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The calcium-dependent protein kinase (PnCDPK1) is involved in Pharbitis nil flowering
Journal of Plant Physiology 169 (2012) 1578–1585
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The calcium-dependent protein kinase (PnCDPK1) is involved in Pharbitis nil flowering Krzysztof Jaworski ∗ , Agnieszka Pawełek 1 , Jan Kopcewicz 2 , Adriana Szmidt-Jaworska 3 Nicolaus Copernicus University, Chair of Plant Physiology and Biotechnology, Gagarina St. 9, PL 87-100 Torun, Poland
a r t i c l e
i n f o
Article history: Received 16 April 2012 Received in revised form 24 May 2012 Accepted 28 May 2012 Keywords: CDPK Development Flower Kinase Pharbitis nil
s u m m a r y Signaling pathways, and specifically the signaling pathway of calcium, have been widely implicated in the regulation of a variety of signals in plants. Calcium-dependent protein kinases (CDPKs) are essential sensor-transducers of calcium signaling pathways, the functional characterization of which is of great interest because they play important roles during growth and in response to a wide range of environmental and developmental stimuli. Here, we report the first evidence of transient and specific elevation of PnCDPK1 transcript level and enzyme activity following conversion of a leaf bud to a flower bud, as well as participation of PnCDPK1 in evocation and flower morphogenesis in Pharbitis nil. Fluorescence microscopy immunolocalization and biochemical analysis confirmed the presence of CDPK in shoot apexes. The protein level was low in leaves, vegetative apexes and increased significantly in apexes after a flowering long-induction night. In the vegetative apex, a very weak PnCDPK1 protein signal was accumulated prominently in the zone of the ground meristem and in external layers of tissues of the cortex. After the dark treatment, the signal in cells of the ground meristem was still present, but a significantly stronger signal appeared in epidermal cells, cortex tissue, and leaf primordium. At the onset of flower meristem development, the PnCDPK1 level diverged significantly. PnCDPK1 mRNA, protein level and enzyme activity were very low at the beginning of flower bud development and gradually increased in later stages, reaching the highest level in a fully open flower. Analysis of flower organs revealed that PnCDPK1 was accumulated mainly in petals and sepals rather than in pistils and stamens. Our results clearly indicate that PnCDPK1 is developmentally regulated and may be an important component in the signal transduction pathways for flower morphogenesis. Findings from this research are important for further dissecting mechanisms of flowering and functions of CDPKs in flowering plants. © 2012 Elsevier GmbH. All rights reserved.
Introduction The most obvious features that distinguish angiosperm plants from others are their reproductive organs, the flowers. In the course of flowering, plants undergo a change from vegetative to reproductive growth, known as the floral transitions. Flowers contain non-reproductive parts: petals and sepals, and reproductive parts: stamens and pistils. Upon fertilization, a subset of carpels develops into fruits. When plants initiate the flowering process, the vegetative shoot apical meristem, which gives rise to all the parts of the shoot, is transformed into a generative meristem (floral meristem),
∗ Corresponding author. Tel.: +48 56 61 14 456; fax: +48 56 61 14 438. E-mail addresses: [email protected] (K. Jaworski), [email protected] (A. Pawełek), [email protected] (J. Kopcewicz), [email protected] (A. Szmidt-Jaworska). 1 Tel.: +48 56 61 14 456; fax: +48 56 61 14 772. 2 Tel.: +48 56 61 14 431; fax: +48 56 61 14 772. 3 Tel.: +48 56 61 14 456; fax: +48 56 61 14 438. 0176-1617/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.jplph.2012.05.025
that gives rise to floral organs. The control mechanisms leading to flower formation are not clear, but it is known that this is a multilayer and multi-stage process that requires external factors such as light, darkness, or temperature, as well as internal ones, e.g. second messengers or hormones. Calcium is a universal second messenger that responds to diverse endogenous and environmental stimuli (Reddy and Reddy, 2004). It is involved in regulating many plant functions, including cell elongation and division, ion fluxes, cellular pH, reproductive development, stress responses and apoptosis (Hepler, 2005; Jain et al., 2011). Additionally, calcium ions represent an important node for the crosstalk involving an array of diverse second messengers in plants. Its ubiquitous presence and versatility as a nutrient and signaling agent is dependent upon regulation of free cytosolic Ca2+ concentration and appropriate perception of variability in amplitude, duration, location, kinetics and frequency of Ca2+ spikes during signal transduction events (Jain et al., 2011). Intracellular calcium fluxes can be sensed and transduced to downstream biological responses through the activation of several
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Fig. 1. Stages of Pharbitis nil flower development. Numbers (1–8) represent the stages of flower growth from first flower bud (20 days after inductive long night) to fully open flower. Bar = 1 cm.
calcium-regulated proteins such as calmodulins, calmodulin-like proteins, calcineurin B-like proteins and calcium-dependent protein kinases (CDPKs) (Poovaiah and Reddy, 1993; Harmon et al., ´ 2000; Harper and Harmon, 2005; Klimecka and Muszynska, 2007). Among Ca2+ binding sensory proteins in plants, CDPKs play the most important role in transducing calcium signal events by reversible phosphorylation of substrate proteins (Patharkar and Cushman, 2000; Ito et al., 2010). This class of enzymes contains members of the serine/threonine protein kinase family that are composed of a single polypeptide chain with a catalytic kinase domain and a calmodulin-like domain (CLD), with four EF-hand Ca2+ binding sites which require micromolar concentra´ tions of free Ca2+ for their activation (Klimecka and Muszynska, 2007). In most cases, CDPKs are encoded by multigene families; for example, in Arabidopsis thaliana and Oryza sativa there are 34 and 31 genes, respectively. Multigene families of CDPKs are also present in other plants, including Solanum lycopersicum, Glycine max and Zea ´ mays (Cheng et al., 2002; Klimecka and Muszynska, 2007; Chang et al., 2009). However, there are some plants with a single copy of the CDPK gene e.g. PaCDPK1 in Phalaenopsis amabilis (Tsai et al., 2007). CDPKs have been reported to be involved in diverse (patho)physiological processes, including the accumulation of storage starch and protein in immature seeds of rice (Asano et al., 2002), root development and regulation of nodule numbers in Medicago truncatula (Ivashuta et al., 2005), stomata closure in Arabidopsis (Mori et al., 2006), pollen tube growth in Petunia (Yoon et al., 2006), flower induction process in Pharbitis nil (Jaworski et al., 2011), regulation of a transcription factor in Nicotiana (Ishida et al., 2008a,b), tolerance to cold, salt and drought stress in various species (Chang et al., 2009; Yu et al., 2010), a defense response to wounding (Kamiyoshihara et al., 2010), and regulation of ROS production in Lycopersicon esculentum (Kobayashi et al., 2007). In light signaling, CDPKs are believed to be important regulators involved in various processes, such as seed germination, development of chloroplasts, stem elongation, and flowering time in photoperiodic plants (Kaczorowski and Quail, 2005; Roig-Villanova et al., 2006; Giammaria et al., 2010; Jaworski et al., 2011). In plant organs grown in light or darkness, differences in expression and activity of some CDPKs have been noted (Klimecka and ´ Muszynska, 2007). For example, darkness elevated the expression level and protein activity of CDPKs in organs of zucchini seedlings, maize, rice and cucumber (Ellard-Ivey et al., 1999; Barker et al., 1998; Ullanat and Javabaskaran, 2002; Frattini et al., 1999; Saijo et al., 1997). Also, in wheat (Sharma et al., 1997) and Japanese morning glory seedlings (Jaworski et al., 2003, 2010, 2011), the activity of CDPKs was higher in etiolated tissues.
To date, little is known about the role of CDPKs in photoperiodic induction of flowering, evocation and flower morphogenesis in short-day plants. Most studies on the flowering process have been carried out on the model long-day plant A. thaliana, in which flowering is subject to a different regulation mechanism than in short-day plants. In the model short-day plant, P. nil, it has been demonstrated that the application of calcium, calcium chelating agents and protein kinase inhibitors on P. nil cotyledons during a flower induction long night affected the number of flower buds (Friedman et al., 1989; Szmidt-Jaworska et al., 2006). Lateral work revealed that some proteins are phosphorylated in a calcium-dependent manner during a dark inductive period, and PnCDPK1 shows down-regulation on both the molecular and biochemical level in response to light (Jaworski et al., 2003, 2010, 2011). At present, our studies are a continuation of the issue of CDPK influences on the flowering process. In this paper, we present PnCDPK1 as a potential element involved in the transition of a vegetative shoot apex to a generative one and the distribution of enzyme changes in the apex after the induction process. We also provide evidence for the participation of CDPK in processes of flower morphogenesis in the short-day plant P. nil. Materials and methods Plant material The investigations were conducted on morning glory (Pharbitis nil L. Chois), the Japanese variety Violet (Marutane Seed Co., Kyoto, Japan). Seeds before imbibition were soaked in concentrated sulfuric acid for 45 min and then washed with running tap water for 3 h. They were left in ddH2 O for 24 h at 25 ± 2 ◦ C in continuous white light (130 mol m−2 s−1 ; cool white fluorescent tubes, Polam, Poland). The imbibed seeds were planted on a mixture of vermiculite and sand (2:1) in plastic pots, covered with Saran Wrap to maintain high humidity and grown at 25 ± 2 ◦ C in continuous light for 5 days and exposed to a 16-h-long night to promote flowering. Subsequently, plants were grown in a growth chamber under continuous light. To see flower buds, all seedlings were allowed to grow for 20 days. Flower buds were collected at various stages of growth, from appearance of flower buds to fully open flower (Fig. 1). To distinguish vegetative buds from flower ones they were examined under a binocular microscope. Flower elements as pistils, stamens, petals, sepals were collected from fully open, mature flower (Fig. 1, stage 8). Thereafter, material was frozen in liquid nitrogen and stored at −80 ◦ C for later use.
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Gene expression analysis Semi-quantitative RT-PCR analysis was performed to analyze the expression of PnCDPK1 gene. Total RNA was isolated from particular plant material with the GeneMATRIX Universal RNA Purification Kit (Eurx) and then the RNA sample (1 g) was reverse transcribed with the MMLV RT enzyme (Epicentre) in 20 ◦ C at 37 ◦ C for 1 h. PCR was conducted at the linearity phase of the exponential reaction for each gene. The gene-specific primer pairs homologous to the 3 UTR of PnCDPK1 cDNA were as follows: for PnCDPK1, forward primer 5 -TTGTGTCGTGTTGCAATGTG-3 and reverse primer 5 -CAGCAGCACAACCTCTCAAA-3 and for actin4 gene, forward primer 5 -GAATTCGATATCCGAAAAGACTTGTATGG3 and reverse primer 5 -GAATTCCATACTCTGCCTTGGCAATC-3 . Actin4 expression level was used as a quantitative control. The specificity of PnCDPK1 RT-PCR product was confirmed by cloning and DNA sequencing. The computer application used for the analysis was Quantity One (BioRad), and for the statistic calculations and graphs we used SigmaPlot 2001 v. 7.0 (SPSS Inc.). Protein extraction Tissues from elements of flowers and different stages of P. nil flower growth were frozen in liquid nitrogen and homogenized using a mortar and pestle. The homogenate was then suspended in extraction buffer (20 mM, Tris–HCl pH 7.5, 2.5 M EDTA, 5 mM NaF, 10 M aprotinin, 10 M leupeptine and 1 M PMSF) and held on ice for 15 min. The crude protein extracts were centrifuged at 16,000 × g at 4 ◦ C for 30 min. The pellet was discarded and the supernatant containing the soluble proteins was used for further experiments. Protein concentration was determined by the method of Bradford (1976) using BSA as standard. In vitro enzyme assay Activity was determined in vitro by measuring the incorporation of 32 P from [␥-32 P] ATP (Polatom, Poland) into endogenous histone III-S. Calcium-dependent protein kinase assays were performed in a total volume of 50 L containing 50 mM Tris–HCl (pH 7.5), 10 mM MgCl2 , 0.5 mg mL−1 histone III-S, 1 mM CaCl2 or 1 mM EDTA. Reactions were initiated by the addition of 10 M [␥-32 P] ATP (1000 cpm pmol−1 ) and assay mixtures were incubated for 10 min at 30 ◦ C. Spotting 40 L on P-81 filter terminated the reaction. The filters were washed with 5% (w/v) phosphoric acid and 95% ethanol, dried and added to scintillation vials containing 4 mL scintillation cocktail and counted in a liquid scintillation counter (Wallac 1407). In a SDS-polyacrylamide gel protein kinase assay, in-gel phosphorylation of histone III-S was carried out according to the method described by Jaworski et al. (2010). Aliquots of the soluble fraction of each homogenate (40 g) were subjected to SDS-PAGE in 10% gel with histone III-S (0.5 mg mL−1 ) added to the separation gel just prior to polymerization. After electrophoresis, SDS was removed by washing the gel for 30 min at room temperature with 20% (v/v) isopropanol in 50 mM Tris–HCl (pH 8.0) and then 2 × 30 min in 50 mM Tris–HCl (pH 8.0), 5 mM -ME (buffer A). Proteins were denaturated by treating the gel with 6 M guanidine–HCl in buffer A for 1 h at room temperature and then renaturated with 0.04% Tween 40 in buffer A at 4 ◦ C over night. Next, the gel was preincubated with 25 mL 50 mM Tris–HCl (pH 7.5) containing 5 mM MgCl2 , 2 mM MnCl2 and 2 mM DTT for 1 h at 30 ◦ C and then in 4 mL of the same buffer 50 Ci [␥-32 P] ATP (3000 Ci mmol−1 ) and 1 mM CaCl2 or 2 mM EGTA for 1 h at 30 ◦ C. The reaction was stopped by washing the gel with 1% (w/v) sodium pyrophosphate in 5% (w/v) trichloroacetic acid. The washed gels were dried and exposed to
Fig. 2. PnCDPK1 activity and protein level in the 5-day-old shoot apex of Pharbitis nil growing in the light (L) and after exposure to 16 h of darkness (L + D). Lanes 1 and 2, in-gel phosphorylation assay of PnCDPK1. Lanes 3 and 4, protein blot analysis using anti-soybean CDPK. Arrow indicates position of PnCDPK1.
X-ray film (Foton). Signals were also scanned with phosphoimager Fuji 5000 and quantified with ImageQuant software. Immunodetection of CDPK using anti-soybean CDPK Resolved proteins on SDS-PAGE were transferred to PVDF membrane by the semi-dry system. After blocking in 3% non-fat dry milk solution, the membrane was incubated overnight at 4 ◦ C with primary polyclonal antibodies against the CLD domain of CDPK from soybean 1:2000 in TBS. After washing, the membrane was incubated for 1 h with secondary horseradish peroxidase conjugated to goat anti-rabbit IgG diluted to 1:10,000 in TBS buffer, and bands were visualized by chemiluminescence using the ECL plus system (GH Healthcare). The computer application used for the analysis was Quantity One (BioRad), and for the calculations and graphs we used SigmaPlot 2001 v. 7.0 (SPSS Inc.) Immunofluorescence labeling Material was fixed in a mixture of 4% paraformaldehyde and 0.25% glutaraldehyde in phosphate-buffered saline (PBS) buffer, pH 7.2, overnight at 4 ◦ C. In the next step, material was dehydrated in increasing ethanol concentrations, supersaturated and then embedded in BMM resin (butyl methacrylate, methyl methacrylate, 0.5% benzoin ethyl ether, 10 mM dithiothreitol; Fluka). The embedded material was cut into semithin sections, which were placed on microscope slides covered with Biobond (British Biocell International, Cardiff, U.K.). CDPK protein was detected by incubating with a primary polyclonal anti-CDPK antibody from soybean in 1% bovine serum albumin (BSA) in PBS (1 in 50 dilution), pH 7.2, overnight at 4 ◦ C, and the secondary goat anti-rabbit antibody Alexa Fluor 488 (Molecular Probes) in 1% BSA in PBS (1 in 500 dilution) for 1 h at 37 ◦ C. The control reaction was performed without the primary antibody. DNA was stained with 4,6-diamidino-2phenylindole (DAPI) (Sigma–Aldrich). Samples were analyzed with a Nikon Eclipse 80i fluorescence microscope. The CPI Plan Fluor 100 (numerical aperture, 1.3) DIC H/N2 oil immersion lens and narrow band filters (UV-2EC, B-2EC, G-2EC) were used. The results were
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Fig. 3. Localization of PnCDPK1 protein in the 5-day-old shoot apex of Pharbitis nil exposed to different light conditions. Panels 1n and 2n presented subcellular protein localization in shoot apexes from green seedlings grown in the light (non inductive); panels 3i–4i show protein localization in shoot apexes from green seedlings after 16 h dark treatment (flowering inductive conditions). Panels 1n and 3i – immunostaining with anti-CDPK antibody from soybean, positive signal is visible in green, panels 2n and 4i present the merging of signals from immunostaining and DAPI fluorescence (blue color). The symbols are: SAM – shoot apical meristem, GR – ground meristem, LP – leaf primordium, L – young leaf. Bar – 140 m.
registered with a Nikon DS-5Mc color cooled digital camera and Nikon NIS Elements software (Nikon Europe, Düsseldorf, Germany). Results Immunolocalization of PnCDPK in the shoot apex In previous reports, we showed that PnCDPK1 is a light regulated enzyme that can be involved in generating the induction signal that appears in cotyledons, as a consequence of a long night and transport to the apical meristem (Jaworski et al., 2010, 2011). Therefore, to fill a gap in the knowledge on this subject, experiments have been planned aimed at evaluating the processes the CDPK isoform undergoes in P. nil apical meristem before and after a long induction night, as well as in subsequent days after induction leading to the formation of particular flower elements. To examine changes in the level of PnCDPK1 in a vegetative and generative shoot apex, we performed the immunolocalization with anti-CDPK antibody from soybean, which cross-reacted with
P. nil CDPK (Jaworski et al., 2003, 2010, 2011). Immunolocalization experiments confirmed the presence of PnCDPK1 in both apexes (Fig. 3). In the vegetative apex (panels 1n and 2n), CDPK protein was accumulated prominently in the zone of the ground meristem. After the dark treatment (panels 3i and 4i), the signal in cells of the ground meristem was much stronger. Moreover, a strong signal was observed in the leaf primordium and apical meristem. Additionally, no major differences were detected in the architecture and size of the shoot meristem in induced and non-induced plants. In-gel assay activity and protein blot analysis showed specificity of the antibody. Moreover, it confirmed the increase in the amount of PnCDPK1 in the shoot apex after darkness (Fig. 2). PnCDPK in flower morphogenesis Gene expression analysis of PnCDPK1 In this study, we used RT-PCR to monitor the level of PnCDPK1 gene expression during evocation and flower morphogenesis as a
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Fig. 4. Semi-quantitative RT-PCR analyses of PnCDPK1 expression in different stages of flower development (1–8). (A) The histogram shows relative expression level of PnCDPK1 transcript. (B) Changes in the transcript levels on 1.7% agarose gel. The InACT4 transcript was amplified as RT-PCR control. Results are the mean values of two independent experiments (each done in triplicate assays); bars indicate standard deviation. There was a significant correlation between stages of flower development and relative gene expression (Sperman Rank Order Correlation: r(S) = 0.83 at p < 0.001).
consequence of the flowering induction. PnCDPK1 transcript accumulation was studied in the flower buds at different stages of growth, from a young generative bud to a fully open flower. The appearance and size of the flower bud, compared to 8 arbitrarily selected standard flower buds at different flower development stages, was a criterion for how advanced its development was (Fig. 1). PnCDPK1 was expressed during evocation, the process immediately preceding the formation of a flower and flower morphogenesis, and the level of its expression was regulated both temporally and spatially. The amount of PnCDPK1 mRNA increased gradually during flower morphogenesis (Fig. 4). The lowest level was observed in the first 3 stages, and ranged from 0.15 to 0.18. In stages 4–7 the transcript levels remained relatively stable, but it was 20% higher when compared to the stages 1–3. The highest level of the tested mRNA was observed in fully developed flowers (stage 8) and it was twice as high as the level of transcript in stage 1. PnCDPK1 gene expression was also examined in all elements of mature, ready for pollination flowers of P. nil (Fig. 1, stage 8). The transcript level was analyzed in the pistils, stamens, petals and sepals. The presence of PnCDPK1 transcript was found in all analyzed parts of the flower (Fig. 5). Whereas the highest level of mRNA was observed in the petals and sepals, at 0.45 and 0.4, respectively, the transcript level in the stamen was lower (0.33). However, the lowest concentration was noted in pistils (0.22). Biochemical analysis of PnCDPK1 To determine the PnCDPK1 activity and protein level during various stages of flower development, in vitro and in-gel kinase assays as well as immunoblot analyses were performed. Enzyme activity was assayed in soluble protein extracts isolated from growing flower buds and elements of mature flowers by measuring incorporation of phosphorus into the substrate histone III-S in the presence or absence of calcium ions, to determine the Ca2+ -dependent phosphorylation. As shown in Fig. 6A and B, both the in vitro and in-gel activity increased gradually during flower growth. The lowest PnCDPK1 activity was observed in the first three stages of bud growth, which gradually increased, reaching the highest level just before opening the flower bud (stage 7) and when the
Fig. 5. Semi-quantitative RT-PCR analyses of PnCDPK1 expression in parts of fully open flower. (A) The histogram shows relative expression level of PnCDPK1 transcript. (B) Changes in the transcript levels on 1.7% agarose gel. The InACT4 transcript was amplified as RT-PCR control. Results are the mean values of two independent experiments (each done in triplicate assays); bars indicate standard deviation. There was a significant effect of localization and relative gene expression (1-way ANOVA, F(4, 13) = 12.120 at p < 0.001).
flower was ready for pollination (stage 8). When histone III-S was used as a substrate, activity of three bands, 43, 56 and 68 kDa in the presence of micromolar concentrations of Ca2+ was observed (Fig. 6B). In the case of two bands, 56 kDa and 68 kDa, activity was completely inhibited in the presence of EDTA, whereas the presence of calcium ions or EDTA had no effect on the activity of 43 kDa protein, clearly indicating that activity of this enzyme is independent of calcium ions (Fig. 6C). The identity of PnCDPK1 and its level was confirmed by protein blot analysis using antibodies against CDL. Only a 56-kD protein cross-reacted with anti-soybean CDPK. This protein concentration was below the detection limit at initial stages of flower bud development (stages 1–3), then gradually increased (stages 4–7), reaching the highest point at stage 8 (Fig. 6D). Finally, the accumulation of PnCDPK1 and its activity in elements of flowers were examined. Fig. 7A and B shows the in vitro and in-gel assays of Ca2+ -dependent phosphorylation of PnCDPK1 in the extracts of pistils, stamens, petals, sepals and intact flowers. We found that histone III-S phosphorylation activity was higher in non-reproductive than in reproductive parts of flowers. In sepals and petals, activity was 2- and 4-fold higher compared to the stamen. Activity was almost nonexistent in pistils, even after a prolonged time of exposure to detection film and after usage of protease inhibitors during the extraction. Protein blot analysis of soluble proteins of P. nil revealed the presence of 56 kDa PnCDPK1 protein only (Fig. 7C) and it correlated with corresponding changes in enzyme activity and CDPK mRNA level (Fig. 5). Discussion The transition of plants from the vegetative to the generative phase is a crucial stage in their life. The processes of evocation, flower morphogenesis, pollination, fertilization, and consequently seed formation, lead to the beginning of new plant generation and maintenance of the species. In this series of consecutive events, many external and internal factors are involved, and light and calcium ions play an important role. Since cloning the cDNA encoding P. nil CDPK isoform, we have attempted to determine its biological roles. With this aim in mind,
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Fig. 7. Activity and protein level of Pharbitis nil PnCDPK1 in parts of fully open flower. (A) In vitro Ca2+ -dependent phosphorylation of histone III-S by soluble protein extract in the presence of 1 mM of CaCl2 or 1 mM EDTA followed by counting of incorporated 32 P in a liquid scintillation counter. (B) In-gel Ca2+ -dependent phosphorylation assay of proteins in the presence of 1 mM of CaCl2 or 1 mM EDTA. Protein extracts were electrophoresed in 10% (w/v) SDS-PAGE containing histone III-S as a substrate. After gel renaturation CDPK activity was detected in the presence of 1 mM of CaCl2 (+) or 1 mM EDTA (−). (C) Protein blot analysis of proteins using anti-soybean CDPK. Bars represent SE.
Fig. 6. PnCDPK activity and protein level during different stages of Pharbitis nil flower development. (A) In vitro Ca2+ -dependent phosphorylation of histone III-S by soluble protein extract in the presence of 1 mM of CaCl2 or 1 mM EDTA followed by counting of incorporated 32 P in a liquid scintillation counter. (B) In-gel Ca2+ dependent phosphorylation assay of proteins in the presence of 1 mM of CaCl2 or (C) 1 mM EDTA. Protein extracts were electrophoresed in 10% (w/v) SDS-PAGE containing histone III-S as a substrate. After gel renaturation CDPK activity was detected in the presence of 1 mM of CaCl2 (+) or 1 mM EDTA (−). (D) Protein blot analysis of proteins using anti-soybean CDPK. Bars represent SE.
we used biochemical, molecular and immunocytochemical techniques to characterize CDPK mRNA level, protein level and kinase activity in various organs and stages of plant growth and development (Jaworski et al., 2010, 2011). We have shown that CDPKs, PnCDPK56, now called PnCDPK1, is the dominant isoform of CDPK in P. nil seedlings, the activity and protein accumulation of which is negatively regulated by light and increased in rapidly proliferating cells and growing tissue, which may suggest its participation in cell proliferation
and differentiation. Moreover, based on differences in PnCDPK1 expression, CDPK level and activity during photoperiodic flower induction, we suggested involvement of this isoform of CDPK in this process (Jaworski et al., 2011). As a consequence of the appearance of the induction factor in cotyledons and its transport to the shoot meristem, a spectrum of irreversible changes occur in the shoot meristem, which further lead to flower formation. Thus, we focused on the elucidation of the role of PnCDPK1 during evocation, differentiation and flower morphogenesis. The P. nil plant is an ideal short-day plant model for the study of the early events in the photoperiodic induction of flowering and flower formation, because just 5-day-old, light-grown seedlings can be induced to flower by exposure to a single 16-h-long night, whereas seedlings cannot be induced to flower under continuous light (LL) or long-day (LD) conditions (Ogawa and King, 1990). Data pointing to a role of Ca2+ in the process of photoperiodic flower induction in cotyledons and leaves (Friedman et al., 1989; Tretyn et al., 1994) have been presented previously. It was proposed that, during the inductive dark period, an increase of intracellular free calcium concentration occurs, whereas the exogenously applied compounds modulating protein and involved in
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calcium homeostasis e.g., calcium channels or calmodulin, had the effect of increasing or reducing the number of flower buds formed (Friedman et al., 1989; Tretyn et al., 1994; Szmidt-Jaworska et al., 2006). Moreover, it was revealed that cotyledons and the shoot meristem after induction had higher cytosolic Ca2+ concentration than other cells (Walczysko et al., 2000). During recent years, great progress has been made in the identification of CDPK genes and biochemical characterization of these kinases as a main calcium sensor. Moreover, some potential substrates for CDPKs have been identified (Reddy and Reddy, 2004; Li et al., 2006). Nevertheless, there has been little progress in the elucidation of their biological functions in growth and development processes, especially during induction and flower formation. One approach to this problem involves the study of cell type-specific and inducible expression profiles. Generally, most CDPK genes are expressed in most of the plant tissues examined. However, some of them display an isoform-specific expression pattern that was not only organ- or tissue-specific, but also dependent on growth conditions (Kawasaki et al., 1993; Estruch et al., 1994; Takezawa et al., 1996; Saijo et al., 1997; Zhang et al., 2005; Chang et al., 2009; Jaworski et al., 2011). Our results suggest a potential role for CDPK in the response of the apical meristem of P. nil to the inductive photoperiod. The CDPK distribution throughout the apical meristem was different in non-induced and induced apexes and the fluorescence intensity was significantly higher in plants after induction. The elevation of CDPK level in the apical meristem of P. nil after photoperiodic flower induction has not been reported thus far. Such changes are no doubt a consequence of changes in cytosolic Ca2+ concentration in cells of the apical meristem. Previous work has shown an elevation of [Ca2+ ]cyt , in the range 100–350 nM, in the apical meristem of Sinapis alba and Chenopodium rubrum plants after photoperiodic flower induction (Havalange, 1989; Walczysko et al., 2000). In S. alba, the increase of [Ca2+ ]cyt in the apical bud begins 18 h after the start of the inductive long night. Particularly in the Sinapis meristem, the level of Ca2+ was found to increase in all cellular compartments, but mainly in the nucleolus, which is related to the increase in the rate of cell proliferation, as the first observed symptom of the flowering inducer reaching the apical meristem and the beginning of flower evocation after photoperiodic flower induction (Havalange, 1989). The author proposed that this increase is related to the intensification of the rate of cell proliferation, which is known to take place in the apical meristem after photoperiodic flower induction. Therefore, there is a correlation between the Ca2+ increase and CDPK level and activity in the apical meristem (Hepler, 2005; Lautner and Fromm, 2010). It is the reason that, in our experiments, PnCDPK1 was presented in the shoot primordium including the apical meristem, leaf primordium and floral buds. Previous data indicate that CDPKs demonstrate a temporal and spatial pattern of expression during flower development, which may point to the importance of this class of enzymes in the physiology of reproduction. In Nicotiana tabacum, NtCDPK1 was mainly expressed in the rapidly proliferating tissues, including the shoot meristem, developing flower buds, lateral root and branch primordia (Lee et al., 2003; Zhang et al., 2005). A large amount of NtCPK4 mRNA was also detected at the stigma surface, vascular and stylar vascular bundle. High expression of NtCPK4 was noted during the early development of the anther as well as in sporogenous cells, the tapetum and anther wall. However, during the process of pollen development, NtCPK4 expression decreased gradually, reaching a low level of mRNA in mature pollen cells (Zhang et al., 2005). The expression of one of the maize CDPK genes is pollen-specific and temporally restricted to both the late stages of pollen development and pollen grain germination and tube elongation (Estruch et al., 1994; Yoon et al., 2006; Ge et al., 2009).
Analysis of particular flower elements revealed a higher transcript level and enzyme activity in vegetative elements of flowers (petals and sepals), than generative ones (pistils, stamens) (Figs. 5 and 7). Such a situation can be caused by the presence of CDPK in other, processes occurring in parallel, such as floral anthocyanin synthesis (accumulation), growth of epidermal hairs, or corolla petal ageing, the vitality of which is very short after flower opening (6 h). However, it should be emphasized that, in some plant species (Anil and Rao, 2000; Llop-Tous et al., 2002; Tsai et al., 2007), no gene expression alteration during the moment of flower element formation has been observed, which may indicate the specificity of the CDPK isoform involved in these processes and/or regulation on the enzymatic level. In summary, the present simultaneous analysis of mRNA, protein expression level, and enzyme activity indicates the associations of this enzyme with flowering stages and flower organ development. Therefore, the following question arises: does the same enzyme phosphorylate single versatile of many different substrates depending on the action of a stimulus? If so, what mechanism for the recognition of a given substrate governs this? Further studies, including differential analysis of potential protein substrates, may reveal the answer.
Acknowledgements We would like to thank Dr. Agnieszka Zienkiewicz (Nicolaus Copernicus University, Torun, Poland) for assistance with fluorescence microscopy. The authors are grateful to Dr. Alice C. Harmon (University of Florida, Gainesville, USA) for generously providing the polyclonal antibodies against the calmodulin-like domain of soybean CDPK.
References Anil VS, Rao SK. Calcium-mediated signaling during sandalwood somatic embryogenesis. Role for exogenous calcium as second messenger. Plant Physiol 2000;123:1301–11. Asano T, Kunieda N, Omura Y, Ibe H, Kawasaki T, Takano M, et al. Rice SPK a calmodulin-like domain protein kinase, is required for storage product accumulation during seed development: phosphorylation of sucrose synthase is a possible factor. Plant Cell 2002;14:619–28. Barker LDP, Templeton MD, Ferguson IB. A 67-kDa plasma-membrane-bound Ca2+ -stimulate protein kinase active in sink tissue of higher plants. Planta 1998;205:197–204. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 1976;72:248–54. Chang WJ, Su HS, Li WJ, Zhang ZL. Expression profiling of a novel calcium-dependent protein kinase gene LeCPK2, from tomato (Solanum lycopersicum) under heat and pathogen-related hormones. Biosci Biotechnol Biochem 2009;73:2427–31. Cheng SH, Willmann MR, Chen HC, Sheen J. Calcium signaling through protein kinases. The Arabidopsis calcium-dependent protein kinase gene family. Plant Physiol 2002;129:469–85. Ellard-Ivey M, Hopkins RB, White TJ, Lomax TL. Cloning, expression and N-terminal myristoylation of CpCPK1, a calcium-dependent protein kinase from zucchini (Cucurbita pepo L.). Plant Mol Biol 1999;39:199–208. Estruch JJ, Kadwell S, Merlin E, Crossland L. Cloning and characterization of maize pollen-specific calcium-dependent calmodulin-independent protein kinase. Proc Natl Acad Sci USA 1994;91:8837–41. Frattini M, Morello L, Breviario D. Rice calcium-dependent protein kinase isoforms OsCDPK2 and OsCDPK11 show different response to light and different expression patterns during seed development. Plant Mol Biol 1999;41:753–64. Friedman H, Goldschmidt EE, Halevy AH. Involvement of calcium in the photoperiodic flower induction process of Pharbitis nil. Plant Physiol 1989;89:530–4. Ge LL, Xie CT, Tian HQ, Russell SD. Distribution of calcium in the stigma and style of tobacco during pollen germination and the tube elongation. Sex Plant Reprod 2009;22:87–96. Giammaria V, Grandellis C, Bachmann S, Gargantini PR, Feingold SE, Bryan G, et al. StCDPK2 expression and activity reveal a highly responsive potato calciumdependent protein kinase involved in light signalling. Planta 2010;233:593–609. Harmon AC, Gribskov M, Harper JF. CDPKs – a kinase for every Ca2+ signal? Trends Plant Sci 2000;5:154–9. Harper JF, Harmon A. Plants, symbiosis and parasites: a calcium signalling connection. Nat Rev Mol Cell Biol 2005;6:555–66.
K. Jaworski et al. / Journal of Plant Physiology 169 (2012) 1578–1585 Havalange A. Levels and ultrastructural localization of calcium in Sinapis alba during the floral transition. Plant Cell Physiol 1989;30:351–8. Hepler PK. Calcium: a central regulator of plant growth and development. Plant Cell 2005;17:2142–55. Ishida S, Yuasa T, Nakata M, Takahashi Y. A tobacco calcium-dependent protein kinase CDPK1, regulates the transcription factor REPRESSION OF SHOOT GROWTH in response to gibberellins. Plant Cell 2008a;20: 3273–88. Ishida S, Yuasa T, Nakata M, Takahashic Y. A tobacco calcium-dependent protein kinase CDPK1, regulates the transcription factor REPRESSION OF SHOOT GROWTH in response to gibberellins. Plant Cell 2008b;20: 3273–88. Ito T, Nakata M, Fukuzawa J, Ishida S, Takahashi Y. Alteration of substrate specificity: the variable N-terminal domain of tobacco Ca2+ -dependent protein kinase is important for substrate recognition. Plant Cell 2010;22:1592–604. Ivashuta S, Liu J, Liu J, Lohar D, Haridas S, Bucciarelli B, et al. RNA interference identifies a calcium-dependent protein kinase involved in Medicago truncatula root development. Plant Cell 2005;17:2911–21. Jain M, Pathak BP, Harmon AC, Tillman BL, Gallo M. Calcium dependent protein kinase (CDPK) expression during fruit development in cultivated peanut (Arachis hypogaea) under Ca2+ -sufficient and -deficient growth regimens. J Plant Physiol 2011;168:2272–7. Jaworski K, Pawełek A, Szmidt-Jaworska A, Kopcewicz J. Expression of calciumdependent protein kinase gene (PnCDPK1) is affected by various light conditions in Pharbitis nil seedlings. J Plant Growth Regul 2010;29:316–27. Jaworski K, Szmidt-Jaworska A, Kopcewicz J. Two calcium dependent protein kinases are differently regulated by light and have different activity patterns during seedling growth in Pharbitis nil. Plant Growth Regul 2011;65:369–79. Jaworski K, Szmidt-Jaworska A, Tretyn A, Kopcewicz J. Biochemical evidences for a calcium-dependent protein kinase in Pharbitis nil and its involvement in photoperiodic flower induction. Phytochemistry 2003;62:1047–55. Kaczorowski KA, Quail PH. Arabidopsis PSEUDO-RESPONSE REGULATOR7 is a signaling intermediate in phytochrome-regulated seedling deetiolation and phasing of the circadian clock. Plant Cell 2005;15:2654–65. Kamiyoshihara Y, Iwata M, Fukaya T, Tatsuki M, Mori H. Turnover of LeACS2, a wound-inducible 1-aminocyclopropane-1-carboxylic acid synthase in tomato, is regulated by phosphorylation/dephosphorylation. Plant J 2010;64: 140–50. Kawasaki T, Hayashida N, Baba T, Shinozaki K, Shimada H. The gene encoding a calcium-dependent protein kinase located near the sbe1 gene encoding starch branching enzyme I is specifically expressed in developing rice seeds. Gene 1993;129:183–9. ´ G. Structure and functions of plant calcium-dependent Klimecka M, Muszynska protein kinases. Acta Biochim Pol 2007;54:219–33. Kobayashi M, Ohura I, Kawakita K, Yokota N, Fujiwara M, Shimamoto K, et al. Calcium-dependent protein kinases regulate the production of reactive oxygen species by potato NADPH oxidase. Plant Cell 2007;19:1065–80. Lautner S, Fromm J. Calcium-dependent physiological processes in trees. Plant Biol 2010;12:268–74. Lee SS, Cho HS, Yoon GM, Ahn JW, Kim HH, Pai HS. Interaction of NtCDPK1 calciumdependent protein kinase with NtRpn regulatory subunit of the 26S proteasome in Nicotiana tabacum. Plant J 2003;33:825–40.
1585
Li RJ, Hua W, Lu YT. Arabidopsis cytosolic glutamine synthetase AtGLN1 is a potential substrate of AtCRK3 involved in leaf senescence. Biochem Biophys Res Commun 2006;342:119–26. Llop-Tous I, Dominguez-Puigjaner E, Vendrell M. Characterization of a strawberry cDNA clone homologous to calcium-dependent protein kinases that is expressed during fruit ripening and affected by low temperature. J Exp Bot 2002;53:2283–5. Mori I, Murata Y, Yang Y, Munemasa S, Wang Y, Andreoli S, et al. CDPKs CPK6 and CPK3 function in ABA regulation of guard cell S-type anion- and Ca2+ -permeable channels and stomatal closure. PLoS Biol 2006;4:e327. Ogawa Y, King RW. The inhibition of flowering by non-induced cotyledons of Pharbitis nil. Plant Cell Physiol 1990;31:129–35. Patharkar OR, Cushman JC. A stress-induced calcium-dependent protein kinase from Mesynbryanthemum crystallinum phosphorylates a two-component pseudoresponse regulator. Plant J 2000;24:679–91. Poovaiah BW, Reddy AS. Calcium and signal transduction in plants. CRC Crit Rev Plant Sci 1993;12:185–211. Reddy VS, Reddy ASN. Proteomics of calcium-signaling components in plants. Phytochemistry 2004;65:1745–76. Roig-Villanova I, Bou J, Sorin C, Devlin PF, Martinez-Garcia JF. Identification of primary target genes of phytochrome signaling early transcriptional control during shade avoidance responses in Arabidopsis. Plant Physiol 2006;141:85–96. Saijo Y, Hata S, Sheen J, Izui K. cDNA cloning and prokaryotic expression of maize calcium-dependent protein kinases. Biochim Biophys Acta 1997;1350:109–14. Sharma VK, Jain PK, Malik MK, Maheshwari SC, Khurana JP. Light- and calciummodulated phosphorylation of proteins from wheat seedlings. Phytochemistry 1997;44:781–6. Szmidt-Jaworska A, Jaworski K, Kopcewicz J. The involvement of cyclic ADPR in photoperiodic flower induction of Pharbitis nil. J Plant Growth Regul 2006;25:233–44. Takezawa D, Patil S, Bhatia A, Poovaiah BW. Calcium-dependent protein kinases genes in corn roots. J Plant Physiol 1996;149:329–35. Tretyn A, Czaplewska J, Cymerski M, Kopcewicz J, Kendrick RE. The mechanism of calcium action on flower induction in Pharbitis nil. J Plant Physiol 1994;144:562–8. Tsai TM, Chen YR, Kao TW, Tsay WS, Wu CP, Huang DD, et al. PaCDPK1, a gene encoding calcium-dependent protein kinase from orchid, Phalaenopsis amabilis, is induced by cold, wounding, and pathogen challenge. Plant Cell Rep 2007;26:1899–908. Ullanat R, Javabaskaran C. Distinct light-, cytokinin and tissue-specific regulation of calcium dependent protein kinase gene expression in cucumber (Cucumber sativus). Plant Sci 2002;162:153–63. Walczysko P, Wagner E, Albrechtová JTP. Use of co-loaded Fluo-3 and Fura Red fluorescent indicators for studying the cytosolic Ca2+ concentrations distribution in living plant tissue. Cell Calcium 2000;28:23–32. Yoon GM, Dowd PE, Gilroy S, McCubbin AG. Calcium-dependent protein kinase isoforms in Petunia have distinct functions in pollen tube growth, including regulating polarity. Plant Cell 2006;18:867–78. Yu J, Tian YS, Peng RH, Xiong AS, Zhu B, Jin XF, et al. AtCPK6, a functionally redundant and positive regulator involved in salt/drought stress tolerance in Arabidopsis. Planta 2010;231:1251–60. Zhang T, Wang Q, Chen X, Tian C, Wang X, Xing T, et al. Cloning and biochemical properties of CDPK gene OsCDPK14 from rice. J Plant Physiol 2005;162:1149–59.
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Journal of Plant Physiology journal homepage: www.elsevier.de/jplph
The calcium-dependent protein kinase (PnCDPK1) is involved in Pharbitis nil flowering Krzysztof Jaworski ∗ , Agnieszka Pawełek 1 , Jan Kopcewicz 2 , Adriana Szmidt-Jaworska 3 Nicolaus Copernicus University, Chair of Plant Physiology and Biotechnology, Gagarina St. 9, PL 87-100 Torun, Poland
a r t i c l e
i n f o
Article history: Received 16 April 2012 Received in revised form 24 May 2012 Accepted 28 May 2012 Keywords: CDPK Development Flower Kinase Pharbitis nil
s u m m a r y Signaling pathways, and specifically the signaling pathway of calcium, have been widely implicated in the regulation of a variety of signals in plants. Calcium-dependent protein kinases (CDPKs) are essential sensor-transducers of calcium signaling pathways, the functional characterization of which is of great interest because they play important roles during growth and in response to a wide range of environmental and developmental stimuli. Here, we report the first evidence of transient and specific elevation of PnCDPK1 transcript level and enzyme activity following conversion of a leaf bud to a flower bud, as well as participation of PnCDPK1 in evocation and flower morphogenesis in Pharbitis nil. Fluorescence microscopy immunolocalization and biochemical analysis confirmed the presence of CDPK in shoot apexes. The protein level was low in leaves, vegetative apexes and increased significantly in apexes after a flowering long-induction night. In the vegetative apex, a very weak PnCDPK1 protein signal was accumulated prominently in the zone of the ground meristem and in external layers of tissues of the cortex. After the dark treatment, the signal in cells of the ground meristem was still present, but a significantly stronger signal appeared in epidermal cells, cortex tissue, and leaf primordium. At the onset of flower meristem development, the PnCDPK1 level diverged significantly. PnCDPK1 mRNA, protein level and enzyme activity were very low at the beginning of flower bud development and gradually increased in later stages, reaching the highest level in a fully open flower. Analysis of flower organs revealed that PnCDPK1 was accumulated mainly in petals and sepals rather than in pistils and stamens. Our results clearly indicate that PnCDPK1 is developmentally regulated and may be an important component in the signal transduction pathways for flower morphogenesis. Findings from this research are important for further dissecting mechanisms of flowering and functions of CDPKs in flowering plants. © 2012 Elsevier GmbH. All rights reserved.
Introduction The most obvious features that distinguish angiosperm plants from others are their reproductive organs, the flowers. In the course of flowering, plants undergo a change from vegetative to reproductive growth, known as the floral transitions. Flowers contain non-reproductive parts: petals and sepals, and reproductive parts: stamens and pistils. Upon fertilization, a subset of carpels develops into fruits. When plants initiate the flowering process, the vegetative shoot apical meristem, which gives rise to all the parts of the shoot, is transformed into a generative meristem (floral meristem),
∗ Corresponding author. Tel.: +48 56 61 14 456; fax: +48 56 61 14 438. E-mail addresses: [email protected] (K. Jaworski), [email protected] (A. Pawełek), [email protected] (J. Kopcewicz), [email protected] (A. Szmidt-Jaworska). 1 Tel.: +48 56 61 14 456; fax: +48 56 61 14 772. 2 Tel.: +48 56 61 14 431; fax: +48 56 61 14 772. 3 Tel.: +48 56 61 14 456; fax: +48 56 61 14 438. 0176-1617/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.jplph.2012.05.025
that gives rise to floral organs. The control mechanisms leading to flower formation are not clear, but it is known that this is a multilayer and multi-stage process that requires external factors such as light, darkness, or temperature, as well as internal ones, e.g. second messengers or hormones. Calcium is a universal second messenger that responds to diverse endogenous and environmental stimuli (Reddy and Reddy, 2004). It is involved in regulating many plant functions, including cell elongation and division, ion fluxes, cellular pH, reproductive development, stress responses and apoptosis (Hepler, 2005; Jain et al., 2011). Additionally, calcium ions represent an important node for the crosstalk involving an array of diverse second messengers in plants. Its ubiquitous presence and versatility as a nutrient and signaling agent is dependent upon regulation of free cytosolic Ca2+ concentration and appropriate perception of variability in amplitude, duration, location, kinetics and frequency of Ca2+ spikes during signal transduction events (Jain et al., 2011). Intracellular calcium fluxes can be sensed and transduced to downstream biological responses through the activation of several
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Fig. 1. Stages of Pharbitis nil flower development. Numbers (1–8) represent the stages of flower growth from first flower bud (20 days after inductive long night) to fully open flower. Bar = 1 cm.
calcium-regulated proteins such as calmodulins, calmodulin-like proteins, calcineurin B-like proteins and calcium-dependent protein kinases (CDPKs) (Poovaiah and Reddy, 1993; Harmon et al., ´ 2000; Harper and Harmon, 2005; Klimecka and Muszynska, 2007). Among Ca2+ binding sensory proteins in plants, CDPKs play the most important role in transducing calcium signal events by reversible phosphorylation of substrate proteins (Patharkar and Cushman, 2000; Ito et al., 2010). This class of enzymes contains members of the serine/threonine protein kinase family that are composed of a single polypeptide chain with a catalytic kinase domain and a calmodulin-like domain (CLD), with four EF-hand Ca2+ binding sites which require micromolar concentra´ tions of free Ca2+ for their activation (Klimecka and Muszynska, 2007). In most cases, CDPKs are encoded by multigene families; for example, in Arabidopsis thaliana and Oryza sativa there are 34 and 31 genes, respectively. Multigene families of CDPKs are also present in other plants, including Solanum lycopersicum, Glycine max and Zea ´ mays (Cheng et al., 2002; Klimecka and Muszynska, 2007; Chang et al., 2009). However, there are some plants with a single copy of the CDPK gene e.g. PaCDPK1 in Phalaenopsis amabilis (Tsai et al., 2007). CDPKs have been reported to be involved in diverse (patho)physiological processes, including the accumulation of storage starch and protein in immature seeds of rice (Asano et al., 2002), root development and regulation of nodule numbers in Medicago truncatula (Ivashuta et al., 2005), stomata closure in Arabidopsis (Mori et al., 2006), pollen tube growth in Petunia (Yoon et al., 2006), flower induction process in Pharbitis nil (Jaworski et al., 2011), regulation of a transcription factor in Nicotiana (Ishida et al., 2008a,b), tolerance to cold, salt and drought stress in various species (Chang et al., 2009; Yu et al., 2010), a defense response to wounding (Kamiyoshihara et al., 2010), and regulation of ROS production in Lycopersicon esculentum (Kobayashi et al., 2007). In light signaling, CDPKs are believed to be important regulators involved in various processes, such as seed germination, development of chloroplasts, stem elongation, and flowering time in photoperiodic plants (Kaczorowski and Quail, 2005; Roig-Villanova et al., 2006; Giammaria et al., 2010; Jaworski et al., 2011). In plant organs grown in light or darkness, differences in expression and activity of some CDPKs have been noted (Klimecka and ´ Muszynska, 2007). For example, darkness elevated the expression level and protein activity of CDPKs in organs of zucchini seedlings, maize, rice and cucumber (Ellard-Ivey et al., 1999; Barker et al., 1998; Ullanat and Javabaskaran, 2002; Frattini et al., 1999; Saijo et al., 1997). Also, in wheat (Sharma et al., 1997) and Japanese morning glory seedlings (Jaworski et al., 2003, 2010, 2011), the activity of CDPKs was higher in etiolated tissues.
To date, little is known about the role of CDPKs in photoperiodic induction of flowering, evocation and flower morphogenesis in short-day plants. Most studies on the flowering process have been carried out on the model long-day plant A. thaliana, in which flowering is subject to a different regulation mechanism than in short-day plants. In the model short-day plant, P. nil, it has been demonstrated that the application of calcium, calcium chelating agents and protein kinase inhibitors on P. nil cotyledons during a flower induction long night affected the number of flower buds (Friedman et al., 1989; Szmidt-Jaworska et al., 2006). Lateral work revealed that some proteins are phosphorylated in a calcium-dependent manner during a dark inductive period, and PnCDPK1 shows down-regulation on both the molecular and biochemical level in response to light (Jaworski et al., 2003, 2010, 2011). At present, our studies are a continuation of the issue of CDPK influences on the flowering process. In this paper, we present PnCDPK1 as a potential element involved in the transition of a vegetative shoot apex to a generative one and the distribution of enzyme changes in the apex after the induction process. We also provide evidence for the participation of CDPK in processes of flower morphogenesis in the short-day plant P. nil. Materials and methods Plant material The investigations were conducted on morning glory (Pharbitis nil L. Chois), the Japanese variety Violet (Marutane Seed Co., Kyoto, Japan). Seeds before imbibition were soaked in concentrated sulfuric acid for 45 min and then washed with running tap water for 3 h. They were left in ddH2 O for 24 h at 25 ± 2 ◦ C in continuous white light (130 mol m−2 s−1 ; cool white fluorescent tubes, Polam, Poland). The imbibed seeds were planted on a mixture of vermiculite and sand (2:1) in plastic pots, covered with Saran Wrap to maintain high humidity and grown at 25 ± 2 ◦ C in continuous light for 5 days and exposed to a 16-h-long night to promote flowering. Subsequently, plants were grown in a growth chamber under continuous light. To see flower buds, all seedlings were allowed to grow for 20 days. Flower buds were collected at various stages of growth, from appearance of flower buds to fully open flower (Fig. 1). To distinguish vegetative buds from flower ones they were examined under a binocular microscope. Flower elements as pistils, stamens, petals, sepals were collected from fully open, mature flower (Fig. 1, stage 8). Thereafter, material was frozen in liquid nitrogen and stored at −80 ◦ C for later use.
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Gene expression analysis Semi-quantitative RT-PCR analysis was performed to analyze the expression of PnCDPK1 gene. Total RNA was isolated from particular plant material with the GeneMATRIX Universal RNA Purification Kit (Eurx) and then the RNA sample (1 g) was reverse transcribed with the MMLV RT enzyme (Epicentre) in 20 ◦ C at 37 ◦ C for 1 h. PCR was conducted at the linearity phase of the exponential reaction for each gene. The gene-specific primer pairs homologous to the 3 UTR of PnCDPK1 cDNA were as follows: for PnCDPK1, forward primer 5 -TTGTGTCGTGTTGCAATGTG-3 and reverse primer 5 -CAGCAGCACAACCTCTCAAA-3 and for actin4 gene, forward primer 5 -GAATTCGATATCCGAAAAGACTTGTATGG3 and reverse primer 5 -GAATTCCATACTCTGCCTTGGCAATC-3 . Actin4 expression level was used as a quantitative control. The specificity of PnCDPK1 RT-PCR product was confirmed by cloning and DNA sequencing. The computer application used for the analysis was Quantity One (BioRad), and for the statistic calculations and graphs we used SigmaPlot 2001 v. 7.0 (SPSS Inc.). Protein extraction Tissues from elements of flowers and different stages of P. nil flower growth were frozen in liquid nitrogen and homogenized using a mortar and pestle. The homogenate was then suspended in extraction buffer (20 mM, Tris–HCl pH 7.5, 2.5 M EDTA, 5 mM NaF, 10 M aprotinin, 10 M leupeptine and 1 M PMSF) and held on ice for 15 min. The crude protein extracts were centrifuged at 16,000 × g at 4 ◦ C for 30 min. The pellet was discarded and the supernatant containing the soluble proteins was used for further experiments. Protein concentration was determined by the method of Bradford (1976) using BSA as standard. In vitro enzyme assay Activity was determined in vitro by measuring the incorporation of 32 P from [␥-32 P] ATP (Polatom, Poland) into endogenous histone III-S. Calcium-dependent protein kinase assays were performed in a total volume of 50 L containing 50 mM Tris–HCl (pH 7.5), 10 mM MgCl2 , 0.5 mg mL−1 histone III-S, 1 mM CaCl2 or 1 mM EDTA. Reactions were initiated by the addition of 10 M [␥-32 P] ATP (1000 cpm pmol−1 ) and assay mixtures were incubated for 10 min at 30 ◦ C. Spotting 40 L on P-81 filter terminated the reaction. The filters were washed with 5% (w/v) phosphoric acid and 95% ethanol, dried and added to scintillation vials containing 4 mL scintillation cocktail and counted in a liquid scintillation counter (Wallac 1407). In a SDS-polyacrylamide gel protein kinase assay, in-gel phosphorylation of histone III-S was carried out according to the method described by Jaworski et al. (2010). Aliquots of the soluble fraction of each homogenate (40 g) were subjected to SDS-PAGE in 10% gel with histone III-S (0.5 mg mL−1 ) added to the separation gel just prior to polymerization. After electrophoresis, SDS was removed by washing the gel for 30 min at room temperature with 20% (v/v) isopropanol in 50 mM Tris–HCl (pH 8.0) and then 2 × 30 min in 50 mM Tris–HCl (pH 8.0), 5 mM -ME (buffer A). Proteins were denaturated by treating the gel with 6 M guanidine–HCl in buffer A for 1 h at room temperature and then renaturated with 0.04% Tween 40 in buffer A at 4 ◦ C over night. Next, the gel was preincubated with 25 mL 50 mM Tris–HCl (pH 7.5) containing 5 mM MgCl2 , 2 mM MnCl2 and 2 mM DTT for 1 h at 30 ◦ C and then in 4 mL of the same buffer 50 Ci [␥-32 P] ATP (3000 Ci mmol−1 ) and 1 mM CaCl2 or 2 mM EGTA for 1 h at 30 ◦ C. The reaction was stopped by washing the gel with 1% (w/v) sodium pyrophosphate in 5% (w/v) trichloroacetic acid. The washed gels were dried and exposed to
Fig. 2. PnCDPK1 activity and protein level in the 5-day-old shoot apex of Pharbitis nil growing in the light (L) and after exposure to 16 h of darkness (L + D). Lanes 1 and 2, in-gel phosphorylation assay of PnCDPK1. Lanes 3 and 4, protein blot analysis using anti-soybean CDPK. Arrow indicates position of PnCDPK1.
X-ray film (Foton). Signals were also scanned with phosphoimager Fuji 5000 and quantified with ImageQuant software. Immunodetection of CDPK using anti-soybean CDPK Resolved proteins on SDS-PAGE were transferred to PVDF membrane by the semi-dry system. After blocking in 3% non-fat dry milk solution, the membrane was incubated overnight at 4 ◦ C with primary polyclonal antibodies against the CLD domain of CDPK from soybean 1:2000 in TBS. After washing, the membrane was incubated for 1 h with secondary horseradish peroxidase conjugated to goat anti-rabbit IgG diluted to 1:10,000 in TBS buffer, and bands were visualized by chemiluminescence using the ECL plus system (GH Healthcare). The computer application used for the analysis was Quantity One (BioRad), and for the calculations and graphs we used SigmaPlot 2001 v. 7.0 (SPSS Inc.) Immunofluorescence labeling Material was fixed in a mixture of 4% paraformaldehyde and 0.25% glutaraldehyde in phosphate-buffered saline (PBS) buffer, pH 7.2, overnight at 4 ◦ C. In the next step, material was dehydrated in increasing ethanol concentrations, supersaturated and then embedded in BMM resin (butyl methacrylate, methyl methacrylate, 0.5% benzoin ethyl ether, 10 mM dithiothreitol; Fluka). The embedded material was cut into semithin sections, which were placed on microscope slides covered with Biobond (British Biocell International, Cardiff, U.K.). CDPK protein was detected by incubating with a primary polyclonal anti-CDPK antibody from soybean in 1% bovine serum albumin (BSA) in PBS (1 in 50 dilution), pH 7.2, overnight at 4 ◦ C, and the secondary goat anti-rabbit antibody Alexa Fluor 488 (Molecular Probes) in 1% BSA in PBS (1 in 500 dilution) for 1 h at 37 ◦ C. The control reaction was performed without the primary antibody. DNA was stained with 4,6-diamidino-2phenylindole (DAPI) (Sigma–Aldrich). Samples were analyzed with a Nikon Eclipse 80i fluorescence microscope. The CPI Plan Fluor 100 (numerical aperture, 1.3) DIC H/N2 oil immersion lens and narrow band filters (UV-2EC, B-2EC, G-2EC) were used. The results were
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Fig. 3. Localization of PnCDPK1 protein in the 5-day-old shoot apex of Pharbitis nil exposed to different light conditions. Panels 1n and 2n presented subcellular protein localization in shoot apexes from green seedlings grown in the light (non inductive); panels 3i–4i show protein localization in shoot apexes from green seedlings after 16 h dark treatment (flowering inductive conditions). Panels 1n and 3i – immunostaining with anti-CDPK antibody from soybean, positive signal is visible in green, panels 2n and 4i present the merging of signals from immunostaining and DAPI fluorescence (blue color). The symbols are: SAM – shoot apical meristem, GR – ground meristem, LP – leaf primordium, L – young leaf. Bar – 140 m.
registered with a Nikon DS-5Mc color cooled digital camera and Nikon NIS Elements software (Nikon Europe, Düsseldorf, Germany). Results Immunolocalization of PnCDPK in the shoot apex In previous reports, we showed that PnCDPK1 is a light regulated enzyme that can be involved in generating the induction signal that appears in cotyledons, as a consequence of a long night and transport to the apical meristem (Jaworski et al., 2010, 2011). Therefore, to fill a gap in the knowledge on this subject, experiments have been planned aimed at evaluating the processes the CDPK isoform undergoes in P. nil apical meristem before and after a long induction night, as well as in subsequent days after induction leading to the formation of particular flower elements. To examine changes in the level of PnCDPK1 in a vegetative and generative shoot apex, we performed the immunolocalization with anti-CDPK antibody from soybean, which cross-reacted with
P. nil CDPK (Jaworski et al., 2003, 2010, 2011). Immunolocalization experiments confirmed the presence of PnCDPK1 in both apexes (Fig. 3). In the vegetative apex (panels 1n and 2n), CDPK protein was accumulated prominently in the zone of the ground meristem. After the dark treatment (panels 3i and 4i), the signal in cells of the ground meristem was much stronger. Moreover, a strong signal was observed in the leaf primordium and apical meristem. Additionally, no major differences were detected in the architecture and size of the shoot meristem in induced and non-induced plants. In-gel assay activity and protein blot analysis showed specificity of the antibody. Moreover, it confirmed the increase in the amount of PnCDPK1 in the shoot apex after darkness (Fig. 2). PnCDPK in flower morphogenesis Gene expression analysis of PnCDPK1 In this study, we used RT-PCR to monitor the level of PnCDPK1 gene expression during evocation and flower morphogenesis as a
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Fig. 4. Semi-quantitative RT-PCR analyses of PnCDPK1 expression in different stages of flower development (1–8). (A) The histogram shows relative expression level of PnCDPK1 transcript. (B) Changes in the transcript levels on 1.7% agarose gel. The InACT4 transcript was amplified as RT-PCR control. Results are the mean values of two independent experiments (each done in triplicate assays); bars indicate standard deviation. There was a significant correlation between stages of flower development and relative gene expression (Sperman Rank Order Correlation: r(S) = 0.83 at p < 0.001).
consequence of the flowering induction. PnCDPK1 transcript accumulation was studied in the flower buds at different stages of growth, from a young generative bud to a fully open flower. The appearance and size of the flower bud, compared to 8 arbitrarily selected standard flower buds at different flower development stages, was a criterion for how advanced its development was (Fig. 1). PnCDPK1 was expressed during evocation, the process immediately preceding the formation of a flower and flower morphogenesis, and the level of its expression was regulated both temporally and spatially. The amount of PnCDPK1 mRNA increased gradually during flower morphogenesis (Fig. 4). The lowest level was observed in the first 3 stages, and ranged from 0.15 to 0.18. In stages 4–7 the transcript levels remained relatively stable, but it was 20% higher when compared to the stages 1–3. The highest level of the tested mRNA was observed in fully developed flowers (stage 8) and it was twice as high as the level of transcript in stage 1. PnCDPK1 gene expression was also examined in all elements of mature, ready for pollination flowers of P. nil (Fig. 1, stage 8). The transcript level was analyzed in the pistils, stamens, petals and sepals. The presence of PnCDPK1 transcript was found in all analyzed parts of the flower (Fig. 5). Whereas the highest level of mRNA was observed in the petals and sepals, at 0.45 and 0.4, respectively, the transcript level in the stamen was lower (0.33). However, the lowest concentration was noted in pistils (0.22). Biochemical analysis of PnCDPK1 To determine the PnCDPK1 activity and protein level during various stages of flower development, in vitro and in-gel kinase assays as well as immunoblot analyses were performed. Enzyme activity was assayed in soluble protein extracts isolated from growing flower buds and elements of mature flowers by measuring incorporation of phosphorus into the substrate histone III-S in the presence or absence of calcium ions, to determine the Ca2+ -dependent phosphorylation. As shown in Fig. 6A and B, both the in vitro and in-gel activity increased gradually during flower growth. The lowest PnCDPK1 activity was observed in the first three stages of bud growth, which gradually increased, reaching the highest level just before opening the flower bud (stage 7) and when the
Fig. 5. Semi-quantitative RT-PCR analyses of PnCDPK1 expression in parts of fully open flower. (A) The histogram shows relative expression level of PnCDPK1 transcript. (B) Changes in the transcript levels on 1.7% agarose gel. The InACT4 transcript was amplified as RT-PCR control. Results are the mean values of two independent experiments (each done in triplicate assays); bars indicate standard deviation. There was a significant effect of localization and relative gene expression (1-way ANOVA, F(4, 13) = 12.120 at p < 0.001).
flower was ready for pollination (stage 8). When histone III-S was used as a substrate, activity of three bands, 43, 56 and 68 kDa in the presence of micromolar concentrations of Ca2+ was observed (Fig. 6B). In the case of two bands, 56 kDa and 68 kDa, activity was completely inhibited in the presence of EDTA, whereas the presence of calcium ions or EDTA had no effect on the activity of 43 kDa protein, clearly indicating that activity of this enzyme is independent of calcium ions (Fig. 6C). The identity of PnCDPK1 and its level was confirmed by protein blot analysis using antibodies against CDL. Only a 56-kD protein cross-reacted with anti-soybean CDPK. This protein concentration was below the detection limit at initial stages of flower bud development (stages 1–3), then gradually increased (stages 4–7), reaching the highest point at stage 8 (Fig. 6D). Finally, the accumulation of PnCDPK1 and its activity in elements of flowers were examined. Fig. 7A and B shows the in vitro and in-gel assays of Ca2+ -dependent phosphorylation of PnCDPK1 in the extracts of pistils, stamens, petals, sepals and intact flowers. We found that histone III-S phosphorylation activity was higher in non-reproductive than in reproductive parts of flowers. In sepals and petals, activity was 2- and 4-fold higher compared to the stamen. Activity was almost nonexistent in pistils, even after a prolonged time of exposure to detection film and after usage of protease inhibitors during the extraction. Protein blot analysis of soluble proteins of P. nil revealed the presence of 56 kDa PnCDPK1 protein only (Fig. 7C) and it correlated with corresponding changes in enzyme activity and CDPK mRNA level (Fig. 5). Discussion The transition of plants from the vegetative to the generative phase is a crucial stage in their life. The processes of evocation, flower morphogenesis, pollination, fertilization, and consequently seed formation, lead to the beginning of new plant generation and maintenance of the species. In this series of consecutive events, many external and internal factors are involved, and light and calcium ions play an important role. Since cloning the cDNA encoding P. nil CDPK isoform, we have attempted to determine its biological roles. With this aim in mind,
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Fig. 7. Activity and protein level of Pharbitis nil PnCDPK1 in parts of fully open flower. (A) In vitro Ca2+ -dependent phosphorylation of histone III-S by soluble protein extract in the presence of 1 mM of CaCl2 or 1 mM EDTA followed by counting of incorporated 32 P in a liquid scintillation counter. (B) In-gel Ca2+ -dependent phosphorylation assay of proteins in the presence of 1 mM of CaCl2 or 1 mM EDTA. Protein extracts were electrophoresed in 10% (w/v) SDS-PAGE containing histone III-S as a substrate. After gel renaturation CDPK activity was detected in the presence of 1 mM of CaCl2 (+) or 1 mM EDTA (−). (C) Protein blot analysis of proteins using anti-soybean CDPK. Bars represent SE.
Fig. 6. PnCDPK activity and protein level during different stages of Pharbitis nil flower development. (A) In vitro Ca2+ -dependent phosphorylation of histone III-S by soluble protein extract in the presence of 1 mM of CaCl2 or 1 mM EDTA followed by counting of incorporated 32 P in a liquid scintillation counter. (B) In-gel Ca2+ dependent phosphorylation assay of proteins in the presence of 1 mM of CaCl2 or (C) 1 mM EDTA. Protein extracts were electrophoresed in 10% (w/v) SDS-PAGE containing histone III-S as a substrate. After gel renaturation CDPK activity was detected in the presence of 1 mM of CaCl2 (+) or 1 mM EDTA (−). (D) Protein blot analysis of proteins using anti-soybean CDPK. Bars represent SE.
we used biochemical, molecular and immunocytochemical techniques to characterize CDPK mRNA level, protein level and kinase activity in various organs and stages of plant growth and development (Jaworski et al., 2010, 2011). We have shown that CDPKs, PnCDPK56, now called PnCDPK1, is the dominant isoform of CDPK in P. nil seedlings, the activity and protein accumulation of which is negatively regulated by light and increased in rapidly proliferating cells and growing tissue, which may suggest its participation in cell proliferation
and differentiation. Moreover, based on differences in PnCDPK1 expression, CDPK level and activity during photoperiodic flower induction, we suggested involvement of this isoform of CDPK in this process (Jaworski et al., 2011). As a consequence of the appearance of the induction factor in cotyledons and its transport to the shoot meristem, a spectrum of irreversible changes occur in the shoot meristem, which further lead to flower formation. Thus, we focused on the elucidation of the role of PnCDPK1 during evocation, differentiation and flower morphogenesis. The P. nil plant is an ideal short-day plant model for the study of the early events in the photoperiodic induction of flowering and flower formation, because just 5-day-old, light-grown seedlings can be induced to flower by exposure to a single 16-h-long night, whereas seedlings cannot be induced to flower under continuous light (LL) or long-day (LD) conditions (Ogawa and King, 1990). Data pointing to a role of Ca2+ in the process of photoperiodic flower induction in cotyledons and leaves (Friedman et al., 1989; Tretyn et al., 1994) have been presented previously. It was proposed that, during the inductive dark period, an increase of intracellular free calcium concentration occurs, whereas the exogenously applied compounds modulating protein and involved in
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calcium homeostasis e.g., calcium channels or calmodulin, had the effect of increasing or reducing the number of flower buds formed (Friedman et al., 1989; Tretyn et al., 1994; Szmidt-Jaworska et al., 2006). Moreover, it was revealed that cotyledons and the shoot meristem after induction had higher cytosolic Ca2+ concentration than other cells (Walczysko et al., 2000). During recent years, great progress has been made in the identification of CDPK genes and biochemical characterization of these kinases as a main calcium sensor. Moreover, some potential substrates for CDPKs have been identified (Reddy and Reddy, 2004; Li et al., 2006). Nevertheless, there has been little progress in the elucidation of their biological functions in growth and development processes, especially during induction and flower formation. One approach to this problem involves the study of cell type-specific and inducible expression profiles. Generally, most CDPK genes are expressed in most of the plant tissues examined. However, some of them display an isoform-specific expression pattern that was not only organ- or tissue-specific, but also dependent on growth conditions (Kawasaki et al., 1993; Estruch et al., 1994; Takezawa et al., 1996; Saijo et al., 1997; Zhang et al., 2005; Chang et al., 2009; Jaworski et al., 2011). Our results suggest a potential role for CDPK in the response of the apical meristem of P. nil to the inductive photoperiod. The CDPK distribution throughout the apical meristem was different in non-induced and induced apexes and the fluorescence intensity was significantly higher in plants after induction. The elevation of CDPK level in the apical meristem of P. nil after photoperiodic flower induction has not been reported thus far. Such changes are no doubt a consequence of changes in cytosolic Ca2+ concentration in cells of the apical meristem. Previous work has shown an elevation of [Ca2+ ]cyt , in the range 100–350 nM, in the apical meristem of Sinapis alba and Chenopodium rubrum plants after photoperiodic flower induction (Havalange, 1989; Walczysko et al., 2000). In S. alba, the increase of [Ca2+ ]cyt in the apical bud begins 18 h after the start of the inductive long night. Particularly in the Sinapis meristem, the level of Ca2+ was found to increase in all cellular compartments, but mainly in the nucleolus, which is related to the increase in the rate of cell proliferation, as the first observed symptom of the flowering inducer reaching the apical meristem and the beginning of flower evocation after photoperiodic flower induction (Havalange, 1989). The author proposed that this increase is related to the intensification of the rate of cell proliferation, which is known to take place in the apical meristem after photoperiodic flower induction. Therefore, there is a correlation between the Ca2+ increase and CDPK level and activity in the apical meristem (Hepler, 2005; Lautner and Fromm, 2010). It is the reason that, in our experiments, PnCDPK1 was presented in the shoot primordium including the apical meristem, leaf primordium and floral buds. Previous data indicate that CDPKs demonstrate a temporal and spatial pattern of expression during flower development, which may point to the importance of this class of enzymes in the physiology of reproduction. In Nicotiana tabacum, NtCDPK1 was mainly expressed in the rapidly proliferating tissues, including the shoot meristem, developing flower buds, lateral root and branch primordia (Lee et al., 2003; Zhang et al., 2005). A large amount of NtCPK4 mRNA was also detected at the stigma surface, vascular and stylar vascular bundle. High expression of NtCPK4 was noted during the early development of the anther as well as in sporogenous cells, the tapetum and anther wall. However, during the process of pollen development, NtCPK4 expression decreased gradually, reaching a low level of mRNA in mature pollen cells (Zhang et al., 2005). The expression of one of the maize CDPK genes is pollen-specific and temporally restricted to both the late stages of pollen development and pollen grain germination and tube elongation (Estruch et al., 1994; Yoon et al., 2006; Ge et al., 2009).
Analysis of particular flower elements revealed a higher transcript level and enzyme activity in vegetative elements of flowers (petals and sepals), than generative ones (pistils, stamens) (Figs. 5 and 7). Such a situation can be caused by the presence of CDPK in other, processes occurring in parallel, such as floral anthocyanin synthesis (accumulation), growth of epidermal hairs, or corolla petal ageing, the vitality of which is very short after flower opening (6 h). However, it should be emphasized that, in some plant species (Anil and Rao, 2000; Llop-Tous et al., 2002; Tsai et al., 2007), no gene expression alteration during the moment of flower element formation has been observed, which may indicate the specificity of the CDPK isoform involved in these processes and/or regulation on the enzymatic level. In summary, the present simultaneous analysis of mRNA, protein expression level, and enzyme activity indicates the associations of this enzyme with flowering stages and flower organ development. Therefore, the following question arises: does the same enzyme phosphorylate single versatile of many different substrates depending on the action of a stimulus? If so, what mechanism for the recognition of a given substrate governs this? Further studies, including differential analysis of potential protein substrates, may reveal the answer.
Acknowledgements We would like to thank Dr. Agnieszka Zienkiewicz (Nicolaus Copernicus University, Torun, Poland) for assistance with fluorescence microscopy. The authors are grateful to Dr. Alice C. Harmon (University of Florida, Gainesville, USA) for generously providing the polyclonal antibodies against the calmodulin-like domain of soybean CDPK.
References Anil VS, Rao SK. Calcium-mediated signaling during sandalwood somatic embryogenesis. Role for exogenous calcium as second messenger. Plant Physiol 2000;123:1301–11. Asano T, Kunieda N, Omura Y, Ibe H, Kawasaki T, Takano M, et al. Rice SPK a calmodulin-like domain protein kinase, is required for storage product accumulation during seed development: phosphorylation of sucrose synthase is a possible factor. Plant Cell 2002;14:619–28. Barker LDP, Templeton MD, Ferguson IB. A 67-kDa plasma-membrane-bound Ca2+ -stimulate protein kinase active in sink tissue of higher plants. Planta 1998;205:197–204. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 1976;72:248–54. Chang WJ, Su HS, Li WJ, Zhang ZL. Expression profiling of a novel calcium-dependent protein kinase gene LeCPK2, from tomato (Solanum lycopersicum) under heat and pathogen-related hormones. Biosci Biotechnol Biochem 2009;73:2427–31. Cheng SH, Willmann MR, Chen HC, Sheen J. Calcium signaling through protein kinases. The Arabidopsis calcium-dependent protein kinase gene family. Plant Physiol 2002;129:469–85. Ellard-Ivey M, Hopkins RB, White TJ, Lomax TL. Cloning, expression and N-terminal myristoylation of CpCPK1, a calcium-dependent protein kinase from zucchini (Cucurbita pepo L.). Plant Mol Biol 1999;39:199–208. Estruch JJ, Kadwell S, Merlin E, Crossland L. Cloning and characterization of maize pollen-specific calcium-dependent calmodulin-independent protein kinase. Proc Natl Acad Sci USA 1994;91:8837–41. Frattini M, Morello L, Breviario D. Rice calcium-dependent protein kinase isoforms OsCDPK2 and OsCDPK11 show different response to light and different expression patterns during seed development. Plant Mol Biol 1999;41:753–64. Friedman H, Goldschmidt EE, Halevy AH. Involvement of calcium in the photoperiodic flower induction process of Pharbitis nil. Plant Physiol 1989;89:530–4. Ge LL, Xie CT, Tian HQ, Russell SD. Distribution of calcium in the stigma and style of tobacco during pollen germination and the tube elongation. Sex Plant Reprod 2009;22:87–96. Giammaria V, Grandellis C, Bachmann S, Gargantini PR, Feingold SE, Bryan G, et al. StCDPK2 expression and activity reveal a highly responsive potato calciumdependent protein kinase involved in light signalling. Planta 2010;233:593–609. Harmon AC, Gribskov M, Harper JF. CDPKs – a kinase for every Ca2+ signal? Trends Plant Sci 2000;5:154–9. Harper JF, Harmon A. Plants, symbiosis and parasites: a calcium signalling connection. Nat Rev Mol Cell Biol 2005;6:555–66.
K. Jaworski et al. / Journal of Plant Physiology 169 (2012) 1578–1585 Havalange A. Levels and ultrastructural localization of calcium in Sinapis alba during the floral transition. Plant Cell Physiol 1989;30:351–8. Hepler PK. Calcium: a central regulator of plant growth and development. Plant Cell 2005;17:2142–55. Ishida S, Yuasa T, Nakata M, Takahashi Y. A tobacco calcium-dependent protein kinase CDPK1, regulates the transcription factor REPRESSION OF SHOOT GROWTH in response to gibberellins. Plant Cell 2008a;20: 3273–88. Ishida S, Yuasa T, Nakata M, Takahashic Y. A tobacco calcium-dependent protein kinase CDPK1, regulates the transcription factor REPRESSION OF SHOOT GROWTH in response to gibberellins. Plant Cell 2008b;20: 3273–88. Ito T, Nakata M, Fukuzawa J, Ishida S, Takahashi Y. Alteration of substrate specificity: the variable N-terminal domain of tobacco Ca2+ -dependent protein kinase is important for substrate recognition. Plant Cell 2010;22:1592–604. Ivashuta S, Liu J, Liu J, Lohar D, Haridas S, Bucciarelli B, et al. RNA interference identifies a calcium-dependent protein kinase involved in Medicago truncatula root development. Plant Cell 2005;17:2911–21. Jain M, Pathak BP, Harmon AC, Tillman BL, Gallo M. Calcium dependent protein kinase (CDPK) expression during fruit development in cultivated peanut (Arachis hypogaea) under Ca2+ -sufficient and -deficient growth regimens. J Plant Physiol 2011;168:2272–7. Jaworski K, Pawełek A, Szmidt-Jaworska A, Kopcewicz J. Expression of calciumdependent protein kinase gene (PnCDPK1) is affected by various light conditions in Pharbitis nil seedlings. J Plant Growth Regul 2010;29:316–27. Jaworski K, Szmidt-Jaworska A, Kopcewicz J. Two calcium dependent protein kinases are differently regulated by light and have different activity patterns during seedling growth in Pharbitis nil. Plant Growth Regul 2011;65:369–79. Jaworski K, Szmidt-Jaworska A, Tretyn A, Kopcewicz J. Biochemical evidences for a calcium-dependent protein kinase in Pharbitis nil and its involvement in photoperiodic flower induction. Phytochemistry 2003;62:1047–55. Kaczorowski KA, Quail PH. Arabidopsis PSEUDO-RESPONSE REGULATOR7 is a signaling intermediate in phytochrome-regulated seedling deetiolation and phasing of the circadian clock. Plant Cell 2005;15:2654–65. Kamiyoshihara Y, Iwata M, Fukaya T, Tatsuki M, Mori H. Turnover of LeACS2, a wound-inducible 1-aminocyclopropane-1-carboxylic acid synthase in tomato, is regulated by phosphorylation/dephosphorylation. Plant J 2010;64: 140–50. Kawasaki T, Hayashida N, Baba T, Shinozaki K, Shimada H. The gene encoding a calcium-dependent protein kinase located near the sbe1 gene encoding starch branching enzyme I is specifically expressed in developing rice seeds. Gene 1993;129:183–9. ´ G. Structure and functions of plant calcium-dependent Klimecka M, Muszynska protein kinases. Acta Biochim Pol 2007;54:219–33. Kobayashi M, Ohura I, Kawakita K, Yokota N, Fujiwara M, Shimamoto K, et al. Calcium-dependent protein kinases regulate the production of reactive oxygen species by potato NADPH oxidase. Plant Cell 2007;19:1065–80. Lautner S, Fromm J. Calcium-dependent physiological processes in trees. Plant Biol 2010;12:268–74. Lee SS, Cho HS, Yoon GM, Ahn JW, Kim HH, Pai HS. Interaction of NtCDPK1 calciumdependent protein kinase with NtRpn regulatory subunit of the 26S proteasome in Nicotiana tabacum. Plant J 2003;33:825–40.
1585
Li RJ, Hua W, Lu YT. Arabidopsis cytosolic glutamine synthetase AtGLN1 is a potential substrate of AtCRK3 involved in leaf senescence. Biochem Biophys Res Commun 2006;342:119–26. Llop-Tous I, Dominguez-Puigjaner E, Vendrell M. Characterization of a strawberry cDNA clone homologous to calcium-dependent protein kinases that is expressed during fruit ripening and affected by low temperature. J Exp Bot 2002;53:2283–5. Mori I, Murata Y, Yang Y, Munemasa S, Wang Y, Andreoli S, et al. CDPKs CPK6 and CPK3 function in ABA regulation of guard cell S-type anion- and Ca2+ -permeable channels and stomatal closure. PLoS Biol 2006;4:e327. Ogawa Y, King RW. The inhibition of flowering by non-induced cotyledons of Pharbitis nil. Plant Cell Physiol 1990;31:129–35. Patharkar OR, Cushman JC. A stress-induced calcium-dependent protein kinase from Mesynbryanthemum crystallinum phosphorylates a two-component pseudoresponse regulator. Plant J 2000;24:679–91. Poovaiah BW, Reddy AS. Calcium and signal transduction in plants. CRC Crit Rev Plant Sci 1993;12:185–211. Reddy VS, Reddy ASN. Proteomics of calcium-signaling components in plants. Phytochemistry 2004;65:1745–76. Roig-Villanova I, Bou J, Sorin C, Devlin PF, Martinez-Garcia JF. Identification of primary target genes of phytochrome signaling early transcriptional control during shade avoidance responses in Arabidopsis. Plant Physiol 2006;141:85–96. Saijo Y, Hata S, Sheen J, Izui K. cDNA cloning and prokaryotic expression of maize calcium-dependent protein kinases. Biochim Biophys Acta 1997;1350:109–14. Sharma VK, Jain PK, Malik MK, Maheshwari SC, Khurana JP. Light- and calciummodulated phosphorylation of proteins from wheat seedlings. Phytochemistry 1997;44:781–6. Szmidt-Jaworska A, Jaworski K, Kopcewicz J. The involvement of cyclic ADPR in photoperiodic flower induction of Pharbitis nil. J Plant Growth Regul 2006;25:233–44. Takezawa D, Patil S, Bhatia A, Poovaiah BW. Calcium-dependent protein kinases genes in corn roots. J Plant Physiol 1996;149:329–35. Tretyn A, Czaplewska J, Cymerski M, Kopcewicz J, Kendrick RE. The mechanism of calcium action on flower induction in Pharbitis nil. J Plant Physiol 1994;144:562–8. Tsai TM, Chen YR, Kao TW, Tsay WS, Wu CP, Huang DD, et al. PaCDPK1, a gene encoding calcium-dependent protein kinase from orchid, Phalaenopsis amabilis, is induced by cold, wounding, and pathogen challenge. Plant Cell Rep 2007;26:1899–908. Ullanat R, Javabaskaran C. Distinct light-, cytokinin and tissue-specific regulation of calcium dependent protein kinase gene expression in cucumber (Cucumber sativus). Plant Sci 2002;162:153–63. Walczysko P, Wagner E, Albrechtová JTP. Use of co-loaded Fluo-3 and Fura Red fluorescent indicators for studying the cytosolic Ca2+ concentrations distribution in living plant tissue. Cell Calcium 2000;28:23–32. Yoon GM, Dowd PE, Gilroy S, McCubbin AG. Calcium-dependent protein kinase isoforms in Petunia have distinct functions in pollen tube growth, including regulating polarity. Plant Cell 2006;18:867–78. Yu J, Tian YS, Peng RH, Xiong AS, Zhu B, Jin XF, et al. AtCPK6, a functionally redundant and positive regulator involved in salt/drought stress tolerance in Arabidopsis. Planta 2010;231:1251–60. Zhang T, Wang Q, Chen X, Tian C, Wang X, Xing T, et al. Cloning and biochemical properties of CDPK gene OsCDPK14 from rice. J Plant Physiol 2005;162:1149–59.