1 Institute of Plant Science, University of Berne, Altenbergrain 21, 3013 Bern,
Switzerland
2 Institute of Applied Physics, University of Berne, Sidlerstrasse 5, 3012 Bern,
Switzerland
Author for correspondence (e-mail:
cris.kuhlemeier{at}ips.unibe.ch)
Accepted 25 October 2004
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SUMMARY |
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Here, we employ new microsurgical techniques to reassess and extend the classical studies on phyllotaxis and leaf polarity. Previous experiments have indicated that the isolation of an incipient primordium by a tangential incision caused a change of divergence angle between the two subsequent primordia, indicating that pre-existing primordia influence further phyllotaxis. Here, we repeat these experiments and compare them with the results of laser ablation of incipient primordia. Furthermore, we explore to what extent the different pre-existing primordia influence the size and position of new organs, and hence phyllotaxis. We propose that the two youngest primordia (P1 and P2) are sufficient for the approximate positioning of the incipient primordium (I1), and therefore for the perpetuation of the generative spiral, whereas the direct contact neighbours of I1 (P2 and P3) control its delimitation and hence its exact size and position. Finally, we report L1-specific cell ablation experiments suggesting that the meristem L1 layer is essential for the dorsoventral patterning of leaf primordia.
Key words: Tomato, Meristem, Phyllotaxis, Laser ablation, Dorsoventral, Patterning, Meristem layer, L1 layer
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Introduction |
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It has been proposed that the primordia are the source of a diffusible
inhibitor of organ formation (Schoute,
1913) (reviewed by Steeves and
Sussex, 1989
). According to this idea, inhibitory fields emanate
from the primordia, thus allowing new primordia to be formed only at certain
minimal distances from pre-existing ones. However, recent evidence has
identified an inverse mechanism in which the primordia act as sinks for the
organ inducer auxin rather than as sources of an inhibitor
(Reinhardt et al., 2003a
). The
result of this scenario is similar: new organs can only be formed at certain
minimal distances from pre-existing ones, thus leading to regular arrangement
of leaves.
Given the proposed role of primordia in phyllotaxis, isolating a young
primordium from the meristem is expected to change the position of subsequent
primordia, allowing them to arise closer to the operated site. It has been
attempted to experimentally interfere with leaf positioning by separating
incipient primordia from the remainder of the meristem through tangential
incisions (Snow and Snow,
1931). When P1 was isolated from the meristem by a
tangential incision, I1 arose at its normal position relative to
P1, but the angle between I1 and I2
increased. This may indicate that the position of I1 was
developmentally fixed at the time of the operation, whereas the position of
I2 could still change once the influence from P1 was
eliminated. I1 has been well characterized by its distinct pattern
of gene expression. It differs from the surrounding cells of the peripheral
zone in that it expresses organ marker genes, such as PINFORMED1
(Vernoux et al., 2000
;
Reinhardt et al., 2003a
),
REVOLUTA (Otsuga et al.,
2001
), LEAFY (Weigel
et al., 1992
), and ZWILLE/PINHEAD
(Moussian et al., 1998
;
Lynn et al., 1999
), whereas
KNOTTED1-type transcription factors, markers for meristem identity, are
repressed (Jackson et al.,
1994
; Long and Barton,
2000
). This indicates that the I1 cells are committed
to organogenesis. Gene expression analysis suggests that I2, and
perhaps even incipient primordia as early as I3 or I4,
is distinct from the surrounding cells by the expression of organ marker genes
(Otsuga et al., 2001
).
However, the Snow experiments have shown that the position of I2
can be changed by the isolation of P1, and therefore these cells
are not determined (even though initial steps of commitment may have been
taken). Surprisingly, when I1 was isolated by similar tangential
incisions, the position of I2 was not affected, while I3
was displaced. So, if I2 is not determined (concluded from
P1 isolation), why did it not change its position after
I1 isolation?
Taken together, the experiments of the Snows supported a negative influence
of pre-existing primordia on I1, but they did not evaluate the
relative influence of the different pre-existing primordia on I1,
and they opened the question of whether P1 affects the positioning
of I1 at all. To address these issues, we decided to reassess the
experiments of the Snows using the tomato in vitro meristem culture system
(Reinhardt et al., 2003b). In
a first set of experiments we repeated the original experiments. Our results
are in line with the old data, but they also uncover an effect on elongation
growth that may require a more cautious interpretation. Therefore, we used
infrared laser technology to precisely ablate incipient primordia and to
reduce the experimental interference to a minimum. Finally, we isolated
meristems from the influence of all primordia but P1, in order to
assess the influence of older primordia on phyllotaxis.
While the young primordia influence organ positioning in the meristem, the
meristem in its turn influences the development of organ primordia after their
initiation. For example, the dorsoventral patterning of the leaves, that is
the formation of different upper and lower leaf tissues, depends on the
activity of the meristem. This was first demonstrated by the finding that the
young or incipient leaf primordia of potato developed as radially symmetric
finger-like structures when they were surgically separated from the meristem
(Sussex, 1951;
Sussex, 1955
). However, these
experiments were controversial at the time
(Snow and Snow, 1954a
;
Snow and Snow, 1954b
;
Sussex, 1954
), and have not
been repeated since, neither in potato nor in other species.
Genetic analysis has identified several putative transcription factors that
are required for the specification and development of the upper (adaxial) and
lower (abaxial) leaf surface (Bowman et
al., 2002). Recent evidence indicates that microRNAs (miRNAs) act
in the abaxial domain of the leaf primordia by silencing adaxializing
transcription factors (Emery et al.,
2003
; Juarez et al.,
2004
; Kidner and Martienssen,
2004
). By contrast, the proposed meristem-borne signal that
instructs adaxial cells to adopt their correct identity remains elusive. Here,
we report on new evidence supporting a function of the meristem in the
specification of the adaxial leaf domain, and we explore the role of the
L1 layer in this process.
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Materials and methods |
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Microsurgery and laser ablations
Separation of the site of incipient primordium formation from the remainder
of the meristem was carried out with small pointed scalpel blades
(Bard-Parker® #11, Becton Dickinson, New Jersey, USA). Removal of the
L1 layer was carried out as described
(Reinhardt et al., 2003b).
Superficial ablation of the L1 layer between the site of primodium
formation and the meristem was carried out with drawn glass needles. The
ultimate tip of the needle was removed, and the sharp edge of the remaining
tip was used to superficially scratch the L1 layer. Laser ablation
of the site of incipient leaf formation was performed essentially as described
(Reinhardt et al., 2003b
). A
Q-switched Er:YAG laser emitting infrared radiation at a wavelength of 2.94
µm was used to direct 10 consecutive pulses (2 Hz) of 1.5 mJ per pulse at a
circular area of approximately 40 µm in diameter on the surface of the
meristem.
Microscopy
Scanning electron microscopic analysis and time-lapse photographic analysis
of living tomato apices was carried out as described
(Reinhardt et al., 2003b).
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Results |
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Before we describe the experiments, it is useful to define the nomenclature used in this study. We designate the youngest primordium as P1, and the older primordia as P2-Pn, according to increasing age. Correspondingly, I1 designates the first incipient primordium, and I2-In designates the following primordia in the succession of their appearance. In order to avoid confusion, this nomenclature was applied to the initial situation at the beginning of the experiment (t0) and remained unchanged during the course of the experiment.
First, we repeated the experiment of the Snows. We dissected tomato apices and separated the region of presumptive leaf formation (I1) from the remainder of the meristem shortly before primordium emergence (indicated by the fact that P1 was well developed; Fig. 1A,B). Despite this interference, 89% of the isolated initials grew out at the expected position (16 out of 18) (Fig. 1B-D). We then determined the successive divergence angles between I1 and subsequent primordia (I2, I3, etc.), each at the time of primordium emergence. This immediate determination avoids errors due to later distortions caused by the interaction with neighbouring leaves, or errors due to wound responses (e.g. callus growth).
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The effect on phyllotaxis of removing all primordia but P1
An important observation after isolation of I1, both in this
study as well as in the study of the Snows, was the fact that I2
did not respond to the isolation of I1. Could the position of
I2 have already been fixed? The fact that I2 was
displaced when P1 was isolated
(Snow and Snow, 1931) does not
support this possibility. If the isolation of I1 has no effect on
the position of I2 (although the latter is not fixed), does this
mean that under natural conditions, primordia are not influenced by their next
older predecessor? If this were the case, which would be the primordia that
determine leaf position?
Two principal scenarios could be envisaged: A new primordium (I1) could be influenced by the two previous primordia (P1 and P2), with P1 having the stronger effect so that I1 comes to lay closer to P2. Alternatively, the position could be determined by the two immediate neighbours, which would be P2 and P3 in the case of tomato. In very large meristems, as in the case of the sunflower capitulum, it is likely that the latter mechanisms applies, as the distance between P1 and I1 is very large compared with the distance of P1 to its immediate contact neighbours. By contrast, in small meristems, such as in tomato and most other plants, the meristem appears small enough to permit an influence of P1 on I1.
To distinguish between these two possibilities, we isolated tomato
meristems from the apex in a way that left only one primordium (P1)
attached to them. Although this operation inevitably creates a large wound, it
differs in two important ways from the tangential incisions carried out by the
Snows and in this study. First, the meristem as such is not damaged, as the
cut was made just below the peripheral zone, and secondly, any wound effect
would act equally on the entire circumference of the meristem. Hence, effects
like a shift of the growth axis or distortion of divergence angles are not
expected to occur. Isolated meristems were cultured on MS medium and further
organogenesis was observed. Eighteen of 64 isolated meristems (28%) developed
in culture and continued to form leaf primordia. The first new primordium
(I1) was formed at the expected position
(Fig. 4B, compare with 4A).
However, in many cases the primordia were oversized (n=10;
Fig. 4B,C). This was true
particularly for P1, which in five cases grew approximately to
double width with two tips (Fig.
4D). Such wide and fused primordia resemble the primordia induced
by the ectopic application of IAA to tomato meristems
(Reinhardt et al., 2000). The
next primordium (I2) was formed approximately at the expected
position, thus the direction of phyllotaxis was not reversed
(Fig. 4C). However, its
divergence angle was more variable, conceivably as a consequence of the
changes in size of P1 and I1. Taken together, these
results indicate that P1 and I1 are sufficient to
determine the approximate region of I2 (and therefore to propagate
the generative spiral), whereas the older primordia
(P2-P4), which are the direct neighbours of
P1 and I1 (Fig.
4E), are necessary to determine the boundaries of P1
and I1, and thereby their exact width and position. The large
increase in the width of P1 demonstrates that it was able to
recruit excess cells, even after the onset of outgrowth, if its contact
neighbours P3 and P4 were absent.
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We surgically separated, by tangential incisions, young leaf primordia (or
the site of incipient leaf formation) from the meristem
(Fig. 1A), and followed their
development. However, before reporting on the effect of this operation, it is
useful to give a detailed description of normal leaf development in tomato
(Fig. 5A). The first obvious
sign of dorsoventral patterning of tomato leaf primordia is their curvature
towards the centre of the meristem (e.g. P1 in
Fig. 1A). Later, the primordia
form characteristic trichomes on the adaxial (towards the stem, upper side of
the mature leaf) and abaxial (away from the stem, lower side of the mature
leaf) surface of the primordium. The abaxial side exhibits three types of
trichomes: long linear, short linear, and few globular trichomes, which are
distributed relatively sparsely (Fig.
1I, Fig. 5A). By
contrast, the adaxial side has only globular trichomes, which are concentrated
in a dense stripe along the central axis of the primordium
(Fig. 5A). Thus, adaxial and
abaxial identity can clearly be distinguished by their trichomes. After the
onset of trichome formation, the leaf primordia initiate lateral leaflets in a
basipetal fashion, i.e. new pairs of leaflets are successively formed at the
base of the primordium (Fig.
1I, Fig. 5A)
(Sinha, 1999). The leaflets
grow towards the adaxial side of the primordium, i.e. they point to the
meristem (Fig. 1I, Fig. 5A).
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The defects in dorsoventral patterning could potentially be caused by the
wound itself, rather than by the isolation from a specific patterning signal.
To control for wound effects, we performed incisions of a similar extent
through the centre of the meristem (Fig.
5G; n=12). Such meristems continued to develop and to
form leaf primordia (Fig.
5H-J). The primordia exhibited normal dorsoventral patterns, even
if they were initiated next to the lesion
(Fig. 5J). This was evident by
their characteristic curvature towards the meristem
(Fig. 5J), and later by the
formation of lateral leaflets and normal trichomes (data not shown). As a
result of the incision, the two halves reorganized into two new meristems. The
result of this control experiment shows that wounding is not sufficient to
induce radialized primordia, indicating that the loss of dorsoventral pattern
in surgically isolated primordia is a consequence of the lack of contact with
the meristem, and is not due to wounding. Thus, our results are in agreement
with those of Sussex (Sussex,
1951; Sussex,
1955
), and similarly, we conclude that dorsoventral patterning is
dependent on a signal from the meristem. Taken together, separation of
primordia from the meristem caused two defects: the loss of lateral leaflets
and the loss of dorsoventral patterning.
A role for the meristem L1 layer in dorsoventral patterning of the leaf primordia
The meristem consists of different subdomains, the central zone (CZ) and
the peripheral zone (PZ), and it can be subdivided in three clonally isolated
cell layers, L1, L2 and L3
(Steeves and Sussex, 1989).
Therefore, a certain region of the meristem could potentially play a principal
role in the production or transmission of the adaxializing signal.
Alternatively, the meristem as a whole could produce and release the signal.
Laser ablations of the central zone (CZ) did not affect dorsoventral
patterning in a measurable way (Reinhardt
et al., 2003b
), therefore, the CZ cannot be the exclusive source
of the adaxializing signal.
The L1 layer, which is clonally separated from the subtending
cell layers by its stereotypical, anticlinal cell division pattern, plays
important roles in meristem function and organ development (Baroux, 2001;
Abe et al., 2003; Reinhardt,
2003a; Reinhardt, 2003b). The cells that give rise to the meristem
L1 layer (and the entire epidermis) are set aside early during
embryogenesis at the dermatogen stage
(Jürgens, 2003
). These
cells attain a specific identity that is characterized by the expression of
L1-specific genes, such as AtMERISTEM LAYER1 (AtML1), FIDDLEHEAD
(FDH), PROTODERMAL FACTOR1 (PDF1) and PDF2,
(Lu et al., 1996
;
Yephremov et al., 1999
;
Abe et al., 2001
;
Abe et al., 2003
). We wanted to
assess the role of the L1 layer in dorsoventral patterning of
primordia.
The L1 layer can be surgically removed from the meristem
(Reinhardt et al., 2003b).
Removal of the entire L1 layer results in a gradual degeneration of
basic meristem functions, and in the inhibition of organ formation
(Reinhardt et al., 2003b
). In
order to assess the role of the meristem L1 layer in dorsoventral
patterning of adjacent leaf primordia, we removed the L1 layer to
various extents, while leaving the youngest primordium (P1) intact.
We then followed in detail the consequences for the development of
P1, and for further primordium formation
(Table 1;
Fig. 6). Removal of up to 50%
of the L1 layer had only local effects. P1 developed
normally and leaf formation continued, but only from the area with an intact
L1 layer. The meristem rapidly shifted its centre away from the
wound, and continued to form normal leaf primordia at a normal rate
(Reinhardt et al., 2003b
).
Owing to the ablation and to the shift of the growth centre, the phyllotactic
angles were sometimes irregular (data not shown). In only one case did a
primordium exhibit a dorsoventral patterning defect after removal of less than
25% of the L1 surface (Table
1). When 50-75% of the L1 layer was removed, 36% of the
apices (8 out of 22) terminated after the formation of one new primordium
(I1). In half of these cases (4 out of 8), this primordium was
radially symmetric (Fig. 6B;
Table 1), while P1
developed normally. Removal of 75-100% of the L1 layer resulted in
termination of almost all meristems (26 out of 27;
Table 1). However, 38% of these
apices (10 out of 26) formed a last primordium (I1) before
termination, which was, in 50% of the cases, radially symmetric (5 out of 10).
Of the P1 primordia, only 11% (3 out of 27) developed as partially
or entirely radialized primordia (Fig.
6C-E; Table 1), whereas the others developed normally (Fig.
6F).
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It could be argued that the loss of dorsoventrality after L1
ablations was simply due to the degeneration of the meristem, and that it does
not suggest a specific role of the L1 layer. However, the similar
range of patterning defects (compare Figs
5,
6), as well as the comparable
frequences of dorsoventral defects between incised and L1-ablated
meristems (Table 1), indicates
that the L1 ablations had an effect equivalent to the surgical
separation, which is likely to be effective immediately. By contrast, the
degeneration of the meristem after L1 ablations proceeded slowly
over several days (Reinhardt et al.,
2003b). These results are compatible with a direct role of the
L1 layer in the establishment and maintenance of the dorsoventral
pattern in leaves. For example, a signal for dorsoventral patterning could be
produced and transported to the developing primordia through the L1
layer.
To test this possibility more directly, we performed narrow, superficial ablations of the L1 layer that left the majority of the L1 layer intact, but separated the L1 layer of I1 or P1 from the L1 layer of the remainder of the meristem by a small corridor (further referred to as corridor ablations; Fig. 7A,B). This operation was expected to have no consequences for meristem function and maintenance, but it would interrupt the adaxializing signal, if it were transported through the L1 layer. We operated 30 apices shortly before (I1), and 10 shortly after, primordium initiation (P1). Four apices operated at P1 (40%) showed varying degrees of dorsoventral patterning defects (Fig. 7C-F). Of the apices operated at I1, 13% showed partial defects in dorsoventral pattern (similar to Fig. 6D,E), and one was completely radialized (Fig. 7G). Because, unexpectedly, the percentage of effects was lower when the corridor ablation was performed at the I1 stage than at the P1 stage, we repeated the experiment with 40 apices from which 20 were operated at the I1 stage and 20 at the P1 stage. To assess the extent of the defects more precisely, we distinguished between partially radialized primordia, which were dorsoventral in their distal portion and radialized in their proximal portion, and fully radialized primordia, which failed to exhibit any sign of dorsoventral patterning and had only abaxial trichomes (Table 2; Fig. 7). Again, primordia operated at the I1 stage showed defects at a lower rate and of weaker severity (35% partially, 5% completely radialized; Fig. 7H), compared with primordia operated at the P1 stage, where 40% were fully, and a further 15% partially, radialized (Table 2; Fig. 7). Interestingly, more than half of the apices operated at I1 initiated an accessory meristem above the primordium (11 of 20; Fig. 7I). In such cases, the primordium was frequently normal (9 cases), and in only two cases was it partially radialized. Such an accessory meristem was only formed once in apices operated at the P1 stage. It thus appears likely that the accessory meristems formed after operations at I1 provided the dorsoventral signal, thereby lowering the frequency of radialized primordia. In conclusion, the fact that corridor ablations provoked similar defects at comparable frequencies to deep tangential incisions or complete L1 ablations demonstrates that the continuity of the L1 layer is essential for the establishment of the adaxial domain of leaf primordia.
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Discussion |
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One general limitation of the Snows' study was that they analyzed only the final outcome of the experiments in transverse sections of the shoot prepared 3-5 weeks after the operation. Hence, the divergence angles were measured several plastochrons after the initiation of the respective primordia. Therefore, some of the reported effects on phyllotaxis may have been influenced by wound reactions. We have detected, within 4 days after the operation, shifts of the meristem centre away from the wound, and this response clearly affected apparent divergence angles post-meristematically (Fig. 1B,D). To record the changes in divergence angles with confidence, we followed in our experiments the divergence angles starting immediately after the operation, thus, secondary distortions of the divergence angles can be ruled out.
Concerning the effects on divergence angles, we obtained results that are
in agreement with the findings of the Snows. However, in addition to changes
in divergence angles, we found major changes in the vertical growth
(Fig. 1 and
Fig. 2). The Snows analyzed the
result of incisions at a time at which even I5 and I6
had developed into leaves with a well-developed leaf blade
(Snow and Snow, 1931). Hence,
initial changes in vertical growth, such as the ones reported here, may well
have occurred in Lupinus albus as well, but could have been obscured
at the time of analysis.
Upon isolation of I1, both in the experiments of the Snows and
in ours, the first angle was normal and only the second primordium was
displaced. One possible explanation is that the position of I2 is
already fixed. This, however, is ruled out by the P1 isolations,
which clearly demonstrate that I2 is not fixed, because it was
displaced in this case (Snow and Snow,
1931). Another possibility is that the inflexibility of
I2 after I1 isolation is related to wound effects that
influence the response of the meristem. The incision causes major damage to
the peripheral zone and thus restricts the space available for a new
primordium. In contrast to the tangential incisions, the laser ablations
deleted I1 with a minimum of tissue damage to the meristem.
Importantly, increased vertical growth was not observed. Thus, laser ablation
did not affect the geometry and organisation of the meristem, and it allowed
us to assess, more directly, the question of how spiral phyllotaxis responds
to the specific elimination of a primordium.
After laser ablation of I1 the next primordium
(I1*) was formed ectopically, either in direct proximity
of the lesion (50%) or at a new position between the lesion and I2
(Fig. 3). Hence, under these
conditions, the position of the first primordium forming after the lesion was
not fixed but displayed remarkable flexibility. Such changes in organ position
are in line with theoretical predictions that elimination of a primordium
should affect further organ formation immediately after ablation. Furthermore,
the tendency of I1* to be formed at a position close to
the original I1 position indicates that I1 normally
suppresses organogenesis in its vicinity, similar to P1
(Reinhardt et al., 2000).
After initial deviations caused by I1 ablations, phyllotactic
patterning rapidly returned to normal spiral phyllotaxis, either in the
original, or in the reverse direction. This demonstrates the strong tendency
to revert to spiral phyllotaxis in the case of disturbance, and underscores
the stability of the spiral pattern, once the disturbance is overcome.
The contribution of the different primordia to organ positioning
Theoretical consideration, as well as experimental evidence, suggests that
the positioning of new primordia is influenced by pre-existing primordia (see
above). However, it is not known to what extent the different primordia
contribute to this mechanism. It seems likely that the influence of a
primordium decreases with its distance to the site of incipient organ
formation (I1). In a large meristem, such as the capitulum of the
sunflower, where organ formation proceeds from the edge towards the centre,
the closest neighbours of a new primordium are not the predecessors in the
generative spiral (P1 and P2), but the direct contact
neighbours. If the capitulum exhibits a 34:55 phyllotactic system, the direct
neighbours are P34 and P55, which are 34 and 55
plastochrons older than I1, respectively. By comparison,
P1 and P2 of such a system are positioned much more
remotely. Therefore, it is likely that I1 is positioned according
to P34 and P55, rather than according to P1
and P2. However, in a phyllotactic system, as in tomato and many
other plants, the meristem is small enough that not only the direct neighbours
(in tomato which exhibits a 2:3 phyllotaxis, this is P2 and
P3), but also P1 could influence the positioning of a
new organ.
In the experiments of the Snows, the first primordium to arise after
operations was not affected. Does this mean that the youngest primordium has
no effect on the positioning of the following one? We have previously proposed
that the two youngest primordia influence the positioning of the following
primordium, with P1 having the stronger effect, so that
I1 comes to lay closer to P2 than P1, thus
resulting in the characteristic divergence angle of 137°
(Reinhardt and Kuhlemeier,
2002). In agreement with this idea, the youngest primordia express
auxin transport proteins in a way that suggests a strong sink function for
auxin (Reinhardt et al.,
2003a
). To distinguish between the influence of the direct
predecessors and that of the direct contact neighbours on organ positioning,
we isolated meristems from all influence but that of P1. The
assumption was that I1, whose position is likely to be determined,
would be formed at its normal position. However, I2 could
potentially respond to the experimental isolation with a shift. If it was
positioned ectopically, this would indicate that the direct neighbours
(P1 and P2) are important for organ positioning. If
however the positioning of I2 were not affected, then the
interpretation would be that the youngest primordia (I1 and
P1) are sufficient for leaf positioning. Taken together, our
results suggest a compromise of the two possibilities. The generative spiral
did not reverse in isolated meristems, indicating that I2 was
always formed in the correct direction. This finding is in accordance with
surgical experiments in which isolated meristems of Primula formed
leaves in continuity with the original phyllotactic pattern
(Wardlaw, 1950
), whereas
similar isolations in Lupinus interrupted the original spiral, and
led to the establishment of a new spiral system
(Ball, 1952
).
After isolation of the meristem, P1 and I1 grew considerably wider than normal (Fig. 4), indicating that in the absence of older primordia, the primordia could recruit more cells than normal. Hence, the following picture emerges for phyllotaxis under natural conditions. Because I1 appears to be fixed, it is at the I2 stage that organ position becomes determined. We propose that I1 and P1 determine the approximate location of I2, thereby dictating the direction of the generative spiral. After the approximate positioning in the meristem, the direct neighbours (P1 and P2) delimit its exact boundaries, hence determining its final size and its precise radial position.
The fact that P1 grew wider in the absence of its contact neighbours indicates that lateral restriction (and fine positioning) is a prolonged process that continues after the initiation of a primordium. Although it may seem counterintuitive at first, the conclusion therefore is that organ positioning and organ outgrowth occur concomitantly, and not sequentially. Such a mechanism would allow for the feedback mechanism that is predicted to operate in phyllotaxis and other models of pattern generation in living organisms (Meinhardt, 1994).
Control of leaflet formation and dorsoventral patterning in tomato leaves
The surgical separation of tomato leaf primordia from the meristem caused
two defects. The loss of lateral leaflets and the loss of dorsoventral
patterning. This raises the question whether the two processes are linked. For
example, it is conceivable that leaflets, like the leaf lamina, can be formed
only where the adaxial (dorsal) and the abaxial (ventral) domain of the
primordium are juxtaposed. This view is compatible with the occurrence of
single leaflets in a central position on the adaxial side of the rachis,
instead of in a lateral position (Fig.
5C, Fig. 6E). A
similar case is represented by the variably sized distal patches of adaxial
identity found on primordia of phantastica mutants in
Antirrhinum. In this case, the lamina of the distal leaf portion is
joined on the adaxial side of the primordium, presumably marking the course of
the adaxial/abaxial boundary (Waites and
Hudson, 1995). This position corresponds to the position of the
single central leaflets (Fig.
5C, Fig. 6E). It is
therefore likely that in such cases, the central leaflet marks the boundary
between the extended abaxial domain, and the reduced adaxial domain in the
distal portion of the primordium. Taken together, these data indicate that
lateral leaflet formation, like lamina outgrowth, occurs only at the boundary
between adaxial and abaxial domains.
Dorsoventral patterning of leaves is thought to be influenced by the
meristem (Bowman et al., 2002).
Evidence for this notion came from early microsurgical analysis in potato
(Sussex, 1951
;
Sussex, 1955
). These
experiments were challenged in the following years, and had not been repeated
in other plant species since. We confirm in the present study, using three
different microsurgical techniques (vertical incision, complete L1
ablation, corridor ablation), that the meristem provides information that is
required for the dorsoventral patterning of the primordia (Figs
5,
6,
7). It has been proposed that a
factor from the meristem induces adaxial identity in the upper part of leaf
primordia, while the lower part adopts abaxial fate by default
(Bowman et al., 2002
). However,
despite the identification of a number of putative transcription factors,
which are required for the establishment of adaxial and abaxial identities
(Bowman et al., 2002
), the
nature of the adaxializing signal remains elusive. Recently, miRNAs have been
implicated in the control of abaxial identity. These are expressed just below
incipient primordia (i.e. on their abaxial side), and in the abaxial side of
the leaves. There, they determine abaxial cell fate by downregulating the
levels of adaxializing proteins, such as PHABULOSA and ROLLED LEAF1 on the
abaxial side of the primordia (Kidner and
Martienssen, 2004
; Juarez et
al., 2004
). Thus, miRNA may represent an abaxializing signal that,
in concert with the adaxializing signal from the meristem, establishes
dorsoventral polarity.
We have previously shown that surgical removal of the L1 layer
from the meristem leads to a progressive degeneration of the meristem
(Reinhardt et al., 2003b).
However, removal of L1 also abolished dorsoventral polarity of the
last one or two primordia before the meristem arrested
(Fig. 6). This radialization
was observed at a similar extent and frequency to in the case of surgical
separation from the meristem (50% versus 65%; compare with
Fig. 5), indicating that
removal of L1 is equivalent to an immediate interruption of the
adaxializing signal. This is in contrast to meristem degeneration, which
proceeded slowly over several days
(Reinhardt et al., 2003b
).
Hence, the loss of dorsoventrality after L1 ablation preceded the
loss of meristem identity in the remaining tissue, rendering it improbable
that the loss of dorsoventrality is due, indirectly, to the degeneration of
the meristem. This evidence suggests a special role for the L1
layer in the determination of dorsoventral polarity. For example, the
adaxializing signal could be transported through the L1 layer to
the young primordia to induce adaxial fate in the adjacent portion of the
primordium. Indeed, our data show that the continuity of the L1
layer between the meristem and the site of primordium formation is relevant
for dorsoventral patterning, as corridor ablation caused defects of a similar
extent and at similar rates to the vertical incisions. Nevertheless, it
remains to be clarified whether the L2 and L3 layer also
contribute, directly or indirectly, to dorsoventral patterning.
It is noteworthy that in all experiments only a minority of the primordia
were completely radialized. In most cases, the young primordia had a
dorsoventral distal portion, and a proximal radialized portion of different
extent. Often, this partial loss of dorsoventral pattern was acccompanied by a
partial or complete loss of lateral leaflets. In tomato, the leaf primordia
develop in a basipetal fashion, i.e. new leaflets are successively formed at
the leaf base (Sinha, 1999).
Therefore, the partially radialized primordia consist of an older distal
portion with normal dorsoventrality, and a younger proximal portion that has
lost its dorsoventral pattern during development. The range of dorsoventral
defects observed included all possible intermediates between completely normal
and completely radialized. This indicates that the adaxializing signal needs
to be present during an extended period of leaf development, and that a loss
of the signal during this process can abolish the dorsoventral pattern in the
proximal parts at various stages of development. Thus, the establishment of
dorsoventral polarity appears to be a continuous process. Such a scenario is
compatible with the genetic models for dorsoventral polarization, which
envisage a self-reinforcing mechanism based on the mutual inhibition of
adaxial and abaxial determinants that leads to the gradual separation of the
domains with adaxial and abaxial identity
(Bowman et al., 2002
).
Furthermore, the frequent occurrence of partially radialized primordia with a
normal distal portion shows that radializations are not due to the destruction
of the (predetermined) adaxial domain of the I1 position. Partially
radialized primordia always exhibit a normal distal portion. As this is the
part of the leaf that is formed first, the primordia must have started with an
intact adaxial domain, which later lost its adaxial identity during the course
of leaf development.
This work and our previous study
(Reinhardt et al., 2003b)
demonstrate that microsurgical techniques continue to be useful tools for
studying plant development. They complement genetic analyses, and they are
particularly useful in cases where it is desirable to restrict functional
interference tightly in space and time. Now the challenge will be to develop
new genetically based tools to elucidate the mechanisms underlying phyllotaxis
and dorsoventral patterning in more detail. Such tools will be developed when
we know more about the nature of the components in the signal chains. For
example, once we know the nature of the adaxializing signal, its production,
transport or destruction could be influenced in a tissue-specific manner.
Similarly, the tissue-specific expression in subdomains of the meristem of
genes involved in auxin biosynthesis, metabolism, transport and perception
will allow us to rigorously test models of phyllotaxis.
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ACKNOWLEDGMENTS |
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Footnotes |
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