Search

Summary 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.

The far-red-impaired response 1 (FAR1) transcription family were initially identified as important factors for phytochrome A (phyA)-mediated far-red light signaling in Arabidopsis; they play crucial roles in controlling the growth and development of plants. The reported reference genome sequences of Arachis, including A. duranensis, A. ipaensis, A. monticola, and A. hypogaea, and its related species Glycine max provide an opportunity to systematically perform a genome-wide identification of FAR1 homologous genes and investigate expression patterns of these members in peanut species. Here, a total of 650 FAR1 genes were identified from four Aarchis and its closely related species G. max. Of the studied species, A. hypogaea contained the most (246) AhFAR1 genes, which can be classified into three subgroups based on phylogenic relationships. The synonymous (Ks) and non-synonymous (Ka) substitution rates, phylogenetic relationship and synteny analysis of the FAR1 family provided deep insight into polyploidization, evolution and domestication of peanut AhFAR1 genes. The transcriptome data showed that the AhFAR1 genes exhibited distinct tissue- and stage-specific expression patterns in peanut. Three candidate genes including Ahy_A10g049543, Ahy_A06g026579, and Ahy_A10g048401, specifically expressed in peg and pod, might participate in pod development in the peanut. The quantitative real-time PCR (qRT-PCR) analyses confirmed that the three selected genes were highly and specifically expressed in the peg and pod. This study systematically analyzed gene structure, evolutionary characteristics and expression patterns of FAR1 gene family, which will provide a foundation for the study of genetic and biological function in the future.

Cultivated peanut (Arachis hypogaea) is an allotetraploid crop planted in Asia, Africa, and America for edible oil and protein. To explore the origins and consequences of tetraploidy, we sequenced the allotetraploid A. hypogaea genome and compared it with the related diploid Arachis duranensis and Arachis ipaensis genomes. We annotated 39 888 A-subgenome genes and 41 526 B-subgenome genes in allotetraploid peanut. The A. hypogaea subgenomes have evolved asymmetrically, with the B subgenome resembling the ancestral state and the A subgenome undergoing more gene disruption, loss, conversion, and transposable element proliferation, and having reduced gene expression during seed development despite lacking genome-wide expression dominance. Genomic and transcriptomic analyses identified more than 2 500 oil metabolism-related genes and revealed that most of them show altered expression early in seed development while their expression ceases during desiccation, presenting a comprehensive map of peanut lipid biosynthesis. The availability of these genomic resources will facilitate a better understanding of the complex genome architecture, agronomically and economically important genes, and genetic improvement of peanut.

In order to obtain more valuable insights into the protein dynamics and accumulation of allergens in seeds during underground development, we performed a proteomic study on developing peanut seeds at seven different stages. A total of 264 proteins with altered abundance and contained at least one unique peptide was detected by matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS). All identified proteins were classified into five functional categories as level 1 and 20 secondary functional categories as level 2. Among them, 88 identified proteins (IPs) were related to carbohydrate/ amino acid/ lipid transport and metabolism, indicating that carbohydrate/amino acid/ lipid metabolism played a key role in the underground development of peanut seeds. Hierarchical cluster analysis showed that all IPs could be classified into eight cluster groups according to the abundance profiles, suggesting that the modulatory patterns of these identified proteins were complicated during seed development. The largest group contained 41 IPs, the expression of which decreased at R 2 and reached a maximum at R3 but gradually decreased from R4. A total of 14 IPs were identified as allergen-like proteins by BLAST with A genome (Arachis duranensis) or B genome (Arachis ipaensis) translated allergen sequences. Abundance profile analysis of 14 identified allergens showed that the expression of all allergen proteins was low or undetectable by 2-DE at the early stages (R1 to R4), and began to accumulate from the R5 stage and gradually increased. Network analysis showed that most of the significant proteins were involved in active metabolic pathways in early development. Real time RT-PCR analysis revealed that transcriptional regulation was approximately consistent with expression at the protein level for 8 selected identified proteins. In addition, some amino acid sequences that may be associated with new allergens were also discussed.

A 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.

In the original article, there was an error. The link of sequencing data was not attached in Supplementary Material section. A correction has been made to the Supplementary Material section: The genome assembly and annotation data were deposited in the DRYAD digital repository (https://datadryad.org/). All the data can be downloaded from DRYAD by searching for doi: 10.5061/dryad.hm5vs13 or by directly accessing the link: https://doi.org/10.5061/dryad.hm5vs13. The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.

Peanut (Arachis hypogaea L.), an important leguminous crop, is widely cultivated in tropical and subtropical regions. Peanut is an allotetraploid, having A and B subgenomes that maybe have originated in its diploid progenitors Arachis duranensis (A-genome) and Arachis ipaensis (B-genome), respectively. We previously sequenced the former and here present the draft genome of the latter, expanding our knowledge of the unique biology of Arachis. The assembled genome of A. ipaensis is ~1.39 Gb with 39,704 predicted protein-encoding genes. A gene family analysis revealed that the FAR1 family may be involved in regulating peanut special fruit development. Genomic evolutionary analyses estimated that the two progenitors diverged ~3.3 million years ago and suggested that A. ipaensis experienced a whole-genome duplication event after the divergence of Glycine max. We identified a set of disease resistance-related genes and candidate genes for biological nitrogen fixation. In particular, two and four homologous genes that may be involved in the regulation of nodule development were obtained from A. ipaensis and A. duranensis, respectively. We outline a comprehensive network involved in drought adaptation. Additionally, we analyzed the metabolic pathways involved in oil biosynthesis and found genes related to fatty acid and triacylglycerol synthesis. Importantly, three new FAD2 homologous genes were identified from A. ipaensis and one was completely homologous at the amino acid level with FAD2 from A. hypogaea. The availability of the A. ipaensis and A. duranensis genomic assemblies will advance our knowledge of the peanut genome.

A characteristic feature of peanut is the subterranean fructification, geocarpy, in which the gynophore ('peg'), a specialized organ that transitions from upward growth habit to downward outgrowth upon fertilization, drives the developing pod into the soil for subsequent development underground. As a step towards understanding this phenomenon, we explore the developmental dynamics of the peanut pod transcriptome at 11 successive stages. We identified 110 217 transcripts across developmental stages and quantified their abundance along a pod developmental gradient in pod wall. We found that the majority of transcripts were differentially expressed along the developmental gradient as well as identified temporal programs of gene expression, including hundreds of transcription factors. Thought to be an adaptation to particularly harsh subterranean environments, both up- and down-regulated gene sets in pod wall were enriched for response to a broad array of stimuli, like gravity, light and subterranean environmental factors. We also identified hundreds of transcripts associated with gravitropism and photomorphogenesis, which may be involved in the geocarpy. Collectively, this study forms a transcriptional baseline for geocarpy in peanut as well as provides a considerable body of evidence that transcriptional regulation in peanut aerial and subterranean fruits is complex.

The peanut is a special plant for its aerial flowering but subterranean fructification. The failure of peg penetration into the soil leads to form aerial pod and finally seed abortion. However, the mechanism of seed abortion during aerial pod development remains obscure. Here, a comparative transcriptome analysis between aerial and subterranean pods at different developmental stages was produced using a customized NimbleGen microarray representing 36,158 unigenes. By comparing 4 consecutive time-points, totally 6,203 differentially expressed genes, 4,732 stage-specific expressed genes and 2,401 specific expressed genes only in aerial or subterranean pods were identified in this study. Functional annotation showed their mainly involvement in biosynthesis, metabolism, transcription regulation, transporting, stress response, photosynthesis, signal transduction, cell division, apoptosis, embryonic development, hormone response and light signaling, etc. Emphasis was focused on hormone response, cell apoptosis, embryonic development and light signaling relative genes. These genes might function as potential candidates to provide insights into seed abortion during aerial pod development. Ten candidate genes were validated by Real-time RT-PCR. Additionally, consistent with up-regulation of auxin response relative genes in aerial pods, endogenous IAA content was also significantly increased by HPLC analysis. This study will further provide new molecular insight that auxin and auxin response genes potentially contribute to peanut seed and pod development. Electronic supplementary material The online version of this article (doi:10.1007/s11103-014-0193-x) contains supplementary material, which is available to authorized users.

Peanut (Arachis hypogaea L.) produces flowers aerially, but the fruit develops underground. This process is mediated by the gynophore, which always grows vertically downwards. The genetic basis underlying gravitropic bending of gynophores is not well understood. To identify genes related to gynophore gravitropism, gene expression profiles of gynophores cultured in vitro with tip pointing upward (gravitropic stimulation sample) and downward (control) at both 6 and 12 h were compared through a high-density peanut microarray. After gravitropic stimulation, there were 174 differentially expressed genes, including 91 upregulated and 83 downregulated genes at 6 h, and 491 differentially expressed genes including 129 upregulated and 362 downregulated genes at 12 h. The differentially expressed genes identified were assigned to 24 functional categories. Twenty pathways including carbon fixation, aminoacyl-tRNA biosynthesis, pentose phosphate pathway, starch and sucrose metabolism were identified. The quantitative real-time PCR analysis was performed for validation of microarray results. Our study paves the way to better understand the molecular mechanisms underlying the peanut gynophore gravitropism.

Background Germin-like superfamily members are ubiquitously expressed in various plant species and play important roles in plant development and defense. Although several GLPs have been identified in peanut (Arachis hypogaea L.), their roles in development and defense remain unknown. In this research, we study the spatiotemporal expression of AhGLPs in peanut and their functions in plant defense. Results We have identified three new AhGLP members (AhGLP3b, AhGLP5b and AhGLP7b) that have distinct but very closely related DNA sequences. The spatial and temporal expression profiles revealed that each peanut GLP gene has its distinct expression pattern in various tissues and developmental stages. This suggests that these genes all have their distinct roles in peanut development. Subcellular location analysis demonstrated that AhGLP2 and 5 undergo a protein transport process after synthesis. The expression of all AhGLPs increased in responding to Aspergillus flavus infection, suggesting AhGLPs' ubiquitous roles in defense to A. flavus. Each AhGLP gene had its unique response to various abiotic stresses (including salt, H2O2 stress and wound), biotic stresses (including leaf spot, mosaic and rust) and plant hormone stimulations (including SA and ABA treatments). These results indicate that AhGLPs have their distinct roles in plant defense. Moreover, in vivo study of AhGLP transgenic Arabidopsis showed that both AhGLP2 and 3 had salt tolerance, which made transgenic Arabidopsis grow well under 100 mM NaCl stress. Conclusions For the first time, our study analyzes the AhGLP gene expression profiles in peanut and reveals their roles under various stresses. These results provide an insight into the developmental and defensive roles of GLP gene family in peanut.

Pre-harvest aflatoxin contamination caused by Aspergillus favus is a major concern in peanut. However, little is known about the resistance mechanism, so the incorporation of resistance into cultivars with commercially-acceptable genetic background has been slowed. To identify resistance-associated genes potentially underlying the resistance mechanism, we compared transcriptome profiles in resistant and susceptible peanut genotypes under three different treatments: well watered, drought stress and both A. flavus and drought stress using a customised NimbleGen microarray representing 36 158 unigenes. Results showed that the profile of differentially expressed genes (DEGs) displayed a similar pattern of distribution among the functional classes between resistant and susceptible peanuts in response to drought stress. Under A. flavus infection with drought stress, a total of 490 unigenes involved in 26 pathways were differentially expressed in the resistant genotype YJ1 uniquely responding to A. flavus infection, in which 96 DEGs were related to eight pathways: oxidation reduction, proteolysis metabolism, coenzyme A biosynthesis, defence response, signalling, oligopeptide transport, transmembrane transport and carbohydrate biosynthesis/metabolism. Pathway analysis based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database showed that eight networks were significantly associated with resistance to A. flavus infection in resistant genotype YJ1 compared with susceptible Yueyou7. To validate microarray analysis, 15 genes were randomly selected for real-time RT–PCR analysis. The results provided in this study may enhance our understanding of the pre-harvest peanut–A. flavus interaction and facilitate to develop aflatoxin resistant peanut lines in future breeding programs.