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MPK6 controls H2O2-induced root elongation by mediating Ca2+ influx across the plasma membrane of root cells in Arabidopsis seedlings Shuan Han, Lin Fang, Xuejian Ren, Wenle Wang and Jing Jiang State Key Laboratory of Cotton Biology, College of Life Sciences, Henan University, Jinming Street, Kaifeng Henan 475004, China

Summary Author for correspondence: Jing Jiang Tel: +86 371 23881387 Email: [email protected] Received: 26 March 2014 Accepted: 17 July 2014

New Phytologist (2014) doi: 10.1111/nph.12990

Key words: abscisic acid (ABA), apoplastic H2O2 increase, AtMPK6, cell wall peroxidase34, cytosolic Ca2+ rise, plasma membrane Ca2+ channels, root elongation.


Mitogen-activated protein kinases (MPKs) play critical roles in signalling and growth, and Ca2+ and H2O2 control plant growth processes associated with abscisic acid (ABA). However, it remains unclear how MPKs are involved in H2O2- and Ca2+-mediated root elongation.
Root elongation in seedlings of the loss-of-function mutant Atmpk6 (Arabidopsis thaliana MPK6) was less sensitive to moderate H2O2 or ABA than that in wild-type (WT) plants. The enhanced elongation was a result of root cell expansion. This effect disappeared when ABAinduced H2O2 accumulation or the cytosolic Ca2+ increase were defective.
Molecular and biochemical evidence showed that increased expression of the cell wall peroxidase PRX34 in Atmpk6 root cells enhanced apoplastic H2O2 generation; this promoted a cytosolic Ca2+ increase and Ca2+ influx across the plasma membrane. The plasma membrane damage caused by high levels of H2O2 was ameliorated in a Ca2+-dependent manner.
These results suggested that there was intensified PRX34-mediated H2O2 generation in the apoplast and increased Ca2+ flux into the cytosol of Atmpk6 root cells; that is, the spatial separation of apoplastic H2O2 from cytosolic Ca2+ in root cells prevented H2O2-induced inhibition of root elongation in Atmpk6 seedlings.

Introduction Abscisic acid (ABA) regulates plant root growth by integrating the control of reactive oxygen species (ROS) with cytosolic Ca2+ homeostasis (Hubbard et al., 2010; Kudla et al., 2010). The main ROS involved in root growth are H2O2 and O2• (superoxide anion). H2O2 plays dual roles in cell extensibility. Moderate levels of H2O2 play a role in cell expansion by cross-linking various cell wall molecules (Rodriguez et al., 2002; Liszkay et al., 2004), which is essential for Arabidopsis seedling root growth (Bai et al., 2007; Dunand et al., 2007; Tsukagoshi et al., 2010). Conversely, excessive H2O2 causes over cross-linking, which inhibits cell extension (Carpin et al., 2001; Dunand et al., 2007) and root elongation (Bai et al., 2007). O2• can be converted into H2O2, or can directly promote plant root cell proliferation in the meristem zone (Tsukagoshi et al., 2010). In plant cells, the generation of ROS is mainly catalysed by NADPH oxidases, known as respiratory burst oxidase homologues (RBOHs) (Kimura et al., 2012), and class III cell wall peroxidases (PRXs) (Cosio & Dunand, 2009). As an inducer of ROS generation, ABA drives the phosphorylation of the Arabidopsis RBOHF protein (Sirichandra et al., 2009) and promotes root growth (Foreman et al., 2003; Bai et al., 2009). In addition, PRXs catalyse apoplastic H2O2 generation in Arabidopsis root cells (Dunand et al., 2007) or cultured cells (Daudi et al., Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

2012); thus, these enzymes are also involved in root elongation (Passardi et al., 2006; Tsukagoshi et al., 2010). Interestingly, H2O2 generated by RBOHD induced the transcription of PRX34 and buffered Arabidopsis cell growth in response to biotic stress (Daudi et al., 2012; O’Brien et al., 2012), indicating that H2O2-mediated cell growth depends on the cooperation between PRXs and RBOHs in stress signalling. Modulation of cytosolic Ca2+ is an essential process for H2O2mediated plant root growth (Hubbard et al., 2010). The cytosolic N-terminus of the RBOH proteins contains two EF-hand motifs (Kudla et al., 2010), which bind Ca2+. Therefore, the promotion of Arabidopsis seedling root growth by H2O2 generated by RBOHD/F depends on Ca2+ influx from the apoplast (Foreman et al., 2003; Kwak et al., 2003). Conversely, damage to Ca2+-permeable channels in the root cell plasma membrane, the predominant Ca2+ acquisition system, disrupts root growth (Li et al., 2008). Unlike RBOHs, PRXs contain a putative Ca2+-pectatebinding domain (Carpin et al., 2001). In these proteins, the Ca2+ ion plays a unique structural role (Conn et al., 2011). In fact, Ca2+ has a strong affinity with negatively charged cross-linked pectate. When PRX- or H2O2-generated cross-linked pectate and Ca2+ ions exceed certain concentrations, deposition of Ca2+-pectate complexes occurs, reducing cell extensibility and growth (Conn et al., 2011; O’Brien et al., 2012). This is accompanied by membrane damage events, reflected by increased New Phytologist (2014) 1 www.newphytologist.com

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malondialdehyde (MDA) content and electrolyte leakage (Laohavisit et al., 2010). Clearly, apoplastic Ca2+ and PRXgenerated H2O2 are both involved in the determination of plant cell expansibility. There is increasing evidence that AtMPK6 plays a role in ROS signalling in plant growth and development (Xing et al., 2009; Wang et al., 2010). An MPK6::GUS construct was actively expressed in Arabidopsis seedling roots (M€ uller et al., 2010). ABA increased AtMPK6 protein levels (Ichimura et al., 2000), whereas H2O2 promoted the phosphorylation of AtMPK6 (Wang et al., 2010). These findings explain why Atmpk6-3 seedling root elongation was less sensitive, whereas that in MPK6-overexpressing plants was sensitive to ABA (Xing et al., 2009). By contrast, Atmpk6-3 mediated ABA-induced H2O2 generation in leaf tissue as a result of inactivation of a specific H2O2-scavenging enzyme, catalase 1 (CAT1) (Xing et al., 2008), but CAT1 was not expressed in Arabidopsis seedling roots (Du et al., 2008). Thus, further research is required to investigate the mechanisms of ABA-induced H2O2 accumulation in Atmpk6 roots. Moreover, an increase in cytosolic Ca2+ was required for AtMPK6 promotion of methyl jasmonate-induced leaf senescence (Yue et al., 2012), and the rise in cytosolic Ca2+ activated by a serine–threonine (Ser/Thr) protein kinase enhanced AtRBOHF/D-mediated generation of ROS in a heterologous expression system in HEK293T cells (Kimura et al., 2012). However, it is still unknown whether the activation of AtMPK6 influences Ca2+ distribution in root cells. Based on the repression of primary root growth by AtMPK6 (Lopez-Bucio et al., 2014), here we determine whether AtMPK6 integrates the signals of Ca2+ homeostasis with H2O2-mediated root growth in Arabidopsis seedlings.

MS containing various additions. Plates were placed vertically. The seedling age (in days) was counted from the day of transfer. Measurement of seedling root elongation and root tip cell length The root length of seedlings grown on MS or MS containing various additions was measured under an FV1000 microscope (Olympus, Tokyo, Japan) every day after transfer, as described by Bai et al. (2009). A ruler was imaged alongside the samples and was used to calibrate the measurements. After staining with 10 lg ml1 propidium iodide (PI) for 10–20 min, the number of epidermal cells from the root apex to the elongation region was counted, and the length of epidermal cells in the elongation region was measured under a confocal microscope. Dye loading and laser scanning confocal microscopy The distribution of H2O2 and Ca2+ in root tissue was examined using H2DCF-DA (20 ,70 -dichlorodihydrofluorescein diacetate; Molecular Probes, Eugene, OR, USA) and Fluo-3 AM (Sigma, St Louis, MO, USA) probes, respectively. The roots were dissected from 12-d-old WT or Atmpk6 seedlings grown on MS. Before further experiments, the roots were pre-incubated in the dark in loading buffer (10 mM Tris + 50 mM KCl; pH 7.18) containing 50 lM H2DCF-DA for c. 12 min, or in buffer (50 mM KCl + 10 mM Tris + 5 mM CaCl2 + 0.5 mM eserine; pH 7.18) containing 30 lM Fluo-3 AM for c. 2 h, and then washed with distilled water to remove excess dye. Examinations of fluorescence intensity were performed using a laser scanning confocal microscope (excitation, 488 nm; emission, 500– 550 nm; Olympus FV1000).

Materials and Methods Detection of H2O2 content in seedling roots Plant materials and growth conditions Arabidopsis thaliana L. ecotype Columbia-0 was used as the wildtype (WT) in this study. The T-DNA insertion lines for A. thaliana MPK6 (Atmpk6; At2g43790), including Atmpk6-2 (Salk_073907) and Atmpk6-3 (Salk_127507), have been used in previous studies in our laboratory (Wang et al., 2010). The T-DNA insertion line for PRX34 (At3g49120) was prx34 (Salk_051769). Seeds of mutant lines were obtained from the Arabidopsis Biological Resource Centre, and the homozygous mutant plants were screened by polymerase chain reaction (PCR) amplification according to the method recommended by the Salk Institute (http://signal.salk.edu). All of the seeds were collected and stored in the same conditions. The seeds were surface sterilized with 0.1% HgCl2 for 5 min, sown on Murashige–Skoog (MS) medium (0.6% agar, 1% sucrose) and kept for 3 d at 4°C in the dark to break dormancy. The plates were then transferred to a culture room (day : night temperature cycle of 22°C : 18°C, c. 70% relative humidity and a 16-h light : 8-h dark photoperiod with a light intensity of c. 100 lmol m2 s1) for a further 7 d, and the seedlings were transferred to fresh MS (1.2% agar, 1% sucrose) or New Phytologist (2014) www.newphytologist.com

The roots were cut from 12-d-old WT or Atmpk6 seedlings grown on MS or on MS with various additions, and their H2O2 content was determined using the peroxidase-coupled assay method described by Veljovic-Jovanovic et al. (2002). The reaction solution contained 0.1 M phosphate buffer (pH 6.5), 3.3 mM 3-(dimethylamino) benzoic acid, 0.07 mM 3-methyl-2benzothiazoline hydrazone and 0.3 U peroxidase. The reaction was initiated by the addition of 200 ll of sample. The change in absorbance at 590 nm was measured at 25°C. For each assay, H2O2 content was quantified by reference to an internal standard. Total RNA extraction, reverse transcription (RT)-PCR and quantitative PCR analysis Total RNA was isolated from root tissue (200 mg) of 12-d-old Atmpk6 or WT seedlings grown on MS or treated with the indicated reagents for 9 h, using Trizol reagent (Invitrogen, Carlsbad, CA, USA). First-strand cDNA was synthesized using Moloney murine leukaemia virus reverse transcriptase (Invitrogen). PCR amplifications were performed according to standard protocols Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

New Phytologist using Taq DNA polymerase or Pyrobest DNA polymerase (TaKaRa, Kyoto, Japan). The mean value of three replicates was normalized against that of ACTIN2, which was amplified using the primers 50 -ATTACCCGATGGGCAAGTCA-30 (forward primer, FP) and 50 -CACAAACGAGGGCTGGAACA-30 (reverse primer, RP) as an internal control. The other gene-specific primers used for PCR amplifications were as follows: PRX34 FP 50 -CTGCTTTGTTAATGGTTGTGACGC-30 , RP 50 -TCGC TCTGGATAAGACCTTTTCGC-30 ; AtRBOHD FP 50 -CTGG ACACGTAAGCTCAGGA-30 , RP 50 -GCCGAGACCTACGA GGAGTA-30 ; and AtRBOHF FP 50 -GATGGTTTAGGCGT AACCTAGTCAA-30 , RP 50 -AACAAATGATGCGAATACCA AAAG-30 . Detection of RBOHF and PRX34 promoter::GUS activity in seedling roots For RBOHF- and PRX34-GUS staining, the RBOHF and PRX34 promoter fragments were amplified using the primers (for AtRBOHF) 50 -CCCCTGCATCGTCGTGTCATTACTAC TCA-30 (PstI site in italic) and 50 -CCCGTCGACAGATC CAAAGTCGGAATTCAAA-30 (SalI site in italic), and (for PRX34) 50 -CGCGGATCCCAAAGAAATAGTCTATAGATC T-30 (BamHI site in italic) and 50 -GGTTCTGCAGAACTAGT TGATTCTGTTTACCC-30 (PstI site in italic). These fragments were cloned into the promoter-less b-glucuronidase (GUS) expression vector pCAMBIA1381. The RBOHF- and PRX34GUS constructs were transferred from Escherichia coli DH5a into Agrobacterium tumefaciens strain GV3101, and then transformed by floral infiltration into Atmpk6 and the WT. Roots of transgenic Atmpk6 and WT seedlings were used for GUS staining assays. Extraction of protein and measurement of enzyme activities in seedling roots Total protein was extracted from the roots of 12-d-old seedlings grown on MS (control) or MS containing various additions. The root tissue (1 g) was frozen in liquid nitrogen, ground and homogenized in extraction buffer (100 mM Tris-HCl, pH 8.0, 20% glycerol and 30 mM dithiothreitol). After centrifugation at 15 000 g for 20 min at 4°C, the supernatant was used as the crude extract for enzyme activity assays according to Lv et al. (2011). For glutathione peroxidase (GPX) activity assays, the reaction mixture contained 100 mM potassium phosphate buffer (pH 7.0), 1 mM guaiacol, 0.3% H2O2 and enzyme extract. The increase in absorbance at 470 nm was recorded for 2 min. One unit of GPX activity was defined as 1 mM guaiacol oxidized per minute under these conditions. For ascorbate peroxidase (APX) activity assays, 50 ll of enzyme extract was added to a 3-ml reaction mixture containing 50 mM potassium phosphate buffer (pH 7.5), 0.25 mM ascorbic acid (AsA) and 0.2 mM H2O2. The reaction was initiated by the addition of H2O2. The decrease in absorbance at 290 nm was measured for 1 min. One unit of APX activity was defined as 1 mM ascorbate oxidized per minute under these conditions. Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

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Construction of aequorin-expressing plants for Ca2+ measurements Transgenic Atmpk6-3 and WT plants constitutively expressing intracellular apoaequorin were generated by transformation with pMAQ2, a gift from Dr M. Knight (Oxford University, UK). The F2 generation was screened and used for the following experiments. The roots were dissected from transgenic seedlings grown on MS for 12 d, and aequorin (Aq) was reconstituted by incubation in 2.5 mM native coelenterazine (Promega) overnight in the dark at c. 25°C. The next day, 20 roots were placed in a transparent plastic cuvette, and then transferred into a TD20/20n digital luminometer (Turner Biosystems, Sunnyvale, CA, USA). Luminescence was recorded every 0.2 s under these conditions. After 20 s, 1.5 mM H2O2 or 20 lM ABA was carefully injected into the cuvette. At the end of each experiment, the remaining Aq was discharged by adding an equal volume of 2 M CaCl2 and 20% ethanol. Luminescence values were converted to Ca2+ concentrations according to Bai et al. (2009). Patch-clamp analyses in seedling root cells Root protoplasts were isolated from 10-d-old seedlings grown on MS. The whole-cell voltage-clamp current in root cells was recorded with an EPC-9 amplifier (HEKA Elektronik, Lambrecht, Germany). The recording conditions were as follows: leak currents were decreased by increasing seal resistances, and the final pipette resistances were at least 12 MΩ; voltage ramps were from +32 to 190 mV; the pipette solution contained 10 mM BaCl2, 0.1 mM dithiothreitol, 4 mM ethylene glycol tetraacetic acid (EGTA), 10 mM Hepes (pH 7.2) with osmolarity at 380 mOsmol kg1 adjusted with D-sorbitol; the bath solution contained 100 mM CaCl2, 0.1 mM dithiothreitol, 10 mM Mes (pH 5.5) with osmolarity at 360 mOsmol kg1 adjusted with D-sorbitol. Hyperpolarization-activated conductance appeared within a few minutes of exposure to each treatment, and whole-cell Ca2+ currents were recorded over 15 min. The data were configured and analysed using the PULSE and PULSEFIT software (version 8.3; HEKA Electronik). The final whole-cell Ca2+ currents were expressed as current density (pA pF1) to account for variations in the cell size or surface area. Assay of MDA content in seedling roots Seedling root tissue (0.5 g) from 12-d-old seedlings grown on MS or MS containing various additions was frozen in liquid nitrogen, ground and then homogenized in 1.5 ml of 20% (w/ v) trichloroacetic acid (TCA). The mixture was centrifuged at 10 000 g for 5 min, and then a 1-ml aliquot of the supernatant was added to 2 ml of thiobarbituric acid solution (0.5% (w/v) in 20% TCA). The mixture was heated at 95°C for 15 min, quickly cooled in an ice bath and then centrifuged at 12 000 g for 10 min. The absorbance of the supernatant was measured spectrophotometrically at 450, 532 and 600 nm (optical New Phytologist (2014) www.newphytologist.com

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density, OD). The concentration of MDA was calculated according to the following equation (Lv et al., 2011):

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Concentration ðlMÞ ¼ 6:45  ðOD532  OD600 Þ  0:56  OD450 : Measurement of electrolyte leakage in seedling roots The roots were harvested from 12-d-old seedlings grown on MS (control) or MS containing various additions. The clean roots were weighed and then immersed in 25 ml of deionized water at 25°C. After 24 h, the conductivity of the solution was measured using a conductivity meter (initial conductivity). The tube was heated in an electric water bath for 15 min at 95°C, immediately cooled in an ice bath and then the conductivity was re-measured (final conductivity). The relative electrolyte leakage was calculated according to the following equation (Lv et al., 2011): Electrolyte leakage ¼

Initial conductivity  100%: Final conductivity

Statistical analysis Differences in various parameters were compared using Student’s t-test. P < 0.05 was considered to be statistically significant.

Results Roots of Atmpk6 seedlings showed greater elongation than those of the WT in response to H2O2 Homozygotes of Atmpk6-2 and Atmpk6-3 were identified, and their root growth phenotype was observed in response to H2 O 2 . In H2O2-free MS conditions (control), root elongation was not significantly different between the WT and mutant seedlings; the mean root lengths of 12-d-old Atmpk6-2, Atmpk6-3 and WT seedlings were 6.88  0.48, 6.96  0.41 and 6.61  0.48 cm, respectively (Fig. 1a,b). However, when the concentration of H2O2 in the MS medium was increased from 0.5 to 1.5 mM, the root lengths of Atmpk6-2 and Atmpk6-3 exceeded that of WT plants after 5 d (Fig. 1d). In particular, on medium containing 1.5 mM H2O2, the mean length of Atmpk6-3 roots (c. 3.89 cm) was c. 2.1-fold that of WT roots (c. 1.82 cm) after 12 d (Fig. 1c, d). These data suggested that root elongation was less sensitive to ≤ 1.5 mM H2O2 treatment in Atmpk6 seedlings than in WT seedlings. To examine this reduced sensitivity to H2O2 accumulation in vivo, the effects of ABA-induced H2O2 generation on root growth were analysed. Root elongation in Atmpk6 seedlings was less sensitive to 20 lM ABA relative to that in WT plants (Fig. 2a,c). However, when the H2O2 scavenger AsA (200 mg l1) was added to MS containing ABA, root elongation in Atmpk6 seedlings was similar to that in the WT (Fig. 2b,c). This suggested that root elongation in Atmpk6 showed reduced New Phytologist (2014) www.newphytologist.com

Fig. 1 Increased root elongation in Atmpk6 seedlings in response to moderate H2O2 levels. Arabidopsis seeds were germinated on Murashige– Skoog (MS) for 4 d, and then transferred to fresh MS or MS containing the indicated additions. The seedling root phenotype/length was observed every day. Representative samples are shown. (a) Root elongation was not significantly different among 12-d-old wild-type (WT), Atmpk6-2 and Atmpk6-3, when all seedlings were grown on free MS. (b) Root lengths of 12-d-old Atmpk6-2, Atmpk6-3 and WT seedlings treated with 0, 0.5, 1.5 and 4.5 mM H2O2. (c) Root lengths of 12-d-old Atmpk6-3 and Atmpk6-2 were longer than that of WT seedlings, when all seedlings were grown on MS containing 1.5 mM H2O2. (d) Root elongation of WT, Atmpk6-3 and Atmpk6-3 seedlings exposed to 1.5 mM H2O2 from 2 to 12 d. (b, d) Values are means  SD (**, P < 0.01) of 90–110 seedlings from five independent experiments.

sensitivity to ABA-induced H2O2 accumulation relative to that in WT plants. Atmpk6 showed the promotion of root cell elongation in response to H2O2 To test how H2O2 affected the elongation of Atmpk6 roots, the length of the extensible region from the primary root apex to the beginning of the root fibril was measured under a microscope. In H2O2-free conditions, the length of this extensible region was the same in WT and Atmpk6-3 seedlings (Fig. 3a). On MS containing H2O2 or ABA, this region was longer in Atmpk6-3 roots than in WT roots. The addition of AsA to MS containing ABA reversed this effect, and the length of this region in Atmpk6-3 roots was again similar to that in WT roots (Fig. 3a). To test whether root cell extension in Atmpk6 seedlings was tolerant of H2O2 treatment, the size of the root cells was determined using PI staining. As the appearance of the root fibril indicates mature root cells, we measured the lengths of root cells that were located near the beginning of the root fibril. In H2O2-free conditions, the root cells of Atmpk6-3 and WT were the same size (Fig. 3b). By contrast, in H2O2-supplemented conditions, the Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

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Fig. 2 Root elongation in Atmpk6 was promoted by the abscisic acid (ABA)-induced increase in H2O2. (a) Root lengths of 12-d-old Arabidopsis Atmpk6-2, Atmpk6-3 and wild-type (WT) seedlings grown on Murashige– Skoog (MS) containing 20 lM ABA. (b) Root elongation of Atmpk6-2 and Atmpk6-3 seedlings was greater than that of WT seedlings when all plants were grown on MS containing 20 lM ABA and 150 mg l1 ascorbic acid (AsA). (c) Statistical analysis of root length of Atmpk6-2 (grey bars), Atmpk6-3 (hatched bars) and WT (white bars) seedlings treated with 20 lM ABA and 20 lM ABA + 150 mg l1 AsA. Values are means  SD (**, P < 0.01) of 30–50 seedlings from three independent experiments.

epidermal cells in Atmpk6-3 roots were clearly elongated compared with those in WT roots (Fig. 3b). The addition of AsA equally shortened the mature epidermal cells in Atmpk6-3 and WT seedling roots (Fig. 3b). The number of epidermal cells in a section of the root distant from the root fibril was counted; there were no significant difference in the number of cells in this region between WT and Atmpk6-3 seedlings, with or without H2O2 (data not shown). These findings suggested that elongation of Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

Fig. 3 Atmpk6 showed enhanced H2O2-mediated root cell elongation. (a) Length of the extensible region from the primary root apex to the beginning of the root fibril was measured under a microscope. Values were compared between Arabidopsis Atmpk6-3 and wild-type (WT) seedlings grown on Murashige–Skoog (MS) (control) or MS containing 1.5 mM H2O2, 20 lM abscisic acid (ABA) or 20 lM ABA + 150 mg l1 ascorbic acid (AsA) for 12 d. The arrows indicate the beginning of the root fibril. (b) Length of root epidermal cells located near the beginning of the root fibril. Cell length was determined by propidium iodide (PI) staining under the conditions described above. The spaces between the two yellow arrows in the same root indicate the length of one root cell.

Atmpk6 seedling roots in response to H2O2 was a result of greater cell elongation, rather than enhanced cell division, compared with WT plants. Atmpk6 was dependent on Ca2+ for H2O2-enhanced root cell elongation The role of Ca2+ in Atmpk6 root elongation was investigated. The addition of CaCl2 (0–20 mM) did not result in a difference in root elongation between Atmpk6-3 and the WT (Fig. 4c). When the Ca2+ chelator EGTA was added to MS containing H2O2, root elongation (Fig. 4a,c) and root cell elongation (Fig. 4d) were equally restricted in Atmpk6-3 and WT seedlings. Conversely, the addition of the Ca2+ ionophore A23187 to MS containing H2O2 resulted in greater root growth (Fig. 4b,c) and root cell elongation (Fig. 4d) in Atmpk6-3 roots than in WT New Phytologist (2014) www.newphytologist.com

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Fig. 4 Atmpk6 was dependent on Ca2+ for H2O2-mediated root or root cell elongation. (a) Seedling root elongation phenotypes of 12-d-old Arabidopsis Atmpk6-3, Atmpk6-2 and wild-type (WT) seedlings grown on Murashige–Skoog (MS) (control) or MS containing 1.5 mM H2O2 and 0.5 mM ethylene glycol tetraacetic acid (EGTA). (b) Root elongation responses of Atmpk6-3, Atmpk6-2 and WT seedlings grown on MS containing 1.5 mM H2O2 and 5 lM A23187. (c) Comparative analysis of root lengths of Atmpk6-3 (hatched bars), Atmpk6-2 (grey bars) and WT (white bars) seedlings treated with 1.5 mM H2O2, 1.5 mM H2O2 + 5 lM A23187, 1.5 mM H2O2 + 0.5 mM EGTA or 5 mM CaCl2. Values are means  SD (**, P < 0.01) of 30–50 seedlings from three independent experiments. (d) Length of root epidermal cells located near the beginning of the root fibril was analysed by propidium iodide (PI) staining. The yellow arrows indicate the edges of the root cell. Values were compared between Atmpk6-3 and WT seedlings grown on MS (control) or MS containing 1.5 mM H2O2, 1.5 mM H2O2 + 5 lM A23187 or 5 mM H2O2 + 0.5 mM EGTA for 12 d.

roots. These observations suggested that Ca2+ was required for H2O2-induced elongation of root cells in Atmpk6. Atmpk6 seedlings showed enhanced ABA-induced H2O2 generation in roots To determine whether root elongation in Atmpk6 could tolerate H2O2 accumulation, the level of H2O2 induced by ABA was determined in seedling roots. Staining with 3,5-diaminobenzidine (DAB) showed that the H2O2 level was higher in the roots of the mutants than in the roots of WT seedlings grown on MS containing ABA (Supporting Information Fig. S1). The O2•-specific stain, nitroblue tetrazolium (NBT), showed that the ABA-induced O2• level in primary roots was the same in the mutants and WT (Fig. S2), which excluded the possibility that O2• in root cells was responsible for Atmpk6 root elongation. Biochemical analyses showed that the H2O2 level was higher in the roots of Atmpk6-2 (1122  39 nmol g1 FW) and Atmpk6-3 (1130  63 nmol g1 FW) seedlings than in the roots of WT (953  52 nmol g1 FW) seedlings grown on MS containing ABA (Fig. 5d). Clearly, the H2O2 content was positively related to root elongation in Atmpk6 seedlings. We further monitored the dynamic distribution of H2O2 in the roots of Atmpk6 seedlings using confocal scanning. All of the primary roots were pre-loaded with the H2O2 probe H2DCFDA. There was no change in 20 ,70 -dichlorofluorescein (DCF) fluorescence intensity in the mutant or WT roots in ABA-free New Phytologist (2014) www.newphytologist.com

conditions. The addition of ABA promoted a greater increase in DCF fluorescence in Atmpk6-2 and Atmpk6-3 roots than in WT roots (Fig. 5a). Importantly, the location of the fluorescence indicated that H2O2 was mainly distributed in the apoplast of the elongated root cells (Fig. 5b). That is, apoplastic H2O2 accumulation was positively related to root cell elongation in Atmpk6 seedlings. ROS-generating enzymes were activated in the roots of Atmpk6 seedlings To uncover the mechanism underlying the modified H2O2 generation in the root cells of Atmpk6, the expression of genes encoding the H2O2-generating enzymes RBOHD/F was determined. RT-PCR (Fig. S3a) and real-time PCR (Fig. 6a) analyses showed that the transcript level of RBOHF was higher than that of RBOHD in the roots of Atmpk6-3 and WT seedlings in response to ABA. When the RBOHF promoter was fused to the GUS gene and introduced into Atmpk6-3 and the WT, the GUS activity was higher in Atmpk6 than in WT roots in the presence of ABA (Fig. 6b). We also evaluated the expression of other H2O2-generating enzymes, PRX34 and PRX57, in the roots using the RT-qPCR method. The abundance of PRX34 mRNA showed no significant difference between Atmpk6-3 (3.8  0.5) and WT (3.6  0.4) roots with or without ABA (Fig. 6c). Treatment with H2O2 induced a c. 7.1-fold increase in the PRX34 mRNA level in Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

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Fig. 5 Atmpk6 intensified abscisic acid (ABA)-induced H2O2 generation within the apoplastic space of root tissue. All roots were cut from 12-d-old Arabidopsis wild-type (WT), Atmpk6-3 and Atmpk6-2 seedlings grown on Murashige–Skoog (MS) and pre-incubated with 50 lM H2DCFDA (20 ,70 dichlorodihydrofluorescein diacetate; H2O2 probe) for 12 min. Then, 20 ,70 -dichlorofluorescein (DCF) fluorescence was monitored by confocal microscopy. (a) Representative images of DCF fluorescence intensity in the absence or presence of 20 lM ABA for 10 min. Samples selected randomly from five independent experiments are shown. (b) Yellow arrows indicate location of H2O2 accumulation marked by DCF fluorescence. H2O2 accumulated within the apoplastic space in root tissue. (c) Scale colours of DCF fluorescence intensity in (a) or (b). (d) Determination of H2O2 content in WT (white bars), Atmpk6-3 (hatched bars) and Atmpk6-2 (grey bars) seedling roots grown on MS with or without 20 lM ABA for 12 d. Values are means  SD (*, P < 0.05) from three independent experiments.

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Fig. 6 Atmpk6 activated root peroxidase34 (PRX34) activity, rather than respiratory burst oxidase homologue F (RBOHF) activity. (a) Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis of expression levels of RBOHD/F in 12-d-old Arabidopsis Atmpk6-3 or wild-type (WT) seedling roots in the absence or presence of abscisic acid (ABA) for 9 h. Samples shown were randomly selected from three independent experiments. Expression of ACTIN2 served as the internal control. (b) Activation of RBOHF promoter-GUS in root tissue from transgenic WT or Atmpk6-3 plants grown on Murashige–Skoog (MS) or MS containing 20 lM ABA for 12 d. (c) Ratio of mRNA levels of PRX34 to ACTIN2 using RT-qPCR analysis of PRX34 gene expression in Atmpk6-3 or WT seedling roots, treated with or without 1.5 mM H2O2 or 20 lM ABA for 9 h. Values are means  SD (**, P < 0.01; *, P < 0.05) from four independent experiments. (d) Activation of PRX34 promoter-GUS in root tissue from transgenic WT or Atmpk6-3 plants grown on MS or MS containing 1.5 mM H2O2 for 12 d. (e, f) Total enzyme activity of glutathione peroxidase (GPX) (e) and ascorbate peroxidase (APX) (f) in Atmpk6-3, Atmpk6-2 and WT seedling roots grown on MS with or without 1.5 mM H2O2 or 20 lM ABA for 12 d. Values are means  SD (**, P < 0.01; *, P < 0.05) from five independent experiments. Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

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Atmpk6-3 roots compared with that in WT roots (Fig. 6c), whereas the transcript levels of PRX57 showed no obvious changes with or without ABA or H2O2 (Fig. S3b). Accordingly, when the PRX34 promoter was fused to the GUS gene and introduced into Atmpk6-3 and the WT, ABA significantly increased GUS activity in Atmpk6-3 roots compared with those of WT seedlings (Fig. 6d). The enzyme activities of GPX and APX were determined. In plants grown on MS containing ABA, the GPX activities in Atmpk6-3 roots (41  2.6 U g1 FW) and Atmpk6-2 roots (40  2.5 U g1 FW) were c. 2.1-fold higher than those in WT roots (28  2.4 U g1 FW) (Fig. 6e). Similarly, H2O2 induced higher APX activity in Atmpk6 roots than in WT roots (Fig. 6f). These findings suggested that PRX34 expression enhanced H2O2 accumulation in Atmpk6 roots compared with that in WT roots. The T-DNA insertion mutant per34 cannot express PRX34. In the roots of per34 seedlings, ABA-induced H2O2 generation and root cell elongation were decreased compared with those in Atmpk6-3 roots; however, these growth retardations could be recovered by low concentrations of H2O2 (Fig. S4). These results suggested that the enzyme activity of PER34 played a role in Arabidopsis root growth. Atmpk6 seedling root cells showed a greater H2O2-induced rise in cytosolic Ca2+ To monitor how H2O2 modified the distribution of Ca2+ in Atmpk6 root cells, the fluorescence of the Ca2+-sensitive dye Fluo-3 AM was analysed by confocal microscopy. The H2O2excited fluorescence intensity was stronger in Atmpk6 roots than in WT roots, and this increased fluorescence was centred in the (a)

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Ca2+ influx across the plasma membrane was promoted in Atmpk6 root cells To explain how H2O2 increased the level of cytosolic Ca2+ in Atmpk6 root cells, we tested Ca2+ influx across the plasma membrane using the whole-cell configuration of the patch-clamp technique. Hyperpolarization did not induce the activity of Ca2+-permeable channels in 10-d-old Atmpk6-3 or WT seedling root cells; the average currents were 30.7  7.1 and 31.2  6.3 pA pF1, respectively, at a membrane potential of 190 mV (Fig. 8). However, the addition of ABA to the bath solution resulted in Ca2+ currents rising to 67.9 

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cytosol of root cells (Fig. 7a–c). That is, H2O2 concentrated Ca2+ in the cytosol in Atmpk6 roots. To monitor the variations in cytosolic Ca2+ in vivo, we created transgenic Atmpk6 and WT plants constitutively expressing the Ca2+-sensitive luminescence protein Aq. The levels of Aq were confirmed to be sufficient for experiments by western blot analysis (Fig. 7e). In the control experiments, the luminescence value of cytosolic Ca2+ remained constant at c. 0.05  0.001 lM in the roots of 12-d-old WT and Atmpk6-3 seedlings (Fig. 7f,g). The application of H2O2 caused an increase in Ca2+ from 40 s, peaking at 45–55 s, with Ca2+ peak values of 0.455  0.03 lM in the roots of transgenic Atmpk6 and 0.314  0.02 lM in WT roots. Likewise, ABA triggered a rise from 20 s in transgenic Atmpk6 and WT roots, with Ca2+ peak values of 0.434  0.03 lM and 0.296  0.02 lM, respectively (Fig. 7g). These results clearly showed that H2O2 was able to induce an increase in cytosolic Ca2+ in the root cells of Atmpk6 seedlings compared with those of WT plants.

Fig. 7 Atmpk6 showed increased H2O2induced Ca2+ accumulation in cytosol of root cells. (a–c) Fluorescence intensity of Fluo-3 AM staining measured by confocal microscopy in roots of Arabidopsis wild-type (WT) or Atmpk6 seedlings treated with or without 1.5 mM H2O2 for 6 h. Representative images randomly selected from five independent experiments are shown. (d) Scale colours of Fluo-3 fluorescence intensity in (a–c). (e) Western blot analysis of aequorin (Aq) protein in WT or Atmpk6-3 seedlings according to Bai et al. (2009). NSB, non-specific band detected by the Aq antibody (loading control). (f, g) Seedling roots were cut from Aq-expressing WT or Atmpk6-3 seedlings grown on free Murashige–Skoog (MS). Cytosolic Ca2+ levels in the roots were measured by monitoring changes in Aq-emitted luminescence over time before and after application of 1.5 mM H2O2 (f) or 20 lM abscisic acid (ABA) (g). Recording started at 20 s after application of H2O2 or ABA. Values shown in the graphs are means from five independent experiments (50 roots; P < 0.05). Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

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Fig. 8 Atmpk6 showed increased H2O2-activated Ca2+ flux across the root cell plasma membrane into the cytosol. Representative traces of whole-cell plasma membrane Ca2+ currents are shown from 10-d-old Arabidopsis wild-type (WT) (a) and Atmpk6-3 (b) seedling root cells in the absence or presence of 20 lM abscisic acid (ABA). Responses of Ca2+ influx in WT (c) and Atmpk6-3 (d) root cells in the absence or presence of 1.5 mM H2O2. Voltage ramps were from +32 to 190 mV. (e, f) Average currents (mean  SD; **, P < 0.01; *, P < 0.05) from (a–d).

15.3 pA pF1 in WT and 77.2  8.5 pA pF1 in Atmpk6 root cells at 190 mV (Fig. 8a,b,f,g). Simultaneously, the H2O2-elevated Ca2+ currents rose to 123.2  10.3 pA pF1 in Atmpk6 and 116.9  11.1 pA pF1 in WT root cells at 190 mV (Fig. 8c,d,f,g). The data showed that there was a substantial increase in ABA- or H2O2-activated Ca2+ influx through the plasma membrane in the root cells of Atmpk6 relative to WT plants. In addition, we evaluated the gene expression of Ca2+ transporters. The results showed that the mRNA levels of plasma membrane-localized genes, such as the cyclic nucleotide-gated channels CNGC10/15 and the glutamate receptors GLR1.3/2.5/ 3.1, were higher in Atmpk6-3 than in WT root tissue in response to ABA or H2O2 (Fig. S5). These results implied that there was an increase in H2O2-triggered redistribution of Ca2+ across the plasma membrane in Atmpk6 root cells. Atmpk6 increased H2O2-mediated membrane integrity in a Ca2+-dependent manner in root cells Next, we investigated how the increased levels of apoplastic H2O2 and cytosolic Ca2+ affected membrane integrity in Atmpk6 root cells. We evaluated membrane integrity by checking the MDA content and electrolyte leakage. Initially, the MDA Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

content increased from 0.8 lmol g FW1 (controls) to 2.2 lmol g FW1 (by ABA addition) or 2.5 lmol g FW1 (by H2O2 addition) in WT roots. However, in Atmpk6-3 roots, the MDA content showed smaller increases, from 0.7 lmol g FW1 (control) to 1.2 lmol g FW1 (by ABA addition) or 1.3 lmol g FW1 (by H2O2 addition) (Fig. 9a). Consistent with these results, ABA or H2O2 addition resulted in much smaller increases in the percentage of ion leakage in Atmpk6-3 roots (c. 16% by ABA addition or 19% by H2O2 addition) than in WT roots (c. 38% by ABA addition or 44% by H2O2 addition) (Fig. 9b). The addition of EGTA to MS containing H2O2 resulted in no significant differences in the MDA level and the percentage of ion leakage between Atmpk6 roots (c. 2.0 lmol g FW1 and 21%, respectively) and WT roots (c. 2.3 lmol g FW1 and 15%, respectively) (Fig. 9a,b). Conversely, A23187 decreased the H2O2-induced MDA content (from 2.4 to 1.8 lmol g FW1) and the percentage of ion leakage (from 45% to 21%) in WT roots. A23187 resulted in even larger decreases in these parameters in Atmpk6 roots; the H2O2-induced MDA content was c. 1.3 lmol g FW1 and the percentage of ion leakage was c. 0.6% (Fig. 9a,b). These results suggested that apoplastic H2O2-induced membrane injury was ameliorated by Ca2+ influx in Atmpk6 root cells relative to those of WT. New Phytologist (2014) www.newphytologist.com

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Fig. 9 Atmpk6 showed Ca2+-dependent alleviation of H2O2 damage to membrane integrity of root cells. (a) Malondialdehyde (MDA) content and (b) electrolyte leakage (%) in root tissue of Arabidopsis wild-type (WT; white bars) or Atmpk6-3 (hatched bars) seedlings grown on Murashige–Skoog (MS) or MS containing 20 lM abscisic acid (ABA), 1.5 mM H2O2, 5 lM A23187 or 0.5 mM ethylene glycol tetraacetic acid (EGTA) for 12 d. Values are means  SE (**, P < 0.01: *, P < 0.05) of five independent experiments.

Discussion In Arabidopsis, MPK6 switches various plant growth responses involved in ROS signalling (Xing et al., 2009; Wang et al., 2010; Chang et al., 2012). The present study suggested that the loss of AtMPK6 function altered Ca2+ distribution across the plasma membrane and changed the seedling root elongation response to H2 O 2 . There is an increasing body of evidence indicating that inactivation of AtMPK6 contributes to H2O2-promoted root growth of Arabidopsis under stress conditions. For example, root elongation was less sensitive in the null mutant Atmpk6-3 than in WT seedlings to the stress hormone ABA (Xing et al., 2009). Inaction of the MKK9-MPK6 kit reversed ABA inhibition of seed germination or NaCl inhibition of seedling root elongation (Alzwiy & Morris, 2007). Here, our results showed that root elongation of Atmpk6 was less sensitive than that of WT seedlings to inhibition by H2O2 (≤ 1.5 mM) or ABA (Figs 1, 2), whereas application of AsA reversed this insensitivity (Fig. 2). These results indicated that H2O2 was required for Atmpk6 seedling root elongation. The increased elongation of roots (Figs 1, 2) and root cells (Fig. 3) in Atmpk6 was dependent on the accumulation of H2O2 (Figs 5, S1) derived from PRX34 activity in roots (Figs 6, S4). Atmpk6 showed increased ABA-induced H2O2 accumulation in both leaf (Xing et al., 2008) and root (Figs 5, S1) tissues, compared with that in WT seedlings. The patterns of RBOH and PRX activities explained the molecular and biochemical mechanisms of H2O2 accumulation in Atmpk6 roots. Atmpk6 showed significantly increased PRX34 transcription and peroxidase

Fig. 10 Model for putative pathway of mitogen-activated protein kinase 6 (MPK6)-mediated root elongation integrating H2O2 and Ca2+ signalling in Arabidopsis. Here, the arrows indicate positive and negative roles, respectively. The dotted lines indicate the MPK6-involved events in Arabidopsis root growth. A detailed description of this model is provided in the text. ABA, abscisic acid; RBOHF, respiratory burst oxidase homologue F. New Phytologist (2014) www.newphytologist.com

activity compared with WT seedling roots, and these increases were more sensitive to H2O2 than to ABA (Fig. 6). These findings support the proposal that H2O2 specifically activates PRX34 expression in Arabidopsis roots (Passardi et al., 2006; Dunand et al., 2007). Considering that PRX34 itself can generate H2O2 (Dunand et al., 2007; Daudi et al., 2012), it is not surprising that H2O2 accumulation was mutually promoted alongside PRX34 activity in Atmpk6 roots. Importantly, the accumulated H2O2 was mainly located in the apoplastic space in Atmpk6 root cells (Fig. 5b), raising the possibility that H2O2 may play a role in stretching root cell walls (Rodriguez et al., 2002; Liszkay et al., 2004). In other studies, H2O2 generated by RBOHD promoted PRX34 expression in cultured Arabidopsis cells (Daudi et al., 2012; O’Brien et al., 2012). In the light of these findings, RBOHF-generated H2O2 could induce PRX34 expression in Atmpk6 roots, as the increase in RBOHF activity (Fig. 6) and the cytosolic Ca2+ level (Figs 7, 8) met the requirements for H2O2 generation by ABA. Consequently, Atmpk6 would employ a mutual positive feed-forward mechanism between the activation of PRX34 and H2O2 generation, thus amplifying H2O2 accumulation and root growth. A compensatory mechanism has been observed in H2O2 maintenance of Atmpk6 root elongation. The equal rise in O2• levels in Atmpk6 and WT roots indicated that Atmpk6 did not show greater root cell proliferation than WT (Fig. S2). This confirmed that H2O2 was responsible for the elongation of Atmpk6 roots, as reported by Tsukagoshi et al. (2010). In addition to apoplastic H2O2 accumulation (Fig. 5a,b), the rise in cytosolic Ca2+ levels (Figs 7, 8) resulted in Atmpk6 root elongation (Fig. 4). The fluorescence labelling and in vivo imaging of Aq-expressing seedling roots showed that H2O2 mimicked ABA to elevate cytosolic Ca2+ levels (Fig. 7). This elevation was more sensitive to H2O2 than to ABA (Figs 7, 8); that is, H2O2 promoted the ABA-induced rise in cytosolic Ca2+ in Atmpk6 root cells. The evidence for this hypothesis was that the rise in cytosolic Ca2+ not only contributed to the generation of H2O2 by RBOHs, but also played a unique structural role during cell extension (Conn et al., 2011). Conversely, inhibition of the rise in cytosolic Ca2+ damaged root cell growth (Li et al., 2008; Kudla et al., 2010) and Atmpk6 root elongation (Fig. 4). The elongation of Atmpk6 roots (Figs 1, 2) and root cells (Fig. 3) was positively correlated with PRX34-activated H2O2 Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

New Phytologist accumulation (Figs 6, S4). This observation was somewhat unexpected, because it is generally acknowledged that excessive PRXs or H2O2 can enhance the deposition of Ca2+-pectate complexes and halt cell expansion (Cosio & Dunand, 2009; O’Brien et al., 2012). Indeed, the H2O2-induced Ca2+ influx across the plasma membrane (Figs 8. S5) resulted in not only a rise in cytosolic Ca2+ (Fig. 6b,c), but also in its spatial separation from apoplastic H2O2 accumulation (Fig. 5a,b) in Atmpk6 root cells. Thus, a possible explanation is that the apoplastic H2O2-induced increase in the cytosolic Ca2+ concentration causes a relative lack of Ca2+ ions within Atmpk6 root cell walls. This lack of Ca2+ ions would restrict Ca2+-pectate deposition within the apoplastic space, maintaining membrane integrity (Fig. 9) and root cell extensibility (Conn et al., 2011; O’Brien et al., 2012). Instead, PRX34-activated H2O2 accumulation inhibited cell expansion in AtMPK6-expressing cells in response to biotic stress (O’Brien et al., 2012). In summary, the loss of AtMPK6 activity modified the accumulation of apoplastic H2O2 and cytosolic Ca2+ in root cells; these changes made the mutant less sensitive to H2O2 inhibition of root elongation. A putative signalling pathway showing the relationships among MPK6, ABA, H2O2 and Ca2+ is summarized in Fig. 10.

Acknowledgements We thank Dr M. R. Knight (Oxford University, UK) for the kind gift of pMAQ2, and Professor Benke Kuai (Fudan University) and Professor Shan Lu (Nanjing University) for the kind gift of prx34 seeds. This work was supported by the National Natural Science Foundation of China (grant nos. 30971509 and 31271510 to J.J.).

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Supporting Information Additional supporting information may be found in the online version of this article. Fig. S1 3,5-Diaminobenzidine (DAB) staining showing greater levels of H2O2 accumulation in Atmpk6 than in wild-type (WT) roots.

Fig. S2 Nitroblue tetrazolium (NBT) staining showing equal and slightly increased O2• levels in Atmpk6 and wild-type (WT) roots. Fig. S3 PCR analysis showing the expression levels of PRX57/34 and RBOHD/F in Atmpk6-3 and wild-type (WT) roots in response to abscisic acid (ABA) or H2O2. Fig. S4 Confocal images showing decreased root cell elongation (propidium iodide (PI) staining) and H2O2 generation (20 ,70 -dichlorodihydrofluorescein diacetate (H2DCF-DA) probe) in prx34 roots compared with those in Atmpk6 and wild-type (WT) roots. Fig. S5 PCR analysis showing the expression levels of plasma membrane-located Ca2+ transporters in Atmpk6-3 and wild-type (WT) roots in response to abscisic acid (ABA) or H2O2. Please note: Wiley Blackwell are not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office.

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