Metal halide lighting.

Phillips advance high-intensity discharge 400 W ballasts were used for full-spectrum lighting experiments, along with metal halide (400 W) lamps (Phillips Advance^®, Rosemont, IL). For growth chamber experiments, ballasts were kept outside of the growth chamber, and wiring was passed through front-mounted instrument ports to allow for lamp mounting inside of the growth chamber. Light intensity was measured using a LI-COR quantum sensor and LI-2189 light meter (LI-COR^® Biosciences, Lincoln, NE).

Light-emitting diode array construction.

Blue and red 5050 light-emitting diodes (LEDs) (5.0 × 5.0 mm) were purchased premounted on flexible metal-core printed circuit strips - 60 LED s·m⁻¹ (Torchstar Inc.^®, La Puente, CA). LED strips were cut into lengths of 40 cm and were attached side by side to sheets of cardboard (150 × 60 × 0.64 cm) using 3M^® 8805 thermally conductive tape (3M^®, Medina, OH). LED strips were wired in parallel, and were powered using a 12 V AC-DC switching power supply (LED Wholesalers^®, Hayward, CA). For experiments combining red and blue light, LED strips were wired to separate dimming units (LED Wholesalers^®, Hayward, CA) to allow for adjustment in spectral quality.

High-power, 10-W FR LEDs (LED Engin^® Inc., San Jose, CA) were mounted on aluminum sheet metal (150 × 60 × 0.16 cm) using thermally conductive tape. Four FR LEDs were wired in parallel with a 15-W 4.7-Ω wire-wound cement-filled resistor (Xicon^® Passive Components, Arlington, TX) soldered in between each negative terminal. Lenses were attached to individual LEDs (LED Engin^® High Uniformity Lighting Lens) using thermal adhesive (Arctic Silver^® Inc. Visalia, CA). Four groups of parallel wired FR LEDs (16 total LEDs) were used for each array, and each group was powered using an AC-DC switching power supply. LED arrays for all experiments were constructed in three replicates. LED arrays were mounted within Conviron E15 growth chambers set to 15 °C (Controlled Environment Ltd., Winnipeg, Canada) by hanging them underneath a pre-existing adjustable light rack. Ventilation fans, that provided a flow rate of 425 m³·h⁻¹ or greater, were placed on top of the light rack and provided a constant flow of air over the LED arrays.

Light measurement.

LED array spectral distribution and quantum flux were measured using a Stellarnet^® BLACK-Comet spectroradiometer, a CR2 cosine receptor, and spectra were analyzed using SpectraWiz^® software (Stellarnet^® Inc., Tampa, FL). Blue LEDs produced a bandwidth of 400–525 nm with a peak wavelength of 453 nm (3.38% of the spectrum). Red LEDs produced a bandwidth of 550–700 nm with a peak wavelength of 635 nm (4.61% of the spectrum). FR LEDs produced a bandwidth of 670–800 nm with a peak wavelength of 732 nm (3.13% of the spectrum).

Light intensity was controlled through LED dimming, and by adjusting the distance between the plant canopy and the LED array. When light intensity and distance were set, canopy temperature was measured using a Kestrel 3000 weather meter (Kestrel Meters, Birmingham, MI). On average, the canopy temperatures under an LED array consisting of 100% red light were 20.5 ± 2.7 °C; under 100% blue light, they were 20.6 °C ± 2.5 °C; and under dichromatic arrays (i.e., 50% red and blue light), they were 20.4 °C ± 2.5 °C (not statistically different when compared using Student’s t test; P = 0.05).

Plant materials and growth conditions.

Rough bluegrass, cv. Havana (J.R. Simplot Company, Boise, ID), plants were grown in 2.5 × 12 cm Cone-tainers™ (Stuewe and Sons, Tangent, OR) using soilless media (Farfard^® 3B Mix, Sungro^® Horticulture, Agawam, MA). However, for experiment 1, plants were grown to maturity (≈3 months old) in standard 15 × 11 cm plastic pots using a seeding rate of 1.8 kg (seed)/93 m² according to the methods of Nangle et al. (2015). Mature plants used in experiments other than experiment 1 were grown under greenhouse conditions, and were maintained as single plants (tillers removed) per Cone-tainer™ rather than a stand of multiple plants. Seedlings 7 d past germination (DPG) were also used in experiments 2–5. Light-grown seedlings were grown within a Conviron E15 growth chamber: 12-h photoperiod, 200 μmol·m⁻²·s⁻¹, and 20 °C day/night temperature. Plants were watered daily, and were not fertilized. Dark-grown seedlings were also grown under the same growth chamber conditions by placing Cone-tainers™ under four layers of black polyester cloth. Dark conditions were verified using a LI-COR quantum sensor and LI-2189 light meter. Two to three seedlings were grown per Cone-tainer™.

Pigment extractions and analysis.

Chlorophylls were extracted using N,N-dimethylformamide, analyzed, and spectrophotometrically quantified (Shimadzu UV1800 spectrophotometer, Shimadzu Corp., Colombia, MD) according to the methods of Wellburn (1994). Total anthocyanins were extracted using 1% acidified methanol (HCl). Fresh shoot tissue was weighed and extracted in 10 mL of extraction solvent for 24 h in the dark at 4 °C (Rabino and Mancinelli, 1986). Aliquots were analyzed spectrophotometrically, and relative anthocyanin content was estimated using the following equation: (Abs₅₃₀ − 0.25 Abs₆₅₇)/g fresh weight (FW) (Rabino and Mancinelli, 1986). Cyanidin-3-glucoside equivalents were determined using the pH differential method (Giusti and Wrolstad, 2001). Tissue extracts for the pH differential method were prepared as previously described (Nangle et al., 2015).

Anthocyanins were extracted and isolated for liquid chromatography–mass spectrometry (LC-MS) as previously described (Nangle et al., 2015). Aliquots of rough bluegrass extract were saponified to determine whether anthocyanins present were acylated using previously described methods (de Pascual-Teresa et al., 2002). Purple corn was used as a standard based on previously identified anthocyanins (de Pascual-Teresa et al., 2002). The following method was used for anthocyanin identification. A Shimadzu 2010EV LC-ESI MS coupled with a Shimadzu SPD-M20A photodiode array was used to identify anthocyanins. A Varian C18-A (150 mm × 4.6 mm I.D.) column was used (Varian Inc., Lake Forest, CA). A binary flow mobile phase consisting of a) 4.5% aqueous formic acid and b) 100% acetonitrile with a flow rate of 0.8 mL·min⁻¹ was used, and 20 µL of sample was injected onto the column. Anthocyanins were separated using the following gradient: 12% B 1–25 min, 35% B 25–30 min, a 35% to 12% decrease in B from 30 to 38 min, and 12% B until minute 45. Ultraviolet-vis spectral profiles, retention times, and mass spectra were compared with those previously published for anthocyanin identification (de Pascual-Teresa et al., 2002; Giusti and Wrolstad, 2001; Nangle et al., 2015).

Experimental design, setup, and statistics.

Five separate experiments were performed to determine the role of light in upregulating anthocyanin content in rough bluegrass. Experiment 1 was designed to evaluate whether high-intensity white light could increase anthocyanin content in a rough bluegrass. Six pots of mature rough bluegrass [15 × 11 cm − (1.8 kg seed/93 m²)] were constantly exposed to 1000 µmol·m⁻²·s⁻¹ of light from two 400-W metal halide lamps placed 61 cm above the plant canopy for 5 d. After treatment, leaf tissue was harvested down to the crown of the plant, and analyzed for cyanidin-3-glucoside equivalents. All pots of rough bluegrass were then transferred back to the greenhouse, and leaves were regrown to 3 cm. Rough bluegrass plants were then treated under high-light conditions again for replication over time, and to simulate a potential production system. Using these methods, experiment 1 was replicated three times in total. For a given replicate, the six rough bluegrass samples treated were extracted and analyzed separately, and the resulting cyanidin-3-glucoside equivalents were averaged for statistical analysis. A growth chamber was used for high-light treatment in this experiment, and the experiment was rotated to a new growth chamber for each replicate. Plants grown under high light were compared with untreated plants that were grown under standard greenhouse conditions.

Experiment 2 was designed to determine which wavelengths of light were able to upregulate anthocyanin production. Light- and dark-grown seedlings (7 DPG) were exposed to either 200 µmol·m⁻²·s⁻¹ blue, red, or FR LED light constantly for 5 d. Dark-grown seedlings were used as an added treatment alongside light-grown seedlings because tissues exhibiting lower quantities of chlorophyll a and b may exhibit different patterns of anthocyanin accumulation (Hughes et al., 2007). In experiment 3, an anthocyanin dose response curve was generated using a range of blue light intensities. Light-grown seedlings (7 DPG) were constantly exposed to 5 d of the following blue LED light treatments: 25, 50, 100, 150, 200, 250, 300, and 350 µmol·m⁻²·s⁻¹ of blue light. Experiment 4 was designed to evaluate coaction between blue and red light by altering spectral quality. Mature (3-month old) and light-grown seedlings (7 DPG) were treated for 5 d with 200 µmol·m⁻²·s⁻¹ of constant LED light consisting of the following blue/red spectral distributions: 0% blue (100% red), 30% blue (70% red), 50% blue (50% red), 70% blue (30% red), or 100% blue (0% red).

Experiment 5 was designed to evaluate the effects of photosynthesis on anthocyanin synthesis by altering spectral distribution, light intensity, and photosynthetic depression using the photosystem II inhibitor amicarbazone [4-amino-N-(tert-butyl)-3-isopropyl-5-oxo-4,5-dihydro-1H-1,2,4-triazole-1-carboxamide]. Amicarbazone was applied at a concentration of 2.8 mg·L⁻¹ using the commercially available product Xonerate (70% amicarbazone by weight, Arysta LifeScience^©, Cary, NC) 4 h before light treatment using an atomizing spray applicator (Dayan et al., 2009). Light- and dark-grown seedlings (7 DPG) were treated for 5 d with 170 µmol·m⁻²·s⁻¹ blue light plus 0, 10, 25, 50, or 100 µmol·m⁻²·s⁻¹ red light using dimmable LED arrays. The experiment was also performed in reverse using 170 µmol·m²·s⁻¹ red light plus 0, 10, 25, 50, or 100 µmol·m⁻²·s⁻¹ blue light. Experiments 2–5 had a total of six plant samples per treatment that were pooled for analysis.

Plants were dark adapted for 24 h before light treatments, and were watered daily. Growth chamber doors were open on average for 5 min/day, and light intensity outside of the growth chamber was ≈3 µmol·m⁻²·s⁻¹. For experiments using more than one LED array treatment, LED arrays were separated in each growth chamber using cardboard sheets covered in aluminum foil. Spectroradiometeric measurements were taken before and after the separation sheet was installed to verify that light from neighboring treatments was not being transmitted or interfering. All experiments were analyzed as randomized complete block designs with three replicates. Separate Conviron E15 growth chambers served as blocking units, and three separate chambers were used simultaneously for experiments other than experiment 1 (replicated over time). All statistics were performed using Minitab software v. 17. Data were analyzed using General Linear Model ANOVA (GLM ANOVA), and Tukey’s honest significant difference test was used for mean separation (P = 0.05).

Anthocyanin upregulation under high-intensity white light.

When exposed to constant high-intensity white light (1000 µmol·m⁻²·s⁻¹ − metal halide lighting), rough bluegrass plants significantly increased anthocyanin concentration compared with untreated plants (Table 1). In untreated plants, the low levels of anthocyanins present were localized primarily to sheath tissue, a trend similar to what has been previously observed in field-grown rough bluegrass (Hurley, 2010). Light-treated plants, however, exhibited an average 117.64-fold increase in anthocyanin content, and accumulated anthocyanins in both leaf blades and sheath tissue (Table 1). These increases were independent of the growth stage of the tissue, and were observed in plants of all ages and developmental stages within the stand. Anthocyanins were identified via LC-MS, and the presence of cyanidin-3-glucoside and cyanidin-3-malonylglucoside were confirmed based on comparison of mass fragmentation data (Table 1), retention times, and ultraviolet-vis spectral profiles (Fig. 1) to a purple corn standard containing both cyanidin-3-glucoside and cyanidin-3-malonylglucoside, as well as comparison with previously published retention times and spectral profiles for turfgrasses (de Pascual-Teresa et al., 2002; Nangle et al., 2015). In rough bluegrass tissue, cyanidin-3-glucoside accounted for 12.7% of the total peak area (ultraviolet-vis 520 nm) and cyanidin-3-malonylglucoside accounted for 89.3% of the total peak area (Table 2). To further confirm the presence of cyanidin-3-malonylglucoside, the acyl group was removed through saponification, and the corresponding release of cyanidin-3-glucoside was measured using LC-MS (Fig. 1). Comparing chromatograms of rough bluegrass, saponified rough bluegrass, and purple corn confirmed mass spectrometry results through compound retention time (Fig. 1A–C). Ultraviolet-vis spectral profiles of both cyanidin-3-glucoside and cyanidin-3-malonylglucoside from rough bluegrass and purple corn also exhibited the same spectral profiles and maxima (Fig. 1D–G).

Table 1.Anthocyanin concentration (cyanidin-3-glucoside eq.) of rough bluegrass after light treatment.

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Table 2.Liquid chromatography–mass spectrometry identification of anthocyanins accumulated in rough bluegrass after light treatment.

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Wavelength specificity.

To determine the primary wavelength(s) of light responsible for upregulating anthocyanin synthesis in rough bluegrass, dark-grown and light-grown rough bluegrass seedlings were exposed to blue, red, and FR LED light. Analysis of chlorophyll content showed that seedlings grown under dark conditions had significantly lower concentrations of chlorophyll a, whereas chlorophyll b content was not different between light- and dark-grown seedlings (Fig. 2A). These results are reflected in the chlorophyll a:b ratios calculated for light- and dark-grown seedlings. Dark-grown seedlings had a significantly lower chlorophyll a:b ratio compared with light-grown seedlings (Fig. 2B).

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When exposed to 200 µmol·m⁻²·s⁻¹ of blue, FR, and red LED light, only blue light increased anthocyanin content in light-grown seedlings (Fig. 2C). Likewise, dark-grown seedlings only showed a significant increase in anthocyanin content when exposed to blue light (Fig. 2D). For both dark-grown and light-grown seedlings, anthocyanins visually accumulated in mesocotyl, sheath, and leaf tissues. Interestingly, in dark-grown seedlings, a majority of pigmentation was concentrated in the mesocotyl region, a trend that was not observed in light-grown seedlings.

Blue light dose response.

Light-grown seedlings were treated with blue LED light ranging in intensity from 25 to 350 µmol·m⁻²·s⁻¹, and results showed that anthocyanins did not begin to accumulate in high quantities until plants were exposed to 150 µmol·m⁻²·s⁻¹ blue light (Fig. 3). We determined that the threshold blue light intensity needed to stimulate anthocyanin synthesis in rough bluegrass lies somewhere between 150 and 250 µmol·m⁻²·s⁻¹. Levels of anthocyanin observed after exposure to 150 µmol·m⁻²·s⁻¹ of blue light were not statistically different from those observed at 100 or 200 µmol·m⁻²·s⁻¹. Further, anthocyanin levels at 200 µmol·m⁻²·s⁻¹ were not statistically different from those seen at 250 µmol·m⁻²·s⁻¹. Taken together, these data indicate that anthocyanin production is stimulated at blue light intensities between 150 and 250 µmol·m⁻²·s⁻¹. Our data also indicate that the response begins to saturate above 250 µmol·m⁻²·s⁻¹ of blue light (Fig. 3).

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Coaction between blue and red light.

To determine whether red light could modulate the blue light response, light-grown seedlings were exposed to 200 µmol·m⁻²·s⁻¹ LED light treatments consisting of 0% blue (100% red), 30% blue (70% red), 50% blue (50% red), 70% blue (30% red), or 100% blue (0% red). Applications of blue and red light together increased anthocyanin synthesis compared with blue light alone (Fig. 4). Specifically, the addition of 30% or 50% red light significantly increased anthocyanin content compared with 100% blue light in light-grown seedlings (Fig. 4A). Experiments were repeated using mature rough bluegrass plants (3-month old), and the results showed a similar trend. In the case of mature plants, however, only the addition of 30% red light increased anthocyanin content compared with 100% blue light (Fig. 4B).

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Photosynthetic contribution to anthocyanin synthesis.

Dark-grown and light-grown seedlings were exposed to LED light treatments with a fixed quantity of blue light (170 µmol·m⁻²·s⁻¹) and increasing quantities of red light (0, 10, 25, 50, or 100 µmol·m⁻²·s⁻¹). A subset of rough bluegrass seedlings were also treated with the photosystem II inhibitor amicarbazone. For light-grown seedlings, anthocyanin content increased as the quantity of red light increased (Fig. 5A). The addition of 100 µmol·m⁻²·s⁻¹ of red light led to a significant increase in anthocyanin concentration compared with all other treatments, suggesting that the response may be due to an increase in photosynthesis rather than the involvement of phytochrome. When light-grown plants were treated with amicarbazone, anthocyanin synthesis saturated at 25 µmol·m⁻²·s⁻¹, supporting a potential role for red light in increasing anthocyanin content through photosynthesis (Fig. 5B).

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Results for dark-grown seedlings showed a similar trend to those observed in light-grown seedlings. Anthocyanin content increased in a linear fashion with increasing red light intensity, and the addition of 100 µmol·m⁻²·s⁻¹ led to a significant increase in anthocyanin concentration compared with 0, 10, and 25 µmol·m⁻²·s⁻¹ red light treatments (Fig. 5C). After treatment with amicarbazone, anthocyanin content saturated at 25 µmol·m⁻²·s⁻¹ and exhibited a sharp decline with increasing quantities of red light, again suggesting photosynthetic regulation (Fig. 5D).

In reverse experiments, when seedlings were exposed to a fixed amount of red light (170 µmol·m⁻²·s⁻¹) and increasing blue light (0, 10, 25, 50, 100 µmol·m⁻²·s⁻¹), light-grown seedlings only increased anthocyanin synthesis when exposed to an additional 100 µmol·m⁻²·s⁻¹ of blue light (Fig. 6A). The application of amicarbazone did not change this trend when applied, 100 µmol·m⁻²·s⁻¹ blue light increased anthocyanin production, although overall concentration was reduced (Fig. 6B). This indicates that while photosynthesis may impact the degree to which blue light stimulates anthocyanin production, blue light alone is sufficient to induce anthocyanin synthesis.

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For dark-grown seedlings, the addition of 100 µmol·m⁻²·s⁻¹ blue light led to an increase in anthocyanin content compared with all other LED treatments (Fig. 6C). Similar to other treatments, when amicarbazone was applied to dark-grown seedlings, only 100 µmol·m⁻²·s⁻¹ treatments accumulated anthocyanins in larger quantities (Fig. 6D). These results indicate that 100 µmol·m⁻²·s⁻¹ of blue light is necessary for the response to occur, and the addition of amicarbazone does not eliminate or reduce the response, as was observed in experiments with increasing red light.