No abstract

In aqueous medium etiolated wheat seedlings release superoxide anion (O2*-). Interaction of a synthetic antioxidant, butylated hydroxytoluene (BHT, ionol), with oxygen in the aqueous medium is accompanied by O2*- formation. This suggests that under certain conditions BHT behaves as a prooxidant. A natural antioxidant, superoxide dismutase (SOD), and also a wound healing preparation, emulsified denatured placenta (EDP), do not exhibit the prooxidant properties. In contrast to BHT, they reduce O2*- production by the etiolated wheat seedling system.

Apoptosis was observed in the coleoptile and initial leaf in 5-8-day-old wheat seedlings grown under normal daylight. Apoptosis is an obligatory event in early wheat plant ontogenesis, and it is characterized by cytoplasmic structural reorganization and fragmentation, in particular, with the appearance in vacuoles of specific vesicles containing intact organelles, chromatin condensation and margination in the nucleus, and internucleosomal fragmentation of nuclear DNA. The earliest signs of programmed cell death (PCD) were observed in the cytoplasm, but the elements of apoptotic degradation in the nucleus appeared later. Nuclear DNA fragmentation was detected after chromatin condensation and the appearance in vacuoles of specific vesicles containing mitochondria. Two PCD varieties were observed in the initial leaf of 5-day-old seedlings grown under normal daylight: a proper apoptosis and vacuolar collapse. On the contrary, PCD in coleoptiles under various growing (light) conditions and in the initial leaf of etiolated seedlings is only a classical plant apoptosis. Therefore, various tissue-specific and light-dependent PCD forms do exist in plants. Amounts of O2*- and H2O2 evolved by seedlings grown under normal daylight are less than that evolved by etiolated seedlings. The amount of H2O2 formed in the presence of sodium salicylate or azide by seedlings grown under normal daylight was increased. Contrary to etiolated seedlings, the antioxidant BHT (ionol) did not inhibit O2*- formation and apoptosis and it had no influence on ontogenesis in the seedlings grown under normal daylight. Thus, in plants grown under the normal light regime the powerful system controlling the balance between formation and inactivation of reactive oxygen species (ROS) does exist and it effectively functions. This system is responsible for maintenance of cell homeostasis, and it regulates the crucial ROS level controlling plant growth and development. In etiolated plants, this system seems to be absent, or it is much less effective.

It was found that production of superoxide (O(2)(*-)) is crucial for normal morphogenesis of etiolated wheat seedlings in the early stages of plant development. The development of etiolated wheat seedlings was shown to be accompanied with cyclic changes in the rate of O(2)(*-) production both in the entire intact seedling and in its separated organs (leaf, coleoptile). First increase in the rate of O(2)(*-) production was clearly observed in the period from two to four days of seedling development, then the rate of O(2)(*-) production decreased to the initial level, and then it increased again for two days to a new maximum. An increase in O(2)(*-) production in the period of the first four days of seedling development correlates with an increase in DNA and protein contents in the coleoptile. The second peak of increased rate of O(2)(*-) production observed on the sixth or seventh day of seedling development coincides with a decrease in DNA and protein contents and apoptotic internucleosomal nuclear DNA fragmentation in the coleoptile. Incubation of seedlings in the presence of the antioxidant BHT (ionol) strongly affects their development but it does not influence the increase in DNA and protein contents for the initial four days of seedling life, and it slows down the subsequent age-dependent decrease in protein content and fully prevents the age-dependent decrease in DNA content in the coleoptile. A decrease in the O(2)(*-) amount induced by BHT distorts the seedling development. BHT retards seedling growth, presumably by suppression of cell elongation, and it increases the life span of the coleoptile. It seems that O(2)(*-) controls plant growth by cell elongation at the early stages of seedling development but later O(2)(*-) controls (induces) apoptotic DNA fragmentation and protein disintegration.

We studied the mechanism of action of gametocides on the male reproductive tissue of plants. Gametocides are known to disturb the normal development of pollen. They are morphogenetic poisons used for treating plants at the stage of initiation of generative organs. Our earlier analysis of a databank on the relationship between the chemical structure and activity of gametocides showed that the gametocidal activity was not correlated with definite types of reactivity or spatial structure of the molecules of these compounds [1]. The results obtained allowed us to assume that the effect of gametocides is determined by a nonspecific response of generative tissues of plants to chemical stress, which is expressed as the induction of apoptosis in these tissues.Mizelle et al. [2] performed an electron microscopic study of the pollen of wheat plants treated with the gametocide phenridazone (I).Changes in mitochondrion ultrastructure were described in the pollen grains of experimental plants. At present, these changes are identified as signs of relatively late stages of apoptosis. We considered it important to demonstrate that a gametocide with a qualitatively different chemical structure and reactivity caused similar structural changes in the cells of the main game-tophyte of experimental plants. In this study, we analyzed the effect of dibuthyl phthalate (DBP, II), a potent gametocide substantially different from compound I in chemical structure. Compound I readily enters oneelectron reduction reactions and, hence, can catalyze the production of the radical. Compound II does not enter redox reactions under mild conditions; however, our data indicate that DBP blocks the initial step of the mitochondrial respiratory chain, which leads to the generation of hydrogen peroxide and, as a result, activation of apoptosis [3]. Therefore, we could expect that, despite the fundamentally different chemical properties, these compounds would exert similar biological effects, including similar changes in the structure of the male gametophyte related to the induction of apoptosis. Here, we report the results of the study on wheat plants treated with DBP at the booting stage.Wheat ( Triticum aestivum L.) cultivar Priokskaya was frown in open ground on small separate plots under the same conditions. At the booting stage, some plants were sprayed with a 1% DBP solution containing also a detergent (dimethyl sulfoxide, 15 g per 100 ml). The treatment was repeated three days later.For electron microscopic study, wheat anthers were fixed with a 4% solution of glutaraldehyde in 0.1 M phosphate buffer solution (pH 7.4) containing sucrose and formalin. Then, postfixation with 1% osmium tetroxide and dehydration with ethanol solutions with increasing concentrations (70% ethanol was saturated with uranyl acetate) were performed. The material was immersed into epoxide resin Epon-812. Ultrathin sections were made with the use of an LKB-III ultramicrotome (Sweden), stained according to Reynolds [4], and examined using HU-11B and HU-12 electron microscope...