Photosynthetic gas exchange
Photosynthetic parameters varied concerning different light treatments (Figure 1). In the current study, photosynthetic rate, transpiration rate, stomatal conductance and water use efficiency ranged from 0.397 – 6.23 µmol m-2 s-1, 0.587 – 3.942 mol m-2 s-1, 0.023 – 0.1833 mol m-2 s-1 and 0.101 – 4.647 µmol mol m-2 s-1, respectively. The higher photosynthetic rate was observed in L4, while the transpiration rate and stomatal conductance were higher in L8 and L2 light treatments. On the other hand, higher water use efficiency was recorded in L11 treatment.
Photosynthesis can be affected by the stomatal density, distribution, and opening status as it regulates diffusion of water vapor and the uptake of carbon dioxide in plants. Besides, many factors can influence stomatal behavior, including light, CO2 concentration, and temperature46. Some previous studies suggested that light intensity can enhance stomatal conductance in plants47–49. Simultaneously, the photosynthetic rate and stomatal conductance can be reduced under both lower and excessive light49. It has also been known that the photosynthetic rate depends on chlorophyll content, and it can be affected by any change in it50,51. Our study also gave a similar pattern of the result as under the treatments L1, L5, L6, L7, L8, and L9 plant attain lower chlorophyll content and lower photosynthetic rate. We also observe a similar pattern of results between photosynthesis and water use efficiency, and stomatal conductance and transpiration rate.
Both photosynthetic rate and water use efficiency were increased under all light treatments except L5 and L8. On the other hand, both transpiration and stomatal conductance significantly increased under all light spectra compare to natural light, except L10 and L11. Plant attained a higher photosynthetic rate and water use efficiency and lower transpiration rate and stomatal conductance under L3, L4, and L11. On the other hand, photosynthetic rate and water use efficiency and higher transpiration rate and stomatal conductance were recorded lower in L5 and L8 treatments.
Influence of LED on lipid peroxidation and hydrogen peroxide
Both malondialdehyde (MDA) and H2O2 level were considerably influenced by different light treatments (Figure 2). Higher MDA was recorded in L6, followed by L1, L5, and L8, while lower MDA was observed in L2, L3, and L4 treatments. On the other hand, plants accumulated higher H2O2 in L7, followed by L6, L5, L2, and L9, while lower H2O2 was observed in L8 and L11 treatments.
In the presence of light, chloroplasts and peroxisomes act as leading ROS producers in plants52. Thylakoids are the membrane-bound compartments inside chloroplasts that harbors the efficient light for light-dependent photosynthesis reactions by PS I and PS II53,54. Light energy at the over-saturation point is responsible for photoinhibition by reducing the light-induced photochemical activity in PS II. These negative changes in the photosynthetic electron transport system are mainly responsible for the generation of ROS12,13,55. In these connections during overexcitation of chlorophyll, 1O2 and O2−• produce from O2 in PS II (during electron transfer to O2 through QA and QB) and PS I (Mehler reaction), respectively56–58. Peroxisomes can generate H2O2 by the activities of flavin oxidase, while O2−• and H2O2 may be generated in mitochondria of the cell by reduction of O2 near the electric transport chain59–61. In the present experiment, under the light treatments, L5, L6, L7, and L9 accumulated higher H2O2 with a lower photosynthetic rate indicating an active production of ROS resulting in photoinhibition and/or overexcitation of chlorophyll. To scavenge the excess ROS produced in the electron transport system plant uses various antioxidative defense mechanisms, including enzymatic and non-enzymatic scavenging procedures, which work synergistically and interactively with each other62,63.
Lipids and proteins are the primary victims of oxidative damage by ROS accumulated in plant cells 64. Lipid peroxidation, considered as an indicator of determining the lipid damage extent, occurs in every organism by the oxidative decomposition of polyunsaturated lipids in the plasma membrane under severe condition65–67. However, constant stress for plant generates redundant ROS that cannot be entirely homeostated by the scavenging system of the cell and exert some physiological actions like lipid peroxidation, nucleic acid oxidation, protein denaturation, enzyme activity inhibition and finally lead to programmed cell death57,64,68. In the present study, under the light treatments L1, L5, L6, and L8 produced higher MDA along with lower photosynthetic rate and water use efficiency, indicating severe lipid damage in the plasma membrane of the plant cell.
Effect of LED spectra on antioxidant enzymes activities
From our study, the highest and lowest SOD activity was recorded in L7 and L5 treatments, respectively (Figure 3). However, a higher increment was observed from L7 (9.1%), followed by L11 (6.76%), L6 (6.58%), L3 (5.94%), and L9 (5.89%) respectively compared to natural light. Higher CAT was recorded in L11 followed by L5, L10, L3, L6 and L7 with 62.88%, 45.49%, 42.66%, 39.11%, 38.49% and 34.9% increment (compare to natural light), respectively. Higher APX was recorded in L6, followed by L9, L5, L3, and L11, with 81.12%, 30.77%, 27.97%, 12.59%, and 5.59% increment respectively with comparison to natural light. On the other hand, a higher reduction of APX activity was also observed in L4 (26.57%) and L8 (25.17%). However, higher activity of GPX was observed in L8, L10, L6, L11, L5, and L7 with 92.6%, 91.6%, 70.32%, 44.3%, 42.8%, and 39.4% increment, respectively compare to natural light.
ROS accumulated under stress conditions can act as signaling molecules and trigger a signal transduction pathway. It is also crucial that despite causing programmed cell death, ROS is inevitable to confer the resistance to stress. Notably, the activated response created by ROS should be rapid and decay as long as the stress disappeared63. The main antioxidant enzymes that play a vital role in detoxifying the ROS are SOD, CAT, and APX. SOD converts O2−• to O2 and H2O2, while CAT, APX, and other peroxidase convert H2O2 to H2O and O219,69. In the present experiment in L6 and L7 light treatment, both H2O2 accumulation and SOD activity was higher, indicating an active mode of stress and plant response to mitigate the ROS compound. Under the light treatment, L1, L5, L6, L8, L10, and L11 plant accumulated higher MDA indicated a secondary damage occurrence is running by lipid peroxidation in the plant cell. At the same time, higher activity of CAT in L3, L5, L6, L10, and L11, higher APX activity from L3, L5, L6 and L9, higher GPX activity from L5, L6, L8 and L10 light spectra were recorded. On the other hand, activity of SOD was found lower in L1, L5, L8 and L10 treatments. Earlier a decreased amount of SOD and increased APX activity with the increasing of MDA accumulation under drought stress was reported70. These results indicate that lipid peroxidation may be activated with the lower activity of SOD and higher activity of peroxidases. Generally, elevated oxidative stress stimulates the production of H2O2 and provokes the increase of antioxidant enzyme activities, which help minimize the negative effect of abiotic stress71. Findings from a previous study stated that a higher irradiance of far-red and red light treatment plants produces higher MDA than that of lower irradiance 20. Higher MDA from L5, L6, L8, L10, and L11 compare to other LED spectra in the present study may be due to the presence of far-red light in those spectra.
Effect of LED spectra on antioxidant activities
Total polyphenol (TPC) and total flavonoid (TFC) varied with the spectral variation (Figure 4). Higher TPC was recorded in L6, while both TFC and DPPH free radical scavenging activity (%) was recorded higher in L7 treatment. Results also showed that both TPC and TFC increased under L2, L6, L7, L8, L9, and L10 treatments compare to natural light, while DPPH free radical scavenging activity (%) increased under L2, L6, L7, L8, and L9 treatments.
Generally, the cytokinin level increased by a red light that can stimulate the synthesis of phenolics compound, where far-red helps increase the plants' antioxidant capacity72,73. A previous study of both phenolic compound and antioxidant capacity decreased under a combination of red and blue compared to monochromatic red, blue, and natural light18. The intensity of red light and its ratio with other light sources may contribute to secondary metabolites production. Further, secondary metabolites and antioxidant capacities can vary with the light intensities and ratio of monochromatic light sources74–76. In our study, both TPC and TFC decreased at ≥ 70% and increased at 50-60%, while it turned to dropped at < 40% red light sources compare to natural light. Supplementary UV radiation can increase flavanols and other secondary metabolites that act as a stress response to protect plants from radiation17,77. In our study, UV A radiation was observed prominent with a 60% red light source. Artificial blue LED and far-red light enhance secondary metabolites, and the nutritional quality of crops, including ascorbate, total phenolic compounds, total flavonoid contents, and antioxidant activity, have been reported76,78. We also found an increment of secondary metabolites with the addition of FR light, but the effect of FR light was found prominent with 50-60% red light sources. A previous study stated an increasing intensity of red to blue increased plant flavonoid, which was found best at 7:3 ratio75. Our research also produces higher flavonoids at L6, L7, L8, and L9 treatments with similar red and blue ratios.
Effect of LED spectra on THC, THCA, CBD, and CBDA
Significant (p < 0.05) variations in the Tetrahydrocannabinol (THC), Tetrahydrocannabinolic acid (THCA), Cannabidiol (CBD), and Cannabidiolic acid (CBDA) were observed under different LED spectra (Figure 5). Plant accumulated higher CBD in L4, L5, and L8 while higher THC in all light spectra compare to natural light. Notably, CBD and THC showing a positive relationship in L4, L5, and L8 spectra where both CBD and THC increased significantly. On the other hand, an opposite relationship was observed in L2, L3, L7, L9, L10, and L11 spectra where THC and CBD showed an increasing and decreasing trend, respectively. Higher CBDA was accumulated under all spectra except L7, and higher THCA was accumulated under all spectra except L10 compared to natural light. Interestingly L7 produced quite an antagonistic relationship while others produced an almost positive relationship between THCA and CBDA accumulation.
In general, CBGA produces by alkylation of two precursors olivetolic acid and geranyl pyrophosphate with the help of geranyl pyrophosphate:olivatolate geranyl transferase79,80, which further can convert to THCA by THCA synthase81,82 and CBDA by CBDA synthase83 in the oxidation process . In this connection, during oxidation of CBGA, it produces hydrogen peroxide and THCA in THCA synthase reaction84, which may play a role in the self-defense of Cannabis plants 82. Light quality may play an essential role in cannabinoid synthesis as light intensity influences cannabis yields strongly40,85. We observed both higher THCA and H2O2 accumulation in L6 and L7 spectra in the present study, but we did not find any clear relation between THC and H2O2 from this observation. THCA also showed a positive relationship with antioxidant activities and antioxidant enzymes in L6, L7, L8, and L9 treatments.
On the other hand, it showed a negative relationship with the photosynthetic rate in the above four treatments. In the present study, except L11, THC accumulation was most prominent in L2 (white), L3 (R8B2), and L4 (R7B2G1) spectra, where we can assume very little influence of FR and UV A light on THC accumulation in cannabis plants. It was also reported that Cannabis plants were grown under blue, and synergy between UV-A and blue light improved cannabinoid and cannabigerol accumulation, respectively86. We also found higher THCA, CBDA, and THC concentrations under UV-A mediated spectral combinations.
On the other hand, CBD and CBDA accumulated higher in L4, L5, L6, and L8. From these results, we can see that green light has a significant role in CBDA synthesis and its conversion to CBD. Notably, FR light also influences CBDA and CBD accumulation along with green light, where white and UV-A play a negative role in this process. In some previous studies, the role of green light was shown negative for THC accumulation 7,87, but its role in CBD and CBDA synthesis was not clear. Despite having some shreds of evidence in the previous studies45,88,89, the complex functions of cannabinoids relate to the defensive role toward biotic and abiotic stresses are not clear. Among the cannabinoids, THC and CBD were most discussed for having their antioxidant properties90. Earlier increasing of THC, THCA, CBD, and CBDA were predicted as stress indicators along with some other secondary metabolites in hemp plant under controlled drought stress89. It was also reported that THCA induces necrotic cell death in Cannabis cells and leaves91. The increasing cannabinoids in the present study also indicated a stress response of the cannabis plant under some controlled LED light spectral treatments.
Hierarchical clustering and heatmap unveiled the connections between variables and treatments
The values of all physiological and biochemical parameters of different light treatments were employed to construct the hierarchical clustering and a heatmap (Figure 6). Hierarchical clustering grouped all the variables into two clusters (cluster-A and cluster-B). Hierarchical clustering and heatmap revealed that all the parameters characterized cluster-A relate to abiotic stress, such as MDA, H2O2, SOD, CAT, APX, GPX, TPC, TFC, DPPH, and THCA. All the cluster-A variables showed minimal values at the natural light, white, R8B2, and R7B2G1, which indicated low comparative stress for hemp, whereas R6:B2:G1:FR1 and R5:B2:W2:FR1 treatments increased this trend. On the other hand, cluster-B represents all photosynthetic attributes (Pn, E, gs, and WUE) and cannabinoids like CBD, CBDA, and THC. All cluster-B variables showed maximum values at R7:B2:G1 followed by R5:B2:G1:FR1:UV1, W, and R8:B2. This result is indicating that CBD and THC have a negligible relationship with stress-producing compounds. On the other hand, CBDA has a small extent of the relationship with stress compounds as it increased a little at stress-producing light like R6:B2:G1:FR1. Interestingly, the treatment RBG exhibited minimum and maximum values of almost all cluster-A and cluster-B parameters, respectively.