Soybean (Glycine max) is an important oil crop, but its growth and yield are severely threatened by abiotic stress, such as drought and salinity. DR1372 gene is involved in extreme stress tolerance of a kind of bacteria named Deinococcus radiodurans. However, the function of DR1372 in response to abiotic stresses in soybean has rarely been reported. DR1372 gene was cloned from D. radiodurans and was overexpressed in soybean. The role of DR1372 was investigated through physiological and biochemical experiments combined with transcriptome analysis. Overexpressed DR1372 soybean plants showed enhanced salt and drought tolerance. Under salt stress, transgenic lines exhibited significantly enhanced antioxidant capacity compared to wild-type (WT), with superoxide dismutase (SOD) and peroxidase (POD) activities increased by approximately 51.8

Nitrogen acquisition is pivotal for plant growth. In soil ecosystems, bacterial interactions promote nitrogen assimilation and rhizosphere colonization. However, the mechanisms underlying the interactions between nitrogen-fixing microorganisms and their neighboring organisms in the environment remain unclear. Here, we demonstrate that Bacillus velezensis BRI3, a rhizosphere-derived strain, forms microbial synergy with Stutzerimonas stutzeri A1501, functioning as a facilitator. This microbial synergy greatly increases the nitrogen-fixation by 3.2-fold and rhizosphere colonization capabilities by 2.3-fold of A1501, collectively promoting plant growth in the rhizosphere. In this study, for the first time, we propose that surfactin produced by BRI3 regulates interactions among this bacterial consortium by stimulating A1501 biofilm formation. This discovery enhances the understanding of metabolic interactions between nitrogen-fixing bacteria and their neighboring organisms. Overall, we propose a strain interaction paradigm that offers a novel framework for improving nitrogen utilization and crop yield.IMPORTANCENitrogen is essential for crop productivity because it directly participates in the construction of proteins and nucleic acids. Associative diazotrophs convert N₂ into NH₄+, yet require root biofilms and stable colonization. Owing to the complexity of the rhizospheric microbiota, a systematic understanding of microbe-microbe interactions and their impact on nitrogen-fixation capacity is still lacking. This study uncovers a novel intergeneric synergism in which B. velezensis BRI3 secretes surfactin that triggers S. stutzeri A1501 biofilm formation and concurrently elevates nif gene expression, thereby facilitating the integration of microbe-microbe interaction, biofilm development, and nitrogen-fixation efficiency into a single linear pathway. This phenomenon also provides a portable molecular-to-phenotypic blueprint for designing composite inoculants. Second, field trials revealed that coinoculation of these strains boosted maize growth, allowing partial synthetic-N replacement without transgenes or high costs, merely via rational strain formulation. This study highlights a transition from focusing on the ecological features of associative bacteria toward the development of deployable technology, offering a theory and prototype for sustainable agriculture.

Abstract Gene-programmable expression element library is rapidly expanding, making the regulation of key genes increasingly convenient. A temperature biosensor system was constructed to utilize environmental temperature conditions for gene expression regulation, thereby reducing reliance on costly chemical inducers and enabling successful application to product synthesis. Existing biosensors based on the temperature response of CI857-PR have shortcomings, which greatly limit their application. In this study, we constructed a dual-dependent promoter library P38X for σ70 and σ38 and combined it with the operator gene R1 in the PR promoter to achieve structural decoupling from the wild-type PR promoter and improve promoter persistence in the stationary phase. The method was successfully applied to the de novo lycopene synthesis in Escherichia coli, obtaining a final yield of 116 mg/L within 48 h under shake-flask fermentation. Protein degradation tags were introduced to address the previously reported accumulation of repressor proteins. In addition, D91 from the degradation tag mutant library was introduced into E. coli to synthesize ergothioneine using a temperature-tuned expression delay timer. The yield of 307 mg/L was obtained in 48 h under shake-flask fermentation and 7.5 g/L in a 2 L bioreactor after optimizing the fermentation conditions and S-adenosylmethionine supply. This study provides a new approach to long-term effective gene expression at lower cost, thus enriching the library of programmable expression elements.

The major challenges to the large-scale application of biological nitrogen fixation are its high energy demand and the acute oxygen sensitivity of nitrogenase. Bioelectrochemical nitrogen fixation (e-BNF) presents a promising strategy to circumvent these energetic constraints, however, extant e-BNF systems, largely restricted to anaerobic or microaerobic conditions, remain unable to reconcile efficient nitrogen fixation with high-density cell cultivation and industrial aerobic fermentation. Herein, a novel aerobic e-BNF system was developed, which elicited significant enhancements in nitrogen fixation performance of Azotobacter vinelandii under aerobic conditions with sucrose as the sole carbon source: biomass and ammonium yield increased by 2.73- and 9.36-fold, respectively. Mechanistic investigations revealed that exogenous electricity specifically activated the Mo‒Fe nitrogenase system, orchestrated through extensive metabolic reprogramming and augmented oxidative phosphorylation. A dual electron transfer mechanism was identified-primarily reinforcing the Rnf1-NifF pathway (>90% activity) with a newly discovered alternative electron transfer bypass restoring ∼ 9.3% activity. Metabolic engineering amplified these effects: heterologous OmcS nanowires increased electrically driven ammonium secretion 7.23-fold; phbB deletion eliminated competing PHB synthesis, reducing carbon consumption by 57.30% and raising cellular protein to 66.72%. This work elucidates synergistic mechanisms of aerobic e-BNF and provides a renewable-energy prototype for green ammonia and single-cell protein production.

Both nitrogen (N) and phosphorus (P) are essential nutrients for plant growth but are often limiting in high-yielding agricultural production systems. This study investigated the synergistic effect of nitrogen fixation and organic phosphate mineralization by rhizospheric microorganisms, thereby promoting the growth of host plants. After assessing phosphate-solubilizing activities of five Pseudomonas strains and identifying putative phosphatase genes in silico, selected genes were expressed in nitrogen-fixing P. stutzeri A1501, and the resulting recombinants were evaluated for growth, nitrogenase activity, organic P solubilization (pure and rice co-culture), root colonization, and rice growth-promotion under different N regimes. Nine heterologous phosphatase genes were introduced into A1501, and the resulting recombinant strains displayed significantly elevated extracellular phosphatase activity. Most engineered strains maintained nitrogenase activity comparable to A1501, except A15NapD (37

Artificial trans-encoded small RNAs (atsRNAs), which repress translation by base pairing with target mRNAs, have proven to be powerful tools for regulation of gene expression for reconstructing cell factories and genetic circuits. Currently, rationally designed atsRNAs use mostly E. coli natural trans-acting sRNAs with an Hfq-binding structure and a Rho-independent terminator as scaffolds. However, owing to the structural differences in Hfq proteins between E. coli and other bacteria, the inhibitory efficacy of atsRNAs may be influenced by heterologous sRNA scaffolds, highlighting the necessity of screening native sRNA scaffolds in various bacteria to develop functional synthetic sRNAs. The root-associated bacterium Pseudomonas stutzeri A1501 is a well-studied strain regarding nitrogen fixation, yet post-transcriptional regulatory platforms for this species remain scarce. In this study, we describe the development of an atsRNA platform by employing the natural Hfq-dependent sRNA CrcZ scaffold from the nitrogen-fixing strain P. stutzeri A1501, which is conserved in the Pseudomonas strain. On the basis of the CrcZ scaffold, two atsRNAs targeting the master nitrogen-fixing regulatory complex encoded by the nifLA operon were developed and found to effectively suppress nifLA gene expression at the posttranscriptional level in P. stutzeri A1501. Further studies demonstrated that the 5' untranslated region of target mRNAs and five Hfq-binding sites are optimal features for designing atsRNAs using CrcZ as a scaffold. The well-established effective design principle for synthetic sRNAs with the CrcZ sRNA scaffold will enable more rational and efficient engineering of synthetic gene networks in Pseudomonas strains.

Soil salinization poses a significant threat to global agriculture, necessitating the development of sustainable strategies to increase crop resilience. Plant growth-promoting rhizobacteria (PGPR) offer a promising solution; however, their effectiveness in saline soils is often limited by their own salt sensitivity. The role of the extracytoplasmic function of the sigma factor AlgU in the salt stress adaptation of the nitrogen-fixing PGPR Pseudomonas stutzeri A1501 was investigated in this study. Through the construction of isogenic algU knockout (ΔalgU) and overexpression (OE-algU) strains, combined with phenotypic assays, transcriptomic profiling, and plant experiments, we demonstrate that AlgU acts as a master regulator of salinity tolerance. AlgU increased bacterial survival under acute salt shock, promoted biofilm formation, and, crucially, protected nitrogenase activity from salt inhibition. RNA-seq analysis revealed that AlgU orchestrates a comprehensive transcriptional reprogramming, upregulating the expression of genes involved in exopolysaccharide synthesis, osmoprotection (otsA), and central carbon metabolism (zwf, fumC). This coordinated response ensures an adequate supply of energy and reducing equivalents while maintaining cellular homeostasis. Consequently, inoculation with the OE-algU strain significantly alleviated salt stress in maize, improving seedling growth in pot experiments and outperforming the wild-type strain by increasing grain yield in saline–alkali field trials. Our findings establish AlgU as a key genetic determinant for engineering salt-tolerant PGPR, providing a mechanistic framework for the development of effective microbial inoculants to improve crop productivity in saline soils.

Fast-growing Vibrio natriegens is now recognized as a next-generation chassis for synthetic biology and biotechnology; however, its low transformation efficiency, limited gene editing methods and high fermentation cost are still the main challenges hampering its industrial application. In this study, we established an efficient electroporation transformation and dual-plasmid CRISPR-Cas9 editing system in V. natriegens. Subsequently, the heterologous ergothioneine biosynthetic pathway involving the combination of the superstrong PLlacO1 promoter and weak RBS7 was constructed in V. natriegens. Multiple genes encoding genes involved in byproduct formation and adenosine triphosphate (ATP) degradation were consecutively deleted, while several key genes involved in the S-adenosylmethionine (SAM) cycle and the ATP synthesis pathway were overexpressed to increase ergothioneine production. Finally, fed-batch fermentation was performed using low-cost sucrose as the sole carbon source under high-salinity, non-sterile conditions, resulting in an ergothioneine titer of 1.2 g/L in a 2-L bioreactor. This study not only provides the first successful example of the ergothioneine biosynthesis with engineered V. natriegens strains but also establishes an efficient and economic platform in which V. natriegens is used to produce other high-value compounds.

Nitrite reductase Nrf, encoded by the nrf operon, plays a central role in bacterial responses to nitrosative and oxidative stress under anaerobic conditions. In this study, we show that the pathogenic strain Aeromonas veronii C4 also expresses nrf and exhibits measurable nitrite reductase activity under aerobic growth conditions. Combined ChIP-seq and RNA-seq analyses identify ArgR as a direct negative regulator of nrf transcription. Biochemical assays further demonstrate that ArgR binds to the ARG box within the nrf promoter to repress its expression. Phylogenetic analyses reveal a correlated evolutionary pattern between ArgR and NrfA, and a conserved ARG box is present in the nrf promoters of most Gammaproteobacteria, suggesting that this regulatory mechanism is broadly conserved within this lineage. Functional assays show that deletion of nrf markedly reduces nitrite reduction and oxidative stress resistance under aerobic conditions. In addition, the expression of nrf and its associated electron-transfer genes (pflB, fdoGHI) varies with growth phase and environmental conditions and correlates with argR expression. ArgR transcription is not affected by nitrite but is responsive to arginine availability and oxidative stress, suggesting that ArgR primarily integrates metabolic and stress-related signals rather than nitrite itself. These observations are consistent with an ArgR–nrf regulatory link connecting nitrogen metabolism with aerobic redox stress adaptation, potentially contributing to bacterial fitness in host-associated environments. ArgR directly represses nrf transcription to couple nitrogen metabolism with aerobic oxidative stress defense in pathogenic Aeromonas veronii, revealing a conserved regulatory axis within Gammaproteobacteria.

Cold-adapted pectin lyases are particularly useful in the extraction and clarification of freshly squeezed fruit juices at low temperatures, as they effectively reduce juice viscosity and improve light transmittance. With the increasing attention on low-temperature pectinase in industrial applications, the exploration of low-temperature pectinase with novel characteristics has become one of the key focuses of research and development. In this study, a 1026 bp gene, pel1Ba, encoding a 42.7 kDa pectin lyase, was cloned from sediment samples collected from the South China Sea and heterologously expressed in Escherichia coli. The purified Pel1Ba exhibited an optimal temperature of 40 °C and an optimal pH of 10, with a total enzyme activity of 5100 U/mL. Notably, Pel1Ba is a cold-adapted enzyme that retains 80% of its relative activity across the temperature range of 0–40 °C. When 20 U/mL purified Pel1Ba was added to orange juice, the juice volume increased by 43.00% and its clarity improved by 37.80%. Meanwhile, site-directed mutagenesis analysis revealed that the residual enzyme activities of the mutants A230I, F253I, and L292I were increased by 22.5%, 34.4%, and 25.1%, respectively, compared to the wild type. This study concludes that the cold-active pectate lyase Pel1Ba exhibits potential for applications in the food industry.

Biological nitrogen fixation (BNF) facilitated by nitrogen-fixing bacteria (NFBac) offers a sustainable alternative to conventional nitrogen fertilizers. However, acidic soil conditions, characterized by reduced pH levels, nutrient deficiencies, and disrupted microbiomes, present a significant challenge to the effectiveness of NFBac. This study proposes a nanoengineering approach to bolster the resilience and functionality of NFBac in acidic soils. Azotobacter Pseudomonas stutzeri A1501 cells are sequentially coated with a pH-responsive copolymer L100-55 and calcium phosphate nanoparticles (CaP NPs), resulting in the development of a robust biofertilizer (A1501@L@CaP). This biofertilizer can effectively buffer acidic pH levels, release nutrients, promote nutrient cycling, enhance soil enzymatic activity, and modulate soil microbiomes, rendering substantial enhancements in soil ecology and plant growth. The engineered NFBac offer a viable strategy for integrating BNF into acidic soils in a sustainable manner.

Here, we report the complete genome sequence of Bradyrhizobium japonicum ACCC 15027 from the Agricultural Culture Collection of China (ACCC). The whole genome of ACCC 15027 is 10.02 Mb long and consists of a circular chromosome and two plasmids.

The RNA chaperone Hfq acts as a global regulator of numerous biological processes, such as carbon/nitrogen metabolism and environmental adaptation in plant-associated diazotrophs; however, its target RNAs and the mechanisms underlying nitrogen fixation remain largely unknown. Here, we used enhanced UV cross-linking immunoprecipitation coupled with high-throughput sequencing to identify hundreds of Hfq-binding RNAs probably involved in nitrogen fixation, carbon substrate utilization, biofilm formation, and other functions. Collectively, these processes endow strain A1501 with the requisite capabilities to thrive in the highly competitive rhizosphere. Our findings revealed a previously uncharted landscape of Hfq target genes. Notable among these is nifM, encoding an isomerase necessary for nitrogenase reductase solubility; amtB, encoding an ammonium transporter; oprB, encoding a carbohydrate porin; and cheZ, encoding a chemotaxis protein. Furthermore, we identified more than 100 genes of unknown function, which expands the potential direct regulatory targets of Hfq in diazotrophs. Our data showed that Hfq directly interacts with the mRNA of regulatory proteins (RsmA, AlgU, and NifA), regulatory ncRNA RsmY, and other potential targets, thus revealing the mechanistic links in nitrogen fixation and other metabolic pathways. IMPORTANCE:Numerous experimental approaches often face challenges in distinguishing between direct and indirect effects of Hfq-mediated regulation. New technologies based on high-throughput sequencing are increasingly providing insight into the global regulation of Hfq in gene expression. Here, enhanced UV cross-linking immunoprecipitation coupled with high-throughput sequencing was employed to identify the Hfq-binding sites and potential targets in the root-associated Pseudomonas stutzeri A1501 and identify hundreds of novel Hfq-binding RNAs that are predicted to be involved in metabolism, environmental adaptation, and nitrogen fixation. In particular, we have shown Hfq interactions with various regulatory proteins' mRNA and their potential targets at the posttranscriptional level. This study not only enhances our understanding of Hfq regulation but, importantly, also provides a framework for addressing integrated regulatory network underlying root-associated nitrogen fixation.

Cotton fiber length is basically determined by well-coordinated gene expression and phosphatidylinositol phosphates (PIPs) accumulation during fiber elongation but the regulatory mechanism governing PIPs transport remains unknown. Here, we report a MYB transcription factor GhMYB30D04 in Gossypium hirsutum that promotes fiber elongation through modulating the expression of PIP transporter gene GhLTPG1. Knockout of GhMYB30D04 gene in cotton (KO) results in a reduction of GhLTPG1 transcripts with lower accumulation of PIPs, leading to shorter fibers and lower fiber yield. Conversely, GhMYB30D04 overexpression (GhMYB30D04-OE) causes richer PIPs and longer cotton fibers, mimicking the effects of exogenously applying PIPs on the ovules of GhMYB30D04-KO and wild type. Furthermore, GhMYB30D04 interacts with GhHD1, the crucial transcription factor of fiber initiation, to form an activation complex stabilized by PIPs, both of which upregulate GhLTPG1 expression. Comparative omics-analysis revealed that higher and extended expressions of LTPG1 in fiber elongation mainly correlate with the variations of the GhMYB30D04 gene between two cotton allotetraploids, contributing to longer fiber in G. babardense. Our work clarifies a mechanism by which GhHD1-GhMYB30D04 form a regulatory module of fiber elongation to tightly control PIP accumulation. Our work still has an implication that GhMYB30D04-GhHD1 associates with development transition from fiber initiation to elongation.

To grow in various harsh environments, extremophiles have developed extraordinary strategies such as biofilm formation, which is an extremely complex and progressive process. However, the genetic elements and exact mechanisms underlying extreme biofilm formation remain enigmatic. Here, we characterized the biofilm-forming ability of Deinococcus radiodurans in vitro under extreme environmental conditions and found that extremely high concentrations of NaCl or sorbitol could induce biofilm formation. Meantime, the survival ability of biofilm cells was superior to that of planktonic cells in different extreme conditions, such as hydrogen peroxide stress, sorbitol stress, and high UV radiation. Transcriptome profiles of D. radiodurans in four different biofilm development stages further revealed that only 13 matched genes, which are involved in environmental information processing, carbohydrate metabolism, or stress responses, share sequence homology with genes related to the biofilm formation of Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. Overall, 64% of the differentially expressed genes are functionally unknown, indicating the specificity of the regulatory network of D. radiodurans. The mutation of the drRRA gene encoding a response regulator strongly impaired biofilm formation ability, implying that DrRRA is an essential component of the biofilm formation of D. radiodurans. Furthermore, transcripts from both the wild type and the drRRA mutant were compared, showing that the expression of drBON1 (Deinococcus radioduransBON domain-containing protein 1) significantly decreased in the drRRA mutant during biofilm development. Further analysis revealed that the drBON1 mutant lacked the ability to form biofilm and DrRRA, and as a facilitator of biofilm formation, could directly stimulate the transcription of the biofilm-related gene drBON1. Overall, our work highlights a molecular mechanism mediated by the response regulator DrRRA for controlling extreme biofilm formation and thus provides guidance for future studies to investigate novel mechanisms that are used by D. radiodurans to adapt to extreme environments.

Background/Objectives: Bacillus velezensis has recently received increased attention as a potential biological agent because of its broad-spectrum antagonistic capacity against harmful bacteria and fungi. This study aims to thoroughly analyze the genomic characteristics of B. velezensis BRI3, thereby providing theoretical groundwork for the agronomic utilization of this strain. Methods: In this work, we evaluated the beneficial traits of the newly isolated strain B. velezensis BRI3 via in vitro experiments, whole-genome sequencing, functional annotation, and comparative genomic analysis. Results: B. velezensis BRI3 exhibits broad-spectrum antifungal activity against various soilborne pathogens, displays inhibitory effects comparable to those of the type strain FZB42, and exhibits particularly effective antagonism against Sclerotinia sclerotiorum (Lib.) de Bary. Whole-genome sequencing and assembly revealed that the genome of BRI3 contains one chromosome and two plasmids, which carry a large amount of genetic information. Moreover, 13 biosynthetic gene clusters (BGCs) involved in the biosynthesis of secondary metabolites were predicted within the BRI3 genome. Among these, two unique BGCs (cluster 11 and cluster 13), which were not previously reported in the genomes of other strains and could potentially encode novel metabolic products, were identified. The results of the comparative genomic analysis demonstrated the genomic structural conservation and genetic homogeneity of BRI3. Conclusions: The unique characteristics and genomic data provide insights into the potential application of BRI3 as a biocontrol and probiotic agent.

DnaJ proteins, also known as HSP40s, play a key role in plant growth and development, and response to environmental stress. However, little comprehensive research has been conducted on the DnaJ gene family in maize. Here, we identify 91 ZmDnaJ genes from maize, which are likely distributed in the chloroplast, nucleus, and cytoplasm. Our analysis revealed that ZmDnaJs were classified into three types, with conserved protein motifs and gene structures within the same type, particularly among members of the same subfamily. Gene duplication events have likely contributed to the expansion of the ZmDnaJ family in maize. Analysis of cis-regulatory elements in ZmDnaJ promoters suggested involvement in stress responses, growth and development, and phytohormone sensitivity in maize. Specifically, four cis-acting regulatory elements associated with stress responses and phytohormone regulation indicated a role in adaptation. RNA-seq analysis showed constitutive expression of most ZmDnaJ genes, some specifically in pollen and endosperm. More importantly, certain genes also responded to salt, heat, and cold stresses, indicating potential interaction between stress regulatory networks. Furthermore, early responses to heat stress varied among five inbred lines, with upregulation of almost tested ZmDnaJ genes in B73 and B104 after 6 h, and fewer genes upregulated in QB1314, MD108, and Zheng58. After 72 h, most ZmDnaJ genes in the heat-sensitive inbred lines (B73 and B104) returned to normal levels, while many genes, including ZmDnaJ55, 79, 88, 90, and 91, remained upregulated in the heat-tolerant inbred lines (QB1314, MD108, and Zheng58) suggesting a synergistic function for prolonged protection against heat stress. In conclusion, our study provides a comprehensive analysis of the ZmDnaJ family in maize and demonstrates a correlation between heat stress tolerance and the regulation of gene expression within this family. These offer a theoretical basis for future functional validation of these genes.

Waste oil pollution and the treatment of oily waste present a challenge, and the exploitation of microbial resources is a safe and efficient method to resolve these problems. Lipase-producing microorganisms can directly degrade waste oil and promote the degradation of oily waste and, therefore, have very significant research and application value. The isolation of efficient oil-degrading strains is of great practical significance in research into microbial remediation in oil-contaminated environments and for the enrichment of the microbial lipase resource library. In this study, Acinetobacter junii WCO-9, an efficient oil-degrading bacterium, was isolated from an oil-contaminated soil using olive oil as the sole carbon source, and its enzyme activity of ρ-nitrophenyl decanoate (ρ-NPD) decomposition was 3000 U/L. The WCO-9 strain could degrade a variety of edible oils, and its degradation capability was significantly better than that of the control strain, A junii ATCC 17908. Comparative pan-genome and lipid degradation pathway analyses indicated that A. junii isolated from the same environment shared a similar set of core genes and that the species accumulated more specific genes that facilitated resistance to environmental stresses under different environmental conditions. WCO-9 has accumulated a complete set of oil metabolism genes under a long-term oil-contamination environment, and the compact arrangement of abundant lipase and lipase chaperones has further strengthened the ability of the strain to survive in such environments. This is the main reason why WCO-9 is able to degrade oil significantly more effectively than ATCC 17908. In addition, WCO-9 possesses a specific lipase that is not found in homologous strains. In summary, A. junii WCO-9, with a complete triglyceride degradation pathway and the specific lipase gene, has great potential in environmental remediation and lipase for industry.

Herbicide tolerance has been the dominant trait introduced during the global commercialization of genetically modified (GM) crops. Herbicide-tolerant crops, especially glyphosate-resistant crops, offer great advantages for weed management; however, despite these benefits, glyphosate-resistant maize (Zea mays L.) has not yet been commercially deployed in China. To develop a new bio-breeding resource for glyphosate-resistant maize, we introduced a codon-optimized glyphosate N-acetyltransferase gene, gat, and the enolpyruvyl-shikimate-3-phosphate synthase gene, gr79-epsps, into the maize variety B104. We selected a genetically stable high glyphosate resistance (GR) transgenic event, designated GG2, from the transgenic maize population through screening with high doses of glyphosate. A molecular analysis demonstrated that single copy of gat and gr79-epsps were integrated into the maize genome, and these two genes were stably transcribed and translated. Field trials showed that the transgenic event GG2 could tolerate 9000 g acid equivalent (a.e.) glyphosate per ha with no effect on phenotype or yield. A gas chromatography-mass spectrometry (GC-MS) analysis revealed that, shortly after glyphosate application, the glyphosate (PMG) and aminomethylphosphonic acid (AMPA) residues in GG2 leaves decreased by more than 90% compared to their levels in HGK60 transgenic plants, which only harbored the epsps gene. Additionally, PMG and its metabolic residues (AMPA and N-acetyl-PMG) were not detected in the silage or seeds of GG2, even when far more than the recommended agricultural dose of glyphosate was applied. The co-expression of gat and gr79-epsps, therefore, confers GG2 with high GR and a low risk of herbicide residue accumulation, making this germplasm a valuable GR event in herbicide-tolerant maize breeding.

G3LEA is a family of proteins that exhibit chaperone-like activity when under distinct stress. In previous research, DosH was identified as a G3LEA protein from model extremophile—Deinococcus radiodurans R1 with a crucial core HD domain consisting of eight 11-mer motifs. However, the roles of motifs participating in the process of resistance to stress and their underlying mechanisms remain unclear. Here, eight different proteins with tandem repeats of the same motif were synthesized, named Motif1–8, respectively, whose function and structure were discussed. In this way, the role of each motif in the HD domain can be comprehensively analyzed, which can help in finding possibly crucial amino acid sites. Circular dichroism results showed that all proteins were intrinsically ordered in phosphate buffer, and changed into more α-helical ordered structures with the addition of trifluoroethanol and glycerol. Transformants expressing artificial proteins had significantly higher stress resistance to oxidation, desiccation, salinity and freezing compared with the control group; E. coli with Motif1 and Motif8 had more outstanding performance in particular. Moreover, enzymes and membrane protein protection viability suggested that Motif1 and Motif8 had more positive influences on various molecules, demonstrating a protective role in a chaperone-like manner. Based on these results, the artificial proteins synthesized according to the rule of 11-mer motifs have a similar function to wildtype protein. Regarding the sequence in all motifs, there are more amino acids to produce H bonds and α-helices, and more amino acids to promote interaction between proteins in Motif1 and Motif8; in addition, considering linkers, there are possibly more amino acids forming α-helix and binding substrates in these two proteins, which potentially provides some ideas for us to design potential ideal stress-response elements for synthetic biology. Therefore, the amino acid composition of the 11-mer motif and linker is likely responsible for its biological function.