Functional Characterization of Arylalkylamine N-Acetyltransferase, a Pivotal Gene in Antioxidant Melatonin Biosynthesis from Chlamydomonas reinhardtii

Arylalkylamine N-acetyltransferase (AANAT) is a pivotal enzyme in melatonin biosynthesis that catalyzes the conversion of serotonin to N-acetylserotonin. Homologs of animal AANAT genes are present in animals, but not in plants. An AANAT homolog was found in Chlamydomonas reinhardtii, but not other green algae. The characteristics of C. reinhardtii AANAT (CrAANAT) are unclear. Here, full-length CrAANAT was chemically synthesized and expressed in Escherichia coli. Recombinant CrAANAT exhibited AANAT activity with a Km of 247 μM and Vmax of 325 pmol/min/mg protein with serotonin as the substrate. CrAANAT was localized to the cytoplasm in tobacco leaf cells. Transgenic rice plants overexpressing CrAANAT (CrAANAT-OE) exhibited increased melatonin production. CrAANAT-OE plants showed a longer seed length and larger second leaf angle than wild-type plants, indicative of the involvement of brassinosteroids (BRs). As expected, BR biosynthesis- and signaling-related genes such as D2, DWARF4, DWARF11, and BZR1 were upregulated in CrAANAT-OE plants. Therefore, an increased endogenous melatonin level by ectopic overexpression of CrAANAT seems to be closely associated with BR biosynthesis, thereby influencing seed size.


Introduction
Arylalkylamine N-acetyltransferase (AANAT) is the penultimate enzyme for melatonin biosynthesis in animals and plants. It catalyzes the conversion of serotonin to N-acetylserotonin, which is the substrate for melatonin synthesis by N-acetylserotonin O-methyltransferase (ASMT) [1,2]. AANAT is also named serotonin N-acetyltransferase (SNAT); therefore, to differentiate them from animal AANAT genes, plant AANAT genes are frequently termed SNAT genes. Both animal AANAT and plant SNAT proteins belong to the GCN5-related N-acetyltransferase superfamily, which transfer an acetyl group from acetyl-coenzyme A (CoA) [3]. However, there is no significant amino sequence homology between animal AANATs and plant SNATs except for a few amino acids in the acetyl-CoA-binding domain [4]. Interestingly, an animal homolog of arylalkylamine N-acetyltransferase (AANAT) is present in the genome of C. reinhardtii, but not in other green algae or higher plants [3,5,6].
CrAANAT was first characterized by Okazaki et al. [22]. CrAANAT transfers an acetyl group to serotonin. Transgenic tomato plants overexpressing CrAANAT had an increased melatonin level. However, the K m and V max values of recombinant CrAANAT and the phenotypes of CrAANAT-overexpressing transgenic plants are unknown. The aim of this work was to determine the enzyme kinetics of CrAANAT and its functional role in melatonin biosynthesis through heterologous expression in rice genome. We purified recombinant CrAANAT and determined the K m and V max values of its AANAT activity. Ectopic overexpression of CrAANAT in the rice genome increased the seed length and upregulated brassinosteroid (BR) (rather than cytokinin)-related gene expression.

Affinity Purification of Various Recombinant C. reinhardtii AANAT Proteins from Escherichia coli Expression
Four different types of Escherichia coli vectors were employed to express the full-length synthetic Chlamydomonas reinhardtii AANAT (CrAANAT) DNA. Two vectors were pET300 (Invitrogen, Carlsbad, CA, USA) and pET28b (Novagen, San Diego, CA, USA) which are designed to express the CrAANAT in either N-terminal-or C-terminal-hexahistidine tagged form. The other two vectors were pET32b (Novagen) and pET60 (Novagen) which are designed to express the CrAANAT in N-terminal fusion proteins of either thioredoxin (Trx) or glutathione-s-transferase (GST). As for pET300, CrAANAT-attB1 forward primer (5 -AAA AAG CAG GCT CCA TGG CTG AGG AGT CGC-3 ) and CrAANAT-attB2 reverse primer (5 -AGA AAG CTG GGT CTA GGC CTC AGC AGC CTC-3 ) were used for PCR amplification with the synthetic CrAANAT gene followed by second PCR using adaptor primers with attB recombination sequences (attB1 adaptor forward primer, 5 -GGG GAC AAG TTT GTA CAA AAA AGC AGG CT-3 ; attB2 adaptor reverse primer, 5 -GGG GAC CAC TTT GTA CAA GAA AGC TGG GT-3 ). The resulting PCR product was gel purified and cloned into the pDONR221 Gateway ® vector (Invitrogen) via BP (between the attB and the attP sites) recombination. The pDONR221:CrAANAT gene entry vector was then recombined with the pET300 Gateway destination vector via LR (between the attL and the attR sites) recombination to form the pET300-CrAANAT vector. A pET28b-CrAANAT was constructed by PCR with NcoI forward primer (5 -ACC ATG GCT GAG GAG TCG CTC-3 ) and XhoI reverse primer (5 -CTC GAG GGC CTC AGC AGC CTC TGC-3 ). To generate pET60-CrAANAT, the CrAANAT-attB1 forward primer and 6×His attB2 reverse primer (5 -AGA AAG CTG GGT TCA GTG GTG GTG GTG GTG-3 ) were used to amplify the CrAANAT with the pET28b-CrAANAT plasmid as a template. The resulting PCR product was further amplified with the attB1 adaptor forward and attB2 adaptor reverse primers followed by BP and LR recombination reactions as described above. A pET32b-CrAANAT was constructed by ligating the NcoI and XhoI insert prepared during the pET28b-CrAANAT vector construction. All plasmids were transformed into E. coli strain BL21(DE3) (Novagen).

Purification of Recombinant CrAANAT Proteins
Each 10 mL of E. coli overnight culture of E. coli containing pET300-CrAANAT, pET28b-CrAANAT, pET60-CrAANAT, and pET32b-CrAANAT plasmid vectors was inoculated into 100 mL of Terrific Broth (20 g/L Bacto-tryptone, 24 g/L Bacto-yeast extract, 4 mL/L glycerol, and phosphate buffer [0.017 M monopotassium phosphate and 0.072 M dipotassium phosphate]) containing with 50 mg/L ampicillin or 50 mg/L kanamycin (pET28b-CrAANAT) and incubated at 37 • C until the optical density at 600 nm reached 1.0 about 3 to 4 h. The culture was added with 1 mM isopropyl-β-D-thiogalactopyranoside (Sigma, St. Louis, MO, USA) and grown at 28 • C with shaking at 180 rpm for 5 h. The protein was purified via affinity nickel ion chromatography according to the column manufacturer's instructions (Qiagen, Tokyo, Japan).

Measurement of Serotonin N-Acetyltransferase (SNAT) Enzyme Activity
Two types of purified recombinant CrAANAT proteins were incubated in 100 µL of 100 mM potassium phosphate (pH 8.8 or various pH values) in the presence of 0.5 mM serotonin and 0.5 mM acetyl-coenzyme A. SNAT enzyme assays were conducted at 45 • C for 30 min (or various temperatures) and stopped by adding 25 µL of methanol. Then, 10 µL aliquots of the reaction mixture were subjected to high-performance liquid chromatography (HPLC) coupled to a fluorescence detector system to detect N-acetylserotonin as described previously [24]. Non-enzymatic reaction products that were generated without the CrAANAT enzymes were deducted. To acquire substrate affinity (K m ) and maximum reaction rate (V max ), various substrates (50 to 2000 µM serotonin) and enzyme concentrations (0.2 to 1 µg) were employed. The K m and V max values were calculated using Lineweaver-Burk plots. Protein concentration was determined using Bradford assays (Bio-Rad, Hercules, CA, USA). The analyses were performed in triplicate.

Subcellular Localization of CrAANAT
The pER-mCherry vector which was kindly donated by Dr. H. Kang (Texas State University, San Marcos, TX, USA) was used for assessing the localization of CrAANAT protein in tobacco leaves. Full-length CrAANAT cDNA was amplified by PCR with two AscI containing primers (CrAANAT AscI forward primer 5 -GGC GCG CCA TGG CTG AGG AGT CGC TCG-3 ; CrAANAT AscI reverse primer 5 -GGC GCG CCG GGC CTC AGC AGC CTC TGC-3 ). The resulting PCR product was first cloned into the T&A cloning vector (T&A:CrAANAT; RBC Bioscience, New Taipei City, Taiwan) from which the AscI insert of CrAANAT was produced and cloned into the binary pER8-mCherry vector at the AscI restriction sites downstream of the estrogen-inducible XVE promoter to generate CrAANAT-mCherry fusion proteins. The plasmid was transformed into Agrobacterium tumefaciens strain GV2260 using the freeze-thaw method. As for a transient expression analysis of CrAANAT-mCherry, the leaves of two-week-old tobacco (Nicotiana benthamiana) plant, a native Australian species, were infiltrated with A. tumefaciens strain GV2260 carrying pER8:CrAANAT-mCherry plasmid. The transformed tobacco leaves were then examined using confocal microscopy to determine the subcellular localization of the CrAANAT-mCherry fusion proteins. Further treatment with β-estradiol (Sigma Aldrich, St. Louis, MO, USA) and confocal microscopy analysis were described previously [24].

Vector Construction and Production of CrAANAT-Overexpressing Transgenic Rice Plants
The pDONR221:CrAANAT gene entry vector harboring the synthetic CrAANAT gene was then recombined with the pIPKb002 destination vector [25] via LR recombination to yield pIPKb002-CrAANAT, which was transformed into Agrobacterium tumefaciens strain LBA4404. We used Agrobacterium-mediated rice transformation with the coculture with rice scutelum-derived calli to generate transgenic rice (Oryza sativa cv. Dongjin, a Korean japonica cultivar) plants as described previously [26].

Plant Growth Conditions
Rice (Oryza sativa cv. Dongjin, a Korean japonica cultivar) seeds of both wild type and CrAANAT overexpression (CrAANAT-OE) were sterilized with 2% sodium hyphochlorite and rinsed with sterile distilled water. Sterilized seeds were grown on half-strength Murashige and Skoog (MS) medium under cool daylight fluorescent lamps (60 µmol m −2 s −1 ) (Philips, Amsterdam, Netherlands) in 14-h light/10-h dark photoperiod at 28 • C/24 • C (day/night) for 7 days. Germinated seeds were grown in a paddy field at the Chonnam National University (35 • 09 N and 126 • 54 W; 53 m a.s.l), Gwangju, Korea in 2021. The distance between the rice plants within a row was 30 cm, and the distance between the rows was 30 cm. Grain length, grain width and 1000-grain weight were measured after harvesting followed by drying for 1 month at room temperature of about 26 • C.

Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) Analysis
Total RNA from rice plants was isolated using a NucleoSpin RNA Plant Kit (Macherey-Nagel, Düren, Germany). First-strand cDNA was synthesized from 2 µg of total RNA using EcoDry TM Premix (Takara Bio USA, Inc., Mountain View, CA, USA). qRT-PCR was performed in a Mic qPCR Cycler system (Biomolecular Systems, Queensland, VIC, Australia) with specific primers and the TB Green ® Premix Ex Taq TM (Takara Bio Inc., Kusatsu, Shiga, Japan). The expression of genes was analyzed using Mic's RQ software v2.2 (Biomolecular Systems) and normalized to actin 1 (ACT1). Reverse transcription (RT)-PCR and quantitative real-time (qRT)-PCR were performed with the primer set (Table S1).

Quantification of Melatonin
Frozen samples (0.1 g) were ground in liquid nitrogen with the use of the TissueLyser II (Qiagen, Tokyo, Japan) and extracted with 1 mL of chloroform. The chloroform extracts were centrifuged for 10 min at 12,000× g, and the supernatants (200 µL) were completely evaporated and dissolved in 0.1 mL of 40% methanol, and 20-µL aliquots were subjected to HPLC using a fluorescence detector system (Waters, Milford, MA, USA) as described previously [27]. In brief, melatonin was detected at 280 nm (excitation) and 348 nm (emission) on a Sunfire C18 column (Waters 4.6 × 150 mm) in the following gradient elution condition: from 42% to 50% methanol in 0.1% formic acid for 27 min, followed by isocratic elution with 50% methanol in 0.1% formic acid for 18 min at a flow rate of 0.15 mL/min. All measurements were performed in triplicate.

Statistical Analyses
The data were analyzed using analysis of variance (ANOVA) using IBM SPSS Statistics 23 software (IBM Corp., Armonk, NY, USA). Means with asterisks indicate significantly different values at p < 0.05, according to a Fisher's least significant difference (LSD) test. All data are presented as mean ± standard deviations.

Codon-Optimized Synthesis of CrAANAT and Its Expression in Escherichia coli
The full-length CrAANAT nucleotide sequence (encoding 191 amino acids) was chemically synthesized based on the codon usage of rice SNAT2 (AK068156) exhibiting a 70% G+C content. Among the 192 codons of CrAANAT, 55 were modified in the synthetic CrAANAT gene, increasing the G+C content from 64% to 67% (Figures 1 and 2).  Synthetic CrAANAT was expressed in E. coli as a fusion protein with an N-or Cterminal hexa-histidine (His6) tag, followed by Ni 2+ affinity purification ( Figure 3). Intact His6-CrAANAT and CrAANAT-His6 were insoluble and so could not be purified using a Ni 2+ affinity column. However, CrAANAT fusions with thioredoxin (Trx) or glutathione-s-transferase (GST) were soluble and subjected to Ni 2+ affinity purification ( Figure  3A,B). Recombinant Trx-CrAANAT-His6 exhibited serotonin N-acetyltransferase (SNAT) activity of 10.1 pkat/mg protein at 45 °C and pH 8.8, compared to 18.7 pkat/mg protein for GST-CrAANAT-His6 ( Figure 3C). Therefore, CrAANAT transfers an acetyl group from acetyl-CoA to serotonin to produce N-acetylserotonin, the final substrate in melatonin biosynthesis [4].  Nucleotide sequence alignments of native (black writing; AB474787) and synthetic CrAANAT (red writing) genes. Identity is denoted by red dashes. The nucleotide sequence of synthetic CrAANAT was codon-optimized with reference to rice SNAT2 (AK068156).

Kinetics of Recombinant CrAANAT
SNAT activity peaked at pH 8.8 and was similar at pH 7.8 ( Figure 4). This high pH optimum is consistent with that of other plant SNAT proteins [8,13,23,28,29] but unlike animal AANAT proteins (pH 6.7) [30,31]. SNAT activity was fourfold lower at pH 6.5 than at pH 8.8. SNAT activity was highest at 45 °C followed by 37 °C. The high level of SNAT activity at 37 °C is similar to other animal AANAT proteins but not plant SNAT proteins [8,30,31]. The Km and Vmax values were 247 μM and 5.4 pkat/mg protein (325 pmol/min/mg protein), respectively. The Km value of CrAANAT is similar to those of rice [24], Arabidopsis [29], and tobacco [8], but lower than that of red algae [32] and cyanobacteria [33]. The Km value of CrAANAT was threefold higher than that of sheep AANAT [7]. In contrast, the Vmax value of CrAANAT was lower than that of plant SNATs and sheep AANATs. Therefore, the kinetics of CrAANAT has similarities to those of plant SNATs and animal AANATs. To be more precise, the CrAANAT is close to plant SNAT at the level of Km value, but to animal AANAT at the level of Vmax value.

Kinetics of Recombinant CrAANAT
SNAT activity peaked at pH 8.8 and was similar at pH 7.8 ( Figure 4). This high pH optimum is consistent with that of other plant SNAT proteins [8,13,23,28,29] but unlike animal AANAT proteins (pH 6.7) [30,31]. SNAT activity was fourfold lower at pH 6.5 than at pH 8.8. SNAT activity was highest at 45 • C followed by 37 • C. The high level of SNAT activity at 37 • C is similar to other animal AANAT proteins but not plant SNAT proteins [8,30,31]. The K m and V max values were 247 µM and 5.4 pkat/mg protein (325 pmol/min/mg protein), respectively. The K m value of CrAANAT is similar to those of rice [24], Arabidopsis [29], and tobacco [8], but lower than that of red algae [32] and cyanobacteria [33]. The K m value of CrAANAT was threefold higher than that of sheep AANAT [7]. In contrast, the V max value of CrAANAT was lower than that of plant SNATs and sheep AANATs. Therefore, the kinetics of CrAANAT has similarities to those of plant SNATs and animal AANATs. To be more precise, the CrAANAT is close to plant SNAT at the level of K m value, but to animal AANAT at the level of V max value.

Subcellular Localization of CrAANAT
Because in silico TargetP analysis of CrAANAT showed no transit or signal sequence [34], we hypothesized that CrAANAT is cytoplasmic. We subcloned CrAANAT into a binary vector to express the CrAANAT-mCherry fusion protein under the control of the estrogen-inducible XVE promoter. Agrobacterium cells harboring the binary vector were infiltrated into tobacco leaves followed by transgene induction by β-estradiol. Confocal microscopy showed that CrAANAT-mCherry exhibited strong mCherry fluorescence, which co-localized with the green fluorescence of cytoplasmic GFP ( Figure 5). Therefore, CrAANAT localizes to the cytoplasm, as does sheep AANAT [35]. CrAANAT in the cytoplasm is unlike the chloroplastic localizations of other plant SNAT proteins [8,13,24]. protein), respectively. The Km value of CrAANAT is similar to those of rice [24], Arab sis [29], and tobacco [8], but lower than that of red algae [32] and cyanobacteria [33 Km value of CrAANAT was threefold higher than that of sheep AANAT [7]. In con the Vmax value of CrAANAT was lower than that of plant SNATs and sheep AAN Therefore, the kinetics of CrAANAT has similarities to those of plant SNATs and a AANATs. To be more precise, the CrAANAT is close to plant SNAT at the level value, but to animal AANAT at the level of Vmax value.

Transgenic Rice Plants Overexpressing CrAANAT (CrAANAT-OE)
To determine whether CrAANAT is functionally coupled to melatonin biosynthesis in vivo, we generated CrAANAT-OE under the control of the maize ubiquitin promoter ( Figure 6A).
Homozygous T2 CrAANAT-OE seeds were of greater length, but lesser width, WT seeds (Figure 7). The 1000-seed weight was similar in the CrAANAT-OE lines wild type. To determine whether the increase in seed length is associated with BRs measured the second leaf angle, a phenotypic marker of BR levels. The second-leaf a was larger in the CrAANAT-OE seedlings than in WT seedlings ( Figure 8A), indica increased BR levels. Cytokinins also regulate seed size in plants [37]. To identify the mones responsible for the increased seed length in the CrAANAT-OE lines, we evalu the expression levels of BR-and cytokinin-related genes. The expression levels of Homozygous T 2 CrAANAT-OE seeds were of greater length, but lesser width, than WT seeds (Figure 7). The 1000-seed weight was similar in the CrAANAT-OE lines and wild type. To determine whether the increase in seed length is associated with BRs, we measured the second leaf angle, a phenotypic marker of BR levels. The second-leaf angle was larger in the CrAANAT-OE seedlings than in WT seedlings ( Figure 8A), indicating increased BR levels. Cytokinins also regulate seed size in plants [37]. To identify the hormones responsible for the increased seed length in the CrAANAT-OE lines, we evaluated the expression levels of BR-and cytokinin-related genes. The expression levels of BR-related genes were significantly increased in the CrAANAT-OE lines (the BR biosynthesis-related genes DWARF [D]2, D4, and D11 and BRASSINOZOLE-RESISTANT 1 (BZR1), encoding a transcription factor that regulates BR-responsive gene expression; Figure 8B) [38,39]. By contrast, cytokinin degradation genes (CKX2, CKX4, and CKX10) and cytokinin biosynthesis genes (LOGL1, LOGL3, and LOGL10) were upregulated. Therefore, the increased lamina angle and seed length in the CrAANAT-OE line compared to wild type are caused by BRs rather than cytokinins. related genes were significantly increased in the CrAANAT-OE lines (the BR biosynthesisrelated genes DWARF [D]2, D4, and D11 and BRASSINOZOLE-RESISTANT 1 (BZR1), encoding a transcription factor that regulates BR-responsive gene expression; Figure 8B) [38,39]. By contrast, cytokinin degradation genes (CKX2, CKX4, and CKX10) and cytokinin biosynthesis genes (LOGL1, LOGL3, and LOGL10) were upregulated. Therefore, the increased lamina angle and seed length in the CrAANAT-OE line compared to wild type are caused by BRs rather than cytokinins.

Discussion
The final two genes in melatonin biosynthesis are AANAT (or SNAT) and ASMT [4]. AANAT was first cloned and characterized in sheep and rats [40,41]. AANAT homologs have been cloned from fish [42], humans [31], yeast [43], and mosquitos [44]. AANAT plays a rate-limiting role in melatonin biosynthesis by acetylating serotonin and 5-MT, thus synthesizing N-acetylserotonin and melatonin, respectively, in animals and plants [8,36]. An animal AANAT homolog was reported in the green alga C. reinhardtii, but not in other green algae or higher plants [5]. Okazaki et al. [22] expressed C. reinhardtii AANAT in E. coli and reported that the purified GST-CrAANAT fusion protein transferred an acetyl-CoA group to serotonin but did not determine its K m and V max values. Furthermore, its overexpression in tomato increased melatonin synthesis, but this did not affect the plant phenotype. The K m and V max values of purified recombinant CrAANAT for serotonin indicated that CrAANAT encodes a SNAT enzyme. Similar to other plant SNAT proteins, CrAANAT prefers a high pH, but an optimum temperature of around 37 • C, like animal AANATs [45]. This optimum temperature of CrAANAT is in contrast to that of other plant SNAT enzymes (45 • C to 55 • C) [8], indicating that CrAANAT possesses characteristics of animal AANAT and plant SNAT proteins.
Melatonin has diverse functions in plant growth, development, and biotic and abiotic stress responses [9,10,[46][47][48][49] by modulating the cellular redox balance [50][51][52] and protein quality control [53]. Additionally, melatonin functions in concert with other plant hormones during growth and under stressful conditions [54,55]. Melatonin directly influences hormone levels in Arabidopsis thaliana, in which exogenous melatonin promotes primary root growth via the indole-3-acetic acid signaling pathway [56]. Indirect effects of melatonin on plant hormones have been reported in plants with up-or down-regulated melatonin synthesis [57,58]. Melatonin did not directly enhance gibberellic acid (GA) synthesis [57] and BR effects such as leaf angle increases [58]. However, melatonin suppression in an A. thaliana knockout mutant (snat1) and SNAT2 RNAi rice plants resulted in decreased levels of GA and BR, respectively. This was caused by decreased starch synthesis, which is promoted by melatonin [57,59,60]. An increased endogenous melatonin level in transgenic rice overexpressing caffeic acid O-methyltransferase (COMT) markedly increased the seed size and rice yield as a result of elevated cytokinin levels [61]. Transgenic rice plants overexpressing SNAT genes from Archaea and rice showed an increase in seed size [62,63]; however, whether this is caused by cytokinins or BRs is unclear. Here, the increased rice seed length caused by an increased endogenous melatonin level was linked to increased BR levels due to upregulation of BR biosynthetic genes (Figure 9), not increased cytokinin levels as in COMT-overexpressing rice [61]. Moreover, the increase in BR levels caused by endogenous melatonin overproduction induces melatonin biosynthesis by activating the expression of melatonin biosynthetic genes such as tryptophan decarboxylase 1 (TDC1), TDC3, and tryptamine 5-hydroxylase (T5H) [64] (Figure 9). In sum, the effect of melatonin on seed size and yield [17,61] and stress tolerance [65,66] suggests that the generation of melatonin-rich rice plants by exogenous application [64] or transgenic approaches [4] would increase yields and resistance to many biotic and abiotic stresses and enable the production of melatonin-rich foods with health benefits [67]. BR levels caused by endogenous melatonin overproduction induces melatonin biosynthesis by activating the expression of melatonin biosynthetic genes such as tryptophan decarboxylase 1 (TDC1), TDC3, and tryptamine 5-hydroxylase (T5H) [64] (Figure 9). In sum, the effect of melatonin on seed size and yield [17,61] and stress tolerance [65,66] suggests that the generation of melatonin-rich rice plants by exogenous application [64] or transgenic approaches [4] would increase yields and resistance to many biotic and abiotic stresses and enable the production of melatonin-rich foods with health benefits [67]. Figure 9. Proposed model for the melatonin-mediated increase in seed length. The increase in melatonin synthesis caused by ectopic overexpression of CrAANAT leads to an increased seed length and leaf angle, key phenotypic markers of increased BR levels in rice. A number of BR biosynthesisand signaling-related genes were upregulated in the CrAANAT-overexpressing rice plants. These data suggest that melatonin is positively associated with BR levels in rice plants. The melatonininduced BR increase is an indirect effect on photosynthesis-melatonin is positively coupled to starch synthesis and photosynthesis, which affect BR biosynthesis [57]. BR triggers melatonin biosynthesis by inducing the expression of melatonin biosynthesis genes such as tryptophan decarboxylase 1 (TDC1), TDC3, and tryptamine 5-hydroxylase (T5H) [64]. Solid arrows, confirmed functions; dashed arrows, steps not yet demonstrated in rice. ↑, upregulation.

Conclusions
CrAANAT had homology to animal AANAT proteins but not to plant SNAT proteins. Its optimum temperature was 37 °C, similar to animal AANAT proteins, but its optimum pH was pH 8.8, similar to plant SNAT proteins, demonstrating that CrAANAT has characteristics of both animal AANAT and plant SNAT proteins. Ectopic overexpression of CrAANAT in the rice genome led to an increase in melatonin content, leaf angle, and seed length, indicative of enhanced BR biosynthesis. An increased BR level in CrAANAT-OE rice plants was indirectly verified by the upregulation of BR biosynthetic genes such as D2, D4, and D11. This is the first report of an increase in BR biosynthesis by the ectopic overexpression of AANAT or SNAT in transgenic plants. Many RNAi transgenic rice plants with downregulated melatonin synthesis show decreased BR levels and leaf angle, suggesting a close relationship between melatonin and BR levels in rice plants [39,61]. The CrAANAT can be used as a source gene for a simultaneous increase of melatonin and BR Figure 9. Proposed model for the melatonin-mediated increase in seed length. The increase in melatonin synthesis caused by ectopic overexpression of CrAANAT leads to an increased seed length and leaf angle, key phenotypic markers of increased BR levels in rice. A number of BR biosynthesisand signaling-related genes were upregulated in the CrAANAT-overexpressing rice plants. These data suggest that melatonin is positively associated with BR levels in rice plants. The melatonininduced BR increase is an indirect effect on photosynthesis-melatonin is positively coupled to starch synthesis and photosynthesis, which affect BR biosynthesis [57]. BR triggers melatonin biosynthesis by inducing the expression of melatonin biosynthesis genes such as tryptophan decarboxylase 1 (TDC1), TDC3, and tryptamine 5-hydroxylase (T5H) [64]. Solid arrows, confirmed functions; dashed arrows, steps not yet demonstrated in rice. ↑, upregulation.

Conclusions
CrAANAT had homology to animal AANAT proteins but not to plant SNAT proteins. Its optimum temperature was 37 • C, similar to animal AANAT proteins, but its optimum pH was pH 8.8, similar to plant SNAT proteins, demonstrating that CrAANAT has characteristics of both animal AANAT and plant SNAT proteins. Ectopic overexpression of CrAANAT in the rice genome led to an increase in melatonin content, leaf angle, and seed length, indicative of enhanced BR biosynthesis. An increased BR level in CrAANAT-OE rice plants was indirectly verified by the upregulation of BR biosynthetic genes such as D2, D4, and D11. This is the first report of an increase in BR biosynthesis by the ectopic overexpression of AANAT or SNAT in transgenic plants. Many RNAi transgenic rice plants with downregulated melatonin synthesis show decreased BR levels and leaf angle, suggesting a close relationship between melatonin and BR levels in rice plants [39,61]. The CrAANAT can be used as a source gene for a simultaneous increase of melatonin and BR which will lead to improved plant growth and stress tolerance conferred by either melatonin or BR alone or a combination of both [38,39,50].