IntroductionAPETALA2/ETHYLENE-RESPONSIVE FACTOR (AP2/ERF) transcription factors mainly exist in plants and were first reported in the Arabidopsis AP2 mutant1. AP2/ERF gene is defined by an AP2 domain containing 60–70 conserved amino acid residues, forming many transcription factors (including 147 genes in Arabidopsis). This family plays an important regulatory role in many biological and physiological processes, such as plant morphogenesis, response mechanisms to various stresses, hormone signal transduction, and metabolite regulation2,3. Based on the number of AP2 and other DNA binding domains, the AP2/ERF family could be divided into four major subfamilies and others, including AP2 (APETALA2), RAV (related to ABI3/VP), ERF (ethylene responsive element binding protein), DREB (dehydration responsive element binding), and a few unclassified factors-Soloist4. AP2 subfamily members play an important regulation role in plant growth and development, such as leaf epidermal cell identity and the development of flowers and ovules. Members of the RAV subfamily are involved in regulating plant development and response to various stresses. Meanwhile, ERF and DREB subfamily members are mainly involved in responding to biotic and abiotic stresses3,4,5,6. According to similarity and the number of DNA-binding domains, the family is diversely involved in the regulation of plant growth, development, metabolism, and stress response7,8.ERFs were initially isolated from tobacco. Those transcription factors regulate biotic and abiotic stresses by binding to the GCC-box or dehydration responsive element (DRE)/C-repeat element (CRT), such as ORA59 and ERF1 gene in Arabidopsis, Tsi1 in tobacco and Pti4 in tomato9,10,11,12,13,14. Furthermore, some ERFs were also identified in pepper. CaERFLP1 could form a specific complex with both the GCC box and DRE/CRT motif and respond to Pseudomonas syringae infection and salt stress in transgenic tobacco plants15. CaCBF1 A, CaCBF1B, CaPF1 and CaDREBLP1 could respond to different stresses, such as low-temperature, dehydration, high salinity, and wounding16,17,18. CaRAV1 was demonstrated to be a transcriptional activator in triggering resistance to Xanthomonas campestris cv. vesicatoria. infection. Virus-induced gene silencing of CaRAV1 and CaRAV1/CAOXR1 confers enhanced susceptibility to high salinity and osmotic stresses19,20. CaAP2 was also considered a candidate gene to control the flowering time in pepper21. ERF family has been identified and analyzed in horticulture crops, such as grape, tomato, cucumber, potato, pineapple, and strawberry22,23,24,25,26,27. Under stress conditions, protein members positively or negatively regulate defense responses, leading to plant adaptation. These protein family members play important modulatory roles in response to ABA-mediated dehydration signaling and are crucial for lateral root development28,29. Lateral roots in model plant A. thaliana originate from preselected pericyclic cells, which divide to form lateral root primordia, eventually growing to form a new root meristem. This process provides a mechanism of resistance to environmental stresses, particularly soil drought and salts.Pepper (Capsicum annuum L.) is a significant and widely cultivated vegetable crop globally. In the cases of salt stress, drought stress and R. solanacearum resistance, specific genetic pathways of tolerance have been addressed30,31,32,33,34. Recent studies have shown that a pepper ERF family member, named CaPTI1, has the highest expression level in roots and responds not only to Phytophthora capsici infection but also to cold and drought stresses35. Drought stress induces a series of injuries in terms of plant physiological, biochemical, and metabolic impacts, resulting in plant growth retardation, cell damage, and loss of crop yield and quality36,37. Hong et al. (2017) reported that CaAIEF1 positively regulates the drought stress response and ABA signaling in pepper7. The pepper AP2 domain-containing transcription factor CaDRAT1, belonging to the ERF subfamily, is significantly induced after exposure to abscisic acid (ABA), mannitol, low temperature, and H2O27. Yang et al. (2021) studied the ERF2 gene, significantly upregulated in both resistant and susceptible tomato cultivars in response to Stemphylium lycopersici38. ERF2 plays a key role in salicylic acid (SA) and jasmonic acid (JA) signaling pathways, conferring resistance to invasion by S. lycopersici. Lee et al. (2020) demonstrated that JA synthesis, JA signaling, and ERF family genes contribute to the chilling response in pepper fruit39. This study can help elucidate the cellular mechanism or identify key factors affecting the chilling sensitivity or insensitivity of peppers following harvest.In this study, we isolated and functionally characterized a member of the ERF subfamily B-4, the CaERF14 gene. CaERF14 affected dehydration resistance through the modulation of ABA biosynthesis and ABA sensitivity expressions of defense response-related genes, contributing to the resistance of tobacco to high salt and drought. Our findings imply that CaERF14 functions as a regulator of plant dehydration and salinity stress response. In the process of global climate change, this study of the AP2/ERF factor provides important bases for understanding plant regulatory mechanisms in molecular breeding.ResultsCloning and sequence analysis of CaERF14Molecular alignment of expressed sequence tags (ESTs) showed that the full-length CaERF14 gene was screened and cloned from pepper cDNA library (Fig. 1A). Domain in comparison with the NCBI website, and the sequence analysis to identify putative the AP2/ERF domain-containing gene were analyzed by DNAMAN software. The CaERF14 gene was 1,572 bp in length, which included an open reading frame (ORF) of 849 bp, and was identified from pepper transcriptome database. The ORF of CaERF14 was predicted to encode a protein of 283 amino acids, with a predicted molecular weight of 31.75 kDa. According to the comparison results on the BLAST website, this gene has 70% homology with Arabidopsis AtERF114 (NP_200995.1), 59% homology with Arabidopsis transcription factor AtERF115 (NP_196348.1), and 57% homology with kiwifruit AdERF14 (adj67443.1) (Fig. 1B; Supplementary Figure S2).Fig. 1A new ERF gene CaERF14 was isolated from pepper. (A) cDNA sequence and deduced amino acid sequence. (B) Multiple sequence alignment analysis of AP2/ERF domain containing CaERF14 transcription factor with other plant homologous proteins. The number on the right represents the sequence position of amino acid residue. The upper line reveals the conserved AP2/ERF domain, and the asterisks indicate the A14 and D19 ERF specific amino acid residue. The black dots represent the conserved WLG motif. The gray boxed sequence represents the Xa subgroup specific motif. AtERF114 and AtERF115 are the AP2 transcription factors of Arabidopsis. AdERF14 is the AP2 transcription factor of kiwifruit. (C) Phylogenetic tree shows the relationships of CaERF14 with other species. The accession number indicates unnamed genes.Full size imageThe expression patterns of the CaERF14 revealed the presence of a conserved AP2/ERF DNA binding domain. Amino acids differences at position 14 th and 19 th in the AP2/ERF domain were also observed in these ERFs (Fig. 1B). The ERF subfamily differed from the unique alanine and aspartic acid of DREB subfamily and contained a landmark conserved WLG motif. At the N-terminal, there was a conserved sequence of CMX-1 specific to the Xa subfamily of the ERF family (Fig. 1B).Furthermore, the evolutionary relationships and multiple sequence alignments of CaERF14 in comparison with ERF proteins of other plants were also investigated (Fig. 1C). The results indicate that the newly obtained CaERF14 gene can be confirmed as the Xa (B-4) subfamily of the ERF family (Fig. 1A-C).Tissue-specific expression of CaERF14 gene in pepper plantsThe expression levels of the CaERF14 gene in different tissues of pepper plants were investigated by the MIQE guidelines: quantitative real-time PCR (qRT-PCR)40. A significant difference in CaERF14 gene expression was observed among different tissues (Fig. 2). The highest expression was detected in floral parts, nearly 7-fold of that of roots. The expression level of CaERF14 gene in stems was slightly lower than that in flowers, approximately 6.5-fold of that of roots. Lower expression was detected in fruits, leaves and roots as compared to the floral and stem parts. Particularly, the CaERF14 gene showed the lowest expression in the roots. The tissue-specific expressions of CaERF14 gene in pepper were significantly different from the background actin gene, indicating that the gene expression results were feasible. In particular, the characteristics of CaERF14 gene showed high expression in aboveground tissues, but lowest expression in underground parts such as roots.Fig. 2Quantitative real-time PCR expression level of CaERF14 in pepper different tissues. The actin gene was used as an internal control. R: Roots; S: Stems; L: Leaves; Fl: Flowers; Fr: Fruits. The vertical bar shows mean ± SD (n = 3). Asterisks indicate statistical significance (*0.01