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Plant Physiol. (1998) 118: 297-304
Hexose Transport in Growing Petunia Pollen Tubes and
Characterization of a Pollen-Specific, Putative Monosaccharide
Transporter1
Bauke Ylstra2, 3,
Dolores Garrido3,
Jacqueline Busscher, and
Arjen J. van Tunen*
Department Cell Biology, Agricultural Research
Department-Centre for Plant Breeding and Reproduction Research,
P.O. Box 16, 6700 AA Wageningen, The Netherlands
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ABSTRACT |
We
investigated the molecular and physiological processes of sugar uptake
and metabolism during pollen tube growth and plant fertilization. In
vitro germination assays showed that petunia (Petunia hybrida) pollen can germinate and grow not only
in medium containing sucrose (Suc) as a carbon source, but also in
medium containing the monosaccharides glucose (Glc) or fructose (Fru). Furthermore, high-performance liquid chromatography analysis
demonstrated a rapid and complete conversion of Suc into equimolar
amounts of Glc and Fru when pollen was cultured in a medium containing 2% Suc. This indicates the presence of wall-bound invertase activity and uptake of sugars in the form of monosaccharides by the growing pollen tube. A cDNA designated pmt1 (petunia
monosaccharide transporter 1), which is highly
homologous to plant monosaccharide transporters, was isolated from
petunia. Pmt1 belongs to a small gene family and is
expressed specifically in the male gametophyte, but not in any other
vegetative or floral tissues. Pmt1 is activated after the first pollen mitosis, and high levels of mRNA accumulate in mature
and germinating pollen. A model describing the transport of sugars to
the style, the conversion of Suc into Glc and Fru, and the active
uptake by a monosaccharide transporter into the pollen tube is
presented.
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INTRODUCTION |
The meiotic division of a pollen mother cell early in the
development of the anther generates four immature male gametophytes. The gametophyte undergoes one mitotic division to generate one vegetative and one generative cell, after which the generative cell
further divides to form two sperm cells (for review, see McCormick,
1993 ). The sperm cells are delivered to the female reproductive cells
by unidirectional growth of the vegetative cell. This pollen tube grows
through the stigma and style toward the ovules in the pistil. Much of
the recent molecular research on the physiology of pollen tube growth
focuses on specialized processes such as the incompatibility reaction,
or on substances such as kinases, pectinases, polygalacturonases, and
flavonols (Mascarenhas, 1993; McCormick, 1993). In contrast to this,
one of the most striking phenomena of plant fertilization, the extreme speed and long-range capacity of pollen tube growth, has been poorly
investigated on a molecular level. To enable the fast growth of the
pollen tube, a rapid synthesis of wall material (Derksen et al., 1995)
and a high energy supply is necessary. Therefore, a high level of sugar
import is required (Schlüpmann et al., 1994).
During maturation in the anther, pollen accumulates high levels of
carbohydrates that represent the major part of the mature grain's dry
weight (Stanley and Linskens, 1974a; Pacini, 1996). After germination
on a compatible stigma, the fast growth of the pollen tube is supported
by the pistil. In the stylar fluids of petunia (Petunia
hybrida) pistils the free sugars Suc, Glc, and Fru are available
to the pollen tube (Konar and Linskens, 1966). After absorption by the
pollen, sugars are utilized as an energy source and are converted to
wall material as pectins, cellulose, and callose (Mascarenhas, 1993;
Derksen et al., 1995). Constant de novo synthesis of cell wall material
is essential because many of the carbohydrates used for wall synthesis
are dissipated for participation in further pollen tube
formation.
The primary source of carbohydrates in the pistil and pollen lies in
the photosynthesizing mature leaves, where assimilation takes place.
After assimilation sugars are transported through the phloem to sink
tissues such as the anther and stylar apoplast, mostly in the form of
Suc (Bush, 1993). Nonetheless, it should be noted that the transmitting
tract cells of the petunia pistils contain chloroplasts (B. Ylstra,
personal observations) and might therefore also contribute directly to
sugars in the stylar apoplast (Jansen et al., 1992). The final
destination of the sugars, however, is the pollen, which requires
translocation from the anther, stigma, and stylar apoplast over the
pollen (tube) membrane.
Several sugar transporters were identified in plants
(Sauer and Tanner, 1989, 1993; Bush, 1993) and their genes were cloned and characterized by transgenic expression in yeast (Sauer and Tanner, 1993; Sauer and Stolz, 1994). The gene products were designated mono- and dissaccharide transmembrane symporters, and actively translocate sugars across plasma membranes driven by a
proton-electrochemical potential (Stadler et al., 1995). The
dissaccharide-symporter genes isolated are especially transcribed in
mature leaves. The monosaccharide transporters reported so far are
primarily transcribed in sink tissues such as young leaves and in
storage and floral organs (Sauer and Tanner, 1993). In terms of
sink-source relations pollen should be regarded as a strong sink, since
it is not able to assimilate but requires high levels of starch and
carbohydrates during maturation, germination, and growth.
In vitro germination assays are helpful in the study of the growth
requirements of pollen tubes. Different chemical constituents, pH, and
viscosity could be related to the quality of pollen development and
quantity of tube growth (Stanley and Linskens, 1974a, 1974b; Jahnen et
al., 1989; Derksen et al., 1995). Suc was most commonly included in the
in vitro germination medium as a carbon source. However, sugar import
into germinating and growing pollen has only been studied to a limited
extent in lily (Deshusses et al., 1981). We present physiological and
biochemical experiments that enable us to describe the uptake of
carbohydrates by pollen tubes in molecular terms.
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MATERIALS AND METHODS |
Plant Material
Petunia (Petunia hybrida var R27) was grown under
standard greenhouse conditions.
Carbohydrates during in Vitro Pollen Tube Growth
Pollen was collected from flowers at anthesis and germinated in
medium according to the method of Jahnen et al. (1989). After 13 h
of in vitro pollen tube growth, 1 mL of culture was centrifuged and
filtered (0.22 µm, Optex, Millipore) to exclude pollen tubes and
remnants. Sugars in the germination medium were quantified by HPLC
analysis on a 6.5- × 300-mm column (Shodex SC-1011, Waters Chromatography Division, Millipore) run at 85°C with ultrapure water
at 0.75 cm3 min 1. Suc,
Glc, and Fru were detected with a 2142 refractor index detector
(Pharmacia). One representative experiment out of three is presented in
Figure 2.
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| Figure 2.
Histogram representing HPLC analysis data of the
sugar content in the germination medium before and after pollen tube
growth. Samples were taken at two different time points, 0 and
13 h after in vitro pollen tube growth. S, Suc (cross-hatch); G,
Glc (left hatch); F, Fru (right hatch).
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To monitor the carbohydrates that can promote in vitro tube elongation,
Suc in the germination medium was substituted by D(+)-Glc monohydrate,
D( )-Fru extra pure (Merck, Darmstadt, Germany), or D(+)-mannitol
(Janssen Chimica, Beerse, Belgium) in a concentration of 2% (w/w).
Photographs of the cultures were made after 8 and 24 h.
Isolation of the Full-Size Pmt1 cDNA
Total RNA from pollen was purified for the mRNA fraction by an
oligo-dT column according to the instructions of the manufacturer (Pharmacia). Subsequently, first-strand cDNA was synthesized using the
oligonucleotide primer PR1,
5 -CCGGATCCTCTAGAGCGGCCGC(T)17-3 and rav-2
reverse transcriptase, according to the instructions of the
manufacturer (Amersham). Together with a second oligonucleotide primer
PR2,
5-ATGGTCGACT (G/T)(G/T/C)GCIAA(A/G)(A/G/C)(G/C)(I/C)(I/C)T(I/C)CC(A/T/C)GG-3, a first PCR was performed (annealing sites of primers are underlined in
Fig. 3). Amplification involved 30 PCR thermal cycles with 1 µg of
degenerated primer, 200 ng of undegenerated primer, 10 mM
of each deoxynucleotide triphosphate, and 5 IU of Taq DNA
polymerase (Boehringer Mannheim) in 50 µL of the manufacturer's PCR
buffer using a thermal DNA cycler (model 480, Perkin Elmer). The
thermal PCR cycle involved denaturating for 30 s at 94°C, a
transition of 30 s, annealing for 60 s at 46°C, another
transition of 60 s, and synthesis for 60 s at 72°C.
Amplified cDNA was fractionated on a 1% agarose gel. A clear fragment
0.6 kb in length was cloned into pEMBL derivates, using the restriction
sites BamHI and SalI present in the primers, and
sequenced (see below).
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| Figure 3.
Pmt1 cDNA, amino acid sequence, and
hydropathic character of the protein. A, Pmt1 cDNA
sequence (numbered left) and deduced amino acid sequence (numbered
right). The annealing sites of the primers used are underlined,
successively PR9, PR3,
PR8, PR7, PR6,
PR5, PR2, PR4,
PR10, and PR1. B,
Hydrophobicity plot of the deduced pmt1 amino acid
sequence with the 12 hydrophobic regions indicated by Roman numerals.
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To obtain an additional 5 cDNA sequence a second PCR cycle was
performed according to the same PCR procedure with the degenerated primer PR3, 5-CGGGATCCGGGTGG(T/C)(T/C)T(C/G)AT(T/C)TT(T/C)GG-3 homologous 5 sequences of earlier published monosaccharide
transporters (Sauer and Tanner, 1993) and PR4,
5-CGGAATTCGAAGAGTGTCATGAGTACC-3, with inverse homology to the 5
region of the 0.6-kb PCR fragment. The restriction enzymes
BamHI and EcoRI were used to clone the new
fragment in pEMBL18, however, only a 300-bp additional sequence was
obtained by this approach. To obtain the complete cDNA sequence, RACE-PCR was performed in two subsequent experiments according to the
manufacturer's protocol (5 AmpliFINDER RACE kit; Clontech Labs, Palo
Alto, CA). The oligonucleotide primer PR5,
5-GCTCTAGACCAGAGTCTTGAAGAGGACAGGGGC-3, was used for first-strand
cDNA synthesis. A nested primer PR6, 5-GCTCTAGAGCACATTGATACCTGTGAGTTGTTGG-3, together with the
AmpliFINDER RACE anchor primer was used for amplification by PCR.
In the second round the oligonucleotide primer PR7,
5-GCTCTAGACCATTAAGGACAGCACCAACAAGG-3, was used for first-strand
cDNA synthesis. Subsequently, the nested primer PR8,
5-GCTCTAGAGCCTTGGTGGTTATTGAAGCTGC-3 together with
the anchor primer were used for PCR amplification. 5
RACE-amplification PCR involved 30 thermal cycles with 200 ng of both
primers, 10 mM of each deoxynucleotide triphosphate, and
0.5 IU of Taq polymerase (HT-Biotechnology, Cambridge, UK) in 50 µL of the manufacturer's PCR buffer. Synthesis time in the thermocycler was elongated to 120 s, after gel electrophoresis fragments of 700 or 600 kb, respectively, were cloned into pEMBL18 using the restriction sites XbaI and EcoRI
present in the 5 regions of the primers for the first RACE or blunt
end (SmaI) for the second RACE.
To obtain the complete monosaccharide transporter cDNA a PCR
amplification on pollen mRNA was made with the primers PR9,
5-GCTCTAGACCATGGCAGGAGGCTTTGCAGCTG-3, and PR10,
5-CGGGATCCAGAATCTGAGGTCATAGTAACATCCGGGAGGGC-3,
complementary to, respectively, the 5 and 3 of the
pmt1-coding region, which yielded a fragment with the
expected size of approximately 1.6 kb. This fragment was ligated in
pEMBL19 and the pmt1-coding region was verified by sequence
analysis. This full-size cDNA served as a probe for further analysis.
DNA and RNA Gel-Blot Analysis
For DNA-blot analysis 10 µg of total plant DNA was isolated from
young leaves, digested with EcoRI, HindIII, or
XbaI, and electrophoresed (Koes et al., 1986). Total RNA was
isolated from different plant parts and organs according to the method
of Verwoerd et al. (1989). The developmental stage of the microspores
in the anthers was determined using propion carmine staining. For
germination, pollen from anthers at anthesis were
germinated at room temperature for 13 or 24 h in germination
medium. An aliquot was counterstained with
4,6-diamidino-2-phenylindole and ethidium bromide to determine the
amount of nuclei in the tubes. Total RNA was denatured by glyoxal/DMSO and electrophoresed on a 1.2% agarose gel in
15 mM sodium phosphate buffer, pH 6.5, according to the
method of Angenent et al. (1992). Gels were blotted onto Hybond
N+ membranes and hybridized at 65°C in 1 M NaCl, 1% SDS, and 10% dextrane sulfate. The full-size
cDNA-coding region (nucleotides 253-1848) served as a probe (see Fig.
3 and previous section). After overnight hybridization, the membranes
were washed in 2× SSC or 0.1× SSC with 0.1% SDS at 50°C or 65°C,
and an autoradiogram was made.
DNA Sequencing and Homology Comparison
The full-size pmt1 cDNA was sequenced in both
directions. Therefore, dideoxy chain termination was used for
sequencing either single- or double-stranded DNA pEMBL plasmid
derivates. Reactions were performed with either dyedeoxy or dye primer
ready-reaction sequencing kits (PRISM, Applied Biosystems) according to
the manufacturer's protocol.
Protein structure was predicted by the program SOAP of the PC gene
software, calculated with a window size of nine amino acids (Kyte and
Doolittle, 1982). Homology comparison with the EMBL databank was
performed using the program BLAST according to the method of Altschul
et al. (1990). Homologies presented in Table I represent the percentage of identity
from the start codon or first Met residue to the stop codon or the
last amino acid, respectively.
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Table I.
Percentage identity between monosaccharide
transporters from a variety of species
Nucleotide sequence homologies (top right, coding region) and deduced
amino acid sequence homologies (bottom left) are
shown.
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RESULTS |
Pollen Use Monosaccharides as a Carbon Source for Tube Growth
To identify which sugars are required and at what point during in
vitro pollen tube growth, Suc in the germination medium was substituted
with the monosaccharides Glc, Fru, or mannitol. In Figure
1 pollen tube growth in these media is
shown. After 8 h of incubation no differences were observed with
regard to the quality and the growth rates of pollen in the four
different media (Fig. 1, A-C). Within the following 16 h, tube
growth in Suc, Fru, and Glc continued and long tubes developed. These
long tubes entangled to wadding-like structures that floated in the media, so the tubes were out of focus when photographed (Fig. 1, D and
E). In the mannitol-containing medium, however, tube growth was
severely retarded after 24 h of culturing. Mannitol-cultured pollen tubes remained at the bottom of the wells and were often dead,
with relatively short tubes (Fig. 1F). Mannitol is not well utilized by
plants, and the initial development in mannitol probably reflects
autonomous growth on internally stored sugars (Fig. 1C). Taken
together, these experiments show that an external carbon source is
essential to maintaining pollen tube growth. However, this assay could
not discriminate whether monosaccharides, disaccharides, or both were
actually imported into growing petunia pollen tubes.
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| Figure 1.
Light-microscopic photographs of pollen tubes
grown in vitro with different carbon sources in 2% Suc after 8 h
(A) and 24 h (D); in 2% Fru after 8 h (B) and 24 h (E)
(pollen tubes grown in 2% Glc revealed an identical picture (not
shown); and in 2% mannitol after 8 h (C) and 24 h (F).
Pollen tubes in D and E are out of focus because they float in the
medium due to their length. A through C, Magnifications ×100; D
through F, magnifications ×64.
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In a subsequent experiment the question was addressed whether the
composition of the sugars in the 2% Suc medium was altered during in
vitro tube growth. An HPLC approach was chosen to determine the amount
of free sugars at the start of the germination assay and after 13 h of culturing. In Figure 2 it can be
seen that at the onset of germination the medium contained 58.4 mM Suc (2%). After 13 h of growth the Suc was
completely hydrolyzed into nearly equimolar amounts of Glc and Fru. The
fact that disaccharides were converted into monosaccharides strongly
suggests uptake of carbohydrates by pollen tubes as monosaccharides.
Sequence Analysis of the Petunia Pmt1 cDNA from Pollen
In a research program aimed at the isolation of genes specifically
active in pollen and homologous to the human androgen receptor, a PCR
experiment was performed using mRNA isolated from mid-binucleate pollen, the poly-dT primer PR1, and the degenerated primer PR2. A
number of PCR products unrelated to human steroid receptors were
isolated and sequenced. One of the cDNA fragments was highly homologous
to the sequence of monosaccharide transporters STP1 and MST1. Since our
physiological data suggested that pollen tubes import monosaccharides
rather than disaccharides (previous section), this previously isolated
cDNA clone was selected for further analysis in this study.
The first cDNA fragment obtained contained a 500-bp open reading frame
and an approximately 100-bp 3-untranslated tail. To obtain additional
5 cDNA sequences a second PCR was performed with the degenerated
primer PR3, which is homologous to earlier published monosaccharide
transporters (Sauer and Tanner, 1993), combined with the specific
primer PR5. This approach resulted in the isolation of a cDNA with only
a 300-bp additional sequence. To obtain the complete sequence two
subsequent RACE-PCR reactions were performed, which revealed a total
cDNA sequence of 1912 bp. Sequence analysis indicated an open reading
frame of 1533 bp, a tail of 127 bp, and a 252-bp untranslated leader,
encoding a full-size protein designated PMT1 (Fig.
3A). Reverse-transcriptase PCR with the
two primers PR9 and PR10, 5 and 3 of the pmt1 open reading
frame, confirmed the presence of this mRNA in pollen. Analysis of the
deduced amino acid sequence displayed 12 hydrophobic regions (Fig. 3B).
This strongly suggests that PMT1 is a membrane-spanning protein with 12 transmembrane regions.
Pmt1 mRNA Is Highly and Specifically Transcribed during Pollen
Maturation and Tube Growth
Pmt1 cDNA was isolated using RNA derived from maturing
bicellular pollen grains. To monitor the expression pattern of the pmt1 gene, RNA isolated from many different plant tissues,
including young seed pods (not shown), root, leaf, stem, and a variety
of reproductive floral tissues, was subjected to northern-blot
analysis. Special attention was given to tissues and organs that are
known to serve an important function in carbohydrate metabolism. Young and old leaves act as, respectively, carbohydrate sink or source, and
therefore their RNAs were isolated separately. The nectaries in the
petunia flower are specialized organs that contain and excrete high
levels of Suc (A.J. Koops and A.J. van Tunen, personal observations).
Furthermore, RNAs from stigmas and styles were separately extracted
because these tissues supply sugars to the growing pollen tubes.
Moreover, RNA was isolated from anthers at different developmental
stages. Finally, RNA was derived from mature and germinated pollen.
Northern analysis revealed transcription of the pmt1 gene
exclusively in mature anthers and pollen (Fig. 4). No transcripts were detected in any
of the other tissues tested. Pmt1 transcription starts to be
detectable in anthers directly after the first pollen mitosis and
rapidly increases to very high levels in mature pollen. After 13 h
of in vitro germination high levels of pmt1 transcripts were
still detected (Fig. 4) and did not decrease after 24 h of
germination, when the second pollen mitosis occurs (data not shown).
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| Figure 4.
Northern-blot analysis of mRNA derived from
vegetative and reproductive tissues, anthers, and pollen. Vegetative
tissues include: root, young leaf (sink leaf), old leaf (source leaf),
and stem. Reproductive tissues (open flowers) include: sepal, petal,
stigma, style, ovule, and nectary. Also shown are anthers with male
generative gametophytes at successive developmental stages: meiosis;
tetrad; unicellular; bicellular, late bicellular, and mature
(immediately before anther dehiscence); and mature dehisced and
germinated pollen (after 13 h of in vitro germination).
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The signal on the northern blot can be regarded as highly specific for
the pmt1 gene, since the hybridization pattern was similar
under less stringent washing conditions (2× SSC at 65°C, data not
shown) and cross-hybridization to other members of the monosaccharide
transporter family in petunia was not observed. This is in line with
the results of the Southern-blot analysis (see below), which showed
that other family members are less than 70% identical to
pmt1.
Pmt1-Related Sequences in the Petunia Genome and Other Species
Southern-blot analysis was performed to determine the presence of
related genes in the genome of petunia. Under low-stringency conditions
(2× SSC at 50°C) four to five bands hybridized to the pmt1 probe in separate digestions (Fig.
5A). This indicates a family of four to
five pmt1-related members in the petunia genome. The same
probe also detected multiple bands with DNA digests from tobacco and
Arabidopsis (data not shown). At high-stringency conditions (0.1× SSC
at 65°C) only a single band was observed for each digestion of the
petunia DNA (Fig. 5B). After washing at moderate stringency (2× SSC at
65°C) one single band was detected (data not shown). Based on this
hybridization pattern, the homology of pmt1 to the other
genes of the family is less than 70% based on DNA level, and indicates
the presence of a single pmt1 copy in the petunia genome.
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| Figure 5.
Genomic organization of the pmt1
gene family. A, Southern-blot analysis under low-stringency conditions
(2× SSC at 50°C). B, Southern-blot analysis under high-stringency
conditions (0.1× SSC at 65°C). Restriction enzymes used were
EcoRI, HindIII, or XbaI.
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A homology search was performed to determine the homology of
pmt1 with other sequences in the databanks. This search
revealed significant levels of nucleotide and amino acid homology to
several monosaccharide transporters from various unicellular,
mammalian, and plant species (Table I). The highest homology was found
with the monosaccharide-H+ symporter STP4 from
Arabidopsis, which was 62% at the amino acid level (Truernit et al.,
1996). A high level of homology was also found to the solanaceous
monosaccharide transporter MST1 and a second Arabidopsis monosaccharide
transporter STP1, with, respectively, 60% or 59% identity to the PMT1
protein (Sauer et al., 1990; Sauer and Stadler, 1993). Furthermore,
moderate but significant homology was found between pmt1 and
the hup1 cDNA from Chlorella kessleri and the Glc
transporter isolated from rat brain (Birnbaum et al., 1986; Sauer and
Tanner, 1989; Table I).
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DISCUSSION |
Pollen tubes require high and rapid sugar uptake to support their
growth. The physiological data presented in this article suggest that
pollen tubes import carbohydrates in the form of monosaccharides rather
than disaccharides. This observation was supported by the isolation of
the cDNA clone pmt1, isolated from petunia pollen.
Pmt1 shared high and significant homology to earlier reported monosaccharide-proton symporters and its mRNA transcripts were
exclusively detected in mature anthers, isolated pollen, and growing
tubes.
In conventional media used for germination assays, the disaccharide Suc
is included as a carbon source (Stanley and Linskens, 1974b; Jahnen et
al., 1989; O'Driscoll et al., 1993; Schlüpmann et al., 1994;
Derksen et al., 1995). The HPLC analysis presented in this article
showed that during the in vitro germination of petunia pollen the Suc
in the medium was rapidly converted into Glc and Fru (Fig. 2). This
rapid and dramatic hydrolysis of Suc during pollen tube growth was most
likely the result of wall-bound invertase activity. Pollen invertase
activity was also reported for in vitro-germinated pollen of the
monocot lily (Singh and Knox, 1984). The fact that pollen exhibits such
invertase activity strongly suggests that pollen imports carbohydrates
in the form of monosaccharides. Nonetheless, alternative ways of sugar
uptake by pollen should not be excluded. O'Driscoll et al. (1993), for example, showed uptake of fluorescein isothiocyanate-labeled dextrans via endocytosis.
The PMT1 protein shared high overall homology to earlier reported
monosaccharide transporters (Table I). PMT1 contains all conserved
amino acids and motifs: Q175, Q293, Q294, HWFW490-493, (R/K)-GR(R/K)343-347, and (V/L)PETKG472-476 (Sauer and Stadler, 1993;
Caspari et al., 1994; Will et al., 1994; Harrison, 1996; Fig. 3A). In
addition, an Asp residue essential for the activity of the C. kessleri HUP1 gene in Schizosaccharomyces pombe was conserved at position 39 of PMT1, as well as the residues V433 and N436
of HUP1, which compared to V428 and N431 of PMT1 (Caspari et al., 1994;
Will et al., 1994). Analogous to the earlier reported transmembrane
sugar transporters, PMT1 contains 12 putative transmembrane regions
(Sauer and Tanner, 1993; Fig. 3B). Taken together, the high overall
homology, the conservation of specific amino acids, and the presence of
12 membrane-spanning domains strongly suggest that pmt1
encodes a monosaccharide-transporter protein.
Pmt1 transcription is pollen specific, and expression starts
after the first pollen mitosis. The mRNA transcripts accumulate to
remarkably high levels in mature pollen and remain present during
pollen tube growth. PMT1 is more closely related to the cruciferous
monosaccharide transporter, STP4 (Truernit et al., 1996), than to the
solanaceous monosaccharide transporter, MST1 (Sauer and Stadler, 1993).
The homology of pmt1 to stp4 is reflected in
their site of transcription. Aside from expression in the anther, stp4 is also expressed in root tips and in response to
stress (Truernit et al., 1996). Arabidopsis monosaccharide-transporter stp1 is more closely related to mst1 from tobacco
(Sauer et al., 1991; Sauer and Stadler, 1993; Table I). This
suggests the presence of an additional monosaccharide-transporter gene
in petunia more homologous to MST1/STP1, and could suggest different
classes of monosaccharide transporters. Arabidopsis contains a family
of at least five functionally active monosaccharide-transporter genes, and seven more homologous genes were isolated (Sauer and Tanner, 1993;
Sauer et al., 1994; Treurnit et al., 1996). Southern-blot analysis
demonstrated the presence of a small multigene family in petunia with
four to five members (Fig. 5).
Based on the data presented in this article and the current knowledge
of carbohydrate transport, we propose a model for carbohydrate supply,
metabolism, and translocation into growing pollen tubes (Fig.
6). Suc is transported from the source
tissues, especially mature leaves, through the phloem toward the
pistils (Bush, 1993). To reach the pollen tube, Suc has first to be
unloaded from the spiral veins into the outer layers of the style, and
is subsequently transported to the stylar apoplast of the transmitting
tract. A low amount of sugars might also be produced by the green
transmitting tract cells (Jansen et al., 1992). In the apoplast, the
sugars encounter the growing pollen tubes that have invertase activity converting Suc into Fru and Glc. Subsequently, these monosaccharides are translocated over the pollen tube membrane by the PMT1 protein. In
their studies on H+ and
[U-14C] uptake of labeled Suc, Deshusses et al.
(1981) proposed a proton-sugar cotransport in lily pollen tubes. Like
transport in lily and like all other monosaccharide transporters, PMT1
would act as a symporter, transferring monosaccharides together with a
proton. This requires proton export driven by ATP. Houlné and
Boutry (1994) isolated a plasma membrane-bound
H+-ATPase, aha9, from Arabidopsis.
Aha9 proved to be anther specific, which could mean that for carbon
loading in pollen, in addition to a specific monosaccharide transporter
present in the plasma membrane, there is also a specific
H+-ATPase gene. After transport over the pollen
tube membrane the monosaccharides are used for callose plug formation,
(callose) tube wall synthesis, and respiration required for successful
fertilization.
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| Figure 6.
Schematic representation of a model for
carbohydrate loading of pollen tubes growing through the transmitting
tract, with PMT1, H+-ATPase (HA), and wall-bound invertase
(inverse highlighted). Left to right, One-half of a tip of a growing
pollen tube, the stylar apoplast with transmitting tract cells (TrT,
shaded), flanked by nongreen stylar cells with phloem cells (spiral
veins). "Sugars" in the TrT cells represent additional sugars
synthesized and eventually exported (dotted arrow) from green stylar
cells. "Sugars" in the pollen tube represent carbohydrates after
transport as monosaccharides over the membrane by PMT1. Small circles
at the tube apex represent membrane vesicles and exocytosis (Derksen et
al., 1995).
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FOOTNOTES |
1
This work was supported in part by a grant from
the Spanish Ministry of Science and Education to D.G.
2
Present address: Plant Gene Expression Center,
U.S. Department of Agriculture-Agricultural Research Station,
University of California-Berkeley, 800 Buchanan Street, Albany, CA
94710.
3
These authors contributed equally to this work.
*
Corresponding author; e-mail A.J.vanTunen{at}cpro.dlo.nl; fax
31-317-418094.
Received January 12, 1998;
accepted May 25, 1998.
The accession number for the sequence reported in this work is
AF061106.
| |
ABBREVIATIONS |
Abbreviation:
RACE, rapid amplification of cDNA ends.
| |
ACKNOWLEDGMENTS |
The authors thank Dr. A.J. Koops for assistance with HPLC
analysis. Dr. A.J. Koops, Prof. Dr. J.N.M. Mol, and Dr. H. Dons are thanked for helpful suggestions and critical reading of the manuscript.
| |
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Abstract
Introduction