Regulation of pollen tube growth by Rac-like GTPases

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1 Journal of Experimental Botany, Vol. 54, No. 380, Plant Reproductive Biology Special Issue, pp. 73±81, January 2003 DOI: /jxb/erg044 Regulation of pollen tube growth by Rac-like GTPases Alice Y. Cheung 1,2,3,5, Christine Y-h. Chen 1,2, Li-zhen Tao 1, Tatyana Andreyeva 1, David Twell 4 and Hen-ming Wu 1,2 1 Department of Biochemistry and Molecular Biology, University of Massachusetts, LGRT, Amherst, MA 01003, USA 2 Molecular and Cell Biology Program, University of Massachusetts, LGRT, Amherst, MA 01003, USA 3 Plant Biology Graduate Program, University of Massachusetts, LGRT, Amherst, MA 01003, USA 4 Department of Biology, Leicester University, Leicester LE1 7RA, UK Received 27 May 2002; Accepted 20 September 2002 Abstract Plant Rac-like GTPases have been classi ed phylogenetically into two major groupsðclass I and class II. Several pollen-expressed class I Rac-like GTPases have been shown to be important regulators of polar pollen tube growth. The functional participation by some of the class I and all of the class II Arabidopsis Rac-like GTPases in pollen tube growth remains to be explored. It is shown that at least four members of the Arabidopsis Rac GTPase family are expressed in pollen, including a class II Rac, AtRac7. However, when over-expressed as fusion proteins with GFP, both pollen- and non-pollen-expressed AtRacs interfered with the normal pollen tube tip growth process. These observations suggest that these AtRacs share similar biochemical activities and may integrate into the pollen cellular machinery that regulates the polar tube growth process. Therefore, the functional contribution by individual Rac GTPase to the pollen tube growth process probably depends to a considerable extent on their expression characteristics in pollen. Among the Arabidopsis Racs, GFP-AtRac7 showed association with the cell membrane and Golgi bodies, a pattern distinct from all previously reported localization for other plant Racs. Over-expressing GFP- AtRac7 also induced the broadest spectrum of pollen tube growth defects, including pollen tubes that are bifurcated, with diverted growth trajectory or a ballooned tip. Transgenic plants with multiple copies of the chimeric Lat52-GFP-AtRac7 showed severely reduced seed set, probably many of these defective pollen tubes were arrested, or reduced in their growth rates that they did not arrive at the ovules while they were still receptive for fertilization. These observations substantiate the importance of Rac-like GTPases to sexual reproduction. Key words: Pollen tube, pro ling of pollen Rac GTPases, signalling of polar growth. Introduction The Ras superfamily of small GTPases is a large group of low molecular weight (20±25 kda), single unit signalling molecules with regulatory GTPase activity. The activity of these small GTPases depends on their association with GTP or GDP. The active GTP-bound form activates downstream effectors and signalling pathways. An intrinsic GTPase activity hydrolyses GTP to GDP whereby inactivating them. The equilibrium between the active and inactive forms of these small GTPases is controlled by positive and negative regulatory proteins in response to different cellular conditions and extracellular stimuli (Hall, 1998; Mackey and Hall, 1998; Valster et al., 2000). Within the Ras superfamily, the Rho family is further comprised of three related subfamilies known as Rho/Rac/Cdc42. In plants, the Rho-type small GTPases are represented by a large number of proteins (Valster et al., 2000; Zheng and Yang, 2000) that are most similar to Rac GTPases on the level of their primary sequence comparison and on their interactions with proteins bearing the `CRIB' domain, which binds speci cally to activated forms of Cdc42/Rac 5 To whom correspondence should be addressed. Fax: acheung@biochem.umass.edu ã Society for Experimental Biology 2003

2 74 Cheung et al. proteins (Burbelo et al., 1995; Wu et al., 2001). Therefore, many Rac-like GTPases have been referred to as Racs (e.g. the rice OsRac1; Kawasaki et al., 1999; the cotton GmRac13; Potikha et al., 1999; and several Arabidopsis Racs; Winge et al., 1997, 2000; Kost et al., 1999; Arabidopsis Genome Initiative, 2000; Lemichez et al., 2001), but Rop (for Rho of plants) is also prominently used in many studies (Zheng and Yang, 2000). In the general discussion, these plant Rac-like GTPases are referred to as Racs according to Winge et al. (1997, 2000), which provides the most complete sequence information and thorough phylogenetic comparison among plant Rac-like GTPases to date. Where speci c examples are cited from the literature, the nomenclature adopted in the cited publications is used; where possible, the analogous nomenclature designated in Winge et al. (2000) will be indicated. Rho GTPases from mammals and yeast are activated by a variety of extracellular stimuli and they interact with cellular target proteins to trigger multiple downstream responses. A major target system regulated by animal and yeast Rho GTPases is the actin cytoskeleton (Hall, 1998). Plant Rac-like GTPases, apparently, also have profound effects on the actin cytoskeleton (Kost et al., 1999; Fu et al., 2001; Lemichez et al., 2001; Chen et al., 2002a). A prominent regulatory role played by plant Rac GTPases was rst noted when the over-production of an Arabidopsis Rac, AtRac2 (equivalent to AtRac6 in Winge et al., 2000), altered the normal polar growth characteristics of pollen tubes into isotropic growth (Kost et al., 1999). Subsequently, another Arabidopsis Rac, Rop1At (Li et al., 1999; Fu et al., 2001) (analogous with AtRac11 in Winge et al., 2000) and a tobacco Rac, NtRac1 (most similar to AtRac11; Chen et al., 2002a) have also been observed to induce similar phenotypes in transformed pollen tubes that over-expressed these proteins. The actomyosin system has long been established as critical to pollen tube growth (Hepler et al., 2001). Typically, long actin laments and cables align along the long axis of elongating pollen tubes, but they are normally excluded from the apical clear zone where only short actin laments have been observed (Fig. 1; Chen et al., 2002b). The actin cables in pollen tubes that over-expressed NtRac1 extend into the apical region and are highly disorganized in the expanded tips (Chen et al., 2002a) (Fig. 1). This and observations in tobacco pollen tubes that over-express Arabidopsis Rac/Rop GTPases (Kost et al., 1999; Fu et al., 2001) are consistent with the idea that the actin cytoskeleton is the target for Rac GTPase-regulated downstream activities. The discovery of a key regulatory role played by Rac-like GTPases in the pollen tube tip growth process suggests considerable conservation in the regulation of actin-based cell motility systems in plants and animals. This conservation apparently extends to intermediary steps in the signalling pathways between Rho Fig. 1. Over-expression of Rac-like GTPases affect the pollen tube actin cytoskeleton. Actin cytoskeleton in normal (upper) and NtRac-1 over-producing (lower) tobacco pollen tubes (Chen et al., 2002b) as imaged by GFP-mTalin, a commonly used actin-binding protein (Kost et al., 1999). Actin cables in the control pollen tube are, in general, arranged as long cables along the shank of the tube, extending to the subapical region. Actin cables in the balloon-tipped pollen tube are more bundled in the shank and these cables extend into the ballooning tip region region. In the laboratory, over-expression of GFP-mTalin (Kost et al., 1998) often resulted in actin cytoskeleton not as organized as that revealed by another actin binding protein, GFP-NtADF1 (Chen et al., 2002b) or by phalloidin staining of chemically xed tubes (Vidali et al., 2001). type GTPases and the target actin via actin depolymerizing factor/co lin (Bamburg, 1999; Arber et al., 1998; Yang et al., 1998; Chen et al., 2002a) and phosphatidylinositol 4,5-bisphosphate (Martin, 1997; Van Aelst and D'Souza- Schorey, 1997; Kost et al., 1999) in both animal and plant cells. Plant Rac-like GTPases have been grouped into two classes, I and II, based on amino acid and nucleotide sequence comparisons (Winge et al., 2000). In addition to a slightly higher overall divergence from class I Rac- GTPases, the class II proteins show signi cant divergence from the class I members in what is known as the `insert region' of these proteins, which has been suggested to be important for effector protein interactions (Winge et al., 2000; Valster et al., 2000). In addition, class II Rac- GTPases have a more extended C-terminal region that is in itself more divergent among the members of this group (Winge et al., 2000). In Arabidopsis, there are seven class I and three class II Rac-GTPases. The Racs that have been shown to play important regulatory roles in pollen tube

3 growth (Kost et al., 1999; Fu et al., 2001; Chen et al., 2002a) are all pollen-expressed members of the class I Racs. It is reported that both class I and class II AtRacs are capable of regulating pollen tube growth. Furthermore, over-expression of GFP-AtRac7, a class II Rac GTPase present in pollen, induces multiple pollen tube phenotype and reduced seed set in transgenic plants. These studies further illustrate the importance of regulated Rac-like GTPase activity for pollen tube growth and sexual reproduction. Materials and methods cdna isolation and chimeric gene construction Arabidopsis AtRac2, 4, 5, 7, 8, 9, 10 cdnas (Winge et al., 2000) were isolated by RT-PCR by gene-speci c primers from total RNA isolated from owers at all developmental stages. These cdnas were inserted behind the coding region of the green uorescent protein (GFP) to create an in-frame N-terminal fusion with the various AtRac proteins. The pollen-predominant Lat52 promoter (Twell et al., 1990) was used to express these GFP-AtRac fusion proteins in pollen. Plant transformation, genetic crosses and segregation analysis The Lat52-GFP-AtRac7 chimeric gene was introduced into a Ti plasmid based vector for Agrobacterium transformation of tobacco (SR1). Seed yield from T1 transgenic plants were determined by weighing seeds from individual seed pots. For genetic crosses, female parental owers were emasculated at stage 10 (Koltunow et al., 1990), and pollinated on the following day. Seeds were germinated under tissue culture conditions in the presence of 50 mg ml ±1 kanamycin to select for transformed seedlings and for transgene segregation analysis. Microprojectile transformation of pollen, in vitro pollen tube growth cultures and observation Tobacco pollen was harvested from freshly dehisced anthers from greenhouse-grown plants. Microprojectile transformation was carried out as described earlier (Chen et al. 2002b). 5 mg of plasmid DNA was used for each microprojectile bombardment. Pollen was cultured immediately after bombardment. Pollen germination and tube growth cultures were as described previously (Chen et al., 2002b). Typically, pollen tubes were observed between 4±8 h after germination. Brefeldin A treatment of pollen tubes were carried out as described previously (Cheung et al., 2002). Pollen tubes were observed either by epi uorescence on a NIKON Eclipse 800 microscope or on a Biorad confocal using the 488 nm line for excitation. Pro ling of AtRac expression in Arabidopsis pollen Plasmids with cdna inserts from a Lambda Hybrid-Zap library constructed from Arabidopsis pollen (D Twell, unpublished data) were excised and ampli ed according to the manufacturer's (Stratagene) protocol. The plasmid library DNA was used as templates for PCR ampli cation (30 cycles) using gene-speci c primers for various AtRacs under strict stringency conditions to estimate the contribution by each AtRacs to the total pool of pollenexpressed Racs present in the library. Rac-like GTPases and pollen tube growth 75 Results and discussion Localization of over-expressed class I and class II GFP-AtRacs in elongating pollen tubes The effect of over-expressing the Arabidopsis pollen Raclike GTPases, AtRac2 (analogous to AtRac6 in Winge et al., 2001) and Rop1At (analogous to AtRac11 in Winge et al., 2001) and a tobacco NtRac1 in transformed tobacco pollen tubes have been examined previously (Kost et al., 1999; Fu et al., 2001; Chen et al., 2002a). The high level of homology among various AtRacs suggests considerable biochemical similarities among these proteins. Therefore, differential expression from individual genes may be a predominant determining factor that governs the biological role for each of these small GTPases. To examine whether other Arabidopsis Rac-like GTPases are active regulators of polar pollen tube growth, the class I AtRac2, 4, 5, 9 and the class II AtRac7, 8, 10 were over-expressed as N-terminal GFP-fusion proteins in transformed tobacco pollen tubes (Figs 2, 3, 4). GFP allowed both transformed pollen to be identi ed and the localization of the GFP-Racs to be observed. Except for GFP-AtRac7, all the over-expressed GFP- AtRacs showed considerable cytosolic presence in transformed pollen tubes (Figs 2, 3). GFP-AtRac9 and GFP- AtRac10 appeared to be sequestered ef ciently into protein bodies of various sizes. GFP-AtRac7 showed a unique localization pattern (Fig. 4) so far reported for any plant Racs. Fluorescence was most predominant at the pollen tube surface and associated with highly motile organelles reminiscent of GFP-labelled Golgi bodies (Cheung et al., 2002). Brefeldin A, which interferes with Golgi stack organization (Satiat-Jeunemaitre and Hawes, 1992; Driouich et al., 1993), dissociated the GFP-AtRac7 labelled particles in these transformed pollen tubes and induced aggregates of uorescence (Fig. 4D) similar to those previously observed for similarly treated GFPlabelled Gogli (Cheung et al., 2002). Interestingly, the cell surface association of GFP- AtRac7 appears to be excluded at the most apical region of actively elongating pollen tubes (Fig. 4A±C). On the other hand, growth-arrested pollen tubes, either spontaneously arrested (Fig. 4E±I) or induced to stop growing by brefeldin A treatment (Fig. 4D), showed strong tip membrane GFP-AtRac7 localization. The functional signi cance of the exclusion of GFP-AtRac7 from the tip membrane of active growing pollen tubes remains to be determined. GFP-AtRac7 also associated with the generative cell surface (Fig. 4A, E, I), consistent with its bearing characteristics, including that for intracellular traf cking, similar to that of the vegetative cell membrane. The localization patterns for the GFP-AtRacs shown here are considerably different from those observed for GFP-AtRac2 described in Kost et al. (1999), GFP-Rop1At described in Li et al. (1999) and for GFP-NtRac1 (AY

4 76 Cheung et al. Fig. 2. Over-expression of GFP-AtRac2 induces a range of defective pollen tube phenotypes. All pollen tubes shown were transiently transformed by microprojectile bombardment. Micrographs were taken at about 6 h after microprojectile bombardment and pollen germination. (A) A control transformed pollen tube that overexpressed GFP only. GFP-expressing pollen tubes were usually still elongating and green uorescence was observed throughout the cytosol. (B±F) Transformed pollen tubes that over-expressed GFP- NtRac2. (B, C) Pollen tubes that have elongated for some distance before vacuoles developed and expanded to ll a considerable volume of the tubes. The upper and lower panels of each of these gures show the uorescent and DIC image of the same tube. Green uorescence was appressed against the pollen tube surface by the large vacuoles. It was hard to discern an obvious preference for membrane association of GFP-AtRac2 in these tubes. Strong green uorescent bodies (arrows with dashed tailed) were also observed, often in close proximity to the vegetative nucleus (arrow with a solid tail). (D) A pollen tube whose tip has ballooned after a brief elongation. (E±F) Pollen tubes that were most severely affected by the over-expressed GFP-AtRac2. Tube tip ballooned (E) or growth was arrested (F) shortly after their emergence. The severity of pollen tube phenotypes, in general, correlated with the level of expression of the GFP-AtRac2. Images in all panels, except the asterisked tube in the right-hand panel of (E) were taken by autoexposure. The asterisked image in (E) was of the same tube shown on the left-hand panel, but taken with the same exposure as the uorescent image in (B) was taken. The saturated exposure seen in the asterisked imaged in (E) re ects extremely high level of expression from GFP-AtRac2 in this tube. Cheung, CY-h Chen, H-m Wu, unpublished results). All of these GFP-tagged proteins showed a preferential cell membrane association, especially at the extending pollen tube tip. The Golgi localization of GFP-AtRac7 may re ect a retarded progression of AtRac7 during the traf cking and modi cation steps that lead the activated GTPase to the cell membrane (Shields et al., 2000). On the other hand, it is also possible that transient or stable association with Golgi membrane is a relevant aspect of AtRac7 function. Additional analysis for AtRac7 activity relative to its localization pattern will be necessary to resolve these possibilities. The majority of the AtRacs examined here, AtRac2, 4, 5, 8, and 9, did not show a strong association with the cell membrane. However, these AtRac GTPases apparently do not accumulate to considerable levels in pollen (see below). A more precise localization analysis for these proteins will need to be carried out in cell types that normally accumulate these small GTPases. The effect of over-expressed GFP-AtRacs on pollen tube growth Pollen tube growth was, in general, negatively affected by the over-expression of each of the GFP-AtRacs (Figs 2±4). Phenotypes were often manifested in the form of highly vacuolated tubes that ranged from very short to those that managed to extend to considerable lengths. The severity of pollen tube defects appeared to correlate directly with the expression level of each of the fusion proteins in individual tubes. Pollen tubes that were highly uorescent were usually the most defective and growth was arrested the earliest (Fig. 2E; Fig. 3, AtRac5 bottom panel). For all the GFP-AtRacs examined, defective pollen tubes were observed as early as around 3 h after germination. By 8 h after germination, more than 70% of pollen tubes transformed by each of the GFP-AtRac constructs showed growth defects. GFP-AtRac2 over-expressing tubes appeared to be the most severely affected among all the transformed pollen tube samples (Fig. 2). Tip growth became distorted and a balloon-tipped morphology developed in many of the transformed tubes (Fig. 2D, E). The most severely affected pollen showed a very high level of green uorescence and barely showed any tube elongation (Fig. 2E,F). Over-expressing GFP-AtRac4 or GFP-AtRac5 induced less defective pollen tube growth phenotypes (Fig. 3) relative to other class I AtRacs (AtRac2 shown in Fig. 2; and those described in Kost et al., 1999; Fu et al., 2001). Pollen tube tip morphology was only mildly affected in most of these transformed tubes and tip-ballooning has not been observed. However, some of the transformed pollen tubes had poor tube growth properties, became highly vacuolated and their growth was arrested early. On the other hand, almost an equal number of pollen tubes were capable of extending for considerable distances before signi cant vacuolation and growth arrest occurred (top panels of GFP-AtRac4 and GFP-AtRac5). Over-expression of GFP-AtRac9 also induced a range of severity in phenotypes, affecting mostly the extent of tube

5 Rac-like GTPases and pollen tube growth 77 Fig. 3. Pollen tube phenotypes induced by the over-expression of GFP-AtRac4, GFP-AtRac5, GFP-AtRac8, GFP-AtRac9, and GFP-AtRac10. All pollen tubes shown were transiently transformed by microprojectile bombardment. Within each group, pollen tubes are shown with increasingly severe phenotype from the top to the bottom panel. All images were taken by auto-exposure except the asterisked image in the bottom panel of the GFP-AtRac5 column. The asterisked image was of the same pollen tube as that shown in the panel above it, but taken with the same exposure time as that used for the top panel of that column. The saturated exposure re ects a very high level of accumulation of GFP-AtRac5 in this severely defective pollen tube. The uorescent patches in GFP-AtRac9 and GFP-AtRac10 over-expressing tubes were most likely protein aggregates of the transgenic protein products. growth and intracellular organization. However, unique among all the AtRacs examined, the green uorescent signal from GFP-AtRac9 was highly concentrated in structures that remained to be determined (Fig. 3). Cell membrane association was not observed and cytosolic background was very low compared with pollen tubes transformed by GFP-tagged forms of all the other Rac-like GTPases examined thus far from Arabidopsis and tobacco. AtRac9 is also unique in the sense that its sequence suggests neither a class I nor a class II classi cation. Its expression and functional properties remain to be explored. The class II AtRac8 and 10 GTPases, in general, induced milder growth phenotypes in transformed pollen (Fig. 3). The majority of GFP-AtRac8 over-expressing tubes elongated relatively normally for at least 6 h after germination. However, about 5% of all transformed pollen tubes, those with the most intense green uorescence, were growth-arrested shortly after emergence. Over-expressed GFP-AtRac10 appeared to have the least effect on pollen tube growth among all the AtRacs examined here (Fig. 3). Occasionally small, punctate, green uorescent structures could be seen in the tubes (middle panel of GFP-AtRac9). These structures could be quite extensive, especially in tubes where growth was retarded or newly arrested (bottom panel of GFP-AtRac9). The balloon-tipped pollen tube phenotype induced by AtRac2 in Kost et al. (1999) has been attributed to involve

6 78 Cheung et al. Fig. 4. Pollen tube phenotypes induced by the over-expression of GFP-AtRac7. Microprojectile bombarded, transiently transformed and Agrobacterium transformed pollen produced similar spectra of pollen tube phenotypes. Pollen tubes shown in (B) to (E) were transiently transformed by microprojectile bombardment, others have developed from pollen grains from Lat52-GFP-AtRac7 transformed plants. (A) A single optical section of a GFP-AtRac7 over-expressing pollen tube taken by confocal microscopy. The remaining images were obtained by epi uorescent microscopy. (A±C) GFP-AtRac7 expressing, normally elongating pollen tubes. Arrowheads indicate the tube apex where GFP-AtRac7 labelling was minimum. (D-1, 2, 3) Transformed pollen tubes treated with brefeldin A (Cheung et al., 2002). The GPF-AtRac7 labelled Golgi bodies coalesced into membrane aggregates that appeared as highly uorescent patches. (D-1, E) Balloon-tipped pollen tubes. (F, G) Pollen tubes that showed diverted growth trajectory (* indicates the original growth front of these tubes). The image in (G) was captured at the same exposure as a less uorescent, but normally elongating, pollen tube similar to those shown in (B) and (C). The saturated expression around the tube tip region re ects the high level of accumulation of GFP-AtRac7. (H, I) Bifurcated pollen tubes. The GFP-AtRac2 labelled Golgi bodies could be seen streaming into one of the two branches of the pollen tube in (H). The shorter of the two branches in (I) has obviously arrested soon after bifurcation. Arrows point to the generative cell. changes in the cellular condition of phosphatidylinositols. The effect of Rop1At on pollen tube tip expansion was suggested to be associated with differences in intracellular [Ca 2+ ] and Ca 2+ uxes across the cell membrane (Li et al., 1999). It has been observed that the depolarized pollen tube growth phenotype induced by NtRac1 occurred via a pathway affecting actin depolymerization factor (Chen et al., 2002a). Pollen tube tip ballooning was not a prevalent phenotype induced by GFP-AtRac4, 5, 8, 9, and 10 over-expressing pollen tubes. Instead, these small GTPases preferentially induced overall growth defects re ected by poor tube morphology and intracellular

7 organization. How these defects were mediated remain to be examined. Over-expression of GFP-AtRac7 induces diverse pollen tube growth phenotypes and reduced seed set Over-expressing GFP-AtRac7 resulted in a range of pollen tube phenotypes. Pollen tubes that showed moderate levels of green uorescence elongated relatively normally for considerable lengths (Fig. 4A±C). Their cytological features observed by green uorescence (Fig. 4A±C) and by Normaski optics (not shown) were similar to control pollen tubes. Transformed tubes that expressed the highest level of transgene expression developed ballooned tips and growth was arrested (Fig. 4D±1, E). Some pollen tubes aborted growth at the original tip, but growth was reinitiated from the ank of the tube (Fig. 4F, G). A new protrusion must have developed at the sub-apical region of the original tip and polar elongation continued at this new tip along the new trajectory while the original tip was arrested. Bifurcated pollen tubes (Fig. 4H, I) were also frequently observed. For these tubes, a new protrusion must have been organized but growth at the original tip also resumed, thus giving rise to the double growth fronts. Growth would ultimately be aborted in one of the two branches, some shortly after tube splitting (Fig. 4H, I). Imaging of the GFP-AtRac7 labelled Golgi bodies showed that after tube splitting, pollen cytoplasm preferentially streamed into one of the two branches, presumably to support its growth there while the other branch became lled with a vacuole and aborted growth (Fig. 4H). Tobacco plants that have been transformed by Lat52- GFP-AtRac7 have been obtained. Several transformed plants showed highly reduced seed set after sel ng (Table 1). Segregation analysis of T-DNA inheritance in the progeny seedlings indicates that plants with the lowest seed set had the highest copy number of the transgene (lines No. 1 and 21 in Table 1). Pollen tubes from these plants expressed a high level of green uorescence when cultured in vitro and they displayed the entire spectrum of Rac-like GTPases and pollen tube growth 79 phenotypes as shown in Fig. 4. Presumably, pollen from these transgenic plants have inherited several T-DNA inserts, thus conferring a high level of GFP-AtRac7 expression. Several lines also produced highly uorescent pollen tubes that showed similar defective phenotypes. Segregation analyses of their selfed progeny showed a segregation ratio signi cantly less than 3:1 of transformed to non-transformed seedlings (lines B, J and 26, Table 1). These observations suggest that these transformed plants probably only harboured one T-DNA insert, but expression from the transgene was strong. The reduced seed set observed in these plants could have resulted from retarded or aborted pollen tube growth within the pistil, although analysis of the pollen tube growth characteristics will be necessary to understand the basis of the observed reduced fertility. AtRac7 is apparently expressed in pollen, though not to the same extent as some class I AtRac genes (see below). It will be interesting to carry out functional analysis to examine how it contributes to the pollen tube growth process. Differential representation of AtRac cdnas among pollen cdnas PCR ampli cation from cdnas from an Arabidopsis pollen cdna library showed that AtRac6, and AtRac11 are predominantly represented (Fig. 5). This observation is consistent with previous reports on anther or pollen Fig. 5. Pro ling of AtRac cdnas among a library of Arabidopsis pollen cdnas. Lanes 1±11 represent ampli ed DNA fragments corresponding to AtRac1-11 (Winge et al., 2001), respectively. For each reaction, equal amounts of templates (total pollen cdnas) and AtRac-speci c primers were subjected to identical PCR conditions and 30 cycles of ampli cation. Table 1. GFP-AtRac7 over-expression in transformed tobacco pollen results in reduced seed set Seeds were obtained from sel ng of the T1 generation. Pollen and pollen tube green uorescence level was the strongest in these lines among more than 30 transformed lines analysed. Plant lines Mg seeds/pot (% of control seed yield) Segregation of transgene Km r Km s Km r /Km s Control (SR1) 120 (100) ± ± ± Lat52-GFP-AtRac7 Line 1 a 15 (12.5) Line 21 b A few seeds Line B a 15 (12.5) Line J a 10 (8.3%) Line 26 a 37 (31%) a Seed yield is average from 10 pots; seeds were from the pooled seeds from these pots. b Seeds were pooled from more than 30 pots. All of the seeds were used in this analysis.

8 80 Cheung et al. expression of these AtRacs. The promoter of AtRac6 is most active in pollen and pollen tubes (Kost et al., 1999) and AtRac11 mrna is highly pollen-predominant by assayed by RT-PCR (Li et al., 1998). AtRac1 (equivalent to Rop3At and previously shown to be present among ower RNA; Li et al., 1998) is shown here to be highly represented among pollen cdnas. PCR also revealed the presence of a detectable level of the class II AtRac7 among pollen cdnas. AtRac2, 3, 4, 5, 8, 9, and 10 cdnas are apparently not adequately represented in the cdna library to be detected by the same number of cycles of PCR as the other Racs (Fig. 5). The mechanisms with which each of the over-expressed AtRacs described here affects pollen tube polar growth remains to be determined. It is, however, apparent from the study described here that, irrespective of their normal presence or absence in pollen, each AtRac is capable of integrating into the pollen cellular constituents, whereby affecting the functioning of the endogenous pollenexpressed Rac GTPases, and leading to defective pollen tube growth. For the AtRacs that are normally not expressed (or expressed to very low levels) in pollen, it is possible that their over-expression affects pollen tube growth by titrating out essential factors that support the activity of those Rac-GTPases that are normally expressed in these polar growth cells. The observation that overexpression of both pollen-expressed and non-expressed AtRacs are capable of interfering with the normal pollen tube growth process suggests that these small GTPases are likely to have similar biochemical activities, consistent with the high level of conservation among their primary structures. The predominant biological roles for each of the AtRacs will be governed by their expression properties. Studies of plant Rac-like GTPases are still at a relatively early stage. However, the signi cance of these signalling molecules to different aspects of plant cell growth, development and stress response (Kost et al., 1999; Fu et al., 2001; Lemichez et al., 2001; Baxter-Burrell et al., 2002; Tao et al., 2002) have been suggested by many reports. The arena will be a fertile ground for studies in plant signalling pathways. Acknowledgements The Lat52 promoter was a gift from Dr S McCormick. The original DNA has been modi ed to accommodate convenient cloning sites on the 3 end of the promoter region. We also thank Dr Maura Cannon for the use of the microprojectile bombardment equipment from her laboratory. We thank Miss Cara Haney (Cornell University) for her participation in the generation of Lat52-GFP-AtRac7 transformed tobacco plants. Miss Haney was a fellow supported by an Undergraduate Summer Research Experience grant from the NSF (NSF DBI ) awarded to the University of Massachusetts. The confocal microscopy was carried out at the University of Massachusetts Central Microscopy facility, which is partially supported by a grant from the NSF (BBS ). The work reported here was partially supported by grants from NIH (GM52953) and DOE (97ER20288). References ArabidopsisGenome Initiative Analysis of the genome sequence of the owering plant Arabidopsis thaliana. Nature 408, 796±813. Arber S, Barbayannis FA, Hanser H, Schindler C, Stanton CA, Bernard O, Caroni P Regulation of actin dynamics through phosphorylation of co lin by LIM kinase. Nature 393, 805±809. Bamburg JR Proteins of the ADF/co lin family: essential regulators of actin dynamics. Annual Review of Cell and Developmental Biology 15, 185±230. 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