Artificial microRNAs reveal cell-specific differences in small RNA activity in pollen

Abstract

Pollen formation, while critical for the success of plant reproduction, also represents an important paradigm for differential cellular development within small groups of cells. In Arabidopsis thaliana pollen, the male meiotic product first divides asymmetrically to form a vegetative and a generative (germ) cell, the latter then dividing to generate two sperm cells. Here we have used artificial microRNAs to study small RNA processing in the different pollen cell types. Our data suggest that translational repression by small RNAs is enhanced in the sperm. This work also provides insights into germline RNA movement and the cell-autonomous action of microRNAs.

Analysis of Complementarity Requirements for Plant MicroRNA Targeting Using a Nicotiana benthamiana Quantitative Transient Assay

“…Our development of a rapid and quantifiable system to measure miRNA target site function in vivo will facilitate further study of the role of mRNA secondary structure and target site positioning. Mechanisms of miRNA-initiated target repression can also differ between cell types (Grant-Downton et al, 2013), and of course the miRNA and the mRNA bearing a potential target site must accumulate in at least some of the same cell type(s) or tissues in order for a site to be measurably functional. Future studies in this area should focus on incorporating all of these parameters to produce a more refined model for the efficacies of miRNA target sites in plants.…”

Intercellular communication in Arabidopsis thaliana pollen discovered via AHG3 transcript movement from the vegetative cell to sperm

“…Although all these physical connections presumably ensure that the vegetative nucleus and the sperm cells move in the pollen tube as a unit (known as the "male germ unit"), they also may provide a route for intercellular communication. It has been proposed that small RNAs move from the vegetative cell to sperm cells (9); however, this notion has been challenged (10). Moreover, the reported mechanism of mRNA movement and small RNA movement in sporophytic tissues is different (11,12).…”

“…However, because the LAT52p promoter was reported to drive weak expression at the unicellular microspore stage (10), the signal detected potentially could result from carry over from early stages of pollen development to sperm cells. To examine this possibility, we used the VCK promoter, a vegetative cell-specific promoter expressed first in late bicellular pollen (10), to drive expression of AHG3 5′ UTR plus the genomic coding sequence (Pro VCK :5UTR-geAHG3-GFP, construct 6 in Fig. 2A).…”

“…To determine if the coding sequence of AHG3 was sufficient for transport, we first used the LAT52 promoter, which is expressed predominantly in the vegetative cell (10,17), to drive expression of the AHG3 genomic coding sequence (Pro LAT52 :geAHG3-GFP; construct 4 in Fig. 2A).…”

Small RNAs in pollen

“…AGO5, which specifically accumulates in SC [16], is a closely related homologue of AGO1 that specifically binds C-initiated small RNAs [21] and could thus be involved in a special miRNA activity in pollen. Moreover, a recent study [22] showed that miRNAs in SCs inhibits expression of targets only at the protein level but not at the mRNA level, which is different from that miRNAs repress targets mainly at the mRNA level in the VC. Taken together, these results clearly indicate that mature miRNAs are present in both the VC and SCs, but the spatial temporal regulation of miRNA activity during SC formation and why the mechanism of miRNA-mediated target repression is distinct between the VC and SCs remain unexplored.…”