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 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.
The active participation of ROS in the reproductive physiology of flowering plants is well-documented in model species, mainly plants with dry stigmas. Here we studied the release of ROS into stigma exudate – a fluid involved in pollen acceptance on a wet stigma. The study involved plants from different systematic groups: Paeonia suffruticosa, Nicotiana tabacum, Lilium hybr., Aristolochia manshuriensis and Berberis vulgaris; in most cases, exudate was collected from intact flowers on the whole plant to minimize the effect of experimental intervention on the sensitive redox balance system. The dynamics of total oxidizing capacity of stigma exudate was assessed by EPR spectroscopy and different stigma staining. The level of hydrogen peroxide and the activity of superoxide dismutase, which catalyzes H2O2 formation, was also evaluated. In all the plants studied, total ROS level increased with stigma maturation, however, H2O2 concentration decreased in studied representatives of eudicots, except for peony. In lily it increased, while in birthwort it remained low, which was apparently due to the absence of SOD activity. In all divergent plants studied, the enzyme was active on stigmas with two isoenzymes. During the fertile period, activity was higher than at an earlier stage. Based on the obtained data, we speculate on primitive and progressive patterns of ROS generation and transformation on stigma and its possible evolution.
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