Camellia sinensis - Springer Link

Appl Biochem Biotechnol (2015) 177:1055–1068 DOI 10.1007/s12010-015-1796-7

Molecular Cloning and Characterization of Spermine Synthesis Gene Associated with Cold Tolerance in Tea Plant (Camellia sinensis) Xujun Zhu 1 & Qinghui Li 1 & Jingyan Hu 1 & Mingle Wang 1 & Xinghui Li 1

Received: 13 April 2015 / Accepted: 2 August 2015 / Published online: 15 August 2015 # Springer Science+Business Media New York 2015

Abstract Spermine synthase (SPMS, EC 2.5.1.22), enzyme of spermine (Spm) biosynthesis, has been shown to be related to stress response. In this study, attempts were made to clone and characterize a gene encoding SPMS from tea plant (Camellia sinensis). The effect of exogenous application of Spm in C. sinensis subjected to low-temperature stress was also investigated. A full-length SPMS complementary DNA (cDNA) (CsSPMS) with an open reading frame of 1113 bp was cloned using reverse transcription-PCR and rapid amplification of cDNA ends (RACE) techniques from cultivar “Yingshuang”. The CsSPMS gene, which encoded a 371 amino acid polypeptide, in four cultivars is highly homologous. Quantitative real-time PCR indicated that the CsSPMS gene shows tissue-specific expression, mainly in the leaf and root of tea plant. The expression analysis demonstrated that the CsSPMS gene is quickly induced by cold stress and had similar trends in four cultivars. Spm-supplemented “Baicha” cultivar contains higher endogenous polyamines compared to the control, coupling with higher expression levels of ADC and SPMS. In addition, activities of peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), as well as free proline content in the Spmsupplemented samples were higher than the control during the experiment course or at a given time point, indicating that Spm exerted a positive effect on antioxidant systems. Moreover, Agrobacterium-mediated expression of CsSPMS in tobacco leaves showed relatively higher cold tolerance. Taken together, these findings will enhance the understanding of the relationships among CsSPMS gene regulatory, polyamines accumulation, and cold tolerance in tea plant. Keywords Cold tolerance . CsSPMS . Gene expression . Polyamine . Tea plant

* Xinghui Li [email protected] 1

College of Horticulture, Nanjing Agricultural University, Weigang No.1, Nanjing, Jiangsu Province 210095, People’s Republic of China

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Appl Biochem Biotechnol (2015) 177:1055–1068

Introduction Plants are often suffered by a number of environmental stresses, such as, high/low temperature, heavy mental, salinity soil, pathogen infection, which adversely affect plant quality, productivity and function as a determinant of geographical distribution and growth [1]. Tea plant (Camellia sinensis) is an evergreen woody plant that distributed from tropical to temperature regions. It is one of the important non-alcoholic beverage crops in the world. Tea plants are often damaged by temperature stress which seriously affects the quality and yield of tea products. Therefore, it is of critical importance to produce cold-hardy cultivars, so as to increase tea yield and meet the increasing needs of healthy beverage. Polyamines (PAs), mainly spermine (Spm), spermidine (Spd), and their diamine precursor putrescine (Put), are low-molecular-weight aliphatic amines found in all living organs [2]. In plants, Put is converted to Spd and Spm via two sequential aminopropyl transferase reactions catalyzed by spermidine synthase (SPDS) and spermine synthase (SPMS), respectively [3]. Two SPDS genes (AtSPDS1 and AtSPDS2) [4], as well as one SPMS gene (AtSPMS) were detected in Arabidopsis thanalia [5]. In addition, the aminopropyl groups are donated by decarboxylated S-adenosyl-Met, a compound synthesized in a reaction catalyzed by Sadenosyl-Met decarboxylase (SAMDC) [6]. Physiological functions of PAs have been verified by gain-of-function and loss-of-function experiments on PA biosynthetic genes; for example, in Arabidopsis plant, overexpressed a SPDS gene from Cucurbita ficifolia, became multi-stress tolerant to freezing, salinity, and drought conditions [7]. Overexpression of SAMDC1 gene in Arabidopsis thaliana increases expression of defense-related genes as well as resistance to Pseudomonas syringae [8]. On the other hand, the tetraamine-deficient Arabidopsis plant (acl5/spms) became more sensitive to high salt and drought compared to wild-type plant [9]. Biological roles of SPMS and Spmsupplementation treatment have also been experimental substantiated by many reports. Overexpression of SPMS genes has been proven to promote the polyamine biosynthesis, leading to better biotic or abiotic stress tolerance [10, 11]. Furthermore, exogenous application of Spm is able to lighten stresses in different type of plants [12, 13]. These data indicated that Spm derived from SPMS pathway might be important for impeding the stresses. Only a few researchers, however, pay attention to the functions of PAs in tea plant during environmental stress. Kakker and Nagar [14] reported that high Put and low Spd and Spm levels were associated with imposition of dormancy. Conversely, high Spd and Spm levels were related with dormancy release. A determination method of biogenic amines (including Put, Spd, and Spm) in tea infusion by HPLC after derivatization has been established by Brückner et al. [15]. In this background, cloning stress-responsive SPMS gene will be of important value for tea plant genetic engineering in an effort to improve stress tolerance. “43 Longjing,” “Longjingchangye,” “Yingshuang,” and “Baicha,” which were local varieties in eastern China, had different adaptability to low-temperature environment. Baicha is the most sensitive cultivar under low (or high)-temperature growth condition, while 43 Longjing is the least. In this study, we cloned the CsSPMS gene from the four tea plant cultivars. The analysis profiles of gene expression in different plant organs under cold stress were also determined. Moreover, the activities of peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), as well as ADC, SPMS expression levels in the Spm-supplemented cultivar Baicha were performed. Finally, Agrobacterium-mediated expression of CsSPMS in tobacco leaves was also assayed to test the cold tolerance. The objective was to elucidate how PAs improve cold tolerance from low-temperature condition on tea plant.


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Materials and Methods Plant Materials and Treatment One-year-old tea plant cultivars (Yingshuang, Baicha, 43 Longjing, and Longjingchangye), which pretreated in a chamber with 12 h/12 h light/dark photoperiod at 25 °C/20 °C day/night for 2 weeks, were subjected to cold treatments. To induce cold stress, we cultivated the plants at 12 °C for 4 h, and then the temperature was subsequently decreased to 4 °C. The 2nd leaf from bud were collected for RNA extraction and stored at −70 °C at 2 h after treatment. Tea plants growing in a stress-free environment were used as a control group. The roots, stems, leaves, flowers, and fruits of tea plants under normal growth conditions were also sampled and stored at −70 °C. For Spm supplementing experiment, Baicha cultivar was incubated for 14 days in liquid 1/ 2GM supplemented with 1 mM of Spm, whereas those incubated only in liquid 1/2GM were considered as control. The experimental plots were arranged in completely randomized blocks with three replicates per treatment, and each treatment involved a total of 36 plants. After 1, 4, 10, and 14 days treatment, both the Spm-treated and the control plants were gently washed with distilled water and quickly filter dried to eliminate the liquid solution on the root surface and free spaces.

Isolation of Full-Length cDNA of Tea Plant SPMS by Rapid Amplification of cDNA Ends (RACE) Firstly, a partial complementary DNA (cDNA) encoding tea plant SPMS from Yingshuang was isolated through reverse transcription-PCR (RT-PCR) using degenerate primers (DF, 5′-TGGCCWGGAGARGCNCAYTC-3′; DR, 5′-ACCATCTTRTCWATYTCACA-3′) designed based on highly conserved region of plant SPMSs. In order to obtain a full-length gene, 5′- and 3′-RACE was implied. Total RNA was isolated using an RNAprep pure plant kit (Tiangen, Beijing, China). The quality of total RNA was determined using Smartspec Plus spectrophotometer (Bio-Rad, California, USA). First-strand cDNA was synthesized using an M-MLV RTase cDNA synthesis kit (TaKaRa, Kyoto, Japan) according to the manufacturer’s instructions, which were then used for the 3′/5′-RACE PCR with 3′ genespecific primer (GSP, 5′-TGGGCAGGACCAACAGG-3′) and 5′ GS P (5′CCTGGTGGTGTTCTTTGT-3′) that were designed on the basis of partial SPMS. The PCR products of expected size were purified, cloned into pMD18-T (Invitrogen, CA, USA), and sequenced by Gen-Script Inc. (Nanjing, China). The putative 3′- and 5′-RACE cDNAs and the partial sequence were overlapped with DNAMAN 6.0 to form a cDNA contig, which was used to determine the putative initiating translation codon (ATG) and open reading frame (ORF). Finally, a pair of full-length primers (FLF, 5′AGGGTTCTGTCCACTATGC-3′; FLR, 5′-GGACGAAGAGCCTTTGCTACCG-3′) designed according to the cDNA contig was employed to obtain a full-length cDNA.

Sequence Analysis The full-length cDNA sequence was used to search homologous sequences via BLAST in NCBI (National Center for Biotechnology Information). The molecular phylogenetic tree among the different plants was built using MEGA6 with the neighbor-joining (NJ) method.

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Construction of Plant Expression Vector and Transient Expression Assay The CsSPMS ORF was amplified by PCR, digested by Kpn I and BamH I and then directionally cloned into the plant expression vector pCAMBIA2300. Agrobacterium tumefaciens strain GV3101 cells carrying pCAMBIA2300-CsSPMS was grown overnight in YPE medium containing 50 μg mL−1 kanamycin. Cells were collected by centrifugation (4,000×g), resuspended to an OD600 value of 1.0 with a buffer containing 10 mM MESKOH, pH 5.6, 10 mM MgCl2, and 100 μM acetosyringone, and infiltrated into fully expanded tobacco leaves.

Physiological Analysis in Tea Plant and Tobacco Plant In tea plant, MDA was measured according to the method of Dhindsa et al. [16], and proline content was assayed as Rensburg et al. [17] described. Chlorophyll content was measured to detect the effect of cold treatment on tobacco leaves according to Alsaadawi et al. [18].

Gene Expression Analysis in Tea Plant and Tobacco Plant Quantitative real-time PCR was performed to evaluate the expression levels of CsSPMS (KJ580429), CsADC (arginine decarboxylase, JQ653274), NbDREB2a (FN649467), and NbICE using the following primer pairs: Spms-F, 5′-GCATCCTATCAATCCTATT-3′; SpmsR, 5′-AATCACGAAGAAGCATAA-3′; Adc-F, 5′-CTTAGGGAAATCTCTCGCC-3′; Adc-R, 5′-TAACAAATCCAATGACGCC-3′; and DREB2a-F, 5′-CAAAGTGGTGGTGCTGTA-3′; DREB2a-R, 5′-AATTCATTGTTTAATTGAT-3′. Each 20 μL of the PCR reaction solution contained 10 μL of 2× SYBR Premix ExTaq (TaKaRa, Kyoto, Japan), 10 ng of diluted cDNA, a n d 0 . 2 μ Μ o f g e n e - s p e c i f i c p r i m e r s . C . s i n e n s i s a c t i n ( a c t i n - F, 5 ′ AGGGTTCTGTCCACTATGC-3′; actin-R, 5′-GGACGAAGAGCCTTTGCTACCG-3′) or Nicotiana benthamiana EF1α (EF1α-F, 5′-TTGATCTGGTCAAGAGCCTCAAG-3′; EF1α-R, 5′-CAATCACAGTGTTGGCTTGC-3′) was used as an internal control for the assays. The amplification conditions of thermocycling were listed as follows: 95 °C for 30 s and 40 cycles of 95 °C for 5 s and 60 °C for 30 s.

Quantification of Free Polyamines by High-Performance Liquid Chromatography (HPLC) Free polyamines were extracted and measured according to Shi et al. [13] and Naka et al. [19] with minor modifications. In brief, about 0.2-g sample was powdered with mortar and pestle in liquid nitrogen. One milliliter 5 % (v/v) cold perchloric acid (PCA) was added and kept on ice for 30 min. After centrifugation of 15,000g for 30 min at 4 °C, the supernatants were combined and filtered using a 0.2 μm filter syringe. Two hundred microliter filtrate was transferred to a tube containing 200 μL 2 N NaOH and 10 μL benzoyl chloride. After 30 min incubation at 25 °C, 2 mL saturated NaCl was added. Thereafter, 2 mL of diethyl ester was added and thoroughly mixed, followed by centrifugation at 3,000g for 5 min. Then, the upper phase was transferred to a new tube and dried under vacuum and the residue was resuspended in 100 μL methanol. The benzoylated PAs were analyzed by a HPLC (Shimadzu, Japan) equipped a C18 reverse-phase column (4.6 × 250 mm, particle size 5.0 μm) with programmed gradient solvents (acetonitrile/water) and detected at 254 nm. One detection cycle is consisting of


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60 min at a flow rate of 1.0 mL min−1 at 30 °C; i.e., 42 % acetonitrile for 25 min, followed increased up to 100 % acetonitrile during 3 min, 100 % acetonitrile for 20 min, decreased down to 42 % acetonitrile during 3 min, then 42 % acetonitrile for 9 min.

Extraction and Analysis of Superoxide Dismutase (SOD), Peroxidase (POD), and Catalase (CAT) For extraction of SOD, POD, and CAT, 0.2-g sample was ground using a cold mortar and pestle under liquid nitrogen and then homogenized in 3.0 mL of extraction buffer composed of 50 mM phosphate buffer (pH 7.8) and 1 % polyvinylpyrrolidone, followed by centrifugation for 20 min at 12,000g. The resulting supernatant was used for enzyme analysis at 25 °C by spectrophotometer. SOD and CAT activity were determined according to the method described by Jiang and Zhang [20]; POD activity was assayed according to Ranieri et al. [21].

Statistical Analysis All of the presented data are mean values of a representative experiment (three plants) and shown as the mean ± SE. All statistical analyses were performed with SPSS 17.0 (windows), and significance tests were determined by Ducan’s test and ANOVA.

Results Cloning the Full-Length CsSPMS cDNAs from Tea Plant A 410 bp partial SPMS fragment from tea plant cultivar Yingshuang was isolated via RT-PCR using degenerate primers. The followed 3′- and 5′-RACE PCR were performed using the corresponding GSPs designed in regarding to the partial SPMS to isolate full length of SPMS gene. The nucleotide sequence of CsSPMS from other cultivars was obtained based on sequence information of cultivar Yingshuang. Furthermore, the nucleotide sequences of CsSPMS from the four cultivars shared high level of identity at approximately 99.82 %. The deduced amino acid sequences of the CsSPMS gene from the four cultivars were nearly 99.46 % identical (Fig. 1a). The CsSPMS genes from four tea plant cultivars contain a 1 116 bp open reading frame (ORF) which encoded 371 amino acid residues. Surprisingly, the CsSPMS gene from the four cultivars had two differences in the 236 and 523 locus of the nucleotide acid sequences (A at 440 bp, A at 899 bp, for Baicha, respectively). Indeed, the two predicted amino acid sequences also showed two different amino acids corresponding to the two gene mutation sites (Fig. 1a).

CsSPMS Encoding Amino Acid Sequence Analysis Sequence analyzing by BLAST in NCBI revealed that the CsSPMS protein grouped to AdoMet_Mtases superfamily with S-adenosylmethionine binding site. The multiple alignments of amino acid sequences among the SPMS-like proteins from plant species and a neighbor-joining phylogenetic tree was constructed was shown in Fig. 1b. The result showed that the four tea plant cultivars clustered together.

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Fig. 1 The phylogenetic relationships between the SPMSs in plants. a Phylogenetic tree of SPMSs proteins from different species. The analysis was performed using the Neighbor-Joining method with 1000 bootstraps in the MEGA6 software. Arabidopsis thaliana (NP_568785), Ipomoea batatas (CDO96633), Gossypium arboretum (KHG00313), Morus notabilis (XP_010103219), Triticum urartu (EMS58513), Lotus japonicas (CAM35498), Medicago truncatula (KEH40403), Malus domestica (BAE19758), Oryza sativa (BAD54209), Zea mays (AAW57523), Populus tomentosa (JGI). b Aliment of amino acid sequences of SPMS proteins from tea plant cultivar “43 Longjing,” “Longjingchangye,” “Yingshuang,” and “Baicha”


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Expression Profiles of CsSPMS Gene in Four Cultivars Expression patterns of CsSPMS gene in different organs, such as, leaf, root, stem, flower, fruit, and bud from Yingshuang, were detected by qRT-PCR. CsSPMS was expressed at the highest level in the root and leaf, followed by bud and stem, whereas lowest level in the fruit and flower (Fig. 2a). Subsequently, the CsSPMS gene expression in Yingshuang largely increased 0.5, 1, 4, 8, 12 h, reaching a peak at 12 h, which was approximately 5fold more than 0 h (Fig. 2b). At the meantime, the spermine contents in Yingshuang during cold stress were also determined by HPLC. The spermine contents were increased gradually by cold treatment, and the accumulation level at 4 h reached the peak, whose content was approximately three times as 0 h (Fig. 2b). Interestingly, the relative expression profiles showed significant difference between normal temperature and cold stress in all four cultivars. However, under cold treatment, the CsSPMS transcript level in Baicha enhanced 1.9-folds more than normal temperature condition, while in other three cultivars, the CsSPMS transcript increased a higher level (>2.8-folds) compared to normal temperature treatment (Fig. 2c).

Proline and MDA Contents in Spm-Supplemented and Control Tea Plants During Cold Treatment Both proline and MDA contents exhibited increases during low-temperature treatment (Fig. 3a, b) of Baicha cultivar. The cold trigged increase in MDA content was repressed by exogenous Spm treatment. Conversely, a further increase in proline content was detected with exogenous Spm treatment of tea plant.

Enzyme Activity of Spm-Supplemented and Control Tea Plants During Cold Treatment SOD, POD, and CAT activities were performed to examine the effect of Spm on the antioxidant enzymes under cold treatment. Both the control plants and the Spm-treated plant had a continuous increasing SOD activity during the course of cold stress. Moreover, Spmtreated plant showed a significant higher level than the control on POD and CAT activities (Fig. 4a). At the beginning of the cold stress, POD activity of the Spm-treated plants was slightly higher than that of the control ones. However, POD activity of the Spm-treated plants became lower conversely (Fig. 4b). CAT activity exhibited an almost same performance with SOD activity (Fig. 4c).

Endogenous Free Polyamines and Genes Expression of Spm-Supplemented and Control Tea Plants Under Cold Stress The endogenous polyamines content of the Baicha cultivar plant treated with or without Spm were measured before and after cold treatment, respectively. Free Put of the Spmsupplemented plants increased slowly until 1 day, and a significant increasing was occurred at 10 days. However, the control plants almost show no change in Put content level (Fig. 5a). Free Spd of the Spm-supplemented plants performed a gradually increased until 10 days, where the content was almost five times more than that at 0 day. However, free Spd of the control plants did not change significantly (Fig. 5b). For free

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Fig. 2 CsSPMS gene expression levels and spermine contents in tea plant. a Relative CsSPMS expression level in the different organs of the tea plant cultivar “Yingshuang.” b Relative CsSPMS expression level and spermine content under cold stress of the tea plant cultivar “Yingshuang.” c Relative CsSPMS expression level of four tea plant cultivars under normal temperature or cold stress

Spm content, no difference was detected at 1 day between control and Spmsupplemented plants. However, the Spm-supplemented plants showed much higher Spm than the control at 10 days (Fig. 5c). Furthermore, expressions of polyamine biosynthetic genes (ADC, SPMS) were performed by qRT-PCR. Under cold stress, ADC was induced quickly at 1 day, followed by an increase at 10 days for control plants. In Spm-supplemented plants, ADC also was induced at 1 day, followed by a decline at 10 days. SPMS mRNA level was continuously up-regulated in Spmsupplemented plants, whereas that of control plants yielded a lower expression at 10 days compared to Spm-supplemented plants (Fig. 5d).


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Fig. 3 Effects of cold treatment on MDA (a) and proline (b) content in “Baicha” cultivar plants with or without exogenous 1 mM Spm. The experiments were repeated three times in duplicate

Chlorophyll Content and Gene Expression of Tobacco Plants Under Cold Stress After Agrobacterium-mediated expression of pCAMBIA2300-CsSPMS clone and pCAMBIA2300 empty vector (EV) in tobacco leaves (Fig. 6a), we placed tobacco plants in cold stress environment. The chlorophyll contents and genes expression both in CsSPMS and EV transient expressed tobacco plants were measured after cold treatment 8 and 24 h, respectively. Both the CsSPMS and EV transient expressed plant had continuous decreasing chlorophyll content during the course of cold stress. However, CsSPMS transient expressed plant showed significant higher levels than the EV one (Fig. 6b). The expression profiles of NbDREB2a were detected under cold stress in two lines of transient expressed plants. The expression levels of NbDREB2a in CsSPMS transient expressed plant were increased, but these expression levels decreased in EV lines (Figs. 6c).

Discussion In this study, the genes encoding SPMS were cloned from tea plant cultivars (43 Longjing, Longjingchangye, Yingshuang, and Baicha) for the first time. The different adaptability to low-temperature environment of these four cultivars might be explained by the differences on CsSPMS gene (Fig.1). The CsSPMS in tea plant contain S-Adenosylmethionine binding site and AdoMet MTases superfamily. S-Adenosylmethionine (SAM-e, SAMe, SAM, SAdenosyl-L-methionine, AdoMet, or ademetionine) is a common co-substrate involved in methyl group transfers. It is made from adenosine triphosphate (ATP) and methionine by methionine adenosyltransferase (EC 2.5.1.6) [22]. SAM is required for cellular growth and repair in plant. It is also involved in the biosynthesis of several hormones and neurotransmitters that affect mood, such as epinephrine. Methyltransferases are also responsible for the addition of methyl groups to the 2′ hydroxyls of the first and second nucleotides next to the 5′ cap in messenger RNA [22, 23]. The results of the CsSPMS gene expression levels in tea plant cultivar Yingshuang indicate that the CsSPMS expression has specificity in different organs. The CsSPMS gene expression in Yingshuang was quickly induced by cold treatment, and had a gradually increasing trend

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Activity of SOD (unit g -1 FW)

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Fig. 4 SOD (a), POD (b), and CAT (c) activities of “Baicha” cultivar plants during 14-day cold treatment with or without exogenous 1 mM Spm. The activities were measured three times in duplicate


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until 12 h, which can be matched by the spermine contents variation at the same time course under cold stress (Fig. 2b). The CsSPMS transcript level in Baicha under cold treatment enhanced 1.9-folds more than control condition, which was much lower than other three cultivars (>2.8-folds). MDA is considered as a biomarker for peroxidation and an indicator of the degree of oxidative stress damage [24]. Proline content is one of the most response indicators exposed to environmental stresses, especially in higher plants under cold stress [25]. From this study, proline contents in exogenous Spm-treated tea plants were significantly higher than control in response to cold stress at all treatment period (except 12-day point). On the other hand, higher

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Fig. 6 Effect of cold stress on chlorophyll content and related gene expression in CsSPMS transient expressed tobacco plant. a Phenotypes of tobacco levels at 24 h after cold treatment. b Chlorophyll content from CsSPMS and EV transient expressed tobacco plant leaves were detected by the spectrophotometric method after 8 h and 24-h cold treatment. c NbDREB2a expression levels during 24-h cold treatment in CsSPMS and EV transient expressed tobacco plants

MDA was detected in control plants than Spm-treated ones. The variation in proline and MDA content in this study indicates that exogenous Spm increases the proline content in tea leaf, and reduces membrane lipid peroxidation, which increases cold tolerance in tea plants pretreated with exogenous Spm. Spm pretreatment accounted for an increase in endogenous Spm content. Meanwhile, Spd and Put content had a slight enhancement. Our result corroborated previous study which suggested that exogenous polyamine application led to endogenous counterpart [13]. Polyamine accumulation levels showed that although both the control and Spm-supplemented plants increased endogenous polyamines under cold stress, the Spm-supplemented ones had higher free polyamines than the control, in particular at 10-day point (Fig.5a–c). In addition, the induction of polyamine biosynthetic genes (including ADC and SPMS) by cold treatment can be detected in both the control and Spm-supplemented plants, especially the higher level of Spm-supplemented one. A positive correlation between SPMS gene expression and Spm content was established only for Spm-supplemented plants, but not the control one (Fig. 5c, d). This phenomenon suggested that polyamine biosynthesis regulation is not determined only by transcriptional level but also by regulation of post-transcriptional or translational level [26]. We obtained transient expressed CsSPMS tobacco lines to elucidate the function of the CsSPMS gene. Results demonstrated that the transient expression of CsSPMS in tobacco plants induced the NbDREB2a level, which contains DRE (dehydration-responsive element) in promoter, to improve cold tolerance [27]. As regulation of cellular polyamine accumulation


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is influenced by many factors, the exact mechanism underlying this study is worth investigating in the future. Acknowledgments This research was supported by the National Natural Science Foundation of China (31400584), the Fundamental Research Funds for the Central Universities (KJQN201545), the Earmarked Fund for Modern Agro-industry Technology Research System (CARS-23), Natural Science Foundation of Jiangsu Province (BK20140714), the Specialized Research Fund for the Doctoral Program of Higher Education (20130097120013), and Jiangsu Doctors Gather Project.

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