Simultaneous stimulation of sedoheptulose 1,7‐bisphosphatase, fructose 1,6‐bisphophate aldolase and the photorespiratory glycine decarboxylase‐H protein increases CO 2 assimilation, vegetative biomass and seed yield in Arabidopsis

Summary In this article, we have altered the levels of three different enzymes involved in the Calvin–Benson cycle and photorespiratory pathway. We have generated transgenic Arabidopsis plants with altered combinations of sedoheptulose 1,7‐bisphosphatase (SBPase), fructose 1,6‐bisphophate aldolase (FBPA) and the glycine decarboxylase‐H protein (GDC‐H) gene identified as targets to improve photosynthesis based on previous studies. Here, we show that increasing the levels of the three corresponding proteins, either independently or in combination, significantly increases the quantum efficiency of PSII. Furthermore, photosynthetic measurements demonstrated an increase in the maximum efficiency of CO 2 fixation in lines over‐expressing SBPase and FBPA. Moreover, the co‐expression of GDC‐H with SBPase and FBPA resulted in a cumulative positive impact on leaf area and biomass. Finally, further analysis of transgenic lines revealed a cumulative increase of seed yield in SFH lines grown in high light. These results demonstrate the potential of multigene stacking for improving the productivity of food and energy crops.


Introduction
The accumulated photosynthate produced over the season determines the yield of a crop, but improvements in photosynthesis have not been used in traditional breeding approaches to identify high-yielding varieties. The reasons for this are twofold, (i) methodologies to make accurate field measurements have only been available in the last 10-20 years, and also, (ii) there is a lack of evidence to determine whether there is a correlation between the rate of photosynthesis on a leaf area basis and final yield of the crop (Evans, 2013;Fischer et al., 1998;Gifford and Evans, 1981). There is now an urgent need to increase crop productivity and yields to meet the nutritional demands of a growing world population, and there is growing evidence that this may be achieved through improvement of photosynthetic energy conversion to biomass (von Caemmerer and Evans, 2010;Ding et al., 2016;Lefebvre et al., 2005;Long et al., 2006Long et al., , 2015Simkin et al., 2015). Evidence from a combination of theoretical studies and transgenic approaches has provided compelling evidence that manipulation of the Calvin-Benson (CB) cycle can improve energy conversion efficiency and lead to an increase in yield potential (Long et al., 2006;Poolman et al., 2000;Raines, 2003Raines, , 2006Raines, , 2011Zhu et al., 2007Zhu et al., , 2010. Previous studies have demonstrated that even small reductions in individual CB cycle enzymes such as sedoheptulose 1,7bisphosphatase (SBPase) and fructose 1,6-bisphosphate aldolase (FBPA) negatively impact on carbon assimilation and growth, indicating that these enzymes exercise significant control over photosynthetic efficiency (Ding et al., 2016;Haake et al., 1998Haake et al., , 1999Harrison et al., 1998Harrison et al., , 2001Lawson et al., 2006;Raines, 2003;Raines and Paul, 2006;Raines et al., 1999). Furthermore, the disruption of the chloroplastic fructose-1,6-bisphosphatase (FBPase) gene was also shown to impact negatively on carbon fixation (Kossmann et al., 1994;Rojas-Gonz alez et al., 2015;Sahrawy et al., 2004). These results strongly suggested that improvements in photosynthetic carbon fixation may be achieved by increasing the activity of individual CB cycle enzymes. Evidence supporting this hypothesis came from transgenic tobacco plants over-expressing SBPase (Lefebvre et al., 2005), the cyanobacterial bifunctional SBPase/FBPase (Miyagawa et al., 2001) or FBPA (Uematsu et al., 2012). These single manipulations resulted in increase in photosynthetic carbon assimilation, enhanced growth and an increase in biomass. More recently, Simkin et al. (2015) demonstrated that the combined over-expression of SBPase and FBPA in tobacco resulted in a cumulative increase in biomass and that these increases could be further en'hanced by the overexpression of the cyanobacterial inorganic carbon transporter B (ictB), proposed to be involved in CO 2 transport, although its function was not established in these plants (Simkin et al., 2015). These results demonstrate the potential for the manipulation of photosynthesis, using multigene stacking approaches, to increase biomass yield (Simkin et al., 2015).
The efficiency of CO 2 fixation by the CB cycle is compromised by the oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) which directly competes with CO 2 fixation at the active site, resulting in the formation of 2phosphoglycolate (2PG) and subsequently significant energy costs and CO 2 losses in the photorespiratory pathway, resulting in significant losses in yield (Bowes et al., 1971;Tolbert, 1997;Walker et al., 2015Walker et al., , 2016. Therefore, a major target to improve photosynthesis has been to reduce photorespiration, either through protein engineering to improve Rubisco catalysis or by limiting the flux through this pathway, none of which have as yet yielded positive results due to both the complexity of the Rubisco catalytic and assembly processes (Cai et al., 2014;Carmo-Silva et al., 2015;Lin et al., 2014a;Orr et al., 2016;Sharwood et al., 2016;Whitney et al., 2011). More ambitious approaches to this problem are now being taken, including the introduction of cyanobacterial or algal CO 2 -concentrating mechanisms, novel synthetic metabolic pathways and the introduction of the C4 pathway into C3 crops (Betti et al., 2016;Lin et al., 2014b;McGrath and Long, 2014;Meyer et al., 2016;Montgomery et al., 2016). However, to date the only successful approach to limiting photorespiration which has resulted in an improvement in photosynthesis has been through the introduction of alternative routes to metabolize 2PG and return CO 2 for use in the CB cycle (Dalal et al., 2015;Kebeish et al., 2007;Maier et al., 2012;Nolke et al., 2014;Peterh€ ansel et al., 2013;Xin et al., 2015). Reductions in the flux through the photorespiratory cycle by targeted knock-down of GDC-P in potato and GDC-H in rice have been shown to lead to reductions in photosynthesis and growth rates (Engel et al., 2007;Heineke et al., 2001;Lin et al., 2016). The opposite approach, namely over-expression of the glycine decarboxylase (GDC)-H protein (GDC-H) and glycine decarboxylase (GDC)-L protein (GDC-L) in Arabidopsis thaliana (Arabidopsis), resulted in an improvement of photosynthesis and increased vegetative biomass when compared to wild-type plants (Timm et al., 2012(Timm et al., , 2015(Timm et al., , 2016. Although the underlying mechanism responsible for this effect has not been fully elucidated, these authors proposed that stimulation of the CB cycle is brought about by the increase in GDC activity, resulting in a reduction in the steady-state levels of photorespiratory metabolites that may negatively impact on the function of the CB cycle (e.g. 2PG, glycolate, glyoxylate or glycine (Anderson, 1971;Kelly and Latzko, 1976;Eisenhut et al., 2007;Lu et al., 2014;Timm et al., 2015Timm et al., , 2016).
In the light of the results from Timm et al. (2012Timm et al. ( , 2015, the aim of this study was to explore the possibility that the simultaneous increase in the activity of enzymes of both the CB cycle and the photorespiratory pathway could lead to a cumulative positive impact on photosynthetic carbon assimilation and yield. To test this, we have taken a proof-of-concept approach using the model plant Arabidopsis in which we have over expressed SBPase, FBPA and GDC-H either alone or in combination. We have shown that the simultaneous manipulation of multiple targets can lead to a cumulative impact on biomass yield under both low-and high-light growing conditions. Interestingly, we have also shown that manipulation of the photorespiratory pathway alone resulted in an increase in vegetation biomass but not seed yield. In contrast, manipulation of both the CB cycle and photorespiratory pathway increased both biomass and seed yield.
Further analysis was carried out on T3 plants grown at 130 lmol/m 2 /s in an 8-h/16-h light/dark cycle and total extractable SBPase and FBPA activity determined in extracts from newly fully expanded leaves. The results are represented as a percentage (%) of total activity for SBPase (6.7 lmol/m 2 /s) and FBP aldolase (22 lmol/m 2 /s) determined in wild type (WT). This analysis showed that these plants had increased levels of SBPase (137%-185%) and FBPA (146%-180%) activity ( Figure 1) compared to WT and nontransformed azygous (A) controls (azygouscontrol plants used in this study were recovered from the segregating population and verified by PCR). Interestingly, a small increase in endogenous FBPA activity (125%-136%) was also observed in SBPase over-expressing lines (Figure 1a), but no significant increase in SBPase activity was observed in lines overexpressing FBPA.
Plants over-expressing SBPase (S), FBPA (F) and the GDC-H protein (H) were generated by crossing two SBPase + FBPA (SF) lines (SF6 and SF12) with two Flaveria pringlei GDC-H protein (Kopriva and Bauwe, 1995) over-expressing lines (FpHL17 and FpHL18) originally generated by Timm et al. (2012) under the control of the leaf-specific and light-regulated Solanum tuberosum ST-LS1 promoter (Stockhaus et al., 1989). Four independent lines (SFH4, SFH20, SFH23 and SFH31) over-expressing SBPase, FBPA and GDC-H (SFH) were identified by PCR and SBPase and FBPA enzyme activities. SBPase and FBPA protein levels were found to be similar to those observed in SF lines ( Figure 1b). No significant difference in SBPase or FBPA activities was observed in lines over-expressing GDC-H alone compared to WT/A controls (C). The full set of assays showing the variation between plants for both SBPase and FBPA activities can be seen in Figure S2.
In addition to total extractable enzyme activity, immunoblot analysis of the T3 progenies of S, F, SF, H and SFH lines was carried out using WT/A as controls (C). This analysis identified a number of plants over-expressing SBPase or FBPA and others with increased levels of both SBPase and FBPA (Figures 1a,b and S3). Interestingly, the over-expression of SBPase in Arabidopsis led to an increase in endogenous FBPA protein levels ( Figure 1a) in agreement with the observed increase in enzyme activity. The original H lines and the newly generated SFH plants were shown to accumulate GDC-H when compared to both nontransformed control plants and other transgenic lines (Figure 1a,b). Given the change in FBPA protein levels in the SBPase over-expressing line, we used immunoblot analysis to determine whether there were any changes in other CB cycle enzymes. No detectable changes in the levels of transketolase (TK), phosphoribulokinase (PRK), fructose-1,6-bisphosphatase (FBPase), Rubisco or the ADP glucose pyrophosphorylase (ssAGPase) small protein were observed when compared to levels in C plants ( Figure 2).

Chlorophyll fluorescence imaging reveals increased photosynthetic efficiency in young over-expressing seedlings
To explore the impact of increased levels of SBPase, FBPA and the GDC-H protein on photosynthesis, plants were grown at 130 lmol/m 2 /s in an 8-h/16-h light/dark cycle and the quantum efficiency of PSII photochemistry (F q '/F m ') analysed using chlorophyll a fluorescence imaging (Baker, 2008;Murchie and Lawson, 2013). Plants over-expressing SBPase and FBPA, either independently or in combination (including with GDC-H), had a significantly higher F q '/F m ' at an irradiance of 200 lmol/m 2 /s when compared to C plants (Figure 3a,b). Plants over-expressing GDC-H alone showed a small increase in the average levels of F q '/F m ' compared to C (P = 0.061). When measurements were made at a higher light level (600 lmol/m 2 /s), all lines analysed, with the exception of SFH, showed a significant increase in F q '/F m ' compared to C plants ( Figure S4a). From images taken as part of the chlorophyll fluorescence analysis, leaf area was determined and shown to be significantly larger for all transgenic lines compared with WT and azygous (A) controls ( Figure 3c). Interestingly, SFH plants showed the greatest leaf area in all experiments. No significant differences in leaf area were observed between WT and A. From this point on, C plants used were the combined data from WT and A plants.

Photosynthetic CO 2 assimilation rates are increased in mature plants grown in low light
To explore the impact of changes in the levels of enzymes in both the CB cycle and photorespiratory pathway, CO 2 assimilation rates were determined as a function of light intensity (Figure 4a, b). From these light response curves, the maximum lightsaturated rate of photosynthesis (A sat ) was shown to be significantly higher in all transgenic plants when compared to C plants ( Figure 4c). Small differences in CO 2 assimilation rates (A) were also observed in the S, F, SF and SFH plants even at light intensities as low as 150 lmol/m 2 /s, which is close to that of the growth conditions ( Figure S5).
We also determined A as a function of internal CO 2 concentration (C i ) in the same plants ( Figure 4d,e). In all transgenic plants, except those over-expressing GDC-H alone, A was significantly greater at C i concentrations above 400 lmol/mol than in C plants (Figure 4d,e). Although A in SFH plants was higher than in the control plants at 400 lmol/mol, it was lower than that observed in the S, F or SF plants. The maximum rate of CO 2 assimilation (A max ) was significantly higher in lines S, F, SF SF and SFH lines used in this study compared to non-transformed control (C). Enzyme assays represent data from 12 to 24 independent plants per group compared to 12-16 C plants. The results are represented as a percentage (%) of total activity for SBPase (6.7 lmol/m 2 /s) and FBP aldolase (22 lmol/m 2 /s) determined in wild type (WT). Enzyme activities per plant can be seen in Figure S2. Columns represent mean values, and standard errors are displayed. Lines that are significantly different to C are indicated (*P < 0.05). and SFH compared to C; however, no significant differences were observed between these lines (Figure 4f). Interestingly, the H plants show no increase in A max when compared to C plants. Further analysis of the A/C i curves using the equations published by von Caemmerer and Farquhar (1981) illustrated that the maximum rate of carboxylation by Rubisco (Vc max: Figure S4b) in lines S, SF and SFH was significantly greater than in C, and Vc max in these lines was also significantly greater than in lines expressing GDC-H alone. Maximum electron transport rates (J max : Figure S4c) were also elevated in lines S, F, SF and SFH compared to C and were also shown to be significantly elevated compared to H.
To further assess the effect of the manipulation of the CB cycle and/or the GDC-H protein, the rates of photosynthetic carbon assimilation and electron transport were determined in mature plants as a function of light intensity at 2% [O 2 ] to eliminate photorespiration ( Figure 5a). Electron transport rates through PSII in H and SFH over-expression plants were significantly greater than in the C and SF plants at light levels above 300 lmol/m 2 /s ( Figure 5b). A sat was also significantly higher, 12%-19%, in all lines compared to C although no significant differences were observed between the different transgenic lines (Figure 5c).
Increased SBPase and FBPA activity and over-expression of the glycine decarboxylase-H protein stimulates growth in low light The growth of the different transgenic and C plants was determined using image analysis of total leaf area over a period of 38 days from planting (Figure 6a), which showed all transgenic lines had a significantly greater leaf area than C, as early as 16 days after planting (Figure 6b). Furthermore, plants overexpressing all three transgenes (SFH) were shown to have a significantly larger leaf area when compared to all other transgenic lines including G and SF, indicating a cumulative advantage from combining these transgenes at this stage in development. This growth trend continued through to 15 days postplanting ( Figure S6a). By 20 days after planting ( Figure S6b), plants over-expressing the glycine decarboxylase-H protein (H) were shown to be significantly bigger than S, F and SF at the same time point, and triple over-expressing lines SFH remained significantly bigger than all other lines studied (Figure 6b).
Plants were allowed to continue growing until harvest at 38 days ( Figure S7). At this stage of development, no significant difference in leaf area or dry weight could be observed between S, F, H or SF lines when compared to each other ( Figure 6c). However, all lines attained a significantly larger leaf area and dry weight when compared to C. Notably, at this stage, the triple over-expressing lines SFH were significantly larger with a higher dry weight (+70%) than all other transgenic and C plants. Furthermore, lines SF and SFH both showed a significant increase in leaf number after 38 days ( Figure S8).
Increased SBPase and FBPA activity and expression of the glycine decarboxylase-H protein impacts on the carbohydrate profile of selected lines To determine how the over-expression of these key proteins impacts on downstream processes, leaf tissue was harvested and starch and sugar content were evaluated. No significant difference in starch levels were detected at the end of the day in any of the transgenic lines compared to C (Figure 7). Interestingly, slightly higher starch levels were detected 1 h before sunrise (dark) in transgenic lines F, H and SFH compared to C. All transgenic lines were shown to have consistently higher levels of sucrose, with these levels being significantly higher than C in F and SF lines. SF lines were also shown to have a significantly higher amount of glucose (Figure 7) compared to C, although other lines were consistently elevated but not significantly so.

Impact of light intensity on biomass and seed yield
A subset of plants was allowed to seed in either low or high light, and final vegetative biomass and seed yield determined per plant. In low-light grown plants, the final vegetative biomass was higher in all of the transgenic lines compared to C; however, no significant differences were observed between the different transgenic lines (Figure 8a). Furthermore, seed yield was increased by 35%-53% in transgenic lines S, SF and SFH ( Figure 8b). Interestingly, no increase in seed yield was observed in the H plants.
We next compared the impact of growth of plants in high light to explore further the potential positive impact of these transgenic manipulations on growth. In high-light grown plants, an increase in vegetative biomass from 14% to 51% was observed (Figures 8c  and S9). Notably, the H and SFH plant produced significantly more vegetative biomass than the S, F, SF or C plants. Furthermore, seed yield in high-light-grown plants was increased by 39%-62% in transgenic lines S, F, SF and SFH, when compared to C (Figure 8d). Although the highest increase in seed yield was observed in lines SFH in high light, no increase in seed yield was observed in the H plants in high-light-grown plants. The seed yield for individual plants can be seen in Figure S10.

Discussion
In this study, we have shown that simultaneously increasing the levels of two enzymes of the CB cycle, SBPase and FBPA, and the H protein of the glycine decarboxylase enzyme of the photorespiratory pathway in the same plant, resulted in a substantial and significant increase in both vegetative biomass and seed yield of Arabidopsis grown in controlled environment conditions. An increase in both biomass and yield was also observed in plants overexpressing SBPase or FBPA alone or in combination. However, although overexpression of GDC-H alone resulted in an increase in vegetative biomass, no increase in seed yield was evident in these plants, grown in either low-or high-light conditions. The reasons for this differential effect on seed yield have not yet been elucidated but may be due to changes in carbon status brought about by altered source/sink allocation which is supported by changes to starch and sucrose levels at the end of the night period in some of these lines. Higher levels of sucrose (and fructose, maltose) have also been observed in GDC-L over-expressers (Timm et al., 2015), and the over-expression of GDC-L enhances the metabolic capacity of photorespiration and is believed to alter the carbon flow through the TCA cycle (Timm et al., 2015).
It was shown in earlier studies that over-expression of FBPA or SBPase alone in tobacco results in a stimulation of photosynthesis and biomass, with the greatest effect being seen in plants grown under elevated CO 2 (Lefebvre et al., 2005;Rosenthal et al., 2011;Uematsu et al., 2012). Furthermore, when FBPA was overexpressed in combination with SBPase in tobacco, this led to a cumulative increase in biomass in plants grown in ambient CO 2 under greenhouse conditions (Simkin et al., 2015). Interestingly, in this current study, we have shown that in Arabidopsis that the over-expression of FBPA alone, under current atmospheric CO 2 levels, results in a stimulation of photosynthesis and increase in biomass on a similar level to that observed by over-expression of SBPase alone. However, contrary to the results obtained in tobacco, the co-expression of SBPase and FBPA in Arabidopsis did not lead to a further significant increase in either leaf area or biomass when compared to plants independently expressing SBPase (resulting in higher endogenous FBPA activity) or FBPA. This lack of differential effect of the co-overexpression of SBPase and FBPA in this study can likely be explained by the fact that over-expression of SBPase in Arabidopsis also led to a small but significant increase in endogenous FBPA protein levels and activity (25%-36%). Given that no increase in SBPase was present in the FBPA plants, this would suggest that in Arabidopsis, the stimulation in the SBPase, FBPA and the SF over-expression lines may be due to increased FBPA activity. This is in contrast to tobacco where over-expression of SBPase alone led to an increase in biomass and no increases in endogenous FBPA activity, highlighting the differences between species (Lefebvre et al., 2005;Rosenthal et al., 2011;Simkin et al., 2015).
Detailed analysis of a range of photosynthesis parameters revealed a similar increase in A sat at low [O 2 ] for all of the transgenic lines studied. The most significant increase was observed in SF lines which showed a 44% increase over control plants, with the lowest increase of 19% being observed in the H plants. An evaluation of the electron transport rates at low [O 2 ] in a subset of these plants showed that lines over-expressing GDC-H (both H and SFH) displayed higher photosynthetic electron transport rates compared to C and plants over-expressing SBPase and FBPA (SF). These results are in keeping with the previous study by Timm et al. (2012). All of the transgenic lines analysed here showed an increase in photosynthesis under high light and ambient CO 2 conditions. However, under high light and saturated levels of CO 2 the rate of assimilation in the H plants was similar to C, and this is in contrast to all other transgenic lines. This observation is in keeping with the proposal that overexpression of the H protein stimulates the flow of carbon through the photorespiratory pathway, thereby reducing steady-state levels of inhibitory photorespiratory metabolites, which in turn stimulates flux through the CB cycle. Whilst this hypothesis is supported by metabolite data and the observation that growth of GDC-H plants is not stimulated when these plants are grown in elevated CO 2 conditions (Timm et al., 2012), the exact mechanism of such feedback from photorespiration to the CB cycle is not yet known. The effect of these manipulations on photosynthesis was also determined at the growth light intensity where small differences in A are observed even at light levels as low as 150 lmol/m 2 /s. This together with the increased leaf area observed at early stages in development provides evidence that the small differences in photosynthesis lead to an increase in leaf area. The cumulative impact of this over time results in increased biomass and yield.

Conclusion
In this proof-of-concept study in Arabidopsis, we have demonstrated that the simultaneous over-expression of two CB cycle enzymes leads to an increase in photosynthesis and an increase in overall biomass and seed yield. We also show that when the transgenic SF lines were crossed with GDC-H overexpressing plants (Timm et al., 2012), the combined effects of these three transgenes (SFH) resulted in a cumulative impact on biomass (+71%) which was significantly higher than H (+50%) and SF (+41%) under low light. Importantly, the work here also allowed a parallel comparative analysis between the different manipulations under different environmental conditions.
Although it is still necessary to address the importance of these manipulations in crop species and also under field conditions, this study provides additional evidence that multigene manipulation of photosynthesis and photorespiration can form an important tool to improve crop yield. These results also provide new information indicating that it will be necessary to tailor the targets for manipulation for different crops and for either biomass or seed yield.
Constructs were generated using Gateway cloning technology and vector pGWPTS1. All transgenes were under the control of the rbcS2B (1150 bp; At5 g38420) promoter. Full details of PTS1-SB, PTS1-FB and PTS1-SBFB construct assembly can be seen in the supplementary data. Construct maps are shown in Figure S1b-d.

Generation of transgenic plants
The recombinant plasmids PTS1-SB, PTS1-FB and PTS1-SBFB were introduced into wild-type Arabidopsis by floral dipping (Clough and Bent, 1998) using Agrobacterium tumefaciens GV3101. Positive transformants were regenerated on MS medium containing kanamycin (50 mg/L) and hygromycin (20 mg/L). Kanamycin-/hygromycin-resistant primary transformants (T1 generation) with established root systems were transferred to soil and allowed to self-fertilize. Plants over-expressing SBPase, FBPA and the GDC-H protein were generated by floral inoculation of two SBPase + FBPA lines (SF6 and SF12) with the pollen from two GDC-H protein over-expressing lines (FpH17 and FpH18) provided by Timm et al. (2012). Lines FpH17 and 18 were originally generated by floral dipping and over-expressing the Flaveria pringlei GDC-H protein (Kopriva and Bauwe, 1995) under the control of the leaf-specific and light-regulated Solanum tuberosum ST-LS1 promoter (Stockhaus et al., 1989). Following initial characterization of generated lines, three lines for SBPase (S3, S8, S12), FBPA (F6, F9, F11) and SF (SF6, SF7, SF12) were selected for further study from all lines generated.

Plant growth conditions
Wild-type T2 Arabidopsis plants resulting from self-fertilization of transgenic plants were germinated in sterile agar medium containing Murashige and Skoog salts (plus kanamycin 50 mg/L for the transformants) and grown to seed in soil (Levington F2, Fisons, Ipswich, UK). Lines of interest were identified by immunoblot and qPCR. For experimental study, T3 progeny seeds from selected lines were germinated on soil in controlled environment chambers at an irradiance of 130 lmol photons/ m 2 /s, 22°C, relative humidity of 60%, in an 8-h/16-h squarewave photoperiod. Plants were sown randomly, and trays rotated daily. Four leaf discs (0.6 cm diameter) from two individual leaves, for the analysis of SBPase and FBPA activities, were taken and immediately plunged into liquid N 2 , and stored at À80°C. Leaf areas were calculated using standard photography and ImageJ software (imagej.nih.gov/ij). Wild-type plants and null segregants (azygous) used in this study were initially evaluated independently. However, once it was determined that no significant difference were observed between these two groups (see figures and supplementary figures), wild-type plants and null segregants were combined (null segregants from the transgenic lines verified by PCR for nonintegration of the transgene) and used as a combined 'control' group (C).

Determination of SBPase activity by phosphate release
SBPase activity was determined by phosphate release. Immediately after photosynthesis measurement, leaf discs were isolated from the same leaves and frozen in liquid nitrogen. For analysis, leaf discs were ground to a fine powder in liquid nitrogen in extraction buffer (50 mM HEPES, pH8.2; 5 mM MgCl 2 ; 1 mM EDTA; 1 mM EGTA; 10% glycerol; 0.1% Triton X-100; 2 mM benzamidine; 2 mM aminocaproic acid; 0.5 mM phenylmethylsulphonylfluoride; 10 mM dithiothreitol), and the resulting solution was centrifuged 1 min at 14 000 g, 4°C. The resulting supernatant was desalted through an NAP-10 column (Amersham) and eluted, aliquoted and stored in liquid nitrogen. For the assay, the reaction was started by adding 20 lL of extract to 80 lL of assay buffer (50 mM Tris, pH 8.2; 15 mM MgCl 2 ; 1.5 mM EDTA; 10 mM dithiothreitol; 2 mM SBP) and incubated at 25°C for 30 min as described previously (Simkin et al., 2015). The reaction was stopped by the addition of 50 lL of 1 M perchloric acid and centrifuged for 10 min at 14 000 g, 4°C. Samples (30 lL) and standards (30 lL, 0.125-4 nmol PO 3À 4 ) in triplicate were incubated 30 min at room temperature following the addition of 300 lL of Biomol Green (Affiniti Research Products, Exeter, UK), and the A 620 was measured using a microplate reader (VERSAmax, Molecular Devices, Sunnyvale, CA).

Determination of FBPA activity
Desalted protein extracts, as described above, were evaluated for FBPA activity as described previously (Haake et al., 1998).

Chlorophyll fluorescence imaging
Measurements were performed on 2-week-old Arabidopsis seedlings that had been grown in a controlled environment chamber providing 130 lmol/mol 2 /s PPFD and ambient CO 2 . Chlorophyll fluorescence parameters were obtained using a chlorophyll fluorescence (CF) imaging system (Technologica, Colchester, UK; Barbagallo et al., 2003;Baker and Rosenqvist, 2004). The operating efficiency of photosystem two (PSII) photochemistry, F q '/F m ', was calculated from the measurements of steady-state fluorescence in the light (F') and maximum fluorescence in the light (F m ') since F q '/F m ' = (F m ' À F')/F m '. Images of F' were taken when fluorescence was stable at 130 lmol/m 2 /s PPFD, whilst images of maximum fluorescence were obtained after a saturating 600 ms pulse of 6200 lmol/m 2 / s PPFD (Baker et al., 2001;Oxborough and Baker, 1997). Parallel measurements of plants grown in high light (390 lmol/mol 2 /s PPFD and ambient CO 2 ) were also performed on 2-week-old Arabidopsis (Supporting Information).

Gas exchange measurements
The response of net photosynthesis (A) to intracellular CO 2 (C i ) was measured using a portable gas exchange system (CIRAS-1, PP Systems Ltd, Ayrshire, UK). Leaves were illuminated with an integral red-blue LED light source (PP systems Ltd) attached to the gas exchange system, and light levels were maintained at saturating photosynthetic photon flux density (PPFD) of 1000 lmol/m 2 /s for the duration of the A/C i response curve. Measurements of A were made at ambient CO 2 concentration (C a ) at 400 lmol/mol, before C a was decreased to 300, 200, 150, 100 and 50 lmol/mol before returning to the initial value and increased to 500, 600, 700, 800, 900, 1000, 1100 and 1200 lmol/mol. Leaf temperature and vapour pressure deficit (VPD) were maintained at 22°C and 1 AE 0.2 kPa, respectively. The maximum rates of Rubisco-(Vc max ) and the maximum rate of electron transport for RuBP regeneration (J max ) were determined and standardized to a leaf temperature of 25°C based on equations from Bernacchi et al. (2001) and McMurtrie and Wang (1993), respectively.

Photosynthetic light response curves
A/Q response curves were measured using a CIRAS-1 portable gas exchange system (PP Systems (CIRAS-1, PP Systems Ltd). Cuvette conditions were maintained at a leaf temperature of 22°C, relative humidity of 50%-60% and ambient growth CO 2 concentration (400 mmol/mol for plants grown in ambient conditions). Leaves were initially stabilized at saturating irradiance 1000 lmol/m 2 /s, after which A and g s were measured at the following PPFD levels: 0,50,100,150,200,250,300,350,400,500,600,800 and 1000 lmol/m 2 /s. Measurements were recorded after A reached a new steady state (1-2 min) and before stomatal conductance (g s ) changed to the new light levels. A/Q analyses were performed at 21% and 2% O 2 .

Determination of sucrose and starch
Carbohydrates and starch were extracted from 20 mg leaf tissue, and samples were collected at two time points, 1 h before dawn (15 h into the dark period) and 1 h before sunset (7 h into the light period). Four leaf discs collected from two different leaves were ground in liquid nitrogen, and 20 mg/FW of tissue was incubated in 80% (v/v) ethanol for 20 min at 80°C and then repeated three times with ethanol 80% (v/v) at 80°C. The resulting solid pellet and pooled ethanol samples were freezedried. Suc was measured from the extracts in ethanol using an enzyme-based protocol (Stitt et al., 1989), and the starch contents were estimated from the ethanol-insoluble pellet according to Stitt et al. (1978), with the exception that the samples were boiled for 1 h and not autoclaved.

Statistical analysis
All statistical analyses were performed by comparing ANOVA, using Sys-stat, University of Essex, UK. The differences between means were tested using the post hoc Tukey test (SPSS, Chicago, IL).

Supporting information
Additional Supporting Information may be found online in the supporting information tab for this article: Figure S1 Schematic representation of the (a) vector pGWPTS1, (b) A. thaliana SBPase (PTS1SB) over-expression construct, and the (c) A. thaliana FBPA (PTS1-FB) over-expression construct, (d) shows the structure of a duel construct for the expression of both SBPase and FBPA (PTS1-SBFB). Figure S2 (a) Complete data set for SBPase enzyme assays in plants analysed. (b) Complete data set for FBP aldolase enzyme assays in plants analysed. Figure S3 Molecular and biochemical analysis of the transgenic plants overexpressing SBPase (S), FBPA (F) or both (SF). Figure S4 (a) The operating efficiency of PSII photochemistry of C and transgenic plants at 600 lmol/m 2 /s light. Capacity determined using chlorophyll fluorescence imaging. (b) the maximum carboxylation activity of Rubisco and (c) J max were derived from A/ C i response curves (Figure 4). Figure S5 Photosynthesis carbon fixation rates determined as a function of light intensity in developing leaves. Figure S6. Complete data set for all transgenic lines evaluated. (a) leaf area at 15 days, (b) Leaf area at 20 days (c) Leaf area at 25 days. Figure S7 Growth analysis of the transgenic and control plants grown in low light. Figure S8 Leaf number in control and transgenic lines. Figure S9 Complete data set for leaf area of all transgenic lines evaluated at high light (390 lmol/m 2 /s). Figure S10 Complete data set for seed yield (g) from all transgenic lines evaluated in (a) low light (130 lmol/m 2 /s) and (b) high light (390 lmol/m 2 /s).