Journal of Experimental Botany, Vol. 54, No. 380, pp. 73-81,
January 1, 2003
© 2003 Oxford University Press
Regulation of pollen tube growth by Rac-like GTPases
Received 27 May 2002; Accepted 20 September 2002
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
5 To whom correspondence should be addressed. Fax: +413 545 3292. E-mail: acheung{at}biochem.umass.edu
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Abstract |
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Plant Rac-like GTPases have been classified phylogenetically into two major groups—class 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, profiling of pollen Rac GTPases, signalling of polar growth.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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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 specifically to activated forms of Cdc42/Rac 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 specific 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 first 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 filaments 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 filaments 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 type GTPases and the target actin via actin depolymerizing factor/cofilin (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.
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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 significant 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 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.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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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-specific primers from total RNA isolated from flowers at all developmental stages. These cDNAs were inserted behind the coding region of the green fluorescent 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 flowers 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 µg 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 µg 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 epifluorescence on a NIKON Eclipse 800 microscope or on a Biorad confocal using the 488 nm line for excitation.
Profiling 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 amplified according to the manufacturer’s (Stratagene) protocol. The plasmid library DNA was used as templates for PCR amplification (30 cycles) using gene-specific primers for various AtRacs under strict stringency conditions to estimate the contribution by each AtRacs to the total pool of pollen-expressed Racs present in the library.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Localization of over-expressed class I and class II GFP-AtRacs in elongating pollen tubes
The effect of over-expressing the Arabidopsis pollen Rac-like 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 identified and the localization of the GFP-Racs to be observed.
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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 efficiently 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 fluorescence (Fig. 4D) similar to those previously observed for similarly treated GFP-labelled 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 significance 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 trafficking, 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 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 reflect a retarded progression of AtRac7 during the trafficking and modification 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 fluorescent 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 fluorescence 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 significant 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 growth and intracellular organization. However, unique among all the AtRacs examined, the green fluorescent 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 classification. 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 fluorescence, 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 fluorescent 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 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 [Ca2+] and Ca2+ fluxes 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 reflected by poor tube morphology and intracellular 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 fluorescence elongated relatively normally for considerable lengths (Fig. 4A–C). Their cytological features observed by green fluorescence (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 re-initiated from the flank 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 filled 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 selfing (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 fluorescence when cultured in vitro and they displayed the entire spectrum of 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 fluorescent pollen tubes that showed similar defective phenotypes. Segregation analyses of their selfed progeny showed a segregation ratio significantly 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.
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Differential representation of AtRac cDNAs among pollen cDNAs
PCR amplification 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 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 flower 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).
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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 pollen-expressed 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 over-expression 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 significance 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.
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Acknowledgements |
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The Lat52 promoter was a gift from Dr S McCormick. The original DNA has been modified 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-0097225) 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 (BBS8714235). The work reported here was partially supported by grants from NIH (GM52953) and DOE (97ER20288).
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D. Bloch, M. Lavy, Y. Efrat, I. Efroni, K. Bracha-Drori, M. Abu-Abied, E. Sadot, and S. Yalovsky Ectopic Expression of an Activated RAC in Arabidopsis Disrupts Membrane Cycling Mol. Biol. Cell, April 1, 2005; 16(4): 1913 - 1927. [Abstract] [Full Text] [PDF]
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C. Y.-h. Chen, A. Y. Cheung, and H.-m. Wu Actin-Depolymerizing Factor Mediates Rac/Rop GTPase-Regulated Pollen Tube Growth PLANT CELL, January 1, 2003; 15(1): 237 - 249. [Abstract] [Full Text] [PDF]
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