Introduction

Plants in nature are continuously facing harsh environmental stress such as drought, fluctuating temperature and pressure, salinity, and many more. These conditions are known as abiotic stress and are known to affect plant growth and cause low yield1. In response, plants have developed numerous signaling mechanisms to deal with these unpredictable conditions where protein phosphatases and kinases play important roles by catalyzing reversible protein phosphorylation to adjust to environmental changes2. Although several studies regarding the function of protein kinases have been investigated and proved their role in stress response, much is still unknown when it comes to protein phosphatases3,4.

Protein phosphatases are enzymes that catalyze a variety of cellular functions by dephosphorylating amino acids that have been phosphorylated by kinases and are prevalent in most of the signaling mechanisms in plants where they are essential for establishing a healthy response for plant growth and stress modulation5,6. Although serine, threonine, tyrosine, histidine, proline, arginine, aspartate, and cysteine residues are known as targets for phosphorylation, in most cases serine, threonine, tyrosine, and histidine residues act as the target site7,8. Phosphorylation, catalyzed by kinase enzymes and phosphatase enzyme-catalyzed dephosphorylation, acts as a balancing mechanism for various normal and adverse conditions in nature7.

Protein phosphatases (PPs), in broad terms, can be divided into two classes, which are based on their capacity to dephosphorylate either protein Tyrosine or Ser/Thr residues9,10. Protein serine-threonine phosphatases can be further divided into three subclasses based on their function and crystal structure: (I) Phosphoprotein phosphatases (PPPs), (II) Metal-dependent protein phosphatases (PPMs), and (III) Aspartate-based phosphatases10. PPPs require cofactors for activation and include seven phosphatases PP1, PP2A, PP2B, and PP4-7, which are regarded as the signature phosphatases5, where the PPM subclass contains two phosphatases: protein phosphatase 2C (PP2C) and suprachiasmatic nucleus circadian oscillatory protein (SCOP)/ pleckstrin homology (PH) domain leucine-rich repeat protein phosphatase (PHLPP), and though this enzyme does not require cofactors, they are dependent on Mg2+/Mn2+ cations for activation11.

PP2C is a metal-dependent monomeric enzyme and compared to other organisms’ plants encode a much higher number of PP2C, which indicates the evolutionary significance of this enzyme and its involvement in various cellular functions4. In recent times, genome-wide studies of several plant species, including potato12, wheat13, maize14, tea15, Arabidopsis16, rice16, poplar17, Medicago18, banana19, soybean20, woodland mulberry21, and turnip22 have shown the presence of a high number of PP2C genes. Studies of complete sequences have established that in plants, PP2C is the major phosphatase enzyme representing 60–65% of total phosphatases6,23. PP2C plays a crucial role in various biotic and abiotic stress responses through positive or negative regulation of signaling pathways, and it is worth mentioning that PP2Cs are present in both prokaryotic and eukaryotic organisms, including humans3,24,25. In plants, PP2C enzymes are divided into 11 to 13 subgroups3,6,16,26. Most of the members of clad A are well characterized and play a key role during drought response in the abscisic acid signaling pathway by negative regulation. On the other hand, clad B PP2Cs are necessary for the regulation of the MAPK signaling pathway during stress through mechanical injury4,27,28,29. POLTERGEIST (POL) and POLTERGEIST-LIKE (PLL) are phosphatases found in Arabidopsis PP2C subfamily C, where POL and PLL1 work together to support stem cell identity and according to genetic research. POL and PLL1 govern the shoot and root meristem, and embryo development, as shown by pol/pll1 double mutant characteristics30,31.

Tomato (S. lycopersicum), a member of the Solanaceae family, is one of the most nutrient-rich foods and a great source of vitamins, minerals, antioxidants, and carotenoids, namely lycopene and phenolic compounds32. It is highly versatile as a food source as it is consumed in raw form as well as part of a variety of canned, frozen, preserved, and dry foods. As a result, it is an essential part of everyday diet in almost every country. A total of 50238.10 square kilometers of farmland was used to produce around 170.75 million metric tons of tomatoes worldwide in 2014, and as a result, tomato plays a significant role in the global food industry32. With the growing population, the need to produce more food is increasing exponentially, which can reach as much as 70% by 2050 1. Abiotic stresses such as extreme temperature, salinity, and drought, however, can impact growth and development, ultimately reducing the yield of food crops. Tomato’s nutritional and economic impact, along with the availability of the whole genome sequence, makes it a great candidate for studying a stress-responsive gene family like PP2C. There is a previous genome-wide study in tomato, that identified 92 SlPP2C and performed expression profiling in response to Ralstonia solanacearum infection33. Moreover, to the best of our knowledge, there is no study available investigating the role of the PP2C gene family in tomato, focusing on their expression profile and enzyme activity under abiotic stress. Thus, a detailed genome-wide identification and characterization study on the PP2C gene family was performed in tomato. A total of 95 PP2C members were identified, which are dispersed in 12 sub-groups. Furthermore, different physiochemical properties, structural analysis, evolutionary relation, duplication events, promoter scanning, and expression patterns in different tissues and adverse conditions were investigated in detail for SlPP2C members. Expression analysis was conducted for both abiotic and biotic stress along with different developmental stages and tissues using microarray data. Additionally, the effects of the five most traumatic abiotic stresses were verified through qRT-PCR for selected SlPP2C genes. This study will provide insight into the role of PP2C genes in tomato during abiotic stress and will create a foundation for future studies in tomato and other plants.

Materials and methods

Gene identification

To identify the candidate PP2C family members in S. lycopersicum, the Phytozome database34 was searched using the InterPro PP2C domain “IPR001932” as the search term. Additionally, BLASTp search was also conducted in the Phytozome database (https://phytozome-next.jgi.doe.gov/) using previously identified Arabidopsis and Rice PP2C protein sequences as a query. Solanum lycopersicum Genome (ITAG release 3.2) was set as the target genome, and all the other parameters were kept as default. Each putative gene was then manually evaluated using SMART (http://smart.embl-heidelberg.de), Pfam (http://pfam.xfam.organd), and online Batch CD-search tool (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) to make sure the presence of PP2C catalytic domain and were removed otherwise. In the Batch CD-search tool, the maximum hit was set to 500, and all other parameters were in default, the result was downloaded in standard mode. Moreover, various information like chromosomal location, CDS length, polypeptide length, and gene length were also collected from the Phytozome 13 database.

All selected genes were named with the prefix “Sl” for Solanum lycopersicum followed by “PP2C” for Protein Phosphatase 2C, and then an increasing numerical number was added to every gene according to their chromosomal position (SlPP2C1, SlPP2C2, SlPP2C3). Physiochemical properties of PP2C proteins like Isoelectric point (PI) and molecular weight were determined using ExPASy ProtParam (https://web.expasy.org/compute_pi/), and for subcellular localization Cello (http://cello.life.nctu.edu.tw/) and Wolf pSORT (https://wolfpsort.hgc.jp/) and PLoc (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/) was utilized. The Phytozome database was also used to download all the Amino Acid sequences, CDS sequences, cDNA sequences, and genomic sequences of PP2C of Solanum lycopersicum.

Phylogenetic analysis

Phylogenetic analysis was performed to understand the evolutionary relationship among five distinct plant species- Arabidopsis thaliana (80 proteins), Medicago truncatula (94 proteins), Oryza sativa (78 proteins), Brachypodium distachyon (86 proteins), and S. lycopersicum (95 proteins) which were collected from respective genome database and published literature. Initially, the sequence alignment of 433 protein sequences was performed using MUSCLE alignment in MEGA-X35 software with its default parameters. Following that, a phylogenetic tree was created using MEGA-X with the neighbor-joining method, 1000 bootstraps, Poisson model, and pair-wise deletion. A phylogenetic tree with only the S. lycopersicum protein sequences was also constructed using the above-mentioned procedure.

Gene structure, domain analysis, and motif identification

To analyze and illustrate the exon-intron structure of the SlPP2C members, the Gene Structure Display Server (GSDS) was used36, where the CDS sequences and corresponding Genomic sequences of candidate PP2C genes were used as input. The presence of the PP2C domain in all 95 SlPP2C genes was validated by searching the Pfam v33.1 database37 and the NCBI conserved domain. Afterward, domains were visualized by the BioSequence structure illustrator tool of TBtoolss v1.09 software38. Multiple EM for Motif Elicitation (MEME) program version 5.4.1 (http://meme-suite.org/), which was used to detect the conserved motifs in SlPP2C protein sequences with default parameters.

Gene duplication and Ka/Ks value calculation

Duplication events among the SlPP2C genes were predicted using the MCScanX and visualized with the advanced circos tool of TBtools software38. Homologous genes were considered tandemly duplicated if two genes were within 100 kb region on the varied chromosome, and duplicated genes beyond 100 kb apart were considered segmentally duplicated. The Ka/Ks calculator in TBtools software was utilized to compute the synonymous (Ks), nonsynonymous (Ka) substitution rate, and Ka/Ks ratio, using the sequences of CDS, protein, and gene pair file as input. Synonymous substitution represents mutations in DNA that do not cause change to the resulting amino acid sequence; nonsynonymous substitution, however, causes changes to the protein sequence. As a result, the Ka/Ks ratio provides crucial insight into the selection pressure acting on a specific gene39. Ka/Ks value < 1 defines purifying selections, Ka/Ks greater than 1 defines positive selection, and Ka/Ks equal to 1 means neutral selection. The formula T = Ks/2x (x = 1.5 × 10−8 substitutions per site per year) was used to determine the divergence period in million years ago (Mya) for every duplicated gene pair40.

Chromosomal location and collinearity analysis

Information about S. lycopersicum, such as chromosome number, chromosome length, and physical coordinates of SlPP2C genes, were obtained from the Phytozome database and analyzed with TBtools software. PhenoGram online program from the Ritchie Lab (https://ritchielab.org/) and Microsoft Office PowerPoint 2016 was used to demonstrate the dispersion of 95 SlPP2C genes on 12 S. lycopersicum chromosomes. To analyze the collinearity within tomato and other plant species (A. thaliana, O. sativa, and Solanum tuberosum) GFF3 file and Genomic file of all four plants were taken from the JGI portal (https://data.jgi.doe.gov/) which were then utilized to perform the collinearity analysis using one step MCScanX and Dual Synteny Plot features of TBtoolss software38.

Promoter sequence analysis

The 1500 bp upstream sequences from the start codon (ATG) and gff3 file were collected from the Phytozome 13 database for all the identified SlPP2C genes34. The presence of cis-elements, responsible for stress and hormone response was identified using PlantCARE41. The results of the analysis were illustrated using TBtools software, and details of the eight hormone-responsive and five abiotic stress-responsive cis-elements are listed.

Expression analysis of SlPP2C genes in different tissues, developmental stages, and stresses

Expression details of SlPP2C genes in various developmental phases (main shoot growth, inflorescence visible, flowering, fruit formation, ripening, and Fruit ripening complete) and fifteen tissues (shoot, leaf, shoot apex, roots, flower, stamen, pollen, pistil, carpel, ovary, fruit, seed, pericarp, anther, and skin) were analyzed using microarray data. Although a microarray dataset of all 95 SlPP2C genes for developmental stages and anatomic tissues was available, for stress response, expression data of 56 SlPP2C genes were unavailable from Genevestigator (https://genevestigator.com/gv/)42. So, expression patterns of 39 SlPP2C genes were analyzed under several biotic and abiotic stresses. The MeV v4.9.0 software package was then used to construct the heatmaps using the hierarchical clustering method to visualize the data43.

Expressions levels were also analyzed using RNA-seq data from the TomExpress server (https://tomexpress.gbfwebtools.fr/) for vegetative (Root, Seedling, and Leaf) and reproductive (Fruit, and Flower) tissues and abiotic (Drought, Heat, and Cold) and biotic (Meloidogyne javanica and Tomato yellow leaf curl virus) stress treatments44. Heat maps were generated using the MeV software package43.

Plant material and stress treatment

S. lycopersicum (BARI tomato 5) seeds were acquired from the Bangladesh Agricultural Research Institute (BARI), Gazipur, Bangladesh. All the experimental research on plants was conducted according to the proper guidelines and legislation of national and international regulations. Seeds were germinated at 26 ± 20C temperature with 16-hour light and 8-hour dark photoperiod until they were ready for stress treatment. 10-day-old seedlings were then transferred to different environments to simulate five abiotic stresses, including salinity, drought, heat, cold, and osmotic. 200mM NaCl solution, 5% Mannitol solution, and 5mM H2O2 solution were used to mimic salinity, drought, and oxidative stress, respectively, while plants were kept in 40C water and in an incubator at 400C to impose the cold and heat stress environment. For control, plants were kept at room temperature. Leaf samples were collected from each category at 0 h, 6 h, 12 h, and 24 h of post-treatment.

RNA extraction, cDNA synthesis, and quantitative real-time PCR analysis

Total RNA was extracted from the 6 h post-treatment harvested frozen leaf samples using the SV Total RNA Isolation System (Promega Corporation, USA). The isolated RNA quantification and quality assessment were performed using a Thermo Scientific NanoDrop instrument and agarose gel run. The first-strand cDNA was synthesized by taking 10 µg of RNA according to the manufacturer’s protocol of GoScript™ Reverse Transcriptase (Promega Corporation, USA) and random primers. Gene-specific primers for the selected SlPP2C genes were designed using the Primer-BLAST (https://ncbi.nlm.nih.gov/tools/primer-blast) and synthesized by Macrogen (https://dna.macrogen.com) (Table S1). A 96-well reaction mixture plate was employed to conduct the real-time PCR analysis in QUANTSTUDIO®3 REAL-TIME PCR SYSTEM with SYBR Green qPCR kits, following the manufacturer’s instructions. The experiments were carried out thrice for each condition and treatment. The relative expression levels for each gene at each condition were determined using the 2−ΔΔCt method.

Total protein extraction and PP2C enzyme activity

To determine the PP2C enzyme activity, total protein was extracted from tomato seedlings using the previously described method45, using extraction buffer containing 100 mM potassium phosphate buffer (pH 7.0), 50% glycerol, 16 mM MgSO4, and 0.5 mM PMSF. Protein samples were always kept on ice, and the extraction buffer and equipment used for protein extraction were also chilled properly before extraction. Bradford method46 was used to quantify the protein. Phosphatase activity was measured using p-nitrophenol Phosphate (pNPP) as substrate, where the PP2C enzyme converts pNPP to p-nitrophenol (pNP)47,48. pNPP does not have any color, but when it loses a phosphate group and converts into pNP, it produces a yellow color. The intensity of this color was measured using a spectrophotometer at 410 nm wavelength, and the enzyme activity was determined using the millimolar extinction coefficient of pNP (18.1 mM−1cm−1)47.

Results

Identification of PP2C gene family members in tomato

The InterPro PP2C domain “IPR001932” was used as a keyword to scan the Phytozome 13 (https://phytozome-next.jgi.doe.gov) database to find the candidates for PP2C family members in S. lycopersicum18. After confirming the existence of the catalytic domain by searching Pfam and NCBI batch CD, a total of 95 PP2C genes (SlPP2C1 to SlPP2C95) were confirmed as tomato PP2C family members. Additionally, the SMART domain identification tool was used to confirm the catalytic domain of SlPP2C proteins. Different fundamental details like amino acid sequence length, gene length, Coding DNA Sequence (CDS) length, and chromosomal locations were collected for each member (Table 1). Analysis of the gathered data revealed that the length of the amino acid sequence fluctuates from the smallest 163 amino acids (SlPP2C25) to the largest (SlPP2C82) with 1312 amino acids, with an average of 440.25 amino acids. Similarly, the molecular weight ranges from 146.82 kDa to 21.03 kDa, with the heaviest SlPP2C82 and the lightest SlPP2C25. Large variation was seen in both pI and molecular weight. The pI varies from 9.50 to 3.91, where SlPP2C75 has the highest value, and SlPP2C12 has the lowest. SlPP2C members are predicted to be localized in the chloroplast, plasma membrane, cytoskeleton, cytoplasm, extracellular, mitochondria, nucleus, vacuole, and endoplasmic reticulum. However, most of the SlPP2Cs (56 out of 95) are predicted to be localized in the cytoplasm, nucleus, and chloroplast, and a few of them are in the vacuole and endoplasmic reticulum.

Table 1 List of identified SlPP2C genes along with their detailed information and subcellular localization.
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Evolutionary analysis between PP2C proteins

Phylogenetic analysis showed that the 95 SlPP2C proteins are dispersed into 12 (A-L) subfamilies except for a few members who are not included in any class and this division is consistent with previous genome-wide studies on other plant species (Fig. 1). Two dicots (A. thaliana, and M. truncatula) and two monocots (O. sativa and Brachypodium distachyon) plants were included in the analysis. As a dicot plant, tomato PP2C members are expected to cluster either with Arabidopsis or Medicago. Among 12 subfamilies, subfamilies- A and D (14 SlPP2Cs) contain the greatest number of SlPP2C members in tomato, which is followed by E and F with 13 and 11 members, respectively. A similar distribution of proteins can be seen in both Arabidopsis and Medicago, where subgroups A, D, E, and F are the groups carrying the most PP2C members. Subfamilies- I, J, and L are the smallest of all the subfamilies with 2 SlPP2Cs each, which is also consistent with the other two dicots included in the study. There were four SlPP2Cs (SlPP2C6, SlPP2C29, SlPP2C27 and SlPP2C75) that remained unclassified. Moreover, all five plant species have similar gene distribution in each subfamily, and every subfamily contains PP2Cs from each of the plants, which indicates that tomato, rice, Arabidopsis, Medicago, and Brachypodium have shared a common ancestor, and the differentiation of subfamilies happened before monocot-dicot separation.

Fig. 1
figure 1

Phylogenetic tree depicting the evolutionary relationship of PP2C members among tomato, rice, Arabidopsis, Medicago, andBrachypodium. Phylogenetic analysis of 433 PP2C protein sequences from five different plant species: Arabidopsis thaliana (80), Medicago truncatula (94), Oryza sativa (78), Brachypodium distachyon (86) and Solanum lycopersicum (95). Protein sequences were aligned using the MUSCLE tool in MEGA-X software with default parameters, and a phylogenetic tree was constructed using the neighbor-joining method with 1000 bootstrap support, Poisson model, and pair-wise deletion. Different colors correspond to different subfamilies of PP2C proteins in plants, and uppercase letters in blue boxes indicate the name of the subfamily.

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Gene structure, conserved domain, and motif analysis

A phylogenetic tree with only the 95 SlPP2C proteins was generated to understand the relationship among the family members (Fig. 2A). The tree displayed that all the subfamilies are comprised of the same members as before (Fig. 1). Both different patterns of gene arrangement and protein domains play a crucial part in the evolution of gene superfamily, thus the variations of exon/intron architecture as well as conserved domains of SlPP2C members were investigated. A high level of similarity was observed within the members of the same subfamily (Fig. 2A) in terms of gene structure, where 13 out of 14 members of group A, for instance, contains 4 exons and 3 introns, only the SlPP2C23 have a slightly different structure with 3 exons and 2 introns. Similar occurrences can be seen in other subfamilies like G, I, and H, where most of the members of these subfamilies contain a similar number of exons of 4, 8, and 10, respectively. However, a high level of diversity can be seen in the exon number among other SlPP2C members of different subfamilies, and the exon number ranges from 1 to 20. Most members of subfamily K (SlPP2C8, SlPP2C61, SlPP2C62, SlPP2C88, and SlPP2C90) consist of 1 exon, and a member in subfamily J- SlPP2C74 carry 20 exons, which is the highest among all the SlPP2Cs. The intron count varies between 0 and 19. All four intron-less SlPP2C members (SlPP2C8, SlPP2C61 SlPP2C62, SlPP2C61) belong to subfamily K. Previous research has revealed that genes with no introns are likely to evolve in a shorter period because of gene duplication events. As a result, gene duplication rates may be linked to the development of the SlPP2C subfamily K25.

Fig. 2
figure 2

Analysis of PP2C domains and exon-intron structure in tomato. A phylogenetic tree with only the Solanum lycopersicum protein sequences was constructed using the MEGA-X software through the neighbor-joining method with 1000 bootstrap support. Horizontal black bars and letters correspond to the 12 subfamilies (A-L) of SlPP2Cs. (A) Exons are represented by yellow boxes, introns by grey lines, and up or downstream regions by blue boxes. According to the scaling at the base, the exon/intron arrangement of each SlPP2C gene was appropriately shown. (B) Green lines represent PP2Cc domains, and yellow lines correspond to PKc domains. The scale at the bottom indicates the size of the gene or the length of the domains.

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Analysis of the protein structure (Fig. 2B) showed that along with the PP2C catalytic (PP2Cc) domain, three protein sequences (SlPP2C41, SlPP2C2, and SlPP2C27) also contain protein kinase catalytic (PKc) domains and SlPP2C70 have three PP2C domains. A total of 19 motifs were found. Among them, motifs 1 and 2 were present in all SlPP2Cs except for SlPP2C66, which indicates the conserved positions and functions of these two motifs (Fig. S1). On the contrary, some motifs are only present in the selected SlPP2C subfamilies, for instance, motif 7 can only be found in subfamily D, while motifs 20 and 15 can be found exclusively in subfamily C. Some motifs occur in multiple subfamilies, for example, motif 11 is present in subgroups A, B, C, E, I, and G, which may indicate these motifs are responsible for specific functions during the regulatory process. The number of motifs ranges from 2 in SlPP2C90 to 31 in SlPP2C82, and the members of a certain subfamily have similar motif distribution.

Fig. 3
figure 3

Chromosomal distribution and duplication events inSlPP2Cgenes in tomato. Distribution of SlPP2C genes on tomato chromosome and duplication event analysis. (A) The colored circles represent different subfamilies, and the size (Mb) of tomato chromosomes is represented by the scale bar on the left. (B) Red lines indicate segmental duplication events between SlPP2C gene pairs, the blue circle represents the tomato chromosomes, and positions are in Mb.

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Analysis of chromosomal distribution and duplication

To investigate the genomic localization of SlPP2C genes, information from the Phytozome database was used to estimate their relative position across each chromosome. All 95 SlPP2C genes were clustered on eleven out of twelve chromosomes with unequal distribution (Fig. 3A). A maximum of 14 SlPP2C members are located on chromosome 1, followed by 12 genes each on chromosomes 3 and 10. Similarly, chromosome 6 has a high gene count with 11 SlPP2Cs. Other chromosomes have a similar number of genes, 8 genes each on chromosomes 5 and 12 and 9 genes each on chromosomes 7 and 8. However, a very low SlPP2C number can be seen on chromosomes 9, 2, and 4, with 3, 4, and 5 genes, respectively. Although chromosome 9 is the second largest chromosome of tomato in terms of length, it contains a very low number of SlPP2C gene count (3), indicating chromosome length doesn’t correspond with SlPP2C number. A total of 18 SlPP2C gene pairs were found to be duplicated via segmental duplications (Table 2) with no tandem duplications (Fig. 3B). Chromosomes 2, 6, 8, and 12Contain three duplicated genes each, followed by 4 each on chromosomes 1 and 3, and other duplicated members are dispersed on chromosomes 4, 5, and 9. Chromosomes 7 and 10 contain the highest number of duplicated genes (5), on the other hand, chromosome 11 does not carry any gene. Ka and Ks ratio indicates the selective constraints on SlPP2C genes, showing their functional conservation in the evolutionary process39. Ka/Ks for the duplicated SlPP2C gene pairs were less than 1 (Table 2), except for the duplication between SlPP2C23 and SlPP2C72, implying that duplicated SlPP2C members evolved mostly through purifying selection. Duplicated SlPP2C gene divergence events occurred about 19.78–84.73 Mya.

Fig. 4
figure 4

Collinearity study of SlPP2C genes with potato, Arabidopsis, and rice genomes. The grey line represents the syntenic block in tomato. The red lines indicate PP2C collinear genes. SlPP2C shows higher collinearity with StPP2C (A) than OsPP2Cs (B) and AtPP2Cs (C). Yellow shapes indicate tomato chromosomes, and green boxes represent rice, Arabidopsis, or potato chromosomes. Tomato and potato showed more similarity as compared to Arabidopsis and rice, although there is at least some homologous of SlPP2C genes in both Arabidopsis and rice. The high similarity of SlPP2C genes with potato and very low collinearity with the monocot plant rice are as expected.

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Table 2 Duplicated SlPP2C gene pairs in the tomato genome.
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Collinearity analysis

Collinearity analysis of SlPP2C genes was performed on the counter PP2C members of potato (Fig. 4A), rice (Fig. 4B), and Arabidopsis (Fig. 4C). Arabidopsis was chosen as a model plant, rice for the comparison with a monocotyledonous plant and potato was chosen as same Solanaceae family member as tomato. The result showed tomato has a very high degree of collinearity with potato, as compared to Arabidopsis and rice. In total, 53 SlPP2C genes have a syntenic relation with 62 potato PP2C genes. On the other hand, only 1 SlPP2C gene has a relation with 2 OsPP2C members, and 2 SlPP2C genes have a collinearity relationship with 4 AtPP2C genes. The least homology of SlPP2C genes in both Arabidopsis and rice, indicates the evolutionary diversity in tomato. The presence of a high density of orthologous gene blocks can be seen between tomato and potato, and a few orthologous genes in rice and Arabidopsis, indicating high structural and functional similarity of PP2C genes in different plant species. These findings may be valuable in future comparative studies of PP2C genes having known functional relevance.

Fig. 5
figure 5

Expression ofSlPP2Cgenes in different developmental stages and tissues. A Heatmap of SlPP2C expression in different developmental stages (A) and tissues (B) was generated using MeV software with hierarchical clustering and Manhattan correlation with microarray data. Gene expression of 89 SlPP2C members was analyzed using RNA-seq data in vegetative and reproductive tissues (C). Here, different colors correspond to different tissues. Blue color (100%) implies a high level of expression, while grey represents the lowest level (0%) of expression. As a result, the color intensity in the heatmap is proportional to the expression level of each SlPP2C member.

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Expression analysis of SlPP2C genes at different developmental stages and tissues

PP2Cs play important roles in plant growth and development4,27,49. The expression levels of 95 SlPP2C genes were analyzed in six developmental stages and fifteen distinct tissues. Analysis revealed two distinct patterns of expression in various stages of development, where one set of genes (e.g. SlPP2C87, SlPP2C70, SlPP2C69, SlPP2C67, SlPP2C37, SlPP2C25, SlPP2C68) display a very low level of expression throughout the entire life cycle, while most of the other transcripts including SlPP2C30, SlPP2C29, SlPP2C15, SlPP2C78, SlPP2C64, SlPP2C40, SlPP2C5, SlPP2C26 show high to moderate levels of expression in multiple stages (Fig. 5A). However, three transcripts including SlPP2C82, SlPP2C83, and SlPP2C84 uniquely maintain high expression levels only in the inflorescence stage but show very low expressions in all other stages indicating their very specific function in the formation of inflorescence organ.

Fig. 6
figure 6

Changes inSlPP2Ctranscripts in response to diverse abiotic and biotic stressors. Microarray data of five abiotic (A) and six biotic (C) stressors were used to examine the expression of 39 SlPP2C transcripts. Additionally, heatmap of gene expression levels of 89 SlPP2C genes under abiotic (B) and biotic (D) stress treatment in different tissues using RNA-seq data. The color scale at the middle of the heatmap depicts the intensity of the changes, with yellow indicating upregulation and blue indicating downregulation, with values ranging from − 2 to 2.

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Microarray expression data suggests three patterns in the expression of SlPP2Cs in various tissues (Fig. 5B). Firstly, there are genes like SlPP2C86, SlPP2C81, SlPP2C56, and SlPP2C85 that are highly expressed in stamen, pollen, and anther, but there is almost no or extremely low expression in other tissues, which shows a high level of tissue specificity of these genes. On the other hand, genes like SlPP2C30, SlPP2C29, SlPP2C10, and SlPP2C15 are expressed in all tissues, but interestingly, these genes are not expressed at all in stamen, pollen, and anthers. Lastly, some genes, such as SlPP2C90, SlPP2C83, SlPP2C37, and SlPP2C25 are not expressed at all in any tissues. The majority of SlPP2C genes are expressed in root and shoot apex to varying degrees, and anther, pollen, and stamen have the least level of expression.

SlPP2C gene expressions were also analyzed in both vegetative and reproductive tissues using RNA-seq data from TomExpress44 (Fig. 5C). Findings showed that tomato PP2C genes had different expression patterns in different tissues. Few SlPP2Cs (SlPP2C25, SlPP2C83, SlPP2C32, SlPP2C61 SlPP2C57, SlPP2C9, SlPP2C72) were not expressed in any tissues where SlPP2C49, SlPP2C65, SlPP2C67, SlPP2C51, SlPP2C63, SlPP2C11, SlPP2C34, SlPP2C53, SlPP2C31, SlPP2C7, SlPP2C60, SlPP2C14, SlPP2C79, SlPP2C28, and SlPP2C23 showed moderate to high expression levels in multiple tissues. However, SlPP2C82, 16, 17, 75, and 70 were expressed exclusively in flower tissue, and SlPP2C53, 44, 10, and 41 were expressed solely in seeds. This tissue-specific expression pattern could indicate that these genes are correlated to flower and seed development. Multiple members of StPP2C family had higher expression levels in multiple tissues, for instance, SlPP2C62, SlPP2C67, SlPP2C51, SlPP2C39, and SlPP2C63 were highly expressed in fruit, flower, and seed tissues.

Expression of SlPP2C genes under various abiotic and biotic stressors

The expression of SlPP2C genes was studied further in exposure to various abiotic and biotic stresses using microarray and RNA-seq expression data. Data for 39 StPP2C were analyzed in response to salinity, drought, wounding, and ammonia stress, while data for the rest of the genes was unavailable due to the lack of specific probes. In the case of abiotic stress, most upregulation can be seen in response to salinity and drought stresses (Fig. 6A). Among the 39 analyzed SlPP2Cs, 27 showed at least some level of upregulation in response to salinity, where SlPP2C52, SlPP2C40, and SlPP2C10 showed stronger upregulation compared to others. Salt stress did not affect the expression levels of other 12 SlPP2C transcripts. Transcripts like SlPP2C10, SlPP2C27, SlPP2C30, SlPP2C60, SlPP2C22, SlPP2C64, and SlPP2C29 were highly upregulated during drought stress. Previous studies have shown that genes belonging to subgroup A play a key role in drought response via the ABA signaling pathway50. Interestingly, SlPP2C10, SlPP2C27, SlPP2C29, SlPP2C60, and SlPP2C64 belong to subfamilies other than A, which indicates there could be other mechanisms by which PP2C enzymes can respond to drought stress. Wounding also provides a fluctuation in the expression of genes, especially four transcripts (SlPP2C58, SlPP2C17, SlPP2C39, and SlPP2C77) showed strong upregulation in response to wound at both green and ripening fruit. Ammonia does not create any significant modulation except for SlPP2C77, which indicates that PP2C enzymes may not be involved in the regulation of ammonia stress in tomato.

Fig. 7
figure 7

Expression of selected eightSlPP2Ctranscripts in response to diverse abiotic stressors. Analysis of expression pattern was done using quantitative RT-PCR of eight selected SlPP2C genes in response to five different abiotic stress (Salinity, drought, oxidative, heat, and cold) conditions. Different colors in the bar diagram correspond to different stress, and the average fold change in expression was calculated against the respective control and the Ubiquitin as an internal control.

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Tomato PP2C gene expression level was analyzed using RNA-seq data from the TomExpress44 for three abiotic stressors including drought, heat, and cold (Fig. 6B). Varied levels of gene expression can be seen in the StPP2Cs, where SlPP2C82 and SlPP2C22 showed higher upregulation in cold stress, while few genes (SlPP2C16, SlPP2C46, and SlPP2C86) showed moderate upregulation. Most of the SlPP2C members had extremely low to no increase in expression under heat stress, except for SlPP2C3 and SlPP2C82. For drought stress, higher upregulation was seen for SlPP2C55, SlPP256, SlPP2C44 and SlPP2C39; with moderate upregulation for SlPP2C95, SlPP261 SlPP2C79, SlPP2C18, SlPP2C36, SlPP2C91 and SlPP2C3. Previous studies have shown the role of PP2C in stress response during drought, and the findings here indicate that drought stress produces the most increase in expression levels, which could indicate that PP2Cs might play a similar role in tomato4,51.

For biotic stress, microarray data suggest that the analyzed SlPP2Cs are mostly upregulated in response to seven biotic infections including B. cinerea, C. michiganensis, G. intraradices, R. solanacearum, P. infestans, and tomato spotted wilt virus (TSWV) (Fig. 6C). SlPP2C12 is upregulated against all the biotic stressors with strong upregulation in response to TSWV, P. infestans, and moderate upregulation against B. cinerea at the red ripe fruit stage. SlPP2C38 and SlPP2C77 are also upregulated in all cases except for the C. michiganensis at the 4-day post-inoculation stage. On the other hand, a few genes named SlPP2C32, SlPP2C39, SlPP2C79, SlPP2C92, and SlPP2C94 showed significant downregulation. Two clusters of genes can be located where one is upregulated in most cases and the other one is downregulated. All these might play a key role in the recognition and modulation of biotic stress response.

Expression levels of StPP2C genes were also analyzed using RNA-seq data for two pathogens, Meloidogyne javanica (2 days, 5 days, and 15 days post-infection) and tomato yellow leaf curl virus (Fig. 6D). Moderate upregulation was observed for SlPP2C90, SlPP2C85, SlPP2C84, SlPP2C75, SlPP2C72, SlPP2C70, SlPP2C62, SlPP2C56, SlPP2C25, SlPP2C21, SlPP2C20, SlPP2C19, and SlPP2C9 across both the species. SlPP2C22 on the other hand was highly upregulated for all three stages of M. javanica infection, but not for yellow leaf curl virus. Interestingly, SlPP2C23 was only upregulated for the 15-day stage of M. javancia. For yellow leaf curl virus only SlPP2C39, SlPP2C3, and SlPP2C59 showed a higher increase in expression levels and the other members showed moderate to low expression levels.

Cis-regulatory element analysis

To further understand the stress-responsive regulation of SlPP2C transcripts, the presence of eight hormone-responsive cis-regulatory elements including ABRE, AuxRR-core and TGA element, GARE, P box, and TATC box, TCA, CGTCA motif, and five abiotic stress-responsive cis-regulatory elements such as MBS, ARE, LTR, TC-rich motif, and GC motif were analyzed in the putative promoter region (Fig. S2, and Table S2). Among all the elements ABRE motif occurs 172 times in the promoter region of SlPP2C, followed by ARE 99 times (Table S3). The promoter of SlPP2C40 carries the greatest number of cis-elements with 18 elements whereas that of SlPP2C4 contains only 1 cis-element. These motifs in the putative SlPP2C promoter sequences that are highly related to stress may directly contribute to the upregulation of SlPP2C genes and overall SlPP2C enzyme activity when stress is present. Previous studies have proven that PP2C enzymes are associated with the regulation of drought response through the ABA signaling pathway, and the presence of a very high number of the abscisic acid-responsive element (ABRE) supports that fact3,4.

Fig. 8
figure 8

Total PP2C enzyme activity in response to a variety of abiotic stressors. Total PP2C enzyme activity was measured in response to a variety of abiotic stressors, including salinity, drought, oxidative, heat, and cold, at four different time points: 0 h, 6 h, 12 h, and 24 h. Total PP2C enzyme activity was assessed and represented as average for control (A), salinity (B), drought (C), oxidative (D), heat (E), and cold (F). The activity was measured in nmoles per minute per milligram of protein. All the studies were carried out three times, and the mean ± standard deviation was plotted. The student’s t-test significance levels, as compared to respective control values, are given as * and **, respectively, with p-values less than 0.05 and 0.01.

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Validation of selected SlPP2C genes under various abiotic stresses

Effects of five different abiotic stresses (Salt, Drought, Oxidative stress, Heat and Cold) were validated by determining the expression levels of eight SlPP2C transcripts using quantitative RT-PCR (Fig. 7). Eight highly stress-responsive genes were selected for the analysis. Interestingly, expression levels fluctuated for different SlPP2C genes at different stimuli. Mostly, all eight selected genes respond to these abiotic stresses with upregulation in expression with few exceptions. SlPP2C13, SlPP2C15, SlPP2C39, and SlPP2C40, for instance, were highly upregulated and only SlPP2C30 is downregulated in response to cold. On the other hand, all eight genes were upregulated in response to salinity consistently. However, high downregulation of some transcripts can be seen in response to drought and heat stress. SlPP2C39 showed significant downregulation, while SlPP2C40 and SlPP2C52 were moderately downregulated in response to drought. Similarly, SlPP2C13, SlPP2C39, SlPP2C40, and SlPP2C52 were downregulated in response to heat stress. Oxidative stress, on the other hand, produced upregulations for all the selected transcripts.

Enzyme activity

Expression analysis showed that a high percentage of SlPP2C transcripts are upregulated during abiotic stress, especially in response to salinity, drought, and oxidative stresses which indicates that the activity of PP2C enzymes should increase due to these abiotic stresses. To further understand the effect of the expression on protein level, the total enzyme activity of PP2Cs was measured in response to five abiotic stress conditions including salinity, drought, oxidative, heat, and cold stresses (Fig. 8). All the data from stressed conditions were then compared to respective controlled conditions (Fig. 8A) where samples were collected at four-time points (0 h, 6 h, 12 h, and 24 h). A strong positive onset of PP2C activity can be seen in response to salinity and oxidative stress (Fig. 8 B, and D). In the case of oxidative stress, the level of enzyme activity gradually increased over 24 h. On the other hand, salinity caused a significant enhancement to enzyme activity after 6 h of treatment and maintained significant upregulation till 24 h of treatment. Similarly, drought and heat stress caused an increase in enzyme activity at the 6-hour mark and subsequently decreased at 12-hour and 24-hour time points, but always remained greater than the corresponding control conditions. In the case of cold stress, the result somewhat differs from the other stressors. The maximum amount of activity can be seen at the 12-hour time point even though PP2C activity initially decreased at the 6-hour mark. PP2C enzyme activity (Fig. 8) results correspond well with the upregulation of SlPP2C transcripts in expression data (Figs. 6 and 7).

Discussion

PP2C encodes one of the most important gene families in eukaryotes, as it regulates growth, development, and stress resistance52. Many studies have been done on the characterization of the PP2C family in the past, including model plants like Medicago18 and Arabidopsis16 and non-model plants like banana19 and potato12 using computational methods and database analysis. However, there is no genome-wide study identifying and characterizing PP2Cs in tomato that focuses on abiotic stress response, even though the whole genome sequence is available for this plant34. The current study was done to obtain a deeper understanding of the functional characteristics and evolutionary variety of the PP2C gene family in tomato with economic and nutritional value32. A comprehensive genome-wide analysis was done, including gene expression profiling, phylogenetic and evolutionary analysis, chromosomal localization, identification, and distribution of genes with structural and evolutionary analysis.

The PP2C gene family is the most diversified in terrestrial plants, with a lengthy history of evolution51. The number of PP2C genes found is quite higher in crop plants (Table 3) than in the lower plants, which corresponds well with other studies done on model plants like Arabidopsis16, Medicago18, and rice16. This increase in number can correlate to a more complex and larger genome size of these higher plants and additionally, this might also indicate the importance of PP2C, in adaptation to various adverse stimuli found in the environment3,22. Several members in a certain SlPP2C subfamily have identical characteristics and intron counts. Assessing synonymous (Ks) and non-synonymous (Ka) substitution ratios helps to anticipate evolutionary processes and provides knowledge about the kind of selection (purifying, positive, and neutral selection) of duplicated gene pairs53. If the Ka/Ks value is less than 1.00, it implies purifying selection; if it is equal to 1.00, it shows neutral selection, however, if the value is more than 1.00, it suggests positive selection51. All SlPP2C duplicated gene pairs were segmentally duplicated and most of them had a Ka/Ks ratio of less than 1.00, implying dominant purifying selection. SlPP2C23||SlPP2C72 gene pair was the only exception with a ratio of more than 1, suggesting positive selection. This kind of selection was also found in the PP2C genes of Brassica rapa22. These results are consistent with previous studies and a high number of segmental duplication events may have contributed to the higher number of PP2C genes and diverse functions of PP2C in plant12,18,22,50,54. Additionally, the divergence period between duplicated SlPP2C gene pairs was calculated in this study. The average duplication period of SlPP2C gene pairs is 42.76 Mya, with divergence times ranging from 19.78 to 84.73 Mya. Compared to Arabidopsis, the average divergence time of SlPP2C is higher than AtPP2Cs which is 19 Mya.

Table 3 Distribution of PP2C genes in higher plants.
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Previous studies done on Arabidopsis have established that multiple PP2C subfamily A proteins (ABI1, ABI2, HAB1) take part in drought response through negative regulation of the ABA pathway3,54. ABI1 and ABI2 are the most important components of drought response. Through multisequence alignment and phylogenetic analysis, 10 SlPP2C genes were identified in tomato and expression analysis of microarray data showed that SlPP2C22, SlPP2C30, and SlPP2C52 are highly upregulated during drought (Fig. 6A), which belong to subfamily A. Phylogenetic analysis also indicated that these three genes are closely related to ABI1 and ABI2 where SlPP2C 30 is homologous to them. Moreover, an increase in the PP2C enzyme activity can be seen during 6 h of drought stress (Fig. 8C). Therefore, these three genes might work similarly in tomato as ABI1 and ABI2 in Arabidopsis29.

According to previous studies on Arabidopsis and Medicago, group B PP2C (AP2C1, 2, 3, 4 and MP2C) are involved in creating responses during abiotic stress such as wounding, and cold; and also modulate innate immunity through negative regulation of MAP kinase signaling pathway3,4. A total of five PP2Cs were identified (SlPP2C25, SlPP2C39, SlPP2C53, SlPP2C44, and SlPP2C45) belonging to subfamily B and phylogenetic analysis shows a close relation to AP2C 1–4. Expression analysis showed that SlPP2C53 was highly expressed due to wounding. Furthermore, enzyme activity analysis shows an increase in SlPP2C activity when exposed to a cold environment (Fig. 8F). These results indicate that PP2C proteins belonging to subfamily A and B play an essential role in plant tolerance during abiotic stress and evolutionary relationships. Other than subfamily A and B members some PP2Cs belong to other subfamilies that have been reported to have a role in abiotic stress response3. BdPP2C5 and BdPP2C13 for instance belong to subfamily F and G, respectively in Brachypodium distachyon and may have similar functions to subfamily A and B PP2Cs in abiotic stress response55. Interestingly, in this study, SlPP2C10 and SlPP2C60 were highly upregulated in drought stress and these two genes are part of subfamily E and F, respectively. Furthermore, a phylogenetic study reveals a tight relationship between SlPP2C60 and BdPP2C13. This information suggests that members of other groups, not just subfamily A and B, may also play significant roles in the response to abiotic stress. Different gene structures of different subfamilies can describe the highly diverse function of the PP2C enzyme in higher plants.

Microarray data analysis indicated that some of the SlPP2C genes had distinct tissue-specific expression patterns, with only a few SlPP2C genes such as SlPP2C10, SlPP2C15, SlPP2C29, and SlPP2C30 are expressing in all tissues, and others expressing in one or two tissues, for instance, SlPP2C56, SlPP2C81, SlPP2C85, and SlPP2C86 that are highly expressed in stamen, pollen, and anther, but there is almost no or extremely low expression in other tissues. The tissue-specificity may indicate that specific PP2C genes might be involved in the development of different tissues. During the developmental process, most of the SlPP2Cs were highly expressed except for SlPP2C25, SlPP2C37, SlPP2C67, SlPP2C69, SlPP2C70, and SlPP2C87. This also corresponds well with the fact that several Arabidopsis PP2Cs like POL ( POLTERGEIST) and KAPP (Kinase Associated Protein Phosphatase) regulate flower growth and development51. In addition, expression analysis of microarray data shows that almost all SlPP2Cs are upregulated during the flowering stage (Fig. 5A) and among them SlPP2C7, SlPP2C10, SlPP2C15, SlPP2C29, SlPP2C30, and SlPP2C80, are highly upregulated, indicating their role in flower development. Furthermore, RNA-seq data was also used for gene expression analysis in vegetative and reproductive tissues. This data showed that some SlPP2Cs had higher expression levels in flower and seed tissues when compared to others, suggesting PP2C in tomato could have a role in flower and seed development. Interestingly SlPP2C67, SlPP2C51, SlPP2C87, and SlPP2C39, were highly expressed in fruit tissues indicating PP2C might play a role in fruit development in tomato similar to strawberry56. RNA-seq data also showed high expression levels several SlPP2C genes in abiotic stresses (drought, cold, and heat), especially during drought stress which is consistent with previous studies done on PP2C51,56.

Plants face many abiotic stress factors in natural conditions which limits the growth and yield of plants to survive these harsh adversities plants develop various complex mechanisms that help them to counteract the effects of the stressors and survive57. Cis-acting regulatory elements in the promoter regions are frequently involved in various functions in plants through hormonal regulation58. ABA-responsive elements (ABREs), for instance, respond to ABA, drought, and salt stimuli, Low-temperature responsiveness and control are aided by LTR59. Every SlPP2C gene has one or more stress-responsive cis-regulatory elements in the promoter regions, establishing a link between SlPP2Cs and stress tolerance. ABRE (172 times) is the most abundant cis element followed by LTR (99 times) found in SlPP2C genes, especially in subgroup A, which is similar to Arabidopsis and rice16. This result is further supported by gene expression analysis where it was clear that SlPP2C genes are most upregulated in response to drought stress. All these findings suggest that SlPP2Cs might have a role in drought and salt tolerance.

Conclusion

Protein phosphatase 2C (PP2C) enzymes perform critical roles within cellular signaling pathways, functioning as primary regulators in stress responses, growth, and development in plants. The study presents an all-encompassing genomic identification and characterization of the PP2C gene family in S. lycopersicum. Analysis was conducted to deduce the evolutionary relationships and divergence patterns of the identified PP2C genes within S. lycopersicum as well as across a wide range of plant species. Moreover, transcript profiling of SlPP2C genes was carried out using transcriptomic data, thus shedding light on their expression patterns in various tissues and under diverse environmental conditions. The findings contribute valuable insights into the genomic landscape, evolutionary dynamics, and expression profiles of SlPP2C genes, thereby establishing a foundation for further functional investigations to unravel their roles in growth, development, and stress responses within this agriculturally significant plant species.