ABSTRACT

This study aimed to determine the effects of titanium dioxide nanoparticle (TiO₂NP) pretreatment on seeds of different safflower cultivars (Balci, Dinçer) under salt and heat stresses. The apparent effects on stress markers (malondialdehyde (MDA), hydrogen peroxide (H₂O₂) and superoxide radical (O₂ ^•⁻) content), as well as changes in germination and physiological parameters (radicle and plumula weight and length measurements), were investigated. TiO₂NP pretreatment caused an increase in radicle length and plumula fresh weight for the Balci cultivar under salinity. Furthermore, plumula dry weight was alleviated with TiO₂NP pretreatment for both cultivars. TiO₂NP pretreatment improved plumula dry and fresh weights for both cultivars under heat stress. In addition, MDA content decreased for both cultivars under heat stress but only for Balci under salt stress. The amount of O₂ ^•⁻ radicals positively affected only the radicle for both cultivars under heat stress. This study is the first to document the alleviation of salt stress damage for the Balci safflower cultivar, and protection for both Balci and Dinçer cultivars under heat stress, using 200 ppm TiO₂NP pretreatment.

Keywords:
Carthamus tinctorious; nanoparticle; reactive oxygen species

INTRODUCTION

Salinity and heat are important abiotic stress factors affecting plant growth, development, and yield potential (Zhang & Dai 2019Zhang Q, Dai W. 2019. Plant response to salinity stress. In: Dai W (ed.). Stress physiology of woody plants. CRC Press. p. 155-173.). Salinity causes osmotic stress by reducing water potential, which disrupts plant water balance and turgor (Navada et al. 2020Navada S, Sebastianpillai M, Kolarevic J et al. 2020. A salty start: Brackish water start-up as a microbial management strategy for nitrifying bioreactors with variable salinity. Science of the Total Environment 739: 139934. ). The entry of high concentrations of salt ions into plant cells causes cell ion imbalance and ionic stress. Depending on both osmotic and ionic stress, reactive oxygen species (ROS) production and accumulation increase, causing oxidative damage. Increased ROS causes lipid peroxidation, membrane disruption, and denaturation of biomolecules such as DNA, proteins, and lipids (Arif et al. 2020Arif Y, Singh P, Siddiqui H, Bajguz A, Hayat S. 2020. Salinity induced physiological and biochemical changes in plants: An omic approach towards salt stress tolerance. Plant Physiology and Biochemistry 156: 64-77. ).

Human activities, especially increased emissions of greenhouse gases, such as carbon dioxide, methane, and chlorofluorocarbons, into the atmosphere, cause increased heat, contributing to global warming. Heat must exceed a particular threshold value for heat stress to occur, with a rapid increase in ambient heat of 10 to 15°C being defined as heat stress (Lipiec et al. 2013Lipiec J, Doussan C, Nosalewicz A, Kondracka K. 2013. Effect of drought and heat stresses on plant growth and yield: A review. International Agrophysics 27: 463-477. ). The duration and intensity of high heat determines the effects of heat stress on plant growth, potentially including permanent damage to plant growth and development (Lal et al. 2022Lal MK, Tiwari RK, Gahlaut V et al. 2022. Physiological and molecular insights on wheat responses to heat stress. Plant Cell Reports 41: 501-518. ). Heat stress causes direct and indirect effects that can result in plant death due to protein and lipid denaturation, mitochondrial death, and disruption of membrane stabilization (Kumar & Kaushik 2021Kumar A, Kaushik P. 2021. Heat stress and its impact on plant function: An update. ).

Nanotechnology is an effective method to increase crop yield and ensure plant sustainability (Mohapatra et al. 2023Mohapatra B, Chamoli S, Salvi P, Saxena SC. 2023. Fostering nanoscience’s strategies: A new frontier in sustainable crop improvement for abiotic stress tolerance. Plant Nano Biology 3: 100026.). Nanoparticles can improve agricultural production by providing effective solutions to many agrarian problems via the active role they play in the interaction between atomic or molecular structures (Chen et al. 2016Chen YW, Lee HV, Juan JC, Phang SM. 2016. Production of new cellulose nanomaterial from red algae marine biomass Gelidium elegans. Carbohydrate Polymers 151: 1210-1219. ; Prasad et al. 2017Prasad R, Bhattacharyya A, Nguyen QD. 2017. Nanotechnology in sustainable agriculture: Recent developments, challenges, and perspectives. Frontiers in Microbiology 8: 1014. ; Shang et al. 2019Shang H, Guo H, Ma C, Li C, Chefetz B, Polubesova T, Xing B. 2019. Maize (Zea mays L.) root exudates modify the surface chemistry of CuO nanoparticles: Altered aggregation, dissolution and toxicity. Science of the Total Environment 690: 502-510. ). Nanoparticles range in size from 1 to 100 nm in diameter and can be synthesized by many methods to have different physico-chemical and biological properties than the same substance at a larger size (Singh et al. 2018Singh J, Dutta T, Kim KH, Rawat M, Samddar P, Kumar P. 2018. ‘Green’synthesis of metals and their oxide nanoparticles: Applications for environmental remediation. Journal of Nanobiotechnology 16: 84. ; Cele 2020Cele T. 2020. Preparation of nanoparticles: Engineered nanomaterials-health and safety. IntechOpen.). Nanoparticles can protect cells against lipid, protein and DNA denaturation and cell membrane damage caused by oxidative stress due to the formation of ROS (Kumar et al. 2017Kumar B, Smita K, Cumbal L, Debut A. 2017. Green synthesis of silver nanoparticles using Andean blackberry fruit extract. Saudi Journal of Biological Sciences 24: 45-50. ). Nanoparticles, when applied to plants, have been reported to regulate the harmful effects of environmental stress and the adaptation mechanisms of plants, as well as seed germination, development, and other positive effects (Paparella et al. 2015Paparella S, Araújo SS, Rossi G, Wijayasinghe M, Carbonera D, Balestrazzi A. 2015. Seed priming: State of the art and new perspectives. Plant Cell Reports 34: 1281-1293. ; Guha et al. 2018Guha T, Ravikumar KVG, Mukherjee A, Mukherjee A, Kundu R. 2018. Nanopriming with zero valent iron (nZVI) enhances germination and growth in aromatic rice cultivar (Oryza sativa cv. Gobindabhog L.). Plant Physiology and Biochemistry 127: 403-413. ; Zulfiqar & Ashraf 2021Zulfiqar F, Ashraf M. 2021. Nanoparticles potentially mediate salt stress tolerance in plants. Plant Physiology and Biochemistry 160: 257-268. ). Titanium dioxide nanoparticles (TiO₂NPs) can have various profound effects on the morphological, physiological, and biochemical properties of some plant species (Gohari et al. 2020Gohari G, Mohammadi A, Akbari A et al. 2020. Titanium dioxide nanoparticles (TiO2 NPs) promote growth and ameliorate salinity stress effects on essential oil profile and biochemical attributes of Dracocephalum moldavica. Scientific Reports 10: 912. ). The application of TiO₂NPs has been reported to improve rubisco and antioxidant enzyme activities, photosynthetic rate, and chlorophyll formation, thereby increasing crop yield (Lateef et al. 2018Lateef A, Folarin BI, Oladejo SM, Akinola PO, Beukes LS, Gueguim-Kana EB. 2018. Characterization, antimicrobial, antioxidant, and anticoagulant activities of silver nanoparticles synthesized from Petiveria alliacea L. leaf extract. Preparative Biochemistry and Biotechnology 48: 646-652. ). Particularly in the agricultural sector, nanoparticle application has been shown to provide salinity tolerance as an effective method to increase productivity under stressful environmental conditions (Ahmad & Akhtar 2019Ahmad I, Akhtar MS. 2019. Use of nanoparticles in alleviating salt stress. In: Akhtar MS (ed.). Salt stress, microbes, and plant ınteractions: Causes and solution. Singapore, Springer. p. 199-215. ; Avestan et al. 2019Avestan S, Ghasemnezhad M, Esfahani M, Byrt CS. 2019. Application of nano-silicon dioxide improves salt stress tolerance in strawberry plants. Agronomy 9: 246. ; Abdoli et al. 2020Abdoli S, Ghassemi-Golezani K, Alizadeh-Salteh S. 2020. Responses of ajowan (Trachyspermum ammi L.) to exogenous salicylic acid and iron oxide nanoparticles under salt stress. Environmental Science and Pollution Research 27: 36939-36953. ; Alabdallah & Alzahrani 2020Alabdallah NM, Alzahrani HS. 2020. The potential mitigation effect of ZnO nanoparticles on [Abelmoschus esculentus L. Moench] metabolism under salt stress conditions. Saudi Journal of Biological Sciences 27: 3132-3137. ; Ye et al. 2020Ye Y, Cota-Ruiz K, Hernandez-Viezcas JA et al. 2020. Manganese nanoparticles control salinity-modulated molecular responses in Capsicum annuum L. through priming: A sustainable approach for agriculture. ACS Sustainable Chemistry & Engineering 8: 1427-1436. ). In addition, TiO₂NPs have been reported to alleviate salt stress in broad bean plants under saline conditions by increasing plant growth; antioxidant enzyme activities; soluble sugars, amino acids, proline, phenolics and leaf chlorophyll contents; antioxidant capacity; and yield, while reducing H₂O₂ and MDA contents (Abdel Latef et al. 2018Abdel Latef AAH, Srivastava AK, El‐sadek MSA, Kordrostami M, Tran LP. 2018. Titanium dioxide nanoparticles improve growth and enhance tolerance of broad bean plants under saline soil conditions. Land Degradation and Development 29: 1065-1073. ). Additionally, Pérez-Zavala et al. (2022Pérez-Zavala FG, Atriztan-Hernandez K, Martínez-Irastorza P, Oropeza-Aburto A, López-Arredondo D, Herrera-Estrella L. 2022. Titanium nanoparticles activate a transcriptional response in Arabidopsis that enhances tolerance to low phosphate, osmotic stress and pathogen infection. Frontiers in Plant Science 13: 994523.) reported that TiO₂NPs activate a transcriptional response to osmotic stress in Arabidopsis plants. Similarly, Thakur et al. (2021Thakur S, Asthir B, Kaur G, Kalia A, Sharma A. 2021. Zinc oxide and titanium dioxide nanoparticles influence heat stress tolerance mediated by antioxidant defense system in wheat. Cereal Research Communications 50: 385-396. ) determined that TiO₂NPs cause an increase in biomass by increasing the adverse effects of heat stress. They reported that TiO₂NPs increase defense mechanisms, including against heat exposure and increased ROS production and some natural antioxidants (fenol and flavonoid) in plants (Zafar et al. 2016Zafar H, Ali A, Ali JS, Haq IU, Zia M. 2016. Effect of ZnO nanoparticles on Brassica nigra seedlings and stem explants: Growth dynamics and antioxidative response. Frontiers in Plant Science 7: 535. ). In addition, it has been shown that TiO₂NPs affect membrane repair and improve growth and development of morphological structures under heat stress, and are effective at healing damage to chloroplasts due to heat stress (Faran et al. 2019Faran M, Farooq M, Rehman A, Nawaz A, Saleem MK, Ali N, Siddique KH. 2019. High intrinsic seed Zn concentration improves abiotic stress tolerance in wheat. Plant and Soil 437: 195-213. ; Younis et al. 2020Younis AA, Khattab H, Emam MM. 2020. Impacts of silicon and silicon nanoparticles on leaf ultrastructure and TaPIP1 and TaNIP2 gene expressions in heat stressed wheat seedlings. Biologia Plantarum 64: 343-352. ; El-Saadony et al. 2021El-Saadony MT, Desoky ESM, Saad AM, Eid RS, Selem E, Elrys AS. 2021. Biological silicon nanoparticles improve Phaseolus vulgaris L. yield and minimize its contaminant contents on a heavy metals-contaminated saline soil. Journal of Environmental Sciences 106: 1-14. ). In contrast, TiO₂NP treatment via foliar spraying did not ameliorate drought stress in Helianthus annuus plants (Ramadan et al. 2022Ramadan T, Sayed SA, Abd-Elaal AK, Amro A. 2022. The combined effect of water deficit stress and TiO2 nanoparticles on cell membrane and antioxidant enzymes in Helianthus annuus L. Physiology and Molecular Biology of Plants 28: 391-409.). Lastly, Kumar et al. (2023Kumar S, Dwivedi A, Pandey AK, Vajpayee P. 2023. TiO2 nanoparticles alter nutrients acquisition, growth, biomacromolecules, oil composition and modulate antioxidant defense system in Mentha arvensis L. Plant Nano Biology 3: 100029.) determined that TiO₂NP (100 µg/ml) application alters nutrient levels, growth and the antioxidant defense system of Mentha arvensis because of its toxicity.

The importance of safflower (Carthamus tinctorious L.) stems from the demand for its seed oil (Zemour et al. 2021Zemour K, Adda A, Labdelli A, Dellal A, Cerny M, Merah O. 2021. Effects of genotype and climatic conditions on the oil content and its fatty acids composition of Carthamus tinctorius L. seeds. Agronomy 11: 2048. ). Safflower oil is rich in oleic acid (omega-9) and reaches up to 75% linoleic acid (C18-2), an essential fatty acid for humans, making it an important food source. Also important for human health is its high α-tocopherol content (Sujatha 2002Sujatha M. 2002. Current status and future prospects of in vitro techniques and biotechnology in safflower breeding. Sesame and safflower Newsletter 17: 92-97.). The safflower plant is very sensitive to salt and heat during germination and early seedling development. The safflower plant adapts easily to the climatic conditions of Turkey and so breeding studies are regularly carried out to obtain high-yield, high-quality, and stress-resistant varieties for agriculture. In addition to these long-term studies, there is a need to better understand the dimensions of the stress factors to which safflower is exposed, how they affect the plant, and what solutions can be taken. Therefore, this study aimed to elucidate the mechanism of action of TiO₂NPs for two different safflower seed cultivars - Balci and Dinçer - when under salt and heat stress.

Materials and Methods

Materials

Balci and Dinçer safflower cultivars were obtained from the Gecit Kuşaği Agricultural Research Institute of the Ministry of Agriculture and Forestry of the Republic of Turkey. Titanium dioxide (TiO₂) nanoparticles (20 nm in diameter, 99.9% purity) were purchased from Nanography Nanotechnology.

Experiment preparation

Seeds were submitted to surface sterilization in 1% sodium hydrochloride solution for 10 minutes, followed by five washes with distilled water. The seeds were then placed on filter paper and dried at room temperature. Next, the seeds were kept for 18 hours in a 200 ppm TiO₂NP solution that was prepared in advance using distilled water. The seeds in solution were then exposed to ultrasonic vibration twice, one hour at the beginning of treatment and 30 minutes at the end, followed by three washes with distilled water and drying on filter paper.

Seeds were sowed in petri plates lined with filter paper and provisioned with 3 ml of distilled water. Ten seeds were sowed per plate, which were placed in a dark environment for germination. After three days, the plates were divided into groups for salt and heat stress treatments, with three replicates per cultivar. Preliminary experiments were performed with nanoparticle concentrations of 100, 200 and 500 ppm TiO₂NPs to determine the most effective salt concentration (50 and 100 mM NaCl) and heat treatment (36,40 and 45 ℃), resulting in the selection of 50 mM NaCl and 45 ℃, resepctively. Safflower seedlings are shown in Figs. 1-4.

Salt stress treatment

Treatments for the salt stress experiment included the following: TiO₂NP treatment (control) = pretreatment of 200 ppm TiO₂NPs; NaCl treatment = 50 mM NaCl concentration; and TiO₂NP + NaCl treatment (interaction) = 200 ppm TiO₂NPs + 50 mM NaCl. Salt and interaction treatments received 3 ml of 50 mM NaCl solution. Length and fresh and dry weight were determined for the radicle and plumula four days after salt stress treatment (Fig. 1).

Heat stress treatment

Treatments for the heat stress experiment included the following: TiO₂NP treatment (control) = pretreatment of 200 ppm TiO₂NPs; 45 ℃ treatment = 45 ℃ for six hours throught two days; and TiO₂NP + 45 ℃ treatment (interaction) = 200 ppm TiO₂NPs + 45 ℃. Length and fresh and dry weight were determined for the radicle and plumula (Fig. 2).

Growth analysis

Growth was analyzed after salt stress and heat stress treatments according to Karagüzel et al. (2004Karagüzel O, Cakmakci S, Ortacesme V, Aydinoglu B. 2004. Influence of seed coat treatments on germination and early seedling growth of Lupinus varius (L.). Pakistan Journal of Botany 36: 65-74.), using length and fresh and dry weight of radical and plumula. Radicle and plumula lengths of six germinated seeds taken randomly from each replicate were measured (caliper) and averaged. Radicle and plumula fresh weights (mg/seed) of six germinated seeds taken randomly from each replicate were weighed (0.001 g precision balance). Radicle and plumula dry weights (mg/seed) of six germinated seeds taken randomly from each replicate were determined by rapid drying at 80°C for 24 hours, then weighing (0.001 g precision balance). Average radicle and plumula fresh and dry weights were calculated for the six seeds of each treatment. All measurements were done in triplicate.

Lipid peroxidation level (MDA)

The amount of MDA, the end product of lipid peroxidation, was determined according to Madhava Rao and Stresty (2000Madhava Rao KV, Stresty TVS. 2000. Antioxidative parameters in the seedlings of pigeonpea (Cajanus cajan (L.) Millspaugh) in response to Zn and Ni stresses. Plant Science 157: 113-128. ), using thiobarbituric acid (TBA) reaction for radicle and plumula samples of six germinated seeds taken randomly from each replicate after salt stress or heat stress treatment. Leaf samples (0.5 g) of each treatment were homogenized by adding 2.5 ml of trichloroacetic acid (TCA). Homogenates were then centrifuged at 10,000 x g for 5 min at 4 ℃. The reaction mixture containing TBA and TCA was then pipetted into test tubes containing the obtained supernatants. All test tubes were heated at 95 ℃ for 30 min. The mixture was then centrifuged at 1,000 x g for 15 min and the absorbance of the formed supernatant read 532 and 600 nm using a Thermo Scientific Genesys (10S UV-VIS) spectrophotometer. MDA concentration was calculated using the extinction coefficient 155 mM^-1cm^-1.

Hydrogen peroxide (H₂O₂) content

The amount of hydrogen peroxide (H₂O₂) was determined according to Velikova et al. (2000Velikova V, Yordanov I, Edreva A. 2000. Oxidative stress and some antioxidant systems in acid rain-treated bean plants: Protective role of exogenous polyamines. Plant Science 151: 59-66. ), by homogenizing samples (0.5 g) of each group by adding 5 ml of TCA. Homogenates were then centrifuged at 12,000 rpm for 15 min. The supernatants were then mixed with 0.5 ml of potassium phosphate buffer (10 mM, pH 7) and 1 ml of KI buffer, and their absorbance read at 390 nm using a Thermo Scientific Genesys (10S UV-VIS) spectrophotometer.

Superoxide (O₂ ^•⁻) radical content

Superoxide anion radical content was determined according to Ke and Sun (2004Ke D, Sun G. 2004. The effect of reactive oxygen species on ethylene production induced by osmotic stress in etiolated mungbean seedling. Plant Growth Regulation 44: 199-206. ) by homogenizing samples (0.5 g) of each group by adding 5 ml of TCA. Next, 1 ml of 1 mM hydroxylammonium chloride solution was added to 0.5 ml of supernatant and incubated at 25^oC for 1 hour. Color change was observed for 20 minutes at 25^oC after the addition of 1 ml of 17 mM 4-aminobenzenesulfonic acid solution and 1 ml of 7 mM naphthylamine. Specific absorbance was read at 530 nm using a Thermo Scientific Genesys (10S UV-VIS) spectrophotometer. Sodium nitrite was used as a standard solution to calculate the amount of superoxide radicals.

Statistical Analysis

All data were analyzed using the SPSS package (SPSS, Version 20.0, SPSS Inc, Chicago, IL, USA). Statistical significance was evaluated by the F-test (P < 0.05) and, when significant, the protected least significant difference (Protected DUNCAN) was used to separate.

RESULTS

Effects of TiO₂NP pretreatment on radicle and plumula lengths under salt stress

Radicle length for the Balci cultivar increased significantly (1.8 fold) under the TiO₂NP + NaCl treatment compared to the NaCl treatment (Fig. 1). Radicle length for the Dinçer cultivar increased significantly (2.7 fold) under the TiO₂NP treatment compared to the NaCl treatment (Fig. 1). Plumula length for both Balci and Dinçer cultivars did not differ significantly between the TiO₂NP + NaCl treatment and the NaCl treatment (Fig. 1).

Effect of TiO₂NP pretreatment on fresh and dry weights of radicle and plumula under salt stress

Radicle fresh weight for the Balci cultivar increased significantly (1.3 fold) under the TiO₂NP treatment compared to the TiO₂NP + NaCl treatment and the NaCl treatment (Fig. 2 A ). On the other hand, radicle fresh weight for the Dinçer cultivar did not differ significantly between the NaCl treatment and the TiO₂NP + NaCl treatment (Fig. 2 A ). Plumula fresh weight for the Dinçer cultivar did not differ significantly between the TiO₂NP + NaCl treatment and the NaCl treatment. Plumula weight of the Balci cultivar increased significantly (1.8 fold) under the TiO₂NP + NaCl treatment compared to the NaCl treatment (Fig. 2 A ).

Radicle dry weight for both Balci and Dinçer cultivars did not differ significantly between the TiO₂NP + NaCl treatment and the NaCl treatment (Fig. 2 B ). Plumula dry weight for the Dinçer cultivar increased significantly (1.3 fold) under the TiO₂NP + NaCl treatment compare to the NaCl treatment. Likewise, plumula dry weight for Balci cultivar increased significantly (1.1 times) under the TiO₂NP + NaCl treatment (0.14) compared to the NaCl treatment (Fig. 2 B ).

Effect of TiO₂NP pretreatment on malondialdehyde content under salt stress

Radicle MDA for the Balci cultivar decreased significantly (1.9 fold) under the TiO₂NP + NaCl treatment compared to NaCl treatment. Plumula MDA for the Balci cultivar decreased significantly (by 7.2%) under the TiO₂NP + NaCl treatment compared to the NaCl treatment. Radicle MDA for the Dinçer cultivar increased significantly (1.5 fold) under the TiO₂NP treatment compared to the NaCl treatment Plumula MDA for the Dinçer cultivar did not differ significantly between the TiO₂NP + NaCl treatment and the NaCl treatment (Fig. 3).

Effect of TiO₂NP pretreatment on hydrogen peroxide content under salt stress

Radicle hydrogen peroxide for the Dinçer cultivar decreased significantly (1.45 fold) under the TiO₂NP + NaCl treatment compared to the NaCl treatment Radicle hydrogen peroxide for the Balci cultivar did not differ significantly between the TiO₂NP + NaCl treatment and the NaCl treatment. Plumula hydrogen peroxide for the Dinçer cultivar increased significantly (1.6 fold) under the TiO₂NP + NaCl treatment compared to the NaCl treatment. Plumula hydrogen peroxide for the Balci cultivar decreased (2.14 fold) under the TiO₂NP + NaCl treatment compared to the NaCl treatment (Fig.4).

Effect of TiO₂NP pretreatment on superoxide radical content under salt stress

Superoxide radical content (O₂ ^•⁻) for the Dinçer cultivar did not differ significantly between the TiO₂NP + NaCl treatment and the NaCl treatment. Similarly, radicle O₂ ^•⁻ content for the Balci cultivar did not differ significantly between the TiO₂NP + NaCl treatment and the NaCl treatment. Plumula O₂ ^•⁻ content for the Dinçer cultivar decreased significantly (3.34 fold) under the TiO₂NP + NaCl treatment compared to the NaCl treatment. Plumula O₂ ^•⁻ content for the Balci cultivar increased significantly (1.93 fold) under the TiO₂NP + NaCl treatment compared to the NaCl treatment (Fig.5).

Effects of TiO₂NP pretreatment on growth parameters under heat stress

Radicle and plumula lengths for both Dinçer and Balci cultivars did not differ significantly between the TiO₂NP + 45 ℃ treatment and the 45 ℃ treatment. (Fig. 6). Radicle fresh weight for both Dinçer and Balci cultivars did not differ significantly among treatments (Fig. 7 A , B). Plumula fresh weight for both Dinçer and Balci cultivars increased with the TiO₂NP treatment compared to the 45 ℃ treatment. Radicle and plumula dry weights for both cultivars did not differ significantly between the 45 ℃ treatment and the TiO₂NP treatment (Fig. 7 B ). Plumula dry weight for both cultivars did not differ among treatment groups (Fig. 7 B ).

Effect of TiO₂NP pretreatment on malondialdehyde content under heat stress

Radicle MDA content for the Dinçer cultivar decreased (by 16.33%) under the TiO₂NP treatment compared to the 45 ℃ treatment (Fig. 8). In addition, radicle MDA content for the Balci cultivar decreased (40.94%) under the TiO₂NP + 45 ℃ treatment compared to the 45 ℃ treatment (10.6). Plumula MDA content for the Dinçer cultivar decreased (22%) under the TiO₂NP + 45 ℃ treatment compared to the 45 ℃ treatment (Fig. 8). Plumula MDA content of the Balci cultivar decreased (4.16%) under the TiO₂NP + 45 ℃ treatment compared to the 45 ℃ treatment.

Effect of TiO₂NP pretreatment on hydrogen peroxide content under heat stress

Radicle hydrogen peroxide content for the Dinçer cultivar decreased (by 43.40%) under the TiO₂NP + 45 ℃ treatment compared to the 45 ℃ treatment. Radicle hydrogen peroxide content for the Balci cultivar increased (3.83 fold) under the TiO₂NP + 45 ℃ treatment compared to the 45 ℃ treatment (Fig. 9). Plumula hydrogen peroxide content for the Dinçer cultivar did not differ significantly between the TiO₂NP + 45 ℃ treatment and the 45℃ treatment. Plumula hydrogen peroxide content for the Balci cultivar decreased (73.61%) under the TiO₂NP + 45 ℃ treatment compared to the 45 ℃ treatment (Fig. 9).

Effect of TiO₂NP pretreatment on superoxide radical content under heat stress

Radicle superoxide radical content for the Dinçer cultivar decreasd (by 35.84%) under the TiO₂NP + 45 ℃ treatment compared to 45 ℃ treatment. Radicle superoxide radical content for the Balci cultivar decreased (51.32%) under the TiO₂NP + 45 ℃ treatment compared to the 45 ℃ treatment (Fig. 10). Plumula superoxide radical contents for both Balci and Dinçer cultivars did not differ significantly between the TiO₂NP + 45 ℃ treatment and the 45 ℃ treatment (Fig. 10).

DISCUSSION

Salt stress treatment

Salt stress causes negative effects on plant growth and development in parallel with increasing amounts of NaCl and other soluble salts in the soil. Increased salt concentration in the soil solution and decreased water potential reduce the osmotic potential of plant cells and cause a series of plant reactions (Acosta-Motos et al. 2017Acosta-Motos JR, Ortuño MF, Bernal-Vicente A, Diaz-Vivancos P, Sanchez-Blanco MJ, Hernandez JA. 2017. Plant responses to salt stress: Adaptive mechanisms. Agronomy 7: 18. ). Therefore, depending on the intensity and duration of salt stress, it can affect many biological events in plants, such as growth, development, germination, cell division and photosynthesis (Farooq et al. 2015Farooq M, Hussain M, Wakeel A, Siddique KH. 2015. Salt stress in maize: Effects, resistance mechanisms, and management. A review. Agronomy for Sustainable Development 35: 461-481.), and limit plant productivity and product quality in agricultural areas (Koca et al. 2007Koca H, Bor M, Ozdemir F, Turkan I. 2007. The effect of salt stress on lipid peroxidation, antioxidative enzymes and proline content of sesame cultivars. Environmental and Experimental Botany 60: 344-351. ).

Nanoparticles, when applied to plants, may cause an elongation of the radicle and plumula after germination. In addition, the concentration and amount of salt entering the plant cell are important factors affecting plant development. Certain concentrations of TiO₂NPs are known to have positive effects on radicle and plumula length against the effects of salt that may come from outside to the plant cell structure, as well as on many other factors such as cell pressure, external pressure, and enzymes (Clément et al. 2013Clément L, Hurel C, Marmier N. 2013. Toxicity of TiO2 nanoparticles to cladocerans, algae, rotifers and plants-effects of size and crystalline structure. Chemosphere 90: 1083-1090. ; Doğaroğlu & Köleli 2016Doğaroğlu ZG, Köleli N. 2016. Effect of titanium dioxide and titanium dioxide-silver nanoparticles on seed germination of lettuce (Lactuca sativa). Çukurova Üniversitesi Mühendislik-Mimarlik Fakültesi Dergisi 31: 193-198. ; Zulfiqar & Ashraf 2021Zulfiqar F, Ashraf M. 2021. Nanoparticles potentially mediate salt stress tolerance in plants. Plant Physiology and Biochemistry 160: 257-268. ). The present study observed increases in radicle length for both Balci and Dinçer cultivars when receiving the TiO₂NP treatment under salinity (Fig. 1). Likewise, Liu et al. (2021Liu LI, Cao Y, Guo QIAOSHENG, Zhu Z. 2021. Nanosized Titanium dioxide seed priming enhances salt tolerance of an ornamental and medicinal plant Paeonia Suffruticosa. Pakistan Journal of Botany 53: 1167-1175. ) reported that the application of TiO₂NPs to peony (P. suffruticosa) plants under salt stress increased the number and length of lateral roots. It was also reported that TiO₂ nanoparticles induced growth by increasing photosynthesis and nitrogen metabolism in fennel (Foeniculum vulgare Mill) and broad bean plants (Khater 2016Khater M. 2016. Effect of TiO2 nanoparticles spraying on fennel plant. Journal of Plant Production 7: 29-34. ). The present results corroborate the findings of these studies, with a TiO₂NP concentration of 200 ppm having a positive effect on radicle length against a NaCl concentration of 50 mM applied to seeds of both safflower cultivars (Fig. 1).

In the present study, radicle and plumula fresh weight for the Balci cultivar increased with TiO₂NP treatment compared to the salt treatment alone, while these values did not change for the Dinçer cultivar (Fig. 2 A ). Similar to the present study, TiO₂NP pretreatment of Dracocephalum moldavica L. plants under salt stress caused an increase in root and stem fresh weights (Gohari et al. 2020Gohari G, Mohammadi A, Akbari A et al. 2020. Titanium dioxide nanoparticles (TiO2 NPs) promote growth and ameliorate salinity stress effects on essential oil profile and biochemical attributes of Dracocephalum moldavica. Scientific Reports 10: 912. ). An interpretation of these results is that TiO₂NP pretreatment of safflower seeds under salt stress increases plant nutrient content by positively affecting the uptake of mineral elements from the soil. Nanoparticles have physicochemical properties and the potential to improve plant metabolism (Giraldo et al. 2014Giraldo JP, Landry MP, Faltermeier SM et al. 2014. Plant nanobionics approach to augment photosynthesis and biochemical sensing. Nature Materials 13: 400-408. ). Due to the compatible structure of nanoparticles, they can increase plant water uptake and cause an increase in fresh weight. It is thought that this increase in fresh weight could be related to increased water uptake.

The present study determined that TiO₂NPs regulate the activity of enzymes involved in nitrogen metabolism, such as nitrate reductase, glutamate dehydrogenase, glutamine synthase, and glutamic-pyruvic transaminase. This helps the plant absorb nitrate and provides the conversion of inorganic nitrogen in protein and chlorophyll structure to organic nitrogen, which increases plant fresh and dry weights (Mishra et al. 2014Mishra V, Mishra RK, Dikshit A, Pandey AC. 2014. Interactions of nanoparticles with plants: An emerging prospective in the agriculture industry. In: Ahmad P, Rasool S (eds.). Emerging technologies and management of crop stress tolerance. Academic Press. p. 159-180 . ). In parallel, salt treatment with nanoparticle treatment increased safflower fresh weight. Similarly, TiO₂NP treatment increased the fresh weight of wheat plants under salt stress (Mustafa et al. 2021b Mustafa N, Raja NI, Ilyas N, Ikram M, Ehsan M. 2021b. Foliar applications of plant-based titanium dioxide nanoparticles to improve agronomic and physiological attributes of wheat (Triticum aestivum L.) plants under salinity stress. Green Processing and Synthesis 10: 246-257. ,)Mustafa, N., Raja, N. I., Ilyas, N., Abasi, F., Ahmad, M. S., Ehsan, M., Proćków J. (2022). Exogenous Application of Green Titanium Dioxide Nanoparticles (TiO2 NPs) to Improve the Germination, Physiochemical, and Yield Parameters of Wheat Plants under Salinity Stress. Molecules, 27(15), 4884.. Another study found that TiO₂ nanoparticle treatment stimulates plant growth and reduces the negative effects of selenium (Marchiol et al. 2016Marchiol L, Mattiello A, Pošćić F, Fellet G, Zavalloni C, Carlino E, Musetti R. 2016. Changes in physiological and agronomical parameters of barley (Hordeum vulgare) exposed to cerium and titanium dioxide nanoparticles. International Journal of Environmental Research and Public Health 13: 332. ). Although radicle dry weight was not changed with TiO₂NP treatment compared to salinity in the present study, plumula dry weight increased for both cultivars (Fig. 2 B ). From these results, the increase in dry weight could be explained by increased organic material accumulation with TiO₂NP treatment of safflower plants.

TiO₂NP treatment decreased radicle and plumula MDA content for the Balci cultivar, while it was not effective for the Dinçer cultivar (Fig. 3). According to the literature, nanoparticle pretreatment applied to seeds has a positive effect for different plants under salt stress (Avestan et al. 2019Avestan S, Ghasemnezhad M, Esfahani M, Byrt CS. 2019. Application of nano-silicon dioxide improves salt stress tolerance in strawberry plants. Agronomy 9: 246. ; Abdoli et al. 2020Abdoli S, Ghassemi-Golezani K, Alizadeh-Salteh S. 2020. Responses of ajowan (Trachyspermum ammi L.) to exogenous salicylic acid and iron oxide nanoparticles under salt stress. Environmental Science and Pollution Research 27: 36939-36953. ; Alabdallah & Alzahrani 2020Alabdallah NM, Alzahrani HS. 2020. The potential mitigation effect of ZnO nanoparticles on [Abelmoschus esculentus L. Moench] metabolism under salt stress conditions. Saudi Journal of Biological Sciences 27: 3132-3137. ). A study similar to that presented here reported that TiO₂NP treatment alleviated the effects of salinity on the cell membrane and stimulated the defense system to reduce MDA (Sheikhalipour et al. 2021Sheikhalipour M, Esmaielpour B, Gohari G et al. 2021. Salt stress mitigation via the foliar application of chitosan-functionalized selenium and anatase titanium dioxide nanoparticles in stevia (Stevia rebaudiana Bertoni). Molecules 26: 4090. ). In a study conducted with fava bean plants under salt stress found that TiO₂NP treatment regulated growth parameters and decreased MDA content, and that this was achieved by increasing antioxidant enzyme activities (Abdel Latef et al. 2018Abdel Latef AAH, Srivastava AK, El‐sadek MSA, Kordrostami M, Tran LP. 2018. Titanium dioxide nanoparticles improve growth and enhance tolerance of broad bean plants under saline soil conditions. Land Degradation and Development 29: 1065-1073. ). In addition, Shah et al. (2021Shah T, Latif S, Saeed F et al. 2021. Seed priming with titanium dioxide nanoparticles enhances seed vigor, leaf water status, and antioxidant enzyme activities in maize (Zea Mays l.) under salinity stress. Journal of King Saud University-Science 33: 101207. ) reported that TiO₂NPs applied to maize seeds with seed-induced proline accumulation (Kishor et al. 2005Kishor PBK, Sangam S, Amrutha RN et al. 2005. Regulation of proline biosynthesis, degradation, uptake and transport in higher plants: Its implications in plant growth and abiotic stress tolerance. Current Science 88: 424-438. ) decreased electrical conductivity and reduced MDA content by adjusting osmotic potential.

Similar to MDA, the hydrogen peroxide results of this study showed an alleviation effect of TiO₂NPs (reduced hydrogen peroxide content) in the plumula of the Balci cultivar under salinity, while this did not occur for the radicle. In addition, while these values were increased in the plumula of the Dinçer cultivar, a reduction was observed in radicle (Fig. 4). In parallel with the present results, the H₂O₂ content of plants under salinity stress and treated with TiO₂NPs was determined to be higher than for plants treated with salt alone (Karami & Sepehri 2018Karami A, Sepehri A. 2018. Effect of nano titanium dioxide and sodium nitroprusside on seed germination, vigor index and antioxidant enzymes of Afzal barley seedling under salinity stress. Iranian Journal of Seed Science and Research 5: 47-61. ). The present study found a negative regulation of H₂O₂ for the plumula of the Dinçer cultivar due to different production sites, so it can be said that when H₂O₂ is increased it acts as a signal molecule. Decreased H₂O₂ and MDA content with TiO₂NP treatment of sugar grass plants (Stevia rebaudiana Bertoni) under salt stress was reported to be due to antioxidant enzyme activity (Sheikhalipour et al. 2021Sheikhalipour M, Esmaielpour B, Gohari G et al. 2021. Salt stress mitigation via the foliar application of chitosan-functionalized selenium and anatase titanium dioxide nanoparticles in stevia (Stevia rebaudiana Bertoni). Molecules 26: 4090. ). Lashkary et al. (2021Lashkary M, Moghaddam M, Asgharzade A, Tatari M. 2021. Titanium dioxide nanoparticle is involved in mitigating NaCl-induced Calendula officinalis L. by activation of antioxidant defense system and accumulation of osmolytes. Plant Physiology and Biochemistry 166: 31-40. ) reported that decreased H₂O₂ and MDA content caused an increase in the antioxidant system and osmolyte and biomass accumulation in the marigold Calendula officinalis L. under salt stress. In addition, it is thought that decreased radicle H₂O₂ content may be achieved by increased enzyme activities of the related defense system.

The results showed that O₂ ^•- radical content only had a healing effect for the Dinçer cultivar when under salinity stress and TiO₂NP pretreatment (Fig. 5). Singh et al. (2021Singh P, Arif Y, Siddiqui H et al. 2021. Nanoparticles enhances the salinity toxicity tolerance in Linum usitatissimum L. by modulating the antioxidative enzymes, photosynthetic efficiency, redox status and cellular damage. Ecotoxicology and Environmental Safety 213: 112020. ), found that ZnNP treatment of flax (Linum usitatissimum) plants under salt stress reduced O₂ ^•- radical content. Evaluation of these results leads to the interpretation that increased H₂O₂ content improves oxidative damage to the cell membrane by increasing superoxide dismutase (SOD) enzyme activity, which ensures water balance in plants under salt stress, provides ionic balance, and decreases electrical conductivity.

Heat stress treatment

TiO₂NP pretreatment increased root length, shoot length, fresh weight, and dry weight of wheat under well-irrigated and drought conditions (Mustafa et al. 2021a Mustafa H, Ilyas N, Akhtar N et al. 2021a. Biosynthesis and characterization of titanium dioxide nanoparticles and its effects along with calcium phosphate on physicochemical attributes of wheat under drought stress. Ecotoxicology and Environmental Safety 223: 112519. ). The current study found no change in radicle and plumula lengths for the Balci and Dinçer cultivars under heat and TiO₂NP treatment, compared to heat alone (Fig. 6). In contrast, a study of wheat determined that Zn/TiO₂NPs improve root length and plant water condition by increasing water uptake under heat stress (32 ℃) (Thakur et al. 2021Thakur S, Asthir B, Kaur G, Kalia A, Sharma A. 2021. Zinc oxide and titanium dioxide nanoparticles influence heat stress tolerance mediated by antioxidant defense system in wheat. Cereal Research Communications 50: 385-396. ). In addition, the application of SiO₂NPs to barley plants under drought stress reduces OH^.- radical content and membrane damage, and increases stem length and relative water content (Yildiz 2018Yildiz S. 2018. Kuraklik Stresi Altindaki Arpa Bitkilerinin Yapraklarina SiO2 Nanopartikül Uygulamasinin Etkilerinin İncelenmesi. Mersin Üniversitesi. Fen Bilimleri Enstitüsü. Biyoteknoloji Anabilim Dali Yüksek Lisans Tezi Mersin: 52.). TiO₂NP treatment of plants has been shown to accelerate growth under normal conditions and drought stress, and has been used in different concentrations for different plants under adverse environmental conditions (Selahvarzi et al. 2020Selahvarzi Y, Kamali M, Ahmadpour Mir H. 2020. Effect of nano and balk titanium dioxide on flowering and morphophysiological traits of Rosa damascena under different irrigation regimes. Journal of Plant Process and Function 9: 143-156. ; Sattari & Khayati 2020Sattari R, Khayati GR. 2020. Prediction of the size of silver nanoparticles prepared via green synthesis: A gene expression programming approach. Scientia Iranica 27: 3399-3411. ). Thakur et al. (2021Thakur S, Asthir B, Kaur G, Kalia A, Sharma A. 2021. Zinc oxide and titanium dioxide nanoparticles influence heat stress tolerance mediated by antioxidant defense system in wheat. Cereal Research Communications 50: 385-396. ) reported that pretreatment with different concentrations of ZnNPs caused increases in root and stem lengths of wheat plants under heat stress. TiO₂NP treatment of plants from seed was found to ameliorate damage caused by heat stress by increasing plant photosynthetic capacity (closure of stomata, transpiration rate, regulation of electron transfer), thus regulating plant growth (Qi et al. 2013Qi M, Liu Y, Li T. 2013. Nano-TiO2 improve the photosynthesis of tomato leaves under mild heat stress. Biological Trace Element Research 156: 323-328. ).

The photosynthesis mechanism is fundamental in plants and one of the structures most affected by abiotic stress factors. TiO₂NP treatment has been reported to increase the rate of photosynthesis by decreasing oxidative damage under stress conditions, with positive effects on plant growth and improved crop yield by increased production (Mohammadi et al. 2014Mohammadi R, Maali-Amiri R, Mantri NL. 2014. Effect of TiO2 nanoparticles on oxidative damage and antioxidant defense systems in chickpea seedlings during cold stress. Russian Journal of Plant Physiology 61: 768-775. ; Singh & Lee 2016Singh J, Lee BK. 2016. Influence of nano-TiO2 particles on the bioaccumulation of Cd in soybean plants (Glycine max): A possible mechanism for the removal of Cd from the contaminated soil. Journal of Environmental Management 170: 88-96. ). In the present study, TiO₂NP pretreatment (200 ppm) of seeds of Balci and Dinçer safflower cultivars under heat stress (45 ℃) did not show any significant differences from the other treatments for radicle fresh weight (Fig. 7 A , B). In contrast, external treatment with TiO₂NPs and ZnNPs caused an increase in fresh and dry weights of plants of two wheat cultivars under water deficit (El-Bassiouny et al. 2022El-Bassiouny HMS, Mahfouze HA, Abdallah MMS, Bakry BA, El-Enany MAM. 2022. Physiological and molecular response of wheat cultivars to titanium dioxide or zinc oxide nanoparticles under water stress conditions. International Journal of Agronomy 2022: 3806574.). The present study found an increase (1.9 times) in plumula fresh weight for the TiO₂NP treatment of the Dinçer cultivar under heat stress compared those not receiving the treatment and under heat stress. Plumula fresh weight increased (1.5 fold) with the heat + TiO₂NPs treatment (Fig. 7 A ). Similar to the present study, foliar TiO₂NP treatment of tomato plants under heat stress was reported to increase photosynthesis rate and leaf water condition (Qi et al. 2013Qi M, Liu Y, Li T. 2013. Nano-TiO2 improve the photosynthesis of tomato leaves under mild heat stress. Biological Trace Element Research 156: 323-328. ).

Considering these results, heat treatment positively affected radicle and plumula MDA content by causing a decrease for both safflower cultivars. Thus, it can be said that the reason for this protection from MDA is that TiO₂NP treatment provides cell membrane repair, ionic balance, and preservation of cell membrane integrity (Faran et al 2019Faran M, Farooq M, Rehman A, Nawaz A, Saleem MK, Ali N, Siddique KH. 2019. High intrinsic seed Zn concentration improves abiotic stress tolerance in wheat. Plant and Soil 437: 195-213. ; Thakur et al. 2021Thakur S, Asthir B, Kaur G, Kalia A, Sharma A. 2021. Zinc oxide and titanium dioxide nanoparticles influence heat stress tolerance mediated by antioxidant defense system in wheat. Cereal Research Communications 50: 385-396. ). The present study found an increase in hydrogen peroxide content in the radicle of the Balci cultivar, but a decrease in its plumula (Fig. 9). In addition, these values were decreased in radicle of the Dinçer cultivar, with no change in its plumula. In parallel with this, hydrogen peroxide has been reported to act as a signal molecule due to differences in the location of its production (Nazir et al. 2020Nazir F, Fariduddin Q, Khan TA. 2020. Hydrogen peroxide as a signalling molecule in plants and its crosstalk with other plant growth regulators under heavy metal stress. Chemosphere 252: 126486. ), and decreased H₂O₂ content is mediated by catalase (CAT) and glutathione peroxidase (GPX) enzyme activities and the involvement of phenol and flavonoids in ROS scavenging (Zafar et al. 2016Zafar H, Ali A, Ali JS, Haq IU, Zia M. 2016. Effect of ZnO nanoparticles on Brassica nigra seedlings and stem explants: Growth dynamics and antioxidative response. Frontiers in Plant Science 7: 535. ).

Based on these results, TiO₂NPs applied to broad bean (Vicia faba L.) plants under water deficit stress caused a decrease in the amount of O₂ ^•- radicals by stimulating the defense system, providing water balance through osmolyte accumulation, and causing an increase in internal nitric oxide (Khan et al. 2020Khan MN, AlSolami MA, Basahi RA, Siddiqui MH, Al-Huqail AA. Abbas Z. K. et al. 2020. Nitric oxide is involved in nano-titanium dioxide-induced activation of antioxidant defense system and accumulation of osmolytes under water-deficit stress in Vicia faba L. Ecotoxicology and Environmental Safety 190: 110-152.). In addition, Shoarian et al. (2020Shoarian N, Jamei R, Pasban Eslam B, Salehi Lisar SY. 2020. Titanium dioxide nanoparticles increase resistance of L. iberica to drought stress due to increased accumulation of protective antioxidants. Iranian Journal of Plant Physiology 10: 3343-3354. ) reported that TiO₂NP treatment under drought stress was effective at scavenging ROS by stimulating SOD enzyme activity. The present study found a decrease in O₂ ^•- radical content for the radicle of both cultivars under heat stress (Fig. 10), which can be explained by the decrease in H₂O₂ content and the increase in SOD enzymatic activity in the scavenging of O₂ ^•- radicals.

CONCLUSION

This study is the first to investigate the effect mechanisms of TiO₂NP application for two safflower cultivars (Balci and Dinçer) under salt and heat stress. TiO₂NP pretreatment positively affected both cultivars under heat stress, but only the Balci cultivar under salt stress. This improvement seems to be achieved by the stimulation of the antioxidant defense system or by preserving cell membrane integrity. Considering the results, how TiO₂NP pretreatment under salt and heat will affect safflower plants at the seedling stage remains a matter of curiosity. In addition, changes in the related antioxidant defense system need to investigate using biochemical and molecular approaches.

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