Genome-Wide DNA Methylation Dynamics During Drought Responsiveness in Tibetan Hulless Barley

Differences in drought stress tolerance within diverse grass genotypes have been attributed to epigenetic modifications. DNA methylation is an important epigenetic alteration regulating responses to drought-stress. However, its effects on drought-tolerance are poorly understood in Tibetan hulless barley. Here, bisulfite sequencing was conducted to profile the DNA methylation patterns of drought-tolerant variety XL and drought-sensitive one DQ under drought and control conditions. A total of 5843 million reads were generated. We found the significant genome-wide changes in CHH methylation rates between XL and DQ, while CG or CHG methylation rates did not. Besides that, the two contrasting varieties do reveal distinct responses to drought-stress in differentially methylated region (DMR) numbers and antioxidant activities. Genes in drought-tolerant varieties XL are rapidly and significantly methylated to alleviate the drought stress. DMR related genes in XL might involve in defense response and response to stimuli, which are confirmed by gene ontology analysis. Then, we focused on 1003 transcription factors and identified 15 specific DMR related transcription factors exhibiting specific methylation changes under drought stimuli. Finally, we identified three DMR related TFs (HVUL6H08680.2, HVUL4 h39100.2, and HVUL2H41931.2) where Arabidopsis homologues involve in responses to drought conditions. Altogether, DNA methylation regulate responsiveness to environmental stimuli, which could be mediated by methylation of transcription factors in hulless barely.

Isolation, sequencing of the HvnHID gene and its role in the purple-grain colour development in Tibetan hulless barley

2-hydroxyisoflavanone dehydratase (HID) plays an important role in isoflavone biosynthesis. In this study, HID was isolated from the seeds of the purple-grained Tibetan hulless barley variety Nerumuzha and the white-grained variety Kunlun 10. The HvnHID gene includes the 981 bp open reading frame and encodes a protein of 327 amino acids. It has a typical Abhydrolase_3 domain (78–306) and belongs to the carboxylesterase (CXE) family of the Abhydrolase_3 (α/β hydrolase) superfamily. There are eight nucleotide differences in the HvnHID coding sequence and two amino acid differences (one in the Abhydrolase_3 domain) between Nerumuzha and Kunlun 10. The HvnHID of hulless barley has the closest relationship with the HID in Hordeum vulgare, and the most distant relationship in Panicum hallii. At the early-mid stage of the seed colour development, the HvnHID expression levels in the purple and black seeds were significantly higher than in the white and blue ones (P < 0.01). During the seed colour development of purple-grained hulless barley, the expression of the key genes (HvnF3'H, HvnDRF, HvnANT1, and HvnGT) in the anthocyanidin biosynthetic pathway increased significantly, while the HvnHID expression decreased significantly (P < 0.01). Thus, it is likely that HvnHID negatively regulates the anthocyanidin biosynthesis. This result provides an important basis for further study of the biological functions of HvnHID in the anthocyanidin biosynthetic pathway.

Qualitative and semi-quantitative assessment of anthocyanins in Tibetan hulless barley from different geographical locations by UPLC-QTOF-MS and their antioxidant capacities

Tibetan hulless barley (“qingke” in Chinese) is a valuable food in Tibet. Purple qingke (PQK) and black qingke (BQK), two special pigmented types of hulless barley, have traditionally been widely cultivated and consumed in Tibet for thousands of years. The composition and contents of anthocyanins of two cultivars are unknown. This study aimed to explore the composition and contents of anthocyanins of two cultivars and their antioxidant capacities. Six anthocyanins were identified by ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry from barley, most of the anthocyanins were acylated by malonyl group. The total anthocyanin contents ranged from 141 to 2,304 μg/g in PQK and from 248.7 to 2902.9 μg/g in BQK. Furthermore, qingke has strong antioxidant activity against DPPH, ABTS˙+, and FRAP. Qingke may be useful for treating or preventing diseases caused by the overproduction of radicals.

Identification and Profiling of microRNAs and Their Target Genes in Tibetan Hulless Barley in Response to Barley Leaf Stripe (BLS) Infection

Background Tibetan hulless barley is widely grown on the Qinghai-Tibet Plateau, where it has served as a staple food for Tibetan people since the 5th century CE. Barley leaf stripe (BLS) is one of the most severe fungal diseases affecting the yield and quality of Tibetan hulless barley. Results Here, we compared the miRNA profiles before and after BLS in two Tibetan hulless barley genotypes: Z1141, a BLS-sensitive wild variety, and Kunlun14, a BLS-tolerant hybrid variety. A total of 36 conserved and 56 novel miRNAs were identified. Of these, 10 conserved and 10 novel miRNAs exhibited significantly changed expression between the normal and infected leaves of Kunlun14, respectively, while 3 conserved and 5 novel miRNAs exhibited significantly changed expression between the normal and infected leaves of Z1141, respectively. A total of 24 miRNAs were found in Z1141 and Kunlun14, and a further 546 putative target genes were predicted. Transcriptome sequencing analysis showed that among the 546 candidate genes, 131 had significant differences in expression between the normal and infected leaves of Kunlun14 and Z1141. Gene ontology, pathway, and Blast analyses indicated 10 candidate target genes that were involved in the barley disease resistance. These 10 candidate target genes may be regulated by 7 miRNAs. According to quantitative real-time PCR results, the 10 targets were negatively correlated with their corresponding miRNAs after infection with BLS. Conclusions The miRNAs and their target genes expressed in Tibetan hulless barley were identified and found to be associated with BLS resistance. Thus, these miRNAs and their target genes may be exploited via breeding programs or genetic engineering to improve BLS resistance in Tibetan hulless barley.

Purification, Preliminary Structural Characterization, and In Vitro Inhibitory Effect on Digestive Enzymes by β-Glucan from Qingke (Tibetan Hulless Barley)

Background and Objective. Qingke (Tibetan hulless barley, Hordeum vulgare L.) contains a high content of β-glucan among all the cereal varieties. Although β-glucan has multiple physiological functions, the physiological function of qingke β-glucan was few studied. In this study, the β-glucan was isolated, purified, determined the structural characterization, and measured the inhibitory activity to enzymes correlating blood sugar and lipid. Methods. β-Glucan was isolated from enzymatic aqueous extract of qingke by using deproteinization, decolorization, DEAE-52 column chromatography, and sepharose CL-4B agarose gel column chromatography. The structure of the β-glucan was determined using FT-IR and 13C-NMR spectra analysis, and molecular mass by use of HPSEC-dRI-LS. The kinematic viscosity was measured. The inhibitory effects of this β-glucan on four enzymes were investigated. Results. This β-glucan had a uniform molecular weight of 201,000 Da with β-(1⟶4) as the main chain and β-(1⟶3) as a side chain. The β-glucan presented a relatively strong inhibitory activity on α-glucosidase, moderate inhibition on invertase, and a weak inhibition on α-amylase, whereas it did not inhibit lipase. Conclusion. The study indicates that the enzymatic β-glucan from qingke has the potential as natural auxiliary hypoglycemic additives in functional medicine or foods.