Planta (1996)200:229 237
9 Springer-Verlag 1996
Studies on the role of the Arabidopsis gene MONOPTEROS
in vascular development and plant cell axialization
Gerhard K.H. Przemeck 1, Jim Mattsson 2'*, Christian S. Hardtke 1, Z. Renee Sung 3, Thomas Berleth 1
Institut ftir Genetik und Mikrobiologie, Lehrstuhl Rir Genetik, Universit~it Miinchen, Maria-Ward-Strasse la,
D-80638 Mtinchen, Germany
z Department of Physiological Botany, University of Uppsala, Villav~igen 6, S-75 236 Uppsala, Sweden
3 Department of Plant Biology, University of California, 111 Koshland Hall, Berkeley, CA 94720, USA
Received: 3 January 1996/Accepted: 19 April 1996
Abstract. In the embryo of Arabidopsis thaliana (L.)
Heynh., formation of the hypocotyl/root axis is initiated
at the early-globular stage, recognizable as oriented ex-
pansion of formerly isodiametric cells. The process de-
pends on the activity of the gene M O N O P T E R O S (MP);
mp mutant embryos fail to produce hypocotyl and radicle.
We have analyzed the morphology and anatomy of mp
mutant plants throughout the Arabidopsis life cycle. Mu-
tants form largely normal rosettes and root systems, but
inflorescences either fail to form lateral flowers or these
flowers are greatly reduced. Furthermore, the auxin trans-
port capacity of inflorescence axes is impaired and the
vascular strands in all analyzed organs are distorted.
These features of the mutant phenotype suggest that the
M P gene promotes cell axialization and cell file formation
at multiple stages of plant development.
Key words: Arabidopsis - Cell axialization MONO-
P T E R O S gene P I N - F O R M E D gene - Polar auxin
transport - Vascular development
Introduction
In dicotyledonous plants, the basic elements of the body
pattern are laid down between fertilization and the forma-
tion of the heart-stage embryo (Jiirgens et al. 1991). Stud-
ies of early embryogenesis show that not more than a few
hundred cells are involved in setting up this early pattern
(Natesh and Rau 1984). An important step in plant em-
bryogenesis is the generation of the hypocotyl/root axis,
first recognized as the emergence of parallel files of elon-
gated cells in the basal part of the early globular embryo.
* Present address: Institut ffir Genetik und Mikrobiologie, Lehrstuhl
ftir Genetik, Universit~it Miinchen, Maria-Ward-Strasse la, D-
80638 Miinchen, Germany
Abbreviation: GUS = ]3-glucuronidase; IAA = indole acetic acid
Correspondence to: T. Berleth; FAX: + 49 (89) 179 198 20;
E-mail: uj44205@sunmail.lrz-muenchen.de
Planta
A defined number of these files later constitute the hypo-
cotyl and most of the radicle.
Continuous files of elongated cells are initiated at
several stages of plant development. The process, com-
monly referred to as attainment of"axiality" or "polarity"
by plant cells, is thought to depend on orienting signals
and cellular response mechanisms (Sachs 1991). The
formation of vascular strands has been used as a model
system to investigate cell axialization mechanisms. It has
been proposed that vascular strands are formed along the
routes of a "canalized" flux of a developmental signal that
is possibly identical with the polar flux of auxin (Sachs
1981).
Mutants with specific alterations in the anatomical
organization of the embryo may reveal mechanisms of
plant cell interaction. As judged from diagnostic cell
shapes in mutant embryos, the Arabidopsis gene M O N O -
P T E R O S (MP) appears to be required specifically for the
initiation of files of elongated cells that give rise to the
hypocotyl/root axis. No general cellular defects have been
observed in mp mutants, and largely normal apical struc-
tures, such as the shoot meristem and the cotyledons, are
formed. However, basal cells of the early mp embryo fail to
undergo oriented expansions, and it has been proposed
that mp embryos lack instructions for the directional
growth of the basal region (Berleth and Jfirgens 1993).
In this study, we have used the capacity of mp mutant
seedlings to form adventitious roots to investigate the
morphology and a n a t o m y of mp mutants throughout the
plant life cycle. Our findings suggest an important func-
tion of the M P gene in cell axialization manifested in the
formation of the embryonic axis, the developing vascular
system and the polar transport of auxins. We also discuss
possible implications for inflorescence development.
Materials and methods
Plant growth conditions, plant strains and genetic crosses. Plants of
Arabidopsis were grown as described in Mayer et al. (1993). Unless
otherwise noted, our descriptions are based on observations in six
mutant alleles induced in two Arabidopsis ecotypes; mp ~2, mp T'~7~
230
mp r~3~ induced in Landsber9 erecta (Ler) and mp ~12, mp ~25, mp G33
induced in Columbia (Col-O). The characterization of the mp mutant
alleles mp ~92, mp r37~ and m p Tu399 has been described (Berleth and
Jiirgens 1993). Alleles mp Gl2, mp ~25 and mp ~33 were isolated on the
basis of seedling phenotype following gamma-ray mutagenesis of
Col-O seeds (Cheng et al. 1995). Complementation tests and
phenotypic classification of these mutants were performed as de-
scribed previously (Berleth and Jiirgens 1993). Depending on the
allele analyzed, either Ler or Col-O plants served as wild-type
controls.
Sterile culture. All sterile culture samples were grown under constant
illumination (6000 lux) at 24~ For adventitious root formation,
seeds were surface-sterilized, germinated on agar and basally bisec-
ted as described previously (Berleth and Jfirgens 1993). After bisec-
tion, the apical parts of wild-type and mp mutant seedlings were
either transferred directly to soil or grown on medium containing
0.7% agar, 50 gg.m1-1 ampicillin and half-strength Murashige-
Skoog basal medium (Murashige and Skoog 1962) supplemented
with 1.5% sucrose, 1 x B5-vitamins (Gamborg et al. 1968) and
3 lag"ml 1 indole butyric acid (Sigma, Munich, Germany) for two
weeks before transfer to soil. Plants that survived transfer to soil
immediately after bisection were analyzed after six weeks to assess
the mp phenotype independent of sterile culture conditions. These
plants were also used for measurements of polar auxin transport in
stem segments.
Phenotypic analysis. Inflorescence phenotypes of rooted mutant and
wild-type plants were analyzed after six weeks on soil. Abnormalities
in flower phenotypes were quantified as described in Fig. 2.
Different types of preparations were used to analyze the anatomy
of individual organs. Cross-sections were used to determine the
presence, normal size, and proper arrangement of all tissue types in
mutant and corresponding wild-type organs. Whole-mount prep-
arations and paradermal sections (parallel to the surface) were used
to analyze the integrity of vascular strands as well as proper cell
orientation and alignment.
Whole-mount preparations of leaves and roots were made as
described for seedlings (Berleth and Jfirgens 1993). Inflorescence
axes were treated similarly, but were rehydrated and incubated in a
solution of 5 lag"ml- 1 propidium-iodide for several hours at room
temperature after ethanol fixation. Stained inflorescence axes were
cut longitudinally with a razor blade and mounted.
To determine the degree of vascularization for mutants with
different mp alleles, five to eight rosette leaves of similar size (5 ram,
+ 1 mm in length, similar shape) were selected from each genotype.
The tracheary pattern, as visible in darkfield optics (Fig. 4), was
projected onto a matrix of roughly 900 squares and the proportion
of squares containing one or more xylem strands was determined.
For histological sections, leaves and roots were fixed in 3.7%
formaldehyde in PBT (50raM sodium phosphate pH 7.2, 0.15%
Tween 20; Sigma) at 4~ for 2 d. After fixation the material was
processed, embedded in Spurr's resin (Spurr 1969), sectioned and
stained as described previously (Berleth and Jtirgens 1993). Phloem-
specific [3-glucuronidase (GUS) expression of the ASUS1-GUS
reporter construct (Martin et al. 1993) was used to improve visualiz-
ation of phloem independent of cell shape. The GUS staining prior
to embedding was performed as described in Martin et al. (1993).
Stem segments were fixed and processed similarly, but embedded in
UNICRYL resin (British BioCell, Cardiff, UK). To ensure full poly-
merization, samples were kept at 55~ for 3 d. Sections (3 lam) were
cut with a Reichert-Jung microtome and placed onto polylysine-
coated microscope slides. Stem sections were first stained with 1%
Astra blue (BDH Laboratory Supplies, Poole, UK) in 2% tartaric
acid for 5 min at 70~ washed several times in distilled water and
then stained in 1% safranin at 70~ After several washes (once in
70% ethanol and several times in water) slides were incubated in
0.5% HC1 in 70% ethanol for 5 s at room temperature and then
washed again in water. All sections were mounted in Spurr's me-
dium. Photographs were taken with an Axiophot microscope-
camera (Zeiss, Oberkochen, Germany).
G.K.H. Przemeck et al.: M O N O P T E R O S gene in plant cell axialization
Assay of polar auxin transport. Polar auxin transport in inflorescence
axes was measured using a modification of the method described by
Okada et al. (1991). Among the mutant alleles used in this study,
only plants of the weak allele mp T M produce adventitious roots with
high frequency in the absence of exogenously applied auxins. Bisec-
ted Ler wild-type and mp ~92 mutant seedlings were rooted and
grown on soil. Two-centimetre-long segments from primary inflor-
escence axes, 1 2 mm in diameter, were taken from near the rosette
between one and two weeks after bolting. Segments were placed in
either normal or inverted orientation in 1.5-ml Eppendorf tubes
containing 40 gl half-strength Murashige-Skoog basal medium
(Murashige and Skoog 1962), supplemented with unlabeled IAA
(Sigma) and tritiated IAA (Amersham International, Amersham,
UK) to a final concentration of 1.5 gM and 500 Bq.g1-1 (total
1.2 x 106 cpm). After incubation in closed Eppendorf tubes at room
temperature for 18 h, 5-mm segments were cut from the upper end of
the stem segment that had not been in contact with the solution,
washed four times in medium, and incubated overnight in universal
scintillation fluid (Rotiszint; Roth, Karlsruhe, Germany). The
amount of transported IAA was determined by measuring the
radioactivity released by the upper stem slice using a Beckman
LS5000TD scintillation counter (Beckman Instruments, Munich,
Germany). No additional radioactivity was released by the stem
segments after three more days in scintillation fluid.
Results
A previous report describes the isolation of thirteen m u -
tant alleles of the M O N O P T E R O S gene as well as the
p h e n o t y p e s of m u t a n t embryos a n d seedlings (Berleth a n d
Jiirgens 1993). M u t a n t seedlings with strong mp alleles
lack the hypocotyl a n d the root (Fig. ld) due to the appar-
ent inability to initiate oriented expansions a n d divisions
of the cells of the "lower tier" in the early g l o b u l a r e m b r y o
(Berleth a n d Jiirgens 1993). These directional divisions
n o r m a l l y result in the f o r m a t i o n of parallel cell files
(Fig. la) which eventually constitute the h y p o c o t y l / r o o t
axis. It seems possible that molecular m e c h a n i s m s that
orient cells in the h y p o c o t y l / r o o t axis could also control
cell-file f o r m a t i o n at later d e v e l o p m e n t a l stages. I n order
to assess potential p o s t - e m b r y o n i c functions of the M P
gene, we generated adult m u t a n t plants, exploiting the
capacity of m u t a n t seedlings to produce adventitious
roots (see M a t e r i a l s and methods). I n the following we
describe first the m o r p h o l o g i c a l a n d physiological, then
the a n a t o m i c a l features of plants carrying mp alleles of
different strength. F o r simplicity, we use the term mor-
phology to describe external features of p l a n t organs as
opposed to i n t e r n a l a n a t o m i c a l aspects. Unless otherwise
noted, all descriptions a n d the examples given in the
figures are based o n similar observations in m u t a n t s of
all six alleles used in this study. F o r two traits, a precise
quantification was possible a n d a weaker p h e n o t y p e of
allele mp ~92 could be distinguished (Figs. 2, 4).
M o r p h o l o y y o f mp m u t a n t plants. M u t a n t plants were able
to form a n elaborated root system (data n o t shown),
a rosette of a highly variable n u m b e r of leaves, a n d a n
inflorescence with cauline leaves, axillary shoots, a n d
flowers (Fig. lf, h). Thus, the M P gene is n o t specifically
required for the f o r m a t i o n of any m a j o r part of the adult
plant. O n the other hand, severe m o r p h o l o g i c a l defects
indicated i m p o r t a n t p o s t - e m b r y o n i c functions of the M P
gene. In this study we have focused o n fully p e n e t r a n t
G.K.H. Przemeck et al.: MONOPTEROS gene in plant cell axialization 231

traits, which were found to be localized and allele-specific.

In addition to fully penetrant abnormalities, mutant
plants were also impaired in their overall viability and
appeared more sensitive to adverse growth conditions, as
for example, low humidity. Non-penetrant abnormalities
as well as traits that could be influenced by culture condi-
tions, are not described further.
Figure if shows an mp mutant plant that has produced
six rosette leaves, a central inflorescence and abnormal
flowers. We observed no specific effects of mp mutations
on the morphology of rosette leaves. Since we have also
failed to detect abnormalities in the root system (data not
shown), we conclude that the m o r p h o l o g y of the vegeta-
tive organs is not markedly affected by mutations in the
M P gene.
In contrast to the largely normal vegetative organs, we
observed specific abnormalities in mutant inflorescences.
Differences in flower m o r p h o l o g y and position between
mp mutant and wild-type plants were particularly pro-
nounced. In the wild-type plant, flowers are invariably
found along the stem (Fig. lg), while in mp mutants of all
alleles flowers were almost exclusively found in terminal
positions on top of the inflorescence axes (Figs. lf, 2b-d).
Frequently, more than one flower was formed at the same
position, or flowers could arise at the base of pre-existing
ones (Fig. 2b). Mutant flowers were reduced, lacking
predominantly organs of the outer whorls. The range of
phenotypes, illustrated in Fig. 2a-d, encompasses inflor-
escence axes devoid of any flower structure (a), strongly
reduced flowers consisting only of pistils (b), nearly com-
plete flowers lacking sepals (c) and female-fertile flowers
(d). Completely normal or male-fertile flowers were not
observed in mutant plants. Although the same plant could
produce flowers belonging to two or more of these
phenotypic classes, the overall severity of the flower
phenotypes correlated with allele strength. Figure 2e
summarizes the classification of more than two hundred
flowers of each genotype, illustrating the prevalence of
certain phenotypic classes a m o n g plants with different mp
alleles.
Wild-type plants rooted under identical conditions
displayed a variety of abnormalities, such as reduced
numbers of rosette leaves and altered apical dominance.
However, wild-type plants never displayed the alterations
in flower position and morphology observed in mp
mutants.

Polar auxin transport in mp mutants. Morphological ab-

normalities of mp mutant inflorescences are shared by

Fig. la-h. Morphology of wild-type and mp mutant Arabidopsis

plants. Schematic view of wild-type (a) and mp mutant (b) embryos at
the early heart-stage. Elongated cells in the wild-type embryo
(shaded) are absent in the mutant embryo (Berleth and Jiirgens
1993). Wild-type (Let in c, e; Col-Oin g) and mp mutant plants (rapT M
in fl and f, and mpT M in h) are shown 4 d (c, fl), three weeks (e, f), and
ten weeks (g, h) after germination. Plants in e-h were basally bisected
at the seedling stage and then transferred to soil either directly (e, f)
or after growth on root-inducing medium for two weeks (g, h). The
mutant plant in f shows the largely normal development of the
rosette contrasted by an abnormal young inflorescence (arrowhead).
Inflorescence morphology: wild-type (g), mp mutant (h)
232
plants mutant for the gene P I N F O R M E D (PIN1; G o t o
et al. 1991), which, in turn, resemble wild-type plants that
have been treated with chemical inhibitors of polar auxin
transport (Okada et al. 1991). Furthermore, O k a d a et al.
(1991) have reported a reduced polar auxin transport
capacity in the inflorescence axes o f p i n l mutants. There-
fore, we measured the polar transport of IAA in the stems
of mp mutant and wild-type plants. To avoid in-vitro
culture effects we used plants of the weak allele mp ~9:
which occasionally form roots upon wounding and can
then be grown on soil (see Materials and methods). One
end of each stem segment was placed in [3H]IAA solution
and transport towards the opposite end was determined
after 18 h. As shown in Fig. 3, this assay specifically
monitored basipetal transport, as less than 5 % of the label
was transported in the opposite (acropetal) direction. In
these tests, mutant stem segments of the weak allele mp ~9:
displayed only 31% of the polar auxin transport capacity
of the wild-type controls (Fig. 3).
Internal organization of mp mutant oryans. To assess the
arrangement and internal organization of tissues, we ana-
lyzed the a n a t o m y of all major organs in mp mutant
plants. Figures 4 and 5 illustrate our observations of
rosette leaves and inflorescence axes. Related organs, such
as cauline leaves and side shoots, displayed qualitatively
similar abnormalities (data not shown). Each of the ana-
G.K.H. Przemeck et al.: MONOPTEROS gene in plant cell axialization
Fig. 2a-e. Inflorescence and flower
morphology in mp r3z~(a, b) and mpT M (e, d)
mutant Arabidopsis plants, a Top of
a mutant inflorescence axis without floral
buds. b-d Mutant flowers at terminal
positions consisting of a single carpel (b),
a single carpel plus several reduced
stamens and petals (e), and nearly normal
siliques derived from female fertile flowers
pollinated with wild-type pollen (d).
e Allele-specifc proportions of mutant
flower phenotypes. Each column
represents the proportion of flowers
classified as follows: male fertile mutant
plants were not observed. Each flower was
classified according to hierarchically
ordered criteria: from female fertile
regardless of the presence of other
perianth organs, to perianth organs
including all flower phenotypes producing
either stamen, petal, or sepal(s), to carpel
structures describing flowers producing
either one or two carpels, to inflorescence
axis phenotypes, without any
recognizable flower buds. Examples of
each class are given in a--d. Black columns,
mpr37~ shaded, mpG92;open, wild type
(Ler). To avoid any preselection, all
flowers of a given plant were classified.
Total numbers of flowers/plants
evaluated: mp rjT~ 219/17; mp~J, 297/21;
wild-type, 1187/56.
tomical features described below is based on observations
of organs of at least five plants of each genotype.
The most conspicuous defect in the internal tissue
organization of mp mutant plants lies in the reticulariz-
ation of the vascular system in all types of leaf organs.
Figure 4a e shows the vascular system in wild-type and
mp mutant rosette leaves. Interestingly, the central mid-
vein was always present, albeit sometimes reduced in
length in the mutant, while the veins in the leaf laminae
were either absent or dramatically reduced, in particular,
vascular strands at the leaf margins were invariably miss-
ing in mutants with strong mp alleles. Leaf venation was
quantified by measuring the relative proportion of equally
sized squares from rosette-leaf blades that contained one
or more fully differentiated xylem strand (for details see
Materials and methods; Fig. 4n). We observed a strong
reduction of leaf venation in mutants with strong mp
alleles and an intermediate phenotype for allele mp G92,
which had been described as a weak allele based on
features of the seedling (Berleth and Jtirgens 1993) as well
as of the inflorescence phenotype (Fig. 2e).
We also analyzed the alignment of different types of
vascular cells. In leaves, even in the wild-type, xylem
elements were generally less perfectly aligned than in cy-
lindrical organs (see below). In particular, cell alignment
in peripheral veins close to the leaf margin often appeared
irregular, but strand interruptions were extremely rare
G.K.H. Przemeck et al.: MONOPTEROS gene in plant cell axialization
30000
(cpm) Ler [ G92
20000
10000
basipetal acropetal basipetal acropetal
Fig. 3. Polar auxin transport in stem segments of wild-type and mp
mutant Arabidopsis plants. Stem segments were placed in either
normal or inverted orientation with one end in contact with nutrient
solution containing [3H]IAA and the amount of 3H accumulated at
the opposite end was determined after 18 h (for details see Materials
and methods). Each column represents the mean value of 12 (for Ler)
and 15 (for mp G92)measurements; the bars in each column indicate
the SE. Let, wild-type Landsber9 erecta; G92, weak mp allele mp ~92.
Mean values/SE were: Let, basipetal: 23443 cpm/2035 cpm; mp 692,
basipetal: 7404cpm/1007cpm; Let, acropetal: 318cpm/95cpm;
m p TM, acropetal: 284 cpm/ll0cpm. The data were subjected to
a statistical analysis using the standard Student's t-test for indepen-
dent sampling. The t-value of the difference in polar auxin transport
between Ler and m p ~92 s t e m s was calculated to be 7.11 with 12.5
degrees of freedom. The difference is thus significant at a confidence
level of > 99.9%
(Fig. 4a, d). In mutant leaves, however, interruptions were
very frequent, vessel elements were often not oriented
towards each other, and vascular strands often consisted
of only a few interconnected cells (Fig. 4b, c,e,f). Using
paradermal sections, we looked more closely at the
interconnections of different types of vascular cells. Para-
dermal sections at the level of the xylem often showed
isolated or improperly aligned vessel elements amidst
procambial cells (Fig. 4k). Furthermore, elongated paren-
chymatic cells of the bundle sheath (arrowhead in Fig. 4i)
were often missing (Fig. 4k). Sections at the level of the
phloem showed insufficiently elongated and interconnec-
ted sieve-tube cells (Fig. 4m) and the sites of phloem inter-
ruptions often coincided with those of the xylem strands.
The longitudinal organization of tissues in stems was
analyzed in whole-mount preparations. In rooted wild-
type plants, the xylem strands formed smooth tubes of
long, perfectly aligned vessel elements flanked by elon-
gated cells of other tissues (Fig. 5c). In mutant stems, these
strands were often improperly aligned or interrupted
(Fig. 5f). At the sites of vascular discontinuity the average
length of differentiated tracheid cells was reduced, and
immature as well as improperly oriented vessel elements
were observed (Fig. 5f). However, it should be noted that
the vascular defects in stems were less severe than those in
leaf blades, and that xylem strands often appeared normal
over a greater distance. As phloem strands are more
233
difficult to visualize in whole-mount preparations, their
integrity could not be assessed along the entire length of
stems. Instead, we analyzed phloem in stem close to sites
of xylem strand interruptions only, and here we regularly
observed phloem strand discontinuities (data not shown).
In contrast to the conspicuous defects in the continuity
of vascular strands we observed largely normal radial
arrangements of different tissue types in the mutant or-
gans. In transverse sections through the blade of mutant
rosette leaves, palisade mesophyll cells were properly
elongated, tightly packed and aligned in a single layer on
top of several layers of less precisely arranged round
spongy mesophyll cells (Fig. 4h). Different types of epider-
mal cells, including stomatal guard cells and trichomes
appeared normal in mp mutants. Vascular bundles, how-
ever, were often reduced in size and occasionally devoid of
enclosing parenchymatic cells.
Inflorescence axes of wild-type and mp mutant plants
were compared in cross-sections at the level of the first
internode. Figure 5a shows the basic radial pattern of the
inflorescence axis in the wild type, consisting of a single
layer of epidermis cells, several layers of cortex cells,
vascular bundles and pith tissue. This pattern was also
found in mutants with strong mp alleles (Fig. 5d). Vascular
bundles could consist of fewer xylem and phloem strands,
but their internal organization was similar to wild-type
(Fig. 5d, e). Thus, all tissues were found in their normal
positions in mutant inflorescence axes. Mutant roots were
also analyzed and showed occasional interruptions of
vascular strands in longitudinal preparations, but no ab-
normalities in cross-sections (data not shown).
In summary, our anatomical survey of all major organs
detects mp-specific defects specifically in the vascular tis-
sues. In all organs analyzed, we observed largely normal
radial patterns contrasted by distortions in the vascular
tissues, which were localized in stems and roots but very
extensive in leaves.
Discussion
This study explores post-embryonic functions of the Ara-
bidopsis gene MP, previously shown to be required for the
initiation of the hypocotyl/root axis in the early embryo.
We show that post-embryonically the M P gene is not
required for the formation of any major organ or tissue
type. Instead, mutant plants display a combination of
defects not described before in Arabidopsis mutants. We
observed characteristic morphological abnormalities and
reduced auxin transport capacity in mutant inflorescence
axes and distortions in vascular strand formation in
all organs. We will first discuss the morphological and
physiological abnormalities and then relate them to the
observed vascular and embryonic defects.
Inflorescence development and polar auxin transport in mp
mutant plants. The mp mutant plants display a phenotype
reminiscent of two other recently described Arabidopsis
mutants, p i n l (Goto et al. 1991) and pinoid (pid; Bennett
et al. 1995). These mutations are not allelic and all three
genes have been localized in genetic maps (Berleth and
Jfirgens 1993; Bennett et al. 1995). In all three mutants,
234 G.K.H. Przemeck et al.: M O N O P T E R O S gene in plant cell axialization

Fig. 4a-n. Tissue organization in wild-

type and mp mutant rosette leaves, a-f
Darkfield analysis of rosette-leaf
venation, a-e Ler background; d, e Col-O
background: wild type (a, d), mp Gg: (b),
mp r~'399(e), and mp ~2 (e). f shows selected
region in b (white square) at higher
magnification to illustrate the
interrupted xylem strands in mutant
leaves, g-m Tissue sections of wild-type
and mutant (mp r37~ rosette leaves, g, h
Cross-sections. Cells of the upper and
lower epidermis (ep) as well as of the
palisade mesophyll (pro) are of similar
shape and arrangement in wild-type (g)
and mutant (h) leaves. The cells of the
mutant spongy mesophyll (sin) are
normal in shape and size. By contrast,
vascular bundles (vb) are reduced in
mutant leaves, i-m Paradermal sections
of wild-type (i, 1) and mutant (k, m)
rosette leaves show improper elongation
and interconnection of vessel elements
(arrowheads in k) and of sieve tubes
(marked by arrowheads in I and m). Note
the absence of elongated cells enclosing
vascular bundles in k as compared to
i (arrowhead in i). Dark staining of the
phloem in I and m is due to GUS
expression from the phloem-specific
ASUS I-GUS reporter construct (Martin
et al. 1993). The bars indicate 500/am in
a-e, 50 lam in f and 25/am in g-m.
n Quantification of the degree of
reticularization of vascular strands in the
rosette leaves of wild-type (Let in a;
Col-O in d) as well as of weak ( mp ~92) and
strong (rap ra3~9, mp ~12, mp ~25 and mp ~33)
mutant plants. Quantification was
performed as described in Materials and
methods; the SE for each genotype is
indicated by the bar in each column.
Note that the degree of vascularization
in leaves with strong mutant alleles is
about 30% of the respective wild-type
values in both ecotypes

v e g e t a t i v e d e v e l o p m e n t is l a r g e l y n o r m a l a n d t r a n s i t i o n p h e n o t y p e c a n n o t be t a k e n as e v i d e n c e for a d i r e c t func-
f r o m a v e g e t a t i v e to a n i n f l o r e s c e n c e m e r i s t e m occurs. t i o n of a g e n e in i n f l o r e s c e n c e d e v e l o p m e n t b e c a u s e
H o w e v e r , i n f l o r e s c e n c e s e i t h e r fail to p r o d u c e flowers o r O k a d a et al. (1991) h a v e s h o w n t h a t it c a n result
give rise to o n l y few, g r e a t l y r e d u c e d flowers. T h i s m u t a n t f r o m t r e a t m e n t w i t h c h e m i c a l i n h i b i t o r s of p o l a r a u x i n
G.K.H. Przemeck et al.: MONOPTEROS gene in plant cell axialization 235

(%) 60 t r a n s p o r t . T h e m e c h a n i s m s b y which the flux of a u x i n

affects the f o r m a t i o n of l a t e r a l flowers are largely un-
50
k n o w n . Recent e x p e r i m e n t s have s h o w n t h a t the effect of
40 a u x i n t r a n s p o r t i n h i b i t o r s on l a t e r a l flower f o r m a t i o n is
reversible, e n a b l i n g critical p e r i o d s to be defined ( O k a d a
30 a n d S h i m u r a 1994). These e x p e r i m e n t s suggest t h a t it is
20
the initiation a n d the very early stages of the flower
m e r i s t e m d e v e l o p m e n t t h a t are p a r t i c u l a r l y sensitive to
10 a l t e r a t i o n s of the a u x i n flow, b u t the m o l e c u l a r details are
n o t u n d e r s t o o d . M u t a t i o n s in pid a p p e a r n o t to affect the
0
Ler G92 T(J399CoI-O G25 G12 G33 t r a n s p o r t itself, but are t h o u g h t to interfere with d o w n -
n s t r e a m processes (Bennett et al. 1995). H o w e v e r , r e d u c e d
Fig. 4n. a u x i n t r a n s p o r t has been m e a s u r e d i n p i n l stem segments,

Fig. 5a-f. Anatomy of wild-type and mutant inflorescence axes of Arabidopsis. a-c Wild-type (Let), d-f mp mutant inflorescence a x e s (mpT370). a, d
Cross-sections at the level of the first internode of wild-type and mutant inflorescence axes do not show marked differences in the arrangement or in
the relative sizes of major tissues, or in the internal organization of the vascular bundles (enlarged view in b, e); ep, epidermis; co, cortex; pi, pith; vb,
vascular bundles, e, f Xylem strands in wild type (c) and mp mutant (f) inflorescence axes. Note that the course of vascular strands is more oblique in
mutant stems. Arrowhead in f marks insufficiently elongated and differentiated vessel element, a, b, d, e Histological sections stained with Astra blue
and safranin, c, f Whole-mount preparations, stained with propidium-iodide, fluorescence optics. Bars = 100 lain (a, c, d, f) and 20 ~tm (b, e)
236
and it has been proposed that mutations in P1N1 primarily
affect the transport (Okada et al. 1991). In this study we
have shown that also in mp mutant stem segments the
polar transport of IAA is markedly reduced, suggesting
that mutations in MP, similar to those in PIN1, affect
inflorescence development through reduced auxin
transport.
The cellular mechanisms of the polar transport of
auxins have been studied extensively (reviewed in
Goldsmith 1977; Estelle and Klee 1994), and transport is
thought to be mediated by membrane-bound influx and
efflux carriers, the latter preferentially located at the basal
end of transporting cells (Depta et al. 1983; Hertel et al.
1983). A genetic approach to identifying transport
molecules can be considered, and it might be tempting
to speculate that the products of genes like PIN1 or MP
could be components of the cellular auxin transport
mechanism. However, it should be kept in mind that
numerous cellular or anatomical abnormalities are likely
to interfere with polar auxin transport. Distinct pheno-
typic differences between pinl and mp mutant seedlings,
in fact, argue against very closely related primary func-
tions (see below).
Vascular tissues in mp mutant plants. In order to assess the
function of the M P gene at the cellular level, we analyzed
the internal organization of all major organs ofmp mutant
plants. Anatomical defects were found to be confined to
the vascular tissues, while other cell types, including those
that undergo extensive cellular morphogenesis, such as
trichome cells or epidermal guard cells, were normal in
shape and spatial arrangement. These observations
further support the notion of a specific as opposed to a
general cellular function of the MP gene in plant develop-
ment. A number of vascular mutants have been described
(Caruso and Cutter 1970; Scheres et al. 1995), but the
vascular defects in mp mutants represent a novel pheno-
type as they affect predominantly one specific aspect of
vascular development, the alignment and interconnection
of vascular cells.
Vascular cells, like several other cell types, elongate
during the course of their maturation. Vascular develop-
ment, however, is unique in that this axialization needs to
be precisely integrated along rows of cells to ultimately
produce continuous tubes rather than isolated cells (Sachs
1991). The intercellular signals directing this oriented dif-
ferentiation of vascular cells are still largely elusive and
mutant analysis may supplement conclusions drawn from
developmental correlations or experimental manipula-
tions (for review, see Sachs 1981; Aloni 1987; Steeves and
Sussex 1989; Lyndon 1990 and references therein). Many
of the experimental data have been integrated into a
canalization hypothesis, according to which vascular
strands are formed along the routes of a shoot-to-root
signal flux which might be synonymous with the polar flux
of auxins (Sachs 1981). The model explains the canaliz-
ation of the flux into discrete rows of cells by proposing
a positive feed-back mechanism, in which differentiating
vascular cells are thought to be particularly good conduits
of the signal. The canalized flux, in turn, is assumed to
promote further vascular differentiation and the course of
vascular strands is thought to monitor the canalization
G.K.H. Przemeck et al.: MONOtiTEROS gene in plant cell axialization
process. Although the canalization model helps to explain
many of the general features of vascular systems, it is
explicitly open to additional cellular mechanisms that
may, for example, refine cell alignment to mediate the
nearly perfect alignment in mature vascular strands
(Sachs 1981).
According to the feed-back mechanism proposed by
the canalization model, a mutant impaired in vascular cell
alignment would also be compromised in auxin transport
capacity and vice versa. Moreover, the canalization model
predicts that distortions should be more severe at peri-
pheral positions, such as lateral veins in leaf blades, than
in the major plant axis, and that sites of gross distortions
in vascular cell alignment and sites incomplete vasculariz-
ation should be spatially correlated. These features are
found in mp mutants, suggesting that mutations in M P
somehow interfere with the canalization process. How-
ever, due to feed-back mechanisms, it might be difficult
to determine genetically the level of this interference
(see below).
The relationship between embryonic and post-embryonic
yenefunctions. The seedling phenotype of mp mutants has
been described as a broad-pattern deletion, encompassing
the entire hypocotyl, intermediate region, and radicle.
A previous study has shown that this deletion in the
seedling pattern is likely to be due to the failure of basal
cells in the early embryo to form the oriented cell files that
give rise to the tissue layers of the hypocotyl and the
radicle (Fig. lb, Berleth and Jiirgens 1993).
The concept of a canalized signal flux has not been
restricted to vascular strand formation, but has also ser-
ved to explain cell orientation in primordia and embryos
(Sachs 1991; Cooke et al. 1993). As a positive feedback
mechanism implies that the initiation of axiality is more
sensitive than its maintenance, it seems possible that dis-
tortions of the flux in the very early embryo could com-
pletely prevent the formation of the hypocotyl/root axis.
A common primary defect underlying both embryonic
and post-embryonic abnormalities is supported by earlier
observations of a gradual dependence of hypocotyl forma-
tion on residual MP gene activity: mutant seedlings with
weak mp alleles, that also do better in vascular strand
formation (Fig. 4b) and adventitious root initiation, can
occasionally also produce short hypocotyl stumps
(Berleth and Jfirgens 1993). The polar flux of auxins in
embryos has been demonstrated (Greenwood Goldsmith
1970; Fry and Wangermann 1976), and genetic dissection
could become feasible, especially if more mutants im-
paired in auxin transport should be isolated, and if auxin
transport could be directly measured in early embryos. At
present, the phenotypes of mp and pinl seedlings and of
Brassica juncacea seedlings from zygotic embryos treated
with transport inhibitors can be compared. Though em-
bryos of all three show variable degrees of cotyledon
fusions, only mp mutant embryos fail to initiate the
hypocotyl/root axis (Berleth and Jfirgens 1993; Liu et al.
1993). Furthermore, inhibitor treatment frequently results
in the production of an extensively vascularized cotyledon
collar, in striking contrast to a single, barely vascularized
cotyledon that is often observed in mp mutants. It should
be stressed that available mutations interfere but do
G.K.H. Przemeck et al.: MONOPTEROS gene in plant cell axialization
n o t b l o c k the c a n a l i z a t i o n process a n d thus m a y define
c o n t r i b u t i n g cellular m e c h a n i s m s . T h e p h e n o t y p i c differ-
ences are indicative of s e p a r a t e p r i m a r y defects a n d it
seems likely t h a t further p h e n o t y p i c dissection a n d
d o u b l e - m u t a n t analysis of m u t a n t s like mp a n d pinl will
help to e l u c i d a t e the role of i n d i v i d u a l genes in p r o m o t i n g
auxin transport.
In conclusion, o u r analysis of the p o s t - e m b r y o n i c roles
of the Arabidopsis gene M P has revealed a novel genetic
function that c o n t r i b u t e s to the o r g a n i z a t i o n of v a s c u l a r
cell files, a d e v e l o p m e n t a l process so far only p o o r l y
c h a r a c t e r i z e d by m u t a n t analysis. F u r t h e r m o r e , two o t h e r
c o n s p i c u o u s a b n o r m a l i t i e s of mp m u t a n t s , r e d u c e d p o l a r
a u x i n t r a n s p o r t a n d " p i n " - s h a p e d inflorescence m o r p h o -
logy, are linked to v a s c u l a r d e v e l o p m e n t in widely accep-
ted feed-back m o d e l s a n d suggest t h a t the M P function
c o n t r i b u t e s to a x i a l i z a t i o n in p l a n t d e v e l o p m e n t , p o s s i b l y
by m e d i a t i n g the c a n a l i z a t i o n of the auxin flux. At present,
the c a n a l i z a t i o n c o n c e p t is largely h y p o t h e t i c a l a n d de-
rived from e x p e r i m e n t a l m a n i p u l a t i o n in suitable species.
Such efforts m i g h t be c o m p l e m e n t e d b y a genetic a p -
p r o a c h e m p l o y i n g a d d i t i o n a l m u t a n t s i m p a i r e d in vascu-
lar d e v e l o p m e n t a n d p o l a r auxin t r a n s p o r t t h a t are likely
to b e c o m e a v a i l a b l e in Arabidopsis.
We thank R. Kahmann (Genetics Institute, University of Munich
(LMU), Germany) and P. Engstr6m (Dept. Physiol. Bot., University
of Uppsala, Sweden) for generous support, F. Assaad, S. Ploense
(Genetics Institute, University of Munich (LMU), Germany), G.
Jiirgens, W. Lukowitz (Inst. Dev. Genet., University of Tiibingen,
Germany), and M.A. Yund (UC, Berkeley, Cal., USA) for helpful
suggestions on the manuscript, and L. Willmitzer (IGF, Berlin,
Germany) for providing the ASUS1-GUS line. C.S.H. was sup-
ported by a predoctoral fellowship from the University of Munich
(LMU), J.M. by a postdoctoral fellowship from the Royal Swedish
Academy of Science. This work was supported by grant Be1374/1-3
from the Deutsche Forschungsgemeinschaft.
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