Chloroplast overexpression of rice caffeic acid O‐methyltransferase increases melatonin production in chloroplasts via the 5‐methoxytryptamine pathway in transgenic rice plants

Recent analyses of the enzymatic features of various melatonin biosynthetic genes from bacteria, animals, and plants have led to the hypothesis that melatonin could be synthesized via the 5‐methoxytryptamine (5‐MT) pathway. 5‐MT is known to be synthesized in vitro from serotonin by the enzymatic action of O‐methyltransferases, including N‐acetylserotonin methyltransferase (ASMT) and caffeic acid O‐methyltransferase (COMT), leading to melatonin synthesis by the subsequent enzymatic reaction with serotonin N‐acetyltransferase (SNAT). Here, we show that 5‐MT was produced and served as a precursor for melatonin synthesis in plants. When rice seedlings were challenged with senescence treatment, 5‐MT levels and melatonin production were increased in transgenic rice seedlings overexpressing the rice COMT in chloroplasts, while no such increases were observed in wild‐type or transgenic seedlings overexpressing the rice COMT in the cytosol, suggesting a 5‐MT transport limitation from the cytosol to chloroplasts. In contrast, cadmium treatment led to results different from those in senescence. The enhanced melatonin production was not observed in the chloroplast COMT lines relative over the cytosol COMT lines although 5‐MT levels were equally induced in all genotypes upon cadmium treatment. The transgenic seedlings with enhanced melatonin in their chloroplasts exhibited improved seedling growth vs the wild type under continuous light conditions. This is the first report describing enhanced melatonin production in chloroplasts via the 5‐MT pathway with the ectopic overexpression of COMT in chloroplasts in plants.     

Cloning of Arabidopsis serotonin N‐acetyltransferase and its role with caffeic acid O‐methyltransferase in the biosynthesis of melatonin in vitro despite their different subcellular localizations

Serotonin N‐acetyltransferase (SNAT) is the penultimate enzyme in melatonin biosynthesis. We cloned SNAT from Arabidopsis thaliana (AtSNAT) and functionally characterized this enzyme for the first time from dicotyledonous plants. Similar to rice SNAT, AtSNAT was found to localize to chloroplasts with peak enzyme activity at 45°C (K_m, 309 μm ; V_max, 1400 pmol/min/mg protein). AtSNAT also catalyzed 5‐methoxytryptamine (5‐MT) into melatonin with high catalytic activity (K_m, 51 μm ; V_max, 5300 pmol/min/mg protein). In contrast, Arabidopsis caffeic acid O‐methyltransferase (AtCOMT) localized to the cytoplasm. Interestingly, AtCOMT can methylate serotonin into 5‐MT with low catalytic activity (K_m, 3.396 mm ; V_max, 528 pmol/min/mg protein). These data suggest that serotonin can be converted into either N‐acetylserotonin by SNAT or into 5‐MT by COMT, after which it is metabolized into melatonin by COMT or SNAT, respectively. To support this hypothesis, serotonin was incubated in the presence of both AtSNAT and AtCOMT enzymes. In addition to melatonin production, the production of major intermediates depended on incubation temperatures; N‐acetylserotonin was predominantly produced at high temperatures (45°C), while low temperatures (37°C) favored the production of 5‐MT. Our results provide biochemical evidence for the presence of a serotonin O‐methylation pathway in plant melatonin biosynthesis.     

Rice histone deacetylase 10 and Arabidopsis histone deacetylase 14 genes encode N‐acetylserotonin deacetylase,which catalyzes conversion of N‐acetylserotonin into serotonin,a reverse reaction for melatonin biosynthesis in plants

In plants, melatonin production is strictly regulated, unlike the production of its precursor, serotonin, which is highly inducible in response to stimuli, such as senescence and pathogen exposure. Exogenous serotonin treatment does not greatly induce the production of N‐acetylserotonin (NAS) and melatonin in plants, which suggests the possible existence of one or more regulatory genes in the pathway for the biosynthesis of melatonin from serotonin. In this report, we found that NAS was rapidly and abundantly converted into serotonin in rice seedlings, indicating the presence of an N‐acetylserotonin deacetylase (ASDAC). To clone the putative ASDAC gene, we screened 4 genes that were known as histone deacetylase (HDAC) genes, but encoded proteins targeted into chloroplasts or mitochondria rather than nuclei. Of 4 recombinant Escherichia coli strains expressing these genes, one E. coli strain expressing the rice HDAC10 gene was found to be capable of producing serotonin in response to treatment with NAS. The recombinant purified rice HDAC10 (OsHDAC10) protein exhibited ASDAC enzyme activity toward NAS, N‐acetyltyramine (NAT), N‐acetyltryptamine, and melatonin, with the highest ASDAC activity for NAT. In addition, its Arabidopsis ortholog, AtHDAC14, showed similar ASDAC activity to that of OsHDAC10. Both OsHDAC10 and AtHDAC14 were found to be expressed in chloroplasts. Phylogenetic analysis indicated that ASDAC homologs were present in archaea, but not in cyanobacteria, which differs from the distribution of serotonin N‐acetyltransferase (SNAT). This suggests that SNAT and ASDAC may have evolved differently from ancestral eukaryotic cells.     

Flavonoids inhibit both rice and sheep serotonin N‐acetyltransferases and reduce melatonin levels in plants

The plant melatonin biosynthetic pathway has been well characterized, but inhibitors of melatonin synthesis have not been well studied. Here, we found that flavonoids potently inhibited plant melatonin synthesis. For example, flavonoids including morin and myricetin significantly inhibited purified, recombinant sheep serotonin N‐acetyltransferase (SNAT). Flavonoids also dose‐dependently and potently inhibited purified rice SNAT1 and SNAT2. Thus, myricetin (100 μmol/L) reduced rice SNAT1 and SNAT2 activity 7‐ and 10‐fold, respectively, and also strongly inhibited the N‐acetylserotonin methyltransferase activity of purified, recombinant rice caffeic acid O‐methyltransferase. To explore the in vivo effects, rice leaves were treated with flavonoids and then cadmium. Flavonoid‐treated leaves had lower melatonin levels than the untreated control. To explore the direct roles of flavonoids in melatonin biosynthesis, we first functionally characterized a putative rice flavonol synthase (FLS) in vitro and generated flavonoid‐rich transgenic rice plants that overexpressed FLS. Such plants produced more flavonoids but less melatonin than the wild‐type, which suggests that flavonoids indeed inhibit plant melatonin biosynthesis.     

Chloroplastic and cytoplasmic overexpression of sheep serotonin N‐acetyltransferase in transgenic rice plants is associated with low melatonin production despite high enzyme activity

Serotonin N‐acetyltransferase (SNAT), the penultimate enzyme in melatonin biosynthesis, catalyzes the conversion of serotonin into N‐acetylserotonin. Plant SNAT is localized in chloroplasts. To test SNAT localization effects on melatonin synthesis, we generated transgenic rice plants overexpressing a sheep (Ovis aries) SNAT (OaSNAT) in their chloroplasts and compared melatonin biosynthesis with that of transgenic rice plants overexpressing OaSNAT in their cytoplasm. To localize the OaSNAT in chloroplasts, we used a chloroplast targeting sequence (CTS) from tobacco protoporphyrinogen IX oxidase (PPO), which expresses in chloroplasts. The purified recombinant CTS:OaSNAT fusion protein was enzymatically functional and localized in chloroplasts as confirmed by confocal microscopic analysis. The chloroplast‐targeted CTS:OaSNAT lines and cytoplasm‐expressed OaSNAT lines had similarly high SNAT enzyme activities. However, after cadmium and butafenacil treatments, melatonin production in rice leaves was severalfold lower in the CTS:OaSNAT lines than in the OaSNAT lines. Notably, enhanced SNAT enzyme activity was not directly proportional to the production of N‐acetylserotonin, melatonin, or 2‐hydroxymelatonin, suggesting that plant SNAT has a role in the homeostatic regulation of melatonin rather than in accelerating melatonin synthesis.     

Caffeic acid O‐methyltransferase is involved in the synthesis of melatonin by methylating N‐acetylserotonin in Arabidopsis

Although a plant N‐acetylserotonin methyltransferase (ASMT) was recently cloned from rice, homologous genes appear to be absent in dicotyledonous plants. To clone an ASMT de novo from a dicotyledonous plant, we expressed eight Arabidopsis thaliana O‐methyltransferase (OMT) cDNAs in Escherichia coli and screened for ASMT activity by measuring melatonin production after the application of 1 mm N‐acetylserotonin (NAS). Among the eight strains harboring the full‐length cDNAs, the OMT3 strain produced high levels of melatonin, suggesting that OMT3 encodes an active ASMT. OMT3 is already known as caffeic acid OMT (COMT), suggesting multiple functions for this enzyme. The purified recombinant A. thaliana COMT (AtCOMT) showed high ASMT activity, catalyzing the conversion of NAS to melatonin. The K_m and V_max values for ASMT activity were 233 μm and 1800 pmol/min/mg protein, while the K_m and V_max values for COMT activity were 103 μm and 564,000 pmol/min/mg protein, respectively. The catalytic efficiency (V_max/K_m) for ASMT activity was 709‐fold lower than for COMT. In vitro, ASMT activity was dramatically decreased by the addition of caffeic acid in a dose‐dependent manner, but the activity of COMT was not altered by NAS. Lastly, the Arabidopsis comt knockout mutant exhibited less production of melatonin than the wild type when Arabidopsis leaves were infiltrated with 1 mm NAS, suggestive of in vivo role of COMT in melatonin biosynthesis in plants.     

Melatonin‐deficient rice plants show a common semidwarf phenotype either dependent or independent of brassinosteroid biosynthesis

Melatonin‐deficient rice with a semidwarf erect‐leaf phenotype was created by suppressing serotonin N‐acetyltransferase 2 (SNAT2). We generated an RNAi transgenic rice that suppressed tryptophan decarboxylase (TDC), which encodes the first TDC enzyme committed step for melatonin biosynthesis in plants catalyzing the conversion of tryptophan into tryptamine, to determine whether other transgenic rice with downregulated melatonin biosynthetic genes exhibited the same erect‐leaf phenotype as the snat2 RNAi rice. The TDC RNAi rice produced significantly less melatonin than the wild type and exhibited a semidwarf phenotype, but no erect‐leaf phenotype was observed. In contrast, tryptamine 5‐hydroxylase (T5H) knockout Sekiguchi rice and caffeic acid O‐methyltransferase (COMT) RNAi rice seedlings were semidwarf phenotypes with erect leaves, as was the snat2 RNAi rice due to a melatonin deficiency. All RNAi rice plants showing erect‐leaf phenotypes had lower expression levels of the DWARF4 gene, which is a key enzyme for brassinosteroid (BR) biosynthesis, leading to lower BR levels than their respective wild types. Suppressing melatonin synthesis did not alter the contents of indole 3‐acetic acid (IAA), suggesting the irrelevance of melatonin deficiency to IAA biosynthesis. These data indicate that a semidwarf seedling is a common rice phenotype by the lack of melatonin synthesis with or without BR suppression in a melatonin biosynthetic gene‐specific manner.     

Melatonin is required for H2O2‐ and NO‐mediated defense signaling through MAPKKK3 and OXI1 in Arabidopsis thaliana

Melatonin influences plant innate immunity through the mitogen‐activated protein kinase (MAPK) pathway. However, the most upstream MAPK component in melatonin signaling and the dependence of generation of a reactive oxygen species (ROS) burst on melatonin synthesis and signaling remain unclear. In this study, treatment of several mekk (alias mapkkk)‐knockout Arabidopsis mutants with melatonin revealed that the MAPKKK3 and OXI1 (oxidative signal‐inducible1) kinases are responsible for triggering melatonin‐induced defense signaling pathways. In addition, melatonin induction upon infection with the avirulent pathogen Pseudomonas syringae DC3000 (avrRpt2) was independent of H₂O₂ and NO individually, but dependent on the combination of H₂O₂ and NO. Moreover, melatonin‐mediated induction of the expression of defense‐related genes, such as PR1 and ICS1, was not altered in the H₂O₂‐deficient rbohD/F‐knockout mutant cotreated with an NO scavenger, indicating that melatonin functions downstream of the ROS and NO burst. Collectively, the data indicate that melatonin‐mediated induction of an innate immune response requires multiple signaling molecules and activation of MAPKKK3 and OXI1, followed by triggering of downstream MAPK cascades, such as MAPK3 and MAPK6.     

Molecular cloning of rice serotonin N‐acetyltransferase,the penultimate gene in plant melatonin biosynthesis

Because of the absence of an arylalkylamine N‐acetyltransferase (AANAT) homolog in the plant genome, the proposal was made that a GCN5‐related N‐acetyltransferase superfamily gene (GNAT) could be substituted for AANAT. To clone rice serotonin N‐acetyltransferase (SNAT), we expressed 31 rice GNAT cDNAs in Escherichia coli and screened SNAT activity by measuring N‐acetyltryptamine after application with 1 mm tryptamine. GNAT5 was shown to produce high levels of N‐acetyltryptamine in E. coli, suggesting a possible rice SNAT. To confirm SNAT activity, the GNAT5 protein was purified through affinity purification from E. coli culture. The purified recombinant GNAT5 showed high SNAT enzyme activity catalyzing serotonin into N‐acetylserotonin. The values for K_m and V_max were 385 μm and 282 pmol/min/mg protein, respectively. An in vitro enzyme assay of purified SNAT showed N‐acetylserotonin formation to be proportional to enzyme concentration and time, with peak activity at pH 8.8. High substrate concentrations above 1 mm serotonin inhibited SNAT activity. Finally, the mRNA level of SNAT was higher in shoots than in roots, but it was expressed constitutively, unlike N‐acetylserotonin methyltransferase (ASMT), the terminal enzyme in melatonin synthesis. These results suggest that ASMT rather than SNAT is the rate‐limiting enzyme of melatonin biosynthesis in plants.     

Overexpression of rice serotonin N‐acetyltransferase 1 in transgenic rice plants confers resistance to cadmium and senescence and increases grain yield

While ectopic overexpression of serotonin N‐acetyltransferase (SNAT) in plants has been accomplished using animal SNAT genes, ectopic overexpression of plant SNAT genes in plants has not been investigated. Because the plant SNAT protein differs from that of animals in its subcellular localization and enzyme kinetics, its ectopic overexpression in plants would be expected to give outcomes distinct from those observed from overexpression of animal SNAT genes in transgenic plants. Consistent with our expectations, we found that transgenic rice plants overexpressing rice (Oryza sativa) SNAT1 (OsSNAT1) did not show enhanced seedling growth like that observed in ovine SNAT‐overexpressing transgenic rice plants, although both types of plants exhibited increased melatonin levels. OsSNAT1‐overexpressing rice plants did show significant resistance to cadmium and senescence stresses relative to wild‐type controls. In contrast to tomato, melatonin synthesis in rice seedlings was not induced by selenium and OsSNAT1 transgenic rice plants did not show tolerance to selenium. T₂ homozygous OsSNAT1 transgenic rice plants exhibited increased grain yield due to increased panicle number per plant under paddy field conditions. These benefits conferred by ectopic overexpression of OsSNAT1 had not been observed in transgenic rice plants overexpressing ovine SNAT, suggesting that plant SNAT functions differently from animal SNAT in plants.     

Melatonin induction and its role in high light stress tolerance in Arabidopsis thaliana

In plants, melatonin is a potent bioactive molecule involved in the response against various biotic and abiotic stresses. However, little is known of its defensive role against high light (HL ) stress. In this study, we found that melatonin was transiently induced in response to HL stress in Arabidopsis thaliana with a simultaneous increase in the expression of melatonin biosynthetic genes, including serotonin N ‐acetyltransferase1 (SNAT 1 ). Transient induction of melatonin was also observed in the flu mutant, a singlet oxygen (¹O₂)‐producing mutant, upon light exposure, suggestive of melatonin induction by chloroplastidic ¹O₂ against HL stress. An Arabidopsis snat1 mutant was devoid of melatonin induction upon HL stress, resulting in high susceptibility to HL stress. Exogenous melatonin treatment mitigated damage caused by HL stress in the snat1 mutant by reducing O₂^? production and increasing the expression of various ROS ‐responsive genes. In analogy, an Arabidopsis SNAT 1 ‐overexpressing line showed increased tolerance of HL stress concomitant with a reduction in malondialdehyde and ion leakage. A complementation line expressing an Arabidopsis SNAT 1 genomic fragment in the snat1 mutant completely restored HL stress susceptibility in the snat1 mutant to levels comparable to that of wild‐type Col‐0 plants. The results of the analysis of several Arabidopsis genetic lines reveal for the first time at the genetic level that melatonin is involved in conferring HL stress tolerance in plants.     

Glutathione‐dependent induction of local and systemic defense against oxidative stress by exogenous melatonin in cucumber (Cucumis sativus L.)

Melatonin is involved in defending against oxidative stress caused by various environmental stresses in plants. In this study, the roles of exogenous melatonin in regulating local and systemic defense against photooxidative stress in cucumber (Cucumis sativus) and the involvement of redox signaling were examined. Foliar or rhizospheric treatment with melatonin enhanced tolerance to photooxidative stress in both melatonin‐treated leaves and untreated systemic leaves. Increased melatonin levels are capable of increasing glutathione (reduced glutathione [GSH]) redox status. Application of H₂O₂ and GSH also induced tolerance to photooxidative stress, while inhibition of H₂O₂ accumulation and GSH synthesis compromised melatonin‐induced local and systemic tolerance to photooxidative stress. H₂O₂ treatment increased the GSH/oxidized glutathione (GSSG) ratio, while inhibition of H₂O₂ accumulation prevented a melatonin‐induced increase in the GSH/GSSG ratio. Additionally, inhibition of GSH synthesis blocked H₂O₂‐induced photooxidative stress tolerance, whereas scavenging or inhibition of H₂O₂ production attenuated but did not abolish GSH‐induced tolerance to photooxidative stress. These results strongly suggest that exogenous melatonin is capable of inducing both local and systemic defense against photooxidative stress and melatonin‐enhanced GSH/GSSG ratio in a H₂O₂‐dependent manner is critical in the induction of tolerance.     

Cloning and characterization of a serotonin N‐acetyltransferase from a gymnosperm,loblolly pine (Pinus taeda)

Serotonin N‐acetyltransferase (SNAT) is the penultimate enzyme in melatonin biosynthesis in both animals and plants. SNAT catalyzes serotonin into N‐acetylserotonin, an immediate precursor for melatonin biosynthesis by N‐acetylserotonin methyltransferase (ASMT). We cloned the SNAT gene from a gymnosperm loblolly pine (Pinus teada). The loblolly pine SNAT (PtSNAT) gene encodes 255 amino acids harboring a transit sequence with 67 amino acids and shows 67% amino acid identity with rice SNAT when comparing the mature polypeptide regions. Purified recombinant PtSNAT showed peak activity at 55°C with the K_m (428 μm ) and V_max (3.9 nmol/min/mg protein) values. As predicted, PtSNAT localized to chloroplasts. The SNAT mRNA was constitutively expressed in all tissues, including leaf, bud, flower, and pinecone, whereas the corresponding protein was detected only in leaf. In accordance with the exclusive SNAT protein expression in leaf, melatonin was detected only in leaf at 0.45 ng per gram fresh weight. Sequence and phylogenetic analysis indicated that the gymnosperm PtSNAT had high homology with SNATs from all plant phyla (even with cyanobacteria), and formed a clade separated from the angiosperm SNATs, suggestive of direct gene transfer from cyanobacteria via endosymbiosis.     

Molecular cloning and functional analysis of serotonin N‐acetyltransferase from the cyanobacterium Synechocystis sp. PCC 6803

Serotonin N‐acetyltransferase (SNAT) catalyzes conversion of serotonin into N‐acetylserotonin, which is a direct precursor for melatonin biosynthesis in all organisms. Molecular cloning of plant SNAT from rice led to a screening for SNAT homolog genes in other species. We identified a cyanobacterium SNAT‐like gene (cSNAT) that showed 56% amino acid homology with the rice SNAT. To confirm whether cSNAT encoded SNAT enzyme activity, we expressed cSNAT DNA in Escherichia coli and purified the cSNAT protein as a C‐terminal His‐tagged form. The purified cSNAT protein exhibited SNAT enzyme activities, transferring the acetyl group into either serotonin or tryptamine substrates. The optimum temperature was 55°C, but it was still highly active at 70°C, suggesting that cSNAT is a thermotolerant enzyme. The K_m and V_max were 823 μm and 1.6 nmol/min/mg protein, respectively. The cSNAT gene is highly conserved in all cyanobacterial taxa and seems to be an origin of SNAT in higher plants. The thermotolerance of cSNAT suggests that melatonin plays a role in the response to high‐temperature stress. Further analysis of this role of melatonin in higher plants is needed.     

Enhanced production of melatonin by ectopic overexpression of human serotonin N‐acetyltransferase plays a role in cold resistance in transgenic rice seedlings

Abstract: Serotonin N‐acetyltransferase (SNA), a rate‐limiting enzyme in melatonin biosynthesis in vertebrates, is responsible for the production of N‐acetylserotonin; this molecule is then converted to melatonin by hydroxyindole‐O‐methyltransferase. We generated transgenic rice plants via expression of the human SNA gene under the constitutive ubiquitin promoter using Agrobacterium‐mediated gene transformation. We investigated the role of SNA in the biosynthesis of melatonin and the physiological role of melatonin in rice plants. The integration and expression of the transgene were confirmed in T₁ transgenic rice seedlings by Southern, Northern, and RT‐PCR analyses. High SNA‐specific enzyme activities were observed in the transgenic rice plants, whereas the wild type revealed a trace level of SNA enzyme activity. The functional expression of SNA protein was closely associated with the elevated synthesis of N‐acetylserotonin and melatonin in the transgenic rice plants. Experiments using both exogenous treatment of serotonin and senescent detached leaves, which contain a pool of serotonin, significantly enhanced melatonin biosynthesis, indicating that endogenous serotonin levels play a bottleneck role in the pathway of melatonin biosynthesis. Finally, the transgenic rice seedlings with high levels of melatonin showed elevated chlorophyll synthesis during cold stress, suggesting a role for melatonin in cold‐stress resistance.     

Coordinated regulation of melatonin synthesis and degradation genes in rice leaves in response to cadmium treatment

We investigated the expression patterns of genes involved in melatonin synthesis and degradation in rice leaves upon cadmium (Cd) treatment and the subcellular localization sites of melatonin 2‐hydroxylase (M2H) proteins. The Cd‐induced synthesis of melatonin coincided with the increased expression of melatonin biosynthetic genes including tryptophan decarboxylase (TDC), tryptamine 5‐hydroxylase (T5H), and N‐acetylserotonin methyltransferase (ASMT). However, the expression of serotonin N‐acetyltransferase (SNAT), the penultimate gene in melatonin biosynthesis, was downregulated, suggesting that melatonin synthesis was counter‐regulated by SNAT. Notably, the induction of melatonin biosynthetic gene expression was coupled with the induction of four M2H genes involved in melatonin degradation, which suggests that genes for melatonin synthesis and degradation are coordinately regulated. The induced M2H gene expression was correlated with enhanced M2H enzyme activity. Three of the M2H proteins were localized to the cytoplasm and one M2H protein was localized to chloroplasts, indicating that melatonin degradation occurs both in the cytoplasm and in chloroplasts. The biological activity of 2‐hydroxymelatonin in the induction of the plant defense gene expression was 50% less than that of melatonin, which indicates that 2‐hydroxymelatonin may be a metabolite of melatonin. Overall, our data demonstrate that melatonin synthesis occurs in parallel with melatonin degradation in both chloroplasts and cytoplasm, and the resulting melatonin metabolite 2‐hydroxymelatonin also acts as a signaling molecule for defense gene induction.     

A rice chloroplast transit peptide sequence does not alter the cytoplasmic localization of sheep serotonin N‐acetyltransferase expressed in transgenic rice plants

Ectopic overexpression of melatonin biosynthetic genes of animal origin has been used to generate melatonin‐rich transgenic plants to examine the functional roles of melatonin in plants. However, the subcellular localization of these proteins expressed in the transgenic plants remains unknown. We studied the localization of sheep (Ovis aries) serotonin N‐acetyltransferase (OaSNAT) and a translational fusion of a rice SNAT transit peptide to OaSNAT (TS:OaSNAT) in plants. Laser confocal microscopy analysis revealed that both OaSNAT and TS:OaSNAT proteins were localized to the cytoplasm even with the addition of the transit sequence to OaSNAT. Transgenic rice plants overexpressing the TS:OaSNAT fusion transgene exhibited high SNAT enzyme activity relative to untransformed wild‐type plants, but lower activity than transgenic rice plants expressing the wild‐type OaSNAT gene. Melatonin levels in both types of transgenic rice plant corresponded well with SNAT enzyme activity levels. The TS:OaSNAT transgenic lines exhibited increased seminal root growth relative to wild‐type plants, but less than in the OaSNAT transgenic lines, confirming that melatonin promotes root growth. Seed‐specific OaSNAT expression under the control of a rice prolamin promoter did not confer high levels of melatonin production in transgenic rice seeds compared with seeds from transgenic plants expressing OaSNAT under the control of the constitutive maize ubiquitin promoter.