J. Pineal Res. 2014; 56:275–282

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Molecular, Biological, Physiological and Clinical Aspects of Melatonin

Doi:10.1111/jpi.12120

Journal of Pineal Research

Elevated production of melatonin in transgenic rice seeds expressing rice tryptophan decarboxylase Abstract: A major goal of plant biotechnology is to improve the nutritional qualities of crop plants through metabolic engineering. Melatonin is a wellknown bioactive molecule with an array of health-promoting properties, including potent antioxidant capability. To generate melatonin-rich rice plants, we first independently overexpressed three tryptophan decarboxylase isogenes in the rice genome. Melatonin levels were altered in the transgenic lines through overexpression of TDC1, TDC2, and TDC3; TDC3 transgenic seed (TDC3-1) had melatonin concentrations 31-fold higher than those of wild-type seeds. In TDC3 transgenic seedlings, however, only a doubling of melatonin content occurred over wild-type levels. Thus, a seed-specific accumulation of melatonin appears to occur in TDC3 transgenic lines. In addition to increased melatonin content, TDC3 transgenic lines also had enhanced levels of melatonin intermediates including 5-hydroxytryptophan, tryptamine, serotonin, and N-acetylserotonin. In contrast, expression levels of melatonin biosynthetic mRNA did not increase in TDC3 transgenic lines, indicating that increases in melatonin and its intermediates in these lines are attributable exclusively to overexpression of the TDC3 gene.

Introduction Melatonin (N-acetyl-5-methoxytryptamine) is an amphiphilic bioactive molecule that has been detected in all cellular compartments [1, 2]. Since its first discovery in animals in 1959 [3], the molecule has been found in almost all organisms [4]. Melatonin is a bioactive molecule with important metabolic functions, including those in circadian rhythmicity [5] and antiaging [6, 7]; it is also an anticarcinogenic factor with potent antioxidant activity [8]. Melatonin was first identified in plants in 1995 [9, 10]. Plant melatonin has functions similar to those in animals, such as delaying senescence and antioxidant activity [11–13]. As melatonin occurs naturally and has potent antioxidant activity, it is now considered to be a major health-promoting natural substance. Accordingly, interest in metabolic engineering procedures aiming to increase melatonin levels in food plants has increased [14]. Intensive studies are investigating the mode of melatonin antioxidant activity in animals in relation to those of other well-known natural compounds such flavonoids, vitamin C, and vitamin E. Since the first recognition of antioxidant and antiradical activities in melatonin in 1993 [15], 3700 scientific investigations have been published on this topic [8]. Melatonin is synthesized and accumulated to varying degrees in all plant tissues, with the highest levels generally occurring in medicinal plants and coffee seeds [16, 17]. However, in most plant species, including tomato, lupin, and rice, tissue concentrations reach only a few nanograms per gram fresh mass, a level lower than that of the plant

Yeong Byeon, Sangkyu Park, Hyoung Yool Lee, Young-Soon Kim and Kyoungwhan Back Department of Biotechnology, Interdisciplinary Program of Bioenergy and Biomaterials, Bioenergy Research Center, Chonnam National University, Gwangju, Korea

Key words: functional foods, melatonin, N-acetylserotonin, transgenic rice, tryptophan decarboxylase Address reprint requests to Kyoungwhan Back, Department of Biotechnology, College of Agriculture and Life Sciences, Chonnam National University, Gwangju 500-757, Korea. E-mail: [email protected] Received December 6, 2013; Accepted January 10, 2014.

hormone indole-3-acetic acid (IAA) [18–20]. All enzymes of the melatonin biosynthetic pathway have recently been cloned and characterized; these enzymes include tryptophan decarboxylase (TDC) [21, 22], tryptamine 5-hydroxylase (T5H) [23, 24], serotonin N-acetyltransferase (SNAT) [25], and N-acetylserotonin methyltransferase (ASMT) [26, 27]. To explore the roles of melatonin biosynthetic genes in melatonin overproduction, we initially attempted to investigate the effects of the first melatonin biosynthetic gene TDC through its overexpression in the rice genome. Rice TDC occurs as three copies of a small gene family located on chromosomes 7 and 8. It was also demonstrated that ectopic overexpression of TDC in rice results in increased tryptamine and serotonin levels [22, 28]. In the present study, we generated transgenic rice plants that overexpressed rice TDC1, TDC2, or TDC3 and compared the levels of melatonin and melatonin intermediates among transgenic types. Melatonin and melatonin intermediate levels were greatly enhanced in rice seeds through overexpression of the TDC3 gene. We thus demonstrated a potential mechanism for improving the nutritional value of crop foods.

Materials and methods Plant material and growth conditions Transgenic rice (Oryza sativa cv. Dongjin) plants overexpressing rice TDC1 and TDC2 genes have been previously generated using pGA1611:AK31 (TDC1) and pGA1611: 275

Byeon et al. AK53 (TDC2) binary vectors [22]. In the present study, we used T4 homozygous TDC1 and TDC2 transgenic rice plants to investigate melatonin levels. To measure melatonin in rice seedlings, we first surfacesterilized the dehusked seeds with 70% ethanol for 1 min and then with 2% sodium hypochlorite for 50 min. The seeds were further washed three times with sterilized water and sowed them on half-strength Murashige and Skoog (MS) medium (MB Cell, Seoul, Korea) in vertically oriented polystyrene square dishes (SPL Life Sciences, Pocheon-si, Korea). Seedlings were grown for 7 days at 28°C under a 12-hr light/12-hr dark photoperiod. Samples were stored at 80°C for later melatonin quantification and the reverse transcription-polymerase chain reaction (RT-PCR). To measure melatonin levels, we dehulled seeds of wild-type and transgenic line rice and subjected them to high-performance liquid chromatography (HPLC). Cloning of TDC3 and initiation of its ectopic overexpression in rice plants Full-length TDC3 cDNA was cloned by RT-PCR using information for the annotated rice TDC3 gene (GenBank Accession Number: NM001067504). Briefly, RT-PCR amplification of the TDC3 transcript was performed by synthesizing first-strand cDNA in 50 lL reaction volumes with 2 lg of total RNA isolated from rice flowers or salicylic acid (SA)-treated rice leaves, 500 ng of oligo (dT)15 primer (Promega, Madison, WI, USA), one unit of SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA), and one unit of RNase inhibitor (Invitrogen). Subsequently, the TDC3 sequence was PCR amplified in 20 lL reaction volumes containing 5 lL (12.5 ng) of diluted cDNA, 2 lL Ex Taq buffer (Takara Biotechniques, Shiga, Japan), 2 lL each of 2.5 mM dATP, dCTP, dGTP, and dTTP, 1 lL each of the 10 pmoles respective primers, and one unit of Ex Taq polymerase (Takara Biotechniques). The PCR program included initial denaturation at 94°C for 3 min, followed by 30 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 40 s, followed by a final extension step at 72°C for 5 min. The PCR primers used were forward 5′-ATG GGG AGC TTG GAC GCC-3′ and reverse 5′-CTA CTT GTC TTC TCC GGC-3′. These PCR-amplified fragments were ligated into the pTOP Blunt V2 (Enzynomics, Daejeon, Korea) vector (creating pTOP Blunt V2:TDC3); we verified this sequence before further use. The TDC3 cDNA fragment was further amplified by PCR with a template pTOP Blunt V2:TDC3 using primers containing the attB recombination sequence (forward primer, 5′-AAA AAG CAG GCT CCA TGG GGA GCT TGG ACG-3′; reverse primer, 5′-AGA AAG CTG GGT CTA CTT GTC TTC TCC-3′). The final PCR product was gel-purified and cloned into the pDONR221 Gatewayâ vector (Invitrogen) via BP recombination (between the attB-flanked PCR product and the donor vector containing attP sites, thus creating an entry clone). The pDONR221:TDC3 gene entry vector was then recombined with the pIPKb002 Gateway destination vector [29] via LR recombination to form pIPKb002-TDC3, which was transformed into Agro276

bacterium LBA4404. We used previously described rice transformation procedures [30]. RT-PCR analysis We isolated total RNA from rice seedlings using an RNeasy Plant Mini Kit (Qiagen, Tokyo, Japan). We performed RT-PCR to estimate mRNA expression levels of melatonin biosynthesis genes. The rice ubiquitin-5 gene (UBQ5) served as a loading control. The following primers (5′?3′) were used: for tryptophan decarboxylase 1 (TDC1), forward GCG AGG GTG AAA CCT TCC A and reverse GCG AGC CGG TGG AGT CC; for tryptophan decarboxylase 2 (TDC2), forward GTG CTG CCT TTA ACA TTG TTG G and reverse CAT GTC ATT GGA CTT TGC TAT CTG T; for tryptophan decarboxylase 3 (TDC3), forward GAC GTC GAG CCC TTC CGC and reverse ACC GTC AGC CGC GTG ATG; for tryptamine 5-hydroxylase (T5H), forward CCT CGT CCT GGA CAT GTT CGT C and reverse ATG GCG AAC GTG TTG ATG AAC AC; for serotonin N-acetyltransferase (SNAT), forward GGG CTG CGG CAA CTT GGT CC and reverse AGA AAG CTG GGT CTA AAA TCT GGG GTA; for N-acetylserotonin methyltransferase 1 (ASMT1), forward TAC CGT CCA TGA CGG CG and reverse CGG CCG CCT TCT CGA CA; and for ubiquitin-5 (UBQ5), forward CCG ACT ACA ACA TCC AGA AGG AG and reverse AAC AGG AGC CTA CGC CTA AGC. RT-PCR-amplified fragments were electrophoresed on 1.2% agarose gel. The gel was stained with ethidium bromide and photographed under UV light. Analysis of tryptophan, 5-OH tryptophan, tryptamine, serotonin, and N-acetylserotonin by HPLC Frozen rice leaves (400 mg) were homogenized to a fine powder using a TissueLyser II (Qiagen) and extracted with 4 mL of MeOH. The extracts were cleared by centrifugation, and then, the supernatants were evaporated to dryness and dissolved in 1 mL of 50 mM sodium phosphate buffer (pH 7.9) and analyzed by HPLC as described previously [31]. The analyses were performed in triplicate. Analysis of melatonin by HPLC Frozen rice samples (200 mg) were ground to a powder in liquid nitrogen using a TissueLyser II (Qiagen) and extracted with 1.5 mL of chloroform for overnight at 4°C. The chloroform extracts were evaporated until dry and dissolved in 1.5 mL of 35% methanol. Aliquots of 10 lL were subjected to HPLC with a fluorescence detector system (Waters, Milford, MA, USA). The samples were separated on a Sunfire C18 column (Waters; 4.6 9 150 mm) with a gradient elution profile (from 42% MeOH to 50% MeOH in 0.1% formic acid containing water for 27 min, then isocratic elution with 50% MeOH in 0.1% formic acid containing water for 18 min, at a flow rate of 0.15 mL/min). Melatonin was detected at 280 nm excitation and 348 nm emission, respectively. In this condition,

Melatonin-rich rice seeds the melatonin was eluted at 31 min. All measurements were conducted in triplicate.

(A)

Statistical analysis One-way ANOVAs and least-significant difference (LSD) tests were used for statistical evaluations. A P-value