Evaluation of relationship between P5CS gene over expression and changes of some non-enzymatic antioxidant in transgenic tobacco under in vitro salt stress condition

Introduction The growth and production of crop plants are strongly affected by various environmental stress. Salinity stress affects all stages of plant development including germination, bud growth, vegetative growth, flowering, and fruiting. During the past few years, several genes that encoding various structural and regulatory proteins have been used to produce tolerant plants of abiotic stress. Tolerant plants have been selected by improving our knowledge about molecular mechanism of stress tolerance in plants. Plants increase organic osmolytes such as proline, glycine betaine, polyols, alcoholic sugars, and soluble sugars to modulate osmotic stress. The P5CS gene (proline-5-carboxylate-synthase) is a key enzyme in the pathway for proline synthesis and this amino acid increases resistance of plants to salinity. Although there are many reports about the key role of accumulation proline in mechanism of salinity tolerance, a little information is available about physiological and non-enzymatic antioxidant response of transgenic plants by P5Cs overexpression in salt stress. Therefore, the aim of this study was to evaluate physiological and non-enzymatic antioxidant responses of transgenic and non-transgenic tobacco to salinity under in vitro culture. Materials and methods In order to achieve this goal, transgenic and non-transgenic plants were selected by PCR experiment with NPTII: P5CS proprietary primers. Consequently, the expression of the P5CS gene in transgenic plants was significantly higher than non-transgenic plants. To investigate the mechanism of salt tolerance in tobacco, transgenic and non-transgenic tobacco plants were grown on MS medium containing 0, 100, 150, 200 mM NaCl. After 4 weeks of treatments, fresh and dry weight, photosynthetic pigments, sodium and potassium, proline, phenol, anthocyanin, flavonoid, ascorbate, and hydrogen peroxide were measured. Results and discussion Based on the results, dry and fresh weight as well as chlorophyll content in transgenic plants decreased less than non-transgenic plant under salt stress. For example, the fresh weight of non-transgenic plants in the medium with 100 and 200 mM NaCl decreased by 47% and 33% and their dry weight decreased by 23 and 33%, respectively compared with transgenic plants. Total chlorophyll content of transgenic plants in the medium with 100, 150 and 200 mM salt was improved by 25%, 22%, and 41% compared with non-transgenic plants, respectively. Also, the leaves of transgenic plants accumulated less sodium than non-transgenic plants in response to salinity stress. By adding 100, 150 and 200 mM salt to medium, the level of sodium in transgenic plants decreased by 50%, 17%, and 18% compared with non-transgenic plants respectively. Moreover, the level of phenolic, anthocyanin and flavonoid compounds in the transgenic plants were less than non-transgenic plants by adding salt to medium. The proline content of both transgenic and non-transgenic plants increased in response to salinity. In addition, there was a significant increase in proline content of transgenic plants compared to non-transgenic plant under salt stress. The ascorbate content in transgenic and non-transgenic plants did not change significantly in response to salinity. However, the hydrogen peroxide decreased significantly in transgenic plants as compared with non-transgenic plants in salt stress. The results showed that the accumulation of hydrogen peroxide in transgenic plants in the medium with 100, 150 and 200 mM salt was 83%, 41%, and 23% lower than non-transgenic plants, respectively. So, it seems that transgenic and non-transgenic tobacco plants were salt tolerant and salt sensitive respectively. It has been suggested that high proline content may lead to salt tolerance in plants. According to our experiment, overexpression of P5CS gene increased proline content in other plants and improved salinity resistance in transgenic plants.