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Thursday, 7 July 2016
The Calcineurin B-Like Ca2+ Sensors CBL1 and CBL9 Function in Pollen Germination and Pollen Tube Growth in Arabidopsis
Published Date
July 2013, Vol.6(4):1149–1162, doi:10.1093/mp/sst095
Open Archive, Elsevier user license
Research Article
Title
The Calcineurin B-Like Ca2+ Sensors CBL1 and CBL9 Function in Pollen Germination and Pollen Tube Growth in Arabidopsis
Author
Anette Mähs a
Leonie Steinhorst a
Jian-Pu Han b
Li-Ke Shen b
Yi Wang b
Jörg Kudla a,b,,
aInstitut für Biologie und Biotechnologie der Pflanzen, Universität Münster, Schlossplatz 4, 48149 Münster, Germany
bState Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, National Plant Gene Research Center (Beijing), China Agricultural University, Beijing 100193, China
Received 1 February 2013. Accepted 22 May 2013. Available online 9 December 2014.
ABSTRACT
Ca2+ has been established as an important second messenger regulating pollen germination and tube growth. However, to date, only a few signaling components have been identified to decode and relay Ca2+ signals in growing pollen tubes. Here, we report a function for the calcineurin B-like (CBL) Ca2+ sensor proteins CBL1 and CBL9 from Arabidopsis in pollen germination and tube growth. Both proteins are expressed in mature pollen and pollen tubes and impair pollen tube growth and morphology if transiently overexpressed in tobacco pollen. The induction of these phenotypes requires efficient plasma membrane targeting of CBL1 and is independent of Ca2+ binding to the fourth EF-hand of CBL1. Overexpression of CBL1or its closest homolog CBL9 in Arabidopsis renders pollen germination and tube growth hypersensitive towards high external K+ concentrations while disruption of CBL1 and CBL9 reduces pollen tube growth under low K+ conditions. Together, our data identify a crucial function for CBL1 and CBL9 in pollen germination and tube growth and suggest a model in which both proteins act at the plasma membrane through regulation of K+ homeostasis.
Key words
calcium
CBL
pollen tube growth
potassium
INTRODUCTION
Sexual reproduction of flowering plants requires polar outgrowth of a particular region in the vegetative cell of the male gametophyte, thereby gradually forming the pollen tube. By exclusively expanding its apex, this pollen tube grows towards the ovule to deliver the sperm cells for fertilization of the egg apparatus (Taylor and Hepler, 1997; Qin and Yang, 2011). This highly polarized process is tightly regulated and relies on a multitude of signaling components of which Ca2+ has been established as being of central importance (Jaffe et al., 1975; Obermeyer and Weisenseel, 1991; Michard et al., 2009). Ca2+ forms a concentration gradient across the length of the pollen tube, with its concentration being highest at the tip and gradually decreasing towards the shank (Jaffe et al., 1975; Hepler et al., 2012). It has been demonstrated that perturbation of this Ca2+ gradient severely affects pollen tube tip growth (Obermeyer and Weisenseel, 1991; Rathore et al., 1991; Pierson et al., 1994). Moreover, changes of the focal point of this gradient result in readjustment of the growth axis (Malhó and Trewavas, 1996; Steinhorst and Kudla, 2012). In many species, this tip-focused Ca2+gradient can exhibit oscillatory characteristics that have been associated with periodic changes in pollen tube growth rates (Pierson et al., 1996; Feijó et al., 2001; Hepler et al., 2012). In recent years, genes that code for Ca2+ permeable channels or pumps have been identified as being expressed in pollen. Mutational analyses of the Ca2+ ATPase (ACA) 9, cyclic nucleotide gated channel (CNGC) 18, or glutamate receptor-like (GLR) 1.2 and 3.7 revealed their crucial function in regulating pollen tube growth rates, tube morphology, and interaction with the female gametophyte (Schiøtt et al., 2004; Frietsch et al., 2007; Michard et al., 2011). These findings underlined the importance of the proper formation of Ca2+ signals for these processes.
Actin polymerization and vesicle trafficking that are essential for maintenance of polarized tip growth are regulated by the tip-focused Ca2+ gradient (Li et al., 1999; Gu et al., 2005). A central integrator of this regulatory mechanism is the small GTPase rho of plants (ROP) 1 which upon interaction with its downstream effector rop-interactive CRIB motif-containing protein (RIC) 3 facilitates Ca2+ influx across the plasma membrane of the pollen tube apex. This provokes F-actin disassembly and vesicle fusion (Gu et al., 2005). Conversely, ROP1 enhances actin polymerization through interaction with a second effector protein RIC4, which results in reduced vesicle fusion (Gu et al., 2005). The regulation of F-actin polymerization by ROP1 is mediated by actin binding proteins (ABPs) which bind F-actin at low cytosolic Ca2+concentrations ([Ca2+]cyt) and prevent its disassembly. Conversely, at high [Ca2+]cyt, this mechanism is blocked (Wang et al., 2008a). These regulatory circuits add an additional level of complexity to the properties of Ca2+ in regulating pollen tube growth.
The presence of a Ca2+ gradient and defined fluctuations of [Ca2+]cyt represent information to the cell which demand the existence of decoding mechanisms by Ca2+binding proteins. These proteins function as Ca2+ sensors that, upon Ca2+ binding, undergo conformational changes and convey the information to downstream targets. One group of Ca2+ sensors that represent candidates for this function in pollen is provided by the family of calcineurin B-like (CBL) proteins, which consists of 10 members in Arabidopsis (Konrad et al., 2011; Steinhorst and Kudla, 2012). CBLs harbor a conserved core region containing four EF-hand motifs for Ca2+ binding that are predicted to possess characteristic Ca2+ affinities (Batistič and Kudla, 2009). Each CBL protein specifically interacts with a subset of 26 serine/threonine kinases designated as CBL-interacting protein kinases (CIPKs). Specific targeting signals in the N-termini of CBLs determine the subcellular localization of CBL–CIPK complexes (Batistič et al., 2010). In this way, distinct CBL–CIPK complexes build up a Ca2+decoding signaling network that can perceive Ca2+ signals from different intra- and extracellular stores (Hedrich and Kudla, 2006; Batistič et al., 2010; Hashimoto and Kudla, 2011).
CBLs are involved in a multitude of cellular processes including regulation of ion homeostasis and stress responses (Qiu et al., 2002; Xu et al., 2006; Cheong et al., 2007; Ho et al., 2009; Weinl and Kudla, 2009; Held et al., 2011; Drerup et al., 2013). One example is the phosphorylation-mediated activation of the K+ channel AKT1 by CIPK23 in complex with either CBL1 or CBL9 which enables plant growth under low K+ conditions (Xu et al., 2006; Cheong et al., 2007). CBL1/CBL9–CIPK23 complexes also regulate NO3– sensing and uptake by phosphorylating the nitrate transporter CHL1 (Ho et al., 2009). Upon interaction with CIPK26, CBL1 and CBL9 activate the NADPH oxidase RBOHF (Drerup et al., 2013). Furthermore, the Ca2+ sensor CBL4/SOS3 in complex with CIPK24/SOS2 regulates the activity of the Na+/H+antiporter SOS1 and thereby contributes to establishing salt tolerance in plants (Liu and Zhu, 1998; Halfter et al., 2000; Qiu et al., 2002). Alternatively, CBL4 can interact with the kinase CIPK6 to regulate the plasma membrane targeting of the K+ channel AKT2 (Held et al., 2011). However, despite the accumulating evidence for important functions of CBL–CIPK complexes in many distinct processes, it has so far not been investigated in Arabidopsis whether CBLs and CIPKs function in pollen or growing pollen tubes.
Here, we identify four CBL proteins as being expressed in pollen and growing pollen tubes and report a function for the plasma membrane localized Ca2+ sensors CBL1 and CBL9 in pollen germination and pollen tube tip growth. Transient overexpression of CBL1 or CBL9 in tobacco pollen tubes reduces pollen tube length and induces tip swelling. Also, stable overexpression of CBL1 or CBL9 in Arabidopsis plants strongly reduces pollen germination rates and alters pollen tube morphology, especially at high external K+ concentrations. Induction of these phenotypes requires efficient plasma membrane targeting of CBL1 that depends on a functional N-terminal myristoylation motif. Conversely, pollen of a cbl1/cbl9 double mutant display reduced tube growth under low K+ conditions. These findings indicate that a faithfully regulated accumulation level of CBL1 and CBL9 at the plasma membrane is crucial for efficient implementation of pollen germination and polar tube growth. Moreover, these data suggest a function of CBL1 and CBL9 in the regulation of ion homeostasis in pollen.
RESULTS
CBL1 Is Expressed in Mature Pollen and Pollen Tubes
In recent years, several transcriptomic studies of pollen and pollen tubes have provided growing evidence for a putative role of Ca2+ sensor proteins like calmodulin or calmodulin-like proteins in pollen germination and tube growth (Pina et al., 2005; Wang et al., 2008b). To identify CBL genes that are expressed in pollen grains or pollen tubes, we analyzed the microarray data set reported by Wang et al., 2008aand Wang et al., 2008b. In this way, we uncovered four out of 10 CBLs, namely CBL1, CBL2, CBL3, and CBL9, as being significantly expressed in mature pollen, hydrated pollen, and pollen tubes (Figure 1A). Out of these four, CBL1 turned out to be specifically up-regulated during pollen tube growth, thus making it a promising candidate for a further functional characterization in this cell type. To confirm the expression of CBL1 in Arabidopsis pollen and pollen tubes, we performed a histochemical staining of transgenic Arabidopsis plants expressing the uidA gene under control of a CBL1 promoter fragment. GUS activity in mature flowers of these plants was mainly detected in the anthers of the stamen as it was published before by D’Angelo et al. (2006) (Figure 1B). A more detailed analysis revealed strong GUS activity to be present in pollen grains and in germinating pollen and pollen tubes (Figure 1B), further suggesting a potential role for CBL1 in pollen germination and/or tube growth.
Figure 1. Expression Analyses of CBL Genes in Mature Pollen and Pollen Tubes.
(A) A microarray transcriptomic data set for gene expression during pollen germination and pollen tube growth (Wang et al., 2008b) was analyzed for expression of CBL genes in mature pollen, hydrated pollen, and pollen tubes. Relative expression values of CBL1, CBL2, CBL3, and CBL9are shown as means ± standard deviation of two biological replicates.
(B) Histochemical staining of flowers (left panel), mature pollen (middle panel, bottom), and pollen tubes (right panel, bottom) expressing the uidA reporter gene driven by the CBL1 promoter. Images are representative for eight analyzed plants from two independent lines. Non-transgenic Col-0 pollen and pollen tubes treated in the same way served as negative controls (middle panel, top, and right panel, top, respectively).
CBL1 Is Localized to the Plasma Membrane and to Small Vesicles in the Apex of Growing Tobacco Pollen Tubes
Previous studies established the localization of CBL1 at the plasma membrane of protoplasts and leaf epidermal cells when transiently expressed in Nicotiana benthamiana (D’Angelo et al., 2006; Cheong et al., 2007; Batistič et al., 2008). To analyze its subcellular localization in pollen tubes, CBL1 was C-terminally fused to mVenus and transiently expressed in Nicotiana tabacum pollen under control of the pollen-specific LAT52 promoter from tomato (Twell et al., 1991). The localization of the fusion protein was analyzed by confocal microscopy 4–6h after pollen transformation and germination on microscope slides (Figure 2). Transient expression of mVenus alone served as a control (Figure 2A).
Figure 2. Localization of CBL1–mVenus in Tobacco Pollen Tubes.
(A, B) Time course of growing tobacco pollen tubes transiently expressing mVenus (A) or CBL1–mVenus (B) under control of the LAT52 promoter from tomato. Confocal images were taken 4–6h after transformation with time intervals of 1min.
(C) Pollen tube expressing CBL1–mVenus (green) co-stained with FM 4–64 (red); yellow color indicates co-localization in merged image. FM 4–64 treatment was performed 4–5h after transformation. Confocal images were taken 30min after application of the dye. Bars represent 10 μm.
In growing pollen tubes, mVenus-tagged CBL1 decorated a sharp line along the pollen tube perimeter which clearly represented the plasma membrane (Figure 2B). In addition, CBL1–mVenus was associated with small vesicles that underwent cytoplasmic streaming and formed a dynamic cone-shaped area in the clear zone of the growing pollen tube tip (Figure 2B). Co-staining with FM 4–64 confirmed that this region indeed contained endocytotic vesicles (Figure 2C). During these analyses, we noted that pollen tubes expressing CBL1–mVenus (Figure 2B) grew more slowly than those expressing mVenus alone (Figure 2A) and frequently changed their growth direction. This bending of the pollen tube was preceded by a relocation of the cloud of endocytotic vesicles containing CBL1–mVenus towards the newly formed growth axis. In pollen tubes that ceased growing, the dynamic region decorated by CBL1–mVenus-associated vesicles slowly increased in size and spread from the pollen tube apex further towards the subapical part of the pollen tube (Figure 2C). These data not only disclose that the Ca2+ sensor CBL1 is localized at the plasma membrane in growing pollen tubes, but also identify CBL1-containing vesicles that are subject to highly dynamic endocytosis.
Transient Overexpression of CBL1 in Tobacco Pollen Tubes Impairs Pollen Tube Growth and Induces Tip Swelling
In the course of our localization analyses, we noted a considerably high number of pollen tubes displaying morphological and growth alterations upon expression of mVenus-tagged CBL1. In order to elucidate this observation in more detail, we transiently expressed CBL1 C-terminally fused to mCherry under control of the LAT52promoter in tobacco pollen and quantitatively monitored pollen tube growth in a larger number of transformed cells (Figure 3). Transient expression of mCherry had no detectable effect on pollen tube morphology and thus served as a negative control (Figure 3A). Pollen tubes expressing CBL1–mCherry (Figure 3B–3D) were considerably shorter than control pollen tubes and often exhibited swollen tips. Frequently, expression of CBL1–mCherry also led to disoriented pollen tube growth, which resulted in the formation of a hook in the apical region of the pollen tube (Figure 3C).
Figure 3. Overexpression of CBL1–mCherry or CBL9–mCherry in Tobacco Pollen Provokes Aberrant Pollen Tube Growth.
mCherry, CBL1–mCherry, or CBL9–mCherry were transiently expressed in tobacco pollen and pollen tube growth was monitored by epifluorescence microscopy (A–C, E) or confocal microscopy (D, F).
(A–F). Representative images of non-growing tobacco pollen tubes transiently expressing mCherry (A), CBL1–mCherry (B–D), or CBL9–mCherry (E, F). Pictures were taken 4–6h after transformation. Bars represent 65 μm (A–C, E), 15 μm (D), or 10 μm (F).
(G, H) Quantification of pollen tube length (G) and width (H) upon expression of mCherry, CBL1–mCherry, or CBL9–mCherry. Left panels: average pollen tube length (G) and width (H) ± standard deviation for at least 62 analyzed pollen tubes. A Student’s t-test indicates significant differences between pollen tubes expressing mCherry and pollen tubes expressing CBL1–mCherry or CBL9–mCherry (P < 0.0001). Right panels: histograms display the range of pollen tube length (G) and width (H) observed upon expression of the indicated proteins. Quantifications were performed at least three times, with similar results.
Since CBL1 shares 89% sequence identity with its closest homolog CBL9 (Kolukisaoglu et al., 2004) and CBL9 is also expressed in pollen tubes (Figure 1A), we included CBL9 in our phenotypical analyses. To this end, we similarly expressed a mCherry-tagged version of CBL9 in tobacco pollen tubes under control of the LAT52promoter (Figure 3E and 3F). Pollen tubes expressing the transgene were shorter than control pollen tubes and often displayed swollen tips. Frequently, pollen tubes underwent disoriented growth, thereby exhibiting the same morphological alterations that were observed in pollen tubes overexpressing CBL1–mCherry. Confocal microscopy revealed plasma membrane localization of the CBL9–mCherry fusion protein and accumulation at some internal membrane structures in non-growing pollen tubes (Figure 3F).
Quantification of the observed phenotypes revealed a significant reduction of the pollen tube length upon expression of CBL1–mCherry or CBL9–mCherry with a mean value of 221.9 and 340.0 μm, respectively, compared to 664.6 μm for the control (Figure 3G, P < 0.0001 according to a Student’s t-test). The longest pollen tube expressing CBL1–mCherry that was found in these experiments had a length of 484.2 μm and thus was shorter than an average pollen tube expressing mCherry alone. Conversely, the tip diameter of pollen tubes was strongly increased upon expression of CBL1–mCherry or CBL9–mCherry with mean values of 17.1 and 14.4 μm, respectively, while expression of mCherry resulted in an average tube width of 8.5 μm (Figure 3H, P < 0.0001 according to a Student’s t-test).
To address the specificity of the observed overexpression phenotypes, we investigated the effects of overexpressing the Ca2+ sensor CBL7 that shares 51% identity with CBL1 (Kolukisaoglu et al., 2004). Transient overexpression of CBL7–mCherry in tobacco pollen tubes had no significant impact on the pollen tube length or the tip diameter (Supplemental Figure 1, P = 0.195 and P = 0.784, respectively, according to a Student’s t-test). This finding supports the conclusion that the phenotypes caused by transient overexpression of the CBL1 and CBL9 fusion proteins specifically depend on the function of these proteins and do not result, for example, from non-specific depletion of Ca2+ upon overexpression of a Ca2+ binding protein. Altogether, these results indicate that CBL1 and CBL9 are involved in regulating crucial processes of pollen tube growth and that a faithfully balanced expression level of both proteins is required for establishing proper polarity and growth in pollen tubes.
Plasma Membrane Localization of CBL1 Is Required for Its Function in Pollen Tube Tip Growth
N-terminal lipid modification of CBL1 by myristoylation and palmitoylation is essential for proper targeting of this protein to the plasma membrane (Batistič et al., 2008). In order to elucidate whether plasma membrane localization of CBL1 is required for its function in pollen tube tip growth, we transiently expressed a mutant version of CBL1 (CBL1G2A) that is efficiently blocked in both lipid modifications in tobacco pollen tubes. In microscopic analyses, CBL1G2A–mCherry was exclusively detected in the cytosol of non-growing pollen tubes (Figure 4A). Furthermore, no tip swelling or reduction of tube length was observed upon expression of CBL1G2A–mCherry (Figure 4B and 4C). Quantification of the tube length revealed no significant difference between pollen tubes expressing CBL1G2A–mCherry or mCherry alone (Figure 4B, P = 0.196 according to a Student’s t-test). Similarly, the mean tip diameter of pollen tubes upon expression of CBL1G2A–mCherry was comparable to the control (Figure 4C, P = 0.174 according to a Student’s t-test), indicating that plasma membrane localization of CBL1 is strictly required for proper functioning of the protein in growing pollen tubes.
Figure 4. The CBL1 Pollen Tube Phenotype Is Dependent on the Plasma Membrane Localization of CBL1.
A non-myristoylated and non-palmitoylated mutant version of CBL1, CBL1G2A–mCherry, was transiently expressed in tobacco pollen tubes and pollen tube growth was monitored.
(A) Confocal image of a non-growing pollen tube transiently expressing CBL1G2A–mCherry. The image was taken 5h after transformation. Bar represents 10 μm.
(B, C) Quantification of pollen tube length and width upon expression of CBL1G2A–mCherry. Left panels: average length (B) and width (C) ± standard deviation of at least 50 analyzed pollen tubes. A Student’s t-test indicates no significant differences between pollen tubes expressing mCherry or CBL1G2A–mCherry (P = 0.196 and P = 0.174, respectively). Right panels: histograms illustrating the range of pollen tube length (B) and width (C) upon expression of mCherry, CBL1–mCherry, or CBL1G2A–mCherry. Quantifications were performed at least three times, with similar results.
Mutation of the Fourth Ca2+ Binding EF-Hand of CBL1 Does Not Impair the Effect of CBL1 Overexpression on Pollen Tube Tip Growth
Crystal structure analyses of several CBL proteins like CBL2 (Nagae et al., 2003) and CBL4/SOS3 (Sánchez-Barrena et al., 2005) indicate a crucial function of the third and fourth EF-hand for Ca2+ binding. In the available crystal structures, Ca2+ was found to be bound to EF4 of both CBL2 and CBL4 (Nagae et al., 2003; Sánchez-Barrena et al., 2005) and is also predicted to form a complex with EF4 of CBL1 based on sequence analyses (Kolukisaoglu et al., 2004). We sought to investigate whether mutation of the –Z position in the fourth EF-hand motif of CBL1, which is required for Ca2+ binding to this EF-hand, would have an effect on CBL1 function in pollen tube tip growth. To this end, we transiently expressed the respective mCherry-tagged mutant version of CBL1, CBL1EF4–mCherry in tobacco pollen tubes (Figure 5). Confocal microscopy indicated plasma membrane localization of the fusion protein in non-growing pollen tubes (Figure 5A). Upon expression of CBL1EF4–mCherry, we noted a high number of pollen tubes displaying morphological alterations like reduced growth and tip swelling, similarly to what we observed for CBL1–mCherry (Figure 3). Quantification of the described phenotypes (Figure 5B and 5C) somewhat surprisingly revealed no significant difference between pollen tubes expressing CBL1–mCherry or CBL1EF4–mCherry regarding their length (Figure 5B) and width (Figure 5C; Student’s t-test P = 0.398 and P = 0.866, respectively). This result suggests that, under the physiological conditions of the pollen tube, high-affinity binding of Ca2+ to the fourth EF-hand of CBL1 is not required to bring about the observed phenotypes in pollen tube tip growth.
Figure 5. Mutation of the Fourth Ca2+ Binding EF-Hand of CBL1 Does Not Impair the Effect of CBL1 Overexpression on Pollen Tube Tip Growth.
CBL1EF4–mCherry carrying a mutation in the fourth EF-hand of CBL1 was transiently expressed in tobacco pollen and pollen tube phenotypes were recorded.
(A) Confocal image of a non-growing pollen tube expressing CBL1EF4–mCherry 7h after transformation. Bar represents 10 μm.
(B, C) Quantification of pollen tube length (B) and width (C) upon expression of CBL1EF4–mCherry. Left panels: mean pollen tube length (B) and width (C) ± standard deviation of at least 31 analyzed pollen tubes 4h after pollen transformation. A Student’s t-test indicates no significant differences between pollen tubes expressing CBL1–mCherry and CBL1EF4–mCherry (P = 0.398 and P = 0.866, respectively). Right panels: histograms showing the range of pollen tube length (B) and width (C) upon expression of the indicated proteins. Quantifications were performed at least three times, with similar results.
Overexpression of CBL1 or CBL9 in Arabidopsis thaliana Reduces Pollen Germination Rates and Tube Length Especially under High K+ Conditions
Having utilized the heterologous tobacco pollen tube as a convenient transient expression system to gain first evidence for a putative role of CBL1 and CBL9 in pollen tube tip growth, we next sought to study the functional relevance of these Ca2+sensors for pollen germination and tube growth in Arabidopsis plants. We decided to follow a similar gain-of-function approach as in tobacco by generating CBL1 and CBL9 overexpression lines (CBL1 oex #13–12 and CBL9 oex #3–2) under control of the constitutive UBQ10 promoter which has been shown to confer stable expression of target genes in different plant tissues including pollen (Krebs et al., 2012). A CBL7overexpression line (CBL7 oex #7–4) driven by the UBQ10 promoter served as a control. The presence of enhanced transcript levels in the CBL oex lines was confirmed by qRT–PCRs which revealed a 34-fold increase for CBL1 in CBL1 oex #13–12, a 28-fold change for CBL9 in CBL9 oex #3–2, and a 246-fold increase for CBL7 in CBL7 oex #7–4 when compared to expression levels of the respective CBLgene in wild-type plants (Figure 6A).
Figure 6. Overexpression of CBL1 or CBL9 in Arabidopsis Reduces Pollen Germination Rates and Perturbs Pollen Tube Growth at High External K+ Concentrations.
(A) qRT–PCRs confirm enhanced transcript levels for CBL1, CBL9, or CBL7 in transgenic Arabidopsis plants expressing the respective CBL genes under control of the constitutive UBQ10promoter. For quantification, expression values in Col-0 were defined as 1. Expression of UBQ10served as an internal reference. Graphs show means ± standard error of three biological replicates.
(B) Quantification of pollen germination rates for Col-0 and CBL1, CBL9, and CBL7 oex lines at different external K+ concentrations. Germination rates were determined after an incubation of 4.5h on solid pollen medium containing the indicated K+ content. The graph shows means ± standard error of four independent experiments. Paired t-tests indicate significant differences between Col-0 and the CBL1 and CBL9 oex lines at all indicated K+ concentrations (P ≤ 0.0491).
(C) Quantification of pollen tube length for Col-0 and CBL1, CBL9, and CBL7 oex lines at different external K+ concentrations. Pollen tube length was determined after 4.5 h of incubation on solid growth medium containing the indicated K+ concentrations. The graph shows means ± standard error of four independent experiments. Paired t-tests indicate significant differences between the pollen tube length of Col-0 and the CBL1 and CBL9 oex lines at 5mM external K+ (P ≤ 0.0285).
For determination of pollen germination rates of Col-0 and the CBL oex lines, pollen were collected and germinated on growth medium supplemented with 0, 0.1, 1, or 5mM K+. After a 4.5-h incubation time, the number of germinated pollen was counted and germination rates were calculated (Figure 6B). Subsequent analysis of the CBL1and CBL9 oex lines revealed a significant reduction of pollen germination rates for all tested K+ concentrations when compared to wild-type levels (Figure 6B; paired t-test P ≤ 0.0491). This effect was more pronounced at higher external K+ concentrations. In contrast, the CBL7 oex line did not show a decrease of pollen germination rates at any K+ concentration tested. These results indicate a hypersensitivity of CBL1 and CBL9 oex lines in pollen germination towards high concentrations of external K+.
To analyze the effect of CBL1 and CBL9 overexpression on pollen tube growth, we measured the pollen tube length of at least 200 germinated pollen from four independent experiments (Figure 6C). At 0mM external K+, pollen tubes of the CBL1and CBL9 oex lines were 16% and 25% shorter than control pollen tubes, respectively, although this effect was only significant for the CBL9 oex line (paired t-test P = 0.1302 and P = 0.0164, respectively). Increasing the K+, content of the growth medium to 5mM resulted in a 25% decrease of pollen tube length for CBL1oex #13–12 and a 28% reduction for CBL9 oex #3–2 as compared with Col-0 (paired t-test P = 0.0285 and P = 0.0059, respectively). However, no reduction of pollen tube length could be observed for CBL7 oex #7–4 under all tested K+ concentrations. These results suggest similar regulatory functions for CBL1 and CBL9 in pollen germination and tube growth of Arabidopsis which appear to involve regulation of K+homeostasis in these cells.
Loss of CBL1 and CBL9 Function Impairs Pollen Tube Growth under Low K+ Conditions
The low pollen germination rates and the impaired pollen tube growth that we observed upon overexpression of either CBL1 or CBL9 encouraged us to investigate whether loss of CBL1 or CBL9 function would have an effect on pollen germination and tube growth. To address this question, we germinated pollen of Col-0 and a cbl1T-DNA insertion line (SALK_110426; Xu et al., 2006) on growth medium supplemented with 0, 0.1, 1, and 5mM K+ to determine pollen tube length (Figure 7). We also included a cbl9 mutant allele (SALK_142774; Xu et al., 2006) and a cbl1/cbl9 double mutant line (Xu et al., 2006) in our analyses, since several studies demonstrated functional overlaps of CBL1 and CBL9 especially with regard to their function in regulating K+ homeostasis (Xu et al., 2006; Cheong et al., 2007). Analysis of pollen tube growth under low K+ conditions revealed a significant reduction of pollen tube length for the cbl1/cbl9 double mutant line at 0 and 0.1mM external K+ as compared to the corresponding wild-type levels (Figure 7; paired t-test P ≤ 0.0404). This effect was not observed for the single mutant lines under the same conditions. Addition of 1 or 5mM K+ to the growth medium attenuated the growth phenotype of the cbl1/cbl9double mutant. Complementary to our analyses of CBL1 and CBL9 overexpression which revealed enhanced phenotypes especially under excess supply of external K+, these data reveal a hypersensitivity of pollen tube growth in cbl1/cbl9 mutants towards low K+ conditions. Together, the results suggest a regulation of K+homeostasis as one of the potential functions that is brought about by CBL1 and CBL9 in pollen and pollen tubes.
Figure 7. Disruption of CBL1 and CBL9 Lowers Pollen Tube Length at Low External K+Concentrations.
Quantitative analysis of pollen tube length for Col-0, cbl1, cbl9, and cbl1/cbl9 T-DNA insertion lines. Pollen tube length was determined after 4.5 h of incubation on solid growth medium containing the indicated K+ concentrations. The graph shows means ± standard error of four independent experiments. Paired t-tests indicate significant differences between Col-0 and cbl1/cbl9 at 0 and 0.1mM external K+ (P ≤ 0.0404).
DISCUSSION
Ca2+ has been established as a central regulator of polarized pollen tube growth (Jaffe et al., 1975; Obermeyer and Weisenseel, 1991; Michard et al., 2009). Established cellular targets of signaling and regulation by Ca2+ in pollen are actin dynamics (Li et al., 1999; Gu et al., 2005; Wang et al., 2008a), vesicle trafficking (Camacho and Malhó, 2003), and other fundamental processes like ion transport and accumulation of reactive oxygen species (ROS) (Becker et al., 2004; Potocký et al., 2012). Importantly, recent studies have molecularly identified first ion channels like GLRs that critically function in generating Ca2+ signals and gradients in pollen tubes (Michard et al., 2011). In contrast, our understanding of how Ca2+ signals in growing pollen tubes are sensed and relayed to trigger downstream responses is limited and, so far, only a few signaling components have been identified to affect pollen germination and/or tube growth in Arabidopsis. In 2009, Myers and colleagues reported a combined function for the Ca2+-dependent protein kinases (CDPKs) CPK17 and CPK34 in these processes. While pollen germination rates were not altered in a cpk17/34 double mutant line, pollen tubes derived from this mutant showed reduced tube length and stunted growth in vitro (Myers et al., 2009). Furthermore, pollen tubes of cpk17/34 failed to locate and fertilize ovules of wild-type plants, suggesting an additional function for both kinases in the response of the male gametophyte to tropism signals and in its interaction with the female tissue (Myers et al., 2009). Moreover, calmodulin (CAM) 2 has been reported to function in pollen germination and tube growth in Arabidopsis (Landoni et al., 2010). A cam2 T-DNA insertion line displayed reduced pollen germination rates and was affected in pollen tube growth in vitro (Landoni et al., 2010), thus implicating a member of the CAM-family in mediating these processes.
In this study, we extend the range of known calcium sensor proteins that contribute to decoding Ca2+ signals in pollen by reporting a function for the calcineurin B-like proteins CBL1 and CBL9 in pollen germination and tube growth. Transient overexpression of CBL1 and CBL9 in tobacco pollen tubes resulted in altered tube morphology marked by a reduction of tube length, tip swelling, and loss of the original growth axis (Figure 3). Consistently, overexpression of CBL1 or CBL9 in Arabidopsisplants reduced pollen germination rates and affected pollen tube growth under high K+ conditions (Figure 6). Together, these findings provide first evidence for a role of CBL proteins in sexual reproduction on the level of the male gametophyte.
Based on the results of our phenotypic analyses of loss-of-function lines, we conclude that the function of CBL1 in pollen germination and tube growth at least partially overlaps with the functional range of CBL9. This implication is especially supported by the observation that, in Arabidopsis, neither cbl1 nor cbl9 single mutant lines were negatively affected in pollen germination or tube growth on low K+ medium, while the cbl1/cbl9 double mutant line displayed shorter pollen tubes under these conditions (Figure 7). Moreover, our data suggest that both Ca2+ sensor proteins exert their function at the plasma membrane of pollen tubes. Mutation of the N-terminal myristoylation motif of CBL1 (CBL1G2A) simultaneously prevented accumulation of this protein at the plasma membrane and induction of any detectable phenotype upon overexpression in pollen tubes. Previous studies have reported a similar targeting mechanism for both proteins and, in line with our results, overexpression of CBL1G2A failed to restore salt tolerance in a cbl1 mutant background (Batistič et al., 2008). Moreover, G2A mutation of CBL1 appeared to abolish accumulation of CBL1 in the endocytotic vesicles in the clear zone. This further supports the assumption of an endocytotic streaming of CBL1 from the plasma membrane into the cell and raises the question about a potential function of CBL1 in this cellular compartment. Remarkably, accumulation of CBL1 in endocytotic vesicles has not been observed in any other cell type of plants and may point to a pollen-specific feature of this protein.
One interesting aspect of our CBL1 localization studies in growing pollen tubes is the observation that CBL1 appeared to be evenly distributed across the pollen tube plasma membrane (Figure 2). This observation implies that CBL1 function is not restricted to a specific plasma membrane region, but instead could exert its function across the whole pollen tube perimeter. However, [Ca2+]cyt in the pollen tube varies from 2–10 μM in the apex to 20–200nM in the distal part (Holdaway-Clarke et al., 1997; Messerli et al., 2000), raising the possibility of a molecular switch for CBL1 activity depending on the amount of Ca2+ present in distinct plasma membrane regions of the growing pollen tube. Moreover, it has been reported that interaction of one CBL protein with different CIPKs can display distinct Ca2+ dependence (Batistič and Kudla, 2004; Weinl and Kudla, 2009). Therefore, it is tempting to speculate that, in different regions of the pollen tube, depending on the local Ca2+ concentration, CBL1 may interact with and activate different CIPKs. This potential switch in information processing deserves further investigation in future studies.
For CBL9, we only determined the localization in non-growing pollen tubes where this protein also accumulated evenly distributed in the plasma membrane. Therefore, we hypothesize that this Ca2+ sensor exerts its function in a similar way to CBL1. The two other CBL-type Ca2+ sensor proteins that are significantly expressed in pollen, CBL2 and CBL3, have been reported as tonoplast localized proteins in other cell types (Batistič et al., 2010, 2012). Their role in pollen tube tip growth has been addressed by transient overexpression analyses in tobacco pollen in a previous study (Zhou et al., 2009). Here, these proteins were detected at the top region of the tube plasma membrane and at internal membranes. Overexpression of either CBL2 or CBL3resulted in reduced pollen tube growth and tip swelling. Considering their distinct localization, it appears conceivable that CBL2 and CBL3 contribute to the regulation of pollen tube growth by different mechanisms than CBL1 and CBL9. Interestingly, the study by Zhou and colleagues (2009) also used this transient overexpression analysis in tobacco to investigate the potential function of CAM or CAM-like proteins (CMLs) in pollen tube growth. However, of the 10 proteins investigated, only one (CML21) caused detectable phenotypes in this study, suggesting a remarkable specificity of this transient assay system.
In the course of our phenotypical analyses, we observed opposite effects of the external K+ concentration on mutant and overexpressing pollen tubes, respectively, in that growth of the cbl1/cbl9 double mutant is hypersensitive towards low K+conditions (Figure 7) while overexpression of CBL1 or CBL9 affects pollen germination and tube growth under high K+ conditions (Figure 6). These results point to a positive regulatory function of CBL1 and CBL9 for K+ uptake in germinating pollen and growing pollen tubes. Our data indicate that K+ homeostasis is crucial for pollen tube growth, since disruption of CBL1 and CBL9 lowers pollen tube growth rates under low K+ conditions. Conversely, overexpression of CBL1 or CBL9 renders pollen tube growth hypersensitive towards high external K+ probably by exceeding a certain level of K+ uptake in the pollen tube, which inhibits growth.
In a previous study, a regulatory function of CBL1 and CBL9 for K+ uptake in young seedlings was reported (Xu et al., 2006). This work identified the K+ channel AKT1 from the Shaker family of Arabidopsis as a target of CBL1/9 that, in combination with their interacting kinase CIPK23, mediate regulation of this channel (Xu et al., 2006). Under low K+ conditions, AKT1 is phosphorylated by the protein kinase CIPK23, which activates the transporter and subsequently enhances K+ uptake (Xu et al., 2006). This phosphorylation/activation mechanism is facilitated by CBL1 and CBL9, which interact with CIPK23 and translocate the protein from the cytosol to the plasma membrane, thereby bringing CIPK23 and its downstream effector AKT1 in close proximity to each other (Xu et al., 2006; Cheong et al., 2007). Consequently, loss of CBL1 and CBL9 function rendered mutant plants hypersensitive towards low K+conditions (Xu et al., 2006; Cheong et al., 2007).
While the Shaker-type K+ channel AKT1 is predominantly expressed in root tissues (Wang and Wu, 2010), another member of the Shaker family, Shaker pollen inward K+channel (SPIK), specifically functions in pollen and pollen tubes (Mouline et al., 2002). Expression of SPIK is restricted to pollen and pollen tubes, and disruption of the gene strongly reduces inwardly rectifying K+ currents in pollen grain protoplasts (Mouline et al., 2002). Moreover, loss of SPIK activity leads to a 40–50% decrease in pollen tube length, while pollen germination rates are not affected (Mouline et al., 2002). Taking into account that SPIK and AKT1 are highly homologous and that the phenotype of the cbl1/cbl9 mutant in part resembles the spik pollen tube phenotype under low K+conditions suggests that SPIK may be somehow subject to regulation by CBL1 and CBL9. However, further investigation of this potential regulatory module for K+transport in pollen will require the identification of the respective CIPK that would bring about CBL-dependent phosphorylation of SPIK. Interestingly, a most recent study identified a Ca2+-dependent regulatory circuit that negatively modulates SPIK activity (Zhao et al., 2013). Here, a CDPK module consisting of CPK11 and CPK24 mediates Ca2+-dependent inhibition of SPIK during pollen tube growth (Zhao et al., 2013). This situation would allow for a model in which, depending on the actual Ca2+concentration, CDPKs negatively regulate this K+ channel, while a CBL1/9–CIPK complex may confer activation of SPIK.
Finally, our data suggest that regulation of K+ homeostasis might not be the only function of CBL1 and CBL9 during pollen germination and tube growth. Both CBL1and CBL9 oex lines display shorter pollen tubes, even at lower K+ concentrations (Figure 6), indicating additional roles for the two Ca2+ sensors at least for pollen tube growth. In this regard, for example, ROS have been reported to constitute important regulators of pollen tube growth and a recent study has demonstrated that CBL1 and CBL9 together with their interacting kinase CIPK26 activate the NADPH oxidase RBOHF (Steinhorst and Kudla, 2012; Drerup et al., 2013). Considering that other members of the NADPH oxidase family are specifically expressed in pollen, it appears conceivable that they may represent additional targets of CBL1 and CBL9. Whether additional processes might be regulated by CBL1 and CBL9 in pollen remains speculative at this point. In any case, the data presented here support a model in which CBL1 and CBL9 regulate important processes at the plasma membrane of pollen and growing pollen tubes that can be explored in detail in future studies.
METHODS
Promoter GUS Analyses
For CBL1 expression analyses, transgenic lines expressing a CBL1 promoter–uidAreporter gene construct published in D’Angelo et al. (2006) were used. Flowers and germinated pollen from four different T2 plants of two independent lines were stained with 5-bromo-4-chloro-3-indolyl-β-D-glucuronide for 12h. Stained flowers were subsequently incubated in 70% ethanol to remove the chlorophyll. Pollen of Col-0 treated in the same way served as a negative control.
Generation of Constructs for Transient Gene Expression in Tobacco Pollen Tubes
Pollen expression vectors were generated by introducing a LAT52 promoter fragment (Twell et al., 1991) into the pUC19 vector backbone as a HindIII/blunt end fragment thereby destroying the XbaI restriction site in the multiple cloning site of the vector backbone. mCherry or mVenus cDNA fragments were cloned into the pUC–LAT52 vector backbone using the XmaI and SstI restriction sites downstream of the multiple cloning site leading to the vectors pUC–LAT52–mCherry and pUC–LAT52–mVenus, respectively. CBL1 was amplified by PCR using the primer combination 5’-tttggatccatgggctgcttccactcaaag-3’/5’-tttctcgagtgtggcaatctcatcg-3’ and integrated as a BamHI/XhoI fragment into the pUC–LAT52–mCherry and the pUC–LAT52–mVenus vectors. CBL9 was amplified using the primer combination 5’-ttttctagaggatcctgaatgggttgtttccattcc-3’/5’-tttcccgggcgtcgcaatctcgtcca-3’ and integrated into pUC–LAT52–mCherry as a BamHI/XmaI fragment. CBL7 was amplified by PCR using the primer combination 5’-tttggatcccatggattcaacaagaaattcagc-3’/5’-tttcccgggggtatcttccacttgc-3’ and introduced as a BamHI/XmaI fragment into pUC19–LAT52–mCherry. CBL1G2A was transferred into the pUC–LAT52–mCherry vector backbone as a BamHI/XmaI fragment from a plasmid carrying the respective cDNA (Batistič et al., 2008). Site-directed mutagenesis of EF4 in a CBL1 cDNA fragment was performed using the site-directed mutagenesis protocol from Stratagene according to the manufacturer’s instructions. To this end, the complete plasmid psHA–CBL1 carrying the respective CBL1 cDNA fragment was amplified with Pfu Turbo Polymerase (Stratagene) using the primer combination 5’-ggaaaaattgataaattacagtggagtgatttcgtaaac-3’/5’-gtttacgaaatcactccactgtaatttatcaatttttcc-’3 to introduce the G514C mutation into the reading frame of CBL1. The resulting PCR reaction was treated with DpnI to remove the non-mutagenized maternal template DNA and used for transformation of Escherichia coli strain SURE. The mutagenized plasmid was isolated from a positive E. coli clone and sequenced for correct exchange of the nucleotide. The respective plasmid psHA–CBL1EF4 served as a template for PCR-amplification of the CBL1EF4fragment using the primer combination 5’-tttggatccatgggctgcttccactcaaag-3’/5’-tttctcgagtgtggcaatctcatcg-3’, which was cloned into the pUC–LAT52–mCherry backbone using the BamHI and XhoI restriction sites.
Transient Gene Expression in Tobacco Pollen Tubes
Mature pollen were collected from seven or eight flowers of 6–8-week-old N. tabacumplants, harvested in growth medium (Read et al., 1993), and filtered onto cellulose acetate filters moisturized with growth medium. Pollen were then transformed by particle bombardment with 3 μg of plasmid DNA coated onto 1 μm gold particles. For this, a helium-driven particle accelerator (PDS-1000/He; Biorad) and 1350-psi rupture discs were used and bombardment was performed at a vacuum of 27 inches of mercury. After transformation, pollen were re-suspended in growth medium and germinated on microscope slides at room temperature in the dark for the indicated time periods. Analysis of growing pollen tubes was performed at room temperature (see below). For quantification of pollen tube phenotypes, samples were covered with coverslips and kept on ice 4h after transformation.
FM 4–64 Staining
FM 4–64 was added to growing pollen tubes at a final concentration of 20 μM 4–5h after transformation and imaged after an incubation of 30min using a Leica DMI 6000B inverted microscope equipped with a Leica TCS SP5 laser scanning device (see below).
Microscopy and Analysis of Transiently Transformed Tobacco Pollen Tubes
For quantification of pollen tube phenotypes, pictures were taken with a Leica DMI 6000B epifluorescence microscope equipped with a cooled charge-coupled device (CCD) camera (SenSys Photometrics, www.photometrics.com) and using a HC PL FLUOTAR 10×/0.30 PH1 air objective and a BP 546/2 and BP 605/75 Filterset for excitation and emission of mCherry fluorescence. Pollen tube length and tip diameter were measured with the imageJ software (rsbweb.nih.gov/ij/).
Confocal microscopy was performed using a Leica DMI 6000B inverted microscope equipped with a Leica TCS SP5 laser scanning device. mVenus was excited at 488nm (Ar laser) and fluorescence was recorded at 500–550nm. mCherry was excited at 561nm (DPSS 561 laser) and fluorescence was recorded at 605–638nm. For co-staining with FM 4–64, mVenus and FM 4–64 were synchronously excited at 514 (Ar laser) and 561nm (DPSS 561 laser) and scanned at 521–554 and 605–638nm, respectively. All images were taken using a HCX PL APO lambda blue 63×/1.20 water-immersion objective and the Leica confocal system software.
Generation of CBL1, CBL7, and CBL9 Overexpression Lines
For generation of the CBL1 oex line, a 642-bp cDNA fragment of CBL1 was amplified by PCR using the primer combination 5’-ttttgggcccaaaatgggctgcttccactcaaag-3’/5’-aaaagtcgactcatgtggcaatctcatcgac-3’ and cloned as an ApaI/SalI fragment into the pGPTVII–Hyg vector backbone containing a UBQ10 promoter fragment (Krebs et al., 2012) upstream of the multiple cloning site. A 642-bp cDNA fragment of CBL9 was amplified by PCR using the primer combination 5’-ttttgggcccaaaatgggttgtttccattccacg-3’/5’-aaaagtcgactcacgtcgcaatctcgtcc-3’ and introduced into the pGPTVII–Hyg–pUBQ10 vector backbone by the ApaI/SalI restriction sites. For generation of the CBL7 oex line, a 639-bp cDNA fragment of CBL7 was amplified by PCR using the primer combination 5’-tttggcgcgccaaaatggattcaacaagaaattcagc-3’/5’-aaaggatcctcaggtatcttccacttgcg-3’ and cloned as an AscI/BamHI fragment into the pGPTVII–Kan vector backbone containing the UBQ10 promoter upstream of the multiple cloning site. The corresponding constructs pGPTVII–Hyg–pUBQ10–CBL1, pGPTVII–Hyg–pUBQ10–CBL9, and pGPTVII–Kan–pUBQ10–CBL7 were used for Agrobacterium tumefaciens(GV3101/pMP90)-mediated transformation of Arabidopsis ecotype Col-0 by the floral dip method (Clough and Bent, 1998). Transformed T1 plants were selected on 0.5 MS supplemented with 0.7% agar and 25 μg ml–1 hygromycin or 25 μg ml–1 kanamycin, respectively. Segregation analysis of the T2 and T3 generation eventually led to homozygous T3 plants, which were used for in vitro pollen germination assays.
RT–PCR Analysis of CBL1, CBL7, and CBL9 Overexpression Lines
Total RNA of 7-day-old seedlings grown on 0.5 MS supplemented with 0.7% agar was extracted with the SV Total RNA isolation system (Promega) according to the manufacturer’s instructions. 200 ng of total RNA was used for cDNA synthesis with the iScriptTM cDNA synthesis kit (Biorad). qRT–PCRs were conducted in triplicate using 2 μl of a 1:10 dilution of the respective cDNA in the case of CBL1 and CBL9 oex lines or 2 μl of undiluted cDNA for the CBL7 oex line in a total volume of 20 μl including 10 μl iQTM SYBR Green Supermix (Biorad) and 1 μl of a primer mix containing 10 pmol μl–1of each primer. The PCR reactions were performed in an iCycler (Biorad) using the following amplification program: 2min at 95°C, followed by 40 cycles of 0:20min at 95°C and 0:30min at 60°C. CBL1 was amplified with the primer combination 5’-cctctgagacagcttttagtgtg-3’/5’-tatattctccctcttccggcttt-3’, CBL7 using 5’-caagacatttgtgcaagctg-3’/5’-actcttcctcatcgatcatcc-3’, and CBL9 using 5’-gacagaatggagcaatttcg-3’/5’-gtcgttatatccctgagatacg-3’. UBQ10 served as an internal standard and was amplified with the primer combination 5’-cacactccacttggtcttgcg-3’/5’-tggtctttccggtgagagtcttca-3’. After determination of Ct values, relative expression was calculated with the REST-mcs software (Pfaffl, 2001). Overall quantification of CBL expression levels was performed with three biological replicates.
Plant Growth Conditions and In Vitro Pollen Germination Assays
Plants were grown in mixed soil in a growth chamber. The light intensity was 120–150 μmol m–2 s–1 for a 16-h daily light period, and day and night temperatures were 22 ± 2°C and 18 ± 2°C, respectively. Plants were watered once every 4 d with tap water, and the relative humidity in the growth chamber was kept near 70%.
In vitro Arabidopsis pollen germination experiments were conducted as described previously (Fan et al., 2001), except that the basic medium was slightly modified as follows: 1mM KCl, 5mM CaCl2, 0.8mM MgSO4, 1.5mM boric acid, 1% (w/v) agarose, 19.8% (w/v) sucrose, 10 μM myo-inositol, and 5mM MES. The pH was adjusted to 5.8 with Tris. The heated solution was poured into small Petri dishes (2.5 ml per dish; dish diameter was 35mm) and cooled down to form a medium layer. The dehisced anthers were carefully dipped onto the surface of the medium to make pollen grains stick. The dishes were incubated for 4.5h in a climate chamber (25 ± 0.2°C, 100% relative humidity), frozen at –20°C for 10min to quickly terminate the pollen germination and pollen tube growth, and kept on ice for counting the number of germinated pollen and measuring pollen tube length. Pollen grains with emerging tubes were considered germinated. All of the experiments were repeated four times. For each replicate, there were more than 400 pollen grains counted in order to calculate the pollen germination rate, and more than 200 pollen tubes were measured for quantification of pollen tube length.
Accession Numbers
Sequence data from this article can be found in the Arabidopsis Genome Initiative database under the following accession numbers: At4g17615 (CBL1), At4g26560 (CBL7), At5g47100 (CBL9).
SUPPLEMENTARY DATA
Supplementary Data are available at Molecular Plant Online.
FUNDING
This work was supported by grants from the DFG (Ku931/7–1, SFB629 and FOR964) to J.K. and the National Natural Science Foundation of China (grant no. 21022095 to J.K. and Y.G.). Moreover, this work was supported by the ‘Program of Introducing Talents of Discipline to Universities from China’ (grant no. B06003).
Acknowledgments
We thank Sibylle Arendt for help in generating the CBL1, CBL7, and CBL9 oex lines and Drs Oliver Batistič and Kenji Hashimoto as well as Kathrin Schlücking for providing plasmids containing CBL1G2A, CBL1EF4, CBL9, CBL1, or CBL7 cDNA. We are very grateful to Drs Ingo Heilmann and Till Ischebeck for support in establishing transient tobacco pollen transformation and thank Drs Dirk Prüfer and Michael Hippler for access to the particle gun. No conflict of interest declared.
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