Characterization of metallothionein genes from Broussonetia papyrifera: metal binding and heavy metal tolerance mechanisms.

BACKGROUND: Broussonetia papyrifera is an economically significant tree with high utilization value, yet its cultivation is often constrained by soil contamination with heavy metals (HMs). Effective scientific cultivation management, which enhances the yield and quality of B. papyrifera, necessitates an understanding of its regulatory mechanisms in response to HM stress. RESULTS: Twelve Metallothionein (MT) genes were identified in B. papyrifera. Their open reading frames ranged from 186 to 372 bp, encoding proteins of 61 to 123 amino acids with molecular weights between 15,473.77 and 29,546.96 Da, and theoretical isoelectric points from 5.24 to 5.32. Phylogenetic analysis classified these BpMTs into three subclasses: MT1, MT2, and MT3, with MT2 containing seven members and MT3 only one. The expression of most BpMT genes was inducible by Cd, Mn, Cu, Zn, and abscisic acid (ABA) treatments, particularly BpMT2e, BpMT2d, BpMT2c, and BpMT1c, which showed significant responses and warrant further study. Yeast cells expressing these BpMT genes exhibited enhanced tolerance to Cd, Mn, Cu, and Zn stresses compared to control cells. Yeasts harboring BpMT1c, BpMT2e, and BpMT2d demonstrated higher accumulation of Cd, Cu, Mn, and Zn, suggesting a chelation and binding capacity of BpMTs towards HMs. Site-directed mutagenesis of cysteine (Cys) residues indicated that mutations in the C domain of type 1 BpMT led to increased sensitivity to HMs and reduced HM accumulation in yeast cells; While in type 2 BpMTs, the contribution of N and C domain to HMs' chelation possibly corelated to the quantity of Cys residues. CONCLUSION: The BpMT genes are crucial in responding to diverse HM stresses and are involved in ABA signaling. The Cys-rich domains of BpMTs are pivotal for HM tolerance and chelation. This study offers new insights into the structure-function relationships and metal-binding capabilities of type-1 and - 2 plant MTs, enhancing our understanding of their roles in plant adaptation to HM stresses.

Physicochemical Properties and Biological Activities of Quinoa Polysaccharides.

Quinoa, known as the "golden grain" for its high nutritional value, has polysaccharides as one of its sources of important nutrients. However, the biological functions of quinoa polysaccharides remain understudied. In this study, two crude polysaccharide extracts of quinoa (Q-40 and Q-60) were obtained through sequential precipitation with 40% and 60% ethanol, with purities of 58.29% (HPLC) and 62.15% (HPLC) and a protein content of 8.27% and 9.60%, respectively. Monosaccharide analysis revealed that Q-40 contained glucose (Glc), galacturonic acid (GalA), and arabinose (Ara) in a molar ratio of 0.967:0.027:0.006. Q-60 was composed of xylose (xyl), arabinose (Ara), galactose, and galacturonic acid (GalA) with a molar ratio of 0.889:0.036:0.034:0.020. The average molecular weight of Q-40 ranged from 47,484 to 626,488 Da, while Q-60 showed a range of 10,025 to 47,990 Da. Rheological experiments showed that Q-40 exhibited higher viscosity, while Q-60 demonstrated more elastic properties. Remarkably, Q-60 showed potent antioxidant abilities, with scavenging rates of 98.49% for DPPH and 57.5% for ABTS. Antibacterial experiments using the microdilution method revealed that Q-40 inhibited the growth of methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli), while Q-60 specifically inhibited MRSA. At lower concentrations, both polysaccharides inhibited MDA (MD Anderson Cancer Center) cell proliferation, but at higher concentrations, they promoted proliferation. Similar proliferation-promoting effects were observed in HepG2 cells. The research provides important information in the application of quinoa in the food and functional food industries.

Identification of TIFY gene family in walnut and analysis of its expression under abiotic stresses.

BACKGROUND: Walnuts (Juglans regia L.) are known for their nutrient-rich nuts and are one of the important economic tree species in the world. However, due to global warming and soil salinization, walnuts suffer from various abiotic stresses. TIFY (TIF[F/Y]XG) proteins play an essential role in the growth and development of plants, signal transduction, and stress response in plants. At present, although the TIFY gene family of a number of plants has been identified and studied, how TIFY takes part in stress tolerance remains obscure and many functions of TIFY require further investigation. RESULT: In this study, twenty-one TIFY transcription factors were identified in the walnut genome database, and they were divided into four subfamilies (TIFY, JAZ, ZML, and PPD) by bioinformatics analysis. Chromosome location revealed tandem duplication of some genes. Phylogenetic tree analysis showed JrTIFYs were closely related to the TIFY gene family of Arabidopsis thaliana (A. thaliana). qRT-PCR (quantitative real-time PCR) analysis revealed the TIFY genes have different expression patterns in 'Qingxiang' and 'Xiangling' walnut varieties under drought, heat, and salt stress. JAZ subfamily was more expressed in different abiotic stress than other subfamilies. The expressions of JrTIFY14 under heat and salt stress were significantly higher than those under drought stress. However, the expression of JrTIFYs was not significant in 'Xiangling'. CONCLUSION: This study reveals the TIFY gene family plays an important role in walnuts facing abiotic stresses and provides a theoretical basis for walnut breeding.

Protocatechuic acid, ferulic acid and relevant defense enzymes correlate closely with walnut resistance to Xanthomonas arboricola pv. juglandis.

BACKGROUND: Juglans regia L. is an important nut tree that has a wide range of distribution in temperate regions of the world. In some walnut orchards, walnut blight can become a problematic disease that affects the growth of walnut trees. To explore the correlation between biochemical response and walnut resistance, we inoculated four walnut cultivars with Xanthomonas arboricola pv. juglandis (Xaj). The walnut cultivars were, namely, 'Xiangling', 'Xiluo 2', 'Yuanfeng' and 'Xifu 2'. Total phenol content (TPC) and total flavonoid content (TFC) were measured, whereby nine major phenolic compounds and several relevant enzymes were identified. RESULTS: The results showed that the most resistant and susceptible walnut varieties were 'Xiluo 2' and 'Xifu 2' respectively. The reaction of walnut to Xaj was characterized by the early accumulation of phenolic compounds in the infected site. After inoculation with Xaj, we found that the resistant variety 'Xiluo 2' show the significant differences with other varieties at different time points through the determination of related antioxidant enzymes such as catalase (CAT) and peroxidase (POD). Meanwhile, the phenylalanine ammonia lyase (PAL) of 'Xiluo 2' increased significantly at 8 day post infection (dpi) and made differences from the control samples, while other varieties changed little. And the polyphenol oxidase (PPO) was significantly higher than in the control at 16 dpi, maintaining the highest and the lowest activity in 'Xiluo 2' and 'Xifu 2' respectively. It was also found that the content of protocatechuic acid in all cultivars increased significantly at 4 dpi, and 'Xiluo 2' was significantly higher than that of the control. In the early stage of the disease, ferulic acid content increased significantly in 'Xiluo 2'. CONCLUSION: Our findings confirmed that the metabolism of phenolic compounds and related defense enzymes are of great significance in the response of walnut to Xaj.

Lactobacillusjohnsonii L531 Protects against Salmonella Infantis-Induced Intestinal Damage by Regulating the NOD Activation, Endoplasmic Reticulum Stress, and Autophagy.

Salmonella enterica serovar Infantis (S. Infantis) is an intracellular bacterial pathogen. It is prevalent but resistant to antibiotics. Therefore, the therapeutic effect of antibiotics on Salmonella infection is limited. In this study, we used the piglet diarrhea model and the Caco2 cell model to explore the mechanism of probiotic Lactobacillus johnsonii L531 (L. johnsonii L531) against S. Infantis infection. L. johnsonii L531 attenuated S. Infantis-induced intestinal structural and cellular ultrastructural damage. The expression of NOD pathway-related proteins (NOD1/2, RIP2), autophagy-related key proteins (ATG16L1, IRGM), and endoplasmic reticulum (ER) stress markers (GRP78, IRE1) were increased after S. Infantis infection. Notably, L. johnsonii L531 pretreatment not only inhibited the activation of the above signaling pathways but also played an anti-S. Infantis infection role in accelerating autophagic degradation. However, RIP2 knockdown did not interfere with ER stress and the activation of autophagy induced by S. Infantis in Caco2 cells. Our data suggest that L. johnsonii L531 pretreatment alleviates the intestinal damage caused by S. Infantis by inhibiting NOD activation and regulating ER stress, as well as promoting autophagic degradation.

Autophagy Plays a Protective Role in Sodium Hydrosulfide-Induced Acute Lung Injury by Attenuating Oxidative Stress and Inflammation in Rats.

Sodium hydrosulfide (NaHS), as an exogenous hydrogen sulfide (H2S) donor, has been used in various pathological models. NaHS is usually considered to be primarily protective, however, the toxic effect of NaHS has not been well elucidated. The aim of this study was to investigate whether NaHS (1 mg/kg) can induce acute lung injury (ALI is a disease process characterized by diffuse inflammation of the lung parenchyma) and define the mechanism by which NaHS-induced ALI involves autophagy, oxidative stress, and inflammatory response. Wistar rats were randomly divided into three groups (control group, NaHS group, and 3-MA + NaHS group), and samples from each group were collected from 2, 6, 12, and 24 h. We found that intraperitoneal injection of NaHS (1 mg/kg) increased the pulmonary levels of H2S and oxidative stress-related indicators (reactive oxygen species, myeloperoxidase, and malondialdehyde) in a time-dependent manner. Intraperitoneal injection of NaHS (1 mg/kg) induced histopathological changes of ALI and inhibition of autophagy exacerbated the lung injury. This study demonstrates that administration of NaHS (1 mg/kg) induces ALI in rats and autophagy in response to ROS is protective in NaHS-induced ALI by attenuating oxidative stress and inflammation.

Ultralow Icing Adhesion of a Superhydrophobic Coating Based on the Synergistic Effect of Soft and Stiff Particles.

A novel superhydrophobic coating composed of soft polydimethylsiloxane microspheres and stiff SiO2 nanoparticles was developed and prepared. This superhydrophobic coating showed excellent superhydrophobicity with a large water contact angle of 171.3° and also exhibited good anti-icing performance and ultralow icing adhesion of 1.53 kPa. Furthermore, the superhydrophobic coating displayed good icing/deicing cycle stability, in which the icing adhesion was still less than 10.0 kPa after 25 cycles. This excellent comprehensive performance is attributed to stress-localization between ice and the surface, resulting from the synergistic effect of soft and stiff particles. This work thus opens a new avenue to simultaneously optimize the anti-icing and icephobic performance of a superhydrophobic surface for various applications.

Walnut ethylene response factor JrERF2-2 interact with JrWRKY7 to regulate the GSTs in plant drought tolerance.

Juglans regia is a world-famous woody oil plant, whose yield and quality are affected by drought stress. Ethylene-responsive factors (ERFs) play vital role in plant stress response. In current study, to comprehend the walnut molecular mechanism of drought stress response, an ERF transcription factor was clarified from J. regia (JrERF2-2) and its potential function mechanism to drought was clarified. The results showed that JrERF2-2 could be induced significantly by drought. The transgenic Arabidopsis over-expression of JrERF2-2 displayed enhanced growth, antioxidant enzyme vitalities, reactive oxygen species scavenging and proline produce under drought stress. Especial the glutathione-S-transferase (GST) activity and most GST genes' transcription were elevated obviously. Yeast one-hybrid (Y1H) and co-transient expression (CTE) methods revealed that JrERF2-2 could recognize JrGST4, JrGST6, JrGST7, JrGST8, and JrGSTF8 by binding to GCC-box, and recognize JrGST11, JrGST12, and JrGSTN2 by binding to DRE motif. Meanwhile, the binding activity was strengthened by drought stress. Moreover, JrERF2-2 could interact with JrWRKY7 to promote plant drought tolerance; JrWRKY7 could also distinguish JrGST4, JrGST7, JrGST8, JrGST11, JrGST12, and JrGSTF8 via binding to W-Box motif. These results suggested that JrERF2-2 could effectively improve plant drought tolerance through interacting with JrWRKY7 to control the expression of GSTs. JrERF2-2 is a useful plant representative gene for drought response in molecular breeding.

Identification and characterization of 5 walnut MYB genes in response to drought stress involved in ABA signaling.

Walnut is a popular nut tree species and usually suffers from drought stress. However, little information is available on the mechanism of walnut responding to drought stress, resulting in lack of basic understanding for its resistance. In order to excavate more functional genes that can respond to stressors, and enrich the theoretical basis for walnut resistance, in this study, 5 MYB genes with complete ORFs were identified from J. regia and the basic bio-information as well as expression patterns in different tissues and response to drought and ABA stresses were confirmed using qRT-PCR assay. The results showed that 2 JrMYB genes belong to R1-MYB subfamily and 3 JrMYBs belong to R2R3-MYB, encoding the proteins from 212 to 362 aa in length. The phylogenetic analysis categorized proteins of 5 JrMYBs and 40 Arabidopsis AtMYBs into 10 subgroups. JrMYBs in the same subgroup exhibited significant similarities in the composition of conserved domains and motifs in amino acid sequences and exon/intron organization in DNA sequences. The results of qRT-PCR analysis revealed that JrMYB genes diversely expressed in various tissues. Moreover, the expression values of JrMYBs were upregulated or downregulated significantly under drought and ABA stresses. Most attractively, in contrast with suffering from drought stress alone, the treatments with drought and additional ABA greatly enhanced the transcript levels of JrMYBs. All these results suggested that JrMYB genes play a vital role in plant biological processes and drought as well as ABA stress response, and possibly perform as ABA-dependent drought response transcription factors in plant. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01008-z.

Genome-wide identification and transcript profiles of walnut heat stress transcription factor involved in abiotic stress.

BACKGROUND: Walnut (Juglans regia) is an important tree cultivated worldwide and is exposed to a series of both abiotic and biotic stress during their life-cycles. The heat stress transcription factors (HSFs) play a crucial role in plant response to various stresses by regulating the expression of stress-responsive genes. HSF genes are classified into 3 classes: HSFA, HSFB, and HSFC. HSFA gene has transcriptional activation function and is the main regulator of high temperature-induced gene expression. HSFB gene negatively regulates plant resistance to drought and NaCl. And HSFC gene may be involved in plant response to various stresses. There are some reports about the HSF family in herbaceous plants, however, there are no reports about the HSFs in walnut. RESULT: In this study, based on the complete genome sequencing of walnut, the bioinformatics method was used and 29 HSF genes were identified. These HSFs covered 18 HSFA, 9 HSFB, and 2 HSFC genes. Phylogenetic analysis of these HSF proteins along with those from Arabidopsis thaliana showed that the HSFs in the two species are closely related to each other and have different evolutionary processes. The distribution of conserved motifs and the sequence analysis of HSF genes family indicated that the members of the walnut HSFs are highly conserved. Quantitative Real-Time PCR (qRT-PCR) analysis revealed that the most of walnut HSFs were expressed in the walnut varieties of 'Qingxiang' and 'Xiangling' under high temperature (HT), high salt and drought stress, and some JrHSFs expression pattern are different between the two varieties. CONCLUSION: The complex HSF genes family from walnut was confirmed by genome-wide identification, evolutionary exploration, sequence characterization and expression analysis. This research provides useful information for future studies on the function of the HSF genes and molecular mechanism in plant stress response.

Anti-inflammatory effects of Lactobacillus johnsonii L531 in a pig model of Salmonella Infantis infection involves modulation of CCR6+ T cell responses and ER stress.

Probiotic pretreatment is an effective non-antibiotic strategy for preventing or controlling Salmonella infections. We found that Lactobacillus johnsonii L531, isolated from the colon of a clinically healthy weaned piglet, effectively prevented infection with Salmonella enterica serovar Infantis in a pig model. Newly weaned piglets were intragastrically administered Lactobacillus johnsonii L531 at 1.0 × 1010 CFU/day for 1 week before S. Infantis challenge. Pretreatment with L. johnsonii L531 lessened the severity of diarrhea and ileal inflammation in S. Infantis-infected piglets. Lactobacilli were more abundant in the ileum than jejunum after L. johnsonii L531 pretreatment. Treatment with L. johnsonii L531 reduced the abundance of total bacteria in the ileal mucosa and the production of lipocalin 2 in the jejunum of piglets challenged with Salmonella. Both intestinal morphology and transmission electron microscopy results indicated that L. johnsonii L531 alleviated intestinal tissue damage following S. Infantis challenge, especially in the villus and endoplasmic reticulum (ER). ER stress induced by S. Infantis was attenuated by L. johnsonii L531 treatment. The number of CD4- CCR6+ T cells decreased following S. Infantis challenge, but the percentage of CCR6- IFNγ+ T cells in peripheral blood increased. In intestinal mesenteric lymph nodes, S. Infantis increased the proportion of CCR6+ IFNγ+ T cells, whereas L. johnsonii L531 induced an increase in the proportion of CD4+ CCR6+ T cells in response to S. Infantis infection. Our data thus suggest that L. johnsonii L531 contributes to the maintenance of intestinal homeostasis by modulating T-cell responses and ER stress.

Integrated Analysis of Seed microRNA and mRNA Transcriptome Reveals Important Functional Genes and microRNA-Targets in the Process of Walnut (Juglans regia) Seed Oil Accumulation.

Walnut (Juglans regia) is known as a promising woody oil crop with abundant polyunsaturated fatty acids in its kernel. However, the regulation mechanism of walnut oil accumulation and fatty acid metabolism is still poorly understood, which restricted the breeding and genetic improvement of high-quality oil-bearing walnuts. To reveal the molecular mechanism of walnut oil accumulation, considering the potential regulation of microRNA (miRNA) in seed development, in this study, the oil content of walnut kernel on the 80th, 100th and 120th day after flowering (DAF) was tested and the corresponding proportions are 11.51%, 40.40% and 53.20%. Between DAF of 80th~120th, the content of stearic acid and oleic acid tended to increase, but the proportion of other fatty acids tended to decrease. Meanwhile, comparative transcriptome and sRNA-seq analysis on three stages (80th, 100th and 120th DAF), found 204 conserved miRNAs and 554 novel miRNAs in walnut kernels, among which 104 key genes related to walnut oil accumulation were screened. The phospholipid:diacylglycerol acyltransferase metabolic pathway may contribute more to oil accumulation in walnut. 16 miRNA-mRNA regulatory modules related to walnut oil accumulation and fatty acid synthesis were constructed. 8 known miRNAs and 9 novel miRNAs regulate 28 genes involved in fatty acid (FA) metabolism and lipid synthesis. Among them, jre-miRn105, jre-miRn434, jre-miR477d and jre-miR156a.2 are key miRNAs that regulate walnut FA synthesis. Jre-miRn411 and jre-miR399a.1 are closely related to oil accumulation. These data provide new insights and lay the foundation for subsequent studies on walnut FA synthesis and oil accumulation.

Multiple transcriptional regulation of walnut JrGSTTau1 gene in response to osmotic stress.

Glutathione S-transferases (GSTs) are important plant proteins involved in biotic and abiotic stress responses. A gene from Juglans regia, JrGSTTau1 was previously cloned and functionally characterized as an enzyme involved in improving cold tolerance in plants. To clarify the functional mechanism of JrGSTTau1 and its role in stress response, here, the JrGSTTau1 promoter including the up-stream regulators was examined using yeast one-hybrid together with transient expression assays, and the osmotic stress response ability was confirmed by comparing with wild-type plants. The 1500 bp JrGSTTau1 promoter displayed high GUS expression activity and was enhanced by mannitol stress. The promoter is composed of abundant cis-elements, some of which were osmotic stress response-related motifs, such as ABRE, DRE and MYB, indicating that the expression of JrGSTTau1 is regulated by potential up-stream regulators under abiotic stress. The transcription factors (TFs) of JrDREB2A, JrMYC2, JrMYB44, JrDof1 and JrWRKY7 were identified, which shared a similar response with JrGSTTau1 when exposed to PEG6000 in walnut leaf and root. These results implied that JrDREB2A, JrMYC2, JrMYB44, JrDof1 and JrWRKY7 may act as up-stream regulators of JrGSTTau1 to regulate or combine functionality with JrGSTTau1 in osmotic stress response. Furthermore, compared with the WT plants, the transgenic tobacco plants that overexpress JrGSTTau1 showed improved tolerance to drought induced by osmotic stress, in which antioxidant enzymes, proline and reactive oxygen species (ROS) are involved. Our results demonstrated the positive role played by JrGSTTau1 in osmotic tolerance, which is regulated by multiple up-stream regulators.

B cell activation factor (BAFF) induces inflammation in the human fallopian tube leading to tubal pregnancy.

BACKGROUND: Tubal pregnancy is recognized as one of the most common ectopic pregnancy types. Salpingitis may result in tubal pregnancy by causing fallopian tube occlusion and hydrosalpinx. B cell activation factor (BAFF) is a proinflammatory cytokine that helps regulate both innate and adaptive immune responses. Our previous study firstly showed that BAFF immunostaining appeared on the cellular membrane and in the cytoplasm of tubal epithelial cells, and both BAFF protein and mRNA in human inflamed fallopian tubes had higher expression levels than those in normal fallopian tubes. This study aimed to elucidate the association between the expression of BAFF gene and the inflammation in the human fallopian tube leading to tubal pregnancy. METHODS: We examined 70 patients undergoing salpingectomy for salpingitis (n = 35) and tubal pregnancy (n = 35). Twenty patients with benign uterine diseases undergoing complete hysterectomy and salpingectomy were recruited into control group. BAFF mRNA and protein in tissue samples were detected by qPCR and Western blotting methods. Furthermore, serum levels of BAFF, tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 were measured using ELISA kits. RESULTS: We found statistically significantly elevated expressions of BAFF mRNA or protein in whole tissue samples, and serum levels of BAFF, TNF-α and IL-6 in whole blood samples from patients with salpingitis and tubal pregnancy, in comparison to the control group. CONCLUSION: Based on the results, high expression of BAFF gene might induce inflammation in the human fallopian tube, suggesting its possible role in the tubal pregnancy process.

New insight into the molecular basis of cadmium stress responses of wild paper mulberry plant by transcriptome analysis.

BACKGROUND: Heavy metal contamination is becoming a limitation to the utilization of soil and the distribution of vegetation. In particular, cadmium (Cd) pollution has had a serious impact on the food chain. Broussonetia papyrifera is a widely distributed pioneer tree species of heavy metal contaminated areas with important economic value. However, little is known about the genomic background of the Cd-tolerance mechanism in B. papyrifera. RESULTS: The CdCl2 responsive physiology was evaluated and proved to be involved in antioxidase activity and active oxygen species (ROS) accumulation. The leaf and root transcriptomes derived from B. papyrifera grown under normal and CdCl2 stress conditions were systematically investigated using the Illumina HiSeq method. A total of 180,678,660 bp (27.1 GB) clean reads were assembled into 589,487 high-quality unigenes, of which 256,025 (43.43% of the total) and 250,251 (42.45% of the total) were aligned in Gene Ontology (GO) and Protein family (Pfam), respectively. A total of 24,414 differentially expressed genes (DEGs) were GO-annotated into 53, 23, 55, and 60 terms from the transcriptomes of root and leaf tissues under Cd stress and control conditions. A total of 117,547 Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthology (KO)-annotated DEGs were enriched in at least 47 KEGG pathway terms among the four comparisons. Many genes encoding important transcription factors (e.g., auxin/indole-3-acetic acid (AUX/IAA), basic helix-loop-helix (bHLH), DNA-binding one zinc finger (Dof), and MYB) and proteins involved in plant-pathogen interactions, phenylpropanoid biosynthesis, plant hormone signal transduction, oxidative phosphorylation, carbon fixation, peroxisomes, flavonoid biosynthesis, and glutathione metabolism, among others, were substantially upregulated under CdCl2 stress. CONCLUSIONS: These genes represent important candidates for studying Cd-response mechanisms and molecular biology of B. papyrifera and related species. Our findings provide a genomic sequence resource for functional genetic assignments in B. papyrifera, which will help elucidate the molecular mechanisms of its Cd-stress responses and facilitate the bioremediation of heavy metal contaminated areas via breeding of new stress-tolerant cultivars.

The walnut JrVHAG1 gene is involved in cadmium stress response through ABA-signal pathway and MYB transcription regulation.

BACKGROUND: Vacuolar H+-ATPase (V-ATPase) is a vital protein complex involved in abiotic stress response in plants. The G subunit of Juglans regia (JrVHAG1) was previously identified as a drought tolerance-related gene involved in the ABA (abscisic acid)-signal pathway. Heavy metal stress is becoming a major detriment for plant growth, development, and production. In order to understand the role of JrVHAG1, the potential function mechanism of JrVHAG1 exposed to CdCl2 stress was confirmed in this study. RESULTS: Transcription of JrVHAG1 was induced by ABA and increased to 58.89-fold (roots) and 7.38-fold (leaves) and by CdCl2 to 2.65- (roots) and 11.42-fold (leaves) relative to control, respectively. Moreover, when treated simultaneously with ABA and CdCl2 (ABA+CdCl2), JrVHAG1 was up-regulated to 110.13- as well as 165.42-fold relative to control in the roots and leaves, accordingly. Compared to the wild type (WT) Arabidopsis plants, the transgenic plants with overexpression of JrVHAG1 (G2, G6, and G9) exhibited increased seed germination rate, biomass accumulation, proline content, and activities of superoxide dismutase (SOD) and peroxidase (POD) under ABA, CdCl2, and ABA+CdCl2 treatments. In contrast, the reactive oxygen species (ROS) staining, malondialdehyde (MDA) content, hydrogen dioxide (H2O2) content, as well as electrolyte leakage (EL) rates of transgenic seedlings were all lower than those of WT exposed to ABA, CdCl2 and ABA+CdCl2 stresses. Furthermore, a 1200 bp promoter fragment of JrVHAG1 was isolated by analyzing the genome of J. regia, in which the cis-elements were identified. This JrVHAG1 promoter fragment showed expression activity that was enhanced significantly when subjected to the above treatments. Yeast one-hybrid assay and transient expression analysis demonstrated that JrMYB2 specifically bound to the MYBCORE motif and shared similar expression patterns with JrVHAG1 under ABA, CdCl2 and ABA+CdCl2 stress conditions. CONCLUSIONS: Our results suggested that the JrVHAG1 gene functions as a CdCl2 stress response regulator by participating in ABA-signal pathway and MYB transcription regulation network. JrVHAG1 gene is a useful candidate gene for heavy metal stress tolerance in plant molecular breeding.

The walnut transcription factor JrGRAS2 contributes to high temperature stress tolerance involving in Dof transcriptional regulation and HSP protein expression.

BACKGROUND: GRAS transcription factor (TF) family is unique and numerous in higher plants with diverse functions that involving in plant growth and development processes, such as gibberellin (GA) signal transduction, root development, root nodule formation, and mycorrhiza formation. Walnut tree is exposed to various environmental stimulus that causing concern about its resistance mechanism. In order to understand the molecular mechanism of walnut to adversity response, a GRAS TF (JrGRAS2) was cloned and characterized from Juglans regia in this study. RESULTS: A 1500 bp promoter fragment of JrGRAS2 was identified from the genome of J. regia, in which the cis-elements were screened. This JrGRAS2 promoter displayed expression activity that was enhanced significantly by high temperature (HT) stress. Yeast one-hybrid assay, transient expression and chromatin immunoprecipitation (Chip)-PCR analysis revealed that JrDof3 could specifically bind to the DOFCOREZM motif and share similar expression patterns with JrGRAS2 under HT stress. The transcription of JrGRAS2 was induced by HT stress and up-regulated to 6.73-~11.96-fold in the leaf and 2.53-~4.50-fold in the root to control, respectively. JrGRAS2 was overexpressed in Arabidopsis, three lines with much high expression level of JrGRAS2 (S3, S7, and S8) were selected for HT stress tolerance analysis. Compared to the wild type (WT) Arabidopsis, S3, S7, and S8 exhibited enhanced seed germination rate, fresh weight accumulation, and activities of catalase (CAT), peroxidase (POD), superoxide dismutase (SOD) and glutathione-S-transferase (GST) under HT stress. In contrast, the Evans blue staining, electrolyte leakage (EL) rates, hydrogen dioxide (H2O2) and malondialdehyde (MDA) content of transgenic seedlings were all lower than those of WT exposed to HT stress. Furthermore, the expression of heat shock proteins (HSPs) in S3, S7, and S8 was significant higher than those in WT plants. The similar results were obtained in JrGRAS2 transient overexpression walnut lines under normal and HT stress conditions. CONCLUSIONS: Our results suggested that JrDof3 TF contributes to improve the HT stress response of JrGRAS2, which could effectively control the expression of HSPs to enhance HT stress tolerance. JrGRAS2 is an useful candidate gene for heat response in plant molecular breeding.

Near-infrared hyperspectral imaging for detection and quantification of azodicarbonamide in flour.

BACKGROUND: The present study aimed to establish a method for the detection and quantification of azodicarbonamide (ADC) in flour using hyperspectral imaging technology. Hyperspectral images of pure flour, pure ADC and flour-ADC mixtures with different concentrations of ADC were collected. F-values of one-way analysis of variance for all possible wavebands within the spectra of the flour and ADC were calculated, and the maximum value indicated that the two wavebands have more significant differences, i.e. the optimal two wavebands. Threshold segmentation was used for band ratio images of two wavebands to create a binary image. This allowed visual identification of ADC-rich pixels in the mixtures. RESULTS: The two wavebands with the largest difference between flour and ADC were 2039 nm and 1892 nm. Using the binary image construction method, different concentrations of ADC in flour were identified. The minimum detected concentration was 0.2 g kg-1 . In the mixtures, the number of ADC-rich pixels detected had a good linear relationship with the ADC concentrations, with a correlation coefficient of 0.9845. CONCLUSION: This study indicated that the band ratio algorithm combination with threshold segmentation for hyperspectral images provides a non-destructive method for detecting and quantifying of ADC in flour. © 2017 Society of Chemical Industry.

Characterization of a vacuolar H+-ATPase G subunit gene from Juglans regia (JrVHAG1) involved in mannitol-induced osmotic stress tolerance.

KEY MESSAGE: JrVHAG1 is an important candidate gene for plant osmotic tolerance regulation. Vacuolar H+-ATPase (V-ATPase) is important for plant responses to abiotic stress; the G subunit is a vital part of V-ATPase. In this study, a G subunit of V-ATPase was cloned from Juglans regia (JrVHAG1) and functionally characterized. JrVHAG1 transcription was induced by mannitol that increasing 17.88-fold in the root at 12 h and 19.16-fold in the leaf at 96 h compared to that under control conditions. JrVHAG1 was overexpressed in Arabidopsis and three lines (G2, G6, and G9) with highest expression levels were selected for analysis. The results showed that under normal conditions, the transgenic and wild-type (WT) plants displayed similar germination, biomass accumulation, reactive oxygen species (ROS) level, and physiological index. However, when treated with mannitol, the fresh weight, root length, water-holding ability, and V-ATPase, superoxide dismutase, and peroxidase activity of G2, G6, and G9 were significantly higher than those of WT. In contrast, the ROS and cell damage levels of the transgenic seedlings were lower than those of WT. Furthermore, the transcription levels of V-ATPase subunits, ABF, DREB, and NAC transcription factors (TFs), all of which are factors of ABA signaling pathway, were much higher in JrVHAG1 transgenic plants than those in WT. The positive induction of JrVHAG1 gene under abscisic acid (ABA) treatments in root and leaf tissues indicates that overexpression of JrVHAG1 improves plant tolerance to osmotic stress relating to the ABA signaling pathway, which is transcriptionally activated by ABF, DREB, and NAC TFs, and correlated to ROS scavenging and V-ATPase activity.

Influence of orally fed a select mixture of Bacillus probiotics on intestinal T-cell migration in weaned MUC4 resistant pigs following Escherichia coli challenge.

Efficient strategies for treating enteritis caused by F4(+) enterotoxigenic Escherichia coli (ETEC)/verocytotoxigenic Escherichia coli (VTEC)/enteropathogenic E. coli (EPEC) in mucin 4 resistant (MUC4 RR; supposed to be F4ab/ac receptor-negative [F4ab/acR(-)]) pigs remain elusive. A low (3.9 × 10(8) CFU/day) or high (7.8 × 10(8) CFU/day) dose of Bacillus licheniformis and Bacillus subtilis spore mixture (BLS-mix) was orally administered to MUC4 RR piglets for 1 week before F4(+) ETEC/VTEC/EPEC challenge. Orally fed BLS-mix upregulated the expression of TLR4, NOD2, iNOS, IL-8, and IL-22 mRNAs in the small intestine of pigs challenged with E. coli. Expression of chemokine CCL28 and its receptor CCR10 mRNAs was upregulated in the jejunum of pigs pretreated with high-dose BLS-mix. Low-dose BLS-mix pretreatment induced an increase in the proportion of peripheral blood CD4(-)CD8(-) T-cell subpopulations and high-dose BLS-mix induced the expansion of CD4(-)CD8(-) T cells in the inflamed intestine. Immunostaining revealed that considerable IL-7Rα-expressing cells accumulated at the lamina propria of the inflamed intestines after E. coli challenge, even in pigs pretreated with either low- or high-dose BLS-mix, although Western blot analysis of IL-7Rα expression in the intestinal mucosa did not show any change. Our data indicate that oral administration of the probiotic BLS-mix partially ameliorates E. coli-induced enteritis through facilitating upregulation of intestinal IL-22 and IκBα expression, and preventing loss of intestinal epithelial barrier integrity via elevating ZO-1 expression. However, IL-22 also elicits an inflammatory response in inflamed intestines as a result of infection with enteropathogenic bacteria.