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Ferns appear in the fossil record some 200 Myr before angiosperms. However, as angiosperm‐dominated forest canopies emerged in the Cretaceous period there was an explosive diversification of modern (leptosporangiate) ferns, which thrived in low, blue‐enhanced light beneath angiosperm canopies. A mechanistic explanation for this transformative event in the diversification of ferns has remained elusive. We used physiological assays, transcriptome analysis and evolutionary bioinformatics to investigate a potential connection between the evolution of enhanced stomatal sensitivity to blue light in modern ferns and the rise of angiosperm‐dominated forests in the geological record. We demonstrate that members of the largest subclade of leptosporangiate ferns, Polypodiales, have significantly faster stomatal response to blue light than more ancient fern lineages and a representative angiosperm. We link this higher sensitivity to levels of differentially expressed genes in blue‐light signaling, particularly in the cryptochrome (CRY) signaling pathway. Moreover, CRYs of the Polypodiales examined show gene duplication events between 212.9–196.9 and 164.4–151.8 Ma, when angiosperms were emerging, which are lacking in other major clades of extant land plants. These findings suggest that evolution of stomatal blue‐light sensitivity helped modern ferns exploit the shady habitat beneath angiosperm forest canopies, fueling their Cretaceous hyperdiversification.

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., Two wild barley populations adapt to hot‐dry Terra Rossa soil, and abutting moist fungi‐rich Basalt soil at the microsite “Evolution Slope”, Tabigha, Israel. Sugar metabolism facilitates edaphic adaptation of wild barley to dry Terra Rossa soil, and phenylpropanoid/phenolamide biosynthesis enables the edaphic adaptation of wild barley to moist and fungi‐rich Basalt soil at epigenomic, genomic, transcriptomic, and metabolomic levels.

Phosphite (PHI) has been used in the management of Phytophthora diseases since the 1970s.We assessed the effect of PHI on controlling the incidence of Xanthomonas oryzae pv.oryzae and Pyricularia grisea. As a result, PHI application significantly inhibited the incidence of the diseases. To clarify the molecular mechanism underlying this, a transcriptome study was employed. In total, 2064 differentially expressed genes (DEGs) were identified between control and PHI treatment. The key DEGs could be classified into phenylpropanoid biosynthesis (ko00940), starch and sucrose metabolism (ko00500), and plant hormone signal transduction (ko04075). The expressions of defense-related genes had a higher expression lever upon PHI treatment. This study provides new insights into the mechanism of protection effect of PHI against pathogens.

Plants will meet various abiotic stresses during their growth and development. One of the important strategies for plants to deal with the stress is involved in metabolic regulation, causing the dramatic changes of metabolite profiles. Metabolomic studies have been intensively conducted to reveal the responses of plants to abiotic stress, but most of them were limited to one or at most two abiotic stresses in a single experiment. In this study, we compared the metabolite profiles of barley seedlings exposed to seven abiotic stresses, including drought, salt stress, aluminum (Al), cadmium (Cd), deficiency of nitrogen (N), phosphorus (P) and potassium (K). The results showed that metabolite profiles of barley under these stresses could be classified into three groups: osmotic stresses (drought and salt); metal stresses (Al and Cd) and nutrient deficiencies (N, P and K deficiencies). Compared with the control, some metabolites (including polyamines, raffinose and pipecolic acid) in plants exposed to all abiotic stresses changed significantly, while some other metabolites showed the specific change only under a certain abiotic stress, such as proline being largely increased by osmotic stress (drought and salinity), the P-containing metabolites being largely decreased under P deficiency, some amino acids (lysine, tyrosine, threonine, ornithine, glutamine and so on) showing the dramatic reduction in the plants exposed to N deficiencies, respectively. The current meta-analysis obtained a comprehensive view on the metabolic responses to various abiotic stress, and improved the understanding of the mechanisms for tolerance of barley to abiotic stress.

Stronger Na+ extrusion and vacuolar sequestration are essential to confer better salt tolerance in bread wheat than in durum wheat. Removal of the root meristems increased salt sensitivity in wheat., The progress in plant breeding for salinity stress tolerance is handicapped by the lack of understanding of the specificity of salt stress signalling and adaptation at the cellular and tissue levels. In this study, we used electrophysiological, fluorescence imaging, and real-time quantitative PCR tools to elucidate the essentiality of the cytosolic Na+ extrusion in functionally different root zones (elongation, meristem, and mature) in a large number of bread and durum wheat accessions. We show that the difference in the root’s ability for vacuolar Na+ sequestration in the mature zone may explain differential salinity stress tolerance between salt-sensitive durum and salt-tolerant bread wheat species. Bread wheat genotypes also had on average 30% higher capacity for net Na+ efflux from the root elongation zone, providing the first direct evidence for the essentiality of the root salt exclusion trait at the cellular level. At the same time, cytosolic Na+ accumulation in the root meristem was significantly higher in bread wheat, leading to the suggestion that this tissue may harbour a putative salt sensor. This hypothesis was then tested by investigating patterns of Na+ distribution and the relative expression level of several key genes related to Na+ transport in leaves in plants with intact roots and in those in which the root meristems were removed. We show that tampering with this sensing mechanism has resulted in a salt-sensitive phenotype, largely due to compromising the plant’s ability to sequester Na+ in mesophyll cell vacuoles. The implications of these findings for plant breeding for salinity stress tolerance are discussed.

Limit dextrinase (LD) and limit dextrinase inhibitor (LDI) are the two important traits affecting malt quality. The genetic variation and controlling of LD activity and LDI content in barley grains and malt are not well understood. In this study, we measured LD activity and LDI content in both grains and malt of 68 cultivated barley genotypes. The results show that there is a wide difference among barley genotypes in both LD activity and LDI content. LD in malt is not correlated with LD in grains, but negatively correlated with LDI in malt. LD in malt is positively correlated with diastatic power (DP), Kolbach index (KI) and soluble nitrogen content (SN), and negatively correlated with viscosity (VC). LDI in malt is positively correlated with DP and total nitrogen (TN), and negatively correlated with KI. Association analysis identifies 5 QTLs associated with LD and 3 QTLs associated with LDI in malt. Three major QTLs controlling LD in malt account for 35.7%, 35.7% and 28.4% of phenotypic variation, respectively. A total of 17 QTLs associated with malt quality are identified. The current results address the importance of both LD and LDI in affecting malt quality and the identified QTLs could be useful in barley breeding.

Phenolic acids have been quite extensively studied in food science research because of their antioxidative effect. In this study, the genotypic difference and genetic control of phenolic acids, and their correlation with malt quality, were investigated in barley. Ferulic acid (FA) and p-coumaric acid (p-CA) were identified as two main phenolic acids, showing wide variations among 68 barley genotypes. The mean content of FA and p-CA were 2.15 μg g(-1) and 1.10 μg g(-1) in grains and 4.07 μg g(-1) and 1.44 μg g(-1) in malt, respectively. After malting, FA and p-CA were increased significantly in 55 and 37 genotypes and were reduced in 2 and 14 genotypes, respectively. Both malt FA and p-CA were positively correlated with soluble N content and Kolbach index and negatively correlated with malt extract and viscosity. The results indicated that the effect of malting on the change of an individual phenolic acid is genotype independent. Association mapping identified that 8 markers on Chromosomes 1H, 2H, 4H, and 7H are associated with grain p-CA and 4 markers on Chromosomes 3H and 7H are linked with grain FA. However, only a single marker on Chromosome 3H was found to be associated with malt FA. Moreover, a lack of overlapping markers between grain and malt indicated the genetic diversity of phenolic acids in barley grain and malt. Our results strengthen the understanding of phenolic acids in barley and their responses to the malting process.

The domestication of cultivated barley has been used as a model system for studying the origins and early spread of agrarian culture. Our previous results indicated that the Tibetan Plateau and its vicinity is one of the centers of domestication of cultivated barley. Here we reveal multiple origins of domesticated barley using transcriptome profiling of cultivated and wild-barley genotypes. Approximately 48-Gb of clean transcript sequences in 12 Hordeum spontaneum and 9 Hordeum vulgare accessions were generated. We reported 12,530 de novo assembled transcripts in all of the 21 samples. Population structure analysis showed that Tibetan hulless barley (qingke) might have existed in the early stage of domestication. Based on the large number of unique genomic regions showing the similarity between cultivated and wild-barley groups, we propose that the genomic origin of modern cultivated barley is derived from wild-barley genotypes in the Fertile Crescent (mainly in chromosomes 1H, 2H, and 3H) and Tibet (mainly in chromosomes 4H, 5H, 6H, and 7H). This study indicates that the domestication of barley may have occurred over time in geographically distinct regions.

Low phosphorus (LP) limits crop growth and productivity in the majority of arable lands worldwide. Here, we investigated the changes in physiological and biochemical traits of Tibetan wild barleys (Hordeum vulgare L. ssp. spontaneum) XZ99 (LP tolerant), XZ100 (LP sensitive), and cultivated barley ZD9 (moderately LP tolerant) under two phosphorus (P) levels during vegetative stage. These genotypes showed considerable differences in the change of biomass accumulation, root/shoot dry weight ratio, root morphology, organic acid secretion, carbohydrate metabolism, ATPase (Adenosine triphosphatase) activity, P concentration and accumulation under LP in comparison with CK (control) condition. The higher LP tolerance of XZ99 is associated with more developed roots, enhanced sucrose biosynthesis and hydrolysis of carbohydrate metabolism pathway, higher APase (Acid phosphatase) and ATPase activity, and more secretion of citrate and succinate in roots when plants are exposed to LP stress. The results prove t...

A hydroponic experiment was conducted to elucidate the difference in growth and cell ultrastructure between Tibetan wild and cultivated barley genotypes under moderate (150 mM NaCl) and high (300 mM NaCl) salt stress. The growth of three barley genotypes was reduced significantly under salt stress, but the wild barley XZ16 (tolerant) was less affected relative to cultivated barley Yerong (moderate tolerant) and Gairdner (sensitive). Meanwhile, XZ16 had lower Na+ and higher K+ concentrations in leaves than other two genotypes. In terms of photosynthetic and chlorophyll fluorescence parameters, salt stress reduced maximal photochemical efficiency (F v/F m), net photosynthetic rate (Pn), stomatal conductance (Gs), and intracellular CO2 concentration (Ci). XZ16 showed relatively smaller reduction in comparison with the two cultivated barley genotypes. The observation of transmission electron microscopy found that fundamental cell ultrastructure changes happened in both leaves and roots of all barley genotypes under salt NaCl stress, with chloroplasts being most changed. Moreover, obvious difference could be detected among the three genotypes in the damage of cell ultrastructure under salt stress, with XZ16 and Gairdner being least and most affected, respectively. It may be concluded that high salt tolerance in XZ16 is attributed to less Na+ accumulation and K+ reduction in leaves, more slight damage in cell ultrastructure, which in turn caused less influence on chloroplast function and photosynthesis.

Abscisic acid (ABA)-driven stomatal regulation reportedly evolved after the divergence of ferns, during the early evolution of seed plants approximately 360 million years ago. This hypothesis is based on the observation that the stomata of certain fern species are unresponsive to ABA, but exhibit passive hydraulic control. However, ABA-induced stomatal closure was detected in some mosses and lycophytes. Here, we observed that a number of ABA signaling and membrane transporter protein families diversified over the evolutionary history of land plants. The aquatic ferns Azolla filiculoides and Salvinia cucullata have representatives of 23 families of proteins orthologous to those of Arabidopsis (Arabidopsis thaliana) and all other land plant species studied. Phylogenetic analysis of the key ABA signaling proteins indicates an evolutionarily conserved stomatal response to ABA. Moreover, comparative transcriptomic analysis has identified a suite of ABA-responsive genes that differentially expressed in a terrestrial fern species, Polystichum proliferum. These genes encode proteins associated with ABA biosynthesis, transport, reception, transcription, signaling, and ion and sugar transport, which fit the general ABA signaling pathway constructed from Arabidopsis and Hordeum vulgare. The retention of these key ABA-responsive genes could have had a profound effect on the adaptation of ferns to dry conditions. Furthermore, stomatal assays have shown the primary evidence for ABA-induced closure of stomata in two terrestrial fern species P. proliferum and Nephrolepis exaltata. In summary, we report, to our knowledge, new molecular and physiological evidence for the presence of active stomatal control in ferns.

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.

Aluminum (Al) and manganese (Mn) toxicity commonly coexists in acid soil, so the crop cultivars suitable for planting in acid soil should show high tolerance to both elements simultaneously. However, it is still not clear if the toxicity of Mn and Al on plant growth is antagonistic or synergistic, and the plants with Al tolerance are also tolerant to Mn toxicity. In this study, three barley genotypes (one Tibetan wild and two cultivated), differing in Al tolerance, were characterized for growth and physiological responses to Al or Mn toxicity as well as the combined treatment of the two toxic elements. Interestingly, it has been found that the combined treatment of both metals was less affected in comparison with Al or Mn treatment alone, in terms of plant growth, Al or Mn concentration in plant tissues, and photosynthetic parameters, indicating antagonistic interaction of Al and Mn for their effect on plant growth and physiological traits. The results also showed that there was a dramatic difference among barley genotypes in Mn toxicity tolerance and XZ16 showed much higher tolerance than other two genotypes. High Mn tolerance is mainly described to less Mn uptake and lower Mn concentration in plants, and Mn tolerance is independent of Al tolerance.

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.

Aluminum (Al3+) toxicity has been considered as a major factor limiting crop production in acid soil, which almost occupies 40% of the world’s arable land. The strategies for maintaining crop production on acid soils include lime application to raise soil pH and use of plants with high tolerance to acid soils. Al toxicity initially inhibits root growth that subsequently interrupts several physiological processes, such as uptake of water and nutrients, normal functions of cell wall and plasma membrane, and signal transduction, which finally leads to metabolic disorder. Plants evolve external resistance and internal tolerance mechanisms. Al-induced organic acid secretion has been considered as an important tolerance mechanism in various plant species. These secretion behaviors are mediated by organic acid transporters, which are encoded by the ALMT and MATE gene family. However, the signal transduction in activation of these transporters is still unclear. Recently, omics technologies including transcriptome, proteome, and metabolome have been applied in studies on plant Al tolerance. The omics results could give a global view on Al resistance mechanisms at transcriptional, protein, and metabolic levels. Traditionally, crossing and backcrossing are commonly used in breeding for Al-tolerant cultivars, while transgenic strategy seems to be a more effective and powerful approach.

Improvement of malt quality is the most important objective in malt barley breeding. The current experiments investigated the variation of malt quality characters among barley genotypes and the difference in genetic variants of HvGlb1, encoding β-glucanase isoenzyme I, between Tibetan annual wild barley and cultivated barley. The correlation between the gene variants and malt quality showed that there was a large difference in the four malt quality parameters, i.e. Kolbach index, diastatic power (DP), viscosity and malt extract, among the analyzed barley cultivars. Kolbach index was negatively and positively correlated with viscosity and malt extract, respectively, while malt extract was negatively correlated with viscosity. Malt β-glucan content was a major determinant of malt quality, and was significantly correlated with Kolbach index (−0.633), malt extract (−0.333) and viscosity (0.672). On the other hand, malt β-glucan content was mainly controlled by malt β-glucanase activity. The correlation analysis showed that the HvGlb1 gene was correlated with malt β-glucan content and three of four main malt quality parameters, except DP. In addition, we also found that the HvGlb1 of Tibetan barley had wider diversity in haplotype than that of the cultivated barley, supporting the hypothesis that Tibet is one of the original centers of cultivated barley.

In previous studies, we found Tibetan wild barley accessions with high tolerance to low K. In this study, ionomics and proteomics analyses were done on two wild genotypes (XZ153, tolerant and XZ141, sensitive), and a cultivar (B1031, tolerance to low K) to understand the mechanism of low-K tolerance. XZ153 was much less affected by low K stress than the other two genotypes in plant biomass and shoot K content. A total of 288 differentially accumulated proteins were identified between low-K and normal K treated plants. Among them, 129 proteins related to low-K tolerance were mainly involved in defense, transcription, signal transduction, energy, and protein synthesis. The analysis of tandem mass tag (TMT) detected 51 proteins which were increased in relative abundance under low K in XZ153, but unaltered or decreased in XZ141. The proteomics results showed that XZ153 is highly capable of rearranging ion homeostasis and developing an antioxidant defense system under low-K stress. Moreover, ethylene response and phenylpropanoid pathways could determine the genotypic difference in low-K tolerance. The current results confirmed the possibility of Tibetan wild barley providing low-K tolerant germplasm and identified some candidate proteins for use in developing the cultivars with low-K tolerance.

Sugar degradation during grain germination is important for malt quality. In malting industry, gibberellin (GA) is frequently used for improvement of malting quality. In this study, the changes of metabolite profiles and starch-degrading enzymes during grain germination, and as affected by GA and abscisic acid (ABA) were investigated using two wild barley accessions XZ72 and XZ95. Totally fifty-two metabolites with known structures were detected and the change of metabolite during germination was time- and genotype dependent. Sugars and amino acids were the most dramatically changed compounds. Addition of GA enhanced the activities of starch-degrading enzymes, and increased most metabolites, especially sugars and amino acids, whereas ABA had the opposite effect. The effect varied with the barley accessions. The current study is the first attempt in investigating the effect of hormones on metabolite profiles in germinating barley grain, being helpful for identifying the factors affecting barley germination or malt quality.

Tibetan wild barley (Hordeum vulgare L. ssp. spontaneum), originated and grown in harsh enviroNment in Tibet, is well-known for its rich germpalsm with high tolerance to abiotic stresses. However, the genetic variation and genes involved in Al tolerance are not totally known for the wild barley. In this study, a genome-wide association analysis (GWAS) was performed by using four root parameters related with Al tolerance and 469 DArT markers on 7 chromosomes within or across 110 Tibetan wild accessions and 56 cultivated cultivars. Population structure and cluster analysis revealed that a wide genetic diversity was present in Tibetan wild barley. Linkage disequilibrium (LD) decayed more rapidly in Tibetan wild barley (9.30 cM) than cultivated barley (11.52 cM), indicating that GWAS may provide higher resolution in the Tibetan group. Two novel Tibetan group-specific loci, bpb-9458 and bpb-8524 were identified, which were associated with relative longest root growth (RLRG), located at 2H and 7H on barely genome, and could explain 12.9% and 9.7% of the phenotypic variation, respectively. Moreover, a common locus bpb-6949, localized 0.8 cM away from a candidate gene HvMATE, was detected in both wild and cultivated barleys, and showed significant association with total root growth (TRG). The present study highlights that Tibetan wild barley could provide elite germplasm novel genes for barley Al-tolerant improvement.

A thorough understanding of the mechanisms underlying barley salt tolerance and exploitation of elite genetic resource are essential for utilizing wild barley germplasm in developing barley varieties with salt tolerance. In order to reveal the physiological and molecular difference in salt tolerance between Tibetan wild barley (Hordeum spontaneum) and cultivated barley (Hordeum vulgare), profiles of 82 key metabolites were studies in wild and cultivated barley in response to salinity. According to shoot dry biomass under salt stress, XZ16 is a fast growing and salt tolerant wild barley. The results of metabolite profiling analysis suggested osmotic adjustment was a basic mechanism, and polyols played important roles in developing salt tolerance only in roots, and high level of sugars and energy in roots and active photosynthesis in leaves were important for barley to develop salt tolerance. The metabolites involved in tolerance enhancement differed between roots and shoots, and also between genotypes. Tibetan wild barley, XZ16 had higher chlorophyll content and higher contents of compatible solutes than CM72, while the cultivated barley, CM72 probably enhanced its salt tolerance mainly through increasing glycolysis and energy consumption, when the plants were exposed to high salinity. The current research extends our understanding of the mechanisms involved in barley salt tolerance and provides possible utilization of Tibetan wild barley in developing barley cultivars with salt tolerance.