Summary: The anther is the male reproductive organ of flowering plants, and the Arabidopsis bHLH transcription factors encoded by DYSFUNCTIONAL TAPETUM1 (DYT1) and ABORTED MICROSPORE (AMS) are required for control of the complex transcriptional networks regulating anther development. Knowledge of the mechanisms by which the bHLH proteins affect this diverse gene expression is quite limited. We examine here three recently duplicated Arabidopsis bHLH genes, bHLH010, bHLH089 and bHLH091, using evolutionary, genetic, morphological and transcriptomic approaches, and uncover their redundant functions in anther development. These three genes are relatively highly expressed in the tapetum of the Arabidopsis anther; single mutants at each of the bHLH010, bHLH089 and bHLH091 loci are developmentally normal, but the various double and triple combinations progressively exhibit increasingly defective anther phenotypes (abnormal tapetum morphology, delayed callose degeneration, and aborted pollen development), indicating their redundant functions in male fertility. Further transcriptomic and molecular analyses suggest that these three proteins act slightly later than DYT1, and also form protein complexes with DYT1, subsequently affecting the correct expression of many DYT1 target genes in the anther development transcriptional network. This study demonstrated that bHLH010, bHLH089 and bHLH091 together are important for the normal transcriptome of the developing Arabidopsis anther, possibly by forming a feed‐forward loop with DYT1.

Three downstream transcription factors enhance the function of a key regulator of anther development by promoting its nuclear localization through protein-prote

Small RNAs are central players in RNA silencing, yet their cytoplasmic compartmentalization and the effects it may have on their activities have not been studied at the genomic scale. Here we report that Arabidopsis microRNAs (miRNAs) and small interfering RNAs (siRNAs) are distinctly partitioned between the endoplasmic reticulum (ER) and cytosol. All miRNAs are associated with membrane-bound polysomes (MBPs) as opposed to polysomes in general. The MBP association is functionally linked to a deeply conserved and tightly regulated activity of miRNAs – production of phased siRNAs (phasiRNAs) from select target RNAs. The phasiRNA precursor RNAs, thought to be noncoding, are on MBPs and are occupied by ribosomes in a manner that supports miRNA-triggered phasiRNA production, suggesting that ribosomes on the rough ER impact siRNA biogenesis. This study reveals global patterns of cytoplasmic partitioning of small RNAs and expands the known functions of ribosomes and ER.

As key regulators of gene expression in eukaryotes, small RNAs have been characterized in many seed plants, and pathways for their biogenesis, degradation, and action have been defined in model angiosperms. However, both small RNAs themselves and small RNA pathways are not well characterized in other land plants such as lycophytes and ferns, preventing a comprehensive evolutionary perspective on small RNAs in land plants. Using 25 representatives from major lineages of lycophytes and ferns, most of which lack sequenced genomes, we characterized small RNAs and small RNA pathways in these plants. We identified homologs of DICER-LIKE (DCL), ARGONAUTE (AGO), and other genes involved in small RNA pathways, predicted over 2600 conserved microRNA (miRNA) candidates, and performed phylogenetic analyses on small RNA pathways as well as miRNAs. Pathways underlying miRNA biogenesis, degradation, and activity were established in the common ancestor of land plants, but the 24-nucleotide siRNA pathway that guides DNA methylation is incomplete in sister species of seed plants, especially lycophytes. We show that the functional diversification of key gene families such as DCL and AGO as observed in angiosperms occurred early in land plants followed by parallel expansion of the AGO family in ferns and angiosperms. We uncovered a conserved AGO subfamily absent in angiosperms. Our phylogenetic analyses of miRNAs in bryophytes, lycophytes, ferns, and angiosperms refine the time-of-origin for conserved miRNA families as well as small RNA machinery in land plants.

Summary: Flower development is a complex process requiring proper spatiotemporal expression of numerous genes. Accumulating evidence indicates that epigenetic mechanisms, including DNA methylation, play essential roles in modulating gene expression. However, few studies have examined the relationship between DNA methylation and floral gene expression on a genomic scale. Here we present detailed analyses of DNA methylomes at single‐base resolution for three Arabidopsis floral periods: meristems, early flowers and late flowers. We detected 1.5 million methylcytosines, and estimated the methylation levels for 24 035 genes. We found that many cytosine sites were methylated de novo from the meristem to the early flower stage, and many sites were demethylated from early to late flowers. A comparison of the transcriptome data of the same three periods revealed that the methylation and demethylation processes were correlated with expression changes of >3000 genes, many of which are important for normal flower development. We also found different methylation patterns for three sequence contexts (^mCG, ^mCHG and ^mCHH) and in different genic regions, potentially with different roles in gene expression.