Changes in SBPase activity influence photosynthetic capacity, growth, and tolerance to chilling stress in transgenic tomato plants

Abstract

Sedoheptulose-1, 7-bisphosphatase (SBPase) is an important enzyme involved in photosynthetic carbon fixation in the Calvin cycle. Here, we report the impact of changes in SBPase activity on photosynthesis, growth and development, and chilling tolerance in SBPase antisense and sense transgenic tomato (Solanum lycopersicum) plants. In transgenic plants with increased SBPase activity, photosynthetic rates were increased and in parallel an increase in sucrose and starch accumulation was evident. Total biomass and leaf area were increased in SBPase sense plants, while they were reduced in SBPase antisense plants compared with equivalent wild-type tomato plants. Under chilling stress, when compared with plants with decreased SBPase activity, tomato plants with increased SBPase activity were found to be more chilling tolerant as indicated by reduced electrolyte leakage, increased photosynthetic capacity, and elevated RuBP regeneration rate and quantum efficiency of photosystem II. Collectively, our data suggest that higher level of SBPase activity gives an advantage to photosynthesis, growth and chilling tolerance in tomato plants. This work also provides a case study that an individual enzyme in the Calvin cycle may serve as a useful target for genetic engineering to improve production and stress tolerance in crops.

Figure 1. A schematic diagram illustrating the functional domains of SlSBPase and multiple alignments of SlSBPase-related proteins.

(a) SDS-PAGE of SlSBPase protein. Arrows indicate the HIS-SlSBPase protein. (b) Structure of SlSBPase. (c) Alignment of deduced amino acid sequences of SBPase from five plant species. Identical amino acid residues are shown on grey backgrounds. The amino acid residues showing the regions involved in redox-regulated activation of SBPase are boxed. F1–F6 amino acid residues are six SBPase fingerprints. The accession numbers of SBPase in GenBank are as follows: Solanum lycopersicum, FJ959073; Arabidopsis thaliana, AEE79443; Cucumis sativus, ACQ82818; Oryza sativa, AAO22558; Triticum aestivum, CAA46507.

Figure 2. SlSBPase mRNA abundance, SBPase activity and protein levels in different tissues and during leaf development in tomato plants.

All measurements were done on 20-leaf tomato plants. (a,d) SlSBPase mRNA abundance was measured by quantitative real-time PCR using total RNA separately isolated from different organs (fully expanded leaves, one-week old fruits, stems, roots) and leaves at different developmental stages including post-maturation leaves (leaf no. 5 from base), mature fully expanded leaves (leaf no. 9, 13), new fully expanded leaves (leaf no. 17) and young expanding leaves (leaf no. 20) and in tomato plants. (b,e) Protein levels. 25 μg protein samples from different tissues were separated by SDS-PAGE. SBPase protein was stained by coomassie blue and was subjected to western blot analysis with an anti-SBPase polyclonal antibody. (c,f) SBPase activity. The same tissues for SlSBPase mRNA analysis were sampled for SBPase activity assay. (g) CO₂ assimilation rate in leaves at the different developmental stages. (h) CO₂ assimilation rate as a function of SBPase activity. The results are the means ± SDs (n = 4).

Figure 3. Diurnal changes in SlSBPase mRNA abundance, SBPase activity, CO₂ assimilation rate, PFD and air temperature measured in greenhouse conditions on three consecutive sunny days.

All measurements were done on 20-leaf tomato plants. (a) SlSBPase mRNA abundance was analyzed by quantitative real-time PCR using total RNA isolated from the fully expanded leaves (leaf no. 13–15 from base) at 2-h intervals. (b) Diurnal variation of PFD in greenhouse. (c) Diurnal variation of air temperature in greenhouse. (d) SBPase activity was assayed using the same tissues for SlSBPase mRNA analysis. (e) Measurements of CO₂ assimilation rate were made on the fully expanded leaves (leaf no. 13–15) at 2-h intervals. (f) CO₂ assimilation rates were plotted against levels of SBPase activity, showing the correlation between them. The results are the means ± SDs (n = 4).

Figure 4. SlSBPase mRNA abundance, SBPae protein level, SBPase activity and CO₂ assimilation rate in different transgenic lines.

All measurements were done on 12-leaf wild-type and T1 progeny of transgenic tomato plants. (a,b) Protein levels. 25 μg protein samples from the fully expanded leaves (leaf no. 8, 9 from base) in wild-type and transgenic tomato plants were separated by SDS-PAGE. SBPase protein was stained by coomassie blue and was subjected to western blot analysis with an anti-SBPase polyclonal antibody. (c) SlSBPase mRNA abundance was analyzed by quantitative real-time PCR using total RNA isolated from the fully expanded leaves (leaf no. 8, 9). (d) SBPase activity was assayed using the same tissues for SlSBPase mRNA analysis. (e) CO₂ assimilation rate measurements were made on the fully expanded leaves (leaf no. 8, 9) in greenhouse conditions. (f) Effect of changes in SBPase activity on CO₂ assimilation rate under greenhouse conditions. The results are the means ± SDs (n = 5). Asterisks indicate significant differences between WT and transgenic plants (* for P < 0.05; ** for P < 0.01).

Figure 5. Carbohydrate content in fully expanded leaves of SBPase sense and antisense tomato plants.

All measurements were done on 12-leaf wild-type and T1 progeny of transgenic tomato plants. (a,b) Contents of sucrose and starch were determined in fully expanded leaves (leaf no. 8, 9 from base) harvested at the end of day. (c) Sucrose level as a function of SBPase activity. (d) Starch content as a function of SBPase activity. (e) Sucrose content as a function of CO₂ assimilation rate. (f) Starch content as a function of CO₂ assimilation rate. (g) Sucrose-to-starch ratios in SBPase sense and antisense tomato plants. The results are the means ± SDs (n = 6). Asterisks indicate significant differences between WT and transgenic plants (* for P < 0.05; ** for P < 0.01).

Figure 6. Biomass of SBPase sense and antisense tomato plants.

All measurements were done on 12-leaf wild-type and T1 progeny of transgenic tomato plants. (a) Total plant biomass. (b) Shoot biomass. (c) Root biomass. (d) Root/shoot ratio. (e) Total biomass as a function of SBPase activity. (f) Shoot biomass as a function of SBPase activity. (g) Root biomass as a function of SBPase activity. (h) Root to shoot ratio as a function of SBPase activity. The results are the means ± SDs (n = 6). Asterisks indicate significant differences between WT and transgenic plants (* for P < 0.05; ** for P < 0.01).

Figure 7. Effects of changes in SBPase activity on chilling tolerance in transgenic tomato plants.

All measurements were done on 12-leaf wild-type and T1 progeny of transgenic tomato plants subjected to chilling stress at 5 °C for 12 h. (a) Changes in SBPase activity in response to chilling stress in transgenic plants. (b) Changes in electrolyte leakage in response to chilling stress in transgenic plants. (c) Changes in CO₂ assimilation rates in response to chilling stress in transgenic plants. (d) Changes in RuBP regeneration rate in response to chilling stress in transgenic plants. (e,f) Changes in the maximum potential quantum efficiency (Fv/Fm) and the actual quantum efficiency (ΦPSII) of photosystem II in response to chilling stress in transgenic plants. The results are the means ± SDs (n > 4).