Art_15408.indd Journal of Applied Botany and Food Quality 94, 7 - 14 (2021), DOI:10.5073/JABFQ.2021.094.002 1Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran 2Department of Biology, Garmsar Branch, Islamic Azad University, Garmsar, Iran 3Antimicrobial Resistance Research Center, Bu-Ali Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran 4Department of Microbiology and Virology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran Seed-priming with cold plasma and supplementation of nutrient solution with carbon nanotube enhanced carotenoid contents and the expression of psy and pds in Bitter melon (Momordica charantia) Fereshteh Sadat Seddighinia1, Alireza Iranbakhsh1*, Zahra Oraghi Ardebili2, Saman Soleimanpour 3, 4 (Submitted: July 29, 2020; Accepted: December 4, 2020) * Corresponding author Summary Recent studies on cold-plasma and nanotechnology in some crop species have shown a potential for application in food, medicine, and crop improvement. Here, the behaviours of Momordica charantia were evaluated, following supplementation of nutrient solution with multi-walled carbon nanotube (CNT) and seed priming with cold plasma treatments. The ultra-structural study of stems confirmed CNT uptake and symplastic transportation. CNT supplementation and seed-priming with plasma synergistically provoked a drastic in- crease in the plant’s early growth and performance. Quantitative real- time PCR analysis confirmed that the applied treatments mediated variations in transcriptions of the phytoene-synthase gene (McPSY ) and phytoene desaturase (McPDS). The McPDS and McPSY genes showed a similar expression trend in which the highest expression levels were observed in CNT50+Plasma 60 group. According to HPLC analysis, the CNT50+Plasma60 treatment was the most effec- tive way to increase concentrations of β-carotene. The applied treat- ments dependent on dose and treatment method increased zeaxanthin concentration. Similarly, CNT50+Plasma 60 and CNT100+Plasma 60 groups had significantly higher α-carotene levels than the other treatment group. Moreover, the statistical analysis confirmed the sig- nificant positive correlations between the expression of target genes and concentrations of carotenoids. Herein, a theoretical basis was gained to exploit in the food, pharmaceutical, and agricultural in- dustries. Keywords: Carbon nanotube; cold plasma; carotenoid; gene expres- sion; secondary metabolites; seed-priming Introduction Momordica charantia L., belonging to the Cucurbitaceae family and commonly known as bitter melon, is a medicinal plant that is widely grown in the tropical and subtropical parts of the world such as India, China, and Indonesia (Jia, 2017). Traditionally, bitter melon has been used in the treatment of diabetes and recently many scien- tific evidence have also verified that it is a promising antidiabetic and therapeutic substance (Joseph and Jini, 2013). Furthermore, bitter melon has some other medicinally useful properties, such as anti- viral (pongthanapisith, 2013), antibacterial (Costa, 2010), anti- sepsis, anticancer (poolperm and Jiraungkoorskul, 2017), anti- hepatotoxic (aparna upadhyay, 2015), antiulcer and antioxidant (gill, 2012). M. charantia contains several different types of pri- mary and secondary metabolites in the whole plant such as phenolic, triterpene, flavonoid, and carotenoid compounds, including alpha and beta-carotene, lycopene, and zeaxanthin (hyun young, 2013; Jia, 2017). Among them, carotenoids have significant roles in many physiological processes in this plant and may play an important role in the prevention of some diseases (Cuong, 2017; ishida, 2004; lee, 2017). For example, carotenoids act as the precursors in the synthesis of abscisic acid, which is mainly involved in the plant development and stress regulations. In addition, they can prevent damaging photo- oxidative processes (photo-inhibition phenomenon) by absorption of light in photosynthetic membranes. Moreover, the associated color in flowers and fruits attracts both pollinators and seed dispersal agents (lee, 2017; tuan, 2011). In humans, the carotenoid derivatives have several medical applications, especially in controlling the chronic and vascular diseases (Fiedor, 2014). They are used as a precursor in vitamin A biosynthesis that is essential to prevent blindness and xerophtalmia (tuan, 2011). In addition, carotenoids can significantly reduce the risk of cataracts, photosensitivity disease, lung cancer, prostate cancer and cardiovascular disease. Due to the inability of humans to biosynthesize essential carotenoids, it is necessary to be obtained through the food sources. Therefore, manipulating the meta- bolic pathway of carotenoids to enhance their synthesis can play an important role in increasing the quality and quantity of the product (Cuong, 2017; tuan, 2011). In the carotenoid biosynthetic pathway, phytoene synthase (PSY) and phytoenedesaturase (PDS) are two key enzymes with a close relationship between the associated expression and the level of carotenoids (Fig. 1) (Cuong, 2017; tuan, 2011). Recently non-thermal (cold) plasma technology has been considered as an innovative eco-friendly safe standalone technology, which could be scaled up and exploited in diverse agricultural-related ac- tivities (Bourke, 2018; iranBakhsh, 2017; iranBakhsh, 2018). During the production of plasma phenomenon, not only UV photons are emitted, but also varieties of active nitrogen and oxygen species, like nitric oxide are generated, which all may individually or simul- taneously act as an efficient epigenetic agent to activate key crucial signaling routes, contributed to modulation of plant growth, physio- logy, productivity, and protection (Bourke, 2018; iranBakhsh, 2017; iranBakhsh, 2018). Also, seed pre-treatment with cold plasma as modern eco-agricultural technology could enhance seed germina- tion, seedling growth, however, limited studies are exploring the as- sociated effect on the gene expression (Bourke, 2018; iranBakhsh, 2017; iranBakhsh, 2018). To gain insight into diverse nanoproducts, more convincing studies are required to figure out their advantage or toxicity, and elucidate the contributed mechanisms, for further exploitation in the related indus- tries (raJaee BehBahani, 2020; sotoodehnia-korani et al., 2020). The different industrial consumptions of carbon nanotubes (CNTs), including single-wall (SWNTs) and multi-wall CNTs (MWNTs) are rapidly increasing (Cano, 2016). Based on the recent evidence, CNTs are suitable candidates to achieve various agricultural and biotech- nological aims, especially as pesticides, nano-encapsulated products, 8 F.S. Seddighinia, A. Iranbakhsh, Z. Oraghi Ardebili, S. Soleimanpour and fertilizers (husen, 2014; kah, 2015; liu, 2015). Studies showed that the application of CNTs influenced the growth-related traits in Triticum aestivum (Joshi, 2018), Zea mays (yan, 2013), and Oryza sativa (yan, 2016). The functionalized carbon nanotubes have been demonstrated to increase the water-retaining capacity, biomass, and fruit yield in plants, which is a significant achievement of nano- technology in recent years (husen, 2014). However, there is limited evidence, especially at the molecular level and further studies are required. On the other hand, some recent studies on carbon nano- tube (CNT) or cold plasma in several crops have shown evidence for increased germination, seedling growth, and physiological activi- ties, including photosynthetic activity, enhancement of the level of key enzymes in the metabolic pathways. These also make positive changes in the gene expression that are essential for cell division and plant development, indicating their potential use in crop improve- ment [Lakshman K. Randeniya, 2015 #33;Ling, 2014 #34;Kole, 2013 #36;Husen, 2014 #19;Khodakovskaya, 2013 #38(haghighi m, 2014). In this study, early growth and the relationship between carotenoid accumulation and the expression of McPSY and McPDS, were inves- tigated, using two treatments of cold plasma and carbon nanotube. Furthermore, the simultaneous effect of both actions was examined in M. Charantia. Materials and methods Nanomaterial and seeds The uniform seeds of M. charantia, PALEE F1, were obtained from the East-West Seed International LTD, Thailand. Laboratory and greenhouse experiments were arranged in a completely randomized design with three replications. In this study, MWCNT was purchased from US research nanomaterials, Inc (3302 Twig Leaf Lane Houston, TX 77084, USA) (Tab. 1). Plasma experimental apparatus The applied experimental apparatus in this study was DBD (Model: PS200, Nik Fanavaran Plasma Co., Iran). The details and schematic of the applied plasma producing device, and the plasma diagnostic data were represented in our previously published papers (Fig. 2A and 2B) (BaBaJani, 2018; iranBakhsh, 2017; iranBakhsh, 2018). Plasma at atmospheric pressure is generated between two glass plates as dielectric barriers (the gap between dielectrics: 4 mm) covering the two powered circular plate copper electrodes (radius = 5.5 cm). Argon was utilized as a functional gas between dielectrics (flow rate of 2 liters per min (l·min-1)). The dielectric acts as a stabilizing mate- rial when the potential across the gap reaches the breakdown vol- tage leading to the formation of a large number of micro-discharges. Moreover, a modified AC high voltage power supply (mp516, Nik Plasma Tech., Iran) was utilized (Fig. 2A). The voltage was quanti- fied by a high voltage probe (Pintek HVP40) linked to an oscillo- scope (Tektronix TDS1012B). Furthermore, the frequency and the voltage of the apparatus were respectively fixed at 13 kHz and 10 kV. The instrument power was 80 W, so for 94.98 cm2 plasma treatment areas, the surface power density was equal to 0.84 W/cm2. Besides, to provide plasma diagnostics data, the optical emission spectro- scopy was carried out (the peaks were compared to the data of the NIST Atomic Spectra Database). The temperature was estimated to be between 27-29 °C. The peaks detected in 320-400 nm refer to the presence of UV, OH, and nitrogen related species, whereas the peaks between 700 and 1000 nm related to Ar (Fig. 2C). Fig. 1: Carotenoid biosynthesis pathway in plants. The activity of phytoene synthase (PSY) and phytoene desaturase (PDS) in the biosynthesis of carotenoids. Blue colour denotes the carotenoids measured in this study by HPLC analysis and red colour indicates enzymatic activi- ties for which gene expression was monitored via real time-PCR. GGDP, geranylgeranyl diphosphate. Tab. 1: Physical properties of the MWCNT-COOH. MWCNT-COOH Young’s modulus (GPa) 1200 Tensile strength (GPa) 150 Density (g/cm3) 2.6 Thermal conductivity (W/m.k) 3000 Electrone conductivity (S/m) 105 - 107 Fig. 2: The plasma experimental apparatus: (A) A schematic design of DBD. (B) The plasma experimental apparatus DBD (Model: PS200, Nik Fanavaran Plasma Co., Iran). (C) Optical emission spectro- scopy-based spectrum to illustrate plasma diagnostic data. Treatment of seeds using CNT and cold plasma and greenhouse experiment Uniform and healthy seeds of M. charantia were surface-sterilized by submerging in a 1% (v/v) solution of commercial sodium hypo- chlorite for 15 min and rinsed twice with sterile distilled water. Some seeds were soaked in distilled water and the other in MWCNT solu- tions (50, 100, and 200 mg L-1) for 48 h. After that, the soaked seeds Seed priming of bitter melon by cold plasma 9 were treated with cold plasma (above described DBD; surface power density of 0.84 W/cm2) at three different exposure times, including 0 s (control), 60 s, and 120 s. In this experiment, six seeds in three independent replications (three pots; two seeds per pot) were con- sidered for each treatment group. In detail, treatment descriptions are presented in Tab. 2. Then, the plasma or CNT-primed seeds and the seeds treated by both plasma and MWCNTs were planted in pots filled with a cocopeat/perlite (1:1, v/v) near the substrate surface (1-2 cm deep). Each treatment was replicated three times. The plants were grown under uniform conditions of temperature (25 ± 2 °C/15 ± 2 °C day/night), relative humidity (60%), and photoperiod (16/8 h light/dark). All pots were irrigated twice a week with Hoagland nutrient solution containing different concentrations of MWCNT (0, 50, 100, and 200 mg L-1) and distilled water in intervals (Tab. 2). Tab. 2: Descriptions of treatment groups. Group name Number Treatment description Control 12 Seeds MWCNT 0 mg L-1 + Plasma 0s CNT50 6 Seeds MWCNT 50 mg L-1 CNT100 6 Seeds MWCNT 100 mg L-1 CNT200 6 Seeds MWCNT 200 mg L-1 Plasma60 6 Seeds Plasma 60 s Plasma120 6 Seeds Plasma 120 s CNT50+Plasma60 6 Seeds MWCNT 50 mg L-1 + Plasma 60s CNT100+Plasma60 6 Seeds MWCNT 100 mg L-1 + Plasma 60s CNT200+Plasma60 6 Seeds MWCNT 200 mg L-1 + Plasma 60s CNT50+Plasma120 6 Seeds MWCNT 50 mg L-1 + Plasma 120s CNT100+Plasma120 6 Seeds MWCNT 100 mg L-1 + Plasma 120s CNT120+Plasma120 6 Seeds MWCNT 200 mg L-1 + Plasma 120s Field emission scanning electron microscopy (FESEM) Field emission scanning electron microscopy (FESEM; model: TESCAN MIRA3-FEG, Czech. Republic) was conducted using gold-plated material to investigate tracing uptake and accumula- tion of the nanoparticles on the 21-day old samples. Cross-sections of stems were prepared. The samples were dehydrated and fixed us- ing a freeze dryer. Then, the prepared samples were gold-coated. A portable Microscope Camera 400x USB (Dino Lite AMH-RUT, Shenzhen, China; portable, handheld) was used to observe the effects of CNTs on imbibition. Isolation of RNA and cDNA synthesis Total RNA from the leaves of Bitter melon were extracted and pu- rified by using the RNeasy Plant Mini Kit (QIAGEN, USA). After extraction, complementary DNA (cDNA) was synthesized from 1 μg of total RNA using QuantiTect Reverse Transcription Kit (QIAGEN, USA) according to the manufacturers’ instructions. Sequence analysis Using sequence data from the sequencing of cDNA libraries obtained from bitter melon seedlings. The genes that demonstrated maximum identity and similarity were selected for further study. Quantitative Real-time PCR analysis of McPSY and McPDS To design primers for quantitative real-time PCR (qRT-PCR), the Bicon designer and Primer 3 program (http://frodo.wi.mit.edu/ primer3) was used based on published gene sequences of the M. charantia PSY (GenBank Accession Number: AY494789) and PDS (GenBank Accession Number: AY494790) cDNA sequences. The M. charantia 18S ribosomal RNA gene (GenBank Accession Number AY900000.1), as a housekeeping gene, was used as an internal re- ference. The sequences of primers for these genes are presented in Tab. 3. The level of each gene expression was showed with relative expression, which is the copy number of each gene compared to that of a housekeeping gene. The qRT-PCR was conducted in 0.5 μM of each set of primers in 20 μl final reaction volume of SYBR Green Real-time PCR Premix Ex Taq™ (Takara Bio Inc, Japan) on a Rotor- Gene Q real-time PCR system (QIAGEN, USA) under the following conditions: 94 ºC for 5 min followed by 40 cycles of 94 ºC for 15 s, 56 ºC for 15 s, and 72 ºC for 20 s. All experiments were performed in duplicate and three times. Relative expression of McPSY and McPSD were calculated using equation 2-∆∆Ct in which ∆Ct was obtained by subtracting the internal control Ct value from the Ct value of McPSY and McPSD (raJaee BehBahani et al., 2020). Tab. 3: Sequences of specific primers used for quantitative real-time PCR. Genes Primer sequences Amplicon size (bp) F GCTTCATCGTTGGTTGTCTCTCT R TGCTCCATTTCTGCCTCTTACTC F TTTGCTTGGATTACCCTAGACCA R TGCACCAGCGATCACTACTTTTA F ATAACTCGATGGATCGCACGG R TCCTCCGGAATCGAACCCTA McPSY 154 McPDS 128 Mc18S rRNA 136 High-performance liquid chromatography (HPLC) analysis of carotenoids Carotenoids were extracted from M. charantia leaf samples (0.1 g) in each group with 3 ml of ethanol containing 0.1% ascorbic acid (w/v). This mixture was vortexed for 20 s, and then incubated in a water bath at 85 ºC for 5 min. Subsequently, 120 μl of potassium hydroxide (80% w/v) was added to saponify any potentially inter- fering oils. After vortexing and incubating at 85 ºC for 10 min, the samples were placed on ice and 1.5 ml of cold deionized water and 0.05 ml of β-Apo-80-carotenal (12.5 μg ml-1), an internal standard, were added. Next, the carotenoids were extracted twice with 1.5 ml of hexane and centrifuged at 1200 g each time to separate the layers. Then, the extracts were freeze-dried under a stream of nitrogen gas and resuspended in 50:50 (v/v) dichloromethane/methanol. The ex- traction method used for carotenoid analysis was similar to that de- scribed (howe and tanumihardJo, 2006). For HPLC analysis, the carotenoids were separated on an Agilent 1100 HPLC system with a Hector-M C18 column (150 × 4.6 mm, 5 μm, P/N: C18-M51001546) and detected with an array detector at 450 nm. Solvent A consisted of methanol/water (92:8 v/v) with 10 mM ammonium acetate. Solvent B consisted of 100% methyl tert-butyl ether. The flow rate was main- tained at 1 ml/min and samples were eluted with the following gra- dient: 0 min, 83% A/17% B; 23 min, 70% A/30% B; 29 min, 59% A/41% B; 35 min, 30% A/70% B; 40 min, 30% A/70% B; 44 min, 83% A/17% B; and 55 min, 83% A/17% B. Identification and peak as- signment of carotenoids were primarily based on comparison of their retention time and UV-visible spectrum data with that of standards, and with guidelines previously presented. Statistical analysis The obtained data were subjected to statistical analyses using SPSS software. All data were expressed as mean ± standard error (SE) val- ues of three independent replicates. Significant mean differences be- tween the treatments were estimated according to Duncan’s multiple range test at the level of P ≤ 0.05. 10 F.S. Seddighinia, A. Iranbakhsh, Z. Oraghi Ardebili, S. Soleimanpour Results Both cold plasma and CNT treatments synergistically provoked con- siderable changes in the early growth of the treated plants, especially the combined treatments, among which the simultaneous treatment of 200 mg L-1 CNT and cold plasma for 60 s has shown the best performance (Fig. 3). There was a considerable linear relationship between the applied concentrations of the CNT and the growth rate of seedlings. It should be noted that CNT supplementations have not caused any toxic effects. The ultra-structure of stems (by FESEM) was photographed to trace the uptake and transportation of MWCNT. Cellular uptake and translocation of the CNT were manifested based on the stem ultra-structure (Fig. 4). Expression levels of the McPSY and McPDS Quantitative real-time PCR analysis was performed to determine the expression levels of McPSY and McPDS in all treated groups of M. charantia. McPSY and McPDS expressed in all treated groups, were examined, and shown a similar expression pattern. The McPSY was expressed at the highest level in the CNT50+ Plasma 60 group (26.6- fold) and CNT50 group (6.69-fold) (Tab. 4). Between CNT treated groups, CNT50 had significantly higher expression levels (6.5-fold) than the others (P<0.001). Among Plasma-treated groups, McPSY in Plasma 60 group (3.65-fold) had significantly higher expression levels (P<0.001) than Plasma 120 and control groups (Tab. 4). The McPDS expression showed a similar expression trend to McPSY, and at the highest expression level in the CNT50 group (58.06-fold) and CNT50+ Plasma 60 group (37.45-fold) (Tab. 4). Between CNT- treat- ed groups, CNT50 (58.06-fold) had significantly higher levels than the other concentrations (P<0.001). However, there is no significant difference between Plasma-treated groups. Finally, between the com- bination groups treated with Plasma and CNT, the CNT50+ Plasma 60, had significantly higher expression levels (37.45-fold) than the others (P<0.001) (Tab. 4). HPLC analyses of carotenoids Carotenoids (β-carotene, zeaxanthin, α-carotene, and lutein) were identified in different treatment groups by HPLC. The carotenoid levels varied in the different treated groups of M. charantia (Tab. 4). Changes in β-carotene content Among Plasma-treated groups, the content of β-carotene in Plasma 60 was 155.5 μg g-1 fw and had higher levels than Plasma 120 and control groups (Tab. 4). In addition, in CNT-treated groups, the CNT50 group with 249.3 μg g-1 fw content of β-carotene had sig- nificantly higher levels (P<0.001) than CNT100, CNT200, and con- trol groups (Tab. 4). Totally, between all treated groups, the CNT50 group and CNT50+Plasma60 group had significantly higher levels of β-carotene content than the other treated groups (Tab. 4). The lowest level of β-carotene was in Plasma 120 and CNT200 groups, with no significant difference from the control group (Tab. 4). Changes in zeaxanthin content The production of zeaxanthin in all groups was less than 9 μg g-1 fw, which is relatively low (Tab. 4). In Plasma-treated groups, the content of zeaxanthin in Plasma 60 was 3.87 μg g-1 fw, with sig- nificantly higher levels (P<0.001) than Plasma 120 and the control groups (Tab. 4). Among CNT-treated groups, the CNT50 group with 8.43 μg g-1 fw content of zeaxanthin had significantly higher levels (P<0.001) than the other CNT and control groups (Tab. 4). Finally, between all treated groups, the CNT50, CNT50+Plasma 60 group, and CNT100+Plasma 60 group had significantly higher zeaxanthin levels (P<0.001) than the other treated groups (Tab. 4). The lowest zeaxanthin level was in Plasma 120 and CNT100 groups and there was no significant difference with the control group (Tab. 4). Changes in α-carotene content The findings show that CNT50+ Plasma 60 group had significantly higher α-carotene levels (p<0.001) than the control and other groups. In CNT-treated groups, the CNT50 group had a significantly higher (p<0.001) α-carotene level than the CNT100, CNT200, and control groups (Tab. 4). However, there were no statistically significant dif- ferences in α-carotene production between plasma-treated groups. Among combination groups, treated with plasma and CNT simulta- neously, both CNT50+ Plasma 60 and CNT100+ Plasma 60 groups had significantly higher α-carotene levels than the other treated groups (p<0.001) (Tab. 4). Meanwhile, the CNT200 group contained the lowest α-carotene level (Tab. 4). Changes in lutein content Tab. 4 shows that the highest lutein level was observed in the CNT50+Plasma 60 group with a content of 171.6 μg g-1 fw. Moreover, Fig. 3: The recorded differences in plant early growth following seed prim- ing with the plasma and the supplementation of carbon nanotubes (CNT), 8 days after the treatments. A-Control; B-CNT of 50 mgl-1; C-CNT of 100 mg l-1; D-CNT of 200 mg l-1; E-plasma of 60 s; F- plasma of 60 s and CNT of 50 mg l-1; G-plasma of 60 s and CNT of 100 mg l-1; H-plasma of 60 s and CNT of 200 mg l-1; I-plasma of 120 s; J-plasma of 120 s and CNT of 50 mg l-1; K-plasma of 120 s and CNT of 100 mg l-1; L-plasma of 120 s and CNT of 200 mg l-1. Seed priming of bitter melon by cold plasma 11 Fig. 4: The ultra-structure images are based on the electron microscopy (FESEM) for tracing the uptake of CNT in shoots of the seedlings treated by the plasma and CNT. Cross-sections of M. charantia main shoots after exposure to (A, B, C, D, and E) control, (F, G, H, I and J) functionalized CNT 200 mgl-1, (K, L, M, N, and O) Plasma of 120 s (P, Q, R, S, and T) Plasma of 120 s + functionalized CNT 200 mgl-1. From left to right panels represent general and detailed information, respectively, concerning selected areas. Tab. 4: Evaluation of PSY and PDS genes expression and carotenoid content (μg g-1 dry weight). Results expressed as mean ±SD (n=3) in different plasma and CNT treated groups of M. charantia*. PSY gene 1±0.001f 6.69±0.19b 0.07±0.03g 3.95±0.14c 4±0.38c 26.62±0.05a 1.12±0.04f 2.21±0.01e 0.36±0.02g 3.03±0.03d 0.14±0.02g 1.1±<0.001f PDS gene 1±0.001g 58.06±0.16a 0.43±0.11gh 5.69±0.02d 0.33±0.02gh 37.45±0.81b 0.28±0.09gh 27.09±0.02c 0.73±0.01gh 3.81±0.005e 0.12±0.03h 2.88±0.16f β-carotene 104.8±8.08d 249.3±20.2a 171.6±12.8bc 121.9±8.14cd 155.5±19.3bcd 245±16.14a 202±12.29ab 152.5±20.9cd 99.6±14.42d 209.6±37.1bc 141.3±17.4cd 113.6±4.1cd zeaxanthin 2.31±0.71bc 8.43±1.72a 2.9±0.86bc 3.97±0.32b 3.87±1.17bc 7.44±1.09a 7.32±0.56a 3.02±0.58bc 1.07±0.25c 4.34±1.12bc 3.98±0.87bc 3±0.43bc α-carotene 3.28±0.48e 11.53±1.74abc 10.3±1.9abcd 4.44±0.87de 5.89±0.95cde 14.34±1.93a 13.18±2.28a 10.6±1.32abc 6.3±1.14cde 8.34±1.5bcde 12.45±1.8ab 7.16±1.61cde Lutein 39.23±6.42a 123.5±19.3ab 73.6±10.1bcd 69.4±10.38cd 45.67±8.51d 171.6±21.5a 118.6±15.6abc 98.37±9.89bc 64.8±7.31cd 108.5±4.98abc 85.2±5.3bcd 98.3±35bcd *Statistical significance of the differences between treated groups was determined using ANOVA followed by paired-group comparisons. The different letters (a, b, c and d) indicate significance at P< 0.05. C on tr ol C N T 50 C N T 10 0 C N T 20 0 P 60 C N T 50 +P 60 C N T 10 0+ P 60 C N T 20 0+ P 60 P 12 0 C N T 50 +P 12 0 C N T 10 0+ P 12 0 C N T 20 0+ P 12 0 12 F.S. Seddighinia, A. Iranbakhsh, Z. Oraghi Ardebili, S. Soleimanpour in CNT-treated groups similar to α-carotene, the CNT50 group had a significantly higher lutein level (p<0.001) than the CNT100, CNT200, and control groups (Tab. 4). However, there were no sig- nificant differences in lutein levels between plasma-treated groups. Meanwhile, the Plasma 60 group contained the lowest level of lutein (Tab. 4). The correlation between carotenoid contents and the expression of McPSY and McPDS Correlation analysis, inter se McPSY and carotenoid contents in all groups revealed significant association between the McPSY with all forms of carotenoid [β-carotene (r=0.602, P< 0.001), zeaxan- thin (r=0.539, P< 0.001), α-carotene (r=0.405, P< 0.014) and lutein (r=0.646, P< 0.001)]. Also, correlation analysis, inter se the McPDS and carotenoid contents in all groups revealed significant association between this gene with all forms of carotenoid [β-carotene (R=0.643, P< 0.001), zeaxanthin (r=0.568, P< 0.001), α-carotene (r=0.442, P< 0.007), and lutein (r=0.587, P<0.001)]. Regarding the coefficients, correlation between the expression of two genes and other variables is significant (p <0.001), showing a direct relationship between them (Tab. 5). several genes, like DAT in Catharanthus roseus (ghasempour, 2019), PAL, TAT, and RAS in Salvia verticillate (rahmani, 2020), and HPPR, RAS, PAL, TAT genes in Satureja khuzistanica (Fatemi, 2019). LC-MS analysis revealed that MWCNT supplementation al- tered total fruit metabolome in tomato crops (mCgehee, 2017). These results manifested that similar to other nanomaterials, CNTs can trigger signalling thereby influencing transcriptions of genes and metabolism. Apart from CNTs, cold plasma is also capable of af- fecting the transcription of genes. Seed priming with cold plasma affected the expression of several genes in diverse plant species, like PAL and USP in Astragalus fridae (moghanloo, 2019), defense- related genes in tomato (adhikari, 2020), and WRKY1 transcrip- tion factor, THCAS, OAC, CBDAS, and OLS in Hemp (iranBakhsh et al., 2020). In sunflower, the cold plasma priming was also asso- ciated with transcriptional responses and substantial variation in phytohormones (mildaziene et al., 2019). The plasma-mediated changes in the transcription of genes can be explained by bioactive signalling agents generated during the plasma generation (BaBaJani, 2018; iranBakhsh, 2017; iranBakhsh, 2018; nasrin saFari, 2017; sheteiwy et al., 2018). Our results along with these recent reports underline this hypothesis that cold plasma perception and signal transduction can modify transcription of genes, thereby improving plant growth and metabolism. However, more molecular studies are required to fill knowledge gaps and elucidate the potentially involved mechanisms in plant responses to cold plasma and CNTs. In the present study, it was shown that these two treatments could have synergistic effects. In this connection, the results of this study also demonstrated that CNT50 + plasma 60 can stimulate the higher expression of McPSY and McPDS, compared to the other treated groups; however, the other had acceptable expression. Thus, it can be concluded that plasma 60 can increase the efficiency of the CNT50. Furthermore, the CNT treatments (long-term), especially CNT50, were more efficient than the individual plasma priming (short-term treatment) in affecting the gene expression or carotenoid levels. It should be noted that the significant correlations were found between the McPSY and McPDS expressions and carotenoids’ concentra- tions. However, the similar regular linear relationship between the CNT concentrations, gene expression levels, carotenoid contents, and growth performance was not found and the individual or combined treatments of the CNT50 provoked the highest expression of McPSY and McPDS, as well as terpenoid derivatives. Interestingly, the CNT200 treatment led to the best plant growth performance, while CNT50 provoked the highest expression. Hence, it can be proposed that CNT can affect diverse signalling pathways, thereby altering growth and metabolism. Therefore, it seems that the individual or combined treatments of the CNT and plasma, differentially affect plant cell at diverse growth, physiological, and molecular levels in a dose-dependent manner. To the best of our knowledge, this experiment provides the first molecular evidence on how cold plasma and CNT treatment may synergistically improve carotenoid metabolism which is an impor- tant mechanism in terms of photosynthesis performance and pro- tection. The observed molecular variations at the transcriptional level may be explained by modulation in redox homeostasis in re- sponse to CNT (Fatemi, 2019; rahmani, 2020) and cold plasma (ghasempour, 2019; iranBakhsh et al., 2020; mildaziene et al., 2019; moghanloo, 2019). Monitoring the potential variation in cel- lular redox status following cold plasma and CNTs is, therefore, re- commended for designing future studies. Conclusions In the present article, we addressed the efficacy of CNT and cold plasma treatments to affect the metabolism of carotenoids. According to our results, the CNT and plasma-mediated modification in the me- Discussion In the present study, the relationship between carotenoid accumula- tions and the gene expression of McPSY and McPDS, under indi- vidual and combined treatments of cold plasma and carbon nanotube, were investigated in M. charantia, which could potentially be used as a source of carotenoid in human nutrition. The seed priming with cold plasma and supplementation of nutrient solution with MWCNT in both individual and combined treatments, not only improved plant early growth but also modified the secondary metabolism through changes in expression patterns of two key genes, McPSY and McPDS, contributed to the synthesis of important terpenoid meta- bolites. In our previous report, we provided comprehensive evidence on how CNT and cold plasma treatments were associated with sig- nificant modifications in plant growth, biomass accumulation, yield, and differentiation of tissues (especially xylem conducting tissue) (seddighinia, 2020). The individual or combined treatments of cold plasma and MWCNTs, especially CNT50+plasma60, were capable of upregulating genes in- volved in carotenoid metabolism. As it is well known, carotenoids contribute to protecting the photosynthesis apparatus through the Xanthophyll cycle. There is limited molecular evidence on tran- scriptional responses to CNTs. In this regard, earlier reports showed that CNT application was associated with changes in growth and development-related genes, including SLR1, RTCS, RTH1, and RTH3 genes in maize (yan et al., 2013) and CycB, NtLRX1, and NtPIP1 in tomato (khodakovskaya et al., 2012). Moreover, several reports indicated that CNTs were associated with upregulation in genes in- volved in secondary metabolism which our results are consistent with their findings. In line with our results, the CNT treatments mediated changes in secondary metabolism through upregulating Tab. 5: Correlation coefficients (R) exhibiting relationship between the ex- pression of two evaluated genes (PSY and PDS) and the measured related carotenoids (lutein, α-carotene, zeaxanthin, and β-carotene). Genes lutein α-carotene zeaxanthin β-carotene McPSY 0.646** 0.405* 0.539** 0.602** McPDS 0.587** 0.442** 0.568** 0.643** *: p≤0.05 **: p≤0.01 Seed priming of bitter melon by cold plasma 13 tabolism of carotenoids can be considered as an important mecha- nism by which these treatments may protect the photosynthesis appa- ratus against photoinhibition. 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