The protein kinase A/protein kinase G/protein kinase C-family (AGC kinase family) of eukaryotes is involved in regulating numerous biological processes. The 3-phosphoinositide- dependent protein kinase 1 (PDK1), is a conserved serine/threonine kinase in eukaryotes. To understand the roles of PDK1 homologous genes in cell death and immunity in tetraploid Nicotiana tabacum, the previuosly generated transgenic CRISPR/Cas9 lines, in which 5-7 alleles of the 4 homologous PDK1 genes (NtPDK1a/1b/1c/1d homologs) simultaneously knocked out, were used in this study. Our results showed that the hypersensitive response (HR) triggered by transient overexpression of active Pto (Pto^Y207D) or soybean GmMEKK1 was significantly delayed, whereas the resistance to Pseudomonas syrangae pv. tomato DC3000 (Pst DC3000) and tobacco mosaic virus (TMV) was significantly elevated in these partial knockout lines. The elevated resistance to Pst DC3000 and TMV was correlated with the elevated activation of NtMPK6, NtMPK3, and NtMPK4. Taken together, our results indicated that NtPDK1s play a positive role in cell death but a positive role in disease resistance, likely through negative regulation of the MAPK signaling cascade.
Nicotiana/virology* ; Disease Resistance/genetics* ; Plant Diseases/immunology* ; Plants, Genetically Modified/genetics* ; Gene Knockout Techniques ; Plant Proteins/genetics* ; CRISPR-Cas Systems ; Protein Serine-Threonine Kinases/genetics* ; 3-Phosphoinositide-Dependent Protein Kinases/genetics* ; Pyruvate Dehydrogenase Acetyl-Transferring Kinase ; Tobacco Mosaic Virus/pathogenicity*

The OsSPL10 gene has previously been reported to positively regulate trichome development and negatively regulate salt and drought stress tolerance in rice. However, it is not clear whether this gene can be used for gene editing to create new germplasm of glabrous leaf and salt-tolerant rice. In this study, we created six rice mutants by CRISPR/Cas9-mediated editing of OsSPL10 from 'Xinfeng 2', 'Xinkedao 31', and 'Xindao 25', the main rice cultivars along the Yellow River. Visual observation and scanning electron microscopy verified that the mutants lacked trichomes on the leaves and glumes, and the expression of glabrous marker genes OsHL6, OsGL6, and OsWOX3B in mutants was down-regulated compared with that in the wild type. The net photosynthetic rate, stomatal conductance, and transpiration rate of flag leaves in the mutants were significantly higher than those in the wild type. In addition, the survival rates of the mutants were much higher than that of the wild type after 7 days of treatment with 200 mmol/L NaCl. The results of quantitative real-time polymerase chain reaction (qRT-PCR) further verified that compared with the wild type, the mutants demonstrated down-regulated expression of the salt stress-related gene OsGASR1 and up-regulated expression of OsNHX2 and OsIDS1. Statistical analysis of agronomic traits showed that the mutants had increased plant height and no significant changes in yield-related traits compared with the wild type. The six spl10 mutants created in this study not only had glabrous leaves and glumes but also demonstrated enhanced tolerance to salt stress, serving as new germplasm resources for directional breeding of rice along the Yellow River.
Oryza/physiology* ; CRISPR-Cas Systems/genetics* ; Salt Tolerance/genetics* ; Gene Editing/methods* ; Plant Proteins/genetics* ; Rivers ; Plant Leaves/genetics* ; Mutation ; Plants, Genetically Modified/genetics* ; China

Cucumber (Cucumis sativus L.) is one of the most widely cultivated vegetables in the world. High temperature and other stress conditions can affect the growth and development of this plant, even leading to the decreases in yield and quality. The mitogen-activated protein kinase (MAPK) family plays a crucial role in plant stress responses. However, the role of MPK4 in the stress response of cucumber remains to be reported. In this study, we cloned CsMPK4, which encoded 383 amino acid residues. The qRT-PCR results showed that the expression level of CsMPK4 was the highest in leaves and flowers, moderate in roots, and the lowest in stems and tendrils. CsMPK4 was located in the nucleus and cytoplasm, and it had a close relationship with CmMPK4 in muskmelon. The cucumber plants overexpressing CsMPK4 became stronger and shorter, with reduced length and quantity of tendrils. Moreover, the transgenic seedlings were more resistant to high temperatures, with decreased malondialdehyde (MDA) content and increased activities of peroxidase (POD) and superoxide dismutase (SOD) in young leaves. Furthermore, the protein-protein interaction between CsMPK4 and CsVQ10, a member of the valine-glutamine family, was confirmed by yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays. The results suggested that CsVQ10 cooperated with CsMPK4 in response to the high temperature stress in cucumber. This study laid a foundation for the further study on the stress response mechanism of CsMPK4 and the breeding of stress-resistant cucumber varieties.
Cucumis sativus/metabolism* ; Mitogen-Activated Protein Kinases/physiology* ; Plant Proteins/metabolism* ; Plants, Genetically Modified/metabolism* ; Gene Expression Regulation, Plant ; Stress, Physiological/genetics* ; Cloning, Molecular

Mercury (Hg)-contaminated soil poses a significant threat to the environment and human health. Hg-resistant microorganisms have the ability to survive under the stress of inorganic and organic Hg and effectively reduce Hg levels and toxicity. Compared to physical and chemical remediation methods, microbial remediation technologies have garnered increasing attention in recent years due to their lower cost, remarkable efficacy, and minimal environmental impact. This paper systematically elucidates the molecular mechanisms of Hg resistance in microbes, with a focus on their potential applications in phytoremediation of Hg-contaminated soils through plant-microbe interactions. Furthermore, it highlights the critical role of microbes in enhancing the effectiveness of transgenic plants for Hg remediation, aiming to provide a theoretical foundation and scientific basis for the bioremediation of Hg-contaminated soils.
Mercury/toxicity* ; Biodegradation, Environmental ; Soil Pollutants/isolation & purification* ; Soil Microbiology ; Plants, Genetically Modified/metabolism* ; Bacteria/genetics*

The fungal bioluminescence pathway (FBP) catalyzes the oxidation of endogenous caffeic acid to produce green bioluminescence through an enzymatic cascade. Genetic engineering of FBP into plants creates autoluminescent specimens that circumvent the substrate limitations of conventional reporter systems. These transgenic plants serve dual functions as aesthetic displays and versatile biosensing platforms, enabling applications in real-time gene expression monitoring, continuous environmental surveillance, and non-invasive bioimaging, offering novel opportunities for horticultural production, environmental conservation, and bioengineering applications. This review synthesizes current advances in plant FBP engineering and explores how machine learning approaches can optimize autoluminescent phenotypes, thereby accelerating innovation in agricultural biotechnology, environmental sensing, and synthetic biology applications.
Fungi/genetics* ; Plants, Genetically Modified/metabolism* ; Genetic Engineering ; Biosensing Techniques ; Luminescent Measurements ; Caffeic Acids/metabolism* ; Luminescence

Flowering and bolting are important agronomic traits in cruciferous crops such as Brassica juncea. Timely flowering can ensure the crop organ yield and quality, as well as seed propagation. The WRKY family plays an important role in regulating plant bolting and flowering, while the function and mechanism of WRKY12 in B. juncea remain unknown. To explore its function and mechanism in bolting and flowering of B. juncea, we cloned and characterized the BjuWRKY12 gene in B. juncea and found that its expression levels were significantly higher in flowers and inflorescences than in leaves. BjuWRKY12 belonged to the Ⅱc subfamily of the WRKY family, and subcellular localization indicated that the protein was located in the nucleus. Ectopic overexpression of BjuWRKY12 in transgenic lines promoted bolting and flowering, leading to significant increases in the expression levels of flowering integrators SOC1 and FUL. Furthermore, yeast one-hybrid and dual luciferase reporter system assays confirmed that BjuWRKY12 directly bound to the promoters of BjuSOC1 and BjuFUL, undergoing protein-DNA interactions. This discovery gives new insights into the regulation network and molecular mechanisms of BjuWRKY12, laying a theoretical foundation for the breeding of high-yield and high-quality varieties of B. juncea.
Mustard Plant/metabolism* ; Flowers/growth & development* ; Plant Proteins/physiology* ; Promoter Regions, Genetic/genetics* ; Gene Expression Regulation, Plant ; Plants, Genetically Modified/genetics* ; Transcription Factors/metabolism* ; MADS Domain Proteins/metabolism*

Microalgae possess high photosynthetic efficiency, robust adaptability, and substantial biomass, serving as excellent biological resources for large-scale cultivation. Malic enzyme (ME), a ubiquitous metabolic enzyme in living organisms, catalyzes the decarboxylation of malate to produce pyruvate, CO₂, and NAD(P)H, playing a role in multiple metabolic pathways including energy metabolism, photosynthesis, respiration, and biosynthesis. In this study, we identified the Scenedesmus quadricauda malic enzyme gene (SqME) and its biological functions, aiming to provide excellent target genes for the genetic improvement of higher plants. Based on the RNA-seq data from S. quadricauda under the biofilm cultivation mode with high CO₂ and light energy transfer efficiency and small water use, a highly expressed gene (SqME) functionally annotated as ME was cloned. The physicochemical properties of the SqME-encoded protein were systematically analyzed by bioinformatics tools. The subcellular localization of SqME was determined via transient transformation in Nicotiana benthamiana leaves. The biological functions of SqME were identified via genetic transformation in Nicotiana tabacum, and the potential of SqME in the genetic improvement of higher plants was evaluated. The ORF of SqME was 1 770 bp, encoding 590 amino acid residues, and the encoded protein was located in chloroplasts. SqME was a NADP-ME, with the typical structural characteristics of ME. The ME activity in the transgenic N. tabacum plant was 1.8 folds of that in the wild-type control. Heterologous expression of SqME increased the content of chlorophyll a, chlorophyll b, and total chlorophyll by 20.9%, 26.9%, and 25.2%, respectively, compared with the control. The transgenic tobacco leaves showed an increase of 54.0% in the fluorescence parameter NPQ and a decrease of 30.1% in Fo compared with the control. Moreover, the biomass, total lipids, and soluble sugars in the transgenic tobacco leaves enhanced by 20.5%, 25.7%, and 9.5%, respectively. On the contrary, the starch and protein content in the transgenic tobacco leaves decreased by 22.4% and 12.2%, respectively. Collectively, the SqME-encoded protein exhibited a strong enzymatic activity. Heterologous expressing of SqME could significantly enhance photosynthetic protection, photosynthesis, and biomass accumulation in the host. Additionally, SqME can facilitate carbon metabolism remodeling in the host, driving more carbon flux towards lipid synthesis. Therefore, SqME can be applied in the genetic improvement of higher plants for enhancing photosynthetic carbon fixation and lipid accumulation. These findings provide scientific references for mining of functional genes from S. quadricauda and application of these genes in the genetic engineering of higher plants.
Nicotiana/genetics* ; Photosynthesis/physiology* ; Malate Dehydrogenase/biosynthesis* ; Plant Leaves/genetics* ; Scenedesmus/enzymology* ; Carbon Cycle/genetics* ; Lipid Metabolism/genetics* ; Plants, Genetically Modified/metabolism*

Stresses induced by the deficiency or excess of trace mineral elements, such as manganese (Mn), represent a common limiting factor for the production of crops like Brassica napus. To identify key genes involved in Mn allocation in B. napus and elucidate the underlying mechanisms, a member of the metal tolerance protein (MTP) family obtained in the previous screening of cDNA library of B. napus under Mn stress was selected as the research subject. Based on the sequence information and phylogenetic analysis, it was named as BnMTP10. It belongs to the Mn-cation diffusion facilitator (CDF) subfamily. Expression of BnMTP10 in yeast significantly improved the tolerance of transformants to excessive Mn and iron (Fe) and reduced the accumulation of Mn and Fe. However, the yeast transformants exhibited no significant changes in tolerance to excess cadmium, boron, aluminum, zinc, or copper. The qRT-PCR results demonstrated that the flowers of B. napus had the highest expression of BnMTP10, followed by roots and leaves. Subcellular localization studies revealed that BnMTP10 was localized in the endoplasmic reticulum (ER). Compared with wild-type plants, transgenic Arabidopsis overexpressing BnMTP10 exhibited enhanced tolerance to excessive Mn stress but showed no significant difference under Fe stress. Correspondingly, under excessive Mn stress, the Mn content in the roots of transgenic Arabidopsis increased significantly. However, under excessive Fe stress, the Fe content in transgenic Arabidopsis did not alter significantly. According to the results, we hypothesize that BnMTP10 may alleviate excessive Mn stress in plants by mediating Mn transport to the ER. This study facilitated our understanding of efficient mineral nutrients, and provided theoretical foundations and gene resources for breeding B. napus.
Brassica napus/genetics* ; Manganese/metabolism* ; Plants, Genetically Modified/genetics* ; Plant Proteins/physiology* ; Arabidopsis/metabolism* ; Gene Expression Regulation, Plant ; Phylogeny ; Cation Transport Proteins/metabolism* ; Stress, Physiological

Synthetic biology, recognized as one of the most revolutionary interdisciplinary fields in the 21st century, has established innovative strategies for the genetic improvement of rice through the integration of multidisciplinary technologies including genome editing, genetic circuit design, metabolic engineering, and artificial intelligence. This review systematically summarizes recent research advancements and breakthrough achievements in the application of synthetic biology in the genetic improvement of rice, focusing on three critical domains: yield improvement, nutritional quality fortification, and reinforcement of disease resistance and abiotic stress tolerance. It elucidates that synthetic biology enables precise genomic and metabolic pathway engineering through modular, standard, and systematic approaches, effectively overcoming the limitations of conventional breeding methods characterized by prolonged cycles and restricted trait modification capabilities. The implementation of synthetic biology has facilitated synergistic improvement of multi-traits, thereby providing critical technical references for developing elite rice cultivars with superior productivity and nutritional value. These technological breakthroughs hold significant implications for ensuring global food security and promoting green and sustainable development of agriculture.
Oryza/growth & development* ; Synthetic Biology/methods* ; Metabolic Engineering ; Plant Breeding/methods* ; Gene Editing ; Genetic Engineering/methods* ; Plants, Genetically Modified/genetics* ; Disease Resistance/genetics*

Geminiviruses cause substantial crop yield losses worldwide. The replication initiator protein (Rep) encoded by geminiviruses is indispensable for geminiviral replication. The Rep protein encoded by beet severe curly top virus (BSCTV, genus Curtovirus, family Geminiviridae) induces VARIANT IN METHYLATION 5 (VIM5) expression in Arabidopsis leaves upon BSCTV infection. VIM5 functions as a ubiquitination-related E3 ligase to promote the proteasomal degradation of methyltransferases, resulting in reduction of methylation levels in the BSCTV C2-3 promoter. However, the specific domains of Rep responsible for VIM5 induction remain poorly characterized. Although Rep proteins from several geminiviruses act as viral suppressors of RNA silencing (VSRs), whether BSCTV Rep also possesses VSR activity remains to be illustrated. In this study, we employed a transient expression system in the 16c-GFP transgenic and the wild-type Nicotiana benthamiana plants to analyze the VSR and the VIM5-inducing activities of different truncated Rep proteins haboring distinct domains. We found that the N-terminal domain (amino acids 1-180) of Rep suppressed GFP silencing in 16c-GFP transgenic N. benthamiana leaves. The minimal N-terminal fragment (amino acids 1-104) induced VIM5 expression upon co-infiltration, while C-terminal truncations lacked VIM5-inducing activity. Our results indicate that the N-terminal domain of Rep encoded by BSCTV mediates the suppression of RNA silencing and induces VIM5 expression. Thus, our findings contribute to a better understanding of interactions between geminiviral Rep and plant hosts.
Geminiviridae/genetics* ; Nicotiana/metabolism* ; Arabidopsis/metabolism* ; RNA Interference ; Viral Proteins/metabolism* ; Arabidopsis Proteins/metabolism* ; Plants, Genetically Modified/metabolism* ; Protein Domains ; Plant Diseases/virology* ; Methyltransferases/metabolism* ; Ubiquitin-Protein Ligases/metabolism* ; DNA Helicases/genetics*