Department of Biology, University of Pennsylvania, Philadelphia, PA 19104-6018, USA
* Author for correspondence (e-mail: spoethig{at}sas.upenn.edu)
Accepted 23 December 2002
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SUMMARY |
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Key words: nucleocytoplasmic transport, Arabidopsis, phase change, exportin
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INTRODUCTION |
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Nucleocytoplasmic transport operates by a similar mechanism in yeast and
mammals (Chook and Blobel,
2001; Görlich and Kutay,
1999
; Kaffman and O'Shea,
1999
; Komeili and O'Shea,
2001
; Macara,
2001
). Karyopherins interact with cargo proteins at specific sites
known as nuclear localization signals (NLS) or nuclear export sequences (NES).
Most karyopherins bind directly to these sequences but a few bind via an
adaptor protein, such as importin
, the adaptor for importin ß.
Nuclear transport is driven by the interaction between karyopherins and the
small GTPase, Ran. Importins bind cargo molecules in the presence of Ran-GDP
and release them upon binding Ran-GTP, whereas exportins bind cargo molecules
in association with Ran-GTP and release them when Ran-GTP is hydrolyzed to
Ran-GDP. Ran-GTP is kept at a relatively low level in the cytoplasm by the
activity of the GTPase-activating protein RanGAP and the nucleotide-exchange
factor RanBP1 (RCC), but is present at a relatively high level in the nucleus
where these factors are absent. Importins bind cargo molecules in the
cytoplasm, where Ran is primarily in its GDP-bound form, and release them in
the nucleus, where the concentration of Ran-GTP is relatively high.
Conversely, exportins bind Ran-GTP and their cargo molecules in the nucleus,
and dissociate from these molecules in the cytoplasm when Ran-GTP is
hydrolyzed to Ran-GDP. Other factors that influence cargo binding include the
phosphorylation state of the cargo and its association with factors that
regulate the accessibility of the NLS or NES.
Although the mechanism of nuclear transport in plants has not been
intensively studied, it is likely to be similar to the mechanism that has been
described in other organisms (Merkle,
2001; Smith and Raikhel,
1999
). Proper localization of proteins in plant cells depends on
the same type of nuclear localization signal
(Hicks and Raikhel, 1995
) and
nuclear export sequence (Haasen et al.,
1999
; Ward and Lazarowitz,
1999
) that fulfill this function in other organisms. Furthermore,
plants possess homologs of many of the proteins known to be involved in
nucleocytoplasmic transport in other organisms. These include homologs of
importin
(Ballas and Citovsky,
1997
; Hubner et al.,
1999
; Jiang et al.,
1998a
; Jiang et al.,
2001
; Smith et al.,
1997a
; Smith et al.,
1997b
), importin ß (Jiang
et al., 1998a
), the exportin Crm1/Xpo1
(Haasen et al., 1999
), Ran1
(Haizel et al., 1997
), RanGAP
(Rose and Meier, 2001
) and
RanBP1 (Kim et al., 2001
).
Plant homologs of importin
(Hubner
et al., 1999
; Jiang et al.,
1998a
; Jiang et al.,
2001
) and importin ß
(Jiang et al., 1998b
) have
also been shown to mediate nuclear transport in animal cell systems, although
with somewhat different properties than the homologous yeast and mammalian
proteins.
The HASTY (HST) gene in Arabidopsis was
originally identified in a screen for mutations that affect the transition
between the juvenile and adult phases of vegetative development
(Telfer and Poethig, 1998),
and has also been identified in screens for mutations affecting leaf
(Berna et al., 1999
;
Serrano-Cartagena et al.,
2000
) and floral (Berna et al.,
1999
; Eshed et al.,
2001
; Serrano-Cartagena et
al., 2000
) morphology. We present a detailed analysis of the
hst phenotype that reveals a role for this gene in many different
aspects of plant development. The basis for this pleiotropic phenotype is
suggested by the discovery that HST encodes a protein similar to the
karyopherins exportin 5 (Xpo5) in mammals and Msn5p in yeast
(Alepuz et al., 1999
;
Bohnsack et al., 2002
;
Brownawell and Macara, 2002
;
Calado et al., 2002
;
Görlich et al., 1997
;
Kaffman et al., 1998
). The
hst loss-of-function phenotype indicates some of the developmental
pathways for which HST is required, and suggests that regulation of
nucleocytoplasmic transport may play an important role in these pathways.
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MATERIALS AND METHODS |
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Phenotypic analysis
Abaxial trichomes were scored by observing seedlings 10-18 days after
planting with a stereomicroscope. Leaf morphology was recorded by photocopying
leaves attached to cardboard with double-sided tape. These photocopies were
subsequently scanned into a computer and manipulated with Photoshop to produce
the images presented here. The rate of leaf initiation was measured using
plants homozygous for a LFY::GUS transgene obtained from D. Weigel
(Salk Institute). Soil-grown plants were harvested on successive days and
stained for GUS activity, and the number of leaf primordia was then counted
with the aid of a compound microscope.
GUS activity was observed by incubating seedlings overnight in 1 mM X-gluc, 50 mM sodium phosphate buffer (pH 7), 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide and 0.1% Triton X-100 at 37°C followed by decolorization in 95% ethanol. They were then dehydrated in 100% ethanol, transferred to xylene and mounted in Permount on microscope slides. Specimens expressing 35S::LFY were stained overnight; specimens expressing 35S::HST-GUS were stained for 2 hours.
Histology
Seedlings were fixed overnight at 4°C in 3% glutaraldehyde in 0.5 M
phosphate buffer (pH 5.7), post-fixed in 1% OsO4 for 2 hours, and
then dehydrated and embedded in Spurr's resin. Sections were stained with 1%
Methylene Blue in 1% sodium borate. Scanning electron microscopy was performed
on flowers fixed in FAA (Jensen,
1962).
Positional cloning
F2 progeny of a cross between hst-1 (Col) and wild-type
Landsberg erecta (Ler) were screened for kanamycin resistance either
by spraying soil-grown plants with 200 µg/ml kanamycin one week after
planting, or by sowing F2 seeds on germination medium containing 50
µg/ml kanamycin. Kanamycin-resistant plants that were homozygous
hst-1 were allowed to set self seed at 18°C. RFLP polymorphisms
were scored using DNA isolated from 10 or more progeny of these recombinant
F2 plants. RLFPs useful for mapping hst-1, as well as
allele-specific RFLPs associated with hst mutations, were identified
by hybridizing radioactively labeled BAC clones to blots of genomic DNA
digested with a variety of restriction enzymes.
HST cDNAs were isolated using standard methods
(Sambrook et al., 1989) from
size-fractionated cDNA libraries (Kieber
et al., 1993
) obtained from the Arabidopsis Biological Resource
Center. Bluescript plasmids (pBS SK) were excised from lambda clones
using a rapid excision kit (Stratagene) and the protocol supplied by the
manufacturer.
Northern analysis
RNA for northern analysis was prepared from leaf and floral bud tissues
from mature plants, and from root tissue from plants grown in culture. RNA was
prepared using TRIzol reagent (GibcoBRL) and poly(A) RNA was isolated using
the Oligotex mRNA kit (Qiagen). PolyA RNA (0.75 µg) was run on a 1.2%
agarose gel containing 3% formaldehyde and then transferred to Hybond
N+ membrane (Amersham). Hybridization was carried out according to
Sambrook et al. (Sambrook et al.,
1989), using a 0.7 kb 5' fragment of the HST
cDNA.
Transgenes
The HST cDNA (AY198396) was placed under the regulation of the
CaMV 35S promoter in pCAMBIA 3301. The HST-coding region was
amplified from a cloned HST cDNA template using primers
5'-GCGGATCCATGGAAGATAGCAACTCCACG-3' and
5'-CGGATCCGCTAGCTCATTGTACGAACTCTTCATCC-3' that included the start
and stop codons, respectively. The 5' primer also included an
NcoI site, and subsequent digestion of the PCR product with
NcoI generated a HST cDNA with an NcoI end and a
blunt end. pCAMBIA 3301 was digested with BstEII to remove the
ß-glucuronidase gene, blunt-ended with Klenow enzyme and digested with
NcoI. The vector fragment was gel-purified and ligated to the
HST cDNA to generate the HST over-expression construct. A
HST-GUS fusion construct was generated by amplifying the
HST-coding region minus the stop codon from the HST cDNA
using primers 5'-GCGGATCCATGGAAGATAGCAACTCCACG-3' and
5'-GCGGATCCATGGATTGTACGAACTCTTCATCC-3', which added NcoI
sites at each end of the amplicon. NcoI-cut PCR product was then
inserted into the NcoI site of vector pCAMBIA 3301, to generate a
translational fusion between the 3' end of the HST gene and the
5' end of the Escherichia coli uidA gene encoding
ß-glucuronidase. Both constructs were used to transform wild-type (Col)
and hst-1 (Col) plants using the floral dip method
(Clough and Bent, 1998), and
transformants were selected by spraying the seedlings produced by these plants
with 250 mg/l glufosinate (AgrEvo).
Two-hybrid interaction between RAN1 and HST
The coding region of Arabidopsis RAN1
(Haizel et al., 1997) was
amplified from first-strand cDNA products generated by reverse transcription
of RNA isolated from Arabidopsis seedlings. The RT-PCR reaction
employed a 5' RAN-specific primer with an EcoRI site,
(5'-GGAATTCATGGCTCTACCTAACCAGCAAACCG-3') and a 3'
RAN-specific primer with a BamHI site
(5'-CGGATCCTTACTCAAAGATATCATCATCGTC-3'). Upon digestion with
EcoRI and BamHI, the PCR product was inserted into
EcoRI/BamHI-digested pGBKT7 (Clontech) to generate the
RAN1:BD vector. To generate the HST:BD vector, the
HST-coding region was amplified from the HST cDNA using
primers flanked by NcoI sites, and was inserted into
NcoI-digested pGADT7. The
N-hst:AD vector, which
contains an 106 N-terminal deletion of HST, was created in a similar
fashion using the primers 5'-AGACCATGGCTCTTAAGAGTCAGTCTGCT-3' and
5'-GCGGATCCATGGATTGTACGAACTCTTCATCC-3'. The hst-3:AD
vector was created by in vitro mutagenesis (Stratagene Quick Change kit) of
the HST-AD vector to introduce a mutation present in hst-3.
Yeast transformation and subsequent two-hybrid analyses were carried out
according to the Matchmaker protocol (Clontech). In addition, we made
constructs to test the reciprocal two-hybrid interaction, i.e. RAN
fused to pGADT7 and HST fused to pGBKT7, but we were unable to
recover RAN-GAD transformants, which suggests that this
construct somehow affected yeast growth.
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RESULTS |
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Although hst-1 accelerates floral induction, it has a more variable effect on bolting and the appearance of the first open flower. hst-1 plants sometimes bolt and produce mature flowers earlier than normal (Table 1), but may flower later than wild-type plants under some conditions. For example, the hst-1 plants illustrated in Fig. 3D produced their first open flower 1 day later than normal (24.3 DAP versus 23.5 DAP) even though they produced floral buds 2 days early. This observation suggests that bolting and floral maturation are either regulated independently of floral initiation in mutant plants, or that hst-1 delays bolting and floral maturation after floral initiation has taken place. Given the effect of hst-1 on root and hypocotyl growth, we believe that the latter explanation is more likely.
Unexpectedly, hst-1 has opposite effects on flowering time in short days (SD). Although mutant plants flower slightly earlier than normal in long days (LD; 16 hours light:8 hours dark) or continuous light, they flower much later and with nearly twice as many leaves and bracts as wild-type plants in SD (8 hours light:16 hours dark) (Table 1). We conclude HST normally acts to repress flowering in LD and to promote flowering in SD.
Organ polarity
The first two leaves of hst-1 plants are generally flat or only
slightly up-rolled, but subsequent leaves curve strongly upward
(Fig. 1B). This effect on leaf
expansion is associated with a loss of tissue polarity within the mesophyll of
the leaf blade. In wild-type leaves, cells in the upper mesophyll are
spherical or slightly cylindrical in shape and are densely packed, whereas
cells in the lower mesophyll layer are irregular in shape and are separated by
large air spaces (Fig. 4A).
Although the upper mesophyll layer of hst-1 appears normal, cells in
the lower spongy layer are more regular in shape and have less intercellular
space than normal (Fig. 4B),
which causes this layer to resemble the upper (adaxial) mesophyll layer. A
role for HST in the regulation of organ polarity is also apparent in the
effect of hst-1 on carpel development. Although the carpels of
hst-1 flowers are quite normal early in the growth of the
inflorescence, older inflorescences typically produce flowers with a laterally
expanded stigma, unfused carpels and external ovules
(Fig. 4D,E). The severity of
this defect varies between plants, but occurs in 30% of flowers
(n=277). The defect in carpel fusion and ovule production is usually
limited to the apical end of the carpels although, in rare cases, it may
extend along their entire length (Fig.
4E). This phenotype is characteristic of adaxializing mutations,
e.g. mutations in members of the YABBY
(Bowman, 2000
;
Siegfried et al., 1999
) and
KANADI (KAN) (Eshed et
al., 2001
; Kerstetter et al.,
2001
) gene families, and suggests that HST may regulate abaxial
polarity in carpels as well as in leaves.
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HST interacts with SQUINT
Mutations in SQUINT (SQN), the Arabidopsis
ortholog of cyclophilin 40, are phenotypically similar to hst
mutations in that they slightly delay leaf initiation (albeit at a different
stage in shoot development), accelerate vegetative phase change and produce
aberrant phyllotaxy in the inflorescence as well as abnormal patterns of
carpel development (Berardini et al.,
2001). To determine the genetic relationship between these two
genes, we examined the phenotype of hst-1, sqn-1 plants. Double
mutant seedlings had a much more severe phenotype than either single mutant
(Fig. 5). In addition to being
significantly smaller than both sqn-1 and hst-1, double
mutants produced large numbers of abaxial trichomes starting with leaf 1. By
contrast, hst-1 and sqn-1 did not produce abaxial trichomes
until leaf 3, and had only a few abaxial trichomes on this leaf. We conclude
that SQN and HST either operate cooperatively in the same
regulatory pathway or operate in parallel pathways.
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To determine the effect of overexpressing HST, the HST cDNA was fused to the CaMV 35S promoter in pCAMBIA 3301 and the resulting construct was introduced into hst-1 plants by Agrobacterium transformation. This transgene was capable of completely rescuing the mutant phenotype of hst-1 (Table 1), demonstrating that it encodes a functional HST protein. Wild-type plants transformed with this construct, and which expressed high levels of HST mRNA (data not shown), were completely normal. In order to ensure that the absence of phenotypically aberrant plants was not due to loss-of-function mutations in the transgene, an insertion that completely rescued the hst-mutant phenotype (Table 1) was crossed into a Col genetic background. Col plants carrying a single copy of this transgene were not obviously different from their wild-type siblings: transgenic plants (n=13) had 4.6±0.2 juvenile leaves and 5.5±0.3 adult leaves, whereas their wild-type siblings (n=29) had 4.4±0.1 juvenile leaves and 5.5±0.2 adult leaves. This result suggests either that plants are insensitive to levels of the HST protein above the wild-type amount, or that HST is post-transcriptionally regulated.
HST is similar to exportin 5
HST encodes a protein consisting of 1202 amino acids (133 kDa)
with approximately 12 regions that have similarity to the HEAT repeats
typically found in karyopherins in the importin ß family
(http://www.embl-heidelberg.de/~andrade/papers/rep/search.html)
(Andrade et al., 2001). BLAST
searches of non-redundant databases at GenBank revealed that HST is similar in
both size and amino acid sequence to the mammalian karyopherin exportin 5
(Xpo5) (1204 amino acids, 136 kDa) and the orthologous protein Msn5p (1224
amino acids, 142 kDa) from Saccharomyces cerevisiae
(Fig. 7C). Arabidopsis
genes encoding importin ß-like proteins were identified by searching the
Arabidopsis genome database
(http://arabidopsis.org/wublast/index2.html)
for proteins with similarity to members of this family in yeast and mammals.
Arabidopsis has at least 17 predicted members of the importin ß
family. These Arabidopsis proteins are much more similar to mammalian
proteins than to their yeast homologs. A cladogram illustrating the
relationship between human importin ß-like proteins and predicted members
of this family in Arabidopsis is shown in
Fig. 8. HST is the most closely
related protein in Arabidopsis to Xpo5 and Msn5p, and is more similar
to Xpo5 than it is to any other protein in Arabidopsis. We conclude
that HST is the Arabidopsis ortholog of
Xpo5/MSN5.
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DISCUSSION |
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The potential cargo molecules of HST are difficult to predict because its
orthologs in yeast and mammals have quite different functions. In yeast, Msn5p
exports phosporylated forms of several different transcription factors
(Blondel et al., 1999;
Boustany and Cyert, 2002
;
DeVit and Johnston, 1999
;
Gorner et al., 2002
;
Kaffman et al., 1998
;
Komeili et al., 2000
) and
imports RPA (Yoshida and Blobel,
2001
), a protein involved in DNA replication and repair.
Loss-of-function mutations in MSN5 cause defects in the processes in
which these factors are involved, demonstrating that Msn5p has an essential
function. The function of Xpo5 is more controversial. Brownawell and Macara
(Brownawell and Macara, 2002
)
concluded that Xpo5 is a general exporter of double-stranded RNA-binding
proteins (dsRNA-BP) because it binds to and exports the dsRNA-BP interleukin
enhancer-binding factor 3 (ILF3), and it interacts in vitro with the
dsRNA-binding domains of several other proteins. Other investigators
(Bohnsack et al., 2002
;
Calado et al., 2002
) have
demonstrated that Xpo5 exports tRNAs and regulates the export of the
translation factor eELF1a indirectly, through the association of eELF1a with
tRNAs. The interaction between Xpo5 and ILF3 is also regulated by dsRNA
(Brownawell and Macara, 2002
)
but the functional significance of this observation remains unclear.
Brownawell and Macara (Brownawell and
Macara, 2002
) conclude that binding of ILF3 to dsRNA occurs in the
cytoplasm and promotes the dissociation of ILF3 from Xpo5, whereas Bohnsack et
al. (Bohnsack et al., 2002
)
propose that the interaction between Xpo5 and ILF3 is actually mediated by
dsRNA and is essential for the export of ILF3. These latter investigators
propose that Xpo5 primarily regulates RNA export, not protein export. This
conclusion is supported by the recent results of Gwizek et al. (Gwizek et al.,
2003), which indicate that Xpo5 preferentially exports RNAs that possess a
20-nucleotide minihelix with a base-paired 5' end and an unpaired
3' end. These investigators suggest that the primary cargo molecules of
Xpo5 may be siRNAs involved in gene silencing and the double-stranded
precursors of miRNAs.
Whatever the cargo molecules of HST may be, the phenotype of hst-1
suggests that they include factors required for plant growth as well as
factors that have more specific regulatory functions. A general requirement
for HST is evident in the relatively small size and reduced growth rate of
essentially all the organs of mutant plants. HST is also required for the
growth and/or organization of the shoot apical meristem (SAM), as demonstrated
by the aberrant morphology of the SAM in mutant seedlings and the transient
delay in leaf initiation after germination. We tested the sensitivity of
hst-1 seedlings to auxin, cytokinin and abscisic acid but observed no
major effect of these hormones on root or hypocotyl growth (M.-Y.P. and
R.S.P., unpublished). Thus, the hormonal basis, if any, for this growth defect
is unclear. An alternative possibility is that this growth defect results from
a change in cell identity. Mutations that disrupt radial patterning in the
root, for example, have an effect on root growth similar to that of
hst-1 (Benfey et al.,
1993). However, we do not think this explains the effect of
hst-1 on root and hypocotyl elongation because hst-1 has no
major effect on the cellular organization of these structures. We are
intrigued by the evidence that Xpo5 exports tRNAs and other double-stranded
RNAs because defects in the export of this class of RNAs would be expected to
have widespread effects on growth, like those observed in hst
mutants.
It is unlikely that HST transports only factors with housekeeping functions
because of the developmental specificity of many aspects of its mutant
phenotype. In a previous study, we showed that hst-1 accelerated
abaxial trichome production and increased the sensitivity of plants to the
floral promoter LFY. This leads to the conclusion that HST normally acts to
promote the expression of the juvenile phase and/or to repress the expression
of the adult phase (Telfer and Poethig,
1998). Because the effect of hst-1 on abaxial trichome
production can also be attributed to a change in leaf polarity, we examined
its effects on two non-polarized heteroblastic traits: adaxial trichome
density and leaf shape (Fig.
3). We found that hst-1 accelerates changes in both of
these traits, which supports our previous conclusion about its role in phase
change. This conclusion is also supported by the fact that the early flowering
phenotype of hst-1 is completely accounted for by its effect on the
duration of the juvenile and intermediate phases of shoot development; under
CL, it has little or no effect on the duration of the adult phase. This
specific effect on the duration of the juvenile phase is unusual. Other
flowering time mutations whose effect on vegetative phase change has been
studied have either no effect on the duration of the juvenile phase
(Martinez-Zapater et al.,
1995
; Melzer et al.,
1999
; Scott et al.,
1999
; Telfer et al.,
1997
; Weigel and Nilsson,
1995
) or affect the duration of both the juvenile and adult phases
of development (Gomez-Mena et al.,
2001
; Soppe et al.,
1999
; Telfer et al.,
1997
). hst-1 is also unusual in that it has a completely
opposite effect on flowering time in SD. Under these conditions, mutant plants
display accelerated vegetative phase change but are extremely late flowering.
One possible interpretation of this phenotype is that HST has different
functions in vegetative phase change and floral induction. Early in shoot
development, HST promotes the juvenile vegetative phase and reproductive
incompetence, whereas during the adult phase it acts to promote flowering
under SD but plays no direct role in floral induction under LD. These
functions may be carried out by different molecules that require HST for their
nucleocytoplasmic transport.
How might HST regulate phase change? Because overexpression of HST
has no effect on the timing of vegetative phase change or floral induction, it
is unlikely that these processes are regulated by a change in the
transcription of HST. One possibility is that phase change is
regulated by post-transcriptional modulation of the amount or activity of HST,
as occurs in the case of CRM1 during Xenopus embryogenesis
(Callanan et al., 2000). A
second possibility is that phase change is regulated by a change in the amount
or character of the cargo molecules transported by HST, not by changes in the
activity of HST itself. In this scenario, HST would have a permissive
function, rather than a regulatory one. We favor the latter hypothesis because
hst-1 has a phenotype in every part of the plant, implying that HST
is active at all times and in all tissues. However, the possibility that HST
is post-transcriptionally regulated in certain tissues cannot be excluded.
Along with promoting the production of trichomes on the abaxial surface of
basal leaves in the rosette, hst-1 reduces the lobing of cells in
lower mesophyll layer of the leaf, causes up-rolling of the leaf blade, and
produces carpels that are partially unfused and bear ovules on their external
(adaxial) margin. hst-1 also interacts synergistically with
kan to increase abaxial trichome production and enhances the polarity
defects in kan; pkl carpels
(Eshed et al., 2001). These
features are typical of adaxialized mutants
(Bowman, 2000
;
Eshed et al., 2001
;
Kerstetter et al., 2001
;
McConnell et al., 2001
), and
suggest that HST may be required for the specification of abaxial cell
identity in both leaves and floral organs. However, whether HST regulates leaf
polarity independently of its role in phase change is unclear. Although
adaxial-abaxial polarity is an inherent feature of all lateral organs, the way
in which this polarity is expressed varies in different organs and at
different stages of shoot development. For example, the first two leaves of
the rosette are usually flat, subsequent rosette leaves curve downward,
inflorescence leaves are flat, and sepals, petals and carpels curve upward
(Griffith et al., 1999
).
Similarly, trichomes are restricted to the adaxial surface of juvenile leaves,
are present on both surfaces of adult leaves and are confined to the abaxial
surface of leaves in the inflorescence. Many polarized traits in other
organisms are also expressed phase specifically
(Kerstetter and Poethig,
1998
). This phenomenon implies that the mechanism that establishes
the adaxial-abaxial polarity of an organ is regulated at some level by the
mechanism that specifies the phase identity of the organ. Thus, although it is
possible that HST regulates organ polarity independently of its role in phase
change, it is also possible that the effect of HST on organ polarity is
actually mediated by its effect on phase change.
The basis for the mutant phenotype of HST will only become
apparent, of course, when its cargo molecules have been identified. This
pleiotropic phenotype suggests that HST transports many different molecules,
some of which have regulatory functions. We are particularly interested in the
possibility that HST regulates the transport of tRNAs and other
double-stranded RNAs and their associated proteins. This hypothesis is not
only suggested by function of Xpo5, but is also consistent with the
observation that mutations in PAUSED, the Arabidopsis
ortholog of the tRNA export receptor exportin t, are phenotypically similar to
hst (C.H. and R.S.P., unpublished)
(Telfer et al., 1997). Further
studies should reveal the types of molecules transported by HST and add to our
understanding of the role of nucleocytoplasmic transport in plant growth and
development.
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ACKNOWLEDGMENTS |
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