Plant Physiol. (1998) 118: 521-529
Changes in Growth CO2 Result in Rapid Adjustments of
Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase
Small Subunit Gene
Expression in Expanding and
Mature Leaves of Rice1
Russ W. Gesch*,
Kenneth J. Boote,
Joseph C.V. Vu,
L. Hartwell
Allen, and
Jr., and George Bowes
Department of Agronomy (R.W.G., K.J.B.), United States Department
of Agriculture-Agricultural Research Service (J.C.V.V., L.H.A.),
and Department of Botany (G.B.), University of Florida, Gainesville,
Florida 32611
 |
ABSTRACT |
The accumulation of soluble
carbohydrates resulting from growth under elevated CO2 may
potentially signal the repression of gene activity for the small
subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase
(rbcS). To test this hypothesis we grew rice
(Oryza sativa L.) under ambient (350 µL
L
1) and high (700 µL L1) CO2
in outdoor, sunlit, environment-controlled chambers and performed a
cross-switching of growth CO2 concentration at the late-vegetative phase. Within 24 h, plants switched to high
CO2 showed a 15% and 23% decrease in rbcS
mRNA, whereas plants switched to ambient CO2 increased 27%
and 11% in expanding and mature leaves, respectively.
Ribulose-1,5-bisphosphate carboxylase/oxygenase total activity and
protein content 8 d after the switch increased up to 27% and
20%, respectively, in plants switched to ambient CO2, but
changed very little in plants switched to high CO2. Plants maintained at high CO2 showed greater carbohydrate pool
sizes and lower rbcS transcript levels than plants kept
at ambient CO2. However, after switching growth
CO2 concentration, there was not a simple correlation
between carbohydrate and rbcS transcript levels. We
conclude that although carbohydrates may be important in the regulation
of rbcS expression, changes in total pool size alone
could not predict the rapid changes in expression that we observed.
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INTRODUCTION |
The inevitability of the increase in atmospheric
CO2 concentration makes understanding its
potential effects on plant growth and development of great importance.
Increased growth and yield responses for many plant species grown under
elevated CO2 are well documented (Kimball, 1983
;
Cure and Acock, 1986). However, many C3 species
when grown for long periods in elevated CO2
exhibit decreased photosynthetic capacity (Delucia et al., 1985; Sage et al., 1989; Besford et al., 1990). This acclimation response to
elevated CO2 is often accompanied by an increase
in soluble carbohydrate pools and a decrease in Rubisco protein
content, activity, and activation state (Bowes, 1993; Drake et al.,
1997).
Carbohydrate source-sink balance under growth at elevated
CO2 is believed to play a major role in the
regulation of photosynthesis through feedback inhibition (Arp, 1991;
Stitt, 1991). Source-sink imbalances may occur during exposure to
elevated CO2 when photosynthetic rate exceeds the
export capacity or the capacity of sinks to use the photosynthate for
growth, resulting in the accumulation of carbohydrates in
photosynthetically active source leaves (Farrar and Williams, 1991;
Stitt, 1991). It is hypothesized that accumulation of soluble
carbohydrates in the cell may signal the repression of Rubisco small
and large subunit gene expression (Sheen, 1994). Regulation of the
expression of several genes coding for key photosynthetic enzymes has
been shown to be influenced by soluble carbohydrate levels in plant
cells (Koch, 1996). Transcription of the small subunit of Rubisco
(rbcS) and to a lesser extent the large subunit (rbcL) has been shown to be strongly repressed by Glc and
Suc (Sheen, 1990; Van Oosten and Besford, 1994). Repression of
rbcS and rbcL occurs in field-grown wheat (Nie et
al., 1995) and in growth-chamber-grown Arabidopsis (Cheng et al., 1998)
exposed to elevated CO2. The signal transduction
pathway for the regulation of sugar-sensing genes may involve the
phosphorylation of hexose sugars via hexokinase (Jang and Sheen, 1997).
However, the contributions that carbohydrate pool sizes or fluctuation
of pools via metabolism make to the regulation of gene activity are
still uncertain.
We reasoned that by nonintrusively modifying the source-sink balance
via a cross-switching of growth [CO2], it
should be possible to rapidly reverse the source-sink balance of plants
grown at either ambient or elevated [CO2]. If
photosynthetic acclimation to elevated CO2
results from carbohydrate feedback inhibition, then switching growth
[CO2] from high to ambient should cause a shift from
sink-limited to source-limited photosynthesis, and thus
feedback-inhibited effects would disappear. Conversely, the opposite
should be observed for plants switched from ambient to high
CO2.
Rice (Oryza sativa L.) shows marked acclimation to growth
under elevated atmospheric CO2 (Rowland-Bamford
et al., 1991) and thus provides a good model system for studying the
processes involved. In the present study rice was grown under
field-like conditions in outdoor, sunlit, environment-controlled
chambers under either ambient (350 µL L1) or
high (700 µL L1) atmospheric
[CO2] until the late-vegetative stage, at which time the cross-switching of growth [CO2] was
imposed. It was hypothesized that altering the source-sink relationship
would result in a change in the amount of rbcS mRNA,
followed by corresponding changes in Rubisco total activity and protein
content. Furthermore, if leaf carbohydrate pool size was a direct
component of the signal transduction pathway, then changes in
rbcS mRNA should correlate with changes in soluble
carbohydrate pools.
| |
MATERIALS AND METHODS |
Plant Material and Growth Conditions
Rice (Oryza sativa L. cv Lemont) was grown in four
sunlit environment-controlled chambers, known as
Soil-Plant-Atmosphere-Research chambers, located in Gainesville,
Florida. The chambers were constructed of an aluminum frame covered
with transparent polyethylene terephthalate (Sixlight, Taiyo Kogyo Co.,
Tokyo, Japan), allowing approximately 90% of incident PAR to
penetrate. Aboveground chamber dimensions were 2.0 × 1.0 m in the
cross-section and 1.5 m in height. Chamber tops were attached to
aluminum vats of 2.0 × 1.0 m in the cross-section and 0.6 m
deep, providing a watertight, flooded rooting environment for rice
culture. The chamber vats were filled with soil to a depth of 0.5 m. Pregerminated rice seed was sown in 1.0-m rows that were 0.17 m
apart on October 3, 1996. Nine days after planting the rice was thinned
to approximately 250 plants m2 and a 0.05-m flood was
applied and maintained for the duration of the experiment. Before
planting, the soil was fertilized with 8.4 and 13.5 g
m2 phosphorous and potassium, respectively.
Nitrogen as urea was applied to the soil at 12.6, 6.3, and 6.3 g
m2 at 8, 18, and 25 d after planting,
respectively.
Two chambers of rice were grown at a daytime atmospheric
[CO2] of 350 mL L1
(ambient CO2) and the other two were grown at 700 mL L1 (high CO2). At the
late-vegetative phase of growth, 34 d after planting, a
cross-switching of growth CO2 concentration was
performed. One chamber of rice at ambient CO2 was
switched to high CO2, whereas another chamber at
high CO2 was switched to ambient
CO2. Dry bulb temperatures were maintained at
28°C/21°C (day/night) and the dew point temperatures were
maintained at 18°C/14°C (day/night). The data for microclimate
conditions, canopy carbon-exchange rate, and evapotranspiration were
collected every 2 s, and 300-s averages were computed and
recorded. Each chamber [CO2] was monitored with a dedicated IR gas analyzer (Ultramat 21P, Siemens, Haguenan, France).
Gas analyzers were calibrated and checked for linearity before and
after the experiment with a range of standard
[CO2] in nitrogen. Calibration was checked
daily with a span gas. Daily printouts of environment-control
conditions were monitored to ensure identical performance of the
chambers throughout the study. Variability around the desired set
points was ±0.25°C for dry bulb temperature, ±0.5°C for dew point
temperature, and ±5 µL L1 for
[CO2]. Details of the methods used for
controlling chamber environmental set points (hardware and control
algorithms) are given by Pickering et al. (1994).
Photosynthesis Measurements
Photosynthesis was measured at the treatment growth
[CO2] on the attached uppermost fully expanded
leaf (mature leaf) and the emerging leaf just above it (expanding leaf)
with a portable photosynthesis system (model LI-6200, Li-Cor, Lincoln,
NE) equipped with a 0.25-L cuvette. The calibration of the instrument
was checked daily with CO2 standards of 200, 400, 700, and 1000 µL L1 CO2
in air. At 32 d after planting, 1 d before the start of the experiment, for each chamber the main culms of approximately 80 to 100 plants at the same developmental stage were tagged. The mature leaf was
number 7, and the expanding leaf was number 8 on the main rice culm.
Emerging leaves were approximately 25% expanded at the start of the
measurements and fully expanded by the time the last measurement was
taken. Photosynthesis was measured between 11:30 AM and
12:30 PM eastern standard time, when the solar PPFD was
saturating at 1200 to 1500 mmol m2
s1. Three mature and three expanding leaves
were measured per chamber on the day before the
CO2 switch (d 1), the day of the
CO2 switch (d 2), and for several days
thereafter.
Leaf Sampling
The mature and expanding leaves were detached at the ligule from
10 to 20 plants per treatment, at d 1, 3, and 10 of the
CO2-switching experiment. Leaves were sampled
between 1 PM and 3 PM eastern standard time.
After detachment, the leaves were immediately immersed in liquid
nitrogen. Sampled leaves were pooled by treatment and developmental
stage (mature or expanding), ground to a fine powder in liquid nitrogen
with a mortar and pestle, and stored in liquid nitrogen until analysis
for Rubisco activity, Rubisco content, rbcS transcripts, and
soluble carbohydrates. Ratios of fresh weight to leaf area were
determined for a subset of plants at the same time that samples were
taken for biochemical and molecular analysis. Leaf area was measured
with a leaf area meter (model LI-3100, Li-Cor).
Rubisco Activity and Content Assays
Rubisco total activity was determined by the method described by
Vu et al. (1997). Approximately 150 mg of liquid-nitrogen-frozen leaf
powder was ground in a prechilled Ten Broek homogenizer at 4°C
containing 3 mL of extraction medium consisting of 50 mM
CO2-free Tricine-NaOH, pH 8.0, 10 mM
MgCl2, 5 mM DTT, 10 mM
isoascorbate, 0.1 mM EDTA, and 2% (w/v) PVP-40. The
homogenate was microcentrifuged for 45 s at 4°C, an aliquot of
the supernatant was immediately assayed for Rubisco activity, and a
0.2-mL aliquot of the supernatant was frozen in liquid nitrogen and
later analyzed for Rubisco content. Assay reactions were performed at
30°C in a volume of 0.5 mL. The reaction mixture consisted of 50 mM CO2-free Tricine-NaOH, pH 8.0, 10 mM MgCl2, 5 mM DTT, 0.1 mM EDTA, 0.5 mM RuBP, and 10 mM
NaH14CO3 (2 GBq
mmol1). A 0.1-mL aliquot of the supernatant was
incubated with the reaction mixture with RuBP omitted for 5 min and the
reaction initiated by the addition of RuBP. Reactions were stopped
after 45 s by adding 0.1 mL of 6 N HCl. The assays
were dried at 60°C and the acid-stable 14C
radioactivity determined by scintillation spectrometry. Blank reactions
consisting of the reaction mixture without the leaf extract were used
to subtract background radioactivity from the assays.
Rubisco content was determined by a radioimmunoprecipitation procedure
described by Vu et al. (1997). To a 0.2-mL aliquot of the leaf extract
obtained from the Rubisco activity assays, NaHCO3
was added to 10 mM and the mixture allowed to incubate on
ice for 20 min to activate Rubisco. A 0.025-mL aliquot of this mixture
was added to 0.05 mL of buffer (100 mM Bicine, pH 7.8, 20 mM MgCl2, and 1 mM EDTA)
containing 4 nmol of
[2-14C] carboxyarabinitol bisphosphate and
0.05 mL of antiserum to purified tobacco Rubisco raised from rabbits.
After incubation at 37°C for 2 h, the precipitate was collected
on 0.45-mm pore-size Millipore cellulose acetate/nitrate filters and
washed with 5 mL of a solution containing 145 mM NaCl and
10 mM MgCl2. The amount of bound
14C was determined by liquid-scintillation
spectrometry. Blanks containing everything except the leaf extract were
used to subtract background radioactivity. Assays were performed in
triplicate on two to three extractions per treatment.
Rubisco Transcript Analysis
Total RNA was isolated from approximately 100 mg of
liquid-nitrogen-frozen leaf tissue using a kit (RNeasy, Qiagen,
Chatsworth, CA) following the manufacturer's instructions, but with
the addition of two phenol/chloroform extraction steps. Absorbance of
individual RNA samples was scanned between 320 and 220 nm and
quantified by its A260. Total RNA (10 µg) was
denatured in 50% (v/v) formamide, 18% (v/v) formaldehyde, and
separated by electrophoresis on a 1.5% agarose gel containing 18%
(v/v) formaldehyde. RNA was transferred by northern blotting to
positively charged nylon membranes (Boehringer Mannheim).
Electrophoresis and northern blotting were carried out using the
procedures of Sambrook et al. (1989). Immediately after blotting,
membranes were baked for 30 min at 120°C to achieve cross-linking. A
duplicate set of samples was run on the same gel and stained with
ethidium bromide (1 mg mL1 stock solution). In
addition, after hybridizations, the membranes were stained with
ethidium bromide to verify integrity and equal loading of RNA.
Detection of mRNA for rbcS was performed by hybridization
with a DIG-labeled DNA probe. A 176-bp probe was synthesized using PCR
with a cDNA of the rice rbcS gene as the template (Xie and Wu, 1988). DIG labeling was performed using a probe-synthesis kit (PCR
DIG, Boehringer Mannheim). Hybridization and detection of the probe
were accomplished using the protocols described by the manufacturer.
Hybridizations were carried out in 5× SSC (1× SSC = 150 mM NaCl, 15 mM sodium citrate), 50 mM sodium phosphate, pH 7.0, 50% (v/v) formamide, 7.0%
(w/v) SDS, 0.1% (w/v) N-lauroylsarcosine, and 2% (w/v)
blocking reagent (Boehringer Mannheim) for 16 h at 50°C. After
hybridization, membranes were washed twice for 15 min at room
temperature in 2× SSC, 0.1% (w/v) SDS, and twice for 15 min at 70°C
in 0.1× SSC, 0.1% (w/v) SDS. The DIG label was detected by
chemiluminescence (CSPD, Tropix, Bedford, MA) and the membranes
were exposed to x-ray film (Fugi, Tokyo, Japan) at room temperature.
Quantification of probe hybridized to rbcS mRNA of rice was
achieved by image-density scanning of blots (IS-1000 version 2.0, Alpha
Innotech Corp., San Leandro, CA). Northern-blot analysis was performed
for two separate RNA extractions with similar results.
Carbohydrate Analysis
Soluble carbohydrates were extracted from approximately 150 mg of
liquid-nitrogen-frozen leaf tissue using 80% (v/v) ethanol at 85°C.
Glc, Fru, and Suc were quantified using the microtiter method described
by Hendrix (1993). Carbohydrate assays were performed in triplicate on
three separate extractions.
Statistical Methods
A repeated-measures analysis of variance was used to model Rubisco
and soluble carbohydrate data according to a split-plot-in-time approach using the Mixed Repeated Measures Analysis procedure (SAS
Institute, Cary, NC) to obtain the estimates for the model. The factors
used for the model were [CO2] treatment, day of
experiment, and [CO2] treatment × day of
experiment.
| |
RESULTS |
Leaf Photosynthesis
Light-saturated rates of photosynthetic CO2
assimilation by both mature and expanding leaves of rice, measured at
their respective growth [CO2], were greatest
for plants growing at high CO2 (700 µL L1
CO2). Photosynthetic rates of mature leaves gradually
declined during the course of the experiment, whereas those of
expanding leaves tended to increase (Fig.
1). Values for rice grown continuously under ambient CO2 ranged from 19 to 28 and 19 to
25 µmol CO2 m2 leaf
area s1 for mature and expanding leaves,
respectively, whereas values for rice grown continuously under high
CO2 were as high as 33 to 37 µmol
m2 s1.
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| Figure 1.
Photosynthetic CO2-assimilation rate
from mature (A) and expanding (B) leaves of rice. The switch in
[CO2] was made on d 2. Values are the means ± SE, n = 3.
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Upon switching growth [CO2] from ambient to
high and vice-versa there was a rapid adjustment in photosynthesis rate
measured at the new [CO2]. Mature leaves of
rice switched to ambient CO2 initially showed a
10% reduction in photosynthesis compared with ambient controls on d 2, the day of the CO2 switch, when both were
measured at ambient [CO2]. By d 3, 1 d
after the switch, the photosynthesis rate had increased to that of the
ambient controls (Fig. 1A). Mature leaves of rice switched to high
CO2 had an 8% higher photosynthetic rate on d 2 and a 15% higher rate on d 3, compared with the
high-CO2 controls. By d 5, the rate had declined to that of the high-CO2 controls. The
photosynthetic rates of expanding leaves of rice switched to either
high or ambient CO2 follow the same pattern as
their respective unswitched controls and were statistically
indistinguishable from them (Fig. 1B).
Stomatal conductances of leaves for plants grown at high
CO2 were 35% to 40% lower than those for plants
grown at ambient CO2 (data not shown). For each
of the CO2 treatments the
Ci/Ca ratios were
determined (data not shown). The
Ci/Ca ratio of
high-CO2 controls was 1% higher than that of the
ambient controls throughout the study. In plants switched to either
high or ambient CO2, there was a slight decline
in Ci/Ca ratio of 3%
compared with ambient controls on d 3, but by d 5 it was again within
1% of that of the ambient controls.
Rubisco Total Activity and Content
Rubisco total activity and content declined with age in mature
leaves, with the greatest reduction being in plants grown continuously under high CO2 (Table
I). During the course of the experiment, mature leaves of plants kept at ambient CO2
showed 28% and 33% decreases in Rubisco activity and content,
respectively, whereas plants kept at high CO2
showed 43% and 41% decreases, respectively (Table I).
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Table I.
Rubisco total activity and protein content for
mature and expanding leaves of rice on d 1, 3, and 10 of the experiment
The switch in [CO2] was made on d 2. Values are the
means ± SE, n = 6 to 9; values
followed by a different letter across treatments on the same day are
significantly different at the P  0.05 level.
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At d 3, Rubisco activity and content in mature leaves of rice switched
to ambient CO2 were significantly greater than
those of high-CO2 controls but less than the
ambient-CO2 controls. By d 10, the plants
switched to ambient CO2 did not differ from those kept at ambient CO2, and had significantly higher
activity and content than those kept at high CO2.
Rubisco activity and content in mature leaves of rice switched to high
CO2 differed little from those of the
ambient-CO2 control, and remained greater than those of the high-CO2-grown plants, indicating a
lack of adjustment despite the change in growth
[CO2].
Expanding leaves of rice kept at ambient or high
CO2 showed no significant differences in Rubisco
total activity and content at d 1 and 3 (Table I). However, at d 10, both total activity and content were significantly less in the
high-CO2 plants. One day after switching to high
or ambient CO2 there were no changes in either
total activity or content. By d 10, total activity and content in
plants switched to ambient CO2 had increased to
levels similar to those of plants kept at ambient
CO2. Conversely, plants switched to high
CO2 appeared to have less Rubisco activity and content than the ambient-CO2 plants, although the
differences were not statistically significant.
Rubisco Small Subunit mRNA
Differences in steady-state rbcS transcript levels
between plants grown at ambient and high CO2 were
detected in both mature and expanding leaves (Fig.
2). Levels of rbcS transcripts
for mature leaves were similar in both ambient- and
high-CO2-grown rice on d 1. However, by d 3 the
amount of rbcS transcripts in plants kept at ambient
CO2 was 13% greater than that of plants kept at
high CO2, and by d 10 it was 43% greater (Table
II).
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| Figure 2.
Northern blots of rbcS mRNA for
mature and expanding leaves of rice on d 1, 3, and 10. The switch in
[CO2] was made on d 2.
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Table II.
Abundance of rbcS transcripts for mature and
expanding leaves of rice on d 1, 3, and 10
The switch in [CO2] was made on d 2. Values are the
means ± SD of two experiments.
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After switching the growth [CO2], changes in
rbcS transcript levels occurred within 24 h. In mature
leaves of rice switched to ambient CO2, the
transcript level increased to that of ambient-CO2 controls by d 3, whereas for plants switched to high
CO2 it decreased to 23% less than that of the
ambient-CO2 controls. At d 10 the level of rbcS
transcripts for rice switched to ambient CO2 was 11% greater than that of plants kept at ambient
CO2, whereas plants switched to high
CO2 showed 13% less.
In expanding leaves, rbcS transcript levels remained about
15% to 30% greater for plants kept at ambient
CO2 compared with plants kept at high
CO2 (Table II). After switching growth
[CO2], expanding leaves showed a similar
response to that of mature rice leaves. At d 3 plants switched to
ambient CO2 showed an increase in transcripts. Conversely,
plants switched to high CO2 showed a 15% decrease compared
with plants kept at ambient CO2. At d 10 plants switched to
high CO2 showed the same transcript level as plants kept at
ambient CO2, whereas those switched to ambient CO2 showed less. Expanding leaves of rice were
approximately 20% and 50% expanded on d 1 and 3, respectively, and
were fully mature by d 10.
Soluble Carbohydrates
Mature leaves of plants kept in high CO2 had
considerably greater Glc and Fru pool sizes than those of plants kept
in ambient CO2 (Table
III). However, Suc pool size did not
differ until d 10, when it was significantly greater in plants kept in
high CO2. Plants kept at ambient
CO2 showed little change in Glc and Fru from d 1 to 10, whereas Glc increased in plants kept at high
CO2. Suc pool sizes for both ambient- and
high-CO2 plants were greatest at d 1 and declined
thereafter.
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Table III.
Glc, Fru, and Suc content for mature and expanding
leaves of rice on d 1, 3, and 10
The switch in [CO2] was made on d 2. Values are the
means ± SE, n = 9; values followed by
a different letter across treatments on the same day are statistically
different at the P 0.05 level.
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Switching the growth [CO2] of rice from high to
ambient resulted in a dramatic decline in soluble carbohydrates the day
after the switch. Glc and Fru pools decreased to the same level as in the plants kept at ambient CO2, whereas Suc
decreased to significantly less. Compared with plants kept at high
CO2, Glc, Fru, and Suc pool sizes for plants
switched to ambient CO2 were 64%, 38%, and 42%
less, respectively. At d 10 soluble sugar pool sizes for plants switched to ambient CO2 increased to levels
greater than those of plants kept at ambient CO2,
but Glc and Suc pools were significantly less than those of
high-CO2 controls.
Mature leaves of rice switched to high CO2 showed
no significant difference in Glc and Fru pool sizes by d 3 compared
with ambient-CO2 controls (Table III). However,
Suc decreased dramatically by d 3, similar to that of plants switched
to ambient CO2. By d 10 soluble sugar pool sizes in leaves
of plants switched to high CO2 were not
significantly different from those of plants kept at ambient
CO2.
Glc and Fru pool sizes of expanding leaves generally exceeded those of
mature leaves (Table III). Suc content increased continuously in all
treatments during the course of the experiment, and by d 10 the
high-CO2 plants and plants switched to high
CO2 showed significantly greater amounts than
plants kept in ambient CO2. By d 10 plants kept at high
CO2 showed 47% and 23% greater Glc and Fru pool
sizes, respectively, than plants kept at ambient CO2. Expanding leaves of plants switched to
ambient CO2 showed a decrease in both Glc and Fru
on d 3, with Glc attaining a level significantly less than that of
plants kept at ambient CO2. From d 3 to 10 there
was little change in the level of Glc and Fru in
ambient-CO2-switched rice, and although Suc
increased, it remained significantly less than in the other three
treatments. Plants switched to high CO2 showed a
small decline in Glc and Fru at d 3. However, by d 10 the amount of
Glc, Fru, and Suc had increased dramatically to levels similar to
that in plants kept at high CO2.
| |
DISCUSSION |
A cross-switching of growth [CO2] for rice
previously grown at either ambient (350 µL L1) or high
(700 µL L1) CO2 resulted in a rapid (24 h)
change in rbcS gene expression. Both expanding and mature
leaves of rice switched to high CO2 showed a
decrease in transcripts with respect to plants maintained at ambient
CO2. In contrast, high-CO2
plants switched to ambient CO2 showed an increase
in rbcS transcripts. Whatever signaled the response in the
expression of rbcS with the switching of growth [CO2] apparently was sensed by both expanding
and mature leaves. This change in transcript levels persisted in mature
leaves at d 10. Rapid responses in rbcS mRNA have been
detected as a consequence of exogenously added sugars in detached
tomato leaves (Van Oosten and Besford, 1994), maize mesophyll
protoplasts (Sheen, 1990), and autotrophic cell-suspension cultures
(Krapp et al., 1993). However, to our knowledge, this is the first
report of a rapid response in rbcS expression for plants
growing under field-like conditions and exposed to different
[CO2].
Rubisco total activity and content measured at midday responded to the
switching of rice to ambient CO2, but this
response was not as rapid nor did it parallel the response in
transcript levels. For expanding leaves, Rubisco total activity and
content among all treatments were not significantly different on d 3, but by d 10 increased significantly for rice switched to ambient CO2 and decreased for plants switched to high
CO2, although the change was not significantly
different from that in plants kept at ambient
CO2. A lag time between rbcS
expression and changes in Rubisco total activity and content has been
shown by others. Krapp et al. (1993) found that when Chenopodium
rubrum cell-suspension cultures were supplied with Glc, the
expression of rbcS transcripts decreased within 6 h,
which was long before significant changes in Rubisco total activity and
content occurred. In all CO2 treatments, mature
leaves showed an overall trend of decreasing Rubisco total activity and
content over time. The decrease was considerably greater in rice
maintained at high CO2. Switching to ambient
CO2 slowed this decline so that it paralleled
that of plants maintained at ambient CO2.
However, switching to high CO2 had no apparent effect on Rubisco total activity and content in mature leaves.
Synthesis of photosynthetically active Rubisco requires the
coordination and assemblage of small and large subunits in the chloroplast. Therefore, changes in Rubisco synthesis may be regulated by changes in transcription of rbcS and rbcL,
transcript stability, translation, and protein turnover, all of which
may potentially be affected by growth [CO2]
(Webber et al., 1994). The level and change in regulation through each
of these steps may also vary with developmental stage (Deng and
Gruissem, 1987). Besford et al. (1990) have shown for tomato leaves
that Rubisco activity and protein content peak and then decline earlier
during leaf expansion in high CO2 than in ambient
CO2. Although transcriptional regulation of
Rubisco small and large subunits has been shown to be affected by
[CO2] in some instances (Winder et al., 1992), the level of regulation by translation and posttranslational turnover is complicated by the fact that photosynthetically competent Rubisco has a relatively slow turnover rate (Peterson et al., 1973).
Our study has shown that upon switching from high to ambient
CO2, expanding leaves up-regulated
rbcS mRNA, which apparently resulted in increased Rubisco
synthesis, whereas switching to high CO2 resulted
in down-regulation of rbcS mRNA and a decrease in Rubisco
synthesis. Cheng et al. (1998) also showed that rbcS mRNA
levels were substantially repressed in Arabidopsis 6 d after transferring them from a growth [CO2] of 360 to
1000 µL L1, followed later by a significant
decrease in Rubisco protein content at 9 and 12 d. The response of
rice leaves to changes in growth [CO2] in our
study is much more rapid than the acclimation to elevated
CO2 reported in some other field studies
(Körner and Diemer, 1994; Jacob et al., 1995).
Regulation of rbcS transcription was also apparent in mature
leaves. Rubisco turnover appeared to be greater than synthesis, because
content declined in all treatments from d 1 to 10. However, plants
switched to ambient CO2 showed an increase in
rbcS transcripts and by d 10 significantly greater Rubisco
content than plants kept at high CO2, suggesting
that synthesis had increased in this treatment. The processes of
synthesis and turnover may have been affected differently by the
switching of growth [CO2] depending on the
developmental stage of the leaf. However, it is not evident from these
data whether the turnover rate was affected by
CO2 switching.
Increased amounts of nonstructural carbohydrates are often associated
with photosynthetic acclimation (Bowes, 1993). The down-regulation of
photosynthesis associated with acclimation has been attributed to an
accumulation of carbohydrates in photosynthetically active source
leaves caused when photosynthetic rate exceeds the capacity of sinks to
use assimilate for growth (Arp, 1991; Farrar and Williams, 1991). In
our study leaf photosynthesis was generally 25% to 30% greater in
mature leaves and 20% to 24% greater in expanding leaves of plants
grown under high CO2. Photosynthetic acclimation
was evident in mature leaves of rice grown at high
CO2. This can be inferred by comparing
photosynthetic rates of plants switched to high
CO2 with those of plants maintained at high
CO2 on the day of the switch and 1 d after,
when both were measured at high CO2.
Photosynthesis was 15% greater in plants switched to high CO2. Also, plants switched to ambient
CO2 showed a 10% lower photosynthetic rate than
plants maintained at ambient CO2 on the day of
the switch.
Growth at elevated CO2 often results in decreased
stomatal conductance (Drake et al., 1997). To determine if stomatal
conductance was a major limitation to leaf photosynthesis, in the
present study the Ci/Ca ratios for various
treatments were compared. Even though stomatal conductivity did
decrease approximately 35% to 40% at high CO2,
the Ci/Ca ratio of
high-CO2 controls was nearly identical to that of
ambient controls (1% higher) throughout the study. In plants switched
to either high or ambient CO2, there was a slight
decline in Ci/Ca ratio on d
3 (3% decrease), but by d 5 it was again within 1% of the ambient
controls. These data indicate that stomatal conductance was not a major
limiting factor to photosynthesis in the
high-CO2-grown plants.
Pool sizes of Glc and Fru for mature leaves of plants kept in high
CO2 were up to 2-fold greater than those of
plants kept in ambient CO2, whereas Suc was only
significantly higher on d 10. Transcript levels for rbcS did
not differ on d 1, but were greater for the ambient controls on d 3 and
10. Differences in soluble carbohydrate pool sizes between ambient- and
high-CO2-grown plants were not as great in
expanding leaves as in mature leaves, at least until d 10. This might
be expected, because at d 1 and 3 these leaves were growing and thus
acting as carbohydrate sinks, whereas by d 10 they were fully expanded.
However, at all three sampling times, plants kept at ambient
CO2 contained a higher level of rbcS
mRNA. Switching from high to ambient CO2 led to a
significant decline in Glc and Fru pool sizes for both mature and
expanding leaves and a dramatic decrease in Suc in mature leaves on d
3. This was accompanied by an increase in rbcS transcripts for both mature and expanding leaves, which suggests that decreased carbohydrate pool sizes may have signaled the activation of
rbcS transcription. However, for plants switched to high
CO2, even though photosynthesis increased and
rbcS transcripts decreased, the pool sizes of Glc, Fru, and
Suc either stayed the same or decreased in comparison with plants kept
at ambient CO2. These data are at odds with the
hypothesis that rbcS expression is regulated by soluble
carbohydrate pool sizes. This does not rule out the possibility that
soluble carbohydrates are involved in the regulation of rbcS
expression, but our data indicate that the overall leaf pool sizes
during leaf development cannot be used to predict the degree of
rbcS expression.
Rice is known to be a Suc accumulator when photosynthetic rates are
relatively high (Rowland-Bamford et al., 1990, 1996). During vegetative
growth, elongating nodes and culms act as a major sink for
photosynthetic assimilate (Yoshida and Aln, 1968). Rowland-Bamford et
al. (1990) have shown during vegetative growth in rice that as growth
[CO2] increases, so does the tendency for recently produced photosynthetic assimilate in leaves to be exported rather than stored. Because photosynthesis increased substantially in
plants switched to high CO2, the most likely
reason for the observed decrease in soluble carbohydrates, or the lack
of accumulation, was an increase in export of recent photosynthetic
assimilate.
The lack of correlation between soluble carbohydrate content and
photosynthetic acclimation during growth under elevated
CO2 seen here and in other studies (Wang and
Nobel, 1996; Moore et al., 1997) suggests that fluctuations in soluble
carbohydrate pools and metabolic cycling of carbon produced through
photosynthesis, rather than total pool sizes themselves, may have a
more vital role in signaling regulation of rbcS expression.
The phosphorylation of hexose sugars via hexokinase has been
hypothesized as a sugar-sensing and signal transduction mechanism for
expression of sugar-sensitive genes in plants (Jang and Sheen,
1994). Cheng et al. (1998) showed that rbcS levels in
Arabidopsis fluctuate diurnally, with a maximum occurring at the
beginning and a minimum at the end of the light cycle; soluble
carbohydrate levels generally followed an inverse pattern. Based on
this and previous evidence (Moore et al., 1997), they suggest that
hexokinase may act as a hexose flux sensor, primarily via nighttime
activity, mediating the repression of rbcS transcription.
Recently, Jang et al. (1997) have shown evidence for the involvement of
hexokinase in the Glc-mediated repression of rbcS and
cab1 (chlorophyll a/b-binding protein) and
activation of nitrate reductase genes in transgenic Arabidopsis plants
with altered hexokinase expression.
In conclusion, this study documents that a nonintrusive modification of
atmospheric growth [CO2] in vegetatively
growing rice under field-like conditions leads to a rapid change in
expression of the rbcS in mature and expanding leaves.
Switching the growth [CO2] eventually led to
alterations of Rubisco protein content and total activity. Both mature
and expanding leaves maintained the ability to up-regulate Rubisco upon
being switched from high to ambient CO2, whereas
exposure to high CO2 resulted in down-regulation of Rubisco. The signal for changing rbcS gene expression as
a consequence of an alteration in [CO2]
evidently is detected soon after switching, certainly within 24 h.
However, changes in total leaf soluble carbohydrate pool sizes alone
could not explain the rapid change in rbcS expression that
we detected. Further work is under way to determine the flux of carbon
through soluble carbohydrate pools under growth at elevated
CO2 in an effort to elucidate the signal for
rbcS expression as a function of
[CO2].
| |
FOOTNOTES |
1
This work was supported by the U.S. Department
of Agriculture/National Research Initiative Competitive Grants Program
Plant Responses to the Environment (grant no. 95-37100-1597). This is Florida Agricultural Experiment Station journal series no. R-06325.
*
Corresponding author; e-mail rwg30{at}nervm.nerdc.ufl.edu; fax
1-352-392-6139.
Received March 23, 1998;
accepted June 29, 1998.
| |
ABBREVIATIONS |
Abbreviations:
Ca, atmospheric CO2
concentration.
Ci, internal CO2 concentration.
DIG, digoxigenin.
RuBP, ribulose-1,5-bisphosphate.
| |
ACKNOWLEDGMENTS |
We thank Joan Anderson for her skillful technical assistance and
Drs. Xie Yong and Ray Wu (Cornell University) for kindly providing the
cDNA for rice rbcS.
| |
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Abstract
Introduction