**3. Ionize radiation, reactive oxygen species (ROS) and defense systems of ROS**

**A B** 

transplanted in field (**Figure 3C**–**F**).

38 Plant Engineering

development (**Figure 2E** and **G**). Superior plants (putative mutants) in terms of salt tolerance were moved to a growth chamber (**Figure 3A** and **B**) for a while, and after that, they were

**C D** 

**E F** 

21 March 2013).

**Figure 3.** General view of survived plants of sainfoin in growth chamber (A and B). Transferred putative mutants against to salt stress in the field (C and D). Putative mutant sainfoin plants in the field after 1.5 months (E) and 2 months (F) (Location: The University of Ankara, Faculty of Agriculture, Department of Field Crops, Turkey, photos were taken on Ionizing radiation causes biological injury in exposed biological materials. The first target of ionizing radiation is water molecule, which is ubiquitous in any organisms. The cell is composed of ∼80% water [24]. As a result of excitation and ionization reactions, water molecule (H2 O) and H• and OH radicals are generated [25]. Gamma rays cause to produce free radicals (free radicals like O2 •− and OH• and nonradicals like H2 O2 and 1 O2 ) as known reactive oxygen species (ROS) through direct interactions of radiation with target macromolecules or via products of water radiolysis [13, 24, 26]. The formation of reactive oxygen species (ROS) occurs in the general metabolism of the plant cell.

However, such as other environmental stress, radiation lead to increase the formation of ROS in plant cell due to damage of cellular homeostasis and cause progressive oxidative damage and finally cell death [27].

Reactive oxygen species (ROS) control many different processes in plants [28]. Plants has two antioxidant machinery, one of them is antioxidative enzymes, including ascorbate peroxidase (APX), glutathione reductase (GR), superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPX), monodehydroascorbate reductase (MDHAR), and dehydroascorbate reductase (DHAR). The other one is nonenzymatic antioxidants like ascorbic acid (AA), reduced glutathione (GSH), α-tocopherol, carotenoids, flavonoids, and the osmolyte proline [26]. ROS depends on ionizing radiation level that causes damage or modification of components in plants, ultimately affecting morphology, physiology, anatomy, and biochemistry of plants [13, 29]. Currently, scientific evidence shows that ROS play an important signaling role in plants and regulate biological activities such as growth, development, and especially response to biotic and abiotic stress factors [26, 27]. ROS can induce injury of cell compartment, but on the other hand, they induce new gene expression in cells [30]. However, Esnault et al. [31] hypothesized that ROS (mainly H2 O2 ) can play a secondary role are in signaling process of cell. And after a first stress, plants can be more tolerant to a new stress synthesis due to secondary metabolites. Moreover, using gamma rays can create a permanent gene expression of antioxidative enzymes for scaving "oxidative stress" start from the first generation of plants. And this provides to improve superior plants varieties against biotic and abiotic stress factors.

**Here in the main question**: Can we use ionize radiations (especially, gamma rays) to generate mutants with desirable characteristics via supply permanent gene expression of antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR), and ascorbate peroxidase (APX) and also osmoprotectants such as proline and transmissible to the progeny in plants?

Although there are lots of works related to changing transcriptional regulation of various types of genes (especially genes of antioxidative enzymes ) due to gamma irradiation, there are limited reports on permanent and transmissible increased transcript levels of genes, which induced by gamma rays, of antioxidative enzymes in plants.

Beyaz et al. [32] reported that permanent production of antioxidant enzymes and proline in M1 plants of sainfoin (*Onobrychis viciifolia* Scop.) under *in vitro* conditions. In the study conducted by Beyaz et al. [32], the aim was to investigate effects of gamma radiation on physiological responses of the M1 sainfoin plants. Seeds of sainfoin ecotype 'Koçaş' were exposed to 0, 400, 500, and 600 Gy from a 60Co source at a dose rate of 0.483 kGy h−1. Irradiated and unirradiated seeds were sown into culture vessels containing MS-basal medium to be cultured for 30 days under *in vitro* conditions. At the end of this period, seedlings, which germinated from the radiated and unirradiated seeds, were transferred into pots in a growth chamber for 30 days more. Chlorophyll contents, activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR), as well as contents of malondialdehyde (MDA) and proline were examined in unirradiated and irradiated 60-day-old seedlings. Overall, the activities of the antioxidant enzymes (SOD, CAT, and GR) and contents of chlorophyll and proline in the leaves tended to increase after irradiation in a dose-dependent manner. By contrast, the activity of APX decreased. The lipid peroxidation characterized by the MDA content remained unchanged, except after irradiation to 500 Gy. The highest CAT activity and the highest proline content were observed after irradiation to the highest dose of 600 Gy. The highest SOD and GR activities were observed after irradiation to the lowest tested dose of 400 Gy. This is the first study that provided basic information on the impact of gamma radiation on physiological responses of sainfoin and its radiosensitivity. These findings will be useful in development of a mutation breeding program of sainfoin.

Also Zaka et al. [33] query the same questions in their investigation and reported that the low chronic ionizing radiation provide genetically transmissible gene expression of antioxidant enzymes such as (SOD, GR, CAT, POD, and GPDH) to the progeny of Spila capillata (Poaceae), which grown two contaminated areas (5.4 and 25 μSv h−1) on the Semipalatinsk nuclear test site in Kazakhstan. They considered evolutionary point of view and answered their observation with the natural populations that can change their genetic structure under environmental constraints and facilitates adaptation. The selective pressure of low radioactive contamination levels (leading to gamma-irradiation dose rates as low as 4.5 and 25 μSv h−1) may have played an important role during 50 years. However, Çelik et al. [25] reported that a high level of ascorbate peroxidase activity (APX) in the leaves generated from irradiated soybean seeds, compared to the unirradiated group. The APX activity increased in the unirradiated group over the experimental period and was increased by 3.6-fold at 14 days and 3.8-fold at 21 days after irradiation (P ≤ 0.05). Kim et al. [34] found that gene expression of antioxidant enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), and peroxidase (POD) can be induced depending on increasing gamma-irradiation dosage level in Triticeae species. Plant cells change their protein metabolism under main stress conditions. Resistant or stress proteins are induced in response to gamma-irradiation stress, and this defense mechanism change patterns of gene expression, especially stress-inducible gene expression, which produces qualitative and quantitative changes in total soluble protein [34, 35]. The different gene expression pattern of antioxidant enzymes can be observed in the irradiated plant species [36].

According to Mohammed et al. [37], a substantial variation of the protein patterns of cowpea seeds occur by gamma rays induced, and this variation has occurred because of the new expression of some polypeptide, the silence of others, and overexpression of a third class polypeptides. Aly and El-Beltagi [38] showed that increase in GST, CAT, SOD, and POD activities in *Vicia faba* L. seeds could be attributed to ionizing irradiation stress. Cho et al. [39] investigated the expression patterns of diverse genes at various time points after gamma irradiation of young tobacco plants and found three different gene expression pattern (increased, decreased, and no response) of antioxidative enzymes (CAT, SOD, and GST). However, Al-Rumaih and Al-Rumaih [40] reported that gamma irradiation affected antioxidant enzyme activities in the three investigated species of wheat. Moreover, the increased activity of antioxidant enzymes (SOD, CAT, POX, APOX) in response to gamma-irradiation treatment in many plant species (Nicotiana, *Triticum aestivum*, sugar cane, *Phaseolus vulgaris*, radish, groundnut, and pepper) were reported [41–46].

As a result of metabolic pathways of aerobic cells, free radicals and ROS are occurred and influenced most of the biological activity. On the other hand, products of oxidation reactions play an important role some biological process such as aging, some degenerative diseases, differentiation, and development, including serving as subcellular messengers in gene regulatory and signal transduction pathways. Several studies reported the hypothesis. The hypothesis is that shifts oxidant/antioxidant equilibrium in cell may also affect developmental pathways in a different of tissues from phylogenetically diverse organisms [47].

Over expression of antioxidant enzyme genes likely arise due to an efficient regulatory mechanism to provide cells with resistance [33, 40]. Sen et al. [48] reported that the activities of several antioxidant enzymes were evaluated in both vegetative and flowering stages, and mutant lines (wheat irradiated by 200 Gy) showing the highest biochemical performance. These studies clearly indicated that gene expression of antioxidant enzymes can be made permanently in genome of progeny of plants by gamma-rays irradiation.
