Identification of the Key Transcription Factors Regulating the Expression of the Genes Associated with Barley Malt Quality during Malting.

Barley malt is produced through a malting process; it begins with steeping followed by germination and kilning, in which dramatic changes happen for a large number of physiological and biochemical traits in barley seeds. The objectives of this study were to comprehensively investigate the phenotypic changes during malting, and identify the key regulators that modulate the expression of genes associated with malt quality traits. The results showed that there was a significant positive correlation between gibberellic acid (GA) content and the activities of some hydrolytic enzymes, including α-amylases, ß-amylases, and limit dextrinase (LD), and a significant negative correlation between GA and ß-glucan content. Starch content had little change, but starch granules were pitted severely during malting. Weighted gene coexpression analysis (WGCNA) identified the genes associated with the greatest changes of the examined malt traits during malting. The correlation analysis and protein-protein interaction (PPI) analysis detected several key transcriptional factor (TF) regulating genes associated with malt quality. These genes and TFs regulating malting traits are potentially useful in barley breeding for malt quality improvement.

Genome-wide identification, expression pattern and genetic variation analysis of SWEET gene family in barley reveal the artificial selection of HvSWEET1a during domestication and improvement.

SWEET (Sugars Will Eventually be Exported Transporter) proteins, an essential class of sugar transporters, are involved in vital biological processes of plant growth and development. To date, systematical analysis of SWEET family in barley (Hordeum vulgare) has not been reported. In this study, we genome-wide identified 23 HvSWEET genes in barley, which were further clustered into four clades by phylogenetic tree. The members belonging to the same clade showed relatively similar gene structures and conserved protein motifs. Synteny analysis confirmed the tandem and segmental duplications among HvSWEET genes during evolution. Expression profile analysis demonstrated that the patterns of HvSWEET genes varied and the gene neofunctionalization occurred after duplications. Yeast complementary assay and subcellular localization in tobacco leaves suggested that HvSWEET1a and HvSWEET4, highly expressed in seed aleurone and scutellum during germination, respectively, functioned as plasma membrane hexose sugar transporters. Furthermore, genetic variation detection indicated that HvSWEET1a was under artificial selection pressure during barley domestication and improvement. The obtained results facilitate our comprehensive understanding and further functional investigations of barley HvSWEET gene family, and also provide a potential candidate gene for de novo domestication breeding of barley.

A splicing site change between exon 5 and 6 of the nuclear-encoded chloroplast-localized HvYGL8 gene results in reduced chlorophyll content and plant height in barley.

The chloroplast is an important cellular organelle and metabolic hub, which is not only responsible for plant photosynthesis but is also involved in the de novo biosynthesis of pigments, fatty acids, and hormone metabolisms. Several genes that are responsible for rice leaf color variations have been reported to be directly or indirectly involved in chlorophyll biosynthesis and chloroplast development, whereas a few genes have been functionally confirmed to be responsible for leaf color changes in barley at the molecular level. In this study, we obtained a yellow leaf and dwarf ygl8 mutant from the progeny of Morex (a variety of barley) seeds treated with EMS. We performed bulked-segregant analysis (BSA) and RNA-seq analysis and targeted a UMP kinase encoding gene, YGL8, which generated a splicing site change between exon 5 and 6 of YGL8 due to a G to A single-nucleotide transition in the 5th exon/intron junction in the ygl8 mutant. The splicing site change between exon 5 and 6 of YGL8 had no effects on chloroplast subcellular localization but resulted in an additional loop in the UMP kinase domain, which might disturb the access of the substrates. On one hand, the splicing site change between exon 5 and 6 of YGL8 downregulated the transcriptional expression of chloroplast-encoded genes and chlorophyll-biosynthesis-related genes in a temperature-dependent manner in the ygl8 mutant. On the other hand, the downregulation of bioactive GA-biosynthesis-related GA20ox genes and cell-wall-cellulose-biosynthesis-related CesA genes was also observed in the ygl8 mutant, which led to a reduction in plant height. Our study will facilitate the understanding of the regulation of leaf color and plant height in barley.

Calmodulin and calmodulin-like gene family in barley: Identification, characterization and expression analyses.

Calmodulin (CaM) and calmodulin-like (CML) proteins are Ca2+ relays and play diverse and multiple roles in plant growth, development and stress responses. However, CaM/CML gene family has not been identified in barley (Hordeum vulgare). In the present study, 5 HvCaMs and 80 HvCMLs were identified through a genome-wide analysis. All HvCaM proteins possessed 4 EF-hand motifs, whereas HvCMLs contained 1 to 4 EF-hand motifs. HvCaM2, HvCaM3 and HvCaM5 coded the same polypeptide although they differed in nucleotide sequence, which was identical to the polypeptides coded by OsCaM1-1, OsCaM1-2 and OsCaM1-3. HvCaMs/CMLs were unevenly distributed over barley 7 chromosomes, and could be phylogenetically classified into 8 groups. HvCaMs/CMLs differed in gene structure, cis-acting elements and tissue expression patterns. Segmental and tandem duplication were observed among HvCaMs/CMLs during evolution. HvCML16, HvCML18, HvCML50 and HvCML78 were dispensable genes and the others were core genes in barley pan-genome. In addition, 14 HvCaM/CML genes were selected to examine their responses to salt, osmotic and low potassium stresses by qRT-PCR, and their expression were stress-and time-dependent. These results facilitate our understanding and further functional identification of HvCaMs/CMLs.

Identification and characterization of HAK/KUP/KT potassium transporter gene family in barley and their expression under abiotic stress.

BACKGROUND: HAK/KUP/KT (High-affinity K+ transporters/K+ uptake permeases/K+ transporters) is the largest potassium transporter family in plants, and plays pivotal roles in K+ uptake and transport, as well as biotic and abiotic stress responses. However, our understanding of the gene family in barley (Hordeum vulgare L.) is quite limited. RESULTS: In the present study, we identified 27 barley HAK/KUP/KT genes (hereafter called HvHAKs) through a genome-wide analysis. These HvHAKs were unevenly distributed on seven chromosomes, and could be phylogenetically classified into four clusters. All HvHAK protein sequences possessed the conserved motifs and domains. However, the substantial difference existed among HAK members in cis-acting elements and tissue expression patterns. Wheat had the most orthologous genes to barley HAKs, followed by Brachypodium distachyon, rice and maize. In addition, six barley HAK genes were selected to investigate their expression profiling in response to three abiotic stresses by qRT-PCR, and their expression levels were all up-regulated under salt, hyperosmotic and potassium deficiency treatments. CONCLUSION: Twenty seven HAK genes (HvHAKs) were identified in barley, and they differ in tissue expression patterns and responses to salt stress, drought stress and potassium deficiency.

Screening of Worldwide Barley Collection for Drought Tolerance: The Assessment of Various Physiological Measures as the Selection Criteria.

Drought is a devastating environmental constraint affecting the agronomic production of barley. To facilitate the breeding process, abundant germplasm resources and reliable evaluation systems to identify the true drought-tolerant barley genotypes are needed. In this study, 237 cultivated and 190 wild barley genotypes, originating from 28 countries, were screened for drought tolerance under the conditions of both water deficit and polyethylene glycol (PEG)-simulated drought at seedling stage. Drought stress significantly reduced the plant growth of all barley genotypes, but no significant difference in drought-induced reduction in the performance of barley seedlings was observed under these two drought conditions. Both cultivated and wild barley subspecies displayed considerable genotypic variability in drought tolerance, which underpinned the identification of 18 genotypes contrasting in drought tolerance. A comparative analysis of drought effects on biomass, water relation, photosynthesis, and osmotic adjustment was undertaken using these contrasting barley genotypes, in order to verify the reliability of the screening and to obtain the credible traits as screening criteria of drought tolerance in barley. As expected, the selected drought-tolerant genotypes showed much less reduction in shoot biomass than drought-sensitive ones under water deficit, which was significantly positively correlated with the results of large-scale screening, confirming the reliability of the screening for drought tolerance under two drought conditions in this study. Likewise, the traits of water relation, photosynthetic activity, and osmotic adjustment differed greatly between the contrasting genotypes under water deficit stress, and they were highly correlated to the growth of barley seedlings, suggesting the potential of them to be the selection criteria for drought tolerance. The analysis of the variable importance of these traits in drought tolerance indicated that sap osmolality and relative water content in the youngest fully-expanded leaf are the suitable selection criteria of screening for drought tolerance in barley at seedling stage.

The Ability to Regulate Transmembrane Potassium Transport in Root Is Critical for Drought Tolerance in Barley.

In this work, the effect of drought on K+ uptake in root and its translocation from root to shoot was investigated using six barley genotypes contrasting in drought tolerance. Results showed that drought conditions caused significant changes in K+ uptake and translocation in a time- and genotype-specific manner, which consequently resulted in a significant difference in tissue K+ contents and drought tolerance levels between the contrasting barley genotypes. The role of K+ transporters and channels and plasma membrane (PM) H+-ATPase in barley's adaptive response to drought stress was further investigated at the transcript level. The expression of genes conferring K+ uptake (HvHAK1, HvHAK5, HvKUP1, HvKUP2 and HvAKT1) and xylem loading (HvSKOR) in roots were all affected by drought stress in a time- and genotype-specific manner, indicating that the regulation of these K+ transporters and channels is critical for root K+ uptake and root to shoot K+ translocation in barley under drought stress. Furthermore, the barley genotypes showed a strong correlation between H+ efflux and K+ influx under drought stress, which was further confirmed by the significant up-regulation of HvHA1 and HvHA2. These results suggested an important role of plasma membrane H+-ATPase activity and/or expression in regulating the activity of K+ transporters and channels under drought stress. Taken together, it may be concluded that the genotypic difference in drought stress tolerance in barley is conferred by the difference in the ability to regulate K+ transporters and channels in root epidermis and stele.

Metabolite Profiling of Barley Grains Subjected to Water Stress: To Explain the Genotypic Difference in Drought-Induced Impacts on Malting Quality.

Grain weight and protein content will be reduced and increased, respectively, when barley is subjected to water stress after anthesis, consequently deteriorating the malt quality. However, such adverse impact of water stress differs greatly among barley genotypes. In this study, two Tibetan wild barley accessions and two cultivated varieties differing in water stress tolerance were used to investigate the genotypic difference in metabolic profiles during grain-filling stage under drought condition. Totally, 71 differently accumulated metabolites were identified, including organic acids, amino acids/amines, and sugars/sugar alcohols. Their relative contents were significantly affected by water stress for all genotypes and differed distinctly between the wild and cultivated barleys. The principal component analysis of metabolites indicated that the Tibetan wild barley XZ147 possessed a unique response to water stress. When subjected to water stress, the wild barley XZ147 showed the most increase of ß-amylase activity among the four genotypes, as a result of its higher lysine content, less indole-3-acetic acid (IAA) biosynthesis, more stable H2O2 homeostasis, and more up-regulation of BMY1 gene. On the other hand, XZ147 had the most reduction of ß-glucan content under water stress than the other genotypes, which could be explained by the faster grain filling process and the less expression of ß-glucan synthase gene GSL7. All these results indicated a great potential for XZ147 in barley breeding for improving water stress tolerance.