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At the microsite "Evolution Slope", Tabigha, Israel, wild barley (Hordeum spontaneum) populations adapted to dry Terra Rossa soil, and its derivative abutting wild barley population adapted to moist and fungi‐rich Basalt soil. However, the mechanisms underlying the edaphic adaptation remain elusive. Accordingly, whole genome bisulfite sequencing, RNA‐sequencing, and metabolome analysis are performed on ten wild barley accessions inhabiting Terra Rossa and Basalt soil. A total of 121 433 differentially methylated regions (DMRs) and 10 478 DMR‐genes are identified between the two wild barley populations. DMR‐genes in CG context (CG‐DMR‐genes) are enriched in the pathways related with the fundamental processes, and DMR‐genes in CHH context (CHH‐DMR‐genes) are mainly associated with defense response. Transcriptome and metabolome analysis reveal that the primary and secondary metabolisms are more active in Terra Rossa and Basalt wild barley populations, respectively. Multi‐omics analysis indicate that sugar metabolism facilitates the adaptation of wild barley to dry Terra Rossa soil, whereas the enhancement of phenylpropanoid/phenolamide biosynthesis is beneficial for wild barley to inhabit moist and fungi pathogen‐rich Basalt soil. The current results make a deep insight into edaphic adaptation of wild barley and provide elite genetic and epigenetic resources for developing barley with high abiotic stress tolerance. [ABSTRACT FROM AUTHOR]

Highlights • Tibetan wild barley (Hordeum spontaneum) accession XZ16 is higher salt tolerant than the cultivar Golden Promise. • In roots, miR393a, miR156d and miR172b regulating HvAFB2 / HvTIR1 , UGTs and HvAP2 are responsible for salt tolerance. • In shoots, miR319a/miR396e module, miR159a and miR172b regulating GRFs , MYB33 and HvAP2 might contribute to salt tolerance. Abstract A deep understanding of the mechanisms underlying salt tolerance should be helpful for breeding of salt-tolerant crop cultivars. Our previous study identified some wild barley accessions with high salt tolerance. In this study, small RNA and degradome sequencing was performed to identify salt-responsive microRNAs (miRNAs) and their target genes in a Tibetan wild barley accession XZ16 and a cultivar Golden Promise (GP), differing in salt tolerance. Under salt stress, XZ16 showed higher salt tolerance than GP in terms of biomass and shoot Na+ accumulation. A total of 278 and 320 miRNAs were found in roots and shoots of both genotypes, respectively. Among them, 40 and 51 miRNAs were salt-responsive in roots and shoots, respectively. In roots, miR156d, miR164a, miR393a, miR319a and miR172b targeting UGTs , NAC079 , HvAFB2 / HvTIR1 , TCP4 and HvAP2 , respectively, are probably responsible for salt tolerance. In shoots, miR159a, miR169i, miR319a/miR396e module and miR172b regulating MYB33 , NHX1/LEA7 , TCP4 , GRFs and HvAP2 , respectively, might contribute to salt tolerance. Compared with GP, XZ16 showed differential expression of miR156d, miR164a, miR169i, miR172b, miR319a, miR393a and a novel miRNA PC-miR124. We proposed that these differentially expressed miRNAs could account for the difference in salt tolerance of the two genotypes and drawn up a putative regulatory network of miRNAs to reveal molecular mechanisms of salt tolerance in barley. [ABSTRACT FROM AUTHOR]

Barley ( Hordeum vulgare ) and rice ( Oryza sativa ) differ greatly in their salt tolerance, although both species belong to the Poaceae family. To understand the mechanisms in the difference of salt tolerance between the two species, the responses of ionome, metabolome and gene expression of Na and K transporters to the different salt treatments were analyzed using 4 barley and 4 rice genotypes differing in salt tolerance. In comparison with 4 rice genotypes, four barley genotypes showed better plant growth, lower shoot Na concentration and higher K concentration at the 9 day after salt treatments. There was a dramatic difference in absolute expression levels of SOS , HKT and NHX family genes between barley and rice, which might account for their difference in Na/K homeostasis and salt tolerance. Moreover, rice leaves accumulated excess Na under salt treatments, which caused serious damages to physiological metabolisms based on metabolomic analysis, but barley leaves had lower Na concentration and small changes in the most metabolites. These results provide useful insights into the molecular mechanism in the difference of salt tolerance between rice and barley. [ABSTRACT FROM AUTHOR]

In our previous study, Tibetan wild barley ( Hordeum spontaneum L.) has been found to be rich in the elite accessions with strong abiotic stress tolerance, including salt stress tolerance. However, the molecular mechanism of salt tolerance underlying the wild barley remains to be elucidated. In this study, two Tibetan wild barley accessions, XZ26 (salt-tolerant) and XZ169 (salt-sensitive), were used to investigate ionomic, metabolomic and proteomic responses in roots when exposed to 0, 200 (moderate) and 400 mM (high) salinity. XZ26 showed stronger root growth and maintained higher K concentrations when compared with XZ169 under moderate salinity, while no significant difference was found between the two accessions under high salinity. A total of 574 salt-regulated proteins and 153 salt-regulated metabolites were identified in the roots of both accessions based on quantitative proteomic (iTRAQ methods) and metabolomic (GC-TOF/MS) analysis. XZ26 developed its root adaptive strategies mainly by accumulating more compatible solutes such as proline and inositol, acquiring greater antioxidant ability to cope with ROS, and consuming less energy under salt stress for producing biomass. These findings provide a better understanding of molecular responses of root adaptive strategies to salt stress in the wild barley. [ABSTRACT FROM AUTHOR]

A thorough understanding of ionic detoxification and homeostasis is imperative for improvement of salt tolerance in crops. However, the homeostasis of elements and their relationship to metabolites under salt stress have not been fully elucidated in plants. In this study, Tibetan wild barley accessions, XZ16 and XZ169, differing in salt tolerance, and a salt-tolerant cultivar CM72 were used to investigate ionomic profile changes in tissues in response to 150 and 300 mM NaCl at the germination and seedling stages. At the germination stage, the contents of Ca and Fe significantly decreased in roots, while K and S contents increased, and Ca and Mg contents decreased in shoots, after 10 d of treatment. At the seedling stage, the contents of K, Mg, P and Mn in roots and of K, Ca, Mg and S in shoots decreased significantly after 21 d of treatment. Moreover, Na had a significant negative correlation with metabolites involved in glycolysis, α-ketoglutaric acid, maleic acid and alanine in roots, and metabolites associated with the tricarboxylic acid (TCA) cycle, sucrose, polyols and aspartate in leaves. The salt-tolerant genotypes XZ16 and CM72 showed a lower Na content in tissues, and less reduction in Zn and Cu in roots, of Ca, Mg and S in leaves, and shoot DW than the sensitive genotype XZ169, when exposed to a higher salt level. The results indicated that restriction of Na accumulation and rearrangement of nutrient elements and metabolites in barley tissues are possibly attributable to development of salt tolerance. [ABSTRACT FROM PUBLISHER]

Gene editing using clustered regularly interspaced short palindromic repeat/CRISPR-associated proteins (CRISPR/Cas) has revolutionized biotechnology and provides genetic tools for medicine and life sciences. However, the application of this technology to miRNAs, with the function as negative gene regulators, has not been extensively reviewed in plants. Here, we summarize the evolution, biogenesis, and structure of miRNAs, as well as their interactions with mRNAs and computational models for predicting target genes. In addition, we review current advances in CRISPR/Cas for functional analysis and for modulating miRNA genes in plants. Extending our knowledge of miRNAs and their manipulation with CRISPR will provide fundamental understanding of the functions of plant miRNAs and facilitate more sustainable and publicly acceptable genetic engineering of crops. miRNAs are special regulators for plant growth and its adaption to environmental cues. The conserved biogenesis and structure of miRNAs, and their coevolution with their target genes, suggest their key roles in the evolution, adaptation, and diversification of green plants. Effective approaches for MIR gene editing mediated by clustered regularly interspaced short palindromic repeat (CRISPR) are useful for fundamental plant biology research. The evolving CRISPR toolkit has great potential for crop improvement towards better yield, stress tolerance, and nutritional quality. [ABSTRACT FROM AUTHOR]

• Barley accession XZ26 is more salt tolerant than rice cultivar Nipponbare. • Transcriptomic analysis identified 606 and 186 DEGs in the roots and shoots for XZ26, and 4667 and 2817 DEGs for Nipponbare, respectively. • Alternative splicing analysis identified 40 and 33 AS genes (salt-responsive DEGs) in the roots and shoots of XZ26. Both barley (Hordeum vulgare) and rice (Oryza sativa) belong to Poaceae family, but differ greatly in salt tolerance. In order to understand molecular mechanisms in the difference of salt tolerance between the two species, the responses of transcriptomic profiles to salt stress were compared between rice (cultivar Nipponbare) and barley (accession XZ26) to reveal how alternative splicing (AS) coordinates with transcriptional regulation in adaptation to salt stress. Physiological study showed that XZ26 had higher salt tolerance than Nipponbare, as reflected by less growth inhibition, lower shoot Na+ concentration and higher K+/Na+ ratio when exposed to salt stress. Transcriptomic analysis identified 606 and 186 DEGs in the roots and shoots for XZ26, and 4667 and 2817 DEGs for Nipponbare, respectively. While alternative splicing analysis identified 40 and 33 AS genes (salt-responsive DEGs) in the roots and shoots of XZ26. Moreover, AS genes related to ion transporters and transcription factors could enhance and amplify K+/Na+ homeostasis in barley. The current results suggest that higher salt tolerance of barley accession XZ26 is attributed to its superior K+/Na+ homeostasis, tissue detoxification and less energy consumption. [ABSTRACT FROM AUTHOR]

Background: Barley is relatively sensitive to Aluminum (Al) toxicity among cereal crops, but shows a wide genotypic difference in Al tolerance. The well-known Al-tolerant mechanism in barley is related to Al exclusion mediated by a citrate transporter HvAACT1 (Al-activated citrate transporter 1). A 1-kb insertion in the promoter region of HvAACT1 gene results in a dramatic increase of its expression level, which only occurs in some Al-tolerant cultivars. However, Al-tolerant Tibetan wild barley accession XZ29 did not have the 1-kb insertion. Results: We confirmed that the expression of HvAACT1 and secretion of citrate and other organic acids did not explain the difference in Al-tolerant wild barley XZ29 and Al-sensitive cultivated barley Golden Promise. To identify microRNAs (miRNAs) and their target genes responsive to Al stress in barley roots, eight small RNA libraries with two biological replicates from these two genotypes exposed to control and Al-treated conditions were constructed and submitted to deep sequencing. A total of 342 miRNAs were identified in Golden Promise and XZ29, with 296 miRNAs being commonly shared in the two genotypes. Target genes of these miRNAs were obtained through bioinformatics prediction or degradome identification. Comparative analysis detected 50 miRNAs responsive to Al stress, and some of them were found to be exclusively expressed in XZ29 and associated with Al tolerance. Conclusions: miRNAs exclusively expressing in the wild barley were identified and found to be associated with Al stress tolerance. The current results provide a model of describing the roles of some special miRNAs associated with Al tolerance in the Tibetan wild barley. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Salt stress is one of the major environmental restricts for crop production and food safety. Barley (Hordeum vulgare L.) is the most salt-tolerant cereal crop, which could be the pioneer for shifting agricultural crop production to marginal saline lands. However, probably due to high genetic complexity of salinity tolerance trait, the progress in the identification of salt-tolerant locus or genes of barley roots moves slowly. Here, we determined physiological and ionic changes in mini-core barley accessions under salt conditions. Na+ content was lower in whole-plant but higher in roots of the salt tolerant genotypes than sensitive ones under salt stress. Genome-wide association study (GWAS) analysis identified 43 significant SNPs out of 12,564 SNPs and 215 candidate genes (P < 10−3) in the roots of worldwide barley accessions, highly associated with root relative dry weight (RDW) and Na+ content after hydroponic salinity in greenhouse and growth chamber. Meanwhile, transcriptomic analysis (RNA-Seq) identified 3217 differentially expression genes (DEGs) in barley roots induced by salt stress, mainly enriched in metabolism and transport processes. After GWAS and RNA-Seq integrating analysis, 39 DEGs were verified by qRT-PCR as salt-responding genes, including CYPs , LRR-KISS and CML genes, mostly related to the signal regulation. Taken together, current results provide genetic map-based genes or new locus useful for improving salt tolerance in crop and contributing to the utilization of saline soils. [Display omitted] • GWAS identified 43 significant SNPs associated with RDW and Na under salt stress. • RNA-Seq identified 3217 DEGs induced by salt stress in barley roots. • Prioritized 39 salt-responding genes by integrating GWAS, RNA-seq and qRT-PCR. • Provided map-based candidate genes and a working model for root salt response. [ABSTRACT FROM AUTHOR]

Aluminum (Al) toxicity is a major abiotic stress that restricts crop production in acid soils. Plants have evolved internal and external mechanisms of tolerance, and among them it is well known that AtSTOP1 and OsART1 are key transcription factors involved in tolerance through regulation of multiple downstream genes. Here, we identified the closest homolog of these two proteins in barley, namely HvATF1, Al-tolerance Transcription Factor 1, and determined its potential function in Al stress. HvATF1 is expressed in the nucleus, and functions in transcriptional activation. The transcription of HvATF1 was found to be constitutive in different tissues, and was little affected by Al stress. Knockdown of HvATF1 by RNAi resulted in increased Al sensitivity. Transcriptomics analysis identified 64 differently expressed genes in the RNAi lines compared to the wild-type, and these were considered as candidate downstream genes regulated by HvATF1. This study provides insights into the different molecular mechanisms of Al tolerance in barley and other plants. [ABSTRACT FROM AUTHOR]

The N6-methyladenosine (m6A) modifications play crucial regulatory roles in plant yield development, nutritional growth and stress adaption. Rice (Oryza sativa) is relatively sensitive to salt stress among cereal crops, while its regulatory mechanism conferring salt tolerance is still unclear. Here, we performed transcriptome-wide m6A analysis in roots of rice cultivar Nipponbare under salt conditions (0 and 75 mM NaCl). After salt treatments for 4 d, a total of 18,115 m6A peak callings of 12,516 genes and 22,052 peaks of 14,235 genes were identified under salt and control conditions, respectively. These m6A peaks were mostly enriched in CDS and 3′UTR regions. Totally, 346 m6A differentially peaks (DPs) and 1522 differentially expressed genes (DEGs) were identified in response to salt stress, which were mainly enriched in "MAPK signaling module" and "metal ion binding". Among them, the m6A related genes including 2 writers (MTB and TRM4A), 1 reader (CPSF30) and 1 eraser (ALKBH9A). The m6A modified salt-responding genes including HAK4 , CIPK06 , RBOHH , Myb10 , ERF067 and other transcription factors might participate in transcriptional regulation or multiple Ca2+, ABA and ROS signal pathways. The m6A level and gene expression of these genes were further confirmed by m6A-IP-qPCR and qRT-PCR, respectively, which showed a high consistency with the m6A-seq and transcriptome data. This study provides epigenetic responses of molecular regulatory mechanisms of salt tolerance in rice. ● Identified 346 salt-induced m6A DPs and 1522 DEGs in rice roots. ● Most m6A DPs were enriched in stop codon and CDS related to gene length. ● Proposed the roles of m6A modified salt-responding genes in transcriptional regulation and multiple signal pathways. ● Performed m6A-IP-qPCR and qRT-PCR experiments to validate the accuracy of data. [ABSTRACT FROM AUTHOR]

Barley is an important cereal crop worldwide. Its genetic transformation is now limited to very few cultivars because of the high genotype dependence of embryogenic callus. To reveal the key genes or factors controlling the callus induction and plantlet regeneration in barley, we compared the transcriptomic profiles of immature embryos of Golden Promise and ZU9, which differed dramatically in the efficiency of the genetic transformation. The samples were taken at 0, 5, 10 and 20 days of the culture, respectively. In total, 5386 up-regulated and 6257 down-regulated genes were identified in Golden Promise. Several genes, identified exclusively in GP callus, were selected for further investigation. These genes were mainly involved in protein metabolism, energy metabolism, stress response, detoxification and ubiquitin–proteasome. Four YUCCA flavin monooxygenases, one PIN-FORMED, one tryptophan aminotransferase related, three small auxin up RNA, three indole-3-acetic acid and one adenylate isopentenyl transferase, seven cytokinin oxidase/dehydrogenase, three Arabidopsis histidine kinase, three Arabidopsis histidine phosphotransfer protein, and one Arabidopsis response regulator were differentially expressed in the calli of the two barley genotypes, suggesting that biosynthesis, response and transport of auxin and cytokinin might be associated with cell reprogramming during callus induction. The current results provide insights into molecular mechanisms of callus induction at an early developmental stage and are helpful for optimizing the tissue culture system in barley. [ABSTRACT FROM AUTHOR]

Cadmium (Cd) pollutants restrict crop yield and food security in long-term agricultural activities. Crops have evolved adaptive strategies under Cd condition, however, the transcriptional regulatory mechanism of Cd-tolerant genes remains to be largely illustrated. In this study, barley roots were exposed to 5 µM CdCl 2 for physiological response and transcriptome-wide m6A methylation profile. Cd stress inhibited root growth after 7 d Cd treatment, which is mainly associated with inhibited absorption of Mn. After Cd treatment, 8151 significantly modified m6A sites and 3920 differentially expressed genes were identified. Transcriptome-wide m6A hypermethylation was widely induced by Cd stress and enriched near the stop codon and 3′ UTR regions. Among 435 m6A modified DEGs, 319 hypermethylated genes were up-regulated and 84 hypomethylated genes were down-regulated, respectively, indicating a positive correlation of m6A methylation and expression. But well-known Cd transporter genes (HvNramp5, HvIRT1, HvHMA3 , etc.) were not modified by m6A methylation, except for ABC transporters. We further found key Cd-responding regulatory genes were positively modulated with m6A methylation, including MAPK , WRKY and MYB members. This study proposed a transcriptional regulatory network of Cd stress response in barley roots, which may provide new insight into gene manipulation of controlling low Cd accumulation for crops. [Display omitted] • Identified 8151 m6A modified DPs and 3920 DEGs by Cd stress in barley roots. • 435 DEGs with DPs are likely to be core regulatory machinery in Cd stress response. • Some key DEGs with DPs were regulatory genes, rather than Cd transporter genes. • Proposed a working model of Cd response on plant root epitranscriptome. • Provided new insights into gene manipulation of low Cd accumulation in crop. [ABSTRACT FROM AUTHOR]