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.

Polar growth of root hairs is critical for plant survival and requires fine-tuned Rho of plants (ROP) signaling. Multiple ROP regulators participate in root hair growth. However, protein S-acyl transferases (PATs), mediating the S-acylation and membrane partitioning of ROPs, are yet to be found. Using a reverse genetic approach, combining fluorescence probes, pharmacological drugs, site-directed mutagenesis and genetic analysis with related root-hair mutants, we have identified and characterized an Arabidopsis PAT, which may be responsible for ROP2 S-acylation in root hairs. Specifically, functional loss of PAT4 resulted in reduced root hair elongation, which was rescued by a wild-type but not an enzyme-inactive PAT4. Membrane-associated ROP2 was significantly reduced in pat4, similar to S-acylation-deficient ROP2 in the wild type. We further showed that PAT4 and SCN1, a ROP regulator, additively mediate the stability and targeting of ROP2. The results presented here indicate that PAT4-mediated S-acylation mediates the membrane association of ROP2 at the root hair apex and provide novel insights into dynamic ROP signaling during plant tip growth.

Pollen viability depends on dynamic vacuolar changes during pollen development involving increases and decreases of vacuolar volume through water and osmolite accumulation and vacuolar fission. Mutations in to, the genes encoding phosphatidylinositol 3,5-bisphosphate [PI(3,5)P]-converting kinases, are male gametophyte lethal in Arabidopsis () due to defective vacuolar fission after pollen mitosis I, suggesting a key role of the phospholipid in dynamic vacuolar organization. However, other genetic components that regulate the production of PI(3,5)P and its involvement in pollen germination and tube growth are unknown. Here, we identified and characterized Arabidopsis VAC14, a homolog of the yeast and metazoan VAC14s that are crucial for the production of PI(3,5)P is constitutively expressed and highly present in developing pollen. Loss of function of was male gametophyte lethal due to defective pollen development. Ultrastructural studies showed that vacuolar fission after pollen mitosis I was compromised in mutant microspores, which led to pollen abortion. We further showed that inhibiting the production of PI(3,5)P or exogenous application of PI(3,5)P mimicked or rescued the pollen developmental defect of the mutant, respectively. Genetic interference and pharmacological approaches suggested a role of PI(3,5)P in pollen germination and tube growth. Our results provide insights into the function of VAC14 and, by inference, that of PI(3,5)P in plant cells.

Protein S-acyl transferases (PATs) play critical roles in plant developmental and environmental responses by catalyzing S-acylation of substrate proteins, most of which are involved in cellular signaling. However, only few plant PATs have been functionally characterized. We recently demonstrated that Arabidopsis PAT4 mediates root hair elongation by positively regulating the membrane association of ROP2 and actin microfilament organization. Here, we show that apex-associated re-positioning of nucleus during root hair elongation was impaired by PAT4 loss-of-function. Results presented here pose a significant question concerning the molecular machinery mediating nuclear migration during root hair growth.