Transgenic Plants Research Articles

Epigenetic control of T-DNA during transgenesis and pathogenesis.

Mobile elements known as T-DNAs are transferred from pathogenic Agrobacterium to plants and reprogram the host cell to form hairy roots or tumors. Disarmed non-oncogenic T-DNAs are extensively used to deliver transgenes in plant genetic engineering. Such T-DNAs were the first known targets of RNA silencing mechanisms, which detect foreign RNA in plant cells and produce small RNAs that induce transcript degradation. These T-DNAs can also be transcriptionally silenced by the deposition of epigenetic marks such as DNA methylation and the dimethylation of lysine 9 (H3K9me2) in plants. Here, we review the targeting and the roles of RNA silencing and DNA methylation on T-DNAs in transgenic plants as well as during pathogenesis. In addition, we discuss the crosstalk between T-DNAs and genome-wide changes in DNA methylation during pathogenesis. We also cover recently discovered regulatory phenomena, such as T-DNA suppression and RNA silencing-independent and epigenetic-independent mechanisms that can silence T-DNAs. Finally, we discuss the implications of findings on T-DNA silencing for the improvement of plant genetic engineering.

A Novel Single Base Mutation in OsSPL42 Leads to the Formation of Leaf Lesions in Rice

Rice spotted-leaf mutants serve as valuable resources for studying plant programmed cell death (PCD) and disease resistance mechanisms, making them crucial for research on disease resistance in rice. Map-based cloning was used to identify and clone the spotted-leaf gene OsSPL42. Then, functional complementation and CRISPR/Cas9 techniques were also employed to further validate the function of this gene. By applying leaf clippings for bacterial blight (BB) inoculation, the BB resistance of different rice lines was assessed. The results in this study were as follows: The OsSPL42 behaved as a recessive nuclear gene and was narrowed down to a 111 kb region on chromosome 8. All T0 transgenic rice plants in the complementation experiments exhibited a wild-type phenotype, without any lesion spots on the rice leaves. This suggests that the LOC_Os08g06100 encoding O-methyltransferase is the candidate gene for the mutant spl42. The OsSpl42 is widely expressed and the OsSPL42-GFP protein is mainly localized in the cytoplasm. OsSPL42 overexpression lines are more susceptible to BBs, which indicates that OsSPL42 may act as a negative regulator of rice resistance to BB. In summary, we speculate that OsSPL42 plays an important role in the regulation of pathogen response, providing new insights into plant defense mechanisms.

A Detailed Proteomics and Metabolomics Landscape Sheds Light on the Mechanistic Insights Into the Resistance Response of Transgenic Pigeon Pea Against Wilt Stress.

Pigeon pea, vital for farmers in semi-arid regions, suffers yield losses from Fusarium wilt caused by Fusarium udum. This study demonstrates that introducing the rice oxalate oxidase 4 (Osoxo4) gene significantly boosts wilt resistance. Enhanced resistance in transgenic lines was confirmed through gene expression analysis, enzyme activity assays, biochemical assessments, histochemical staining and in vitro and in vivo bioassays, including spore germination tests. We performed proteomics and metabolomics analyses to investigate mechanisms of enhanced resistance. LC-MS/MS-based label-free proteomics of wilt-infected transgenic and wild-type pigeon pea leaves identified 2386 proteins, with 1048 showing significant abundance changes-738 upregulated and 310 downregulated-in transgenic plants. Notably, proteins such as HMG1/2-like protein, Putative nucleosome assembly protein C364.06, DEAD-box ATP-dependent RNA helicase 3, Lipoxygenase 1, Annexin D1 and Annexin-like protein RJ4 were significantly upregulated, indicating their potential role in developing wilt-resistant cultivars. Metabolomic analysis showed elevated levels of amino acids, sugars, oxalic acid, sugar alcohols and myo-inositol in transgenic pigeon pea, with upregulated pathways in Sugar and Starch Metabolism and Inositol Phosphate Metabolism, indicating enhanced resilience to wilt stress. This study highlights unique regulatory proteins and metabolites, offering insights into stress adaptation and guiding genetic interventions for breeding disease-resistant pigeon pea varieties.

Vascular bundle cell-specific expression of a phosphate transporter improves phosphate use efficiency of transgenic Arabidopsis without detrimental effects

Constitutive overexpression of phosphate (Pi) transporter family 1 often results in the accumulation of toxic levels of Pi, which causes growth retardation in plants. In contrast, we had previously reported that root epidermis-specific overexpression of the phosphate transporter TaPT2 in Arabidopsis leads to improved growth and Pi use efficiency. In the present study, we used promoters AtHKT1;1 and SKOR, which are predominantly expressed in the vascular bundle tissues, to overexpress TaPT2. Transgenic lines exhibited increased shoot growth compared to wild type plants under normal- and low-Pi conditions, along with elevated root Pi and total P content, and higher xylem sap Pi concentration, specifically under low-Pi conditions. This was attributed to moderate Pi accumulation in the xylem parenchyma cells, enhancing the Pi uploading capacity to the xylem. SKOR-TaPT2, however, did not complement pho1 mutant, which was defective in uploading Pi to the xylem. The transcriptional levels of VPT1 and VPT3, which are responsible for transporting excess Pi into a vacuole, were upregulated in SKOR promoter lines under normal-Pi conditions. Our results suggested that root vascular bundle-specific expression of TaPT2 is another promising strategy for increasing biomass production, Pi uptake, and Pi use efficiency while preventing growth retardation in transgenic plants.

Flavonoids Mitigate Nanoplastic Stress in Ginkgo biloba.

Microplastics/nanoplastics are a top global environmental concern and have stimulated surging research into plant-nanoplastic interactions. Previous studies have examined the responses of plants to nanoplastic stress at various levels. Plant-specialized (secondary) metabolites play crucial roles in plant responses to environmental stress, whereas their roles in response to nanoplastic stress remain unknown. Here, we systematically examined the physiological and biochemical responses of Ginkgo biloba, a species with robust metabolite-driven defenses, to polystyrene nanoplastics (PSNPs). PSNPs negatively affected seedling growth and induced phytotoxicity, oxidative stress, and nuclear damage. Notably, PSNPs caused significant flavonoid accumulation, which enhances plant tolerance and detoxification against PSNP stress. To determine whether this finding is universal in plants, we subjected Arabidopsis, poplar, and tomato to PSNP stress and verified the common response of enhanced flavonoids across these species. To further confirm the role of flavonoids, we employed genetic transformation and staining techniques, validating the importance of flavonoids in mitigating excessive oxidative stress induced by NPs. Matrix analysis of transgenic plants with enhanced flavonoids further demonstrated altered downstream pathways, allocating more energy towards resilience against nanoplastic stress. Collectively, our results reveal the flavonoid multifaceted roles in enhancing plant resilience to nanoplastic stress, providing new knowledge about plant responses to nanoplastic contamination.

Innovations in Phytoremediation: Application of Artificial Wetlands for the Contaminated Water Treatment and Heavy Metals Removal

Objective: The objective of this study was a review to compile research on phytoremediation methods—including phytoextraction, rhizofiltration, phytostabilisation, phytodegradation, and phytovolatilisation—using primary sources to address heavy metal contamination in water. Theoretical Framework: The study is a basic investigation that includes a contextualization regarding the water issue, as well as the treatment of wastewater. A description of the characteristics and quality indicators of water was carried out, along with the importance of the removal of contaminants such as heavy metals using different technologies like phytoextraction, rhizofiltration, phytostabilization, phytodegradation, and phytovolatilization. This led to a detailed description of artificial wetland techniques as a viable and sustainable alternative. Method: The methodology adopted for this research includes data collection and compilation of existing information; regarding the different techniques related to wastewater treatment with artificial wetlands, these systems utilize natural processes involving vegetation, soil, and microbial activity to remove contaminants from water. More than 100 articles have been legally downloaded, and systematised in the database to paraphrase sentences and cite appropriately. Results and Discussion: Phytoremediation is an environmentally friendly technical choice because of its low cost to remediate water and soil contamination through methods of phytoextraction, rhizofiltration, phytostabilisation, phytodegradation and phytovolatilisation for organic contaminants, pesticides, oil waste, heavy metals, antibiotics, etc. It is relevant to choose transgenic plant methods to insert DNA genes and conduct molecular mechanisms through experimental optimisation studies for lightened growth and hyperaccumulation of heavy metals, generating space for metal and biomass storage, and prior knowledge of the physiology and biochemical processes of the plants. Research Implications: The results can be applied or influence practices in environmental protection using Phytoremediation, which is based on a natural technique of plants and their microbiota associated with roots and tissues, endophytic bacteria as Aeromonas sp, Pseudomonas sp, Bacillus sp, Sinorhizobium meliloti, Chryseomonas sp, Curtobacterium sp, Sphingomonas sp, Erwinia sp, Flavimonas sp retain contaminants. On the other hand, aquatic macrophytes such as Lemna spp, Typha spp, Potamogeton spp, Azolla filiculoides, Ceratophyllum demersm, Eichhornia crassipes, and Phragmites australis constitute an effective and feasible complementary alternative for the treatment of contaminated waters. Originality/Value: This study contributes to the literature There are different phytoremediation methods such as phytoextraction, rhizofiltration, phytostabilisation, phytodegradation, and hytovolatilisation for organic contaminants, pesticides, petroleum waste, heavy metals, pharmaceutical antibiotics, etc. Each of the elements has specific applications depending on the conditions of the place and the type of contaminants in an ecosystem.

GmTRAB1, a Basic Leucine Zipper Transcription Factor, Positively Regulates Drought Tolerance in Soybean (Glycine max. L)

The basic leucine zipper (bZIP) transcription factors play crucial roles in plant resistance to environmental challenges, but the biological functions of soybean bZIP members are still unclear. In this study, a drought-related soybean bZIP gene, GmTRAB1, was analyzed. The transcript of GmTRAB1 was upregulated under drought, ABA, and oxidative stresses. Overexpression of GmTRAB1 improved the osmotic stress tolerance of transgenic Arabidopsis and soybean hairy roots associated with increased proline content and activity of antioxidant enzymes and reduced accumulations of malonaldehyde and reactive oxide species. However, RNA interference silencing of GmTRAB1 in the soybean hairy roots improved drought sensitivity. Furthermore, GmTRAB1 increased the sensitivity of transgenic plants to ABA and participated in modulating ABA-regulated stomatal closure upon drought stress. In addition, GmTRAB1 stimulated the transcript accumulation of drought-, ABA-, and antioxidant-related genes to respond to drought. Collectively, this research will contribute to understanding the molecular mechanisms of bZIP transcription factors in soybean’s resistance to drought.

An Anthocyanin-Based Visual Reporter System for Genetic Transformation and Genome Editing in Cassava

Cassava (Manihot esculenta Crantz) is a staple crop in tropical and subtropical regions, valued for its high starch content in roots. Effective genetic transformation and genome editing of cassava require efficient screening methods for transgenic and edited plants. In this study, a visual selection marker system using an R2R3-MYB transcription factor anthocyanin 1 gene (HbAN1, LOC110667474) from a rubber tree (Hevea brasiliensis Müll. Arg.) has been developed to facilitate the identification of transgenic cassava plants. Transgenic cassava lines expressing HbAN1 accumulated anthocyanins in their leaves, allowing for easy visual identification without the need for destructive assays or specialized equipment. Importantly, the accumulation of anthocyanins did not affect the regeneration or transformation efficiency of cassava. Additionally, the AR-CRISPR/Cas9-gRNA system with the HbAN1 gene as a marker produced MeCDD4 gene-edited cassava mutants with purple leaves, demonstrating successful editing. This anthocyanin-based visual reporter (AR) system will provide an effective tool for genetic transformation and genome editing in cassava.

Wheat TaPYL9-involved signalling pathway impacts plant drought response through regulating distinct osmotic stress-associated physiological indices.

The abscisic acid (ABA) signalling pathway plays a crucial role in plants' response to drought stress. In this study, we aimed to characterize the impact of an ABA signalling module, which consisted of TaPYL9 and its downstream partners in Triticum aestivum, on plant drought adaptation. Our results showed that TaPYL9 protein contains conserved motifs and targets plasma membrane and nucleus after being sorted by the endoplasmic reticulum. In addition, TaPYL9 transcripts in both roots and leaves were significantly upregulated in response to drought stress. We conducted glucuronidase (GUS) histochemical staining analysis for transgenic plants carrying a truncated TaPYL9 promoter, which suggested that cis-elements associate with ABA and drought response, such as ABRE, DRE and recognition sites MYB and MYC, regulating the gene transcription under drought conditions. Using protein interaction assays (i.e., yeast two-hybrid, bimolecular fluorescence complementation (BiFC), co-immunoprecipitation (Co-IP) and in vitro pull-down), we demonstrated interactions between the intermediate segment of TaPYL9, the intermediate segment of TaPP2C6, the N-terminus of TaSnRK2.8 and the C-terminus of the transcription factor TabZIP1 in wheat, indicating the involvement of TaPYL9 in the constitution of an ABA signalling module, namely TaPYL9/TaPP2C6/TaSnRK2.8/TabZIP1. Transgene analysis revealed that TaPYL9, TaSnRK2.8 and TabZIP1 positively regulated drought response, while TaPP2C6 negatively regulated it, and that these genes were closely associated with the regulation of stomata movement, osmolyte accumulation and ROS homeostasis. Electrophoretic mobility shift (EMSA) and transcriptioal activation assays indicated that TabZIP1 interacted promoters of TaP5CS2, TaSLAC1-1 and TaCAT2 and activated transcription of these genes, which regulated proline biosynthesis, stomata movement and ROS scavenging upon drought signalling, respectively. Furthermore, we found that the transcripts of TaPYL9 and stress-responsive genes were positively correlated with yields in wheat cultivars under field drought conditions. Altogether, our findings suggest that the TaPYL9-involved signalling pathway significantly regulates drought response by modulating osmotic stress-associated physiological processes in T. aestivum.

CPK28-mediated phosphorylation enhances nitrate transport activity of NRT2.1 during nitrogen deprivation.

Nitrate (NO3 -) serves as the primary inorganic nitrogen source assimilated by most terrestrial plants. The acquisition of nitrate from the soil is facilitated by NITRATE TRANSPORTERS (NRTs), with NRT2.1 being the key high-affinity nitrate transporter. The activity of NRT2.1, which has multiple potential phosphorylation sites, is intricately regulated under various physiological conditions. Here, we discovered that CALCIUM-DEPENDENT PROTEIN KINASE 28 (CPK28) positively regulates nitrate uptake under nitrogen deprivation conditions. We found CPK28 as the kinase targeted by immunoprecipitation followed by mass spectrometry and examined the in-planta phosphorylation status of NRT2.1 in cpk28 mutant plants by employing quantitative MS-based phosphoproteomics. Through a combination of in vitro phosphorylation experiment and immunoblotting using phospho-specific antibody, we successfully demonstrated that CPK28 specifically phosphorylates NRT2.1 at Ser21. Functional analysis conducted in Xenopus oocytes revealed that co-expression of CPK28 significantly enhanced high-affinity nitrate uptake of NRT2.1. Further investigation using transgenic plants showed that the phosphomimic variant NRT2.1S21E, but not the nonphosphorylatable variant NRT2.1S21A, fully restored high-affinity 15NO3 - uptake ability in both nrt2.1 and cpk28 mutant backgrounds. This study clarifies that the kinase activity of CPK28 is promoted during nitrogen deprivation conditions. These significant findings provide valuable insights into the intricate regulatory mechanisms that govern nitrate-demand adaptation.

A Novel Solid Media-Free In-Planta Soybean (Glycine max. (L) Merr.) Transformation Approach

Soybean’s lengthy protocols for transgenic plant production are a bottleneck in the transgenic breeding of this crop. Explants cultured on a medium for an extended duration exhibit unanticipated modifications. Stress-induced somaclonal variations and in vitro contaminations also cause substantial losses of transgenic plants. This effect could potentially be mitigated by direct shoot regeneration without solid media or in-planta transformation. The current study focused primarily on developing a rapid and effective media-free in-planta transformation technique for three soybean genotypes (Wm82) and our newly developed two hybrids, designated as ZX-16 and ZX-3. The whole procedure for a transgenic plant takes the same time as a stable grown seedling. Multiple axillary shoots were regenerated on stable-grown soybean seedlings without the ectopic expression of developmental regulatory genes. An approximate amount of 200 µL medium with a growth regulator was employed for shoot organogenesis and growth. The maximal shoot regeneration percentages in the Wm82 and ZX-3 genotypes were 87.1% and 84.5%, respectively. The stable transformation ranged from 3% to 8.0%, with an average of 5.5%. This approach seems to be the opposite of the hairy root transformation method, which allowed transgenic shoots to be regenerated on normal roots. Further improvement regarding an increase in the transformation efficiency and of this technique for a broad range of soybean genotypes and other dicot species would be extremely beneficial in achieving increased stable transformation.

LkERF6 enhances drought and salt tolerance in transgenic tobacco by regulating ROS homeostasis

The transcription factor Ethylene Responsive Factor (ERF) is crucial for responding to various environmental stressors. Proteins containing the ERF-associated amphiphilic repression (EAR) motif often inhibit gene expression. However, the functions of LkERF, an EAR motif-containing protein from Larix kaempferi, especially in reactive oxygen species (ROS) homeostasis, are not well understood. In the present research, we introduce a novel transcription factor, LkERF6, which contains an EAR motif and positively regulates gene expression, thereby enhancing drought and salt tolerance in tobacco. LkERF6 is classified within the ERF-B1 subfamily due to its conserved AP2/ERF domain and EAR motif. Subcellular localization assays demonstrated LkERF6 is primarily localized in the nucleus. Further analysis revealed that LkERF6 interacts with GCC and DRE elements and is significantly induced by NaCl and PEG6000. Moreover, LkERF6 transgenic tobacco plants exhibit lower ROS accumulation and higher levels of antioxidant enzyme activities. Additionally, correlation analysis identified a strong association between LkERF6 and three genes: LkSOD, LkCCS, and LkCAT. Y1H, EMAS, and DLR assays confirmed that LkERF6 directly interacts with the promoters of these genes through GCC-box and DRE-box to activate their expression. These findings shed new light on the function of EAR motif-containing transcription factors and highlight LkERF6’s crucial role in enhancing abiotic stress resistance by activating multiple ROS clearance genes.

An effector essential for virulence of necrotrophic fungi targets plant HIRs to inhibit host immunity

Phytopathogens often secrete effectors to enhance their infection of plants. In the case of Sclerotinia sclerotiorum, a necrotrophic phytopathogen, a secreted protein named SsPEIE1 (Sclerotinia sclerotiorum Plant Early Immunosuppressive Effector 1) plays a crucial role in its virulence. During the early stages of infection, SsPEIE1 is significantly up-regulated. Additionally, transgenic plants expressing SsPEIE1 exhibit increased susceptibility to different phytopathogens. Further investigations revealed that SsPEIE1 interacts with a plasma membrane protein known as hypersensitive induced reaction (HIR) that dampens immune responses. SsPEIE1 is required for S. sclerotiorum virulence on wild-type Arabidopsis but not on Arabidopsis hir4 mutants. Moreover, Arabidopsis hir2 and hir4 mutants exhibit suppressed pathogen-associated molecular pattern-triggered reactive oxygen species (ROS) bursts and salicylic acid (SA)-associated immune gene induction, all of which are phenocopied by the SsPEIE1 transgenic plants. We find that the oligomerization of AtHIR4 is essential for its role in mediating immunity, and that SsPEIE1 inhibits its oligomerization through competitively binding to AtHIR4. Remarkably, both Arabidopsis and rapeseed plants overexpress AtHIR4 display significantly increased resistance to S. sclerotiorum. In summary, these results demonstrate that SsPEIE1 inhibits AtHIR4 oligomerization-mediated immune responses by interacting with the key immune factor AtHIR4, thereby promoting S. sclerotiorum infection.

Optimization of a rapid, sensitive, and high throughput molecular sensor to measure canola protoplast respiratory metabolism as a means of screening nanomaterial cytotoxicity

Nanomaterial-mediated plant genetic engineering holds promise for developing new crop cultivars but can be hindered by nanomaterial toxicity to protoplasts. We present a fast, high-throughput method for assessing protoplast viability using resazurin, a non-toxic dye converted to highly fluorescent resorufin during respiration. Protoplasts isolated from hypocotyl canola (Brassica napus L.) were evaluated at varying temperatures (4, 10, 20, 30 ˚C) and time intervals (1–24 h). Optimal conditions for detecting protoplast viability were identified as 20,000 cells incubated with 40 µM resazurin at room temperature for 3 h. The assay was applied to evaluate the cytotoxicity of silver nanospheres, silica nanospheres, cholesteryl-butyrate nanoemulsion, and lipid nanoparticles. The cholesteryl-butyrate nanoemulsion and lipid nanoparticles exhibited toxicity across all tested concentrations (5-500 ng/ml), except at 5 ng/ml. Silver nanospheres were toxic across all tested concentrations (5-500 ng/ml) and sizes (20–100 nm), except for the larger size (100 nm) at 5 ng/ml. Silica nanospheres showed no toxicity at 5 ng/ml across all tested sizes (12–230 nm). Our results highlight that nanoparticle size and concentration significantly impact protoplast toxicity. Overall, the results showed that the resazurin assay is a precise, rapid, and scalable tool for screening nanomaterial cytotoxicity, enabling more accurate evaluations before using nanomaterials in genetic engineering.

Transcriptome and metabolome analyses reveal that GA3ox regulates the dwarf trait in mango (Mangifera indica L.)

BackgroundMango is a tropical fruit with high economic value. The selection of suitable dwarf mango varieties is an important aspect of mango breeding. However, the mechanisms that regulate mango dwarfing remain unclear.ResultsIn this study, we compared the transcriptomes and metabolomes of mango varieties Guiqi (a dwarfed variety) and Jinhuang (an arborized variety). A total of 4,954 differentially expressed genes and 317 differentially abundant metabolites were identified between the two varieties, revealing the molecular mechanism of the gibberellin 3β-hydroxylase gene GA3ox in regulating dwarfing traits in mangoes using joint transcriptome and metabolome analyses. The results showed that differentially expressed genes were enriched in the diterpenoid biosynthesis pathway and that differentially abundant metabolites were annotated to their upstream pathway, the terpenoid backbone biosynthesis. A gene regulation network based on these two pathways was constructed, indicating the upregulation of the GA3ox gene and the accumulation of gibberellin in dwarfed mangoes. We then transferred the GA3ox gene to tobacco plants following the application of gibberellin, and the morphology and height of the transgenic tobacco plants largely recovered the phenotype.ConclusionsThese results demonstrated that GA3ox plays a role in the regulation of dwarf traits. Our study provides an important theoretical basis for studying the regulatory mechanisms underlying mango dwarfism to facilitate mango breeding.

Arabidopsis DREB26/ERF12 and its close relatives regulate cuticular wax biosynthesis under drought stress condition.

Land plants have evolved a hydrophobic cuticle on the surface of aerial organs as an adaptation to ensure survival in terrestrial environments. Cuticle is mainly composed of lipids, namely cutin and intracuticular wax, with epicuticular wax deposited on plant surface. The composition and permeability of cuticle have a large influence on its ability to protect plants against drought stress. However, the regulatory mechanisms underlying cuticular wax biosynthesis in response to drought stress have not been fully elucidated. Here, we identified three AP2/ERF transcription factors (DREB26/ERF12, ERF13 and ERF14) involved in the regulation of water permeability of the plant surface. Transmission electron microscopy revealed thicker cuticle on the leaves of DREB26-overexpressing (DREB26OX) plants, and thinner cuticle on the leaves of transgenic plants expressing SRDX repression domain-fused DREB26 (DREB26SR). Genes involved in cuticular wax formation were upregulated in DREB26OX and downregulated in DREB26SR. The levels of very-long chain (VLC) alkanes, which are a major wax component, increased in DREB26OX leaves and decreased in DREB26SR leaves. Under dehydration stress, water loss was reduced in DREB26OX and increased in DREB26SR. The erf12/13/14 triple mutant showed delayed growth, decreased leaf water content, and reduced drought-inducible VLC alkane accumulation. Taken together, our results indicate that the DREB26/ERF12 and its closed family members, ERF13 and ERF14, play an important role in cuticular wax biosynthesis in response to drought stress. The complex transcriptional cascade involved in the regulation of cuticular wax biosynthesis under drought stress conditions is discussed.

Rehmannia glutinosa RgMATE35 Participates in the Root Secretion of Phenolic Acids and Modulates the Development of Plant Replant Disease

Phenolic allelochemicals from root exudates dominate rhizosphere formation, lead to autotoxicity in plants subjected to continuous monoculture (CM) stress and induce the emergence of replant disease. However, the regulatory mechanisms governing the transport of phenolics from plant roots to the rhizosphere remain poorly understood. A potential phenolic efflux transporter from Rehmannia glutinosa, designated RgMATE35, has been preliminarily characterized. The objective of this study was to elucidate the molecular function of RgMATE35 in the secretion of phenolics and to investigate its role in the development of plant replant disease using quantitative real-time PCR (qRT-PCR), genetic transformation, HPLC-Q-TOF-MS and other analytical techniques. A tissue expression pattern analysis of RgMATE35 revealed that it is highly expressed in plant roots. Transient expression analysis confirmed the localization of the protein in plasma membranes. An assessment of the transport activity of RgMATE35 in Xenopus oocytes indicated that it plays a role in facilitating the efflux of labeled ferulic acid ([2H3]-FA) and trans-p-coumaric acid [2H6]-pCA. The results of functional studies in R. glutinosa demonstrated that RgMATE35 positively mediates the secretion of FA and pCA from plant roots into the rhizosphere. A molecular and physiological analysis of RgMATE35 transgenic plants subjected to CM stress revealed that the overexpression or repression of RgMATE35 resulted in notable changes in the degree of autotoxic injury in plants. These findings demonstrate that RgMATE35 plays a positive role in the development of replant disease through the secretion of phenolic acids from plant roots. They also provide a fundamental framework for elucidating the molecular regulatory mechanism through which MATEs regulate replant disease through the root secretion of allelochemicals.

Genome-wide computational analysis of the dirigent gene family in Solanum lycopersicum

BackgroundDirigent (DIR) genes play a key role in the development of organic products in plants. They confer conformational influence on processes that lack stereoselectivity and regioselectivity through processes that are mostly understood. They are required to produce lignans, which are a unique and widely distributed family of plant secondary metabolites with intriguing pharmacological characteristics and potential role in plant development. DIR genes are implicated in the process of lignification and protect plants from environmental stresses, including biotic and abiotic stresses. Nevertheless, no research has been performed on the DIR gene family in Solanum lycopersicum. This study provides detailed information on the DIR gene family in S. lycopersicum.Methods and resultsThe conserved domain analysis, phylogenetic analysis, evolutionary adaptation, cis-acting elements, proteomic analysis, signal peptide detection, transmembrane potential analysis, sequence identity and similarity analysis, gene assembly, genomic localization, duplication of gene analysis, and evolutionary linkage of 31 potential DIR genes were studied. All these analyses provide a deep understanding of DIR genes in the S. lycopersicum genome that will provide a useful reference for further functional analysis of the DIR genes in S. lycopersicum.ConclusionThis research provides an in-depth and comprehensive explanation of the detailed process and structural characterization of DIR genes in the genome of S. lycopersicum, laying the groundwork for future plant genetic engineering and crop development exploration. This work will provide valuable information for identifying DIR genes in higher plants and support future research on the DIR gene family.

The GRAS transcription factor PtrPAT1 of Poncirus trifoliata functions in cold tolerance and modulates glycine betaine content by regulating the BADH-like gene

Abstract GRAS, termed after GAI (gibberellic acid insensitive), RGA (repressor of GA1), and SCR (scarecrow), is a plant-specific transcription factor crucial for plant development and stress response. However, understanding of the functions played by the GRAS members and their target genes in citrus is limited. In this study, we identified a cold stress-responsive GRAS gene from Poncirus trifoliate, designated as PtrPAT1, by yeast one-hybrid library screening using the promoter of PtrBADH-l, a betaine aldehyde dehydrogenase (BADH)-like gene. PtrPAT1, belonging to the PAT1 subfamily, was localized in the nucleus and plasma membrane, exhibited transactivation activity and showed a remarkable upregulation under cold stress. Overexpression of PtrPAT1 elevated BADH activity, increased glycine betaine (GB) accumulation and conferred enhanced cold tolerance in transgenic tobacco plants compared with wild type, while downregulation in trifoliate orange by virus-induced gene silencing (VIGS) resulted in opposite trends. Furthermore, the activities of two antioxidant enzymes, including peroxidase (POD) and superoxide dismutase (SOD), were significantly increased in the overexpression plants, but remarkably decreased in the VIGS line, consistent with accumulation patterns of the reactive oxygen species (ROS). PtrPAT1 was demonstrated to interact with and activate the PtrBADH-l promoter through the putative PAT1-binding motif with the core sequence of TTTCATGT, indicating that PtrBADH-l is a target gene of PtrPAT1. Taken together, these results demonstrate that PtrPAT1 positively affects cold tolerance through the regulation of GB biosynthesis by modulating PtrBADH-l expression.

Overexpression of the RAV Transcription Factor OsAAT1 Confers Enhanced Arsenic Tolerance by Modulating Auxin Hemostasis in Rice.

Characterization of arsenic (As)-responsive genes is fundamental to solving the issue of As contamination in rice. Herein, we establish the involvement of an RAV transcription factor OsAAT1 (Arsenic Accumulation and Tolerance 1) in regulating As response in rice. The expression of OsAAT1 is significantly higher in roots and stems of rice seedlings and is clearly upregulated by higher concentrations of arsenite [As(III)]. Compared with wild-type (WT) plants, OsAAT1-overexpressed transgenic lines (OE-OsAAT1) exhibit tolerance, while OsAAT1-knockout mutants (Osaat1) are sensitive to As(III) stress. Notably, the application of exogenous 1-naphthylacetic acid (NAA) greatly enhances the As tolerance of WT and transgenic lines, with stronger effects on OE-OsAAT1. The change in OsAAT1 expression leads to the alteration of As and auxin accumulation in transgenic plants by regulating the expression of OsLsi1, OsLsi2, OsCRL4, and OsAUX1 genes. Moreover, overexpression of OsAAT1 accelerates ROS scavenging and phytochelatins (PCs) synthesis, especially with the addition of exogenous NAA. OsAAT1 localizes in the nucleus and works as a transcriptional suppressor. OsGH3-12, belonging to the auxin-responsive GH3 gene family, is the downstream target gene of OsAAT1, whose expression is extensively downregulated by As(III). These findings provide new insights into As response via auxin signaling pathway in rice.