A B-box transcription factor NtBBX46 negatively regulates cold tolerance in tobacco

Tobacco (Nicotiana tabacum L.) is a profound global economic crop originating from tropical or subtropical regions, with optimal growth in warm climates. Low temperatures can impair the growth and development of tobacco, leading to reduced yield and compromised commercial value. B-box (BBX) transcription factors act as key regulators in plant growth, development, and response to diverse abiotic and biotic stresses. To date, 49 BBX proteins have been identified in tobacco, but the functional characteristics of the Group V members (NtBBX41-NtBBX49) and the molecular mechanisms underlying their regulatory functions remain largely unclear. In this study, we characterized the functional response of NtBBX46 to cold stress (4 °C). Subcellular localization assays confirmed that NtBBX46 is exclusively localized in the cell nucleus. Cold stress significantly induced NtBBX46 transcription. Notably, NtBBX46-silenced (NtBBX46i) tobacco displayed enhanced cold tolerance compared to wild-type (WT) plants. Under cold stress, NtBBX46i tobacco accumulated lower levels of malondialdehyde (MDA) and reactive oxygen species (ROS), thereby alleviating oxidative damage to cellular membranes. Furthermore, RNA-seq analysis identified 264 differentially expressed genes (DEGs) between NtBBX46i and WT. Silencing NtBBX46 strengthened stress resistance by upregulating various abiotic stress-responsive genes, including CBFs and activating alternative cold-tolerance pathways. In conclusion, we propose a regulatory model wherein NtBBX46 functions as a transcriptional repressor and negatively regulates cold stress tolerance by suppressing the expression of NtCBF5.