The LBD gene family members in sour jujube were identified using two methods. Initially, 43 LBD genes from Arabidopsis thaliana and 57 LBD genes from poplar were downloaded from the Arabidopsis Information Resource (TAIR, http://www.Arabidopsis.org/) and the Phytozome database (http://rice.plantbiology.msu.edu/), respectively. A blastp search (E < 1 × 10^-5) was conducted against the Telomere-to-Telomere (T2T) genome of sour jujube (Li et al., 2024) using the 43 Arabidopsis LBD genes as queries. Subsequently, the Hidden Markov Model (HMM) file for the LOB domain (PF03195) was downloaded from the Pfam database (http://pfam.sanger.ac.uk/), and the sour jujube LBD protein sequences were obtained using the HMMER program (version 3.2.1). The program was run with default settings and a cutoff value of 10^-5. Finally, an intersection of the LBD proteins identified by both methods was taken, retaining only those protein sequences encoded by the longest transcripts of the same gene. The LOB domain in ZjLBDs were verified using InterPro (http://www.ebi.ac.uk/interpro/) and SMART (http://smart.embl-heidelberg.de/) according to Letunic et al. (2012).

Six AtLBD and six PtLBD sequences were selected as reference sequences from different subgroups. The full-length LBD protein sequences in sour jujube were aligned using DNAMAN (version 6.0) to delineate the LOB domain. A phylogenetic tree was constructed using the neighbor-joining method with 1000 replicates in MEGA (version X). Based on phylogenetic relationships, the LBD proteins in sour jujube were categorized into distinct classes and subgroups, following the classification methods of AtLBD (Shuai et al., 2002) and PtLBD (Zhu et al., 2007).

The open reading frame (ORF) length, molecular weight (MW), and isoelectric point (pI) of LBD proteins in sour jujube were calculated using the ExPASy website (http://web.expasy.org/protparam/). The conserved motifs of LBD proteins in sour jujube were identified using MEME (version 5.1.0, http://meme-suite.org/tools/meme), with the maximum number of motifs set to 20 (Bailey et al., 2009).

The chromosomal localization of ZjLBD genes was determined using MapInspect (http://www.softsea.com/review/MapInspect.html) and aligned to the genomic chromosomes. The selective pressure on ZjLBD genes was assessed through Ka/Ks ratios. The exon-intron structure of ZjLBD genes was predicted by comparing the coding sequence (CDS) with their corresponding full-length DNA sequences using the Gene Structure Display Server (http://gsds.cbi.pku.edu.cn) (Hu et al., 2015). Visualization was performed using TBtools (version 1.0) (Chen et al., 2020).

A 2.0-kb sequence upstream of the transcription start site of ZjLBD was extracted from the sour jujube genome and submitted to the PlantCARE website (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) to identify cis-regulatory elements involved in hormone signaling, defense and stress responses, and growth and development regulation (Lescot et al., 2002).

To identify the potential TF genes binding with ZjLBDs promoters, the promoter sequences of ZjLBDs were retrieved. Following the method described by Tian et al. (2020), the binding site prediction tool available on the PlantRegMap platform (https://plantregmap.gao-lab.org/index.php) we utilized to scan for TF binding sites with a threshold p-value ≤ 1e^-4. The potential TF genes identified through this process were further subjected to Gene Ontology (GO) enrichment analysis. GO terms with a p-value < 0.01 were selected as significantly enriched terms.

To investigate the expression patterns of ZjLBD genes across various tissues, RNA-seq datasets from 9 distinct tissue types—namely root, leaves, flower, stem, branch, young fruit, white mature fruit, half-red fruit, and fully red fruit—were retrieved from the National Center for Biotechnology Information (NCBI) database under Sequence Read Archive (SRP046073) (Liu et al., 2014). After filtering the raw data, SRA files were converted to FASTQ format through SRA Toolkit (version 2.8.2). Quantitative gene expression analysis was conducted using Cufflinks (version 2.2.1) on the aligned read files, with gene expression levels being determined based on the Fragments Per Kilobase per Million mapped reads (FPKM).

To further confirm the differential expression levels of ZjLBD genes identified in RNA-seq in different tissues, samples of root, leaves, flower, stem, branch, and fruit were collected from three ‘Dongzao’ jujube trees for experiments in 2024.

To analyze the expression levels of ZjLBD genes under abiotic stresses, 45-day-old rooted sour jujube seedlings were transferred to Hoagland nutrient solution for 1 week and then subjected to simulated drought and salt conditions. For drought treatment, seedlings were placed in a 20% PEG6000 solution, and leaves were collected after 6 h, 12 h, and 48 h of treatment. For salt treatment, seedlings were placed in Hoagland nutrient solution containing 150 mmol/L NaCl, and leaves were collected after 24 h and 48 h of treatment. Untreated seedlings served as control group for both drought and salt treatments.

For cold stress, branches with consistent growth vigor were selected from three Dongzao jujube trees. These branches were then exposed to temperatures of 4 °C, -10°C, -20°C, -30°C, and -40°C for 10 h at a cooling rate of 5 °C/h, and the branches treated at 4 °C were used as control samples. Then the xylem of branches was collected.

All experiments were conducted with three biological replicates. After collection, samples were flash-frozen in liquid nitrogen and stored at -80°C for qRT-PCR analysis.

RNA extraction was performed using the Aidlab RNA Extraction Kit (Aidlab Biotechnologies Co., Ltd, Beijing, China). Following quantification and integrity assessment of the extracted RNA, qRT-PCR analysis was conducted using a Roche Light Cycler 96 instrument with the 2^-ΔΔCt method (Livak and Schmittgen, 2001). The primers used are listed in Supplementary Table 1 .

A combination of two bioinformatics approaches was employed to identify members of the LBD gene family in sour jujube. Initially, we performed a BLASTP search using 43 LBD TFs from Arabidopsis thaliana as queries, identifying 38 candidate LBD proteins in sour jujube. Subsequently, we utilized a HMM based on the LOB domain (Pfam PF03195) to identify 38 LBD proteins. By intersecting the results from both methods and removing redundant sequences, we ultimately identified 37 unique LBD genes for further analysis ( Table 1 ).

Based on the positional information of LBD genes on chromosomes, the 37 LBD genes were designated as ZjLBD1-ZjLBD37. A comprehensive analysis was conducted on these genes and their encoded proteins, encompassing gene location, ORF length, CDS length, MW, and pI. As shown in Table 1 and Figure 1 , ZjLBDs are distributed across 11 chromosomes. The CDS lengths range from 468 bp (ZjLBD25) to 1002 bp (ZjLBD16), while the ORF lengths of the LBD proteins vary from 155 amino acids (ZjLBD25) to 333 amino acids (ZjLBD16). The MW of the proteins span from 17.72 kDa (ZjLBD25) to 37.55 kDa (ZjLBD16), and the predicted pI range from 5.08 (ZjLBD10) to 9.17 (ZjLBD1).

Fifteen homologous gene pairs were detected among nine chromosomes in the ZjLBDs, representing inter-chromosomal collinearity events. Notably, ZjLBD23 located on Chr6 and ZjLBD34 on Chr9 were identified as high-frequency genes for collinearity events, each occurring three times.

To assess the selective pressure on these colinear ZjLBD genes during the evolutionary process, the ratio of the number of nonsynonymous substitutions per nonsynonymous site (Ka) to the number of synonymous substitutions per synonymous site was analyzed ( Supplementary Table 2 ). The results indicated that for 13 colinear events involving 19 ZjLBD genes, 11 pairs of ZjLBD genes exhibited Ka/Ks ratios less than 1, suggesting that these genes were under purifying selection.

A phylogenetic analysis was conducted on 43 Arabidopsis and 57 poplar LBD proteins along with the LBD family proteins of sour jujube ( Figure 2 ). These proteins were classified into two major groups, Class I and Class II, which were further divided into four (subgroups Ia to Id) and two subgroups (subgroups IIa to IIb), respectively. Class I contained 31 ZjLBD proteins, while Class II included only 6. Similarly, the number of LBD proteins in Arabidopsis and poplar was higher in Class I than in Class II.

Six AtLBD proteins (AT4G37540-LBD39, AT1G68510-LBD42, AT1G07900-LBD1, AT2G42430-LBD16, AT1G36000-LBD5, AT5G63090-LOB) and six PtLBD proteins (Potri.005G145500, Potri.008G120600, Potri.010G217700, Potri.002G041100, Potri.001G196400, Potri.007G039500) were randomly selected from the six subgroups. Their LOB domains were used as references to identify the domains in ZjLBDs. As shown in Figure 3 , all 37 ZjLBD proteins possess a conserved N-terminal motif composed of cysteine residues (CX2CX6CX3C) indicating their DNA-binding capabilities. The C-terminus of Class I contains a leucine zipper-like motif (LX6LX3LX6L), which is associated with protein dimerization. Additionally, subgroups Ia, Ib, and Ic contain a conserved glycine-alanine-serine (GAS) domain near the C-terminus, which includes conserved proline and glycine residues.

Structural analysis of the ZjLBD genes and their encoded proteins revealed distinct motif compositions and gene structures. As depicted in Figures 4A, B , ZjLBD proteins contain 10 motifs, with motif 1 and motif 2 consistently present at the N-terminus. Class I and Class II proteins exhibit different motif compositions, with motif 3 being unique to Class II. Notably, all Class I proteins, except for ZjLBD1 and ZjLBD18, contain motif 6. Additionally, unique motifs (motifs 5, 7, 8, 9, and 10) are found in only 2 to 6 ZjLBD proteins within Class I, suggesting that these proteins may have distinct functions.

The coding and non-coding regions of the ZjLBD genes were also analyzed. The lengths of all ZjLBD genes were within 4500bp, with the numbers of coding region varying from one (ZjLBD1, ZjLBD2, ZjLBD3, ZjLBD5, ZjLBD6, ZjLBD12, ZjLBD15, ZjLBD20, ZjLBD30) to three (ZjLBD25, ZjLBD26). As shown in Figure 4C , the structures of Class I and Class II genes were distinctly different, with more than half of the ZjLBD genes in Class I exhibiting longer intron sequences compared to those in Class II.

A total of 100 types of cis-acting elements were detected in the ZjLBDs promoters, including 8 unnamed elements, which were annotated as having 43 functions. The TATA-box was the most frequently detected element, present in all ZjLBDs promoters. For further analysis, 14 types of functional annotations were selected. As shown in Figure 5 , the anaerobic induction element (ARE) and the abscisic acid response element (ABRE) were present in all ZjLBDs. Specifically, 8, 7, and 6 ABREs were found in the promoters of ZjLBD28, ZjLBD29, and ZjLBD35, respectively. At least one of the five hormone response elements was present in each ZjLBD promoter, and eight methyl jasmonate responsiveness elements have been identified in the promoter of ZjLBD11. Notably, drought induction element coexisted with ABRE in 14 ZjLBDs promoters. Of the 13 ZjLBDs promoters containing auxin response elements, four types were identified: AuxRR-core, TGA-element, TGA-box, and AuxRE. Except for ZjLBD5 and ZjLBD12, all of these ZjLBDs promoters co-occur with elements annotated in growth-related pathways. The promoters of 24 ZjLBDs contain 6 types of development-related elements associated with cell cycle regulation, seed-specific expression, meristem and endosperm development, and circadian rhythm control, with meristem expression-related element identified in 11 of these promoters. Additionally, 9 types of site-binding elements were identified in the ZjLBDs promoters, among which the MYB binding site involved in drought-inducibility, was the most common. These findings suggest that ZjLBD genes promoters harbor cis-acting elements associated with hormone responsiveness, stress induction, and growth-related functions, highlighting their potential roles in stress adaptation and developmental regulation.

We conducted a comprehensive analysis of transcriptional regulatory sites in the promoters of ZjLBDs, and identified 350 potential upstream genes encoding 39 types of TFs that collectively have 11,261 binding sites across the promoter sequences of 37 ZjLBDs. Among these ZjLBDs, the promoter of ZjLBD36 was predicted to possess the highest number of TF binding sites, totaling 516, and it may interact with 166 upstream TF genes. The Dof has the most binding sites with the ZjLBDs promoters. Additionally, the highest numbers of genes encoding ERF and MYB were predicted, with 44 and 41 genes, respectively. As shown in Figures 6A–F , the interaction regulatory network analysis of upstream TF binding to the promoters of ZjLBDs in six subgroups revealed that subgroup Ia is centered around ZjLBD19, which is likely regulated by 87 genes encoding TFs. Subgroup Ib includes ZjLBD14 and ZjLBD30, which may be regulated by 103 and 112 TF genes, respectively. Subgroup Ic is represented by ZjLBD29, regulated by 187 TF genes, while subgroup Id is represented by ZjLBD26, regulated by 172 TF genes. Subgroup IIa is centered around ZjLBD34, regulated by 147 TF genes, and subgroup IIb is centered around ZjLBD17, regulated by 189 TF genes. These findings reveal the intricate and diverse transcriptional regulatory modules of ZjLBDs across different subgroups, providing a basis for elucidating their functional divergence.

The TF genes potentially binding to the ZjLBDs promoters were subjected to Gene Ontology (GO) enrichment analysis, revealing that 342 TF genes were enriched in 415 GO terms ( Figure 6G ). The GO enrichment analysis indicated that the upstream TF genes of ZjLBDs in each subgroup were significantly enriched in the Biological Process (BP) terms regulation of nitrogen compound metabolic process and response to stimulus. Notably, the enriched p-values for response to stimulus and response to abiotic stimulus in the upstream TF genes of ZjLBD genes in Class II were higher than those in Class I. Meanwhile, the annotation count of upstream TF genes in response to osmotic stress and response to salt stress was lower in Class II than in Class I, yet the number remains greater than 18. Except for subgroup Ia, which lacked upstream gene annotations for response to cold, all other subgroups had more than 10 genes annotated in this pathway. Additionally, approximately 10 genes were annotated in pathways related to xylem development and phloem or xylem histogenesis. Five subgroups on average possess 27, 19 and 4 upstream TF genes annotated in fruit development, meristem development, and formation of organ boundary. In the Molecular Function (MF) category, the most abundant term was DNA binding. Significant differences were observed between Class I and II in terms of protein dimerization activity and specific DNA-binding functions. Specifically, subgroup IIa has more than 40 fewer genes annotated in transcription factor activity, sequence-specific DNA binding compared to Class I, and no genes were annotated in transcription factor binding. Finally, no significant differences were observed in the Cellular Component (CC) annotations of the potential upstream genes across the six subgroups. Therefore, although the upstream TF genes of ZjLBDs in different subgroups exhibit similar functions, these factors form modules that may exhibit hierarchical or intensity-dependent characteristics in tissue development and stress adaptation.

Based on publicly available RNA-seq data, the expression levels of ZjLBD genes were analyzed across six different tissues: root, leaves, flower, stem, branch and fruit ( Figure 7A ). The results revealed that different ZjLBDs showed distinct expression patterns across various plant tissues. Notably, 11 ZjLBD genes from subgroups Ia, Ib, Id, and IIa exhibited higher expression levels in flowers compared to other tissues. The root tissue contained 7 highly expressed ZjLBD genes, including ZjLBD7 (subgroup Ia) and ZjLBD22 (subgroup IIb), whose expression levels were 312-fold (for semi-red fruits) and 16-fold (for fully red fruits) higher than those in the least expressing tissue, respectively. Furthermore, ZjLBD12 and ZjLBD23 were highly expressed in flowers, whereas ZjLBD15 was predominantly expressed in stems, and ZjLBD19 and ZjLBD37 were highly expressed in branches. Notably, there was almost no high expression of ZjLBD genes in leaves. The expression levels of ZjLBD genes in jujube fruits were generally low, with only 6 ZjLBD genes exhibiting FPKM values exceeding 20. When comparing the expression of ZjLBD genes across four developmental stages of fruit, 8 ZjLBD genes from five subgroups showed elevated expression during the white mature stage. These ZjLBD genes included members from subgroup IIb, as well as ZjLBD11 (subgroup Ia) and ZjLBD37 (subgroup IIa).

To validate the expression levels of these genes across different tissues, ZjLBD7, ZjLBD11, ZjLBD13, ZjLBD14, ZjLBD17, ZjLBD22, ZjLBD23 and ZjLBD28 were further selected for qRT-PCR analysis ( Figures 7B–I ). The expression level of ZjLBD17 was significantly lower in leaves, while it was relatively higher in fruits. In contrast, ZjLBD7 and ZjLBD11 exhibited lower expression levels in fruits compared to in other tissues. Notably, ZjLBD11, ZjLBD13, ZjLBD22, and ZjLBD28 had higher expression levels in roots, while ZjLBD7 and ZjLBD23 were more highly expressed in flowers. During the four developmental stages of fruit development, ZjLBD14 showed a higher expression level in young fruit, where it reached the highest expression, with the maximum fold change being 45 times higher than that in stems. Meanwhile, ZjLBD13, ZjLBD14, and ZjLBD23 exhibited distinct differential expression patterns. Specifically, ZjLBD13 and ZjLBD14 showed a pattern of initial decrease, followed by an increase, and then a decrease again, while ZjLBD23 demonstrated a pattern of initial increase followed by a sharp decline, with its expression level dropping 84-fold from the white mature stage to the semi-red stage. The expression patterns of these genes in different tissues reveal their potential roles in the development of specific tissues.

Based on the analysis of promoter and upstream transcription factor functions, 12 ZjLBD genes—ZjLBD9, ZjLBD11, ZjLBD13, ZjLBD14, ZjLBD17, ZjLBD19, ZjLBD22, ZjLBD23, ZjLBD24, ZjLBD28, ZjLBD33 and ZjLBD35—from six subgroups were selected for analysis of their expression levels under three independent stress treatments: low temperature, drought, and salt conditions ( Figures 8A–L ). The results indicated that, except for ZjLBD14, the other ZjLBD genes exhibited distinct expression patterns under different low-temperature environments. Specifically, ZjLBD22 was down-regulated at -10°C, with a 3.5-fold decrease compared to its expression at 4°C, while ZjLBD9 showed a 6-fold increase at -10°C relative to at 4°C. At -30°C, ZjLBD28 exhibited an approximately 383-fold increase in expression compared to 4°C, whereas ZjLBD13 showed a 9-fold decrease. Furthermore, ZjLBD19 expression was downregulated at -20°C and remained low even at -40°C. Under drought stress, the expression level of ZjLBD14 remains significantly higher for 48 h of drought treatment than that under normal conditions. The expression level of ZjLBD23 significantly decreased by 2-fold within 6 h of drought stress but returned to normal levels thereafter. Meanwhile, the expression of ZjLBD33 was upregulated by 5-fold after 6 h of drought treatment. Additionally, after 48 h of drought treatment, two genes—ZjLBD9 and ZjLBD35—showed significant upregulation compared to the control group, with increases of 3-fold and 4-fold, respectively. Meanwhile, ZjLBD11 exhibited a significant reduction. In response to salt stress, the expression levels of ZjLBD13, ZjLBD22, ZjLBD23, ZjLBD24, ZjLBD28, and ZjLBD35 gradually increased with the duration of salt stress. Notably, after 48 h of salt stress, the expression level of ZjLBD22 was 21-fold that of the control group. Specifically, the expression of ZjLBD9 was approximately halved after 24 h of salt stress compared to the untreated control group. The expression patterns of these genes reveal their regulatory function under different stress conditions, providing important clues for understanding gene functions.