The role of reactive oxygen species in pollen germination in Picea pungens (blue spruce)

Abstract

Endogenous ROS, including those produced by NADPH oxidase, are required for spruce pollen germination and regulate membrane potential in pollen tubes; [Formula: see text] and H O are unevenly distributed along the tube. Recently, the key role of reactive oxygen species (ROS) in plant reproduction has been decisively demonstrated for angiosperms. This paper is dedicated to the involvement of ROS in pollen germination of gymnosperms, which remained largely unknown. We found that ROS are secreted from pollen grains of blue spruce during the early stage of activation. The localization of different ROS in pollen tube initials and pollen tubes demonstrated the accumulation of HO in pollen tube apex. Colocalization with mitochondria-derived [Formula: see text] showed that HO is produced in mitochondria and amyloplasts in addition to its apical gradient in the cytosol. The necessity of intracellular ROS and, particularly, [Formula: see text] for pollen germination was demonstrated using different antioxidants. ·OH and extracellular ROS, on the contrary, were found to be not necessary for germination. Exogenous hydrogen peroxide did not affect the germination efficiency but accelerated pollen tube growth in a concentration-dependent manner. The optical measurements of membrane potential showed that in spruce pollen tubes there is a gradient which is controlled by H-ATPase, potassium- and calcium-permeable channels, anion channels and ROS, as demonstrated by inhibitory analysis. An important role of NADPH oxidase in the regulation of ROS balance in particular, and in germination in general, has been demonstrated by inhibiting the enzyme, which leads to the reduction in ROS release, depolarization of pollen tube plasma membrane, and blocking of pollen germination.

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

“…Thus, this ROS can be regarded as a normal component of the environment for pollen on stigma, which induces germination in angiosperms. Interestingly, for gymnosperms this pattern is not characteristic (Maksimov et al 2018).…”

“…Hypothesis (2) has convincing evidence obtained using simplified in vitro model system (Potocký et al 2007;Smirnova et al 2009a): ROS quenching with Mn-TMPP (100-300 µM) significantly lowers the growth rate (Potocký et al 2007). Adding moderate concentrations of Mn-TMPP (H 2 O 2 and O 2 -• quencher) as well as DPI suppressed pollen germination both in tobacco and spruce (Smirnova et al 2009a;Maksimov et al 2018). On the other hand, the addition of exogenous ROS in moderate concentrations (100 µM for tobacco) activates germination (Smirnova et al 2009a); Despite this rather convincing data, detailed studies and comparison of pollen grain physiology in the presence of both ROS and the stigma exudate were needed to clarify the function of ROS on the receptive stigma surface.…”

“…Mapping of MP in pollen tubes was performed as described earlier (Maksimov et al 2018) with Di-4-ANEPPS (Sigma) which allows to detect accurately spatiotemporal variations of MP. Pollen tubes were stained with 2 μM Di-4-ANEPPS and immediately used for microscopy.…”

Oxygen radicals and cytoplasm zoning in growing lily pollen tubes

“…The distribution of inorganic ions is also strictly subjected to the principle of zoning, including gradient distribution of ionic currents through the plasma membrane as well as free inorganic ions in the cytoplasm (Michard et al 2017;Feijó and Wudick 2018;Zheng et al 2019). The latter is maintained due to the uneven allocation of ion transport systems and their activitywhich also results in the gradient distribution of membrane potential values, which has been to date revealed in 3 species, including lily (Breygina et al 2010;Maksimov et al 2018;Podolyan et al 2019).…”

“…Model experiments on protoplasts with exogenous ROS highlighted several aspects of pollen physiology related to redox-signaling, the main of which is ion homeostasis, as ion transport across the membrane is regulated by Н 2 О 2 Maksimov et al 2016). As Н 2 О 2 is the most stable ROS, all studies in vitro were confined to Н 2 О 2 , which was shown to have multiple effects in pollen tubes, both angiosperm (Podolyan et al 2019) and gymnosperm (Maksimov et al 2018). Studies on somatic plant cells that have been exposed to stress lead to the opinion that Н 2 О 2 doesn't affect "targets" that had been attributed to this ROS but rather binds to transition metal binding sites converting it to OH • (Demidchik 2015).…”