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First published online November 2, 2007; 10.1105/tpc.107.053413 The Plant Cell 19:3705-3722 (2007) © 2007 American Society of Plant Biologists Profilin Is Essential for Tip Growth in the Moss Physcomitrella patens[W]
a Biology Department, University of Massachusetts, Amherst, Massachusetts 01003 1 Address correspondence to bezanilla{at}bio.umass.edu.
The actin cytoskeleton is critical for tip growth in plants. Profilin is the main monomer actin binding protein in plant cells. The moss Physcomitrella patens has three profilin genes, which are monophyletic, suggesting a single ancestor for plant profilins. Here, we used RNA interference (RNAi) to determine the loss-of-function phenotype of profilin. Reduction of profilin leads to a complete loss of tip growth and a partial inhibition of cell division, resulting in plants with small rounded cells and fewer cells. We silenced all profilins by targeting their 3' untranslated region sequences, enabling complementation analyses by expression of profilin coding sequences. We show that any moss or a lily (Lilium longiflorum) profilin support tip growth. Profilin with a mutation in its actin binding site is unable to rescue profilin RNAi, while a mutation in the poly-L-proline binding site weakly rescues. We show that moss tip growing cells contain a prominent subapical cortical F-actin structure composed of parallel actin cables. Cells lacking profilin lose this structure; instead, their F-actin is disorganized and forms polarized cortical patches. Plants expressing the actin and poly-L-proline binding mutants exhibited similar F-actin disorganization. These results demonstrate that profilin and its binding to actin are essential for tip growth. Additionally, profilin is not needed for formation of F-actin, but profilin and its interactions with actin and poly-L-proline ligands are required to properly organize F-actin.
Tip growth in plants is required for the development of an essential subset of plant cells, including pollen tubes and root hairs in seed plants, the filamentous tissues of mosses and ferns (protonemata), and algal rhizoids. Tip growth is a complex process that involves the orchestration of many cellular events (Hepler et al., 2001  ) and is thought to depend on differential cell wall extensibility and turgor-driven cell expansion (Hepler et al., 2001; Harold, 2002). The actin cytoskeleton has emerged as a central component for polarization and cell growth in plants. Various studies using inhibitors of the actin cytoskeleton indicate that actin dynamics are essential for tip growth (Mascarenhas and Lafountain, 1972; Doonan et al., 1988; Gibbon et al., 1999; Hepler et al., 2001; Vidali and Hepler, 2001; Vidali et al., 2001). However, few molecular mechanisms behind actin dynamics and tip growth in plants are known.
Controlling actin dynamics revolves around proper maintenance of the balance between monomeric and filamentous actin and higher-order organization of actin filaments in the cell. This control is achieved by the interaction of actin with a multitude of actin binding proteins, whose effects include regulating the available monomeric actin pool, the availability of polymerizable ends, the creation of new actin filaments, disassembly of old actin filaments, and the assembly of filaments into bundles. Studies have demonstrated the consequences of altering the levels of key actin binding proteins in tip growth in plants. For example, overexpression of the actin depolymerizing protein ADF/cofilin inhibits pollen tube and root hair elongation (Dong et al., 2001
One of the key regulators of the actin cytoskeleton is the small actin monomer binding protein, profilin. Profilin is essential for viability in eukaryotes ranging from fungi to animals (Magdolen et al., 1988
In addition to actin, profilin can bind to phosphoinositides and poly-L-proline (polyproline) stretches. Analysis in yeast has demonstrated that actin binding and polyproline binding sites are essential for viability (Wolven et al., 2000
Profilin is essential in fungi and animals since its absence leads to the inhibition of critical processes, such as cytokinesis and cell migration (Balasubramanian et al., 1994
To study profilin's role in tip growth, we analyzed the loss of profilin function in the tip-growing protonemal cells of the moss Physcomitrella patens (Menand et al., 2007
P. patens Has Three Profilin Genes In general, plants contain many profilin isoforms; for example, Arabidopsis has five (Christensen et al., 1996 ; Kandasamy et al., 2002), maize (Zea mays) has at least five (Staiger et al., 1993; Gibbon et al., 1998; Kovar et al., 2001a), and tobacco (Nicotiana tabacum) has at least three (Mittermann et al., 1995). To identify how many profilin genes are present in P. patens, we searched the P. patens genome (http://genome.jgi-psf.org//Phypa1_1/Phypa1_1.home.html) and found three profilin genes. We used information from two different EST collections (Nishiyama et al., 2003) to construct gene models. The models contain four exons and three introns each. The distribution of exons and introns is similar to that of profilin genes from Arabidopsis, but the three P. patens profilin genes have an additional 5' untranslated region (UTR) exon (Figure 1A
, exon 0). Coding sequence exon/intron junctions were confirmed by amplifying the coding sequence from cDNA and sequencing. A high degree of conservation is evident since the relative position of junction sites between exons is identical in P. patens and Arabidopsis profilins.
To determine which profilin gene is expressed in protonemata, we performed comparative RT-PCR using protonemal total RNA. We found that the most abundant isoform is PRFa, followed by PRFc, with PRFb exhibiting a very low level of expression (Figure 1B). Thus, to ensure complete profilin loss of function, it is necessary to suppress the expression of all three profilin genes. To compare the P. patens profilins between themselves and other profilins and to identify residues critical for moss profilin function, we constructed an amino acid alignment of several well-characterized profilins, which included sequences from maize, Schizosaccharomyces pombe, Saccharomyces cerevisiae, and humans (Figure 1C, Table 1 ). As expected, we found that residues in the actin and polyproline binding sites are highly conserved (Figure 1C). Based on this alignment, we predict that moss profilins do not enhance actin's ATP exchange. As is the case for other plant profilins, moss profilins contain an Arg at position 85 (Figure 1C, black arrow), which prevents profilin's enhancement of ATP exchange on actin (Perelroizen et al., 1996 ; Kovar et al., 2000; Lu and Pollard, 2001).
Because several profilin isoforms are present in most vascular plants (Huang et al., 1996
Knockdown of Profilin Inhibits Tip Growth and Proliferation We tested the effect of profilin RNAi on protonemal growth using an RNAi construct containing a highly conserved sequence, including part of the coding sequence of all three profilin isoforms (CDS-RNAi). The identity in the coding sequence region of this construct is 89% with PRFb and 75% with PRFc (see Supplemental Figure 2 online). Protonemal cells of GFP-negative plants transformed with CDS-RNAi do not exhibit tip growth, as evaluated by total plant area estimated from the area of chlorophyll autofluorescence (Figures 2A and 2B ).
To demonstrate that the profilin RNAi phenotype is specific to loss of profilin function, we tested for rescue of the profilin RNAi phenotype by expression of wild-type profilin. To do this, the expression construct must be insensitive to the silencing effects of the RNAi construct. Thus, we generated a second profilin RNAi construct targeting the 3'UTR of all three moss profilins. This construct consists of the 3'UTR of PRFb, which is almost identical to that of PRFa (82% identity), fused to the 3'UTR of PRFc (UTR-RNAi; see Supplemental Figure 2 online). To determine whether the UTR-RNAi construct phenocopies the CDS-RNAi construct, we compared the efficiency of these constructs at disrupting tip growth of protonemal cells. The two RNAi constructs are indistinguishable with respect to inhibition of growth (Figure 2). To isolate individual plants for scanning electron microscopy and immunofluorescence analysis, we modified the established protoplast regeneration protocol by substituting liquid medium for the top agar medium. The number of protoplasts regenerating under these conditions was reduced by approximately half, but the plants regenerated and grew faster (compare GUS-RNAi control in Figures 2B and 6B). Because of the faster regeneration, we were able to isolate single plants undergoing gene silencing at just a week after transformation.
When analyzed with the scanning electron microscope, 1-week-old, control GUS-RNAi plants have protonemata that are branched and filamentous (Figures 3A and 3B ). By contrast, UTR-RNAi plants are small and have spherical cells that form clusters (Figures 3C and 3D). Some of these round cells can expand isotropically but no longer undergo tip growth (Figure 4 ; see Supplemental Movies 1 and 2 online).
The smaller profilin RNAi plants presumably result from smaller cells but could also be due to an inhibition of cell division. To determine if profilin RNAi plants have similar numbers of cells compared with GUS-RNAi transformed plants, we counted the nuclei in both silenced and control plants using 4',6-diamidino-2-phenylindole (DAPI) as a nuclear stain. Similar to control plants, we found that the large majority of profilin RNAi cells contains only one nucleus (Figure 5A , bottom row), suggesting that mitosis and some form of cytokinesis is possible in the profilin RNAi plants. Nevertheless, profilin RNAi plants have 25% of the number of nuclei present in control plants (Figure 5B). We interpret this result as an indication that cell division is coupled to cell growth in moss protonema; thus, cells with impaired growth are delayed in cell division. Actively silenced profilin RNAi plants are unable to grow for more than 2 weeks after transformation, suggesting that long-term profilin deficiency is lethal.
The Profilin Loss-of-Function Phenotype Can Be Complemented with All Three Moss Profilins To complement the UTR-RNAi loss-of-function phenotype, we cotransformed UTR-RNAi with a plasmid driving PRFa expression from the strong constitutive maize ubiquitin promoter. Under these conditions, plants lacking nuclear GFP form long cells with abundant branches indistinguishable from control plants, while profilin RNAi plants are small and contain spherical cells (Figure 6A ). We also tested the two other moss profilins for their ability to complement. Both PRFb and PRFc rescued the RNAi phenotype, demonstrating that a single moss profilin is sufficient for tip growth (Figure 6A).
To have a quantitative and statistical estimate for the levels of complementation between different constructs, we focused our analysis on three morphological parameters deduced from the chlorophyll autofluorescence of each plant. The plants are analyzed by automated morphometry using digital images and computer algorithms (see Methods). This approach generates a relatively large sample size, which permits robust statistical analyses. The parameters are overall size (area), the degree of polarized extension (circularity), and the degree of polarization and branching (solidity) of the plant. These parameters are described fully in Methods; briefly, circularity reflects the ratio of plant area to plant perimeter, and solidity reflects the presence of concavities in the plant. For circularity, a value of one represents a perfect circle, while values approaching zero have a more linear structure; for solidity, a plant that has no branches is solid and has a value of one, whereas a plant with many branches has empty space between the branches, lowering the solidity value. Between these three factors it is possible to determine if the cells in a plant are undergoing normal tip growth, in particular when compared with control plants. Fully rescued plants attained values of all three parameters that are statistically indistinguishable from control GUS-RNAi plants (Figures 6B to 6D, Table 2 ), indicating normal tip growth. By contrast, there were highly significant differences between the UTR-RNAi plants and the control in all three parameters analyzed (Table 2). To determine if a distantly related profilin from a seed plant can substitute for endogenous moss profilins, we tested whether lily (Lillium longiflorum) profilin rescues the profilin RNAi phenotype. We found that lily PRF1 rescues as efficiently as the moss profilins as estimated by all three morphological parameters (Figure 6, Table 2).
Profilin Binding Sites for Actin and Polyproline Are Required for Tip Growth
To abolish profilin binding to actin, we introduced a K87E mutation. The analogous mutation in S. pombe greatly reduced the affinity of profilin for actin without destabilizing the protein or affecting polyproline binding (Lu and Pollard, 2001
To test for in vivo significance of the polyproline binding site of profilin, we introduced a Y6D mutation in the PRFa expression construct. Similar mutations have been shown to greatly decrease the affinity of profilin for polyproline without disrupting its affinity for actin or destabilizing the protein (Kovar et al., 2001a
Endogenous Levels of Profilin Are Reduced by RNAi
We used the antibody to immunostain moss protonemata expressing either a control GUS-RNAi construct or profilin UTR-RNAi. Compared with the GUS-RNAi control, plants expressing UTR-RNAi stain weakly for profilin (Figure 7B). Complementation of UTR-RNAi with PRFa restored a high level of staining, as does coexpression with K87E and Y6D. These results demonstrate that profilin protein levels are reduced in the profilin RNAi plants and that the levels are increased by coexpression with profilin constructs. Note that confirming these results by protein gel blot analysis was not attempted because silenced plants used for immunostaining, preselected based on the loss of the nuclear GFP signal, were too limited in number and mass for reliable protein extraction. To evaluate profilin levels, we measured fluorescence intensity of the immunostained plants. All values were background subtracted and normalized to GUS-RNAi levels (Figure 7C, Table 3 ). This quantification confirmed that expression of all moss profilin constructs was similar. Plants complemented by expression of lily PRF1 gave a stronger immunofluorescence signal (Figure 7, Table 3), which presumably reflects the fact that the polyclonal antibodies were generated against profilins from lily pollen.
The F-Actin Network Is Disrupted in Profilin RNAi Plants
Profilin RNAi cells also contain abundant cortical F-actin. However, the actin filaments are no longer longitudinally oriented, appearing shorter and less organized (Figure 8). To quantify the degree of disorganization, we analyzed the orientation of the actin filaments using a method developed to analyze the orientation of structures in the cell wall (Marga et al., 2005 ). In this method, the image of the cell is subjected to fast Fourier transform (FFT), and the elliptical shape of the transform is analyzed: the more eccentric the ellipse (value approaching one) the greater the orientational order. On average, the FFT of F-actin images from GUS-RNAi cells exhibited ellipses with high eccentricity, whereas profilin RNAi images were more circular (Figure 9
), indicating that the F-actin is more axially organized in GUS-RNAi cells and randomized in orientation in profilin RNAi cells.
Despite the fact that profilin RNAi cells are round, many observed cells contain F-actin patches, cortical structures containing actin filaments that are polarized to one end of the cell (Figure 8, arrowheads). Interestingly, this polarization is always toward the pole that is opposite to the wall of the neighboring cell. Profilin RNAi plants complemented with the PRFa construct have the same F-actin distribution as GUS-RNAi plants (Figures 8 and 9); the caulonemal and chloronemal tip cells have clear fringe structures, and the side branches exhibit a concentration of apically localized cortical F-actin (Figure 8, +PRFa). In addition, the degree of axial orientation is restored (Figure 9). RNAi plants expressing profilin containing the K87E mutation that disrupts the actin binding site have a similar F-actin distribution to profilin RNAi cells; F-actin is present but is not organized (Figure 9), and many cells have polarized cortical patches of F-actin (Figure 8, +K87E left and right panels). RNAi plants expressing profilin containing the Y6D mutation also contain F-actin. Some of these cells show a slight amount of polarized growth; in those cells, the actin is slightly axially oriented (Figure 8, +Y6D right panel). However, on average, the actin filament organization is randomized in Y6D expressing cells to the same extent as profilin RNAi and K87E expressing cells (Figure 9). Other cells expressing profilin with the Y6D mutation have a more rounded morphology and also show polarized patches of F-actin (Figure 8, +Y6D middle panel). Occasionally when a side branch forms, the branch contains apical F-actin similar to control cells (Figure 8, +Y6D left panel).
Our results demonstrate that profilin is required for tip cell growth in plants. Using RNAi to reduce the levels of all profilin genes in moss protonemal cells, we reproducibly observed that profilin RNAi plants are dramatically smaller than control plants, and individual cells are small and rounded. This phenotype is observed with either the CDS-RNAi construct or the UTR-RNAi construct. In addition, the immunofluorescence data support that profilin levels were reduced (Figure 7). Since the CDS-RNAi construct contains a region of sequence from PRFa and the UTR-RNAi construct contains regions of sequence from PRFb and PRFc, we are confident that all profilin function is greatly reduced in these RNAi studies. Thus, the strategy of using one sequence to knock down multiple family members is valid. Furthermore, compared with gene knockouts, this transient RNAi approach is much more rapid. In fact, gene knockouts may not be possible to obtain, since our results strongly suggest that profilin function is essential for plant survival.
By observing profilin-RNAi plants using time-lapse microscopy (Figure 4B; see Supplemental Movie 2 online), we found that the profilin-RNAi cells grow diffusely instead of by focusing growth at their apex. Thus, profilin is essential for maintaining a site for tip growth. To investigate how the absence of profilin affects the actin cytoskeleton, we used fluorescent phalloidin to stain F-actin. In control plants, we found a prominent subapical structure reminiscent of the collar or fringe described in pollen tubes (Kost et al., 1998
In the absence of profilin, moss cells round up and the actin cytoskeleton loses its axial orientation. An organized cortical fringe was not detected in profilin RNAi cells, suggesting that this structure is required for tip growth. We were able to detect abundant actin filaments in profilin RNAi plants. Qualitatively, the amount of F-actin in profilin RNAi plants was not very different from control plants. The staining method we employed requires the continuous presence of fluorescent phalloidin, which complicates a quantitative analysis of F-actin levels. Further optimization of this method will be required to estimate the levels of F-actin. Nevertheless, the presence of F-actin in profilin RNAi cells is similar to what has been reported in other systems where profilin levels have been reduced or abolished (Haarer et al., 1990 Surprisingly, the profilin RNAi cells are able to polarize a subset of actin structures. These structures are composed of short actin filaments that are often tightly opposed to the plasma membrane. These filaments accumulate in the external pole of the cell, which is the region distal to the neighboring cell. The ability of F-actin to polarize in the absence of profilin raises an important question as to how polarity is established and maintained. It also suggests that profilin functions downstream of an F-actin polarizing mechanism and may be necessary to assemble the cortical F-actin structure required for polarized growth. To investigate the molecular basis of how profilin participates in tip growth, we took advantage of the ability to complement the profilin-RNAi phenotype in planta. We have been able to complement plants by simultaneous transformation of the UTR-RNAi plasmid and a profilin-expression plasmid. Using this cotransformation assay, the number of plants with the profilin-RNAi phenotype was reduced to undetectable levels. In addition, this complementation result verifies the specificity of the assay, since expression of wild-type profilin rescues all the phenotypes observed, ruling out the possibility that another gene is being affected by the RNAi construct. Notably, all three moss profilins complement the RNAi phenotype with similar efficiency (Figure 6), indicating that there is a strong conservation of function between profilin isoforms. Furthermore, the capacity of lily pollen profilin to complement shows that this conservation extends to distantly related plant species. The complementation studies in planta are ideal for undertaking a detailed molecular analysis of protein function. Based on the conservation of profilin structure and function, we selected mutations in profilin that are known to specifically ablate actin or polyproline binding in other organisms. We selected two residues that have been well characterized in fission yeast, budding yeast, humans, and maize. The mutated residues are highly conserved across all profilins (Figure 1), and the introduced mutations, K87E and Y6D, should abolish binding to actin and polyproline, respectively.
Previous work in S. pombe has shown that a change to Glu in the equivalent position to K87 in moss rendered the protein nonfunctional in vivo, while maintaining normal protein stability and binding to polyproline in vitro (Lu and Pollard, 2001 Our results show that, in moss protonemata, profilin harboring the K87E mutation is unable to complement profilin-RNAi, demonstrating that profilin's actin binding site is essential for tip growth. The role of profilin could be to maintain a pool of unpolymerized actin or to enhance actin polymerization at newly formed barbed ends via interactions with formins. The organization of the actin cytoskeleton in these cells was similar to that observed in profilin RNAi cells. F-actin was still present, and some polarization of small F-actin structures could be observed. This indicates that the actin binding site of profilin is essential for profilin to organize the actin cytoskeleton.
Profilin interaction with formins is known to be mediated by polyproline binding. Thus, we investigated the effect of disrupting polyproline binding. Mutations equivalent to position Y6 were previously tested in S. pombe (Lu and Pollard, 2001
Plants expressing profilin with the Y6D mutation show an interesting intermediate phenotype, indicating that an intact polyproline binding site is an important aspect of profilin function in vivo. With this mutation, we predict that the actin monomer binding activity of profilin remains intact, but profilin would be unable to enhance actin polymerization in combination with polyproline-containing proteins, such as the formins. Formins have been shown to be important for cell polarization in other systems (Evangelista et al., 1997 One potential limitation of our complementation assay is that either the absence of the expression plasmid or lack of protein expression could result in the inability to rescue, thus producing a false negative result. To control for this, we selected actively silenced plants (by their lack of nuclear GFP fluorescence) and performed immunofluorescence using an anti-lily profilin antibody that cross-reacts with moss profilins to test for the levels of profilin mutants in the transformed plants, regardless of whether the plants were complemented. Further confirming the specificity of the immunostaining, we observed an additional increase in signal in the plants transformed with the lily profilin construct. This is expected since the antibody was generated against lily pollen profilin and should therefore have a higher affinity for lily profilin. These results confirm that the analyzed plants expressed the transformed constructs and that the lack of complementation was fully due to mutations in the binding sites.
Previous work in pollen tubes using microinjection has shown that the actin binding site of profilin was necessary for profilin to inhibit pollen tube tip growth and that the polyproline binding site of profilin was not critical for this inhibition (McKenna et al., 2004
RT-PCR We isolated total RNA from 6- to 7-d-old moss protonemal tissue using the RNeasy plant mini kit from Qiagen. One microgram of total RNA was used as template for reverse transcription (Thermoscript RT from Invitrogen) and primed with an oligo(dT) primer. Equivalent amounts of cDNA template were used for amplification of small fragments of PRFa, PRFb, and PRFc (Figure 1). Primers used for amplification are listed in Table 4 .
Protein Sequence Alignment and Phylogenetic Analysis Protein sequences for profilins from a variety of plants were downloaded form the Swiss-Prot repository (http://ca.expasy.org/sprot/). Sequences for all three Physcomitrella patens profilins were deduced from cDNA sequences. The amino acid alignment presented in Figure 1 was initially done using the structural alignment of Swiss-PdbViewer (Guex and Peitsch, 1997 ) using profilin structures available in the Protein Data Bank (http://www.pdb.org/); subsequent alignment for other profilins was done manually. For the phylogenetic analysis presented in Supplemental Figure 1 online, alignments were done initially with ClustalW and manually aligned based on the structural alignment from Figure 1 (alignment presented in Supplemental Figure 3 online). The Phylip suite of programs was used to infer a parsimony-based phylogeny. Parsimony was run with a 1000 bootstrap for the final tree. Schizosaccharomyces pombe profilin was used as an outgroup to root the presented tree. A similar tree was obtained using neighbor-joining algorithms.
Tissue Culture and Protoplast Transformation
Construct Generation
The resulting PCR product was cloned into pENT-TOPO (Invitrogen) using the manufacturer's recommendations and subsequently sequenced. The PRFa CDS-RNAi fragment was transferred to the destination vector pUGGi (Bezanilla et al., 2005 To generate UTR-RNAi, we compared the 3'UTR regions of the three profilin cDNAs and selected a 249-bp region of PRFb that is almost identical between PRFa and PRFb, this region starts 400 bp after the ATG and ends 649 bp after the ATG. This region was fused to a corresponding region of PRFc, starting 400 bp after the ATG and ending 680 bp after the ATG, which is 280 bp long (see Supplemental Figure 1 online), using PCR amplification as follows. The PCR fragment of PRFb was amplified from genomic DNA with a BamHI site engineered into the 3' end of the product using primers PRFbUT-F and PRFbUTBam-R (Table 4). The PCR fragment of PRFc was amplified from genomic DNA with a BamHI site designed into the 5' end of the product using primers PRFcUTBam-F and PRFcUT-R (Table 4). The two products were ligated together and further amplified using the PRFb forward primer and PRFc reverse primer, for a total length of 529 bp. This product was cloned in pENT-TOPO, sequenced, and transferred into pUGGi as described above.
Expression constructs were generated using a similar strategy as outlined above: coding sequences for PRFa, PRFb, and PRFc were amplified from P. patens cDNA using specific primers (Table 4) containing CACC at the 5' end for oriented cloning into pENT-TOPO. Lily (Lilium longiflorum) profilin was subcloned from a cDNA clone (McKenna et al., 2004
Scanning Electron Microscopy
Time-Lapse Microscopy
Nuclear Counting
Morphometric Analysis
Plants without a nuclear GFP signal were selected from the images using a 500 x 500 pixel cropping square. The red channel of the color images corresponding to chlorophyll fluorescence was digitally separated. The resulting 8-bit image was manually thresholded and the total area estimated as the number of pixels selected; the same threshold setting was used for all plants from a single experiment. Two more morphometric parameters were evaluated: circularity defined as 4
Immunostaining and Protein Gel Blotting Immunostaining was used to estimate the levels of profilin in the RNAi and complemented plants. Every plant undergoing active silencing was selected by the absence of nuclear GFP signal under a stereomicroscope equipped with epifluorescence optics; there was no additional selection. Plants were lifted with a sharp metal needle immobilized to a glass rod and immediately immersed into fixative (2% formaldehyde, 25 mM PIPES, pH 6.8, 5 mM MgCl2, and 1 mM CaCl2). The plants were collected in containers constructed with a 20-µm nylon mesh immobilized to the cap of a PCR tube; this system allows for the change of solutions without disrupting the cells while using small volumes in 96-well plates. Plants were fixed for 30 min and washed three times in PME buffer 1 (25 mM PIPES, pH 6.8, 5 mM MgCl2, and 5 mM EGTA). The membranes were permeabilized with 0.1% Triton X-100 in PME for 30 min, the cell walls digested with 0.2% driselase (Sigma-Aldrich) in PME for 30 min, and then subsequently washed two times in PME and two times in TBST (125 mM NaCl, 25 mM Tris-HCl, pH 8, and 0.05% Tween 20). Nonspecific sites were blocked in TBST + 5% BSA for 1 h and incubated overnight with a 1:100 dilution of antiprofilin antibody or preimmune serum. The next day, plants were washed three times for 10 min each in TBST and incubated for 3 h in 1:200 dilution of CY3 goat anti-rabbit (Jackson Immuno Research). After three washes with TBST, the plants were incubated in 10 µg/mL of DAPI in TBST and mounted in this solution for observation. Image pairs were taken with the CY3 filter setting (excitation 545/30, dichroic 570 long pass, emission 620/60) and chlorophyll setting (excitation 480/40, dichroic 505 long pass, emission 510 long pass) on a Leica stereomicroscope. All settings and exposure times were kept constant. For quantification of the fluorescent signal, we used threshold segmentation. A threshold value was determined that included all background signal from the preimmune serum–treated plants. This threshold value was used to calculate average fluorescence values (total fluorescence/area) under all other conditions. An average of the background was calculated from the preimmune serum values and subtracted from the other measurements. The value obtained for the GUS-RNAi plants after background subtraction was used to normalize the rest of the values, which are presented as a fraction of the control (GUS-RNAi). The experiment was performed four times and the mean values for all plants calculated.
Fluorescent Phalloidin Staining of Actin Filaments The identified plants were collected with a 20-µL pipette tip and transferred to the well of a 96-well plate containing 250 µL of PME buffer. Excess PME buffer was removed for a final volume of 90 µL. Saponin (Calbiochem) was added directly to the well from a 10% stock in water to a final concentration of 1%, and Alexa-488 phalloidin (Invitrogen) was added from a 66.6 µM stock in methanol to a final concentration of 0.666 µM. Plants were stained from 2 to 24 h. Plants were mounted in 30 µL of the same staining solution between a glass slide and a cover slip using hot wax as a sealant. Plants were visualized immediately after mounting using the 488 argon laser of a Nikon confocal microscope (Nikon D-Eclipse-C1) on an inverted stand (Nikon Eclipse-TE2000-S) using a x60 oil immersion 1.4–numerical aperture objective and a pixel size of 83 nm. Several confocal sections, 0.5 µm apart, were acquired for each cell, and most optical sections consisted of the bottom half of the cell including the cortical area that contained the majority of the F-actin structures. Further image processing was done with AutoDeblurGold Cf (MediaCybernetics) using five three-dimensional deconvolution iterations and displayed as a maximal Z-projection.
Quantification of Actin Filament Alignment with the FFT
Statistical Analyses
Accession Numbers
Supplemental Data
We thank Pierre-Francois Perroud and Ralph Quatrano for kindly providing the pTHUBI-Gate plasmid and providing insightful discussion (supported by National Science Foundation Grant IBN-0112461). We also thank Margaret Riley and Michelle Lizotte-Waniewski for their help with the phylogenetic analysis of plant profilins. We thank Wei-Lih Lee for instruction on the construction of the time-lapse imaging chamber and Dale Callaham for assistance with the preparation and analysis of material for scanning electron microscopy. Peter Hepler, Tobias Baskin, and Patricia Wadsworth provided useful comments on the writing of the manuscript. This work was supported by the National Science Foundation (MCB-0516702 and MCB-0640530).
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Magdalena Bezanilla (bezanilla{at}bio.umass.edu).
[W] Online version contains Web-only data. www.plantcell.org/cgi/doi/10.1105/tpc.107.053413 Received June 5, 2007; Revision received October 5, 2007. accepted October 10, 2007.
Ashton, N.W., Grimsley, N.H., and Cove, D. (1979). Analysis of gametophytic development in the moss, Physcomitrella patens, using auxin and cytokinin resistant mutants. Planta 144: 427–435.[CrossRef][Web of Science] Balasubramanian, M.K., Hirani, B.R., Burke, J.D., and Gould, K.L. (1994). The Schizosaccharomyces pombe cdc3+ gene encodes a profilin essential for cytokinesis. J. Cell Biol. 125: 1289–1301. Bezanilla, M., Pan, A., and Quatrano, R.S. (2003). RNA interference in the moss Physcomitrella patens. Plant Physiol. 133: 470–474. Bezanilla, M., Perroud, P.F., Pan, A., Klueh, P., and Quatrano, R.S. (2005). An RNAi system in Physcomitrella patens with an internal marker for silencing allows for rapid identification of loss of function phenotypes. Plant Biol (Stuttg) 7: 251–257.[CrossRef][Medline] Chen, C.Y., Wong, E.I., Vidali, L., Estavillo, A., Hepler, P.K., Wu, H.M., and Cheung, A.Y. (2002). The regulation of actin organization by actin-depolymerizing factor in elongating pollen tubes. Plant Cell 14: 2175–2190. Cheung, A.Y., and Wu, H.M. (2004). Overexpression of an Arabidopsis formin stimulates supernumerary actin cable formation from pollen tube cell membrane. Plant Cell 16: 257–269. Christensen, H.E., Ramachandran, S., Tan, C.T., Surana, U., Dong, C.H., and Chua, N.H. (1996). Arabidopsis profilins are functionally similar to yeast profilins: Identification of a vascular bundle-specific profilin and a pollen-specific profilin. Plant J. 10: 269–279.[CrossRef][Web of Science][Medline] Cooley, L., Verheyen, E., and Ayers, K. (1992). Chickadee encodes a profilin required for intercellular cytoplasm transport during Drosophila oogenesis. Cell 69: 173–184.[CrossRef][Web of Science][Medline] Cvrckova, F., Novotny, M., Pickova, D., and Zarsky, V. (2004). Formin homology 2 domains occur in multiple contexts in angiosperms. BMC Genomics 5: 44.[CrossRef][Medline] Deeks, M.J., Cvrckova, F., Machesky, L.M., Mikitova, V., Ketelaar, T., Zarsky, V., Davies, B., and Hussey, P.J. (2005). Arabidopsis group Ie formins localize to specific cell membrane domains, interact with actin-binding proteins and cause defects in cell expansion upon aberrant expression. New Phytol. 168: 529–540.[CrossRef][Web of Science][Medline] Ding, Z., Lambrechts, A., Parepally, M., and Roy, P. (2006). Silencing profilin-1 inhibits endothelial cell proliferation, migration and cord morphogenesis. J. Cell Sci. 119: 4127–4137. Dong, C.H., Xia, G.X., Hong, Y., Ramachandran, S., Kost, B., and Chua, N.H. (2001). ADF proteins are involved in the control of flowering and regulate F-actin organization, cell expansion, and organ growth in Arabidopsis. Plant Cell 13: 1333–1346. Doonan, J.H., Ccove, D.J., and Lloyd, C.W. (1988). Microtubules and microfilaments in tip growth: Evidence that microtubules impose polarity on protonemal growth in Physcomitrella patens. J. Cell Sci. 89: 533–540. Eads, J.C., Mahoney, N.M., Vorobiev, S., Bresnick, A.R., Wen, K.K., Rubenstein, P.A., Haarer, B.K., and Almo, S.C. (1998). Structure determination and characterization of Saccharomyces cerevisiae profilin. Biochemistry 37: 11171–11181.[CrossRef][Web of Science][Medline] Evangelista, M., Blundell, K., Longtine, M.S., Chow, C.J., Adames, N., Pringle, J.R., Peter, M., and Boone, C. (1997). Bni1p, a yeast formin linking cdc42p and the actin cytoskeleton during polarized morphogenesis. Science 276: 118–122. Evangelista, M., Pruyne, D., Amberg, D.C., Boone, C., and Bretscher, A. (2002). Formins direct Arp2/3-independent actin filament assembly to polarize cell growth in yeast. Nat. Cell Biol. 4: 260–269.[CrossRef][Medline] Gibbon, B.C., Kovar, D.R., and Staiger, C.J. (1999). Latrunculin B has different effects on pollen germination and tube growth. Plant Cell 11: 2349–2363. Gibbon, B.C., Zonia, L.E., Kovar, D.R., Hussey, P.J., and Staiger, C.J. (1998). Pollen profilin function depends on interaction with proline-rich motifs. Plant Cell 10: 981–993. Guex, N., and Peitsch, M.C. (1997). SWISS-MODEL and the Swiss-PdbViewer: An environment for comparative protein modeling. Electrophoresis 18: 2714–2723.[CrossRef][Web of Science][Medline] Haarer, B.K., Lillie, S.H., Adams, A.E., Magdolen, V., Bandlow, W., and Brown, S.S. (1990). Purification of profilin from Saccharomyces cerevisiae and analysis of profilin-deficient cells. J. Cell Biol. 110: 105–114. Harold, F.M. (2002). Force and compliance: Rethinking morphogenesis in walled cells. Fungal Genet. Biol. 37: 271–282.[CrossRef][Web of Science][Medline] Harries, P.A., Pan, A., and Quatrano, R.S. (2005). Actin-related protein2/3 complex component ARPC1 is required for proper cell morphogenesis and polarized cell growth in Physcomitrella patens. Plant Cell 17: 2327–2339. Haugwitz, M., Noegel, A.A., Karakesisoglou, J., and Schleicher, M. (1994). Dictyostelium amoebae that lack G-actin-sequestering profilins show defects in F-actin content, cytokinesis, and development. Cell 79: 303–314.[CrossRef][Web of Science][Medline] Hepler, P.K., Vidali, L., and Cheung, A.Y. (2001). Polarized cell growth in higher plants. Annu. Rev. Cell Dev. Biol. 17: 159–187.[CrossRef][Web of Science][Medline] Higgs, H.N. (2005). Formin proteins: A domain-based approach. Trends Biochem. Sci. 30: 342–353.[CrossRef][Web of Science][Medline] Huang, S., McDowell, J.M., Weise, M.J., and Meagher, R.B. (1996). The Arabidopsis profilin gene family. Evidence for an ancient split between constitutive and pollen-specific profilin genes. Plant Physiol. 111: 115–126.[Abstract] Kandasamy, M.K., McKinney, E.C., and Meagher, R.B. (2002). Plant profilin isovariants are distinctly regulated in vegetative and reproductive tissues. Cell Motil. Cytoskeleton 52: 22–32.[CrossRef][Web of Science][Medline] Kost, B., Spielhofer, P., and Chua, N.H. (1998). A GFP-mouse talin fusion protein labels plant actin filaments in vivo and visualizes the actin cytoskeleton in growing pollen tubes. Plant J. 16: 393–401.[CrossRef][Web of Science][Medline] Kovar, D.R. (2006). Molecular details of formin-mediated actin assembly. Curr. Opin. Cell Biol. 18: 11–17.[CrossRef][Web of Science][Medline] Kovar, D.R., Drobak, B.K., Collings, D.A., and Staiger, C.J. (2001a). The characterization of ligand-specific maize (Zea mays) profilin mutants. Biochem. J. 358: 49–57.[CrossRef][Web of Science][Medline] Kovar, D.R., Drobak, B.K., and Staiger, C.J. (2000). Maize profilin isoforms are functionally distinct. Plant Cell 12: 583–598. Kovar, D.R., Harris, E.S., Mahaffy, R., Higgs, H.N., and Pollard, T.D. (2006). Control of the assembly of ATP- and ADP-actin by formins and profilin. Cell 124: 423–435.[CrossRef][Web of Science][Medline] Kovar, D.R., Yang, P., Sale, W.S., Drobak, B.K., and Staiger, C.J. (2001b). Chlamydomonas reinhardtii produces a profilin with unusual biochemical properties. J. Cell Sci. 114: 4293–4305. Kramer, C.Y. (1956). Extension of multiple range tests to group means with unequal numbers of replications. Biometrics 12: 309–310. Lambrechts, A., Jonckheere, V., Dewitte, D., Vandekerckhove, J., and Ampe, C. (2002). Mutational analysis of human profilin I reveals a second PI(4,5)-P2 binding site neighbouring the poly(L-proline) binding site. BMC Biochem. 3: 12.[CrossRef][Medline] Lovy-Wheeler, A., Wilsen, K.L., Baskin, T.I., and Hepler, P.K. (2005). Enhanced fixation reveals the apical cortical fringe of actin filaments as a consistent feature of the pollen tube. Planta 221: 95–104.[CrossRef][Web of Science][Medline] Lu, J., and Pollard, T.D. (2001). Profilin binding to poly-L-proline and actin monomers along with ability to catalyze actin nucleotide exchange is required for viability of fission yeast. Mol. Biol. Cell 12: 1161–1175. Magdolen, V., Oechsner, U., Muller, G., and Bandlow, W. (1988). The intron-containing gene for yeast profilin (PFY) encodes a vital function. Mol. Cell. Biol. 8: 5108–5115. Marga, F., Grandbois, M., Cosgrove, D.J., and Baskin, T.I. (2005). Cell wall extension results in the coordinate separation of parallel microfibrils: Evidence from scanning electron microscopy and atomic force microscopy. Plant J. 43: 181–190.[CrossRef][Web of Science][Medline] Mascarenhas, J.P., and Lafountain, J. (1972). Protoplasmic streaming, cytochalasin B, and growth of the pollen tube. Tissue Cell 4: 11–14.[Web of Science][Medline] McKenna, S.T., Vidali, L., and Hepler, P.K. (2004). Profilin inhibits pollen tube growth through actin-binding, but not poly-L-proline-binding. Planta 218: 906–915.[CrossRef][Web of Science][Medline] McKinney, E.C., Kandasamy, M.K., and Meagher, R.B. (2001). Small changes in the regulation of one Arabidopsis profilin isovariant, PRF1, alter seedling development. Plant Cell 13: 1179–1191. Menand, B., Calder, G., and Dolan, L. (2007). Both chloronemal and caulonemal cells expand by tip growth in the moss Physcomitrella patens. J. Exp. Bot. 58: 1843–1849. Michelot, A., Guerin, C., Huang, S., Ingouff, M., Richard, S., Rodiuc, N., Staiger, C.J., and Blanchoin, L. (2005). The formin homology 1 domain modulates the actin nucleation and bundling activity of Arabidopsis FORMIN1. Plant Cell 17: 2296–2313. Mittermann, I., Swoboda, I., Pierson, E., Eller, N., Kraft, D., Valenta, R., and Heberle-Bors, E. (1995). Molecular cloning and characterization of profilin from tobacco (Nicotiana tabacum): Increased profilin expression during pollen maturation. Plant Mol. Biol. 27: 137–146.[CrossRef][Web of Science][Medline] Nishiyama, T., Fujita, T., Shin, I.T., Seki, M., Nishide, H., Uchiyama, I., Kamiya, A., Carninci, P., Hayashizaki, Y., Shinozaki, K., Kohara, Y., and Hasebe, M. (2003). Comparative genomics of Physcomitrella patens gametophytic transcriptome and Arabidopsis thaliana: Implication for land plant evolution. Proc. Natl. Acad. Sci. USA 100: 8007–8012. Paavilainen, V.O., Bertling, E., Falck, S., and Lappalainen, P. (2004). Regulation of cytoskeletal dynamics by actin-monomer-binding proteins. Trends Cell Biol. 14: 386–394.[CrossRef][Web of Science][Medline] Perelroizen, I., Didry, D., Christensen, H., Chua, N.H., and Carlier, M.F. (1996). Role of nucleotide exchange and hydrolysis in the function of profilin in action assembly. J. Biol. Chem. 271: 12302–12309. Perroud, P.F., and Quatrano, R.S. (2006). The role of ARPC4 in tip growth and alignment of the polar axis in filaments of Physcomitrella patens. Cell Motil. Cytoskeleton 63: 162–171.[CrossRef][Web of Science][Medline] Pruyne, D., Gao, L., Bi, E., and Bretscher, A. (2004). Stable and dynamic axes of polarity use distinct formin isoforms in budding yeast. Mol. Biol. Cell 15: 4971–4989. Ramachandran, S., Christensen, H.E., Ishimaru, Y., Dong, C.H., Chao-Ming, W., Cleary, A.L., and Chua, N.H. (2000). Profilin plays a role in cell elongation, cell shape maintenance, and flowering in Arabidopsis. Plant Physiol. 124: 1637–1647. Rogers, S.L., Wiedemann, U., Stuurman, N., and Vale, R.D. (2003). Molecular requirements for actin-based lamella formation in Drosophila S2 cells. J. Cell Biol. 162: 1079–1088. Sagot, I., Rodal, A.A., Moseley, J., Goode, B.L., and Pellman, D. (2002). An actin nucleation mechanism mediated by Bni1 and profilin. Nat. Cell Biol. 4: 626–631.[Web of Science][Medline] Severson, A.F., Baillie, D.L., and Bowerman, B. (2002). A formin homology protein and a profilin are required for cytokinesis and Arp2/3-independent assembly of cortical microfilaments in C. elegans. Curr. Biol. 12: 2066–2075.[CrossRef][Web of Science][Medline] Sonobe, S., and Shibaoka, H. (1989). Cortical fine actin-filaments in higher-plant cells visualized by rhodamine-phalloidin after pretreatment with m-maleimidobenzoyl N-hydroxysuccinimide ester. Protoplasma 148: 80–86.[CrossRef][Web of Science] Staiger, C.J., Goodbody, K.C., Hussey, P.J., Valenta, R., Drobak, B.K., and Lloyd, C.W. (1993). The profilin multigene family of maize: Differential expression of three isoforms. Plant J. 4: 631–641.[CrossRef][Web of Science][Medline] Tewinkel, M., Kruse, S., Quader, H., Volkmann, D., and Sievers, A. (1989). Visualization of actin filament pattern in plant-cells without pre-fixation - A comparison of differently modified phallotoxins. Protoplasma 149: 178–182.[CrossRef][Web of Science] Vidali, L., and Hepler, P.K. (1997). Characterization and localization of profilin in pollen grains and tubes of Lilium longiflorum. Cell Motil. Cytoskeleton 36: 323–338.[CrossRef][Web of Science][Medline] Vidali, L., and Hepler, P.K. (2001). Actin and pollen tube growth. Protoplasma 215: 64–76.[CrossRef][Web of Science][Medline] Vidali, L., McKenna, S.T., and Hepler, P.K. (2001). Actin polymerization is essential for pollen tube growth. Mol. Biol. Cell 12: 2534–2545. Weiner, M.P., Costa, G.L., Schoettlin, W., Cline, J., Mathur, E., and Bauer, J.C. (1994). Site-directed mutagenesis of double-stranded DNA by the polymerase chain reaction. Gene 151: 119–123.[CrossRef][Web of Science][Medline] Witke, W. (2004). The role of profilin complexes in cell motility and other cellular processes. Trends Cell Biol. 14: 461–469.[CrossRef][Web of Science][Medline] Witke, W., Sutherland, J.D., Sharpe, A., Arai, M., and Kwiatkowski, D.J. (2001). Profilin I is essential for cell survival and cell division in early mouse development. Proc. Natl. Acad. Sci. USA 98: 3832–3836. Wolven, A.K., Belmont, L.D., Mahoney, N.M., Almo, S.C., and Drubin, D.G. (2000). In vivo importance of actin nucleotide exchange catalyzed by profilin. J. Cell Biol. 150: 895–904. Yi, K., Guo, C., Chen, D., Zhao, B., Yang, B., and Ren, H. (2005). Cloning and functional characterization of a formin-like protein (AtFH8) from Arabidopsis. Plant Physiol. 138: 1071–1082. This article has been cited by other articles:
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ABSTRACT
INTRODUCTION
-helices and β-strands in the secondary structure, respectively. Residues important for binding to polyproline and actin are indicated by P and A, respectively. The arrow indicates the Arg present in plant profilins that prevents the enhancement of ATP exchange on actin. Residues mutated for this study are indicated with a boxed asterisk (Tyr-6 and Lys-87). See 
*area/perimeter2 and solidity defined as area/convex hull area. All image analysis was done using macros written for ImageJ (
