The maternal-to-zygotic transition (MZT) is an essential developmental turning point in both plants and animals. In plants, the timing of MZT and parental contributions to the zygotic transcriptome remain unclear. Here, by overcoming technical limitations, we characterize the Arabidopsis egg cell, zygote, and embryo transcriptomes across multiple stages. Using these datasets, we demonstrate that MZT occurs during zygote development and is a two-step interrelated process of rapid maternal transcript degradation followed by large-scale de novo transcription. Parental contributions to the zygotic transcriptome are stage-dependent: the spherical zygote is characterized by a maternally dominated transcriptome, whereas the elongated zygote transcriptome shows equal parental contributions. Our results show that plant MZT is similar to that in animals, showing a typical two-step process, and that zygotic genome activation is required for zygote elongation and division, indicating that de novo transcripts are essential for the establishment of zygote polarity and embryogenesis promotion.

WUSCHEL-related homeobox (WOX) gene is a plant-specific clade of homeobox transcription factors. Increasing evidences reveal that WOXs play critical roles in early embryogenesis, which involves zygote development, initiation of zygote division, and apical or basal cell lineage establishment. However, how WOXs regulate these developmental events remains largely unknown, and even detailed expression pattern in gametes and early proembryos is not yet available. Here, 13 WOX family genes were identified in Nicotiana tabacum genome. Comparative analysis of 13 WOX family genes with their homologs in Arabidopsis thaliana reveals relatively conserved expression pattern of WUS and WOX5 in shoot/root apical meristem. Whereas variations were also found, e.g., lacking homolog of WOX8 (a marker for suspensor cell) in tobacco genome and the expression of WOX2/WOX9 in both apical cell and basal cell. Transient transcriptional activity analysis revealed that WOXs in WUS clade have repressive activities for their target's transcription, whereas WOXs in ancient and intermediate clade have activation activities, giving a molecular basis for the phylogenetic classification of tobacco WOXs into three major clades. Expression pattern analysis revealed that some WOXs (e.g., WOX 13a) expressed in both male and female gametes and some WOXs (e.g., WOX 11 and WOX 13b) displayed the characteristics of parent-of-origin genes. Interestingly, some WOXs (e.g., WOX2 and WOX9), which are essential for early embryo patterning, were de novo transcribed in zygote, indicating relevant mechanism for embryo pattern formation is only established in zygote right after fertilization and not carried in by gametes. We also found that most WOXs displayed a stage-specific and cell type-specific expression pattern. Taken together, this work provides a detailed landscape of WOXs in tobacco during fertilization and early embryogenesis, which will facilitate the understanding of their specific roles in these critical developmental processes of embryogenesis.

In Arabidopsis, a zygote undergoes asymmetrical cell division that establishes the first two distinct cell types of early proembryos, apical and basal cells. However, the genome-wide transcriptional activities that guide divergence of apical and basal cell development remain unknown. Here, we present a comprehensive transcriptome analysis of apical and basal cell lineages, uncovering distinct molecular pathways during cell lineage specification. Selective deletion of inherited transcripts and specific de novo transcription contribute to the establishment of cell lineage-specific pathways for cell fate specification. Embryo-related pathways have been specifically activated in apical cell lineage since 1-cell embryo stage, but quick transcriptome remodeling toward suspensor-specific pathways are found in basal cell lineage. Furthermore, long noncoding RNAs and alternative splicing isoforms may be involved in cell lineage specification. This work also provides a valuable lineage-specific transcriptome resource to elucidate the molecular pathways for divergence of apical and basal cell lineages at genome-wide scale.

Plant zygote divides asymmetrically into an apical cell that develops into the embryo proper and a basal cell that generates the suspensor, a vital organ functioning as a conduit of nutrients and growth factors to the embryo proper. After the suspensor has fulfilled its function, it is removed by programmed cell death (PCD) at the late stages of embryogenesis. The molecular trigger of this PCD is unknown. Here we use tobacco (Nicotiana tabacum) embryogenesis as a model system to demonstrate that the mechanism triggering suspensor PCD is based on the antagonistic action of two proteins: a protease inhibitor, cystatin NtCYS, and its target, cathepsin H-like protease NtCP14. NtCYS is expressed in the basal cell of the proembryo, where encoded cystatin binds to and inhibits NtCP14, thereby preventing precocious onset of PCD. The anti-cell death effect of NtCYS is transcriptionally regulated and is repressed at the 32-celled embryo stage, leading to increased NtCP14 activity and initiation of PCD. Silencing of NtCYS or overexpression of NtCP14 induces precocious cell death in the basal cell lineage causing embryonic arrest and seed abortion. Conversely, overexpression of NtCYS or silencing of NtCP14 leads to profound delay of suspensor PCD. Our results demonstrate that NtCYS-mediated inhibition of NtCP14 protease acts as a bipartite molecular module to control initiation of PCD in the basal cell lineage of plant embryos.

Imprinted genes display biased expression of paternal and maternal alleles and are only found in mammals and flowering plants. Compared to several hundred imprinted genes that are functionally characterized in mammals, very few imprinted genes were confirmed in plants and even fewer of them have been functionally investigated. Here, we report a new imprinted gene, NUWA, in plants. NUWA is an essential gene, because loss of its function resulted in reduced transmission through the female gametophyte and defective cell/nuclear proliferation in early Arabidopsis embryo and endosperm. NUWA is a maternally expressed imprinted gene, as only the maternal allele of NUWA is transcribed and translated from gametogenesis to the 16-cell globular embryo stage after fertilization, and the de novo transcription of the maternal allele of NUWA starts from the zygote stage. Different from other identified plant imprinted genes whose encoded proteins are mostly localized to the nucleus, the NUWA protein was localized to the mitochondria and was essential for mitochondria function. Our work uncovers a novel imprinted gene of a previously unidentified type, namely, a maternal-specific expressed nuclear gene with its encoded protein localizing to and controlling the function of the maternally inherited mitochondria. This reveals a unique mechanism of maternal control of the mitochondria and adds an extra layer of complexity to the regulation of nucleus-organelle coordination during early plant development.

Cystatins are tightly bound and reversible inhibitors of cysteine proteases in C1A and C13 peptidase families, which have been identified in several species and shown to function in vegetative development and response to biotic/abiotic stresses in plants. Recent work revealed their critical role in regulating programmed cell death during embryogenesis in tobacco and suggested their more comprehensive roles in the process of sexual plant reproduction, although little is known about cystatin family genes in the processes. Here, 10 cystatin family genes in Nicotiana tabacum were identified using an expressed sequence tag (EST)-based gene clone strategy. Analysis of their biochemical properties showed that nine of them have the potency to inhibit the activities of both commercial cathepsin L-like proteases and extracted cysteine proteases from seeds, but with different K i values depending on the types of proteases and the developmental stages of the seed tested. This suggests that cystatin-dependent cathepsin L-like proteolytic pathways are probably important for early seed development. Comprehensive expression profile analysis revealed that cystatin family genes showed manifold variations in their transcription levels in different plant cell types, including the sperm, egg, and zygote, especially in the embryo and seed at different developmental stages. More interestingly, intracellular localization analysis of each cystatin revealed that most members of cystatin families are recognized as secretory proteins with signal peptides that direct them to the endoplasmic reticulum. These results suggest their widespread roles in cell fate determination and cell-cell communication in the process of sexual reproduction, especially in gamete and embryo development, as well as in seed formation.