Plant 22-nt siRNAs mediate translational repression and stress adaptation

Small interfering RNAs (siRNAs) are critical for proper development and immunity in eukaryotes1. Plants produce siRNAs with lengths of 21-, 22-, or 24- nucleotides (nt), wherein the 21- and 24-nt siRNAs mediate mRNA cleavage and DNA methylation2,3, respectively. However, the biological functions of 22-nt siRNAs remain elusive. Here we report the identification and characterization of a group of endogenous 22-nt siRNAs generated from the action of DICER-LIKE 2 (DCL2). When cytoplasmic RNA decay and DCL4 are deficient, the massive accumulation of 22-nt siRNAs causes pleiotropic growth disorders, including severe dwarfism, meristem defect, and pigmentation. Notably, two genes that encode nitrate reductases, NIA1 and NIA2, produce nearly half of the total of 22-nt siRNAs. Production of 22-nt siRNA triggers explosive self-amplification that leads to a small RNA storm, and induces dramatic translational repression both gene-specifically and globally. 22-nt siRNAs are also found to preferentially accumulate upon nitrogen deficiency, which acts to restrain plant growth and promote stress responses. Thus, our research uncovers the unique properties of 22-nt siRNAs, a previously unexplored class of plant siRNAs, and highlights the length of small RNA as a major functional determinant. Col-0, ein5-1 dcl4-2 and ein5-1 dcl4-2 dcl2-1were grown on the MS medium for 6 days and then were transferred to the soil for another 14 days. Aerial parts of 20-day-old plant were collected at 15:00 for AGO1-associated small RNA isolation. Two biological replicates were prepared for each genotype. Library preparation and high-throughput sequencing were conducted in accordance with the manufacturers’ instructions. Total RNA was extracted using the Trizol and precipitated by ethonal. Sequencing libraries were prepared using the NEBNext® Small RNA Library Prep Set for Illumina. RNA quality and library quality were examined by a Bioanalyzer 2100 instrument (Agilent), and paired-end, 150-bp deep sequencing was performed on an Illumina Nova6000 platform.