Growth, Photosynthesis, and Physiological Responses of Ornamental Plants to Complementation with Monochromic or Mixed Red-Blue LEDs for Use in Indoor Environments

Inch (Tradescantia zebrina) and spider (Chlorophytum comosum) plants were grown in a growth chamber for two months in plastic containers to evaluate the effects of different light treatments (TO Tube luminescent Dunn (TLD) lamps or control), TB (TLD lamps + blue light emitting diodes (LEDs)), TR (TLD lamps + red LEDs), and TBR (TLD lamps + blue and red LEDs) on biomass, photosynthesis, and physiological parameters. Total dry weight and water content were evaluated at the end of the experimental period. After two months, pigment concentrations and the photosynthetic rate were assessed in both species. The total soluble sugar, starch, and proline concentrations in the leaf as physiological parameters were studied at the end of the experiment. Both species had increased root, shoot, and total dry weight under blue LEDs conditions. The chlorophyll concentration showed a specific response in each species under monochromic or mixed red-blue LEDs. The highest photosynthetic rate was measured under the addition of mixed red-blue LEDs with TLD lamps. At the physiological level, each species triggered different responses with respect to total soluble sugars and the proline concentration in leaves under monochromic or mixed red-blue LEDs. Our study demonstrated that the addition of blue LEDs is advisable for the production of these ornamental foliage species.


Introduction
Foliage plant production represents an important agricultural industry worldwide with a net wholesale value of 50 billion € [1,2]. Nowadays, the use of foliage plants for removing indoor air pollutants in buildings is a fashion trend among consumers since these species are air-filters for volatile compounds such as benzene [3]. The production of indoor foliage plants with an additional capacity of pollutant removal is an added value that should be considered in ornamental horticulture. The choice of species with a well-known capacity to remove pollutants should be done following the recommendations given by Cathey and Campbell [4], who established the lighting requirements for indoor foliage plants. For instance, Chlorophytum comosum is well known to removal volatile compounds such as benzene [5], and Tradescantia sp. removes formaldehyde [6].
In ornamental indoor horticulture, the use of artificial lighting has been related to the improvement of the commercial value which depends on the visual quality characterized by different factors such as stem elongation, compactness, branching, and flowering [7]. Nowadays, light emitting diodes (LEDs) are the most frequently used lighting sources for plant growth under controlled conditions due to their durability, long lifetime, high radiant efficiency, and relatively narrow emission spectra [8,9]. Moreover, LEDs also provide options to select specific wavelengths for a targeted plant response which can be desirable for the production of indoor ornamental plants [10].
We investigated the responses of two different ornamental foliage plants to different light treatments. Tradescantia zebrina (inch plant) is an ornamental foliage plant native to eastern Mexico belonging to the family Commelinaceae. It is prized among interiorscapers for its attractive variegated foliage and tolerance to a wide range of growing conditions [11]. Chlorophytum comosum (spider plant) is an evergreen horticultural plant native to southern Africa belonging to the family Asparagaceae. It is characterized by high biomass production, easy cultivation, intense competitive ability, and wide geographic distribution [12].
There are several studies focused on the effects of mixed lights of fluorescent lamps and LEDs on horticultural crops such as radish and spinach [13], as well as ornamental plants such as marigold and salvia [14]. Nevertheless, there is little information concerning the effects of these mixtures of lights on our targeted foliage species. Therefore, in this trial, a pot experiment with inch and spider plants was established in order to determine the effects of different light treatments on their biomass, photosynthesis, and physiological parameters.

Plant Material and Pre-Cultivation
Rooted inch and spider cuttings (plants) were obtained from plants growing in a multitunnel greenhouse at Almería (lat. 36 • 49 N, long. 2 • 24 W) and were transplanted into 2.0 L polyethylene pots containing a mixture of sphagnum peat-moss, vegetable compost, and perlite (Natura Universal substrate, Projar, Valencia, Spain). The cuttings of each species were rooted under tunnel propagation for one month (from January to February) with the following microclimatic conditions: ranges of temperatures from 25 to 30 ºC and relative humidity higher than 90% monitored continuously with a data logger (HOBO model H 08-004-02, Onset Computer Corp., Bourne, MA) under a natural photoperiod (11/13 h light/dark). Both species were watered every two days during the rooting period with a 60% Steiner nutrient solution. Then, rooted cuttings (plants) were scheduled for follow-up in a growth chamber of l.5 × 2.5 × 2.5 = 9.38 m 3 divided into four compartments (2.16 m 3 ) with a black polyethylene film to prevent interference between neighboring light treatments. Each compartment was assigned to each light treatment during the entire experiment (two months). In each compartment, there were four blocks with six plants per block and species that were randomly positioned. The rooted cuttings of inch plants had an average dry weight of 1.34 g, and spider plants of 0.28 g on the day of transfer to the growth chamber. The growth chamber was set at a constant (day/night) temperature of 25 • C and a relative humidity of 55% to minimize any potentially confounding effect of a diurnal temperature difference or relative humidity on extension growth. To further minimize any edge or position effects within each treatment, the pots were separated with covers and rearranged every other day. During the experimental period, both species were watered manually every day with a Steiner Agronomy 2020, 10, 284 3 of 10 nutrient solution in order to avoid any water or nutrient limitation. The plants were grown under a 12/12 h light/dark photoperiod according to the light requirement of these species [15].

Experimental Set-Up and Light Quality Treatments
The experiment consisted of four light treatments: T O (Tube luminescent Dunn (TLD) lamps) (control treatment) (fluorescent TLD 18W (4 light Philips TCS097 × 2 lamps, Philips Lighting Spain, Madrid, Spain) with a power of 144 W), T B (TLD lamps + blue LEDs) (fluorescent TLD 18W (4 light Philips TCS097 × 2 lamps, Philips Lighting Spain, Madrid, Spain) and 4 lines of ALUM 40*25 blue LEDs × 9 W with console DN-RGB FIBER LIGHT, Modular Signs, Murcia, Spain) with a total power of 180 W, T R (TLD lamps + red LEDs) (fluorescent TLD 18W (4 light Philips TCS097 × 2 lamps, Philips Lighting Spain, Madrid, Spain) and 4 lines of ALUM 40*25 red LEDs × 9 W with console DN-RGB FIBER LIGHT, Modular Signs, Murcia, Spain) with a total power of 180 W, and T BR (TLD lamps + blue and red LEDs) (fluorescent TLD 18W (4 light Philips TCS097 × 2 lamps, Philips Lighting Spain, Madrid, Spain) and 2 lines of ALUM 40*25 red LEDs × 9 W and 2 lines of ALUM 40*25 blue LEDs × 9 W with console DN-RGB FIBER LIGHT, Modular Signs, Murcia, Spain) with a total power of 180 W. The spectral distribution scans were recorded at 400-1100 nm with 1-nm steps of the different light treatments with a calibrated spectroradiometer (LI-COR 1800, Lincoln, Ne, USA) at the canopy level. With these measurements, agronomic characterization of each light treatment was assessed following the methodology established by Baille et al. [16]. The experimental design consisted of four light treatments, four random blocks, and six plants (one plant per pot) per block giving a total of 96 plants per species plus border plants per experiment. The selection of these light treatments was done following the recommendations given by local ornamental growers.

Biomass Parameters
At the end of the experimental period, six plants per species and treatment were harvested, the substrate was gently washed from the roots, and the root surface was dried with blotting paper. The plants were divided into roots (R) and shoots (S), and the respective fresh weights (FW) were measured. Roots and shoots were then oven-dried at 60 • C until they reached a constant weight to provide the respective dry weights (DW). The fresh and dry weights of roots and shoots were used to calculate the water content (WC-g water per g fresh weight) of each organ as indicated by Ben Amor et al. [17].

Photosynthetic Parameters
To determine the concentrations of pigments (chlorophylls and carotenoids) in leaves, six plants of each species were randomly selected per treatment at harvest. Extraction of chlorophyll a and b (Chl a and Chl b) and carotenoids was performed by submerging 0.2 g of fresh leaves in methanol (10 mL) in the dark at room temperature (15 • C) for 24 h. The supernatant was removed, and the photosynthetic pigment concentrations were determined colorimetrically at their respective wavelengths using a Shimadzu ultraviolet-1201 spectrophotometer (Shimadzu Scientific Instruments, Columbia, MD): Chl a (λ = 666 nm), Chl b (λ = 653 nm), and carotenoids (λ = 470 nm) following the methodology of Wellburn [18]. Pigment concentrations were expressed as mg·g −1 FW. The leaf photosynthetic rate of each species was measured at the end of the experiment under the compartments used to assess the different light treatments using a leaf portable chamber photosynthesis meter (LC Pro, ADC Bioscientific, Hoddesdon, UK) (leaf temperature 25 • C, RH of 55%, PPFD corresponding to the photosynthetically active radiation (PAR) in each case (107.49, 113.33, 111.64, and 117.17 b for T 0 , T B , T R, and T BR ; respectively) and ambient CO 2 concentration). The values presented are the mean of 10 measurements and are expressed as µmol CO 2 ·m −2 ·s −1 .

Physiological Parameters
To determine the concentrations of total soluble sugars (TSS), starch, and proline in leaves, six plants of each species were randomly selected per treatment at harvest. The free proline, TSS, and starch were determined in an alcoholic extract (supernatant). The free proline concentration was determined by the ninhydrin reagent method. Free proline concentration was expressed as µg·g −1 FW. The total soluble sugar concentration was determined by the anthrone reagent method. The starch concentration was determined using the oven-dried residue from the total soluble sugar determination. The total soluble sugar and starch concentrations were expressed as mg glucose equivalent·g −1 FW [19].

Statistical Analysis
The experiment had a completely randomized block design, and the values obtained for each plant and each variable were considered independent replicates. The data were analyzed through one-way analysis of variance (ANOVA) and least significant difference (LSD) tests (p < 0.05) in order to assess the differences between treatments. All statistical analyses were done with Statgraphic Plus for Windows (version 5.1; Statpoint Technologies, Warrenton, VA).

Spectral Distribution
To evaluate the effectiveness of different light regimes, we used the spectral distribution highlighting the different peaks found and compared the integrated photon flux of each agronomic region of interest and its ratios. The spectral distribution of the different light treatments showed common peaks at 404, 436, 486, 544, 580, 612, and 704 nm ( Figure 1). The agronomic characterization of light treatments (Table 1) showed different trends according to the spectral region studied. The values of PAR were slightly higher under the LED-supplemented conditions. The T BR treatment had the highest values of PAR and total radiation. The T B and T BR treatments had the highest value in the blue (B) region. Similarly, T R and T BR had the highest value in the red (R) region. All treatments presented the same values of far red radiation (FR) and near infrared radiation (NIR). As far as the ratios was concerned, there were no differences in the ratio PAR:TOTAL between light treatments, i.e., all light treatments had the same light efficiency for agricultural production. The T B treatment had the highest value for the ratio B:R, and T BR had the highest PAR:NIR value. Therefore, the T BR treatment should cause less damage to the foliage due to the high temperature. The ratio B:FR had the highest values in T B and T BR , whereas the ratio R:FR had the highest values in T R and T BR . Table 1. Agronomic characterization of light treatments (T O (TLD lamps), T B (TLD lamps + blue LEDs), T R (TLD lamps + red LEDs), and T BR (TLD lamps + blue and red LEDs)).

Spectral Distribution
To evaluate the effectiveness of different light regimes, we used the spectral distribution highlighting the different peaks found and compared the integrated photon flux of each agronomic region of interest and its ratios. The spectral distribution of the different light treatments showed common peaks at 404, 436, 486, 544, 580, 612, and 704 nm (Figure 1). The agronomic characterization of light treatments (Table 1) showed different trends according to the spectral region studied. The values of PAR were slightly higher under the LED-supplemented conditions. The TBR treatment had the highest values of PAR and total radiation. The TB and TBR treatments had the highest value in the blue (B) region. Similarly, TR and TBR had the highest value in the red (R) region. All treatments presented the same values of far red radiation (FR) and near infrared radiation (NIR). As far as the ratios was concerned, there were no differences in the ratio PAR:TOTAL between light treatments, i.e., all light treatments had the same light efficiency for agricultural production. The TB treatment had the highest value for the ratio B:R, and TBR had the highest PAR:NIR value. Therefore, the TBR treatment should cause less damage to the foliage due to the high temperature. The ratio B:FR had the highest values in TB and TBR, whereas the ratio R:FR had the highest values in TR and TBR.

Biomass Parameters
Throughout the experiment, there were no mortalities or any visual damage evident on the plants in response to the different light treatments. In both species, root, stem, and total dry weights had the highest value under T B . The water content in the roots and shoots remained unchanged in both species under different light treatments (Table 2).

Photosynthetic Parameters
Chlorophyll a concentrations of inch and spider plant leaves in all treatments were higher than the respective chl b concentrations. In inch plants, the highest chl a and a+b concentration was found in plants grown under T B whereas the control treatment (T 0 ) had the highest chl b concentration. In spider plants, the highest chl a and a+b concentration was found in plants grown under the control treatment whereas growth under T BR resulted in the highest chl b concentration. No significant differences were observed in the carotenoid concentration regardless of the light treatment in both species. The photosynthetic rate had the highest value in plants of both species grown under T BR (Table 3).

Physiological Parameters
In inch plants, the highest concentration of total soluble sugars and proline in leaves was found in plants grown under T BR and T B , respectively. In spider plants, the control treatment (T 0 ) had the highest value of leaf total soluble sugars whereas the highest leaf proline concentration was found in plants grown under T BR . There were no significant differences in starch concentration at the end of the experiment in inch plants but in spider plants, the highest concentration was found under T BR (Table 4).

Discussion
The monochromic or mixed red-blue LEDs had different effects on the biomass and biochemical parameters according to the species. After 60 days of exposure, inch and spider plants had the highest root dry weight under the addition of blue LEDs. These results may be related to the fact that under blue light conditions, there is a higher generation of auxins by plants, which improves root growth as reported by Horwitz [20]. In addition, these findings indicate that blue light signals perceived aboveground directly contribute to the regulation of growth and development of roots through the internal light-conducting system from stem to roots as reported by Sun et al. [21]. Nevertheless, the results obtained in this experiment for both species were not in line with the results obtained by Randall and Lopez [22], who carried out an experiment with ornamental plants such as vinca, impatiens, and geraniums under varying proportions (%) of red:blue light (from 87:13 to 70:30), resulting in a decrease in root dry weight.
The lack of variation in the root dry weight in both species in our experiment under red light conditions was in line with the results obtained by Randall and Lopez [23], who reported that snapdragon, Madagascar periwinkle, zonal geranium, and French marigold plants did not show significant changes in root dry weight under red light conditions compared to the control treatment.
The incorporation of blue LEDs increased the shoot and total dry weights in both species compared to plants grown under only TLD lamps. Contradictory and variable results can be found in the literature concerning the effects of blue light on the biomass in different species. For instance, different researchers have reported an increase in dry weight under blue light conditions in crops such as spinach [24]. Nevertheless, other researchers have reported a reduction in dry weight under blue light in other crops such as red leaf lettuce [25] and Chrysanthemum morifolium [26]. The results obtained in our experiment suggest a strong morphogenetic effect since the addition of blue LEDs to TLD lamps in both species resulted in the production of plants that were more saleable mainly due to the increase in biomass.
In our experiment, the water content in the shoots and roots of both species was not affected by the addition of red, blue or the mixed red and blue LEDs to the control treatment. These results disagree with those reported by Almansa et al. [27], who noted that the addition of red and blue LEDs to fluorescent lamps resulted in a decrease in the water content of tomato seedlings. It is possible that if the experiment was extended for a longer period (>60 days), the water content will probably have been affected.
As far as pigment concentration was concerned, there were different trends between the species. For instance, inch plants had the highest concentrations of Chl a and Chl (a+b) under blue light conditions, whereas spider plants had the highest values of Chl a and Chl (a+b) in the control treatment without the addition of any type of LED. It is worth mentioning that a high chlorophyll concentration in plants, which causes a dark green coloration of leaves, is a desirable feature, especially in ornamental plants [28]. In the case of inch plants, the highest concentrations of Chl a and Chl (a+b) under blue LED light may be the result of the positive and synchronized influence of blue light on both nuclear and plastid genomes and could have played a vital role in the formation of chlorophyll and chloroplast development as reported by Akoyunoglou and Anni [29]. Similar results have been reported by other researchers for other crops such as grapes [30] and chrysanthemum [31]. The increase in the chlorophyll concentration under blue light conditions may be also ascribed to the enhancement of the 5-aminolevulinic acid synthesizing activity, which in turn mediates the biosynthesis of tetrapyrroles Agronomy 2020, 10, 284 8 of 10 such as chlorophylls, as reported by Kamiya et al. [32]. With respect to spider plants, the decrease in pigment concentrations in our experiment under monochromic or mixed red-blue LEDs may suggest chlorophyll degradation under changes in irradiance wavelengths as reported by Taiz and Zeiger [33].
In our experiment, the addition of blue, red or mixed red and blue LEDs to TLD lamps did not affect the carotenoid concentration compared to the plants grown under only TLD lamps, for both species. These results suggest that the carotenoid concentration did not appear to be influenced by the wavelengths in the light environment, coinciding with the results reported by Zheng and Van Labeke [7], who noted that carotenoid concentrations in cabbage tree and weeping fig did not vary under different light treatments.
The highest photosynthetic rate reported in both species in our experiment under the combination of blue and red LEDs is in line with the results reported by different researchers for other ornamental species such as Withania somnifera [34], Rosa sp. [35], and Petunia x hybrida [36] grown under a combination of red and blue lights. These results can be related to the fact that plants grown under mixed red and blue LEDs absorb the most efficient wavelengths for photosynthesis [37] since the peaks of B and R LEDs coincide with the peaks of the relative quantum efficiency curve [38].
Light quality may regulate carbohydrate metabolism of plants as well as being involved in other physiological processes [39]. In our present study, we investigated the effect of spectral quality of different LEDs on the total soluble sugar, starch, and proline concentrations in the leaves of two indoor foliage plants. In inch plants, the highest leaf total soluble sugar concentration under mixed red-blue LEDs may be ascribed to the high photosynthetic rate and the compactness of plants due to the ratios R/FR and B/R already explained agreeing with the results reported by Almansa et al. [40]. In the case of spider plants, the lower values of the leaf total soluble sugar concentration under monochromic or mixed red-blue LEDs may be attributed to an accumulation of starch [33]. With respect to starch concentration, the highest concentration reported under mixed red-blue LEDs was related to the high photosynthetic rate. These results are similar to those obtained by Heo et al. [41] in an experiment carried out with different species of bedding plants such as floss flower, Mexican marigold, and scarlet sage under different light treatments. They reported that all the species studied were sensitive to the different mixtures of radiation, and the accumulation of starch in leaves increased under blue and red-light conditions. Similar to the leaf total soluble sugar concentration, the leaf proline concentration showed a differential response in both species according to the type of LED. For example, the addition of blue LEDs to TLD lamps resulted in the highest leaf proline concentration in inch plants whereas in spider plants, the highest leaf proline concentration was found under the combination of red and blue LEDs with TLD lamps. There are few reports concerning the effects of light quality on the leaf proline concentration. The result of this experiment showed that the increase in the leaf proline concentration in inch plants under the addition of blue LEDs was in line with the results obtained by Zheng and Van Labeke [42] for chrysanthemum plants.

Conclusions
The addition of monochromic or mixed red-blue LEDs triggered different responses in each species. At the end of the experimental period, the highest root, shoot, and total dry weights were found in both species grown under the blue LEDs with TLD lamps. There were different responses at the photosynthetic and physiological levels in both species under the different light regimes. Under mixed-red blue conditions, there was a higher accumulation of carbohydrates as total soluble sugars in inch plants and as starch in spider plants. These results suggest the importance of studying the effects of different light conditions to establish the light requirements of these two foliage species in indoor environments. The addition of blue LEDs to TLD lamps improved the biomass of both species, resulting in more saleable plants to growers and gardeners.