Tree Physiology 25, 317–324
© 2005 Heron Publishing—Victoria, Canada
Aluminum-induced decrease in CO2 assimilation in citrus seedlings is
unaccompanied by decreased activities of key enzymes involved in CO2
assimilation
LI-SONG CHEN,1,2 YI-PING QI,3 BRANDON RHETT SMITH4 and XING-HUI LIU1
1
College of Horticulture, Fujian Agriculture and Forestry University, Fuzhou, 350002, P.R. China
2
Corresponding author ([email protected])
3
Fujian Provincial Institute of Medical Sciences, Fuzhou, 350001, P.R. China
4
Department of Horticulture, Cornell University, Ithaca, NY 14853, USA
Received April 13, 2004; accepted September 3, 2004; published online January 4, 2005
Summary ‘Cleopatra’ tangerine (Citrus reshni Hort. ex
Tanaka) seedlings were irrigated daily for 8 weeks with 1/4
strength Hoagland’s nutrient solution containing 0 (control) or
2 mM aluminum (Al). Leaves from Al-treated plants had decreased CO2 assimilation and stomatal conductance, but increased intercellular CO2 concentrations compared with control leaves. On a leaf area basis, 2 mM Al increased activities of
key enzymes in the Calvin cycle, including ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), NADP-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoribulokinase (PRK), stromal fructose-1,6-bisphosphatase
(FBPase), and a key enzyme in starch synthesis, ADP-glucose
pyrophosphorylase (AGPase), compared with control leaves.
Aluminum had no effect on cytosolic FBPase activity, but it decreased sucrose phosphate synthase (SPS) activity. Aluminum
had no effect on area-based concentrations of carbohydrates,
glucose-6-phosphate (G6P) and fructose 6-phosphate (F6P) or
the G6P:F6P ratio, but it decreased the area-based concentration of 3-phosphoglycerate (PGA). Photochemical quenching
coefficient (qP) and electron transport rate through PSII were
greatly reduced by Al. Non-photochemical quenching coefficient (NPQ) was less affected by Al than qP and electron transport rate through PSII. We conclude that the reduced rate of
CO2 assimilation in Al-treated leaves was probably caused by a
combination of factors such as reduced electron transport rate
through PSII, increased closure of PSII reaction centers and increased photorespiration.
Keywords: ADP-glucose pyrophosphorylase (AGPase), Citrus
reshni, ‘Cleopatra’ tangerine, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), sucrose phosphate synthase
(SPS).
Introduction
Aluminum (Al) toxicity is a major factor limiting crop productivity in many acid soils throughout the tropics and subtropics.
It is also considered to be the primary toxic agent in acid-rain-
induced forest decline. The molecular and physiological bases
of Al toxicity and tolerance have been extensively studied
(Kochian 1995, Matsumoto 2000, Kochian et al. 2002). Most
research, however, has focused on phenomena occurring at the
plant root level, because inhibition of root growth is one of the
earliest and most easily recognized symptoms of Al injury
(Foy et al. 1978). Less is known about the effects of Al toxicity
on leaves (Peixoto et al. 2002).
Aluminum inhibits CO2 assimilation in many plant species,
including Citrus spp. (Pereira et al. 2000), Pinus massoniana
Lamb. (Cao et al. 1991), longan (Dimocarpus longan Lour.)
(Xiao 2002), Thinopyrum bessarabicum Savul & Rayss
(Moustakas et al. 1996), sorghum (Sorghum bicolor L.
Moench) (Peixoto et al. 2002), tomato (Lycopersicon esculentum Mill.) (Simon et al. 1994) and maize (Zea mays L.)
(Lidon et al. 1999). It has been proposed that the Al-induced
inhibition of CO2 assimilation results from both stomatal and
non-stomatal factors (Simon et al. 1994, Moustakas et al.
1995, 1996). Simon et al. (1994) suggested that stomatal closure was at least partially responsible for the Al-induced reduction in CO2 assimilation, because intercellular CO2
concentration decreased in response to Al. Pereira et al. (2000)
suggested that the Al-induced decrease in CO2 assimilation
was associated with structural damage to the thylakoids, as
shown by a decrease in the ratio of variable fluorescence (Fv )
to initial fluorescence (Fo ). Lidon et al. (1999) associated the
Al-induced decrease in photosynthesis with a reduction in
photosynthetic electron transport associated with PSI,
whereas Moustakas et al. (1995) concluded that Al caused a
decline in photosynthesis as a result of the closure of PSII reaction centers and a reduction in PSII electron transport rate. It
was also suggested that a combination of factors such as reduced pigment content, impaired PSII photochemistry and the
distribution of enzymatic machinery could account for the
Al-induced decrease in CO2 assimilation (Peixoto et al. 2002).
Many studies have shown that heavy metals affect CO2 assimilation by inhibiting different enzymes involved in the Calvin cycle (Malik et al. 1992, Krupa and Baszyñski 1995,
318
CHEN, QI, SMITH AND LIU
Krupa 1999, Chugh and Sawhney 1999, Romanowska 2002);
however, other studies have shown little or no change in the activities of Calvin cycle enzymes accompanying a heavy-metalinduced decline in CO2 assimilation (Sheoran et al. 1990).
Therefore, it is unclear whether Calvin cycle enzymes are affected by Al.
Simon et al. (1994) reported that accumulation of Al in the
roots of tomato caused tissue damage, resulting in reduced sucrose utilization, probably causing an accumulation of leaf
carbohydrates and feedback inhibition of photosynthesis. Unfortunately, carbohydrates were not determined in this study
and so it remains to be established if the Al-induced decrease
in CO2 assimilation is associated with feedback inhibition of
photosynthesis resulting from the accumulation of leaf carbohydrates.
We reexamined Al toxicity in relation to leaf carbohydrate
metabolism. Our specific objective was to determine the responses to Al of the following in citrus leaves: CO2 assimilation; key enzymes in the Calvin cycle and starch and sucrose
syntheses; nonstructural carbohydrates; and photosynthetic
intermediates.
Materials and methods
Plant culture and Al treatments
Seeds of ‘Cleopatra’ tangerine (Citrus reshni Hort. ex Tanaka)
were germinated in plastic trays containing MetroMix 360
(Scotts, Marysville, OH) and were irrigated when necessary
with 1/4 strength Hoagland’s nutrient solution. Four weeks after germination, uniform seedlings with a single stem were
each transplanted to a 1.7-l plastic pot containing MetroMix
360 and grown in a greenhouse maintained at a diurnal temperature of 22/27 °C in a natural photoperiod. Seedlings were
supplied twice weekly with 150 ml of 1/4 strength Hoagland’s
nutrient solution. Five weeks after transplanting, seedlings
were supplied daily with 150 ml of 1/4 strength Hoagland’s
nutrient solution containing 0 (control) or 2 mM Al from
Al2(SO4)3·18H2O. The pH was adjusted to 4.1 with 0.1 M HCl
or 0.1 M NaOH. There were 30 trees per Al treatment in a
completely randomized design. Eight weeks after the beginning of the Al treatments, recently expanded leaves were chosen for measurements.
tance were made from 400 to 700 nm at 1-nm intervals. The
sample scan was divided by its corresponding reference scan,
and integrated over the wavelength range to obtain the mean
reflectance or transmittance. Leaf absorptance was calculated
as: 1 − reflectance − transmittance.
Chlorophyll (Chl) fluorescence was measured with a pulsemodulated fluorometer FMS2 (Hansatech Instruments, Norfolk, U.K.), both at midday in full sun (1020 µmol m –2 s –1
PPF) and at predawn, on the same leaves used for gas exchange measurements. The fiber optic of the FMS2 was held at
a 60° angle above the upper surface of the leaf with the
PPF/temperature leaf clip, and the distance between the fiber
optic and the leaf surface was kept constant for both the predawn and the midday measurements. Maximum (Fm ) and
minimum fluorescence (Fo ) of dark-adapted leaves were measured at predawn. For the measurements at midday, steadystate fluorescence (Fs ) was monitored to ensure it was stable
before a reading was taken. Maximum fluorescence (Fm′) in
natural light was obtained by imposing a 1-s saturating flash of
about 6000 µmol m –2 s –1 PPF at the leaf surface to reduce all
the PSII centers. To determine minimum fluorescence (Fo′) in
natural light, the leaf was covered with a black cloth while a
far-red light was switched on to oxidize PSII rapidly by drawing electrons from PSII to PSI. Thermal energy dissipation
was estimated from non-photochemical quenching (NPQ) as:
Fm /Fm′ − 1 (Bilger and Björkman 1990). Maximum PSII efficiency of dark-adapted leaves was calculated as: Fv /Fm = (Fm –
Fo )/Fm (van Kooten and Snel 1990). The ratio of variable fluorescence (Fv ) to initial fluorescence (Fo ) was calculated as:
Fv /Fo = (Fm – Fo )/Fo. The photochemical quenching coefficient (qP) was calculated as: (Fm′ − Fs )/(Fm′ − Fo′). Efficiency
of excitation transfer to open PSII centers in natural light was
calculated as: Fv′/Fm′ = (Fm′ − Fo′)/Fm′. We calculated actual
quantum yield of PSII electron transport (Φ PSII ) as: (Fm′ − Fs)/
Fm′ (Genty et al. 1989).
Electron transport rate through PSII was estimated from
Φ PSII (PPF)(LA/2) (Schreiber et al. 1994), where LA is leaf absorptance and the rate of PSI photochemistry was assumed to
match that of PSII (Genty et al. 1990). The rate of excess energy
production was estimated from (1 − qP)(Fv′/Fm′)(PPF)(LA /2)
(Kato et al. 2003).
Assays of leaf enzymes associated with the Calvin cycle and
carbohydrate metabolism
Leaf gas exchange measurements
Gas exchange measurements were made with a CIRAS-1 portable photosynthesis system (PP Systems, Herts, U.K.) at ambient CO2 concentration (360 µmol mol –1) with a natural
photosynthetic photon flux (PPF) of 1020 ± 34 µmol m –2 s –1
between 1100 and 1200 h on a clear day. During measurements, leaf temperature and ambient vapor pressure were 28 ±
0.1 °C and 1.48 ± 0.01 kPa, respectively.
Leaf absorptance and chlorophyll fluorescence
Leaf reflectance and transmittance were measured with an
LI-1800 spectroradiometer and an 1800-12S integrating
sphere attachment (Li-Cor, Lincoln, NE). For each leaf, both a
reference scan and a sample scan of reflectance or transmit-
Leaf disks (1 cm 2) were taken from the same seedlings used
for gas exchange measurements at midday in full sun
(1020 µmol m –2 s –1 PPF), frozen in liquid N2 and stored at
–80 °C until extracted.
Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC4.1.1.39), NADP-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC1.2.1.12), phosphoribulokinase (PRK,
EC2.7.1.19), fructose-1,6-bisphosphatase (FBPase, EC3.1.3.
11) and sucrose phosphate synthase (SPS, EC2.4.1.14) were
extracted according to Chen and Cheng (2003). Three frozen
leaf disks were ground with a pre-cooled mortar and pestle in
1.5 ml of extraction buffer containing 50 mM Hepes-KOH
(pH 7.5), 10 mM MgCl2, 2 mM ethylenediaminetetraacetic
TREE PHYSIOLOGY VOLUME 25, 2005
ALUMINUM DECREASES CO 2 ASSIMILATION IN CITRUS
acid (EDTA), 10 mM dithiothreitol (DDT), 1% (v/v) Triton
X-100, 5% (w/v) insoluble polyvinylpolypyrrolidone (PVPP),
1% (w/v) bovine serum albumin (BSA) and 10% (v/v) glycerol. The extract was centrifuged at 13,000 g for 5 min, and the
supernatant was assayed immediately.
Total Rubisco activity was assayed after incubating the leaf
extract in assay solution without ribulose-1,5-biphosphate
(RuBP) for 15 min at room temperature (Cheng and Fuchigami 2000).
Activities of GAPDH and PRK were determined according
to Leegood (1990).
Stromal FBPase was assayed in a mixture (1 ml) containing
50 mM Tris-HCl (pH 8.2), 10 mM MgCl2, 1 mM EDTA,
0.1 mM fructose 1,6-bisphosphate (FBP), 0.5 mM NADP,
4 units of phosphoglucose isomerase (PGI, EC5.3.1.9) and
2 units of glucose-6-phosphate dehydrogenase (G6PDH,
EC1.1.1.49). The reaction was initiated by adding enzyme extract (Leegood 1990, Holaday et al. 1992). Cytosolic FBPase
was assayed according to Holaday et al. (1992) with some
modifications. The enzyme activity was assayed in 1 ml of reaction mixture containing 50 mM Hepes-NaOH (pH 7.0),
2 mM MgCl2, 0.1 mM FBP, 0.5 mM NADP, 4 units of PGI and
2 units of G6PDH. The reaction was initiated by adding enzyme extract.
We assayed SPS according to Grof et al. (1998). We incubated 60 µl of extract for 15 min at 30 °C with 100 mM HepesKOH (pH7.5), 100 mM KCl, 6 mM EDTA, 30 mM uridine
5′-diphosphoglucose (UDPG), 10 mM fructose-6-phosphate
(F6P) and 40 mM glucose-6-phosphate (G6P) in a total volume of 100 µl. At the end of the 15-min incubation period, the
reaction was stopped by adding 100 µl of ice-cold 1.2 M
HClO4 and holding the mixture on ice for 15 min. The reaction
mixture was neutralized by adding 60 µl of 2 M KHCO3, held
on ice for 15 min, then centrifuged at 13,000 g for 1 min. An
aliquot (130 µl) of the supernatant was assayed for uridine
5′-diphosphate (UDP) by coupling with the oxidation of
NADH with lactate dehydrogenase (LDH, EC1.1.1.27) and
pyruvate kinase (EC2.7.1.40). The reaction mixture (1 ml)
contained 50 mM Hepes-NaOH (pH7.0), 5 mM MgCl2,
0.3 mM NADH, 0.8 mM phosphenolpyruvate (PEP), 14 units
LDH and 4 units pyruvate kinase. The reaction was started by
adding pyruvate kinase (Stitt et al. 1988). For each sample,
controls without F6P and G6P were carried through the assay
procedure.
The enzyme ADP-glucose pyrophosphorylase (AGPase,
EC2.7.7.27) was extracted and assayed according to Chen and
Cheng (2003). The only modification was that reduced glutathione (GSH) was excluded from the extraction buffer.
Assays of leaf G6P, F6P and 3-phosphoglycerate (PGA)
Forty 1-cm 2 leaf disks (about 1 g) were taken at midday in full
sun (1020 µmol m –2 s –1, PPF), frozen in liquid N2 and stored at
–80 °C until extracted. We extracted and assayed G6P, F6P
and PGA according to Chen et al. (2002), with some modifications (Chen and Cheng 2003). The recoveries of G6P, F6P and
PGA were 93–103%.
319
Analysis of leaf nonstructural carbohydrates
Three leaf disks (total of 3 cm 2 ) were taken at dusk and at predawn from the same leaf, frozen in liquid N2 and stored at
–80 °C until extracted. Sucrose, glucose, fructose and starch
were extracted three times with 80% (v/v) ethanol at 80 °C
(Chen et al. 2001, Chen and Cheng 2003). Sucrose, fructose
and glucose were determined spectrophotometrically by a
modified enzymatic method (Jones et al. 1977). Starch was determined enzymatically as glucose equivalents with G6PDH
and hexokinase (EC2.7.1.1) (Jones et al. 1977). The recoveries
of starch, sucrose, fructose and glucose were 91–99%.
Analysis of leaf Chl, total soluble protein and Al
Leaf Chl was extracted and measured according to Arnon
(1949). Total soluble protein was determined according to
Bradford (1976) with BSA as standard. Leaf Al was assayed
by ICP emission spectrometry (Lin and Myhre 1991).
Measurements of root, stem and leaf fresh and dry mass, and
leaf mass ratio
Eight weeks after the beginning of the Al treatments, eight
plants per Al treatment were harvested. The plants were divided into roots, stems and leaves, and the fresh mass of each
part was determined. The plant material was dried at 80 °C for
48 h and dry mass (DM) measured. Leaf mass ratio (LMR; gDM
m –2) was determined as described by Syvertsen et al. (1980).
Statistical analysis
Experiments were performed with 6–8 replicates (one plant
per replicate). Results are presented as means ± SD for n = 6 or
8. The unpaired t-test was applied for comparison between
means.
Results
Growth characteristics
Aluminum-treated plants had decreased leaf, stem and root
fresh and dry mass, but increased leaf mass per leaf area (leaf
mass ratio; LMR) compared with control plants. Leaf and stem
fresh and dry mass decreased to a greater degree than root
fresh and dry mass in response to Al (Table 1).
Leaf Chl, total soluble protein and Al concentrations
Area-based total Chl and Chl b concentrations were slightly
lower in Al-treated leaves than in control leaves (Table 2),
whereas there was no significant difference in area-based
Chl a concentration between Al-treated leaves and control
leaves. Because Chl b decreased more than Chl a in response
to Al, the Chl a/b ratio was slightly greater in Al-treated leaves
than in control leaves. The Al treatment increased area-based
total soluble protein concentration and area- and mass-based
Al concentrations, but had no effect on mass-based total soluble protein concentration.
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CHEN, QI, SMITH AND LIU
Table 1. Leaf, stem and root fresh and dry mass, and leaf mass ratio in aluminum (Al)-treated and control plants (n = 8). Within a column, values
followed by different letters are significantly different at P < 0.05.
Treatment
Al
Control
Dry mass (g plant –1)
Fresh mass (g plant –1)
Leaves
Stems
Roots
Leaves
Stems
Roots
Leaf mass ratio
(g DM m –2)
2.00 a
3.46 b
1.05 a
1.92 b
0.57 a
0.81 b
5.07 a
9.54 b
4.08 a
6.54 b
3.66 a
4.49 b
88.0 a
72.5 b
Leaf gas exchange
Both CO2 assimilation and stomatal conductance decreased
significantly in response to 8 weeks of Al treatment (Table 3).
Because Al decreased CO2 assimilation more than stomatal
conductance, the intercellular CO2 concentration increased in
Al-treated leaves.
Activities of key enzymes associated with the Calvin cycle
and carbohydrate metabolism
On a leaf area basis, activities of key enzymes in the Calvin cycle, including Rubisco, GAPDH, PRK, stromal FBPase (Figures 1A–D), and a key enzyme in starch synthesis, AGPase
(Figure 1F), were significantly higher in Al-treated leaves than
in control leaves. There was no significant difference in cytosolic FBPase activity between Al-treated and control leaves
(Figure 1E); however, SPS activity was significantly lower in
Al-treated leaves than in control leaves (Figure 1G). When expressed on a leaf soluble protein basis, activities of GAPDH,
PRK and AGPase were higher in Al-treated leaves than in control leaves (Figures 1I, 1J and 1M), whereas the specific activities of cytosolic FBPase and SPS were lower (Figures 1L and
1N). Aluminum had no effect on the specific activities of
Rubisco and stromal FBPase (Figures 1H and 1K). The effects
of Al on these key enzymes associated with the Calvin cycle
and carbohydrate metabolism were similar when enzymatic
activities were expressed on a leaf dry mass basis rather than
on a leaf soluble protein basis (data not shown).
Leaf concentrations of G6P, F6P and PGA
Aluminum had no effects on area-based G6P and F6P concentrations or the G6P:F6P ratio, but it increased area-based PGA
concentration. When expressed on a leaf dry mass basis, G6P
and PGA concentrations were lower in Al-treated leaves than
in control leaves, whereas Al had no effect on F6P concentration (Table 4).
Leaf concentrations of nonstructural carbohydrates at dusk
and predawn
On a leaf area basis, there were no differences in the concentrations of glucose, fructose, sucrose, starch and total nonstructural carbohydrates (TNC) at either predawn or dusk
between Al-treated and control leaves (Figures 2A–E). Concentrations of sucrose, starch and TNC in control leaves were
higher at dusk than at predawn (Figures 2C–E), whereas glucose and fructose concentrations did not change with measurement time (Figures 2A and 2B). No difference was found in
the concentrations of glucose, fructose, starch and TNC in
Al-treated leaves between measurement times (Figures 2A,
2B, 2D and 2E), whereas sucrose concentration was greater at
dusk than at predawn (Figure 2C). When expressed on a leaf
dry mass basis, concentrations of fructose, sucrose, starch and
TNC at dusk and concentrations of glucose and sucrose at predawn were slightly higher in control leaves than in Al-treated
leaves, whereas the other mass-based results were similar to
the area-based results (Figures 2A–J).
Leaf absorptance and Chl fluorescence variables
Aluminum treatment decreased leaf absorptance, non-photochemical quenching coefficient (NPQ), maximal PSII quantum efficiency (Fv /Fm ) of dark-adapted leaves at predawn,
ratio of variable to initial Chl fluorescence (Fv /Fo ), photochemical quenching coefficient (qP), actual quantum yield of
PSII electron transport (Φ PSII ) and electron transport rate
through PSII by 1, 19, 4, 19, 44, 46 and 47%, respectively,
whereas it increased excess energy by 23%. There was no significant difference in the efficiency of excitation transfer
(Fv′/Fm′) between Al-treated leaves and control leaves (Table 5).
Discussion
We found that CO2 assimilation decreased in response to Al
(Table 3), which is in agreement with previous results obtained
Table 2. Chlorophyll (Chl) concentrations, the Chl a/b ratio, and area- and mass-based aluminum (Al) and total soluble protein concentrations in
Al-treated and control leaves (n = 6). Within a column, values followed by different letters are significantly different at P < 0.05.
Treatment
Al
Control
Chl
(mg m – 2 )
Chl a
(mg m – 2 )
Chl b
(mg m – 2 )
Chl a/b
590 a
643 b
452 a
480 a
139 a
164 b
3.26 a
2.93 b
Al
Soluble protein
Area-based
(g m –2)
Mass-based
(mg g –1
DM )
Area-based
(g m –2)
Mass-based
(mg g –1
DM )
2.74 a
1.70 b
31.1 a
23.4 b
9.04 a
7.27 b
103 a
100 a
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ALUMINUM DECREASES CO 2 ASSIMILATION IN CITRUS
321
Table 3. Assimilation of CO2, stomatal conductance and intercellular
CO2 concentration in aluminum (Al)-treated and control leaves (n =
6). Within a column, values followed by different letters are significantly different at P < 0.05.
Treatment
CO2
Stomatal
assimilation
conductance
(µmol m –2 s –1) (mmol m –2 s –1)
Intercellular CO2
concentration
(µmol mol –1)
Al
Control
3.95 a
6.55 b
242 a
229 b
70.2 a
101.5 b
for four Citrus spp. rootstocks (Pereira et al. 2000), and two
Triticosecale cultivars (Nunes et al. 1995). The reduced rate of
CO2 assimilation in Al-treated leaves (Table 3) was not caused
by stomatal limitation, because the intercellular CO2 concentration was higher in Al-treated leaves than in control leaves
(Table 3). Both Pereira et al. (2000) and Peixoto et al. (2002)
found higher intercellular CO2 concentrations in Al-treated
leaves than in control leaves, whereas Moustakas et al. (1996)
observed a 10% decrease in intercellular CO2 concentration in
plants treated with 1 mM Al. The Al-induced reduction in
stomatal conductance (Table 3) is in agreement with results reported in wheat (Triticum aestivum L.) (Moustakas et al.
1996). In contrast, stomatal conductance of ‘Carvo’ lemon
(Citrus limonia) and ‘Volkamer’ lemon (Citrus volkameriana)
seedlings was not significantly affected by Al concentrations
up to 400 µM (Pereira et al. 2000). Peixoto et al. (2002) observed that Al decreased stomatal conductance to a similar degree in both Al-tolerant and Al-sensitive cultivars of sorghum
on Days 8 and 10 after the beginning of Al treatment, respectively; thereafter, however, there was no consistent effect of Al
on stomatal conductance in either cultivar. Thus, it appears
that the influence of Al on stomatal conductance and intercellular CO2 concentration depends on Al concentration, time
of exposure to Al, and the species or cultivar.
The decrease in Chl concentration in response to the Al
treatment (Table 2) was probably not the primary factor limiting CO2 assimilation, because there was a greater excess of absorbed PPF in Al-treated leaves than in control leaves (Table 5). The slight increase in the Chl a/ b ratio in Al-treated
seedlings most likely resulted from the preferential photodestruction of Chl b, because Chl b decreased more than Chl a
(Table 2). There are mixed reports about the effects of Al on
Chl a/b ratio; Al increased the ratio in longan seedlings (Xiao
2002), but decreased it in sorghum (Peixoto et al. 2002).
In our study, the reduction in CO2 assimilation in Al-treated
leaves cannot be attributed to a decrease in activities of Calvin
cycle enzymes, because Al caused an increase or no change in
the activities of enzymes involved in the Calvin cycle, depending on the basis (area, mass or protein) used to express enzymatic activity (Figures 1A–D and 1H–K; data expressed on a
mass basis not shown). This contrasts with previous findings
that heavy metals affect CO2 assimilation by inhibiting enzymes in the Calvin cycle (Malik et al. 1992, Krupa and
Baszyñski 1995, Krupa 1999, Chugh and Sawhney 1999, Romanowska 2002). Area-based activities of Rubisco, GAPDH,
Figure 1. Activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, A and H), NADP-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, B and I), phosphoribulokinase (PRK, C and
J), stromal fructose-1,6-bisphosphatase (FBPase, D and K), cytosolic
FBPase (E and L), ADP-glucose pryphosphorylase (AGPase, F and
M) and sucrose phosphate synthase (SPS, G and N) expressed on a
leaf area or a leaf soluble protein basis in aluminum (Al)-treated and
control leaves. Bars represent means ± SE (n = 6). Different letters
above the bars indicate a significant difference at P < 0.05.
PRK and stromal FBPase were significantly higher in
Al-treated leaves than in control leaves (Figures 1A–D). The
higher activities of Rubisco and stromal FBPase in Al-treated
leaves (Figures 1A and D) could be associated with increased
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CHEN, QI, SMITH AND LIU
Table 4. Area- and mass-based concentrations of glucose-6-phosphate (G6P), fructose-6-phosphate (F6P) and 3-phosphoglycerate (PGA), and
G6P:F6P ratio in aluminum (Al)-treated and control leaves (n = 6). Within a column, values followed by different letters are significantly different
at P < 0.05.
Treatment
G6P
F6P
–2
Al
Control
µmol m
nmol
37.8 a
39.8 a
429 a
549 b
g –1
DM
µmol m
PGA
–2
10.9 a
11.0 a
area-based total soluble protein concentration (Table 2), because the specific activities of these enzymes remained unchanged (Figures 1H and 1K). In contrast, Al increased specific activities of GAPDH and PRK (Figures 1I and 1J). It is
Figure 2. Glucose (A and F), fructose (B and G), sucrose (C and H),
starch (D and I) and total nonstructural carbohydrate (TNC, E and J)
concentrations expressed on a leaf area or a leaf dry mass basis at predawn (open columns) and at dusk (solid columns) in Al-treated leaves
and control leaves. Bars represent means ± SE (n = 5). Different lowercase letters above the bars indicate significant differences at P < 0.05
between predawn and dusk values within a treatment. Different capital letters above the bars indicate significant differences at P < 0.05
between values in the control and aluminum (Al) treatments within
each schedule.
nmol
g –1
DM
124 a
152 a
µmol m
146 a
189 b
G6P:F6P
–2
µmol
g –1
DM
1.66 a
2.60 b
3.48 a
3.67 a
unclear whether these two enzymes were stimulated or induced by Al. Because no significant difference was found in
mass-based total soluble protein concentration between
Al-treated leaves and control leaves (Table 2), the higher
area-based total soluble protein concentration in Al-treated
leaves was probably associated with the increase in specific
leaf mass (Table 1).
The principal end products of leaf photosynthesis are starch
and sucrose. When triose phosphate utilization cannot keep
pace with triose phosphate production, a feedback mechanism
is triggered to down-regulate light energy input and Rubisco
(Sharky 1990). In our experiment, CO2 assimilation in Altreated leaves could not have been limited by end-product synthesis, because AGPase activity (Figure 1F) was increased,
cytosolic FBPase and SPS activities were little affected (Figures 1E and G) and starch concentration was not increased by
Al (Figures 2D and 2I).
Aluminum did not cause accumulation of leaf carbohydrates (Figures 2A–J), suggesting that feedback inhibition by
carbohydrate accumulation did not contribute to the decrease
in CO2 assimilation in Al-treated leaves (Table 3). If the export
of carbon from starch degradation during the night represents
the sink activity, our results indicate that the demand for carbohydrates decreased in Al-treated leaves (Figures 2D and 2I). It
could be that Al decreases both the utilization of carbohydrates for growth and the translocation of carbohydrates from
the leaves, which would explain why Al had little effect on carbohydrate concentration despite causing severe impairment of
CO2 assimilation. Although AGPase activity was higher in
Al-treated leaves than in control leaves (Figures 1F and 1M),
starch concentration was not increased by Al (Figures 2D and
2I). It is unclear whether AGPase was in excess or does not catalyze a rate-limiting step in citrus leaves.
Dark-adapted Fv /Fm was only slightly decreased by Al (Table 5), suggesting that the potential maximum photochemical
efficiency of PSII was little affected by Al. This is in agreement with Lidon et al. (1999), who found that Fv /Fm did not
significantly change in maize in responses to Al, but contrasts
with the findings of Peixoto et al. (2002), who found that
Fv /Fm was reduced by Al in sorghum. We found that the Fv /Fo
ratio was decreased by only 19% in Al treatment (Table 5) and
was less affected than CO2 assimilation, suggesting that PSII
photochemical efficiency was largely unrelated to the decrease
in CO2 assimilation.
Actual quantum yield of PSII electron transport (Φ PSII ),
which indicates photochemical efficiency under steady-state
light conditions, is the product of qP and the efficiency of exci-
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ALUMINUM DECREASES CO 2 ASSIMILATION IN CITRUS
323
Table 5. Leaf absorptance, non-photochemical quenching (NPQ), maximum PSII efficiency of dark-adapted leaves (Fv /Fm ), ratio of variable to
initial chlorophyll fluorescence (Fv /Fo ), photochemical quenching coefficient (qP ), efficiency of excitation transfer (Fv′/Fm′), actual quantum
yield of PSII electron transport (Φ PSII ), electron transport rate through PSII and excess energy in aluminum (Al)-treated and control leaves (n = 6).
Within a column, values followed by different letters are significantly different at P < 0.05.
Treatment
Absorptance
(%)
NPQ
Fv /Fm
Fv /Fo
qP
Fv′/Fm′
Φ PSII
Electron transport
rate (µmol m –2 s –1)
Excess energy
(µmol m –2 s –1)
Al
Control
92 a
93 b
0.55 a
0.68 b
0.80 a
0.83 b
3.98 a
4.92 b
0.24 a
0.42 b
0.62 a
0.66 a
0.15 a
0.27 b
69 a
129 b
223 a
181 b
tation capture Fv′/Fm′ by open PSII centers under non-photorespiratory conditions (Genty et al. 1989). Because there was
more excess absorbed energy in Al-treated leaves than in control leaves, Φ PSII should be down-regulated to match photosynthetic electron transport. There are two ways in which Φ PSII
can be down-regulated. One is to decrease the efficiency with
which excitation energy is delivered to PSII centers by thermal
dissipation of excitation energy in the antenna pigment complexes (Genty et al. 1990). The alternative is to close PSII reaction centers by over-reducing the electron acceptors of PSII.
Because there was no significant difference in Fv′/Fm′ between
Al-treated and control leaves (Table 5), the decrease in ΦPSII in
our Al-treated leaves must have resulted from an increase in
closed PSII reaction centers, as indicated by the decrease in
qP. We conclude that the main factor leading to a decrease in
electron transport rate through PSII in the presence of Al was
the decrease in Φ PSII, because leaf absorptance (Table 5) was
little affected by Al.
Aluminum slightly reduced NPQ (Table 5), suggesting that
thermal dissipation did not have a central role in dissipating
excess excitation energy in Al-treated leaves. Thus, it is likely
that both the water–water cycle and photorespiration, two processes involved in dissipating excess excitation through electron transport (Asada 1999), are up-regulated to cope with the
increased excess excitation energy in response to Al. This suggestion is supported by our finding that activities of enzymes
and concentrations of antioxidant metabolites involved in the
water–water cycle all increased significantly in response to Al
(data not shown). Activity of catalase (CAT, EC 1.15.1.1), an
enzyme involved in scavenging the bulk H2O2 generated by
photorespiration (Willekens et al. 1995), was increased significantly by Al (data not shown). The significant increase in
area-based Rubisco activity (Figure 1A) suggests that the
higher CAT reflects a higher rate of photorespiration in Altreated leaves.
Aluminum had no effect on leaf malondialdehyde (MDA, a
marker of peroxidative damage) concentration (data not
shown), suggesting that Al has no effect on the membrane lipid
in the chloroplast. Therefore, decreased CO2 assimilation in
Al-treated leaves cannot be attributed to photo-oxidative damage.
We conclude that Al decreased CO2 assimilation, but either
increased or had no effect on the activities of enzymes involved in the Calvin cycle, depending on the basis used for expressing enzyme activity. The Al-induced decrease in CO2 assimilation capacity was not associated with feedback inhibi-
tion by carbohydrate accumulation, but was probably caused
by a combination of factors, including a reduced rate of electron transport through PSII, increased closure of PSII reaction
centers and increased photorespiration.
Acknowledgments
This work was supported by the National Natural Science Foundation
of China (30270930). We acknowledge the invaluable help of Dr. L.
Cheng (Cornell University, USA) during the study.
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