Video_1_Heat stress responses vary during floret development in European spring barley cultivars.mp4

The Poaceae, or grasses, include many agriculturally important cereal crops such as rice (Oryza sativa), maize (Zea mays), barley (Hordeum vulgare) and bread wheat (Triticum aestivum). Barley is a widely grown cereal crop used for stock feed, malting and brewing. Abiotic stresses, particularly global warming, are the major causes of crop yield losses by affecting fertility and seed set. However, effects of heat stress on reproductive structures and fertility in barley have not been extensively investigated. In this study we examined three commercial European spring barley varieties under high temperature conditions to investigate the effects on floret development. Using a combination of fertility assays, X-ray micro computed tomography, 3-dimensional modelling, cytology and immunolabelling, we observed that male reproductive organs are severely impacted by increased temperature, while the female reproductive organs are less susceptible. Importantly, the timing of stress relative to reproductive development had a significant impact on fertility in a cultivar-dependent manner, this was most significant at pollen mitosis stage with fertility ranged from 31.6-56.0% depending on cultivar. This work provides insight into how heat stress, when applied during male pollen mother cell meiosis and pollen mitosis, affects barley fertility and seed set, and also describes complementary invasive and non-invasive techniques to investigate floret development. This information will be used to identify and study barley cultivars that are less susceptible to heat stress at specific stages of floral development.</p

DataSheet_1_Natural Variation in Ovule Morphology Is Influenced by Multiple Tissues and Impacts Downstream Grain Development in Barley (Hordeum vulgare L.).pdf

The ovule plays a critical role in cereal yield as it is the site of fertilization and the progenitor of the grain. The ovule primordium is generally comprised of three domains, the funiculus, chalaza, and nucellus, which give rise to distinct tissues including the integuments, nucellar projection, and embryo sac. The size and arrangement of these domains varies significantly between model eudicots, such as Arabidopsis thaliana, and agriculturally important monocotyledonous cereal species, such as Hordeum vulgare (barley). However, the amount of variation in ovule development among genotypes of a single species, and its functional significance, remains unclear. To address this, wholemount clearing was used to examine the details of ovule development in barley. Nine sporophytic and gametophytic features were examined at ovule maturity in a panel of 150 European two-row spring barley genotypes, and compared with grain traits from the preceding and same generation. Correlations were identified between ovule traits and features of grain they produced, which in general highlighted a negative correlation between nucellus area, ovule area, and grain weight. We speculate that the amount of ovule tissue, particularly the size of the nucellus, may affect the timing of maternal resource allocation to the fertilized embryo sac, thereby influencing subsequent grain development.</p

Data_Sheet_1_Manipulation of Barley Development and Flowering Time by Exogenous Application of Plant Growth Regulators.docx

Matching flowering time to the optimal flowering period in Mediterranean cropping zones is pivotal to maximize yield. Aside from variety selection and sowing date, growers have limited options to alter development in season. Plant hormones and growth regulators are used in perennial horticultural systems to manipulate development and floral initiation. In this study, a range of plant hormonal products were tested to analyze their effects on barley (Hordeum vulgare L) development by exogenous spray applications. Plants were grown in controlled conditions under long and short photoperiods with different vernalization treatments. The gibberellin (GA) products demonstrated the greatest potential for altering development. The GA inhibitor trinexapac-ethyl was able to delay the time to flowering in genetically divergent barley cultivars by up to 200 degree days under controlled conditions. A similar delay in flowering could be achieved via application at both early (GS13) and late (GS33) stages, with higher rates delaying flowering further. Notably, trinexapac-ethyl was able to extend the duration of pre-anthesis phases of development. By contrast, GA3 was unable to accelerate development under extreme short (8 h) or long (16 h) day lengths. There was also little evidence that GA3 could reproducibly accelerate development under intermediate 10–12 h day lengths. In addition, sprays of the cytokinin 6-benzyladenine (6-BA) were unable to reduce the vernalization requirement of the winter genotype Urambie. The present study provides baseline data for plant growth regulator treatments that delay cereal development. These treatments might be extended in field studies to align flowering of early sown crops to the optimal flowering period.</p

DataSheet_2_Natural Variation in Ovule Morphology Is Influenced by Multiple Tissues and Impacts Downstream Grain Development in Barley (Hordeum vulgare L.).pdf

The ovule plays a critical role in cereal yield as it is the site of fertilization and the progenitor of the grain. The ovule primordium is generally comprised of three domains, the funiculus, chalaza, and nucellus, which give rise to distinct tissues including the integuments, nucellar projection, and embryo sac. The size and arrangement of these domains varies significantly between model eudicots, such as Arabidopsis thaliana, and agriculturally important monocotyledonous cereal species, such as Hordeum vulgare (barley). However, the amount of variation in ovule development among genotypes of a single species, and its functional significance, remains unclear. To address this, wholemount clearing was used to examine the details of ovule development in barley. Nine sporophytic and gametophytic features were examined at ovule maturity in a panel of 150 European two-row spring barley genotypes, and compared with grain traits from the preceding and same generation. Correlations were identified between ovule traits and features of grain they produced, which in general highlighted a negative correlation between nucellus area, ovule area, and grain weight. We speculate that the amount of ovule tissue, particularly the size of the nucellus, may affect the timing of maternal resource allocation to the fertilized embryo sac, thereby influencing subsequent grain development.</p

Image_1_Heat stress responses vary during floret development in European spring barley cultivars.pdf

The Poaceae, or grasses, include many agriculturally important cereal crops such as rice (Oryza sativa), maize (Zea mays), barley (Hordeum vulgare) and bread wheat (Triticum aestivum). Barley is a widely grown cereal crop used for stock feed, malting and brewing. Abiotic stresses, particularly global warming, are the major causes of crop yield losses by affecting fertility and seed set. However, effects of heat stress on reproductive structures and fertility in barley have not been extensively investigated. In this study we examined three commercial European spring barley varieties under high temperature conditions to investigate the effects on floret development. Using a combination of fertility assays, X-ray micro computed tomography, 3-dimensional modelling, cytology and immunolabelling, we observed that male reproductive organs are severely impacted by increased temperature, while the female reproductive organs are less susceptible. Importantly, the timing of stress relative to reproductive development had a significant impact on fertility in a cultivar-dependent manner, this was most significant at pollen mitosis stage with fertility ranged from 31.6-56.0% depending on cultivar. This work provides insight into how heat stress, when applied during male pollen mother cell meiosis and pollen mitosis, affects barley fertility and seed set, and also describes complementary invasive and non-invasive techniques to investigate floret development. This information will be used to identify and study barley cultivars that are less susceptible to heat stress at specific stages of floral development.</p

Data_Sheet_2_Identification of candidate MYB transcription factors that influence CslF6 expression in barley grain.PDF

(1,3;1,4)-β-Glucan is a non-cellulosic polysaccharide required for correct barley grain fill and plant development, with industrial relevance in the brewing and the functional food sector. Barley grains contain higher levels of (1,3;1,4)-β-glucan compared to other small grain cereals and this influences their end use, having undesirable effects on brewing and distilling and beneficial effects linked to human health. HvCslF6 is the main gene contributing to (1,3;1,4)-β-glucan biosynthesis in the grain. Here, the transcriptional regulation of HvCslF6 was investigated using an in-silico analysis of transcription factor binding sites (TFBS) in its putative promoter, and functional characterization in a barley protoplast transient expression system. Based on TFBS predictions, TF classes AP2/ERF, MYB, and basic helix-loop-helix (bHLH) were over-represented within a 1,000 bp proximal HvCslF6 promoter region. Dual luciferase assays based on multiple HvCslF6 deletion constructs revealed the promoter fragment driving HvCslF6 expression. Highest HvCslF6 promoter activity was narrowed down to a 51 bp region located −331 bp to −382 bp upstream of the start codon. We combined this with TFBS predictions to identify two MYB TFs: HvMYB61 and HvMYB46/83 as putative activators of HvCslF6 expression. Gene network analyses assigned HvMYB61 to the same co-expression module as HvCslF6 and other primary cellulose synthases (HvCesA1, HvCesA2, and HvCesA6), whereas HvMYB46/83 was assigned to a different module. Based on RNA-seq expression during grain development, HvMYB61 was cloned and tested in the protoplast system. The transient over-expression of HvMYB61 in barley protoplasts suggested a positive regulatory effect on HvCslF6 expression.</p

Data_Sheet_1_Identification of candidate MYB transcription factors that influence CslF6 expression in barley grain.PDF

(1,3;1,4)-β-Glucan is a non-cellulosic polysaccharide required for correct barley grain fill and plant development, with industrial relevance in the brewing and the functional food sector. Barley grains contain higher levels of (1,3;1,4)-β-glucan compared to other small grain cereals and this influences their end use, having undesirable effects on brewing and distilling and beneficial effects linked to human health. HvCslF6 is the main gene contributing to (1,3;1,4)-β-glucan biosynthesis in the grain. Here, the transcriptional regulation of HvCslF6 was investigated using an in-silico analysis of transcription factor binding sites (TFBS) in its putative promoter, and functional characterization in a barley protoplast transient expression system. Based on TFBS predictions, TF classes AP2/ERF, MYB, and basic helix-loop-helix (bHLH) were over-represented within a 1,000 bp proximal HvCslF6 promoter region. Dual luciferase assays based on multiple HvCslF6 deletion constructs revealed the promoter fragment driving HvCslF6 expression. Highest HvCslF6 promoter activity was narrowed down to a 51 bp region located −331 bp to −382 bp upstream of the start codon. We combined this with TFBS predictions to identify two MYB TFs: HvMYB61 and HvMYB46/83 as putative activators of HvCslF6 expression. Gene network analyses assigned HvMYB61 to the same co-expression module as HvCslF6 and other primary cellulose synthases (HvCesA1, HvCesA2, and HvCesA6), whereas HvMYB46/83 was assigned to a different module. Based on RNA-seq expression during grain development, HvMYB61 was cloned and tested in the protoplast system. The transient over-expression of HvMYB61 in barley protoplasts suggested a positive regulatory effect on HvCslF6 expression.</p

Table_5_Identification of candidate MYB transcription factors that influence CslF6 expression in barley grain.XLS

(1,3;1,4)-β-Glucan is a non-cellulosic polysaccharide required for correct barley grain fill and plant development, with industrial relevance in the brewing and the functional food sector. Barley grains contain higher levels of (1,3;1,4)-β-glucan compared to other small grain cereals and this influences their end use, having undesirable effects on brewing and distilling and beneficial effects linked to human health. HvCslF6 is the main gene contributing to (1,3;1,4)-β-glucan biosynthesis in the grain. Here, the transcriptional regulation of HvCslF6 was investigated using an in-silico analysis of transcription factor binding sites (TFBS) in its putative promoter, and functional characterization in a barley protoplast transient expression system. Based on TFBS predictions, TF classes AP2/ERF, MYB, and basic helix-loop-helix (bHLH) were over-represented within a 1,000 bp proximal HvCslF6 promoter region. Dual luciferase assays based on multiple HvCslF6 deletion constructs revealed the promoter fragment driving HvCslF6 expression. Highest HvCslF6 promoter activity was narrowed down to a 51 bp region located −331 bp to −382 bp upstream of the start codon. We combined this with TFBS predictions to identify two MYB TFs: HvMYB61 and HvMYB46/83 as putative activators of HvCslF6 expression. Gene network analyses assigned HvMYB61 to the same co-expression module as HvCslF6 and other primary cellulose synthases (HvCesA1, HvCesA2, and HvCesA6), whereas HvMYB46/83 was assigned to a different module. Based on RNA-seq expression during grain development, HvMYB61 was cloned and tested in the protoplast system. The transient over-expression of HvMYB61 in barley protoplasts suggested a positive regulatory effect on HvCslF6 expression.</p

Table_2_Identification of candidate MYB transcription factors that influence CslF6 expression in barley grain.XLSX

(1,3;1,4)-β-Glucan is a non-cellulosic polysaccharide required for correct barley grain fill and plant development, with industrial relevance in the brewing and the functional food sector. Barley grains contain higher levels of (1,3;1,4)-β-glucan compared to other small grain cereals and this influences their end use, having undesirable effects on brewing and distilling and beneficial effects linked to human health. HvCslF6 is the main gene contributing to (1,3;1,4)-β-glucan biosynthesis in the grain. Here, the transcriptional regulation of HvCslF6 was investigated using an in-silico analysis of transcription factor binding sites (TFBS) in its putative promoter, and functional characterization in a barley protoplast transient expression system. Based on TFBS predictions, TF classes AP2/ERF, MYB, and basic helix-loop-helix (bHLH) were over-represented within a 1,000 bp proximal HvCslF6 promoter region. Dual luciferase assays based on multiple HvCslF6 deletion constructs revealed the promoter fragment driving HvCslF6 expression. Highest HvCslF6 promoter activity was narrowed down to a 51 bp region located −331 bp to −382 bp upstream of the start codon. We combined this with TFBS predictions to identify two MYB TFs: HvMYB61 and HvMYB46/83 as putative activators of HvCslF6 expression. Gene network analyses assigned HvMYB61 to the same co-expression module as HvCslF6 and other primary cellulose synthases (HvCesA1, HvCesA2, and HvCesA6), whereas HvMYB46/83 was assigned to a different module. Based on RNA-seq expression during grain development, HvMYB61 was cloned and tested in the protoplast system. The transient over-expression of HvMYB61 in barley protoplasts suggested a positive regulatory effect on HvCslF6 expression.</p

Table_1_Identification of candidate MYB transcription factors that influence CslF6 expression in barley grain.XLSX

(1,3;1,4)-β-Glucan is a non-cellulosic polysaccharide required for correct barley grain fill and plant development, with industrial relevance in the brewing and the functional food sector. Barley grains contain higher levels of (1,3;1,4)-β-glucan compared to other small grain cereals and this influences their end use, having undesirable effects on brewing and distilling and beneficial effects linked to human health. HvCslF6 is the main gene contributing to (1,3;1,4)-β-glucan biosynthesis in the grain. Here, the transcriptional regulation of HvCslF6 was investigated using an in-silico analysis of transcription factor binding sites (TFBS) in its putative promoter, and functional characterization in a barley protoplast transient expression system. Based on TFBS predictions, TF classes AP2/ERF, MYB, and basic helix-loop-helix (bHLH) were over-represented within a 1,000 bp proximal HvCslF6 promoter region. Dual luciferase assays based on multiple HvCslF6 deletion constructs revealed the promoter fragment driving HvCslF6 expression. Highest HvCslF6 promoter activity was narrowed down to a 51 bp region located −331 bp to −382 bp upstream of the start codon. We combined this with TFBS predictions to identify two MYB TFs: HvMYB61 and HvMYB46/83 as putative activators of HvCslF6 expression. Gene network analyses assigned HvMYB61 to the same co-expression module as HvCslF6 and other primary cellulose synthases (HvCesA1, HvCesA2, and HvCesA6), whereas HvMYB46/83 was assigned to a different module. Based on RNA-seq expression during grain development, HvMYB61 was cloned and tested in the protoplast system. The transient over-expression of HvMYB61 in barley protoplasts suggested a positive regulatory effect on HvCslF6 expression.</p