Effect of arginine, putrescine and spermidine on the polyamine, proline and chlorophyll content of tobacco (Nicotiana tabacum L.)

of plants, but, accordingly to previous experiments, spermidine has the main effect on the enhancement of salt tolerance. Nicotiana tabacum L. plants were grown in vitro on MS medium, the treatments were as follows: arginine (150 mg l), putrescine (10 mg l), spermidine (10 mg l). Proline, chlorophyll a, b and polyamine contents were measured. The obtained results show that the arginine decarboxylase and the


INTRODUCTION
Polyamines (PAs), such as spermidine (Spd) spermine (Spm) and their direct precursor, the diamine putrescine (Put) are vital and essential aliphatic amines which are also present in plants (Tun et al., 2006). They can be found in all compartments of the plant, also including the nucleus, which clearly shows their importance in the basic processes of the cells (Bouchereau et al., 1999;Galston et al., 1997;Walden et al. 1997). Polyamine levels are highly dependent upon the plant species, the organ and tissue type, and the developmental stage (Kuznetsov and Shevyakova, 2007). Polyamines are not classified as phytohormones, but similarly to them, they take part in several physiological processes (like growth, senescence, stress response) (Nambeesan et al., 2008).
As we previously mentioned (Mendel et al., 2018), though ethylene and polyamines are also involved in fruit ripening, the participating genes must operate in other biosynthetic pathways too.
Ethylene regulates the growth and development of plants, but in the case of stresses, it behaves as a stress hormone (Abeles et al., 1992). Levels of ethylene and its direct precursor 1-aminocyclopropane-1-carboxylic acid (ACC) become higher to the impact of abiotic stresses (Morgan and Drew, 1997).
In addition to the ethylene, polyamines play an important role in developing salt stress tolerance, and in responses for biotic and abiotic stresses (Kasukabe et al., 2004;Alcázar et al., 2010). Proline and polyamines are the most important nitrogen containing osmolytes in plants (Tarezynski et al., 1993). Polyamines form covalent bonds with macromolecules, such as nucleic acids and (Gill and Tuteja, 2010). Via osmotic regulation, they improve membrane stability, help the removal of free radicals, and influence the actions of stoma in the cases of abiotic stresses (Liu et al., 2007). Polyamine uptake at the cellular level is a very active and fast process, it reaches the saturation within 1-2 minutes and the cell store them mostly in the vacuoles (Bagni and Pistocchi, 1991).
Spermidine prevents the activation of the superoxide-generating NADPH-oxidase (Shen et al., 2000). Exogenous polyamines were successfully used to enhance salt tolerance in plants. Exogenous putrescine decreased the accumulation of Na + and Clions in different organs of salt stressed Datura stramonium L. (Ali, 2000). It affected positively the rooting of cucumber seedlings (Cucumis sativus L.) exposed to 100 mM NaCl stress, as it decreased the Na + uptake and increased the potassium accumulation (Shi et al., 2008).
In addition to the putrescine, exogenous spermidine enhances the salt tolerance of plants through bonding free radicals and maintain the Na + /K + balance (Saleethong et al., 2011), moreover decreases the Na + uptake, preventing K + loss (Zhao et al., 2007). According to Zhu et al., (2006) exogenous spermidine blocks Na + transport from the roots to the direction of leaves.
Adding exogenous spermine to salt stressed cucumbers increased the activity of some antioxidant enzymes, such as superoxide-dismutase, peroxidase and ascorbate-peroxidase, which resulted in decreased superoxide anion and malonaldehyde levels (Shu et al., 2013).
Consequently, exogenous application of all three main polyamine forms (Put, Spd, Spm) increased salt tolerance of plants, but accordingly to previous experiments, spermidine has the main effect on the enhancement of salt tolerance.

Plant material and treatments
In our experiment, Nicotiana tabacum L. seeds were germinated in vitro on MS medium in Petridishes. 21 days after germination plantlets were transferred into half strength MS medium with or without the supplements quoted below, three plants in each container. The plants were kept in 16 h light and 8 h dark periods at 23 °C. S Treats were as follows: arginine (150 mg l -1 ), putrescine (10 mg l -1 ), spermidine (10 mg l -1 ). Distilled water was used as control. Plants were treated three weeks after germination, samples were collected four weeks later. Samples from three containers per treatments were bulked up, therefore every measurement represents nine plants. Every measurement is performed with these samples.

Chlorophyll a, b content measurements
Chlorophyll content was determined after the method of Porra et al. (1989). 200 mg of leaf samples were pulverized in liquid nitrogen, then suspended in 2 ml of 80% (v/v) ice-cold acetone. After this, the suspensions were centrifuged at 4 ºC with 12000 g speed for 20 minutes. The upper phase was recentrifuged for 5 minutes at 4 ºC with 12000 g. 1 ml 1 M Tris (pH 8.0) was added to 1 ml of supernatant.

Determination of proline content
For proline measurement, the following method was used: 2 g leaf was extracted in 3% sulfosalicylic acid. 2 ml of ninhydrin reagent (2.5% ninhydrin dissolved in 60% glacial acetic acid, and 40% of 6 M phosphoric acid) and 2 ml of glacial acetic acid was added to 2 ml of aliquot, incubated at 100 °C for 40 minutes. Then samples were chilled on ice. After this 5 ml of toluene was added, vortexed, and incubated at 23 °C for 24 hours. The absorbance was measured at 520 nm. L-proline was used as standard (Bates et al., 1973). Proline was determined in the same HPLC examination as polyamines.

Polyamine analysis
The quantity of free polyamines was determined by the method described by Németh et al. (2002). 200 mg of samples were homogenized in liquid nitrogen, and extracted with 2 ml 0.2 M ice-cold perchloric-acid (HClO4), then chilled on ice for 20 minutes. After this, samples were centrifuged at 4 °C for 20 minutes with 10000 g.
In the case of free polyamine fraction 100 μl supernatant was used for preparing dansyl-chloride derivative as Smith and Davies (1985) described: In a 2 ml Eppendorf tube 200 μl saturated sodiumcarbonate and dansyl-chloride (5 mg ml -1 ) dissolved in 400 μl acetone were added to 100 μl sample. After homogenization, samples were incubated at 60 °C for 60 minutes in dark, then 100 μl of proline solution (100 mg ml -1 ) were added and incubated in dark at room temperature for 30 more minutes. After this, dansylderivatives were extracted with 500 μl of toluene for 30 seconds, and the organic upper phase were transferred to 1.5 ml Eppendorf tubes using a Pasteur-pipette and evaporated in vacuum. Dansylated polyamines were dissolved in 1 ml of 100% methanol and filtered via tephlone membrane filter (0.2 μm). Samples were examined by a WATERS W 2690 (Milford, USA) HPLC device, carrier phase was acetonitrile. Polyamines (putrescine, spermidine and spermine) from Sigma-Aldrich Ltd. were dansylated by the previously described method, and used as standards.

Statistical analysis
The obtained results were statistically evaluated using the standard deviation and ANOVA methods and these represent the mean values of at least 3 measurements.

RESULTS AND DISCUSSION
Measured quantities were calculated per gram of fresh weight, summarized in Table 1. It can be seen the graph, that the amount of the polyamines (and their ratio) has appreciably changed only on the effect of spermidine treatment, while at the other treatments the changes are minor. The concentration of proline decreased in all of the three treatments, which can indicate the reduced sensitivity to stress of the plants. This supposed is promoted by the fact, that the concentration of total chlorophyll a and b content increased in these cases.
The Figure 2 shows the measured putrescine, spermidine, spermine, proline and chlorophyll a, b concentrations altered by the treatments in the percentage variance of those in the control plants. We can see, that the amount of putrescine decreased by 29%, 16% and 21% on the effect of the exogenous arginine, putrescine and spermidine treatments, respectively. The change of spermidine content is not this obvious at all: decreased by 16% on the effect of arginine, increased by 2% in the putrescin treatment, decreased by 49% while spermidine was added. The amount of spermine decreased in arginine treatment, but increased in the effect of putrescine and spermidine treatments (2% and 12%, respectively). Taken all around, the total content of polyamines (last columns) showed 20%, 4% and 19% decrease on the effect of the three exogenous compounds. According to Pérez-Amador et al. (1995), polyamine metabolism in under strict regulation, and is hard to achieve alterations. Minor differences in polyamine-concentrations are available due to catabolic enzymes. Disintegration of polyamines is catalyzed by the copper-containing diamine-oxidase and the flavoprotein-dependent polyamine-oxidase (Kuznetsov and Shevyakova, 2007). Contrary to these, the concentration of the stress indicating proline showed a 6-6% decrease to arginine and putrescine addition, and decreased extremely (30%) to spermidine addition. Accordingly, the amount of chlorophyll a and b content increased by 26% 4% and 9%, which can indicate extended vitality. Presence of polyamines is needed for excessive growth and survival of plants, and for forming a strong abiotic stress tolerance. (Kovács et al., 2020;Kusano et al., 2008).

CONCLUSIONS
If we compare the measured amounts of putrescine, spermidine, spermine, proline and chlorophyll a, b it turns out, that the exogenous addition of arginine and polyamines also increased the vitality of the tobacco plants, decreased the stress sensitivity without the significant increase of endogenous polyamine levels. Hence, it has lightened that not only the increased amount of polyamines caused the stress tolerance, the effect is also indirect. The results show that activity of the argininedecarboxylase and the spermidine-synthase genes involved in polyamine metabolism, cannot be enhanced by exogenous addition of their precursor molecules. On the contrary, the spermine-synthase gene has a positive reaction to the lower-class forms of polyamines. For better understanding of mode of action of these genes further investigations are needed.