Metabolic modulation of Basil ( Ocimum basilicum L.): an insight into growth, metabolomics and antioxidant activity under varying temperature and light conditions

Background Temperature and light are two critical environmental stimuli that greatly impact our agricultural indus‑ try. We explored the effects of varying temperature and light conditions on the metabolomics of basil, a plant widely used in cuisine and for medicinal purposes worldwide. Basil plants were subjected to elevated temperatures of 30 °C, 35 °C, and 40 °C, along with two light intensities (D11 and D23). Results Our results showed that the maximum shoot length (42.98 cm) was achieved at T30 and D23, the maximum leaf area (6.92 cm) at T30/D11, and the highest number of leaves (472) at T40/D11. However, at the higher tempera‑ ture of T40, the number of leaves increased, but shoot length and leaf size significantly decreased (p < 0.05). Maxim shoot biomass (81 gm) and root biomass (91.2 gm) was recorded at T35/D23 treatments. Metabolomic studies of basil revealed that rosmarinic acid (RA), and linalool and eugenol were the major phenolic and volatile organic compounds (VOCs) under different growth conditions. RA levels were higher at 30 °C, correlated with elevated shikimate levels, indicating a carbon supply through the TCA cycle. In contrast, the growth conditions of 35 °C/D11 (μmol/m 2 /d) up‑ regulated the glyoxylate cycle (fumaric acid and malic acid) as part of stomatal closure, preventing ROS accumulation and resulting in increased VOC emission. Exposure to the higher temperature of 40 °C induced higher amino acid accumulation, improving temperature stresstolerance. Conclusions This study suggests that elevated temperature and light intensity affected the overall health of basil plants by modulating underlying growth mechanisms. Furthermore, elevated temperature plays a vital role in plant defense mechanisms by mediating the production of secondary metabolites associated with plant defense responses.


Introduction
Plants are sensitive to environmental stimuli and adaptable to changes in their surroundings [1].Temperature and light are the most critical environmental cues that influence the growth and development of plants by providing seasonal information [2].The interrelated and complex effects of temperature and light trigger various plant responses.Light significantly contributes to acclimation responses to both low and high temperatures and can influence the extent of stress damage [3].Plant photoreceptors, like phytochrome and cryptochrome, crucially recognize the quality, quantity, and direction of light, as well as temperature sensing.They differentially affect phytohormone signaling based on temperature levels [1,4,5].Photoreceptors are responsible for both light quality and high temperatures, showing that photo-and thermo-morphogenesis share a core signaling pathway, indicating their interdependence [6][7][8][9][10][11].While normal conditions tend to counteract light signals in stem growth adjustment, a temporary temperature increase supports the light-induced inhibition of hypocotyl growth [12].Karayekov, Sellaro [12] demonstrated that the hightemperature response in Arabidopsis depends on the presence or absence of light, with a significant effect in response to light but not in the dark.Additionally, various developmental processes, such as seed germination, seedling de-etiolation, vegetative growth, and flowering, are induced by unfavorable conditions [6,7].These responses are considered part of the defense mechanism activated by plants to perceive environmental stimuli and minimize damage from challenging conditions [2,13].
Under unfavourable environmental conditions, such as heat stress, plants experience reduced productivity and shortened life cycles due to diminished photosynthetic efficiency and compromised photochemical reactions [14].Additionally, secondary metabolite accumulation contributes to environmental adaptation and acts as a defense mechanism against external stimuli [15].Temperature stress can also lead to the accumulation of reactive oxygen species (ROS) in plant cells, resulting in lipid peroxidation, enzyme inactivation, nucleic acid and protein degradation [16,17].To counteract these stresses, plants activate various non-enzymatic antioxidants, such as phenolics, flavonoids, carotenoids, and glucosinolates, serving as a non-enzymatic defense mechanism against both biotic and abiotic stressors [14,18].These well-documented secondary metabolites are of high value due to their utility, and efforts have been made to enhance their biosynthesis pathways and content [19].Rosmarinic acid (RA) found in Lamiaceae plants is renowned for its value in plant defense mechanisms and pharmacological activities.RA directly scavenges ROS and indirectly suppresses various disease mechanisms, including cancer, diabetes, aging, cardiovascular disease, and inflammation [20].Up-regulated genes involved in RA biosynthesis under abiotic stress, such as tyrosine aminotransferase and rosmarinic acid synthase, have been reported [21][22][23][24].In addition to RA, VOCs act as stress response metabolites.In basil, produced VOCs are essential constituents participating in antimicrobial activity [24], antioxidant capacity [25], and anti-fungal ability [26].These small molecules (less than 300 Da) derive from diverse biosynthetic pathways related to fats, lipids, amino acids, and proteins [27].They are emitted from the cuticle, stomata, or wounded tissues and serve as physiological defense signals, with biogenic VOCs being emitted more under stressed conditions [28].Stress-induced VOC emissions can interact with neighboring plants to signal stress occurrence and affect plant phenotypic responses [29].When exposed to mild stress, plants perceive it as a signal and activate various defense systems.These defense systems involve the production of antioxidant enzymes and secondary metabolites, playing critical roles in their adaptation and defense against stress [30,31].
The hypothesis of this study was based on the induction of secondary metabolite biosynthesis at elevated temperatures would differ depending on the quality of light intensity.Previous research by Walters and Currey [32] on basil demonstrated that yield was proportional to temperature up to 30 °C, but a decrease in node appearance rate and fresh weight occurred at 35 °C.However, this finding was based on experiments conducted under specific light intensity conditions, and the effects of the interaction between light intensity and temperature on basil productivity and functionality were not thoroughly explored.To investigate the impact of temperature and light intensity as mild stressors, this study examined various temperature conditions (normal, 30 °C; mild heat, 35 °C; and extreme heat, 40 °C) and daily light integral (day/night) and within a relative humidity range of 50-80%.

Plant growth condition
Germinated basil seedlings with 6-7 leaves were transplanted into a small-sized vertical farm.The farm was equipped with real-time environmental monitoring using a CO 2 sensor module (KCD-HP100, Korea Digital Co., LTD, Seoul, South Korea), a temperature and humidity sensor module (KSH-7310, Korea Digital Co., LTD, Seoul, South Korea), and a cooling and heating system to regulate the desired temperature conditions.The lighting conditions were set to a 14:10 h light/dark cycle, and the light intensity was controlled by LEDs consisting of a mixture of 11 white LEDs, 4 blue LEDs (454 nm), and 7 red LEDs (658 nm) (KLB-40-2C; KAST Engineering, Gumi, South Korea).For specific planting densities and cultivation system configurations, please refer to Fig. 1.To investigate the optimal growth conditions for basil, different daily light integral (DLI) and temperatures regimen were applied as follows.DLI was calculated using the following formula: (DLI; low, 11 μmol/m 2 /d; and high, 23 μmol/m 2 /d) on basil.

Plant cultivation
Basil (Ocimum basilicum L.) seeds were obtained from World Seed Company in Gwangju, South Korea.Seeds were sown individually in 200-cell plug trays filled with rock wool plugs manufactured by Grodan Rockwool BV in the Netherlands.To maintain the seed moisture contents, the rock wool plugs were watered for the initial 14 days, and then the electric conductivity (EC) was gradually increased.The nutrient solution used consisted of 3.16 mM NO 3− , 0.77 mM NH 4 , 0.32 mM PO 4− , 5.99 mM K, 4.00 mM Ca, 2.00 mM Mg, and 0.67 mM SO 4− .Before transplanting, the EC levels were incrementally adjusted, maintaining them at 0.5 dS/m from 13 to 19 days after sowing (DAS) and at 0.

Growth measurements
Plant growth parameters, including shoot length (cm) and leaf size (width and length in cm), as well as the number of leaves were measured weekly from five plants per treatment.At the end of the experimental period i.e. after 9 weeks of cultivation under various growth conditions, plants that had been subjected to different treatments for five weeks were harvested, and their fresh shoot and root weights (grams) were measured.

Sample preparation for HPLC-DAD analysis
The basil shoot samples were freeze-dried at minus 80 °C for one week and grounded into fine powder using an IKA ® A11 basic mill (IKA-Werke, Staufen, Germany).For extraction purpose, 500 mg of lyophilized basil powder was mixed with 40 mL of 70% ethanol.The mixture was sonicated at 60 °C in an ultrasonic water bath (UCP-10, Lab companion, Korea) for 1 h, followed by centrifugation at 3500 rpm for 15 min.The supernatant extracts were filtered through a 0.45 μm filter (Smartpor-II, PTFE syringe filter) and then evaporated using a nitrogen concentrator (Allsheng MD 200, Hangzhou Allsheng Instrument Co., Ltd., Hangzhou, China).The resulting dried crude concentrates were re-dissolved in dimethyl sulfoxide (DMSO) and filtered through a 0.2 μm filter (Whatman, PVDF syringe filter) before HPLC-DAD analysis.

Determination of total phenolics content (TPC)
TPC was determined using the Folin-Ciocalteu method with some modification [33], with gallic acid (Sigma Aldrich, St. Louis, MO) as quantification standard.
To create a calibration curve, the same reaction was conducted with gallic acid at concentrations ranging from 40 to 500 μg/mL, and the results were expressed as milligrams of gallic acid equivalent (mg GAE) per gram of dry weight (DW).In sample preparation, 10 mg/mL of the samples was mixed with 2% Na 2 CO 3 for 3 min.Afterward, 10 μL of 1 N Folin-Ciocalteu reagent was added and allowed to react for 30 min at room temperature.The absorbance of the samples at 750 nm was measured using a multi-detection microplate reader, Synergy HT (BioTek Instruments, Winooski, VT, USA).

Quantification of rosmarinic acid content (RAC)
RAC was quantified using an Agilent 1260 Infinity HPLC system (Agilent Technologies) equipped with a diode array detector (DAD).RA in basil extracts was separated using a YMC Triart C18 column

Determination of antioxidant activities
The antioxidant activities of basil extracts were determined by measuring 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and 2,2'-azino-bis-(3-ethylbenzothiazoline 6-sulphonic acid) (ABTS) radical scavenging activity.The DPPH radical scavenging activity was measured by previous method with some modifications [34].Sample extracts (10 μL) were mixed with 190 μL of DPPH reagent, and the mixture was incubated for 30 min in the dark.The absorbance was then measured at 517 nm using a multi-detection microplate reader (Synergy HT, BioTek Instruments, Winooski, VT, USA).The ABTS radical scavenging activity was determined as described previously with some modifications [35].The ABTS radical scavenging activity was determined by mixing 190 μL of ABTS + solution with 10 μL of sample extracts, also in the dark.The absorbance was measured at 734 nm after 10 min using the same microplate reader.The RC 50 value, representing the concentration of the sample required to reduce the initial radical concentration by 50%, was calculated and expressed in μg/mL.

Profiling of VOCs by HS-SPME-GC-TOF-MS
VOCs analysis was performed following an established protocol [36] using head space (HS)-solid phase microextraction (SPME) coupled with GC-TOF-MS (LECO Pegasus GC HRT, Leco Corp., St. Joseph, MI, USA).The freeze-dried basil was finely ground, and 20 mg of basil was mixed with 1 mL of a saturated 30% NaCl solution, with 2 μL of 0.2 mg/mL 3-pentanol serving as the internal standard.Sampling was done using an SPME holder equipped with a 50/30 μm fiber (DVB/CAR/PDMS, model 57,348-U, Supelco, Bellefonte, PA, USA).Prior to use, the fiber was conditioned at 250 °C for 5 min.VOCs were then extracted through the SPME fiber at 70 °C with a stirring rate of 500 rpm for 20 min.Subsequently, GC-TOF-MS analysis was performed on a capillary column (Rtx-5MS, 30 m length, 0.25 mm diameter, 0.25 μm thickness, 5% diphenyl, 95% dimethyl polysiloxane, Restek, PA, USA) under a split mode (30:1).The front inlet and transfer line temperatures were set at 240 °C and 250 °C, respectively.The mass range covered 36 to 450 m/z.The ion source temperature was maintained at 250 °C with an ionization voltage of 70 eV, and helium was used as the carrier gas at a flow rate of 3 mL/min.The oven temperature program was set as follows: 60 °C for 1 min, followed by 150 °C at a rate of 13 °C/min, and then 180 °C at a rate of 8 °C/min.Subsequently, the temperature was programmed to increase to 200 °C at a rate of 10 °C/min and then further to 245 °C at a rate of 30 °C/ min.Finally, it was held at 245 °C for 3 min.The relative quantity of VOCs was calculated based on the concentration of the internal standard.Retention indices (RI) were determined from the retention times of a series of n-alkanes (saturated C7-C40) using linear interpolation.The VOCs were identified based on RI and compared with the recorded mass spectra of each compound using a mass spectrum library search (NIST).

Profiling of primary metabolite through GC-TOF-MS
To extract and derivatize the primary metabolites, 50 mg of freeze-dried basil samples were mixed with 1 mL of a solvent mixture (chloroform/methanol/water, 1:2.5:1, v/v/v), with the addition of ribitol (0.2 mg/mL) as an internal standard [37].The mixture was sonicated at 37 °C in an ultrasonic water bath (UCP-10, Lab companion, Korea) for 30 min and then centrifuged at 13,000 rpm for 5 min.After centrifugation, 500 μL of the supernatant was dried using a nitrogen concentrator, and the dried residue was mixed with 50 μL of methoxyamine hydrochloride (40 mg/mL in pyridine) and heated at 37 °C for 90 min.Subsequently, 50 μL of N-Methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA) reagent was added for derivatization, followed by incubation at 37 °C for 30 min.Prior to analysis, the samples were filtered through a 0.2 μm PVDF syringe filter.
Analysis of primary metabolites in basil was performed using an Agilent 7890B GC coupled with a Pegasus HT TOF-MS (Leco Corporation, St. Joseph, MI, USA).Derivatized metabolites were separated on a RESTEK Rtx-5MS column (30 m × 0.25 mm I.D., 0.25 μm film thickness), and helium gas was used as the carrier gas at a constant flow rate of 1 mL/min.The oven temperature was initially set at 80 °C for 2 min, and then increased to 180 °C at a rate of 10 °C/min.It was further programmed to reach 200 °C at a rate of 2 °C/min and then increased at a rate of 12 °C/min up to 300 °C, where it was held for 2 min.The ion source and transfer line temperatures were set at 200 °C and 250 °C, respectively.The injection volume of the sample was 1 μL in split mode (30:1, v/v), and the mass scan range covered 24-500 m/z.Five biological replicates were prepared for each sample, and the relative quantity and identification of primary metabolites were determined using the same method as for VOCs.

Statistical analysis
Descriptive statistics for the studied characteristics were presented as mean ± standard deviation.ANOVA (Analysis of Variance) was performed to determine whether there was a difference between the groups in terms of considered characteristics.Post-hoc Tukey's Honestly Significant Difference (HSD) test was used to determine significant differences.Statistical significance level was considered as 5% (p < 0.05) and R Studio was used for all statistical computations.Heatmap analysis was conducted using MetaboAnalyst V5.0.Each treatment comprised five replications.

Influence of elevated temperature and light on growth attributes of Basil
In this study, the growth parameters exhibited a steady increase during the cultivation period, with differences becoming more pronounced across all parameters as the growth progressed (Table 1).Shoot length did not show significant differences until 58 days after sowing (DAS).However, at 65 DAS, a significant difference was observed (p < 0.05).Notably, under normal conditions (T30), there was a positive correlation between shoot length and the increase in daily light integral (DLI).In contrast, changes in DLI had no significant effect on shoot length under mild and extreme temperature stresses (T35 and T40).Moreover, significantly larger leaf area indicators (leaf length and width) were observed in T30 under the same DLI levels, and these measurements showed a moderate positive correlation with internode length (r = 0.4272 and 0.5008) (Fig. 2).Temperature and DLI also had an impact on production.Under T30 and T35, production increased with rising DLI.However, at extreme temperature stress (T40), high light intensity (D23) resulted in a slight decrease in yield.These observations indicated distinct morphological and yield changes resulting from long-term treatments of DLI and temperature.

Analysis of TPC and RAC under elevated temperature and light
The rosmarinic acid (RA) was identified as the major component in the basil used in this experiment, as presented in Additional file 1: Fig. S1.We observed that RAC significantly contributed to TPC, and a similar trend between RAC and TPC was evident, with a very high correlation coefficient (r) of 0.959 (p < 0.05) determined.When averaged across temperatures (Fig. 3A,  B), it was found that TPC and RAC were higher at lower temperatures, with significantly higher values observed in T30 (TPC, 62.2 mg GAE/g; RAC, 37.0 mg/g) compared to T35 (TPC, 52.8 mg GAE/g; RAC, 29.0 mg/g) and T40 (TPC, 37.6 mg GAE/g; RAC, 13.0 mg/g).Overall, the effect of DLI on TPC (D11, 62.9 mg GAE/g; D23, 61.6 mg GAE/g) and RAC (D11, 38.9 mg/g; D23, 35.1 mg/g) was insignificant under T30 (Fig. 3B, D).

Analysis of antioxidant activities
DPPH and ABTS radical assays have been developed to spectrophotometrically monitor antioxidant activities as such assays are considered effective for screening and identifying the natural bioactivity of complex mixtures.We recorded higher activity in the ABTS assay (RC50 = 65.7 ± 15.8 μg/mL) compared to the DPPH assay (RC50 = 133.2± 3.2 μg/mL) as shown in Fig. 3. Notably, temperature was found a significant factor in antioxidant activity.Based on the temperature grouping (Fig. 3C, D), the T30 treatment exhibited stronger DPPH (RC50 = 107.1 ± 19.4 μg/mL) and ABTS radical scavenging activities (RC50 = 55.2 ± 5.7 μg/ mL) compared to T35 (DPPH RC50 = 141.6 ± 29.7 μg/ Fig. 2 Heatmap plot representing the correlation matrix between variables.It was constructed by diverse variables related to growth parameters [9], phenolics and antioxidant activities [4], organic acids [20], amino acids [18], sugars [9], fatty acids [6], and VOCs [50].Significance levels were expressed using numbers for significant results (p < 0.05) and blanks for non-significant result.Positive correlations were depicted in red, while negative correlations were shown in blue, as indicated by the color scale bar located at the right of the heatmap    Under the same DLI conditions, higher activity was observed at lower temperatures, and this temperaturedependent trend became more pronounced at lower DLI levels, regardless of the experimental method.Therefore, strong correlations (p < 0.0001) were observed between phenolic contents and antioxidant activities (Fig. 2), as follows: TPC-DPPH: r = 0.6666; TPC-ABTS: r = 0.6632; RAC-DPPH: r = 0.5960; and RAC-ABTS: r = 0.5803.Considering the results categorized by temperature conditions, the highest functional value, as judged by TPC, RAC, and antioxidant activities of basil grown under T30 conditions, was clearly demonstrated.

ANOVA analysis
A two-way ANOVA analysis was performed to evaluate the effects of variables (temperature and DLI) on growth patterns, secondary metabolites (TPC, RAC, and VOCs), and antioxidant activities.While the production based on shoot weight was significantly influenced by the combination of temperature and DLI, not by single factors, a significant effect of temperature was observed in most cases (p < 0.05).In particular, a highly significant effect of temperature on TPC, RAC, and total VOCs was determined (p < 0.005).TPC and RAC showed a p value of less than 0.005 only with temperature as a factor, but total VOCs significantly changed with temperature alone and when combined with DLI.
Experiment was conducted five biological replications and data were expressed as µg/20 mg (means ± standard deviation).Same lower cases represent nonsignificant difference and different lower cases (a-d) in the same row represent statistically significant differences among the groups according to Tukey HSD Test (p < 0.05)

Profiling of primary metabolites
Based on the secondary metabolite analysis and twoway ANOVA, it was elucidated that temperature primarily regulates the secondary metabolism of basil.
We also conducted an analysis of primary metabolites in this study.The variables within the same category (such as organic acids, amino acids, sugars, and fatty acids) displayed positive correlations with one another, as indicated in Fig. 2. Furthermore, the results revealed that primary metabolites exhibited variations based on temperature, as illustrated in Fig. 5. Consequently, we averaged the results by temperature to facilitate interpretation.The total organic content and total sugar content decreased with rising temperatures, while the trend for total amino acids was the opposite.Notably, the highest total organic acids content was observed in the T30 condition (128.5 ± 21.3 μg/10 mg), which was approximately 1.17-and 1.29-times higher than the T35 (110.2 ± 16.2 μg/10 mg) and T40 (55.2 ± 16.4 μg/10 mg) conditions.The content of all observed organic acids (fumaric acid, malic acid, shikimic acid, and citric acid) related to the TCA cycle contributed significantly to the total organic acids content, with positive correlations (r = 0.7180, 0.8260, 0.8860, and 0.5643, respectively).In the T30 condition, it was observed that as DLI increased, the content of these organic acids and sugars also increased.Similarly, the total sugar content followed the same trend.T30 (90.5 ± 32.8 μg/10 mg) exhibited 1.64-and 3.64-times higher content compared to T35 (55.2 ± 16.4 μg/10 mg) and T40 (24.9 ± 3.5 μg/10 mg), respectively.Most sugar components, except for sucrose, displayed significantly positive correlations with total sugar content.In particular, glucose, which represented over 50% of the total sugar content, exhibited a very strong correlation with total sugar content (r value of 0.999).This is why the highest total sugar content was determined in T30/D23 (120.6 ± 8.0 μg/10 mg), where the glucose content was the highest (99.0 ± 6.9 μg/10 mg).Conversely, the total amino acids content exhibited a significant correlation within the total amino acids content.All detected amino acids, except for 5-oxoproline, showed positive correlations.The content of total amino acids was found to be 1.33 times higher in T40 (27.5 ± 2.2 μg/10 mg) compared to T30 (20.6 ± 2.7 μg/10 mg) and 1.32 times higher compared to T35 (20.8 ± 7.0 μg/10 mg).

Discussions
It is widely known that the preferred temperature range for most plant species typically falls within the range of 20 to 30 °C [38].However, when plants are exposed to temperatures exceeding their optimal growth range by more than 5 °C, they experience stress, which triggers a variety of metabolic defense mechanisms and leads to the accumulation of secondary metabolites [39].It's important to note that prolonged exposure to stressful conditions can lead to an acclimation process in plants.This acclimation results in increased stress tolerance, which is characterized by higher survival rates and enhanced production [3].Therefore, there have been ongoing efforts to improve both production and functionality by modulating these factors.In this study, basil plants were subjected to elevated temperatures and light intensities (D11 and D23), and their responses were comprehensively analyzed through the evaluation of various metabolites, including both primary and secondary metabolites.
Plants within the Lamiaceae family, especially those with high levels of phenolic compounds, are considered valuable sources of natural substances for healthcare [40].Basil, a member of the Lamiaceae family, contains various phenolic compounds such as caffeic acid, chicoric acid, caftaric acid, p-coumaric acid, and RA.The distribution of these phenolic compounds varies among basil species and cultivars [41,42].Among these compounds, RA has been identified as the most prevalent phenolic compound in sweet basil, as well as in other Lamiaceae species like thyme, marjoram, sage, rosemary, and lemon balm [43,44].RA content significantly influences the antioxidant properties of these plants, and a strong correlation has been found between phenolic content and antioxidant activities in various plant species [45,46].RA has also been reported to have various health-related properties, such as inducing melanogenesis, photoprotective effects, and anti-staphylococcus aureus and anti-inflammatory activities.These properties make RA valuable for industrial applications, such as in antiacne products [47,48].Therefore, enhancing the content of RA in basil is crucial to make it more valuable with higher economic returns.Researchers have conducted various experiments to enhance the content of these substances in basil, including modifying cultivation conditions like drought stress, silver treatment, heat stress, and phytohormone treatments [49][50][51][52].Basil exposed to temperature stress activates the accumulation of RA, which serves as a main component of the plant's defense mechanism [53].These findings are consistent with the previous results, where the highest TPC and RAC were observed under specific temperature conditions (T30).According to factorial ANOVA analysis, temperature alone showed a significant effect on TPC and RAC, while the combination of temperature and DLI did not have a significant effect.For the metabolomics aspect, prior research has also suggested that light intensity does not significantly affect the content of certain compounds in basil [54,55].This is in line with previous results where DLI alone or combined with temperature did not have a significant impact on TPC and RAC.Additionally, the non-significant combined effect was consistent with the previously mentioned decreased complex influence in tomatoes [56].Furthermore, the elevated temperature induced significant changes in the VOCs contents.VOCs have numerous functions, such as mediating plant-plant interactions, facilitating plant-pathogen communication, repelling insects through chemotaxis, and attracting pollinators by releasing pheromones [57].The production of VOCs in plants is a metabolic activity that varies in composition and quantity depending on the function, developmental stage, genotypes, species, site of biosynthesis, and environmental conditions [58].A similar previous conducted on the effect of drought stress severity on the biosynthesis of phenylpropanoids and essential oils in basil reported a decreased gene expression (C4H, cinnamate 4-hydroxylase) in the upstream biosynthesis pathway and an increased gene expression (EOMT, eugenol O-methyltransferase; CVOMT, chavicol O-methyltransferase) [59].Additionally, Rezaie, Abdollahi Mandoulakani [60] described that prolonged cold stress (over 48 h) did not show any difference in their expression compared to untreated plants.These previous studies highlighted the effect of certain plant VOCs that can be released in response to various abiotic and biotic stresses.For basil, two phenylpropanoids (eugenol and methyl chavicol) and two terpenoids (1,8-cineole and linalool) have been reported as the major compounds determining its flavor [61].In this study, the main VOCs emitted by basil plants were linalool (monoterpene) and eugenol (phenylpropene), and significantly higher VOCs content was observed at the specific temperature of T35 conditions.
Primary metabolism is involved in a complex defense system by interacting with secondary metabolites and participating in reactions against environmental stressors.This involvement occurs through the regulation of carbohydrates, proteins, and lipids in plants [62].Due to differences in secondary metabolism responses to light and temperature treatments, an attempt was made to confirm the mechanism differences through primary metabolite analysis.Temperature stress has been reported to alter respiration and photosynthesis in plants, resulting in a shortened life cycle and decreased plant productivity [63].It also affects the activity of enzymes involved in the carbon flux of the chloroplast stroma [64].However, it should be noted that the impact of stress can vary depending on the type, intensity, duration, and developmental stage of the plant [65].In unstressed plants, normal photosynthetic processes occur, and glucose produced serves as an energy source through glycolysis [66].However, when plants are exposed to photosynthetic stress, such as temperature stress, drought stress, or excessive light, the equilibrium between the production and scavenging of ROS is disrupted, leading to excessive ROS accumulation.Consequently, this is considered the initial stress response and can result in oxidative stress and damage to cell components [67].This primarily occurs in chloroplasts, mitochondria, and peroxisomes in plants.To prevent excess ROS accumulation, the process of photorespiration is activated, dissipating excess reducing equivalents and energy through both direct and indirect mechanisms [68].As part of these mechanisms, alternative metabolic pathways are engaged to restore cellular homeostasis.Malic acid, through the malate valve shuttle, plays a significant role in reducing excess equivalents in the chloroplasts [69,70].Moreover, under stress conditions, plants accumulate amino acids.The TCA cycle in plants becomes suppressed under stress, and the previously generated energy is redirected to accumulate amino acids, enhancing stress tolerance [71][72][73].Shikimate, a core compound in the biosynthesis of aromatic amino acids like phenylalanine, tyrosine, and tryptophan, is particularly important [74].Thus, metabolites related to the TCA cycle are crucial for interpreting the stress response and synthesizing secondary metabolites in plants, owing to their role in producing ATP and providing carbon skeletons for secondary metabolites [75].In our current study, a significant increase in shikimate production was observed in basil plants that were grown under two conditions: T30 (T30/D11: 5.1 ± 6.9 μg/10 mg; T30/D23: 10.5 ± 0.8 μg/10 mg) and RA (T30/D11: 38.9 ± 6.9 mg/g; T30/D23: 35.1 ± 5.7 mg/g).Shikimate, which is the final product of the shikimate pathway, was also found highly quantified under the T30 condition.This observation is supported by a correlation coefficient (R-value) of 0.6906.These results suggest that photosynthesis under the T30 condition was functioning effectively and generating carbon skeletons that contributed to the production of the RA precursor.The superior photosynthetic efficiency under T30 conditions compared to other conditions can also be explained through identifiable growth patterns.Photosynthesis serves as a crucial determinant of plant biomass, yield, and metabolism.It enables the capture of solar energy, subsequently converting it into chemical molecules [76].Carbohydrates produced through photosynthesis play dual roles, acting as signals and providing carbon for various plant metabolic processes and growth, including leaf development [77].As shown in Table 4, carbohydrate (glucose) content was highly observed in T30 and T35 samples (27.7-99.0µg/10 mg) but the T40 condition, which was extreme temperature stress, exhibited below 20 µg/10 mg content of glucose.Carbohydrate content provides insight into the potential impact of temperature variations on glucose production.Not only growth parameters linked to photosynthesis but also showed high in T30 conditions.In T30 conditions, both leaf length and leaf width, relative to lateral area, exhibited an expansion as light intensity decreased, and under consistent light conditions, with decreasing temperature.Furthermore, shoot length and internode length were markedly higher under T30 conditions, following a discernible trend of increase with decreasing temperature (Table 4).
The gradual reduction in leaf area and shoot length observed from T30 was considered to be a plant acclimatization strategy against temperature stress at T35 and T40.Various plant species have been documented to initiate acclimation responses during prolonged exposure to high temperatures, involving changes in morphological, physiological, and biochemical characteristics [78].These morpho-physiological alterations, including diminished leaf area and photosynthesis, subsequently have a negative effect on vegetative stress and productivity [78].In our study, the T35/D11 condition was identified as being under stress, as indicated by significantly elevated levels of various primary metabolites, including fumaric acid and malic acid.These changes can be attributed to the increased occurrence of photorespiration and the glyoxylate cycle, as documented in previous studies [69,70].It is important to note that secondary compounds, such as terpenes, can be synthesized to facilitate photorespiration, which serves as a protective mechanism for plants against oxidative stress and photodamage [79].This observation aligns with our current findings.
As the stress intensity increased, particularly under the T40 extreme temperature stress condition, plants exhibited a notable accumulation of amino acids, which can act as a means of temperature stress resistance [80,81].Amino acids, being constituents of proteins, play diverse regulatory roles in abiotic stress responses.They can serve as signaling molecules, precursors for secondary metabolites, protein chaperones, and osmotic protectants [71].Under temperature stress, plants accumulate specific amino acids, which can function as osmoprotectants to help maintain optimal balance, preserve redox homeostasis, and support metabolic processes [53,71].Aromatic amino acids, including phenylalanine, tyrosine, and tryptophan, are crucial precursors in protein synthesis and the synthesis of various natural products [82].These amino acids are synthesized using the carbon skeleton provided by the shikimate pathway [83,84].Consequently, phenylalanine and tyrosine, as products of the shikimate pathway and intermediates in the RA (rosmarinic acid) and VOCs biosynthesis pathway, were found highly quantified in both the T40 condition (both in D11 and D23).This resulted in a negative correlation between amino acids and RAC (phenylalanine, r = − 0.4073; tyrosine, r = − 0.6695) and shikimate (phenylalanine, r = − 0.4249; tyrosine, r = − 0.5928).Furthermore, glutamine and glutamic acid, which contribute to balance homeostasis in plants by exporting into the cytoplasm and converting to GABA (gamma-aminobutyric acid) [85], also exhibited a significant increase under the T40 condition.
In plants, continuous stress treatments lasting for days or weeks lead to an acclimation process [86].In this study, both the maintenance/enhancement of growth and the enhancement of functional components were achieved by inducing adaptive mechanisms in basil through longterm treatments.The responses to temperature and light intensity were interpreted based on metabolic changes.

Conclusion
In plants, continuous stress treatments lasting for days or weeks lead to an acclimation process that enhances stress tolerance, resulting in higher survival rates and increased production.Therefore, long-term cultivation conditions with different temperatures (T30, T35, and T40) and DLI (D11 and D23) were applied to basil cultivation and morphological and biochemical (primary and secondary metabolites) investigations in plants were performed to obtain a understanding.As growth progressed, the growth parameters gradually displayed differences, indicating that treatment conditions induced morphological changes in basil plants, and it was interpreted as associated with metabolites.The larger leaf area, longer shoot length, and internode length of T30 were attributed to effective photosynthesis, resulting in a higher amount of carbohydrates and these served as generating carbon skeletons for the TCA cycle.Consequently, higher organic acids, in particular shikimate, are attributed to RA precursor.Meanwhile, along with the temperature increase, reduced leaf area was observed and as the assimilation process, plants stimulated a balance in homeostasis against the accumulated reactive oxygen species (ROS).As a part of this response, plants stimulate transpiration, consequently, fumaric acid and malic acid, which are involved in the glyoxylate pathway, and emission of VOCs were increased under T35/T11.Furthermore, under the more intense heat stress conditions of T40, an extreme accumulation of amino acids was observed, suggesting an adaptation mechanism for survival.

mol
75 dS/m from 20 to 27 DAS.During the germination period, the plants were exposed to a light intensity of 200 ± 11 μmol/m 2 /s from fluorescent lamps (Philips Korea, Seoul, South Korea) on a 14:10 h light/dark cycle at temperatures of 18/25 °C DLI = Basils were cultivated under diverse conditions by varying DLI and temperatures.T means the temperature and the numbers following T indicates the temperature level.D indicates the DLI and the numbers following D represent the DLI value.

Fig. 3
Fig. 3 Effects of temperature and DLI on the TPC, RAC, and antioxidant activities.A TPC (mg GAE/g), B RAC (mg/g), C DPPH radical scavenging activity (RC50; µg/mL), and D ABTS radical scavenging activity (RC50; µg/mL) were expressed based on temperature grouping (a) and within each group (b).In box-plots, each group consisted of five biological replicates represented by individual scores, and different light conditions were indicated by color (D11, orange; D23, blue).Statistical differences between temperature groups were determined using Student's T-test (*p < 0.05; ***p < 0.005; NS, non-significant).The bar graph represents the means ± standard deviation of five biological replicates in each group, and non-significant differences are indicated by the same letters according to Tukey HSD Test (p < 0.05)

Fig. 5
Fig. 5 Heatmap analysis between cultivation conditions and metabolites.Visulization was conducted un including (A) growth parameters, (B) organic acids, (C) fatty acids, (D) amino acids, (E) sugars, (F) phenolics and antioxidants, and (G) VOCs for each sample.The data were normalized, and a color-bar positioned above the heatmap indicated the Pearson correlation coefficient, with blue representing lower content and red representing higher content.Rows represented the variables, while columns represented the samples, which were color-coded according to the respective growth conditions: red for T30/D11, orange for T30/D23, blue for T35/D11, pink for T35/D23, green for T40/D11, and yellow for T40/ D23 AbbreviationsANOVA Analysis of variance DLI Daily light integral GC-TOF-MS Gas chromatogram-time of flight-mass HPLC High performance liquid chromatogram HS Head space PPFD Photosynthetic photon flux density RA Rosmarinic acid RAC Rosmarinic acid content ROS Reactive oxygen species SPME Solid-phase microextraction TCA Tricarboxylic acid TPC Total phenolics content VOCs Volatile organic compounds

Table 1
Growth attributes of Basil cultivated under elevated temperatures and DLIEach treatment comprised five replicates and average of three for internodes.Values are the means ± standard deviation.Same lower cases represent non-significant difference and different lower cases (a-d) in the same row represent statistically significant differences among the groups according to Tukey HSD Test (p < 0.05).DAS, days after sowing

Table 2
HS-SPME-GC-TOF-MS analysis for VOCs profiling and dynamic changes of basil

Table 3
Summary table of two-way (factorial) ANOVA

Table 4
Primary metabolites of basil cultivated under different temperature and DLI

Table 4
(continued)Each group used five individuals as biological replicates and values are the means ± standard deviation (µg/10 mg).Same lower cases represent non-significant difference and different lower cases (a-d) in the same row represent statistically significant differences among the groups according to Tukey HSD Test (p < 0.05).RI, retetion index; ND, not detected