Daily UV-B treatments and elevated CO₂ increases pigment concentrations and net photosynthesis of basil (Ocimum basilicum L.)

Since the 1950s, there has been a steady increase in ultraviolet radiation to the Earth's surface due to anthropogenic emissions and degradation of the ozone layer^[1]. Of the three types of ultraviolet radiation, ultraviolet-B (UV-B) radiation (wavelengths 280−315 nm) is the most harmful type that reaches the Earth's surface, with intensity impacted by altitude, latitude, and time of day^{[1, 2]}. In addition, the effects of UV-B light on plant photosynthesis have proven to lower net gains and efficiencies^[3]. However, these effects have been variable and highly dependent on the length of time plants are exposed to UV-B^[4].

The reduction in photosynthesis is coupled with a decrease in quantum yield efficiency^[5] and attributed to the decrease in stomatal conductance^[6] and the onset of decreased CO₂ assimilation^[5,7]. In conjunction with the decline of physiological performance, light-absorbing chlorophylls and protecting carotenoids are reduced in most plants with higher UV-B exposure^[6,8]. However, low levels of UV-B radiation can be beneficial to plants. For example, low levels of UV-B can stimulate the production of jasmonate and phenolic compounds offering protection against pathogens and insects^[2,9]. Consequently, high levels of UV-B radiation can induce plant stress by increasing the concentrations of reactive oxygen species leading to irreversible cellular and DNA damage^[10,11]. Other anthropogenic factors that have led to the increase in ultraviolet radiation have also caused the increase in atmospheric CO₂ concentrations^[12]. Elevated CO₂ concentrations have been shown to increase plant photosynthesis and growth, yet when combined with UV-B radiation, it can reduce and even negates these advantages^[13].

Al Jaouni et al.^[14] identified that basil grown under elevated CO₂ had an increased accumulation of biomass, increased plant photosynthetic rates, and elevated primary and secondary metabolite accumulation. Being a widely used culinary, medicinal, and ornamental plant, basil has exhibited variability in growth and photosynthesis when produced under different light conditions^[15]. Although used to protect the crop from light, the use of glazing materials and coverings in the greenhouse production of basil results in reduction in flavor and nutritional properties^[16,17]. Basil also responds to environmental stressors like ultraviolet radiation as these stimulate oil gland filling, improving nutritional content^[18], increasing regulatory enzymes affecting plant nutrition and growth^[17], and increasing phenolic compounds that protect the photosynthesis apparatus^[11]. These studies demonstrated that UV-B radiation is essential to maintain the nutritional content of basil, although it may have negative effects on its morphology. Other studies have analyzed variations of UV-B radiation applications on basil^[11,19,20]. However, those studies have not investigated continuous UV-B radiation applications when combined with elevated CO₂ concentrations and its effects on basil. Thus, the current study's objective is to determine the individual and interactive effects of UV-B radiation and elevated CO₂ concentration on basil photosynthetic and biochemical parameters when grown under ambient light.

In June−July 2019, an experiment to determine how UV-B and CO₂ affect basil biochemical and photosynthesis parameters was set up in four sunlit controlled environmental chambers, called soil-plant-atmosphere-research (SPAR) units. The SPAR units are located at the Rodney Foil Plant Science research facility of Mississippi State University, Mississippi State, MS, USA. Details of the SPAR unit operations are described in Reddy et al.^[21], Zhao et al.^[22], and Wijewardana et al.^[23]. Average daily solar radiation outside SPAR units ranged from 4.20 to 35.5 MJ m⁻² d⁻¹ with an average value of 25.86 ± 0.92 MJ m⁻² d⁻¹, measured with a pyranometer (Model 4-8; The Eppley Laboratory, Inc., Newport, RI, USA). The Plexiglass chambers on the SPAR units are completely opaque to UV radiation (wavelengths less than 385 nm) and transmit over 95% of incoming PAR (400−700 nm).

A 3:1 sand/soil (sandy loam; 87% sand, 2% clay, and 11% silt) was used as the soil medium for the polyvinyl-chloride pots (15.2 cm diameter by 30.5 cm height) with 500 g of gravel in the bottom of the pots. Six seeds of basil 'Genovese' (Johnny's Selected Seeds, Winslow, ME, USA) were sown in the pots and then thinned to one plant per pot approximately seven days after emergence. A two-by-two factorial arrangement was used to organize the pots with UV-B and CO₂ treatments, with each chamber having three rows of pots and 10 pots per row. Basil plants were randomly assigned to each chamber consisting of 0 kJ m⁻² d⁻¹ and 10 kJ m⁻² d⁻¹ combined with ambient (437 ppm) or elevated (725 ppm) CO₂ concentrations. UV-B radiation treatments were initiated 14-days after sowing, or 0 days of treatment (DAT).

Eight fluorescent lamps (UV-313 lamps, Q-Panel Company, Cleveland, OH, USA), with peak wavelength at 313 nm, attached to dimmable 40w ballasts were positioned 0.5 m above the plant canopy, wrapped in calcium diacetate films and changed routinely to filter UV-C radiation, were used to impose UV-B treatments. UV-B treatments occurred from 8:00 to 16:00 daily, with the interception of radiation at the canopy being measured daily with a UVX digital radiometer (UVP Inc., San Gabriel California, CA, USA) and calibrated against an Optronic Laboratories Model 754 spectroradiometer (Optronic Laboratories, Orlando, FL, USA). Non-illuminated bulbs and frames were used in chambers without UV-B radiation treatment.

Pure CO₂ from compressed gas cylinders was individually supplied to each chamber through pressure regulators, solenoids, and needle valves with calibrated flow meter^[21] and constantly measured by a dedicated infrared gas analyzers (LI-6252, LI-COR Biosciences, Lincoln, NE, USA).

Except for UV-B radiation and CO₂, all environmental growth conditions were kept the same throughout the experiment. The daytime temperature of 30 °C was initiated at sunrise, and the night time temperature of 22 °C was initiated 1 h after sunset. Plants were irrigated three times per day (7:00, 12:00, and 17:00) using an automated computer-controlled drip system with full-strength Hoagland's nutrient solution^[24] based on treatment-based evapotranspiration values detailed by McKinion & Hodges^[25], and modified as described by Reddy et al.^[21].

Gas exchange and chlorophyll fluorescence

Photosynthesis and fluorescence parameters were measured between 10:00 and 12:00 with an LI-6400XT portable photosynthesis system (LI-COR Biosciences, Lincoln, NE, USA) equipped with an integrated fluorescence chamber head (LI-6400-40, LI-COR Biosciences, Lincoln, NE, USA) at 18 DAT. The light intensity (PAR) in the measuring chamber was set to 1,500 μmol m⁻² s^−1, and the relative humidity was set to 50%. The measurement chamber temperature was kept at 30 °C. The CO₂ concentration of the leaf chamber was set to the chamber CO₂ level, with the flow rate adjusted to 500 mol s⁻¹. When the total coefficient of variation (% CV) reached < 0.5%, measurements were recorded. By considering incoming and outgoing flow rates and leaf area, the instrument provides the data for transpiration (E), stomatal conductance (g_sw), internal CO₂ concentration (C_i), and electron transport rate (ETR). The internal to external CO₂ ratio was calculated by the relationship C_i/C_a.

Chlorophyll fluorescence measurements were obtained by providing a saturating flash of light > 8,000 μmol m⁻² s⁻¹ for 0.8 s followed by a dark-flash for 6 s with far-red light pulses to drive photosystem I, draining the electrons of photosystem II. Light-adapted maximal quantum yield of photochemistry (F_v'/F_m') was calculated from chlorophyll fluorescence (F_m' – F_o')/F_m', where F_o' and F_m' are minimal and maximal fluorescence of light saturated leaves. The effective quantum yield of photosystem II photochemistry (Φ_PSII) was calculated from chlorophyll fluorescence as (F_m' − F_s)/F_m', where F_s is the steady-state fluorescence, and F_m' is the maximal fluorescence, of light saturated leaves. The effective quantum yield of gas exchange measurements ($\text{Φ}_{\rm{CO_2}} $) was calculated from fluorescence as (A − A_dark)/I∙α_leaf where A is assimilation rate, A_dark is dark assimilation rate, I is incidence of PAR, and α_leaf is leaf absorptance. Photochemical quenching (q_P) and non-photochemical quenching (q_N) were calculated from chlorophyll fluorescence where (F_m' − F_s)(F_m' − F_o') and (F_m − F_m')/(F_m − F_o'), respectively, where F_m is dark-adapted maximal fluorescence.

Pigment analysis

Plant pigments such as chlorophylls and carotenoids were extracted from freeze-dried tissues according to Kopsell et al. with modifications as described by Brazel et al. using an Agilent 1260 high-performance liquid chromatography (Agilent Technologies, Santa Clara, CA, USA)^{[26, 27]}.

Antioxidant and oxidative analysis

Malondialdehyde (MDA)

Lipid peroxidation of membranes was estimated from MDA content, a lipid peroxidation product, using the method described by Heath & Packer^[28].

Hydrogen peroxide (H₂O₂)

The content of H₂O₂ was estimated by the method of Mukherjee & Choudhuri^[29].

Superoxide dismutase (SOD)

The activity of SOD was measured following the method of Dhindsa et al.^[30].

Ascorbic acid (ASC)

The estimation of ASC was done according to the method of Mukherjee & Choudhuri^[29].

Trehalose

Trehalose concentration was estimated according to the method of Trevelyan & Harrison^[31] and the anthrone method of Brin^[32]. The enzymes associated with trehalose metabolism were assayed as per the procedures of Pramanic & Imai^[33], with few changes. Trehalose-6-phosphate synthase (TPS) activity was assayed, according to Hottiger et al.^[34], which determines the release of UDP from UDP-glucose, involving glucose-6-phosphate. Trehalose-6-phosphate phosphatase (TPP) activity was assayed according to the method of Klutts et al.^[35] by measuring the release of inorganic phosphate from trehalose-6-phosphate. Trehalose activity was determined by activating phosphorylation using cyclic adenosine monophosphate and assayed by measuring the glucose concentration^[36].

Glutathione

Reduced glutathione (GLTH) was estimated according to the method of Griffith^[37]. GLTH content was calculated from a standard graph as described by Griffith^[37] and expressed as nmol g^–1 DM.

Statistical Analysis

Statistical analysis was performed using SAS (version 9.4; SAS Institute, Cary, NC). PROC GLIMMIXED analysis of variance (ANOVA) followed by mean separation was used to analyze the data. Fixed effects for the experiment consisted of two UV-B light and two CO₂ treatments, with replications as random effects. Pooled error terms from the ANOVA table were used as the base for standard errors. UV-B and CO₂ treatment differentiation classification was determined by using Duncan's multiple range test (P ≤ 0.05) based on significant F-values for main effects. Model-based values were used to reflect statistical testing when compared to unequal standard errors from data calculations. Treatment variances were tested to be statistically equal prior to pooling.

The UV-B radiation and elevated CO₂ treatments modified plant photosynthesis and fluorescence traits of basil (Tables 1 & 2). The intercellular CO₂ concentration for plants grown at elevated CO₂ concentrations was more than 40% greater compared to ambient CO₂ concentrations. Leaf temperature of plants was influenced by UV-B radiation treatment, and CO₂ concentration, where leaf temperatures of plants treated with UV-B were at least 1 °C warmer when compared to non-treated plants. Elevated CO₂ treatments also increased leaf temperature slightly. There was a UV-B by CO₂ concentration interaction for net photosynthesis (P_NET), light adapted-maximal quantum yield of photochemistry (F_v'/F_m'), effective quantum yield of gas exchange ($\Phi_{\rm{CO_2}} $), photochemical quenching (q_P), and non-photochemical quenching (q_N). Net photosynthesis and $\Phi_{\rm{CO_2}} $ values were reduced by 22.6 and 20.5%, respectively, when treated with UV-B radiation only under ambient CO₂ concentrations, but no differences existed under elevated CO₂ concentrations. For both F_v'/F_m' and q_N, UV-B radiation treatments at ambient CO₂ concentrations did not impact values. In contrast, UV-B radiation treatments on plants at elevated CO₂ concentrations caused a reduction of 7.6% for both values compared against non-UV-B treated plants. Photochemical quenching (q_P) was reduced by UV-B radiation treatment under ambient CO₂ concentration and was increased by UV-B radiation treatment under elevated CO₂ concentration.

Pigment concentrations

A UV-B by CO₂ interaction existed for all pigment concentrations and ratios evaluated (Table 3). Both zeaxanthin and antheraxanthin exhibited a decrease in concentration when exposed to UV-B radiation only under elevated CO₂. The content of neoxanthin and β-carotene decreased when CO₂ was elevated without UV-B treatment, while UV-B treatment caused a dissimilar content increase for both CO₂ concentrations. The lutein content decreased when CO₂ concentration was elevated under no UV-B treatment, while UV-B treatment increased the content by 15.3% and 63.3% of ambient and elevated CO₂ treatments, respectively. The concentrations of violaxanthin, chlorophyll B, chlorophyll A, and total chlorophylls were elevated by exposure to UV-B radiation, with elevated CO₂ further increasing pigment concentrations only for UV-B treated plants by 31.4%, 42.2%, 43.0%, and 43.0%, respectively. The total xanthophylls content was elevated for plants treated with UV-B only under ambient CO₂ concentration, with no difference under elevated CO₂ concentration. The xanthophyll ratio decreased under ambient CO₂ when exposed to UV-B treatment, while elevating CO₂ further reduced the ratio of UV-B treatment. CO₂ did not affect the xanthophyll ratio of plants without UV-B treatment.

Antioxidant and oxidative concentrations

No differences existed for the hydrogen peroxide, trehalose, or glutathione concentrations of plant samples subjected to CO₂ concentrations and UV-B treatment (Table 4). Superoxide dismutase exhibited increased activity when subjected to elevated CO₂ regardless of UV-B treatment compared to ambient CO₂ levels. Similarly, ascorbate showed a rise in concentration under elevated CO₂, but only for plants not subjected to UV-B treatment. For malondialdehyde concentration, a UV-B by CO₂ interaction existed where UV-B treatment increased malondialdehyde concentration under ambient CO₂, but no difference existed under elevated CO₂.

DISCUSSION

The use of the SPAR units to conduct studies on multiple environmental stresses in a controlled environment is indispensable to expanding our knowledge and research capabilities. Using the SPAR units enables data collection that will help develop crop growth and development models and various physiological responses using field conditions while simultaneously collecting data in a controlled environment. The SPAR units' ability to create an environment of the projected global anthropogenic emission that is estimated to occur in Mississippi by the end of the century with 1,000 ppm CO₂ concentration and average UV-B radiation of 10 to 15 kJ m⁻² d⁻¹ allows for a unique insight into current and future environmental conditions^[22,38,39].

Gas exchange and chlorophyll fluorescence

For most plants, UV-B radiation can cause a reduction in stomatal conductance that reduces transpiration and photosynthetic processes^[40]. Our results indicate that stomatal conductance and transpiration were unaffected by UV-B radiation, which may be due to the phenolic compounds that basil naturally produces in response to UV-B radiation. The phenylpropanoid pathway and associated enzymes are activated when exposed to increased UV-B radiation. This can lead to the production of numerous antioxidants and phenylpropanoids that fill the oil glands and help to protect the plant by absorbing UV-B radiation^[18].

Another effect of UV-B radiation is the decrease in P_NET, which was observed only under ambient CO₂. The reduced P_NET under ambient CO₂ may be a result of the impairment of the photosystem II electron transport system, diminished ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity and regeneration, the damaging structure of chloroplasts, and the thylakoid membrane^[1]. However, no single process, rather a culmination of numerous minor variations under ambient CO₂, can be identified as the reason for UV-B reduction based on the non-significant results of Φ_PSII, ETR, and chlorophyll fluorescence measurements.

Elevating CO₂ resulted in greater P_NET regardless of UV-B treatment, increasing internal CO₂ (C_i) and $\Phi_{\rm{CO_2}} $ of the leaves compared to ambient CO₂. Yet, no difference existed in the C_i/C_a ratio. These results indicate that the amount of C_i for basil plants is directly correlated with ambient (environmental) CO₂ within this range. High concentrations of CO₂ are stimulatory of higher P_NET and suppression of photorespiration due to the increased substrate availability for Rubisco to prefer CO₂ over O₂^[41]. While low C_i concentrations would be inhibitory of Rubisco regeneration, high concentrations can also be inhibitory as Rubisco regeneration rate would become limited by electron transport rate, and further by triose phosphates^[41,42]. Although photorespiration is considered an overall energy wasting process, Takahashi & Badger^[43] identify that while it limits plant photosynthesis, this limitation may confer protection against damages due to reductions in ETR preventing excessive ROS generation under stressful conditions and dissipating excessive light energy.

While we observed no reductions in ETR rate, we did observe that supplemental CO₂ causes an increase in P_NET, negating some of the harmful effects of UV-B on the photosynthetic apparatus. In contrast, some harmful effects still occur, like those observed with a significant decrease in F_v'/F_m' and q_N of UV-B treated plants at elevated CO₂. Although a decline in Φ_PSII would indicate a significant decline in overall photosynthesis, we did observe the decline in F_v'/F_m' and q_N in UV-B and elevated CO₂ treatments, which is indicative of some reduction in photosynthetic performance and efficiency. Previous research by Romanatti et al.^[44] also demonstrated a sharp decline in dark-adapted quantum efficiency and q_N for eggplants (Solanum melongena) when subjected to UV-B radiation.

Pigment concentrations

Mosadegh et al.^[45] found that juvenile basil, when grown at similar UV-B levels (8.5 kJ m⁻² d⁻¹), led to no changes in chlorophyll concentrations but caused decreases in carotenoid concentrations. The reduction in carotenoid concentrations is a typical response in most plants as ultraviolet radiation indirectly affects plant pigments by either inhibiting their synthesis or impacting enzymes in their biosynthetic pathway^[40]. However, the results of other researchers are very different from the overall increase among all concentrations of individual pigments that we observed from daily UV-B treatments and may be linked with the age of plants. Previous research by Mosadegh et al.^[45] found significant effects of UV-B radiation on plant pigments after only 72 h of exposure, rather than the 28 days of treatment in the current study. Additionally, the long-term exposure of square-wave radiation may have enabled plants to become acclimated, like in the current study. Thus, when plants become acclimated, they may activate protective mechanisms, improving overall plant health and performance, which may enhance plant pigment production.

North et al.^[46] identified in Arabidopsis thaliana that ABA production directly affects the concentrations of neoxanthin and violaxanthin with little to no influence on other carotenoid pigments. This is due to both neoxanthin and violaxanthin being direct precursors needed for the synthesis of ABA, and both displayed significant increases under UV-B treatment. This increase observed in our study may result from increased demand for abscisic acid (ABA) used to confer UV-B stress protection, which was also observed and concluded as one of the defensive mechanisms against UV-B damage in Nicotiana attenuate^[47].

Antioxidant and oxidative concentrations

Malondialdehyde (MDA) is a molecule used to estimate the relative concentration of lipid peroxidation, which is created as a byproduct of reactive oxygen species (ROS) and is a sign of harmful ROS accumulation in plants signifying cellular membrane damage^[48,49]. UV-B's effects on MDA accumulation in basil have not been studied, but in cucumber UV-B treatments, increased MDA concentrations as well as marked increases in membrane leakage and reductions in seedling growth significantly reducing crop productivity^[49]. Our study only observed an extreme rise in MDA concentration under ambient CO₂, UV-B treatment combination, indicating large amounts of lipid peroxidation had occurred and is most likely the result of the UV-B application. However, the lack of elevated MDA under the elevated CO₂ and UV-B treatment combination does not necessarily indicate no UV-B damage. Morales & Munné-Bosch^[48] identify that MDA accumulation in plants does not always signify stress as it accumulates during periods of plant acclimation to new conditions, and more so is dependent on the length of its accumulation to cause damage in plant membranes.

Of the remaining antioxidant and oxidative concentrations evaluated, only superoxide dismutase and ascorbate displayed differences among treatments, both of which play a role in removing ROS protecting plants^[50]. Yet, the current study's results did not show elevated levels of ROS different between treatments. Elevated levels of these antioxidants would occur when plants are under oxidative stress and confer protection and removal of ROS. Our results show basil plants were only under slight oxidative stress when increasing CO₂ concentration, regardless of UV-B treatment; however, like MDA, these concentrations are temporal and change as plants mature and acclimate^[51].

Although UV-B radiation confers harmful effects on plants, it is essential for crops like basil, which stimulates plant protective mechanisms and pathways to fill oil glands and produce numerous other healthful compounds. Additionally, when UV-B radiation is combined with elevated CO₂, at projected levels to be reached by the end of the century due to anthropogenic emissions, it can help to mitigate most of the harmful effects and even improve plant photosynthesis. Thus, when CO₂ is combined with environmental stressors like UV-B radiation in our study, it displays the complex nature of plant responses. Furthermore, when combined with information from other sources, the current study's results from analyzing MDA conclude that data from a single point of time is insufficient to determine overall oxidative stress. From these observations, antioxidants and oxidative reactions can change temporally. Therefore, analyzing antioxidants and oxidative reactions will need to be measured multiple times throughout the plant's life cycle to accurately determine its overall oxidative stress response. Thus, while UV-B radiation is essential for basil production but can also harm the plant process, future studies analyzing antioxidant and oxidative concentrations should focus on temporal changes in UV-B radiation rather than single measurements.