High relative humidity improves leaf burn resistance in flowering Chinese cabbage seedlings cultured in a closed plant factory

Plant material and growth conditions

All experiments in this study, from seed treatment to vegetable crop growth, were carried out in a small, closed plant factory with artificial light in the College of Horticulture, South China Agricultural University. The seedling module and the culture module (Sananbio China, Xiamen, Fujian, China), were installed in the plant factory; both of them are multi-layer vertical deep flow hydroponic systems. The main parameters of modules and light sources are shown in Table S1 and Fig. S1. The temperature, ventilation and the RH in the plant factory were controlled by an air conditioner system (KFR-120TW/(1256S) NhBa-2, Gree, China) and dehumidifiers (DY-620EB, Deye, Ningbo, Zhejiang, China). A constant temperature of 24 °C was set to ensure that the plant canopy temperature was stayed around at 26 °C when the lights were turned on. RH was set to 70  ± 10%. The canopy air velocity in plant factory was almost zero.

The seeds were imbibed in 55 °C warm water for 15 min, then soaked for 2 h with water, and germinated for the radicle to emerge under dark conditions. The radicle-emerging seeds were sowed into the seedling tray (60 cm × 29.5 cm × 4.9 cm) containing a sponge block with water, and then placed on the seedling module for 14 days. Once the cotyledons were flattened, the water was replaced with 1-strength Hoagland’s nutrient solution every three days in the nursery tray.

Cultivation tests of vegetable species and cultivars in plant factory

Thirty types of common vegetable crops, from 25 species and eight families, were selected for this part. The 14-day-old seedlings grown on the seedling module were transplanted to the culture module for further hydroponics. The photoperiod was set to 12 h/12 h (day/night). The sampling time was determined according to the growth rate of different crops and the incidence of “leaf burn”, and photographs were taken accordingly.

Based on the degree of leaf burn of the different vegetable species, the degree of leaf burn was categorized into three grades: LV1, the plant does not show leaf burn during the whole growth period, and its growth and development are normal; LV2, the plant has a slight leaf burn, and the rate of new healthy leaf production is higher than that of the leaf burn of the lower leaves, i.e., the number of healthy leaves is always more than burnt leaves, and the plants could grow continuously until harvest. LV3, severe leaf burn occurs in the plant, and the leaf burn rate of the lower leaves is higher than the new leaf production rate, the plant does not grow properly and eventually die.

Light intensity, and photoperiod treatment

In order to analyze whether leaf burn occurs due to light conditions, two flowering Chinese cabbage varieties, Sijiu-19 (Sijiu, early-maturity cultivar) and Chixin No. 2 (Chixin, late-maturity cultivar), were examined to investigate the leaf burn incidence at various light intensities and photoperiod conditions in plant factory. The seedlings were planted in seedling trays, and then placed on the seedling module for 14 days. Shading nets were used to adjust the light intensity, and tin foil was used as seedling tray cover to adjust the photoperiod. The light treatments are shown in Table 1.

Relative air humidity treatment

Considering insignificant improvement of light conditions on the leaf burn occurrence of two flowering Chinese cabbage cultivars, we extended the analysis of environment factor RH on the prevention of leaf burn occurrence. The 70 ± 10% of canopy RH was set as the control treatment. The sealed seedling tray cover was used to increase the canopy RH to 90–99% (daily average VPD 0.16 kPa). The control seedlings were covered with seedling tray lid with only the top and four corners. All the seedlings were grown in seedling trays, and then placed in the seedling module with a photoperiod of 12 h/12 h L to D and a constant temperature of 24 °C within 16 days. Seventeen days after sowing, the seedlings were transplanted to culture module with hydroponic system for continuous growth until the harvest of early-maturity cultivar ‘Sijiu’ with a photoperiod of 12 h/12 h L to D and a constant temperature of 24 °C. Unfortunately, the seedlings gradually died grown at 70% RH; at 90% RH, both two cultivars grew healthy. Chlorophyll content and leaf temperature were examined on the 14th day after sowing. Leaf water potential, stomatal density, stomatal aperture, and photosynthetic gas exchange parameters were studied on the 12th, 14th, and 16th days after sowing. Growth-related parameters were determinated on the 14th and 35th days, and quality indicators were measured on 35th days.

Statistics of leaf burn incidence and measurement of growth parameters

The leaf burn incidence is defined as the ratio of the number of plants with leaf burn to total number of plants (leaf burn incidence = numbers of plants with burnt leaves in any extent/total number of plants). The fresh weight of the whole plant was weighed with electronic balance. The plant dry weight was obtained after drying the fresh plants in an oven at 100 °C for 30 min, followed by drying at 80 °C to constant weight.

Determination of chlorophyll pigment content

The chlorophyll pigment contents were examined referring to the method of Wang et al. (2022). Briefly, 0.2 g fresh leaf samples were soaked in 20 mL of an acetone-ethanol mixture (acetone: ethanol = 2:1, v/v) for 24 h at 25 °C in the dark till the leaves turned white. The absorbance of the supernatant was determined by UV-spectrophotometer (Shimadzu UV-16A, Shimadzu, Corporation, Kyoto, Japan) at 645 nm, 663 nm, and 440 nm, respectively.

Determination of quality index

The quality index, including vitamin C (Vc), soluble sugar and soluble protein, was determined in the flower stalk of cultivar ‘Sijiu’, and in the rosette leaves of cultivar ‘Chixin’. The leaves were separated from the flower stalk, and the two parts were used for analysis respectively in cultivar ‘Sijiu’.

Vc content was determined by molybdenum blue colorimetric method (Tan et al., 2021). Briefly, fresh leaf and flower stalk samples (1.0 g) were extracted using 25 mL of oxalic acid EDTA solution, and then one mL of supernatant was mixed with 0.5 mL of phosphate-acetic acid, two mL of 5% ammonium molybdate. After incubation at 30  °C for 15 min, the absorbance was recorded at 760 nm using a UV-spectrophotometer (Shimadzu UV-16A, Shimadzu, Corporation, Kyoto, Japan).

Soluble sugar content was determined using anthrone-sulfuric acid colorimetric method (Kohyama & Nishinari, 1991). Fresh leaf and flower stalk samples were heated in a boiling water bath with 25 mL of distilled water for 30 min. Then, 0.5 mL of supernatant was mixed with 1.5 mL distilled water, 0.5 mL anthrone ethyl acetate, and five mL vitriol. The absorbance was recorded at 630 nm using a UV-spectrophotometer (Shimadzu UV-16A, Shimadzu, Corporation, Kyoto, Japan).

Soluble protein content was measured by Coomassie brilliant blue G-250 dye method (Bradford, 1976). Fresh leaf and flower stalk samples (1.0 g) were homogenized with eight mL distilled water. The homogenate was centrifuged at 8,000 ×g for 10 min at 4 °C; then, 0.2 mL the supernatant was mixed with 0.8 mL distilled water and five mL 0.1 g/L Coomassie brilliant blue G-250 solution. The absorbance was measured at 595 nm using a UV-spectrophotometer (Shimadzu UV-16A, Shimadzu, Corporation, Kyoto, Japan) after standing for 15 min.

Determination of leaf water potential

Leaf water potential was examined using a WP4C dewpoint potential meter (Original Decagon, Pullman, Washington, USA) (Lu et al., 2021). Briefly, the leaves were sampled and quickly placed into the sample chamber to make sure the leaves to cover the full bottom of the sample chamber. The difference between the sample temperature and the block chamber temperature was kept below −0.5 °C.

Observation of stomatal aperture and density

The scotch tape method was used to obtain the leaf lower epidermis. Stomata image from an epifluorescence microscope (Olympus BX53, Olympus Corporation, Tokyo, Japan) was analyzed by ImageJ software (Rasband, W.S., ImageJ 1.8.0 172, U.S.A) to count the number of stomata and measure the aperture size (Lawrence et al., 2018).

Determination of photosynthetic parameters

The seedlings were adapted about 0.5 h under darkness to estimate the maximum quantum yield of photosystem II photochemistry (Fv/Fm) using an Imaging-PAM chlorophyll fluorometer (PlantExplorer, PhenoVation, Wageningen, Netherlands). Gas exchange measurements were performed using a portable photosynthesis system (LI-6400, LI-COR, USA). The net photosynthetic rate, intercellular CO₂ concentration, stomatal conductance, and transpiration rate of the first true leaf were measured after turning on the light for 6 h in the morning. photosynthetic photon flux density (PPFD) set as 200 µmol m⁻² s⁻¹. The portable leaf area meter (RX-YMJ, TuoPu, Zhejiang, China) was used to determine the actual blade area enclosed in the leaf chamber, and the gas exchange area were recalculated (Carriquí, Nadal & Flexas, 2021).

Data analysis

Leaf burn incidence and growth determinations were performed with 45 and 15 replicates per treatment, respectively. Leaf water potential, leaf temperature and quality index were carried out with five replicates. Leaf stomatal parameters were performed with six replicates. Fv/Fm was done with 18 replicates. Photosynthetic parameters were performed with three replicates. All experiment was conducted based on a randomized complete block design with biological repeats. The data are presented as the mean ± SEs and were analyzed by SPSS 20 statistics software (IBM Corp., Armonk, NY, USA), the experimental data were analyzed with Duncan’s multiple range test at a significance level of 0.05 and 0.01.

Differences in the degree of leaf burn among vegetable species and varieties under the closed plant factory

Among the 30 vegetable varieties studied, 10 varieties performed normal growth and development, including Cucurbitaceae family plants, Lagenaria siceraria Stand var. siceraria, Benincasa hispida Cogn. var. chieh-qua How, and Luffa acutangular Roxb; the Asteraceae family plants, Chrysanthemum coronarium L. var. spatiosum Bailey and Lactuca sativa L. var. longifolia Lam; a Solanaceae family plant, Solanum lycopersicum L.; an Amaranthaceae family plant, Amaranthus mangostanus L.; a Chenopodiaceae family plant, Beta vulgaris L. var. cicla Koch.; an Lamiaceae family plant, Ocimum basilum L. No leaf burn (LV1) was observed in these vegetables.

The remaining 20 vegetable varieties all showed different degrees of leaf burn. Occurrence of leaf burn in the cruciferous vegetables was the most common and severe, except Brassica campestris ssp. chinensis var. communis Tsen et Lee (LV2 degree), all other cruciferous vegetables showed severe leaf margin browning and death (LV3), and could not grow and develop normally, and eventually died. Cucurbitaceae species showed obviously different leaf burn resistance. Momordica charantia L., and Cucumis melon L. exhibited minor leaf burn symptoms (LV2), while Cucumis sativus L. and Cucurbita moschata Duch ex Poir had severe leaf burn (LV3) (Table 2, Fig. 1).

The incidence of leaf burn in flowering Chinese cabbage under different light intensity and photoperiod conditions

Under different light conditions, cultivar ‘Chixin’ was more resistance to leaf burn compared with the cultivar ‘Sijiu’. Under PPFD 150 µmol m⁻² s⁻¹ and 12 h d⁻¹ photoperiod (T1), two flowering Chinese cabbage cultivars exhibited different degrees of leaf burn symptom, and leaf burn incidence reached 28.89% and 18.52% in ‘Sijiu’ and ‘Chixin’, respectively (Fig. 2). Decreasing light intensity from PPFD 150 µmol m⁻² s⁻¹ (T1) to 105 µmol m⁻² s⁻¹ (T2) reduced the incidence to 15.21% in ‘Sijiu’, and absolutely inhibited leaf burn occurrence in ‘Chixin’ (Fig. 2B). When the photoperiod was shortened from 12 to 8 h d⁻¹ at 105 µmol m⁻²s⁻¹, leaf burn incidence gradually reduced in ‘Sijiu’. The cultivar ‘Chixin’ did not show any leaf burn symptom at 12 h d⁻¹ (T2) and 8 h d⁻¹ (T4) photoperiod and exhibited mild leaf damage at 10 h d⁻¹ (T3), with 5.88% of leaf burn incidence. Although leaf burn occurrence was affected to different degrees under different light conditions, all plants grew slowly and bad in general.

The effect of RH on the leaf burn occurrence and physiological characteristics of flowering Chinese cabbage

Because the minor alleviation of light conditions on the leaf burn occurrence and plant growth in flowering Chinese cabbage, we attempted to explore the effect of environment factor RH. Fortunately, both in ‘Sijiu’ and ‘Chixin’, increasing RH from 70% to 90% by putting a cover on the seedling tray not only absolutely prevented the occurrence of leaf burn (Figs. 3A∼3C), but also improved plant growth; as a result, plant fresh weight and dry weight were greatly promoted. Fourteen days after sowing, fresh weight of plants cultivated at high RH level increased by 38.79% and 69.76%, and dry weight of plants increased by 20.76% and 62.21% respectively in ‘Sijiu’ and ‘Chixin’ compared with the plants cultivated at low RH (Figs. 3D∼3E). Fifteen days after sowing, ‘Chixin’ and ‘Sijiu’ were transplanted to the culture module for continuous growth. At high RH level, two flowering Chinese cabbage cultivars grew healthy and developed normally without leaf burn symptom (Fig. 3B); thirty-five days after sowing, the early-maturing cultivar ‘Sijiu’ formed flower stalks, and the late-maturing cultivar ‘Chixin’ was still at seedling stage. In ‘Sijiu’, shoot fresh weight was 48.53 g plant⁻¹, and the contents of soluble sugar, Vc and soluble protein were 1.89 mg gFW⁻¹, 133.71 mg 100 gFW⁻¹ and 62.89 mg gFW⁻¹ respectively in the leaves, and 1.12 mg gFW⁻¹, 51.63 mg 100gFW⁻¹ and 30.35 mg gFW⁻¹ respectively in the stalks on days 35 after sowing under high RH conditions; In ‘Chixin’, shoot fresh weight was 32.43 g plant⁻¹, and the contents of soluble sugar, Vc and soluble protein were 5.27 mg gFW⁻¹, 206.91 mg 100gFW⁻¹ and 86.24 mg gFW⁻¹ respectively in the leaves (Figs. 3G∼3I). At low RH, both the cultivar ‘Sijiu’ and ‘Chixin’ gradually died after transplantation to the culture module.

We extended the analysis of leaf water potential using a WP4C dewpoint potential meter. At low RH, the water potential in the leaves of two cultivars gradually decreased with the extension of the experiment, which decreased faster in ‘Sijiu’ than ‘Chixin’. On day 16 after sowing, the leaf water potential in ‘Sijiu’ was significantly lower than that in ‘Chixin’. Conversely, the leaf water potential both in ‘Sijiu’ and ‘Chixin’ kept relatively stable under high RH conditions. Compared with the plants grown at low RH level, the significantly higher water potential was maintained in the leaves of ‘Sijiu’ grown at high RH level on 16 days after sowing, and in the leaves of ‘Chixin’ on days 14 and 16 after sowing (Fig. 4A). On day 14 after sowing, the significantly lower T_leaf was observed in the plants at high RH than the plants at low RH regardless of plant varieties (Fig. 4B).

The stomatal aperture and density of plants were generally reduced in response to the increased RH. On 16 days after sowing, high RH significantly reduced the stomatal aperture and density in ‘Sijiu’ leaves and stomatal aperture in ‘Chixin’ leaves. Except that on day 14 after sowing, high RH slightly increased the stomatal density in ‘Chixin’ leaves and stomatal aperture in ‘Sijiu’ leaves compared to low RH treatment (Fig. 5).

The photosynthetic performance of flowering Chinese cabbage grown at 90% RH was significantly improved compared with those at 70% RH. The high RH increased maximum quantum yield of PS II (Fv/ Fm), the levels of chlorophyll a, chlorophyll b, carotenoids, and total chlorophyll in comparison to low RH treatment on day 14 after sowing (Figs. 6A∼6E). Compared with the plants cultivated at low RH, the stomatal conductance in ‘Chixin’ leaves cultivated at high RH increased by 76.77%, 90.74%, and 150.74% respectively, and increased by 52.58%, 85.95% and 85.63% respectively in ‘Sijiu’ leaves 12, 14 and 16 days after sowing; the transpiration rate in ‘Chixin’ leaves treated with high RH increased by 49.29%, 37.06% and 53.15%, and increased by 11.17%, 75.17% and 42.54% in ‘Sijiu’ leaves respectively; the net photosynthetic rate (Pn) in ‘Chixin’ leaves increased by 93.08%, 280.59%, and 182.52%, and increased by 67.84%, 553.71% and 101.00% in ‘Sijiu’ leaves respectively. Under two RH levels, no significant difference of internal CO₂ (C_i) was observed within the same cultivar at the same sample time; C_i was maintained at about 250 µmol CO₂.mol⁻¹ (Figs. 6F∼6I). In general, no great difference of the photosynthetic performance was observed between two cultivars regardless of canopy RH, although higher Fv/Fm, chlorophyll b and transpiration rate in ‘Chixin’ leaves at low RH were found than those in ‘Sijiu’.