Search Results (105)

Genetic transformation is an essential tool for investigating gene function and editing genomes. Kiwifruit, recognized as a significant global fresh fruit crop, holds considerable economic and nutritional importance. However, current genetic transformation techniques for kiwifruit are impeded by low efficiency, lengthy culture durations (a minimum of six months), and substantial labor requirements. In this research, we established an efficient system for shoot regeneration and the stable genetic transformation of the ‘Hayward’ cultivar, utilizing leaf explants in conjunction with two strains of Agrobacterium that harbor the expression vector pBI121-35S::GFP, which contains the green fluorescent protein (GFP) gene as a visible marker within the T-DNA region. Our results show that 93.3% of leaf explants responded positively to the regeneration medium, producing multiple independent adventitious shoots around the explants within a six-week period. Furthermore, over 71% of kanamycin-resistant plantlets exhibited robust GFP expression, and the entire transformation process was completed within four months of culture. Southern blot analysis confirmed the stable integration of GFP into the genome, while RT-PCR and fluorescence microscopy validated the sustained expression of GFP in mature plants. This efficient protocol for regeneration and transformation provides a solid foundation for micropropagation and the enhancement of desirable traits in kiwifruit through overexpression and gene silencing techniques. Full article

The SEPALLATA3 (SEP3)-like MADS-box genes play crucial roles in determining petal identity and development in the petunia and tomato of Solanaceae. Solanum nigrum is a self-pollinating plant in the Solanaceae family, and produces white flowers. However, the mechanisms controlling the transition from green to white petals during flower development remain poorly understood. In this study, we isolated a flower-specific SEP3-like gene, SnMADS37, from S. nigrum, and investigated its potential role in chlorophyll metabolism during petal development. Our results show that quantitative RT-PCR analysis demonstrates that SnMADS37 is exclusively expressed in petals and stamens during early floral bud development. Overexpression of SnMADS37 clearly enhanced the number of petals, promoting the formation of additional petal-like tissues in stamens and extra organs in some fruits. Moreover, fully opened transformed petals exhibited notable chlorophyll accumulation at their tips and veins, whereas silencing of Snmads37 clearly inhibited petal expansion and reduced green pigmentation in early flower buds. Additionally, the transformed green petals exhibited distinct conical epidermal cells in the green regions, similar to wild type (WT) petals. Our results demonstrate that SnMADS37 plays a critical role in regulating petal identity, expansion, and chlorophyll metabolism during petal development. These findings provide new insights into the functional diversification of SEP3-like MADS-box genes in angiosperms. Full article

Objective: This study aimed to elucidate the regulatory mechanism of an auxin amide hydrolase gene (IAA-Leucine Resistant1-like Hydrolase, RhILL1) in the petal pigmentation of rose (Rosa hybrida), providing theoretical insight into the hormonal regulation of flower coloration at the molecular level. Methods: Using petals at Stage 3 (S3) of the cut rose cultivar ‘Pink Floyd’ as experimental material, we cloned the rose auxin amide hydrolase gene RhILL1 and validated its function via virus-induced gene silencing (VIGS). The expression levels of anthocyanin biosynthetic genes, anthocyanin content, and auxin (IAA) levels were analyzed to assess the role of RhILL1 in petal pigmentation. Results: The full-length open reading frame (ORF) of RhILL1 was cloned, spanning 1326 bp and encoding a 441-amino-acid protein harboring two conserved domains, Peptidase_M20 and M20_dimer, characteristic of the ILL1 protein family. Functional characterization was performed using VIGS. Quantitative real-time PCR (qRT-PCR) revealed that RhILL1 expression progressively increased from the Green (G) stage to S3, correlating with intensified petal coloration. Silencing RhILL1 resulted in visibly lighter petals, the reduced expression of anthocyanin biosynthetic genes, and a significant decrease in endogenous indole-3-acetic acid (IAA) levels compared with controls. Moreover, exogenous application of 10 μM naphthaleneacetic acid (NAA) to petals significantly preserved petal pigmentation. Conclusion: These findings suggest that RhILL1 contributes to the development and maintenance of petal coloration in rose, likely by modulating IAA levels, thereby influencing the expression of anthocyanin biosynthesis-related genes. Full article

Lesion mimic phenotypes, characterized by leaf spots formed in the absence of pathogens or pests, are often associated with reactive oxygen species (ROS) accumulation and cell necrosis. This study identified a novel and stable homozygous spotted phenotype (HSP) from the F₈ population of common wheat (XN509 × N07216). The yellow spots that appeared at the booting stage were light-sensitive, and accompanied by cell necrosis and H₂O₂ accumulation. Compared with homozygous normal plants (HNPs), HSPs exhibited enhanced resistance to stripe rust and powdery mildew without compromising yield. RNA-Seq analysis at three stages revealed that differentially expressed genes (DEGs) between HSPs and HNPs were significantly enriched in KEGG pathways related to photosynthesis and photosynthesis-antenna proteins. GO analysis highlighted chloroplast and light stimulus-related down-regulated DEGs. Fine mapping identified TaSpl1 within a 0.91 Mb interval on chromosome 3DS, flanked by the markers KASP188 and KASP229, using two segregating populations comprising 1117 individuals. The candidate region contained 42 annotated genes, including 14 DEGs based on previous BSR-Seq data. PCR amplification and qRT-PCR verification identified the expression of TraesCS3D02G022100 was consistent with RNA-Seq data. Gene homology analysis and silencing experiments confirmed that TraesCS3D02G022100 was associated with stay-green traits. These findings provide new insights into the genetic regulation of lesion mimics, photosynthesis, and disease resistance in wheat. Full article

Pepper golden mosaic virus (PepGMV) is a bipartite begomovirus of pepper and tomato from North America. In ‘Anaheim’ pepper plants PepGMV-Mo strain (Mo) causes systemic yellow foliar mosaic symptoms, while PepGMV-D strain (D) causes distortion of 1st–6th expanding leaves, and asymptomatic infection of subsequently developing leaves, like other known ‘recovery’ phenotypes. Infections established with DNA-A Mo and D components expressing red-shifted green fluorescent protein in place of coat protein and in situ hybridization, showed PepGMV-Mo localized to phloem and mesophyll cells, while -D was mesophyll restricted. Alignment of PepGMV-Mo and -D DNA-B components revealed three indels upstream of the BC1 gene that encodes the movement protein (MP). To determine if this non-coding region (*BC1) D-strain MP putative promoter contributed to ‘recovery’, plants were inoculated with chimeric DNA-B Mo/D components harboring reciprocally exchanged *BC1, and wild-type DNA-A Mo and D components. Symptoms were reminiscent but not identical to wild-type -Mo or -D infection, respectively, suggesting ‘recovery’ cannot be attributed solely to the *BC1. Both BC1 and D*BC1 were targeted by post-transcriptional gene silencing; however, ‘recovered’ leaves accumulated fewer transcripts and 21–24 nt vsiRNAs. Thus, inefficient in planta movement of PepGMV-D is associated with a non-pepper-adapted ‘defective’ BC1 that facilitates hyper-efficient PTGS, leading to BC1 transcript degradation that in turn limits virus spread, thereby recapitulating disease ‘tolerance’. Full article

Heme oxygenase (HO) metabolizes heme into ferrous iron, carbon monoxide (CO), and biliverdin-IXα (BV), the latter being reduced into bilirubin-IXα (BR) by the biliverdin reductase-A (BVR). Heme oxygenase exists as two isoforms, HO-1, inducible and involved in the cell stress response, and HO-2, constitutive and committed to the physiologic turnover of heme and in the intracellular oxygen sensing. Many studies have identified genetic variants of the HO/BVR system and suggested their connection in free radical-induced diseases. The most common genetic variants include (GT)n dinucleotide length polymorphisms and single nucleotide polymorphisms. Gain-of-function mutations in the HO-1 and HO-2 genes foster the ventilator response to hypoxia and reduce the risk of coronary heart disease and age-related macular degeneration but increase the risk of neonatal jaundice, sickle cell disease, and Parkinson’s disease. Conversely, loss-of-function mutations in the HO-1 gene increase the risk of type 2 diabetes mellitus, chronic obstructive pulmonary disease, and some types of cancers. Regarding BVR, the reported loss-of-function mutations increase the risk of green jaundice. Unfortunately, the physiological role of the HO/BVR system does not allow for the hypothesis gene silencing/induction strategies, but knowledge of these mutations can certainly facilitate a medical approach that enables early diagnoses and tailored treatments. Full article

MYB (myeloblastosis) is one of the most abundant transcription factors in plants which regulates various biological processes. The molecular characteristics and function of R2R3-MYB transcription factors in amaranth remain unclear. In this study, 73 R2R3-MYB members were identified from the amaranth genome database and we further analyzed their chromosome position, conserved motifs, physiological and biochemical features, collinearity relationships, gene structure, phylogeny and cis-acting element. Based on the phylogenetic and expression pattern analysis, 14 candidate R2R3-MYB genes might be involved in the betalain synthesis. Amongst the 14 candidate R2R3-MYB genes, the expression level of AtrMYB72 was higher in ‘Suxian No.1’ than ‘Suxian No.2’, and also higher in the red section than in the green section of the same leaf in Amaranthus. The overexpression vector pCambia1301-AtrMYB72-GUS and VIGS (virus-induced gene silencing) vector pTRV2- AtrMYB72 were transferred into leaves of ‘Suxian No.1’ via an Agrobacterium-mediated method. The results showed that AtrMYB72 overexpression could promote betalain synthesis. A yeast one-hybrid assay and dual luciferase reporter gene assay demonstrated that AtrMYB72 could bind to the AtrCYP76AD1 promoter to promote betalain synthesis. These results indicated that AtrMYB72 promoted betalain biosynthesis in amaranth by activating the AtrCYP76AD1 transcription. Our results could provide new insights into the betalain biosynthesis in amaranth. Full article

The color of the rind is one of the most crucial agronomic characteristics of watermelon (Citrullus lanatus L.). Its genetic analysis was conducted to provide the identification of genes regulating rind color and improving the quality of watermelon appearance. In this study, a mapping population of 505 F₂ plants, derived from a cross between green (CG058) and light-green (CG265) rinds, along with a high-density genetic linkage (average 0.9 cM distance between bin markers), was used to map and identify possible candidate genes. The green rind trait was determined to be regulated by a single Mendelian locus and was precisely located within a 110 kb genomic site on chromosome nine (Chr 9). In the respective region, two potential genes, Cla97C09G175170 and Cla97C09G175180, were substantially downregulated in the light-green rind in comparison to the green rind. Previous studies revealed that Cla97C09G175170, encoding a two-component response regulator-like protein (APRR2), is possibly involved in the green rind trait in watermelon. Virus-induced gene silencing (VIGS) assay confirmed that ClAPRR2 is a key gene responsible for green rind color. Moreover, qRT-PCR analysis revealed that the transcription levels of multiple key genes in the chlorophyll (Chl) biosynthesis pathway were downregulated in the light-green rind relative to the green rind. The current findings have the potential to clarify the regulatory mechanisms that underlie the color of the watermelon rind. These data would provide valuable insights for the targeted molecular design and development of watermelon rinds. Full article

B-box (BBX) transcription factors play crucial roles in plant growth, development, and defense responses to biotic and abiotic stresses. In this study, we cloned a BBX transcription factor gene, CsCOL9I, from cucumber and analyzed its role in the plant’s defense against the feeding of Bemisia tabaci. CsCOL9 is expressed throughout all developmental stages in cucumber, with the highest expression in the leaves. CsCOL9 is induced by B. tabaci feeding, salicylic acid (SA), methyl jasmonate (MeJA), and hydrogen peroxide (H₂O₂). Cucumber plants with CsCOL9 silence (TRV2-CsCOL9) and overexpression (1301-CsCOL9) were obtained and analyzed. After CsCOL9 silencing, survival rates and host selectivity for B. tabaci increased; however, the expression levels of genes encoding enzymes (CsSOD, CsRBOH, CsPOD), activities of superoxide dismutase (SOD) and peroxidase (POD), and content of H₂O₂ in plants were all reduced. CsCOL9 overexpression led to decreased survival rates and host selectivity for B. tabaci. Conversely, the expression levels of genes (CsSOD, CsRBOH and CsPOD), activities of SOD and POD, and content of H₂O₂ increased after CsCOL9 overexpression in plants. Collectively, our results demonstrate CsCOL9 positively regulates cucumber resistance to B. tabaci by activating reactive oxygen species bursts. This study lays a theoretical foundation for the application of CsCOL9 in cucumber resistance breeding and green pest control of B. tabaci. Full article

Background/Objectives: Bringing small interfering RNA (siRNA) into the cell cytosol to achieve specific gene silencing is an attractive but also very challenging option for improved therapies. The first step for successful siRNA delivery is the complexation with a permanent cationic or ionizable compound. This protects the negatively charged siRNA and enables transfection through the cell membrane. The current study explores the performance of the innovative, ionizable lipid 2-Tetradecylhexadecanoic acid-(2-bis{[2-(2,6-diamino-1-oxohexyl)amino]ethyl}aminoethyl)-amide (T14diLys), in combination with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), for siRNA delivery and the impact of the production method (sonication vs. extrusion) on the particle properties. Methods: Liposomes were produced either with sonication or extrusion and characterized. The extruded liposomes were combined with siRNA at different N/P ratios and investigated in terms of size zeta potential, encapsulation efficiency, lipoplex stability against RNase A, and knockdown efficiency using enhanced green fluorescent protein (eGFP)-marked colon adenocarcinoma cells. Results: The liposomes prepared by extrusion were smaller and had a narrower size distribution than the sonicated ones. The combination of siRNA and liposomes at a nitrogen-to-phosphate (N/P) ratio of 5 had optimal particle properties, high encapsulation efficiency, and lipoplex stability. Gene knockdown tests confirmed this assumption. Conclusions: Liposomes produced with extrusion were more reproducible and provided enhanced particle properties. The physicochemical characterization and in vitro experiments showed that an N/P ratio of 5 was the most promising ratio for siRNA delivery. Full article

Di(2-ethhylhexyl) phthalate (DEHP) is a common plastic rubberizer. DEHP leaches from plastic matrices and is under increasing scrutiny as numerous studies have linked it to negative human health manifestations. Coxsackievirus B3 (CVB) is a human pathogen that typically causes subclinical infections but can sometimes cause severe diseases such as pancreatitis, myocarditis, and meningoencephalitis. Though CVB infections are common, severe illness is relatively rare, and it is unclear what factors mediate disease severity. In this study, we sought to determine the effects that DEHP has on CVB infection in a variety of human cell types to evaluate whether this plastic-derived pollutant could represent a proviral environmental factor. Methods: HeLa cervical cancer cells, human induced pluripotent stem cell-derived brain-like endothelial cells (iBECs), and Caco-2 colon carcinoma cells were exposed to 40 µg/mL DEHP for 24 h prior to infecting with enhanced green fluorescent protein (EGFP)-expressing CVB. The severity of the infection was evaluated via fluorescence microscopy and flow cytometry-based viral EGFP detection, viral plaque assay on tissue culture media, and Western blotting to detect VP1 viral capsid protein. Interferon-associated proteins such as interferon regulatory factor (IRF) 3, IRF7, interferon-induced transmembrane (IFITM) 2, and IFITM3 were measured by Western blotting. The roles of IFITM2 and IFITM3 in the context of CVB infection were evaluated via siRNA silencing. Results: We found that DEHP drastically increased CVB infection in each of the cell types we tested, and, while the cellular processes underlying DEHP’s proviral properties were not entirely clear, we observed that DEHP may subvert CVB-induced interferon signaling and elevate levels of IFITMs, which appeared to bolster CVB infection. Conclusions: DEHP may represent a major environmental factor associated with the severity of CVB infection. Further understanding of how DEHP exacerbates infection may better elucidate its potential role as a proviral environmental factor. Full article

Phytoalexins are plant defense metabolites that are biosynthesized transiently in response to pathogens. Despite that their biosynthesis is highly restricted in plant tissues, the transcription factors that negatively regulate phytoalexin biosynthesis remain largely unknown. Glyceollins are isoflavonoid-derived phytoalexins that have critical roles in protecting soybean crops from the oomycete pathogen Phytophthora sojae. To identify regulators of glyceollin biosynthesis, we used a transcriptomics approach to search for transcription factors that are co-expressed with glyceollin biosynthesis in soybean and stilbene synthase phytoalexin genes in grapevine. We identified and functionally characterized the WRKY family protein GmWRKY72, which is one of four WRKY72-type transcription factors of soybean. Overexpressing and RNA interference silencing of GmWRKY72 in the soybean hairy root system decreased and increased expression of glyceollin biosynthetic genes and metabolites, respectively, in response to wall glucan elicitor from P. sojae. A translational fusion with green fluorescent protein demonstrated that GFP-GmWRKY72 localizes mainly to the nucleus of soybean cells. The GmWRKY72 protein directly interacts with several glyceollin biosynthetic gene promoters and the glyceollin transcription factor proteins GmNAC42-1 and GmMYB29A1 in yeast hybrid systems. The results show that GmWRKY72 is a negative regulator of glyceollin biosynthesis that may repress biosynthetic gene expression by interacting with transcription factor proteins and the DNA of glyceollin biosynthetic genes. Full article

A corporate carbon information disclosure strategy is essentially an environmental responsibility manifestation of “inconsistency between words and deeds”. It has two forms:, green “silence” and green “catering”, both of which restrict the externalization of green productivity and affect the high-quality development of enterprises. This study shows that ① there is a U-shaped relationship between carbon information disclosure strategies and the high-quality development of enterprises. Green “silence” positively affects the high-quality development of enterprises, and the impact of green “catering” on the high-quality development of enterprises changes from negative to positive. ② Green “silence” affects the high-quality development of enterprises by increasing R&D investment, reducing tax burdens, and intensifying financing constraints, while green “catering” affects the high-quality development of enterprises by decreasing R&D investment, increasing the tax burden, and easing financing constraints. ③ If the competition in the industry is fierce, the green “silence” strategy should be adopted. When there is monopoly in the industry, the green “catering” strategy is dominant. The findings of this study not only provide management suggestions for enterprises on how to correctly treat the carbon information disclosure strategies that have been implemented or planned to promote their own high-quality development, but also provide policy inspiration for relevant regulatory authorities to complete the transition from voluntary disclosure to mandatory disclosure. Full article

Leaf color mutants serve as ideal materials for studying photosynthesis, chlorophyll metabolism, and other physiological processes. Here, we identified a spontaneous yellow-leaf mutant (yl1) with chlorophyll-reduced leaves from G. hirsutum L. cv ZM24. Compare to wild type ZM24 with green leaves, yl1 exhibited patchy yellow leaves and reduced chlorophyll content. To further explore the mechanisms of the patchy yellow phenotype of the mutant plant, the transcriptomics and proteomics profiles were conducted for the mutant and wild types. A total of 9247 differentially expressed genes (DEGs) and 1368 differentially accumulated proteins (DAPs) were identified. Following gene ontology (GO) annotation and KEGG enrichment, the DEGs/DAPs were found to be significantly involved in multiple important pathways, including the obsolete oxidation-reduction process, photosynthesis, light-harvesting, the microtubule-based process, cell redox homeostasis, and the carbohydrate metabolic process. In photosynthesis and the light-harvesting pathway, a total of 39 DAPs/DEGs were identified, including 9 genes in the PSI, 7 genes in the PS II, 9 genes in the light-harvesting chlorophyll protein complex (LHC), 10 genes in the PsbP family, and 4 genes in the cytochrome b6/f complex. To validate the reliability of the omics data, GhPPD1, a DAPs in the PsbP family, was knocked down in cotton using the TRV-based VIGS system, and it was observed that the GhPPD1-silenced plants exhibited patchy yellow color, accompanied by a significant decrease in chlorophyll content. In conclusion, this study integrated transcriptomic and proteomic approaches to gain a deeper understanding of the mechanisms underlying the chlorophyll-reduced leaf phenotype. Full article

Tobacco mosaic virus (TMV) was the first virus to be studied in detail and, for many years, TMV and other tobamoviruses, particularly tomato mosaic virus (ToMV) and tobamoviruses infecting pepper (Capsicum spp.), were serious crop pathogens. By the end of the twentieth and for the first decade of the twenty-first century, tobamoviruses were under some degree of control due to introgression of resistance genes into commercial tomato and pepper lines. However, tobamoviruses remained important models for molecular biology, biotechnology and bio-nanotechnology. Recently, tobamoviruses have again become serious crop pathogens due to the advent of tomato brown rugose fruit virus, which overcomes tomato resistance against TMV and ToMV, and the slow but apparently inexorable worldwide spread of cucumber green mottle mosaic virus, which threatens all cucurbit crops. This review discusses a range of mainly molecular biology-based approaches for protecting crops against tobamoviruses. These include cross-protection (using mild tobamovirus strains to ‘immunize’ plants against severe strains), expressing viral gene products in transgenic plants to inhibit the viral infection cycle, inducing RNA silencing against tobamoviruses by expressing virus-derived RNA sequences in planta or by direct application of double-stranded RNA molecules to non-engineered plants, gene editing of host susceptibility factors, and the transfer and optimization of natural resistance genes. Full article