The WRKY gene family in the halophyte Limonium bicolor: identification, expression analysis, and regulation of salt stress tolerance.

KEY MESSAGE: 63 L. bicolor WRKY genes were identified and their informatics was analyzed. The results suggested that the LbWRKY genes involved in the development and salt secretion of salt glands in L. bicolor. Salt stress, as a universal abiotic stress, severely inhibits the growth and development of plants. WRKY transcription factors play a vital role in plant growth and development, as well as in response to various stresses. Nevertheless, little is known of systematic genome-wide analysis of the WRKY genes in Limonium bicolor, a model recretohalophyte. In this study, 63 L. bicolor WRKY genes were identified (LbWRKY1-63), which were unevenly distributed across seven chromosomes and one scaffold. Based on the structural and phylogenetic characteristics, 63 LbWRKYs are divided into three main groups. Cis-elements in the LbWRKY promoters were related to growth and development, phytohormone responses, and stress responses. Colinearity analysis showed strong colinearity between LbWRKYs and GmWRKYs from soybean (Glycine max). Therefore, LbWRKY genes maybe have similar functions to GmWRKY genes. Expression analysis showed that 28 LbWRKY genes are highly expressed in roots, 9 in stems, 26 in leaves, and 12 in flowers and most LbWRKY genes responded to NaCl, ABA, and PEG6000. Silencing LbWRKY10 reduced salt gland density and salt secretion ability of leaves, and the salt tolerance of the species. Consistent with this, genes associated with salt gland development were markedly down-regulated in the LbWRKY10-silenced lines. Our findings suggested that the LbWRKY genes involved in the development and salt secretion of salt glands in L. bicolor. Our research provides new insights into the functions of the WRKY family in halophytes.

Obesity induced disruption on diurnal rhythm of insulin sensitivity via gut microbiome-bile acid metabolism.

The disruption of the diurnal rhythm has been recognized as a significant contributing factor to metabolic dysregulation. The important role of gut microbiota and bile acid metabolism has attracted extensive attention. However, the function of the gut microbiota-bile acid axis in regulating the diurnal rhythms of metabolic homeostasis remains largely unknown. Herein, we aimed to investigate the interplay between rhythmicity of host metabolism and gut microbiota-bile acid axis, as well as to assess the impact of obesity on them. We found that high fat diet feeding and Leptin gene deficiency (ob/ob) significantly disturbed the rhythmic patterns of insulin sensitivity and serum total cholesterol levels. The bile acid profiling unveiled a conspicuous diurnal rhythm oscillation of ursodeoxycholic acid (UDCA) in lean mice, concomitant with fluctuations in insulin sensitivity, whereas it was absent in obese mice. The aforementioned diurnal rhythm oscillations were largely desynchronized by gut microbiota depletion, suggesting the indispensable role of gut microbiota in diurnal regulation of insulin sensitivity and bile acid metabolism. Consistently, 16S rRNA sequencing revealed that UDCA-associated bacteria exhibited diurnal rhythm oscillations that paralleled the fluctuation in insulin sensitivity. Collectively, the current study provides compelling evidence regarding the association between diurnal rhythm of insulin sensitivity and gut microbiota-bile acid axis. Moreover, we have elucidated the deleterious effects of obesity on gut microbiome-bile acid metabolism in both the genetic obesity model and the diet-induced obesity model.

Ootheca mantidis mitigates renal fibrosis in mice by the suppression of apoptosis via increasing the gut microbe Akkermansia muciniphila and modulating glutamine metabolism.

Renal interstitial fibrosis (RIF), a progressive process affecting the kidneys in chronic kidney disease (CKD), currently lacks an effective therapeutic intervention. Traditional Chinese medicine (TCM) has shown promise in reducing RIF and slowing CKD progression. In this study, we demonstrated the dose-dependent attenuation of RIF by Ootheca mantidis (SPX), a commonly prescribed TCM for CKD, in a mouse model of unilateral ureteral obstruction (UUO). RNA-sequencing analysis suggested that SPX treatment prominently downregulated apoptosis and inflammation-associated pathways, thereby inhibiting the fibrogenic signaling in the kidney. We further found that transplantation of fecal microbiota from SPX-treated mice conferred protection against renal injury and fibrosis through suppressing apoptosis in UUO mice, indicating that SPX ameliorated RIF via remodeling the gut microbiota and reducing apoptosis in the kidneys. Further functional exploration of the gut microbiota combined with fecal metabolomics revealed increased levels of some probiotics, including Akkermansia muciniphila (A. muciniphila), and modulations in glutamine-related amino acid metabolism in UUO mice treated with SPX. Subsequent colonization of A. muciniphila and supplementation with glutamine effectively mitigated cell apoptosis and RIF in UUO mice. Collectively, these findings unveil a functionally A. muciniphila- and glutamine-involved gut-renal axis that contributes to the action of SPX, and provide important clue for the therapeutic potential of SPX, A. muciniphila, and glutamine in combatting RIF.

Development of a simultaneous quantification method for the gut microbiota-derived core nutrient metabolome in mice and its application in studying host-microbiota interaction.

The gut microbiota interacts with the host via production of various metabolites of dietary nutrients. Herein, we proposed the concept of the gut microbiota-derived core nutrient metabolome, which covers 43 metabolites in carbohydrate metabolism, glycolysis, tricarboxylic acid cycle and amino acid metabolism, and established a quantitative UPLC-Q/TOF-MS method through 3-nitrophenylhydrazine derivatization to investigate the influence of obesity on the gut microbiota in mice. All metabolites could be simultaneously analyzed via separation on a BEH C18 column within 18 min. The lower limits of quantification of most analytes were less than 1 µM. Validation results demonstrated suitability for the analysis of mouse fecal samples. The method was then applied to detect the gut microbiota-derived nutrient metabolome in the feces of high-fat diet induced obese (DIO) and ob/ob (leptin-deficient) mice, as well as obesity-prone (OP) and obesity-resistant (OR) mice. Compared to the control groups, there were 13, 23 and 10 differentially abundant metabolites detected in ob/ob, DIO and OP groups, respectively. Among them, amino acids including leucine, isoleucine, glycine, methionine, tyrosine and glutamine were co-downregulated in the obese or OP mice and exhibited inverse association with body weight. 16S rDNA analysis revealed that the genera Lactobacillus and Dubosiella were also inversely associated with body weight and positively correlated with fecal amino acids. Collectively, our work provides an effective and simplified method for simultaneous quantifying the gut microbiota-derived core nutrient metabolome in mouse feces, which could assist various future studies on host-microbiota metabolic interaction.

Limosilactobacillus reuteri and caffeoylquinic acid synergistically promote adipose browning and ameliorate obesity-associated disorders.

OBJECTIVE: High intake of caffeoylquinic acid (CQA)-rich dietary supplements, such as green coffee bean extracts, offers health-promoting effects on maintaining metabolic homeostasis. Similar to many active herbal ingredients with high pharmacological activities but low bioavailability, CQA has been reported as a promising thermogenic agent with anti-obesity properties, which contrasts with its poor oral absorption. Intestinal tract is the first site of CQA exposure and gut microbes might react quickly to CQA. Thus, it is of interest to explore the role of gut microbiome and microbial metabolites in the beneficial effects of CQA on obesity-related disorders. RESULTS: Oral CQA supplementation effectively enhanced energy expenditure by activating browning of adipose and thus ameliorated obesity-related metabolic dysfunctions in high fat diet-induced obese (DIO) mice. Here, 16S rRNA gene amplicon sequencing revealed that CQA treatment remodeled the gut microbiota to promote its anti-obesity actions, as confirmed by antibiotic treatment and fecal microbiota transplantation. CQA enriched the gut commensal species Limosilactobacillus reuteri (L. reuteri) and stimulated the production of short-chain fatty acids, especially propionate. Mono-colonization of L. reuteri or low-dose CQA treatment did not reduce adiposity in DIO mice, while their combination elicited an enhanced thermogenic response, indicating the synergistic effects of CQA and L. reuteri on obesity. Exogenous propionate supplementation mimicked the anti-obesity effects of CQA alone or when combined with L. reuteri, which was ablated by the monocarboxylate transporter (MCT) inhibitor 7ACC1 or MCT1 disruption in inguinal white adipose tissues to block propionate transport. CONCLUSIONS: Our data demonstrate a functional axis among L. reuteri, propionate, and beige fat tissue in the anti-obesity action of CQA through the regulation of thermogenesis. These findings provide mechanistic insights into the therapeutic use of herbal ingredients with poor bioavailability via their interaction with the gut microbiota. Video Abstract.

Hepatic cytochrome P450 8B1 and cholic acid potentiate intestinal epithelial injury in colitis by suppressing intestinal stem cell renewal.

Although disrupted bile acid (BA) homeostasis is implicated in inflammatory bowel disease (IBD), the role of hepatic BA metabolism in the pathogenesis of colitis is poorly understood. Here, we found that cholic acid (CA) levels were increased in patients and mice. Cytochrome P450 8B1 (CYP8B1), which synthesizes CA, was induced in livers of colitic mice. CA-treated or liver Cyp8b1-overexpressing mice developed more severe colitis with compromised repair of the mucosal barrier, whereas Cyp8b1-knockout mice were resistant to colitis. Mechanistically, CA inhibited peroxisome proliferator-activated receptor alpha (PPARα), resulting in impeded fatty acid oxidation (FAO) and impaired Lgr5+ intestinal stem cell (ISC) renewal. A PPARα agonist restored FAO and improved Lgr5+ ISC function. Activation of the farnesoid X receptor (FXR) suppressed liver CYP8B1 expression and ameliorated colitis in mice. This study reveals a connection between the hepatic CYP8B1-CA axis and colitis via regulating intestinal epithelial regeneration, suggesting that BA-based strategies might be beneficial in IBD treatment.

Melatonin increases growth and salt tolerance of Limonium bicolor by improving photosynthetic and antioxidant capacity.

BACKGROUND: Soil salinization is becoming an increasingly serious problem worldwide, resulting in cultivated land loss and desertification, as well as having a serious impact on agriculture and the economy. The indoleamine melatonin (N-acetyl-5-methoxytryptamine) has a wide array of biological roles in plants, including acting as an auxin analog and an antioxidant. Previous studies have shown that exogenous melatonin application alleviates the salt-induced growth inhibition in non-halophyte plants; however, to our knowledge, melatonin effects have not been examined on halophytes, and it is unclear whether melatonin provides similar protection to salt-exposed halophytic plants. RESULTS: We exposed the halophyte Limonium bicolor to salt stress (300 mM) and concomitantly treated the plants with 5 µM melatonin to examine the effect of melatonin on salt tolerance. Exogenous melatonin treatment promoted the growth of L. bicolor under salt stress, as reflected by increasing its fresh weight and leaf area. This increased growth was caused by an increase in net photosynthetic rate and water use efficiency. Treatment of salt-stressed L. bicolor seedlings with 5 µM melatonin also enhanced the activities of antioxidants (superoxide dismutase [SOD], peroxidase [POD], catalase [CAT], and ascorbate peroxidase [APX]), while significantly decreasing the contents of hydrogen peroxide (H2O2), superoxide anion (O2•-), and malondialdehyde (MDA). To screen for L. bicolor genes involved in the above physiological processes, high-throughput RNA sequencing was conducted. A gene ontology enrichment analysis indicated that genes related to photosynthesis, reactive oxygen species scavenging, the auxin-dependent signaling pathway and mitogen-activated protein kinase (MAPK) were highly expressed under melatonin treatment. These data indicated that melatonin improved photosynthesis, decreased reactive oxygen species (ROS) and activated MAPK-mediated antioxidant responses, triggering a downstream MAPK cascade that upregulated the expression of antioxidant-related genes. Thus, melatonin improves the salt tolerance of L. bicolor by increasing photosynthesis and improving cellular redox homeostasis under salt stress. CONCLUSIONS: Our results showed that melatonin can upregulate the expression of genes related to photosynthesis, reactive oxygen species scavenging and mitogen-activated protein kinase (MAPK) of L. bicolor under salt stress, which can improve photosynthesis and antioxidant enzyme activities. Thus melatonin can promote the growth of the species and maintain the homeostasis of reactive oxygen species to alleviate salt stress.

Development and characterization of EST-SSR markers via transcriptome sequencing in Brainea insignis (Aspleniaceae s.l.).

PREMISE OF THE STUDY: Brainea insignis (Aspleniaceae) is an endangered tree fern in China whose wild populations have been seriously damaged due to overexploitation. Expressed sequence tag-simple sequence repeat (EST-SSR) primers were developed to investigate its genetic diversity and provide resources for future conservation studies. METHODS AND RESULTS: We obtained 72,897 unigenes of B. insignis using transcriptome sequencing and detected 15,006 SSRs in 12,058 unigenes. Based on these results, we designed 100 EST-SSR primer pairs and successfully amplified 52 of them in six individuals; 27 demonstrated polymorphisms after amplification against 72 individuals across three populations. Allele numbers ranged from three to 10, and the observed and expected heterozygosities ranged from 0.105 to 1.000 and from 0.523 to 0.865, respectively, in the tested populations. Most of these primers could be successfully amplified in two other fern species (Blechnumorientale and Chieniopteris harlandii). CONCLUSIONS: These selected EST-SSRs are valuable for genetic diversity and conservation studies in B. insignis and other related fern species.

Genetic diversity and community structure of rhizobia nodulating Sesbania cannabina in saline-alkaline soils.

Sesbania cannabina is a plant that grows naturally along the seashores in Rudong County, China (RDC) and it has been introduced into the Yellow River Delta (YRD) as a pioneer plant to improve the saline-alkaline soils. In order to investigate the diversity of S. cannabina rhizobia in these soils, a total of 198 rhizobial isolates were characterized and phylogenetic trees were constructed based on data from multilocus sequence analysis (MLSA) of the housekeeping genes recA, atpD and glnII, as well as 16S rRNA. Symbiotic features were also studied by establishing the phylogeny of the symbiotic genes nodA and nifH, and by performing nodulation assays. The isolates had highly conserved symbiotic genes and were classified into nine genospecies belonging to the genera Ensifer, Agrobacterium, Neorhizobium and Rhizobium. A unique community structure was detected in the rhizobia associated with S. cannabina in the saline-alkaline soils that was characterized by five novel genospecies and four defined species. In addition, Ensifer sp. I was the predominant rhizobia in YRD, whereas Ensifer meliloti and Neorhizobium huautlense were the dominant species in RDC. Therefore, the study demonstrated for the first time that this plant strongly selected the symbiotic gene background but not the genomic background of its microsymbionts. In addition, biogeographic patterns existed in the rhizobial populations associated with S. cannabina, which were mainly correlated with pH and salinity, as well as the mineral nutrient contents. This study provided novel information concerning the interaction between soil conditions, host plant and rhizobia, in addition to revealing the diversity of S. cannabina rhizobia in saline-alkaline soils.

[Genetic diversity of the Rhizobia and screening of high-efficient growth-promoting strains isolated from Sesbania cannabina in Rudong County].

OBJECTIVE: To study the genetic diversity and phylogeny of Rhizobia isolated from Sesbania cannabina growing on the tidal flat in Rudong County and screen high-efficient growth-promoting strains as Rhizobia inoculator to S. cannabina. METHODS: Phylogenetic analyses were based on 16S rRNA gene, housekeeping genes (recA, atpD, glnII) and symbiotic genes (nodA, nifH). The growth-promoting efficiency was tested by plant inoculation assay on S. cannabina in greenhouse. RESULTS: The 32 isolates belonged to Ensifer, Neorhizobium, Rhizobium, and most closely related to E. meliloti, N. huautlense, R. pusense. The phylogenies of nodA and nifH were congruent, and most closely related to E. saheli. The 7 representative isolates were resistant to high concentration of NaCl (5%, W/V), and YIC5082 grew well in TY medium with 6% NaCl. In plant inoculation assay, all the 7 representative isolates were effective on symbiotic nitrogen fixation, and 6 out of the 7 isolates significantly enhanced the fresh weight and height of plants. CONCLUSION: Rhizobia isolated from S. cannabina growing on the tidal flat in Rudong County showed rich genetic diversity. N. huautlense and E. meliloti were the dominant species. Most of the isolates showed fine growth-promoting efficiency and salt tolerance. YIC5077 showed the best growth-promoting efficiency, good nodulation and nitrogen fixation abilities, which has promising potential applications as Rhizobia inoculator to S. cannabina.

Hydrofluoric acid etched stainless steel wire for solid-phase microextraction.

Stainless steel wire has been widely used as the substrate of solid-phase microextraction (SPME) fibers to overcome the shortcomings of conventional silica fibers such as fragility, by many researchers. However, in previous reports various sorbent coatings are always required in conjunction with the stainless steel wire for SPME. In this work, we report the bare stainless steel wire for SPME without the need for any additional coatings taking advantage of its high mechanical and thermal stability. To evaluate the performance of stainless steel wire for SPME, polycyclic aromatic hydrocarbons (PAHs), benzene, toluene, ethylbenzene, chlorobenzene, n-propylbenzene, aniline, phenol, n-hexane, n-octane, n-decane, n-undecane, n-dodecane, chloroform, trichloroethylene, n-octanol, and butanol were tested as analytes. Although the stainless steel wire had almost no extraction capability toward the tested analytes before etching, it did exhibit high affinity to the tested PAHs after etching with hydrofluoric acid. The etched stainless steel wire gave a much bigger enhancement factor (2541-3981) for the PAHs than the other analytes studied (< or = 515). Etching with hydrofluoric acid produced a porous and flower-like structure with Fe(2)O(3), FeF(3), Cr(2)O(3), and CrF(2) on the surface of the stainless steel wire, giving high affinity to the PAHs due to cation-pi interaction. On the basis of the high selectivity of the etched stainless steel wire for PAHs, a new SPME method was developed for gas chromatography with flame ionization detection to determine PAHs with the detection limits of 0.24-0.63 microg L(-1). The precision for six replicate extractions using one SPME fiber ranged from 2.9% to 5.3%. The fiber-to-fiber reproducibility for three parallel prepared fibers was 4.3-8.8%. One etched stainless steel wire can stand over 250 cycles of SPME without significant loss of extraction efficiency. The developed etched stainless steel wire is very stable, highly selective, and reproducible for the SPME of PAHs.