Research Article

Cytoskeleton DOI 10.1002/cm.21174

Fluorescent protein-based reporters of the actin cytoskeleton in living plant cells: fluorophore variant, actin binding domain and promoter considerations Julia Dyachoka,1,2, J. Alan Sparksa, 1,2, Fuqi Liaob, Yuh-Shuh Wangc and Elison B. Blancaflora a

Plant Biology Division and bDepartment of Computing Services, The Samuel Roberts

Noble Foundation, 2510 Sam Noble Parkway, Ardmore, Oklahoma, 73401, USA c

Plant Signal Research Group, Institute of Technology, University of Tartu, Nooruse 1,

Tartu 50411, Estonia 1

Current address: Department of Pharmacology, University of Texas Southwestern

Medical Center, 6001 Forest Park Road Dallas, Texas, 75390-9041 2

These authors contributed equally to this article

Address correspondence to: Elison B. Blancaflor, Plant Biology Division 2510 Sam Noble Parkway, The Samuel Roberts Noble Foundation, Ardmore, OK 73401, USA E-mail: [email protected] Phone: (580) 224-6687 Fax: (580) 224-6692

Running title: Imaging actin in living plants

Additional Supporting Information may be found in the online version of this article. This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as Dyachok, et al., (2013), Fluorescent protein-based reporters of the actin cytoskeleton in living plant cells: fluorophore variant, actin binding domain and promoter considerations. Cytoskeleton, doi: 10.1002/cm.21174 © 2014 Wiley-Blackwell, Inc. Received: May 23, 2013; Revised: Feb 27, 2014; Accepted: Mar 19, 2014

Cytoskeleton

Abstract Genetically encoded filamentous actin (F-actin) reporters designed based on fluorescent protein fusions to F-actin binding domains of actin regulatory proteins have emerged as powerful tools to decipher the role of the actin cytoskeleton in plant growth and development. However, these probes could interfere with the function of endogenous actin binding proteins and in turn impact actin organization and plant growth. We therefore surveyed F-actin labeling and compared organ growth in Arabidopsis thaliana lines expressing a variety of F-actin markers. Here we show that the variant of fluorescent protein, type of actin binding domain, and the promoter that drives reporter expression can influence the quality of F-actin labeling particularly in stable plant lines. For example, older red fluorescent protein (RFP)-based probes such as DsRed2 and mOrange induced more aberrant labeling compared to the newer RFPbased, mCherry, GFP, and GFP-derived fluorophores such as YFP and CFP. Moreover, qualitative and quantitative analyses revealed differences in F-actin organization in seedlings expressing Talin- and Lifeact-based reporters in some cell types compared to the fimbrin actin binding domain 2 (ABD2)-based reporters. Finally, the use of the ubiquitin10 (UBQ10) promoter to drive expression of the GFP-ABD2-GFP probe minimized loss of fluorescence and growth defects observed in the 35S-driven version. Taken together, this study shows that care must be taken in the interpretation of data derived from stable expression of certain F-actin reporters and that using alternative promoters such as UBQ10 can overcome some of the pitfalls that accompany the use of in vivo F-actin probes in plants.

Key words: Actin Cytoskeleton, Arabidopsis, Fluorescent Proteins, Live Cell Microscopy, Plants

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Introduction Plants rely on a dynamic actin cytoskeleton to drive basic cellular processes that are essential for their normal growth and development. These include processes associated with cell division, cell expansion, and membrane remodeling (Hussey et al., 2006). In concert with a multitude of regulatory proteins, globular actin (G-actin) monomers assemble into higher order filamentous actin (F-actin) structures. F-actin networks not only provide trafficking routes for the delivery of cell wall precursorcontaining vesicles required for polarized cell growth but also serve as molecular tracks that guide the intracellular movement of organelles during plant responses to environmental stimuli such as light and gravity (Blanchoin et al., 2010; Morita, 2010; Kong and Wada, 2011; Blancaflor 2013). Actin-mediated signaling cascades have also been implicated in dictating how plants defend themselves against pathogens and in facilitating their response to abiotic stresses (Wang et al., 2010; Day et al., 2011). The advances in understanding the role of the actin cytoskeleton in plant development have come in large part from the improved ability to document its dynamics and organization in living cells. Although actin imaging in living plant cells was already achieved many years ago through microinjection of fluorescent actin analogs or F-actin binding probes such as fluorescently-tagged phalloidin (Schmit and Lambert, 1990; Ren et al., 1997), the technical expertise and specialized equipment needed to implement such methods have hindered their widespread application. With the discovery of the green fluorescent protein (GFP; Tsein, 1998), live actin imaging in plants has now become routine in many laboratories. Through simple molecular cloning techniques, a number of genetically encoded F-actin binding probes have been generated (e.g. Kost et al., 1998; Sheahan et al., 2004; Wang et al., 2004; 2008; Voigt et al., 2005; Cheung et al., 2008; Vidali et al., 2009; Era et al., 2009). These genetically encoded reporters have led to new insights on actin function in plant virus movement, cell division and expansion, cell polarity establishment, responses to hormones and microorganisms, heat shock and recovery, and organelle dynamics (Liu et al., 2005; Sano et al., 2005; Voigt et al., 2005, Müller et al., 2007; Rahman et al., 2007; Tian et al., 2009; Dyachok et al., 2008; 2011; Yoo et al., 2012). Furthermore, the use of live F-actin probes in combination with improved imaging approaches has led to the identification of 3 John Wiley & Sons

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mechanisms underlying actin remodeling that appear to be unique to plant cells (Staiger et al., 2009; Reymann et al., 2011; Henty et. al., 2011). Increasing application of fluorescent protein-based F-actin probes in plants has in turn revealed problems associated with their use. Perturbed actin organization and dynamics resulting in growth defects have been reported for each of the F-actin markers developed for plants. However, a more thorough survey of the problems with live F-actin probes has been limited in the scientific literature, making end users of these probes unaware of potential limitations in the type of F-actin data they can collect. For example, it has been shown that F-actin binding GFP probes inhibited the growth of some Arabidopsis cell types and exhibited loss of fluorescence, particularly in the root meristematic zone and mature leaves (e.g. Ketelaar et al., 2004; Wang et al., 2008; Labuz et al., 2010). Detrimental effects on the growth of moss protonema have also been documented (Vidali et al., 2009). In one case, however, no obvious effects on organ growth were observed in Arabidopsis plants (Voigt et al., 2005). With the increased popularity of fluorescent-based F-actin reporters for plant research, there is a need to draw more attention to the aforementioned problems so that further improvements in their design can be made, and caution can be exercised in the interpretation of information derived from their use. In this paper we revisit some of the issues that we encountered while attempting to stably express different variants of the fimbrin Actin Binding Domain 2 (ABD2) reporter in Arabidopsis. In addition to the loss of fluorescence in roots and cell growth defects that we reported previously (Wang et al., 2004; 2008), we found that the type of fluorescent protein variant used in generating the actin reporter led to some differences in F-actin patterns among cells and tissues of young Arabidopsis seedlings. One example was the tendency of the red-emitting probe, DsRed2 to induce F-actin bundles in transient and stable expression studies. Also, we show that Arabidopsis seedlings expressing a GFP-Talin or a Lifeact-Venus probe under the control of the cauliflower mosaic virus 35S promoter had some cell types with less dense and more bundled Factin than ABD2-based probes. Furthermore, we report that the loss of fluorescence in the root meristem and transition zone, and inhibitory effects of the GFP-ABD2-GFP reporter on cell and organ growth (Wang et al., 2008) can be minimized by replacing the 4 John Wiley & Sons

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strong constitutive 35S promoter with the ubiquitin10 (UBQ10) promoter (Norris et al., 1993; Geldner et al., 2009). Although the use of the UBQ10 promoter is only a minor improvement, we report here that it could minimize some of the artifacts that researchers might encounter when using ABD2-based and other F-actin reporters for studying actin dynamics and organization in living plants.

Results

F-actin Organization in Arabidopsis Seedlings Expressing Red-Emitting Fluorescent Proteins Fused to ABD2 and Talin Fluorescent proteins that emit in the red region of the visible light spectrum are valuable for live cell microscopy because they provide the ideal partner for GFP in double labeling studies. We therefore generated transgenic Arabidopsis plants expressing two types of red-emitting F-actin reporters: monomeric (m) Orange or DsRed2 (Baird et al. 2000; Shaner et al., 2004). To compare these red-emitting F-actin markers with their corresponding GFP versions, mOrange was fused to the C and N termini of ABD2 under the control of the constitutive 35S promoter (35S::mOrangeABD2-mOrange). This construct is similar to the 35S::GFP-ABD2-GFP reporter described previously (Wang et al., 2008). The other red-emitting F-actin probe we generated was 35S::DsRed2-Talin, which is similar to the previously described 35S::GFP-Talin reporter (Wang et. al., 2004). Arabidopsis plants expressing 35S::mOrange-ABD2-mOrange were significantly smaller compared to wild-type plants or plants expressing 35S::GFP-ABD2-GFP. Such differences in growth stature were most apparent in mature plants (Supporting Information, Fig. S1A, B) but not in young seedlings growing on the agar surface. In fact, primary root length of 8 day old 35S::mOrange-ABD2-mOrange seedlings was slightly longer than wild-type (Supporting Information, Fig. S1C). Furthermore, all of the independent 35S::mOrange-ABD2mOrange lines that we selected had distinct fluorescence that was confined to cells of the root maturation zone and hypocotyl cells close to the root-hypocotyl junction with very weak or no fluorescence in the growing zone of primary roots and epidermal cells of the cotyledons (Supporting Information, Fig. S1D). 5 John Wiley & Sons

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The pattern of F-actin labeling in cells of the seedling where expression of the 35S::mOrange-ABD2-mOrange reporter was detected differed significantly from that of 35S::GFP-ABD2-GFP. For example, the 35S::GFP-ABD2-GFP reporter labeled an extensive network of F-actin in epidermal cells of hypocotyls, cotyledons and the root maturation zone consistent with our previous results (Fig. 1A-C; Wang et. al., 2008). The 35S::mOrange-ABD2-mOrange seedlings on the other hand displayed thick fluorescent aggregates and diffuse fluorescence in epidermal cells of hypocotyls (Fig. 1D) and mostly diffuse fluorescence in epidermal pavement cells of cotyledons (Fig. 1E). A few filamentous structures could be detected in hypocotyls and cotyledon pavement cells in 35S::mOrange-ABD2-mOrange lines. However, distinct F-actin structures were only consistently observed in epidermal cells of the root maturation zone. In these cell types, F-actin structures decorated by the 35S::mOrange-ABD2mOrange probe ranged from transverse bundles that accumulated within a confined region of the cell (Fig. 1F) or a few thick longitudinal bundles (Fig. 1G). While imaging the 35S::mOrange-ABD2-mOrange seedlings, we also noticed that fluorophore bleaching occurred very rapidly and could explain the often grainy images obtained from this line. We next examined F-actin patterns in seedlings expressing 35S::GFP-Talin and 35S::DsRed2-Talin. Compared to 35S::GFP-ABD2-GFP, 35S::GFP-Talin labeled a population of thicker F-actin cables in epidermal cells of hypocotyls, cotyledons and the root maturation zone (Fig. 1H-J). On the other hand, we could only detect short fluorescent aggregates and bundles in epidermal cells of hypocotyls, cotyledons and primary roots of 35S::DsRed2-Talin-expressing seedlings (Fig. 1K-M). The apparent bundling of F-actin observed in stable plant lines was also observed when 35S::DsRed2-Talin was expressed transiently in tobacco leaf epidermal cells (Supporting Information, Fig. S2A). Only a few 35S::DsRed2-Talin transformed cells showed an F-actin network with both fine and thick arrays (Supporting Information, Fig. S2B). Seedlings and mature plants expressing 35S::DsRed2-Talin reporter did not exhibit significant growth defects (Supporting Information, Fig. S2C, D). To quantitatively assess differences in F-actin patterns among seedlings expressing the four types of F-actin reporters, we used two parameters to measure the 6 John Wiley & Sons

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extent of bundling (skewness) and filament density (occupancy) based on the methods of Higaki et al. (2010) (Supporting information, Fig. S3 and see Materials and Methods). Consistent with our qualitative visual observations, seedlings expressing the 35S::GFPABD2-GFP reporter had significantly higher percentage occupancy values than seedlings expressing the 35S::mOrange-ABD2-mOrange probe in cells of the hypocotyl and root maturation zone (Fig. 2A). Furthermore, seedlings expressing 35S::GFPABD2-GFP showed significantly lower skewness values in hypocotyl cells than 35S::mOrange-ABD2-mOrange lines. Although the average skewness in the root maturation zone of 35S::GFP-ABD2-GFP lines was also lower than the mOrange version, the differences were not statistically significant (Fig. 2B). The predominantly diffuse fluorescence observed in cotyledons of the 35S::mOrange-ABD2-mOrange lines prevented us from extracting reliable occupancy and skewness values from this cell type (see Fig. 1E). Furthermore, because there were generally very few filaments in the 35S::DsRed2-Talin-expressing lines (Fig.1K-M), it is unclear whether the extracted skewness and occupancy values from these lines represent anything relevant. Nonetheless, quantitative analysis based on established metrics verified that 35S::GFPABD2-GFP reporter labeled a more dense and less bundled network of F-actin compared to the mOrange version. Occupancy and skewness analyses also showed that F-actin in epidermal cells of the cotyledon and root maturation zone labeled by the ABD2 probe were denser and less bundled than the Talin probe regardless of whether Talin was fused to GFP or DsRed2 (Fig. 2A, B). 35S::GFP-Talin lines also had higher occupancy values than 35S::DsRed2-Talin lines in cells of the hypocotyl and root maturation zone but skewness between the two lines were not significantly different (Fig. 2B).

F-actin Organization in Arabidopsis Seedlings Expressing Yellow and Cyan Emitting F-actin Reporters The problems with the red-emitting F-actin reporters described above indicate their limited use for reliable F-actin labeling particularly in stable plant lines. We therefore tried another color variant of GFP namely the yellow fluorescent protein (YFP) fused to the N and C termini of ABD2. Seedlings expressing this 35S::YFP-ABD2-YFP 7 John Wiley & Sons

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reporter were compared to seedlings expressing a circularly permutated derivative of YFP called Venus fused to Lifeact, an F-actin binding probe consisting of the first 17 amino acids of the yeast actin binding protein 170 (35S:: Lifeact-Venus; Era et al., 2009). We also generated seedlings expressing cyan fluorescent protein (CFP) fused to the N and C termini of ABD2 to determine if this blue-green emitting variant could be useful for F-actin imaging in transgenic seedlings. When transiently expressed in tobacco leaf epidermal cells either by particle bombardment or Agrobacterium infiltration, 35S::YFP-ABD2-YFP and 35S::CFP-ABD2-CFP exhibited similar patterns of F-actin labeling as 35S::GFP-ABD2-GFP, and did not display the extensive bundling issues induced by transiently expressing 35S::DsRed2-Talin (Supporting Information, Fig. S4A, B). We next examined F-actin structures in seedlings expressing the YFP-, CFPand Venus-based F-actin probes. In order to minimize the artifacts due to overexpression, we selected for comparison, stable lines with similar protein expression levels and similar generations for several fluorescent actin reporters (Fig. 3). Thus, differences in the levels of fluorescent proteins did not exceed two-fold between stable lines expressing 35S::GFP-ABD2-GFP, 35S::Lifeact-Venus, 35S::YFP-ABD2-YFP and 35S::CFP-ABD2-CFP (Fig. 3B, D). On the other hand, the levels of fluorescent proteins were thirty-to-forty fold higher in 35S::GFP-Talin compared to other stable lines (Fig. 3B, D). We found that in epidermal cells of hypocotyls, cotyledons and the root maturation zone, the 35S::YFP-ABD2-YFP and 35S::CFP-ABD2-CFP reporters labeled a fine and dense network of F-actin that was similar to that decorated by the 35S::GFPABD2-GFP probe (compare Fig. 1N-S to Fig. 1A-C). Although 35S::CFP-ABD2-CFPexpressing plants exhibited similar F-actin organization as the YFP and GFP versions, they often had patches of cells with higher background fluorescence in the cytoplasm and required increased line averaging with the confocal microscope to obtain highly resolved F-actin images. On the other hand, F-actin patterns in the 35S::Lifeact-Venus lines appeared to resemble those of the 35S::GFP-Talin lines (compare Fig. 1T-V to Fig. 1H-J). Indeed, quantitative analysis of occupancy and skewness revealed statistically significant differences in F-actin organization among lines expressing 8 John Wiley & Sons

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35S::YFP-ABD2-YFP, 35S::CFP-ABD2-CFP and 35S::Lifeact-Venus. For example, occupancy values of epidermal cells of hypocotyls and cotyledons were higher in 35S::CFP-ABD2-CFP and 35S::YFP-ABD2-YFP lines compared to 35S::Lifeact-Venus indicating that F-actin in seedlings expressing the latter probe were less dense (Fig. 2A). In regard to skewness, 35S::Lifeact-Venus had significantly higher values than the 35S::YFP-ABD2-YFP and 35S::CFP-ABD2-CFP in all three cell types, which is indicative of more extensive F-actin bundling (Fig. 2B). Quantitative analysis also revealed statistically higher occupancy values indicative of a denser network of F-actin in epidermal cells of hypocotyls and cotyledons of 35S::CFP-ABD2-CFP seedlings compared to 35S::YFP-ABD2-YFP (Fig. 2A). In the root meristem, 35S::YFP-ABD2-YFP, 35S::CFP-ABD2-CFP labeled similar F-actin structures as 35S::Lifeact-Venus lines including phragmoplasts and cortical Factin networks at the transverse cell ends (Supporting Information, Fig. S5A-C). In the root transition zone, however, fluorescent signals that clearly marked the transverse end walls in 35S::YFP-ABD2-YFP and 35S::CFP-ABD2-CFP diminished in 35S::LifeactVenus-expressing seedlings (Supporting Information, Fig. S5D-F). Growing root hairs in all three lines displayed similar gradients of fine F-actin at the tip to longitudinal cables toward the base (Supporting Information, Fig. S5G-I). Like cells in other above ground organs, F-actin in guard cells of the cotyledon of 35S::Lifeact-Venus lines were less dense and appeared to be more bundled than 35S::YFP-ABD2-YFP and 35S::CFPABD2-CFP lines (Supporting Information, Fig. S5J-L).

Comparison of F-actin Organization Between Immunolabeled Roots and Roots Expressing Live Cell F-actin Reporters The qualitative and quantitative analyses described above indicate that ABD2 fused to GFP, YFP and CFP variants might offer the best alternative for live cell imaging of F-actin in stable Arabidopsis seedlings. To address this issue further, we labeled Factin in fixed whole mount Arabidopsis seedlings by indirect immunofluorescence (Dyachok et al., 2009) so we could compare the resulting actin patterns with seedlings expressing the various live cell F-actin reporters. Using our whole mount seedling processing techniques, however, only the meristem and transition zone of fixed 9 John Wiley & Sons

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Arabidopsis roots were labeled adequately possibly because of low permeability problems of other cell and tissue types for the antibodies. Therefore, only these root regions are shown for comparison. In primary roots of seedlings, it was found that structures in the meristem of fixed roots such as phragmoplasts and cortical F-actin at the transverse cell ends of the root meristem and transition zone were similar to the structures in the live roots of seedlings expressing 35S::GFP-ABD2-GFP, 35S::LifeactVenus, 35S::YFP-ABD2-YFP and 35S::CFP-ABD2-CFP (Fig. 4; Supporting Information, Fig. S5). Phragmoplasts were also detected in the meristem of 35S::GFP-Talinexpressing lines. However, many cells in the meristem and transition zone of the 35S::GFP-Talin line had predominantly diffuse fluorescence as opposed to the distinct fine F-actin structures observed in other lines (Fig. 4; Supporting Information, Fig. S5).

Other Notable Observations in Seedlings Expressing Various Live Cell F-actin Reporter Constructs As previously reported, seedlings expressing the 35S::GFP-ABD2-GFP construct showed loss of fluorescence in files of cells within the meristematic region and transition zone of their roots (Fig. 4D; Wang et. al., 2008). We attributed this lack of fluorescence to silencing; therefore, we introduced the 35S::GFP-ABD2-GFP into plants mutated in the SGS3 gene that has been shown to reduce transgene-induced silencing (Muangsan et al., 2004). Stable sgs3 35S::GFP-ABD2-GFP lines had approximately 30% less fluorescent proteins compared to stable wild-type 35S::GFP-ABD2-GFP plants (Fig. 4C, D). However, sgs3 mutant seedlings expressing 35S::GFP-ABD2-GFP still exhibited the patchy fluorescence in the root meristem and transition zone, similar to wild-type plants expressing the same construct (Supporting information, Fig. S6A, B). It was also observed that elongating root hairs of 35S::Lifeact-Venus and 35::GFP-ABD2-GFP seedlings exhibited similar F-actin patterns with thick longitudinal bundles in the base and finer F-actin at the tip. The pattern of F-actin labeling in actively elongating root hairs was similar to other lines such as 35S::YFP-ABD2-YFP and 35S::CFP-ABD2-CFP (Supporting Information, Fig. S7B, C compare to Fig. S5G, H). However, as reported previously, this gradient of F-actin in actively growing root hairs was not observed in 35S:: GFP-Talin lines (Supporting Information, Fig. S7A). Growing 10 John Wiley & Sons

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root hairs of the 35S:: GFP-Talin line exhibited intense cytoplasmic-like fluorescence that was enriched at the extreme tip and reminiscent of the pattern of Talin-GFP fluorescence reported previously (Supporting Information, Fig. S7A; Baluska et al., 2000; Wang et al., 2004; Supporting Information, Movie S1). Both 35S::Lifeact-Venus and 35S::GFP-ABD2-GFP root hairs clearly showed visible and highly dynamic fine Factin at the tip (Supporting Information, Movies S2 and S3). In contrast to actively growing root hairs, F-actin organization in mature, non-growing root hairs of 35S::GFPABD2-GFP, 35S::Lifeact-Venus and 35S:: -GFP-Talin was very similar in that thick bundles of F-actin protruded all the way to the non-growing root hair tip (Supporting Information, Fig. S7D-F).

Stable Expression of UBQ10::GFP-ABD2-GFP Minimizes Loss of Fluorescence in Growing Cells of the Root and Growth Defects in Arabidopsis Seedlings Most of the F-actin reporters for stable expression in plants have been driven by the strong constitutive 35S promoter. However, as documented above, seedlings expressing these 35S-driven F-actin reporters often displayed loss of fluorescence particularly in the root meristem and transition zone where active cell proliferation and expansion are occurring (Fig. 4; Wang et. al., 2008). Because expression of the 35S::GFP-ABD2-GFP in the sgs3 mutant did not appear to eliminate this patchy loss of fluorescence (Supporting information, Fig. S6A, B), we expressed the GFP-ABD2-GFP construct under the control of the UBQ10 promoter (Norris et al., 1993; Geldner et. al., 2009), which was recently shown to confer moderate GFP expression in Arabidopsis (Krebs et al., 2011). Also, because none of the red-emitting F-actin reporters appeared to adequately label F-actin in the root meristem and transition zone (see Fig. 1), we recently made an mCherry-ABD2-mCherry construct under the control of the UBQ10 promoter (UBQ10::mCherry-ABD2-mCherry). We first transformed Arabidopsis plants with the UBQ10::GFP-ABD2-GFP constructs. Compared to 35S::GFP-ABD2-GFP seedlings, UBQ10::GFP-ABD2-GFP seedlings had approximately 30% less fluorescent proteins and exhibited about 60% lower fluorescence intensity (Fig. 3C, D and Supporting information, Fig. S8). The signal from UBQ10::GFP-ABD2-GFP plants required increasing the gain settings of the 11 John Wiley & Sons

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confocal microscope detector to acquire images of comparable brightness to that of 35S::GFP-ABD2-GFP plants. We found that T3 seedling roots expressing the UBQ10::GFP-ABD2-GFP construct had mostly uniform expression in the meristem and transition zone where loss of fluorescence in the 35S::GFP-ABD2-GFP seedlings was typically observed (Fig. 5A, C compare to Fig. 4C, D and Supporting information, Fig. S6A, B). Furthermore, the pattern of F-actin labeling in stable UBQ10::GFP-ABD2-GFP lines in root hairs generally mirrored that of 35S::GFP-ABD2-GFP (Fig. 5E compare to Supporting information, Fig. S7B). Similar F-actin patterns to those of 35S::GFP-ABD2GFP lines were also observed in epidermal cells of light-grown hypocotyls, epidermal pavement cells of the cotyledons, guard cells, and epidermal cells of the root maturation zone (Fig. 5G, I, K compare to Fig. 1A-C). Comparison of occupancy and skewness between 35S::GFP-ABD2-GFP and UBQ10::GFP-ABD2-GFP was mostly consistent with our qualitative observations. Occupancy and skewness values in epidermal cells of hypocotyls and the root maturation zone were not significantly different between the two lines. However, in epidermal cells of cotyledons, occupancy values in UBQ10::GFP-ABD2-GFP lines were significantly lower compared to the 35S::GFP-ABD2-GFP lines. On the other hand, skewness values were significantly higher in UBQ10::GFP-ABD2-GFP lines compared to 35S::GFP-ABD2-GFP lines (Fig. 6A,B). The UBQ10::mCherry-ABD2-mCherry construct was imaged from seedlings at the T1 generation. From our initial imaging results, it was found that T1 seedlings expressing UBQ10::mCherry-ABD2-mCherry had very similar patterns of F-actin labeling to T3 lines expressing UBQ10::GFP-ABD2-GFP (Fig. 5). Consistent with qualitative observations, quantitative analyses showed that occupancy between UBQ10::GFP-ABD2-GFP and UBQ10::mCherry-ABD2-mCherry were not significantly different in epidermal cells of cotyledons and the root maturation zone. Skewness values between the two lines were also not significantly different in epidermal cells of hypocotyls and the root maturation zone. However, in epidermal cells of hypocotyls, occupancy was significantly lower in UBQ10:mCherry-ABD2-mCherry lines compared to UBQ10::GFP-ABD2-GFP lines. On the other hand, skewness in epidermal cells of

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cotyledons was significantly higher in UBQ10:mCherry-ABD2-mCherry lines compared to UBQ10::GFP-ABD2-GFP lines (Fig. 6A, B). An example of how the UBQ10::GFP-ABD2-GFP lines can be used for dynamic F-actin imaging can be seen in a root hair transitioning from the initiation stage to the onset of tip growth (Supporting Information, Movie S4). Like 35S::GFP-ABD2-GFP and 35S::Lifeact-Venus lines, fine dynamic F-actin could be visualized in UBQ10::GFPABD2-GFP and UBQ10::mCherry-ABD2-mCherry lines (Supporting Information, Movie S5 and S6). Another problem reported with 35S-driven F-actin reporters was their inhibitory effects on cell growth (Keteelar et al., 2004; Wang et al., 2008; Vidali et al., 2009). We compared the growth of organs and selected cell types of 35S::GFP-ABD2-GFP, 35S::Talin-GFP and 35S::Lifeact-Venus lines with that of UBQ10::GFP-ABD2-GFP under different environmental conditions (Fig. 7). Under diurnal light, roots of 35S::TalinGFP and 35S::GFP-ABD2-GFP were shorter compared to non-transgenic controls, whereas 35S::Lifeact-Venus and UBQ10::GFP-ABD2-GFP roots were similar to those of non-transgenic controls (Fig. 7A, B). Root hair lengths were wild type-like in plants expressing 35S::Talin-GFP but shorter in 35S::Lifeact-Venus and 35S::GFP-ABD2-GFP plants. Although UBQ10::GFP-ABD2-GFP seedlings were significantly shorter than wild-type root hairs, the extent of root hair length reduction was not as dramatic as the 35S-driven version (Fig. 7C). In addition, a large percentage of root hair cells were branched in 35S::Lifeact-Venus lines and 35S::GFP-ABD2-GFP lines (Fig. 7D, E). The percent of root hairs that were branched in 35S::Talin-GFP and UBQ10::GFP-ABD2GFP were not significantly different from wild-type (Fig. 7E). Defects in root hair growth in the 35S-driven F-actin reporters observed here were most pronounced when the primary root was grown on the surface of agar media with their root hairs exposed to air. In root hairs embedded in the agar media, there were no dramatic defects in root hair growth and morphology in 35S::Talin-GFP and 35S::GFP-ABD2-GFP lines (Supporting Information, Fig. S9; see also Wang et al., 2008). However, we noted that 35S::LifeactVenus seedlings had generally shorter and wavy root hairs when embedded in the agar medium (Supporting Information, Fig. S9).

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We also compared transgenic seedlings to non-transgenic controls under dark conditions. Hypocotyls and roots of dark-grown 35S::GFP-ABD2-GFP were inhibited compared to hypocotyls of non-transgenic controls (Fig. 7F-H) but no hypocotyl growth inhibition was observed in UBQ10::GFP-ABD2-GFP lines. Roots but not hypocotyls of 35S::Talin-GFP seedlings were inhibited in the dark, similar to root growth inhibition in 35S::Talin-GFP plants grown under diurnal light (Fig. 7A, F). Hypocotyls of dark-grown 35S::Lifeact-Venus were dramatically inhibited whereas roots of these plants were, surprisingly, longer compared to roots of non-transgenic controls. Such differential elongation of hypocotyls and roots was observed only in 35S::Lifeact-Venus expressing seedlings but not in seedlings expressing other F-actin markers (Fig. 7F-H).

F-actin Dynamics in Hypocotyls of Light-Grown Seedlings Differ Among the Reporter Lines As noted, quantitative analysis of static F-actin organization and seedling growth showed differences among the various reporter lines. We next asked whether the measured differences in static actin parameters (i.e. skewness and occupancy; Fig. 2 and 6) correlate with global F-actin dynamics. To quantify changes in F-actin organization over time, we focused on epidermal cells of hypocotyls from light-grown seedlings and utilized the metrics of pixel difference values developed by Vidali et al (2010) to analyze overall F-actin behavior among UBQ10::GFP-ABD2-GFP, 35S::GFPABD2-GFP, 35S:: GFP-Talin and 35S::Lifeact-Venus lines (Fig. 8A; see Supporting Information, Movies S7 – S10 for representative movie sequences). The intensity difference between images, captured every 2 seconds, for all possible time intervals were calculated according to Vidali et. al. (2010). It was found that changes in difference values over time were more pronounced in UBQ10::GFP-ABD2-GFP lines compared to 35S::GFP-ABD2-GFP, 35S::Talin-GFP and 35S::Lifeact-Venus lines. This indicates that F-actin is more dynamic in hypocotyl epidermal cells of UBQ10::GFP-ABD2-GFP lines compared to the other three lines (Fig. 8B). Vidali et al (2010) also used correlation values between two images as a way to assess global changes in F-actin dynamics with a slower decay in correlation values indicative of enhanced dynamics. Using this metric, it was found that 35S::Talin-GFP lines showed the slowest decay in correlation 14 John Wiley & Sons

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coefficient values while the decay in correlation coefficient values among UBQ10::GFPABD2-GFP, 35S::GFP-ABD2-GFP and 35S::Lifeact-Venus followed roughly the same pattern at various time intervals (Fig. 8C). Statistical analyses of changes in difference and correlation coefficient decay at 20, 40 and 60 sec intervals are shown in Table 1. Based on the assessment of correlation coefficient decay and changes in pixel difference from more than 10 movie sequences of hypocotyl epidermal cells, 35S::GFPTalin has the least dynamic F-actin network among the four lines.

Discussion In this paper we surveyed a collection of Arabidopsis lines expressing a variety of F-actin reporters to gain additional insights into their suitability for investigating actindependent biological processes. Some of the F-actin–expressing lines described here were the result of our previous efforts to obtain an F-actin marker optimal for stable expression in the model plant Arabidopsis (Wang et al., 2004; 2008). At the same time we wanted to create F-actin reporter variants that emit at a region of the visible light spectrum for potential double labeling studies with other cellular components. The plant lines in this survey included those expressing ABD2 and Talin fusions to mutant variants of GFP from Aequorea victoria (YFP and CFP) and red fluorescent protein (RFP) from Discosoma striata (DsRed2, mOrange and mCherry). We compared these reporters to Lifeact-Venus, which is the most recent in vivo F-actin marker used in plants. While conducting this survey, a range of results were obtained that showed similarities and differences in F-actin patterns depending on the variant of fluorescent protein, type of F-actin binding domain, the promoter used to drive expression, and whether expression of the reporter was transient or stable. We realize, however, that in conducting this survey, not all comparisons are exact equivalents due to the large number and variability of available plant lines and constructs, differences in how the reporters were made (e.g. length of the linker between the fluorescent protein and the F-actin binding protein), cells types where fluorescent signal was detected, the number of fluorescent proteins fused (e.g. two GFPs versus one GFP), and non-equivalent imaging parameters (e.g. different laser lines/confocal microscope gain settings). Thus, these caveats must be considered in the interpretation of our results. For example, even 15 John Wiley & Sons

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though the F-actin networks in seedlings labeled by the 35S::Lifeact-Venus and 35S:: GFP-Talin probes appeared more bundled than that decorated by the 35S::GFP-ABD2GFP reporter, we cannot discount the possibility that the ABD2- reporter is failing to bind to the thick actin bundles. Another important caveat to consider is the possibility that some of the redemitting fluorescent proteins (e.g. DsRed2 and mOrange) affect the affinity of the probe for F-actin as compared to the probe with GFP, Venus or mCherry. Without performing corresponding biochemical assays, it is impossible to determine whether probe affinity or expression levels are the main factors affecting visualization of F-actin organization. Direct comparison of protein levels of the red- and green-emitting F-actin binding probes is not possible because RFPs do not cross react with GFP antibodies (Fig. 3). A further complicating factor is that ABD2, Talin, and Lifeact could have different affinities for Factin. Therefore, the expression levels required for each reporter to obtain similar binding in the cell to F-actin will be different. In fact, a recent study by van der Honing et al. (2011) showed that less Lifeact expression is required to obtain similar dynamic actin parameters as that obtained with ABD2 (with a single GFP). Thus, Lifeact can be a good F-actin probe as long as it is expressed at levels lower than ABD2 (van der Honing et al. 2011). Perhaps the most equivalent comparisons made in this survey are data shown in Fig. 1 where seedlings expressing 35S::GFP-ABD2-GFP were compared to 35S::mOrange-ABD2-mOrange and 35S::GFP-Talin was compared to 35S::DsRed2Talin. From this comparison, it was clear that the red-emitting fluorophores triggered more problems regardless of whether the reporters were stably- or transientlyexpressed. Although red-emitting F-actin probes have been used for double labeling studies in plants in transient expression assays (Liu et al., 2005; Ju et al., 2005; Hofmann et al., 2009; Martiniere et al 2011), stable expression of such probes has been rare and to the best of our knowledge, documented only in Bright Yellow 2 tobacco suspension cells (Vanstraelen et al., 2006) and in Physcomitrella patens (Van Gisbergen et. al., 2012). The scarcity of red-emitting F-actin expression in stable plant lines could in large part be due to the problems observed here such as severe growth inhibition resulting in the inability to recover adequate amount of seeds, non-uniform 16 John Wiley & Sons

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labeling of F-actin, rapid bleaching (e.g. mOrange-ABD2-mOrange), and artificial bundling (e.g. DsRed2-Talin). The bundling of DsRed2-Talin could be the result of the tendency of the DsRed2 protein to form tetramers to generate a functional fluorophore (Baird et al., 2000; Yarbrough et al., 2001). On the other hand, the tendency of mOrange to bleach rapidly (Shaner et al., 2004) could be tied to the release of toxic byproducts that might impact overall plant growth. Although data were obtained only from seedlings at the T1 generation, it was intriguing that the UBQ10::mCherry-ABD2mCherry construct mirrored that of T3 seedlings expressing UBQ10::GFP-ABD2-GFP given the very different F-actin patterns observed between 35S::GFP-ABD2-GFP and 35S::mOrange-ABD2-mOrange lines (see Fig. 1 and Fig. 5). mOrange and mCherry differ by ten amino acids (Shaner et. al., 2004), and this could be sufficient to account for the differences in F-actin labeling patterns between the two fluorophores. Perhaps the moderate expression conferred by the UBQ10 promoter allowed the plant to tolerate any adverse effects of the mCherry probe. Qualitative and quantitative analysis showed that CFP-ABD2-CFP and YFPABD2-YFP decorated abundant F-actin networks in seedlings. Because CFP has a lower quantum yield than GFP and YFP (Tsein, 1998), and requires excitation with short wavelength, higher energy light, bleaching would occur more rapidly in CFP-ABD2-CFP expressing lines, which explains the need to modify imaging parameters needed to acquire data. Nonetheless, the CFP-ABD2-CFP lines described here could still potentially be used in transient assays as a pair with YFP for double labeling studies. Previous papers have shown that stable expression of ABD2- and Lifeactbased probes did not significantly impact overall plant development and growth of individual cells such as root hairs (Wang et al., 2008; van der Honing et al., 2011). In this study, however, we found that subjecting these lines to previously untested environmental conditions can trigger some cell and organ growth defects to manifest themselves. For example, dark-grown plants expressing 35S::Lifeact-Venus had inhibited hypocotyls that were more pronounced than the inhibited hypocotyls in seedlings expressing 35S::GFP-ABD2-GFP. The differences in hypocotyl elongation mirror different labeling patterns among the F-actin reporters. In 35S::GFP-Talin hypocotyls, for example, cells exhibited stronger cytoplasmic fluorescence and fewer 17 John Wiley & Sons

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filaments compared to 35S::Lifeact-Venus and 35S::GFP-ABD2-GFP hypocotyls. It is possible that overexpression of these markers in darkness, which induces rapid growth in hypocotyls, has a stronger negative impact on ABD2 and Lifeact lines because of the tendency of the aforementioned reporters to decorate more of the F-actin networks. This in turn could adversely impact the activity of other actin regulatory proteins that regulate actin dynamics (Keteelar et al., 2004). Surprisingly, unlike short roots of darkgrown plants expressing 35S::GFP-Talin or 35S::GFP-ABD2-GFP, roots of 35S::LifeactVenus were longer in darkness compared to non-transgenic wild-type. Opposite effects of 35S::Lifeact-Venus expressed at high levels on hypocotyls and roots of dark-grown plants suggest different mechanisms regulating F-actin in these organs. Indeed, accelerated hypocotyl growth in the dark is accompanied by abundant cortical F-actin (Staiger et al., 2009) whereas darkness inhibits cortical F-actin accumulation and growth in roots (Dyachok et al., 2011). Thus, Lifeact-Venus could be useful in investigating the differences between the subpopulations of F-actin in hypocotyls and roots of dark-grown plants. Recently, it was shown that seedlings expressing 35S::Lifeact-Venus but not 35S::GFP-ABD2 allowed the visualization of fine F-actin at the tips of growing root hairs (van der Honing et al., 2011). Here, we show that like the Lifeact-Venus construct, fine F-actin at the growing root hair tip can be observed in 35S::GFP-ABD2-GFP and UBQ10::GFP-ABD2-GFP seedlings (Supporting Information Movies S2, S3 and S5). Reduced F-actin reorganization rate in 35S::Lifeact-Venus lines was one reason provided for the improved ability to visualize fine actin networks at the root hair tip (van der Honing et al., 2011). It is possible that the extra GFP tag on the GFP-ABD2-GFP reporter resulted in a decline in F-actin reorganization rate making it possible to observe fine F-actin at the root hair tip as opposed to plants expressing ABD2 with a single GFP. However, this scenario presents another set of complications because if F-actin reorganization is slowed down by the extra GFP tag of the reporter, the true rate of Factin reorganization might not be reflected in the GFP-ABD2-GFP lines. Indeed, the shorter root hair length in GFP-ABD2-GFP and Lifeact-Venus-expressing seedlings could be explained by the reduced F-actin dynamics at the tip. Perhaps stable Arabidopsis lines expressing UBQ10::Lifeact-mGFP will help resolve this issue. 18 John Wiley & Sons

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By simply substituting UBQ10 for the 35S promoter, we show that the loss of fluorescence in the root tip and growth defects associated with the overexpression of GFP-ABD2-GFP can be minimized. Although the intensity of UBQ10::GFP-ABD2-GFP fluorescence was less than half of the fluorescence of 35S::GFP-ABD2-GFP lines, both constructs illuminated similar F-actin networks in several plant cells and tissues, including roots and hypocotyls (Compare Fig. 1 and Fig. 5). In epidermal pavement cells, however, substituting UBQ10 for 35S promoter in driving the expression of GFPABD2-GFP diminished the portion of fluorescent signal associated with the cell periphery and could explain differences in calculated filament density and bundling in cotyledons between the two lines (Fig. 6). It is possible that the fluorescence associated with the cell periphery represents the fraction of the fluorescent marker that accumulated in a thin layer of perimembrane cytoplasm due to overexpression of 35S::GFP-ABD2-GFP. Cytoplasmic accumulation of fractions of ABD2 to GFP fusions not bound to F-actin has been reported before (Sheahan et al., 2004; Wilsen et al., 2006). The advantage of UBQ10 over 35S promoter in driving the expression of F-actin reporters can also be appreciated from the analysis of changes in global F-actin dynamics. It was found that changes over time in pixel difference values in hypocotyl epidermal cells of UBQ10::GFP-ABD2-GFP lines were higher than 35S::GFP-ABD2GFP lines (Fig. 8; Table 1) despite the absence of significant differences in static F-actin parameters between the two lines (Fig. 6). This indicates that the lower expression of GFP-ABD2-GFP result in a more dynamic F-actin network, which in turn could explain the absence of adverse growth defects in UBQ10::GFP-ABD2-GFP lines (Fig. 7). We also found that in some lines, the measured static F-actin parameters correlated well with measurements of global F-actin dynamics. For instance, the increased bundling and reduced occupancy in hypocotyls of 35S::GFP-Talin lines was associated with slower decay in correlation coefficient and smaller pixel difference over time (Fig. 8B, C), indicating that the more bundled the F-actin network, the less dynamic it is (see Supplemental Movies, S7-10). In other cases, however, such tight correlations between static F-actin parameters and global F-actin dynamics were not always apparent. For instance, whereas the higher average skewness values in hypocotyls of 35S::Lifeact19 John Wiley & Sons

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Venus lines correlated well with smaller changes in pixel difference over time (Fig. 8B), decay in correlation coefficient did not (Fig. 8C). Perhaps a tighter link between static Factin organization and F-actin dynamics is more pronounced at the level of individual actin filaments or in other cell types. For the future, it would be interesting to measure global F-actin dynamics in other cell/tissue types and determine how these various lines differ in regard to stochastic properties of individual actin filaments (Staiger et al., 2009). In conclusion, our survey of various in vivo F-actin reporters has generated a number of new questions and avenues for improvement. For instance, the UBQ10::GFP-ABD2-GFP provides a valuable addition to the in vivo F-actin imaging toolkit because it minimizes the harmful side effects in Arabidopsis seedling growth and loss of primary root fluorescence observed with the 35S-driven version. UBQ10::GFPABD2-GFP lines also had more dynamic changes in global F-actin organization, which could partly explain their more robust seedling growth compared to other lines. Thus, exploring alternative promoters that drive moderate expression provide a way to optimize the use of genetically encoded fluorescent F-actin markers in plants. For the future, it would be interesting to determine if Lifeact- or Talin-based probes would work better for stable plant lines under the control of the UBQ10 promoter.

Materials and Methods

Plant Material, Construction of F-actin Reporters, and Generation of Transgenic Arabidopsis Plants All plants used in this study were in the Columbia background. 35S::Talin-GFP and 35S::GFP-ABD2-GFP plants were described earlier (Wang et al., 2004; 2008). Seeds of 35S::Lifeact-Venus (Era et al., 2009) were kindly provided by Dr.Takashi Ueda (University of Tokyo). All other F-actin reporters were constructed in a modified pCAMBIA-1390 vector with an introduced cauliflower mosaic virus (CaMV) 35S promoter as described in Wang et al. (2004; 2008). The CFP-ABD2-CFP, YFP-ABD2-YFP, mOrange-ABD2-mOrange, and mCherry-ABD2-mCherry variants were prepared similarly as the GFP version 20 John Wiley & Sons

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(Wang et al., 2008), by simply replacing GFP with CFP, YFP (Clontech, CA, USA), mOrange and mCherry (Shaner et al., 2004; kind gift of Dr. Roger Tsein (University of California, San Diego). The DsRed2-Talin was prepared by replacing GFP from the GFP-Talin construct (Wang et al., 2004) with a DsRed2 (Clontech, CA, USA). The UBQ10::GFP-ABD2-GFP and UBQ10::mCherry-ABD2-mCherry were generated by replacing the 35S promoter in the original GFP-ABD2-GFP construct with the UBQ10 promoter (Geldner et al,, 2009) using a two step cloning procedure. Only minimal linkers (one or two amino acids) between the actin-binding domains (ABD2 and Talin) and the flourescent proteins were introduced due to the cloning strategy, whereas in the Lifeact-Venus fusion a four-amino acid-linker (GlyGlySerGly) was designed (Era et al., 2009). In the case of FP-ABD2-FP fusions, extra amino acids consisting of ThrMet and Phe were introduced at the N- and C-terminal of ABD2, respectively. A GluPhe linker was introduced in the GFP-Talin and DsRed2-Talin constructs. Arabidopsis plants were transformed with the aforementioned reporter constructs using the floral dip method (Clough & Bent, 1998). Seeds were harvested and screened on media containing 20mg/L hygromycin B and 100mg/L cefotaxime (Nakazawa & Matsui, 2003). Fluorescent hygromycin-resistant seedlings were further selected using an SZX12 stereomicroscope (Olympus America, NY, USA). Seeds used in this study were obtained from plants grown at 80 µmol m-2 s-1 intensity of fluorescent light on a 18 hour light/6 hour dark cycle at 22°C. Surface-sterilized seeds were dried on Whatman filter paper for 7-10 days before use. Seeds were sown on ½ MS medium supplemented with 1% sucrose, solidified with 1% agar (Sigma). After stratification at 4°C for 2 days, vertically oriented plates with seedlings were grown either at 80 µmol m-2 s-1 intensity of fluorescent light on a 16 hour light/8 hour dark cycle (diurnal light conditions), or in darkness at 21°C for up to 7 days.

Transient Expression Assays in Tobacco Leaves and Immunolabeling of Whole Mount Arabidopsis Seedlings F-actin reporters were transiently expressed in tobacco leaves by biolistic transformation as described in Wang et al. (2004) or by agroinfiltration (Vaghchhipawala et al., 2011). 21 John Wiley & Sons

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Actin immunolocalization in roots of 4-day-old seedlings was carried out as described by Dyachok et al. (2008; 2009). Briefly, seedlings were fixed for 1 h in PME buffer (50 mM PIPES, pH 7.2, with 20 mM EGTA and 20 mM MgSO4) containing 2% paraformaldehyde, 0.1%Triton X-100 and 400 mm Maleimidobenzoyl-Nhydroxysuccinimide ester (Pierce/ThermoFisher Scientific), permeabilized for 1 h in 1% Triton X-100 in PME. Wall digestion was for 20 min in 0.05% Pectolyase Y-23 in PME with 0.1%Triton X-100, 1% bovine serum albumin, 0.4 M mannitol. Samples were then permeabilized for 10 min in cold methanol (-20°C). Mouse monoclonal anti-chicken gizzard actin (“C4,” Chemicon, Temecula, CA) was used at a dilution of 1:1,000 and AlexaFluor 488 goat antimouse (InVitrogen, Eugene, OR) at 1:200. Samples were mounted in Vectashield (Vector Laboratories, Burlingame, CA) prior to imaging.

Quantification of Seedling F-actin Fluorescence, Density and Bundling, and Global F-actin Dynamics For quantitative analysis of GFP-ABD2-GFP fluorescence, seedling primary roots were imaged with a 20X objective of a Nikon Optiphot 2 fluorescence microscope. Images were acquired using NIS-Elements F3.0 and processed with ImageJ. The rectangular selection marquee tool of ImageJ was used to measure average pixel intensities in eight randomly selected areas per root. Average fluorescence intensity was then calculated for each individual root. To quantify percent occupancy (density) and skewness (bundling), we utilized the method described by Higaki et al. (2010). Projected images of single cells from the hypocotyl, cotyledon and root maturation zone were processed by software that was built using MATLAB R2013a (The Mathworks). Briefly, the original single cell images from the confocal microscope were first contrast enhanced, converted into binary images, and skeletonized into one bit widths. The skeletonized images were used to delineate filaments from gray scale images generated from the original confocal images. To calculate density, filament occupancy was defined as the total pixel numbers of all marked filaments, and cell area was determined from the cell outline. Density was calculated as the ratio of filament occupancy and cell area (Supporting information, Fig. S3A; Higaki et al., 2010). 22 John Wiley & Sons

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For skewness, the intensity distribution of all the marked filaments was measured. The skewness of the intensity distribution was calculated with the histograms representing the intensity distribution of the marked filaments (Higaki, et al. 2010; Supporting information, Fig. S3B). Statistical analysis was conducted in MATLAB with statistical toolbox. For each of the image groups, the mean and standard error (S.E.) were calculated. The comparison of skewness and density among various cell types of the reporter lines was performed by using one way ANOVA and Tukey’s test. Quantification and statistical analyses of global F-actin dynamics was conducted following the methods of Vidali et al., (2010) using 512 X 512 images of hypocotyl epidermal cells from 9-15 time-lapse movie sequences.

Protein analyses For protein analyses, approximately 100 mg of tissue from 3-day-old seedlings were extracted in 200 µl 1xLDS sample buffer (Invitrogen) supplemented with 1 mM DTT and 1/100 plant protease inhibitor cocktail (Sigma). Total protein extracts were heated at 95°C for 10 min and cleared by centrifugation at 16,000g for 10 min in a table top centrifuge. Similar amounts of proteins (according to the Coomassie-stained SDSPAGE gel) were loaded onto the 10% acrylamide gels for immunoblotting. Proteins were electrophoresed using Tris-glycine-SDS running buffer, and transferred to nitrocellulose membrane. Immunoblotting of protein extracts was carried out using mouse anti-GFP antibodies (Zymed/Invitrogen) at 0.1 µg/ml followed by IRDye 800CW– conjugated goat anti-mouse IgG (Licor) diluted1:10,000. Imaging and quantitation of immunoblots were performed using the Licor Odyssey Infrared imaging system with manufacturer’s reagents and procedures.

Growth Measurements For root and hypocotyl length measurements, plates with seedlings were imaged using Nikon DXM1200 camera controlled by the Nikon ACT1-C software. Root and hypocotyl lengths were measured using ImageJ software version 1.42o (http://rsb.info.nih.gov/ij/). Root hair cells were imaged using Nikon Optiphot 2

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microscope equipped with Nikon Digital Sight DS-Ri1 Camera, controlled by Nikon NISElements F3.0 software.

Confocal Microscopy Seedlings were mounted in water on slides and examined for fluorescence using Leica TCS SP2 AOBS confocal laser scanning microscope (Leica Microsystems, Exton, PA) equipped with a 63× HCX Plan-Apo water immersion objective (numerical aperture 1.20). Individual images were acquired using Leica LCS confocal software. For imaging F-actin dynamics in root hair cells and hypocotyl epidermal cells, seedlings were grown inside approx. 1-mm layer of 0.5% agar supplemented with ½ MS medium and 1% sucrose on 62 × 48-mm glass coverslips as described previously (Dyachok et al., 2009). Fine F-actin networks in root hair tips were imaged using a Perkin Elmer UltraView ERS spinning-disc confocal microscope equipped with a 63X Zeiss C-Apochromat water immersion objective (numerical aperture 1.20) and Hamamatsu EM-CCD digital camera. Individual images were acquired at 300 msec to 2 sec intervals, and movies were assembled using Volocity 4.3.1 software (Improvision, Lexington, MA).

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Cheung AY, Duan QH, Costa SS, de Graaf BH, Di Stilio VS, Feijo J, Wu HM. 2008. The dynamic pollen tube cytoskeleton: live cell studies using actin-binding and microtubule-binding reporter proteins. Mol Plant. 2008 Jul;1(4):686-702. Clough, S.J., and A.F. Bent. 1998. Floral dip: a simplified method for Agrobacteriummediated transformation of Arabidopsis thaliana. Plant J 16:735-743. Day B, Henty JL, Porter KJ, Staiger CJ 2011.The pathogen-actin connection: a platform for defense signaling in plants. Annu Rev Phytopathol. 2011;49:483-506 Dyachok, J., M.R. Shao, K. Vaughn, A. Bowling, M. Facette, S. Djakovic, L. Clark, and L. Smith. 2008. Plasma membrane-associated SCAR complex subunits promote cortical F-actin accumulation and normal growth characteristics in Arabidopsis roots. Mol Plant 1:990-1006. Dyachok J, Yoo C M, Palanichelvam K. Blancaflor EB. 2009. Sample preparation for fluorescence imaging of the cytoskeleton in fixed and living plant roots. In: Gavin RH, editor. Cytoskeleton Methods and Protocols. Totowa, New Jersey: Humana Press. Methods in Molecular Biology series. Vol. 586; 157-169. Dyachok, J., L. Zhu, F. Liao, J. He, E. Huq, and E.B. Blancaflor. 2011. SCAR mediates light-induced root elongation in Arabidopsis through photoreceptors and proteasomes. Plant Cell 23: 3610-3626. Era, A., M. Tominaga, K. Ebine, C. Awai, C. Saito, K. Ishizaki, K.T. Yamato, T. Kohchi, A. Nakano, and T. Ueda. 2009. Application of Lifeact reveals F-actin dynamics in Arabidopsis thaliana and the liverwort, Marchantia polymorpha. Plant Cell Physiol 50:1041. Geldner N, Dénervaud‐Tendon V, Hyman DL, Mayer U, Stierhof YD, Chory J. 2009. Rapid, combinatorial analysis of membrane compartments in intact plants with a multicolor marker set. Plant J 59:169-178. Henty JL, Bledsoe SW, Khurana P, Meagher RB, Day B, Blanchoin L, Staiger CJ. 2011. Arabidopsis actin depolymerizing factor4 modulates the stochastic dynamic behavior of actin filaments in the cortical array of epidermal cells. Plant Cell 23: 3711-26. Higaki T, Kutsuna N, Sano T, Kondo N, Hasezawa S. 2010. Quantification and cluster analysis of actin cytoskeletal structures in plant cells: role of actin bundling in 25 John Wiley & Sons

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Voigt, B., A.C.J. Timmers, J. Samaj, J. Muller, F. Baluska, and D. Menzel. 2005. GFPFABD2 fusion construct allows in vivo visualization of the dynamic actin cytoskeleton in all cells of Arabidopsis seedlings. Eur J Cell Biol 84:595-608. Wang, Y.S., C.M. Motes, D.R. Mohamalawari, and E.B. Blancaflor. 2004. Green fluorescent protein fusions to Arabidopsis fimbrin 1 for spatio-temporal imaging of F-actin dynamics in roots. Cell Motil Cytoskeleton 59:79-93. Wang, Y.S., C.M. Yoo, and E.B. Blancaflor. 2008. Improved imaging of actin filaments in transgenic Arabidopsis plants expressing a green fluorescent protein fusion to the C‐and N‐termini of the fimbrin actin‐binding domain 2. New Phytologist 177:525-536. Wang C, Zhang L, Yuan M, Ge Y, Liu Y, Fan J, Ruan Y, Cui Z, Tong S, Zhang S. 2010. The microfilament cytoskeleton plays a vital role in salt and osmotic stress tolerance in Arabidopsis. Plant Biol (Stuttg) 12:70-78. Wilsen, K.L., A. Lovy-Wheeler, B. Voigt, D. Menzel, J.G. Kunkel, and P.K. Hepler. 2006. Imaging the actin cytoskeleton in growing pollen tubes. Sexual Plant Reproduction. 19:51-62. Yarbrough D, Wachter RM, Kallio K, Matz MV, Remington SJ. 2001. Refined crystal structure of DsRed, a red fluorescent protein from coral, at 2.0-A resolution. Proc Natl Acad Sci U S A. 98:462-467. Yoo CM, Quan L, Cannon AE, Wen J., Blancaflor EB (2012). AGD1, a class 1 ARFGAP, acts in common signaling pathways with phosphoinositide metabolism and the actin cytoskeleton in controlling Arabidopsis root hair polarity. Plant J 69:1064–1076.

Acknowledgments We thank Dr. Roger Tsien (University of California, San Diego) for the mOrange and mCherry vectors and Dr Takashi Ueda (University of Tokyo) for the 35S::VenusLifeact expressing seed. This work was supported by the National Aeronautics and Space Administration (NASA grants NNX10AF43G and NNX12 AM94G to E.B.B.), Oklahoma Center for the Advancement of Science and Technology (OCAST grant PSB 29 John Wiley & Sons

Cytoskeleton

10-004 to E.B.B.) and The Samuel Roberts Noble Foundation. Confocal microscopes used in this study were obtained through multi-user instrumentation grants from the National Science Foundation (NSF grants DBI-0400580 and 0722635). Y.S.W. was supported by the Estonian Ministry of Science and Education, and the European Regional Fund (IUT2-21 and Center of Excellence Environment). The authors declare no conflict of interests.

Figure Legends

Fig. 1. Organization of F-actin in seedlings expressing 35S::GFP-ABD2-GFP, 35S::mOrange-GFP-ABD2-mOrange (35S::mO-GFP-mO), 35S::GFP-Talin, 35S::DsRed2-Talin, 35S::YFP-ABD2-YFP, 35S::CFP-ABD2-CFP and 35S::LifeactVenus . Representative images of epidermal cells in the hypocotyl (A, D, H, K, N, Q, T), epidermal pavement cells of the cotyledon (B, E, I, L, O, R, U) and epidermal cells in the root maturation zone (C, F, G, J, M, P,S, V). Seedlings were grown under 14h/10h light/dark conditions for 5 days before imaging. Images are projections of twenty, 1 µm Z-stacks. Bars= 20 µm.

Fig. 2. Quantitative analysis of F-actin density and bundling in seedlings expressing 35S::GFP-ABD2-GFP, 35S::mOrange-GFP-ABD2-mOrange (35S::mOGFP-mO), 35S::GFP-Talin, 35S::DsRed2-Talin, 35S::YFP-ABD2-YFP, 35S::CFPABD2-CFP and 35S::Lifeact-Venus. The average percent of occupancy or F-actin density (A) and extent of F-actin bundling or skewness (B) was measured in epidermal cells of hypocotyls, cotyledons and root maturation zone according to the methods of Higaki et al., (2010). Statistical significance was determined by one way ANOVA. Means (n=15-33 cells) ±S.E. with different letters are significantly different (P

Fluorescent protein-based reporters of the actin cytoskeleton in living plant cells: fluorophore variant, actin binding domain, and promoter considerations.

Genetically encoded filamentous actin (F-actin) reporters designed based on fluorescent protein fusions to F-actin binding domains of actin regulatory...
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