Caffeic Acid O-Methyltransferase Gene Family in Mango (Mangifera indica L.) with Transcriptional Analysis under Biotic and Abiotic Stresses and the Role of MiCOMT1 in Salt Tolerance

Abstract

Caffeic acid O-methyltransferase (COMT) participates in various physiological activities in plants, such as positive responses to abiotic stresses and the signal transduction of phytohormones. In this study, 18 COMT genes were identified in the chromosome-level reference genome of mango, named MiCOMTs. A phylogenetic tree containing nine groups (I-IX) was constructed based on the amino acid sequences of the 71 COMT proteins from seven species. The phylogenetic tree indicated that the members of the MiCOMTs could be divided into four groups. Quantitative real-time PCR showed that all MiCOMT genes have particularly high expression levels during flowering. The expression levels of MiCOMTs were different under abiotic and biotic stresses, including salt and stimulated drought stresses, ABA and SA treatment, as well as Xanthomonas campestris pv. mangiferaeindicae and Colletotrichum gloeosporioides infection, respectively. Among them, the expression level of MiCOMT1 was significantly up-regulated at 6-72 h after salt and stimulated drought stresses. The results of gene function analysis via the transient overexpression of the MiCOMT1 gene in Nicotiana benthamiana showed that the MiCOMT1 gene can promote the accumulation of ABA and MeJA, and improve the salt tolerance of mango. These results are beneficial to future researchers aiming to understand the biological functions and molecular mechanisms of MiCOMT genes.

Keywords: COMT gene family; salt tolerance; transcriptional analysis; transient overexpression.

Figure 1

Phylogenetic relationship of COMTs in different species using complete protein sequences. The neighbor-joining (NJ) phylogenetic tree was constructed with the Poisson model with 1000 bootstrap replicates using MEGA 11 software. The tree was generated from an amino acid sequence alignment of M. indica (Mi) (18 MiCOMTs), A. thaliana (At) (17 members), M. esculenta (Me) (10 members), N. tabacum (Nt) (8 members), S. lycopersicum (Sl) (5 members), O. sativa (Os) (1 member), and P. bretschneideri (Pb) (12 members). The phylogenetic tree was categorized into nine groups, highlighted using different colors. The numerical value on the branch indicates the genetic distance. I–IX indicates grouping.Red star means emphasized that the later MiCOMT1 is the main body of research in this article.

Figure 2

The structural and motif analysis of the MiCOMTs: (A) The phylogenetic tree was constructed with MEGA 11 software using protein sequences of the 18 MiCOMT proteins. The motifs were characterized using the MEME website and TBtools v1.098768 software with the number of motifs set to 8, and the motif discovery mode was classic mode. The eight motifs are named Motif 1 to Motif 8, and are represented with different color boxes. (B) The consensus sequence of 8 motifs.

Figure 3

The collinearity and Ka/Ks of COMT proteins: (A) Collinearity analysis of the COMT gene family in mango. Each colored square on the edge of the circle represents mango chromosomes. The gray lines indicate collinear pairs of all mango genes, and the red lines represent collinear pairs of MiCOMTs. (B) Distribution of the Ka/Ks values of MiCOMTs. (C) Collinearity analysis of COMT genes among mango and Arabidopsis. The gray lines represent the collinear blocks in the genomes of mango and Arabidopsis, and the red line represents the collinear COMT gene pairs.

Figure 4

The expression of MiCOMT genes in different mango tissues. The colors spanning from blue to orange represent increasing levels of gene expression. The level of expression in root was used as a reference to determine up- or down-regulation in the other plant tissues. The horizontal axis represents the different tissue parts of the mango, and the vertical axis represents the MiCOMT genes. “#” represents significantly up-regulated gene expression. “*” represents significantly down-regulated gene expression.

Figure 5

Expression patterns of MiCOMT genes under salt and drought stresses. Mango seedlings were treated with 300 mmol·L⁻¹ NaCl (A) and 30% PEG6000 (B) for 0, 3, 6, 12, 24, 48, and 72 h. The level of expression at 0 h was used as a reference to determine up- or down-regulation at other time points. The colors spanning from blue to orange represent increasing levels of gene expression. “#” represents significantly up-regulated gene expression. “*” represents significantly down-regulated gene expression.

Figure 6

Expression patterns of MiCOMT genes under ABA and SA treatment. Mango seedling leaves were treated with 5 mmol·L⁻¹ ABA (A) and 5 mmol·L⁻¹ SA (B) for 0, 3, 6, 12, 24, 48, and 72 h. The level of expression at 0 h was used as a reference to determine up- or down-regulation at other time points. The colors spanning from blue to orange represent increasing levels of gene expression. “#” represents significantly up-regulated gene expression. “*” represents significantly down-regulated gene expression.

Figure 7

Expression patterns of MiCOMT genes during infection with X. campestris pv. mangiferaeindicae and C. gloeosporioides. Mango seedling leaves were treated with 2 × 10⁶ cfu·mL⁻¹ of X. campestris pv. mangiferaeindicae suspension (A) and 2 × 10⁷ conidia·mL⁻¹ suspension of C. gloeosporioides (B) for 0, 3, 6, 12, 24, 48, and 72 h. The level of expression at 0 h was used as a reference to determine up- or down-regulation at other time points. The colors spanning from blue to orange represent increasing levels of gene expression. “#” represents significantly up-regulated gene expression. “*” represents significantly down-regulated gene expression.

Figure 8

At 3 dpi, gene-overexpressing N. benthamiana leaves were used for physiological and biochemical analysis. (A) Schematic diagram of the control (vector) and 35S::MiCOMT1. (B–J) Each experiment had three replicates and six N. benthamiana seedlings per replicate. Oligomeric Proanthocyanidin (OPC), chlorophyll (CHL), hydrogen peroxide (H₂O₂), methyl jasmonic acid (MeJA), salicylic acid (SA), malondialdehyde (MDA), abscisic acid (ABA), ethylene (ETH), and auxin (IAA) contents of N. benthamiana seedlings were treated as described. All data represent mean ± SE for three biological replications. Statistical significance was determined using Duncan’s test, and “a and b” represent significant differences.

Figure 9

Overexpression of MiCOMT1 confers salt tolerance in N. benthamiana: (A–I) MiCOMT1 was overexpressed in N. benthamiana seedlings for three days, and then treated with 300 mmol·L⁻¹ NaCl solution. Each experiment had three replicates and six N. benthamiana seedlings per replicate. Oligomeric Proanthocyanidin (OPC), chlorophyll (CHL), hydrogen peroxide (H₂O₂), methyl jasmonic acid (MeJA), salicylic acid (SA), malondialdehyde (MDA), abscisic acid (ABA), ethylene (ETH), and auxin (IAA) contents of N. benthamiana seedlings were treated as described. All data represent mean ± SE for three biological replications. Statistical significance was determined via Duncan’s test, and “a, b, c, d, and e” represent significant differences.