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.

In vertebrates, vitamin K is a cofactor for the gamma-glutamyl carboxylase (GGCX) involved in the carboxylation of glutamic acid residues. During the vitamin K cycle, vitamin K is oxidised by GGCX, and then reduced by vitamin K epoxide reductase (VKOR), which is inhibited by the synthetic coumarin warfarin. GGCX and VKOR are present in Drosophila melanogaster, but the existence of a vitamin K cycle remains unproven. Semi-lethal concentrations (LC₅₀) of K₃, menadione sodium bisulfite (MSB), and warfarin to neutralise the negative effect of MSB were selected for the Drosophila cultivation medium. LC-MS analysis was used for vitamin K measurement in flies’ extracts. The EPR method and RT-PCR were used for ROS level measurement and gene transcription assessment, respectively. The LC₅₀ of MSB in the medium resulted in a more than 20-fold increase in endogenous K₂ in flies, demonstrating the mechanism of K₃-to-K₂ conversion. Administration of 1 mM warfarin in the medium with MSB completely neutralised its negative effect on viability. Developed flies had decreased K₂ level, confirming the existence of a vitamin K cycle, and both reduced ROS level and hsp22 gene transcription. The biochemical pathways affected by elevated K₂ concentrations involves both elements of the vitamin K cycle and the adaptive mitochondrial antioxidant system.

Nicotiana tabacum is a classic model for studying pollination on wet stigma. Reactive oxygen species (ROS) and nitric oxide (NO) production are closely related to stigma fertility and depend on the activity of redox enzymes. This study is devoted to the comparison of two tobacco lines differing in physiological parameters and reproductive success. Samsun is a tobacco variety that is widely used in research due to its low demands; however, the reproductive potential of the variety is quite low. Based on this variety, a new line was obtained, called “Fortune”; the plants are externally similar to the Samsun plants, but are more successful in reproduction. The total production of ROS + NO on the stigmas of the Fortune plants is lower than the Samsun plants, but their ROS production is higher, and the main decrease occurs due to NO. Superoxide dismutase activity differs between the two lines at all stages of stigma development except the fertile stage, while ascorbate peroxidase activity is higher in “Fortune” at all stages. Additional isoforms of ascorbate peroxidase are detected in developing stigmas of the Fortune variety. Presumably due to differences in redox metabolism, Fortune plants produce more seeds, their fruit are larger, and their leaves and flowers are also larger compared to the Samsun plants. In this study, we investigated both redox homeostasis parameters and plant productivity using tobacco as the model plant and suggested that there is a correlation between these groups of parameters, which may be important for breeding highly productive plants.

Tobacco is a plant with a wet stigma, which produces reactive oxygen species (ROS) and abscisic acid (ABA) which is important for in vivo pollen germination. Furthermore, ROS can be linked to growth processes, stimulating or inhibiting them. However, to what extent do differences in the redox environment and ABA level on the stigma and in pistil tissues correlate with flower growth, pollination success and resulting fruit parameters? We investigated redox homeostasis and ABA concentrations in stigma exudates of two tobacco lines (“Samsun” and “Fortune”) with different floral organ size and seed production. Fortune has longer flowers, larger fruits, and more seeds than Samsun. We report here that Samsun has a higher total oxidative capacity in stigma exudate, and possibly also higher NO level, than Fortune, as estimated by electron paramagnetic resonance spectroscopy. Fortune has a higher ascorbate peroxidase (APX) content in stigma tissues, as determined by Western blot analysis, and a higher ABA concentration in stigma exudate. Analyzing ROS levels and enzyme activity during the elongation stage in buds, we found that shorter Samsun styles had higher ROS levels, but they also had higher superoxide dismutase (SOD) and APX activity. The results of this study may help in developing approaches to a targeted increase in flower size and seed productivity.