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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. [ABSTRACT FROM AUTHOR] more...

Data on ABA involvement in osmoregulation of in vitro germinating petunia ( Petunia hybrida L.) male gametophyte were obtained. Two potential targets of ABA action in a pollen tube (PT) are identified. These are represented by (1) plasma membrane (PM) H-ATPase, electrogenic proton pump participating in PM polarization, and (2) Ca-dependent K-channels localized in the same membrane. It was established that a stimulatory effect of ABA on electrogenic activity of H-ATPase is mediated by the increase in free Ca level in the cytosol of a PT and reactive oxygen species (ROS) generation. Based on the results obtained on the role of K ions in the hormonal control of water transport-driving forces in a PT, the hypothesis suggesting that ABA stimulated pollen grains (PGs) germinating and PT growth by activating K-channels was put forward. The revealed ABA-induced shift in cytoplasmic pH (pH) is suggested to be involved in a cascade of the events of the progamic phase of fertilization, including pH-dependent K-channels functioning. It was established that ABA abolishes the inhibitory effects of ethylene receptors blocker, 1-methylcyclopropene (1-MCP), and blockers of ACC and ABA synthesis (aminooxyacetic acid, AOA, and fluridone, respectively) on PT germination and growth, whereas ethrel blocks the inhibitory effect of fluridone on PT growth. In stigmas pretreated with ABA and AOA before pollination, this phytohormone was found to suppress inhibitory effect of AOA on ACC synthesis in the pollen-pistil system. All these findings, taken together, led us to the conclusion that ABA is involved in petunia male gametophyte osmoregulation interacting with ethylene at the level of ACC synthesis in the progamic phase of fertilization. [ABSTRACT FROM AUTHOR] more...

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. [ABSTRACT FROM AUTHOR] more...

In the pollen-pistil system of petunia ( Petunia hybrida L.) self-compatible and self-incompatible clones within 7 h after self-pollination, we determined the content of ACC (1-aminocyclopropane-1-carboxylic acid), the activity of two enzymes (ACC synthase and ACC oxidase), and the rate of 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 different levels of ACC and ethylene release. The pollen-pistil system of the self-compatible clone contained twice more ACC than in the self-incompatible clone, whereas the pollen-pistil system in the self-incompatible clone produced 4-5 times more ethylene than in the self-compatible clone. For both types of pollination, ACC and ethylene were predominantly produced in the stigma tissues. The rate of ethylene production therein was 50 times greater than in the styles and ovaries, and the content of ACC was 100 times higher than in the styles and ovaries. Germination of male gametophyte after both types of pollination was accompanied by elevated ACC synthase activity (especially in the case of compatible pollination), whereas notable increase in ACC oxidase activity was manifested in growing pollen tubes after self-incompatible pollination [ABSTRACT FROM AUTHOR] more...

The effects of exogenous phytohormones (IAA, ABA, and GA3) on the intracellular (cytoplasmic) pH (pHc) in ungerminating and germinating petunia ( Petunia hybrida L.) pollen grains were studied. The pHc values were measured with fluorescein diacetate. In ungerminating pollen grains, all phytohormones reduced pHc relatively rapidly; after 10–15 min, initial value was restored. In germinating pollen grains, IAA and ABA induced a relatively rapid cytosol alkalization, which was not reversed during experiment. GA3 acidified the cytosol, i.e., exerted the effect similar to that in ungerminating pollen grains. Sodium orthovanadate suppressed completely the hormone-induced pHc shift toward alkaline values in germinating pollen grains, whereas this inhibitor did not affect pHc in the absence of phytohormones. Sodium orthovanadate also slowed the recovery of pHc after hormone-induced cytoplasm acidification in ungerminating pollen grains, reduced pHc in control ungerminating grains, and weakened substantially the effects of all phytohormones on these pollen grains. On the basis of these results, we suggested that physiological activities of phytohormones in this system were mediated by pHc modulation, namely, a transient disturbance in the cytosolic pH homeostasis, which could trigger further phytohormone-induced cell responses. We concluded that a hormone-induced cytoplasm alkalization in pollen grains was mediated by the activity of their plasma membrane H+-ATPase and that this proton pump was involved in the control of pHc in both germinating and ungerminating pollen grains. [ABSTRACT FROM AUTHOR] more...

The data obtained characterize the changes in the contents of endogenous phytohormones (IAA, cytokinins, GA, and ABA) in germinating pollen grains and growing pollen tubes of a self-compatible clone of petunia ( sPetunia hybrida L.) within an 8-h period under in vitro conditions. The hydration and initiation of germination of pollen grains brought the ABA content down to a zero level, while the levels of GA, IAA, and cytokinins increased 1.5–2-fold. Later, in the growing pollen tubes, the GA content increased twofold, while the levels of IAA and cytokinins decreased. The exogenous ABA and GA3 considerably promoted pollen germination and pollen tube growth; however, only the treatment with GA3 produced the maximum length of pollen tubes. The exogenous IAA promoted and the exogenous cytokinins hindered the growth of pollen tubes. The membrane potential, as assessed with a potential-sensitive dye diS-C3-(5), considerably increased in the pollen grains treated with ABA and benzyladenine, whereas IAA and GA3 did not practically affect it. The authors conclude that the mature pollen grains contain the complete set of hormones essential for pollen germination and pollen tube growth. ABA, GA, and IAA together with cytokinins control the processes of pollen grain hydration, germination, and pollen tube growth, respectively. [ABSTRACT FROM AUTHOR] more...

Presents the results of a clinical study on the generation of calcium (Ca) signal in petunia pollen grains. Determination of the localization at the cellular level of the Ca2+ transporting system; Temperature where spectrophotometric and spectrofluorometric measurements were performed; Confirmation on the activating effect of depolarization of pollen grains on Ca2+. more...