С. М. Романчук
Институт ботаники имени Н. Г.Холодного НАН Украины
ВЛИЯНИЕ ИОНИЗИРУЮЩЕГО ИЗЛУЧЕНИЯ НА АКТИВНОСТЬ Β-ГЛЮКОЗИДАЗЫ В ПРОРОСТКАХ ARABIDOPSIS THALIANA (L.) HEYNH.
Исследовано влияние рентгеновских лучей в дозах 0.5, 1, 2, 4, 6, 8, 10 и 12 Гр на активность β-глюкозидазы в проростках Arabidopsis thaliana. Показано, что рентгеновское облучение изменяет активность β-глюкозидазы. Выявлены расхождения в показателях β-глюкозидазной активности при различных дозах рентгеновских лучей. Рассмотрены показатели активности β-глюкозидазы как молекулярный маркер на действие ионизирующей радиации.
Ключевые слова: рентгеновское облучение, Arabidopsis thaliana, активность β-глюкозидазы
S. M. Romanchuk
М. G. Kholodny Institute of Botany of NASof Ukraine
INFLUENCE OF IONIZING RADIATION ON THE β-GLUCOSIDASE
The article deals with the influence of X-ray radiation on the β-glucosidase activity of Arabidopsis thaliana 3- and 13-day-old seedlings grown in the stationary conditions. The Brassicaceae family is characterized by the presence of ER bodies in plant cells, which are derivative of granular endoplasmic reticulum. The research demonstrates that an enzyme β-glucosidase with an ER retention signal selectively accumulates in A. thaliana ER bodies. β-glucosidase (EC 184.108.40.206) catalyzes the hydrolysis of aryl- and alkyl-β-glucosides, releasing glucose andaglycone.This enzyme is known to perform a protective function in responses to different unfavorable factors. The study has proved that ER bodies are highly susceptible and sensitive to injuries, mechanical pressure, influence of toxic substances, pathogen lesions, insect bites andclinorotation. It has been recently reported that formation of ER bodies in A.thaliana roots is sensitive to the influence of X-ray radiation since their quantity and size increase under X-ray radiation. In addition, ER bodies develop greater variability after X-ray radiation at a dose of 8Gy.The level of β-glucosidase in the ER bodies has increased. The family Brassicaceae is quite resistant to irradiation. Therefore, the primary concern of this research is to studythe effect of X-ray radiation on the β-glucosidase actiion off A. thaliana seedlings. Seeds of A. thaliana (line Columbia) were sterilized and then sewn on the МS'mineral medium. Seedlings have been growing for 3 and 13 days. Some of the 3-day-old seedlings were treated with X-ray radiation in dose of 0.5, 1, 2, 4, 6, 8, 10 and 12 Gy on the unit RUM-17 (Russia) (dose rate 0.43 cGr/sec). Seedlings were examined for two hours (3-day-old seedlings) and 10 days (13-day-old seedlings) as exposed to X-ray radiation. The control group was composed of seedlings of the same age but without such an exposure. To determine β-glucosidase activity we applied a modified method of Matsuura et al. (1989), using the synthetic substrate p-nitrophenyl-β-D-glucopiranoside. The absorbance was measured in a spectrophotometer Specord M40, at 420 nm. Protein determinations were performed colorimetrically (Bradford, 1976). A unit of enzyme action was defined as the amount of enzyme which would release 1.0 mg of 4-nitrophenol in 40 minutes of reaction in 100 mg of A. thaliana sample. All experiments were carried out in three biological and three analytical repetitions. The conducted research showed thatleaves of 3-day-old A. thaliana seedlings from control group were small, round-shaped and of dark green color.The main root showed emerging lateral roots. In control group, the rosette of 13-day-old seedlings was ofproper form. Besides, it was made up of four leaves, each more than 1 mm in size.These leaves were oval-shaped, serrated on the edges and of rich green color. The root included a main root and branching lateral roots. Organs of 3- and 13-day-old A. thaliana seedlings did not show any signs of stress after X-ray radiation. The effect of β-glucosidase on 3-day-old seedlings was 0.42 units in a control group. After a two-hour exposure to X-ray radiation of different doses the effect of β-glucosidasehas changed. Results demonstated no correlation between a dose of irradiation and enzyme activity.The highest β-glucosidase activity was detected at doses of 0.5, 8 and 12 Gy. It has showed more than two-fold increase as compared to control group. β-glucosidase activity after exposure to X-ray radiation was: 0.92 units at dose of 0,5 Gy; 0.97 units at dose of 8 Gy; and 0.93 units at dose of 12 Gy. Other doses, however, had almost no influence on activity. Such a variability suggests that the difference could be due to genetic factors. Cells of A. thaliana seedling interpret a dose in 0.5 Gy as a signal to utilize adaptive mechanisms and, above all, to activate reparative system. The measured effect of β-glucosidase on 13-day-old seedlings was 0.38 units in a control group. After a 10-day exposure to X-ray radiation of different doses the β-glucosidase activity did not differ greatly from seedlings under control. The highest β-glucosidase activity was detected at doses of 8Gy. It has demonstrated more than 1.5-fold increase compared to a control group. β-glucosidase activity after X-ray radiationdose of 8 Gy was 0.59 units. It was first recorded that the most reactive dose was 8 Gy, with the effect of which lasting for 10 days after exposure to X-ray radiation. Plant cells respond actively to the influence of ionizing radiation. One of these reactions is the change in enzyme activity, hydrolytic in particular. Provided that β-glucosidase is the main component of ER bodies, we may assume that ER bodies are the original depositories of this protein under the influence of irradiation as the indicators of the activity of β-glucosidase after X-ray radiation at a dose of 0.5, 1, 2, 4, 6, 8, 10 and 12 Gy as a molecular marker of ionizing radiation have been considered.
Keywords: X-ray radiation, Arabidopsis thaliana, β-glucosidase activity