Transformation and plant growth conditions
The Dwarf gene was PCR amplified from the cDNA of tomato (Solanum lycopersicum L. cv Condine Red) using forward primer 5’-GGGGTACCCCATGGCCTTCTTC-3’ and reverse primer 5’-GCTCTAGAGCTTAGTGAGCTGAAAC-3’ based on the published sequence (Sol genomic network accession Solyc02g089160.2). For transformation, we used the binary vector pMV2, which carries the spectinomycin resistance gene for bacterial selection and the neomycin phosphotransferase II gene for selection of transformed plants [21]. The binary vector was constructed by inserting the Dwarf cDNA between the KpnI and XbaI sites in the sense orientation driven by the cauliflower mosaic virus 35S promoter. Agrobacterium tumefaciens strain C58 was used to mediate introducing the vector into callus of tomato cultivar Condine Red. After screening for regenerated shoots on selection medium containing kanamycin, the transgenic plants were further verified by PCR using genomic DNA as template and 35S forward and gene-specific reverse primers. Two lines (DWF:OX2, and DWF:OX3) were chosen for the experiments.
To compare the photosynthetic capacity, wild type Condine Red (CR), the BR biosynthesis mutant d
im (in the background of CR), and DWF:OX2 and DWF:OX3 were used. The d
im mutant was obtained from the Tomato Genetics Resource Center (University of California, Davis, CA, USA, accession LA0571). These seeds were germinated and grown in a mixture of peat and vermiculite (1:1, v/v) under a 16 h light (200 μmol m−2 s−1; at 25 °C) and 8 h dark (at 20 °C) cycle. Two-month-old plants were used for the experiments. For assay of Calvin cycle enzyme activity, leaf discs were harvested from different genotypes, frozen immediately in liquid nitrogen, and stored at -80 °C prior to analysis. For activity of antioxidant enzymes, the samples were collected based on fresh weight, whereas for gene expression, the whole leaflets were collected.
Leaf gas exchange and chlorophyll fluorescence analysis
Gas exchange analysis was conducted on the 6th leaf from tomato plants using an open gas exchange system (LI-6400; LI-COR, Lincoln, NE, USA). The measurements were taken from 8 to 11 am in the morning. The assimilation versus intercellular CO2 concentration (A/Ci) curve was determined according to von Caemmerer and Farquhar [22]. The maximum ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) carboxylation rates (V
c,max) and maximum ribulose-1,5-bisphosphate (RuBP) regeneration rates (J
max) were estimated from the A/Ci curves using the method described by Ethier and Livingston [23].
Chlorophyll fluorescence parameters were determined by analyzing the slow kinetics curve using Dual-PAM-100 system (Walz, Germany). The analysis started after the plants were dark-adapted for 30 min. The initial fluorescence (Fo) was obtained after switching on the measuring beam, and then the maximum fluorescence (Fm) was obtained after applying a 0.8 s saturating pulse (>10000 μmol m−2 s−1). After the fluorescence signal decayed for 20s, the actinic light (280 μmol m−2 s−1) was switched on for 300 s, during which the saturating pulse was applied every 20s. The steady state fluorescence (Fs) and maximum fluorescence under illumination (Fm’) were recorded before and during the saturating pulse, respectively. The minimal fluorescence under illumination (Fo’) was calculated according to Baker [24]. Maximum quantum yield of PSII (Fv/Fm), actual quantum yield of PSII (ФPSII), efficiency of antenna excitation transfer (Fv’/Fm’), and photochemical quenching coefficient (qP) were calculated as (Fm-Fo)/Fm, (Fm’-Fs)/Fm’, (Fm’-Fo’)/Fm’, and (Fm’-Fs)/(Fm’-Fo’), respectively.
Measurement of endogenous BRs, total chlorophyll and soluble protein contents
Endogenous BRs were determined using solid-phase extraction with double-layered cartridge followed by high-performance liquid chromatography–tandem mass spectrometry [25]. For the determination of BRs and their metabolites, (2H3) castasterone (D-Cs) and (2H3) brassinolide (D-BL) were spiked into the extraction solution with 1 g of leaf sample. Total chlorophyll content was determined by the method of Arnon [26]. Total soluble protein content was measured using Bradford reagent (Bio-Rad, Hercules, California).
Western-blot analysis of rbcL, rbcS and RCA
Proteins were extracted from leaf samples as described previously [20]. For Western-blot analysis, proteins were separated by SDS-PAGE using 12.5 % (w/v) acrylamide gels and electrophoretically transferred to nitrocellulose membranes (Millipore, Saint-Quentin, France). The proteins were detected with commercial antibodies raised against rbcL, rbcS and RCA (Agrisera, Vannes, Sweden).
Non-reducing SDS-PAGE and western blot analysis of 2-cystein peroxiredoxin
For detection of 2-cystein peroxiredoxin (2-CP), the samples were extracted with buffer containing 100 mM HEPES, pH 7.5, 5 mM EDTA, 5 mM EGTA, 10 mM Na3VO4, 10 mM NaF, 50 mM β-glycerophosphate, 1 mM phenylmethylsulphonyl fluoride, 10 % glycerol, 7.5 % polyvinylpolypyrrolidone (PVP), 10 mM dithiothreitol (DTT) and 10 mM N-ethylmaleimide (NEM, thiol-blocking reagent). The grinded samples were centrifuged at 13 000 g for 20 min. For analysis of the redox status of 2-CP, DTT was omitted in the extraction buffer as described previously [27]. Protein samples (15 μg) supplemented with 5× loading buffer [225 mM Tris–HCl, pH 6.8, 5 % (w/v) SDS, 50 % glycerol, 0.05 % bromophenol blue] were separated via 12 % SDS-PAGE, and 2-CP was detected through western blot as described previously [28] with a polyclonal antibody against 2-CP (Beijing Protein Innovation, Beijing, China).
Determination of RuBisCO, RuBisCO activase (RCA), and FBPase activity
RuBisCO activity was measured spectrophotometrically by coupling 3-phosphoglyceric acid formation with NADH oxidation according to Ward and Keys [29] with some modifications. Leaf discs were homogenized with extraction buffer (50 mM HEPES, pH 8.0, 1 mM EDTA, 10 mM MgCl2, 2 % insoluble PVPP and 10 mM β-mercaptoethanol). The homogenate was centrifuged at 4 °C for 15 min at 15000 g. Total activity was assayed after the crude extract had been activated in a 0.1 ml activation mixture containing 50 mM HEPES (pH 8.0), 26.6 mM MgCl2 and 16.6 mM NaHCO3 for 15 min at 28 °C. The measurement of initial RuBisCO activity were carried out in 0.1 ml of reaction medium containing 50 mM HEPES-NaOH (pH 8.0), 10 mM NaHCO3, 20 mM MgCl2, 1 U creatine phosphokinase, 1 U 3-phosphoglyceric phosphokinase, 1 U glyceraldehydes 3-phosphate dehydrogenase, 0.5 mM ATP, 0.015 mM NADH, 0.5 mM phosphocreatine and 0.06 mM RuBP. The change in absorbance at 340 nm was monitored for 90 s. RCA activity was determined using a RuBisCO Activase Assay Kit (Genmed Scientifics, Washington, DC, USA). Briefly, 0.2 g leaf sample was rapidly ground in a 15-mL tube with liquid N2. Then, 500 μL lysis buffer was added. The mixture was vortexed and homogenized. The homogenate was transferred to a 1.5-mL eppendorf tube and centrifuged at 4 °C for 5 min at 300 g. Aliquot of supernatant was transferred to a new eppendorf tube and centrifuged at 4 °C for 10 min at 1000 g. The supernatant was removed and the pellet was suspended with 200 μL lysis buffer. The suspension was used for RCA activity assay. RCA activity was measured based on RuBisCO activity after incubating inactivated RuBisCO with enzyme extract in the presence or absence of ATP. RuBisCO activity was measured by the coupled spectrophotometric assay.
FBPase activity was determined by monitoring the absorption at 340 nm, using an extinction coefficient of 6.2 mM−1 cm−1 [30]. Total activity was assayed after the crude extract had been activated in a 0.1 ml activation mixture containing 100 mM DTT, 2 mM fructose-1,6-bisphosphate (FBP), 10 mM MgCl2, and 0.1 M Tris–HCl (pH 8.0). The initial activity was assayed immediately after homogenization. The assay mixture consisted of 0.1 M HEPES (pH 8.0), 0.5 mM Na2EDTA, 10 mM MgCl2, 0.3 mM NADP+, 0.6 mM FBP, 0.6U of glucose-6-phosphate dehydrogenase from baker’s yeast (Sigma, Santa Clara, CA, USA), 1.2U of glucose phosphate isomerase from baker’s yeast (Sigma, Santa Clara, CA, USA), and 100 μl of enzyme extract in a final volume of 1 ml.
Measurements of glutathione and ascorbate contents
For the measurement of reduced glutathione (GSH) and oxidized glutathione (GSSG), plant leaf tissue (0.3 g) was homogenized in 2 ml of 6 % metaphosphoric acid containing 2 mM EDTA and centrifuged at 4 °C for 10 min at 12 000 g. After neutralization with 0.5 M phosphate buffer (pH 7.5), 0.1 ml of the supernatant was added to a reaction mixture containing 0.2 mM NADPH, 100 mM phosphate buffer (pH 7.5), 5 mM EDTA, and 0.6 mM 5,5’-dithio-bis (2-nitrobenzoic acid). The reaction was initiated by adding 3U of glutathione reductase (GR) from yeast (Sigma, Santa Clara, CA, USA) and was monitored by measuring the changes in absorbance at 412 nm for 1 min. For the GSSG assay, GSH was masked by the addition of 40 μl of 2-vinylpyridine to the neutralized supernatant, whereas 40 μl of water was added for the total glutathione assay. The GSH concentration was obtained by subtracting the GSSG concentration from the total concentration [31].
Determination of the activity of enzymes involved in the AsA-GSH cycle
To determine the activities of enzymes involved in the AsA-GSH cycle, leaf tissue (0.3 g) was ground in 3 ml of ice-cold buffer containing 25 mM HEPES (pH 7.8), 0.2 mM EDTA, 2 mM ascorbic acid, and 2 % PVP. The homogenates were centrifuged at 4 °C for 20 min at 12 000 g, and the resulting supernatants were used to determine the enzymatic activity. The dehydroascorbate reductase (DHAR) activities were evaluated by measuring the increase in absorbance at 265 nm, as described by Nakano and Asada [32]. GR activity was measured according to the decrease of absorbance at 340 nm based on the method described by Halliwell and Foyer [33]. All spectrophotometric analyses were conducted in a SHIMADZU UV-2410PC spectrophotometer (Shimadzu Corporation, Kyodo, Japan).
Total RNA extraction and gene expression analysis
Total RNA was isolated from tomato leaves using the TRIZOL reagent (Sangon, Shanghai, China) according to the instructions supplied by the manufacturer. After extraction, the total RNA was dissolved in RNase-free water. The cDNA template for qRT-PCR was synthesized from 2 μg of total RNA using the ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan).
For qRT-PCR analysis, PCR products were amplified using iQ SYBR Green SuperMix (Bio-Rad, Hercules, CA, USA) in 25 μl assays. PCR was performed using the iCycleriQ 96-well real-time PCR Detection System (Bio-Rad, Hercules, CA, USA), and the cycling conditions consisted of denaturation at 95 °C for 3 min, followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at 58 °C for 30 s, and extension at 72 °C for 30 s. The tomato actin gene was used as an internal control. Primers used for the qRT-PCR analysis were listed in the Additional file 1: Table S1. Relative gene expression was calculated as described by Livak and Schmittgen [34].
Statistical analysis
The experimental design was a completely randomized block design with four replicates. Each replicate contained ten plants. Statistical analysis were performed by SPSS statistical software (ver.19.0, SPSS Inc., Chicago, IL, USA), using one-way analysis of variance (ANOVA). To evaluate the treatment effects, a Tukey’s test (P < 0.05) was performed.