Enhancing cold resistance in Banana (Musa spp.) through EMS-induced mutagenesis, L-Hyp pressure selection: phenotypic alterations, biomass composition, and transcriptomic insights.

BACKGROUND: The cultivation of bananas encounters substantial obstacles, particularly due to the detrimental effects of cold stress on their growth and productivity. A potential remedy that has gained attention is the utilization of ethyl mesylate (EMS)-induced mutagenesis technology, which enables the creation of a genetically varied group of banana mutants. This complex procedure entails subjecting the mutants to further stress screening utilizing L-Hyp in order to identify those exhibiting improved resistance to cold. This study conducted a comprehensive optimization of the screening conditions for EMS mutagenesis and L-Hyp, resulting in the identification of the mutant cm784, which exhibited remarkable cold resistance. Subsequent investigations further elucidated the physiological and transcriptomic responses of cm784 to low-temperature stress. RESULTS: EMS mutagenesis had a substantial effect on banana seedlings, resulting in modifications in shoot and root traits, wherein a majority of seedlings exhibited delayed differentiation and limited elongation. Notably, mutant leaves displayed altered biomass composition, with starch content exhibiting the most pronounced variation. The application of L-Hyp pressure selection aided in the identification of cold-resistant mutants among seedling-lethal phenotypes. The mutant cm784 demonstrated enhanced cold resistance, as evidenced by improved survival rates and reduced symptoms of chilling injury. Physiological analyses demonstrated heightened activities of antioxidant enzymes and increased proline production in cm784 when subjected to cold stress. Transcriptome analysis unveiled 946 genes that were differentially expressed in cm784, with a notable enrichment in categories related to 'Carbohydrate transport and metabolism' and 'Secondary metabolites biosynthesis, transport, and catabolism'. CONCLUSION: The present findings provide insights into the molecular mechanisms that contribute to the heightened cold resistance observed in banana mutants. These mechanisms encompass enhanced carbohydrate metabolism and secondary metabolite biosynthesis, thereby emphasizing the adaptive strategies employed to mitigate the detrimental effects induced by cold stress.

A functional study reveals CsNAC086 regulated the biosynthesis of flavonols in Camellia sinensis.

MAIN CONCLUSION: CsNAC086 was found to promote the expression of CsFLS, thus promoting the accumulation of flavonols in Camellia sinensis. Flavonols, the main flavonoids in tea plants, play an important role in the taste and quality of tea. In this study, a NAC TF gene CsNAC086 was isolated from tea plants and confirmed its regulatory role in the expression of flavonol synthase which is a key gene involved in the biosynthesis of flavonols in tea plant. Yeast transcription-activity assays showed that CsNAC086 has self-activation activity. The transcriptional activator domain of CsNAC086 is located in the non-conserved C-terminal region (positions 171-550), while the conserved NAC domain (positions 1-170) does not have self-activation activity. Silencing the CsNAC086 gene using antisense oligonucleotides significantly decreased the expression of CsFLS. As a result, the concentration of flavonols decreased significantly. In overexpressing CsNAC086 tobacco leaves, the expression of NtFLS was significantly increased. Compared with wild-type tobacco, the flavonols concentration increased. Yeast one-hybrid assays showed CsNAC086 did not directly regulate the gene expression of CsFLS. These findings indicate that CsNAC086 plays a role in regulating flavonols biosynthesis in tea plants, which has important implications for selecting and breeding of high-flavonols-concentration containing tea-plant cultivars.

The Difference of Milk-Derived Extracellular Vesicles from Cow Colostrum and Mature Milk on miRNAs Expression and Protecting Intestinal Epithelial Cells against Lipopolysaccharide Damage.

Intestinal epithelial cells (IECs) play crucial roles in forming an essential barrier, providing host defense against pathogens and regulating nutrients absorption. Milk-derived extracellular vesicles (EVs) within its miRNAs are capable of modulating the recipient cell function. However, the differences between colostrum and mature milk EVs and their biological function in attenuating intestinal epithelial cell injury remain poorly understood. Thus, we carried out the present study to characterize the difference between colostrum and mature milk-derived miRNA of EVs and the effect of colostrum and mature milk EVs on the proliferation, apoptosis, proinflammatory cytokines and intestinal epithelial barrier related genes in IEC-6 induced by LPS. Differential expression of 329 miRNAs was identified between colostrum and mature milk EVs, with 185 miRNAs being downregulated and 144 upregulated. In addition, colostrum contains a greater number and protein concentration of EVs than mature milk. Furthermore, compared to control, EVs derived from colostrum significantly inhibited the expression of apoptosis- (Bax, p53, and caspase-3) and proinflammatory-related genes (TNFα, IL6, and IL1ß). EVs derived from mature milk did not affect expression of apoptosis-related genes (Bax, p53, bcl2, and caspase-3). The EVs derived from mature milk significantly inhibited the expression of proinflammatory-related genes (TNFα and IL6). Western blot analysis also indicated that colostrum and mature milk EVs significantly decreased the apoptosis of IEC-6 cells. The EdU assay results showed that colostrum and mature milk EVs significantly increased the proliferation of IEC-6 cells. The expression of intestinal barrier-related genes (TJP1, CLDN1, OCLN, CDX2, MUC2, and IGF1R) was significantly promoted in IEC-6 cells after colostrum and mature milk EVs addition. Importantly, colostrum and mature milk EVs significantly relieved the LPS-induced inhibition of proliferation and intestinal barrier-related genes expression and attenuated apoptosis and proinflammatory responses induced by LPS in IEC-6 cells. Flow cytometry and Western blot analysis also indicated that colostrum and mature milk EVs significantly affect the apoptosis of IEC-6 cells induced by LPS. The results also indicated that EVs derived from colostrum had better effects on inhibiting the apoptosis- and proinflammatory cytokines-related genes expression. However, the EVs derived from mature milk exhibited beneficial effects on intestinal epithelial barrier protection. The present study will provide a better understanding of the role of EVs derived from colostrum and milk in dairy cows with different responses in the regulation of intestinal cells function, and also presents new evidence for the change of EVs cargos during various stages of lactation.

Effects of glycated serum protein, homocysteine, and cystatin-C levels on pregnancy outcomes in patients with gestational diabetes mellitus.

Objective: To explore the effects of serum glycated serum protein (GSP), homocysteine (Hcy) and cystatin-C (Cys-C) levels on pregnancy outcomes in patients with gestational diabetes mellitus (GDM). Methods: Retrospective selection of 247 pregnant women who underwent normal prenatal examinations in The Yan'an People's Hospital from January 2020 to May 2022 were included in this retrospective study. Among them, 119 were pregnant women with diabetes (GDM-group) and 128 were pregnant women with normal blood glucose (Normal-group). The levels of serum GSP, HCY, CYS-C, and incidence of adverse pregnancy outcomes were compared between the two groups. The clinical value of levels of serum GSP, Hcy, and Cys-C in predicting adverse pregnancy outcomes were analyzed. Results: Compared with the Normal-group, the overall incidence of adverse pregnancy outcomes, serum GSP, Hcy, and Cys-C levels in the GDM-group were significantly higher (p<0.05). Logistic regression analysis showed that the levels of GSP, Hcy, and Cys-C were independent risk factors for adverse pregnancy outcomes in the GDM-group (p<0.05). Receiver operating characteristic (ROC) curve showed that the area under the curve (AUC) for diagnosing adverse pregnancy outcomes in pregnant women with GDM using serum GSP, Hcy, and CysC levels alone were 0.817, 0.843, and 0.775, respectively. The AUC of the three indicators combined was 0.921, indicating that this combination has a good predictive value for diagnosing adverse outcomes in GDM-complicated pregnancies. Conclusions: GDM is associated with a high risk of adverse pregnancy outcomes. Levels of serum GSP, Hcy, and Cys-C are higher in patients with GDM. The higher the levels of GSP, Hcy, and Cys-C, the greater the risk of adverse pregnancy outcomes. Combining these three indicators can effectively predict maternal pregnancy outcomes.

Structural basis for the arsenite binding and translocation of Acr3 antiporter with NhaA folding pattern.

The arsenical resistance-3 (ACR3) family constitutes the most common pathway that confers high-level resistance to toxic metalloids in various microorganisms and lower plants. Based on the structural model constructed by AlphaFold2, the Acr3 antiporter from Bacillus subtilis (Acr3Bs ) exhibits a typical NhaA structure fold, with two discontinuous helices of transmembrane (TM) segments, TM4 and TM9, interacting with each other and forming an X-shaped structure. As the structural information available for these important arsenite-efflux pumps is limited, we investigated the evolutionary conservation among 300 homolog sequences and identified three conserved motifs in both the discontinuous helices and TM5. Through site-directed mutagenesis, microscale thermophoresis (MST), and fluorescence resonance energy transfer (FRET) analyses, the identified Motif C in TM9 was found to be a critical element for substrate binding, in which N292 and E295 are involved in substrate coordination, while R118 in TM4 and E322 in TM10 is responsible for structural stabilization. In addition, the highly conserved residues on Motif B of TM5 are potentially key factors in the protonation/deprotonation process. These consensus motifs and residues are essential for metalloid compound translocation of Acr3 antiporters, by framing the core domain and the typical X-shaped of NhaA fold.

Characterization and functional analysis of a novel C-type lectin in blunt snout bream (Megalobrama amblycephala).

C-type lectins, one of the pattern recognition receptors (PRRs), play significant roles in innate immune responses through binding to the pathogen-associated molecular patterns (PAMPs) presented on surfaces of microorganisms. Here, a novel C-type lectin (named as MaCTL) from blunt snout bream (Megalobrama amblycephala) was cloned and characterized. The open reading frame (ORF) of MaCTL is 573 bp long encoding a putative protein of 190 amino acids (aa), which contains a typical feature of signal peptide at 1-23 aa, a characteristic CRD domain at 45-178 aa and a WND/EPN motif that is required for carbohydrates-binding specificity. Phylogenetic analysis indicated that MaCTL is a novel member of CTL family and possessed the highest similarity to that of grass carp (92.11%). The qRT-PCR analysis revealed that MaCTL expressed widely in all examined normal tissues, including heart, liver, spleen, kidney, head-kidney, gill, intestine and muscle, with the higher expression in the spleen, liver and muscle. The expression of MaCTL in spleen was significantly elevated, peaking at 9 h and 6 h after LPS stimulation and Aeromonas hydrophila challenge, respectively, suggesting its association with involvement in innate immune response. The recombinant MaCTL protein (rMaCTL) agglutinated markedly both Gram-positive (Staphylococcus aureus) and Gram-negative bacteria, including Escherichia coli, Vibrio anguillarum, Vibrio vulnificus and Aeromonas hydrophila, in a Ca2+-dependent manner. Meanwhile, rMaCTL showed the binding effects on the five bacteria and four carbohydrates, such as glucose, surose, LPS and PGN. Moreover, rMaCTL could remarkably inhibit the growth of three types of bacteria in vitro. Overall, the results obtained above demonstrated firmly that MaCTL binds to carbohydrates on the surface of diverse pathogens as a PRR and elicits antimicrobial responses, which shed new light on a better understanding of antibacterial functions of CTLs in teleost fish.

Influence of Estrus on Dairy Cow Milk Exosomal miRNAs and Their Role in Hormone Secretion by Granulosa Cells.

Estrus is crucial for cow fertility in modern dairy farms, but almost 50% of cows do not show the behavioral signs of estrus due to silent estrus and lack of suitable and high-accuracy methods to detect estrus. MiRNA and exosomes play essential roles in reproductive function and may be developed as novel biomarkers in estrus detection. Thus, we analyzed the miRNA expression patterns in milk exosomes during estrus and the effect of milk exosomes on hormone secretion in cultured bovine granulosa cells in vitro. We found that the number of exosomes and the exosome protein concentration in estrous cow milk were significantly lower than in non-estrous cow milk. Moreover, 133 differentially expressed exosomal miRNAs were identified in estrous cow milk vs. non-estrous cow milk. Functional enrichment analyses indicated that exosomal miRNAs were involved in reproduction and hormone-synthesis-related pathways, such as cholesterol metabolism, FoxO signaling pathway, Hippo signaling pathway, mTOR signaling pathway, steroid hormone biosynthesis, Wnt signaling pathway and GnRH signaling pathway. Consistent with the enrichment signaling pathways, exosomes derived from estrous and non-estrous cow milk both could promote the secretion of estradiol and progesterone in cultured bovine granulosa cells. Furthermore, genes related to hormonal synthesis (CYP19A1, CYP11A1, HSD3B1 and RUNX2) were up-regulated after exosome treatment, while exosomes inhibited the expression of StAR. Moreover, estrous and non-estrous cow-milk-derived exosomes both could increase the expression of bcl2 and decrease the expression of p53, and did not influence the expression of caspase-3. To our knowledge, this is the first study to investigate exosomal miRNA expression patterns during dairy cow estrus and the role of exosomes in hormone secretion by bovine granulosa cells. Our findings provide a theoretical basis for further investigating milk-derived exosomes and exosomal miRNA effects on ovary function and reproduction. Moreover, bovine milk exosomes may have effects on the ovaries of human consumers of pasteurized cow milk. These differential miRNAs might provide candidate biomarkers for the diagnosis of dairy cow estrus and will assist in developing new therapeutic targets for cow infertility.

Effects of oral alkali drug therapy on clinical outcomes in pre-dialysis chronic kidney disease patients: a systematic review and meta-analysis.

BACKGROUND: Metabolic acidosis accelerates the progression of chronic kidney disease (CKD) and increases the mortality rate. Whether oral alkali drug therapy benefits pre-dialysis CKD patients is controversial. We performed a meta-analysis of the effects of oral alkali drug therapy on major clinical outcomes in pre-dialysis CKD patients. METHODS: We systematically searched MEDLINE using the Ovid, EMBASE, and Cochrane Library databases without language restriction. We included all eligible clinical studies that involved pre-dialysis CKD adults and compared those who received oral alkali drug therapy with controls. RESULTS: A total of 18 eligible studies, including 14 randomized controlled trials and 4 cohort studies reported in 19 publications with 3695 participants, were included. Oral alkali drug therapy led to a 55% reduction in renal failure events (relative risk [RR]: 0.45; 95% confidence interval [CI]: 0.25-0.82), a rate of decline in the estimated glomerular filtration rate (eGFR) of 2.59 mL/min/1.73 m2 per year (95% CI, 0.88-4.31). There was no significant effect on decline in eGFR events (RR: 0.34; 95% CI: 0.09-1.23), proteinuria (standardized mean difference: -0.32; 95% CI: -1.08 to 0.43), all-cause mortality events (RR: 0.90; 95% CI: 0.40-2.02) and cardiovascular (CV) events (RR: 1.03; 95% CI: 0.32-3.37) compared with the control groups. CONCLUSION: Based on the available and low-to-moderate certainty evidence, oral alkali drug therapy might potentially reduce the risk of kidney failure events, but no benefit in reducing all-cause mortality events, CV events, decline in eGFR and porteninuria.

Effects of parity and days in milk on milk composition in correlation with ß-hydroxybutyrate in tropic dairy cows.

The current study was conducted to evaluate the effect of parity and days in milk on milk yield and milk production traits and their correlation with ß-hydroxybutyrate (BHB) concentrations in milk of Chinese tropic Holstein dairy cows which are adapted to a humid subtropical climate in central China. About 3055 milking records of Holstein cows were obtained from three farms in the hot region in the center of China. The records were classified according to parity to 4 categories: first parity, second parity, third parity, and greater than third parity. According to days in milk, there were 4 groups, first group from (1-100 days), second group from (101-200 days), third group from (201-305 days), and fourth group (>305 days). Milk samples collected between April and November 2019 from the three farms were routinely checked for milk components including BHB using mid-infrared spectroscopy a MilkoScan FT+ (Foss, Hillerød, Denmark). Data were analyzed by multivariate analysis of variance (generalized linear model, GLM). Pearson's correlation coefficients were used to measure the correlation between SCC and BHB with milk yield and milk production traits. Results showed the significant effect of parity and days in milk on milk yield and milk production traits. There was a negative effect of parity and days in milk on milk quality, with increasing parity and days in milk being associated with higher somatic cell count (SCC) (P <0.001). Days in milk significantly affected (P=0.001) BHB. It was concluded that with increasing parity and prolonged days in milk, there was a negative effect on milk quality and udder health of the tropic dairy cows in central China. Based on the results of the current study, sampling milk for specific metabolites, somatic cell count, and quality are sufficient to asses herd health.

Acetylcholine ameliorated TNF-α-induced primary trophoblast malfunction via muscarinic receptors†.

Oxidative stress and apoptosis of trophoblasts are involved in preeclampsia (PE). Numerous studies have shown that acetylcholine (ACh), the principal vagal neurotransmitter, plays a crucial role in attenuating oxidative stress, inflammation, and apoptosis in a variety of human diseases. However, the role of ACh in PE management remains unclear. Here, we aimed to determine the effects of ACh on TNF-α-treated human primary trophoblast cells. Western blotting, CCK-8, DHE, TUNEL immunofluorescence staining, transwell assays, and wound-healing assays were performed to evaluate the role of ACh in vitro. We found that both TNF-α expression and the apoptotic index were higher in placentas from preeclamptic women than in normal placentas. TNF-α enhanced oxidative stress and increased the number of TUNEL-positive nuclei, Bax/Bcl-2 ratio, and the cleaved caspase-3/caspase-3 ratio while decreasing cell viability in primary human trophoblast cells. TNF-α promoted cell migration and invasion. PDTC, a selective NF-κB inhibitor, significantly blunted TNF-α-induced effects. ACh treatment attenuated oxidative stress and apoptosis while further promoting migration and invasion of TNF-α-treated primary trophoblast cells. The effects of ACh could be reversed by the muscarinic receptor antagonist atropine. Overall, our findings indicate that ACh significantly ameliorates TNF-α-induced oxidative stress and apoptosis of human primary trophoblast cells via muscarinic receptors. This is the first time that the improvement of vagal activity served as a therapeutic strategy for PE-like trophoblasts, suggesting its potential value in clinical practice.

Construction of photonic crystals with thermally adjustable pseudo-gaps.

Photonic crystals (PCs) are periodic dielectric structures with photonic bandgaps and they can be used to control and manipulate photons effectively. Novel photonic crystal materials with tunable bandgaps can be prepared by changing the refractive index of the dielectric or lattice parameters under external stimuli, while using temperature to adjust the photonic band gap is a simple and convenient method. In this paper, silica PCs having different pseudo-gaps in the range of 450-750 nm were prepared with colloidal SiO2 spheres of different sizes. Thermo-sensitive PNIPAM hydrogel was then infiltrated into the PCs to obtain PNIPAM-PCs, whose pseudo-gap blue-shifted when the temperature was changed from 24 to 34 °C and exhibited good reversibility. The PCs with tunable bandgaps are significant for the development of integrated photonic devices, sensors, and in detection and other technologies.

Hydrogen-rich water alleviates cyclosporine A-induced nephrotoxicity via the Keap1/Nrf2 signaling pathway.

Oxidative stress induced by long-term cyclosporine A (CsA) administration is a major cause of chronic nephrotoxicity, which is characterized by tubular atrophy, tubular cell apoptosis, and interstitial fibrosis in the progression of organ transplantation. Although hydrogen-rich water (HRW) has been used to prevent various oxidative stress-related diseases, its underlying mechanisms remain unclear. This study investigated the effects of HRW on CsA-induced nephrotoxicity and its potential mechanisms. After administration of CsA (25 mg/kg/day), rats were treated with or without HRW (12 mL/kg) for 4 weeks. Renal function and vascular activity were investigated. Histological changes in kidney tissues were analyzed using Masson's trichrome and terminal deoxynucleotidyl transferase dUTP nick-end labeling stains. Oxidative stress markers and the activation of the Kelch-like ECH-associated protein 1 (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway were also measured. We found that CsA increased the levels of reactive oxygen species (ROS) and malonaldehyde (MDA), but it reduced glutathione (GSH) and superoxide dismutase (SOD) levels. Such alterations induced vascular dysfunction, tubular atrophy, interstitial fibrosis, and tubular apoptosis. This was evident secondary to an increase in urinary protein, serum creatinine, and blood urea nitrogen, ultimately leading to renal dysfunction. Conversely, HRW decreased levels of ROS and MDA while increasing the activity of GSH and SOD. This was accompanied by an improvement in vascular and renal function. Moreover, HRW significantly decreased the level of Keap1 and increased the expression of Nrf2, NADPH dehydrogenase quinone 1, and heme oxygenase 1. In conclusion, HRW restored the balance of redox status, suppressed oxidative stress damage, and improved kidney function induced by CsA via activation of the Keap1/Nrf2 signaling pathway.

Pollution status and ecological risk of heavy metals in the soils of five land-use types in a typical sewage irrigation area, eastern China.

Sewage irrigation has been widespread in the water shortage area of eastern China and inevitably tends to result in heavy metal accumulation in soils. A total of 148 surface soil samples from five land-use types were collected in Longkou, a typical sewage irrigation area of China, and As, Cd, Cu, Pb, and Zn concentrations were determined. The Nemerow index method and improved fuzzy comprehensive evaluation method were used to examine the pollution status of heavy metals. The potential ecological risk was evaluated by the Hakanson model by adjusting the assessment threshold, and its spatial distribution was interpolated using geostatistical techniques. As, Cd, Cu, Pb, and Zn accumulated in different amounts in the five land-use types. Urban industrial land and mining land were moderately polluted, irrigated land was slightly polluted, orchards were minimally polluted, and bare land was at a safe level of pollution. Cd exhibited high percentages of strong and severe levels of potential ecological risks. For Cd, irrigated land, orchard, and bare land mainly presented moderate risks, whereas urban industrial land and mining land mainly presented high risks. The comprehensive ecological risk of the five heavy metals was at a severe level for all tested land-use classes except for bare land.

Alkaline Response of a Halotolerant Alkaliphilic Halomonas Strain and Functional Diversity of Its Na+(K+)/H+ Antiporters.

Halomonas sp. Y2 is a halotolerant alkaliphilic strain from Na+-rich pulp mill wastewater with high alkalinity (pH >11.0). Transcriptome analysis of this isolate revealed this strain may use various transport systems for pH homeostasis. In particular, the genes encoding four putative Na+/H+ antiporters were differentially expressed upon acidic or alkaline conditions. Further evidence, from heterologous expression and mutant studies, suggested that Halomonas sp. Y2 employs its Na+/H+ antiporters in a labor division way to deal with saline and alkaline environments. Ha-NhaD2 displayed robust Na+(Li+) resistance and high transport activities in Escherichia coli; a ΔHa-nhaD2 mutant exhibited growth inhibition at high Na+(Li+) concentrations at pH values of 6.2, 8.0, and 10.0, suggesting its physiological role in osmotic homeostasis. In contrast, Ha-NhaD1 showed much weaker activities in ion exporting and pH homeostasis. Ha-Mrp displayed a combination of properties similar to those of Mrp transporters from some Bacillus alkaliphiles and neutrophiles. This conferred obvious Na+(Li+, K+) resistance in E. coli-deficient strains, as those ion transport spectra of some neutrophil Mrp antiporters. Conversely, similar to the Bacillus alkaliphiles, Ha-Mrp showed central roles in the pH homeostasis of Halomonas sp. Y2. An Ha-mrp-disrupted mutant was seriously inhibited by high concentrations of Na+(Li+, K+) but only under alkaline conditions. Ha-NhaP was determined to be a K+/H+ antiporter and shown to confer strong K+ resistance both at acidic and alkaline stresses.

Co-Immobilization of Enzymes and Magnetic Nanoparticles by Metal-Nucleotide Hydrogelnanofibers for Improving Stability and Recycling.

In this paper we report a facile method for preparing co-immobilized enzyme and magnetic nanoparticles (MNPs) using metal coordinated hydrogel nanofibers. Candida rugosa lipase (CRL) was selected as guest protein. For good aqueous dispersity, low price and other unique properties, citric acid-modified magnetic iron oxide nanoparticles (CA-Fe3O4 NPs) have been widely used for immobilizing enzymes. As a result, the relative activity of CA-Fe3O4@Zn/AMP nanofiber-immobilized CRL increased by 8-fold at pH 10.0 and nearly 1-fold in a 50 °C water bath after 30 min, compared to free CRL. Moreover, the immobilized CRL had excellent long-term storage stability (nearly 80% releative activity after storage for 13 days). This work indicated that metal-nucleotide nanofibers could efficiently co-immobilize enzymes and MNPs simultaneously, and improve the stability of biocatalysts.

Biochemical and transcriptomic analyses reveal different metabolite biosynthesis profiles among three color and developmental stages in 'Anji Baicha' (Camellia sinensis).

BACKGROUND: The new shoots of the albino tea cultivar 'Anji Baicha' are yellow or white at low temperatures and turn green as the environmental temperatures increase during the early spring. 'Anji Baicha' metabolite profiles exhibit considerable variability over three color and developmental stages, especially regarding the carotenoid, chlorophyll, and theanine concentrations. Previous studies focused on physiological characteristics, gene expression differences, and variations in metabolite abundances in albino tea plant leaves at specific growth stages. However, the molecular mechanisms regulating metabolite biosynthesis in various color and developmental stages in albino tea leaves have not been fully characterized. RESULTS: We used RNA-sequencing to analyze 'Anji Baicha' leaves at the yellow-green, albescent, and re-greening stages. The leaf transcriptomes differed considerably among the three stages. Functional classifications based on Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes enrichment analyses revealed that differentially expressed unigenes were mainly related to metabolic pathways, biosynthesis of secondary metabolites, phenylpropanoid biosynthesis, and carbon fixation in photosynthetic organisms. Chemical analyses revealed higher ß-carotene and theanine levels, but lower chlorophyll a levels, in the albescent stage than in the green stage. Furthermore, unigenes involved in carotenoid, chlorophyll, and theanine biosyntheses were identified, and the expression patterns of the differentially expressed unigenes in these biosynthesis pathways were characterized. Through co-expression analyses, we identified the key genes in these pathways. These genes may be responsible for the metabolite biosynthesis differences among the different leaf color and developmental stages of 'Anji Baicha' tea plants. CONCLUSIONS: Our study presents the results of transcriptomic and biochemical analyses of 'Anji Baicha' tea plants at various stages. The distinct transcriptome profiles for each color and developmental stage enabled us to identify changes to biosynthesis pathways and revealed the contributions of such variations to the albino phenotype of tea plants. Furthermore, comparisons of the transcriptomes and related metabolites helped clarify the molecular regulatory mechanisms underlying the secondary metabolic pathways in different stages.

Expression and functional analysis of two NhaD type antiporters from the halotolerant and alkaliphilic Halomonas sp. Y2.

Na(+)/H(+) antiporters play important roles in ion and pH homeostasis. In this study, two NhaD homologues that effectively catalyze Na(+)/H(+) antiporter were identified from Halomonas sp. Y2, a halotolerant and alkaliphilic strain isolated from sodium enriched black liquor. They exhibited high sequence identity of 72 % and similar binding affinities for Na(+) and Li(+) translocation, while having different pH profiles. Ha-NhaD1 was active at pH 6.0 and most active at pH 8.0-8.5, whereas Ha-NhaD2 lacked activity at pH 6.0 but exhibited maximum activity at pH 9.5 or higher. Based on multiple alignments, 11 partially conserved residues were selected and corresponding mutants were generated for Ha-NhaD1. As expected, replacement of most of the hydrophobic residues abolished the cation exchange activities. Three serine residues at positions 200, 282 and 353 in Ha-NhaD1 were replaceable by alanines with partial retention of activity. The S353A mutant exhibited significantly reduced binding affinity for Na(+) and Li(+), while S282 mutant exhibited an alkaline shift of about 1.5 pH units, as compared to the wild type Ha-NhaD1. Serine at position 282 was predicted to be located in transmembrane segment VIII and was found to be important in regulating pH sensitivity in concert with flanking residues.

Global transcriptome and gene regulation network for secondary metabolite biosynthesis of tea plant (Camellia sinensis).

BACKGROUND: Major secondary metabolites, including flavonoids, caffeine, and theanine, are important components of tea products and are closely related to the taste, flavor, and health benefits of tea. Secondary metabolite biosynthesis in Camellia sinensis is differentially regulated in different tissues during growth and development. Until now, little was known about the expression patterns of genes involved in secondary metabolic pathways or their regulatory mechanisms. This study aimed to generate expression profiles for C. sinensis tissues and to build a gene regulation model of the secondary metabolic pathways. RESULTS: RNA sequencing was performed on 13 different tissue samples from various organs and developmental stages of tea plants, including buds and leaves of different ages, stems, flowers, seeds, and roots. A total of 43.7 Gbp of raw sequencing data were generated, from which 347,827 unigenes were assembled and annotated. There were 46,693, 8446, 3814, 10,206, and 4948 unigenes specifically expressed in the buds and leaves, stems, flowers, seeds, and roots, respectively. In total, 1719 unigenes were identified as being involved in the secondary metabolic pathways in C. sinensis, and the expression patterns of the genes involved in flavonoid, caffeine, and theanine biosynthesis were characterized, revealing the dynamic nature of their regulation during plant growth and development. The possible transcription factor regulation network for the biosynthesis of flavonoid, caffeine, and theanine was built, encompassing 339 transcription factors from 35 families, namely bHLH, MYB, and NAC, among others. Remarkably, not only did the data reveal the possible critical check points in the flavonoid, caffeine, and theanine biosynthesis pathways, but also implicated the key transcription factors and related mechanisms in the regulation of secondary metabolite biosynthesis. CONCLUSIONS: Our study generated gene expression profiles for different tissues at different developmental stages in tea plants. The gene network responsible for the regulation of the secondary metabolic pathways was analyzed. Our work elucidated the possible cross talk in gene regulation between the secondary metabolite biosynthetic pathways in C. sinensis. The results increase our understanding of how secondary metabolic pathways are regulated during plant development and growth cycles, and help pave the way for genetic selection and engineering for germplasm improvement.

A new class of cuprous bromide cluster-based hybrid materials: direct observation of the stepwise replacement of hydrogen bonds by coordination bonds.

Although a variety of functional metal-organic frameworks (MOFs) have been synthesized, post-modified, and applied in various areas, there is little knowledge about how molecular cluster building units are stepwise evolved into MOFs via intermediates. Coordination bonds are generally stronger than hydrogen bonds, and thus equivalent replacement of X-H···Y hydrogen bonds by X-M-Y coordination bonds can transform hydrogen bond networks into MOFs. In this work, solvothermal in situ reduction reactions of CuBr2 and 1,4-diazoniabicyclo[2,2,2]octane (DABCO) generated a myriad of tunable photoluminescent cuprous body-centered cubic bromide cluster-based networks with the general formula [Cu4+xH4-xBr6(DABCO)4](HCO2)2·S (x = 0, 0.56, 0.81, 1.27, 1.39, 2.56, 2.78, and 4 for compounds 1-8, respectively). All of these compounds crystallize in the cubic space group with the largest volume difference being only 5.2%, but they belong to three remarkably different kinds of crystals. Complex 1 is a molecular crystal and consists of tetrahedral [Cu4Br6(HDABCO)4](2+) clusters with monodentate HDABCO groups that are supported via N-H···Br synthons in the hydrogen bond network. Compound 8 is a [Cu8Br6](2+) cube cluster-based MOF with bridged DABCO ligands. Complexes 2-7 are seemingly impossible Cu/H-substituted solid solutions of 1 and 8. The CuBr framework components in 1-8 are Cu4Br6, Cu4.56Br6, Cu4.81Br6, Cu5.27Br6, Cu5.39Br6, Cu6.56Br6, Cu6.78Br6, and Cu8Br6, respectively. Crystallization kinetics studies revealed that the [Cu4Br6(HDABCO)4](2+) cluster-based hydrogen bond network (1) was initially formed such that N-H···Br hydrogen bonds could be stepwise replaced by N-Cu-Br coordination bonds to form the [Cu8Br6](2+) cube cluster-based MOF (8) via solid solutions. These observations directly reveal the equivalence and transformation between the N-H···Br hydrogen bond and the N-Cu-Br coordination bond and the evolutionary mechanism of a molecular crystal to a MOF via solid solutions, which is of fundamental importance in materials but has never before been revealed. DFT calculations suggest that equivalent replacement of a N-H···Br hydrogen bond by a N-Cu-Br coordination bond is exothermic and exergonic, which also supports the transformation from molecule 1 to MOF 8.

Transcriptome analysis of the Ophiocordyceps sinensis fruiting body reveals putative genes involved in fruiting body development and cordycepin biosynthesis.

Ophiocordyceps sinensis is a highly valuable and popular medicinal fungus used as a tonic and roborant for thousands of years in traditional Asian medicine. However, unsustainable harvesting practices have endangered this species and very little is known about its developmental programming, its biochemistry and genetics. To begin to address this, the transcriptome of the medicinal O. sinensis fruiting body was analyzed by high-throughput. In this O. sinensis 454-EST dataset, four mating type genes and 121 genes that may be involved in fruiting body development, especially in signal transduction and transcription regulation, were discovered. Moreover, a model was developed for the synthesis of the primary medicinal compound, cordycepin, and the putative biosynthetic enzymes identified. This transcriptome dataset provides a significant new resource for gene discovery in O. sinensis and dissection of its valuable biosynthetic and developmental pathways.