Premeiotic, 24-Nucleotide Reproductive PhasiRNAs Are Abundant in Anthers of Wheat and Barley But Not Rice and Maize.

Sebastien Bélanger,Kirk Czymmek,Blake C Meyers,Suresh Pokhrel

doi:10.1104/pp.20.00816

Abstract

Two classes of premeiotic (21-nucleotides [nt]) and meiotic (24-nt) phased small interfering RNAs (phasiRNAs) and their patterns of accumulation have been described in maize (Zea mays) and rice (Oryza sativa) anthers. Their precise function remains unclear, but studies have shown that they support male fertility. The important role of phasiRNAs in anthers underpins our present study to characterize these small RNAs in wheat (Triticum aestivum) and barley (Hordeum vulgare) anthers. We staged anthers at every 0.2 mm of development for one wheat and two barley varieties. We isolated premeiotic (0.2, 0.4, and 0.6 mm), meiotic (0.8, 1.0, and 1.4 mm), and postmeiotic (1.8 mm) anthers, for which we then investigated accumulation patterns of RNAs, including reproductive phasiRNAs. We annotated a total of 12,821 and 2,897 PHAS loci in the wheat and barley genomes, respectively. By comparing the total number of PHAS loci in genomes of maize, rice, barley, and wheat, we identified an expansion of reproductive PHAS loci in the genomes of Poaceae subfamilies from Panicoideae to Oryzoideae and to Poideae. In addition to the two classes of premeiotic (21-nt) and meiotic (24-nt) phasiRNAs, previously described in maize and rice anthers, we characterized a group of 24-nt phasiRNAs that accumulate in premeiotic anthers. The absence of premeiotic 24-nt phasiRNAs in maize and rice suggests a divergence in grass species of the Poideae subfamily. Additionally, we performed a gene coexpression analysis describing the regulation of phasiRNA biogenesis in wheat and barley anthers. We highlight Argonaute 9 (AGO9) and Argonaute 6 (AGO6) as candidate binding partners of premeiotic and meiotic 24-nt phasiRNAs, respectively.

Highlights

21 Plants produce small RNAs of typically 21 to 24 nucleotides
Among the novel AGO proteins discovered in barley, we have annotated new copies for AGO1, AGO2, AGO5 and AGO10. 155 Some interesting observations emerged from our phylogenetic analysis
We showed that all the machinery needed to process phasiRNAs is expressed in anthers of both species and, phasiRNA biogenesis and functional activity should be active. 203 Myriad Stage-Specific phasiRNAs in Wheat and Barley Anthers To allow accurate and sensitive identification of phasiRNAs that accumulate in anthers, we analyzed the 63 sRNA libraries generated from seven sequential stages of anther development

Summary

Introduction

21 Plants produce small RNAs (sRNAs) of typically 21 to 24 nucleotides (nt). The biogenesis and regulation of sRNAs requires RNA polymerases (Pol), Dicer-like (DCL) proteins, double-stranded RNA-binding (DRB) proteins, RNA-directed RNA polymerases (RDRs) and Argonaute (AGO) proteins (Borges and Martienssen, 2015; Yu et al, 2018). Among the sRNA expressed in plants, phased siRNAs, or “phasiRNAs”, are a distinct group of sRNAs characterized as a product of processive cleavage of double-stranded RNAs in regular increments (duplexes of 21 nt or 24 nt) from a well-defined terminus (Axtell and Meyers, 2018). Accumulation of two distinct groups of pre-meiotic (21-nt) and meiotic (24-nt) reproductive phasiRNAs was found to occur in maize (Zea mays; Zhai et al, 2015) and rice (Oryza sativa; Komiya et al, 2014; Fei et al, 2016) anthers. 42 Nonomura et al (2007) identified the MEIOSIS ARRESTED AT LEPTOTENE 1 (MEL1) gene, which encodes an AGO protein, in a male-sterile rice mutant. A mel loss-of-function mutant is characterized by an arrest of chromosome condensation at early meiotic stages as well as by irregularly sized, multi-nucleated and vacuolated pollen mother cells (Nonomura et al, 2007)

Methods

Results

Discussion

Conclusion