HvHOX9, a novel homeobox leucine zipper transcription factor, positively regulates aluminum tolerance in Tibetan wild barley.

Aluminum (Al) toxicity is the primary limiting factor of crop production on acid soils. Tibetan wild barley germplasm is a valuable source of potential genes for breeding barley with acid and Al tolerance. We performed microRNA and RNA sequencing using wild (XZ16, Al-tolerant; XZ61, Al-sensitive) and cultivated (Dayton, Al-tolerant) barley. A novel homeobox-leucine zipper transcription factor, HvHOX9, was identified as a target gene of miR166b and functionally characterized. HvHOX9 was up-regulated by Al stress in XZ16 (but unchanged in XZ61 and Dayton) and was significantly induced only in root tip. Phylogenetic analysis showed that HvHOX9 is most closely related to wheat TaHOX9 and orthologues of HvHOX9 are present in the closest algal relatives of Zygnematophyceae. Barley stripe mosaic virus-induced gene silencing of HvHOX9 in XZ16 led to significantly increased Al sensitivity but did not affect its sensitivity to other metals and low pH. Disruption of HvHOX9 did not change Al concentration in the root cell sap, but led to more Al accumulation in root cell wall after Al exposure. Silencing of HvHOX9 decreased H+ influx after Al exposure. Our findings suggest that miR166b/HvHOX9 play a critical role in Al tolerance by decreasing root cell wall Al binding and increasing apoplastic pH for Al detoxification in the root.

Identification of microRNAs in response to aluminum stress in the roots of Tibetan wild barley and cultivated barley.

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.

Association mapping for total polyphenol content, total flavonoid content and antioxidant activity in barley.

BACKGROUND: The interest has been increasing on the phenolic compounds in plants because of their nutritive function as food and the roles regulating plant growth. However, their underlying genetic mechanism in barley is still not clear. RESULTS: A genome-wide association study (GWAS) was conducted for total phenolic content (TPC), total flavonoid content (FLC) and antioxidant activity (AOA) in 67 cultivated and 156 Tibetan wild barley genotypes. Most markers associated with phenolic content were different in cultivated and wild barleys. The markers bPb-0572 and bPb-4531 were identified as the major QTLs controlling phenolic compounds in Tibetan wild barley. Moreover, the marker bPb-4531 was co-located with the UDP- glycosyltransferase gene (HvUGT), which is a homolog to Arabidopsis UGTs and involved in biosynthesis of flavonoid glycosides . CONCLUSIONS: GWAS is an efficient tool for exploring the genetic architecture of phenolic compounds in the cultivated and Tibetan wild barleys. The DArT markers applied in this study can be used in barley breeding for developing new barley cultivars with higher phenolics content. The candidate gene (HvUGT) provides a potential route for deep understanding of the molecular mechanism of flavonoid synthesis.

Tolerance to Drought, Low pH and Al Combined Stress in Tibetan Wild Barley Is Associated with Improvement of ATPase and Modulation of Antioxidant Defense System.

Aluminum (Al) toxicity and drought are two major constraints on plant growth in acidic soils, negatively affecting crop performance and yield. Genotypic differences in the effects of Al/low pH and polyethyleneglycol (PEG) induced drought stress, applied either individually or in combination, were studied in Tibetan wild (XZ5, drought-tolerant; XZ29, Al-tolerant) and cultivated barley (Al-tolerant Dayton; drought-tolerant Tadmor). Tibetan wild barley XZ5 and XZ29 had significantly higher H⁺-ATPase, Ca2+Mg2+-ATPase, and Na⁺K⁺-ATPase activities at pH 4.0+Al+PEG than Dayton and Tadmor. Moreover, XZ5 and XZ29 possessed increased levels in reduced ascorbate and glutathione under these conditions, and antioxidant enzyme activities were largely stimulated by exposure to pH 4.0+PEG, pH 4.0+Al, and pH 4.0+Al+PEG, compared to a control and to Dayton and Tadmor. The activity of methylglyoxal (MG) was negatively correlated with increased levels of glyoxalase (Gly) I and Gly II in wild barley. Microscopic imaging of each genotype revealed DNA damage and obvious ultrastructural alterations in leaf cells treated with drought or Al alone, and combined pH 4.0+Al+PEG stress; however, XZ29 and XZ5 were less affected than Dayton and Tadmor. Collectively, the authors findings indicated that the higher tolerance of the wild barley to combined pH 4.0+Al+PEG stress is associated with improved ATPase activities, increased glyoxalase activities, reduced MG, and lower reactive oxygen species levels (like O2- and H2O2) due to increased antioxidant enzyme activities. These results offer a broad comprehension of the mechanisms implicated in barley's tolerance to the combined stress of Al/low pH and drought, and may provide novel insights into the potential utilization of genetic resources, thereby facilitating the development of barley varieties tolerant to drought and Al/low pH stress.

Physiological and molecular analysis on root growth associated with the tolerance to aluminum and drought individual and combined in Tibetan wild and cultivated barley.

MAIN CONCLUSION: The drought-stimulated gene expression of NCED, SUS, and KS - DHN and ABA signal cross-talk with other phytohormones maintains barley root growth under drought stress at pH 4.0 plus polyethylene glycol plus aluminum. Aluminum (Al) toxicity and drought are two major factors that limit barley production. In this work, the individual and combined effects of Al/acid and polyethylene glycol (PEG 6000) induced drought stress that suppressed root growth and caused oxidative damage as characterized by increased H2O2 and O2(.-) accumulation. The wild-barley genotypes, XZ5 and XZ29, exhibited a higher tolerance than the two cultivars Dayton (Al tolerant) and Tadmor (drought tolerant) under combined stress (pH 4.0 + PEG + Al). The oxidative damage induced by PEG was more severe at pH 4.0 than at pH 6.0. In XZ29, the highest root secretion of malate and citrate was recorded, and the least Al uptake in the four genotypes. In XZ5, a peak accumulation of ABA and minor synthesis of zeatin riboside and ethylene were found being essential in maintaining primary root elongation and root hair development. PEG-induced drought stress repressed Al uptake in root tips, with a lower increase in callose formation and HvMATE (Hordeum vulgare multidrug and toxic compound exudation) expression compared to Al-induced callose production. Stress by pH 4.0 + PEG + Al up-regulated 9-cis-epoxycarotenoid dioxygenase (NCED) which is involved in ABA biosynthesis. Such treatment stimulated the regulation of ABA-dependent genes sucrose synthase (SUS) and KS-type dehydrin (KS-DHN) in root tips. Our results suggest that the tolerance ranking to pH 4.0 + PEG + Al stress in Tibetan wild barley by gene expression is closely correlated to physiological indices. The results show that acclimatisation to pH 4.0 + PEG + Al stress involves specific responses in XZ5 and XZ29. The present study provides insights into the effects of Al/acid and drought combined stress on the abundance of physiological indices in the roots of barley varieties.

HvEXPB7, a novel ß-expansin gene revealed by the root hair transcriptome of Tibetan wild barley, improves root hair growth under drought stress.

Tibetan wild barley is a treasure trove of useful genes for crop improvement including abiotic stress tolerance, like drought. Root hair of single-celled structures plays an important role in water and nutrition uptake. Polyethylene-glycol-induced drought stress hydroponic/petri-dish experiments were performed, where root hair morphology and transcriptional characteristics of two contrasting Tibetan wild barley genotypes (drought-tolerant XZ5 and drought-sensitive XZ54) and drought-tolerant cv. Tadmor were compared. Drought-induced root hair growth was only observed in XZ5. Thirty-six drought tolerance-associated genes were identified in XZ5, including 16 genes specifically highly expressed in XZ5 but not Tadmor under drought. The full length cDNA of a novel ß-expansin gene (HvEXPB7), being the unique root hair development related gene in the identified genes, was cloned. The sequence comparison indicated that HvEXPB7 carried both DPBB_1 and Pollon_allerg_1 domains. HvEXPB7 is predominantly expressed in roots. Subcellular localization verified that HvEXPB7 is located in the plasma membrane. Barley stripe mosaic virus induced gene silencing (BSMV-VIGS) of HvEXPB7 led to severely suppressed root hairs both under control and drought conditions, and significantly reduced K uptake. These findings highlight and confer the significance of HvEXPB7 in root hair growth under drought stress in XZ5, and provide a novel insight into the genetic basis for drought tolerance in Tibetan wild barley.

Genome resequencing and transcriptome profiling reveal molecular evidence of tolerance to water deficit in barley.

INTRODUCTION: 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. OBJECTIVES: To identify the genetic basis and unique mechanisms for drought tolerance in Tibetan wild barley. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

The Barley S-Adenosylmethionine Synthetase 3 Gene HvSAMS3 Positively Regulates the Tolerance to Combined Drought and Salinity Stress in Tibetan Wild Barley.

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.

Ionomic, metabolomic and proteomic analyses reveal molecular mechanisms of root adaption to salt stress in Tibetan wild barley.

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.

Ionomic and physiological responses to low nitrogen stress in Tibetan wild and cultivated barley.

In a previous study, we identified the low-nitrogen (LN) tolerant accessions from the Tibetan wild barley (Hordeum vulgare subsp. spontaneum). In this study, two wild barley genotypes (XZ149, LN-tolerant and XZ56, LN-sensitive) and a barley cultivar ZD9 (H. vulgare) were used to determine the LN tolerant mechanism underlying the wild barley in the ionomic and physiological aspects. XZ149 exhibited higher LN tolerance with highest relative dry weight and N accumulation among three barley genotypes under LN stress. When exposed to LN stress, XZ149 had more N transportation from roots to leaves, and remained relatively higher activities of nitrate reductase (NR, EC.1.7.1.1) and glutamine synthetase (GS, EC.6.3.1.2) in leaves than other two genotypes, ensuring its higher capacity of N assimilation and utilization. The ionome analysis showed that LN stress had a significant effect on tissue ionome and the effect was genotypic and tissue-specific difference. On the whole, XZ149 maintained more stable Mn and Cu contents in roots, and less reduction of root P, K and Ca contents than XZ56 and ZD9 when exposed to LN stress. It may be assumed that more N movement into shoots, greater N assimilating capacity and specific rearrangement of nutrient element levels in tissues under LN stress are attributed to LN tolerance in XZ149.

Metabolic analysis of two contrasting wild barley genotypes grown hydroponically reveals adaptive strategies in response to low nitrogen stress.

Nitrogen (N) is an essential macronutrient for plants. The increasingly severe environmental problems caused by N fertilizer application urge alleviation of N fertilizer dependence in crop production. In previous studies, we identified the Tibetan wild barley accessions with high tolerance to low nitrogen (LN). In this study, metabolic analysis was done on two wild genotypes (XZ149, tolerant and XZ56, sensitive) to understand the mechanism of LN tolerance, using a hydroponic experiment. Leaf and root samples were taken at seven time points within 18 d after LN treatment, respectively. XZ149 was much less affected by low N stress than XZ56 in plant biomass. A total of 51 differentially accumulated metabolites were identified between LN and normal N treated plants. LN stress induced tissue-specific changes in carbon and nitrogen partitioning, and XZ149 had a pattern of energy-saving amino acids accumulation and carbon distribution in favor of root growth that contribute to its higher LN tolerance. Moreover, XZ149 is highly capable of producing energy and maintaining the redox homeostasis under LN stress. The current results revealed the mechanisms underlying the wild barley in high LN tolerance and provided the valuable references for developing barley cultivars with LN tolerance.

Identification of Quantitative Trait Loci for the Phenolic Acid Contents and Their Association with Agronomic Traits in Tibetan Wild Barley.

Phenolic acids have been of considerable interest in human nutrition because of their strong antioxidative properties. However, even in a widely grown crop, such as barley, their genetic architecture is still unclear. In this study, genetic control of two main phenolic acids, ferulic acid (FA) and p-coumaric acid (p-CA), and their associations with agronomic traits were investigated among 134 Tibetan wild barley accessions. A genome-wide association study (GWAS) identified three DArT markers (bpb-2723, bpb-7199, and bpb-7273) associated with p-CA content and one marker (bpb-3653) associated with FA content in 2 consecutive years. The contents of the two phenolic acids were positively correlated with some agronomic traits, such as the first internode length, plant height, and some grain color parameters, and negatively correlated with the thousand-grain weight (TGW). This study provides DNA markers for barley breeding programs to improve the contents of phenolic acids.

Genetic diversity and QTL mapping of thermostability of limit dextrinase in barley.

Limit dextrinase (LD) is an essential amylolytic enzyme for the complete degradation of starch, and it is closely associated with malt quality. A survey of 51 cultivated barley and 40 Tibetan wild barley genotypes showed a wide genetic diversity of LD activity and LD thermostability. Compared with cultivated barley, Tibetan wild barley showed lower LD activity and higher LD thermostability. A doubled haploid population composed of 496 DArT and 28 microsatellite markers was used for mapping Quantitative Trait Loci (QTLs). Parental line Yerong showed low LD activity and high LD thermostability, but Franklin exhibited high LD activity and low LD thermostability. Three QTLs associated with thermostable LD were identified. The major QTL is close to the LD gene on chromosome 7H. The two minor QTLs colocalized with previously reported QTLs determining malt-extract and diastatic power on chromosomes 1H and 2H, respectively. These QTLs may be useful for a better understanding of the genetic control of LD activity and LD thermostability in barley.

Differences in physiological features associated with aluminum tolerance in Tibetan wild and cultivated barleys.

Aluminum (Al) toxicity is a major limiting factor for plant production in acid soils. Wild barley germplasm is a treasure trove of useful genes and offers rich sources of genetic variation for crop improvement. Al-stress-hydroponic-experiments were performed, and the physiochemical characteristic of two contrasting Tibetan wild barley genotypes (Al-resistant XZ16 and Al-sensitive XZ61) and Al-resistant cv. Dayton were compared. Ultrastructure of chloroplasts and root cells in XZ16 was less injured than that in Dayton and XZ61. Moreover, XZ16 secreted significantly more malate besides citrate and exhibited less Al uptake and distribution than both of XZ61 and Dayton in response to Al stress, simultaneously maintained higher H⁺-, Ca²âºMg²âº- and total-ATPase activities over XZ61. The protein synthesis inhibitor cycloheximide reduced citrate secretion from XZ16, but not from Dayton. In Tibetan wild barley, our findings highlight the significant correlations between Al tolerance, ATPase activity and citrate secretion, providing some insights into the physiological basis for Al-detoxification.

Differential changes in grain ultrastructure, amylase, protein and amino acid profiles between Tibetan wild and cultivated barleys under drought and salinity alone and combined stress.

Grain phytochemical profiles were compared in Tibetan wild barley XZ5 (drought-tolerant), XZ16 (salinity/aluminum-tolerant) and cv CM72 (salinity-tolerant) in response to drought and salinity alone and combination (D+S) during anthesis. Total antioxidant capacity assessed by determining ferric-reducing antioxidant potential (FRAP) in grains increased significantly as follows: D+S>drought>salinity, and XZ5>XZ16>CM72. A marked increase in the total phenol (TP) from individual and combined stresses was observed in XZ5, while a decrease occurred in CM72. Moreover, the activity of α-/ß-amylase in the grains under combined stress was 81.8%/16.9% in XZ5 and 48.6%/18.7% in XZ16 higher than that of CM72. Increases in amino acids, protein content and protein fractions of albumin, globulin, hordein and glutelin were maximised under D+S, with larger values in the Tibetan wild genotypes. Observation with a scanning electron microscopy showed a distinct genotypic difference under D+S; for example, XZ5 and XZ16 maintained a relatively integral starch granule with a greater protein deposit/matrix, while CM72 degraded by pitting. This research expands our understanding of barley drought and salt-tolerance mechanisms and provides possibility of Tibetan wild barley in developing barley cultivars with both tolerance to drought and salinity.