Melatonin represses oil and anthocyanin accumulation in seeds.

Previous studies have clearly demonstrated that the putative phytohormone melatonin functions directly in many aspects of plant growth and development. In 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 A. thaliana 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.

tissues of wild-type plants. SNAT1 was highly expressed in various tissues 139 except for stems (Figures 2A, B). COMT was widely distributed in different 140 tissues, and its transcript level was much higher in roots, flower buds, open 141 flowers, and developing seeds than in stems, rosette leaves, and cauline 142 leaves ( Figures 2D, E). During seed development, the expression of SNAT1 143 and COMT exhibited a similar pattern and increased rapidly from 8 days after 144 pollination (DAP) to the maximal level at 10 DAP, and then decreased gradually 145 afterwards ( Figures 2B, E). We determined the melatonin levels in developing siliques at 12 DAP 182 between wild type plants and various single and double mutants of SNAT1 and 183 COMT genes. As illustrated in Figure 3D, the three single mutants of snat1-1, 184 comt-1, and comt-2 contained much less melatonin than wild-type plants, and 185 the double mutant of snat1-1 comt-1 accumulated much less than their 186 corresponding single mutants. The snat1-1 comt-1 mutant still produced 187 melatonin ( Figure 3D  We measured the quantities of the major FA compositions and total FAs 192 per microgram of mature seeds between wild-type plants and the single and 193 double mutants of SNAT1 and COMT genes. As shown in Figure 4A and Table  194 S1, the seed FA contents in all three single mutants of snat1-1, comt-1, and 195 comt-2 were about 6% higher than that of wild type plants, and the significant 196 increase of FA contents was accompanied by an increase in all detected FA 197 compositions. The FA content of snat1-1 comt-1 seeds was much higher than 198 that of their corresponding single mutants and was 17% higher than that of 199 wild-type plants ( Figure 4A; Table S1). These results indicated that SNAT1 and 200 COMT have an additive effect in the repression of FA accumulation in A. 201 thaliana seeds. 202 We also analyzed the contents of anthocyanins in seeds of wild-type plants 203 and various single and double mutants of SNAT1 and COMT genes. The loss 204 of function of either SNAT1 or COMT resulted in a significant increase in the 205 accumulation of anthocyanins in seeds, and the comt mutation accumulated 206 more anthocyanins than the snat1-1 mutation ( Figure 4C; Table S2). However, 207 no obvious difference was observed in the seed anthocyanin content between 208 the comt mutants and the double mutant snat1-1 comt-1 ( Figure 4C; Table S2). 209 These results suggested that SNAT1 and COMT have a non-additive effect on 210 the accumulation of anthocyanins in seeds, and COMT is more important than 211 SNAT1 for seed anthocyanin biosynthesis. 212 To further confirm the function of SNAT1 and COMT on the accumulation of 213 FAs and anthocyanins, we transformed snat1-1 and comt-1 mutants with the 214 genomic constructs of gSNAT1 and gCOMT, respectively. Among more than 215 15 independent lines regenerated for each construct, at least three 216 homozygous progenies for each construct containing a single transgene were 217 selected based on a 3:1 Mendelian segregation ratio on 218 glufosinate-ammonium-containing medium. Examination of the representative 219 lines, snat1-1 gSNAT1#1 and comt-1 gCOMT#1, showed that the expression 220 levels of SNAT1 and COMT were restored to wild-type levels ( Figure S2), and 221 the lower melatonin content in both snat1-1 and comt-1 was also fully rescued 222 to wild-type levels ( Figure 3D) in their corresponding rescued lines. Thus, the 223 representative transformants of snat1-1 gSNAT1#1 and comt-1 gCOMT#1 224 were utilized for further experiments. We found that the higher contents of both 225 FAs and anthocyanins in snat1-1 and comt-1 seeds were fully restored to 226 The results showed that exogenous application of melatonin on wild-type plants 233 led to a significant decrease of both oil ( Figure 4B; Table S1) and anthocyanin 234 ( Figure 4D; Table S2) levels in seeds. Under exogenous melatonin treatment, 235 the seed oil content of the single and double mutants was almost the same as 236 that of wild-type plants ( Figure 4B; Table S1), whereas the seed oil content of 237 the single mutants was slightly lower than that of the double mutant, and slightly 238 higher than that of wild-type plants ( Figure 4B; Table S1). These findings 239 showed that SNAT1 and COMT repress FA accumulation in an independent 240 and additive manner, but mainly by influencing melatonin biosynthesis, in A. 241 thaliana seeds. 242 In addition, under exogenous melatonin treatment, the anthocyanin content 243 in snat1-1 seeds was the same as that of wild-type plants ( Figure 4D; Table  244 S2), whereas the seed anthocyanin contents of comt1-1 and snat1-1 comt-1 245 mutants were the same, and higher than that of wild-type plants ( Figure 4D; 246 Table S2). These findings indicated that SNAT1 inhibits seed anthocyanin 247 deposition only by affecting melatonin biosynthesis, whereas COMT represses 248 seed anthocyanin accumulation not only by itself, but also by influencing 249 melatonin biosynthesis. 250 No obvious differences in seed coat color, size, and weight were observed 251 among the single and double mutants of SNAT1 and COMT, the transgenic 252 plants of snat1-1 gSNAT1#1 and comt-1 gCOMT#1, wild-type plants applied 253 with exogenous melatonin, or their corresponding controls ( Figure S3). 254 Overall, we demonstrated that, through blocking endogenous melatonin 255 biosynthesis by knocking out SNAT1 and/or COMT and by exogenous 256 application of melatonin, melatonin represses the accumulation of both oil and 257 anthocyanins. In addition, SNAT1 and COMT, independent of melatonin, 258 exhibit distinct roles in the inhibition of oil and anthocyanin biosynthesis in A. 259 thaliana seeds. 260 261 Genome-wide analysis of DEGs in developing seeds at 12 DAP between 262 wild type and snat1-1 comt-1 plants 263 In A. thaliana developing seeds, FAs start to accumulate at 6 DAP, and 264 increase linearly from 8 to 18 DAP during seed maturation (Baud and Lepiniec, 265 2009, 2010). The double mutant snat1-1 comt-1 accumulated much more seed 266 FAs than wild type and single mutants of SNAT1 and COMT ( Figure 4A; Table  267   S1). In addition, 12 DAP is the key stage for the biosynthesis of seed 268 flavonoids, including anthocyanins, during seed maturation (Routaboul et al., 269 2012). Therefore, we utilized developing seeds at 12 DAP to compare the 270 expression profiles at a genome-wide level between wild type and snat1-1 271 comt-1 plants. These profiles would provide information on the downstream 272 targets of melatonin that contribute to FA and anthocyanin accumulation, as 273 well as facilitate a better understanding of the regulatory network underlying 274 melatonin-mediated metabolites biosynthesis in A. thaliana seeds. 275 RNA-seq analysis identified 243 differentially expressed genes (DEGs), 276 among which 119 were up-regulated (Table S3) and 124 were down-regulated 277 (Table S4) in snat1-1 comt-1 developing seeds at 12 DAP. Functional analysis 278 discovered that 12 (4.9%) and six (2.5%) of the DEGs were related to oil and 279 anthocyanin metabolisms, respectively (Tables S2 and S3). However, the 280 expression of other genes that play major roles in oil and anthocyanin 281 accumulation was not altered in snat1-1 comt-1 seeds compared to wild-type 282 seeds (Table S5). Up to nine (7.6%) up-regulated genes and no 283 down-regulated genes were related to carbohydrate metabolism (Tables S3  284 and S4). Multiple up-regulated (16, 13.4%) and down-regulated (30, 24.2%) 285 genes were involved in general protein metabolism in snat1-1 comt-1 seeds 286 (Tables S3 and S4 precursors (2S1 to 2S5), between wild-type and snat1-1 comt1-1 seeds (Table  292 S5). Consistently, there was no substantive difference in the content of seed 293 storage proteins between wild type and snat1-1 comt1-1 plants ( Figure S4). It is 294 worth mentioning that the number of DEGs involved in the stress/defense 295 response and other biological processes accounts for the largest proportion of 296 all the DEGs in snat1-1 comt-1 seeds (Tables S3 and S4). 297 Therefore, simultaneous knockout of SNAT1 and COMT, essential for 298 melatonin biosynthesis, regulates a series of genes important for oil and 299 anthocyanin accumulation and many genes involved in other biological 300 processes during seed maturation. 301 302 Verification of regulated genes involved in oil and anthocyanin 303 biosynthesis at different developmental stages in snat1-1 comt-1 seeds 304 To confirm the regulation of DEGs involved in oil and anthocyanin 305 biosynthesis in snat1-1 comt-1 developing seeds at 12 DAP, and to extensively 306 explore expression alterations of these genes, we performed RT-qPCR to 307 compare their expression patterns at the seed maturation stages (12-16 DAP) 308 between wild type and snat1-1 comt-1 plants. 309 For the highly up-regulated genes related to oil accumulation, we chose 310 one regulatory gene, WRINKLED1 (WRI1), and five structural genes, BIOTIN 311  Table 1). The expression levels 316 of all six genes from 12 to 16 DAP were always significantly higher in the 317 snat1-1 comt-1 mutant than in wild type ( Figure 5). As detailed in Figure 5 Table 2). Except for 331 GPT2 expression at 16 DAP, from 12 to 16 DAP the expression levels of all six 332 genes were dramatically altered in the snat1-1 comt-1 mutant compared to wild 333 type ( Figure 6). Compared to wild type, the relative expression of KFB39 was 334 always significantly lower, and the relative expression levels of UGT73B2, 335 KAN4, and GPT2 gradually declined in snat1-1 comt-1 developing seeds from 336 12 to 16 DAP ( Figure 6). The relative expression levels of 4CL1 and CHI 337 increased from 12 DAP to the peaks at 14 DAP and then decreased afterwards 338 in the snat1-1 comt-1 mutant compared to wild type ( Figure 6). 339 Taken together, simultaneous knockout of SNAT1 and COMT, essential for 340 melatonin biosynthesis, inhibits seed oil and anthocyanin accumulation by 341 regulating a range of genes contributing to oil and anthocyanin biosynthesis, 342 respectively, during seed maturation.  (Table S5). On the other hand, exogenous application of 361 melatonin to wild-type plants did not alter the accumulation of seed coat 362 mucilage ( Figure S5). The results suggested that melatonin has no effect on 363 seed coat mucilage biosynthesis, although SNAT1 and COMT antagonistically 364 affect its production. 365 To investigate how SNAT1 and COMT separately regulate seed coat 366 mucilage production, we carried out RT-qPCR to compare the expression of 367  their seeds than wild-type plants ( Figures 8B, C). Moreover, the higher 396 amounts of PAs in snat1-1 and comt-1 were fully rescued by the introduction of 397 gSNAT1 and gCOMT, respectively ( Figures 8B, C). It is worthy to note that 398 levels of both total and solvent-soluble PAs in the comt seeds were higher than 399 those of snat1-1 seeds, and comparable with snat1-1 comt-1 seeds (Figure 8). Thus, these regulated carbohydrate metabolism genes (Table S3)   thus the much lower expression of the two GDSL-type lipase genes 513 (AT2G30310 and AT5G45670) observed in our study is helpful for 514 understanding the higher oil content in snat1-1 comt-1 seeds (Table S4).  (Table S4) 518 together assist in promoting seed oil accumulation ( Figure 4A; Table S1) in 519 snat1-1 comt-1 developing seeds. 520 Anthocyanin biosynthesis starts from the phenylpropanoid pathway 521     Table S2) and PAs (Figure 8), and mucilage 585 (Figures 7 and S1). These interesting questions need further investigation. 586 Even so, as exogenous application of melatonin and loss of function of SNAT1 587 and COMT exhibited opposite effects on seed oil and anthocyanin 588 accumulation (Figure 4; Tables S1 and S2), and SNAT1 and COMT had a 589 common and additive role in melatonin biosynthesis in developing siliques 590 seeds; an underlying mechanism is proposed in Figure 9. 593 In summary, the present study provides significant and fresh information in 594 several ways. First, this study demonstrates that melatonin represses seed oil 595 and anthocyanin accumulation during seed maturation by inhibiting the 596 expression of important genes involved in oil and anthocyanin biosynthesis, 597 respectively. Second, in A. thaliana seeds, the two essential melatonin 598 biosynthetic genes SNAT1 and COMT, independent of melatonin, have distinct 599 functions on different metabolites, including oil, flavonoids inclusive of 600 anthocyanins and PAs, and mucilage, which might be due to their differential 601 distribution among subcellular fractions. Third, seed metabolite accumulation is 602 controlled by a coordinated regulatory network, which is not only pertinent to 603 major steps of their metabolic pathways but also requires the partitioning of and Level-4, respectively. The different levels of melatonin solutions were 624 applied to ten individual plants (Col-0, snat1-1, comt-1, and snat1-1 comt-1) Table S6. 657

Microscopic observation of A. thaliana seed traits 658
Mature seeds of different A. thaliana lines were harvested from major 659 inflorescences, specifically from siliques at the basal region, and then randomly 660 selected to be photographed with an OLYMPUSSZ61 stereomicroscope 661 (Tokyo, Japan) for seed traits, including color, size, and seed coat mucilage 662 and PAs. 663 The ruthenium red staining of seed coat mucilage was performed as 664

Measurement of seed anthocyanins and PAs 726
The anthocyanin content was measured as previously described (Li et al.,  Tables S3 and  766 S4. 767

Analysis of gene expression 768
The sampling of developing seeds used for gene expression was the same 769 as that described for the RNA-seq experiment. Other tissues were harvested  Table S1.          Three independent biological replicates were carried out. C, Reverse transcription 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 days after pollination from wild type (Col-0), the single mutants of snat1-1, comt-1, and comt-2, the double mutant snat1-1 comt-1, and   Different letters within various lines represent significant differences at P ≤ 0.05 (Tukey's highly significant difference test). PAs, proanthocyanidins. DW, dry weight.
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