The role of watermelon caffeic acid O-methyltransferase (ClCOMT1) in melatonin biosynthesis and abiotic stress tolerance

Abstract

Melatonin is a pleiotropic signaling molecule that regulates plant growth and responses to various abiotic stresses. The last step of melatonin synthesis in plants can be catalyzed by caffeic acid O-methyltransferase (COMT), a multifunctional enzyme reported to have N-acetylserotonin O-methyltransferase (ASMT) activity; however, the ASMT activity of COMT has not yet been characterized in nonmodel plants such as watermelon (Citrullus lanatus). Here, a total of 16 putative O-methyltransferase (ClOMT) genes were identified in watermelon. Among them, ClOMT03 (Cla97C07G144540) was considered a potential COMT gene (renamed ClCOMT1) based on its high identities (60.00-74.93%) to known COMT genes involved in melatonin biosynthesis, expression in almost all tissues, and upregulation under abiotic stresses. The ClCOMT1 protein was localized in the cytoplasm. Overexpression of ClCOMT1 significantly increased melatonin contents, while ClCOMT1 knockout using the CRISPR/Cas-9 system decreased melatonin contents in watermelon calli. These results suggest that ClCOMT1 plays an essential role in melatonin biosynthesis in watermelon. In addition, ClCOMT1 expression in watermelon was upregulated by cold, drought, and salt stress, accompanied by increases in melatonin contents. Overexpression of ClCOMT1 enhanced transgenic Arabidopsis tolerance against such abiotic stresses, indicating that ClCOMT1 is a positive regulator of plant tolerance to abiotic stresses.

Fig. 1. Chromosomal locations and phylogenetic analysis of ClOMT genes in watermelon.

A The chromosomal location was drawn using MapChart2.32. The tandem duplicated genes are boxed. B The phylogenetic tree of the ClOMT genes was constructed using MEGA 7.0.21, and the ClOMT genes were placed into groups I, II and III. Bar, 0.2 substitutions per site; Chr, chromosome; ClOMT Citrullus lanatus O-methyltransferase

Fig. 2. The expression of ClOMT genes in different tissues and in response to abiotic stresses.

Heat maps of ClOMT genes in (A) different tissues and (B) in response to cold, drought, or osmotic stress. The RNA-seq data were obtained from the CuGenDB (http://cucurbitgenomics.org/). Color coding was done according to the scale given. Boxes filled with dots indicate no detection. In (A), watermelon tissues included fruit flesh (FF), fruit rind (FR) 10, 18, 26, and 34 days after pollination, seeds 49 days after pollination, phloem, vascular bundle, root, and leaf tissues. Transcript levels were calculated as log2-transformed Reads Per Kilobase of exon model per Million mapped reads (RPKM). In (B), the watermelon seedlings were exposed to 4 °C for 6 h, unwatered for eight days, or treated with PEG 6000 (20%) on roots for 6 h. Leaf tissues were harvested after cold and drought, while root samples were taken after PEG 6000 treatment. The values were calculated as log2-transformed RPKM ratios of Treatment/Control. ClOMT Citrullus lanatus O-methyltransferase

Fig. 3. Comparison of the ClCOMT1 protein sequence with those of AtCOMT, SlCOMT1, and OsCOMT.

The putative N-acetylserotonin (NAS)-binding domains are shown in the red box. The phenolic substrate-binding sites are marked with asterisks. The S-adenosyl-L-methionine (SAM)-binding sites are underlined. At Arabidopsis thaliana, Cl Citrullus lanatus, Os Oryza sativa, Sl Solanum lycopersicum, COMT caffeic acid O-methyltransferase

Fig. 4. Subcellular localization of ClCOMT1 in watermelon protoplasts.

A, E Bright-field image of watermelon protoplasts. B, F Red fluorescence of chlorophyll autofluorescence. C, G Green fluorescence of GFP. D, H Merged view of two fluorescence images (B + C, F + G). Bars = 18.2 µm. ClCOMT Citrullus lanatus caffeic acid O-methyltransferase, GFP green fluorescent protein

Fig. 5. Effects of ClCOMT1 gene-editing on endogenous melatonin contents in watermelon calli.

A Two sgRNAs (Target 1 and Target 2) in the 5’-region of ClCOMT1. B Schematic diagram for constructing two sgRNA cassettes in the binary vector PBSE402. C Mutation types and rates in the transgenic watermelon calli after CRISPR/Cas9-mediated gene editing. D Melatonin contents in transgenic and wild-type (WT) calli. In (D), values are means ± SD (n = 3). Asterisk (*) indicates significant difference at P < 0.05. ClCOMT Citrullus lanatus caffeic acid O-methyltransferase

Fig. 6. Effects of ClCOMT1 overexpression on endogenous melatonin contents in watermelon calli.

A Schematic diagram of the pCambia1305.4-ClCOMT1 vector. B, C RT-PCR and qRT-PCR analysis of ClCOMT1 transcripts in overexpression lines and wild-type (WT) calli. D Melatonin contents in overexpression lines and WT calli. β-actin, watermelon internal reference gene. In (C, D), values are means ± SD (n = 3). Asterisk (*) indicates significant difference at P < 0.05. ClCOMT Citrullus lanatus caffeic acid O-methyltransferase

Fig. 7. Melatonin contents and ClCOMT1 expression in response to cold, drought, or salt stress.

Watermelon seedlings with three true leaves were exposed to cold at 4 °C, unwatered, or irrigated with 300 mM NaCl (80 mL per plant). Leaf samples were taken after cold, drought, or salt treatment for 24 h, 4 d, or 2 d, respectively. Values are means ± SD (n = 3). An asterisk (*) indicates significant difference at P < 0.05. CK control check, ClCOMT, Citrullus lanatus caffeic acid O-methyltransferase

Fig. 8. Effects of ClCOMT1 overexpression on melatonin contents and abiotic stress tolerance in Arabidopsis.

A Schematic diagram of the pGREEN-ClCOMT1 vector. B RT-PCR analysis of ClCOMT1 expression in transgenic and wild-type (WT) Arabidopsis. C Melatonin contents in transgenic and WT Arabidopsis. D, E Phenotypes and survival rates of transgenic (line #4) and WT Arabidopsis after freezing, mannitol, or NaCl treatment. For freezing treatment, two-week-old Arabidopsis seedlings were exposed to −10 °C for 1 h, followed by recovery at 22 °C for 4 d. For drought and salt stress, Arabidopsis seeds were sown and grown on ½ MS containing 250 mM D-Mannitol and 75 mM NaCl, respectively. In (C, E), values are means ± SD (n = 3). Asterisk (*) indicates significant difference at P < 0.05. CK, control check; ClCOMT, Citrullus lanatus caffeic acid O-methyltransferase