The genome and gene editing system of sea barleygrass provide a novel platform for cereal domestication and stress tolerance studies.

The tribe Triticeae provides important staple cereal crops and contains elite wild species with wide genetic diversity and high tolerance to abiotic stresses. Sea barleygrass (Hordeum marinum Huds.), a wild Triticeae species, thrives in saline marshlands and is well known for its high tolerance to salinity and waterlogging. Here, a 3.82-Gb high-quality reference genome of sea barleygrass is assembled de novo, with 3.69 Gb (96.8%) of its sequences anchored onto seven chromosomes. In total, 41 045 high-confidence (HC) genes are annotated by homology, de novo prediction, and transcriptome analysis. Phylogenetics, non-synonymous/synonymous mutation ratios (Ka/Ks), and transcriptomic and functional analyses provide genetic evidence for the divergence in morphology and salt tolerance among sea barleygrass, barley, and wheat. The large variation in post-domestication genes (e.g. IPA1 and MOC1) may cause interspecies differences in plant morphology. The extremely high salt tolerance of sea barleygrass is mainly attributed to low Na+ uptake and root-to-shoot translocation, which are mainly controlled by SOS1, HKT, and NHX transporters. Agrobacterium-mediated transformation and CRISPR/Cas9-mediated gene editing systems were developed for sea barleygrass to promote its utilization for exploration and functional studies of hub genes and for the genetic improvement of cereal crops.

Transcriptome-wide m6A methylation profile reveals regulatory networks in roots of barley under cadmium stress.

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 CdCl2 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.

Vacuolar H+-pyrophosphatase HVP10 enhances salt tolerance via promoting Na+ translocation into root vacuoles.

Vacuolar H+-pumping pyrophosphatases (VPs) provide a proton gradient for Na+ sequestration in the tonoplast; however, the regulatory mechanisms of VPs in developing salt tolerance have not been fully elucidated. Here, we cloned a barley (Hordeum vulgare) VP gene (HVP10) that was identified previously as the HvNax3 gene. Homology analysis showed VP10 in plants had conserved structure and sequence and likely originated from the ancestors of the Ceramiales order of Rhodophyta (Cyanidioschyzon merolae). HVP10 was mainly expressed in roots and upregulated in response to salt stress. After salt treatment for 3 weeks, HVP10 knockdown (RNA interference) and knockout (CRISPR/Cas9 gene editing) barley plants showed greatly inhibited growth and higher shoot Na+ concentration, Na+ transportation rate and xylem Na+ loading relative to wild-type (WT) plants. Reverse transcription quantitative polymerase chain reaction and microelectronic Ion Flux Estimation results indicated that HVP10 likely modulates Na+ sequestration into the root vacuole by acting synergistically with Na+/H+ antiporters (HvNHX1 and HvNHX4) to enhance H+ efflux and K+ maintenance in roots. Moreover, transgenic rice (Oryza sativa) lines overexpressing HVP10 also showed higher salt tolerance than the WT at both seedling and adult stages with less Na+ translocation to shoots and higher grain yields under salt stress. This study reveals the molecular mechanism of HVP10 underlying salt tolerance and highlights its potential in improving crop salt tolerance.

Identification of microRNAs Responding to Aluminium, Cadmium and Salt Stresses in Barley Roots.

Plants are frequently exposed to various abiotic stresses, including aluminum, cadmium and salinity stress. Barley (Hordeum vulgare) displays wide genetic diversity in its tolerance to various abiotic stresses. In this study, small RNA and degradome libraries from the roots of a barley cultivar, Golden Promise, treated with aluminum, cadmium and salt or controls were constructed to understand the molecular mechanisms of microRNAs in regulating tolerance to these stresses. A total of 525 microRNAs including 198 known and 327 novel members were identified through high-throughput sequencing. Among these, 31 microRNAs in 17 families were responsive to these stresses, and Gene Ontology (GO) analysis revealed that their targeting genes were mostly highlighted as transcription factors. Furthermore, five (miR166a, miR166a-3p, miR167b-5p, miR172b-3p and miR390), four (MIR159a, miR160a, miR172b-5p and miR393) and three (miR156a, miR156d and miR171a-3p) microRNAs were specifically responsive to aluminum, cadmium and salt stress, respectively. Six miRNAs, i.e., miR156b, miR166a-5p, miR169a, miR171a-5p, miR394 and miR396e, were involved in the responses to the three stresses, with different expression patterns. A model of microRNAs responding to aluminum, cadmium and salt stresses was proposed, which may be helpful in comprehensively understanding the mechanisms of microRNAs in regulating stress tolerance in barley.

GWAS and transcriptomic integrating analysis reveals key salt-responding genes controlling Na+ content in barley roots.

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.

Transcriptome analysis reveals the tolerant mechanisms to cobalt and copper in barley.

Cobalt (Co) and copper (Cu) co-exist commonly in the contaminated soils and at excessive levels, they are toxic to plants. However, their joint effect and possible interaction have not been fully addressed. In this work, a hydroponic experiment was performed to investigate the combined effects of Co and Cu on two barley genotypes at transcriptional level by RNA-seq analysis. The results identified 358 genes inclusively expressed in both genotypes under single and combined treatments of Co and Cu, with most of them being related to metal transport, stress response and transcription factor. The combined treatment induced more differently expressed genes (DEGs) than the single treatment, with Yan66, a metal tolerant genotype having more DEGs than Ea52, a sensitive genotype. The pathways associated with anthocyanin biosynthesis, MAPK signaling, glutathione biosynthesis, phenylalanine metabolism, photosynthesis, arginin biosynthesis, fatty acid elongation, and plant hormone signal transduction biosynthesis were induced and inhibited in Yan66 and Ea52, respectively. Furthermore, flavonoid biosynthesis was much more largely enhanced and accordingly more free flavonoid components (naringin, narirutin and neohesperidin) were accumulated in Yan66 than in Ea52. It may be suggested that high tolerance to both Co and Cu in Yan66 is attributed to its high gene regulatory ability.

The Influence of Nitrogen Application Level on Eating Quality of the Two Indica-Japonica Hybrid Rice Cultivars.

Indica-japonica hybrid rice cultivars show great yield potential but poor eating quality and require more nitrogen (N) input relative to japonica rice. However, the effect of N levels on the eating quality of indica-japonica hybrid rice is little known. A field experiment was carried out to investigate the effects of four N levels on two indica-japonica hybrid rice cultivars (Yongyou12 and Yongyou17) differing in eating quality. The results showed that the contents of amylose chains and water-insoluble storage proteins, especially prolamin, increased largely under a high N level, leading to deterioration of the rice-eating quality, although a low N level (100 N kg/ha) had a less negative effect on the eating quality. Moreover, both of the indica-japonica hybrids had high ratios of inferior grains (IG), and the ratio of IG increased with the N level. Grain weight and the immature ratio of IG were reduced and increased with the N level, respectively, which are also factors for deterioration of the eating quality. The two cultivars differed greatly in the responses of eating quality to the N level, with Yongyou17 being more sensitive than Yongyou12. The current results indicated that a high N level deteriorates the eating quality of indica-japonica hybrid rice mainly due to a large increase of IG.

High accumulation of phenolics and amino acids confers tolerance to the combined stress of cobalt and copper in barley (Hordeum vulagare).

Cobalt (Co) and copper (Cu) co-exist in the metal contaminated soils and cause the serious toxicity to crops, while their interactive effect on plant growth and development is still poorly understood. In this work, a hydroponic experiment was carried out to reveal the interactive effect of Co and Cu on photosynthesis and metabolite profiles of two barley genotypes differing in metal tolerance. The results showed that both single and combined treatments of Co and Cu caused a significant reduction in chlorophyll content and photosynthetic rate of the two barley (Hordeum vulgare) genotypes, with the effect being greater for the combined treatment and the sensitive genotype (Ea52) being more affected than the tolerant genotype (Yan66). Compared to Cu or Co treatment alone, the combined treatment significantly increased the levels of phenolic components, including cinnamic derivatives (caffeic, chlorogenic, ferullic, p-coumaric); benzoic derivatives (p-hydroxybenzoic, vanillic, syringic, sallicilic, protocatechuic acid) as well as free amino acids, with Yan66 having more accumulation than Ea52. Meanwhile, under the combined treatment, the phenylalanine ammonialyase-related gene (HvPAL) was highly regulated along with the genes involved in the synthesis of malate (HvMDH) and citrate (HvCSY), with Ya66 showing the higher expression of these genes than Ea52. It can be concluded that higher Cu and Co stress tolerance in Yan66 is attributed to more accumulation of the metabolites including phenolics and amino acids.

Calmodulin HvCaM1 Negatively Regulates Salt Tolerance via Modulation of HvHKT1s and HvCAMTA4.

Calcium (Ca2+) signaling modulates sodium (Na+) transport in plants; however, the role of the Ca2+ sensor calmodulin (CaM) in salt tolerance is elusive. We previously identified a salt-responsive calmodulin (HvCaM1) in a proteome study of barley (Hordeum vulgare) roots. Here, we employed bioinformatic, physiological, molecular, and biochemical approaches to determine the role of HvCaM1 in barley salt tolerance. CaM1s are highly conserved in green plants and probably originated from ancestors of green algae of the Chlamydomonadales order. HvCaM1 was mainly expressed in roots and was significantly up-regulated in response to long-term salt stress. Localization analyses revealed that HvCaM1 is an intracellular signaling protein that localizes to the root stele and vascular systems of barley. After treatment with 200 mm NaCl for 4 weeks, HvCaM1 knockdown (RNA interference) lines showed significantly larger biomass but lower Na+ concentration, xylem Na+ loading, and Na+ transportation rates in shoots compared with overexpression lines and wild-type plants. Thus, we propose that HvCaM1 is involved in regulating Na+ transport, probably via certain class I high-affinity potassium transporter (HvHKT1;5 and HvHKT1;1)-mediated Na+ translocation in roots. Moreover, we demonstrated that HvCaM1 interacted with a CaM-binding transcription activator (HvCAMTA4), which may be a critical factor in the regulation of HKT1s in barley. We conclude that HvCaM1 negatively regulates salt tolerance, probably via interaction with HvCAMTA4 to modulate the down-regulation of HvHKT1;5 and/or the up-regulation of HvHKT1;1 to reduce shoot Na+ accumulation under salt stress in barley.

Physiological and molecular mechanisms of cobalt and copper interaction in causing phyto-toxicity to two barley genotypes differing in Co tolerance.

The combined effects of cobalt (Co) and copper (Cu) in their toxicity to plants is poorly studied although these two metals co-exist commonly in soil. In this study, a hydroponic experiment was carried out to investigate the effect of longer exposure of two barley genotypes differing in Co tolerance to the combined Co and Cu stress. The results confirmed the previous findings that Co accumulation in plant tissues was reduced by Cu presence, while Cu accumulation was stimulated by Co presence. Moreover, both single and combined treatments of Co and Cu reduced the mineral (Mn, Zn and K) uptake. Co and Cu applied alone or in combination at rate of 50 µM resulted in the significant reduction of plant growth and increase of oxidative stress (ROS and MDA), and meanwhile the capacity of scavenging active oxygen species (AOS) was enhanced, reflected by increased phytochelatin (PC) and glutathione (GSH and GSSG) content, as well as expression of the related genes (HvPCS1 and HvGR1). Yan66, a Co tolerant genotype was less affected in oxidative stress, and had higher AOS scavenging capacity in comparison with Ea52, a Co sensitive one. Among three HvSOD isoforms, only HvFeSOD expression was up-regulated in the combined treatment relative to control as well as the treatment of Co or Cu alone, while HvCuZnSOD and HvMnSOD were down-regulated and unaffected, respectively. In addition, the expressions of metal transporter genes (HvHMA2, HvHMA3 and HvHMA5) varied with genotype and metal treatments, with the extent being greater in Yan66 on the whole. The results suggest that upon longer exposure to the combined stress of Co and Cu, the greater phyto-toxicity than each element alone is associated with more Cu accumulation stimulated by Co and that, the higher regulation of transporter genes observed in Yan66 could in part explain for its higher metal tolerance ability.

OsC2DP, a Novel C2 Domain-Containing Protein Is Required for Salt Tolerance in Rice.

Salt stress is one of the major factors limiting crop production globally, including rice (Oryza sativa). Although a number of genes involved in salt tolerance have been functionally identified, the mechanism underlying salt tolerance in rice is still poorly understood. Here, we reported a novel C2 domain-containing protein, OsC2DP required for salt tolerance in rice. OsC2DP was predominately expressed in the roots and its expression was repressed by salt stress. Transient expression of OsC2DP in rice protoplast cells showed that it was localized in the cytosol. Immunostaining further showed that OsC2DP was able to translocate from the cytosol to plasma membrane under salt conditions. Knockout of OsC2DP did not affect Na+ concentration in the roots, but increased shoot Na+ concentration, resulting in a significant sensitivity of rice to salt stress. Furthermore, the quantitative Real-time PCR and transcriptomic analysis showed that the expression level of some genes related to salt tolerance were indirectly regulated by OsC2DP, especially OsSOS1 and OsNHX4. These results indicate that OsC2DP has an important role in salt tolerance and these findings provide new insights into the regulation of OsC2DP gene for rice breeding with high salt tolerance.

Copper alleviates cobalt toxicity in barley by antagonistic interaction of the two metals.

Cobalt (Co) commonly co-exists with copper (Cu) in natural soils, but the information about their combined effects on plants is poorly available. In this study, we hydroponically investigated the combined effects of Co and Cu on two barley genotypes differing in Co toxicity tolerance to reveal the interaction pattern of these two metals. The results showed that single treatment of Co or Cu at the dose of 100 µM led to a significant decrease of growth and photosynthetic rate, and a significant increase of lipid peroxidation, ROS radicals as well as anti-oxidative enzyme (SOD, CAT and GR) activities and glutathione content, with the extent of effect being less in Yan66 than Ea52. The combined treatment of Co and Cu alleviated the toxicity of both metals in comparison with each metal treatment alone, as reflected by improved growth and photosynthesis, and much slight oxidative stress. The alleviation of metal toxicity upon combined treatment is mainly attributed to a drastic reduction of Co uptake and its translocation from roots to shoots. It may be suggested that interaction of Co and Cu on their uptake and movement in plants is antagonistic.

Metabolite profiling and gene expression of Na/K transporter analyses reveal mechanisms of the difference in salt tolerance between barley and rice.

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.

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.

Alleviating effects of calcium on cobalt toxicity in two barley genotypes differing in cobalt tolerance.

Cobalt (Co) contamination in soils is becoming a severe issue in environment safety and crop production. Calcium (Ca), as a macro-nutrient element, shows the antagonism with many divalent heavy metals and the capacity of alleviating oxidative stress in plants. In this study, the protective role of Ca in alleviating Co stress was hydroponically investigated using two barley genotypes differing in Co toxicity tolerance. Barley seedlings exposed to 100µM Co showed the significant reduction in growth and photosynthetic rate, and the dramatic increase in the contents of reactive oxygen species (ROS), malondialdehyde (MDA), reduced glutathione (GSH) and oxidized glutathione (GSSG), and the activities of anti-oxidative enzymes, with Ea52 (Co-sensitive) being much more affected than Yan66 (Co-tolerant). Addition of Ca in growth medium alleviated Co toxicity by reducing Co uptake and enhancing the antioxidant capacity. The effect of Ca in alleviating Co toxicity was much greater in Yan66 than in Ea52. The results indicate that the alleviation of Co toxicity in barley plants by Ca is attributed to the reduced Co uptake and enhanced antioxidant capacity.

Multi-omics analysis reveals molecular mechanisms of shoot adaption to salt stress in Tibetan wild barley.

BACKGROUND: Tibetan wild barley (Hordeum spontaneum L.) has been confirmed to contain elite accessions in tolerance to abiotic stresses, including salinity. However, molecular mechanisms underlying genotypic difference of salt tolerance in wild barley are unknown. RESULTS: In this study, two Tibetan wild barley accessions (XZ26 and XZ169), differing greatly in salt tolerance, were used to determine changes of ionomic, metabolomic and proteomic profiles in the shoots exposed to salt stress at seedling stage. Compared with XZ169, XZ26 showed better shoot growth and less Na accumulation after 7 days treatments. Salt stress caused significant reduction in concentrations of sucrose and metabolites involved in glycolysis pathway in XZ169, and elevated level of tricarboxylic acid (TCA) cycle, as reflected by up-accumulation of citric acid, aconitic acid and succinic acid, especially under high salinity, but not in XZ26. Correspondingly, proteomic analysis further proved the findings from the metabolomic study. CONCLUSION: XZ26 maintained a lower Na concentration in the shoots and developed superior shoot adaptive strategies to salt stress. The current result provides possible utilization of Tibetan wild barley in developing barley cultivars for salt tolerance.