Plant Cell Physiol. 45(10): 1529–1536 (2004)
JSPP © 2004
Short Communication
Spatial and Temporal Tracing of Vessel Differentiation in Young Arabidopsis
Seedlings by the Expression of an Immature Tracheary Element-specific
Promoter
Hyunjin Pyo 1, 2, 3, Taku Demura 2 and Hiroo Fukuda 1, 2
1
2
Department of Biological Sciences, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033 Japan
Plant Science Center, RIKEN, 1-7-22 Suehiro, Tsurumi-ku, Yokohama-shi, Kanagawa, 230-0045 Japan
;
The vascular system is a complex tissue composed of
several vascular cell types. However, little is known about
the differentiation process of each vascular cell in situ. In
this study, we found that the expression of the Zinnia
cysteine protease 4 (ZCP4) promoter is restricted to only
immature tracheary elements (TEs) in situ. Therefore, we
monitored the early TE differentiation process in young
Arabidopsis seedlings using a fusion gene of the ZCP4 promoter and the β-glucuronidase gene as a molecular marker.
This approach revealed unique processes of vessel differentiation during early seedling development, in which discontinuous initiation of vessel element differentiation occurs at
distinct regions, followed by the simultaneous differentiation of protoxylem vessels and bidirectional differentiation
of metaxylem vessels to form a vessel in the plant body.
Keywords: Arabidopsis — Cysteine protease — Metaxylem
vessel — Programmed cell death — Protoxylem vessel — Tracheary element differentiation.
Abbreviations: GUS, β-glucuronidase; HAI, hour after imbibition; PCD, programmed cell death; TE, tracheary element.
The vascular system forms the structural architecture
throughout the plant body for the transport of nutrients, water
and signal molecules. Therefore, vascular system formation is
regulated strictly in temporal and spatial manners. During primary vascular development, the pattern of vascular tissue is
first visualized with the appearance of the procambial cells.
The procambium of dicotyledonous embryos becomes evident
at the late globular stage as elongated cells in the center of the
embryo that are distinct from the nearly isodiametric surrounding ground tissue cells (West and Harada 1993, Jürgens 1994).
Consistent with a histological basis, the expression of a marker
gene, Athb-8, for the procambial state in Arabidopsis plants,
has successfully visualized the putative procambium of globular- to heart-shaped staged embryos (Baima et al. 1995). After
germination, the procambial cells subsequently differentiate
3
into the xylem and the phloem cells that constitute each vascular bundle (Dharmawardhana et al. 1992, Dolan et al. 1993,
Busse and Evert 1999a, Busse and Evert 1999b).
Vascular formation is an important issue in view of pattern formation in plants. In Arabidopsis, several mutants with
aberrant vascular systems that lack continuity have been
reported. Analyses of these mutants revealed the involvement
of polar auxin transport and the auxin response in vascular pattern formation (Hardtke and Berleth 1998, Hamann et al. 1999,
Hobbie et al. 2000, Deyholos et al. 2000, Geldner et al. 2004,
Hardtke et al. 2004). In addition, the identification of causal
genes of such mutants provided a new insight into the involvement of sterols (Choe et al. 1999, Diener et al. 2000, Jang et al.
2000, Carland et al. 2002, Schrick et al. 2002, Souter et al.
2002), small peptides (Casson et al. 2002) and cytokinins
(Mahonen et al. 2000, Inoue et al. 2001) in vascular patterning.
The vascular system is a complex tissue composed of
xylem and phloem cells that consist of several cell types such
as conductive elements, parenchyma cells, fiber cells and
cambium/procambium cells. However, little is known about the
differentiation process of each vascular cell in situ. Anatomical
and histological studies using an optical or electron microscope allowed us to follow the differentiation process, but only
after morphological cell changes. Moreover, the overall and
coordinated differentiation pattern of each vascular tissue in the
whole plant is difficult to understand because observations of
vascular tissues are made with thin tissue sections. As a result,
molecular markers are extremely useful for detecting cells at a
specific differentiation stage and for following the differentiation process of certain vascular cells. For example, the preferential expression of an Arabidopsis homeobox gene, Athb-8, in
procambial cells enabled us to use a fusion gene of this promoter and the GUS gene as a marker of procambium formation
(Baima et al. 1995). Indeed, studies with this molecular marker
have contributed to an understanding of procambium formation in plants (Koizumi et al. 2000, Kang and Dengler 2002,
Kang et al. 2003, Carland and Nelson 2004). However, we need
more molecular markers to understand the differentiation process of other vascular cells.
Corresponding author: E-mail, [email protected]; Fax, +81-3-3812-4929.
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Tracing Arabidopsis seedling vessel development
Fig. 1 GUS expression pattern in Arabidopsis seedlings harboring
the ZCP4 promoter::GUS gene. (A, B) Two-week-old seedlings. Magnified image of the part boxed by white lines in (A) is shown in (B).
(C, D, E) The rosette leaf of 1-month-old seedlings. Magnified images
of GUS expression in immature TEs are shown in (D) and (E). Circle,
the leaf primodia; asterisk, shoot apical meristem. Bars: (A) 1 mm; (B)
200 µm; (C) 0.5 mm; (D, E) 100 µm.
The in vitro cell culture system of Zinnia is an excellent
model with which to analyze the processes of vascular differentiation (Fukuda and Komamine 1980). In this system, about
half of the isolated mesophyll cells are induced to transdifferentiate into tracheary elements (TEs). Using this system, a
number of TE differentiation-related genes have been identified (Lin and Northcote 1990, Demura and Fukuda 1993, Ye
and Varner 1993, Demura and Fukuda 1994, Ye and Droste
1996, Aoyagi et al. 1998). Construction of a large-scale EST
database and comprehensive gene expression profiling using
this culture system have allowed the identification of many
developmental stage-specific genes that are related to auxin
function, signal transduction, cell wall formation and degradation, and programmed cell death (PCD) (Milioni et al. 2001,
Demura et al. 2002, Milioni et al. 2002).
To understand the differentiation process of a specific vascular cell, we considered the ZCP4 gene as an efficient molecular marker of TE differentiation. ZCP4 encodes a papain-like
cysteine protease, and its transcript is transiently induced to
dramatically high levels just before autolysis of TE PCD in
vitro (Yamamoto et al. 1997) and in vivo (Demura et al. 2002).
In this study, we first isolated the ZCP4 promoter from
Zinnia and performed a detailed analysis of the expression pattern of the ZCP4 promoter::GUS gene in transgenic Arabidopsis. This analysis clearly showed that expression of the
promoter occurred strictly in immature TEs, and not in mature
TEs or other cells, and that expression was early, occurring
before visible secondary cell wall formation. This demon-
strated that ZCP4 expression marks vessel differentiation prior
to morphological changes and temporally before cell contents
are lost. Therefore, we followed the TE differentiation process
using the ZCP4 promoter as a molecular marker. As a result,
we succeeded in presenting spatiotemporal patterns of vessel
differentiation during primary vascular development.
ZCP4 (GenBank accession no. AB091070) was isolated
from a cDNA library prepared from Zinnia cultured cells that
transdifferentiated into xylem cells (Demura et al. 2002). To
determine the spatial and developmental changes in the expression profile of ZCP4, we used inverse PCR to isolate a ZCP4
genomic DNA fragment that contained 1829 bp of the ZCP4
promoter region and the 27 bp coding region. The fragment
was translationally fused to the β-glucuronidase (GUS) gene,
and then introduced into Arabidopsis with Agrobacterium
tumefaciens. GUS expression, which was driven by the ZCP4
promoter in the shoot apical region of 2-week-old transgenic
plants, was exclusively located in the differentiating vessels,
but not in mature vessels, shoot apices or leaf primordia (Fig.
1A, B). The GUS expression in rosette leaves of 1-month-old
plants was also restricted to nascent vessels of higher order
veins and free-ending veinlets (Fig. 1C–E). Similarly, the ZCP4
promoter induced transient GUS expression that was specific to
the differentiating vessels of all organs examined. This
included cotyledons, hypocotyls, roots, stems and flowers (Fig.
2, data not provided). This staining pattern was the same as that
driven by the shorter promoter region (429 bp) (data not provided). The strict restriction of expression to developing vessels
and early expression (occurring before visible secondary wall
formation) imply that the ZCP4 promoter::GUS gene can be an
efficient marker for analyzing vessel development, rather than
morphological features.
Using the ZCP4 promoter::GUS gene, we tried to visualize the temporal and spatial distribution of vessel differentiation during the development of Arabidopsis seedlings. We
harvested Arabidopsis seedlings between 81 and 120 h after
imbibition (HAI) at 3 h intervals, and categorized their GUS
expression patterns as shown in Fig. 2, 3.
Initiation of the first protoxylem vessel differentiation
At 81 HAI, when the radicle protruded from the ruptured
seed coat, the first GUS expression was observed in the
proximal region of the cotyledon midveins and/or the distal
region of the vascular strands of the hypocotyls (Fig. 2, 3A1–
A3). At this time, we could not detect any developing TEs with
secondary cell walls (Fig. 2a, b). Interestingly, the proportion
of type A3 seedlings with GUS expression at the two regions
was about 20% through the early development of the seedling
(up to 99 HAI) (Fig. 4A). This result suggests that differentiation of the first protoxylem vessels initiates at the proximal
region of the cotyledon midvein and/or the distal region of the
hypocotyls.
Tracing Arabidopsis seedling vessel development
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Fig. 2 Typical pattern types of GUS expression driven by the ZCP4 promoter during the
development of transgenic Arabidopsis seedlings. GUS staining of transgenic Arabidopsis
seedlings between 81 and 120 HAI were categorized as follows. Early development of cotyledon midvein and hypocotyl-root axis: (A1)
proximal region of the cotyledon midvein;
(A2) distal region of the hypocotyl; (A3) combination of (A1) and (A2); (A4-1 and -2) discontinuous cell files through the cotyledon
midvein and hypocotyl-root axis; (A5) continuous cell files through the length of the cotyledon midvein and hypocotyl-root axis.
Magnified images of the regions boxed in (A1)
and (A2) are shown in (a) and (b), respectively. Subsequent development at the
hypocotyl-root axis: (B1) protoxylem vessels
at the most distal part of the root; (B2) protoxylem vessels at the most distal part of the roots
and metaxylem vessels at the hypocotyl-root
junction; (B3) in addition to the region
described in (B2), metaxylem vessels at the
hypocotyl-cotyledon junction; (B4) protoxylem vessels at the most distal part of the root,
metaxylem vessels around the cotyledonhypocotyl junction, and the upper and lower
regions of the hypocotyl-root junction. (C1)
shows the cotyledon with GUS expression at
the distal and proximal regions of the distal
secondary veins and at junctions between the
distal and proximal secondary veins. Magnified images of the regions boxed in (C1) are
shown in (c) and (d). (A1–A3) Seedlings at
84 HAI; (A4-1 and -2) seedlings at 90 HAI;
(A5) seedlings at 99 HAI; (B1) seedlings at
105 HAI; (B2) seedlings at 117 HAI; (B3)
seedlings at 114 HAI; (B4) seedlings at
120 HAI; (C1) seedlings at 111 HAI. Bars:
(A1–B4) 200 µm; (C1) 200 µm; (a, b) 30 µm;
(c, d) 50 µm.
Progress of protoxylem vessel differentiation
At 87 HAI, discontinuous and almost evenly distributed
GUS expression was observed in the cotyledon midvein and
along the hypocotyl-root axis (Fig. 2, 3A4). The first second-
ary cell wall formation was recognized only in some cells with
GUS staining at this time. Seedlings with this pattern markedly
increased until 93 HAI, and decreased thereafter (Fig. 4A).
However, type A5 seedlings with GUS expression through the
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Tracing Arabidopsis seedling vessel development
Fig. 3 Schematic diagram of expression patterns of the ZCP4 promoter::GUS gene in transgenic Arabidopsis seedlings. Gray, blue, red and
striped lines indicate procambial cells, protoxylem vessels with GUS expression, metaxylem vessels with GUS expression and mature vessels,
respectively.
length of the cotyledon midvein and hypocotyl-root axis
appeared at 96 and 99 HAI (Fig. 4A). This observation implies
that the differentiation of protoxylem vessels starts at the discontinuous region and progresses simultaneously to form continuous strands in cotyledon midveins, hypocotyls and roots.
Initiation of metaxylem differentiation in hypocotyls and roots
As development of the seedlings advanced, GUS expression in mature protoxylem vessels of the hypocotyls and roots
immediately disappeared and was restricted to immature protoxylem vessels, which were next to the previously differentiated cells in the same cell file of the xylem pole in roots (Fig. 2,
3B1). Thereafter, continuous protoxylem vessels were always
formed close to the root tip during seedling development (Fig.
2, 3B2–B4).
GUS expression in metaxylem vessels just inside the protoxylem vessels along the hypocotyl-root axis was first
detected at 105 HAI (Fig. 4B2). Expression was found only at
the hypocotyl-root junction (B2), or at both the hypocotyl-root
junction and the cotyledon-hypocotyl junction (B3) (Fig. 2, 3).
GUS expression restricted to the cotyledon-hypocotyl junction
was rarely observed. While type B2 seedlings were most common at 108 HAI, type B3 seedlings were most common at
111 HAI (Fig. 4B). These results indicate that metaxylem vessel differentiation along the hypocotyl-root axis initiates at the
hypocotyl-root and cotyledon-hypocotyl junctions, and also suggests that differentiation at the hypocotyl-root junction occurs
slightly before differentiation at the cotyledon-hypocotyl
junction.
Progress of metaxylem vessel differentiation in hypocotyls and
roots
Type B4 seedlings, which have mature metaxylem vessels
at the hypocotyl-root junction and GUS expression in immature
vessels both above and below the mature metaxylem vessels,
appeared at 108 HAI and increased until 120 HAI (Fig. 4B).
Fig. 4 Changes in the proportion of GUS expression patterns in
transgenic Arabidopsis plants. GUS expression patterns of more than
20 seedlings were observed at 3 h intervals from 81 to 120 HAI and
categorized into A0–A5 or B0–B4, as described in Fig. 3, 4. (A) A0–
A5 (B) B0–B4.
This result implies that metaxylem vessel differentiation that
was initiated at the hypocotyl-root junction progresses bipetally.
Vessel differentiation in cotyledon secondary veins
The temporal patterns of GUS expression in the secondary veins of cotyledons varied greatly in each seedling, which
allowed us to analyze changes in GUS expression patterns only
qualitatively. There were no seedlings with GUS expression in
Tracing Arabidopsis seedling vessel development
Fig. 5 Schematic diagram of the spatial pattern model of vessel differentiation during the early development of Arabidopsis seedlings.
The first protoxylem vessels differentiate at two separate regions: the
cotyledon-hypocotyl junction and the proximal region of the cotyledon (yellow). Subsequently, vessel differentiation occurs almost simultaneously in the cotyledon and hypocotyl-root axis (blue). Following
this, metaxylem vessels differentiate bipetally from the cotyledonhypocotyl and hypocotyl-root junctions (red arrows). Vessel differentiation in the distal secondary veins of the cotyledons begins at four
regions (red). Subsequently, vessel differentiation occurs almost simultaneously in four regions (green). Gray represents proximal secondary
vein at the procambial stage. mv, midvein region; ds, distal secondary
vein region; ps, proximal secondary vein region.
the secondary veins of cotyledons until 102 HAI. GUS expression in the secondary veins was first observed in the distal
region of cotyledons at 105 HAI (Fig. 2, 3C1). Between 108
and 120 HAI, GUS expression was observed mainly at both the
distal and the proximal regions that crossed the midvein, and
sometimes at the junctions of loops of the secondary veins (Fig.
2, 3C1). Subsequently, a closed loop of GUS-expressing cells
appeared along the secondary veins of some seedlings (see cotyledons in Fig. 2B2, 3C2).
In plants, increased gene expression of the papain-like
cysteine proteases is associated with protein remobilization
during seed germination (Tranbarger and Misra 1996), organ
senescence (Lohman et al. 1994, Coupe et al. 2003) and numerous plant cell suicide programs (Solomon et al. 1999, Fukuda
2000, Hayashi et al. 2001, Wan et al. 2002). The final process
of TE differentiation involves developmental PCD, during
which time cell components are autolyzed (Fukuda 2000).
ZCP4 transcripts accumulate transiently just before the autolysis of TE PCD in vitro (Yamamoto et al. 1997) and in vivo
(Demura et al. 2002). Consistent with such previous studies, its
homologs, Zinnia p48h-17, Arabidopsis XCP1, XCP2 and Zea
1533
AI734319, have been reported to be expressed preferentially in
immature xylem cells (Ye and Varner 1996, Funk et al. 2002)
and vascular cells (Nakazono et al. 2003). These results
strongly suggest that ZCP4-type cysteine proteases function in
vessel development.
The expression of most vascular differentiation-related
genes that have been isolated so far is not restricted to vessel
cells (Igarashi et al. 1998, Li et al. 1999, Chen et al. 2000,
Lauvergeat et al. 2002, Groover et al. 2003). In this study, we
demonstrated that expression of the ZCP4 promoter occurred
strictly in immature vessels, and not in mature vessels or other
cells such as xylem parenchyma cells and meristem cells. Nor
was the ZCP4 promoter induced by wounding (data not provided). These results suggested the possibility of using the
ZCP4 promoter as a marker of the temporal and spatial distribution of cells undergoing vessel differentiation. Therefore, we
used a chimeric ZCP4 promoter::GUS gene to monitor vessel
development in young Arabidopsis seedlings after germination, and succeeded in revealing the formation of the spatiotemporal vessel pattern in seedlings.
The pattern of vessel differentiation in the young Arabidopsis seedlings is summarized in Fig. 5. Protoxylem vessel
differentiation is initiated discontinuously (yellow lines) and
then progresses almost simultaneously along the cotyledon,
hypocotyl and root (blue lines and blue arrow). In the root, new
protoxylem vessel elements are added acropetally onto the previously differentiated elements in the same cell file, and consequently, a continuous, uninterrupted protoxylem vessel is
established close to the root tip (blue arrow). Similar to the
cotyledon midvein, vessel differentiation is initiated discontinuously at vessel junctions (red lines) and progresses simultaneously to form continuous secondary veins (green lines).
Metaxylem vessel differentiation also discontinuously starts at
the cotyledon-hypocotyl and hypocotyl-root junctions (red
arrows). Interestingly, metaxylem vessels subsequently differentiate bidirectionally from the junctions.
Previous anatomical studies suggested that differentiation
and lignification of the protoxylem vessels proceed acropetally
in the cotyledon midvein and basipetally in the hypocotyl during the early development of Arabidopsis seedlings (Dharmawardhana et al. 1992, Busse and Evert 1999a). In this study,
however, we demonstrated that protoxylem vessel differentiation is initiated discontinuously and then progresses almost
simultaneously along the cotyledon midvein, hypocotyl and
root. This new finding might be due to the use of an efficient
molecular marker, with which we could easily follow the early
stages of vessel differentiation throughout the plant in detail. In
contrast, morphological criteria such as secondary cell wall
thickening and lignification, which are the markers not only of
differentiating vessel cells, but also of mature vessel cells,
might not precisely mirror the sequence and timing of cell differentiation. Little is known about spatial and temporal patterns of metaxylem vessel differentiation. A molecular marker,
the chimeric ZCP4 promoter::GUS gene, also allowed us to fol-
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Tracing Arabidopsis seedling vessel development
low the development of metaxylem differentiation, and to
determine the bidirectional development of metaxylem vessels.
We indicated that new vessel differentiation starts at
restricted regions in young Arabidopsis seedlings; that is, the
proximal region of the midveins in cotyledons, the distal region
of hypocotyls for the first protoxylem vessel differentiation
(Fig. 5, yellow), and the cotyledon-hypocotyl and hypocotylroot junctions for metaxylem vessel differentiation (Fig. 5, red
arrow). In the secondary veins of cotyledons, vessel differentiation started at four vein crosses (Fig. 5, red). What initiates the
differentiation of xylem vessel elements at the restricted
regions? The initiation might be coupled with phytohormonal
action. Auxins and cytokinins are well known to be involved in
the initiation of vessel differentiation (Sachs 1981, Fukuda
1992, Aloni 1987, Aloni 1995). In Zinnia culture cells, vessel
cell differentiation is induced only in culture medium that contains both auxins and cytokinins (Fukuda and Komamine
1980). Indeed, the ZCP4 gene was induced only when cells were
cultured in auxin-cytokinin-containing medium (Yamamoto et
al. 1997). Recent progress using molecular markers and specific monoclonal antibodies has enabled the visualization of
patterns of production, movement, and the accumulation of free
auxin in developing plant tissues (Sieburth 1999, AvsianKretchmer et al. 2002, Friml et al. 2002, Marchant et al. 2002,
Kamada et al. 2003, Aloni et al. 2003). Visualization of auxin
distribution using an auxin-inducible promoter during Arabidopsis leaf development indicated that free auxin accumulates
in the region at which the midvein and secondary veins of
rosette leaves meet (Aloni et al. 2003). The leaf-stem junction
is suggested to be the local barrier that slows down auxin flow
and results in the increasing levels of auxin concentrations
found there (Aloni 2001). Kamada et al. (2003) suggested that
the root-shoot junction is a source of auxin signals for gravimorphogenesis (peg formation) in cucumber seedlings. These
results strongly suggest that the initiation sites of vessel differentiation in young Arabidopsis seedlings might be the sites at
which free auxin accumulates. If this is true, further vessel differentiation from the initiation sites might also be controlled by
auxins. We found bidirectional differentiation of metaxylem
vessels from the initiation sites. This phenomenon cannot be
explained simply by the polar transport of auxins. However,
recent advance in studies on PIN auxin efflux carrier proteins
revealed dynamic changes in inter- and intracellular localization of the PIN proteins and subsequent changes in the direction of auxin flow (Friml et al. 2003). Therefore, to understand
the mechanism of the initiation and further development of vessel cell differentiation in young seedlings, we need to reveal the
accumulation and flow of auxins in vascular tissues.
A 1,625 bp genomic fragment DNA, which corresponded
to the ZCP4 cDNA, was obtained by PCR from Zinnia
genomic DNA using primers for the 5′ and 3′ ends of the
cDNA (5′-CAACAATGGCATTCATATTCTCTTCCAAAA-3′
and 5′-GCTGAAAAATGATGGATTTATTCATTCCAA-3′, re-
spectively). The PCR product was cloned into a pGEM-T Easy
vector (Promega, Madison, WI, U.S.A.), yielding pZCP4-G.
The ZCP4 promoter region flanking the coding region was obtained using the inverse PCR method (Ochman et al. 1988)
with a set of specific primers for the ZCP4 cDNA (5′-GATTGAAAACTCGTGGGCTAAAGCC-3′ and 5′-ACATAGGTTTGAGATCTTCATGGAC-3′). The resulting PCR product was
cloned into pGEM-T Easy vector, yielding pZCP4-P.
Using pZCP4-P, a 1,829 bp and a 429 bp promoter fragment were amplified using primers that incorporated an XbaI
site (underlined) at the 5′ end (5′-GGTCTAGACTCAAGACATTTCTTACTTATAGAC-3′) and (5′-GGTCTAGATCCACCATCCCTTATAATGTAATAT-3′) and a BamHI site at the 3′
end (5′-CCGGATCCCTTTTTGGAAGAGAATATGAATGCC3′). The resulting PCR products were subcloned into pGEM-T
Easy and subsequently digested with XbaI and BamHI. The
XbaI–BamHI fragments were inserted in-frame with the GUS
reporter gene in the binary vector, yielding pZCP4-GUS. The
pZCP4-GUS binary vector was used to transform A. tumefaciens, strain C58C1. Four-week-old Arabidopsis plants were
infected with the transformed Agrobacterium strain, according
to the vacuum infiltration method (Bechtold and Pelletier
1998). Transgenic plants were selected by plating on medium
that contained hygromycin B (20 µg ml–1). T1 plants and
homozygous T3 seeds were used in this study.
For the tracing vessel differentiation, homozygous T3
seeds were surface-sterilized by immersion in a solution of 1%
(v/v) sodium hypochlorite and 0.1% (w/v) Triton X-100 for
10 min, and rinsed five times with sterile deionized water prior
to plating. Plates were sealed with gas-permeable tape, incubated for 72 h in the dark at 4°C, and then incubated at 24°C
under constant illumination. Seedlings were harvested at the
indicated HAI to measure development.
Histochemical GUS staining was performed according to
the method of Jefferson et al. (1987). Seedlings were fixed in
90% (v/v) acetone for more than 1 day at –20°C. After washing in 100 mM sodium phosphate buffer (pH 7.0) at least three
times, the seed coats were removed from the seedlings. Samples were immersed in a reaction buffer (1 mM 5-bromo-4chloro-3-indolyl glucuronide, 0.5 mM potassium ferricyanide
and 0.5 mM potassium ferrocyanide) in 100 mM sodium phosphate buffer (pH 7.0), and then incubated at 37°C for 1 h in the
dark. After GUS staining, the samples were cleared in a clearing solution (8 g chloral hydrate, 1 ml glycerol and 2 ml water),
and then observed under a light microscope equipped with
Nomarski optics (Olympus, Tokyo, Japan).
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(Received June 28, 2004; Accepted August 8, 2004)