52 ACTA BOT. CROAT. 82 (1), 2023 Acta Bot. Croat. 82 (1), 52–59, 2023 CODEN: ABCRA 25 DOI: 10.37427/botcro-2023-001 ISSN 0365-0588 eISSN 1847-8476 Effect of excess boron on growth, membrane stability, and functional groups of tomato seedlings Abeer A. Radi1, Hussein Kh. Salam2, Afaf M. Hamada1*, Fatma A. Farghaly1 1 Botany and Microbiology Department, Faculty of Science, Assiut University, Assiut 71516, Egypt 2 Biology Department, Faculty of Applied Science, Thamar University, Dhamar, Yemen Abstract - With the scarcity of good quality water, plants like tomatoes will be more susceptible to excess boron (EB) in Mediterranean regions. The effects of EB on the growth, free, semi-bound, and bound boron (B) concentrations, and macromolecules of the Solanum lycopersicum L. cultivar Castle Rock, were investigated in this study. Seedlings were exposed to four levels of EB using boric acid. The results showed that EB inhibited tomato growth, total water content, and photosynthetic pigments. EB harmed membrane stability, as seen by increased potassium (K) leakage, UV absor- bance metabolites, and electrolyte conductivity (EC) in leaf disc solution. EB raised concentrations of free, semi-bound, and bound forms of B in seedlings. Fourier-transform infrared spectroscopy (FTIR) data revealed that EB induced un- even wax deposition, altered the shape of cell walls, and lowered cellulose synthesis in seedlings. EB affected the amide I and amide II proteins indicating damage to the protein pools. These results provide new insights into understanding the specific effects of EB on the functional groups of different macromolecules of tomato seedlings. Keywords: excess boron, FTIR analysis, membrane stability, photosynthetic pigments, plant growth, tomato Introduction Boron (B) performs vital tasks in plant life at ideal levels, whereas excess boron (EB) has negative consequences. The difference in B insufficiency and toxicity levels is minimal (Fang et al. 2016). B-rich soils can be found all over the world and are prevalent in arid and semi-arid areas (Ardıc et al. 2009). With reduced precipitation in the Mediterra- nean area (Cervilla et al. 2012) and irrigation water shortage due to new dams, demand for desalinated water for agricul- ture is expected to rise, potentially raising the level of B in irrigation water. Moreover, rising sea levels (Mediterranean Sea) can pollute groundwater, resulting in higher B levels in irrigation water (Princi et al. 2016). In Egypt the cultivated area is suitable for intensive cultivation and this, along with anthropogenic activity, may lead to B contamination ( Elbehiry et al. 2017). EB produces different physiological and morphological changes in plants, resulting in decreased plant growth, leaf chlorophyll, membrane stability ( El-Shazoly et al. 2019), and ultimately reduced production (Metwally et al. 2018). Tomatoes are grown all over the Mediterranean region, where there is a disturbance with EB in the soil. Tomatoes are among the most important vegetable crops in Egypt throughout the year, with a total production of 6,729,004 tons and a total cultivation area of 166,206 hectares (FAO- STAT 2017). EB has led to alterations in tomatoes, including biomass, membranes, photosynthetic pigments, phenolic compounds, and antioxidant enzymes (Cervilla et al. 2012, Farghaly et al. 2022b), leading to reduced yields (Kaya et al. 2009). The advantage of Fourier-transform infrared spectros- copy (FTIR) is its ability to produce spectra on different samples such as powders and liquids with minimal sample preparation, which reduces analysis time (Canteri et al. 2019). FTIR is an appropriate analytical tool for biological macromolecules, assessing the composition of organic com- ponents (Wu et al. 2017). Absorption outlines show fixed peaks area that identifies modest modifications of metabo- lites related to physiological processes after infrared spectra (400–4000 cm–1) pass through plant samples (Renuka et al. 2016). The peak areas, positions, and bandwidth values are critical to changes in plant macromolecules (Renuka et al. 2016). However, to our knowledge, there are no reports on the use of this technique to assess physiological changes produced by excess boron on tomato seedlings. In this study, we aimed to focus on how EB treatments alter tomato macromolecules, assessing the composition of * Corresponding author e-mail: hamada@aun.edu.eg, afafhamada@yahoo.com mailto:hamada@aun.edu.eg mailto:afafhamada@yahoo.com BORON TOXICITY AND TOMATO SEEDLING GROWTH ACTA BOT. CROAT. 82 (1), 2023 53 organic components using FTIR analysis. Additionally, we measured the growth, photosynthetic pigments, membrane stability, and B forms concentration in tomato seedlings. The findings reveal a fresh understanding of the various struc- tural responses of tomato seedlings when exposed to EB. Materials and methods Growth conditions Vegetables Department, Faculty of Agriculture, Assiut University gave seeds of Solanum lycopersicum L. (tomato), cultivar Castle Rock. Under a laminar airflow hood, seeds were surface sterilized for 15 minutes with a 5% NaClO so- lution and rinsed four times with sterile water. We wanted to achieve data without any extraneous influences and re- peat the experiment under the same settings, so we did it in vitro. Sterilized seeds were grown on sterile, half-strength Murashige and Skoog (MS) medium (Murashige and Skoog 1962). The medium was supplemented with 2.2 g L–1 MS, 3% sucrose, various concentrations (0, 2, 4, and 6 mM) of H3BO3, and 0.3% gelrite added after adjusting the pH to 5.7. The medium was sterilized for 15 min at 121 °C, pressed at 105 kPa, and allowed to cool to room temperature. Seed- lings were cultured in a growth chamber at 25 ± 1 °C, 65–70% relative humidity, and a photoperiod of 16/8 h with 30 μM m–2 S–1 illumination. After 20 days, some seedlings were separated into shoots and roots, weighed quickly to estimate the fresh weight (FW), and stored at –80 °C. Other seedlings were oven- dried at 60 °C to determine the dry weight (DW). The total water content (TWC) of shoots and roots was determined using the following formula: TWC = FW – DW Photosynthetic pigments Using a spectroscopic approach, photosynthetic pig- ments, including chlorophyll a (chl a), chlorophyll b (chl b), and carotenoids (cars), were determined (Lichtenthaler 1987). 0.1 g of a fresh leaf was dropped in 5 mL of 95% eth- anol at 60 °C until colorless, and the volume was then fin- ished to 10 mL using 95% ethanol. Using a spectrophotom- eter (Unico UV-2100), the concentrations of carotenoids and chlorophylls were determined using formulas: chl a = (13.36 × A663) – (5.19 × A644) chl b = (27.49 × A644) – (8.12 × A663) cars = {(1000 × A452) – (2.13 × chl a) – (9.76 × chl b)}/209. Results were expressed as mg g–1 FW. Cell membrane stability Different parameters were assessed, including electrical conductivity (EC%), potassium leakage (K leakage), and UV-absorbing metabolites (metabolite leakage) for deter- mining the cell membrane stability. The percentage of injury (electrical conductivity; EC) was measured according to the Premachandra et al. (1992) method. Fresh leaf discs (2.1 cm) were soaked in 10 mL of distilled water for 24 h at 10 °C. After measuring the initial electrical conductivity (EC1) of all test tubes at 25 °C, the leaf discs were autoclaved for 15 min, cooled to 25 °C, and then the last EC2 was measured again. The cell membrane stability index was estimated using a percentage of damage: Electrical conductivity (%) = (EC1/EC2) × 100 Potassium leakage was measured using a flame photom- eter in the same conductivity solution before and after ster- ilization (Williams and Twine 1960). Metabolite leakage was assessed using the Navari-lzzo et al. (1989) method in the same solution of conductivity measurements. Boron analysis Boron forms were extracted, according to Du et al. (2002) and Li et al. (2017). 5 mL of distilled water was added to 0.2 g of powdered dry seedlings, shaken at 25 °C for 24 h, filtered, and the free B was measured. The residue was shak- en at 25 °C for 24 h in a plastic tube with 1 M NaCl, filtered, and then semi-bound B was quantified in the filtrate. Fi- nally, the residue was shaken at 25 °C for 24 hours in a plastic tube with HCl (1 M), filtered, and then the bound B was quantified in the filtrate. According to Mohan and Jones (2018), B concentration was quantified using the curcumin-acetic acid method and detected at 550 nm. The curcumin-acetic acid (1 mL) solu- tion was added to 1 mL of filtrates and 0.25 mL of concen- trated H2SO4, shaken for 30 min, and diluted with 95% eth- anol to 5 mL after 30 min read at 550 nm using H3BO3 as a reference. Fourier-transform infrared spectroscopy (FTIR) analysis To analyze macromolecular alteration, we employed Fourier-transform infrared spectroscopy (Nicolet IS 10 FTIR) in Chemistry Department. A translucent, homogeneous tablet was prepared by a tablet-making machine using a little amount of the finely powdered sample (approximately 100 μg) mixed with KBr (1: 100 p/p). The absorbance of spectra was measured (400–4000 cm–1) against an ordinary KBr pellet (blank), then the resolution was 4 cm–1. The functional groups of the sample were determined by comparison of the spectroscopic result with a reference. Statistical analysis The studies (25 jars/each treatment) were repeated at least twice, with the findings being an average ± standard deviation (SD) of four biological replicates. Charts were gen- erated by Origin 8.6 and Microsoft Excel 2010. Using SPSS RADI A.A., SALAM H.KH., HAMADA A.M., FARGHALY F.A. 54 ACTA BOT. CROAT. 82 (1), 2023 Statistical Package 22.0, a one-way analysis of variance test was performed and followed by a Tukey’s test for significant differences (P � 0.05) to compare the means. The correla- tion between the mean values of different parameters of to- matoes under EB treatments was determined using Pear- son’s correlation analysis. Results Growth EB affected all growth parameters of the tested tomato seedlings, including FW, DW, and TWC of shoots and roots (Figs. 1A-D, On-line Suppl. Fig. 1). Compared to control, tomatoes treated with 2 mM B showed a slight or consider- able increase in FW, DW, and TWC of shoots and roots. In contrast, treatments with 4 and 6 mM B lowered FW, DW, and TWC of shoots and roots. After exposure to 6 mM B, the highest DW reductions of 56.90% and 86.52% were re- corded in shoots and roots, respectively. Further, treatment with EB showed a significant negative association between shoots and roots, FW, DW, TWC, and an increase in free, semi, and bound B contents. However, the shoot/root ratio was positively and strongly associated with free, semi- bound, and bound B concentrations in the shoots (0.907**, 0.922**, and 0.646*, respectively, On-line Suppl. Tab. 1 and Tab. 2). Although the correlations between bound B and growth parameters were significant, they were the weakest of all the growth criteria associations. Photosynthetic pigments EB had varied effects on the contents of chl a, b, a+b, and cars pigments in leaves (Fig. 2A). Compared to control, EB at a low-level (2 mM) stimulated chl a content in leaves by 31.28%, but at a high-level (6 mM), it significantly reduced it by 48.34%. In the shoots, there were strong negative as- sociations between chl a and free, semi-bound, and bound B content (–0.768**, –0.822**, and –0.812**, respectively, On-line Suppl. Tab. 1). Low EB treatments promoted the synthesis of chl b in tomato leaves. Compared to control, the rise in chlorophyll b content at low EB levels (2 and 4 mM) was considerable (129.45% and 66.23%, respectively), but there was no sig- nificant increase at a high EB level (6 mM). Insignificant relationships between chl b and free, semi-bound, and bound B levels in shoots confirmed these findings (–0.224, –0.311, and –0.341, respectively, On-line Suppl. Tab. 1). 6 mM B reduced chl a + b concentration by 42.69% com- pared to control. The moderate treatment (4 mM B) showed a lower increase in the chl a + b content (0.63%) than expo- sure to 2 mM B, which resulted in a 40.66% rise compared to control. Moreover, results revealed a strong negative as- sociation between chl a + b and free, semi-bound, and bound B content in shoots (–0.704*, –0.767**, and –0.675**, respectively, On-line Suppl. Tab. 1). Regarding carotenoids, EB boosted cars content by 37.18% at a low-level (2 mM) but lowered it by 44.72% at a Fig. 1. Fresh (A), dry weight (B), total water content (C), and shoot/root ratio (D) in tomato seedlings grown under 0, 2, 4, and 6 mM boron for 20 days. The data are means ± SD (n = 4). Different letters, capital for roots and small for shoots, indicate statistically sig- nificant differences (P ≤ 0.05). BORON TOXICITY AND TOMATO SEEDLING GROWTH ACTA BOT. CROAT. 82 (1), 2023 55 high level (6 mM) compared to control. Carotenoids and free, semi-bound, and bound B levels had negative associa- tions (–0.655*, –0.593*, and –0.686*, respectively), like chlo- rophylls (On-line Suppl. Tab. 1). Moreover, the data revealed a significant and positive relationship between chl a, a + b, cars, and shoot DW (0.669*, 0.734**, and 0.785**, respec- tively), except for chl b, which was not. Boron concentrations The most important factor in measuring a plant’s toler- ance to EB is the B concentration in its tissues. Therefore, the B forms in tomatoes grown under various EB treatments were measured (Fig. 2B). Our results indicated that free B content was higher than the content of semi-bound and bound B content in seedlings. Our results indicated that free B content was higher than semi-bound and bound B content in seedlings. With increasing EB concentrations, the accu- mulation of all B forms also increased. Compared with op- timal B concentration, EB at the low level (2 mM) increased free, semi-, and bound B by 25.41%, 37.40%, and 88.61%, while the high level (6 mM) increased it by 149.69%, 134.98%, and 367.93%, respectively. Fig. 2. Photosynthetic pigments (chl a; chl b; chl a + b; cars; A) and boron concentration (free, semi-bound; and bound B) in tomato seedlings grown under 0, 2, 4, and 6 mM boron for 20 days. The data are means ± SD (n = 4). Different letters, capital for chl a, free B and small for chl b, semi-bound B, small1 for chl a + b, bound B, small2 for cars, indicate statistically significant differences (P ≤ 0.05). Fig. 3. Electrical conductivity (EC; A), potassium leakage (K leakage; B), and UV absorbing metabolites (metabolite leakage; C) in to- mato seedlings grown under 0, 2, 4, and 6 mM boron for 20 days. The data are means ± SD (n = 4). Different letters indicate statisti- cally significant differences (P ≤ 0.05). RADI A.A., SALAM H.KH., HAMADA A.M., FARGHALY F.A. 56 ACTA BOT. CROAT. 82 (1), 2023 Membrane stability To quantify the degree of membrane integrity under EB stress, the EC, K leakage, and metabolite leakages in seed- lings undergoing various treatments were measured (Figs. 3A-C and On-line Suppl. Tab. 1 and Tab. 2). 6 mM B raised the EC and incidence of K and UV metabolites in leaves by 67.22%, 101.54%, and 91.99%, respectively. Furthermore, EC (0.931**, 890**, and 0.724**, respectively), K (0.943**, 0.915**, and 0.828**, respectively), and metabolite leakages (0.971**, 0.937**, and 0.687*, respectively) were shown to be strongly connected with free, semi-bound, and bound B levels in shoots. FTIR analysis We employed FTIR analysis to assess the effect of EB on seedling ultrastructure (Fig. 4 and Tab. 1). EB did not induce extensive alterations within the four peaks at 3405.17 cm–1, 2927.25 cm–1, 1384.45 cm–1, and 825.42 cm–1. Treatment with 4 mM B raised the peak intensity of 3405.17 cm–1, but expo- sure to 2 and 6 mM B lowered it compared to control. More- over, treatments with 4 and 6 mM B raised the peak inten- sity at 2927.25 cm–1 and 1384.45 cm–1, respectively, while exposure to 2 mM B lowered them compared to control. However, EB levels raised the peak intensity of 825.45 cm–1 relative to control. Regarding the peak at 1653.21 cm–1 (control), 2 mM B treatment did not significantly alter its transmission, while 4 and 6 mM concentrations lowered it by –6.34 cm–1 and –12.48 cm–1, respectively. Moreover, 4 mM B raised this peak intensity, although other B treatments did not. Mean- while, the peak at 1540.60 cm–1 (control) disappeared under B treatments (2, 4, and 6 mM). The peak recorded at 1058.10 cm–1 (control) was nega- tively shifted by –3.20 cm–1, –4.27 cm–1, and –4.18 cm–1 upon exposure to 2, 4, and 6 mM B, respectively. Moreover, treatments with 2 mM and 4 mM B stimulated this peak intensity, but treatment with 6 mM EB reduced it. Compared to the peak recorded at 617.04 cm–1 (control), EB treatments increased from the transmission area by 1.04 cm–1, 5.31 cm–1, and 3.75 cm–1 at 2, 4 and 6 mM concentra- tions, respectively. Furthermore, low levels of EB (2 and 4 mM) increased the peak intensity, while the treatment with 6 mM B decreased it. The peaks recorded at 540.52 cm–1, 536.78 cm–1, and 537.63 cm–1 appeared under treatments with 2, 4, and 6 mM B, respectively. Under the high levels of EB (4 and 6 mM), the 483.52 cm–1 and 459.94 cm–1 peaks disappeared, while the exposure to 2 mM B stimulated their values by 0.74 cm–1 and 4.26 cm–1, respectively. Discussion The shoot/root ratio of seedlings was enhanced at the lowest EB concentration, indicating the higher growth (Figs. 1A-D). Moreover, lower growth at high levels of EB was Fig. 4. Fourier-transform infrared spectroscopy (FTIR) spectra (range 4000–400 cm–1; A, and 0–500 cm–1 region expanded; B) of tomato seedlings grown under 0, 2, 4, and 6 mM boron for 20 days. Tab. 1. Fourier-transform infrared spectroscopy (FTIR) spectra showing observed peaks in tomato seedlings grown under 0, 2, 4, and 6 mM boron for 20 days. Frequency (cm–1) Excess boron (mM) Peak 0 2 4 6 1 3405.17 3404.98 3405.43 3406.01 2 2927.25 2927.25 2926.60 2926.75 3 1653.21 1656.79 1646.87 1640.73 4 1540.60 – – – 5 1384.45 1384.28 1384.34 1384.30 6 1058.10 1054.9 1053.83 1053.92 7 825.42 825.55 825.68 825.42 8 617.04 618.08 622.35 620.79 9 – 540.52 536.78 537.63 10 483.52 484.26 – – 11 459.94 464.20 – – 12 – 453.29 – 453.74 BORON TOXICITY AND TOMATO SEEDLING GROWTH ACTA BOT. CROAT. 82 (1), 2023 57 linked to the concentration of B forms within the seedlings, demonstrating that B buildup was limiting growth. As dem- onstrated by the strong positive relationships between shoot/root ratio and B level within shoots, the negative B effect was more evident in tomato roots than in shoots. EB has a comparable negative effect on tomato growth, accord- ing to Kaya et al. (2020). Cell division (Liu and Yang 2000), cell expansion, cell numbers (Choi et al. 2007), water con- tent (Metwally et al. 2018), and cell wall matrix stiffness (Farghaly et al. 2022b) are all linked to decreased seedling growth. Conversely, promoting growth at a low EB level may be associated with active B influx, which lowers intra- cellular B levels (Reid et al. 2004, Ardic et al. 2009). The primary organelles damaged by EB are the chloro- plasts (Landi et al. 2019). The deficiency of photosynthetic pigments found in this study (Fig. 2A) could be due to a structural damage to thylakoids as a result of abnormal spongy parenchyma growth (Papadakis et al. 2004), oxida- tion of chlorophyll and chloroplast membranes (Aftab et al. 2012), and a reduction in three types of thylakoid-related proteins (Sang et al. 2015). Our results match the findings of wheat and tomato, which are vulnerable to EB (El- Shazoly et al. 2019, Kaya et al. 2020). Thus, EB has a variety of con- sequences on photosynthetic processes, including changes in photosynthetic pigment levels, lower CO2 assimilation, impaired photosystem II performance, and a decreased electron transport rate (Landi et al. 2019). High content of photosynthetic pigments at a low EB level suggests seedling tolerance. Furthermore, the chloro- plasts were less vulnerable to EB since the DW was high at this level of 2 mM EB. Additionally, strong positive relation- ships between pigment contents and shoot DW revealed that pigment preservation is necessary to stimulate seedling growth. Accordingly, Eraslan et al. (2007) found no signifi- cant changes in chl a and b concentration in carrot plants when exposed to EB. Boron amount in plants is considered the main physi- ological feature utilized to examine tolerance to EB in an environment. Our findings revealed that all B forms were significantly increased in EB-stressed seedlings, explaining the symptoms of increased EB (Fig. 2B). Likewise, during exposure to EB, an accumulation of B forms was previous- ly observed in tomato calli (Farghaly et al. 2021, 2022b). Free B demonstrated the ability to cross cell membranes and showed promise as being immediately accessible for poten- tial physiological roles in the cell, according to Dannel et al. (1998). In this study, the content of semi-bound and bound B forms varied from about 6%–76% and 2%–119%, respec- tively. These data may reveal that a small amount of EB was attached to the cell walls in exchange for increased B avail- ability, but this amount was too low to actively participate in EB detoxification, as indicated by increased free B ( Dannel et al. 1998, Farghaly et al. 2022b). Ionic solutes and cellular metabolites are widely applied to assess membrane integrity (Palta et al. 1977, Navari-Izzo et al. 1993). According to our findings presented in Figs. 3A-C, the membrane damage was more severe as EB levels in the medium increased. These findings showed that EB had a sig- nificant impact on the permeability of tomato membranes, revealed by the EC and leakage of K and UV metabolites, which were all confirmed in a prior work with wheat ( Metwally et al. 2012). FTIR spectra revealed further information about the in- fluence of EB on seedling macromolecules (Fig. 4 and Tab. 1). Türker-Kaya and Huck (2017) correlate the first peak, recorded at 3405.17 cm–1 in control, with O-H and N-H re- lated to alcohol, carbohydrates, phenols, and proteins. EB did not affect the wavenumber, indicating that the lack of alterations in cell wall components and the reduction in bound-B in seedlings may clarify these findings. Further- more, EB lowered peak intensity, suggesting that EB may change the pattern in binding between alcohols, carbohy- drates, proteins, phenols, and components of walls. Riaz et al. (2021) demonstrated that EB increased lignin and suber- in levels in rice plants, perhaps leading to cell wall stiffness. Otherwise, changes in peak intensity can be referred to as changes in cell wall shape (Zuverza-Mena et al. 2016). The peak found around 3000–2800 cm–1 was assigned the C-H stretching area of lipids, wax, and fats (Legner et al. 2018). EB did not affect this peak’s value (3000–2800 cm–1), but the intensity of the peak increased at 4 and 6 mM EB. These data indicate that no changes were made to wall wax amount, while the shape of wall wax may only change un- der EB (Morales et al. 2013). Mesquita et al. (2016) found ir- regular wax deposition on the surface of citrus leaves under EB, which might support our findings. The peak in the region of 1700–1600 cm–1 is characteris- tic of the C=O of the amide I (proteins) (Dumas and Miller 2003). The amide II peak in the region of 1480–1580 cm–1 is a mixture of N-H and C-N vibrations that aid in ionic reac- tion response, although it is less well understood (Zhao and Wang 2016). The amide I peak value was reduced by high levels of EB (4 and 6 mM), demonstrating that the protein’s structure is changed to chelate EB, and this explanation might be confirmed by the finding of Farghaly et al. (2022a). According to Riaz et al. (2021), EB significantly affected the amide protein, amide II, and amide III, indicating damage to the protein pools. The disappearance of the amide II peak under EB treatments can disclose the binding of B ions to nitrogen amide to chelate the EB, and plants can use this claw to withstand B toxicity. Dunbar et al. (2012) reported the disappearance of amide II (around 1550 cm–1; bending of amide N-H) owing to iminol structural coordination be- tween the amide II and magnesium, nickel, and cobalt. Wei et al. (2015) assigned the peaks between 1500–1000 cm–1 fingerprint regions of the amide III, nucleic acid func- tional groups, and carbohydrates. Our results revealed that EB treatments induce a change in the intensity of the peak recorded at 1384.45 cm–1. The increasing peak intensity might reveal that the additional B has changed the finger- print region’s components and linked EB with proteins. RADI A.A., SALAM H.KH., HAMADA A.M., FARGHALY F.A. 58 ACTA BOT. CROAT. 82 (1), 2023 EB reduced the 1058.10 cm–1 peak, which was attributed to cellulose (Wu et al. 2017), indicating a reduction in cel- lulose production in seedlings under EB. This peak inten- sity was lowered by EB at its maximum level, indicating a decrease in cellulose synthesis. Similarly, Farghaly et al. (2022b) discovered that EB decreased the cellulose content of tomato calli in their study. The peak recorded at 825.42 cm–1 in control, which was not affected by EB treatments, was assigned to the trisac- charide (D-(+)-raffinose pentahydrate) with α-glycosidic bonds (Wiercigroch et al. 2017). The intensity of this peak was increased, indicating B binding to the pentahydrate. EB also boosted the 617.04 cm–1 peak, which was assigned to D- (+)-glucose (Wiercigroch et al. 2017), showing the degrada- tion of cellulose or sucrose into simple monosaccharides. This explanation might confirm a decrease in the cellulose wavelengths. Under EB, the appearance of additional peaks, recorded at 540.52 cm–1, 536.78 cm–1, and 537.63 cm–1, may also demonstrate glucose buildup (Farghaly et al. 2022b). Furthermore, at high EB levels, the disappearance of ribose peaks (484 cm–1 and 460 cm–1; Wiercigroch et al. 2017) sug- gested EB binding to ribose, and this confirmed the ability of EB to stabilize ribose to create a nucleotide of a borate ester (Grew et al. 2011, Scorei 2012). In conclusion, EB treatments exhibited unfavorable in- fluences on FW, DW, TWC, and photosynthetic pigments of tomato seedlings. EB also caused a reduction in mem- brane integrity, as seen by higher EC, and K and UV-me- tabolite leakage. B absorption matched the B content in the nutritional medium, resulting in increased accumulation of various B forms in seedlings. EB inhibited cellulose synthe- sis in seedlings and altered wax deposition in cell walls. Moreover, EB affected the amide I and amide II indicating damage to the protein pools. Finally, our results reveal that decreased tomato growth under EB might be related to al- terations in photosynthetic pigments, membrane stability, and macromolecules. Acknowledgments The authors thank Assiut University for providing the laboratory equipment. References Aftab, T., Khan, M.M., Naeem, M., Idrees, M., Moinuddin, A.S., Teixeira da Silva, J.A., Ram, M., 2012: Exogenous nitric ox- ide donor protects Artemisia annua from oxidative stress generated by boron and aluminum toxicity. Ecotoxicology and Environmental Safety 80, 60–68. Ardic, M., Sekmen, A.H., Tokur, S., Ozdemir, F., Turkan, I., 2009: Antioxidant responses of chickpea plants subjected to boron toxicity. Plant Biology 11, 328–338. Canteri, M.H., Renard, C.M., Le Bourvellec, C., Bureau, S., 2019: ATR-FTIR spectroscopy to determine cell wall composition: Application on a large diversity of fruits and vegetables. Car- bohydrate Polymers 212, 186–196. Cervilla, L.M., Blasco, B., Ríos, J.J., Rosales, M.A., Sánchez- Rodríguez, E., Rubio-Wilhelmi, M.M., Romero, L., Ruiz, J.M., 2012: Parameters symptomatic for boron toxicity in leaves of tomato plants. Journal of Botany 726206, 1–17. Choi, E.Y., Kolesik, P., Mcneill, A., Collins, H., Zhang, Q., Huynh, B., Graham, R., 2007: The mechanism of boron tol- erance for maintenance of root growth in barley (Hordeum vulgare L.). Plant, Cell and Environment 30, 984–993. Dannel, F., Pfeffer, H., Romheld, V., 1998: Compartmentation of B in roots and leaves of sunflower as affected by B supply. Journal of Plant Physiology 153, 615–622. Du, C.W., Wang, Y.H., Xu, F.S., Yang, Y.H., Wang, H.Y., 2002: Study on the physiological mechanism of boron utilization efficiency in rape cultivars. Journal of Plant Nutrition 25, 231–244. Dumas, P., Miller, L., 2003: The use of synchrotron infrared mi- crospectroscopy in biological and biomedical investigations. Vibrational Spectroscopy 32, 3–21. Dunbar, R.C., Steill, J.D., Polfer, N.C., Berden, G., Oomens, J., 2012: Peptide bond tautomerization induced by divalent metal ions: characterization of the iminol configuration. Angewandte Chemie International Edition in English 51, 4591–4593. Elbehiry, F., Elbasiouny, H., El-Henawy, A., 2017: Boron: spatial distribution in an area of North Nile Delta, Egypt. Commu- nications in Soil Science and Plant Analysis 48, 294–306. El-Shazoly, R.M., Metwally, A.A., Hamada, A.M., 2019: Salicyl- ic acid or thiamin increases tolerance to boron toxicity stress in wheat. Journal of Plant Nutrition 42, 702–722. Eraslan, F., Inal, A., Gunes, A., Alpaslan, M., 2007: Boron toxic- ity alters nitrate reductase activity, proline accumulation, membrane permeability, and mineral constituents of toma- to and pepper plants. Journal of Plant Nutrition 30, 981–994. Fang, K., Zhang, W., Xing, Y., Zhang, Q., Yang, I., Cao, Q., Qin, L., 2016: Boron toxicity causes multiple effects on Malus domestica pollen tube growth. Frontiers of Plant Science 7, 208. FAOSTAT, 2017: Food and Agriculture Organization of the United Nations (FAO). FAOSTAT Database. Retrieved from http://www.fao.org/faostat/en/#data/QC. Farghaly, F.A., Hamada, A.M., Radi, A.A., 2022a: Phyto-reme- dial of excessive copper and evaluation of its impact on the metabolic activity of Zea mays. Cereal Research Communi- cations 50, 973–985. Farghaly, F.A., Salam, H.Kh, Hamada, A.M., Radi, A.A., 2021: The role of benzoic acid, gallic acid and salicylic acid in pro- tecting tomato callus cells from excessive boron stress. Sci- entia Horticulturae 278, 109867. Farghaly, F.A., Salam, H.Kh, Hamada, A.M., Radi, A.A., 2022b: Alleviating excess boron stress in tomato calli by applying benzoic acid to various biochemical strategies. Plant Physi- ology and Biochemistry 182, 216–226. Grew, E.S., Bada, J.L., Hazen, R.M., 2011: Borate minerals and origin of the RNA world. Origins of Life and Evolution of Biospheres 41, 307–316. Kaya, C., Levent, Tuna, A.L., Dikilitas, M., Ashraf, M., Koskeroglu, S., Guneri, M., 2009: Supplementary phosphorus can alleviate boron toxicity in tomato. Scientia Horticulturae 121, 284– 288. Kaya, C., Sarıŏglu, A., Ashraf, M., Alyemeni, M.N., Parvaiz Ah- mad, P., 2020: Gibberellic acid-induced generation of hydro- gen sulfide alleviates boron toxicity in tomato (Solanum lycopersicum L.) plants. Plant Physiology and Biochemistry 153, 53–63. Landi, M., Margaritopoulou, T., Papadakis, I.E., Araniti, F., 2019: Boron toxicity in higher plants: an update. Planta 250, 1011–1032. BORON TOXICITY AND TOMATO SEEDLING GROWTH ACTA BOT. CROAT. 82 (1), 2023 59 Legner, N., Meinen, C., Rauber, R., 2018: Root differentiation of agricultural plant cultivars and proveniences using FTIR spectroscopy. Frontiers of Plant Science 9, 748. Li, M., Zhao, Z., Zhang, Z., Zhang, W., Zhou, J., Xu, F., Liu, X., 2017: Effect of boron deficiency on anatomical structure and chemical composition of petioles and photosynthesis of leaves in cotton (Gossypium hirsutum L.). Scientific Reports 7, 4420. Lichtenthaler, H.K., 1987: Chlorophylls and carotenoids: pig- ments of photosynthetic biomembranes. Methods in Enzy- mology 148, 183–350. Liu, P., Yang, Y.A., 2000: Effects of molybdenum and boron on membrane lipid peroxidation and endogenous protective systems of soybean leaves. Acta Botanica Sinica 42, 461– 466. Mesquita, G.L., Zambrosi, F.C., Tanaka, F.A., Boaretto, R.M., Quaggio, J.A., Ribeiro, R.V., Jr. Mattos, D., 2016: Anatomical and physiological responses of citrus trees to varying boron availability are dependent on rootstock. Frontiers of Plant Science 7, 224. Metwally, A.M., El-Shazoly, R.M., Hamada, A.M., 2012. Effect of boron on growth criteria of some wheat cultivars. Journal of Biology and Earth Sciences 2, 1–9. Metwally, A.M., Radi, A.A., El-Shazoly, R.M., Hamada, A.M., 2018. The role of calcium, silicon and salicylic acid treatment in protection of canola plants against boron toxicity stress. Journal of Plant Research 131, 1015–1028. Mohan, T.C., Jones, A.M., 2018: Determination of boron content using a simple and rapid miniaturized curcumin assay. Bio- Protocol 8, e2703. Morales, M.I., Rico, C.M., Hernandez-Viezcas, J.A., Nunez, J.E., Barrios, A.C., Tafoya, A., Flores-Marges, J.P., Peralta-Videa, J.R., Gardea-Torresdey, J.L., 2013: Toxicity assessment of ce- rium oxide nanoparticles in cilantro (Coriandrum sativum L.) plants grown in organic soil. Journal of Agricultural and Food Chemistry 61, 6224–6230. Murashige, T., Skoog, F., 1962: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physio- logia Plantarum 15, 473–497. Navari-Izzo, F., Quartacci, M.F., Melfi, D., Izzo, R., 1993: Lipid composition of plasma membranes isolated from sunflower seedlings grown under water-stress. Physiologia Plantarum 87, 508–514. Navari-Izzo, F., Izzo, R., Quartacci, M.F., Lorenzini, G., 1989: Growth and solute leakage in Hordeum vulgare exposed to long-term fumigation with low concentration of CO2. Phys- iologia Plantarum 76, 445–450. Palta, J.P., Levitt, J., Stadelmann, E.J., 1977: Freezing injury in onion bulb cells. I. Evaluation of the conductivity method and analysis of ion and sugar efflux from injured cells. Plant Physiology 60, 393–397. Papadakis, I., Dimassi, K., Bosabalidis, A., Therios, I., Patakas, A., Giannakoula, A., 2004: Boron toxicity in ‘Clementine’ mandarin plants grafted on two rootstocks. Plant Science 166, 539–547. Premachandra, G.S., Saneoka, A.H., Fujta, K., Ogata, S., 1992: Leaf water relations, osmotic adjustment, cell membrane sta- bility, epicuticular wax load and growth as affected by in- creasing water deficits in sorghum. Journal of Experimental Botany 43, 1569–1576. Princi, M.P., Lupini, A., Araniti, F., Longo, C., Mauceri, A., Sunseri, F., Abenavoli, M.R., 2016: Boron toxicity and toler- ance in plants: Recent advances and future perspectives. In: Ahmad, P. (ed.), Plant Metal Interaction; 115–147. Elsevier, Amsterdam, The Netherlands. Reid, R.J., Hayes, J.E., Post, J.C., Stangoulis, A., Graham, R.D., 2004: A critical analyses of the caises of boron toxicity in plants. Plant, Cell and Environment 25, 1405–1414. Renuka, B., Sanjeev, B., Ranganathan, D., 2016: Evaluation of phytoconstituents of Caralluma nilagirina by FTIR and UV- Vis spectroscopic analysis. Journal of Pharmacognosy and Phytochemistry 5, 105–108. Riaz, M., Kamran, M., El-Esawi, M., Hussain, S., Wang, X., 2021: Boron-toxicity induced changes in cell wall components, bo- ron forms, and antioxidant defense system in rice seedlings. Ecotoxicology and Environmental Safety 216, 112192. Sang, W., Huang, Z.R., Qi, Y.P., Yang, L.T., Guo, P., Chen, L.S., 2015: An investigation of boron-toxicity in leaves of two cit- rus species differing in boron-tolerance using comparative proteomics. Journal of Proteomics 123, 128–146. Scorei, R., 2012: Is boron a prebiotic element? A mini-review of the essentiality of boron for the appearance of life on earth. Origins of Life and Evolution of Biospheres 42, 3–17. Türker-Kaya, S., Huck, C.W., 2017: A review of mid-infrared and near-infrared imaging: principles, concepts and applications in plant tissue analysis. Molecules 22, 168. Wei, Z., Jiao, D., Xu, J., 2015: Using Fourier transform infrared spectroscopy to study effects of magnetic field treatment on wheat (Triticum aestivum L.) seedlings. Journal of Spectros- copy 570190, 1–6. Wiercigroch, E., Szafraniec, E., Czamara, K., Pacia, M.Z., Majzner, K., Kochan, K., Kaczor, A., Baranska, M., Malek, K., 2017: Raman and infrared spectroscopy of carbohydrates: A review. Spectrochimica Acta Part A: Molecular and Bio- molecular Spectroscopy 185, 317–335. Williams, C.H., Twine, J.R., 1960: Flame photometric method for sodium, potassium and calcium. In: Peach K., Tracey M.V. (eds.), Modern methods of plant analysis, vol 5, 3–5. Springer, Berlin. Wu, X.W., Muhammad, R., Yan, L., Du, C.Q., Liu, Y.L., Jiang, C.C., 2017: Boron deficiency in trifoliate orange induces changes in pectin composition and architecture of compo- nents in root cell walls. Frontiers of Plant Science 8, 1882. Zhao, J., Wang, J., 2016: Uncovering the sensitivity of amide-II vibration to peptide-ion interactions. The Journal of Physi- cal Chemistry B 120, 9590–9608. Zuverza-Mena, N., Armendariz, R., Peralta-Videa, J.R., Gardea- Torresdey, J.L., 2016: Effects of silver nanoparticles on radish sprouts: root growth reduction and modifications in the nu- tritional value. Frontiers of Plant Science 7, 90.