The Plant Cell, Vol. 14, 3009–3028, December 2002, www.plantcell.org © 2002 American Society of Plant Biologists
OSM1/SYP61: A Syntaxin Protein in Arabidopsis Controls
Abscisic Acid–Mediated and Non-Abscisic Acid–Mediated
Responses to Abiotic Stress
Jianhua Zhu,a Zhizhong Gong,b Changqing Zhang,b Chun-Peng Song,b Barbara Damsz,a Günsu Inan,a
Hisashi Koiwa,c Jian-Kang Zhu,b Paul M. Hasegawa,a and Ray A. Bressana,1
a Center
for Plant Environmental Stress Physiology, Department of Horticulture and Landscape Architecture, Purdue
University, West Lafayette, Indiana 47907-1165
b Department of Plant Sciences, University of Arizona, Tucson, Arizona 85721
c Department of Horticultural Sciences, Texas A&M University, College Station, Texas 77843-2133
To identify the genetic loci that control salt tolerance in higher plants, a large-scale screen was conducted with a bialaphos marker–based T-DNA insertional collection of Arabidopsis ecotype C24 mutants. One line, osm1 (for osmotic
stress–sensitive mutant), exhibited increased sensitivity to both ionic (NaCl) and nonionic (mannitol) osmotic stress in a
root-bending assay. The osm1 mutant displayed a more branched root pattern with or without stress and was hypersensitive to inhibition by Na, K, and Li but not Cs. Plants of the osm1 mutant also were more prone to wilting when
grown with limited soil moisture compared with wild-type plants. The stomata of osm1 plants were insensitive to both
ABA-induced closing and inhibition of opening compared with wild-type plants. The T-DNA insertion appeared in the
first exon of an open reading frame on chromosome 1 ( F3M18.7, which is the same as AtSYP61). This insertion mutation cosegregated closely with the osm1 phenotype and was the only functional T-DNA in the mutant genome. Expression of the OSM1 gene was disrupted in mutant plants, and abnormal transcripts accumulated. Gene complementation
with the native gene from the wild-type genome completely restored the mutant phenotype to the wild type. Analysis of
the deduced amino acid sequence of the affected gene revealed that OSM1 is related most closely to mammalian syntaxins 6 and 10, which are members of the SNARE superfamily of proteins required for vesicular/target membrane fusions. Expression of the OSM1 promoter::-glucuronidase gene in transformants indicated that OSM1 is expressed in all
tissues except hypocotyls and young leaves and is hyperexpressed in epidermal guard cells. Together, our results demonstrate important roles of OSM1/SYP61 in osmotic stress tolerance and in the ABA regulation of stomatal responses.
INTRODUCTION
It is now well recognized that plants respond to abiotic
stresses by altering the expression of many genes and that
this altered expression is a major mechanism leading to adaptation and survival during periods of stress (Hasegawa et
al., 2000). In the past several years, considerable effort has
been expended to determine the particular genes that control the perception of environmental stresses and the subsequent activation of gene expression contributing to adaptation (Zhu et al., 1997; Hasegawa et al., 2000; Bohnert and
Bressan, 2001; Kawasaki et al., 2001).
Although not all genes induced in plant responses to osmotic stress require abscisic acid (ABA) mediation (Grillo et
1 To whom correspondence should be addressed. E-mail bressan@
hort.purdue.edu; fax 765-494-0391.
Article, publication date, and citation information can be found at
www.plantcell.org/cgi/doi/10.1105/tpc.006981.
al., 1995; Shinozaki and Yamaguchi-Shinozaki, 1997), this
important plant hormone plays a crucial role in adaptation to
abiotic stress, and the level of expression of several genes
has been shown to be affected by exogenous application of
ABA or correlated with the amount of endogenous ABA, including some genes thought to be involved in osmotic or
dehydration tolerance (Oliver, 1996; Oliver and Bewley,
1997; Leung and Giraudat, 1998). Also, specific cis elements of ABA-targeted genes and trans factors that interact
with and activate promoters with such cis elements have
been identified (Shinozaki and Yamaguchi-Shinozaki, 1997;
Leung and Giraudat, 1998). The role of ABA in the adaptation to environmental stress can be attributed to its effects
on different plant tissues and stages of development and
physiological processes. In particular, these include effects
on seed dormancy and germination (Leung and Giraudat,
1998), seedling and plant growth (Leung and Giraudat, 1998;
Sharp, 2001), and stomatal function (Leung and Giraudat,
1998).
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Although effects of ABA on gene expression in epidermal
guard cells have not been excluded, overwhelming evidence
indicates that ABA helps to control osmotic stress tolerance
through direct (nontranscriptional) effects on guard cell water relations that control stomatal opening and closing
(Leung and Giraudat, 1998; Schroeder et al., 2001). Specifically, ABA has been shown to play a crucial role in stomatal
function through Ca2-dependent and at least partially independent mechanisms that affect the activities of the guard
cell plasma membrane and tonoplast-localized ion channels, principally S-type anion channels and inward- and outward-rectifying K channels (Leung and Giraudat, 1998;
Schroeder et al., 2001). In addition, ABA induces the alkalinization of the guard cell cytosol, which increases K channel
efflux activity (Leung and Giraudat, 1998; Schroeder et al.,
2001).
Using exogenously applied ABA at concentrations capable of inhibiting the germination of wild-type Arabidopsis
seeds, several ABA-insensitive mutants have been isolated, and the affected loci for ABI1 to ABI5 have been
identified as encoding two protein phosphatase 2Cs ( ABI1
and ABI2) and B3 (ABI3), AP2 (ABI4), and basic domain/
Leu zipper (ABI5) transcription factors (Giraudat et al.,
1992; Finkelstein et al., 1998; Finkelstein and Lynch, 2000;
Merlot et al., 2001). The 2C phosphatases encoded by
ABI1 and ABI2 also control stomatal responses to ABA.
Through mutational analysis of Arabidopsi s, a number of
other loci have been identified that control stomatal and
other responses to ABA (e.g., ERA1, ERA2, ERA3, FUS3,
LEC1, GCA, and GPA) (Leung and Giraudat, 1998; Schroeder
et al., 2001). Most recently, unexpected loci that encode
proteins involved in RNA metabolism (Hugouvieux et al., 2001;
Xiong et al., 2001a) and a RHO-like GTPase (Hugouvieux et
al., 2001; Lemichez et al., 2001) that controls guard cell
cytoskeleton responses to ABA also have been found to
mediate stomatal function. In addition, genetic loci in
other species have been found to affect ABA-mediated responses, including those of stomata (Leung and Giraudat,
1998).
One of the most intriguing genes that has been linked with
ABA-mediated responses encodes a SNARE-like protein
(for soluble NSF [N-ethylmaleimide–sensitive factor] adaptor
protein receptor) from tobacco (Leyman et al., 1999). Using
a gain-of-function assay involving the transfer of plant
mRNA to frog oocytes and subsequently isolating a specific
mRNA that is required for ABA-mediated Cl channel activity, the SNARE mRNA was identified (Leyman et al., 1999).
Its role in the ABA-mediated control of plant guard cell Cl channels was confirmed by observing that pharmacological
inhibition of SNARE function also inhibited the ABA-induced
Cl channel current in isolated guard cell protoplasts (Leyman
et al., 1999).
SNARE proteins are elements of a core complex that is
membrane anchored and required for the fusion of vesicles
with target membranes and as such can be categorized
within the SNARE superfamily as v (vesicle)-SNAREs and t
(target)-SNAREs (Jahn and Südhof, 1999). The v-SNAREs
are represented by the well-characterized synaptobrevin/
VAMP (vesicle-associated membrane protein) commonly
found on nerve synaptic vesicles, and the t-SNAREs are
represented by syntaxins (Sanderfoot and Raikhel, 1999)
that are present on target membranes. The function of
SNARE proteins in membrane fusion is complex and occurs
in association with numerous other cofunctional elements
(Springer and Schekman, 1998; Jahn and Südhof, 1999;
Almers, 2001; Pelham, 2001). However, in addition to the
role of SNARE proteins in vesicular fusion control, they have
been shown to participate in the activation of both Ca 2
and Cl channels in eukaryotic systems (Naren et al., 1998;
Catterall, 2000). The ability of SNARE proteins to control
membrane Ca2 and Cl ion channel activity is consistent
with the finding of Leyman et al. (1999) that the tobacco
syntaxin SNARE protein (which is most closely related by
sequence comparison to the Arabidopsis gene At-SYR1,
which is the same as AtSYP121/F26K24.11) (Sanderfoot et
al., 2000) is required for the ABA-mediated Cl flux in oocytes and apparently in stomatal guard cell membranes as
well.
The completion of the Arabidopsis genome sequence has
afforded a complete view of the syntaxin family of proteins
from a plant species. Sanderfoot et al. (2000) have classified
the 24 Arabidopsis syntaxins into eight groups, SYP1 to
SYP8 (for syntaxin from plants) based on sequence identity.
Of the large group of Arabidopsis syntaxins, few have been
associated with particular roles in phenotypes using genetic
analyses. The KNOLLE locus encodes a syntaxin (AtSYP111)
that is required for cytokinesis (Lukowitz et al., 1996), and
disruptions of syntaxin genes from families 2 and 4 are lethal (Sanderfoot et al., 2001b).
In a screen for Arabidopsis insertion-tagged mutants with
altered responses to salinity stress, we isolated a particular
mutant, osm1, with impaired root growth in the presence of
140 mM NaCl. Identification of the mutated locus that is responsible for this altered phenotype revealed that it encodes a member of the SNARE superfamily, syntaxin SYP61
(Sanderfoot et al., 2000). The osm1 mutant displays several
interesting features, including decreased root growth and
seed germination in the presence of NaCl. Roots of osm1
are more highly branched than wild-type roots, even in the
absence of NaCl stress. When osm1 plants are grown in soil
with limited water availability or with added NaCl, they display severe wilting symptoms and decreased growth and
survival. The stomata of osm1 plants exhibit both impaired
induction of closing and inhibition of opening by exogenously
added ABA. Promoter fusion studies with OSM1/SYP61
promoter::-glucuronidase (GUS) indicate that OSM1/SYP61
is highly expressed in both root and leaf tissues but is expressed even more intensely in epidermal guard cells. These
results are consistent with the evidence presented by Leyman
et al. (1999) that syntaxin proteins can function in plants to
mediate the ABA control of guard cell ion channel activity. In
addition, our findings indicate that OSM1/SYP61 also medi-
Syntaxin Controls ABA and Stress Responses
ates the development and growth sensitivity of roots to osmotic stress.
RESULTS
Identification of the osm1 Mutant in a
T-DNA–Tagged Population
We generated a large (300,000) T-DNA–tagged population
of Arabidopsis mutants by transforming plants (ecotype C24
carrying a homozygous RD29A::luciferase [LUC] transgene)
using the binary vector pSKI015. Zhu et al. (1998) reported
that screens for sos (salt overly sensitive) mutants were saturated using low concentrations ( 100 mM) of NaCl. Therefore, our screen was conducted using a higher concentration (140 or 160 mM) of NaCl in an attempt to prevent the
reisolation of sos mutants. Seeds of 65,000 independent T2
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lines were screened in a root-bending assay (Wu et al.,
1996). From these insertion lines, we identified 100 saltsensitive isolates, which were found to be stable genetic
mutants. One of the stable mutants was named osm1 (for
osmotic stress–sensitive mutant). Seedlings of osm1 bent and
grew normally on control Murashige and Skoog (1962) (MS)
medium but formed more branches and root hairs. On medium
supplemented with 140 mM NaCl, osm1 mutant seedlings exhibited greatly reduced growth (Figures 1A and 1B).
osm1 Is Sensitive to General Osmotic Stress
To determine the specificity of the salt sensitivity of the
osm1 mutant, the root elongation of seedlings was examined in the presence of several different ions and in medium
containing mannitol as the osmotic chemical. The osm1
plants were sensitive to NaCl, KCl, and LiCl but not to CsCl
(Figure 2). Because osm1 seedlings had increased sensitivity to 10 mM LiCl but not to 10 mM CsCl, the OSM1 gene
Figure 1. Isolation of osm1 by the Root-Bending Assay.
Four-day-old seedlings were transferred from MS medium to MS medium containing 140 mM NaCl, and the seedlings (with roots upside down)
were allowed to grow for an additional 7 days. Mutant sos3 plants (Liu and Zhu, 1997) in the ecotype Columbia background were used as positive controls.
(A) Control treatment (0 mM NaCl).
(B) Treatment with 140 mM NaCl.
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Figure 2. The osm1 Mutant Is Sensitive to Na, K, and Li but Not to Cs.
Four-day-old seedlings grown on MS agar plates placed vertically were transferred to MS medium or MS medium supplemented with different
concentrations of NaCl (A), KCl (B), LiCl (C), or CsCl (D). Root elongation was measured after an additional 7 days of growth and is shown as a
percentage of relative growth (compared with growth on normal MS medium). Closed circles, wild type; open circles, osm1. Error bars represent
standard deviations (n 15).
appears to affect sensitivity to cations rather than to Cl .
Figures 3A and 3B show that osm1 also was sensitive to
general osmotic stress induced by mannitol. Together, the
results shown in Figures 2, 3A, and 3B indicate that the osm1
mutation leads to a defective osmotic stress response.
osm1 Seed Germination and Seedling Growth Are
Sensitive to Inhibition by NaCl but Not by ABA
The sensitivity of plants to salt stress often depends on the
stage of development (Wu et al., 1996). Therefore, we tried
to determine if osm1 seed germination also is sensitive to
NaCl. Germination of osm1 seeds was found to be more
sensitive to NaCl than germination of wild-type seeds at
concentrations of 10 mM or higher (Figure 3C). The ability of
osm1 seeds to germinate in the presence of ABA also was
examined, and we found that ABA inhibited both osm1 and
wild-type seed germination similarly (Figure 3D). Likewise,
the root growth of wild-type and osm1 seedlings was inhibited equally by ABA (Figure 4).
osm1 Mutant Plants Have Increased Sensitivity to Salt
and to Soil Desiccation
The growth and development of osm1 plants in soil in the
greenhouse in the absence of stress appeared normal, as
shown in Figures 5A and 5B. When the soil was allowed to
dry by withholding water, osm1 plants became wilted 10
days after the withdrawal of water, whereas wild-type plants
did not begin to wilt until 14 days after watering was discontinued (Figure 5C and data not shown). Under conditions of
soil water deficit, the number of wilting plants and the number of plants that recovered after water was given again
were quantified, as shown in Figure 5D. These results indicate that osm1 plants had substantially increased sensitivity
to water deficit stress. Plants of osm1 and the wild type also
were examined in the greenhouse for sensitivity to NaCl.
Compared with wild-type plants, osm1 plant growth was reduced much more by watering with solutions containing
NaCl (Figures 6A and 6B), exhibiting reduced vigor and
growth at NaCl concentrations as low as 150 mM (data not
shown) and at higher NaCl concentrations (Figure 6C).
Syntaxin Controls ABA and Stress Responses
OSM1 Does Not Appear to Function Directly in the
SOS Pathway
The most well-defined signal pathway that mediates the
response of plants to osmotic stress has been described
from the characterization of several mutants of Arabidopsis
that display sensitivity to NaCl. The sos mutants have led
to the identification of a Ca 2-mediated response that appears to activate a Na efflux mechanism at the plasma
membrane (Zhu, 2001a, 2001b). Because all of the sos mutants have impaired growth on low-potassium medium, we
sought to determine whether osm1 plants are affected
similarly; if so, they could have a defect in the same response pathway as sos mutants. Wild-type and osm1
seedlings grown on MS medium were transferred to modified MS medium containing various levels of KCl. Root
growth was measured 9 days after the transfer. Root
growth in the first 2 days was not included to minimize the
effect of residual K carried from the seedlings transferred
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from the MS medium. Figure 7A shows that wild-type and
osm1 mutant seedlings required a similar level of potassium to reach maximum growth. Na and K contents also
were measured, as shown in Figures 7B and 7C. Together,
these data indicate that the osm1 mutation does not affect
the sos pathway directly and does not lead to altered uptake of K or Na ions.
Expression of RD29A::LUC and Other
Stress-Responsive Genes in osm1 Mutant Plants
The osm1 mutant was isolated from a collection of plants
carrying a chimeric transgene, RD29A::LUC. The RD29A
gene is responsive to ABA and the osmotic related stresses
NaCl, cold, and desiccation (Shinozaki and YamaguchiShinozaki, 1997). We examined whether RD29A::LUC expression is altered in the mutant plants. Results presented in
Figure 8 demonstrate no detectable difference between
Figure 3. The osm1 Mutation Affects General Osmotic Stress Sensitivity and Sensitivity of Seed Germination to NaCl but Not to ABA.
(A) Time course of root growth on 250 mM mannitol. Closed circles, wild type; open circles, osm1.
(B) Relative root growth on different levels of mannitol. Four-day-old seedlings grown on MS agar plates placed vertically were transferred to MS
medium or MS medium supplemented with different levels of mannitol. Root elongation was measured after an additional 7 days of growth and
is shown as a percentage of relative growth (compared with growth on normal MS medium). Closed circles, wild type; open circles, osm1.
(C) and (D) Seeds were sown on normal MS medium or MS medium supplemented with the indicated concentrations of NaCl (C) or ABA (D).
The percentage of seeds that had germinated 7 days after sowing was determined. Open bars, wild type; closed bars, osm1.
Error bars represent standard deviations (n 15 for [A] and [B]; n 3 for [C] and [D]).
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Identification of the OSM1/SYP61 Gene
Figure 4. Growth Sensitivity to ABA Is Similar in Wild-Type and
osm1 Mutant Plants.
Four-day-old seedlings grown on MS medium were transferred to
MS medium supplemented with the indicated concentrations of
ABA. Growth of roots was determined 7 days after transfer. Open
bars, wild type; closed bars, osm1. Error bars represent standard
deviations (n 15).
wild-type and osm1 mutant plants in RD29A::LUC expression.
This finding indicates either that OSM1 is not involved in the
signal transduction pathway(s) that controls RD29A gene
expression or that its role is redundant to that of another
component. However, the induction of endogenous RD29A
by ABA treatment was delayed in osm1 plants (Figure 9).
The expression of two other salt-induced genes, RD22 and
COR15a, was not significantly different between osm1 and
wild-type plants (Figure 9).
Genetic Analysis of osm1 Mutant Plants
The osm1 mutant was backcrossed to the wild type. Phenotype analysis of the F1 progeny showed that the osm1 mutation is recessive. Selfed F2 seedlings segregated 3:1 for
wild-type:osm1 phenotypes (Table 1). Therefore, the osm1
phenotype is caused by a single recessive nuclear mutation.
All of the F1 progeny were resistant to bialaphos (the activation-tagging vector carries the bialaphos-resistant gene),
and all (263) of the plants selected from the segregating F2
progeny with the osm1 phenotype were bialaphos resistant
(Table 1). Of 1635 seedlings tested, 1205 were resistant to
bialaphos and 430 were sensitive to bialaphos (Table 1).
These results are consistent with the conclusion that there is
only one functional T-DNA in the genome of osm1 plants
and that it closely cosegregates with the locus affected in
osm1 mutant plants.
Molecular cloning of the genomic DNA flanking the left border of the T-DNA insert of osm1 plants was achieved by
thermal asymmetric interlaced PCR (TAIL-PCR). A 0.6-kb
genomic DNA flanking the T-DNA left border was isolated,
and the TAIL-PCR product was sequenced. Database searches
located this sequence to a region within a sequenced BAC
(F3M18) on Arabidopsis chromosome 1. The T-DNA tag was
located at nucleotide 25,953 in BAC F3M18, a position 526
bp upstream of the predicted ATG translation start site of
gene F3M18.7/AtSYP61 (At1g28490; Sanderfoot et al., 2000),
with its right border closer to this gene (Figure 10A). We further analyzed the genomic DNA flanking the right border of
the T-DNA insert in osm1 plants by performing PCR and sequencing the PCR product (see Methods for details). There
is an 82-bp deletion of the genomic DNA (nucleotides 25871
to 25952 in BAC clone F3M18) and a 46-bp deletion inside
the T-DNA insert (nucleotides 4249 to 4294), including the
T-DNA right border (nucleotides 4270 to 4294). These deletions probably resulted from the T-DNA integration event.
Database searches revealed an EST and a full-length cDNA
clone (R19864) available for the OSM1/SYP61 gene. Analysis of the sequences of the OSM1/SYP61 EST and the fulllength cDNA indicated that the T-DNA insertion that was
identified in the osm1/syp61 mutant genome eliminated the
original transcription start site (nucleotide 25,923 in BAC
clone F3M18) of OSM1/SYP61.
We then designed two pairs of PCR primers to determine whether this T-DNA insert is homozygous or heterozygous on chromosome 1 of plants that are homozygous for the osm1/syp61 mutant phenotype. Figure 10B
shows the scheme of these PCR results. Using a combination of forward and reverse primers that were both 250
bp outside of the T-DNA integration site, PCR yielded an
appropriately sized band from the genomic DNA template
from wild-type DNA but not from osm1/syp61 mutant genomic DNA. Using the combination of a reverse primer and
the primer from the T-DNA left border (LB3; see Methods
for details), PCR did not produce a band from wild-type
template DNA but did produce a slightly smaller specific
band from osm1/syp61 template DNA (Figure 10C). We
performed this type of PCR with genomic DNA prepared
from 20 plants that displayed the osm1/syp61 mutant phenotype from a segregating F2 population and found no evidence for the presence of a wild-type OSM1/SYP61 allele
(Figure 10C). These results indicate that the T-DNA insert
is homozygous on chromosome 1 and cosegregates
closely with the locus responsible for the osm1/syp61 mutant phenotype.
Complementation of the osm1/syp61 Mutation
To unequivocally prove that the osm1/syp61 phenotype is
the consequence of the T-DNA insertion, we cloned the ge-
Syntaxin Controls ABA and Stress Responses
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Figure 5. osm1 Mutant Plants Are Sensitive to Soil Moisture Deficit.
Three-week-old greenhouse-grown plants were fully watered, and the number of wilted plants 16 days after watering was discontinued was
scored and is represented as the percentage of the total plants (from 30 plants of each genotype and four replicates each). Wilted plants then
were given water again (on day 17 after water was withheld), and the number of recovered plants (fully regained turgor and resumed growth) was
scored after an additional 4 days (day 21) and is represented as the percentage of the total plants wilted.
(A) Three-week-old wild-type (left) and osm1 (right) plants grown in soil before watering was discontinued.
(B) and (C) The positions of plants are the same as in (A) after an additional 16 days of continued watering (controls) (B) and 17 days after water
was withheld (C).
(D) Percentage of plants scored as wilted on day 17 and of plants scored as surviving after watering was resumed for 4 additional days (day 21;
recovered). Open bars, wild type; closed bars, osm1. Error bars represent standard deviations.
nomic fragment of F3M18.7 with its own promoter into binary vector pCAMBIA 1303 and introduced this construct
into osm1/syp61 mutant plants. We found that this fragment
was sufficient to reverse the salt sensitivity of root growth of
osm1/syp61 seedlings (Figure 11A). Of 50 independent lines
from the selfed T1 plants that had been transformed with
the wild-type F3M18.7 gene, 45 lines exhibited a clear wildtype phenotype on agar plates containing 140 mM NaCl.
Growth measurements indicate that the addition of a new
wild-type allele of this locus was able to completely complement the mutant phenotype (Figures 11B and 11C). Introduction of the empty expression vector alone did not cause
any phenotypic change of the osm1/syp61 mutant (data not
shown). Plants of the osm1/syp61 mutant that were transformed with the wild-type allele of OSM1/SYP61 also displayed the wild-type stomatal transpiration phenotype (see
Figure 13B). Therefore, the F3M18.7 gene corresponds to
the OSM1/SYP61 locus.
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The T-DNA Insertion Disrupts the Normal Expression of
the OSM1/SYP61 Gene
Figure 6. osm1 Plants Are Sensitive to Salt Treatment in Soil.
One-month-old plants were watered with different concentrations of
NaCl every 3 days for 3 additional weeks (25 plants of each genotype). Photographs were taken at the end of NaCl treatment, and
shoot fresh weight was measured.
(A) Wild-type plants treated with (from left to right) 0, 200, 300, or
400 mM NaCl.
(B) osm1 mutant plants treated as in (A).
(C) Shoot fresh weight after each treatment. Open bars, wild type;
closed bars, osm1. Error bars represent standard deviations (n 20).
Because the T-DNA insertion site is inside the open reading frame of F3M18.7, we examined whether the expression of this open reading frame is altered. The T-DNA insertion resulted in an altered pattern of OSM1/SYP61
transcript accumulation (Figure 9). The major transcript
that accumulates in osm1/syp61 mutant plants is smaller
than the one that accumulates in wild-type plants. The
osm1/syp61 mutant also appears to accumulate a small
amount of mRNA that is slightly larger than that observed
in wild-type plants (as observed on 2.5% agarose gels;
data not shown). Performing 5 rapid amplification of
cDNA ends (RACE) on both wild-type and osm1/syp61
mutant plants revealed that the transcription of OSM1/
SYP61 in wild-type plants starts from nucleotide 25,923 in
BAC clone F3M18, as indicated by the full-length cDNA
R19864, which gives rise to the normal transcript of
OSM1/SYP61, as we observed with RNA gel blots. RACE
analysis also revealed that the transcription of OSM1/
SYP61 in osm1/syp61 mutant plants starts from nucleotides 25,852 and 25,847 on BAC clone F3M18, respectively, which could explain the presence of both slightly
shorter and longer transcripts of OSM1/SYP61 in our RNA
gel blot analysis. The transcript in osm1/syp61 plants that
starts from nucleotide 25,852 on BAC clone F3M18 includes the first intron, but the DNA sequences from nucleotides 25,818 to 25,731 are not present in this transcript.
This would produce a transcript 240 bp longer than the
transcript found in wild-type plants. The transcript in
osm1/syp61 plants that starts from nucleotide 25,847 on
BAC clone F3M18 could produce a transcript 76 bp
shorter than that found in the wild type, because RACE
analysis determined that the first intron is spliced normally. OSM1/SYP61 mRNA levels in wild-type plants were
increased only slightly by NaCl, LiCl, and ABA treatments.
Interestingly, the abnormally smaller transcript of osm1/
syp61 mutant plants was induced much more strongly by
NaCl, LiCl, ABA, and especially cold treatment than it was
in wild-type plants (Figure 9). The function, if any, of the
detected transcripts is uncertain. Considering this possibility, and the possibility that undetected normal protein
still could be present, osm1/syp61 plants are not necessarily null mutants.
OSM1/SYP61 Encodes a Syntaxin Protein
Analysis of the full-length cDNA and the EST sequences
also indicated that OSM1/SYP61 is composed of eight exons and seven introns. OSM1/SYP61 encodes a 245–amino
acid protein with a predicted molecular mass of 27,734.18 D
(Figure 12A). The OSM1/SYP61 protein has been studied and
characterized by Sanderfoot et al. (2001a), and comparison
of the predicted OSM1/SYP61 amino acid sequence with
Syntaxin Controls ABA and Stress Responses
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those of other gene products in the database revealed
that OSM1/SYP61 shares greatest sequence similarities with
t-SNARE–type syntaxins from mammals (Figure 12B) (Jahn
and Südhof, 1999; Sanderfoot et al., 2000). The OSM1/SYP61
sequence also predicts structural characteristics similar to
those of the syntaxin family of vesicle transport proteins
(Bock et al., 1996; Sanderfoot et al., 2000). It has a predicted
transmembrane domain (outside to inside helix, from amino
acids 225 to 243; 85% probability) at the C-terminal end. The
protein also is predicted to have a relatively low pI of 4.97.
OSM1/SYP61 contains sequences with high identity to the
conserved t-SNARE coiled-coil domain (Figure 12C). Being
clearly a member of the plant syntaxin family further supports
the possibility that osm1/syp61 may not be a null mutant, because null mutants of other syntaxin family members were
found to be lethal (Sanderfoot et al., 2001b).
OSM1/SYP61 Controls Stomatal Responses to ABA
The wilty phenotype (Figures 5B and 6B) of osm1/syp61 plants
indicates that they have impaired stomatal function. In fact,
when detached shoots were allowed to dehydrate slowly, the
rate of water loss from osm1/syp61 mutant plants was consistently higher than that from wild-type plants, and this difference
was abolished by complementation of the mutant with the
wild-type allele (Figure 13B). This finding suggests that during
dehydration, the stomata of osm1/syp61 mutant plants fail to
respond to water deficit–induced ABA. Indeed, treatment of
the epidermis of osm1/syp61 plants with ABA resulted in incomplete stomatal closure compared with wild-type plants
Figure 7. Low-Potassium Response and Ion Content of osm1 Plants.
(A) Wild-type and osm1 plants were grown on modified MS agar
medium containing K as KCl at the indicated concentrations. Root
length was determined after 9 days. Closed circles, wild type; open
circles, osm1. Error bars represent standard deviations (n 15).
(B) Na content in osm1 and wild-type seedlings as a function of external NaCl concentration in the medium. Open bars, wild type;
closed bars, osm1. Error bars represent standard deviations (n 3).
(C) K content in osm1 and wild-type seedlings as a function of external NaCl concentration in the medium. Open bars, wild type;
closed bars, osm1. Error bars represent standard deviations (n 3).
Figure 8. RD29A::LUC Expression in osm1 and Wild-Type Plants.
Relative luminescence of RD29A::LUC plants is shown as photometric counts (Ishitani et al., 1997). Treatments were as follows:
control (room temperature without treatment), cold (0C for 48 h),
ABA (100 M ABA for 3 h), and NaCl (300 mM NaCl for 5 h). Data
represent the average of 20 individual seedlings for each treatment.
Open bars, wild type; closed bars, osm1. Error bars represent standard deviations.
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The Plant Cell
Figure 9. Expression of Stress-Controlled Genes in osm1 and Wild-Type Plants.
Total RNA was extracted from whole seedlings after different treatments as indicated at top. Treatments were as follows: CN (control, no treatment), NaCl
(300 mM NaCl), LiCl (40 mM LiCl), ABA (100 M ABA), Man (600 mM mannitol), and cold (4C in the dark). Numbers indicate the period of treatment
(hours). Total RNA (20 g) was loaded in each lane. The bottom panels show ethidium bromide–stained total RNA gels as a loading control. WT, wild type.
(Figure 13A). The osm1/syp61 mutation clearly results in an
impaired response of guard cells to ABA. In addition, guard
cells of osm1/syp61 plants did not respond normally to ABA
application during the induction of stomatal opening (Figure
13A). Compared with wild-type plants, ABA failed to fully
block stomatal opening in osm1/syp61 plants. Thus, the
OSM1/SYP61 protein appears to play a crucial role in ABAmediated guard cell turgor control.
nomic sequence upstream of the OSM1/SYP61 translation start
site) was fused to the GUS reporter gene, and the resultant construct was introduced into wild-type Arabidopsis plants. GUS
expression was observed in all plant tissues examined except
for hypocotyls and young leaves (Figure 14), which is consistent
with the observations of Sanderfoot et al. (2001a). In all tissues,
expression was stronger in the vasculature (Figure 14). Very
strong expression was observed in roots and in guard cells of
the leaf epidermis (Figures 14C and 14F).
Tissue Expression Pattern of
OSM1/SYP61 Promoter::GUS
Subcellular Localization of Green Fluorescent Protein
Fused to OSM1/SYP61
To determine the tissue and cell type expression pattern of
OSM1/SYP61, the OSM1/SYP61 promoter (2.38 kb of the ge-
To determine which membrane compartment OSM1/SYP6
is associated with, OSM1/SYP61 cDNA was fused in frame
Table 1. Genetic Analysis of the osm1 Mutant
Cross
Generation
Plants Tested (No.)
Wild Typea
Mutanta
Resistantb
Sensitiveb
Wild type osm1
F1
F2
146
1083
1653d
146
820
0
263c
146
263c
1205
0
0c
430
a Mutant
and wild-type phenotypes were determined using the root-bending assay with 140 mM NaCl.
or sensitivity was determined by either spraying the plants with bialaphos solution or treating the plants with bialaphos in vitro.
c These plants with the mutant phenotype were transferred to soil and sprayed with bialaphos solution (P 0.2).
d These seedlings were scored only for bialaphos resistance in vitro to estimate the T-DNA copy number (P 0.2).
b Resistance
Syntaxin Controls ABA and Stress Responses
3019
at its N terminus to the green fluorescent protein (GFP)
marker gene. GFP fused to OSM1/SYP61 was expressed
under the control of the 35S promoter of Cauliflower mosaic
virus in transgenic Arabidopsis plants. The subcellular localization of GFP fused to OSM1/SYP61 was examined by
green fluorescence imaging using a confocal microscope.
The green fluorescent images were detected predominantly
in vesicle-like structures and along the cell wall inner surface
(data not shown), indicating a subcellular localization consistent with the report of Sanderfoot et al. (2001a) that
OSM1/SYP61 may be localized to the trans-Golgi network
and the prevacuolar compartment.
DISCUSSION
In recent years, it has been firmly established that ABA mediates its effects on stomatal movement through altered
guard cell turgor brought about mainly by the control of a
network of ion channels localized in the guard cell plasma
membrane and tonoplast. The major effectors of turgor
changes are the inward- and outward-rectifying K channels, the slow-activating and rapid transient anion channels
that when activated by increased cytosolic Ca 2 lead to net
anion efflux and membrane depolarization, which in turn activates outward-rectifying K channels. The Ca2-stimulated
overall efflux of both anions and cations results in reduced
guard cell turgor and stomatal closure.
Although phospholipase-mediated, ABA-induced Ca 2 increase in guard cell cytoplasm is recognized as an event
that is central to the ion movements that drive water fluxes
and stomatal function, the receptor molecules that perceive
ABA remain unknown. However, several important signaling
intermediates that transduce the perception of ABA to allow
increased cytosolic Ca2 concentration have been identified
and include inositol 1,4,5-triphosphate, cyclic ADP-Rib, and
reactive oxygen species (Schroeder et al., 2001). ABA induces a rapid increase in reactive oxygen species in guard
cells, which activates Ca2 channels and is dependent on a
functional GCA gene (Pei et al., 2000). ABA-induced cytosolic Ca2 concentration also is mediated by protein phosphatase 2Cs encoded by the ABI1 and ABI2 genes (Merlot
et al., 2001; Schroeder et al., 2001). Several other protein kinases, second messenger scaffolding, and other signaling
proteins have been implicated in the control of stomatal
function (Schroeder et al., 2001).
In an attempt to isolate an ABA receptor, Leyman et al.
(1999) used a heterologous gene expression system involving Xenopus laevis oocytes because this system has been
used successfully to isolate animal cell receptors. When oocytes were injected with mRNAs from water-stressed tobacco leaves, ABA was able to induce a Ca 2-dependent
Cl current (Leyman et al., 1999). Using a process of elimination, Leyman et al. (1999) determined that the ABA-induced
Cl current required a particular mRNA encoding Nt-Syr1, an
Figure 10. OSM1 Locus Determined by TAIL-PCR and T-DNA Insertion Diagnostic PCR of osm1 Plants.
(A) Physical map of the OSM1 locus and the insertion site of the T-DNA.
The solid line represents a fragment of BAC clone F3M18; closed
boxes indicate the coding region of a gene; arrows under the closed
boxes represent the predicted transcription direction; the upward
arrow indicates the insertion site of the T-DNA in the osm1/syp61
mutant; and black triangles near the right border of the T-DNA
insert indicate the four 35S enhancers. LB, left border; RB, right
border.
(B) Scheme of T-DNA diagnostic PCR. F, forward primer; LB3,
primer specific to the T-DNA left border; R, reverse primer.
(C) Diagnostic PCR of the T-DNA inserted in osm1/syp61. Lane 1,
wild type; lane 2, osm1/syp61; lanes 3 and 4, two representative
lines of osm1/syp61 from segregating F2 progeny. Lanes 5 to 8
were labeled in the same order used for lanes 1 to 4. M, molecular
mass markers.
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The Plant Cell
Figure 11. The osm1 Mutation Is Complemented Completely by a
Genomic Fragment of the OSM1/SYP61 Promoter and Amino Acid–
Coding DNA.
(A) Seedlings of wild type (left), osm1/syp61 mutant (center), and
complemented mutant osm1/syp61 (osm1/syp61 OSM1/SYP61;
right) transformed with a genomic fragment (see Methods) corresponding to OSM1/SYP61 grown on MS agar medium supplemented with 140 mM NaCl.
(B) and (C) Quantitation of root growth of wild-type, osm1/syp61,
and osm1/syp61 OSM1/SYP61 plants on control MS medium (B)
and MS medium supplemented with 120 mM NaCl (C). Root growth
was measured 3 days (open bars), 6 days (hatched bars), and 9 days
(closed bars) after transfer from MS medium to either MS medium
(B) or MS supplemented with 120 mM NaCl (C). Error bars represent
standard deviations (n 15 for each genotype).
apparent homolog of yeast syntaxin. Curiously, the Nt-Syr1
mRNA also was able to constitutively activate Cl currents
in oocyte membranes without any added ABA, which may
reflect the nonspecificity of a heterologous system. The Arabidopsis syntaxin family most closely resembling Nt-Syr1
from tobacco is AtSYP121. In additional pharmacological
studies, Leyman et al. (1999) further implicated Nt-Syr1 and
its ortholog, AtSYR1 (AtSYP121), in ABA-mediated stomatal
behavior. They were able to demonstrate that ABA control
of guard cell ion channels could be inhibited by the injection
of guard cell protoplasts with a dominant-negative-acting
truncated Nt-Syr1 protein (Sp2) or with neurotoxin type C
of Clostridium botulinum (Bot N/C), an inhibitor of syntaxin-dependent vesicle fusion (Leyman et al., 1999, and
references therein).
Beyond these pharmacological effects, we present here
clear genetic evidence that a plant syntaxin protein plays an
important role in ABA-mediated stomatal control. osm1/syp61
mutant plants displayed not only impaired ABA-induced
stomatal closure, but ABA treatment of osm1/syp61 mutant
plants was unable to inhibit stomatal opening, as it did in
wild-type plants (Figure 13A), two processes that are not
simply the reverse of each other (Schroeder et al., 2001;
Wang et al., 2001). In addition, the effects of the osm1/
syp61 mutation on stomatal behavior are reflected clearly in
significant changes in water loss characteristics and increased sensitivities of osm1/syp61 mutant plants to salinity
and water deficit stress (Figures 5 and 6).
According to the classification of Arabidopsis syntaxin
genes by Sanderfoot et al. (2000), the tobacco Nt-Syr1 syntaxin that is required for ABA-induced Cl current in oocytes
(Leyman et al., 1999) most closely matches the sequence of
the Arabidopsis AtSYP121. The fact that our screen identified AtSYP61 and not AtSYP121 could be explained by a
number of possibilities. We simply could have failed to
screen enough lines, or the osm1/syp61 mutation may have
a stronger phenotype, or mutation in AtSYP121 may have
little or no effect on the root phenotype examined in the
root-bending assay. It also is quite possible that OSM1/
SYP61 is more functionally related to Nt-Syr1 than is
AtSYP121.
Both OSM1/SYP61 and Nt-Syr1 contain the conserved
H3 helical cytoplasmic domains required for SNARE function in membrane fusion. However, the three sites necessary
for binding and cleavage by Bot N/C (Schiavo et al., 1995;
O’Connor et al., 1997), which are present on Nt-Syr1 (Leyman
et al., 1999) and associated with sensitivity to the toxin, are
poorly conserved in OSM1/SYP61 (Figure 12B). Although it
remains unproven that OSM1/SYP61 is not sensitive to Bot
N/C, this finding suggests that there may be another Arabidopsis SNARE protein that has a similar function, because
Leyman et al. (1999) were able to inhibit ABA-induced Cl
channel current in tobacco guard cells with Bot N/C treatment. Apparently, such a toxin-sensitive protein, if it exists,
does not substantially substitute for OSM1/SYP61 function,
thereby allowing recognition of the altered phenotype of osm1/
Syntaxin Controls ABA and Stress Responses
3021
Figure 12. Sequence Analysis of OSM1/SYP61.
(A) Scheme of the OSM1/SYP61 gene structure. Positions are relative to the initiation codon. Closed boxes represent exons, and lines between
closed boxes represent introns.
(B) Comparison of predicted amino acid sequences of OSM1/SYP61 and other eukaryotic syntaxins. The predicted amino acid sequences of
OSM1/SYP61 and its orthologs were compared using CLUSTAL W (http://searchlauncher.bcm.tmc.edu:9331/multi-align/multi-align.html) with
the default settings and BOXSHADE 3.21. Amino acids identical in at least two proteins are shaded black. Residues shaded gray are considered
similar by the following convention: R and K; Q and N; T and S; E and D; and V, I, L, F, and M. Sources of the compared proteins are as follows:
OSM1/SYP61, Arabidopsis; STX6_MOUSE, syntaxin 6 from mouse; STX6_HUMAN, syntaxin 6 from human; STX10_HUMAN, syntaxin 10 from
human. Sequences shown above the aligned sequences are two predicted Clostridium botulinum toxin C1 recognition sites (FMEEFFEQVE and
ELEDMLESGN, respectively) and a cleavage site (AVKYQSKAR) from squid syntaxin (Schiavo et al., 1995; O’Connor et al., 1997).
(C) Sequence alignment in the t-SNARE coiled-coil domain. STX6_RAT, syntaxin 6 from rat; C15C7, hypothetical protein C15C7 from Caenorhabditis elegans; STX8_HUMAN, syntaxin 8 from human; SNAP-29, synaptosomal-associated protein 29 from rat.
syp61 mutant in our screen. The H3 domain also is required
for the syntaxin-mediated control of Ca2 channels in eukaryotic systems (Bezprozvanny et al., 2000). It is tempting to
speculate that the conserved H3 domain of OSM1/SYP61
may interact with guard cell Ca2 channels to regulate ion
fluxes and subsequent water movement that drives guard cell
turgor changes and stomatal pore size (Schroeder et al.,
2001). It is interesting as well that syntaxin appears to be required for tethering to membrane sites of the Ca 2 channel
that affects neurotransmitter release, ensuring G-protein
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The Plant Cell
Figure 13. Stomata Movement and Water Loss.
(A) Stomata opening and closing on epidermal peels with and without ABA treatment. Control represents epidermal peels from plants
kept for 12 h in the dark and then incubated under light in buffer only
for 3 h before aperture measurements. Opening represents epidermal peels from plants kept for 12 h in the dark and then incubated
under light in buffer plus 2 M ABA for 3 h before aperture measurements. Closing represents epidermal peels from plants kept for 12 h
in the dark and then incubated under light in buffer for 3 h and then
treated with 2 M ABA for 3 h before aperture measurements. Open
bars, wild type; closed bars, osm1/syp61. Error bars represent standard errors (n 30).
(B) Transpiration water loss from detached shoots. Closed circles,
wild type; open circles, osm1/syp61; closed diamonds, osm1/syp61
complemented with the wild-type gene. Error bars represent standard deviations (n 7).
mediation of Ca2 channel activity (Stanley and Mirotznik,
1997). In fact, SNAREs also can interact directly with an
orphan G-protein–coupled receptor, CIRL (Lelianova et al.,
1998).
Guard cell channel regulation by a G-protein also has
been reported for Arabidopsis, and mutation of the G-protein (GPA) results in a similar stomatal dysfunction that is
observed in osm1/syp61 mutant plants, suggesting that
OSM1/SYP61 and GPA may function in the same ABAmediated pathway (Wang et al., 2001). Genetic additivity analysis of OSM1/SYP61 and GPA could help resolve this possibility. Most studies on the regulation of guard cell water flux
have focused on the role of Ca2 and other ion fluxes at the
plasma membrane. Although the localization of SYP61 protein at the trans-Golgi network and the prevacuolar compartment by Sanderfoot et al. (2001a) suggests an important
role for vesicle trafficking in stomal function, these observations still may not be inconsistent with a role for SYP61 in
controlling Ca2 ion flux, because such regulatory mechanisms to control water flux to and from the cell vacuole are
likely to operate at the tonoplast as well, where SYP61 may
have been undetected previously.
Leyman et al. (1999) reported that Nt-Syr SNARE transcript
from tobacco could be induced rapidly within 2 h to a level
nearly 10-fold greater after ABA treatment and 48 h after
desiccation treatment. However, we did not observe any
dramatic induction of the OSM1/SYP61 transcript after osmotic stress or ABA treatment (Figure 9). Although significant induction of the abnormally small transcript in osm1/
syp61 mutant plants was observed, this smaller transcript
apparently is not functionally equivalent to the normal transcript, because complementation with the wild-type gene
was required to restore the normal phenotype. The location
of the T-DNA insertion upstream of the amino acid coding
start site could have affected transcription regulation or
mRNA stability, resulting in the accumulation of abnormal
mRNA.
OSM1/SYP61 apparently plays a role in the natural development of root tissues, because the osm1/syp61 mutation
clearly results in altered root growth and morphology characterized by more extensive branching and increased sensitivity to osmotic stress. Because K transport plays such a
crucial role in plant cell expansion and tissue growth and
development (Keller and Van Volkenburgh, 1996; Claussen
et al., 1997), it is not unexpected to observe growth and
morphology changes in mutations that can alter K transport characteristics. It is possible that these changes are related to the same biochemical effects of the osm1/syp61
mutation on guard cells that are predicted from the results
of Leyman et al. (1999), that is, altered ion flux activities. In
fact, mutations of genes that encode specific K transporters result in altered cell growth and tissue morphologies,
such as those observed in mutations of TRH1/KT3/KUP4
(Rigas et al., 2001) and SHY3/KT2/KUP2 (Elumalai et al.,
2002).
Recently, Geelen et al. (2002) reported that overexpression of the Nt-Syr1 protein and expression of the negativeacting (Leyman et al., 1999) Sp2 fragment of Nt-Syr1 result
in alterations in the morphology and, interestingly, growth of
tobacco roots. Several interesting hypotheses connecting
the previously observed role of Nt-Syr1 in the ABA-medi-
Syntaxin Controls ABA and Stress Responses
Figure 14. Tissue Expression of OSM1/SYP61 Promoter-GUS.
Histochemical GUS staining of transgenic Arabidopsis plants harboring the OSM1/SYP61 promoter-GUS fusion protein.
(A) Whole seedling.
(B) Mature leaf.
(C) Epidermis of leaf.
(D) Hypocotyl.
(E) Young leaves.
(F) Root.
(G) Silique.
(H) Flower.
(I) Close-up of part of a flower.
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The Plant Cell
ated control of guard cell ion channels were put forth by
Geelen et al. (2002). However, because expression of the
dominant-negative-acting Sp2 fragment of Nt-Syr1 in transgenic plants apparently had no effect on stomatal function,
there was no genetic evidence presented by Geelen et al.
(2002) that would confirm the biochemical experiments with
Sp2 protein reported by Leyman et al. (1999) and connect
all of these possible functions of a single SNARE protein.
Our results provide considerably more support to this possibility, because disruption of a single gene, OSM1/SYP61,
resulted in the loss of the responsiveness of stomata to ABA
and altered seed germination and root growth characteristics that appear to be independent of ABA action.
It is tempting to speculate, like Geelen et al. (2002), that
the functions of the SNARE proteins in controlling guard cell
ion channel activity and, especially, cell growth and development, are related at least partly to their function in vesicular
trafficking. This possibility is based indirectly on the knowledge of the role of SNAREs in the complex biochemistry of
vesicle trafficking, and Geelen et al. (2002) presented arguments for and against this, essentially concluding that it is
more likely that the role of the syntaxin protein in guard cell
ion channel control is direct but that a vesicular trafficking
effect, especially on cell growth and development, remains
conceivable. It is intriguing that a single SNARE proteincoding gene (OSM1/SYP61) when mutated was able to disrupt both ABA-mediated and non-ABA-mediated responses
to stress (Figures 2, 3, and 13). Thus, the OSM1/SYP61 protein also may represent a crucial signal component connecting well-recognized ABA-dependent and ABA-independent pathways leading to important stress responses
(Shinozaki and Yamaguchi-Shinozaki, 1997), perhaps by a
dual essential function as a signaling element and as part of
the membrane-trafficking machinery (Leyman et al., 1999;
Blatt, 2000; Geelen et al., 2002).
Other genes that control guard cell signaling, in addition
to OSM1/SYP6, have been shown to be expressed in several tissues and have pleiotropic effects on plant phenotype
(Leung and Giraudat, 1998; Schroeder et al., 2001). To date,
little has been done to determine if these signal components
participate in multiple response pathways that are designed
to marshal a coordinated protective phenotypic response
during exposure to soil water deficits, such as closing stomata more and favoring root growth over shoot growth. Additional experiments using these mutants and their combinations will shed more light on this intriguing possibility.
[pMP90RK]) T-DNA transformation using the activation-tagging vector pSKI015 (provided by D. Weigel, Salk Institute for Biological
Studies, La Jolla, CA). Seeds from T2 plants that are resistant to bialaphos (30 mg/L) were used for subsequent experiments. T2 seeds
were surface-sterilized in a solution of 70% ethanol briefly and
placed directly into a 5% Clorox plus 0.1% Triton X-100 solution for
10 to 15 min and rinsed five times with sterile water. The seeds were
resuspended in sterile 0.4% (w/v) low-melting-point agarose before
being sown in rows onto agar plates for germination. The agar medium contained Murashige and Skoog (1962) (MS) salts with 3% (w/v)
Suc and 1.2% (w/v) agar, pH 5.7. The plates then were stored at 4C
for 48 h to ensure uniform germination before being placed nearly
vertically (to prevent separation of the seedlings from the cellophane
membrane [see below]) (Bio-Rad, Hercules, CA) in a growth room for
germination. When appropriate, seedlings (10 to 20 days old) were
transferred to soil (Metro-Mix, Grace Sierra Horticultural Products,
Milpitas, CA) and grown to maturity in a growth chamber. Plants
were watered twice per week with one-quarter-strength Hoagland
solution. The growth chamber temperature was 23 2C. Light provided by cool-white fluorescent bulbs was 50 to 70 E·m2·s1 (constant) for seedlings on agar plates and 100 E·m2·s1 (16 h of light
and 8 h of dark) for plotted plants.
Mutant Isolation
Approximately 65,000 T2 mutated lines were screened for salthypersensitive mutants using a root-bending assay as described by
Wu et al. (1996) with the slight modification of using a membrane
overlay to facilitate the transfer of the seedlings from germination
medium to medium containing NaCl. Four-day-old seedlings with 1to 1.5-cm-long roots were transferred together with the cellophane
membrane from the germination medium into a second medium supplemented with 140 mM NaCl. Putative sensitive or resistant mutants
then were transferred to soil to grow to maturity as described above.
Seeds from the putative mutants were screened again using the
same modified root-bending assay on agar plates with 140 mM NaCl
to confirm the phenotypes.
Growth Measurements
For growth measurements, 4-day-old seedlings from germination
medium were transferred and placed with roots pointing downward
on agar plates (placed vertically) supplemented with various salts.
Each plate contained six mutant and six wild-type seedlings. Three
replicate plates were used for each treatment. Increase in root length
was measured with a transparent ruler every day for 7 days unless
specified otherwise.
Seed Germination
METHODS
Plant Materials and Growth Conditions
Arabidopsis thaliana plants (ecotype C24) carrying the homozygous
transgene RD29A promoter::LUC (Ishitani et al., 1997) were mutagenized by Agrobacterium tumefaciens–mediated (strain GV3101
Wild-type and osm1 seeds (at least 100 seeds each) were sterilized
and kept for 4 days at 4C in the dark to break dormancy. The seeds
then were sown directly on the surface of filter paper soaked with either different NaCl solutions or solutions containing various levels of
abscisic acid (ABA) in the same Petri dish and incubated at 22 2C
with a 16-h light photoperiod. The number of germinated seeds was
expressed as a percentage of the total number of seeds plated. Unless indicated otherwise, three replicate plates were used for each
treatment.
Syntaxin Controls ABA and Stress Responses
Low-Potassium Response and Ion Content Measurements
To determine the K requirement of mutant plants, seeds were sown
on vertically positioned agar plates, and 4 days after germination,
they were transferred to a modified MS medium supplemented with
various levels of KCl, as described by Liu and Zhu (1997). To measure the ion contents, 100 to 150 seeds were sterilized and incubated in a 500-mL flask containing 150 mL of medium (half-strength
MS salts and 2% Suc, pH 5.5). The flasks were shaken at 120 rpm in
a growth room with cool-white fluorescent illumination at 22 1C
(16 h of light and 8 h of dark). After 12 days, 5 M NaCl was added to
give the desired NaCl concentration, and the seedlings were allowed
to continue to grow for 3 days. The seedlings then were collected,
washed extensively with several changes of distilled water, weighed,
and dried at 65C for 48 h. After the dry weight measurements, the
seedlings were ground to fine powder and extracted with 0.1 mM nitric acid overnight. The extraction mixture was filtered, and the recovered solution was used for ion content measurements with an
atomic absorption spectrophotometer (Wu et al., 1996).
LUC Imaging
RD29A promoter::LUC expression in response to low temperature,
exogenous ABA, or osmotic stress was examined in 7-day-old seedlings grown on 0.7% agar plates containing full-strength MS and 3%
Suc. After 24 h at 0C, 3 h after treatment with 100 M ABA, or 5 h after treatment with 300 mM NaCl, seedlings were examined with a luminescence imaging system as described by Ishitani et al. (1997).
Genetic Analysis
Mutant plants were backcrossed to the wild type as described by Wu
et al. (1996). Seedlings of F1 and F2 progeny were scored for salt
sensitivity by a modified root-bending assay (Wu et al., 1996; this
study). Three-week-old plants of F1 and F2 progeny also were tested
for bialaphos resistance by spraying the bialaphos solution once
each day for 3 days, and leaf tissues from these plants were collected to determine the presence of homozygous or heterozygous insertion DNA. To test the resistance of seedlings to bialaphos in vitro,
seeds of the F2 progeny were surface-sterilized and plated on MS
medium supplemented with 4.5 g/mL bialaphos.
RNA Gel Blot Analysis
Ten-day-old seedlings grown on agar plates were treated with NaCl,
LiCl, ABA, mannitol, or low temperature as indicated in Figure 9. Total RNA was isolated from seedlings, and 20 g from each sample
was separated on 1.2% (w/v) agarose formaldehyde gels and transferred to Hybond-N nylon membranes (Amersham Pharmacia Biotech), as described by Gong et al. (1997). The probe for OSM1 consisted of the entire open reading frame of this gene, and transcript
abundance was determined by PCR amplification using cDNA prepared from mRNA of salt-treated Arabidopsis plants as a template.
OSM1 primers were 5-ATGTCTTCAGCTCAAGATCC-3 (forward)
and 5-TCAAACCAGCAACAAGACC-3 (reverse). Gene-specific probes
for RD29A, RD22, and Cor15a were made from EST clones
BE845342, BE845022, and BE845371 respectively, as described
(Gong et al., 2000).
3025
Cloning of OSM1 and 5 Rapid Amplification of cDNA
Ends Analysis
DNA flanking the left border of the inserted T-DNA in osm1 plants
was isolated by thermal asymmetric interlaced PCR (Liu et al., 1995;
Liu and Whittier, 1995) and subcloned into cloning vector pBluescript
SK (Stratagene, La Jolla, CA) pGEM-T and pGEM-T Easy Vector
System (Promega, Madison, WI) according to the manufacturer’s instructions. The entire isolated fragment was sequenced. The primers
used in the thermal asymmetric interlaced PCR were specific primers
for the T-DNA left border (LB1, 5-ATACGACGGATCGTAATTTGTC-3;
LB2, 5-TAATAACGCTGCGGACATCTAC-3; and LB3, 5-TTGACCATCATACTCATTGCTG-3) and degenerate primers (DEG1, 5-WGCNAGTNAGWANAA-3; and DEG2, 5-AWGCANGNCWGANATA-3).
A DNA fragment including plant genomic DNA flanking the right border of the inserted T-DNA and a DNA fragment inside the T-DNA was
amplified by PCR using osm1 mutant genomic DNA with the following primer pair: T7, 5-GTAATACGACTCACTATAGGGC-3 (T7 is
from nucleotides 2829 to 2850 inside the T-DNA insert); and OSM1RBFlank, 5-CATCTCATTGGCAGGGATTGTTC-3 (from nucleotides
25,538 to 25,560 in BAC clone F3M18). The PCR product was separated on a 1.5% gel, recovered from the gel using the QIAquick Gel
Extraction Kit (Qiagen, Valencia, CA), and sequenced. We also designed the following primer pair to determine whether the T-DNA
insert is homozygous or heterozygous on chromosome 1 of plants
that are homozygous for the osm1 mutant: forward primer, OSM1RBFlank; reverse primer, 5-AACAATCATTTATAGTAGCATCGG-3.
For 5 rapid amplification of cDNA ends (RACE) analysis, total RNA
from both nontreated wild-type and osm1 mutant plants was extracted using the Absolutely RNA reverse transcriptase–mediated
PCR miniprep kit (Stratagene) according to the manufacturer’s instructions. First-strand cDNA synthesis was performed using the
SMART RACE cDNA amplification kit (Clontech, Palo Alto, CA), and
5-RACE reactions were performed using a combination of the
SMART RACE cDNA amplification kit (Clontech) and the Advantage
2 PCR enzyme system (Clontech) according to the manufacturer’s
instructions. The following primer pairs were used. For the first-round
reaction, the forward primer was Universal Primer Mix A (provided
with the SMART RACE cDNA amplification kit) and the reverse
primer (also called Gene-Specific Primer 1) was 5-CCAGCTCATCTACCTGCCACTCAATGC-3 (nucleotides 270 to 296 in the fulllength cDNA clone R19864). For the second-round reaction, the forward primer was Nested Universal Primer A (provided with the
SMART RACE cDNA amplification kit) and the reverse primer (also
called Nested Gene-Specific Primer 1) was 5-CCACATGTGCTTGATCACCCATGTCGG-3 (nucleotides 216 to 242 in the full-length
cDNA clone R19864). The PCR products from the second-round 5
RACE reaction using nested primers were purified from the gel and
subcloned into cloning vector pBluescript SK by T/A cloning. The entire inserts in the positive clones were sequenced.
Gene Complementation of the osm1 Mutation
For gene complementation, a genomic DNA fragment corresponding
to the OSM1 gene with its own promoter (including 2.38 kb upstream
of the start codon of OSM1) was amplified by PCR using C24 genomic DNA. The primers used for this PCR were as follows: OSM1g-F, 5-CTGACTCGAGCGCAGAGATTCCCCGACGGTTGTC-3; and
OSM1-g-R, 5-TTCAGGTTCTCTAGAGATCACACAGCC-3. The PCR
product was purified from an agarose gel and then subcloned into
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The Plant Cell
pBluescript SK vector. The identity of the OSM1 gene was confirmed
by sequencing the entire gene. The resulting plasmid was digested
with XbaI and KpnI and ligated into expression vector pCAMBIA
1303, which was digested with XbaI and KpnI. The recombinant
plasmid was used to transform Agrobacterium strain GV3101. The
resulting transformant was used to transform osm1 mutant plants.
Agrobacterium strain GV3101 transformed with pCAMBIA 1303
empty vector also was used to transform the osm1 mutant plants as
a control.
of wild-type, osm1 mutant, and complemented plants at the rosette
stage were placed on weighing trays and allowed to dry slowly at
constant temperature (25C) and humidity (50%). Weights of shoots
were determined over a 3-h period.
Upon request, all novel materials described in this article will be
made available in a timely manner for noncommercial research purposes.
Accession Numbers
Construction of the OSM1/SYP61 Promoter-GUS Fusion and
GFP Fused to OSM1/SYP61
Two primers were used to construct a GFP fused to the OSM1/
SYP61 fusion vector: forward, 5-CCGAATTCATGTCTTCAGCTCAAGATCCATTCTACATTG-3 (containing an EcoRI site); and reverse,
5- CCGGATCCCAAGAAGACAAGAACGAATAGGATGATGAAC-3
(containing a BamHI site). The template for the PCR was OSM1
cDNA in subcloning vector pBluescript SK. The PCR conditions
were as follows: 30 cycles of 94C for 2 min, 94C for 30 s, 62C for
30 s, and 72C for 1.5 min, followed by 72C for 10 min. The PCR
product was precipitated by ethanol and digested with EcoRI and
BamHI (Gibco BRL, Life Technologies). The digested PCR product
was purified from an agarose gel and subcloned into pEGAD vector
that was digested with EcoRI and BamHI. The cDNA sequence was
confirmed, and the resulting plasmid was used to transform Agrobacterium strain GV3101. These strains were used to transform Arabidopsis ecotype Columbia gl1 plants. Primary transformants were
selected on MS medium containing 3 mg/L bialaphos and transferred to soil at maturity. Images of the GFP fused to OSM1/SYP6
were made using a confocal microscope (Cutler et al., 2000).
For the OSM1/SYP61 promoter-GUS fusion, the following primers
were used: forward, 5-CACAGGAAGCTATGGGTGATGGTGC-3; and
reverse, 5-CGGGATCCTACCCTATACACCAAATAACACAGCTG-3.
The genomic fragment in the vector used for complementation
analysis was used as the template for PCR. The PCR conditions
were the same as those described above. The PCR product was
subcloned into binary vector pCAMBIA 1381. The resulting plasmid
was transformed to Agrobacterium strain GV3101. These strains
were used to transform Arabidopsis ecotype Columbia gl1 plants.
Primary transformants were selected on MS medium supplemented
with 25 mg/L hygromycin and transferred to soil for selfing. Histochemical assays of GUS activity were performed using 5-bromo4-chloro-3-indolyl glucuronide (Duchefa, Haarlem, The Netherlands)
as a substrate (Jefferson et al., 1987) and were performed on intact
seedlings. The samples were treated with 70% ethanol for 2 to 6 h to
remove chlorophyll from tissues when necessary.
Stomatal Aperture Bioassays and Stomatal Water Loss
Plants were grown in potting soil in a growth chamber (16 h of light
and 8 h of darkness at 22C and 80% RH). Leaves were harvested
from 4- to 5-week-old plants kept for 12 h in the dark. Paradermal
sections of abaxial epidermis were placed cuticle up in Petri dishes
containing 10 mM KCl, 7.5 mM potassium iminodiacetate, and 10
mM KOH, pH 6.15, and incubated at room temperature (Leymarie et
al., 1998; this study). The aperture of the stomatal pores was determined using a calibrated ocular micrometer on a Nikon Optiphot microscope (Nikon, Nippon Kogaku, Tokyo, Japan). Detached shoots
The accession numbers for the sequences mentioned in this article
are AC016795 (At-SYR1, which is the same as AtSYP121/
F26K24.11), AC010155 (BAC F3M18), AF187951 (T-DNA insert),
AI995303 (EST), AY072115 (clone R19864), and AAL59937 (OSM1/
SYP61). Accession numbers for the protein sequences shown in
Figure 12 are as follows: STX6_MOUSE, syntaxin 6 from mouse,
NP_067408; STX6_HUMAN, syntaxin 6 from human, O43752;
STX10_HUMAN, syntaxin 10 from human, XP_009197; squid syntaxin, CAA74913; STX10_HUMAN, syntaxin 10 from human,
AAC05087; STX6_RAT, syntaxin 6 from rat, Q63635; C15C7, hypothetical protein C15C7 from Caenorhabditis elegans, AAK39171;
STX8_HUMAN, syntaxin 8 from human, AAD20831; and SNAP-29, synaptosomal-associated protein 29 from rat, Q9Z2P6.
ACKNOWLEDGMENTS
We sincerely thank David W. Ehrhardt for providing the pEGAD vector.
We also acknowledge Terri Kirk and P.A. Hammer for their excellent
technical assistance. This work was supported by National Science
Foundation Plant Genome Award DBI9813360. This is Purdue University Agricultural Program Paper 16,753.
Received August 9, 2002; accepted September 17, 2002.
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OSM1/SYP61: A Syntaxin Protein in Arabidopsis Controls Abscisic Acid−Mediated and
Non-Abscisic Acid−Mediated Responses to Abiotic Stress
Jianhua Zhu, Zhizhong Gong, Changqing Zhang, Chun-Peng Song, Barbara Damsz, Günsu Inan, Hisashi
Koiwa, Jian-Kang Zhu, Paul M. Hasegawa and Ray A. Bressan
Plant Cell 2002;14;3009-3028; originally published online November 26, 2002;
DOI 10.1105/tpc.006981
This information is current as of June 15, 2017
References
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