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 [. When plants are subjected to various stresses, they rapidly release calcium ions (Ca2+) from storage compartments (such as vacuole, endoplasmic reticulum) into the cytosol. Transient increases of free Ca2+ in cytosolic are perceived and decoded through different Ca2+ sensors and Ca2+ binding proteins, such as calcium-dependent protein kinases (CDPKs), calmodulin-like proteins, calmodulins and calcineurin B-like proteins. CDPKs distinguished from other calcium-sensing proteins, as they not only can decode and translate the increase of Ca2+ concentration into improvement of protein kinase activity but also can activate downstream effectors [.

CDPKs exist in protists, oomycetes, green algae and plants, but not in animals [. Genome-wide analysis of different plant species showed that they are encoded by a large multigene family. For example, Oryza sativa, Zea mays, Malus domestica, Populus trichocarpa, and Arabidopsis thaliana were identified 31, 35, 37, 30 and 34 CDPK genes in their genomes, respectively [. CDPKs have a conserved modular structure including a variable N-terminal domain, a kinase domain, an auto-inhibitory domain or junction domain and a regulatory domain or CaM-like domain, which canonically contains four EF-hands [. In the absence or low concentration of cytoplasmic Ca2+, auto-inhibitory domain blocks the kinase domain and inhibits its activity [. When plants perceive stimuli, an immediate increase of the concentration of Ca2+ in plant intracellular promotes Ca2+ binding to EF-hand motifs, which will induce molecular conformation changes and activate enzyme activities, leading to phosphorylation of the targeted substrates as well as CDPK autophosphorylations [. The phosphorylated proteins probably participate in plant defense reactions, ethylene synthesis, cytoskeleton organization, carbon and nitrogen metabolism, and stress responses [ Knowledge about CDPK functions and mechanisms of the responses to environmental stress is increasing. Substantial experimental evidences indicate CDPKs play important roles in response to abiotic/biotic stress. For example, Arabidopsis CPK28 acts as a positive regulator in response to osmotic stress [. OsCPK9 in rice plays a positive role in drought, osmotic, and dehydration stress responses [. Overexpressing of OsCPK4, OsCPK12 in rice exhibited increased salt/drought stress tolerance and rice blast disease resistance [. CaCDPK15 in pepper (Capsicum annum) positively regulates response to Ralstonia solanacearum [ In Arabidopsis, overexpression of SiCDPK24 enhanced drought tolerance [. OsCDPK1 positively regulates salt and drought tolerance in rice [, meanwhile it acts as a positive regulator of OsPR10a participating in the defense signaling pathway [. Conversely, some CDPKs are negative regulators of stress response because transgenic plants overexpressing them are more sensitive to abiotic/biotic stresses. Arabidopsis thaliana cpk23 mutant increased endurance to drought and salt stresses, while AtCPK23 overexpressing plants reduced the resistance to drought and salt stresses [. Overexpression of ZmCPK1 in maize mesophyll protoplasts suppressed the expression of the cold-induced marker gene Zmerf3, and ectopic expression of ZmCPK1 in Arabidopsis reduces plants adaption to the cold tolerance, suggesting ZmCPK1 act as a negative regulator of cold stress signalling in maize [. The Arabidopsis CPK28 plays as a negative regulator of immune signaling that continually buffers immune signaling by controlling the turnover of BIK1, an important convergent substrate of multiple pattern recognition receptor (PRR) complexes [. Thus, CDPKs are implicated in both positive and negative regulation of plant abiotic/biotic stress adaptation.

However, the research on function of CDPKs in apple has been rarely reported. This study focused on the function of MdCPK1a, a CDPK gene from M. domestica, in response to abiotic stresses. MdCPK1a-overexpressed N.benthamiana plants were investigated to different abiotic stress conditions. Experimental results showed that overexpression of MdCPK1a in N.benthamiana confers it resistance to salt and cold stresses. Furthermore, the mechanism of enhancement of cold tolerance in the transgenic plants was disclosed in this research.

Materials and methods

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. [. 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.embl-heidelberg.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) [. 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 pCAMBIA1300 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 [. 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 [. 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 [. 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 [. 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 [. 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 H2O2 and O2- 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 [. 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® Premix ExTaq™ qRT-PCR kits (TaKaRa, Dalian, China). Gene-specific primers were designed by Primer 5.0 (S1 Table). PCR mixtures contained 10.0 μL of 2×SYBR Premix, 1.0 μL of cDNA template, 200 nM of each primer, then added ddH2O up to a total volume of 20.0 μL. PCR reaction was performed as follows: denaturation at 94°C for 3 min followed by 40 cycles at 94°C for 20 s, 60°C for 20 s, and 72°C for 40 s. After that, melting curves were determined as follows: 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s. qPCR was performed three independent biological repeats for each sample and three technical repeats for each reaction. Expression values were normalized with NtTubulin gene (Accession No: EF051136). The relative expression of a gene was calculated by using 2-ΔΔCt method [ΔΔCt = (Cttarget gene− Cttubulin gene) treatment−(Cttarget gene–Cttubulin gene) control].

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.

Results

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. MdCPK1a protein possesses the characteristics as other plant CDPKs: an N-terminal variable domain (107aa) preceding a Ser/Thr protein kinase catalytic domain (259 aa), a junction domain (42 aa), a CaM-like domain containing four EF hand Ca2+-binding motifs (142 aa) and a C-terminal variable domain (16 aa). A possible ATP-binding site and active site in the N-terminal region and 15 invariant amino acid residues for eukaryotic Ser/Thr protein kinase in the N-terminal of kinase domain were shown in Fig 1. The putative post-translational modifications of MdCPK1a protein was predicted by the software GPS-Lipid, showing that there are one myristoylation (Gly at the 2nd residue from the N-terminus) and two palmitoylation (Cys at the 5th and 136th residues from the N-terminus) in the protein (Fig 1).

Fig 1

Protein sequence analysis of MdCPK1a.

Kinase domains, Junction domain, and EF hand loops of CaM-LD domain of CDPK are marked. The 15 invariant amino acid residues for eukaryotic Ser/Thr protein kinase are indicated by asterisks. Protein kinase ATP-binding site and active site are highlighted in dark orange and green, respectively. The positions of predicted S-Palmitoylation (Pal) and N-myristoylation (Myr) are indicated in the diagram.

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 stress-related 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 [ 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.

Fig 2

Subcellular location of MdCPK1a.

(a) Schematic representations of the vector constructs of 35S::GFP and 35S::MdCPK1a-GFP. (b) Subcellular localization of MdCPK1a-GFP fusion protein was conducted by transient expression experiment in N. benthamiana cells. Images were taken by using Leica confocal microscopy at 72 hours post agroinfiltration (GFP: fluorescence, green; Bright: visible light image; Merge: merged images of above two images). Bars = 20 μm. (c) Subcellular localization of MdCPK1a-GFP fusion protein was detected by western blot. Total protein extract (T), cell membrane fraction (Cm), cytosolic fraction (Cy), and cell nucleus fraction (Cn) isolated from 35S::GFP-expressing (left) 35S::MdCPK1a-GFP-expressing (right) tobacco cells were immunoblotted with anti-GFP antibody.

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. MdCPK1a mRNA displayed the highest level in A4 and the lowest level in A2 (S2 Fig).

Stress tolerance of MdCPK1a-overexpressed N. benthamiana plants

For salt stress analysis, 6-week-old of WT and T2 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.

Fig 3

Phenotype and stress tolerance of the MdCPK1a-OX plants under salt stress.

(a) Photographs of wild-type and MdCPK1a-OX plants (line A4, A36, and A2) under 200 mM NaCl. The left shows the phenotype of aerial part of tobacco plants, the right shows the phenotype of shoot and root. (b) Shoot and root dry weight of MdCPK1a-OX plants after the salt-stress treatment. Error bars indicate the standard error of the mean (SEM) of three independent experiments. Significant differences between the WT and transgenic plants are indicated by asterisks (*p< 0.05, **p < 0.01).

Fig 4

Photographs of wild-type and MdCPK1a-OX plants under drought stress.

4-week-old seedlings were transplanted into soil with sufficient water in the chamber at 25°C for 14 d. They were cultivated for 25 d without watering for draught stress, and then re-watered for 10 d for recovery. Photographs of representative plants of WT and MdCPK1a-OX plants (A4, A36, and A2) were taken before and after the treatment of drought stress, and 10 days after re-watering, respectively. Twenty plants of each line were used for the experiments. Bars in picture = 10 cm.

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.

Fig 5

Overexpression of MdCPK1a enhances cold tolerance in transgenic tobacco.

(a) Phenotype of seedlings of WT and MdCPK1a-OX plants (A4, A36 and A2) at the normal growth temperature and low temperature (4°C) for 10 d on MS medium after germinated; (b) Responses to cold stress of 4-week-old WT and MdCPK1a-OX plants; (c) Root lengths of seedlings after germination for 10 d on MS medium at 4°C; (d) Survival rates of WT and MdCPK1a-OX plants. Values are the mean ± SE. Thirty plants of each line were used for statistics; (e) Detection of MDA content in WT and MdCPK1a-OX plants. FW means Fresh weight. Data represent the means ± SE of at least three replicates; (f) Electrolyte leakage in WT and MdCPK1a-OX tobacco plants; Significant differences between the WT and transgenic plants are indicated by asterisks (*p< 0.05, **p < 0.01).

Analysis of ROS levels and antioxidant enzyme activities in transgenic N. benthamiana

Under abiotic stresses, reactive oxygen species (ROS) such as hydroxyl radical (·HO), superoxide radical (O2-.) or hydrogen peroxide (H2O2), are excessively accumulated in plants, which act as important signal molecules and also are toxic by-products leading to oxidative damage [. 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 and DAB staining were used to detect the accumulation of O2-. or H2O2 in leaves, respectively. Before the cold treatment, the leaves had similar dyeing degree in the transgenic plants and WT, indicating O2-. or H2O2 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).

Fig 6

The accumulation of reactive oxygen species (ROS) in WT and MdCPK1a-OX plants by histochemical staining.

(a) Representative photographs show in situ accumulation of H2O2 in the leaves before (upper panel) and after the cold treatment via nitro blue tetrazolium (NBT) staining. (b) in situ accumulation of O2- in WT and MdCPK1a-OX plants before and after the cold stress by 3,3'-diaminobenzidine (DAB). (c) Evaluation of DAB staining in the leaves of plants before and after cold stress. (d) Evaluation of NBT staining in the leaves of plants before and after cold stress. The relative staining intensities were calculated based on the staining intensity of WT plants. Error bars indicate the SEM (n = 4); *P < 0.05.

Fig 7

Analysis of antioxidant enzyme activities in the WT and MdCPK1a-OX plants before and after cold treatment.

(a–c) Activities of SOD, POD and CAT, respectively. Data represent the means ± SE of at least three replicates. The significant differences between WT and MdCPK1a-OX plants are indicated by asterisks (*p< 0.05, **p < 0.01).

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 WT and the transgenic plants before and after cold treatments. The gene transcriptional levels of NtSOD, NtGPX and NtCAT were remarkably higher in transgenic tobacco whether under normal condition or cold stress, except NtrbohD which was lower under normal condition and significantly lower after cold stress in the transgenic tobacco. The mRNA levels of cold-responsive genes (NtLEA5, NtSPS, NtDREB3, except NtERD10C) were higher in the transgenic tobacco plants than those of WT plants under normal and cold stress condition. The expression of NtERD10C in the transgenic tobacco plants was similar with that in WT under normal condition, but higher under cold stress (Fig 8).

Fig 8

The expression of the ROS-related and cold-responsive genes in WT and MdCPK1a-OX plants.

Data represent the means ± SE of at least three replicates. The significant differences between the WT and MdCPK1a-OX plants are indicated by asterisks (*p< 0.05, **p < 0.01).

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 [ however, the biological functions of which mostly remain unclear. In this study, MdCPK1a was identified in apple and characterized in transgenic tobacco. The sequence alignment of MdCPK1a with different plant CDPKs shows high similarity with stress-responsive CDPK genes such as AtCPK1 [ OsCDPK7 [ and ZmCPK1 [. It suggests that MdCPK1a might be involved in stress tolerance.

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 [ 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 [. A recent study revealed that OsCPK17 has five alternative splicing (AS) forms with different subcellular localization [. 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 post-translational 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 [. The plasma membrane- or nucleus- localized CDPKs involved in abiotic stress response were also reported in other plants, such as ZoCDPK1 in Zingiber officinale [, SiCDPK24 [ 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 T2 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 Ca2+ derived from influx from the apoplastic space and release from internal stores [. Ca2+ binding proteins can sense the transient increases of cytosolic Ca2+, 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 [, 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 [. Furthermore, the transgenic Arabidopsis plants overexpressing VaCPK20, a CDPK from Vitis amurensis and PeCPK10 from Populus euphratica improved freezing resistance [, while in Zea mays, ZmCPK1 negatively regulate cold tolerance [ 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 [. 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 [. These results suggest that apple MdCPK1a like CDPK genes in other species has overlapping functions [

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 [. 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 [, on the other hand, they are also toxic byproducts that can cause oxidative destruction of cell [. In general, CDPKs seem to function as positive regulators of ROS production in biotic stress signaling [, , 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) [. 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) [ However, overexpression OsCPK12 decreases ROS accumulation by increasing the expression of OsAPx2, OsAPx8 and OsrbohI and confers increased tolerance to salt stress in rice [ Overexpression of OsCPK4 in rice confers salt and drought tolerance by preventing cellular membranes from stress-induced oxidative damage [. 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.

Primer sequences used for cloning, subcellular localization, vector construction, transgenic confirmation and expression analysis.

(TIF)

Click here for additional data file.

Phylogenetic relationship between MdCPK1a and other CDPK proteins.

The unrooted tree was generated using MEGA 6.0 program (http://www.megasoftware.net/) by the neighbor-joining method. Bootstrap supports from 500 replicates are indicated at each branch.

(TIF)

Click here for additional data file.

The identification of transgenic tobacco.

(a) Schematic representations of the vector constructs of pYH455-MdCPK1a. (b) Ten T1 lines of transgenic tobacco were confirmed by PCR with specific primers (PST1). (c) Six T1 lines of transgenic tobacco were confirmed by RT-PCR with specific primers (PST1). (d) Three T2 lines of transgenic tobacco were confirmed by RT-PCR with specific primers. (e) Quantification the expression of MdCPK1a mRNAs in the transgenic tobacco plants performed by real-time RT-PCR. RNA was extracted from the leaves of WT and MdCPK1a-transformed tobacco plant lines (A4, A36, and A2). Transcript abundance was normalized against the Nttubulin gene expression level. Data represent means and standard errors of three replicates. Significant differences between the WT and transgenic plants are indicated by asterisks(*p< 0.05, **p < 0.01).

(TIF)

Click here for additional data file.

(ZIP)

Click here for additional data file.

11 Sep 2019

PONE-D-19-19438

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

PLOS ONE

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Reviewer #1: Authors identified CPK1a from apple and characterized it. In addition, this research revealed that MdCPK1a might play important roles in the acclimation of plants to cold stress as well as salt and heat stress via activation of ROS scavenging systems. It might be important research leading to the elucidation of the roles of CDPK in crops, however, the points below should be addressed for the publication in this journal.

Authors indicated the nuclear and cytosolic localization of MdCPK1a by GFP fusion protein. But, nuclear localization should be confirmed by DAPI staining.

In the picture taken from the top (“a” on the left panel) in Fig.3, A2 plants seem to be much larger than WT plants. However, shoot and root dry weight of A2 are comparable with that of WT plants. This discrepancy between picture and graph should be explained.

In this study, the results presented in Figure 7 are essential to support the main conclusions. Authors therefore should quantify amount of ROS with more accurate way. Especially, the ways to quantify H2O2 by fluorescent dye or kit have been established.

Although the stress response phenotypes of the transgenic plants are shown in this study, the effects of MdCPK1a expression on growth of transgenic plants under non-stressed conditions are not clearly shown. The growth phenotypes of transgenic plants need to be analyzed and presented.

Reviewer #2: The manuscript by Dong et al describes the role of a calcium-dependent protein kinase from apple, MdCPK1a, in abiotic stress tolerance, using ectopic overexpression in tobacco plants. The transgenic lines appear more tolerant to salt and cold while they behave like wild-type under drought stress. The authors further studied the cold responses to link CDPK-mediated cold tolerance and ROS detoxification, by measuring ROS level, activity of detoxifying enzymes and gene expression. However, some results are not consistent.

Major comment:

1. The 3 transgenic lines should behave similarly to link the phenotype to the expression of MdCPK1a, or at least follow the overexpression level. Yet, it is often not the case. For heat stress, the higher MdCPK1a is expressed, the lower the tolerance is induced (Fig 5) and the authors claim in the abstract l.16 that “Ectopic expression of MdCPK1a in Nicotiana benthamiana increased its salt, heat and cold resistance” while in the discussion, they state l.303-304: “ectopic expression of MdCPK1a improved tobacco cold tolerance and also exhibit slightly increased salt tolerance, but no obvious improvement of heat and drought tolerance”. For cold, lines A4 and A36 already appear much bigger than WT in control conditions when grown on plates (Fig 6a), which questions whether the increased tolerance to cold is specific or just a consequence of initial bigger size. Moreover, the analyzed molecular parameters, i.e. enzyme activity and gene expression, are not consistent in the 3 transgenic lines, which makes it difficult to link those responses to MdCPK1a overexpression.

2. In Fig 2, the fluorescent signal of MdCPK1a-GFP being very weak, it is difficult to conclude that MdCPK1a localizes to plasma membrane and not cytosol. The authors should improve the quality of the pictures and check by western-blot that the signal corresponds to the fusion protein and not to GFP alone.

3. The literature is not always relevant. For example, l. 33 ref 52 is more relevant than ref 1. L. 45, ref 6 and 10 are not relevant here. L. 263, ref 51 doesn’t deal with AtCPK1. Instead, the authors should cite ref 48 and Gao et al Plos Pathogen 2013 vol 9: e1003127. And Ref 52 is not relevant there. L. 298, the authors should include OsCPK24 (Liu et al 2018 Journal of Integrative Plant Biology vol 2 p.173-188). L. 326, ref 74 is not relevant. Instead, the authors should cite Boudsocq et al Nature 2010 vol 464 p. 418-22; Dubiella et al, PNAS 2013 vol 110 p. 8744-8749; Gao et al Plos Pathogen 2013; Kadota et al Molecular Cell 2014 vol 54, p. 43-55.

Minor comments:

1. In Fig 3a, on the left panel, line A2 seems to be as tolerant as lines A4 and A36, which is different in the right panel. Moreover, the 2 panels are not well explained in the legend. The statistics are missing in fig 3b.

2. In Fig 7, the ROS should be quantified.

3. the stress protocols are not similar in methods and results. They should be clarified.

4. the English should be improved.

5. Figure legends are inverted in Fig 6 between panels c, d, e and f l. 403-407; sup fig S1 and S2 are inverted l.427, 437.

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21 Oct 2019

Response to the editors:

1. When submitting your revision, we need you to address these additional requirements. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming.

-------We changed the revised manuscript style according to PLOS ONE’s style requirements.

2. For reproducibility reasons we would recommend that you amend your methods section to include the source and/or deposition numbers of all the plants and seeds used in your study. We would also recommend providing some minimal details regarding the protocols references in section "Physiological measurements and histochemical staining protocol details" to allow researchers that may not have access to these references to reproduce your findings.

--------In the revised manuscript, we provided more details in the section “Physiological measurements and histochemical staining”

3. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files.

In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions.

-------- We have provided raw blot/gel images in supporting information.

4. Thank you for stating in your Funding Statement: [This work was supported by the National Natural Science Foundation of China (31872076, 31560551).].

* Please provide an amended statement that declares *all* the funding or sources of support (whether external or internal to your organization) received during this study, as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now. Please also include the statement “There was no additional external funding received for this study.” in your updated Funding Statement. Please include your amended Funding Statement within your cover letter. We will change the online submission form on your behalf.

------- We have provided all the funding. According the instruction, we included “There was no additional external funding receives for this study” in our updated funding statement.

Reviewer #1:

1. Authors indicated the nuclear and cytosolic localization of MdCPK1a by GFP fusion protein. But, nuclear localization should be confirmed by DAPI staining.

--------- Thank you for the reviewer’s suggestion! There is no doubt that the nuclear localization confirmed by DAPI staining can further guarantee the credibility of our result. We repeated the subcellular localization experiment for several times and the same results were obtained. We regretted that we did not take the photos of the DAPI staining. However, DAPI staining is not necessary for confirming nuclear localization. For example, a recent study revealed that some alternative splicing (AS) forms of OsCPK17 located in cytoplasm and nucleus. In this report, they constructed GFP-fused OsCPK17 AS isoform vectors (GFP-OsCPK17.1/2, GFP-OsCPK17.3, GFP-OsCPK17.4, and GFP-OsCPK17.5 or otherwise free GFP as a control). They used particle bombardment to identify OsCPK17 AS isoform subcellular localization in onion epidermal cells, and showed the subcellullar localization results in the Figure 3, in which they presented fluorescence projection, differential interference contrast (DIC) and merge images, but lack of DAPI staining or other nuclear markers (Almadanim et al, 2018). The similar case can also be found in the article Li et al.(2009), Plant Cell,21(2):429-441, including its correction at January 01, (2018). Plant Physiology, 177(3):1339-1341. In other case, to confirm the localization of some proteins on small organelles, such as on Golgi , Microsome, et al. It requires the organelle-markers (Almadanim et al, 2018).

Reference:

(1) Almadanim, M. C., Goncalves, N. M., Rosa, M. T., Alexandre, B. M., Cordeiro, A. M., Rodrigues, M., ... & Abreu, I. A. (2018). The rice cold-responsive calcium-dependent protein kinase OsCPK17 is regulated by alternative splicing and post-translational modifications. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 1865(2), 231-246.

(2) Li, S., Lauri, A., Ziemann, M., Busch, A., Bhave, M., & Zachgo, S. (2009). Nuclear activity of ROXY1, a glutaredoxin interacting with TGA factors, is required for petal development in Arabidopsis thaliana. The Plant Cell, 21(2), 429-441.

(3) Plant Physiology Jul 2018, 177 (3) 1339-1341; DOI: 10.1104/pp.18.0066

2. In the picture taken from the top (“a” on the left panel) in Fig.3, A2 plants seem to be much larger than WT plants. However, shoot and root dry weight of A2 are comparable with that of WT plants. This discrepancy between picture and graph should be explained.

------Thank for your careful reading. After salt stress, A2 plants seem to be much larger than WT plants,while the shoot and dry weight of A2 are comparable with that of WT plants. It may be that WT plants displayed yellowing and wilted leaves under salt stress, while the leaves of transgenic plants were green and leaves fully extended, so 3 transgenic lines all seem to be larger than WT plants from the overall looking, but for the individual plants, the plant high and root size between WT and A2 plants have no significant difference, which can be seen in Fig.3(“a” on the right panel).

3. In this study, the results presented in Figure 7 are essential to support the main conclusions. Authors therefore should quantify amount of ROS with more accurate way. Especially, the ways to quantify H2O2 by fluorescent dye or kit have been established.

------ We agreed the reviewer’s comment. If we measured the content of ROS, it can provide the direct evidence, but now, for lack of the growing transgenic tobacco plants, it is difficult for us to supplement the experiment in the short time. Actually, based on the histochemical staining to determine the level of the accumulation of H2O2 and O2- has been used in many papers, such as Shen et al (2016), Trevisan et al.(2019) and Sahoo et al.(2019). For better describing the degree of staining, we supplement the column diagram of the percentage of staining area according to the statistic results of five staining leaves for each treatment in Figure 7 of the revised manuscript, which could quantify the amount of ROS indirectly.

Figure 7

Reference:

Shen, L., Yang, S., Yang, T., Liang, J., Cheng, W., Wen, J., ... & Shi, W. (2016). CaCDPK15 positively regulates pepper responses to Ralstonia solanacearum inoculation and forms a positive-feedback loop with CaWRKY40 to amplify defense signaling. Scientific reports, 6, 22439.

Trevisan, Sara, et al. Nitrate affects transcriptional regulation of UPBEAT1 and ROS localisation in roots of Zea mays L. Physiologia plantarum 166.3 (2019): 794-811.

Sahoo, S., Saha, B., Awasthi, J. P., Omisun, T., Borgohain, P., Hussain, S., ... & Panda, S. K. (2019). Physiological introspection into differential drought tolerance in rice cultivars of North East India. Acta Physiologiae Plantarum, 41(4), 53.

4. Although the stress response phenotypes of the transgenic plants are shown in this study, the effects of MdCPK1a expression on growth of transgenic plants under non-stressed conditions are not clearly shown. The growth phenotypes of transgenic plants need to be analyzed and presented.

--------The growth phenotypes of transgenic plants has been presented in this paper actually. The Fig 6a shows the phenotype of WT, A4, A36 and A2 at the normal growth temperature for 10 days on MS medium after germinated. It showed that the root length of transgenic plants A4 and A36 is longer, but only of A4 is significant longer than that of WT. In the panel of “before drought” of Figure 4 , the growth of transgenic plants under the normal conditions are also shown. It shows that the aerial part of WT plants is little higher than that of the transgenic plants under normal condition. We also speculated that MdCPK1a might participate in the regulation of plant development.

Thus，in the discussion of revised manuscript, in line 449-454, we added the discussion as below: “Additionally, the root length of transgenic plants A4 and A36 is longer than that of WT when the seedlings of them cultured at 25℃ for 10 d on MS medium( Fig 6a) . The aerial part of WT plants is a little higher than that of the transgenic plants under normal condition (Fig 4). We speculated that MdCPK1a might also participate in the regulation of plant development.”

Reviewer #2:

Major comment:

1. The 3 transgenic lines should behave similarly to link the phenotype to the expression of MdCPK1a, or at least follow the overexpression level. Yet, it is often not the case. For heat stress, the higher MdCPK1a is expressed, the lower the tolerance is induced (Fig 5) and the authors claim in the abstract l.16 that “Ectopic expression of MdCPK1a in Nicotiana benthamiana increased its salt, heat and cold resistance” while in the discussion, they state l.303-304: “ectopic expression of MdCPK1a improved tobacco cold tolerance and also exhibit slightly increased salt tolerance, but no obvious improvement of heat and drought tolerance”. For cold, lines A4 and A36 already appear much bigger than WT in control conditions when grown on plates (Fig 6a), which questions whether the increased tolerance to cold is specific or just a consequence of initial bigger size. Moreover, the analyzed molecular parameters, i.e. enzyme activity and gene expression, are not consistent in the 3 transgenic lines, which makes it difficult to link those responses to MdCPK1a overexpression.

--------We apologized for confusing the reader because of our carelessness. Ectopic expression of MdCPK1a improved tobacco cold and salt tolerance, but no obvious improvement of heat and drought tolerance.The description in the summary is incorrect and has been modified.

AS for the comment “whether the increased tolerance to cold is specific or just a consequence of initial bigger size?” CDPK regulating development in plant has been reported in some studies ( see review by Singh et al. 2017 ). In our study, we also supposed that MdCPK1a might involve in plant development (Fig 6a). We added the discussion in L449-L454. On the other hand, we don’t think that the bigger size of transgenic plants will influence on the cold resistance ability in this experiment. We think the cold stress related parameters, such as the content of MDA，the electrolyte leakage，the enzyme activities, et al. do not change with the plant size.

In our research, it was no doubt that the stress related enzyme activities and the stress related gene expression of the transgenic plants were higher that those of control plant. The transgenic tobacco plants are more resistant to cold and salt stresses as result of overexpression of MdCPK1a. Why are the stress related enzyme activities and gene expression not consistent with the gene expression of MdCPK1a ? We explained that the posttranscriptional or posttranslational modification of MdCPK1a might exist in MdCPK1a -OX plants, which might be influenced by the insertion locus of MdCPK1a.

Reference:

Singh, A., Sagar, S., & Biswas, D. K. (2017). Calcium dependent protein kinase, a versatile player in plant stress management and development. Critical reviews in plant sciences, 36(5-6), 336-352.

2. In Fig 2, the fluorescent signal of MdCPK1a-GFP being very weak, it is difficult to conclude that MdCPK1a localizes to plasma membrane and not cytosol. The authors should improve the quality of the pictures and check by western-blot that the signal corresponds to the fusion protein and not to GFP alone.

------- Indeed, the fluorescent signal of MdCPK1a-GFP is weak on plasma membrane. We also believe it will make our conclusion more convincing if we conducted western-blot. The similar comment was given on the subcelluar localization by Reviewer #1. Please refer to our response to the comment to Reviewer #1. According to the reviewer’s suggestion, we improved the quality of the image. It became more clearly.

3. The literature is not always relevant.

------Thank you very much for careful reading! According the reviewer’s suggestion, we corrected the references accordingly.

For example, l. 33 ref 52 is more relevant than ref 1.

------We corrected it.

L. 45, ref 6 and 10 are not relevant here.

------We deleted the two reference.

L. 263, ref 51 doesn’t deal with AtCPK1. Instead, the authors should cite ref 48 and Gao et al Plos Pathogen 2013 vol 9: e1003127. And Ref 52 is not relevant there.

-------We corrected it.

L. 298, the authors should include OsCPK24 (Liu et al 2018 Journal of Integrative Plant Biology vol 2 p.173-188).

-------We added OsCPK24 in the manuscript, and cited the reference by Liu et al (2018).

L. 326, ref 74 is not relevant. Instead, the authors should cite Boudsocq et al Nature 2010 vol 464 p. 418-22; Dubiella et al, PNAS 2013 vol 110 p. 8744-8749; Gao et al Plos Pathogen 2013; Kadota et al Molecular Cell 2014 vol 54, p. 43-55.

-------We corrected it.

Minor comments:

1. In Fig 3a, on the left panel, line A2 seems to be as tolerant as lines A4 and A36, which is different in the right panel. Moreover, the 2 panels are not well explained in the legend. The statistics are missing in fig 3b.

-------The comment was also given by the Reviewer #1. Please refer to our response above. Thank you for your careful reviewing! The shoot dry weight of A4 and A36 (except A2) was significantly higher than that of WT; The root dry weight of A4 (except A36 and A2)was significantly higher than that of WT. We added the statistics on the Fig.3b.

2. In Fig 7, the ROS should be quantified.

-------We responded the same comment to the Reviewer #1 on the above.

3. the stress protocols are not similar in methods and results. They should be clarified.

-------We read carefully and already kept the stress protocol of methods and results consistent.

4. the English should be improved.

------- We polished the English.

5. Figure legends are inverted in Fig 6 between panels c, d, e and f l. 403-407; sup fig S1 and S2 are inverted l.427, 437.

------- Thank you so much for your careful reading! We corrected it .

Submitted filename: Response to reviewers.docx

Click here for additional data file.

12 Dec 2019

PONE-D-19-19438R1

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

PLOS ONE

Dear Prof. Wang,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: (No Response)

Reviewer #2: Partly

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Basically, authors properly answered the comments by explaining the detail. However, I found another point to be modified. Fig.4 might be important pictures that also show difference in growth even under normal condition. Scale bars should be indicated to further provide information of plant size.

Reviewer #2: The authors answered part of the initial comments but some points still need clarification.

Major comments:

1. The part on heat tolerance is confusing since the authors conclude l. 434-436 that “ectopic expression of MdCPK1a exhibit…no obvious improvement of heat and drought tolerance” while in the results, they state l.310-311 “the survival rates of…A36 (24%) and A2 (98%) were remarkably higher than WT”. Indeed, the thermotolerance is inversely correlated with MdCPK1 expression level in the transgenics, which makes it difficult to conclude without any additional investigation. Thus, the whole part on heat stress tolerance should be deleted from this manuscript.

2. The picture of MdCPK1 localization has been more contrasted. However, the membrane localization is still not demonstrated. Indeed, some cytoplasmic strands are visible and MdCPK1 could be located in cytosol and/or plasma membrane. This experiment is important to correlate with the acylation prediction of MdCPK1. Either the authors balance their statement, or they provide additional data such as co-localization with known plasma membrane protein, or western-blot of proteins extracted in the presence/absence of triton X100 which will extract total proteins including the membrane ones (presence) or only soluble ones (absence). Moreover, a western-blot will additionally prove that the fluorescence observed is due to MdCPK1-GFP fusion and not GFP alone. Finally, “plasma membrane” should be deleted l.265-266 since free GFP doesn’t go to membrane. It only diffuses to cytosol and nucleus.

Minor comments:

1. In Fig3, the discrepancy between pictures (a, left panel) and graphs (b, left panel) is surprising. Maybe measuring fresh weight would have been more relevant here. Nevertheless, the authors could comment on that: it suggests that under salt stress, MdCPK1 transgenics retain water better than WT.

2. In Fig7, the authors added quantification of the ROS staining. The corresponding legend is missing l.358-362, the panels c and d should be cited in the results l.352, and the way they quantified the staining should be explained in the methods.

3. In the discussion l.447-448, the authors state that “the aerial part of WT plants is little higher than that of transgenics under normal conditions”. However, the picture in Fig4 shows that WT plants are actually smaller than the transgenics in control conditions. This should be corrected.

4. The stress protocols are still different in methods and figures. In results, salt stress (l.285) and drought (l.303) are performed for 25d on 4-week-old plants while in the methods, 4-week-old plants are further grown for 14d before applying stress, ie 6-week-old before stress (l.169-170). Also correct accordingly if needed the legend l.305-307.

5. L.86, Ralstonia solanac should be Ralstonia solanacearum. L.402, predicated should be predicted.

6. English still needs some improvement, especially l.89-91, l.113-115, l.178-180.

7. L.251, the sentence should be rephrased by “the selected CDPK proteins were clustered into three subgroups” because the authors just didn’t include CDPKs from subgroup IV. L.252, ZmCDPK1 should be “ZmCPK1”.

8. In FigS2, the qRT-PCR (panel e) is missing.

9. Fig6 has been reorganized but the labels are now wrong: a and b are inverted, c and d, and e and f, as well. Moreover, the genotype labelling is missing in panel b (which should be “a” with MS and MS+4°C).

10. L.375-378, the sentence should be modified because NtSPS and NtLEA5 are already strongly induced in MdCPK1 transgenics in normal conditions.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

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28 Sep 2020

Thank you for the two reviewers and the editors

We are grateful to your critical reading. Your comments are invaluable for the manuscript conforming to the requirement for publishing. The comments and responses were listed as follows. We hope the revised manuscript will meet the requirements for publication on Plos One.

Reviewer #1: Basically, authors properly answered the comments by explaining the detail. However, I found another point to be modified. Fig.4 might be important pictures that also show difference in growth even under normal condition. Scale bars should be indicated to further provide information of plant size.

Reply：Thank you for the reviewer’s suggestion！We have indicated the scale bars in the figure 4 and the legends. Bars =10 cm.

Reviewer #2: The authors answered part of the initial comments but some points still need clarification.

Major comments:

1.The part on heat tolerance is confusing since the authors conclude l. 434-436 that “ectopic expression of MdCPK1a exhibit…no obvious improvement of heat and drought tolerance” while in the results, they state l.310-311 “the survival rates of…A36 (24%) and A2 (98%) were remarkably higher than WT”. Indeed, the thermotolerance is inversely correlated with MdCPK1 expression level in the transgenics, which makes it difficult to conclude without any additional investigation. Thus, the whole part on heat stress tolerance should be deleted from this manuscript.

Reply: Thank you for the reviewer’s suggestion. We deleted the whole part of heat stress in the manuscript.

2. The picture of MdCPK1 localization has been more contrasted. However, the membrane localization is still not demonstrated. Indeed, some cytoplasmic strands are visible and MdCPK1 could be located in cytosol and/or plasma membrane. This experiment is important to correlate with the acylation prediction of MdCPK1. Either the authors balance their statement, or they provide additional data such as co-localization with known plasma membrane protein, or western-blot of proteins extracted in the presence/absence of triton X100 which will extract total proteins including the membrane ones (presence) or only soluble ones (absence). Moreover, a western-blot will additionally prove that the fluorescence observed is due to MdCPK1-GFP fusion and not GFP alone. Finally, “plasma membrane” should be deleted l.265-266 since free GFP doesn’t go to membrane. It only diffuses to cytosol and nucleus.

Reply: Thank you for your suggestion. We conducted the supplementary experiment of Western Blot to further confirm the subcellular localization of MdCPK1 according to the reviewer’ suggestion.

The Western blot assay showed that MdCPK1a-GFP was exclusively detected in the cell membrane and nucleus fractions, but not in the cytosolic by immunoblotting with anti-GFP antibody (Figure 2c). The results indicated that MdCPK1a protein was localized to the nucleus and cell membrane. The result was added in the revised manuscript and Figure 2 was modified also accordingly..

Minor comments:

1. In Fig3, the discrepancy between pictures (a, left panel) and graphs (b, left panel) is surprising. Maybe measuring fresh weight would have been more relevant here. Nevertheless, the authors could comment on that: it suggests that under salt stress, MdCPK1 transgenics retain water better than WT.

Reply: we agreed with the reviewer’s comments that fresh weight would have been more relevant here. However, we believed that dry weight might more accurately reflect difference of the growth and development of lines. A transgenic line will increase more biomass and accumulate more dry matter if it has higher adaptability under certain condition, accordingly, the dry weight of the plant is higher than the others. We don’t think the picture a(left) and graph of b(left) is discrepant.

2. In Fig7, the authors added quantification of the ROS staining. The corresponding legend is missing l.358-362, the panels c and d should be cited in the results l.352, and the way they quantified the staining should be explained in the methods.

Reply: We apologized for our carelessness. The way of quantification for the staining have been added in the methods. The corresponding legend has been added for the Fig7 (in the revised manuscript, it was renamed Fig.6) and the panels c and d were cited in the results.

3. In the discussion l.447-448, the authors state that “the aerial part of WT plants is little higher than that of transgenics under normal conditions”. However, the picture in Fig4 shows that WT plants are actually smaller than the transgenics in control conditions. This should be corrected.

Reply: We apologized for our carelessness, we have corrected the mistake.

4. The stress protocols are still different in methods and figures. In results, salt stress (l.285) and drought (l.303) are performed for 25d on 4-week-old plants while in the methods, 4-week-old plants are further grown for 14 d before applying stress, ie 6-week-old before stress (l.169-170). Also correct accordingly if needed the legend l.305-307.

Reply: Thank you for the reviewer’s careful reading. We corrected the mistyping. The transgenic lines were all treated at 6 weeks old.

5. L.86, Ralstonia solanac should be Ralstonia solanacearum. L.402, predicated should be predicted.

Reply: Thank so much for your comments! we have corrected the error.

6. English still needs some improvement, especially l.89-91, l.113-115, l.178-180.

Reply: Thank you! We proofread the whole manuscript and improved the three sentences as follows:

L89-91: Conversely, some CDPKs play negative regulators of stress response for transgenic plants overexpressed them show more sensitive to abiotic/biotic stresses.

L113-115: 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.

L.178-180: 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.

7. L.251, the sentence should be rephrased by “the selected CDPK proteins were clustered into three subgroups” because the authors just didn’t include CDPKs from subgroup IV. L.252, ZmCDPK1 should be “ZmCPK1”.

Reply: Thank you for your suggestion! We have rephrased the sentence in the revised manuscript.

8. In FigS2, the qRT-PCR (panel e) is missing.

Reply: We added the qPCR (panel e) in the new FigS2.

9. Fig6 has been reorganized but the labels are now wrong: a and b are inverted, c and d, and e and f, as well. Moreover, the genotype labelling is missing in panel b (which should be “a” with MS and MS+4°C).

Reply: Thank you! We have reorganized the Fig5 and re-labelled (the original figure 6 was revised as figure 5 in the revised manuscript).

10. L.375-378, the sentence should be modified because NtSPS and NtLEA5 are already strongly induced in MdCPK1 transgenics in normal conditions.

Reply: we have modified the sentence.

The mRNA levels of cold-responsive genes except NtERD10C were higher in the transgenic tobacco than those of WT plants under normal and cold stress condition (Fig 8). The expression of NtERD10C in the transgenic tobacco was similar with that in WT under normal condition, but higher under cold stress.

Submitted filename: response to the reviewers.doc

Click here for additional data file.

28 Oct 2020

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

PONE-D-19-19438R2

Dear Dr. Wang,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Hidenori Sassa

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I think authors properly answered the comments from reviewers. Indeed, the manuscript was much improved after the revisions. For me, this manuscript is currently sufficient for the publication in this journal.

Reviewer #2: The authors answered almost to all my comments. There are just few minor editing points left:

1. English still needs improvement. L. 87-88 could be “Conversely, some CDPKs are negative regulators of stress responses because transgenic plants overexpressing them are more sensitive to abiotic/biotic stresses.” L.352-354 could be “To know whether MdCPK1a regulates ROS levels in cold response, we compared the ROS levels in the overexpressing tobacco lines and WT plants after suffering cold stress.”

2. l.191, “3 ml” has been replaced by “equal volume”. But this would correspond to 1 ml, and not 3 ml. Please correct or confirm the good value.

3. l.259, and l.399, ZmCDPK1 should be ZmCPK1.

4. L.469, ref 48 should be ref 49.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

3 Nov 2020

PONE-D-19-19438R2

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

Dear Dr. Wang:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Hidenori Sassa

Academic Editor

PLOS ONE