Fine-Tuning of MiR528 Accumulation Modulates Flowering Time in Rice

In plants,microRNA (miRNA) functions in the post-transcriptional repression of target mRNAs have been well explored.However,the mechanisms regulating the accumulation of miRNAs remain poorly under.stood.Here,we report that distinct mechanisms regulate accumulation of a monocot-specific miRNA,rice (Oryza sativa) miR528.At the transcriptional level,miR528 accumulated to higher levels in older plants than in young seedlings and exhibited aging-modulated gradual accumulation and diurnal rhythms in leaves;at the post-transcriptional level,aging also modulated miR528 levels by enhancing pri-miR528 alter.native splicing.We found that miR528 promotes rice flowering under long-day conditions by targeting RED AND FAR-RED INSENSITIVE2 (OsRFI2).Moreover,natural variations in the MIR528 promoter region caused differences in miR528 expression among rice varieties,which are correlated with their different binding affinities with the transcription factor OsSPL9 that activates the expression of miR528.Taken together,our findings reveal rice plants have evolved sophisticated modes fine-tuning miR528 levels and provide insight into the mechanisms that regulate MIRNA expression in plants.

Degradome sequencing reveals endogenous small RNA targets in rice (Oryza sativa L. ssp. indica)

MicroRNAs(miRNAs)and small interfering RNAs(siRNAs)regulate gene expression in eukaryotes.Plant miRNAs modulate their targets mainly via messenger RNA(mRNA)cleavage.Small RNA(sRNA)targets have been extensively investigated in Arabidopsis using computational prediction,experimental validation,and degradome sequencing.However,small RNA targets are largely unknown in rice(Oryza sativa).Here,we report global identification of small RNA targets using high throughput degradome sequencing in the rice indica cultivar 93-11(Oryza sativa L.ssp.indica).One hundred and seventy-seven transcripts targeted by a total of 87 unique miRNAs were identified.Of targets for the conserved miRNAs between Arabidopsis and rice,transcription factors comprise around 70%(58 in 82),indicating that these miRNAs act as masters of gene regulatory nodes in rice.In contrast,non-conserved miRNAs targeted diverse genes which provide more complex regulatory networks.In addition,5 AUXIN RESPONSE FACTORs(ARFs)cleaved by the TAS3 derived ta-siRNAs were also detected.A total of 40 sRNA targets were further validated via RNA ligasemediated 5′rapid amplification of cDNA ends(RLM 5′-RACE).Our degradome results present a detailed sRNAtarget interaction atlas,which provides a guide for the study of the roles of sRNAs and their targets in rice.

An Integrated Workflow for DNA Methylation Analysis

The analysis of cytosine methylation provides a new way to assess and describe epigenetic regulation at a whole-genome level in many eukaryotes.DNA methylation has a demonstrated role in the genome stability and protection,regulation of gene expression and many other aspects of genome function and maintenance.BS-seq is a relatively unbiased method for profiling the DNA methylation,with a resolution capable of measuring methylation at individual cytosines.Here we describe,as an example,a workflow to handle DNA methylation analysis,from BS-seq library preparation to the data visualization.We describe some applications for the analysis and interpretation of these data.Our laboratory provides public access to plant DNA methylation data via visualization tools available at our ＂Next-Gen Sequence＂ websites（http：//mpss.udel.edu）,along with small RNA,RNA-seq and other data types.