Transcriptomics analysis of hulless barley during grain development with a focus on starch biosynthesis.

Hulless barley, with its unique nutritional value and potential health benefits, has increasingly attracted attentions in recent years. However, the transcription dynamics during hulless barley grain development is not well understood. In the present study, we investigated the transcriptome changes during barley grain development using Illumina paired-end RNA-sequencing. Two datasets of the developing grain transcriptomes from two barley landraces with the differential seed starch synthesis traits were generated, and comparative transcriptome approach in both genotypes was performed. The results showed that 38 differentially expressed genes (DEGs) were found co-modulated in both genotypes during the barley grain development. Of those, the proteins encoded by most of those DGEs were found, such as alpha-amylase-related proteins, lipid-transfer protein, homeodomain leucine zipper (HD-Zip), NUCLEAR FACTOR-Y, subunit B (NF-YBs), as well as MYB transcription factors. More interestingly, two genes Hvulgare_GLEAN_10012370 and Hvulgare_GLEAN_10021199 encoding SuSy, AGPase (Hvulgare_GLEAN_10033640 and Hvulgare_GLEAN_10056301), as well as SBE2b (Hvulgare_GLEAN_10018352) were found to significantly contribute to the regulatory mechanism during grain development in both genotypes. Moreover, six co-expression modules associated with specific biological processes or pathways (M1 to M6) were identified by consensus co-expression network. Significantly enriched pathways of those module genes showed difference in both genotypes. These results will expand our understanding of the complex molecular mechanism of starch synthesis during barley grain development.

Transcriptome analysis revealed the drought-responsive genes in Tibetan hulless barley.

BACKGROUND: Hulless barley, also called naked barley, is an important cereal crop worldwide, serving as a healthy food both for human consumption and animal feed. Nevertheless, it often suffered from drought stress during its growth and development, resulting in a drastic reduction in barley yields. Therefore, study on molecular mechanism of hulless barley drought-tolerance is very important for increasing barley production. To investigate molecular mechanism of barley drought-resistance, this study examined co-regulated mRNAs that show a change in expression pattern under early well water, later water deficit and finally water recovery treatments, and to identify mRNAs specific to water limiting conditions. RESULTS: Total of 853 differentially expressed genes (DEGs) were detected and categorized into nine clusters, in which VI and VIII were apparently up-regulated under low relative soil moisture content (RSMC) level. The majority of genes in these two clusters was relevant to abiotic stress responses in abscisic acid (ABA) dependent and independent signaling pathway, including NCED, PYR/PYL/RCAR, SnRK2, ABF, MYB/MYC, AP2/ERF family, LEA and DHN. In contrast, genes within clusters II and IV were generally down-regulated under water stress; cluster IX genes were up-regulated during water recovery response to both low and high RSMC levels. Genes in implicated in tetrapyrrole binding, photosystem and photosynthetic membrane were the most affected in cluster IX. CONCLUSION: Taken together, our findings indicate that the responses of hulless barley to drought stress shows differences in the pathways and genes activated. Furthermore, all these genes displayed different sensitivities to soil water deficit and might be profitable for future drought tolerance improvement in barley and other crops.

Culture dependent and independent characterization of endophytic bacteria in the seeds of highland barley.

Endophytes in the seeds of plants have shown plant growth promoting (PGP) properties. Highland barley is an economically important crop and a major part of the local diet in the Tibetan Plateau, China, with potential health benefits. We applied culture-dependent and culture-independent methods to study endophytic bacteria in the seeds of eight Highland barley varieties. Based on the seed properties, the variety Ali was clearly separated from the other varieties except the variety CM. Most of the 86 isolates were assigned into genus Bacillus. Approximately half of the isolates showed PGP properties in vitro. Compared to the not-inoculated plants, inoculation with the isolate Bacillus tequilensis LZ-9 resulted in greater length and number of roots, and in bigger aboveground and root weights. Based on the 16S rRNA gene sequencing, the seed microbiome was majorly affiliated with the phylum Proteobacteria and the family Enterobacteriaceae. Overall, the bacterial community compositions in the different varieties were different from each other, yet the between variety differences in community composition seemed relatively small. The differences in community compositions were associated with differences in the total and reducing sugar contents and viscosity of the seeds, thus possibly connected to differences in the osmotic pressure tolerance of the endophytes. The results suggested that the seed endophytes are likely to promote the growth of Highland barley since germination.

Quantitative Proteome Profiling Provides Insight into the Proteins Associated with ß-Glucan Accumulation in Hull-less Barley Grains.

The hull-less barley (Qingke) is widely planted as a staple food crop in the Tibetan area, China, and the grains contains high content of ß-glucan (BG). The mechanisms of BG synthesis and accumulation in qingke has not been studied at the protein level. This study characterized the proteins associated with BG synthesis and accumulation during qingke seed development. The proteome profiles of qingke seeds taken at 20, 30, and 40 days after flowering were compared using the TMT-based quantitative proteomics. A total of 4283 proteins were identified, with 759 being differentially expressed (DEPs) throughout seed development. Comparisons of protein expression pattern, functions, and pathway enrichment tests highlight cell wall modification, carbon and energy metabolism, polysaccharide metabolism, post-transcriptional modifications, and vesicular transport as critical biological processes related to qingke BG accumulation. Furthermore, induction of starch synthase, starch branching enzyme, pectin acetyl esterases, beta-glucosidases, beta-amylases, 1,4-beta-xylan, xyloglucan, α-amylase inhibitors, and glycosyltransferases underpinned BG synthesis. The results also indicated that the proteins involved in glycolytic, gluconeogenesis, and glyoxylate bypass pathways provided energy and reducing power for BG storage. Parallel reaction monitoring (PRM) and quantitative real-time PCR (qPCR) analyses confirmed the expression profile of the proteins obtained by TMT-based proteomics. The current results provided an insight into the mechanisms of BG synthesis and accumulation during qingke seed development.

An improved high-quality genome assembly and annotation of Tibetan hulless barley.

Hulless barley (Hordeum vulgare L. var. nudum) is a barley variety that has loose husk cover of the caryopses. Because of the ease in processing and edibility, hulless barley has been locally cultivated and used as human food. For example, in Tibetan Plateau, hulless barley is the staple food for human and essential livestock feed. Although the draft genome of hulless barley has been sequenced, the assembly remains fragmented. Here, we reported an improved high-quality assembly and annotation of the Tibetan hulless barley genome using more than 67X PacBio long-reads. The N50 contig length of the new assembly is at least more than 19 times larger than other available barley assemblies. The new genome assembly also showed high gene completeness and high collinearity of genome synteny with the previously reported barley genome. The new genome assembly and annotation will not only remove major hurdles in genetic analysis and breeding of hulless barley, but will also serve as a key resource for studying barley genomics and genetics.

Genome-wide Dissection of Co-selected UV-B Responsive Pathways in the UV-B Adaptation of Qingke.

Qingke (Tibetan hulless barley) has long been cultivated and exposed to long-term and strong UV-B radiation on the Tibetan Plateau, which renders it an ideal species for elucidating novel UV-B responsive mechanisms in plants. Here we report a comprehensive metabolite profiling and metabolite-based genome-wide association study (mGWAS) using 196 diverse qingke and barley accessions. Our results demonstrated both constitutive and induced accumulation, and common genetic regulation, of metabolites from different branches of the phenylpropanoid pathway that are involved in UV-B protection. A total of 90 significant mGWAS loci for these metabolites were identified in barley-qingke differentiation regions, and a number of high-level metabolite trait alleles were found to be significantly enriched in qingke, suggesting co-selection of various phenylpropanoids. Upon dissecting the entire phenylpropanoid pathway, we identified some key determinants controlling natural variation of phenylpropanoid content, including three novel proteins, a flavone C-pentosyltransferase, a tyramine hydroxycinnamoyl acyltransferase, and a MYB transcription factor. Our study, furthermore, demonstrated co-selection of both constitutive and induced phenylpropanoids for UV-B protection in qingke.

Quantitative proteomics analysis reveals proteins and pathways associated with anthocyanin accumulation in barley.

The aim of the present study was to explore the underlying mechanisms involved in anthocyanin biosynthesis in purple, blue, and white barley using quantitative proteomics analysis. We identified the differences in protein expression and related functions involved in anthocyanin biosynthesis in purple, blue, and white barley (named H, M, and L groups, respectively, based on their anthocyanin content) using TMT-liquid chromatography/mass spectroscopy-based proteomic methods. Totally, 297, 300, 254, and 1421 differentially expressed proteins (DEPs) were found in H vs. L, H vs. M, L vs. M, and H vs. L vs. M groups, respectively. Six clusters of proteins from the 1421 DEPs were mainly involved in carbon metabolism, amino acid and secondary metabolite biosynthesis, and metabolic pathways. Several proteins were validated using parallel reaction monitoring. The proteins involved in amino acid biosynthesis, carbon metabolism, metabolic pathways, and phenylpropanoid biosynthesis were responsible for the color differences in the three barley varieties.

Metabolite profiling in two contrasting Tibetan hulless barley cultivars revealed the core salt-responsive metabolome and key salt-tolerance biomarkers.

Salinity stress represents one of the most harmful abiotic stresses for agricultural productivity. Tibetan hulless barley is an important economic crop widely grown in highly stressful conditions in the Qinghai-Tibet Plateau and is often challenged by salinity stress. To investigate the temporal metabolic responses to salinity stress in hulless barley, we performed a widely targeted metabolomic analysis of 72 leaf samples from two contrasting cultivars. We identified 642 compounds 57 % of which were affected by salt stress in the two cultivars, principally amino acids and derivatives, organic acids, nucleotides, and derivatives and flavonoids. A total of 13 stress-related metabolites including piperidine, L-tryptophan, L-glutamic acid, L-saccharopine, L-phenylalanine, 6-methylcoumarin, cinnamic acid, inosine 5'-monophosphate, aminomalonic acid, 6-aminocaproic acid, putrescine, tyramine and abscisic acid (ABA) represent the core metabolome responsive to salinity stress in hulless barley regardless of the tolerance level. In particular, we found that the ABA signalling pathway is essential to salt stress response in hulless barley. The high tolerance of the cultivar 0119 is due to a metabolic reprogramming at key stress times. During the early salt stress stages (0-24 h), 0119 tended to save energy through reduced glycolysis, nucleotide metabolism and amino acid synthesis, while increased antioxidant compounds such as flavonoids. Under prolonged stress (48-72 h), 0119 significantly enhanced energy production and amino acid synthesis. In addition, some important compatible solutes were strongly accumulated. By comparing the two cultivars, nine salt-tolerance biomarkers, mostly unreported salt-tolerance compounds in plants, were uncovered. Our study indicated that the salt tolerant hulless barley cultivar invokes a tolerance strategy which is conserved in other plant species. Overall, we provide for the first time some extensive metabolic data and some important salt-tolerance biomarkers which may assist in efforts to improve hulless barley tolerance to salinity stress.

Variation in denitrifying bacterial communities along a primary succession in the Hailuogou Glacier retreat area, China.

BACKGROUND: The Hailuogou Glacier is located at the Gongga Mountain on the southeastern edge of the Tibetan Plateau, and has retreated continuously as a result of global warming. The retreat of the Hailuogou Glacier has left behind a primary succession along soil chronosequences. Hailuogou Glacier's retreated area provides an excellent living environment for the colonization of microbes and plants, making it an ideal model to explore plant successions, microbial communities, and the interaction of plants and microbes during the colonization process. However, to date, the density of the nitrogen cycling microbial communities remain unknown, especially for denitrifiers in the primary succession of the Hailuogou Glacier. Therefore, we investigated the structural succession and its driving factors for denitrifying bacterial communities during the four successional stages (0, 20, 40, and 60 years). METHODS: The diversity, community composition, and abundance of nosZ-denitrifiers were determined using molecular tools, including terminal restriction fragment length polymorphism and quantitative polymerase chain reactions (qPCR). RESULTS: nosZ-denitrifiers were more abundant and diverse in soils from successional years 20-60 compared to 0-5 years, and was highest in Site3 (40 years). The denitrifying bacterial community composition was more complex in older soils (40-60 years) than in younger soils (≤20 years). The terminal restriction fragments (T-RFs) of Azospirillum (90 bp) and Rubrivivax (95 bp) were dominant in soisl during early successional stages (0-20 years) and in the mature phase (40-60 years), respectively. Specific T-RFs of Bradyrhizobium (100 bp) and Pseudomonas (275 bp) were detected only in Site3 and Site4, respectively. Moreover, the unidentified 175 bp T-RFs was detected only in Site3. Of the abiotic factors that were measured in this study, soil available phosphorus, available potassium and denitrifying enzyme activity (DEA) correlated significantly with the community composition of nosZ-denitrifiers (P < 0.05 by Monte Carlo permutation test within RDA analysis).

Time-Course Comparative Metabolite Profiling under Osmotic Stress in Tolerant and Sensitive Tibetan Hulless Barley.

Tibetan hulless barley is widely grown in the extreme environmental conditions of the Qinghai-Tibet Plateau which is characterized by cold, high salinity, and drought. Osmotic stress always occurs simultaneously with drought and its tolerance is a vital part of drought tolerance. The diversity of metabolites leading to osmotic stress tolerance was characterized using widely-targeted metabolomics in tolerant (XL) and sensitive (D) accessions submitted to polyethylene glycol. XL regulated a more diverse set of metabolites than D, which may promote the establishment of a robust system to cope with the stress in XL. Compounds belonging to the group of flavonoids, amino acids, and glycerophospholipids constitute the core metabolome responsive to the stress, despite the tolerance levels. Moreover, 8 h appeared to be a critical time point for stress endurance involving a high accumulation of key metabolites from the class of nucleotide and its derivative which provide the ultimate energy source for the synthesis of functional carbohydrates, lipids, peptides, and secondary metabolites in XL. This intrinsic metabolic adjustment helped XL to efficiently alleviate the stress at the later stages. A total of 22 diverse compounds were constantly and exclusively regulated in XL, representing novel stress tolerance biomarkers which may help improving stress tolerance, especially drought, in hulless barley.

The complete chloroplast genome of Tibetan hulless barley.

We report complete chloroplast genome of the Tibetan hulless barely using IIlumina Hiseq 2000 sequencing technology (IIlumina Inc., San Diego, CA). The chloroplast genome size is 136 462 bp in length that includes two inverted repeats (IRs) of 10 528 bp, which are separated by the large single copy (LSC 101 375 bp) and small single copy (SSC 14 030 bp). Hulless barely chloroplast genome encodes 76 protein-coding genes, four rRNA genes, and 29 tRNA genes. The maximum-likelihood (ML) phylogenetic tree of the nine complete chloroplast genomes was selected from Poaceae family using Oryza sativa japonica as the out-group, supporting that hulless barely is closely related to the Hordeum vulgare subsp. vulgare.

Comparative Transcriptome Analysis Revealed Genes Commonly Responsive to Varied Nitrate Stress in Leaves of Tibetan Hulless Barley.

Nitrogen (N) deprivation or excess can lead to dramatic phenotype change, disrupt important biological processes, and ultimately limit plant productivity. To explore genes in Tibetan hulless barley responsive to varied N stress, we utilized a comparative transcriptomics method to investigate gene expression patterns under three nitrate treatments. The transcriptome of the control (optimal-nitrate, ON) sample was compared with that of free-nitrate (FN), low-nitrate (LN), and high-nitrate (HN) treatment samples, identifying 2428, 1274, and 1861 genes, respectively, that exhibited significant differences in transcript abundance. Among these, 9 genes encoding ribulose bisphosphate carboxylases exhibited up-regulated expression under varied N stress. We further compared FN vs. ON and LN vs. ON to investigate the impact of stress degree on gene expression. With the aggravation of stress, more genes were differentially expressed and thus possibly involved in the response to nitrogen deficiency. Cluster and functional enrichment analysis indicated that the differentially expressed genes (DEGs) in FN were highly enriched in response to stress, defense response, and gene expression regulation. Comprehensive comparison analysis further suggested that Tibetan hulless barley could respond to varied N stress by regulating multiple common biological processes and pathways such as nitrogen metabolism, carbon metabolism, and photosynthesis. A large number of specific DEGs involved in diverse biological processes were also detected, implying differences in the potential regulatory patterns of low- and high-N stress response. Notably, we also identified some NIN-like proteins and other transcription factors significantly modulated by these stresses, suggesting the involvement of these transcription factors in N stress response. To our knowledge, this study is the first investigation of the Tibetan hulless barley transcriptome under N stress. The identified N-stress-related genes may provide resources for genetic improvement and promote N use efficiency.