Hydrogen peroxide in tobacco stigma exudate affects pollen proteome and membrane potential in pollen tubes

Abstract

ROS are known to be accumulated in stigmas of different species and can possibly perform different functions important for plant reproduction. Here we tested the assumption that one of their functions is to control membrane potential and provoke synthesis of unique proteins in germinating pollen. We used spectrofluorometry and spectrophotometry to detect H2O2 in stigma exudate, quantitative fluorescent microscopy of pollen tubes and flow cytometry of pollen protoplasts to reveal effects on membrane potential, and a label‐free quantification approach to study pollen proteome changes after H2O2 treatment. We found that in both growing pollen tubes and pollen protoplasts exudate causes plasmalemma hyperpolarization similar to that provoked by H2O2. This effect is abolished by catalase treatment and the ROS quencher, MnTMPP. Inhibitory analysis indicates probable participation of Ca2+‐ and K+‐conducting channels in the observed hyperpolarization. For a deeper understanding of pollen response, we analysed proteome alterations in H2O2‐treated pollen grains. We found 50 unique proteins and 20 differently accumulated proteins that are mainly involved in cell metabolism, energetics, protein synthesis and folding. Observed hyperpolarization and proteome alterations agree well with previously reported stimulation of pollen germination by H2O2 and sensitivity of Ca2+‐ and K+‐conducting channels to this ROS. Thus, H2O2 is one of the active substances in tobacco stigma exudate that stimulates various physiological processes in germinating pollen.

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

“…This means that, first of all, hydrogen peroxide is crucial for normal pollen germination, and O • 2 − cannot replace it. These data are in good agreement with the result previously obtained in vitro, where the effect of the stigma exudate on tobacco pollen protoplasts disappeared after its treatment with catalase [ 17 ].…”

“…Moderate activity of some ROS-regulating enzymes, in particular peroxidases [ 13 , 14 , 15 ], has been shown in several species from Asteracea and Solanaceae ; in sunflower, SOD activity was also visualized [ 15 ]. On the other hand, pollen exine and pollen suspension have been found to have antioxidant properties [ 16 , 17 ]. Thus, the final balance between ROS production and elimination during in vivo germination is the result of a complex interaction between sporophyte and male gametophyte, which includes both low molecular weight components and antioxidant enzymes [ 18 ].…”

“…It turned out that hydrogen peroxide is present in tobacco stigma exudate in significant amounts. It causes membrane hyperpolarization in pollen tubes and protoplasts; treatment with catalase or ROS quencher MnTMPP abolishes this effect [ 17 ]. However, not only hydrogen peroxide can affect the male gametophyte.…”

Receptor for Activated C Kinase1B (OsRACK1B) Impairs Fertility in Rice through NADPH-Dependent H2O2 Signaling Pathway

“…ROS, in particular H 2 O 2 , has shown to be involved in pollen grain activation in kiwifruit [ 106 , 107 ], hyperpolarization of lily, and tobacco pollen grain protoplast [ 108 , 109 , 110 ]. Several reports suggest that a tightly controlled spatio-temporal regulation of ROS homeostasis is crucial for pollen development.…”

Pollen Germination and Pollen Tube Growth in Gymnosperms

“…Redox homeostasis is one of the main regulatory systems during pollen germination and tube growth in flowering plants, and it has been actively studied in recent years, especially regarding pollination and pollen behavior in vivo [ 89 , 90 , 91 , 92 ]. However, much less is known about the role of this system in gymnosperms [ 89 ].…”