Cell, Vol. 115, 591–602, November 26, 2003, Copyright 2003 by Cell Press
Local, Efflux-Dependent Auxin Gradients
as a Common Module for Plant Organ Formation
growing shoots or roots bearing a secondary meristem
at the tip. Aerial organs such as leaves and secondary
shoots, including flowers, originate from primordia on
Eva Benkova´ ,1,2,3,
* Marta Michniewicz,1
Michael Sauer,1
Thomas Teichmann,2
Daniela Seifertova´ ,1
Gerd Ju¨ rgens,1
and Jirˇı´ Friml1,
*
1
Zentrum fu¨ r Molekularbiologie der Pflanzen the flanks of the shoot meristem. Flower primordia give
rise to lateral organs, such as sepals, petals, stamens,Universita¨ t Tu¨ bingen
72076 Tu¨ bingen and carpels. Carpels fuse to form the gynoecium, which
produces organs—ovules—along the placenta of theGermany
2
Max-Delbru¨ ck-Laboratorium ovary. Ovules, in turn, initiate integuments as lateral
primordia (Esau, 1977). The situation is less complex inder Max-Planck-Gesellschaft
50829 Ko¨ ln the root where lateral root primordia are initiated from
localized cell groups within the pericycle (Dubrovsky etGermany
3
Institute for Biophysics al., 2000).
The morphological diversity of plant organs dependsASCR
61265 Brno on the developmental origin of their primordia: shoot
meristems generate leaves, floral meristems floral or-Czech Republic
gans, pistils ovules, ovules integuments, and roots make
lateral roots. The difference between leaves and floral
organs, for example, can be attributed to the expressionSummary
of floral homeotic genes in the latter but not the former
organs (Honma and Goto, 2001). A common feature ofPlants, compared to animals, exhibit an amazing
adaptability and plasticity in their development. This all shoot-derived, as opposed to root-derived, organ
primordia is the expression of the AINTEGUMENTA tran-is largely dependent on the ability of plants to form
new organs, such as lateral roots, leaves, and flowers scription factor (Elliott et al., 1996). Another example is
the involvement of the WUSCHEL homeobox protein induring postembryonic development. Organ primordia
develop from founder cell populations into organs by both flower and ovule development (Gross-Hardt et al.,
2002). By contrast, underground organs such as lateralcoordinated cell division and differentiation. Here, we
show that organ formation in Arabidopsis involves dy- roots are developmentally distinct from aerial organs in
terms of primordium initiation, subsequent developmentnamic gradients of the signaling molecule auxin with
maxima at the primordia tips. These gradients are me- as well as regulatory gene expression (Malamy and Benfey,
1997). These observations suggested at least twodiated by cellular efflux requiring asymmetrically localized
PIN proteins, which represent a functionally re- different regulatory mechanisms for postembryonic organ
formation in plants.dundant network for auxin distribution in both aerial
and underground organs. PIN1 polar localization un- Studies focused on the initiation and positioning of
organs, such as leaves (Reinhardt et al., 2000), flowersdergoes a dynamic rearrangement, which correlates
with establishment of auxin gradients and primordium (Okada et al., 1991; Oka et al., 1999), and lateral roots
(Laskowski et al., 1995), have implicated the plant-sig-development. Our results suggest that PIN-dependent,
local auxin gradients represent a common mod- naling molecule auxin. Auxin is unique among plant hormones
in being actively and directionally transportedule for formation of all plant organs, regardless of their
mature morphology or developmental origin. from the place of synthesis in young apical parts. Current
models propose that members of the PIN protein family
of auxin efflux regulators represent an important part ofIntroduction
a network for auxin distribution throughout the plant
(reviewed in Friml, 2003). PIN proteins have been shownPlasticity and adaptability of development represent the
most important life strategy of plants. Unlike animals, to mediate various developmental processes such as
which can escape unfavorable conditions by behavioral vascular tissue and flower development (PIN1; Ga¨ lweiler
responses, plants compensate for their sessile lifestyle et al., 1998), tropisms (PIN2, PIN3; Mu¨ ller et al., 1998,
by changing their development in response to external Friml et al., 2002b), as well as patterning of the root
cues. Whereas animals establish the entire body organi- (PIN4; Friml et al., 2002a). Cellular polarities of PIN prozation
during embryogenesis, the adult life form of plants tein localization correlate with directions of auxin flux
is largely dependent on postembryonic development. (reviewed in Friml, 2003). The flexibility of the transport
Self-maintaining stem cell systems—the meristems, system may result, at the cellular level, from the continuwhich
are established at both ends of the apical-basal ous cycling of PIN proteins between the plasma memembryonic
axis—are able to perpetuate existing organs brane and endosomes (Geldner et al., 2001, 2003a),
and initiate new organ primordia (Steeves and Sussex, allowing for rapid changes in PIN polar localization and
1989). Some primordia develop into determinate organs hence redirection of auxin fluxes in development. So
with finite cell numbers, whereas others develop into far, this has been demonstrated for PIN3 relocation in
response to gravity stimulation (Friml et al., 2002b). It
remains to be determined whether PIN relocation for*Correspondence: jiri.friml@zmbp.uni-tuebingen.de (J.F.), eva.
benkova@zmbp.uni-tuebingen.de (E.B.) auxin redistribution could also provide a general mecha-
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592
Figure 1. Distribution of Auxin and Its Response
in Lateral Root Primordia
(A) Spatial pattern of DR5::GUS expression
during primordium development: the DR5 activity
gradient with the maximum at the primordium
tip is gradually established. OL and
IL, outer and inner layers; central cells out-
lined.
(B) DR5::GUS in columella initial region of the
mature lateral root.
(C) 2,4-D treatment: loss of DR5 gradient and
induction of DR5 activity in the entire pri-
mordium.
(D) NPA treatment: impaired DR5 gradient
after inhibition of auxin efflux.
(E) Immunolocalization of IAA during primordium
development: the IAA gradient with the
maximum at the tip is gradually established.
Inset shows increased signal, however no
change of the gradient after the IAA
treatment.
(F) IAA accumulation in columella initial region
of the mature lateral root. Inset shows
negative control, no signal without IAA fix-
ation.
Division planes marked by arrowheads and
developmental stages indicated in the upper
right corner: I, II, III, IV, V, and e – emergence.
GUS signal in blue (A–D), IAA signal in brown
(E and F).
nism for other developmental processes including or- activity was highest in columella initials and surrounding
cells (Figure 1B), identical to the reported pattern ingan formation.
Here, we analyze the role of auxin in the formation of primary roots (Sabatini et al., 1999).
The activity of the DR5 reporter does not necessarilydiverse organs during Arabidopsis development. In each
case, developmentally regulated changes in PIN polar reflect auxin levels in all cases because of its own sensitivity
threshold and saturation of the auxin-signalinglocalization redirect auxin fluxes to create local auxin
gradients required for the establishment of primordia pathway. Therefore, we examined whether DR5 can be
used in lateral root primordia as a reporter of endoge-and their development into mature organs. Our data
suggest that efflux-mediated auxin gradients represent nous auxin levels. The exogenously applied naturally
occurring auxin IAA or its synthetic analog NAA (up toa common module that operates in the formation of all
plant organs, regardless of their developmental origin 10 ␮M, 12 hr) increased the intensity of the DR5 signal
and fate. in the primordia without changing its spatial pattern
(data not shown) similar like it has been shown in the
primary root (Friml et al., 2002a). In contrast, the sameResults
treatment with another auxin analog 2,4-D, which can
be only poorly transported by auxin efflux (Delbarre etAuxin and Auxin Response Gradients
al., 1996) or metabolized, induced DR5 activity in allin Lateral Root Primordia
lateral root primordium cells (Figure 1C). In addition,The synthetic auxin responsive promoters such as DR5
inhibition of efflux by the phytotropin NPA interfered(Ulmasov et al., 1997) have become useful tools for monwith
the auxin response gradient (Figure 1D). These dataitoring auxin response in planta. It has been shown that
demonstrate an active auxin efflux in lateral root primor-activity of these reporters correlates with auxin content
dia, which can handle exogenously applied auxin andin roots (Casimiro et al., 2001). We examined auxin rein
this manner maintain the endogenous auxin distribu-sponse during lateral root formation using DR5::GUS
tion. It also suggests that the spatially restricted signals(Figure 1A) and DR5rev::PEH A, which gave identical
in untreated roots reflect differences in auxin levels. Toresults. DR5 activity was absent from the entire differenobtain
additional confirmation, the distribution of auxintiated root and was detected only within the pericycle
itself was visualized using an anti-IAA antibody, whichin the presumptive founder cells of primordia (stage 0).
is specific to IAA (Caruso et al., 1995). In developingAfter the formation of short initial cells by anticlinal divilateral
roots, IAA was detected already at the earliestsions (stage I), staining was detected in all these cells.
primordium stage in short initial cells, subsequently, theWhen outer and inner layers were established by periclistaining
was confined to central cells, and later the maxi-nal divisions (stage II), staining was confined to the cenmum
in IAA accumulation was established at the primor-tral cells of both layer derivatives. During the progresdium
tip (Figure 1E). In the mature lateral root, IAA accu-sion to later stages (III to emergence, e), a gradient in
mulated around the columella initials (Figure 1F). TheDR5 activity with its maximum at the primordium tip was
gradually established. In the mature lateral root, DR5 incubation of roots with IAA led to a visible increase in
Auxin Gradients in Plant Organogenesis
593
the signal intensity (Figure 1E, inset), whereas experiments
omitting either the primary antibody or the IAA
fixation by EDAC (Figure 1F, inset) did not yield any
distinct staining pattern. Thus, the IAA accumulation
pattern mirrored the DR5 activity pattern. In summary,
these results suggest a dynamic auxin gradient during
lateral root primordia development that is mediated by
auxin transport.
PIN Expression and Localization
in Lateral Root Primordia
The best-characterized molecular components of auxin
transport are the PIN auxin efflux regulators. The entire
PIN family in Arabidopsis consists of 8 members. PIN5
and PIN8 lack the entire middle hydrophilic region present
in all other characterized PIN proteins and might be
therefore functionally divergent. To examine which PIN
proteins may play a role in auxin distribution during
primordia development, we first analyzed PIN expression
in lateral roots using PIN1, 2, 3, 4, 6::GUS and
PIN7:GUS transgenic plants and confirmed the staining
patterns by protein localization experiments (Figure 2).
All analyzed PIN genes were expressed from earliest
developmental stages on, except PIN2, which was expressed
only after primordium emergence (e) with PIN2
localized in epidermis cells toward the base of the lateral
root (Figures 2B and 2C). In contrast, PIN1 expression
was detected from stage II on only in the derivatives of
inner layer cells (Figure 2A). PIN3 was expressed at the
base of the primordium and from stage V on additionally
in columella precursors of the newly forming meristem
(Figures 2D and 2E). The PIN4 expression pattern was
partially overlapping, although more restricted to the
margins (Figures 2F and 2G). Interestingly, a newly isoFigure
2. PIN Expression and Localization Patterns during Laterallated PIN gene, the primordia-specific PIN6, was also
Root Formationexpressed from the earliest stages on. Later, its expres(A)
PIN1::GUS expression in derivatives of inner cells. Arrowheadsion was detected exclusively in primordium margins
marks the border between inner and outer cells.
(Figures 2H and 2I). PIN7 displayed a similar expression
(B) PIN2::GUS in outer cells.
pattern in the margins; however at later stages, PIN7 (C) PIN2 polar localization pointing away from the tip (arrows).
expression became restricted to inner, provascular (D) PIN3::GUS at the base and later appearing in the forming meristem
(arrowhead).cells, mirroring PIN1 expression (data not shown). Thus,
(E) PIN3 localization in the columella precursors of forming lateralat least 6 PIN genes are expressed during lateral root
root meristem.primordium development in specific, partially overlap(F)
PIN4::GUS at the base, in the margins, and later in the meriping
patterns. This represents the molecular basis for
stem (arrowhead).
a transport system that can mediate auxin redistribution (G) PIN4 localization in margins and the meristem.
during primordium development. (H) PIN6::GUS in margins.
(I) PIN6::GUS, face view.
Developmental stages are indicated in the upper right corner.
DR5 Activity Gradients and Lateral Root GUS staining in blue (A, B, D, F, H, and I), protein localization signal
in red (C and G) or green (E), nuclear counterstain blue (C and G).Formation in pin Mutants
To test whether differentially expressed PIN proteins
play any role in auxin gradients and lateral root formation,
we analyzed primordium initiation and develop- suggests the requirement of various PIN proteins for the
lateral root initiation.ment as well as the spatial pattern of DR5 activity in pin
mutants and in 35S::PIN1 transgenic plants. 35S::PIN1 In order to assess the developmental rate of primordia,
we determined the proportion of primordia, whichoverexpressed PIN1 in all cells of the lateral root primordium
(Figure 4C). The density of initiated lateral root had reached emergence stage in 6-day-old plants.
About 20% of primordia in control plants (21%; n ϭ 159)primordia (from stage I on) in 5- to 10-days old seedlings
was scored. Pin3 (54%; Student’s t-test: P Ͻ 0.002), or pin2 mutants (29%; n ϭ 296) reached the emergence
stage, whereas this proportion was significantly lowerpin7 (170%; P Ͻ 0.008) and 35S::PIN1 (153%; P Ͻ 0.02)
seedlings showed significant changes in number of initi- in pin1 mutants (7%; P Ͻ 0.001) and 35S::PIN1 (14%;
P Ͻ 0.003), indicating slower primordia development.ated lateral roots compared to control (100%; n ϭ 147),
pin1 (91%; n ϭ 76) or pin2 (93%; n ϭ 32) seedlings. This Also, the pattern of DR5 activity was analyzed in primor-
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594
Figure 3. In Vivo Monitored Correlation between
Individual Primordium Development
and the DR5 Gradient
(A and B) Wild-type (A) and pin2 (B) primordium
development with correctly established
DR5 gradient.
(C) Retarded pin1 primordium development
without established DR5 gradient.
(D) Retarded 35S::PIN1 primordium development,
no DR5 gradient.
(E) Postembryonic phenotype of pin1 pin3
pin4 pin7 mutant: very short root and cotyledon
defects.
(F–H) Induction of lateral root development
by NAA treatment. Normal primordia development
in wild-type (F) seedlings. Affected
primordia development in pin3 pin7 (G) and
pin1 pin3 pin4 (H) mutants.
Numbers indicate time points during primordium
development (A–D) or period of NAA
treatment (F–H). GUS staining in blue (A–D).
dia between stage IV and emergence. In control plants, mutants (Figure 3C; 43%, n ϭ 88) and 35S::PIN1 (Figure
most primordia at these stages displayed the typical 3D; 65%; n ϭ 54), highly increased frequencies of priDR5
pattern with maximum at the tip (see Figures 1A mordia with retarded growth were found. In addition, in
and 3A). However, some primordia (27%; n ϭ 138) did pin1 (29%) but not in 35S::PIN1 (7%) plants the propornot
establish the DR5 gradient at this stage. Interest- tion of primordia with irregular shape was increased.
ingly, in pin2 seedlings the DR5 gradient was formed in Interestingly, pin2 primordia (Figure 3B; 8%; n ϭ 36)
all primordia at these stages (100%; n ϭ 24; Figure 3B). typically developed faster than those of control plants.
By contrast, more than half of the pin1 (62%, n ϭ 95) Regardless of seedling genotypes, the individual primoror
35S::PIN1 primordia (77%, n ϭ 56) displayed rather dia, which developed normally, displayed normal DR5
diffuse DR5 patterns (Figures 3C and 3D). These data activity distribution, whereas abnormal development
demonstrate that the differentially expressed PIN pro- was accompanied by defects in DR5 activity distribution
teins play distinct roles in both the initiation and devel- (Figures 3A–3D). In conclusion, these findings support
opment of primordia as well as in the establishment of a correlation between PIN-dependent auxin gradients
the auxin gradient during lateral root formation. and lateral root primordia development. Notably, also
in control plants certain variability in the rate of lateral
root development was observed, with some primordiaIn Vivo Analysis of Lateral Root Development
showing less DR5 signal accumulation at the tip andWe established an experimental system for in vivo analyslower
development. It is well known that the initiationsis of primordium development, which enabled us to
of the lateral roots as well as their outgrowth is variablestudy lateral root formation in relation to the PIN-depenand
largely depends on the availability of the nutrientsdent auxin distribution in more detail. Four hours timeand
minerals (Dubrovsky et al., 2000). It is possible thatlapse observation of individual primordia revealed that
the adaptation mechanism regulating the rate of lateralthey typically developed within 24 hr from stage II–III to
roots development is based on the balanced auxin sup-stage VII or emergence (Figure 3A). A smaller proportion
ply to the tip and its PIN2-dependent retrieval throughof primordia (16%, n ϭ 91) developed more slowly. Few
primordia (6%) displayed an irregular shape. In pin1 outer layers of the primordium.
Auxin Gradients in Plant Organogenesis
595
Figure 4. Correlation of PIN1 Relocation, DR5 Gradients, and Primordium Development
(A and B) PIN1:GFP (A) and PIN1 (B) localization in developing lateral root primordium with gradual establishment of PIN1 at the lateral
cell membranes.
(C) Nonpolar PIN1 localization in all cells of 35S::PIN1 lateral root primordium.
(D–F) PIN1:GFP rearrangement in primordium initiated after IAA treatment (D), no relocation to lateral sides after IAA ϩ BFA (E) or IAA ϩ NPA
treatments (F).
(G–O) Misexpression of molecular markers after inhibition of PIN1 relocation. Left image after treatment with IAA, right image after IAA ϩ NPA
(n) or BFA (b). Auxin response: DR5rev::PEH A (G); cell division: CycB1:GUS (H); margins: CUC3::GUS (I); PIN6::GUS (J) and M0223 (K); inner
layer: Q0990 (L); pericycle (arrowheads): M0171 (M); columella and protophloem: AUX1::GUS (N); stele of the primary root: Q0680 (O).
Arrowheads indicate PIN1 polar localization (A,B, D–F). GUS and PEH A signal in blue (G–J and N), GFP signal in green (A, D–F, K–M, and O),
PIN1 signal in yellow (B) or red (C), nuclear counterstain blue (C).
PIN Functional Redundancy NAA application on impaired primary roots. In control
roots, lateral root formation was initiated throughoutin Lateral Root Formation
The fact that defects in lateral root formation are only the whole pericycle; regularly spaced primordia were
established and developed into mature lateral roots (Fig-mild in single pin mutants together with the partially
overlapping expression pattern of various PIN proteins ure 3F). By contrast, in pin3 pin7 (Figure 3G), pin4 pin7,
and increasingly strong in pin1 pin4 pin7 or pin1 pin3suggested functional redundancy between PIN proteins.
Therefore, we analyzed multiple pin mutants with re- pin7 (data not shown) mutants, less well-defined primordia
developed. In pin1 pin3 pin4 seedlings (Figure 3H),spect to the lateral root development. The lateral root
phenotypes of the multiple mutants were either additive only massive division of pericycle cells without any trace
of defined primordia was detected. Exactly the sameor difficult to interpret, since also the primary root development
was impaired few days after germination. For defects were found in wild-type seedlings upon inhibition
of auxin transport (Figures 4G–4O) or in weak gnomexample, pin1 pin3 roots initiated fewer primordia (58%,
P Ͻ 0.01), which developed more slowly (only 6% mutant alleles (Geldner et al., 2003b). These increasingly
stronger defects in multiple pin mutants demonstrateemerged roots compared to 21% in controls, P Ͻ 0.001).
On the other hand, pin1 pin3 pin4 primary roots dis- that auxin efflux, which is dependent on functionally
redundant PIN proteins, is essential for primordium de-played defects a few days after germination (data not
shown) and the pin1 pin3 pin4 pin7 quadruple mutants velopment.
showed strong defects already in embryonic development
(Figure 3E). To analyze specific defects in lateral Rearrangement of PIN1 Polarity
in Lateral Root Primordiaroot formation in multiple pin mutants and to better
distinguish defects in initiation and development, lateral Asymmetric subcellular localization of various PIN proteins
has been shown to correspond to the direction ofroot formation was artificially initiated by exogenous
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596
auxin flux (reviewed in Friml, 2003). We examined the various molecular markers. All these markers showed
in the IAA-induced primordia the same expression pat-polarity of PIN1 localization during primordium development.
To gain insight into its dynamics, we constructed tern as in untreated seedlings with exception of margin
marker M0223, which was undetectable in untreated rootstransgenic plants carrying functional PIN1:GFP, which
showed a pattern of expression and localization identi- (data not shown). The cell division marker CYCB::GUS
confirmed that in differentiated root parts after IAA treat-cal to the endogenous PIN1 (Figures 4A and 4B). At the
earliest stage, PIN1:GFP was detected exclusively on ment, cell division activity was confined exclusively to
developing primordia (Figure 4H, left). In contrast, inthe transverse (anticlinal) sides of the short initial cells.
From stage II on, the GFP signal was found in addition NPA or BFA treated seedlings, CYCB::GUS expression
was detected along the whole pericycle (Figure 4H,at lateral (periclinal) sides and later the polarity pointed
more and more toward the primordium tip (Figure 4A). right). Stele markers Q0680 (Figure 4O) and Q1630 (data
not shown) discontinued expression in the pericycle ofIn the emerged lateral roots, PIN1 localization pointed
predominantly toward the root apex, giving the same the primary root at positions of developing primordia,
but were homogeneously expressed upon NPA and BFAlocalization pattern, which was reported for the primary
root (Friml et al., 2002a). The same relocation of PIN1 treatment. The establishment of primordium margins
was analyzed using three different markers—CUC3::GUS,occurred also in primordia, which were initiated after
exogenous IAA or NAA applications (Figure 4D). Interest- PIN6::GUS, and M0223. After IAA treatment, these markers
were specifically expressed only in primordiumingly, the PIN1 relocation to the lateral sides was prevented
by substances, which have been shown to inter- boundaries (Figures 4I–4K, left). After concomitant NPA
or BFA treatment, however, CUC3::GUS (Figure 4I) orfere with the subcellular movement of PIN proteins
(Geldner et al., 2001), such as the protein trafficking PIN6::GUS (Figure 4J) showed a homogenous expression,
whereas M0223 expression was not initiated (Fig-inhibitor brefeldin A (BFA) or phytotropins such as NPA
(Figures 4E and 4F). It has been shown previously that ure 4K). Also the coordinated differentiation of various
cell types in developing primordia, such as stele (Q0990;GNOM, a regulator of BFA-sensitive vesicle trafficking,
mediates the establishment of PIN1 polarity in em- Figure 4L), pericycle (M0171; Figure 4M), or vasculature
and lateral root cap (AUX1::GUS; Figure 4N), did notbryogenesis (Steinmann et al., 1999). We tested whether
GNOM also mediates the PIN1 relocation during lateral occur in NPA- or BFA-treated seedlings. Thus, molecular
markers further confirmed that in absence of coordi-root formation. This is indeed the case since in transgenic
plants expressing an engineered BFA-resistant nated PIN-dependent auxin transport, dividing pericycle
cells do not develop into lateral root primordium-likeversion of GNOM (GNOMM696L
-myc), BFA did not perturb
PIN1 relocation (data not shown, similar to Figure 4D). structures, they do not define boundaries and various
primordium-specific cell types.Thus, GNOM-dependent rearrangement of PIN1 localization
occurs during lateral root development suggesting
a gradually established auxin flux through the PIN-Dependent Auxin Distribution
inner cells toward the primordium tip. in Cotyledon Formation
We analyzed whether a PIN-mediated auxin gradient
dependent mechanism operates in other organogeneticPIN1 Relocation Correlates with DR5 Gradients
and Primordium Development processes. In Arabidopsis, two symmetrically positioned
embryonic leaves (cotyledons) are initiated at theThe PIN1 polarity rearrangement from the transverse to
the lateral sides of cells correlated with the establish- triangular stage of embryogenesis and develop subsequently
(Mayer et al., 1991). From the triangular stagement of the auxin gradient with its maximum at the
primordium tip. To analyze this relationship and its con- on, beside an apical-basal auxin gradient, additional
sites of DR5rev::GFP activity (Figure 5A) as well as IAAsequences for primordium development, we induced
synchronized initiation of lateral root primordia by exog- accumulation (Figure 5B) were detected at the tips of
incipient cotyledons. As reported, PIN1 was localizedenous IAA or NAA application. In control experiments,
primordia were initiated, PIN1 relocated (Figure 4D), DR5 to the apical ends of the cells in outer embryo layers
(Steinmann et al., 1999). Analysis of PIN1:GFP revealedgradients properly formed (Figure 4G, left), and primordia
developed normally (up to stage VII within 48 hr). In that its polar localization pointed exactly toward the
position of auxin accumulation in incipient cotyledoncontrast, when PIN1 relocation was prevented either by
BFA or NPA treatment (Figures 4E and 4F), initiation still tips (Figure 5N). Inside the primordium, PIN1 localization
was gradually established at the basal side of futureoccurred, nonetheless, no DR5 gradients were established
(Figure 4G, right) and only a massive division of vasculature cells during cotyledon development (Figures
5H–5L). In analogy to lateral root development, NPApericycle cells without clear separation into primordia
was observed similar to multiple pin mutants (see Figure and BFA interfered with the gradual establishment of
PIN1 polarity (Figure 5M) as well as with DR5 gradient3H). In the GNOMM696L
-myc line, however, only NPA (data
not shown, but identical to Figures 4F and 4G, right) but and cotyledon establishment in in vitro cultured embryos
(Figure 5D). Similar defects were observed alsonot BFA (data not shown, identical to 4D and 4G, left)
interfered with PIN1 relocation and primordium develop- in gnom mutant embryos (Figure 5C). As expected in
GNOMM696L
-myc embryos, the BFA effect on PIN1 local-ment. This data shows that under all testable conditions
the coordinated rearrangement of PIN1 correlates with ization was not observed and hence the DR5 gradients
and cotyledons formed normally (Figure 5E compare toDR5 gradients and correct primordium development.
For a detailed analysis of the NPA- or BFA-induced 5D). Moreover, pin1 (Figure 5F; Okada et al., 1991), pin4
pin7 (Figure 5G), and pin1 pin3 pin4 pin7 (Figure 3E)defects in primordium development, we made use of
Auxin Gradients in Plant Organogenesis
597
Figure 5. PIN-Dependent Auxin Gradients in Cotyledon Formation
(A) DR5rev::GFP in triangular embryo. Signals (arrowheads) visible in the basal region and in the tips of incipient cotyledons.
(B) IAA immunolocalization mirroring the DR5 pattern in heart stage embryo.
(C) Defects in the DR5 gradients and cotyledon formation in the gnom embryo.
(D and E) BFA treatment: defects in DR5 gradient and cotyledon formation in control embryo (D). No BFA effects on DR5 gradients and
cotyledon formation in BFA resistant GNOMM696L
-myc embryo (E).
(F and G) Defects in cotyledon formation in pin1 (F) and pin4 pin7 (G) mutants.
(H–J) PIN1 localization in embryos. In the globular embryo stage, the basal PIN1 localization in cells below incipient cotyledons is not established
yet (H). Gradual establishment of PIN1 basal localization at triangular (I) and early heart (J) stages.
(K and L) Schematics of putative PIN1-dependent auxin fluxes (arrows) at globular (K) and triangular (L) stage.
(M) NPA treatment prevents the establishment of PIN1 basal localization in provascular cells.
(N) PIN1:GFP polarity in the epidermis of heart embryos pointing toward the incipient cotyledon tips.
Arrowheads indicate PIN1 polar localization (H–J, M, and N). GFP signals in green (A, C–E, and N). IAA signal in brown (B) and PIN1 signal in
red (H–J, and M). Nuclear counterstain in blue (H–J, and M).
mutants displayed increasingly stronger defects in coty- DR5 activity, PIN1 localization as well as consequences
of auxin efflux inhibition on these processes. Usingledon formation. These data demonstrate, in analogy
with lateral roots, a correlation between the GNOM- DR5rev::GFP plants, we detected local DR5 activity gradients
with a maximum at the tip of primordia in alldependent establishment of PIN1 polar localization,
auxin gradients, and cotyledon formation. However, the inspected organs, such as leaves or flowers (Figure 6A),
all floral organs (Figure 6C), ovules (Figure 6L), as welldirection of auxin flux is opposite to that in lateral root
primordia. In incipient cotyledons, the auxin gradient is as integuments (Figure 6N). Also PIN1 and PIN1:GFP
localizations were detected in all primordia, such asestablished by transport through the outer layer toward
the primordium tip. From there auxin appears to be those flanking the shoot (Figure 6E) or floral (Figure 6F)
meristems, as well as those of floral organs (Figures 6G“drained” into the primordium interior and through the
future vascular system toward the basal embryo pole. and 6H), ovules (Figures 6M and 6P), or ovules with
integuments (Figure 6O). In all cases, PIN1 was expressed
in the outer cell layer with its polarity pointingPIN-Dependent Auxin Distribution in Shoottoward
the primordium tip (Figures 6H and 6F, insets).Derived Organogenesis
In the inner future vascular cells, PIN1 localization wasVarious shoot-derived organs such as leaves, flowers
gradually established toward the basal ends during pri-with different floral organs, and ovules are formed postmordium
development (Figure 6E, inset). Inhibition ofembryonically, contributing largely to the final shape of
the mature plant. We analyzed the spatial pattern of auxin efflux interfered with the DR5 gradients in the
Cell
598
Figure 6. PIN-Dependent Auxin Gradients in Shoot-Derived Organ Formation
(A and B) DR5rev::GFP in the apical meristem. Accumulation of signals visible at the position of incipient primordia and their tips (A). Auxin
efflux inhibition by NPA interferes with the DR5 signal at tips (B).
(C and D) DR5rev::GFP in developing flowers. Signals visible at the tips of all floral organ primordia (C). Auxin efflux inhibition by NPA interferes
with the DR5 signals at tips (D).
(E and F) PIN1 localization in apical (E) and secondary floral (F) meristems. In the incipient vasculature below the primordia, PIN1 basal
localization is gradually established (inset in E). PIN1 localizes in the epidermis cells toward primordia tips (inset in F).
(G) PIN1:GFP in epidermis and incipient vasculature in developing flowers.
(H) PIN1:GFP polar localization (inset) pointing toward the tips of floral organ primordia.
(I) Pin-like inflorescence in wild-type plant grown on NPA.
(J) pin1 inflorescence without the initiated lateral organs.
(K) Defective flowers in pin3 pin7 mutants. Missing stamens and carpels indicated (arrowheads). Inset shows example with missing sepals,
petals, and fused petals.
(L and M) Young ovule primordium. DR5rev::GFP signal at the tip (L). PIN1:GFP polar localization in outer layer (M).
(N and O) Older ovule primordium. DR5rev::GFP signal at the tip and at tips of developing integuments (arrowheads) (N). PIN1:GFP polar
localization in outer layer and in inner cells (O).
(P) PIN1 signals in developing ovule primordia in wild-type gynoecium.
(Q) pin1 gynoecium, no or entirely misshaped (arrowhead) ovules develop.
Arrowheads mark incipient primordia and their tips (A–E, and N) or PIN1 polar localization (F, H, and M). GFP signal in green (A–D, F–H, and
L–O) and PIN1 signal in yellow (E and P).
Auxin Gradients in Plant Organogenesis
599
primordia. For example, treatment of the shoot apical Interference with either PIN-dependent auxin transport
or PIN relocation causes defects in both auxin gradientmeristem (Figure 6B) or developing flowers (Figure 6D)
with NPA or BFA resulted in withdrawal of the DR5 signal and lateral root formation. All the available correlative
evidence from both genetic and physiological experi-from primordium tips and its accumulation in central
parts below the apex. The long-term inhibition of auxin ments taken together with what is known about the
mechanism of auxin transport, suggest the followingefflux by these substances led to the formation of a pinlike
inflorescence (Figure 6I)—a plant strikingly similar model of how primordium development is regulated.
First, auxin accumulates at the position of the futureto the pin1 mutant (Figure 6J; Okada et al., 1991). Loss
of PIN1 function was shown to cause defects in all post- primordia, cell division is activated and subsequently,
endogenous signals mediate retargeting of continuouslyembryonic organogenetic processes such as development
of leaves, flowers, and floral organs (Okada et al., cycling PIN proteins. As a consequence, auxin transport
is redirected, providing auxin from the root vasculature1991). We found also ovule defects, since in flowers,
which occasionally develop, the gynoecium resembled via the interior of the primordium into the tip from where
auxin is transported away through the outer layer (“foun-hollow tubes with no ovules or few malformed structures
(Figure 6Q, compare to 6P). The strong defects in the tain” model; Figure 7A). Thus, the combined action of
differentially expressed and localized PIN proteins re-pin1 mutant after transition to flowering render PIN1 as
the main and nonredundant regulator of organogenesis sults in the formation of the auxin gradient, which mediates
proper lateral root development.at this stage. Nonetheless, flower defects were found
also in other pin mutants, such as pin3 pin7, which
developed abnormal flowers with fused petals, no sta- PIN-Dependent Auxin Gradients in Aerial
mens, and occasionally no sepals (Figure 6K). All these Organs Formation
observations suggest that also in shoot-derived organ Plant aerial organs such as cotyledons, leaves, and floral
formation, PIN-dependent efflux mediates primordium organs have long been regarded as homologous strucdevelopment
by supplying auxin to the tip through the tures (Esau, 1977). In all aerial organ formation, we obouter
layer, from where auxin is drained through the served similar correlations between the PIN-dependent
primordium interior into the vascular network. auxin efflux, auxin gradients, and primordia development
as for lateral root formation. However, in contrast
to the “fountain” flow in the root, the direction of auxinDiscussion
transport appears to be reversed. In aerial organs, auxin
is supplied through the outer layer and accumulates atThe architecture of the adult plant is largely dependent
on the formation of a variety of new organs during post- the primordium tip, from which it is drained into the
interior of the primordium and transported downwardembryonic development. These organs are initiated as
lateral primordia and subsequently attain diverse mor- through its middle (“reverse fountain”; Figure 7B). Following
this route, new vasculature differentiates andphologies, due to the expression of different sets of
regulatory genes. Thus, developmental fates of organ thus, the newly formed organ is connected to the existing
vascular strands in more mature parts of the plant.primordia are conditioned by their developmental history,
ultimately going back to the fundamental decision Another distinguishing feature seems to be a dominant
role of PIN1 as regulator of both local auxin distributionof shoot versus root in embryogenesis.
We have addressed the developmental issue of organ and organogenesis in the shoot apex, based on the
strong defects in the pin1 mutant after transition to flow-formation in general to identify common underlying
mechanisms, regardless of the developmental origin ering. Despite these differences, a common auxin gradient-dependent
mechanism seems to underlie primor-and fate of individual primordia.
dium development of both aerial and underground
organs.PIN-Dependent Auxin Gradients
in Lateral Root Formation
In Arabidopsis, lateral roots are initiated within the peri- A Common Developmental Module
for Organogenesis in Plantscycle, developing primordia display a nearly invariant
cell division pattern and proceed through several well- During organ formation, first a site of primordium initiation
has to be selected and then a new growth axis hasdefined stages. Eventually, a new meristem is formed,
which is anatomically identical to the primary root meri- to be established. Our results suggest that redirection
of auxin flow and local accumulations of auxin play astem (Malamy and Benfey, 1997). Auxin promotes lateral
root initiation, as was demonstrated by exogenous auxin fundamental role in these processes. As a first step,
auxin accumulates at the site of initiation. Already thisapplication (Blakely and Evans, 1979) and by the analysis
of mutants affected in auxin biosynthesis (Barlier et step might be mediated by PIN-dependent auxin transport,
as evidenced by organ initiation and positioningal., 2000) or response (Fukaki et al., 2002). We found that
auxin accumulates at the position of future primordia. At defects observed in pin mutants or caused by manipulation
of local auxin distribution (Reinhardt et al., 2000;later stages, an auxin gradient is gradually established
with its maximum at the primordium tip. This gradient Laskowski et al., 1995). In a second step, cell division
is activated and the auxin transport machinery reorga-is dependent on auxin efflux mediated by differentially
expressed, functionally redundant PIN proteins. More- nizes. Regardless of the type of primordia, the new direction
of PIN-mediated auxin transport determines theover, the polar localization of PIN1 gradually changes to
the cell surface facing the future tip. This rearrangement growth axis of the developing organ, establishing an
auxin gradient with its maximum at the tip.correlates with and is required for the auxin gradient.
Cell
600
Figure 7. Model for Auxin Transport and Distribution
in Root- and Shoot-Derived Organ
Primordia
(A) Lateral root primordium: auxin is provided
by PIN-dependent auxin transport through
the primordium interior toward the tip, where
it accumulates. From here, part of the auxin
is retrieved by a PIN2-dependent auxin route
through the outer layers.
(B) Aerial organ primordium: PIN1 is a major
component for auxin distribution. Auxin is
provided to the primordium tip through the
outer layers. From the tip, auxin is drained
through a gradually established transport
route toward the vasculature.
Places of auxin accumulation are depicted in
green. Presumptive routes of auxin transport
are depicted by red arrows.
signal.salk.edu/cgi-bin/tdnaexpress) mutant lines have sequence-Local, efflux-dependent auxin gradients as a common
indexed insertions at positions 1349 and Ϫ51 from ATG, respec-module for organ development may also apply to other
tively. The pin1, 3, 4, 7 multiple mutants were generated from pin1,
plants. Higher plants form an evolutionarily closely repin3-5,
pin4-3 and pin7-1 single mutant alleles. 35S::PIN1 plants
lated group of species, they share the same set of organs were generated by fusion of the CaMV35S promoter with the PIN1
(Esau, 1977), and organ development is affected by coding sequence. PIN1:GFP was generated by insertion of mGFP4
into the PIN1 genomic fragment (nucleotides Ϫ2320 to 3508 fromauxin and auxin efflux inhibitors treatment in all species
ATG) at position 1510. Its functionality was verified by crossing oftested (for overview see Lomax et al., 1995). Moreover,
three independent transgenic lines into pin1 mutants, in total 350PIN-related sequences have been found in both dicot
F2 plants were analyzed for rescue. The anti-IAA (1: 500; agdia,
and monocot plant species. Therefore, it is likely that http://www.agdia.com), -PIN1 (1: 300; Ga¨ lweiler et al., 1998), -PIN2
the auxin transport-dependent mechanism, which we (1: 200; Mu¨ ller et al., 1998), -PIN3 (1: 50), and -PIN4 (1: 400; Friml
demonstrated for Arabidopsis, also operates in organ et al., 2002a, 2002b) antibodies have been described. Alkaline phosphatase-conjugated
antimouse (Novagen), FITC-, and CY3-conju-formation in other higher plants.
gated antirabbit secondary antibodies (Dianova) were dilutedAlthough developmental strategies differ markedly
1:1000, 1:200, and 1:600, respectively.
between plants and animals, both groups of organisms
make use of developmental modules. In Drosophila, for Growth Conditions
example, imaginal discs give rise to morphologically Arabidopsis plants and seedlings were grown as described (Friml
diverse organs, such as antennae, legs, wings, or geni- et al., 2002a). Exogenous drug application was performed by incubation
of 5 days old seedlings in 0.5 ϫ MS liquid medium supplementedtals. Organs of one type can be transformed into another
with 1–25 ␮M IAA, NAA, 2,4-D, NPA, and BFA for up to 60 hr. NPAtype, either by mutation or transdetermination, revealing
treatment of floral primordia was described (Nemhauser et al., 2000).
the developmental similarity of organ primordia (Maves For in vitro embryo culture, excised ovules were placed on 0.5 ϫ
and Schubiger, 1999). The underlying patterning pro- MS media containing 2% sucrose, 400 mg/l glutamine, and 0.3%
cess involves gradients of morphogens, such as Deca- Phytagel supplemented with 10–20 ␮M BFA or NPA. Plates were
kept in the dark at 22ЊC for up to 4 days. For ovule developmentpentaplegic or Wingless, that regulate development of
analysis, carpels were dissected, fixed in ethanol/acetic acid (3:1),all organs, regardless of their origin and fate (Cadigan,
and mounted in chloralhydrate. For phenotypic analyses of lateral
2002). Thus, modular development in organ formation
root initiation and development, at least 20 seedlings were proand
patterning seems to be a general principle of higher cessed (Malamy and Benfey, 1997) and inspected. For in vivo analylife
forms. sis of individual lateral root primordia development, 5-day-old seedlings
were transferred on slides with a thin layer of 0.5 ϫ MS, 0.5%
Experimental Procedures agarose medium, covered, supplemented with 0.5 ϫ MS liquid medium,
and incubated over night in a humid chamber. For all compariUsed
Materials sons, at least three independent experiments were performed giving
The DR5::GUS (Ulmasov et al., 1997); gnom (Mayer et al., 1993); the same statistically significant results. Data were statistically eval-
GNOMM696L
-myc (Geldner et al., 2003a); CYCb::GUS (Ferreira et al., uated using ProStat 1.02 (PolySoftware International).
1994); CUC3::GUS (Vroemen et al., 2003); AUX1::GUS (Marchant et
al., 1999); pin1-1 (Okada et al., 1991); pin1::En134 (Ga¨ lweiler et al., Expression and Localization Analysis
1998); pin2 (Mu¨ ller et al., 1998); PIN2::GUS (Friml et al., 2003); Histochemical staining for GUS activity, whole-mount PIN immunoPIN3::GUS,
pin3-2, pin3-3 (Friml et al., 2002b); DR5rev::PEH A, localization (Friml et al., 2003; Steinmann et al., 1999) as well as
PIN4::GUS, and pin4-3 (Friml et al., 2002a) have been described. immunolocalization at sections (Ga¨ lweiler et al., 1998) were perThe
pin1-1 and pin2 (eir1-1) mutant lines with introduced DR5::GUS formed as described. IAA immunolocalization was done after prehave
been generated previously (Sabatini et al., 1999). DR5rev::GFP fixation with 3% EDAC for 1 hr. GFP was visualized in 5% glycerol
has been generated by replacing PEH A in DR5rev::PEH A by ER- without fixation. Microscopy was done on a Zeiss Axiophot
targeted eGFP coding sequence (Clontech). GFP-based marker equipped with an Axiocam HR CCD camera. For confocal laser
lines were isolated from the library based on the GAL4/UAS trans- scanning microscopy, a Leica TCS SP2 was used. Images were
activation system (Haseloff, 1999). PIN1::GUS and PIN6::GUS processed in Adobe Photoshop 7.0 (Adobe Inc.).
(AF087819) constructs were generated by fusing PCR-amplified
fragments (nucleotides Ϫ1289 to Ϫ5 and Ϫ1794 to Ϫ1) with the Acknowledgments
uidA gene. The PIN7:GUS (AF087820) translational fusion was generated
by fusing uidA gene to the C terminus of the PIN7 coding We thank K. Palme for enabling E.B. to accomplish part of this work
in his laboratory. We are grateful to M.L.O. Mendes, K. Nettesheim,sequence. pin7-1 (http://genetrap.cshl.org/) and pin3-5 (http://
Auxin Gradients in Plant Organogenesis
601
R. Schwab, O. Tietz, J. Wang, and D. Weijers for technical assis- Auxin transport inhibitors block PIN1 cycling and vesicle trafficking.
tance; K. Cornelis, M. Guttenberger, and D. Weijers for critical read- Nature 413, 425–428.
ing of the manuscript; and N. Geldner for sharing unpublished data
Geldner, N., Anders, N., Wolters, H., Keicher, J., Kornberger, W.,
and helpful discussions. We thank M. Gutenberger for the expert
Muller, P., Delbarre, A., Ueda, T., Nakano, A., and Ju¨ rgens, G.
help with the statistic evaluation. Sequence-indexed Arabidopsis
(2003a). The Arabidopsis GNOM ARF-GEF mediates endosomal reinsertion
mutants were obtained from the Salk Institute Genomic
cycling, auxin transport, and auxin-dependent plant growth. Cell
Analysis Laboratory and Cold Spring Harbor Laboratory. This work
112, 219–230.
was supported by the Volkswagenstiftung to J.F. (J.F., M.M., and
Geldner, N., Richter, S., Vieten, A., Marquardt, S., Torres-Ruiz, R.,D.S.) and the Margarete von Wrangell-Habilitationsprogramm (E.B).
Mayer, U., and Ju¨ rgens, G. (2003b). Partial loss-of-function alleles
reveal a role for GNOM in auxin transport related, post-embryonicReceived: August 11, 2003
development of Arabidopsis. Development, in press.Revised: November 3, 2003
Accepted: November 3, 2003
Gross-Hardt, R., Lenhard, M., and Laux, T. (2002). WUSCHEL signalPublished
online: November 13, 2003
ing functions in interregional communication during Arabidopsis
ovule development. Genes Dev. 16, 1129–1138.
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