Understanding the molecular regulation of flavonoid 3'-hydroxylase in anthocyanin synthesis: insights from purple qingke.

BACKGROUND: The Flavonoid 3'-hydroxylase gene(F3'H) is an important structural gene in the anthocyanin synthesis pathway of plants, which has been proven to be involved in the color formation of organs such as leaves, flowers, and fruits in many plants. However, the mechanism and function in barley are still unclear. RESULTS: In order to explore the molecular mechanism of the grain color formation of purple qingke, we used the cultivated qingke variety Nierumzha (purple grain) and the selected qingke variety Kunlun 10 (white grain) to conduct transcriptomic sequencing at the early milk, late milk and soft dough stage. Weighted Gene Co-expression Network Analysis (WGCNA) was used to construct weighted gene co-expression network related to grain color formation, and three key modules (brown, yellow, and turquoise modules) related to purple grain of qingke were selected. F3'H (HORVU1Hr1G094880) was selected from the hub gene of the module for the yeast library, yeast two-hybrid (Y2H), subcellular localization and other studies. It was found that in purple qingke, HvnF3'H mainly distributed in the cytoplasm and cell membrane and interacted with several stress proteins such as methyltransferase protein and zinc finger protein. CONCLUSIONS: The results of this study provide reference for the regulation mechanism of anthocyanin-related genes in purple grain qingke.

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.

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.

Genome-wide identification of WD40 transcription factors and their regulation of the MYB-bHLH-WD40 (MBW) complex related to anthocyanin synthesis in Qingke (Hordeum vulgare L. var. nudum Hook. f.).

BACKGROUND: WD40 transcription factors, a large gene family in eukaryotes, are involved in a variety of growth regulation and development pathways. WD40 plays an important role in the formation of MYB-bHLH-WD (MBW) complexes associated with anthocyanin synthesis, but studies of Qingke barley are lacking. RESULTS: In this study, 164 barley HvWD40 genes were identified in the barley genome and were analyzed to determine their relevant bioinformatics. The 164 HvWD40 were classified into 11 clusters and 14 subfamilies based on their structural and phylogenetic protein profiles. Co-lineage analysis revealed that there were 43 pairs between barley and rice, and 56 pairs between barley and maize. Gene ontology (GO) enrichment analysis revealed that the molecular function, biological process, and cell composition were enriched. The Kyoto Encyclopedia of Genes and Genomes (KEGG) results showed that the RNA transport pathway was mainly enriched. Based on the identification and analysis of the barley WD40 family and the transcriptome sequencing (RNA-seq) results, we found that HvWD40-140 (WD40 family; Gene ID: r1G058730), HvANT1 (MYB family; Gene ID: HORVU7Hr1G034630), and HvANT2 (bHLH family; Gene ID: HORVU2Hr1G096810) were important components of the MBW complex related to anthocyanin biosynthesis in Qingke, which was verified via quantitative real-time fluorescence polymerase chain reaction (qRT-PCR), subcellular location, yeast two-hybrid (Y2H), and bimolecular fluorescent complimentary (BiFC) and dual-luciferase assay analyses. CONCLUSIONS: In this study, we identified 164 HvWD40 genes in barley and found that HvnANT1, HvnANT2, and HvWD40-140 can form an MBW complex and regulate the transcriptional activation of the anthocyanin synthesis related structural gene HvDFR. The results of this study provide a theoretical basis for further study of the mechanism of HvWD40-140 in the MBW complex related to anthocyanin synthesis in Qingke.

Accumulation and regulation of anthocyanins in white and purple Tibetan Hulless Barley (Hordeum vulgare L. var. nudum Hook. f.) revealed by combined de novo transcriptomics and metabolomics.

BACKGROUND: Colored barley, which may have associated human health benefits, is more desirable than the standard white variety, but the metabolites and molecular mechanisms underlying seedcoat coloration remain unclear. RESULTS: Here, the development of Tibetan hulless barley was monitored, and 18 biological samples at 3 seedcoat color developmental stages were analyzed by transcriptomic and metabolic assays in Nierumuzha (purple) and Kunlun10 (white). A total of 41 anthocyanin compounds and 4186 DEGs were identified. Then we constructed the proanthocyanin-anthocyanin biosynthesis pathway of Tibetan hulless barley, including 19 genes encoding structural enzymes in 12 classes (PAL, C4H, 4CL, CHS, CHI, F3H, F3'H, DFR, ANS, ANR, GT, and ACT). 11 DEGs other than ANR were significantly upregulated in Nierumuzha as compared to Kunlun10, leading to high levels of 15 anthocyanin compounds in this variety (more than 25 times greater than the contents in Kunlun10). ANR was significantly upregulated in Kunlun10 as compared to Nierumuzha, resulting in higher contents of three anthocyanins compounds (more than 5 times greater than the contents in Nierumuzha). In addition, 22 TFs, including MYBs, bHLHs, NACs, bZips, and WD40s, were significantly positively or negatively correlated with the expression patterns of the structural genes. Moreover, comparisons of homologous gene sequences between the two varieties identified 61 putative SNPs in 13 of 19 structural genes. A nonsense mutation was identified in the coding sequence of the ANS gene in Kunlun10. This mutation might encode a nonfunctional protein, further reducing anthocyanin accumulation in Kunlun10. Then we identified 3 modules were highly specific to the Nierumuzha (purple) using WGCNA. Moreover, 12 DEGs appeared both in the putative proanthocyanin-anthocyanin biosynthesis pathway and the protein co-expression network were obtained and verified. CONCLUSION: Our study constructed the proanthocyanin-anthocyanin biosynthesis pathway of Tibetan hulless barley. A series of compounds, structural genes and TFs responsible for the differences between purple and white hulless barley were obtained in this pathway. Our study improves the understanding of the molecular mechanisms of anthocyanin accumulation and biosynthesis in barley seeds. It provides new targets for the genetic improvement of anthocyanin content and a framework for improving the nutritional quality of barley.

Increased serum LOXL2 concentration in pelvic inflammatory disease with pelvic adhesion.

BACKGROUND: Lysyl oxidase-like 2 (LOXL2) belongs to a family of the LOX secretory enzyme, which involves the cross-linkage of extracellular matrix (ECM) proteins. Here, we aimed to analyze the correlation between serum LOXL2 and pelvic adhesion in chronic pelvic inflammatory disease (PID). METHODS: A total of 143 patients with PID and 130 healthy controls were included in this study. The serum levels of LOXL2 were measured using enzyme-linked immunosorbent assay (ELISA) kits. The patients were divided into non-adhesion group (102 cases) and adhesion group (41 cases). RESULTS: It was found that the serum level of LOXL2 expression was elevated in PID patients compared with healthy controls, and was elevated in PID patients with pelvic adhesion compared to patients without adhesion. In all PID patients, serum LOXL2 level was positively correlated with matrix metalloproteinases-9 (MMP-9), transforming growth factor-ß (TGF-ß1), whole blood viscosity (WBV) at low shear rate (LSR), WBV at high shear rate (HSR), and hematocrit (HcT). Multivariate logistic regression analysis showed that serum LOXL2 level was an independent risk factor for pelvic adhesion in PID patients (OR = 1.058; 95% CI = 1.030-1.086, P < 0.001). CONCLUSIONS: Serum LOXL2 level not only predicts the presence of PID, but serum LOXL2 concentration is also associated with the presence of pelvic adhesions.

Inhibitory effects of glucagon-like peptide-1 receptor on epilepsy.

Glucagon-like peptide-1 (GLP-1) and its receptor, GLP-1R, are valuable tools in the therapy of type 2 diabetes mellitus. Although GLP-1R stimulation is also potentially applicable to neurological disorders, few investigators have evaluated its beneficial effects in neurological disease models. Thus, we aimed to look into the antiepileptic effects of GLP-1R on epilepsy and its underlying mechanisms. The cerebral cortex of 22 patients with temporal lobe epilepsy (TLE) and 16 patients with trauma were collected to the epilepsy and control groups, respectively. Seizures were induced by pentylenetetrazole (PTZ) in rats. Liraglutide was used to up-regulate GLP-1R, and exendin fragment 9-39 (ex9-39) was used to down-regulate GLP-1R. The motor responses and scalp electroencephalograms of rats were recorded, and the interaction between GLP-1R and neuronal receptors (GABAARß2/3, GluA1-4, GluNR1, GluN2A and GluN2B) was evaluated by coimmunoprecipitation. GLP-1R expression was investigated by immunohistochemistry and immunofluorescence staining, and the levels of GLP-1R and neuronal receptors were evaluated by western blotting. The results indicated that GLP-1R was decreased in patients with TLE and in PTZ-treated rats and the administration of liraglutide decreased seizure severity, which indicates that liraglutide exerts antiepileptic effects. Moreover, liraglutide significantly up-regulated GLP-1R and GABAARß2/3 and down-regulated GluA1-4, GluNR1, GluN2A and GluN2B. In addition, ex9-39 exerted adverse effects and weakened the effects of liraglutide. Therefore, GLP-1R might suppress seizures by regulating the levels of neuronal receptors.

Comparison of the Analgesic and Anti-Inflammatory Effects of Xiaoyuningkun Decoction with Cynanchum Paniculatum and Fukeqianjin in a Mouse Model of Pelvic Inflammatory Disease.

BACKGROUND Preclinical and clinical studies have shown that the extract of Cynanchum paniculatum (bunge) kitag and the fukeqianjin formulation have beneficial effects in pelvic inflammatory disease (PID). This study aimed to compare the effects of Cynanchum paniculatum and fukeqianjin with a new decoction, xiaoyuningkun, consisting of Melia toosendan, Angelica biserrata, and Cynanchum paniculatum, in a mouse model of PID. MATERIAL AND METHODS The mouse model of PID included injection of the upper genital tract with hydrochloric acid (HCl) and lipopolysaccharide (LPS). The control group underwent sham treatment with 0.9% physiological saline. Cynanchum paniculatum, fukeqianjin, and xiaoyuningkun decoction were administered orally for 15 days. Acetic acid-induced writhing and thermal nociception hot plate tests evaluated the analgesic effects of treatment. Mouse uterus and Fallopian tubes were examined histologically to evaluate the degree of inflammation. Immunohistochemistry was used to measure the protein expression of intercellular adhesion molecule-1 (ICAM-1) and vascular endothelial growth factor (VEGF). Enzyme-linked immunosorbent assay (ELISA) measured serum levels of inflammatory cytokines. RESULTS Treatment with xiaoyuningkun decoction significantly reduced the pain threshold in the mouse model of PID and the degree of inflammation in the uterus and Fallopian tubes compared with Cynanchum paniculatum and fukeqianjin. Cynanchum paniculatum decoction significantly reduced the serum levels of interleukin-1ß (IL-1ß), tumor necrosis factor-alpha (TNF-alpha), ICAM-1, and VEGF, and the expression of ICAM-1 and VEGF in the mouse uterus and Fallopian tubes. CONCLUSIONS The new xiaoyuningkun decoction had analgesic and anti-inflammatory effects in the mouse model of PID, possibly by inhibiting ICAM-1, VEGF, and inflammatory cytokines.

Construction of a high-density genetic map: genotyping by sequencing (GBS) to map purple seed coat color (Psc) in hulless barley.

BACKGROUND: Colored hulless barley are more suitable in food processing compared to normal (yellow) varieties because it is rich in bioactive compounds and produces higher extraction pearling fractions. Therefore, seed coat color is an important agronomic trait for the breeding and study of hulless barley. RESULTS: Genotyping-by-sequencing single-nucleotide polymorphism (GBS-SNP) analysis of a doubled haploid (DH) mapping population (Nierumuzha × Kunlun10) was conducted to map the purple seed coat color genes (Psc). A high-density genetic map of hulless barley was constructed, which contains 3662 efficient SNP markers with 1129 bin markers. Seven linkage groups were resolved, which had a total length of 645.56 cM. Chromosome length ranged from 60.21 cM to 127.21 cM, with average marker density of 0.57 cM. A total of five loci accounting for 3.79% to 23.86% of the observed phenotypic variation for Psc were detected using this high-density map. Five structural candidate genes (F3'M, HID, UF3GT, UFGT and 5MAT) and one regulatory factor (Ant1) related to flavonoid or anthocyanin biosynthesis were identified.. CONCLUSIONS: Five structural candidate genes and one regulatory factor related to flavonoid or anthocyanin biosynthesis have been identified using a high-density genetic map of hulless barley. This study lays the foundation for map-based cloning of Psc but provides a valuable tool for studying marker-trait associations and its application to marker-assisted breeding of hulless barley.

Inhibition of Calcineurin A by FK506 Suppresses Seizures and Reduces the Expression of GluN2B in Membrane Fraction.

FK506, a calcineurin inhibitor, shows neuroprotective effects and has been associated with neurodegenerative diseases. Calcineurin A (CaNA), a catalytic subunit of calcineurin, mediates the dephosphorylation of various proteins. N-methyl-D-aspartate receptor (GluN) is closely related to epileptogenesis, and various phosphorylation sites of GluN2B, a regulatory subunit of the GluN complex, have different functions. Thus, we hypothesized that one of the potential anti-epileptic mechanisms of FK506 is mediated by its ability to promote the phosphorylation of GluN2B and reduce the expression of GluN2B in membrane fraction by down-regulating CaNA. CaNA expression was increased in the cortex of patients with temporal lobe epilepsy and pentylenetetrazol (PTZ)-induced epileptic models. CaNA was shown to be expressed in neurons using immunofluorescence staining. According to our behavioral observations, epileptic rats exhibited less severe seizures and were less sensitive to PTZ after a systemic injection of FK506. The levels of phosphorylated GluN2B were decreased in epileptic rats but increased after the FK506 treatment. Moreover, there was no difference in the total GluN2B levels before and after FK506 treatment. However, the expression of GluN2B in membrane fraction was suppressed after FK506 treatment. Based on these results, FK506 may reduce the severity and frequency of seizures by reducing the expression of GluN2B in membrane fraction.

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.

Nanoswimmers statistical mechanics: Unlocking whole-blood viscosity sensing for tumor microenvironment.

BACKGROUND AND OBJECTIVE: The ability to sense the biological microenvironment surrounding early-stage tumor tissues is critical for tumorigenesis tracing and tumor detection and treatment. An efficient tumor microenvironment (TME) sensing strategy remains a significant challenge. We propose a novel "seeing is sensing" approach that has the potential to discern the whole-blood viscosity (WBV) information of the TME by using a swarm of nanoswimmers (NS). METHODS: In this study, we employ statistical mechanics methods to derive the relationship between the aggregation of NS in the microscale and their macroscopic concentration distribution. We utilize the finite difference method to develop a discrete numerical model of the NS diffusion in the blood. We further develop a novel model for TME sensing that can dynamically detect the WBV by analyzing the kinematic motion of the NS swarm, which enables real-time detection of WBV by observing the Full Width at Half Maximum (FWHM) of the NS swarm motion. RESULTS: The viscosity value obtained with our sensing method is benchmarked against the gold standard results obtained from the Brookfield viscometer. The measurements obtained from both methods exhibit an excellent correlation, with a coefficient of determination (R2) of 0.9617. Furthermore, the constructed Bland-Altman plot reveals that the majority of observed data points lie within the limits of agreement of the 95% clinical confidence interval (lower limit of agreement = -0.0660, upper limit of agreement = 0.1130), thus validating the feasibility of our sensing strategy. CONCLUSIONS: We present a new sensing strategy that utilizes the diffusion dynamics of the NS swarm within the vascular network to infer variations in WBV. Comparative analysis with gold standard data substantiates the accuracy and applicability of this method in assessing WBV parameters in the vicinity of tumor tissues. Our work demonstrates relevant prospects for visualizing, comprehending, and evaluating the pathological progression of blood-related disorders in real-time.

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.

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.

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.

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.

Potassium indole-3-butyric acid affects rice's adaptability to salt stress by regulating carbon metabolism, transcription factor genes expression, and biosynthesis of secondary metabolites.

Soil salinity pollution is increasing worldwide, seriously affecting plant growth and crop production. Existing reports on how potassium indole-3-butyric acid (IBAK) regulates rice salt stress adaptation by affecting rice carbon metabolism, transcription factor (TF) genes expression, and biosynthesis of secondary metabolites still have limitations. In this study, an IBAK solution at 40 mg L-1 was sprayed on rice leaves at the seedling stage. The results showed that the IBAK application could promote shoot and root growth, decrease sucrose and fructose content, increase starch content, and enhance acid invertase (AI) and neutral invertase (NI) activity under salt stress, indicating altered carbon allocation. Furthermore, the expression of TF genes belonging to the ethylene responsive factor (ERF), WRKY, and basic helix-loop-helix (bHLH) families was influenced by IBAK. Many key genes (OsSSIIc, OsSHM1, and OsPPDKB) and metabolites (2-oxoglutaric acid, fumaric acid, and succinic acid) were upregulated in the carbon metabolism pathway. In addition, this study highlighted the role of IBAK in regulating the biosynthesis of secondary metabolites pathway, potentially contributing to rice stress adaptability. The results of this study can provide new sustainable development solutions for agricultural production.

Cloning, subcellular localization and expression of phosphate transporter gene HvPT6 of hulless barley.

Deficiency of phosphate (Pi) is one of the main growth-limiting factors for crops. Generally, phosphate transporters play a key role in the uptake of P in the crops. However, current knowledge regarding the molecular mechanism underlying Pi transport is still limited. In this study, a phosphate transporter (PT) gene, designated HvPT6, was isolated from a cDNA library constructed from hulless barley "Kunlun 14." The promoter of HvPT6 showed a large number of elements related to plant hormones. The expression pattern also indicated that HvPT6 was highly induced by low phosphorus, drought, abscisic acid, methyl jasmonate and gibberellin. Phylogenetic tree analysis revealed that HvPT6 belongs to the same subfamily of the major facilitator superfamily as OsPT6 from Oryza sativa. Subcellular localization of HvPT6:GFP using transient expression of Agrobacterium tumefaciens showed the green fluorescent protein signal in the membrane and nucleus of the Nicotiana benthamiana leaves. Overexpressing HvPT6 led to a longer and higher lateral root length and dry matter yield in the transgenic Arabidopsis lines under low Pi conditions, indicating that HvPT6 improves plant tolerance under Pi-deficient conditions. This study will lay a molecular basis for phosphate absorption mechanism in barley and breeding barley with high-efficient phosphate uptake.

Comparative transcriptome analysis of major lodging resistant factors in hulless barley.

Hulless barley (Hordeum vulgare L. var. nudum Hook. f.), belonging to the genus Gramineae, has high and steady output and thus considered as a principal food crop by Tibetan people. Hulless barley grain can be used for food, brewing, and functional health product development, while its straw serves as an essential supplementary forage and is a crucial cereal crop. Lodging can reduce the yield and quality of barley grain and straw, and it hinders mechanical harvesting. It is a significant factor affecting high and stable yields of barley. Unlike other Poaceae plants (such as rice, wheat), hulless barley is mainly grown in high-altitude regions, where it is susceptible to low temperatures, strong winds, and heavy rainfall. As a result, its stem lodging resistance is relatively weak, making it prone to lodging during the growth period. In this study, we observed that the lignin concentration and the contents of lignin monomers (H, S, and G), and neutral detergent fibre of the lodging-resistant variety Kunlun14 were substantially greater than those of the lodging-sensitive variety Menyuanlianglan. We performed the weighted gene co-expression network analysis (WGCNA) and Short Time-series Expression Miner (STEM) analysis of both the lodging-resistant and lodging-sensitive varieties. Through transcriptome sequencing analysis at different developmental stages, combined with the previously annotated genes related to lodging resistance, a total of 72 DEGs were identified. Among these DEGs, 17 genes were related to lignin, cellulose, and hemicellulose synthesis or regulation, including five transcription factors about NAC, MYB and WRKY. Our results provide a basis for further exploring the molecular mechanism of stem lodging resistance in hulless barley and provide valuable gene resources for stem lodging resistance molecular breeding.

Genome-Wide Association Mapping of Hulless Barely Phenotypes in Drought Environment.

Drought stress is one of the main factors restricting hulless barley (Hordeum vulgare L. var. nudum Hook. f.) yield. Genome-wide association study was performed using 269 lines of hulless barley to identify single-nucleotide polymorphism (SNP) markers associated with drought-resistance traits. The plants were cultured under either normal or drought conditions, and various quantitative traits including shoot fresh weight, shoot dry weight, root fresh weight, root dry weight, leaf fresh weight, leaf saturated fresh weight, leaf dry weight, ratio of root and shoot fresh weight, ratio of root and shoot dry weight, shoot water loss rate, root water loss rate, leaf water content and leaf relative water content, and field phenotypes including main spike length, grain number per plant, grain weight per plant, thousand grain weight (TGW), main spike number, plant height, and effective spike number of plants were collected. After genotyping the plants, a total of 8,936,130 highly consistent population SNP markers were obtained with integrity > 0.5 and minor allele frequency > 0.05. Eight candidate genes potentially contributed to the hulless barley drought resistance were obtained at loci near significant SNPs. For example, EMB506, DCR, and APD2 genes for effective spike number of plants, ABCG11 gene for main spike number (MEN), CLPR2 gene for main spike length, YIP4B gene for root and shoot dry weight (RSWD), and GLYK and BTS genes for TGW. The SNPs and candidate genes identified in this study will be useful in hulless barley breeding under drought resistance.