Spatiotemporal Production Of ROS By NADPH Oxidase Is Critical For Tapetal Programmed Cell Death And Pollen Development In Arabidopsis

Programmed Cell Death(PCD) is a vital and universal biological phenomenon in eukaryotes. PCD refers to the process of cellular self-degradation and disappearance and is regulated by complex genetic networks in which transcriptional cascades and proteolytic activities play key roles. Tapetum is a layer of cells adjacent and of vital importance to the production of viable pollen by providing proteins, lipids and sporopollenin through a PCD process in angiosperms. Interference with the proper timing of tapetal degradation results in pollen abortion and male sterility. Because its wide applications in the agricultural industry, tapetum-mediated male sterility in angiosperms has been extensively studied.It was demonstrated that an evolutionarily conserved transcriptional cascades regulate the tapetal PCD process, among which DYSFUNCTIONAL TAPETUM1(DYT1), DEFECTIVE IN TAPETAL DEVELOPMENT AND FUNCTION1(TDF1/MYB35), ABORTED MICROSPORES(AMS), and MYB80(also known as MYB103) play key roles. Mutations of DYT1, TDF1, and AMS resulted in delayed tapetum degradation while the myb80 mutants showed an early tapetum degeneration, both of which led to pollen abortion and male sterility. Reactive oxygen species(ROS) are universal signals for developmental PCD. It was recently shown that failures in ROS scavenging resulted in precocious degeneration of tapetum and pollen developmental defects, suggesting that ROS play a key role in the tapetal PCD process. However, it is unclear from what sources ROS are generated, whether there is temporal ROS amplitude, how the ROS-producing system interacts with the tapetal transcriptional network, and its downstream signaling pathway contributing to tapetal PCD. Preliminary studies in our laboratory showed that two Arabidopsis RESPIRATORYBURST OXIDASE HOMOLOG(RBOH) genes, RBOHE and RBOHC are spatiotemporally expressed in tapetum during anther development stage 6-10. Functional loss of these RBOH genes resulted in pollen abortion due to delayed tapetal degeneration. By contrast, overexpression of RBOH under a tapetum-specific promoter led to pollen abortion due to precocious tapetal PCD. These results indicated that spatiotemporal ROS in tapetum was mainly controlled by NADPH oxidases. We also found that the expression of RBOHE was down-regulated in the dyt1, tdf1, and ams mutants whereas up-regulated in the myb80 mutant, implying a complex transcriptional regulation of RBOHE by these transcription factors. Therefore, that NADPH oxidase-mediated temporal ROS production may control pollen development in an evolutionarily conserved manner. Studies shown here provide the theoretical basis for genetic manipulation of male fertility in various crop species.The main results and conclusions of this study are as follows:(1) Spatiotemporal expression of Arabidopsis RBOHE in the tapetumTo verify the expression pattern of RBOHE, we applied RNA in situ analysis and generated ProRBOHE:GUS reporter lines and analyzed GUS signals by histochemical analysis. The results showed that the expression of RBOHE started at anther stage 6-7, gradually increased to reach an expression peak at stage 8-9. Later, the expression of RBOHE gradually decreased till none detectable at stage10-11. Similar results were obtained by analyzing the expression of RBOHC, although at a weaker level. The spatiotemporal expression of RBOHE and RBOHC implied their importance for tapetal PCD.(2) Both RBOHE loss-of-function and gain-of-function interfered with the timing of tapetum PCD.To determine the function of RBOHE in the tapetum layer, we took a reverse genetics approach by characterizing its T-DNA insertional mutants. Transcriptional analysis verified that both were null mutants for RBOHE. Under green house conditions, the rbohe mutants showed reduced seed sets and significantly increased pollen abortion rates. We showed that the rbohe mutants were delayed in tapetal degradation as well as tapetal PCD by histological analysis, transmission electron microscopy(TEM), as well as terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling(TUNEL) analyses.On the other hand, the enhanced expression of RBOHE in a spatiotemporal pattern by using a tapetum-specific promoter whose expression pattern is similar to that of RBOHE also resulted in pollen abortion and male sterility. However, pollen abortion of RBOHE gain-offunction plants was due to precocious tapetal degeneration and PCD, in contrast to that of RBOHE loss-of-function.(3) RBOHE and RBOHC were functionally redundant during tapetum PCDBecause both RBOH genes are expressed in a spatiotemporal way during tapetal PCD, we then generated double mutant in which both genes were null mutants. Indeed, the pollen abortion rate was increased by about 60% and tapetum PCD was significantly delayed in the rbohe rbohc double mutant than that in single mutants. These results demonstrated that RBOHE and RBOHC are functionally redundant during tapetum PCD and pollen maturation.(4) RBOHE and RBOHC regulate the timing of tapetum PCD by the production of ROS.Because RBOHE and RBOHC encode NADPH oxidases that localized at the plasma membrane to generate ROS, we analyzed the ROS level of anthers at different developmental stages by nitrotetrazolium blue chloride(NBT) staining and 29,79-dichlorodihydro?uorescein diacetate(H2DCF-DA) staining. Our results showed that developing Arabidopsis anthers displayed a dynamic ROS level, correlating with the temporal expression of RBOHE. The ROS level was increased from stage 6-7 to highest at stage 8-9, then gradually decreased at stage10-11. By contrast, ROS levels were significantly reduced in either rbohe-2 or rbohe-2 rbohc whereas overexpression of RBOHE displayed significantly increased ROS levels at anther developmental stage 6-7 and interfered the temporal ROS pattern seen in wild type. These results suggest the importance of spatiotemporal ROS pattern for the timing of tapetum PCD.(5) RBOHE is positive regulated by AMS but negative regulated by MYB80The expression of RBOHE was reduced in ams but enhanced in ms188-1. We further verified the transcriptional changes of RBOHE in these mutants by RNA in situ hybridization and ProRBOHE:GUS reporter lines. All results supported the idea that RBOHE is positive regulated by AMS but negative regulated by MYB80. Indeed, genetic analysis showed that introducing rbohe-2 into ms188-1 partially rescued the precocious tapetal PCD phenotype of the ms188-1 mutants, confirming their genetic epistasis.In summary, our research demonstrated that RBOH-encoded NADPH oxidases are critical components regulating the proper timing of tapetal PCD by generating a dynamic ROS level during anther development. The spatiotemporal expression of RBOHs is regulated by the evolutionarily conserved tapetal transcriptional network, suggesting similar mechanisms may exist in other plant species as well. Studies shown here provide theoretical basis for generating male sterile plants for agricultural applications.