Transcriptome and Metabolite Insights into Domestication Process of Cultivated Barley in China

The domestication process of cultivated barley in China remains under debate because of the controversial origins of barley. Here, we analyzed transcriptomic and non-targeted metabolic data from 29 accessions together with public resequencing data from 124 accessions to explore the domestication process of cultivated barley in China (Cb-C). These analyses revealed that both Cb-C and Tibetan wild barley (Wb-T) were the descendants of wild barley from the Near East Fertile Crescent (Wb-NE), yielding little support for a local origin of Wb-T. Wb-T was more likely an intermediate in the domestication process from Wb-NE to Cb-C. Wb-T contributed more genetically to Cb-C than Wb-NE, and was domesticated into Cb-C about 3300 years ago. These results together seem to support that Wb-T may be a feralized or hybrid form of cultivated barley from the Near East Fertile Crescent or central Asia. Additionally, the metabolite analysis revealed divergent metabolites of alkaloids and phenylpropanoids and these metabolites were specifically targeted for selection in the evolutionary stages from Wb-NE to Wb-T and from Wb-T to Cb-C. The key missense SNPs in the genes HORVU6Hr1G027650 and HORVU4Hr1G072150 might be responsible for the divergence of metabolites of alkaloids and phenylpropanoids during domestication. Our findings allow for a better understanding of the domestication process of cultivated barley in China.

Mechanistic Insights into Potassium-Conferred Drought Stress Tolerance in Cultivated and Tibetan Wild Barley: Differential Osmoregulation, Nutrient Retention, Secondary Metabolism and Antioxidative Defense Capacity

Keeping the significance of potassium (K) nutrition in focus, this study explores the genotypic responses of two wild Tibetan barley genotypes (drought tolerant XZ5 and drought sensitive XZ54) and one drought tolerant barley cv. Tadmor, under the exposure of polyethylene glycol-induced drought stress. The results revealed that drought and K deprivation attenuated overall plant growth in all the tested genotypes; however, XZ5 was least affected due to its ability to retain K in its tissues which could be attributed to the smallest reductions of photosynthetic parameters, relative chlorophyll contents and the lowest Na+/K+ ratios in all treatments. Our results also indicate that higher H+/K+-ATPase activity (enhancement of 1.6 and 1.3-fold for shoot; 1.4 and 2.5-fold for root), higher shoot K+ (2 and 2.3-fold) and Ca2+ content (1.5 and 1.7-fold), better maintenance of turgor pressure by osmolyte accumulation and enhanced antioxidative performance to scavenge ROS, ultimately suppress lipid peroxidation (in shoots: 4% and 35%; in roots 4% and 20% less) and bestow higher tolerance to XZ5 against drought stress in comparison with Tadmor and XZ54, respectively. Conclusively, this study adds further evidence to support the concept that Tibetan wild barley genotypes that utilize K efficiently could serve as a valuable genetic resource for the provision of genes for improved K metabolism in addition to those for combating drought stress, thereby enabling the development of elite barley lines better tolerant of abiotic stresses.

Single Nucleotide Polymorphisms in Bmy1 Intron III Alleles Conferring the Genotypic Variation in β-amylase Activity under Drought Stress Between Tibetan Wild and Cultivated Barley

β-amylase activity is related to the polymorphism of Bmy1 intron III; however, no attention has been given to such relationship under environmental stresses like drought. In this study, 73 cultivated barley genotypes and 52 Tibetan wild barley accessions were used to test the association between Bmy1 gene intron III polymorphisms and β-amylase activity under drought stress. Our results showed that three alleles, Bmy1.a, Bmy1.b and Bmy1.c, existed in the examined barley genotypes. Tibetan wild barley had higher proportion of Bmy1.b, whereas cultivated barley showed higher proportion of Bmy1.a. Impressively, barley genotypes with Bmy1.b showed significant increase in β-amylase activity under drought stress, compared with those with Bmy1.a or Bmy1.c, indicating that Bmy1.b allele might provide more chances for developing barley cultivars with higher β-amylase activity under water stress than both Bmy1.a and Bmy1.c alleles. Furthermore, the Tibetan wild barley XZ147, belonging to Bmy1.b allele type, showed significant higher β-amylase activity than the cultivar Triumph under drought stress. This might result from the unique amino acid substitution M527 or the amino acid composition of R115, D165, A233, S347 and M527 of XZ147.

Transcriptome and Metabolite Insights Into Domestication Process of Cultivated Barley in China

Background Barley is one of the earliest domesticated crops and regarded as one of the founder of Neolithic transition in the Near East Fertile Crescent. Domestication process of cultivated barley (especially east-Asian cultivated barley) has been under debate because of the controversial origin centers of barley, which caused by widely dispersal of wild barley. What’s more, no comprehensive study regarding alteration in metabolism during domestication has been delineated in barley so far. Results Transcriptomic and non-targeted metabolic analyses were performed for two wild barley populations (wild barley of Near East Fertile Crescent (Wb-NE), and wild barley of Tibetan Plateau (Wb-T)), and one cultivated barley population (cultivated barley of China (Cb-C)), the results revealed two stages of the domestication process of Cb-C, first from Wb-NE to Wb-T, and then from Wb-T to Cb-C. The Wb-T played an important intermediate role in the domestication from Wb-NE to Cb-C, and had made more genetic contribution than Wb-NE to Cb-C. Meanwhile, we found continuous gene flow, a large number of selective genes and metabolites during domestication. Divergent metabolites of alkaloids and phenylpropanoids were specific targeted in stages from Wb-NE to Wb-T and from Wb-T to Cb-C, respectively. The key missense SNPs in genes HORVU6Hr1G027650 and HORVU4Hr1G072150 might be related to the divergence of metabolites of alkaloids and phenylpropanoids during domestication. Conclusions Our results revealed that two stages of the domestication process of Cb-C, and distinct sets of metabolites were targeted by selection during the evolution from wild barley of the Near East Fertile Crescent to Tibetan wild barley to cultivated barley of China. Our findings not only provided genetic and metabolic insights into domestication process of barley but also highlighted the power of combining omics data for trait dissection.

Genotypic Difference in the Responses to Nitrogen Fertilizer Form in Tibetan Wild and Cultivated Barley

Nitrogen (N) availability and form have a dramatic effect on N uptake and assimilation in plants, affecting growth and development. In the previous studies, we found great differences in low-N tolerance between Tibetan wild barley accessions and cultivated barley varieties. We hypothesized that there are different responses to N forms between the two kinds of barleys. Accordingly, this study was carried out to determine the response of four barley genotypes (two wild, XZ16 and XZ179; and two cultivated, ZD9 andHua30) under 4Nforms (NO3−, NH4+, urea and glycine). The results showed significant reduction in growth parameters such as root/shoot length and biomass, as well as photosynthesis parameters and total soluble protein content under glycine treatment relative to other N treatments, for both wild and cultivated barley, however, XZ179 was least affected. Similarly, ammonium adversely affected growth parameters in both wild and cultivated barleys, with XZ179 being severely affected. On the other hand, both wild and cultivated genotypes showed higher biomass, net photosynthetic rate, chlorophyll and protein in NO3− treatment relative to other three N treatments. It may be concluded that barley undisputedly grows well under inorganic nitrogen (NO3−), however in response to the organic N wild barley prefer glycine more than cultivated barely.

Physiological and antioxidant responses of cultivated and wild barley under salt stress

Saline soil is a critical environmental problem affecting crop yield worldwide. Tibetan wild barley is distinguished for its vast genetic diversity and high degree of tolerance to abiotic stress, including salinity. The present study compared the response of antioxidant defense system in the XZ16 wild and CM72 cultivated barleys to salt stress. Wild barley was relatively more tolerant than cultivated CM72, salt-tolerant cultivar, with less Na⁺ uptake and more K⁺, Ca²⁺, and Mg²⁺ retention in plant tissues. The results of diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining showed that XZ16 had significantly lower H₂O₂ and O₂⁻ concentrations than a salt-sensitive cultivar Gairdner, suggesting that the salt-tolerant genotype suffer from less oxidative damage. Moreover, XZ16 and Gairdner had the highest and lowest anti-oxidative enzyme activities and proline content in plant tissues. In addition, the microscopic examination revealed that DNA damage in cv. Gairdner was closely correlated to oxidative stress, representing that more reactive oxygen species accumulation in plants tissues leads to subsequent DNA damage. The present results show that higher salt tolerance of wild barley XZ16 is attributed to less Na⁺ accumulation and stronger anti-oxidative capacity.

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.

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.

Genome-Wide Identification and Characterization of Drought Stress Responsive microRNAs in Tibetan Wild Barley

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.