CRISPR/Cas9-mediated editing of GmDWF1 brassinosteroid biosynthetic gene induces dwarfism in soybean.

KEY MESSAGE: The study on the GmDWF1-deficient mutant dwf1 showed that GmDWF1 plays a crucial role in determining soybean plant height and yield by influencing the biosynthesis of brassinosteroids. Soybean has not adopted the Green Revolution, such as reduced height for increased planting density, which have proven beneficial for cereal crops. Our research identified the soybean genes GmDWF1a and GmDWF1b, homologous to Arabidopsis AtDWF1, and found that they are widely expressed, especially in leaves, and linked to the cellular transport system, predominantly within the endoplasmic reticulum and intracellular vesicles. These genes are essential for the synthesis of brassinosteroids (BR). Single mutants of GmDWF1a and GmDWF1b, as well as double mutants of both genes generated through CRISPR/Cas9 genome editing, exhibit a dwarf phenotype. The single-gene mutant exhibits moderate dwarfism, while the double mutant shows more pronounced dwarfism. Despite the reduced stature, all types of mutants preserve their node count. Notably, field tests have shown that the single GmDWF1a mutant produced significantly more pods than wild-type plants. Spraying exogenous brassinolide (BL) can compensate for the loss in plant height induced by the decrease in endogenous BRs. Comparing transcriptome analyses of the GmDWF1a mutant and wild-type plants revealed a significant impact on the expression of many genes that influence soybean growth. Identifying the GmDWF1a and GmDWF1b genes could aid in the development of compact, densely planted soybean varieties, potentially boosting productivity.

Loss of polarity protein Par3 in the intestinal epithelium promotes colitis-associated colorectal cancer progression by damaging tight junction assembly.

Partitioning defective 3 (Par3) is a polarity protein critical in establishing epithelial cell polarity and tight junctions (TJs). Impaired intestinal epithelial barrier integrity is closely associated with colitis-associated colorectal cancer (CRC) progression. According to the GEO and TCGA database analyses, we first observed that the expression of Par3 was reduced in CRC patients. To understand how Par3 is related to CRC, we investigated the role of Par3 in the development of CRC using an in vivo genetic approach. Our results show that the intestinal epithelium-specific PAR3 deletion mice demonstrated a more severe CRC phenotype in the context of azoxymethane/dextran sodium sulfate (AOM/DSS) treatment, with a corresponding increase in tumor number and inflammatory cytokines profile. Mechanistically, loss of Par3 disrupts the TJs of the intestinal epithelium and increases mucosal barrier permeability. The interaction of Par3 with ZO-1 prevents intramolecular interactions within ZO-1 protein and facilitates the binding of occludin to ZO-1, hence preserving TJs integrity. Our results suggest that Par3 deficiency permits pathogenic bacteria and their endotoxins to penetrate the intestinal submucosa and activate TLR4/MyD88/NF-κB signaling, promoting inflammation-driven CRC development and that Par3 may be a novel potential molecular marker for the diagnosis of early-stage CRC.

RNA-Seq and Comparative Transcriptomic Analyses of Asian Soybean Rust Resistant and Susceptible Soybean Genotypes Provide Insights into Identifying Disease Resistance Genes.

Asian soybean rust (ASR), caused by Phakopsora pachyrhizi, is one of the most destructive foliar diseases that affect soybeans. Developing resistant cultivars is the most cost-effective, environmentally friendly, and easy strategy for controlling the disease. However, the current understanding of the mechanisms underlying soybean resistance to P. pachyrhizi remains limited, which poses a significant challenge in devising effective control strategies. In this study, comparative transcriptomic profiling using one resistant genotype and one susceptible genotype was performed under infected and control conditions to understand the regulatory network operating between soybean and P. pachyrhizi. RNA-Seq analysis identified a total of 6540 differentially expressed genes (DEGs), which were shared by all four genotypes. The DEGs are involved in defense responses, stress responses, stimulus responses, flavonoid metabolism, and biosynthesis after infection with P. pachyrhizi. A total of 25,377 genes were divided into 33 modules using weighted gene co-expression network analysis (WGCNA). Two modules were significantly associated with pathogen defense. The DEGs were mainly enriched in RNA processing, plant-type hypersensitive response, negative regulation of cell growth, and a programmed cell death process. In conclusion, these results will provide an important resource for mining resistant genes to P. pachyrhizi infection and valuable resources to potentially pyramid quantitative resistance loci for improving soybean germplasm.

Identification of Quantitative Trait Locus and Candidate Genes for Drought Tolerance in a Soybean Recombinant Inbred Line Population.

With global warming and regional decreases in precipitation, drought has become a problem worldwide. As the number of arid regions in the world is increasing, drought has become a major factor leading to significant crop yield reductions and food crises. Soybean is a crop that is relatively sensitive to drought. It is also a crop that requires more water during growth and development. The aim of this study was to identify the quantitative trait locus (QTL) that affects drought tolerance in soybean by using a recombinant inbred line (RIL) population from a cross between the drought-tolerant cultivar 'Jindou21' and the drought-sensitive cultivar 'Zhongdou33'. Nine agronomic and physiological traits were identified under drought and well-watered conditions. Genetic maps were constructed with 923,420 polymorphic single nucleotide polymorphism (SNP) markers distributed on 20 chromosomes at an average genetic distance of 0.57 centimorgan (cM) between markers. A total of five QTLs with a logarithm of odds (LOD) value of 4.035-8.681 were identified on five chromosomes. Under well-watered conditions and drought-stress conditions, one QTL related to the main stem node number was located on chromosome 16, accounting for 17.177% of the phenotypic variation. Nine candidate genes for drought resistance were screened from this QTL, namely Glyma.16G036700, Glyma.16G036400, Glyma.16G036600, Glyma.16G036800, Glyma.13G312700, Glyma.13G312800, Glyma.16G042900, Glyma.16G043200, and Glyma.15G100700. These genes were annotated as NAC transport factor, GATA transport factor, and BTB/POZ-MATH proteins. This result can be used for molecular marker-assisted selection and provide a reference for breeding for drought tolerance in soybean.

Overexpression of GmMYB14 improves high-density yield and drought tolerance of soybean through regulating plant architecture mediated by the brassinosteroid pathway.

MYB transcription factors (TFs) have been reported to regulate the biosynthesis of secondary metabolites, as well as to mediate plant adaption to abiotic stresses, including drought. However, the roles of MYB TFs in regulating plant architecture and yield potential remain poorly understood. Here, we studied the roles of the dehydration-inducible GmMYB14 gene in regulating plant architecture, high-density yield and drought tolerance through the brassinosteroid (BR) pathway in soybean. GmMYB14 was shown to localize to nucleus and has a transactivation activity. Stable GmMYB14-overexpressing (GmMYB14-OX) transgenic soybean plants displayed a semi-dwarfism and compact plant architecture associated with decreased cell size, resulting in a decrease in plant height, internode length, leaf area, leaf petiole length and leaf petiole angle, and improved yield in high density under field conditions. Results of the transcriptome sequencing suggested the involvement of BRs in regulating GmMYB14-OX plant architecture. Indeed, GmMYB14-OX plants showed reduced endogenous BR contents, while exogenous application of brassinolide could partly rescue the phenotype of GmMYB14-OX plants. Furthermore, GmMYB14 was shown to directly bind to the promoter of GmBEN1 and up-regulate its expression, leading to reduced BR content in GmMYB14-OX plants. GmMYB14-OX plants also displayed improved drought tolerance under field conditions. GmBEN1 expression was also up-regulated in the leaves of GmMYB14-OX plants under polyethylene glycol treatment, indicating that the GmBEN1-mediated reduction in BR level under stress also contributed to drought/osmotic stress tolerance of the transgenic plants. Our findings provided a strategy for stably increasing high-density yield and drought tolerance in soybean using a single TF-encoding gene.

Genome-Wide Investigation and Expression Profiling Under Abiotic Stresses of a Soybean Unknown Function (DUF21) and Cystathionine-ß-Synthase (CBS) Domain-Containing Protein Family.

Cystathionine-ß-synthase (CBS) domain-containing proteins (CDCPs) constitute a large family in plants, and members of this family have been implicated in a variety of biological processes. However, the precise functions and the underlying mechanisms of most members of this family in plants remain to be elucidated. CBSDUF proteins belong to the CDCP superfamily, which contains one domain of unknown function (DUF21) and an N terminus that is adjacent to two intracellular CBS domains. In this study, a comprehensive genome database analysis of soybean was performed to investigate the role(s) of these CBSDUFs and to explore their nomenclature, classification, chromosomal distribution, exon-intron organization, protein structure, and phylogenetic relationships; the analysis identified a total of 18 putative CBSDUF genes. Using specific protein domains and phylogenetic analysis, the CBSDUF gene family was subdivided into eight groups. The soybean CBSDUF genes showed an uneven distribution on 12 chromosomes of Glycine max. RNA-seq transcriptome data from different tissues in public databases revealed tissue-specific and differential expression profiles of the GmCBSDUFs, and qPCR analysis revealed that certain groups of soybean CBSDUFs are likely involved in specific stress responses. In addition, GmCBSDUF3 transgenic Arabidopsis was subjected to phenotypic analysis under NaCl, PEG, and ABA stress treatments. The overexpression of GmCBSDUF3 could enhance tolerance to drought and salt stress in Arabidopsis. This study presents a first comprehensive look at soybean CBSDUF proteins and provides valuable resources for functionally elucidating this protein subgroup within the CBS domain-containing protein family.

Genome-Wide Identification and Characterization of Soybean GmLOR Gene Family and Expression Analysis in Response to Abiotic Stresses.

The LOR (LURP-one related) family genes encode proteins containing a conserved LOR domain. Several members of the LOR family genes are required for defense against Hyaloperonospora parasitica (Hpa) in Arabidopsis. However, there are few reports of LOR genes in response to abiotic stresses in plants. In this study, a genome-wide survey and expression levels in response to abiotic stresses of 36 LOR genes from Glycine max were conducted. The results indicated that the GmLOR gene family was divided into eight subgroups, distributed on 14 chromosomes. A majority of members contained three extremely conservative motifs. There were four pairs of tandem duplicated GmLORs and nineteen pairs of segmental duplicated genes identified, which led to the expansion of the number of GmLOR genes. The expansion patterns of the GmLOR family were mainly segmental duplication. A heatmap of soybean LOR family genes showed that 36 GmLOR genes exhibited various expression patterns in different tissues. The cis-acting elements in promoter regions of GmLORs include abiotic stress-responsive elements, such as dehydration-responsive elements and drought-inducible elements. Real-time quantitative PCR was used to detect the expression level of GmLOR genes, and most of them were expressed in the leaf or root except that GmLOR6 was induced by osmotic and salt stresses. Moreover, GmLOR4/10/14/19 were significantly upregulated after PEG and salt treatments, indicating important roles in the improvement of plant tolerance to abiotic stress. Overall, our study provides a foundation for future investigations of GmLOR gene functions in soybean.

Identification of soybean drought-tolerant genotypes and loci correlated with agronomic traits contributes new candidate genes for breeding.

KEY MESSAGE: Drought tolerance level of 136 soybean genotypes, the correlations among traits were evaluated, and several important drought-tolerant genotypes, traits, SNPs and genes were possibly useful for soybean genetic breeding. Drought is an adverse environmental factor affecting crops growth, development, and yield. Promising genotypes and genes with improved tolerance to drought are probably effective ways to alleviate the situation. In this study, our main task was to determine drought tolerance level of 136 soybean genotypes, the correlations among physiological and agronomic traits under drought, and drought-tolerant single nucleotide polymorphism (SNPs) and genes. In this study, twenty-six varieties were identified as excellent tolerant genotypes to stress among which S14, S93 and S135 with high drought-tolerant index (DTI > 1.3) and yield (Y > 300 kg). Fourteen varieties were identified as drought-sensitive genotypes, such as S25, S45 and S58, with low drought-tolerant index (DTI < 0.5). 422 SNPs and 302 genes correlated with seed number per plant (SNPP), maturity (M), number of seeds per pod (NSPP), node number of main stem (NNMS), Stem diameter (SD) and pull stem (PS) were detected under well-watered and drought conditions by genome-wide association study (GWAS). Among them, we found SNPs (Chr 3:1758920-1958934) between drought-tolerant and sensitive genotypes. 13 genes (Glyma.03G017800, Glyma.03G018000, Glyma.03G018200, Glyma.03G018400, Glyma.03G018500, Glyma.03G018600, Glyma.03G018700, Glyma.03G018800, Glyma.03G018900, Glyma.03G019000, Glyma.03G019100, Glyma.03G019200, Glyma.03G019300) correlated with NNMS were detected. By qRT-PCR, the expression level of Glyma.03G018000 and Glyma.03G018900 in drought-tolerant varieties was significantly increased, but low or no expression in sensitive varieties under drought stress. This study provides important drought-tolerant genotypes, traits, SNPs and potential genes, possibly useful for soybean genetic breeding.

Genome-wide survey of soybean papain-like cysteine proteases and their expression analysis in root nodule symbiosis.

BACKGROUND: Plant papain-like cysteine proteases (PLCPs) are a large class of proteolytic enzymes and play important roles in root nodule symbiosis (RNS), while the whole-genome studies of PLCP family genes in legume are quite limited, and the roles of Glycine max PLCPs (GmPLCPs) in nodulation, nodule development and senescence are not fully understood. RESULTS: In the present study, we identified 97 GmPLCPs and performed a genome-wide survey to explore the expansion of soybean PLCP family genes and their relationships to RNS. Nineteen paralogous pairs of genomic segments, consisting of 77 GmPLCPs, formed by whole-genome duplication (WGD) events were identified, showing a high degree of complexity in duplication. Phylogenetic analysis among different species showed that the lineage differentiation of GmPLCPs occurred after family expansion, and large tandem repeat segment were specifically in soybean. The expression patterns of GmPLCPs in symbiosis-related tissues and nodules identified RNS-related GmPLCPs and provided insights into their putative symbiotic functions in soybean. The symbiotic function analyses showed that a RNS-related GmPLCP gene (Glyma.04G190700) really participate in nodulation and nodule development. CONCLUSIONS: Our findings improved our understanding of the functional diversity of legume PLCP family genes, and provided insights into the putative roles of the legume PLCPs in nodulation, nodule development and senescence.

CRISPR/Cas9-mediated targeted mutagenesis of GmSPL9 genes alters plant architecture in soybean.

BACKGROUND: The plant architecture has significant effects on grain yield of various crops, including soybean (Glycine max), but the knowledge on optimization of plant architecture in order to increase yield potential is still limited. Recently, CRISPR/Cas9 system has revolutionized genome editing, and has been widely utilized to edit the genomes of a diverse range of crop plants. RESULTS: In the present study, we employed the CRISPR/Cas9 system to mutate four genes encoding SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors of the SPL9 family in soybean. These four GmSPL9 genes are negatively regulated by GmmiR156b, a target for the improvement of soybean plant architecture and yields. The soybean Williams 82 was transformed with the binary CRISPR/Cas9 plasmid, assembled with four sgRNA expression cassettes driven by the Arabidopsis thaliana U3 or U6 promoter, targeting different sites of these four SPL9 genes via Agrobacterium tumefaciens-mediated transformation. A 1-bp deletion was detected in one target site of the GmSPL9a and one target site of the GmSPL9b, respectively, by DNA sequencing analysis of two T0-generation plants. T2-generation spl9a and spl9b homozygous single mutants exhibited no obvious phenotype changes; but the T2 double homozygous mutant spl9a/spl9b possessed shorter plastochron length. In T4 generation, higher-order mutant plants carrying various combinations of mutations showed increased node number on the main stem and branch number, consequently increased total node number per plants at different levels. In addition, the expression levels of the examined GmSPL9 genes were higher in the spl9b-1 single mutant than wild-type plants, which might suggest a feedback regulation on the expression of the investigated GmSPL9 genes in soybean. CONCLUSIONS: Our results showed that CRISPR/Cas9-mediated targeted mutagenesis of four GmSPL9 genes in different combinations altered plant architecture in soybean. The findings demonstrated that GmSPL9a, GmSPL9b, GmSPL9c and GmSPL9 function as redundant transcription factors in regulating plant architecture in soybean.

Identification and Comparative Analysis of CBS Domain-Containing Proteins in Soybean (Glycine max) and the Primary Function of GmCBS21 in Enhanced Tolerance to Low Nitrogen Stress.

Nitrogen is an important macronutrient required for plant growth, and is a limiting factor for crop productivity. Improving the nitrogen use efficiency (NUE) is therefore crucial. At present, the NUE mechanism is unclear and information on the genes associated with NUE in soybeans is lacking. cystathionine beta synthase (CBS) domain-containing proteins (CDCPs) may be implicated in abiotic stress tolerance in plants. We identified and classified a CBS domain-containing protein superfamily in soybean. A candidate gene for NUE, GmCBS21, was identified. GmCBS21 gene characteristics, the temporal expression pattern of the GmCBS21 gene, and the phenotype of GmCBS21 overexpression in transgenic Arabidopsis thaliana under low nitrogen stress were analyzed. The phenotypes suggested that the transgenic Arabidopsis thaliana seedlings performed better under the nitrogen-deficient condition. GmCBS21-overexpressing transgenic plants exhibit higher low nitrogen stress tolerance than WT plants, and this suggests its role in low nitrogen stress tolerance in plants. We conclude that GmCBS21 may serve as an excellent candidate for breeding crops with enhanced NUE and better yield.

Genetic analysis and molecular mapping of resistance gene to Phakopsora pachyrhizi in soybean germplasm SX6907.

KEY MESSAGE: In this study, Rpp6907, a novel resistance gene/allele to Phakopsora pachyrhizi in soybean, was mapped in a 111.9-kb region, including three NBS-LRR type predicted genes, on chromosome 18. Soybean rust caused by Phakopsora pachyrhizi Sydow has been reported in numerous soybean-growing regions worldwide. The development of rust-resistant varieties is the most economical and environmentally safe method to control the disease. The Chinese soybean germplasm SX6907 is resistant to P. pachyrhizi and exhibits immune reaction compared with the known Rpp genes. These characteristics suggest that SX6907 may carry at least one novel Rpp gene/allele. Three F2 populations from the crosses of SX6907 (resistant) and Tianlong 1, Zhongdou40, and Pudou11 (susceptible) were used to map the Rpp gene. Three resistance responses (immune, red-brown, and tan-colored lesion) were observed from the F2 individuals. The segregation follows a ratio of 1(resistance):2(heterozygous):1(susceptible), indicating that the resistance in SX6907 is controlled by a single incomplete dominant gene (designated as Rpp6907). Results showed that Rpp6907 was mapped on soybean chromosome 18 (molecular linkage group G, MLG G) flanked by simple sequence repeat (SSR) markers SSR24 and SSR40 at a distance of 111.9 kb. Among the ten genes marked within this 111.9-kb region between the two markers, three genes (Glyma18g51930, Glyma18g51950, and Glyma18g51960) are nucleotide-binding site and leucine-rich repeat-type genes. These genes may be involved in recognizing the presence of pathogens and ultimately conferring resistance. Based on resistance spectrum analysis and mapping results, we inferred that Rpp6907 is a novel gene different from Rpp1 in PI 200492, PI 561356, PI 587880A, PI 587886, and PI 594538A, or a new Rpp1-b allele.

ZnGa2O4 nanorod arrays decorated with Ag nanoparticles as surface-enhanced Raman-scattering substrates for melamine detection.

The availability of sensitive, reproducible, and stable substrates is critically important for surface-enhanced Raman spectroscopy (SERS)-based applications, but it presently remains a challenge. In this work, well-aligned zinc gallate (ZnGa2O4) nanorod arrays grown on a Si substrate by chemical vapor deposition were used as templates to fabricate SERS substrates by deposition of Ag nanoparticles onto the ZnGa2O4 nanorod surfaces. The coverage of the Ag nanoparticles on the ZnGa2O4 nanorod surfaces was easily controlled by varying the amount of AgNO3. SERS measurements showed that the number density of Ag nanoparticles on the ZnGa2O4 nanorod surfaces had a great effect on SERS activity. The SERS signals collected by point-to-point and SERS mapping images showed that as-prepared SERS substrates exhibited good spatial uniformity and reproducibility. Detection of melamine molecules at low concentrations (1.0×10(-7) M) was used as an example to show the possible application of such a substrate. In addition, the effect of benzoic acid on the detection of melamine was also investigated. It was found that the SERS signal intensity of melamine decreased greatly as the concentration of benzoic acid was increased.

Ganoderic acid D attenuates gemcitabine resistance of triple-negative breast cancer cells by inhibiting glycolysis via HIF-1α destabilization.

BACKGROUND: Gemcitabine (GEM) resistance is the primary reason why combination chemotherapy is limited in triple-negative breast cancer (TNBC). Ganoderic acid D (GAD), a natural triterpenoid compound obtained from Ganoderma lucidum, has been shown to have antitumor activities. However, whether GAD can reverse GEM resistance in TNBC requires further investigation. PURPOSE: This study investigated whether and how GAD could reverse GEM resistance in TNBC as an antitumor adjuvant. METHODS: The effects of GAD on cell proliferation, cell cycle, and glycolysis were studied in vitro using a GEM-resistant (GEM-R) TNBC cell model. We enriched key pathways affected by GAD using proteomics techniques. Western blotting and qPCR were used to detect the expression of glycolysis-related genes after GAD treatment. A mouse resistance model was established using GEM-R TNBC cells, and hematoxylin-eosin staining and immunohistochemistry were used to assess the role of GAD in reversing resistance in vivo. RESULTS: Cellular functional assays showed that GAD significantly inhibited proliferation and glucose uptake in GEM-R TNBC cells. GAD reduces HIF-1α accumulation in TNBC cells under hypoxic conditions through the ubiquitinated protease degradation pathway. Mechanistically, GAD activates the p53/MDM2 pathway, promoting HIF-1α ubiquitination and proteasomal degradation and downregulating HIF-1α-dependent glycolysis genes like GLUT1, HK2, and PKM2. Notably, GAD combined with gemcitabine significantly reduced the growth of GEM-R TNBC cells in a subcutaneous tumor model. CONCLUSIONS: This study reveals a novel antitumor function of GAD, which inhibits glycolysis by promoting HIF-1α degradation in GEM-R TNBC cells, offering a promising therapeutic strategy for TNBC patients with GEM resistance.

Comparative genomic and transcriptomic analyses provide new insight into symbiotic host specificity.

Host specificity plays important roles in expanding the host range of rhizobia, while the genetic information responsible for host specificity remains largely unexplored. In this report, the roots of four symbiotic systems with notable different symbiotic phenotypes and the control were studied at four different post-inoculation time points by RNA sequencning (RNA-seq). The differentially expressed genes (DEGs) were divided into "found only in soybean or Lotus," "only expressed in soybean or Lotus," and "expressed in both hosts" according to the comparative genomic analysis. The distributions of enriched function ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways vary significantly in different symbiotic systems. Host specific genes account for the majority of the DEGs involved in response to stimulus, associated with plant-pathogen interaction pathways, and encoding resistance (R) proteins, the symbiotic nitrogen fixation (SNF) proteins and the target proteins in the SNF-related modules. Our findings provided molecular candidates for better understanding the mechanisms of symbiotic host-specificity.

An pair of an atypical NLR encoding genes confer Asian soybean rust resistance in soybean.

Asian soybean rust (ASR), caused by Phakopsora pachyrhizi, is a devastating disease that is present in all major soybean-producing regions. The limited availability of resistant germplasm has resulted in a scarcity of commercial soybean cultivars that are resistant to the disease. To date, only the Chinese soybean landrace SX6907 has demonstrated an immune response to ASR. In this study, we present the isolation and characterization of Rpp6907-7 and Rpp6907-4, a gene pair that confer broad-spectrum resistance to ASR. Rpp6907-7 and Rpp6907-4 encode atypic nucleotide-binding leucine-rich repeat (NLR) proteins that are found to be required for NLR-mediated immunity. Genetic analysis shows that only Rpp6907-7 confers resistance, while Rpp6907-4 regulates Rpp6907-7 signaling activity by acting as a repressor in the absence of recognized effectors. Our work highlights the potential value of using Rpp6907 in developing resistant soybean cultivars.

A Mild Hyperthermia Hollow Carbon Nanozyme as Pyroptosis Inducer for Boosted Antitumor Immunity.

The immune checkpoint blockade (ICB) antibody immunotherapy has demonstrated clinical benefits for multiple cancers. However, the efficacy of immunotherapy in tumors is suppressed by deficient tumor immunogenicity and immunosuppressive tumor microenvironments. Pyroptosis, a form of programmed cell death, can release tumor antigens, activate effective tumor immunogenicity, and improve the efficiency of ICB, but efficient pyroptosis for tumor treatment is currently limited. Herein, we show a mild hyperthermia-enhanced pyroptosis-mediated immunotherapy based on hollow carbon nanozyme, which can specifically amplify oxidative stress-triggered pyroptosis and synchronously magnify pyroptosis-mediated anticancer responses in the tumor microenvironment. The hollow carbon sphere modified with iron and copper atoms (HCS-FeCu) with multiple enzyme-mimicking activities has been engineered to induce cell pyroptosis via the radical oxygen species (ROS)-Tom20-Bax-Caspase 3-gasdermin E (GSDME) signaling pathway under light activation. Both in vitro and in vivo antineoplastic results confirm the superiority of HCS-FeCu nanozyme-induced pyroptosis. Moreover, the mild photothermal-activated pyroptosis combining anti-PD-1 can enhance antitumor immunotherapy. Theoretical calculations further indicate that the mild photothermal stimulation generates high-energy electrons and enhances the interaction between the HCS-FeCu surface and adsorbed oxygen, facilitating molecular oxygen activation, which improves the ROS production efficiency. This work presents an approach that effectively transforms immunologically "cold" tumors into "hot" ones, with significant implications for clinical immunotherapy.

Heterologous expression of the Glycine soja Kunitz-type protease inhibitor GsKTI improves resistance to drought stress and Helicoverpa armigera in transgenic Arabidopsis lines.

Kunitz-like protease inhibitors (KTIs) have been identified to play critical roles in insect defense, but evidence for their involvement in drought stress is sparse. The aim of this study was to identify and functionally characterize a Kunitz-like protease inhibitor, GsKTI, from the wild soybean (Glycine soja) variety ED059. Expression patterns suggest that drought stress and insect herbivory may induce GsKTI transcript levels. Transgenic Arabidopsis lines overexpressing GsKTI have been shown to exhibit enhanced drought tolerance by regulating the ABA signaling pathway and increasing xylem cell number. Transgenic Arabidopsis leaves overexpressing GsKTI interfered with insect digestion and thus had a negative effect on the growth of Helicoverpa armigera. It is concluded that GsKTI increases resistance to drought stress and insect attack in transgenic Arabidopsis lines.

Roles for a soybean RAV-like orthologue in shoot regeneration and photoperiodicity inferred from transgenic plants.

The soybean gene Glyma10g34760 appears to encode a RAV2-like transcription factor orthologue (DQ147914; hereafter GmRAV) based on sequence similarity. The gene is a member of the ERF/AP2 transcription factor family that has been shown to be increased in transcript abundance by cytokinins (CKs). Transgenic GmRAV-overexpressing (-ox) tobacco plants exhibited increased CK signalling-related phenotypes including dwarfism, reduced apical dominance, extreme longevity, vigorous outgrowth of lateral buds, small and dark green leaves, reduced root growth, repressed flowering under both long- and short-day conditions, and altered sensitivity to daylength. In contrast, inhibition (-i) of GmRAV in soybean displayed the opposite phenotypic alterations which were consistent with defects in CK signalling. Phenotypes included earlier time of emergence; reduced numbers of branches, leaves, and flower buds; increased plant height; increased apical dominance; and earlier flowering and maturity. GmRAV-i soybean was less sensitive to cytokinin in hypocotyls and root growth inhibition assays. GmRAV-i soybean showed decreased frequency of adventious shoot formation in tissue culture in the presence of CKs, which might be attributed to the significantly decreased activities of CUC2, STM, and WUS involved in shoot meristem specification. GmRAV protein was localized in the nucleus in leaves. The GmRAV promoter-ß-glucuronidase (GUS) fusion was largely expressed in a meristematic region of the shoot apex, which was consistent with expressed sequence tag and microarray data. GmRAV was inferred to play a key role in CK and photoperiod signalling that subsequently regulated plant development.