Arabidopsis embryos lacking DICER-LIKE1 (DCL1), which is required for microRNA (miRNA) biogenesis, arrest early in development. To assess the functions of embryonic miRNAs, we determined the developmental and molecular consequences of DCL1 loss. We found that DCL1 is required for cell differentiation events as early as the eight-cell stage and soon thereafter for proper division of the hypophysis and subprotoderm cells. By the early globular (~32-cell) stage, dcl1-null mutant embryos overexpress~50 miRNA targets. In dcl1 eight-cell embryos, the two most up-regulated targets are those of miR156 and encode SPL10 and SPL11 transcription factors. SPL10 and SPL11 are derepressed >150-fold in dcl1 embryos and are redundantly required for the dcl1 early patterning defects. Moreover, as early as the eight-cell stage, miR156-mediated repression of zygotic SPL transcripts prevents premature accumulation of transcripts from genes normally induced during the embryonic maturation phase. Thus, the first perceptible molecular function of plant embryonic miRNAs is the opposite of that in vertebrates; in vertebrates, miRNAs sharpen the first developmental transition, whereas in plants, they forestall developmental transitions by repressing mRNAs that act later. We propose that, by preventing precocious expression of differentiation-promoting transcription factors, miRNAs enable proper embryonic patterning.

Summary In animals, maternal gene products deposited into eggs regulate embryonic development before activation of the zygotic genome1. In plants, an analogous period of prolonged maternal control over embryogenesis is thought to occur based on gene-expression studies2–6. However, other gene-expression studies and genetic analyses show that some transcripts must derive from the early zygotic genome7–14, implying that the prevailing model does not fully explain the nature of zygotic genome activation in plants. To determine the maternal, paternal and zygotic contributions to the early embryonic transcriptome, we sequenced the transcripts of hybrid embryos from crosses between two polymorphic inbred lines of Arabidopsis thaliana and used single-nucleotide polymorphisms (SNPs) diagnostic of each parental line to quantify parental contributions. Although some transcripts appeared to be either inherited from primarily one parent or transcribed from imprinted loci, the vast majority of transcripts were produced in near-equal amounts from both maternal and paternal alleles, even during the initial stages of embryogenesis. Results of reporter experiments and analyses of transcripts from genes that are not expressed in sperm and egg indicate early and widespread zygotic transcription. Thus, in contrast to early animal embryogenesis, early plant embryogenesis is mostly under zygotic control.

A major goal of global gene expression profiling in plant seeds has been to investigate the parental contributions to the transcriptomes of early embryos and endosperm. However, consistency between independent studies has been poor, leading to considerable debate. We have developed a statistical tool that reveals the presence of substantial RNA contamination from maternal tissues in nearly all published Arabidopsis thaliana endosperm and early embryo transcriptomes generated in these studies. We demonstrate that maternal RNA contamination explains the poor reproducibility of these transcriptomic data sets. Furthermore, we found that RNA contamination from maternal tissues has been repeatedly misinterpreted as epigenetic phenomena, which has resulted in inaccurate conclusions regarding the parental contributions to both the endosperm and early embryo transcriptomes. After accounting for maternal RNA contamination, no published genome-wide data set supports the concept of delayed paternal genome activation in plant embryos. Moreover, our analysis suggests that maternal and paternal genomic imprinting are equally rare events in Arabidopsis endosperm. Our publicly available software (https://github.com/ Gregor-Mendel-Institute/tissue-enrichment-test) can help the community assess the level of contamination in transcriptome data sets generated from both seed and non-seed tissues.