TY - JOUR
AU - Geitmann, Anja
AB - Abstract The actin cytoskeleton plays a crucial role in many aspects of plant cell development. During male gametophyte development, the actin arrays are conspicuously remodeled both during pollen maturation in the anther and after pollen hydration on the receptive stigma and pollen tube elongation. Remodeling of actin arrays results from the highly orchestrated activities of numerous actin binding proteins (ABPs). A key player in actin remodeling is the actin depolymerizing factor (ADF), which increases actin filament treadmilling rates. We prepared fluorescent protein fusions of two Arabidopsis pollen-specific ADFs, ADF7 and ADF10. We monitored the expression and subcellular localization of these proteins during male gametophyte development, pollen germination and pollen tube growth. ADF7 and ADF10 were differentially expressed with the ADF7 signal appearing in the microspore stage and that of ADF10 only during the polarized microspore stage. ADF7 was associated with the microspore nucleus and the vegetative nucleus of the mature grain during less metabolically active stages, but in germinating pollen grains and elongating pollen tubes, it was associated with the subapical actin fringe. On the other hand, ADF10 was associated with filamentous actin in the developing gametophyte, in particular with the arrays surrounding the apertures of the mature pollen grain. In the shank of elongating pollen tubes, ADF10 was associated with thick actin cables. We propose possible specific functions of these two ADFs based on their differences in expression and localization. Introduction The actin cytoskeleton fulfills various functions in plant cells, some of which differ significantly from their animal counterparts. Actin arrays are the structural basis for cyclosis, the rapid motion of organelles within the plant cytoplasm (Woods et al. 1984), they are associated with the phragmoplast, a cytoskeletal configuration regulating plant cytokinesis (Higaki et al. 2008) and they seem to be involved in the perception of mechanical stimuli such as those leading to gravitropism (Kordyum et al. 2009, Stanga et al. 2009). Actin filaments also play a crucial role in the regulation of cell shape generation and the initiation of local growth events that lead to the morphogenesis of the complex shapes characterizing certain plant cell types (Smith and Oppenheimer 2005). Whereas in mammalian cells actin-mediated morphogenesis is accomplished by a direct effect of the forces exerted by actin polymerization and contraction on the surrounding plasma membrane, the morphogenetic role of actin in plant cells is thought to be exerted through the targeting of cell wall material to defined surface domains designated for cell expansion (Mathur 2006, Geitmann and Dumais 2009). How exactly this plant-specific mechanism operates is poorly understood, however. Therefore, although actin dynamics is known to be a crucial feature during plant development, its precise regulatory role in many of these developmental processes remains elusive. The spatial configuration of actin arrays and their dynamic behavior are largely controlled by the activity of proteins that influence actin filament polymerization, depolymerization, branching and bundling. Many of the proteins identified in mammalian cells are also known to be expressed in plant tissues with a varying degree of similarity in amino acid sequence and 3D structure (McCurdy et al. 2001, Hussey et al. 2006, Yokota and Shimmen 2006). One of the protein families involved in the control of plant actin dynamics is the actin depolymerizing factor (ADF)/cofilin. ADF is phylogenetically conserved in plants, animals and fungi (Hussey et al. 2002) and it is known to specifically bind the ADP-bound form of both monomeric and filamentous actin. ADF binding to filamentous actin occurs preferentially at the pointed ends where this interaction causes a change in the helical twist of the actin filament and accelerates the dissociation of actin subunits (Lopez et al. 1996, Carlier et al. 1997, Jiang et al. 1997b, McGough et al. 1997, Hussey et al. 1998, Bamburg 1999, Bowman et al. 2000, Cooper and Schafer 2000, Bamburg and Bernstein 2008). Under conditions of limited actin monomer supply, the resulting increased availability of the monomer promotes actin filament polymerization at the barbed end thus accelerating treadmilling (Carlier et al. 1997, Michelot et al. 2007). ADF/cofilin is also capable of severing actin filaments, which reduces filament length but simultaneously increases the number of available barbed ends that serve as nucleators for more polymerization activity (Hayden et al. 1993, Blanchoin and Pollard 1999, Staiger and Blanchoin 2006, Staiger et al. 2009). ADF/cofilin activity is concentration dependent—it promotes actin severing at low concentrations and induces actin nucleation and actin assembly at higher concentrations (Yeoh et al. 2002, Andrianantoandro and Pollard 2006). The ability of ADF to depolymerize actin is also pH dependent with alkaline conditions favoring this process (Carlier et al. 1997, Allwood et al. 2001, Chen et al. 2002). ADF activity is controlled by the phosphorylation state of a serine residue present at the N-terminal region of the protein (Smertenko et al. 1998, Allwood et al. 2001, Chen et al. 2003). The phosphorylation of ADF, and therefore ADF activity, was found to be controlled by a calcium-stimulated protein kinase present in plant cells (Smertenko et al. 1998). ADF can also be inactivated by phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-biphosphate (PIP2) through their binding to the actin binding domain of ADF (Yonezawa et al. 1990, 1991, Gungabissoon et al. 1998, Kusano et al. 1999). ADF activity was also found to be controlled by Rop GTPases (Chen et al. 2003), a plant member of the Rho family of GTP binding proteins, thus providing a downstream element through which many regulatory pathways are likely to act on cellular morphogenesis. The functional analysis of plant ADF through downregulation has been challenging because of the presence of numerous isoforms in the plant genome. While unicellular eukaryotes typically possess only one ADF gene and one ADF and two cofilin genes are found in most vertebrate genomes, several ADF genes exist in most higher plant species analyzed so far (Maciver and Hussey 2002, Bamburg and Bernstein 2008). The ADF family in Arabidopsis thaliana comprises 11 genes (Arabidopsis.org) that are divided into four subclasses (Mun et al. 2000, Ruzicka et al. 2007). A member of subclass 1, ADF2, was shown to be involved in the regulation of plant cell growth and differentiation, since RNAi knockdown interfered with plant development (Clement et al. 2009). The members of subclass 2, ADF7, ADF8, ADF10 and ADF11, are suspected to have roles in tip growth, a type of highly polarized growth activity that is regulated by the actin cytoskeleton. Transcriptomics and proteomics, promoter–GUS assays and immunolocalization data have shown that ADF8 and ADF11 are expressed in trichoblasts and root hairs (Ruzicka et al. 2007), whereas ADF7 and ADF10 are specifically expressed in pollen and pollen tubes (Becker et al. 2003, Honys and Twell 2003, 2004, Noir et al. 2005, Pina et al. 2005, Hruz et al. 2008, Wang et al. 2008b, Qin et al. 2009, Zou et al. 2009). This is consistent with the expression of ADF during male gametogenesis in rice (Hobo et al. 2008). Because of the expression of multiple ADF isoforms even within individual cell types, the precise functions of the proteins specific to tip-growing cells remain elusive. However, downregulation of ADF in the tip-growing moss Physcomitrella, which only has a single gene coding for ADF, severely interferes with tip growth (Augustine et al. 2008), whereas partial antisense silencing increased the actin dynamics and root hair elongation in Arabidopsis (Dong et al. 2001). This suggests that the regulation of actin dynamics through ADF activity is likely to be a crucial component of the tip growth process in higher plants. Several functional investigations of ADF in plant cells have exploited the pollen tube, a tip-growing plant cell type that grows fastest and that can therefore be expected to have an extremely dynamic actin cytoskeleton. This cylindrical, cellular protuberance is formed by the pollen after contact with a receptive stigma and can grow at rates of up to 3 mm/h. Its purpose is to deliver the immotile sperm cells from the pollen grain to the female gametophyte, which is nestled deep within the pistillar tissues. The importance of the actin cytoskeleton for the growth process is readily demonstrated by drugs resulting in actin depolymerization such as latrunculin B (LatB) or cytochalasin, which halt the process immediately (Gibbon et al. 1999, Miller et al. 1999, Vidali et al. 2001, Gossot and Geitmann 2007). A principal function of actin in pollen tubes is logistic in nature. Pollen tube growth requires significant amounts of membrane and cell wall material to be delivered to the growing surface domain in order to sustain continuous elongation over distances as long as many centimeters (Franklin-Tong 1999). This material is transported by secretory vesicles from Golgi bodies located anywhere in the cytoplasm to the small region on the cellular surface where it is needed, the growing tip (Bove et al. 2008, Zonia and Munnik 2008). Vesicles and other organelles are therefore shuttled rapidly along the tube in a bidirectional movement that is largely myosin mediated (Vidali and Hepler 2001) and occurs on actin filaments oriented in opposite directions in the periphery and central regions of the cytoplasm (Lenartowska and Michalska 2008). Close to the apex, a fine mesh of actin filaments forms a cortical fringe (Lovy-Wheeler et al. 2005) that colocalizes with an alkaline cytoplasmic region (Feijó et al. 1999). There are almost no actin filaments at the very tip of the pollen tube where vesicles accumulate during a transition phase between forward and backward movement (Kroeger et al. 2009). The guidance of vesicles to a precisely determined annular region around the pole of the cell is thought to be crucial for the generation of a perfectly cylindrical tube, since exocytosis must be under tight spatial control for geometrically correct morphogenesis (Cardenas et al. 2008, Geitmann and Dumais 2009, Fayant et al. 2010) as well as for the control of growth direction (our own unpublished data) and invasive activity (Gossot and Geitmann 2007). The crucial role of the subapical actin fringe for morphogenesis can be demonstrated experimentally since drug- or mutation-induced alterations in the cytoskeletal functioning are known to cause apical swelling and hence a loss of the perfectly polar growth activity (Hepler et al. 2001, Cheung and Wu 2008, Yang 2008, Zerzour et al. 2009). Importantly, the subapical fringe and pollen tube elongation are more sensitive to actin-depolymerizing drugs than the long-distance organelle transport occurring in the cylindrical shank of the cell (Vidali et al. 2001). For the actin arrays in the rapidly growing pollen tube to maintain their well-defined spatial configuration and to respond to external signals, their dynamics need to be tightly regulated. Actin filament polymerization, depolymerization, branching, capping and bundling must be finely tuned in order for the cell to accomplish specific functions. ADF is among the actin binding proteins (ABPs) known to operate in the subapical actin fringe as demonstrated on tobacco and lily pollen tubes (Chen et al. 2002, Lovy-Wheeler et al. 2006, Wilsen et al. 2006). This role is clearly critical since overexpression of NtADF1 inhibits tobacco pollen tube growth in a concentration-dependent manner (Chen et al. 2002, 2003). Similarly, the overexpression of a pollen-specific ADF from cotton (GhADF7) decreases pollen viability and reduces pollen tube growth (Li et al. 2010). Not only pollen tube growth but also the earlier phases of male gametophyte development are characterized by a precisely coordinated remodeling of the actin cytoskeleton (Heslop-Harrison et al. 1986, Tiwari and Polito 1988, Heslop-Harrison and Heslop-Harrison 1992, Tanaka and Wakabayashi 1992, Derksen et al. 1995, Taylor and Hepler 1997, Cai et al. 2005). Without this reorganization of the cytoskeleton, pollen germination inevitably fails and fertilization becomes impossible (Gibbon et al. 1999). In order to characterize the potential roles of ADF in this cytoskeletal reorganization, we examined the subcellular localization of ADF7 and ADF10 coupled to the cyan fluorescent protein (CFP) and the yellow fluorescent protein (YFP), respectively, during the different stages of pollen development in A. thaliana. Results Expression pattern of ADF7 and ADF10 in Arabidopsis Transcriptomic and proteomic data have shown that in Arabidopsis, ADF7 and ADF10 seem to be expressed specifically in pollen (Becker et al. 2003, Honys and Twell 2003, 2004, Noir et al. 2005, Pina et al. 2005, Ruzicka et al. 2007, Hruz et al. 2008, Wang et al. 2008b, Zou et al. 2009). ADF7 and ADF10 share 94% similarity in their amino acid sequences when using NCBI blast (Altschul et al. 1997). ADF7 shares between 77% and 92% similarity with the other members of the Arabidopsis ADF family members while for ADF10 this similarity is somewhat lower with 74–90%. ADF7 and ADF10 share >90% similarity in their amino acid sequence with the other two members of the subclass 2 ADFs, i.e. ADF8 and ADF11. A prediction search for possible nuclear export signals using the NetNES 1.1 server of the Technical University of Denmark (la Cour et al. 2004) yielded a positive peak at amino acid 21 for both ADFs with a 0.592 score for ADF10 and a 0.743 score for ADF7. In order to visualize ADF7 and ADF10, we transformed A. thaliana with chimeric ADF7 and ADF10 genes tagged with the genes for CFP and YFP, respectively, under the control of their respective native promoter and terminator sequences. To assess the expression pattern of the fusion proteins in the transformed plants, we examined CFP and YFP expression in roots, root hairs, stems, leaves, trichomes and all flower organs. With the exception of the male gametophyte, we did not observe any fluorescence above the background level in any of these organs. This confirms that Arabidopsis ADF7 and ADF10, when expressed as fluorescent protein chimeras under the control of their own promoters, are indeed pollen specific. To assert that the chimeric genes were not significantly overexpressed in the mutants, we assessed their expression in the inflorescence using semi-quantitative RT–PCR. The presence of both chimeric genes caused slightly reduced expression of the respective native genes. Expression of ADF7–CFP was at a similar level (albeit slightly higher) than the endogene and expression of ADF10–YFP was significantly lower than that of the respective endogene (Supplementary Fig. S1). ADF7 and ADF10 localization during male gametophyte development To determine the subcellular localization of ADF7 and ADF10 during the development of the male gametophyte, we harvested different stages of pollen from transformed Arabidopsis plants and observed them by confocal microscopy. The developmental stages were annotated based on the description of gametophyte morphology by Twell and coworkers (Borg et al. 2009). In order to visualize the nuclei, pollen grains were also stained with 4'-6-diamidino-2-phenylindole (DAPI) prior to observation. ADF7–CFP expression appeared for the first time during the early microspore stage just after tetrad separation. Prior to this stage no significant label was visible in the developing gametophyte (not shown). After tetrad separation, ADF7–CFP-labeled microspores displayed diffuse fluorescence in the cytoplasm and slightly more intense label in the nucleus (Fig. 1A). At this stage, ADF10–YFP expression was not yet visible (not shown). At the polarized microspore stage, ADF7–CFP was still present in the nucleus (Fig. 1B) and ADF10–YFP started to appear in short, rod-shaped aggregates in the cytoplasm (Fig. 2A). Just before the bicellular stage, ADF7–CFP disappeared from the nucleus and diffusely labeled the cytoplasm (Fig. 1C), while ADF10–YFP was associated with longer filamentous structures in the cytoplasm (Fig. 2B). At the bicellular stage, some ADF7–CFP accumulated around the vegetative nucleus and slightly denser aggregates started appearing in the periphery of the cytoplasm (Fig. 1D). At this developmental stage, ADF10–YFP appeared to be associated with longer filamentous structures, a portion of which was aggregated around the vegetative nucleus but most were concentrated at the periphery of the cell (Fig. 2C). At the mature pollen stage, short filamentous elements labeled for ADF7–CFP were present around the vegetative nucleus while longer and more densely packed filaments dominated the periphery of the cytoplasm, which also contained diffuse CFP fluorescence (Fig. 1E). ADF10–YFP at this stage displayed a very similar localization to that in the bicellular stage with filamentous structures around the vegetative nucleus and a dense mesh of filaments mostly at the periphery of the cytoplasm (Fig. 2D). Just before anthesis, ADF7–CFP formed a dense mesh of long filaments in the periphery of the cytoplasm and diffuse fluorescence was present throughout the cytoplasm and in the vegetative nucleus (Fig. 1F). Label intensity was higher at the apertures. Before anthesis, ADF10–YFP on the other hand more specifically targeted filamentous structures in the periphery of the cytoplasm with very little diffuse label and no fluorescence in the nucleus (Fig. 2E). In pollen from open flowers, ADF7–CFP labeled the vegetative nucleus and was present as a dense filamentous mesh in the periphery with high concentration at the apertures (Fig. 1F). ADF10–YFP label assumed the shape of longer and thicker filaments located at the apertures and oriented parallel to their long axes (Fig. 2F). Optical sections and surface rendering demonstrated that the label was located in the periphery of the cytoplasm (Fig. 3) and it was not detectable in the central regions of the cytoplasm. Fig. 1 View largeDownload slide ADF7–CFP expression during different stages of male gametophyte development. The first column shows maximum projections of Z-stack images acquired with the confocal microscope and the second column shows the corresponding median optical sections. The third column represents single optical sections of the same cells labeled with DAPI and the fourth column represents the corresponding brightfield images. The microspore nucleus and the vegetative nuclei are indicated with an arrow. Scale bars, 10 µm. Fig. 1 View largeDownload slide ADF7–CFP expression during different stages of male gametophyte development. The first column shows maximum projections of Z-stack images acquired with the confocal microscope and the second column shows the corresponding median optical sections. The third column represents single optical sections of the same cells labeled with DAPI and the fourth column represents the corresponding brightfield images. The microspore nucleus and the vegetative nuclei are indicated with an arrow. Scale bars, 10 µm. Fig. 2 View largeDownload slide ADF10–YFP expression during different stages of male gametophyte development. The first column represents projections of Z-images taken on the confocal microscope. The first column shows maximum projections of Z-stack images acquired with the confocal microscope and the second column shows the corresponding median optical sections. The third column represents single optical sections of the same cells labeled with DAPI and the fourth column represents the corresponding brightfield images. Vegetative nuclei are shown with an arrow. Scale bars, 10 µm. Fig. 2 View largeDownload slide ADF10–YFP expression during different stages of male gametophyte development. The first column represents projections of Z-images taken on the confocal microscope. The first column shows maximum projections of Z-stack images acquired with the confocal microscope and the second column shows the corresponding median optical sections. The third column represents single optical sections of the same cells labeled with DAPI and the fourth column represents the corresponding brightfield images. Vegetative nuclei are shown with an arrow. Scale bars, 10 µm. Fig. 3 View largeDownload slide Surface rendering image of the pollen grain at the open flower stage expressing ADF10–YFP shown in Fig. 2F. Only the upper half of the Z-stack has been used to reveal ADF localization to the peripheral region of the grain. The two groups of long filaments are located at the two apertures present in the half of the grain shown here. Scale bar, 5 µm. Fig. 3 View largeDownload slide Surface rendering image of the pollen grain at the open flower stage expressing ADF10–YFP shown in Fig. 2F. Only the upper half of the Z-stack has been used to reveal ADF localization to the peripheral region of the grain. The two groups of long filaments are located at the two apertures present in the half of the grain shown here. Scale bar, 5 µm. ADF7 and ADF10 target the actin cytoskeleton To identify the filamentous structures associated to which ADF7 and ADF10 label was observed, we labeled mature pollen expressing ADF7–CFP and ADF10–YFP from open flowers with rhodamine–phalloidin following chemical fixation (Fig. 4A, B, G, H). The spatial configuration of the filamentous structures labeled for the ADF and for actin were near identical. Surprisingly, the filamentous structures labeled for ADF7 were frequently longer than the corresponding structures labeled with phalloidin (Fig. 3G, H). A possible explanation may be that ADF7–CFP sterically blocks potential phalloidin binding sites on the actin filaments. Moreover, it has been shown that ADF-saturated actin filaments lose their phalloidin binding sites due to changes in the actin filament twist (Ressad et al. 1998, Bamburg 1999). Fig. 4 View largeDownload slide Subcellular localization of fluorescent ADF and actin labeled with rhodamine–phalloidin in mature pollen from open flowers. The first column represents ADF label, the second column shows actin labeled with rhodamine–phalloidin and last column is the corresponding brightfield image. (D–F, J–L) Pollen grains were treated with LatB prior to fixation and phalloidin label. Certain longer filaments labeled by ADF7 were not labeled with phalloidin (arrow). All fluorescence micrographs are maximum projections of Z-stacks acquired with the confocal microscope. Arrowheads indicate vegetative nuclei. Scale bars, 10 µm. Fig. 4 View largeDownload slide Subcellular localization of fluorescent ADF and actin labeled with rhodamine–phalloidin in mature pollen from open flowers. The first column represents ADF label, the second column shows actin labeled with rhodamine–phalloidin and last column is the corresponding brightfield image. (D–F, J–L) Pollen grains were treated with LatB prior to fixation and phalloidin label. Certain longer filaments labeled by ADF7 were not labeled with phalloidin (arrow). All fluorescence micrographs are maximum projections of Z-stacks acquired with the confocal microscope. Arrowheads indicate vegetative nuclei. Scale bars, 10 µm. In order to further confirm the nature of the filamentous structures labeled with ADF7 and ADF10, we administered LatB to pollen grains. LatB is a toxin isolated from the red sea sponge Latrunculia magnifica that sequesters G-actin leading to F-actin depolymerization due to the ongoing disassembly at the minus ends of the filaments (Gibbon et al. 1999). Following LatB treatment, the filamentous structures labeled with phalloidin disappeared partly or entirely (Fig. 4E, K). Similarly, label for ADF7 and ADF10 lost its filamentous configuration (Fig. 4D, J). Any remaining filamentous structures coincided with structures labeled by phalloidin (Fig. 4D, E, J, K). Diffuse fluorescence of ADF7 in the cytoplasm was strongly enhanced as a result of the LatB treatment (Fig. 4D). Interestingly, after treatment of pollen from dehiscent flowers with LatB, ADF10–YFP was associated with the vegetative nucleus (Fig. 4D). Actin arrays are typically highly dynamic. In order to assess whether the ADF-labeled structures behaved similarly, we acquired time-lapse series of hydrated pollen grains from open flowers of ADF7–CFP and ADF10–YFP mutants. The structures labeled with CFP and YFP were highly dynamic (Supplementary videos 1, Supplementary Data), as would be consistent with the behavior of actin filaments. ADF7 and ADF10 localization in the germinating pollen and the pollen tube Pollen germination requires the delivery of secretory vesicles to the aperture (Cresti et al. 1977, 1985) and the actin cytoskeleton is known to form characteristic arrays prior to and during germination (Heslop-Harrison et al. 1986, Tiwari and Polito 1988, Heslop-Harrison and Heslop-Harrison 1992, Tanaka and Wakabayashi 1992, Derksen et al. 1995, Taylor and Hepler 1997, Cai et al. 2005). In order to assess whether ADF7 or ADF10 is involved in the remodeling of the actin cytoskeleton prior to and during pollen germination, we placed mature pollen in germination medium for 1 h before observation. When the pollen grain started germinating, the filamentous elements labeled by ADF7–CFP disappeared from the grain and appeared associated with long filamentous cables seemingly winding around the tip of the newly emerging tube (Fig. 5A). In newly developed pollen tubes (<60 µm), filaments labeled with ADF7–CFP were associated with the tip of the tube and a significant amount of diffuse label was visible in the shank of the cell (Fig. 5B) and in the pollen grain. In older pollen tubes (>60 µm), ADF7–CFP targeted long filamentous cables in the subapical region of the tube and throughout the shank region (Fig. 5C). Fig. 5 View largeDownload slide Localization of ADF7–CFP in germinating Arabidopsis pollen grain (A), in short pollen tube (B) and in long pollen tube (C). All micrographs are maximum projections of Z-stacks acquired with the confocal microscope. Scale bars, 10 µm. Fig. 5 View largeDownload slide Localization of ADF7–CFP in germinating Arabidopsis pollen grain (A), in short pollen tube (B) and in long pollen tube (C). All micrographs are maximum projections of Z-stacks acquired with the confocal microscope. Scale bars, 10 µm. In germinating pollen grains expressing ADF10–YFP, fluorescence was confined to the periphery of the emerging pollen tube (Fig. 6A). Once the tube had almost attained a length that corresponded to the diameter of the grain, most of the label was found inside the tube and almost no label was left in the grain. The label was associated with longer filamentous structures that were densely packed (Fig. 6B) and could be observed to form loops (Fig. 6C). Compared with ADF7–CFP, the number of individual filaments labeled by ADF10–YFP seemed much higher and their arrangement denser (compare Fig. 4A with Fig. 5B). As the tube continued to grow, the fluorescence label for ADF10–YFP became more concentrated in the subapex where it formed shorter filaments (Fig. 6D). Pollen grains with longer tubes displayed a few, thick cables that continued from the grain into the tube (Fig. 6E, F). Less pronounced but clearly visible label was associated with the subapical fringe in longer-tube pollen tube (Fig. 6G, H). To confirm the nature of the filamentous structure in the pollen tubes, ADF10–YFP-expressing pollen tubes were fixed and labeled for actin with rhodamine–phalloidin. The label patterns for YFP and rhodamine fluorescence were identical (Fig. 7) confirming that ADF is associated with the actin cytoskeleton. Fig. 6 View largeDownload slide Localization of ADF10–YFP in germinating Arabidopsis pollen grain (A, B, C), in short pollen tube (D, F) and in long pollen tubes (E, G). (C) is a pollen grain with two emerging pollen tubes. (H) is a magnified and contrast-enhanced image of the tip of the pollen tube in (G) to show the actin fringe. All micrographs are maximum projections of Z-stacks acquired with the confocal microscope. Scale bars, 10 µm. Fig. 6 View largeDownload slide Localization of ADF10–YFP in germinating Arabidopsis pollen grain (A, B, C), in short pollen tube (D, F) and in long pollen tubes (E, G). (C) is a pollen grain with two emerging pollen tubes. (H) is a magnified and contrast-enhanced image of the tip of the pollen tube in (G) to show the actin fringe. All micrographs are maximum projections of Z-stacks acquired with the confocal microscope. Scale bars, 10 µm. Fig. 7 View largeDownload slide Arabidopsis pollen tube expressing ADF10–YFP (B) labeled with rhodamine–phalloidin (A). Images are maximum projections of Z-stacks of images taken with the Zeiss Apotome. Scale bar, 10 µm. Fig. 7 View largeDownload slide Arabidopsis pollen tube expressing ADF10–YFP (B) labeled with rhodamine–phalloidin (A). Images are maximum projections of Z-stacks of images taken with the Zeiss Apotome. Scale bar, 10 µm. Discussion Actin remodeling is of great importance in the process of plant fertilization since the movement of secretory vesicles, and hence cell wall assembly, in the growing pollen tube is based on acto-myosin transportation (DePina and Langford 1999, Geitmann and Steer 2006, Yokota and Shimmen 2006). Actin remodeling occurs during male gametophyte development, pollen grain hydration and continues during pollen germination (Heslop-Harrison et al. 1986, Tiwari and Polito 1988, Heslop-Harrison and Heslop-Harrison 1992, Tanaka and Wakabayashi 1992, Derksen et al. 1995, Taylor and Hepler 1997, Cai et al. 2005). Actin dynamics relies on the activities of several ABPs including members of the ADF family, several of which are expressed in or are specific to pollen. Given that male gametophyte expresses several ADF isoforms, we suspected that they might have distinct functions and differ in their temporal expression profile or subcellular localization. We focused on ADF7 and ADF10, which are both specifically expressed in pollen but whose functions have not been characterized. We opted for a technique for the construction of chimeric genes with intrinsically fluorescent proteins that respects three conditions. First, our strategy conserved the native expression levels of the proteins by using their native promoter and terminator sequences. This was important since changing the expression level, for example by using a highly expressing promoter such as Lat52, can alter protein function and affect cell growth especially in highly dynamic and sensitive tip growth of the pollen tube. Overexpression of several genes in the pollen tube has been demonstrated to cause swelling, reduced growth rates, abnormal morphology and altered subcellular organization (Kost et al. 1999, Li et al. 1999, Fu et al. 2001, Chen et al. 2002, 2003, Gu et al. 2003, Cheung and Wu 2004, Bosch and Hepler 2005, Yoon et al. 2006, Chang et al. 2007, Frietsch et al. 2007, Ischebeck et al. 2008, Röckel et al. 2008, Sousa et al. 2008, Wang et al. 2008a, Ye et al. 2009). Second, we used the entire sequence of the gene including the introns. Introns were shown to have an enhancement effect on gene expression (Rose 2008), specifically in actin genes (McElroy et al. 1990, Jeong et al. 2009) and ABPs (Jeong et al. 2006). Expression of Petunia ADF in Arabidopsis was particularly enhanced by the presence of the first intron (Mun et al. 2002, Jeong et al. 2007). Third, in order to conserve binding sites, functional domains and proper targeting of the proteins, insertion of the fluorescent protein in these particular sites was avoided. The technique used here, high-throughput fluorescent tagging of full-length protein (FTFLP) (Tian et al. 2004) respected these three conditions and was clearly successful since pollen grains germinated and formed perfectly shaped tubes whose morphology was indistinguishable from that of wild-type plants. Importantly, the expression levels of the transgenes was not significantly above those of the corresponding native genes in the wild-type plants (Supplementary Fig. S1). Furthermore, the gene expression pattern was consistent with that predicted by transcriptomic, proteomic and protein immunolabeling data (Honys and Twell 2003, 2004, Pina et al. 2005, Ruzicka et al. 2007) with exclusive expression in the male gametophyte and absence in all other organs. This confirms that our transformation strategy preserved native expression levels and developmental temporal and spatial expression profiles. Confocal laser scanning microscopy of different stages during gametophyte development revealed that ADF7 and ADF10 displayed different patterns of expression and subcellular localization. ADF7 was expressed earlier, starting from the microspore stage immediately after tetrad separation, whereas ADF10 was only visible at the polarized microspore stage. ADF7 showed nuclear localization at these early stages and at the late pollen grain stage. It is unclear what role ADF plays in the nucleus. ADF/cofilin have been shown to enter the nucleus in animal cells subjected to stress (Sanger et al. 1980, Nishida et al. 1987, Lida et al. 1992, Yahara et al. 1996) and in maize root cells treated with cytochalasin D (Jiang et al. 1997b). However, while vertebrate ADF and cofilin have a nuclear localization sequence that allows them to chaperone actin into the nucleus (Bamburg and Bernstein 2008), these sequences have not been found in plant ADFs. Nevertheless, ADF localization to the plant nucleus has been reported using immunolabeling (Jiang et al. 1997a, Ruzicka et al. 2007, Augustine et al. 2008). Two explanations for the nuclear localization have been proposed. ADF in the nucleus may serve to protect actin and reduce ATP loss due to actin dynamics. Alternatively, actin and ADF enter the nucleus to accomplish chromatin remodeling, to ensure structural stability of the nucleus and to contribute to proper gene expression through the possible effect of ADF on actin and therefore on RNA polymerase activity (Jockusch et al. 2006). Our data showed that the association of ADF7 with the nucleus was limited to the microspore and to the polarized microspore stages. During the subsequent developmental stages, ADF7 was not associated with the nucleus any more, until it returned to the nucleus during late mature stages. This time course suggests that ADF7 is absent from the nucleus during high metabolic activity associated with cell division and it is present in the nucleus during stages of low metabolic activity such as the microspore and the late mature or dormant pollen. This is consistent with the notion that the nuclear localization may serve as a storage form of either ADF or ADF-bound actin that can be recruited when cell division or pollen germination requires it. Unlike ADF7, ADF10 was not localized in the nucleus at any of the developmental stages observed here, but it entered the nucleus upon LatB treatment. Since our sequence search showed that both ADF7 and ADF10 possess a positive peak for nuclear export signals with a score of 0.743 and 0.592, respectively, it is possible that the ADF10 score is below the threshold for normal nuclear export and allows ADF10 nuclear targeting only during high-stress conditions. The stress-induced behavior of ADF10 is consistent with the notion that ADF inhibits actin denaturation, supporting the hypothesis that actin is packed into the nucleus to protect it during stress and make it available after the stress is removed (Hayden et al. 1993). ADF10 was not expressed in the Arabidopsis male gametophyte until the polarized microspore stage. Label for ADF10 was associated much more frequently with longer actin filaments than ADF7. It also displayed a denser distribution around the apertures (compare Fig. 6G with F). These differences suggest that ADF10 has a different function from ADF7 and that it might rather be involved in regulating actin dynamics required for the physical process of pollen tube germination, possibly by ensuring that the actin array correctly directs vesicles toward the site of pollen tube emergence. This is consistent with the fact that different ADF genes expressed in the same cell type show variable expression levels that might be related to functional diversity (Zhang et al. 2007). Our data are consistent with the dramatic changes observed in expression levels of numerous genes during microgametophyte development (Fujita et al. 2010). In short pollen tubes, ADF7 seemed to be more abundant in the subapical region of the pollen tube tip than ADF10 indicating an important role for ADF7 in the initial phase of the cell expansion process occurring at the apex of the emerging pollen tube. In pollen tubes of the same developmental stage, ADF10 was more concentrated on thick bundles of actin filaments in the shank of the pollen tube and in the grain, suggesting that ADF10 has the function to mark older filaments for turnover. It remains unknown where the actin monomers liberated from these distal filaments are used again. They would certainly be expected to be consumed at the pollen tube tip since the elongating tube requires a continuous advancement of the subapical fringe and hence substantial actin polymerization activity. However, even in the shank region of the tube polymerization of actin may be ongoing. An increased number of filaments could serve to produce thicker actin bundles, which could ensure efficient long-distance organelle transport and continuous vesicle supply toward the apical zone. This is supported by the notion that acto-myosin-mediated vesicular transport is known to happen more quickly on highly bundled actin filaments (Holweg 2007). The expression level of ADFs within a given cell might influence the subcellular localization and therefore modulate specific subcellular functions. Transient expression of NtADF1 in tobacco pollen under the control of the Lat52 promoter was observed to cause this protein to target thicker actin cables in highly expressing pollen grains, whereas in grains with lower expression level label was associated with thinner cables (Chen et al. 2002). The preferential association of ADF10 with thick actin bundles in the shank of the pollen tubes might therefore be a consequence of its relatively high expression level compared with ADF7. It must be pointed out that the interpretation of ADF expression profiles and subcellular localization needs to be done cautiously, since the presence of ADF does not necessarily mean that the protein is active. Phosphorylation is able to deactivate ADF (Smertenko et al. 1998, Allwood et al. 2001, Chen et al. 2003) even if it remains bound to actin. On the other hand, phosphoinositides are also able to inactivate ADF (Yonezawa et al. 1990, 1991, Gungabissoon et al. 1998, Kusano et al. 1999) by binding to the actin-specific site of free ADF. The fact that diffuse cytoplasmic label was more abundant for ADF7 than ADF10 could be a result of either a lower affinity of ADF7 for actin, a different steady-state equilibrium between bound and free ADF or a different control mechanism based on phosphoinositide binding. Other proteins could also regulate the activities of ADF7 and ADF10 differently. Lily ADF has for example been shown to be activated by actin interacting protein 1 (AIP1) (Allwood et al. 2002). Finally, although we qualitatively compared fluorescence intensity of ADF7–CFP and ADF10–YFP, direct quantitative comparison is proscribed, of course, since different lasers and light channels were used for image acquisition. Our data clearly show that ADF7 and ADF10 display different spatial profiles, with ADF7 showing nuclear localization during stages of lower cell activity whereas ADF10 appears in the nucleus only after exposure to stress. This suggests that ADF7 is responsible for ensuring the presence of an actin–ADF stock in the nucleus that can be activated when required for cell division or pollen tube formation, whereas ADF10 protects actin from denaturation during stress conditions. Although ADF7 labels actin filaments in the periphery of the pollen grain, ADF10 showed stronger localization in the peripheral cytoplasm of the developing gametophyte, at the apertures, in the grain after germination and at the tip of the young elongating pollen tube. In elongating pollen tubes, ADF10 seemed to be more highly expressed than ADF7 and it was associated with older actin filaments, likely marking them for recycling. ADF7 on the other hand seemed to be involved in the rapid turnover that characterizes the subapical actin fringe and that is crucial for vesicle targeting to the growing surface (Kroeger et al. 2009). The dynamics of actin filaments in the fringe is known to be crucial for pollen tube growth since its loss results in a loss of polarity and eventual growth arrest (Cardenas et al. 2005) and the ability of the pollen tube to redirect its growth upon application of external triggers is compromised (our own unpublished data). The differences in expression profiles and subcellular localization between ADF7 and ADF10 are summarized in Fig. 8. The different profiles suggest that there is a division of labor between different ADF isoforms in the Arabidopsis male gametophyte. Proof of this concept awaits experimental evidence on whether the suppression of either protein causes differential effects on pollen development, pollen tube elongation and the control of polarized and directional growth. Similarly, it will be important to determine whether the individual ADF isoforms are the targets of different signaling pathways. Fig. 8 View largeDownload slide ADF7–CFP (cyan) and ADF10–YFP (yellow) distribution in the developing male gametophyte, germinating pollen and elongating pollen tube of A. thaliana. Elements are not drawn to scale. The dashed line indicates the position of one of the three apertures. For simplicity, nuclei are not shown in the germinated pollen. Fig. 8 View largeDownload slide ADF7–CFP (cyan) and ADF10–YFP (yellow) distribution in the developing male gametophyte, germinating pollen and elongating pollen tube of A. thaliana. Elements are not drawn to scale. The dashed line indicates the position of one of the three apertures. For simplicity, nuclei are not shown in the germinated pollen. Materials and methods Fluorescent tagging and native expression of ADF To reproduce the expression level and pattern of the target gene, the construct included the 5' untranslated region (UTR) and promoter sequences (about 1000 bp), the coding region with introns, and the 3' UTR and the terminator region. To minimize further effects of the fluorescent tag on native subcellular localization and function of the chimeric protein, the location of the tag relative to the target gene was determined based on computer-assisted predictions of protein folding and functional domains. Arabidopsis plants expressing ADF7 (At4g25590) and ADF10 (At5g52360) were generated using the FTFLP technique (Tian et al. 2004). Two sets of primers, P1–P2 and P3–P4, were designed to amplify each of the target genes in two fragments. P1 and P4 contained attB1 and attB2 recombination sites respectively in addition to gene-specific sequences. P2 and P3 contained gene-specific sequences and fluorescent tag-specific sequences. ADF7 was labeled with the cyan variant of the yellow fluorescent protein (CFP) while ADF10 was labeled with YFP. Both tags contained a glycine-rich linker peptide at the N-terminal side and an alanine rich linker peptide at the C-terminal region to reduce interference with protein folding (Tian et al. 2004). ADF7 primer sequences: P1, 5' gctcgatccacctaggctatcgctgaaacgaggaacagaaag 3'; P2, 5' cacagctccacctccacctccaggccggcccactgccatccccgacgc 3'; P3, 5' tgctggtgctgctgcggccgctggggccgaggacgagtgcaagctgaag 3' and P4, 5' cgtagcgagaccacaggatcctttctaatgtgcgttgtggtt 3'. ADF10 primer sequences: P1, 5'-gctcgatccacctaggctcaatctgtttgcgctttcttttatt-3'; P2, 5'-cacagctccacctccacctccaggccggcccaccgccatccccgacgc-3'; P3, 5’-tgctggtgctgctgcggccgctggggccgaggacgagtgtaagctgaag-3' and P4, 5'-cgtagcgagaccacaggacgaaagtgagctattacacgagaa-3'. The fluorescent tag was combined with the two PCR fragments using a triple template PCR with a forward primer containing the attB1 site 5'-ggggacaagtttgtacaaaaaagcaggctgctcgatccacctaggct-3' and a reverse primer containing the attB2 site 5'-ggggaccactttgtacaagaaagctgggtcgtagcgagaccacagga-3'. Individual PCR fragments were amplified using Phusion (Finnzymes) DNA polymerase and the triple template PCR was performed using ExTaq (TaKaRa) DNA polymerase. DNA was extracted from gel using QIAquick (Qiagen) gel extraction kit. Final PCR fragments were introduced into pDONR Zeo (Invitrogen) entry vector using a BP (Invitrogen) recombination reaction according to the industrial manual. Sequencing was used to verify the positive clones. The chimeric gene was introduced into pBIN-GW (Tian et al. 2004) destination vector using LR (Invitrogen) recombination reaction according to the manufacturer's manual. Plasmid extraction from bacteria was done with QIAprep spin (Qiagen) miniprep kit. Plant material and pollen tube growth A. thaliana Col-0 plants were grown in soil at 22–20°C day/night temperatures, at 60% relative humidity in growth chambers with 16 h light/8 h dark light cycle. For pollen tube growth in vitro, pollen was collected from flowers at anthesis. Germination was conducted in Arabidopsis pollen tube growth medium for 5 h as described in Bou Daher et al. (2009). For microspore collection at different stages of development, flowers were dissected under a stereomicroscope and only gametophytes from long anthers were mounted for observation. Actin and DNA labeling After 5 h of growth, Arabidopsis pollen tubes were chemically fixed for 40 s in the microwave oven (PELCO Cold Spot® Biowave 34700) under 150 W in 3% formaldehyde, 0.5% glutaraldehyde and 0.05% Triton X-100 solution in a buffer composed of 100 mM PIPES, 5 mM MgSO4 and 0.5 mM CaCl2 at pH 9. Pollen tubes were then washed three times for 1 min each in the same buffer then incubated overnight at 4°C in rhodamine–phalloidin (Molecular Probes) in a buffer composed of 100 mM PIPES, 5 mM MgSO4, 0.5 mM CaCl2 and 10 mM EGTA at pH 7. Next day, pollen was washed five times for 1 min each in the same buffer. All washing steps were conducted in the microwave at 150 W. Pollen was then mounted on glass slides in a drop of Citifluor (Electron Microscopy Sciences), covered with a cover slip, sealed and immediately observed in the microscope. DNA was labeled by placing the gametophytes in 1 µg/ml DAPI solution in Arabidopsis pollen medium. For LatB treatment, pollen was incubated in Arabidopsis medium containing 100 nM LatB before fixation and actin labeling. Microscopic observations Arabidopsis gametophytes from lines expressing the fluorescent proteins were observed in a Zeiss Imager-Z1® microscope equipped for structured illumination microscopy (Apotome) and with a Zeiss AxioCam MRm camera. A filter set of BP 450–490 excitation, FT 510 beamsplitter and BP 515–565 emission was used. For confocal imaging, a Zeiss LSM 510 META/LSM 5 LIVE/Axiovert 200M system confocal microscope was used. A 488 nm argon laser was used for YFP and 458 nm for CFP excitation. For surface rendering and to show a view from inside the pollen grain, the upper half of the Z-stack was used to produce a 3D reconstruction of half a grain using the inside 4D function of AxioVision 4.8 (Zeiss) software. Semi-quantitative RT-PCR Total RNA was extracted from Arabidopsis flowers (wild type, ADF7–CFP and ADF10–YFP) using Trizol reagent (Invitrogen). Five micrograms were used to synthesize cDNA using the SuperScript® III First-Strand Synthesis SuperMix (Invitrogen). ADF7 and ADF10 endogenes were amplified using as a forward primer: 5'-GAACGCGGCGTCGGGGATG-3' and as reverse primers: 5'-GAGCTTGCATACACCATCTTC-3' and 5'-GAGCTCGCATACACCATCTTC-3', respectively. In order to amplify only the transgene, the corresponding forward primer was chosen on the fluorescent protein: 5'-CACTACCTGAGCACCCAGT-3' and 5'-GCAGCGTGCAGCTCGCCGAC-3' for ADF7–CFP and ADF10–YFP, respectively. Amplified fragments were obtained from 25 cycles using 58°C for annealing and 45 s of elongation time. A total of 5 µl of each reaction was loaded on a preparative 1% (w/v) agarose gel stained with ethidium bromide and band intensity on the photographs was quantified using ImageJ software (http://rsbweb.nih.gov/ij/). The endogene expression levels in the transgenic lines were used as a reference value for expression after normalization based on the actin expression profile. Funding This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Fonds Québécois de la Recherche sur la Nature et les Technologies (FQRNT) to A.G. Acknowledgments The authors would like to thank professor Natasha Raikhel for kindly providing the p-Citrine3, p-CFP and pMN-GW plasmids. Abbreviations Abbreviations ADF actin depolymerizing factor CFP cyan fluorescent protein DAPI 4'-6-diamidino-2-phenylindole LatB latrunculin B YFP yellow fluorescent protein. 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TI - Spatial and Temporal Expression of Actin Depolymerizing Factors ADF7 and ADF10 during Male Gametophyte Development in Arabidopsis thaliana
JF - Plant and Cell Physiology
DO - 10.1093/pcp/pcr068
DA - 2011-06-01
UR - https://www.deepdyve.com/lp/oxford-university-press/spatial-and-temporal-expression-of-actin-depolymerizing-factors-adf7-3cbQHEeTFB
SP - 1177
EP - 1192
VL - 52
IS - 7
DP - DeepDyve
ER -