As the second largest family of transcription factors (TFs) in plants, basic helix–loop–helices (bHLHs) play key roles in regulating plant growth and development and responding to environmental stress. As the fastest growing Citrus variety in China in recent years, Orah mandarin has vital economic and nutritional value. Although a comprehensive genome-wide analysis of the bHLH TF family has been performed in many plants, a systematic study of the genes of this family has not been carried out in Orah mandarin. In this study, 114 bHLH TFs were identified in Orah mandarin via genome-wide analysis and were classified into 27 subfamilies according to the evolutionary tree. The gene expression profile revealed that five genes were significantly upregulated at 12 h and 24 h after low-temperature stress treatment. In addition, soluble sugars, soluble proteins, and proline contents increased with increasing low-temperature stress, which promoted the expression of the CrbHLH46 gene, thus mediating the interconversion pathway of pentose and glucose to improve the cold tolerance of Orah mandarin. The results help explore the characteristics and functions of CrbHLH genes and provide a basis for further research on the Orah mandarin resistance to low-temperature stress.

Dehydration response element binding proteins (DREBs) have been identified as major regulators of cold acclimatization in many angiosperms. Cold stress is one of the primary abiotic stresses affecting kiwifruit growth and development. However, kiwifruit is currently one of the most widely consumed fruits worldwide because of its high nutritional value. 5-Aminolevulinic acid (5-ALA) is a nonprotein amino acid known for its distinct promotional effects on plant resistance, growth, and development. However, studies on the function of the kiwifruit DREB gene in alleviating low-temperature stress in its seedlings via exogenous 5-ALA have not been reported. Therefore, in this study, we performed a genome-wide identification of DREB gene family members in kiwifruit and analyzed the regulatory effects of exogenous 5-ALA on kiwifruit DREB genes under low-temperature stress. A total of 193 DREB genes were identified on 29 chromosomes. Phylogenetic analysis classified these genes into six subfamilies. Although there were some differences in cis-elements among subfamilies, all of them contained more biotic or abiotic stresses and hormone-related cis-acting elements. GO and KEGG enrichment analyses revealed that AcDREB plays an essential role in hormone signaling, metabolic processes, and the response to adverse stress. Under low-temperature stress, the application of exogenous 5-ALA inhibited the accumulation of APX and DHAR, promoted an increase in chlorophyll, and increased the accumulation of enzymes and substances such as 5-ALA, MDHAR, GR, ASA, GAH, and GSSH, thereby accelerating ROS scavenging and increasing the cold hardiness of kiwifruits. Functional analysis revealed that 46 differentially expressed DREB genes, especially those encoding AcDREB69, AcDREB92, and AcDREB148, which are involved in ethylene signaling and defense signaling, and, after the transcription of downstream target genes is activated, are involved in the regulation of low-temperature-stressed kiwifruits by exogenous 5-ALA, thus improving the cold tolerance of kiwifruits. Notably, AcDREB69, AcDREB92, and AcDREB148 could serve as key genes for cold tolerance. This study is the first to investigate the function of AcDREB genes involved in the role of exogenous 5-ALA in regulating low-temperature stress, revealing the regulatory mechanism by which DREB is involved in the ability of exogenous 5-ALA to alleviate low-temperature stress.