Transgenic Plants Research Articles

Overexpression of vacuolar H+-pyrophosphatase from a recretohalophyte Reaumuria trigyna enhances vegetative growth and salt tolerance in transgenic Arabidopsis thaliana

Reaumuria trigyna, a wild and endangered salt-secreting small shrub, is distributed in arid and semi-arid areas of Inner Mongolia, China. An H+-pyrophosphatase gene (RtVP1) was isolated from R. trigyna according to transcriptomic data, which encoded a plasma membrane and tonoplast-localized protein. RtVP1 was quickly upregulated by NaCl and exogenous abscisic acid treatment and rescued the sucrose deficiency sensitive phenotype of the AtVP1 mutant (avp1). Transgenic Arabidopsis overexpressing RtVP1 exhibited a higher leaf area, plant height, fresh weight, root length, and soluble carbohydrate accumulation compared to the wild type (WT) under normal conditions. RtVP1 overexpression increased the seed germination rate and decreased the reduction rate of fresh weight, root length, and chlorophyll content in transgenic plants under salt stress. Catalase enzyme activity, proline content, relative water content, and soluble sugar content were significantly increased in transgenic Arabidopsis under salt stresses, but the malondialdehyde content was dramatically decreased. More K+ and less Na+ were accumulated in transgenic Arabidopsis leaves, resulting in a relatively lower Na+/K+ ratio. In transgenic Arabidopsis roots, K+ was unchanged, but Na+ and the Na+/K+ ratios were reduced compared to those in WT. More Na+ and K+ were accumulated in the intracellular of transgenic yeast, and the Na+/K+ ratio was significantly reduced compared to the control. These results showed that R. trigyna RtVP1 promotes the vegetative growth of plants, mainly by regulating carbohydrate metabolism, and confers salt tolerance in transgenic Arabidopsis by maintaining Na+/K+ homeostasis and enhancing the antioxidant and osmotic regulatory capacity. These results indicated that RtVP1 can serve as an important candidate gene for genetic improvement of crop yield and salt tolerance.

OsbHLH5 Synergically Regulates Phenolamide and Diterpenoid Phytoalexins Involved in the Defense of Rice Against Pathogens

Rice (Oryza sativa) produces phenolamides and diterpenoids as major phytoalexins. Although the biosynthetic pathways of phenolamides and diterpenoids in plants have been revealed, knowledge of their accumulation regulatory mechanisms remains limited, and, in particular, no co-regulatory factor has been identified to date. Here, using a combined co-expression and evolutionary analysis, we identified the basic helix–loop–helix (bHLH) transcription factor OsbHLH5 as a positive bifunctional regulator of phenolamide and diterpenoid biosynthesis in rice. Metabolomic analysis revealed that OsbHLH5 significantly increased the content of phenolamides (such as feruloyl tryptamine (Fer-Trm) and p-coumaroyl tyramine (Cou-Tyr)) and diterpenoid phytoalexins (such as momilactones A, momilactones B) in the overexpression lines, while their content was reduced in the OsbHLH5 knockout lines. Gene expression and dual-luciferase assays revealed that OsbHLH5 activates phenolamide biosynthetic genes (including putrescine hydroxycinnamoyltransferase 3 (OsPHT3), tyramine hydroxycinnamoyltransferases 1/2 (OsTHT1/2), and tryptamine benzoyltransferase 2 (OsTBT2)) as well as diterpenoid biosynthetic genes (including copalyl diphosphate synthase 4 (OsCPS4) and kaurene synthase-like 4/7/10/11 (OsKSL4/7/10/11)). Furthermore, we have demonstrated that OsbHLH5 is induced by jasmonic acid (JA), while pathogen inoculation assays indicated that the overexpression of OsbHLH5 in transgenic rice plants leads to enhanced resistance to Xanthomonas oryzae pv. oryzae (Xoo). Overall, we have identified a positive regulator of phenolamide and diterpenoid biosynthesis and have demonstrated that biotic stress induces phytoalexin accumulation partly in an OsbHLH5-dependent manner, providing new insights into the metabolic interactions involved in pathogen response and offering valuable gene resources for the development, through genetic improvement, of new rice varieties that are resistant to diseases.

Physiological and biochemical response of AtEXPA1 transgenic tobacco plants to salinity stress

Physiological and biochemical response of AtEXPA1 transgenic tobacco plants to salinity stress

ABA Affects Distinctive Rice Caryopses Physicochemical Properties on Different Branches

Abscisic acid (ABA) plays an important regulatory role in the grain filling process, which in turn will affect the final yield and quality of rice. The ABA biosynthesis genes of OsNCED3 and degradation gene OsABA8ox3 affect the ABA content, and then further regulate the ABA signaling. During the development of rice panicle, compared with primary grains (superior grains) growing on primary branches, secondary grains (inferior grains) growing on secondary branches exhibit characteristics. However, little is reported on the physicochemical characteristics of starch between superior and inferior grains in ABA related transgenic lines. In this study, OsNCED3 and OsABA8ox3 transgenic plants were used as materials. The results showed that compared with the WT, the OsNCED3-RNAi lines on grain weight was consistent with the trend of superior and inferior grains, while the OsABA8ox3-RNAi lines affected superior or inferior grains. The total starch and soluble sugar content of grains decreased in both OsNCED3-RNAi and OsABA8ox3-RNAi lines, and the total starch content of superior and inferior grains in OsABA8ox3-RNAi lines decreased. The starch granule size distribution of all samples showed a bimodal and increased proportion of starch grains with large granule size, in which the influence on inferior grains was greater than that of superior grains, which eventually led to a significant increase in their average granule size. The apparent amylose content of inferior grains increased significantly in most lines. The swelling power of the superior grains decreased significantly, while that of the inferior grains increased significantly. Fourier analysis showed that the order degree of starch granule surface decreased in the superior grains of the RNAi line, while it increased in the inferior grains of the OsABA8ox3-RNAi line but decreased in the OsNCED3-RNAi lines. In the superior grains, the relative crystallinity of starch decreased in the OsNCED3-RNAi lines, but remained unchanged or increased in the OsABA8ox3-RNAi line. In inferior grains, the relative crystallinity of starch decreased in the ABA synthesis RNAi line, but increased in the OsABA8ox3-RNAi line. In summary, the influence of ABA on the physicochemical properties of inferior grains is greater than that of superior grains.

The potato sugar transporter SWEET1g affects apoplasmic sugar ratio and phloem-mobile tuber- and flower-inducing signals.

The main phloem loader in potato, sucrose transporter StSUT1, is co-expressed with two members of the SWEET gene family: StSWEET11b, a clade III member of SWEET carriers assumed to be involved in sucrose efflux, and StSWEET1g, a clade I member involved in glucose efflux into the apoplast that physically interacts with StSUT1. We investigated the functionality of SWEET carriers via uptake experiments with fluorescent glucose or sucrose analogs. Inhibition or overexpression of StSWEET1g/SlSWEET1e affected tuberization and flowering in transgenic potato plants. Isolation of the apoplasmic fluid by vacuum infiltration centrifugation revealed changes in the apoplasmic hexose composition and mono-to-disaccharide ratio, affecting sink strength. Down-regulation of StSWEET1g expression affected the expression of SP6A, a tuberigen, and miR172 under LD conditions, leading to early flowering and tuberization. A systematic screen for StSWEET1g-interacting protein partners revealed several proteins affecting cell wall integrity and strengthening. StSWEET1g and the main interaction partners were strongly down-regulated during tuber development. We discuss whether StSWEET1g activity might be linked to cell wall remodeling during tuber development and the switch from apoplasmic to symplasmic phloem unloading.

Molecular cloning and characterization of a brassinosteriod biosynthesis-related gene PtoCYP90D1 from Populus tomentosa

Brassinosteroids (BRs), one of the major classes of phytohormones are essential for various processes of plant growth, development, and adaptations to biotic and abiotic stresses. In Arabidopsis, AtCYP90D1 acts as a bifunctional cytochrome P450 monooxygenase, catalyzing C-23 hydroxylation in the brassinolide biosynthetic pathway. The present study reports the functional characterizations of PtoCYP90D1, one of the AtCYP90D1 homologous genes from Populus tomentosa. The qRT-PCR analysis showed that PtoCYP90D1 was highly expressed in roots and old leaves. Overexpression of PtoCYP90D1 (PtoCYP90D1-OE) in poplar promoted growth and biomass yield, as well as increased xylem area and cell layers. Transgenic plants exhibited a significant increase in plant height and stem diameter as compared to the wild type. In contrast, the CRISPR/Cas9-generated mutation of PtoCYP90D1 (PtoCYP90D1-KO) resulted in significantly decreased biomass production in transgenic plants. Further studies revealed that cell wall components increased significantly in PtoCYP90D1-OE lines but not in PtoCYP90D1-KO lines, as compared to wild-type plants. Overall, the findings indicate a positive role of PtoCYP90D1 in improving growth rate and elevating biomass production in poplar, which will have positive implications for its versatile industrial or agricultural applications.

Application of an Efficient Enhancer in Gene Function Research

Although great progress has been made in transgenic technology, increasing the expression level and thus promising the expected phenotypes of exogenous genes in transgenic plants is still a crucial task for genetic transformation and crop engineering. Here, we conducted a comparative study of the enhancing efficiency of three putative translational enhancers, including Ω (natural leader from a plant virus), OsADH 5′ (natural leader from a plant gene), and ARC (active ribosomal RNA complementary), using the transient gene expression systems of Nicotiana benthamiana and Chirita pumila. We demonstrate that three tandem repeats of ARC (3 × ARC) are more efficient than other enhancers in expression. The enhancing efficiency of 6 × ARC is further increased, up to 130 times the expression level without the insertion of enhancers. We further evaluated the enhancing efficiency of 6 × ARC under agrobacterium-mediated transformation systems. In C. pumila, 6 × ARC significantly amplifies the phenotypic effect of CpCYC1 and CpCYC2 in repressing stamen development and yellow pigmentation. In Arabidopsis thaliana, 6 × ARC and the AtAP1 promoter work together to promote the accumulation of anthocyanin pigments in vegetative and reproductive organs. Most significantly, the fusion of 6 × ARC in a CpCYC1/2 transgenic system in C. pumila fully reveals that these genes have the complete function of repressing the yellow spots, displaying an advantage in manifesting the function of exogenous genes. This study highlights the application potential of the enhancer 6 × ARC in gene function research in plants.

Heterologous Expression of MYB Gene (Rosea1) or bHLH Gene (Delila) from Antirrhinum Increases the Phenolics Pools in Salvia miltiorrhiza

Phenolic acids have health-promoting properties, however, but their low concentrations in Salvia miltiorrhiza limit broader medicinal applications. MYB and bHLH transcription factors activate multiple target genes involved in phenylpropanoid metabolism, thereby enhancing the production of various secondary metabolites. We introduced the MYB transcription factor Antirrhinum Rosea1 (AmROS1) or Delila (AmDEL) into S. miltiorrhiza and observed that antioxidant activity in transgenic plants increased by 1.40 to 1.80-fold. The total content was significantly higher in transformants compared to the controls. Furthermore, heterologous expression of AmROS1 or AmDEL triggered moderate accumulations of rosmarinic acid and salvianolic acid at various growth stages. Levels of total phenolics, total flavonoids, and anthocyanins were significantly elevated. These biological and phytochemical alterations were correlated with the upregulated expression of genes involved in phenolic acid biosynthesis. Our findings demonstrate that AmROS1 and AmDEL function as a transcriptional activator in phenolic acids biosynthesis. This study offers further insights into the heterologous or homologous regulation of phenolics production, potentially enabling its engineering in S. miltiorrhiza.

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.

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.

Genome-wide identification of shaker K+ channel gene family in sugar beet (Beta vulgaris L.) and function of BvSKOR in response to salt and drought stresses

Potassium (K+) is the most abundant cation in plants, which is absorbed by roots and distributed throughout the plants and within plant cells, and is involved in various cellular processes. Shaker K+ channel plays crucial roles in the absorption and distribution of K+ and in the response to abiotic stress in plants. Herein, a total of six shaker K+ channel genes, BvKAT1, BvKAT3, BvAKT1, BvAKT2, BvAKT5, and BvSKOR, were identified in the genome of sugar beet (Beta vulgaris L.). The coding domain sequences (CDS) of these genes ranged from 2232 to 2739bp, and protein lengths were varied from 743 to 912 aa. The shaker K+ channel genes contained hormone-related and light responsiveness cis-acting regulatory elements. The phylogenetic analysis showed that BvSKOR was highly conserved and contained six transmembrane structures. The expression patterns of BvSKOR under salt and osmotic stress were analyzed by qRT-PCR, and found that the expression level of BvSKOR under low concentration salt and osmotic stress at short period of treatment were significantly higher than that of the control group. The function of BvSKOR was further verified in tobacco (Nicotiana tabacum), and the results showed that under salt and osmotic stress, the roots of transgenic plants were significantly stronger than those of wild type (WT) plants, and the relative water content (RWC), chlorophyll, proline, soluble sugar, soluble proteins contents and antioxidant enzyme activity were significantly higher than those of WT plants. These results indicated that overexpression of BvSKOR can significantly enhance the salt and drought tolerance in transgenic tobacco plants. This study could provide theoretical support and genetic resources for genetic improvement of crops stress resistance.

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.

Heterologous biosynthesis of betanin triggers metabolic reprogramming in tobacco

Engineering of a specialized metabolic pathway in plants is a promising approach to produce high-value bioactive compounds to address the challenges of climate change and population growth. Understanding the interaction between the heterologous pathway and the native metabolic network of the host plant is crucial for optimizing the engineered system and maximizing the yield of the target compound. In this study, we performed transcriptomic, metabolomic and metagenomic analysis of tobacco (Nicotiana tabacum) plants engineered to produce betanin, an alkaloid pigment that is found in Caryophyllaceae plants. Our data reveals that, in a dose-dependent manor, the biosynthesis of betanin promotes carbohydrate metabolism and represses nitrogen metabolism in the leaf, but enhances nitrogen assimilation and metabolism in the root. By supplying nitrate or ammonium, the accumulation of betanin increased by 1.5–3.8-fold in leaves and roots of the transgenic plants, confirming the pivotal role of nitrogen in betanin production. In addition, the rhizosphere microbial community is reshaped to reduce denitrification and increase respiration and oxidation, assistant to suppress nitrogen loss. Our analysis not only provides a framework for evaluating the pleiotropic effects of an engineered metabolic pathway on the host plant, but also facilitates the development of novel strategies to balance the heterologous process and the native metabolic network for the high-yield and nutrient-efficient production of bioactive compounds in plants.