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 maintenance of genome integrity over cell divisions is critical for plant development and the correct transmission of genetic information to the progeny. A key factor involved in this process is the STRUCTURAL MAINTENANCE OF CHROMOSOME5 (SMC5) and SMC6 (SMC5/6) complex, related to the cohesin and condensin complexes that control sister chromatid alignment and chromosome condensation, respectively. Here, we characterize NON-SMC ELEMENT4 (NSE4) paralogs of the SMC5/6 complex in Arabidopsis (Arabidopsis thaliana). NSE4A is expressed in meristems and accumulates during DNA damage repair. Partial loss-of-function nse4a mutants are viable but hypersensitive to DNA damage induced by zebularine. In addition, nse4a mutants produce abnormal seeds, with noncellularized endosperm and embryos that maximally develop to the heart or torpedo stage. This phenotype resembles the defects in cohesin and condensin mutants and suggests a role for all three SMC complexes in differentiation during seed development. By contrast, NSE4B is expressed in only a few cell types, and loss-of-function mutants do not have any obvious abnormal phenotype. In summary, our study shows that the NSE4A subunit of the SMC5-SMC6 complex is essential for DNA damage repair in somatic tissues and plays a role in plant reproduction.

Androgenic responsiveness in anther (AC) and isolated microspore cultures (MC) was analysed using 15 lines of Polish winter rye (Secale cereale L.). The effect of low temperature (LT) alone or in combination with osmotic stress induced by mannitol treatment (MAN) and/or with reduced glutathione (GSH) on the effectiveness of the process was studied. Interestingly, each treatment had a different effect on microspore (mcs) vitality and capability to divide symmetrically. The first criterion for successful embryogenesis was to exceed the threshold number of at least 25% dividing microspores, which determined ‘embryogenic suspension culture’. In some configurations a spectacular effect was achieved, especially in lines highly recalcitrant to androgenesis induction. Relatively high effectiveness of androgenesis induction (up to 4.58 AS per 10⁵ mcs per spike in MC and 21.29 AS per spike in AC) showed that the developed protocol with GSH and/or MAN tiller pre-treatments overcomes the genotypic barrier for androgenesis initiation in rye. Moreover, relatively high, spontaneous genome diploidization (55%) of regenerated plants demonstrated that the described protocols could be effectively integrated into conventional rye breeding programmes.

Enhanced antioxidant defence plays an essential role in plant survival under stress conditions. However, excessive antioxidant activity sometimes suppresses the signal necessary for the initiation of the desired biological reactions. One such example is microspore embryogenesis (ME)—a process of embryo-like structure formation triggered by stress in immature male gametophytes. The study focused on the role of reactive oxygen species and antioxidant defence in triticale (×Triticosecale Wittm.) and barley (Hordeum vulgare L.) microspore reprogramming. ME was induced through various stress treatments of tillers and its effectiveness was analysed in terms of ascorbate and glutathione contents, total activity of low molecular weight antioxidants and activities of glutathione–ascorbate cycle enzymes. The most effective treatment for both species was a combination of low temperature and exogenous application of 0.3 M mannitol, with or without 0.3 mM reduced glutathione. The applied treatments induced genotype-specific defence responses. In triticale, both ascorbate and glutathione were associated with ME induction, though the role of glutathione did not seem to be related to its function as a reducing agent. In barley, effective ME was accompanied by an accumulation of ascorbate and high activity of enzymes regulating its redox status, without direct relation to glutathione content.

Nucleus, chromatin, and chromosome organization studies heavily rely on fluorescence microscopy imaging to elucidate the distribution and abundance of structural and regulatory components. Three-dimensional (3D) image stacks are a source of quantitative data on signal intensity level and distribution and on the type and shape of distribution patterns in space. Their analysis can lead to novel insights that are otherwise missed in qualitative-only analyses. Quantitative image analysis requires specific software and workflows for image rendering, processing, segmentation, setting measurement points and reference frames and exporting target data before further numerical processing and plotting. These tasks often call for the development of customized computational scripts and require an expertise that is not broadly available to the community of experimental biologists. Yet, the increasing accessibility of high- and super-resolution imaging methods fuels the demand for user-friendly image analysis workflows. Here, we provide a compendium of strategies developed by participants of a training school from the COST action INDEPTH to analyze the spatial distribution of nuclear and chromosomal signals from 3D image stacks, acquired by diffraction-limited confocal microscopy and super-resolution microscopy methods (SIM and STED). While the examples make use of one specific commercial software package, the workflows can easily be adapted to concurrent commercial and open-source software. The aim is to encourage biologists lacking custom-script-based expertise to venture into quantitative image analysis and to better exploit the discovery potential of their images. Abbreviations: 3D FISH: three-dimensional fluorescence in situ hybridization; 3D: three-dimensional; ASY1: ASYNAPTIC 1; CC: chromocenters; CO: Crossover; DAPI: 4',6-diamidino-2-phenylindole; DMC1: DNA MEIOTIC RECOMBINASE 1; DSB: Double-Strand Break; FISH: fluorescence in situ hybridization; GFP: GREEN FLUORESCENT PROTEIN; HEI10: HUMAN ENHANCER OF INVASION 10; NCO: Non-Crossover; NE: Nuclear Envelope; Oligo-FISH: oligonucleotide fluorescence in situ hybridization; RNPII: RNA Polymerase II; SC: Synaptonemal Complex; SIM: structured illumination microscopy; ZMM (ZIP: MSH4: MSH5 and MER3 proteins); ZYP1: ZIPPER-LIKE PROTEIN 1.