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Single-cell RNA-seq (scRNA-seq) has been highlighted as a powerful tool for the description of human cell transcriptome, but the technology has not been broadly applied in plant cells. Herein, we describe the successful development of a robust protoplast cell isolation system in the peanut leaf. A total of 6,815 single cells were divided into eight cell clusters based on reported marker genes by applying scRNA-seq. Further, a pseudo-time analysis was used to describe the developmental trajectory and interaction network of transcription factors (TFs) of distinct cell types during leaf growth. The trajectory enabled re-investigation of the primordium-driven development processes of the mesophyll and epidermis. These results suggest that palisade cells likely differentiate into spongy cells, while the epidermal cells originated earlier than the primordium. Subsequently, the developed method integrated multiple technologies to efficiently validate the scRNA-seq result in a homogenous cell population. The expression levels of several TFs were strongly correlated with epidermal ontogeny in accordance with obtained scRNA-seq values. Additionally, peanut AHL23 (AT-HOOK MOTIF NUCLEAR LOCALIZED PROTEIN 23), which is localized in nucleus, promoted leaf growth when ectopically expressed in Arabidopsis by modulating the phytohormone pathway. Together, our study displays that application of scRNA-seq can provide new hypotheses regarding cell differentiation in the leaf blade of Arachis hypogaea. We believe that this approach will enable significant advances in the functional study of leaf blade cells in the allotetraploid peanut and other plant species.

Genome editing, a powerful technology for precise manipulation of DNA sequences, has revolutionized the field of agricultural biotechnology. In recent years, there has been increasing interest in applying genome editing techniques to improve important crop plants, such as peanut ( Arachis hypogaea ). Peanuts are a vital source of oil and protein, and they play a crucial role in global food security. However, peanut crops face numerous challenges, including susceptibility to diseases, pests, and environmental stressors. The advent of clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing tools has provided researchers with a rapid, efficient, and precise method to edit the peanut genome. Despite being a polyploid crop, several successful applications of genome editing in peanuts have been reported. For instance, CRISPR/Cas9-mediated gene editing has been used to increase oleic acid content in oil and allergen reduction in peanut varieties through precise genome modifications. However, despite these advancements, challenges remain in the widespread adoption of genome editing in peanuts, off-target effects, and unintended consequences. Advancements in CRISPR-based genome editing holds great promise for the improvement of peanuts by addressing its various prospective traits like phytate reduction, fresh seed dormancy, aflatoxin resistance and abiotic stress tolerance. However, careful consideration of issues, such as safety assessment, and public acceptance, is essential for the successful application and commercialization of genome-edited peanut varieties. Future research and collaborations are needed to overcome these challenges and fully harness the potential of genome editing in improving peanut crops for sustainable agriculture and global food security. Graphical abstract

Nanomaterials are used in practically every aspect of modern life, including agriculture. The aim of this study was to evaluate the effectiveness of iron oxide nanoparticles (Fe2O3 NPs) as a fertilizer to replace traditional Fe fertilizers, which have various shortcomings. The effects of the Fe2O3 NPs and a chelated-Fe fertilizer (ethylenediaminetetraacetic acid-Fe; EDTA-Fe) fertilizer on the growth and development of peanut (Arachis hypogaea), a crop that is very sensitive to Fe deficiency, were studied in a pot experiment. The results showed that Fe2O3 NPs increased root length, plant height, biomass, and SPAD values of peanut plants. The Fe2O3 NPs promoted the growth of peanut by regulating phytohormone contents and antioxidant enzyme activity. The Fe contents in peanut plants with Fe2O3 NPs and EDTA-Fe treatments were higher than the control group. We used energy dispersive X-ray spectroscopy (EDS) to quantitatively analyze Fe in the soil. Peanut is usually cultivated in sandy soil, which is readily leached of fertilizers. However, the Fe2O3 NPs adsorbed onto sandy soil and improved the availability of Fe to the plants. Together, these results show that Fe2O3 NPs can replace traditional Fe fertilizers in the cultivation of peanut plants. To the best of our knowledge, this is the first research on the Fe2O3 NPs as the iron fertilizer.

Key messageA first creation of high oleic acid peanut varieties by using transcription activator-like effecter nucleases (TALENs) mediated targeted mutagenesis of Fatty Acid Desaturase 2 (FAD2).Transcription activator like effector nucleases (TALENs), which allow the precise editing of DNA, have already been developed and applied for genome engineering in diverse organisms. However, they are scarcely used in higher plant study and crop improvement, especially in allopolyploid plants. In the present study, we aimed to create targeted mutagenesis by TALENs in peanut. Targeted mutations in the conserved coding sequence of Arachis hypogaea fatty acid desaturase 2 (AhFAD2) were created by TALENs. Genetic stability of AhFAD2 mutations was identified by DNA sequencing in up to 9.52 and 4.11% of the regeneration plants at two different targeted sites, respectively. Mutation frequencies among AhFAD2 mutant lines were significantly correlated to oleic acid accumulation. Genetically, stable individuals of positive mutant lines displayed a 0.5–2 fold increase in the oleic acid content compared with non-transgenic controls. This finding suggested that TALEN-mediated targeted mutagenesis could increase the oleic acid content in edible peanut oil. Furthermore, this was the first report on peanut genome editing event, and the obtained high oleic mutants could serve for peanut breeding project.

Significance A great challenge for humanity is feeding its growing population while minimizing ecosystem damage and climate change. Here, we uncover the global benefits arising from the introduction of one wild species accession to peanut-breeding programs decades ago. This work emphasizes the importance of biodiversity to crop improvement: peanut cultivars with genetics from this wild accession provided improved food security and reduced use of fungicide sprays. However, this study also highlights the perilous consequences of changes in legal frameworks and attitudes concerning biodiversity. These changes have greatly reduced the botanical collections, seed exchanges, and international collaborations which are essential for the continued diversification of crop genetics and, consequently, the long-term resilience of crops against evolving pests and pathogens and changing climate.

Background Intercropping, an essential cultivation pattern in modern agricultural systems, increases crop yields and soil quality. Cassava and peanut intercropping systems exhibit advantages in solar utilization and cadmium absorption, etc. However, the inner mechanisms need to be elucidated. In this study, Illumina MiSeq platform was used to reveal the rhizospheric microbes and soil quality in cassava/peanut intercropping systems, and the results provided a reference for the application of this method in studying other intercropping systems. Results Both intercropping cassava/peanut (IP) and intercropping peanut/cassava (IC) systems significantly increased available N, available K, pH value, and urease activity, comparing with that in monocropping cassava (MC) and monocropping peanut (MP) system. However, there were few effects on the total N, total P, total K, available P, organic matter, protease activity, catalase activity, sucrase activity, and acid phosphatase activity. Both IP and MP soils contained more bacteria and fungi than those in the IC and MC soils, which were mainly made of Proteobacteria and Actinobacteria. Intercropping remarkably increased the number of Nitrospirae in IP and IC soils comparing those in MC and MP soils. Redundancy analysis (RDA) revealed that the abundances of DA101 , Pilimelia , and Ramlibacter were positively correlated to the soil quality. These results suggest that intercropping enhances the available nitrogen content of soil through increasing the quantity of rhizospheric microbes, especially that of DA101 and Pilimelia . Conclusions The cassava/peanut intercropping system improves soil quality through increasing the available nitrogen content and abundance of DA101 , Pilimelia , and Ramlibacter in the soil.

Background Peanut is an important legume crop growing worldwide. With the published allotetraploid genomes, further functional studies of the genes in peanut are very critical for crop improvement. CRISPR/Cas9 system is emerging as a robust tool for gene functional study and crop improvement, which haven’t been extensively utilized in peanut yet. Peanut plant forms root nodules to fix nitrogen through a symbiotic relationship with rhizobia. In model legumes, the response of plants to rhizobia is initiated by Nod factor receptors (NFRs). However, information about the function of NFRs in peanut is still limited. In this study, we applied the CRISPR/Cas9 tool in peanut hairy root transformation system to explore the function of NFR genes. Results We firstly identified four AhNFR1 genes and two AhNFR5 genes in cultivated peanut ( Tifrunner ). The gene expression analysis showed that the two AhNFR1 and two AhNFR5 genes had high expression levels in nodulating (Nod+) line E5 compared with non-nodulating (Nod-) line E4 during the process of nodule formation, suggesting their roles in peanut nodulation. To further explore their functions in peanut nodulation, we applied CRISPR technology to create knock-out mutants of AhNFR1 and AhNFR5 genes using hairy root transformation system. The sequencing of these genes in transgenic hairy roots showed that the selected AhNFR1 and AhNFR5 genes were successfully edited by the CRISPR system, demonstrating its efficacy for targeted mutation in allotetraploid peanut. The mutants with editing in the two AhNFR5 genes showed Nod- phenotype, whereas mutants with editing in the two selected AhNFR1 genes could still form nodules after rhizobia inoculation. Conclusions This study showed that CRISPR-Cas9 could be used in peanut hairy root transformation system for peanut functional genomic studies, specifically on the gene function in roots. By using CRISPR-Cas9 targeting peanut AhNFR genes in hairy root transformation system, we validated the function of AhNFR5 genes in nodule formation in peanut.

Summary Cultivated peanut (Arachis hypogaea L.) is an important oil and cash crop. Pod size is one of the major traits determining yield and commodity characteristic of peanut. Fine mapping of quantitative trait locus (QTL) and identification of candidate genes associated with pod size are essential for genetic improvement and molecular breeding of peanut varieties. In this study, a major QTL related to pod size, qAHPS07, was fine mapped to a 36.46 kb interval on chromosome A07 using F2, recombinant inbred line (RIL) and secondary F2 populations. qAHPS07 explained 38.6%, 23.35%, 37.48%, 25.94% of the phenotypic variation for single pod weight (SPW), pod length (PL), pod width (PW) and pod shell thickness (PST), respectively. Whole genome resequencing and gene expression analysis revealed that a RuvB‐like 2 protein coding gene AhRUVBL2 was the most likely candidate for qAHPS07. Overexpression of AhRUVBL2 in Arabidopsis led to larger seeds and plants than the wild type. AhRUVBL2‐silenced peanut seedlings represented small leaves and shorter main stems. Three haplotypes were identified according to three SNPs in the promoter of AhRUVBL2 among 119 peanut accessions. Among them, SPW, PW and PST of accessions carrying Hap_ATT represent 17.6%, 11.2% and 26.3% higher than those carrying Hap_GAC，respectively. In addition, a functional marker of AhRUVBL2 was developed. Taken together, our study identified a key functional gene of peanut pod size, which provides new insights into peanut pod size regulation mechanism and offers practicable markers for the genetic improvement of pod size‐related traits in peanut breeding.

Intercropping systems have been studied as a sustainable agricultural planting pattern to increase soil quality and crop yields. However, the relationships between metabolites and soil physicochemical properties remain poorly understood under sugarcane/peanut intercropping system. Thus, we determined the rhizosphere soil physicochemical properties, and analyzed rhizosphere soil metabolites and root metabolites by metabolomics method under monoculture and intercropping patterns of sugarcane and peanut. The results showed that pH, the contents of total phosphorus (P), total potassium (K), available nitrogen (N), available phosphorus (P), and available potassium (K) were higher in rhizosphere soil of intercropping peanut than monoculture peanut, and the content of total P was higher in rhizosphere soil of intercropping sugarcane than monoculture sugarcane. Sugarcane/peanut intercropping also significantly increased the activities of acid phosphatase and urease in rhizosphere soil. The metabolomics results showed that 32 metabolites, mainly organic acids and their derivatives (25.00%), nucleotides and their metabolites (18.75%), were detected in root and rhizosphere soil samples. In the MP-S (rhizosphere soil of monoculture peanut) vs. IP-S (rhizosphere soil of intercropping peanut) comparison, 47 differential metabolites (42 upregulated) were screened, including glycerolipids (19.15%), organic acids and their derivatives (17.89%), and amino acids and their metabolites (12.77%). In the MS-S (rhizosphere soil of monoculture sugarcane) vs. IS-S (rhizosphere soil of intercropping sugarcane) comparison, 51 differential metabolites (26 upregulated) were screened, including heterocyclic compounds (15.69%), glycerolipids (11.76%), and organic acids and their derivatives (9.80%). The metabolite species from MP-S, MS-S, IP-S, and IS-S were similar, but some metabolite contents were significantly different, such as adenine, adenosine, maltotriose, thermozeaxanthin-13 and PE-NMe (20:0/24:0). Adenine and adenosine were detected in root and rhizosphere soils, and their levels were increased in the intercropping treatment, which were mainly related to enhanced purine metabolism in root and rhizosphere soils under the sugarcane/peanut intercropping system. Importantly, adenine and adenosine were significantly positively correlated with total P and total K contents, acid phosphatase and urease activities, and pH. This study clarified that the sugarcane/peanut intercropping system could improve soil nutrients and enzymes and was related to purine metabolism.

Drought is a major environmental constraint limiting global peanut productivity. Wild peanut species, characterized by greater genetic diversity, represent valuable resources for improving drought resilience in cultivated peanut. However, the molecular mechanisms underpinning drought tolerance in wild peanut species remain largely unexplored. This study evaluated the drought tolerance of three wild-type peanut accessions from two different species, Arachis dardani GK12946, Arachis dardani V7215, and Arachis ipaënsis K30076. Physiological measurements such as fresh weight and dry weight revealed statistically non-significant differences between drought-stressed and well-watered conditions, indicating strong inherent drought tolerance. Transcriptome analysis revealed that 3272, 3648, and 1181 genes in leaf samples of A. dardani GK12946, A. dardani V7215, and A. ipaënsis K30076 were differentially expressed, respectively. In root samples, 3014, 3472, and 2033 genes were differentially expressed in the same accessions. Notably, differentially expressed genes (DEGs) and set intersection (Venn) analysis suggests A. dardani V7215 exhibited the highest number of DEGs (1155) uniquely expressed in leaves, and 899 DEGs uniquely expressed in roots, suggesting accession-specific gene expression. Gene Ontology enrichment revealed that upregulated genes were associated with abiotic stress responses, temperature stimulus, heat stress, and DNA-binding transcription factor activity. Co-expression network analysis using WGCNA identified key drought-responsive modules, enriched for GO terms like stress regulation, protein folding, as well as GST family amino acid metabolic processes. Overall, this study provides comprehensive insights into the molecular basis of drought tolerance in wild peanut accessions. Our findings establish a valuable resource for functional genomics and crop improvement under water-limited conditions.