Aliphatic alcohols naturally exist in many organisms as important cellular components; however, their roles in extracellular polymer biosynthesis are poorly defined. We report here the isolation and characterization of a rice (Oryza sativa) male-sterile mutant, defective pollen wall (dpw), which displays defective anther development and degenerated pollen grains with an irregular exine. Chemical analysis revealed that dpw anthers had a dramatic reduction in cutin monomers and an altered composition of cuticular wax, as well as soluble fatty acids and alcohols. Using map-based cloning, we identified the DPW gene, which is expressed in both tapetal cells and microspores during anther development. Biochemical analysis of the recombinant DPW enzyme shows that it is a novel fatty acid reductase that produces 1-hexadecanol and exhibits >270-fold higher specificity for palmiltoyl-acyl carrier protein than for C16:0 CoA substrates. DPW was predominantly targeted to plastids mediated by its N-terminal transit peptide. Moreover, we demonstrate that the monocot DPW from rice complements the dicot Arabidopsis thaliana male sterile2 (ms2) mutant and is the probable ortholog of MS2. These data suggest that DPWs participate in a conserved step in primary fatty alcohol synthesis for anther cuticle and pollen sporopollenin biosynthesis in monocots and dicots.

In male reproductive development in plants, meristemoid precursor cells possessing transient, stem cell–like features undergo cell divisions and differentiation to produce the anther, the male reproductive organ. The anther contains centrally positioned microsporocytes surrounded by four distinct layers of wall: the epidermis, endothecium, middle layer, and tapetum. Here, we report that the rice (Oryza sativa) basic helix-loop-helix (bHLH) protein TDR INTERACTING PROTEIN2 (TIP2) functions as a crucial switch in the meristemoid transition and differentiation during early anther development. The tip2 mutants display undifferentiated inner three anther wall layers and abort tapetal programmed cell death, causing complete male sterility. TIP2 has two paralogs in rice, TDR and EAT1, which are key regulators of tapetal programmed cell death. We revealed that TIP2 acts upstream of TDR and EAT1 and directly regulates the expression of TDR and EAT1. In addition, TIP2 can interact with TDR, indicating a role of TIP2 in later anther development. Our findings suggest that the bHLH proteins TIP2, TDR, and EAT1 play a central role in regulating differentiation, morphogenesis, and degradation of anther somatic cell layers, highlighting the role of paralogous bHLH proteins in regulating distinct steps of plant cell–type determination.

The accumulation of a number of small RNAs in plants is affected by abscisic acid (ABA) and abiotic stresses, but the underlying mechanisms are poorly understood. The miR168-mediated feedback regulatory loop regulates ARGONAUTE1 (AGO1) homeostasis, which is crucial for gene expression modulation and plant development. Here, we reveal a transcriptional regulatory mechanism by which MIR168 controls AGO1 homeostasis during ABA treatment and abiotic stress responses in Arabidopsis (Arabidopsis thaliana). Plants overexpressing MIR168a and the AGO1 loss-of-function mutant ago1-27 display ABA hypersensitivity and drought tolerance, while the mir168a-2 mutant shows ABA hyposensitivity and drought hypersensitivity. Both the precursor and mature miR168 were induced under ABA and several abiotic stress treatments, but no obvious decrease for the target of miR168, AGO1, was shown under the same conditions. However, promoter activity analysis indicated that AGO1 transcription activity was increased under ABA and drought treatments, suggesting that transcriptional elevation of MIR168a is required for maintaining a stable AGO1 transcript level during the stress response. Furthermore, we showed both in vitro and in vivo that the transcription of MIR168a is directly regulated by four abscisic acid-responsive element (ABRE) binding factors, which bind to the ABRE cis-element within the MIR168a promoter. This ABRE motif is also found in the promoter of MIR168a homologs in diverse plant species. Our findings suggest that transcriptional regulation of miR168 and posttranscriptional control of AGO1 homeostasis may play an important and conserved role in stress response and signal transduction in plants.

After meiosis, tapetal cells in the innermost anther wall layer undergo program cell death (PCD)-triggered degradation. This step is essential for microspore development and pollen wall maturation. We identified a key gene, Defective Tapetum Cell Death 1 (DTC1), that controls this degeneration by modulating the dynamics of reactive oxygen species (ROS) during rice male reproduction. Mutants defective in DTC1 exhibit phenotypes of an enlarged tapetum and middle layer with delayed degeneration, causing male sterility. The gene is preferentially expressed in the tapetal cells during early anther development. In dtc1 anthers, expression of genes encoding secretory proteases or lipid transporters is significantly reduced, while transcripts of PCD regulatory genes, e.g. UDT1, TDR1, and EAT1/DTD, are not altered. Moreover, levels of DTC1 transcripts are diminished in udt1, tdr, and eat1 anthers. These results suggest that DTC1 functions downstream of those transcription factor genes and upstream of the genes encoding secretory proteins. DTC1 protein interacts with OsMT2b, a ROS scavenger. Whereas wild-type plants accumulate large amounts of ROS in their anthers at Stage 9 of development, those levels remain low during all stages of development in dtc1 anthers. These findings indicate that DTC1 is a key regulator for tapetum PCD by inhibiting ROS-scavenging activity.

Background: Many eukaryotic genomes encode cis-natural antisense transcripts (cis-NATs). Sense and antisense transcripts may form double-stranded RNAs that are processed by the RNA interference machinery into small interfering RNAs (siRNAs). A few so-called nat-siRNAs have been reported in plants, mammals, Drosophila, and yeasts. However, many questions remain regarding the features and biogenesis of nat-siRNAs. Results: Through deep sequencing, we identified more than 17,000 unique siRNAs corresponding to cis-NATs from biotic and abiotic stress-challenged Arabidopsis thaliana and 56,000 from abiotic stress-treated rice. These siRNAs were enriched in the overlapping regions of NATs and exhibited either site-specific or distributed patterns, often with strand bias. Out of 1,439 and 767 cis-NAT pairs identified in Arabidopsis and rice, respectively, 84 and 119 could generate at least 10 siRNAs per million reads from the overlapping regions. Among them, 16 cis-NAT pairs from Arabidopsis and 34 from rice gave rise to nat-siRNAs exclusively in the overlap regions. Genetic analysis showed that the overlapping double-stranded RNAs could be processed by Dicer-like 1 (DCL1) and/or DCL3. The DCL3-dependent nat-siRNAs were also dependent on RNA-dependent RNA polymerase 2 (RDR2) and plant-specific RNA polymerase IV (PolIV), whereas only a fraction of DCL1-dependent nat-siRNAs was RDR- and PolIV-dependent. Furthermore, the levels of some nat-siRNAs were regulated by specific biotic or abiotic stress conditions in Arabidopsis and rice. Conclusions: Our results suggest that nat-siRNAs display distinct distribution patterns and are generated by DCL1 and/or DCL3. Our analysis further supported the existence of nat-siRNAs in plants and advanced our understanding of their characteristics.

The cytoplasmic hydroxycinnamoyl-CoA:ω-hydroxy fatty acid transferase DPW2 plays a fundamental role in male reproduction via the biosynthesis of key components