Exogenous L-Carnitine Promotes Plant Growth and Cell Division by Mitigating Genotoxic Damage of Salt Stress

L-carnitine is a fundamental ammonium compound responsible for energy metabolism in all living organisms. It is an oxidative stress regulator, especially in bacteria and yeast and lipid metabolism in plants. Besides its metabolic functions, l-carnitine has detoxification and antioxidant roles in the cells. Due to the complex interrelationship of l-carnitine between lipid metabolism and salinity dependent oxidative stress, this study investigates the exogenous l-carnitine (1 mM) function on seed germination, cell division and chromosome behaviour in barley seeds (Hordeum vulgare L. cv. Bulbul-89) under different salt stress concentrations (0, 0.25, 0.30 and 0.35 M). The present work showed that l-carnitine pretreatment could not be successful to stimulate cell division on barley seeds under non-stressed conditions compared to stressed conditions. Depending on increasing salinity without pretreatment with l-carnitine, the mitotic index significantly decreased in barley seeds. Pretreatment of barley seeds with l-carnitine under salt stress conditions was found promising as a plant growth promoter and stimulator of mitosis. In addition, pretreatment of barley seeds with l-carnitine alleviated detrimental effects of salt stress on chromosome structure and it protected cells from the genotoxic effects of salt. This may be caused by the antioxidant and protective action of the l-carnitine. Consequently, this study demonstrated that the exogenous application of 1 mM l-carnitine mitigates the harmful effects of salt stress by increasing mitosis and decreasing DNA damage caused by oxidative stress on barley seedlings.

of studies describe changes to fatty acid, polar lipids and sterols as being influential in salt stress 4,6 . Although there is no direct evidence between carnitine and fatty acid metabolism, carnitine acyltransferase enzymes is formed from carnitine esters from acyl CoAs which is responsible from the mitochondrial β-oxidation in the avocado plant and pea seedlings 14,[25][26][27][28] .
Salinity presents a serious environmental problem as it affects life cycle parameters such as plant fertility, growth and production. Salinity affects approximately 20% of the world's total agricultural land [29][30][31][32] . Salinity causes oxidative stress, which in turn modifies the composition of fatty acids and the lipid content of organisms. The ability of plants to adapt to different environmental conditions is directly affected by the composition of fatty acids and cell membrane lipids [33][34][35][36] . Depending on plant species and intensity of the salt stress to which the plant is exposed, lipids are key to regulating selective cell membrane permeability 37,38 . According to Shaya-Khmetova et al. 39 , after applying exogenous salt stress to wheat species, the total lipid content had decreased in a week. The change in the concentration of salt modulates the activity of ion-transporting proteins 40 and the synthesis of osmolytes 41 , reflecting changes in the fatty acid composition of cell membrane lipids and lipid metabolism 42 . Furthermore, the levels of carnitine correlates with tolerance to abiotic stresses in plants 30 . Considering the positive effects of l-carnitine on lipid metabolism, fatty acid composition, cell membrane permeability and apoptosis under biotic and abiotic stress this study designed to determine how mitotic cycle is affected by l-carnitine and how l-carnitine alleviate detrimental effects of different concentrations of exogenous salt stress. The study was designed to determine whether l-carnitine could be effective to increase cell division and might be suppressor on programmed cell death under salt stress conditions; it also evaluates whether the compound can alleviate genotoxic effects of salt stress on DNA level and the chromosome structure in the meristematic root tip cells of barley (Hordeum vulgare L.).

Effects of L-carnitine on cell division under salt stress.
A preliminary study was performed to determine appropriate concentrations of NaCl to use; 0.25 M, 0.30 M and 0.35 M were deemed suitable to initiate salt stress responses in barley seeds. The salt concentrations preventing germination of seeds to a great extent (above 50% germination rate) were used to determine the NaCl concentrations. As a result of the preliminary study to identify which dose of the l-carnitine is effective for alleviating salt stress from 1000 mM to 0.1 mM concentrations were tested. As shown in the Fig. 1, the percentage of seed germination showed the same effect for 5 mM (84%) and 1 mM (84%) in 0.25 M salt. Depending on the increased salt stress concentration, the percentage of the germination regularly decreased in the 5 mM LC level (28% at 0.30 M and 10% at 0.35 M). Also, the experimental studies showed that l-carnitine solution must be prepared before used to get the same effects on seed germination. Finally, 1 mM l-carnitine concentration was found to be the most effective dose to offset the damage of salt stress stimulated to seed germination and the regulation of plant growth.
L-carnitine pretreatment (positive control, 1 mM LC) could not be successful to stimulate cell division on barley seeds by alone. Generally, mitotic index of barley seeds germinated in distilled water (negative control, 0 M NaCl) showed a higher rate (0.17) than pre-treated seeds group with l-carnitine (0.07) ( Table 1). In addition, the number of anaphase and telophase cells were very low in both groups. However, most of the cells were observed in prophase stage in the negative control group. There is a statistically significant difference was determined in the prophase and metaphase indices between pre-treated and untreated groups with LC (p ≤ 0.05) ( Table 1). Depending on the increasing salt stress concentration, the mitotic index significantly decreased in barley seeds. For instance, although the mitotic index value was calculated 0.17 in negative control group, it was 0.16 at 0.25 M NaCl, 0.14 at 0.30 M NaCl and 0.12 at 0.35 M NaCl concentrations. However, mitotic index of the pretreatment with l-carnitine under salt stress conditions was found promising as a plant growth promoter. In other words, pretreatment seeds with 1 mM l-carnitine to overcome detrimental effects of salt stress on cell division had effective results with the 0.28 mitotic index value at 0.25 M NaCl, 0.24 at 0.30 M NaCl and 0.14 at 0.35 M salinity conditions ( Table 1). As seen from the phase indices after pretreatment with l-carnitine, the mitotic cycle was stimulated by increasing the prophase stage under salt stress conditions.  5i) were also observed and noticed as statistically significant (p ≤ 0.05) compared to most common chromosome aberrations. Also, screening the DNA damage on nuclei to find more proof about genotoxicity, all threated samples were examined using Comet Assay (Table 3). Based on the genotoxicity index, depending on the salt stress concentration from 0 to 0.35 M, the percentage of tail DNA was increased from 0.17 ± 0.02 to 74.24 ± 2.70 (Fig. 6). Indeed, the 0 M NaCl concentration showed no head DNA (99.83%) damage. However, 0.35 M NaCl concentration showed significant differences on head DNA level (25.76%) (p ≤ 0.05). On the other hand, the application of 1 mM l-carnitine to barley seeds showed almost no damage on tail DNA (0.45%). Furthermore, the most protective role of l-carnitine on DNA level on barley seeds was observed in the highest concentration (0.35 M) of salt, after pretreatment with l-carnitine the percentage of tail DNA decreased almost three-fold from 74.24 ± 2.70 to almost 27.03 ± 1.30. Figure 7 also shows the changes on the nucleus on DNA damage due to the concentration levels.

Discussion
In the present study, cytogenetic and genotoxic effects of l-carnitine as a plant growth promoter germinated in different salt stress concentrations (0, 0.25, 0.30 and 0.35 M) was reported by determining the putative effects on seed germination, cell division and DNA damage in the meristematic root tip cells of H. vulgare L.
Pretreatment of barley seeds with l-carnitine made a mitodeppresive effect on cell cycle compared to the negative control group germinated seeds in distilled water. While mitotic index was counted 17% in the negative control group cells it was observed in the positive control group cells as 7% (see Tables 1 and 2). Chromosomal alterations were not observed in the root tip cells of barley seeds germinated in distilled water and the genotoxicity index was 0.00% (see Table 2, Fig. 2). However, the barley seeds pre-treated with 1 mM l-carnitine showed 8% genotoxicity index and different types of chromosome aberrations such as multinucleated cells and disorderly prophase (see Table 2, Fig. 3). According to the comet assay genotoxicity results, percentage of tail DNA in positive  www.nature.com/scientificreports www.nature.com/scientificreports/ control group cells increased 2.65-fold compared with the negative control group cells. Furthermore, 1 mM concentration of l-carnitine showed more genotoxic effects under unstressed conditions which might be cytotoxic on the mitotic chromosome structure in barley seeds. Otherwise, there are limited reports about l-carnitine levels in plant metabolism 43,44 . The only report for Panter and Mudd 45 presented endogenous l-carnitine levels in some higher plants and it was observed that barley seeds did not contain any level of l-carnitine. Hence, barley seeds were used to determine the exogenous effects in all studied parameters pre-treated with l-carnitine for 24 hours. These results indicated that exogenous l-carnitine was not effective to stimulate cell cycle for mitosis in the absence of stress (see Table 1).
Salinity is a global and major problem reducing agricultural crop production all over the world. It is reported that ~50% of the world's arable land will be affected from the increasing soil salinity by 2050 60,61 . Salinity also has a very strong relationship with osmotic and oxidative stress acting osmolytes, reactive oxygen species and membrane proteins 53,62-64 . Salt accumulation in the root systems generates osmotic stress by inhibiting water and essential elements uptake 65 and high concentration of salt stress cause repressive effects on DNA, RNA and protein synthesis, cell cycle progression, seed germination, plant growth and production 66,67 . To survive under salt stress, plants adapt their mechanism via activating genes and protein kinases associated with stresses, signal transduction involved in cell division 24,68 . To increase plant resistance to salt stress, researchers or breeders focus on identifying new nutrients and fertilizers given to plants exogenously for improving plant production. As an exogenous supplement in plant metabolism, these findings suggest that l-carnitine could be a potential salt stress inhibitor stimulating progression of cell cycle via increasing mitosis under salt stress conditions (see Table 1), because l-carnitine has antioxidant action by activating antioxidant enzymes responsible from the synthesis of protective molecules reported by numerous studies in mammals under stress conditions 59 . Similarly, antioxidants influence specific cell cycle transitions to control cell cycle and protect plant cells from oxidative stress damages 53,[69][70][71] . According to Charrier et al. 29 exogenous carnitine stimulates development and protects Arabidopsis thaliana seedlings from the oxidative and salt stress damage 29 . Likewise, the results of this study, as a new plant growth stimulator by promoting seed germination, increasing cell division and decreasing the damage on DNA caused by salt stress, 1 mM l-carnitine is recommended as the appropriate dose to protect cells from the oxidative damage.  [72][73][74] . Similarly, the highest chromosome alteration percentage was observed in the highest salt stress concentration (84%, 0.35 M) in this study. The sensitivity of the genotype to salt stress was statistically significant and different. Disorderly prophase (12 and 9%, respectively in 0.30 and 0.35 M NaCl) was the most common chromosome aberration in barley seeds germinated in salt stresses. Moreover, ring chromosomes (2%) (see Table 2, Fig. 4c,d) were the second significant aberration in 0.30 M salt stress concentration. Salinity side effects on chromosome structure were dependent to salt stress level, where a low level of salt stress has fewer side effects in contrast to high level stress application in all studied treatment group cells (see Table 2). Pretreatment with l-carnitine also significantly decreased the percentage of the genotoxicity index or chromosome aberrations and DNA damage on tail DNA in the meristematic root tips cells of barley (by ~5-fold in 0.25 M, ~4-fold in 0.30 M and ~6-fold in 0.35 M NaCl) compared to the absence of l-carnitine under salt stress (see Table 3). This could also be due to the www.nature.com/scientificreports www.nature.com/scientificreports/ regulation of the protective effect as well as DNA repair capability of l-carnitine 58,75,76 . These findings showed that l-carnitine was effective at different degrees to alleviate detrimental effects of salt stress on chromosome structure (see Table 2). However, different types of chromosomal alterations were also counted in pretreatment with 1 mM l-carnitine such as micronucleus, stickiness, alignment anaphase, fault polarization, anaphase/telophase bridges, vagrant chromosomes and multinucleated cells in interphase cells in the meristematic root tip cells of barley seeds under salt stress conditions. Interestingly, multinucleated cells were only observed in the pre-treated l-carnitine groups both the absence of salt stress and under salt stress concentrations (see Table 2). So, it may result from the l-carnitine and may cause formation of multinucleated cells in interphase level. Similarly, some researchers reported that a significant decrease in DNA damage and chromosome aberrations pre-treated with l-carnitine alleviates negative effects of oxidative stress 56,77 and inhibition of tumor growth 78,79 and cancer cell death 80 in animal cells. According to Missihoun et al. 81 the regulation of carnitine biosynthesis was affected by the salt stress levels in A. thaliana wild and transgenic plants that expression level of the betaine aldehyde dehydrogenase www.nature.com/scientificreports www.nature.com/scientificreports/ genes has an important role on the early growing stage of the seedlings to mitigate the salt stress in young tissues. Compared to the results of this study, it was determined that l-carnitine might have a cytoprotective effect to balance the overexpression level of the genes depending on increasing levels of salt stress. Due to the physiological functions of the carnitine and other betaines such as protection of enzyme structure and increasing membrane stability depending on the stress level have been reported as non-toxic even in high concentrations 82 . Otherwise, positive control group (1 mM l-carnitine) found to be genotoxic on the barley chromosome structure compared to the negative control group (0 mM l-carnitine). However, the presented study here suggests that l-carnitine has a protective effect on chromosome structure and DNA level against the genotoxic effects under the salt stress especially in high concentrations in barley cells.
In summary, the results of this study imply that the level of l-carnitine pretreatment (1 mM) was species specific under the tolerance levels to salt stress of H. vulgare L. Testing the putative cytoprotective effects of l-carnitine as an exogenous supplement were identified on cell cycle proliferation, genotoxicity and the level of DNA damage under salinity. A deeper understanding of these protective role of l-carnitine on DNA level related to putative salt stress sensors might help marker assisted breeding and genetic engineering to cope with various abiotic stresses.    Fig. 2b) is one of the world's oldest and most often grown cultivated crop plant. Its success and popularity for agriculture is based on its high adaptive capacity to withstand abiotic stress. Because of its innate ability to endure drought, fungal infection and salinity, barley is a model plant; thus, it is an ideal model organism for in biological research to study plant stress responses. This study used barley (H. vulgare L. cv. Bulbul-89) seeds to explore the cytogenetic response of l-carnitine (C 7 H 15 NO 3 , MA: 161.2 g/mol) in salt stress conditions. Three biological replicates of each twenty-five uniformly sized barley seeds were prepared for each application by pre-treating with 1 mM of l-carnitine or distilled water (negative control), then leaving them at room temperature for 24 h. Then the solutions were removed and the seeds were vacuum-dried 83 . For each application, the seeds were put into 10 cm petri dishes and covered with two sheets of filter paper; sheets were moistened with 10 ml of distilled water (0 M salt) and the others with one of the predetermined concentrations of salt (0.25 M, 0.30 M or 0.35 M). To allow the seeds to germinate, the petri dishes were incubated at 20 ± 1 °C for 7 days. The root tips of the germinated seeds were cut off once they were 1-1.5 cm long, and they were pre-treated for 4 hours with paradichlorobenzene. The root tips were fixed in Carnoy solution [(ethanol (99%): glacial acetic acid (3:1)] for 24 h. Then they were stored at 4 °C in 70% ethanol until required. The root tips were hydrolyzed for 17 min. using 1 N HCl and stained using the Feulgen protocol for at least 1 h and before squashing the tips on slides with a little 45% acetic acid. At least 3.000 cells per treatment (1.000 per slide) were evaluated, and the mitotic index, phase indices (I) of dividing cells and chromosome aberrations were scored. To calculate the mitotic index (%), the number of cells undergoing mitotic division was divided by the total number of cells × 100 84 . For a more detailed evaluation of cell division, we calculated the indices (I) of the separate phases, prophase (IP), metaphase (IM), anaphase (IA) and telophase (IT). In accordance with Ivanova et al. 85  An Olympus CX-41 research microscope (100X objective) was used to observe the chromosome structure; they were photographed using a C-5060 WZ camera. commet assay. Microscope slides were pre-heated and coated with 1% normal melting point agarose and dehydrated at room temperature. The meristematic root tips of barley seedlings were chopped in 600 μl nucleus isolation buffer (4 mM MgCl 2 .6H 2 O, 0.5% Triton TM X-100 and 0.2 M Tris, pH 7.5) for 30 s. The buffer containing nuclei was transferred to a clean microcentrifuge tubes for each treatment and the tubes were centrifuged at 12,000 rpm for 15 min. at 4 °C. Following centrifugation, 50 μl of suspension of nuclei were placed on each slide mixed with 50 μl 1% low melting point agarose at 55 °C, covered with 20 × 50 mm cover glass and stated on ice for 5 min. Then, the samples were subjected to electrophoresis buffer in high salt solution (300 mM NaOH, 1 mM EDTA, pH > 13) for 30 min. at 4 °C. After that, they were exposed to 25 V for 20 min. and 300 mA and cooled on ice for 5 min. For neutrolization step, the slides were kept in 100 mM Tris-HCl for 5 min. Each slide stained with 70 μl ethidium bromide solution and kept at 4 °C for 5 min. to photograph the DNA damage on nuclei under fluorescence microscope on 200 cells/comets for each concentration using OpenComet v1.3.1 Software.