The basic laucena zipper becomes the core to multifunctional activities. Previous, the bZIP transcriptional factor in Eukaryotes has reported in various studies as biologically and physiologically a driving force of plant toward growth and bring about a successive plant development(Huang et al. 2019). The bZIP trasncriptional factor in poplar plant shows different expressions pattern in various tissues such as roots, stem, and leaves, which demonstrates directly involvement in metabolite biosynthesis and secondary metabolic activities, resulting in the growth and development of poplar(Zhao et al. 2021). Additionally, the bZIP transcription factor is reported to play a important role in controlling biotic stress(Wang et al. 2021). For instance, the upregulation of StbZIP-65 in potatoes in response to salinity is a direct postive results of bZIP trascnription factor on abiotic stress signals (Zhao et al. 2020). Furthermore, among one of the Arabidopsis representative groups specifically group A, the function of bZIP trascription factor in ABA activities and drought stress response has been revealed((Joo et al. 2021). Although the genomic activities and expression profile of the bZIP transcription factor in forestry species still have uncertain(Jing et al. 2022), here in our study we aim to highlight the shade region and paving a way for new knowledge. We have looked into various biochemical and molecular activities of the bZIP transcription factor in response to abiotic stress together with responsible process for growth and development in Phoebe bournei.
We analyzed the bZIP genes in P. bournei using whole-genome identification and systematic bioinformatics techniques. The P. bournei genome database was used to identify and thoroughly investigate 71 members of the PbbZIP gene family. The theoretical isoelectric point (PI) of all the PbbZIPs, except PbbZIP 21, is 4.78. A few members, namely PbbZIP 1, PbbZIP 2, PbbZIP 5, PbbZIP 10, PbbZIP 11, PbbZIP 18, PbbZIP 19, PbbZIP 23, PbbZIP 24, PbbZIP 25, PbbZIP 31, PbbZIP 32, PbbZIP 37, PbbZIP 59, PbbZIP 62, PbbZIP 63, PbbZIP 65, PbbZIP 69, and PbbZIP 71, have PIs ranging from 5.89 to 6.4; the remaining members have PIs greater than 7, signifying that they are alkaline proteins made up of basic amino acids (Table 1). Moreover,we examining conserved motifs offers a theoretical foundation for investigating the roles and categorization of the PbbZIP gene family. After a prediction of the PbbZIP protein sequence motifs using MEME, 25 related conserved motifs were discovered. All members of the gene family shared the conserved motif 7 of the PbbZIP domain, according to a schematic diagram of the full-length PbbZIP protein have distinct conserved motifs (Fig. 3a). The variation in motif number indicates that there may have been a splicing error in the annotation or that the sequence of these genes was lost or deleted during the gene's long-term evolution, which could provide more evidence for the functional of PbbZIP gene in various subfamilies. According to additional examination of the intron–exon structure, every gene has both exons and introns, suggesting some degree of structural conservation. Variations exist in the locations, counts, and lengths of exons and introns among the various PbbZIP gene members (Fig. 3b ), indicating that the family has essential biological fuction such as signal transduction and gene regulation which is essential for organism to survive(Gao et al. 2021). The unequal distribution of exons and introns; these results showed that the PbbZIP family's divergent functions are linked to the intron and exon structures' divergence(Sawada and Mitaku 2011; Manzoor et al. 2021). To bolster our theory regarding the close relationship between Phoebe bournei and A. thaliana, an ancestral tree was constructed. Of these, 71 PbbZIP gene families and 75 AtbZIP gene families were found, with the PbbZIP in P. bournei being divided into 12 groups. The findings demonstrated how closely these two species have evolved. In particular, A. thaliana is more closely related to PbbZIP groups S, A, D, I, and E than it is to PbbZIP gene family groups F, G, C, and A. Furthermore, 90% of the genes seemed to form gene clusters, and it was hypothesized that PbbZIP's function was conserved throughout evolution. This hypothesis is supported by the finding that P. bournei and A. thaliana have a high degree of gene homology, as determined by a collinear analysis of the localization of genes in chromosomes. P. bournei shared approximately 11 of its 12 chromosomes with A. thaliana genes, with chromosome positions ranging from 003076.9 to 00307.1 in the genomic sequence. This suggested that P. bournei is widespread throughout species and originated before the formation of the species. Every PbbZIP gene family member is physically mapped onto each of the 12 chromosomes(Fig. 1). The greatest number of PbbZIPs are found on chromosome 5, while the fewest PbbZIPs are found on chromosomes 7 and 8, which have two PbbZIP genes. The distribution of PbbZIP genes across chromosomes showed that certain regions had a higher frequency of gene clusters than others. On chromosomes 3 and 4, the PbbZIP gene family are uniformly distributed throughout the chromosome, whereas PbbZIP genes on chromosomes 1, 2, and 5 appear to congregate at the lower and upper ends of the arms. Moreover, it was shown that PbbZIP gene family members duplicated in tandem. Genes are tandemly duplicated on chromosomes 1, 4, 5, and 6. Only four pairs of tandem duplicates have been found overall, which suggests that tandem duplication has a small role in the expansion of gene families(Wang et al. 2011; Baloglu et al. 2014). These genes are separated by a distance ranging from 25 to 140 megabases (Mb). Next, we identified segmentally duplicated bZIP genes in P. bournei genome. Twelve segmentally duplicated P. bournei bZIP genes in total were found; these comprise approximately sixteen percent (12/71) of PbbZIP genes. An important mechanism thought to be responsible for the expansion of gene families is segmental duplication in which indicate that gene is heavly coserved. Furthermore, a few cis-acting elements were found to relate to abiotic stress hormone regulation, growth and development, for example, auxin, abscisic acid, gibberellin, and jasmonate-responsive elements. Abiotic stress response required a number of cis-acting elements, while the encompassed elements—such as the high-temperature response—were crucial to the defense and stress responses. Related elements like light-responsive elements, salicylic-acid-response elements, and seed-specific regulatory response elements are necessary for plant growth and development. A more recent study discovered that an MYB gene could attach to the PbbZIP genes. This gene regulates the synthesis of flavonoids, as well as light and drought tolerance in plants. It is feasible that the MYB gene and the PbbZIP protein interact to create a regulatory network (Mitra 2018; Jiang et al. 2022).
Lastly, the PbbZIP gene family in leaves were determined using qRT-PCR. The PbbZIP gene family exhibited a significant peak at various time points (0, 6, 12 and 24 h). Further analysis was conducted on the PbZIP expression intensity in various tissues (Fig. 6). The findings show that the genes.were expressed in the leaves, root xylem, root, stem bark, and stem xylem. The PbbZIP gene family, however, is highly expressed in the stem and roots and, except for a few members, poorly expressed in the stem, xylem, and leaves. High expression was found in the leaves for PbbZIP 33, PbbZIP 49, PbbZIP 71, PbbZIP 61, PbbZIP 57, PbbZIP 54, PbbZIP 40, PbbZIP 58, PbbZIP 30, PbbZIP 63, PbbZIP 41, PbbZIP 55, PbbZIP 60, PbbZIP 36, and PbbZIP 66. This suggested that P. bournei's growth and development depend heavily on the bZIP gene family (Wang et al. 2022; Dong et al. 2023). Additionally, the average length of the core conserved structure domain in PbbZIP members reveals the amino acid residues constituting the PbbZIP domain. The basic domain comprises 25 amino acids, characterized by numerous arginine and lysine residues. Its primary structure, N-R-X8-R/K-X-R/K, is situated within the site region 10–25, as depicted in (Fig. 5). Furthermore, within the leucine zipper structure domain, delineated by the site region 26–65 in (Fig. 5), there exist 41 amino acid residues, including leucine (X6-L).The basic region has a distinctive motif (N-X7-R/K-X9) that is important for nuclear localization and DNA binding. It also deviates from bZIP's overall distribution of amino acids. However, as previously reported, the leucine zipper forms an amphipathic surface that meditates on particular recognition and dimerization (Li et al. 2016). Such variation may alter this protein’s DNA binding specificity and subcellular localization(Dröge-Laser et al. 2018). These predictions align with the PbbZIP protein's variable subcellular localization signal in plants, which may be important for activation (Jin et al. 2014). Thus, phylogenetic analysis, transcriptomic data, and RT-qPCR all showed that five PbbZIP transcriptomic factors (PbbZIP 14, PbbZIP 26, PbbZIP 32, PbbZIP 67, and PbbZIP 69) are quickly activated and play key roles in response to abiotic stress (Fig. 7). In this study, we observed upregulation of these representative genes after exposure to a high temperature of 40°C and salt stress. Under salt stress, PbbZIP 14, PbbZIP 67, and PbbZIP 69 showed rapid induction and reached peak expression levels at 6 and 12 hours. This suggests their potential involvement in mechanisms contributing to salinity tolerance of P. bournei. The PbbZIP gene family is also upregulated when the temperature is high, with peak expression at 6 and 12 hours (mostly PbbZIP 14, PbbZIP 67, and PbbZIP 69). However, no clear patterns were seen when the plant was under drought stress; as we observed previously, each gene showed varying degrees of upregulation or downregulation(Ramakrishna et al. 2018). These findings provide preliminary evidence of the important functions of the five PbbZIP genes in adapting to abiotic stressors such as heat, drought, and salinity.