The role of red and white light in optimizing growth and accumulation of plant specialized metabolites at two light intensities in medical cannabis (Cannabis sativa L.)

Growth conditions during vegetative and generative phase

At day 21 of the propagation phase, a uniform selection of 128 plants was transplanted into 15 x 15 x 15 cm stone wool blocks (Hugo Blocks; Grodan) and grown at a planting density of 16 plants m^-2 for 14 days under long days (18 h photoperiod); plants achieved a height of 30 cm. Subsequently, plants were grown for 56 days at a planting density of 9 plants m^-2 during the short-day phase (12 h photoperiod), to induce flower development.

Twenty-four hours prior to transplanting, stone wool plugs and blocks were pre-soaked in a nutrient solution (Supplementary Table S1) with electrical conductivity (EC) of 1.5 and 2.2 dS m^-1, respectively. The pH of the nutrient solution was ~5.8. Stone wool plugs were irrigated on day 14 of the propagation phase by ebb- and flow. A drip irrigation system administered the nutrient solution six and four times daily for the long-day and short-day phase, respectively at a rate of 60 mL min^-1 and a duration between two and four minutes, per stone wool block, depending on the irrigation demand for healthy plant growth. EC values of these nutrient solutions were 2.2 and 2.5 dS m^-1 for the long-day and short-day phase, respectively (Supplementary Table S1).

At day 7 of the long-day phase, four secondary branches per plant were retained, to improve crop uniformity and reduce apical dominance, by removing the apical meristem at the seventh node and removing the two lowest secondary branches. At day 10 of the short-day phase, plants were pruned to promote airflow and reduce a high relative humidity in the canopy’s microclimate by removing the bottom 20 cm of leaves and tertiary branches (Figure 2); pruned plant material was collected for inclusion in total plant dry matter production. RH was 75% and decreased to 70% on day 7 of the long-day phase to promote transpiration. For the short-day phase, relative humidity was set to 65% and subsequently decreased by 5% weekly until it reached 55% to promote transpiration and thus water uptake, and to prevent infections and infestations such as Botrytis (Botrytis cinerea) and powdery mildew (Golovinomyces ambrosiae and Podosphaera macularis). Air temperature was set to 28/24°C, 27/22°C, 26/22°C, and 25/22°C during the long-day phase, and on days 0-28, 29-42, and day 49-56 of the short-day phase, respectively. This temperature regime aimed to facilitate generative growth. [CO₂] was set to 800/400 and 1000/400 ppm (day/night) during the long-day and short-day phase, respectively.

Light treatments

The PPFD at canopy height was 600 and 1200 µmol m^-2 s^-1 (26 and 52 mol m^-2 d^-1, respectively), provided by LEDs (ams OSRAM, Munich, Germany) mounted in VYPR fixtures (Fluence, Texas, Austin, USA). Four spectra were applied at both PPFD: two low-white (7B-20G-73R/Narrow and 6B-19G-75R/2Peaks) and two high-white (15B-42G-43R/Narrow and 17B-40G-43R/Broad) spectra (Figure 3). The blue-green-red ratios of the two low-white spectra were approximately equivalent, as well as the ratios of the two high-white spectra (Table 1). The low-white spectra either contained a single peak wavelength at 660 nm (7B-20G-73R/Narrow) or dual peak wavelengths at 640 and 660 nm (6B-19G-75R/2Peaks). The high-white spectra differed in broadness of the white spectrum: narrowband spectrum (42G-43R/Narrow), featuring peak wavelengths at 450 nm and 660 nm, and broadband spectrum (17B-40G-43R/Broad), which displayed a more uniform light distribution across a wide range of wavelengths, approximately spanning 400-750 nm.

During the long-day phase, the PPFD was initially set at 400 µmol m^-2 s^-1 and gradually increased to 600 µmol m^-2 s^-1 by day 12. In the short-day phase, the PPFD was further increased to 1200 µmol m^-2 s^-1 on day 7 for half of the plots. Weekly quantum sensor measurements (MQ-610, Apogee Instruments Inc., Logan, CA, USA) were conducted across nine points per plot to ensure uniform PPFD at canopy height.

Destructive measurements

Per treatment, seven plants per plot were destructively measured at the transition from the long-day to the short-day phase (14 days after transplanting), and nine plants were measured at the end of the experiment (70 days after transplanting). Dry weights of inflorescences, leaves that had been trimmed from the inflorescences, regular leaves, and stems were quantified. Inflorescence weights were determined after trimming inflorescence leaves with an industrial trimmer (MT Tumbler 200; Master Products, Girona, Spain). Leaf area of regular leaves was determined using a LI-3100C area meter (LI-COR Inc., Lincoln, Nebraska, USA). Dry weight was determined using a ventilated oven (24h at 70°C, followed by 48h at 105°C). Inflorescence weight at 10% moisture content was calculated from the oven dry weight of the inflorescence by multiplication with 1.10. Inflorescence length and width were measured on each of the four branches per plant to calculate inflorescence volume (assuming a cylinder shape) by =ℎ**(ℎ2)2 . The inflorescence is identified as the complete inflorescence structure on a single branch (Spitzer-Rimon et al., 2019). Inflorescence density was calculated by dividing inflorescence dry weight by inflorescence volume. Light use efficiency (LUE) was determined by dividing inflorescence or total plant dry weight by the cumulative incident photosynthetically active radiation (PAR) at canopy height, across both the long-day and short-day phases (total light integral; TLI).

Leaf light absorptance, transmittance, and reflectance

Leaf light absorptance was measured in accordance to (Taylor et al., 2019), which involved the use of a dark enclosure equipped with two integrating spheres to determine leaf light transmission and reflection. Per treatment, leaf samples were collected from six randomly selected plants, with one leaf per plant, to quantify leaf light absorptance. Selected leaves were fully expanded, containing five or more leaflets, and positioned within 20 cm from the apex, ensuring full exposure to the light. The calculation of absorbed PAR (PAR_abs) involved multiplying incident PAR by leaf light absorptance.

Leaf photosynthesis measurements

Leaf photosynthesis was measured using a LI-6800 photosynthesis system (LI-COR) on six randomly selected plants per treatment (6 replicate plants per plot). Gas-exchange measurements were conducted on leaves that were selected on similar criteria as for leaf light absorptance. Data were collected during the fourth and seventh week of the short-day phase. Measurements of leaf photosynthesis light-response curves and operational photosynthesis were conducted within a seven-hour window per measurement day, starting one hour after the lights turned on. Measurements were alternated between treatments to reduce possible time-of-day effects. The infrared gas analyzers were matched between measurements on different plants. Conditions within the fluorometer cuvette were set to 27°C, 60% RH, a fan speed of 10000 rpm, a flow rate of 400 µmol s^-1, 2000 ppm [CO₂], and spectrum of 20B:80R. Following a 15-minute light acclimation period to 3000 µmol m^-2 s^-1, A was stabilized and recorded for 120-180 s, depending on the stabilization of A. Sequential PPFD were set to: 3000, 2500, 2000, 1500, 1000, 800, 600, 400, 200, 100, and 0 µmol m^-2 s^-1. A non-rectangular hyperbola was fitted to the light response curve data (Thornley, 1977), and the parameters maximum net assimilation rate at saturating light (A _max), quantum yield (α_LRC), light compensation point (LCP), and dark respiration rate (R_d) were obtained.

Measurements of operational photosynthesis (A _op) were obtained using a transparent leaf cuvette at the incident PAR at canopy height. Environmental conditions within the leaf cuvette were set equal to the climate room environment. Quantum yield of photosynthesis (α_op) was calculated as α=(+) , where R_d is the average respiration rate per plot, estimated from the light-response curve.

Gas chromatography-mass spectrometry analysis

Cannabinoid and terpenoid concentrations were quantified in inflorescences located at the apical inflorescence above the canopy, within 5 cm from the apical inflorescence. Per treatment, three pooled samples were collected, each three samples at four different times, 0, 5, 10, and 15 days before harvest (DBH). Each pooled sample consisted of three inflorescence clusters, each harvested from a randomly selected plant of a given treatment, totaling approximately 1 g per pooled sample. Bleached inflorescences, which were exclusively found at the tip of the apical inflorescences in the 6B-19G-75R/2Peaks treatment at 1200 µmol m^-2 s^-1, were individually harvested and analyzed, with each sample weighing approximately 0.4 g. Inflorescence samples were stored at -80°C until further processing. Per sample, 0.2 ± 0.01 g were measured into a glass tube, into which 2 mL of n-Hexane with 1 mg L^-1 squalene (Thermo Fisher Scientific, Waltham, Massachusetts, USA) as an internal standard was added. Extraction of PSM was performed for 10 minutes, using an ultrasonic bath without elevated temperatures (Branson 2800; Branson Ultrasonics Corporation, Danbury, CT, USA). The resulting extract was then passed through a filtration column, containing of a Pasteur’s pipet filled with glass-wool and anhydrous sodium sulphate (Biosolve B.V., Valkenswaard, the Netherlands), and collected in a 2 ml glass vial for Gas Chromatography-Mass Spectrometry (GC-MS) analysis.

The PSM analysis was conducted using an Agilent Gas Chromatography (GC) Model 7890 (Agilent Technologies, Inc., Santa Clara, CA, USA) system fitted with a 30 x 0.25 mm i.d., 0.25-µm film thickness Zebron 5MS Column (Phenomenex Inc., Torrance, CA, USA), and a Model 5972A Mass Selective (MS) Detector (Hewlett-Packard, Palo Alto, CA, USA). The GC was programmed at an initial temperature of 60°C for two minutes, increased by 5°C min⁻¹ until reaching 250°C, accelerated at 10°C min⁻¹ to 280°C, and kept at this temperature for 5 min. The temperatures of the injection port, interface, and MS source were set to 250°C, 290°C, and 180°C, respectively. Helium inlet pressure was electronically controlled to sustain a constant column flow rate of 1.0 ml min⁻¹. Ionization was conducted at a potential of 70 eV, and mass scanning ranged from 45 to 400 amu with a scan rate of 5 scans min^-1. Samples were diluted 5-fold (i.e. 0.2 ml extract combined with 0.8 ml n-hexane) and one µL of each sample was injected and analyzed in split less mode.

Identification of terpenoid and cannabinoid compounds was based on their respective GC-MS retention times, and spectral comparisons against the NIST11 Mass Spectral Library (National Institute of Standards and Technology, Gaithersburg, MD, USA), the Adams essential oil library (Sparkman, 2005) and a comprehensive in-house spectral library generated with authentic standards. For semi-quantification of compounds, areas under the curve (AUC) were computed relative to the AUC of the internal standard (Squalene) and normalized for dilution and sample weight. For each treatment, data are presented as the mean ± SEM derived from three replicates. Concentrations of THC and CBD were determined using calibration curves from authentic standards, while additional cannabinoids and terpenoids were quantified in units relative to the internal standard. Initial quantifications were based on fresh weight, which were then normalized to a 10% moisture content, which reflects the market-standard weight for saleable inflorescences, by accounting for dry matter in the inflorescences. The THC and CBD yields were determined by multiplying their respective cannabinoid concentrations by the dry weight of the inflorescence.

Plant specialized metabolites

Spectrum or PPFD did not affect total cannabinoid concentration, nor that of any specific cannabinoid (Figure 6B and Supplementary Table S2). White light with a dual red peak of 640 and 660 nm compared to white light with a single red peak at 660 nm increased total terpenoid concentrations at high PPFD (Figure 6A). Neither increasing the white fraction nor spectrum broadness, irrespective of PPFD, affected total terpenoid concentrations. Total terpenoid concentration was manifested predominantely by β-Myrcene, α-Pinene, β-Pinene, Limonene, and Germacrene D (Supplementary Table S2), and was highest 5 days before harvest (DBH) (Supplementary Figure S3B). Bleached inflorescences were exclusively found at the tip of apical inflorescences in white light with a dual red peak of 640 and 660 nm at 1200 µmol m^-2 s^-1, and not in the other treatments. Bleached inflorescences exhibited increased total cannabinoid concentrations compared to green inflorescences, primarily attributed to CBD as THC was not affected (Figure 6E). The type of inflorescence did not influence total terpenoid concentrations.

White light with dual red peaks at 640 and 660 nm increases inflorescence weight through increased plant dry matter production compared to white light with single red peak At 660 nm

White light with a dual red peak of 640 and 660 nm (6B-19G-75R/2Peaks) increased inflorescence weight (Figure 4A) and light use efficiency (LUE; Figure 4C and Supplementary Figure S2B), compared to white light with a single red peak at 660 nm (7B-20G-73R/Narrow). Similar results were obtained by (Wollaeger and Runkle, 2013) in various ornamental crops. In their study, crops were grown at 125 and 250 µmol m^-2 s^-1 PPFD with various combinations of 634 and 664 nm, making up 80% of the spectrum, with 10% blue (446 nm) and 10% green (516 nm). They observed that shoot fresh weight was higher when grown at 40% 634 and 40% 664 nm in comparison to other spectrum combinations, with leaf chlorophyll concentrations being higher under this treatment at low PPFD. Although the light treatment in our study with dual red peak (640 and 660 nm), and single red peak (660 nm) had an equivalent red fraction, the inclusion of two maximum absorption peaks at 640 and 660 nm appeared to drive photosynthesis (α_LRC and α_op) and plant dry matter production more effectively than a single maximum absorption peak at 660 nm (Figure 7C, D). This effect may be attributed to the fact that, within the red waveband, Chl b and Chl a have their maximum absorption peaks around 642 nm and 663 nm, respectively (Zhu et al., 2008; Chazaux et al., 2022). Chl b is specifically bound to light-harvesting complexes while Chl a is bound to both photosystem core- and light-harvesting complexes (Caffarri et al., 2011; Zhao et al., 2017; Pan et al., 2018). Chl b is critical in regulating the size of the light-harvesting complexes, absorbing light energy that would otherwise cause photoinhibition when directly absorbed by the photosystem core complexes (Voitsekhovskaja and Tyutereva, 2015). Distributing the light energy over both Chl b and Chl a likely allowed for more efficient light energy absorption and conversion to chemical energy, preventing photoinhibition due to excessive light energy. However, Chl a and Chl b coexist alongside both photosynthetic and non-photosynthetic pigments. This assortment of pigments influences the efficacy of various light wavelengths in driving photosynthesis (Walla et al., 2014; Smith et al., 2017).

The use of 660 nm light may trigger phytochrome activation, inhibiting flowering in short-day plants like strawberries (Takeda and Newell, 2006). This phenomenon could explain the reduced inflorescence weights observed under white light with a single red peak at 660 nm compared to white light with a dual red peak of 640 and 660 nm, likely resulting from a prolonged flower induction phase. Park and Runkle (2018) observed a similar response, where inflorescence buds appeared earlier in begonia (Begonia spp.), geranium (Pelargonium spp.), petunia (Petunia spp.), and snapdragon (Antirrhinum majus) under 100% white, or 75% white with 25% red light, compared to a combination of 15% blue and 85% red. Although we did not measure the phytochrome stationary state or the precise moment of flower induction, these factors merit consideration in future research exploring the effects of 640 and 660 nm wavelengths on inflorescence development of medical cannabis.

Larger fraction of white light improves dry matter partitioning to the inflorescences, but did not increase plant dry matter production

White fraction did not affect inflorescence weight. Increasing the white fraction in our study caused increases in both blue and green fractions and decrease in red fraction. All these changes in fraction blue, green, and red or their mutual ratios could have contributed to the observed treatment effects. Our study, along with similar research in the field, was conducted at low to average PPFD compared to conventional medical cannabis cultivation (Lumigrow, 2017; Fluence, 2020). We found that leaf-level A still increased at PPFD >1200 µmol m^-2 s^-1, suggesting the potential for further exploration at higher PPFD. While high PPFD can overexcite the photosystems and induce stress responses that give rise to destructive reactive-oxygen-species (ROS) (Demmig-Adams and Adams, 1992; Asada, 2006), increasing the white fraction at high PPFD may alleviate this stress due to a larger green fraction, which penetrates deeper in the leaf and thus distributes light more evenly among the chloroplasts, referred to as the ‘detour’ effect (Terashima et al., 2009; Brodersen and Vogelmann, 2010; Slattery et al., 2016; Smith et al., 2017). In support of this, Liu & Van Iersel (2021) observed higher quantum yields in lettuce under low-white light at 200 µmol m^-2 s^-1, and under a combination of red and green light at 1000 µmol m^-2 s^-1. Similarly, substituting up to 24% of red+blue LED light with green light increased both shoot fresh and dry weight, which was attributed to green light penetrating deeper within folded lettuce leaves (Kim et al., 2004; Bian et al., 2016).

When PPFD increases, light energy is rarely a limiting factor for plant dry matter production. Nevertheless, overexcitation of pigments can lead to the formation of ROS, potentially causing photooxidative damage to the photosystems and ultimately photoinhibition (Bassi and Dall’Osto, 2021). Up to 90% of red and blue light can be absorbed by the chloroplasts located within the upper 20% of the leaf’s profile (Nishio et al., 1993). Supplementing saturating white halogen light with monochromatic green light enhanced A in Helianthus annuus more efficiently than monochromatic red light (Terashima et al., 2009). As such, increasing the green fraction is especially relevant in crops which form dense canopies, and in crops that can be grown at very high PPFD, such as medical cannabis (Smith et al., 2017).

Decreasing the white fraction led to an increase in plant height, which is associated with an increased inflorescence weight. Increased plant height results in a more open plant structure. Such open structures have been associated with increased yields and the production of plant specialized metabolites in several plant species (Bugbee, 2016; Danziger and Bernstein, 2021b). The increase in plant height, which led to a more open plant structure, likely increased light distribution in the canopy and photon capture, thereby increasing both whole-crop photosynthesis and plant dry matter production (Takenaka, 1994; Sarlikioti et al., 2011). This factor is particularly vital for medical cannabis, a crop with a dense canopy. A larger white fraction increased dry matter allocation to the inflorescences. This finding is consistent with Magagnini et al. (2018), who reported a lower harvest index with increased red fraction. The effect was ascribed to increased dry matter partitioning to the stems, correlating with an increased plant height. This response aligns with findings by Danziger and Bernstein (2021a), who noted a similar response in plants grown under a high red fraction.

Total cannabinoid concentrations were unaffected by spectrum and PPFD. These observations contradict with those of Islam et al. (2022), who reported increased cannabinoid concentrations under spectra with an increased blue-to-red ratio at a PPFD of 300 µmol m^-2 s^-1. Furthermore, in Mentha spp., Sabzalian et al. (2014) reported that a combination of red and blue light led to increased essential oil concentrations compared to white light. Studies by Hawley et al. (2018) and Namdar et al. (2018) associated higher blue fractions with increased cannabinoid concentrations. Nevertheless, due to differing experimental conditions, including lower PPFD and shorter durations of the short-day phase, a direct comparison with our findings warrants caution. Westmoreland et al. (2021) and Wei et al. (2021) observed no significant impact of blue fraction on cannabinoid concentrations, and suggested that photoreceptor saturation at high PPFD might underlie these observations. Magagnini et al. (2018) demonstrated that the influence of spectrum on concentrations of THC, CBD, and CBG is cultivar dependent. For instance, Danziger and Bernstein (2021a) observed varying effects on the naturally occurring forms of cannabinoids—Cannabigerolic Acid (CBG), Cannabidiolic Acid (CBD), and Tetrahydrocannabinolic Acid (THC)—across three cultivars when comparing various LED spectra with high-pressure sodium (HPS) lights.

We hypothesize that low-white spectra at high PPFD could overexcite the photosystems, potentially leading to bleached inflorescences, which have been compared to photoinhibition of the leaves, potentially caused by production of reactive oxygen species. A somewhat similar response was observed by (Massa et al., 2008), where white tissue development was observed in peppers grown under 85% red and 15% blue light, specifically on inflorescence sepals. The precise mechanism behind this phenomenon is still uncertain and warrants further investigation. In our research, bleached inflorescences had higher total cannabinoid concentrations, primarily due to more CBD. This could be due to cannabinoids being proposed as potent antioxidants (Mukhopadhyay et al., 2011; Raja et al., 2020), possibly accumulating in greater amounts in tissues with higher concentrations of ROS, to maintain a balance in light-harvesting and energy utilization (Islam et al., 2022).

We would like to thank the students Luc Rademakers and Beertje Douven for their data collection efforts. We also would like to thank the followings staff members of Wageningen University and Research for their technical support; Gerrit Stunnenberg, David Brink, Jannick Verstegen, Dieke Smit, Chris van Asselt, Sean Geurts, Martijn Verweij, and Jonathan Hovenkamp. We also thank Francel Verstappen for his assistance with the GC-MS analysis. Additionally, we acknowledge the technical support on the lighting system provided by the ams OSRAM System Solution Engineering (SSE) team: Horst Varga, Marcus Hofmann, Chew Wui Chai, Mardiana Bt. Khalid.