Identification and characterization of Nonexpressor of Pathogenesis-Related (NPR) genes in peanut and functional analysis of AhNPRs involved in cold stress

Abstract

Peanut (Arachis hypogaea) is a significant industrial crop that plays a vital role in both agricultural and economic sectors worldwide. The yield of peanuts is often affected by cold damage when cultivated in high-latitude and high-altitude regions. Identifying cold-tolerance-related genes is of great significance for understanding the cold resistance mechanisms of peanuts and for developing low-temperature tolerant varieties. NON-EXPRESSER OF PATHOGENESIS-RELATED GENES 1 (NPR1) represents a master regulator of plant responses to pathogens, conferring immunity in a wide range of crops. However, research on how it responds to abiotic stress is relatively restricted. Herein, eight AhNPR genes in peanuts were identified and analyzed. The neighbor-joining method was used to analyze the evolutionary relationships, and based on chromosome localization, gene duplication events and structural characteristics, the close genetic relationship between peanut NPR genes and other leguminous plants was revealed, as well as their unique evolution and distribution patterns in gene families. Cis-element and transcription factor analyses of AhNPR genes revealed a diverse array of regulatory elements and potential regulators, suggesting their significant functions in peanut growth, hormone signaling, and stress responses. Analyses of the transcriptome and qRT-PCR of AhNPR genes demonstrated unique expression profiles that were responsive to a variety of tissues, different abiotic stress conditions, and hormone treatments. Compared to wild-type plants, functional analysis of AhNPR3A/4 A in Arabidopsis overexpressing plants demonstrated enhanced cold stress tolerance, as indicated by improved growth and lowered oxidative damage. The VIGS system demonstrated that reduced expression of AhNPR3A/4 A resulted in increased sensitivity to cold stress, as evidenced by greater wilting, higher hydrogen peroxide accumulation, diminished antioxidant enzyme activity, and elevated malondialdehyde levels in gene-silenced plants compared to wild-type and control plants. These outcomes reveal a novel role for NPR genes in the peanut's response to low temperatures, positioning them as a key integrator of cold and pathogen signaling, thereby enhancing the plant's adaptability to a changing environment.