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

Abstract

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.

Pattern of ROS generation and interconversion on wet stigmas in basal and divergent angiosperms

“…In both cases, the decrease in SOD activity correlated with a decline in reproductive success. The importance of SOD for successful tobacco pollination was confirmed by the application of SOD inhibitor to stigma, which led to a decrease in both the number of tubes ingrown into the style and the seed set (Breygina et al 2022). It can be assumed that SOD activity on stigma is important both for ROS interconversion in preparation for pollination and oxidative stress management.…”

“…To determine the concentration of hydrogen peroxide and level of ROS generation on stigma, exudate was collected by the "cap method", which was developed by us earlier (Breygina et al 2022). Briefly, a pipette tip containing a certain amount (corresponding to pistil size) of distilled water and/or spin trap solution (0.5 mM САТ1Н (see below), 0.1 EDTA) was put on the pistil without damaging any parts of the flower (except anthers, which in some cases were removed) and incubated for 30 min (except for peony, as mentioned further).…”

“…Superoxide dismutase (EC 1.5.1.1) activity was detected as described earlier (Breygina et al 2022). Briefly, stigmas were collected from fresh flowers and homogenized at 0 °C in Tris-HCl buffer (50 mM, pH 7.0) with 50 mM NaCl, 0.05% Tween-20 and 0.1% protein inhibitor cocktail (Sigma, Darmstadt, Germany).…”

“…Moderate levels of H 2 O 2 stimulated tobacco pollen germination in vitro (Smirnova et al 2013). The application of superoxide dismutase (SOD) inhibitor to receptive tobacco stigmas markedly reduced the efficiency of pollen germination and seed formation (Breygina et al 2022). Thus, in tobacco, the conversion of superoxide to peroxide, a SOD-controlled step in redox metabolism, is one of the keys to maintaining pollen germination in vivo.…”

“…We have recently developed a methodological approach to semi-quantitatively assess dynamics of different ROS, which is applicable to wet stigmas. It is based on EPR spectroscopic and spectrophotometric analysis of stigma exudate (Breygina et al 2022) and has been so far applied to one model species -tobacco. Here we use this approach to compare flowering plants from different taxonomic groups, phylogenetically distant from each other.…”