Transgenic Plants Research Articles

Different concentrations of carbon nanotubes promote or inhibit organogenesis of Arabidopsis explants by regulating endogenous hormone homeostasis.

Carbon nanotubes concentration modulates endogenous hormone balance, influencing callogenesis and organogenesis efficiency, with potential for optimizing plant transformation programs. A unique feature of plant somatic cells is their remarkable ability to regenerate new organs and even an entire plant in vitro. In this work, we investigated how an important group of environmental factors, carbon nanotubes (CNTs) (both single-walled nanotubes as SWCNTs and multi-walled nanotubes as MWCNTs), affect the regenerative capacity of plants and the underlying molecular mechanisms. Our data show that both the induction of pluripotent callus from Arabidopsis root explants and the frequency of de novo shoot regeneration were influenced by the concentration, but not the type of CNTs. Raman analyses show that CNTs can be transported and accumulate in the callus tissue and in the newly formed seedlings. The contrasting effects of CNTs at 0.1mgL-1 and 50mgL-1 were reflected not only in the concentrations of endogenous auxin and trans-zeatin (tZT), but also in the changes in the expression levels of positive cell cycle regulators and transcriptional regulators that control callus pluripotency and the establishment of shoot apical meristem (SAM). Since most existing plant transformation strategies involve the conversion of dedifferentiated calli into regenerated plantlets and are very time consuming and inefficient, this work suggests that CNTs could be used as an additive to optimize plant micropropagation and genetic engineering systems by modulating hormone balance and stimulating the intrinsic totipotency of plants, thus overcoming organogenic recalcitrance.

A split ribozyme system for in vivo plant RNA imaging and genetic engineering.

RNA plays a central role in plants, governing various cellular and physiological processes. Monitoring its dynamic abundance provides a discerning understanding of molecular mechanisms underlying plant responses to internal (developmental) and external (environmental) stimuli, paving the way for advances in plant biotechnology to engineer crops with improved resilience, quality and productivity. In general, traditional methods for analysis of RNA abundance in plants require destructive, labour-intensive and time-consuming assays. To overcome these limitations, we developed a transformative innovation for in vivo RNA imaging in plants. Specifically, we established a synthetic split ribozyme system that converts various RNA signals to orthogonal protein outputs, enabling in vivo visualisation of various RNA signals in plants. We demonstrated the utility of this system in transient expression experiments (i.e., leaf infiltration in Nicotiana benthamiana) to detect RNAs derived from transgenes and tobacco rattle virus, respectively. Also, we successfully engineered a split ribozyme-based biosensor in Arabidopsis thaliana for in vivo visualisation of endogenous gene expression at the cellular level, demonstrating the feasibility of multi-scale (e.g., cellular and tissue level) RNA imaging in plants. Furthermore, we developed a platform for easy incorporation of different protein outputs, allowing for flexible choice of reporters to optimise the detection of target RNAs.

Wo interacts with SlTCP25 to regulate type I trichome branching in tomato

Abstract Plant trichomes serve as a protective barrier against various stresses. Although the molecular mechanisms governing the initiation of trichomes have been extensively studied, the regulatory pathways underlying the trichome branching in tomato remain elusive. Here, we found that Woolly (Wo) mutant and its overexpression transgenic plants displayed branched type I trichomes. The expression level of SlTCP25, a transcription factor of type TB1 of the TCP subfamily, was obviously decreased in Wo mutant and Wo overexpressing lines. Knockout of SlTCP25 resulted in the formation of type I trichome branches on the hypocotyls. Genetic evidence showed that SlTCP25 is epistatic to Wo in the branched trichome formation. Biochemical data further indicated that Wo can directly bind to the L1-box cis-element in the SlTCP25 promoter and repress its transcription. We further determined that SlTCP25 interacts with Wo to weaken Wo-regulated the expression of SlCycB2, a trichome branching inhibitor. In addition, the number of trichome branches was significantly increased in Sltcp25Slcycb2 double mutant, suggesting that SlTCP25 and SlCycB2 coordinately repress trichome branching in wild type. In conclusion, we elucidate a molecular network governing the morphogenesis of multicellular trichomes in tomato.

Manipulation of the brown glume and internode 1 gene leads to alterations in the colouration of lignified tissues, lignin content and pathogen resistance in wheat.

Lignin is a crucial component of the cell wall, providing mechanical support and protection against biotic and abiotic stresses. However, little is known about wheat lignin-related mutants and their roles in pathogen defence. Here, we identified an ethyl methanesulfonate (EMS)-derived Aegilops tauschii mutant named brown glume and internode 1 (bgi1), which exhibits reddish-brown pigmentation in various tissues, including internodes, spikes and glumes. Using map-based cloning and single nucleotide polymorphism (SNP) analysis, we identified AET6Gv20438400 (BGI1) as the leading candidate gene, encoding the TaCAD1 protein. The mutation occurred in the splice acceptor site of the first intron, resulting in a premature stop codon in BGI1. We validated the function of BGI1 using loss-of-function EMS and gene editing knockout mutants, both of which displayed reddish-brown pigmentation in lignified tissues. BGI1 knockout mutants exhibited reduced lignin content and shearing force relative to wild type, while BGI1 overexpression transgenic plants showed increased lignin content and enhanced disease resistance against common root rot and Fusarium crown rot. We confirmed that BGI1 exhibits CAD activity both in vitro and in vivo, playing an important role in lignin biosynthesis. BGI1 was highly expressed in the stem and spike, with its localisation observed in the cytoplasm. Transcriptome analysis revealed the regulatory networks associated with BGI1. Finally, we demonstrated that BGI1 interacts with TaPYL-1D, potentially involved in the abscisic acid signalling pathway. The identification and functional characterisation of BGI1 significantly advance our understanding of CAD proteins in lignin biosynthesis and plant defence against pathogen infection in wheat.

Advancements in genome editing tools for genetic studies and crop improvement

The rapid increase in global population poses a significant challenge to food security, compounded by the adverse effects of climate change, which limit crop productivity through both biotic and abiotic stressors. Despite decades of progress in plant breeding and genetic engineering, the development of new crop varieties with desirable agronomic traits remains a time-consuming process. Traditional breeding methods often fall short of addressing the urgent need for improved crop varieties. Genome editing technologies, which enable precise modifications at specific genomic loci, have emerged as powerful tools for enhancing crop traits. These technologies, including RNA interference, Meganucleases, ZFNs, TALENs, and CRISPR/Cas systems, allow for the targeted insertion, deletion, or alteration of DNA fragments, facilitating improvements in traits such as herbicide and insect resistance, nutritional quality, and stress tolerance. Among these, CRISPR/Cas9 stands out for its simplicity, efficiency, and ability to reduce off-target effects, making it a valuable tool in both agricultural biotechnology and plant functional genomics. This review examines the functional mechanisms and applications of various genome editing technologies for crop improvement, highlighting their advantages and limitations. It also explores the ethical considerations associated with genome editing in agriculture and discusses the potential of these technologies to contribute to sustainable food production in the face of growing global challenges.

Overexpression of the Vitis amurensis Ca2+-binding protein gene VamCP1 in Arabidopsis thaliana and grapevine improves cold tolerance.

Calcium ions (Ca2+) are important second messengers and are known to participate in cold signal transduction. In the current study, we characterized a Ca2+-binding protein gene, VamCP1, from the extremely cold-tolerant grape species Vitis amurensis. VamCP1 expression varied among organs but was highest in leaves following cold treatment, peaking 24 h after treatment onset. VamCP1 was found to localize to the plasma membrane and nucleus and the gene showed transcriptional autoactivation activity. Overexpression of VamCP1 in Arabidopsis thaliana and grapevine (V. vinifera) resulted in transgenic plants that were more tolerant to cold stress than the wild type. This correlated with reduced accumulation of reactive oxygen species (ROS), elevated activity of antioxidant enzymes and proline content, as well as lower levels of malondialdehyde and electrolyte leakage. Additionally, the expression of genes related to cold tolerance, including C-repeat binding factors (CBF) and cold-regulated (COR) genes, was higher in the transgenic lines. Taken together, our results indicate that overexpression of VamCP1 enhanced cold tolerance in plants by promoting the upregulation of genes related to cold tolerance and scavenging of excessive ROS. These findings provide a foundation for the molecular breeding of cold-tolerant grapevine.

Essential role of rice ERF101 in the perception of TAL effectors and immune activation mediated by the CC-BED NLR Xa1

Key messageRice CC-BED NLR Xa1 recognizes TAL effectors through the interaction between ERF101 and TAL effectors.The rice Xa1 gene encodes a nucleotide-binding leucine-rich repeat receptor with an N-terminal coiled coil-zinc finger BED (CC-BED) domain. Xa1 recognizes the transcription activator-like (TAL) effectors of Xanthomonas oryzae pv. oryzae (Xoo) in the nucleus, triggering a number of immune responses, including hypersensitive cell death. We previously discovered that the rice transcription factor ERF101 directly interacts with Xa1, and functions as a positive regulator of Xa1-dependent immunity. However, the involvement of ERF101 in Xa1-induced immunity remains unclear. We herein demonstrated that the expression of the CC-BED domain in rice protoplasts inhibited Xa1-induced cell death. However, the CC-BEDC165A,C168A domain which has mutations of cysteine residues conserved in the zinc-finger motifs of BED domains and is essential for forming tetrahedral coordination geometry, failed to inhibit cell death or interact with ERF101. Therefore, Xa1-induced cell death appears to depend on the interaction between the BED domain and ERF101. In addition, we generated transgenic plants overexpressing N-terminal or C-terminal FLAG-tagged ERF101. FLAG-ERF101 transgenic plants exhibited reduced levels of Xa1-mediated immunity against Xoo, even though the overexpression of ERF101-FLAG or non-tagged ERF101 enhanced immunity. This result was consistent with the CC-BED domain interacting with C-terminal tagged ERF101, but not N-terminal tagged ERF101, whereas N-terminal and C-terminal tagged ERF101 both interacted with TAL effectors. Therefore, the interaction between the BED domain and ERF101 appears to be essential for the recognition of TAL effectors by Xa1.

Genome-wide identification of the bHLH gene family and the mechanism regulation of anthocyanin biosynthesis by ChEGL1 in Cerasus humilis.

Cerasus humilis is a fruit tree with enormous potential economic value, and its fruit is rich in various bioactive substances. The basic helix loop helix (bHLH) gene family plays an important role in the biosynthesis of plant anthocyanins. However, there was no research on the ChbHLH gene family in C. humilis. In this study, 114 ChbHLH genes were identified from the C. humilis genome and divided into 17 subgroups. Then, evolutionary relationships, conserved motifs, gene structures, and cis-acting elements were analyzed. By predicting the interaction network between ChbHLH proteins and ChMYB1, it was found that ChbHLH44 (here named as ChEGL1) was located at the core of the interaction network. Further experiments revealed that ChEGL1 and ChMYB1 could interact with each other both in vivo and in vitro. In addition, ChEGL1 significantly increased the anthocyanin content in transgenic tomato plants. This study provides a comprehensive understanding of the ChbHLH gene family and supports further enrichment of the regulation mechanism of anthocyanin biosynthesis in C. humilis fruit.

OsbZIP23 delays flowering by repressing OsMADS14 expression in rice

Flowering time is a fundamental factor determining the global distribution and final yield of rice (Oryza sativa L.). The initiation of the floral transition process signifies the beginning of the reproductive phase. The florigens Heading Date 3a (Hd3a) and Rice Flowering Locus T 1 (RFT1) combine with GF14 proteins and OsFD-like basic leucine zipper (bZIP) transcription factors to form florigen activation/repressor complexes (FACs/FRCs) that regulate the transition to flowering. We herein report that a bZIP transcription factor (OsbZIP23) functions as a flowering repressor. Transgenic plants overexpressing OsbZIP23 exhibited delayed flowering, which was in contrast to the slightly early flowering of the osbzip23 mutants, under natural short-day and long-day conditions. Molecular and biochemical analyses indicated that OsbZIP23 can bind to the 5′ untranslated region of OsMADS14 and suppress expression. Moreover, it delays the floral transition probably by interacting with OsFTL1/Hd3a/RFT1 and 14-3-3 proteins to form FRCs. Our findings have further elucidated the molecular mechanisms regulating the flowering time in rice.

Identification of the regulatory role of SsMYBS25-4 in salt stress from MYB-related transcription factors in sugarcane (Saccharum spontaneum).

Sugarcane is a highly valued crop known for its significant production of sugar and biomass. MYB transcription factors (TFs) are critical regulators in plant growth and stress tolerance, but MYB-related genes, an atypical subset of the MYB family, remain less explored. In this study, we identified 119 MYB-related genes in the genome of wild sugarcane (S. spontaneum). We thoroughly investigated their phylogenetic relationships, chromosomal locations, motif compositions, and three-dimensional (3D) protein structures by bioinformatic methods. Moreover, the expression patterns of these genes demonstrated significant diversity in plant growth and under salt stress. One of the genes, SsMYBS25-4, exhibited a significantly up-regulated expression in response to salt stress and was selected for further functional elucidation. It was found that the overexpression (OE) of SsMYBS25-4 in Arabidopsis can improve the salt stress tolerance of transgenic plants. Interestingly, the expression of some marker genes related to salt stress was significantly up-regulated in OE plants compared to wide-type plants. The SsMYB25-4 protein was localized in the nucleus and was proven to be directly bound to the promoter of the AtDR29B gene. We proposed a mechanism for SsMYB25-4 that enhances salt stress tolerance, contributing to the understanding and application of MYB-related genes in sugarcane breeding.

Global identification and regulatory network analysis reveal the significant roles of lncRNAs during anther and pollen development in Arabidopsis.

A high-throughput sequencing identified 1283 lncRNAs in anthers at different stages in Arabidopsis and their relationship with protein-coding genes and miRNAs during anther and pollen development were analyzed. Long non-coding RNAs (lncRNAs) are important regulatory molecules involved in various biological processes. However, their roles in male reproductive development and interactions with miRNAs remained elusive. In this study, a high-throughput sequencing of anthers at different developmental stages in Arabidopsis identified 1283 lncRNAs including 524 differentially expressed lncRNAs (DELs). Most of these DELs exhibited positive correlations with the expression patterns of adjacent protein-coding genes. Weighted gene co-expression network analysis (WGCNA) revealed that protein-coding genes targeted by DELs in four modules related to the tetrad stage were associated with functions such as pollen wall formation, pollen germination, or pollen tube growth, respectively. Furthermore, five, 10, and 11 lncRNAs were predicted as miRNAs' endogenous target mimics (eTMs), precursors, and natural antisense transcripts of pri-miRNA, respectively. Remarkably, the lncRNA, host gene of ath-miR167a (ath-miR167aHG), predicted as the precursor of miR167a, was selected for function validation. Its overexpression resulted in the up-regulation of miR167a and the subsequent down-regulation of miR167a's target genes ARF6 and ARF8, demonstrating a functional interaction between ath-miR167aHG and miR167a. The transgenic plants showed delayed flowering, shorter filaments, abnormal anther dehiscence, and undeveloped siliques ultimately, suggesting a role of ath-miR167aHG in male reproductive development. Collectively, our research shed new light on the functions of lncRNAs in male reproductive development and uncovered the unique interactions between lncRNAs and miRNAs.

GmGGDR Gene Confers Abiotic Stress Tolerance and Enhances Vitamin E Accumulation in Arabidopsis and Soybeans

Vitamin E, comprising tocopherols and tocotrienols, is a crucial fat-soluble antioxidant that helps maintain intracellular redox homeostasis in plants when they are under stress. Soybeans are a significant source of natural vitamin E. GGDR catalyzes the formation of phytyl diphosphate (PDP), a key vitamin E precursor, and it is involved in chlorophyll degradation. The GmGGDR gene, identified via RNA-seq in soybean germplasms with high and low vitamin E contents, encodes GGDR, a key enzyme involved in both vitamin E synthesis and chlorophyll degradation. This study shows that the GmGGDR-encoded protein is hydrophilic and stable, predominantly expressed in leaves, and markedly responsive to gibberellins. The GmGGDR gene enhances the tolerance of transgenic Arabidopsis and soybean plants to salt and drought stresses; transgenic soybeans overexpressing GmGGDR exhibited an approximately 8-fold increase in POD activity, with no significant changes in SOD and CAT activities. Moreover, the GmGGDR gene enhances the levels of α-, γ-, δ-, and total tocopherol content in transgenic soybean and Arabidopsis plants and also increases the chlorophyll a levels in the leaves of these transgenic plants. The increases in α-tocopherol, γ-tocopherol, and δ-tocopherol and total tocopherol in transgenic Arabidopsis seeds ranged from 177.8% to 600.0%, 42.9% to 90.0%, 17.6% to 292.9%, and 71.4% to 127.3% over the control, respectively. Similarly, transgenic soybeans exhibited a minimum increase of 42.9%, 27.8%, 7.1%, and 25.0% in these tocopherol fractions. Overexpression of GmGGDR also significantly elevated chlorophyll a levels in the leaves of these transgenic plants by 33.3–112.5%. This study preliminarily elucidated the function of the GmGGDR gene. It provides a theoretical foundation for further research. It presents a novel strategy for the genetic enhancement of soybean vitamin E content.

TaWI12 may be involved in pistillody and leaf cracking in wheat

TaWI12 is a member of the wound-induced (WI) protein family, which has been implicated in plant stress responses and developmental processes. Wheat (Triticum aestivum L.) is a crucial staple crop upon which human sustenance relies. Consequently, investigating the developmental mechanisms of pistils and stamens in wheat is profoundly significant for enhancing wheat characteristics and boosting productivity. In this study, we cloned TaWI12, from common wheat and observed a significant resemblance among the three homoeologs of TaWI12. The open reading frames (ORFs) of TaWI12-4A, TaWI12-4B and TaWI12-4D were 408 bp, 417 bp and 417 bp, respectively, and encoded 135, 138 and 138 amino acids, respectively. The phylogenetic tree revealed a high degree of homology between the protein sequences of TaWI12 and the wound-induced proteins of Hordeum vulgare (KAI4994568) and Aegilops tauschii (XP_020196548). To clarify the characteristics and functions of TaWI12 homoeologs, we obtained transgenic positive plants of Arabidopsis thaliana and observed significant filament shortening and decrease. Simultaneously, we used the CRISPR/Cas9 system to generate mutant plants via the modification of three homoeologs of TaWI12 in wheat. We noticed two distinct phenotypic differences in the knockout mutant. First, we observed the different degrees of homologous conversion of stamens to pistils in the single mutant TaWI12-4D. Second, we observed leaf cracking in both the single mutant TaWI12-4A and the double mutants TaWI12-4A and TaWI12-4D. Our findings further revealed that TaWI12 plays an important role in flower development, which is important for revealing the molecular mechanisms of pistil and stamen development in wheat and has important application value for high-yield wheat breeding.

Cytokinin negatively regulates tomato fruit ripening by influencing the ethylene pathway.

Reducing endogenous CK levels accelerates fruit ripening in tomato by regulating ethylene biosynthesis and signalling pathway. Tomato is a typical climacteric fruit and is recognized as one of the most important horticultural crops globally. The ripening of tomato fruits is a complex process, highly regulated by phytohormones. Cytokinin (CK) is a hormone that primarily impacts the early development of fruit, however its influence on fruit ripening has not been thoroughly investigated. In this study, we used both wild-type Micro-Tom and transgenic tomato plants that overexpress AtCKX2, a CK degradation gene driven by the fruit-specific promoter Tfm7, to investigate the effect of CK on tomato fruit ripening. Our findings revealed that reducing endogenous CK levels in transgenic plants can accelerate the ripening process of tomato fruits. Premature activation of ethylene biosynthetic genes and ripening regulator genes was upregulated in CK-deficient fruits. Moreover, the application of exogenous ethylene inhibitors resulted in delayed fruit ripening in CK-deficient fruits. These results together suggest that CK plays a negative role in tomato fruit ripening by affecting the ethylene pathway.

α-Copaene is a potent repellent against the Asian Citrus Psyllid Diaphorina citri

The Asian Citrus Psyllid (ACP), Diaphorina citri, severely threatens citrus production worldwide by transmitting the greening (= Huanglongbing)—causing bacterium Candidatus Liberibacter asiaticus. There is growing evidence that the push-pull strategy is suitable to partially mitigate HLB by repelling ACP with transgenic plants engineered to produce repellents and attracting the vector to plants with a minimal disease transmission rate. Species that pull ACP away from commercial citrus plants have been identified, and transgenic plants that repel ACP have been developed. The concept of a repellent-producing plant was first demonstrated with an Arabidopsis line engineered to overexpress a gene controlling the synthesis of β-caryophyllene and other sesquiterpenes. We have analyzed the volatile organic compounds released by this Arabidopsis line and identified α-humulene, α-copaene, and trace amounts of β-elemene, in addition to β-caryophyllene. Behavioral measurements demonstrated that α-copaene repels ACP at doses ca. 100× lower than those needed for β-caryophyllene repellence. In contrast, α-humulene is innocuous at the level emitted by the transgenic plant. We confirmed that a mixture of the three sesquiterpenes in the ratio 1:100:10 repels ACP. Likewise, a commercial sample of copaiba oil containing the three sesquiterpenes, in a proportion similar to that in the transgenic plant, repelled ACP.

Effect of transgene on salt tolerance of tobacco.

To explore the effects of salt-tolerance gene accumulation on salt tolerance in transgenic plant, we used four types of plant expression vector (N27, N28, N29, and N30) carrying mtlD, mtlD + gutD, mtlD + gutD + BADH, mtlD + gutD + BADH + sacB genes respectively, to transform tobacco through Agrobacterium-mediated method. Transgenic lines were identified through polymerase chain reaction (PCR) detection. Transgenic lines and non-transgenic plant (CK) were subjected to 6‰ sodium chloride solution stress; then, fluorescence quantitative PCR (FQ-PCR) and salt tolerance indexes were used to assess characteristics. PCR showed the exogenous genes had been integrated into the tobacco genome. FQ-PCR showed under clean water treatment the target genes were expressed in all transgenic plants at the transcriptional level. The transcript abundances of target genes changed with the number of genes increased, and improved following salt stress. Comparative analyses of salt tolerance indexes showed height growth, biomass (except for N29), chlorophyll content, net photosynthetic rate, Fv/Fm, and PI of all transgenic plants and CK were lower under salt stress than under clean water treatment, to varying degrees. However, the descent ratio was smaller in transgenic plants. A comprehensive evaluation of multiple salt-tolerance indicators performed using the membership function method showed the average salt tolerance of each vector transgenic line was higher than that of CK, and salt tolerance was greater in transgenic polyvalent gene lines than in transgenic monovalent gene lines. The average salt tolerance was N29 > N28 > N30 > N27 > CK. This study provides a theoretical and practical reference for salt tolerance breeding in other plants.

A Comprehensive Method to Generating and Identifying Transgenic Tobacco Lines with a Single Transgene Integration Locus for Functional Analysis

Tobacco is a popular model plant used for studying gene function. The generation of transgenic tobacco is tremendously essential in functional genomics. The generated transgenic plants must undergo careful selection and analysis before being used. However, most published protocols for generating transgenic tobacco for functional genomics are not comprehensive and involve sophisticated equipment. This study demonstrates an efficient and comprehensive method for developing and selecting transgenic tobacco lines without involving sophisticated equipment. Transgene was delivered into the genome of a tobacco plant via Agrobacterium tumefaciens. Polymerase Chain Reaction (PCR) was performed to verify the integration of transgenes in the putative primary transformants. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) examined transgene expressions. The number of transgene integration loci (TIL) was determined by transgene segregation analysis. PCR results revealed that ≈97% of the primary transformants were positive. The transgene was highly expressed in the transgenic plants. Segregation analysis showed that 47.6%–66.7% of the transgenic plants contained a single TIL, and the T1 and T2 progenies inherited the transgene. Homozygous transgenic lines with a single TIL were successfully developed by using our method. This manuscript encompasses detailed guidance on genetic transformation, molecular analysis, seed production, and transgene segregation analysis. It serves as a guideline for the researchers to produce transgenic tobacco lines that can be used for functional analysis. The procedures described here can be conducted in standard laboratories as they require no high-end equipment. This comprehensive and efficient method for generating transgenic tobacco will foster functional genomics.

Identification and functional characterization of the MYB transcription factor GmMYBLJ in soybean leaf senescence

Leaf senescence is an important agronomic trait that significantly influences the quality and yield of soybeans. v-Myb avian myeloblastosis viral oncogene homolog (MYB) transcription factors are considered crucial regulators governing leaf senescence, which can be utilized to improve agronomic traits in crops. However, our knowledge regarding the functional roles of soybean MYBs in leaf senescence is extremely limited. In this study, GmMYBLJ, a CCA1-like MYB, was identified and functionally characterized with respect to leaf senescence. The GmMYBLJ protein is localized in the nucleus, and a high accumulation of its transcripts was observed in nodules and embryos. Notably, GmMYBLJ was highly expressed in soybean senescent leaves and was transcriptionally induced by dark or NaCl treatment, as confirmed by histochemical GUS staining analysis. Ectopic overexpression of GmMYBLJ in Arabidopsis not only led to earlier leaf senescence, reduced chlorophyll content, and increased MDA accumulation but also promoted the expression of several WRKY family transcription factors and senescence-associated genes, such as SAG12 and ORE1. Further investigation showed that overexpression of GmMYBLJ accelerated Arabidopsis leaf senescence under darkness and in response to Pst DC3000 infection. Moreover, transgenic soybean plants overexpressing GmMYBLJ grew faster and exhibited accelerated senescence under salt stress. DAB staining analysis showed that GmMYBLJ induced ROS accumulation in soybean hairy roots and Arabidopsis leaves. Collectively, our results provided useful information into the functional roles of GmMYBLJ in both age-dependent and stress-induced senescence.

Plant cross-fertilization for production of dual-specific antibodies targeting both Ebola virus-like particles and HER2 protein in F1 plants.

This study explores the cross-fertilization of transgenic tobacco plants to produce dual-specific monoclonal antibodies (mAbs) targeting Ebola virus-like particles and HER2 proteins. We generated F1 plants by hybridizing individual transgenic lines expressing the anti-HER2 breast cancer VHH mAb (HV) and the H-13F6 human anti-Ebola large single chain mAb (EL). Hybridizing transgenic plants to express dual-antibodies between different structures VHH and LSCK indicate the potential of transgenic plants as a cost-effective and scalable production system for dual targeting mAbs. We performed polymerase chain reaction (PCR) analysis to confirm the integration of EL and HV genes in the F1 progeny. The reverse-transcription (RT)-PCR and immunoblotting were performed to confirm the expression of transgenes. Indirect enzyme-linked immunosorbent assay was conducted to confirm the functionality of purified EL and HV mAb. A PCR analysis confirmed the successful integration of both EL and HV mAb genes in the F1 progeny. Additionally, (RT)-PCR and immunoblotting validated the expression of these transgenes, with EL and HV mAbs purified from the F1 plants. Indirect enzyme-linked immunosorbent assay (ELISA) demonstrated that EL × HV mAb proteins maintained binding activity to Ebola virus-specific antigens, comparable to that of the EL mAb protein, while also exhibiting binding activity against HER2 proteins similar to that of the HV mAb. This study indicates the potential for transgenic plants to produce dually targeting mAbs, suggesting a promising application in enabling the co-expression of antibodies targeting two different diseases in a single plant.

Development of robust constitutive synthetic promoter using genetic resources of plant pararetroviruses.

With the advancement of plant synthetic biology, complex genetic engineering circuits are being developed, which require more diverse genetic regulatory elements (promoters) to operate. Constitutive promoters are widely used for such gene engineering projects, but the list of strong, constitutive plant promoters with strength surpassing the widely used promoter, the CaMV35S, is limited. In this work, we attempted to increase the constitutive promoter library by developing efficient synthetic promoters suitable for high-level gene expression. To do that, we selected three strong pararetroviral-based promoters from Mirabilis mosaic virus (MMV), Figwort mosaic virus (FMV), and Horseradish latent virus (HRLV) and rationally designed and combined their promoter elements. We then tested the newly developed promoters in Nicotiana benthamiana and found a highly active tri-hybrid promoter, MuasFuasH17 (MFH17). We further used these promoter elements in generating random mutant promoters by DNA shuffling techniques in an attempt to change/improve the MFH17 promoter. We further evaluated the activity of the MFH17 promoter in Oryza sativa seedlings and studied the effect of as-1 elements present in it. Finally, we tested the efficacy and tissue specificity of the MFH17 promoter in planta by developing transgenic Nicotiana tabacum and Arabidopsis thaliana plants and found it highly constitutive and efficient in driving the gene throughout the plant tissues. Overall, we conclude that this tripartite synthetic promoter MFH17 is a strong, highly constitutive, and dual-species (dicot and monocot) expressing promoter, which can be a valuable addition to the constitutive plant promoter library for plant synthetic biology.