Overexpression of the Melatonin Synthesis-Related Gene SlCOMT1 Improves the Resistance of Tomato to Salt Stress

Abstract

Melatonin can increase plant resistance to stress, and exogenous melatonin has been reported to promote stress resistance in plants. In this study, a melatonin biosynthesis-related SlCOMT1 gene was cloned from tomato (Solanum lycopersicum Mill. cv. Ailsa Craig), which is highly expressed in fruits compared with other organs. The protein was found to locate in the cytoplasm. Melatonin content in SlCOMT1 overexpression transgenic tomato plants was significantly higher than that in wild-type plants. Under 800 mM NaCl stress, the transcript level of SlCOMT1 in tomato leaf was positively related to the melatonin contents. Furthermore, compared with that in wild-type plants, levels of superoxide and hydrogen peroxide were lower while the content of proline was higher in SlCOMT1 transgenic tomatoes. Therefore, SlCOMT1 was closely associated with melatonin biosynthesis confers the significant salt tolerance, providing a clue to cope with the growing global problem of salination in agricultural production.

Keywords: SlCOMT1; genetical transformation; melatonin; salt stress; tomato.

Figure 1

Bioinformatic analysis of the SlCOMT1 protein. (A) Two domains of the SlCOMT1 protein. (B) Phylogenetic tree was constructed using the five tomato COMT proteins and Arabidopsis AtCOMT protein. (C) Comparison of predicted SlCOMT1 protein sequence with AtCOMT. SlCOMT1 (XP_004235028.1), AtCOMT (NP_200227.1). Sl, Solanum lycopersicum; At, Arabidopsis thaliana.

Figure 2

Induction of the SlCOMT1 protein in vitro. Lane 1, bacterial solution with no IPTG (control); lane 2, bacterial solution with IPTG cultured for 6 h at 37 °C; lane 3, supernatant derived from pET32a-SlCOMT cell lysate; and lane 4, precipitated SlCOMT1 protein.

Figure 3

Evolution relationship of SlCOMT1 with other COMT proteins. (A) Comparison of amino acid sequences between tomato SlCOMT1 and COMT from other species, including In, Ipomoea nil (BAE94400.1); Oe, Olea europaea (XP_022844536.1); Vv, Vitis vinifera (XP_003634161.1); Rs, Rauvolfia serpentine (AOZ21153.1); Cc, Capsicum chinense (BAR88175.1); Sp, Solaum pennellii (XP_015070697.1); and Cs, Camellia sinensis (ADN27527.1); (B) Five conserved domains of the COMT proteins.

Figure 4

Phylogenetic analysis of the SlCOMT1 protein and its homologs. The proteins in the phylogenetic tree include Bp, Betula pendula (FJ667539.2); Jr, Juglans regia (XP_018828596.1); Pt, Populus Table 002321948. Me, Manihot esculenta (XP_021627291.1); Dz, Durio zibethinus (XP_022736469.1); Ga, Gossypium arboretum (XP_017611038.1); Ls, Liquidambar styraciflua (AF139533.1); Tt, Thalictrum tuberosum (AF064694.1); Na, Nicotiana attenuata (OIT03318.1); St, Solanum tuberosum (XP_015164331.1); Si, Sesamum indicum (XP_011075886.2); Dc, Daucus carota (XM_017381671.1); As, Anthriscus sylvestris (AB820126.1); and Ca, Capsicum annuum (NP_001311774.1).

Figure 5

Structure prediction of the SlCOMT1 protein. (A) The secondary structure prediction of the SlCOMT1 protein. The blue indicates α-helices; purple indicates random coils; red indicates extended strands; green indicates β-turns; and the horizontal numbers indicate the positions of the amino acids. (B): The predicted three-dimensional structure of the SlCOMT1 protein.

Figure 6

Localization of SlCOMT1. (A) Green fluorescence of SlCOMT1-PRI. (B) Bright-field image of Agrobacterium-infiltrated tobacco leaf. (C) The merged fluorescent images. The tobacco leaves were injected with the transgenic Agrobacterium liquid, and then cultivated in the culture chamber for 2 days and observed by confocal microscopy. GFP, Green Fluorescence Protein; DIC, Diascoptic Lighting Channel.

Figure 7

Expression levels of SlCOMT1 in different tomato tissues.

Figure 8

(A) Melatonin content in WT, OE-1, and OE-2 tomato plants. The same letter in the same growing season means no significant differences among three biological replicates (p < 0.05). Error bars represent standard error. (B) Growth status of WT, OE-1, and OE-2 after 800 mM NaCl treatment. WT, wild-type; OE, overexpression transgenic tomato.

Figure 9

(A–C) were respectively the contents of superoxide, hydrogen peroxide, and proline in WT, OE-1, and OE-2 tomato plants, respectively. The same letter in the same growing season means no significant differences among three biological replicates (p < 0.05). Error bars represent SE.