Unveiling the mysteries of HvANS: a study on anthocyanin biosynthesis in qingke (hordeum vulgare L. var. Nudum hook. f.) seeds.

BACKGROUND: Based on our previous research, a full-length cDNA sequence of HvANS gene was isolated from purple and white Qingke. The open reading frame (ORF) in the purple variety Nierumuzha was 1320 base pairs (bp), encoding 439 amino acids, while the ORF in the white variety Kunlun 10 was 1197 bp, encoding 398 amino acids. A nonsynonymous mutation was found at the position of 1195 bp (T/C) in the coding sequence (CDS) of the HvANS gene. We carried out a series of studies to further clarify the relationship between the HvANS gene and anthocyanin synthesis in Qingke. RESULTS: The conservative structural domain prediction results showed that the encoded protein belonged to the PLN03178 superfamily. Multiple comparisons showed that this protein had the highest homology with Hordeum vulgare, at 88.61%. The approximately 2000 bp promoter sequence of the HvANS gene was identical in both varieties. The real-time fluorescence PCR (qRT-PCR) results revealed that HvANS expression was either absent or very low in the roots, stems, leaves, and awns of Nierumuzha. In contrast, the HvANS expression was high in the seed coats and seeds of Nierumuzha. Likewise, in Kunlun 10, HvANS expression was either absent or very low, indicating a tissue-specific and variety-specific pattern for HvANS expression. The subcellular localization results indicated that HvANS was in the cell membrane. Metabolomic results indicated that the HvANS gene is closely related to the synthesis of three anthocyanin substances (Idaein chloride, Kinetin 9-riboside, and Cyanidin O-syringic acid). Yeast single hybridization experiments showed that the HvANS promoter interacted with HvANT1, which is the key anthocyanin regulatory protein. In a yeast two-hybrid experiment, we obtained two significantly different proteins (ZWY2020 and POMGNT2-like) and verified the results by qRT-PCR. CONCLUSIONS: These results provide a basis for further studies on the regulatory mechanism of HvANS in the synthesis of anthocyanins in Qingke purple grains.

Investigating the regulatory role of HvANT2 in anthocyanin biosynthesis through protein-motif interaction in Qingke.

Background: Currently, there are no reports on the HvbHLH gene family in the recent barley genome (Morex_V3). Furthermore, the structural genes related to anthocyanin synthesis that interact with HvANT2 have yet to be fully identified. Methods: In this study, a bioinformatics approach was used to systematically analyze the HvbHLH gene family. The expression of this gene family was analyzed through RNA sequencing (RNA-seq), and the gene with the most significant expression level, HvANT2, was analyzed using quantitative reverse transcription polymerase chain reaction (qRT-PCR) in different tissues of two differently colored varieties. Finally, structural genes related to anthocyanin synthesis and their interactions with HvANT2 were verified using a yeast one-hybrid (Y1H) assay. Results: The study identified 161 bHLH genes, designated as HvbHLH1 to HvbHLH161, from the most recent barley genome available. Evolutionary tree analysis categorized barley bHLH TFs into 21 subfamilies, demonstrating a pronounced similarity to rice and maize. Through RNA-Seq analysis of purple and white grain Qingke, we discovered a significant transcription factor (TF), HvANT2 (HvbHLH78), associated with anthocyanin biosynthesis. Subsequently, HvANT2 protein-motifs interaction assays revealed 41 interacting motifs, three of which were validated through Y1H experiments. These validated motifs were found in the promoter regions of key structural genes (CHI, F3'H, and GT) integral to the anthocyanin synthesis pathway. These findings provide substantial evidence for the pivotal role of HvANT2 TF in anthocyanin biosynthesis.

Comparative Analysis of Hulless Barley Transcriptomes to Regulatory Effects of Phosphorous Deficiency.

Hulless barley is a cold-resistant crop widely planted in the northwest plateau of China. It is also the main food crop in this region. Phosphorus (P), as one of the important essential nutrient elements, regulates plant growth and defense. This study aimed to analyze the development and related molecular mechanisms of hulless barley under P deficiency and explore the regulatory genes so as to provide a basis for subsequent molecular breeding research. Transcriptome analysis was performed on the root and leaf samples of hulless barley cultured with different concentrations of KH2PO4 (1 mM and 10 µM) Hoagland solution. A total of 46,439 genes were finally obtained by the combined analysis of leaf and root samples. Among them, 325 and 453 genes had more than twofold differences in expression. These differentially expressed genes (DEGs) mainly participated in the abiotic stress biosynthetic process through Gene Ontology prediction. Moreover, the Kyoto Encyclopedia of Genes and Genomes showed that DEGs were mainly involved in photosynthesis, plant hormone signal transduction, glycolysis, phenylpropanoid biosynthesis, and synthesis of metabolites. These pathways also appeared in other abiotic stresses. Plants initiated multiple hormone synergistic regulatory mechanisms to maintain growth under P-deficient conditions. Transcription factors (TFs) also proved these predictions. The enrichment of ARR-B TFs, which positively regulated the phosphorelay-mediated cytokinin signal transduction, and some other TFs (AP2, GRAS, and ARF) was related to plant hormone regulation. Some DEGs showed different values in their FPKM (fragment per kilobase of transcript per million mapped reads), but the expression trends of genes responding to stress and phosphorylation remained highly consistent. Therefore, in the case of P deficiency, the first response of plants was the expression of stress-related genes. The effects of this stress on plant metabolites need to be further studied to improve the relevant regulatory mechanisms so as to further understand the importance of P in the development and stress resistance of hulless barley.

Identification and functional analysis of long non-coding RNA (lncRNA) and metabolites response to mowing in hulless barley (Hordeum vulgare L. var. nudum hook. f.).

BACKGROUND: Hulless barley (Hordeum vulgare L. var. nudum Hook. f.) is a significant cereal crop and a substantial source of forage for livestock. Long non-coding RNAs (lncRNAs) and metabolites play crucial roles in the nutrient accumulation and regeneration of hulless barley plants following mowing. The study aimed to identify differentially expressed lncRNAs and metabolites in hulless barley plants by analyzing transcriptomic and metabolomic datasets at 2 h, 24 h, and 72 h following mowing. RESULTS: The study revealed that 190, 90, and 438 lncRNA genes were differentially expressed at the 2 h, 24 h, and 72 h time points compared to the non-mowing control. We identified 14 lncRNA genes-11 downregulated and 3 upregulated-showing consistently significant differential expression across all time points after mowing. These differentially expressed lncRNAs target genes involved in critical processes such as cytokinin signaling, cell wall degradation, storage protein accumulation, and biomass increase. In addition, we identified ten differentially expressed metabolites targeting diverse metabolic pathways, including plant hormones, alkaloids, and flavonoids, before and after mowing at various time points. Endogenous hormone analysis revealed that cytokinin most likely played a crucial role in the regeneration of hulless barley after mowing. CONCLUSIONS: This study created a comprehensive dataset of lncRNAs, metabolites, and hormones in hulless barley after mowing, revealing valuable insights into the functional characteristics of lncRNAs, metabolites, and hormones in regulating plant regeneration. The results indicated that cytokinin plays a significant role in facilitating the regeneration process of hulless barley after mowing. This comprehensive dataset is an invaluable resource for better understanding the complex mechanisms that underlie plant regeneration, with significant implications for crop improvement.

The CDPK-related protein kinase HvCRK2 and HvCRK4 interact with HvCML32 to negatively regulate drought tolerance in transgenic Arabidopsis thaliana.

Calcium-dependent protein kinase (CDPK) as one of calcium sensors were play important roles in stress responses. CDPK-related protein kinase (CRK) was identified as subgroup III of CDPK has been characterized in many plants, but the members and functions of CRK genes in hulless barley (Hordeum vulgare L.) has not been clarified. Here, we identified four HvCRK genes and named HvCRK1-4 according to chromosomes localization. Moreover, the physiological function of highly induced genes of HvCRK2 and HvCRK4 were investigated in drought stress tolerance by examining their overexpression transgenic lines functions generated in Arabidopsis thaliana. Under drought stress, both overexpression HvCRK2 and HvCRK4 displayed reduced drought resistance, and accompanied by higher accumulation levels of ROS. Notably, overexpression of HvCRK2 and HvCRK4 reduced sensitivity to exogenous ABA, meanwhile the expression of ABA-responsive genes in transgenic plants were down-regulated compared to the wild type in response to drought stress. Furthermore, the physically interaction of HvCRK2 and HvCRK4 with calmodulin (CaM) and calmodulin-like (CML) proteins were determined in vivo, the further results showed that HvCML32 binds to HvCRK2/4 S_TKC structural domains and negatively regulates drought tolerance. In summary, this study identified HvCRK members and indicated that HvCRK2 and HvCRK4 genes play negative roles in drought tolerance, and provide insight into potential molecular mechanism of HvCRK2 and HvCRK4 in response to drought stress.

Bioinformatics, expression analysis, and functional verification of allene oxide synthase gene HvnAOS1 and HvnAOS2 in qingke.

Allene oxide synthase (AOS) is a key enzyme involved in the jasmonic acid (JA) synthesis pathway in plants. To explore its function on the regulatory mechanism of JA synthesis, we screened and identified two AOS genes HvnAOS1 and HvnAOS2 in qingke. Both HvnAOS1 and HvnAOS2 contained conserved heme-binding motif, which is most closely related to AtsAOS2, indicating controlled dehydration of fatty acid hydroperoxides to allene oxides. Molecular docking simulations identified the key amino acid sites that were important for heme binding and interaction with 13(S)-HPOT, respectively. The expression pattern also indicated that HvnAOS1 and HvnAOS2 were highly induced by JA, abscisic acid, and salicylic acid. Subcellular localization of HvnAOS1 and HvnAOS2 using transient expression of Agrobacterium tumefaciens showed the green fluorescent protein signal in the cell cytoplasm of the N. benthamiana leaves. Overexpression of HvnAOS1 and HvnAOS2 in Arabidopsis aos mutant restored male fertility and plant resistance to Botrytis cinerea, indicating that HvnAOS1 and HvnAOS2 can restore the functions of AOS in Arabidopsis aos mutant.

Genome-wide identification of the CNGC gene family and negative regulation of drought tolerance by HvCNGC3 and HvCNGC16 in transgenic Arabidopsis thaliana.

Cyclic nucleotide-gated ion channels (CNGCs), as non-selective cation channels, play essential roles in plant growth and stress responses. However, they have not been identified in Qingke (Hordeum vulgare L.). Here, we performed a comprehensive genome-wide identification and function analysis of the HvCNGC gene family to determine its role in drought tolerance. Phylogenetic analysis showed that 27 HvCNGC genes were divided into four groups and unevenly located on seven chromosomes. Transcription analysis revealed that two closely related members of HvCNGC3 and HvCNGC16 were highly induced and the expression of both genes were distinctly different in two extremely drought-tolerant materials. Transient expression revealed that the HvCNGC3 and HvCNGC16 proteins both localized to the plasma membrane and karyotheca. Overexpression of HvCNGC3 and HvCNGC16 in Arabidopsis thaliana led to impaired seed germination and seedling drought tolerance, which was accompanied by higher hydrogen peroxide (H2O2), malondialdehyde (MDA), proline accumulation and increased cell damage. In addition, HvCNGC3 and HvCNGC16-overexpression lines reduced ABA sensitivity, as well as lower expression levels of some ABA biosynthesis and stress-related gene in transgenic lines. Furthermore, Yeast two hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays revealed that HvCNGC3 and HvCNGC16 interacted with calmodulin/calmodulin-like proteins (CaM/CML), which, as calcium sensors, participate in the perception and decoding of intracellular calcium signaling. Thus, this study provides information on the CNGC gene family and provides insight into the function and potential regulatory mechanism of HvCNGC3 and HvCNGC16 in drought tolerance in Qingke.