Plant Hormone and Fatty Acid Screening of Nicotiana tabacum and Lilium longiflorum Stigma Exudates.

Pollen germination in vivo on wet stigmas is assisted by the receptive fluid-stigma exudate. Its exact composition is still unknown because only some components have been studied. For the first time, hormonal screening was carried out, and the fatty acid (FA) composition of lipid-rich (Nicotiana tabacum) and sugar-rich (Lilium longiflorum) exudates was studied. Screening of exudate for the presence of plant hormones using HPLC-MS revealed abscisic acid (ABA) in tobacco stigma exudate at the two stages of development, at pre-maturity and in mature stigmas awaiting pollination, increasing at the fertile stage. To assess physiological significance of ABA on stigma, we tested the effect of this hormone in vitro. ABA concentration found in the exudate strongly stimulated the germination of tobacco pollen, a lower concentration had a weaker effect, increasing the concentration did not increase the effect. GC-MS analysis showed that both types of exudate are characterized by a predominance of saturated FAs. The lipids of tobacco stigma exudate contain significantly more myristic, oleic, and linoleic acids, resulting in a higher unsaturation index relative to lily stigma exudate lipids. The latter, in turn, contain more 14-hexadecenoic and arachidic acids. Both exudates were found to contain significant amounts of squalene. The possible involvement of saturated FAs, ABA, and squalene in various exudate functions, as well as their potential relationship on the stigma, is discussed.

Fatty Acid Composition of Dry and Germinating Pollen of Gymnosperm and Angiosperm Plants.

A pollen grain is a unique haploid organism characterized by a special composition and structure. The pollen of angiosperms and gymnosperms germinate in fundamentally similar ways, but the latter also have important features, including slow growth rates and lower dependence on female tissues. These features are, to some extent, due to the properties of pollen lipids, which perform a number of functions during germination. Here, we compared the absolute content and the fatty acid (FA) composition of pollen lipids of two species of flowering plants and spruce using GC-MS. The FA composition of spruce pollen differed significantly, including the predominance of saturated and monoene FAs, and a high proportion of very-long-chain FAs (VLCFAs). Significant differences between FAs from integumentary lipids (pollen coat (PC)) and lipids of gametophyte cells were found for lily and tobacco, including a very low unsaturation index of the PC. The proportion of VLCFAs in the integument was several times higher than in gametophyte cells. We found that the absolute content of lipids in lily pollen is almost three times higher than in tobacco and spruce pollen. For the first time, changes in the FA composition were analyzed during pollen germination in gymnosperms and angiosperms. The stimulating effect of H2O2 on spruce germination also led to noticeable changes in the FA content and composition of growing pollen. For tobacco in control and test samples, the FA composition was stable.

Dynamics of endogenous levels and subcellular localization of ABA and cytokinins during pollen germination in spruce and tobacco.

We used the enzyme-linked immunosorbent assay (ELISA) to assess the level of endogenous hormones in spruce pollen, and immunolocalization and confocal microscopy to study hormone localization in spruce and tobacco pollen. During pollen activation, the levels of ABA, zeatin, and its riboside significantly decreased. After the initiation of polar growth, the levels of all cytokinins increased sharply; ABA level also increased. In dormant spruce pollen grains, zeatin and ABA were localized uniformly throughout the cytoplasm. Zeatin was not detected in the nuclei, and the antheridial cell showed higher levels than the vegetative cell; ABA signal was detected in the cytoplasm and the nuclei. In germinating pollen, both hormones were detected mainly in plastids. The similar pattern was found in growing pollen tubes; signal from ABA also had a noticeable level in the cytosol of the tube cell, and was weaker in the antheridial cell. Zeatin fluorescence, on the other hand, was more pronounced in the antheridial cell. In non-germinated grains of tobacco, zeatin was localized mainly in organelles. ABA in dormant pollen grains demonstrated uniform localization, including the nuclei and cytoplasm of both cells. After germination, zeatin was accumulated in the plasmalemma or cell wall. ABA signal in the cytoplasm decreased; in the nuclei, it remained high. In growing tubes, the strongest zeatin and ABA signals were observed at the plasma membrane. The differences in ABA and cytokinin localization between species and dynamic changes in their level in spruce pollen highlight the key spatial and temporal parameters of hormonal regulation of gymnosperm pollen germination.

The Balance between Different ROS on Tobacco Stigma during Flowering and Its Role in Pollen Germination.

The concept of ROS as an important factor controlling pollen germination and tube growth has become generally accepted in the last decade. However, the relationship between various ROS and their significance for the success of in vivo germination and fertilization remained unexplored. For the present study, we collected Nicotiana tabacum stigma exudate on different stages of stigma maturity before and after pollination. Electron paramagnetic resonance (EPR) and colorimetric analysis were used to assess levels of O•2- and H2O2 on stigma. Superoxide dismutase activity in the stigma tissues at each stage was evaluated zymographically. As the pistil matured, the level of both ROS decreased markedly, while the activity of SOD increased, and, starting from the second stage, the enzyme was represented by two isozymes: Fe SOD and Cu/Zn SOD, which was demonstrated by the in-gel inhibitory analysis. Selective suppression of Cu/Zn SOD activity shifted the ROS balance, which was confirmed by EPR. This shift markedly reduced the rate of pollen germination in vivo and the fertilization efficiency, which was estimated by the seed set. This result showed that hydrogen peroxide is a necessary component of stigma exudate, accelerates germination and ensures successful reproduction. A decrease in O•2- production due to NADPH oxidase inhibition, although it slowed down germination, did not lead to a noticeable decrease in the seed set. Thus, the role of the superoxide radical can be characterized as less important.

Pollen Germination and Pollen Tube Growth in Gymnosperms.

Pollen germination and pollen tube growth are common to all seed plants, but these processes first developed in gymnosperms and still serve for their successful sexual reproduction. The main body of data on the reproductive physiology, however, was obtained on flowering plants, and one should be careful to extrapolate the discovered patterns to gymnosperms. In recent years, physiological studies of coniferous pollen have been increasing, and both the features of this group and the similarities with flowering plants have already been identified. The main part of the review is devoted to physiological studies carried out on conifer pollen. The main properties and diversity of pollen grains and pollination strategies in gymnosperms are described.

Oxygen radicals and cytoplasm zoning in growing lily pollen tubes.

KEY MESSAGE: Differential modulation of ROS content of the microenvironment (O ¯/MnTMPP/OH·) affects growth speed and morphology in lily pollen tubes. Oxygen radicals influence ionic zoning: membrane potential and pH gradients. Recently, redox-regulation of tip growth has been extensively studied, but differential sensitivity of growing cells to particular ROS and their subcellular localization is still unclear. Here, we used specific dyes to provide mapping of H2O2 and O·2¯ in short and long pollen tubes. We found apical accumulation of H2O2 and H2O2-producing organelles in the shank that were not colocalized with O·2¯-producing mitochondria. Differential modulation of ROS content of the germination medium affected both growth speed and pollen tube morphology. Oxygen radicals affected ionic zoning: membrane potential and pH gradients. OH· caused depolarization all along the tube while O·2¯ provoked hyperpolarization and cytoplasm alkalinization. O·2¯accelerated growth and reduced tube diameter, indicating that this ROS can be considered as pollen tube growth stimulator. Serious structural disturbances were observed upon exposure to OH· and ROS quencher MnTMPP: pollen tube growth slowed down and ballooned tips formed in both cases, but OH· affected membrane transport and organelle distribution as well. OH·, thus, can be considered as a negative regulator of pollen tube growth. Pollen tubes, in turn, are able to reduce OH· concentration, which was assessed by electron paramagnetic resonance spectroscopy (EPR).

Dynamics of Pollen Activation and the Role of H+-ATPase in Pollen Germination in Blue Spruce (Picea pungens).

Pollen is a highly specialized structure for sexual plant reproduction. Early stages of pollen germination require the transition from dormant state to active metabolism. In particular, an important role during this early phase of angiosperm pollen germination is played by H+-ATPase. Very little is known about pollen activation in gymnosperm species, and information on the involvement of H+-ATPase is lacking. We tracked four indicators characterizing the physiological state of pollen: membrane potential, intracellular pH, anion efflux and oxygen uptake, in order to monitor the dynamics of activation in Picea pungens. Based on pH dynamics during activation, we assumed the important role of H+-ATPase in spruce pollen germination. Indeed, germination was severely suppressed by P-type ATPase inhibitor orthovanadate. In spruce pollen tubes, a pronounced pH gradient with a maximum in the apical zone was found, which was different from the pollen tubes of flowering plants. Using orthovanadate and fusicoccin, we found that the proton pump is largely responsible for maintaining the gradient. Immunolocalization of the enzyme in pollen tubes showed that the distribution of H+-ATPase generally coincides with the shape of the pH gradient: its maximum accumulation is observed in the apical zone.

ROS and Ions in Cell Signaling during Sexual Plant Reproduction.

Pollen grain is a unique haploid organism characterized by two key physiological processes: activation of metabolism upon exiting dormancy and polar tube growth. In gymnosperms and flowering plants, these processes occur in different time frames and exhibit important features; identification of similarities and differences is still in the active phase. In angiosperms, the growth of male gametophyte is directed and controlled by its microenvironment, while in gymnosperms it is relatively autonomous. Recent reviews have detailed aspects of interaction between angiosperm female tissues and pollen such as interactions between peptides and their receptors; however, accumulated evidence suggests low-molecular communication, in particular, through ion exchange and ROS production, equally important for polar growth as well as for pollen germination. Recently, it became clear that ROS and ionic currents form a single regulatory module, since ROS production and the activity of ion transport systems are closely interrelated and form a feedback loop.

Redox-regulation of ion homeostasis in growing lily pollen tubes.

The pollen tube is characterized by cytoplasm compartmentalization typical for cells with polar growth. This concept includes "ion zoning", i.e. gradient distribution of ionic currents across the plasma membrane and free inorganic ions in the cytoplasm. One of the putative mechanisms for maintaining "ion zoning" is indicated by the sensitivity of the ion transport systems to reactive oxygen species (ROS). Here we test the possibility of redox regulation of ionic gradients and membrane potential (MP) gradient in growing pollen tubes using quantitative fluorescence microscopy. ROS quencher MnTMPP and exogenic H2O2 cause different alterations of intracellular Ca2+ gradient, pH gradient and MP gradient during short-term exposure. MnTMPP significantly shifts the gradients of Ca2+ and MP at low concentrations while high concentration cause growth alterations (ballooned tips) and cytoplasm acidification. H2O2 at 0,5 and 1 mM affects ion homeostasis as well (MP, Ca2+, pH) but doesn't decrease viability or alter shape of the tubes. Here we present original quantitative data on the interconnection between ROS and ion transport during tip growth.

The role of reactive oxygen species in pollen germination in Picea pungens (blue spruce).

KEY MESSAGE: Endogenous ROS, including those produced by NADPH oxidase, are required for spruce pollen germination and regulate membrane potential in pollen tubes; [Formula: see text] and H 2 O 2 are unevenly distributed along the tube. Recently, the key role of reactive oxygen species (ROS) in plant reproduction has been decisively demonstrated for angiosperms. This paper is dedicated to the involvement of ROS in pollen germination of gymnosperms, which remained largely unknown. We found that ROS are secreted from pollen grains of blue spruce during the early stage of activation. The localization of different ROS in pollen tube initials and pollen tubes demonstrated the accumulation of H2O2 in pollen tube apex. Colocalization with mitochondria-derived [Formula: see text] showed that H2O2 is produced in mitochondria and amyloplasts in addition to its apical gradient in the cytosol. The necessity of intracellular ROS and, particularly, [Formula: see text] for pollen germination was demonstrated using different antioxidants. ·OH and extracellular ROS, on the contrary, were found to be not necessary for germination. Exogenous hydrogen peroxide did not affect the germination efficiency but accelerated pollen tube growth in a concentration-dependent manner. The optical measurements of membrane potential showed that in spruce pollen tubes there is a gradient which is controlled by H+-ATPase, potassium- and calcium-permeable channels, anion channels and ROS, as demonstrated by inhibitory analysis. An important role of NADPH oxidase in the regulation of ROS balance in particular, and in germination in general, has been demonstrated by inhibiting the enzyme, which leads to the reduction in ROS release, depolarization of pollen tube plasma membrane, and blocking of pollen germination.

Effects of Ni2+ and Cu2+ on K+ and H+ currents in lily pollen protoplasts.

Heavy metals affect plant development and reproduction if they are present in excessive amounts, a situation that is becoming increasingly common. Pollen is a convenient object for pollution assessment as it is in most cases a 2- or 3-cellular organism exposed to the environment. At the same time, pollen is a key stage in the life cycle of seed plants; pollen viability and efficiency of germination are crucial for reproductive success and crop yield. In the present study we reveal for the first time, to our knowledge, targets for heavy metals (Cu2+ and Ni2+) in the pollen grain plasma membrane using the patch-clamp technique. Ni2+ dramatically decreases K+ current in pollen grain protoplasts, whereas Cu2+ does not alter the current density. Instead, Cu2+ strongly enhances H+ current driven by H+-ATPase, whereas Ni2+ fails to affect this current. The short-term treatment with Cu2+ also leads to reactive oxygen species (ROS) accumulation in pollen grain protoplasts but intracellular pH and membrane potential remain unchanged. Ni2+ had no significant effect on ROS content or membrane potential. Thus, plasmalemma K+ channels in pollen grains are sensitive to Ni2+ and H+-ATPase is sensitive to Cu2+, possibly, in a ROS-mediated way. Both metals leave pollen viable since membrane potential is maintained at the control level.

Periplasmic multilamellar membranous structures in Nicotiana tabacum L. pollen grains treated with Ni²âº or Cu²âº.

Essential trace elements Ni(2+) and Cu(2+) can block pollen germination without causing cell death. Mechanisms of this effect remain unclear. Using TEM, we studied the effects of Ni(2+) or Cu(2+) treatment on the ultrastructure of the aperture regions in tobacco pollen preparing to germinate in vitro, since in these zones, the main fluxes of water, ions, and metabolites cross the plasmalemma. Neither Ni(2+) nor Cu(2+) altered the cytoplasm ultrastructure, but both affected the reorganization of apertural periplasm during pollen activation. Numerous multilamellar membranous structures continuous with the plasma membrane could be seen in hydrated but not yet activated pollen. When the normal activation was completed, the structures disappeared and the plasmalemma became smooth. In the presence of 1 mM Ni(2+) or 100 µM Cu(2+), these structures preserved its original appearance. It is assumed to be the storage form for the membrane material, which is to provide an initial phase of the pollen tube growth. Ni(2+) and Cu(2+) affect the utilization of these membranes, thereby, blocking the pollen germination.

Ni(2+) effects on Nicotiana tabacum L. pollen germination and pollen tube growth.

To investigate the mechanisms of Ni(2+) effects on initiation and maintenance of polar cell growth, we used a well-studied model system-germination of angiosperm pollen grains. In liquid medium tobacco pollen grain forms a long tube, where the growth is restricted to the very tip. Ni(2+) did not prevent the formation of pollen tube initials, but inhibited their subsequent growth with IC(50) = 550 µM. 1 mM Ni(2+) completely blocked the polar growth, but all pollen grains remained viable, their respiration was slightly affected and ROS production did not increase. Addition of Ni(2+) after the onset of germination had a bidirectional effect on the tubes development: there was a considerable amount of extra-long tubes, which appeared to be rapidly growing, but the growth of many tubes was impaired. Studying the localization of possible targets of Ni(2+) influence, we found that they may occur both in the wall and in the cytoplasm, as confirmed by specific staining. Ni(2+) disturbed the segregation of transport vesicles in the tips of these tubes and significantly reduced the relative content of calcium in the aperture area of pollen grains, as measured by X-ray microanalysis. These factors are considered being critical for normal polar cell growth. Ni(2+) also causes the deposition of callose in the tips of the tube initials and the pollen tubes that had stopped their growth. We can assume that Ni(2+)-induced disruption of calcium homeostasis can lead to vesicle traffic impairment and abnormal callose deposition and, consequently, block the polar growth.