LETTER doi:10.1038/nature13620 Non-equivalent contributions of maternal and paternal genomes to early plant embryogenesis Gerardo Del Toro-De Leo ´n 1 , Marcelina Garcı ´a-Aguilar 1 & C. Stewart Gillmor 1 Zygotic genome activation in metazoans typically occurs several hours to a day after fertilization, and thus maternal RNAs and pro- teins drive early animal embryo development 1 . In plants, despite sev- eral molecular studies of post-fertilization transcriptional activation, the timing of zygotic genome activation remains a matter of debate. For example, two recent reports that used different hybrid ecotype combinations for RNA sequence profiling of early Arabidopsis em- bryo transcriptomes came to divergent conclusions. One identified paternal contributions that varied by gene, but with overall maternal dominance 2 , while the other found that the maternal and paternal genomes are transcriptionally equivalent 3 . Here we assess paternal gene activation functionally in an isogenic background, by perform- ing a large-scale genetic analysis of 49 EMBRYO DEFECTIVE genes and testing the ability of wild-type paternal alleles to complement phenotypes conditioned by mutant maternal alleles. Our results dem- onstrate that wild-type paternal alleles for nine of these genes are com- pletely functional 2 days after pollination, with the remaining 40 genes showing partial activity beginning at 2, 3 or 5 days after pollination. Using our functional assay, we also demonstrate that different hybrid combinations exhibit significant variation in paternal allele activation, reconciling the apparently contradictory results of previous transcrip- tional studies 2,3 . The variation in timing of gene function that we observe confirms that paternal genome activation does not occur in one early discrete step, provides large-scale functional evidence that maternal and paternal genomes make non-equivalent contributions to early plant embryogenesis, and uncovers an unexpectedly profound effect of hybrid genetic backgrounds on paternal gene activity. In animals, maternal RNAs and proteins drive early embryo develop- ment. The transition to zygotic control requires both clearing of maternal transcripts and zygotic genome activation, a phenomenon collectively known as the maternal-to-zygotic transition 1 . Research in plants has focused almost exclusively on zygotic genome activation, and has led to differing conclusions on its timing. Studies in maize and in tobacco sug- gested that large-scale zygotic transcription does not begin until 3–4 days after fertilization 4,5 . In agreement with this, an Arabidopsis RNA sequence profiling and reporter line study 2 and three smaller-scale mole- cular studies 6–8 also came to the conclusion that many paternal alleles are either quiescent or transcribed at lower levels than corresponding mater- nal alleles in the first few days after fertilization. Other work has suggested a contrary view, namely that zygotic tran- scription begins immediately or soon after fertilization. Significant dif- ferences have been found between the transcript populations of egg cells and two-celled embryos in wheat 9 , and equivalent maternal and pater- nal expression for 25 different genes was observed in maize zygotes at 1 day after pollination 10 . In Arabidopsis, three genes have been shown by genetic means to be expressed from the paternal allele in early embryo- genesis 11–13 , and a recent genome-wide transcriptional study found biparental transcription for nearly all of the ,8,000 genes that could be assayed at the one- to two-cell zygote stage 3 . Even taking into account that timing of zygotic genome activation may differ among plant species, it has been difficult to reconcile these conflicting results. However, when evaluating the two RNA profiling experiments arguing for 3 or against 2 immediate zygotic genome activation in Arabidopsis, it is important to note that each study used a unique combination of polymorphic strains to distinguish maternal and paternal transcripts, such that the results are based on gene expression in embryos hybrid for different combina- tions of ecotypes. To study paternal allele activation in an isogenic background, we used a genetic assay with strict functional criteria: a test of the ability of wild- type (WT) paternal alleles to complement early embryo mutant pheno- types conditioned by mutant maternal alleles. While this assay is necessarily limited in the number of genes that can be tested (and in the corresponding conclusions that can be drawn), it presents two significant advantages over RNA sequencing approaches: by design, we only evaluate genes with a proven role in embryo development, leading to biologically relevant results. Second, we avoid the complex effects on quantitative traits—such as gene expression values—that necessarily result from crossing poly- morphic, distantly related ecotypes (the basis for allele specific profil- ing by RNA sequencing) 14–17 . We screened a collection of mutants in several hundred EMBRYO DEFECTIVE (EMB) genes required for early embryo development in Arabidopsis 18,19 , and selected genes that were associated with clearly recognizable anatomical defects segregating in emb/1 plants at pre- globular stages (Supplementary Table 1 and Supplementary Data Set 1). For 49 different EMB genes, we then crossed heterozygous mutant plants with WT pollen, and scored the resulting embryos for mutant pheno- types at 2, 3, 5 and ,14 days after pollination (Fig. 1; complete data shown in Supplementary Table 2). We identified nine genes where the WT pater- nal allele complemented the mutant maternal allele as soon as there was a requirement for the gene product, resulting in no observed mutant phe- notypes at 2 days after pollination or later (AtSWI3A shown as an example in Fig. 1d–f, j). For the remaining 40 genes, the WT paternal allele gradual- ly complemented the mutant phenotype conferred by the mutant mater- nal allele (EMB2804 shown as an example in Fig. 1g–i, j). To determine more precisely the time point of the onset of paternal allele activation, we studied a subset of 13 genes in more detail (Supplementary Table 3). Comparison of mutant ratios resulting from hand-pollinated emb/13 emb/1 and emb/13 Col crosses identified genes whose activation began at either 2, 3 or 5 days after pollination. By contrast, Col 3 emb/1 control crosses resulted in no mutant phenotypes, showing that the delayed com- plementation we observed does not reflect haploinsufficiency expressed only in the first few days of embryogenesis, and that the maternal allele for these genes is absolutely required in early embryogenesis, while the paternal allele is not (Supplementary Table 3). We next sought to correlate function and expression, selecting the GCT and CCT genes as examples 20 . Figure 2a–g, i–o shows that mater- nally derived gGCT-GUS and pCCT::GUS reporter constructs drove embryo expression at 2 days after pollination, whereas paternally derived constructs drove embryo expression beginning at 3 days after pollina- tion, and reached full expression at 4 days after pollination. Phenotypic complementation of gct and cct mutant maternal alleles by the WT paternal allele began at 5 days after pollination (Fig. 2h, p), 1 day after 1 Laboratorio Nacional de Geno ´ mica para la Biodiversidad (Langebio), Unidad de Geno ´ mica Avanzada, Centro de Investigacio ´ n y de Estudios Avanzados del Instituto Polite ´ cnico Nacional (CINVESTAV-IPN), Irapuato, Guanajuato 36821, Me ´ xico. 624 | NATURE | VOL 514 | 30 OCTOBER 2014 Macmillan Publishers Limited. All rights reserved ©2014