Multiple factors and features dictate the selective production of ct-siRNA in Arabidopsis.

Coding transcript-derived siRNAs (ct-siRNAs) produced from specific endogenous loci can suppress the translation of their source genes to balance plant growth and stress response. In this study, we generated Arabidopsis mutants with deficiencies in RNA decay and/or post-transcriptional gene silencing (PTGS) pathways and performed comparative sRNA-seq analysis, revealing that multiple RNA decay and PTGS factors impede the ct-siRNA selective production. Genes that produce ct-siRNAs often show increased or unchanged expression and typically have higher GC content in sequence composition. The growth and development of plants can perturb the dynamic accumulation of ct-siRNAs from different gene loci. Two nitrate reductase genes, NIA1 and NIA2, produce massive amounts of 22-nt ct-siRNAs and are highly expressed in a subtype of mesophyll cells where DCL2 exhibits higher expression relative to DCL4, suggesting a potential role of cell-specific expression of ct-siRNAs. Overall, our findings unveil the multifaceted factors and features involved in the selective production and regulation of ct-siRNAs and enrich our understanding of gene silencing process in plants.

Plant 22-nt siRNAs mediate translational repression and stress adaptation.

Small interfering RNAs (siRNAs) are essential for proper development and immunity in eukaryotes1. Plants produce siRNAs with lengths of 21, 22 or 24 nucleotides. The 21- and 24-nucleotide species mediate cleavage of messenger RNAs and DNA methylation2,3, respectively, but the biological functions of the 22-nucleotide siRNAs remain unknown. Here we report the identification and characterization of a group of endogenous 22-nucleotide siRNAs that are generated by the DICER-LIKE 2 (DCL2) protein in plants. When cytoplasmic RNA decay and DCL4 are deficient, the resulting massive accumulation of 22-nucleotide siRNAs causes pleiotropic growth disorders, including severe dwarfism, meristem defects and pigmentation. Notably, two genes that encode nitrate reductases-NIA1 and NIA2-produce nearly half of the 22-nucleotide siRNAs. Production of 22-nucleotide siRNAs triggers the amplification of gene silencing and induces translational repression both gene specifically and globally. Moreover, these 22-nucleotide siRNAs preferentially accumulate upon environmental stress, especially those siRNAs derived from NIA1/2, which act to restrain translation, inhibit plant growth and enhance stress responses. Thus, our research uncovers the unique properties of 22-nucleotide siRNAs, and reveals their importance in plant adaptation to environmental stresses.