Melatonin Represses Oil and Anthocyanin Accumulation in Seeds

Abstract

Previous studies have clearly demonstrated that the putative phytohormone melatonin functions directly in many aspects of plant growth and development. In Arabidopsis (Arabidopsis thaliana), the role of melatonin in seed oil and anthocyanin accumulation, and corresponding underlying mechanisms, remain unclear. Here, we found that serotonin N-acetyltransferase1 (SNAT1) and caffeic acid O-methyltransferase (COMT) genes were ubiquitously and highly expressed and essential for melatonin biosynthesis in Arabidopsis developing seeds. We demonstrated that blocking endogenous melatonin biosynthesis by knocking out SNAT1 and/or COMT significantly increased oil and anthocyanin content of mature seeds. In contrast, enhancement of melatonin signaling by exogenous application of melatonin led to a significant decrease in levels of seed oil and anthocyanins. Further gene expression analysis through RNA sequencing and reverse-transcription quantitative PCR demonstrated that the expression of a series of important genes involved in fatty acid and anthocyanin accumulation was significantly altered in snat1-1 comt-1 developing seeds during seed maturation. We also discovered that SNAT1 and COMT significantly regulated the accumulation of both mucilage and proanthocyanidins in mature seeds. These results not only help us understand the function of melatonin and provide valuable insights into the complicated regulatory network controlling oil and anthocyanin accumulation in seeds, but also divulge promising gene targets for improvement of both oil and flavonoids in seeds of oil-producing crops and plants.

Figure 1.

Subcellular localization of the SNAT1 and COMT proteins in N. benthamiana leaves. Subcellular distribution of the SNAT1 (A) and COMT (B) proteins fused with GFP (35S:SNAT1-GFP or 35S:COMT-GFP). DAPI, Fluorescence of 4′, 6-diamino-2-phenylindole; Merge 1, merge of GFP, DAPI, and bright-field images; Merge 2, merge of chlorophyll, GFP, DAPI, and bright-field images.

Figure 2.

Tissue-specific analyses of SNAT1 and COMT expression patterns. A and D, RT-qPCR analysis of SNAT1 (A) and COMT (D) expression in various tissues of wild-type (Col-0) plants. Rt, Roots; St, stems; RL, rosette leaves; CL, cauline leaves; FB, flower buds; OF, open flowers. Values are means ± sd (n = 3). B and E, RT-qPCR analysis of SNAT1 (B) and COMT (E) expressions in developing seeds of wild type (Col-0) plants. Values are means ± sd (n = 3). C and F, Representative GUS staining of pSNAT1:GUS (C) and pCOMT:GUS (F) transgenic plants show SNAT1 and COMT expression levels, respectively, in vegetative and reproductive tissues in wild-type (Col-0) plants. C, Upper photos successively (from left to right) indicate 9-d–old seedlings (C1), rosette leaves (C2), stems and cauline leaves (C3), and flower buds and open flowers (C4), and lower photos successively (from left to right) represent developing seeds at different developmental stages (C5–C8). F, Upper photos successively (from left to right) indicate 8-d–old seedlings (F1), rosette leaves (F2), stems, cauline leaves, and flower buds (F3), and open flowers (F4), and lower photos successively (from left to right) represent developing seeds at different developmental stages (F5–F8). The RT-qPCR results were normalized against the expression of EF1αA4 as an internal control. Scale bars = 2 mm (C1–C4 and F1–F4), except for seeds, where scale bars = 100 μm (C5–C8 and F5–F8).

Figure 3.

Melatonin quantification in developing siliques from various lines of SNAT1 and COMT. A, Structure of the COMT (AT5G54160) gene showing the position of T-DNA insertions in SALK_002373 (comt-1) and SALK_020611C (comt-2) mutants. The coding and untranslated regions are represented by black and gray boxes, respectively, and introns and other genomic regions are represented by open boxes. Translation start site (ATG) and stop codon (TAA) are indicated. The arrow indicates the left border of the T-DNA. B, PCR-based DNA genotyping of the homozygous mutants of the COMT gene. LP and RP refer to the gene-specific primers, and BP refers to T-DNA right-border primer. Three independent biological replicates were carried out. C, RT-PCR analysis of COMT transcript in wild type (Col-0) and their corresponding mutants. EF1αA4 was used as an internal control. Three independent biological replicates were conducted. D, Melatonin levels in the developing siliques at 12 DAP from wild type (Col-0); the single mutants of snat1-1, comt-1, and comt-2; the double mutant snat1-1 comt-1; and the transgenic plants of snat1-1 gSNAT1-1 and comt-1 gCOMT-1. Values are means ± sd (n = 3). Different lowercase letters within various lines of the SNAT1 and COMT genes indicate significant differences at P ≤ 0.05 (Tukey’s honest significant difference test). FW, Fresh weight.

Figure 4.

Effect of endogenous deficiency and exogenous application of melatonin on seed FA and anthocyanin accumulation. A and C, Total FA (A) and anthocyanin (C) contents in seeds from wild type (Col-0); the single mutants of snat1-1, comt-1, and comt-2; the double mutant snat1-1 comt-1; and the transgenic plants of snat1-1 gSNAT1-1 and comt-1 gCOMT-1. Different lowercase letters within various lines of the SNAT1 and COMT genes indicate significant differences at P ≤ 0.05 (Tukey’s honest significant difference test). B and D, Total FA (B) and anthocyanin (D) contents in seeds of wild type (Col-0), and snat1-1, comt-1, and snat1-1 comt-1 exogenously applied with different concentrations of melatonin solutions (0, 100, and 200 μm). Different letters within each treatment indicate significant differences at P ≤ 0.05 (Tukey’s honest significant difference test); lowercase letters compare with each other, capital letters compare with each other, and Greek letters compare with each other. Asterisks denote statistically significant differences between the indicated samples (Student’s t test, P ≤ 0.05). A to D, Values are means ± sd (n = 5). DW, Dry weight.

Figure 5.

Dynamic expression analysis of genes related to seed oil accumulation in developing seeds of wild-type (Col-0) and snat1-1 comt-1 plants. Gene expression was normalized against the expression of EF1αA4 as an internal control, and the expression level in wild type was set to 1 (dotted line). Values are means ± sd (n = 3). Asterisks indicate significant differences in gene expression levels in snat1 comt-1 plants compared with those in wild-type plants (two-tailed paired Student’s t test, *P ≤ 0.05).

Figure 6.

Dynamic expression analysis of genes contributing to seed anthocyanin accumulation in developing seeds of wild-type (Col-0) and snat1-1 comt-1 plants. Gene expression was normalized against the expression of EF1αA4 as an internal control, and the expression level in wild type was set to 1 (dotted line). Values are means ± sd (n = 3). Asterisks indicate significant differences in gene expression levels in snat1-1 comt-1 plants compared with those in wild-type plants (two-tailed paired Student’s t test, *P ≤ 0.05).

Figure 7.

Effect of SNAT1 and COMT on seed-coat mucilage deposition. A, Comparison of the mucilage layer attached to the seed coat among wild type (Col-0); the single mutants of snat1-1, comt-1, and comt-2; the double mutant snat1-1 comt-1; and the transgenic plants of snat1-1 gSNAT1-1 and comt-1 gCOMT-1. Scale bars = 500 μm. B, Comparison of the dynamic expression of DF1 and MUM4 in developing seeds from 8 to 12 DAP among wild type (Col-0), the single mutant snat1-1, and the transgenic plant snat1-1 gSNAT1-1. C, Comparison of the dynamic expression of DF1 and MUM4 in developing seeds from 8 to 12 DAP among wild type (Col-0), the single mutant comt-1, and the transgenic plant comt-1 gCOMT-1. Gene expression was normalized against the expression of EF1αA4 as an internal control, and the expression level in wild type was set to 1. In B and C values are means ± sd (n = 3). Asterisks indicate significant differences in gene expression levels in snat1-1 or comt-1 plants compared with those in wild-type plants (two-tailed paired Student’s t test, *P ≤ 0.05).

Figure 8.

Effect of SNAT1 and COMT on the accumulation of PAs in seeds. A, Seeds stained with DMACA for 16 h among wild type (Col-0); the single mutants of snat1-1, comt-1, and comt-2; the double mutant snat1-1 comt-1; and the transgenic plants of snat1-1 gSNAT1-1 and comt-1 gCOMT-1. Scale bars = 500 μm. B and C, Analysis of soluble (B) and insoluble (C) PAs by acidic hydrolysis among wild type (Col-0); single mutants of snat1-1, comt-1, and comt-2; the double mutant snat1-1 comt-1; and the transgenic plants of snat1-1 gSNAT1-1 and comt-1 gCOMT-1. In B and C, values are means ± sd (n = 5). Different letters within various lines represent significant differences at P ≤ 0.05 (Tukey’s honest significant difference test). DW, Dry weight.

Figure 9.

A proposed working model shows that the deficiency of melatonin by knocking out SNAT1 and/or COMT represses the accumulation of oil and anthocyanins by regulating the expression of key genes that control the biosynthesis of oil and anthocyanins, respectively, in Arabidopsis seeds. Arrows and T bars indicate promoting and inhibitory effects, respectively.