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[Display omitted] • First report whole genome wide comparative analysis of drought tolerant wild barley vs cultivated barley. • Drought tolerance in Tibetan wild barley attributes to candidate genes with unique genetic variants and expression patterns. • Wild barley employs effective regulators to activate various responsive pathways to adapted to water deficit conditions. • Novel functional genes HvZIFL2 and HvPOD11 positively regulate drought tolerance. Frequent climate change-induced drought events are detrimental environmental stresses affecting global crop production and ecosystem health. Several efforts have facilitated crop breeding for resilient varieties to counteract stress. However, progress is hampered due to the complexity of drought tolerance; a greater variety of novel genes are required across varying environments. Tibetan annual wild barley is a unique and precious germplasm that is well adapted to abiotic stress and can provide elite genes for crop improvement in drought tolerance. To identify the genetic basis and unique mechanisms for drought tolerance in Tibetan wild barley. Whole genome resequencing and comparative RNA-seq approaches were performed to identify candidate genes associated with drought tolerance via investigating the genetic diversity and transcriptional variation between cultivated and Tibetan wild barley. Bioinformatics, population genetics, and gene silencing were conducted to obtain insights into ecological adaptation in barley and functions of key genes. Over 20 million genetic variants and a total of 15,361 significantly affected genes were identified in our dataset. Combined genomic, transcriptomic, evolutionary, and experimental analyses revealed 26 water deficit resilience-associated genes in the drought-tolerant wild barley XZ5 with unique genetic variants and expression patterns. Functional prediction revealed Tibetan wild barley employs effective regulators to activate various responsive pathways with novel genes, such as Zinc-Induced Facilitator-Like 2 (HvZIFL2) and Peroxidase 11 (HvPOD11), to adapt to water deficit conditions. Gene silencing and drought tolerance evaluation in a natural barley population demonstrated that HvZIFL2 and HvPOD11 positively regulate drought tolerance in barley. Our findings reveal functional genes that have been selected across barley's complex history of domestication to thrive in water deficit environments. This will be useful for molecular breeding and provide new insights into drought-tolerance mechanisms in wild relatives of major cereal crops. [ABSTRACT FROM AUTHOR]

Summary: The considerable drought tolerance of wild cereal crop progenitors has diminished during domestication in the pursuit of higher productivity. Regaining this trait in cereal crops is essential for global food security but requires novel genetic insight.Here, we assessed the molecular evidence for natural variation of drought tolerance in wild barley (Hordeum spontaneum), wild emmer wheat (Triticum dicoccoides), and Brachypodium species collected from dry and moist habitats at Evolution Canyon, Israel (ECI).We report that prevailing moist vs dry conditions have differentially shaped the stomatal and photosynthetic traits of these wild cereals in their respective habitats. We present the genomic and transcriptomic evidence accounting for differences, including co‐expression gene modules, correlated with physiological traits, and selective sweeps, driven by the xeric site conditions on the African Slope (AS) at ECI. Co‐expression gene module 'circadian rhythm' was linked to significant drought‐induced delay in flowering time in Brachypodium stacei genotypes. African Slope‐specific differentially expressed genes are important in barley drought tolerance, verified by silencing Disease‐Related Nonspecific Lipid Transfer 1 (DRN1), Nonphotochemical Quenching 4 (NPQ4), and Brassinosteroid‐Responsive Ring‐H1 (BRH1).Our results provide new genetic information for the breeding of resilient wheat and barley in a changing global climate with increasingly frequent drought events. [ABSTRACT FROM AUTHOR]

Summary: Plant K+ uptake typically consists low—affinity mechanisms mediated by Shaker K+ channels (AKT/KAT/KC) and high‐affinity mechanisms regulated by HAK/KUP/KT transporters, which are extensively studied. However, the evolutionary and genetic roles of both K+ uptake mechanisms for drought tolerance are not fully explored in crops adapted to dryland agriculture. Here, we employed evolutionary bioinformatics, biotechnological and electrophysiological approaches to determine the role of two important K+ transporters HvAKT2 and HvHAK1 in drought tolerance in barley. HvAKT2 and HvHAK1 were cloned and functionally characterized using barley stripe mosaic virus‐induced gene silencing (BSMV‐VIGS) in drought‐tolerant wild barley XZ5 and agrobacterium‐mediated gene transfer in the barley cultivar Golden Promise. The hallmarks of the K+ selective filters of AKT2 and HAK1 are both found in homologues from strepotophyte algae, and they are evolutionarily conserved in strepotophyte algae and land plants. HvAKT2 and HvHAK1 are both localized to the plasma membrane and have high selectivity to K+ and Rb+ over other tested cations. Overexpression of HvAKT2 and HvHAK1 enhanced K+ uptake and H+ homoeostasis leading to drought tolerance in these transgenic lines. Moreover, HvAKT2‐ and HvHAK1‐overexpressing lines showed distinct response of K+, H+ and Ca2+ fluxes across plasma membrane and production of nitric oxide and hydrogen peroxide in leaves as compared to the wild type and silenced lines. High‐ and low‐affinity K+ uptake mechanisms and their coordination with H+ homoeostasis play essential roles in drought adaptation of wild barley. These findings can potentially facilitate future breeding programs for resilient cereal crops in a changing global climate. [ABSTRACT FROM AUTHOR]

Aluminum (Al) toxicity in acidic soil is a major factor affecting crop productivity. The extensive genetic diversity found in Tibetan wild barley germplasms offers a valuable reservoir of alleles associated with aluminum tolerance. Here, resequencing of two Al-tolerant barley genotypes (Tibetan wild barley accession XZ16 and cultivated barley Dayton) identified a total of 19,826,182 and 16,287,277 single nucleotide polymorphisms (SNPs), 1628,052 and 1386,973 insertions/deletions (InDels), 61,532 and 57,937 structural variations (SVs), 248,768 and 240,723 copy number variations (CNVs) in XZ16 and Dayton, respectively, and uncovered approximately 600 genes highly related to Al tolerance in barley. Comparative genomic analyses unveiled 71 key genes that contain unique genetic variants in XZ16 and are predominantly associated with organic acid exudation, Al sequestration, auxin response, and transcriptional regulation. Manipulation of these key genes at the genetic and transcriptional level is a promising strategy for developing optimal haplotype combinations and new barley cultivars with improved Al tolerance. This study represents the first comprehensive examination of genetic variation in Al-tolerant Tibetan wild barley through genome-wide profiling. The obtained results make the deep insight into the mechanisms underlying barley adaptation to Al toxicity, and identified the candidate genes useful for improvement of Al tolerance in barley. [Display omitted] • First report genome-wide profiling of genetic variations in Al-tolerant Tibetan wild barley. • Resequencing identified valuable genetic variations associated with Al-tolerance in barley. • Unique Al tolerance of XZ16 comes from effect genetic variations in multiple pathways. • Comparative genomic assay disclosed key Al tolerance genes/mechanism in Tibetan XZ16. [ABSTRACT FROM AUTHOR]

• Tibetan wild barley drought-responsive lncRNAs firstly studied vs cv. Tadmor. • The 535 lncRNAs differently expressed in wild barely XZ5, XZ54 and Tadmor. • Expression patterns of drought-responsive lncRNAs validated by qRT-PCR. • Identified 10 drought-tolerant related lncRNAs in XZ5 and their target genes. • Detected novel drought-tolerant-related lncRNAs with such target as SMG1 in XZ5. Drought is the most common and frequently occurring abiotic stress limiting crop yield and quality. Long non-coding RNAs (lncRNA) play crucial roles in regulating expression of genes in plants under various abiotic stress. The participation of lncRNAs in plant drought tolerance in barley is largely unknown. Here, we compared the lncRNA transcriptome in roots of two Tibetan wild barley genotypes XZ5 (drought-tolerant) and XZ54 (drought-sensitive), and one drought tolerant cv. Tadmor in response to polyethylene glycol-induced drought stress. In total, 23,651 novel lncRNAs were identified, among which 535 were characterized as drought responsive according to their different expression profile among XZ5, and XZ54 and Tadmor in response to drought stress. LncRNA target prediction revealed 1279 potential lncRNA-messenger RNA (mRNA) pairs, among which 503 pairs function in cis-acting mode and 776 in trans. Functional enrichment analysis showed that the targets were significantly enriched in molecular functions related to plant hormone signal transduction, diterpenoid biosynthesis, and ascorbate/aldarate metabolism. Multiple promising targets of drought-responsive lncRNAs were discovered, including the SPL3 , BRI1 gene. Further analysis of genotypic difference in transcriptome we identified 10 drought-tolerance associated lncRNAs in XZ5, being exclusively induced by drought stress in XZ5 among the three genotypes, and sequencing data were confirmed by RT-qPCR. The lncRNA-mRNA interaction-based functional role showed these lncRNAs mediated the regulation of kinase mediated signal transduction, a serine/threonine-protein kinase SMG1 is likely responsible for drought tolerance in XZ5. These findings extend our knowledge of lncRNAs as important regulators in drought tolerance. [ABSTRACT FROM AUTHOR]

Cadmium (Cd) in the food chain poses a serious hazard to human health. Therefore, a greenhouse hydroponic experiment was conducted to examine the potential of exogenously strigolactone GR24 in lessening Cd toxicity and to investigate its physiological mechanisms in the two barley genotypes, W6nk2 (Cd-sensitive) and Zhenong8 (Cd-tolerant). Exogenous application of 1 μM GR24 (strigol analogue) reduced the suppression of growth caused by 10 μM Cd, lowered plant Cd contents, increased the contents of other nutrient elements, protected chlorophyll, sustained photosynthesis, and markedly reduced Cd-induced H 2 O 2 and malondialdehyde accumulation in barley. Furthermore, exogenous GR24 markedly increased NO contents and nitric oxide synthase activity in the Cd-sensitive genotype, W6nk2, effectively alleviating the Cd-induced repression of the activities of superoxide dismutase and peroxidase, increasing reduced glutathione (GSH) and ascorbic acid (AsA) pools and activities of AsA-GSH cycle including ascorbate peroxidase, glutathione peroxidase, glutathione reductase, dehydroascorbate reductase and monodehydroascorbate reductase. The findings of the present study indicate that GR24 could be a candidate for Cd detoxification by decreasing Cd contents, balancing nutrient elements, and protecting barley plants from toxic oxidation via indirectly eliminating reactive oxygen species (ROS), consequently contributing to reducing the potential risk of Cd pollution. Image 1 • External strigolactone GR24 significantly alleviates Cd-toxicity in barley. • GR24 reduced Cd content, improved photosynthesis performance and uptake of Zn, Cu, Mn and Fe. • GR24 alleviated Cd-induced oxidative stress, induced AsA/GSH, and activities of AsA-GSH cycle. • GR24 mitigated the Cd-induced depression of NO and NOS in Cd-sensitive genotype. • NO and AsA-GSH cycle involved in GR24-induced Cd tolerance in barley. [ABSTRACT FROM AUTHOR]

Drought and salinity are two of the most frequently co-occurring abiotic stresses. Despite recent advances in the elucidation of the effects of these stresses individually during the vegetative stage of plants, significant gaps exist in our understanding of the combined effects of these two frequently co-occurring stresses. Here, Tibetan wild barley XZ5 (drought tolerant), XZ16 (salt tolerant), and cultivated barley cv. CM72 (salt tolerant) were subjected to drought (D), salinity (S), or a combination of both treatments (D+S). Protein synthesis is one of the primary activities of the green part of the plant. Therefore, leaf tissue is an important parameter to evaluate drought and salinity stress conditions. Sixty differentially expressed proteins were identified by mass spectrometry (MALDI-TOF/TOF) and classified into 9 biological processes based on Gene Ontology annotation. Among them, 21 proteins were found to be expressed under drought or salinity alone; however, under D+S, 7 proteins, including S-adenosylmethionine synthetase 3 (SAMS3), were exclusively upregulated in drought-tolerant XZ5 but not in CM72. HvSAMS3 carries both N-terminal and central domains compared with Arabidopsis and activates the expression of several ethylene (ET)-responsive transcription factors. HvSAMS3 is mainly expressed in the roots and stems, and HvSAMS3 is a secretory protein located in the cell membrane and cytoplasm. Barley stripe mosaic virus-based virus-induced gene silencing (BSMV-VIGS) of HvSAMS3 in XZ5 severely compromised its tolerance to D+S and significantly reduced plant growth and K+ uptake. The reduced tolerance to the combined stress was associated with the inhibition of polyamines such as spermidine and spermine, polyamine oxidase, ethylene, biotin, and antioxidant enzyme activities. Furthermore, the exogenous application of ethylene and biotin improved the tolerance to D+S in BSMV-VIGS:HvSAMS3-inoculated plants. Our findings highlight the significance of HvSAMS3 in the tolerance to D+S in XZ5. [ABSTRACT FROM AUTHOR]

Drought stress is a major obstacle to agricultural production. Tibetan wild barley with rich genetic diversity is useful for drought-tolerant improvement of cereals. MicroRNAs (miRNAs) play critical roles in controlling gene expression in response to various environment perturbations in plants. However, the genome-wide expression profiles of miRNAs and their targets in response to drought stress are largely unknown in wild barley. In this study, a polyethylene glycol (PEG) induced drought stress hydroponic experiment was performed, and the expression profiles of miRNAs from the roots of two contrasting Tibetan wild barley genotypes XZ5 (drought-tolerant) and XZ54 (drought-sensitive), and one cultivated barley Tadmor (drought-tolerant) generated by high-throughput sequencing were compared. There were 69 conserved miRNAs and 1574 novel miRNAs in the dataset of three genotypes under control and drought conditions. Among them, seven conserved miRNAs and 36 novel miRNAs showed significantly genotype-specific expression patterns in response to drought stress. And 12 miRNAs were further regarded as drought tolerant associated miRNAs in XZ5, which mostly participate in gene expression, metabolism, signaling and transportation, suggesting that they and their target genes play important roles in plant drought tolerance. This is the first comparation study on the miRNA transcriptome in the roots of two Tibetan wild barley genotypes differing in drought tolerance and one drought tolerant cultivar in response to PEG treatment. Further results revealed the candidate drought tolerant miRNAs and target genes in the miRNA regulation mechanism in wild barley under drought stress. Our findings provide valuable understandings for the functional characterization of miRNAs in drought tolerance. [ABSTRACT FROM AUTHOR]

Tibetan wild barley has been identified to show large genetic variation and stress tolerance. A genome-wide association (GWA) analysis was performed to detect quantitative trait loci (QTLs) for drought tolerance using 777 Diversity Array Technology (DArT) markers and morphological and physiological traits of 166 Tibetan wild barley accessions in both hydroponic and pot experiments. Large genotypic variation for these traits was found; and population structure and kinship analysis identified three subpopulations among these barley genotypes. The average LD (linkage disequilibrium) decay distance was 5.16 cM, with the minimum on 6H (0.03 cM) and the maximum on 4H (23.48 cM). A total of 91 DArT markers were identified to be associated with drought tolerance-related traits, with 33, 26, 16, 1, 3, and 12 associations for morphological traits, H+K+-ATPase activity, antioxidant enzyme activities, malondialdehyde (MDA) content, soluble protein content, and potassium concentration, respectively. Furthermore, 7 and 24 putative candidate genes were identified based on the reference Meta-QTL map and by searching the Barleymap. The present study implicated that Tibetan annual wild barley from Qinghai–Tibet Plateau is rich in genetic variation for drought stress. The QTLs detected by genome-wide association analysis could be used in marker-assisting breeding for drought-tolerant barley genotypes and provide useful information for discovery and functional analysis of key genes in the future. [ABSTRACT FROM AUTHOR]

Cadmium (Cd) is one of the most harmful and widespread toxic heavy metal contaminants. Long non-coding RNAs (lncRNAs) play vital roles in response to abiotic stress by participating in the gene regulatory network. Here, we performed a transcriptome analysis of lncRNAs in roots of the two contrasting barley genotypes, namely Zhenong8 (ZN, Cd-tolerant) and W6nk2 (Cd-sensitive). In total, 9937 novel lncRNAs were identified, and 5758 lncRNA-target pairs were predicted to be cis-acting and 4159 to be trans-acting. Further comparison of genotypic differences revealed 195 novel lncRNAs as differentially expressed between the two genotypes in response to Cd stress. The miRanda prediction and expression relationship analyses among miRNA, lncRNA, and mRNA revealed that 8 lncRNAs may act as target mimics of miRNAs in barley. Among them, HvGAMYB was predicted to be the target gene of miR319a_1 and miR319b-3p, with LTCONS_00177709 and LTCONS_00185175 acting as endogenous competitors. Barley stripe mosaic virus-induced gene silencing (BSMV-VIGS) of HvGAMYB in ZN8 led to hypersensitivity to Cd stress with significantly increased Cd concentration in both roots and shoots as well as Cd accumulation in shoots. Moreover, silencing HvGAMYB suppressed photosynthesis and antioxidant-related genes but stimulated the production of reactive oxygen species (ROS) after Cd exposure, leading to exacerbated Cd-induced MDA accumulation and cell death. Our findings provide new knowledge and insights into the regulatory functions of lncRNAs in plant Cd tolerance. • Barley Cd-responsive lncRNAs firstly studied by comparing two barley genotypes ZN8 (Cd-tolerant) and W6nk2 (Cd-sensitive). • Identified 195 novel lncRNAs differently expressed in the two genotypes, including 21 Cd tolerance-associated lncRNAs. • lncRNA-miRNA-mRNA-based interaction analysis revealed 8 Cd tolerance-related key lncRNAs with targets such as HvGAMYB. • Silencing of HvGAMYB led to increased Cd concentration in barley and hypersensitivity to Cd stress. [ABSTRACT FROM AUTHOR]