To identify the CKX gene family members in tomatoes, we conducted a comparison of gene sequences with the tomato genome database found on the Sol Genomics Network website (https://solgenomics.net/), which is derived from the Ensembl Plants database (https://plants.ensembl.org/index.html). This analysis allowed us to establish the total count of tomato CKX gene family members, which were then assigned the names SlCKX1-9.

The Expasy website (https://web.expasy.org/protparam/) was utilized to analyze the physicochemical characteristics of members of the SlCKXs family. This analysis encompassed a range of properties such as molecular weights, theoretical isoelectric points, protein instability indexes, and fat indexes (which represent the relative values of aliphatic amino acid content) [28]. Subcellular localizations were predicted using Cell-Ploc 2.0 (www.csbio.sjtu.edu.cn).

We used the Expasy website (https://swissmodel.expasy.org/interactive) to analyze the secondary structure of the SlCKX proteins and predict their tertiary structures. Additionally, we constructed protein tertiary structure models.

The MapChart tools (v 2.3.2) were used to visually show the localization of SlCKX genes on the chromosome [29]. By utilizing the MEME online software (https://meme-suite.org/meme/tools/meme), conserved motifs (motif) in the SlCKX protein sequences were identified and then visualized with TBtools.

We used MEGA 7.0 (www.megasoftware.net) to determine the evolutionary relationships of the SlCKX tree [30], and the results were displayed using evolutionary trees from the online website iTOL (https://itol.embl.de/itol.cgi) [31].

We utilized the MapChart tool (v. 2.3.2) [29] for mapping the SlCKX genes on chromosomes. Furthermore, we employed MCScanX software [32] to study the evolutionary relationship of homologous CKX genes among tomato and Arabidopsis, eggplant, and cabbage. Subsequently, KaKs_Calculator 2.0 was applied to determine the nonsynonymous (Ka) and synonymous (Ks) substitutions for each iteration of the SlCKX gene [33]. The divergence time was calculated using the formula T = Ks/2R, with R representing 1.5 × 10⁸ synonymous substitutions per year.

To investigate the cis-acting elements found in the promoter regions of SlCKX family members, 2000-base pair DNA sequences upstream of the start codon of SlCKX family members were obtained through TBtools software. Subsequently, PlantCARE, an online tool available at http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ [34], was employed for analyzing cis-elements.

To establish a sterile seedling culture, seeds of ‘Micro Tom’ were immersed in sterilized water at 55°C for 30 min, and the water temperature was lowered to 30°C for germination by immersion. Under aseptic conditions, the seeds were soaked in 75% alcohol for 30 s, rinsed three times with sterile water, dried on filter paper, soaked in 4%–5% NaClO for 10 min, rinsed four to five times with sterile water, and blotted on filter paper to dry the seed surface. The sterile seeds were placed on MS solid medium (0.7% agar) amended with 3% sucrose and allowed to germinate and grow for 10 days in a constant incubator (25°C, 4,000 lux light intensity, and 12/12 h day/night rhythm).

Young leaves of 10-day-old tomato seedlings were cut into 0.5 cm × 0.5 cm squares on an ultra-clean bench, then inoculated onto the MS co-culture medium (composition: MS nutrition powder 4.42 g·L⁻¹, IAA 1.0 mg·L⁻¹, agar 7 g·L⁻¹, sucrose 30 g·L⁻¹; pH 5.8) supplemented with five different concentrations of ZT (0.1, 0.5, 1.0, 1.5 and 2.0 mg·L⁻¹). 20 explants were cultured in one vial, each replicated three times in a completely randomized design. Whole explants were kept at 25°C, 12 h photoperiod, and the light intensity was adjusted to 3,000 lux. According to the induction of the callus, the growth of the callus was counted regularly.

Callus explants with different ZT concentrations were selected to extract RNA from tomato calli by following the method of the Vazyme Plant Total RNA Extraction Kit (RC401), and the reverse transcription kit (KR118) of the Tiangen Company was then used to synthesize cDNA using RNA as a template. Genstar Biological 2 × RealStar Green Power Mixture (A311-01) was used for the quantitative procedures, and the website Sangon.com was employed to design specific primers for qRT-PCR analysis, with the gene sequence provided in Supplementary S1. A LightCycler 96 fluorescence quantitative PCR system from Roche was utilized for the PCR, with 20 μL of SlActin serving as the reference gene. The gene-specific primers can be found in Table 1. The PCR protocol comprised denaturation at 95°C for 15 s, annealing/extension at 60°C for 15 s, repeated for 40 cycles, followed by the generation of a melting curve. The relative gene expressions were calculated using the 2^−∆∆Ct method [35]. Each treatment was conducted with three biological replicates.

Statistical analysis was performed on the collected data using a one-way analysis of variance (ANOVA), followed by Tukey’s test with SPSS (IBMSPSS Statistical Version 24). Statistical significance was determined by a p-value of <0.05. The graphs were created using GraphPad Prism 8 (San Diego, CA, USA).

To identify the CKX family proteins in tomatoes, we conducted a query of the tomato genome (v4.0) (https://solgenomics.net/ftp/tomato_genome/assembly/build_4.00/). This search yielded nine SlCKX sequences: SlCKX1 (Solyc04g080820.2.1), SlCKX2 (Solyc12g008900.2.1), SlCKX3 (Solyc04g016430.3.1), SlCKX4 (Solyc10g079870.3.1), SlCKX5 (Solyc01g088160.4.1), SlCKX6 (Solyc08g061920.3.1), SlCKX7 (Solyc08g061930.4.1), SlCKX8 (Solyc10g017990.2.1), and SlCKX9 (Solyc12g008920.3.1). All these sequences contain the typical flavin adenine dinucleotide (FAD) binding and CK-binding domains, which are unique features of CKX family members. Table 2 presents basic information on SlCKXs including loci, chromosome positioning, and open reading frame (ORF) length, the nine members of the SlCKXs gene family exhibit various physicochemical properties, The analysis revealed that these proteins consist of amino acid sequences ranging from 453 to 553 residues, with corresponding relative molecular weights falling within the range of 51.661 to 62.494 kD, and isoelectric points varying from 5.77 to 8.59. Among them, SlCKX1, 4, 5, 6, 7, and 9 are acidic proteins, while SlCKX2, 3, and 8 are basic proteins. The stability coefficient of SlCKX1, 4, 5, 6, 7, and 8 is less than 40, indicating that they are all stable proteins. However, SlCKX2, 3, and 9 have coefficients greater than 40, suggesting that they are unstable proteins.

To explore the structural characteristics of SlCKX proteins, we used the online tool SOPMA for secondary structure prediction. As shown in Table 3, we determined that all SlCKX proteins β-sheets, extended chains, and random coils. The proportion of α-helices, β-sheets, extended chains, and random coils ranged from 32.26% to 36.98%, from 5.47% to 6.98%, from 17.54% to 20.87%, and from 38.11% to 42.94%, respectively. These structural elements were found to differ among the nine SlCKX proteins. Following the assessment of the secondary structure, we analyzed the tertiary structure (Fig. 1). Protein modeling indicated that the global model quality estimation (GMQE) scores for the SlCKX1-9 protein sequences were all close to 1, indicating a high degree of consistency within the protein family and greater reliability of the related sequences.

Based on tomato genome annotation, we identified nine members that belonged to the SlCKXs gene family and were distributed across five tomato chromosomes (Fig. 2). Eight of these genes were evenly located on four chromosomes, with SlCKX5 being the only gene located on chromosome 1. Furthermore, SlCKX1 and SlCKX3 were located on chromosome 4, SlCKX6 and SlCKX7 on chromosome 8, and SlCKX4 and SlCKX8 on chromosome 10. Additionally, SlCKX2 and SlCKX9 were found on chromosome 12. Notably, the proximity of SlCKX6 and SlCKX7 on chromosome 8, as well as SlCKX2 and SlCKX9 on chromosome 12, suggests that these genes may be orthologs resulting from segmental duplication events.

We identified 15 conserved motifs of SlCKX proteins by using MEME software. As depicted in Fig. 3, we observed some similarity in the number and arrangement of conserved motifs among the nine protein sequences of SlCKX. Each protein within the same subfamily exhibited a unique motif pattern, where SlCKX8 lacked motif9, SlCKX9 lacked motif4 and motif7, and SlCKX4 lacked motif2. All nine members of the SlCKX family possessed six highly conserved motifs, including motif1, motif3, motif5, motif6, motif8, and motif10. Furthermore, motif10 was located closer to the 5′-end, whereas motif5 and motif7 were located closer to the 3′-end, suggesting the high conservation of these six motifs during evolution.

All the nine members of the SlCKXs family possess conserved domains for FAD-binding and CK-binding 2, respectively. The FAD-binding domains are located at the 5′-end, whereas the CK-binding domains are situated at the 3′-end. Members within the SlCKXs family exhibit variations in untranslated regions and coding sequences (CDS), with a number of these CDS ranging between 4 and 6. This diversity may arise from evolutionary changes in CKX genes.

To better understand the phylogenetic relationships of CKX genes in tomato, Arabidopsis, eggplant, and nonbulb cabbage, we constructed a phylogenetic tree. This tree was based on conserved domains of the CKX genes, utilizing the neighbor-joining (NJ) method and multiple sequence alignments. Analysis of the results revealed that the 34 CKX genes can be classified into four distinct subgroups: I, II, III, and IV (Fig. 4). Among these groups, groups I and II were the largest, each comprising 12 members. Subgroup I consisted of three tomatoes, two Arabidopsis, four nonbulbous cabbage, and three eggplant species, whereas subgroup II consisted of three tomatoes, three Arabidopsis, five nonbulbous cabbage, and one eggplant species. The phylogeny diagram indicated a high homology between tomato and eggplant likely due to their shared membership in the Solanaceae family. Overall, members in the same subfamily are likely to share similar functions, allowing for further exploration into the potential biological roles of the SlCKX family.

To delve deeper into the evolutionary relationships of the tomato CKX family, a comparative collinear analysis was conducted between the CKX family members of Arabidopsis and tomato. This analysis identified strong collinear relationships between the nine SlCKX family genes and other species and identified the presence of a strong collinear region between tomato and Arabidopsis (Fig. 5). Moreover, The collinearity analysis of AtCKXs and SlCKX revealed that three tomato chromosomes were collinear with five Arabidopsis chromosomes. Moreover, SL4.0ch12 corresponded to three Arabidopsis chromosomes, demonstrating high homology and providing valuable insights into the genetic relationships and gene functions of these species.

We analyzed cis-acting elements located in the promoter region 2000 bp upstream of the SlCKX gene. We found that the SlCKX gene family primarily comprises four categories of cis-acting elements (Fig. 6A): light-response elements, hormone-response elements, stress-response elements, and growth-response elements. The details are provided in the Supplementary S2. The number of hormone-response elements in the SlCKX gene family was five (Fig. 6B), distributed across all eight members except for SlCKX6. The most widely distributed element was the ABA-response element, with the highest number found in SlCKX1 and SlCKX9. Auxin-response elements were found in SlCKX1, 2, and 8; methyl jasmonate-response elements were detected in SlCKX1, 3, 8 and 9; ABA-response elements in SlCKX1, 2, 3, 4, 5, 7, 8, and 9; salicylic acid-response elements in SlCKX2, 5, 7, and 9; and gibberellin-response elements in SlCKX5, 8, and 9, these cis-elements indicated the functional diversity of SlCKX genes, particularly in hormone response, suggesting the involvement of SlCKX in plant growth regulation.

To further examine the expression patterns of the SlCKX gene family in tomato (Fig. 7), We isolated RNA from the calli exposed to varying concentrations of trans-ZT and subsequently reverse-transcribed into cDNA for quantitative real-time PCR. The findings revealed that the transcriptional activity of SlCKX5 and SlCKX9 genes initially increased and then decreased, whereas the transcriptional activity of SlCKX2, 3, 4, 6, 7 and 8 genes initially increased and then decreased at a concentration of 2.0 mg·L⁻¹. The most pronounced difference was observed in the medium supplemented with 1.0 mg·L⁻¹ IAA and 1.0 mg·L⁻¹ ZT, suggesting that a hormonal concentration of 1.0 mg·L⁻¹ is optimal for the growth of tomato calli.