville, TN 37996,
USA

Received May 26, 2008; accepted July 8, 2008; published online October 1, 2008

Summary Seedlings of Citrus grandis (L.) Osbeck cv. Introduction

Tuyou were irrigated daily for 5 months with nutrient solution In acidic soils, aluminum (Al) occurs in soluble forms that are
containing 0 (control), 0.2, 0.6 or 1.6 mM aluminum (Al) from phytotoxic, whereas in mildly acidic or neutral soils, it is
AlCl3·6H2O. Shoot growth was more sensitive to Al toxicity largely insoluble and biologically inactive. In many acid soils
than root growth, gas exchange, chlorophyll (Chl) concentra-
throughout the tropics and subtropics, Al toxicity is a major
tion, polyphasic Chl a fluorescence (OJIP) induction and re-
factor limiting crop productivity. Citrus spp. are evergreen
lated parameters. Leaves of Al-treated plants showed de-
subtropical fruit trees that show poor growth and a shortened
creased CO2 assimilation and Chl concentration, yet inter-
lifespan when grown in soil with a low pH and a high Al con-
cellular CO2 concentration increased and ribulose-1,5-bis-
centration (Lin and Myhre 1990).
phosphate carboxylase/oxygenase (Rubisco) activity was
Earlier studies have shown that Al decreases CO2 assimila-
unchanged. Chlorophyll a fluorescence induction analysis of
tion in many plant species including Citrus spp. (Pereira et al.
Al-stressed leaves showed a large rise at the O-step and a large
2000, Chen et al. 2005a, 2005b), longan (Dimocarpus longan
depression at the P-step, accompanied by two new bands at
Lour.; Xiao 2002), Thinopyrum bessarabicum Savul & Rayss
300 µs (K-band) and at about 150 µs (L-band). Maximum fluo-
(Moustakas et al. 1996), sorghum (Sorghum bicolor L.
rescence, maximum quantum yield of primary photochem-
Moench; Peixoto et al. 2002), tomato (Lycopersicon escu-
istry, oxygen-evolving complex (OEC), quantum yield of elec-
lentum Mill.; Simon et al. 1994), and maize (Zea mays L.;
tron transport, quantum yield of electron transport from QA– to
Lidon et al. 1999). Because Photosystem II (PSII) is the com-
the Photosystem I end electron acceptors, IP phase and total
ponent of the photosynthetic apparatus that is most sensitive to
performance index were decreased in leaves of Al-treated
environmental stress, it is considered to play a key role in the
plants, whereas minimum fluorescence, relative variable fluo-
response of photosynthesis to environmental perturbations.
rescence at the J-step and I-step, and dissipated energy were in-
Peixoto et al. (2002) associated the Al-induced decrease in
creased. We propose that impaired electron transport capacity
photosynthesis with impaired PSII photochemistry. Pereira et
accompanied by lack of reducing equivalents were the main
al. (2000) observed that Al induced a decrease in the ratio of
factors contributing to decreased CO2 assimilation in Al-
maximum variable fluorescence (Fv ) to minimum fluores-
treated plants. Aluminum-induced photoinhibition occurring
cence (Fo ). Moustakas et al. (1995) concluded that Al caused a
at both the donor (i.e., the OEC) and the acceptor sides of
decline in photosynthesis as a result of the closure of PSII re-
Photosystem II may be associated with growth inhibition. Be-
action centers (RCs) and a reduction in PSII electron transport
sides decreased light absorption due to reduced Chl concentra-
rate. Chen et al. (2005b) reported that the reduced CO2 assimi-
tion, enhanced energy dissipation protected the leaves of
lation rate in Al-treated citrus plants was probably caused by a
Al-treated plants from photo-oxidative damage in high light.
combination of factors such as reduced electron transport rate
Keywords: aluminum, Citrus grandis, CO2 assimilation, through PSII, increased closure of PSII RCs and increased
photoinhibition, Photosystem II. photorespiration. However, little is known about how PSII ab-
sorption flux, trapped energy flux, electron transport flux (do-
1864 JIANG, CHEN, ZHENG, HAN, TANG AND SMITH
nor and acceptor sides) and dissipated energy flux are affected
by Al.
Monitoring chlorophyll (Chl) a fluorescence is a common
method for investigating the behavior of PSII because it is
noninvasive, highly sensitive, reliable, fast and easily mea-
sured, and can provide important information about the photo-
synthetic apparatus. Time-resolved fluorescence measure-
ment provides detailed information on the fast kinetics of the
fluorescence rise, because the measurement can be made
within 10 µs. All oxygenic photosynthetic materials investi-
gated so far by this method show that the fast kinetics of Chl a
fluorescence emitted by dark-adapted photosynthetic samples
upon high irradiance with actinic light follow a polyphasic in-
crease, denoted as OJIP (Strasser et al. 1995, 2000, 2004). The
OJIP transient is highly sensitive to environmental stresses and
changes in shape according to changes in environmental con-
ditions (Strasser et al. 2000, 2004, Appenroth et al. 2001). The
JIP test analysis of the OJIP transient provides information for
quantifying the behavior of both PSII, including absorption
flux, trapped energy flux, electron transport flux and dissi-
pated energy flux (Strasser et al. 2000, 2004), and Photosys-
tem I (PSI), including the reduction of end electron acceptors
(Schansker et al. 2005, Tsimilli-Michael and Strasser 2008).
We investigated changes in CO2 assimilation, the OJIP tran-
sient and related parameters in leaves of citrus plants grown in
the presence of Al. Our objective was to determine how PSII
photochemistry is affected by Al at the levels of the RCs, en-
ergy absorption, energy dissipation, excitation energy trap-
ping, electron transport and reduction of end electron accep-
tors.
Materials and methods
Plant culture and aluminum treatments
Seeds of Citrus grandis (L.) Osbeck cv. Tuyou were germi-
nated in sand in plastic trays, and irrigated when necessary
with a nutrient solution containing 0.5 mM KNO3, 0.5 mM
Ca(NO3 )2, 0.1 mM KH2PO4, 0.2 mM MgSO4, 50 µM KCl,
46 µM H3BO3, 2 µM MnCl4, 1 µM ZnSO4, 0.3 µM CuSO4,
0.5 µM NaMoO4 and 20 µM Fe-EDTA (Pereira et al. 2000).
Five weeks after germination, uniform seedlings with a single
stem were selected and transplanted to 6-l pots containing
sand. Seedlings, three to a pot, were grown in a greenhouse in a
natural photoperiod at Fujian Agriculture and Forestry Uni-
versity. Each pot was supplied with 500 ml of nutrient solution
every two days. Six weeks after transplanting and until the end
of the experiment, each pot was supplied daily with a nutrient
solution containing 0 (control), 0.2, 0.6 or 1.6 mM Al from
AlCl3·6H2O until the sand was saturated. The pH of the solu-
tion was adjusted to 4.1 with 0.1 M HCl or 0.1 M NaOH. There
were 20 pots per Al treatment in a completely randomized
design. Five months after the beginning of the Al treatments,
recent fully expanded leaves from each replicate and Al treat-
ment were measured. For the determination of ribulose-1,5-
bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39)
and Chl concentration, leaf discs (0.61 cm2 in size) were col-
TREE PHYSIOLOGY VOLUME 28, 2008
lected at noon in full sun, frozen in liquid nitrogen, and stored
at –80 °C until assayed.
Leaf chlorophyll a fluorescence transient
The OJIP transient was measured with a Handy Plant Effi-
ciency Analyser (Handy PEA, Hansatech Instruments, Nor-
folk, U.K.) according to Strasser et al. (1995). The OJIP tran-
sient was induced by red light of about 3400 µmol m –2 s –1 pro-
vided by an array of three light-emitting diodes (peak 650 nm)
that focused on the leaf surface to give homogeneous illumina-
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tion over the exposed area of the leaf (4 mm in diameter). Ini-
tially, data were sampled at 10-µs intervals for the first 300 µs.
The time resolution of digitization was then switched to lower
acquisition rates as the kinetics of the fluorescence signal
slowed. All measurements were made at room temperature on
plants adapted to darkness for 3 h.
JIP test
The OJIP transient was analyzed according to the JIP test.
From the OJIP transient, the measured parameters (Fo, Fm,
F50µs, F100µs, F300µs, FJ, FI, tFm ) led to the calculation and deriva-
tion of a range of parameters as described previously (Strasser
et al. 2000, 2004, Appenroth et al. 2001, Tsimilli-Michael and
Strasser 2008; Table 1).
Leaf gas exchange
Leaf gas exchange measurements were made with a CI-301PS
portable photosynthesis system (CID, WA) at ambient CO2
concentration and a photosynthetic photon flux (PPF) of
1100 µmol m –2 s –1 between 1030 and 1200 h on a clear day
(Cheng and Fuchigami 2000, Chen et al. 2005b). During mea-
surements, leaf temperature and ambient vapor pressure were
29 ± 0.1 °C and 1.53 ± 0.01 kPa, respectively.
Assay of leaf total Rubisco activity
Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)
was extracted according to Chen et al. (2005b). Two frozen
leaf discs from the same leaf were ground with a pre-cooled
mortar and pestle in 1 ml of extraction buffer containing
50 mM Hepes-KOH (pH 7.5), 10 mM MgCl2, 2 mM ethylene-
diaminetetraacetic acid (EDTA), 10 mM dithiothreitol
(DDT), 1% (v/v) Triton X-100, 5% (w/v) insoluble poly-
vinylpolypyrrolidone (PVPP), 1% (w/v) bovine serum albu-
min (BSA) and 10% (v/v) glycerol. The extract was centri-
fuged at 13,000 g for 40 s at 2 °C, and the supernatant was used
immediately for assays of Rubisco activity.
Total Rubisco activity was assayed according to Cheng and
Fuchigami (2000) with some modifications. Total Rubisco ac-
tivity was measured after incubating the leaf extract in the
assay solution without ribulose-1,5-biphosphate (RuBP) for
15 min at room temperature to activate Rubisco. The assay so-
lution (final volume of 1 ml) contained 100 mM Hepes-KOH
(pH 8.0), 25 mM KHCO3, 20 mM MgCl2, 3.5 mM ATP, 5 mM
phosphocreatine, 5 units NAD-glyceraldehyde-3-phosphate
dehydrogenase (NAD-GAPDH, EC 1.2.1.12), 5 units 3-phos-
 ALUMINUM-INDUCED EFFECTS ON PSII IN CITRUS 1865

Table 1. Parameters and formulae and their description using data extracted from the chlorophyll a fluorescence (OJIP) transient.

Fluorescence parameter Description

Extracted parameters
Ft Fluorescence at time t after onset of actinic illumination
Fo Minimum fluorescence, when all PSII reaction centers (RCs) are open
Fm Maximum fluorescence, when all PSII RCs are closed
F50µs , F100µs and F300µs Fluorescence intensities at 50, 100 and 300 µs, respectively
FJ and FI Fluorescence intensities at the J-step (2 ms) and at the I-step (30 ms), respectively
tFm Time (in ms) to reach Fm
Area Total complementary area between fluorescence induction curve and F = Fm

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Derived parameters
Selected OJIP parameters
Vt = (Ft − Fo )/(Fm − Fo )
VJ = (F2ms − Fo )/(Fm − Fo )
VI = (F30ms − Fo )/(Fm − Fo )
Mo = 4(F300µs − Fo )/(Fm − Fo )
Sm = ECo /RC = Area/(Fm − Fo )
ECo /ABS = (ECo /RC)(RC/ABS)
Yields or flux ratios
ϕPo = TRo /ABS = 1 – Fo /Fm = Fv /Fm
ϕEo = ETo /ABS = (Fv /Fm )(1 – VJ )
ψEo = ETo /TRo = 1 – VJ
ϕDo = 1− ϕPo = Fo /Fm
δRo = REo /ETo = (1 – VI )/(1 – VJ )
ϕRo = REo /ABS = ϕPoψEoδRo
ρRo = REo /TRo = ψEoδRo
Specific fluxes or activities per reaction center
ABS/RC = Mo /VJ /ϕPo
TRo /RC = Mo /VJ
ETo /RC = (Mo /VJ )ψEo = (Mo /VJ )(1 − VJ )
DIo /RC = ABS/RC – TRo /RC
REo /RC = (REo /ETo )(ETo /RC)
Phenomenological fluxes or activities per excited cross section
ABS/CSo ≈ Fo
ETo /CSo = (ABS/CSo )ϕEo
TRo /CSo = (ABS/CSo )ϕPo
DIo /CSo = ABS/CSo – TRo /CSo
REo /CSo = (REo /ETo )(ETo /CSo )
Density of reaction centers
RC/CSo = ϕPo(ABS/CSo )(VJ /Mo )
Performance index
PIabs = (RC/ABS) (ϕPo /(1 − ϕPo )) (ψEo /(1 − ψEo ))
PIabs,total = (RC/ABS)(ϕPo /(1 − ϕPo ))·
(ψEo /(1 − ψEo )) (δRo /(1 − δRo ))
Relative variable fluorescence at time t
Relative variable fluorescence at the J-step (2 ms)
Relative variable fluorescence at the I-step (30 ms)
Approximated initial slope (in ms –1 ) of the fluorescence transient V = f(t)
Normalized total complementary area above the OJIP transient (reflecting multiple-
turnover QA reduction events) or total electron carriers per RC
Electron carriers per ABS at t = 0
Maximum quantum yield of primary photochemistry at t = 0
Quantum yield for electron transport at t = 0
Probability (at time 0) that a trapped exciton moves an electron into the electron
transport chain beyond QA–
Quantum yield at t = 0 for energy dissipation
Efficiency with which an electron can move from the reduced intersystem electron
acceptors to the PSI end electron acceptors
Quantum yield for the reduction of end acceptors of PSI per photon absorbed
Efficiency with which a trapped exciton can move an electron into the electron
transport chain from QA– to the PSI end electron acceptors
Absorption flux per RC
Trapped energy flux per RC at t = 0
Electron transport flux per RC at t = 0
Dissipated energy flux per RC at t = 0
Reduction of end acceptors at PSI electron acceptor side per RC at t = 0
Absorption flux per cross section (CS) at t = 0
Electron transport flux per CS at t = 0
Trapped energy flux per CS at t = 0
Dissipated energy flux per CS at t = 0
Reduction of end acceptors at PSI electron acceptor side per CS at t = 0
Amount of active PSII RCs per CS at t = 0
Performance index (PI) on absorption basis
Total PI, measuring the performance up to the PSI end electron acceptors

phoglyceric phosphokinase (PCK, EC 2.7.2.3), 17.5 units
creatine phosphokinase (EC 2.7.3.2), 0.25 mM NADH,
0.5 mM RuBP and 50 µl of sample extract. The reaction was
started by adding 50 µl 10 mM RuBP 15 min after the sample
extract was combined with the assay solution. The oxidation
of NADH was followed by measuring the decrease in
absorbance at 340 nm for 40 s.
Assays of leaf chlorophyll and aluminum
Leaf Chl was extracted and measured according to Arnon
(1949). Leaf Al was determined colorimetrically by the
aluminon method (Hsu 1963).
Root and shoot dry mass
At the end of the experiment, 15 plants per treatment from five

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1866 JIANG, CHEN, ZHENG, HAN, TANG AND SMITH

pots (one pot per replicate) were harvested. The plants were di-
vided into roots and shoots. The plant material was dried at
80 °C for 48 h and dry mass (DM) measured (Chen et al.
2005b).

Statistical analysis
Experiments were performed with 4–18 replicates (one plant
per pot, except for the dry mass measurements). Results are
presented as means ± SE. Differences among treatments were
separated by the least significant difference (LSD) test at

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P < 0.05.

Results

Leaf aluminum and plant growth

Compared with untreated control leaves, Al-treated leaves had
increased Al concentration (Figure 1A) and decreased shoot
dry mass (Figure 1B). The presence of up to 0.6 mM Al in the
nutrient solution had no significant effect on root dry mass, but
root dry mass was significantly lower in plants in the 1.6 mM
Al treatment than in control plants (Figure 1C).

Leaf gas exchange, Chl concentration and Rubisco activity

Leaf CO2 assimilation was similar in plants in the 0.2 to
0.6 mM Al treatments, but it was 60% lower in plants in the
1.6 mM Al treatment than in control plants. Compared with
control values, stomatal conductance increased in plants in the
0.2 and 0.6 mM Al treatments, and decreased 36% in plants in
the 1.6 mM Al treatment. Intercellular CO2 concentration in-
creased with increasing Al supply (Table 2).
Foliar Chl concentration did not change significantly as Al
supply increased from 0 to 0.6 mM, but it was 34% lower in
plants in the 1.6 mM Al treatment compared with control Figure 1. Effects of aluminum (Al) treatments on mean (+ SE, n = 5)
plants. None of the Al treatments had a significant effect on ei- (A) leaf Al concentration and (B) shoot and (C) root dry mass (DM)
of Citrus grandis seedlings. Different letters indicate significant dif-
ther Chl a/b ratio or Rubisco activity (Table 2). ferences between treatments (P < 0.05).
Chlorophyll a fluorescence transient and related parameters
Both control and Al-treated leaves showed a typical poly- The OJIP data are presented in Figure 3 as the kinetics of
phasic rise in Chl a fluorescence (i.e., OJIP) (cf. Strasser et al. relative variable fluorescence at any time, Vt = (Ft – Fo )/(Fm –
(1995). As shown in Figure 2, Al-treated leaves showed a large Fo ), and as a difference kinetic profile (i.e., the normalized
increase at the O-step and a large depression at the P-step. Het- Al-treated transient minus the control transient). The differ-
erogeneity in the OJIP transient increased among samples as ence kinetic profiles revealed three distinct trends: (1) an in-
Al supply increased (Figure 2). crease in the K-band (300 µs); (2) an increase in the 2 to 4 ms

Table 2. Mean (± SE, n = 4–5) CO2 assimilation, stomatal conductance, intercellular CO2 concentration, chlorophyll (Chl) concentration, Chl a/b
ratio, and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity of aluminum (Al)-treated and control leaves of Citrus grandis.
Different letters within a column indicate significant differences between treatments (P < 0.05).

Al treatment CO2 assimilation Stomatal Intercellular CO2 Chl Chl a/b Rubisco
(mM) (µmol m –2 s –1 ) conductance concentration concentration ratio activity
(mmol m –2 s –1 ) (µmol mol –1 ) (mg m –2 ) (µmol m –2 s –1 )
0 (control) 7.35 ± 0.55 a 44.7 ± 4.4 ab 133 ± 11 b 424 ± 49 a 2.90 ± 0.23 a 29.7 ± 2.9 a
0.2 7.67 ± 0.73 a 54.9 ± 9.4 a 150 ± 21 b 392 ± 9 a 2.94 ± 0.18 a 22.9 ± 2.8 a
0.6 6.97 ± 0.66 a 59.1 ± 13.3 a 183 ± 32 ab 438 ± 23 a 2.84 ± 0.19 a 23.5 ± 3.0 a
1.6 2.94 ± 0.35 b 28.6 ± 3.9 b 221 ± 16 a 281 ± 19 b 3.19 ± 0.16 a 23.2 ± 0.6 a

TREE PHYSIOLOGY VOLUME 28, 2008

 ALUMINUM-INDUCED EFFECTS ON PSII IN CITRUS
range J-step; and (3) an increase in the 30 to 100 ms range
I-step. To make the K-band clearer, the relative variable fluo-
rescence between Fo and FJ called W = (Ft – Fo )/(FJ – Fo ) for
the different Al treatments was calculated and is presented in
Figure 3B. Plotting the difference spectrum (the normalized
Al-treated transient minus the control transient) revealed a
positive K-band that was a function of Al concentration. Fig-
ure 3C shows the relative variable fluorescence between Fo
and F300µs. The difference spectrum showed a clear L-band at
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1867
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Figure 2. Effects of aluminum (Al)
treatments on the high irradiance
actinic-light-induced chlorophyll a
fluorescence (OJIP) transient of
dark-adapted Citrus grandis leaves
plotted on a logarithmic time scale
(0.01 ms to 1 s). Gray circles are
single measurements and black
circles are mean transients of all
measured samples.
about 150 µs in Al-treated leaves (Figure 3C). As shown in
Figure 3D, Al-treated leaves had a decreased IP phase and an
increased IP rise time, and the end value was typically lowered
by Al.
The presence of up to 0.6 mM Al in the nutrient solution had
no significant effects on Fo, Fm, F50µs, F100µs, F300µs, FJ, FI, tFm,
and Area. Leaves of plants in the 1.6 mM Al treatment had in-
creased Fo, F50µs, F100µs, F300µs and tFm, but decreased Fm, FI and
Area compared with control values (Table 3).
Figure 3. Mean chlorophyll a fluo-
rescence kinetics (Ft ) expressed as
the kinetics of relative variable fluo-
rescence: (A) between Fo and Fm:
Vt = (Ft – Fo )/(Fm – Fo ); (B) be-
tween Fo and FJ: Wt = (Ft – Fo )/(FJ
– Fo ); (C) between Fo and F300µs:
(Ft – Fo )/(F300µs – Fo ); and (D) be-
tween Fo and FI: (Ft – Fo )/(FI – Fo ).
In each of the plots (A), (B) and
(C), the differences of the four sam-
ples to the reference sample of
non-stressed control Citrus grandis
plants are plotted with an amplifica-
tion of 5, 6.5 and 6, respectively.
The K- and L-bands are clearly re-
vealed in (B) and (C), respectively.
The IP phase normalized on the Fo
to FI phase = (Fm – Fo )/(FI – Fo ) –
(FI – Fo )/(FI – Fo ) = (Fm – Fo )/(FI –
Fo ) – 1.
1868 JIANG, CHEN, ZHENG, HAN, TANG AND SMITH

Figure 4 shows the behavior of 24 biophysical parameters of

PSII. For each parameter, the values were normalized against
the control value. Leaves of Al-treated plants had increased
ABS/CSo, DIo /CSo, TRo /CSo, ϕDo, TRo /RC, DIo /RC,
ABS/RC and CSo /RC but decreased Sm, PIabs,total, PIabs, ρRo,
δRo, ψEo, RC/CSo, ETo /CSo, REo /CSo, RC/ABS, ϕPo, ϕEo, ϕRo,
ECo /ABS, REo /RC and ETo /RC compared with control
leaves.

Aluminum supply, shoot dry mass, leaf Al concentration, IP

phase and CO2 assimilation

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As Al supply increased, relative leaf Al concentration in-
creased linearly, whereas shoot dry mass, leaf IP phase from
F80ms to Fm and CO2 assimilation decreased linearly. Shoot DM
decreased more than both IP phase and CO2 assimilation in re-
sponse to Al stress, and shoot growth was more sensitive to Al
toxicity than IP phase from F80ms to Fm and CO2 assimilation
(Figure 3A). Shoot DM decreased linearly with increasing
leaf Al concentration (Figure 5B). Leaf CO2 assimilation in-
creased linearly with increasing leaf IP phase from F80ms to Fm
(Figure 5C).
Discussion
Aluminum affected shoot dry mass more than root dry mass
(Figures 1B and 1C), as previously found for ‘Cleopatra’ tan-
gerine (Chen et al. 2005b) and ‘Nemaguard’ peach (Prunus
persica (L.) Batsch; Graham 2001). However, Al did not affect
the shoot dry mass/root dry mass ratio of ‘Swingle’ citrumelo
(Citrus paradisi Mcf. × Poncirus trifoliate Raf.) seedlings (dos
Santos et al. 2000). Xiao et al. (2002) reported that the relative
sensitivity of roots and shoots of longan seedlings to Al de-
pended on the Al concentration in the nutrient solution. Thus it
appears that the relative sensitivity of roots and shoots to Al
depends on Al concentration and plant species.
The decrease in leaf CO2 assimilation in response to Al
treatment was primarily caused by non-stomatal factors be-
cause the decrease in assimilation rate was accompanied by an
increase in intercellular CO2 concentration (Table 2). Similar
results have been reported for ‘Cleopatra’ tangerine (Chen et
al. 2005b), Citrus spp. (Pereira et al. 2000), and sorghum (Sor-
ghum bicolor L.; Peixoto et al. 2002). Because there was no
significant difference in Rubisco activity among the Al treat-
ments (Table 2) and because the Rubisco activation state in
leaves of Citrus grandis plants treated with 1.2 mM Al did not
Figure 4. Effects of aluminum (Al) treatments on Citrus grandis
plants, evaluated by the behavior of 24 structural and functional pa-
rameters of Photosystem II, derived by the JIP-test from the OJIP
transient exhibited by dark-adapted leaves of plants grown with 0
(control), 0.2, 0.6 and 1.6 mM Al. In each case the parameters are de-
rived from the corresponding mean transient. All the values were ex-
pressed relative to the control (set as 1).
change significantly even though CO2 assimilation decreased
(data not shown), the reduction in CO2 assimilation in leaves
of Al-treated plants could not be attributed to decreased activi-
ties of Calvin-Benson cycle enzymes. This inference is also
supported by the observation that the Al-induced decrease in
CO2 assimilation in ‘Cleopatra’ tangerine leaves was unac-
companied by decreased activities of enzymes involved in the
Calvin-Benson cycle (Chen et al. 2005b).
We found that ϕEo, which is the product of ϕPo and ψEo, de-
creased in Al-treated leaves (Figure 4). The Al-induced inhibi-
tion of electron transport was due more to changes in ψEo than
in ϕPo, because ψEo decreased more than ϕPo in response to the
Al treatments (Figure 4). Similar results were obtained with
Cr-treated Spirodela polyrhiza leaves (Appenroth et al. 2001).
Aluminum decreased the total electron acceptor capacity of
leaves, as indicated by decreased Sm, which measures the pool
of electron transporters between PSII and the acceptor side of

Table 3. Effects of aluminum treatments (Al, mM) reflected in data extracted from the recorded chlorophyll a fluorescence transient of Citrus
grandis. Values are means ± SE (n = 9–12). Different letters within a column indicate significant differences between treatments (P < 0.05). Refer
to Table 1 for parameter definitions.

Al Fo Fm F50µs F100µs F300µs FJ FI tFm Area

0 574 ± 18 b 3094 ± 43 a 743 ± 25 b 903 ± 34 b 1470 ± 47 b 2293 ± 41 a 2750 ± 45 a 351 ± 23 b 51309 ± 1538 a
0.2 553 ± 8 b 3125 ± 46 a 734 ± 12 b 906 ± 22 b 1505 ± 41 b 2323 ± 34 a 2780 ± 38 a 303 ± 10 b 49567 ± 1616 a
0.6 587 ± 20 b 3103 ± 57 a 774 ± 32 b 954 ± 47 b 1556 ± 70 b 2350 ± 58 a 2796 ± 54 a 354 ± 21 b 47800 ± 2373 a
1.6 957 ± 71 a 2597 ± 140 b 1144 ± 77 a 1357 ± 91 a 1839 ± 94 a 2284 ± 115 a 2478 ± 131 b 500 ± 25 a 19644 ± 2558 b

TREE PHYSIOLOGY VOLUME 28, 2008

 ALUMINUM-INDUCED EFFECTS ON PSII IN CITRUS
Figure 5. (A) Effects of aluminum (Al) treatments on relative (to the
control) leaf IP phase from F80ms to Fm (䉭, medium dash; y = 107.0 −
48.5x, r 2 = 0.937, P = 0.021), shoot dry mass (DM, 䉱, short dash; y =
97.2 − 47.5x, r 2 = 0.9868, P = 0.004), CO2 assimilation (䊊, long dash;
y = 109.2 − 40.6x, r 2 = 0.882, P = 0.040) and Al concentration (䊉,
solid line; y = 19.5 + 51.1x, r 2 = 0.894, P = 0.036) in Citrus grandis.
(B) Relative shoot DM in relation to relative leaf Al concentration.
(C) Relative leaf CO2 assimilation in relation to relative leaf IP phase
from F80ms to Fm. The IP phase from F80ms to Fm is normalized on the
Fo to FI phase = (Fm – Fo )/(FI – Fo ) – (F80ms – Fo )/(FI – Fo ).
PSI (Pinior et al. 2005). Leaves of Al-treated plants also had a
lower ETo /CSo than control plants, but an increased TRo /CSo
(Figure 4), indicating that Al affects the activity of the electron
transport chain (cf. Chen et al. 2005b). We found that the IP
phase from F80ms to Fm decreased with increasing Al supply,
indicating that Al slowed the fractional reduction of the PSI
end electron acceptors. The finding that CO2 assimilation rate
increased linearly with the increasing IP phase from F80ms to
Fm (Figure 5C) corroborated previous suggestions that the am-
plitude of the IP phase is a measure of the amount of reduced
TREE PHYSIOLOGY ONLINE at http://heronpublishing.com
1869
end acceptors at the PSI acceptor side and that the IP phase
represents the last and rate-limiting step of the photosynthetic
electron transport chain (Schansker et al. 2005). Our results
demonstrated that Al decreased yields (δRo, ϕRo and ρRo ) and
fluxes (REo /RC and REo /CSo ) and damaged all of the photo-
chemical and non-photochemical redox reactions that we
measured, as indicated by the decreases in PIabs and PIabs,total
(Figure 4). We hypothesize that impaired electron transport
capacity accompanied by the lack of reducing equivalents are
the main factors contributing to decreased CO2 assimilation in
response to Al.
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According to the Grouping Concept (Strasser 1978) and the
JIP-test (Strasser et al. 2004), the presence of a positive L-band
in leaves of Al-treated plants indicates that the OJIP transient
in Al-treated leaves is less sigmoidal than in control leaves,
implying that the PSII units are less tightly grouped, or less en-
ergy is being exchanged between independent PS II units. Be-
cause the grouped conformation is more stable than the
ungrouped conformation, the decreased cooperativity implies
that the PSII units of Al-treated plants have lost stability and
become more fragile.
Aluminum decreased Fv /Fm (ϕPo Figure 4) and altered the
OJIP transient (Figure 2), indicating that photoinhibition oc-
curs in leaves exposed to Al (Baker et al. 1994, Maxwell and
Johnson 2000). The reduction in Fv /Fm was caused by both a
decline of Fm and an increase in Fo (Table 3). Because Al treat-
ment decreased RC/CSo (Figure 4), the increase in Fo may
have been induced by inactivation of some of the PSII RCs
(Yamane et al. 1997), or have been related to the accumulation
of reduced QA (Bukhov et al. 1990), because the fractional re-
duction of QA to QA– (per total QA ), as indicated by the increase
in Mo (Figure 3A), increased in Al-treated leaves. Because the
Al treatments had no significant effects on Rubisco activity,
Al-induced accumulation of reduced QA was probably not
caused by an over-reduction of the photosystem in response to
a slowing of the dark reactions resulting from a decrease in the
activities of Rubisco and other photosynthetic enzymes.
Leaves of Al-treated plants showed a decreased JI phase but a
similar J level to leaves of control plants. This was not com-
pensated for by an increase in IP phase, and therefore resulted
in a decreased P level (Table 3, Figure 2). The lower Fm ob-
served in Al-treated leaves may be associated with
photoinhibitory quenching (qI) (Müller et al. 2001, Force et al.
2003) rather than with xanthophyll cycle-dependent thermal
energy dissipation (Demmig-Adams and Adams 1992), which
was lower in 2 mM Al-treated ‘Cleopatra’ tangerine leaves
than in control leaves (Chen et al. 2005a). We observed that
Al-treated leaves had decreased RC/CSo but increased
DIo /CSo (Figure 4). It is known that inactive PSII RCs can pre-
vent further damage to themselves and protect neighboring ac-
tive PSII RCs by acting as sinks for excitation energy (Öquist
et al. 1992). Therefore, qI probably arises from both the for-
mation of inactive PSII RCs (Krause 1988) and PSII photo-
damage (Müller et al. 2001). Horton et al. (1996) suggested
that the origin of qI was similar to that of energy dependent
quenching (qE), with qI being a longer lasting, more stably
protonated light-harvesting complex associated with a PSII
1870 JIANG, CHEN, ZHENG, HAN, TANG AND SMITH
state with a high ratio of zeaxanthin (Z) to violaxanthin (V). In
our study, qI also may arise from “qE-like” qI because of the
larger change in ψEo compared with Fv /Fm (Figure 3, Force et
al. 2003). This is supported by data showing that leaves of
‘Cleopatra’ tangerine treated with 2 mM Al have a higher con-
version of V to antheraxanthin (A) and Z (Chen et al. 2005a)
and higher Z:V ratio than control leaves (Chen et al. unpub-
lished data). Photoinhibition is considered to be more accu-
rately identified by an increase in DIo /RC and a decline in ψEo
than by a decline in Fv /Fm (Force et al. 2003). Our Al-treated
leaves had increased DIo /RC and decreased ψEo (Figure 4),
supporting the view that photoinhibition occurred in the
Al-treated leaves.
The Al-induced K-step in the OKJIP at 300 µs agrees with
the results obtained with Cr-treated Lemna gibba L. (Ali et al.
2006) and Spirodela polyrhiza (L.) Schleid. (Susplugas et al.
2000). Copper-induced inhibition of Zea mays L. photosyn-
thesis was reported to be caused by the alteration of proteins
having a role in the oxygen-evolving complex (OEC, Barün et
al. 1995). Cadmium inhibition of PSII activity in Pisum
sativum L. leaves was caused by a change in D1 protein, indi-
cating that the inhibitory site was on the reducing side of PSII
(Franco et al. 1999). Therefore, the Al-induced K-step may be
caused by an inhibition of electron donation from water to the
secondary electron donor of PSII (YZ ) resulting from OEC in-
activation due to the release of Mn from OEC (Strasser 1997,
Hakala et al. 2005). This is also supported by the data showing
that Al-treated leaves had decreased OEC (Fraction of OEC =
[1 – (VK /VJ )]treated sample/[1 – (VK /VJ )]control, see Appentrol et al.
2001) (data not shown).
Our Al-treated leaves had increased VI compared with con-
trol leaves (Figure 3A), indicating that Al inhibits on the ac-
ceptor side of PSII ( Lu and Zhang 1999, Chen et al. 2004). We
note that VI is a derived ratio parameter (Table 1) and its in-
crease can be caused by both an increase in FI or a decrease in
Fm, or both. Because FI and Fm were lower in Al-treated leaves
than in control leaves (Table 3), the increased VI in Al-treated
leaves may indicate a relative change in the proportion of
QB-non-reducing PSII RCs, rather than an increase in the ab-
solute amount of the QB-non-reducing PSII RCs. Alumi-
num-induced inhibition of the acceptor side is supported by
the finding that Fv was reduced in Al-treated leaves along with
an increase in Fo (Table 3), which is characteristic of inhibition
of the acceptor side (Setlik et al. 1990).
Because CO2 assimilation in Al-treated leaves decreased to
a greater degree than Chl concentration (Table 2) and
Al-treated leaves had a decreased probability of energy con-
servation of the excitation energy trapped by the RCs, as indi-
cated by the positive J-step (Figure 3A), only a small fraction
of the absorbed light energy was used in electron transport, as
indicated by the decreases in Sm, ϕEo and ϕRo (Figure 4), com-
pared with the control. As a result, more excess excitation en-
ergy existed in Al-treated leaves than in control leaves in high
light (Chen et al. 2005a). Correspondingly, energy dissipation,
as indicated by DIo /CSo, DIo /RC and ϕDo, increased in
Al-treated leaves (Figure 4).
To conclude, our results demonstrated that shoot growth
TREE PHYSIOLOGY VOLUME 28, 2008
was more sensitive to Al toxicity than root growth, gas ex-
change, Chl concentration, the OJIP transient and related pa-
rameters, and that the photosynthetic machinery is regulated at
the levels of the RCs, energy absorption, energy dissipation,
excitation energy trapping, electron transport and reduction of
end electron acceptors. The impaired electron transport capac-
ity accompanied by the lack of reducing equivalents appeared
to be the main factors contributing to the decreased CO2 assim-
ilation in Al-treated leaves. Aluminum-induced photo-
inhibition occurring at both the donor (i.e., the OEC) and the
acceptor sides of PSII was likely associated with growth inhi-
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bition. In addition to decreasing light absorption by lowering
Chl concentration, Al treatment enhanced energy dissipation,
thereby protecting leaves from photo-oxidative damage in
high light.
Acknowledgments
The study was supported financially by the National Natural Science
Foundation of China (Nos. 30270930 and 30771487), the Agricul-
tural Commonweal Industrial Special Fund Program of Department
of Agriculture, China (nyhyzx07-023) and the Natural Science Foun-
dation of Fujian Province of China (Nos. B0710011 and 2007J0050).
We thank Dr. Reto J. Strasser, University of Geneva, Switzerland for
editing the manuscript.
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