Maternal siRNAs mediate spatial and temporal regulation of gene expression and seed development in Arabidopsis
In Arabidopsis seed development involves maternal small interfering RNAs (siRNAs) that induce RNA-directed DNA methylation (RdDM) through the NRPD1 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 spatio-temporal activity of the NRPD1 pathway on seed development using 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 that were 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 the sensor AGL40:GUS and AGL91:GUS constructs we demonstrate that spatial and temporal expression patterns of these genes in the endosperm were regulated by the NRPD1-mediated pathway irrespective of complete silencing (AGL91) or incomplete silencing (AGL40) of these target genes. Modified expression of these siRNA-targeted genes affects seed size and 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. Overall design: 1. Examination of small RNAs in embryo, endosperm and seed coat of Arabidopsis seeds. 2. Examiniation of small RNAs in reciprocal crosses between wild type and nrpd1a mutant as well as rdr2 mutant. 7 crosses were made including 3 self pollination of parent strains.