The Role of Nano-selenium in Alleviating the Effects of Salt Stress in Date Palm Trees (Phoenix dactylifera L.): a Fourier Transform Infrared (FTIR) Spectroscopy Study

Fourier transform infrared (FTIR) spectroscopy was used to determine the biochemical changes in date palm leaves which induced different NaCl concentrations (2.5 (control), 5, 10 and 20 dS m−1). It was also used to determine the potential role of selenium nanoparticles (Se NPs) at concentrations of 80 and 160 ppm in alleviating salinity stress of date palm (Phoenix dactylifera L.) trees. Results showed the appearance of a new peak at 2850 cm−1 in the lipid region (2800–3000 cm−1) when date palm trees were exposed to salinity stress. This peak was not observed in the control treatment or when salinity was combined with foliar spraying of Se NPs. Furthermore, a clear and distinct peak at 1735 cm−1 was only seen in plants exposed at 10 or 20 dS m−1 salinity. This peak was attributed to membrane lipid compounds that contain carbonyl ester groups. In addition, the findings demonstrated that the treatments affected the secondary structure of proteins (1500–1800 cm−1) and carbohydrates (1200–1500 cm−1). This was evident by the appearance and disappearance of some characteristic peaks in these regions.


Introduction
Date palm is one of the oldest and most valuable trees grown mainly in the southern part of Iraq. It has nutritional, economic and social importance and is also used as a design for landscape decoration [1]. Salinity in the southern part of Iraq is caused by insufficient rainfall and over-irrigation with brackish or salty water [2]. Date palm can resist high soil salt concentrations. However, irrigation with brackish water results in high salt levels that significantly reduce fruit production and decrease the number of date palm trees that are still alive [3]. The soil salinity tolerance varies amongst date palm cultivars. Certain date palm cultivars can tolerate a high soil salt level of 12.8 dS m −1 (1 dS m −1 = 640 mg l −1 ) without any discernible effects on the seedling phenotype [4]. Meanwhile, some cultivars can tolerate soil salt levels of up to 9 dS m −1 [5]. Salt stress is a harmful environmental stressor because it simultaneously produces ionic toxicity and osmotic and oxidative stress [6]. The metabolic equilibrium of plants is interrupted and altered when they are grown in stressful environments. For example, the activities of several genes and proteins are drastically altered when the salt level is increases, thus altering plant metabolism [7]. With the existence of nanoparticles (NPs), nanotechnology has attracted the interest of scientists across various fields. It plays a vital role in various areas of our lives, including health, manufacturing, agriculture, electronics, energy and the environment [8]. Recently, nano-compound materials have attracted the interest of agricultural experts because of their unique features [9]. As NPs have a larger surface area than bulk materials, they have greater solubility and surface reactivity [10]. Despite being an essential element of animal and human nutrition, selenium (Se) is not regarded as a necessary element for higher plants. [11]. Recently, selenium nanoparticles (Se NPs) have been employed as a foliar treatment to protect plants from extreme stress by improving antioxidant defence systems, photosynthetic indices and secondary metabolism [12]. Some studies have conducted foliar spraying of nano-selenium on coriander [13] and strawberry [14] and common bean (Phaseolus vulgaris) [12] to improve their tolerance to salinity and mitigate its harmful effects. The application of metabolomic technologies for a more thorough investigation of cellular metabolites provides a reasonable way to look into the wide ramifications of these metabolic changes [15]. Metabolomics employs a range of analytical technologies, including Fourier transform infrared (FTIR) technology. FTIR spectroscopy is more sensitive than other analytical methods. This non-destructive approach recognises functional groups and offers structural and chemical changes in response to biotic or abiotic stress [16]. FTIR spectroscopy is used to assess the changes in plant response to salt stress [17,18]. However, to our knowledge, no study has used FTIR spectroscopy to detect the response of date palm to salinity stress. In the current study, FTIR spectroscopy was used to detect the changes in functional groups in date palm leaves as a response to salinity stress with or without a foliar spray of nano-selenium.

Materials and Methods
This study was conducted in a private date palm orchard in Basrah, Iraq. A total of 36 date palm trees (6 years old, Barhi cultivar) were selected and then divided into three blocks, with each block containing a replication. The experiment consisted of 12 treatments, which were the combination of four levels of saline solution (2.5, 5, 10 and 20 dS m −1 ) applied with irrigation water and two concentrations of Se NPs (80 and 160 ppm) applied as foliar spray. Each salinity level was applied alone or in combination with one of the tested concentrations of Se NPs. The treatments were applied from January 1, 2021, to June 30, 2022. Saline irrigation treatments were applied to the experiment plants every 15 days, whereas Se NPs spraying treatments were applied monthly. Fruit bunches were covered to protect them from spray solutions.

Preparation of Saline Solutions
A volumetric flask containing 80 ml of distilled water was used to create 2.5, 5, 10 and 20 dS m −1 saline solutions. To create 2.5 dS m −1 saline solution, 1.6 g of NaCl (HiMedia, India) was added to the volumetric flask. Once the salt had completely dissolved, water was added to reach a volume of 1000 ml. The remaining saline solutions (5, 10 and 20 dS m −1 ) were then created by dissolving 3.2, 6.4 and 12.8 g of NaCl, respectively. Afterwards, the electrical conductivity (Ec) of the solution was read to confirm the needed level.

Preparation of Nano-selenium
Two concentrations of Se NPs (Se, purity, 99.9%; APS, < 80 nm) solutions were produced (80 and 160 ppm). Se NP treatments were used as foliar sprays and applied on leaves, and each level of salinity was added to the soil irrigated with water. The experimental treatments were continued up to 180 days, after which samples were obtained for FTIR analysis.

FTIR Spectroscopy
After 180 days of experimental treatments, samples from the three replicates of treatments were utilised for FTIR analysis. The samples were oven-dried for 2 days at 60 °C. Leaf powder (2 mg) was mixed with KBr (1:100 p/p) in an agate mortar to make tablets for FTIR spectroscopy, and the absorbance spectra between 400 and 4000 cm −1 were obtained. The spectra were collected using an FTIR spectrometer (Jasco FTIR 4200, USA).

Results and Discussion
FTIR technology was used to monitor the biochemical changes in date palm leaves exposed to various salinity levels and the possible involvement of Se NPs in reducing salinity stress. The functional group region, which comprises the stretching vibrations or group frequencies of typical functional groups, extends the high-frequency range from 3000 to 1200 cm −1 . Therefore, according to [16], the lipids region, proteins and carbohydrates were allocated at a range of 3000-2000 cm −1 , 1800-1500 cm −1 and 1500-1200 cm −1 , respectively.

3000-2000 cm −1 Lipid Region
A peak at 2920 cm −1 was detected in the lipid region in the control treatment (Fig. 1a), whereas another peak at 2850 cm −1 was observed at all levels of salinity treatments (Fig. 1b-d). The peak at 2850 cm −1 disappeared when salt stress was combined with foliar sprays of Se NPs at 80 or 160 ppm (Figs. 2 and 3a-d).
The asymmetric and symmetric stretching vibrations of (CH 2 ) groups are assigned to the bands at 2920 and 2850 cm −1 , respectively, and correspond to the methylene groups in fats, wax and lipids [17,19]). The lipid content changes in stressed plants indicate a decrease in − CH 2 /peroxide and hydrogen peroxide, which can be used as a biomarker for lipid peroxidation in the primary cell membrane component [20]. In a previous study [17], the absorption strength of around 2850 cm −1 was enhanced in saltstressed jojoba plants compared with control plants.

1800-1500 cm −1 Protein Region
Bands linked with protein secondary structures are seen in the 1800-1500 cm −1 range, particularly the amide I and II bands  [21]. A peak appeared at 1735 cm −1 in the spectra of plants that were only exposed to 10 or 20 dS m −1 salinity stress. The C = O group has an absorption band at approximately   [22]. The amide I band is closely tied to the backbone conformation and is mainly involved with the C = O stretching vibration (70%-80%) [19]. In this work, a peak was observed in the amide I region, which appeared between 1616 and 1644 cm −1 (Figs. 1-3). According to a previous study [23], the bands at 1605-1620, 1620-1630, 1630-1637 and 1638-1646 cm −1 are attributed to side chains, cross β-sheets, parallel β-sheets and unordered structures, respectively. Table 1 illustrates the peaks that appeared in the 1700-1600 cm −1 region after treatments and the spectral components associated with various secondary structures. Amide II is produced by N-H bending vibrations (40%-60%) and C-N stretching vibrations (18%-40%) [19]. Figures 1-3 show amide II (1500-1600 cm −1 ) bands in the spectra of date palm leaves. Each treatment had a peak that varied between 1520 and 1515 cm −1 except for foliar spray with Se NPs at 160 ppm paired with t 2.5 (control) and 10 dS m −1 salinity and at 80 ppm coupled with 20 dS m −1 salinity, where the band in this region disappeared. The N-H bending in the plane, C-N stretching and C-H in plane bending of phenyl rings (tyrosine) generated the bands in this location (amide II) [24].
Amide I and II bands can effectively detect protein IR absorption changes when they are exposed to salt. The amide I region (1700-1600 cm −1 ) mostly represents polypeptide C = O stretching vibrations, which can detect changes in the overall protein structure and content [17]. In addition, the changes in proteins in the amide I region might be produced by their composition changes under salt stress [18].

1500-1200 cm −1 Carbohydrate Region
This region is known as 'local symmetry', mainly consisting of deformational vibrations of groups with local symmetry, such as CH 2 and various C-OH deformations found in carbohydrates [25]. Two peaks were found in this study. One peak ranged between 1554 and 1437 cm −1 and appeared in all treatment samples, except for those collected from water irrigation with a 20 dS m −1 salinity combined with foliar spray of 80 ppm Se NPs and 2.5 dS m −1 salinity coupled with foliar spray of 160 ppm Se NPs (Figs. 1-3). The other peak ranged between 1269 and 1247 cm −1 . This band appeared only in six different treatments levels. Notably, this band did not exist in the control treatment [18]. However, it appeared at 1269, 1251 and 1247 cm −1 in samples treated with 5, 10 and 20 dS m −1 salinity alone, respectively. Furthermore, this band appeared at 1253 and 1251 cm −1 in samples treated with foliar spray of Se NPs at 80 ppm combined with 2.5 and 10 dS m −1 salinity, respectively. It also appeared at 1255 cm −1 in samples treated with foliar spray of Se NPs at 160 ppm coupled with 5 dS m −1 salinity. Peaks appearing or disappearing in this region indicate an alteration in carbohydrate structure caused by the effect of treatments. Previously, FTIR spectroscopy was used to detect changes in carbohydrates under salinity stress [13], 15].

Conclusions
The biochemical response of date palm plants to salt stress and the possible function of Se NPs in reducing the adverse effects of salinity were studied using FTIR spectroscopy. The current study demonstrated the importance of FTIR spectroscopy as a quick and efficient tool for identifying the effects of salinity stress on date palm trees. It also demonstrated the role of Se NPs in mitigation salinity by comparing different variations in the characteristic peaks of the regions of macromolecules in the FTIR spectrum, particularly the lipid region. The results suggest that moderate doses of Se NPs spraying may be used to reduce the detrimental impacts of salt on date palms. However, more research is required to determine the toxicity of this nanomaterial in the fruits.