CabHLH55 Negatively Regulates Cold Tolerance in Pepper by Modulating Reactive Oxygen Species Homeostasis and Cold-Responsive Gene Expression

Low temperature is a major abiotic stress that constrains the early-spring and late-season cultivation of pepper (Capsicum annuum L.). Basic helix–loop–helix (bHLH) transcription factors play important roles in plant stress responses; however, their functions in pepper cold tolerance remain poorly understood. In this study, we characterized CabHLH55 and investigated its role in cold stress through bioinformatic analysis, subcellular localization, quantitative reverse transcription PCR (RT-qPCR), heterologous overexpression in Arabidopsis, and virus-induced gene silencing (VIGS) in pepper. CabHLH55 encodes a 257-amino-acid nuclear protein containing a conserved bHLH domain. Its promoter contains multiple cis-acting elements associated with phytohormone and stress responses. Expression analysis showed that CabHLH55 responded to multiple abiotic stresses and was markedly downregulated under cold stress. Under cold conditions, CabHLH55-overexpressing Arabidopsis plants exhibited reduced chlorophyll content and increased relative electrolyte leakage and malondialdehyde (MDA) levels compared with WT plants, indicating enhanced cold-induced damage. Conversely, silencing CabHLH55 in pepper alleviated cold injury, reduced reactive oxygen species (ROS) accumulation and MDA levels, and increased peroxidase (POD) activity. Moreover, the expression of the cold-responsive genes CaCBF1a, CaKIN, and CaCOR47 was significantly increased in CabHLH55-silenced plants under cold stress. Collectively, these results indicate that CabHLH55 functions as a negative regulator of cold tolerance in pepper, potentially by affecting ROS homeostasis and cold-responsive gene expression. This study provides a basis for further elucidating bHLH-mediated cold stress responses in pepper and may facilitate the molecular breeding of cold-tolerant pepper cultivars.