Skip to main content

The role of walnut bZIP genes in explant browning



Basic leucine zipper (bZIP) proteins are important transcription factors in plants. To study the role of bZIP transcription factors in walnut explant browning, this study used bioinformatics software to analyze walnut bZIP gene family members, along with their transcript levels in different walnut tissues, to evaluate the transcriptional expression of this gene family during the primary culture of walnut explants and to reveal the mechanism of action of walnut bZIP genes in walnut explant browning.


The results identified 65 JrbZIP genes in the walnut genome, which were divided into 8 subfamilies and distributed on 16 chromosomes. The results of transcriptome data analysis showed that there were significant differences in the expression of four genes, namely, JrbZIP55, JrbZIP70, JrbZIP72, and JrbZIP88, under both vermiculite and agar culture conditions. There were multiple hormone (salicylic acid, abscisic acid, auxin, and gibberellin) signaling and regulatory elements that are responsive to stress (low temperature, stress, and defense) located in the promoter regions of JrbZIP55, JrbZIP70, JrbZIP72, and JrbZIP88. The walnut JrbZIP55 protein and Arabidopsis bZIP42 protein are highly homologous, and the proteins interacting with Arabidopsis bZIP42 include the AT2G19940 oxidoreductases, which act on aldehyde or oxygen-containing donors.


It is speculated that JrbZIP55 may participate in the regulation of browning in walnut explants.

Peer Review reports


bZIP (basic leucine zipper) genes encode one of the largest and most highly conserved families of transcription factors and repressor proteins in eukaryotes [1]. bZIP proteins in plants generally contain 60 to 80 amino acid residues and include 2 structural units: a highly conserved DNA binding region and a variable leucine zipper dimer region [2]. DNA structural regions typically contain 16 amino acid residues with a highly conserved N-X7-R/K motif. The leucine zipper is typically characterized by 1 leucine per 7 amino acids as well as hydrophobic amino acids at the 3rd and 4th positions [3]. To date, bZIP genes have been identified in the genomes of various plants, including Arabidopsis [4], maize [5], tomato [6], rice [2], apple [7], kiwi [8], date palm [9], tobacco [10] and wheat [11], and it has been shown that this family of genes is involved in plant biotic and abiotic stress responses [6, 12], seed germination [13], anthocyanin accumulation [14], lignin synthesis [15] and the regulation of growth and developmental processes [4] and that bZIP transcription factors participate in plant responses to ABA, light and developmental signals by regulating functional gene expression [16, 17]. The main function of bZIP transcription factors is to regulate the intensity of target gene expression in response to exogenous hormones and environmental stresses [16]. Under stressful environmental conditions, bZIP transcription factors can regulate the expression of downstream target genes by binding to the promoter regions of ABA-inducible genes, and ABA signaling plays a crucial role in plant responses to abiotic stress, such as defense against pathogens or the control of spatiotemporally specific expression of target genes [18].

Browning, also known as the browning reaction, is a phenomenon that occurs in all life stages of a plant and results in the creation of a brown polymer through a series of reactions [19]. Plant tissues produce large amounts of unstable phenolics that are secreted after damage, and the formation of brown quinones from these phenolics is then catalyzed by a variety of oxidative enzymes. This enzymatic browning is the main cause of explant browning [20]. Browning can lead to the death of explants in tissue culture, affect the experimental process, reduce the efficiency of plant genetic transformation, and reduce the quality of fruits by browning during postharvest transport [21]. Therefore, the study of the effects of plant browning inhibition is particularly important. Some studies have found that overexpression of StSPI128 significantly alleviated potato browning and increased phenolic content [22]. Browning inhibition in mushrooms can be achieved by using nano-PM to inhibit TYR activity, which in turn delays the conversion of GHB to GDHB and tyrosine to DOPA, ultimately reducing melanogenesis [23], and the addition of ascorbic acid to DKW medium significantly reduced the browning of the medium and the explants [19]. Aloe vera gel can be used as an edible coating (dry environment) and as a gel solution (aqueous environment: a new method) to prevent browning during the refrigeration of fresh Persian walnut kernels [24]. However, the regulatory mechanism of plant tissue browning needs to be further investigated.

Juglans regia L., also known as English walnut, is a deciduous tree of the genus Walnut in the family Walnutaceae. It is native to southeastern Europe, western Asia and southwestern China [25]. China has a long history of walnut cultivation and ranks first in the world in terms of the cultivated area and production of walnut [25]; however, because walnuts are rich in polyphenols, their explants are highly susceptible to browning, and the difficulty of seedling rooting has become a limiting factor in walnut factory nurseries [26]. In recent years, with the surge in demand for woody food and oil plants, the demand for walnut seedlings in domestic and international markets has also expanded each year [27]. Therefore, overcoming explant browning and speeding up the process of factory seeding are important issues in the walnut industry. Higher compatible solute levels in the leaves of in vitro plants do not contribute to water conservation during ex vitro acclimation, and low foliar ion levels in in vitro plants can occur due to the low transpiration rate of plants as a result of in vitro production [28]. Walnut tissue is rich in phenolic substances [29], causing it to brown particularly easily after injury. It is also susceptible to browning during tissue culture. Thus, we used vermiculite as the primary medium, which effectively reduced the browning rate of explants, mainly because of the air permeability of vermiculite, which can mitigate damage to the explants, resulting in a high survival rate [30]. Studies have shown that bZIP genes in tomato are involved in defense responses during tissue damage [6], but whether bZIP genes are also involved in regulatory processes during the culture of walnut explants is unclear. Therefore, this study was conducted to mine and identify the bZIP family members of the walnut genome and analyze the changes in the expression of bZIP genes during explant culture. The aim was to clarify the regulatory role of the bZIP gene family in tissue damage and to lay the foundation for resolving the mechanism of browning in walnut explants.


Identification and basic information of bZIP gene family members of walnut

Hidden Markov models were constructed using Arabidopsis bZIP family protein sequence information, bZIP genes in the walnut genome were searched using the Hmmer tool, and 91 walnut bZIP family genes were screened and tentatively named JrbZIP1-JrbZIP91. Using the Pfam database and the results of prediction analysis with online SMART software, after the removal of redundancy [31], 65 walnut bZIP transcription factor family gene members were finally identified [32]. The physical and chemical properties of the walnut bZIP family members were analyzed (Table 1), and the results showed that the theoretical isoelectric point (PI) of JrbZIP ranged from 4.85 (JrbZIP72) to 10.04 (JrbZIP59), gene length ranged from 453 bp (JrbZIP51) to 23704 bp (JrbZIP58), CDS length ranged from 399 bp (JrbZIP49) to 2352 bp (JrbZIP70), relative molecular masses ranged from 15.08 kD (JrbZIP49) to 84.52 kD (JrbZIP70), and the amino acid length of reads ranged from 132 aa (JrbZIP49) to 783 aa (JrbZIP70).

Table 1 Basic information on members of the walnut bZIP transcription factor family

Physicochemical characterization of member proteins of the walnut bZIP transcription factor family

The 91 bZIP transcription factor protein sequences were identified as corresponding to 65 walnut bZIP transcription factor family genes (Table 2) using bioinformatic methods such as Hmmer 3.0 combined with the Pfam database and sequence comparison using MEGAX [33]. Based on known sequence information, the online software ExPasy (, the TMHMM ( method, the SignalP3.0 server ( dk/services/SignalP) and other online software were employed to predict the affinity, instability coefficients, transmembrane regions, signal peptides, glycosylation sites, phosphorylation sites, and subcellular localization of bZIP family genes [34]. The results showed that among the 65 walnut bZIP genes, the shortest length was 453 bp, the longest was 23,704 bp, and the CDS length ranged from 438 to 2352 bp. The translated JrbZIP proteins were all between 145 and 783 amino acids in length, with molecular weights ranging from 16.36 to 84.52 KD; the isoelectric points ranged from 4.85 to 10.04 The average hydrophobicity values varied in the range -0.306 ~ -1.158, and the hydrophobicity of all proteins was less than 0, indicating that the JrbZIP proteins were all hydrophilic. Few transmembrane structures were identified; only JrbZIP70, JrbZIP71, JrbZIP72, and JrbZIP73 had transmembrane structures, and 95.6% of the bZIP family protein members were not transmembrane proteins. Relatively few proteins contained signal peptides; only JrbZIP41, JrbZIP45, JrbZIP57, JrbZIP63, and JrbZIP91 contained signal peptides, and 94.5% of the bZIP family protein members were not secretory proteins. Subcellular localization analysis revealed that all JrbZIP proteins localized to the nucleus. Therefore, it was hypothesized that the bZIP proteins of walnut mainly functioned in the nucleus (Table 2).

Table 2 Basic information on the walnut bZIP transcription factor protein family

Evolutionary tree of the walnut bZIP gene family

To investigate the evolutionary relationships of bZIP family members in walnut and Arabidopsis, a maximum likelihood phylogenetic tree of the bZIP family proteins in walnut (65) and Arabidopsis (73) was constructed and is shown in Fig. 1. The bZIP family genes of walnut and Arabidopsis showed some homology between the two plants. We classified the bZIP gene families in walnut into ten subfamilies, A, B, C, D, E, F, G, H, I, and S, according to the Arabidopsis bZIP gene family classification. Among these genes, subfamily S was the largest, with 16 members (showing significant gene duplication and amplification), followed by subfamily A, with 15 members, while subfamily F was the smallest, with only 1 member. Members of subfamilies E and I clustered on the same branch, and members of subfamilies D and F clustered together on another branch, indicating that members of these subfamilies were more closely related.

Fig. 1
figure 1

Phylogenetic analysis of bZIP proteins in Arabidopsis thaliana and Juglans regia. Areas of different colors represent different subfamilies. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches

Conserved motif and gene structure analyses of the walnut bZIP gene family

To confirm all of the putative bZIP gene structures and conserved protein domains [35], we acquired gene exon numbers and positions in the genome as well as motifs and conserved domains of proteins (Fig. 2). We used the online analysis tool Gene Structure Display Server to obtain the gene structure map of bZIP transcription factor family members [36] (Fig. 2A), with the coding region (CDS) in yellow and the 5' noncoding and 3' noncoding regions in the left and right blue regions, respectively. The number of exons in ten families exhibited distinct differences, ranging from 1 to 12. While the members of the G and D subgroups showed 8–12 introns, those of the S and F subgroups had only 1 intron, the B subgroup members had only 1 exon, and the members of the other subgroups presented 3–6 introns. According to our analysis, all of the JrbZIP proteins had motif 1 and motif 5, and the distribution of conserved motifs in the protein sequences of members of the same subgroup was highly similar (Fig. 2B). The motif compositions of subgroups C, S and E were the same, indicating that the functions of different members of the three groups may also be the same.

Fig. 2
figure 2

Conserved motif analysis and gene structure analysis of walnut bZIP members. A Conservative motif analysis. B Gene structure analysis. Number represents branch series. The motifs in the bZIP proteins were identified using MEME. Ten conserved motifs were identified in 65 species and are shown in different colors

Chromosomal analysis and collinearity analysis of walnut bZIP members

The positions of the 65 bZIP genes on the chromosomes were determined based on their genomic distributions. The results showed that the 65 bZIP genes were unevenly distributed on 16 chromosomes (Fig. 3A). Chr11, Chr13, Chr8 and Chr5 contained 12, 8, 7 and 6 bZIP genes, respectively, while Chr7 and Chr16 harbored 5 bZIP genes each, and the remaining chromosomes contained 1–4 bZIP genes.

Fig. 3
figure 3

Chromosome mapping and collinearity analysis of the bZIP gene family in walnut. A Chromosome mapping. Sixty-five members of the bZIP gene family in walnut are distributed on 16 chromosomes. Different colored regions represent different chromosomes. B Collinearity analysis diagram. Fifty-one pairs of collinear genes were found in 65 members of the walnut gene family, and no tandem repeats were found

In the process of evolution, gene families are amplified by means of large segment replication and tandem duplication to improve the adaptability of plants to the environment [37]. In this study, we analyzed the synteny relationships of the bZIP gene family (Fig. 3B) within the walnut genome and identified a total of 51 pairs of fragment duplication events. This situation was considered to indicate large fragment replication, and no tandem duplication events were found [38], suggesting that large fragment replication events were the main driving force for the amplification of the walnut bZIP gene family.

Differences in JrbZIP gene expression in different walnut tissues

We analyzed the expression of 65 bZIP genes in seven different tissues of walnut: bud, pistil, catkin, mature leaves, young leaves, yang stem, and root. We obtained the expression levels of bZIP genes in each walnut tissue and plotted a heatmap based on the RPKM values of the JrbZIP genes (Fig. 4). From the heatmap, it can be seen that the 65 genes were expressed to varying degrees in 7 tissues. Among them, four genes, JrbZIP24, JrbZIP34, JrbZIP61, and JrbZIP73, presented relatively high expression levels in young leaves (YL), while in mature leaves (ML), they showed relatively low expression levels, indicating that these four genes may participate in the regulation of leaf maturity, as their expression levels decrease with the maturation of walnut leaves. In young stems (YS), the expression levels of six genes, JrbZIP1, JrbZIP31, JrbZIP34, JrbZIP55, JRbZIP56, and JrbZIP59, were relatively high, suggesting that they may be specifically expressed during the development of young stems.

Fig. 4
figure 4

Changes in JrbZIP expression in different tissues. Each value represents the mean ± SE of six biological replicates. (ML: mature leaves; YL: young leaves; YS: young stems.)

Transcriptional expression analysis of bZIP family members in walnut explants under different culture conditions

Based on the transcriptome analysis of different tissues of walnut, the browning of 'Qingxiang' explants was observed after 12 h, 24 h and 48 h of culture in different media, and it was found that the explants showed significant browning after 48 h of culture in agar medium, while browning was not obvious in vermiculite medium (Fig. 5). The relevant physiological indicators also showed significant differences in the pivotal enzyme activities and total phenolic content of the explants cultured in different medium. (Additional file 8). Further transcriptomic analysis of 'Qingxiang' explants after 12 h, 24 h and 48 h of incubation in both types of media using FPKM as a measure of gene expression showed different degrees of change in all walnut bZIP family gene members (Fig. 6) [39]. Through differential screening, it was found that among 'Qingxiang' JrbZIP8, JrbZIP11, JrbZIP24, JrbZIP28, JrbZIP29, JrbZIP45, JrbZIP47, JrbZIP48, JrbZIP50, JrbZIP54, JrbZIP54, JrbZIP55, JrbZIP61, JrbZIP62, JrbZIP70, JrbZIP72, JrbZI76, JrbZIP79, and JrbZIP88, 18 of the genes were differentially expressed. The expression of the JrbZIP55 gene in explants in agar and vermiculite media was not significantly different before 24 h but showed a significant increase in vermiculite after 48 h. The expression of three genes, JrbZIP70, JrbZIP72 and JrbZIP88, showed an increasing and then decreasing trend in vermiculite and a significant increase in agar. These results show that the expression of the four genes differed significantly under the two culture conditions. Additionally, since 'Clear Scent' explants in agar medium gradually began to brown with increasing culture time, it was speculated that these four genes, JrbZIP55, JrbZIP70, JrbZIP72 and JrbZIP88, might be related to the browning of walnut explants. The results were found to be consistent with the transcriptomic data, as verified by real-time quantitative (Additional file 9).

Fig. 5
figure 5

Browning of explants in different media. A: agar treatment, V: vermiculite treatment

Fig. 6
figure 6

Changes in the expression of JrbZIPs in different media cultures. Each value represents the average ± SE of three biological replicates

Cis element analysis and protein interaction prediction

The 2000 bp [40] promoter region elements upstream of the start codons of JrbZIP55, JrbZIP70, JrbZIP72 and JrbZIP88 were analyzed by PlantCARE (Fig. 7), and multiple hormone (salicylic acid, abscisic acid, growth hormone and gibberellin) signaling and stress (low temperature, stress and defense) regulatory elements were identified in their promoter regions. Among these regions, JrbZIP55 contained cis-acting elements involved in gibberellin and abscisic acid reactions and regulatory elements in response to stress and defense; JrbZIP72 contained only cis-acting elements involved in abscisic acid reactions; JrbZIP88 contained cis-acting elements involved in growth hormone and gibberellin reactions and regulatory elements involved in the response to low temperature stress; and JrbZIP70 contained cis-acting elements involved in erythromycin and salicylic acid reactions as well as regulatory elements involved in stress and defense responses.

Fig. 7
figure 7

Analysis of the cis-regulatory element of the promoter. Using the Plantcare website, we conducted promoter cis-acting element analysis on the four selected genes, JrbZIP55, JrbZIP70, JrbZIP72, and JrbZIP88. Different colors represent different components

Thus, the walnut JrbZIP55 protein was identified as being highly homologous to the Arabidopsis bZIP42 protein, the walnut bZIP72 protein was identified as being highly homologous to the Arabidopsis bZIP60 protein, the walnut bZIP70 protein was identified as being highly homologous to Arabidopsis bZIP28, and the walnut bZIP88 protein was identified as being highly homologous to Arabidopsis AHBP-1B (Fig. 8). These Arabidopsis proteins were used as references for protein interaction prediction. The results showed that the protein highly homologous to walnut JrbZIP55, Arabidopsis bZIP42, interacted with the oxidoreductase class AT2G19940 proteins, which are enzymes that act on aldehyde or oxygen-containing group donors, and it is hypothesized that JrbZIP55 is associated with the browning of walnut explants.

Fig. 8
figure 8

Protein interaction prediction. A The walnut bZIP55 protein and Arabidopsis bZIP42 protein are highly homologous proteins. B The walnut bZIP72 protein is highly homologous to the Arabidopsis bZIP60 protein. C The walnut bZIP70 protein is highly homologous to the Arabidopsis bZIP28 protein. D The walnut bZIP88 protein is highly homologous to the Arabidopsis AHBP-1B protein


Compared to Arabidopsis [41] (75 members with an average length of 321 amino acids, divided into 10 subfamilies (A, B, C, D, E, F, G, H, I and G) based on protein structural features), rice [42] (89 members with an average length of 311 amino acids), poplar [43] (86 members, divided into 12 subfamilies based on Arabidopsis as a reference), and castor [44] (49 members, divided into 11 subfamilies (I-XI) based on their gene structures, DNA-binding sites, conserved motifs, and phylogenetic relationships), walnut contains 65 bZIP transcription factors with an average length of 463 amino acids, and they have been classified into ten subfamilies (A, B, C, D, E, F, G, H, I and S) using Arabidopsis as a reference. In comparison with the conserved motifs found in bZIP transcription factors in other plants, a total of one motif (motif 1) in walnut and Arabidopsis and two motifs (motifs 3 and 4) in walnut and poplar were identified, indicating that these additional motifs outside the bZIP domain might be conserved among plant species. However, some additional motifs are variable among species and may be species-specific in plants.

Previous studies showed that BjubZIP54, a member of the S2 subfamily of the bZIP family in Capsella [45], was upregulated under both high temperature and drought stresses, presumably to regulate the expression of downstream related genes involved in abiotic stress response. The bZIP family transcription factor AcePosF21 in kiwifruit [46] mediates the regulation of AceGP3 expression and AsA biosynthesis, thereby reducing oxidative damage caused by cold stress. PhebZIP47 in moso bamboo [47] positively regulates drought tolerance in transgenic plants by regulating differentially expressed genes (DEGs), including genes in the drought tolerance regulatory pathway and ABA signaling pathway. It is evident that bZIP transcription factors regulate downstream related genes as well as the response to various stresses through hormonal signaling in most plants [44, 46]. In this study, based on family analysis and transcriptome data analysis, multiple hormone (salicylic acid, abscisic acid, growth hormone and gibberellin) signaling and stress (low temperature, stress and defense) response elements were also identified in the genes encoding four walnut transcription factors, JrbZIP55, JrbZIP70, JrbZIP72 and JrbZIP88, by promoter cis-acting element analysis, as were reciprocal protein prediction regulatory elements. It is hypothesized that bZIP genes may act as transcriptional regulators involved in stress response regulation in walnut plants.

The browning of plants after damage occurs mainly due to a decrease in the ascorbic acid content and increases in phenylalanine deaminase and lipoxygenase activities [48]. Some studies have found that there was a strong linear relationship between total phenolic compound content of microshoots and increasing antioxidant activity [26]. In contrast, AT2G19940 is present in the Arabidopsis chloroplast stroma as a donor reductase that acts on aldehydes or oxidative groups as an acceptor to participate in the amino acid metabolic synthesis process with NAD and NADP as acceptors [49]. In this study, AT2G19940 and AtbZIP42 were predicted to interact with each other. PgbZIP131 in ginseng [50] belongs to the S subfamily, which shows a strong response to abiotic stresses, where AtbZIP44 plays an important role in drought stress [51] and belongs to the same subfamily as AtbZIP42. Transgenic Arabidopsis thaliana overexpressing SibZIP8 from cereals [52] shows a higher germination rate under salt stress than the wild type. AtbZIP42 in Arabidopsis is the only protein that shows reasonable homodimeric activity in yeast [53]. AtbZIP42 and JrbZIP55 are homologous proteins, and it is speculated that the JrbZIP55 transcription factor may regulate the browning of walnut explants. Concerning the specific role the JrbZIP55 gene plays in walnut browning and which downstream structural genes are regulated to cause alterations in synthetic pathways that affect walnut exine browning, further elucidation of the molecular mechanisms of these specific regulatory syntheses involved in plant water stress is needed.


Sixty-five bZIP genes were identified in the walnut genome, distributed on sixteen chromosomes and divided into ten subfamilies. The expression of four genes, JrbZIP55, JrbZIP70, JrbZIP72, and JrbZIP88, showed significant differences in different media, and there were multiple signaling and regulatory elements in the promoter regions of these genes. The walnut JrbZIP55 protein and Arabidopsis bZIP42 protein are highly homologous, and the proteins interacting with Arabidopsis bZIP42 include the AT2G19940 oxidoreductases, which act on aldehyde or oxygen-containing donors. This suggests that JrbZIP55 may be involved in the regulation of walnut explant browning.


Plant materials and treatment

The test material 'Qingxiang' walnut (Juglans regia L.) was grown in the specimen garden of Hebei Agricultural University, rooted from cuttings, and was used at 6 years of age, with normal growth and fruiting. The identification of the 'Qingxiang' cultivar was performed by the Hebei Provincial Forestry Species Validation and Approval Committee in 2003, with accession number Ji S-ETS-JR-007–2003. No special permission was necessary to collect such samples. In mid-April, developing branches of uniform thickness and robust growth were collected from the middle and upper parts of the trees, cut into 3 cm stem segments, with each segment containing 1 or 2 full buds, and subsequently washed in the following sequence: laundry detergent water, water, 75% alcohol for 30 s for surface sterilization, 0.1% HgCl2 for 8 min for sterilization, and sterile water (6 times), after which they were transferred to vermiculite and agar medium containing DKW [54]. The materials were then grown at 25°C under a light intensity of 3000 lx and a 16 h light and 8 h dark cycle. Samples were collected at 0 h, 12 h, 24 h, and 48 h and snap frozen in liquid nitrogen for RNA-Seq. Seven different tissues were taken from leaf buds, female flowers, male flowers, mature leaves, young leaves, roots and fruits at different times of the growing season, with the older leaves taken on July 15 and the other six tissues taken on April 23. Three stem sections were mixed as a single sample, and three replicates were performed. RNA-Seq was carried out by Novogene Co. The experimental method of this study is shown in a flowchart (Additional file 10).

Identification and bioinformatics analysis of the bZIP gene family in walnut

Walnut whole-genome data were downloaded from the NCBI database ( Accessed 20 May 2023). The hidden Markov model files for bZIP transcription factors were downloaded from the Pfam database ( Accessed 20 May 2023), and these hidden Markov model files were used as search criteria. A program in hmmer3.0 software was used to search for walnut protein sequences (E ≤ 1 × 10 − 10), the obtained results were deduplicated, and extraction was performed by using the SMART ( Accessed 20 May 2023) and NCBI-CDD ( Accessed 21 May 2023) databases for further identification and screening, and the walnut bZIP family protein sequences were finally obtained (Additional file 2) [55]. The isoelectric point and molecular weight of walnut bZIP protein sequences were analyzed using the online tool ProtParam ( Accessed 21 May 2023).

We used the alignment result to construct a phylogenetic tree via the neighbor-joining method in MEGA7 software ( Accessed 22 May 2023). GSDS v2.0 ( Accessed on 26 May 2023) was used to predict the inline-exon structure of the bZIP gene family in walnut [56]. MEME ( Accessed on 26 May 2023) was used to analyze the motif composition of JrbZIP proteins (motif size setting: 6–50, number setting: 20, default values for the other parameters), and the results were visualized using the program “Gene Structure View (Advanced)” in TBtools software. The protein sequences of walnut were downloaded from GenBank (accession GCA 001411555.2). The types, numbers and functions of the cis-acting elements of the walnut bZIP gene promoters were analyzed using the PlantCARE ( Accessed on 26 May 2023) website. Finally, the results were visualized using the program “Simple Biosequence Viewer” from TBtools software. Information on the chromosomal position of each JrbZIP gene was obtained from the walnut gene GFF file, and duplication events in the genome were analyzed using MCScanX software (Additional file 5). The chromosomal localization of the walnut bZIP gene family was determined using the TBtools software program (Gene Location Visualize from GTF/GFF).

Physicochemical analysis of walnut JrbZIP protein

The isoelectric point and molecular weight of walnut bZIP protein sequences were analyzed using the online tool ProtParam ( Accessed on 26 May 2023). The online software Plant-mPLoc 2.0 ( Accessed on 26 May 2023) was used to predict and analyze the subcellular localization of walnut bZIP proteins. Protein interaction analysis was performed using STRING ( Accessed on 29 May 2023).

Transcriptional expression analysis of the JrbZIP gene in different tissues of walnut

We analyzed the expression of walnut bZIP genes in seven different tissues: buds, female flowers, male flowers, mature leaves, young leaves, roots, and fruits. We also used RNA-Seq data to screen differentially expressed genes using FPKM values as a gene expression measure and padj < 0.05 and log2(golden change > 0) as thresholds for screening differential expression. Data were also visualized using TBtools software to illustrate the content of walnut bZIP genes in buds, female flowers, male flowers, mature leaves, young leaves, roots, and fruits. The sequence data were deposited in GSA (

Transcriptional expression analysis of the JrbZIP gene in walnut explants

We analyzed the genes that were specifically expressed in stem segments based on RNA-Seq data from different tissues and analyzed the changes in the expression of bZIP genes associated with explants cultured in different media for 0 h, 12 h, 24 h and 48 h based on RNA-Seq explant data. The sequence data were deposited in GSA (

We used a kit extraction method for RNA extraction. The kits used for RNA extraction and DNA extraction were from Tiangen (product numbers: DP441 and DP350, respectively). The reagents used for reverse transcription, PCR and real-time PCR were from Takara (product numbers: RR047A, RR901A and RR820A, respectively). For RNA isolation experiments, all samples were immediately frozen in liquid nitrogen and extracted using a Tiangen Plant RNA Extraction Kit according to the manufacturer’s protocol. First-strand cDNA was synthesized with a Tiangen Inverse Transcription Kit [57].

Availability of data and materials

The RNA-Seq datasets are available in the GSA (submission number: CRA011411,; submission number: CRA011462, The CDS and genome sequences of bZIPs in walnut were retrieved from the whole walnut genome database (accession GCA_001411555.2) in NCBI. All data and materials are presented in the main paper and additional file.



Basic Leucine Zipper


Vermiculite-DKW medium


Agar-DKW medium


Differential gene expression


Mature leaves


Young leaves


Young stems


  1. Niu X, Renshaw-Gegg L, Miller L, Guiltinan MJ. Bipartite determinants of DNA-binding specificity of plant basic leucine zipper proteins. Plant Mol Biol. 1999;41(1):1–3.

    Article  CAS  PubMed  Google Scholar 

  2. Zg E, Zhang YP, Zhou JH, Wang L. Mini review roles of the bZIP gene family in rice. Genet Mol Res. 2014;13(2):3025–36.

    Article  Google Scholar 

  3. Lu ZX, Chang TJ, Liu X, Zhu Z. Research progress of plant basic leucine zipper (bZIP) protein (I) - structure, classification, distribution and homology analysis. Hereditas. 2001;(6):564–570.

    Google Scholar 

  4. Marc J, Bernd W, Wolfgang D, Jesus Vicente-Carba J, Jens T, Thomas K, François P. bZIP transcription factors in Arabidopsis. Trends Plant Sci. 2002;7(3):106–111.

    Article  Google Scholar 

  5. Kusano T, Berberich T, Harada M, Suzuki N, Sugawara K. A maize DNA-binding factor with a bZIP motif is induced by low temperature. Mol Gen Genet. 1995;248(5):507–517.

    Article  CAS  PubMed  Google Scholar 

  6. Stanković B, Vian A, Henry-Vian C, Davies E. Molecular cloning and characterization of a tomato cDNA encoding a systemically wound-inducible bZIP DNA-binding protein. Planta. 2000;212(1):60–66.

    Article  PubMed  Google Scholar 

  7. An JP, Qu FJ, Yao JF, Wang XN, You CX, Wang XF, et al. The bZIP transcription factor MdHY5 regulates anthocyanin accumulation and nitrate assimilation in apple. Hortic Res. 2017;4:17023

  8. Han X, Mao L, Lu W, Wei X, Ying T, Luo Z. Positive regulation of the transcription of AchnKCS by a bZIP transcription factor in response to ABA-stimulated suberization of kiwifruit. J Agric Food Chem. 2019;67(26):7390–7398.

    Article  CAS  PubMed  Google Scholar 

  9. Zhang Y, Gao W, Li H, Wang Y, Li D, Xue C, et al. Genome-wide analysis of the bZIP gene family in Chinese jujube (Ziziphus jujuba Mill.). BMC Genomics. 2020;21(1):483.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Xue C, Qiu S, Li H, Li SJ, Li X, Shang J, et al. Identification of tobacco bZIP gene family and expression analysis of subgroup A under ABA treatment. Mol Plant Breed. 2020;18(17):5607–21.

    Google Scholar 

  11. Sornaraj P, Luang S, Lopato S, Hrmova M. Basic leucine zipper (bZIP) transcription factors involved in abiotic stresses: a molecular model of a wheat bZIP factor and implications of its structure in function. Biochim Biophys Acta. 2016;1860(1 Pt A):46–56.

    Article  CAS  PubMed  Google Scholar 

  12. Alves MS, Dadalto SP, Gonçalves AB, De Souza GB, Barros VA, Fietto LG. Plant bZIP transcription factors responsive to pathogens: a review. Int J Mol Sci. 2013;14(4):7815–7828.

    Article  PubMed  PubMed Central  Google Scholar 

  13. Wu J, Zhu C, Pang J, Zhang X, Yang C, Xia G, et al. OsLOL1, a C2C2-type zinc finger protein, interacts with OsbZIP58 to promote seed germination through the modulation of gibberellin biosynthesis in Oryza sativa. Plant J. 2014;80(6):1118–1130.

    Article  CAS  PubMed  Google Scholar 

  14. Liu H, Su J, Zhu Y, Yao G, Allan AC, Ampomah-Dwamena C, et al. The involvement of PybZIPa in light-induced anthocyanin accumulation via the activation of PyUFGT through binding to tandem G-boxes in its promoter. Hortic Res. 2019;6:134.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Tu M, Wang X, Yin W, Wang Y, Li Y, Zhang G, et al. Grapevine VlbZIP30 improves drought resistance by directly activating VvNAC17 and promoting lignin biosynthesis through the regulation of three peroxidase genes. Hortic Res. 2020;7:150.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Reeves WM, Lynch TJ, Mobin R, Finkelstein RR. Direct targets of the transcription factors ABA-Insensitive(ABI)4 and ABI5 reveal synergistic action by ABI4 and several bZIP ABA response factors. Plant Mol Biol. 2011;75(4):347–363.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Chattopadhyay S, Ang LH, Puente P, Deng XW, Wei N. Arabidopsis bZIP protein HY5 directly interacts with light-responsive promoters in mediating light control of gene expression. Plant Cell. 1998;10(5):673–683.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Lu ZX, Chang TJ, Liu X, Zhu Z. Research progress of plant basic leucine zipper (bZIP) protein (II) - DNA binding characteristics, gene expression, function and application. Hereditas. 2002;02:182–189.

    Google Scholar 

  19. Bhat SN, Khalil A, Nazir N, Mir MA, Khan I, Mubashir SS, et al. In vitro prevention of browning in persian walnut (Juglans regia L.) cv. sulaiman. Int J of Plant Bio. 2022;13(3):330–342.

    Article  CAS  Google Scholar 

  20. Saltveit ME. Wound induced changes in phenolic metabolism and tissue browning are altered by heat shock. Postharvest Bio Technol. 2000;21(1):61–69.

    Article  CAS  Google Scholar 

  21. Su YL, Gao XM, Zhang XZ, Yang J, Wang L, Wang SK, et al. Fine mapping and candidate gene analysis of flesh browning in pear (Pyrus L.). Sci Horticulturae. 2022;302:111140.

    Article  CAS  Google Scholar 

  22. Dong TT, Cao Y, Li GC, Wang QG. Enhanced expression of serine protease inhibitor StSPI128 alleviates the enzymatic browning in fresh-cut potatoes via increasing antioxidant abilities. Postharvest Bio Tec. 2022;192:112022.

    Article  CAS  Google Scholar 

  23. Zhang PR, Fang DL, Pei F, Wang C, Jiang W, Hu QH, Ma N. Nanocomposite packaging materials delay the browning of Agaricus bisporus by modulating the melanin pathway. Postharvest Bio Tec. 2022;192:112014.

    Article  CAS  Google Scholar 

  24. Habibi A, Yazdani N, Chatrabnous N, Koushesh Saba M, Vahdati K. Inhibition of browning via aqueous gel solution of Aloe vera: a new method for preserving fresh fruits as a case study on fresh kernels of Persian walnut. J Food Sci Tech. 2022;59:2784–2793.

    Article  CAS  Google Scholar 

  25. Zhang ZH, Pei D. Walnut Science. Beijing: China Agricultural Publishing House; 2018.

    Google Scholar 

  26. Cheniany M, Ebrahimzadeh H, Vahdati K, Preece J, Masoudinejad A, Mirmasoumi M. Content of different groups of phenolic compounds in microshoots of Juglans regia cultivars and studies on antioxidant activity. Acta Physiol Plant. 2013;35:443–450.

    Article  CAS  Google Scholar 

  27. Lee A. Climate change brings need for new walnut. nut breeder says. Western Farm Press. 2021.

  28. Asayesh ZM, Vahdati K, Aliniaeifard S. Investigation of physiological components involved in low water conservation capacity of in vitro walnut plants. Sci Hortic. 2017;224:1–7.

    Article  Google Scholar 

  29. Cosmulescu S, Trandafir I. Anti-oxidant activities and total phenolics contents of leaf extracts from 14 cultivars of walnut (Juglans regia L.). J Hortic Sci Biotech. 2012;87(5):504–508.

    Article  Google Scholar 

  30. Zhao S, Wang H, Liu K, Li L, Yang J, An X, et al. The role of JrPPOs in the browning of walnut explants. BMC Plant Biol. 2021;21(1):9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Ye FT, Pan XF, Mao ZJ, Li ZW, Fan K. Molecular evolution and functional analysis of water lily transcription factor bZIP family. J Integr Agric. 2021;54(21):4694–708.

    Google Scholar 

  32. Tian WW, Yan ZX, Wang C, Yuan Q, Hua H, Liu L, et al. Analysis of WRKY transcription factor family based on full-length transcriptome sequencing in Polygonatum cyrtonema. China J Chin Mater Med. 2023;48(4):939–50.

    Google Scholar 

  33. Lindsay S, Hunt L, Gray J, Hetherington A. Analysis of the R2R3-MYB transcription factor family identifies genes involved in stomatal function. Comp. Biochem. Phys. A. 2008;150(3S):S195.

  34. Xu P, Wu F, Ma T, Yan Q, Zong X, Li J, et al. Analysis of six transcription factor families explores transcript divergence of cleistogamous and chasmogamous flowers in Cleistogenes songorica. DNA Cell Biol. 2020;39(2):273–288.

    Article  CAS  PubMed  Google Scholar 

  35. Hu JT, Ruan Y, Gan LP. Identification and expression analysis of the B-box transcription factor family in chili peppers. Hortic Plant J. 2021;48(05):987–1001.

    Google Scholar 

  36. Li P, Zheng TC, Li LL, Wang J, Cheng TR, Zhang QX. Genome-wide investigation of the bZIP transcription factor gene family in Prunus mume: Classification, evolution, expression profile and low-temperature stress responses. Hortic Plant J. 2022;8(2):230–342.

    Article  CAS  Google Scholar 

  37. Huang S, Yu J, Li Y, Wang J, Wang X, Qi H, et al. Identification of soybean genes related to fatty acid content based on a soybean genome collinearity analysis. J Agric Food Chem. 2019;67(1):258–274.

    Article  CAS  PubMed  Google Scholar 

  38. Zhong S, Ma L, Fatima SA, Yang J, Chen W, Liu T, et al. Collinearity analysis and high-density genetic mapping of the wheat powdery mildew resistance gene Pm40 in PI 672538. PLoS ONE. 2016;11(10):e0164815.

    Article  PubMed  PubMed Central  Google Scholar 

  39. Liu BY, Wang L, Wang SM, Li WJ, Liu D, Guo XF, et al. Transcriptomic analysis of bagging-treated‘Pingguo’pear shows that MYB4-like1, MYB4-like2, MYB1R1 and WDR involved in anthocyanin biosynthesis are up-regulated in fruit peels in response to light. Sci Hortic. 2019;244:428–434.

    Article  CAS  Google Scholar 

  40. Zhang Y, Zou BH, Lu S, Ding Y, Liu H, Hua J. Expression and promoter analysis of the OsHSP16.9C gene in rice. Biochem Bioph Res Co. 2016;479(2):260–265.

    Article  CAS  Google Scholar 

  41. Jakoby M, Weisshaar B, Dröge-Laser W, Vicente-Carbajosa J, Tiedemann J, Kroj T, et al. bZIP transcription factors in Arabidopsis. Trends Plant Sci. 2002;7(3):106–111.

    Article  CAS  PubMed  Google Scholar 

  42. Nijhawan A, Jain M, Tyagi AK, Khurana JP. Genomic survey and gene expression analysis of the basic leucine zipper transcription factor family in rice. Plant Physiol. 2008;146(2):333–350.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Zhao K, Chen S, Yao W, Cheng Z, Zhou B, Jiang T. Genome-wide analysis and expression profile of the bZIP gene family in poplar. BMC Plant Biol. 2021;21(1):122.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Jin Z, Xu W, Liu A. Genomic surveys and expression analysis of bZIP gene family in castor bean (Ricinus communis L.). Planta. 2014;239(2):299–312.

    Article  CAS  PubMed  Google Scholar 

  45. Dai CY, Zhang XH, Wang XL, Gong Q, Su X, Cheng YH, et al. Identification and expression analysis of mustard bZIP gene family. Molec plant breed. 2023;1–17.

  46. Liu X, Bulley SM, Varkonyi-Gasic E, Zhong C, Li D. Kiwifruit bZIP transcription factor AcePosF21 elicits ascorbic acid biosynthesis during cold stress. Plant Physiol. 2023.

    Article  PubMed  PubMed Central  Google Scholar 

  47. Lan YG, Pan F, Zhang KM, Wang LN, Liu HX, Jiang CZ, et al. PhebZIP47, a bZIP transcription factor from moso bamboo (Phyllostachys edulis), positively regulates the drought tolerance of transgenic plants. Ind Crops Prod. 2023;197:116538.

    Article  CAS  Google Scholar 

  48. Zhang L, Wang ZQ, Zeng SX, Yuan SZ,Yuexz, Tian T, et al. Browning mechanism in stems of fresh-cut lettuce. Food Chem. 2023;405:134575.

    Article  CAS  Google Scholar 

  49. Rutschow H, Ytterberg AJ, Friso G, Nilsson R, van Wijk KJ. Quantitative proteomics of a chloroplast SRP54 sorting mutant and its genetic interactions with CLPC1 in Arabidopsis. Plant Physiol. 2008;148(1):156–175.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Wang SJ, Sun JY, Liu MQ, Ran MC, Yu XX, Liu XB, et al. Bioinformatics analysis of ginseng bZIP gene family. Chinese Traditional and Herbal Drugs. 2022;53(9):2786–2794.

    Google Scholar 

  51. Weltmeier F, Rahmani F, Ehlert A, Dietrich K, Schütze K, Wang X, et al. Expression patterns within the Arabidopsis C/S1 bZIP transcription factor network: availability of heterodimerization partners controls gene expression during stress response and development. Plant Mol Biol. 2009;69(1–2):107–119.

    Article  CAS  PubMed  Google Scholar 

  52. Liu P, Wu YM, Wu QQ, Li XY. Identification of bZIP gene family in millet and green bristlegrass and functional study of SibZIP8 gene in millet. J Shanxi Agri. Sci. 2020;48(9):1361-1370

  53. Ehlert A, Weltmeier F, Wang X, Mayer CS, Smeekens S, Vicente-Carbajosa J, et al. Two-hybrid protein-protein interaction analysis in Arabidopsis protoplasts: establishment of a heterodimerization map of group C and group S bZIP transcription factors. Plant J. 2006;46(5):890–900.

    Article  CAS  PubMed  Google Scholar 

  54. Wang T, Yan T, Shi J, Sun Y, Wang Q, Li Q. The stability of cell structure and antioxidant enzymes are essential for fresh-cut potato browning. Food Res Int. 2023;164:112449.

    Article  CAS  PubMed  Google Scholar 

  55. Zhu XL, Wang BQ, Wei XH. Identification and expression analysis of the CqSnRK2 gene family and a functional study of the CqSnRK2.12 gene in quinoa (Chenopodium quinoa Willd.). BMC Genomics. 2022;23(1):134575.

    Google Scholar 

  56. Yan L, Wang CP, Chen JW, Qiao GX, Li J. Analysis of MYB transcription factor family of Lycium barbarum based on transcriptome information. J Integr Agric. 2017;50(20):3991–4002.

    Google Scholar 

  57. Liu K, Zhao SG, Wang S, Wang HX, Zhang ZH. Identification and analysis of the FAD gene family in walnuts (Juglans regia L.) based on transcriptome data. BMC genomics. 2020;21:299

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references


We would also like to thank Dr. Xiuhong An, Dr. Yi Tian, and Dr. Hongxia Wang for valuable suggestions on the data analysis and manuscript.


This work was supported by the Natural Science Foundation of Hebei Province (C2019204270), the Science and Technology Research Project of the Universities of Hebei Province (QN2019159), and Research Foundations for Returned Scholars from Overseas of the Human Resources Dept. of Hebei Province (C20190345). Funds were used for the design of the study, sample collection, analysis or interpretation of data and in writing the manuscript.

Author information

Authors and Affiliations



HW and SZ designed the research. HW, SZ and JP performed the experiments, analyzed the data and wrote the paper. YL, LX,and WD participated in the data analysis. All authors read and approved the final the manuscript.

Corresponding author

Correspondence to Shugang Zhao.

Ethics declarations

Ethics approval and consent to participate

All plant materials were used following national and international standards and local laws and regulations. Using all plant materials does not pose any risk to other species in nature. No specific permission is required to collect all samples described in this study.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1: 

bZIP protein sequence of Arabidopsis thaliana.

Additional file 2: 

bZIP protein sequence of Juglans regia.

Additional file 3: 

bZIP CDS Sequence of Juglans regia.

Additional file 4:

 bZIP gene sequence of Juglans regia.

Additional file 5: 

Collinearity data of JrbZIP gene family.

Additional file 6: Table S1.

Information  of JrbZIP gene family.

Additional file 7: Table S2.

Primers used in real-time PCR.

Additional file 8: Figure S1.

Enzymatic activities of explant during culture in different medium.

Additional file 9: Figure S2.

Changes in the expression of some genes.

Additional file 10: Figure S3.

Experimental method flowchart.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wang, H., Peng, J., Li, Y. et al. The role of walnut bZIP genes in explant browning. BMC Genomics 24, 377 (2023).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: