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First published online April 29, 2005; 10.1104/pp.104.055046 Plant Physiology 138:451-460 (2005) © 2005 American Society of Plant Biologists Increased Sedoheptulose-1,7-Bisphosphatase Activity in Transgenic Tobacco Plants Stimulates Photosynthesis and Growth from an Early Stage in Development1Department of Biological Sciences, University of Essex, Colchester CO4 3SQ, United Kingdom
Activity of the Calvin cycle enzyme sedoheptulose-1,7-bisphosphatase (SBPase) was increased by overexpression of an Arabidopsis (Arabidopsis thaliana) cDNA in tobacco (Nicotiana tabacum) plants. In plants with increased SBPase activity, photosynthetic rates were increased, higher levels of Suc and starch accumulated during the photoperiod, and an increase in leaf area and biomass of up to 30% was also evident. Light saturated photosynthesis increased with increasing SBPase activity and analysis of CO2 response curves revealed that this increase in photosynthesis could be attributed to an increase in ribulose 1,5-bisphosphate regenerative capacity. Seedlings with increased SBPase activity had an increased leaf area at the 4 to 5 leaf stage when compared to wild-type plants, and chlorophyll fluorescence imaging of these young plants revealed a higher photosynthetic capacity at the whole plant level. Measurements of photosynthesis, made under growth conditions integrated over the day, showed that mature plants with increased SBPase activity fixed 6% to 12% more carbon than equivalent wild-type leaves, with the young leaves having the highest rates. In this paper, we have shown that photosynthetic capacity per unit area and plant yield can be increased by overexpressing a single native plant enzyme, SBPase, and that this gives an advantage to the growth of these plants from an early phase of vegetative growth. This work has also shown that it is not necessary to bypass the normal regulatory control of SBPase, exerted by conditions in the stroma, to achieve improvements in carbon fixation.
The photosynthetic carbon reduction (Calvin) cycle is the primary pathway for fixation of atmospheric CO2. This cycle plays a central role in plant metabolism, providing intermediates not only for starch and Suc biosynthesis, but also for isoprenoid metabolism and shikimic acid biosynthesis (Geiger and Servaites, 1994  ). Recently, a major focus has been to identify the individual steps that control carbon flux through the Calvin cycle. To address this question, antisense plants have been produced in which the levels of individual enzymes in the cycle have been reduced and the response of photosynthesis in these plants has been measured. This has allowed the contribution that individual enzymes exert over the control of flux through the Calvin cycle to be quantified (for review, see Stitt and Schulze, 1994; Raines, 2003). A number of interesting and surprising results have emerged from these analyses. Firstly, it was shown that for plants grown in moderate light and temperature conditions, more than 50% of wild-type levels of Rubisco could be removed before any significant effect on ambient photosynthesis was observed. However, under conditions of high light, reductions in photosynthesis were proportionate to decreases in Rubisco activity (Quick et al., 1991; Stitt et al., 1991; Hudson et al., 1992; Krapp et al., 1994; Stitt and Schulze, 1994). Reductions of greater than 50% in the levels of glyceraldehyde-3-P dehydrogenase, Fru-1,6-bisphosphatase (FBPase), phosphoribulokinase (PRKase), and plastid aldolase were also needed before photosynthetic capacity was affected (Kossmann et al., 1994; Stitt and Schulze, 1994; Paul et al., 1995; Price et al., 1995; Haake et al., 1998). Although the level of control exerted by any single enzyme in the cycle varied with environmental conditions and developmental status, the data from the transgenic plants strongly indicated that a number of enzymes in the Calvin cycle are present in excess. The implication from this is that the levels of Rubsico, glyceraldehyde-3-P dehydrogenase, FBPase, and PRKase are not close to limiting carbon fixation through this cycle and therefore would not be useful targets for overexpression to increase photosynthetic capacity.
In contrast, photosynthesis has been shown to be sensitive to small reductions in the levels of the enzymes transketolase and sedoheptulose-1,7-bisphosphatase (SBPase; Harrison et al., 1998
Production and Selection of Tobacco Transformants
A full-length Arabidopsis (Arabidopsis thaliana) SBPase cDNA (Willingham et al., 1994
The Impact of Increased SBPase Activity on Photosynthesis, Carbohydrate Levels, and Growth in Plants Grown in Controlled Environment Conditions
Fluorescence Imaging Analysis of Photosynthetic Capacity in Young Seedlings
CO2 Assimilation Rates in Leaves at Different Stages of Development Gas exchange measurements were used to investigate whether the increased photosynthetic capacity observed in young seedlings was maintained in mature tobacco plants. CO2 assimilation rates were determined, under the light level and CO2 concentration in which the plants were grown, for young expanding leaves (12 and 13), a leaf approaching full expansion (11), and new fully expanded leaves (9 and 10) in wild-type and SBPase sense plants when 25 to 27 leaves had been produced (Fig. 2A ). In wild-type plants, photosynthetic activity was lowest in the youngest leaves and increased to a maximum in the newest fully expanded leaves. A similar pattern of development of photosynthetic capacity was observed in the leaves of SBPase sense plants. However, in the SBPase overexpressing plants, the photosynthetic rate in the youngest leaves (12 and 13) was higher than wild type, but in the fully expanded leaves (9 and 10) no significant increase in photosynthesis was evident (Fig. 2A). In leaf 11 (close to full expansion), photosynthesis was only increased in the plants with the highest levels of SBPase activity. This may reflect the fact that leaf 11 is making the transition to full expansion where no significant increase in photosynthesis is evident, even in the group of plants with the highest increase in SBPase activity.
Diurnal Carbohydrate Levels In parallel with photosynthetic analysis, diurnal patterns of carbohydrate accumulation and turnover were determined (Fig. 2B). In both the wild-type and SBPase sense plants, Suc and starch accumulated during the light period in both young expanding and newly fully expanded leaves. A correlation between increased carbohydrate accumulation and increased SBPase activity was evident, and lines with the highest SBPase activity accumulated up to 50% more Suc and starch than equivalent wild-type plants. Suc and starch levels at the end of the night were low in both the wild-type and SBPase overexpressing plants, indicating that the carbohydrate accumulated in the light was being used and/or remobilized at night (Fig. 2B). Although photosynthetic capacity of the mature leaves (9 and 10) was not increased, the levels of both Suc and starch were higher in the transgenic plants with the highest SBPase activities.
Effect of Increased SBPase Activity on Growth
Determination of Photosynthetic Capacity and Growth of Plants Grown in Greenhouse Conditions Previous work on transgenic plants with altered Calvin cycle enzyme activity has shown that the environmental conditions under which the plants are grown influences the impact of any changes in enzyme activity on photosynthesis and growth. To assess the changes in photosynthetic parameters of the SBPase sense plants in a more natural environment, two separate cohorts of T4 SBPase sense plants were grown in a controlled environment greenhouse where they were subject to normal daily changes in light and temperature.
Response of Photosynthesis to Changes in CO2
The data from the A/Ci curve were used to calculate the apparent in vivo maximum Rubisco activity (Vc, max) and Jmax for all of the SBPase sense and wild-type plants using the equations developed by von Caemmerer and Farquhar (1981 ; von Caemmerer, 2000). In the plants with increased SBPase activity, the Jmax was increased by as much as 30% in response to increasing SBPase activity (Fig. 5A
). Interestingly, Vc, max was 10% above that for wild-type plants (Fig. 5B).
Daily Progression of Photosynthesis The data obtained from the A/Ci response analysis revealed that under saturating light a stimulation of photosynthesis was evident in the newest fully expanded leaves. To investigate this further, the in situ response of photosynthesis to increased SBPase activity was determined by measuring photosynthesis under the prevailing light and CO2 conditions in a young expanding and newly fully expanded leaf on wild-type and transgenic plants throughout the photoperiod. The SBPase sense lines had slightly higher rates of photosynthesis over the course of the day than equivalent wild-type plants (Fig. 6 ). This stimulation of photosynthesis was greatest in the young leaves, in agreement with the analysis of the plants grown in the controlled environment cabinet (Fig. 2; Table I). Integrating ambient photosynthesis measurements over the day revealed that in the SBPase sense plants the daily carbon fixed in young leaves was increased by 12% (P < 0.05) and, even in the mature leaves, a 6% increase was evident. No significant differences in SBPase activity were evident between the young and old leaves of the wild-type, or of SBPase sense, plants.
Growth Response to Increased SBPase Activity In the SBPase sense plants grown in greenhouse conditions, only small differences in shoot biomass were found when plants were harvested at the floral bud stage of development (Fig. 7A ). However, although stem height was increased significantly (Fig. 7C), only small changes were seen in stem and leaf dry weights and these were not statistically significant (Fig. 7, B and D). Increases in total leaf area and leaf biomass increased in parallel in the SBPase sense plants by 10% (Fig. 7, D and E), resulting in no change in specific leaf area (Fig. 7F).
In addition to analysis of total leaf area and biomass, the area and weight of all of the individual leaves were determined for the wild-type and SBPase sense plants. The average area and weight of leaves on the SBPase sense plants produced during early- to mid-vegetative phase (I, II) was about 50% greater than those of wild-type plants. During phase III, small increases in both leaf area and leaf weight were evident but the level of significance was just above P = 0.05. In contrast, the average area and weight of leaves produced later in the vegetative phase and just prior to flowering (IV) were similar to those of the wild-type plants (Fig. 8, A and B ).
Transgenic plants expressing an Arabidopsis SBPase sense construct were produced and a number of lines identified with increased SBPase activity. Photosynthesis and growth rates were increased in the SBPase overexpressing plants grown in both controlled environment chambers and the greenhouse. Light-saturated rates of photosynthesis, measured under prevailing and saturating CO2 concentrations, increased linearly in response to increased SBPase activity. However, this increase in photosynthesis was not directly proportionate to the change in total extractable SBPase activity, indicating that the relationship between SBPase activity and photosynthesis is not simple. This could reflect regulatory properties of the system, resulting in photosynthetic carbon fixation being limited at another point in the Calvin cycle (Stitt and Schulze, 1994 ; Raines, 2003). This may be due, in part, to the fact that SBPase activity is subject to regulatory feedback by its product and also by redox via thioredoxin (Raines et al., 1999). Analysis of A/Ci response curves in the SBPase sense plants, using the equations developed by von Caemmerer and Farquhar (1981), showed that the increase in photosynthetic capacity could be attributed to an increase in the rate of Jmax in the SBPase sense lines as compared to wild-type plants. These results fit well with the location of SBPase in the regenerative part of the Calvin cycle and also with our previous results showing that Jmax declined linearly with reductions in SBPase activity (Harrison et al., 2001). Interestingly, the values for Vc, max were also increased by about 10%. Rubisco activation state and RuBP levels were also shown to be higher in transgenic plants where photosynthetic capacity has been increased by the overexpression of a bifunctional cyanobacterial FBPase/SBPase (Miyagawa et al., 2001).
Our data have shown clearly that, under light-saturating conditions, photosynthetic rates on a leaf area basis were increased in response to increased SBPase activity. In addition, using fluorescence imaging of whole plants, we have also shown that young seedlings (4–5 leaf stage) had significantly higher photosynthetic capacity than wild-type plants. These data show that whole-plant photosynthesis can be increased during the early stages of vegetative growth by increasing SBPase activity. Analysis of the daily progression of photosynthesis, measured under the same light and CO2 concentrations that the plants were grown in, revealed an increase in total carbon fixed during the photoperiod in the SBPase sense lines. Interestingly, this increase in photosynthesis was greater in young expanding leaves (12%) than in fully expanded leaves (6%) in the SBPase sense plants compared to the equivalent leaves on wild-type plants. This difference in the response of photosynthesis to increased SBPase activity (between young expanded and newly fully expanded leaves) was also observed for the plants grown in controlled environment chambers. This result was somewhat unexpected and cannot be accounted for by differential increases in SBPase activity in the young leaves. It is possible that, in the mature leaves of SBPase overexpressing lines, additional limiting factors, such as the availability of water, control the flux of carbon through the Calvin cycle. These differences might be expected from previous studies of Calvin cycle antisense plants that have also shown that the relative importance of any individual enzyme over carbon fixation is not fixed and will vary depending on growth condition and development (for review, see Stitt and Schulze, 1994 Analysis of diurnal changes in carbohydrate accumulation revealed that the levels of both Suc and starch increased at the end of the day in SBPase sense plants grown in controlled environment conditions. In expanding leaves (12 and 13; Fig. 2A), a clear correlation was evident between SBPase activity, increased photosynthesis, and increased accumulation of Suc and starch. In the fully expanded leaves (9 and 10) of the transgenic plants with the greatest increase in SBPase activity, increased levels of both Suc and starch were also found; however, no significant increase in photosynthesis was observed in these leaves (Fig. 2, A and B). This apparent discrepancy may be due to the fact that these photosynthetic measurements were made at a single time point in the day, but the measurements of Suc and starch were at steady state, giving the total daily accumulation. In support of this suggestion, the daily time course measurements of photosynthesis in the fully expanded leaves of the SBPase sense plants revealed a small increase (6%) in the daily carbon fixed in the mature leaves (Fig. 6).
In the young expanding and newly fully expanded leaves of both the wild-type and SBPase sense plants, only low levels of sugar and starch remained in the predawn samples. These results showed that the additional carbohydrate produced in the SBPase sense plants was being used in the dark period, possibly for growth. Indeed, the total shoot biomass and total leaf area of the SBPase sense plants grown in the controlled environment chamber were increased by up to 25%, compared to wild-type plants. A similar trend toward increased growth was evident in the greenhouse-grown plants; however, in this case, the impact was less substantial and an increase of between 7% and 12% in leaf area and leaf biomass was evident. This increase could be attributed to an increase in leaf area and biomass that occurred in the leaves produced during the early- to mid-vegetative phase of the life cycle, when the average leaf area and mass was increased by 25% compared to equivalent leaves on the wild-type plants. In contrast, the leaves produced later in the vegetative phase and just prior to flowering had a similar biomass and leaf area to that of wild-type plants. These analyses suggested that increases in SBPase activity impact photosynthesis and growth from an early stage of seedling development. This finding is in contrast with data from plants expressing a bifunctional FBPase/SBPase enzyme where increases in growth were most evident in older plants. It is possible that these differences could be due to the fact that these plants were grown in hydroponic culture, under low light and/or that the tobacco cultivar used was xanthii (Miyagawa et al., 2001
The relationship between photosynthetic capacity and yield is not clear, and in fact, photosynthesis per unit leaf area has remained constant even in high-yielding crop varieties (Evans and Fischer, 1999
Generation of the Transgenic Plants
An Arabidopsis (Arabidopsis thaliana) full-length SBPase cDNA (Willingham et al., 1994
Wild-type tobacco plants and T1 progeny resulting from self-fertilization of transgenic plants were germinated in sterile agar medium containing Murashige and Skoog salts supplemented with 1% (w/v) Suc (plus hygromycin 300 mg L–1 for the transformants). Seeds were germinated in controlled environment chambers at an irradiance of 40 µmol photons m–2 s–1, 22°C, relative humidity of 60%, in a 16-h photoperiod. After 2 weeks, plants were transferred to soil (Levington F2, Fisons, Ipswich, UK) and grown in either a controlled environment chamber with a 16-h photoperiod, 500 to 600 µmol m–2 s–1, at 25°C day/20°C night, or a controlled environment greenhouse (16-h photoperiod, 25°C–30°C day/20°C night, and natural light supplemented with high-pressure sodium light bulbs, giving between 600–1,600 µmol m–2 s–1 from the pot level to the top of the plant, respectively). Positions of the plants were changed daily and watered with a nutrient medium (Hoagland and Arnon, 1950
Leaf areas were determined immediately after harvest using a Scan Jet 5370C (Hewlett-Packard, Palo Alto, CA). Leaves, stems, and roots were dried at 60°C until a constant weight was obtained (4 d) and final dry weights determined.
A/Ci response curves were made using a portable gas exchange system (LI-COR 6400; LI-COR, Lincoln, NE). The gas exchange system was zeroed daily using anhydrous calcium carbonate (Drierite, W.A. Hammond Drierite, Xenia, OH) to remove water and using soda lime (sofnolime granules, Morgan Medical, Kent, UK) to remove CO2 from the air entering the cuvette. Leaf temperatures were set at 25°C for all measurements, though actual temperature ranged from 25°C to 30°C. Leaves were illuminated using a red-blue light source attached to the gas-exchange system, and light levels were maintained at 1,200 µmol m–2 s–1 for the duration of the A versus Ci response curve. Leaf vapor pressure deficits were maintained between 0.5 to approximately 1.6 kPa. Measurements of photosynthetic carbon assimilation (A) were made starting at 400 µmol mol–1 CO2 surrounding the leaf, decreased stepwise to 50 µmol mol–1, returned to 400 µmol mol–1, and increased stepwise to 1,600 µmol mol–1 CO2. Each complete curve consisted of at least nine separate measurements. Values for A and Ci were calculated using the equations of von Caemmerer and Farquhar (1981)
The diurnal response of leaf photosynthesis was measured beginning at 6:30 AM and finishing at 6:30 PM; photosynthesis of a newly fully expanded leaf and a young expanding leaf were measured for all plants at approximately 3-h intervals. Measurements were made using a portable gas exchange system (LI-COR 6400). Light levels at each time point were set to reflect the ambient light over the day: 6:30 AM to 8 AM, 400 µmol m–2 s–1; 9:30 AM to 11 AM, 1,000 µmol m–2 s–1; 12.30 PM to 2 PM, 1,500 µmol m–2 s–1; 4:30 PM to 6:30 PM, 500 µmol m–2 s–1. Photosynthesis measurements were recorded at steady state.
Chlorophyll fluorescence measurements were performed on 3-week-old tobacco seedlings that had been grown in a controlled environment chamber providing 500 to 600 µmol m–2 s–1 light and a CO2 concentration of 360 µmol mol–1. Chlorophyll fluorescence parameters were obtained using a CF Imager chlorophyll fluorescence imaging system (Technologica, Colchester, UK; Barbagallo et al., 2003
Leaf discs sampled as described above were ground in liquid nitrogen and protein quantification determined (Harrison et al., 1998
SBPase activity was determined by phosphate release (Harrison et al., 1998
Carbohydrates and starch were extracted from leaf discs sampled 2 times, 10 h into the light period and 7 h into the dark period, on leaves 9, 10, 12, and 13 of 7-week-old plants. The leaf discs were incubated in 80% (v/v) ethanol for 30 min at 80°C and then washed 4 times with ethanol 80% (v/v). Suc was measured from the extracts in ethanol using an enzyme-based protocol (Stitt et al., 1989
All statistical analyses were done by comparing ANOVA, using Sys-stat, University of Essex, UK. The differences between means were tested using the Post hoc Tukey test (SPSS, Chicago).
We thank Professor Neil Baker for advice on fluorescence imaging analysis and Professor Susanne von Caemmerer for helpful discussions. Received October 19, 2004; returned for revision December 21, 2004; accepted January 17, 2005.
1 This work was supported by the Biotechnology and Biological Sciences Research Council (grant no. 84/P15748).  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.055046. * Corresponding author; e-mail rainc{at}essex.ac.uk; fax 44–(1206)–872592.
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ABSTRACT
RESULTS
) and sense plants with increased SBPase activity of 25% to 50% (
) and 60% to 100% (
). Values represent the mean of at least three plants ± SE. B, The relationship between SBPase activity (determined in the same leaf area used for photosynthesis) and light-saturated photosynthetic carbon assimilation rates at 400 µmol mol–1 (white symbols) and 1,200 µmol mol–1 (black symbols) external CO2 derived from the A/Ci curves. Individual measurements of photosynthesis are shown for each plant, and SBPase activity is the mean ± SE of triplicate assays on 2 extracts from each plant, line 6 (
), line 11 (
).
0.125–4 nmol) were incubated 25 min at room temperature with 300 µL of Biomol Green (Affiniti Research Products, Exeter, UK) and the A620 was measured using a microplate reader (VERSAmax, Molecular Devices, Sunnyvale, CA). 
