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 Rice^{<a href="#FN1" rid="FN1" class=" fn">1</a>}

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 m ^{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 m phosphorous and potassium, respectively. Nitrogen as urea was applied to the soil at 12.6, 6.3, and 6.3 g m at 8, 18, and 25 d after planting, respectively.}

Two chambers of rice were grown at a daytime atmospheric [CO₂] of 350 mL L ^{(ambient CO}₂) and the other two were grown at 700 mL L ^{(high CO}₂). 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 CO_2, 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 L ^{for [CO}₂]. 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 L ^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 m ^{s. Three mature and three expanding leaves were measured per chamber on the day before the CO}₂ 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 NaH^CO₃ (2 GBq mmol^{). 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 ^{C 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-^{C] 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 m}m NaCl and 10 mm MgCl₂. The amount of bound ^{C 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 mL ^{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.

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 L ^CO₂). Photosynthetic rates of mature leaves gradually declined during the course of the experiment, whereas those of expanding leaves tended to increase (Fig. ​(Fig.1).1). Values for rice grown continuously under ambient CO₂ ranged from 19 to 28 and 19 to 25 μmol CO₂ m ^{leaf area s for mature and expanding leaves, respectively, whereas values for rice grown continuously under high CO}₂ were as high as 33 to 37 μmol m ^s.

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.

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. ​(Fig.1A).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. ​(Fig.11B).

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 C_i/C_a ratios were determined (data not shown). The C_i/C_a 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 C_i/C_a 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 ​(TableI).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 ​(TableI). I).

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 ​(TableI).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. ​(Fig.2).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 ​(TableII). II).

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.

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 ​(TableII).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 CO_2, 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.

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.