2024 Volume 89 Issue 4 Pages 297-306
Using the Allium test, the various toxicities caused by three distinct dosages (10, 50, and 100 mM) of the hazardous non-protein amino acid L-canavanine (L-CAN) were examined in this work. Indicators of toxicity included cytogenetic [micronucleus (MN) frequency, chromosomal abnormalities (Cas), mitotic index (MI)], physiological [germination percentage (GP), root number (RN), root length (RL), and fresh weight (FW)], biochemical [free proline (PR) level, malondialdehyde (MDA) level, catalase (CAT) activity, and superoxide dismutase (SOD) activity], and anatomical parameters. Four sets of Allium cepa L. bulbs were created: one for control (C) and three for treatments. For 7 days, the bulbs in the treatment groups were germinated with three different doses of L-CAN, whereas the bulbs in the C group were germinated with tap water. Consequently, at all three levels, exposure to L-CAN resulted in a reduction in every physiological parameter measured. In addition, every L-CAN dosage resulted in a rise in the frequency of MN and CAs together with a decrease in MI. L-CAN produced CAs such as notched nuclei, micronuclei accumulation, bilobulated and trilobulated nuclei with bud, C-metaphase, chromosomal stickiness, vagrant chromosome, and chromatid bridge in the root meristem cells. Through the induction of oxidative stress in the cells, L-CAN also produced toxicity. L-CAN exposure resulted in dose-related increases in the levels of free PR, MDA, CAT, and SOD in the root. L-CAN exposure induced anatomical harms such as deformations of the epidermal cells, development of micronucleus, accumulation of certain chemical substances, abnormal position of the epidermal cell nucleus, giant cell nucleus, and vacuole formation in the nucleus of the root tip meristem cells. Due to its inhibitory impact on Allium cepa L. test material, L-CAN induced comprehensive toxicity; the Allium test proved to be a valuable tool in identifying this toxicity.
Non-protein amino acids (NPAAs) are widely distributed in the plant kingdom. At least 300 NPAAs from plants and about 700 from natural sources have been identified. They are mostly present in Fabaceae, Cucurbitaceae, Aceraceae, Sapindaceae, seaweeds, and fungi. These amino acids are also found in commonly consumed animal feeds and human foods. Therefore, they enter human bodies through the food chain and are integrated into proteins that have harmful consequences in addition to preventing nerve transmission, urea synthesis, and protein synthesis (Nunn et al. 2010). NPAAs have allelopathic activity and play an active role in protecting. Due to their allelopathic action, NPAAs actively defend plants against diseases, insects, predators, and other plant species that threaten them (Huang et al. 2011). Moreover, some NPAAs are used as drugs or raw materials in human and veterinary medicine (Bell 2003).
Canavanine (CAN), also known as 2-amino-4-guanidinooxybutyric acid, is the guanidinooxy structural analog of the L-arginine (L-ARG) amino acid (Rosenthal 1977). L-CAN is especially found in species belonging to the Leguminosae family. Approximately 1,500 species belonging to 246 legume genera contain L-CAN (Ekanayake et al. 2007). However, varying levels of L-CAN are also present in several genera of Lotoideae, and more specifically in the subfamily Papilionoideae (Bell 1971; Weaks and Hunt 1974). This non-protein amino acid is a potent plant growth inhibitor and acts as an allelochemical compound that suppresses the growth of competing plants (Shibuya 1994). It is also the main nitrogen reserve metabolite for many legume plants and seeds (Bence et al. 2003; Van Wyk and Albrecht 2008). L-CAN has anti-cancer, anti-viral, anti-fungal, and anti-bacterial activity in humans (Ekanayake et al. 2007). Therefore, this amino acid is a valuable pharmaceutical product. The pharmacological activity of L-CAN is due to its structure similar to L-ARG (Hwang et al. 1996). Therefore, the mode of action of L-CAN is closely related to L-ARG metabolism, and concomitant administration of L-ARG significantly reduces the inhibitory effect of L-CAN (Van Wyk 2008; Van Wyk and Albrecht 2008).
One of the most widely consumed and farmed vegetables worldwide is the onion, A. cepa L., which is a member of the Amaryllidaceae family and the Allioideae subfamily (Peruzzi et al. 2017). Its antioxidant, antidiabetic, and antibacterial properties have reportedly led to its usage as a medicinal plant from ancient times in addition to its nutritional worth (Marrelli et al. 2019). A. cepa L., a herb that is mostly biennial or perennial, can reach a height of around half a meter. Its bulb may be oval, extended, or spherical. It has a hollow, cylindrical body. The leaves range in color from yellow to bluish-green. They are cylindrical, hollow, fleshy, and have a single flat side. Their seeds have a glossy black color. It has greenish-white, hermaphrodite blooms (Ashwini and Sathishkumar 2014; Lim 2015). Besides, for a variety of reasons, including its ease of availability and cultivation, low count of chromosomes and huge chromosomes, and ease of measuring biochemical processes, it is favored as a bioindicator in experimental studies (Yalçın et al. 2021).
Toxicological and mutagenic properties are assessed using the Allium test (Tedesco and Laughinghouse 2012). Inhibition of root growth is used to evaluate toxicity, and rates of chromosomal abnormalities are used to measure mutagenicity. Many researchers who deal with in vitro animal organism tests have approved cytotoxicity experiments employing in vivo plant test systems, such as A. cepa L., and the reported results are identical (Teixeira et al. 2003; Chaparro et al. 2010). Because this test is an alternative, reliable, short-term, and cost-effective, it has been certified by the United Nations Environment Program (UNEP) and the International Chemical Safety Program (IPCS) as an efficient biotest for tracking mutagenesis impacts (Grant 1999).
The cyto- and genotoxic effects of exogenous L-CAN administration during A. cepa L. bulb germination under normal conditions have not been studied in the literature. Consequently, the purpose of this investigation was to ascertain the level of toxicity of 10, 50, and 100 mM L-CAN dosages on different (physiological/cytogenetic/biochemical/anatomical) parameters of onion bulbs.
In this work, the test plant for the experimental phase was A. cepa bulbs. Bulbs were bought from a greengrocery in Isparta-Turky and L-canavanine/L-CAN (CAS number: 543-38-4) was acquired from Sigma-Aldrich Corporation. The determined L-CAN concentrations were based on the found EC50 dose. Fifty millimolars was determined to be the EC50 dosage. The lowest dose of 10 mM and a dose of 100 mM, which is double the EC50 level, were preferred.
Determination of physiological changes and treatment procedureRoughly equivalent-sized, healthy, and plump bulbs were selected. Bulbs were divided into four separate groups. Twenty bulbs from each identified group were put into sterile plastic 1.7-L containers with a perforated cover with the root sections inside and the remaining parts outside. The bulbs were then allowed to germinate for 168 h (7 days) in the dark in an incubator kept at 20°C. First group control bulbs were kept in tap water medium until the end of the study; second group bulbs were kept in 10 mM L-CAN medium; third group bulbs were kept in 50 mM L-CAN medium; and fourth group bulbs were germinated in 100 mM L-CAN treated medium.
The hairy roots (root number/RN) of the germinated bulbs in the control and treatment groups were counted after the 168-h application period. Root lengths (RL) were measured in mm using a ruler fitted with a millimetric scale. Precision balance was used to determine fresh weights (FW) in grams and Eq. (1) was utilized to express the germination percentages (GP) in percentage. The germination threshold was determined to be the radicle’s 10 mm protrusion from the testa. In this investigation, every trial was set up in triplicate to enable statistical analysis of the collected data.
 | (1) |
Materials cut to a length of 1–2 cm from the onion root tips were immersed in saturated paradichlorobenzene for 4 h, fixed in a mixture of 3 parts ethyl alcohol and 1 part acetic acid, and then stored in 70% ethyl alcohol to identify chromosomal damage. In order to prepare the root tips permanently, they were first hydrolyzed in 1 M HCl at 60°C for 17 min, then stained for 1 to 1.5 h with Feulgen, crushed in 45% acetic acid on a slide, covered with a coverslip, balm applied around the coverslip, and photographed using a microscope (Sharma and Gupta 1982). Thirty thousand cells were counted for each root tip from the prepared preparations to calculate the mitotic index (MI), and Eq. (2) was used to determine the percentage of cells entering mitosis. In order to calculate chromosomal abnormalities (CAs), 2,000 dividing cells were counted.
 | (2) |
Fifty millimolars cold sodium phosphate buffer (pH 7.8) was used to homogenize 0.5 g of root sample. After passing through coarse filter paper, the homogenate was centrifuged for 20 min at 10,000 rpm. The activity of the enzymes catalase (CAT) and superoxide dismutase (SOD) were measured spectrophotometrically using the supernatant.
Nitroblue tetrazolium chloride (NBT) photochemical reduction at 560 nm was used to measure SOD activity. 1.5 mL of 0.05 M sodium phosphate buffer (pH 7.8), 750 µM NBT, 130 mM L-methionine, 0.1 mM EDTA-Na2, 20 µM riboflavin, 4% polyvinylpyrrolidone, supernatant, and deionized water were used for the reaction. The reaction mixture was incubated for 10 min under 15-W fluorescent light after riboflavin was added last in the dark (Beauchamp and Fridovich 1971). U mg−1 FW was used to express SOD activity (Zou et al. 2012).
CAT activity was determined by monitoring the absorbance drop at 240 nm. In 200 mM pH 7.8 sodium phosphate buffer, 0.1 M H2O2, supernatant, and deionized water were incubated for 2 min at 37°C. The reaction was then stopped with 1 M HCl. The amount of enzyme needed to break down 1 µmol of H2O2 was defined as one unit of enzyme activity. The expression for CAT activity was OD 240 nm min g−1 FW (Beers and Sizer 1952).
Identification of lipid peroxidationThe expression for lipid peroxidation is the concentration of malondialdehyde (MDA). After homogenizing a 0.5 g sample of onion roots with 10 mL of 5% trichloroacetic acid (TCA), the homogenate was centrifuged for 15 min at 12,000 rpm and 24°C. One milliliter of the clear portion of the centrifuged sample was extracted, and to it was added 4 mL of 20% TCA diluted in 0.5% thiobarbituric acid (TBA). The mixture was rapidly chilled in an ice bath after being maintained at 96°C for 25 min and centrifuged at 10,000 rpm for 5 min. Next, the absorbance from the clear portion was measured at 532 nm, and the MDA concentration was computed using the extinction coefficient of 155 M−1 cm−1 and represented as µmol (Unyayar et al. 2006).
Identification of free proline (PR) accumulationA 0.5 g fresh root sample was homogenized using 10 mL of 3% sulfosalicylic acid. After that, Whatman filter paper was used to filter the root samples. A volume of 2 mL was extracted, followed by an equal volume of acid ninhydrin and glacial acetic acid. The combination was stored for 1 h in a water bath at 100°C and for 5 min in an ice bath. To create two phases, 5 mL of toluene was added to the reaction mixture, vortexed for 15 to 20 s, and then allowed to settle. Using a micropipette, the upper phase was obtained, and the absorbance values were measured in the spectrophotometer at 520 nm in comparison to the pure toluene control. The outcomes of the L-PR standard were contrasted with the examples’ results. With the use of Eq. 3, the amount of free PR was determined, and µg g−1 is represented as fresh weight (Bates et al. 1973).
 | (3) |
To get rid of the residues on the onion roots’ surface, the root tips were cleaned with purified water. After cutting cross-sections from the tips of the roots with the help of a razor and dyeing them with 2% methylene blue, the stained samples from each group were inspected under a 500× magnification light microscope.
Analysis of dataUsing “the SPSS 23 analytical software” for Windows, statistical analyses of the collected data were performed, and the variations in the outcomes were shown as “mean ± standard deviation.” It was examined using a one-way ANOVA and the Duncan test at the p＜0.05 significant level.
Figures 1 and 2 depict the alterations in the onion bulbs’ physiological characteristics. A GP of 95±0.0% was found in the control (C) bulbs. The GP rates were 76±2.4%, 40±1.2%, and 24±0.0% at 10 mM, 50 mM, and 100 mM L-CAN doses, respectively. So, the GP reduced by 19% at 10 mM, 55% at 50 mM, and 71% at 100 mM L-CAN concentration according to the C (Group I). In the same way, a substantial reduction in the fresh weight (FW), radicle length (RL), and radicle number (RN) of the bulbs was seen with increasing L-CAN doses (p＜0.05). The C group had the highest RL measurement (80.2±1.7 mm), whereas Group IV, which was exposed to 100 mM L-CAN, had the lowest RL measurement (13.1±0.5 mm). As per the C (Group I), there was a reduction of 30.5 mm in RL in Group II, 51.4 mm in Group III, and 67.1 mm in Group IV. The average RN of the C’s bulbs was counted as 55.3±1.0. This parameter was counted as an average of 40.2±0.8 in 10 mM dose, an average of 29.7±0.7 in 50 mM dose, and an average of 16.4±0.3 in 100 mM dose of L-CAN. At 100 mM L-CAN dose, the RN reduced by 3.3 times compared to the C. The FW of the bulbs was 17.1±0.6 g in the C group. The FW values for Group II, Group III, and Group IV exposed to L-CAN were 11.6±0.5 g, 9.5±0.3 g, and 6.0±0.2 g, respectively.
(A) Germination (%). (B) Root length (m). (C) Root number. (D) Fresh weight (g/seedling). Group I (control) was treated with tap water; Group II was treated with 10 mM L-CAN; Group III was treated with 50 mM L-CAN; Group IV was treated with 100 mM L-CAN; for each determined group, 20 bulbs were studied and the experiments were carried out in triplicate; standard deviation (±SD) shown with error bars; different letters(a–d) indicate average p＜0.05 is important.
Group I (tap water), Group II (10 mM L-CAN), Group III (50 mM L-CAN), Group IV (100 mM L-CAN).
While a few researches have been done on the effects of exogenous L-CAN administration on seed germination and seedling growth under normal, or stress-free, circumstances, no work has been done to investigate its impact on bulb germination and growth. The mentioned study is also quite significant as it is the first study in the literature to investigate the influences of L-CAN on bulb germination and growth. On the other hand, the research results reporting that externally applied L-CAN reduces the germination of seeds and growth of seedlings in various plant species (Rosenthal et al. 1975; Wilson and Bell 1978; Schwartz et al. 1997; Staszek et al. 2019) support the current study’s findings.
The decreases in physiological parameters brought on by L-CAN exposure could have a variety of causes. Inadequate water and mineral material intake by the roots may be the cause of the decline in the FW and water content of seedlings cultivated in L-CAN medium (Fig. 1). The absence of essential minerals and water for cells also adversely impresses the mitotic activity (Fig. 3) and reduces in the RL and RN (Fig. 1) become inevitable. In addition, L-CAN application can cause suppression of ATP generation and modifications in the cell membrane’s electrochemical gradient by promoting lipid peroxidation (Fig. 5) in the membranes of root cells. As a result, a breakdown in the membrane structure could result in less water and mineral substance absorption as well as a delay in the onion bulb’s germination and growth.
(A) Mitotic index (%). (B) Micronucleus frequency (%). (C) Chrosome aberretion (%). Group I (control) was treated with tap water; Group II was treated with 10 mM L-CAN; Group III was treated with 50 mM L-CAN; Group IV was treated with 100 mM L-CAN; for each determined group, fresh root samples of 20 bulbs were studied; 30,000 cells were counted to calculate the MI and 2,000 dividing cells were counted to calculate CAs, and the experiments were carried out in triplicate; standard deviation (±SD) shown with error bars; different letters(a–d) indicate average p＜0.05 is important.
The impacts of exogenous L-CAN on cytogenetic parameters, including chromosomal aberrations (CAs), micronucleus (MN) frequency, and mitotic index (MI), are displayed in Fig. 3. The MI was determined in the root cells of the bulbs in the control group (Group I) at 9.0±0.5%, in Group II at 7.9±0.3%, in Group III at 5.7±0.1%, and in Group IV at 3.8±0.1%, which were the treatment groups. In comparison to the C group, the MI declined by about 12%, 37%, and 58% in the treatment groups. These results demonstrate that a higher L-CAN dose has a negative impact on MI. It becomes lethal when test organisms’ MI falls below 22% of the C (Antosiewicz 1990). Because the lowest MI value was only 3.8% at the highest L-CAN concentration (100 mM) across all applied L-CAN concentrations, a lethal effect was not seen in the current investigation. This value was approximately 48% of the C (Fig. 3).
Similarly, higher doses of L-CAN encouraged the development of MN in the bulb’s root tip meristem cells. MN formation increased obviously in the L-CAN applied groups; this parameter was 15.3±1.5% in Group II, 32.7±1.7% in Group III, and 46.1±2.0% in Group IV, whereas MN number was determined to be 0.7±0.9% in the Group I. When compared to the Group I, the number of MN increased by 45.4 times in Group IV, which received the greatest dose of L-CAN. It has also been shown that L-CAN increases the number of CAs in the root tip meristem cells. As a result of microscopic examination, the CAs were observed as 0.8±0.7% in the Group I bulbs’ root tip cells, while the count of these aberrations attained 15.4±1.0% at 10 mM dose, 38.1±1.4 at 50 mM dose and 55.4±2.2 at 100 mM dose. Especially at a 100 mM dose of L-CAN, this value increased by 54.6 times compared to the Group I (Fig. 3).
Figure 4 illustrates the root tip cells’ normal mitotic phases and abnormal mitotic stages caused by L-CAN exposure. L-CAN application promoted CAs such as the notched nuclei (Fig. 4E), accumulation of micronuclei in the cell (Fig. 4F), bilobulated nucleus (Fig. 4G), trilobulated nucleus (Fig. 4H), formation of bud (Fig. 4G, H), C-metaphase (Fig. 4I), metaphase with chromosomal stickiness (Fig. 4J), go into the division of metaphase plate (Fig. 4K), anaphase with polar slip (Fig. 4L), anaphase/telophase with vagrant chromosomes (Fig. 4M, N, P), chromatid bridges in anaphase (Fig. 4N), and alignment anaphase (Fig. 4O) in the root tip cells.
(A) prophase. (B) metaphase 2n=16 chromosome. (C) anaphase. (D) telophase. (E) notched nuclei. (F) accumulation of micronuclei in cell (arrows). (G) bilobulated nucleus with bud (arrow). (H) trilobulated nucleus with bud (arrow). (I) C-metaphase. (J) metaphase with chromosomal stickiness. (K) go into division of metaphase plate. (L) anaphase with polar slip. (M) anaphase with vagrant chromosomes (arrows). (N) chromatid bridges in anaphase with vagrant chromosomes (arrow). (O) alignment anaphase (arrow). (P) telophase with vagrant chromosome (arrow). Scale bar=10 µm.
Reactive oxygen species (ROS) accumulation in the cells of the root following L-CAN administration is hypothesized to be the cause of the MI value reduction and the MN and CA numbers rise. Because ROS formed by the effect of L-CAN can cause damage to DNA and microtubule structures that are essential for cell division. Notable increases in CAs and MN formation show that L-CAN is a genotoxic substance that affects A. cepa. As a result of the literature review, very old and limited studies were found on external L-CAN application’s impacts on the CAs and MI. Weaks (1974) and Weaks and Hunt (1974) reported that L-CAN administration at low doses (1.8×10−4 M) did not affect the mitosis and did not cause the CAs in the root cells of Phaseolus vulgaris L. Results from these studies and the present study revealed that exogenous L-CAN application was ineffective on the cytogenetic characteristics such as CAs, MN frequency, and MI at low doses, but was extremely effective at high doses.
Biochemical impacts of exogenous L-CAN exposureThe cytotoxic chemicals known as ROS have extremely harmful effects and function as intermediary signaling molecules that control the expression of genes linked to antioxidant defense mechanisms. Antioxidant enzymes make up a sizable portion of the antioxidant systems found in plants and reduce the harm caused by ROS (Srivastava and Singh 2020). SOD and CAT are two of these enzymes that are the most functioning. SOD is a metalloenzyme, whereas CAT is a tetrameric homoprotein. The cellular microbodies known as glyoxisomes and peroxisomes contain these two antioxidant enzymes (Soares et al. 2010).
Figure 5 depicts the dose-dependent effects of exogenous L-CAN on the CAT and SOD activities in the bulb root cells. Both SOD and CAT activity in bulb root cells markedly increased in tandem with the rise in L-CAN concentrations. Group I, also referred to as the C group, had SOD activity measurement of 60±1.0 U mg−1 FW; Group II had 83±1.5 U mg−1 FW; Group III had 104±2.2 U mg−1 FW; and Group IV had 142±3.2 U mg−1 FW. Meanwhile, Groups I, II, III, and IV’s CAT activity measurements were 1.0±0.2 OD 240 nm/min. gFW, 1.9±0.4 OD 240 nm min−1. gFW, 2.7±0.5 OD 240 nm min−1. gFW, and 3.5±0.5 OD 240 nm min−1. gFW, respectively. Abnormally high intrinsic levels of these two enzymes show that the treatment of L-CAN caused the root cells to produce an excessive amount of ROS and that the antioxidant-oxidant balance was thrown off, favoring oxidative stress. Staszek et al. (2019) reported that the CAT and SOD activity decreased in the roots of tomato plants exposed to 10 µM and 50 µM L-CAN for 24–72 h and this finding is inconsistent with the results of the present study. As a result, depending on the type of plant, the dosage of administration, and the duration of administration, L-CAN can have varying effects on the activity of the antioxidant enzymes.
(A) SOD (U/mg FW). (B) CAT (OD 240 nm/min gFW). (C) MDA (µmol/gFW). (D) PR (µmol/gFW). Group I (control) was treated with tap water; Group II was treated with 10 mM L-CAN; Group III was treated with 50 mM L-CAN; Group IV was treated with 100 mM L-CAN; for each determined group, 0.5 g fresh root samples of 20 bulbs were studied and the experiments were carried out in triplicate; standard deviation (±SD) shown with error bars; different letters(a–d) indicate average p＜0.05 is important.
Lipid peroxidation is one of the key metabolic pathways that contribute to the oxidative degradation of lipids, polyunsaturated fatty acids, malondialdehyde/MDA, and ROS production (Odjegba and Adeniran 2015). A rise in MDA concentration (Fedina and Benderliev 2000), an oxidative product of membrane lipids and a biomarker of oxidative stress (Janero 1990), is brought on by damage to the cell membrane. ROS causes damage to the peroxidation of a number of substances, including proteins, lipids, and nucleic acids (Dinakar et al. 2010). Furthermore, they impact DNA, which results in mutations in chromosomes and nucleic acids (Yarsan 2014). Major rises in MDA content were seen with increasing dosages of exogenous L-CAN. Group I’s root cells had an average MDA concentration of 6.6±0.8 µmol g−1 FW, but Group II, III, and IV bulbs had mean MDA contents of 13.7±0.9 µmol g−1 FW, 19.5±1.2 µmol g−1 FW, and 25.2±1.7 µmol g−1 FW, respectively (Fig. 5). An indication of destructive damage to cell membranes and deterioration in antioxidant-oxidant dynamics are the increases in MDA levels brought on by exogenous L-CAN treatment.
Osmotic potential and turgor are protected by PR, an essential osmolite that is produced in plants under osmotic stress (Munns and Tester 2008). Produced by plants from ornithine and glutamate (Celik and Atak 2012), this osmolite stabilizes proteins and cellular membranes (Wang and Han 2009), and protects cells. Free PR content prominently increased with increasing exogenous L-CAN dosages. When Group IV bulbs were grown in 100 mM L-CAN medium, the free PR content rose to 50.4±3.0 µmol g−1 FW, the highest level. However, in Group I (the C group) bulbs grown in a tap water medium, the free PR content was 18.5±1.6 µmol g−1 FW, the lowest level. Moreover, exogenous L-CAN administration increased the free PR content approximately1.4-fold at 10 mM, 2.1-fold at 50 mM, and 2.7-fold at 100 mM compared to the C (Fig. 5). Free PR accumulation under L-CAN treatments could be due to an increased synthesis or to an inhibition in the oxidation of PR. In this work, it was found that there was a favorable association between MDA and PR accumulation (Fig. 5). This demonstrates that PR efficiently contributes to the removal of generated ROS, thereby protecting cells from oxidative damage. Although there are few studies about the effects of L-CAN externally applied to plants grown under normal conditions on the SOD and CAT enzyme activity in the root cells, there is no study about its effects on the MDA and free PR content of the roots. Therefore, the biochemical findings obtained from this study are very important.
Anatomical impacts of exogenous L-CAN exposureFigure 6 displays microscopic findings of the alterations and deteriorations brought on by externally administered L-CAN in the root anatomical structure of onion bulbs. The root anatomy of the Group I bulbs showed no changes or detrimental effects (Fig. 6A, D). Nevertheless, detriments and alters such as deformation of the epidermal cells (Fig. 6B), formation of micronuclei (Fig. 6C), accumulation of some chemical compounds (Fig. 6C), abnormal position of epidermal cell nucleus (Fig. 6C), giant cell nucleus (Fig. 6E) and vacuole formation in the cell nucleus (Fig. 6F) were observed in the roots of the groups applied with L-CAN. Furthermore, it was shown that the roots of the bulbs grown in 100 mM L-CAN medium had higher anatomical alterations and damages than the roots of the bulbs grown in other L-CAN concentrations.
(A) normal view of the epidermal cells in the control group. (B) the epidermal cell deformations in L-CAN treated groups. (C) the micronucleus formation (arrow on top), accumulation of some chemical compounds (arrow top right) and abnormal position of epidermal cell nucleus (arrows at the bottom) at epidermal cell in L-CAN treated groups. (D) normal view of the cell nucleus in the control group. (E) the giant cell nucleus in L-CAN treated groups. (F) the micronucleus formation (arrow at the bottom) and vacuoles formation in cell nucleus (arrow on top) in L-CAN treated group. Scale bar=10 µm.
According to information found in the literature, plants use carrier proteins to store these compounds in specific areas so they can tolerate the impacts of hazardous agents (Baker 1981). The accumulation of specific chemicals in the root’s epidermal cells caused by L-CAN administration is not a result of harm, but rather an anatomical response to chemical exposure (Fig. 6C). The accumulation of the exposed chemical in the epidermal tissue prevents the transport to other tissues and provides the development of tolerance and resistance (Bahmani et al. 2015). In addition, a shift in intracellular pressure and the epidermal cell nuclei displacement may arise as a consequence of the cytological, physiological, and biochemical reactions brought on by chemical substance buildup in epidermal tissue cells (Fig. 6C).
On the other hand, deterioration of DNA double helix structure, DNA volume, and nuclear protein concentration may lead to changes in the nucleus’s volume and shape (Dahl et al. 2008; Dauer and Worman 2009), such as the giant cell nucleus (Fig. 6E) and vacuole formation in the cell nucleus (Fig. 6F). In the literature, no data have been reported on the effects of L-CAN applied externally at different doses on the root anatomical structure of plants. Therefore, because the anatomical results of the current study about L-CAN are being presented for the first time, it is quite significant.
This study examined in detail the multifaceted harmful roles of the non-protein amino acid L-CAN on a range of physiological, cytogenetic, biochemical, and anatomical characteristics of the onion plant—a major global food source. While L-CAN decreased GP, RL, RN, FW, and MI, it caused increases in MN and CAs numbers, MDA and free PR contents, and activities of SOD and CAT enzymes. L-CAN application also promoted different types of chromosomal and anatomical damage in root meristem cells. No research has been done on the toxicity of high L-CAN dosages in onion plants in the literature. This is the first study to address the toxic effects of L-CAN on eukaryotic plant cells by using a variety of factors. For this reason, we believe that it will make an important contribution to the literature and will shed light on future studies.
