Barley Grass Juice Attenuates Hydrodynamic Transfection-Induced HCC Initiation in Mice.

Barley (Hordeum vulgare L.) grass has been recognized as a functional food with a wide spectrum of health-promoting properties. Supplementation with barley grass has the potential to prevent chronic diseases, such as cancer. Here, we investigated whether barley grass could protect against hepatocellular carcinoma (HCC). Our data showed that administration of barley grass juice attenuates tumor development in a hydrodynamic gene delivery-induced model of HCC. The expression levels of the immune cell markers Ptprc and Adgre1 were upregulated in the barley grass juice-treated and normal groups, compared to those in the vehicle group in the HCC model. Immune cells (CD45+, F4/80+, and CLEC4F + iNOS + cells) infiltration in the liver increased following barley grass juice administration. Our results indicate that barley grass could be beneficial for HCC alleviation, partly by regulating immune cell infiltration. The ingredients of barley grass affect immune cell infiltration in HCC, and the detailed mechanism requires further study.

Rapid Generation of Barley Homozygous Transgenic Lines Based on Microspore Culture: HvPR1 Overexpression as an Example.

Obtaining homozygous lines from transgenic plants is an important step for phenotypic evaluations, but the selection of homozygous plants is time-consuming and laborious. The process would be significantly shortened if anther or microspore culture could be completed in one generation. In this study, we obtained 24 homozygous doubled haploid (DH) transgenic plants entirely by microspore culture from one T0 transgenic plant overexpressing the gene HvPR1 (pathogenesis-related-1). Nine of the doubled haploids grew to maturity and produced seeds. qRCR (quantitative real-time PCR) validation showed that the HvPR1 gene was expressed differentially even among different DH1 plants (T2) from the same DH0 line (T1). Phenotyping analysis suggested that the overexpression of HvPR1 inhibited nitrogen use efficiency (NUE) only under low nitrogen treatment. The established method of producing homozygous transgenic lines will enable the rapid evaluation of transgenic lines for gene function studies and trait evaluation. As an example, the HvPR1 overexpression of DH lines also could be used for further analysis of NUE-related research in barley.

Genome-wide identification of sucrose nonfermenting-1-related protein kinase (SnRK) genes in barley and RNA-seq analyses of their expression in response to abscisic acid treatment.

BACKGROUND: Sucrose nonfermenting-1 (SNF1)-related protein kinases (SnRKs) play important roles in regulating metabolism and stress responses in plants, providing a conduit for crosstalk between metabolic and stress signalling, in some cases involving the stress hormone, abscisic acid (ABA). The burgeoning and divergence of the plant gene family has led to the evolution of three subfamilies, SnRK1, SnRK2 and SnRK3, of which SnRK2 and SnRK3 are unique to plants. Therefore, the study of SnRKs in crops may lead to the development of strategies for breeding crop varieties that are more resilient under stress conditions. In the present study, we describe the SnRK gene family of barley (Hordeum vulgare), the widespread cultivation of which can be attributed to its good adaptation to different environments. RESULTS: The barley HvSnRK gene family was elucidated in its entirety from publicly-available genome data and found to comprise 50 genes. Phylogenetic analyses assigned six of the genes to the HvSnRK1 subfamily, 10 to HvSnRK2 and 34 to HvSnRK3. The search was validated by applying it to Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) genome data, identifying 50 SnRK genes in rice (four OsSnRK1, 11 OsSnRK2 and 35 OsSnRK3) and 39 in Arabidopsis (three AtSnRK1, 10 AtSnRK2 and 26 AtSnRK3). Specific motifs were identified in the encoded barley proteins, and multiple putative regulatory elements were found in the gene promoters, with light-regulated elements (LRE), ABA response elements (ABRE) and methyl jasmonate response elements (MeJa) the most common. RNA-seq analysis showed that many of the HvSnRK genes responded to ABA, some positively, some negatively and some with complex time-dependent responses. CONCLUSIONS: The barley HvSnRK gene family is large, comprising 50 members, subdivided into HvSnRK1 (6 members), HvSnRK2 (10 members) and HvSnRK3 (34 members), showing differential positive and negative responses to ABA.

Comparative transcriptome analysis reveals compatible and recalcitrant genotypic response of barley microspore-derived embryogenic callus toward Agrobacterium infection.

BACKGROUND: The Agrobacterium mediated transformation has been routinely used in lots of plant species as a powerful tool to deliver genes of interest into a host plant. However, the transformation of elite and commercially valuable cultivar is still limited by the genotype-dependency, and the efficiency of Agrobacterium infection efficiency is crucial for the success of transformation. RESULTS: In this study, the microspore-derived embryogenic calli (MDEC) of barley elite cultivars and breeding lines were employed as unique subjects to characterize the genotypic response during Agrobacterium infection process. Our results identified compatible barley genotypes (GanPi 6 and L07, assigned as GP6-L07 group) and one recalcitrant genotype (Hong 99, assigned as H99) for the Agrobacterium strain LBA4404 infection using GUS assay. The accumulation trend of reactive oxygen species (ROS) was similar among genotypes across the time course. The results of RNA-seq depicted that the average expressional intensity of whole genomic genes was similar among barley genotypes during Agrobacterium infection. However, the numbers of differentially expressed genes (DEGs) exhibited significant expressional variation between GP6-L07 and H99 groups from 6 to 12 h post-inoculation (hpi). Gene ontology (GO) enrichment analysis revealed different regulation patterns for the predicted biological processes between the early (up-regulated DEGs overrepresented at 2 hpi) and late stages (down-regulated DEGs overrepresented from 6 to 24 hpi) of infection. KEGG analysis predicted 12 pathways during Agrobacterium infection. Among which one pathway related to pyruvate metabolism was enriched in GP6 and L07 at 6 hpi. Two pathways related to plant hormone signal transduction and DNA replication showed expressional variation between GP6-L07 and H99 at 24 hpi. It was further validated by qRT-PCR assay for seven candidate genes (Aldehyde dehydrogenase, SAUR, SAUR50, ARG7, Replication protein A, DNA helicase and DNA replication licensing factor) involved in the three pathways, which are all up-regulated in compatible while down-regulated in recalcitrant genotypes, suggesting the potential compatibility achieved at later stage for the growth of Agrobacterium infected cells. CONCLUSIONS: Our findings demonstrated the similarity and difference between compatible and recalcitrant genotypes of barley MDEC upon Agrobacterium infection. Seven candidate genes involved in pyruvate metabolism, hormonal signal transduction and DNA replication were identified, which advocates the genotypic dependency during Agrobacterium infection process.

Rapid Visual Detection Method for Barley Yellow Mosaic Virus Using Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP).

Barley yellow mosaic disease, caused mainly by Barley yellow mosaic virus (BaYMV) and Barley mild mosaic virus, is a devastating disease of barley and is a threat to Eurasian barley production. Early detection is essential for effective management of the pathogens and to assure food security. In this study, a simple, rapid, specific, sensitive, and visual method was developed to detect BaYMV using loop-mediated isothermal amplification (LAMP). Two pairs of oligonucleotide primers (inner and outer primers) were designed to amplify the gene encoding the coat protein of BaYMV. The optimal conditions for the LAMP method were determined, and a one-step reverse transcription (RT)-LAMP method was also developed. Subsequently, the fastest processing time for RT-LAMP was determined. Among eight plant viruses examined using the LAMP method, only BaYMV was detectable, suggesting that the assay was highly specific. The RT-LAMP method was 10 times more sensitive than the RT-PCR method in the sensitivity test. To further shorten the virus detection process, a dye was added to the RT-LAMP products, and positive reactions were simply read by the naked eye via a color change (from orange to light green) under visible light. Barley samples from the middle and lower reaches of the Yangtze River basin, where BaYMV broke out very seriously in 1970s, were detected by the newly established RT-LAMP method. The results showed that all samples were positive for BaYMV, indicating the potential risk of the virus in these areas. This newly established LAMP/RT-LAMP method could be a promising tool for barley protection and food security control.

Novel low-nitrogen stress-responsive long non-coding RNAs (lncRNA) in barley landrace B968 (Liuzhutouzidamai) at seedling stage.

BACKGROUND: Reducing the dependence of crop production on chemical fertilizer with its associated costs, carbon footprint and other environmental problems is a challenge for agriculture. New solutions are required to solve this problem, and crop breeding for high nitrogen use efficiency or tolerance of low nitrogen availability has been widely considered to be a promising approach. However, the molecular mechanisms of high nitrogen use efficiency or low-nitrogen tolerance in crop plants are still to be elucidated, including the role of long non-coding RNAs (lncRNAs). RESULTS: In this study, we identified 498 lncRNAs in barley (Hordeum vulgare) landrace B968 (Liuzhutouzidamai), of which 487 were novel, and characterised 56 that were responsive to low-nitrogen stress. For functional analysis of differentially-expressed lncRNAs, the gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment of co-expressed and co-located protein-coding genes were analyzed, and interactions with annotated co-expressed protein coding genes or micro RNAs (miRNAs) were further predicted. Target mimicry prediction between differentially-expressed lncRNAs and miRNAs identified 40 putative target mimics of lncRNAs and 58 target miRNAs. Six differentially-expressed lncRNAs were further validated by qPCR, and one in particular showed consistent differential expression using both techniques. Expression levels of most of the lncRNAs were found to be very low, and this may be the reason for the apparent inconsistency between RNA-seq and qPCR data. CONCLUSIONS: The analysis of lncRNAs that are differentially-expressed under low-nitrogen stress, as well as their co-expressed or co-located protein coding genes and target mimics, could elucidate complex and hitherto uncharacterised mechanisms involved in the adaptation to low-nitrogen stress in barley and other crop plants.

Transcriptome Analysis Identifies Candidate Genes and Functional Pathways Controlling the Response of Two Contrasting Barley Varieties to Powdery Mildew Infection.

Powdery mildew caused by Blumeria graminis f. sp. hordei (Bgh) is one of the most serious diseases in barley. The numerous barley varieties across China provide valuable genetic resources to screen the resistant germplasm and to discover the primary genes of resistance to powdery mildew. In this study, Chinese barley variety Feng 7 was identified as a highly resistant genotype which limited Bgh colonization by cell apoptosis using leaf staining assay, while another variety Hua 30 showed high susceptibility. The performance of high resistance to Bgh in F1 plants from the two varieties suggested dominant gene(s) controlled the resistance to powdery mildew in Feng 7. To understand the host transcriptional response to Bgh infection, these two barley varieties Feng 7 and Hua 30 were inoculated with Bgh, and their transcriptional profiling using RNA sequencing (RNA-seq) at four time points (12 h post-inoculation (hpi), 24 hpi, 48 hpi, and 72 hpi) were compared. 4318 differentially expressed genes (DEGs), including 2244 upregulated and 2074 downregulated genes, were detected in Feng 7, compared with Hua 30 at 12 hpi. 4907 DEGs (2488 upregulated and 2419 downregulated) were detected at 24 hpi. 4758 DEGs (2295 upregulated and 2463 downregulated) were detected at 48 hpi. 3817 DEGs (2036 upregulated and 1781 downregulated) were detected at 72 hpi. The results showed the number of DEGs between two varieties peaked at 24 hpi (for the upregulated) or 48 hpi (for the downregulated), which is matched with the processing of Bgh infection. In addition, the number of upregulated DEGs involved in the functional pathways of plant defense (mitogen-activated protein kinase (MAPK) pathway and plant hormone signal transduction) is elevated remarkably at 24 hpi. Six candidate genes (PR13, glutaredoxin, alcohol dehydrogenase, and cytochrome P450) were identified in Feng 7. All of them present continuous expression at higher levels upon Bgh infection, compared with the performance in Hua 30, which revealed the potential contribution to Feng 7 mediate resistance to Bgh. In conclusion, the candidate genes and relevant pathways provided key information towards understanding the defense of barley to Bgh attack and the molecular mechanisms of different genetic resistance to powdery mildew.

Transient Overexpression of HvSERK2 Improves Barley Resistance to Powdery Mildew.

Somatic embryogenesis receptor-like kinases (SERKs) play an essential role in plant response to pathogen infection. Here we identified three SERK genes (HvSERK1/2/3) from barley, and aimed to determine their implication in defense responses to barley powdery mildew (Bgh). Although HvSERK1/2/3 share the characteristic domains of the SERK family, only HvSERK2 was significantly induced in barley leaves during Bgh infection. The expression of HvSERK2 was rapidly induced by hydrogen peroxide (H2O2) treatment, but not by treatment with salicylic acid (SA), methyl jasmonate (MeJA), ethephon (ETH), or abscisic acid (ABA). Bioinformatics analysis of the cloned HvSERK2 promoter revealed that it contains several elements responsible for defense responses against pathogens. Promoter functional analysis showed that the HvSERK2 promoter was induced by Bgh and H2O2. Subcellular localization analysis of HvSERK2 indicated that it is mainly located on the plasma membrane. Transient overexpression of HvSERK2 in epidermal cells of the susceptible barley cultivar Hua 30 reduced the Bgh haustorium index from 58.6% to 43.2%. This study suggests that the HvSERK2 gene plays a positive role in the improvement of barley resistance to powdery mildew, and provides new insight into the function of SERK genes in the biotic stress response of plants.

Transcriptome analysis reveals translational regulation in barley microspore-derived embryogenic callus under salt stress.

KEY MESSAGE: Transcriptome analysis of barley embryogenic callus from isolated microspore culture under salt stress uncovered a role of translation inhibition and selective activation of stress-specific proteins in cellular defense. Soil salinity is one of the major abiotic stresses which constrains the plant growth and reduces the productivity of field crops. In this study, it was observed that the salt stress in barley isolated microspore culture impacted not only on the quantity of embryogenic callus but also on the quality for later differentiation. The barley microspore-derived embryogenic callus, a transient intermediate form linked cells and plants, was employed for a global transcriptome analysis by RNA sequencing to provide new insights into the cellular adaptation or acclimation to stress. A total of 596 differentially expressed genes (DEGs) were identified, in which 123 DEGs were up-regulated and 473 DEGs were down-regulated in the embryogenic callus produced from microspore culture under salt stress as compared to the control conditions. KEGG pathway analysis identified 'translation' (27 DEGs; 12.56 %) as the largest group and followed by 'folding, sorting and degradation' (25 DEGs; 11.63 %) in 215 mapped metabolic pathways. The results of RNA-Seq data and quantitative real-time polymerase chain reaction validation showed that the genes related to translation regulation (such as eIF1A, RPLP0, RPLP2, VARS) were down-regulated to control general protein synthesis, and the genes related to endoplasmic reticulum stress response (such as small heat shock protein genes) were selectively up-regulated against protein denaturing during microspore embryogenesis under continuous salt stress. These transcriptional remodeling might affect the essential protein synthesis for the cell development to fulfill totipotency under salt stress.

Efficient isolated microspore culture protocol for callus induction and plantlet regeneration in japonica rice (Oryza sativa L.).

BACKGROUND: Isolated microspore culture is a useful biotechnological technique applied in modern plant breeding programs as it can produce doubled haploid (DH) plants and accelerate the development of new varieties. Furthermore, as a single-cell culture technique, the isolated microspore culture provides an excellent platform for studying microspore embryogenesis. However, the reports on isolated microspore culture are rather limited in rice due to the low callus induction rate, poor regeneration capability, and high genotypic dependency. The present study developed an effective isolated microspore culture protocol for high-frequency androgenesis in four japonica rice genotypes. Several factors affecting the isolated microspore culture were studied to evaluate their effects on callus induction and plantlet regeneration. RESULTS: Low-temperature pre-treatment at 4 â„ƒ for 10-15 days could effectively promote microspore embryogenesis in japonica rice. A simple and efficient method was proposed for identifying the microspore developmental stage. The anthers in yellow-green florets located on the second type of primary branch on the rice panicle were found to be the optimal stage for isolated microspore culture. The most effective induction media for callus induction were IM2 and IM3, depending on the genotype. The optimal concentration of 2, 4-D in the medium for callus induction was 1 mg/L. Callus induction was negatively affected by a high concentration of KT over 1.5 mg/L. The differentiation medium suitable for japonica rice microspore callus comprised 1/2 MS, 2 mg/L 6-BA, 0.5 mg/L NAA, 30 g/L sucrose, and 6 g/L agar. The regeneration frequency of the four genotypes ranged from 61-211 green plantlets per 100 mg calli, with Chongxiangjing showing the highest regeneration frequency. CONCLUSIONS: This study presented an efficient protocol for improved callus induction and green plantlet regeneration in japonica rice via isolated microspore culture, which could provide valuable support for rice breeding and genetic research.

A chromosome-scale genome assembly of Dasypyrum villosum provides insights into its application as a broad-spectrum disease resistance resource for wheat improvement.

Dasypyrum villosum is one of the most valuable gene resources in wheat improvement, especially for disease resistance. The mining of favorable genes from D. villosum is frustrated by the lack of a whole genome sequence. In this study, we generated a doubled-haploid line, 91C43DH, using microspore culture and obtained a 4.05-GB high-quality, chromosome-scale genome assembly for D. villosum. The assembly contains39 727 high-confidence genes, and 85.31% of the sequences are repetitive. Two reciprocal translocation events were detected, and 7VS-4VL is a unique translocation in D. villosum. The prolamin seed storage protein-coding genes were found to be duplicated; in particular, the genes encoding low-molecular-weight glutenin at the Glu-V3 locus were significantly expanded. RNA sequencing (RNA-seq) analysis indicated that, after Blumeria graminearum f.sp tritici (Bgt) inoculation, there were more upregulated genes involved in the pattern-triggered immunity and effector-triggered immunity defense pathways in D. villosum than in Triticum urartu. MNase hypersensitive sequencing (MH-seq) identified two Bgt-inducible MH sites (MHSs), one in the promoter and one in the 3' terminal region of the powdery mildew resistance (Pm) gene NLR1-V. Each site had two subpeaks and they were termed MHS1 (MHS1.1/1.2) and MHS2 (MHS2.1/2.2). Bgt-inducible MHS2.2 was uniquely present in D. villosum, and MHS1.1 was more inducible in D. villosum than in wheat, suggesting that MHSs may be critical for regulation of NLR1-V expression and plant defense. In summary, this study provides a valuable genome resource for functional genomics studies and wheat-D. villosum introgression breeding. The identified regulatory mechanisms may also be exploited to develop new strategies for enhancing Pm resistance by optimizing gene expression in wheat.

Integrated analysis of transcriptome and metabolome reveals molecular mechanisms of salt tolerance in seedlings of upland rice landrace 17SM-19.

Salt stress is a major abiotic stress that threatens global rice production. It is particularly important to improve salt tolerance in upland rice because of its growth environment. Upland rice landrace 17SM-19 with high salt tolerance was obtained from a previous study. In this study, an integrated analysis of transcriptome and metabolome was performed to determine the responses of the rice seedling to salt stress. When treated with 100 mm NaCl, the rice seedling growth was significantly inhibited at 5 d, with inhibition first observed in shoot dry weight (SDW). Changes in potassium (K+) content were associated with changes in SDW. In omics analyses, 1,900 differentially expressed genes (DEGs) and 659 differentially abundant metabolites (DAMs) were identified at 3 d after salt stress (DAS), and 1,738 DEGs and 657 DAMs were identified at 5 DAS. Correlation analyses between DEGs and DAMs were also conducted. The results collectively indicate that salt tolerance of upland rice landrace 17SM-19 seedlings involves many molecular mechanisms, such as those involved with osmotic regulation, ion balance, and scavenging of reactive oxygen species.

Rapid Generation and Analysis of a Barley Doubled Haploid Line with Higher Nitrogen Use Efficiency Than Parental Lines by F1 Microspore Embryogenesis.

Creating varieties with high nitrogen use efficiency (NUE) is crucial for sustainable agriculture development. In this study, a superior barley doubled haploid line (named DH45) with improved NUE was produced via F1 microspore embryogenesis with three rounds of screening in different nitrogen levels by hydroponic and field experiments. The molecular mechanisms responsible for the NUE of DH45 surpassing that of its parents were investigated by RNA-seq analysis. A total of 1027 differentially expressed genes (DEGs) were identified that were up- or down-regulated in DH45 under low nitrogen conditions but showed no significant differences in the parents. GO analysis indicated that genes involved in nitrogen compound metabolic processes were significantly enriched in DH45 compared with the parents. KEGG analysis showed the MAPK signaling pathway plant to be highly enriched in DH45 relative to its parents, as well as genes involved in alanine, aspartate and glutamate metabolism, and arginine biosynthesis. In conclusion, our study revealed the potential to fix trait superiority in a line by combining crossing with F1 microspore culture technologies in future crop breeding and also identified several candidate genes that are expressed in shoots and may enable barley to cope with low-nitrogen stress.

Comparative Analysis of Morphology, Photosynthetic Physiology, and Transcriptome Between Diploid and Tetraploid Barley Derived From Microspore Culture.

Polyploids play an important role in the breeding of plant for superior characteristics, and many reports have focused on the effects upon photosynthesis from polyploidization in some plant species recently, yet surprisingly little of this is known for barley. In this study, homozygous diploid and tetraploid plants, derived from microspore culturing of the barley cultivar "H30," were used to assess differences between them in their cellular, photosynthetic, and transcriptomic characteristics. Our results showed that tetraploid barley has the distinct characteristics of polyploids, namely thicker and heavier leaves, enlarged stomata size or stomatal guard cell size, and more photosynthetic pigments and improved photosynthesis (especially under high light intensity). This enhanced photosynthesis of tetraploid barley was confirmed by several photosynthetic parameters, including net photosynthetic rate (Pn), stomatal conductance (Gs), intercellular CO2 concentration (Ci), transpiration rate (Tr), maximum net photosynthetic rate (Pmax), light saturation point (LSP), maximum RuBP saturated rate carboxylation (Vcmax), and maximum rate of electron transport (Jmax). Transcriptomic analyses revealed that just ~2.3% of all detected genes exhibited differential expression patterns [i.e., differentially expressed genes (DEGs)], and that most of these - 580 of 793 DEGs in total - were upregulated in the tetraploid barley. The follow-up KEGG analysis indicated that the most enriched pathway was related to photosynthesis-antenna proteins, while the downregulation of DEGs was related mainly to the light-harvesting cholorophyII a/b-binding protein (Lhcb1) component, both validated by quantitative PCR (qPCR). Taken together, our integrated analysis of morphology, photosynthetic physiology, and transcriptome provides evidences for understanding of how polyploidization enhances the photosynthetic capacity in tetraploids of barley.

Karrikin Improves Osmotic and Salt Stress Tolerance via the Regulation of the Redox Homeostasis in the Oil Plant Sapium sebiferum.

Karrikins are reported to stimulate seed germination, regulate seedling growth, and increase the seedling vigor in abiotic stress conditions in plants. Nevertheless, how karrikins alleviate abiotic stress remains largely elusive. In this study, we found that karrikin (KAR1) could significantly alleviate both drought and salt stress in the important oil plant Sapium sebiferum. KAR1 supplementation in growth medium at a nanomolar (nM) concentration was enough to recover seed germination under salt and osmotic stress conditions. One nanomolar of KAR1 improved seedling biomass, increased the taproot length, and increased the number of lateral roots under abiotic stresses, suggesting that KAR1 is a potent alleviator of abiotic stresses in plants. Under abiotic stresses, KAR1-treated seedlings had a higher activity of the key antioxidative enzymes, such as superoxide dismutase, peroxidase, catalase, and ascorbate peroxidase, in comparison with the control, which leads to a lower level of hydrogen peroxide, malondialdehyde, and electrolyte leakage. Moreover, the metabolome analysis showed that KAR1 treatment significantly increased the level of organic acids and amino acids, which played important roles in redox homeostasis under stresses, suggesting that karrikins might alleviate abiotic stresses via the regulation of redox homeostasis. Under abiotic stresses, applications of karrikins did not increase the endogenous abscisic acid level but altered the expression of several ABA signaling genes, such as SNF1-RELATED PROTEIN KINASE2.3, SNF1-RELATED PROTEIN KINASE2.6, ABI3, and ABI5, suggesting potential interactions between karrikins and ABA signaling in the stress responses. Conclusively, we not only provided the physiological and molecular evidence to clarify the mechanism of karrikins in the regulation of stress adaptation in S. sebiferum but also showed the potential value of karrikins in agricultural practices, which will lay a foundation for further studies about the role of karrikins in abiotic stress alleviation in plants.

Rapid Generation of Barley Mutant Lines With High Nitrogen Uptake Efficiency by Microspore Mutagenesis and Field Screening.

In vitro mutagenesis via isolated microspore culture provides an efficient way to produce numerous double haploid (DH) lines with mutation introduction and homozygosity stabilization, which can be used for screening directly. In this study, 356 DH lines were produced from the malt barley (Hordeum vulgare L.) cultivar Hua-30 via microspore mutagenic treatment with ethyl methane sulfonate or pingyangmycin during in vitro culture. The lines were subjected to field screening under high nitrogen (HN) and low nitrogen (LN) conditions, and the number of productive tillers was used as the main screening index. Five mutant lines (A1-28, A1-84, A1-226, A16-11, and A9-29) with high numbers of productive tillers were obtained over three consecutive years of screening. In the fifth year, components related to N-use efficiency (NUE), including N accumulation, utilization, and translocation, were characterized for these lines based on N uptake efficiency (NUpE), N utilization efficiency (NUtE), and N translocation efficiency (NTE). The results show that the NUpE of four mutant lines (A1-84, A1-226, A9-29, and A16-11) improved significantly under HN, whereas that of two lines (A1-84 and A9-29) improved under LN. As a result, their NUE improved greatly. No improvement in NUtE was observed in any of the five mutant lines. A1-84 and A9-29 were selected as an enhanced genotype in N uptake, and A1-28 showed improved NTE at the grain-filling stage. Our results imply that high-NUpE mutants can be produced through microspore mutagenesis and field screening, and that improvement of NUE in barley depends on enhancement of N uptake.

Expression Analysis of Nitrogen Metabolism-Related Genes Reveals Differences in Adaptation to Low-Nitrogen Stress between Two Different Barley Cultivars at Seedling Stage.

The excess use of nitrogen fertilizers causes many problems, including higher costs of crop production, lower nitrogen use efficiency, and environmental damage. Crop breeding for low-nitrogen tolerance, especially molecular breeding, has become the major route to solving these issues. Therefore, in crops such as barley (Hordeum vulgare L.), it is crucial to understand the mechanisms of low-nitrogen tolerance at the molecule level. In the present study, two barley cultivars, BI-04 (tolerant to low nitrogen) and BI-45 (sensitive to low nitrogen), were used for gene expression analysis under low-nitrogen stress, including 10 genes related to primary nitrogen metabolism. The results showed that the expressions of HvNIA2 (nitrite reductase), HvGS2 (chloroplastic glutamine synthetase), and HvGLU2 (ferredoxin-dependent glutamate synthase) were only induced in shoots of BI-04 under low-nitrogen stress, HvGLU2 was also only induced in roots of BI-04, and HvGS2 showed a rapid response to low-nitrogen stress in the roots of BI-04. The expression of HvASN1 (asparagine synthetase) was reduced in both cultivars, but it showed a lower reduction in the shoots of BI-04. In addition, gene expression and regulation differences in the shoots and roots were also compared between the barley cultivars. Taken together, the results indicated that the four above-mentioned genes might play important roles in low-nitrogen tolerance in barley.

Genotypes-Independent Optimization of Nitrogen Supply for Isolated Microspore Cultures in Barley.

To establish a high-efficiency system of isolated microspore culture for different barley genotypes, we investigated the effects of nitrogen sources and concentrations on callus induction and plant regeneration in different barley genotypes. The results showed that the organic nitrogen sources greatly increased the callus induction, and the great reduction of total nitrogen sources would significantly decrease the callus induction. And the further optimization experiments revealed that the increasing of organic nitrogen sources was much important in callus induction while it seemed different in plant regeneration. Based on the great effects of organic nitrogen on callus induction, the medium of N6-ANO1/4-2000 might be the best choice for the microspore culture system. In addition, the phylogenetic analysis indicated that there were clear differences of genetic backgrounds among these barley genotypes, and it also suggested that this medium for microspore culture had widespread utilization in different barley genotypes.

Comparative Transcriptional Profiling of Two Contrasting Barley Genotypes under Salinity Stress during the Seedling Stage.

Salinity is one of the major abiotic stresses that affect crop productivity. Identification of the potential novel genes responsible for salt tolerance in barley will contribute to understanding the molecular mechanism of barley responses to salt stress. We compared changes in transcriptome between Hua 11 (a salt-tolerant genotype) and Hua 30 (a salt sensitive genotype) in response to salt stress at the seedling stage using barley cDNA microarrays. In total, 557 and 247 salt-responsive genes were expressed exclusively in the shoot and root tissue of the salt-tolerant genotype, respectively. Among these genes, a number of signal-related genes, transcription factors and compatible solutes were identified and some of these genes were carefully discussed. Notably, a LysM RLK was firstly found involved in salt stress response. Moreover, key enzymes in the pathways of jasmonic acid biosynthesis, lipid metabolism and indole-3-acetic acid homeostasis were specifically affected by salt stress in salt tolerance genotype. These salt-responsive genes and biochemical pathways identified in this study could provide further information for understanding the mechanisms of salt tolerance in barley.