Natural variation in CYCLIC NUCLEOTIDE-GATED ION CHANNEL 4 reveals a novel role of calcium signaling in vegetative phase change in Arabidopsis.

The timing of vegetative phase change (VPC) in plants is regulated by a temporal decline in the expression of miR156. Both exogenous cues and endogenous factors, such as temperature, light, sugar, nutrients, and epigenetic regulators, have been shown to affect VPC by altering miR156 expression. However, the genetic basis of natural variation in VPC remains largely unexplored. Here, we conducted a genome-wide association study on the variation of the timing of VPC in Arabidopsis. We identified CYCLIC NUCLEOTIDE-GATED ION CHANNEL 4 (CNGC4) as a significant locus associated with the diversity of VPC. Mutations in CNGC4 delayed VPC, accompanied by an increased expression level of miR156 and a corresponding decrease in SQUAMOSA PROMOTER BINDING-LIKE (SPL) gene expression. Furthermore, mutations in CNGC2 and CATION EXCHANGER 1/3 (CAX1/3) also led to a delay in VPC. Polymorphisms in the CNGC4 promoter contribute to the natural variation in CNGC4 expression and the diversity of VPC. Specifically, the early CNGC4 variant promotes VPC and enhances plant adaptation to local environments. In summary, our findings offer genetic insights into the natural variation in VPC in Arabidopsis, and reveal a previously unidentified role of calcium signaling in the regulation of VPC.

Binding mechanism of oseltamivir and influenza neuraminidase suggests perspectives for the design of new anti-influenza drugs.

Oseltamivir is a widely used influenza virus neuraminidase (NA) inhibitor that prevents the release of new virus particles from host cells. However, oseltamivir-resistant strains have emerged, but effective drugs against them have not yet been developed. Elucidating the binding mechanisms between NA and oseltamivir may provide valuable information for the design of new drugs against NA mutants resistant to oseltamivir. Here, we conducted large-scale (353.4 µs) free-binding molecular dynamics simulations, together with a Markov State Model and an importance-sampling algorithm, to reveal the binding process of oseltamivir and NA. Ten metastable states and five major binding pathways were identified that validated and complemented previously discovered binding pathways, including the hypothesis that oseltamivir can be transferred from the secondary sialic acid binding site to the catalytic site. The discovery of multiple new metastable states, especially the stable bound state containing a water-mediated hydrogen bond between Arg118 and oseltamivir, may provide new insights into the improvement of NA inhibitors. We anticipated the findings presented here will facilitate the development of drugs capable of combating NA mutations.

Development of full-length cell-culture infectious clone and subgenomic replicon for a genotype 3a isolate of hepatitis C virus.

Hepatitis C virus (HCV) genotype 3 is widely distributed, and genotype 3-infected patients achieve a lower cure rate in direct-acting antiviral (DAA) therapy and are associated with a higher risk of hepatic steatosis than patients with other genotypes. Thus, the study of the virology and pathogenesis of genotype 3 HCV is increasingly relevant. Here, we developed a full-length infectious clone and a subgenomic replicon for the genotype 3a isolate, CH3a. From an infected serum, we constructed a full-length CH3a clone, however, it was nonviable in Huh7.5.1 cells. Next, we systematically adapted several intergenotypic recombinants containing Core-NS2 and 5'UTR-NS5A from CH3a, and other sequences from a replication-competent genotype 2 a clone JFH1. Adaptive mutations were identified, of which several combinations facilitated the replication of CH3a-JFH1 recombinants; however, they failed to adapt to the full-length CH3a and the recombinants containing CH3a NS5B. Thus, we attempted to separately adapt CH3a NS5B-3'UTR by constructing an intragenotypic recombinant using 5'UTR-NS5A from an infectious genotype 3a clone, DBN3acc, from which L3004P/M in NS5B and a deletion of 11 nucleotides (Δ11nt) downstream of the polyU/UC tract of the 3'UTR were identified and demonstrated to efficiently improve virus production. Finally, we combined functional 5'UTR-NS5A and NS5B-3'UTR sequences that carried the selected mutations to generate full-length CH3a with 26 or 27 substitutions (CH3acc), and both revealed efficient replication and virus spread in transfected and infected cells, releasing HCV of 104.2 f.f.u. ml-1. CH3acc was inhibited by DAAs targeting NS3/4A, NS5A and NS5B in a dose-dependent manner. The selected mutations permitted the development of subgenomic replicon CH3a-SGRep, by which L3004P, L3004M and Δ11nt were proven, together with a single-cycle virus production assay, to facilitate virus assembly, release, and RNA replication. CH3acc clones and CH3a-SGRep replicon provide new tools for the study of HCV genotype 3.

Production of H2S with a Novel Short-Process for the Removal of Heavy Metals in Acidic Effluents from Smelting Flue-Gas Scrubbing Systems.

Direct sulfidation using a high concentration of H2S (HC-H2S) has shown potential for heavy metals removal in various acidic effluents. However, the lack of a smooth method for producing HC-H2S is a critical challenge. Herein, a novel short-process hydrolysis method was developed for the on-site production of HC-H2S. Near-perfect 100% efficiency and selectivity were obtained via CS2 hydrolysis over the ZrO2-based catalyst. Meanwhile, no apparent residual sulfur/sulfate poisoning was detected, which guaranteed long-term operation. The coexistence of CO2 in the products had a negligible effect on the complete hydrolysis of CS2. H2S production followed a sequential hydrolysis pathway, with the reactions for CS2 adsorption and dissociation being the rate-determining steps. The energy balance indicated that HC-H2S production was a mildly exothermic reaction, and the heat energy could be maintained at self-balance with approximately 80% heat recovery. The batch sulfidation efficiencies for As(III), Hg(II), Pb(II), and Cd(II) removal were over 99.9%, following the solubilities (Ksp) of the corresponding metal sulfides. CO2 in the mixed gas produced by CS2 hydrolysis did not affect heavy metals sulfidation due to the presence of abundant H+. Finally, a pilot-scale experiment successfully demonstrated the practical effects. Therefore, this novel on-site HC-H2S production method adequately achieved heavy metals removal requirements in acidic effluents.

Recent progress on the molecular breeding of Cucumis sativus L. in China.

KEY MESSAGE: Molecular breeding of Cucumis sativus L. is based on traditional breeding techniques and modern biological breeding in China. There are opportunities for further breeding improvement by molecular design breeding and the automation of phenotyping technology using untapped sources of genetic diversity. Cucumber (Cucumis sativus L.) is an important vegetable cultivated worldwide. It bears fruits of light fragrance, and crisp texture with high nutrition. China is the largest producer and consumer of cucumber, accounting for 70% of the world's total production. With increasing consumption demand, the production of Cucurbitaceae crops has been increasing yearly. Thus, new cultivars that can produce high-quality cucumber with high yield and easy cultivation are in need. Conventional genetic breeding has played an essential role in cucumber cultivar innovation over the past decades. However, its progress is slow due to the long breeding period, and difficulty in selecting stable genetic characters or genotypes, prompting researchers to apply molecular biotechnologies in cucumber breeding. Here, we first summarize the achievements of conventional cucumber breeding such as crossing and mutagenesis, and then focus on the current status of molecular breeding of cucumber in China, including the progress and achievements on cucumber genomics, molecular mechanism underlying important agronomic traits, and also on the creation of high-quality multi-resistant germplasm resources, new variety breeding and ecological breeding. Future development trends and prospects of cucumber molecular breeding in China are also discussed.

Modulation of miR156 to identify traits associated with vegetative phase change in tobacco (Nicotiana tabacum).

After germination, plants progress through juvenile and adult phases of vegetative development before entering the reproductive phase. The character and timing of these phases vary significantly between different plant species, which makes it difficult to know whether temporal variations in various vegetative traits represent the same, or different, developmental processes. miR156 has been shown to be the master regulator of vegetative development in plants. Overexpression of miR156 prolongs the juvenile phase of development, whereas knocking-down the level of miR156 promotes the adult phase of development. Therefore, artificial modulation of miR156 expression is expected to cause corresponding changes in vegetative-specific traits in different plant species, particularly in those showing no substantial difference in morphology during vegetative development. To identify specific traits associated with the juvenile-to-adult transition in tobacco, we examined the phenotype of transgenic tobacco plants with elevated or reduced levels of miR156. We found that leaf shape, the density of abaxial trichomes, the number of leaf veins, the number of stomata, the size and density of epidermal cells, patterns of epidermal cell staining, the content of chlorophyll and the rate of photosynthesis, are all affected by miR156. These newly identified miR156-regulated traits therefore can be used to distinguish between juvenile and adult phases of development in tobacco, and provide a starting point for future studies of vegetative phase change in the family Solanaceae.

Research progress of non-coding RNAs in vegetable responses to abiotic stresses.

Vegetable crops play a crucial role in agricultural production, providing essential vitamins and minerals necessary for a healthy diet. Recently, there has been growing interest in cultivating vegetable varieties with outstanding agricultural and economic traits. However, vegetable production is often exposed to various abiotic stresses like soil drought, temperature fluctuations, and heavy metal stress, which can negatively impact yield and quality. While previous research has investigated the physiological responses of vegetable crops to such stressors, less attention has been given to genetic networks. Plants respond to environmental stress mainly by adapting first and then reacting, thereby enhancing their resistance to stress. Typically, different abiotic stresses trigger epigenetic changes, which can regulate non-coding RNAs. Therefore, studying the epigenetic mechanisms of vegetable crop responses to abiotic stress can provide insights into the molecular response mechanisms of plants under stress. This knowledge has practical applications in breeding vegetable crops for resistance. This article summarizes the primary research findings on the regulation of non-coding RNAs and their expression levels in vegetable crops exposed to abiotic stresses to guide molecular breeding approaches for vegetable crops.

Genome-wide identification of miR156 and SPL family genes and phenotypic analysis of vegetative phase change in pepper (Capsicum annuum L.).

After germination, plants go through two phases of vegetative development - juvenile and adult - before entering the reproductive phase. These phases have varying characteristics and timing across plant species, making it challenging to determine if different vegetative traits correspond to the same or distinct developmental processes. miR156 has been identified as the primary regulator of vegetative phase change in plants, with the miR156-SPLs (SQUAMOSA Promoter Binding Protein-Likes) module playing a crucial role in regulating age-related agronomic traits in various crops. Such traits include disease resistance, optimal plant breeding, and secondary metabolism regulation. However, it is unknown whether miR156-SPLs contribute to the critical agronomic traits of pepper (Capsicum annuum L.). Thus, this study seeks to identify miR156 and SPLs genes in pepper, analyze their evolutionary links with model plants, and confirm their expression patterns using gene expression assays. The study also examines the relationship between miR156 expression levels in two cultivars of pepper and specific traits associated with the juvenile-to-adult transition. The results indicate that leaf shape and the number of leaf veins are correlated to the timing expression of miR156. Our study represents an important resource for identifying age-dependent agronomic traits in pepper and lays the foundation for future systematic regulation of miR156-SPLs to advance pepper development.

Coordinated regulation of vegetative phase change by brassinosteroids and the age pathway in Arabidopsis.

Vegetative phase change in plants is regulated by a gradual decline in the level of miR156 and a corresponding increase in the expression of its targets, SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes. Gibberellin (GA), jasmonic acid (JA), and cytokinin (CK) regulate vegetative phase change by affecting genes in the miR156-SPL pathway. However, whether other phytohormones play a role in vegetative phase change remains unknown. Here, we show that a loss-of-function mutation in the brassinosteroid (BR) biosynthetic gene, DWARF5 (DWF5), delays vegetative phase change, and the defective phenotype is primarily attributable to reduced levels of SPL9 and miR172, and a corresponding increase in TARGET OF EAT1 (TOE1). We further show that GLYCOGEN SYNTHASE KINASE3 (GSK3)-like kinase BRASSINOSTEROID INSENSITIVE2 (BIN2) directly interacts with and phosphorylates SPL9 and TOE1 to cause subsequent proteolytic degradation. Therefore, BRs function to stabilize SPL9 and TOE1 simultaneously to regulate vegetative phase change in plants.

The Formation of Fruit Quality in Cucumis sativus L.

Cucumber is one of the most widely grown vegetables in China and an indispensable fresh fruit in the diet. With the development of society, the demand of people for cucumber quality is higher and higher. Therefore, cultivating high-quality cucumber varieties is one of the main goals of cucumber breeding. With the rapid development of biotechnology such as molecular marker, cucumber quality control network is becoming clear. In this review, we describe the formation mechanism of cucumber fruit quality from three aspects: (1) the commercial quality of cucumber fruit, (2) nutritional quality formation, and (3) flavor quality of cucumber fruit. In addition, the determinants of cucumber fruit quality were summarized from two aspects of genetic regulation and cultivation methods in order to provide ideas for cucumber researchers and cultivators to improve fruit quality.

RNA-Seq Identification of Cd Responsive Transporters Provides Insights into the Association of Oxidation Resistance and Cd Accumulation in Cucumis sativus L.

Greenhouse vegetable production (GVP) has grown rapidly and has become a major force for cucumber production in China. In highly intensive GVP systems, excessive fertilization results in soil acidification, increasing Cd accumulation and oxidative stress damage in vegetables as well as increasing health risk of vegetable consumers. Therefore, enhancing antioxidant capacity and activating the expression level of Cd transporter genes seem to be feasible solutions to promote plant resistance to Cd stress and to reduce accumulated Cd concentration. Here, we used transcriptomics to identify five cucumber transporter genes (CsNRAMP1, CsNRAMP4, CsHMA1, CsZIP1, and CsZIP8) in response to cadmium stress, which were involved in Cd transport activity in yeast. Ionomics, gene expression, and REDOX reaction level association analyses have shown that the transcript of CsNRAMP4 was positively correlated with Cd accumulation and antioxidant capacity of cucumber roots. The expression level of CsHMA1 was negatively correlated with Cd-induced antioxidant capacity. The overexpression of CsHMA1 significantly relieved Cd stress-induced antioxidant activities. In addition, shoots with high CsHMA2 expression remarkably presented Cd bioaccumulation. Grafting experiments confirmed that CsHMA1 contributed to the high antioxidant capacity of cucumber, while CsHMA2 was responsible for the transport of Cd from the roots to the shoots. Our study elucidated a novel regulatory mechanism for Cd transport and oxidative damage removal in horticultural melons and provided a perspective to regulate Cd transport artificially by modulating Cd accumulation and resistance in plants.

Regulation of cadmium tolerance and accumulation by miR156 in Arabidopsis.

Plants have evolved effective strategies to cope with heavy metals Cd toxicity, but the regulatory mechanism underlying Cd tolerance and accumulation are still poorly understood. miR156 has been shown to be the master regulator of development and stress response in plants. However, whether miR156 is also involved in plant Cd stress response remains unknown. Here, we show that plants overexpressing miR156 (miR156OE) accumulated significantly less Cd in the shoot, and conferred enhanced tolerance to Cd stress. Plants with a knocked-down level of miR156 (MIM156) were sensitive to Cd stress, and accumulated significantly higher Cd. Under Cd stress, miR156OE had significantly longer primary root length, higher biomass and chlorophyll content, increased activities of antioxidative enzymes and lower levels of endogenous reactive oxygen species (ROS), while MIM156 had the opposite phenotype. To investigate the underlying mechanism of miR156-mediated Cd stress response in Arabidopsis, we profiled the expression of several Cd transporter genes. The expression of Cd uptake transporter of AtZIP1、AtZIP2 and vacuole segregated transporter AtABCC1 was significantly elevated in miR156OE, whereas it was significantly reduced in MIM156. MIM156 also led to an elevated level of AtHMA4 responsible for transporting Cd from the root to the shoot. Our results indicate that miR156 acts as a positive regulator of plant tolerance to Cd stress by modulating ROS levels and Cd uptake/transport genes expression. Therefore, our study adds a new layer of regulatory mechanism for Cd transport and tolerance in plants, and provides a perspective to regulate Cd transport artificially by modulating plant vegetative growth and development using miR156.

Identification of nucleotides in the 5'UTR and amino acids substitutions that are essential for the infectivity of 5'UTR-NS5A recombinant of hepatitis C virus genotype 1b (strain Con1).

Genotype 1b strain Con1 represents an important reference in the study of hepatitis C virus (HCV). Here, we aimed to develop an advanced infectious Con1 recombinant. We found that previously identified mutations A1226G/F1464L/A1672S/Q1773H permitted culture adaption of Con1 Core-NS5A (C-5A) recombinant containing 5'UTR and NS5B-3'UTR from JFH1 (genotype 2a), thus acquired additional mutations L725H/F886L/D2415G. C-5A containing all seven mutations (C-5A_7m) replicated efficiently in Huh7.5 and Huh7.5.1 cells and had an increased infectivity in SEC14L2-expressing Huh7.5.1 cells. Incorporation of Con1 NS5B was deleterious to C-5A_7m, however Con1 5'UTR was permissive but attenuated the virus. Nucleotides G1, A4, and G35 primarily accounted for the viral attenuation without affecting RNA translation. C-5A_7m was inhibited dose-dependently by simeprevir and daclatasvir, and substitutions at A4, A29, A34, and G35 conferred resistance to miR-122 antagonism. The novel Con1 5'UTR-NS5A recombinant, adaptive mutations, and critical nucleotides described here will facilitate future studies of HCV culture systems and virus-host interaction.

Development of an Infectious Cell Culture System for Hepatitis C Virus Genotype 6a Clinical Isolate Using a Novel Strategy and Its Sensitivity to Direct-Acting Antivirals.

Hepatitis C virus (HCV) is classified into seven major genotypes, and genotype 6 is commonly prevalent in Asia, thus reverse genetic system representing genotype 6 isolates in prevalence is required. Here, we developed an infectious clone for a Chinese HCV 6a isolate (CH6a) using a novel strategy. We determined CH6a consensus sequence from patient serum and assembled a CH6a full-length (CH6aFL) cDNA using overlapped PCR product-derived clones that shared the highest homology with the consensus. CH6aFL was non-infectious in hepatoma Huh7.5 cells. Next, we constructed recombinants containing Core-NS5A or 5'UTR-NS5A from CH6a and the remaining sequences from JFH1 (genotype 2a), and both were engineered with 7 mutations identified previously. However, they replicated inefficiently without virus spread in Huh7.5 cells. Addition of adaptive mutations from CH6a Core-NS2 recombinant, with JFH1 5'UTR and NS3-3'UTR, enhanced the viability of Core-NS5A recombinant and acquired replication-enhancing mutations. Combination of 22 mutations in CH6a recombinant with JFH1 5'UTR and 3'UTR (CH6aORF) enabled virus replication and recovered additional four mutations. Adding these four mutations, we generated two efficient recombinants containing 26 mutations (26m), CH6aORF_26m and CH6aFL_26m (designated "CH6acc"), releasing HCV of 104.3-104.5 focus-forming units (FFU)/ml in Huh7.5.1-VISI-mCherry and Huh7.5 cells. Seven newly identified mutations were important for HCV replication, assembly, and release. The CH6aORF_26m virus was inhibited in a dose- and genotype-dependent manner by direct-acting-antivirals targeting NS3/4A, NS5A, and NS5B. The CH6acc enriches the toolbox of HCV culture systems, and the strategy and mutations applied here will facilitate the culture development of other HCV isolates and related viruses.

[Growth characteristics of Campylobacter jejuni on different culture media and their expression of outer membrane proteins].

OBJECTIVE: To evaluate the biological characteristics of Campylobacter jejuni (CJ) cultured on different culture media and their expression abundance of outer membrane proteins (OMPs). METHODS: CJ was cultured on the improved Bull's medium yolk agar, improved Bull's blood agar or improved Bull's agar for 48 h. The biological characteristics of the bacteria, including the colony feature, morphology, motility, biochemistry, and results of indirect fluorescence test were observed and compared. OMP of the cultured CJ was extracted using 0.2 mol/L and glycine-hydrochloride buffered solution (pH 2.2) and identified by SDS-PAGE to compare the expression abundance of the OMPs with molecular weight of 28-31 kD. RESULTS: CJ exhibited typical biological characteristics with larger cell body and more rapid growth on improved Bull's medium yolk agar than those on improved Bull's blood agar and improved Bull's agar. The bacteria grown on improved Bull's medium yolk agar showed also greater expression abundance of the OMPs with molecule mass between 28 kD and 31 kD. CONCLUSION: Improved Bull's medium yolk agar allows rapid growth of CJ with typical biological characteristics and enhanced expression of the OMPs with molecular weight of 28 -31 kD, and can be widely used in CJ subunit vaccine development, CJ epidemiological survey, CJ food safety examination, and CJ quarantine.