Peanut embryo development is a complex process involving a series of gene regulatory pathways and is easily affected by various elements in the soil. Calcium deficiency in the soil induces early embryo abortion in peanut, which provides an opportunity to determine the mechanism underlying this important event. MicroRNA (miRNA)-guided target gene regulation is vital to a wide variety of biological processes. However, whether miRNAs participate in peanut embryo abortion under calcium deficiency has yet to be explored. In this study, with the assistance of a recently established platform for genome sequences of wild peanut species, we analyzed small RNAs (sRNAs) in early peanut embryos. A total of 29 known and 132 potential novel miRNAs were discovered in 12 peanut-specific miRNA families. Among the identified miRNAs, 87 were differentially expressed during early embryo development under calcium deficiency and sufficiency conditions, and 117 target genes of the differentially expressed miRNAs were identified. Integrated analysis of miRNAs and transcriptome expression revealed 52 differentially expressed target genes of 20 miRNAs. The expression profiles for some differentially expressed targets by gene chip analysis were consistent with the transcriptome sequencing results. Together, our results demonstrate that seed/embryo development-related genes such as TCP3, AP2, EMB2750, and GRFs; cell division and proliferation-related genes such as HsfB4 and DIVARICATA; plant hormone signaling pathway-related genes such as CYP707A1 and CYP707A3, with which abscisic acid (ABA) is involved; and BR1, with which brassinosteroids (BRs) are involved, were actively modulated by miRNAs during early embryo development. Both a number of miRNAs and corresponding target genes likely playing key roles in the regulation of peanut embryo abortion under calcium deficiency were identified. These findings provide for the first time new insights into miRNA-mediated regulatory pathways involved in peanut embryo abortion under calcium deficiency.

GOLDEN2-LIKE (GLK) is a member of the myeloblastosis (MYB) family transcription factor and it plays an important role in the regulation of plastid development and stress tolerance. In this study, a gene named AhGLK1b was identified from a cultivated peanut showing down-regulation in response to low calcium with a complete open reading frame (ORF) of 1212 bp. The AhGLK1b has 99.26% and 96.28% sequence similarities with its orthologs in Arachis ipaensis and A. duranensis, respectively. In the peanut, the AhGLK1b was localized in the nucleus and demonstrated the highest expression in the leaf, followed by the embryo. Furthermore, the expression of AhGLK1b was induced significantly in response to a bacterial pathogen, Ralstonia solanacearum infection. Ectopic expression of AhGLK1b in Arabidopsis showed stronger resistance against important phytopathogenic fungi S. sclerotiorum. It also exhibited high resistance to infection of the bacterial pathogen Pst DC3000. AhGLK1b-expressing Arabidopsis induced defense-related genes including PR10 and Phox/Bem 1 (PBI), which are involved in multiple disease resistance. Taken together, the results suggest that AhGLK1b might be useful in providing dual resistance to fungal and bacterial pathogens as well as tolerance to abiotic stresses.

Peanut (Arachis hypogaea L.) is an important food and feed crop worldwide and is affected by various biotic and abiotic stresses. The cellular ATP levels decrease significantly during stress as ATP molecules move to extracellular spaces, resulting in increased ROS production and cell apoptosis. Apyrases (APYs) are the nucleoside phosphatase (NPTs) superfamily members and play an important role in regulating cellular ATP levels under stress. We identified 17 APY homologs in A. hypogaea (AhAPYs), and their phylogenetic relationships, conserved motifs, putative miRNAs targeting different AhAPYs, cis-regulatory elements, etc., were studied in detail. The transcriptome expression data were used to observe the expression patterns in different tissues and under stress conditions. We found that the AhAPY2-1 gene showed abundant expression in the pericarp. As the pericarp is a key defense organ against environmental stress and promoters are the key elements regulating gene expression, we functionally characterized the AhAPY2-1 promoter for its possible use in future breeding programs. The functional characterization of AhAPY2-1P in transgenic Arabidopsis plants showed that it effectively regulated GUS gene expression in the pericarp. GUS expression was also detected in flowers of transgenic Arabidopsis plants. Overall, these results strongly suggest that APYs are an important future research subject for peanut and other crops, and AhPAY2-1P can be used to drive the resistance-related genes in a pericarp-specific manner to enhance the defensive abilities of the pericarp.

The TEOSINTE-BRANCHED1/CYCLOIDEA/PROLIFERATING-CELL-FACTOR (TCP) gene family, a plant-specific transcription factor family, plays pivotal roles in various processes such as plant growth and development regulation, hormone crosstalk, and stress responses. However, a comprehensive genome-wide identification and characterization of the TCP gene family in peanut has yet to be fully elucidated. In this study, we conducted a genome-wide search and identified 51 TCP genes (designated as AhTCPs) in peanut, unevenly distributed across 17 chromosomes. These AhTCPs were phylogenetically classified into three subclasses: PCF, CIN, and CYC/TB1. Gene structure analysis of the AhTCPs revealed that most AhTCPs within the same subclade exhibited conserved motifs and domains, as well as similar gene structures. Cis-acting element analysis demonstrated that the AhTCP genes harbored numerous cis-acting elements associated with stress response, plant growth and development, plant hormone response, and light response. Intraspecific collinearity analysis unveiled significant collinear relationships among 32 pairs of these genes. Further collinear evolutionary analysis found that peanuts share 30 pairs, 24 pairs, 33 pairs, and 100 pairs of homologous genes with A. duranensis, A. ipaensis, Arabidopsis thaliana, and Glycine max, respectively. Moreover, we conducted a thorough analysis of the transcriptome expression profiles in peanuts across various tissues, under different hormone treatment conditions, in response to low- and high-calcium treatments, and under low-temperature and drought stress scenarios. The qRT-PCR results were in accordance with the transcriptome expression data. Collectively, these studies have established a solid theoretical foundation for further exploring the biological functions of the TCP gene family in peanuts, providing valuable insights into the regulatory mechanisms of plant growth, development, and stress responses.