The prohibitins (PHB) gene family in tomato: Bioinformatic identification and expression analysis under abiotic and phytohormone stresses

Abstract

The prohibitins (PHB) are SPFH domain-containing proteins found in the prokaryotes to eukaryotes. The plant PHBs are associated with a wide range of biological processes, including senescence, development, and responses to biotic and abiotic stresses. The PHB proteins are identified and characterized in the number of plant species, such as Arabidopsis, rice, maize, and soybean. However, no systematic identification of PHB proteins was performed in Solanum lycopersicum. In this study, we identified 16 PHB proteins in the tomato genome. The analysis of conserved motifs and gene structure validated the phylogenetic classification of tomato PHB proteins. It was observed that various members of tomato PHB proteins undergo purifying selection based on the Ka/Ks ratio and are targeted by four families of miRNAs. Moreover, SlPHB proteins displayed a very unique expression pattern in different plant parts including fruits at various development stages. It was found that SlPHBs processed various development-related and phytohormone responsive cis-regulatory elements in their promoter regions. Furthermore, the exogenous phytohormones treatments (Abscisic acid, indole-3-acetic acid, gibberellic acid, methyl jasmonate) salt and drought stresses induce the expression of SlPHB. Moreover, the subcellular localization assay revealed that SlPHB5 and SlPHB10 were located in the mitochondria. This study systematically summarized the general characterization of SlPHBs in the tomato genome and provides a foundation for the functional characterization of PHB genes in tomato and other plant species.

Keywords: Tomato; expression; phylogeny; prohibitins; stress; synteny.

Figure 1.

Chromosomal location and synteny of PHB genes in the tomato genome. (a) The chromosome location of tomato SLPHB genes. The scale of chromosomes is in megabases (MB). (b) Circos plot presenting gene segmental duplication events of PHB genes. Segmental duplication pairs are indicated with different color lines.

Figure 2.

The phylogeny of the PHB proteins. An unrooted neighbor-joining phylogenetic tree of PHB proteins from Arabidopsis, rice, maize, soybean, and tomato was generated in the MEGA program with a bootstrap value set as 1000 replicates. The tree was clustered into various clades and subclades. The black dots represent tomato SlPHB proteins.

Figure 3.

Phylogeny, gene exon/intron distribution, and conserved motif analysis of 16 tomato SlPHB genes. (a) An unrooted neighbor-joining phylogenetic tree of PHB proteins with bootstrap set at 1000 replicates and clustered into different clades and subclades. (b) Tomato SlPHB gene intron and exon distribution. The scale at the bottom is corresponding to gene size in kb. (c) The putative conserved motifs in 16 tomato PHB proteins identified using the MEME suite. A total of ten motifs (1 to 10) were identified and each color of the box is corresponding to a motif. The scale at the bottom represents the protein size in kb.

Figure 4.

The putative cis-regulatory sequences were identified in 16 tomato SlPHB genes by submitting their corresponding promoter sequences to the PlantCARE database. Different cis-regulatory elements circadian control (circadian), meristem development (CAT-box), endosperm development (GCN4_motif), zein metabolism regulation (O2-site), MYB binding site involved in drought-inducibility (MBS), WRKY binding site involved in abiotic stress and defense response (W-box), anaerobic induction element (ARE), defense- and stress-responsive element (TC-rich repeats), low-temperature-responsive element (LTR), wound-responsive element (WUN-motif), element for maximal elicitor-mediated activation (AT-rich sequence) ethylene (ERE), gibberellin (GARE-motif), methyl jasmonate (MeJA, CGTCA-motif), abscisic acid (ABRE), and salicylic acid (TCA-element) and son on was detected.

Figure 5.

The endogenous expression profile of 16 tomato SlPHB genes in various plant parts including root, leaves, FB (flower bud), FF (fully opened flower), 1/2/3 cm fruit, mature green fruit (MG_F), breaker fruit (B_F), and 10 days breaker fruit (B10_F). A log2 transformed heatmap was generated using heatmapper program. Blue, white, and red color is corresponding to low, moderate, and high expressions. The genes were clustered by applying the Euclidean method.

Figure 6.

Abiotic stress-induced expression profile of SlPHB genes. (a) salt (b) drought (PEG) induced expression profile at 0 h, 3 h, 6 h, 12 h, and 24 h time points. A log2 transformed heatmap was generated using heatmapper program. Blue, white, and red color is corresponding to low, moderate, and high expressions. The genes were clustered by applying the Euclidean method.

Figure 7.

Phytohormone induced expression profile of SlPHB genes. (a) abscisic acid (ABA), (b) gibberellin (GA3), (c) auxin (IAA), (d) methyl jasmonate (MeJA) induced expression profile at 0 h, 3 h, 6 h, 12 h, and 24 h time points. A log2 transformed heatmap was generated using heatmapper program. Blue, white, and red color is corresponding to low, moderate, and high expressions. The genes were clustered by applying the Euclidean method.

Figure 8.

Subcellular localization images of SlPHB5 and SlPHB10 in Arabidopsis protoplasts. The full-length sequences of SlPHB5 and SlPHB10 were fused in the pro35S vector to generate p35S-SlPHBs/GFP constructs. The images were observed via confocal laser scanning microscopy. The LoTPS3 form Lilium ‘Siberia’ was used as red mitochondrial control for SlPHB5 and SlPHB10. The green, red, merged and BF represents the GFP fluorescence, chlorophyll autofluorescence, combined chlorophyll autofluorescence, and GFP fluorescence and bright field respectively. Scale bars 5 µm.