Maternal small RNAs mediate spatial-temporal regulation of gene expression, imprinting, and seed development in Arabidopsis

Abstract

Arabidopsis seed development involves maternal small interfering RNAs (siRNAs) that induce RNA-directed DNA methylation (RdDM) through the NRPD1-mediated pathway. To investigate their biological functions, we characterized siRNAs in the endosperm and seed coat that were separated by laser-capture microdissection (LCM) in reciprocal genetic crosses with an nrpd1 mutant. We also monitored the spatial-temporal activity of the NRPD1-mediated pathway on seed development using the AGO4:GFP::AGO4 (promoter:GFP::protein) reporter and promoter:GUS sensors of siRNA-mediated silencing. From these approaches, we identified four distinct groups of siRNA loci dependent on or independent of the maternal NRPD1 allele in the endosperm or seed coat. A group of maternally expressed NRPD1-siRNA loci targets endosperm-preferred genes, including those encoding AGAMOUS-LIKE (AGL) transcription factors. Using translational promoter:AGL::GUS constructs as sensors, we demonstrate that spatial and temporal expression patterns of these genes in the endosperm are regulated by the NRPD1-mediated pathway irrespective of complete silencing (AGL91) or incomplete silencing (AGL40) of these target genes. Moreover, altered expression of these siRNA-targeted genes affects seed size. We propose that the corresponding maternal siRNAs could account for parent-of-origin effects on the endosperm in interploidy and hybrid crosses. These analyses reconcile previous studies on siRNAs and imprinted gene expression during seed development.

Abundant expression of maternal siRNAs is a conserved feature of seed development

“…In B. rapa, loss of maternal RdDM causes severe seed development defects and high rates of seed abortion regardless of the genotype of the filial tissues (Grover et al, 2018), suggesting that siren siRNA production in the developing seed coat might be required for seed development. However, silencing by siren RNAs is not required in A. thaliana, as nrpd1 mutant mothers have only subtle developmental phenotypes (Grover et al, 2018;Kirkbride et al, 2019). Furthermore, rapid evolution of siren loci argues against a conserved role in regulation of developmental genes.…”

“…To determine whether siren loci are specific to B. rapa, or are present in more diverse species, we utilized public data from A. thaliana and rice (Autran et al, 2011;Baldrich et al, 2019;Huang et al, 2019;Kirkbride et al, 2019;Rodrigues et al, 2013). Using these datasets we identified 16,422 small RNA loci in A. thaliana and 122,661 loci in rice.…”

“…To eliminate the possibility that maternal bias of siren siRNAs in the endosperm was due to contamination by maternal seed coat (Schon and Nodine, 2017), we measured siren accumulation in publicly-available A. thaliana libraries, including laser-capture microdissected endosperm (Kirkbride et al, 2019). Reciprocal crosses between nrpd1 or rdr2 and wild type demonstrate that siRNA accumulation at siren loci in whole seeds or seed coats is dependent on maternal alleles of NRPD1 and RDR2 ( Figure 7C).…”

“…The seed coat surrounds and protects the embryo and endosperm, is entirely maternal and sporophytic in origin, and is descended from the integument tissue surrounding the female gametes. Several studies indicate that 24-nt small interfering (si)RNAs and RNA-directed DNA Methylation (RdDM) are important during early seed development, although a biological role for RdDM during seed development has remained elusive (Erdmann et al, 2017;Kirkbride et al, 2019;Lu et al, 2012;Martinez et al, 2018;Satyaki and Gehring, 2019).…”

“…However, the maternal genome is the source for the majority of 24-nt siRNAs in the developing seed, including a class of siRNAs which exclusively accumulate in reproductive tissues (Mosher et al, 2009;Pignatta et al, 2014). Maternal siRNA accumulation in the seed is linked to expression of developmental regulators in A. thaliana endosperm (Kirkbride et al, 2019;Lu et al, 2012). It has been hypothesized that siRNAs are imprinted in the endosperm (Lu et al, 2012;Mosher et al, 2009;Rodrigues et al, 2013), though perturbation of known imprinting pathways did not change their apparent uniparental expression pattern (Mosher et al, 2011), and this interpretation has been challenged (Pignatta et al, 2014).…”