MicroRNA activity in the Arabidopsis male germline

Abstract

Most of the core proteins involved in the microRNA (miRNA) pathway in plants have been identified, and almost simultaneously hundreds of miRNA sequences processed in the Arabidopsis sporophyte have been discovered by exploiting next-generation sequencing technologies. However, there is very limited understanding about potentially distinct mechanisms of post-transcriptional regulation between different cell lineages. In this review the focus is on the Arabidopsis male gametophyte (pollen), where the germline differentiates after meiosis giving rise to the male gametes. Based on comparative analysis of miRNAs identified in sperm cells by in-depth sequencing, their possible functions during germ cell specification and beyond fertilization are discussed. In addition, 25 potentially novel miRNAs processed in sperm cells and pollen were identified, as well as enriched variations in the sequence length of known miRNAs, which might indicate subfunctionalization by association with a putative germline-specific Argonaute complex. ARGONAUTE 5 (AGO5), by close homology to AGO1 and localizing preferentially to the sperm cell cytoplasm in mature pollen, may be part of such a complex.

Fig. 1.

Argonaute protein family in Arabidopsis. Phylogenetic tree illustrating the 10 Argonaute proteins in Arabidopsis, subdivided into the three main functional classes based on sequence homology: miRNA-guided slicing and translational repression of target transcripts, trans-acting siRNA (ta-siRNA) activity, and chromatin remodelling by siRNA-directed DNA methylation (RdDM). AGO2, AGO3, AGO8 (red), and AGO5 (green) have unknown function. AGO5 is a close relative of AGO1 and AGO10, and could be involved in a novel miRNA pathway in the germline (see Fig. 2). (adapted from Yigit et al., 2006).

Fig. 2.

AGO5 expression in Arabidopsis pollen. (A) Transgene expression of AGO5 protein using the native promoter region (1000 bp upstream of the 5'-untranslated region) and the genomic coding sequence translationally fused to eGFP. AGO5p::AGO5–eGFP expression in mature pollen localizes preferentially in the sperm cell (SC) cytoplasm (B) remaining during pollen tube growth. (C) A magnification of the male germ unit shows that AGO5–GFP localizes in the SC cytoplasm and not in the nucleus, extending through the cytoplasmic connection that links the SCs with the vegetative nucleus (VN). DAPI-stained DNA shows SCs and VNs in A and C.

Fig. 3.

Venn diagram illustrating miRNA families detected in sperm cells and pollen. Overlap between known miRNA families detected in sperm cells (83) and pollen (75) by Illumina sequencing (Slotkin et al., 2009), and a 454 sequencing data set of pollen small RNAs (31) reported by Grant-Downton et al. (2009b). Numbers in parentheses represent total miRNA families identified in each data set.

Fig. 4.

MiRNA families and variations in sequence length. (A) Relative abundance of known miRNAs detected in sperm cell (SC), pollen, and inflorescence data sets. (B) MiRNAs 156h and 158b have isoforms of 21 nt length with nucleotide extensions in the 5' terminus, for which the number of reads in SCs and pollen is higher than the annotated 20 nt isoforms. miR158a reads are presented to exemplify that these variations do not accumulate significantly for the majority of other miRNAs. (C) The 22 nt miRNAs that may function as siRNA triggers are differentially accumulated in SC, pollen, and inflorescence tissues.