From the Julius-von-Sachs-Institut, Lehrstuhl
Molekulare Pflanzenphysiologie und Biophysik, Universität
Würzburg, Julius-von-Sachs-Platz 2, D-97082 Würzburg and
§ Zentrum für Biochemie und Molekularbiologie,
Christian-Albrechts-Universität zu Kiel, Leibnizstrasse 11,
D-24098 Kiel, Germany
Received for publication, December 13, 2002, and in revised form, February 27, 2003
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ABSTRACT |
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In search of K+ channel genes
expressed in the leaf of the C4 plant Zea mays,
we isolated the cDNA of KZM1 (for
K+ channel Zea
mays 1). KZM1 showed highest
similarity to the Arabidopsis K+ channels KAT1
and KAT2, which are localized in guard cells and phloem. When expressed
in Xenopus oocytes, KZM1 exhibited the characteristic
features of an inward-rectifying, potassium-selective channel. In
contrast to KAT1- and KAT2-type K+ channels, however, KZM1
currents were insensitive to external pH changes. Northern blot
analyses identified the leaf, nodes, and silks as sites of
KZM1 expression. Following the separation of
maize leaves into epidermal, mesophyll, and vascular fractions, quantitative real-time reverse transcriptase-PCR allowed us to localize
KZM1 transcripts predominantly in vascular strands and the
epidermis. Cell tissue separation and KZM1 localization
were followed with marker genes such as the bundle sheath-specific ribulose-1,5-bisphosphate carboxylase, the phloem K+
channel ZMK2, and the putative sucrose transporter
ZmSUT1. When expressed in Xenopus oocytes,
ZmSUT1 mediated proton-coupled sucrose symport. Coexpression of ZmSUT1
with the phloem K+ channels KZM1 and ZMK2 revealed that
ZMK2 is able to stabilize the membrane potential during phloem
loading/unloading processes and KZM1 to mediate K+ uptake.
During leaf development, sink-source transitions, and diurnal changes,
KZM1 is constitutively expressed, pointing to a
housekeeping function of this channel in K+ homeostasis of
the maize leaf. Therefore, the voltage-dependent K+-uptake channel KZM1 seems to mediate K+
retrieval and K+ loading into the phloem as well as
K+-dependent stomatal opening.
Since the first isolation of a plant K+ channel gene
10 years ago, plant science has focused on their cell-specific
localization and structure-function relationship. Therefore, new
insights into the physiological role of the different K+
channel genes have been gained. The Arabidopsis thaliana
genome contains at least 15 K+ channel genes (1). Among
them, the Shaker family of Arabidopsis K+ channels consists of nine members (for review see Ref.
2). According to their localization, structure, and function, these genes can be assigned to different subfamilies. In 1992, the first plant K+ channel genes isolated were AKT1 and
KAT1 (3, 4). Both proteins represent K+-uptake
channels (5, 6). AKT1-like channels are involved in K+
uptake into growing roots (AKT1) (7, 8) and pollen tubes (SPIK) (9),
and KAT1 plays a role in Arabidopsis guard cells (10-12).
Recently, the inward rectifier KAT2 could be characterized as the
closest relative to KAT1 (13). KAT2 is expressed in guard cells, too, but in contrast to KAT1,
KAT2 transcripts were identified in the phloem parenchyma of
the leaf. A coding sequence of the channel gene AKT5 (AKT1
subfamily) could be isolated from hypocotyl tissue,1 but its function
still remains unknown. The AtKC1 gene splits into another
subfamily and together with AKT1 subunits seems to generate the
functional properties of the root hair K+-influx channel
(14, 15). In contrast to the mentioned inward rectifiers within the
AKT1 and KAT1 family, members of the AKT2/3 subfamily are characterized
by weak voltage dependence, a Ca2+ and H+
block, and seem to control phloem function (16-19). The K+
channel genes SKOR, localized in xylem vessels of the root
(20), and GORK, in guard cells, vasculature, and roots
(14, 21), build the subfamily of outward-rectifying
K+ channels in Arabidopsis.
Because the Arabidopsis genome reveals the complete set of
Shaker-like K+ channel genes in plants, we can
assign the orthologs from different plant species to the respective
subfamilies. So far K+ channel genes have been isolated
from 12 different plants (compare with Ref. 22) including the
C4 plant Zea mays. In maize, the two
K+ channel genes ZMK1 and ZMK2,
isolated from the coleoptile, belong to the AKT1- and AKT2/3-type
subfamilies, respectively (23). ZMK1 is involved in auxin-induced
K+ uptake, coleoptile growth, and tropisms. ZMK2
displays the voltage-independent features of the AKT2/3-type
K+ channels and thus seems to serve phloem-associated
functions (24). Besides rice, the maize plant is not only a model
system for monocotyledonous crops but C4 photosynthesis as
well. This involves a special anatomic feature called Kranz anatomy:
mesophyll cells, involved in the pre-fixation of CO2,
transport C4 compounds to the bundle sheath cells, which
surround the vascular strands and finally fix CO2 in the
Calvin cycle (reviewed in Ref. 25). Due to this cell and chloroplast
dimorphism, C4 plants are characterized by a better
water-use efficiency than C3 plants. The carbohydrate transport between mesophyll, bundle sheath, and vascular parenchyma cells of the maize leaf is accomplished by numerous plasmodesmata (26-28). The loading of sucrose from the vascular parenchyma to the
thin-walled sieve tubes, representing the site of assimilate export,
however, is thought to involve an apoplastic step (reviewed in Refs. 29
and 30). To characterize the sugar import machinery, Aoki et
al. (31) isolated ZmSUT1, a putative sucrose transporter from
source leaves of maize. By heterologous expression in
Xenopus oocytes, we showed that ZmSUT1 indeed represents a
sucrose/H+ symporter under the voltage control of the
AKT2/3 ortholog ZMK2.
Because the AKT2/3-type channels such as AKT2/3 from
Arabidopsis, VFK1 from Vicia faba, and ZMK2 from
Z. mays seem to play an important role in the control of
phloem sucrose loading and unloading (16, 17, 24, 32), we here studied
K+ channels expressed in the dimorphic structure of the
maize leaf. In addition to ZMK2 (23), we isolated the cDNA of
KZM1. KZM1 represents the maize ortholog to
KAT2 from Arabidopsis. Like
KAT2 we found this new maize K+ channel
gene expressed in vascular/bundle sheath strands as well as
guard cell- and subsidiary cell-enriched epidermal fractions of the
maize leaf. However, KZM1 is characterized by unique functional properties that enabled us to discriminate between the function of KAT2
in the dicotyledonous plant Arabidopsis and KZM1
in the monocotyledonous C4 plant maize. The
K+-uptake channel KZM1 is able to mediate phloem
K+ loading and retrieval as well as
K+-dependent stomatal movement. The function of
the inward-rectifier KZM1 in combination with ZMK2 and the
sucrose/H+ symporter ZmSUT1 as well as its expression
pattern point to a housekeeping function of KZM1 for K+
homeostasis in the phloem of the maize leaf.
Plant Material--
Cloning, Northern blot
procedures, and quantitative real-time
RT2-PCR analysis were
performed on tissues isolated from maize plants (Z. mays L.,
hybrid corn cv. "Oural FA0230," Deutsche Saatveredelung, Lippstadt, Germany). Seeds were sown in soil and grown in a greenhouse with a 16-h light (25 °C) and 8-h dark (18 °C) cycle. The white light used had a photon-flux density of 210 µmol·m Cloning of KZM1 cDNA--
Degenerated oligonucleotide
primers, directed toward homologous regions of known plant
inward-rectifying K+ channels, were used to amplify a
corresponding region of potassium channels from reverse-transcribed
maize leaf RNA (RT-PCR). By using the SMART RACE cDNA Amplification
kit (Clontech, Heidelberg, Germany) in
combination with gene-specific primers, we amplified overlapping N- and
C-terminal K+ channel fragments according to the RACE
technique. The corresponding full-length cDNA was generated in a
single PCR step using primers flanking the 5'- and 3'-ends of the
coding sequence of KZM1 and ligated into pCRII-TOPO TA
vector (Invitrogen). Besides the N- and C-terminal clones of
KZM1, three identical full-length clones of the channel
cDNA were sequenced using the LiCOR 4200 sequencer (LiCOR, Bad
Homburg, Germany).
Northern Blot Analysis--
Total RNA was isolated from the
respective maize organs using the Plant RNeasy Extraction kit (Qiagen,
Hilden, Germany). Poly(A)+ RNA was purified from total RNA
using Dynabeads (Dynal, Hamburg, Germany) and subjected to Northern
blot analysis as described (33). The blotted poly(A)+ RNA
was hybridized against 32P-radiolabeled full-length
cDNA probes of the K+ channel genes KZM1 and
ZMK2 as described in Philippar et al. (23). For
the sucrose transporter ZmSUT1, a 339-bp-long 3'-terminal cDNA fragment, amplified between the primers ZmSUT LCfw
(5'-cccacaaaggcaaac-3') and ZmSUT LCrev (5'-tggtgtgggtgacg-3'), served
as a probe. Each probe exhibited specific signals at 2.5 kb for
KZM1, 2.8 kb for ZMK2, and 2.0 kb for
ZmSUT1. To standardize transcript abundance, 15 ng of dotted
poly(A)+ was hybridized against a
[ Separation of Leaf Tissues--
Tissue from the last
fully developed leaf of 5-week-old maize plants was separated into
epidermis, mesophyll cells, and vascular strands by a procedure
modified according to Keunecke and Hansen (34). The central vascular
strand was excised, and the lower epidermis was collected in 1 mM CaCl2, 5 mM Mes/KOH, pH 6.5, adjusted with mannitol to 530 mosmol kg Quantitative Real-time RT-PCR--
For real-time RT-PCR
experiments, total RNA from the fractionated maize leaves was isolated
using the Plant RNeasy Extraction kit (Qiagen, Hilden, Germany). To
minimize DNA contaminations, mRNA was purified twice with the
Dynabeads mRNA Direct kit (Dynal, Hamburg, Germany), or total RNA
was subjected to digestion with RNase-free DNase and purified by
phenol/chloroform extraction (33). Mesophyll protoplast mRNA was
directly purified with the Dynabeads mRNA Direct kit (Dynal).
First-strand cDNA synthesis and quantitative real-time RT-PCR were
performed as described before (12) using a LightCycler (Roche Molecular
Biochemicals). The following K+ channel-specific primers
were used: KZM1 LCfw (5'-aagaagcatggttgttac-3'), KZM1 LCrev
(5'-tgaaaccaaagaagtctc-3'), ZMK2 LCfw (5'-gacggctcaggttcag-3'), and
ZMK2 LCrev (5'-gagaaggcgttgatcg-3'). For detection of the coding
sequence of the small subunit of the ribulose-1,5-bisphosphate carboxylase (ZmRuBPCsu, GenBankTM accession number X06535)
and the 3'-untranslated region of the C4 form
phosphoenolpyruvate carboxylase (ZmC4-PEPC,
GenBankTM accession number X15238), we used the primer
pairs RuBPCssu LCfw (5'-caacaagaagttcgagacg-3'), RuBPCssu LCrev
(5'-cgggtaggatttgatggc-3'), and C4-PEPC LCfw
(5'-ggcttctcttcactcacc-3'), C4-PEPC LCrev
(5'-tccaatgggctgggata-3'), respectively. All quantifications were
normalized to the signal of actin cDNA fragments generated by the
primers ZmAct 81/83fw (5'-acacagtgccaatct-3') and ZmAct 81/83rev
(5'-actgagcacaatgttac-3'), which amplified cDNA from the maize
actins ZmAct 81 (GenBankTM accession number AAB40106) and
ZmAct 83 (GenBankTM accession number AAB40105). The
relative amount of channel cDNA was calculated from the correlation
2(n actin Two-electrode Voltage Clamp Experiments--
For
heterologous expression in Xenopus laevis
oocytes, the cDNAs of KZM1 in pCRII and
ZmSUT1 in pBS SK( Patch Clamp Experiments--
For patch clamp experiments,
devitellinized oocytes were placed in a bath solution containing 100 mM KCl, 2 mM MgCl2, 1 mM CaCl2, and 10 mM Tris/Mes, pH
7.5. Pipettes were filled with solution containing 100 mM
KCl, 2 mM MgCl2, 1 mM
CaCl2, and 10 mM Tris/Mes, pH 7.5. Currents
were recorded in the cell-attached configuration using an EPC-9
amplifier (HEKA, Lambrecht, Germany) as described previously (37).
KZM1 Represents a KAT1-type Shaker K+ Channel
Gene--
To study the role of K+ channels in
C4 leaves, characterized by Kranz anatomy, we isolated
KZM1 from maize leaf cDNA via RT-PCR and RACE
techniques. The cloning strategy took advantage of highly conserved
regions in the Shaker gene family of plant K+
channels (for review see Ref. 2). Sequence analysis of the open reading
frame of the KZM1 cDNA (2274 bp) revealed the basic features of the KAT1 subfamily (Fig.
1A) as follows: six putative transmembrane domains (S1-S6) with a proposed voltage sensor in segment 4 and a K+-selective pore (P), formed by the
amphiphilic linker between S5 and S6 (for structure-function analysis
of plant K+ channels see Refs. 38 and 39). The deduced KZM1
protein spans 758 amino acids with a predicted molecular mass of 86.7 kDa. When compared on the amino acid level to the
Arabidopsis K+ channels of the Shaker
family, KZM1 showed highest similarity to KAT2 (48% identity, Ref. 13)
and KAT1 (47% identity, Ref. 3), whereas the identity to the
previously identified maize K+ channels ZMK1 and ZMK2 (23)
was only 36 and 34%, respectively. Thus, KZM1 represents a member of
the KAT1 subfamily of plant K+ channels (Fig.
1B). The proposed cytoplasmic C terminus of KZM1 contains a
region, which shares structural homologies to cyclic nucleotide binding
domains (Fig. 1A). In contrast to ZMK2 (five ankyrin
repeats), the sequence of KZM1 did not contain an ankyrin binding
domain (Fig. 1A), a feature that is conserved among
K+ channels of the KAT1 subfamily (1), beside the SIRK
protein from Vitis vinifera (22) and KPT1
from Populus tremula (GenBankTM
accession number AJ344623, for details see "Discussion"). In the
5'-region of the open reading frame of KZM1, we could
identify two possible translational start positions ("ATG"), a
structural element also found with the Arabidopsis ortholog
KAT2 (Fig. 1A). In addition, plant-specific
hydrophobic and acidic C-terminal domains, involved in plant
K+ channel clustering (40, 45), could be identified. Based
on Southern blot analysis with maize DNA, we characterized
KZM1 as a single copy gene within the maize genome (not
shown). Besides KZM1 we could also identify
KZM2,3 the second
maize member of the KAT1 subfamily (Fig. 1B), most likely
representing the ortholog to KAT1 from
Arabidopsis.
KZM1 Is a Voltage-dependent K+-uptake
Channel--
When expressed in Xenopus oocytes, the gene
product of KZM1 showed the characteristic properties of a
voltage-dependent, inward-rectifying plant K+
channel (Fig. 2). In two-electrode
voltage clamp experiments, KZM1 activated upon hyperpolarization to
membrane potentials negative to
In agreement with a K+-selective channel, K+
currents through KZM1 increased as a function of the external
K+ concentration (not shown) with the current reversal
potential following the Nernst potential for potassium (60.8 ± 4.7 mV per 10-fold change in external K+ concentration,
Fig. 3A). Replacing
K+ by Rb+ (100 mM) caused a drop in
the inward current (at
KAT1-like channels are stimulated by external acidification (13, 22,
47). When we analyzed the sensitivity of KZM1 to extracellular pH
changes in Xenopus oocytes, however, the relative open
probabilities (Po), obtained at different
external proton concentrations, rendered KZM1 pH-insensitive (Fig.
3D). Thus, KZM1 seems to represent the first KAT1-type
K+ channel not affected by external pH changes (for details
see "Discussion"). In contrast, K+ currents through
KZM1 increased upon cytoplasmic acidification of the oocyte in response
to 10 mM sodium acetate, pH 5.6 in the bath solution (Fig.
3E, compare with Refs. 18 and 38). As shown in Fig.
3F, the cytosolic acid activation of KZM1 results from a
shift of the half-maximal activation voltage U1/2 ( KZM1 Is Expressed in Vascular Strands and Epidermis of the Maize
Leaf--
To identify KZM1-expressing tissues, mRNA
from different organs of the maize plant was isolated. In Northern
analyses KZM1 transcripts were found in developing
(1-week-old) and mature (5-week-old) leaves (not shown). High
transcript levels were also detected in nodes, husks, and silks,
whereas KZM1 mRNA was rare in internodes and not
detectable in young cobs and developing tassels (not shown). To study
KZM1 expression within the C4 leaf in more
detail, we fractionated the last fully developed leaf of 5-week-old
maize plants into epidermal tissue, mesophyll protoplasts, and vascular strands (Fig. 4A). During
enzymatic digestion, the bundle sheath cells remained attached to the
vascular strands (compare with Ref. 34). In the following, the latter
fraction will be addressed as "vascular/bundle sheath strands." To
estimate contaminations of this fraction, the small subunit of the
ribulose-1,5-bisphosphate carboxylase (ZmRuBPCssu), specifically
expressed in maize bundle sheath cells, was used as marker for
vascular/bundle sheath strands. In addition the C4 form of
the phosphoenolpyruvate carboxylase (ZmC4-PEPC) served as
marker for mesophyll cells (compare with Refs. 25 and 48). By using
quantitative real-time RT-PCR to determine the transcript density of
those two marker genes, we could show that the epidermis and mesophyll
protoplast fractions were contaminated by less than 1% of the
vascular/bundle sheath RuBPCsu transcripts (Fig. 4B). In
contrast, 35 and 40% of the mesophyll-specific C4-PEPC
were detected in epidermis and vascular/bundle sheath strands, showing
that these tissue preparations contained residual mesophyll fractions,
probably from protoplasts still attached to the epidermal strips or
vascular strands (for separation of mesophyll and bundle sheath cells
compare Refs. 49 and 50). We cannot, however, exclude that the
C4 form of PEPC is expressed in guard cells and/or
subsidiary cells as well (for discussion see Refs. 51 and 52).
Quantitative real-time RT-PCR on mRNA from the three different
samples demonstrated that KZM1 expression in the maize leaf is restricted to vascular/bundle sheath strands and the epidermis (Fig.
4B). The transcripts were about 13 times more abundant in the vascular/bundle sheath strands than in the epidermis. In mesophyll protoplasts the KZM1 mRNA level was at the detection
limit of the real-time RT-PCR method. Upon peeling of the epidermis,
common epidermal cells rupture, whereas viable guard and subsidiary
cells, visualized by neutral red staining (Fig. 4A,
inset, compare with Ref. 53), survive this mechanical
treatment. Thus, KZM1 expression in the maize leaf is
restricted to guard/subsidiary cells and the phloem-enriched
vascular/bundle sheath strands. As a phloem marker we used the
K+ channel gene ZMK2 (23, 24), which was
detected in vascular bundles only (not shown, compare for phloem
localization of AKT2/3 (16, 18, 19)). These results suggest that
KZM1 displays an expression pattern similar to its
Arabidopsis ortholog KAT2 (phloem tissue and
guard cells (13), for differences see "Discussion").
Expression of KZM1 during Development, Sink-Source Transitions, and
Diurnal Changes--
To explore the role of KZM1 in phloem
physiology, we followed the expression of this K+ channel
gene along different developmental stages of the primary leaf and the
4th leaf of the maize plant (Fig. 5).
Here the juvenile, just emerging organs of the leaves represent
carbohydrate sinks (54), whereas the mature leaves serve as source
tissue. In contrast to the broad pattern of KZM1 expression,
transcripts of the phloem K+ channel gene ZMK2
were restricted to sink tissues such as young leaves (Fig. 5) and
coleoptiles (23, 24). The sucrose transporter gene ZmSUT1
(31), however, was prominent in the RNA fraction isolated from mature
source leaves (Fig. 5). Because ZmSUT1 expressed in oocytes
mediates proton-coupled sucrose uptake (compare Fig. 8), this may
indicate a source-specific function of ZmSUT1 for sucrose loading into
the phloem.
The tip region of the maize leaf contains the oldest cells and mostly
minor veins, in which thin-walled sieve tubes in combination with
companion cells serve as the source site of apoplastic phloem loading
(25, 29). In contrast, the leaf base is dominated by young cells and
thick vascular bundles. In young leaves the latter are involved in
phloem unloading (sink) to support growth in this expanding leaf zone,
and in mature leaves they mediate long distance transport of
carbohydrates in the thin-walled sieve tubes (compare with Refs. 28 and
54). In contrast to the leaf blade, characterized by sucrose production
and phloem loading in a C4-specific manner, the sheath of
the maize leaf does not show the C4 intrinsic Kranz anatomy
(55). Here the sheath contains mostly large vascular bundles with
thin-walled sieve tubes, considered to mediate transport of
photosynthates out of the leaf. Moreover, veins of the leaf blade
contain thick-walled sieve tubes, not present in the leaf sheath (55).
Thick-walled sieve tubes in combination with vascular parenchyma cells
are thought to mediate retrieval of solutes leaking out of the xylem
vessels (56, 57). Northern blot analyses with mRNA extracted from 6 different zones of a 20-day-old maize leaf blade showed KZM1
to be evenly expressed from tip to base. With leaf sheath mRNA,
however, no signal was obtained (Fig. 6),
pointing to a role for KZM1 in K+ retrieval from xylem
vessels of the leaf blade via phloem parenchyma and thick-walled sieve
tubes. As noticed for leaf development before (Fig. 5), ZMK2
was predominantly expressed in the sink and transport regions of the
leaf, represented by the leaf base and sheath (Fig. 6). In addition
ZMK2 mRNA was seen in the very tip. As was shown by Aoki
et al. (31), transcripts of the source-specific sucrose
transporter ZmSUT1 in expanding leaf blades increase from the unexpanded base (sink) toward the expanded tip region (source).
During the day, ZmSUT1 transcript levels in the leaf
increase, reach a maximum at the end of the light period, and decrease during the night (31). Thus, the expression pattern of this sugar
transporter correlates with carbohydrate synthesis and phloem loading.
In line with a constitutively active gene, KZM1 expression in the leaf blade was not affected by diurnal changes (Fig.
7). In contrast, ZMK2
transcripts accumulated during the dark period, again pointing to an
important function of this phloem K+ channel in sink
control.
These studies show that KZM1 expression in contrast to the
source- (ZmSUT1) and sink-specific (ZMK2) genes
is not affected by leaf development, sink-source transitions, or
diurnal changes. However, expression of KZM1 is clearly
associated with K+ retrieval by phloem parenchyma cells of
the leaf blade and not with long distance transport of photosynthates
in the leaf sheath or internodes. Based on this expression pattern, one
would conclude that KZM1 has a housekeeping function in the vascular
bundles of the maize leaf, controlling K+ retrieval into
the phloem and K+ homeostasis.
Cooperation of Phloem K+ Channels and Sucrose
Transporters--
To gain insight into the feedback control between
K+ channels and sucrose transporters in the phloem of the
maize leaf, we studied the functional properties of the sucrose
transporter ZmSUT1 alone and in the presence of either KZM1 or ZMK2 in
Xenopus oocytes (Fig. 8). When
the phloem-specific sucrose/H+ co-transporter ZmSUT1 (31)
was expressed in Xenopus oocytes, a sucrose-induced
depolarization of the membrane was monitored. Properties like the
sucrose specificity of ZmSUT1, as well as its concentration and pH
dependence (not shown), were well in agreement with studies on the
phloem sucrose/H+ symporter ortholog AtSUC2 from
Arabidopsis (17). However, when oocytes expressing both the
voltage-independent phloem K+ channel ZMK2 (23) and
ZmSUT1 were challenged with sucrose, the drop in membrane potential was
prevented (Fig. 8). This indicates that ZMK2, because of its peculiar
kinetics and voltage dependence (compare Refs. 23 and 24), stabilizes
the membrane potential in the presence of the sucrose-fueled
sugar/H+ symporter. In vivo this phloem
K+ channel thus very likely repolarizes the membrane rather
than catalyzing the bulk flow of potassium, a function of which KZM1 is
capable (see below). In contrast to ZMK2, KZM1 is activated at
hyperpolarizing potentials only (compare Fig. 2). Coexpression of the
sucrose/H+ symporter with the inward rectifier KZM1
therefore did not prevent the sucrose-dependent
depolarization of the membrane potential. Thus, voltage-independent,
proton-blocked K+ channels like ZMK2, which clamp the
membrane to the Nernst potential for K+, during phloem
loading and unloading processes interact with the sucrose transporter
ZmSUT1 via the membrane potential and extracellular pH. The
voltage-dependent but pH-insensitive inward rectifier KZM1
is able to maintain the K+ homeostasis of the leaf phloem.
Under physiological conditions the phloem H+-ATPase at the
source site often hyperpolarizes the membrane negative to In this study we have focused on KZM1, a new
K+ channel gene from Z. mays, belonging to the
Shaker family of plant K+ channels.
KZM1 displays structural, functional, and expression patterns reminiscent of the KAT2 K+ channel from
Arabidopsis. Thus, KZM1 represents the first KAT-type K+ channel isolated from a C4 species. In
Arabidopsis the KAT1 subfamily consists of two members, KAT1
and KAT2 (compare with Fig. 1). With the isolation of
KZM2,3 another KAT-type K+ channel
gene from maize, most likely representing the orthologous gene to
KAT1, the genome of Z. mays also seems to harbor
two KAT1 subfamily members.
The KZM1 protein is characterized by the absence of an ankyrin repeat
domain, a feature that was assigned to be specific for the KAT1-type
Shaker K+ channels (1). Only very recently it
was reported that SIRK, a KAT-like K+ channel from
grapevine (V. vinifera), contains an
ankyrin repeat (22). Because this ankyrin repeat is truncated (one
repeat and two half-motifs) compared with complete ankyrin binding
domains of the AKT1-, AKT2/3-, and SKOR-like channels (five to six
repeats), it was concluded that this atypical feature of SIRK gives
insight into the evolution of the plant Shaker
K+ channel family. The KAT-type channel KPT1 from poplar
trees (P. tremula) contains such a truncated ankyrin
domain as well,4 indicating
that the KAT1 subfamily of woody species may be identified by this
motif. KZM1 from the monocot Z. mays, however, is more closely related to KAT1, KAT2 from Arabidopsis,
and KST1 from Solanum tuberosum, all characterized by the
absence of ankyrin repeat motifs. Like KAT2 (13) and
AKT2 (18), KZM1 contains two possible
translational start positions within the 5'-region of the open reading
frame. The cRNA including both methionines was functional in
Xenopus oocytes. Future experiments similar to
those performed with AKT2 will address the role of the
second possible translational start position (compare with Ref.
18).
When expressed in Xenopus oocytes, KZM1 showed the
characteristic properties of a voltage-dependent,
inward-rectifying plant K+ channel. Differences between the
monocotyledonous KZM1 and the dicotyledonous KAT-type K+
channels KAT1, KAT2, KST1, and SIRK could be assigned to the higher
single channel conductance of KZM1 (20 pS) and the fact that the gating
of KZM1 required potassium. The latter property could recently be
recognized for AKT3, another phloem K+ channel (for
discussion see Ref. 39). These channels, sensing the K+
concentration in the sink and source and along the transport phloem,
are able to control K+ release into growing tissue and
resorption from mature or even senescing leaves.
Another unique feature of KZM1 is displayed by its insensitivity to
external pH changes. All previously characterized KAT1-like K+ channels are activated by an increase in the
extracellular proton concentration (13, 22, 38), whereas channels from
the AKT2/3 and SKOR subfamilies are proton blocked (19, 21, 58). Thus, structural elements of KZM1 might provide a new molecular tool for
future structure-function experiments to access the external pH sensor
of plant K+ channels. This proton sensor of the potato
guard cell K+ channel KST1 could be assigned to two
extracellular histidine residues in the outer pore (His-271) and
between the linker of the transmembrane helices S3 and S4 (His-160) of
the protein (36). The histidine of the outer pore is unique to all
plant K+ channels. However, KZM1 lacks the histidine
residue in the linker between S3 and S4 but possesses a more positive
charged arginine at this site. When the adequate mutation H160R was
introduced into KST1, the protein lost its pH sensitivity almost
completely (36). Therefore, it is tempting to speculate that the
absence of histidine residue 160 might contribute to the insensitivity of KZM1 to external pH changes. In contrast, we found the histidine of
the internal pH sensor, originally identified for the
Arabidopsis KAT1 (59), located in the intracellular loop
between the transmembrane helices S2 and S3 of KZM1, very likely
responsible for the sensitivity of KZM1 to internal protons. The
insensitivity of KZM1 toward changes in the extracellular proton
concentration clearly distinguishes this channel from its
Arabidopsis ortholog KAT2 and therefore might reflect a
feature required to operate a monocotyledonous plant like maize,
characterized by Kranz anatomy and C4-acid metabolism (see below).
By quantitative real-time RT-PCR analysis, we could identify guard
cells, subsidiary cells, and vascular/bundle sheath strands as sites of
KZM1 expression. By using the patch clamp technique, KZM1-like, inward-rectifying K+ channels have been
identified in maize guard cells (60, 61) and subsidiary cells (53).
Thus KZM1, together with KZM2,3 seems to carry a major part
of the inward K+ current in Z. mays guard cells
and subsidiary cells during stomatal movement (compare Refs. 12 and
13). Likewise in bundle sheath cells, KZM1 might contribute to
the inward K+ currents recorded by Keunecke and
Hansen (34) and Keunecke et al. (62).
Because KZM1 expression was most pronounced in vascular/bundle sheath
strands, we investigated the role of this channel protein for
carbohydrate export and import during leaf development, sink-source transitions, and diurnal changes. Sugar transport from the bundle sheath cells of the C4 plant Z. mays to the
phloem parenchyma cells occurs symplastically (26-28), whereas the
subsequent loading to the thin-walled sieve element-companion cell
complex is believed to involve an apoplastic step (29, 30, 56,
57). Aoki et al. (31) provided evidence for a role of
the sucrose transporter ZmSUT1 during phloem loading of carbohydrates
exported from source leaf blades. This hypothesis is supported by
our finding that ZmSUT1 encodes a
H+/sucrose cotransporter, and its expression is
restricted to source tissues (e.g. mature leaves). In
contrast, the K+ channel gene ZMK2 displayed an
inverse expression pattern pointing to a more sink-specific function.
The phloem K+ channel ZMK2 belongs to the AKT2/3-type
subfamily of Shaker K+ channels (compare Fig.
1B). In Arabidopsis, AKT2/3 is
expressed predominantly in source organs and plays a role in sugar
loading of the phloem (16, 17). In contrast to
AKT2/3 but in line with ZMK2, their
ortholog VFK1 from V. faba is found in sink
tissues and during transition from source to sink and therefore related to phloem unloading (32). However, we localized ZMK2
expression also in the tip of the leaf (source), indicating that ZMK2
can be involved in phloem loading as well, as discussed for AKT2/3-like channels (18, 32).
The expression of KZM1 in the maize leave was highest in the
vascular/bundle sheath. In contrast to ZmSUT1 or
ZMK2 this gene was constitutively expressed during leaf
development, sink-source transitions, and diurnal changes. The tip of
the maize leaf, responsible for loading of sucrose to the phloem,
alkalinizes the apoplast, whereas the expansion growth of young cells
in the leaf base results in an acidification of the extracellular
medium (63). Moreover during phloem unloading in sink tissues, the
apoplastic pH increases, activating K+ channels like ZMK2
to control the membrane potential (for discussion see Ref. 32). In such
an environment, the K+-uptake channel KZM1 is insensitive
to external pH changes, providing a mechanism that is robust to
sink-source changes, day-night cycles, and even development and thus
maintains phloem K+ uptake and homeostasis. Testing the
potential feedback loops between the maize phloem K+
channels and the sucrose/H+ symporter ZmSUT1 in
Xenopus oocytes, we could show that the voltage-independent ZMK2 prevents a collapse of membrane potential during
H+/sucrose transport via ZmSUT1. In contrast, KZM1 in the
presence of ZmSUT1 is able to mediate the bulk flow of potassium into
the maize phloem. At hyperpolarized potentials and acidic apoplast following enhanced H+-ATPase activity, KZM1 is able to
substitute ZMK2 in control of phloem potential. In addition,
KZM1 gene expression is linked to K+ retrieval
from xylem vessels in the leaf blade and not to long distance transport
in the leaf sheath or stem internodes. The orthologous K+
channel gene KAT2 in Arabidopsis shows a similar
expression pattern as KZM1 but is characterized by distinct
functional properties such as activation by external protons,
K+-independent gating, and a smaller single channel
conductance (13). Up to now data on the regulation and phloem function
of the KAT2 gene are lacking. Here we could show that KZM1
in contrast is equipped with unique functional characteristics
(K+-dependent gating, insensitivity to external
pH, and high single channel conductance) and a specific expression
pattern in the leaf phloem, all pointing to a housekeeping function of
KZM1 for K+ homeostasis in the phloem of a C4
leaf and K+ transport required for the related organic
acid-based metabolism.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
2·s
1 (LiCOR Quantum
Sensor LI-250, Walz GmbH, Effeltrich, Germany). After
harvesting, all maize tissues were stored in liquid nitrogen prior to
RNA extraction. The age of plants or organs is denoted in days after sowing.
-32P]dATP end-labeled oligo(dT) probe as described
(23).
1, and frozen in
liquid nitrogen for mRNA extraction. To test the vitality of
epidermal cells before freezing, an aliquot of the epidermal fraction
was stained with neutral red (see Fig. 4). To isolate mesophyll
protoplasts, the remaining leaf sections were incubated for 90 min at
30 °C in enzyme solution containing 1.5% cellulase (Cellulase R-10,
Yakult Honsha Co., Tokyo, Japan), 2% pektinase (Sigma), 10 mM KCl, 10 mM Mes/KOH, pH 6.2, adjusted with
D-sorbitol to 480 mosmol kg
1. The digestion
was stopped before the bundle sheath cells were released from the
vascular strands. Isolated vascular/bundle sheath strands were pooled
and frozen in liquid nitrogen. Mesophyll protoplasts were sedimented
at 60 × g for 5 min at 4 °C and frozen in liquid nitrogen.
n
channel) with n = threshold cycle of the
respective PCR product. To identify contaminating genomic DNA, the
primers for ZMK2 were selected to flank an intron.
) were subcloned as XhoI/SpeI and BamHI/XhoI
fragments, respectively, into the pGEMHE vector (35). Expression of
ZMK2 in oocytes was performed as described (23). The
respective cRNA was generated by in vitro transcription
(T7-Megascript kit, Ambion Inc., Austin, TX) and injected into
Xenopus oocytes (CRBM, CNRS, Montpellier, France) using a
PicospritzerII microinjector (General Valve, Fairfield, NJ). Two to 6 days following injection, double-electrode voltage clamp recordings
were performed with a Turbotec-01C amplifier (NPI Instruments, Tamm,
Germany). The electrodes were filled with 3 M KCl and had
typical input resistance of about 2-4 megaohm. Solutions were composed
of 30 mM KCl, 2 mM MgCl2, 1 mM CaCl2, and 10 mM
Tris/Mes, pH 7.5, 10 mM Mes/Tris, pH 5.6, and 10 mM citrate/Tris, pH 4.5, respectively. Acidification of the
cytosolic pH was accomplished by perfusion with 30 mM KCl,
2 mM MgCl2, 1 mM CaCl2
and 10 mM Mes/Tris, pH 5.6, as well as 10 mM
NaAc. The control solution contained 10 mM NaCl instead of
NaAc. When recording KZM1-mediated currents at 100, 30, 10, and 3 mM external K+ concentrations, the ionic
strength was adjusted with Na+. In K+-free
solutions, K+ was substituted with Na+ or
Li+ as indicated. All media were adjusted to a final
osmolality of 215-235 mosmol kg
1 with
D-sorbitol. Analyses of voltage and pH dependence were
performed as described previously (19, 36). Membrane potential
measurements with ZmSUT1-expressing oocytes and coexpression of ZmSUT1
with KZM1 and ZMK2 were performed as described for AtSUC2, KAT2, and AKT2/3 in Deeken et al. (17).
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
KZM1 belongs to the KAT1 subfamily of plant
Shaker potassium channels. A, sequence
comparison of Shaker K+ channels from maize and
Arabidopis. Alignment of the deduced amino acid sequences of
KZM1 cDNA (GenBankTM accession number
AJ421640) with the K+ channels KAT2 from
Arabidopis and ZMK2 from maize is shown. The start and stop
codons of KZM1 were used as end positions of the alignment. Amino acids
identical in all 3 channel proteins are shown as black-boxed
letters, and residues conserved in 2 sequences are shown as
gray-boxed letters. The predicted transmembrane regions (S1
to S6) and the pore region (P) are marked with solid
lines. The C-terminal region of all 3 channels contains a
conserved cyclic nucleotide binding motive (cNMP,
dashed line), whereas only ZMK2 exhibits a putative ankyrin
binding domain (ANK, asterisks). Blocks denote
hydrophobic (KH) and acidic (KA)
core sequences according to Ref. 40. The alignment was generated using
ClustalX (41) and GeneDoc 2.0 (42), and protein domains were identified
with InterPro (43). B, phylogenetic tree, demonstrating that
KZM1 is a member of the KAT1-type plant K+ channel
subfamily. The 9 Shaker-type K+ channels from
A. thaliana KAT1 (M86990), KAT2 (AJ288900), AKT1 (X62907),
SPIK (AJ309323), AKT5 (AJ249479), AtKC1 (Z83202), AKT2/3 (AJ243703,
U44745), SKOR (AJ223357), and GORK (AJ279009) group into 5 subfamilies
(highlighted by gray backgrounds). A. thaliana
members, which named the subfamilies, are underlined.
Whereas ZMK1 (Y07632) from maize is similar to AKT1 and ZMK2 (AJ132686)
is the ortholog to AKT2/3, KZM1 and KZM23 belong to the
KAT1-type subfamily. GenBankTM accession numbers of the
respective channels are shown in parentheses. The alignment
was generated using ClustalX (41); the tree was drawn with TreeView
(44).
60 mV (Fig. 2A). The
steady-state current-voltage curve of the data shown in Fig.
2A underlines the strong inward rectification of KZM1 (Fig.
2B). From activation curve analyses, a half-maximal activation voltage U1/2 of
105.4 ± 6.9 mV
(n = 5) was calculated. Recordings in the cell-attached patch clamp configuration allowed us to resolve single KZM1
channel-fluctuations (Fig. 2C). The channel amplitude and
time-dependent activity increased with increasing negative
voltages. From the current-voltage relationship of the single channels
(Fig. 2D), a unitary conductance of 20 ± 0.7 pS
(n = 3, mean ± S.E. with 100 mM
K+ in the pipette) was deduced. Thus, KZM1 exhibits a
2-4-fold higher conductance than the previously characterized
K+ channels of the KAT1 subfamily (6.7 pS for KAT2 (13), 5 pS for KAT1 (37), 7 pS for KST1 (46), and 13 pS for SIRK (22)).
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Fig. 2.
KZM1 is an inward-rectifying K+
channel. A and B, two-electrode voltage
clamp recordings. A, representative macroscopic recordings
of inward currents recorded from KZM1-injected
Xenopus oocytes. Inward currents of KZM1 were elicited in
response to 2-s voltage pulses from +40 to 180 mV (
20-mV steps)
from a holding potential of
50 mV. The bath solution was composed of
30 mM KCl, 1 mM CaCl2, 2 mM MgCl2, and 10 mM Tris/Mes, pH
7.5. B, steady-state currents (ISS) at the end
of the voltage pulses were normalized to ISS (
150 mV) and
plotted against the membrane voltage (U) as mean ± S.D. (n = 6). C and D, patch
clamp recordings. C, single channel fluctuations recorded at
50,
75, and
100 mV in the cell-attached patch clamp
configuration. Closed (c) and open channel states
(o1 and o2) are
indicated. D, single channel current-voltage relationship.
Linear regression on 3 different patches (circles,
squares, and triangles) reveal the single channel
conductance of 20 ± 0.7 pS (mean ± S.E., n = 3).
150 mV,
IRb+/IK+ = 0.190 ± 0.041, n = 4). Comparison of the reversal potential in either
K+ or Rb+ solutions allowed us to
determine the permeability ratio
PRb/PK = 0.437 ± 0.085, n = 4. In contrast to other KAT1-like channels, the permeability ratios for Na+ and Li+ ions
could not be determined, because KZM1 did not even conduct outward currents in Na+- or Li+-based media
(Fig. 3, B and C). Similar results were obtained with N-methyl-D-glucamine solution, pointing to
gating properties shared with the AKT3 channel (compare with Ref.
39).
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Fig. 3.
KZM1 is K+-selective and
independent on external pH. A, shift in reversal
potential (Urev) in response to changes in
extracellular K+ concentration. In tail-current
experiments, KZM1-expressing oocytes were challenged with an
activating prepulse to 150 mV. In subsequent voltage jumps to
potentials ranging from +40 to
180 mV, tail currents were elicited
that reversed direction (Urev) around the
predicted Nernst potential for K+. Changing the
K+ concentration 10-fold caused a shift in
Urev of 60.8 ± 4.7 mV. Error
bars indicate S.D. (n
4). B,
tail-current recordings of KZM1-injected oocytes at 20 mV after a 1-s
preactivating pulse to
130 mV in 100 mM K+ or
Li+, respectively. The bath solution was composed of 100 mM KCl/LiCl, 1 mM CaCl2, 2 mM MgCl2, and 10 mM Tris/Mes, pH
7.5. Note that the outward currents in Li+ decay, although
the driving force for K+ release increases. C,
relative instantaneous tail-current amplitudes (rel.
IT) plotted against the membrane voltage (U)
revealed outward currents positive from the reversal potential in 100 mM K+ (closed circles), but not in
100 mM Li+ or Na+ (closed
diamonds and open circles, respectively).
KZM1-expressing oocytes were challenged with an activating prepulse to
130 mV. In subsequent voltage jumps to potentials from +30 to
150
mV in 10-mV decrements, relative instantaneous tail-current amplitudes
were measured at t = 0. IT was normalized to
the values in 100 mM K+ at
140 mV. Results
represent mean ± S.D., n = 6. D,
Boltzmann analysis of voltage-dependent gating at various
external pH values (pH 7.5, 5.6, and 4.5, open and
closed circles and squares, respectively)
normalized to the maximal conductance of 1.0 obtained by Boltzmann
fittings. The relative open probabilities (rel. Po)
were plotted against the membrane voltage (U). Solid
lines represent best Boltzmann fits to the data (gating
parameters: half-maximal activation voltage U1/2,
gating charge Z: pH 7.5: U1/2 =
105.4 ± 6.9 mV, Z = 1.17 ± 0.12; pH 5.6: U1/2 =
108.8 ± 11.4 mV, Z = 1.05 ± 0.13; pH 4.5:
U1/2 =
110.1 ± 10.1 mV, Z = 0.97 ± 0.07). Error bars indicate S.D. (n
5).
pH changes had no effect on the gating of KZM1 channels. E,
lowering the internal proton concentration by perfusion with 10 mM NaAc at pH 5.6 (+NaAc) the inward currents,
elicited by 2-s voltage pulses to
160 mV, increased with respect to
those in the absence of acetate (
NaAc). F, the
voltage-dependent gating in response to internal
acidification was analyzed with a Boltzmann function as described in
B (gating parameters: half-maximal activation voltage
U1/2, gating charge Z: +NaAc (open
circles): U1/2 =
84.8 ± 3.5 mV, Z = 1.08 ± 0.17;
NaAc (closed circles):
U1/2 =
121.6 ± 7.1 mV, Z = 1.22 ± 0.22). Error bars indicate S.D. (n = 3).
KZM1 is activated by internal acidification due to a positive-going
shift of the half-maximal activation voltage (36.9 ± 10.3 mV).
U1/2 = 36.9 ± 10.3 mV, n = 3) toward more positive values.
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Fig. 4.
KZM1 transcripts in the leaf originate
from vascular strands and epidermis. A, photographs of
fractionated maize leaf tissues and cells. Top,
cross-section through a 5-week-old leaf. Epidermal, mesophyll, and
vascular/bundle sheath sections are indicated by solid
lines. Left, epidermal strips, excised from maize
leaves. Inset, neutral red viability stain of guard and
subsidiary cells. Middle, mesophyll protoplast
suspension. Inset, single mesophyll protoplast at higher
magnification. Right, vascular strands with bundle sheath
cells attached. B, quantitative real-time RT-PCR analysis on
mRNA from tissues of the last fully developed leaf from 5-week-old
maize plants. Top, quantification of ZmRuBPCssu and
ZmC4-PEPC transcripts relative to actin (n = 3) in the tissue fractions shown in A. The transcript
content of ZmRuBPCssu and ZmC4-PEPC was set to 100% in
vascular/bundle sheath strands and mesophyll protoplasts, respectively.
Bottom, quantification of KZM1 transcripts
relative to actin (n = 6, mean ± S.E.) in
epidermis, mesophyll protoplasts, and vascular/bundle sheath strands as
shown in A. The transcript content measured in a total leaf
fraction was set to 1.0 (arbitrary units).
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Fig. 5.
KZM1 is constitutively expressed during leaf
development. Northern blot with 0.7 µg mRNA, isolated from young
(7 days (7d)) and mature (11 days (11d)) maize
primary leaves (left) and from the 4th leaf of the maize
plant (right) at juvenile (15 days (15d)),
intermediate (19 days (19d)), and mature stage (28 days
(28d)). Young leaves were just emerging from the plant (sink
tissue), and the mature leaves were characterized by a visible leaf
collar (source tissue). The RNA was blotted against radiolabeled
cDNA probes of KZM1, ZMK2, and
ZmSUT1 (GenBankTM accession number AB008464). To
standardize mRNA levels, 15 ng of dotted mRNA were hybridized
against a radiolabeled oligo(dT) probe (lower panel).
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Fig. 6.
KZM1 is evenly expressed in the leaf
blade. Northern blot with 0.7 µg mRNA from the 4th leaf of the
maize plant (20 days), dissected into 5 zones (10 cm each) from tip to
base, and the leaf sheath (sh, upper panel). The RNA was
blotted against radiolabeled cDNA probes of KZM1 and
ZMK2, and 15 ng of dotted mRNA were standardized with a
radiolabeled oligo(dT) probe (lower panel).
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Fig. 7.
KZM1 is constitutively expressed throughout
the day-night cycle. Northern blot with 0.7 µg mRNA from the 4th
leaf of the maize plant (33 days) at different times of a 16-h day
(25 °C, white bar) and 8-h night (18 °C, dark
bar) cycle. The light period lasted from 6 a.m. to 10 p.m. Leaf samples were collected at 9 time points as indicated. The RNA
was hybridized against radiolabeled cDNA probes of KZM1
and ZMK2, and 15 ng of dotted mRNA were standardized
with a radiolabeled oligo(dT) probe (lower panel).
80 mV.
Membrane polarization in the long run is accompanied by a drop in
external pH. The latter effect will inhibit ZMK2, but not KZM1 which is
activated negative to
60 mV (compare Fig. 2A). Thus, the
inward rectifier provides for K+ uptake as well as for a
membrane control unit at more hyperpolarized potentials. With the
voltage-independent, H+-blocked ZMK2 and the
voltage-dependent, H+-insensitive KZM1 working
hand in hand, membrane potential and K+ homeostasis can be
controlled over a broad voltage and pH range.
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Fig. 8.
Coexpression of ZmSUT1 with KZM1 and ZMK2 in
Xenopus oocytes. Membrane potential
(VM) measurements with ZmSUT1
injected and ZmSUT1/KZM1,
ZmSUT1/ZMK2 coinjected oocytes, respectively.
The external solution was composed of 30 mM KCl, 1 mM CaCl2, 1.5 mM MgCl2,
and 10 mM Mes/Tris, pH 5.6. The arrow indicates
the start of perfusion with 10 mM sucrose. ZmSUT1 mediates
a sucrose-dependent depolarization of the membrane
potential (upper trace), whereas ZmSUT1 in the presence of
ZMK2 is not able to collapse the membrane potential (lower
trace). ZMK2 stabilizes the membrane potential by balancing the
H+ influx by K+ efflux. Under these conditions,
the depolarization of the membrane potential through ZmSUT1 is not
prevented by coinjection of ZmSUT1 with the inward rectifier
KZM1.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* This work was supported in part by grants from the Deutsche Forschungsgemeinschaft (to R. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Supported by an EMBO long term fellowship. Present address: Biochimie et Physiologie Moléculaire des Plantes, UMR 5004 Agro-M/CNRS/INRA/UMII, Place Viala, 34060 Montpellier Cedex 1, France.
To whom correspondence should be addressed. Tel.:
49-931- 888-6101; Fax: 49-931-888-6158; E-mail:
hedrich@botanik.uni- wuerzburg.de.
Published, JBC Papers in Press, February 27, 2003, DOI 10.1074/jbc.M212720200
1 S. Scheuermann, unpublished results.
3 K. Büchsenschütz, unpublished results.
4 K. Langer, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: RT, reverse transcriptase; RACE, rapid amplification of cDNA ends; Mes, 2-morpholinoethanesulfonic acid; NaAc, sodium acetate; RuBPCssu, ribulose-1,5-bisphosphate carboxylase small subunit; PEPC, phosphoenolpyruvate carboxylase.
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