Increased sedoheptulose-1,7-bisphosphatase activity in transgenic tobacco plants stimulates photosynthesis and growth from an early stage in development

Abstract

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.

Figure 1.

Production and selection of SBPase sense transgenic tobacco plants. A, The sense construct contained a full-length Arabidopsis SBPase cDNA (1,350 bp) driven by the CaMV 35S promoter and the nopaline synthase termination sequence. B, Western-blot analysis of primary transformants (T₀); 25 μg of leaf protein samples from the newest fully mature leaves were separated by SDS-PAGE and polyclonal antibodies used to detect SBPase protein. The plants marked with an asterisk were selected for further analysis. C, Western-blot analysis of wild-type and individual T₁ progeny of SBPase sense plants. The same blots were stripped and then reprobed using polyclonal antibodies raised against PRKase and chloroplastic FBPase. Each lane represents a sample taken from one individual plant, the first number denotes the line and the second number indicates an individual segregant from that line. D, Increase in total SBPase activity in individual transformants of first (T₁) and fourth (T₄) generation transgenic plants as compared to wild type (100%). E, Leaf area of wild-type and transgenic seedlings after 3 weeks of growth in a controlled environment chamber. The results are the mean ± se for wild-type (n = 5) and SBPase sense (n > 4) plants (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Figure 2.

Ambient photosynthesis and diurnal carbohydrate accumulation in wild-type and T₁ SBPase sense tobacco plants. Plants were grown in a controlled environment chamber with a light level of 600 μmol m⁻² s⁻¹ and ambient CO₂ concentration until 25 leaves were produced. A, Total SBPase activity was determined in newly fully expanded leaves (9 and 10) on each plant and for subsequent analysis plants were divided into 2 groups with increases in SBPase activity of 10% to 35% and 45% to 65%. Values represent activities ± se (n = 4 separate plants). Photosynthesis was determined under growth conditions in 5 different leaves (numbers 9, 10, 11, 12, and 13) of wild-type (black square) and SBPase sense plants (white circle, activity increased 10%–35%; white triangle, activity increased 45%–65%). Data points represent the mean ± se of four different plants. B, Suc and starch levels were determined in 2 newly fully expanded leaves (9 and 10, black symbols) and 2 young expanding leaves (12 and 13, white symbols) 10 h after the onset of the light period (end of day) and at the end of the night. Symbols are as used in A. The data points represent the mean ± se (n = 4 separate plants).

Figure 3.

Growth analysis of controlled-environment-grown wild-type and SBPase sense plants. Plants grown in the same conditions as for Figure 2 were harvested after 7 weeks of growth. Results are means ± se (n = 4); plants with increased SBPase activity were grouped as for Figure 2. Asterisks indicate that mean values are significantly different between wild-type and SBPase sense plants (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Figure 4.

Photosynthetic capacity of greenhouse-grown wild-type and SBPase sense plants. Plants were grown in a controlled-environment greenhouse with light levels of 600 to 1,600 μmol m⁻² s⁻¹ and measurements made after 7 weeks of growth. A, Photosynthetic carbon fixation rates were determined in the newest fully expanded leaf, as a function of increasing CO₂ concentration at saturating-light levels (1,200 μmol m⁻² s⁻¹; A/Ci response curve). Wild-type plants (▪) 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 (▵), line 60 (○), and wild type (□).

Figure 5.

Rubisco activity and Jmax derived from the A/Ci response curves shown in Figure 4 using the equations of von Caemmerer and Farquhar (1981). A, Maximum velocity of carboxylation (Vc, max). B, Maximum in vivo rates of Jmax. Values are the mean ± se in wild-type plants (n = 7) and SBPase sense plants (increase in activity 25%–50%, n = 6; increase in activity 50%–100%, n = 7). Asterisks indicate the mean values that are significantly different between wild-type SBPase sense plants (**, P < 0.01).

Figure 6.

Diurnal progression of leaf photosynthesis in greenhouse-grown SBPase sense plants. Plants were grown in a controlled-environment greenhouse with light levels of 600 to 1,600 μmol m⁻² s⁻¹ photosynthetic carbon fixation rates determined under ambient conditions in 2 different leaves. A, Young expanding leaf. B, Newly fully expanded leaf of wild-type and SBPase sense plants. For the wild type (▪), each symbol represents the mean ± se of eight plants. For the transgenic plants (○), each symbol represents the mean ± se of 16 plants. The SBPase activity in the young leaves of the wild-type plants was 12.57 ± 0.67 and 16.6 ± 0.64 for the transgenic plants and in mature leaves of wild-type plants was 13.76 ± 1.18 and 18 ± 0.42 for the transgenic plants.

Figure 7.

Growth analysis of greenhouse-grown wild-type and SBPase sense plants. Plants grown in a controlled-environment greenhouse (and analyzed in Figs. 4 and 5) were harvested when the first flower buds appeared. Values are the mean ± se in wild-type plants (n = 8) and SBPase sense plants (increase in activity 25%–50%, n = 6; increase in activity 60%–100%, n = 7). Asterisks indicate that mean values are significantly different between wild-type and SBPase sense plants (*, P < 0.05; **, P < 0.01).

Figure 8.

Leaf area profiles for wild-type and SBPase sense plants grown in a controlled-environment greenhouse. A, Average leaf area and (B) average leaf dry weight were determined for I, leaves 3 to 6; II, leaves 7 to 10; III, leaves 11 to 16; and IV, leaves 17 to 32 in the same plants used for the analyses in Figures 4 to 6. Values are the mean ± se in wild-type plants (black bar, n = 7) and SBPase sense plants (hatched bar, increase in activity 25%–50%, n = 6; white bar, increase in activity 60%–100%, n = 7). Asterisks indicate that mean values are significantly different between wild-type and SBPase sense plants (*, P < 0.05; **, P < 0.01; ***, P < 0.001).