J. Pineal Res. 2014; 57:408–417
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Molecular, Biological, Physiological and Clinical Aspects of Melatonin
Doi:10.1111/jpi.12180
Journal of Pineal Research
Overexpression of MzASMT improves melatonin production and enhances drought tolerance in transgenic Arabidopsis thaliana plants Abstract: Melatonin is a potent naturally occurring reactive oxygen species (ROS) and reactive nitrogen species (RNS) scavenger in plants. Melatonin protects plants from oxidative stress and, therefore, it improves their tolerance against a variety of environmental abiotic stressors. N-acetylserotonin-Omethyltransferase (ASMT) is a specific enzyme required for melatonin synthesis. In this report, an ASMT gene was cloned from apple rootstock (Malus zumi Mats) and designated as MzASMT1 (KJ123721). The MzASMT1 expression was induced by drought stress in apple leaves. The upregulation of MzASMT1 in the apple leaf positively relates to melatonin production over a 24-hr dark/light cycle. Purified MzASMT1 protein expressed in E. coli converted its substrates to melatonin with an activity of approximately 5.5 pmol/min/mg protein. The transient transformation in tobacco identified that MzASMT1 is located in cytoplasm of the cell. When MzASMT1 gene driven by 35S promoter was transferred to Arabidopsis, melatonin levels in transgenic Arabidopsis plants were 2–4 times higher than those in the wild type. The transgenic Arabidopsis plants had significantly lower intrinsic ROS than the wild type and therefore these plants exhibited greater tolerance to drought stress than that of wild type. This is, at least partially, attributed to the elevated melatonin levels resulting from the overexpression of MzASMT1. The results elucidated the important role that membrane-located melatonin synthase plays in drought tolerance. These findings have significant implications in agriculture.
Introduction As melatonin (N-acetyl-5-methoxytryptamine) was first reported in plants in 1995 [1, 2], it has been found that almost every plant tested contains melatonin [3–11]. The major functions of melatonin in plants are considered to be a universal reactive oxygen species (ROS)/reactive nitrogen species (RNS) scavengers and it provides protection against oxidative injury of roots, leaves, flowers, fruits, and seeds [12–17]. Plants are unavoidably exposed to a variety of environmental stressors including abiotic and biotic ones. These stressors induce ROS generation in cells and organs, resulting in molecular damage. ROS are also signaling molecules for plant development, flowering, ripening, and early stress responses [17–24]. But excessive ROS production leads to plant injury including programmed cell death [25] and eventually to plant death [26]. Different from the classic anti-oxidants, melatonin is an amphiphilic molecule and it enters all cellular compartments including membrane, cytoplasm, nucleus, and mitochondria to detoxify ROS [27, 28]. Moreover, its metabolites also scavenge ROS thereby amplifying the anti-oxidative capacity of melatonin [29]. The ability of melatonin and its metabolites to neutralize ROS is referred as the scavenging 408
Bixiao Zuo1,*, Xiaodong Zheng1,*, Pingli He2, Lin Wang1, Qiong Lei1, Chao Feng1, Jingzhe Zhou1, Qingtian Li1, Zhenhai Han1 and Jin Kong1 1
Institute for Horticultural Plants, China Agricultural University, Beijing, China; 2State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing, China
Key words: apple, drought tolerance, melatonin, MzASMT1, ROS scavenger, transgenic Arabidopsis Address reprint requests to Jin Kong and Zhenhai Han, College of Agriculture and Biotechnology, China Agricultural University, No.2 Yuan Mingyuan Xi Road, 100193 Beijing, China. E-mails: [email protected] and rschan@cau. edu.cn *These authors contributed equally to the paper. Received September 6, 2014; Accepted September 19, 2014.
cascade reaction [30]. As a result, melatonin has been successfully used to protect plants against various environmental stressors including drought [16], cold [31, 32], salt [33], extreme temperature [11, 34–39], etc. The synthetic pathway of melatonin in plant was not elucidated until 2012 [40]. This pathway is different from that in animals. For example, in the initial step, tryptophan is decarboxylated to form tryptamine rather than being hydroxylated to 5-hydroxytryptophan, as it occurs in animals. Tryptamine then is hydroxylated to form serotonin. Thereafter, the remaining processes of melatonin production are similar to those that exist in animals. These steps involve serotonin N-acetyltransferase (SNAT) [41] and N-acetylserotonin-O-methyltransferase (ASMT) [42, 43]. Previous studies have observed that transgenic tomatoes overexpressing ovine ASMT gene exhibit elevated melatonin production and enhanced drought tolerance [44]. To date, there are no reports on the outcome of transgenic plants which carry the plant ASMT gene. Herein, a drought-inducible ASMT gene from apple rootstock (Malus zumi Mats) was cloned and transferred to Arabidopsis. The responses of transgenic Arabidopsis to drought stress were systematically studied. The results indicate that MzASMT1 is a key factor in regulating the response of plants to abiotic stressors.
MzASMT and melatonin in drought tolerance
Materials and methods Isolation and bio-informative analysis of MzASMT1 gene Using BLAST search of apple genome database (http:// www.rosaceae.org/species/malus/malus_x_domestica) based on the amino acid sequence of rice ASMT1, apple MzASMT1 was found to share high homology (39.7%) with rice ASMT1. MzASMT1-specific primers were designed to amplify its coding sequence. Total RNA was extracted from leaves of Malus zumi Mats using the TRIzol reagent (CWBIO, Beijing, China) and immediately used for cDNA synthesis with a firststrand cDNA synthesis kit (TaKaRa, Shiga, Japan) according to the manufacturer’s instructions. The open reading frame (ORF) of MzASMT1 (KJ123721) was amplified using the 2 9 ES Taq MasterMix (CWBIO) and MzASMT1-specific primers designed by Premier 5.0 software (Premier, Palo Alto, CA, USA). The primers (MzASMT1 forward: 50 - GGATCCATGGAGGGAGAT GAAGCA-30 , and MzASMT1 reverse: 50 - CTCGAGAT CATATATATCACATGGTTGC-30 ) were applied to amplify MzASMT1 gene with PCR program at 94°C for 5 min; 30 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min; and 72°C for 10 min. The PCR product was cloned into the PMD19-T Simple vectors (TaKaRa) following the manufacturer’s instructions. The cloned MzASMT1 was sequenced. The conserved domain of MzASMT1 as well as its molecular weight was predicted on line at http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi and http://www.bio-soft.net/sms/, respectively. The isoelectric point was analyzed on line at http://web.expasy.org/compute_pi/. Phylogenic tree construction and the multiple alignments of MzASMT1 To find the evolutionary derivation for MzASMT1, a phylogenic tree was constructed using its homologous protein of AK072740 (Rice, Oryza sativa. ASMT1), KJ123721 (Apple, Malus zumi. ASMT1), BankIt 1690408 (Apple, Malus zumi. ASMT9), AFZ23489 (Cyanobacteria, Cylindrospermum stagnale PCC 7417 ASMT). Because there are few functional ASMT were isolated from plant, we used nearly all the functional ASMT we knew from plant and also the ASMT from the possible ancestor of chloroplast, cyanobacteria. Multiple alignments of MzASMT1 were conducted using DNAMAN to identify the shared domains among cyanobacteria, rice, and apple. The amino acid identities between MzASMT1 and other homologous proteins are 39.41% (Rice, Oryza sativa. ASMT1), 37.13% (Apple, Malus zumi. ASMT9), 21.07% (Cyanobacteria, Cylindrospermum stagnale PCC 7417 ASMT), respectively. Circadian rhythm of melatonin production and MzASMT1 expression in apple leaves To identify the circadian associations of melatonin production and MzASMT1 expression, the leaves of apple trees were collected at eight points during a 24-hr light/
dark cycle (16:8 light/dark), that is, the leaves were collected at 8:30, 11:30, 14:30, 17:30, 20:30, 23:30 on 27 April 2014 and at 02:30, 05:30 on 28 April 2014 at the China Agricultural University (latitude 40°000 N, longitude 116°350 W). A total of 8 g leaves were collected at each time point; they were immediately frozen in liquid nitrogen and stored at 80°C until analysis. To evaluate the impact of light on melatonin production, light intensity was measured at every sampling time using TES-1335 digital light meter (TES, Taiwan, China). Drought treatment for expression pattern detection of MzASMT1 For drought stress treatment, 3-wk-old apple seedlings were transferred to 1/2 Hoagland’s solutions under 12/12 hr-light/dark cycle at room temperature (20–22°C) for 1 wk. Then, they were transferred to complete Hoagland’s solutions for 2 wk. A total of 40 seedlings were used for this treatment, and 40 control seedlings were also used. The leaves from these seedlings were collected respectively after 8 hr’ treatment with 20% polyethylene glycol, and then frozen in liquid nitrogen for RNA preparation. RNA extraction and semi-quantitative RT-PCR analysis To analyze the expression of MzASMT1 gene, total RNA was extracted from apple leaves using the EASYspin Plant RNA Rapid Extraction Kit (Biomed, Beijing, China). The first-strand cDNA was synthesized following the protocol of Kit (Promega, Madison, WI, USA). The primers (F: 50 CATTGGTTGATGTTGGTGGTG-30 , R: 50 - AGCTTTGC TTCGGTTATTTCAT-30 ) were designed according to the MzASMT1 sequences, respectively, by Primer 5.0 software and checked by BLAST search in the apple genome of Computational Biology Web Resources (http://genomics. research.iasma.it/). The semi-quantitative RT-PCRs were performed as follows: 94°C 5 min; 94°C 30 s, 55°C 30 s, 72°C 30 s, 28 cycles, 72°C 10 min, The 20 lL PCR system contained 10 lL of 2 9 Taq MasterMix, 2 lL of cDNA, 6 lL of ddH2O, 0.5 lM primer, respectively. The PCR product (5 lL) of the 340-bp MzASMT1 fragment was detected in a 1% TAE-agarose gels. The ACTIN gene was used as the internal standard. The intensity of amplified ACTIN and MzASMT1 was captured by the Image J software (http://en.wikipedia.org/wiki/ImageJ). Melatonin and malondialdehyde (MDA) analysis A total of 1 g leaves were ground to a fine powder in liquid nitrogen with a mortar, which was buffered in 10 mL methanol and ultrasonicated (80 Hz) for 35 min at 45°C. After centrifugation at 9600 g (4°C) for 15 min, the supernatants were collected and dried by nitrogen gas. The sample preparation and HPLC detection of melatonin were performed as described by Zhao et al. [17]. A total of 1 g apple leaves were used for the extraction and determination of MDA. The MDA detection was performed as described by Zhao et al. [17]. Both the melatonin and the MDA detection were repeated three times. 409
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Identification of subcellular localization of MzASMT1 To find the subcellular localization of MzASMT1, the MzASMT1 gene was fused with GFP (green fluorescent protein) gene. The coding frame was amplified with genespecific primers (F: 50 -GGATCCATGGAGGGAGATG AAGCA-30 , and R: 50 -CTCGAGATCATATATATCAC ATGGTTGC-30 ), which was inserted into the C terminal of GFP in pc1390-UBQpro-n-mCherry with BamHI/XhoI. The GFP–MzASMT1 plasmids were introduced into the Agrobacterium tumefaciens strain GV3101 using the freeze–thaw method. One-month-old Nicotiana benthamiana leaves were used for infiltration with GV3101 carrying UBQ-GFP-MzASMT1. The subcellular localization of MzASMT1 was identified using a 9 400 objective Olympus confocal microscope 3 days after transient transformation. The images were processed with the FV10-ASM software version 3.0. Expression and purification of MzASMT1 protein in Escherichia coli The coding frame without stop codon of MzASMT1 was fused with Glutathione S-transferase and inserted into the pGEX-6p-1 by enzyme sites BamHI/XhoI. The MzASMT1 410
Fig. 1. The multiple alignments and phylogenetic tree of MzASMT1. (A) The multiple alignments were constructed using DNAMAN. The conserved amino acid sequences shared by OMT in different species are highlighted by different colors. The GenBank accession numbers are AK072740 (Rice ASMT1), KJ123721 (Apple, Malus zumi. ASMT1), KJ156531 (Apple, Malus zumi. OMT9), AFZ23489 (Blue algae, Cylindrospermum stagnale PCC 7417 OMT). The black color highlights the same sequence, while the pink color identifies the sequence with only one amino acid difference and blue with more than one amino acid. (B) The phylogenetic tree was constructed using the neighbor-joining method and a bootstrap test with 1000 iterations, using MEGA5.2 software.
expression was induced by 0.2 mmol/l Isopropyl-beta-Dthiogalactopyranoside in E. coli strain BL21 at 23°C for 8 hr. The bacteria were centrifuged at 6200 g for 10 min. The sediment was suspended and subjected to ultrasonication (80 Hz); the samples were then centrifuged at 9600 g for 50 min at 4°C. The MzASMT1 protein was purified by precipitation with GSTrapTM column (CWBIO) which was equilibrated with a quintuple volume of PBS buffer. The purified MzASMT1 protein was confirmed by SDS–PAGE. Measurement of MzASMT1 enzyme activity For MzASMT1 enzyme activity measurement, a total of 50 lg MzASMT1 protein purified from E. coli was incubated in the reaction system (100 lL of 100 mM potassium phosphate buffer (pH 7.8), 0.5 mM S-adenosyl-L-methionine, and 0.5 mM N-acetylserotonin). The 1-hr reaction at 30°C was stopped with 50 lL methanol. Then, a total of 200 lL acetonitrile were added to precipitate the protein. The mixture was centrifuged at 9600 g for 10 min at 4°C. The supernatant was injected into the HPLC for melatonin analysis. The enzyme activity of MzASMT1 was determined by multiplying melatonin content with 0.4/60 min/ 50 lg.
MzASMT and melatonin in drought tolerance (A)
frozen in liquid nitrogen immediately, which were then stored at 80°C until analysis. A total of three homozygous T3 lines with single insertion and high expression level of MzASMT1 gene were selected for melatonin detection [17]. Drought stress treatment for MzASMT1 transgenic Arabidopsis
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Fig. 2. The melatonin levels, MDA contents, and MzASMT1 expression in leaves of apple trees over 24-hr light/dark cycle. (A) The levels of melatonin and MDA. (B) The gel image of MzASMT1 and actin expression. Empty bar represents light, and black bar represents dark. (C) The light intensity over 24-hr light/ dark cycle from 8:30, 27 April to 5:30, 28 April.
Surface-sterilized Arabidopsis seeds of wild type (Col-0) and transgenic lines overexpressing MzASMT1 were sown on MS growth media subjected to a 3 days’ dark treatment at 4°C to synchronize germination. The seedlings grew for 1 wk in a growth chamber at 22°C with a 16-hrlight, 8-hr-dark cycle before treatment. For drought stress, seedlings were then transferred to MS growth medium containing 300 mM D-Mannitol in the square petri dish and placed in a growth chamber at 22°C with a 8-hr-light, 16-hr-dark cycle. A total of 48 plants of transgenic or wild-type plants were divided into six groups, respectively. Three groups of plants were subjected to stress treatment and the other three groups growing on normal MS medium were used as control. For the drought treatment in soil (1:1 vermiculite and nutrient soil) conducted at 22°C on a 8-hr-light, 16-hr-dark cycle, a total of 12 transgenic plants were subjected to drought stress in soil and 12 wildtype plants were used as control. The 4-wk-old seedlings received no water for 12 days; thereafter, both the transgenic and the control plants were re-watered to observe their phenotype. ROS detection
Fig. 3. MzASMT1 expression in apple leaves before and after 8-hr’ drought stress.
Overexpressing MzASMT1 in transgenic Arabidopsis thaliana The open reading frame sequence of the MzASMT1 gene was amplified with primers having BamHI/XhoI restriction sites as described above and ligated to plant expression vector pBIN438 to make it driven by the CaMV35S promoter. The pBIN438-MzASMT1 plasmid was transferred into Agrobacterium tumefaciens strain GV3101 and then transformed into col-0 Arabidopsis plants using the floral dip method. Transgenic plants were placed on 1/2 Murashige-Skoog (MS) growth media containing 25 mg/L kanamycin. The PCR confirmed transgenic seedlings were transplanted into soil for seed collection in a growth chamber at 22°C in a 16/8 hr light/dark cycle. The RT-PCR using MzASMT1-specific primers mentioned above were applied to detect its expression in single-insertion and homologous T3 transgenic lines. Measurement of melatonin production in transgenic Arabidopsis overexpressing MzASMT1 Two-month-old transgenic Arabidopsis plants overexpressing MzASMT1 and wild-type plants were collected and
The 7-day-old seedlings grown on vertically oriented MS medium were transferred to MS medium supplemented with 300 mM D-mannitol for drought treatment. A week later, photographs were taken and seedlings were collected to measure ROS levels. In addition, the leaves of 1-monthold Arabidopsis plants subjected to drought treatment in soil for 12 days were also collected for in vivo ROS detection. The samples were incubated with 5-(and 6)-chlorom0 0 ethyl-2 -7 -dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA) for 20 min and washed with distilled H2O to remove excess CM-H2DCFDA. For the ROS detection in medium-cultured seedling, CM-H2DCFDA was introduced into these seedlings using vacuum filtration at a pressure of -1 Mpa for 10 min. The fluorescence images were obtained with a Leica stereoscope (Leica, Wetzlar, Germany) (910). Measurement of IAA content in transgenic Arabidopsis plants Surface-sterilized Arabidopsis seeds from wild type (Col0) and transgenic Arabidopsis lines overexpressing MzASMT1 were sown on MS growth media and subjected to a 3-days dark treatment at 4°C to synchronize germination. The seedlings were grown in a growth chamber at 22°C under a 16-hr-light, 8-hr-dark cycle. One-month-old seedlings were collected to measure IAA content. A total of 0.5 g aerial parts of wild-type and transgenic plants 411
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(D) Fig. 4. The subcellular localization of MzASMT1 in plasma membrane and nucleus. (A) The bright-field image of Agrobacterium tumefaciens-infiltrated tobacco leaves; (B) Red fluorescence of chlorophyll; (C) Green fluorescence of GFP-MzASMT1; (D) The merged fluorescent images. Tobacco leaves infiltrated with Agrobacterium tumefaciens harboring GFP-MzASMT1 driven by UBQ promoter were visualized by confocal microscopy (9400) 2 days after transformation.
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Fig. 5. HPLC spectra of the melatonin levels in wild type and three transgenic Arabidopsis lines overexpressing MzASMT1. (A) HPLC spectrum of melatonin standard sample (retention time: 8.6). (B) HPLC spectrum of Arabidopsis extracts. The retention time of melatonin extracts from wild type and three transgenic lines was the same as the melatonin standard sample. The peak area of wild type and three transgenic lines was 48.18, 226.06, 274.92, 323.96, respectively. (C) and (D) Melatonin and MzASMT1 expression levels in wild type and three transgenic Arabidopsis lines. All of the three lines had high levels of MzASMT1 expression shown by RT-PCR resulting in significantly higher melatonin levels compared with the wild type. The data are means S.D. of triplicate experiments. Asterisks (*) indicate significant differences from the control (P < 0.05).
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MzASMT and melatonin in drought tolerance (A)
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Fig. 6. MzASMT1 conferred drought tolerance to transgenic Arabidopsis plants overexpressing MzASMT1. (A) and (B) Three transgenic Arabidopsis lines overexpressing MzASMT1 had more lateral roots and fresh weight than wild type under drought stress on medium containing 300 mM D-Mannitol. The data are means S.D. of triplicate experiments. Asterisks (*) indicate significant differences from the control (P < 0.05). (C) Transgenic Arabidopsis line 2 and line 3 plants were more tolerant to drought stress than the wild type in soil.
were ground to a fine powder in liquid nitrogen, with a mortar and pestle. The samples were then suspended in precooled 2.5 mL acetonitrile containing 1 mM 2,6-Ditert-butyl-4-methylphenol (BHT) and placed on ice for 24 hr. Following sample preparation, HPLC detection of IAA was performed as described by Wang et al. [44]. IAA detection was repeated three times in transgenic lines and wild-type plants.
Results The open reading frame of MzASMT1 was 1077 bases long. It encodes a protein of 358 amino acids with a predicted molecular weight of 39.72 KD. Its isoelectric point was approximately 5.34. MzASMT1 has a conserved
domain (from a.a. 93 to a.a.332) shared by rice ASMT1 (Fig. 1A). The phylogenetic tree showed that MzASMT1 has the closest relation with rice ASMT1 than other O-methyltransferase found in apple and cyanobacteria. In the 24-hr light/dark cycle, the highest light intensity happened at 14:30. A total of two melatonin peaks occurred in apple leaves. One peak was at 14:30 and the other occurred at 5:30 in the early morning (Fig. 2A). As to the changes on MDA, there were also ‘dual peaks’ in leaves, but they occurred in advance of the melatonin peaks. This suggested that the oxidative stress might induce the biosynthesis of melatonin in leaves (Fig. 2B). To investigate the role of MzASMT1 in melatonin biosynthesis, RT-PCR was used to detect the expression of MzASMT1. The results showed that the expression of 413
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MzASMT1 was in good agreement with melatonin content. It preceded melatonin change in the 24-hr light/dark cycle. It was apparent that when the apple seedlings were under drought stress, the expression of MzASMT1 was induced. The MzASMT1 expression after 8-hr drought treatment was much higher than that of control (Fig. 3). This suggests that MzASMT1 positively responses to drought stress. When transient transformation mediated by agrobacterium GV3101 with GFP-MzASMT1 driven by UBQ promoter occurred in tobacco leaves, GFP-MzASMT1 was observed to be located in cytoplasm as observed in rice ASMT and sheep serotonin N-acetyltransferase [45, 46] when examined with the confocal microscope (Fig. 4). The MzASMT1 fused with GST was purified from E. coli by GST tag. After the cocultivation of the ASMT1 protein with its substrates (S-adenosyl-L-methionine and N-acetylserotonin), melatonin was detected by HPLC. This proved that MzASMT1 could synthesize melatonin in vitro with enzyme activity of around 5.5 pmol/min/mg protein. A total of three single insertional homozygous transgenic lines with high expression of MzASMT1 gene were selected for melatonin detection (Fig. 5D). The melatonin levels in the three lines were 68.82 ng/g, 90.19 ng/g, and 98.63 ng/g FW, respectively, which were 2–4 times higher than wild type (22 ng/g FW). The highest amount (98.63 ng/g FW) was almost four times higher than that in the wild type (22 ng/g FW) (Fig. 5C). HPLC spectra of melatonin detection were shown in Fig. 5A,B. On the medium, the three transgenic Arabidopsis lines that developed more lateral roots and fresh weight were more tolerant to drought stress than wild type after 7-days’ drought treatment (Fig. 6A,B). Coincidentally, the transgenic Arabidopsis plants were also more tolerant to drought stress than wild type in soil. It is shown in Fig. 6C; thus, more wild-type plants wilted compared with transgenic Arabidopsis after 12-days’ drought treatment. After re-watering, the transgenic Arabidopsis plants exhib414
Fig. 7. The ROS levels in wild type and transgenic Arabidopsis overexpressing MzASMT1 under drought stress. (A) The ROS levels presented by green fluorescence in the leaves of wild type and the transgenic Arabidopsis overexpressing MzASMT1 under drought stress in soil. More ROS accumulated in the leaf trichomes of wild type than in the transgenic plant. (B) The green fluorescence shows the ROS levels in wild type and the transgenic Arabidopsis plants under drought stress on medium containing in 300 mM D-mannitol growth medium. The wild-type plants emitted stronger fluorescence than three transgenic lines. The fluorescence images were obtained with a Leica stereoscope (Leica Microsystems) (910).
Fig. 8. The IAA contents of the aerial part of 1-month-old transgenic line 3 and wild-type plants. The data are means S.D. of triplicate experiments. Asterisks (*) indicate significant differences from the control (P < 0.05).
ited renewed growth, while some wild-type plants died. Obviously, overexpression of MzASMT1 enhanced the resistance against drought stress. The ROS levels in wild type and the transgenic Arabidopsis overexpressing MzASMT1 were detected when they were under drought. The fluorescent dye 5-(and 6)-chlo0 0 romethyl-2 -7 -dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA) was used to visualize in vivo ROS in leaves of plants growing in soil and aerial parts of the plants growing on medium under drought stress. In the leaves of the stressed plant in soil, the ROS accumulation in leaf trichomes was observed. The wild type obviously accumulated more ROS than the transgenic Arabidopsis plants under drought stress. Only few positive leaf trichomes were observed in transgenic leaves, while many of the leaf trichomes of wild type exhibited a bright green fluorescence under drought stress (Fig 7A). Coincidentally, in the case of the aerial part of the plants growing on medium for drought treatment, the wild type also produced more ROS than did the transgenic plants. The wild-type plants had very bright green color, while the transgenic plants had only a weak signal (Fig. 7B). But we failed to dye the leaf of Arabidopsis plants growing in soil, probably because the structures of the mature plants prevent the dyes from entering into the leaves. These results confirmed
MzASMT and melatonin in drought tolerance that the increased melatonin resulting from MzASMT1 overexpression effectively scavenges ROS under abiotic stress. We measured the IAA contents in the aerial parts of the transgenic and the wild-type plants with three repeats. The IAA levels in wild type were respectively 103.92 ng/g FW, 114.09 ng/g FW, and 100.45 ng/g FW. By contrast, the IAA contents in transgenic plants were 63.47 ng/g FW, 74.44 ng/g FW, and 88.17 ng/g FW. The results shown in Fig. 8 are the average values. It showed a significant difference between the transgenic plants and the wild type in IAA levels. The IAA content in aerial parts of transgenic plants (75.36 ng/g FW) was just 70% that of the wild type (106.15 ng/g FW) (Fig. 8).
increased melatonin production could result in reduced synthesis of IAA. There was no significant height difference between transgenic plants and wild type. This has been reported by Wang et al. [44] in transgenic tomato overexpressing oASMT gene. Collectively, the results indicate that MzASMT1 may be a key factor in regulating in the response of plants against abiotic stressors by elevating melatonin production in plants.
Acknowledgements Our research was supported by 973 Program (National Basic Research Program of China, Project No: 2012CB114200) and the 863 Project (Project No: 2011AA100204).
Discussion Many studies have shown that exogenously applied melatonin effectively enhances the capacity of plants to tolerate unfavorable environmental insults. These include heat [38, 39], drought [16], cold [31, 32], excessive water [47], long periods of dark exposure [48–50], UV irradiation [17, 37, 51], and chemical pollutants [33, 52]. Several studies have found that transgenic plants overexpressing animal SNAT or ASMT also improve their tolerance to abiotic stressors [44, 53, 54]. To date, little is known about the outcomes of transgenic plants overexpressing plant melatonin synthetic genes. In the current study, an apple ASMT gene (MzASMT1) was cloned for this purpose. We observed that MzASMT1 was drought inducible in apple leaves. This implies that MzASMT1 may be a candidate for the regulation of melatonin synthesis under drought conditions. The phylogenetic analysis shows that MzASMT1 is more closely related to the ASMT of rice rather than to the other O-methyltransferase in apple. This suggests that the MzASMT1 is an ortholog of rice ASMT despite the low amino acid identity (39.7%). Similar to the rice ASMT, MzASMT1 was also identified to be distributed in the cytoplasm. To further understand the physiological role of MzASMT1 in the stress response, it was transferred into Arabidopsis. It was found that the transgenic Arabidopsis overexpressing MzASMT1 produces 2- to 4-fold higher levels of melatonin than those of wild type. Accordingly, the transgenic plants were found to have a greater tolerance to drought than the wild type. The mechanistic exploration unambiguously proved that the elevated drought tolerance in transgenic Arabidopsis is directly associated with the anti-oxidant capacity of melatonin. Transgenic Arabidopsis have significantly lower levels of ROS and higher levels of melatonin than does the wild type under drought (Fig. 7). The injuries in plants caused by drought are mainly mediated by oxidative stress [55]. Scavenging ROS and reducing oxidative stress are key factors to enhance the tolerance of plants to abiotic stressors [56]. Melatonin is an important anti-oxidant to reduce oxidative stress and protect plants from drought stress. In the current study, the levels of indole 3-acetic acid (IAA) of transgenic Arabidopsis plants were lower than that of wild type. This is not unexpected as IAA and melatonin share the same precursor. Due to substrate competition,
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© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Molecular, Biological, Physiological and Clinical Aspects of Melatonin
Doi:10.1111/jpi.12180
Journal of Pineal Research
Overexpression of MzASMT improves melatonin production and enhances drought tolerance in transgenic Arabidopsis thaliana plants Abstract: Melatonin is a potent naturally occurring reactive oxygen species (ROS) and reactive nitrogen species (RNS) scavenger in plants. Melatonin protects plants from oxidative stress and, therefore, it improves their tolerance against a variety of environmental abiotic stressors. N-acetylserotonin-Omethyltransferase (ASMT) is a specific enzyme required for melatonin synthesis. In this report, an ASMT gene was cloned from apple rootstock (Malus zumi Mats) and designated as MzASMT1 (KJ123721). The MzASMT1 expression was induced by drought stress in apple leaves. The upregulation of MzASMT1 in the apple leaf positively relates to melatonin production over a 24-hr dark/light cycle. Purified MzASMT1 protein expressed in E. coli converted its substrates to melatonin with an activity of approximately 5.5 pmol/min/mg protein. The transient transformation in tobacco identified that MzASMT1 is located in cytoplasm of the cell. When MzASMT1 gene driven by 35S promoter was transferred to Arabidopsis, melatonin levels in transgenic Arabidopsis plants were 2–4 times higher than those in the wild type. The transgenic Arabidopsis plants had significantly lower intrinsic ROS than the wild type and therefore these plants exhibited greater tolerance to drought stress than that of wild type. This is, at least partially, attributed to the elevated melatonin levels resulting from the overexpression of MzASMT1. The results elucidated the important role that membrane-located melatonin synthase plays in drought tolerance. These findings have significant implications in agriculture.
Introduction As melatonin (N-acetyl-5-methoxytryptamine) was first reported in plants in 1995 [1, 2], it has been found that almost every plant tested contains melatonin [3–11]. The major functions of melatonin in plants are considered to be a universal reactive oxygen species (ROS)/reactive nitrogen species (RNS) scavengers and it provides protection against oxidative injury of roots, leaves, flowers, fruits, and seeds [12–17]. Plants are unavoidably exposed to a variety of environmental stressors including abiotic and biotic ones. These stressors induce ROS generation in cells and organs, resulting in molecular damage. ROS are also signaling molecules for plant development, flowering, ripening, and early stress responses [17–24]. But excessive ROS production leads to plant injury including programmed cell death [25] and eventually to plant death [26]. Different from the classic anti-oxidants, melatonin is an amphiphilic molecule and it enters all cellular compartments including membrane, cytoplasm, nucleus, and mitochondria to detoxify ROS [27, 28]. Moreover, its metabolites also scavenge ROS thereby amplifying the anti-oxidative capacity of melatonin [29]. The ability of melatonin and its metabolites to neutralize ROS is referred as the scavenging 408
Bixiao Zuo1,*, Xiaodong Zheng1,*, Pingli He2, Lin Wang1, Qiong Lei1, Chao Feng1, Jingzhe Zhou1, Qingtian Li1, Zhenhai Han1 and Jin Kong1 1
Institute for Horticultural Plants, China Agricultural University, Beijing, China; 2State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing, China
Key words: apple, drought tolerance, melatonin, MzASMT1, ROS scavenger, transgenic Arabidopsis Address reprint requests to Jin Kong and Zhenhai Han, College of Agriculture and Biotechnology, China Agricultural University, No.2 Yuan Mingyuan Xi Road, 100193 Beijing, China. E-mails: [email protected] and rschan@cau. edu.cn *These authors contributed equally to the paper. Received September 6, 2014; Accepted September 19, 2014.
cascade reaction [30]. As a result, melatonin has been successfully used to protect plants against various environmental stressors including drought [16], cold [31, 32], salt [33], extreme temperature [11, 34–39], etc. The synthetic pathway of melatonin in plant was not elucidated until 2012 [40]. This pathway is different from that in animals. For example, in the initial step, tryptophan is decarboxylated to form tryptamine rather than being hydroxylated to 5-hydroxytryptophan, as it occurs in animals. Tryptamine then is hydroxylated to form serotonin. Thereafter, the remaining processes of melatonin production are similar to those that exist in animals. These steps involve serotonin N-acetyltransferase (SNAT) [41] and N-acetylserotonin-O-methyltransferase (ASMT) [42, 43]. Previous studies have observed that transgenic tomatoes overexpressing ovine ASMT gene exhibit elevated melatonin production and enhanced drought tolerance [44]. To date, there are no reports on the outcome of transgenic plants which carry the plant ASMT gene. Herein, a drought-inducible ASMT gene from apple rootstock (Malus zumi Mats) was cloned and transferred to Arabidopsis. The responses of transgenic Arabidopsis to drought stress were systematically studied. The results indicate that MzASMT1 is a key factor in regulating the response of plants to abiotic stressors.
MzASMT and melatonin in drought tolerance
Materials and methods Isolation and bio-informative analysis of MzASMT1 gene Using BLAST search of apple genome database (http:// www.rosaceae.org/species/malus/malus_x_domestica) based on the amino acid sequence of rice ASMT1, apple MzASMT1 was found to share high homology (39.7%) with rice ASMT1. MzASMT1-specific primers were designed to amplify its coding sequence. Total RNA was extracted from leaves of Malus zumi Mats using the TRIzol reagent (CWBIO, Beijing, China) and immediately used for cDNA synthesis with a firststrand cDNA synthesis kit (TaKaRa, Shiga, Japan) according to the manufacturer’s instructions. The open reading frame (ORF) of MzASMT1 (KJ123721) was amplified using the 2 9 ES Taq MasterMix (CWBIO) and MzASMT1-specific primers designed by Premier 5.0 software (Premier, Palo Alto, CA, USA). The primers (MzASMT1 forward: 50 - GGATCCATGGAGGGAGAT GAAGCA-30 , and MzASMT1 reverse: 50 - CTCGAGAT CATATATATCACATGGTTGC-30 ) were applied to amplify MzASMT1 gene with PCR program at 94°C for 5 min; 30 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min; and 72°C for 10 min. The PCR product was cloned into the PMD19-T Simple vectors (TaKaRa) following the manufacturer’s instructions. The cloned MzASMT1 was sequenced. The conserved domain of MzASMT1 as well as its molecular weight was predicted on line at http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi and http://www.bio-soft.net/sms/, respectively. The isoelectric point was analyzed on line at http://web.expasy.org/compute_pi/. Phylogenic tree construction and the multiple alignments of MzASMT1 To find the evolutionary derivation for MzASMT1, a phylogenic tree was constructed using its homologous protein of AK072740 (Rice, Oryza sativa. ASMT1), KJ123721 (Apple, Malus zumi. ASMT1), BankIt 1690408 (Apple, Malus zumi. ASMT9), AFZ23489 (Cyanobacteria, Cylindrospermum stagnale PCC 7417 ASMT). Because there are few functional ASMT were isolated from plant, we used nearly all the functional ASMT we knew from plant and also the ASMT from the possible ancestor of chloroplast, cyanobacteria. Multiple alignments of MzASMT1 were conducted using DNAMAN to identify the shared domains among cyanobacteria, rice, and apple. The amino acid identities between MzASMT1 and other homologous proteins are 39.41% (Rice, Oryza sativa. ASMT1), 37.13% (Apple, Malus zumi. ASMT9), 21.07% (Cyanobacteria, Cylindrospermum stagnale PCC 7417 ASMT), respectively. Circadian rhythm of melatonin production and MzASMT1 expression in apple leaves To identify the circadian associations of melatonin production and MzASMT1 expression, the leaves of apple trees were collected at eight points during a 24-hr light/
dark cycle (16:8 light/dark), that is, the leaves were collected at 8:30, 11:30, 14:30, 17:30, 20:30, 23:30 on 27 April 2014 and at 02:30, 05:30 on 28 April 2014 at the China Agricultural University (latitude 40°000 N, longitude 116°350 W). A total of 8 g leaves were collected at each time point; they were immediately frozen in liquid nitrogen and stored at 80°C until analysis. To evaluate the impact of light on melatonin production, light intensity was measured at every sampling time using TES-1335 digital light meter (TES, Taiwan, China). Drought treatment for expression pattern detection of MzASMT1 For drought stress treatment, 3-wk-old apple seedlings were transferred to 1/2 Hoagland’s solutions under 12/12 hr-light/dark cycle at room temperature (20–22°C) for 1 wk. Then, they were transferred to complete Hoagland’s solutions for 2 wk. A total of 40 seedlings were used for this treatment, and 40 control seedlings were also used. The leaves from these seedlings were collected respectively after 8 hr’ treatment with 20% polyethylene glycol, and then frozen in liquid nitrogen for RNA preparation. RNA extraction and semi-quantitative RT-PCR analysis To analyze the expression of MzASMT1 gene, total RNA was extracted from apple leaves using the EASYspin Plant RNA Rapid Extraction Kit (Biomed, Beijing, China). The first-strand cDNA was synthesized following the protocol of Kit (Promega, Madison, WI, USA). The primers (F: 50 CATTGGTTGATGTTGGTGGTG-30 , R: 50 - AGCTTTGC TTCGGTTATTTCAT-30 ) were designed according to the MzASMT1 sequences, respectively, by Primer 5.0 software and checked by BLAST search in the apple genome of Computational Biology Web Resources (http://genomics. research.iasma.it/). The semi-quantitative RT-PCRs were performed as follows: 94°C 5 min; 94°C 30 s, 55°C 30 s, 72°C 30 s, 28 cycles, 72°C 10 min, The 20 lL PCR system contained 10 lL of 2 9 Taq MasterMix, 2 lL of cDNA, 6 lL of ddH2O, 0.5 lM primer, respectively. The PCR product (5 lL) of the 340-bp MzASMT1 fragment was detected in a 1% TAE-agarose gels. The ACTIN gene was used as the internal standard. The intensity of amplified ACTIN and MzASMT1 was captured by the Image J software (http://en.wikipedia.org/wiki/ImageJ). Melatonin and malondialdehyde (MDA) analysis A total of 1 g leaves were ground to a fine powder in liquid nitrogen with a mortar, which was buffered in 10 mL methanol and ultrasonicated (80 Hz) for 35 min at 45°C. After centrifugation at 9600 g (4°C) for 15 min, the supernatants were collected and dried by nitrogen gas. The sample preparation and HPLC detection of melatonin were performed as described by Zhao et al. [17]. A total of 1 g apple leaves were used for the extraction and determination of MDA. The MDA detection was performed as described by Zhao et al. [17]. Both the melatonin and the MDA detection were repeated three times. 409
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Identification of subcellular localization of MzASMT1 To find the subcellular localization of MzASMT1, the MzASMT1 gene was fused with GFP (green fluorescent protein) gene. The coding frame was amplified with genespecific primers (F: 50 -GGATCCATGGAGGGAGATG AAGCA-30 , and R: 50 -CTCGAGATCATATATATCAC ATGGTTGC-30 ), which was inserted into the C terminal of GFP in pc1390-UBQpro-n-mCherry with BamHI/XhoI. The GFP–MzASMT1 plasmids were introduced into the Agrobacterium tumefaciens strain GV3101 using the freeze–thaw method. One-month-old Nicotiana benthamiana leaves were used for infiltration with GV3101 carrying UBQ-GFP-MzASMT1. The subcellular localization of MzASMT1 was identified using a 9 400 objective Olympus confocal microscope 3 days after transient transformation. The images were processed with the FV10-ASM software version 3.0. Expression and purification of MzASMT1 protein in Escherichia coli The coding frame without stop codon of MzASMT1 was fused with Glutathione S-transferase and inserted into the pGEX-6p-1 by enzyme sites BamHI/XhoI. The MzASMT1 410
Fig. 1. The multiple alignments and phylogenetic tree of MzASMT1. (A) The multiple alignments were constructed using DNAMAN. The conserved amino acid sequences shared by OMT in different species are highlighted by different colors. The GenBank accession numbers are AK072740 (Rice ASMT1), KJ123721 (Apple, Malus zumi. ASMT1), KJ156531 (Apple, Malus zumi. OMT9), AFZ23489 (Blue algae, Cylindrospermum stagnale PCC 7417 OMT). The black color highlights the same sequence, while the pink color identifies the sequence with only one amino acid difference and blue with more than one amino acid. (B) The phylogenetic tree was constructed using the neighbor-joining method and a bootstrap test with 1000 iterations, using MEGA5.2 software.
expression was induced by 0.2 mmol/l Isopropyl-beta-Dthiogalactopyranoside in E. coli strain BL21 at 23°C for 8 hr. The bacteria were centrifuged at 6200 g for 10 min. The sediment was suspended and subjected to ultrasonication (80 Hz); the samples were then centrifuged at 9600 g for 50 min at 4°C. The MzASMT1 protein was purified by precipitation with GSTrapTM column (CWBIO) which was equilibrated with a quintuple volume of PBS buffer. The purified MzASMT1 protein was confirmed by SDS–PAGE. Measurement of MzASMT1 enzyme activity For MzASMT1 enzyme activity measurement, a total of 50 lg MzASMT1 protein purified from E. coli was incubated in the reaction system (100 lL of 100 mM potassium phosphate buffer (pH 7.8), 0.5 mM S-adenosyl-L-methionine, and 0.5 mM N-acetylserotonin). The 1-hr reaction at 30°C was stopped with 50 lL methanol. Then, a total of 200 lL acetonitrile were added to precipitate the protein. The mixture was centrifuged at 9600 g for 10 min at 4°C. The supernatant was injected into the HPLC for melatonin analysis. The enzyme activity of MzASMT1 was determined by multiplying melatonin content with 0.4/60 min/ 50 lg.
MzASMT and melatonin in drought tolerance (A)
frozen in liquid nitrogen immediately, which were then stored at 80°C until analysis. A total of three homozygous T3 lines with single insertion and high expression level of MzASMT1 gene were selected for melatonin detection [17]. Drought stress treatment for MzASMT1 transgenic Arabidopsis
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Fig. 2. The melatonin levels, MDA contents, and MzASMT1 expression in leaves of apple trees over 24-hr light/dark cycle. (A) The levels of melatonin and MDA. (B) The gel image of MzASMT1 and actin expression. Empty bar represents light, and black bar represents dark. (C) The light intensity over 24-hr light/ dark cycle from 8:30, 27 April to 5:30, 28 April.
Surface-sterilized Arabidopsis seeds of wild type (Col-0) and transgenic lines overexpressing MzASMT1 were sown on MS growth media subjected to a 3 days’ dark treatment at 4°C to synchronize germination. The seedlings grew for 1 wk in a growth chamber at 22°C with a 16-hrlight, 8-hr-dark cycle before treatment. For drought stress, seedlings were then transferred to MS growth medium containing 300 mM D-Mannitol in the square petri dish and placed in a growth chamber at 22°C with a 8-hr-light, 16-hr-dark cycle. A total of 48 plants of transgenic or wild-type plants were divided into six groups, respectively. Three groups of plants were subjected to stress treatment and the other three groups growing on normal MS medium were used as control. For the drought treatment in soil (1:1 vermiculite and nutrient soil) conducted at 22°C on a 8-hr-light, 16-hr-dark cycle, a total of 12 transgenic plants were subjected to drought stress in soil and 12 wildtype plants were used as control. The 4-wk-old seedlings received no water for 12 days; thereafter, both the transgenic and the control plants were re-watered to observe their phenotype. ROS detection
Fig. 3. MzASMT1 expression in apple leaves before and after 8-hr’ drought stress.
Overexpressing MzASMT1 in transgenic Arabidopsis thaliana The open reading frame sequence of the MzASMT1 gene was amplified with primers having BamHI/XhoI restriction sites as described above and ligated to plant expression vector pBIN438 to make it driven by the CaMV35S promoter. The pBIN438-MzASMT1 plasmid was transferred into Agrobacterium tumefaciens strain GV3101 and then transformed into col-0 Arabidopsis plants using the floral dip method. Transgenic plants were placed on 1/2 Murashige-Skoog (MS) growth media containing 25 mg/L kanamycin. The PCR confirmed transgenic seedlings were transplanted into soil for seed collection in a growth chamber at 22°C in a 16/8 hr light/dark cycle. The RT-PCR using MzASMT1-specific primers mentioned above were applied to detect its expression in single-insertion and homologous T3 transgenic lines. Measurement of melatonin production in transgenic Arabidopsis overexpressing MzASMT1 Two-month-old transgenic Arabidopsis plants overexpressing MzASMT1 and wild-type plants were collected and
The 7-day-old seedlings grown on vertically oriented MS medium were transferred to MS medium supplemented with 300 mM D-mannitol for drought treatment. A week later, photographs were taken and seedlings were collected to measure ROS levels. In addition, the leaves of 1-monthold Arabidopsis plants subjected to drought treatment in soil for 12 days were also collected for in vivo ROS detection. The samples were incubated with 5-(and 6)-chlorom0 0 ethyl-2 -7 -dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA) for 20 min and washed with distilled H2O to remove excess CM-H2DCFDA. For the ROS detection in medium-cultured seedling, CM-H2DCFDA was introduced into these seedlings using vacuum filtration at a pressure of -1 Mpa for 10 min. The fluorescence images were obtained with a Leica stereoscope (Leica, Wetzlar, Germany) (910). Measurement of IAA content in transgenic Arabidopsis plants Surface-sterilized Arabidopsis seeds from wild type (Col0) and transgenic Arabidopsis lines overexpressing MzASMT1 were sown on MS growth media and subjected to a 3-days dark treatment at 4°C to synchronize germination. The seedlings were grown in a growth chamber at 22°C under a 16-hr-light, 8-hr-dark cycle. One-month-old seedlings were collected to measure IAA content. A total of 0.5 g aerial parts of wild-type and transgenic plants 411
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(D) Fig. 4. The subcellular localization of MzASMT1 in plasma membrane and nucleus. (A) The bright-field image of Agrobacterium tumefaciens-infiltrated tobacco leaves; (B) Red fluorescence of chlorophyll; (C) Green fluorescence of GFP-MzASMT1; (D) The merged fluorescent images. Tobacco leaves infiltrated with Agrobacterium tumefaciens harboring GFP-MzASMT1 driven by UBQ promoter were visualized by confocal microscopy (9400) 2 days after transformation.
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Fig. 5. HPLC spectra of the melatonin levels in wild type and three transgenic Arabidopsis lines overexpressing MzASMT1. (A) HPLC spectrum of melatonin standard sample (retention time: 8.6). (B) HPLC spectrum of Arabidopsis extracts. The retention time of melatonin extracts from wild type and three transgenic lines was the same as the melatonin standard sample. The peak area of wild type and three transgenic lines was 48.18, 226.06, 274.92, 323.96, respectively. (C) and (D) Melatonin and MzASMT1 expression levels in wild type and three transgenic Arabidopsis lines. All of the three lines had high levels of MzASMT1 expression shown by RT-PCR resulting in significantly higher melatonin levels compared with the wild type. The data are means S.D. of triplicate experiments. Asterisks (*) indicate significant differences from the control (P < 0.05).
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MzASMT and melatonin in drought tolerance (A)
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Fig. 6. MzASMT1 conferred drought tolerance to transgenic Arabidopsis plants overexpressing MzASMT1. (A) and (B) Three transgenic Arabidopsis lines overexpressing MzASMT1 had more lateral roots and fresh weight than wild type under drought stress on medium containing 300 mM D-Mannitol. The data are means S.D. of triplicate experiments. Asterisks (*) indicate significant differences from the control (P < 0.05). (C) Transgenic Arabidopsis line 2 and line 3 plants were more tolerant to drought stress than the wild type in soil.
were ground to a fine powder in liquid nitrogen, with a mortar and pestle. The samples were then suspended in precooled 2.5 mL acetonitrile containing 1 mM 2,6-Ditert-butyl-4-methylphenol (BHT) and placed on ice for 24 hr. Following sample preparation, HPLC detection of IAA was performed as described by Wang et al. [44]. IAA detection was repeated three times in transgenic lines and wild-type plants.
Results The open reading frame of MzASMT1 was 1077 bases long. It encodes a protein of 358 amino acids with a predicted molecular weight of 39.72 KD. Its isoelectric point was approximately 5.34. MzASMT1 has a conserved
domain (from a.a. 93 to a.a.332) shared by rice ASMT1 (Fig. 1A). The phylogenetic tree showed that MzASMT1 has the closest relation with rice ASMT1 than other O-methyltransferase found in apple and cyanobacteria. In the 24-hr light/dark cycle, the highest light intensity happened at 14:30. A total of two melatonin peaks occurred in apple leaves. One peak was at 14:30 and the other occurred at 5:30 in the early morning (Fig. 2A). As to the changes on MDA, there were also ‘dual peaks’ in leaves, but they occurred in advance of the melatonin peaks. This suggested that the oxidative stress might induce the biosynthesis of melatonin in leaves (Fig. 2B). To investigate the role of MzASMT1 in melatonin biosynthesis, RT-PCR was used to detect the expression of MzASMT1. The results showed that the expression of 413
Zuo et al. (A)
(B)
MzASMT1 was in good agreement with melatonin content. It preceded melatonin change in the 24-hr light/dark cycle. It was apparent that when the apple seedlings were under drought stress, the expression of MzASMT1 was induced. The MzASMT1 expression after 8-hr drought treatment was much higher than that of control (Fig. 3). This suggests that MzASMT1 positively responses to drought stress. When transient transformation mediated by agrobacterium GV3101 with GFP-MzASMT1 driven by UBQ promoter occurred in tobacco leaves, GFP-MzASMT1 was observed to be located in cytoplasm as observed in rice ASMT and sheep serotonin N-acetyltransferase [45, 46] when examined with the confocal microscope (Fig. 4). The MzASMT1 fused with GST was purified from E. coli by GST tag. After the cocultivation of the ASMT1 protein with its substrates (S-adenosyl-L-methionine and N-acetylserotonin), melatonin was detected by HPLC. This proved that MzASMT1 could synthesize melatonin in vitro with enzyme activity of around 5.5 pmol/min/mg protein. A total of three single insertional homozygous transgenic lines with high expression of MzASMT1 gene were selected for melatonin detection (Fig. 5D). The melatonin levels in the three lines were 68.82 ng/g, 90.19 ng/g, and 98.63 ng/g FW, respectively, which were 2–4 times higher than wild type (22 ng/g FW). The highest amount (98.63 ng/g FW) was almost four times higher than that in the wild type (22 ng/g FW) (Fig. 5C). HPLC spectra of melatonin detection were shown in Fig. 5A,B. On the medium, the three transgenic Arabidopsis lines that developed more lateral roots and fresh weight were more tolerant to drought stress than wild type after 7-days’ drought treatment (Fig. 6A,B). Coincidentally, the transgenic Arabidopsis plants were also more tolerant to drought stress than wild type in soil. It is shown in Fig. 6C; thus, more wild-type plants wilted compared with transgenic Arabidopsis after 12-days’ drought treatment. After re-watering, the transgenic Arabidopsis plants exhib414
Fig. 7. The ROS levels in wild type and transgenic Arabidopsis overexpressing MzASMT1 under drought stress. (A) The ROS levels presented by green fluorescence in the leaves of wild type and the transgenic Arabidopsis overexpressing MzASMT1 under drought stress in soil. More ROS accumulated in the leaf trichomes of wild type than in the transgenic plant. (B) The green fluorescence shows the ROS levels in wild type and the transgenic Arabidopsis plants under drought stress on medium containing in 300 mM D-mannitol growth medium. The wild-type plants emitted stronger fluorescence than three transgenic lines. The fluorescence images were obtained with a Leica stereoscope (Leica Microsystems) (910).
Fig. 8. The IAA contents of the aerial part of 1-month-old transgenic line 3 and wild-type plants. The data are means S.D. of triplicate experiments. Asterisks (*) indicate significant differences from the control (P < 0.05).
ited renewed growth, while some wild-type plants died. Obviously, overexpression of MzASMT1 enhanced the resistance against drought stress. The ROS levels in wild type and the transgenic Arabidopsis overexpressing MzASMT1 were detected when they were under drought. The fluorescent dye 5-(and 6)-chlo0 0 romethyl-2 -7 -dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA) was used to visualize in vivo ROS in leaves of plants growing in soil and aerial parts of the plants growing on medium under drought stress. In the leaves of the stressed plant in soil, the ROS accumulation in leaf trichomes was observed. The wild type obviously accumulated more ROS than the transgenic Arabidopsis plants under drought stress. Only few positive leaf trichomes were observed in transgenic leaves, while many of the leaf trichomes of wild type exhibited a bright green fluorescence under drought stress (Fig 7A). Coincidentally, in the case of the aerial part of the plants growing on medium for drought treatment, the wild type also produced more ROS than did the transgenic plants. The wild-type plants had very bright green color, while the transgenic plants had only a weak signal (Fig. 7B). But we failed to dye the leaf of Arabidopsis plants growing in soil, probably because the structures of the mature plants prevent the dyes from entering into the leaves. These results confirmed
MzASMT and melatonin in drought tolerance that the increased melatonin resulting from MzASMT1 overexpression effectively scavenges ROS under abiotic stress. We measured the IAA contents in the aerial parts of the transgenic and the wild-type plants with three repeats. The IAA levels in wild type were respectively 103.92 ng/g FW, 114.09 ng/g FW, and 100.45 ng/g FW. By contrast, the IAA contents in transgenic plants were 63.47 ng/g FW, 74.44 ng/g FW, and 88.17 ng/g FW. The results shown in Fig. 8 are the average values. It showed a significant difference between the transgenic plants and the wild type in IAA levels. The IAA content in aerial parts of transgenic plants (75.36 ng/g FW) was just 70% that of the wild type (106.15 ng/g FW) (Fig. 8).
increased melatonin production could result in reduced synthesis of IAA. There was no significant height difference between transgenic plants and wild type. This has been reported by Wang et al. [44] in transgenic tomato overexpressing oASMT gene. Collectively, the results indicate that MzASMT1 may be a key factor in regulating in the response of plants against abiotic stressors by elevating melatonin production in plants.
Acknowledgements Our research was supported by 973 Program (National Basic Research Program of China, Project No: 2012CB114200) and the 863 Project (Project No: 2011AA100204).
Discussion Many studies have shown that exogenously applied melatonin effectively enhances the capacity of plants to tolerate unfavorable environmental insults. These include heat [38, 39], drought [16], cold [31, 32], excessive water [47], long periods of dark exposure [48–50], UV irradiation [17, 37, 51], and chemical pollutants [33, 52]. Several studies have found that transgenic plants overexpressing animal SNAT or ASMT also improve their tolerance to abiotic stressors [44, 53, 54]. To date, little is known about the outcomes of transgenic plants overexpressing plant melatonin synthetic genes. In the current study, an apple ASMT gene (MzASMT1) was cloned for this purpose. We observed that MzASMT1 was drought inducible in apple leaves. This implies that MzASMT1 may be a candidate for the regulation of melatonin synthesis under drought conditions. The phylogenetic analysis shows that MzASMT1 is more closely related to the ASMT of rice rather than to the other O-methyltransferase in apple. This suggests that the MzASMT1 is an ortholog of rice ASMT despite the low amino acid identity (39.7%). Similar to the rice ASMT, MzASMT1 was also identified to be distributed in the cytoplasm. To further understand the physiological role of MzASMT1 in the stress response, it was transferred into Arabidopsis. It was found that the transgenic Arabidopsis overexpressing MzASMT1 produces 2- to 4-fold higher levels of melatonin than those of wild type. Accordingly, the transgenic plants were found to have a greater tolerance to drought than the wild type. The mechanistic exploration unambiguously proved that the elevated drought tolerance in transgenic Arabidopsis is directly associated with the anti-oxidant capacity of melatonin. Transgenic Arabidopsis have significantly lower levels of ROS and higher levels of melatonin than does the wild type under drought (Fig. 7). The injuries in plants caused by drought are mainly mediated by oxidative stress [55]. Scavenging ROS and reducing oxidative stress are key factors to enhance the tolerance of plants to abiotic stressors [56]. Melatonin is an important anti-oxidant to reduce oxidative stress and protect plants from drought stress. In the current study, the levels of indole 3-acetic acid (IAA) of transgenic Arabidopsis plants were lower than that of wild type. This is not unexpected as IAA and melatonin share the same precursor. Due to substrate competition,
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