CBL-interacting protein kinase 25 contributes to root meristem development

CIPK25, a calcium-regulated protein kinase, plays an important role in the co-ordination of auxin and cytokinin signaling in root meristem development.

Abstract

Co-ordination of auxin and cytokinin activities determines root meristem size during post-embryonic development. Calcineurin B-like proteins (CBLs) and their interacting protein kinases (CIPKs) constitute signaling modules that relay calcium signals. Here we report that CIPK25 is involved in regulating the root meristem size. Arabidopsis plants lacking CIPK25 expression displayed a short root phenotype and a slower root growth rate with fewer meristem cells. This phenotype was rescued by restoration of CIPK25 expression. CIPK25 interacted with CBL4 and -5, and displayed strong gene expression in the flower and root, except in the cell proliferation domain in the root apical meristem. Its expression in the root was positively and negatively regulated by auxin and cytokinin, respectively. The cipk25 T-DNA insertion line was compromised in auxin transport and auxin-responsive promoter activity. The cipk25 mutant line showed altered expression of auxin efflux carriers (PIN1 and PIN2) and an Aux/IAA family gene SHY2. Decreased PIN1 and PIN2 expression in the cipk25 mutant line was completely restored when combined with a SHY2 loss-of-function mutation, resulting in recovery of root growth. SHY2 and PIN1 expression was partially regulated by cytokinin even in the absence of CIPK25, suggesting a CIPK25-independent cytokinin signaling pathway(s). Our results revealed that CIPK25 plays an important role in the co-ordination of auxin and cytokinin signaling in root meristem development.

Introduction

Roots exhibit indeterminate growth which is regulated by external factors (Schenk and Jackson, 2002; Chapman et al., 2012). Root cells are produced in the root apical meristem (RAM) region. The quiescent center (QC), a group of cells located at the rootward proximal border of the RAM, is considered as the organizing center. The cells that surround the QC constitute the stem cell niche (SCN) that gives rise to the different cell files of the root by cell proliferation. Each stem cell divides and the daughter cells undergo additional cycles of division and move longitudinally towards the shoot. Eventually, the proliferation rate of these cells decreases as they move upwards; at one point they show a very low probability of division and begin to elongate rapidly at the elongation zone (EZ). For practical convenience, the region between the QC and the first elongated cortex cell was considered as the RAM in several studies. A small domain of the RAM bordering the EZ shows a very low probability of cell division, but is yet to start cell elongation and is referred to as the transition domain (TD). The cells in this domain of the RAM grow at the same relative rate as the cells in the proliferative domain and pass to the EZ. As the rate of cell division is different for each cell type, the boundary of the TD towards the QC cannot be well demarcated (Verbelen et al., 2006; Dello Ioio et al., 2007, 2008; Perilli and Sabatini, 2010; Ivanov and Dubrovsky, 2013). Thus, a balance between cell division and differentiation is required for proper maintenance of meristem size.

Various phytohormones such as auxin, cytokinin, gibberellin, ethylene, and abscisic acid (ABA) play vital roles in maintenance of this balance (reviewed in Pacifici et al., 2015). Auxin promotes cell proliferation in the RAM in various ways including long- and short-distance signals mediated by the polar auxin transporters Pin-Formed (PIN), essential for creating auxin gradients (Tanaka et al., 2006). A high concentration of auxin promotes SCF^TIR1-mediated degradation of the Aux/IAA family of transcriptional repressors that form heterodimeric complexes with auxin response factors (ARFs). Aux/IAA degradation releases ARFs from Aux/IAA-mediated repression, resulting in activation of auxin-responsive target genes (Kepinski and Leyser, 2005; Mockaitis and Estelle, 2008; Boer et al., 2014). On the other hand, cytokinin drastically reduces growth of the RAM primarily by promoting cell differentiation (Beemster and Baskin, 2000; Dello Ioio et al., 2008). Cytokinin signaling is mediated through a two-component system composed of membrane-localized Arabidopsis histidine kinases (AHKs) (Hwang and Sheen, 2001; Inoue et al., 2001; To and Kieber, 2008). The signal is transmitted through phosphorelay to activate Arabidopsis response regulators (ARRs) that promote expression of cytokinin target genes (To et al., 2004; To and Kieber, 2008). Transition from mitotic cells in the RAM to elongated cells in the EZ is accompanied by DNA polyploidization of cells (Dello Ioio et al., 2007; Ishida et al., 2010; Adachi et al., 2011). Cytokinin activates expression of CCS52A1, which promotes DNA polyploidization through ARR2, and thereby regulates meristem size (Takahashi et al., 2013). It is proposed that the position of the boundary between dividing and elongating zones is determined by the antagonistic interaction of auxin and cytokinin. Cytokinin-activated ARR1 directly activates expression of SHY2/IAA3, resulting in repression of ARF activity and down-regulation of auxin transporter PIN genes, causing suppression of cell division. On the other hand, a high concentration of auxin promotes degradation of SHY2 and releases ARFs from repression to promote PIN expression and cell division (Dello Ioio et al., 2008;Mockaitis and Estelle, 2008). It has recently been shown that in addition to cytokinin-mediated modulation of auxin distribution by regulating PIN expression, cytokinin-dependent auxin degradation also plays an important role in positioning the TD by creating a region of auxin minima (Di Mambro et al., 2017).

Calcium ions (Ca²⁺) plays a pivotal role in perception of external signals. An increase in free cytosolic Ca²⁺ ([Ca²⁺]_cyt) is recognized by an array of Ca²⁺ sensors including calcineurin B-like proteins (CBLs). CBLs in concert with interacting kinases CIPKs (CBL-interacting protein kinases) relay the signal by phosphorylation of various substrates (Luan, 2009). Altered [Ca²⁺]_cyt homeostasis inhibits root cell elongation (Bai et al., 2009; Wu et al., 2017). The role of Ca²⁺ and calcium-regulated kinases in auxin signaling has been described in several reports (Thimann and Schneider, 1939; Fuglsang et al., 2007; Duby et al., 2009; Monshausen et al., 2011; Takahashi et al., 2012; Li et al., 2016; Wang et al., 2016; Santin et al., 2017). In this study, we observed that Arabidopsis CIPK25 T-DNA insertion lines exhibited a short root phenotype resulting from reduced RAM size. Restoring expression of CIPK25 in one of these lines complemented the root phenotype, suggesting that this gene plays a role in root development. CIPK25 expression was responsive to external application of auxin and cytokinin. A mutant lacking CIPK25 expression displayed compromised auxin transport and low expression of PIN proteins. The short root phenotype of the cipk25 line was restored in the SHY2 loss-of-function background. Our data suggest that CIPK25 plays a role in balancing auxin and cytokinin signaling in root development.

Materials and methods

Plant materials, growth conditions, and treatments

Arabidopsis thaliana wild-type (ecotype Columbia; Col-0) and mutant lines (SALK_079011 and SALK_029271) with T-DNA insertions in the exon of AtCIPK25 (At5g25110) were procured from the Arabidopsis Biological Research Center. Homozygous lines with T-DNA insertions were screened using primers specific for T-DNA borders and the CIPK25 gene. Both SALK_079011 and SALK_029271 lines were used for the initial phenotyping study. The remaining experiments were performed with SALK_079011 which was designated as the cipk25 mutant. P_PIN1::PIN1-GFP, DR₅::GUS, and shy2-24 lines were kindly provided by Dr Jason W. Reed (University of North Carolina, USA). P_PIN2::PIN2-GFP and arr1 arr12 lines were provided by Dr Ashverya Laxmi (National Institute of Plant Genome Research, India). The above-mentioned reporter lines were crossed with the cipk25 mutant. F₃/F₄ homozygous plants were selected by genotyping and used in the experiments. For stable line generation, 35S::CIPK25, P_CIPK25::GUS, and P_SHY2::GUS constructs were introduced into Col-0 plants by the Agrobacterium-mediated floral dip method as described before (Clough and Bent, 1998). The P_CIPK25::CIPK25 construct was introduced into cipk25 mutant plants to generate the complementation line. T₃ homozygous P_SHY2::GUS-expressing plants were crossed with the cipk25 mutant for further studies. All seeds were surface-sterilized, stratified, and sown on half-strength Murashige and Skoog medium (1/2 MS) plates supplemented with 1% sucrose and 0.8% agar. Arabidopsis plants were grown on an agropeat:vermiculite (3:1) mixture in growth chambers with 120 µmol µm^–2 s^–1 light under a 16 h light/8 h dark cycle at 21 ± 1 °C. For root phenotyping, all plants were grown vertically on 1/2 MS medium plates as mentioned above. Root length was marked on plates from 4 days post-germination (4 dpg) onwards. Images of 10-day-old seedlings were captured for all the plates; root length and per day growth rate were measured for individual seedling using ImageJ software (Abramoff et al., 2004).

For analyzing fold expression of CIPK25 in response to hormones, Col-0 seedlings were transferred to liquid 1/2 MS medium, 12 h prior to the treatment for acclimatization. Auxin [indole acetic acid (IAA)] at 5 µM and t-zeatin at 5 µM final concentration were added to liquid medium. Root tissues were collected from treated seedlings at different time intervals. For CIPK25 expression studies at different developmental stages, root tissues were collected from seedlings grown on 1/2 MS plates. CIPK25 promoter activites were analyzed in P_CIPK25::GUS lines in different tissues and at different developmental stages. Forty-five-day-old soil-grown plants were taken as matured plants. To determine promoter–reporter activity by staining, seedlings were grown on vertical 1/2 MS plates and treated with hormones for various periods before staining and microscopy.

Expression analysis by real-time PCR

Plant tissues were ground to a fine powder with liquid N₂, and total RNA was isolated using the TRIzol Reagent (Invitrogen) according to the manufacturer’s protocol. DNA-free total RNA (1 µg) was converted into single-stranded cDNA using oligo(dT₁₈) primers and a High Fidelity cDNA synthesis kit (Roche Diagnostics GmbH, Germany). Gene-specific primers were designed using the NCBI primer design tool (http://www.ncbi.nlm.nih.gov/tools/primer-blast). For quantitative real-time PCR (qRT-PCR), primers were designed to produce 100–150 bp amplicons. qRT-PCR was done in optical 96-well plates on a Vii A 7 Real-Time PCR System (Applied Biosystems, CA, USA) using 2× Power SYBR Green PCR master mix (Applied Biosystems). A dissociation curve analysis was performed for all primer pairs, and all experimental samples yielded a single sharp peak at the amplicon’s melting temperature. ACTIN2 (At3g18780) was used as internal control. Fold induction values of target genes were calculated with the ΔΔCT equation (Rao et al., 2013) and are presented relative to the mRNA level of target genes in control conditions. Technical and biological replicates were used in all the assays. The primer pairs used are listed in Supplementary Table S1 at JXB online.

Yeast two-hybrid assay

The CIPK25 full-length coding sequence (CDS) was fused to the GAL 4 DNA-binding domain in the pGBKT7-BD vector (Clontech Laboratories Inc.). The CDS of all CBLs (CBL1–CBL10) of Arabidopsis were cloned into the pGADT7-AD (Clontech Laboratories Inc.) to be expressed as a fusion with the GAL4 activation domain. The pGBKT7-BD and PGADT7-AD constructs were co-introduced into the Y2H Gold strain of yeast using the Matchmaker Gold yeast Two-Hybrid system kit (Clontech Laboratories Inc.). The yeast colonies harboring both the plasmids were selected on double drop-out (DDO) medium lacking leucine and tryptophan and were confirmed by colony PCR. Positive colonies selected on DDO plates were grown in liquid DDO media for 2 d, adjusted to an OD₆₀₀ of 1, 0.1, and 0.01. A 10 µl aliquot of each dilution was spotted on quadruple drop-out medium (-Leu-Trp-Ade-His) with 2.5 mM 3-AT (3-amino-1,2,4-triazole). Plates were incubated at 30 °C for 5 d.

Transient expression of fusion proteins and microscopic visualization

The CIPK25 CDS was cloned between the NcoI and SpeI sites of the pCAMBIA1305 vector to generate overexpression lines. To generate the CIPK25 expression line under its native promoter, 2.6 kb of the promoter and coding region with the stop codon was amplified from genomic DNA and cloned in the SmaI site of the pBI101.2 promoter-less vector. For the promoter–β-glucuronidase (GUS) fusion construct, 2.6 kb fragments upstream of the translation start codon of the corresponding genes were amplified from genomic DNA and were cloned in the pBI101.2 vector at the BamHI site (for the CIPK25 promoter) and SalI/XbaI sites (SHY2 promoter). The 35S::CIPK25-YFP construct generated by Gateway Technology (Invitrogen) in the pEG101 binary vector was introduced into Agrobacterium tumefaciens (GV3101) and infiltrated in Nicotiana benthamiana leaves for transient expression. For agro infiltration, overnight-grown cell cultures were collected and resuspended in 1 ml of infiltration buffer (10 mM MES-KOH, pH 5.6, 10 mM MgCl₂, and 150 µM acetosyringone) to an optical density at 600 nm (OD₆₀₀) of 0.6. The working suspensions were prepared by mixing the Agrobacterium culture harboring the 35S::CIPK25-YFP construct with an organelle marker for the plasma membrane (PM-mCherry) (Nelson et al., 2007) in a 1:1 ratio, incubated at room temperature for 3 h, and infiltrated onto the abaxial surface of fully developed leaves. Plants were cultivated for 2–3 d under growth room conditions. Subcellular fractionation of CIPK25–yellow fluorescent protein (YFP)-expressing plants was done by using the Qproteome Cell Compartment kit (Qiagen GmbH, Germany) by following the manufacturer’s instructions. Cytosolic, membrane, and nuclear protein fractions were separated by SDS–PAGE followed by western blot, and probed with anti-green fluorescent protein (GFP) (Abcam, UK) antibody. The blotted membrane was probed with the antibodies against fraction-specific marker proteins, for example anti-histone H3 (nuclear fraction) (Sigma-Aldrich, Saint Louis, MO, USA), anti-plasma membrane H⁺ ATPase (membrane fraction), and anti-cytosolic fructose-1,6-bisphosphatase antibodies (cytosolic fraction) (Agrisera, Sweden).

For GUS reporter staining, Arabidopsis seedlings and different plant tissues were vacuum infiltrated with GUS staining solution [50 mM sodium phosphate buffer (pH 7.0), 2 mM EDTA, 0.12% Triton, 0.4 mM ferrocyanide, 0.4 mM ferricyanide, 1.0 mM 5-bromo-4-chloro-3-indolyl-β-d-glucuronide cyclohexylammonium salt] for 5–15 min and incubated in the dark at 37 °C. The staining time varied between 2 h and 12 h depending on the tissue type. Tissues were cleared of chlorophyll by treatment with 70% ethanol at 65 °C for 1 h, and analyzed by light microscopy.

For bimolecular fluorescence complementation (BiFC) assay, the full-length cDNA fragments (without the stop codon) of CBL4, CBL5, and CBL9 were amplified using PCR primers having attB adaptors and cloned into the Gateway entry vector pDONR 207 by BP clonase reaction (Invitrogen). The CIPK25 CDS was cloned into pSITE-nEYFP-C1 (CD3-1648) (Martin et al., 2009) to generate the YFP^N–CIPK25 construct. CBL4, CBL5, and CBL9 were cloned similarly into pSITE-cEYFP-N1 (CD3-1651) to generate CBL4–YFP^C, CBL5–YFP^C, and CBL9–YFP^C constructs. Agrobacterium strain GV3101 cultures harboring BiFC constructs were infiltrated in N. benthamiana leaves as discussed above. For fluorescence quantification, 6 mm leaf discs were cut out of the infiltrated areas and put into a black 96-well plate. Fluorescence intensities of leaf discs were measured in a The FLUOstar^® Omega plate reader (BMG LABTECH, Germany) using a 485 ± 12 nm excitation and a 520 nm emission filter. To examine root meristem cells, seedlings were incubated in 10 µg ml^–1 propidium iodide for 5–10 min, mounted in water, and visualized by a Leica TCS SP5 (Leica Microsystems, Wetzlar, Germany) laser-scanning confocal microscope at 535 nm excitation and 617 nm emission for propidium iodide. GFP was visualized at 395 nm excitation and 509 nm emission in P_PIN1::PIN1-GFP- and P_PIN2::PIN2-GFP-expressing roots of Col-0, cipk25, and CIPK25OX seedlings. Agroinfiltrated N. benthamiana leaf cells for subcellular localization of CIPK25–YFP and plasma membrane marker fused with mCherry were analyzed at 514–527 nm and 587–610 nm for YFP and mCherry, respectively.

Site-directed mutagenesis, bacterial expression, and kinase assay

Site-directed mutagenesis was done by replacing threonine (T) with aspartic acid (D) at the 201th position in CIPK25 as described (Meena et al., 2015). The coding regions of CIPK25 and CIPK25T/D²⁰¹ were cloned into the pGEX-4T2 vector (Amersham Pharmacia Biotech) at the SmaI site and transformed into Escherichia coli BL21 (DE3)-CodonPlus cells (Stratagene, La Jolla, CA, USA). Recombinant proteins were purified from the bacterial lysates by glutathione–Sepharose affinity chromatography as described before (Gong et al., 2002a, b). In vitro protein autophosphorylation assays were performed in a 40 µl reaction mixture comprising purified recombinant glutathione S-transferase (GST) proteins incubated in kinase buffer (10 µCi of [γ-³²P]ATP, 10 µM ATP, 20 mM Tris–HCl pH 8.0, 5 mM MnCl₂, 1 mM CaCl₂, 0.1 mM EDTA, and 1 mM DTT) for 30 min at 30 °C. For substrate phosphorylation, MBP (myelin basic protein) was incubated with immunoprecipitated CIPK25 protein from Col-0 seedlings in kinase buffer. The reaction was stopped by addition of 4× SDS sample buffer. Reaction samples were fractionated by electrophoresis and analyzed by autoradiography. CIPK25 protein was detected by a specific antibody generated against the CIPK25 immunogenic peptide.

Auxin transport assay

Auxin transport was measured essentially as previously described (Shin et al., 2005). Five-day-old vertically grown seedlings were used for the experiment. Briefly, agar blocks of 1 mm in diameter containing 7.7 × 10⁻⁸ M [³H]IAA (Amersham) were applied at different positions. Agar blocks were placed at the root tip for basipetal (shootward) and the hypocotyl–root junction for acropetal (rootward) auxin transport. After incubation for 3 h, a 0.5 mm section of the root close to the agar block was removed under the microscope. Two consecutive 2 mm segments below the incision line were then collected separately from 15 roots and were pooled and placed into glass scintillation vials containing 5 ml of scintillation fluid. The radioactivities in these two pools of root segments were measured using a Perkin Elmer Tri-Carb 2800TR Scintillation counter (Waltham, MA, USA).

Statistical analysis

Statistical differences between different groups were detected by Student’s t-test and one-way ANOVA with post-hoc Student–Newman–Keuls (SNK) test in SigmaStat 2.03. Asterisks or different letters indicate significant differences between genotypes and treatments. Relative root growth rate in the presence of external IAA was given as the percentage growth rate (per day) of the same line in control conditions (without IAA).

Results

CIPK25 is involved in root meristem development

We previously reported that overexpression of the chickpea CIPK25 (CaCIPK25) in tobacco (Nicotiana tabacum) enhanced root growth (Meena et al., 2015). Arabidopsis CIPK25 (At5g25110.1) is an 1697 bp intron-less gene with a CDS of 1467 bp located between base pairs 8 657 626 and 8 659 322 on chromosome 5. Screening of T-DNA insertion lines (Alonso et al., 2003) to investigate the role of Arabidopsis CIPK25 identified two SALK lines, SALK_079011 (cipk25-1) and SALK_029271 (cipk25-2), with a T-DNA insertion in the CIPK25 coding region having a root length shorter by ~40% and ~32%, respectively, as compared with wild-type (Col-0) plants at the 8 dpg stage (Fig. 1A). Sequencing of PCR products with T-DNA- and gene-specific primers identified the T-DNA insertions at 902 bp and 1392 bp downstream of the translation initiation site in cipk25-1 and cipk25-2, respectively. Gene expression analyses by qRT-PCR showed that both mutant lines did not express a significant amount of CIPK25 mRNA as compared with that in Col-0 plants (Fig. 1B). The SALK_079011 line was used for further study and designated as cipk25. Expression of the CIPK25 full-length CDS under the 2.6 kb long native promoter in cipk25 plants (cipk25 P_CIPK25::CIPK25) functionally complemented the short root phenotype as observed in plants at 10 dpg. CIPK25-overexpressing [under the Cauliflower mosaic virus (CaMV) 35S promoter] Col-0 lines (CIPK25OX, line 1 and 2) displayed ~20% longer roots (Fig. 1C–E), suggesting that CIPK25 is required for root development in Arabidopsis. CIPK25OX line 1 was used for further studies as it showed comparatively better expression and root growth than the other line.

Fig. 1.

The cipk25 mutant exhibits a shorter root length phenotype. (A) Primary root length phenotype of 8 dpg Col-0, cipk25-1 and cipk25-2 mutants grown vertically on 1/2 MS medium. The lower panel shows root length measurement of three replicate experiments with 15 seedlings for each replicate of each genotype. Error bars are ±SD. Statistically significant differences between root lengths of Col-0 and cipk25 mutants were analyzed by unpaired t-test: *P≤0.05, **P≤0.001. (B) Schematic representation of the CIPK25 gene structure and T-DNA insertion sites of cipk25-1 and cipk25-2 mutants. The lower panel shows the relative transcript level of CIPK25 in 8 dpg Col-0 and cipk25 mutants by qRT-PCR. ACTIN2 was used as the control. Error bars indicate ±SD of three replicates. Significant differences were calculated by unpaired t-test: **P≤0.001. (C) Root length phenotype of 10 dpg Col-0, cipk25, cipk25 P_CIPK25::CIPK25, CIPK25OX-1, and CIPK25OX-2 lines. (D) CIPK25 relative transcript levels in 10 dpg Col-0, cipk25, cipk25 P_CIPK25::CIPK25, CIPK25OX-1, and CIPK25OX-2 seedlings. ACTIN2 was used as the endogenous control. Different letters indicate significant differences (ANOVA; P≤0.05). (E) Root length comparison of 10 dpg Col-0, cipk25, cipk25 P_CIPK25::CIPK25, CIPK25OX-1, and CIPK25OX-2 seedlings. Error bars are ±SD, n=30.

Root lengths and growth rates of the Col-0, cipk25, and CIPK25OX plants were compared over a period of 4–9 dpg. No apparent differences were observed in germination rates and germination periods. Measurement of five replicates of 15 seedlings each showed that the average root length of the mutant line was shorter by 30–36% as compared with the Col-0 plants, whereas that of the CIPK25OX line was >11% longer at 9 dpg (Fig. 2A). Root growth rate, as measured by the increase in the primary root length per day, of the mutant line was ~25% less than that of the Col-0 plants when measured between the fourth and fifth day (Fig. 2B). During the same period, the root growth rate of the CIPK25OX line was similar to that of the Col-0 plant. Root growth rates of cipk25 and CIPK25OX were ~26% less and ~17% more than that of the wild-type plants, respectively, when measured between the eighth and ninth day. To investigate the reason for the shorter root length in the mutant line, the number of root meristem cells was counted following the method of Perilli and Sabatini (2010). Propidium iodide-stained roots showed ~18–22% lower root meristem cell numbers in the cipk25 line as compared with the wild type at 5–9 dpg. During the same period, the root meristem cell numbers for the CIPK25OX line were higher by ~10–20%. However, organization of cells in the root meristematic zone was apparently unperturbed in the mutant line (Fig. 2C, D).

Fig. 2.

The cipk25 mutant exhibits a slower root growth rate and a lower number of root meristem cells. (A and B) Primary root length and growth rate (mm d^–1) measurements of Col-0, cipk25, and CIPK25OX seedlings grown vertically on 1/2 MS medium. Values are ±SD of 75 samples for each genotype. Different letters indicate significant differences (ANOVA; P≤0.05) in seedlings at 9 and 8–9 dpg for root length and growth rate. (C) Meristem cell number count in Col-0, cipk25, and CIPK25OX in 5, 7, and 9 dpg seedlings. Error bars are ±SD of n=20. Different letters indicate statistically significant differences among 9 dpg seedlings (ANOVA; P≤0.05). (D) Propidium iodide-stained primary roots of 5 dpg Col-0, cipk25, and CIPK25OX seedlings. Lower and upper arrows indicate the quiescent center (QC) and the first elongated cortex cell, respectively.

CIPK25 encodes an active protein kinase and interacts with CBL4 and CBL5

To investigate its biochemical character, CIPK25 protein fused with GST was expressed in bacteria. GST–CIPK25 protein showed autophosphorylation activity in an in vitro reaction. Replacement of a conserved threonine residue with aspartic acid in the activation loop has been shown to increase autophosphorylation activity for various CIPKs (Gong et al., 2002a, b). Similarly, substitution of the conserved Thr201 residue with aspartic acid resulted in an ~3-fold enhancement in the autophosphorylation activity of CIPK25. CIPK25 protein immunoprecipitated from Col-0 seedling protein extract showed in vitro kinase activity with MBP as substrate, implying that the plant CIPK25 protein is an active kinase (Fig. 3A). To explore the subcellular localization of CIPK25 protein, the CIPK25–YFP construct was agroinfiltrated in N. benthamiana leaves along with the plasma membrane marker PM-mCherry (Nelson et al., 2007). Fluorescence overlay showed that CIPK25 was localized in both the plasma membrane and the cytosol (Fig. 3B). To substantiate the localization of CIPK25, subcellular fractionation of transiently expressed CIPK25–YFP in N. benthamiana leaves was performed. The presence of CIPK25 was detected by western blot using anti-GFP antibody mostly in the membrane and much less in cytosolic fractions, but not in the nuclear fraction (Fig. 3C).

Fig. 3.

CIPK25, an active protein kinase, interacts with CBL4 and 5 in planta. (A) Autophosphorylation assay of bacterially expressed GST–CIPK25 and CIPK25T/D²⁰¹ proteins. Below is the Coomassie blue-stained gel. The lower panel shows substrate phosphorylation of MBP by immunoprecipitated CIPK25 protein from Col-0 and cipk25 mutant seedlings. Western blot of CIPK25 is shown. (B) Visualization of CIPK25–YFP fusion protein in agroinfiltrated leaves of N. benthamiana co-infiltrated with the plasma membrane marker PM-mCherry. The magnified view of the merged image in the lower panel shows CIPK25 localization at the plasma membrane and cytoplasm. (C) Western blot showing subcellular fractions of CIPK25–YFP-expressing N. benthamiana leaves. The blot was probed with antibodies against GFP for CIPK25, plasma membrane (PM) H⁺-ATPase, cytosolic fructose-1,6-bisphosphatase (cFBPase), and H3 histone. (D) Yeast two-hybrid interaction study of CIPK25 and CBLs. Y2H gold yeast cells were co-transformed with pGBKT7-CIPK25 and pGADT7-CBL1-10, and auxotrophic selection was carried out to detect protein–protein interaction. (E) Interactions between CIPK25 and CBL4/CBL5 in planta were shown by BiFC assay. CIPK25 fused with the N-terminal domain of YFP (YFP^N) and CBL4/CBL5/CBL9 fused with the C-terminal domain of YFP (YFP^C) were transiently expressed in N. benthamiana by agroinfiltration. Images show YFP-mediated fluorescence derived from the protein–protein interaction. Scale bar=100 µm. Infiltration of CBL9–YFP^C with YFP^N–CIPK25 was used as the negative control. Expression of the proteins is shown in Supplementary Fig. S1. (F) Fluorescence intensity within leaf discs expressing YFP^N–CIPK25 with CBL4–YFP^C, CBL5–YFP^C, or CBL9–YFP^C. Different letters denote significant differences among samples based on Student–Newman–Keuls test (n=8, ANOVA; P≤0.05).

Multiple CBL proteins can interact with a specific CIPK depending on context, and this is thought to define distinct signaling pathways (Kolukisaoglu et al., 2004). A yeast two-hybrid assay was conducted to identify the interacting CBL partners of CIPK25. The full-length CIPK25 protein was found to interact only with CBL4 and CBL5 (Fig. 3D). In planta interaction of CIPK25 with CBL4 and CBL5 was investigated by BiFC assay in N. benthamiana leaves. Fluorescence due to reconstitution of YFP was observed when combinations of YFP^N–CIPK25 with CBL4–YFP^C or CBL5–YFP^C were infiltrated in N. benthamiana leaves, whereas the combinations of YFP^N–CIPK25 and CBL9–YFP^C did not show any fluorescence (Fig. 3E). Expression of the fused protein in the infiltrated leaf was verified by western blot (see Supplementary Fig. S1). To assess the strength of BiFC signals from YFP^N–CIPK25/CBL4–YFP^C and YFP^N–CIPK25/CBL5–YFP^C pairs, the fluorescence signal intensity of the leaf discs infiltrated with the BiFC constructs were quantified using a spectrofluorometer. No significant differences were observed between the BiFC fluorescence signals of YFP^N–CIPK25 with CBL4–YFP^C and CBL5–YFP^C, while the signals fromYFP^N–CIPK25 and CBL9–YFP^C were negligibly low (Fig. 3F).

Tissue-specific expression of CIPK25

Expression of CIPK25 was investigated in different tissues and root growth stages, and in response to auxin and cytokinin treatments by qRT-PCR. Expression of CIPK25 was the lowest in leaves of the mature plant and highest in flowers, followed by the root and stem (Fig. 4A). Expression of CIPK25 in the root increased gradually from the fourth to the 10th day by ~3 fold, and decreased to ~2.5 fold on the 12th day (Fig. 4B). To investigate the in planta expression pattern, the GUS reporter gene was cloned under a 2.6 kb 5' upstream regulatory region (including the 5'-untranslated region) of CIPK25 (P_CIPK25) and expressed in Arabidopsis. GUS activity could be detected throughout the radicles in the germinated seed. GUS activity was observed in cotyledons and hypocotyl, except in the proliferative region of the RAM in a 2-day-old seedling. In a 5-day-old seedling, GUS expression initiated at the TD and continued to the root–shoot junction. GUS activity in the aerial part of a 10-day-old seedling was not well pronounced where the blades of young leaves showed low levels of GUS expression. GUS activity progressively increased with age in the primary and lateral roots, except in the root tip. P_CIPK25::GUS showed strong expression in the floral organs in adult plants. A high level of CIPK25 expression was detected in carpel, petals, and stamens. The ovule was dissected and a strong CIPK25 promoter activity was observed in ovule integuments, but not in the embryo (Fig. 4C). The plant growth hormone auxin (IAA) positively regulated CIPK25 expression in root. The transcript level was increased by ~4-fold within 1 h of treatment with 5 µM IAA and then slowly decreased to ~1.8-fold within 5 h and remained at a similar level till 10 h. In contrast, cytokinin (5 µM t-zeatin) treatment down-regulated CIPK25 gene expression by almost 10-fold within 5 h (Fig. 4D). Activation and repression of the CIPK25 promoter by auxin and cytokinin treatments was also validated by promoter–reporter assay (Fig. 4E).

Fig. 4.

CIPK25 gene expression profiles in different tissues, root growth stages, and in response to hormone treatments. CIPK25 gene expression in different tissues (A) and in different root growth stages (B) analyzed by qRT-PCR. ACTIN2 expression was used for normalization. Error bars show ±SD, n=3. Different letters indicate significant differences among samples (ANOVA; P≤0.05). (C) CIPK25 promoter activities were investigated with promoter::reporter gene (GUS) constructs expressed in transgenic plants. Histochemical GUS stainings is shown for (i) the radicle of a germinating seedling; (ii) a 2 dpg seedling; (iii) the primary root tip of a 5 dpg seedling (the arrow indicates the first elongated cortex cell); (iv) a 10 dpg seedling; (v) flower; (vi) gynoecium; (vii) immature seeds with embryo; (viii) embryo. (D) Fold change in CIPK25 expression as analyzed by qRT-PCR in roots of 10 dpg Col-0 seedlings treated with 5 µM IAA and 5 µM t-zeatin for different time intervals. ACTIN2 expression was used for normalization. Error bars show ±SD, n=3. Different letters indicate significant differences among samples (ANOVA; P≤0.05). (E) pCIPK25::GUS expression was visualized in primary roots of 10 dpg seedlings treated with 5 µM IAA for 1 h and with 5 µM t-zeatin for 5 h.

The cipk25 mutant showed compromised auxin transport and auxin-responsive promoter activity

Cell division and patterning in the root are tightly controlled by the local concentration of auxin, which is regulated by a process commonly referred to as polar auxin transport. Acropetal (rootward) and basipetal (shootward) modes of auxin transport in root were assayed using radiolabeled IAA. The mutant line exhibited reduced acropetal and basipetal auxin transport by ~32% and ~38%, respectively, in comparison with Col-0 plants, while the CIPK25OX line displayed similar auxin transport to that in Col-0 by both modes (Fig. 5A).

Fig. 5.

Auxin transport and auxin-responsive promoter assays in cipk25. (A) Comparison of acropetal (rootward) and basipetal (shootward) root auxin transport in 5 dpg Col-0, cipk25, and CIPK25OX seedlings in a root segment 2 mm below (in acopetal) or above (in basipetal) the site of [³H]IAA application. Data are presented as the percentage of auxin transport relative to Col-0. Error bars are ±SD, t-test (*P≤0.05) of three measurements of 15 seedlings each. (B) Auxin-mediated root growth rate retardation of Col-0 and cipk25 in response to 0.1 µM and 0.5 µM auxin treatments. Col-0 and cipk25 mutant seedlings grown on 1/2 MS medium were transferred at 4 dpg to IAA-free or IAA-containing medium, and grown for an additional 5 d. Relative root growth rate in the presence of external IAA is presented as a percentage of the growth rate (per day) of the same line in control conditions (without IAA). The error bars represent ±SD, t-test (*P≤0.05), with three replicates of 10 seedlings each. (C) Auxin-responsive promoter activity using DR₅::GUS in Col-0 and the cipk25 mutant. Seedlings at 5 dpg were treated with 5 µM and 10 µM IAA for 2 h and stained for GUS activity.

External treatment with auxin is known to retard root growth (Eliasson et al., 1989). To ascertain the effect of external auxin, 4-day-old seedlings of cipk25 and Col-0 were transferred to media containing 0.1 µM and 0.5 µM IAA, and incubated for 5 d. Exogenous application of 0.1 µM IAA caused relative root growth rate inhibition in Col-0 seedlings by 65–70%. In contrast, relative root growth rate inhibition of cipk25 seedlings was 48–60%, suggesting that the mutant was less responsive to auxin-mediated root growth retardation, and a higher concentration of auxin was required for a similar effect to that in Col-0. With higher concentration (0.5 µM) of IAA, the difference in relative root growth rate retardation between Col-0 and cipk25 was not significant (Fig. 5B).

Auxin-responsive gene expression in roots was assessed by the activity of the auxin-responsive promoter–reporter DR₅::GUS. Col-0 plants with DR₅::GUS were crossed with cipk25 to develop a homozygous mutant line expressing DR₅::GUS (cipk25 DR₅::GUS). GUS activity in the RAM of 5-day-old cipk25 seedlings was much lower than in Col-0, implying reduced auxin activity in the RAM of the mutant line. Further, external application of auxin (5 µM and 10 µM IAA for 2 h) caused weaker induction of the auxin-responsive promoter in the cipk25 root as compared with that in Col-0, suggesting lower auxin transport/signaling in the mutant line (Fig. 5C). A similar contrasting pattern of DR₅::GUS activity was observed in the case of the agr1-5 pin2 mutant, which was impaired in basipetal auxin transport (Shin et al., 2005).

Reduced expression of auxin-responsive genes including auxin efflux carriers in the cipk25 root

The PIN auxin efflux carriers control growth and patterning in Arabidopsis root (Blilou et al., 2005). ARFs, such as ARF6 and ARF8, were shown to be the positive regulators of adventitious root initiation (Gutierrez et al., 2012). MONOPTEROS/ARF5 is required for primary root formation and vascular development (Berleth and Jürgens, 1993; Przemeck et al., 1996). ARF7 and ARF19 were shown to regulate lateral root formation (Okushima et al., 2007). Expression levels of PINs and ARFs were analyzed by qRT-PCR in roots of Col-0, cipk25, and CIPK25OX plants (Fig. 6A). PIN1, PIN2, and PIN3 expression levels were down-regulated by ~3-fold in cipk25 seedlings, while an ~2-fold reduction was observed for PIN7. Out of five ARFs, expression levels of ARF5, ARF6, and ARF8 were down-regulated by >2-fold in cipk25 as compared with wild-type plants, whereas expression levels of ARF7 and ARF19, involved in lateral root formation, did not show a significant change in cipk25. ARF and PIN genes, which showed reduced expression in the cipk25 line, exhibited ~1.5- to 2-fold higher expression levels in the roots of the CIPK25OX line. Auxin is known to be transported through the central root tissue towards the tip and then redistributed to the cortical tissue and transported basipetally through the epidermis (Swarup and Bennett, 2003; Petrásek and Friml, 2009). The PIN protein auxin efflux carriers facilitate this polar auxin transport. PIN1 is thought to promote auxin movement towards the root tip (acropetal) (Blilou et al., 2005), whereas basipetal auxin transport is dependent on PIN2 (Müller et al., 1998; Abas et al., 2006). To examine the expression of PIN proteins in the absence of CIPK25 expression, cipk25 mutant lines were crossed with reporter lines expressing PIN1 and PIN2 proteins fused with GFP under their native promoters (P_PIN1::PIN1-GFP and P_PIN2::PIN2-GFP). Fluorescence imaging of roots of 5-day-old seedlings showed that expression of PIN1 protein was considerably reduced in the stele of the root in the cipk25 background. Similarly, the absence of CIPK25 caused a significant reduction in PIN2 protein expression in the epidermis. Expression of GFP-fused PIN1 and PIN2 was higher in CIPK25OX plants (Fig. 6B). All these results suggested that CIPK25 was required for optimal expression of some members of auxin efflux carriers and ARFs.

Fig. 6.

Expression of auxin response factors (ARFs) and efflux carriers (PINs) in cipk25. (A) Relative transcript levels of auxin response factors (ARF5, ARF6, ARF7, ARF8, and ARF19) and efflux carriers (PIN1, PIN2, PIN3, and PIN7) were analyzed by qRT-PCR in roots of 5 dpg Col-0, cipk25, and CIPK25OX seedlings. ACTIN2 expression was used for normalization. Error bars represent ±SD of three biological replicates with 15 seedlings for each genotype. (B) Visualization of expression of auxin efflux carriers PIN1 and PIN2. Fluorescence images from the GFP fusion proteins expressed through the native promoters of individual genes in the Col-0, cipk25, and CIPK25OX background. Seedlings at 5 dpg were stained with propidium iodide to explore meristem cell profiles and analyzed by confocal microscopy. Scale bars in confocal images=100 µm. Lower and upper arrows indicate the quiescent center (QC) and the first elongated cortex cell, respectively.

SHY2 loss of function rescued the short root phenotype of cipk25

The number of cells in the root meristem is regulated by auxin and cytokinin, which promote cell division and cell elongation, respectively. SHY2/IAA3 was proposed to act as a point of intersection of auxin- and cytokinin-mediated signaling to regulate root meristem size. Cytokinin represses PIN1, PIN3, and PIN7 expression to influence root meristem maintenance in a SHY2-dependent manner (Tian et al., 2002; Blilou et al., 2005; Vieten et al., 2005; Dello Ioio et al., 2008). As CIPK25 was found to influence root meristem size and PIN expression, the SHY2 expression level was investigated in cipk25 and was found to be ~3-fold higher than in the roots of wild-type seedlings. This observation was substantiated by in planta expression of the SHY2 promoter–GUS fusion construct (P_SHY2::GUS). The GUS reporter gene was cloned under a 2.6 kb long SHY2 promoter and was expressed in wild-type Arabidopsis. It was crossed with the cipk25 line to generate the cipk25 P_SHY2::GUS line. The SHY2 promoter showed a very weak activity in the TD and upwards in the wild-type background, whereas a high SHY2 promoter activity was observed in the cipk25 background (Fig. 7A).

Fig. 7.

Genetic interaction of CIPK25 and SHY2. (A) Relative transcript abundance of SHY2 in 5 dpg Col-0 and cipk25 mutant roots was analyzd by qRT-PCR and semi-quantitative RT-PCR. ACTIN2 was used as the internal control. The right-hand panel shows histochemical GUS staining of primary roots of 5 dpg seedlings expressing the GUS reporter under the SHY2 promoter. The arrow shows the first elongated cortex cell. (B) Primary root length phenotype of 10 dpg Col-0, cipk25, shy2-24, and cipk25 shy2-24 mutants grown vertically on 1/2 MS medium. The right-hand panel shows root length measurement. Error bars represent ±SD, n=24. (C) Relative transcript levels of auxin efflux carriers were analyzed by qRT-PCR in roots of 5 dpg Col-0, cipk25, shy2-24, and cipk25 shy2-24 seedlings. Different letters indicate statistically significant differences by ANOVA, P≤0.05. Error bars are ±SD, n=3. (D) qRT-PCR analysis of the expression levels of CIPK25 in roots of 10 dpg Col-0 and arr1 arr12 seedlings treated with 5 µM t-zeatin for different time intervals. ACTIN2 expression was used as the internal control. Error bars are ±SD, n=3.

We explored the influence of SHY2 on CIPK25 expression by assessing it in the SHY2 loss-of-function mutant shy2-24. In shy2-24, a stop codon was introduced at amino acid position 61, which caused truncation of the protein, and the mutant was considered as a null mutant for SHY2 (Tian and Reed, 1999). CIPK25 expression in shy2-24 was equivalent to that in Col-0 (Supplementary Fig. S2). To investigate the genetic interaction between CIPK25 and SHY2, a cipk25 shy2-24 double mutant was generated. The short root phenotype of the cipk25 plants was completely recovered in the cipk25 shy2-24 mutant, resulting in a similar root length to that of the wild-type plants (Fig. 7B). PIN1, PIN2, and PIN3 expression levels in cipk25 shy2-24 were found to be equivalent to those of the wild-type plants (Fig. 7C), indicating that CIPK25 is genetically epistatic to SHY2. Cytokinin treatment down-regulated expression of CIPK25 (Fig. 4D). Cytokinin control of root meristem size is mediated through ARR1 and ARR12 expression in the TD (Dello Ioio et al., 2007, 2008). Absence or overexpression of CIPK25 did not cause appreciable changes in ARR1 and ARR12 expression (Supplementary Fig. S3). Moreover, cytokinin treatment did not change CIPK25 expression in the arr1 arr12 background, suggesting that cytokinin-mediated down-regulation of CIPK25 expression was dependent on ARR1/ARR12 (Fig. 7D). An analysis of the CIPK25 promoter using PlantPan2.0 (Chow et al., 2015) showed a number of potential ARR1- and ARR12-binding sites (Supplementary Table S2). All these observations suggested an involvement of CIPK25 in cytokinin signaling mediated through ARR1/ARR12.

Cytokinin-mediated down-regulation of PIN1 expression in roots was shown to be dependent on SHY2 (Dello Ioio et al., 2008). To determine whether down-regulation of PIN1 expression by cytokinin is completely dependent on CIPK25, wild-type (Col-0) and cipk25 plants expressing P_PIN1::PIN1-GFP were treated with 5 μM t-zeatin. Cytokinin treatment resulted in marked down-regulation of PIN1 protein expression in the wild-type background. A reduction in PIN1 expression in cipk25 roots in control conditions was already shown earlier. Treatment with cytokinin further reduced PIN1 expression in the cipk25 root (Fig. 8A). This observation was substantiated by PIN1 gene expression in the cipk25 root. Cytokinin treatment resulted in an ~3-fold reduction in PIN1 expression in the wild-type root. A similar (~3-fold lower) PIN1 expression level was observed in control cipk25. Cytokinin treatment caused further reduction in the PIN1 expression level in cipk25 by 1.8-fold (Fig. 8B). Additional reduction in meristematic cell number in cipk25 after cytokinin treatment corroborated reduced PIN1 expression (Fig. 8C). This result suggested that cytokinin regulates root meristem size by both CIPK25-dependent and -independent pathways.

Fig. 8.

Cytokinin-responsive PIN1 expression in cipk25. (A) Fluorescence images of auxin efflux carrier PIN1–GFP fusion protein driven by its native promoter in Col-0 and cipk25 seedlings in control conditions and in response to cytokinin treatment (5 μM t-zeatin, 6 h). Roots of 5 dpg seedlings were stained with propidium iodide (PI) for visualizing root cells. Lower and upper arrows indicate the quiescent center (QC) and the first elongated cortex cell, respectively. Scale bar=100 µm. (B) Relative transcript levels of PIN1 were analyzed by qRT-PCR in roots of 5 dpg Col-0 and cipk25 seedlings treated or not with cytokinin (5 μM t-zeatin, 6 h). Error bars are ±SD of three biological replicates, ANOVA, P≤0.05. (C) Meristem cell number count in 5 dpg Col-0 and cipk25 seedlings in control conditions and in response to cytokinin treatment (5 μM t-zeatin, 12 h). Values are the mean ±SD of 20 samples each. Different letters indicate significant differences among samples (ANOVA; P≤0.05).

Discussion

Post-embryonic root growth is maintained by the RAM. Highly proliferative cells of the RAM move longitudinally towards the shoot and eventually differentiate. Hence, cell division and differentiation rates must be equal in order to maintain root meristem size and ultimately overall root development. It has been proposed that a balance of auxin and cytokinin signaling regulates the transition of dividing cells to differentiated cells by activating expression of SHY2 at the TD, which subsequently decreases expression of PIN genes (Dello Ioio et al., 2008) by suppressing auxin signaling and defines the position of the TD by reducing auxin transport and, additionally, by auxin degradation (Di Mambro et al., 2017). It was reported that SHY2 expression in the root was minimal at 3 dpg in an actively growing meristem and reached its highest level at 5 dpg, when the meristem attained its final size (Moubayidin et al., 2010). By the same analogy, SHY2 expression must also be restricted to maintain an equal rate of cell division and rate of transition to elongation. Our results suggest that CIPK25 negatively regulates SHY2 expression to maintain root meristem size. CIPK25 expression initiated at the TD and was intense in the differentiated zone, and its expression level was highest when SHY2 attained its highest expression level after the establishment of the meristem. SHY2 expression is induced by cytokinin treatment in an ARR1-dependent manner (Moubayidin et al., 2010), while cytokinin treatment decreased CIPK25 expression in an ARR1/ARR12-dependent manner. A high SHY2 expression was observed in the absence of CIPK25. All of these observations suggested an antagonistic relationship between SHY2 and CIPK25. However, application of cytokinin caused a further reduction in PIN1 expression and meristem size in the cipk25 mutant, suggesting that cytokinin also functions in CIPK25-independent pathways. The CIPK25-dependent pathway seems to be totally dependent on SHY2, as a SHY2 loss-of-function mutant complemented the short root phenotype of cipk25 and restored expression of PIN1, PIN2, and PIN3. Therefore, CIPK25 may be considered as one of the candidate molecules that balance activities of auxin and cytokinin during root development.

As reported before (Schmid et al., 2005), CIPK25 displayed the highest expression in flowers (Fig. 4). Interestingly, like ARF6 and ARF8, CIPK25 was also expressed in the stamen and gynoecium, and its expression was greatly reduced in arf6 arf8 flowers (Reeves et al., 2012). ARF6 and ARF8 showed reduced expression in cipk25 roots. ARF6 and ARF8 are required for flower maturity and growth of the ovule integument (Nagpal et al., 2005; Wu et al., 2006). CIPK25 also displayed a high expression in ovule integuments. We did not observe any abnormal phenotype in the flowers of cipk25 plants. This might be due to the fact that expression of ARF6 and ARF8 was only reduced, but not abolished, in cipk25.

CBL4 and CBL5 interacted with CIPK25 in yeast two-hybrid and in planta BiFC assays. It is known from several previous reports that an individual CBL protein can interact with multiple CIPKs and vice versa, depending on context (Luan, 2009). It is believed that this flexibility of interaction and their subcellular localizations are crucial in sensing and responding to specific signals. CBL4 was previously shown to interact with CIPK24 and CIPK6 in the context of salinity stress to modulate the activity of the Na⁺/H⁺ antiporter (Qiu et al., 2002) and plasma membrane targeting of the potassium channel AKT2 (Held et al., 2011). Both CBL4 and CBL5 possess conserved domains for myristoylation and acylation to localize in cell membranes (Batistič et al., 2010; Saito et al., 2018). Arabidopsis CIPK25 protein, although it possesses an MG-motif, does not undergo N-myristoylation (Saito et al., 2018). It is possible that a major pool of the expressed Arabidopsis CIPK25 protein interacted with the corresponding CBL(s) of N. benthamiana and was targeted to the plasma membrane. CBL sequences possess evolutionarily conserved motifs (Mohanta et al., 2015). Cross-species interaction of CIPKs and CBLs was reported before (Tripathi et al., 2009; Wang et al., 2012). Plasma membrane localization might be essential for CIPK25 function. CIPK11 interacts with CBL5, and myristoylation and acylation of CBL5 were shown to be required for the CBL5–CIPK11 complex to phosphorylate the guard cell anion channel SLAC1 (Saito et al., 2018).

Auxin and cytokinin together regulate various aspects of a plant’s growth and development. The balance between their interactions is maintained by negative regulation of one signaling pathway by the other. On the other hand, they regulate each other’s biosynthesis (Nordström et al., 2004; Jones et al., 2010). The cytokinin biosynthetic gene ATP/ADP isopentyl transferase 5 (IPT5) is rapidly induced by auxin and further induced in the SHY2 loss-of-function background, whereas auxin-dependent induction of P_IPT5::GUS was totally abolished in the SHY2 gain-of-function background (Dello Ioio et al., 2008). Relative enhancement of cytokinin activity at the root transition domain might reduce CIPK25 expression, leading to increased SHY2 expression resulting in reduced auxin signaling. We propose CIPK25 functions genetically above SHY2. Cytokinin-mediated suppression of CIPK25 expression was dependent on ARR1/ARR12. ARR1 is able to regulate SHY2 expression directly by binding to and activating the SHY2 promoter (Dello Ioio et al., 2008). It is noteworthy that cytokinin was able to suppress PIN1 expression even in the absence of CIPK25, suggesting a partial role for CIPK25 in balancing auxin–cytokinin signaling in root meristem development. Presently, it is not clear how CIPK25 functions in auxin/cytokinin signaling. Auxin and cytokinin are involved in many interconnected developmental processes through various cross-connected signaling pathways. Therefore, a role for CIPK25 in other pathways related to root development cannot be ruled out.

It is known that extracellular application of calcium inhibits cell growth in the TD (Ishikawa and Evans, 1992; Legué et al., 1997; Fasano et al., 2002). Ca²⁺ plays a vital role in polarity and rate of auxin transport through PIN proteins by modulating endo- and exocytosis and the direction of PIN localization in the plasma membrane (reviewed in Vanneste and Friml, 2013). In this study, we proposed that a Ca²⁺-regulated protein kinase, CIPK25, is involved in regulation of root meristem size by regulating auxin transport through modulating PIN protein expression.

Supplementary data

Supplementary data are available at JXB online.

Table S1. Primers used in this study.

Table S2. A list of auxin- and cytokinin-responsive elements in the CIPK25 promoter.

Fig. S1. Detection of YFP^N–CIPK25, CBL4–YFP^C, and CBL9–YFP^C fusion proteins in BiFC assay.

Fig. S2. qRT-PCR analysis of the expression levels of CIPK25 in the shy2-24 line.

Fig. S3. Relative transcript abundance of ARR1 and ARR12 in cipk25 and CIPK25OX seedlings.