Boron Toxicity Tolerance in Barley through Reduced
Expression of the Multifunctional
Aquaporin HvNIP2;11[W]
Thorsten Schnurbusch2,3, Julie Hayes2, Maria Hrmova, Ute Baumann, Sunita A. Ramesh,
Stephen D. Tyerman, Peter Langridge, and Tim Sutton*
Australian Centre for Plant Functional Genomics (T. Schnurbusch, J.H., M.H., U.B., P.L., T. Sutton), and
School of Agriculture, Food and Wine (S.A.R., S.D.T.), University of Adelaide, Waite Campus, Urrbrae, South
Australia 5064, Australia
Boron (B) toxicity is a significant limitation to cereal crop production in a number of regions worldwide. Here we describe the
cloning of a gene from barley (Hordeum vulgare), underlying the chromosome 6H B toxicity tolerance quantitative trait locus. It
is the second B toxicity tolerance gene identified in barley. Previously, we identified the gene Bot1 that functions as an efflux
transporter in B toxicity-tolerant barley to move B out of the plant. The gene identified in this work encodes HvNIP2;1, an
aquaporin from the nodulin-26-like intrinsic protein (NIP) subfamily that was recently described as a silicon influx transporter
in barley and rice (Oryza sativa). Here we show that a rice mutant for this gene also shows reduced B accumulation in leaf
blades compared to wild type and that the mutant protein alters growth of yeast (Saccharomyces cerevisiae) under high B.
HvNIP2;1 facilitates significant transport of B when expressed in Xenopus oocytes compared to controls and to another NIP
(NOD26), and also in yeast plasma membranes that appear to have relatively high B permeability. We propose that tolerance to
high soil B is mediated by reduced expression of HvNIP2;1 to limit B uptake, as well as by increased expression of Bot1 to
remove B from roots and sensitive tissues. Together with Bot1, the multifunctional aquaporin HvNIP2;1 is an important determinant of B toxicity tolerance in barley.
Barley (Hordeum vulgare) is an annual grain, ranked
fourth among the cereals in terms of total world
production (FAOSTAT; http://faostat.fao.org). Cultivated barleys are not well adapted to high soil boron
(B), although toxicity to B has been known for some
time (Christensen, 1934; Cartwright et al., 1986). B is an
essential micronutrient for higher plants (Sommer and
Lipman, 1929), and there is only a very small difference between deficient and toxic concentrations of B
in plant tissues (Eaton, 1944). Early genetic studies of
B toxicity tolerance in a barley mapping population
identified favorable alleles at four genetic loci on
chromosomes 2H, 3H, 4H, and 6H (Jefferies et al.,
1999). Tolerance to B toxicity is associated primarily
1
This work was supported by the Australian Research Council,
the South Australian Government, and the Grains Research and
Development Corporation. T. Schnurbusch was partly supported by
an Alexander-von-Humboldt Fellowship (Feodor-Lynen Postdoctoral Program), Germany.
2
These authors contributed equally to the article.
3
Present address: Leibniz-Institute of Plant Genetics and Crop
Plant Research (IPK), Genebank Department, Corrensstr. 3, D–06466
Gatersleben, Germany.
* Corresponding author; e-mail [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy
described in the Instructions for Authors (www.plantphysiol.org) is:
Tim Sutton ([email protected]).
[W]
The online version of this article contains Web-only data.
www.plantphysiol.org/cgi/doi/10.1104/pp.110.158832
1706
with reduced B accumulation, but also with increased
root growth and dry matter production and reduced
leaf symptom expression. In all cases, the favorable
alleles are derived from the highly B toxicity-tolerant
Algerian landrace barley Sahara 3771 (Sahara). We
recently identified a gene (Bot1) underlying the tolerance quantitative trait locus (QTL) on chromosome 4H
of barley, that is a putative integral trans-membrane B
transporter with similarity to bicarbonate transporters
in animals (Sutton et al., 2007). In roots of Sahara,
active efflux of B and high Bot1 expression at least
partially control lower B accumulation in roots and
shoots of barley plants (Hayes and Reid, 2004; Reid,
2007; Sutton et al., 2007).
A second class of proteins capable of B transport
belongs to the superfamily of major intrinsic proteins
(MIPs) or aquaporins. Aquaporins have previously
been suggested to be involved in B transport in higher
plants (Dordas et al., 2000; Fitzpatrick and Reid, 2009)
and are required for normal growth of Arabidopsis
(Arabidopsis thaliana) under B deficiency conditions
(Takano et al., 2006; Tanaka et al., 2008). However,
aquaporins have not been shown to be involved in
B toxicity tolerance. Five major subgroups of aquaporins in plants are now recognized (Danielson and
Johanson, 2008; Sade et al., 2009; Shelden et al., 2009).
In particular, members of one of these subgroups, the
nodulin-26-like intrinsic proteins (NIPs), were recently
described as functional equivalents of aquaglyceroporins (Bhattacharjee et al., 2008). These channel pro-
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Aquaporin-Mediated Boron Toxicity Tolerance in Barley
teins usually enable water transport but recent studies
have demonstrated that they also facilitate transport of
other small, uncharged solutes under physiological
conditions including glycerol and lactic, boric (B),
silicic (Si), arsenious (As), antimonious (Sb), and germanic (Ge) acids (Ma et al., 2006, 2008; Takano et al.,
2006; Choi and Roberts, 2007; Bienert et al., 2008; Chiba
et al., 2008; Isayenkov and Maathuis, 2008; Tanaka
et al., 2008; Kamiya et al., 2009; Mitani et al., 2009).
Here we report that HvNIP2;1, a member of the NIP
subfamily, underlies a B toxicity tolerance QTL on
chromosome 6H of barley associated with reduced B
uptake (Jefferies et al., 1999). This gene was recently
identified and described in barley as the Si influx
transporter HvLsi1, largely on the basis of the observed
characteristics of the putative orthologous protein
OsNIP2;1 (OsLsi1) in rice (Oryza sativa; Chiba et al.,
2008). HvLsi1 showed transport activity for Si when
expressed heterologously and immunohistological
staining in barley revealed that the protein is expressed on the distal side of epidermal and cortical
cells in the basal part of seminal roots, embedded in
the plasma membrane (Chiba et al., 2008). However,
expression was poorly correlated with Si uptake in
barley (Chiba et al., 2008). In this work, our molecular
models suggested that HvNIP2;1 (HvLsi1) could facilitate the transport of a range of small, uncharged
solutes including Si and also B. We expressed
HvNIP2;1 protein in yeast (Saccharomyces cerevisiae)
and Xenopus oocytes and functionally characterized its
B transport properties. Furthermore, we observed
lower shoot B accumulation in a rice mutant displaying a point mutation Ala-132Thr in OsNIP2;1 (lsi1; Ma
et al., 2006; Mitani et al., 2008). Higher tolerance to B in
Sahara appears to be mediated through lower transcript levels of HvNIP2;1 in basal root segments of
barley, possibly owing to a repeat insertion in the
promoter region of HvNIP2;1, approximately 2 kb
upstream of the start codon. Based on these results
we propose that reduced expression of HvNIP2;1
lowers the passive influx of B into roots under high
soil B concentration, contributing to lower shoot B
accumulation and thus, higher B tolerance in Sahara.
RESULTS
Genetic Mapping of HvNIP2;1
Previous QTL mapping in a barley doubled haploid
population identified a B toxicity tolerance locus on
chromosome 6H, with the favorable allele derived
from the B toxicity-tolerant landrace Sahara (Jefferies
et al., 1999). Prior to F3 population development for
fine mapping of the 6H locus gene, we assessed the
effect of alternative chromosome 4H-Bot1 alleles on
leaf blade B concentration, determined by segregation
of 6H gene alleles (Fig. 1A). In both the 4H Clipper
(Bot1:C) and 4H Sahara (Bot1:S) allele backgrounds,
the 6H Sahara allele contributed positively to reduced
Figure 1. HvNIP2;1 genetic analysis. A, Phenotypic effect of chromosome 4H-Bot1 and chromosome 6H-HvNIP2;1 Clipper and Sahara
allele combinations on leaf blade B concentration in barley. Individual
F2 plants derived from a cross of two Clipper 3 Sahara doubled haploid
plants were marker tested to determine Bot1 and HvNIP2;1 genotypes.
Plants homozygous at these loci were grown in hydroponics solution
supplemented with 2 mM B and leaf blade B concentration was
measured after 21 d. The boundaries of the box indicate the 25th and
75th percentiles and a line within the box marks the median. Whiskers
above and below the box indicate the 90th and 10th percentiles.
Outliers are shown as black circles. Data for Clipper and Sahara are
from five plants; for all others 28 to 30 plants were analyzed. Tenth and
90th percentiles were unable to be calculated for Clipper and Sahara
due to the smaller numbers of individuals analyzed. B, Genetic
mapping of the chromosome 6H B toxicity tolerance QTL on the
long arm of chromosome 6H. Numbers above the line indicate
recombinants identified for each marker interval and mapped markers
are listed below the line. B toxicity tolerance was mapped to the xCSM1-xRAT-3 interval by genetic and phenotypic analysis of F4 progeny
families derived from F3 recombinant plants.
leaf blade B concentration. In the presence of 4H-Bot1:
C, plants carrying the 6H Sahara allele (HvNIP2;1:S)
accumulated 25% less B in leaf blades than those
carrying the 6H Clipper allele (HvNIP2;1:C). Similarly,
in the presence of 4H-Bot1:S, plants carrying the 6H
Sahara allele (HvNIP2;1:S) accumulated 40% less B in
leaf blades than those carrying the 6H Clipper allele
(HvNIP2;1:C). Furthermore, this analysis also demonstrated that a mapping population fixed for the 4H
Sahara allele (Bot1:S) provided the phenotypic discrimination (i.e. largely nonoverlapping distribution)
required for accurate mapping of the 6H QTL (Fig.
1A). We subsequently developed an F3 mapping population consisting of 192 plants derived from a cross
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Schnurbusch et al.
between two Clipper 3 Sahara doubled haploid lines
that differed for alleles at the B tolerance locus on
chromosome 6H, but not for alleles at the other known
B toxicity tolerance loci (Jefferies et al., 1999). The
population was fixed for the Sahara allele of the
chromosome 4H tolerance gene Bot1.
Gene-derived PCR markers were developed using
synteny to rice and were mapped in this F3 population. We were able to narrow the 6H QTL interval by
characterizing F4 progeny individuals of F3 lines
recombinant in the region, both for molecular markers
and leaf blade B accumulation in hydroponics (Supplemental Fig. S1). This analysis positioned the 6H
tolerance locus between markers xCSM-1 and xRAT-3
(Fig. 1B). The corresponding interval in rice was approximately 474 kb, between rice genes Os02g0740300
at position 30,977,102 bp and Os02g0748100 at position
31,451,191 bp in chromosome 2 (GenBank accession
no. NC_008395). We considered one of the rice genes
in the mapped syntenic rice interval (OsNIP2;1; GenBank accession no. AB222272) to be a strong candidate based on its known transport properties (Ma et al.,
2006). We isolated the Clipper and Sahara barley
orthologs of OsNIP2;1 and developed the cleaved
amplified polymorphic sequence marker xHvNIP2;1
from this sequence, which cosegregated with B toxicity
tolerance in our F3 mapping population. This confirmed HvNIP2;1 as a candidate for the B toxicity
tolerance gene in barley. While we considered an
ortholog of OsNIP2;1 to be the likely candidate tolerance gene in barley, we are unable to completely rule
out the possibility of a role of other tolerance cosegregating genes acting either independently or in conjunction with HvNIP2;1 in B tolerance in barley.
Characteristics of HvNIP2;1 in Clipper and Sahara Barley
The open reading frame sequences for HvNIP2;1
from Clipper and Sahara barley contain a single nucleotide polymorphism (C to G), which does not alter
the encoded amino acid residue (Val-283; Supplemental Fig. S2). HvNIP2;1 shares 82% sequence identity
with OsNIP2;1 at the amino acid level (Supplemental
Fig. S2; Chiba et al., 2008).
Leaf blade and root northern hybridization analyses
in Clipper and Sahara detected expression of HvNIP2;1
in roots only, which was unresponsive to an increase in
B concentration (Fig. 2A). Higher transcript levels of
HvNIP2;1 were evident in Clipper roots compared to
Sahara. We also observed significant varietal transcript
differences when we analyzed cDNAs from root sections using quantitative (Q)-PCR (Fig. 2B). Transcript
levels of HvNIP2;1 increased for both lines along the
length of the root from the apex toward the basal root
regions, with up to 15-fold higher transcript levels
observed in roots of the B-sensitive barley variety
Clipper (Fig. 2B).
We determined the nucleic acid sequences of
HvNIP2;1 from Clipper and Sahara barley genotypes
upstream of the ATG start codon and compared them
Figure 2. Expression of HvNIP2;1 mRNA in the B-tolerant variety
Sahara and B-intolerant variety Clipper. A, HvNIP2;1 mRNA levels in
whole roots (lanes 2–11) and leaf blades (lanes 12–21) of Clipper and
Sahara barley grown in hydroponic solution supplemented with additional B to 0.02 mM, 0.05 mM, 0.5 mM, and 1 mM for 14 d. Lanes 2, 7,
12, and 17 (0 mM B) are from plants grown in base solution with no
supplemental B added. Total RNA was visualized with ethidium
bromide to indicate loading and analyzed by northern hybridization
using a probe specific for the 3# untranslated region of HvNIP2;1
(Supplemental Table S1). A single band corresponding to a transcript
size of approximately 1.36 kb is shown. B, HvNIP2;1 mRNA levels
analyzed by Q-PCR in 10-mm segments taken along the root of Sahara
(white circles) and Clipper (black circles) barley seedlings treated with
1 mM B for 7 d. Data are means (n = 3) 6 SE and are presented on a log2
scale.
using the Web-based promoter analysis tool PlantPAN
(Chang et al., 2008). The first 1,377 bp upstream of the
ATG start codon are identical between Clipper and
Sahara. Further upstream we found six single nucleotide polymorphisms between 1,378 and 2,128 bp in
the putative promoter region. Beyond this region the
two sequences became dissimilar and we found an
insertion in Sahara relative to Clipper that contained a
repeat sequence beginning at position 2,311 bp upstream from the start ATG codon. We also found a
non-long terminal repeat transposon classified as an
EMMA repeat, inserted in the promoter region of
Sahara, between 3,097 and 3,151 bp, upstream of the
start ATG codon (Supplemental Fig. S3). Further
sequencing upstream of the currently obtained promoter regions in Clipper and Sahara should reveal
the extent and significance of these changes in alleles
of HvNIP2;1.
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Aquaporin-Mediated Boron Toxicity Tolerance in Barley
Molecular Modeling of HvNIP2;1
To investigate structural determinants in HvNIP2;1,
and how transport of B and other metalloids is mediated across a membrane, molecular modeling studies
were performed. An archeal aquaporin from Methanobacterium thermoautotrophicum (AqpM; Lee et al., 2005)
was chosen as a structural template for modeling,
since it showed the highest amino acid sequence
identity/similarity (36%/72%) with HvNIP2;1. Evaluation of molecular models (parameters are specified
in “Materials and Methods”) indicated that AqpM
was a suitable template for modeling and that the
constructed models were of satisfactory quality. The
molecular model of HvNIP2;1 revealed up-down bundle protein folding, housing six membrane helices and
two reentrant helices (Fig. 3). The latter structural
elements generated an additional membrane span,
resembling an hourglass arrangement (Engel et al.,
2000; Borgnia and Agre, 2001; Lee et al., 2005; Krissinel
and Henrick, 2007). Analysis of the buriedness of the
pore of HvNIP2;1 showed that the hourglass-shaped
channel spanned the entire length of the membrane
and that buriedness increased progressively toward
the center of the pore. Calculations indicated that the
pore volume of AqpM was approximately 1,113 cubic
Å, while that of HvNIP2;1 varied between 1,149 and
1,392 cubic Å. The likely homotetrameric organization of HvNIP2;1 (Krissinel and Henrick, 2007) could
be simulated, and it is expected that this higher
structural organization could permeate metalloids
and solutes more effectively (Engel et al., 2000; Borgnia
and Agre, 2001; Krissinel and Henrick, 2007).
Molecular models of HvNIP2;1 allowed the disposition of interacting amino acid residues to be defined
in the pore with respect to various metalloids. The
positions of two highly conserved NPA (Asn-Pro-Ala)
motifs in HvNIP2;1 and AqpM were almost identical.
Superposition of residues in the channels of both
porins indicated that four of the six key residues in
the constriction of AqpM were also conserved in
HvNIP2;1. One B, As, Si, or Ge acid with four water
molecules were modeled in the pore of HvNIP2;1 (Fig.
3). All solutes were positioned favorably in the channel and did not generate steric clashes with neighboring residues. The models showed that Asn side chains
in the two NPA motifs were critical for mediating
interactions of all four solutes near the hourglass
constriction. Water molecules in the HvNIP2;1 pore
could form hydrogen bonds of 3.2 to 3.5 Å with all
metalloids. The aromatic/Arg (ar/R) selectivity filter
in aquaporins is considered to be a major determinant
for solute permeability (Beitz et al., 2006). We found
that while the ar/R filter in AqpM was up to 6.5 Å
wide, it was up to 8 Å wide in HvNIP2;1, primarily
due to the substitution of a bulky Phe in AqpM with
Gly in HvNIP2;1 on the a-helix H2. This substitution
could lead to a more open and polar character of the
HvNIP2;1 pore.
Similar pore diameter values were also observed in
a model of OsNIP2;1 in complex with B. When comparing amino acid residues that line the pore of
OsNIP2;1 with those in HvNIP2;1, the only residue
that was found to protrude into the OsNIP2;1 pore was
Ser-74. At this position in OsNIP2;1, the Gly-Ser-Asp
motif is substituted with an Ala-His-Asp motif; these
components of the aquaporin sequences form highly
variable regions. Thus, while the residue Ser-74 could
potentially protrude into the cavity of the pore of
OsNIP2;1, the overall transport properties of OsNIP2;1
and HvNIP2;1 are not expected to differ greatly.
Heterologous HvNIP2;1 Expression Studies
Heterologous HvNIP2;1 protein expression increased the sensitivity of yeast to high B concentrations in the media (Fig. 4A; Supplemental Fig. S4).
Growth of HvNIP2;1-expressing yeast was inhibited
slightly even without addition of B to the media (Supplemental Fig. S4), suggesting either that HvNIP2;1
expression altered the membrane permeability of
yeast to a range of solutes affecting growth, or that
sufficient B was present in the basal medium to restrict
growth. To confirm that the observed growth sensitivity under high and low B was due to expression of
Figure 3. Cross-sectional views of 3D molecular
models of HvNIP2;1. A, The clip shows an updown bundle fold (rainbow colors) of HvNIP2;1.
Accessibility of B acid (atomic colors, arrow) and
four water molecules (dark-blue spheres) are
illustrated in the central cavity of the pore. The
molecular surface and internal surface of the pore
are in yellow. B to E, Four zoom-in views show
positions of B (B), As (C), Si (D), and Ge (E) acids
in the pore, indicated by arrows.
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Schnurbusch et al.
Figure 4. Heterologous expression of HvNIP2;1 in yeast. A to C,
Growth of yeast expressing HvNIP2;1 in the presence of B (A), Ge (B),
and As (C) on Gal minimal media. On each plate are three replicates
transformed with empty vector (top), together with three replicates
expressing HvNIP2;1 (bottom). For each, 10-fold serial dilutions were
spotted onto the plates (from right to left). D, Stopped-flow analysis of
the B permeability of yeast SY1 vesicles, demonstrating different rate
constants for vesicles isolated from cells either expressing HvNIP2;1 or
containing empty vector (P , 0.05, unpaired t test). Data are means 6
SE (n = 9).
HvNIP2;1, Gal was replaced with Glc in minimal
media to repress transcription that is under the control
of the GAL1 promoter (West et al., 1984). Under these
conditions, phenotypic variation was not observed
between the HvNIP2;1-transformed and control yeast
cells (Supplemental Fig. S5), clearly indicating that the
sensitivity of yeast to B grown in the presence of Gal is
due to expression of HvNIP2;1. Si added to agar media
at soluble concentrations did not affect the growth of
yeast and we were therefore unable to determine
whether HvNIP2;1 has Si transport activity, as has
recently been shown in Xenopus and rice (Chiba et al.,
2008). However, Ge is a toxic metalloid, which is often
used as a chemical analog for comparatively nontoxic
Si in plant studies of Si transport (Nikolic et al., 2007).
We found that HvNIP2;1 expression increased the sensitivity of yeast to both Ge (Fig. 4B) and the metalloid As (Fig. 4C).
The ability of the HvNIP2;1 protein to facilitate B
transport was demonstrated by heterologous expression in both yeast and Xenopus oocytes. In yeast we
expressed HvNIP2;1 in the SY1 mutant strain (Ruetz
and Gros, 1987; Nakamoto et al., 1991) and used
stopped-flow analysis of isolated vesicles to measure
permeability to B (Dordas and Brown, 2000). The rate
constant of vesicle reswelling induced by an inwardly
directed B concentration gradient was significantly
faster for vesicles derived from HvNIP2;1-expressing
yeast compared to empty vector controls (t test P ,
0.05, n = 9 independent yeast cultures; Fig. 4D). Particle size analysis (dynamic light scattering) demonstrated that vesicles from all yeast preparations were
of the same diameter (99.7 nm 6 10.9 nm, six independent vesicle preparations), and we could therefore
calculate the B permeability (PB = k 3 r/3, where r is
vesicle radius and k is the rate constant for vesicle
reswelling) of the two isolates. For HvNIP2;1 vesicles,
PB was 1.6 3 1026 cm s21, while for empty vector
controls PB was 0.98 3 1026 cm s21.
In Xenopus, we compared the transport properties of
HvNIP2;1 with GmNOD26 from soybean (Glycine
max), the archetype of the NIP subfamily of aquaporins with well-characterized water and glycerol transport properties (Rivers et al., 1997). Oocytes were
injected with either HvNIP2;1 or GmNOD26 capped
RNA (cRNA). A further set of oocytes were injected
with water to act as controls. We measured oocyte
swelling in response to an inwardly directed B concentration gradient (160 mM) in a solution of the same
osmotic pressure as the original bath solution (see
“Materials and Methods”). The influx of B down its
concentration gradient induces swelling due to osmotically induced water influx. The rate of swelling can be
used as a measure of B permeability since water influx
is not rate limiting. Water transport in the oocytes was
also measured after replacing the incubation solution
with a 5-fold diluted solution. Swelling measurements showed that expression of both HvNIP2;1 and
GmNOD26 increased the permeability of Xenopus
oocytes to water (Fig. 5A), whereas only expression
of HvNIP2;1 increased the permeability of the oocytes
to B (Fig. 5B). Water-injected oocytes showed limited
permeability to water and were not permeable to B
(Fig. 5).
OsNIP2;1 Facilitates B Transport
We measured B accumulation in leaf blades of wildtype rice (cv Oochikara) and an OsNIP2;1 (lsi1) rice
mutant line with an Ala-132Thr mutation that results
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Aquaporin-Mediated Boron Toxicity Tolerance in Barley
support the observed reduction in leaf B content
of OsNIP2;1 mutant plants compared to wild-type
Oochikara rice grown hydroponically.
DISCUSSION
The Algerian barley landrace Sahara carries at least
four known loci for B toxicity tolerance, of which
two are related to lower leaf blade B concentrations.
These QTL are located on chromosomes 4H and 6H
(Jefferies et al., 1999). We recently identified the gene
underlying the 4H QTL as an efflux-type borate anion
transporter. Here we have identified a gene underlying the chromosome 6H QTL, the other locus contributing to the reduced shoot B concentration in barley.
We show that the gene, HvNIP2;1, facilitates B transport and that differential expression between Sahara
and Clipper barley may contribute to the reduced
levels of shoot B in Sahara. HvNIP2;1 is a member of
the MIP, or aquaporin superfamily and has recently
been described as a Si influx transporter (HvLsi1;
Chiba et al., 2008). Interestingly, Sahara barley is also
reported to have reduced levels of shoot Si compared
to Clipper barley (Nable et al., 1990).
Figure 5. Heterologous expression of HvNIP2;1 in Xenopus oocytes.
Increase in relative volume (V/V0) of Xenopus oocytes injected with
HvNIP2;1 cRNA, GmNod26 cRNA, or water (negative control), after
replacing the incubation solutions with A, a 5-fold diluted solution, or
with B, a 5-fold diluted solution supplemented with B. Data are means
(n = 5) 6 SD.
in loss of function as determined by measurements of
Si transport (Ma et al., 2006). Hydroponically grown
leaf blades of OsNIP2;1 mutant rice had significantly
lower levels of leaf B compared to wild-type Oochikara rice, both when supplied with low and excessive
concentrations of B in the growth solution (Fig. 6A).
We also expressed OsNIP2;1 mutant protein in yeast
to analyze the effect of the Ala-132Thr mutation on
B transport in yeast. While it is known that the Ala132Thr mutation disrupts Si transport through
OsNIP2;1 (Ma et al., 2006), it is unknown if this
mutation similarly affects B transport. Yeast expressing Ala-132Thr OsNIP2;1 mutant protein were more
sensitive to high concentrations of B than cells with an
empty vector (control cells), but grew better than yeast
expressing the OsNIP2;1 wild-type protein (Fig. 6B).
These data demonstrate that the mutation in OsNIP2;1
partially affects B transport through OsNIP2;1 and
Figure 6. Phenotypic effect of the OsNIP2;1 Ala-132Thr mutation. A,
Leaf blade B concentration of wild-type (cv Oochikara) rice plants and
OsNIP2;1 (lsi1) mutant plants after 14 d growth in hydroponics solution
containing 0 mM B or supplemental B to 1 mM. Data are means (n = 6) 6
SE. B, Heterologous expression of Oochikara OsNIP2;1 and mutant
OsNIP2;1 (lsi1) protein in yeast on minimal Gal media containing 5 mM
B. Two replicates of each strain are shown, with 10-fold serial dilutions
spotted from right to left across the plate.
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Schnurbusch et al.
HvNIP2;1 belongs to the NIP subfamily of plant
MIPs, members of which have been identified to
transport numerous small, uncharged solutes including B (Takano et al., 2006; Tanaka et al., 2008), Si (Ma
et al., 2006; Chiba et al., 2008; Mitani et al., 2009), As
(Bienert et al., 2008; Ma et al., 2008), Sb (Bienert et al.,
2008), and lactic (Choi and Roberts, 2007) acids, as well
as glycerol (Wallace and Roberts, 2005) and water.
Recent phylogenetic studies have classified plant NIPs
into three broad subgroups (Mitani et al., 2008; Rougé
and Barre, 2008), based on key amino acids forming
the ar/R selectivity filter. The archetype of the NIP
subfamily, GmNOD26, is unable to transport B (Fig. 5)
and falls into NIP subgroup I, while the Arabidopsis B
transport proteins, AtNIP5;1 and AtNIP6;1, cluster in
subgroup II and have a wider filter (Rougé and Barre,
2008). HvNIP2;1 and OsNIP2;1 have an even larger,
more open pore and are members of a third subgroup.
In fact, in a recent modeling study, several NIP subfamily members had the largest ar/R filter among 70
maize (Zea mays) and rice aquaporins (Bansal and
Sankararamankrishnan, 2007). Our molecular modeling indicates that the pore formed by HvNIP2;1 when
embedded in a lipid bilayer may be up to 8 Å wide at
the constriction, and is sufficiently large to allow for
the passage of B, As, Si, and Ge acids (Fig. 3).
Furthermore, we functionally characterized the
protein and used heterologous protein expression in
yeast and in Xenopus oocytes to show that HvNIP2;1
can facilitate the transport of B, Ge, As, and water
(Figs. 4 and 5), while Si transport has also been
demonstrated (Chiba et al., 2008). The B permeability we measured in yeast vesicles not expressing
HvNIP2;1 (1.0 3 1026 cm s21) is at the higher end of
the range of values reported in the literature for plant
membranes and lipid vesicles (Dordas and Brown,
2000; Dordas et al., 2000; Stangoulis et al., 2001) and it
is likely that a component of this permeability could
be attributed to other B transporters in the native
yeast membrane (Nozawa et al., 2006) in addition to
the lipid permeability that can be high depending on
lipid composition (Dordas and Brown, 2000). Despite
this intrinsically high permeability, HvNIP2;1 expression afforded a substantial increase in permeability
to 1.6 3 1026 cm s21. HvNIP2;1 appears to be able to
facilitate the transport of a broad range of small, near
apolar solutes, and we are currently introducing sitespecific mutations into HvNIP2;1 to investigate the
relationship between pore size and discrimination
between B and the more toxic As and Ge acid metalloids. It has also been reported that the NIP aquaporins
function in the acquisition of B under B-limiting
conditions (Takano et al., 2006), which raises the
question of whether the tolerance allele of HvNIP2;1
may confer a disadvantage to Sahara barley under
conditions of low B supply. This hypothesis could be
tested in material genetically fixed for other loci in
Sahara known to be involved in B uptake (Jefferies
et al., 1999). While this may be of low importance in
areas of high soil B, such as North Africa where the
Sahara landrace originated, it may be of more relevance in the target environments of modern barley
breeding programs where localized B deficiency may
be more common.
For in planta analyses we measured B accumulation
in a rice mutant (lsi1) defective in OsNIP2;1 and found
lower levels of shoot B in the mutant relative to wildtype Oochikara rice (Fig. 6A). We also demonstrated
that the Ala-132Thr mutation in OsNIP2;1 affects
growth of yeast differently to cells expressing the
wild-type OsNIP2;1 protein. The growth phenotype
we observed in yeast expressing mutant OsNIP2;1
was intermediate between yeast expressing wild-type
OsNIP2;1 and the empty vector control, suggesting
that the Ala-132Thr mutation did not completely abolish transport.
Northern and Q-PCR analyses of HvNIP2;1 RNA
levels in barley leaf blade and root tissues demonstrated that HvNIP2;1 is specifically expressed in roots,
and that there are higher levels of RNA in roots of the
B-sensitive barley variety, Clipper (Fig. 2). Significantly, we found higher expression in basal root segments, which was particularly evident in Clipper. Our
transcript data support the hypotheses that higher
HvNIP2;1 expression in Clipper increases the permeability of the roots to B, and that significant expression
is only found in mature regions of the root, where
apoplastic transport of solutes is inhibited by the
development of suberized/lignified Casparian bands
(Steudle and Peterson, 1998). Our observations of increased transcript levels of HvNIP2;1 in mature roots is
consistent with those of RNA levels of OsNIP2;1 in
roots of rice (Yamaji and Ma, 2007), and subsequent
observations of barley HvNIP2;1 expression (Chiba
et al., 2008). Immunohistological investigations showed
that expression of HvNIP2;1 protein in barley was
limited to the distal sides of cortical, epidermal, and
hypodermal cells, in regions of the root where a suberized layer was present (Chiba et al., 2008).
The observed differences in transcript levels of
HvNIP2;1 in Clipper and Sahara barley may be explained by a repeat insertion into the cis-regulatory
region, approximately 2 kb upstream of the translational start site (Supplemental Fig. S3). Repeat insertions in noncoding regions of genes have the ability to
alter gene expression in plants and animals, and result
in subtle or dramatic phenotypic changes. They are
considered to be a rich source of functional diversification (Kidwell and Lisch, 1997). Recently, a study of
rice genes containing insertions in putative promoter
regions found a positive relationship between the
distance of the repeat sequence from the start codon
and the likelihood of gene expression (Krom et al.,
2008). We cannot rule out the possibility that the repeat
insertion in the promoter region of Sahara barley
might cause altered tissue- or cell-specific expression
of HvNIP2;1 in the root. Together these findings indicate that reduced HvNIP2;1 expression should contribute to lower leaf blade B accumulation and greater
tolerance of barley to excessive soil B.
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Copyright © 2010 American Society of Plant Biologists. All rights reserved.
Aquaporin-Mediated Boron Toxicity Tolerance in Barley
B toxicity is a significant agronomic problem in a
number of cereal growing regions across the world,
including West Asia, Northern Africa, and Southern
Australia. The discovery of key genes responsible for B
toxicity tolerance in cereals presents opportunities for
the development of elite lines adapted for growth in
high B soils, through either genetic engineering or
conventional breeding. HvNIP2;1 is the second B toxicity tolerance gene to be identified in barley. Tolerance
to high B may be achieved by a loss of function of this
gene and, therefore, there is significant potential for
the development of B-tolerant germplasm derived
from identification of individuals with mutations in
HvNIP2;1. We are currently undertaking a search for
suitable mutants for this purpose.
sequences were used as input parameters to generate three-dimensional
(3D) models of HvNIP2;1 in complex with B, As, Si, and Ge acids and four
water molecules, using modeller 9v6 (Sali and Blundell, 1993). A 3D model of
OsNIP2;1 in complex with B acid was also constructed. The coordinates of B
and As acids were taken from the Protein Data Bank, while tetragonally
coordinated Si and Ge acids were generated from Si acid diester and
dimethylammonium gallotrigermanate, respectively, deposited in the Cambridge Crystallographic Data Centre. Models with the lowest value of the
modeller 9v5 objective function were chosen for evaluation. The overall
G-factors (estimates of stereochemical parameters) as evaluated by PROCHECK (Laskowski et al., 1993), were 0.45 for AqpM and between 0.03 and
0.08 for HvNIP2;1 in complex with metalloids. The z-score values (Sippl,
1993), reflecting combined statistical potential energy, for AqpM and the
HvNIP2;1 models were 27.98 and between 25.23 and 25.47, respectively. The
super algorithm in PyMol (DeLano, 2009) was used to determine the rootmean-square-deviation values in the Ca positions between the models and
their template. The root-mean-square-deviation values, excluding three deletions of modeled HvNIP2;1, were 0.31 to 0.36 Å over 208 superposed residues.
Buriedness of HvNIP2;1 was calculated by PocketPicker plug-in (Weisel et al.,
2007) in PyMol. Molecular graphics were generated with PyMol (DeLano,
2009).
MATERIALS AND METHODS
Plant Growth and Analyses
We used the parent barley (Hordeum vulgare) lines Sahara and Clipper, and
lines derived from Clipper 3 Sahara F1 doubled haploids. The rice (Oryza
sativa) cv Oochikara and mutant line lsi1 were obtained from Prof. J.F. Ma
(Okayama University, Japan). Seeds of barley or rice were germinated on filter
paper and grown hydroponically in a base nutrient solution as described
previously (Sutton et al., 2007). The seedlings were grown in controlled
conditions (15°C night/23°C day with a 14-h photoperiod for barley, and 24°C
night/28°C day with a 16-h photoperiod for rice). Plant material was
oven dried and analyzed by inductively coupled plasma optical emission
spectrometry.
Genetic Mapping of HvNIP2;1
The F3 mapping population was derived from a cross between two Clipper 3 Sahara F1-derived doubled haploids, which differed for alleles at the B
tolerance locus on 6H but not for alleles at other known B tolerance loci. The
population was fixed for the Sahara allele of Bot1 on chromosome 4H. F2
plants selected as heterozygous for the 6H B toxicity tolerance locus region
were used to generate F3 seed for recombinant screening. Cleaved amplified
polymorphic sequence markers were developed using barley ESTs related to
genes from the corresponding interval on rice chromosome 2. Markers are
listed in Supplemental Table S1. F3 recombinants for the region were marker
selected and their tolerance phenotype was determined by measuring leaf
blade B accumulation in F4 progeny individuals. To enable precise scoring of
the QTL, the same progeny plants were scored for markers to follow the
inheritance of recombinant and nonrecombinant 6H chromosomes, to confirm
when the observed variation in B accumulation was controlled by segregation
at the 6H locus.
Q-PCR and Northern Hybridization
Q-PCR assays were performed as described previously (Sutton et al., 2007).
Primers (Supplemental Table S1) contained no mismatches to the corresponding HvNIP2;1 sequences. Northern hybridization was performed using standard methods.
Construction of a Three-Dimensional Molecular Model
of HvNIP2;1
We used an archaeal aquaporin, AqpM, from Methanobacterium thermoautotrophicum (Protein Data Bank accession no. 2evu; Lee et al., 2005) as a
structural template for modeling. The template structure was aligned with
HvNIP2;1 using MUSTER (Wu and Zhang, 2008), after truncation of the
HvNIP2;1 sequence at the NH2- and COOH-termini to eliminate nonstructural
sequence. The alignment was checked manually to maintain the integrity of
secondary structural elements, using Robetta (Kim et al., 2004) and hydrophobic cluster analysis (Callebaut et al., 1997). The structurally aligned
Heterologous Expression
NIP2;1 cDNA sequences were subcloned into the Gateway vector pCR8
and transferred via recombination to pYES-DEST52 (Invitrogen) for Galinducible expression in yeast (Saccharomyces cerevisiae). We used the yeast
strains INVSc2 (MATahis3-D1ura3-52) and SY1 (MATa,ura3-52,leu2-3,112,his4619,sec6-4,GAL) in this study, and lithium-acetate transformation (Gietz and
Woods, 2002). Yeast was routinely cultured in liquid or solid minimal media
containing 2% (w/v) Glc or 2% (w/v) Gal as described previously (Sutton
et al., 2007). For assessment of phenotypes, 10-fold dilutions of precultured
yeast cells were deposited onto solid media containing B (pH 5.5), GeO2
(Aldrich), or As2O3 (Sigma). Alternatively, precultured cells were added to
liquid medium supplemented with B, with growth assessed by measuring
A600 of samples using a spectrophotometer. For measurement of permeability
to B, we used SY1, a temperature-sensitive sec6-4 mutant yeast strain.
Galactose cultures of SY1, transformed with or without HvNIP2;1, were
grown at 22°C to an A600 of approximately 1, harvested by centrifugation,
resuspended in fresh medium, and incubated for 2 h at 37°C to induce the
accumulation of vesicles. Vesicles were isolated as described previously
(Coury et al., 1999) and resuspended in a buffer of 0.3 M Suc and 25 mM
Tris-HCl (pH 7.0) with added protease inhibitor (Sigma P8125; 3.8 mL mL21).
Permeability of the vesicle preparations to B was measured using a stopped
flow spectrophotometer (DX.17MV, Applied Photophysics), where reswelling
of vesicles (at 21.5°C) was measured by 90° light scattering (500 nm) upon
rapidly mixing with a solution that created an inwardly directed concentration gradient for B in 25 mM Tris-HCl buffer (pH 7.0), containing 0.3 M Suc and
0.4 M H3BO3. The reswelling kinetics was fitted to a single exponential
function, from which the rate constant was obtained. The diameter of the
vesicles was determined using dynamic light scattering (NICOMP 380,
Particle Sizing Systems) operating in a vesicle mode and was weighted on
numbers of vesicles.
We performed cRNA transcription for Xenopus oocyte expression, using a
T7 RNA polymerase kit (mMessage mMachine, Ambion). Xenopus oocytes
were surgically removed and defolliculated, then injected with 30 ng cRNA or
water, using a microinjector (Drummond Nanoject II automatic nanolitre
injector, Drummond Scientific). The oocytes were allowed to recover for 3 d at
18°C in ND96 in 5 mM HEPES-NaOH buffer, pH 7.4 containing 96 mM NaCl,
2 mM KCl, 1.8 mM CaCl2, and 1 mM MgCl2 (osmolarity 199 mOsmol kg21), with
added antibiotics (tetracycline at 5 mg mL21, penicillin, and streptomycin at
10 mg mL21, all from Sigma).
To measure water permeability, oocytes were transferred to a 5-fold diluted
solution of ND96 (osmolarity 47 mOsmol kg21). To assess permeability to B,
oocytes were transferred to a 5-fold diluted solution of ND96 supplemented
with 160 mM B (osmolarity 201 mOsmol kg21). Oocyte volume change was
derived from images captured at 5 s intervals for 6 min under a dissecting
microscope, and data were analyzed using GLOBAL LAB Image/2 software
(Data Translation Inc.).
Sequence data from this article can be found in the GenBank/EMBL data
libraries under accession numbers GQ496519 to GQ496522.
Plant Physiol. Vol. 153, 2010
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Copyright © 2010 American Society of Plant Biologists. All rights reserved.
Schnurbusch et al.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Determination of chromosome 6H B toxicity
tolerance locus genotypes.
Supplemental Figure S2. Protein sequence alignment of HvNIP2;1 and
OsNIP2;1.
Supplemental Figure S3. Alignment of Clipper and Sahara HvNIP2;1
nucleotide sequences immediately upstream of the 5# ATG start site.
Supplemental Figure S4. Growth of yeast containing empty vector (black
circles) compared to yeast expressing HvNIP2;1 (white circles) in liquid
Gal minimal media, containing A, no added B, or B, 5 mM B.
Supplemental Figure S5. Growth of yeast containing empty vector and
those transformed with vector containing HvNIP2;1 on solid Glc media
(to repress transcription from GAL1 promoter) containing A, no added
B, B, 5 mM B, and C, 10 mM B.
Supplemental Table S1. Primers used in this work.
ACKNOWLEDGMENTS
We thank M. Pallotta, A. Hay, A. Okada, T. Oz, and N. Shirley for technical
assistance; N. Collins for propagation of mapping lines; and J.F. Ma for
provision of the rice mutant lsi1.
Received May 4, 2010; accepted June 21, 2010; published June 25, 2010.
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