BACKGROUND
biochemical models of c <dig> photosynthesis  predict that a is limited by the slowest of three processes: the maximum carboxylation capacity of the enzyme rubisco , the regeneration of ribulose-5-phosphate  via whole chain electron transport , or the inorganic phosphate release from the utilization of triose phosphates   <cit> .
at current atmospheric , and under non stressed conditions, light saturated a operates at the transition between rubisco and rubp regeneration limitation.
globally,  is expected to increase from current levels of  <dig> ppm  <cit>  to over  <dig> ppm by the middle of this century  <cit> .
elevating  stimulates c <dig> photosynthesis by increasing the substrate for carboxylation, co <dig>  and by reducing photorespiration  <cit> .
therefore, as atmospheric carbon dioxide concentration increases, the control of photosynthesis will shift away from rubisco limitation toward rubp regeneration limitation.
although photosynthetic stimulation at  <dig> ppm  could in theory increase production by 34%, the observed increase in field c <dig> crops is only 15%  <cit> .
additional future increases in yield potential of the world's major crops through an increase in the proportion of biomass allocated to grain or an increase in the efficiency of light capture will be small, as conventional breeding programs are reaching the theoretical maximum with diminishing returns  <cit> .
in contrast, model simulations demonstrate that increasing photosynthetic efficiency under current  by optimizing the biochemistry of photosynthesis could increase the energy conversion efficiency of a given crop in less time than conventional breeding programs  <cit> .
at current levels of crop productivity, global food requirements may outpace current crop production by the middle of this century  <cit> .
taken together, these observations suggest that direct improvements in photosynthetic efficiency will be needed if we are to meet global food needs in the future.
a common acclimation response of plants grown at elevated  is to allocate fewer resources to rubisco, thereby downregulating maximum carboxylation capacity .
this so called photosynthetic acclimation makes more resources available for other metabolic processes  <cit> .
the implication is that plants could reallocate resources in the photosynthetic carbon reduction  cycle to increase the efficiency of n use in elevated   <cit> .
in practice, however, plants' photosynthetic resources are not optimally allocated for current  nor is their acclimation response optimal in elevated   <cit> .
theoretically, and by reference to a biochemical model of photosynthesis , a plant with a 15% decrease in rubisco content and 15% increase in rubp regeneration capacity could translate to a 40% increase in a and photosynthetic efficiency of nitrogen use at elevated  .
it follows that plants engineered with an increased capacity for rubp regeneration would have a greater increase in productivity in elevated  when compared to wild type plants  <cit> .
while  <dig> enzymes are involved in the pcr cycle, modeling and metabolic control analyses have consistently demonstrated that four enzymes are expected to exert the greatest control of flux in the cycle: ribulose bisphosphate carboxylase-oxygenase , sedoheptulose- <dig> -bisphosphatase , aldolase and transketolase  <cit> .
two enzymes, rubisco and sbpase, are predicted to have the greatest control over carbon assimilation  <cit> .
rubisco is well known to be highly abundant, containing 25% of leaf nitrogen   <cit>  and may in some cases account for up to half of leaf n  <cit> .
all attempts to improve photosynthesis by manipulating rubisco expression, activity, or specificity have yielded poor results, in part because of inherent tradeoffs between activity and specificity of the enzyme and limited capacity to add more of this highly abundant protein  <cit> .
an additional hurdle to engineering "better" rubsico is that the functional enzyme requires the coordinated assembly of eight plastid encoded and eight nuclear encoded subunits to form the large  and small  units of the hexadecameric enzyme <cit> .
with the exception of rubisco, the other enzymes exerting the greatest control on photosynthesis all function in the rubp regeneration portion of the pcr cycle.
thus, near term future improvements in photosynthetic biochemistry in c <dig> plants are more likely to be achieved by improving content or activity of enzymes other than rubisco .
sedoheptulose- <dig> -bisphosphatase  is positioned at the branch point between regenerative  and assimilatory  portions of the pcr cycle.
it functions to catalyze the irreversible dephosphorylation of sedoheptulose <dig> -bisphosphate  to sedoheptulose-7-phosphate .
transketolase then catalyzes the transfer for a two carbon ketol group from s7p to glyceraldehyde-3-phoshpate  to yield xylulose-5-phosphate  or ribose-5-phosphate   <cit> .
sbpase is therefore critical for maintaining the balance between the carbon needed for rubp regeneration and that leaving the cycle for biosynthesis  <cit> .
previous experiments have demonstrated that tobacco transformants overexpressing sbpase accumulated more biomass than wt in controlled environment chambers at ambient co <dig> <cit> .
smaller increases in biomass were reported for mature sbpase overexpressing plants grown in greenhouse conditions  <cit> .
additionally, overexpression of sbpase in rice did not increase biomass relative to wt for plants grown at ambient co <dig> levels in two controlled environments  <cit> .
the variance in the realized benefit of sbpase overexpression coupled with the fact that rubp regeneration is highly sensitive to environmental conditions underscores the need to test the response of plants with this single gene manipulation in agronomically relevant conditions  <cit> .
moreover, models predict that as atmospheric  increases so will the benefit of increasing rubp regeneration capacity in plants  <cit> .
therefore, we compared wt and sbpase overexpressing plants under field conditions at ambient and elevated  , and we tested the prediction that transformants would exhibit greater stimulation of photosynthesis and yield than wt plants when grown under fully open air co <dig> fumigation.
methods
plant material
wild type tobacco  and sense tobacco plants  overexpressing a full length arabidopsis thaliana sbpase cdna, driven by camv 35s promoter and the nopaline synthase termination sequence  <cit> , were germinated in petri dishes and transferred to soil when true leaves emerged.
sense plants  were germinated on hygromycin  medium.
one individual from each of two transgenic lines overexpressing sbpase with varying sbpase levels and several randomly selected wild type  individuals were selected for the experiments.
individuals were subsequently clonally propagated by rooting cuttings in peat pots on misting benches and then planted directly in the field at soyface on july  <dig>  <dig>
soyface site
the soyface facility is located in the experimental research station of the university of illinois at urbana-champaign  <cit> .
soybean  is grown in eight plots  located within a typically managed soybean field of ca.
<dig> hectares .
four rings are fumigated with pure  and four rings are non-fumigated controls.
six cuttings of each sbpase genotype  and six of wt were planted in subplots within each ring.
ambient atmospheric  at the beginning of the  <dig> field season was ca.
<dig> ppm and the target  for elevated rings in  <dig> was  <dig> ppm .
in the fumigated rings, 89% of  values recorded every ten minutes from june  <dig> to september  <dig>   <dig>  were within 10% of the target value of  <dig> ppm.
the mean daily  in elevated rings at soyface during that time was  <dig>  ±  <dig>   ppm.
elevated rings were fumigated using a modification of the method of miglietta et al.
<cit> .
leaf protein and western blotting
prior to planting, leaf discs were collected from cuttings and immediately frozen in liquid nitrogen to confirm that sense plants had greater sbpase content than wt.
protein quantifications and western blots were performed following  <cit> .
sample lanes were loaded on an equal protein basis, separated using 10%  sds-page, transferred to polyvinylidene difluoride membrane, and probed using antibodies raised against sbpase and transketolase.
antibody target proteins were detected using horseradish peroxidase conjugated to the secondary antibody and ecl chemiluminescence detection reagent .
western blots were quantified by densiometry using the molecular imaging gel doc xr system  and imaging software.
in situ measurements of gas exchange and photosynthetic parameters
the diurnal course of photosynthesis at the soyface site was measured on two young fully expand leaves from each genotype at ambient conditions at both normal   and elevated   at five time points on two dates in august,  <dig>  to ensure that each plant was measured in similar environmental conditions, the leds of the controlled environment cuvettes of the gas exchange system  were set to deliver the same ambient light ppfd.
temperature and relative humidity were similarly set to ambient conditions and kept constant for the duration of each measurement period in the diurnal course.
to estimate the total daily carbon gain , photosynthesis was assumed to increase linearly from  <dig> μmol co <dig> m- <dig> s- <dig> at dawn  to the first measured value and decrease linearly from the last measured values to  <dig> μmol co <dig> m- <dig> s- <dig> at dusk .
sunrise and sunset data were determined using the us naval observatory website: http://aa.usno.navy.mil/data/docs/rs_oneyear.php.
dew on the leaves prevented us from measuring photosynthesis until about 10: <dig> h. we estimated a' for each block by integration using the trapezoidal rule and then performed analyses on the integrals  <cit> .
in vivo values of three photosynthetic parameters: maximum carboxylation capacity , maximum linear electron transport through photosystem ii  and respiration in the light  were determined by measuring the response of a to intercellular   on august  <dig> and august  <dig>  <dig>  a vs. ci curves were measured in situ on one young fully expanded leaf of each genotype in all blocks of each treatment  with an open gas exchange system .
initially, plants were allowed to reach steady state photosynthesis at their growth   at a saturating light level of  <dig> μmol m- <dig> s- <dig>  mean leaf to air vapor pressure deficit  was  <dig>  ±  <dig>  , and mean leaf temperature was  <dig> ± 1°c .
once steady state was reached, photosynthetic uptake rate  and chlorophyll fluorescence parameters were recorded at the growth ; then  was decreased in  <dig> or  <dig> uniform steps to  <dig> ppm, returned to growth , and then increased in  <dig> or  <dig> uniform steps to  <dig> ppm .
a minimum of  <dig> data points were collected for each plant following the methods outlined by long and bernacchi  <cit> .
curves were measured in the morning to avoid confounding treatment and genotype effects with transient decreases in water potential, decreases in chloroplast inorganic phosphate concentration or decreases in maximum photosystem ii  efficiency .
electron transport rate , the actual flux of photons driving psii, and fv'/fm' were calculated using fluorescence parameters, fs, fm', fo',  <cit> .
fluorescence parameters were estimated using a licor  <dig> integrated gas exchange system equipped with a fluorescence and light source accessory .
fs is the steady state light adapted fluorescence, fm' is the maximal fluorescence of a light adapted leaf following a saturating light pulse, and fo' is the minimal fluorescence of a light adapted leaf that is darkened.
etr=fm′-fs′fmfiαleaf
where f, is the fraction of photons absorbed by psii, assumed be  <dig>  for c <dig> plants; i is the incident photon flux density ; and α is leaf absorptance which was constant .
a vs. ci curves were fitted using a biochemical model of photosynthesis  <cit>  including the temperature response functions determined by bernacchi et al.
<cit>  and were solved for the parameters vc,max, jmax and rd.
the kinetic constants for rubisco, ko, kc and Γ* in tobacco are taken from  <cit> .
data below the inflection point of the curve were used to solve for vc,max and rd using the equation for rubisco limited photosynthesis  <cit>  and following the method of  <cit> .
data above the inflection point of the a vs. ci curve were similarly used to solve for jmax using the equation for rubp limited photosynthesis  <cit> .
leaf traits and final biomass
leaf disks  were collected from plants on august  <dig> during the midday gas exchange measurements.
leaf disks were sealed in pre-cooled vials, placed in coolers and disk fresh weights were determined the same afternoon.
leaf disks were dried at 60°c for  <dig> hours and then re-weighed.
dry and wet weights were used to determine specific leaf area  and specific leaf weight .
these same disks were then ground to a fine powder and used to determine leaf carbon  and nitrogen  content by total combustion .
statistical analyses were performed using sas  and jump .
trait and parameter means of sbpase transformant lines were statistically indistinguishable so the lines were pooled for subsequent anovas.
simple effect tests as implemented in sas  were used to determine if there were significant differences 1) between types within treatments  or 2) between treatments within types .
the diurnals at soyface were analyzed as a repeated measures mixed model analysis of variance .
as above, sbpase lines were statistically indistinguishable during the time course and were pooled in anovas.
type , co <dig> concentration  , and time of day  were fixed factors.
each block contained one ambient and one elevated co <dig> plot and was considered a random factor.
as there were only  <dig> blocks, significant probability was set at p <  <dig>  a priori to reduce the possibility of type ii errors  <cit> .
RESULTS
protein quantification
sbpase content was 150%  greater in transformants and more uniform relative to wt plants .
sbpase overexpressing lines did not differ from each other in terms of the sbpase protein content .
transketolase content was similar in wt and transformants .
diurnal course of gas exchange and electron transport rate
diurnal trends of photosynthesis and fluorescence parameters were measured at their respective growth   on july  <dig> and august  <dig>   <dig> .
on july  <dig>  photosynthetic rate  was significantly higher in transformants, due to significant differences around midday at elevated   .
on average, electron transport rate   was significantly higher for transformants at elevated  .
differences in etr between transformants and wt were driven by significantly lower values for wt plants at midday in elevated  on july  <dig>  on august  <dig>  a was significantly greater at elevated co <dig> for both wt and transformants , however, there were no detectable differences in photosynthesis between wt and transformants.
etr was similar for transformants and wt plants in ambient and elevated co <dig> on august  <dig> .
diurnal measurements were collected on july  <dig> and august  <dig>   <dig>
on july  <dig>  elevating  increased a' for wt and transformants .
transformants had significantly greater a' than wt in elevated  , but in ambient  they were not significantly different .
on july  <dig>  a' increased 14% for transformants but only 8% for wt.
in contrast, on august  <dig>  elevating  increased a' by 6% for transformants but by 11% for wt .
there were no detectable differences in a' between transformants and wt in ambient or elevated  on august  <dig> .
photosynthetic biochemical parameters
a vs. ci curves were measured in the field the morning following each diurnal  under similar meteorological conditions as the diurnals.
on august 1st vc,max tended to be lower in elevated   than in ambient   but the trend was not significant .
there was a type by  interaction for the response of jmax .
further analysis revealed that growth at elevated  significantly increased jmax of transformants but not wt  on august  <dig>  consequently, the ratio of vc,max to jmax  was similar between wt and transformants at ambient .
elevating  significantly reduced v/j in transformants  but not in wt plants on august  <dig> .
growth at elevated  significantly increased respiration in the light  and transformants had significantly higher rd than wt in both ambient  and elevated    on august  <dig>
parameters were derived from a vs  curves measured in the field see methods for details.
only three blocks could be measured on august  <dig>
on august  <dig>  both vc,max and jmax were significantly lower for plants grown under elevated than ambient  .
transformants had significantly greater jmax than wt at ambient  but not in elevated  .
elevating  significantly decreased v/j in transformants and wt .
elevating  significantly increased rd for wt and transformants .
leaf traits and final biomass
specific leaf area  was significantly lower at elevated  compared to ambient, and transformants had significantly lower sla than wt plants .
further analysis revealed that transformant sla was lower than wt sla in elevated  .
elevating  significantly decreased leaf nitrogen content ; consequently, the carbon to nitrogen ratio  of leaves increased significantly in elevated  .
transformant c:n increased more than wt .
above ground biomass  was greater for plants grown in elevated  and transformant biomass was greater than wt plants .
biomass increased more for transformants than wt following growth in elevated   .
discussion
the goal of our experiments was to test the hypothesis that tobacco plants transformed to over express the pcr cycle enzyme sbpase would exhibit greater stimulation of carbon assimilation than wt plants when grown at elevated  under field conditions .
transformant biomass increases more than wt at elevated
when grown under fully open air co <dig> fumigation, sbpase overexpressing plants displayed up to 14% greater light saturated photosynthetic rates  and up to 21% more linear electron flux through psii  than wt plants.
moreover, after  <dig> weeks of growth at elevated , harvested biomass increased by 13% in wt plants and more than 22% in transformants when compared to plants grown in ambient .
in a prior experiment, the same transformants grown in a greenhouse under prevailing light conditions at ambient  accumulated 12% more biomass than wt plants  <cit> .
here, at ambient   under field conditions, transformants also yielded 12% more biomass than wt plants  consistent with the lefebvre et al  <cit>  greenhouse study.
taken together, these results support our hypothesis and clearly show the benefit of overexpressing sbpase in field grown plants at both current and future levels of atmospheric .
wt biomass was 13% greater in elevated  when compared to ambient grown wt plants, which is somewhat lower than the average increase in biomass for c <dig> crops in face experiments .
growth at elevated  alters plant insect interaction and increases palatability of crops  <cit> ; thus it is possible that yield stimulations were slightly lower because of aphid and hornworm herbivory .
in tobacco in particular, aphid infestation significantly reduced the stimulatory effect of  on biomass  <cit> .
nevertheless, transformant biomass increased more than wt at elevated   and more than the average for c <dig> crops in face experiments.
lefebvre et al.
<cit>  reported that the greatest differences between transformants and wt photosynthetic rates occurred prior to flowering in greenhouse plants and during early development in chamber grown plants.
the differences between young expanding and fully expanded leaves could not be accounted for by differential sbpase activity .
we show that in ambient and elevated  plots, carbon uptake was enhanced more for transformants during the vegetative phase  than when plants were starting to flower .
when plants were beginning to flower, differences between transformants and wt were no longer detectable, yet carbon uptake was consistently stimulated for plants growing in elevated .
ultimately, even though the realized increase in a and a' between wt and transformants falls well short of the theoretical 40% increase in assimilation predicted if plants were to reallocate 15% of photosynthetic resources from rubisco to rubp regeneration , increases in the carbon uptake of transformants early in growth and prior to flowering were sufficiently large to increase final biomass.
several studies demonstrate that changing expression and activity level of sbpase directly impacts carbon assimilation, growth, and biomass accumulation in tobacco growing at current ambient    <cit> .
while the positive relationship between sbpase activity and carbon assimilation was clearly shown in wt and transformants  <cit> , overexpression of sbpase in rice and tobacco has not always increased biomass for plants grown at ambient  levels in controlled environments  <cit> .
for instance, lefebvre et al.
noted that no increase in photosynthesis or plant yield was evident for tobacco transformants grown in winter when days were shorter and light levels were lower <cit>  .
the observations of lefebvre et al.
<cit>  and this study are also consistent with the notion that sbpase exerts control over co <dig> fixation under light saturating conditions.
by definition, the amount of spbase would not affect the light limited rate of photosynthesis which depends on the rate of production of nadph and atp on the photosynthetic membrane.
our diurnal measurements are consistent with these expectations, as transformants with increased sbpase activity showed the greatest increases in carbon assimilation relative to wild type plants around midday when light levels were highest.
in contrast, there was no difference in assimilation rates between the sbpase overexpressing and wild type plants at the beginning or end of the day .
acclimation to  increases nutrient use efficiency more for transformants than wt
both wt and transformants showed evidence of a similar decrease in vc,max after a month of growth at elevated ,indicating photosynthetic acclimation via down regulation of in vivo rubisco capacity.
photosynthetic acclimation to growth in elevated  is presumed to be a biochemical adjustment to optimize nitrogen use  <cit> .
as  increases so does the catalytic rate of rubisco, therefore less n needs to be invested in rubisco to fix carbon.
reallocation of n is then, for instance, available to upregulate respiratory metabolism in response to growth at elevated   <cit> .
sbpase represents less than 1% of the n contained in the enzymes of photosynthetic carbon metabolism  <cit> .
it is therefore remarkable that ca.
50% increase in the amount of this protein in transformants results in detectable increases in co <dig> assimilation.
the relatively large increase in co <dig> assimilation at elevated  was associated with a significant decrease in leaf n per unit mass .
thus for a small increase in protein, transformants had a significantly greater increases in nitrogen use efficiency than wt at elevated .
the results are consistent with numerous other face studies showing that will stimulate growth in spite of photosynthetic acclimation and that growth at elevated increases nitrogen use efficiency .
transformants and wt plants grown in elevated  tended to have higher respiration in the light  than plants in ambient plots.
leaves of plants grown under elevated  accumulate larger concentrations of non-structural carbohydrates   <cit> , and this may underlie higher respiration  <cit> .
recently, leakey et al.
<cit>  demonstrated that the acclimation response of respiration to elevated  was mediated via transcriptional upregulation of respiratory enzymes.
we speculate that the reportedly greater sucrose and starch accumulation in transformants  <cit>  stimulates additional acclimation of respiration to elevated  and may therefore also diminish the benefit of overexpressing sbpase.
alternatively, higher rd in transformants may be a result of the unregulated overexpression of the enzyme.
either way, higher rd, the requirement for high light, and unmeasured natural stresses all would contribute to a lower realized benefit to overexpressing sbpase in the field.
CONCLUSIONS
the data presented in this paper have demonstrated that transgenic tobacco plants with increased sbpase have the potential for greater stimulation of photosynthesis and biomass production relative to wild type tobacco when grown at elevated .
differences between theoretical and realized increases in carbon assimilation are to be expected as studies of pcr cycle antisense plants have demonstrated that the relative importance of any one pcr cycle enzyme is not fixed and will vary according to environmental and developmental conditions .
nevertheless, our findings are consistent with the notion that elevating  increases the metabolic control of rubp-regeneration and decreases the control exerted by rubisco at light saturation  <cit> .
though smaller than theoretically predicted, the increases in photosynthetic stimulation at elevated  demonstrated here are indicative that c <dig> crop plants can be engineered to meet a rapidly changing environment.
authors' contributions
dr conceived and designed the experiment, acquired and analyzed the data, and wrote the paper.
al aided in data acquisition and analysis, revised the paper, and gave final approval of the manuscript.
mk aided in data acquisition, data analysis and gave final approval of the manuscript.
cr provided the transformants, provided technical support, revised the paper, and gave final approval of the manuscript.
sl and do conceived and aided in the design of the experiment, revised the manuscript, and gave final approval of the manuscript.

 biochemical models of c photosynthesis predict that a is limited by the slowest of three processes: the maximum carboxylation capacity of the enzyme rubisco, the regeneration of ribulose-5-phosphate via whole chain electron transport, or the inorganic phosphate release from the utilization of triose phosphates. at current atmospheric, and under non stressed conditions, light saturated a operates at the transition between rubisco and rubp regeneration limitation. globally, is expected to increase from current levels of ppm to over ppm by the middle of this century. elevating stimulates c photosynthesis by increasing the substrate for carboxylation, co and by reducing photorespiration. therefore, as atmospheric carbon dioxide concentration increases, the control of photosynthesis will shift away from rubisco limitation toward rubp regeneration limitation. although photosynthetic stimulation at ppm could in theory increase production by 34%, the observed increase in field c crops is only 15%. additional future increases in yield potential of the world's major crops through an increase in the proportion of biomass allocated to grain or an increase in the efficiency of light capture will be small, as conventional breeding programs are reaching the theoretical maximum with diminishing returns. in contrast, model simulations demonstrate that increasing photosynthetic efficiency under current by optimizing the biochemistry of photosynthesis could increase the energy conversion efficiency of a given crop in less time than conventional breeding programs. at current levels of crop productivity, global food requirements may outpace current crop production by the middle of this century. taken together, these observations suggest that direct improvements in photosynthetic efficiency will be needed if we are to meet global food needs in the future. a common acclimation response of plants grown at elevated is to allocate fewer resources to rubisco, thereby downregulating maximum carboxylation capacity. this so called photosynthetic acclimation makes more resources available for other metabolic processes. the implication is that plants could reallocate resources in the photosynthetic carbon reduction cycle to increase the efficiency of n use in elevated. in practice, however, plants' photosynthetic resources are not optimally allocated for current nor is their acclimation response optimal in elevated. theoretically, and by reference to a biochemical model of photosynthesis, a plant with a 15% decrease in rubisco content and 15% increase in rubp regeneration capacity could translate to a 40% increase in a and photosynthetic efficiency of nitrogen use at elevated. it follows that plants engineered with an increased capacity for rubp regeneration would have a greater increase in productivity in elevated when compared to wild type plants. while enzymes are involved in the pcr cycle, modeling and metabolic control analyses have consistently demonstrated that four enzymes are expected to exert the greatest control of flux in the cycle: ribulose bisphosphate carboxylase-oxygenase, sedoheptulose- -bisphosphatase, aldolase and transketolase. two enzymes, rubisco and sbpase, are predicted to have the greatest control over carbon assimilation. rubisco is well known to be highly abundant, containing 25% of leaf nitrogen and may in some cases account for up to half of leaf n. all attempts to improve photosynthesis by manipulating rubisco expression, activity, or specificity have yielded poor results, in part because of inherent tradeoffs between activity and specificity of the enzyme and limited capacity to add more of this highly abundant protein. an additional hurdle to engineering "better" rubsico is that the functional enzyme requires the coordinated assembly of eight plastid encoded and eight nuclear encoded subunits to form the large and small units of the hexadecameric enzyme. with the exception of rubisco, the other enzymes exerting the greatest control on photosynthesis all function in the rubp regeneration portion of the pcr cycle. thus, near term future improvements in photosynthetic biochemistry in c plants are more likely to be achieved by improving content or activity of enzymes other than rubisco. sedoheptulose- -bisphosphatase is positioned at the branch point between regenerative and assimilatory portions of the pcr cycle. it functions to catalyze the irreversible dephosphorylation of sedoheptulose -bisphosphate to sedoheptulose-7-phosphate. transketolase then catalyzes the transfer for a two carbon ketol group from s7p to glyceraldehyde-3-phoshpate to yield xylulose-5-phosphate or ribose-5-phosphate. sbpase is therefore critical for maintaining the balance between the carbon needed for rubp regeneration and that leaving the cycle for biosynthesis. previous experiments have demonstrated that tobacco transformants overexpressing sbpase accumulated more biomass than wt in controlled environment chambers at ambient co. smaller increases in biomass were reported for mature sbpase overexpressing plants grown in greenhouse conditions. additionally, overexpression of sbpase in rice did not increase biomass relative to wt for plants grown at ambient co levels in two controlled environments. the variance in the realized benefit of sbpase overexpression coupled with the fact that rubp regeneration is highly sensitive to environmental conditions underscores the need to test the response of plants with this single gene manipulation in agronomically relevant conditions. moreover, models predict that as atmospheric increases so will the benefit of increasing rubp regeneration capacity in plants. therefore, we compared wt and sbpase overexpressing plants under field conditions at ambient and elevated, and we tested the prediction that transformants would exhibit greater stimulation of photosynthesis and yield than wt plants when grown under fully open air co fumigation. methods plant material wild type tobacco and sense tobacco plants overexpressing a full length arabidopsis thaliana sbpase cdna, driven by camv 35s promoter and the nopaline synthase termination sequence, were germinated in petri dishes and transferred to soil when true leaves emerged. sense plants were germinated on hygromycin medium. one individual from each of two transgenic lines overexpressing sbpase with varying sbpase levels and several randomly selected wild type individuals were selected for the experiments. individuals were subsequently clonally propagated by rooting cuttings in peat pots on misting benches and then planted directly in the field at soyface on july soyface site the soyface facility is located in the experimental research station of the university of illinois at urbana-champaign. soybean is grown in eight plots located within a typically managed soybean field of ca. hectares. four rings are fumigated with pure and four rings are non-fumigated controls. six cuttings of each sbpase genotype and six of wt were planted in subplots within each ring. ambient atmospheric at the beginning of the field season was ca. ppm and the target for elevated rings in was ppm. in the fumigated rings, 89% of values recorded every ten minutes from june to september were within 10% of the target value of ppm. the mean daily in elevated rings at soyface during that time was ± ppm. elevated rings were fumigated using a modification of the method of miglietta et al.. leaf protein and western blotting prior to planting, leaf discs were collected from cuttings and immediately frozen in liquid nitrogen to confirm that sense plants had greater sbpase content than wt. protein quantifications and western blots were performed following. sample lanes were loaded on an equal protein basis, separated using 10% sds-page, transferred to polyvinylidene difluoride membrane, and probed using antibodies raised against sbpase and transketolase. antibody target proteins were detected using horseradish peroxidase conjugated to the secondary antibody and ecl chemiluminescence detection reagent. western blots were quantified by densiometry using the molecular imaging gel doc xr system and imaging software. in situ measurements of gas exchange and photosynthetic parameters the diurnal course of photosynthesis at the soyface site was measured on two young fully expand leaves from each genotype at ambient conditions at both normal and elevated at five time points on two dates in august, to ensure that each plant was measured in similar environmental conditions, the leds of the controlled environment cuvettes of the gas exchange system were set to deliver the same ambient light ppfd. temperature and relative humidity were similarly set to ambient conditions and kept constant for the duration of each measurement period in the diurnal course. to estimate the total daily carbon gain, photosynthesis was assumed to increase linearly from μmol co m- s- at dawn to the first measured value and decrease linearly from the last measured values to μmol co m- s- at dusk. sunrise and sunset data were determined using the us naval observatory website: http://aa.usno.navy.mil/data/docs/rs_oneyear.php. dew on the leaves prevented us from measuring photosynthesis until about 10: h. we estimated a' for each block by integration using the trapezoidal rule and then performed analyses on the integrals. in vivo values of three photosynthetic parameters: maximum carboxylation capacity, maximum linear electron transport through photosystem ii and respiration in the light were determined by measuring the response of a to intercellular on august and august a vs. ci curves were measured in situ on one young fully expanded leaf of each genotype in all blocks of each treatment with an open gas exchange system. initially, plants were allowed to reach steady state photosynthesis at their growth at a saturating light level of μmol m- s- mean leaf to air vapor pressure deficit was ±, and mean leaf temperature was ± 1°c. once steady state was reached, photosynthetic uptake rate and chlorophyll fluorescence parameters were recorded at the growth; then was decreased in or uniform steps to ppm, returned to growth, and then increased in or uniform steps to ppm. a minimum of data points were collected for each plant following the methods outlined by long and bernacchi. curves were measured in the morning to avoid confounding treatment and genotype effects with transient decreases in water potential, decreases in chloroplast inorganic phosphate concentration or decreases in maximum photosystem ii efficiency. electron transport rate, the actual flux of photons driving psii, and fv'/fm' were calculated using fluorescence parameters, fs, fm', fo',. fluorescence parameters were estimated using a licor integrated gas exchange system equipped with a fluorescence and light source accessory. fs is the steady state light adapted fluorescence, fm' is the maximal fluorescence of a light adapted leaf following a saturating light pulse, and fo' is the minimal fluorescence of a light adapted leaf that is darkened. etr=fm′-fs′fmfiαleaf where f, is the fraction of photons absorbed by psii, assumed be for c plants; i is the incident photon flux density; and α is leaf absorptance which was constant. a vs. ci curves were fitted using a biochemical model of photosynthesis including the temperature response functions determined by bernacchi et al. and were solved for the parameters vc,max, jmax and rd. the kinetic constants for rubisco, ko, kc and Γ* in tobacco are taken from. data below the inflection point of the curve were used to solve for vc,max and rd using the equation for rubisco limited photosynthesis and following the method of. data above the inflection point of the a vs. ci curve were similarly used to solve for jmax using the equation for rubp limited photosynthesis. leaf traits and final biomass leaf disks were collected from plants on august during the midday gas exchange measurements. leaf disks were sealed in pre-cooled vials, placed in coolers and disk fresh weights were determined the same afternoon. leaf disks were dried at 60°c for hours and then re-weighed. dry and wet weights were used to determine specific leaf area and specific leaf weight. these same disks were then ground to a fine powder and used to determine leaf carbon and nitrogen content by total combustion. statistical analyses were performed using sas and jump. trait and parameter means of sbpase transformant lines were statistically indistinguishable so the lines were pooled for subsequent anovas. simple effect tests as implemented in sas were used to determine if there were significant differences 1) between types within treatments or 2) between treatments within types. the diurnals at soyface were analyzed as a repeated measures mixed model analysis of variance. as above, sbpase lines were statistically indistinguishable during the time course and were pooled in anovas. type, co concentration, and time of day were fixed factors. each block contained one ambient and one elevated co plot and was considered a random factor. as there were only blocks, significant probability was set at p < a priori to reduce the possibility of type ii errors.  protein quantification sbpase content was 150% greater in transformants and more uniform relative to wt plants. sbpase overexpressing lines did not differ from each other in terms of the sbpase protein content. transketolase content was similar in wt and transformants. diurnal course of gas exchange and electron transport rate diurnal trends of photosynthesis and fluorescence parameters were measured at their respective growth on july and august. on july photosynthetic rate was significantly higher in transformants, due to significant differences around midday at elevated. on average, electron transport rate was significantly higher for transformants at elevated. differences in etr between transformants and wt were driven by significantly lower values for wt plants at midday in elevated on july on august a was significantly greater at elevated co for both wt and transformants, however, there were no detectable differences in photosynthesis between wt and transformants. etr was similar for transformants and wt plants in ambient and elevated co on august. diurnal measurements were collected on july and august on july elevating increased a' for wt and transformants. transformants had significantly greater a' than wt in elevated, but in ambient they were not significantly different. on july a' increased 14% for transformants but only 8% for wt. in contrast, on august elevating increased a' by 6% for transformants but by 11% for wt. there were no detectable differences in a' between transformants and wt in ambient or elevated on august. photosynthetic biochemical parameters a vs. ci curves were measured in the field the morning following each diurnal under similar meteorological conditions as the diurnals. on august 1st vc,max tended to be lower in elevated than in ambient but the trend was not significant. there was a type by interaction for the response of jmax. further analysis revealed that growth at elevated significantly increased jmax of transformants but not wt on august consequently, the ratio of vc,max to jmax was similar between wt and transformants at ambient. elevating significantly reduced v/j in transformants but not in wt plants on august. growth at elevated significantly increased respiration in the light and transformants had significantly higher rd than wt in both ambient and elevated on august parameters were derived from a vs curves measured in the field see methods for details. only three blocks could be measured on august on august both vc,max and jmax were significantly lower for plants grown under elevated than ambient. transformants had significantly greater jmax than wt at ambient but not in elevated. elevating significantly decreased v/j in transformants and wt. elevating significantly increased rd for wt and transformants. leaf traits and final biomass specific leaf area was significantly lower at elevated compared to ambient, and transformants had significantly lower sla than wt plants. further analysis revealed that transformant sla was lower than wt sla in elevated. elevating significantly decreased leaf nitrogen content; consequently, the carbon to nitrogen ratio of leaves increased significantly in elevated. transformant c:n increased more than wt. above ground biomass was greater for plants grown in elevated and transformant biomass was greater than wt plants. biomass increased more for transformants than wt following growth in elevated. discussion the goal of our experiments was to test the hypothesis that tobacco plants transformed to over express the pcr cycle enzyme sbpase would exhibit greater stimulation of carbon assimilation than wt plants when grown at elevated under field conditions. transformant biomass increases more than wt at elevated when grown under fully open air co fumigation, sbpase overexpressing plants displayed up to 14% greater light saturated photosynthetic rates and up to 21% more linear electron flux through psii than wt plants. moreover, after weeks of growth at elevated, harvested biomass increased by 13% in wt plants and more than 22% in transformants when compared to plants grown in ambient. in a prior experiment, the same transformants grown in a greenhouse under prevailing light conditions at ambient accumulated 12% more biomass than wt plants. here, at ambient under field conditions, transformants also yielded 12% more biomass than wt plants consistent with the lefebvre et al greenhouse study. taken together, these results support our hypothesis and clearly show the benefit of overexpressing sbpase in field grown plants at both current and future levels of atmospheric. wt biomass was 13% greater in elevated when compared to ambient grown wt plants, which is somewhat lower than the average increase in biomass for c crops in face experiments. growth at elevated alters plant insect interaction and increases palatability of crops; thus it is possible that yield stimulations were slightly lower because of aphid and hornworm herbivory. in tobacco in particular, aphid infestation significantly reduced the stimulatory effect of on biomass. nevertheless, transformant biomass increased more than wt at elevated and more than the average for c crops in face experiments. lefebvre et al. reported that the greatest differences between transformants and wt photosynthetic rates occurred prior to flowering in greenhouse plants and during early development in chamber grown plants. the differences between young expanding and fully expanded leaves could not be accounted for by differential sbpase activity. we show that in ambient and elevated plots, carbon uptake was enhanced more for transformants during the vegetative phase than when plants were starting to flower. when plants were beginning to flower, differences between transformants and wt were no longer detectable, yet carbon uptake was consistently stimulated for plants growing in elevated. ultimately, even though the realized increase in a and a' between wt and transformants falls well short of the theoretical 40% increase in assimilation predicted if plants were to reallocate 15% of photosynthetic resources from rubisco to rubp regeneration, increases in the carbon uptake of transformants early in growth and prior to flowering were sufficiently large to increase final biomass. several studies demonstrate that changing expression and activity level of sbpase directly impacts carbon assimilation, growth, and biomass accumulation in tobacco growing at current ambient. while the positive relationship between sbpase activity and carbon assimilation was clearly shown in wt and transformants, overexpression of sbpase in rice and tobacco has not always increased biomass for plants grown at ambient levels in controlled environments. for instance, lefebvre et al. noted that no increase in photosynthesis or plant yield was evident for tobacco transformants grown in winter when days were shorter and light levels were lower. the observations of lefebvre et al. and this study are also consistent with the notion that sbpase exerts control over co fixation under light saturating conditions. by definition, the amount of spbase would not affect the light limited rate of photosynthesis which depends on the rate of production of nadph and atp on the photosynthetic membrane. our diurnal measurements are consistent with these expectations, as transformants with increased sbpase activity showed the greatest increases in carbon assimilation relative to wild type plants around midday when light levels were highest. in contrast, there was no difference in assimilation rates between the sbpase overexpressing and wild type plants at the beginning or end of the day. acclimation to increases nutrient use efficiency more for transformants than wt both wt and transformants showed evidence of a similar decrease in vc,max after a month of growth at elevated,indicating photosynthetic acclimation via down regulation of in vivo rubisco capacity. photosynthetic acclimation to growth in elevated is presumed to be a biochemical adjustment to optimize nitrogen use. as increases so does the catalytic rate of rubisco, therefore less n needs to be invested in rubisco to fix carbon. reallocation of n is then, for instance, available to upregulate respiratory metabolism in response to growth at elevated. sbpase represents less than 1% of the n contained in the enzymes of photosynthetic carbon metabolism. it is therefore remarkable that ca. 50% increase in the amount of this protein in transformants results in detectable increases in co assimilation. the relatively large increase in co assimilation at elevated was associated with a significant decrease in leaf n per unit mass. thus for a small increase in protein, transformants had a significantly greater increases in nitrogen use efficiency than wt at elevated. the results are consistent with numerous other face studies showing that will stimulate growth in spite of photosynthetic acclimation and that growth at elevated increases nitrogen use efficiency. transformants and wt plants grown in elevated tended to have higher respiration in the light than plants in ambient plots. leaves of plants grown under elevated accumulate larger concentrations of non-structural carbohydrates, and this may underlie higher respiration. recently, leakey et al. demonstrated that the acclimation response of respiration to elevated was mediated via transcriptional upregulation of respiratory enzymes. we speculate that the reportedly greater sucrose and starch accumulation in transformants stimulates additional acclimation of respiration to elevated and may therefore also diminish the benefit of overexpressing sbpase. alternatively, higher rd in transformants may be a result of the unregulated overexpression of the enzyme. either way, higher rd, the requirement for high light, and unmeasured natural stresses all would contribute to a lower realized benefit to overexpressing sbpase in the field.  the data presented in this paper have demonstrated that transgenic tobacco plants with increased sbpase have the potential for greater stimulation of photosynthesis and biomass production relative to wild type tobacco when grown at elevated. differences between theoretical and realized increases in carbon assimilation are to be expected as studies of pcr cycle antisense plants have demonstrated that the relative importance of any one pcr cycle enzyme is not fixed and will vary according to environmental and developmental conditions. nevertheless, our findings are consistent with the notion that elevating increases the metabolic control of rubp-regeneration and decreases the control exerted by rubisco at light saturation. though smaller than theoretically predicted, the increases in photosynthetic stimulation at elevated demonstrated here are indicative that c crop plants can be engineered to meet a rapidly changing environment. authors' contributions dr conceived and designed the experiment, acquired and analyzed the data, and wrote the paper. al aided in data acquisition and analysis, revised the paper, and gave final approval of the manuscript. mk aided in data acquisition, data analysis and gave final approval of the manuscript. cr provided the transformants, provided technical support, revised the paper, and gave final approval of the manuscript. sl and do conceived and aided in the design of the experiment, revised the manuscript, and gave final approval of the manuscript. 
there was evidence of photosynthetic acclimation to elevated via downregulation of vc,max in both wt and transformants. we tested the hypothesis that tobacco transformed to overexpressing sbpase will exhibit greater stimulation of a than wild type tobacco when grown under field conditions at elevated under fully open air fumigation. nevertheless, greater carbon assimilation and electron transport rates for transformants led to greater yield increases than wt at elevated compared to ambient grown plants. these results provide proof of concept that increasing content and activity of a single photosynthesis enzyme can enhance carbon assimilation and yield of c crops grown at expected by the middle of the 21st century. biochemical models predict that photosynthesis in c plants is most frequently limited by the slower of two processes, the maximum capacity of the enzyme rubisco to carboxylate rubp, or the regeneration of rubp via electron transport. in the future, leaf photosynthesis should be increasingly limited by rubp regeneration, as is predicted to exceed ppm by the c cycle enzyme sedoheptulose- bisphosphatase has been shown to exert strong metabolic control over rubp regeneration at light saturation. climate changephotosynthetic carbon reduction cyclec plantsrubp regenerationelectron transportimproving photosynthesis
 biochemical models predict that photosynthesis in c plants is most frequently limited by the slower of two processes, the maximum capacity of the enzyme rubisco to carboxylate rubp, or the regeneration of rubp via electron transport. at current atmospheric levels rubisco is not saturated; consequently, elevating increases the velocity of carboxylation and inhibits the competing oxygenation reaction which is also catalyzed by rubisco. in the future, leaf photosynthesis should be increasingly limited by rubp regeneration, as is predicted to exceed ppm by the c cycle enzyme sedoheptulose- bisphosphatase has been shown to exert strong metabolic control over rubp regeneration at light saturation.  we tested the hypothesis that tobacco transformed to overexpressing sbpase will exhibit greater stimulation of a than wild type tobacco when grown under field conditions at elevated under fully open air fumigation. growth under elevated stimulated instantaneous a and the diurnal photosynthetic integral more in transformants than wt. there was evidence of photosynthetic acclimation to elevated via downregulation of vc,max in both wt and transformants. nevertheless, greater carbon assimilation and electron transport rates for transformants led to greater yield increases than wt at elevated compared to ambient grown plants.  these results provide proof of concept that increasing content and activity of a single photosynthesis enzyme can enhance carbon assimilation and yield of c crops grown at expected by the middle of the 21st century. climate changephotosynthetic carbon reduction cyclec plantsrubp regenerationelectron transportimproving photosynthesis 