Pollen Specific Expression of Maize Genes Encoding Actin Depolymerizing Factor-Like Proteins

Pollen Specific Expression of Maize Genes Encoding Actin Depolymerizing Factor-LikeProteinsAuthor(s): Imelda Lopez, Richard G. Anthony, Sutherland K. Maciver, Chang-Jie Jiang, SafinaKhan, Alan G. Weeds and Patrick J. HusseySource: Proceedings of the National Academy of Sciences of the United States of America,Vol. 93, No. 14 (Jul. 9, 1996), pp. 7415-7420Published by: National Academy of SciencesStable URL: http://www.jstor.org/stable/39600 .

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Proc. Natl. Acad. Sci. USA Vol. 93, pp. 7415-7420, July 1996 Plant Biology

Pollen specific expression of maize genes encoding actin depolymerizing factor-like proteins

(cytoskeleton/actophorin/cofilin/pollen tube)

IMELDA LOPEZ*t, RICHARD G. ANTHONY*, SUTHERLAND K. MACIVERt, CHANG-JIE JIANG*, SAFINA KHAN*, ALAN G. WEEDS*, AND PATRICK J. HUSSEY*?

*School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey TW20 OEX, United Kingdom; and tMedical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, United Kingdom

Communicated by J. Heslop-Harrison, Hertfordshire, United Kingdom, March 25, 1996 (received for review December 8, 1995)

ABSTRACT In pollen development, a dramatic reorganization of the actin cytoskeleton takes place during the passage of the pollen grain into dormancy and on activation of pollen tube growth. A role for actin-binding proteins is implicated and we report here the identification of a small gene family in maize that encodes actin depolymerizing factor (ADF)-like proteins. The ADF group of proteins are believed to control actin polymerization and depolymerization in response to both intracellular and extracellular signals. Two of the maize genes ZmABPI and ZmABP2 are expressed specifically in pollen and germinating pollen suggesting that the protein products may be involved in pollen actin reorganization. A third gene, ZmABP3, encodes a protein only 56% and 58% identical to ZmABPI and ZmABP2, respectively, and its expression is suppressed in pollen and germinated pollen. The fundamental biochemical characteristics of the ZmABP proteins has been elucidated using bacterially expressed ZmABP3 protein. This has the ability to bind monomeric actin (G-actin) and filamentous actin (F-actin). Moreover, it decreases the viscosity of polymerized actin solutions consistent with an ability to depolymerize filaments. These biochemical characteristics, taken together with the sequence comparisons, support the inclusion of the ZmABP proteins in the ADF group.

Microfilaments perform essential functions in eukaryotic cells and are responsible, with other cytoskeletal elements, for the structural integrity of the cell and for many of the cellular movements including cytokinesis and cytoplasmic streaming. One of the most dramatic reorganizations of the microfilament network to be studied in higher plants is observed in pollen development. In the pollen grains of the Liliaceae, for example, pollen activation and germination are accompanied by the gradual replacement of large F-actin aggregates present in the vegetative cytoplasm by a filamentous network that converges on the germinal aperture and eventually ramifies the pollen tube. This arrangement of microfilaments is required for particle movement and contributes to tip growth by directing vesicle traffic to the tip (1, 2).

So far there is very little known about the actin-binding proteins that are involved in this dramatic reorganization or the signals controlling them. The recent characterization of pollen-specific profilins suggests an integral role for this actin-binding protein (3, 4). Although profilins sequester actin monomers in vitro, recent research on the interaction of profilin with actin has suggested that they may play an important role in regulating filament assembly in vivo (5, 6). Also, plant profilin has been shown to bind phosphatidylinositol 4,5-bisphosphate (PIP2) indicating that, like mammalian profilins, plant profilin may be part of and/or regulated in the phosphoinositide signal transduction pathway (7-10).

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. ?1734 solely to indicate this fact.

More recently, a few plant genes have been identified, including the maize gene ZmABP] (11, 12), whose predicted amino-acid sequences showed considerable identity with actin depolymerizing factor (ADF) (13), also known as destrin (14, 15). This protein is a member of a group of proteins, the ADF group, that includes also cofilin (16-18), depactin in starfish (19), and actophorin in Acanthamoeba castellani (20). These proteins interact with both monomeric and filamentous actin in vitro, depending on the conditions, and they sever filaments (15, 21-23). Binding to actin is regulated by phosphoinositides (24) and phosphorylation (25-27). In the presence of the actin filament crosslinking protein a-actinin, both actophorin and cofilin promote the formation of actin filament bundles (28, 29). The effects of these proteins on cytoskeletal organization in cells has also been studied. Microinjection of cofilin induced major cytoskeletal reorganization in cultured cells (30) and overexpression of Dictyostelium cofilin in Dictyostelium discoi- deum stimulated cell movement and membrane ruffling (29). Thus, the ADF group of proteins appear to play an important role as stimulus responsive modulators of actin dynamics (31).

In this paper we report the distinct expression patterns of three maize genes (ZmABP], ZmABP2, and ZmABP3) that encode proteins similar to members of the ADF group. The ZmABP] and ZmABP2 genes are specifically expressed in pollen. Taken together with the biochemical properties of the ADF group, it is reasonable to suggest that the ZmABP1 and ZmABP2 proteins may play a significant role in actin reorganization in pollen activation and germination. The third gene, ZmABP3, exhibits an expression pattern quite different from ZmABP] and ZmABP2. ZmABP3 is expressed in every maize tissue examined with the exception of pollen. In support of the amino acid sequence comparisons, biochemical characterization of the protein product of the ZmABP3 cDNA clone has been used to demonstrate the fundamental actin-binding and actin-depolymerizing activity of these proteins.

MATERIALS AND METHODS Plant Material. The Zea mays inbred line A188 was used

throughout. Roots and shoots were harvested from 4- to 7-day-old germinated seedlings. Leaves were from greenhouse grown plants with six leaves. Cobs and pollen were collected from plants grown in the greenhouse or in the field. Embryos were obtained by dissection of kernels 28 days post-pollination. Staged spikelets were harvested from greenhouse grown plants. Pollen was germinated at room temperature for 45 min

Abbreviation: ADF, actin depolymerizing factor. Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. X97725 ( ZmABP2) and X97726 (ZmABP3)). TPresent address: Departamento de Biotecnologia, Instituto de Inves- tigaciones Biomedicas, Universidad Nacional Autonoma de Mexico, Apartado Postal 70228, Mexico 04510 D.F. Mexico. ?To whom reprint requests should be addressed.

7415



7416 Plant Biology: Lopez et al. Proc. Natl. Acad. Sci. USA 93 (1996)

on solid medium containing 17% (wt/vol) sucrose, 0.3 g/liter CaCl2, 0.1 g/liter H3BO3, 1% (wt/vol) agarose (Sigma).

Isolation of cDNA Clones. A pollen cDNA library was used for the isolation of ZmABPI (11) and ZmABP2 cDNA clones. The library was constructed in the vector Bluescribe M13+ (Stratagene) (32). Approximately 60,000 colonies of the library were screened sequentially with radiolabeled shoot and pollen cDNAs using the Zetaprobe (Bio-Rad) colony hybridization procedure and the following hybridization conditions: 50% (vol/vol) formamide, 5x SSPE (standard saline phosphate/EDTA), 50 mM Tris HCl (pH 7.4), 1% (wt/vol) SDS, 5x Denhardt's solution, 0.1 mg/ml calf thymus DNA, and 50 pLg/ml poly(A) at 42?C. The filters were washed three times for 20 min in 0.5x SSC (standard saline citrate), 1% (wt/vol) SDS at 65?C. The colonies showing a greater degree of hybridization to the pollen cDNA probes compared with the shoot cDNA probes were picked into microtiter dishes containing 2x YT (2X YT medium: 16 g/liter tryptone/10 g/liter yeast extract/5 g/liter NaCl), and 0.1 mg/ml ampicillin. A total of 1101 clones were picked into individual wells of 16 microtiter dishes. Cross-hybridization experiments (data not shown) revealed that these 1101 clones represented approximately 300 different clones. The different clones were sequenced at the 5' end and one, ZmABP1, showed identity with the deduced amino acid sequence of human ADF. Using a microtiter dish replicating tool, all 16 microtiter plates were imprinted on nylon membrane (Zetaprobe, Bio-Rad), and the colonies screened as above, but using radiolabeled ZmABP1 as probe. ZmABP2 was 1 of 28 positives from this screen. ZmABP3 was a kind gift from Keith Edwards (Long Ashton Research Station, Bristol, U.K.) who identified the cDNA clone in an "expressed sequence tag" collection from a maize leaf cDNA library.

DNA Sequencing. All sequencing was done using the dideoxy chain termination method (33) and an adaptation thereof for double- stranded templates (34). The cDNA sequences were analyzed using both the Staden and Genetics Computer Group (Madison, WI) packages (35) and the DNASIS package (LKB).

Southern and Northern Blotting. Maize genomic DNA was isolated (36), 15 ,ug aliquots were digested with either EcoRI or HindlIl, and the restriction fragments separated on 0.8% (wt/vol) agarose gels. Transfer of DNA from the gel to a nylon filter (Zetaprobe, Bio-Rad) was as described by Southern (37) with a modification of an ammonium acetate buffer (38). All Southern blots were prehybridized and hybridized as described for the colony hybridizations. The filters were washed in 2x SSC/0.1% (wt/vol) SDS, at room temperature in the case of the ZmABPI and ZmABP3 coding region probes, and at 50?C in the case of the 3' noncoding region (gene specific) probes, with three 20-min washes. The filters were stripped between probings by incubation at 75-80?C for 15 min in 5 mM Tris HCl (pH 7.5), 0.2 mM EDTA, and 0.1% (wt/vol) SDS.

Total RNA was prepared (32) and fractionated on 1.75% (wt/vol) formaldehyde agarose gels and transferred to nylon membranes (Nytran, Schleicher & Schuell) using lOX SSC as the transfer buffer. The filters were prehybridized, hybridized, and washed as described for the Southern blot hybridizations. Each lane on the gel contained 10 ,ug of RNA as quantified by A260 spectrophotometric assays supplemented by comparison of ribosomal RNAs on ethidium bromide stained agarose gels.

Preparation of Radioactive Probes. The ZmABPI and ZmABP2 coding sequence probes were labeled by random priming using an oligolabeling kit (Pharmacia). The 3' noncoding region (gene specific) probes were labeled from gel- purified PCR products using a primer extension method. Fifty nanograms of fragment were denatured by heating for 5 min at 900C in the presence of the appropriate oligonucleotides and lx medium salts buffer (50 mM NaCl/l0 mM Tris HCl, pH 7.5/10 mM MgCl2/1 mM DTT) and cooled to 37?C. Labeling was done with 5 units of Klenow at 37?C with 12 mM DTT, 30

/iM dCTP, 30 /iM dTTP, 30 /iM dGTP, and 50 pLCi of [ca-32P]dATP (3000 Ci/mmol; Amersham; 1 Ci = 37 GBq) for 1 hr. Approximate specific activities for all the probes were 108 cpm4Kg.

PCR. For the production of gene specific probes, primers were designed to the 5' and 3' ends of each of the 3' noncoding regions of the three cDNA clones and used to amplify these regions by PCR. Ten nanograms of template DNA was am- plified in the presence of 300 ng of primer, 0.5 mM dNTPs, 1.5 mM MgCl2, 50 mM KCI, 10 mM TrisHCl (pH 8.3), 0.01% (wt/vol) gelatine, and 2.5 units of AmpliTaq (Perkin-Elmer/ Cetus) for 30 cycles at 60?C using a Hybaid Omnigene ther- mocycling apparatus. PCR products to be used for radioactive labeling were purified on 1.5% (wt/vol) agarose gels and the DNA was electroeluted, phenol/chloroform extracted, and ethanol precipitated.

Protein Purification. ZmABP3 protein was purified as described in the Results. Actin from rabbit skeletal muscle was prepared as described in ref. 39.

G-Actin-Binding Assay. Binding to G-actin was monitored by native gel electrophoresis as described (40). Ten percent polyacrylamide gels were run at 200 V in the presence of 2 mM DTT and 0.2 mM ATP in both the gels and the running buffer. Proteins were loaded at a final concentration of 12 ,uM.

Cosedimentation Assay. F-actin binding was measured by sedimentation at 100,000 X g for 15 min in a Beckman TLA100 centrifuge (41). F-actin and ZmABP3 protein were incubated together (10 ,uM) for 20 min in 12 mM Tris HCl (pH 7.0), 50 mM KCI, 1 mM MgCl2, 1 mM EGTA, 0.2 mM ATP, 0.5 mM DTT, 0.2 mM CaCl2, and 1 mM NaN3. Aliquots from the supernatant and pellet, resuspended in a volume of buffer equal to the supernatant, were analyzed by SDS/PAGE as described in ref. 42.

Falling Ball Viscometry. Falling ball viscometry was used as an indirect assay for severing activity (39). Actin (10 ,uM) was mixed with different concentrations of ZmABP3 protein and volumes made to 180 ,l in buffer containing 10 mM Tris HCl (pH 8.0), 0.1 mM ATP, 0.2 mM CaCl2, 0.2 mM DTT, and 1 mM NaN3. Twenty microliters of 10 x KME (0.5 M KCl/10 mM MgSO4/10 mM EGTA) was added, the solution briefly vortexed, and then taken up in 100-,ul capillaries that were then sealed with "plasticene." After approximately 1 hr, the viscosity was measured by timing the velocity of steel ball bearings (Atlas Ball-Bearing, Birmingham, U.K.) falling through the solutions.

RESULTS

Isolation of cDNA Clones. The ZmABP] cDNA clone is an expressed sequence tag from a collection of 1101 pollen clones derived from a differentially screened pollen cDNA library using radiolabeled pollen and shoot cDNAs as described in Materials and Methods. The ZmABP] clone was identified as encoding a putative actin depolymerizing factor by comparison of the deduced amino acid sequence on current databases (11). The ZmABP2 cDNA clone was isolated by probing nylon filters containing the 1101 pollen clones with radiolabeled full-length ZmABP1 cDNA. The ZmABP3 cDNA clone is an expressed sequence tag from a maize leaf cDNA library identified as showing nucleic acid sequence identity with ZmABP1.

ZmABP Sequences. Full-length cDNA clones corresponding to the ZmABP], ZmABP2, and ZmABP3 genes were sequenced using oligonucleotide primers generated to sequence approximately 150 bp apart along the clones. All three ZmABP clones encode proteins of 139 amino acids. The deduced amino acid sequences of the three cDNA clones are aligned with those of the putative lily ADF (12), vertebrate ADF (13), and cofilin (43) in Fig. 1. The plant sequences show between 28-33% identity (67-70% similarity) with the vertebrate sequences. The boxed area in Fig. 1 is an alignment of the



Plant Biology: Lopez et al. Proc. Natl. Acad. Sci. USA 93 (1996) 7417

1 50 ZmABP1 MANSSSGLAV NDECKVKFRE LKSRRTFRFI VFR................. ZmABP2 MANSSSGLAV SDECKVKFRD LKARRSFRFI VFR. LilADF MANSSSGMAV DDECKLKFME LKAKRNFRFI VFK ....... ..........

ZmABP3 MANARSGVAV NDECMLKFGE LQSKRLHRFI TFK.................

PigADF ... MASGVQV ADEVCRIFYD MKVRKCSTPE EIKKRKKAVI FCLSADKKCI PigCof ... MASGVAV SDGVIKVFND MKVRKSSTPE EVKKRKKAVL FCLSEDKKNI CONSEN ..... ..V ..... F .. .......... .......... ..........

51 100 ZmABP1 IDDTDMEIKV DRLGEP.NQG YGDFTDSLPA NECRYAIYDL DFTTIENCQK ZmABP2 IDDKDMEIKV DRLGEP.NQG YGDFTDSLPA DECRYAIYDL DFTTVENCQK LilADF IEEKVQQVTV ERLGQP.NES YDDFTECLPP NECRYAVFDF DFVTDENCQK

ZmABP3 MDDKFKEIVV DQVGDR.ATS YDDFTNSLPE NDCRYAIYDF DFVTAEDVQK

PigADF IVEEGKEILV GDVGVTITDP FKHFVGMLPE KDCRYALYDA SFETKE.SRK PigCof ILEEGKEILV GDVGQTVDDP YATFVKMLPD KDCRYALYDA TYETKE.SKK CONSEN . V ... ...... F ... .LP. . .CRYA..D. ...T.E .. K

101 150

ZmABP1 SKIFFFS WSPDTARTRSKMLYASSKDRFRREL DGIQCEIQATDPSEMSLD ZmABP2 SKIFFFS WSPDTARTRSKMLYASSKDRFRREL DGIQCEIQATDPSEMSLD LilADF SKIFFIS WSPDTSRVRSKMLYASTKDRFKREL DGIQVELQATDPSEMSMD

ZmABP3 SRIFYIS WSPSSAKVKSKMLYASSNQKFKSGL NGIQVELQATDASEISLD

PigADF EELMFFL WAPELAPLKSKMIYASSKDAIKKKF QGIKHECQANGPEDL. NR PigCof EDLVFIF WAPECAPLKSKMIYASSKDAIKKKL TGIKHELQANCYEEVKDR CONSEN . ...... .P...... SIU.YA8 . ........ GI..E.QA.

151 170 ZmABP1 IVRSRTN ... .......... ZmABP2 IVKSRTN. LilADF IIKARAF .

ZmABP3 EIKDRAR ... ..........

PigADF ACIAEKLGGS LIVAFEGCPV PigCof CTLAEKLGGS AVISLEGKPL CONSEN .......... ..........

FIG. 1. An alignment of the deduced amino acid sequences of ZmABPI (11), ZmABP2, and ZmABP3 with a putative lily actin depolymerizing factor (LilADF) (12) and with vertebrate ADF (13) and cofilin (43). The boxed area indicates the actin-binding domain in vertebrate cofilin and the equivalent region in the related sequences. The five-point star denotes the Ser-3 and the Lys-112/Lys-114 shown to play important roles in the activity of human ADF and cofilin (22, 26, 27). The consensus relates to those amino acids that are identical in all six sequences. Only nine of these amino acids (indicated in bold) show 100% identity at the equivalent position in all ADF-like sequences published to date.

sequences corresponding to pig cofilin Trp-104 to Leu-128. Chemical crosslinking experiments, mutational analysis, and competition studies using synthetic peptides have demonstrated that this amino acid region (Trp-104 to Leu-128) in cofilin is likely to be a multifunctional domain binding both phosphoinositides and actin (22, 44, 45). This amino acid region is highly conserved between the members of the ADF group and with the plant sequences. All of the plant sequences show 52% identity (92% similarity) with the cofilin sequence in this region.

A comparison of the plant amino acid sequences reveals that ZmABP1 and ZmABP2 proteins are 94% identical. These are deduced protein sequences from pollen cDNAs and more identity is shown between these and the lily sequence from an anther cDNA (70% and 71%, respectively) than to that of ZmABP3 (56% and 58%, respectively), which is from a leaf cDNA.

Only 9 amino acids show 100% identity at the equivalent position in all 12 ADF-like sequences (including the sequences in this paper) published to date. Two of these amino acids Ser-3 and Lys-114 on the pig ADF sequence are known to play pivotal roles in the actin depolymerizing activity of ADF or cofilin (22, 26, 27).

Divergent Members of a Gene Family. Gene specific DNA probes were designed to distinguish between the ZmABP genes on maize genomic Southern blots. These probes were prepared by PCR amplification of the 3' noncoding regions of the ZmABPl, ZmABP2, and ZmABP3 cDNA clones. Fig. 2 shows the same Southern blot hybridized, stripped, and reprobed with several DNA probes as indicated. One band in each of the

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FIG. 2. Southern blot of EcoRI- or HindlIl-digested maize genomic DNA (15 jig) probed, stripped, and reprobed with gene specific probes prepared for ZmABPI, ZmABP2, and ZmABP3 and coding region probes for ZmABPI and ZmABP3.

two lanes for the Southern blot probed with either ZmABPI or ZmABP2 gene specific probes indicates that each represents a single gene. However, the coding sequences of both genes are closely related (showing 97% identity) and this is shown by the cross-hybridization of the ZmABPI coding sequence probe with the ZmABP2 gene on the Southern blot. The Southern blot probed with the ZmABP3 gene specific probe identifies two bands in each lane and, because there are no internal EcoRI and HindlIl sites within the probe, this would indicate that there are two ZmABP3 genes. The ZmABP3 coding region probe gives the same result as the corresponding gene specific probe. There is no cross-hybridization of ZmABP3 with ZmABPJ or ZmABP2 under our low-stringency conditions demonstrating the low identity (67% over the coding sequence) between these sequences and indicating that the ZmABP gene family is composed of at least two divergent subfamilies of genes with ZmABPJ and ZmABP2 in one subfamily and the pair of ZmABP3 genes in another.

Differential Expression of the ZmABPI, ZmABP2, and ZmABP3 Genes. A Northern blot containing RNA from various maize tissues has been probed, stripped and reprobed consecutively with the ZmABPJ, ZmABP2 and ZmABP3 gene specific probes. The Northern blot in Fig. 3 contains 10 gug of total RNA from a spikelet and pollen developmental series of tissues (2.5 mm, 5.0 mm, 7.0 mm, 10 mm spikelets, pollen and germinating pollen) and root, shoot, leaf, cob and embryo. Aceto-carmine staining of the anthers in the spikelets revealed that the 2.5 mm spikelet contained premeiotic sporogenous cells, the 5 mm and 7 mm spikelet anthers contained cells at mid-prophase 1 and the tetrad stage in meiosis, respectively, and the 10 mm spikelet anthers contained maturing pollen grains. It must be noted that due to the developmental



7418 Plant Biology: Lopez et al. Proc. Natl. Acad. Sci. USA 93 (1996)

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ZmABP2 Gene specific 1I:,

ZmABP3 Gene specific

FIG. 3. Northern blots containing 10 .tLg of total RNA from a maize spikelet and pollen developmental series and various maize organs demonstrating the differential expression of the three ZmABP genes. The Northern blot was probed, stripped, and reprobed with the gene specific probes of ZmABPJ, ZmABP2, and ZmABP3.

variation between the two florets within individual maize spikelets there will be some overlap between the consecutive staged spikelet tissue used for RNA isolation. The results show that the ZmABPJ and ZmABP2 transcripts only accumulate in mature pollen grains and germinated pollen indicating that gene expression takes place after pollen mitosis. In contrast, ZmABP3 transcript is abundant in every tissue examined with the exception of pollen and germinated pollen. In conclusion, this analysis shows that ZmABPl and ZmABP2 are expressed specifically in pollen whereas expression of ZmABP3 is only suppressed in pollen. These experiments were repeated using coding sequence probes with the same results (data not shown).

Purification of Recombinant ZmABP3 Protein. ZmABP3 was expressed in Escherichia coli strain BL21(DE3) by trans- fection of a cDNA fragment encoding ZmABP3 in the pMW172 vector (15). One liter of medium (Luria broth) with 0.1 mg/ml ampicillin was inoculated with a single colony of cells containing the vector grown on a bacterial plate of the same media. The flask was shaken at 37?C until the A600 was 0.6. Isopropyl ,B-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM and growth continued for 90 min. Cells were harvested by centrifugation at 10,000 x g for 10 min and the pellet solubilized in 25 ml of buffer: 50 mM Tris HCl (pH 7.5), 1 mM EGTA, 10% (wt/vol) sucrose, 1 mM NaN3, 1 mM DTT. The slurry was sonicated by 30 intermittent bursts over 1 min. The insoluble material was pelleted at 10,000 x g for 1 hr at 4?C and 5 ml of the supernatant (Fig. 4, lane 1) applied to a 1-ml Mono-Q column (Pharmacia) equilibrated in the same buffer. The protein was added and eluted with a 0-200 mM KCl linear gradient. The fractions containing the ZmABP3 protein, as assessed by SDS/PAGE (Fig. 4, lane 2) were pooled and applied directly to an hemagglutinin-Ultrogel column (IBF, Villeneuve-la-Garenne, France) equilibrated with the same column buffer but without EGTA. After a wash in this buffer, proteins were eluted with 0-200 mM phosphate buffer at pH 6.9. The resulting ZmABP3 gave a single band on SDS/PAGE (Fig. 4, lane 3).

Actin Binding and Depolymerizing Activity of ZmABP3 Protein. Native gel electrophoresis was used to demonstrate complex formation between ZmABP3 protein and G-actin. Both G-actin and ZmABP3 protein migrate as single bands on

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FIG. 4. Purification of bacterially expressed ZmABP3 protein in two steps. Lanes: 1, total protein extract of IPTG induced E. coli harboring the ZmABP3 cDNA in pMW172 vector; 2, fraction containing ZmABP3 protein after the Mono-Q column; 3, fraction containing ZmABP3 protein after the hemagglutinin-Ultrogel column.

nondenaturing gels in the presence of ATP, whereas a mixture of the two proteins gives predominantly a band of intermediate mobility with some excess actin migrating at the same position as the control (Fig. 5A). Similar experiments using actophorin showed complex formation under these conditions (40). Bind- ing of ZmABP3 protein to F-actin was demonstrated in sedimentation experiments (Fig. 5B). On its own, ZmABP3 protein is not pelleted at 100,000 X g, whereas in the presence of an equimolar concentration of F-actin, most of it cosediments with the actin. Comparison of the sedimentation profiles of actin in the presence and absence of the ZmABP3 protein shows that a small proportion of the actin is released into the supernatant when ZmABP3 protein is present. This is consistent with a low level of severing activity to produce short filaments that do not pellet under these conditions. Fig. 5C shows a dramatic reduction in the viscosity of F-actin when it is polymerized in the presence of low concentrations of ZmABP3 protein. Similar behavior has been reported for actin in the presence of actophorin, which provides further evidence for severing activity (20, 21).

DISCUSSION In pollen grains of the Liliaceae, large F-actin aggregates are distributed throughout the vegetative cytoplasm. Such aggregates are likely to be a storage form of actin ready for activation and germination. As a grain is activated, the spiculate aggregates dissociate and at a later stage more elongated actin fibrils can be seen converging on the germinal aperture. In the germinated pollen grain, actin cables ramify within the tube cytoplasm and run essentially parallel to the length of the tube and are continuous with the filamentous array in the pollen grain (46). In the grasses this reorganization is less clear partly

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Plant Biology: Lopez et al. Proc. Natl. Acad. Sci. USA 93 (1996) 7419

A ATP-Actin ZmABP3 ZmABP3 +

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FIG. 5. (A) Native gel electrophoresis shows a complex formation between G-actin and ZmABP3 protein. G-actin and ZmABP3 protein migrate as single bands, whereas a mixture of the two gives a new component of mobility intermediate between the two. (B) ZmABP3 protein binds F-actin. Sedimentation of ZmABP3 protein and 10 ,uM F-actin separately or as a mixture. At 100,000 x g, ZmABP3 protein remains in the supernatant (S), whereas all the actin is pelleted (P). When mixed together most of the ZmABP3 protein cosediments with F-actin, but a small proportion of the actin is released into the supernatant. (C) ZmABP3 protein reduces the low shear viscosity of F-actin. Actin (10 ,uM) was polymerized in the presence of increasing concentrations of ZmABP3 protein for 1 hr and the viscosity was measured as described.

mature grass pollen is that actin cables converge on a dense disk of F-actin at the germinal aperture. As germination

proceeds the disk decreases in size (47); presumably this actin is forming cables for tube extension. It is almost certain that actin- binding proteins will mediate such dramatic changes in actin organization and a specialized role for pollen-specific actin-binding proteins is not unlikely.

We report here the specific patterns of expression of three maize genes, ZmABP1, ZmABP2, and ZmABP3, that encode proteins related to the ADF group of proteins. The ZmABP1 and ZmABP2 genes are expressed specifically in pollen, whereas ZmABP3 is only suppressed in pollen. The pollen- specific expression of ZmABPI and ZmABP2 is similar to that previously observed for three maize profilin genes (3). More- over, the ZmABPI and ZmABP2 genes now join the relatively small group of proteins active in the male gametophyte but not in the sporophyte (48).

The deduced amino acid sequences of ZmABP1, ZmABP2, and ZmABP3, share between 28% and 33% identity (67 and 70% similarity) with mammalian cofilin and ADF. Biochem- ical analysis of the ZmABP3 protein confirms that it has actin-binding properties similar to members of the ADF group. It forms complexes with G-actin, cosediments with filamentous actin at pH 7.0, and markedly reduces the low shear viscosity of actin under polymerizing conditions that is consistent with actin severing activity. Sequences within the cofilin molecule identified as important for actin interaction (45) are highly conserved in the maize proteins (boxed in Fig. 1), including the lysine residue (Lys-114), mutation of which inhibited both filament binding and depolymerizing activity (22). These data confirm the fact that the ZmABP proteins have phenotypic properties expected of a member of the ADF group of actin severing proteins.

The ZmABP] and ZmABP2 pollen cDNA clones share 97% nucleic acid and 94% amino acid sequence identity, whereas they show only 67% nucleic acid and 56-58% amino acid sequence identity with the leaf cDNA clone ZmABP3, indicating that the genes expressed in pollen and leaves have evolved under different selective pressures. If we assume that all three related genes code for proteins with similar activities, it will be important to find out whether there are distinctive differences in their specific activities in the tissues where they are located. Certainly, the actin cytoskeletal reorganization that occurs in male gametophyte development has no parallel in sporophytic tissues examined to date, and may require a specialized ADF-like protein both for the passage of the pollen grain into dormancy and the activation of pollen tube growth. In this context, it is interesting that a lily gene (12), also similar to the ADF group of proteins and preferentially expressed in anthers, shows more identity with ZmABP1/ZmABP2 compared with ZmABP3. Perhaps, within the monocots at least, there has been some functional constraint on the evolution of these genes expressed specifically in the male gametophyte.

It has been established that actin depolymerizing factors reversibly regulate actin polymerization and depolymerization (8, 9, 31). The biochemical data obtained for the ZmABP3 protein clearly demonstrate that a similar activity is likely to occur in plant cells. It is also known that the vertebrate ADF-actin interaction is inhibited by PIP2 (24). This implies that this group of proteins could be involved in, or regulated by, the phosphoinositide signal transduction pathway. How- ever, vertebrate ADF binds other polyphosphoinositides indicating that the ADF-PIP2 interaction may not be as specific as it is, for example, for profilin (9). Also, the activity of mammalian ADF and cofilin is regulated by pH (15, 16). The fact that cytoplasmic pH does play a vital role in the regulation of many cellular processes (49-51) and that pH gradients do exist in germinated pollen (52) would suggest that alterations in pH may contribute to controlling the activity of ZmABP1/ ZmABP2 proteins. Recently, it has been shown that phosphorylation of human ADF and cofilin inactivates these proteins and that the regulatory phosphorylation site is the serine



7420 Plant Biology: Lopez et al. Proc. Nat!. Acad. Sci. USA 93 (1996)

at position 3 (MASGV) (26, 27). This serine and its surround- ing sequence does not form a consensus for any known serine kinase and it is therefore likely that a specific ADF regulatory serine kinase is responsible (26). This serine, together with the adjacent glycine, is conserved in all ADF-like sequences so far published, including the maize ZmABP sequences, indicating that similar regulatory control will be exerted in plants. In conclusion, the significance of these findings is that they show, in principle, how the maize ZmABP proteins could be mod- ified by signaling pathways that thereby modulate actin biology.

Further work will be needed to demonstrate how the ZmABP proteins are involved in actin reorganization in pollen. However, one possibility can be extrapolated from the work of Maciver et al. (28). They have shown that when polymerizable actin is mixed with the actin bundling protein, a-actinin, together with the actin depolymerizing factor, actophorin, rigid bundles of actin filaments appear that form a viscous gel. A similar experiment, but in the absence of actophorin, resulted in a much looser homogeneous gel as expected from a-actinin activity. Maciver et al. (28) proposed that actophorin can promote bundling by shortening actin filaments enough to allow them to rotate into positions for lateral interactions with each other via a-actinin. This may suggest that the F-actin aggregates in the lily pollen or the F-actin disk in the grass pollen is the result of the cooperative interaction of at least the actin bundling proteins and actin depolymerizing factors. Once the actin depolymerizing factors are inactivated by one or more of the pathways detailed above the actin is available to form its near homogeneous array that extends into the pollen tube. It is therefore important to characterize these plant actin depolymerizing factors in greater detail and also to identify what other classes of actin-binding protein show pollen-specific expression.

This work was supported in part by a British Council scholarship (I.L.), by the Biotechnology and Biological Sciences Research Council (R.G.A. and C.-J.J.), and by the Medical Research Council (S.K.M.).

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Pollen Specific Expression of Maize Genes Encoding Actin Depolymerizing Factor-Like Proteins