Martin Kovacik

In cereals, the persistent endosperm accumulates starch and storage proteins, making their grains a major source of food and feed ^1–3 . Early grain development is epigenetically controlled through the parent-of-origin-specific expression of imprinted genes in the endosperm that regulate endosperm proliferation, nutrient accumulation and embryo nourishment ^4–6 . Imprinted genes have been identified in several major cereals, and a few have been functionally characterized ^7–14 . However, it is unclear how imprinted genes regulate the accumulation of storage compounds in cereal grains. Through spatiotemporal transcriptome analysis of 255 imprinted genes in barley ( Hordeum vulgare ), we identify endosperm transfer cells in grains as a hotspot of expression of imprinted genes. Furthermore, we show that the transcription factor LYS3 dynamically regulates grain filling in barley through early expression of the maternal allele, followed by partial activation of the paternal allele. One-sentence summary: We identified imprinted genes in developing barley endosperm and characterized transcription factor LYS3 as a temporally maternally expressed gene, uniparentally regulating barley grain filling.

Summary paragraph: Imprinted genes do not act alone to shape seed development, but as a complex network — just like any other gene. Yet, the molecular context in which they are embedded, i.e. their gene network, remains largely understudied. To address this knowledge gap, we characterized the imprintome of Arabidopsis arenosa at the species-level and used gene regulatory network analyses. We show that genomic imprinting preferentially affects only a few pathways, offering candidates for dosage sensitive processes and the molecular arena of parental conflict. In these pathways, some imprinted genes act as hub genes, among which NRPE1 , highlighting the importance of epigenetics in endosperm development. The interaction between parental genomes was rather one-sided: paternally expressed regulators preferentially targeted PEGs, while maternally expressed regulators targeted both PEGs and MEGs indiscriminately. This aligns with a self-promoting paternal influence and a maternal buffer under a parental conflict scenario. Shared paternal and maternal regulation of downstream targets was nevertheless common, and we reveal novel molecular interactions between imprinted regulators. Overall, our work shows how genomic imprinting may act as a molecular means for parental conflict, but also highlights the importance of parental molecular coordination in endosperm development.

Summary: In plants, a key example of convergence is the repeated evolution of floral traits associated with the transition from outcrossing to self‐fertilization, often resulting in the ‘selfing syndrome’ (e.g. reduced flower size and loss of scent). However, potentially overlooked changes concern the pollen coat, which plays a role in different aspects of outcrossing strategy. To investigate this, we compared pollen coat thickness and pollen proteomes between selfing and outcrossing species in the Arabidopsis and Capsella genera, representing three independent selfing transitions. Pollen coat area diminished with the age of the selfing transition, with significant reductions in A. thaliana and Capsella rubella , but not in A. lyrata (recent transition, no selfing syndrome). Moreover, specific differentially abundant pollen coat proteins were shared across selfer–outcrosser contrasts more than expected by chance, even though the direction of changes was not consistent across the three comparisons. These specific proteins, while annotated as pathogen response due to their structure, are likely involved in pollen–pistil interactions instead. Overall, our findings reveal limited convergent evolution of pollen coat thickness and protein composition following selfing transitions. Our work identified a group of pollen coat proteins that are largely understudied, but whose abundance appears to be a hotspot for evolutionary changes.

SUMMARY: Polyploidy is a key evolutionary force in plants; among its many consequences, hybridization between diploids and polyploids is restricted due to the “triploid block”. While the molecular mechanisms of this postzygotic barrier are extensively studied in the model species Arabidopsis thaliana , our understanding of the triploid block in natural systems remains limited. Here, we investigated the transcriptome of failing triploid seeds in Arabidopsis arenosa , a close relative of A. thaliana with diploid and autotetraploid populations meeting in nature. We also identified A. arenosa imprinted genes. Triploid seeds showed preferential misregulation of imprinted genes, which parallels the parent-of-origin features of the triploid block. Tissue-specific transcriptomic analyses revealed pathogen defense-like response being recurrently affected in the endosperm and seed coat. This pathway is commonly misregulated in all species with triploid seed transcriptomes studied to date. The associated genes however are likely more involved in cell-cell signaling rather than pathogen defense per se . Altogether, this study depicts a thorough molecular landscape of the triploid block for the first time in natural systems. Combining data on the understudied maternal excess transcriptome, a new imprintome, a tissue-specific focus, and a cross-species comparison, this study also advances our understanding of the triploid block and seed development.

Cereal grains are an important source of food and feed. To provide comprehensive spatiotemporal information about biological processes in developing seeds of cultivated barley (Hordeum vulgare L. subsp. vulgare), we performed a transcriptomic study of the embryo, endosperm, and seed maternal tissues collected from grains 4–32 days after pollination. Weighted gene co-expression network and motif enrichment analyses identified specific groups of genes and transcription factors (TFs) potentially regulating barley seed tissue development. We defined a set of tissue-specific marker genes and families of TFs for functional studies of the pathways controlling barley grain development. Assessing selected groups of chromatin regulators revealed that epigenetic processes are highly dynamic and likely play a major role during barley endosperm development. The repressive H3K27me3 modification is globally reduced in endosperm tissues and at specific genes related to development and storage compounds. Altogether, this atlas uncovers the complexity of developmentally regulated gene expression in developing barley grains.

Rabl organization is a type of interphase chromosome arrangement with centromeres and telomeres clustering at opposite nuclear poles. Here, we analyzed nuclear morphology and chromosome organization in cycling and endoreduplicated nuclei isolated from embryo and endosperm tissues of developing barley seeds. We show that endoreduplicated nuclei have an irregular shape, less sister chromatid cohesion at 5S rDNA loci, and a reduced amount of centromeric histone CENH3. While the chromosomes of the embryo and endosperm nuclei are initially organized in Rabl configuration, the centromeres and telomeres are intermingled within the nuclear space in the endoreduplicated nuclei with an increasing endoreduplication level. Such a loss of chromosome organization suggests that Rabl configuration is introduced and further reinforced by mitotic divisions in barley cell nuclei in a tissue and seed age-dependent manner.

Wild barley is abundant, occupying large diversity of sites, ranging from the northern mesic Mediterranean meadows to the southern xeric deserts in Israel. This is also reflected in its wide phenotypic heterogeneity. We investigated the dynamics of DNA content changes in seed tissues in ten wild barley accessions that originated from an environmental gradient in Israel. The flow cytometric measurements were done from the time shortly after pollination up to the dry seeds. We show variation in mitotic cell cycle and endoreduplication dynamics in both diploid seed tissues (represented by seed maternal tissues and embryo) and in the triploid endosperm. We found that wild barley accessions collected at harsher xeric environmental conditions produce higher proportion of endoreduplicated nuclei in endosperm tissues. Also, a comparison of wild and cultivated barley strains revealed a higher endopolyploidy level in the endosperm of wild barley, that is accompanied by temporal changes in the timing of the major developmental phases. In summary, we present a new direction of research focusing on connecting spatiotemporal patterns of endoreduplication in barley seeds and possibly buffering for stress conditions.

Seeds are complex biological systems comprising three genetically distinct tissues: embryo, endosperm, and maternal tissues (including seed coats and pericarp) nested inside one another. Cereal grains represent a special type of seeds, with the largest part formed by the endosperm, a specialized triploid tissue ensuring embryo protection and nourishment. We investigated dynamic changes in DNA content in three of the major seed tissues from the time of pollination up to the dry seed. We show that the cell cycle is under strict developmental control in different seed compartments. After an initial wave of active cell division, cells switch to endocycle and most endoreduplication events are observed in the endosperm and seed maternal tissues. Using different barley cultivars, we show that there is natural variation in the kinetics of this process. During the terminal stages of seed development, specific and selective loss of endoreduplicated nuclei occurs in the endosperm. This is accompanied by reduced stability of the nuclear genome, progressive loss of cell viability, and finally programmed cell death. In summary, our study shows that endopolyploidization and cell death are linked phenomena that frame barley grain development.