S-RNase disrupts tip-localized reactive oxygen species and induces nuclear DNA degradation in incompatible pollen tubes of Pyrus pyrifolia

Abstract

SummaryPear (Pyrus pyrifolia L.) has an S-RNase-based gametophytic self-incompatibility (SI) mechanism, and S-RNase has also been implicated in the rejection of self-pollen and genetically identical pollen. However, RNA degradation might be only the beginning of the SI response, not the end. Recent in vitro studies suggest that S-RNase triggers mitochondrial alteration and DNA degradation in the incompatible pollen tube of Pyrus pyrifolia, and it seems that a relationship exists between self S-RNase, actin depolymerization and DNA degradation. To further uncover the SI response in pear, the relationship between self S-RNase and tip-localized reactive oxygen species (ROS) was evaluated. Our results show that S-RNase specifically disrupted tip-localized ROS of incompatible pollen tubes via arrest of ROS formation in mitochondria and cell walls. The mitochondrial ROS disruption was related to mitochondrial alteration, whereas cell wall ROS disruption was related to a decrease in NADPH. Tip-localized ROS disruption not only decreased the Ca 2+ current and depolymerized the actin cytoskeleton, but it also induced nuclear DNA degradation. These results indicate that tip-localized ROS disruption occurs in Pyrus pyrifolia SI. Importantly, we demonstrated nuclear DNA degradation in the incompatible pollen tube after pollination in vivo. This result validates our in vitro system in vivo. Journal of Cell Science Results S-RNase disrupts tip-localized ROS in incompatible pollen tubesTo assess the effect of ROS on pollen tube growth, the pollen was grown in the basal medium for 1 hour at 25°C, then the NADPH oxidase inhibitor diphenylene iodonium chloride (DPI) and ROS scavenger 10,15,21H,23H-porphin (TMPP) were added to the medium. As expected, DPI and TMPP obviously inhibited pollen tube growth (Fig. 1A), which indicated that ROS are necessary for pear pollen tube growth. Furthermore, to evaluate the effect of S-RNase on tip-localized ROS, pollen tubes were stained with the ROS fluorescence probe, 5-(and 6-)chloromethyl-2Ј,7Ј-dichlorodihydrofluorescein diacetate (CM-H 2 DCFDA). Two types of pollen tubes were stained with CM-H 2 DCFDA (Fig. 1B): pollen tubes with strongest fluorescence in the tip (from subapical domain to apex) were regarded as normal, whereas pollen tubes with uniform fluorescence suggested that ROS in the tip-localized pollen tube were disrupted. The sample was stained with CM-H 2 DCFDA at 30 minutes after S-RNase, DPI or TMPP challenge to count the pollen tube with strongest fluorescence in tip (Fig. 1C). In the control, the proportion of pollen tubes with strongest fluorescence in the tip relative to the total number of tubes was 52.7±2.1% (means ± s.e.; n>100), and with the DPI or TMPP treatments, only 32.4±3.4% (n>100) and 35.9± 4.4% (n>100), respectively. In the compatible treatment, similarly to the control, 52.0±1.4% (n>100) of pollen tubes had strongest fluorescence in the tip. However, in the incompatible treatment, this was only 28.8±1.7% (n>100), nearly half the control value.Nitroblue tetrazo...

Organisation and regulation of the cytoskeleton in plant programmed cell death

“…A more recent study suggests that ROS may be involved in mediating actin depolymerisation in Pyrus pollen tubes. 34 …”

A cascade signal pathway occurs in self-incompatibility ofPyrus pyrifolia

“…[9][10][11][12] Supporting this, the actin-depolymerizing agent, cytochalasin B (CB) or the actin-stabilization agent, phalloidin induces degradation of nuclear DNA of pollen tubes, a hallmark feature of apoptosis. 3,13 Moreover, we have demonstrated that nuclear DNA of incompatible pollen tube was degraded under SI challenge in vitro or in vivo. 3,13 Most importantly, we have demonstrated that nuclear DNA were degraded in advanced of pollen tube viability compromise, 13 which indicate that nuclear DNA degradation were the cause, not the result, of growth arrest of the pollen tubes.…”

“…3,13 Moreover, we have demonstrated that nuclear DNA of incompatible pollen tube was degraded under SI challenge in vitro or in vivo. 3,13 Most importantly, we have demonstrated that nuclear DNA were degraded in advanced of pollen tube viability compromise, 13 which indicate that nuclear DNA degradation were the cause, not the result, of growth arrest of the pollen tubes.…”

“…2 Recently, we published a paper 3 describing that tiplocalized ROS disruption and Ca 2+ currents decrease in incompatible pollen tube of Pyrus pyrifolia in vitro. 3 Otherwise, these changes happened before pollen tube viability compromise, which indicated these changes are the cause, not the consequence, of pollen tube growth inhibition. Moreover, we find that tiplocalized ROS disruption decrease the Ca 2+ current and depolymerises the actin cytoskeleton, finally degrading the nuclear DNA.…”

“…Moreover, we find that tiplocalized ROS disruption decrease the Ca 2+ current and depolymerises the actin cytoskeleton, finally degrading the nuclear DNA. 3 Actually, it is reported that ROS and Ca 2+ have cross talk. On one hand, ROS can stimulate the activity of plasma membrane hyperpolarization-activated Ca 2+ channels; 4 on the other hand, Ca 2+ channel blockers can inhibit ROS formation in turn via inhibition activity of NADPH oxidise.…”