* Laboratory of Plant Molecular Biology, The Rockefeller University, New York 10021-6399; Laboratory of Plant Cell Biology,
Institute of Molecular Agrobiology, National University of Singapore, Singapore 117604; § Division of Signal Transduction, Beth
Israel Deaconess Medical Center, Boston, Massachusetts 02115; and
Department of Cell Biology and ¶ Department of Medicine,
Harvard Medical School, Boston, Massachusetts 02215
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Abstract |
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Pollen tube cells elongate based on actin-
dependent targeted secretion at the tip. Rho family
small GTPases have been implicated in the regulation
of related processes in animal and yeast cells. We have
functionally characterized Rac type Rho family proteins that are expressed in growing pollen tubes. Expression of dominant negative Rac inhibited pollen
tube elongation, whereas expression of constitutive active Rac induced depolarized growth. Pollen tube Rac
was found to accumulate at the tip plasma membrane
and to physically associate with a phosphatidylinositol
monophosphate kinase (PtdIns P-K) activity. Phosphatidylinositol 4, 5-bisphosphate (PtdIns 4, 5-P2), the
product of PtdIns P-Ks, showed a similar intracellular
localization as Rac. Expression of the pleckstrin homology (PH)-domain of phospholipase C (PLC)-1, which
binds specifically to PtdIns 4, 5-P2, inhibited pollen tube
elongation. These results indicate that Rac and PtdIns
4, 5-P2 act in a common pathway to control polar pollen
tube growth and provide direct evidence for a function
of PtdIns 4, 5-P2 compartmentalization in the regulation of this process.
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Introduction |
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RHO, Rac, and Cdc42 homologues, which constitute
the Rho family of Ras related small GTPases, are
signaling molecules with key roles in the regulation
of a variety of cellular processes in animal and yeast cells
(Hall, 1998). They act as molecular switches by transducing signals in the GTP-bound conformation and by returning to an inactive GDP-bound state after GTP hydrolysis (Bourne et al., 1991
). The best characterized function of
Rho family proteins is the regulation of actin organization.
Rho, Rac, and Cdc42 homologues each control the formation of distinct actin structures in different cell types (Hall,
1998
). Drastic rearrangement of the cortical actin cytoskeleton accompanies the induction of secretion in animal
cells (Aunis, 1998
). Rho and Rac were found to regulate
mast cell secretion, both via controlling actin organization
and in an actin-independent manner (Price et al., 1995
;
Mariot et al., 1996
; Norman et al., 1996
). Saccharomyces cerevisiae Cdc42 plays an essential role in bud formation
during vegetative reproduction. Together with a group of
other proteins, including several Rho homologues, Cdc42
is thought to control the assembly of polarized, actin-containing complexes at the cell surface that mediate targeted
secretion required for bud growth (Mata and Nurse, 1998
).
Although the function of Schizosaccharomyces pombe
Rho homologues is less well characterized, these proteins are also known to localize to the cell cortex specifically at growth sites (Arellano et al., 1997
; Hirata et al., 1998
).
Phosphatidylinositol monophosphate kinases (PtdIns
P-Ks),1 which synthesize phosphatidylinositol 4, 5-bisphosphate (PtdIns 4, 5-P2), are among the proteins that have
been identified as Rac and Rho effectors (Chong et al.,
1994; Hartwig et al., 1995
). Stimulation of PtdIns 4, 5-P2
synthesis appears to be a pathway that could allow Rho
family GTPases to control targeted secretion by regulating actin organization and exocytotic membrane traffic in a coordinated manner (Martin, 1997
; Van Aelst and
D'Souza-Schorey, 1997
). PtdIns 4, 5-P2 is a ligand for a
large number of regulatory proteins, including actin-binding proteins and proteins with pleckstrin homology (PH-)
or C2-domains. Binding to PtdIns 4, 5-P2 may affect the activities of these proteins and/or recruit them to target membranes (Lemmon et al., 1997
; Kopka et al., 1998
;
Toker, 1998
). It is well established that PtdIns 4, 5-P2 regulates actin organization via its interaction with key actin-binding proteins such as profilin, gelsolin, and vinculin
(Janmey, 1994
). Synthesis of PtdIns 4, 5-P2 has also been
demonstrated to be essential for regulated secretion in animal cells (Hay et al., 1995
). Although the precise function
of PtdIns 4, 5-P2 in this process is not known, Hay and co-workers suggested that it acts by controlling actin organization, by altering the lipid composition of membrane microdomains, and/or by recruiting proteins that mediate
membrane fusion. Compartmentalized synthesis of PtdIns
4, 5-P2 and other membrane lipids was proposed to be a
key step in the organization of localized signaling events (Carpenter and Cantley, 1996
; Martin, 1997
; Irvine, 1998
).
However, only limited direct evidence for the accumulation of particular lipids in specific membrane domains has
been reported to date.
A surprisingly large number of homologues of Rho family small GTPases has been cloned from different plant
species. Interestingly, most of the cloned genes encode
proteins that are closely related to each other and to mammalian Rac (Winge et al., 1997; Li et al., 1998
). Little information is currently available on the cellular functions of
these proteins. A pea Rac homologue (Rop1Ps) was found
to be specifically expressed in pollen and in pollen tubes. Microinjection of an antibody against this protein inhibited pea pollen tube elongation (Lin and Yang, 1997
). Using immunofluorescence techniques, Rop1Ps was determined to localize to the pollen tube plasma membrane in
the tip region (Lin et al., 1996
). These results indicated a
possible role of Rac homologues in the regulation of pollen tube growth.
Pollen tubes are highly specialized, extremely elongated
cells with a diameter of 10-20 µm and length of up to several centimeters. Male generative cells are enclosed in the
pollen tube cytoplasm. Growing through the style, pollen
tubes transport generative cells from the stigma to the
ovule, where fertilization occurs. Pollen tubes extend in a
strictly polar manner with rates of several micrometers per
minute and are among the fastest growing cells known to
exist (Bedinger et al., 1994).
The structure of pollen tube cells has been examined in
numerous studies (Pierson and Cresti, 1992; Taylor and
Hepler, 1997
). The living protoplast is located in a 1-2-mm-
long region at tip, whereas the rest of the pollen tube consists of nothing but cell wall material. The pollen tube protoplast shows a characteristic longitudinal zonation. An
apical cytoplasmic "clear zone" packed with post-Golgi
secretory vesicles is followed by more granular cytoplasm
that contains other cell organelles and male generative cells. Large vacuoles are located at the basal end. Longitudinally oriented, thick actin bundles are found throughout the cytoplasm with the exception of the apex, where
actin filaments are sparse and fine (Miller et al., 1996
; Kost
et al., 1998
).
The following mechanisms have been implicated in polar pollen tube extension (Derksen et al., 1995; Taylor and
Hepler, 1997
). Pollen tube elongation is thought to be
based on a process known as "tip growth." Post-Golgi
secretory vesicles are believed to fuse with a small area of
the plasma membrane at the tube apex and to deliver cell
membrane as well as cell wall material required for growth
exclusively to this location. As observed in other tip growing cells, rapid translocation of pollen tube organelles along the longitudinal axis ("cytoplasmic streaming") is
essential for sustained secretion and tube elongation.
Pollen tube growth is very sensitive to drugs that interfere with actin polymerization and insensitive to microtubule inhibitors. Actin is believed to mediate cytoplasmic
streaming and may also have a function in the secretory
processes at the pollen tube tip.
Several reports have demonstrated an important role
of Ca2+ in the regulation of pollen tube growth. A tip-focused Ca2+ gradient with highest concentrations in the
cell cortex at the extreme apex has been detected in pollen
tube cells. The steepness of the gradient was found to correlate with the speed of pollen tube elongation (Pierson et al.,
1996). Interestingly, reorientation of pollen tube growth
was induced by photoactivated local release of caged Ca2+
in the flanks of the apical dome (Malhó and Trewavas,
1996
). Pharmacological experiments in combination with
analysis of effects of local release of caged inositol 1, 4, 5-triphosphate (Ins-P3) have provided evidence suggesting
that phospholipase C (PLC)-mediated Ins-P3 production
from PtdIns 4, 5-P2 is involved in Ca2+ regulation in pollen
tubes (Franklin-Tong et al., 1996
).
Here, we describe the cloning of an Arabidopsis thaliana Rac type small GTPase that is preferentially expressed in pollen and in pollen tubes. The function of this protein and its tobacco homologues was analyzed in cultured tobacco pollen tubes. Our results indicate that Rac proteins and PtdIns 4, 5-P2 both localize to the tip plasma membrane and act in a common pathway to regulate polar pollen tube growth. Possible links between Rac and Ca2+ signaling in growing pollen tubes are discussed.
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Materials and Methods |
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Cloning of At-Rac cDNA and Genomic Sequences
An At-Rac1 cDNA (Xia et al., 1996) probe was used to screen an A.
thaliana whole-plant Uni-ZAP XR cDNA library (Stratagene). From
phages containing hybridizing sequences, pBluescriptSK
phagemids
were excised according to the manufacturer's protocol. By sequencing, a
phagemid with an insert containing a full-length cDNA encoding an At-Rac1 homologue was identified. The sequence of this cDNA was deposited in the database (GenBank accession number AF107663) and the encoded protein was designated At-Rac2. The MegAlign software package
(DNASTAR Inc.) and the Clustal method were used to align the amino
acid sequences of At-Rac2 and of related proteins.
An A. thaliana genomic library (CD 4-8; Voytas et al., 1990; obtained
from the Arabidopsis Biological Resource Center, Columbus, OH) was
screened with At-Rac1 and At-Rac2 cDNA probes. Genomic fragments
that specifically hybridized with each probe were subcloned and partially
sequenced. Clones containing sequences identical to At-Rac1 and At-Rac2 coding sequences were identified. These clones were later found to
overlap with BAC clones sequenced as part of the ESSAII project. Clones
M4E13 (GenBank accession number AL022023) and T19K4 (GenBank
accession number AL022373) contain the genomic sequences of At-Rac1
or At-Rac2, respectively.
Expression Constructs
pUCAP-GUS was constructed by cloning an expression cassette consisting of the 35S promoter, a GUS (-glucuronidase) coding sequence interrupted by an intron (GUS-intron), and the nos poly A addition signal as
an SalI fragment from pTJK136 (Kapila et al., 1997
) into pUCAP (van
Engelen et al., 1995
). Sequences upstream of the At-Rac1 and At-Rac2
coding regions were amplified by PCR from genomic clones using a polymerase with proofreading capability (Pfu polymerase; Stratagene). Primers were designed to amplify the entire At-Rac1 (~1.1 kb) or At-Rac2
(~1.9 kb) promoter regions from the ends of adjacent open reading
frames (ORFs) to the beginning of the coding sequences and to introduce
NcoI sites at the start codons. Amplified At-Rac promoters were fused
via the NcoI site to the GUS-intron sequence by cloning restricted PCR
fragments into pUCAP-GUS. Insertion of the At-Rac promoters replaced the 35S promoter originally present in this construct. The resulting plasmids were used to analyze promoter activity in growing pollen tubes
in transient expression experiments. At-Rac1::GUS-intron::nos and At-Rac2::GUS-intron::nos expression cassettes were subcloned from these
constructs as HindIII or XbaI fragments, respectively, into the binary vector pPZP211 (Hajdukiewicz et al., 1994
). Resulting plasmids were used to
generate stably transformed A. thaliana lines.
Sequences encoding At-Rac2 and At-Rac2CSIL were amplified by
PCR from the At-Rac2 cDNA in pBluescriptSK
. A DNA fragment encoding the NH2-terminal catalytic domain of Clostridium difficile toxin B
(TcdB; amino acids 1-546) was also amplified by PCR using the construct
"CDB1-546 in pGEX-2T" (Hofmann et al., 1997
) as a template. Primers
were designed to create an ATG context optimal for gene expression in
dicot plants (AACAATG; Lütcke et al., 1987
) and to introduce stop
codons at the 3' ends of the At-Rac2
CSIL and TcdB sequences. PCR
fragments, after confirmation of error-free amplification by sequencing,
as well as a GUS cDNA (derived from pBI121; CLONTECH Laboratories Inc.) were inserted into the pUCAP-based expression vector
pLAT52MCS (Kost et al., 1998
) between the lat52 promoter and the nos
poly A addition signal. QuikChangeTM (Stratagene) PCR-based mutagenesis was used to create mutant sequences encoding G15V-At-Rac2, T20N-At-Rac2, and Q64E-At-Rac2. After sequence confirmation, mutated At-Rac2 cDNAs were subcloned into nonamplified pLAT52MCS.
The cloning of sequences encoding green fluorescent protein (GFP)
and GFP fused to the NH2 terminus of the mouse talin f-actin binding domain into pLAT52MCS was described earlier (Kost et al., 1998). Into the
same expression vector, sequences were inserted that encoded GFP fused
to the NH2 terminus of the following polypeptides: At-Rac2, At-Rac2
CSIL, and G15V-At-Rac2 (sequences subcloned from the vectors
described above); the PH-domain of human PLC-
1 (amino acids 2-175 of
PLC-
1; sequence subcloned from "PLC-
1-PH in Hiro3"; Stauffer et al.,
1997
); and the PH-domain of ADP-ribosylation factor nucleotide-binding
site opener (ARNO) (amino acids 262-399 of ARNO; sequence amplified from "ARNO in pEGFP-C1", Venkateswarlu et al., 1998
. The DraIII-SalI fragment at the 3' end of the PCR product was found to contain a point
mutation and was replaced by corresponding wild-type sequences from
"ARNO-PH in pEGFP-C1"; the final construct was free of sequence errors). The GFP-PLC-
1-PH sequence in pLAT52MCS was altered by QuikChangeTM mutagenesis to sequences encoding GFP-PLC-
1-PH-K32L and GFP-PLC-
1-PH-K32E. Correct amplification of the regions encoding mutated versions of PLC-
1-PH was confirmed by sequencing.
A. thaliana Transformation
Transgenic A. thaliana ecotype Landsberg erecta lines were generated as
described in Kost et al. (1998) using the Agrobacterium vacuum-infiltration method developed by Bechtold et al. (1993)
. T1 seeds were plated on
MS medium (Murashige and Skoog, 1962
) containing 50 mg/liter kanamycin and 100 mg/liter cefotaxime. In vitro grown, kanamycin-resistant T1
plants at different developmental stages as well as pollen collected from
such plants were analyzed for GUS expression as described below.
Transient Gene Expression in Cultured Tobacco Pollen Tubes
For transient expression, genes were transferred into cultured tobacco
pollen tubes by particle bombardment as described in Kost et al. (1998).
Coexpression of two genes was achieved by coating particles with equal
amounts of each expression vector.
Histochemical Analysis of GUS Expression
To assay for GUS activity, A. thaliana plants and pollen grains were immersed for 12-18 h in a X-Gluc substrate solution. Assayed plants were extracted with ethanol to remove chlorophyll and improve visibility of the blue GUS reaction product. Destained plants were examined under a dissection microscope. Plant organs and pollen grains were mounted in water between slides and coverslips for observation at higher magnifications.
To visualize transient GUS expression, X-Gluc solution (1 ml per plate) was evenly distributed on the surface of solid culture medium covered with pollen tubes grown from bombarded grains. After 2 h of incubation, squares of solid medium were cut out and flipped upside-down (pollen tubes in direct contact with the glass) onto coverslips.
Stained plant organs, pollen grains, and pollen tubes were analyzed by bright-field transmitted light microscopy using an Axioscope (Carl Zeiss Inc.) microscope. Images were taken by 35-mm photography (64T film; Eastman Kodak Co.).
Incubation in X-Gluc solution was performed at 37°C. All X-Gluc solutions were buffered with 0.1 M sodium phosphate at pH 7.0. Plants were incubated in a solution containing 0.2% X-Gluc (Jersey Lab and Gloves Supply), 0.1% Triton X-100, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, and 6% N,N-dimethyl-formamide (DMF). The same solution supplemented with 5% mannitol was used for pollen tubes. Pollen grains were assayed in 0.1% X-Gluc, 0.1% Triton X-100, 3% DMF, and 5% mannitol.
Fluorescence Microscopy
Epifluorescent images of GFP-expressing pollen tubes were taken
through a 4× lens using a standard FITC filter set and an Axioscope microscope equipped with a 100-W mercury lamp. All images were obtained
by exposing 400 ASA 35 mm film (EliteII; Eastman Kodak Co.) for 10-15 s.
Confocal analysis of GFP expression was performed using an LSM410 inverted confocal microscope (Carl Zeiss Inc.) as outlined in Kost et al.
(1998). Methods used to determine pollen tube growth rates and techniques used for digital image processing are described in the same report.
Intensity plots were created using the Scion Image software package
(Scion Corp.).
Preparation of Recombinant Protein
Recombinant glutathione S-transferase (GST) and GST fusion proteins
were prepared as described by Lemichez et al. (1997) with some modifications. Synthesis of recombinant protein in Escherichia coli BL21 was induced by treatment with 0.5 mM IPTG for 2 h at 30°C. Cells were lysed
and recombinant proteins were purified in PB buffer (50 mM Tris-HCl at
pH 7.4; 250 mM NaCl, 5 mM MgCl2, 0.1 mM DTT). After purification, recombinant proteins on agarose beads were resuspended in PB buffer containing 50% glycerol, frozen in liquid nitrogen, and stored at
80°C. For
GTP binding and kinase binding assays, recombinant proteins on agarose
beads were loaded with nucleotides for 20 min at room temperature in
loading buffer (50 mM Tris-HCl at pH 7.4; 25 mM NaCl, 1 mM EDTA,
0.5 mM DTT) containing 0.1 mM [
-32P]GTP (6,000 Ci/mmol; New England Nuclear), GTP
S, or GDP
S (both Boehringer Mannheim), followed by addition of MgCl2 to a final concentration of 20 mM. GTP
binding and GTPase activity assays were performed using protocols established by Self and Hall (1995a
and 1995b, respectively).
Preparation of Pollen Tube Extracts
Tobacco pollen tubes (1 g fresh weight) grown for 3 h in liquid PT medium
(Kost et al., 1998) were washed twice with a solution containing 150 mM
potassium phosphate (pH 7.2), 0.4 M mannitol, and 150 mM NaCl. After
resuspension in 5 ml lysis buffer, pollen tubes were passed twice through a
Dounce homogenizer. If nothing else is indicated, a solution containing 10 mM Hepes (pH 7.4) and 0.8 M sorbitol was used as lysis buffer. All lysis
buffers used were supplemented with a protease inhibitor cocktail (CompleteTM, EDTA-free; Boehringer Mannheim) according to the manufacturer's instructions.
Preparation of Affinity-purified Anti-At-Rac Antibody
(-At-Rac), SDS-PAGE, and Immunoblotting
Polyclonal anti-At-Rac antiserum was obtained from rabbits immunogenized with GST-At-Rac1. Standard methods described in Harlow and
Lane (1988) were used to affinity purify specific anti-At-Rac antibodies
(
-At-Rac) on nitrocellulose membranes loaded by blotting with thrombin-treated, GST-free At-Rac1. Pollen tube and recombinant proteins
were resolved by SDS-PAGE according to Laemmli (1970)
under reducing conditions using 12% gels. For immunoblotting, proteins were transferred onto PVDF membranes (Schleicher & Schuell). Membranes were
incubated with primary antibodies, affinity-purified
-At-Rac (diluted
1:200) or a monoclonal mouse anti-actin antibody (Boehringer Mannheim;
diluted 1:2,500), and secondary antibodies, peroxidase-conjugated goat
anti-rabbit or goat anti-mouse antibodies (Boehringer Mannheim; diluted 1:5,000), for 1 h at room temperature in Tris-buffered saline (TBS;
25 mM Tris-HCl at pH 7.4; 137 mM NaCl, 5 mM KCl) supplemented with
4% BSA (A-3059; Sigma Chemical Co.). After incubation with primary
and secondary antibodies, membranes were washed twice for 10 min with
TBS containing 4% BSA and 0.02% Tween 20. After the final wash,
membrane-associated peroxidase activity was visualized using the ECL kit
(Boehringer Mannheim) according to the manufacturer's instructions.
TcdB Glucosylation Assays
Pollen tube extracts were prepared as described above with homogenization performed in FB (50 mM triethanolamine at pH 7.8; 150 mM KCl, 1 mM
DTT, 5 mM MgCl2, protease inhibitors). TcdB glucosylation reactions
were carried out as described in Just et al. (1995). In brief, 0.5 µg TcdB
(obtained from Ingo Just, Albert-Ludwigs-Universität, Freiburg, Germany) and 0.3 µCi UDP-[14C]-glucose (300 mCi/mmol; New England Nuclear) in FB buffer were added to extract containing 10 µg total pollen
protein or to PB containing 1 µg recombinant GST-At-Rac2 to obtain a final volume of 20 µl. The reaction mix was scaled up to a total volume of 200 µl for immunoprecipitation experiments. Reactions were carried out
at 37°C for 1 h. Products were resolved by SDS-PAGE either directly or
after immunoprecipitation with affinity-purified
-At-Rac (diluted 1:20).
Gels were incubated for 10 min in 1 M salicylic acid before drying.
Pollen Tube Fractionation
Pollen tube extracts were centrifuged for 10 min at 10,000 g to obtain a post-nuclear supernatant (PNS) with a protein concentration of ~0.25 mg/ml. The resulting PNS was centrifuged at 100,000 g for 1 h (SW-55 Ti rotor; Beckman Instruments Inc.) to separate cytoplasmic (supernatant) and membrane (pellet) fractions. Pelleted membranes were resuspended in lysis buffer. Proteins contained in 20 µl of each PNS, cytoplasmic, and resuspended membrane fractions were analyzed by immunoblotting. TcdB glucosylation assays were carried out as described above using 5 µl of each fraction.
Kinase Binding Assays
Kinase binding assays were performed as described by Ren et al. (1996)
with some modifications. Pollen tube extracts were prepared as described
above with homogenization performed in buffer A (50 mM Tris-HCl at
pH 7.4; 1% Triton X-100, 100 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 1 mM
DTT, protease inhibitors). Extracts were clarified by centrifugation at
13,000 g for 15 min at 4°C. Agarose beads bound to GST (25 µg protein)
or to GST-At-Rac2 (2.5 µg protein) loaded with nucleotides as described
above were added to 500 µl clarified extract (0.5 mg/ml total protein) and
incubated for 2 h at 4°C. After incubation, beads were washed three times
in buffer B (50 mM Tris-HCl at pH 7.4; 0.1% Triton X-100, 50 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 1 mM DTT) and once in buffer C (50 mM
Tris-HCl at pH 7.4; 0.02% Triton X-100, 100 mM NaCl, 5 mM MgCl2, 0.1 mM EGTA, 1 mM DTT). After resuspension in buffer C during the last washing step, one-tenth of the total volume of each sample was analyzed by
SDS-PAGE and Coomassie brilliant blue staining to verify concentration
and quality of the recombinant proteins used. Washed beads were resuspended in 35 µl buffer C. Samples used to investigate effects of phosphatidic acid on At-Rac2-associated kinase activity were treated similarly,
but Triton X-100 was omitted from all buffers except for buffer A. Beads
in 35 µl buffer C were supplemented with 20 µg lipid substrates in 10 µl
10 mM Tris-HCl, pH 7.4, and incubated at room temperature for 10 min.
Either PtdIns 4-P (P-9628; Sigma Chemical Co.) or a mix of phosphoinositides (P-6023; Sigma Chemical Co.) was used as a substrate. Some
samples were supplemented with phosphatidic acid (P-9511; Sigma Chemical Co.) to a final concentration of 40 µM. Kinase reactions were started
by adding 5 µl ATP buffer (50 mM Tris-HCl at pH 7.4; 5 mM MgCl2, 0.5 mM
ATP, 10-25 µCi [
-32P]ATP, 6,000 Ci/mmol; New England Nuclear) to
each sample. After 7 min, reactions were stopped with 80 µl HCl (1 N).
Lipids were extracted in 160 µl methanol/chloroform (1:1) by vigorous
mixing for 1 min. Organic and aqueous phases were separated by centrifugation. The lower organic phase was washed with 260 µl HCl (1 N)/methanol/chloroform (48:47:3), concentrated under N2 to a volume of 20 µl and
spotted on Oxalate-EDTA-impregnated silica gel plates (LK6D; Whatman Inc.). Chromatography was performed as described by Pignataro and
Ascoli (1990)
.
Radioactivity Detection
Radioactivity emitted from membranes, gels, or TLC plates was visualized using a PhosphorImager detection system (Molecular Dynamics).
HPLC Analysis
32P-labeled products of kinase binding assays performed as described
above using a mix of phosphoinositides as lipid substrate were deacylated,
mixed with 3H-labeled standards, and analyzed by anion-exchange HPLC
using a Partisphere SAX column (Whatman) as described by Serunian et al. (1991). Elution of assay products and standards was detected simultaneously using an on-line continuous flow scintillation detector (Radiomatic® FSA; Packard Instrument Co.).
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Results |
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Cloning of Sequences Encoding an A. thaliana Rac Homologue That Is Preferentially Expressed in Growing Pollen Tubes
Previously, we reported the cloning of At-Rac1, which was
identified in a screen for A. thaliana cDNAs that cause
morphological changes when expressed in yeast (Xia et al.,
1996). Using At-Rac1 as a probe, At-Rac2 was isolated
from an A. thaliana cDNA library. The corresponding genomic sequences were cloned by screening an A. thaliana
library with At-Rac1 or At-Rac2 cDNA probes. Genomic sequences upstream of the At-Rac1 and At-Rac2 ORFs, including the ends of adjacent ORFs and the entire At-Rac
5' untranslated regions, were fused to a cDNA encoding
GUS (Jefferson et al., 1987
). Transgenic A. thaliana lines
containing the resulting At-Rac1 or At-Rac2 promoter-
GUS fusion constructs (At-Rac1::GUS, At-Rac2::GUS) were generated and histochemically analyzed for GUS expression. Four independent lines transformed with At-Rac2::GUS showed GUS expression confined to pollen
(Fig. 1 a), to growing pollen tubes, to stipules, and to a
small region of the vascular tissue below the cotyledons
(data not shown). In contrast, six lines transformed with
At-Rac1::GUS were all found to express GUS in the vascular tissues of all organs. Only one of these lines showed a
weak GUS expression in pollen (data not shown).
|
GUS is known to be a stable protein with a slow turnover rate in plant cells (Martin et al., 1992). To confirm
that GUS activity detected in transgenic pollen tubes resulted at least partially from gene expression during pollen
germination and tube growth, promoter-GUS fusion constructs were also transiently expressed in growing tobacco
(Nicotiana tabacum) pollen tubes. Under these conditions,
the At-Rac2 promoter was found to confer strong GUS expression (Fig. 1 c) at a level similar to that obtained with the pollen-specific lat52 promoter (Twell et al., 1989
; Fig. 1 e). No GUS expression was observed in pollen tubes transiently transfected with At-Rac1::GUS (Fig. 1 d) or with
a control construct containing a cDNA encoding GFP
(Prasher et al., 1992
) fused to the lat52 promoter (Fig. 1 f).
These results indicate that At-Rac2, but not At-Rac1, is
preferentially expressed in pollen and in growing pollen tubes.
At-Rac1 and At-Rac2 encode proteins of 21 kD that
show high homology to human HsRac1 (62.0 and 61.3%
identical amino acids, respectively; Fig. 2). Recombinant
At-Rac1 and At-Rac2 were both found to bind and to hydrolyze GTP (data not shown), confirming that the two
proteins are indeed functional small GTPases. They belong to a large family of Rac-like proteins with unknown
or poorly characterized cellular functions whose genes
have been cloned recently from different plant species.
The protein sequences of Arac1 (Winge et al., 1997) and
Rop1At (Li et al., 1998
), two A. thaliana Rac-like proteins
that share very high sequence homology with At-Rac2
(98.5 and 95.4% identical amino acids, respectively), are
shown in Fig. 2. Interestingly, Rop1At has been shown to
be expressed in pollen and was proposed to have a function in the regulation of pollen tube growth (Li et al.,
1998
).
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We found that in situ analysis of At-Rac2 function in
cultured A. thaliana pollen tubes is not feasible. These
cells elongate slowly, show severe morphological abnormalities, and cease growing only a few hours after germination (Taylor and Hepler, 1997; Kost, B., and N.-H.
Chua, unpublished observations). By contrast, tobacco pollen tubes can grow rapidly and morphologically normal
in vitro for up to 48 h (Read et al., 1993
). Methods have
been established that allow analysis of transient gene expression in cultured tobacco pollen tubes after gene transfer into germinating pollen grains by particle bombardment (Kost et al., 1998
). We have used these methods,
along with biochemical techniques, to study the function of At-Rac2 and its tobacco homologues in the regulation
of pollen tube growth.
Growing Tobacco Pollen Tubes Express at Least One At-Rac2 Homologue with an Essential Function in Tube Elongation
An antibody raised against At-Rac1 (-At-Rac) was
found to have similar affinities to recombinant At-Rac1
and At-Rac2 (data not shown). Immunoblot analysis
showed that in tobacco pollen tube extracts
-At-Rac
binds to at least one protein with an apparent molecular
mass of 21 kD (Fig. 3 a), which corresponds to the size of
Rac. Clostridium difficile toxin B (TcdB) is known to glucosylate and thereby inactivate specifically mammalian
Rho family proteins including Rac (Aktories, 1997
).
Treatment with TcdB and UDP-[14C]-glucose resulted in
glucosylation and labeling of recombinant At-Rac2 (data
not shown), showing that plant Rac homologues can serve
as substrates of this toxin. Radiolabeled proteins of 21 kD
were detected in tobacco pollen tube extracts assayed for
glucosylation by TcdB, but not in control samples treated
with UDP-[14C]-glucose alone (Fig. 3 b). To investigate
whether the detected pollen tube TcdB targets are recognized by
-At-Rac, proteins interacting with this antibody
were immunoprecipitated from tobacco pollen tube extracts assayed for glucosylation by TcdB. In these experiments, at least one 21-kD pollen tube protein glucosylated by TcdB was found to interact with
-At-Rac (Fig. 3 c)
and was therefore identified as an At-Rac2 homologue.
No radiolabeled proteins were precipitated from assayed
extracts treated with preimmune serum (Fig. 3 c).
|
A cDNA encoding the catalytic domain of TcdB (Hofmann et al., 1997) was transiently expressed under the
control of the lat52 promoter in tobacco pollen tubes. Fluorescence emitted by coexpressed GFP was used to identify successfully targeted pollen tubes among an excess of
untransformed, nonfluorescent tubes. Transient expression of marker genes did not affect pollen tube growth.
Control pollen tubes transfected with expression vectors
containing GFP and GUS coding sequences fused to the
lat52 promoter showed normal morphology (Fig. 4, a-d)
and elongated at the same rate as untransformed pollen
tubes (data not shown). By contrast, transient expression of the catalytic domain of TcdB clearly inhibited tobacco
pollen tube growth (Fig. 4, e-h). Together, these results indicate that tobacco pollen tubes contain at least one At-Rac2 homologue with an essential function in tube elongation.
|
Transient Expression of Wild-Type and Mutant Forms of At-Rac2 Interferes Drastically with the Polar Extension of Tobacco Pollen Tubes
The function and the intracellular localization of Ras related small GTPases can be predictably altered by introducing specific mutations in conserved domains (Diekmann et al., 1991; Farnsworth and Feig, 1991
; Chardin,
1993
). Sequences encoding wild-type and mutant forms of
At-Rac2 (Fig. 2) were transiently expressed in growing tobacco pollen tubes under the control of the lat52 promoter. Coexpression of GFP was used to identify targeted
tubes. Expression of dominant negative T20N-At-Rac2
strongly inhibited pollen tube extension (Fig. 4, i-l). After
reaching a total length (tip to grain) of several 100 µm, T20N-At-Rac2-expressing pollen tubes stopped elongating. By contrast, expression of constitutive active G15V-At-Rac2 or Q64E-At-Rac2 caused germinating pollen
tubes to form large, spherical balloons instead of elongating tubes (Fig. 4, m-p, and data not shown, respectively).
Apparently, a complete loss of polarity of cell extension
was induced. Wild-type At-Rac2 had similar although somewhat weaker effects (Fig. 4, q-t). After germination,
pollen tubes elongated normally for a short while before
tips started to form balloons. The balloons often had irregular shapes instead of being spherical, presumably because
the potential for polarized growth was not completely
abolished. At-Rac2
CSIL, lacking the COOH-terminal
consensus prenylation domain known to be required for
the membrane association of Ras related proteins, still induced depolarized tip growth but was clearly less effective
than wild-type At-Rac2 (Fig. 4, u-x). These results demonstrate a key role of At-Rac2 homologous proteins in the
regulation of polar pollen tube growth and indicate that
membrane localization is essential for their activity.
The best characterized function of Rho family proteins
in animal and yeast cells is the regulation of actin organization (Hall, 1998). Therefore, effects of transient expression of mutant At-Rac2 on the tobacco pollen tube actin
cytoskeleton were examined. G15V-At-Rac2 or T20N-At-Rac2 were coexpressed in pollen tubes with a GFP-mouse
talin fusion protein, which we have shown to label plant
actin filaments in vivo in a specific and noninvasive manner (Kost et al., 1998
). The actin cytoskeleton in normally
growing tobacco pollen tubes consists of thick, longitudinally oriented actin bundles in the shank and fine filamentous structures close to the tip (Fig. 5 a; Kost et al., 1998
).
An excessive number of thick actin cables arranged in a
helical pattern was observed in balloons formed by G15V-At-Rac2-expressing pollen tubes (Fig. 5 b). Expression of
constitutive active At-Rac2 apparently induced actin polymerization or bundling and, possibly, reorientation of actin cables in the pollen tube tip. By contrast, actin bundles
in pollen tubes expressing dominant negative T20N-At-Rac2 were generally finer and less organized than in control tubes, indicating that inhibition of pollen tube Rac activity may reduce actin bundling (Fig. 5 c). As in other cell
types, one of the functions of pollen tube Rac appears to
be the regulation of the actin cytoskeleton.
|
Pollen Tube Rac Localizes to the Plasma Membrane at the Tip and to the Cytoplasm
Analysis of pollen tube extracts revealed that a significant
portion of the 21-kD tobacco pollen tube protein that interacts with the -At-Rac antibody cofractionates with
membranes, whereas the majority of the protein remains
in the cytoplasmic fraction (Fig. 6 a). A similar distribution
of radiolabeled protein with the same size was observed
when pollen tube extracts assayed for glucosylation by
TcdB were fractionated (Fig. 6 c). In control experiments,
pollen tube actin showed a typical distribution between cytoplasmic and membrane fractions (Fig. 6 b).
|
To investigate the intracellular localization of pollen tube Rac in more detail, sequences encoding wild-type and mutant forms of At-Rac2 fused to the COOH terminus of GFP were transiently expressed in tobacco pollen tubes under the control of the lat52 promoter. A GFP cDNA was also expressed from the same promoter. Although somewhat weaker, effects of expressing the fusion proteins on pollen tube growth (Fig. 7, a-c) were very similar to those observed with untagged wild-type or mutant At-Rac2 (Fig. 4, m, q, and u). This indicates that the fusion proteins were functional and that analysis of their localization provided relevant information on the intracellular distribution of pollen tube Rac proteins.
|
Tobacco pollen tubes expressing GFP or GFP fused to
different forms of At-Rac2 were examined by confocal microscopy. GFP was found to be evenly distributed in the
pollen tube cytoplasm (Fig. 4, b and c, and Fig. 7, h and l).
Confocal optical sections through pollen tubes expressing
GFP fused to constitutive active (Fig. 7, e and i) or to wild-type (Fig. 7, f and j) At-Rac2 revealed that these fusion
proteins accumulated at the cell cortex exclusively in the
tube tip, indicating that they associated with the plasma membrane specifically in this pollen tube region. The rest
of the fusion proteins was evenly distributed in the cytoplasm (Fig. 7, e, f, i, and j). GFP fused to At-Rac2CSIL
did not show membrane association (Fig. 7, g and k) and
was localized to the cytoplasm similar to GFP (Fig. 7, h
and l).
In summary, the results described here indicate that At-Rac2 homologues localize specifically to the plasma membrane at the pollen tube tip and confirm that the COOH-terminal prenylation domain is essential for their proper localization and function.
Pollen Tube Rac Physically Associates with a PtdIns P-K Activity That Produces PtdIns 4, 5-P2
PtdIns P-Ks and PtdIns 4, 5-P2 were identified as effectors
of mammalian Rho family small GTPases (Van Aelst and
D'Souza-Schorey, 1997). Rac and Rho, both in the GTP-
and in the GDP-bound conformation, have been demonstrated to physically interact with PtdIns P-K activity in
mammalian cell extracts (Ren et al., 1996
; Tolias et al.,
1998
).
We have tested the possibility that PtdIns P-Ks and PtdIns
4, 5-P2 act as Rac effectors also in pollen tubes. Experiments were performed to investigate whether At-Rac2
and its tobacco homologues bind to PtdIns P-K activity
present in tobacco pollen tube extracts. Recombinant At-Rac2 fused to the COOH terminus of GST was loaded
with the nonhydrolyzable nucleotide analogues GTPS or
GDP
S. Proteins interacting with nucleotide-bound GST-At-Rac2 were purified from tobacco pollen tube extracts
and assayed for lipid kinase activity using [
-32P]ATP and
a mix of phosphoinositides as substrates. An excess of
GST (Fig. 8 a, bottom) was used in control experiments.
Labeled lipids produced in kinase assays were analyzed by
TLC and autoradiography. Fig. 8 a (top) shows that significant amounts of radiolabeled PtdIns P2 were synthesized
when proteins associated with GTP
S- or GDP
S-loaded
GST-At-Rac2 were assayed, whereas minimal levels of
PtdIns P2 production were detected in GST control samples. Similar results were obtained when purified PtdIns 4-P
was used as a substrate instead of a mixture of phosphoinositides or when proteins coimmunoprecipitated with
At-Rac2 homologues from tobacco pollen tube extracts by
the
-At-Rac antibody were assayed (data not shown).
|
The different PtdIns P2 isoforms identified to date in
plant and/or animal cells, including PtdIns 4, 5-P2, PtdIns
3, 4-P2 and PtdIns 3, 5-P2 (Fruman et al., 1998; Munnik et al.,
1998
), are difficult to separate on TLC plates. Therefore,
32P-labeled lipid products of kinase binding assay performed as described above using a mix of phosphoinositides as substrates were deacylated and subjected to HPLC.
Under conditions that allow clear separation of deacylated PtdIns P2 isoforms (glycerophospho-inositol-bisphosphates, GroPIns P2s; Rameh et al., 1997
), 32P-labeled,
deacylated assay products coeluted from HPLC columns with 3H-labeled GroPIns 4, 5-P2 used as a standard (Fig. 8 b).
The described results demonstrate that recombinant At-Rac2, both in the GTP- and in the GDP-bound conformation, as well as at least one of its tobacco homologues, physically associates with tobacco pollen tube PtdIns P-K activity, which synthesizes specifically PtdIns 4, 5-P2.
Expression of GFP Fused to the PH-Domain of PLC-1
Inhibits Tobacco Pollen Tube Growth and Labels the
Plasma Membrane at the Tube Tip
The isolated PH-domain of PLC-1 has been shown to
bind PtdIns 4, 5-P2 integrated into lipid membranes specifically and with high affinity, both in vitro (Lemmon et al.,
1995
, Tall et al., 1997
) and in vivo (Stauffer et al., 1997
;
Várnai and Balla, 1998
). Under the control of the lat52
promoter, a sequence encoding a PLC-
1 PH-domain
GFP fusion protein (GFP-PLC-
1-PH) was transiently expressed in tobacco pollen tubes. Consistent with a key role of PtdIns 4, 5-P2 in the regulation of pollen tube growth,
moderate levels of GFP-PLC-
1-PH expression were sufficient to strongly inhibit tobacco pollen germination and
tube growth (Fig. 9 a). Only weakly fluorescent GFP-PLC-
1-PH-expressing pollen tubes were able to grow normally. Fluorescence emitted by these pollen tubes was too
weak to be visible on micrographs like the one shown in
Fig. 9 a. Confocal optical sections through such pollen
tubes revealed that GFP-PLC-
1-PH accumulated at the
plasma membrane in the tube tip (Fig. 9, e and i). Although GFP-PLC-
1-PH appeared to label a somewhat
smaller area, its localization in tobacco pollen tubes was
strikingly similar to that of transiently expressed GFP-At-Rac2 fusion proteins (Fig. 7). Pollen tubes with GFP-PLC-
1-PH labeling as shown in Fig. 9 e were indistinguishable
from untransformed control tubes in terms of morphology
(Fig. 9 e; data not shown) and growth rate (the average
growth rate of 20 weakly fluorescent, GFP-PLC-
1-PH-
expressing pollen tubes was 5 µm/s, which is identical to
the average growth rate of untransformed pollen tubes),
demonstrating that the observed localization of the fusion
protein provides information on a physiologically normal
situation.
|
A number of control experiments supported the view
that the specific interaction of GFP-PLC-1-PH with PtdIns 4, 5-P2 caused inhibition of pollen tube growth and was
responsible for the localization of the fusion protein to the
plasma membrane at the tip. The free amino group of the
lysine residue at position 32 in the PLC-
1 PH-domain has
been shown to form a direct hydrogen bond to the 4-phosphate of PtdIns 4, 5-P2 (Ferguson et al., 1995
). Replacement of this basic lysine residue by neutral leucine (K32L)
or even by glutamic acid (K32E) was demonstrated to abolish the ability of PLC-
1 to bind PtdIns 4, 5-P2 in membranes (Yagisawa et al., 1998
). Neither GFP-PLC-
1-PH-K32L (Fig. 9, f and j) nor GFP-PLC-
1-PH-K32E (Fig. 9, g
and k) detectably accumulated at the tip membrane when
transiently expressed in tobacco pollen tubes. Whereas
high level expression of GFP-PLC-
1-PH-K32L still severely inhibited pollen tube growth (Fig. 9 b), even brightly fluorescent tubes expressing GFP-PLC-
1-PH-K32E could elongate rapidly and showed an essentially
normal morphology (Fig. 9 c; data not shown). The PH-domain of ARNO has been demonstrated to bind with a
high degree of specificity to PtdIns 3, 4, 5-P3 (Irvine, 1998
;
Venkateswarlu et al., 1998
), a lipid that has not been identified in plant cells to date (Munnik et al., 1998
). Transient
expression from the lat52 promoter of a cDNA sequence
encoding the ARNO PH-domain fused to GFP did not significantly affect tobacco pollen tube growth (Fig. 9 d). No
accumulation of the GFP-ARNO-PH fusion protein at the
plasma membrane was observed (Fig. 9, h and l).
These results provide strong evidence for an essential function of PtdIns 4, 5-P2 in pollen tube elongation. Rac proteins, which physically interact with PtdIns 4, 5-P2 synthesizing PtdIns P-K activity, and PtdIns 4, 5-P2 both localize to the plasma membrane at the pollen tube tip. This suggests that Rac proteins, PtdIns P-K activity, and PtdIns 4, 5-P2 act together in a common pathway to regulate polar pollen tube growth.
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Discussion |
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Rac Homologues Control Polar Pollen Tube Growth
Transient expression of wild-type or constitutive active
At-Rac2 in tobacco pollen tubes resulted in the formation
of large balloons instead of elongated tips. The cell wall of
pollen tubes, similar to that of most plant cells, must withstand turgor pressure built up in the protoplast by osmotic
water uptake. Certain treatments, including incubation in
hypotonic media, can induce some swelling of pollen tubes
at the tip, where the newly formed cell wall is relatively
elastic and has not yet attained its ultimate rigidity. However, pollen tube tips generally burst before the swelling induced by such treatments results in a substantial increase
of their diameter (Steer and Steer, 1989; Benkert et al.,
1997
; Kost, B., and N.-H. Chua, unpublished observations). Extensive balloon formation as induced by Rac
overexpression requires the formation of rigid cell wall
structures which depends on constant deposition of new
cell wall material. Balloons induced by Rac overexpression clearly resulted from organized but depolarized
growth. Whereas Rac overexpression depolarized pollen
tube extension, inhibition of endogenous Rac activity by
transient expression of a dominant negative At-Rac2 or of
TcdB completely inhibited the growth of these cells. These
observations demonstrate that pollen tube Rac is essential
for growth and plays a key role in the determination of
growth polarity.
Intracellular Localization of Pollen Tube Rac
A pea pollen tube Rac homologue was determined to be
localized to the plasma membrane at the tip using immunofluorescence techniques (Lin et al., 1996). Chemical fixation and permeabilization required for such experiments
are known to severely change the structure of pollen tube
cells (He and Wetzstein, 1995
; Doris and Steer, 1996
) and
may affect Rac localization. We have chosen to use GFP
as a tag to investigate the intracellular localization of At-Rac2 in living pollen tubes. This technique has been successfully employed to analyze the intracellular distribution
of related small GTPases in different cell types (Larochelle et al., 1997
; Hirata et al., 1998
; Vasudevan et al.,
1998
).
Transient expression of GFP-At-Rac2 fusion proteins and of corresponding untagged At-Rac proteins had similar effects on pollen tube growth. This demonstrates that the fusion proteins were functional and valid indicators of At-Rac2 localization. A few hours after particle bombardment, when effects on pollen tube morphology started to become apparent, GFP-At-Rac2 fusion proteins were associated with an extended area of the plasma membrane at the tip. At this stage, their localization was very similar to the intracellular distribution of pea pollen tube Rac as observed by immunofluorescence. At later stages, the fusion proteins localized to the plasma membrane throughout the balloons formed (Kost, B., and N.-H. Chua, unpublished observations).
Pollen tubes transiently transformed with GFP-At-Rac2
sequences emitted fluorescence of varying intensities, indicating that they expressed the fusion proteins at different
levels. Using a truncated lat52 promoter (Bate et al., 1996)
or the 35S promoter, which both confer lower expression
in pollen tubes as compared with the full-length lat52 promoter (Lonsdale et al., 1995
; Bate et al., 1996
; Wilkinson
et al., 1997
; Kost, B., and N.-H. Chua, unpublished observations), resulted in a reduction of the total number of pollen tubes emitting detectable fluorescence. However, independent of the promoter used, all fluorescent pollen
tubes analyzed displayed depolarized growth and localization of GFP-At-Rac2 fusion proteins as described above
(Kost, B., and N.-H. Chua, unpublished observations).
This indicates that expression of the fusion proteins at
minimal levels required for visualization by fluorescence
microscopy is sufficient to affect pollen tube growth. Because fluorescence detection may require relatively high
concentrations of GFP fusion proteins, it is possible that
endogenous Rac in normally growing pollen tubes localizes to a more restricted area of the tip plasma membrane
as compared with GFP-At-Rac2 fusion proteins in transiently transformed tubes. Additional experiments, e.g.,
immunogold labeling of sections through physically fixed pollen tubes, may be required to determine the exact extension of the plasma membrane area that is Rac associated in normally growing pollen tubes. Nevertheless, our
results and the earlier immunolocalization study clearly
demonstrate that Rac localizes to pollen tube plasma
membrane specifically in the tip region.
Pollen Tube Rac Regulates Actin Organization
In normally elongating pollen tubes, the actin cytoskeleton
consists essentially of longitudinally oriented thick actin
cables that mediate cytoplasmic streaming and of fine actin structures in the tube apex that may have a direct function in polarized secretion (Miller et al., 1996; Kost et al.,
1998
). Transient expression of mutant Rac was found to
alter pollen tube actin organization. The clearest effect observed was the formation of extensive actin cables in growing balloons induced by expression of constitutive active
Rac. Expression of dominant negative Rac resulted in a
reduction of actin bundling. Interfering with the activity of
Rho type small GTPases in fibroblasts is known to have
comparable effects. In these cells, Rho activation leads to
the formation of actin cables, whereas its inactivation results in the disappearance of thick actin bundles (Hall,
1998
). As its animal and yeast homologues, pollen tube
Rac functions in the regulation of actin organization.
However, the observed effects on the actin cytoskeleton
alone are unlikely to account for the dramatic changes
in pollen tube growth induced by the expression of mutant Rac.
Pollen Tube Rac May Control Targeted Secretion in the Pollen Tube Tip
Pollen tube elongation is thought to be based on polarized
secretion restricted to the apex (Steer and Steer, 1989;
Taylor and Hepler, 1997
). As suggested for some of its animal and yeast homologues, pollen tube Rac may control
directed secretion possibly via the coordinated regulation
of actin organization and of exocytotic membrane traffic.
In normally elongating pollen tubes, activated endogenous
Rac associated with the plasma membrane in a restricted area at the tip may organize directed secretion to this site. Inactivation of endogenous Rac by transient expression of
TcdB or of dominant negative mutant Rac was found to
inhibit pollen tube growth, presumably by blocking secretion. By contrast, expression of constitutive active Rac led
to the formation of balloons instead of elongated tips, conceivably because it resulted in an extension of the membrane area associated with activated Rac, which caused depolarized secretion and growth. Overexpressed wild-type Rac was apparently partially activated by endogenous factors and had similar, although somewhat weaker,
effects. Deletion of the COOH-terminal CAAX-domain
clearly reduced, but did not complete abolish, the potential of wild-type Rac to induce depolarized growth. Even in the absence of a membrane targeting domain, local concentrations of At-Rac2
CSIL at the tip plasma membrane
achieved by transient expression of a sequence encoding
this protein under the control of the strong lat52 promoter
were apparently high enough for some stimulation of ectopic secretion.
Estimations based on simple geometric calculations revealed that the total surface of pollen tubes transiently expressing constitutive active Rac was about four times smaller than that of control pollen tubes at the time of analysis (12-18 h after particle bombardment). Whereas this appears to be in contradiction with a role of activated Rac in the stimulation of secretion, it likely results from the disruption of cytoplasmic organization caused by transient expression of constitutive active Rac. The total volume of the pollen tube cytoplasm, which remains essentially constant after pollen germination, was estimated to be ~20 times smaller than the volume of balloons formed by constitutive active At-Rac2-expressing pollen tubes. As a consequence, the cytoplasm could only form a thin layer at the inner surface of these balloons, with the remainder of the volume filled by large vacuoles. It is conceivable that the proceeding drastic disruption of cytoplasmic organization during balloon formation increasingly interfered with the efficient delivery of secretory vesicles to the plasma membrane.
Rac, PtdIns P-K, and PtdIns 4, 5-P2 Act in a Common Pathway to Regulate Pollen Tube Growth
Mammalian Rho family small GTPases in the GTP-bound
conformation have been found to stimulate PtdIns P-K activity and synthesis of PtdIns 4, 5-P2 in permeabilized cells
and in cell lysates (Chong et al., 1994; Hartwig et al., 1995
).
Recombinant as well as endogenously produced mammalian Rac and Rho were shown to physically interact with a
PtdIns P-K activity in cell extracts (Tolias et al., 1995
; Ren
et al., 1996
). Here, we present compelling evidence that
pollen tube Rac, PtdIns P-K, and PtdIns 4, 5-P2 cooperate
in a common pathway to regulate polar pollen tube
growth. Our results indicate that PtdIns P-K and PtdIns 4, 5-P2 may act as Rac effectors in pollen tubes, as they do in
mammalian cells. Rac inactivation by transient expression
of dominant negative mutant forms of this protein inhibited pollen tube growth. The same effect was observed when the interaction of PtdIns 4, 5-P2 with its downstream
targets was disrupted by transient expression of GFP-PLC-
1-PH, which binds strongly and specifically to this
lipid. Recombinant At-Rac2 and its endogenous tobacco
homologues were demonstrated to physically associate in
pollen tube extracts with PtdIns P-K activity that synthesizes specifically PtdIns 4, 5-P2. Rac and PtdIns 4, 5-P2 were both observed to localize to the plasma membrane
specifically at the pollen tube tip.
Interestingly, recombinant mammalian Rac and Rho
(Tolias et al., 1995; Ren et al., 1996
) as well as pollen tube
At-Rac2 were found to interact with a PtdIns P-K activity
both in the activated GTP-bound and in the inactive GDP-bound form. In a recent report, evidence was presented indicating that the COOH-terminal end of mammalian Rac
is mainly responsible for the interaction of this protein
with PtdIns P-K and not the NH2-terminally localized effector domain, which is known to undergo drastic conformational changes upon GTP binding (Tolias et al., 1998
).
As was suggested for its mammalian homologues, activated pollen tube Rac may stimulate PtdIns P-K activity
and PtdIns 4, 5-P2 synthesis via GTP-dependent binding of
an additional, unidentified cofactor (Ren et al., 1996
) or by
translocating a constitutively associated PtdIns P-K from the cytoplasm to the plasma membrane (Tolias et al.,
1998
). In vivo and in vitro experiments with mutant At-Rac2 are currently being performed to further characterize the interaction between Rac and PtdIns P-K activity in
pollen tubes.
Recent results have established that two different
PtdIns P-K-dependent pathways contribute to the synthesis of PtdIns 4, 5-P2 in mammalian cells. In addition to
phosphorylation of PtdIns 4-P by PtdIns 4-P 5-K, which
represents a well established pathway, phosphorylation of
PtdIns 5-P at position 4 of the inositol ring was found to be
catalyzed by PtdIns 5-P 4-Ks, formerly known as type II
PtdIns 4-P 5-K (Rameh et al., 1997). Rac-associated pollen tube lipid kinase activity generated PtdIns 4, 5-P2 when
PtdIns 4-P was used as a substrate. Because commercially
available PtdIns 4-P preparations may contain traces of
PtdIns 5-P (Rameh et al., 1997
), this does not entirely rule
out the possibility that pollen tube Rac interacts with
PtdIns 5-P 4-Ks activity. However, the activity of the Rac-associated pollen tube lipid kinase could be stimulated by
phosphatidic acid (Lemichez, E., and N.-H. Chua, unpublished observation), which is considered to be characteristic for PtdIns 4-P 5-Ks in mammalian systems (Fruman et
al., 1998
; Toker, 1998
). Therefore, it appears likely that we
have detected an interaction between Rac and PtdIns 4-P
5-Ks activity in pollen tubes.
Function of Compartmentalized PtdIns 4, 5-P2 in the Regulation of Pollen Tube Growth
Signaling appears to often involve recruitment of regulatory proteins to specific membrane domains where these
proteins form complexes that organize local cellular responses. A large number of regulatory proteins is known
to bind PtdIns 4, 5-P2 or other phosphoinositides specifically and with high affinity. This has led to the idea that localized phosphoinositide synthesis may have a key function in spatially restricted signaling events. However, only
limited evidence for lipid compartmentalization in cells
has been generated to date and the mechanisms involved in the regulation of localized synthesis of particular membrane lipids are unknown (Martin, 1997; Irvine, 1998
;
Toker, 1998
).
Our results provide direct evidence showing that PtdIns
4, 5-P2 accumulates in the plasma membrane of living pollen tubes specifically at the tip and indicate that the observed PtdIns 4, 5-P2 compartmentalization is controlled
by Rac homologues. By causing translocation of actin-binding proteins, tip-localized PtdIns 4, 5-P2 may induce
uncapping and bundling of actin filaments, which could
stimulate elongation of longitudinally oriented actin cables in growing pollen tubes, and control the formation of
actin structures present in the apical dome. In addition to
its effect on actin organization, PtdIns 4, 5-P2 may directly
control exocytosis at the pollen tube tip, either by recruiting proteins that regulate membrane fusion or by locally
altering membrane lipid composition. PtdIns 4, 5-P2 potentially represents the main effector of activated Rac in
pollen tubes. It may initiate the formation of complexes of
regulatory proteins at the plasma membrane in the tube
apex which control actin organization, targeted secretion,
and polar growth. Activated Rac possibly stabilizes these
complexes as well as their interaction with PtdIns 4, 5-P2.
GFP-PLC-1-PH did not detectably accumulate at the tip
plasma membrane of pollen tubes showing depolarized
growth induced by coexpressed constitutive active At-Rac2 (Kost, B., and N.-H. Chua, unpublished observations). In such pollen tubes, access of the GFP-PLC-
1-PH
fusion protein to PtdIns 4, 5-P2 may have been blocked by
tightly bound regulatory proteins.
In addition to acting as an effector itself, PtdIns 4, 5-P2
at the pollen tube tip may also function as a precursor for
the generation of other signaling molecules. Hydrolysis of
PtdIns 4, 5-P2 by PLC, which results in the formation of
inositol 1, 4, 5-triphosphate (Ins-P3) and diacylglycerol,
followed by Ins-P3-induced Ca2+ release from intracellular stores, is a key element of many known signaling events
(Clapham, 1995). Recently published results have indicated an essential role of this pathway in poppy pollen
tube elongation (Franklin-Tong et al., 1996
). PLC-mediated hydrolysis of tip localized PtdIns 4, 5-P2 and Ins-P3-
induced Ca2+ influx into the cytoplasm may be involved in
the establishment of the tip-focused Ca2+ gradient, which
is known to have an important function in the regulation
of pollen tube growth. ER elements are present in the clear zone at the pollen tube tip (Taylor and Hepler, 1997
)
and could function as Ins-P3-sensitive Ca2+ stores. Alternatively, putative Ins-P3-regulated Ca2+ channels, which
allow Ca2+ influx from the extracellular matrix, may be
present in the pollen tube plasma membrane.
Conclusions
Our results strongly suggest that Rac homologues act in a
common pathway with a PtdIns P-K (probably PtdIns 4-P
5-K) activity and PtdIns 4, 5-P2 to regulate polar pollen
tube growth. We present direct evidence for PtdIns 4, 5-P2
compartmentalization in the plasma membrane at the pollen tube tip, which appears to be derived from Rac-controlled local activation of a PtdIns P-K. PtdIns 4, 5-P2
localized to the plasma membrane at the tip could potentially act as the main Rac effector in pollen tubes by directly regulating actin-mediated targeted secretion and
polarized growth. It may also serve as a substrate for Ins-P3 production by PLC activity, which could have a function in establishing the tip-focused Ca2+ gradient known to
be involved in the regulation of pollen tube elongation
(Malhó and Trewavas, 1996; Pierson et al., 1996
).
![]() |
Footnotes |
---|
Address correspondence to Nam-Hai Chua, Laboratory of Plant Molecular Biology, The Rockefeller University, 1230 York Ave., New York, NY 10021-6399. Tel.: (212) 327-8126. Fax: (212) 327-8327. E-mail: chua{at}rockvax.rockefeller.edu
Received for publication 5 January 1999 and in revised form 15 March 1999.
The work presented here was supported by the following organizations:
DOE (grant DOE94ER20143 to N.-H. Chua), Swiss National Science
Foundation (grants 823A-050394 and 823A-046686 to P. Spielhofer and B. Kost), Human Frontier Science Program (grant LT-256/97 to E. Lemichez), and the National Institutes of Health (grant GM54389 to C. Carpenter).
Benedikt Kost's present address is Laboratory of Plant Cell Biology, Institute of Molecular Agrobiology, National University of Singapore, Singapore 117604.
We would like to thank Yang-Sun Chan and Eric Marechal for help with plasmid construction; Ingo Just for supplying C. difficile toxin B preparations; Klaus Aktories, Geert Angenon, Peter Cullen, Fred Hofmann, Jyoti Kapila, Pal Maliga, Tobias Meyer, Thomas Stauffer, Willem Stiekema, and Kanamarlapudi Venkateswarlu for providing cDNAs or vectors; Patrice Boquet for helpful discussions; and Gregory Jedd, Te Piao King, Ulrich Klahre, and Tim Kunkel for critical assessment of the manuscript.
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Abbreviations used in this paper |
---|
ARNO, ADP-ribosylation factor nucleotide-binding site opener;
-At-Rac, affinity-purified polyclonal
anti-At-Rac antibody;
GFP, green fluorescent protein;
GST, glutathione
S-transferase;
GUS,
-glucuronidase;
GroPIns, glycerophospho-inositol;
Ins, inositol;
K, kinase;
ORF, open reading frame;
PH, pleckstrin homology;
PLC, phospholipase C;
PNS, post-nuclear supernatant;
PtdIns, phosphatidylinositol;
TcdB, Clostridium difficile toxin B.
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