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

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 H₂ O₂ 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 H₂ O₂ treatment. We found that in both growing pollen tubes and pollen protoplasts exudate causes plasmalemma hyperpolarization similar to that provoked by H₂ O₂ . 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 H₂ O₂ -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 H₂ O₂ and sensitivity of Ca²⁺ - and K⁺ -conducting channels to this ROS. Thus, H₂ O₂ is one of the active substances in tobacco stigma exudate that stimulates various physiological processes in germinating pollen.