Spatiotemporal Production of Reactive Oxygen Species by NADPH Oxidase Is Critical for Tapetal Programmed Cell Death and Pollen Development in Arabidopsis

Abstract

Male sterility in angiosperms has wide applications in agriculture, particularly in hybrid crop breeding and gene flow control. Microspores develop adjacent to the tapetum, a layer of cells that provides nutrients for pollen development and materials for pollen wall formation. Proper pollen development requires programmed cell death (PCD) of the tapetum, which requires transcriptional cascades and proteolytic enzymes. Reactive oxygen species (ROS) also affect tapetal PCD, and failures in ROS scavenging cause male sterility. However, many aspects of tapetal PCD remain unclear, including what sources generate ROS, whether ROS production has a temporal pattern, and how the ROS-producing system interacts with the tapetal transcriptional network. We report here that stage-specific expression of NADPH oxidases in the Arabidopsis thaliana tapetum contributes to a temporal peak of ROS production. Genetic interference with the temporal ROS pattern, by manipulating RESPIRATORY-BURST OXIDASE HOMOLOG (RBOH) genes, affected the timing of tapetal PCD and resulted in aborted male gametophytes. We further show that the tapetal transcriptional network regulates RBOH expression, indicating that the temporal pattern of ROS production intimately connects to other signaling pathways regulated by the tapetal transcriptional network to ensure the proper timing of tapetal PCD.

MONENSIN SENSITIVITY1 (MON1)/CALCIUM CAFFEINE ZINC SENSITIVITY1 (CCZ1)-Mediated Rab7 Activation Regulates Tapetal Programmed Cell Death and Pollen Development

“…In plants, tapetal PCD is a prerequisite for normal pollen development and successful reproduction (Xie et al, 2014;Zhang et al, 2014;Dresselhaus et al, 2016). Through tapetal PCD process, tapetum provides its cellular contents, including lipids and proteins, for pollen coat formation .…”

“…In the wild type, tapetal cells undergo PCD for tapetum degeneration from late stage 10 ( Fig. 6G; Xie et al, 2014). By contrast, in the mon1 mutant, weak TUNEL-positive signals began to be detected at stage 11 and 12, showing delayed PCD in mon1 (Fig.…”

“…For semithin sections, anthers at various developmental stages were prefixed and embedded as described previously (Xie et al, 2014). Semithin sections of 2 mm were cut using a UC6 ultramicrotome (Leica) and stained with 1% toluidine blue O (Sigma-Aldrich).…”

“…Pollen in vitro germination and in vivo analysis were performed as described previously (Samuel et al, 2009;Xie et al, 2014;H. Wang et al, 2016).…”

“…The development of fertile pollen requires nutritive contributions from the surrounding cell layers (Xie et al, 2014;Zhang et al, 2014). The tapetum is of particular importance in this regard by providing proteins, lipids, and other materials.…”