Regulation of Vegetative Phase Change by SWI2/SNF2 Chromatin Remodeling ATPase BRAHMA.

Plants progress from a juvenile vegetative phase of development to an adult vegetative phase of development before they enter the reproductive phase. miR156 has been shown to be the master regulator of the juvenile-to-adult transition in plants. However, the mechanism of how miR156 is transcriptionally regulated still remains elusive. In a forward genetic screen, we identified that a mutation in the SWI2/SNF2 chromatin remodeling ATPase BRAHMA (BRM) exhibited an accelerated vegetative phase change phenotype by reducing the expression of miR156, which in turn caused a corresponding increase in the levels of SQUAMOSA PROMOTER BINDING PROTEIN LIKE genes. BRM regulates miR156 expression by directly binding to the MIR156A promoter. Mutations in BRM not only increased occupancy of the -2 and +1 nucleosomes proximal to the transcription start site at the MIR156A locus but also the levels of trimethylated histone H3 at Lys 27. The precocious phenotype of brm mutant was partially suppressed by a second mutation in SWINGER (SWN), but not by a mutation in CURLEY LEAF, both of which are key components of the Polycomb Group Repressive Complex 2 in plants. Our results indicate that BRM and SWN act antagonistically at the nucleosome level to fine-tune the temporal expression of miR156 to regulate vegetative phase change in Arabidopsis.

DICER-LIKE2 plays a primary role in transitive silencing of transgenes in Arabidopsis.

Dicer-like (DCL) enzymes play a pivotal role in RNA silencing in plants, processing the long double-stranded RNA (dsRNA) that triggers silencing into the primary short interfering RNAs (siRNAs) that mediate it. The siRNA population can be augmented and silencing amplified via transitivity, an RNA-dependent RNA polymerase (RDR)-dependent pathway that uses the target RNA as substrate to generate secondary siRNAs. Here we report that Arabidopsis DCL2-but not DCL4-is required for transitivity in cell-autonomous, post-transcriptional silencing of transgenes. An insertion mutation in DCL2 blocked sense transgene-induced silencing and eliminated accumulation of the associated RDR-dependent siRNAs. In hairpin transgene-induced silencing, the dcl2 mutation likewise eliminated accumulation of secondary siRNAs and blocked transitive silencing, but did not block silencing mediated by primary siRNAs. Strikingly, in all cases, the dcl2 mutation eliminated accumulation of all secondary siRNAs, including those generated by other DCL enzymes. In contrast, mutations in DCL4 promoted a dramatic shift to transitive silencing in the case of the hairpin transgene and enhanced silencing induced by the sense transgene. Suppression of hairpin and sense transgene silencing by the P1/HC-Pro and P38 viral suppressors was associated with elimination of secondary siRNA accumulation, but the suppressors did not block processing of the stem of the hairpin transcript into primary siRNAs. Thus, these viral suppressors resemble the dcl2 mutation in their effects on siRNA biogenesis. We conclude that DCL2 plays an essential, as opposed to redundant, role in transitive silencing of transgenes and may play a more important role in silencing of viruses than currently thought.

The N-terminal ATPase AT-hook-containing region of the Arabidopsis chromatin-remodeling protein SPLAYED is sufficient for biological activity.

The SNF2-like chromatin-remodeling ATPase SPLAYED (SYD) was identified as a co-activator of floral homeotic gene expression in Arabidopsis. SYD is also required for meristem maintenance and regulates flowering under a non-inductive photoperiod. SNF2 ATPases are structurally and functionally conserved from yeast to humans. In addition to the conserved protein features, SYD has a large unique C-terminal domain. We show here that SYD is present as two forms in the nucleus, full-length and truncated, with the latter apparently lacking the C-terminal domain. The ratio of the two forms of endogenous SYD differs in juvenile and in adult tissues. Furthermore, an SYD variant lacking the C-terminal domain (SYDDeltaC) rescues the syd null mutant, indicating that the N-terminal ATPase AT-hook-containing region of SYD is sufficient for biological activity. Plants expressing SYDDeltaC show molecular and morphological phenotypes opposite to those of the null mutant, suggesting that the construct results in increased activity. This increased activity is at least in part due to elevated SYD protein levels in these lines. We propose that the C-terminal domain may control SYD accumulation and/or specific activity in the context of the full-length protein. The presence of the C-terminal domain in rice SYD suggests that its role is probably conserved in the two classes of flowering plants.

A pathway for the biogenesis of trans-acting siRNAs in Arabidopsis.

The Arabidopsis genes, TAS2 and TAS1a, produce structurally similar noncoding transcripts that are transformed into short (21-nucleotide [nt]) and long (24-nt) siRNAs by RNA silencing pathways. Some of these short siRNAs direct the cleavage of protein-coding transcripts, and thus function as trans-acting siRNAs (ta-siRNAs). Using genetic analysis, we defined the pathway by which ta-siRNAs and other short siRNAs are generated from these loci. This process is initiated by the miR173-directed cleavage of a primary poly(A) transcript. The 3' fragment is then transformed into short siRNAs by the sequential activity of SGS3, RDR6, and DCL4: SGS3 stabilizes the fragment, RDR6 produces a complementary strand, and DCL4 cleaves the resulting double-stranded molecule into short siRNAs, starting at the end with the miR173 cleavage site and proceeding in 21-nt increments from this point. The 5' cleavage fragment is also processed by this pathway, but less efficiently. The DCL3-dependent pathway that generates long siRNAs does not require miRNA-directed cleavage and plays a minor role in the silencing of these loci. Our results define the core components of a post-transcriptional gene silencing pathway in Arabidopsis and reveal some of the features that direct transcripts to this pathway.

SGS3 and SGS2/SDE1/RDR6 are required for juvenile development and the production of trans-acting siRNAs in Arabidopsis.

Higher plants undergo a transition from a juvenile to an adult phase of vegetative development prior to flowering. Screens for mutants that undergo this transition precociously produced alleles of two genes required for posttranscriptional gene silencing (PTGS)--SUPPRESSOR OF GENE SILENCING3 (SGS3) and SUPPRESSOR OF GENE SILENCING2(SGS2)/SILENCING DEFECTIVE1 (SDE1)/RNA-DEPENDENT POLYMERASE6 (RDR6). Loss-of-function mutations in these genes have a phenotype similar to that of mutations in the Argonaute gene ZIPPY (ZIP). Epistasis analysis suggests that ZIP, SGS3, SGS2/SDE1/RDR6, and the putative miRNA export receptor, HASTY (HST), operate in the same pathway(s). Microarray analysis revealed a small number of genes whose mRNA is increased in ZIP, SGS3, and SGS2/SDE1/RDR6 mutants, as well as genes that are up-regulated in SGS3 and SGS2/SDE1/RDR6 mutants, but not in ZIP mutants. One of these latter genes (At5g18040) is silenced posttranscriptionally in trans by the sRNA255 family of endogenous, noncoding, small interfering RNAs (siRNAs). The increase in At5g18040 mRNA in SGS3 and SGS2/SDE1/RDR6 mutants is attributable to the absence of sRNA255-like siRNAs in these mutants. These results demonstrate a role for endogenous siRNAs in the regulation of gene expression, and suggest that PTGS plays a central role in the temporal control of shoot development in plants.