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.

The VERNALIZATION1 (VRN1) gene encodes a MADS-box transcription factor and plays an important role in the cold-induced transition from the vegetative to reproductive stage. Allelic variability of VRN1 homoeologs has been associated with large differences in flowering time. The aim of this study was to investigate the genetic variability of VRN1 homoeologs (VRN-A1, VRN-B1 and VRN-D1). We performed an in-depth sequence analysis of VRN1 homoeologs in a panel of 105 winter and spring varieties of hexaploid wheat. We describe the novel allele Vrn-B1f with an 836 bp insertion within intron 1 and show its specific expression pattern associated with reduced heading time. We further provide the complete sequence of the Vrn-A1b allele, revealing a 177 bp insertion in intron 1, which is transcribed into an alternative splice variant. Copy number variation (CNV) analysis of VRN1 homoeologs showed that VRN-B1 and VRN-D1 are present in only one copy. The copy number of recessive vrn-A1 ranged from one to four, while that of dominant Vrn-A1 was one or two. Different numbers of Vrn-A1a copies in the spring cultivars Branisovicka IX/49 and Bastion did not significantly affect heading time. We also report on the deletion of secondary structures (G-quadruplex) in promoter sequences of cultivars with more vrn-A1 copies.

In temperate cereals, vernalization regulates the transition from vegetative to reproductive growth in response to low temperatures. A central component of this pathway is VERNALIZATION 1 ( VRN1 ). In winter wheat cultivars, the recessive vrn1 allele is transcriptionally activated by cold and promotes the transition to flowering. In contrast, spring cultivars carry dominant VRN1 alleles expressed in a cold-independent manner. This is caused by mutations in the cis -regulatory elements in the promoter or in the “critical region” of intron 1. The widespread spring bread wheat allele Vrn-A1a carries a promoter mutation but retains a winter-like allele intron 1. Intriguingly, Vrn-A1a is partially responsive to cold, suggesting regulatory element(s) beyond the promoter mutation. Here, we investigated the effect of combined promoter and first intron mutations on Vrn-A1a expression and flowering time in bread wheat. We deleted a 901-bp segment of the Vrn-A1a so-called “critical region”, thus generating a novel combination of the promoter and intron 1 mutations not found in natural germplasm. Plants carrying both mutations exhibited Vrn-A1 transcript levels and flowering times comparable to those of the wild-type under both vernalizing and non-vernalizing conditions. Hence, partial cold responsiveness of Vrn-A1a is controlled by regulatory element(s) outside the removed intron 1 segment. Overall, our study advances the understanding of VRN1 transcriptional regulation and highlights the complexity of vernalization control in wheat.

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.