Introduction

Plant growth and development relies on complex phytohormonal regulatory networks that include ethylene1,2, jasmonic acid3,4, cytokinins5, gibberellins6, abscisic acid7,8, and brassinosteroids (BRs)9. Ethylene, a gaseous phytohormone, governs seed dormancy, organ development, stress responses, and notably fruit ripening. Ethylene also serves as a crucial mediator between external environmental changes and internal plant developmental responses10,11,12. In climacteric fruits, ripening involves synchronized ethylene production spikes and respiratory bursts, directly controlling sugar accumulation, color change and texture modification13. Intriguingly, previous studies found that ethylene plays an important role in controlling ripening of non-climacteric fruit (like jujube), which maintain low ethylene content14,15,16,17. Comprehending the molecular mechanisms driving ethylene biosynthesis and its regulation is crucial for deciphering the orchestration of fruit maturation.

The ethylene biosynthesis pathway requires two committed enzymatic reactions. In the first progress, S-adenosyl-L-methionine (SAM) is converted to ACC and 5’-methylthioadenosine (MTA) by the enzyme ACS. In the second, ACC is converted to ethylene, CO2 and cyanide by the enzyme ACO18. These rate-limiting enzymes are evolutionarily conserved across plants, with gene family expansions reflecting functional specialization. In plants, the coding genes and the functions of ACS and ACO enzymes have been extensively investigated18,19. In Arabidopsis thaliana, there are 10 AtACSs and 5 AtACOs, and nine of the AtACS (AtACS1, AtACS2, AtACS4-9, and AtACS11) exhibit catalytic functions, only AtACS3 functions as a pseudogene, while AtACS10 and AtACS12 exhibit aminotransferase activity. These AtACS proteins are divided into 3 types based on which interaction domains are in their sequences. Type 1 AtACS proteins have target sites for mitogen-activated and calcium-dependent protein kinases (MAPKs and CDPKs, found in AtACS1, AtACS2, and AtACS6, respectively), Type 2 AtACS (AtACS4, AtACS5, AtACS8, AtACS9, and AtACS11) are targets for CDPKs and E3 ligases, and the Type 3 AtACS contains no known target sites (AtACS7)20. Likewise, the AtACO protein family can be divided into three types based on amino acid sequence similarity of the conserved dioxygenase-specific RXS motif: Type 1 (AtACO2, AtACO3, AtACO4), Type 2 (AtACO1), and Type 3 (AtACO5)21. In tomato, there are 14 SlACS genes and 6 SlACO genes. SlACS2, SlACS4, SlACO1 and SlACO4 showed distinct expression patterns during fruit ripening and are the main SlACS and SlACO supporting ripening-associated ethylene production22. In strawberry, 5 FaACSs and 4 FaACOs were identified and showed different expression tendencies during fruit ripening23. In pear, 11 PuACSs and 8 PuACOs were expressed during different stages of ripening, and 4 PuACSs and 3 PuACOs genes could be induced by ethephon and inhibited by 1-MCP treatment24.

Additionally, diverse regulatory mechanisms have been extensively documented for the roles that ACSs and ACOs play in fruit maturation. Several studies have revealed that the expression of ACO and ACS was regulated by multiple TFs, including Ethylene Response Factor (ERF), NAM/ATAF/CUC2 (NAC), and WRKY. In apple, MdERF2 negatively regulates the synthesis of ethylene and the fruits ripening by inhibiting the transcription of MdACS125. Furthermore, the TFs MdERF008 and MdNAC71 bound to the promoter region of MdACS1 and directly control its transcription, thereby controlling ethylene biosynthesis in apple fruits26. In tomato, SlNAC4 and SlNAC9 bind with LeACS2, LeACS4, and LeACO1 promoters, affecting ripening of tomato fruits27. In kiwifruit, AcWRKY4 is involved in postharvest kiwi ripening by binding to the promoters of AcACS1 and AcACS2 to activate their expression28. Despite these advances in climacteric fruits, functional studies remain scarce for non-climacteric species beyond basic expression profiling in strawberry29, sand pear30, and grape31, highlighting a critical knowledge gap in ethylene regulation diversity.

The fresh jujube cultivar ‘Dongzao’ is appreciated by consumers for its flavor, crispness, and nutritional value, and is cultivated worldwide for its economic value32,33. However, postharvest fresh jujube fruits exhibit high respiration rates and rapid water loss, resulting in a very short shelf life. When stored at ambient temperatures, shelf life remains marketable for only 3–5 days, severely limiting their transport and market reach34. Therefore, elucidating the regulatory mechanisms underlying jujube fruit ripening is critical for extending postharvest storage, enhancing fruit quality, optimizing harvest timing, and advancing varietal improvement to boost economic benefits. The role of ethylene in regulating non-climacteric fruit ripening has long been debated. However, emerging studies suggest that ethylene, despite maintaining low levels, plays a significant regulatory role in the maturation of non-climacteric fruits14,15,16,17. As a non-climacteric fruit, the ZjACS and ZjACO gene families, key enzymes for ethylene synthesis in ‘Dongzao’ jujube, have not been adequately explored, resulting in a restricted comprehension of the ZjACS and ZjACO gene families. To minimize this comprehension gap, we first performed a genome-wide analysis to identify the ZjACS and ZjACO genes in ‘Dongzao’. Then we extensively investigated the evolutionary relationships, gene structures, conserved motifs, collinearity relationships, and cis-regulatory elements of the ZjACS and ZjACO genes. Additionally, we investigated their expression profiles and co-expression networks with various fruit ripening-associated transcription factors during fruit maturation. Our findings provide light on the rich variety of the ZjACS and ZjACO gene families, as well as their possible functions in ethylene biosynthesis and fruit maturation in jujube.

Materials and methods

Plant materials

Fruits were sampled from healthy, 10-year-old jujube trees of the cultivar ‘Dongzao’ in Anyang City, Henan Province, China (geographical location: 34°51′35′′ N, 112°42′51′′ E) at 40, 55, 70, 85, 100, 115 and 130 days after pollination (DAP). The seven sampling time points were between mid-July and the end of October in 2023, and named as S1, S2, S3, S4, S5, S6 and S7. At each stage, twenty fruits were mixed as one biological replicate, and each stage had three replicates. After removing the seed, sampls were quickly frozen in liquid nitrogen and kept at -80 °C for extracting RNA.

Extraction and determination of ACS and ACO activities

The activities of ACS and ACO enyzemes in jujube were assessed using ELISA detection kits for Plant ACC Synthase (ACS) and Plant ACC Oxidase (ACO) from Shanghai Enzyme-linked Biotechnology Co., Ltd. (mlbio, Shanghai, China). Fresh jujube samples from different stages (− 100 mg) were crushed in liquid nitrogen before being transferred to a 2-mL centrifuge tube, kept on ice, and mized with 0.9 mL of homogenizing solution (PBS, pH 7.4, 0.01 mol/L). The mixture was centrifuged at 8000 g for 15 min. The supernatant was collected for analysis.

Identification of ACS and ACO genes in ‘Dongzao’ genome

To identify the ACS and ACO proteins from three Ziziphus jujuba cultivars, ‘Dongzao’, ‘Suanzao’ and ‘Junzao’, the newest jujube genomes with gene annotation were acquired from NCBI (https://www.ncbi.nlm.nih.gov/) by a global search35. Phytozome (http://phytozome.jgi.doe.gov/pz/portal.html) provided the ACS and ACO protein sequences for Arabidopsis, Fragaria × ananassa, Malus × domestica, and Solanum lycopersicum. The protein basic local alignment search tool (BLASTp) was used to search for these sequences, with a threshold E-value of 10− 10. Furthermore, HMM profiles were created utilizing conserved ACS and ACO domain sequences derived from ACS (PF00155) and ACO (PF14226, PF03171)36. The putative jujube ACS and ACO proteins in jujube were found through HMM profiles and HMMER software (version 3.0) utilizing a standard E-value. Redundant sequences were removed to guarantee that the unique ACS and ACO genes. Calculation of the molecular weights and the isoelectric points (PIs) for the presumed jujube ACS and ACO proteins utilized ExPASy (http://web.expasy.org/compute_pi/). Furthermore, the WoLF PSORT site (https://wolfpsort.hgc.jp/) served the purpose of forecasting subcellular positioning.

Phylogenetic analysis

The phylogenetic tree of the ACO and ACS proteins was built with the Neighbor-Joining (NJ) technique and a bootstrap value of 1000 in MEGA version 11.0 and was visualized using Evolview 3.0 (https://evolgenius.info//evolview-v2).

Analysis of predicted ACS and ACO genes from ‘Dongzao’ jujube

The exons/introns of the ZjACS and ZjACO genes were determined using the Gene Structure Display Server (GSDS2.0: https://gsds.gao-lab.org/Gsds_help.php) based on the coding and corresponding genomic sequences37.

Conserved motifs within the predicted ZjACS and ZjACO proteins were determined through the online tool Motif Elicitation (MEME) version 5.5.7 (https://meme-suite.org/meme/)38, adhering to these criteria: (1) motif occurrences were restricted to 0 or 1 per sequence; (2) a maximum of 10 motifs were considered; (3) the optimum motif width was set between 6 and 50 amino acids; and (4) only motifs with an E-value below 0.05 were considered significant39.

The upstream promoter regions (2000 bp) of each ACS and ACO genes from ‘Dongzao’ genome were extracted with TBtools software and upload to PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/), which could examine the cis-regulatory elements40. Classification of the cis-acting elements was determined by their predicted function.

Chromosomal distribution and gene duplication analysis

Chromosomal locations of the ZjACSs and ZjACOs were obtained from the annotation information of the ‘Dongzao’ genome database and analyzed through the Gene Location Visualization feature of TBtools41. For illustrating the synteny of the orthologous ACS and ACO genes founded in ‘Dongzao’ and other chosen genomes, we examined gene duplication occurrences using the Multicollinearity Scanning Toolkit (MCScanX)42 with its standard settings, and then employed the Circos and Dual Synteny Plot results in TBtools. Calculations for the evolutionary rates Ka, Ks, and the Ka/Ks ratio calculated in KaKs_Calculator 3.017,43.

Expression profiles analysis based on RNA-seq data

Transcriptome sequencing data from seven samples (Z. jujuba ‘Dongzao’, at 40, 55, 70, 85,100, 115 and 130 days after pollination), were generated (SMART BIOTECH, Illumina HiSeq 4000) and deposited in NCBI (PRJNA1173509). The raw data were individually aligned with the jujube genome (NCBI, ASM3175591v1) via HISAT2 v2.0.5 using standard settings44, and gene expression was ascertained through the fragment count per kilobase per million mapped exons (FPKM) technique. Genes exhibiting differential expression were additionally pinpointed through the DESeq2 R package in R (version 1.16.1), ensuring p < 0.05 and absolute log2 (fold change) values exceeded 145. Ultimately, heatmaps were created by TBtools41.

Co-expression network analysis

Based on the transcriptome and qRT-PCR datasets, the Pearson’s correlation coefficients (PC) were calculated between two genes through R package. Genes with expression correlation coefficients greater than 0.9 were identified as co-expressed. Co-expression between ACS and ACO genes and fruit ripening related transcription factors were analyzed and a co-expression network was created and shown using Cytoscape v.3.7.146.

qRT-PCR analysis of ZjACSs and ZjACOs

Total RNA extraction was performed using a plant RNA extraction kit (Vazyme, Nanjing, China). Utilizing a HiScript II Q RT SuperMix for qPCR (+ gDNA wiper) (R223-01, Vazyme, Nanjing, China), the initial strand of cDNA was produced as 1000 ng of total RNA. In addition, qRT-PCR was done utilizing a ChamQ Universal SYBR qPCR Master Mix (Q711-02, Vazyme, Nanjing, China), totaling 20 µL, including 2.0 µL cDNA, 10 µL SYBR premix solution, 0.4 µL forward/reverse primers and 7.2 µL ddH2O. The procedure for the PCR thermal program was established in this manner: 95 °C for 60s, then 40 cycles of amplification for 10s at 95 °C, 30 s at 59 °C, and a standard dissociation phase in a Bio-Rad CFX Connect system. The 2−ΔΔCT method was employed to calculate the comparative expression levels of the ZjACSs and ZjACOs47, with t-tests applied for statistical evaluation. The primers used for the qRT-PCR analysis are listed in Table S6.

Statistical analysis

All data are the average of three duplicates. GraphPad software version 10.0 was used to statistical analysis. The student’s t-test and one-way ANOVA were employed to assess the variances at a significance level of p < 0.05.

Results

Ethylene biosynthesis enzyme activity during jujube fruit development

Numerous studies have showed that ethylene is essential to the growth and maturity of jujube fruit and that the activities of ACC synthase (ACS) and ACC oxidase (ACO) restrict the production of ethylene18,21,48. ‘Dongzao’ fruit samples were collected 40, 55, 70, 85, 100, 115, and 130 days after pollination (DAP, named as S1, S2, S3, S4, S5, S6, and S7), and the ACS and ACO activities were examined (Fig. 1). There is an increasing trend in ACS activity throughout jujube fruit development. In contrast, ACO activity increased from S1 to S4, followed by a slight decline from S5 to S6, and then another increase from S6 to S7. These results revealed the likely roles of ACS and ACO in ‘Dongzao’ fruit development.

Fig. 1
figure 1

Changes in fruit phenotype and activities of ACC synthase (ACS) and ACC oxidase (ACO) enzymes during the ripening of ‘Dongzao’ jujube. (a) Phenotype of ‘Dongzao’ jujube at different developmental stages. Scale bar, 3 cm. (b, c) ACS and ACO enzymes activity analysis. Variations in letters signify statistically notable disparities at various phases, as determined by one-way ANOVA (P < 0.05). The standard deviation of the duplicates is denoted by the error bars.

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Identification and characterization of ACS and ACO genes in ‘Dongzao’ jujube genome

To clarify the function of ACS and ACO enzymes in ‘Dongzao’, the ACS and ACO genes were identified through a genome-wide analysis. A total of 7 ACS genes and 36 ACO genes were identified in the ‘Dongzao’ genome (Fig. 2, Table S1). Comparing the ACS and ACO genes across various plant species, there are fewer ACS genes predicted in jujube compared with Arabidopsis, while there are significantly more ACO genes (Fig. 2a). The physicochemical properties of the ACS and ACO proteins were predicted (Fig. 2b), showing that the 7 ACS genes encoded proteins ranging from 470 (ZjACS5) to 561 (ZjACS7) amino acids in length, and 52,407.22 Da (ZjACS5) to 60,554.1 Da (ZjACS7) in size. The proteins encoded by the 36 ACO genes were predicted to contain between 216 (ZjACO31) and 416 (ZjACO29) amino acids, with molecular weights (MWs) varying between 24,822.25 Da (ZjACO31) and 48,037.36 Da (ZjACO29). The ACS and ACO proteins were predicted to have high aliphatic amino acid index (AI) values, with thermal stability values for ACS ranging from 78.96 (ZjACS4) to 96.33 (ZjACS6) and for ACO ranging from 77.45 (ZjACO6) to 97.94 (ZjACO22). Prediction of subcellular localization indicated that ACS proteins would primarily be found in the chloroplast, with a few (ZjACS2 and ZjACS4) localized in cytoplasm, while all ACO proteins were expected to be located in the cytoplasm. The variety of subcellular localization indicates different biological roles for ‘Dongzao’ jujube ACS and ACO gene families49.

Fig. 2
figure 2

Identification and physicochemical properties of ZjACSs and ZjACOs genes in ‘Dongzao’ jujube. (a) The number of ACS and ACO genes identified in the Ziziphus jujuba, Arabidopsis thaliana, Malus × domestica, Solanum lycopersicum, and Fragaria × ananassa genomes. (b) Predicted physicochemical properties of ACS and ACO proteins in ‘Dongzao’ jujube.

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Phylogenetic analysis of ‘Dongzao’ ACS and ACO genes

To explore the evolutionary relationships of the ACS and ACO gene families between three jujube cultivars (‘Dongzao’, ‘Junzao’, ‘Suanzao’), with sequences from Arabidopsis as an outlier, phylogenetic trees were constructed (Fig. 3). As seen in the phylogenetic trees, the ACS genes could be divided into four subtypes (Fig. 3a). Types I, II, and IV each contained 2 ZjACS genes, while there was one ACO gene in type III, from ‘Dongzao’. Furthermore, the type IV genes were grouped with the greatest bootstrap values, followed by type III.

The ACO proteins could be classified into three major types, with the type III genes further subdivided into four subtypes (Fig. 3b). Among the major types, type I contained genes only from ‘Dongzao’ jujube, suggesting that these genes might be unique to ‘Dongzao’. In contrast, the classes II and III-4 did not contain any sequences from ‘Dongzao’ jujube, while the other types included genes from all three jujube cultivars and Arabidopsis. In subclasses III-2 and III-3, each jujube cultivar has the same number of genes as its corresponding subfamily in Arabidopsis (1 ZjACO, 1 JzACO, 1 SzACO and 1 AtACO), while the number of type III-1 in Arabidopsis was 1.5 times that in each jujube cultivar (2 ZjACOs, 2 JzACOs, 2 SzACOs and 3 AtACOs).

Fig. 3
figure 3

Phylogenetic trees of ACS and ACO family genes. (a) ACS genes from three jujube cultivars (‘Dongzao’ ▲, ‘Junzao’ ●, ‘Suanzao’ ■) and Arabidopsis (★). (b) ACO genes from three jujube cultivars and Arabidopsis. Using neighbor-joining (NJ) with 1000 bootstrap replicates, the tree’s construction was executed in MEGA 11 software. Gene family members from ‘Dongzao’, ‘Junzao’, ‘Suanzao’, and Arabidopsis are denoted by a red triangle, a blue circle, a teal square, and a green star, respectively. Branches of varying colors symbolize diverse gene subfamilies.

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Gene structure and conserved motif analyses of the ZjACS and ZjACO family genes in ‘Dongzao’

The exon and intron structures as well as the diversity of motifs in a sequence, can provide insights into the evolution of family members. Therefore, the gene structures and conserved motifs of the ACS and ACO gene families in ‘Dongzao’ jujube were analyzed combination with their phylogenetic relationships (Fig. 4). In ZjACS proteins, motif composition and exon/intron structures exhibited a consistent pattern within a subfamily, while different subfamilies displayed markedly divergent structures (Fig. 4a). The count of preserved motifs varied between 9 and 10, exhibiting variations in their composition (Fig. 4b; Table S2). Among them, ZjACS7 did not contain motif 9, while all other ZjACSs contained motif 1–10. The exon/intron structures revealed that 7 ZjACS genes contained introns, of which 6 contained three introns, 1 (ZjACS5) contained one intron (Fig. 4c). For ZjACO proteins, proteins with similar motif distributions and gene structure were clustered (Fig. 4d). All ZjACO proteins contained four of the motifs (motifs 1, 2, 7 and 9). Motifs 3, 5, 6, and 10 were only found in type I ACOs, whereas motifs 4 and 8 were present in all ZjACO proteins except for ZjACO4 and ZjACO8. ZjACO proteins in class I had different conserved motif compositions, whereas type III-1, III-2, and III-3 ZjACO proteins shared the same conserved motif composition, suggesting that some of the family members exhibited high conservation while others had notable variation (Fig. 4e). All 36 ZjACO genes contained exons and introns, and that 32 ZjACO genes contained UTRs, except ZjACO22, ZjACO29, ZjACO31, ZjACO32. All type I ZjACO genes had no more than three introns, except for ZjACO31 and ZjACO29, while both type III-1 members contained three introns (Fig. 4f).

Fig. 4
figure 4

The phylogenetic relationships, conserved motifs and gene structures of ZjACSs and ZjACOs. (a, d) The phylogenetic trees of ZjACSs and ZjACOs were built by MEGA11 utilizing the Neighbor-Joining (NJ) technique, incorporating 1000 bootstrap duplicates. Subtypes are distinguished by different colors. (b, e) Distribution of conserved motifs in ZjACSs and ZjACOs. Every enzyme family had 10 predicted motifs. (c, f) The gene structures of ZjACSs and ZjACOs included introns (illustrated by black lines), exons (depicted as yellow rectangles), and untranslated regions (UTRs, indicated by green rectangles).

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Promoter region cis-acting regulatory elements

To predict the regulatory behavior of different ZjACS and ZjACO genes in ‘Dongzao’ jujube, the cis-acting regulatory elements (CREs) within the 2-kb promoter regions of the ZjACS and ZjACO genes were analyzed (Table S3). A variety of CREs related to phytohormones were found in the upstream sequences (Fig. 5). Response elements related to six types of hormones, namely ETH, ABA, MeJA, gibberellin, auxin, and salicylic acid, were found.

In the promoter regions of the ZjACS genes (Fig. 5b and c), all seven ZjACS genes contained CREs associated with ABA, MeJA, ETH, salicylic acid, gibberellin, and auxin responsiveness, indicating that ZjACS proteins were likely subject to regulation by various hormone signals. We also noticed that ZjACS2 contained the largest number of cis-elements related to hormones.

ZjACOs gene promoters were particularly enriched with ABA-responsive elements (ABRE) and MeJA-responsive elements (CGTCA-motif and TGACG-motif) (Fig. 5e and f). Specifically, CREs associated with abscisic acid response were present in 27 out of 36 ZjACOs across all subfamilies. MeJA-responsive CREs were predominantly observed in certain subfamilies, while approximately 75% of all ZjACOs (20 out of 36), particularly in subfamilies I, III-1, and III-2, contained auxin-responsive CREs (CGTCA-motif and TGACG-motif) in their promoter regions. Additionally, 8 of the 36 ZjACOs, namely ZjACO1, ZjACO2, ZjACO7, ZjACO13, ZjACO18, ZjACO22, ZjACO28, and ZjACO34, contained ethylene-responsive CREs (DRE1 and DRE core) in their promoter regions. Furthermore, several ZjACOs promoter regions contained multiple elements that respond to the same hormone, indicating the potential for more rapid and intense hormonal responses. Conversely, some ZjACOs, such as ZjACO13, ZjACO23, ZjACO28, and ZjACO31, contained diverse hormone response elements, indicating their possible involvement in multiple regulatory networks.

Fig. 5
figure 5

Analysis of cis-elements in the promoters of the ZjACS and ZjACO genes. (a, d) The phylogenetic trees of ZjACS and ZjACO proteins were built by MEGA11 utilizing the Neighbor-Joining (NJ) method, with 1000 bootstrap replicates. Subtypes are distinguished by different colors. (b, e) Distribution of cis-acting elements identified in the 2000-bp promoter regions upstream of the ZjACS and ZjACO genes. (c, f) Heatmap of various cis-acting elements.

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Chromosomal location and collinearity analysis of ZjACS and ZjACO genes

To study locus relationships among the ZjACS and ZjACO genes in jujube, the distribution and collinearity of the ZjACS and ZjACO genes in the ‘Dongzao’ genome were analyzed. The 7 ZjACS genes and 36 ZjACO genes were unequally distributed on twelve jujube linkage groups (Fig. 6a). The largest number of ZjACS genes occurred on NC_083384.1 (2 ACS genes), and all 7 ZiACS genes were segmentally duplicated genes. The largest number of ZjACO genes occurred on NC_083381.1 (23 ACO genes), implying that this location may have significantly influence on the expansion of the relatively large ZjACO gene family in ‘Dongzao’. Among the 36 ACO genes, 30 ZjACO genes were derived from tandem duplication in ‘Dongzao’ jujube, while the remaining 6 ZjACO genes were derived from segmental duplication. The MCScanX approach was utilized to further evaluate the gene duplication events in the ‘Dongzao’ genome, and uncover 5 gene pairs presumably originated from gene duplication events were found (Fig. 6a). These results suggested that these ACS and ACO genes may have arisen through gene duplication. The Ka/Ks ratio, which compares the amounts of nonsynonymous substitutions at each nonsynonymous site (Ka) with the total synonymous substitutions at each synonymous site (Ks), was determined for all five duplicated gene pairs (Table S4). Merely one pair (ZjACO20- ZjACO1) exhibited a Ka/Ks ratio exceeding one, suggesting that they received strong and positive selection throughout the development of the ‘Dongzao’ ancestors. All the Ka/Ks ratios for the remaining duplicated gene pairs fell below one, suggesting that most might have undergone evolutionary purifying selection to preserve their structure.

To further illustrate the collinearity of the ACS and ACO genes and deduce their evolutionary links, we chose three representative species for comparison analysis with ‘Dongzao’, encompassing two jujube cultivars (‘Suanzao’ and ‘Junzao’) and Arabidopsis thaliana (Fig. 6b). A total of 11 ZjACS and 14 ZjACO genes exhibited collinear relationships with ‘Suanzao’ and ‘Junzao’, followed by Arabidopsis (12 ZjACSs and 8 ZjACOs) (Table S5). These findings revealed that the collinearity count between ‘Junzao’, ‘Suanzao’, and ‘Dongzao’ were higher than that between Arabidopsis and ‘Dongzao’. Individual homologous genes exhibited either one-to-many or many-to-one homozygosity. Six ACS and three ACO genes (ZjACS1, ZjACS2, ZjACS3, ZjACS5, ZjACS6, ZjACS7, ZjACO1, ZjACO6, and ZjACO20) exhibited collinear relationships across all three chosen cultivars, indicating that these genes in the ZjACS and ZjACO gene families may have remained closely related throughout evolution.

Fig. 6
figure 6

Chromosome distribution and intraspecific interspecific collinearity analysis. (a) The distribution and collinearity of ZjACS and ZjACO genes on chromosomes. Black-marked genes do not exhibit collinearity, but red-marked genes do. Black rectangles that symbolize tandem repeat gene sets around tandem repeat genes. Circle A: The 12 chromosomes; B: gene density in liner plot; C: gene density in heatmap. (b) Synteny analysis of ACS and ACO genes of ‘Dongzao’ and 3 representative plants, namely. Arabidopsis thaliana, and jujube cultivars ‘Junzao’, and ‘Suanzao’.

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Expression profiles of ZjACSs and ZjACOs across different fruit developmental stages

To explore the possible functions of individual ZjACS and ZjACO genes, the expression patterns of the 7 ZjACSs and 36 ZjACOs across fruit growth were characterized using RNA-seq data (Fig. 7). Among the ZjACS genes (Fig. 7a), three ZjACS genes were up-regulated during fruit maturation processes, whereas others, such as ZjACS6 and ZjACS7, were down-regulated. The expression of ZjACS1 and ZjACS5 peaked during the intermediate stage of fruit ripening. Among the ZjACO genes (Fig. 7b), 16 ZjACOs showed preferential expression during the later stages of fruit ripening, whereas 7 ZjACOs were higher during the inital stages of fruit ripening. The remaining 13 genes showed little expression during fruit ripening. The higher ACO expression levels at later stages of fruit development may be conducive to fruit ripening.

For confirming the reliability of the transcriptome data, the gene expression pattern of four ZjACSs and eight ZjACOs were chosen randomly for qRT-PCR analysis (Fig. 7c; Table S6). The results align with the transcriptome data, suggesting their credibility.

Fig. 7
figure 7

Expression profiles and qRT-PCR expression analysis of ZjACS and ZjACO genes in cultivar ‘Dongzao’. (a, b) Heatmap of the transcript levels of ZjACS (a) and ZjACO (b) genes. (c) Transcript levels of selected ZjACS and ZjACO genes in ‘Dongzao’ jujube fruit, sampled 40–130 days after full bloom (DAFB). Which gene was used as control and set to 0 or 1. ‘qRT-PCR’ is not really a unit for the y-axis – should have arbitrary units or set to 1 of some standards.

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Co-expression of ACS and ACO genes and fruit ripening related transcription factors

Previous studies have revealed that members of the ERF50, NAC51, and WRKY13 transcription factor families play important roles during fruit ripening. To further investigate the regulation of the ZjACS and ZjACO genes in ‘Dongzao’ jujube, co-expression networks for both the ZjACS and ZjACO genes were individually constructed. Firstly, we identified fruit ripening related transcription factors (ERF, NAC, and WRKY) in ‘Dongzao’ jujube from previous transcriptome data. Based on expression profiles, key TFs from the ERF family (15 in total), the NAC family (12), and the WRKY family (20) were selected, and the Pearson’s correlation coefficients (PCs) between these TFs and the 7 ZjACS genes and 36 ZjACO genes were calculated. After gene pairs with |PCs| < 0.9 were removed, the remaining gene pairs were used to build a co-expression network (Table S7). We identified 4 ZjACS genes, 9 ZjACO genes, 20 ERF TFs, 13 NAC TFs, and 12 WRKY TFs. Co-expression relationships were identified for 37 pairs between ZjACS genes and TFs and 128 pairs between ZjACO genes and TFs. More than 40% of these co-expressed ZjACS genes correlated with fruit ripening-related ERF and WRKY TFs, and 28.57% of ZjACS genes correlated with NAC TFs. For the ZjACO genes, about 20% of the ZjACO genes correlated with ERF and NAC TFs, and only 13.89% of the co-expressed ZjACO genes correlated with WRKY TFs (Table 1). Notably, ZjERF2 had the most co-expression relationships and was highly correlated with 6 ZjACOs. Among them, ZjACO2 had the highest correlation with ZjERF2.

Table 1 The numbers of ZjACS and ZjACO genes co-expressed with fruit ripening-related TFs.
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Notes for Table 1. Co-expression analyses between the ZjACS and ZjACO genes and 15 ERF TFs, 12 NAC TFs, and 20 WRKY TFs were conducted.

Two networks were built to show the co-expression relationships between ZjACSs, ZjACOs genes and TFs involved in fruit ripening in the ERF, NAC, and WRKY families (Fig. 8). In the ZjACSs network, 31 TFs were found to have positive correlations with ZjACS genes, including 10 ERF TFs, 11 NAC TFs, and 10 WRKY TFs. ZjACS2 and ZjACS3 exhibited extensive co-expression relationships. Specifically, the expression pattern of ZjACS2 demonstrated the strongest correlation with ZjWRKY2 (PC = 0.9845), while ZjACS3 showed the highest correlation with ZjWRKY1 (PC = 0.9679). In the ZjACOs network, the most commonly associated TFs belonged to the ERF family (17 TFs), followed by the NAC family (13 TFs) and then WRKY family (10 TFs). The majority of ZjACO genes displayed positive correlations with transcription factors, among which ZjACO2/4/8/33 exhibited extensive co-expression relationships. ZjACO2/4 is highly correlated with the expression of ZjNAC2 (PC = 0.9886/ 0.9965). ZjACO8/33 exhibited highly correlations with ZjWRKY1 (PC = 0.9745) and ZjERF10 (PC = 0.9834), respectively.

Fig. 8
figure 8

Co-expression networks of fruit ripening related transcription factor families ERF, NAC, WRKY and ethylene synthesis related genes ZjACSs and ZjACOs. (a) Co-expression networks of ZjACSs and ERF, NAC, WRKY TFs using transcriptome data. (b) Co-expression networks of ZjACOs and ERF, NAC, WRKY TFs using transcriptome data. Reported fruit ripening related transcription factor families ERF, NAC, and WRKY were found in ‘Dongzao’ jujube and are denoted by various colored circle nodes, purple for ERF, pink for NAC, blue for WRKY. A red line connects two points, signifying a positive link (0.9 ≤ r ≤ 1) among the two genes, whereas a blue line depicts a negative link (− 0.9 ≤ r ≤ − 1) between the genes. The dimension of the nodes indicates the extent of relationships in co-expression. (c) Correlation of selected ZjACSs, and ZjACOs genes with ERF, NAC, and WRKY TFs using qRT-PCR data.

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Discussion

ACS and ACO enzymes drive ethylene biosynthesis18,52, with gene family expansions varing across species, such as Arabidopsis (12 ACS genes, 5 ACO genes)18,30, pear (13 ACS, 11 ACO)24, tomato (7 ACS, 11 ACO)53 and watermelon (8 ACS and 8 ACO)16. In this study, a total of 7 ZjACS and an outstanding 36 ZjACO genes were identified within the genome of ‘Dongzao’ jujube. The numbers of ZjACS and ZjACO genes in ‘Dongzao’ jujube were about 0.6- and 3.3-folds of those in Arabidopsis, respectively, implying a lack of expansion of the ACS family and an obvious expansion of the ACO family in ‘Dongzao’ jujube. The variance observed is attributed to the evolutionary process and duplication in plants49,54. Consistent with the classification of the Arabidopsis ACS genes in previous studies18,30, the 7 ZjACS genes in ‘Dongzao’ jujube were categorized into 4 types (type I, II, III, IV). While 36 ZjACOs formed four subgroups (type I, III-1, III-2, III-3) including a novel type I clade absent in model plants (Fig. 3). This specific expansion pattern reflected functional diversification through gene duplication events, particularly prominent in the ACO gene family.

The process of gene duplication plays a crucial part in the expansion of gene families and the formation of gene clusters through tandem duplication, whereas segmental duplications lead to the creation of similar genes, increasing the overall gene count and facilitating genetic drift55. In ‘Dongzao’ jujube, ZjACS genes exclusively arose from segmental duplication (7 genes), whereas ZjACO expansion primarily resulted from tandem duplication (30/36 genes) (Fig. 6). The ZjACO family was largely expanded in ‘Dongzao’ jujube when compared to Arabidopsis, which might be attributed to complex gene duplication of the ZjACO family that has not occurred in other lineages. Collinearity analysis identified 5 duplicated pairs (3 ZjACSs, 2 ZjACOs), with only ZjACO20-ZjACO1 showing strong positive selection (Ka/Ks > 1), signifying a robust, favorable evolutionary selection of these genes. Cross-species comparisons revealed strongest synteny with ‘Suanzao’/‘Junzao’ cultivars and weakest with A. thaliana, highlighting lineage-specific evolutionary paths. These patterns explain the ACO family’s exceptional expansion in jujube compared to model plants, suggesting divergent selection pressures shaped ethylene biosynthesis genes during domestication56.

The exon-intron structure and motif composition can be used to understand the evolutionary relationships among genes or organisms57,58. The exon–intron (Fig. 4) structure revealed that all 7 ZjACS and 36 ZjACO genes have introns. Introns ranged in number from 1 to 3 in ZjACS genes, and ranged from 2 to 4 in ZjACO genes. Notably, several ZjACOs genes (ZjACO22, ZjACO29, ZjACO31, and ZjACO32) lack untranslated regions (UTRs). This discrepancy may be associated with evolutionary gene loss and alternative splicing mechanisms59,60,61. Furthermore, previous studies have suggested that UTRs may play critical roles in transcriptional regulation62,63. In this study, the genes ZjACO22, ZjACO29, ZjACO31, and ZjACO32, which lack UTRs, exhibited low expression level (Fig. 7). It is speculated that their transcription might be impaired, although specific roles remain to be elucidated through further research. Motif analysis (Fig. 4) showed that motif 9 in the predicted ZjACS proteins was common in all except the type IV gene encoding ZjACS7. Among the ZjACO proteins, motif 5 was present only in proteins encoded by type I genes. Typically, closely related gene members exhibit comparable structural traits, as seen in plants like Arabidopsis64, and sand pear30, indicating that these structures are evolutionarily preserved. These structural studies provided independent support for the phylogeny and categorization conclusions.

The promoters of genes contain diverse cis-elements that mediate the accurate binding of effector proteins to template DNA, which either activates or impedes gene transcription. Consequently, gene function can be deduced through the identification of cis-elements65. Plant hormones have been shown to regulate gene expression in several plants66. Promoter analysis of ZjACS and ZjACO genes revealed abundant hormone-responsive cis-elements (ABA, MeJA, GA, SA, auxin and ethylene) (Fig. 5), indicating their integration into phytohormone signaling networks. Notably, ZjACS2 and ZjACO13 included many MeJA-responsive elements, suggesting that ZjACS2 and ZjACO13 may be induced by MeJA or abiotic stress. Moreover, the ZjACO2 and ZjACO11 promoters contained ethylene-responsive elements, and these genes exhibited higher expression in later stages of fruit ripening. The ZjACS6 promoter also contained ethylene-responsive elements, and exhibited a higher expression during the initial phases of fruit ripening. Together, these results suggested that transcriptional regulation of ethylene biosynthesis genes through hormonal cross-talk, particularly through ABA and ethylene signals.

To analyse the roles of ZjACS and ZjACO genes in ‘Dongzao’ jujube during ripening, we profiled their expression patterns throughout the growth and maturation phases of the fruit (Fig. 7). Over 60% of the ZjACS and ZjACO genes were expressed differently throughout fruit growth and ripening, suggesting significant influence of the ACS and ACO families in regulating ethylene-mediated fruit ripening in ‘Dongzao’. The expression levels of genes controlling ethylene biosynthesis (ZjACS2, ZjACO2/4/8/11/33) remained low at the early maturation stage in ‘Dongzao’ jujube, but increased significantly at the half-red and full-red stages. This expression pattern aligned with the elevated ethylene production during fruit ripening reported by Zhang et al.67 in ‘Dongzao’ jujube, suggesting that these genes may respond to the increase in endogenous ethylene emission. In non-climacteric blueberry fruits, the expression of ACS1 was up-regulated, preceding the ethylene increase. ACO2 transcript level increased significantly during later maturation stages, indicating that both ACS1 and ACO2 mediate ethylene accumulation to participate in fruit maturation68. Notably, type IV ACS-like genes, which are derived from the AATase gene were prominent64. In Arabidopsis, AtACS10 and AtACS12 are assumed to be amino acid transferases without ACS activity64. However, Zhang et al.30 have reported that, while low, the expression of PpyACS12 and PpyACS13 were constitutive during sand pear development. Thus, ACS-like gene expression patterns in sand pear indicate that ACS-like genes might have functions in the biosynthesis of ethylene. In our study, the type IV ZjACS6 and ZjACS7 exhibited obvious opposite expression trends during fruit ripening. Therefore, we also speculated that these two genes might play a role in ethylene biosynthesis and were negatively correlated with fruit ripening.

Co-expression analyses (Fig. 8) showed that ZjACS and ZjACO genes were tightly co-expressed with known fruit ripening-related TFs (ERF, NAC, and WRKY). Among them, ZjACS3-ZjWRKY1, ZjACO2-ZjNAC2/ZjERF2, ZjACO4-ZjNAC2, ZjACO8-ZjWRKY1, and ZjACO33-ZjERF10 exhibited highly correlations. It suggested a coordinated regulatory module governing ethylene biosynthesis during ‘Dongzao’ jujube ripening. In kiwifruit, AdACSs and AdACOs were regulated by ERF and NAC TFs to amplified ethylene biosynthesis69. The similar relationships (ZjACS3/ZjACO8-ZjWRKY1, ZjACO2/4-ZNAC2) in ‘Dongzao’ jujube, implies that ethylene signaling may operate through conserved transcriptional hubs. In banana, MaERF11 regulated the expression of MaEXP2/7/8 and MaACO1 by binding to the GCC-box motif within their promoter regions70. Wei et al.71 reported that the NAC TF MaNAC083 affected fruit ripening by inhibiting the transcription of five ethylene biosynthesis genes (MaACS1 and MaACO1/4/5/8). In Arabidopsis thaliana, AtWRKY29 positively regulated the expression of AtACS5/6/8/11 and AtACO5 and promoted ethylene biosynthesis in roots72. Among the co-expression network, ZjERF2/10, ZjNAC2 and ZjWRKY1 were candidate TFs for regulation of ethylene biosynthesis, and it was speculated that they played a similar regulatory role in ‘Dongzao’ jujube. The specific regulatory mechanisms require further experimental validation, and the following approaches could be used in subsequent studies: (1) investigating the regulatory effects of target genes on ethylene biosynthesis through virus-induced gene silencing (VIGS) or transient over-expression; (2) characterizing transcription factor interactions with putative target genes using dual-luciferase reporter assays or electrophoretic mobility shift assays (EMSA); (3) given the limitations of the jujube genetic transformation system, assessing functional conservation via heterologous expression in model plant systems.

Conclusion

In this study, we provide a systemic analysis on the ACS and ACO genes of ‘Dongzao’ jujube. A total of 7 ZjACS and 36 ZjACO genes were identified and characterized. Furthermore, phylogenetic relationships, chromosomal distribution, duplication events, conserved motifs, promoter region cis-regulatory elements and expression levels during different fruit developmental stages were all investigated. These analyses helped to classify these genes and provide insights into the evolution of the ACS and ACO gene families. The expression pattern of the ZjACSs and ZjACOs may provide a preliminary knowledge of their function during fruit ripening. Moreover, we predicted key ZjACSs, ZjACOs and related TFs that may play roled in fruit ripening through co-expression analysis. Their functions will be investigated in further studies to better understand the regulation of fruit ripening in ‘Dongzao’ jujube. Together, this extensive genomic examination of ACS and ACO genes in ‘Dongzao’ jujube provides new perspectives for upcoming functional analyses of fruit maturation.