Aminooxyacetic acid (АОА), inhibitor of 1-aminocyclopropane-1-carboxilic acid (AСС) synthesis, suppresses self-incompatibility-induced programmed cell death in self-incompatible Petunia hybrida L. pollen tubes.

Self-incompatibility (SI) is genetically determined reproductive barrier preventing inbreeding and thereby providing the maintenance of plant species diversity. At present, active studies of molecular bases of SI mechanisms are underway. S-RNAse-based SI in Petunia hybrida L. is a self-/non-self recognition system that allows the pistil to reject self pollen and to accept non-self pollen for outcrossing. In the present work, using fluorescent methods including the TUNEL method allowed us to reveal the presence of markers of programmed cell death (PCD), such as DNA fragmentation, in growing in vivo petunia pollen tubes during the passage of the SI reaction. The results of statistical analysis reliably proved that PCD is the factor of S-RNAse-based SI. It was found that preliminary treatment before self-pollination of stigmas of petunia self-incompatible line with aminooxyacetic acid (AOA), inhibitor of ACC synthesis, led to stimulation of pollen tubes growth when the latter did not exhibit any hallmarks of PCD. These data argue in favor of assumption that ethylene controls the passage of PCD in incompatible pollen tubes in the course of S-RNAse-based SI functioning. The involvement of the hormonal regulation in SI mechanism in P. hybrida L. is the finding observed by us for the first time.

ABA and IAA control microsporogenesis in Petunia hybrida L.

The formation of fertile male gametophyte is known to require timely degeneration of polyfunctional tapetum tissue. The last process caused by the programmed cell death (PCD) is a part of the anther program maturation which leads to sequential anther tissue destruction coordinated with pollen differentiation. In the present work, distribution of abscisic acid (ABA) and indole-3-acetic acid (IAA) in developing anthers of male-fertile and male-sterile lines of petunia (Petunia hybrida L.) was analyzed by using the immunohistochemical method. It was established that the development of fertile male gametophyte was accompanied by monotonous elevation of ABA and IAA levels in reproductive cells and, in contrast, their monotonous lowering in tapetum cells and the middle layers. Abortion of microsporocytes in the meiosis prophase in the sterile line caused by premature tapetum degeneration along with complete maintenance of the middle layers was accompanied by dramatic, twofold elevation in the levels of both the phytohormones in reproductive cells. The data obtained allowed us to conclude that at the meiosis stage ABA and IAA are involved in the PCD of microsporocytes.

[Auxin Abolishes Inhibitory Effects of Methylcyclopropen and Amino Oxyacetic Acid on Pollen Grain Germination, Pollen Tube Growth, and the Synthesis of ACC in Petunia].

As established by us earlier, ethylene behaves as a regulator of germination, development, and growth of male gametophyte during the progamic phase of fertilization. However, the mechanisms of the regulation of these processes remain so far unstudied. It is believed that the main factor providing variety of the ethylene responses is its interaction with other phytohormones. According to our working hypothesis, ethylene controls germination of pollen grains (PGs) and growth of pollen tubes (PTs) by interacting with auxin, which, as the available data indicate, is likely a key regulator of plant cell polarization and morphogenesis and one of the factors modulating the biosynthesis of ethylene at the level of ACC-synthase gene expression. In the present work, on germinating in vitro male gametophyte and the pollen-stigma system for petunia (Petunia hybrida L.) effects of phytohormones (ethylene and IAA) and known blockers repressing ethylene reception (1-methylcyclopropene, 1-MCP), the synthesis of ACC (amino oxyacetic acid, AOA) and transport IAA (triyodbenzoynaya acid, TYBA) on PGs germination, PTs growth and the synthesis of ACC were investigated. According to the data obtained, exogenous ethylene and IAA stimulated both PGs germination and PTs growth. 1-MCP and TYBA completely inhibited the first process, whereas IAA abolished the inhibitory action of 1-MCP and AOA on both the above processes. Etrel only partially weakened the inhibitory effect of TYBA. Examination of ACC synthesis modulation with AOA showed that IAA does not affect the level of ACC in germinating in vitro male gametophyte and nonpollinated stigmas, while this phytohormone insignificantly raised the level of ACC and abolished the inhibitory effect of AOA on its synthesis in the pollenstigma system. Pollination of stigmas with the pollen preliminarily treated with 1-MCP led to 2.5-fold decline in both the rate of PT growth and the level of ACC. At the same time, IAA abolished the inhibitory action of 1-MCP recovering the synthesis of ACC and growth of PTs to the control values. All these results, taken together, provide evidence for the interaction of the signal transduction pathways of ethylene and auxin at the level of ACC biosynthesis in the course of germination and growth of petunia male gametophyte during the progamic phase of fertilization.

[Exogenous IAA and ABA Stimulate Germination of Petunia Male Gametophyte by Activating Ca(2+)-Dependent K(+)-Channels and by Modulating the Activity of Plasmalemma H(+)-ATPase and Actin Cytoskeleton].

To date, the molecular mechanisms underlying the osmoregulation of pollen grains (PGs) related to the maintenance of their water status and allowing pollen tubes (PTs) to regulate concentrations in them of osmolytes and transmembrane water transport remain to be not so far characterized. In the present work, the data on the participation of IAA and ABA in the osmoregulation of germinating in vitro petunia male gametophyte were obtained. It has been established that the growth-stimulating effect of these phytohormones is due to their action on intracellular pH (pHc), the membrane potential of plasmalemma (PM), the activity of PM H(+)-ATPase, K(+)-channels in the same membrane and organization of actin cytoskeleton (AC). Two possible targets of the action of these compounds are revealed. These are represented by (1) PM H(+)-ATPase, electrogenic proton pump responsible for polarization of this membrane, and (2) Ca(2+)-dependent K+-channels. The findings of the present work suggest that the hormone-induced pHc shift is involved in cascade of the events including the functioning of pH-dependent K+-channels. It was shown that the hormoneinduced hyperpolarization of the PM is a result of stimulation of electrogenic activity of PM H(+)-ATPase and the hormonal effects are mediated by transient elevation in the level of free Ca(2+) in the cytosol and generation of reactive oxygen species (ROS). The results on the role of K(+) ions in the control of water-driving forces for transmembrane water transport allowed us to formulate the hypothesis that IAA and ABA stimulate germination of PGs and growth of PTs by activating K(+)-channels. In addition, the studies performed showed that the AC of male gametophyte is sensitive to the action of exogenous phytohormones, with to more extent to the action of IAA. As judged by the action of latrunculin B (LB) the AC may serve as the determinant of the level of endogenous phytohormones that most likely participate in the regulation of the polar growth of PTs impacting on the pool of F-actin in their apical and subapical zones.

[Role of ethylene in the control of gametophyte-sporophyte interactions in the course of the progamic phase of fertilization].

We investigated dynamics of the content of 1-aminocyclopropane-l-carboxylic acid (ACC) and ethylene production in male gametophyte development and germination in fertile (self-compatible and self-incompatible) and sterile clones of petunia. Fertile male gametophyte development was accompanied by two peaks of ethylene production by anther tissues. The first peak occurred during the microspore development simultaneously with the degeneration of both the tapetal tissues and the middle layers of the anther wall. The second peak coincided with dehydration and maturation of pollen grains. In the anther tissues of the sterile line of petunia, tenfold higher ethylene production was observed at the meiosis stage compared with that in fertile male gametophytes. This fact correlated with the degeneration of both microsporocytes and tapetal tissues. Exogenously applied ethylene (1-100 ppm) induced a degradation of the gametophytic generation at the meiosis stage. According to the obtained data, ethylene synthesis in germinating male gametophyte is provided by a 100-fold ACC accumulation in mature pollen grains. The male gametophyte germination, both in vitro, on the culture medium, and in vivo, on the stigma surface, was accompanied by an increase in ethylene production. Depending on the type of pollination, germination of pollen on the stigma surface and the pollen tube growth in the tissues of style were accompanied by various levels ofACC and ethylene release. The male gametophyte germination after self-compatible pollination was accompanied by higher content of ACC as compared with the self-incompatible clone, whereas, after the self-incompatible pollination, we observed a higher level of ethylene production compared with compatible pollination. For both types of pollination, ACC and ethylene were predominantly produced in the stigma tissues. Inhibitor of ethylene action, 2,5-norbornadiene (NBN), blocked both the development and germination of the male gametophyte. These results suggest that ethylene is an important factor in male gametophyte development, germination, and growth at the progamic phase of fertilization.

[An automated thermographic complex: new possibilities of the diagnosis of ORL disease]. / Avtomatizirovannyi termograficheskii kompleks: novye vozmozhnosti diagnostiki zabolevanii LOR-organov.

The Kiev Research Institute of Otorhinolaryngology has built an automatic diagnostic system that includes a Raduga thermograph and a CM-1403 computer. The diagnostic algorithms are based on the thermosemiotics of ENT inflammatory diseases developed by the authors. The advantages of the system are: production of a hard copy of thermograms, registration and long storage of information, mathematical treatment of thermal images, data base and rapid search for the thermogram needed, screening diagnosis based on the "healthy-sick" principle.