The role of calcium-dependent protein kinase in hydrogen peroxide, nitric oxide and ABA-dependent cold acclimation

The role of calcium-dependent protein kinase in hydrogen peroxide, nitric oxide and ABA-dependent... Abstract Cold acclimation-induced cold tolerance is associated with the generation of reactive oxygen species (ROS), nitric oxide (NO), and mitogen-activated protein kinases (MPKs) in plants. Here, we hypothesized that calcium-dependent protein kinases (CPKs) induce a crosstalk among ROS, NO, and MPKs, leading to the activation of abscisic acid (ABA) signaling in plant adaptation to cold stress. Results showed that cold acclimation significantly increased the transcript levels of CPK27 along with the biosynthesis of ABA in tomato (Solanum lycopersicum). Silencing of CPK27 compromised acclimation-induced cold tolerance, generation of hydrogen peroxide (H2O2) in the apoplast, NO and ABA accumulation, and the activation of MPK1/2. Crosstalk among H2O2, NO, and MPK1/2 contributes to the homeostasis of H2O2 and NO, activation of MPK1/2, and cold tolerance. ABA is also critical for CPK27-induced cold tolerance, generation of H2O2 and NO, and the activation of MPK1/2. These results strongly suggest that CPK27 may function as a positive regulator of ABA generation by activating the production of ROS and NO as well as MPK1/2 in cold adaptation. Abscisic acid, calcium-dependent protein kinase, cold acclimation, hydrogen peroxide, mitogen-activated protein kinase, nitric oxide, Solanum lycopersicum Introduction Plants undergo continuous exposure to various biotic and abiotic stresses in their growth environment. Cold stress adversely affects the growth and development of plants and significantly constrains their spatial distribution and agricultural productivity. To survive under stressful conditions, plants have evolved intricate mechanisms to perceive external signals, allowing optimal responses to environmental conditions. For example, many plants show decreased sensitivity to cold stress when they have undergone prior exposure to suboptimal low temperatures, a phenomenon called cold acclimation. Cold acclimation involves the remodeling of cell and tissue structures and the reprogramming of metabolism and gene expression (Thomashow, 1998; Chinnusamy et al., 2007). The molecular basis of this acquired cold tolerance is not completely understood. Generally, a stress signal is first perceived by a membrane receptor, followed by the up-regulation of the cytoplasmic Ca2+ ion level (McAinsh and Hetherington, 1998). This change in cytoplasmic Ca2+ is sensed by calcium sensors such as calcium-dependent protein kinases (CPKs), which interact with downstream signaling components including hormones such as abscisic acid (ABA), reactive oxygen species (ROS), and mitogen-activated protein kinases (MPKs), leading to an increased tolerance to various stresses (Boudsocq and Sheen, 2013). CPKs play an important role in signal transduction, and the functions of several CPKs in Arabidopsis and rice have recently been identified (Almadanim et al., 2017; Jin et al., 2017). Activated CPKs participate in different signal transduction pathways through the phosphorylation of specific substrates (Kobayashi et al., 2012; Santin et al., 2017). Recently, several CPK substrates have been identified. For instance, MPK5 is directly phosphorylated by CPK18 to regulate stress responses and disease resistance in rice, while StCDPK5 and AtCPK1/2/4/5/11 can phosphorylate and thereby activate NADPH oxidase to promote the production of ROS in response to abiotic and biotic stimuli (Kobayashi et al., 2007; Dubiella et al., 2013; Gao et al., 2013; Xie et al., 2014). The tight regulation of steady-state levels of ROS is important for many cellular processes in plants (Fujita et al., 2006). While excess ROS accumulation induces oxidative stress in cells, ROS also function as signaling molecules (Fujita et al., 2006). Recent studies suggest that the NADPH-dependent respiratory burst oxidase homolog (RBOH) genes are involved in cold acclimation-induced cold tolerance in several plants. Silencing of RBOH1 in tomato increased the sensitivity of plants to cold and attenuated the protective role of cold acclimation (Zhou et al., 2012). Similar to ROS, nitric oxide (NO) also participates in the regulation of cold response in several plant species (Zhao et al., 2009; Diao et al., 2017). In particular, nitrate reductase (NR)-dependent NO participates in the regulation of the cold acclimation process (Zhao et al., 2009; Lv et al., 2017). In a number of cases, generation of NO and H2O2 occurs in parallel or in rapid succession, and these molecules can act synergistically or independently (Bright et al., 2006; Asai et al., 2008; Shi et al., 2015; Deng et al., 2016). In addition, increased generation of ROS/NO is frequently accompanied by the activation of MPKs. Silencing of MPK1 and MPK2 compromised cold acclimation-induced chilling tolerance and the activation of several antioxidant enzymes in tomato (Lv et al., 2017). However, the role of CPKs in regulating the generation of ROS/NO and the activation of MPKs is unclear. As a phytohormone, ABA is extensively involved in the responses to abiotic stresses such as drought, low temperature, and osmotic stress. In response to cold stress, plants usually accumulate an increased amount of ABA. Many stress-inducible genes are regulated by the endogenous ABA that accumulates during stress (Shinozaki et al., 2003). Plants deficient or impaired in ABA biosynthesis or signaling components show a reduced response to cold acclimation (Gilmour and Thomashow, 1991; Mantyla et al., 1995). ABA can also act as a secondary signal to induce changes in Ca2+ levels that eventually impact cold signaling. ABA-enhanced stress tolerance is associated with the induction of diverse signaling molecules, such as Ca2+, calmodulin (CaM) (Hu et al., 2007), H2O2 (Jiang and Zhang, 2002), NO (Neill et al., 2008), MPK (Zhang et al., 2006; Xing et al., 2008), and CPK (Ding et al., 2013; Zhu et al., 2007; Zou et al., 2015), suggesting an intensive crosstalk among ROS, NO, and MPKs. However, how plants transduce calcium signaling to affect ROS, NO, and MPK signaling remains largely unclear, as does the relation of CPKs to ABA in cold acclimation. Here, we hypothesized that CPKs induce a crosstalk among ROS, NO, and MPKs, leading to the activation of ABA signaling in plant adaptation to cold stress. To this end, we identified the critical CPKs in the cold response and examined their relation to the induction of H2O2, NO, and ABA signaling, and the activation of MPK1/2. Results showed that CPK27-mediated crosstalk between H2O2, NO, and MPK1/2 contributed to cold acclimation-induced ABA biosynthesis and cold tolerance in tomato plants. We also found that crosstalk between CPK27 and ABA was essential for the tight control of the interactions of H2O2 with NO and MPK1/2. Materials and methods Plant material and growth conditions In the current study, tomato (Solanum lycopersicum L.) cultivar Condine Red was used for the majority of experiments. Seedlings were grown in a mixture of vermiculite and peat at the ratio of 3:1 (v/v), receiving Hoagland’s nutrient solution daily. The growth conditions were as follows: 12 h photoperiod, temperature of 25/20 °C (day/night), and photosynthetic photon flux density (PPFD) of 600 µmol m−2 s−1. To examine the significance of endogenous ABA levels in cold acclimation-induced cold tolerance, seeds of wild type (WT, cv. Ailsa Craig) and an ABA biosynthesis mutant, notabilis (not), in the Ailsa Craig background were used. Virus-induced gene silencing (VIGS) was performed on the fully expanded cotyledons of 15-day-old tomato seedlings as described previously (Lv et al., 2017). The fragment for silencing CPK27 was PCR-amplified using the gene-specific primer listed in Supplementary Table S1 at JXB online, and the vectors for silencing NR and RBOH1 and co-silencing MPK1/2 were constructed as described previously (Kandoth et al., 2007; Zhou et al., 2014b; Lv et al., 2017). The infiltrated plants were grown at 21 °C and used in experiments at the five-leaf stage. qRT-PCR analysis was performed to evaluate gene silencing efficiency (Supplementary Fig. S1). Cold acclimation and cold stress treatments All experiments were carried out in environmentally controlled growth chambers using tomato plants at the five-leaf stage. For the cold acclimation treatment, plants were exposed to an aerial temperature of 12 °C with a 12/12 h light/dark cycle under 600 µmol m−2 s−1 PPFD and 85% humidity from 07.00 h. The cold acclimation treatment lasted for 3 d. Then, acclimated and non-acclimated plants were challenged with a cold stress treatment of 4 °C with 12/12 h light/dark cycle for 5 d under 600 μmol m−2 s−1 PPFD. There were four replicates in each treatment, and each replicate consisted of 16 plants. To determine the roles of H2O2, NO, and ABA in cold acclimation-induced cold tolerance, tomato seedlings at the five-leaf stage were sprayed with 10 mM H2O2, 500 μM sodium nitroprusside (SNP; a donor of NO) or 50 μM ABA. H2O2 and SNP were diluted with water while ABA was dissolved in a very low concentration of ethanol (0.01%, v/v) in all working solutions including control water solution. Twelve hours after the pretreatment, the plants were transferred to growth chambers for the cold acclimation treatment at 12 °C for 3 d and then exposed to 4 °C for 5 d. Evaluation of cold tolerance An Imaging-PAM Chlorophyll Fluorimeter equipped with a computer-operated PAM-control unit (IMAG-MAXI; Heinz Walz, Effeltrich, Germany) was used to detect the maximum quantum yield of PSII (Fv/Fm), as previously described (Zhou et al., 2012). The air temperature, relative humidity, CO2 concentration and PPFD were maintained at 25 °C, 85%, 380 μmol mol−1 and 1200 μmol m−2 s−1, respectively. Relative electrolyte leakage (REL, %) was determined following protocols described previously (Cao et al., 2007). Measurements of endogenous NO level and NR activity NO accumulation in leaves was monitored using the NO-sensitive dye 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-2DA) as reported previously (Lv et al., 2017). Leaf sections were infiltrated with 10 µM DAF-2DA prepared in 10 mM Tris–HCl (pH 7.4) and incubated in the dark at 37 °C for 30 min before observation. Fluorescence from DAF-2T, the reaction product of DAF-2DA with NO, was observed using a fluorescence stereomicroscope (Leica TCS SL; Leica Microsystems, Wetzlar, Germany) equipped with a charge-coupled device camera. The excitation was at 488 nm, and emission images at 525 nm were obtained with a constant acquisition time. The treatments were repeated 10 times. The signal intensities of green fluorescence in the images were quantified by ImageJ software (NIH, Bethesda, MD, USA). Values were corrected for background. Data were presented as the means of fluorescence intensity relative to those of control plants. In addition, NO accumulation was also determined by colorimetric assay with Griess reagent. Briefly, 0.3 g tomato leaves were homogenized using 50 mM glacial acetic acid (pH3.6) in an ice bath and centrifuged at 12000 g for 15 min. An aliquot of supernatant was mixed with Griess reagent (Sigma-Aldrich, USA) and kept at 25 °C for 30 min for reaction. The content of NO was calculated based on the absorbance of the reaction mixture at 540 nm. NO content was calculated by comparison with a standard curve of NaNO2. NR was assayed as described by Foyer et al. (1998) with small modifications. Briefly, 0.3 g leaf tissue was homogenized with 1.5 ml of extraction buffer [10 mM HEPES–KOH, pH 7.5, 5 mM DTT, 1 mM Na2MoO4, 10 mM FAD, 2 mM β-mercaptoethanol, 5 mM EDTA, and 1% polyvinylpolypyrrolidone (PVP)]. After centrifuging at 4 °C for 15 min at 12000 g, 0.3 ml of the supernatant was used for the NR activity assay. The reaction mixture (0.7 ml) consisted of 100 mM HEPES–KOH buffer, pH 7.5, 5 mM KNO3, and 0.25 mM NADH (freshly prepared). The reaction was terminated after 25 min by adding an equal volume of sulfanilamide [1% (w/v) in 3 M HCl] and then N-(1-naphthyl)-ethylenediamine dihydrochloride [0.02% (w/v)] to the reaction mixture, and the absorbance was measured using a spectrophotometer at 540 nm. The protein content was determined following the Coomassie blue staining method as previously described (Bradford, 1976). The NR activity was expressed as nmol of NO2− produced per minute and per milligram of protein. Measurements of endogenous H2O2 levels and NADPH oxidase activity H2O2 was extracted from leaf tissue according to Doulis et al. (1997) and measured as described in our previous study (Xia et al., 2011). For the determination, 0.3 g FW leaf material was homogenized with 3 ml of 0.2 M HClO4 on ice. After centrifuging at 6000 g for 5 min at 4 °C, the supernatant was adjusted to pH 6.5 with 4 M KOH, and centrifuged at 12 000 g for 5 min at 4 °C. After filtering through an AG1 × 8 prepacked column (Bio-Rad, Hercules, CA, USA), 800 µl of extracts was mixed with 400 µl reaction buffer containing 4 mM 2,2′-azino-di (3-ethylbenzthiazoline-6-sulfonic acid) and 100 mM potassium acetate at pH 4.4, 400 µl deionized water and 0.25 U of horseradish peroxidase (HRP). H2O2 content was measured at optical density at 412 nm. H2O2 was visualized at the subcellular level using CeCl3 for localization (Zhou et al., 2012). Tissue pieces (1–2 mm2) were cut from the leaves and incubated in freshly prepared 5 mM CeCl3 in 50 mM MOPS at pH 7.2 for 1 h. The leaf sections were then fixed in 1.25% (v/v) glutaraldehyde and 1.25% (v/v) paraformaldehyde in 50 mM sodium cacodylate buffer (pH 7.2) for 1 h. After fixation, tissues were washed twice for 10 min in the same buffer and postfixed for 45 min in 1% (v/v) osmium tetroxide and then dehydrated in a graded ethanol series (30–100%; v/v) and embedded in Eponaraldite (Agar Aids). After 12 h in pure resin, followed by a change of fresh resin for 4 h, the samples were polymerized at 60 ℃ for 48 h. Blocks were sectioned (70–90 nm) on a Reichert-Ultracut E microtome and mounted on uncoated copper grids (300 mesh). The CeCl3 deposits formed in the presence of H2O2 were examined using a transmission electron microscope (H7650, Hitachi, Tokyo, Japan) at an accelerating voltage of 75 kV (Bestwick et al., 1997). For the determination of NADPH oxidase activity, leaf plasma membranes were isolated using a two-phase aqueous polymer partition system (Xia et al., 2009). Briefly, leaves (5 g) were homogenized in 20 ml extraction buffer [50 mM Tris–HCl, pH 7.5, 0.25 M sucrose, 1 mM ascorbic acid (AsA), 1 mM EDTA, 0.6% PVP, and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. The homogenate was filtered through four layers of cheesecloth, and the resulting filtrate was centrifuged at 10000 g for 15 min. Microsomal membranes were pelleted from the supernatant by centrifugation at 100000 g for 30 min. The pellet was suspended in 0.33 M sucrose, 3 mM KCl, and 5 mM potassium phosphate, pH 7.8. The plasma membrane fraction was isolated by adding an aqueous two-phase polymer system to give a final composition of 6.2% (w/w) dextran T500, 6.2% (w/w) polyethylene glycol 3350, 0.33 M sucrose, 3 mM KCl, and 5 mM potassium phosphate, pH 7.8. Three successive rounds of partitioning yielded the final upper phase. The upper phase was diluted 5-fold in Tris–HCl dilution buffer (10 mM, pH 7.4) containing 0.25 M sucrose, 1 mM EDTA, 1 mM DTT, 1 mM AsA, and 1 mM PMSF. The fractions were centrifuged at 120000 g for 1 h. The pellets were then resuspended in Tris–HCl dilution buffer and used immediately for further analysis. The protein content was determined following the Coomassie blue staining method as previously described (Bradford, 1976). The NADPH-dependent O2−-generating activity in isolated plasma membrane vesicles was determined as described previously (Xia et al., 2009). The rates of O2− generation were calculated using an extinction coefficient of 21.6 mM−1 cm−1. Total RNA extraction and qRT-PCR analysis Total RNA was extracted using an RNAsimple Total RNA Kit (Tiangen, Beijing, China) following the manufacturer’s protocols. After removing residual DNA with a DNase Mini Kit (Qiagen, Hilden, Germany), total RNA was reverse transcribed using a ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan), following the manufacturer’s instructions. The gene-specific primer pairs used to amplify the 29 CPK genes were taken from a previously published study (Hu et al., 2016), and additional primers are shown in Supplementary Table S2. qRT-PCR was performed using a Roche LightCycler 480 real-time PCR machine (Roche, Basel, Switzerland). Relative transcript levels were calculated according to the method of Livak and Schmittgen (2001). The tomato ACTIN2 gene was used as an internal reference. Total protein extraction and western blot analysis Total protein extracts were obtained by homogenizing frozen powdered leaves in an extraction buffer (100 mM HEPES, pH 7.5, 5 mM EDTA, 5 mM EGTA, 10 mM DTT, 10 mM Na3VO4, 10 mM NaF, 50 mM β-glycerophosphate, 1 mM PMSF, 5 μg ml−1 antipain, 5 μg ml−1 aprotinin, 5 μg ml−1 leupeptin, 10% glycerol, and 7.5% PVP). The extracts were centrifuged at 12000 g for 20 min before quantifying total protein content. For immunoblots, protein extracts were resolved on a 12.5% SDS-PAGE gel and later transferred to polyvinylidene fluoride membranes (Bio-Rad). The activated state of MPK1/2 was detected using an anti-p42/44 MPK primary antibody (1:1000, Cell Signaling Technology, Boston, MA, USA) followed by anti-rabbit HRP-conjugated secondary antibodies (Cell Signaling Technology, Boston, MA, USA) (Zhou et al., 2014b). Accumulation of MPK1/2 was quantified using Quantity One software (Bio-Rad). To obtain a loading control, membranes were stained with Ponceau S solution. Measurement of ABA levels ABA extraction and analysis from tomato leaves were performed using previously described procedures (Wang et al., 2016). Briefly, 0.1 g of frozen leaf material was spiked with D6-ABA (OlChemIm Ltd, Czechoslovakia) as an internal standard to a final concentration of 100 ng ml−1 before extraction with 1 ml of ethyl acetate. Following shaking for 12 h in the dark at 4 °C and centrifuging at 18000 g for 10 min at 4 °C, the supernatant was collected and evaporated to dryness under N2 gas. The residue was then resuspended in 0.5 ml of 70% methanol (v/v) and centrifuged at 18000 g for 2 min at 4 °C, and the supernatant was analysed by high-performance liquid chromatography (HPLC)–mass spectrometry (MS) on an Agilent 1290 Infinity HPLC system (including a vacuum degasser, a binary pump, a column oven, and an autosampler) coupled to an Agilent 6460 Triple Quadrupole LC/MS system (Agilent Technologies, Heilbronn, Germany). The levels of ABA in tomato leaves were expressed as nanograms per gram fresh weight leaf material. Statistical analysis The experimental design was a completely randomized block with four replicates. Data were statistically analysed by one-way ANOVA and the means were compared using Tukey’s multiple comparison test at the P<0.05 level. Results CPK27 is critical for cold acclimation-induced cold tolerance and ABA biosynthesis In our previous study, we identified 29 CPK genes in tomato (Hu et al., 2016). To examine the potential roles of these CPKs in cold tolerance, we examined the changes in the transcript levels of these 29 CPKs in response to cold acclimation in the leaves by qRT-PCR. After cold acclimation at 12 °C for 3 d, followed by a cold stress treatment at 4 °C for 12 h, 14 out of the 29 CPKs genes showed increased transcript accumulation in the leaves, ranging from 2- to 12-fold (see Supplementary Fig. S2A). Among them, the transcript accumulation of CPK27 (accession number: Solyc11g065660.1.1) was most highly induced, with an increase of 12-fold relative to that of plants grown at 25 °C. A time course further revealed that the transcript level of CPK27 was increased within 0.5 d after cold exposure and remained high throughout the cold acclimation process (Fig. 1A). Moreover, a further increase in the CPK27 transcript level was found in plants when they were subsequently exposed to cold at 4 °C for 12 h. However, after exposure to 4 °C for 5 d, the CPK27 transcript level returned to values close to those of the controls at day 0. In comparison, the CPK27 transcript level in non-acclimated plants was up-regulated around 4-fold at 12 h after being exposed to 4 °C and then decreased to values lower than those of the controls at 5 d after the onset of cold stress at 4 °C (Fig. 1A). Fig. 1. View largeDownload slide CPK27 plays a vital role in cold acclimation-induced cold tolerance. (A) Time course of changes in transcript levels of CPK27 in response to cold stress with or without cold acclimation. (B) Relative electrolyte leakage (REL). (C) Maximum photochemical efficiency of photosystem II (Fv/Fm). (D) ABA content in the leaves of control (25 °C) and chilled (4 °C) tomato plants with or without cold acclimation. At the five-leaf stage, tomato plants were either cold acclimated (12 °C for 3 d) or kept at normal temperature (25 °C) before the imposition of cold stress (4 °C for 5 d). CPK27 gene expression was analysed at different time points as indicated in (A). REL and Fv/Fm values were determined at 5 d, while ABA content was assayed at 12 h after commencement of the cold stress treatment. The false color code depicted at the bottom of each image ranges from 0 (black) to 1.0 (purple), representing the level of leaf damage. Data are the means (±SD) of four biological replicates, except for Fv/Fm, which represents the mean of 15 leaves from independent plants. Different letters indicate significant differences (P<0.05) according to Tukey’s test. Fig. 1. View largeDownload slide CPK27 plays a vital role in cold acclimation-induced cold tolerance. (A) Time course of changes in transcript levels of CPK27 in response to cold stress with or without cold acclimation. (B) Relative electrolyte leakage (REL). (C) Maximum photochemical efficiency of photosystem II (Fv/Fm). (D) ABA content in the leaves of control (25 °C) and chilled (4 °C) tomato plants with or without cold acclimation. At the five-leaf stage, tomato plants were either cold acclimated (12 °C for 3 d) or kept at normal temperature (25 °C) before the imposition of cold stress (4 °C for 5 d). CPK27 gene expression was analysed at different time points as indicated in (A). REL and Fv/Fm values were determined at 5 d, while ABA content was assayed at 12 h after commencement of the cold stress treatment. The false color code depicted at the bottom of each image ranges from 0 (black) to 1.0 (purple), representing the level of leaf damage. Data are the means (±SD) of four biological replicates, except for Fv/Fm, which represents the mean of 15 leaves from independent plants. Different letters indicate significant differences (P<0.05) according to Tukey’s test. To determine the role of CPK27 in the cold response, we silenced CPK27 (pTRV-CPK27) by the VIGS approach, which reduced its transcript level by ca. 70% (see Supplementary Fig. S1C). In the CPK27-silenced plants, the expression of CPK22 and CPK26, two close homologs of CPK27, was not significantly changed. Under optimal growth conditions, the empty vector plants (pTRV) and the pTRV-CPK27 plants showed similar plant growth, maximum quantum yields of photosystem II (PSII) (Fv/Fm) and relative electrolyte leakage (REL) (Fig. 1B, C; Supplementary Fig. S2B). Notably, cold acclimation increased cold tolerance, as evidenced by fewer wilting symptoms, increased Fv/Fm, and decreased REL values relative to those of non-acclimated plants. However, no significant difference in REL was found between non-acclimated pTRV plants and non-acclimated pTRV-CPK27 plants while Fv/Fm was higher in pTRV plants than that in pTRV-CPK27 plants (Fig. 1B, C). Importantly, CPK27 silencing attenuated the effects of cold acclimation on cold tolerance, as evidenced by a lower Fv/Fm and an increased REL relative to those of pTRV plants after the cold stress at 4 °C. In addition, ABA accumulation was increased during the cold acclimation process and subsequent cold treatment in pTRV plants, and this increase in ABA accumulation was partly attenuated in pTRV-CPK27 plants (Fig. 1D). However, sudden cold treatment induced ABA accumulation to a lesser extent compared with cold acclimation in both pTRV plants and pTRV-CPK27 plants. CPK27 triggers the generation of H2O2 and NO and the activation of MPK1/2 NO, H2O2, and MPKs are all involved in cold acclimation-induced cold tolerance (Zhao et al., 2009; Cantrel et al., 2011; Zhou et al., 2012). We thus examined whether cold acclimation-induced accumulation of NO and H2O2 and the activation of MPK were CPK27 dependent. At 25 °C, no significant differences in the transcript levels of RBOH1, NR, MPK1, and MPK2 (see Supplementary Fig. S3A–D), the activities of NADPH oxidase and NR (Fig. 2A, C), or the accumulation levels of H2O2 and NO (Fig. 2B, D; Supplementary Fig. S3E–G) were found between pTRV plants and pTRV-CPK27 plants. Both cold acclimation and cold stress increased the transcript levels of RBOH1, NR, MPK1, and MPK2, the activities of NADPH oxidase and NR, and content of H2O2 and NO in pTRV plants (Fig. 2A–D; Supplementary Fig. S3). Subsequent exposure to cold stress further increased the transcript levels of RBOH1, NR, MPK1, and MPK2, the activities of NADPH oxidase and NR, and the accumulation levels of H2O2 and NO in acclimated plants (Fig. 2A–D; Supplementary Fig. S3). However, silencing CPK27 significantly attenuated the cold acclimation-induced transcript levels of RBOH1, NR, MPK1, and MPK2, the activities of NADPH oxidase and NR, and the accumulation levels of H2O2 and NO (Fig. 2A–D; Supplementary Fig. S3). MPK1/2 activation (the upper lane) was induced by cold acclimation but not by sudden cold at 4 °C in pTRV and pTRV-CPK27 plants (Fig. 2E). However, cold acclimation-induced MPK1/2 activation was attenuated in CPK27-silenced plants. In addition, we found that the application of H2O2 or SNP increased cold acclimation-induced cold tolerance not only in pTRV plants but also in pTRV-CPK27 plants (Supplementary Fig. S4). All these results suggest that CPK27 is involved in the cold acclimation-induced accumulation of H2O2 and NO and MPK1/2 activation and CPK27 appears to act upstream of H2O2 and NO in cold adaptation. Fig. 2. View largeDownload slide Silencing of CPK27 attenuates NADPH-oxidase-induced H2O2 production, nitrate reductase (NR)-induced nitric oxide (NO) production and MPK1/2 activation level in tomato plants. NADPH oxidase activity (A), H2O2 accumulation in the apoplast (B), NR activity (C), NO accumulation (D), and MPK1/2 activation level (E) in the leaves of control (25 °C) and chilled (4 °C) tomato plants with or without cold acclimation. H2O2 accumulation in the apoplast was assayed by observing CeCl3 precipitates located on the membrane using a transmission electron microscope. Blue arrows indicate apoplastic H2O2 accumulation; scale bar: 1 μm. NO accumulation in leaves was visualized using an NO-specific fluorescent probe, 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-2DA), and fluorescence was photographed with a laser scanning confocal microscope (LSCM-500, Zeiss, Germany). Representative images selected from 10 replicates of each treatment are shown in (D); scale bar: 50 μm. The numbers above the images in (E) indicate the relative intensity of MPK1/2 (the upper lane). All parameters in this figure were assayed at 12 h after commencement of the cold stress treatment. Data are the means (±SD) of four biological replicates. Different letters indicate significant differences (P<0.05) according to Tukey’s test. Fig. 2. View largeDownload slide Silencing of CPK27 attenuates NADPH-oxidase-induced H2O2 production, nitrate reductase (NR)-induced nitric oxide (NO) production and MPK1/2 activation level in tomato plants. NADPH oxidase activity (A), H2O2 accumulation in the apoplast (B), NR activity (C), NO accumulation (D), and MPK1/2 activation level (E) in the leaves of control (25 °C) and chilled (4 °C) tomato plants with or without cold acclimation. H2O2 accumulation in the apoplast was assayed by observing CeCl3 precipitates located on the membrane using a transmission electron microscope. Blue arrows indicate apoplastic H2O2 accumulation; scale bar: 1 μm. NO accumulation in leaves was visualized using an NO-specific fluorescent probe, 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-2DA), and fluorescence was photographed with a laser scanning confocal microscope (LSCM-500, Zeiss, Germany). Representative images selected from 10 replicates of each treatment are shown in (D); scale bar: 50 μm. The numbers above the images in (E) indicate the relative intensity of MPK1/2 (the upper lane). All parameters in this figure were assayed at 12 h after commencement of the cold stress treatment. Data are the means (±SD) of four biological replicates. Different letters indicate significant differences (P<0.05) according to Tukey’s test. Relation of NO, H2O2, and MPK1/2 in cold acclimation We next investigated how NO, H2O2, and MPK1/2 were involved in the acclimation-induced tolerance to cold. Plants with RBOH1 silencing (pTRV-RBOH1) showed increased sensitivity to cold stress despite the cold acclimation treatment as evidenced by increased REL and decreased Fv/Fm (Fig. 3A; Supplementary Fig. S5A). pTRV-RBOH1 plants showed decreased accumulation of NO and reduced activation of MPK1/2 relative to those in pTRV plants after the cold acclimation (Fig. 3B, C; Supplementary Fig. S5B, C). However, foliar application of exogenous H2O2 enhanced cold tolerance as indicated by the lower REL, higher Fv/Fm, and increased accumulation of NO, with the effects being more significant in pTRV-RBOH1 plants (Fig. 3A, B; Supplementary Fig. S5A–C). Exogenous H2O2 increased the activation of MPK1/2 in both pTRV plants and pTRV-RBOH1 plants (Fig. 3C). On the other hand, plants with NR silencing (pTRV-NR) showed increased REL and decreased Fv/Fm (Fig. 3D; Supplementary Fig. S5D), which was followed by decreased accumulation of H2O2 and reduced activation of MPK1/2 relative to pTRV after the cold acclimation (Fig. 3E, F; Supplementary Fig. S5E). Application of SNP enhanced cold tolerance with lower REL, higher Fv/Fm, and increased accumulation of H2O2 in the apoplast, with the effects being more significant in pTRV-NR plants (Fig. 3E; Supplementary Fig. S5E). In addition, SNP application further increased the activation of MPK1/2 in both pTRV plants and pTRV-NR plants (Fig. 3F). These results not only demonstrated the role of RBOH1 and NR in the MPK1/2 activation and cold tolerance, but also indicated the interdependency of the production of H2O2 and that of NO during the cold acclimation. Fig. 3. View largeDownload slide NADPH-oxidase-dependent H2O2 and nitrate reductase (NR)-dependent NO are essential for cold acclimation-induced MPK1/2 activation and cold tolerance. (A, D) REL in RBOH1- (A) and NR- (D) silenced plants. (B, E) NO accumulation in RBOH1-silenced plants (B) and H2O2 accumulation in the apoplast in NR-silenced plants (D). (C, F) MPK1/2 activation level in RBOH1- (C) and NR- (F) silenced plants. H2O2 at 10 mM and SNP at 500 μM were applied 12 h before the cold acclimation treatment. H2O2 and NO accumulation in leaves were visualized using the same protocols as shown in Fig. 2. Scale bars: 50 μm (B) and 1 μm (E). The numbers above the images in (C, F) indicate the relative intensity of MPK1/2 (the upper lane). REL was determined at 5 d, while the other parameters were assayed at 12 h after commencement of the cold stress treatment. Data are the means (±SD) of four biological replicates. Different letters indicate significant differences (P<0.05) according to Tukey’s test. Fig. 3. View largeDownload slide NADPH-oxidase-dependent H2O2 and nitrate reductase (NR)-dependent NO are essential for cold acclimation-induced MPK1/2 activation and cold tolerance. (A, D) REL in RBOH1- (A) and NR- (D) silenced plants. (B, E) NO accumulation in RBOH1-silenced plants (B) and H2O2 accumulation in the apoplast in NR-silenced plants (D). (C, F) MPK1/2 activation level in RBOH1- (C) and NR- (F) silenced plants. H2O2 at 10 mM and SNP at 500 μM were applied 12 h before the cold acclimation treatment. H2O2 and NO accumulation in leaves were visualized using the same protocols as shown in Fig. 2. Scale bars: 50 μm (B) and 1 μm (E). The numbers above the images in (C, F) indicate the relative intensity of MPK1/2 (the upper lane). REL was determined at 5 d, while the other parameters were assayed at 12 h after commencement of the cold stress treatment. Data are the means (±SD) of four biological replicates. Different letters indicate significant differences (P<0.05) according to Tukey’s test. To examine the respective contribution of H2O2 and NO in acclimation-induced cold tolerance and the activation of MPK1/2, we applied either H2O2 or SNP to pTRV-NR and pTRV-RBOH1 plants before they were exposed to the cold acclimation and cold stress treatments. We found that the application of H2O2 induced cold tolerance not only in pTRV plants but also in pTRV-NR plants as evidenced by the decreased REL (Fig. 4A). In comparison, the application of SNP decreased REL in both pTRV and pTRV-RBOH1 plants. In agreement with these results, the application of H2O2 induced the activation of MPK1/2 in both pTRV and pTRV-NR plants, while the application of SNP induced the activation of MPK1/2 in both pTRV and pTRV-RBOH1 plants (Fig. 4B). These results demonstrated that RBOH1-dependent H2O2 production and NR-dependent NO production both play a critical role in cold acclimation-induced MPK1/2 activation and cold tolerance in tomato plants. Moreover, the results indicated that H2O2 and NO could independently regulate cold acclimation-induced cold tolerance and MPK1/2 activation. Fig. 4. View largeDownload slide Crosstalk between H2O2 and NO and its role in the regulation of MPK1/2 activation in tomato plants. (A, B) REL (A) and MPK1/2 activation (B) in RBOH1- or NR-silenced plants sprayed with 500 μM SNP or 10 mM H2O2, respectively. pTRV plants sprayed with water at 25 °C are shown as control. The numbers above the images in (B) indicate the relative intensity of MPK1/2 (the upper lane). REL and MPK1/2 activation were assayed at 5 d and 12 h, respectively, after commencement of the cold stress treatment. Data are the means (±SD) of four biological replicates. Different letters indicate significant differences (P<0.05) according to Tukey’s test. Fig. 4. View largeDownload slide Crosstalk between H2O2 and NO and its role in the regulation of MPK1/2 activation in tomato plants. (A, B) REL (A) and MPK1/2 activation (B) in RBOH1- or NR-silenced plants sprayed with 500 μM SNP or 10 mM H2O2, respectively. pTRV plants sprayed with water at 25 °C are shown as control. The numbers above the images in (B) indicate the relative intensity of MPK1/2 (the upper lane). REL and MPK1/2 activation were assayed at 5 d and 12 h, respectively, after commencement of the cold stress treatment. Data are the means (±SD) of four biological replicates. Different letters indicate significant differences (P<0.05) according to Tukey’s test. We then determined whether cold acclimation-activated MPK1/2 in turn affects the homeostasis of H2O2 and NO in response to cold. The results showed that MPK1/2 silencing not only attenuated the acclimation-induced cold tolerance with higher REL and lower Fv/Fm, but also decreased the accumulation of H2O2 and NO (Fig. 5; Supplementary Fig. S6). Exogenous application of H2O2 and SNP restored cold tolerance, as evidenced by the decreased REL and increased Fv/Fm in pTRV-MPK1/2 plants (Fig. 5A; Supplementary Fig. S6A). Furthermore, exogenous application of SNP and H2O2 induced the accumulation of H2O2 and NO, respectively, in pTRV-MPK1/2 plants (Fig. 5B, C; Supplementary Fig. S6B–D). Fig. 5. View largeDownload slide Influence of MPK1/2 co-silencing and exogenous SNP or H2O2 on REL (A), apoplastic H2O2 (B) or NO level (C) with regard to cold acclimation-induced cold tolerance in tomato plants. SNP at 500 μM and H2O2 at 10 mM were applied 12 h before the cold acclimation treatment. Leaf samples were taken at 5 d after commencement of the cold stress treatment for the determination of REL. H2O2 and NO accumulation levels were estimated as described in Fig. 2. Scale bars: 50 μm (C) and 1 μm (B). Data are the means (±SD) of four biological replicates. Different letters indicate significant differences (P<0.05) according to Tukey’s test. Fig. 5. View largeDownload slide Influence of MPK1/2 co-silencing and exogenous SNP or H2O2 on REL (A), apoplastic H2O2 (B) or NO level (C) with regard to cold acclimation-induced cold tolerance in tomato plants. SNP at 500 μM and H2O2 at 10 mM were applied 12 h before the cold acclimation treatment. Leaf samples were taken at 5 d after commencement of the cold stress treatment for the determination of REL. H2O2 and NO accumulation levels were estimated as described in Fig. 2. Scale bars: 50 μm (C) and 1 μm (B). Data are the means (±SD) of four biological replicates. Different letters indicate significant differences (P<0.05) according to Tukey’s test. H2O2, NO, and MPK1/2 are involved in cold acclimation-induced ABA biosynthesis ABA is known to play important roles in plant responses to cold stress. Although ABA accumulation was induced by cold acclimation, this induction was mitigated in pTRV-RBOH1, pTRV-NR, and pTRV-MPK1/2 plants (Fig. 6). We then investigated whether the increased accumulation of ABA was essential for the acclimation-induced cold tolerance. The tomato notabilis (not) mutant, which is deficient in ABA, showed increased cold sensitivity, as indicated by the increased REL from the leaf cells and decreased Fv/Fm following exposure to cold acclimation for 3 d and cold stress conditions for 5 d (Fig. 7A; Supplementary Fig. S7A). Foliar application of exogenous ABA enhanced cold tolerance in both wild-type (WT) and not plants, as evidenced by the decreased REL and increased Fv/Fm. Cold acclimation-induced increases in CPK27 transcript levels, accumulation of H2O2 and NO, and MPK1/2 activation were considerably attenuated in not plants (Fig. 7B–E; Supplementary Fig. S7B–D). Additionally, application of exogenous ABA enhanced the levels of CPK27 transcript, H2O2 and NO, as well as MPK1/2 activation, in both WT and not plants. Fig. 6. View largeDownload slide Silencing of RBOH1, NR, or MPK1/2 attenuates cold acclimation-induced ABA accumulation. Leaf samples were taken at 12 h after commencement of the cold stress treatment for the determination of ABA content. Data are the means (±SD) of four biological replicates. Different letters indicate significant differences (P<0.05) according to Tukey’s test. Fig. 6. View largeDownload slide Silencing of RBOH1, NR, or MPK1/2 attenuates cold acclimation-induced ABA accumulation. Leaf samples were taken at 12 h after commencement of the cold stress treatment for the determination of ABA content. Data are the means (±SD) of four biological replicates. Different letters indicate significant differences (P<0.05) according to Tukey’s test. Fig. 7. View largeDownload slide Cold acclimation-induced transcript levels of CPK27, accumulation of H2O2 and NO, and MPK1/2 activation are dependent on ABA levels in plants. REL (A), relative expression of CPK27 (B), NO (C), H2O2 in the apoplast (D), and activation of MPK1/2 (E) in the wild type (WT) and the ABA biosynthetic mutant, notabilis (not), with foliar application of ABA or H2O. ABA at 50 μM was applied 12 h before the cold acclimation treatment. The numbers above the images in (E) indicate the relative intensity of MPK1/2 (the upper lane). Leaf samples were taken at 5 d for the determination of REL, while the other assays were performed at 12 h after commencement of the cold stress treatment. H2O2 and NO accumulation levels were estimated as described in Fig. 2. Scale bars: 50 μm (C) and 1 μm (D). Data are the means (±SD) of four biological replicates. Different letters indicate significant differences (P<0.05) according to Tukey’s test. Fig. 7. View largeDownload slide Cold acclimation-induced transcript levels of CPK27, accumulation of H2O2 and NO, and MPK1/2 activation are dependent on ABA levels in plants. REL (A), relative expression of CPK27 (B), NO (C), H2O2 in the apoplast (D), and activation of MPK1/2 (E) in the wild type (WT) and the ABA biosynthetic mutant, notabilis (not), with foliar application of ABA or H2O. ABA at 50 μM was applied 12 h before the cold acclimation treatment. The numbers above the images in (E) indicate the relative intensity of MPK1/2 (the upper lane). Leaf samples were taken at 5 d for the determination of REL, while the other assays were performed at 12 h after commencement of the cold stress treatment. H2O2 and NO accumulation levels were estimated as described in Fig. 2. Scale bars: 50 μm (C) and 1 μm (D). Data are the means (±SD) of four biological replicates. Different letters indicate significant differences (P<0.05) according to Tukey’s test. CPK27 is required for ABA-induced cold tolerance We then examined whether cold acclimation-induced ABA accumulation regulates CPK27-dependent events as a feedback mechanism. Thus, we analysed the changes in cold acclimation-induced and CPK27-dependent accumulation of NO and H2O2, as well as MPK1/2 activation and cold tolerance, in response to exogenous ABA and cold acclimation. Application of ABA enhanced cold tolerance as indicated by the decreased REL and increased Fv/Fm (Fig. 8A; Supplementary Fig. S8A), increased the accumulation of H2O2 in the apoplast and NO in the leaves, and induced MPK1/2, but these effects were greatly tempered in plants with CPK27 silencing (Fig. 8B–D; Supplementary Fig. S8B–D). These results indicated that CPK27 is vital for the ABA-induced production of H2O2 in the apoplast and NO in the leaves and for the induction of MPK1/2. Fig. 8. View largeDownload slide CPK27 is essential for ABA-induced cold tolerance, H2O2 and NO accumulation, and MPK1/2 activation in tomato plants. REL (A), NO (B), apoplastic H2O2 (C), and activation of MPK1/2 (D) in pTRV control plants and CPK27-silenced plants with foliar application of ABA or H2O. ABA at 50 μM was applied 12 h before the cold acclimation treatment. The numbers above the images in (D) indicate the relative intensity of MPK1/2 (the upper lane). Leaf samples were taken at 5 d for the determination of REL, while other assays were performed at 12 h after commencement of the cold stress treatment. H2O2 and NO accumulation levels were estimated as described in Fig. 2. Scale bars: 50 μm (B) and 1 μm (C). Data are the means (±SD) of four biological replicates. Different letters indicate significant differences (P<0.05) according to Tukey’s test. Fig. 8. View largeDownload slide CPK27 is essential for ABA-induced cold tolerance, H2O2 and NO accumulation, and MPK1/2 activation in tomato plants. REL (A), NO (B), apoplastic H2O2 (C), and activation of MPK1/2 (D) in pTRV control plants and CPK27-silenced plants with foliar application of ABA or H2O. ABA at 50 μM was applied 12 h before the cold acclimation treatment. The numbers above the images in (D) indicate the relative intensity of MPK1/2 (the upper lane). Leaf samples were taken at 5 d for the determination of REL, while other assays were performed at 12 h after commencement of the cold stress treatment. H2O2 and NO accumulation levels were estimated as described in Fig. 2. Scale bars: 50 μm (B) and 1 μm (C). Data are the means (±SD) of four biological replicates. Different letters indicate significant differences (P<0.05) according to Tukey’s test. Discussion CPKs play critical roles in regulating growth, development, and stress responses in plants (Boudsocq and Sheen, 2013). There is evidence that CPKs participate in the regulation of plant response to cold stress (Martín and Busconi, 2001; Böehmer and Romeis, 2007; Komatsu et al., 2007). However, the mechanisms underlying CPK-dependent cold tolerance induced by cold acclimation remain mostly elusive. Here, we present several lines of evidence supporting the role of CPK27 in the cold response. Cold acclimation-induced CPK27 contributes to early signal transduction processes and is connected to NO, H2O2, and MPK1/2 activation in ABA signaling. This study not only delineated the NO–H2O2–MPK1/2 loop as an integrated signaling mechanism for modulating ABA-dependent cold tolerance but also suggested a positive impact of ABA on CPK27-dependent cold tolerance through a feedback loop in cold acclimation. CPK27 is a positive regulator of cold tolerance induced by cold acclimation Several CPKs have been shown to constitute a complicated regulation network, functioning positively and negatively in plant adaptation to cold stress. For example, OsCPK7, OsCPK13, and OsCPK17 in Oryza sativa (Saijo et al., 2000; Komatsu et al., 2007; Almadanim et al., 2017), AtCPK1 in Arabidopsis (Böhmer and Romeis, 2007), PeCPK10 in Populus euphratica (Chen et al., 2013), and VaCPK20 in Vitis amurensis (Dubrovina et al., 2015) have been characterized as positive regulators of cold stress tolerance, while ZmCPK1 in Zea mays has been shown to act as a negative regulator of cold stress signaling (Weckwerth et al., 2015). Here, we provide multiple lines of evidence that CPK27 is a positive regulator of the cold response in tomato plants. First, transcript levels of CPK27 were significantly increased by cold acclimation (Fig. 1A; Supplementary Fig. S2A). Second, knockdown of CPK27 transcripts attenuated cold acclimation-induced cold tolerance and compromised ABA-induced cold tolerance (Figs 1B, C, 8A). Third, CPK27 silencing reduced the cold acclimation-induced accumulation of ABA, H2O2, and NO, as well as the activation of MPK1/2 (Figs 1D, 2B, D, E; Supplementary Fig. S3E–G), all of which are positive regulators of cold response in plants. These results strongly suggest that CPK27 is not only important for cold response but also involved in the regulation of multiple signaling pathways, including ABA, ROS, NO, and MPK signaling, in response to cold. Evidence is increasing for the roles of NO, apoplastic H2O2, and MPKs in the cold response in plants (Diao et al., 2017; Kim et al., 2017; Si et al., 2017). In agreement with our earlier results, silencing of RBOH1 or NR or co-silencing of MPK1 and MPK2 differentially compromised cold acclimation-induced cold tolerance (Figs 3A, D, 5A). Interestingly, the transcript levels of RBOH1, NR, MPK1, and MPK2 were all subject to regulation by CPK27 (see Supplementary Fig. S3A–D). Silencing of CPK27 attenuated cold acclimation-induced H2O2 accumulation in the apoplast, NO accumulation in the leaves, and activation of MPK1/2 (Fig. 2B, D, E; Supplementary Fig. S3E–G). These results provided convincing evidence for the role of CPK27 in cold acclimation-triggered ROS signaling, NO signaling, and MPK signaling in the cold response. Several studies have demonstrated that CPKs can phosphorylate the N-terminal regions of plasma membrane RBOH proteins (NADPH oxidase) and participate in RBOH-mediated ROS bursts (Kobayashi et al., 2012). Consistent with this, we found that the cold acclimation activation of NADPH oxidase was largely abolished in pTRV-CPK27 plants (Fig. 2A), suggesting that the N-terminal region of NADPH oxidase is likely phosphorylated by CPK27. Evidence also exists that CPKs can activate MPK signaling by activating MPKs. For example, CPK18 in rice was identified as an upstream kinase of MAPK (MPK5) and was shown to phosphorylate and activate MPK5 (Xie et al., 2014). In our present study, CPK27 silencing greatly attenuated the cold acclimation-induced activation of MPK1/2 (Fig. 2E), suggesting a potential role of CPK27 in the activation of MPKs in tomato plants. Interestingly, cold acclimation-induced NO accumulation was also attenuated in CPK27-silenced plants (Fig. 2D; Supplementary Fig. S3F, G), implying the involvement of CPK27 in the regulation of NO homeostasis by indirectly activating NR. Until now, there is no clear evidence for the direct regulation of NR by CPKs in plants in vivo. Further protein–protein interaction experiments will shed light on the mechanisms underlying CPK27-induced activation of ROS signaling, NO signaling, and MPK signaling in response to cold. CPK27-activated crosstalk among H2O2, NO, and MPK1/2 in cold acclimation Several studies have demonstrated the complicated interactions between NO and H2O2, NO and MPKs, and H2O2 and MPKs in response to various stresses or stimuli (Bright et al., 2006; Asai et al., 2008; Takahashi et al., 2011; Ye et al., 2013; Zhou et al., 2014b; Li et al., 2017). Here, we provide several lines of evidence for the existence of a feedback loop among H2O2, MPKs, and NO in the cold response. First, silencing of RBOH1 or NR not only abolished the accumulation of NO or H2O2, respectively, but also decreased the accumulation of activated MPK1/2 induced by cold acclimation (Fig. 3B, C, E, F). Second, foliar application of SNP to pTRV-RBOH1 plants and foliar application of H2O2 to pTRV-NR plants both resulted in an increased accumulation of activated MPK1/2 (Fig. 4B). Third, silencing of MPK1/2 decreased cold acclimation-induced accumulation of both H2O2 and NO (Fig. 5B, C). Fourth, silencing of RBOH1, NR, or MPK1/2 attenuated cold acclimation-induced ABA accumulation (Fig. 6). These results allow us to argue that the crosstalk among H2O2, MPK1/2, and NO is critical to maintain the homeostasis of H2O2 and NO and the activated state of MPK1/2. However, how cold acclimation initiates this loop is unclear, as both NADPH oxidase and MPKs could be phosphorylated or activated by CPKs in plants in vivo (Kobayashi et al., 2007; Xie et al., 2014). The H2O2–MPK1/2–NO feedback loop is likely to be important for the homeostasis of H2O2 and NO and the activation of MPK1/2. Data from the present study show that H2O2 and NO could function independently in the cold response. This finding is substantiated by the increased cold tolerance caused in pTRV-RBOH1 by the foliar application of SNP, in pTRV-NR plants by the foliar application of H2O2, and in pTRV-MPK1/2 plants by the foliar application of SNP or H2O2 (Figs 4A, 5A). While ROS, NO, and MPKs share many similarities in target genes or proteins, they can also act independently in the regulation of many physiological or metabolic processes in plants (Yoshioka et al., 2011; Xu et al., 2014). Studies to date support a role of ROS in the regulation of gene transcripts and protein functions by cysteine modification (Akter et al., 2015). In comparison, NO and MPKs affect signaling cascades, mostly by S-nitrosylation and phosphorylation, respectively (Wang et al., 2015; Kim et al., 2017). Accordingly, the coordination of MPK1/2, H2O2, and NO likely contributes greatly to the cold acclimation-induced cold response by altering a variety of physiological processes, as substantiated by the results of RNAseq and metabolite analysis (Barrero-Gil et al., 2016). Relationship between CPK27 and ABA in cold acclimation Although Ca2+ and CPKs have been suggested as early signals in stress response, evidence for CPK activity in the induction of ABA biosynthesis or signaling is still lacking. Here, we found that CPK27 plays a critical role in ABA accumulation. We observed that cold acclimation-induced ABA accumulation was greatly attenuated in pTRV-CPK27 plants (Fig. 1D). CPK27 is important for the acclimation-induced transcript of RBOH1, NR, and MPK1 and MPK2, the generation of ROS and NO and the activation of MPK1/2 (Fig. 2; Supplementary Fig. S3). However, silencing of RBOH1, NR, or MPK1/2 resulted in decreased accumulations of ABA during the cold acclimation (Fig. 6). While RBOH1-dependent NADPH oxidase is involved in the generation of ABA in response to heat and oxidative stresses, interaction of ROS and NO results in the induction of ABA biosynthesis (Zhao et al., 2001; Xing et al., 2004; Zhou et al., 2014a). Therefore, CPK27 may function as a positive regulator of ABA generation by activating the production of ROS and NO as well as MPK1/2. The results of the present study demonstrated that CPK27-induced generation of ROS and NO and activation of MPK1/2 were subject to regulation by ABA (Fig. 7B–E). Silencing of CPK27 abolished the ABA-induced increase in accumulation of H2O2 and NO, and activation of MPK1/2 in response to cold acclimation (Fig. 8B–D). Many CPKs, including CPK27 in tomato, are ABA responsive (Hu et al., 2016). Arabidopsis CPK4 and CPK11 have been identified as important positive regulators in CPK/calcium-mediated ABA signaling (Zhu et al., 2007). ABA-induced transcriptional reprogramming via ABA-responsive ABF transcription factors is likely to be a key feature of CPK signaling. For example, AtCPK32 activates ABF4 in vivo, resulting in the induction of ABF4 target genes (Choi et al., 2005). Taken together, these results suggest that crosstalk between ABA and CPK27 is important not only for the activation of MPK1/2, ROS signaling, and NO signaling but also for the maintenance of ABA signaling and thus for cold tolerance. Supplementary data Supplementary data are available at JXB online. Fig. S1. Efficiency of gene silencing by virus-induced gene silencing Fig. S2. Effects of cold acclimation on transcripts of the tomato CPK family genes and plant phenotype after cold stress in tomato plants. Fig. S3. Effects of CPK27 silencing on the transcript levels of RBOH1, NR, MPK1, and MPK2 and accumulation levels of H2O2 and NO in leaves of control (25 °C), cold-acclimated and non-acclimated tomato plants. Fig. S4. Exogenous H2O2 or SNP partly rescues the chilling-sensitive phenotype due to CPK27 silencing. Fig. S5. Effects of RBOH1 or NR silencing and exogenous H2O2 or SNP on cold acclimation-induced cold tolerance and NO or H2O2 accumulation in control (25 °C) and cold-acclimated tomato plants. Fig. S6. Effects of MPK1/2 co-silencing and exogenous SNP or H2O2 on Fv/Fm, accumulation of H2O2 or NO in control (25 °C) and cold-acclimated tomato plants. Fig. S7. ABA and cold acclimation-induced changes in Fv/Fm and accumulation of H2O2 and NO in the wild type and the ABA-deficient mutant not. Fig. S8. ABA and cold acclimation-induced changes in Fv/Fm and accumulation of H2O2 and NO in pTRV control plants and CPK27-silenced plants. Table S1. PCR primer sequences used for vector construction. Table S2. List of primer sequences used for qRT-PCR analysis. Acknowledgements The authors are grateful to the Tomato Genetics Resource Center at the California University for tomato seeds. This work was supported by the National Natural Science Foundation of China (grant nos 31372109, 31430076) and the Fundamental Research Funds for the Central Universities (2016XZZX001-07). References Akter S , Huang J , Waszczak C , Jacques S , Gevaert K , Van Breusegem F , Messens J . 2015 . Cysteines under ROS attack in plants: a proteomics view . Journal of Experimental Botany 66 , 2935 – 2944 . Google Scholar CrossRef Search ADS PubMed Almadanim MC , Alexandre BM , Rosa MTG , Sapeta H , Leitão AE , Ramalho JC , Lam TT , Negrão S , Abreu IA , Oliveira MM . 2017 . Rice calcium-dependent protein kinase OsCPK17 targets plasma membrane intrinsic protein and sucrose-phosphate synthase and is required for a proper cold stress response . Plant, Cell & Environment 40 , 1197 – 1213 . Google Scholar CrossRef Search ADS PubMed Asai S , Ohta K , Yoshioka H . 2008 . MAPK signaling regulates nitric oxide and NADPH oxidase-dependent oxidative bursts in Nicotiana benthamiana . The Plant Cell 20 , 1390 – 1406 . Google Scholar CrossRef Search ADS PubMed Barrero-Gil J , Huertas R , Rambla JL , Granell A , Salinas J . 2016 . Tomato plants increase their tolerance to low temperature in a chilling acclimation process entailing comprehensive transcriptional and metabolic adjustments . Plant, Cell & Environment 39 , 2303 – 2318 . Google Scholar CrossRef Search ADS PubMed Bestwick CS , Brown IR , Bennett MH , Mansfield JW . 1997 . Localization of hydrogen peroxide accumulation during the hypersensitive reaction of lettuce cells to Pseudomonas syringae pv phaseolicola . The Plant Cell 9 , 209 – 221 . Google Scholar CrossRef Search ADS PubMed Böhmer M , Romeis T . 2007 . A chemical-genetic approach to elucidate protein kinase function in planta . Plant Molecular Biology 65 , 817 – 827 . Google Scholar CrossRef Search ADS PubMed Boudsocq M , Sheen J . 2013 . CDPKs in immune and stress signaling . Trends in Plant Science 18 , 30 – 40 . Google Scholar CrossRef Search ADS PubMed Bradford MM . 1976 . A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding . Analytical Biochemistry 72 , 248 – 254 . Google Scholar CrossRef Search ADS PubMed Bright J , Desikan R , Hancock JT , Weir IS , Neill SJ . 2006 . ABA-induced NO generation and stomatal closure in Arabidopsis are dependent on H2O2 synthesis . The Plant Journal 45 , 113 – 122 . Google Scholar CrossRef Search ADS PubMed Cantrel C , Vazquez T , Puyaubert J , Rezé N , Lesch M , Kaiser WM , Dutilleul C , Guillas I , Zachowski A , Baudouin E . 2011 . Nitric oxide participates in cold-responsive phosphosphingolipid formation and gene expression in Arabidopsis thaliana . New Phytologist 189 , 415 – 427 . Google Scholar CrossRef Search ADS PubMed Cao WH , Liu J , He XJ , Mu RL , Zhou HL , Chen SY , Zhang JS . 2007 . Modulation of ethylene responses affects plant salt-stress responses . Plant Physiology 143 , 707 – 719 . Google Scholar CrossRef Search ADS PubMed Chen J , Xue B , Xia X , Yin W . 2013 . A novel calcium-dependent protein kinase gene from Populus euphratica, confers both drought and cold stress tolerance . Biochemical and Biophysical Research Communications 441 , 630 – 636 . Google Scholar CrossRef Search ADS PubMed Chinnusamy V , Zhu J , Zhu JK . 2007 . Cold stress regulation of gene expression in plants . Trends in Plant Science 12 , 444 – 451 . Google Scholar CrossRef Search ADS PubMed Choi HI , Park HJ , Park JH , Kim S , Im MY , Seo HH , Kim YW , Hwang I , Kim SY . 2005 . Arabidopsis calcium-dependent protein kinase AtCPK32 interacts with ABF4, a transcriptional regulator of abscisic acid-responsive gene expression, and modulates its activity . Plant Physiology 139 , 1750 – 1761 . Google Scholar CrossRef Search ADS PubMed Deng XG , Zhu T , Zou LJ , Han XY , Zhou X , Xi DH , Zhang DW , Lin HH . 2016 . Orchestration of hydrogen peroxide and nitric oxide in brassinosteroid-mediated systemic virus resistance in Nicotiana benthamiana . The Plant Journal 85 , 478 – 493 . Google Scholar CrossRef Search ADS PubMed Diao Q , Song Y , Shi D , Qi H . 2017 . Interaction of polyamines, abscisic acid, nitric oxide, and hydrogen peroxide under chilling stress in tomato (Lycopersicon esculentum Mill.) seedlings . Frontiers in Plant Science 8 , 203 . Google Scholar CrossRef Search ADS PubMed Ding Y , Cao J , Ni L , Zhu Y , Zhang A , Tan M , Jiang M . 2013 . ZmCPK11 is involved in abscisic acid-induced antioxidant defence and functions upstream of ZmMPK5 in abscisic acid signalling in maize . Journal of Experimental Botany 64 , 871 – 884 . Google Scholar CrossRef Search ADS PubMed Doulis AG , Debian N , Kingston-Smith AH , Foyer CH . 1997 . Differential localization of antioxidants in maize leaves . Plant Physiology 114 , 1031 – 1037 . Google Scholar CrossRef Search ADS PubMed Dubiella U , Seybold H , Durian G , Komander E , Lassig R , Witte CP , Schulze WX , Romeis T . 2013 . Calcium-dependent protein kinase/NADPH oxidase activation circuit is required for rapid defense signal propagation . Proceedings of the National Academy of Sciences, USA 110 , 8744 – 8749 . Google Scholar CrossRef Search ADS Dubrovina AS , Kiselev KV , Khristenko VS , Aleynova OA . 2015 . VaCPK20, a calcium-dependent protein kinase gene of wild grapevine Vitis amurensis Rupr., mediates cold and drought stress tolerance . Journal of Plant Physiology 185 , 1 – 12 . Google Scholar CrossRef Search ADS PubMed Foyer CH , Valadier MH , Migge A , Becker TW . 1998 . Drought-induced effects on nitrate reductase activity and mRNA and on the coordination of nitrogen and carbon metabolism in maize leaves . Plant Physiology 117 , 283 – 292 . Google Scholar CrossRef Search ADS PubMed Fujita M , Fujita Y , Noutoshi Y , Takahashi F , Narusaka Y , Yamaguchi-Shinozaki K , Shinozaki K . 2006 . Crosstalk between abiotic and biotic stress responses: a current view from the points of convergence in the stress signaling networks . Current Opinion in Plant Biology 9 , 436 – 442 . Google Scholar CrossRef Search ADS PubMed Gao X , Chen X , Lin W , et al. 2013 . Bifurcation of Arabidopsis NLR immune signaling via Ca²⁺-dependent protein kinases . PLoS Pathogens 9 , e1003127 . Google Scholar CrossRef Search ADS PubMed Gilmour SJ , Thomashow MF . 1991 . Cold acclimation and cold-regulated gene expression in ABA mutants of Arabidopsis thaliana . Plant Molecular Biology 17 , 1233 – 1240 . Google Scholar CrossRef Search ADS PubMed Hu X , Jiang M , Zhang J , Zhang A , Lin F , Tan M . 2007 . Calcium-calmodulin is required for abscisic acid-induced antioxidant defense and functions both upstream and downstream of H2O2 production in leaves of maize (Zea mays) plants . New Phytologist 173 , 27 – 38 . Google Scholar CrossRef Search ADS PubMed Hu Z , Lv X , Xia X , Zhou J , Shi K , Yu J , Zhou Y . 2016 . Genome-wide identification and expression analysis of calcium-dependent protein kinase in tomato . Frontiers in Plant Science 7 , 469 . Google Scholar PubMed Jiang M , Zhang J . 2002 . Water stress-induced abscisic acid accumulation triggers the increased generation of reactive oxygen species and up-regulates the activities of antioxidant enzymes in maize leaves . Journal of Experimental Botany 53 , 2401 – 2410 . Google Scholar CrossRef Search ADS PubMed Jin Y , Ye N , Zhu F , Li H , Wang J , Jiang L , Zhang J . 2017 . Calcium-dependent protein kinase CPK28 targets the methionine adenosyltransferases for degradation by the 26S proteasome and affects ethylene biosynthesis and lignin deposition in Arabidopsis . The Plant Journal 90 , 304 – 318 . Google Scholar CrossRef Search ADS PubMed Kandoth PK , Ranf S , Pancholi SS , Jayanty S , Walla MD , Miller W , Howe GA , Lincoln DE , Stratmann JW . 2007 . Tomato MAPKs LeMPK1, LeMPK2, and LeMPK3 function in the systemin-mediated defense response against herbivorous insects . Proceedings of the National Academy of Sciences, USA 104 , 12205 – 12210 . Google Scholar CrossRef Search ADS Kim SH , Kim HS , Bahk S , An J , Yoo Y , Kim JY , Chung WS . 2017 . Phosphorylation of the transcriptional repressor MYB15 by mitogen-activated protein kinase 6 is required for freezing tolerance in Arabidopsis . Nucleic Acids Research 45 , 6613 – 6627 . Google Scholar CrossRef Search ADS PubMed Kobayashi M , Ohura I , Kawakita K , Yokota N , Fujiwara M , Shimamoto K , Doke N , Yoshioka H . 2007 . Calcium-dependent protein kinases regulate the production of reactive oxygen species by potato NADPH oxidase . The Plant Cell 19 , 1065 – 1080 . Google Scholar CrossRef Search ADS PubMed Kobayashi M , Yoshioka M , Asai S , Nomura H , Kuchimura K , Mori H , Doke N , Yoshioka H . 2012 . StCDPK5 confers resistance to late blight pathogen but increases susceptibility to early blight pathogen in potato via reactive oxygen species burst . New Phytologist 196 , 223 – 237 . Google Scholar CrossRef Search ADS PubMed Komatsu S , Yang G , Khan M , Onodera H , Toki S , Yamaguchi M . 2007 . Over-expression of calcium-dependent protein kinase 13 and calreticulin interacting protein 1 confers cold tolerance on rice plants . Molecular Genetics and Genomics 277 , 713 – 723 . Google Scholar CrossRef Search ADS PubMed Li FC , Wang J , Wu MM , Fan CM , Li X , He JM . 2017 . Mitogen-activated protein kinase phosphatases affect UV-B-induced stomatal closure via controlling NO in guard cells . Plant Physiology 173 , 760 – 770 . Google Scholar CrossRef Search ADS PubMed Livak KJ , Schmittgen TD . 2001 . Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔC(T) method . Methods 25 , 402 – 408 . Google Scholar CrossRef Search ADS PubMed Lv X , Ge S , Jalal Ahammed G , Xiang X , Guo Z , Yu J , Zhou Y . 2017 . Crosstalk between nitric oxide and MPK1/2 mediates cold acclimation-induced chilling tolerance in tomato . Plant & Cell Physiology 58 , 1963 – 1975 . Google Scholar CrossRef Search ADS PubMed Mantyla E , Lang V , Palva ET . 1995 . Role of abscisic acid in drought-induced freezing tolerance, cold acclimation, and accumulation of LT178 and RAB18 proteins in Arabidopsis thaliana . Plant Physiology 107 , 141 – 148 . Google Scholar CrossRef Search ADS PubMed Martín ML , Busconi L . 2001 . A rice membrane-bound calcium-dependent protein kinase is activated in response to low temperature . Plant Physiology 125 , 1442 – 1449 . Google Scholar CrossRef Search ADS PubMed McAinsh MR , Hetherington AM . 1998 . Encoding specificity in Ca2+ signalling systems . Trends in Plant Science 3 , 32 – 36 . Google Scholar CrossRef Search ADS Neill S , Barros R , Bright J , Desikan R , Hancock J , Harrison J , Morris P , Ribeiro D , Wilson I . 2008 . Nitric oxide, stomatal closure, and abiotic stress . Journal of Experimental Botany 59 , 165 – 176 . Google Scholar CrossRef Search ADS PubMed Saijo Y , Hata S , Kyozuka J , Shimamoto K , Izui K . 2000 . Over-expression of a single Ca2+-dependent protein kinase confers both cold and salt/drought tolerance on rice plants . The Plant Journal 23 , 319 – 327 . Google Scholar CrossRef Search ADS PubMed Santin F , Bhogale S , Fantino E , Grandellis C , Banerjee AK , Ulloa RM . 2017 . Solanum tuberosum StCDPK1 is regulated by miR390 at the posttranscriptional level and phosphorylates the auxin efflux carrier StPIN4 in vitro, a potential downstream target in potato development . Physiologia Plantarum 159 , 244 – 261 . Google Scholar CrossRef Search ADS PubMed Shi K , Li X , Zhang H , Zhang G , Liu Y , Zhou Y , Xia X , Chen Z , Yu J . 2015 . Guard cell hydrogen peroxide and nitric oxide mediate elevated CO2-induced stomatal movement in tomato . New Phytologist 208 , 342 – 353 . Google Scholar CrossRef Search ADS PubMed Shinozaki K , Yamaguchi-Shinozaki K , Seki M . 2003 . Regulatory network of gene expression in the drought and cold stress responses . Current Opinion in Plant Biology 6 , 410 – 417 . Google Scholar CrossRef Search ADS PubMed Si T , Wang X , Wu L , et al. 2017 . Nitric oxide and hydrogen peroxide mediate wounding-induced freezing tolerance through modifications in photosystem and antioxidant system in wheat . Frontiers in Plant Science 8 , 1284 . Google Scholar CrossRef Search ADS PubMed Takahashi F , Mizoguchi T , Yoshida R , Ichimura K , Shinozaki K . 2011 . Calmodulin-dependent activation of MAP kinase for ROS homeostasis in Arabidopsis . Molecular Cell 41 , 649 – 660 . Google Scholar CrossRef Search ADS PubMed Thomashow MF . 1998 . Role of cold-responsive genes in plant freezing tolerance . Plant Physiology 118 , 1 – 8 . Google Scholar CrossRef Search ADS PubMed Wang F , Guo Z , Li H , Wang M , Onac E , Zhou J , Xia X , Shi K , Yu J , Zhou Y . 2016 . Phytochrome A and B function antagonistically to regulate cold tolerance via abscisic acid-dependent jasmonate signaling . Plant Physiology 170 , 459 – 471 . Google Scholar CrossRef Search ADS PubMed Wang P , Du Y , Hou YJ , Zhao Y , Hsu CC , Yuan F , Zhu X , Tao WA , Song CP , Zhu JK . 2015 . Nitric oxide negatively regulates abscisic acid signaling in guard cells by S-nitrosylation of OST1 . Proceedings of the National Academy of Sciences, USA 112 , 613 – 618 . Google Scholar CrossRef Search ADS Weckwerth P , Ehlert B , Romeis T . 2015 . ZmCPK1, a calcium-independent kinase member of the Zea mays CDPK gene family, functions as a negative regulator in cold stress signalling . Plant, Cell & Environment 38 , 544 – 558 . Google Scholar CrossRef Search ADS PubMed Xia XJ , Wang YJ , Zhou YH , Tao Y , Mao WH , Shi K , Asami T , Chen Z , Yu JQ . 2009 . Reactive oxygen species are involved in brassinosteroid-induced stress tolerance in cucumber . Plant Physiology 150 , 801 – 814 . Google Scholar CrossRef Search ADS PubMed Xia XJ , Zhou YH , Ding J , Shi K , Asami T , Chen Z , Yu JQ . 2011 . Induction of systemic stress tolerance by brassinosteroid in Cucumis sativus . New Phytologist 191 , 706 – 720 . Google Scholar CrossRef Search ADS PubMed Xie K , Chen J , Wang Q , Yang Y . 2014 . Direct phosphorylation and activation of a mitogen-activated protein kinase by a calcium-dependent protein kinase in rice . The Plant Cell 26 , 3077 – 3089 . Google Scholar CrossRef Search ADS PubMed Xing H , Tan LL , An LH , Zhao ZG , Wang SM , Zhang CL . 2004 . Evidence for the involvement of nitric oxide and reactive oxygen species in osmotic stress tolerance of wheat seedlings: inverse correlation between leaf abscisic acid accumulation and leaf water loss . Plant Growth Regulation 42 , 61 – 68 . Google Scholar CrossRef Search ADS Xing Y , Jia W , Zhang J . 2008 . AtMKK1 mediates ABA-induced CAT1 expression and H2O2 production via AtMPK6-coupled signaling in Arabidopsis . The Plant Journal 54 , 440 – 451 . Google Scholar CrossRef Search ADS PubMed Xu J , Xie J , Yan C , Zou X , Ren D , Zhang S . 2014 . A chemical genetic approach demonstrates that MPK3/MPK6 activation and NADPH oxidase-mediated oxidative burst are two independent signaling events in plant immunity . The Plant Journal 77 , 222 – 234 . Google Scholar CrossRef Search ADS PubMed Ye Y , Li Z , Xing D . 2013 . Nitric oxide promotes MPK6-mediated caspase-3-like activation in cadmium-induced Arabidopsis thaliana programmed cell death . Plant, Cell & Environment 36 , 1 – 15 . Google Scholar CrossRef Search ADS PubMed Yoshioka H , Mase K , Yoshioka M , Kobayashi M , Asai S . 2011 . Regulatory mechanisms of nitric oxide and reactive oxygen species generation and their role in plant immunity . Nitric Oxide 25 , 216 – 221 . Google Scholar CrossRef Search ADS PubMed Zhang A , Jiang M , Zhang J , Tan M , Hu X . 2006 . Mitogen-activated protein kinase is involved in abscisic acid-induced antioxidant defense and acts downstream of reactive oxygen species production in leaves of maize plants . Plant Physiology 141 , 475 – 487 . Google Scholar CrossRef Search ADS PubMed Zhao MG , Chen L , Zhang LL , Zhang WH . 2009 . Nitric reductase-dependent nitric oxide production is involved in cold acclimation and freezing tolerance in Arabidopsis . Plant Physiology 151 , 755 – 767 . Google Scholar CrossRef Search ADS PubMed Zhao ZG , Chen GC , Zhang CL . 2001 . Interaction between reactive oxygen species and nitric oxide in drought-induced abscisic acid synthesis in root tips of wheat seedlings . Australian Journal of Plant Physiology 28 , 1055 – 1061 . Zhou J , Wang J , Li X , Xia XJ , Zhou YH , Shi K , Chen Z , Yu JQ . 2014a. H2O2 mediates the crosstalk of brassinosteroid and abscisic acid in tomato responses to heat and oxidative stresses . Journal of Experimental Botany 65 , 4371 – 4383 . Google Scholar CrossRef Search ADS PubMed Zhou J , Wang J , Shi K , Xia XJ , Zhou YH , Yu JQ . 2012 . Hydrogen peroxide is involved in the cold acclimation-induced chilling tolerance of tomato plants . Plant Physiology and Biochemistry 60 , 141 – 149 . Google Scholar CrossRef Search ADS PubMed Zhou J , Xia XJ , Zhou YH , Shi K , Chen Z , Yu JQ . 2014b. RBOH1-dependent H2O2 production and subsequent activation of MPK1/2 play an important role in acclimation-induced cross-tolerance in tomato . Journal of Experimental Botany 65 , 595 – 607 . Google Scholar CrossRef Search ADS PubMed Zhu SY , Yu XC , Wang XJ , et al. 2007 . Two calcium-dependent protein kinases, CPK4 and CPK11, regulate abscisic acid signal transduction in Arabidopsis . The Plant Cell 19 , 3019 – 3036 . Google Scholar CrossRef Search ADS PubMed Zou JJ , Li XD , Ratnasekera D , Wang C , Liu WX , Song LF , Zhang WZ , Wu WH . 2015 . Arabidopsis CALCIUM-DEPENDENT PROTEIN KINASE8 and CATALASE3 function in abscisic acid-mediated signaling and H2O2 homeostasis in stomatal guard cells under drought stress . The Plant Cell 27 , 1445 – 1460 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Experimental Botany Oxford University Press

The role of calcium-dependent protein kinase in hydrogen peroxide, nitric oxide and ABA-dependent cold acclimation

Loading next page...
 
/lp/ou_press/the-role-of-calcium-dependent-protein-kinase-in-hydrogen-peroxide-7OP0DBRIhK
Publisher
Oxford University Press
Copyright
© The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Experimental Biology.
ISSN
0022-0957
eISSN
1460-2431
D.O.I.
10.1093/jxb/ery212
Publisher site
See Article on Publisher Site

Abstract

Abstract Cold acclimation-induced cold tolerance is associated with the generation of reactive oxygen species (ROS), nitric oxide (NO), and mitogen-activated protein kinases (MPKs) in plants. Here, we hypothesized that calcium-dependent protein kinases (CPKs) induce a crosstalk among ROS, NO, and MPKs, leading to the activation of abscisic acid (ABA) signaling in plant adaptation to cold stress. Results showed that cold acclimation significantly increased the transcript levels of CPK27 along with the biosynthesis of ABA in tomato (Solanum lycopersicum). Silencing of CPK27 compromised acclimation-induced cold tolerance, generation of hydrogen peroxide (H2O2) in the apoplast, NO and ABA accumulation, and the activation of MPK1/2. Crosstalk among H2O2, NO, and MPK1/2 contributes to the homeostasis of H2O2 and NO, activation of MPK1/2, and cold tolerance. ABA is also critical for CPK27-induced cold tolerance, generation of H2O2 and NO, and the activation of MPK1/2. These results strongly suggest that CPK27 may function as a positive regulator of ABA generation by activating the production of ROS and NO as well as MPK1/2 in cold adaptation. Abscisic acid, calcium-dependent protein kinase, cold acclimation, hydrogen peroxide, mitogen-activated protein kinase, nitric oxide, Solanum lycopersicum Introduction Plants undergo continuous exposure to various biotic and abiotic stresses in their growth environment. Cold stress adversely affects the growth and development of plants and significantly constrains their spatial distribution and agricultural productivity. To survive under stressful conditions, plants have evolved intricate mechanisms to perceive external signals, allowing optimal responses to environmental conditions. For example, many plants show decreased sensitivity to cold stress when they have undergone prior exposure to suboptimal low temperatures, a phenomenon called cold acclimation. Cold acclimation involves the remodeling of cell and tissue structures and the reprogramming of metabolism and gene expression (Thomashow, 1998; Chinnusamy et al., 2007). The molecular basis of this acquired cold tolerance is not completely understood. Generally, a stress signal is first perceived by a membrane receptor, followed by the up-regulation of the cytoplasmic Ca2+ ion level (McAinsh and Hetherington, 1998). This change in cytoplasmic Ca2+ is sensed by calcium sensors such as calcium-dependent protein kinases (CPKs), which interact with downstream signaling components including hormones such as abscisic acid (ABA), reactive oxygen species (ROS), and mitogen-activated protein kinases (MPKs), leading to an increased tolerance to various stresses (Boudsocq and Sheen, 2013). CPKs play an important role in signal transduction, and the functions of several CPKs in Arabidopsis and rice have recently been identified (Almadanim et al., 2017; Jin et al., 2017). Activated CPKs participate in different signal transduction pathways through the phosphorylation of specific substrates (Kobayashi et al., 2012; Santin et al., 2017). Recently, several CPK substrates have been identified. For instance, MPK5 is directly phosphorylated by CPK18 to regulate stress responses and disease resistance in rice, while StCDPK5 and AtCPK1/2/4/5/11 can phosphorylate and thereby activate NADPH oxidase to promote the production of ROS in response to abiotic and biotic stimuli (Kobayashi et al., 2007; Dubiella et al., 2013; Gao et al., 2013; Xie et al., 2014). The tight regulation of steady-state levels of ROS is important for many cellular processes in plants (Fujita et al., 2006). While excess ROS accumulation induces oxidative stress in cells, ROS also function as signaling molecules (Fujita et al., 2006). Recent studies suggest that the NADPH-dependent respiratory burst oxidase homolog (RBOH) genes are involved in cold acclimation-induced cold tolerance in several plants. Silencing of RBOH1 in tomato increased the sensitivity of plants to cold and attenuated the protective role of cold acclimation (Zhou et al., 2012). Similar to ROS, nitric oxide (NO) also participates in the regulation of cold response in several plant species (Zhao et al., 2009; Diao et al., 2017). In particular, nitrate reductase (NR)-dependent NO participates in the regulation of the cold acclimation process (Zhao et al., 2009; Lv et al., 2017). In a number of cases, generation of NO and H2O2 occurs in parallel or in rapid succession, and these molecules can act synergistically or independently (Bright et al., 2006; Asai et al., 2008; Shi et al., 2015; Deng et al., 2016). In addition, increased generation of ROS/NO is frequently accompanied by the activation of MPKs. Silencing of MPK1 and MPK2 compromised cold acclimation-induced chilling tolerance and the activation of several antioxidant enzymes in tomato (Lv et al., 2017). However, the role of CPKs in regulating the generation of ROS/NO and the activation of MPKs is unclear. As a phytohormone, ABA is extensively involved in the responses to abiotic stresses such as drought, low temperature, and osmotic stress. In response to cold stress, plants usually accumulate an increased amount of ABA. Many stress-inducible genes are regulated by the endogenous ABA that accumulates during stress (Shinozaki et al., 2003). Plants deficient or impaired in ABA biosynthesis or signaling components show a reduced response to cold acclimation (Gilmour and Thomashow, 1991; Mantyla et al., 1995). ABA can also act as a secondary signal to induce changes in Ca2+ levels that eventually impact cold signaling. ABA-enhanced stress tolerance is associated with the induction of diverse signaling molecules, such as Ca2+, calmodulin (CaM) (Hu et al., 2007), H2O2 (Jiang and Zhang, 2002), NO (Neill et al., 2008), MPK (Zhang et al., 2006; Xing et al., 2008), and CPK (Ding et al., 2013; Zhu et al., 2007; Zou et al., 2015), suggesting an intensive crosstalk among ROS, NO, and MPKs. However, how plants transduce calcium signaling to affect ROS, NO, and MPK signaling remains largely unclear, as does the relation of CPKs to ABA in cold acclimation. Here, we hypothesized that CPKs induce a crosstalk among ROS, NO, and MPKs, leading to the activation of ABA signaling in plant adaptation to cold stress. To this end, we identified the critical CPKs in the cold response and examined their relation to the induction of H2O2, NO, and ABA signaling, and the activation of MPK1/2. Results showed that CPK27-mediated crosstalk between H2O2, NO, and MPK1/2 contributed to cold acclimation-induced ABA biosynthesis and cold tolerance in tomato plants. We also found that crosstalk between CPK27 and ABA was essential for the tight control of the interactions of H2O2 with NO and MPK1/2. Materials and methods Plant material and growth conditions In the current study, tomato (Solanum lycopersicum L.) cultivar Condine Red was used for the majority of experiments. Seedlings were grown in a mixture of vermiculite and peat at the ratio of 3:1 (v/v), receiving Hoagland’s nutrient solution daily. The growth conditions were as follows: 12 h photoperiod, temperature of 25/20 °C (day/night), and photosynthetic photon flux density (PPFD) of 600 µmol m−2 s−1. To examine the significance of endogenous ABA levels in cold acclimation-induced cold tolerance, seeds of wild type (WT, cv. Ailsa Craig) and an ABA biosynthesis mutant, notabilis (not), in the Ailsa Craig background were used. Virus-induced gene silencing (VIGS) was performed on the fully expanded cotyledons of 15-day-old tomato seedlings as described previously (Lv et al., 2017). The fragment for silencing CPK27 was PCR-amplified using the gene-specific primer listed in Supplementary Table S1 at JXB online, and the vectors for silencing NR and RBOH1 and co-silencing MPK1/2 were constructed as described previously (Kandoth et al., 2007; Zhou et al., 2014b; Lv et al., 2017). The infiltrated plants were grown at 21 °C and used in experiments at the five-leaf stage. qRT-PCR analysis was performed to evaluate gene silencing efficiency (Supplementary Fig. S1). Cold acclimation and cold stress treatments All experiments were carried out in environmentally controlled growth chambers using tomato plants at the five-leaf stage. For the cold acclimation treatment, plants were exposed to an aerial temperature of 12 °C with a 12/12 h light/dark cycle under 600 µmol m−2 s−1 PPFD and 85% humidity from 07.00 h. The cold acclimation treatment lasted for 3 d. Then, acclimated and non-acclimated plants were challenged with a cold stress treatment of 4 °C with 12/12 h light/dark cycle for 5 d under 600 μmol m−2 s−1 PPFD. There were four replicates in each treatment, and each replicate consisted of 16 plants. To determine the roles of H2O2, NO, and ABA in cold acclimation-induced cold tolerance, tomato seedlings at the five-leaf stage were sprayed with 10 mM H2O2, 500 μM sodium nitroprusside (SNP; a donor of NO) or 50 μM ABA. H2O2 and SNP were diluted with water while ABA was dissolved in a very low concentration of ethanol (0.01%, v/v) in all working solutions including control water solution. Twelve hours after the pretreatment, the plants were transferred to growth chambers for the cold acclimation treatment at 12 °C for 3 d and then exposed to 4 °C for 5 d. Evaluation of cold tolerance An Imaging-PAM Chlorophyll Fluorimeter equipped with a computer-operated PAM-control unit (IMAG-MAXI; Heinz Walz, Effeltrich, Germany) was used to detect the maximum quantum yield of PSII (Fv/Fm), as previously described (Zhou et al., 2012). The air temperature, relative humidity, CO2 concentration and PPFD were maintained at 25 °C, 85%, 380 μmol mol−1 and 1200 μmol m−2 s−1, respectively. Relative electrolyte leakage (REL, %) was determined following protocols described previously (Cao et al., 2007). Measurements of endogenous NO level and NR activity NO accumulation in leaves was monitored using the NO-sensitive dye 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-2DA) as reported previously (Lv et al., 2017). Leaf sections were infiltrated with 10 µM DAF-2DA prepared in 10 mM Tris–HCl (pH 7.4) and incubated in the dark at 37 °C for 30 min before observation. Fluorescence from DAF-2T, the reaction product of DAF-2DA with NO, was observed using a fluorescence stereomicroscope (Leica TCS SL; Leica Microsystems, Wetzlar, Germany) equipped with a charge-coupled device camera. The excitation was at 488 nm, and emission images at 525 nm were obtained with a constant acquisition time. The treatments were repeated 10 times. The signal intensities of green fluorescence in the images were quantified by ImageJ software (NIH, Bethesda, MD, USA). Values were corrected for background. Data were presented as the means of fluorescence intensity relative to those of control plants. In addition, NO accumulation was also determined by colorimetric assay with Griess reagent. Briefly, 0.3 g tomato leaves were homogenized using 50 mM glacial acetic acid (pH3.6) in an ice bath and centrifuged at 12000 g for 15 min. An aliquot of supernatant was mixed with Griess reagent (Sigma-Aldrich, USA) and kept at 25 °C for 30 min for reaction. The content of NO was calculated based on the absorbance of the reaction mixture at 540 nm. NO content was calculated by comparison with a standard curve of NaNO2. NR was assayed as described by Foyer et al. (1998) with small modifications. Briefly, 0.3 g leaf tissue was homogenized with 1.5 ml of extraction buffer [10 mM HEPES–KOH, pH 7.5, 5 mM DTT, 1 mM Na2MoO4, 10 mM FAD, 2 mM β-mercaptoethanol, 5 mM EDTA, and 1% polyvinylpolypyrrolidone (PVP)]. After centrifuging at 4 °C for 15 min at 12000 g, 0.3 ml of the supernatant was used for the NR activity assay. The reaction mixture (0.7 ml) consisted of 100 mM HEPES–KOH buffer, pH 7.5, 5 mM KNO3, and 0.25 mM NADH (freshly prepared). The reaction was terminated after 25 min by adding an equal volume of sulfanilamide [1% (w/v) in 3 M HCl] and then N-(1-naphthyl)-ethylenediamine dihydrochloride [0.02% (w/v)] to the reaction mixture, and the absorbance was measured using a spectrophotometer at 540 nm. The protein content was determined following the Coomassie blue staining method as previously described (Bradford, 1976). The NR activity was expressed as nmol of NO2− produced per minute and per milligram of protein. Measurements of endogenous H2O2 levels and NADPH oxidase activity H2O2 was extracted from leaf tissue according to Doulis et al. (1997) and measured as described in our previous study (Xia et al., 2011). For the determination, 0.3 g FW leaf material was homogenized with 3 ml of 0.2 M HClO4 on ice. After centrifuging at 6000 g for 5 min at 4 °C, the supernatant was adjusted to pH 6.5 with 4 M KOH, and centrifuged at 12 000 g for 5 min at 4 °C. After filtering through an AG1 × 8 prepacked column (Bio-Rad, Hercules, CA, USA), 800 µl of extracts was mixed with 400 µl reaction buffer containing 4 mM 2,2′-azino-di (3-ethylbenzthiazoline-6-sulfonic acid) and 100 mM potassium acetate at pH 4.4, 400 µl deionized water and 0.25 U of horseradish peroxidase (HRP). H2O2 content was measured at optical density at 412 nm. H2O2 was visualized at the subcellular level using CeCl3 for localization (Zhou et al., 2012). Tissue pieces (1–2 mm2) were cut from the leaves and incubated in freshly prepared 5 mM CeCl3 in 50 mM MOPS at pH 7.2 for 1 h. The leaf sections were then fixed in 1.25% (v/v) glutaraldehyde and 1.25% (v/v) paraformaldehyde in 50 mM sodium cacodylate buffer (pH 7.2) for 1 h. After fixation, tissues were washed twice for 10 min in the same buffer and postfixed for 45 min in 1% (v/v) osmium tetroxide and then dehydrated in a graded ethanol series (30–100%; v/v) and embedded in Eponaraldite (Agar Aids). After 12 h in pure resin, followed by a change of fresh resin for 4 h, the samples were polymerized at 60 ℃ for 48 h. Blocks were sectioned (70–90 nm) on a Reichert-Ultracut E microtome and mounted on uncoated copper grids (300 mesh). The CeCl3 deposits formed in the presence of H2O2 were examined using a transmission electron microscope (H7650, Hitachi, Tokyo, Japan) at an accelerating voltage of 75 kV (Bestwick et al., 1997). For the determination of NADPH oxidase activity, leaf plasma membranes were isolated using a two-phase aqueous polymer partition system (Xia et al., 2009). Briefly, leaves (5 g) were homogenized in 20 ml extraction buffer [50 mM Tris–HCl, pH 7.5, 0.25 M sucrose, 1 mM ascorbic acid (AsA), 1 mM EDTA, 0.6% PVP, and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. The homogenate was filtered through four layers of cheesecloth, and the resulting filtrate was centrifuged at 10000 g for 15 min. Microsomal membranes were pelleted from the supernatant by centrifugation at 100000 g for 30 min. The pellet was suspended in 0.33 M sucrose, 3 mM KCl, and 5 mM potassium phosphate, pH 7.8. The plasma membrane fraction was isolated by adding an aqueous two-phase polymer system to give a final composition of 6.2% (w/w) dextran T500, 6.2% (w/w) polyethylene glycol 3350, 0.33 M sucrose, 3 mM KCl, and 5 mM potassium phosphate, pH 7.8. Three successive rounds of partitioning yielded the final upper phase. The upper phase was diluted 5-fold in Tris–HCl dilution buffer (10 mM, pH 7.4) containing 0.25 M sucrose, 1 mM EDTA, 1 mM DTT, 1 mM AsA, and 1 mM PMSF. The fractions were centrifuged at 120000 g for 1 h. The pellets were then resuspended in Tris–HCl dilution buffer and used immediately for further analysis. The protein content was determined following the Coomassie blue staining method as previously described (Bradford, 1976). The NADPH-dependent O2−-generating activity in isolated plasma membrane vesicles was determined as described previously (Xia et al., 2009). The rates of O2− generation were calculated using an extinction coefficient of 21.6 mM−1 cm−1. Total RNA extraction and qRT-PCR analysis Total RNA was extracted using an RNAsimple Total RNA Kit (Tiangen, Beijing, China) following the manufacturer’s protocols. After removing residual DNA with a DNase Mini Kit (Qiagen, Hilden, Germany), total RNA was reverse transcribed using a ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan), following the manufacturer’s instructions. The gene-specific primer pairs used to amplify the 29 CPK genes were taken from a previously published study (Hu et al., 2016), and additional primers are shown in Supplementary Table S2. qRT-PCR was performed using a Roche LightCycler 480 real-time PCR machine (Roche, Basel, Switzerland). Relative transcript levels were calculated according to the method of Livak and Schmittgen (2001). The tomato ACTIN2 gene was used as an internal reference. Total protein extraction and western blot analysis Total protein extracts were obtained by homogenizing frozen powdered leaves in an extraction buffer (100 mM HEPES, pH 7.5, 5 mM EDTA, 5 mM EGTA, 10 mM DTT, 10 mM Na3VO4, 10 mM NaF, 50 mM β-glycerophosphate, 1 mM PMSF, 5 μg ml−1 antipain, 5 μg ml−1 aprotinin, 5 μg ml−1 leupeptin, 10% glycerol, and 7.5% PVP). The extracts were centrifuged at 12000 g for 20 min before quantifying total protein content. For immunoblots, protein extracts were resolved on a 12.5% SDS-PAGE gel and later transferred to polyvinylidene fluoride membranes (Bio-Rad). The activated state of MPK1/2 was detected using an anti-p42/44 MPK primary antibody (1:1000, Cell Signaling Technology, Boston, MA, USA) followed by anti-rabbit HRP-conjugated secondary antibodies (Cell Signaling Technology, Boston, MA, USA) (Zhou et al., 2014b). Accumulation of MPK1/2 was quantified using Quantity One software (Bio-Rad). To obtain a loading control, membranes were stained with Ponceau S solution. Measurement of ABA levels ABA extraction and analysis from tomato leaves were performed using previously described procedures (Wang et al., 2016). Briefly, 0.1 g of frozen leaf material was spiked with D6-ABA (OlChemIm Ltd, Czechoslovakia) as an internal standard to a final concentration of 100 ng ml−1 before extraction with 1 ml of ethyl acetate. Following shaking for 12 h in the dark at 4 °C and centrifuging at 18000 g for 10 min at 4 °C, the supernatant was collected and evaporated to dryness under N2 gas. The residue was then resuspended in 0.5 ml of 70% methanol (v/v) and centrifuged at 18000 g for 2 min at 4 °C, and the supernatant was analysed by high-performance liquid chromatography (HPLC)–mass spectrometry (MS) on an Agilent 1290 Infinity HPLC system (including a vacuum degasser, a binary pump, a column oven, and an autosampler) coupled to an Agilent 6460 Triple Quadrupole LC/MS system (Agilent Technologies, Heilbronn, Germany). The levels of ABA in tomato leaves were expressed as nanograms per gram fresh weight leaf material. Statistical analysis The experimental design was a completely randomized block with four replicates. Data were statistically analysed by one-way ANOVA and the means were compared using Tukey’s multiple comparison test at the P<0.05 level. Results CPK27 is critical for cold acclimation-induced cold tolerance and ABA biosynthesis In our previous study, we identified 29 CPK genes in tomato (Hu et al., 2016). To examine the potential roles of these CPKs in cold tolerance, we examined the changes in the transcript levels of these 29 CPKs in response to cold acclimation in the leaves by qRT-PCR. After cold acclimation at 12 °C for 3 d, followed by a cold stress treatment at 4 °C for 12 h, 14 out of the 29 CPKs genes showed increased transcript accumulation in the leaves, ranging from 2- to 12-fold (see Supplementary Fig. S2A). Among them, the transcript accumulation of CPK27 (accession number: Solyc11g065660.1.1) was most highly induced, with an increase of 12-fold relative to that of plants grown at 25 °C. A time course further revealed that the transcript level of CPK27 was increased within 0.5 d after cold exposure and remained high throughout the cold acclimation process (Fig. 1A). Moreover, a further increase in the CPK27 transcript level was found in plants when they were subsequently exposed to cold at 4 °C for 12 h. However, after exposure to 4 °C for 5 d, the CPK27 transcript level returned to values close to those of the controls at day 0. In comparison, the CPK27 transcript level in non-acclimated plants was up-regulated around 4-fold at 12 h after being exposed to 4 °C and then decreased to values lower than those of the controls at 5 d after the onset of cold stress at 4 °C (Fig. 1A). Fig. 1. View largeDownload slide CPK27 plays a vital role in cold acclimation-induced cold tolerance. (A) Time course of changes in transcript levels of CPK27 in response to cold stress with or without cold acclimation. (B) Relative electrolyte leakage (REL). (C) Maximum photochemical efficiency of photosystem II (Fv/Fm). (D) ABA content in the leaves of control (25 °C) and chilled (4 °C) tomato plants with or without cold acclimation. At the five-leaf stage, tomato plants were either cold acclimated (12 °C for 3 d) or kept at normal temperature (25 °C) before the imposition of cold stress (4 °C for 5 d). CPK27 gene expression was analysed at different time points as indicated in (A). REL and Fv/Fm values were determined at 5 d, while ABA content was assayed at 12 h after commencement of the cold stress treatment. The false color code depicted at the bottom of each image ranges from 0 (black) to 1.0 (purple), representing the level of leaf damage. Data are the means (±SD) of four biological replicates, except for Fv/Fm, which represents the mean of 15 leaves from independent plants. Different letters indicate significant differences (P<0.05) according to Tukey’s test. Fig. 1. View largeDownload slide CPK27 plays a vital role in cold acclimation-induced cold tolerance. (A) Time course of changes in transcript levels of CPK27 in response to cold stress with or without cold acclimation. (B) Relative electrolyte leakage (REL). (C) Maximum photochemical efficiency of photosystem II (Fv/Fm). (D) ABA content in the leaves of control (25 °C) and chilled (4 °C) tomato plants with or without cold acclimation. At the five-leaf stage, tomato plants were either cold acclimated (12 °C for 3 d) or kept at normal temperature (25 °C) before the imposition of cold stress (4 °C for 5 d). CPK27 gene expression was analysed at different time points as indicated in (A). REL and Fv/Fm values were determined at 5 d, while ABA content was assayed at 12 h after commencement of the cold stress treatment. The false color code depicted at the bottom of each image ranges from 0 (black) to 1.0 (purple), representing the level of leaf damage. Data are the means (±SD) of four biological replicates, except for Fv/Fm, which represents the mean of 15 leaves from independent plants. Different letters indicate significant differences (P<0.05) according to Tukey’s test. To determine the role of CPK27 in the cold response, we silenced CPK27 (pTRV-CPK27) by the VIGS approach, which reduced its transcript level by ca. 70% (see Supplementary Fig. S1C). In the CPK27-silenced plants, the expression of CPK22 and CPK26, two close homologs of CPK27, was not significantly changed. Under optimal growth conditions, the empty vector plants (pTRV) and the pTRV-CPK27 plants showed similar plant growth, maximum quantum yields of photosystem II (PSII) (Fv/Fm) and relative electrolyte leakage (REL) (Fig. 1B, C; Supplementary Fig. S2B). Notably, cold acclimation increased cold tolerance, as evidenced by fewer wilting symptoms, increased Fv/Fm, and decreased REL values relative to those of non-acclimated plants. However, no significant difference in REL was found between non-acclimated pTRV plants and non-acclimated pTRV-CPK27 plants while Fv/Fm was higher in pTRV plants than that in pTRV-CPK27 plants (Fig. 1B, C). Importantly, CPK27 silencing attenuated the effects of cold acclimation on cold tolerance, as evidenced by a lower Fv/Fm and an increased REL relative to those of pTRV plants after the cold stress at 4 °C. In addition, ABA accumulation was increased during the cold acclimation process and subsequent cold treatment in pTRV plants, and this increase in ABA accumulation was partly attenuated in pTRV-CPK27 plants (Fig. 1D). However, sudden cold treatment induced ABA accumulation to a lesser extent compared with cold acclimation in both pTRV plants and pTRV-CPK27 plants. CPK27 triggers the generation of H2O2 and NO and the activation of MPK1/2 NO, H2O2, and MPKs are all involved in cold acclimation-induced cold tolerance (Zhao et al., 2009; Cantrel et al., 2011; Zhou et al., 2012). We thus examined whether cold acclimation-induced accumulation of NO and H2O2 and the activation of MPK were CPK27 dependent. At 25 °C, no significant differences in the transcript levels of RBOH1, NR, MPK1, and MPK2 (see Supplementary Fig. S3A–D), the activities of NADPH oxidase and NR (Fig. 2A, C), or the accumulation levels of H2O2 and NO (Fig. 2B, D; Supplementary Fig. S3E–G) were found between pTRV plants and pTRV-CPK27 plants. Both cold acclimation and cold stress increased the transcript levels of RBOH1, NR, MPK1, and MPK2, the activities of NADPH oxidase and NR, and content of H2O2 and NO in pTRV plants (Fig. 2A–D; Supplementary Fig. S3). Subsequent exposure to cold stress further increased the transcript levels of RBOH1, NR, MPK1, and MPK2, the activities of NADPH oxidase and NR, and the accumulation levels of H2O2 and NO in acclimated plants (Fig. 2A–D; Supplementary Fig. S3). However, silencing CPK27 significantly attenuated the cold acclimation-induced transcript levels of RBOH1, NR, MPK1, and MPK2, the activities of NADPH oxidase and NR, and the accumulation levels of H2O2 and NO (Fig. 2A–D; Supplementary Fig. S3). MPK1/2 activation (the upper lane) was induced by cold acclimation but not by sudden cold at 4 °C in pTRV and pTRV-CPK27 plants (Fig. 2E). However, cold acclimation-induced MPK1/2 activation was attenuated in CPK27-silenced plants. In addition, we found that the application of H2O2 or SNP increased cold acclimation-induced cold tolerance not only in pTRV plants but also in pTRV-CPK27 plants (Supplementary Fig. S4). All these results suggest that CPK27 is involved in the cold acclimation-induced accumulation of H2O2 and NO and MPK1/2 activation and CPK27 appears to act upstream of H2O2 and NO in cold adaptation. Fig. 2. View largeDownload slide Silencing of CPK27 attenuates NADPH-oxidase-induced H2O2 production, nitrate reductase (NR)-induced nitric oxide (NO) production and MPK1/2 activation level in tomato plants. NADPH oxidase activity (A), H2O2 accumulation in the apoplast (B), NR activity (C), NO accumulation (D), and MPK1/2 activation level (E) in the leaves of control (25 °C) and chilled (4 °C) tomato plants with or without cold acclimation. H2O2 accumulation in the apoplast was assayed by observing CeCl3 precipitates located on the membrane using a transmission electron microscope. Blue arrows indicate apoplastic H2O2 accumulation; scale bar: 1 μm. NO accumulation in leaves was visualized using an NO-specific fluorescent probe, 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-2DA), and fluorescence was photographed with a laser scanning confocal microscope (LSCM-500, Zeiss, Germany). Representative images selected from 10 replicates of each treatment are shown in (D); scale bar: 50 μm. The numbers above the images in (E) indicate the relative intensity of MPK1/2 (the upper lane). All parameters in this figure were assayed at 12 h after commencement of the cold stress treatment. Data are the means (±SD) of four biological replicates. Different letters indicate significant differences (P<0.05) according to Tukey’s test. Fig. 2. View largeDownload slide Silencing of CPK27 attenuates NADPH-oxidase-induced H2O2 production, nitrate reductase (NR)-induced nitric oxide (NO) production and MPK1/2 activation level in tomato plants. NADPH oxidase activity (A), H2O2 accumulation in the apoplast (B), NR activity (C), NO accumulation (D), and MPK1/2 activation level (E) in the leaves of control (25 °C) and chilled (4 °C) tomato plants with or without cold acclimation. H2O2 accumulation in the apoplast was assayed by observing CeCl3 precipitates located on the membrane using a transmission electron microscope. Blue arrows indicate apoplastic H2O2 accumulation; scale bar: 1 μm. NO accumulation in leaves was visualized using an NO-specific fluorescent probe, 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-2DA), and fluorescence was photographed with a laser scanning confocal microscope (LSCM-500, Zeiss, Germany). Representative images selected from 10 replicates of each treatment are shown in (D); scale bar: 50 μm. The numbers above the images in (E) indicate the relative intensity of MPK1/2 (the upper lane). All parameters in this figure were assayed at 12 h after commencement of the cold stress treatment. Data are the means (±SD) of four biological replicates. Different letters indicate significant differences (P<0.05) according to Tukey’s test. Relation of NO, H2O2, and MPK1/2 in cold acclimation We next investigated how NO, H2O2, and MPK1/2 were involved in the acclimation-induced tolerance to cold. Plants with RBOH1 silencing (pTRV-RBOH1) showed increased sensitivity to cold stress despite the cold acclimation treatment as evidenced by increased REL and decreased Fv/Fm (Fig. 3A; Supplementary Fig. S5A). pTRV-RBOH1 plants showed decreased accumulation of NO and reduced activation of MPK1/2 relative to those in pTRV plants after the cold acclimation (Fig. 3B, C; Supplementary Fig. S5B, C). However, foliar application of exogenous H2O2 enhanced cold tolerance as indicated by the lower REL, higher Fv/Fm, and increased accumulation of NO, with the effects being more significant in pTRV-RBOH1 plants (Fig. 3A, B; Supplementary Fig. S5A–C). Exogenous H2O2 increased the activation of MPK1/2 in both pTRV plants and pTRV-RBOH1 plants (Fig. 3C). On the other hand, plants with NR silencing (pTRV-NR) showed increased REL and decreased Fv/Fm (Fig. 3D; Supplementary Fig. S5D), which was followed by decreased accumulation of H2O2 and reduced activation of MPK1/2 relative to pTRV after the cold acclimation (Fig. 3E, F; Supplementary Fig. S5E). Application of SNP enhanced cold tolerance with lower REL, higher Fv/Fm, and increased accumulation of H2O2 in the apoplast, with the effects being more significant in pTRV-NR plants (Fig. 3E; Supplementary Fig. S5E). In addition, SNP application further increased the activation of MPK1/2 in both pTRV plants and pTRV-NR plants (Fig. 3F). These results not only demonstrated the role of RBOH1 and NR in the MPK1/2 activation and cold tolerance, but also indicated the interdependency of the production of H2O2 and that of NO during the cold acclimation. Fig. 3. View largeDownload slide NADPH-oxidase-dependent H2O2 and nitrate reductase (NR)-dependent NO are essential for cold acclimation-induced MPK1/2 activation and cold tolerance. (A, D) REL in RBOH1- (A) and NR- (D) silenced plants. (B, E) NO accumulation in RBOH1-silenced plants (B) and H2O2 accumulation in the apoplast in NR-silenced plants (D). (C, F) MPK1/2 activation level in RBOH1- (C) and NR- (F) silenced plants. H2O2 at 10 mM and SNP at 500 μM were applied 12 h before the cold acclimation treatment. H2O2 and NO accumulation in leaves were visualized using the same protocols as shown in Fig. 2. Scale bars: 50 μm (B) and 1 μm (E). The numbers above the images in (C, F) indicate the relative intensity of MPK1/2 (the upper lane). REL was determined at 5 d, while the other parameters were assayed at 12 h after commencement of the cold stress treatment. Data are the means (±SD) of four biological replicates. Different letters indicate significant differences (P<0.05) according to Tukey’s test. Fig. 3. View largeDownload slide NADPH-oxidase-dependent H2O2 and nitrate reductase (NR)-dependent NO are essential for cold acclimation-induced MPK1/2 activation and cold tolerance. (A, D) REL in RBOH1- (A) and NR- (D) silenced plants. (B, E) NO accumulation in RBOH1-silenced plants (B) and H2O2 accumulation in the apoplast in NR-silenced plants (D). (C, F) MPK1/2 activation level in RBOH1- (C) and NR- (F) silenced plants. H2O2 at 10 mM and SNP at 500 μM were applied 12 h before the cold acclimation treatment. H2O2 and NO accumulation in leaves were visualized using the same protocols as shown in Fig. 2. Scale bars: 50 μm (B) and 1 μm (E). The numbers above the images in (C, F) indicate the relative intensity of MPK1/2 (the upper lane). REL was determined at 5 d, while the other parameters were assayed at 12 h after commencement of the cold stress treatment. Data are the means (±SD) of four biological replicates. Different letters indicate significant differences (P<0.05) according to Tukey’s test. To examine the respective contribution of H2O2 and NO in acclimation-induced cold tolerance and the activation of MPK1/2, we applied either H2O2 or SNP to pTRV-NR and pTRV-RBOH1 plants before they were exposed to the cold acclimation and cold stress treatments. We found that the application of H2O2 induced cold tolerance not only in pTRV plants but also in pTRV-NR plants as evidenced by the decreased REL (Fig. 4A). In comparison, the application of SNP decreased REL in both pTRV and pTRV-RBOH1 plants. In agreement with these results, the application of H2O2 induced the activation of MPK1/2 in both pTRV and pTRV-NR plants, while the application of SNP induced the activation of MPK1/2 in both pTRV and pTRV-RBOH1 plants (Fig. 4B). These results demonstrated that RBOH1-dependent H2O2 production and NR-dependent NO production both play a critical role in cold acclimation-induced MPK1/2 activation and cold tolerance in tomato plants. Moreover, the results indicated that H2O2 and NO could independently regulate cold acclimation-induced cold tolerance and MPK1/2 activation. Fig. 4. View largeDownload slide Crosstalk between H2O2 and NO and its role in the regulation of MPK1/2 activation in tomato plants. (A, B) REL (A) and MPK1/2 activation (B) in RBOH1- or NR-silenced plants sprayed with 500 μM SNP or 10 mM H2O2, respectively. pTRV plants sprayed with water at 25 °C are shown as control. The numbers above the images in (B) indicate the relative intensity of MPK1/2 (the upper lane). REL and MPK1/2 activation were assayed at 5 d and 12 h, respectively, after commencement of the cold stress treatment. Data are the means (±SD) of four biological replicates. Different letters indicate significant differences (P<0.05) according to Tukey’s test. Fig. 4. View largeDownload slide Crosstalk between H2O2 and NO and its role in the regulation of MPK1/2 activation in tomato plants. (A, B) REL (A) and MPK1/2 activation (B) in RBOH1- or NR-silenced plants sprayed with 500 μM SNP or 10 mM H2O2, respectively. pTRV plants sprayed with water at 25 °C are shown as control. The numbers above the images in (B) indicate the relative intensity of MPK1/2 (the upper lane). REL and MPK1/2 activation were assayed at 5 d and 12 h, respectively, after commencement of the cold stress treatment. Data are the means (±SD) of four biological replicates. Different letters indicate significant differences (P<0.05) according to Tukey’s test. We then determined whether cold acclimation-activated MPK1/2 in turn affects the homeostasis of H2O2 and NO in response to cold. The results showed that MPK1/2 silencing not only attenuated the acclimation-induced cold tolerance with higher REL and lower Fv/Fm, but also decreased the accumulation of H2O2 and NO (Fig. 5; Supplementary Fig. S6). Exogenous application of H2O2 and SNP restored cold tolerance, as evidenced by the decreased REL and increased Fv/Fm in pTRV-MPK1/2 plants (Fig. 5A; Supplementary Fig. S6A). Furthermore, exogenous application of SNP and H2O2 induced the accumulation of H2O2 and NO, respectively, in pTRV-MPK1/2 plants (Fig. 5B, C; Supplementary Fig. S6B–D). Fig. 5. View largeDownload slide Influence of MPK1/2 co-silencing and exogenous SNP or H2O2 on REL (A), apoplastic H2O2 (B) or NO level (C) with regard to cold acclimation-induced cold tolerance in tomato plants. SNP at 500 μM and H2O2 at 10 mM were applied 12 h before the cold acclimation treatment. Leaf samples were taken at 5 d after commencement of the cold stress treatment for the determination of REL. H2O2 and NO accumulation levels were estimated as described in Fig. 2. Scale bars: 50 μm (C) and 1 μm (B). Data are the means (±SD) of four biological replicates. Different letters indicate significant differences (P<0.05) according to Tukey’s test. Fig. 5. View largeDownload slide Influence of MPK1/2 co-silencing and exogenous SNP or H2O2 on REL (A), apoplastic H2O2 (B) or NO level (C) with regard to cold acclimation-induced cold tolerance in tomato plants. SNP at 500 μM and H2O2 at 10 mM were applied 12 h before the cold acclimation treatment. Leaf samples were taken at 5 d after commencement of the cold stress treatment for the determination of REL. H2O2 and NO accumulation levels were estimated as described in Fig. 2. Scale bars: 50 μm (C) and 1 μm (B). Data are the means (±SD) of four biological replicates. Different letters indicate significant differences (P<0.05) according to Tukey’s test. H2O2, NO, and MPK1/2 are involved in cold acclimation-induced ABA biosynthesis ABA is known to play important roles in plant responses to cold stress. Although ABA accumulation was induced by cold acclimation, this induction was mitigated in pTRV-RBOH1, pTRV-NR, and pTRV-MPK1/2 plants (Fig. 6). We then investigated whether the increased accumulation of ABA was essential for the acclimation-induced cold tolerance. The tomato notabilis (not) mutant, which is deficient in ABA, showed increased cold sensitivity, as indicated by the increased REL from the leaf cells and decreased Fv/Fm following exposure to cold acclimation for 3 d and cold stress conditions for 5 d (Fig. 7A; Supplementary Fig. S7A). Foliar application of exogenous ABA enhanced cold tolerance in both wild-type (WT) and not plants, as evidenced by the decreased REL and increased Fv/Fm. Cold acclimation-induced increases in CPK27 transcript levels, accumulation of H2O2 and NO, and MPK1/2 activation were considerably attenuated in not plants (Fig. 7B–E; Supplementary Fig. S7B–D). Additionally, application of exogenous ABA enhanced the levels of CPK27 transcript, H2O2 and NO, as well as MPK1/2 activation, in both WT and not plants. Fig. 6. View largeDownload slide Silencing of RBOH1, NR, or MPK1/2 attenuates cold acclimation-induced ABA accumulation. Leaf samples were taken at 12 h after commencement of the cold stress treatment for the determination of ABA content. Data are the means (±SD) of four biological replicates. Different letters indicate significant differences (P<0.05) according to Tukey’s test. Fig. 6. View largeDownload slide Silencing of RBOH1, NR, or MPK1/2 attenuates cold acclimation-induced ABA accumulation. Leaf samples were taken at 12 h after commencement of the cold stress treatment for the determination of ABA content. Data are the means (±SD) of four biological replicates. Different letters indicate significant differences (P<0.05) according to Tukey’s test. Fig. 7. View largeDownload slide Cold acclimation-induced transcript levels of CPK27, accumulation of H2O2 and NO, and MPK1/2 activation are dependent on ABA levels in plants. REL (A), relative expression of CPK27 (B), NO (C), H2O2 in the apoplast (D), and activation of MPK1/2 (E) in the wild type (WT) and the ABA biosynthetic mutant, notabilis (not), with foliar application of ABA or H2O. ABA at 50 μM was applied 12 h before the cold acclimation treatment. The numbers above the images in (E) indicate the relative intensity of MPK1/2 (the upper lane). Leaf samples were taken at 5 d for the determination of REL, while the other assays were performed at 12 h after commencement of the cold stress treatment. H2O2 and NO accumulation levels were estimated as described in Fig. 2. Scale bars: 50 μm (C) and 1 μm (D). Data are the means (±SD) of four biological replicates. Different letters indicate significant differences (P<0.05) according to Tukey’s test. Fig. 7. View largeDownload slide Cold acclimation-induced transcript levels of CPK27, accumulation of H2O2 and NO, and MPK1/2 activation are dependent on ABA levels in plants. REL (A), relative expression of CPK27 (B), NO (C), H2O2 in the apoplast (D), and activation of MPK1/2 (E) in the wild type (WT) and the ABA biosynthetic mutant, notabilis (not), with foliar application of ABA or H2O. ABA at 50 μM was applied 12 h before the cold acclimation treatment. The numbers above the images in (E) indicate the relative intensity of MPK1/2 (the upper lane). Leaf samples were taken at 5 d for the determination of REL, while the other assays were performed at 12 h after commencement of the cold stress treatment. H2O2 and NO accumulation levels were estimated as described in Fig. 2. Scale bars: 50 μm (C) and 1 μm (D). Data are the means (±SD) of four biological replicates. Different letters indicate significant differences (P<0.05) according to Tukey’s test. CPK27 is required for ABA-induced cold tolerance We then examined whether cold acclimation-induced ABA accumulation regulates CPK27-dependent events as a feedback mechanism. Thus, we analysed the changes in cold acclimation-induced and CPK27-dependent accumulation of NO and H2O2, as well as MPK1/2 activation and cold tolerance, in response to exogenous ABA and cold acclimation. Application of ABA enhanced cold tolerance as indicated by the decreased REL and increased Fv/Fm (Fig. 8A; Supplementary Fig. S8A), increased the accumulation of H2O2 in the apoplast and NO in the leaves, and induced MPK1/2, but these effects were greatly tempered in plants with CPK27 silencing (Fig. 8B–D; Supplementary Fig. S8B–D). These results indicated that CPK27 is vital for the ABA-induced production of H2O2 in the apoplast and NO in the leaves and for the induction of MPK1/2. Fig. 8. View largeDownload slide CPK27 is essential for ABA-induced cold tolerance, H2O2 and NO accumulation, and MPK1/2 activation in tomato plants. REL (A), NO (B), apoplastic H2O2 (C), and activation of MPK1/2 (D) in pTRV control plants and CPK27-silenced plants with foliar application of ABA or H2O. ABA at 50 μM was applied 12 h before the cold acclimation treatment. The numbers above the images in (D) indicate the relative intensity of MPK1/2 (the upper lane). Leaf samples were taken at 5 d for the determination of REL, while other assays were performed at 12 h after commencement of the cold stress treatment. H2O2 and NO accumulation levels were estimated as described in Fig. 2. Scale bars: 50 μm (B) and 1 μm (C). Data are the means (±SD) of four biological replicates. Different letters indicate significant differences (P<0.05) according to Tukey’s test. Fig. 8. View largeDownload slide CPK27 is essential for ABA-induced cold tolerance, H2O2 and NO accumulation, and MPK1/2 activation in tomato plants. REL (A), NO (B), apoplastic H2O2 (C), and activation of MPK1/2 (D) in pTRV control plants and CPK27-silenced plants with foliar application of ABA or H2O. ABA at 50 μM was applied 12 h before the cold acclimation treatment. The numbers above the images in (D) indicate the relative intensity of MPK1/2 (the upper lane). Leaf samples were taken at 5 d for the determination of REL, while other assays were performed at 12 h after commencement of the cold stress treatment. H2O2 and NO accumulation levels were estimated as described in Fig. 2. Scale bars: 50 μm (B) and 1 μm (C). Data are the means (±SD) of four biological replicates. Different letters indicate significant differences (P<0.05) according to Tukey’s test. Discussion CPKs play critical roles in regulating growth, development, and stress responses in plants (Boudsocq and Sheen, 2013). There is evidence that CPKs participate in the regulation of plant response to cold stress (Martín and Busconi, 2001; Böehmer and Romeis, 2007; Komatsu et al., 2007). However, the mechanisms underlying CPK-dependent cold tolerance induced by cold acclimation remain mostly elusive. Here, we present several lines of evidence supporting the role of CPK27 in the cold response. Cold acclimation-induced CPK27 contributes to early signal transduction processes and is connected to NO, H2O2, and MPK1/2 activation in ABA signaling. This study not only delineated the NO–H2O2–MPK1/2 loop as an integrated signaling mechanism for modulating ABA-dependent cold tolerance but also suggested a positive impact of ABA on CPK27-dependent cold tolerance through a feedback loop in cold acclimation. CPK27 is a positive regulator of cold tolerance induced by cold acclimation Several CPKs have been shown to constitute a complicated regulation network, functioning positively and negatively in plant adaptation to cold stress. For example, OsCPK7, OsCPK13, and OsCPK17 in Oryza sativa (Saijo et al., 2000; Komatsu et al., 2007; Almadanim et al., 2017), AtCPK1 in Arabidopsis (Böhmer and Romeis, 2007), PeCPK10 in Populus euphratica (Chen et al., 2013), and VaCPK20 in Vitis amurensis (Dubrovina et al., 2015) have been characterized as positive regulators of cold stress tolerance, while ZmCPK1 in Zea mays has been shown to act as a negative regulator of cold stress signaling (Weckwerth et al., 2015). Here, we provide multiple lines of evidence that CPK27 is a positive regulator of the cold response in tomato plants. First, transcript levels of CPK27 were significantly increased by cold acclimation (Fig. 1A; Supplementary Fig. S2A). Second, knockdown of CPK27 transcripts attenuated cold acclimation-induced cold tolerance and compromised ABA-induced cold tolerance (Figs 1B, C, 8A). Third, CPK27 silencing reduced the cold acclimation-induced accumulation of ABA, H2O2, and NO, as well as the activation of MPK1/2 (Figs 1D, 2B, D, E; Supplementary Fig. S3E–G), all of which are positive regulators of cold response in plants. These results strongly suggest that CPK27 is not only important for cold response but also involved in the regulation of multiple signaling pathways, including ABA, ROS, NO, and MPK signaling, in response to cold. Evidence is increasing for the roles of NO, apoplastic H2O2, and MPKs in the cold response in plants (Diao et al., 2017; Kim et al., 2017; Si et al., 2017). In agreement with our earlier results, silencing of RBOH1 or NR or co-silencing of MPK1 and MPK2 differentially compromised cold acclimation-induced cold tolerance (Figs 3A, D, 5A). Interestingly, the transcript levels of RBOH1, NR, MPK1, and MPK2 were all subject to regulation by CPK27 (see Supplementary Fig. S3A–D). Silencing of CPK27 attenuated cold acclimation-induced H2O2 accumulation in the apoplast, NO accumulation in the leaves, and activation of MPK1/2 (Fig. 2B, D, E; Supplementary Fig. S3E–G). These results provided convincing evidence for the role of CPK27 in cold acclimation-triggered ROS signaling, NO signaling, and MPK signaling in the cold response. Several studies have demonstrated that CPKs can phosphorylate the N-terminal regions of plasma membrane RBOH proteins (NADPH oxidase) and participate in RBOH-mediated ROS bursts (Kobayashi et al., 2012). Consistent with this, we found that the cold acclimation activation of NADPH oxidase was largely abolished in pTRV-CPK27 plants (Fig. 2A), suggesting that the N-terminal region of NADPH oxidase is likely phosphorylated by CPK27. Evidence also exists that CPKs can activate MPK signaling by activating MPKs. For example, CPK18 in rice was identified as an upstream kinase of MAPK (MPK5) and was shown to phosphorylate and activate MPK5 (Xie et al., 2014). In our present study, CPK27 silencing greatly attenuated the cold acclimation-induced activation of MPK1/2 (Fig. 2E), suggesting a potential role of CPK27 in the activation of MPKs in tomato plants. Interestingly, cold acclimation-induced NO accumulation was also attenuated in CPK27-silenced plants (Fig. 2D; Supplementary Fig. S3F, G), implying the involvement of CPK27 in the regulation of NO homeostasis by indirectly activating NR. Until now, there is no clear evidence for the direct regulation of NR by CPKs in plants in vivo. Further protein–protein interaction experiments will shed light on the mechanisms underlying CPK27-induced activation of ROS signaling, NO signaling, and MPK signaling in response to cold. CPK27-activated crosstalk among H2O2, NO, and MPK1/2 in cold acclimation Several studies have demonstrated the complicated interactions between NO and H2O2, NO and MPKs, and H2O2 and MPKs in response to various stresses or stimuli (Bright et al., 2006; Asai et al., 2008; Takahashi et al., 2011; Ye et al., 2013; Zhou et al., 2014b; Li et al., 2017). Here, we provide several lines of evidence for the existence of a feedback loop among H2O2, MPKs, and NO in the cold response. First, silencing of RBOH1 or NR not only abolished the accumulation of NO or H2O2, respectively, but also decreased the accumulation of activated MPK1/2 induced by cold acclimation (Fig. 3B, C, E, F). Second, foliar application of SNP to pTRV-RBOH1 plants and foliar application of H2O2 to pTRV-NR plants both resulted in an increased accumulation of activated MPK1/2 (Fig. 4B). Third, silencing of MPK1/2 decreased cold acclimation-induced accumulation of both H2O2 and NO (Fig. 5B, C). Fourth, silencing of RBOH1, NR, or MPK1/2 attenuated cold acclimation-induced ABA accumulation (Fig. 6). These results allow us to argue that the crosstalk among H2O2, MPK1/2, and NO is critical to maintain the homeostasis of H2O2 and NO and the activated state of MPK1/2. However, how cold acclimation initiates this loop is unclear, as both NADPH oxidase and MPKs could be phosphorylated or activated by CPKs in plants in vivo (Kobayashi et al., 2007; Xie et al., 2014). The H2O2–MPK1/2–NO feedback loop is likely to be important for the homeostasis of H2O2 and NO and the activation of MPK1/2. Data from the present study show that H2O2 and NO could function independently in the cold response. This finding is substantiated by the increased cold tolerance caused in pTRV-RBOH1 by the foliar application of SNP, in pTRV-NR plants by the foliar application of H2O2, and in pTRV-MPK1/2 plants by the foliar application of SNP or H2O2 (Figs 4A, 5A). While ROS, NO, and MPKs share many similarities in target genes or proteins, they can also act independently in the regulation of many physiological or metabolic processes in plants (Yoshioka et al., 2011; Xu et al., 2014). Studies to date support a role of ROS in the regulation of gene transcripts and protein functions by cysteine modification (Akter et al., 2015). In comparison, NO and MPKs affect signaling cascades, mostly by S-nitrosylation and phosphorylation, respectively (Wang et al., 2015; Kim et al., 2017). Accordingly, the coordination of MPK1/2, H2O2, and NO likely contributes greatly to the cold acclimation-induced cold response by altering a variety of physiological processes, as substantiated by the results of RNAseq and metabolite analysis (Barrero-Gil et al., 2016). Relationship between CPK27 and ABA in cold acclimation Although Ca2+ and CPKs have been suggested as early signals in stress response, evidence for CPK activity in the induction of ABA biosynthesis or signaling is still lacking. Here, we found that CPK27 plays a critical role in ABA accumulation. We observed that cold acclimation-induced ABA accumulation was greatly attenuated in pTRV-CPK27 plants (Fig. 1D). CPK27 is important for the acclimation-induced transcript of RBOH1, NR, and MPK1 and MPK2, the generation of ROS and NO and the activation of MPK1/2 (Fig. 2; Supplementary Fig. S3). However, silencing of RBOH1, NR, or MPK1/2 resulted in decreased accumulations of ABA during the cold acclimation (Fig. 6). While RBOH1-dependent NADPH oxidase is involved in the generation of ABA in response to heat and oxidative stresses, interaction of ROS and NO results in the induction of ABA biosynthesis (Zhao et al., 2001; Xing et al., 2004; Zhou et al., 2014a). Therefore, CPK27 may function as a positive regulator of ABA generation by activating the production of ROS and NO as well as MPK1/2. The results of the present study demonstrated that CPK27-induced generation of ROS and NO and activation of MPK1/2 were subject to regulation by ABA (Fig. 7B–E). Silencing of CPK27 abolished the ABA-induced increase in accumulation of H2O2 and NO, and activation of MPK1/2 in response to cold acclimation (Fig. 8B–D). Many CPKs, including CPK27 in tomato, are ABA responsive (Hu et al., 2016). Arabidopsis CPK4 and CPK11 have been identified as important positive regulators in CPK/calcium-mediated ABA signaling (Zhu et al., 2007). ABA-induced transcriptional reprogramming via ABA-responsive ABF transcription factors is likely to be a key feature of CPK signaling. For example, AtCPK32 activates ABF4 in vivo, resulting in the induction of ABF4 target genes (Choi et al., 2005). Taken together, these results suggest that crosstalk between ABA and CPK27 is important not only for the activation of MPK1/2, ROS signaling, and NO signaling but also for the maintenance of ABA signaling and thus for cold tolerance. Supplementary data Supplementary data are available at JXB online. Fig. S1. Efficiency of gene silencing by virus-induced gene silencing Fig. S2. Effects of cold acclimation on transcripts of the tomato CPK family genes and plant phenotype after cold stress in tomato plants. Fig. S3. Effects of CPK27 silencing on the transcript levels of RBOH1, NR, MPK1, and MPK2 and accumulation levels of H2O2 and NO in leaves of control (25 °C), cold-acclimated and non-acclimated tomato plants. Fig. S4. Exogenous H2O2 or SNP partly rescues the chilling-sensitive phenotype due to CPK27 silencing. Fig. S5. Effects of RBOH1 or NR silencing and exogenous H2O2 or SNP on cold acclimation-induced cold tolerance and NO or H2O2 accumulation in control (25 °C) and cold-acclimated tomato plants. Fig. S6. Effects of MPK1/2 co-silencing and exogenous SNP or H2O2 on Fv/Fm, accumulation of H2O2 or NO in control (25 °C) and cold-acclimated tomato plants. Fig. S7. ABA and cold acclimation-induced changes in Fv/Fm and accumulation of H2O2 and NO in the wild type and the ABA-deficient mutant not. Fig. S8. ABA and cold acclimation-induced changes in Fv/Fm and accumulation of H2O2 and NO in pTRV control plants and CPK27-silenced plants. Table S1. PCR primer sequences used for vector construction. Table S2. List of primer sequences used for qRT-PCR analysis. Acknowledgements The authors are grateful to the Tomato Genetics Resource Center at the California University for tomato seeds. This work was supported by the National Natural Science Foundation of China (grant nos 31372109, 31430076) and the Fundamental Research Funds for the Central Universities (2016XZZX001-07). References Akter S , Huang J , Waszczak C , Jacques S , Gevaert K , Van Breusegem F , Messens J . 2015 . Cysteines under ROS attack in plants: a proteomics view . Journal of Experimental Botany 66 , 2935 – 2944 . Google Scholar CrossRef Search ADS PubMed Almadanim MC , Alexandre BM , Rosa MTG , Sapeta H , Leitão AE , Ramalho JC , Lam TT , Negrão S , Abreu IA , Oliveira MM . 2017 . Rice calcium-dependent protein kinase OsCPK17 targets plasma membrane intrinsic protein and sucrose-phosphate synthase and is required for a proper cold stress response . Plant, Cell & Environment 40 , 1197 – 1213 . Google Scholar CrossRef Search ADS PubMed Asai S , Ohta K , Yoshioka H . 2008 . MAPK signaling regulates nitric oxide and NADPH oxidase-dependent oxidative bursts in Nicotiana benthamiana . The Plant Cell 20 , 1390 – 1406 . Google Scholar CrossRef Search ADS PubMed Barrero-Gil J , Huertas R , Rambla JL , Granell A , Salinas J . 2016 . Tomato plants increase their tolerance to low temperature in a chilling acclimation process entailing comprehensive transcriptional and metabolic adjustments . Plant, Cell & Environment 39 , 2303 – 2318 . Google Scholar CrossRef Search ADS PubMed Bestwick CS , Brown IR , Bennett MH , Mansfield JW . 1997 . Localization of hydrogen peroxide accumulation during the hypersensitive reaction of lettuce cells to Pseudomonas syringae pv phaseolicola . The Plant Cell 9 , 209 – 221 . Google Scholar CrossRef Search ADS PubMed Böhmer M , Romeis T . 2007 . A chemical-genetic approach to elucidate protein kinase function in planta . Plant Molecular Biology 65 , 817 – 827 . Google Scholar CrossRef Search ADS PubMed Boudsocq M , Sheen J . 2013 . CDPKs in immune and stress signaling . Trends in Plant Science 18 , 30 – 40 . Google Scholar CrossRef Search ADS PubMed Bradford MM . 1976 . A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding . Analytical Biochemistry 72 , 248 – 254 . Google Scholar CrossRef Search ADS PubMed Bright J , Desikan R , Hancock JT , Weir IS , Neill SJ . 2006 . ABA-induced NO generation and stomatal closure in Arabidopsis are dependent on H2O2 synthesis . The Plant Journal 45 , 113 – 122 . Google Scholar CrossRef Search ADS PubMed Cantrel C , Vazquez T , Puyaubert J , Rezé N , Lesch M , Kaiser WM , Dutilleul C , Guillas I , Zachowski A , Baudouin E . 2011 . Nitric oxide participates in cold-responsive phosphosphingolipid formation and gene expression in Arabidopsis thaliana . New Phytologist 189 , 415 – 427 . Google Scholar CrossRef Search ADS PubMed Cao WH , Liu J , He XJ , Mu RL , Zhou HL , Chen SY , Zhang JS . 2007 . Modulation of ethylene responses affects plant salt-stress responses . Plant Physiology 143 , 707 – 719 . Google Scholar CrossRef Search ADS PubMed Chen J , Xue B , Xia X , Yin W . 2013 . A novel calcium-dependent protein kinase gene from Populus euphratica, confers both drought and cold stress tolerance . Biochemical and Biophysical Research Communications 441 , 630 – 636 . Google Scholar CrossRef Search ADS PubMed Chinnusamy V , Zhu J , Zhu JK . 2007 . Cold stress regulation of gene expression in plants . Trends in Plant Science 12 , 444 – 451 . Google Scholar CrossRef Search ADS PubMed Choi HI , Park HJ , Park JH , Kim S , Im MY , Seo HH , Kim YW , Hwang I , Kim SY . 2005 . Arabidopsis calcium-dependent protein kinase AtCPK32 interacts with ABF4, a transcriptional regulator of abscisic acid-responsive gene expression, and modulates its activity . Plant Physiology 139 , 1750 – 1761 . Google Scholar CrossRef Search ADS PubMed Deng XG , Zhu T , Zou LJ , Han XY , Zhou X , Xi DH , Zhang DW , Lin HH . 2016 . Orchestration of hydrogen peroxide and nitric oxide in brassinosteroid-mediated systemic virus resistance in Nicotiana benthamiana . The Plant Journal 85 , 478 – 493 . Google Scholar CrossRef Search ADS PubMed Diao Q , Song Y , Shi D , Qi H . 2017 . Interaction of polyamines, abscisic acid, nitric oxide, and hydrogen peroxide under chilling stress in tomato (Lycopersicon esculentum Mill.) seedlings . Frontiers in Plant Science 8 , 203 . Google Scholar CrossRef Search ADS PubMed Ding Y , Cao J , Ni L , Zhu Y , Zhang A , Tan M , Jiang M . 2013 . ZmCPK11 is involved in abscisic acid-induced antioxidant defence and functions upstream of ZmMPK5 in abscisic acid signalling in maize . Journal of Experimental Botany 64 , 871 – 884 . Google Scholar CrossRef Search ADS PubMed Doulis AG , Debian N , Kingston-Smith AH , Foyer CH . 1997 . Differential localization of antioxidants in maize leaves . Plant Physiology 114 , 1031 – 1037 . Google Scholar CrossRef Search ADS PubMed Dubiella U , Seybold H , Durian G , Komander E , Lassig R , Witte CP , Schulze WX , Romeis T . 2013 . Calcium-dependent protein kinase/NADPH oxidase activation circuit is required for rapid defense signal propagation . Proceedings of the National Academy of Sciences, USA 110 , 8744 – 8749 . Google Scholar CrossRef Search ADS Dubrovina AS , Kiselev KV , Khristenko VS , Aleynova OA . 2015 . VaCPK20, a calcium-dependent protein kinase gene of wild grapevine Vitis amurensis Rupr., mediates cold and drought stress tolerance . Journal of Plant Physiology 185 , 1 – 12 . Google Scholar CrossRef Search ADS PubMed Foyer CH , Valadier MH , Migge A , Becker TW . 1998 . Drought-induced effects on nitrate reductase activity and mRNA and on the coordination of nitrogen and carbon metabolism in maize leaves . Plant Physiology 117 , 283 – 292 . Google Scholar CrossRef Search ADS PubMed Fujita M , Fujita Y , Noutoshi Y , Takahashi F , Narusaka Y , Yamaguchi-Shinozaki K , Shinozaki K . 2006 . Crosstalk between abiotic and biotic stress responses: a current view from the points of convergence in the stress signaling networks . Current Opinion in Plant Biology 9 , 436 – 442 . Google Scholar CrossRef Search ADS PubMed Gao X , Chen X , Lin W , et al. 2013 . Bifurcation of Arabidopsis NLR immune signaling via Ca²⁺-dependent protein kinases . PLoS Pathogens 9 , e1003127 . Google Scholar CrossRef Search ADS PubMed Gilmour SJ , Thomashow MF . 1991 . Cold acclimation and cold-regulated gene expression in ABA mutants of Arabidopsis thaliana . Plant Molecular Biology 17 , 1233 – 1240 . Google Scholar CrossRef Search ADS PubMed Hu X , Jiang M , Zhang J , Zhang A , Lin F , Tan M . 2007 . Calcium-calmodulin is required for abscisic acid-induced antioxidant defense and functions both upstream and downstream of H2O2 production in leaves of maize (Zea mays) plants . New Phytologist 173 , 27 – 38 . Google Scholar CrossRef Search ADS PubMed Hu Z , Lv X , Xia X , Zhou J , Shi K , Yu J , Zhou Y . 2016 . Genome-wide identification and expression analysis of calcium-dependent protein kinase in tomato . Frontiers in Plant Science 7 , 469 . Google Scholar PubMed Jiang M , Zhang J . 2002 . Water stress-induced abscisic acid accumulation triggers the increased generation of reactive oxygen species and up-regulates the activities of antioxidant enzymes in maize leaves . Journal of Experimental Botany 53 , 2401 – 2410 . Google Scholar CrossRef Search ADS PubMed Jin Y , Ye N , Zhu F , Li H , Wang J , Jiang L , Zhang J . 2017 . Calcium-dependent protein kinase CPK28 targets the methionine adenosyltransferases for degradation by the 26S proteasome and affects ethylene biosynthesis and lignin deposition in Arabidopsis . The Plant Journal 90 , 304 – 318 . Google Scholar CrossRef Search ADS PubMed Kandoth PK , Ranf S , Pancholi SS , Jayanty S , Walla MD , Miller W , Howe GA , Lincoln DE , Stratmann JW . 2007 . Tomato MAPKs LeMPK1, LeMPK2, and LeMPK3 function in the systemin-mediated defense response against herbivorous insects . Proceedings of the National Academy of Sciences, USA 104 , 12205 – 12210 . Google Scholar CrossRef Search ADS Kim SH , Kim HS , Bahk S , An J , Yoo Y , Kim JY , Chung WS . 2017 . Phosphorylation of the transcriptional repressor MYB15 by mitogen-activated protein kinase 6 is required for freezing tolerance in Arabidopsis . Nucleic Acids Research 45 , 6613 – 6627 . Google Scholar CrossRef Search ADS PubMed Kobayashi M , Ohura I , Kawakita K , Yokota N , Fujiwara M , Shimamoto K , Doke N , Yoshioka H . 2007 . Calcium-dependent protein kinases regulate the production of reactive oxygen species by potato NADPH oxidase . The Plant Cell 19 , 1065 – 1080 . Google Scholar CrossRef Search ADS PubMed Kobayashi M , Yoshioka M , Asai S , Nomura H , Kuchimura K , Mori H , Doke N , Yoshioka H . 2012 . StCDPK5 confers resistance to late blight pathogen but increases susceptibility to early blight pathogen in potato via reactive oxygen species burst . New Phytologist 196 , 223 – 237 . Google Scholar CrossRef Search ADS PubMed Komatsu S , Yang G , Khan M , Onodera H , Toki S , Yamaguchi M . 2007 . Over-expression of calcium-dependent protein kinase 13 and calreticulin interacting protein 1 confers cold tolerance on rice plants . Molecular Genetics and Genomics 277 , 713 – 723 . Google Scholar CrossRef Search ADS PubMed Li FC , Wang J , Wu MM , Fan CM , Li X , He JM . 2017 . Mitogen-activated protein kinase phosphatases affect UV-B-induced stomatal closure via controlling NO in guard cells . Plant Physiology 173 , 760 – 770 . Google Scholar CrossRef Search ADS PubMed Livak KJ , Schmittgen TD . 2001 . Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔC(T) method . Methods 25 , 402 – 408 . Google Scholar CrossRef Search ADS PubMed Lv X , Ge S , Jalal Ahammed G , Xiang X , Guo Z , Yu J , Zhou Y . 2017 . Crosstalk between nitric oxide and MPK1/2 mediates cold acclimation-induced chilling tolerance in tomato . Plant & Cell Physiology 58 , 1963 – 1975 . Google Scholar CrossRef Search ADS PubMed Mantyla E , Lang V , Palva ET . 1995 . Role of abscisic acid in drought-induced freezing tolerance, cold acclimation, and accumulation of LT178 and RAB18 proteins in Arabidopsis thaliana . Plant Physiology 107 , 141 – 148 . Google Scholar CrossRef Search ADS PubMed Martín ML , Busconi L . 2001 . A rice membrane-bound calcium-dependent protein kinase is activated in response to low temperature . Plant Physiology 125 , 1442 – 1449 . Google Scholar CrossRef Search ADS PubMed McAinsh MR , Hetherington AM . 1998 . Encoding specificity in Ca2+ signalling systems . Trends in Plant Science 3 , 32 – 36 . Google Scholar CrossRef Search ADS Neill S , Barros R , Bright J , Desikan R , Hancock J , Harrison J , Morris P , Ribeiro D , Wilson I . 2008 . Nitric oxide, stomatal closure, and abiotic stress . Journal of Experimental Botany 59 , 165 – 176 . Google Scholar CrossRef Search ADS PubMed Saijo Y , Hata S , Kyozuka J , Shimamoto K , Izui K . 2000 . Over-expression of a single Ca2+-dependent protein kinase confers both cold and salt/drought tolerance on rice plants . The Plant Journal 23 , 319 – 327 . Google Scholar CrossRef Search ADS PubMed Santin F , Bhogale S , Fantino E , Grandellis C , Banerjee AK , Ulloa RM . 2017 . Solanum tuberosum StCDPK1 is regulated by miR390 at the posttranscriptional level and phosphorylates the auxin efflux carrier StPIN4 in vitro, a potential downstream target in potato development . Physiologia Plantarum 159 , 244 – 261 . Google Scholar CrossRef Search ADS PubMed Shi K , Li X , Zhang H , Zhang G , Liu Y , Zhou Y , Xia X , Chen Z , Yu J . 2015 . Guard cell hydrogen peroxide and nitric oxide mediate elevated CO2-induced stomatal movement in tomato . New Phytologist 208 , 342 – 353 . Google Scholar CrossRef Search ADS PubMed Shinozaki K , Yamaguchi-Shinozaki K , Seki M . 2003 . Regulatory network of gene expression in the drought and cold stress responses . Current Opinion in Plant Biology 6 , 410 – 417 . Google Scholar CrossRef Search ADS PubMed Si T , Wang X , Wu L , et al. 2017 . Nitric oxide and hydrogen peroxide mediate wounding-induced freezing tolerance through modifications in photosystem and antioxidant system in wheat . Frontiers in Plant Science 8 , 1284 . Google Scholar CrossRef Search ADS PubMed Takahashi F , Mizoguchi T , Yoshida R , Ichimura K , Shinozaki K . 2011 . Calmodulin-dependent activation of MAP kinase for ROS homeostasis in Arabidopsis . Molecular Cell 41 , 649 – 660 . Google Scholar CrossRef Search ADS PubMed Thomashow MF . 1998 . Role of cold-responsive genes in plant freezing tolerance . Plant Physiology 118 , 1 – 8 . Google Scholar CrossRef Search ADS PubMed Wang F , Guo Z , Li H , Wang M , Onac E , Zhou J , Xia X , Shi K , Yu J , Zhou Y . 2016 . Phytochrome A and B function antagonistically to regulate cold tolerance via abscisic acid-dependent jasmonate signaling . Plant Physiology 170 , 459 – 471 . Google Scholar CrossRef Search ADS PubMed Wang P , Du Y , Hou YJ , Zhao Y , Hsu CC , Yuan F , Zhu X , Tao WA , Song CP , Zhu JK . 2015 . Nitric oxide negatively regulates abscisic acid signaling in guard cells by S-nitrosylation of OST1 . Proceedings of the National Academy of Sciences, USA 112 , 613 – 618 . Google Scholar CrossRef Search ADS Weckwerth P , Ehlert B , Romeis T . 2015 . ZmCPK1, a calcium-independent kinase member of the Zea mays CDPK gene family, functions as a negative regulator in cold stress signalling . Plant, Cell & Environment 38 , 544 – 558 . Google Scholar CrossRef Search ADS PubMed Xia XJ , Wang YJ , Zhou YH , Tao Y , Mao WH , Shi K , Asami T , Chen Z , Yu JQ . 2009 . Reactive oxygen species are involved in brassinosteroid-induced stress tolerance in cucumber . Plant Physiology 150 , 801 – 814 . Google Scholar CrossRef Search ADS PubMed Xia XJ , Zhou YH , Ding J , Shi K , Asami T , Chen Z , Yu JQ . 2011 . Induction of systemic stress tolerance by brassinosteroid in Cucumis sativus . New Phytologist 191 , 706 – 720 . Google Scholar CrossRef Search ADS PubMed Xie K , Chen J , Wang Q , Yang Y . 2014 . Direct phosphorylation and activation of a mitogen-activated protein kinase by a calcium-dependent protein kinase in rice . The Plant Cell 26 , 3077 – 3089 . Google Scholar CrossRef Search ADS PubMed Xing H , Tan LL , An LH , Zhao ZG , Wang SM , Zhang CL . 2004 . Evidence for the involvement of nitric oxide and reactive oxygen species in osmotic stress tolerance of wheat seedlings: inverse correlation between leaf abscisic acid accumulation and leaf water loss . Plant Growth Regulation 42 , 61 – 68 . Google Scholar CrossRef Search ADS Xing Y , Jia W , Zhang J . 2008 . AtMKK1 mediates ABA-induced CAT1 expression and H2O2 production via AtMPK6-coupled signaling in Arabidopsis . The Plant Journal 54 , 440 – 451 . Google Scholar CrossRef Search ADS PubMed Xu J , Xie J , Yan C , Zou X , Ren D , Zhang S . 2014 . A chemical genetic approach demonstrates that MPK3/MPK6 activation and NADPH oxidase-mediated oxidative burst are two independent signaling events in plant immunity . The Plant Journal 77 , 222 – 234 . Google Scholar CrossRef Search ADS PubMed Ye Y , Li Z , Xing D . 2013 . Nitric oxide promotes MPK6-mediated caspase-3-like activation in cadmium-induced Arabidopsis thaliana programmed cell death . Plant, Cell & Environment 36 , 1 – 15 . Google Scholar CrossRef Search ADS PubMed Yoshioka H , Mase K , Yoshioka M , Kobayashi M , Asai S . 2011 . Regulatory mechanisms of nitric oxide and reactive oxygen species generation and their role in plant immunity . Nitric Oxide 25 , 216 – 221 . Google Scholar CrossRef Search ADS PubMed Zhang A , Jiang M , Zhang J , Tan M , Hu X . 2006 . Mitogen-activated protein kinase is involved in abscisic acid-induced antioxidant defense and acts downstream of reactive oxygen species production in leaves of maize plants . Plant Physiology 141 , 475 – 487 . Google Scholar CrossRef Search ADS PubMed Zhao MG , Chen L , Zhang LL , Zhang WH . 2009 . Nitric reductase-dependent nitric oxide production is involved in cold acclimation and freezing tolerance in Arabidopsis . Plant Physiology 151 , 755 – 767 . Google Scholar CrossRef Search ADS PubMed Zhao ZG , Chen GC , Zhang CL . 2001 . Interaction between reactive oxygen species and nitric oxide in drought-induced abscisic acid synthesis in root tips of wheat seedlings . Australian Journal of Plant Physiology 28 , 1055 – 1061 . Zhou J , Wang J , Li X , Xia XJ , Zhou YH , Shi K , Chen Z , Yu JQ . 2014a. H2O2 mediates the crosstalk of brassinosteroid and abscisic acid in tomato responses to heat and oxidative stresses . Journal of Experimental Botany 65 , 4371 – 4383 . Google Scholar CrossRef Search ADS PubMed Zhou J , Wang J , Shi K , Xia XJ , Zhou YH , Yu JQ . 2012 . Hydrogen peroxide is involved in the cold acclimation-induced chilling tolerance of tomato plants . Plant Physiology and Biochemistry 60 , 141 – 149 . Google Scholar CrossRef Search ADS PubMed Zhou J , Xia XJ , Zhou YH , Shi K , Chen Z , Yu JQ . 2014b. RBOH1-dependent H2O2 production and subsequent activation of MPK1/2 play an important role in acclimation-induced cross-tolerance in tomato . Journal of Experimental Botany 65 , 595 – 607 . Google Scholar CrossRef Search ADS PubMed Zhu SY , Yu XC , Wang XJ , et al. 2007 . Two calcium-dependent protein kinases, CPK4 and CPK11, regulate abscisic acid signal transduction in Arabidopsis . The Plant Cell 19 , 3019 – 3036 . Google Scholar CrossRef Search ADS PubMed Zou JJ , Li XD , Ratnasekera D , Wang C , Liu WX , Song LF , Zhang WZ , Wu WH . 2015 . Arabidopsis CALCIUM-DEPENDENT PROTEIN KINASE8 and CATALASE3 function in abscisic acid-mediated signaling and H2O2 homeostasis in stomatal guard cells under drought stress . The Plant Cell 27 , 1445 – 1460 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

Journal

Journal of Experimental BotanyOxford University Press

Published: Jun 1, 2018

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off