Plant Science 166 (2004) 1565–1573
The carbohydrate metabolism enzymes sucrose-P synthase and
ADG-pyrophosphorylase in phaseolus bean leaves are up-regulated at
elevated growth carbon dioxide and temperature
P.V. Vara Prasad a,∗ , Kenneth J. Boote a , Joseph C.V. Vu b , L. Hartwell Allen, Jr. b
b
a Agronomy Department, University of Florida, 304 Newell Hall, Gainesville, FL 32611, USA
Agricultural Research Service, United States Department of Agriculture, Gainesville, FL 32611, USA
Received 1 November 2003; received in revised form 13 February 2004; accepted 13 February 2004
Abstract
Kidney bean (Phaseolus vulgaris L. cv. Montcalm) plants were grown under daytime maximum/nighttime minimum temperatures of
28/18, 34/24 and 40/30 ◦ C at ambient carbon dioxide concentration (CO2 ; 350 ␮mol mol−1 ), and 28/18, 31/21, 34/24, 37/27 and 40/30 ◦ C
at elevated (twice-ambient) CO2 , to characterize how increases in growth CO2 and temperature affected kidney bean leaf photosynthesis
and carbohydrate metabolism. Elevated CO2 enhanced leaf photosynthetic rates by about 57% across the temperature regimes, compared
with plants grown an ambient CO2 . As growth temperature increased from 28/18 to 40/30 ◦ C, leaf photosynthetic rates decreased at both
ambient and elevated CO2 . Growth at either elevated temperature or CO2 decreased activity, protein content and activation of the primary
photosynthetic enzyme ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco). Elevated CO2 increased activities of sucrose-phosphate
synthase (SPS) and adenosine-5 -diphosphoglucose pyrophosphorylase (AGP) and accumulation of soluble sugars and starch across all
temperatures, compared with plants grown at ambient CO2 . At elevated CO2 , growth temperatures above 34/24 ◦ C significantly increased
leaf carbohydrates (total soluble sugars and starch) and activity of AGP. The up-regulation of leaf carbohydrate metabolism enzymes under
elevated CO2 plus temperature would be beneficial for growth and productivity of kidney bean in future climates.
© 2004 Elsevier Ireland Ltd. All rights reserved.
Keywords: Carbon dioxide; Carbohydrate; SPS; AGP; Rubisco; Photosynthesis
1. Introduction
At the present rates of greenhouse gas emissions, atmospheric carbon dioxide concentration (CO2 ) is expected to
reach more than twice the pre-industrial concentration by
the end of this century [1]. In addition, climate models predict that the doubling of CO2 will increase the global average surface air temperatures by 1.4 to 5.8 ◦ C [1,2]. These
changes in CO2 and temperature will not only influence climate but also the physiological processes, including photosynthesis, carbohydrate metabolism, growth and yield of
agricultural crop plants.
Leaf photosynthesis in C3 plants is influenced by the
primary carbon fixation enzyme ribulose-1,5-bisphosphate
∗ Corresponding author. Tel.: +1-352-3921811x232;
fax: +1-352-392-1840.
E-mail addresses: [email protected], [email protected]
(P.V.V. Prasad).
carboxylase-oxygenase (Rubisco) as well as by the accumulation and metabolism of carbohydrates in the leaf
and source-sink balance [3,4]. Long-term exposure to elevated CO2 often results in down-regulation of Rubisco [3].
Detailed survey by Long and Drake [5] showed that, on
average, elevated CO2 decreased both Rubisco content and
activity in the range of 8 to 15% in plants grown in pots and
large rooting volumes. Similarly, increases in air temperatures to the extent of 5–6 ◦ C above present ambient levels
is known to influence photosynthesis, Rubisco content and
carbohydrate metabolism [3,6].
Phaseolus bean is an important grain legume crop and a
rich source of protein and carbohydrates for human populations across the globe. Elevated CO2 caused increases in
leaf photosynthetic rates of bean in some studies [7,8], while
others have observed no beneficial effects [9–11]. In potted
bean plants, CO2 enrichment (700 ␮mol mol−1 ) compared
to ambient (360 ␮mol CO2 mol−1 ) increased net carbon assimilation rates early in the season, but from 25 days after
0168-9452/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.plantsci.2004.02.009
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P.V.V. Prasad et al. / Plant Science 166 (2004) 1565–1573
germination there was a steady decline to almost similar levels to those measured in plants grown under ambient CO2
[12]. These differential responses of photosynthesis to CO2
enrichment could be attributed to varying experimental techniques, methodologies to elevate CO2 during plant growth
[13], restricted root volumes [8,14], age of the crop or cultivar differences. Leaf photosynthetic rates of bean were optimum at 25 ◦ C [15], and increasing temperature from 25
to 45 ◦ C for a 5 h period significantly decreased subsequent
carbon exchange rates of bean by 40–50% [16]. Influence
of high temperatures on Rubisco activity and carbohydrate
metabolism of bean has received less attention. However, for
soybean (Glycine max L. Merr.) Rubisco activity and Rubisco protein content at temperatures from 28/18 to 40/30 ◦ C
were similar, but declined at 44/34 or 48/38 ◦ C under both
ambient and elevated CO2 [17].
The declines in leaf photosynthetic capacities are often
associated with increases in leaf carbohydrates, which occurred under both large and small rooting volumes even
when grown under sufficient nitrogen supply [5]. One of
the explanations for declines in Rubisco is the imbalance in
source-sink capacities, especially insufficient sink capacity
for the excess carbohydrates [18,19]. There is a greater accumulation of carbohydrates in plants grown under elevated
CO2 due to increased photosynthetic rates [17]. Studies have
shown that soluble carbohydrates particularly glucose and
sucrose signal the repression of genes which code for Rubisco small and large subunits [20] which could result in
lower Rubisco protein. Therefore, if process of resource optimization is involved, the down-regulation of carbon gain
may cause up-regulation of the capacity for carbon utilization and export.
In the advent of climate change increases in CO2 will
be associated with elevated temperature and both could
have significant influence on leaf photosynthesis as well
as carbohydrate metabolism and related enzyme activities
[17,21,22]. Although a few studies have investigated influence of CO2 [7,9,11,12] and temperature [16] separately on
photosynthesis, independent or combined effects of elevated
CO2 and high temperature on carbohydrate metabolism,
photosynthetic capacity and related enzyme activities have
received less attention for major crops and particularly
for bean. The key enzymes involved in carbon utilization,
i.e. synthesis of sucrose and starch are sucrose-phosphate
synthase (SPS) and adenosine-5 -diphosphoglucose pyrophosphorylase (AGP). Studies have shown elevated CO2
increases the activities of SPS in rice [22] and that of
SPS and AGP in soybean when compared to ambient CO2
conditions [17]. There is indeed a need for understanding
leaf carbohydrate metabolism in response to both rising
atmospheric CO2 and temperature.
The objectives of the present research were (a) to determine the season long effects of elevated CO2 and/or
temperature on leaf photosynthesis, Rubisco activity and
protein content, carbohydrate accumulation, and carbohydrate metabolism enzyme activities in bean leaves; and (b)
to test the hypotheses that growth at elevated CO2 and/or
temperature leads to an up-regulation of the carbohydrate
metabolizing enzymes SPS and AGP to accommodate for
export of the excess carbohydrates resulting from increased
photosynthetic rates.
2. Materials and methods
2.1. Plant material and growth conditions
Uniform seeds of red kidney bean (Phaseolus vulgaris
L. cv Montcalm) were inoculated with Rhizobium (Nitragin; Lipha Tech, Inc., Milwaukee, Wisconsin, USA) and
sown at a depth of 3 cm in north–south rows at a spacing of 33 cm × 10 cm (six, 0.9 m long rows per chamber
and nine hills per row) on 15 August 2000 in eight sunlit
controlled environment chambers. Each chamber consisted
of polyethylene telphtalate tops and sides on an aluminum
frame 2 m long, 1 m wide and 1.5 m high. Temperature in
each chamber was controlled on a diurnal sinusoidal wave
between the diel maximum and minimum (gradual increase
or decrease in temperature) as described by Prasad et al.
[23]. The dewpoint temperature was maintained 5 ◦ C below
the target day and night air temperatures. The air temperature in each chamber was measured above the crop canopy
by using shielded, aspirated thermocouples. Readings were
taken every second and means of successive 5 min periods
were stored using a data logger (CR10, Campbell Scientific Inc., North Logan, Utah, USA). Photosynthetic photon
flux density was measured on top of each chamber using
calibrated photovoltaic cells (Panasonic, Atlanta, Georgia,
USA).
Carbon dioxide concentrations in the chambers were measured and maintained either at ambient (350 ␮mol mol−1 )
or elevated (700 ␮mol mol−1 ) levels as per treatment by injecting pure CO2 with mass flow controllers (Brooks Instruments, Hatfield, Pennsylvania, USA) from high-pressure
cylinders. The CO2 concentration in each chamber was measured by infrared gas analyzer (Siemens Corporation, New
York, USA), and controlled and recorded by the CR10 data
loggers. The actual mean day and night air temperatures
were ±0.3 ◦ C of the target temperatures. Similarly, the actual CO2 concentration was ±5 ␮mol CO2 mol−1 of the target CO2 levels at all temperature treatments. The details of
the chamber characteristics, their function, specific methods
for controlling set environments, and quality of environment
controls are described elsewhere [23,24].
Plants in all eight chambers were grown at a daytime
maximum/nighttime minimum air temperature regime of
31/21 ◦ C from sowing to emergence (i.e. 8 days after sowing, DAS). Thereafter, plants in each chamber were exposed
at air temperature regimes of 28/18, 31/21, 34/24, 37/27 and
40/30 ◦ C at elevated CO2 , and 28/18, 34/24 and 40/30 ◦ C
at ambient CO2 until maturity. Plants were irrigated with
overhead sprinklers from sowing to 20 DAS and thereafter
P.V.V. Prasad et al. / Plant Science 166 (2004) 1565–1573
they were dependent on subsurface irrigation provided by
constant water table at 0.5 m below the soil surface. This
worked well because the Kendrick fine sand soil had good
hydraulic capillary flow. Thinning was done at 10 days after emergence, leaving one plant per hill (i.e. nine plants per
row) and a plant density of 27 plants per m2 . The crop was
kept weed-free and healthy throughout the season by manual hand weeding and release of biological predator green
lacewing (Chysoperla spp.) to control aphids, thrips, white
flies and red spider mites.
The crop was fertilized with 60 g m−2 each of N, P and K
as a basal application by broadcasting the fertilizer and incorporating to 15 cm soil depth. At planting the organic nematicide Nem-A-cide [a.i. Chitin (poly-N-acetyl-d-glucosamine)
protein; Voluntary Purchasing Groups Inc, Bonham, Texas,
USA] was incorporated into the soil at 250 g m−2 to protect
from nematode damage. At 60 DAS, plants were fertilized
with 40 g m−2 each of N, P and K with water-soluble fertilizer. Black polypropylene shades were placed around the
sides of the crop canopy to create an environment similar to
that of border rows in the field.
2.2. Leaf sampling
At 42 DAP, on a clear sunny day, eight to 10 uppermost
fully-expanded leaves from each treatment were randomly
collected from 10 different plants at midday (1300 h) when
PPFD was 1600–1800 ␮mol m−2 s−1 . Leaf samples were
immediately immersed in liquid N2 , ground to a fine powder in liquid N2 and then stored in liquid N2 until further
analyses. Most of the hard midrib vein was removed while
grinding the samples. There were three replications (three
separate extractions) of leaf powder and each replication was
assayed three times (i.e. total of nine observations) for carbohydrate contents, activities of SPS and AGP, and activity
and content of Rubisco. Leaf samples were also collected
at the same time for determination of leaf area and leaf dry
weight and used for expression of data based on unit leaf
area.
2.3. Photosynthesis measurement
On the same day of leaf sampling, photosynthetic rates
of individual attached leaves were measured between
1100 and 1400 h, using a LI-6200 Portable Photosynthesis System (LICOR, Lincoln, USA). Each observation
was repeated three times on three different randomly selected, fully-exposed leaves from three different plants,
after the measuring cuvette had equilibrated to the temperature and CO2 in the growth chamber and when the
solar photosynthetic photon irradiance was between 1600
and 1800 ␮mol m−2 s−1 . The duration of each measurement typically lasted 45 s. Leaflets from the photosynthesis
measurements were marked and their area and dry weights
recorded. Photosynthetic rates were expressed on a leaf area
basis.
1567
2.4. Assays of soluble sugars and starch
Microplate technique as described by Cairns [25] and
Hendrix [26] with slight modifications was used for analysis
of carbohydrates. For extraction of carbohydrates, approximately 100 mg of liquid N2 frozen leaf powder was added to
tubes containing 80% (v/v) ethanol and placed in an 85 ◦ C
water bath for about 1 h with frequent decantation and addition of ethanol. Finally, the volume of ethanol-soluble supernatant was adjusted to 10 ml with addition of 80% (v/v)
ethanol, and mixed with 130 mg of finely divided activated
charcoal. The tubes were gently shaken and refrigerated until
assay. The ethanol-insoluble pellet was oven-dried at 60 ◦ C
and saved for starch analysis. The charcoal purification step
was essential to remove ethanol-soluble materials in plant
extracts which interfere with subsequent enzyme-coupled
assays [26,27]. Sugars were unaffected by this processes as
the charcoal treatment was performed prior to evaporation of
ethanol and dilution in water [28]. Our data from controlled
experiments with and without activated charcoal using standard glucose (Sigma G-7528) and sucrose (Sigma S-7903)
solutions showed full recovery of sugars and there was no
effect of charcoal on sugar recovery.
To assay soluble sugars (glucose, fructose and sucrose),
the refrigerated extract was centrifuged at 12,000 × g for
15 min. Twenty ␮l aliquots of the supernatant were added
to the micro-plate wells and heated at 70 ◦ C for 5 min to
evaporate the ethanol. Then 20 ␮l of water was added followed by 100 ␮l of assay mixture. The micro-plate was gently mixed, covered with aluminum foil and kept in darkness
for 15–30 min at room temperature. The optical density of
each well was then determined at 490 nm.
For the starch analysis, the dry pellet from each sample
was digested with 1 ml of 0.2 N KOH in a boiling water
bath for 30 min. After cooling to room temperature, 0.2 ml
of acetic acid and 2 ml of acetate buffer (pH 4.6) containing
␣-amyloglucosidase (six units) were added, and the tubes
were incubated at 55 ◦ C for 60 min to complete starch hydrolysis. Then 2 ml of water was added and the tubes were
mixed and centrifuged. To assay starch, 20 ␮l of supernatant
was added to the wells of a standard micro-plate for glucose
analysis. Glucose equivalents from standard curves were determined to estimate starch concentration, as described by
Hendrix [26].
2.5. Assay of leaf Rubisco activity and protein content
The initial and total activity of Rubisco (carboxylase) (EC
4.1.1.39) was assayed as described by Vu et al. [21] with
slight modifications. About 150 mg of the ground frozen leaf
powder was transferred to a pre-chilled (2 ◦ C). Ten Broeck
homogenizer and ground on ice in 3 ml ice-cold medium
containing 50 mM of Tris–HCl (pH 8.5), 5 mM MgCl2 ,
5 mM DTT, 0.1 mM EDTA and 1.5% (w/v) PVP-40. The
homogenate was micro-centrifuged at 12,000 × g for 30 s
at 2 ◦ C, and an aliquot of the supernatant was immediately
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P.V.V. Prasad et al. / Plant Science 166 (2004) 1565–1573
assayed for initial and total activity of Rubisco at 30 ◦ C.
The initial activity was measured by injecting 100 ␮l of
supernatant into the reaction mixture (500 ␮l of total volume) containing 50 mM Tris–HCl (pH 8.5), 5 mM DTT,
10 mM MgCl2 , 0.1 mM EDTA, 0.5 mM RuBP and 10 mM
of NaH14 CO3 . The assay reaction was terminated after 45 s
with 0.1 ml of 6 N HCl.
The total activity of Rubisco was also measured in a manner similar to that of initial activity, except that the reaction mixture did not contain RuBP. After a 5 min activation
period, the reaction was initiated by adding RuBP and the
reaction was terminated after 45 s by adding HCl. In the
control assays, RuBP was not added to ensure that no other
carboxylase was active in the crude extract. After the assay,
each mixture was dried at 60 ◦ C and the acid-stable 14 C radioactivity was determined by liquid scintillation spectrometry. Activation of Rubisco was calculated as the ratio of the
initial to the corresponding total activity.
For determination of Rubisco protein content, about
100 mg of leaf powder stored in liquid N2 was ground in
2.5 ml of extraction mix containing 100 mM Bicine–NaOH
buffer (pH 7.8), 5 mM DTT, 0.1 mM EDTA, 10 mM MgCl2 ,
10 mM NaHCO3 and 2% (w/v) PVP-40. The homogenate
was micro-centrifuged at 12,000 × g for 1 min at 2 ◦ C.
A 25 ␮l aliquot of the supernatant was then added to
a 50 ␮l assay mixture [100 mM Bicine–NaOH, 20 mM
MgCl2 and 1 mM EDTA at pH 7.8] containing 4 nmol
2-14 C-carboxyarabinitol bisphosphate (1.92 TBq mol−1 )
and 50 ␮l of antiserum to purified tobacco Rubisco raised
from rabbits (antiserum was supplied by Kel Farm, Alachua,
Gainesville, FL, USA). After incubation for 2 h at 37 ◦ C,
the precipitate was collected on Millipore filtration apparatus with 0.45 ␮m filter and washed with 5 ml of a 0.85%
(w/v) NaCl solution containing 10 mM MgCl2 . The filter
was then transferred to a scintillation vial and bound 14 C
was determined by a liquid scintillation counter. For each
replication blanks were run in parallel.
Total soluble protein in the supernatant was determined
using Bio-Rad protein reagent (Bio-Rad Laboratories, Inc,
California) against a bovine serum albumin standard. Total
leaf nitrogen was estimated using standard micro-Kjeldahl
method. Specific leaf weight is defined as leaf dry weight
per unit leaf area; and specific leaf nitrogen is defined as
leaf N content per unit leaf area.
2.6. Assays of SPS and AGP
Frozen leaf samples from midday harvested material
were extracted and assayed for activities of SPS (EC
2.4.1.14) and AGP (EC 2.7.7.27) with some modifications
to the procedures originally described by Huber et al. [29]
and Nakamura et al. [30], respectively. For extraction of
SPS, 150 mg of frozen ground leaf powder was homogenized in a medium consisting of 50 mM MOPS–NaOH
(pH 7.5), 15 mM MgCl2 , 1 mM EDTA, 2.5 mM DTT,
0.1% Triton X-100, 2% PVP-40, 10 ␮M leupeptin and
1 mM PMSF. The extract was centrifuged at 12,000 × g
for 45 s and the supernatant was immediately desalted on
a Sephadex G-25 column equilibrated with the extraction
buffer at 2 ◦ C. For the SPS assay, 25 ␮l of the desalted
enzyme extract was incubated at 30 ◦ C with an assay mixture consisting of 50 mM MOPS–NaOH (pH 7.5), 15 mM
MgCl2 , 2.5 mM DTT, 10 mM UDPG, 10 mM F6P and
40 mM G6P, in a total volume of 200 ␮l. After a 10 min
incubation, the reaction was terminated by the addition
of 70 ␮l of 1 N NaOH. Placing the tubes in boiling water for 10 min destroyed the un-reacted F6P. Thereafter,
0.25 ml of 0.1% resorcinol in 95% ethanol and 0.75 ml of
30% HCl were added, and the mixture was incubated at
80 ◦ C for 8 min. After cooling, absorbance at 520 nm was
measured.
For extraction of AGP, approximately 100 mg of liquid N2
frozen powdered leaf tissue was homogenized at 2 ◦ C in 3 ml
of extraction buffer containing 50 mM HEPES–NaOH (pH
7.0), 10 mM MgCl2 , 0.1 mM EDTA, 5 mM DTT and 10%
(v/v) glycerol. The extracts were centrifuged at 12,000 × g
at 2 ◦ C for 60 s and assayed for AGP. Activities in crude
extract were assayed in two stages. Stage 1 assay included
100 mM HEPES–NaOH (pH 7.4), 5 mM MgCl2 , 4 mM DTT,
3 mM PGA, 3 mM NaPPi and 2 mM AGP. The assay was
initiated by adding 20 ␮l of the extract in a total volume of
250 ␮l. After 10 min at 30 ◦ C, the reaction was terminated
by boiling for 1 min. Thereafter the samples were frozen
at −20 ◦ C. The following day during Stage 2, the samples
were thawed and diluted by addition of 350 ␮l of water and
centrifuged at 12,000 × g for 5 min. Afterwards 500 ␮l of
supernatant was mixed with 15 ␮l of 10 mM NADP and the
initial absorbance at 340 nm was recorded. The reaction was
initiated by adding 2 ␮l of coupling enzymes; one unit each
of phosphoglucomutase and glucose-6-phosphate dehydrogenase, and increase in optical density at 340 nm was measured. Activities of AGP were calculated by reference to a
G1P standard generated for bean. Blanks for each replication were run in parallel using complete assays reaction mix
with enzyme denatured by boiling.
2.7. Data analysis
Means and standard errors of means of all the measured
and computed variables were derived from a total of nine observations (i.e. three replications and three measurements).
The significant difference between the treatment means was
tested at 95% confidence interval using t-test and two-tail P
(probability) values using data analyses techniques in SAS.
3. Results
3.1. Leaf photosynthetic rates
Elevated CO2 , when compared to ambient CO2 , increased
leaf photosynthetic rates by 57% when averaged across
10
5
0
350 µmol CO2 mol
700 µmol CO2 mol
-1
-1
28/18 31/21 34/24 37/27 40/30
Air temperature (˚C)
(daytime maximum/nighttime minimum)
Fig. 1. Effects of ambient (䊉, 350 ␮mol mol−1 ) and elevated (䊊,
700 ␮mol mol−1 ) CO2 at different temperature regimes on midday leaf
photosynthetic rates of kidney bean measured on a cloudless day between
1100 and 1400 h when PPFD was 1600–1800 ␮mol m−2 s−1 . Vertical bars
indicate ± standard error and are shown where they exceed the size of
the symbol.
all growth temperature treatments (Fig. 1). The percentage
increases in leaf photosynthetic rates due to elevated CO2
were smaller at warmer temperatures (34/24 and 40/30 ◦ C)
compared to cooler temperature (28/18 ◦ C). Increases in
growth temperatures from 28/18 to 40/30 ◦ C, linearly decreases leaf photosynthetic rates under both ambient and
elevated CO2 .
3.2. Rubisco activity and protein content
Under high light conditions at midday, elevated CO2 decreased the initial Rubisco activity across all temperature,
and the percentage decrease was greater at 28/18 ◦ C (33%)
than at 34/24 ◦ C (25%) and 40/30 ◦ C (9%) (Fig. 2A). Increase in temperature from 28/18 to 40/30 ◦ C decreased
initial Rubisco activities under both ambient and elevated
CO2 treatments. Similar to initial Rubisco activity, total
Rubisco activity was decreased by elevated CO2 , as compared to ambient CO2 , at growth temperature of 28/18
and 34/24 ◦ C, but not at 40/30 ◦ C (Fig. 2B). The percentage decrease in total Rubisco activity due to elevated CO2
was, however, smaller at higher temperatures. At ambient CO2 , increases in growth temperature from 28/18 to
40/30 ◦ C linearly decreased total Rubisco activity. In contrast, there was no effect of growth temperature on total
Rubisco activity at elevated CO2 , with an exception at
37/27 ◦ C which was significantly higher than those at other
temperatures. There was no effect of elevated CO2 on Rubisco activation at growth temperatures (Fig. 2C). However,
increase in growth temperatures from 28/18 to 40/30 ◦ C decreased Rubisco activation under both ambient and elevated
CO2 .
Elevated CO2 decreased Rubisco protein content by
38% at 28/18 ◦ C and 24% at 34/24 ◦ C, but not at 40/30 ◦ C
(Fig. 3A). At ambient CO2 , growth temperature from 28/18
had higher Rubisco protein content when compared to
other growth temperatures. Total soluble protein was not
(µmol m-2 leaf area s-1)
15
Initial Rubisco activity
20
Total Rubisco activity
25
(µmol m-2 leaf area s-1)
30
Rubisco activation
(%)
Leaf photosynthetic rate
(µmol m-2 leaf area s-1)
P.V.V. Prasad et al. / Plant Science 166 (2004) 1565–1573
60
1569
(A)
-1
350 µmol CO2 mol
-1
700 µmol CO2 mol
45
30
15
0
60
(B)
45
30
15
0
100
(C)
90
80
70
60
28/18 31/21 34/24 37/27 40/30
Air temperature (˚C)
(daytime maximum/nighttime minimum)
Fig. 2. Effects of ambient (䊉, 350 ␮mol mol−1 ) and elevated (䊊,
700 ␮mol mol−1 ) CO2 at different temperature regimes on (A) initial
Rubisco activity; (B) total Rubisco activity; and (C) activation of Rubisco in kidney bean. Leaves were sampled at 1300 h, when PPFD was
1600–1800 ␮mol m−2 s−1 . Vertical bars indicate ± standard error and are
shown where they exceed the size of the symbol.
affected by elevated CO2 or temperature (Fig. 3B), but the
ratio of Rubisco protein to total soluble protein decreased
at elevated CO2 across all temperatures (Fig. 3C). At ambient CO2 , there was no effect of temperature increase from
28/18 to 34/24 ◦ C, but further increase in temperature to
40/30 ◦ C decreased the ratio of Rubisco to total soluble
protein.
3.3. Leaf nitrogen
Total leaf nitrogen concentration was decreased by elevated CO2 at all growth temperatures (Fig. 4A). But specific leaf N was increased by elevated CO2 by about 37%
at growth temperatures of 28/18 and 40/30 ◦ C (Fig. 4B)
as a result of increased specific leaf weight (Fig. 4C). In
general, there was no effect of growth temperature on total leaf N concentration, specific leaf N or specific leaf
weight at ambient CO2 . However, at elevated CO2 increase
in growth temperature from 28/18 to 34/24 ◦ C decreased
leaf N concentration, specific leaf N and specific leaf
weight, but further increase in temperature to 37/27 and
40/30 ◦ C increased the respective values to similar levels at
28/18 ◦ C.
P.V.V. Prasad et al. / Plant Science 166 (2004) 1565–1573
(A)
-1
350 µmol CO2 mol
-1
700 µmol CO2 mol
2.0
1.5
1.0
0.5
(g m-2 leaf area)
8
(B)
80
4
2
0
(C)
Specific leaf weight
-2
(g m leaf area)
30
15
0
28/18 31/21 34/24 37/27 40/30
20
0
3.4. Carbohydrate metabolism
Elevated CO2 significantly increased leaf sucrose
(Fig. 5A) and total soluble sugars (Fig. 5B) by 40% when
averaged across 28/18, 34/24 and 40/30 ◦ C growth temperatures. Increasing temperature from 28/18 to 34/24 ◦ C at
ambient or elevated CO2 decreased sucrose and total soluble sugars. But further increase of growth temperature to
40/30 ◦ C increased sucrose by 31 and 52% and total soluble
sugars by 10 and 36% at ambient and elevated CO2 , respectively. There were substantial increases in hexose sugars
under elevated CO2 particularly at higher growth temperatures of 37/27 and 40/30 ◦ C. Elevated CO2 increased starch
contents at all temperatures, but the response was greater
at the two highest growth temperatures (Fig. 5C). At ambient CO2 , starch content decreased linearly as growth
temperature increased from 28/18 to 40/30 ◦ C. In contrast,
at elevated CO2 , starch content was similar at growth temperature up to 34/24 ◦ C (mean = 14.8 g m−2 leaf area),
but further increase in temperature to 37/27 and 40/30 ◦ C
increased starch to 36 and 27 g m−2 leaf area, respectively.
There were positive correlations between activity of SPS
and accumulation of sucrose (r2 = 0.49; n = 8; P < 0.05),
and activity of AGP and accumulation of starch (r2 = 0.88;
n = 8; P < 0.01) (data not shown).
(B)
3
2
1
0
(C)
60
40
20
0
28/18 31/21 34/24 37/27 40/30
Air temperature (˚C)
(daytime maximum/nighttime minimum)
Fig. 3. Effects of ambient (䊉, 350 ␮mol mol−1 ) and elevated (䊊,
700 ␮mol mol−1 ) CO2 at different temperature regimes on (A) Rubisco
protein; (B) total soluble protein; and (C) ratio of Rubisco protein to total soluble protein in kidney bean. Leaves were sampled at 1300 h, when
PPFD was 1600 to 1800 ␮mol m−2 s−1 . Vertical bars indicate ± standard
error and are shown where they exceed the size of the symbol.
-1
350 µmol CO2 mol
-1
700 µmol CO2 mol
40
80
45
(A)
60
4
6
60
(%)
Rubisco/Total
Total soluble protein
0.0
Specific leaf nitrogen
-2
(g m leaf area)
Rubisco protein
(g m-2 leaf area)
2.5
Total leaf nitrogen
-1
(mg g leaf dry weight)
1570
Air temperature (˚C)
(daytime maximum/nighttime minimum)
Fig. 4. Effects of ambient (䊉, 350 ␮mol mol−1 ) and elevated (䊊,
700 ␮mol mol−1 ) CO2 at different temperature regimes on (A) total leaf
nitrogen concentration; (B) specific leaf nitrogen content; and (C) specific
leaf weight in kidney bean. Leaves were sampled at 1300 h, when PPFD
was 1600 to 1800 ␮mol m−2 s−1 . Vertical bars indicate ± standard error
and are shown where they exceed the size of the symbol.
Elevated CO2 compared to ambient CO2 increased SPS
activity by 48% at 34/24 ◦ C, and 58% at 40/30 ◦ C (Fig. 6A).
There was no effect of increasing temperature on SPS activity at elevated CO2 , while at ambient CO2 , activity of the
enzyme decreased as temperature increased from 28/18 to
34/24 ◦ C and 40/30 ◦ C. Elevated CO2 increased AGP activity across all temperatures, however, the percentage increase
was greater at lowest (28/18 ◦ C) and highest (40/30 ◦ C)
(Fig. 6B). At ambient CO2 , there was no effect of elevated
temperature on AGP activity. Whereas, at elevated CO2 ,
AGP activity decreased with increase in temperature from
28/18 to 34/24, but further increase in temperature to 37/27
and 40/30 ◦ C increased AGP activity.
4. Discussion
Overall elevated growth CO2 enhanced kidney bean leaf
photosynthesis by 57% across all temperatures. As growth
temperature increased from 28/18 to 40/30 ◦ C, there was a
small but significant decrease in leaf photosynthetic rates at
both CO2 treatments (Fig. 1). Nevertheless, midday leaf photosynthetic rates of elevated CO2 -grown plants at the highest temperature (40/30 ◦ C) was still 35% greater than that
P.V.V. Prasad et al. / Plant Science 166 (2004) 1565–1573
Sucrose
-2
(g m leaf area)
10
-1
350 µmol CO2 mol
-1
700 µmol CO2 mol
(A)
7.5
5.0
2.5
Total soluble sugars
-2
(g m leaf area)
0
10
7.5
5.0
2.5
0
40
Starch
-2
(g m leaf area)
(B)
(C)
30
20
10
0
28/18 31/21 34/24 37/27 40/30
Air temperature (˚C)
(daytime maximum/nighttime minimum)
Fig. 5. Effects of ambient (䊉, 350 ␮mol mol−1 ) and elevated (䊊,
700 ␮mol mol−1 ) CO2 at different temperature regimes on (A) sucrose
(B) total leaf soluble sugars; and (C) starch in kidney bean. Leaves were
sampled at 1300 h, when PPFD was 1600–1800 ␮mol m−2 s−1 . Vertical
bars indicate ± standard error and are shown where they exceed the size
of the symbol.
SPS activity
(µmol m-2 leaf area s-1)
10
350 µmol CO2 mol
(A)
-1
700 µmo l CO2 mol
-1
8
6
4
2
AGP activity
(µmol m-2 leaf area s-1)
0
25
(B)
20
15
10
5
0
28/18
31/21
34/24
37/27
40/30
Air temperature (˚C)
(daytime maximum/nighttime minimum)
Fig. 6. Effects of ambient (䊉, 350 ␮mol mol−1 ) and elevated (䊊,
700 ␮mol mol−1 ) CO2 at different temperature regimes on activities of (A)
sucrose-phosphate synthase (SPS); and (B) adenosine-5 -diphosphoglucose
pyrophosphorylase (AGP) in kidney bean. Leaves were sampled at 1300 h,
when PPFD was 1600–1800 ␮mol m−2 s−1 . Vertical bars indicate ± standard error and are shown where they exceed the size of the symbol.
1571
of ambient CO2 plants at the lowest temperature (28/18 ◦ C),
indicating that the deleterious effects of high temperature
on photosynthesis could be compensated by elevated CO2 .
The beneficial interactions of CO2 and high temperature on
leaf photosynthesis, however, were not reflected on reproductive processes and seed yields, especially at above optimum growth temperatures in this study on kidney bean [31]
and other C3 species such as peanut [23], soybean [32] and
cotton [33].
Growth at elevated CO2 increased leaf carbohydrate
metabolism and accumulation of carbohydrates in leaves
across all temperatures (Figs. 5 and 6). However, carbohydrate metabolism showed a threshold temperature at
34/24 ◦ C, above which the positive response was greater.
This response was due mainly to the fact that there were
severe reproductive failures at temperatures above 34/24 ◦ C
[31]. No seeds were produced at growth temperatures
≥37/27 ◦ C, causing no sink demand for the carbohydrates,
resulting in greater carbohydrate accumulation in leaves.
This was more prominent under elevated CO2 because of
relatively higher photosynthetic rates. Furthermore, elevated
CO2 did not increase the individual leaf areas, resulting in
thicker leaves as apparent from greater specific leaf weight
(Fig. 4C).
For many plant species, long-term growth at elevated CO2
results in a down-regulation of Rubisco [10,13,21,34]. Both
“coarse” control, through lowering the protein content of the
enzyme and “fine” control, through decreasing the enzyme
activation state, play a role in the elevated CO2 -mediated
down-regulation of Rubisco [21]. Because less Rubisco is
required under elevated growth CO2 , the “coarse” control
suggests a redistribution of the nitrogen resources away from
Rubisco to increase the efficiency of nitrogen use [13,34],
as well as optimization of CO2 acquisition with utilization
of the fixed carbon [19]. Growth at elevated CO2 caused
declines in activity and protein content of kidney bean Rubisco. The decline in Rubisco activity represents one of the
most consistent features in a variety of C3 species when
grown at elevated CO2 [13,34]. Decrease in Rubisco activity could be a result of decrease in Rubisco protein content,
Rubisco activation, or a decline in RuBP regeneration. In
the present study, decreased activity of Rubisco under elevated CO2 was related to lower Rubisco protein content,
but not Rubisco activation. Long et al. [35] concluded that
a decrease in Rubisco protein content at elevated CO2 can
occur even when the supply of nitrogen and rooting volumes are high as in our study. At elevated growth temperature, decreases in midday Rubisco activities of kidney bean
were due mainly to lower activation and partially to less
protein content the enzyme (Figs. 2 and 3). Studies on soybean and rice showed that elevated growth CO2 and temperatures decreased Rubisco activity associated with both
decreases in protein content and activation of the enzyme
[21].
In kidney bean, down-regulation of Rubisco protein at
elevated CO2 was associated with declines in the ratio of
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P.V.V. Prasad et al. / Plant Science 166 (2004) 1565–1573
Rubisco protein to total soluble protein but not total soluble protein (Fig. 3). Since the amount of total leaf soluble
protein was not affected by elevated growth CO2 , a partial allocation of nitrogen was likely diverted away from
Rubisco, for up-regulation in activities of the carbohydrate
metabolism enzymes SPS and AGP (Fig. 6), as a part of
resource optimization. The implication on enhancement
in SPS and AGP, however, is not complete without direct
quantification of their protein content. SPS is key regulatory
enzyme that is involved in synthesis, partitioning, utilization
and mobilization of carbohydrates, while AGP is primarily responsible for starch synthesis. Up-regulations of SPS
and AGP activities were associated with increased sugars
and starch accumulation, respectively, (Figs. 5 and 6). Our
study supports the hypothesis that increased SPS activities
are associated with increased translocation of photoassimilates, associated with increased photosynthetic activities
under elevated CO2 . Previous studies on rice [22], soybean
[17], and orchids (Oncidium goldiana [36] also showed that
down-regulation of Rubisco was associated with increased
SPS activity and sucrose production under at elevated CO2 .
Despite observed down-regulation of Rubisco activity and
protein content kidney bean leaves maintained greater photosynthetic rate at elevated CO2 . This is partly because
even with a down-regulation, the activity and content of
Rubisco protein were still adequate to maintain greater photosynthesis at elevated CO2 . An enhancement of photosynthesis is typically still observed despite a down-regulation
of the Rubisco protein [5,34]. This suggests a decreased
requirement for Rubisco and/or a higher Rubisco efficiency and photosynthetic N-use efficiency at elevated CO2
[34,37].
In conclusion, growth at elevated CO2 up-regulated activities of the carbohydrate metabolizing enzymes SPS and
AGP, resulting in greater accumulation and export of carbohydrates associated with photosynthetic activities, despite
declines in Rubisco activity and Rubisco protein content. To
take advantage of increased carbohydrate metabolism and
photosynthesis under climate change conditions, future research should be aimed at identifying and/or modifying crop
cultivars to use excess non-structural carbohydrates more efficiently to produce greater seed yield at elevated CO2 and
temperatures.
Acknowledgements
We sincerely thank Ms. Joan Anderson for her excellent
technical help in assays of carbohydrates and enzymes, Dr.
Julia Reiskind for providing antiserum and facilities for Rubisco assays and Dr. Jean Thomas for assisting with photosynthesis measurements. We also thank Messrs Wayne Wynn
and Andy Frenock for engineering support. This research
is a contribution of the University of Florida and approved
for publication as Florida Agricultural Experiment Station
Journal Series No: R-08501.
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