The CRT family members in the four tomato species were identified in the protein sequences retrieved from the Solanaceae Genomics Network (https://solgenomics.net/, accessed November 15, 2023). The HMM profile of the CRT domain (PF00262) was downloaded from Pfam (http://pfam.xfam.org/, accessed November 15, 2023), and searched against downloaded protein sequences by using HMMER search (https://www.ebi.ac.uk/Tools/hmmer/, accessed November 15, 2023). All the predicted CRT family members were further confirmed through hmmscan and SMART database (http://smart.embl.de/, accessed November 17^th, 2023) for the presence of CRT domains. The CRT protein’s physicochemical parameters, such as amino acid number, molecular weight, theoretical pI, isoelectric point, grand average of hydropathicity (GRAVY) and instability index were computed through the EXPASY ProtParam Tool (http://www.expasy.org/tools/protparam.html, accessed November 19, 2023) and the proteins subcellular localization were predicted with the SubCELlular Localization Predictor Tool (http://cello.life.nctu.edu.tw/, accessed November 19, 2023).

The CDS and genomic sequences of CRT family members were used to survey the exon/intron organization and analyze the gene structure by the genes structure display Server program (GSDS) online server (http://gsds.cbi.pku.edu.cn, accessed November 22, 2023). For the evaluation of conserved motifs and domain, the CRT protein sequences were searched using the Multiple Expectation Maximization for Motif Elicitation (MEME) (http://meme-suite./org/tools/meme, accessed November 22, 2023) and HMMER online tools, respectively. Further, the Gene Structure View package in the TBtools toolkit was used for visualization (Chen et al., 2020).

In addition to four tomato species, amino acid sequences of five other monocot and dicot plants were used for the phylogenetic analysis ( Supplementary Table S1 ). The CRT protein sequences of these species were retrieved using HMMER search. All the protein sequences were aligned with the MUSCLE method and the phylogenetic tree was constructed in Mega 11 using the neighbor-joining method with 1000 bootstrap values. Further, the tree was designed with the ITOL tree online tool (https://itol.embl.de/, accessed November 25, 2023).

The chromosomal position of the 21 candidate CRT genes was retrieved from the genome annotation file and visualized using the Show Genes on Chromosomes- Gene Location Visualize package in TBtools (Chen et al., 2020). The MCScanX toolkit (https://github.com/wyp1125/MCScanX, accessed November 28, 2023) was used to calculate the gene duplication for tomato CRT genes (Wang et al., 2012). The homologous gene duplication and divergence events and the selection pressure on duplicated genes indicated by Ks and Ka, respectively were determined in the TBtools software. The times of duplications in each gene pair were calculated through the formula T = Ks/2λ, where λ= 1.5 x10^-8 substitutions/synonymous site/year (Blanc and Wolfe, 2004). The degree of homology and evolutionary divergence tree for the CRT genes of the tomato and six other plant species was computed and visualized using the Dual Synteny Plotter implemented package in the TBtools.

To predict the cis−acting regulatory elements in the promoter region, sequences of 2000bp (upstream of the transcription start site) were extracted for the CRT members of both cultivated and wild-type tomato species. The acquired sequences were examined in PlantCARE database (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed November 23, 2023) and the obtained results were visualized and classified.

The Phyre² online portal (http://www.sbg.bio.ic.ac.uk/phyre2/html, accessed December 09, 2023) at the intensive modeling mode was used to predict the structure of the of CRT proteins in the cultivated tomato (Kelley et al., 2015). To further depict the role of the CRT protein family, the functional interactions between SlCRTs were obtained using amino acid sequences for protein-protein in STRING database (https://string-db.org/, accessed December 10, 2023).

Expression profiles for the SlCRT genes were carried out from the Tomato Functional Genomics Database (http://ted.bti.cornell.edu/cgi-bin/TFGD/digital/home.cgi, accessed December 15, 2023). The expression patterns of SlCRTs were investigated in various tissues including leaf, root, bud, flower and fruit stages (1cm, 2 cm, 3 cm, mature green, breaker, and breaker+ 10). TBTools software was utilized to visualize and create a heat map for analysis of the data.

Tomato (Solanum lycopersicum) cv. M82 was used to study the expression patterns of SlCRT genes. First, the seeds were washed and soaked with warm distilled water and then placed in water-saturated filter paper for germination. The healthy germinated seeds were transferred to seedling trays containing a mixture of soil, peat, and vermiculite and placed in the growth room under control conditions at 25°C/18°C, 16 h light/8 h dark photoperiod with a relative humidity 60-70%. One-month-old seedlings were exposed to ABA treatment and abiotic stress conditions. In ABA treatment, 100 µM solution was sprayed on seedlings until the solution dripped from the leaves and an equal amount of mock solution was used as a control. The drought and salt stresses were imposed by applying 15% polyethylene glycol (PEG-6000) and 200 mM sodium chloride solutions, respectively. The composite leaf samples were collected from five uniform seedlings at 0, 3, 6, 12 and 24 hours. The samples were immediately frozen in liquid nitrogen and stored at -80 °C for further analysis.

Total RNA Extraction Kit, Polysaccharide Polyphenol Plant (DP441, Tiangen, Beijing, China) was utilized to extract the RNA from the selected samples, following the manufacturer’s instructions. The first strand of cDNA was synthesized using 5 × All-In-One RT MasterMix (G492, ABM, Vancouver, Canada) according to the labeled instructions. NCBI primer design tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast) was used to design SlCRT genes qRT-PCR specific primers ( Supplementary Table S2 ). The total reaction mixture was 20 µl containing 1 µl of cDNA template, 10 µl of SYBR qPCR Master Mix (Q711, Vazyme, Nanjing, China), 0.5 µl each primer and 8 µl of sterile distilled water. Quantitative RT-PCR was carried out using LightCycler^® real-time fluorescent quantitative PCR system (Roche, Basel, Switzerland) with PCR conditions: at 95 °C for 5 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30s. The relative expressions of the genes were calculated using the 2^−ΔΔCt method (Livak and Schmittgen, 2001) with the SlActin gene as a normalized control.

SPSS (IBM, Armonk, New York, USA) software was used for data analysis. One-way ANOVA post hoc Least Significant Difference (LSD) test was used for multiple comparisons with a level of significance p ≤ 0.05 and were expressed as the mean ± standard error (SE) of three replicates.

The hidden Markov model (HMMER) 3.0 was used to identify the tomato CRT family candidate genes. The predicted amino acid sequences were verified using hmmscan and SMART for the presence of the CRT domain “PF00262”. Finally, 5, 5, 5 and 6 CRT genes were identified in S. lycopersicum, S. pennellii, S. pimpinellifolium, and S. lycopersicoides, respectively. The identified genes were named SlCRT1 to SlCRT5, SpiCRT1 to SpiCRT5, SpCRT1 to SpCRT5, and SlydCRT1 to SlydCRT6 based on their chromosomal location ( Supplementary Table S3 ).

Important characteristics of 21 CRT proteins from four tomato species are presented in Table 1 . In case of SlCRT, the CDS length ranged from 1242 (SlCRT3) to 1617 (SlCRT2), with an average length of about 1400 bp. The length of amino acids ranged from 413 (SlCRT3) to 538 (SlCRT2), molecular weight in the range of 47. 60 to 48.54, isoelectric point values in the range of 4.50 to 6. 55, an aliphatic index in the range of 56.64 to 72.34 and an instability index between 35. 53 to 44.30. Similarly, the CDS length varied from 645 to 1617 in the other three species and the amino acid length from 214 to 538. Whereas the molecular weight, instability index and aliphatic index in the three wild species varied from 24.97 to 60.97, 34.03 to 43.1 and 56.64 to 98.41. Proteins CRT1 and CRT3 were stable in nature with lower instability index in all four species. The results of isoelectric points indicated that all CRT proteins are acidic with low pi (pI < 7) values except SpiCRT3 (8.37). Hydrophilicity analysis showed that all 21 CRT proteins are hydrophilic with negative GRAVY values. The subcellular localization prediction of each member of CRT was performed and it was found that CRT proteins were located in the endoplasmic reticulum, nucleus and cytoplasm ( Table 1 ). CRT1 and CRT2 proteins of cultivated and wild tomato species were predicted to be located in the endoplasmic reticulum and CRT3 in the nucleus, except SpiCRT3, which is in the cytoplasm.

To investigate the evolutionary history of the CRT family members, all the CRT protein sequences, 21 from tomato and 29 from other monocot and dicot species were utilized to construct an unrooted phylogenetic tree with the MEGA 11.0 software. Based on the bootstrap and the tree’s topology, the CRT genes were categorized into three groups designated as I, II and III ( Figure 1 ). Group III harbored 20 CRTs, represented the biggest and groups I and II contained 17 and 13 CRTs respectively. The CRT members from all selected plant species were distributed in all three groups, among which cultivated tomato SlCRT2 and SlCRT5 were distributed in group I, SlCRT1 in group II and SlCRT3 and SlCRT4 in group III. The wild tomato CRT genes showed a similar distribution pattern as the cultivated tomato, except for S. lycopersicoides in which SlydCRT2 and SlydCRT6 clustered in group I, SlydCRT1 in group II and SlydCRT3, SlydCRT4 and SlydCRT5 in group III. The cultivated tomato SlCRT genes are more closely related to the members of the CRT gene families of its wild progenitors and the clustering of different species CRT members into the same group suggested that they have similar origins, evolutionary history and relationships.

To study the structural diversity of CRT genes of different tomato species, we compared the intron and exon structures with their corresponding genomic DNA sequences by constructing an unrooted phylogenetic tree of CRT genes ( Figure 2A ). The analysis showed that the CRT gene members in four tomato species were clustered into three groups. Group III was the dominant with 9 members followed by group I (8 genes) and group II had 4 genes. The intron and exon numbers varied in each group, ranging from 4 to 13 and 5 to 14 respectively ( Figure 2B ). Furthermore, members clustered in the same group shared similar exon-intron structures, including the number of exons and introns. For instance, all the genes in group one contained 7 introns and 6 exons. In contrast, CRTs in group III showed diversity in the exon and intron numbers with SlydCRT5 containing only 5 exons and 4 introns. Protein structure analysis of the CRTs revealed that the length and position of the calreticulin domain were highly conserved among different tomato species and phylogenetic groups ( Figure 2C ). The domain positions in group one, group two and most of the group three members were slightly towards the N-terminal, while in group three the SlydCRT4 and SlydCRT5 candidates domains were more towards N-terminal and C-terminal than other members, respectively. Furthermore, the identity percentage of all the CRT protein sequences in the four tomato species was 51.77%, which varied in each group, with 97.72%, 89.87% and 64.1% in I, II and III groups, respectively. To further describe the structural diversity of CRT members from the four tomato species, the analysis of the conserved motif using MEME tool depends on the amino acid sequences with twenty motifs ( Figure 2D ; Supplementary Table S4 ). The detected motifs length ranged from 11 to 50 amino acids and almost all CRT genes studied here contained motifs 1 and motif 3. Further, members within group one and group two shared exactly similar motif numbers and patterns. In both groups, motifs 12 and 15 were distributed at the C-terminus and N-terminus of motif patterns, respectively. However, it was found that the variation in the domain position of the two members (SlydCRT4 and SlydCRT5) changed the motif numbers and patterns in group three. In conclusion, CRT genes from both cultivated and wild types showed a high degree of homology in structure and motif, implying that they may be closely related and have similar functions.

Cis-regulatory elements are essential in the transcriptional initiation of various developmental, hormonal and stress-related genes (Marand et al., 2023). In order to explore the cis-elements in the CRT genes of the four tomato species, 2000 bp upstream promoter regions were retrieved from the Sol Genomics Network. The promoter sequences were then uploaded to the PlantCARE database to identify the putative cis-acting elements. Overall, 47 types of elements were detected in the 21 genes of four tomato species ( Figure 3 ). Based on their regulatory and biological functions, the elements were further classified into four categories: light-responsive (17), stress-responsive (11), phytohormone-responsive (10), and plant growth and development cis-elements (9). Two light-responsive cis-elements (Box-4 and G-box), three stress-responsive (ARE, MYC and MYB), three phytohormones responsive (ERE, ABRE, and TGACG-motif), and one plant growth and development cis-element (as-1) were identified in the high ratio in the promoter regions of the different tomato species CRTs genes. Here, the presence of various types and numbers of cis-elements at different positions in the gene promoter region reveals the potential function of the gene.

To examine the chromosomal distribution, each CRT from one cultivated and three wild species was searched and mapped on the respective chromosomes according to the number and position of the chromosome. The results demonstrated that twenty-one CRTs were positioned on six of the twelve tomato chromosomes including chromosomes 1, 3, 4, 5, and 6 ( Figure 4 ). Among the species, the distribution of CRT on S. lycopersicum, S. pennellii and S. pimpinellifolium was exactly similar with one gene on each chromosome, while one extra gene of S. lycopersicoides mapped on chromosome 5. Almost all the genes of the four species were distributed on one side of the chromosomes except CRT3 which was centrally mapped.

Gene duplication events are the key factors involved in the gene amplification and expansion of gene families during the genome’s expansion (Cannon et al., 2004). In this study, we did not find any tandemly duplicated gene pair in the four tomato species, while eight CRTs were found to exhibit four pairs (single pair for each species) of segmental duplication events positioned on the different chromosomes ( Figure 5 ). The matching pair genes CRT2-CRT5 were common in S. lycopersicum, S. pimpinellifolium and S. pennellii, while different in S. lycopersicoides (CRT2-CRT6). Further, the species evolution assessment revealed that the Ka/Ks ratios for all segmentally duplicated CRTs gene pairs were less than 1, which implies that during the evolution process, tomato CRTs have gone through pure selection ( Supplementary Table S5 ). The predicted divergence time showed that duplication of SlCRT2 & SlCRT5 in S. lycopersicum, SpiCRT2 & SpiCRT5 in S. pimpinellifolium, SpCRT2 & SpCRT5 in S. pennellii, and SlydCRT2 & SlydCRT6 in S. lycopersicoides occurred approximately 24.55, 24.46, 24.78 and 23.52 Mya, respectively. Segmental duplication is suggested to play a crucial role in amplifying the CRT family in tomato species.

Further, interspecies collinearity analysis was performed to identify the homologs of the CRT genes between four tomato species, two monocots (O. sativa and Z. mays) and four dicots (A. thaliana, S. melongena, S. tuberosum and Vitis vinifera) species. The results showed that five tomato CRT genes were collinear with S. tuberosum, four were collinear with S. melongena and only two were with other plant species ( Figure 6 ). However, the divergence time revealed higher intraspecific homology among different Solanaceae members and a distant relationship with other plant species. This homology was probably because of their close kinship.

The five predicted models of the SlCRTs were generated using the Phyre2 online tool based on c3rg0A and c1jhnA templates with a 100% probability ( Figure 7A ). The prediction results showed that SlCRT1, SlCRT3 and SlCRT4 shared similar protein structures, however, SlCRT4 and SlCRT5 have more coiled structures compared to the other members. The protein interaction results showed different interactions of SlCRTs, where the total number of nodes was 11 with 8.91 average node degree. The STRING analysis revealed 49 edges and three representative local network clusters: CL: 4111, CL: 4113 and CL: 4117. The SlCRTs showed interactions with heat shock proteins (Hsps) SlHSP90 and SlHSP70 and lumenal binding protein (Bip/GRP78) ( Figure 7B ). Moreover, the protein analysis exhibited a common calreticulin domain (PF00262) in all five members.

To observe the expression profiles, already published RNA-seq data was used to examine the expression of SlCRTs genes in various plant tissues and development stages. The result showed that all five tomato SlCRT genes were differentially expressed among the examined tissues ( Figure 8 ; Supplementary Table S6 ). SlCRT1 showed a significantly higher expression level than the other SlCRTs in all detected tissues, whereas SlCRT5 exhibited the lowest transcript accumulation in most tissues except the bud. Furthermore, SlCRT1, SlCRT2 and SlCRT3 showed maximum expression patterns in roots, buds and different fruit developmental stages, while SlCRT4 and SlCRT5 were highly expressed in fruits and buds, respectively. Interestingly, the transcript level of all the CRT genes was lowered in the leaf tissue than in other tissues except for SlCRT3, which was expressed at a high level in leaf tissues.

To determine the expression profiling of tomato CRT genes, quantitative RT-PCR analysis was performed at different time intervals under drought, salt and ABA treatments ( Figure 9 ). In drought stress, the expression of SlCRT1 and SlCRT2 significantly up-regulated and reached a maximum of 7 and 13 folds at 24 h and 12 h respectively. The SlCRT3 showed a similar expression to that of control at 1 h, however, increased at 6, 12 and 24 h and peaked 10 folds at 3 h treatment. SlCRT4 expression plummeted at 1 h but after that steadily increased and rose to 2 folds at 24 h treatment. Interestingly, a gradual decrease was observed in the expression of SlCRT5 at 3, 6 and 12 h before recovering at 24 h. Under salt stress treatment, the expressions of SlCRT2 and SlCRT3 showed similar expression trends as both were initially up-regulated and then fell back. The maximum expression level of SlCRT2 (3.5 folds) and SlCRT3 (4 folds) was recorded at 6 h treatment. The expression level of SlCRT1, SlCRT4 and SlCRT5 significantly downregulated under salt treatment. In SlCRT1, the transcript level was lowered at all treatment time points in comparison to control, however, SlCRT4 and SlCRT5 expressions were recovered and induced up to 2 folds at 24 h. In ABA treatment, the expression of SlCRT1, SlCRT2 and SlCRT4 exhibited similar expression to that of control at 3 h before reaching a maximum of 18, 16 and 6 folds, at 12, 24 and 6 h, respectively. Additionally, the expression of SlCRT3 initially downregulated at 3h, however, significantly increased at 6, 12 and 24 h with a maximum expression of 5 folds. The SlCRT5 showed a gradually low expression level from 3 to 12h time points, but significantly induced (2 folds) at 24 h compared to the control. The differential expression and response to abiotic stresses and ABA treatments implied the potential role of SlCRT genes in mediating the tolerance mechanism of tomato.