From the Department of Biology and Center for Molecular Genetics, University of California, San Diego, La Jolla, California 92093-0116
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ABSTRACT |
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Potassium is an important macronutrient required
for plant growth, whereas sodium (Na+) can be toxic
at high concentrations. The wheat K+ uptake transporter
HKT1 has been shown to function in yeast and oocytes as a high affinity
K+-Na+ cotransporter, and as a low affinity
Na+ transporter at high external Na+. A
previous study showed that point mutations in HKT1, which confer
enhancement of Na+ tolerance to yeast, can be isolated by
genetic selection. Here we report on the isolation of mutations in new
domains of HKT1 showing further large increases in Na+
tolerance. By selection in a Na+ ATPase deletion mutant of
yeast that shows a high Na+ sensitivity, new
HKT1 mutants at positions Gln-270 and Asn-365 were
isolated. Several independent mutations were isolated at the Asn-365
site. N365S dramatically increased Na+ tolerance in yeast
compared with all other HKT1 mutants. Cation uptake
experiments in yeast and biophysical characterization in Xenopus oocytes showed that the mechanisms underlying the
Na+ tolerance conferred by the N365S mutant were: reduced
inhibition of high affinity Rb+ (K+) uptake at
high Na+ concentrations, reduced low affinity
Na+ uptake, and reduced Na+ to K+
content ratios in yeast. In addition, the N365S mutant could be clearly
distinguished from less Na+-tolerant HKT1
mutants by a markedly decreased relative permeability for
Na+ at high Na+ concentrations. The new
mutations contribute to the identification of new functional domains
and an amino acid in a loop domain that is involved in cation
specificity of a plant high affinity K+ transporter and
will be valuable for molecular analyses of Na+ transport
mechanisms and stress in plants.
Potassium is an important macronutrient required for plant growth.
K+ uptake into plant root cells is generally thought to be
mediated by transport systems with high and low affinities for
K+ (1-5). A cDNA named HKT1 was isolated
from wheat roots and was characterized as a member of high affinity
K+ transport systems from plants (6). HKT1 is
highly expressed in the root cortex and in cells surrounding the
vasculature in leaves (6) and shows homology to the yeast plasma
membrane high affinity K+ uptake transporters
TRK1 and TRK2 (6, 7). HKT1 has been shown to
mediate high affinity Na+-K+ symport (8),
suggesting that additional high affinity K+ uptake pathways
should exist in roots (9) (see "Discussion").
Detailed biophysical studies showed that HKT1 is highly selective for
the alkali cations K+ and Na+ (9). When the
extracellular concentrations of K+ and Na+ are
similar, HKT1 functions as a Na+-coupled K+
uptake transporter. HKT1 is modelled to have a high affinity K+-coupling site with an apparent affinity for
K+ of 3 µM, and a high affinity
Na+-coupling site with an apparent affinity for
Na+ of 175 µM (8, 9). At toxic, high
millimolar, extracellular Na+ concentrations, however,
Na+ competes with K+ at the high affinity
K+-coupling site, leading to a block of high affinity
K+ uptake; large rates of detrimental low affinity
Na+ uptake are mediated via HKT1 in yeast and oocytes (8,
9). The apparent affinity of the high affinity K+-coupling
site for Na+ is approximately 5 mM (8, 9).
These findings suggest that HKT1 may be one of the pathways for
Na+ transport across plant root and leaf membranes that is
important for salt toxicity in plants.
Characterization of the molecular mechanisms underlying Na+
transport in plants and identifying structural components that allow
Na+ uptake via these transporters are required for a
fundamental understanding of Na+ homeostasis. It has been
proposed that important Na+ entry pathways into plant cells
represent K+ uptake transporters (10, 11), and multiple
Na+ uptake pathways are likely to exist (12, 13).
Salinization of irrigated lands in arid regions is affecting
agriculture world-wide, reducing production because most crop plants
are glycophytes and sensitive to high millimolar NaCl concentrations
that occur in saline soils (14). Plant responses to salt stress are a
complex phenomenon involving several processes such as
osmoprotectant accumulation (14, 15), enzyme sensitivity
to Na+ (16, 17), and ion transport across different
plant membranes (14, 18-20).
Structural studies of the wheat transporter HKT1 led to the
identification of point mutations located in the 6th hydrophobic domain
that decrease low affinity Na+ uptake and confer
Na+ tolerance to yeast (8). Here we report on the isolation
of new Na+-tolerant HKT1 mutations in novel
domains, by using yeast mutants with increased Na+
sensitivity. These new HKT1 mutants cause greatly enhanced
Na+ tolerance in yeast compared with previous mutants. The
mechanisms by which mutants cause Na+ tolerance were
characterized, and mechanistic distinctions among Na+-tolerant HKT1 mutants are revealed. The new
mutations contribute to the identification of a new site in a loop
domain, N365, that is involved in cation specificity of HKT1 and in
HKT1-mediated Na+ tolerance. These findings contribute to a
molecular physiological understanding of HKT1 structure and function.
Furthermore, highly tolerant HKT1 mutants can be used to
determine contributions of HKT1 to plant Na+
transport in molecular physiological studies.
Yeast Growth--
Saccharomyces cerevisiae strains
CY162 (Mat a, ura3-52, his3 Random HKT1 Mutagenesis and Mutant Selection--
The
HKT1 cDNA cloned in the pYES2 vector (Invitrogen, La
Jolla, CA) was mutagenized randomly by error PCR as described elsewhere (26). Two different sets of DNA fragments generated by error PCR that
covered the coding region of the HKT1 cDNA were
employed: the first reaction produced DNA fragments from position Yeast Uptake Experiments--
The K+
uptake-deficient yeast strain CY162 described above (21) and the same
strain transformed with a plasmid containing the wild type
HKT1 cDNA or the mutated HKT1 cDNA under
the control of the yeast PMA1 gene promoter (8, 28) were
used for uptake experiments. Cells were grown in arginine phosphate
medium (23) supplemented with 30 mM K+. For
Na+ uptake experiments in the low affinity range of
concentrations (1-300 mM Na+), cells were used
directly. For Rb+ and Na+ uptake experiments in
the high affinity range of concentrations (1-300 µM
Rb+ or Na+), cells were previously starved of
K+ for 5-6 h (29). For the uptake experiments, cells were
harvested and resuspended in uptake buffer (10 mM Mes, 0.1 mM MgCl2, 2% glucose brought to pH 6.0 with
Ca(OH)2) and incubated at 30 °C. For high affinity
Rb+ and Na+ uptake experiments,
86Rb+ or 22Na+ (NEN
Life Science Products) were added at 0.05 µCi nmol Internal K+ and Na+ Content Determination
in Yeast--
Yeast cells expressing HKT1 or the
Na+-tolerant HKT1 mutants were incubated in AP medium
supplemented with 0.1 mM K+ and 300 or 500 mM Na+. After 24 h, samples were collected
on filters as described above and the internal K+ and
Na+ contents determined by atomic emission
spectrophotometry. Error bars denote standard error of the mean.
Statistical analyses for uptake experiments and ionic content
determination in yeast were performed with the Instat3 software (GraphPad Software Inc., San Diego, CA) using an unpaired t
test. The significance level was p < 0.05.
Xenopus Oocyte Expression and
Electrophysiology--
HKT1 and HKT1 mutant
mRNA synthesis and injection (20 ng) into oocytes, voltage clamp
recordings with a Dagan Cornerstone TEV-200 voltage clamp amplifier
(Dagan, Minneapolis, MN) one day after mRNA injection, and data
acquisition and analysis with the program Axotape (Axon Instruments,
Foster City, CA) were performed as described previously (6). Data were
low pass filtered at 20 Hz. Oocytes were impaled with electrodes filled
with 1 M KCl and were bathed in a solution containing (in
mM): 6 MgCl2, 1.8 CaCl2, 10 Mes-Tris, pH 5.5, osmolality 240-260 mosmol kg pK+/pNa+ Permeability Ratio
Determinations--
To calculate the
pK+/pNa+ permeability
ratios, the Goldman equation (Eq. 1) for ion channels was employed.
In addition, and to more accurately reflect the
pK+/pNa+ permeability
ratios of just the K+-coupling site for the HKT1 model with
two separate Na+ and K+-coupling sites (8), an
expanded version of the Goldman equation was employed (Eq. 2).
Eq. 2 consists of the addition of two terms that consider the
permeability of HKT1 for the K+-coupling site (m
term) and the permeability of HKT1 for the Na+ coupling
(n term). To solve for
pK+/pNa+ in the expanded
Goldman equation (Eq. 2), the K+ permeability of the high
affinity Na+-coupling site was approximated to be
negligible (9), and the stoichiometries were set to m = 2 and n = 1 (8).
For all equations, Erev is the reversal
potential, R is the gas constant, T is the
absolute temperature, z is the charge of the ion,
F is the Faraday constant, pNa+ is
the relative permeability of the K+-coupling site of HKT1
for Na+, pK+ is the relative
permeability of the K+-coupling site of HKT1 for
K+. p'Na+ and
p'K+ refer to the Na+-coupling site.
The ionic concentrations are given in brackets (o = external, i = internal). The internal K+
and Na+ concentrations were assumed to be 92.5 mM K+ and 6.2 mM Na+ as
has been determined for Xenopus oocytes (30).
Isolation of New Na+ Tolerant HKT1 Mutants--
A
nonbiased random selection approach was used to identify new amino acid
positions in HKT1 that affect Na+ transport. To isolate new
HKT1 mutants that conferred even larger Na+
tolerance than those found previously (8), we used a yeast strain, 9.3, which is more Na+ sensitive than the CY162 strain. The 9.3 yeast strain has a deletion in the four ENA1 to
ENA4 genes that encode plasma membrane
Na+-extruding ATPases (31, 32). In addition, the yeast
K+ uptake transporter genes TRK1 and
TRK2 were deleted in the 9.3 strain. Mutants were isolated
at 0.1 mM K+ where strain 9.3 does not grow
because it lacks the endogenous high affinity K+ uptake
system, thus including selective pressure for maintaining high affinity
K+ uptake in HKT1 mutants, and 400 mM Na+ to select for Na+ tolerance.
The HKT1 cDNAs were randomly mutagenized by error-prone
PCR amplification (see "Experimental Procedures"). By screening
5,000 clones, putative HKT1 mutants were isolated that
showed increased growth in the presence of 0.1 mM
K+ plus 400 mM Na+. Subsequent
plasmid isolation and retransformation of yeast was performed to retest
HKT1 mutants for Na+ tolerance. Sequencing of
the strongest Na+ tolerance-conferring HKT1
mutants showed that five new HKT1 mutants had been isolated
(Table I). Interestingly, four of five
mutants were altered in the Asn-365 residue in independent PCR
reactions, indicating that this residue is important for
Na+ tolerance.
The single base Q270L and N365S HKT1 mutants were analyzed
in detail here, because they conferred the highest degree of
Na+ tolerance (Table I). Fig.
1A shows that at high
Na+, the yeast strain 9.3 expressing HKT1 did
not grow, whereas 9.3 expressing the HKT1 mutants Q270L,
N365S, and the former mutant A240V grew. To quantify the
Na+ tolerance conferred by the two new mutations, the
growth of the 9.3 strain expressing HKT1 and the
Na+-tolerant HKT1 mutants was followed in liquid
culture at several Na+ concentrations. The HKT1
mutant A240V was included for comparison because it was the more
Na+-tolerant of the two previously described
HKT1 mutants (8). At 150 mM Na+, 9.3 cells expressing HKT1 did not grow and 9.3 cells expressing the HKT1 mutants A240V, Q270L, and N365S grew well (Fig.
1B). The control strain 9.3 did not show growth because the
medium contained low K+ and high Na+
concentrations.
When the Na+ concentration was increased to 250 mM Na+, only yeast cells expressing the N365S
mutation showed large growth rates (Fig. 1C; N365S doubling
time 10 ± 0.7 h). Thus, this mutant dramatically increased
Na+ tolerance compared with the previously isolated
Na+-tolerant HKT1 mutant A240V. According to
hydrophobicity analysis of HKT1, the new N365S and Q270L mutations lie
in the loop between the 8th and 9th hydrophobic domains (N365S) and in
the 7th hydrophobic domain (Q270L) (Fig. 1D). These results
provide evidence that HKT1 domains in addition to the 6th hydrophobic
domain are involved in HKT1-mediated Na+ tolerance.
Kinetic Characterization of Rb+ and Na+
Transport in the Na+-tolerant HKT1 Mutants--
To
investigate whether the two new HKT1 mutations Q270L and
N365S affected high affinity transport characteristics under
non-Na+ stress conditions, Rb+ and
Na+ uptake experiments at micromolar Rb+ and
Na+ concentrations were carried out in the yeast strain
CY162 expressing the HKT1 mutants. Micromolar
Na+ concentrations produced a similar activation of high
affinity Rb+ uptake in HKT1 and in the N365S and Q270L
mutants (data not shown). Rb+ uptake was measured in the
presence of 1 mM Na+ for maximal activation of
high affinity Rb+ uptake (8). Fig.
2A shows that CY162 expressing
wild type HKT1 and the mutants Q270L and N365S displayed
approximately similar rates of high affinity Rb+ uptake.
Detailed analysis of uptake kinetics showed a slight 1.5-fold increase
in the affinity for Rb+ in the Na+-tolerant
N365S mutant. The Km (Rb+) values for
Rb+ uptake in the presence of 1 mM
Na+ were 70.2 ± 8 µM for HKT1,
49.8 ± 5 µM for Q270L, and 45.8 ± 9 µM for N365S, and the Vmax (nmol
Rb+ mg
High affinity Na+ uptake experiments were performed in the
presence of 100 µM K+ for maximal activation
of high affinity Na+ uptake (8). The two mutations showed
small effects on high affinity Na+ uptake (Fig.
2B). The Km (µM
Na+) values for Na+ uptake in the presence of
100 µM K+ were 65.3 ± 6.9 µM for HKT1, 36.5 ± 13.3 µM for
Q270L, and 33.2 ± 9.8 µM for N365S, and the
Vmax (nmol mg
An earlier study showed that high Na+ concentrations
inhibited high affinity Rb+ uptake through HKT1 (8). The
effect of millimolar Na+ concentrations on high affinity
Rb+ uptake was investigated for the new HKT1
mutants, Q270L and N365S. Fig.
3A shows the rates of
Rb+ uptake from a 100 µM Rb+
solution in yeast cells expressing HKT1 mutants as a
function of increasing millimolar Na+ concentrations in the
range from 1 to 400 mM Na+. High
Na+ concentrations inhibited high affinity K+
(Rb+) uptake to a lesser extent in cells expressing the
Na+-resistant HKT1 mutants than in cells
expressing wild type HKT1 (Fig. 3A). The three
mutations N365S, Q270L, and A240V showed a similar reduction in the
inhibition of high affinity Rb+ uptake by high
Na+ concentrations. The Na+ concentrations
(mM Na+) that produced 50% inhibition of
Rb+ uptake were 40 ± 4.3 for HKT1, 90 ± 2.6 for
N365S (p = 0.0044), 90 ± 1.7 for Q270L
(p = 0.0054), and 112 ± 2.9 for A240V
(p = 0.0016). These data show that the ability of
Na+ to bind to a modelled high affinity
K+-coupling site (9) is reduced in the HKT1
mutants. However, the 50% inhibition constants show that
Na+ block of K+ uptake alone cannot account for
the increased Na+ tolerance of the N365S mutant with
respect to A240V (Figs. 3A and 1C).
In addition to high affinity Na+-coupled K+
uptake, HKT1 also promotes low affinity Na+ uptake in yeast
and oocytes, with an apparent Km of approximately 5 mM Na+ (8, 9). High and low
affinity components of Na+ uptake mediated by HKT1 could be
clearly separated by Eadie-Hofstee analyses (Fig. 3B). This
is consistent with previous studies showing two distinct transport
mechanisms for high and low affinity Na+ uptake by HKT1 (8,
9). Low affinity Na+ uptake was investigated and kinetic
parameters were compared in the Na+-tolerant mutants. Fig.
3C shows that CY162 cells expressing the more
Na+-tolerant mutants A240V and N365S showed a substantial
reduction in low affinity Na+ uptake compared with the wild
type HKT1. The A240V mutant showed a significant lower
Vmax value and the N365S showed significant lower affinity and lower Vmax of low affinity
Na+ uptake (Table II). The
results described above illustrate that HKT1 mutants become
more Na+ resistant by simultaneously reducing
Na+ inhibition of Rb+ uptake (Fig.
3A) and by reducing low affinity Na+ uptake
(Fig. 3C, Table II). This dual effect of the mutations cannot be explained by a reduction in the expression level of the
mutated transporter in yeast. To further test this hypothesis, we
measured the internal K+ and Na+ contents of
yeast cells expressing HKT1 and the HKT1 mutants after incubation in
media with high Na+ concentrations (Fig.
4). When the yeast cells were incubated for 24 h in medium with 0.1 mM K+ and 500 mM Na+, the internal
Na+/K+ ratios were 6.8 ± 1.4 for HKT1
(n = 6), 2.7 ± 0.4 for A240V (n = 6, p = 0.013), 1.9 ± 0.2 for Q270L
(n = 6, p = 0.0065), and 0.6 ± 0.1 for N365S (n = 6, p = 0.0025). The
most Na+-tolerant mutant N365S showed the lowest
internal Na+/K+ ratio at both 300 and 500 mM Na+ (Fig. 4).
Electrophysiological Characterization of the
Na+-tolerant HKT1 Mutants--
Differences in kinetic
parameters between the two most Na+-tolerant N365S and
A240V mutations were observed as described above. However, a strong
differentiation between the N365S and A240V mutations was not apparent,
except for low affinity Km (Na+) values
(Table II) and Na+ to K+ content ratios in
yeast. The stronger Na+ resistance of the N365S mutant when
compared with the A240V mutant was quantitatively characterized further
by studying biophysical transport characteristics. Yeast uptake
experiments suggest that Na+ toxicity of HKT1 is mediated
by the large rate of low affinity Na+ uptake recorded at
toxic millimolar Na+ concentrations (Fig. 3C)
and by Na+ inhibition of K+ nutrition (Fig.
3A). To allow a more quantitative biophysical characterization of this Na+ uptake component at millimolar
Na+ concentrations in N365S, two electrode voltage-clamp
experiments were performed in Xenopus oocytes expressing
wild type HKT1 and the Na+-tolerant
HKT1 mutants.
We first investigated Na+-coupled K+ currents
induced by increasing external K+ or Na+
concentrations in the range of 1 to 10 mM while maintaining
the other cation at a concentration of 1 mM. Voltage ramps
were applied to oocytes expressing HKT1 or the mutant N365S (Fig.
5). When the external Na+
concentration was maintained at a constant value of 1 mM
Na+ and K+ was increased from 1 to 10 mM K+, no significant differences for the
shifts in the current-voltage relationships were observed for HKT1 and
N365S expressing oocytes (HKT1: n = 4; N365S: n = 5; p = 0.42) (Fig. 5, A and B).
In complementary experiments (Fig. 5, C and D), a
10-fold increase in the external Na+ concentration from 1 to 10 mM Na+ at a constant K+
concentration of 1 mM produced reversal potential shifts of
+23.1 ± 0.9 mV for HKT1 (n = 4) and +24.6 ± 2.6 mV for N365S (n = 4). This indicated that the N365S
mutation did not greatly affect Na+-coupled K+
uptake mediated by HKT1, which was also deduced from uptake studies in
yeast at micromolar K+ and Na+
concentrations.
Experiments were then carried out to study the currents induced by high
millimolar Na+ concentrations in oocytes expressing HKT1
and N365S to examine the low affinity Na+ uptake mode of
HKT1 (8, 9) (Fig. 6, A and
B). In wild type HKT1 expressing oocytes
(n = 10), the 100 mM
Na+-induced inward currents were much larger than the 1 mM K+ + 1 mM
Na+-induced currents (Fig. 6A). In
N365S-expressing oocytes (n = 10), the size of the
currents induced by 100 mM Na+ were smaller
than those induced by 1 mM K+ + 1 mM Na+ (Fig. 6B), confirming
suppression of low affinity Na+ uptake by N365S (Fig.
3C). Note that the magnitude of the 1 mM K+ + 1 mM Na+-induced currents is
higher in N365S than in HKT1-expressing oocytes (
We also investigated the currents induced by 1 mM
K+ + 1 mM Na+ and compared these to
currents mediated by 100 mM Na+ in oocytes
expressing the A240V, Q270L, and L247F mutants to allow a comparison
among these mutants. In A240V- and Q270L-expressing oocytes, the
magnitude of the 100 mM Na+-induced currents
were smaller than the 1 mM K+ + 1 mM Na+-induced currents, similar to
N365S-expressing oocytes. We calculated the ratio of the current
induced by 100 mM Na+ over the current induced
by 1 mM K+ + 1 mM Na+
at a membrane potential of
The results described above indicate that the HKT1 mutations
had decreased the permeability for Na+ at high
Na+ concentrations. Changes in permeabilities of cations
would affect the reversal potential of the transporter (33). To
directly analyze this hypothesis, and to quantitatively determine
whether the low affinity Na+ transport mode of HKT1 mutants
can be biophysically distinguished among mutants, we measured the
reversal potentials of currents in the presence of 10 mM or
100 mM Na+ in oocytes expressing HKT1 and the
Na+-tolerant mutants. Fig. 7
shows the shift in the reversal potential of HKT1-mediated currents in
response to an increase in the external Na+ concentration
from 10 to 100 mM (equivalent to an 8.5-fold
Na+ activity shift after correction for ionic activities).
Wild type HKT1 expression produced the previously described large
shifts in the reversal potential of +48.2 ± 0.9 mV
(n = 6) upon increasing the extracellular
Na+ concentration (8). The more Na+-tolerant
mutants, N365S and A240V, showed dramatic reductions in reversal
potential shifts, whereas wild type HKT1 and the
Na+-tolerant mutants, L247F (n = 6) and
Q270L (n = 5), showed large shifts of
We analyzed the permeability ratio for K+ over
Na+ using the Goldman equation for ion channels (see
"Experimental Procedures"). Because in contrast to ion channels,
HKT1 has been modelled to possess two independent ion-coupling sites,
one of which is highly selective for Na+ and the other is
selective for K+, we also calculated the K+ to
Na+ permeability ratios using an expansion of the Goldman
equation (see "Experimental Procedures") to more accurately reflect
the pK+/pNa+ permeability
ratios of just the K+-coupling site. Using the Goldman
equation, the permeability ratios pK+/pNa+ for HKT1 and the
Na+-tolerant mutants derived from absolute reversal
potential measurements in the presence of 100 mM external
Na+ were 5.3 ± 0.8 (n = 4) for HKT1,
12.1 ± 2.3 (n = 7) for A240V, and 15.5 ± 1.8 (n = 5) for N365S. Using the expanded model for two
ion-coupling sites (see "Experimental Procedures"),
pK+/pNa+ permeability
ratio values for the K+ selective site were 51 for HKT1,
187 for A240V, and 257 for N365S. The high relative permeabilities of
the K+-coupling site for K+ over
Na+ derived from the two-site model are consistent with the
predicted high selectivity of this site for K+ over other
cations (8, 9). Permeability ratio analyses show a K+ to
Na+ selectivity sequence of N365S > A240V > wild type HKT1, independent of the model used.
Expression of the HKT1 protein in yeast was shown to confer
typical characteristics of high affinity K+ uptake,
including a micromolar apparent K+ affinity
(Km Flux studies have shown both high and low affinity Na+
uptake pathways in plants and suggested that high affinity
Na+ uptake is mediated by K+ transporters (10,
11). However, Na+ toxicity related to salt stress has been
specifically attributed to the low affinity components of
Na+ uptake (11, 14, 19). Therefore, the finding that HKT1
mediates low affinity Na+ uptake at millimolar
Na+ concentrations indicates a putative contribution of
HKT1 as one of several components mediating low affinity
Na+ uptake during salt stress in plants (12, 13). RNA
in situ hybridizations show HKT1 expression in
root and leaf tissues that are relevant to Na+ transport
and stress (6). Expression of HKT1 in leaf cells indicates a
role for Na+ transport in leaves (6), where Na+
is particularly deleterious to plant metabolism and growth. Note that
Na+ uptake studies in roots show several low affinity
Na+ uptake components (12). Recently, several plant
cDNAs encoding cation transporters have been isolated that may
encode additional low affinity Na+ uptake components
depending on the ionic conditions, including LCT1 from wheat
(34) and HvHAK1 from barley (35). The
Na+-coupled high affinity K+ uptake mechanism
of HKT1 suggested that additional high affinity K+ uptake
pathways should exist in plant cells (8, 9). Both the
Arabidopsis, AtKT, or AtKUP cDNAs and the
homologous barley HvHAK1 cDNA have been shown to mediate
K+ uptake (35-38).
Na+-tolerant mutants in HKT1 were isolated here
to identify and characterize important amino acid residues and
mechanisms involved in Na+ transport and to isolate
HKT1 mutants with reduced Na+ uptake for
molecular physiological studies to determine contributions of HKT1 to
Na+ transport. Wheat HKT1 is highly selective for the
alkali cations K+ and Na+, and micromolar
K+ concentrations strongly inhibit high affinity
Rb+ uptake.2 At a
concentration of Identification of HKT1 Mutants with Reduced Na+
Permeability--
In the present study, the use of a yeast strain
deficient in the ENA family of Na+-extruding
ATPases allowed a stringent screen to isolate highly Na+-tolerant HKT1 mutations. The N365S mutation
that showed the strongest increase in Na+ tolerance was
analyzed in detail here. The N365S mutation is a relatively
conservative substitution. Interestingly, the Asn-365 amino acid is
conserved in the Arabidopsis HKT1
homologue.3 Four of the five
newly identified mutants showed a mutation at position Asn-365,
indicating that this site is of central importance for determining the
interaction of HKT1 with Na+ (Table I).
Consistent with the above model, the Na+-tolerant
HKT1 mutants characterized here show a reduced block of
Rb+ uptake by Na+ (Fig. 3A). At the
same time, the apparent Km of low affinity
Na+ uptake was increased, and the
Vmax for Na+ decreased (Fig.
3C, Table II). Note that the Km for low
affinity Na+ uptake of the Na+-tolerant HKT1
mutants (Fig. 3C) does not coincide with the Na+
concentration that inhibits high affinity Rb+ uptake (Fig.
3A). The higher value obtained for Na+
Ki (Fig. 3A) compared with the low
affinity Na+ Km (Fig. 3C) may
be a consequence of the micromolar Rb+ concentration (100 µM) present in the Ki experiment (Fig.
3A), which will compete at the high affinity
K+-coupling site, reducing the affinity of the transporter
for millimolar Na+. However, the existence of additional
low affinity Na+ binding sites in HKT1 involved in
activation, inhibition, or transport of Na+ cannot be ruled
out. Although N365S displayed the lowest apparent affinity for low
affinity Na+ uptake (Km = 10.2 mM Na+) and also had a reduced maximal uptake
rate (Fig. 3C, Table II), these parameters alone did not
appear to resolve the strikingly higher Na+ resistance of
N365S as compared with the A240V mutant.
An additional effect of the N365S mutation is a lower Na+
permeability of HKT1. The selectivity for Na+ of wild type
HKT1 and the mutants was determined by measuring current reversal
potentials in oocytes when external Na+ concentrations were
changed from 10 mM Na+ to 100 mM
Na+ (Fig. 7). Indeed, the N365S mutant showed a dramatic
reduction in the reversal potential shift (16.6 mV versus
48.2 mV), reflecting a markedly reduced Na+ permeability in
the N365S mutant. Calculations of relative K+ to
Na+ permeability ratios with 100 mM
Na+ using either a channel model or a two-site coupled
transporter model confirmed that the N365S mutant has the highest
selectivity for K+ over Na+ of all mutants
isolated so far. Calculated K+ to Na+
selectivity values were lower for the channel model, because both
K+ and Na+ transport sites are treated as one
entity. The model for the two-site coupled transporter quantifies the
previously predicted high K+ to Na+ selectivity
of the K+-coupling site (8, 9), with K+ to
Na+ permeability ratios ranging from 51 for HKT1 to 257 for
N365S. A recent study has identified point mutations at other sites in HKT1 that affect the Na+-coupling site (39), which differs
mechanistically from the K+-coupling site mutations
described here. The reduction in relative Na+ permeability
of the N365S mutant leads to a reduction in low affinity
Na+ uptake as shown in oocyte (Fig. 6) and yeast (Fig.
3B) experiments and to increased K+ content (Fig.
4). Note that permeability ratio values of HKT1 will depend on
conditions, because of competition of ionic interactions in HKT1
(9).
The N365S mutation affects several important parameters in HKT1
function: (i) reduction in low affinity Na+ uptake, (ii)
reduction in Na+ to K+ permeability, and (iii)
reduction in the inhibition of high affinity K+ uptake by
millimolar Na+ concentrations. The combined effects of
these changes, integrated over physiological time periods, contribute
to maintain a low internal Na+/K+ ratio during
long-term uptake studies (Fig. 4), resulting in Na+-resistant cell growth. The results presented here
suggest a model to explain the increase in Na+ tolerance
conferred by the N365S mutation. In this mutant, the selectivity
between micromolar K+ and millimolar Na+ at the
high affinity K+-coupling site is increased in comparison
with wild type HKT1 and also with the A240V mutant. As a result, the
internal Na+/K+ concentration ratio is kept
below the threshold level that would inhibit yeast growth.
Interestingly, Asn-365 is not located in a hydrophobic domain but in a
hydrophilic loop domain. In the case of K+ channels the
selectivity filter has been ascribed largely to a short hydrophilic
loop ("P-domain") rather than hydrophobic domains (40-43). In the
bacterial high affinity K+ pump, Kdp, 33 independent
mutations affecting 13 amino acid residues were identified that reduced
K+ affinity (44). Of these 13 residues, 12 were located in
hydrophilic loop domains (44). The Asn-365 site in a loop domain of
HKT1 substantially affects Na+ sensitivity, Na+
transport, and Na+ permeability, which correlates well with
findings in K+ channels and in the bacterial K+
pump Kdp on the importance of loop domains for cation selectivity.
Physiological Significance of K+ versus Na+
Uptake--
In yeast, high affinity Na+ uptake via the
K+-Na+ symport activity of HKT1 (Fig.
2B), does not cause Na+ toxicity. This is most
likely because of the low Na+ uptake rates under high
affinity uptake conditions. At these low rates, transfer of
Na+ from the cytosol into the vacuole would be sufficiently
rapid to prevent toxic levels of Na+ in the cytosol. In
addition, because the stoichiometry of high affinity uptake by HKT1 at
micromolar cation concentrations was found to be approximately 1.7 K+ per Na+ ion (8), the low levels of
Na+ uptake would be sufficiently balanced by K+
uptake to prevent displacement of K+ by Na+ in
the cytosol. (Note that because of competition, the exact K+ to Na+ uptake stoichiometry of HKT1 was
shown to depend on ionic conditions (9, 45).) It has recently been
proposed that the HKT1 protein may be located in the vacuolar membrane
rather than in the plasma membrane (46). Here we report on HKT1 mutants
that reduce HKT1-mediated Na+ transport into the cytosol.
These findings would be relevant even if HKT1 were targeted to an
endomembrane in plants because Na+ transport by HKT1 would
affect the cytoplasmic Na+/K+ content ratio.
Sodium-tolerant HKT1 mutants increased the ratio of
K+ to Na+ content in yeast (8). In plants,
analysis of the Na+-sensitive Arabidopsis mutant
sos1 supports the model that the balance of cytosolic
K+ versus Na+ concentrations is an
important determinant of Na+ toxicity. The sos1
mutant shows strongly reduced high affinity K+ uptake and a
reduction in the ratio of K+ to Na+ content
(47, 48). Interestingly, sos1 at the same time also shows
reduced low affinity Na+ uptake. The
sos1-affected low affinity Na+ uptake component
is induced in wild type Arabidopsis roots in response to
K+ starvation, which also induces high affinity
K+ uptake systems (5, 49). These data indicate
a genetic relationship of high affinity K+ uptake and low
affinity Na+ uptake in Arabidopsis roots (48).
Note that the sos1 mutation appears to affect several
different transporters and therefore probably has a regulatory function
(47, 48).
Conclusions--
The identification and characterization of the
strongly Na+-tolerant N365S HKT1 mutant
described here provides a new tool to study contributions of HKT1
transporters to Na+ sensitivity, Na+ transport,
regulation of Na+ transport, and K+
versus Na+ content ratios in transgenic plants.
New HKT1 sites that reduce toxic effects of Na+
in yeast were identified and characterized by a combination of random
mutagenesis, functional selection in yeast, and flux and biophysical
transport studies. Mutants maintain the physiologically beneficial
function of Na+-coupled high affinity K+ uptake
because they were selected in K+ uptake-deficient yeast
mutant backgrounds (21). A new critical site in HKT1,
Asn-365, and new biophysical transport parameters were identified that
can be altered to enhance Na+ resistance in yeast. In
plants, toxic low affinity Na+ uptake is thought to be
mediated by multiple Na+ uptake transporters (see above and
Ref. 12). The use of an Na+-ATPase yeast deletion mutant
line (9.3 ena
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
200, his44-15, trk1
,
trk2::pCK64) (21) and 9.3 (Mat a,
ena1
:HIS3::ena4
, leu2, ura3-1, trp1-1, ade2-1,
trk1
, trk2::pCK64) kind gifts of Dr.
Richard Gaber (Northwestern University) and Dr. Alonso
Rodríguez-Navarro (Universidad Politécnica de Madrid,
Spain), respectively, were used for selection and physiological
characterization of HKT1 mutants. Standard minimal medium
(22) and arginine phosphate medium
(AP)1 (23) supplemented with
KCl and NaCl as indicated were used for yeast growth. Yeast
transformation was carried out by the polyethylene glycol method as
described previously (24). Standard procedures were used for
Escherichia coli growth and transformation and DNA
manipulations (25).
36
to 1152, and the second reaction from position 657 to 1570. The DNA fragments were cotransformed into yeast to allow homologous
recombination (27) with a gapped pDR195-based plasmid containing the
yeast PMA1 gene promoter (28) and the HKT1
cDNA lacking the BgII (387 position) to the
BclI (939 position) fragment for the first set of PCR
reactions or the BclI (939 position) to the XbaI
(1478 position) fragment for the second set of PCR reactions.
URA+ transformants were first selected on standard minimal
medium lacking uracil and supplemented with 100 mM
K+ and then replica-plated to AP medium supplemented with
0.1 mM K+ plus 400 mM
Na+, where strain 9.3 containing the wild type HKT1 did not
grow because of Na+ toxicity. Growing colonies were
selected, and their plasmids were isolated and reintroduced into yeast
to retest for Na+ tolerance. Clones conferring the highest
Na+ tolerance were chosen for further characterization.
1 at
time 0. Samples were filtered at different time points through a
Millipore membrane (0.8 µm) and washed with a 10 mM RbCl
or NaCl solution. The internal Rb+ or Na+
content was calculated from the external activity, and the counts accumulated in the cells. Samples were counted in a Beckman Ls-230 scintillation counter. For low affinity Na+ uptake
experiments, NaCl was added at time 0. Samples were collected as
described above, washed with a 20 mM MgCl2
solution, and extracted with acid. The internal Na+ content
was determined by atomic emission spectrophotometry using a
Perkin-Elmer 5000 spectrophotometer. Initial rates of Rb+
or Na+ uptake are reported and represent averages of at
least three independent experiments. Error bars denote standard error
of the mean.
1 with
D-sorbitol. K+ and Na+ were added as glutamate
salts. Tris+ (as glutamate) was added in experiments where
the total alkali cation concentration varied. Error bars denote
standard error of the mean.
(Eq. 1)
(Eq. 2)
where
(Eq. 3)
(Eq. 4)
RESULTS
Mutations in HKT1 that confer Na+ tolerance
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Fig. 1.
The new HKT1 mutations N365S
and Q270L confer Na+ tolerance to yeast.
Panel A, growth of the yeast strain in which
Na+-extruding ATPases were deleted (9.3 strain) was used to
isolate and assay the Na+-tolerant HKT1 mutants
N365S, Q270L, and A240V. Yeast cells were grown on an AP medium
supplemented with 0.1 mM K+ plus 350 mM Na+. Panel B, liquid
culture growth of the yeast strain 9.3 expressing wild type
HKT1 ( ) and the mutants N365S (
), Q270L (
), A240V
(
), and untransformed 9.3 strain (
) in liquid AP medium
supplemented with 0.1 mM K+ plus 150 mM Na+. The ability of
Na+-resistant HKT1 mutant expressing lines to
grow in high Na+ is evident. At time 0, media were
inoculated with 106 cells/ml of each strain and the optical
density (OD) at 600 nm of the culture was recorded.
Panel C, increasing the Na+
concentration to 250 mM shows the strong Na+
resistance of the N365S mutant. Conditions were the same as in
panel B but in the presence of 0.1 mM
K+ plus 250 mM Na+. Panel
D, according to the Kyte and Doolittle algorithm, HKT1 comprises
12 hydrophobic domains. Mutants A240V and L247F are located in the
6th hydrophobic domain, mutant Q270L in the 7th hydrophobic domain, and
mutant N365S in the loop between the 8th and 9th hydrophobic
domains.
1 min
1) values 2.7 ± 0.1 for HKT1, 2.2 ± 0.1 for Q270L, and 2.0 ± 0.1 for
N365S. Statistical analyses of the data showed that there were not
significant differences among the Rb+ Km
values (p values were 0.079 for the HKT1, N365S pair and
0.084 for the HKT1, Q270L pair). The untransformed control strain CY162
did not show Rb+ uptake in the range of Rb+
concentrations used in these experiments because of the deletion of the
high affinity K+ uptake systems encoded by TRK1
and TRK2 genes.
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Fig. 2.
HKT1 and the Na+-tolerant
HKT1 mutants Q270L and N365S display high affinity
Rb+ and Na+ uptake. Panel
A, initial rates of Rb+ uptake in the presence
of 1 mM Na+ as a function of the external
Rb+ concentration were analyzed in CY162 yeast cells
expressing wild type HKT1 ( ), the HKT1 mutants
Q270L (
) and N365S (
), and untransformed strain CY162 (
).
Panel B, initial rates of Na+ uptake
in the presence of 100 µM K+ as a function of
the external Na+ concentration. In panels
A and B high affinity Rb+ and
Na+ uptake were analyzed in the K+
uptake-deficient yeast strain CY162 (trk1
,
trk2
) expressing the indicated HKT1 mutants.
1 min
1)
values 6.8 ± 0.2 for HKT1, 5.5 ± 0.2 for Q270L, and
5.0 ± 0.2 for N365S. Statistical analyses of the data showed that
the Na+ Km values were not significantly
different (p = 0.054 for the HKT1, N365S pair and
p = 0.11 for the HKT1, Q270L pair), whereas the
Na+ Vmax values were significantly
different (p = 0.005 for the HKT1, N365S pair and
p = 0.009 for the HKT1, Q270L pair). The untransformed control strain CY162 did not show Na+ uptake in the range
of Na+ concentrations used in these experiments. The
Na+-tolerant mutants showed significantly lower
Vmax values for high affinity Rb+
and Na+ uptake. This could be a consequence of a lower
expression of the mutants in yeast as compared with wild type HKT1.
However, a reduced expression level would decrease the rate at which
yeast expressing the mutants grow in low K+ medium, which
was not observed (data not shown). Although the lower
Vmax for high affinity Na+ uptake
showed by the mutants could contribute to Na+ tolerance, we
hypothesized that other substantial changes in transport
parameters would be required in the HKT1 mutants to achieve the observed dramatic increase in Na+ tolerance
(Fig. 1C).
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Fig. 3.
Toxic concentrations of Na+
inhibit high affinity Rb+ uptake to a lesser extent and
promote a reduced low affinity Na+ uptake in the
Na+-tolerant HKT1 mutants when compared
with wild type HKT1. A, initial rates of
Rb+ uptake at 100 µM Rb+ were
determined in the presence of 1-400 mM Na+ in
the K+ uptake-deficient yeast strain CY162
(trk1 , trk2
). A clear enhanced
resistance to high Na+ was found in cells expressing the
Na+ resistant mutants N365S (
), Q270L (
), and A240V
(
), when compared with cells expressing wild type HKT1
(
). The Rb+ uptake rates are expressed as a percentage
of the Rb+ uptake rate at 1 mM Na+
(100%). B, high and low affinity HKT1-mediated
Na+ uptake can be clearly distinguished by Eadie-Hofstee
analysis. For Na+ uptake experiments in the micromolar
range of concentrations, 0.1 mM K+ was added.
C, Na+-tolerant HKT1 mutants show
reduced toxic low affinity Na+ uptake. Initial rates of
Na+ uptake at external Na+ concentrations in
the range 0-100 mM Na+ were measured in the
K+ uptake-deficient yeast strain CY162
(trk1
, trk2
) expressing wild
type HKT1 (
) and the HKT1 mutants N365S (
), A240V (
), Q270L
(
), and the control strain CY162 (
).
Km and Vmax values for low affinity Na+ uptake
in yeast cells expressing HKT1, Na+-tolerant HKT1 mutants and
the control yeast strain CY162
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Fig. 4.
The internal Na+/K+
ratio of yeast cells expressing HKT1 is higher than in yeast cells
expressing the Na+-tolerant HKT1 mutants. Yeast cells
expressing HKT1 or the Na+-tolerant HKT1 mutants were
incubated in AP media supplemented with 0.1 mM
K+ and 300 mM Na+ (solid
bars) or 500 mM Na+ (open
bars). After 24 h of incubation, the internal Na+
and K+ content was determined. The more
Na+-tolerant mutant N365S showed the lower internal
Na+/K+ ratio. At 500 mM
Na+, the internal K+ and Na+
concentrations (nmol mg 1) were 14 ± 1.7 K+ and 94 ± 2.9 Na+ for HKT1-expressing
cells, 37.7 ± 4.6 K+ and 99.7 ± 4.6 Na+ for A240V-expressing cells, 193 ± 11 K+ and 107.7 ± 7 Na+ for N365S-expressing
cells, and 58 ± 1.7 K+ and 113 ± 7.2 Na+ for Q270L-expressing cells.
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Fig. 5.
Wild type HKT1 and the mutant N365S show
similar reversal potential shifts for Na+-K+
symport. Increasing external K+ concentrations from 1 to 10 mM in the presence of 1 mM
Na+ resulted in similar shifts toward more positive
potentials of the current-voltage relationships of HKT1-mediated
currents (A) and N365S-mediated currents (B) in
oocytes. Increasing external Na+ concentrations from 1 to
10 mM in the presence of 1 mM K+
produced similar shifts toward more positive potentials of the
current-voltage relationships of HKT1 (C) and N365S
(D) in oocytes. The above treatments did not produce
measurable currents in uninjected oocytes (not shown). Voltage ramps of
40 s duration from 40 to
120 mV were applied. Illustrated
recordings show raw current data after filtering and use of the
indicated symbols for clarity.
2.06 ± 0.4 µA for N365S and
1.13 ± 0.07 µA for HKT1, at a membrane potential of
120 mV), suggesting that a reduced expression of the
N365S mutant in oocytes is not the cause of the reduction in the
currents induced by 100 mM Na+.
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Fig. 6.
The Na+-tolerant HKT1
mutant N365S shows a dramatic reduction in Na+ uptake
current induced by 100 mM Na+ when compared
with wild type HKT1. The large Na+ uptake (negative)
currents induced by 100 mM Na+ ( ) in wild
type HKT1-expressing Xenopus oocytes (panel
A) are repressed by the N365S point mutation
(panel B). Panels A and B,
magnitude of HKT1-induced currents as a function of the imposed
membrane potentials recorded from oocytes expressing wild type
HKT1 (panel A) or the HKT1
mutant N365S (panel B) exposed to 1 mM K+ plus 1 mM Na+
(
) or to 100 mM Na+ (
). The above
treatments did not elicit currents in uninjected control oocytes
(panel B, inset).
120 mV. The ratio was 2.56 ± 0.12 for HKT1-expressing oocytes (n = 20), 1.55 ± 0.12 for L247F-expressing oocytes (n = 11), 0.82 ± 0.05 for A240V-expressing oocytes (n = 17), 0.78 ± 0.1 for N365S-expressing oocytes (n = 11), and
0.65 ± 0.16 for Q270L-expressing oocytes (n = 6).
For the less Na+-resistant L247F HKT1 mutant
(n = 4), 100 mM Na+-induced
currents were larger than the 1 mM K+ + 1 mM Na+-induced currents, but were smaller than
wild type HKT1 currents. This result correlates to the intermediate
level of Na+ resistance conferred by L247F (8). These data
illustrate the strong reduction in low affinity Na+ uptake
in the more Na+-tolerant HKT1 mutants.
50 mV. The
most Na+-tolerant N365S mutant showed the smallest shift in
the reversal potential of +16.6 ± 4.2 mV (n = 7),
which correlates with the strong Na+-tolerant phenotype of
this mutant when compared with all other mutants. The A240V mutation,
which shows less Na+ resistance than N365S, also showed an
intermediate shift in the reversal potential of +26.1 ± 3.3 (n = 11), revealing a larger Na+
permeability than the N365S mutant (Fig. 7).
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Fig. 7.
The Na+-tolerant N365S mutant
shows a reduction in reversal potential shifts induced by increasing
external Na+ from 10 to 100 mM. Average
shifts in reversal potentials ( Erev) of
HKT1-mediated currents in response to an increase in the external
Na+ concentration from 10 to 100 mM. Reversal
potentials of steady-state currents of HKT1 and HKT1 mutants
were recorded as in Fig. 6 (8, 9). Data from 5 to 11 cells (±S.E.)
were averaged for each HKT1 construct (see text).
DISCUSSION
3 µM K+) and
saturation of uptake at micromolar K+ and Rb+
concentrations (6, 8, 9). High millimolar Na+
concentrations have two important effects on cation transport by HKT1
leading to toxicity in yeast (8). First, high affinity K+ uptake via HKT1 is blocked by millimolar Na+
concentrations (Fig. 3A). Second, at these millimolar
Na+ concentrations, HKT1 functions as a low affinity
transporter mediating high rates of Na+ uptake (Fig.
3C) (8, 9).
15 µM, K+ produces a
15-fold higher uptake rate than Rb+ (9). However, when
K+ is present at high millimolar concentrations,
K+ partially impairs high affinity Na+-coupled
K+ uptake (9).
) in combination with uptake
deficiency (e.g. trk
) to select for
Na+-tolerant mutants, as presented for HKT1 here, can be
applied to other plant Na+ uptake transporters and could
provide a potent approach to identify strong Na+-tolerant
mutants in individual Na+ transporters in the future.
Isolation and analysis of additional HKT1 mutants should
allow insights into further structural sites and mechanisms that affect
Na+ transport. Mutations characterized here provide
molecular tools to test physiological contributions of HKT1 to
Na+ transport in complex multicellular systems such as
plant tissues.
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ACKNOWLEDGEMENTS |
---|
We thank Judie Murray for assistance in preparing the manuscript, Eugene Kim for comments on the manuscript, Alonso Rodríguez-Navarro for providing the 9.3 yeast strain, and Richard F. Gaber for providing the CY162 strain.
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FOOTNOTES |
---|
* This research was supported by grants from the United States Department of Agriculture (to J. I. S.) and by a Ministerio de Educación y Ciencia (Spain) postdoctoral fellowship (to F. R.).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.
Present address: Departamento de Biotecnología, Escuela
Técnica Superior de Ingenieros Agrónomos, 28040 Madrid, Spain.
§ Present address: Zentrum f. Molekulare Neurobiologie, Martinistr. 52, 20246 Hamburg, Germany.
¶ Present address: Dept. of Plant Biology, 111 Koshland Hall, University of California, Berkeley, CA 94720-3102.
To whom correspondence should be addressed. Tel.: 619-534-7759 or 619-534-6296; Fax: 619-534-7108; E-mail:
Julian{at}biomail.ucsd.edu.
2 F. Rubio, D. P. Schachtman, W. Gassmann, and J. I. Schroeder, unpublished material.
3 N. Uozumi, E. J. Kim, F. Rubio, S. Muto, and J. I. Schroeder, unpublished material.
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ABBREVIATIONS |
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The abbreviations used are: AP, arginine phosphate; PCR, polymerase chain reaction; Mes, 4-morpholineethanesulfonic acid.
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REFERENCES |
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