Overexpression of MdCPK1a gene, a calcium dependent protein kinase in apple, increase tobacco cold tolerance via scavenging ROS accumulation

Calcium-dependent protein kinases (CDPKs) are important calcium receptors, which play a crucial part in the process of sensing and decoding intracellular calcium signals during plant development and adaptation to various environmental stresses. In this study, a CDPK gene MdCPK1a, was isolated from apple (Malus×domestica) which contains 1701bp nucleotide and encodes a protein of 566 amino acid residues, and contains the conserved domain of CDPKs. The transient expression and western blot experiment showed that MdCPK1a protein was localized in the nucleus and cell plasma membrane. Ectopic expression of MdCPK1a in Nicotiana benthamiana increased the resistance of the tobacco plants to salt and cold stresses. The mechanism of MdCPK1a regulating cold resistance was further investigated. The overexpressed MdCPK1a tobacco plants had higher survival rates and longer root length than wild type (WT) plants under cold stress, and the electrolyte leakages (EL), the content of malondialdehyde (MDA) and reactive oxygen species (ROS) were lower, and accordingly, antioxidant enzyme activities, such as superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) were higher, suggesting the transgenic plants suffered less chilling injury than WT plants. Moreover, the transcript levels of ROS-scavenging and stress-related genes were higher in the transgenic plants than those in WT plants whether under normal conditions or cold stress. The above results suggest that the improvement of cold tolerance in MdCPK1a-overexpressed plants was due to scavenging ROS accumulation and modulating the expression of stress-related genes.


Introduction
Abiotic stresses, such as drought, high salinity, cold or submergence, are serious threats to crop productivity. Plants have evolved fine signaling strategies enabling them to overcome these stresses and other harmful conditions. Among the strategies adopted by plants, calcium signals are important regulators in many crucial and sophisticated cellular processes [1].

Cloning, sequencing and phylogenetic analysis of MdCPK1a
The fourth and fifth young leaves were taken from the annual branches of the Malus domestica cv.'Jonathan' growing in the greenhouse. Total RNA was extracted by using CTAB method. [35]. Based on the released sequence of MdCPK1a (MDP0000153100) from Phytozome (https://phytozome. jgi.doe.gov/pz/portal.html), a pair of primers GSP1 was designed for gene amplification by RT-PCR (S1 Table). The PCR amplification was performed in a total 50 μL reaction volume containing 300 ng cDNA, 1×TransStart Fast Pfu buffer, 0.25 mM dNTPs, 0.4 mM of each primer and 2.5 units of TransStart Fast Pfu DNA polymerase. PCR conditions were set as follows: initial denaturation at 95˚C for 2 min; 40 cycles of 95˚C for 20 s, 55˚C for 20 s, and 72˚C for 60 s, and followed by a final extension at 72˚C for 5 min. The construction of PCR products ligation with pMD19-T vector were named pMD19T-MdCPK1a, and sequenced by Invitrogen (Shanghai, China). The domain was identified through PROSITE and Smart™ databases (http://smart.emblheidelberg.de/); Molecular weight and theoretical isoelectric point (pI) were calculated by ExPASy software (http://www.expasy.org/); The position of S-Palmitoylation and N-Myristoylation were predicted using the online tool GPS-Lipid (http://lipid.biocuckoo.org/presult.php) [36][37][38]. The homologous proteins were searched by BLASTp program (http://www.ncbi.nlm. nih.gov/) using the deduced amino acid sequence of MdCPK1a. Multialignment was performed by DNAMAN software (http://www.lynnon.com/). A phylogenetic tree was built through the neighbor-joining (NJ) method under the MEGA 6.0 program with Poisson-corrected distances with 500 bootstrap replicates.

Subcellular localization analysis
The coding sequence of MdCPK1a with termination codon removal was amplified from pMD19T-MdCPK1a using primer GSP3 (S1 Table). Amplification products were digested with Xba I and BamH I, and cloned into the downstream of CaMV 35S promoter in pCAM-BIA1300 vector resulting in MdCPK1a C-terminal in-frame fusion with GFP gene to form a plasmid 35S::MdCPK1a-GFP. The 35S::MdCPK1a-GFP and 35S::GFP (control) constructs were transiently transformed into N. benthamiana leaves described by Sheludko [39]. GFP fluorescence was imaged under a laser confocal fluorescence microscopy (Zeiss TCS SP8) with an excitation wavelength of 488 nm and a 505-530 nm bandpass filter.

Protein extraction and western blot
Tobacco leaves that transiently expressed 35S::MdCPK1a-GFP and 35S::GFP (control) were homogenized in liquid nitrogen. The nuclear proteins, cytoplasmic proteins and plasma membrane(PM) proteins were extracted with the Plant Nuclear, Cytoplasmic and Membrane Proteins Extraction Kit (BestBio, Shanghai, China) from plant tissues, respectively. Total proteins were extracted with extraction buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1%SDS, 1%Triton X-100), and followed by centrifugation at 12000 rpm for 15min, and the supernatants were collected.
Following standardization of protein concentrations using BCA Protein Assay kit (BestBio, Shanghai, China), Equal amounts of protein were employed in 10% SDS-PAGE and transferred to the NC membrane. After blocking with 5% skimmed milk powder in PBST (0.5% Tween in PBS) at room temperature for 2 h, the membrane was incubated with Anti-GFP rabbit polyclonal antibody (Sangon Biotech, Shanghai, China) at 4˚C overnight. After this, the membrane was rinsed three times with PBST for 5 min and then incubated with the HRP-conjugated Goat Anti-Rabbit IgG (Sangon Biotech, Shanghai, China) for 1 h. Subsequently, the membrane was washed with PBST, visualized by enhanced chemiluminescence and then detected in the Tanon 2500 chemiluminescence imaging system (Shanghai, China).

Overexpression of MdCPK1a in N.benthamiana
The full length ORF of MdCPK1a flanking BamH I and Sac I at 5' and 3' respectively was amplified by the primers GSP2 (S1 Table). The PCR products were double-digested with BamH I and Sac I, then ligated into the pYH455 vector at downstream of CaMV35S promoter (S2 Fig), generating a plasmid pYH455-MdCPK1a. Subsequently, it was transferred into EHA105. Tobacco transformation was conducted using leaf disk method [40]. Transgenic tobacco plants were selected on MS medium supplement 50 mg�L -1 kanamycin. The kanamycin-resistance plants were further confirmed by PCR and RT-PCR respectively with the control of non-transformed tobacco plants cultured on MS medium.

Abiotic tolerance analysis of the transgenic tobacco plants
Three independent lines (A2, A4 and A36) and wild type (WT) plants were used to analyze abiotic tolerance. After being surface disinfected, the seeds of A2, A4, A36 and WT were sown on MS medium (for transgenic lines MS medium supplemented 50 mg�L -1 kanamycin). N. benthamiana were cultured under long day conditions 16 h light at 23-25˚C and 8 h dark at 18-20˚C.
To assess cold resistance, we grew the seedlings under cold stress (4˚C) for 10 d after seeds germinating on MS medium and measured the root length after cold treatment. Meanwhile, four-week-old plants growing in medium were stressed at 4˚C for 10 d, and then the survival rates were calculated after recovering at 25˚C for 14 d according to the number of green plants.
For salt or drought tolerance assays, 4-week-old plants were transplanted into soil with sufficient water under a normal environmental chamber at 25˚C for 14 d. They were then watered with 200 mM NaCl solution in soil for salt stress analysis, or cultured without irrigation for 25 d, and then recovered by re-watering for 10 d for drought stress analysis. The biomass and phenotype were investigated after the treatments.

Physiological measurements and histochemical staining
Sixty-day-old plants treated at 4˚C for 48 h were used as material. Malondialdehyde (MDA) contents were measured using the thiobarbituric acid (TBA)-based colorimetric method [41]. Leaf samples (0.5 g) were homogenized in 2 mL 20% trichloroacetic acid with the aid of some sand, and then the homogenate was centrifuged at 16,000 g for 20 min at 4˚C. The supernatant (1 mL) was mixed with equal volume of 0.5% (w/v) TBA. The mixture was heated at 95˚C for 30 min and then quickly cooled in an ice bath. After centrifugation at 10,000 g for 10 min, absorbancy was measured at 532 nm corrected for nonspecific turbidity by subtracting the absorbancy at 600 nm. The MDA content was calculated using its molar extinction coefficient (155 mM -1 cm -1 ), and the value was expressed as μmol MDA� mg -1 fresh weight (FW). Electrolyte leakages (EL) were detected using the protocol according to [42]. The leaf segments from at least three plants of each line were placed in deionized water for 2 h at 25˚C. Total electrolyte content was measured after autoclaving the leaf segments for 15 min and taken as 100% leakage. Activities of catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) were analyzed according to [43]. Leaf samples (0.2 g) were homogenized in liquid nitrogen adding 2 mL precooled 50 mM pH 7.8 phosphate buffer (containing 0.1 mM EDTA and 1% PVP) and ground into homogenate in an ice bath. Add extraction medium to rinse the mortar for 2-3 times and make the final volume 8.0 mL. The supernatant was centrifuged at 12 000 × g for 15 min at 4˚C and stored in the ice bath for the detection of SOD, POD, CAT activities. The accumulation of H 2 O 2 and O 2 was tested by histochemical staining with nitroblue tetrazolium (NBT) and 3, 3'-diaminobenzidine (DAB) respectively. Leaves were incubated in the NBT solution (0.1 mg�mL -1 ) and DAB solution (1.0 mg�mL -1 , pH 3.8) for 24 h at 25˚C in the dark. Then, the leaves were soaked in 95% ethanol overnight to remove the chlorophyll [44,45]. DAB/NBT-stained leaves were scanned, and the pixel intensity of the DAB/NBT stain was quantified using Adobe PHOTOSHOP CS4 software.

Quantitative RT-PCR analysis of gene expression in transgenic plants
The expression level of stress-related genes was monitored by quantitative RT-PCR (qPCR) on an ABI7300 Detection System using SYBR 1 Premix ExTaq™ qRT-PCR kits (TaKaRa, Dalian, China). Gene-specific primers were designed by Primer 5.0 (S1 Table).

Statistical analysis
Every experiment was repeated three times, and the value was got from an average from three independent replicates and shown with error bar representing with standard error (SE). All statistical analyses were performed using SPSS software and based on Duncan's multiple range tests, statistical differences were compared and p values <0.05 or <0.01 were used as the thresholds for significant or extremely significant differences, respectively.

Cloning and bioinformatics analysis
The full length open reading frame (ORF) of MdCPK1a was isolated from apple, which consisted of 1701 nucleotides encoding a 566-amino acid polypeptide with the predicted molecular weight 62.86 kDa and the isoelectric point 5. 16 showing that there are one myristoylation (Gly at the 2 nd residue from the N-terminus) and two palmitoylation (Cys at the 5 th and 136 th residues from the N-terminus) in the protein (Fig 1).
Multiple sequence alignments showed the deduced amino acid sequence of MdCPK1a with 72.73% similarity to OsCDPK7 (BAB16888), 76% to AtCPK1 (NP_196107), and 71.75% to ZmCPK1 (BAA12338). The phylogenetic relationships between MdCPK1a and several stressrelated CDPKs are presented in S1 Fig. The selected CDPK proteins were clustered into three subgroups including I, II, III. MdCPK1a, along with OsCDPK7, AtCPK1, and ZmCPK1 which were reported to regulate abiotic and biotic stress tolerances [33, 46,47], belongs to the subgroups I, which hints that MdCPK1a may participate in stress responses in apple.

Subcellular localization
The subcellular location of a protein determines or is closely correlated with its function. To investigate the subcellular location of MdCPK1a protein, we cloned the full-length ORF sequence of MdCPK1a into pCAMBIA1300 vector under CaMV35S promoter, constructing an in-frame fusion protein plasmid 35S::MdCPK1a-GFP (Fig 2A). The construct was transformed to N. benthamiana leaves by agro-infiltration for transient expression analysis. The subcellular location of MdCPK1a-GFP was detected by laser scanning confocal microscopy, with the leaves transiently transformed 35S::GFP as the control. The tobacco cells expressing the 35S::MdCPK1a-GFP emitted fluorescence both in nucleus and plasma membrane, whereas in expressing the 35S::GFP tobacco cells, the fluorescence filled the entire cytoplasm, plasma membrane and nucleus (Fig 2B). We further verified the subcellular localization by western blot by immunoblotting with anti-GFP antibody. MdCPK1a-GFP was detected exclusively in the fractions of plasma membrane and cell nucleus but not in the fraction of cytosol (Fig 2C). These results indicated that MdCPK1a protein was localized to the nucleus and cell plasma membrane.

Overexpression of MdCPK1a gene in tobacco
The overexpression construct of MdCPK1a (pYH455-MdCPK1a) was introduced into N. benthamiana by A. tumefaciens-mediated transformation. Ten transgenic lines were obtained and further identified by PCR using gene-specific primers (GSP1). Six lines were randomly selected for gene transcription analysis. The result showed MdCPK1a was expressed constitutively in these lines, among which three independent lines (A4, A36, and A2) were used for analysis of the resistance to abiotic stresses. The levels of MdCPK1a mRNA in the three transgenic lines were quantified by qPCR.

Stress tolerance of MdCPK1a-overexpressed N. benthamiana plants
For salt stress analysis, 6-week-old of WT and T 2 plants of A4, A36 and A2 were irrigated with 200 mM NaCl solution in the soil once a week. After suffering from salt stress for 25 d, WT plant leaves turn yellow, the MdCPK1a-overexpressed (MdCPK1a-OX) transgenic tobacco plants grew better and more vigorously compared with WT, (Fig 3A). The dry weight of shoots and roots of the transgenic lines, except A2, was significant higher than that of WT (Fig 3B), which suggests that MdCPK1a-OX tobacco plants increased the tolerance to salt stress. However, the similar symptoms between WT and the transgenic lines were observed under drought stress. The transgenic plants and WT displayed slow-growing, rolled and wilted leaves without irrigation for 25 d and showed no significant difference after re-watering for 10 d (Fig 4), suggesting that there were no obvious differences of drought resistance between WT and the transgenic lines.
The cold tolerance of seedlings of WT and the transgenic plants was monitored on MS medium at 4˚C. Both of them showed severe growth inhibition, however, the root length of WT was significantly shorter than that of the transgenic plants (Fig 5A and 5C). Meanwhile, we also analyzed the cold resistance of them at 4 weeks old. They were stressed in 4˚C for 10 d then recovered in normal conditions (25˚C) for 14 d. WT plants suffered from chilling injury more severely than A4, A36, and A2. Only 17% of WT plants survived after cold treatment, while 60-96% of the transgenic lines survived (Fig 5B and 5D). Physiological analysis showed that the transgenic plants had lower MDA content and less electrolyte leakage (EL) than WT plants under cold stress (Fig 5E and 5F), indicating that the transgenic plants were less injured compared with WT plants.

PLOS ONE
act as important signal molecules and also are toxic by-products leading to oxidative damage [48]. To reduce the damage of excessive production of ROS, plants have developed a scavenging mechanism allowed them to overcome ROS toxicity. To know whether MdCPK1a regulates ROS levels in cold response, we compared with the ROS levels in the overexpressing tobacco lines and WT plants after suffering cold stress. NBT  accumulation was similar in both plants. However, lower dyeing degree was detected in the transgenic plants whether by NBT (Fig 6A and 6C) or DAB (Fig 6B and 6D) staining under cold stress, suggesting less ROS accumulated in the transgenic plants than that in WT. Furthermore, compared with WT, the enzyme activities of CAT, POD and SOD of the transgenic lines were higher before treatment and significantly higher after 48h cold treatment. POD activity was still significantly higher in the transgenic plants while there was no significant difference in CAT and SOD activities after 72 h cold treatment (Fig 7).

Expression analysis of the stress related genes in transgenic N. benthamiana
The transcriptional levels of stress-responsive and ROS-related genes (NtSOD, NtGPX, NtCAT, and one ROS-producing NADPH oxidase gene, NtrbohD) were analyzed by qPCR in

Discussion
Calcium-dependent protein kinases respond to abiotic stress and play important roles in calcium signaling pathways. In apple, CDPKs are encoded by a multigene family consisting of 37 genes [9], however, the biological functions of which mostly remain unclear. In this study, MdCPK1a was identified in apple and characterized in transgenic tobacco.

Apple MdCPK1a protein localization
CDPK function is dependent on specific subcellular localization. Previous research has shown that CDPK proteins are found in cytoplasm, nucleus, the plasma membrane, oil bodies, mitochondrial outer membrane, peroxisome, and endoplasmic reticulum [6], suggesting their different functions. The N-terminal domain of CDPKs is important to subcellular localization. It is reported that membrane association is mediated by N-terminal acylation. The membrane localized CDPKs harbour a predicted N-myristoylation site and cysteine residues which would allow further palmtoylation in their N-terminus [51]. A recent study revealed that OsCPK17 has five alternative splicing (AS) forms with different subcellular localization [52]. In our experiment, MdCPK1a protein has been proved to be localized in the plasma membrane and nucleus, which is conformity with the prediction that MdCPK1a is putatively myristoylated and palmitoylated at its N-terminal (Fig 1). It indicates the posttranslational modifications might allow targeting MdCPK1a protein to the plasma membrane. The location of MdCPK1a protein suggests it might participate in early signaling pathways of environmental stress by phosphorylation and activation of downstream genes [53]. The plasma membrane-or nucleus-localized CDPKs involved in abiotic stress response were also reported in other plants, such as ZoCDPK1 in Zingiber officinale [54], SiCDPK24 [29] in Setaria italica response to drought stress. Our results further indicate that CDPKs might have multiple subcellular localizations and involved in multiple signal transduction pathways.

Apple MdCPK1a involves in response to abiotic stresses
To further understand MdCPK1a function, the T 2 plants of MdCPK1a overexpressing tobacco were used to study their responses to abiotic stresses. After 200 mM NaCl treatment, transgenic tobacco A4 and A36 lines showed more tolerant to salt stress, while the dry weight of shoots and roots of the transgenic lines A2 was comparable to that of the wild type, indicating that the tolerance of transgenic lines to salt stress is positive correlated to the ectopic expression levels of MdCPK1a in tobacco (Figs 3 and S2). One of early responses to low temperature or other abiotic stresses in plant cell is a transient increase in cytosolic Ca 2+ derived from influx from the apoplastic space and release from internal stores [55]. Ca 2+ binding proteins can sense the transient increases of cytosolic Ca 2+ , and then transmit signals to its target protein. CDPKs are the main responders in combining calcium signal with particular protein phosphorylation cascades. Although some studies showed that several CDPK mRNA are responsive to cold stress [8, [56][57][58], only a few of them were made further functional identification. In plants, as far as we know, OsCPK7, OsCPK13, OsCPK17 and OsCPK24 in rice and AtCPK1 in Arabidopsis have been reported that participated in the response to cold stress [46, [59][60][61][62]. Furthermore, the transgenic Arabidopsis plants overexpressing VaCPK20, a CDPK from Vitis amurensis and PeCPK10 from Populus euphratica improved freezing resistance [63,64], while in Zea mays, ZmCPK1 negatively regulate cold tolerance [33]. In our research, ectopic expression of MdCPK1a improved tobacco cold tolerance and also exhibit slightly increased salt tolerance, but no obvious improvement of drought tolerance. The cold-responsive genes, such as NtDREB3(dehydration-responsive element binding protein), NtERD10C (early response to dehydration 10C), NtLEA5 (late embryogenesis abundant protein) and NtSPS (Suc-P synthase) were significantly up-regulated in the overexpressing MdCPK1a transgenic tobacco plants compared with the WT plants. It is known that DREBs are important transcription factors by regulating the expression of stress-responsive genes, including ERD10C, LEA and SPS, and so on [65,66]. Overexpression of MdCPK1a increased cold-responsive genes in tobacco suggest that MdCPK1a may function upstream of DREBs as a positive regulator participated in the response to cold stress. Additionally, the root length of transgenic plants A4 and A36 is longer than that of WT when the seedlings of them cultured at 25˚C for 10 d on MS medium (Fig 5A). The aerial part of WT plants is little lower than that of the transgenic plants under normal condition (Fig 4). We speculated that MdCPK1a might also participate in the regulation of plant development. Our previous research showed that MdCPK1a was also induced by biotic stress [9]. These results suggest that apple MdCPK1a like CDPK genes in other species has overlapping functions [33, 67, 68].
Overexpressing MdCPK1a enhanced tolerance to cold stress in the transgenic tobacco by scavenging ROS Plants produced less ROS in organelles under optimal growth conditions, but under abotic stress, the rate of ROS production is significantly elevated. ROS was produced by two major sources under abiotic stress: one is as a consequence of disruptions in metabolic activity and another is as signaling ROS which produced by NADPH oxidase [69]. ROS accumulation is a double-edged sword for plants response to abiotic stress: on the one hand, they are signaling molecules of the abiotic stress-response signal transduction network [70], on the other hand, they are also toxic byproducts that can cause oxidative destruction of cell [48,71]. In general, CDPKs seem to function as positive regulators of ROS production in biotic stress signaling [49, 72-75], while some researches showed that CDPKs decrease ROS accumulation in abiotic stress by increasing the expression of ROS scavenging enzymes such as ascorbate peroxidase (APX), superoxide dismutase(SOD), catalase(CAT), and glutathione peroxidase(GPX) [76,77]. For example, overexpression of the constitutively active form of oilseed rape BnaCPK2 induces ROS accumulation and cell death through interacting with NADPH oxidase-like respiratory burst oxidase homolog D (RbohD) [78]. However, overexpression OsCPK12 decreases ROS accumulation by increasing the expression of OsAPx2, OsAPx8 and OsrbohI and confers increased tolerance to salt stress in rice [25]. Overexpression of OsCPK4 in rice confers salt and drought tolerance by preventing cellular membranes from stress-induced oxidative damage [26]. In this study, overexpression of MdCPK1a in tobacco promoted the tolerance to cold stress by decreasing the expression of NtrbohD and increasing the expression of NtSOD, NtCAT and NtGPX. Compared with WT plants, the enzyme activities of CAT, POD, and SOD is higher and the accumulation of ROS was less in transgenic tobacco plants under cold stress. Collectively, these results suggest that MdCPK1a plays roles in abiotic stresses, and ectopic expression of MdCPK1a gene in tobacco enhances the tolerance to cold stress, which contributes to increasing the transcription levels of stress-relative genes and regulating the expression of APX, CAT, SOD and rbohD to reduce the damage to plants caused by ROS accumulation.

Conclusion
In this research, a CDPK gene MdCPK1a from apple was characterized. MdCPK1a protein was found to localize the plasma membrane and the nucleus. Overexpression MdCPK1a in tobacco plants showed significantly improved their cold and salt stress tolerance than the wild type. Furthermore, Tobacco plants transfected with MdCPK1a showed increased resistance to cold stress by scavenging ROS accumulation and modulating the expression of stress-related genes. These results will be useful to further explore the function of MdCPK1a in apple.