Effect of Daily Light Integral on Cucumber Plug Seedlings in Artificial Light Plant Factory

In a controlled environment, in an artificial light plant factory during early spring or midsummer, vegetable seedlings can be uniform, compact, and high quality. Appropriate light parameters can speed up the growth of seedlings and save on production costs. Two experiments were carried out in this study: (1) cucumber seedling growth under different daily light integrals (DLIs) (5.41–11.26 mol·m−2·d−1) and optimum DLI for seedling production were explored (experiment 1: Exp. 1); (2) under the same DLI selected by Exp. 1, the effects of different light intensities and photoperiods on cucumber seedlings were investigated (experiment 2: Exp. 2). The root biomass, root-to-shoot ratio, seedling index, and shoot dry matter rate increased as the DLI increased from 5.41 to 11.26 mol·m−2·d−1, while the shoot biomass and leaf area decreased in Exp. 1. The cucumber seedlings became more compact as DLI increased, but more flowers developed after transplanting when the DLI was 6.35 mol·m−2·d−1. Under the optimal DLI (6.35 mol·m−2·d−1), the optimal intensity was 110–125 μmol·m−2·s−1, and the optimal photoperiod was 14–16 h, in which plant biomass, shoot dry matter rate, seedling index, and photochemical efficiency were higher.


Introduction
The daily light integral (DLI) is the total amount of photosynthetic light delivered to plants each day [1,2]. The DLI is an important environmental parameter for plants and displays stronger correlation with vegetative growth parameters than with photoperiod and light intensity [3,4]. Many studies have shown that a suitable DLI could promote the growth and development of plants, such as the germination rate of photoblastic seeds [5], shoot and root biomass [6][7][8][9], stem diameter [10,11], leaf area and leaf number [12], and the differentiation of flower buds [4,13,14].
The production of high-quality seedlings with characteristics of higher biomass, compactness and adequate leaf area in a short cultivation period is the target of producers. However, this constitutes a significant challenge under insufficient scrutiny [15,16]. Many operations use supplemental lighting to increase DLI under low irradiance to accelerate growth and improve the morphology of seedlings in typical greenhouses [17][18][19]. Cucumber seedlings became more compact and vigorous, with characteristics of a short hypocotyl length, large leaf area, and high biomass under a high DLI [19,20]. Increases in DLI during the seedling stage have been also reported to accelerate subsequent floral initiation and decrease the time to the first flowering after transplant [13,21,22]. The number of tomato floral buds and days to flowering increased corresponding to the decrease in the shading before anthesis [23].
However, higher DLI is correlated with more illuminating apparatus and electric energy consumption. Additionally, superfluous DLI with a high light intensity or long photoperiod may cause damage to the photosynthetic system and inhibits the production In both Exp. 1 and 2, seeds of cucumber 'Yuexiu No.3 were place onto humid cotton in an incubator for 2 days. The temperature and relative humidity were set to 25 • C and 80%, respectively. The germinated seeds were sown into plug trays (50-cell size (28 mL)) in a substrate with peat, coconut coir and perlite (6: 3: 1, v: v: v) and placed into a walk-in growth chamber at the College of Horticulture of South China Agricultural University, Guangzhou. Each tray was a repeat. Each experiment conducted four treatments, in which three repeats were concluded. Before cotyledons were fully expanded after sowing, 800 ± 50 mL tap water was added to each tray every second day. Once cotyledons were fully expanded, 800 ± 50 mL 1/4 strength modified Hoagland and Arnon's nutrient solution was added to each tray every second day to provide (in mg·L −1 ) 53 nitrogen (N), 40 calcium (Ca), 59 potassium (K), 8 phosphorus (P), 25 magnesium (Mg), 33 sulphur (S), 0.56 iron (Fe), 0.5 boron (B), 0.5 manganese (Mn), 0.05 zinc (Zn), 0.01 molybdenum (Mo) and 0.005 copper (Cu) until the emergence of the second true leaf. Then, 800 ± 50 mL 1/2 strength modified Hoagland and Arnon's nutrient solution was added to each tray every second day until sampling.

Growth Chamber Environment
All the plug trays were placed on steel shelves in two vertical layers in a walk-in growth chamber, with an average daily temperature of 25 • C, relative humidity (RH) of 75%, and average carbon dioxide (CO 2 ) concentration of 400 µmol·mol −1 .
All the plug trays were placed on steel shelves in two vertical layers in a walk-in growth chamber, with an average daily temperature of 25 °C, relative humidity (RH) of 75%, and average carbon dioxide (CO2) concentration of 400 μmol•mol −1 .

Morphology and Growth Measurement
The morphology and growth of cucumber plug seedlings in both Exp. 1 and 2 were measured on the 26th day after sowing. Six seedlings of each repeat (i.e., 18 seedlings per DLI treatment) were randomly selected and measured to determine the hypocotyl length, stem diameter, total leaf area, shoot fresh mass, shoot dry mass, root fresh mass, and root dry mass. The fresh mass of seedlings was recorded by an electronic balance, and the dry mass was recorded after seedlings were heated to de-enzyme at 105 • C, then dried at 80 • C until steady.

Physiological Characteristics and Photosynthesis Pigment Content
The soluble sugar content was extracted and analyzed by anthrone colorimetry. A 0.5 g amount of leaf fresh sample was added to a test tube with 10 mL of distilled water, and then it was extracted in boiling water for 30 min. The extract was filtered into a 25 mL volumetric flask, the test tube and residue were rinsed repeatedly, and the volume was maintained to scale. An amount of 0.1 mL of the sample extract was absorbed into a 20 mL graduated test tube, followed by adding 1.9 mL of distilled water, 0.5 mL of ethyl anthrone acetate reagent and 5 mL of concentrated sulfuric acid. The solution was fully oscillated and cooled down to room temperature. The absorbance was measured at 630 nm by using a UV-spectrophotometer [35].
The soluble protein content was extracted and analyzed by Coomassie Brilliant Blue methods. Amount of 0.5 g of fresh leaf sample and 5 mL of distilled water were ground into a homogenate and centrifuged at 10,000× g for 10 min at 4 • C. After mixing 0.3 mL of supernatant and 0.7 mL of distilled water, 5 mL of Coomassie Brilliant Blue G-250 solution (0.1 g/L) was added to the mixture. After 2 min, the absorbance at 595 nm was determined by using a UV-spectrophotometer [35].
The content of photosynthesis pigment was determined by an acetone-ethanol blend method. An amount of 0.2 g of leaf with removed veins was placed into a test tube containing 20 mL of acetone and ethanol (1:1, v:v), and extracted under dark conditions. The absorbance at 663, 645 and 440 nm was determined by using a UV-spectrophotometer after the leaves turned white. Chlorophyll a (Chl a) content (mg·g −1 ) = (12.70

Chlorophyll Fluorescence
Chlorophyll fluorescence was measured by MINI-PAM-II (WALZ company, Germany), and the actinic illumination was set at 286 µmol·m −2 ·s −1 . Leaves were dark-acclimated for 20 min with the manufacturer's plastic and foam clips before measurements were recorded. Six seedlings of each repeat were randomly selected and measured, and the locus was the second true leaf of each seedling. The photochemical quenching indexes (qP and qL), the non-photochemical quenching indexes (qN and NPQ), photic injury nonphotochemical quenching quantum yield (Y(NO)), photoprotection non-photochemical quenching quantum yield (Y(NPQ)) and PSII conversion efficiency of light energy (Φ PSII ), and maximal photochemical efficiency of PSII in the dark (Fv/Fm) were measured.

Flowering in Cucumber Plant after Transplant
As a supplementary experiment, only the run 2 in Exp. 1 was carried out for flowering statistics. After DLI treatments, nine cucumber seedlings from each DLI treatment were transplanted into a growth bag (15 cm diameter, 15 cm height) filled with substrate of peat, coconut coir and perlite (6:3:1, v:v:v). Plants were watered with 1/2 strength Hoagland and Arnon's nutrient solution before the first flower bloom, and then full strength nutrient solution. The days to first flower bloom from transplant, node of the first male flower, male flower number, node of first female flower, female flower number on main stem, number of lateral branches, and total female flower number under the 15th node were recorded at 60 days after the transplant.

Statistical Analysis
Data were analyzed by one-way ANOVA using PASW Statistics 18.0. Duncan's multiple range test at p < 0.05 was used to determine significant treatment differences. The plotting and fitting of monomial and binomial response functions were conducted using Origin 8.6.

Experiment 1 3.1.1. Morphology and Growth of Cucumber Plug Seedlings
The morphology and growth of cucumber plug seedlings were affected by DLI. The hypocotyl length and leaf area of cucumber plug seedlings showed the same trend as DLI increased. Hypocotyl length decreased from 8.28 to 3.55 cm, while the leaf area decreased from 123.15 to 56.07 cm 2 , as DLI increased from 5.41 to 11.26 mol·m −2 ·d −1 (Figure 2a,c). The stem diameter of cucumber plug seedlings was not affected by DLI (Figure 2b). Shoot fresh mass and dry mass decreased as DLI increased from 5.41 to 11.26 mol·m −2 ·d −1 . The root fresh mass and dry mass first increased and then decreased as the DLI increased, peaking at 8.43 mol·m −2 ·d −1 (Figure 3a). The root-to-ratio and seedling index first increased and then remained steady with the increase in DLI. At a lower DLI, the leaf area ratio decreased significantly with an increased DLI, but it remained at a low value under higher DLIs. The shoot dry mass rate increased from 0.073 to 0.095 as the DLI increased from 5.41 to 11.26 mol·m −2 ·d −1 (Figure 3b).

Morphology and Growth of Cucumber Plug Seed
The morphology and growth of cucumber plug se hypocotyl length and leaf area of cucumber plug seedli increased. Hypocotyl length decreased from 8.28 to 3.55 from 123.15 to 56.07 cm 2 , as DLI increased from 5.41 to 1 stem diameter of cucumber plug seedlings was not a fresh mass and dry mass decreased as DLI increased f root fresh mass and dry mass first increased and then peaking at 8.43 mol•m −2 •d −1 (Figure 3a). The root-to-r creased and then remained steady with the increase in ratio decreased significantly with an increased DLI, bu higher DLIs. The shoot dry mass rate increased from 0 from 5.41 to 11.26 mol•m −2 •d −1 (Figure 3b). The soluble sugar content of cucumber plug seedlings increased by 8.72 m increased from 5.41 to 11.26 mol•m −2 •d −1 (Figure 4a). When the DLI was in mol•m −2 •d −1 , the soluble protein remained high but then fell dramatically 4b). All the photosynthesis pigment contents were negatively correlated w

Physiological Characteristics and Photosynthesis Pigment Content of Cucumber Plug Seedlings
The soluble sugar content of cucumber plug seedlings increased by 8.72 mg·g −1 linearly as the DLI increased from 5.41 to 11.26 mol·m −2 ·d −1 (Figure 4a). When the DLI was in the range of 6.35-8.52 mol·m −2 ·d −1 , the soluble protein remained high but then fell dramatically beyond this range (Figure 4b). All the photosynthesis pigment contents were negatively correlated with DLI ( Figure 5).

Flowering in Cucumber Plant after Transplant
The days to the first flower bloom from transplant, and the node of the first male and female flowers (data not shown) were unaffected by DLI. When DLI was about 6.35 mol•m −2 •d −1 , more male flowers, female flowers and lateral branches appeared (Table 3). * Presented values are means ± SEs (n = 9). a,b Different lowercase letters indicate statistically differences among treatments (p < 0.05).

Morphology and Growth of Cucumber Plug Seedlings
Under the same DLI, the hypocotyl length, stem diameter and shoot fresh mass were independent of changes in illumination strength and time (supplementary section and Figure 6a). The shoot dry mass, root fresh mass and root dry mass decreased by 50.24, 310.00 and 15.13 mg, respectively, as the light intensity increased from 111 to 177 μmol•m −2 •s −1 and the photoperiod decreased from 16 to 10 h (Figure 6a). Two sets of low light intensities and long photoperiods (T1 and T2) had a higher root-to-shoot ratio, seedling index, shoot dry matter rate and lower leaf area ratio (Figure 6b).

Flowering in Cucumber Plant after Transplant
The days to the first flower bloom from transplant, and the node of the first male and female flowers (data not shown) were unaffected by DLI. When DLI was about 6.35 mol·m −2 ·d −1 , more male flowers, female flowers and lateral branches appeared (Table 3). * Presented values are means ± SEs (n = 9). a,b Different lowercase letters indicate statistically differences among treatments (p < 0.05).

Experiment 2 3.2.1. Morphology and Growth of Cucumber Plug Seedlings
Under the same DLI, the hypocotyl length, stem diameter and shoot fresh mass were independent of changes in illumination strength and time (supplementary section and Figure 6a). The shoot dry mass, root fresh mass and root dry mass decreased by 50.24, 310.00 and 15.13 mg, respectively, as the light intensity increased from 111 to 177 µmol·m −2 ·s −1 and the photoperiod decreased from 16 to 10 h (Figure 6a). Two sets of low light intensities and long photoperiods (T1 and T2) had a higher root-to-shoot ratio, seedling index, shoot dry matter rate and lower leaf area ratio (Figure 6b). Horticulturae 2021, 7, x FOR PEER REVIEW 9 of 14 (a) (b) Figure 6. The effects of different light intensities and photoperiods on (a) shoot fresh and dry mass, root fresh and dry mass (b) root-to-shoot ratio, seedling index, leaf area ratio, and shoot dry matter rate of cucumber seedlings. Different letters indicate statistically significant differences among treatments (p ≤ 0.05). Error bars show SEs (n = 18).

Physiological Characteristics of Cucumber Plug Seedlings
The soluble sugar content was the highest in T1, and there were no significant differences in the other three treatments. The soluble sugar content in T1 was 25.66%, 27.95%, and 35.42% higher than in T2, T3, and T4, respectively (Figure 7a). There was no obvious pattern between the content of soluble protein with the variation of illumination parameters under the same DLI (Figure 7b). Figure 6. The effects of different light intensities and photoperiods on (a) shoot fresh and dry mass, root fresh and dry mass (b) root-to-shoot ratio, seedling index, leaf area ratio, and shoot dry matter rate of cucumber seedlings. Different letters indicate statistically significant differences among treatments (p ≤ 0.05). Error bars show SEs (n = 18).

Physiological Characteristics of Cucumber Plug Seedlings
The soluble sugar content was the highest in T1, and there were no significant differences in the other three treatments. The soluble sugar content in T1 was 25.66%, 27.95%, and 35.42% higher than in T2, T3, and T4, respectively (Figure 7a). There was no obvious pattern between the content of soluble protein with the variation of illumination parameters under the same DLI (Figure 7b

Photosynthesis Pigment Content and Chlorophyll Fluorescence of Cucumber Plug Seedlings
When the light intensity increased and the photoperiod decreased, the contents of chlorophyll and carotenoid tended to increase (data shown in supplementary section). As the light intensity increased and the photoperiod decreased, the photochemical quenching indexes (qP and qL) and ΦPSII all showed a downwards trend, and the decreased range from T3 to T4 was sharp, and there were significant differences among the four treatments. The non-photochemical quenching indexes (qN and NPQ) and Y(NPQ) increased first then decreased later, which was the largest in T3. Y(NO) showed an increasing trend with the increase in light intensity and shortening of the photoperiod, and T4 was 146.84%, 144.89%, and 117.27% higher than T1, T2, and T3, respectively. The Fv/Fm of T4 was the smallest, and there was no difference among T1, T2, and T3 (Table  4).

Discussion
Under low irradiance conditions, plants extend stems and leaves to capture more light energy, and are prone to thin branches and leaves [34,37]. Increasing DLI along with low irradiance can effectively inhibit plants spindling growth (elongated hypocotyl, thin stem diameter) of cucumber seedling [17]. As the DLI increased, the hypocotyl length and leaf area decreased in this study, but the stem diameter remained at 3-3.3 cm.
With the increased DLI, cucumber seedling quality improved in some respects (Figure 3b). High-quality vegetable seedlings are usually compact and fully rooted transplants with a higher seedling index ratio and root-to-shoot ratio [15]. Hurt et al. [38] found that when DLI was increased to about 10 mol•m −2 •d −1 , the seedling quality index of five annual bedding plants was significantly improved. The root-to-shoot ratio of new

Photosynthesis Pigment Content and Chlorophyll Fluorescence of Cucumber Plug Seedlings
When the light intensity increased and the photoperiod decreased, the contents of chlorophyll and carotenoid tended to increase (data shown in supplementary section). As the light intensity increased and the photoperiod decreased, the photochemical quenching indexes (qP and qL) and Φ PSII all showed a downwards trend, and the decreased range from T3 to T4 was sharp, and there were significant differences among the four treatments. The non-photochemical quenching indexes (qN and NPQ) and Y(NPQ) increased first then decreased later, which was the largest in T3. Y(NO) showed an increasing trend with the increase in light intensity and shortening of the photoperiod, and T4 was 146.84%, 144.89%, and 117.27% higher than T1, T2, and T3, respectively. The Fv/Fm of T4 was the smallest, and there was no difference among T1, T2, and T3 (Table 4).

Discussion
Under low irradiance conditions, plants extend stems and leaves to capture more light energy, and are prone to thin branches and leaves [34,37]. Increasing DLI along with low irradiance can effectively inhibit plants spindling growth (elongated hypocotyl, thin stem diameter) of cucumber seedling [17]. As the DLI increased, the hypocotyl length and leaf area decreased in this study, but the stem diameter remained at 3-3.3 cm.
With the increased DLI, cucumber seedling quality improved in some respects (Figure 3b). High-quality vegetable seedlings are usually compact and fully rooted transplants with a higher seedling index ratio and root-to-shoot ratio [15]. Hurt et al. [38] found that when DLI was increased to about 10 mol·m −2 ·d −1 , the seedling quality index of five annual bedding plants was significantly improved. The root-to-shoot ratio of new guinea impatiens, geranium, and petunia cuttings were increased as DLI increased [11]. In this study, the root-to-shoot ratio and seedling index increased as DLI increasing from 5.4 to 8.5 mol·m −2 ·d −1 , then, became stable due to the decrease of root dry mass with DLI continuing to increase. A lower leaf area ratio and a higher shoot dry matter rate are also regarded as high quality, which represents a higher proportion of structural materials and carbohydrates per of unit fresh weight [34]. As the DLI increased, the leaf area ratio of eight kinds of bedding plants (i.e., ageratum, begonia, impatiens, marigold, petunia, salvia, vinca, and zinnia) decreased, while the shoot dry matter rate increased [34]. This phenomenon also occurred with the Tecoma stans [16], impatiens cuttings [37], Heuchera americana [39], lettuce [40], and brassica microgreens [41].
High-quality seedlings exhibit luxuriant growth both before and post-transplant growth, which is mainly due to a higher shoot fresh and dry mass. Generally, increasing DLI can promote the accumulation of shoot biomass [41,42]. However, there was a negative correlation between DLI and biomass in this study. It is possible that higher DLIs exceeded the amount required for cucumber seedlings. As in Ji's [20] experiment, when DLI exceeded 14.4 mol·m −2 ·d −1 , the correlation between the fresh mass and dry mass of the shoot in three cucumber seedlings and DLI changed from positive to negative. The promotion of DLI to flower development is usually accompanied by an increased shoot biomass [23,28,34,43]. Leaf area reached 500 cm 2 , and dry mass reached 3 g, which were prerequisite for the flowering of tomato seedlings, revealing that some level of photoassimilates is necessary for floral initiation [23]. In this study, the decreased shoot biomass and leaf area with a higher DLI might have slowed down the development of cucumber seedlings, which stunted flower development after transplant. Therefore, the optimal DLI for cucumber seedlings at the nursery stage was 6.35 mol·m −2 ·d −1 , in which there is a higher shoot biomass and better floral initiation.
Under the optimal DLI (6.35 mol·m −2 ·d −1 ) screened by Exp. 1, a further exploration of appropriate lighting parameters combining different light intensities and photoperiods of cucumber seedlings was conducted. Under the same DLI, the variation of the light intensity and photoperiod mainly influences dry matter accumulation [44]. In this study, the shoot dry mass decreased with the increased light intensity and the reduction in the photoperiod, while there was no significant change in shoot fresh mass. Additionally, the combined treatment of a high light intensity and short photoperiod was not conducive to thicker leaves and stronger seedlings (Figure 6b).
In Exp. 2, the chlorophyll fluorescence parameters were consistent with the growth conditions, and also demonstrated that the combination of a low light intensity and long photoperiod was more favorable for the growth of cucumber seedlings. Under the same photoperiod, as PPFD increased, Φ PSII decreased in three species (Ipomea batatas, Lactuca sativa, and Epipremnum aureum) [45]. This phenomenon was also found in lettuce and mizuna under the same DLI, which meant that most PSII reaction centers were closed at high PPFDs [31,46]. In this study, Φ PSII decreased as PPFD increased, resulting in a decline in photosynthate. NPQ and Y(NPQ) increased first and then decreased, indicating that with the increase in light intensity and the shortening of the photoperiod, the photoprotection mechanism of cucumber seedlings was activated, but the excessive light intensity destroyed the PSII and reduced the photoprotection ability of cucumber seedlings [47]. Meanwhile, Y(NO) showed that the degree of light damage was severe in T4. Due to the low light intensity requirements, these combinations (low light intensities and long light durations) can also reduce capital expenses by reducing the cost of light fixtures [18,31].
With the increased DLI, the photosynthetic pigment showed a linear declining trend, which confirmed that leaves gained more light capture ability by increasing the chlorophyll content under lower DLIs [48,49]. The relative chlorophyll content of cucumber leaves of different ages decreased upon increasing the DLI, and the apparent quantum yield decreased according to the light response curve [50]. The chlorophyll content was positively correlated with shoot biomass in Exp. 1 and negatively correlated with shoot biomass in Exp. 2, which reveal that the chlorophyll content only represents the adaptability of plants to environmental changes and had no direct relationship with the amount of photosynthate produced by plants [49].
Plant leaves adapt to light conditions not only anatomically and morphologically, but also biochemically [48]. The increase of soluble sugar content increased the plant resistance to some extent [51]. Previous research has shown that increasing DLI promotes the accumulation of soluble sugar in lettuce [40]. Higher DLIs and longer photoperiods may be more beneficial to improve the resistance of cucumber seedlings. These might be future research perspectives.

Conclusions
In conclusion, within the range of 5.41-11.26 mol·m −2 ·d −1 , the increment in DLI was conducive to a compact morphology but inhibited the shoot biomass of cucumber plug seedlings. The optimal DLI in an artificial light condition was 6.35 mol·m −2 ·d −1 , for which the leaf area and shoot biomass were relatively high, and floral initiation was accelerated in the subsequent stage. Under the condition of optimal DLI (6.35 mol·m −2 ·d −1 ), the combinations of low light intensities and long photoperiods are more conducive to the accumulation of root growth and whole plant dry matter, with a relatively high seedling index and thicker leaves. Therefore, the optimal light intensity was 110-125 µmol·m −2 ·s −1 , and the optimal photoperiod was 14-16 h for cucumber plug seedlings.