Molecular Plant Advance Access published October 11, 2013 Molecular Plant
RESEARCH ARTICLE
A Calcium-Dependent Protein Kinase Interacts with and Activates A Calcium Channel to Regulate Pollen Tube Growth Liming Zhoua,b,2, Wenzhi Lana,c,2, Yuanqing Jianga, Wei Fangb, and Sheng Luana,1
ABSTRACT Calcium, as a ubiquitous second messenger, plays essential roles in tip-growing cells, such as animal neurons, plant pollen tubes, and root hairs. However, little is known concerning the regulatory mechanisms that code and decode Ca2+ signals in plants. The evidence presented here indicates that a calcium-dependent protein kinase, CPK32, controls polar growth of pollen tubes. Overexpression of CPK32 disrupted the polar growth along with excessive Ca2+ accumulation in the tip. A search of downstream effector molecules for CPK32 led to identification of a cyclic nucleotidegated channel, CNGC18, as an interacting partner for CPK32. Co-expression of CPK32 and CNGC18 resulted in activation of CNGC18 in Xenopus oocytes where expression of CNGC18 alone did not exhibit significant calcium channel activity. Overexpression of CNGC18 produced a growth arrest phenotype coupled with accumulation of calcium in the tip, similar to that induced by CPK32 overexpression. Co-expression of CPK32 and CNGC18 had a synergistic effect leading to more severe depolarization of pollen tube growth. These results provide a potential feed-forward mechanism in which calcium-activated CPK32 activates CNGC18, further promoting calcium entry during the elevation phase of Ca2+ oscillations in the polar growth of pollen tubes. Key words: pollen tubes; calcium-dependent protein kinase; cyclic nucleotide-gated channel; Ca2+ oscillations.
Introduction Pollen tube growth is a model system for studying cell polarity control. As pollen germinates in the culture, pollen extrudes a tube that elongates by extreme polar growth, resulting in a uniformly shaped cylindrical cell (Zheng and Yang, 2000; Krichevsky et al., 2007; Yang, 2008; Johnson, 2012). It is well known that Ca2+ serves as a key element in the control of polar growth in pollen tubes (Miller et al., 1992; Pierson et al., 1994; Malho and Trewavas, 1996; Hepler, 1997; Messerli and Robinson, 1997). A steep tip-focused Ca2+ gradient has been detected at the tip of elongating pollen tubes (Rathore et al., 1991; Pierson et al., 1994; Hepler, 1997; Hepler et al., 2011). Perturbation of tip-focused Ca2+ gradient results in a reversible cessation of pollen tube tip growth, whereas elevation of internal Ca2+ levels induces bending of the growth axis toward the zone of higher cytoplasmic calcium concentration ([Ca2+]cyt) (Malho et al., 1994; Bibikova et al., 1997; Hepler, 1997; Li et al., 1999; Cheung and Wu, 2008). In addition, excessive Ca2+ has been found to promote F-actin disassembly, perhaps via Ca2+ -dependent actin-binding proteins (Kovar et al., 2000; Yokota et al., 2000; Huang et al., 2004; Gu et al., 2005).
As a major calcium sensor in plants, the family of calciumdependent protein kinases (CPKs) appears to be candidates for calcium effectors involved in pollen tube growth regulation (Price et al., 2003; Harper et al., 2004; Ludwig et al., 2004; Harper and Harmon, 2005; Wu et al., 2010; Jamin and Yang, 2011), although other calcium sensors may also be involved as shown by a recent study (Mähs et al., 2013). Earlier studies have shown that two CPKs (PiCPK1, PiCPK2) from Petunia inflate may regulate pollen tube growth, as PiCPK1 overexpression increased [Ca2+]cyt at the swelling tip of pollen tubes (Yoon et al., 2006). The Arabidopsis genome encodes 34 CPKs (Harmon et al., 2000). Through a genome-wide survey for the function of CPKs in pollen tubes, our previous study demonstrated that CPK32 overexpression causes severe 1 To whom correspondence should be addressed. E-mail sluan@berkeley. edu, tel. 1-510-6431725. 2
These authors contributed equally to this work.
© The Author 2013. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPB and IPPE, SIBS, CAS. doi:10.1093/mp/sst125 Received 3 June 2013; accepted 24 June 2013
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a Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA b College of Life Sciences, Hebei United University, Tangshan, Hebei 063000, China c NJU–NJFU Joint Institute for Plant Molecular Biology, School of Life Sciences, Nanjing University, Nanjing 210093, China
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tube tip (Yoon et al., 2006). We speculated that Arabidopsis CPK32, as a calcium sensor, may have a similar effect on the level of cytosolic Ca2+ at the tip of the growing tubes. To test this possibility, we performed ratiometric Ca2+ imaging in wildtype (WT) pollen tubes or the tubes overexpressing CPK32–GFP, respectively. As shown in Figure 1, comparing with the normal tip-focused Ca2+ gradient in a WT pollen tube, CPK32 overexpression tube displayed an altered Ca2+ gradient that extended throughout the swollen apex of the tube, indicating that CPK32 may be involved in maintaining Ca2+ homeostasis.
Identification of Calcium Channel CNGC18 as a Potential Target of CPK32 As noted above, CPK32 overexpression induces Ca2+ accumulation in the tip of pollen tubes. Concerning the transporters that control Ca2+ fluxes, evidence supports that CNGCs may serve as non-specific cation channels carrying an inward Ca2+ current across the plasma membrane (PM) (Kaupp and Seifert, 2002; Talke et al., 2003; Dodd et al., 2010). We hypothesized that CPK32 may interact with CNGCs to control tip-focused Ca2+ gradient in the pollen tube. To identify potential downstream targets in the CNGC family, we conducted a yeast twohybrid screen using a constitutively active mutant of CPK32 (CACPK32) as the bait and C-terminal soluble domain of CNGC members in the prey vector. As shown in Figure 2A, CACPK32 specifically interacted with CNGC18 that was also preferentially expressed in mature pollen grains on the basis of microarray data sets at the Genevestigator database. Furthermore,
RESULTS AND DISCUSSION CPK32 Regulates Calcium Signaling in Pollen Tube Tip Growth Through a genome-wide functional survey of CPK family members in pollen tubes, our previous study demonstrated that CPK32 overexpression induced severe depolarization of pollen tube growth, resulting in a short tube with the swelling tip (Zhou et al., 2009). In addition, earlier studies showed that overexpression of Petunia PiCPK1 also causes a severe loss of growth polarity associated with elevated cytosolic Ca2+ in the bulging
Figure 1. CPK32 Overexpression Induces Excessive Ca2+ Accumulation in the Tip of Pollen Tubes. Ratiometric Ca2+ imaging of pollen tubes was performed using the calcium-sensitive dye Indo-1-dextran. Calcium concentrations have been pseudo-color-coded according to the inset scale.
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depolarization in pollen tube growth, leading to formation of a bulge at the tip (Zhou et al., 2009). Disruption of CPK17 and CPK34 in Arabidopsis leads to a significantly reduced growth rate of pollen tubes as compared to wild-type. Most of the pollen tubes fail to target and fertilize ovules (Myers et al., 2009). Channel-mediated Ca2+ influx is largely responsible for the formation of tip-focused Ca2+ gradient during pollen tube growth (Etter et al., 1994; Malho et al., 1995; HoldawayClarke et al., 1997; Yang, 2008; Jammes et al., 2011). Cyclic nucleotide-gated channels (CNGCs) are non-selected cation channels and belong to the superfamily of voltage-gated ion channels (Ma and Berkowitz, 2011; Hedrich, 2012). In plants, CNGCs possess conserved transmembrane domains (S1-S6), predicted C-terminal cyclic nucleotide-binding domain (CNB), and calmodulin-binding domain (CaMB) (Demidchik et al., 2002; Talke et al., 2003). The Arabidopsis genome encodes 20 putative CNGC genes, cataloged into five subfamilies that potentially play distinct functions in specific tissues and organelles (Talke et al., 2003; Ma and Berkowitz, 2011; Hedrich, 2012). Previous studies have demonstrated that CNGCs function as non-specific cation channels that can be activated by cyclic nucleotides and blocked by Ca2+/calmodulin (Hua et al., 2003; Talke et al., 2003; Ali et al., 2006, 2007). For example, AtCNGC2 is a K+ and Ca2+-permeable channel that could be inhibited by CaM (Leng et al., 2002; Ali et al., 2007). AtCNGC4 plays a role in K+ and Na+ transport (Balague et al., 2003). Up to date, several CNGCs characterized from Arabidopsis, such as AtCNGC2, AtCNGC4, AtCNGC11, and AtCNGC12, function in pathogen and abiotic stress responses (Clough et al., 2000; Balague et al., 2003; Ali et al., 2007). Particularly relevant to this study, a loss-of-function mutant for AtCNGC18 show defective phenotypes in pollen tube growth and fertilization (Chang et al., 2007; Frietsch et al., 2007). AtCNGC16 has been identified as an important regulator for pollen fertility under conditions of heat stress and drought (Tunc-Ozdemir et al., 2013). In this paper, we show that CPK32 appears to be a crucial element for calcium signaling in pollen tubes growth. Subsequently, CNGC18 was identified as a downstream target for CPK32. CPK32 interacts with and activates CNGC18 channel activity, supporting a model in which CPK32 physically interacts with Ca2+-permeable CNGC18 to couple Ca2+ signaling with tip growth of pollen tubes.
Zhou et al. • CPK32–CNGC18 Interaction and Pollen Tube Growth
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Figure 2. CPK32 Interacts with CNGC18. (A, B) Yeast two-hybrid assay of CACPK32 (or CACPK34) with C-terminal soluble domain of CNGC members. The pGADT7 and pGBKT7 vectors were used as negative control. (C) Overexpression phenotype and subcellular localization for CNGC18. (D) Overexpression of CNGC18 disturbs tip-focused Ca2+ gradient visualized by ratiometric Ca2+ imaging. (E, F) Quantitative analysis of pollen tube phenotypes induced by co-expression of LAT52::CNGC18 with LAT52::GFP. Asterisks represent a significant difference from LAT52::GFP control (p < 0.05; t-test); error bars indicate SD. Data were collected from 50–60 tubes per experiment.
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co-expressing CNGC18 with CPK32 through particle bombardment. As described above, CNGC18 overexpression induced a loss of polarity in pollen tube growth, leading to sock-like phenotype. We reasoned that, if CPK32 acts as a positive regulator of CNGC18, CNGC18-induced phenotype should be further enhanced by co-expressing CPK32. In contrast, CNGC18-induced phenotype should be reversed by co-expressing CPK32 if CPK32 inhibits CNGC18 activity. If CPK32 does not regulate CNGC18 activity, co-expressing CPK32 should have no effect on CNGC18-induced defect in pollen tube. As shown in Figure 4, the co-expression of CPK32 and CNGC8 caused more severe loss of growth polarity than that induced by either of them, suggesting that CPK32 acts as a CNGC18 activator to regulate pollen tube tip growth.
CPK32 Activates CNGC18-Mediated Ca2+ Influx in Xenopus Oocytes To further confirm the relationship between CNGC18 and CPK32, we next co-expressed CNGC18 with CACPK32 or CACPK34 in the Xenopus oocytes and measured CNGC18 activity. Compared with the water-injected control, the oocytes injected with CNGC18 cRNA produced a low, but detectable, current in the presence of 10 mM extracellular Ca2+ (Figure 5A and 5B). The CNGC18 channel currents were enhanced significantly when CNGC18 was co-expressed with CACPK32 (Figure 5A and 5B). By contrast, co-expression with
Co-Expression of CPK32 and CNGC18 Causes a Synergistic Effect on Pollen Tube Growth To assess whether CPK32 functionally interacts with CNGC18, we next analyzed the phenotypes of pollen tubes
Figure 3. FRET Analysis of CACPK32 Interaction with CNGC18. Tobacco pollen tubes co-expressing CNGC18–YFP and CACPK32–CFP or CACPK34–CFP were examined with FRET procedure as described in the ‘Methods’ section. The intensity of FRET signals is displayed in pseudocolor mode (red, highest signal).
Figure 4. Phenotypes Analysis of Pollen Tubes Co-Expressing CNGC18 with CPK32–GFP. (A) Representative confocal images of pollen tubes transiently expressing CPK32–GFP, CNGC18–GFP, or co-expressing CNGC18 with CPK32–GFP. (B) Quantitative analysis of the co-expression phenotype. Data were collected from 50–60 tubes per experiment. Error bars indicate SD.
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previous study also showed that CNGC18 is essential for fertility and pollen tube growth (Frietsch et al., 2007). We next investigated the subcellular localization and overexpression phenotype for CNGC18. YFP-tagged CNGC18 exhibited PM localization at the pollen tube tip (Figure 2C). Compared with YFP control vector, CNGC18 overexpression produced shorter (reduction of the tube length from 531.6 to 283.4 µm) but wider (increase of the tube width from 8.6 to 12.4 µm) pollen tubes (Figure 2E and 2F). To further confirm the interaction between CPK32 and CNGC18 in vivo, fluorescence resonance energy transfer (FRET) analysis was performed by co-expressing CNGC18– YFP with CFP-tagged CACPK32 in tobacco pollen tubes (Figure 3). Co-expression of CNGC18–YFP and CACPK34–CFP was used as negative control, as CNGC18 did not interact with CACPK34 (Figure 2B). FRET signals between CNGC18–YFP and CACPK32–CFP were primarily detected at the apical PM where CNGC18–YFP is localized. By contrast, FRET signals between CNGC18–YFP and CACPK34–CFP were not detected (Figure 3). These results suggest that the interaction of CNGC18 with CPK32 primarily occurs at the apical region of the PM. To evaluate whether CNGC18 functions as a Ca2+-permeable channel to mediate Ca2+ influx across the PM, ratiometric Ca2+ imaging was conducted to measure cytosolic Ca2+ concentration in transformed pollen tubes. Overexpression of CNGC18 increased Ca2+ accumulation in the tip (Figure 2D), as seen in the tubes overexpressing CPK32 (Figure 1). These findings support a model that CNGC18 may be regulated by CPK32 to mediate Ca2+ transport across the PM in response to Ca2+ oscillation.
Zhou et al. • CPK32–CNGC18 Interaction and Pollen Tube Growth
CACPK34 that does not interact with CNGC18 (Figure 2B) had no significant effect on the activity of CNGC18 (Figure 5B). These results indicate that CACPK32 specifically activates the activity of CNGC18 channel expressed in the oocytes.
Concluding Remarks Our results in this report provide new insights into the mechanism underlying Ca2+ regulation in oscillatory tip growth of pollen tubes. Previous studies and evidence presented here show that CPK32 overexpression caused severe loss of growth polarity along with elevated Ca2+ at the tube tip (Figure 1) (Zhou et al., 2009), similar to that caused by overexpression of PM-localized Petunia PiCPK1 (Yoon et al., 2006). This implicates CPK32 as a critical component of a signaling pathway regulating Ca2+ homeostasis. A number of studies indicate that tip-focused Ca2+ gradient is tightly associated with polarized growth of pollen tubes, and tip-localized influx of extracellular Ca2+ is required for this process (Rathore et al., 1991; Pierson et al., 1994; Malho and Trewavas, 1996; Pierson et al., 1996; Holdaway-Clarke et al., 1997; Holdaway-Clarke and Hepler, 2003; Guan et al., 2013). In plants and animals, conventional CNGCs function as non-specific cation channels that are known to be permeable to calcium (Kaupp and Seifert,
2002). Our results in this paper showed that CACPK32 specifically interacts with CNGC18 that is preferentially localized to the PM of pollen tubes, including apical regions where the tip-focused calcium influxes occur (Pierson et al., 1996; Qin and Yang, 2011; Kroeger and Geitmann, 2012). In addition, CNGC18 overexpression produced a short, bulbous, and growth arrest phenotype (Figure 2C–2F), along with accumulation of calcium in the tip. In previous studies, two cngc18-null mutations display tip-growth defects of pollen tubes, resulting in short and thin tubes with non-directional growth (Frietsch et al., 2007). These findings suggest that CNGC18 is one of the calcium-permeable channels responsible for the regulation of the tip-focused cytosolic Ca2+ gradient. Based on available microarray databases, both CPK32 and CNGC18 are preferentially expressed in mature pollen. Their functional interaction was further confirmed by FRET and electrophysiological assays (Figures 3 and 5). Furthermore, CPK32 and CNGC18 display similar apical Ca2+ accumulation in pollen tube when overexpressed (Figures 1 and 2). These results support a possible model that CPK32 and CNGC18 appear to function in the same pathway regulating the tip growth of pollen tubes. Activation of a calcium channel by a calcium-dependent protein kinase provides a positive feedback mechanism by which the calcium level is further increased by the kinase and thus rapidly reaches the peak level. A negative feedback mechanism, currently unknown, may be present to down-regulate the calcium channel and cellular calcium levels, thereby producing the oscillations during pollen tube growth. It is well known that a steep tip-focused Ca2+ gradient is required for polarized cell growth in a number of systems including fungal hyphae, pollen tubes, and root hairs (Yang, 2008; Dodd et al., 2010; Hepler et al., 2011; Kroeger and Geitmann, 2012). It will be of interest to determine whether similar calciumdependent signaling paradigm identified in pollen tubes can be extended to other systems.
METHODS Plant Materials and Growth Conditions Tobacco (Nicotiana tabacum) plants were grown in growth chambers at 22°C under a light regime of 12-h darkness and 12-h light.
Construction of Plasmids The full-length cDNAs for AtCPKs were obtained from the Arabidopsis Biological Resource Center. The coding region of each CPK was amplified by PCR using corresponding primers. PCR-amplified DNA fragments were cloned into pGEM-T Easy vector, and sequenced prior to cloning into PUCLNGFP2 vectors under the LAT52 promoter for expression in pollen or pACT2 for yeast two-hybrid, respectively. For those genes subcloned to PUCLNGFP2 vector, to generate pLAT52::X constructs, the GFP cDNA sequence was transferred from
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Figure 5. CPK32 Activates CNGC18 in the Xenopus Oocytes. (A) Typical current traces recorded from the oocytes injected with water (Control), CACPK32, CNGC18, or CNGC18 plus CACPK32 (CNGC18+CACPK32). The oocytes were perfused with bath solution containing 10 mM CaCl2, 0.1 mM dibutyryl-cAMP, 185 mM mannitol, and 10 mM Mes-Tris (pH 7.4). Dotted lines represent the zero current level. (B) Effects of CACPK32 and CACPK34 on the currents generated by CNGC18 at –150 mV. The pooled current values were at 1.8 s of each voltage-clamp episode and presented as means ± SE, with n = 5 in each case.
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pLAT52::X–GFP by BamHI and BglII digestion, followed by self-ligation. The CACPKs and DNCPKs were constructed using the methods described previously (Harper et al., 1994; McCubbin et al., 2004). CACPKs were produced by truncating each CPKs at the junction between the auto-inhibitory and kinase domains (322N in CPK32). The mutant constructs were cloned into pGADT7 vectors as described above.
Measurements of Cytoplasmic Ca2+ Concentration Pollen tubes were injected with the fluorescent Ca2+ sensitive dye (Indo-1-dextran), and Ca2+ concentration in pollen tubes was visualized by ratio imaging under a confocal laser scanning microscope (Zeiss LSM 510 META) as described previously (Bibikova et al., 1997).
Electrophysiological Procedures Transient Gene Expression in Tobacco Pollen Tubes
Microscopy For analysis of pollen tube morphology, the images of transformed pollen tubes were recorded through a Cooled CCD Camera (model DP70; OLYMPUS) attached on an inverted microscope (model BX51; OLYMPUS). The maximal diameter of the tip region and the length of the pollen tube were measured by Zeiss LSM image browser (Version 3.2). Three independent experiments were performed. All the data were subjected to a statistic Student’s t-test. To reveal subcellular localization, transformed pollen tubes were observed under a confocal laser scanning microscope (Zeiss LSM 510 META). Confocal images were analyzed by Zeiss LSM image browser (Version 3.2) and processed within Photoshop CS3.
Yeast Two-Hybrid Assays The bait and prey vectors were transformed into yeast strain AH109 via the lithium acetate transformation procedure as described in the yeast protocols handbook (Clontech Laboratories, Inc.). A nylon filter assay was performed to measure the β-galactosidase activity. Each assay was performed in three independent experiments. One of the representative assays was shown.
FRET Assay FRET Assay was performed using a confocal laser scanning microscope (Zeiss LSM 510 META) according to a previously described method (Gu et al., 2005). Scan images were sequentially collected according to relevant channel settings below: For the CFP channel, excitation and emission were set at 442 and 450–490 nm; for the YFP channel, excitation at 514 nm, emission at 525–600 nm; for the FRET channel, excitation 442 nm, emission 560–638 nm. Corrected FRET signal was normalized with the acceptor amount (FRET efficiency, % of YFP emission with 10% 514-nm laser excitation). FRET efficiency was measured at three different regions of PM where CPK32– CFP and CNGC18–YFP are co-localized and in cytosolic domains where individual donors and acceptors were enriched.
The concentration of the capped cRNA prepared by the mMESSAGE mMACHINE T7 RNA transcription Kit was determined at A260/A280. The total volume of cRNA injected into freshly isolated Xenopus oocytes was 23 nl and the final concentration was 125 mg l–1. The oocytes 40–50 h after injection were used for two-electrode voltage-clamp experiments. The pipette solution contained 3 M KCl. To record the potential Ca2+ current, the perfusion solution contained 10 mM CaCl2, 0.1 mM lipophilic analog of cAMP (dibutyrylcAMP), 185 mM mannitol, and 10 mM Mes-Tris (pH 7.4). The current was record by hyperpolarized pulses of a 0.2-s prepulse at −40 mV followed by voltage steps of 60 to −150 mV (in 15-mV decrements, 1.8-s duration) followed by a 1.5-s deactivation at 0 mV. Histograms were generated from the pooled currents reached after 1.8 s of each voltage-clamp episode. Data are presented as representative recordings or as mean ± S.E. of n observations with at least five repetitions, in which n is the number of samples. Statistical comparisons were made using Student’s paired as appropriate, and differences were considered to be significant at p < 0.05.
FUNDING This work was supported by a grant from the National Science Foundation (to S.L.) and Tangshan Plan of Science and Technology Research & Development (12120202a, 13130237z, and 12120201a).
Acknowledgments We thank the Arabidopsis Biological Resource Center (ABRC) for providing CPKs cDNA, Yuanqing Jiang for pGADT7– CNGCs for yeast two-hybrid assay, and Fang Chang for LAT52::CNGC18–YFP in transient expression. No conflict of interest declared.
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Zhou et al. • CPK32–CNGC18 Interaction and Pollen Tube Growth
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SUMMARY This study identified a molecular link between calciumdependent protein kinase (CPK32) and Ca2+-permeable channel (CNGC18) in control of pollen tube tip growth, implicating a feed-forward mechanism in the formation of the tipfocused Ca2+ gradient through the CPK32–CNGC18 signaling pathway.
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