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Plant Physiol. (1998) 117: 1143-1152
Characterization of a Maize Tonoplast Aquaporin Expressed in
Zones of Cell Division and Elongation1
François Chaumont,
François Barrieu,
Eliot M. Herman, and
Maarten J. Chrispeels*
Department of Biology, University of California-San Diego, La
Jolla, California 92093-0116 (F.C., F.B., M.J.C.); and Plant Molecular
Biology Laboratory, United States Department of Agriculture,
Agricultural Research Service, Beltsville, Maryland 20705 (E.M.H.)
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ABSTRACT |
We studied aquaporins in maize
(Zea mays), an important crop in which numerous studies
on plant water relations have been carried out. A maize cDNA,
ZmTIP1, was isolated by reverse transcription-coupled PCR using conserved motifs from plant aquaporins. The derived amino
acid sequence of ZmTIP1 shows 76% sequence identity
with the tonoplast aquaporin  -TIP (tonoplast intrinsic protein) from Arabidopsis. Expression of ZmTIP1 in Xenopus laevis
oocytes showed that it increased the osmotic water permeability of
oocytes 5-fold; this water transport was inhibited by mercuric
chloride. A cross-reacting antiserum made against bean  -TIP was used
for immunocytochemical localization of ZmTIP1. These results indicate
that this and/or other aquaporins is abundantly present in the small
vacuoles of meristematic cells. Northern analysis demonstrated that
ZmTIP1 is expressed in all plant organs. In situ
hybridization showed a high ZmTIP1 expression in
meristems and zones of cell enlargement: tips of primary and lateral
roots, leaf primordia, and male and female inflorescence meristems. The
high ZmTIP1 expression in meristems and expanding cells
suggests that ZmTIP1 is needed (a) for vacuole biogenesis and (b) to
support the rapid influx of water into vacuoles during cell expansion.
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INTRODUCTION |
The vacuole is a multifunctional organelle with important roles in
space filling, osmotic adjustment, storage, and digestion. In dividing
cells the vacuolar compartment is represented by small vacuoles that
first increase in number as a result of de novo biogenesis, and then
expand and coalesce to form one or several highly lobed structures that
occupy most of the cellular volume. Vacuole biogenesis and enlargement
require the transport of osmotically active substances across the
tonoplast, followed by the rapid influx of water into the vacuole. This
influx generates the turgor pressure that drives cell expansion and
maintains cell shape. Recent studies (Maurel et al., 1997 ; Niemietz and
Tyerman, 1997) show that the tonoplast is highly permeable to water and
that this high permeability is caused by the presence of
mercuric-chloride-inhibitable water channels that permit the rapid
passage of water with a low energy of activation. Such observations are
consistent with the presence of aquaporins in the tonoplast.
Aquaporins form a large family (Weig et al., 1997) of proteins present
in the plasma membrane (PIPs) and tonoplast (TIPs) that increase the
hydraulic conductivity of the plasma membrane when expressed in
Xenopus laevis oocytes (for review, see Maurel, 1997). They
are 25- to 29-kD membrane proteins with primary sequences similar to
those of the MIP family (Park and Saier, 1996). MIPs have six
transmembrane domains with cytosolic amino and carboxy termini and
short, conserved amino acid motifs, including the signature sequence
SGxHxNPA, which is repeated in the second half of the protein as NPA.
Some of these proteins transport small solutes, others transport small
solutes and water, and still others transport only water (Park and
Saier, 1996).
The expression patterns of specific plant aquaporins are tissue- and
cell-type specific. The aquaporin -TIP from common bean accumulates
during seed maturation (Johnson et al., 1989; Melroy and Herman, 1991),
and the aquaporins -TIP and  -TIP from Arabidopsis are
preferentially expressed in elongating root cells and in the parenchymal cells of vascular tissues, respectively (Ludevid et al.,
1992; Daniels et al., 1996). The plasma membrane aquaporin RD28 from
Arabidopsis is found in all plant organs, but is absent from seeds
(Daniels et al., 1994). Several other studies have revealed the organ-
and cell-type-specific expression patterns of TIP and PIP aquaporins
(Yamamoto et al., 1991; Kammerloher et al., 1994; Opperman et al.,
1994; Kaldenhoff et al., 1995; Yamada et al., 1995). The variety of the
expression patterns suggests that aquaporins may function in
long-distance transport (xylem and phloem loading and unloading), in
short-distance transcellular water flow, and in intracellular osmotic
adjustment.
Maize (Zea mays) has been used extensively to study water
transport and its regulation by environmental parameters (Westgate and
Boyer, 1985; Sharp et al., 1988; Zhu and Steudle, 1991), and for this
reason we decided to characterize its aquaporins and study their
expression patterns. The presence of conserved sequence motifs in plant
MIPs allows their cDNAs to be isolated by RT-PCR. In this paper we
report the isolation and properties of a highly expressed maize
tonoplast aquaporin cDNA, ZmTIP1, and document its
expression in tissues of maize that are actively dividing and beginning
to elongate: meristems of primary roots and lateral roots, leaf
primordia, and male and female inflorescence meristems. We interpret
our results to indicate that TIPs may be needed for vacuole biogenesis
and enlargement in cells that are still dividing and beginning to
expand.
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MATERIALS AND METHODS |
Plant Growth Conditions
Maize (Zea mays, Oh43 line) was grown in a greenhouse
under a 16-h light/8-h dark photoperiod. Seedlings were grown on
moistened filter paper at 30°C in the dark.
RNA Extraction
Total RNA was obtained from seeds, embryos, and endosperm at
19 d after pollination from shoots and roots of germinating
seedlings, from leaves of 1- to 2-week-old plants, and from developing
ears and tassels approximately 2 cm in size. Endosperm samples were isolated from developing seeds by cutting off the top of the seed coat,
extracting the seed contents with a small spatula, and removing the
embryo from the endosperm. All tissue samples were frozen in liquid
N2 after isolation and stored at  70°C. Total
RNA was extracted as previously described (Cone et al., 1986).
Poly(A+) RNA was isolated from total RNA using
the Poly(A+) Tract Kit (Promega) following the
instructions of the manufacturer.
Identification of MIP cDNAs by RT-PCR
cDNA was synthesized from 0.5 µg of seed mRNA using
oligo(dT)12-18 as a primer and Moloney murine
leukemia virus RT (GIBCO-BRL). Partial ZmTIP1 cDNA was
amplified by PCR using degenerate TIP2 and TIP4 primers (Weig et al.,
1997), and the reaction products were separated and cloned as described
previously (Weig et al., 1997).
ZmTIP1 cDNA Cloning
Full-length ZmTIP1 cDNA was obtained using the
5 /3 RACE kit (Boehringer Mannheim) following the instructions
of the manufacturer. For the ZmTIP1 5/3 RACE, three
antisense- and one sense-specific primers (MRACE3,
5-GCGATGGTGCCCAGGCTGCC-3; MRACE7, 5-GGTCCACCGCCGTGGCGTAC-3; MRACE10, 5-CAGCACGTGCGCCACCCAGTA-3; and MRACE5,
5-GCAGGCCACGGGCACCTTCG-3) were used. The PCR products were cloned
into pCRII (TA cloning kit, Invitrogen) and sequenced. The full-length
ZmTIP1 cDNA was amplified using Pfu
polymerase (Stratagene) with proofreading activity and specific primers
to the 5- and 3-noncoding regions (ZMTIP1-1,
5-CGGAATTCTCCAGCTCCAATCACAGTC-3; and ZMTIP1-2,
5-CGGAATTCACGGTTACAAGCAG-3) incorporating EcoRI sites
on both ends, and subcloned into EcoRI site of
Bluescript II SK+ (Stratagene).
Plasmid Constructions and in Vitro RNA Synthesis
cDNA encoding ZMTIP1 was amplified by PCR with specific primers
(ZMTIP1-3, 5-GGCGGATCCTACCATGCCGATCAATAGGAT-3; and ZMTIP1-4, 5-CGATGGATCCACGTGCACGAG-3) incorporating BamHI
sites on both ends, and subcloned into the BglII site of
a pSP64T-derived Bluescript vector carrying 5- and 3-untranslated
sequences of a  -globin gene from Xenopus laevis
(Preston et al., 1992). The orientation of the insert was determined by
restriction mapping and sequencing. Capped complementary RNA encoding
ZMTIP1 was synthesized in vitro using T3 RNA polymerase, and was
purified as described by Preston et al. (1992).
The 3-untranslated region of ZmTIP1 was amplified by
PCR with specific primers (ZMTIP1-5, 5-CACCGGATCCTAAAAGCCGAAG-3; and ZMTIP1-2) incorporating BamHI and EcoRI
sites on the ends, and subcloned into the corresponding sites of
pBluescript II SK+ (Stratagene)
(pBS3-ZmTIP1).
Part of ZmTIP1 cDNA encoding the carboxy-terminal 62 amino acid residues of ZMTIP1 was amplified by PCR with T7 and
ZMTIP1-7 (5-GGCGGCGAATTCGACGGCGC-3) primers. The PCR product was
digested with EcoRI and SalI and
subcloned in the corresponding sites of pGEX-4T-1 (Pharmacia)
(pGEX-C-Zmtip1).
Osmotic Water-Permeability Assay
X. laevis oocytes were prepared and injected as
previously described (Daniels et al., 1996), and the osmotic water
permeability of the plasma membrane was determined (Weig et al., 1997).
DNA Gel-Blot Analysis
Total DNA was extracted from leaf tissue as described previously
(Schmidt et al., 1987). DNA blots and hybridizations were as described
previously (Evola et al., 1986). For probe synthesis, the
3-untranslated region of ZmTIP1 cDNA was gel purified and radiolabeled using a kit (Rediprime, Amersham) following the
instructions of the manufacturer. Hybridizations were performed at
42°C in 50% formamide. Washes were performed four times for 15 min
each in 0.1× SSC (1× SSC is 150 mM NaCl and 15 mM
Na3C6H5O7)
and 0.1% SDS at 60°C.
RNA Gel-Blot Analysis
Total RNA samples (20 µg each) were fractionated by
electrophoresis on a Hepes-formaldehyde 1.5% agarose gel following the protocol of Tsang et al. (1993), and were transferred to Hybond-N nylon
membranes (Amersham) using standard blotting techniques (Sambrook et
al., 1989). Ethidium-bromide-stained rRNAs were used as the internal
loading control. The RNA was bound to the membrane with UV illumination
and baking at 80°C for 1 to 2 h. The prehybridization and
hybridization were performed at 42°C in 50% formamide, 5× SSPE (1×
SSPE is 180 mM NaCl, 1 mM EDTA, and 10 mM Na2HPO4, pH 7.7), 5× Denhardt's solution (1× Denhardt's solution is 0.02% [w/v] BSA, 0.02% [w/v] Ficoll, 0.02% [w/v] PVP), 0.5% SDS, and 100 µg mL 1 of yeast tRNA. The
random-primer-labeled probes were generated using the Rediprime kit
following the instructions of the manufacturer. Hybridized membranes
were washed under high-stringency conditions (0.2× SSPE, 0.2 SDS at
65°C for 20 min), and then exposed to radiographic film with
intensifying screens at 70°C.
GST-C-ZMTIP1 Expression in Escherichia coli and
Immunodetection
pGEX-C-ZmTIP1 plasmid was introduced in the M15 bacterial strain
(Qiagen, Santa Clarita, CA), and GST-C-ZMTIP1 expression was induced by
2 mM isopropyl -D-thiogalactopyranoside for
2 h. Appropriate quantities of total protein extract were
fractionated by 12.5% SDS-PAGE, transferred to nitrocellulose, and the
proteins detected using the rabbit antisera raised against Arabidopsis -TIP (Höfte et al., 1992) and bean -TIP (Johnson et al.,
1989). Goat anti-rabbit IgG coupled to horseradish peroxidase (Bio-Rad) was used as the secondary antibody.
Immunocytochemical Localization
The immunocytochemical localization of ZmTIP1 in the embryo after
1 d of germination was performed with antiserum raised against bean seed -TIP, as described previously (Melroy and Herman, 1991).
RNA in Situ Hybridization
Maize tissues were fixed in 50% (v/v) ethanol, 5% (v/v) acetic
acid, and 3.7% (v/v) formaldehyde at room temperature for 4 h
with occasional degassing under a vacuum for 15 min. After fixation, the tissues were dehydrated through an alcohol series and embedded in
Paraplast Plus (Oxford Labware, St. Louis, MO). The tissues were
sectioned into 8- to 10-µm slices, dewaxed with Histoclear (National
Diagnostics, Atlanta, GA), and hydrated by passing through an alcohol
series to water. Sections were prepared for in situ hybridization as
described previously (Marrison and Leech, 1994).
The in situ hybridizations were performed as described previously
(Marrison and Leech, 1994) with some modifications. ZmTIP1 sense- and antisense-labeled probes were generated using
pBS3-ZmTIP1 linearized with EcoRI or
BamHI and transcribed using a digoxigenin RNA-labeling
mixture (Boehringer Mannheim) with either T3 or T7 RNA polymerase
(Promega), respectively. The probes were hybridized to the tissue
sections overnight at 50°C at a concentration of 200 to 400 ng
mL1 in 40 µL of hybridization buffer (6×
SSC, 3% [w/v] SDS, 50% [v/v] formamide, and 100 µg
mL1 tRNA). After hybridization, the sections
were incubated twice in wash buffer (2× SSC and 50% [v/v]
formamide) at 50°C for 90 min; treated with RNase A (10 µg
mL1 in 2× SSC) at 37°C for 30 min; and
washed at 50°C for 1 h in wash buffer. The sections were
incubated in a blocking solution (Boehringer Mannheim, 0.5% in TBS)
for 1 h; in 1% (w/v) BSA and 0.3% (v/v) Triton X-100 in TBS for
30 min; and in the same solution containing alkaline
phosphatase-conjugated antibodies (Boehringer Mannheim) at a 1/1000
dilution for 90 min. Unbound antibody conjugate was removed and
ZmTIP1 transcripts were detected according to the method of
Marrison and Leech (1994).
Photographs were made using a light microscope (Optiphot-2, Nikon). The
slides were digitized using a slide scanner (CoolScan, Nikon).
Brightness and contrast were adjusted using Photoshop 3.0 (Adobe
Systems, Mountain View, CA). Composite figures were prepared in Canvas
3.5 (Deneba Software, Miami, FL) and printed using a dye-sublimation
color printer (Phaser IIsdx, Tektronix, Wilsonville, OR).
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RESULTS |
Isolation of ZmTIP1 cDNA
A comparison of plant aquaporin amino acid sequences showed the
presence of several conserved regions. Two of them, HI/VNPAVT and
WI/VF/YWVGP, were used to design degenerate oligonucleotide primers for
RT-PCR (Weig et al., 1997). Using these primers with cDNAs prepared
from maize seeds 19 d after pollination, we obtained a
PCR-amplified fragment (0.42 kb) containing a sequence homologous to
plant TIP aquaporins. The corresponding full-length cDNA was recovered
by 5/3 RACE with RNA from maize seeds and roots and named
ZmTIP1 (accession no. AF037061).
The ZmTIP1 cDNA consists of 1097 bp upstream of the
poly(A+) tail, which includes a 93-bp leader sequence,
followed by 753 bp of open reading frame encoding 250 amino acids, and,
finally, a 251-bp 3 noncoding region. ZmTIP1 has a calculated
Mr of 25,820 and contains the MIP family
signature sequence SGxHxNPAVT, which is repeated in the second half of
the protein as NPA. A comparison of the amino acid sequences of six
other TIPs is shown in Figure 1. ZmTIP1
has the highest sequence identity at the amino acid level with two
other monocot TIPs from rice (Liu et al., 1994) and barley
(Schünmann and Ougham, 1996) (95.2% and 90.4%, respectively). ZmTIP1 is also related to the known vacuolar aquaporins BobTIP26 from
cauliflower (Barrieu et al., 1998; F. Barrieu, D. Marty-Mazars, F. Chaumont, M. Chrispeels, and F. Marty, unpublished data) and -TIP
from Arabidopsis (Maurel et al., 1993) (77.3% and 76.3% identity,
respectively). These proteins cluster together on a dendogram, whereas
other TIP aquaporins such as -TIP from Arabidopsis (Daniels et al.,
1996) and seed -TIP from bean (Johnson et al., 1990) are more
distant (61.1% and 52.7% identity with ZmTIP1, respectively) (Fig.
2).
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| Figure 1.
Comparison of ZmTIP1 sequence with other
plant TIPs. Amino acid sequences were compared with the Clustal W
multiple alignment program (Thompson et al., 1994). The amino acid
sequences were obtained from the following sources: ZmTIP1 (this work);
OsTIP1 (Liu et al., 1994); HvTIP1 (Schünmann and Ougham, 1996);
BobTIP26 (Barrieu et al., 1998); At-TIP (Hofte et al., 1992);
At-TIP (Daniels et al., 1996); and Pv-TIP (Johnson et al., 1990).
Identical amino acid residues common to at least three sequences are
shaded. Numbering refers to the respective amino acid sequence. The
position of the Cys residue responsible for the mercury sensitivity of
At-TIP and At-TIP is noted by an arrowhead in the consensus
line.
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| Figure 2.
Dendogram of the comparison between ZmTIP1 and
other plant TIPs. Amino acid sequences from Figure 1 were compared
using the program PILEUP (Genetics Computer Group, Madison, WI).
Underlined sequences have been identified as aquaporins.
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ZmTIP1 Forms Water Channels in X. laevis Oocytes
In vitro-transcribed cRNA encoding ZmTIP1 was injected into
X. laevis oocytes and osmotically driven water transport
into the oocytes was investigated 3 d after injection. Oocytes
were exposed to hypoosmotic conditions by diluting the culture medium, and the changes in cell volume were recorded. In these conditions water-injected oocytes swelled slowly. In contrast, oocytes injected with ZmTIP1 cRNA rapidly increased their volume,
indicating the presence of a facilitated water-transport pathway. The
osmotic water-permeability coefficient (Pf) of the oocyte membrane
increased 4- to 5-fold over the control value (Fig.
3).
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| Figure 3.
Osmotic water permeability (Pf) values of
individual ZmTIP1 cRNA-injected oocytes derived from
volume change measurements made over two independent preparations of
oocytes. White bars, Control (no mercuric chloride); black bars, assay
performed in the presence of 3 mM mercuric chloride with a
10-min preincubation. Data are expressed as the mean ± SE,
with the number of replicates indicated next to each bar in
parentheses.
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Mercuric chloride is a characteristic inhibitor of many water-channel
proteins (Preston et al., 1992; Maurel et al., 1993). The
mercury-sensitive sites of Arabidopsis -TIP and -TIP have been
identified as Cys-118 and Cys-116, respectively, at a conserved position in a presumed membrane-spanning domain (Daniels et al., 1996).
This Cys residue is conserved among the TIPs, including ZmTIP1, with
the exception of -TIP from bean (see arrow in Fig. 1). Water
transport through ZmTIP1 is inhibited 70% by 3 mM mercuric chloride (Fig. 3). These results support the interpretation that ZmTIP1
forms channels in oocyte membranes that facilitate water transport.
ZmTIP1 Cross-Reacts with Different Aquaporin Antisera
In the past our laboratory has raised antisera against the
30 carboxy-terminal amino acid residues of Arabidopsis -TIP
(Höfte et al., 1992) and the whole -TIP from bean (Johnson et
al., 1989). To determine if these antisera cross-react with ZmTIP1, we
fused the sequence encoding the carboxy-terminal 62 amino acid residues of ZmTIP1 to the GST gene in the pGEX-4T-1 plasmid vector, and the
fusion protein was expressed in E. coli (see ``Materials and Methods''). When the induced culture was allowed to express the
fusion protein for 2 h, a strong band of 34 kD, corresponding to
the GST-C-ZmTIP1 polypeptide, was produced as observed by SDS-PAGE
analysis (Fig. 4, lane 2).
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| Figure 4.
Coomassie blue-stained gel and immunoblot of
extracts of E. coli expressing GST-C-ZmTIP1. Total
E. coli protein extract before (lanes 1, 3, and 5) and
after (lanes 2, 4, and 6) 2 h of induction of GST-C-ZmTIP1
expression by 2 mM isopropyl
-D-thiogalactopyranoside was fractionated by
SDS-PAGE. Polypeptides were visualized with Coomassie blue (lanes 1 and
2) or transferred to nitrocellulose and immunostained using Arabidopsis
-TIP antiserum (lanes 3 and 4) or bean -TIP (lanes 5 and 6). The
positions of molecular mass standards are indicated.
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Immunoblot analysis of the same bacterial extract using the
Arabidopsis -TIP antiserum showed that this serum detects the GST-C-ZmTIP1 polypeptide and cross-reacts with two E. coli
proteins (Fig. 4, lanes 3 and 4). In the same way, the bean -TIP
antiserum cross-reacted with GST-C-ZmTIP (Fig. 4, lane 6). The
reactivity of these two sera with ZmTIP1 came from the ZmTIP1
polypeptide (and not from the GST) because neither -TIP or -TIP
antisera elicited an immunostaining reaction with expressed GST protein (data not shown). Sequence identity in this carboxy-terminal region of
ZmTIP1 with Arabidopsis -TIP and bean -TIP was 73% and 55%, respectively (Fig. 1). These data indicate that antigenic epitopes recognized by -TIP and -TIP antisera are conserved in TIPs from monocots and dicots.
A ZmTIP1 Cross-Reacting Serum Labels the Tonoplast
The cross-reactivity of ZmTIP1 with -TIP antiserum allowed us
to examine the subcellular localization of ZmTIPs by
immunocytochemistry. We chose maize embryos for this localization
because ZmTIP1 is highly expressed there (see below).
Meristematic cells of embryos are characterized by the presence of
numerous small vacuoles, which subsequently fuse and enlarge. Figure
5 shows abundant colloidal gold labeling
of the interface between the cytoplasm and the vacuole where the
tonoplast is located. This method of fixation, which minimizes the
destruction of protein epitopes, does not allow for the visualization
of the tonoplast. There was no specific labeling of the vacuolar
content (Fig. 5) or of the plasma membrane (data not shown). These
results indicate that ZmTIPs are localized in the tonoplast of maize
vacuoles. We do not know if the labeling was caused by the presence of
ZmTIP1 alone or by the presence of other aquaporins or MIPs that
cross-react with this serum
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| Figure 5.
Immunocytochemical localization of ZmTIP1 in maize
embryos. The gold particles are primarily at the interface of the
cytoplasm and the vacuole (V). The vacuoles apparently contain
aggregated protein. Magnification is ×60,000.
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Expression of ZmTIP1 in Different Tissues during
Development
To analyze the expression pattern of ZmTIP1, a
203-bp DNA fragment from the 3-untranslated region of
ZmTIP1 cDNA was used as a probe. The specificity of the
probe was tested by Southern hybridization (Fig.
6). In this experiment, only restriction
enzymes that do not cut the 3-untranslated sequence
(EcoRI, HindIII, and XbaI)
were used to digest genomic DNA samples. Hybridization at
high-stringency conditions (0.1× SSC, 0.1% SDS, and 60°C) revealed only one band for each of the restriction digests. This result suggests
that the probe is likely to be ZmTIP1 gene specific.
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| Figure 6.
Genomic Southern analysis. Total maize genomic DNA
(15 µg per lane) was digested with EcoRI,
HindIII, and BamHI and hybridized with
labeled 3-untranslated region of ZmTIP1 cDNA. The
positions of the Mr markers are indicated.
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To characterize the pattern of expression of ZmTIP1,
gel-blot analysis of total RNA from different maize tissues was
performed. ZmTIP1 transcripts with a size of 1.15 kb
were observed in all of the expanding tissues studied, from the embryo
to the flower organs (Fig. 7A, lanes
3-8). The transcripts were absent from the endosperm, which represents
more than 90% of the total seed extract (Fig. 7A, lane 2). To
determine if the high ZmTIP1 expression seen in the
expanding tissues persists in older organs, we performed an RNA-blot
hybridization with total RNA obtained from leaves of light-grown
seedlings at the three-leaf stage of development (Fig. 7, B-D). In
these seedlings the first plumular leaf (leaf no. 1) was fully
expanded, leaf no. 2 was close to the end of its growth, and the
youngest leaf (leaf no. 3) was still growing rapidly (Fig. 7B). The
ZmTIP1 transcript level was highest in the youngest
expanding leaf (Fig. 7C), indicating a possible role of ZmTIP1 in leaf
expansion. ZmTIP1 transcript abundance in the different
leaves was lower in these green leaves compared with the level in the
developing etiolated shoot.
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| Figure 7.
Gel-blot analysis of ZmTIP1 mRNA in
different vegetative and reproductive organs. Total RNA (20 µg) was
extracted from the indicated organs (A, C, and D) and from 10-d-old
maize plantlet leaves (1-3 in B-D), and separated by gel
electrophoresis in the presence of ethidium bromide (D). After transfer
the blots were hybridized with ZmTIP1 probe (A and C).
Total shoot RNA was used as a control to compare the signal intensity
in the different blots.
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To more precisely determine the localization of ZmTIP1
expression in developing organs, the patterns of ZmTIP1
mRNA localization were determined by in situ hybridization of
digoxigenin-labeled RNA probes using longitudinal sections through
various organs (Fig. 8). The intensity of
the red color indicates the abundance of mRNA. The controls probed with
sense cRNA were white (Fig. 8, C, E, and I). In the primary root of a
maize seedling the highest expression of ZmTIP1 was
detected in the apical meristem and the cell-elongation zone (Fig. 8A).
Cells close to the vascular bundles stained more intensely than the
cortical cells. No transcripts were detected in the root cap or the
quiescent center. At more distal regions from the root tip,
ZmTIP1 expression decreased dramatically, and seemed to
be restricted to the epidermis and a zone surrounding the vascular
cylinder. More distally, strong signals were found in the new lateral
root primordia at the periphery of the vascular cylinder (Fig. 8B). A
weak expression was still detectable around the vascular bundle.
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| Figure 8.
Localization of ZmTIP1 mRNA by in
situ hybridization. The controls, hybridized with sense RNA, are shown
in C, E, and I; all other panels were hybridized with antisense RNA. A,
Longitudinal section of 3-d-old root tip; B, longitudinal section in
the zone of lateral root initiation; C, control, same section as shown
in A; D and E, median sections of 3-d-old plumule; F, median section of
an immature tassel; G, close-up of F; H and I, median section of an
immature ear. Ca, Root cap; DZ, division zone; EZ, elongation zone; Gl,
glume; Le, lemma; LF, lower floret; Lo, lodicule; LP, leaf primordium;
S, stamen; VB, vascular bundle; TZ, transition zone; UF, upper floret.
White and black arrows in B indicate RNA transcript signal in the
lateral root meristems and the vascular bundle, respectively.
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Figure 8D shows ZmTIP1 transcripts in the shoot apical
region of a seedling plumule, where ZmTIP1 was most
strongly expressed in leaf primordia and expanding leaves. No
transcripts were detected in the coleoptile at this stage of seedling
development (data not shown). In immature male and female
inflorescences, ZmTIP1 expression was mainly localized
in the developing spikelets (Fig. 8, F-H), but transcripts were also
present around the vascular bundles. In the tassel spikelet, expression
was highest in the stamen and lodicule primordia and in the adjoining
vessel bundles, but signal was also present in the developing glume and
lemma surrounding the florets (Fig. 8G). In the ear spikelet,
ZmTIP1 expression was seen mainly in the upper and lower
floret primordia (Fig. 8H).
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DISCUSSION |
The discovery of water-channel proteins in the membranes of plant
cells allows the formulation of new mechanisms that may be used by
plants to control water transport and osmotic adjustment (Maurel,
1997). The presence of highly conserved motifs in plant aquaporins
permitted us to identify and clone by RT-PCR and RACE a tonoplast
aquaporin cDNA from maize, ZmTIP1, which is closely related
to the Arabidopsis -TIP aquaporin (76% amino acid identity). -TIP is an integral TIP expressed in the vegetative body of
Arabidopsis (Höfte et al., 1992) that can form water channels in
X. laevis oocyte membranes (Maurel et al., 1993). In the
same way, ZmTIP1 increased the water membrane permeability of X. laevis oocytes. Both aquaporins are sensitive to mercuric
chloride. On a dendogram, the ZmTIP1 amino acid sequence clusters with
TIP homologs from two monocots, rice (Liu et al., 1994) and barley
(Schünmann and Ougham, 1996), and together these three form a
larger group with the cauliflower BobTIP26 and Arabidopsis -TIP, two
dicot aquaporins. This group diverges from other identified tonoplast
aquaporins such as the Arabidopsis -TIP (Daniels et al., 1996) and
bean -TIP (Johnson et al., 1990), which have different expression patterns. The clustering of -TIP homologs in two groups according to
the plant classes suggests that a common but already specialized -TIP ancestor diverged during the evolution of the monocots and dicots.
ZmTIP1 Is Highly Expressed in Dividing Cells
Detailed analysis of ZmTIP1 transcript localization
by in situ hybridization showed a high expression in zones of cell
division and elongation of the roots, leaves, and reproductive organs. The high level of expression observed in conducting tissues is discussed in the accompanying paper (Barrieu et al., 1998). Dividing cells contain numerous small vacuoles in different stages of
development. Stereological measurements with meristematic cells of
Vicia faba showed that the combined volume of the
spherical vacuoles represents 27% of the cell volume and that the
combined surface area of these vacuoles is as large as that of the
plasma membrane area (Steer, 1981). Meristematic cells must
generate equal amounts of tonoplast and plasma membrane between rounds
of cell division, a process that requires the synthesis of new membrane
components. Vacuole biogenesis proceeds through the formation of
provacuoles that fuse in an autophagic process (for review, see Marty,
1997).
Because TIPs are abundant in the tonoplast, one might expect a high
level of TIP transcripts in dividing cells. The presence of TIPs in
meristematic cells has been previously shown in root and shoot of beet
(Marty-Mazars et al., 1995), in barley and pea root tips (Paris et al.,
1996), and in cauliflower florets (Barrieu et al., 1998). Transcripts
of the root-specific TobRB7 were also detected in
meristematic cells but it is not known if this gene encodes a tonoplast
or plasma membrane aquaporin (Yamamoto et al., 1991). The expression of
ZmTIP1 reported here for dividing cells differs
substantially from our previous finding with the close Arabidopsis
homolog -TIP (Ludevid et al., 1992). Analysis of -TIP
promoter-GUS gene fusion-transformed plant and
whole-mount in situ hybridizations carried out with seedlings showed
expression in vascular bundles and other tissues, but not in meristems.
With respect to the GUS fusions it is likely that regulatory elements were missing from the promoter-GUS fusion construct.
ZmTIP1 Is Highly Expressed in Expanding Cells
In addition to the zone of cell division, root tips have a zone of
cell elongation, and between these two there is a transition zone or
distal elongation zone, in which the cells expand isodiametrically, growing in width as much as in length (Fig. 7A) (Ishikawa and Evans,
1995; Baluska et al., 1996). In this transition zone cells have to
develop the necessary synthetic machinery for the biogenesis of new
tonoplast and plasma membranes, cell wall components, new enzymatic
complexes, and cytoplasmic structures that support the rapid growth in
the elongation zone (Baluska et al., 1996). Cells of the distal
elongation zone respond to a variety of signals, such as auxin
(Ishikawa and Evans, 1993), water stress (Sharp et al., 1988) and
gravistimulation (Ishikawa et al., 1991), differently from the cells in
the main elongation zone. For instance, the elongation of cells in this
zone is unaffected by reducing the water potential, whereas the rate of
elongation in the main elongation zone is inhibited (Sharp et al.,
1988). As observed by in situ hybridization experiments, the expression
of the tonoplast aquaporin ZmTIP1 is high in these cells. It would be
interesting to analyze the pattern of expression of
ZmTIP1 and/or other aquaporin genes in this zone in
response to external signals.
Rapid elongation of plant cells is based on an extensive uptake of
solutes coupled with the uptake of water, resulting in the formation of
a prominent vacuolar compartment. This mechanism maintains the turgor
pressure that drives cell expansion. Rapid cell expansion may require a
high hydraulic permeability of the tonoplast to support water entry
into the vacuole. An important role for TIPs during this process was
initially suggested by the observation that Arabidopsis -TIP is
highly expressed in the zone of cell elongation in roots, hypocotyls,
and leaves (Ludevid et al., 1992). Arabidopsis -TIP expression was
shown to be up-regulated after application of GA3 in
ga1, a GA-deficient dwarf mutant (Phillips and Huttly,
1994). Also, HvTIP1 transcripts were increased in the
slender mutant of barley, characterized by a faster elongation rate of
the leaves (Schünmann and Ougham, 1996). Maize ZmTIP1 expression is also high in young, developing leaves and zones of cell
elongation in roots, as observed by RNA gel-blot analysis and in situ
hybridization.
The high water permeability of the plant tonoplast was recently
demonstrated directly with tonoplast vesicles of cultured tobacco cells
(Maurel et al., 1997) and wheat roots (Niemietz and Tyerman, 1997).
Tonoplast vesicles have channels that transport water with a low energy
of activation and that are inhibited by mercuric chloride, whereas
plasma membrane vesicles either do not have such channels or the
channels are inactive. Together these experiments support the
interpretation that TIPs permit the rapid influx of water into the
vacuole of elongating cells. The much lower permeability of plasma
membrane vesicles observed in the same studies (Maurel et al., 1997;
Niemietz and Tyerman, 1997) may indicate that cells regulate the influx
of water at the plasma membrane.
| |
FOOTNOTES |
1
This work was supported by a cell biology grant
from the National Science Foundation. F.C. was supported by a European
Molecular Biology Organization fellowship.
*
Corresponding author; e-mail mchrispeels{at}ucsd.edu; fax
1-619-534-4052.
Received January 12, 1998;
accepted March 30, 1998.
| |
ABBREVIATIONS |
Abbreviations:
GST, glutathione S transferase.
MIP, major
intrinsic protein.
PIP, plasma membrane intrinsic protein.
RACE, rapid
amplification of cDNA ends.
RT-PCR, reverse transcription-coupled PCR.
TIP, tonoplast intrinsic protein.
| |
ACKNOWLEDGMENTS |
We thank Dr. R.J. Schmidt and his collaborators for helpful
discussions, and members of Dr. M.F. Yanofsky's laboratory for help
with the in situ hybridization and use of their equipment.
| |
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Abstract
Introduction