1 National Institute for Basic Biology, Okazaki 444-8585, Japan
2 Department of Molecular Biomechanics, The Graduate University for Advanced
Studies, Okazaki 444-8585, Japan
Author for correspondence (e-mail:
mhasebe{at}nibb.ac.jp)
Accepted 2 June 2003
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SUMMARY |
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Key words: Homeobox, Leucine zipper, Epidermal cell, Rhizoid, Auxin, Chloroplast, Physcomitrella patens, Cell differentiation
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Introduction |
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Molecular genetic studies of individual specialized cell types in
angiosperms have provided information on the mechanism behind the patterns of
cell differentiation (Brownlee,
2000; Hülskamp et al.,
1994
; Marks,
1997
). Positioning of presumptive cells is likely regulated by
cell-cell communication via molecular signals, such as the phytohormone
ethylene, which functions in root hair formation
(Tanimoto et al., 1995
), and
the serine protease subtilisin, which participates in stomatal guard cell
differentiation (Berger and Altmann,
2000
). The factors involved in trichome differentiation, as well
as additional determinants of root hair and stomatal guard cell
differentiation, are unknown. Communication usually occurs between clonally
unrelated cells in root hairs and trichomes, while the determination of
stomatal guard cells occurs via cell-cell interactions among clonally related
cells (Brownlee, 2000
;
Glover, 2000
). Such cell-cell
communication triggers signal transduction cascades and induces transcription
of downstream genes involved in later events of cell differentiation. Various
classes of transcription factors are involved in these cascades
(Lee and Schiefelbein, 1999
;
Oppenheimer et al., 1991
;
Payne et al., 2000
;
Wada et al., 1997
).
GLABRA2 (GL2), a member of the homeodomain-leucine zipper
(HD-Zip) gene family, functions in the promotion and repression of
specialization in trichome and root hair cells, respectively
(Di Cristina et al., 1996
;
Rerie et al., 1994
), in
co-operation with other transcription factors
(Lee and Schiefelbein, 1999
;
Oppenheimer et al., 1991
;
Payne et al., 2000
;
Wada et al., 1997
).
The HD-Zip genes, characterized by a homeodomain and an adjacent leucine
zipper motif, form four subfamilies (Sessa
et al., 1994) and are found only in green plants [the HD-Zip I-IV
subfamilies (Sakakibara et al.,
2001
)]. In addition to GL2 of the HD-Zip IV subfamily,
some members of the HD-Zip gene family are involved in cell differentiation.
Overexpression of the HD-Zip I gene Athb-1 produces pleiotropic
effects on leaf cell differentiation
(Aoyama et al., 1995
), and most
members of the HD-Zip III subfamily play roles in the cellular differentiation
of stems (Baima et al., 2001
;
Zhong and Ye, 1999
) and leaves
(McConnell et al., 2001
).
Rhizoids are widely observed in green plants, but their development has not
been studied at molecular level, mainly because useful model systems were
lacking. The moss Physcomitrella patens is a plant suitable to study
rhizoid development, since techniques for transformation and gene targeting
have been established (Cove et al.,
1997; Schaefer,
2001
; Schaefer and Zrÿd,
1997
). The rhizoids of Physcomitrella patens are
multicellular filamentous structures that develop from the epidermal cells of
the stem of a leafy shoot (gametophore). The rhizoids function in the
attachment of leafy shoots to the substratum and in the uptake of nutrients
(Bates and Bakken, 1998
;
Duckett et al., 1998
). Auxin
is reported to increase the number of rhizoids
(Ashton et al., 1979
), although
the details of their spatial patterns are unknown.
In this study, we report that rhizoid development can be divided into two processes, determination and differentiation. The cell-cell communication via auxin and unknown factor(s) regulates the former process, while a member of the HD-Zip I subfamily, Pphb7 induced by auxin is involved only in the latter process.
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Materials and methods |
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Microscopy
The histochemical detection of ß-glucuronidase (GUS) activity followed
the method of Nishiyama et al. (Nishiyama
et al., 2000). Leaves were observed after clearing with chloral
hydrate (Tsuge et al.,
1996
).
Images of rhizoid cells were digitized with a CCD camera (CoolSNAP, Roper Scientific Photometrics, Germany) to quantify rhizoid pigmentation. Light transmittance was calculated as the ratio of light intensity in each, red, green and blue channel on the rhizoid to that outside the rhizoid. The light intensity was determined as the mean in an area of 2500 pixels. In order to quantify chloroplast size, an outline of the chloroplast was traced on the image and the area was measured.
For the sectioning of resin-embedded tissues, gametophores were fixed in 2.5% formaldehyde and 2% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.0), dehydrated in a graded ethanol series, and embedded in Technovit 7100 (Heraeus Kulzer, Wehrheim, Germany). Sections (5 µm thick) were made with a microtome and stained with 0.05% (w/v) Toluidine Blue in 0.1 M sodium phosphate buffer (pH 7.0).
For scanning electron microscopy (SEM), gametophores were fixed with
modified Karnovsky's fixative (3% glutaraldehyde, 1.5% (w/v) paraformaldehyde
in 0.1 M sodium phosphate buffer, pH 7.4)
(Karnovsky, 1965) for 2 hours,
and soaked in 1% (w/v) tannic acid overnight. The samples were then fixed in
2% osmium tetroxide for 2 hours and in 1% uranyl acetate overnight before
dehydration through a graded ethanol series. The fixed preparations were
soaked once in an isoamyl acetate and ethanol mixture (1:1) for 30 minutes and
twice in isoamyl acetate for 30 minutes, and were critical-point dried using
carbon dioxide. The dried tissues were mounted on stubs and coated with gold
with an ion spatter. The preparations were observed using a scanning electron
microscope (S-800; Hitachi Ltd., Tokyo, Japan).
For transmission electron microscopy, rhizoids were fixed in the Karnovsky's fixative for 2 hours and 2% osmium tetroxide for 2 hours, stained with 1% uranyl acetate en bloc before dehydration, and embedded with Epon. A JEM 100CX (JEOL Ltd, Akishima, Japan) was operated at 100 kV.
Gene targeting
Most of the Pphb7 genomic DNA (2851 bp) amplified using Pphb7-52
(5'-TAGCGGCCGCGGAAAGGGGAGGGAAGGGTGTAA-3') and Pphb7-32
(5'-TAGCGGCCGCTCAGGGACGCACAACAGCGACAA-3') primers were cloned into
pGEM3z (Promega, WI), thereby generating pgPphb7.
A SalI site was added to the end of the coding sequence of pgPphb7
with the SalI-Pphb7S primer (5'-ACGCGTCGACTCCGAGCTCGATGGTTAAG-3').
The 693 bp SalI fragment from the internal SalI site to the
new SalI site was cloned into the SalI site of the
pGUS-NPTII-2 plasmid, which contained the coding sequence of uidA,
nopaline synthase polyadenylation signal (nos-ter) and NPTII cassette (nptII;
Nishiyama et al., 2000),
thereby creating an in-frame fusion of the Pphb7 and uidA
genes (Fig. 4B). A 1.2 kb
XbaI fragment of pgPphb7 that contained the 3' region was
inserted into this plasmid and the recombinant plasmid was designated as
pPphb7-GUS. This plasmid was linearized with ApaI and NotI
for gene targeting (Fig.
4B).
|
A 4.4 kb fragment that encompassed uidA, nos-ter, and nptII was
cloned into the PmaCI site of pgPphb7. The plasmid was linearized
with NotI for gene targeting (Fig.
4D). PEG-mediated transformation followed the method of Hiwatashi
et al. (Hiwatashi et al.,
2001).
RNA and DNA gel blot analyses
Poly(A)+ RNA and DNA extraction, blotting and hybridization
followed the method of Hiwatashi et al.
(Hiwatashi et al., 2001). A
BamHI-digested fragment of the plasmid p3Pphb7
(Sakakibara et al., 2001
) was
used as the cPphb7 probe for northern analysis
(Fig. 4A). A fragment amplified
with the Pphb7-52 and Pphb7-32 primers using pgPphb7 plasmid as template was
used as the gPphb7 probe (Fig.
4A). A GAPDH (Leech
et al., 1993
) cDNA fragment was amplified with the PpgapC5'
(5'-GAGATAGGAGCATCTGTACCGCTTGTGC-3') and PpgapC3'
(5'-CATGGTGGGATCGGCTAAGATCAAGGTC-3') primers, using pPpGapC
(Hiwatashi et al., 2001
) as
template. Radioactivity of hybridization signals was quantified with FUJIX
BAS2000 Bio Analyzer (Fuji Photo Film Co. Ltd., Tokyo, Japan).
Reverse transcription-polymerase chain reaction (RT-PCR)
Rhizoids were collected from gametophores cultured on the G medium for 6
weeks under continuous light. Total RNA was extracted using RNeasy Plant mini
kit (QIAGEN, CA). cDNA was synthesized with a mixture of dT20
primer and chloroplast gene specific primers
(5'-CAGATGGCTCGATTCGAGCAA-3',
5'-CAGCAGCTAATTCAGGACTCCA-3', and
5'-TCTAGAGGGAAGTTGTGAGCGT-3'). PCR was performed using
PfuTurbo Hotstart DNA polymerase. The products were analyzed by
electrophoresis and directly sequenced. The primer pairs were as follows: for
PpPOR1, 5'-CATTCGGGCTCAGGGTGTTG-3' and
5'-CCCATCAGCATATTAGCCAAGAGGA-3'; for PpPOR2,
5'-GCCAGCGTACAATCATCAGCA-3' and
5'-TGAGGACAGATCACAGTGCATCA-3'; for PprbcS1,
5'-TTGGCTGCATTGCCCTTGCGAT-3' and
5'-ATCAAAGCTACTGCTACCCGACC-3'; for chlB,
5'-AGTAATTCCTGAAGGAGGCTCTGT-3' and
5'-CGAGTTATTGAAGCTGCGTGAGT-3'; for rbcL (AB066207),
5'-TACCCATTAGATTTATTTGAAGAAGGTTC-3' and
5'-CGTTCCCCTTCAAGTTTACCTACTACAGT-3'; and for psbA,
5'-CTTGCTACATGGGTCGTGAGTG-3' and
5'-TGCTGATACCTAATGCAGTGAACC-3'.
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Results |
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Effect of exogenous auxin on rhizoid development
Ashton et al. (Ashton et al.,
1979) described increase in the number of rhizoids by exogenous
auxin. In this study, we observed spatial patterns of increased rhizoids. When
adult gametophores with 12-16 leaves were cultured in 0-, 0.1-, 1.0-, or 10
µM NAA for 1 week, the numbers of mid-stem rhizoids per gametophore
increased relative to NAA concentration
(Table 1;
Fig. 2A). With exogenous auxin,
the uppermost rhizoids formed in the more apical part of gametophores, and the
number of leaves above the uppermost mid-stem rhizoid decreased
(Table 1). The additional
rhizoids developed from stem epidermal cells in the cell files of the midrib
cells, as was the case with non-treated gametophores
(Fig. 2B). Adventitious
gametophores were formed from gametophore stems, and several rhizoids were
observed on the adventitious gametophores
(Fig. 2A).
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In order to characterize the detailed expression pattern of Pphb7,
the coding sequence of ß-glucuronidase (GUS)
(Jefferson et al., 1987) was
inserted in-frame just before the stop codon of Pphb7 by homologous
recombination (Fig. 4B),
enabling histochemical detection of GUS activity of the Pphb7-GUS fusion
protein. Stable transformants were obtained, two of which had the fusion
targeted to the Pphb7 locus, as confirmed by Southern analysis
(Fig. 4F). The blot showed that
one line (Pphb7-GUS-1) contained a single copy of the fusion inserted in the
Pphb7 locus (Fig.
4B,F), which was confirmed by sequencing the region. The second
transformant contained multiple copies of the fusion construct
(Fig. 4F). Since the GUS
activity patterns of the two Pphb7-GUS lines were not distinguishable, the
results for Pphb7-GUS-1 are shown (Fig.
5).
|
The subcellular localization of the protein was not readily detected in the
histochemical GUS staining of Pphb7-GUS lines, because subcellular membranes
were broken with the detergent. To determine the subcellular localization, the
GFP-Pphb7 fragment, in which the coding sequence of sGFP
(Chiu et al., 1996) was
inserted in-frame just before the start codon of Pphb7, was
introduced into P. patens (Fig.
4C). Southern analysis showed that one line (GFP-Pphb7-1)
contained a single copy of the insertion in the Pphb7 locus, while
other tested lines contained multiple copies of the fusion construct
(Fig. 4C,G). The intracellular
localization of GFP-Pphb7-2 and -3 was not distinguishable from that of
GFP-Pphb7-1.
Green fluorescence was detected in the nucleus and cytoplasm of apical
cells (Fig. 6A), but the
nuclear signal was stronger. The nuclear GFP signal was equally strong in the
second cell (Fig. 6B), but
weaker in subsequent cells (Fig.
6C,D). GFP fluorescence was detected in both the nucleus and
cytoplasm of a rhizoid in which the intact GFP was expressed under the control
of the GH3 promoter (Li et al.,
1999) (Sakaguchi, Fujita, and Hasebe unpublished)
(Fig. 6E-G). The wild-type
rhizoid emitted very weak fluorescence
(Fig. 6H-J).
|
|
As dehydration and osmotic stress induce expression of several HD-Zip I
genes (Lee et al., 1998; Söderman et
al., 1996; Söderman et
al., 1999
), gametophores with rhizoids were left without water or
were soaked in 6% mannitol (Fig.
7C). Dehydration induced Pphb7 expression in 1 and 6
hours, while 6% mannitol treatment did not induce Pphb7
expression.
Disruption of Pphb7 does not affect the number and position
of rhizoids with or without exogenous auxin
A DNA fragment that contained the NPTII cassette upstream from the homeobox
of Pphb7 was introduced into P. patens to obtain
Pphb7 disruptants (Fig.
4D). Five of 20 randomly selected stable lines in 107 independent
stable transformants were found by PCR analysis to have a disrupted
Pphb7 locus (data not shown). Southern analysis showed that two lines
(Pphb7dis-2 and -3) contained a single insertion in the Pphb7 locus,
while the other lines contained multiple copies
(Fig. 4H).
To examine whether Pphb7 is involved in the determination of rhizoids, the number and position of rhizoids in the wild type and in Pphb7 disruptants were compared (Fig. 8A). The number of mid-stem and basal rhizoids per gametophore with 11-14 leaves did not differ (Table 2). The number of leaves above the uppermost rhizoid was similar in the wild type and disruptants (Table 1), indicating that the position of the uppermost mid-stem rhizoid of Pphb7 disruptants was indistinguishable from wild type. The relationship between the leaf trace and mid-stem rhizoid cell of the Pphb7 disruptant was identical to that of the wild type (Fig. 8B-D).
|
Pphb7 disruption alters rhizoid differentiation
The size and shape of rhizoids were similar in wild type and disruptants
(data not shown). The wild-type rhizoids contained brown pigment, while those
of both Pphb7 disruptants were greener
(Fig. 3, Fig. 8A). Light transmittance
in the red, green and blue channels was estimated for rhizoid cells using CCD
images. The mean light transmittance of Pphb7dis-2 (data not shown) and
Pphb7dis-3 (Fig. 9A) at the
fifth cell was significantly higher than that of wild type
(P<0.001 by Student's t-test), indicating that rhizoids
of Pphb7 disruptants were less pigmented than those of the wild
type.
|
The project areas of the chloroplasts were measured to quantify chloroplast size. The mean chloroplast size was significantly larger in Pphb7 disruptant rhizoids than in those of wild type (P<0.01; Fig. 9C). To compare the structure of chloroplasts between the wild-type and Pphb7 disruptants, nucleoids were observed by staining with SYBR Green I (Fig. 8H,I). The number and size of nucleoids of wild type and of Pphb7 disruptants were similar. The internal structure of chloroplasts was further observed with electron microscopy. Well-developed thylakoid membranes with grana were observed in both wild type and Pphb7 disruptants (Fig. 8J,K).
To see the effects of auxin in rhizoid differentiation, the light transmittance and the number and size of chloroplasts in wild-type and disruptant rhizoids cultured in 10-µM NAA were compared (Fig. 9). The light transmittance and the number of chloroplasts were decreased by the NAA treatment in both wild type and Pphb7 disruptants (Fig. 9A,B). The size of chloroplasts increased in both wild type and disruptants (Fig. 9C). These results indicate that auxin is involved in both determination and differentiation of rhizoids.
To further characterize the effects of Pphb7 and auxin on
chloroplasts, mRNA expression of the following genes involved in the
photosynthesis was compared between wild-type and Pphb7-disruptant
rhizoids by RT-PCR: a ribulose-1,5-bisphosphate carboxylase/oxygenase large
subunit gene (rbcL) and a small subunit gene (PprbcS1) as
Calvin cycle enzymes (reviewed by Hartman
and Harpel, 1994), NADPH-protochlorophyllide oxidoreductase genes
(PpPOR1 and 2) and light-independent protochlorophyllide
reductase gene (chlB) as the key enzymes in chlorophyll biosynthesis
(reviewed by Armstrong, 1998
)
and the psbA gene encoding D1 protein as a component of photosystem
II (reviewed by Zhang and Aro,
2002
). Levels of rbcL and PprbcS1 expression
were higher in disruptants than in the wild type, while levels of PpPOR1,
PpPOR2, chlB and psbA did not differ from those of the wild type
(Fig. 10). The expression
levels of each gene in auxin-treated rhizoids were not different from those
without auxin (Fig. 10).
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Discussion |
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The number of mid-stem rhizoids increased with exogenous NAA (Tables 1, 2), indicating that auxin promotes mid-stem rhizoid development from stem epidermal cells below adult leaves. The number of basal rhizoids increased when juvenile gametophores were cultured with 0.1-µM NAA (Table 2), but did not increase when adult gametophores were cultured in NAA (Table 1). This indicates that auxin promotes basal rhizoid development from epidermal cells below juvenile leaves, before the cells are fully matured. While auxin plays inductive roles in both mid-stem and basal rhizoids, the patterns of additional rhizoid development differ. The increased mid-stem rhizoids always developed from stem epidermal cells that lay adjacent to a leaf trace (Fig. 1K), whereas additional basal rhizoids originated from epidermal cells in a random manner (Fig. 1M). These results suggest that auxin is sufficient for the induction of basal rhizoids, but that an unknown factor related to leaf traces is required for mid-stem rhizoid induction (Fig. 11A).
The midrib differentiates in the adult leaf, and probably functions as a
conducting tissue (Ligrone et al.,
2000). The aforementioned unknown factor may be produced in the
leaf and pass through the midrib and the leaf trace as a signal, which, in
conjunction with auxin, directs the epidermal cells around the leaf trace to
form rhizoids. In the absence of NAA, the uppermost mid-stem rhizoid is
usually associated with the eighth leaf from the gametophore apex, which
implies that it takes a set amount of time for the unknown factor to
accumulate in sufficient amounts to induce rhizoid development. Although
ectopic induction of root hairs by ethylene or its precursor ACC has been
reported (Tanimoto et al.,
1995
), ACC did not increase the number of rhizoids (data not
shown), suggesting that different signals are involved in rhizoid
induction.
Gametophores grown without NAA formed several mid-stem rhizoids around the
first mid-stem rhizoid during later stages of development
(Fig. 1L). Rose and Bopp
(Rose and Bopp, 1983) reported
basipetal auxin transport in rhizoids. The auxin transport from the rhizoid to
the stem, together with the unknown factor from the leaf trace, may enhance
the induction of mid-stem rhizoids. During Arabidopsis trichome
differentiation, previously differentiated trichomes inhibit surrounding cells
to differentiate into trichomes (Marks,
1997
). However, mid-stem rhizoids promote subsequent rhizoid
development.
Two explanations are possible for the differences in patterning between mid-stem and basal rhizoids. First, the different patterns may reflect differences in the distribution of the unknown factor (Fig. 11B). If the unknown factor is abundant in the basal part of a gametophore and is restricted around the leaf trace in the middle, every epidermal cell in the basal part can form a rhizoid in response to auxin, whereas only the epidermal cells around the leaf trace can form rhizoids. The structural differences between juvenile and adult leaves probably account for differences in the distribution of the unknown factor. In juvenile leaves, the unknown factor emanates from leaves, passes through the entire juvenile leaf, and broadly diffuses in epidermal cells, below the juvenile leaves without a midrib. Second, the differences in patterning may reflect different mechanisms for the determination of basal and mid-stem rhizoids. When basal rhizoids develop, the epidermal cells are formed while the gametophore is juvenile, but when mid-stem rhizoids develop, the epidermal cells are formed after the gametophore reaches adulthood. Different mechanisms could be involved during different developmental stages. Therefore, the unknown factor that is required for mid-stem rhizoid development may not be essential for basal rhizoid development.
Auxin and Pphb7 function in rhizoid cell
differentiation
The specific expression of Pphb7 was observed in the rhizoid
initial and rhizoid cells (Fig.
5), indicating that Pphb7 is involved in rhizoid
development. Since the Pphb7 disruptants formed rhizoids in positions
indistinguishable from those in wild-type plants
(Fig. 8), it appears that
Pphb7 is not involved in the positional determination of rhizoids,
but is involved in their differentiation
(Fig. 11A).
Once an epidermal cell had acquired a rhizoid initial cell fate,
Pphb7 regulates various features of the rhizoids, including
pigmentation and the size and number of chloroplasts in the rhizoid cell.
Chloroplasts in wild-type rhizoids are smaller than those in protonemata and
gametophores (data not shown), consistent with the notion that rhizoids are
not predominant photosynthetic organs
(Duckett et al., 1998).
However, the structure of rhizoid plastids resembles that of chloroplasts in
photosynthetic organs (Kasten et al.,
1997
). Rhizoid plastids possess chlorophylls, as observed by UV
excitation (Fig. 3),
well-developed thylakoid membranes with grana stacks at maturity
(Fig. 8J,K) and randomly
distributed plastid nucleoids (Fig.
8H,I). Rhizoid chloroplasts of Pphb7 disruptants had all
the three characteristics observed in wild-type rhizoid chloroplasts, although
the size and number of chloroplasts in disruptants were increased
(Fig. 3E-F,
Fig. 9B,C). This indicates that
Pphb7 does not affect plastid differentiation, but regulates
chloroplast size and number in rhizoid subapical cells. This is concordant
with the results in Arabidopsis, in which different genes regulate
these two processes. The double mutant of AtGlk1 and AtGlk2
exhibits reduction in granal thylakoids, but not in the number of plastids
(Fitter et al., 2002
). The
accumulation and replication of chloroplasts (arc) mutants
are affected in chloroplast division, but the chloroplasts develop normally in
these loss-of-function mutants (Pyke,
1999
).
The size and number of chloroplasts are negatively correlated in many
Arabidopsis mutants, suggesting that the genes are involved in
chloroplast division (Marrison et al.,
1999; Pyke, 1997
;
Pyke, 1999
). The phenotype of
Pphb7 disruptants clearly differs from these mutants. Both the size
and number of plastids were increased in the rhizoids of disruptants;
therefore, Pphb7 is considered as a new class of regulator that
down-regulates total chloroplast mass per cell. When the gametophytes were
treated with NAA, the expression of Pphb7 mRNA increased within 1
hour and further increased up to 6 hours
(Fig. 7A). The kinetics of
induction was comparable to auxin-responsive genes, such as PpIAA1
(Imaizumi et al., 2002
),
suggesting that Pphb7 is regulated by auxin fairly directly.
Histochemical staining of GUS activity in the Pphb7-GUS line confirmed that
the expression was up-regulated in rhizoid cells
(Fig. 5). As auxin induces
Pphb7, which is involved in rhizoid differentiation, auxin is likely
a positive regulator of both rhizoid differentiation and determination
(Fig. 11A). Furthermore, auxin
is likely involved in rhizoid differentiation in another way, which can be the
major auxin response pathway promoting rhizoid differentiation, because
exogenous auxin enhanced all the three rhizoid characters, pigmentation,
chloroplast number and chloroplast size even in the Pphb7 disruptant
(Fig. 9). These responses were
slightly stronger in Pphb7 disruptant, which may indicate that
Pphb7 could function as negative regulator in such a second,
independent pathway.
BA also induced Pphb7 expression as determined by the northern
analysis (Fig. 7B). Microscopic
observation showed that new adventitious buds differentiated after BA
treatment, and new rhizoids formed at the bases of the newly formed
adventitious buds. The GUS signals of the Pphb7-GUS lines were visible at the
bases of the adventitious buds, but those in rhizoid cells were not different
from the controls. Therefore, Pphb7 induction by BA is likely caused
by increase of rhizoid initials accompanying adventitious bud formation. While
exogenous cytokinin induces chloroplast division in protonemata
(Abel et al., 1989), the
numbers of plastids in wild-type and Pphb7-disrupted rhizoids treated
with 10-µM BA for 48 hours were similar to that in non-treated rhizoids
(data not shown). This implies that the regulation of plastid division in
rhizoids differs from that in protonemata.
Other phytohormones (ABA and GA), dehydration and osmotic stress did not
induce Pphb7 more than water, which induced Pphb7 three
fold. This is concordant with the observation that rhizoids are induced by low
nutrient condition in some mosses
(Duckett, 1994). Although
submersion is known to induce stress response mediated by ethylene, we could
not detect the Pphb7 induction with the ethylene precursor ACC. ABA
and GA likely reduce Pphb7 expression, and further studies on
regulation of Pphb7 by phytohormones are necessary.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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