Search Results (239)

Fe deficiency in apple trees can lead to leaf chlorosis and impede root development, resulting in significant alterations in signaling, metabolism, and genetic functions, which severely restricts fruit yield and quality. It is well established that WRKY transcription factors (TFs) are of vital significance in mediating plant responses to abiotic stress. Real-time quantitative fluorescence (RT-qPCR) analysis displayed that Fe deficiency stress can significantly induce WRKY69 TF gene expression. However, the potential mechanisms by which the WRKY69 gene involved in Fe deficiency stress remains to be investigated. To address this limitations, the WRKY69 gene (MD09G1235100) was successfully isolated from apple rootstock Malus halliana and performed both homologous and heterologous expression analyses in apple calli and tobacco to elucidate its functional role in response to Fe deficiency stress. The findings indicated that transgenic tobacco plants exhibited enhanced growth vigor and reduced chlorosis when subjected to Fe deficiency stress compared to the wild type (WT). Additionally, the apple calli that were overexpressed WRKY69 also exhibited superior growth and quality. Furthermore, the overexpression of the WRKY69 gene enhanced the ability of tobacco to Fe deficiency stress tolerance by stimulating the synthesis of photosynthetic pigments, increasing antioxidant enzyme activity, and facilitating Fe reduction. Additionally, it increased the resistance of apple calli to Fe deficiency stress by enhancing Fe reduction and elevating the activity of antioxidant enzymes. For example, under Fe deficiency stress, the proline (Pro) contents of the overexpression lines (OE-2, OE-5, OE-6) were 26.18 mg·g⁻¹, 26.13 mg·g⁻¹, and 26.27 mg·g⁻¹, respectively, which were 16.98%, 16.76%, and 17.38% higher than the proline content of 22.38 mg·g⁻¹ in the wild-type lines, respectively. To summarize, a functional analysis of tobacco plants and apple calli displayed that WRKY69 TF serves as a positive regulator under Fe deficiency stress, which provides candidate genetic resources for cultivating apple rootstocks or varieties with strong stress (Fe deficiency) resistance. Full article

Dryopteris fragrans (L.) Schott synthesizes volatile sesquiterpenes through the mevalonate pathway (MVA), in which 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) serves as the key rate-limiting enzyme. Although HMGR plays a crucial role in terpenoid biosynthesis, its functional characteristics in D. fragrans and its involvement in stress responses remain unclear. This study identified three HMGR genes (DfHMGR1/2/3) from the transcriptome data of D. fragrans. Bioinformatics analysis revealed that the encoded proteins are localized to the endoplasmic reticulum and share high sequence similarity with fern homologs. Under abiotic stress conditions, DfHMGRs exhibited differential expression patterns, with marked upregulation under salt and drought stress. To validate the functions of these genes, we generated transgenic Nicotiana tabacum L. plants overexpressing DfHMGRs. Compared with wild-type controls, the transgenic lines showed enhanced tolerance to drought and heat stress. Promoter analysis identified functional regulatory regions controlling DfHMGR expression, and co-expression network analysis predicted 21 potential transcriptional regulators. This study validates the function of D. fragrans HMGRs in a heterologous system and provides candidate genes for improving stress resistance in plants. Full article

The plant cuticle, a protective barrier against external stresses, and abscisic acid (ABA), a key phytohormone, are crucial for plant growth and stress responses. Heterologous expression of AtNCED3 in plants has been widely studied. In this research, by comparing the japonica rice cultivar Zhonghua 10 and its AtNCED3 over-expressing lines during the vegetative growth stage through multiple methods, we found that AtNCED3 over-expression increased leaf ABA content, enhanced epidermal wax and cutin accumulation, modified wax crystal density, and thickened the cuticle. These changes reduced leaf epidermal permeability and the transpiration rate, thus enhancing drought tolerance. This study helps understand the role of endogenous ABA in rice cuticle synthesis and its mechanism in plant drought tolerance, offering potential for genetic improvement of drought resistance in crops. Full article

The investigation of phosphorus metabolism and regulatory mechanisms is conducive to maintaining stable production of crops within a low-phosphorus environment. In phosphorus signal transduction, a few phosphorus starvation response (PHR) transcription factors were identified to bind to the characteristic cis-element, namely the PHR1 binding sequence (P1BS). While the molecular function of the PHR transcription factor has been intensively elucidated, here, we explore a novel transcription factor, BnaA01.KAN3, that undergoes specific binding to the P1BS by yeast one-hybrid and electrophoretic mobility shift assays, and its expression is induced with low-phosphorus stress. BnaA01.KAN3 possessed transcriptional activation and was located in the nucleus. The spatiotemporal expression pattern of BnaA01.KAN3 exhibited tissue specificity in developmental seed, and its expression level was especially high 25–30 days after pollination. Regarding the phenotype analysis, the independent heterologous overexpression lines of BnaA01.KAN3 in Arabidopsis thaliana exhibited not only significantly longer taproots but also an increased number of lateral roots compared to that of the wild type undergoing low-phosphorus treatment, while no differences were seen under normal phosphorus conditions. Furthermore, these lines showed higher anthocyanin and inorganic phosphorus contents with normal and low-phosphorus treatment, suggesting that BnaA01.KAN3 could enhance phosphorus uptake or remobilization to cope with low-phosphorus stress. In summary, this study characterized the transcription factor BnaA01.KAN3 that modulates low-phosphate adaptation and seed development, providing insights for improving phosphorus use efficiency and yield traits in Brassica napus. Full article

Background/Objectives: ALK+ Anaplastic Large Cell Lymphoma (ALCL) is an aggressive T-cell lymphoma that is characterized by expression of the Anaplastic Lymphoma Kinase (ALK), which is induced by the t(2;5) chromosomal rearrangement, leading to the expression of the NPM-ALK fusion oncogene. Most previous preclinical models of ALK+ ALCL were based on overexpression of the NPM-ALK cDNA from heterologous promoters. Due to the enforced expression, this approach is prone to artifacts arising from synthetic overexpression, promoter competition and insertional variation. Methods: To improve the existing ALCL models and more closely recapitulate the oncogenic events in ALK+ ALCL, we employed CRISPR/Cas-based chromosomal engineering to selectively introduce translocations between the Npm1 and Alk gene loci in murine cells. Results: By inducing precise DNA cleavage at the syntenic loci on chromosome 11 and 17 in a murine IL-3-dependent Ba/F3 reporter cell line, we generated de novo Npm-Alk translocations in vivo, leading to IL-3-independent cell growth. To verify efficient recombination, we analyzed the expression of the NPM-ALK fusion protein in the recombined cells and could also show the t(11;17) in the IL-3 independent Ba/F3 cells. Subsequent functional testing of these cells using an Alk-inhibitor showed exquisite responsiveness towards Crizotinib, demonstrating strong dependence on the newly generated ALK fusion oncoprotein. Furthermore, a comparison of the gene expression pattern between Ba/F3 cells overexpressing the Npm-Alk cDNA with Ba/F3 cells transformed by CRISPR-mediated Npm-Alk translocation indicated that, while broadly overlapping, a set of pathways including the unfolded protein response pathway was increased in the Npm-Alk overexpression model, suggesting increased reactive changes induced by exogenous overexpression of Npm-Alk. Furthermore, we observed clustered expression changes in genes located in chromosomal regions close to the breakpoint in the new CRISPR-based model, indicating positional effects on gene expression mediated by the translocation event, which are not part of the older models. Conclusions: Thus, CRISPR-mediated recombination provides a novel and more faithful approach to model oncogenic translocations, which may lead to an improved understanding of the molecular pathogenesis of ALCL and enable more accurate therapeutic models of malignancies driven by oncogenic fusion proteins. Full article

Hioredoxins are small proteins crucial for maintaining cellular redox balance and are involved in various biological processes, including growth, photosynthesis, development, and stress responses. This study aims to conduct a genome-wide analysis of the typical Thioredoxin (TRX) gene family in hexaploid Camellia oleifera and explore the role of the CoTRX25 gene in flowering. Through bioinformatics approaches, we identified 27 typical TRX gene family members in the C. oleifera genome and analyzed their phylogenetic relationships, gene structures, conserved motifs, and chromosomal distributions. Transcriptomic analysis across different tissues was performed to determine the expression patterns of these genes. Additionally, the CoTRX25 gene was cloned and heterologously overexpressed in Arabidopsis thaliana to investigate its functional role in flowering. The 27 TRX genes were mainly located on 11 chromosomes, with multiple gene duplication events identified, indicating that gene duplication has played a significant role in the expansion of the TRX family. Transcriptomic analysis revealed that most typical TRX genes are highly expressed in embryos, suggesting their potential importance in seed development. Overexpression of CoTRX25 in A. thaliana led to delayed flowering, implying that this gene may be involved in flowering regulation. This study provides a theoretical basis for understanding the functions of typical TRX genes in C. oleifera growth and development, particularly highlighting the role of CoTRX25 in flowering regulation. Full article

Background: β-ketolipoyl coenzyme A synthase (KCS) is an essential limiting catalyst involved in carbon chain elongation during fatty acid biosynthesis, characterized by strict substrate specificity. C18:1 (oleic acid) plays a vital role in cell membranes and is essential for nutrient storage and stress defense. There are indications of significant accumulation and rapid synthesis of C18:1 during the early growth stages of Ginkgo biloba L. episperm. The KCS gene family in G. biloba has yet to be analyzed, and the role of KCS in oleic acid synthesis remains unexplored. Methods: In this study, this issue was investigated using transcriptomic and metabolomic data, bioinformatics analysis to screen a key gene from the KCS gene family, and dual validation using yeast and Arabidopsis thaliana expression systems to probe its function. Results: A total of 11 members of the GbKCS gene family were identified, and the dynamics of these genes were analyzed during exocarp development in the G. biloba genome. Among them, the gene designated GbKCS7 showed a highly direct association with the content of C18:1. Heterologous expression of GbKCS7 in yeast increased C18:1N12 and C18:1 content by 3.18-fold and 2.07-fold, respectively. Overexpression of GbKCS7 in Arabidopsis showed that C18:1 was increased by 27.70% and 31.43% in GbKCS7-OE-1 and GbKCS7-OE-2 strains, correspondingly, in juxtaposition to the non-transgenic plants. In addition, the content of VLCFAs increased to varying degrees. Conclusions: These outcomes offer important insights for investigating the role of KCS genes in fatty acid synthesis to further improve G.biloba resistance. Full article

Flavonoids are an important type of bioactive substance contained in jujubes. Flavonol synthase (FLS) is a key enzyme for the synthesis of flavonoids such as flavonols and anthocyanins. To study the biological functions of FLS in jujubes, we cloned the ZjFLS gene; analyzed its physicochemical properties and evolutionary relationships; and then conducted an expression characteristic analysis, subcellular localization, prokaryotic expression and heterologous overexpression in Arabidopsis thaliana. The results showed that the length of ZjFLS is 951 bp, and it encodes 316 amino acids. A sequence analysis revealed that ZjFLS exhibited a high degree of conservation in evolution. The results of a qRT-PCR analysis indicated that the ZjFLS gene could be expressed in different tissues of jujube: the expression level was the highest in the leaves, followed by the flowers, and the lowest was in the fruits. Within these expression levels, it was higher in young leaves than in mature leaves and higher in the white-ripe-stage fruits than in the semi-red-stage fruits. Subcellular localization indicated that the ZjFLS gene was located in the nucleus, cytoplasmic matrix, and cytoplasmic membrane. Our research findings show that the ZjFLS protein can be induced and obtained in the prokaryotic expression system and successfully purified. It mainly exists in the form of inclusion bodies and has a relatively low content in the soluble supernatant. The total flavonoid content of Arabidopsis thaliana strains with a heterologous overexpression of the ZjFLS gene was significantly higher than that of the wild type, confirming that the ZjFLS gene can promote the biosynthesis of flavonoid substances. Full article

Tomato bacterial wilt, caused by Ralstonia solanacearum (G⁻), is one of the most devastating plant diseases. Developing effective resistance against this pathogen remains a major challenge in plant disease management. In this study, we constructed a fusion gene BPI-LY by combining the gene encoding the lipophilic functional domains of human bactericidal/permeability-increasing protein (BPI) with the gene of human lysozyme (LY). The recombinant gene BPI-LY was heterologously expressed in yeast and tomato. Preliminary in vitro assays in yeast demonstrated that BPI enhances LY’s antibacterial activity against G⁻ bacteria. Furthermore, overexpression of BPI-LY in tomato delayed onset of the disease in the transgenic lines and lowered the degree of tissue damage and the number of bacteria present in the stems relative to those in the wild-type plant. Additionally, the expression levels of the SlSOD, SlPOD, SlPAL, SlPR5, SlPR10, and SlPR-NP24 genes were indirectly upregulated in the transgenic plants following R. solanacearum inoculation. Collectively, these findings demonstrate that BPI-LY enhances the resistance of transgenic tomato against bacterial wilt caused by R. solanacearum. Full article

Background:Salmonella Typhimurium is a major zoonotic pathogen, in which type 1 fimbriae play a crucial role in intestinal colonization and immune modulation. This study aimed to improve the protective immunity of a previously developed growth-deficient strain—a double auxotroph for D-glutamate and D-alanine—by engineering the inducible expression of type 1 fimbriae. Methods: P_tetA-driven expression of the fim operon was achieved by λ-Red mutagenesis. fimA expression was quantified by qRT-PCR, and fimbriation visualized by transmission electron microscopy. Adhesive properties were evaluated through FimH sequence analysis, yeast agglutination, mannose-binding/inhibition assays, and HT-29 cell adherence. BALB/c mice were immunized orogastrically with IRTA ΔΔΔ or IRTA ΔΔΔ P_tetA::fim. Safety and immunogenicity were assessed by clinical monitoring, bacterial load, fecal shedding, ELISA tests, and adhesion/blocking assays using fecal extracts. Protection was evaluated after challenging with wild-type and heterologous strains. Results: IRTA ΔΔΔ P_tetA::fim showed robust fimA expression, dense fimbrial coverage, a marked mannose-sensitive adhesive phenotype and enhanced HT-29 attachment. Fimbrial overexpression did not alter intestinal colonization or translocation to mesenteric lymph nodes (mLNs). Immunization elicited a mixed IgG1/IgG2a, significantly increased IgA and IgG against type 1 fimbriae-expressing Salmonella, and enhanced the ability of fecal extracts to inhibit the adherence of wild-type strains. Upon challenge (IRTA wild-type/20220258), IRTA ΔΔΔ P_tetA::fim reduced infection burden in the cecum (−1.46/1.47-log), large intestine (−1.35/2.17-log), mLNs (−1.32/0.98-log) and systemic organs more effectively than IRTA ΔΔΔ. Conclusions: Inducible expression of type 1 fimbriae enhances mucosal immunity and protection, supporting their inclusion in next-generation Salmonella vaccines. Future work should assess cross-protection and optimize FimH-mediated targeting for mucosal delivery. Full article

Plant-specific WUSCHEL (WUS)-related homeobox (WOX) family of transcription factors are involved in apical meristem maintenance, embryogenesis, lateral organ development, and hormone signaling. Among the members of this family, WOX1 is known to play essential roles in many species. However, the function of the peony ‘Feng Dan’ (Paeonia ostii L.) WOX1 (PoWOX1) remains unknown. The initial bioinformatic analysis revealed that PoWOX1 belongs to the modern clade of the WOX gene family and has a highly conserved homeodomain (HD), the WUS motif, the STF-box, and the MAEWEST/WOX4-box. Subsequent heterologous overexpression in Arabidopsis thaliana revealed that PoWOX1 promotes root growth, early shoot initiation, and flowering. The root vascular tissues, especially the arrangement and size of xylem cells, were different between the PoWOX1-overexpressing transgenics and the wild-type plants, and the pericycle cells adjacent to the xylem divided more easily in the transgenics than in the wild type. Furthermore, under in vitro conditions, the transgenic leaf explants exhibited more callus induction and differentiation than the wild-type leaf explants. Thus, the study’s findings provide novel insights into the role of PoWOX1 in promoting root development and callus tissue induction and differentiation, serving as a reference for developing an efficient regeneration system for the peony. Full article

The yeast metabolic and evolutionary engineering, especially Saccharomyces cerevisiae, plays a significant role in the enhancement of its industrial applications in food, beverage, and biofuel production. This review integrates genetic engineering, systems biology, and evolutionary principles to optimize yeast performance, adaptability, and productivity. The key strategies which enable targeted genome modifications to improve substrate utilization, stress tolerance, and the biosynthesis of valuable metabolites such as flavor compounds, organic acids, vitamins, and antioxidants, including precise gene editing, notably CRISPR-Cas9. The metabolic pathway optimization through gene overexpression, deletion, and heterologous pathway integration, supported by multi-omics analyses and the Subcellular compartmentalization of metabolic pathways, which enhances biosynthetic efficiency. This review then discusses evolutionary engineering and global transcription machinery engineering by leveraging natural selection and global gene regulation to improve complex traits. The exploration of non-Saccharomyces species and genome shuffling expands the genetic toolkit for strain development. Emerging approaches, including machine learning and synthetic biology, are accelerating rational strain design. By critically synthesizing these diverse methodologies, this review highlights current advancements, identifies key challenges, and outlines future directions in engineering robust yeast strains for sustainable food biotechnology. Full article

Isodon suzhouensis, also known as “Wangzaozi”, is an edible and medicinal plant belonging to the Lamiaceae family. Its main functional constituents are the tetracyclic diterpenoids known as wangzaozins. Wangzaozins have a strong structural similarity to gibberellins (GAs), which are synthesized via the diterpenoid biosynthetic pathway (map00904). The formation of the diterpenoid skeleton is regulated by copalyl diphosphate synthase (CPS) and kaurene synthase (KS). In order to identify and study the KS gene involved in wangzaozins biosynthesis, a transcriptomic and metabolomic analysis of Isodon suzhouensis was performed. The IsKS1 gene, which was highly expressed in leaves, was successfully cloned. The binding mode and sites of IsKS1 with its catalyzed substrate, ent-copalyl diphosphate (ent-CPP), were predicted using AutoDock. The docking results revealed hydrophobic interactions, hydrogen bonds, and salt bridges between them. Furthermore, overexpression of IsKS1 in Arabidopsis thaliana resulted in a significant increase in gibberellin content, as well as the up-regulation of GA2(KS) and GA3OX1 genes. These results suggest that the IsKS1 gene is involved in gibberellin biosynthesis and may potentially contribute to the biosynthesis of wangzaozins. Full article

In plants, ferric-chelate reductase (FRO) plays a critical role in mediating extracellular iron (Fe) reduction, a process essential for cellular Fe homeostasis and abiotic stress tolerance. However, the biological functions and regulatory mechanisms of FRO proteins in fruit crops remain poorly characterized. Here, six VvFRO genes were identified in the table grape cultivar ‘Yanhong’. Transcriptional analysis revealed that root expression of these genes was mainly induced under Fe deficiency, Fe depletion, NaCl stress, and PEG-induced drought stress, respectively, but remained unchanged by low temperature (4 °C) or heat treatment (45 °C). Among them, VvFRO3 exhibited the highest constitutive expression, predominantly in leaves, and was significantly up-regulated under Fe deficiency, Fe depletion, or NaCl treatment. Functional complementation assays demonstrated that heterologous overexpression of VvFRO3 in the Arabidopsis thaliana fro2 knockout mutant rescued its growth retardation phenotype, particularly under Fe-deficient conditions. This study advances our understanding of Fe uptake, transport, and homeostasis mechanisms in perennial fruit crops. Full article

Gossypol is a polyphenolic toxic compound present in cotton plants. To determine whether the candidate cytochrome P450BM3 enzymes could reduce gossypol in vitro, functional recombinant cytochrome P450BM3 enzymes were successfully expressed in E. coli. Site-directed mutagenesis generated mutants (R162H, R162K, Q129H, Q129N) to explore structural determinants of catalytic efficiency. Both wild-type P450BM3 and mutants exhibited significant ability to reduce gossypol levels, with R162H and R162K showing 33.4% and 24.2% reduced catalytic efficiency compared with the wild-type enzyme, respectively. Q129H and Q129N mutants maintained comparable catalytic efficiency to the wild type. Metabolomic profiling revealed two distinct reducing pathways catalyzed by wild-type P450BM3 and its mutants (R162H/Q129H), involving decarboxylation, hydroxylation, and C-C bond cleavage. This study demonstrated the feasibility of P450BM3 as a highly efficient biocatalyst for reducing gossypol levels, speculated that Arg162 might be a critical active residue, and hypothesized the potential pathways by which P450BM3 catalyzes the reduction of gossypol content, thereby providing a theoretical foundation for the enzymatic reduction of gossypol. Full article