Search Results (100)

Plant aquaporin proteins (AQPs) are categorized into seven distinct families, among which, plasma membrane intrinsic proteins (PIPs) play pivotal roles in plant growth and physiological processes. In this study, we identified 11 CpPIP genes in wintersweet (Chimonanthus praecox (L.) Link) based on bioinformatic characterization of gene structural organization, chromosomal localization, and phylogenetic relationships. Subsequent phylogenetic reconstruction resolved two evolutionarily distinct CpPIP subclasses. We focused on the isolation and characterization of CpPIP1;1, which showed the highest expression in floral organs. During flowering, a significant increase was observed in the expression of the CpPIP1;1 gene in response to a gradual reduction in environmental temperature. Moreover, the overexpression of CpPIP1;1 in Arabidopsis thaliana resulted in early flowering and an enhanced tolerance to salt, drought, and cold stress. We subsequently transcriptionally fused the CpPIP1;1 promoter containing MYC and MYB low-temperature response elements to the β-glucuronidase (GUS) reporter gene and introduced this construct into Nicotiana tabacum. GUS activity assays of the transgenic plants revealed that the CpPIP1;1 promoter was effectively expressed in flowers. Furthermore, the promoter transcriptional activity was enhanced in response to salt, drought, cold, gibberellic acid, and methyl jasmonate treatments. Collectively, our findings in this study revealed that CpPIP1;1 plays a key role in the regulation of flowering and stress tolerance in wintersweet plants. Full article

β-glucuronidase is an important hydrolase, which plays an important role in drug metabolism, clinical diagnostics, and biotransformation. This study focuses on the heterologous expression, isolation, purification, and its enzymatic properties of β-glucuronidase CpGUS from Clostridium perfringens, as well as its application in the whole-cell transformation of unconjugated bilirubin from pig bile. A recombinant E. coli BL21(DE3)/pET-28a-CpGUS was constructed for the heterologous expression of CpGUS, with the majority of the expressed enzyme being soluble. Enzymatic analysis showed that CpGUS displayed optimal activity at pH 5.0 and 45 °C, and it rapidly lost activity at pH < 4.5. Metal ions, such as Mg²⁺ and Fe²⁺, enhanced CpGUS catalysis, while Zn²⁺, K⁺, Fe³⁺, Mn²⁺, Cu²⁺, and Na⁺ inhibited it. Notably, Cu²⁺ and Fe³⁺ can significantly inhibit β-glucuronidase, resulting in the complete loss of its activity. The results of the whole-cell transformation experiment show that when E.coli BL21(DE3)/ pET-28a-CpGUS at an OD₆₀₀ of 10 was incubated at pH 5.0, a temperature of 45 °C, and a rotation speed of 200 rpm for 12 h, the hydrolysis rate of the conjugated bilirubin in pig bile reached 81.1%, the yield of unconjugated bilirubin was 76.8%, and the purity of unconjugated bilirubin was 98.2%. The three-dimensional structure of CpGUS was predicted using AlphaFold2 (AlphaFold v2.0, DeepMind Technologise Limited, London, UK), and p-Nitrophenyl-β-D-Glucuronide (pNPG) and conjugated bilirubin were then docked to the CpGUS protein model using SWISSDOCK. The best docked conformations of the CpGUS–pNPG and CpGUS–conjugated bilirubin complex systems were simulated by independent 500 ns molecular dynamics (MD) runs with the RSFF2C force field, and the binding dynamic and catalytic mechanism of each system were obtained. The results indicated that π-π stacking, hydrogen bonding, and hydrophobic interactions between the key residue Tyr472 and the benzene ring of pNPG molecules are crucial for its catalytic process. Similarly, for the binding and catalysis of conjugated bilirubin by CpGUS, the π-π stacking and hydrogen bonding and hydrophobic interactions between the sidechains of residues Phe368 and Tyr472 and the benzene ring of conjugated bilirubin play a synergistic role during its catalytic process. Their total binding free energy (∆G_bind) values were calculated to be as high as −65.05 ± 12.66 and −86.70 ± 17.18 kJ/mol, respectively. These results suggest that CpGUS possesses high binding and catalytic hydrolysis properties for both pNPG and conjugated bilirubin. Full article

Arabinogalactan proteins (AGPs) constitute a diverse class of hydroxyproline-rich glycoproteins implicated in various aspects of plant growth and development. However, their functional characterization in cotton (Gossypium spp.) remains limited. As a globally significant economic crop, cotton serves as the primary source of natural fiber, making it essential to understand the genetic mechanisms underlying its growth and development. This study aims to perform a comprehensive genome-wide identification and characterization of the AGP gene family in Gossypium spp., with a particular focus on elucidating their structural features, evolutionary relationships, and functional roles. A genome-wide analysis was conducted to identify AGP genes in Gossypium spp., followed by classification into distinct subfamilies based on sequence characteristics. Protein motif composition, gene structure, and phylogenetic relationships were examined to infer potential functional diversification. Subcellular localization of a key candidate gene, GhAGP50, was determined using fluorescent protein tagging, while gene expression patterns were assessed through β-glucuronidase (GUS) reporter assays. Additionally, hormonal regulation of GhAGP50 was investigated via treatments with methyl jasmonate (MeJA), abscisic acid (ABA), indole-3-acetic acid (IAA), and gibberellin (GA). A total of 220 AGP genes were identified in Gossypium spp., comprising 19 classical AGPs, 28 lysine-rich AGPs, 55 AG peptides, and 118 fasciclin-like AGPs (FLAs). Structural and functional analyses revealed significant variation in gene organization and conserved motifs across subfamilies. Functional characterization of GhAGP50, an ortholog of AGP18 in Arabidopsis thaliana, demonstrated its role in promoting epidermal hair formation in leaves and stalks. Subcellular localization studies indicated that GhAGP50 is targeted to the nucleus and plasma membrane. GUS staining assays revealed broad expression across multiple tissues, including leaves, inflorescences, roots, and stems. Furthermore, hormonal treatment experiments showed that GhAGP50 expression is modulated by MeJA, ABA, IAA, and GA, suggesting its involvement in hormone-mediated developmental processes. This study presents a comprehensive genome-wide analysis of the AGP gene family in cotton, providing new insights into their structural diversity and functional significance. The identification and characterization of GhAGP50 highlight its potential role in epidermal hair formation and hormonal regulation, contributing to a deeper understanding of AGP functions in cotton development. These findings offer a valuable genetic resource for future research aimed at improving cotton growth and fiber quality through targeted genetic manipulation. Full article

Solanum betaceum Cav. (tamarillo) has a strong biotechnological potential given the ease of obtaining cell lines from it that can be genetically transformed. However, genetic transformation of tamarillo cell suspension cultures has not yet been described. This study presents a simple method for Agrobacterium-mediated transformation of these cells and demonstrates the successful insertion of the β-glucuronidase gene (gusA) and the yellow fluorescent protein gene (eyfp) in their genome. For the success of this protocol, the selection of actively growing sub-cultured callus as explant and isolation of bacterial colonies with a cell density OD₆₀₀ of 0.6–0.8 were key steps. Also, the inoculation of the callus in a bacteria liquid culture, the use of sonication, and the addition of antioxidants were essential. The transient expression of the gusA gene in tamarillo callus was confirmed and quantified, and no significant differences were observed between using LBA4404 or EHA105 strains. Finally, the insertion of the eyfp gene in the tamarillo genome enabled the in vivo confirmation of the transformation success. The present study showed that tamarillo cell suspension cultures can be genetically modified, opening the way for metabolite production in transformed cells and future scaling-up in bioreactors. Full article

Gut bacterial β-glucuronidase is an important molecular target in several therapeutic applications. β-glucuronidase inhibitors can effectively alleviate gastrointestinal toxicity caused by certain drugs. Licorice, a traditional Chinese medicine, harmonizes various herbs and mitigates the toxicity of hundreds of herbs. In this study, a comprehensive computational strategy was employed to evaluate four licorice flavonoids (liquiritigenin, isoliquiritigenin, liquiritin, and isoliquiritin) as potential Escherichia coli β-glucuronidase (EcGUS) inhibitors. Density functional theory was used to determine their geometries, thermal parameters, dipole moments, polarizabilities, and molecular electrostatic potentials. The inhibitory mechanisms of these four flavonoids on EcGUS were investigated using molecular docking, molecular dynamics simulations, and free energy calculations. The results show that all four flavonoids stably bind to EcGUS. Moreover, all molecules, except liquiritigenin, are potent and selective inhibitors of EcGUS. Further calculations suggest that isoliquiritin exhibits the strongest binding affinity for EcGUS among the four licorice flavonoids. Thus, isoliquiritin is a promising candidate for the development of EcGUS inhibitors. These findings will aid in designing and developing novel flavonoid-based inhibitors of EcGUS to alleviate gastrointestinal toxicity caused by drugs. Full article

AP2/ERF (APETALA2/Ethylene Responsive Factor) transcription factors, a class of plant-specific transcription factors, play a pivotal role in plant growth, development, metabolism, and stress response. The pineapple (Ananas comosus (L.) Merr.), a perennial fruit, belongs to the Bromeliaceae family. It is an economically important crop worldwide, which is consumed as fresh fruit, canned fruit, a fiber source, and even pharmaceutical raw material. We identified 75 AcoAP2/ERF genes in the pineapple genome, with four manually curated. They were distributed evenly on 23 chromosomes, except on LG20 and LG23. Sequence lengths, molecular weights, and intron numbers were diverse. The majority of pineapple AcoAP2/ERF genes were localized in nuclear while seven AcoERFs were located in mitochondrial or chloroplast. All pineapple AcoAP2/ERF genes possess an AP2 domain and are divided into 10 clades. Most originate from whole-genome or segmental duplication instead of transposon events. Utilizing pineapple calluses as experimental material, qRT–PCR analysis revealed that the expression of the majority of AcoAP2/ERF genes was induced in response to abscisic acid (ABA), gibberellic acid (GA), ethylene (ET), and naphthalene acetic acid (NAA). In this study, we cloned the promoter sequence of the AcoERF24 gene and divided it into three fragments to construct individual vectors. These vectors were subsequently introduced into Arabidopsis thaliana for β-glucuronidase (GUS) activity analysis, revealing variations in activity levels among the different fragments. This study not only deepens our understanding of the AcoAP2/ERF genes family in olives but also provides an important basis for subsequent studies on the regulation of AcoERF24 gene expression and biological functions. Full article

Baicalein is a unique flavonoid compound with important pharmacological activities, derived from Scutellaria baicalensis Georgi. Baicalein, as the aglycone of baicalin, is a key form for exerting pharmacological activity in vivo. β-glucuronidases (GUSs) are the enzymes involved in the conversion of baicalin to baicalein. In this study, the content of baicalein in S. baicalensis was significantly increased by 20.44% after treatment with 5% PEG6000. Seven GUSs from the glycoside hydrolase 79 family were identified through comparative transcriptome analysis. Among them, GUS1 and GUS2 were confirmed to have catalytic activity in converting baicalin to baicalein in prokaryotic and eukaryotic systems. The correlation analysis further revealed a significant positive correlation of 0.962 (p < 0.01) between the expression of GUS2 and baicalein content in six different sources of S. baicalensis. Interestingly, the presence of variable sites in the GUS1 and GUS2 genes significantly affected their catalytic efficiency in the S. baicalensis samples from the six geographic origins. These findings also provide valuable GUS biological enzyme resources for the effective synthesis of baicalein and offer new insights into the accumulation pattern of baicalein in S. baicalensis. Full article

SMALL AUXIN UP-REGULATED RNA (SAURs) genes are acknowledged as auxin-responsive genes that play crucial roles in modulating adaptive growth under abiotic stress conditions. Low temperatures constitute a primary limiting factor that significantly impairs the development, growth, and fruit quality of watermelon plants during the winter and spring seasons. Despite their potential importance, SAURs have not yet been thoroughly investigated or characterized in watermelon. In this study, we identified a positive regulator of the chilling stress response among watermelon SAURs, designated as ClSAUR1. Subcellular localization analysis demonstrated that the protein is directed to both the nucleus and cytoplasm. Quantitative real-time PCR (qRT-PCR) analysis indicated that ClSAUR1 is ubiquitously expressed across various watermelon tissues, with pronounced expression in the roots and leaves. Moreover, qRT-PCR and promoter::β-glucuronidase (GUS) staining assays revealed that the expression of ClSAUR1 is significantly upregulated in response to exogenous abscisic acid (ABA) and chilling stress. The overexpression of ClSAUR1 in tobacco lines was contrasted and analyzed, revealing an increased tolerance to chilling stress. This was evidenced by a reduced degree of wilting and chlorosis compared to wild-type (WT) plants. Furthermore, the overexpressed lines showed reduced reactive oxygen species (ROS) accumulation and increased antioxidant enzyme activity. The qRT-PCR results further indicated that the expression levels of genes associated with abscisic acid (ABA), antioxidant enzymes, and CBF–COR cold-responsive pathways were upregulated in the transgenic tobacco lines. This study provides new insights into the role of ClSAURs in enhancing the cold resistance of watermelon. Full article

Pinus koraiensis is classified as a second-class protected wild plant in China, recognized for its considerable economic and ecological importance. However, progress in functional research and breeding applications for this species has been hindered by the lack of an effective genetic transformation system. The purpose of this study was to develop a reliable and efficient genetic transformation system for a Pinus koraiensis embryonic callus using somatic embryogenesis technology. The Pinus koraiensis embryonic callus and β-glucuronidase (GUS) were employed as the reporter gene in an Agrobacterium-mediated transformation to investigate critical transformation factors, including antibiotic type and concentration, Agrobacterium bacterial solution concentration, infiltration, and co-cultivation times. The findings indicated that the proliferation of the Pinus koraiensis embryonic callus was substantially inhibited by 10 mg·L⁻¹ of Hygromycin (Hyg), and a remarkable 93.42 ± 2.13% efficiency was achieved with an OD600 absorbance value of 0.6 during transformation. Two days of optimal co-cultivation yielded a transformation rate of 82.61%, with the resistant embryonic callus exhibiting a high GUS staining rate of 88.89%. Resistant somatic embryos were effectively obtained following the optimized protocol. This research contributes to the advancement of seed resource breeding and genetic enhancement for Pinus koraiensis, establishing a solid foundation for the investigation of gene functions specific to this species. Full article

Microbes produce various bioactive metabolites that can influence plant growth and stress tolerance. In this study, a plant growth-promoting rhizobacterium (PGPR), strain S14, was identified as Micrococcus luteus (designated as MlS14) using de novo whole-genome assembly. The MlS14 genome revealed major gene clusters for the synthesis of indole-3-acetic acid (IAA), terpenoids, and carotenoids. MlS14 produced significant amounts of IAA, and its volatile organic compounds (VOCs), specifically terpenoids, exhibited antifungal activity, suppressing the growth of pathogenic fungi. The presence of yellow pigment in the bacterial colony indicated carotenoid production. Treatment with MlS14 activated the expression of β-glucuronidase (GUS) driven by a promoter containing auxin-responsive elements. The application of MlS14 reshaped the root architecture of Arabidopsis seedlings, causing shorter primary roots, increased lateral root growth, and longer, denser root hairs; these characteristics are typically controlled by elevated exogenous IAA levels. MlS14 positively regulated seedling growth by enhancing photosynthesis, activating antioxidant enzymes, and promoting the production of secondary metabolites with reactive oxygen species (ROS) scavenging activity. Pretreatment with MlS14 reduced H₂O₂ and malondialdehyde (MDA) levels in seedlings under drought and heat stress, resulting in greater fresh weight during the post-stress period. Additionally, exposure to MlS14 stabilized chlorophyll content and growth rate in seedlings under salt stress. MlS14 transcriptionally upregulated genes involved in antioxidant defense and photosynthesis. Furthermore, genes linked to various hormone signaling pathways, such as abscisic acid (ABA), auxin, jasmonic acid (JA), and salicylic acid (SA), displayed increased expression levels, with those involved in ABA synthesis, using carotenoids as precursors, being the most highly induced. Furthermore, MlS14 treatment increased the expression of several transcription factors associated with stress responses, with DREB2A showing the highest level of induction. In conclusion, MlS14 played significant roles in promoting plant growth and stress tolerance. Metabolites such as IAA and carotenoids may function as positive regulators of plant metabolism and hormone signaling pathways essential for growth and adaptation to abiotic stress. Full article

Eremosparton songoricum (Litv.) Vass. is a desert legume exhibiting extreme drought tolerance and the ability to withstand various harsh environments, making it a good candidate for investigating stress tolerance mechanisms and exploring valuable stress-resistant genes. However, the absence of a genetic transformation system for E. songoricum poses significant limitations for functionally validating these stress-resistant genes in situ. In this study, we developed an Agrobacterium-mediated transient transformation system for E. songoricum utilizing the β-glucuronidase (GUS) gene as a reporter. We investigated three types of explants (seedlings, assimilated branches and callus) and the effects of different Agrobacterium strains, seedling ages, OD₆₀₀ values, acetosyringone (AS) concentrations, sucrose concentrations and infection times on the transformation efficiency. The results reveal that the optimal transformation system was infecting one-month-old regenerating assimilated branches with the Agrobacterium strain C58C1. The infection solution comprised 1/2 MS medium with 3% sucrose and 200 μM AS at an OD₆₀₀ of 0.8, infection for 3 h and then followed by 2 days of dark cultivation, which achieving a maximum transformation rate of 97%. The maximum transformation rates of the seedlings and calluses were 57.17% and 39.51%, respectively. Moreover, we successfully utilized the assimilated branch transient transformation system to confirm the role of the previously reported transcription factor EsDREB2B in E. songoricum. The overexpression of EsDREB2B enhanced drought tolerance by increasing the plant’s reactive oxygen species (ROS) scavenging capacity in situ. This study established the first transient transformation system for a desert legume woody plant, E. songoricum. This efficient system can be readily applied to investigate gene functions in E. songoricum. It will expedite the exploration of genetic resources and stress tolerance mechanisms in this species, offering valuable insights and serving as a reference for the transformation of other desert plants and woody legumes. Full article

Medicago sativa L. (Alfalfa) is a globally recognized forage legume that has recently gained attention for its high protein content, making it suitable for both human and animal consumption. However, due to its perennial nature and autotetraploid genetics, conventional plant breeding requires a longer timeframe compared to other crops. Therefore, genetic engineering offers a faster route for trait modification and improvement. Here, we describe a protocol for achieving efficient transient gene expression in alfalfa through genetic transformation with the Agrobacterium tumefaciens pCAMBIA1304 vector. This vector contains the reporter genes β-glucuronidase (GUS) and green fluorescent protein (GFP), along with a selectable hygromycin B phosphotransferase gene, all driven by the CaMV 35s promoter. Various transformation parameters—such as different explant types, leaf ages, leaf sizes, wounding types, bacterial concentrations (OD_600nm), tissue preculture periods, infection periods, co-cultivation periods, and different concentrations of acetosyringone, silver nitrate, and calcium chloride—were optimized using 3-week-old in vitro-grown plantlets. Results were attained from data based on the semi-quantitative observation of the percentage and number of GUS spots on different days of agro-infection in alfalfa explants. The highest percentage of GUS positivity (76.2%) was observed in 3-week-old, scalpel-wounded, segmented alfalfa leaf explants after 3 days of agro-infection at a bacterial concentration of 0.6, with 2 days of preculture, 30 min of co-cultivation, and the addition of 150 µM acetosyringone, 4 mM calcium chloride, and 75 µM silver nitrate. The transient expression of genes of interest was confirmed via histochemical GUS and GFP assays. The results based on transient reporter gene expression suggest that various factors influence T-DNA delivery in the Agrobacterium-mediated transformation of alfalfa. The improved protocol can be used in stable transformation techniques for alfalfa. Full article

Alignment of picornavirus proteinase/polymerase sequences reveals this family evolved into five ‘supergroups’. Interestingly, the nature of the 2A region of the picornavirus polyprotein is highly correlated with this phylogeny. Viruses within supergroup 4, the Paavivirinae, have complex 2A regions with many viruses encoding multiple 2A^NPGP sequences. In vitro transcription/translation analyses of a synthetic polyprotein comprising green fluorescent protein (GFP) linked to β-glucuronidase (GUS) via individual 2A^NPGPs showed two main phenotypes: highly active 2A^NPGP sequences—similar to foot-and-mouth disease virus 2A^NPGP—and, surprisingly, a novel phenotype of some 2A^NPGP sequences which apparently terminate translation at the C-terminus of 2A^NPGP without detectable re-initiation of downstream sequences (GUS). Probing databases with the short sequences between 2A^NPGPs did not reveal any potential ‘accessory’ functions. The novel, highly active, 2A-like sequences we identified substantially expand the toolbox for biomedical/biotechnological co-expression applications. Full article

Promoters are powerful tools for breeding new varieties using transgenic technology. However, the low and unstable expression of target genes is still a limiting factor in Larix kaempferi (Lamb.) Carr (Japanese larch) genetic transformation. In this study, we analyzed L. kaempferi transcriptome data, screened out highly expressed genes, cloned their promoters, and constructed plant expression vectors containing the β-glucuronidase (GUS) reporter gene driven by these promoters. Recombinant vectors were introduced into the L. kaempferi embryogenic callus by means of the Agrobacterium-mediated transient or stable genetic transformation method, and the promoter activity was then determined by measuring GUS expression and its enzyme activity in the transformed materials. Four highly expressed genes were identified: L. kaempferi Zhang Chen Yi-1 (LaZCY-1), Zhang Chen Yi-2 (LaZCY-2), Translationally Controlled Tumor Protein (LaTCTP), and ubiquitin (LaUBQ). The 2000 bp fragments upstream of ATG in these sequences were cloned as promoters and named pLaZCY-1, pLaZCY-2, pLaTCTP, and pLaUBQ. Semi-quantitative and quantitative RT-PCR analyses of transient genetic transformation materials showed that all four promoters could drive GUS expression, indicating that they have promoter activities. Semi-quantitative and quantitative RT-PCR analyses and the histochemical staining of stable genetic transformation materials showed that the pLaUBQ promoter had higher activity than the other three L. kaempferi promoters and the CaMV35S promoter. Thus, the pLaUBQ promoter was suggested to be used in larch genetic transformation. Full article

The primary constituents of the essential oil derived from Santalum album L. are (Z)-α-santalol, (Z)-β-santalol, (Z)-α-exo-bergamotol, and (Z)-epi-β- santalol. SaCYP736A167 plays a pivotal role in the biosynthesis of these sesquiterpene alcohols within S. album, but the mechanisms governing the expression of the SaCYP736A167 gene is far from being deciphered. In this research, a promoter sequence of the SaCYP736A167 gene, spanning 2808 base pairs, was isolated from S. album. A bioinformatics analysis of the 2384-bp SaCYP736A167 promoter (PSaCYP736A167) showed that abundant stress-inducible cis-acting elements were distributed in different regions of PSaCYP736A167. The histochemical β-glucuronidase (GUS) staining of T1 transgenic Nicotiana tabacum plants harboring PSaCYP736A167 demonstrated that the predominant GUS activity was exhibited in the parenchyma cells of the stem cortex and phloem, suggesting that PSaCYP736A167 is a tissue-specific expression promoter. GUS fluorometric assays of transiently transgenic N. benthamiana leaves revealed that seven distinct segments of PSaCYP736A167 exhibited notably varied levels of GUS activity. A 936-base pair sequence upstream of the transcription initiation codon ATG constitutes the core promoter section of PSaCYP736A167. Our findings shed light on the regulatory mechanisms controlling the transcription of the SaCYP736A167 gene, potentially serving as a novel tissue-specific promoter for applications in transgenic plant biotechnology. Full article