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.

Contribution of apoplast to short-term copper uptake by wheat and mung bean roots.

In this study we addressed the controversial issue of contribution of cell walls (CWs) to Cu binding in plant roots. We compared short-term Cu uptake at different solution Cu levels by mung bean (Vigna radiata (L.) R. Wilczek) and wheat (Triticum aestivum L., cv. Inna) and by root CWs isolated from either Cu-treated or non-treated plants. Twenty four hours of plant exposure to Cu affected Cu-binding capacity of mung bean root CWs but not wheat CWs. Amounts of Cu associated with CWs and roots increased with Cu concentration. The Cu accumulated in CWs could account for total Cu content of roots (except for wheat in highest Cu treatment). Pectin content of the CWs and their Cu-sorption capacity were positively correlated. The accumulation of Cu in root CWs is a principal response of wheat and mung bean plants to excess Cu, limiting symplastic Cu uptake in roots in short-term treatment. The contribution of CWs to Cu absorption by plant roots depends on Cu level in the medium and plant species.

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.