ROS in tobacco stigma exudate affect pollen proteome and provoke membrane hyperpolarization

Abstract

ROS are known to be accumulated in stigmas of different species and can possibly perform different functions in plant reproduction. Here we confirm the assumption that they affect pollen by altering ion transport through the plasma membrane; as a more deferred effect, pollen proteome is modified. We detected ROS in stigma exudate, found hyperpolarization in exudatetreated growing pollen tubes and used flow cytometry of pollen protoplasts to compare the effects of fresh exudate and exogenous H 2 O 2 on pollen tube plasmalemma. Exudate causes plasmalemma hyperpolarization similar to the one provoked by H 2 O 2 , which is abolished by catalase treatment and ROS quencher MnTMPP. Inhibitory analysis indicates the participation of Ca 2+ -and K + -conducting channels in the observed hyperpolarization, linking obtained data with previous patch-clamp studies in vitro. For a deeper understanding of pollen response to ROS we analyzed proteome alterations in H 2 O 2 -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. Thus, pollen is getting ready for effective resource usage, construction of cellular components and rapid growth. Highlights The active substance in stigma exudate is H 2 O 2  H 2 O 2 causes hyperpolarization mediated by the activation of cation channels.  H 2 O 2 affects pollen proteome; we found 50 unique proteins.