Download Multiple Regulatory Elements Contribute to the Vascular

Plant Cell Physiol. 46(8): 1400–1410 (2005)
doi:10.1093/pcp/pci153, available online at www.pcp.oupjournals.org
JSPP © 2005
Multiple Regulatory Elements Contribute to the Vascular-specific Expression
of the Rice HD-Zip Gene Oshox1 in Arabidopsis
Enrico Scarpella 1, 2, Erik J. Simons 1 and Annemarie H. Meijer 1, *
1
2
Insitute of Biology, Leiden University, Clusius Laboratory, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands
Department of Biological Sciences, University of Alberta, CW405 Biological Sciences Building, Edmonton AB, Canada T6G 2E9
;
The primary vascular tissues of plants differentiate
from a single precursor tissue, the procambium. The role of
upstream regulatory sequences in the transcriptional
control of early vascular-specific gene expression is largely
unknown. The onset of expression of the rice homeodomain-leucine zipper (HD-Zip) gene Oshox1 marks
procambial cells that have acquired their distinctive anatomical features but do not yet display any overt signs of
terminal vascular differentiation. The expression pattern of
Oshox1 in rice appears to be mainly controlled by the activity of the 1.6 kb upstream promoter region. Here, we show
that the Oshox1 promoter directs vascular, auxin- and
sucrose-responsive reporter gene expression in Arabidopsis
plants in a fashion comparable with that in rice. This is the
case not only during normal development but also upon
experimental manipulation, suggesting that the cis-acting
regulatory elements that are instrumental in Oshox1 expression pattern are conserved between rice and Arabidopsis.
Finally, through analysis of reporter gene expression profiles conferred by progressive 5′ deletions of the Oshox1
promoter in transgenic Arabidopsis, we have identified
upstream regulatory regions required for auxin and sucrose
inducibility, and for cell type-, tissue- and organ-specific
aspects of Oshox1 expression. Our study suggests that
Oshox1 embryonic vascular expression is mainly achieved
through suppression of expression in non-vascular tissues.
Keywords: Arabidopsis — Cis-elements — Procambium —
Rice — Transcriptional regulation — Vascular marker.
Abbreviations: DAG, d after germination; GM, ground meristem;
GUS, β-glucuronidase; HD-Zip, homeodomain-leucine zipper; NAA,
naphthalene-1-acetic acid; β-NAA, naphthalene-2-acetic acid.
Introduction
The vascular tissues of plants are organized in a continuous network of vascular strands that extend through each organ
and throughout the entire plant, functionally connecting all
parts of the shoot with the root system (Esau 1965, Steeves and
Sussex 1989). Plant vascular tissues play crucial functions in
long-distance transport of water and nutrients, and confer
*
rigidity to the plant body. Furthermore, plant vascular tissues
are a source of signals that act locally, to organize surrounding
tissues, and across the whole plant, to coordinate the initiation
of new shoot organs with that of new roots (Nelson and
Dengler 1997, Berleth and Sachs 2001, Dengler 2001, Turner
and Sieburth 2002, Ye 2002, Berleth and Scarpella 2004,
Fukuda 2004). As such, the patterned differentiation of
vascular tissues is tightly interconnected with embryo development and post-embryonic organ formation (Berleth and Sachs
2001, Dengler 2001, Dengler and Kang 2001, Berleth and
Chatfield 2002, Turner and Sieburth 2002, Ye 2002, Berleth
and Scarpella 2004, Fukuda 2004). Experimental manipulation
of vascular development suggests that the vascular pattern is
stably established with the formation of the tissue precursor
of all primary vascular cells, the procambium (Sachs 1989,
Mattsson et al. 1999). The study of procambium formation is
therefore central to the understanding of vascular patterning.
However, procambial strands cannot be readily visualized in
large numbers of samples because of their internal position and
variable arrangement in developing organs (Nelson and
Dengler 1997, Turner and Sieburth 2002, Ye 2002, Scarpella
and Meijer 2004). Thus, the isolation of marker lines displaying early vascular reporter gene expression from different
enhancer and gene trap collections (Sundaresan et al. 1995,
Malamy and Benfey 1997, Clay and Nelson 2002, Holding and
Springer 2002, Scarpella et al. 2004) and the identification of
genes specifically expressed at early stages of vascular differentiation (Demura and Fukuda 1994, Baima et al. 1995,
Hardtke and Berleth 1998, Hiwatashi and Fukuda 2000,
Scarpella et al. 2000, Elge et al. 2001, Clay and Nelson 2002,
Hamann et al. 2002, Kang and Dengler 2002, Ohashi-Ito et al.
2002, Groover et al. 2003, Ohashi-Ito and Fukuda 2003,
Carland and Nelson 2004, Kang and Dengler 2004, Scarpella et
al. 2004) have generated invaluable tools to visualize procambium formation. For some of these genes, functional analysis
has supported a role in regulation of vascular development
(Berleth and Jürgens 1993, Przemeck et al. 1996, Carland et al.
1999, Hamann et al. 1999, Scarpella et al. 2000, Baima et al.
2001, Clay and Nelson 2002, Scarpella et al. 2002). However,
little is known of the role that upstream regulatory sequences
play in the transcriptional control of these early vascularspecific genes. The expression profile of the homeodomainleucine zipper (HD-Zip) gene Oshox1 of rice offers the con-
Corresponding author: E-mail, meijer@rulbim.leidenuniv.nl; Fax, +31-71-5274999.
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Cis-elements in Oshox1 vascular expression
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Results
1N). In siliques, expression was restricted to vascular strands
(Fig. 1O). Finally, in mature embryos, Oshox1-GUS expression was observed in procambial strands of the cotyledons, but
not in the procambial cylinder of the embryonic axis (Fig. 1P).
Unlike in rice, Oshox1-GUS was not expressed in stomata or
pollen of Arabidopsis (Fig. 1D, I–K, N).
In rice, Oshox1 is ectopically induced in non-vascular
cells of the root tip in response to auxin and sucrose (Scarpella
et al. 2000). To test whether the cis-elements that are involved
in auxin- and sucrose-regulated Oshox1 gene expression are
conserved in a dicot species, we tested Oshox1-GUS inducibility by auxin and sucrose in Arabidopsis. Oshox1-GUS was
ectopically induced in non-vascular cells of the Arabidopsis
root tip by naphthalene-1-acetic acid (NAA) (Fig. 1Q), IAA
and 2,4-D, but not by naphthalene-2-acetic acid (β-NAA) (an
inactive auxin analog). A similar Oshox1-GUS induction pattern was observed in the presence of 500 mM sucrose (Fig.
1R), but not upon a 500 mM mannitol treatment, thus excluding an induction due to osmotic stress.
In conclusion, the Oshox1 promoter is able to drive vascular, auxin- and sucrose-responsive reporter gene expression in
transgenic Arabidopsis.
Oshox1 promoter-driven expression pattern in Arabidopsis
To investigate whether the cis-elements that are instrumental for the vascular-specific expression of the rice HD-Zip
gene Oshox1 are conserved in a dicot species, we analyzed in
Arabidopsis the expression pattern of the β-glucuronidase
(GUS) reporter gene when driven by the 1.6 kb Oshox1 promoter fragment (Oshox1-GUS).
In post-embryonic root tips of Arabidopsis seedlings harboring the Oshox1-GUS transgene, reporter gene activity was
detected in the central vascular cylinder with the exclusion of
its most distal part (Fig. 1A, 6R). In mature regions of the root,
Oshox1-GUS expression remained confined to the vascular
cylinder, and expression was never detected in cortex or epidermis (Fig. 1B). Oshox1-GUS was expressed in all the cell types
of the vascular cylinder, including the pericycle (Fig. 1C). In
seedling hypocotyls, Oshox1-GUS expression was detected in
all the cell types of the vascular cylinder, including the pericycle (Fig. 1D, E). In leaves, expression was detected in procambial strands before any detectable signs of cell wall
lignification (Fig. 1I, J), and persisted in all the cell types of the
completely differentiated vascular strands of mature leaves,
including bundle sheath cells (Fig. 1K, L). Further, Oshox1GUS expression was detected in leaf trichomes (Fig. 6X). In
the basal part of the stems of mature plants, Oshox1-GUS
expression was detected in the interfascicular and vascular
cambium, and in the phloem (Fig. 1G, H). In hypocotyls of
plants at the same stage, expression was detected in the cambial zone and in the phloem (Fig. 1F). Oshox1-GUS expression was detected in immature vascular strands of floral buds
(Fig. 1M), and expression continued to be present in the completely differentiated vascular strands of mature flowers (Fig.
Oshox1-GUS expression in Arabidopsis leaf development
To examine the dynamics of the Oshox1-GUS expression
pattern, and to compare them with those of procambium formation, we simultaneously analyzed Oshox1-GUS and ET1335GUS expression in Arabidopsis first leaf development. At all
stages of leaf development, the onset of ET1335-GUS expression coincides precisely with overt procambium formation,
and remains strong in fully differentiated vascular strands
(Scarpella et al. 2004).
At 2 d after germination (DAG), the first two vegetative
leaf primordia were recognizable as semi-spherical bulges
flanking the shoot apical meristem (Fig. 2A, B). ET1335-GUS
expression marking the formation of the primary procambial
strand became visible in the elongated primordia at 3 DAG
(Fig. 2C). At 4 DAG, lamina formation was initiated, and
ET1335-GUS expression was additionally, but transiently,
detected in ground meristem (GM) cells at the leaf tip (compare Fig. 2E and I). At this stage, Oshox1-GUS expression was
first detected in GM cells at the leaf tip (Fig. 2F). ET1335-GUS
expression labeling the appearance of the first loops of secondary procambial strands was detected at 5 DAG (Fig. 2G).
Oshox1-GUS expression was detected in the distal portion of
the primary procambial strand in 5 DAG primordia (Fig. 2H).
At 6 DAG, ET1335-GUS expression marked the formation of
the second loops of secondary procambial strands (Fig. 2I), and
Oshox1-GUS expression in the primary procambial strand
extended basally, and was additionally detected in the distal
portion of the first loops of secondary procambial strands (Fig.
2J). At 7 DAG, ET1335-GUS expression labeled third order
procambial strands in the first and second intercostal areas
(Fig. 2K). At this stage, Oshox1-GUS expression in the first
crete possibility to approach this issue experimentally. In all
rice organs and at all developmental stages of the rice plant,
Oshox1 is first expressed in procambial cells that have already
acquired their distinctive anatomical properties, but that have
not yet entered terminal differentiation pathways (Scarpella et
al. 2000). Additionally, Oshox1 expression in rice is responsive to auxin and sucrose, and all these features are largely
controlled by the activity of its own promoter (Scarpella et al.
2000).
In this study, we show that the Oshox1 promoter directs
reporter gene expression in Arabidopsis during the undisturbed
course of development and under experimentally challenged
conditions in a fashion comparable with that reported in rice.
Further, we show that the Oshox1 promoter confers auxin and
sucrose responsiveness to reporter gene expression in
Arabidopsis. Finally, by means of progressive 5′ deletions, we
show that the Oshox1 promoter has a complex and modular
structure. Our study suggests that Oshox1 embryonic vascular
expression is mainly attained through suppression of expression in non-vascular tissues.
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Cis-elements in Oshox1 vascular expression
Fig. 1 Histochemical localization of Oshox1-GUS activity in transgenic Arabidopsis. (A, C, E, H, K, L, N, P) Bright-field optics. (D, F, G, J, M,
O) Dark-field illumination. (B, I, Q, R) DIC microscopy. (A) Apical region of the primary root of a 7-d-old seedling. (B) Basal region of the primary root of a 14-d-old seedling (C) Detail of a transverse section through the basal region of the primary root of a 14-d-old seedling. (D)
Hypocotyl of a 7-d-old seedling. (E) Detail of a transverse section through the hypocotyl of a 14-d-old seedling. (F) Transverse section through
the hypocotyl of a plant at the green-silique stage (Altamura et al. 2001). (G) Transverse section through the basal part of the stem of a plant at the
green-silique stage. (H) Detail of a transverse section through the basal part of the stem of a plant at the green-silique stage. (I) Detail of the tip of
the first leaf primordium of a 6-d-old seedling. (J) Abaxial view of the first leaf of a 8-d-old seedling. (K) Abaxial view of the fully differentiated
third leaf of a 21-d-old seedling. (L) Detail of a secondary vein in a transverse section through a fully differentiated first leaf of a 14-d-old-seedling. (M) Floral bud (stage 9 according to Smyth et al. 1990). (N) Mature flower (stage 15 according to Smyth et al. 1990). (O) Silique. (P)
Mature embryo. (Q, R) Ectopic induction of Oshox1-GUS expression in the apical region of the primary root of a 7-d-old seedling in response to
1 µM NAA (Q) or 500 mM sucrose (R). Lignified cell walls appear white in (J) because of light reflection, and red in (H) because of phloroglucinol staining. 1, primary procambial strand; 2, secondary procambial strand; b, bundle sheath; c, vascular cambium; cz, cambial zone; e, endodermis; ic, interfascicular cambium; if, interfascicular fibres; ls, lignified vascular strands; p, phloem; pc, pericycle; ps, procambial strands; rh, root
hair; vc, vascular cylinder; vs, vascular strands; x, xylem. Scale bars: (A, D, F, G, J) 200 µm (B, H, Q, R) 50 µm (C, E, I, L) 10 µm (K) 1 mm (M,
P) 100 µm (N, O) 500 µm.
Fig. 2 Expression of Oshox1GUS, ET1335-GUS and GT5211GUS in Arabidopsis first leaf
development. (A–D) Lateral view.
(E–R) Abaxial view. DIC microscopy. (N, P, R) Enlargements of
the areas boxed in M, O and P,
respectively. Upper right: marker
line identity. Lower left: primordium age in d after germination
(DAG). Numbers refer to vein
orders (for clarity, higher-order
veins have not been labeled).
Arrows in E and F indicate
marker expression in GM cells at
the primodium tips. Scale bars:
(A–H) 25 µm (I, J) 50 µm (K, L,
N, P and R) 100 µm (M, O and Q)
1 mm.
Cis-elements in Oshox1 vascular expression
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Fig. 3 Histochemical localization of Oshox1-GUS
activity during vascular dedifferentiation and redifferentiation in explanted transgenic Arabidopsis hypocotyls.
(A–C) Hypocotyl explants after 3 (A), 7 (B) or 10 (C) d
of culture. (D–F) Details of longitudinal sections through
calli originated from the vascular cylinder of hypocotyl
explants cultured for 7 (D, E) or 10 (F) d. (D) The callus
does not show anatomical signs of vascular differentiation
nor Oshox1-GUS expression. (E) Vascular tissues have
started to differentiate within the callus. Note Oshox1GUS expression in procambial-like cells (pc) and in differentiating tracheary elements (te). Arrows indicate epidermal and cortical cells detaching from the vascular
cylinder of the hypocotyls explants. c, callus tissue originating from the vascular cylinder of the hypocotyls
explants; e, epidermis; v, vascular tissues. Scale bars: (A–
C) 250 µm (D, F) 25 µm (E) 50 µm.
loops of secondary procambial strands extended basally, and
was additionally detected in the distal portion of the second
loops of secondary procambial strands, and in third order procambial strands in the first intercostals areas (Fig. 2L). Throughout leaf development, Oshox1-GUS expression domains always
appeared in continuity with pre-existing vasculature.
High levels of GT5211-GUS expression mark procambium formation (Scarpella et al. 2004). GT5211-GUS does not
seem to be expressed in any type of mature vascular tissues, as
it disappears completely from mature higher order veins, which
consist exclusively of fully differentiated cells (Scarpella et al.
2004). Unlike GT5211-GUS expression (Fig. 2Q, R), both
ET1335-GUS and Oshox1-GUS expression persisted in veins of
all orders in fully differentiated leaves at 14 DAG (Fig. 2M–P).
In summary, at all stages of leaf development, Oshox1GUS expression appeared in procambial strands after their
overt formation was labeled by ET1335-GUS expression. Further, Oshox1-GUS was transiently expressed in GM cells at the
tip of 4–8 DAG primordia (Fig. 1J, 2F). Finally, Oshox1-GUS
expression appeared to proceed continuously and progressively within individual procambial strands, and persisted in
fully differentiated veins.
Oshox1-GUS expression during vascular dedifferentiation and
redifferentiation in Arabidopsis
We next asked whether the association between Oshox1GUS expression and vascular differentiation observed in
Arabidopsis also persisted under perturbed experimental conditions. In Arabidopsis and other dicots, cell proliferation can be
reinitiated in vascular cells of hypocotyl explants that are cultured in the presence of a suitable combination of hormones
(Guzzo et al. 1994, Guzzo et al. 1995, Ozawa et al. 1998). The
dedifferentiation of vascular cells in this culture system gives
rise to callus tissue, within which new vascular tissues subsequently differentiate. As such, this system conveniently allows
testing of the dynamics of Oshox1-GUS expression during
experimentally induced vascular dedifferentiation and redifferentiation in Arabidopsis.
To this aim, hypocotyls of Oshox1-GUS Arabidopsis
transgenic seedlings were explanted on medium containing the
synthetic auxin 2,4-D and the cytokinin kinetin. After 3 d of
culture, Oshox1-GUS expression was detected in all cell types
of the swelling hypocotyls (Fig. 3A). After 7 d, the enlarged
epidermal and cortical cells started to detach in layers from the
central vascular cylinder, and small calli formed at both cut
ends upon dedifferentiation of vascular cells (Fig. 3B).
Oshox1-GUS expression was down-regulated in those vascular cells that had reacquired meristematic activity (Fig. 3B, D),
and reappeared only in close association with new vascular
cells within the callus tissue (Fig. 3B, E). After 10 d, epidermal and cortical cells were almost completely lost, and cell
division was also resumed in vascular cells along the hypocotyl
axis, giving rise to small calli lacking Oshox1-GUS expression
(Fig. 3C). Also in these calli, Oshox1-GUS activity later reappeared in newly differentiated vascular elements (Fig. 3F).
In summary, in the Arabidopsis vascular dedifferentiation
and redifferentiation system, Oshox1-GUS expression was first
ectopically induced following excision. Expression was then
silenced in vascular cells that had reacquired meristematic
properties, and reappeared in cells that were undergoing vascular differentiation. Therefore, Oshox1-GUS expression in
Arabidopsis is associated with vascular differentiation during
both undisturbed and experimentally challenged development.
Expression patterns conferred by progressive 5′ -end deletions
of the Oshox1 promoter
Because the 1.6 kb Oshox1 promoter region directs GUS
reporter gene expression in transgenic Arabidopsis in a fashion
that mimics the expression pattern of Oshox1 in rice (see Discussion), we took advantage of the short life cycle of
Arabidopsis and its faster transformation procedure to map the
cis-acting elements responsible for the Oshox1 expression pattern. To this aim, we generated a series of 5′ deletions of the
Oshox1 promoter (Fig. 4). These fragments were fused to the
GUS gene, and their expression patterns were analyzed in
transgenic Arabidopsis.
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Cis-elements in Oshox1 vascular expression
Similarly, expression of all constructs, except HN-GUS, was
observed in the vascular cylinder of the basal, mature region of
the root (Fig. 6G–L), although the level of expression varied
among the different constructs. None of the Oshox1 promoter
fragments, except for the full-length promoter, was able to
drive reporter gene expression in the region of the vascular cylinder close to the root apex (Fig. 6M–R). In young developing
first leaves, expression of all constructs, except HN-GUS, was
detected at the apical hydathode (Fig. 6S–X). Furthermore, the
PN-, DN- and Oshox1-GUS constructs were also expressed in
trichomes (Fig. 6V–X), but only the DN-GUS construct could
additionally confer the expression in differentiating vascular
strands typical of the Oshox1-GUS construct (Fig. 6W, X).
Finally, none of the Oshox1 promoter fragments, except for the
full-length promoter (Fig. 1), was able to confer auxin or
sucrose responsiveness to reporter gene expression.
Fig. 4 Schematic representation of the Oshox1 promoter 5′ deletion
series. The full-length promoter construct (Oshox1-GUS) is shown at
the top. The GUS gene (black box) is not drawn to scale with respect
to the promoter region (white box). Restriction sites: C, SacI, D, DraI,
H, HindIII, P, SspI, M, MluI, S, SphI, N, NcoI. Position +1 represents
the start codon of the Oshox1 open reading frame.
In mature embryos, Oshox1-GUS expression was detected
in primary and secondary procambial strands of cotyledons,
with stronger expression in the apical region of the strands
(Fig. 1L, 5F, L), and expression was absent from the embryonic axis (Fig. 1L, 5R). Reporter gene expression was never
detected in the lines obtained from the transformation with the
shortest promoter construct of the deletion series, HN-GUS
(Fig. 5A, G, M). In cotyledons of the SN-GUS mature
embryos, expression was detected in the basal region of primary procambial strands and in surrounding GM tissue (Fig.
5B, H). In MN-GUS mature embryonic cotyledons, expression
was much stronger and was observed in both primary and secondary procambial strands and in neighboring GM tissue (Fig.
5C, I). In cotyledons of PN-GUS mature embryos, the expression pattern was very similar to that conferred by the fulllength promoter, but expression was stronger in the basal
region of procambial strands (Fig. 5D, J). Finally, in DN-GUS
mature embryonic cotyledons, expression was observed in both
primary and secondary procambial strands, with stronger
expression in the apical region of the strands, as in Oshox1GUS embryos (Fig. 5E, K). However, unlike in Oshox1-GUS
and PN-GUS embryos, DN-GUS expression was also detected
in GM cells surrounding the apical region of the procambial
strands (Fig. 5E, K). Like the full-length promoter, none of the
promoter deletion fragments could drive reporter gene expression in the axis of mature Arabidopsis embryos (Fig. 5M–Q).
As in embryos, expression of the shortest promoter construct, HN-GUS, was never detected at the seedling stage (Fig.
6A, G, M, S). In cotyledons of 1-week-old seedlings, expression of all other constructs was detected in correspondence
with both primary and secondary vascular strands (Fig. 6B–F).
Discussion
The role of upstream regulatory sequences in the
transcriptional control of early vascular-specific genes, and
the evolutionary conservation of such elements, is largely
unknown. Studies in Arabidopsis and other species have identified a number of genes expressed at early stages of vascular
development (Demura and Fukuda 1994, Baima et al. 1995,
Hardtke and Berleth 1998, Hiwatashi and Fukuda 2000,
Scarpella et al. 2000, Elge et al. 2001, Clay and Nelson 2002,
Hamann et al. 2002, Kang and Dengler 2002, Ohashi-Ito et al.
2002, Groover et al. 2003, Ohashi-Ito and Fukuda 2003,
Carland and Nelson 2004, Kang and Dengler 2004, Scarpella et
al. 2004). Among them, the rice gene Oshox1 is one of the few
for which promoter sequences have been shown to mimic the
endogenous pattern of expression (Scarpella et al. 2000). In
this study, we have analyzed the dynamics of Oshox1 promoter-driven reporter gene expression in Arabidopsis, and
found them to be similar to those reported for rice. Taking
advantage of this correlation, we have explored the role of
upstream regulatory sequences of Oshox1 in the control of its
early vascular gene expression in Arabidopsis.
As in rice, the onset of Oshox1-GUS expression in
Arabidopsis seems to coincide with a specific stage of procambium development at which cells have already acquired anatomical conspicuity, but do not yet display overt signs of
terminal vascular differentiation. First, in both rice and
Arabidopsis mature embryos, Oshox1-GUS activity was
detected in procambial strands of the cotyledons, but not in
those of the embryonic axis. Second, in both rice and
Arabidopsis, procambial cells closest to the post-embryonic
root apex did not express Oshox1-GUS, whereas expression
appeared in procambial cells further away from the root tip.
Third, Oshox1-GUS expression marked procambial strands
before any signs of terminal vascular differentiation in both
rice and Arabidopsis leaves. At all stages of Arabidopsis leaf
development, expression of Oshox1-GUS was invariably
Cis-elements in Oshox1 vascular expression
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Fig. 5 Histochemical localization of
GUS expression driven by different
Oshox1
promoter
deletions
in
Arabidopsis mature embryos. (A–R)
DIC microscopy. (A–F) Cotyledons,
abaxial view. (G–L) Details of procambial cells (outlined) and surrounding GM cells in cotyledons of mature
embryos. (M–R) Root–hypocotyl axis,
lateral view. 1, primary vein; 2, secondary vein; vc, vascular cylinder. For
clarity, anatomical features have only
been labeled in F and R. Scale bars:
(A–L, M–R) 100 µm (G–L) 10 µm.
Fig. 6 Histochemical localization of
GUS expression driven by different
Oshox1 promoter deletions in 1-weekold Arabidopsis seedlings. (A–X) DIC
microscopy. (A–F, S–X) Adaxial view.
(G–R) Lateral view. (A–F) Cotyledon.
(G–L) Basal region of the root. (M–R)
Root apex. (S–X) First leaf. Insets: leaf
trichomes. The arrow indicates leaf apical hydathode. 1, primary vein; 2, secondary vein; vc, vascular cylinder. For
clarity, anatomical features have only
been labeled in F, L, R and X. Scale
bars: 100 µm.
initiated after that of ET1335-GUS, which is expressed simultaneously with the appearance of procambial cell features
(Scarpella et al. 2004). Therefore, we suggest that Oshox1GUS expression, in combination with previously described
reporter gene expression markers of early vascular development (e.g. Clay and Nelson 2002, Hamann et al. 2002,
Groover et al. 2003, Ohashi-Ito and Fukuda 2003, Carland and
Nelson 2004, Scarpella et al. 2004), can be used to visualize
distinct stages of procambial development that cannot be identified by anatomical features. Fourth, as in rice, Oshox1-GUS
expression in Arabidopsis persisted in completely differentiated vascular strands of all organs.
Rice plants do not undergo secondary growth. However,
Oshox1-GUS expression was detected in the interfascicular
and vascular cambium of Arabidopsis, indicating that the regulatory sequences in the Oshox1 promoter are sufficient to direct
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Cis-elements in Oshox1 vascular expression
Fig. 7 Schematic representation of the cis-regulatory elements of the
full-length Oshox1 promoter. Restriction sites: C, SacI, D, DraI, H,
HindIII, P, SspI, M, MluI, S, SphI, N, NcoI. Signal-responsive, cell
type- or tissue-specific expression: a, auxin; g, ground tissue; h,
hydathode; pI, primary procambial strand; pII, secondary procambial
strand; s, sucrose; t, trichome; v, differentiating or differentiated vascular tissues. Organ-specific expression (subscript): c, cotyledon; l, leaf;
r, root. –, reducer of expression. Different cis-regulatory elements
between each pair of respective restriction sites are indicated in alphabetical order. Position +1 represents the start codon of the Oshox1
open reading frame.
expression in secondary vascular meristems. Further, in rice,
Oshox1-GUS expression was also detected in trichomes, stomata and pollen (Scarpella et al. 2000). In Arabidopsis,
Oshox1-GUS was expressed in trichomes, but we never
observed expression in stomata or pollen, probably reflecting
differences in upstream regulatory events or in specific celltype development. Finally, in both rice and Arabidopsis roots,
Oshox1-GUS expression was ectopically induced by auxin and
sucrose.
If the congruence of Oshox1-GUS expression in rice and
Arabidopsis is more than a coincidence, it should also be
observed under altered experimental conditions. In monocots,
root tips can be regenerated after excision by dedifferentiation
and subsequent redifferentiation of cells of the vascular cylinder (Feldman 1976). In rice, Oshox1-GUS expression was
ectopically induced in epidermal and cortical cells of the root at
the site of tip excision (Scarpella et al. 2000). Expression subsequently was rapidly down-regulated in the regenerated root
tip, where Oshox1-GUS expression again became confined to
the central vascular cylinder (Scarpella et al. 2000). Both
Oshox1-GUS ectopic induction and root tip regeneration were
found to be strictly dependent on polar auxin transport
(Scarpella et al. 2000). In Arabidopsis and other dicots,
vascular cells of hypocotyl explants can be induced to dedifferentiate through the action of a suitable combination of
hormones (Guzzo et al. 1994, Guzzo et al. 1995, Ozawa et al.
1998). The dedifferentiation of vascular cells in this culture
system gives rise to callus tissue, within which new vascular
cells differentiate. We found that Oshox1-GUS expression
was ectopically induced in epidermal and cortical cells of
the cultured hypocotyl explants before vascular cells had
resumed division. Oshox1-GUS expression was subsequently
down-regulated in dedifferentiated, proliferating vascular cells.
Expression of the transgene reappeared within the meristematic tissues in close association with newly differentiating
vascular cells. Oshox1-GUS ectopic induction and vascular cell
dedifferentiation were both found to be dependent on the presence of the synthetic auxin 2,4-D in the culture medium (not
shown). Therefore, the expression dynamics of Oshox1-GUS
in these experiments paralleled Oshox1 expression during root
tip regeneration in rice (Scarpella et al. 2000).
The conservation of Oshox1-GUS expression profiles
both during unchallenged development and after experimental
interference in rice and Arabidopsis prompted us to exploit the
shorter life cycle of Arabidopsis and its faster transformation
procedure to investigate the role of cis-acting regulatory
sequences in the control of early vascular gene expression. Our
analysis of different truncated versions of the Oshox1 promoter in transgenic Arabidopsis plants implicates a series of
cell type-, tissue- and organ-specific regulatory elements in the
control of the Oshox1 expression profile (Fig. 7). Further, our
study suggests that Oshox1 embryonic vascular expression is
mainly achieved through suppression of expression in nonvascular tissues. No expression was ever detected when the
reporter gene was solely under the control of the sequence of
the Oshox1 promoter located between position –118 and the
start codon of the Oshox1 open reading frame (position +1; HN
fragment). This region probably represents a minimal promoter unable to direct transcription per se at detectable levels.
The sequence of the Oshox1 promoter between position –244
and +1 (SN fragment) was sufficient to direct reporter gene
expression in primary procambial strands and neighboring GM
tissue of mature embryonic cotyledons. Since the region from
–118 to +1 was not able to drive any reporter gene expression,
the elements responsible for the embryonic expression of the
SN-GUS construct are likely to be located in the region
between position –244 and –118. Furthermore, in this region,
one or more elements capable of driving expression in vascular strands of seedling cotyledons and root also seem to be
present. Finally, this region also seems to contain a regulatory
element capable of driving expression in leaf hydathodes.
Promoter functional studies have implicated CCA/TGG repeats
and CCCC stretches in positive control of vascular gene expression (Hauffe et al. 1993, Hatton et al. 1995, Torres-Schumann
et al. 1996, Yin et al. 1997, Lacombe et al. 2000). Consistent
with a function in vascular expression, the SH fragment of the
Oshox1 promoter contains three CCA/TTG repeats and four
CCCC stretches. With respect to the slightly shorter SN
promoter fragment, the region of the Oshox1 promoter from
position –290 to +1 (MN fragment) seems to contain an element that, in mature embryonic cotyledons, directs additional
expression in secondary procambial strands and in surrounding
GM tissue. In comparison with the shorter MN promoter fragment, the Oshox1 promoter region that spans position –528 to
+1 (PN fragment) seems to contain a regulatory element that
suppresses the expression in GM tissue of mature embryonic
cotyledons that is conferred by the elements present in the
region between –290 and –118. Furthermore, the region of the
Oshox1 promoter from –528 to –244 seems to contain an
element capable of directing reporter gene expression in leaf
trichomes. Deletion of the AACA negative regulatory element in a number of vascular promoters results in ectopic
activity in cell types other than the vascular ones (Keller and
Cis-elements in Oshox1 vascular expression
Baumgartner 1991, Hauffe et al. 1993, Hatton et al. 1995,
Hatton et al. 1996, Lacombe et al. 2000, Liu et al. 2003). Consistent with a role in suppressing expression in non-vascular
cells, the PM fragment of the Oshox1 promoter contains two
AACA motifs. Further, this region contains 12 CCA/TTG
repeats and four CCCC stretches, both of which have been
implicated in positive control of vascular expression (Hauffe et
al. 1993, Hatton et al. 1995, Torres-Schumann et al. 1996, Yin
et al. 1997, Lacombe et al. 2000). The Oshox1 promoter
sequence between position –898 and +1 (DN fragment) drives
expression in GM tissue of cotyledons of mature embryos. This
suggests either that the sequence between –898 and –528 contains a positive regulatory element that drives expression in this
tissue or that it contains an element that counteracts the negative effects on GM tissue expression that the element located
between position –528 and –290 seems to have. Furthermore,
the sequence of the Oshox1 promoter located between –898
and –528 additionally contains an element capable of directing
expression in differentiating vascular strands of developing
leaves. In agreement with a role in vascular expression, the DP
region of the Oshox1 promoter contains 13 repeats of the CCA/
TTG positive vascular regulatory element. Finally, the fulllength Oshox1 promoter confers procambial expression in
mature embryonic cotyledons. The sequence of the Oshox1
promoter from position –1,621 to –898 is thus likely to contain
an element that suppresses the GM tissue-specific element
present in the region between position –898 and –528. Furthermore, the sequence from position –1,621 to –898 seems also to
contain an element capable of driving expression in the vascular cylinder close to the post-embryonic root apex. Consistent
with a role in driving vascular expression, and in suppressing
expression in non-vascular cells, this region of the Oshox1 promoter contains 35 repeats of the CCA/TTG positive vascular
regulatory element, and three copies of the AACA negative
regulatory element of non-vascular expression.
Promoter functional analysis of auxin-regulated genes has
identified a number of cis-elements involved in auxin responsiveness: the (G/T)GTCCCAT and TGTCTC elements
(reviewed in Guilfoyle et al. 1998), the related GGTCCAT
sequence (Sakai et al. 1996), and the AAGG and AAGG Dof
elements (Kim et al. 1994, Liu and Lam 1994, Qin et al. 1994,
Ulmasov et al. 1994, Zhang and Singh 1994, van der Zaal et al.
1996, Kisu et al. 1998, Baumann et al. 1999, Kang and Singh
2000). The sequence of the Oshox1 promoter between position
–1,621 and –898 is necessary for auxin responsiveness. In
agreement with such a role, this region contains 11 Dof elements and one GGTCCAT element. In addition, these motifs
might contribute to the procambial expression conferred by the
full-length Oshox1 promoter. In fact, mutations in Dof elements abolish both auxin responsiveness and procambial
expression (Baumann et al. 1999), and specific Dof transcription factors are expressed in the procambium (Baumann et al.
1999, Gualberti et al. 2002).
1407
To date, five different types of cis-elements have been
implicated in sucrose-regulated gene expression: the G-box
(Giuliano et al. 1988), the B-box (Grierson et al. 1994, Zourelidou et al. 2002), the SURE (Grierson et al. 1994), the SP8
element (Ishiguro and Nakamura 1994) and the TGGACGG
sequence (Maeo et al. 2001). The sequence of the Oshox1 promoter from position –1,621 to –898 is necessary to confer
sucrose responsiveness. Consistent with such function, this
region contains four SP8 elements (TACTATT), three SUREs
(TACTATT, TCACTATT and TACTAT) and one B-box
(CTAAAC).
A growing body of evidence is highlighting the importance of combinatorial control of transcriptional regulation in
plants (Singh 1998). The mosaic of regulatory elements present
in the promoter of Oshox1 implies that the control of its expression is a complex process involving both multiple DNA–protein and protein–protein interactions. It is conceivable that
distinct transcription factor binding to the different regulatory
elements of the Oshox1 promoter may operate in a combinatorial fashion to specify the temporal and spatial aspects of
Oshox1 gene expression. The isolation and analysis of these
proteins will help to elucidate the molecular mechanisms by
which gene expression at early stages of vascular development
is regulated.
Materials and Methods
Vector construction
The Oshox1-GUS construct containing the 1.6-kb region
upstream of the start codon of the Oshox1 open reading frame fused to
the GUS coding sequence in pCAMBIA-1391z (AF234312) has been
described previously (Scarpella et al. 2000). The HN-GUS plasmid
was constructed by digesting Oshox1-GUS with HindIII and by subsequently religating the vector thus obtained. The SN promoter fragment was excised from Oshox1-GUS with SphI and NcoI and
subcloned into the corresponding sites of pUC21 (AF223641). The
promoter fragment was subsequently excised with BglII and NcoI, and
cloned into the BamHI and NcoI sites of pCAMBIA-1391z to give rise
to the SN-GUS construct. The MN promoter fragment was excised
from the pUC21-Oshox1 promoter (Scarpella et al. 2000) with MluI
and NcoI, subcloned into the corresponding sites of pUC21, re-excised
as an NcoI–BamHI fragment and cloned into the NcoI and BamHI sites
of pCAMBIA-1391Z to give rise to the MN-GUS construct. The PN
promoter fragment was excised from the pUC21-Oshox1 promoter
with SspI and NcoI, and cloned into the SmaI and NcoI sites of pCAMBIA-1391z to give rise to the PN-GUS construct. Finally, the DN promoter fragment was excised from the pUC21-Oshox1 promoter with
DraI and NcoI, and cloned into the SmaI and NcoI sites of pCAMBIA1391z to give rise to the DN-GUS construct.
Plant transformation and growth conditions
Arabidopsis thaliana (L.) Heynh ecotypes C24 (Oshox1-GUS)
and Col-0 (Oshox1-, DN-, PN-, MN-, SN- and HN-GUS) were transformed using the vacuum infiltration method (Clough and Bent 1998).
The progeny of 20 independent primary transformants per ecotype per
transgene were selected for preliminary expression pattern analysis.
The GUS expression profile was identical in all tested lines, except for
two Oshox1-GUS lines, one of which showed additional expression in
1408
Cis-elements in Oshox1 vascular expression
the root cap, while the other showed patchy expression not restricted to
vascular tissues. Except for the HN-GUS lines, which never showed
reporter gene expression, 2–4 lines per ecotype per transgene were
either considerably weaker or stronger than the remaining lines. More
detailed analyses were performed on the progeny of three (Oshox1GUS) or five (DN-, PN-, MN-, SN- and HN-GUS) representative,
independent primary transformants per ecotype per transgene. Representative lines were selected based on medium level of expression
(except for HN-GUS lines, in which reporter expression was never
detected), and insertion of one copy of the transgene. Seeds were surface sterilized (McCourt and Keith 1998) and plated on MA medium
(Masson and Paszkowski 1992) containing 25 mg l–1 hygromycin
(Duchefa Biochemie, Amsterdam, The Netherlands). Plates were incubated in the dark at 4°C for 4 d and then moved to a 16 h light : 8 h
dark cycle at 21°C. Induction with auxins (1 µM IAA; 1 µM NAA;
1 µM β-NAA; 1 µM 2,4-D) and sugars (500 mM sucrose; 500 mM
mannitol) in MA medium was performed as described (Scarpella et al.
2000).
Vascular dedifferentiation and redifferentiation assay
Hypocotyls from 2-week-old seedlings of three independent
Oshox1-GUS C24 lines were aseptically excised and cultured up to
14 d at a 16 h light : 8 h dark cycle at 25°C on callus induction
medium (Vergunst et al. 1998), in which 2% glucose was replaced by
2% sucrose to enhance the frequency of callus formation (Iwami and
Goto 1990). This concentration of sucrose (∼50 mM) has no effect on
Oshox1-GUS expression (not shown). Material was harvested for GUS
analysis at 3, 7, 10 and 14 d after transfer to callus induction medium.
Results are representative of three independent experiments, each performed on 10 hypocotyls per transgenic line per time point.
Microtechniques and microscopy
Histochemical detection of Oshox1-GUS activity was performed
on whole seedlings, freshly dissected plant organs or hand sections as
described (Scarpella et al. 2004). Briefly, samples were permeabilized
in 90% acetone for 1 h at –20°C, washed twice for 5 min with 100 mM
phosphate buffer pH 7.5–7.7, and incubated at 37°C for 16 h in
100 mM sodium phosphate buffer pH 7.5–7.7, 10 mM sodium EDTA,
2 mM 5-bromo-4-chloro-3-indolyl-β–D-glucuronic acid (Biosynth AG,
Staad, Switzerland), 5 mM potassium ferrocyanide and 5 mM potassium ferricyanide. High concentrations of potassium ferrocyanide and
potassium ferricyanide reduce diffusion of the intermediate of the
GUS staining reaction at the expense of sensitivity of detection (Lojda
1970). The absence of detectable Oshox1-GUS expression in certain
organs at specific developmental stages, such as distal root tips, young
leaf primordia, immature embryos and embryo axis of mature
embryos, could thus be due to high stringency of staining conditions.
Therefore, for those organs and stages, we performed additional stainings, in which the concentrations of potassium ferricyanide and potassium ferrocyanide were reduced to 0.5 mM. No additional GUS
expression domains were detected under those conditions, thereby
confirming the genuine absence of expression in those organs and
stages. Histochemical detection of ET1335-GUS and GT5211-GUS
activities was performed on whole seedlings as described (Scarpella et
al. 2004). The reaction was stopped by fixing the samples in ethanol :
acetic acid 3 : 1 for 1–16 h at room temperature, depending on the age
of the seedlings. Samples were stored in 70% ethanol at 4°C. Rehydrated samples were cleared in chloral hydrate : glycerol : water 8 : 3 :
1 before microscopic observation. For histological analysis, rehydrated GUS-stained samples were fixed overnight in 2% glutaraldehyde and embedded in glycol methacrylate as described (Scarpella
et al. 2000). Sections (10 µm) were dried onto slides at 37°C and counterstained with 0.5% Safranin O in water before mounting in DPX for
microscopic observation. Samples were viewed with a Leica MZ12
stereomicroscope (Leica Microsystems, Wetzlar, Germany) equipped
with a Sony 3CCD Digital Photo Camera DKC-5000 (Sony Corporation, Tokyo, Japan); with a Wild TYPE 376788 stereomicroscope
(Wild, Heerbrug, Switzerland) equipped with a Nikon DXM1200 digital camera (Nikon, Tokyo, Japan); with a Zeiss Axioplan 2 Imaging
microscope (Carl Zeiss, Oberkochen, Germany) equipped with a Sony
3CCD Digital Photo Camera DKC-5000 (Sony Corporation, Tokyo,
Japan); or with an Olympus BX51 microscope (Olympus Corporation,
Tokyo, Japan) equipped with a Photometrix CoolSnap fx digital camera (Roper Scientific, Trenton, NJ, USA) and a MicroColor liquid
crystal tunable RGB filter (Cambridge Research and Instrumentation,
Inc., Woburn, MA, USA). Images were assembled using Adobe
Photoshop 7.0 (Adobe Systems, Mountain View, CA, USA), and figures were labeled using Canvas 8 (ACD Systems Ltd, Saanichton, BC,
Canada).
Computational analysis
Promoter sequence analysis was performed by using the Plant
CARE (http://oberon.fvms.ugent.be:8080/PlantCARE/index.html) (Lescot
et al. 2002) and PLACE (http://www.dna.affrc.go.jp/PLACE/) (Higo et
al. 1999) databases of plant regulatory elements.
Acknowledgments
The authors would like to thank Dolf Weijers and René Benjamins for advice on Arabidopsis procedures during the initial phase of
this work, Wenzi Ckurshumova for precious help with Arabidopsis
transformation, Mike Deyholos for allowing the use of his microscopy
equipment, Thomas Berleth for seeds and for invaluable discussion
during the preparation of this manuscript, and Pieter Ouwerkerk,
Naden Krogan and Nancy Dengler for critically reading the manuscript. E.S. was supported by a European Commission Training and
Mobility of Researchers ‘Marie Curie’ Grant (ERBFMBICT972716),
and by a Discovery Grant of the Natural Sciences and Engineering
Research Council of Canada.
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(Received January 31, 2005; Accepted June 8, 2005)