Herbaceous peony tryptophan decarboxylase confers drought and salt stresses tolerance

Environmental and Experimental Botany 162 (2019) 345–356

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Herbaceous peony tryptophan decarboxylase confers drought and salt stresses tolerance Daqiu Zhao, Xiayan Zhang, Rong Wang, Ding Liu, Jing Sun, Jun Tao

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Jiangsu Key Laboratory of Crop Genetics and Physiology, College of Horticulture and Plant Protection, Yangzhou University, Yangzhou 225009, PR China

A R T I C LE I N FO

A B S T R A C T

Keywords: Herbaceous peony Melatonin Tryptophan decarboxylase Drought Salt

Herbaceous peony (Paeonia lactiflora Pall.) is known as the king of herbaceous flowers. Drought stress seriously restricts P. lactiflora growth and reduces its ornamental value, but little is known about its underlying mechanism. In this study, one tryptophan decarboxylase gene (TDC), which is the first enzyme gene for melatonin biosynthesis, was obtained, and its expression level was found to be positively related to melatonin production during drought stress. In P. lactiflora, the first TDC that was isolated was comprised of 1849 bp with no intron, and was designated as PlTDC (KY765554). It encoded a 502-amino acid protein with a molecular weight of 56 kDa, which was localized in the cytoplasm of cells and catalyzed the reaction converting tryptophan into tryptamine. When PlTDC was transferred into tobacco, the transgenic plants produced approximately 1.67-fold higher levels of melatonin than that of wild-type control. Moreover, the transgenic plants had enhanced tolerance to drought and salt stresses, potentially due to the reduction in hydrogen peroxide (H2O2) and superoxide anion free radical (O2%−) accumulation. In addition, the enhanced H2O2 and O2%− scavenging under drought stress led to decreased cell membrane damage, increased potential photosynthetic capacity, and delayed plant senescence. RNA-Seq data correlated well with the protective role of melatonine because it was found an increase of gene expression involved in the oxidation-reduction process and photosynthesis. These results together suggested that PlTDC was a key factor in increasing melatonin production, to confer drought and salt stress tolerance in P. lactiflora.

1. Introduction Melatonin (N-acetyl-5-methoxytryptamine) is an important indoleamine compound that is widely present in almost all organisms. Since first being detected in plants in 1995 (Dubbels et al., 1995; Hattori et al., 1995), melatonin has been found in a number of plant species, and is now considered a major health-promoting natural substance because of its important roles in delaying senescence and antioxidant activity (Wang et al., 2012; Park et al., 2013). Additionally, melatonin plays key roles in plant development, including fruit ripening (Xu et al., 2018), root growth (Zhang et al., 2014), flowering (Kolář et al., 2003), leaf senescence (Liang et al., 2015), and plant stress defense, by improving resistance to temperature and drought (Bajwa et al., 2014; Antoniou et al., 2017), enhancing salt tolerance (Li et al., 2017), alleviating damage from heavy metal (Li et al., 2016), protecting plants from UV-B radiation (Afreen et al., 2006), improving their defenses against pathogen infection (Zhao et al., 2015), and so on. These physiological functions are being intensively explored in plants using exogenous melatonin treatment, but this method has to be judged with

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caution as a pharmacological dose of melatonin does not necessarily reflect the physiological levels of endogenous melatonin (Reiter et al., 2016). To expedite the unraveling of unknown functions of melatonin in plants and compensate for the disadvantages of pharmacological studies, molecular genetic approaches are essential. In plants, the melatonin biosynthetic pathway is well documented as follows: firstly, tryptophan is decarboxylated to tryptamine by tryptophan decarboxylase (TDC, E.C. 4.1.1.28); subsequently, tryptamine 5-hydroxylase (T5H, E.C. 1.14.16.4) hydroxylates the C-5 position of tryptamine to form serotonin; next, serotonin is converted via serotonin N-acetyltransferase (SANT, E.C. 2.3.1.87) into N-acetylserotonin; and finally, Nacetylserotonin-O-methyltransferase (ASMT, E.C. 2.1.1.4) or hydroxyindole-O-methytransferase (HIOMT, E.C. 2.1.1.4) executes the final formation of melatonin (Back et al., 2016). Among these enzymes, TDC serves as the first rate-limiting enzyme in melatonin biosynthesis (Zhao et al., 2013), and since a cDNA clone encoding TDC was first isolated from Catharanthus roseus (De Luca et al., 1989), many TDC genes from a range of plant species have been isolated, including for Camptotheca

Corresponding author. E-mail address: [email protected] (J. Tao).

https://doi.org/10.1016/j.envexpbot.2019.03.013 Received 18 January 2019; Received in revised form 14 March 2019; Accepted 14 March 2019 Available online 15 March 2019 0098-8472/ © 2019 Elsevier B.V. All rights reserved.

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peroxide (H2O2), superoxide anion free radical (O2%−), and relative electric conductivity (REC). In addition, soil water content was also determined.

acuminata (López-Meyer and Nessler, 1997), Ophiorrhiza pumila (Yamazaki et al., 2003), Withania coagulans (Jadaun et al., 2017), and Mitragyna speciosa (Charoonratana et al., 2013). Its expression pattern demonstrates that it is closely related to melatonin biosynthesis (Lei et al., 2013; Zhao et al., 2013), and the ectopic overexpression in Oryza sativa results in the increase of tryptamine and serotonin levels (Kang et al., 2007; Kanjanaphachoat et al., 2012). Moreover, transgenic O. sativa plants overexpressing O. sativa TDC3 not only have 31-fold elevated melatonin, but also enhanced levels of melatonin intermediates, including 5-hydroxytryptophan, tryptamine, serotonin, and N-acetylserotonin in seeds (Byeon et al., 2014). Inversely, TDC-overexpressing transgenic plants with elevated melatonin production showing significant tolerance to any type of stress have not been observed until now. Herbaceous peony (Paeonia lactiflora Pall.) is known to be elegant and gorgeous as tree peony (Paeonia suffruticosa Andr.), and is known as the king of herbaceous flowers. Because of its huge, brightly coloured and fragrant flowers, P. lactiflora is regarded as one of the most rewarding plants to grow, and is mainly used for urban landscaping, garden cultivation, potted admiration, and as a high-grade cut flower worldwide (Holloway and Buchholz, 2013). P. lactiflora is resistant to drought, but soil with excessively insufficient water inhibits its growth and reduces its ornamental value, especially in the case of potted P. lactiflora, which cannot absorb water from the soil directly and thus is more vulnerable to drought stress. However, until now, few studies had preliminarily clarified the physiological mechanism underlying drought stress (Wang et al., 2014), and little is known about its underlying molecular mechanisms, which makes breeding drought-resistant P. lactiflora varieties impossible by means of plant genetic engineering. Melatonin has important defense-related functions, as mentioned above, and several transgenic plants expressing plant melatonin biosynthetic genes have been demonstrated to have enhanced tolerance to drought stress. For example, overexpression of Malus zumi SNAT5 and ASMT in Arabidopsis has been reported to elevate the melatonin level, and confer drought stress tolerance in transgenic plants (Zuo et al., 2014; Wang et al., 2017). Here, only one TDC was identified from previous transcriptomic database of P. lactiflora under drought stress (SRA: SRP131648). Its expression pattern and melatonin level revealed that it was a close model for drought stress in P. lactiflora. Moreover, the full-length of the TDC cDNA was isolated, and then its subcellular localization in O. sativa protoplasts, heterologous expression in Escherichia coli, and enzymatic activity were investigated. Furthermore, because P. lactiflora’s genetic regeneration system has not yet been established to date, we generated transgenic tobacco plants that overexpressed P. lactiflora TDC to compare the melatonin levels, and systematically study the responses of transgenic plants to multiple abiotic stresses including drought and salt. Our data suggested that P. lactiflora TDC was a key factor in improving melatonin production and conferring drought and salt stress tolerance, which could lay a theoretical foundation for breeding drought-resistant P. lactiflora varieties.

2.2. Isolation and bioinformatics analysis of PlTDC Total RNA was extracted using a MiniBEST Plant RNA Extraction Kit (TaKaRa, Japan). The full-length cDNA of TDC was isolated by rapidamplification of cDNA ends (RACE) technology, which was performed using a 3′ full RACE Core Set Ver. 2.0 (TaKaRa, Japan) and SMARTer™ RACE cDNA Amplification Kit (Clontech, Japan) with gene-specific primers (Supplementary material Table S1). The PCR product was subjected to agarose gel electrophoresis to separate it, and then sent to be sequenced. Total DNA was extracted using a MiniBEST Plant Genomic DNA Extraction Kit (TaKaRa, Japan). Genomic DNA sequence of TDC was obtained according to our previous study, with gene-specific primers (Supplementary material Table S1) (Zhao et al., 2019a). DNAMAN 5.2.2 was used for sequence comparison and MEGA5.05 was used to construct a phylogenetic tree. 2.3. Subcellular localization of PlTDC The subcellular localization of PlTDC was determined by confocal laser microscopy (LSM510/ConfoCor2, Zeiss, Germany) in O. sativa protoplasts. The p35S:PlTDC-GFP gene fusions were constructed to obtain the PlTDC product. The open reading frame (ORF) of PlTDC was amplified by primers containing BsaI/Esp3I restriction sites (forward 5′CAGT GGTCTCACAACATGGGTAGTCTTGAACATCC-3′, reverse 5′-CAG TCGTCTCATACAAC ACCCCTTGAGTATTTTAT-3′), which was digested and the product ligated into the expression vectors with T4 DNA ligase (TaKaRa) to generate a set of pBWA(V)HS-PlTDC-GFP fusions. These were subsequently sequenced for verification, and the specific operations of the transformation of p35S:PlTDC-GFP observed according to our previous study (Zhao et al., 2019a). 2.4. Heterologous expression of PlTDC in E. coli and purification of its protein The expression plasmid of PlTDC was constructed. The full-length of PlTDC was amplified by primers containing NotI/CpoI restriction sites (forward 5′-GATCCGGTCCGATGGGATTATCTTTTTACAAAAACAA TTT-3′, reverse 5′-TGCAGCGGCCGCTTAACACCCCTTGAGTATTTTATC TACTC-3′) and ligated into the pET-sumo vector using the Champion™ pET SUMO Expression System (Thermo Fisher Scientific, USA). The ligation (pET-PlTDC-sumo) mixture was chemically transformed into E. coli Top 10 competent cells and inoculated in LB medium (50 μg/mL Kanamycin sulfate). After 16 h of growth at 37 °C, the single colony recombinant plasmid was inoculated in 3 mL LB (50 μg/ mL kanamycin sulfate). This growth was also carried out at 37 °C for 16 h, and isopropyl-β-D-thiogalactopyranoside (IPTG) was added at an OD600 of 0.6 until the final concentration was 1.0 mM. Growth continued for 3 h at 37 °C, and then 0.15 mL of the solution was centrifuged at 12,000 × g for 120 s. The pellet was collected and re-suspended in 40 μL 1 × loading buffer, and detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In addition, 100 μL recombinant plasmid was inoculated in 100 mL LB (50 μg/mL Kanamycin sulfate) and shake cultured. After one night, 100 μL of the solution was inoculated in 2000 mL LB and allowed to grow at 37 °C until OD600 = 0.6. IPTG was then added to a final concentration of 0.1 mM, and the culture temperature was adjusted to 30 °C. After 8 h, it was centrifuged at 8000 x g for 180 s. The pellet was then re-suspended in 50 mL of NTA-0 buffer and ice bathed for 30 min. Ultrasonic treatment was then performed at 200 W for 30 min, with pulse on/off pauses of 3 × 4 s. The disrupted cells were centrifuged at 16,000 × g for 50 min at 4 °C, and the supernatant was collected and detected by SDS-PAGE. Finally, the

2. Materials and methods 2.1. Drought stress treatment of P. lactiflora Five-year-old P. lactiflora ‘Da Fugui’ in potting soil (loam:peat:coarse sand, 1:1:1) were obtained from the germplasm repository of Horticulture and Plant Protection College, Yangzhou University, Jiangsu Province, China (32°30′ N, 119°25′ E). The above plants were used as experimental materials and the water was permeated, and then the plants were divided into two groups. One group was watered daily as the control and the other group was subjected to natural drought treatment (Fig. 1). After determination of photosynthetic parameters and chlorophyll fluorescence parameters, all samples were collected on 0, 7, 14 and 21 days after treatment. The samples were used for measurement of other indices, including leaf water content, hydrogen 346

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Fig. 1. Phenotype of P. lactiflora under drought stress. Plants were watered daily as the control and the treatment was subjected to natural drought during July and August 2017.

2.7. Abiotic stress treatment for PlTDC transgenic tobacco

purification of PlTDC protein was subsequently performed according to the method of our previous study (Sun et al., 2018).

Tobacco cultivation soil was prepared with a ratio of 1:1 of vermiculite and nutrient soil. The transgenic tobacco plants and wild type were cultivated for 3 months before being used for abiotic stress treatment. The temperature of the drought stress treatment was 22 °C, the light condition was a 10 h light / 14 h dark cycle, and then drought stress was carried out for 18 days in the absence of water. The treated plants were first subjected to chlorophyll fluorescence parameter measurement, anatomical observation, and determination of relevant physiological indicators including H2O2, O2%−, REC and water content. Immediately after the measurement, the leaves were stored at −80 °C with liquid nitrogen to facilitate subsequent measurement of melatonin content, RNA-Seq and gene expression analysis. RNA-Seq and data analysis of tobacco were performed by Novogene Co. (Beijing, China). The differentially expressed genes (DEGs) were defined by a fold change of ≥2.0 and an adjusted P-value of ≤ 0.05. These DEGs were validated by quantitative real-time PCR (qRT-PCR) with gene-specific primers (Supplementary material Table S2). The confirmed DEGs were subjected to GO functional analysis. In the salt stress treatment, leaf discs were removed from healthy wild-type and transgenic tobacco plants using a puncher and the leaf discs were placed in 0 mM, 200 mM, 400 mM, 600 mM and 800 mM NaCl solutions for 36 h. These leaf discs were used for phenotype, H2O2 and O2%− accumulation observation.

2.5. Assay for PlTDC activity PlTDC activity was detected using high performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC–MS/MS) (Agilent 1200-6460 QQQ, USA). Firstly, 1 mL of the reaction mixture contained 100 mM Tris−HCl (pH 8.0), 1 mM pyridoxal 5′-phosphate, 3 mM dithiothreitol, 1 mM L-tryptophan and 0.01 mg PlTDC protein. The mixture was kept at 37 °C for 1 h and then terminated by adding 2 mL methanol. The filtrate was passed through 0.22 μm membrane filters (Shanghai ANPEL Scientific Instrument Co., Ltd., China). The HPLC column was Agilent C18 (2.1 mm × 150 mm, 5 μm) with a flow rate of 0.3 mL/min. The mobile phase consisted of acetonitrile as solvent A, and 0.1% aqueous formic acid as solvent B (0.1:99.9; v / v, HCOOH : H2O). The linear gradient profile was 5% A at 0 min, 95% A at 5 min, 95% A at 6 min, 5% A at 7 min, and then returned to 5% A at 15 min. The analysis conditions of the mass spectrometry multi-reactions monitoring (MRM) technology were as follows: parent ion of tryptamine, 161.2; daughter ion of tryptamine, 144.1; positive ionization mode; gas temperature, 300 °C; gas flow rate, 10 L/min; nebulizer pressure, 15 Pa; sheath gas temperature, 250 °C; sheath gas flow rate, 7 L/min; capillary voltage, 4.0 Kv (positive mode) and 3.5 Kv (negative mode).

2.8. Melatonin content measurement 2.6. Overexpressing PlTDC in transgenic tobacco Melatonin was detected according to the guidelines of an ELISA kit (Shanghai Qiaodu Biotechnology Co., Ltd., China), and its content was obtained by a SpectraMax M5 plate reader (Molecular Devices Corporation, USA). The specific operations were carried out according to our previous study (Zhao et al., 2018). Firstly, equilibrate the kit for 20 min at room temperature, then add 10 μL of the sample to be tested to the sample well and add 40 μL sample10-fold dilution. Secondly, 100 μL of horseradish peroxidase (HRP)-labeled detection antibody was added to the sample well, and the reaction well was sealed with a sealing plate and a water bath at 37 °C for 60 min. Discard the liquid, pat dry on absorbent paper, and repeat the plate wash 5 times. Then, add 50 μL of the substrates A and B to each well, and incubate at 37 °C for 15 min in the dark. Finally, after incubation, 50 μL of stop solution

The ORF sequence of PlTDC was amplified with primers which included BamHI/KpnI restriction sites (forward 5′-CGCGGATCCATGGGT AGTCTTGAAC-3′, reverse 5′-GGGTACCCTAACACCCCTTGAGTAT-3′). This sequence was ligated into the expression vector pCAMBIA1301 (the vector was located after the cauliflower mosaic virus (CaMV) 35S promoter). The pCAMBIA1301-PlTDC plasmid was introduced into the Agrobacterium tumefaciens strain EHA105 via the freeze-thaw method. The strains were transformed into tobacco (Nicotiana tabacum ‘k236’) using a leaf disc transformation method. The T0-generation of the transformed plants was identified by PCR and qRT-PCR. Wild type (WT) and transgenic tobacco were cultured under the same conditions, and phenotypic differences were observed. 347

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fluorescence parameters, respectively. Malonaldehyde (MDA) content was determined by a kit (Nanjing Jianshe Bioengineering Co., Ltd.), using the sodium thiobarbital (TBA) method. Proline (Pro) content was determined by a colorimetric method using a kit (Nanjing Jianshe Bioengineering Co., Ltd.), and the specific operation depended upon the specification. 2.10. Anatomy observation The surfaces of leaves were observed by the environmental scanning electron microscopy (Philips XL-30 ESEM, Holland), and the specific operations were conducted according to the method of our previous study (Zhao et al., 2019b). 2.11. PCR and gene expression analysis To identify the transformed plants, tobacco leaves were used to extract total RNA and DNA, and the cDNA was synthesized using a PrimeScript® RT reagent Kit with gDNA Eraser (TaKaRa, Japan). PCR and qRT-PCR were used to identify the transformed plants and gene transcript levels, and the specific operations were performed according to the method of our previous study, with gene-specific primers (Supplementary material Table S3) (Zhao et al., 2019a). 2.12. Statistical analysis All experiments were repeated three times and randomly arranged, with all data being the average of three replicates with plus standard deviation. The variance was analyzed by the SAS / STAT statistical analysis package (version 6.12, SAS Institute, Cary, NC, USA). Fig. 2. Relative expression level of TDC and melatonin content of P. lactiflora under drought stress. The values represented the mean ± SD, and different letters indicate significant differences according to Duncan’s multiple range test (P < 0.05).

3. Results 3.1. Cloning and characteristics of PlTDC Based on the previous transcriptomic database of P. lactiflora under drought stress (SRA: SRP131648), we found only one TDC gene. To identify whether TDC plays an important role in protecting P. lactiflora from drought stress, its expression pattern and downstream melatonin production were analyzed (Fig. 2). During drought stress, the relative expression level of TDC was positively related to melatonin production in P. lactiflora. They all presented the same tendency, increasing first and then decreasing, and peaking on day 7. Meanwhile, the physiological changes of P. lactiflora in response to drought stress were analyzed. Analysis of leaf oxidative damage revealed that REC and MDA were significantly enhanced by 82.87% and 160% (on day 21) respectively, in response to drought stress. Pro content was also significantly increased under drought stress, especially on day 21 when it was enhanced by 13 times compared with the control (Fig. 3A). In comparison with the control, H2O2 and O2%− accumulated dramatically and were significantly higher under drought stress, especially on day 21 (Fig. 3B, C). These results were relative to the TDC expression pattern and melatonin level. On this basis, the full-length cDNA of the TDC gene was successfully isolated from P. lactiflora by RACE technology. The 1849 bp TDC cDNA contained an untranslated region (UTR) of 53 bp at the 5′ end, a 1515 bp ORF encoding a 504 amino acid protein, a 3′-UTR of 281 bp and a poly (A) tail. The genomic DNA sequence of TDC was amplified by the PCR method, but no intron was identified in this gene (Supplementary material Fig. S1). Amino acid sequence comparison revealed that this protein shared more than 75% of its identity with TDC from C. acuminata (AON76721), O. prostrata (ABU40982), O. pumila (BAC41515), M. speciosa (AEQ01059), Tabernaemontana elegans (AEY82396), Rauvolfia verticillata (ABP96805), Vinca minor (AEY82397) and Hordeum vulgare (AB162961), and it also contained a highly conservative sequencebinding domain, the DDC/GAD/HDC/TyrDC pyridoxal 5′-phosphate

was added to each well, and then placed in a microplate reader to measure the OD value of each well at a wavelength of 450 nm. 2.9. Physiological indices measurement Diaminobenzidine (DAB) staining was used to detect hydrogen peroxide (H2O2) accumulation (Tian et al., 2013). The leaves were immersed in 0.1 mg/mL DAB in 50 mM Tris-acetate buffer (pH 5.0), and placed in a dark environment at 25 °C. After 24 h, the sample was taken out and boiled in 95% (v/v) ethanol for 15 min or more, and then photographed using a camera (Canon 50D, Japan). Superoxide anion free radical (O2%−) accumulation was detected by a reagent kit (Shanghai Haling Biotechnology Co., Ltd., China). A total of 100 μL of the cleaning solution (Regant A) was dropped on the slide, and the blade placed on the filter paper was quickly cut by two stainless steel double-sided razor blades (Gillette) to avoid the main vein. A sample of the blade was dipped between the two blades with a fine brush, and placed it on the slide. After replacing the cleaning solution, 10 μL of the fluorescent dye dihydrobromide (DHE) was added. Then, the samples were placed in a 37 °C incubator for 20 min, DHE was removed, and the samples were cleaned with a cleaning solution. The samples were imaged with a fluorescent microscope at a 540 nm excitation wavelength and 590 nm emission wavelength (Axio Imager D2, ZEISS, Germany). ZEN software (ZEISS, Germany) was used to gather the fluorescent signals. Relative electrical conductivity (REC) was determined according to the method in our previous study (Yang et al., 1996). In addition, a portable photosynthesis system (Li-Cor LI-6400, USA) and a chlorophyll fluorescence spectrometer (Heinz Walz GmbH 91090 Effeltrich, Germany) were used to measure photosynthetic and chlorophyll 348

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Fig. 3. Physiological changes of P. lactiflora under drought stress. (A) Relative electrical conductivity (REC), malonaldehyde (MDA) and proline (Pro) contents. (B) H2O2 accumulation was detected by DAB staining. The deeper brown showed in leaf, the more H2O2 accumulated. (C) O2%− accumulation was detected by a fluorescence probe. The higher fuorescence intensity showed in section, the more O2%− accumulated. The values represented the mean ± SD, and different letters indicate significant differences according to Duncan’s multiple range test (P < 0.05).

Fig. 4. Subcellular localization of PlTDC in rice protoplasts. GFP excitation and emission wavelengths were 480 nm and 510 nm, respectively.

(Fig. 4). Meanwhile, heterologous expression of the recombinant PlTDC plasmid (pET-PlTDC-sumo rose) was constructed and transformed in E. coli cells. The recombinant E. coli cells were induced using IPTG, the cells were harvested, and the protein was isolated. The soluble fractions from the recombinant strains differed from those of non IPTG-induced pET-PlTDC-sumo rose and the empty vector of pET-sumo rose. The apparent molecular weight of the PlTDC protein plus the GST-Tag protein (16 kDa) was approximately 72 kDa, thus, the molecular weight of the PlTDC protein was approximately 56 kDa (Fig. 5A). Following a western blot experiment, the results also showed that the size of the PlTDC

attachment site (SLSLSPHKWLLSYLDCCCLWVK) (Supplementary material Fig. S2A). The phylogenetic tree showed that these TDC from plants could be classified into monocotyledon and dicotyledon, and C. acuminata was the most similar to P. lactiflora (Supplementary material Fig. S2B). These results were consistent with the traditional classification. Therefore, this sequence could be confirmed in the TDC gene in P. lactiflora, which was appointed to PlTDC with the GenBank accession number KY765554. Subsequently, subcellular localization of PlTDC was observed. When the transient transformation with PlTDC-GFP, driven by the 35S promoter, occurred in O. sativa protoplasts, PlTDC-GFP fluorescence showed it was localized in the cytoplasm of the cells

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Fig. 5. Heterologous expression of PlTDC and its catalytic activity. (A) Heterologous expression of PlTDC protein in E. coli. M and 3: Protein marker; 1: IPTG induced pET-PlTDC-sumo rose; 2: Non IPTG-induced soluble fraction of E. coli pET-PlTDC-sumo rose; 4: Empty vector of pET-sumo rose. The gel corresponds to protein staining with coomassie blue. (B) Western Blot was carried out and detected by HIS antibody. M: Protein marker; 1: PlTDC protein; 2: control of HIS antibody. HIS antibody dilution was 1:1000. (C) Western Blot was carried out and detected by sumo antibody. M: Protein marker; 1: PlTDC protein. sumo antibody dilution was 1:1000. (D) HPLC chromatograms of tryptamine and the resulting product of PlTDC assay.

control (Fig. 7B). Additionally, O2%− accumulated dramatically in the transgenic lines, as shown by the significant increase in fluorescence intensity in transgenic tobacco leaves compared to the wild-type control (Fig. 7C). Moreover, REC, leaf water content and Fv/Fm were determined. Among them, transgenic tobacco lines REC was decreased, whereas water content and Fv/Fm were both increased compared with the wild-type control, and significant differences of these three indices were all detected between wild-type and transgenic tobacco lines (Fig. 7D). Additionally, the expression levels of related protective enzyme genes and senescence-associated genes were examined. Protective enzyme genes, including the superoxide dismutase [Cu-Zn] gene (Cu/ ZnSOD), peroxidase gene (POD), CAT and ascorbate peroxidase gene (APX), were highly expressed in the transgenic lines, whereas senescence-associated genes including senescence-associated gene 21 (SAG), harpin inducing protein 18 gene (HIN1), cysteine protease 1 gene (CP1) and cysteine protease 2 gene (CP2) had low expression in the transgenic lines. There was a significant difference in the expression levels of the tested genes between wild-type and transgenic tobacco lines, except POD and CP2 (Fig. 7E). Obviously, overexpression of PlTDC enhanced the resistance against drought stress by enhancing melatonin production. Additionally, the microstructure of the leaf surface was observed by environmental scanning electron microscopy, and the opening degree of stomata of transgenic lines was higher than that of wild-type plants (Fig. 8). To systematically analyze the contribution of the enhanced melatonin production to the resistance against drought stress, RNA-Seq was performed. Compared with transcripts of wild-type plants, transgenic tobacco lines had 1428 DEGs with alterations of twofold or more, including 85 up-regulated DEGs and 613 down-regulated DEGs (Fig. 9A), and these results were validated by qRT-PCR (Fig. 9B). Subsequently, DEGs were assigned to GO categories. For up-regulated DEGs, obvious over-representation was observed for the oxidation-reduction process in the biological process category (Fig. 9C, Supplementary material Table S4). The oxidation reduction process, with 78 DEGs, was dominant in

band was approximately 72 kDa, by HIS antibody (Fig. 5B) and sumo antibody (Fig. 5C). Further, it was demonstrated that the purification of the PlTDC protein was successful. The PlTDC protein was incubated with tryptophan as a substrate and pyridoxal 5′-phosphate as a cofactor. As shown in Fig. 5D, the PlTDC protein catalyzed the reaction, converting tryptophan into tryptamine. 3.2. Functions of PlTDC on conferring drought and salt stresses tolerance To further investigate whether PlTDC could improve tolerance to drought stress by enhancing melatonin production, a 35S:PlTDC vector was constructed and introduced into tobacco plants via A. tumefaciensmediated transformation. Polymerase chain reaction (PCR) analysis of PlTDC mRNA confirmed the homozygous transgenic tobacco lines contained PlTDC. PlTDC mRNA was expressed constitutively in the transgenic tobacco lines, but not in the control WT plant (Fig. 6B). Additionally, qRT-PCR analysis revealed that the transgenic lines had significantly higher transcript levels of PlTDC, which was on average 856 times that of wild-type plant (Fig. 6D). However, no visible phenotypic differences were observed between wild-type and transgenic tobacco lines under the same culture conditions (Fig. 6A). Subsequently, their melatonin content was detected, and the results indicated that it was in good agreement with the expression of PlTDC in wild-type and transgenic tobacco lines. The melatonin content in transgenic the tobacco lines was 2435.17 ng/g FW, 2593.29 ng/g FW and 2708.25 ng/ g FW, respectively, which was approximately 1.67-fold higher than in the wild-type control (1536.78 ng/g FW) (Fig. 6C). Subsequently, the wild-type and transgenic tobacco plants were stressed by drought without water for 18 d, The wild-type plant wilted, whereas the transgenic tobacco lines exhibited normal growth (Fig. 7A). H2O2 content showed significant differences between wildtype and transgenic tobacco leaves, and a light color was observed in the transgenic tobacco leaves compared to the wild-type control, suggesting the accumulation of H2O2 was considerably in the wild-type 350

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Fig. 6. Melatonin content and relative expression level of PlTDC in wide-type and transgenic tobacco lines. (A) Phenotype of wide-type and transgenic lines. (B) PCR analysis of PlTDC mRNA. (C) Melatonin content of leaves. (D) Relative expression level of PlTDC in leaves. WT: wild-type. The values represented the mean ± SD, and different letters indicate significant differences according to Duncan’s multiple range test (P < 0.05).

melatonin might play an important role in the H2O2 and O2%− scavenging process. Moreover, quite a number of DEGs responsible for chloroplast structure (thylakoid, thylakoid part, thylakoid membrane) and photosynthesis (photosynthesis, photosystem, photosynthetic membrane, photosystem II, photosystem II oxygen evolving complex,

the main category of biological processes (Fig. 9D, Supplementary material Table S5), while 28 and 8 DEGs, respectively, were involved in oxidoreductase activity. The oxidoreductase complex was dominant in the main categories of molecular function and cellular components. The DEGs associated with oxidation/reduction activity implied that

Fig. 7. Effect of drought stress on wide-type and transgenic tobacco lines overexpressing PlTDC. (A) Phenotype of wide-type and transgenic lines. (B) H2O2 accumulation was detected by DAB staining. (C) O2%− accumulation was detected by a fluorescence probe. (D) Other physiological indices. (E) Relative expression levels of related protective enzyme genes and senescence-associated genes. Tobacco plants were stressed by drought without water for 18 d. WT: wild-type. The values represented the mean ± SD, and different letters indicate significant differences according to Duncan’s multiple range test (P < 0.05). 351

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Fig. 8. Effect of drought stress on stomatal characteristics in wide-type and transgenic tobacco lines overexpressing PlTDC. The lower scanning electron micrograph was partial enlargement in the upper micrograph marked by an arrow.

stress, and melatonin is a unique antioxidant that could directly scavenge free radicals (Su et al., 2018). Currently, melatonin is shown to be associated with drought stress: both exogenously applied melatonin and endogenously produced melatonin can enhance resistance to drought stress in plants (Zuo et al., 2014; Li et al., 2017; Wang et al., 2017; Gao et al., 2018; Liang et al., 2019). However, whether this factor is involved in the response to drought stress in P. lactiflora has yet to be identified. In present study, we found one TDC gene from the previous transcriptomic database of P. lactiflora under drought stress (SRA: SRP131648). Consistent with those earlier conclusions (Lei et al., 2013; Zhao et al., 2013), we found that TDC plays an important role in melatonin biosynthesis, according to its expression patterns and melatonin production in P. lactiflora. However, the relative expression level of TDC and melatonin production under drought stress presented first with an increase and then a decrease compared with the control, with a peak on day 7, and this result was basically consistent with TDC expression of two Malus species and TDC activity of C. roseus under drought stress (Zhang et al., 2012; Li et al., 2015). This might be because first mild drought stress could induce TDC expression and a large amount of melatonin synthesis to resist stress and relieve plants damage, but with increasing drought stress, H2O2 and O2%− were excessively produced when melatonin was quickly consumed and TDC expression was decreased sharply, which eventually resulted in plants damage and death. This implied that TDC may be a candidate for the regulation of melatonin synthesis, and be involved in protecting P. lactiflora from drought stress. To further validate this hypothesis, TDC was isolated from P. lactiflora by the RACE technique, based on RNA-Seq, which was the first study on P. lactiflora TDC to our knowledge. The full-length of PlTDC cDNA was 1849 bp, encoding 504 amino acids, which was basically similar to findings in Withania coagulans (Jadaun et al., 2017) and M. speciosa (Charoonratana et al., 2013). PCR of the genomic DNA sequence had confirmed that there was no intron in this gene. Heterologous expression of the PlTDC protein in E. coli suggested its molecular weight was 56 kDa, which was consistent with a previous report on W. coagulans (Jadaun et al., 2017). Sequence comparison and phylogenetic tree analysis indicated that this protein shared more than 75% of its identity with other plants, and contained a highly conserved sequencebinding domain, the DDC/GAD/HDC/TyrDC pyridoxal 5′-phosphate attachment site, which was used for decarboxylation of tryptophan through TDC. Catalytic activity analysis also confirmed PlTDC could

photosystem I, photosystem I reaction center) were upregulated, suggesting that melatonin was functional in these processes. Among those that were down-regulated, DEGs involved in response to inorganic substances (8 DEGs), response to water (5 DEGs), and response to acid chemicals (5 DEGs) were notable (Fig. 9C, Supplementary material Table S5). Moreover, the effect of salt stress on detached leaves from wild-type and transgenic tobacco lines was also observed. As shown in Fig. 10A, the transgenic tobacco lines were more tolerant to salt stress than the wild-type control after 36-h of NaCl solution treatment. When leaf discs were treated with 0 and 200 mM NaCl solutions, the wild-type and transgenic tobacco lines had similar phenotypes, in that we did not observe much of a difference in leaf colouration between the transgenic and untransformed lines showing green. When the concentration of NaCl solution was more than 200 mM, the damage to the wild-type leaf discs became gradually more serious, and the green area gradually become smaller with increasing concentration. At 800 mM, the wildtype leaf discs all turned brown. Conversely, the transgenic tobacco leaf discs only turned brown locally at 800 mM, and most of them remained green. When the concentration of the NaCl solution increased, H2O2 and O2%− accumulation increased, but it was apparent that their levels were lower in transgenic tobacco lines than in the wild-type control (Fig. 10B, C). 4. Discussion Because of the sessile nature of plants, they are exposed to particular environmental stress which animals can sometimes avoid. Thus, plants can suffer more stress-mediated oxidative damage (Zheng et al., 2017). P. lactiflora is stressed by adverse environments, such as drought conditions. Herein, Wang et al. (2014) considered that P. lactiflora damage caused by drought stress was associated with damaged cell membranes and increased membrane permeability. This speculation was supported by the findings in this study, in which REC, MDA, Pro, H2O2 and O2%− were significantly enhanced in response to drought stress. In this situation, an elaborate system of enzymatic and nonenzymatic antioxidants has evolved to protect plants against oxidative stress, including enzymatic (SOD, POD, CAT, APX and glutathione peroxidase) and nonenzymatic mechanisms (ascorbic acid, flavonoid, carotenoids, glutathione and vitamin E, etc.) (Ahmad et al., 2010). Besides these classic antioxidants, plants have other mechanisms to protect against 352

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Fig. 9. RNA-seq analysis of wide-type and transgenic tobacco lines overexpressing PlTDC under drought stress. (A) Volcano plot of DEGs. The X axis represents log2 transformed fold change, the Y axis represents −log10 transformed significance, the red points represent up-regulated DEGs, the blue points represent downregulated DEGs and the black points represent non-DEGs. (B) Correlation of RNA-Seq (y axis) and qRT-PCR data (x axis). The correlation assay is carried out for 20 DEGs with log2 ratios ≥ 1.00 or ≤ 1.00. (C) Histogram of gene ontology annotation. URGs: upregulated genes. DRGs: downregulated genes. (D) The heat map of differential expression genes involved in the oxidation-reduction process. The annotation information of genes can be found in Additional file 5: Table S4 (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

in the transgenic plants than in the wild-type control. The accumulation of melatonin was highly correlated with the expression level of PlTDC in different transgenic lines, indicating that the increase of melatonin levels in transgenic tobacco plants directly resulted from the ectopic expression of PlTDC. This result was consistent with that reported by Zuo et al. (2014). It also demonstrated that genetic manipulation to elevate melatonin production by introduction of genes from other species, even with low homology, is feasible. In terms of the phenotype, serotonin accumulation in transgenic O. sativa by overexpressing TDC resulted in a dark brown phenotype and stunted growth (Kanjanaphachoat et al., 2012). However, our PlTDC transgenic tobacco lines showed the normal phenotype, which was in agreement with the TDC3 transgenic lines in

catalyze the reaction converting tryptophan into tryptamine. Furthermore, PlTDC was identified to be distributed in the cytoplasm of cells according to subcellular localization, which is consistent with several previous reports (De Luca and Cutler, 1987; Facchini and St-Pierre, 2005). Subsequently, PlTDC was transformed into tobacco because the P. lactiflora genetic regeneration system had not yet been established to date; tobacco is a model plant for plant gene engineering operations, and as it naturally has almost undetectable levels of melatonin it that is useful for the functional study of melatonin in plants (Hattori et al., 1995). After A. tumefaciens-mediated transformation, tobacco plants overexpressing PlTDC were obtained. Similar to observations in other studies (Byeon et al., 2014), the melatonin content was 1.67-fold higher 353

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Fig. 10. Effect of salt stress on detached leaves from wide-type and transgenic tobacco lines overexpressing PlTDC. (A) Phenotypes of wide-type and transgenic lines. (B) H2O2 accumulation was detected by DAB staining. (C) O2%− accumulation was detected by a fluorescence probe. Tobacco plants were stressed by NaCl solution with different concentration for 36 h. WT: wild-type. The values represented the mean ± SD, and different letters indicate significant differences according to Duncan’s multiple range test (P < 0.05).

Gao et al., 2018). Moreover, compared with the wild-type control, Fv/ Fm and the expression levels of the protective enzyme genes Cu/ZnSOD, POD, CAT and APX increased, whereas the expression levels of the senescence-associated genes SAG, HIN1, CP1 and CP2 markedly decreased in transgenic tobacco plants under drought stress. The opening degree of leaf stomata of the transgenic tobacco plants was higher than that of the wild-type plants. Additionally, genome-wide expression profiling by RNA-Seq found quite a number of DEGs responsible for the oxidation reduction process, chloroplast structure, photosynthesis and stress responses, which also revealed that melatonin ameliorated H2O2 and O2%− accumulation. This result was in agreement with the obtained data from O. sativa (Liang et al., 2015). The above results all demonstrated that the enhanced melatonin content ameliorated H2O2 and O2%− accumulation, which decreased cell membrane damage (as indicated by REC and MDA accumulation) and increased potential photosynthetic capacity (as indicated by Fv/Fm values) under drought stress. This ultimately delayed plant senescence (as indicated by senescence-associated genes expression) (Fig. 11).

O. sativa (Byeon et al., 2014). To date, no studies on the function of melatonin in TDC transgenic plants have been carried out. In order to confirm PlTDC is involved in protecting P. lactiflora from drought stress, and in view of the role of melatonin in alleviating stress damage, transgenic tobacco plants were used to examine multiple abiotic stresses. Accordingly, it was found that the transgenic tobacco plants had greater tolerance to drought and salt stress compared with the wild-type control, and the mechanistic exploration unambiguously proved that the enhanced tolerance was directly associated with the antioxidant capacity of melatonin. These results are similar to the observations made in transgenic Arabidopsis thaliana overexpressing the M. zumi ASMT gene and Triticum aestivum caffeic acid 3-O-methyltransferase gene (COMT), which also had enhanced drought tolerance (Zuo et al., 2014; Yang et al., 2019). Plant senescence induced by an adverse environment was accompanied by excessive accumulation of reactive oxygen species (ROS) (BuchananWollaston et al., 2003). In current study, histochemical staining and fluorescence probes revealed rapid production of both H2O2 and O2%− in the wild-type control. Moreover, the excessive accumulation of H2O2 and O2%− led to membrane lipid peroxidation, which could be assessed by REC (Bhattacharjee, 2014). Here, the rapid accumulation of REC was also found in the wild-type control, but in transgenic tobacco plants, the accumulation of H2O2, O2%− and REC all significantly decreased, which might be due to the H2O2 and O2%− scavenging effect of melatonin. These results were agreement with previous studies (Wang et al., 2016;

5. Conclusions In the present study, we have demonstrated that PlTDC was a key factor in increasing melatonin production to confer drought and salt stress tolerance. Overexpression of PlTDC ameliorated H2O2 and O2%− accumulation, decreased cell membrane damage, and increased 354

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Fig. 11. A proposed model of PlTDC-mediated melatonin biosynthesis and enhancing tolerance to multiple abiotic stresses. Drought stress could affect PlTDC expression and decreased H2O2 and O2%− scavenging. Overexpression of PlTDC in tobacco plants improved the melatonin content, which enhanced H2O2 and O2%− scavenging leading to acquire salt tolerance. Furthermore, the reduced H2O2 and O2%− accumulation enhanced drought tolerance through decreasing cell membrane damage, increasing photosynthetic capacity and delaying leaf senescence.

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