Indole Acetic Acid Distribution Coincides with
Vascular Differentiation Pattern during Arabidopsis
Leaf Ontogeny1
Orna Avsian-Kretchmer, Jin-Chen Cheng2, Lingjing Chen, Edgar Moctezuma3, and Z. Renee Sung*
Department of Plant and Microbial Biology, University of California, 111 Koshland Hall, Berkeley,
California 94720
We used an anti-indole acetic acid (IAA or auxin) monoclonal antibody-based immunocytochemical procedure to monitor
IAA level in Arabidopsis tissues. Using immunocytochemistry and the IAA-driven ␤-glucuronidase (GUS) activity of
Aux/IAA promoter::GUS constructs to detect IAA distribution, we investigated the role of polar auxin transport in vascular
differentiation during leaf development in Arabidopsis. We found that shoot apical cells contain high levels of IAA and that
IAA decreases as leaf primordia expand. However, seedlings grown in the presence of IAA transport inhibitors showed very
low IAA signal in the shoot apical meristem (SAM) and the youngest pair of leaf primordia. Older leaf primordia accumulate
IAA in the leaf tip in the presence or absence of IAA transport inhibition. We propose that the IAA in the SAM and the
youngest pair of leaf primordia is transported from outside sources, perhaps the cotyledons, which accumulate more IAA
in the presence than in the absence of transport inhibition. The temporal and spatial pattern of IAA localization in the shoot
apex indicates a change in IAA source during leaf ontogeny that would influence flow direction and, consequently, the
direction of vascular differentiation. The IAA production and transport pattern suggested by our results could explain the
venation pattern, and the vascular hypertrophy caused by IAA transport inhibition. An outside IAA source for the SAM
supports the notion that IAA transport and procambium differentiation dictate phyllotaxy and organogenesis.
In 1880, Darwin stated: “Some influence moves from
the tip of an oat coleoptile to the region below the tip
where it controls elongation.” This moving influence—later shown to be indole acetic acid (IAA; Went,
1926; Kogl and Haagen-Smit, 1931)—is the first description of polar auxin transport. Polar auxin transport is ubiquitous among higher plants. Efficient
transport of IAA modulates cell shape and differentiation and is necessary for normal organogenesis and
vascular patterning (Sachs, 1989, 1991; Mattsson et al.,
1999; Sieburth, 1999).
Vascular tissues are conduits for water and nutrients throughout the plant body. They are generated
during embryogenesis and organogenesis, expanding along the growth axis of the organ. Vascular
development begins with the differentiation of provascular tissue, the procambium (Esau, 1965),
through periclinal cell division, cell elongation, and
cell alignment. The procambium of dicotyledonous
embryos such as Arabidopsis becomes evident at
1
This work was supported by the National Science Foundation
(grant no. DBI–9813361 to Z.R.S.).
2
Present address: College of Literature, Science, and the Arts,
University of Michigan, Ann Arbor, MI 48109.
3
Present address: U.S. Department of Agriculture-Agricultural
Research Service, Beltsville, MD 20705.
* Corresponding author; e-mail [email protected];
fax 510 – 642– 4995.
Article, publication date, and citation information can be found
at www.plantphysiol.org/cgi/doi/10.1104/pp.003228.
early heart stage as elongated cells in the center of the
embryo distinct from the nearly isodiametric surrounding ground tissue cells (West and Harada,
1993). As the embryo matures, the procambial cells
differentiate into phloem and xylene elements (Aloni,
1995). Vascular tissues connect the leaves and other
parts of the shoot with the roots, enabling efficient
long-distance transport between organs.
The vascular network is particularly extensive in
leaves, with primary, secondary, tertiary (or 1°, 2°,
and 3°, respectively), and higher order veins. The
veins arise at different times and are arranged in a
pattern, referred to as the venation pattern, reflecting
the ontogeny and structural organization of the leaf.
Arabidopsis leaves are pinnate with a single 1° vein
(midvein) from which arise all the 2° veins that rejoin
the 1° vein, forming a series of prominent arches
(Hickey, 1979). The 3° veins form bridges between 2°
veins, whereas quaternary veins extend from 3° veins
and end blindly in areoles (Mattsson et al., 1999). The
hierarchical differentiation of 1°, 2°, 3°, and higher
order veins provides an excellent system to study the
mechanism of vascular differentiation and pattern
formation (Nelson and Dengler, 1997).
Vascular differentiation is related to auxin flux
(Aloni, 1995). Auxin transport appears to be mediated by specific cellular influx and efflux proteins
(Lomax et al., 1995; Estelle, 1998). The directionality
of auxin flow is attributed to polar distribution of the
efflux carrier molecules in the plant cell membrane
(Galweiler et al., 1998). Two models, canalization of
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Avsian-Kretchmer et al.
Figure 1. IAA immunolocalization in Arabidopsis tissues. A through D, Cross sections of inflorescence stem. A, Stem tissues
prefixed with ethyl-3(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC), embedded in paraffin, sectioned, and
reacted with the anti-IAA antibody followed by anti-mouse IgG secondary antibody conjugated with alkaline phosphatase.
There is a high level of IAA signal in the epidermal and cortical tissues and around vascular bundles: B through D, controls,
showing very low levels of IAA signal; B, no EDAC prefixation; C, no primary antibody; D, no secondary antibody; E through
K, longitudinal section of young siliques; E, eight-cell embryo; F, early globular stage embryo, showing high IAA signal in
the embryo and endosperm cells (b) and a lower IAA level in the suspensor (a) and ovule cells (c); G, globular stage embryo
with the omission of the primary anti-IAA antibody; and H, torpedo stage embryo with the omission of the secondary
antibody. These controls show very low levels of the IAA signal: I, heart stage embryo; J, torpedo stage embryo; and K,
walking stick stage embryo, showing high levels of IAA in the embryo. The suspensor (a), endosperm (b), and ovule cells (c)
are indicated. L and M, GUS activity in embryos of DR5::GUS transgenic plants. L, Heart stage embryo; M, torpedo stage
embryo showing high level of IAA in the embryos. Bar ⫽ 100 ␮m in A through D, 10 ␮m in E and F, 5 ␮m in G, 20 ␮m
in H through J, 50 ␮m in K, 20 ␮m in L, and 50 ␮m in M.
auxin flow and reaction-diffusion prepattern, have
been proposed to explain the pattern of vascular
differentiation (Nelson and Dengler, 1997). The canalization of signal flow hypothesis is based on a
positive feedback mechanism: a proposed gradual
restriction of IAA flow from a field to specialized
files of cells, resulting in provascular, and later vascular, differentiation (Sachs, 1981). IAA-induced de
novo vascular differentiation (Jacobs, 1952) and the
effect of changing IAA flow on vascular pattern
200
(Mattsson et al., 1999) support the IAA flowdependent canalization hypothesis (Sachs, 1989,
1991). However, some investigators (Carland et al.,
1999; Koizumi et al., 2000) have argued for the reaction diffusion theory based on observations such as
the fragmented vascular strands in some vascular
mutants. This theory emphasizes generation of stable
patterns autonomously in an initially homogenous
field by interacting substances with different diffusion rates (Meinhardt, 1996). Both theories predict
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Plant Physiol. Vol. 130, 2002
Indole Acetic Acid Distribution and Vascular Differentiation
vascular development based on a leaf autonomous
signal source (Dengler and Kang, 2001).
When the polar auxin transport inhibitor 1-Nnaphthylphtalamic acid (NPA) is used to block IAA
flow, vascular development is impaired (Mattsson et
al., 1999). NPA caused central and marginal vascular
hypertrophy—a general increase in the number and
size of veins. NPA treatment also interferes with
organogenesis, inhibiting both lateral root development (Reed et al., 1998) and the formation of new leaf
primordia (Reinhardt et al., 2000).
Although auxin transport is implicated in a variety
of growth and differentiation processes (Aloni, 1995;
Lomax et al., 1995), little is known about the site of
IAA production or its route of transport. It has been
generally believed that IAA is produced in the shoot
apex (Avery, 1935; Bartel, 1997) and in the tips of
older leaves (Aloni, 2001) and transported basipetally, but the site of IAA production and its distribution in plant tissue have not been characterized.
Methods for detecting IAA in plant tissues are being
developed. Constructs containing an IAA-inducible
promoter (Aux/IAA) fused to the ␤-glucuronidase
(GUS) reporter gene can detect IAA in situ (Oono et
al., 1988; Gil and Green, 1997; Ulmasov et al., 1997; Yi
et al., 1999). Monoclonal antibodies against IAA (Leverone et al., 1991; Caruso et al., 1995) have been
used to localize IAA in maize (Zea mays; Shi et al.,
1993; Kerk and Feldman, 1995) and peanut (Arachis
hypogaea) tissues (Moctezuma, 1999).
In this work, we show that immunocytochemistry
with a monoclonal anti-IAA antibody can detect free
IAA in Arabidopsis tissues. While studying IAA distribution in growing organs, we found that NPA
prevents accumulation of IAA in the shoot apical
meristem (SAM) and the youngest pair of leaf primordia, but not in older leaf primordia. The implications of our findings for the direction of IAA flow
and vascular differentiation are discussed below.
RESULTS
IAA Immunolocalization in Arabidopsis Tissues
The production and isolation of monoclonal antibodies highly specific for IAA (Leverone et al., 1991;
Caruso et al., 1995) provided the means to localize
and evaluate in situ IAA levels in maize (Shi et al.,
1993; Kerk and Feldman, 1995) and peanut tissues
(Moctezuma, 1999). We prefixed Arabidopsis tissue
samples with EDAC, which cross-links the carboxyl
group of IAA to structural proteins in the plant tissues, creating the epitope recognized by this antiIAA monoclonal antibody (Leverone et al., 1991; Caruso et al., 1995). The prefixed tissues were
processed, sectioned, and reacted first with the
monoclonal anti-IAA antibody then with the secondary antibody, anti-mouse IgG conjugated with alkaline phosphatase, before the enzymatic reaction was
carried out to obtain the color signal.
Plant Physiol. Vol. 130, 2002
IAA signal was low in pith but high in the epidermal and cortical tissues and vascular bundles of inflorescence stems (Fig. 1A). We verified the effectiveness of the immunolocalization technique and the
specificity of the antibody with several controls. Because the monoclonal antibody was raised against
free IAA cross-linked to bovine serum albumin (BSA)
through its carboxyl group (Leverone et al., 1991), we
tested unfixed tissues for color reaction. We observed
no color comparable with the prefixed stem section
(Fig. 1, A and B), indicating both that prefixation with
EDAC is essential for free IAA detection by this
antibody and that the antibody does not recognize
other epitopes in these tissue sections. No signal was
detected when the primary (Fig. 1C) or secondary
antibody (Fig. 1D) was omitted, indicating that the
color reaction is dependent on the presence of these
antibodies on the tissue section and demonstrating
again that the background color is very low.
We used the anti-IAA monoclonal antibody to
study IAA distribution in growing tissues, embryos,
leaf primordial, and SAMs (Figs. 1 and 2), where IAA
is expected to be high. Immunocytochemistry on longitudinal sections of young siliques with different
stages of embryo development is shown in Figure 1,
E, F, and I through K. The signal is high in all embryo
cells and lower in the suspensor and the ovule.
Lower IAA signal may reflect more disperse cytoplasm in the more vacuolated suspensor and ovule
cells. High signal is detected in the endosperm surrounding the suspensor in the early embryonic stages
(Fig. 1, E and F).
Embryonic stages in which procambium can be
detected, i.e. heart stage (Fig. 1I), torpedo stage (Fig.
1J), and walking stick stage (Fig. 1K), have high IAA
signal evenly distributed in all embryonic cells with
no difference in signal level between procambium
and ground tissue. No IAA signal was detected when
the primary anti-IAA antibody (Fig. 1G) or the secondary antibody (Fig. 1H) was omitted, demonstrating again that the immunocytochemical reaction is
specific and that background color is very low.
The IAA signal pattern was consistent across embryos with only minor variation. All 42 pro-embryos
and globular stage embryos examined showed high
IAA signal in the embryo and surrounding endosperm and lower signal in the suspensor. Thirtyfive of 39 heart stage embryos analyzed had the
uniform immunostaining shown in Figure 1I,
whereas four showed high signal only in the distal
end of the cotyledons and not uniformly in all the
embryonic cells. Seventeen of 18 torpedo stage embryos showed the signal pattern in Figure 1J. Figure
1K shows the high IAA signal seen in all 19 walking
stick stage embryos examined.
To verify the anti-IAA antibody signal, we used the
GUS activity of transgenic plants carrying the
DR5::GUS construct, composed of the IAA-inducible
promoter (Aux/IAA) fused to a GUS reporter gene
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Avsian-Kretchmer et al.
Figure 2. IAA immunolocalization in leaf primordia and SAM. A and B, Cross sections of the shoot apex of 4-d-old seedlings
were treated as described in the legend to Figure 1. A, Shoot apex of an untreated seedling, showing high IAA signal in the
pair of first node leaf primordial. B, Shoot apex of a seedling grown in the presence of 40 ␮M NPA, showing little IAA signal
in the leaf primordial. C and D, Longitudinal sections of 4-d-old seedlings. C, SAM of an untreated seedling, showing high
IAA level. D, SAM of a seedling grown in the presence of 40 ␮M NPA, showing low IAA level. E through H, Serial cross
sections of the shoot apex of 5-d-old seedlings that were treated as described in the legend to Figure 1. Drawing on the left
depicts the sites where the four sections were made through the shoot apex including the first node (1) and second node (2)
leaf primordia. Higher IAA levels are detected in the upper than in the lower sections. C, Petiole of the cotyledons. Bar ⫽
20 ␮m in A and B, 10 ␮m in C and D, and 25 ␮m in E through H.
202
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Plant Physiol. Vol. 130, 2002
Indole Acetic Acid Distribution and Vascular Differentiation
Figure 3. IAA distribution and venation pattern in transgenic plants grown in the presence and absence of 40 ␮M NPA.
DR5::GUS activity in transgenic plants: A, 4-d-old shoot apex, showing the GUS-positive first true leaf and the stipules (white
arrows); B, 4-d-old shoot apex in a seedling grown in the presence of NPA, showing very low levels of IAA; C, 5-d-old shoot
apex showing the GUS-positive true leaves and their stipules (white arrows); D, 5-d-old shoot apex in a seedling grown in
the presence of NPA, showing some GUS signal in the distal end of the leaf; E, 6-d-old shoot apex showing first node leaf
primordium with declining GUS activity and second node leaves (red arrow) and stipules (white arrows) with high level of
IAA; F, 6-d-old shoot apex in seedling grown in the presence of NPA, showing more GUS signal in the leaf tip and the
emerging marginal veins; G, 8-d-old shoot apex showing the GUS activity in the second node leaves (red arrows) and
stipules (white arrows), whereas the signal in the first node true leaves decreased and is concentrated in the leaf tip; H,
8-d-old shoot apex in a seedling grown in the presence of NPA, showing increased GUS signal and the expanding veins
along the leaf margin; I, IAA distribution in the subsequent leaf nodes of a 10-d-old seedling, showing high IAA signal in the
third node leaves (red arrows) and the stipules (white arrows) and lower signals in the second node leaves; J, second rosette
leaves of 10-d-old seedlings grown in the presence of NPA, showing no IAA signal; K and L, GUS activity in the first true
leaf of a 10-d-old seedling (K) and a 10-d-old seedling grown in the presence of 40 ␮M NPA (L); M and N, venation pattern
and IAA distribution in the first true leaf of 10-d-old seedling. Seedlings were fixed in 6:1 (v/v) ethanol:acetic acid for 4 h
at room temperature and then rinsed and whole mounted as described in “Materials and Methods”; M, venation pattern of
the first true leaf, showing 1o, 2o, and 3o veins; N, venation pattern of a first true leaf of seedling grown in the presence of 40 ␮M
NPA, showing the marginal and central hypertrophy; 1, first node leaves; 2, second node leaves; and 3, third node leaves. Bar ⫽
20 ␮m in A and B, 50 ␮m in C and D, 100 ␮m in E and F, 200 ␮m in G through J, and 400 ␮m in K through N.
(Ulmasov et al., 1997). Figure 1, L and M, show GUS
activity throughout heart and torpedo stage embryos. The walking stick stage embryo has less GUS
activity, which diminishes as the embryo matures
and becomes dormant. The overall GUS pattern is
consistent with the immunological signals during
embryogenesis. For unknown reasons, there are a
small percentage, about 10%, of the torpedo stage
and walking stick stage embryos that show higher
GUS activity in the root cap and the tips of the
cotyledons (data not shown).
IAA Signal in the Shoot Apex
The immunocytochemistry technique was used to
examine IAA distribution in shoot apices of Arabidopsis seedlings. The first node rosette leaves (Fig.
2A) and SAM (Fig. 2C) of 4-d-old seedlings had high
levels of IAA signal. Figure 2A shows high levels of
IAA signal in a cross section of the first node rosette
leaves, and Figure 2C shows high levels of IAA in a
longitudinal section of the SAM. The IAA signals in
Figure 2 were representative of 48 leaf primordia and
17 SAMs examined.
Plant Physiol. Vol. 130, 2002
We also determined the GUS activity of transgenic
seedlings carrying the DR5::GUS construct (Ulmasov
et al., 1997). As shown in Figure 3A, high levels of
GUS activity were detected in the first node leaf and
stipule of 4-d-old seedlings. These results are similar
to the IAA immunolocalization findings, demonstrating that the IAA inducibility of the DR5::GUS construct is a useful reporter of the endogenous IAA
levels in the shoot apex. Figure 3, A, C, E, and G
show the first true leaf at different ages. As the leaf
primordium grows, only the distal end of the leaf
primordium maintains high GUS activity (Fig. 3, A,
C, E, and G). High GUS activity in the distal end of
the leaf primordia was confirmed with the immunological assay for IAA. Figure 2, E through H, show a
series of four cross sections of a 5-d-old shoot apex
that has high anti-IAA antibody signal in the sections
from the distal end of the first node leaf primordia,
particular in the adaxial side of the leaf. Leaf primordia of subsequent nodes have a similar spatial and
temporal pattern of GUS activity: high levels in the
young leaf primordia that decline with growth (for
example, see the second node leaf in Fig. 3, E, G, and
I, and the third node leaf in Fig. 3I).
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Avsian-Kretchmer et al.
Table I. Leaf lengths of Arabidopsis seedlings growing with or without NPA
Data represent mean (⫾SE) of 10 leaves. Measurements represent leaf blade ⫹ petiole. Lengths are in ␮m.
DAGa
4
5
6
8
10
a
First Node Leaves (⫺NPA ⫹ NPAb)
120 ⫾ 15
270 ⫾ 30
450 ⫾ 15
1,450 ⫾ 35
4,500 ⫾ 200
Days after germination.
b
100 ⫾ 10
165 ⫾ 5
370 ⫾ 5
950 ⫾ 55
1,400 ⫾ 50
40 ␮M NPA.
c
Second Node Leaves (⫺NPA ⫹ NPAb)
–
–
120 ⫾ 30
400 ⫾ 40
1,400 ⫾ 70
Third Node Leaves (⫺NPA ⫹ NPAb)
–
–
–
–
140 ⫾ 15
–
–
–
120 ⫾ 20
400 ⫾ 35
–
–
–
–
–
–, Not measurable on intact seedlings.
Effect of NPA on IAA Distribution during
Leaf Ontogeny
To study the effect of inhibition of IAA transport
on IAA distribution, we germinated Arabidopsis
seedlings in media supplemented with 40 ␮m NPA
(concentration chosen based on studies of Mattsson
et al., 1999) and sectioned shoot apices for IAA immunolocalization studies. The IAA signal in the leaf
primordia (Fig. 2B) and in the SAM (Fig. 2D) of
4-d-old seedlings grown in the presence of NPA was
very low relative to the untreated control (Fig. 2, A
and C, respectively). (Three of 44 leaf primordia from
seedlings grown on NPA showed a slightly higher
IAA signal than in Fig. 2B.)
Similarly, DR5::GUS activity was barely detectable
in shoot apices of seedlings grown in the presence of
40 ␮m NPA (Fig. 3, B and D). However, IAA signal
was detectable in the apical area of the first node leaf
primordium in 5- to 6-d-old NPA-grown seedlings
(Fig. 3, D and F) and increased in intensity in leaf
primordia of 8-d-old (Fig. 3H) and 10-d-old (Fig. 3L)
seedlings, in the presence of NPA. No GUS activity
was detected in the stipules or the SAM (Fig. 3, B, D,
F, H, and J).
The second node leaves of NPA-grown seedlings
showed the same spatial and temporal distribution of
the GUS activity. No IAA signal was detected in
newly emerged, 140-␮m leaf primordia (Table I; Fig.
3J) until d 5 to 6, when IAA signal appeared at the
leaf tip (data not shown). In contrast, control leaf
primordia of the same length showed GUS signal
throughout the entire leaf (second node leaf in Fig. 3E
and third node leaf in Fig. 3I).
Effect of NPA on Organogenesis and Venation Pattern
NPA treatment altered the venation pattern and
leaf and root shape. Leaf primordia grown in the
presence of NPA (Fig. 3, D, F, H, and L) have broad
leaf blades and broad, short petioles relative to leaves
grown in the absence of the IAA transport inhibitor
(Fig. 3, C, E, G, and I). Relative to the control root
(Fig. 4N), the NPA-treated seedling root is shorter,
lateral roots are inhibited, and the root tip is wider
(Fig. 4O). The first node leaf primordia of untreated
plants triple in length every 2 d. The leaves of NPAtreated seedlings grow much slower (Table I). NPA
204
c
also slows the rate of emergence of leaves from subsequent nodes (Table I).
As reported in Mattsson et al. (1999), NPA treatment affects the venation pattern. For example, a
5-d-old leaf has a differentiated midvein, but leaves
of seedlings grown in the presence of NPA do not
(Fig. 3, F and H). Instead, the first veins form laterally
along the margins of the leaf primordia at the distal
end of the leaf in 6-d-old seedlings (Fig. 3F), then
expand along the leaf margins to form a broad band
of vascular tissue, called the marginal hypertrophy
(Fig. 3H). By 8 d after germination, multiple files of
veins, called the central hypertrophy (Fig. 3N), are
formed in the center of the leaf blade, extending into
the petiole but not connecting to the vascular system
of the hypocotyl. This is consistent with the findings
of Mattsson et al. (1999) and Sieburth (1999). It is
worth noting that the area of IAA-driven DR5::GUS
activity (Fig. 3, F and H) coincides with the site of
NPA-induced vascular differentiation, especially in
the marginal hypertrophy.
IAA Distribution in Different promoter::GUSContaining Seedlings Grown in the Presence and
Absence of NPA
Because the expression pattern of DR5::GUS depends on tissue-specific promoter activity as well as
the presence of IAA, we tested two other Aux/IAA
response promoter::GUS constructs: PSIAA4/5 BA::GUS
(Oono et al., 1988) and SAUR-AC1::GUS (Gil and
Green, 1997). BA is the PSIAA4/5 promoter region that
contains two auxin-responsive domains (AuxRD A
and AuxRD B). Domain A contains a highly conserved
sequence found in various IAA-inducible genes that
behaves as a major auxin-responsive element. Domain
B functions as an enhancer element. The two domains,
which act cooperatively to stimulate transcription
(Ballas et al., 1995), were fused to the GUS reporter
gene and introduced into Arabidopsis (Oono et al.,
1988). The SAUR-AC-1 genes (Gee et al., 1991) encode
auxin-inducible small RNAs. High promoter activity
was reported by Gil and Green (1997) in vascular
tissues of transgenic plants harboring the SAUR-AC1:GUS construct.
The GUS expression patterns in seedling shoot
apex of the BA::GUS (Fig. 4, C and D) and the
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Plant Physiol. Vol. 130, 2002
Indole Acetic Acid Distribution and Vascular Differentiation
Figure 4. IAA distribution in seedlings containing a different IAA-inducible promoter::GUS and in a DR5::GUS-containing
line treated with three different IAA transport inhibitors. A through F, GUS activity in 4-d-old transgenic seedlings containing
different Aux/IAA promoter::GUS. A, DR5::GUS transgenic line; B, DR5::GUS transgenic line grown in the presence of 40
␮M NPA; C, BA::GUS transgenic line; D, BA::GUS transgenic line grown in the presence of 40 ␮M NPA; E, SAUR-AC1::GUS
transgenic line; and F, SAUR-AC1::GUS transgenic line grown in the presence of 40 ␮M NPA. G through K, GUS activity in
a 5-d-old DR5::GUS transgenic line grown with a different IAA transport inhibitor; G, seedlings grown without any inhibitor,
showing high level of GUS activity; H, seedlings grown in the presence of 20 ␮M NPA, showing low level of the GUS activity;
I, seedlings grown in the presence of 40 ␮M NPA; J, seedlings grown in the presence of 20 ␮M 2-chloro-9-hydroxyfluorene9-carboxylic acid (HFCA); K, seedlings grown in the presence of 40 ␮M 2,3,5-triiodobenzoic acid (TIBA); L, 5-d-old
cotyledon, showing lower level of the GUS activity than a 5-d-old cotyledon of seedling grown in the presence of NPA (M);
and N, 4-d-old root tip and O, 4-d-old root tip of seedling grown in the presence of 40 ␮M NPA, both showing high level
of GUS activity. Bar ⴝ 50 ␮m in A through F, 100 ␮m in G through K, 200 ␮m in L and M, and 100 ␮m in N and O.
Plant Physiol. Vol. 130, 2002
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Avsian-Kretchmer et al.
SAUR-AC-1::GUS (Fig. 4, E and F) transgenic plants
are similar to that of the DR5::GUS plants (Fig. 4, A
and B), with high GUS activities in the shoot apex of
4-d-old seedlings and reduced GUS activities in the
shoot apex of seedlings grown in the presence of
NPA. Little GUS activity is detected in the SAM, leaf
primordial, or stipules of NPA-grown seedlings. The
faint GUS activity in the shoot apex of the NPAgrown SAUR-AC-1::GUS transgenic plants (Fig. 4F) is
much lower than in non-treated SAUR-AC-1::GUS
transgenic plants (Fig. 4E). In general, these results
confirm the IAA distribution pattern during Arabidopsis leaf ontogeny determined by DR5::GUS activity and by IAA immunocytochemistry. It is presumed that NPA prevents IAA transport to the shoot
apex, resulting in the absence of the IAA necessary
for inducing GUS activity.
IAA Distribution in DR5::GUS Seedlings Grown in the
Presence of Three Different IAA Transport Inhibitors
To confirm that NPA affects IAA transport to the
shoot apex, we grew DR5::GUS transgenic plants in
the presence of two other IAA transport inhibitors,
TIBA and HFCA. Seedlings grown in the presence of
these two inhibitors had reduced IAA signal in their
shoot apices (Fig. 4, J and K) relative to the untreated
control (Fig. 4G), as did the NPA-grown seedlings
(Fig. 4, H and I). The congruence of results with these
three IAA transport inhibitors confirms the conclusion that the reduced IAA signal in the shoot apex is
due to lack of auxin transport. Thus, the IAA in the
untreated shoot apex comes from an outside source.
In an attempt to identify this outside source, we
examined GUS activity in the cotyledons and root tip
of seedlings grown in the presence of NPA. The
signal detected in root tips of NPA-grown seedlings
was similar to the control (Fig. 4, N and O). The
cotyledons of seedlings grown in the presence of
NPA (Fig. 4M), TIBA, and HFCA (data not shown)
have higher GUS activity than those grown in the
absence of inhibitors (Fig. 4L). This result implicates
the cotyledons as a probable source of the shoot apex
IAA of germinating seedlings.
DISCUSSION
Because polar auxin transport plays a major role in
plant development, characterization of the IAA distribution pattern in relation to the processes that are
regulated by IAA will contribute to our overall understanding of how organs communicate with each
other to coordinate the growth of the whole plant. To
elucidate the role of polar auxin transport in vascular
differentiation, we investigated the temporal and
spatial pattern of IAA localization in the shoot apex
of Arabidopsis seedlings. We found that inhibition of
auxin transport prevented IAA accumulation in the
SAM and the youngest pair of leaves, but not in older
206
leaf primordia. The temporal pattern of IAA localization implies a switch in IAA transport direction that
is consistent with the mechanism of vascular differentiation proposed in the canalization of IAA flux
hypothesis.
IAA Immunocytochemistry
IAA distribution in Arabidopsis was determined
using the anti-IAA monoclonal antibody employed
to detect IAA in maize (Kerk and Feldman, 1995) and
peanut (Moctezuma, 1999) tissues. This monoclonal
antibody, raised in mice (Mus musculus) against IAA
conjugated to albumin through its carboxyl group
(Leverone et al., 1991), shows maximal crossreactivity to the methyl ester of IAA in radioimmuno
assay and ELISA (Caruso et al., 1995; Gao et al.,
1999). The antibody does recognize IAA conjugates
(Gao et al., 1999), but Arabidopsis has little IAA-Glu
conjugate, IAA-Asp conjugate, or IAA-Glc conjugate
(Tam et al., 2000).
We used EDAC to cross-link the exposed carboxyl
group of free IAA to the free amino groups of structural proteins in Arabidopsis cells. IAA conjugates
lack a free carboxyl and cannot be cross-linked to
structural proteins by EDAC, which precludes antibody reaction with any conjugates that might be
present in the tissues. The free carboxyl group of Trp
can be cross-linked by EDAC, but this monoclonal
anti-IAA antibody has a very low cross-reactivity
with Trp (Pence and Caruso, 1988). Positive controls,
including adding exogenous IAA to tissues and radioactive IAA blotting assay to test the EDAC crosslinking of IAA, verified the specificity of the antibody
for free IAA (Moctezuma, 1999). The antibody failed
to detect any signal in tissues not prefixed with
EDAC (Fig. 1B), further confirming the specificity of
the antibody to cross-linked IAA. Moreover, the immunological signal was absent from shoot apical tissues when IAA transport was inhibited, indicating
that the signal is detecting IAA and not other compounds, such as Trp. Finally, the anti-IAA monoclonal antibody and the three different Aux/IAA
promoter::GUS constructs produced similar patterns,
indicating that immunocytochemistry is a reliable
method for identifying IAA in Arabidopsis tissues.
IAA immunocytochemistry will be an important tool
for detecting IAA in very small tissues such as the
SAM, in tissues where the Aux/IAA promoters are not
expressed, and in plant species such as monocots in
which promoter::reporter constructs are not yet
available.
IAA Production and Transport in Shoot Apex
Using in situ IAA determination, we have found
that Arabidopsis rosette shoot apex, i.e. the SAM and
the youngest set of leaf primordia, do not accumulate
free IAA if IAA transport is inhibited with NPA. We
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Plant Physiol. Vol. 130, 2002
Indole Acetic Acid Distribution and Vascular Differentiation
observed similar depletion of IAA in seedlings
treated with two other IAA transport inhibitors, confirming that the reduced IAA in the NPA-treated
seedlings results from lack of IAA transport. Thus,
IAA is likely transported into the shoot apex, not
produced there. However, as leaf primordia grow
and mature, IAA is found at the distal end of the leaf
regardless of IAA transport inhibition. The presence
of IAA in older leaf primordia of NPA-grown seedlings indicates that IAA can be produced in the presence of NPA. Elevated IAA in cotyledons of seedlings grown in the presence of NPA (Fig. 4, L and M)
suggests that IAA is normally transported out of the
cotyledons and that they might be the source of the
IAA in the first pair of true leaves. IAA accumulation
at the distal end of transport-blocked leaves implies
IAA production at the leaf tip. IAA produced in the
leaf tip must drain out of the leaf (Mattsson et al.,
1999). Based on these results, we propose the “IAA
flow model” to describe the temporal and spatial pattern of IAA flow in the shoot apex (Fig. 5). During
leaf development, the IAA source changes from extrinsic to intrinsic, which would change IAA flow
direction and affect the pattern of vascular differentiation (see below) and probably cell and organ differentiation as well. The correspondence of the patterns of IAA flux and the leaf vascular formation, e.g.
as seen in Figure 3, E and H, supports Sachs’ canalization hypothesis (1991) and is in good agreement
Figure 5. “IAA flow model” in the shoot apex of Arabidopsis seedling. The youngest leaf primordia (marked “2” for second node
leaves) and SAM do not produce IAA. Rather, IAA is being acropetally transported into this region. IAA is produced at the tip and
marginal regions of the older leaf primordia (marked “1” for first node
leaves) and basipetally drained, primarily through the midvein. Arrows represent flow direction of IAA. The IAA source for the youngest
pair of leaf primordia and SAM may be the cotyledons (C), older
leaves, and shoot tissues underneath the SAM. The acropetal flow of
the IAA into the leaf primordia can explain the acropetal formation of
the midvein. The time of IAA appearance in the distal end of the leaf
corresponds to the time of secondary vein differentiation along the
leaf margin, consistent with the notion that inability to drain IAA from
the leaf tip basipetally through the midvein would cause marginal
hypertrophy (Mattsson et al., 1999).
Plant Physiol. Vol. 130, 2002
with the leaf venation hypothesis recently proposed
by Aloni (2001).
Role of IAA Flux on Venation Pattern
There are at least three orders of veins in the Arabidopsis rosette leaf: 1o (or midvein), 2o, and 3o,
which form in a hierarchical fashion (Esau, 1965). The
midvein is usually seen growing acropetally from the
hypocotyl vasculature into the emerging leaf primordium (Sieburth, 1999). The extrinsic IAA source and
acropetal flow pattern suggested by our results is
consistent with the acropetal differentiation of midvein procambium at the onset of leaf ontogeny. If the
5- to 6-d-old leaf begins to produce IAA at the distal
end, IAA could then flow basipetally into the plant.
Seedlings germinated on NPA do not form the prominent midvein. Instead, files of veins appear, first
along the leaf margins (Fig. 3, F and H), and afterward toward the leaf base (Fig. 3N), that are not
connected to the hypocotyl vascular system
(Sieburth, 1999). This is again consistent with the
canalization hypothesis: Without the midvein, there
is no constant basipetal flow of IAA from the leaf tip.
The IAA produced at the leaf tip, the presumed IAA
source of 6-d and older leaves, cannot itself be cannalized into specific cell files and cause differentiation of a vascular bundle connected to the plant
vascular system. Prior differentiation of the midvein
by flow of auxin into the leaf appears to be required
for the later flow from the leaf tip to generate the
normal venation pattern.
The three pairs of secondary veins in the young
rosette leaves appear as arches (Mattsson et al., 1999).
As the leaf primordium grows laterally, the first pair
forms as a loop from the tip of the midvein along the
leaf margin that rejoins the midvein at a basal location. The secondary veins may join the midvein again
because IAA flows toward the midvein, which drains
the IAA out of the leaf. In the absence of a midvein in
NPA-treated leaves, IAA flows along the growing
leaf margin rather than toward the center of the leaf.
Thus, the pair of arches is replaced by a band of
vascular tissues along the leaf margin forming the
marginal hypertrophy (Mattsson et al., 1999). The
mechanism of IAA flows along the leaf margin is
unknown, but flow is apparently independent of the
IAA transport mechanism that can be disrupted by
NPA, TIBA, or HFCA.
Role of Localized IAA Production and IAA
Transport in Organogenesis
Chemical inhibition of polar auxin transport alters
venation pattern and leaf shape. The accumulation of
IAA and vasculature in the upper one-half of the leaf
may promote lateral leaf growth and inhibit longitudinal growth, resulting in broad leaves with short
petioles (Fig. 3, C, D, G, and H).
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207
Avsian-Kretchmer et al.
It has been generally believed that IAA, which
plays a major role in regulating tissue differentiation
and organogenesis, is produced in the SAM (Avery,
1935; Bartel, 1997). Recently, a very different concept
was proposed—IAA is transported to, not produced
in, the shoot meristem proper. It was further proposed that, because of the acropetally advancing procambial strand from the stem, IAA from an extrinsic
source might dictate the position of new leaf primordia, phyllotaxis, and initiate organogenesis (Kuhlemeier and Reinhardt, 2001). Our results provide the
first evidence in support of the concept that IAA
flows from the plant to the SAM. Import of IAA into
the SAM would explain impairment of organogenesis when IAA flow is blocked. Growth observed in
NPA-treated, IAA-depleted shoot apices may be supported by very low levels of IAA or by other forms of
auxin such as indole butyric acid (Bartel, 1997).
In conclusion, finding both extrinsic and intrinsic
sources of IAA in leaf primordia is novel, providing
both the first evidence for changing IAA flow pattern
and insights into the mechanism of IAA fluxmediated venation pattern. Our results establish a
foundation from which to pursue the IAA signaling
pathways of phyllotaxis and organogenesis.
MATERIALS AND METHODS
Excised tissue samples were immediately prefixed in 3% (w/v) aqueous
solution of EDAC (Sigma) and postfixed in FAA (3.7% formaldehyde:50%
ethanol:5% glacial acetic acid [v/v]) for 16 h at 4°C, dehydrated with a
graded ethanol series, embedded in paraffin, and sectioned to 10-␮m slices.
Sections were affixed onto slides (Probe-On Plus, Fisher Scientific, Pittsburgh). After overnight drying at 42°C, sections were deparaffinized with
xylene and hydrated in an ethanol-water series. Slides were processed as
described in Moctezuma (1999) with some modifications: Slides were incubated in a blocking solution containing 10 mm phosphate-buffered saline
(PBS; 2.68 mm KCl, 0.15 m Na2HPO4, and 0.086 m KH2PO4), 0.1% (v/v)
Tween 20, 1.5% (v/v) Gly, and 5% (w/v) BSA, for 45 min at 22°C, then
rinsed in a regular salt rinse solution (10 mm PBS, 0.88% [w/v] NaCl, 0.1%
[v/v] Tween 20, and 0.8% [w/v] BSA), and washed briefly with 10 mm PBS
and 0.8% (w/v) BSA solution to remove the Tween 20. Fifty microliters of
1:200 (w/v) anti-IAA antibody (1 mg mL⫺1) were placed on each slide,
covered with coverslips, and incubated overnight in a humidity chamber at
22°C. Two 10-min vigorous washes with high-salt rinse solution, 10 mm
PBS, 2.9% (w/v) NaCl, 0.1% (v/v) Tween 20, and 0.1% (v/v) BSA were
followed by a 10-min wash with a regular salt rinse and a brief rinse with 10
mm PBS and 0.8% (v/v) BSA. Fifty microliters of 1:100 (w/v) dilution of the
1 mg mL⫺1 anti-mouse IgG-alkaline phosphatase-conjugate (Promega, Madison, WI) were added to each slide, which was covered with coverslip and
incubated for 4 to 6 h in a humidity chamber at 22°C. Two 15-min washes
with a regular salt rinse were followed by a 15-min wash with water. Two
hundred microliters of ready-to-use Western Blue (Promega) were added to
each slide; each slide was then covered with a coverslip and incubated in the
dark for 15 to 30 min. When blue/purple color was observed, slides were
rinsed with water and then dehydrated in a graded water-ethanol series,
ethanol-xylene, xylene. Slides were mounted with Permount (Fisher Scientific), dried overnight in a 42°C oven, and observed under an Axiophot
microscope (Zeiss, Jena, Germany). Photomicrographs were taken by a
video camera attached to the microscope and processed with the Scion
Image 1.60. The figures were arranged using Adobe Photoshop version 5.5
(Adobe, Mountain View, CA).
Plant Material and Growth Conditions
Arabidopsis ecotype Columbia-0 was used in the study. Transgenic seeds
containing the promoter::GUS constructs were kindly provided by:
DR5::GUS, Dr. Tom Guilfoyle (University of Missouri, Columbia); PSIAA4/5
BA::GUS, Dr. Anastasios Theologis (The Plant Gene Expression Center, U.S.
Department of Agriculture, Albany, CA); and SAUR-AC1::GUS, Dr. Pamela
Green (Michigan State University, East Lansing).
Seeds were surface sterilized in 70% (v/v) ethanol for 1 min, followed by
10% (v/v) commercial bleach for 15 min, washed three times in sterile
distilled water, and plated in 0.4% (w/v) molten agar on top of a solid
germinating medium in 9-cm petri dishes. Germinating medium contained
0.5⫻ Murashige and Skoog basal salts (Murashige and Skoog, 1962), 1.5%
(w/v) Suc, and 0.8% (w/v) agar. Plates were sealed with parafilm, incubated at 6°C in the dark for 2 d, and then transferred to a growth chamber
set at 22°C for a 16-h-light (120–150 ␮mol m⫺2 s⫺1), 8-h-dark cycle (long-day
conditions). The time of transfer to growth chamber was considered the
starting point of all the experiments. Plants were transplanted into soil 2
weeks later and were grown under long-day conditions until seeds were
harvested. For the inflorescence stem tissues, seedlings were germinated as
described above and were grown for 2 weeks at 22°C in an 8-h-light
(120–150 ␮mol m⫺2 s⫺1), 16-h-dark cycle (short-day conditions), then transferred to soil and grown in the greenhouse under long-day conditions
(16-h-light, 8-h-dark cycle). Four- to 10-cm inflorescence stems were collected for the IAA immunocytochemical studies.
Auxin transport inhibitors NPA (Chem Service, West Chester, PA), TIBA
(Sigma, St. Louis), and HFCA (Sigma) were dissolved in dimethyl sulfoxide
(Sigma). The concentration of the dimethyl sulfoxide in the growth media
never exceeded 0.1% (v/v).
IAA Immunocytochemical Localization
The monoclonal anti-IAA antibody used in the immunolocalization studies was kindly provided to us by Dr. John L. Caruso (Department of
Biological Sciences, University of Cincinnati). The antibody was raised
against free IAA that was cross-linked to BSA at the carboxyl group in mice
(Mus musculus) (Leverone et al., 1991).
208
GUS Activity
To assay GUS activity, dissected samples were incubated with 5-bromo4-chloro-3-indolyl-␤-d-glucuronide (X-Gluc) solution as described by Cheng
et al. (2000). Excised samples were vacuum infiltrated in the X-Gluc solution
for 10 min at room temperature and then incubated at 37°C in the dark for
16 h. Samples were rinsed with 50 mm sodium phosphate buffer, pH 7.0,
and then fixed in ethanol:acetic acid (9:1 [v/v]) for 4 h at room temperature.
X-Gluc-treated samples were rinsed with 95% (v/v) ethanol and transferred to 70% (v/v) ethanol. Tissue samples were whole mounted on microscope slides in a clearing solution of chloral-hydrate:glycerol:water (8:1:2
[v/v]) as described by Berleth and Jurgens (1993). The samples were covered and observed with a Zeiss Axiophot microscope. Photomicrographs
were taken as described above.
For GUS staining of the zygotic embryos, ovules were dissected from
siliques. To minimize wounding the embryo, a hole was punctured to allow
the penetration of the X-Gluc solution into each ovule. After 16 h of incubation, embryos were dissected from the broken ovule for fixation, clearing,
and photography. 35S::GUS embryos were used as control to ensure proper
penetration of the X-Gluc solution through the damaged ovule.
Upon request, all novel materials described in this publication will be
made available in a timely manner for noncommercial research purposes.
ACKNOWLEDGMENTS
We thank Prof. Tsvi Sachs (Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Israel) for helpful comments and
discussion, and Dr. John Caruso (Department of Biological Sciences, University of Cincinnati) for the monoclonal anti-IAA antibodies and helpful
information. We also thank Dr. Denise Schichnes and Dr. Steven Ruzin
(Biological Imaging Facility, College of Natural Resources, University of
California, Berkeley) for their help and support with the microscopy work.
Received January 30, 2002; returned for revision May 17, 2002; accepted May
28, 2002.
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Plant Physiol. Vol. 130, 2002
Indole Acetic Acid Distribution and Vascular Differentiation
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