Reactive oxygen species are involved in regulation of pollen wall cytomechanics

Abstract

Production and scavenging of reactive oxygen species (ROS) in somatic plant cells is developmentally regulated and plays an important role in the modification of cell wall mechanical properties. Here we show that H2O2 and the hydroxyl radical ((•)OH) can regulate germination of tobacco pollen by modifying the mechanical properties of the pollen intine (inner layer of the pollen wall). Pollen germination was affected by addition of exogenous H2O2, (•)OH, and by antioxidants scavenging endogenous ROS: superoxide dismutase, superoxide dismutase/catalase mimic Mn-5,10,15,20-tetrakis(1-methyl-4-pyridyl)21H, 23H-porphin, or a spin-trap α-(4-pyridyl-1-oxide)-N-tert-butylnitrone, which eliminates (•)OH. The inhibiting concentrations of exogenous H2O2 and (•)OH did not decrease pollen viability, but influenced the mechanical properties of the wall. The latter were estimated by studying the resistance of pollen to hypo-osmotic shock. (•)OH caused excess loosening of the intine all over the surface of the pollen grain, disrupting polar growth induction. In contrast, H2O2, as well as partial removal of endogenous (•)OH, over-tightened the wall, impeding pollen tube emergence. Feruloyl esterase (FAE) was used as a tool to examine whether H2O2-inducible inter-polymer cross-linking is involved in the intine tightening. FAE treatment caused loosening of the intine and stimulated pollen germination and pollen tube growth, revealing ferulate cross-links in the intine. Taken together, the data suggest that pollen intine properties can be regulated differentially by ROS. (•)OH is involved in local loosening of the intine in the germination pore region, while H2O2 is necessary for intine strengthening in the rest of the wall through oxidative coupling of feruloyl polysaccharides.

EXO70A2 is critical for the exocyst complex function in Arabidopsis pollen

“…Also mutants in two other exocyst subunits, sec3a and sec8, exhibited severe defects in their germination efficiency (Bloch et al, 2016;Cole et al, 2005). One of the crucial regulatory modules in pollen germination and pollen tube growth is the ROS production (Potocký et al, 2007;Potocký et al, 2012;Smirnova et al, 2014). Two Arabidopsis pollen-specific NADPH oxidases, RBOHH and RBOHJ, localized at the PM are necessary for ROS accumulation in pollen grain cell wall upon pollination and later in pollen tube tips.…”

“…Double mutants in these genes exhibited much lower ROS level resulting in collapsing pollen tubes (Lassig et al, 2014;Kaya et al, 2015). Interestingly, data of Smirnova et al (2014) suggest that targeted production of extracellular ROS is required in order to change mechanical properties of intine pollen grain cell wall to ensure germination. Since we observed a significant decrease in ROS production in exo70a2 germinating pollen grains, we suggest that the pollen exocyst may function in the delivery of NADPH oxidases to specific sites at the PM during germination.…”

“…Even after 20 hours the exo70a2 pollen still showed 8-fold lower germination efficiency than WT pollen. Since high concentrations of extracellular reactive oxygen species (ROS) were previously reported at the onset of the pollen germination process (Smirnova et al, 2014), we analyzed the ROS production in WT and exo70a2 pollen grains 20 min after imbibition using histochemical staining of superoxide anion radicals by nitroblue tetrazolium (NBT). The ROS levels were dramatically reduced in mutant pollen grains, indicating their different physiological status (Fig.…”

Genetic and Biochemical Mechanisms of Pollen Wall Development

“…Reactive oxygen species (ROS) affect pollen intine properties [98], thus mads3-4 exhibits disorganized intine owing to the loss of temporal ROS kinetics [99]. RESPI-RATORY-BURST OXIDASE HOMOLOG E (RBOHE), a PM-localized NADPH oxidase that is crucial for the temporal ROS pattern in tapetum, also affects exine formation [100].…”

Pollen wall degradation in the Brassicaceae permits cell emergence after pollination

“…Pollen and stigmas are, in fact, often richly pigmented with red and yellow carotenoids, as in saffron (Negri et al, 2011). Such antioxidants protect against radiation (He et al, 2007), pathogen response (Baker and Orlandi, 1995), and especially against oxidative damage from the hydrogen peroxide that covers mature, receptive stigmas in many species (McInnis et al, 2006a; Smirnova, et al, 2014). Along with antioxidants and hydrogen peroxide, a number of peroxidases are found at very high levels in stigmas (McInnis et al, 2006a, b; Smirnova et al, 2014).…”