Plant material and climate conditions
The experiment was performed from June to October, 2016 in a Chinese solar greenhouse (CSG) and an artificial climate chamber (ACC, Zhejiang Qiushi Environment Co., Zhejiang, China) at the Horticultural Research Center, Shandong Agricultural University, P. R. China. After immersing sweet pepper (Capsicum annuum L. cv. Hongqijian) seeds (Jinan Weili Seeds Co., Ltd., Shandong, China) in water for 15 minutes at the temperature of 55 oC and soaking it in cold water (4 oC) for 24 h. The seeds were sown into 50-cell plug trays (54.0 × 30.0 × 4.4 cm) filled with a mixture of peat (Floragard Seed 2, Floragard Co., Oldenburg, Germany) and vermiculite (2:1, v/v) in the CSG. All seedlings were watered daily with half-strength Yamazaki’s pepper nutrient solution. Three weeks later, when their second true leaf had fully expanded, the seedlings were transplanted into plastic pots (8 cm long, 8 cm wide and 10 cm deep, one seedling per pot) containing the same substrate and watered with full-strength nutrient solution. Then, 480 seedlings in total were chosen, transferred into the ACC and cultured while receiving four kinds of light quality treatments for 28 d. Each light treatment was repeated three times in the same ACC and there were 40 plants for per replication per treatment. Five plants were randomly sampled at 7, 14, 21 and 28 DAT from each replication each treatment and were subjected to morphological and biochemical analyses. There was ventilation in the controlled environment, so the CO2 level was the same as the CO2 level of atmosphere outside. The relative humidity (RH) was kept at 70 ± 10 %, with a 12 h photoperiod and a temperature of 26 ± 1 oC during the daytime and 18 ± 1 oC at night.
Light treatments
All the mixed LEDs had a uniform spectrum for R and B light and were designed by Chunying Optoelectronics Technology Co., Ltd., Guangdong, China. The cultivation rack in the ACC was a steel frame structure with an LED light source placed at the top. The different treatments were insulated from one another by silver shading material. The plants were grown under the following light conditions: monochromatic B light with a maximum intensity at 457 nm, R light or mixed R and B light (3:1, RB: 75% R light witht a wavelength of 657 nm and 25% B light with a wavelength of 457 nm) has a maximum intensity at 657 nm. There was a multi-wavelength W light treatment as control (Supplementary Fig. 1). The light intensity, expressed as PPFD at the canopy level, was set at 300 μmol/m2·s, which was measured using a quantum sensor (LI-250, LI-COR Inc., Lincoln, NE, USA) and maintained by adjusting the distance of the LEDs from the canopies. The LEDs was approximately 10 cm far away from the canopy. A spectroradiometer (Unispec-SC Spectral Analysis System, PP Systems Inc., Haverhill, MA, USA) was used to measure the spectral photon flux density distributions (SPDs) of the LEDs.
Biomass analysis
Five seedlings, including leaves and roots, were removed from each replication each treatment at 28 DAT and dried in an oven at 105 °C for 30 min. The oven temperature was changed to 75 °C and the plants were dried to a constant weight. Then, the DWs of leaves and roots were measured using an electronic balance (precision: ± 0.1 g, Model LA16001S, Sartorius Co., Hamburg, Germany).
Leaf anatomy
Leaf anatomy was measured on the fully expanded second leaves from five pepper seedlings at a similar position for each replication each treatment [46] on 28 DAT. Leaf segments of 5 mm × 5 mm were taken from the central leaf blade next to the main vein, fixed with formalin-acetic acid-alcohol (FAA) fixative, dehydrated in an alcohol and xylene series, embedded in paraffin, cross-sectioned to a thickness of 10 μm, and stained with red-solid green. The total thickness of the whole leaf and the thickness of the upper epidermis, lower epidermis, PT and SPT were measured under a transmission light microscope (DP71, Olympus Inc., Tokyo, Japan). Images were collected using a digital camera (Camedia C4040, Olympus Inc., Tokyo, Japan) and analyzed by AnalySIS 5.0 (Olympus Inc., Tokyo, Japan).
Photosynthetic light- and CO2-response curves
Between 09:00 am and 14:00 pm, the measurement of photosynthetic light-response curves and CO2-response curves was made on the second leaf fully-unfolded using a portable photosynthesis systems machine (LI-6400XT, Li-COR, Lincoln, NE, USA) at 28 DAT. The measurement technique was based on a modified method described by Pan et al. [52]. In the leaf chambers, the temperature was 26 ± 1 ∘C, air relative humidity was 65 ± 5 % and the flow rate was 300 μmol/s. The measurement of light-response curves was made under different graded PPFD series of 1800, 1500, 1200, 1000, 800, 600, 400, 300, 200, 150, 100, 50, 20 and 0 μmol/m2·s. When the CO2-response curve measurements were taken, the light intensity and CO2 concentration of the leaf cuvette were set to 1000 μmol/m2·s and 400 μmol/mol, respectively, for 30 min. After reaching a steady state, the curves of CO2 response were measured by a CO2 mixer under a graded Ci value series of 400, 300, 200, 100, 50, 100, 200, 300, 400, 600, 800, 1000, 1200, 1500 and 1800 μmol·CO2/mol. The leaf chamber spends 120 to 180 seconds in adjusting its new microclimate each time. According to a previous report, three times of measurement were made for each curve, which was suitable for a non-linear regression equation [73, 74], so that the LCP, LSP, Pnmax, CCP, CSP and the maximum RuBP regeneration rate. The starting slope of the curve of light response was the AQY, and the starting slope of the curve of CO2 response was the CE.
Chlorophyll fluorescence and chlorophyll fluorescence transients
The Chl fluorescence measurements were performed using a portable pulse modulation fluorometer (FMS-II, Hansatech Instruments Ltd., King’s Lynn, Norfolk, UK). The second fully expanded leaves of five seedlings from each replication each treatment were dark adapted for 20 min, and the Fo (original fluorescence yield) and Fm (maximum fluorescence yield) were determined. Then, the leaves were put under natural light for 1 h, and the measurements of F'o, F'm and Fs values was made under the activating light of 800 μmol/m2·s. With the saturation pulse intensity of 3000 μmol/m2·s and the duration of 0.8 s, F'o and F'm respectively stand for the minimum and maximum fluorescence yields of an illuminated leaf, which were measured by applying the method of saturation pulse. Fs means the steady fluorescence yield. The maximum photochemical efficiency of PSII was calculated using Fv/Fm = (Fm – Fo) / Fm, actual PSII photochemical efficiency was calculated using (ΦPSII) = (F'm – Fs) / F'm and maximum photochemical efficiency of PSII under light adaptation was calculated using (F'v/F'm) = (F'm – F'o) / F'm.
A plant efficiency analyzer (Handy PEA, Hansatech Instruments Ltd., King’s Lynn, Norfolk, UK) was used to measure the OJIP on the second leaves. Strasser’s method was employed to calculate the JIP-test formulae and glossary of terms [75, 76]. The following derivative parameters were determined according to Lin et al. [61] and Miao et al. [30]: RC/ABS, Sm, DIo/RC, TRo/RC, PIABS, PItotal, ΦRo and δRo.
Calvin cycle enzymes activity
After being sampled at 7, 14, 21 and 28 DAT, the second leaves selected from top 15 plants of each treatment were used to determine the enzyme activities. Leaf tissue (0.5 g) was homogenized in 4 mL of ice-cold extraction buffer: (25 mM Hepes (K+), pH 7.5, 10 mM MgSO4, 5 mM dithiothreitol (DTT), 1 mM Na2EDTA, 1 mM phenylmethanesulfonyl fluoride (PMSF), 5% (w/v) insoluble polyvinylpyrrolidone (PVP) and 0.05% (v/v) Triton X-100). The homogenate was filtered through muslin cloth and centrifuged at 14,000 × g for 5 min at 4 °C. The supernatant was used as the enzyme extract for the enzyme activity assays [77].
An ELISA kit (Shanghai Yanji Biological Technology Ltd., Shanghai, China) was employed to determine the Rubisco (EC 4.1.1.39), FBPase (EC 3.13.11), FBA (EC 4.1.2.13), GAPDH (EC 1.2.1.12) and TK (EC 2.2.1.1) activities, and the extraction approach for these enzymes were modified based on Rao and Terry [78] and Wang et al. [36]. After grounding the frozen leaf samples (0.5 g) to fine powder in a liquid nitrogen with a mortar and pestle, the powder was put into a centrifuge tube and extracted to the precool extraction buffer (5 mL). The centrifugation of enzyme extraction solution was made at 12,000 × g for 15 min at the temperature of 4 oC. The activity assay of Calvin cycle enzymes used the supernatant. Afterwards, a microplate absorbance reader (Bio-Tek ELX800, Bio-Tek Instruments, Winooski, VT, USA) was used to determine the activities of the Calvin cycle enzymes under an absorbance of 450 nm based on the instructions of the manufacturer.
The measurement of the protein concentration of each enzyme extraction solution was made based on Bradford [79]. The results of the measurement were showed as U/g of protein.
Gene expression
Quick RNA Isolation Kit was used to extract total RNA according to the supplier’s instructions (Huayueyang Biotech Co., Ltd., Beijing, China). A ReverTra Ace qPCR RT-Kit (Toyobo Bio-Technology, Co., Ltd., Osaka, Japan) was applied to make reverse transcription. Real-time PCR was employed to conduct the gene expression analysis with 18S rRNA as an internal control. The thermal cycler procedure was cycled once for 2 min at the temperature of 94 °C and cycled for 40 times at the temperature of 94 °C for 10 s, 60 °C for 20 s and 72 °C for 30 s. The method described in Livak and Schmittgen was used to analyze relative gene expressions [80]. The specific gene primers used for real-time PCR analysis of the genes involved in the PS complexes are shown in Supplementary Table 1.
Data analysis
The experiment had a totally random design. Values presented are the mean ± standard deviation (SD) of three replicates. One-way variance analysis (ANOVA) was employed to analyze the data, and the differences between the means were tested by Duncan’s multiple range test (P < 0.05). The charts were created using Origin (version 8.5, Microcal Software Inc., Northampton, MA, USA).