The Characteristics and Expression Analysis of the Tomato SlRBOH Gene Family under Exogenous Phytohormone Treatments and Abiotic Stresses

Abstract

Respiratory burst oxidase homologs (RBOHs), also known as NADPH oxidases, contribute significantly to the production of ROS in plants, alongside other major sources such as photosynthesis and electron transport in chloroplasts. It has been shown that plant RBOHs play an active role in plant adversity response and electron transport. However, the phylogenetic analysis and characterization of the SlRBOH gene family in tomatoes have not been systematically studied. This study identified 11 SlRBOH genes in the tomato genome using a genome-wide search approach. The physicochemical properties, chromosomal localization, subcellular localization, secondary structure, conserved motifs, gene structure, phylogenetics, collinear relationships, cis-acting elements, evolutionary selection pressures, tissue expressions, and expression patterns under exogenous phytohormones (ABA and MeJA) and different abiotic stresses were also analyzed. We found that the SlRBOHs are distributed across seven chromosomes, collinearity reflecting their evolutionary relationships with corresponding genes in Arabidopsis thaliana and rice. Additionally, all the SlRBOH members have five conserved domains and 10 conserved motifs and have similar gene structures. In addition, the results of an evolutionary selection pressure analysis showed that SlRBOH family members evolved mainly by purifying selection, making them more structurally stable. Cis-acting element analyses showed that SlRBOHs were responsive to light, hormone, and abiotic stresses. Tissue expression analysis showed that SlRBOH family members were expressed in all tissues of tomato to varying degrees, and most of the SlRBOHs with the strongest expression were found in the roots. In addition, the expressions of tomato SlRBOH genes were changed by ABA, MeJA, dark period extension, NaCl, PEG, UV, cold, heat, and H₂O₂ treatments. Specifically, SlRBOH4 was highly expressed under NaCl, PEG, heat, and UV treatments, while SlRBOH2 was highly expressed under cold stress. These results provide a basis for further studies on the function of SlRBOHs in tomato.

Keywords: abiotic stress; gene family; respiratory burst oxidase; tissue expression profiling.

Figure 1

(A) Conserved domain of the SlRBOH genes in tomato. Domains of the NADPH_Ox, Ferric_reduct, FAD_binding_8, NAD_binding_6 motif, and EF_hand_7. (B) Chromosomal localization of the SlRBOH gene in tomato. Chromosome positioning was based on the physical location of the 11 tomato SlRBOHs. Gene names are indicated in black. The scale bar is on the left. (C) Collinearity analysis of RBOH gene family in tomato, rice, and A. thaliana. The red line indicates the SlRBOH1 gene, the black line indicates the SlRBOH2 gene, the blue line indicates the SlRBOH5 gene, the yellow line indicates the SlRBOH6 gene, the purple line indicates the SlRBOH7 gene, the orange line indicates the SlRBOH10 gene, and the brown line indicates the SlRBOH11 gene. Gray lines in the background indicate collinear blocks within the tomato, rice, and A. thaliana genes.

Figure 2

The exon–intron structure of the SlRBOH gene family in tomato. The evolutionary tree was constructed based on the full lengths of tomato SlRBOH protein sequences using MEGA11.0 (v11.0.13). The exon–intron graph of tomato SlRBOH genes was drawn using TBtools software (v1.09876). The untranslated regions (UTRs) are indicated by thick green boxes. The exons are indicated by thick yellow boxes. The introns are indicated by black lines.

Figure 3

Unrooted phylogenetic tree of tomato, A. thaliana, rice, and eggplant RBOH gene families. A phylogenetic tree containing 11 tomato, 10 A. thaliana (At), 9 rice (Os), and 8 eggplant (Sm) RBOH proteins was constructed using the maximum likelihood method. These six subgroups have different colors. The four different colored shapes represent RBOH proteins from four species. The black square is tomato, the blue triangle is A. thaliana, the red star is rice, and the green circle is eggplant. The numbers on the nodes in the phylogenetic tree indicate the percentage of confidence in the bootstrap validation for that branch.

Figure 4

Motif composition and distribution of tomato SlRBOH proteins. (A) Colored boxes represent different conserved motifs, and (B) 1–10 motifs are shown. (B) Amino acid sequences of different conserved motifs, represented by stacked letters at each position. The total height of the stack represents the information content of the relative amino acid in the position of each letter in the motif in bits. The height of the individual letter in a stack was calculated by the probability of the letter at that position times the total information content of the stack. The X- and Y-axes represent the width and the bits of each letter, respectively.

Figure 5

Analysis of cis-acting elements of the SlRBOH genes family in tomato. Different colored wedges represent different cis-elements. The length and position of each SlRBOH gene were mapped to scale. The scale bar represents the length of the DNA sequence.

Figure 6

The number of cis-acting elements in tomato SlRBOH genes. The different colors and numbers of the grid indicate the numbers of different cis-acting regulatory elements in these SlRBOH genes.

Figure 7

Evolutionary selection pressure analysis of the SlRBOH homologous gene pairs. The X-axis represents the Ka value, the Y-axis represents the Ks value, and the Z-axis represents the ratio of Ka to Ks. The color scale represents the fold change normalized by the log2-transformed data.

Figure 8

Expression levels of SlRBOH genes of different plant tissues in a vegetative growth period. The expression patterns of SlRBOH1-SlRBOH11 in different tissues are shown in A–K, respectively. Error bars represent the standard error (SE) of three replicates. The relative expression of each gene in different tissues is expressed as mean ± SE (n = 3). Bars with different lowercase letters were significantly different according to Duncan’s multiple range tests (p < 0.05).

Figure 9

Expression levels of SlRBOH genes of different tissues in a reproductive growth period. The expression patterns of SlRBOH1-SlRBOH11 in different tissues are shown in A–K, respectively. Error bars represent the standard error (SE) of three replicates. The relative expression of each gene in different tissues is expressed as mean ± SE (n = 3). Bars with different lowercase letters were significantly different according to Duncan’s multiple range tests (p < 0.05).

Figure 10

Expression levels of SlRBOH genes under (A) ABA and (B) MeJA. The asterisk (*) indicates that the expression level of the stress group is significantly different from that of the control group (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, one-way ANOVA, and Tukey test). The samples in the 0 h treatment were used as controls.

Figure 11

(A) NaCl, (B) PEG, (C) cold, (D) heat, (E) H₂O₂, (F) UV, and (G) dark period extension treatments. The asterisk (*) indicates the corresponding gene that was significantly up- or down-regulated compared with the 0 h status (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, one-way ANOVA, and Tukey test). The samples in the 0 h treatment were used as controls.