Materials and treatments
The experiment was carried out in the light quality culture room at the College of Horticulture Science and Engineering, Shandong Agricultural University, Shandong, China (longitude: 117.12°E; latitude: 36.19°N) during October and November 2018. We used the Welsh onion variety ‘Yuanzang’, which was originally sourced from the Tai’an Taishan Seed Industry Technology Co., Ltd. The seeds were plants in 50-hole trays, and the cultivation substrate was a 6:3:1 mixture of charcoal, perlite, and vermiculite. The seedlings were watered with 1/2 Hoagland nutrient solution every 3 days after sowing. When the seedling height was approximately 5 cm, the plants were thinned so that only one seedling per hole remained. When the seedling height was approximately 15 cm, 2–3 pieces of leaves were sampled and placed in the LED light treatments. The light treatments used dimming plant lamps (Huizhou Kedao Technology Co., Ltd.) of five different wavelengths: W light (control group), B light, G light, Y light, and R light. The spectral characteristics of the LED sources were measured with a UNSPEC-DCTM spectrum analyzer (PP-SYSTEMS, UK), with a bandwidth of 300–1100 nm and a 3.3 nm scanning interval. The spectral characteristics of each light treatment are shown in Fig. 1A-B.
The light intensity was maintained at 301.6 ± 12.7 μmol/m2·s. The day/night temperature was maintained at 25 °C/18 °C, respectively, the relative humidity was 65.2 ± 4.5 %, and the light/dark (L/D) photoperiod was set to 12 h/12 h. Each treatment contained 20 plants, and all treatments and assays were repeated 5 times.
Measurement of morphological and physiological characteristics
The Welsh onion plants grown in different light treatments were randomly sampled and measured 30 days after planting. The measurements included the leaf number, LA, plant height, cauloid diameter, leaf FW, cauloid FW, root FW, and aboveground dry matter content. The plant height and cauloid diameter were measured with a ruler and Vernier caliper, respectively. The LA was determined using a LI-3000C leaf area meter (LI-COR Biosciences, USA). For the biomass measurements, the samples were divided into two parts: the shoot and the roots. The two parts were placed in a box, dried at 75 °C for 48 h, and then weighed for the shoot and root DW, total DW, and root/shoot ratio in dry weight basis (R/S). From these measures, we calculated DQI as follows [28]: (see Equation 1 in the Supplementary Files)
Measurement of photosynthetic pigment content
The Chl content of the leaves was determined by 80 % acetone extraction. A fresh 0.2 g sample of the third leaf blade was weighed and placed in a 20 mL test tube containing 5 mL of absolute ethanol and 5 mL of 80 % acetone and incubated in darkness for 24 h. The optical density (OD) was measured using a UV-1200 spectrophotometer (Shimadzu, Japan) at 470 nm (OD470) for carotenoids, 663 nm (OD663) for Chl a, and 645 nm (OD645) for Chl b. These measurements were used to calculate the content of each respective pigment in the leaves using the following formulas [67, 68]:
Chl a (mg g-1) = (12.72 OD663 nm - 2.59 OD645 nm) V/1 000 W;
Chl b (mg g-1) = (22.88 OD645 nm - 4.67 OD663 nm) V/1 000 W;
Carotenoids (mg g-1) = (1 000 OD470 nm - 3.27 Chl a - 104 Chl b) V/(229 × 1 000 W),
where V is the total volume of acetone extract (ml), and W is the FW (g) of the sample.
Measurement of photosynthetic characteristics and chlorophyll fluorescence
On day 30 (after planting), the functional third leaves of the plants were sampled and Pn, Gs, Ci, and E were measured using a Li-6800 portable photosynthetic apparatus (Li-COR, USA) following the methods of Li [69], with slight modifications. To measure the CO2 fixation by photosynthesis under different light conditions, the gas exchange characteristics of the functional leaves were measured under each light source. The leaf chamber temperature and leaf CO2 concentration were maintained at 25 °C and 400 μmol/m2·s, respectively, and the vapor pressure deficit in the leaf chamber was kept at 1.0 kPa. When the Pn reached steady state (after about 5 min), it was recorded. The measurements were repeated 5 times for each light treatment, and the average value was calculated for each photosynthetic parameter. The RuBPCase activity of RuBisCO was determined using an enzyme-linked immunosorbent assay kit (Suzhou Keming).
The Chl fluorescence of the third fully expanded functional leaf was measured using an M-series modulated Chl fluorescence imaging system (MINI-IMAGING-PAM, Walz, Effeltrich, Germany). To do so, the fluorescence parameters were first determined after dark adaptation for 20 min. Initial fluorescence (Fo) was measured after induction by a weak modulation (0.05 μmol/m2·s), followed by excitation with a strong saturation pulse (6 000 μmol/m2·s, pulse time = 2 s) to produce and measure the maximum fluorescence (Fm). Next, for light adaptation, the Fo and Fm′ (the maximum fluorescence yield obtained when the light-adapted sample was exposed to the saturation pulse) were directly measured under each LED light before the actinic light was turned on, followed by a series of saturation pulses under each LED light. Multiple strong saturated flash pulses were applied (6 000 μmol/m2·s, pulse time = 2 s), and the fluorescence yield (Ft) and Fm′ under light adaptation with each LED light were measured every 20 s until pulse termination. We calculated the average values of the last six flashes (after a substantially steady state was reached after 10 flashes), and the measurements from five plants were averaged for each treatment. The measured indicators included Fo, Fm, and Ft. Other fluorescence parameters were calculated according to Genty [70]:
Maximum photochemical efficiency of photosystem II (PSII) under dark adaptation (Fv/Fm) = (Fm-Fo)/Fm;
Maximum photochemical efficiency of PSII under light adaptation (Fv′/Fm′) = (Fm′ - Fo′)/Fm′;
Actual photochemical efficiency (ΦPSII) = (Fm′ - Fs)/Fm′;
Non-photochemical quenching coefficient (NPQ) = 1 - (Fm′ - Fo′)/(Fm - Fo);
Photochemical quenching coefficient (qP) = (Fm′ - Ft)/(Fm′ - Fo′);
Apparent ETR = ΦPSII·PAR·0.5·0.84, where PAR is 300 μmol/m2·s.
Observation of the leaf anatomy and chloroplast ultrastructure of Welsh onion
On day 30, paraffin sections (5 mm × 5 mm) of the samples were taken, fixed with a formalin–acetic acid–alcohol fixative, dehydrated in an alcohol and xylene series, embedded in paraffin, cross-sectioned to a thickness of 10 μm, and red–solid green stained. The total thickness of the transverse sections, as well as the thickness of the upper epidermis, palisade mesophyll tissue, and spongy mesophyll tissue, was measured under a light microscope using a micrometer.
Pieces of the functional leaves were sampled (1 mm × 1 mm), quickly placed in a 2.5 % glutaraldehyde fixative solution, and evacuated with a vacuum pump. After the pieces sank to the bottom of the fixative solution, they were maintained at room temperature (25 °C) for 2 h, and then transferred to a refrigerator and stored at 4 °C. The samples were rinsed three times with 0.1 M phosphate buffer (PB, pH = 7.4) for 15 min each, fixed with 1 % citric acid in 0.1 M phosphate-buffered saline (pH = 7.4) at room temperature (25 °C) for 5 h, and rinsed again three times with 0.1 M PB (pH = 7.4) for 15 min each. The leaf tissue was sectioned on a dehydration-infiltration-embedding-slicer (Leica, LeicaUC7) and imaged using a section-staining-transmission electron microscope (HITACHI, HT7700).
Data analysis
All plants were randomly sampled in this study. The data were processed, plotted, and statistically analyzed in Excel 2016 and DPS software. The differences among treatments were tested using Duncan’s new multiple range test at a significance level of P ≤ 0.05.