University of Toronto, Department of Botany, 25 Willcocks Street, Toronto, Ontario M5S 3B2, Canada
* Author for correspondence (e-mail: berleth{at}botany.utoronto.ca)
Accepted 18 March 2004
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Arabidopsis thaliana, Procambium, Leaf development, Vascular patterning
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In leaves, all vascular cells differentiate from a vascular meristematic
tissue, the procambium. Procambial cells become apparent as narrow,
cytoplasm-dense cells emerging from the subepidermal tissue of the leaf
primordium [ground meristem; GM (Foster,
1952; Esau,
1965
)]. Procambial cells are characteristically arranged in
continuous strands and acquire their narrow shape through coordinated,
oriented divisions, parallel to the axis of the emerging strand. The
mechanisms by which procambial cells are selected from the GM cell population
in the young leaf primordium is not known, but the high degree of plasticity
of the patterning process in most species suggests that positional cues
influenced by growth conditions are of critical importance. The molecular
details of positional specification of procambial cell fate are particularly
intriguing since the underlying mechanism must reconcile the plasticity of
vein patterns with the stringent requirement to keep all parts of the vascular
system interconnected (Berleth et al.,
2000
).
Alternative models to explain vascular tissue patterning have been derived
from the variability in natural vascular patterning, responses of vascular
patterns to experimental interference and from the phenotypes of vascular
mutants (reviewed by Nelson and Dengler,
1997; Turner and Sieburth,
2002
). These mutants have provided evidence for the involvement of
sterols (Szekeres et al.,
1996
; Choe et al.,
1999
; Jang et al.,
2000
; Carland et al.,
2002
; Souter et al.,
2002
; Willelmsen et al., 2003), small peptides
(Casson et al., 2002
),
cytokinin (Mähönen et al.,
2000
; Inoue et al.,
2001
) and auxin (Hardtke and
Berleth, 1998
; Hamann et al.,
2002
; Hellmann et al.,
2003
) in vascular patterning, but the number of vascular pattern
mutants is surprisingly small, which may in part be due to the scarcity of
criteria by which abnormalities in vascular mutants can be discerned.
Mutant screens and the molecular genetic analysis of cell patterning
processes are highly dependent upon the swift and precise recognition of cell
types and differentiation stages, referred to as cell states. While this is
usually unproblematic for anatomically conspicuous cells in exposed positions,
genetic dissection of cell identity acquisition in internal tissues may only
become feasible if distinct cell states are specifically visualized through
gene expression markers. Marking individual cell states along vascular
differentiation pathways should in principle be an easy task, as gene
expression profiles associated with specific differentiation states have been
identified (Ye, 2000; Turner and Sieburth,
2002). However, most of these gene expression profiles are
associated with the terminal differentiation of various vascular cell types
and very few are available to mark early stages prior to procambium
formation.
In this study we have searched for reporter gene expression profiles suitable for high-resolution analyses of preprocambial patterning and procambium formation in young leaf primordia. In particular, we were interested in assigning these markers to specific preprocambial and procambial stages, and in visualizing very early stages of vein formation in different vein orders. Four marker gene expression profiles indicate striking similarities in the ontogeny of veins of different orders. When visualized at an early preprocambial stage, all veins seem to be generated as parts of a common reiterative branching pattern, which becomes terminated by negative interference with mesophyll differentiation.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Because of the different meanings associated with the term `provascular' in
the literature (Esau, 1965;
Steeves and Sussex, 1989
;
Clay and Nelson, 2002
;
Holding and Springer, 2002
),
we have decided to exclusively use the terms `procambial' and `procambium' in
this manuscript to refer to anatomically identifiable precursor cells of
vascular tissues (illustrated in Fig.
2A-H). `Ground meristem' (GM) refers to all immature subepidermal
tissue in the leaf (Foster,
1952
).
|
Microtechniques and microscopy
For histochemical detection of ß-glucuronidase (GUS) activity, whole
seedlings or detached leaves were permeabilized in cold (20°C) 90%
(v/v) acetone for 1 hour at 20°C
(Hemerly et al., 1993), washed
twice for 5 minutes with 100 mM phosphate buffer pH 7.7, and incubated in the
dark at 37°C in freshly prepared 100 mM sodium phosphate buffer pH 7.7, 10
mM sodium EDTA, 2 mM 5-bromo-4-chloro-3-indolyl-ß-D-glucuronic acid
(X-gluc; BioShop Canada Inc., Burlington, ON, Canada). To facilitate the
uptake of the X-gluc substrate, acetone treatment is indispensable, but should
be limited to 1 hour at 20°C to reduce structural damage.
Best preservation of anatomical structures is achieved by limiting the GUS
staining reaction to less than 2 hours. The incubation buffer was supplemented
with either 1 (GT5211, ET1335 and 553-643) or 5 (Athb8-GUS) mM of both
potassium ferrocyanide and potassium ferricyanide. Higher concentrations
accelerate the oxidative dimerization of the reaction intermediate into the
blue insoluble product and thereby reduce diffusion of the intermediate at the
expense of sensitivity (Lojda,
1970). The indicated molarities account for the differential
expression levels of the reporter genes and ensured that each was easily
detectable after 2 hours of incubation.
Under these conditions, we have not observed diffusion of the intermediate, which typically results in a gradient of blue staining with a maximum towards a neighboring strongly stained cell, as opposed to uniform staining at all intensity levels resulting from genuine GUS expression.
The enzymatic reaction was terminated by fixing in ethanol:acetic acid (3:1, v/v) for 1-16 hours at room temperature, depending on the age of the seedlings. Samples were stored in 70% (v/v) ethanol at 4°C. For visualization of GUS activity in leaves older than 10 DAI, the enzymatic reaction was extended to 16 hours and the concentrations of both ferrocyanide and ferricyanide salts were increased to 10 mM (Athb8-GUS), 5 mM (553-643) or 2 mM (ET1335 or GT5211). For microscopy, rehydrated samples were dissected under water, mounted abaxial side up in chloral hydrate:glycerol:water (8:3:1, w/v/v) and viewed under differential interference contrast (DIC) optics or dark-field illumination (Olympus AX70 microscope, Olympus Optical Co., Tokyo, Japan). Images were acquired with a Fujifilm FinePix S1 Pro digital camera (Fuji Photo Film Co., Tokyo, Japan).
For confocal laser scanning (CLS) microscopy, LTI6b::YFP seedlings [line
29-1 (Cutler et al., 2000)]
were mounted in water and observed with a Zeiss Axiovert 100M microscope (Carl
Zeiss, Oberkochen, Germany) equipped with a Zeiss LCM510 laser module confocal
unit (Carl Zeiss, Oberkochen, Germany). LTI6b::YFP signal was visualized using
the 488 nm line of an argon laser and a BP 505-530 filter at the
photomultiplier. Chloroplast autofluorescence was detected with the 543 nm
line of a helium-neon laser and a LP 585 filter at the photomultiplier.
Optical slices (<1.6 µm) were obtained with a pinhole diameter of 129
µm (2.00 Airy units).
Images were assembled using Adobe Photoshop 7.0 (Adobe Systems, Mountain View, CA, USA), and figures were labeled using Canvas 8 (ACD Systems Ltd, Saanichton, BC, Canada).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
First leaf development and ET1335-GUS expression
The development of Arabidopsis leaves has previously been
described (Telfer and Poethig,
1994; Kinsman and Pyke,
1998
; Candela et al.,
1999
; Donnelly et al.,
1999
; Mattsson et al., 1999;
Kang and Dengler, 2002
;
Mattsson et al., 2003
). The
schemes in Fig. 1 summarize the
temporal sequence of vascular development events in first rosette leaf
primordia to which we refer in Figs
2,
3,
4 and in the Figs S1 to S3,
http://dev.biologists.org/supplemental/.
The often oblique course of procambial strands was followed through various
focal planes under DIC optics. Only one focal plane can be shown in Figs
2,
3,
4 and in Figs S1-S3, so that
not all cellular features present are visible within a given marker gene
expression domain. At all stages, the onset of ET1335-GUS expression coincided
precisely with the onset of procambium formation
(Fig. 2), except that
ET1335-GUS was also transiently expressed in a larger domain at the tip of
3.5-5.5 DAG primordia (Fig.
3D-G). Because a gene expression marker that accurately monitored
procambium formation would be very helpful, we rigorously reassessed the
congruence of ET1335-GUS expression with procambium initiation at various
stages and found it confirmed (Fig.
2). The following description therefore refers to the emergence of
both patterns.
At 2 DAG, the first two vegetative leaf primordia become recognizable as semispherical protrusions flanking the shoot apical meristem (Fig. 1A, Fig. 2A). At 2.5 DAG, leaf primordia begin to elongate along the proximodistal axis (Fig. 1B, Fig. 2B). Elongated cells of the 1st order procambial strand and expression of ET1335-GUS become visible in the further elongated primordia at 3 DAG (Fig. 1C, Fig. 2A,B, Fig. 3C). At 4 DAG, lamina formation is initiated (Fig. 1D, Fig. 2E), followed by the formation of the first loops of 2nd order procambial strands at 5 DAG (Fig. 1E, Fig. 2C,D, Fig. 3F). At 5.5 DAG (Fig. 1F, Fig. 2E,F, Fig. 3G) the second loops and at 8 DAG (Fig. 1J, Fig. 2J) the third loops of 2nd order procambial strands appear. At 6.5, 7 and 8 DAG, the 3rd order procambial strands appear in the first, second and third intercostal area (IA), respectively (Fig. 1H-J, Fig. 2G,H, Fig. 3H-J). Differentiation of xylem reaches completion in the 1st order vein at 6.5 DAG (Fig. 1H) and in the first, second and third loops of 2nd order veins at 7 (Fig. 1I), 8 (Fig. 1J, Fig. 2K) and 10 DAG (data not shown), respectively. Xylem in 3rd order veins is initiated in the first IA at 7 DAG and then progresses towards higher-order veins as well as in more proximal IAs (Fig. 1I,J) until at 14 DAG xylem is continuous in veins of all orders (Fig. 1K). ET1335-GUS expression was initiated precisely with overt procambium formation but remained strong in differentiating vascular strands and was still visible even in the fully extended leaf at 14 DAG (Fig. 3L).
Expression of Athb8-GUS
Extremely weak Athb8-GUS expression was detected in the innermost GM cells
of the 2 DAG primordium (Fig.
4A). At 2.5 DAG, Athb8-GUS expression was considerably stronger
and now visible in a central file of GM cells
(Fig. 4B,C). At 3 DAG,
Athb8-GUS was strongly expressed in the 1st order procambial strand and weakly
in GM cells at the tip of the primordium
(Fig. 4D). In slightly older
but still cylindrical primordia (3.25 DAG), Athb8-GUS expression was
reproducibly detected in single cells at the side of the 1st order procambial
strand (arrow in Fig. 4E). With
the beginning of lamina growth at 3.5 DAG, Athb8-GUS marked reproducibly a
looped 2nd order preprocambial domain extending from proximal to distal
(Fig. 4F). Expression of
Athb8-GUS in the two loops of the first pair of 2nd order veins was
consecutive rather than simultaneous (Fig.
4G), but both loops were complete by 4.25 DAG
(Fig. 4H). Simultaneously with
the completion of the first loop expression domain, Athb8-GUS expression
marking the incipient second loops of 2nd order procambial strands became
recognizable. As for the first loops, expression was initiated proximally in a
domain extending from the 1st order procambial strand
(Fig. 4I). From there,
expression progressed distally to form a loop that eventually joined the first
loop of 2nd order procambial strands in 4.75 and 5 DAG primordia
(Fig. 4J-L). Athb8-GUS
expression in the third loops of 2nd order vein loops followed the same
temporal pattern as described for the first and second loops (not shown). The
earliest 3rd order Athb8-GUS expression domains became visible in the first
IAs in a significant portion (23/50) of 5 DAG primordia and were visible in
this position in all primordia by 6 DAG
(Fig. 4M,N). Formation of 3rd
order Athb8-GUS expression domains in the second IAs followed about 1 day
later (Fig. 4O,P). Immediately
after their initiation, nearly all higher-order Athb8-GUS expression domains
ended blindly (Fig. 4M-P),
suggesting a polar progression of Athb8-GUS expression domains also in the
formation of higher-order veins (see below).
In summary, Athb8-GUS presages procambium formation with remarkable
accuracy, except for a domain of transient expression at the tips of 3-5 DAG
primordia. Expression of Athb8-GUS marks earlier preprocambial stages than any
of the other markers in this study. Most conspicuously, Athb8-GUS expression
progresses in a polar fashion from initiation points connected to pre-existing
vasculature (see below and Discussion). Consistent with the auxin inducibility
of the Athb8 gene (Baima et al., 1995;
Mattsson et al., 2003),
Athb8-GUS expression domains are spatially correlated with the expression
domains of the DR5-GUS auxin response marker in early leaf primordia (Fig.
S3O-R,
http://dev.biologists.org/supplemental/).
In contrast to Athb8-GUS, however, the temporal expression profile of DR5-GUS
is somewhat variable and the expression has been shown to be heterogeneous
along individual veins (Mattsson et al.,
2003
). This marker is therefore unlikely to be correlated with a
specific preprocambial cell state.
Reporter gene expressions in line GT5211 and in line 553-643
Two more of the surveyed twelve lines displayed remarkably invariant
reporter gene expression patterns prior to procambium formation. As summarized
in Fig. 6A, the onset of
expression of both markers, gene-trap marker GT5211 and enhancer-trap marker
553-643, was delayed relative to Athb8-GUS expression, but clearly preceded
the appearance of procambium. Expression of marker GT5211 strongly increased
as cells adopted procambial shape. A detailed description of both expression
profiles is provided in the supplementary material (see Figs S1, S2 at
http://dev.biologists.org/supplemental/).
|
|
|
As shown in Fig. 2C-F and Fig. 3F,G,J, our results confirm earlier reports (Mattsson et al., 1999) that 2nd order vein procambium emerges simultaneously along the entire length of each loop. In higher-order veins, it is impossible to conclude from the expression pattern of a single marker whether the cells in an incipient vein acquire procambial identity simultaneously or progressively, since the final extension of a vein cannot be predicted. To determine the temporal pattern of procambium formation in higher-order veins, we took advantage of the marker Athb8-GUS, which marks the emerging vein at both the preprocambial and the procambial stage. We reasoned that if procambium is formed progressively, we should observe Athb8-GUS expression domains composed of stretches of procambial and GM cells. By contrast, if procambium is formed simultaneously throughout the full length of an Atbh8-GUS expression domain, procambial cells should be either visible along the entire domain or not at all. Among 314 Athb8-GUS domains, we did not find a single domain of composite appearance. We found five ambiguous domains, in which few cells showed aligned division axes, but these cells were still of the same maximum extension as surrounding GM cells.
In summary, the temporal expression patterns of early vascular gene expression markers and of procambium formation suggest a common pattern of differentiation in all vein orders, in which cells in a given vein acquire a preprocambial identity (marked by Athb8-GUS) progressively, while they acquire procambial identity simultaneously or extremely rapidly. The formal similarity of all vein orders and the extension of each vein from a single branch point is reminiscent of a typical reiterative branching pattern as opposed to a closed network pattern that could arise from dispersed differentiation zones.
Manipulation of vein orders
If veins of all orders are initiated as freely ending veins in a
reiterative branching process and connecting veins are the products of later
vein fusions, it might be possible to convert connecting into freely ending
veins and vice versa by manipulating the duration of the reiterative branching
process. Interruption of this reiterative process would result in reduced
numbers or even disappearance of higher-order veins (`venation complexity'
below) and may simultaneously be associated with an increased proportion of
freely ending veins in the remaining orders. We therefore assessed whether we
could change the fate of defined orders of veins.
Differentiation in a leaf primordium proceeds in a distal-to-proximal direction and therefore all processes potentially terminating vein formation are probably not synchronized across the whole leaf. Therefore in order to look at a nearly homogenous group of veins, in each assay we monitored higher-order veins within a particular IA only (Table 1). In leaf primordia grown under our standard condition (condition A), less than half of all 4th order veins in the first IA were freely ending (Table 1). By contrast, in leaf primordia grown at a higher temperature (25°C) and under continuous illumination (condition B), 4th order veins in the first IA were less abundant and invariably ended freely (Table 1). Therefore, an incipient 4th order vein is not predisposed to become a freely ending vein, as it might appear in leaves grown under condition B. In the more complex higher-order venation pattern of the second IA, a change of growth condition from A to B is associated with a similar reduction in venation complexity. Veins of the 5th order, which are produced under condition A, are absent under condition B and the propensity of 4th order veins to become connected is significantly reduced (Table 1). Therefore, in both IAs, veins of the 4th order are becoming connected, if reiterative branching is allowed to proceed as under condition A.
The closed lobes of 2nd order veins are hallmarks of the brochidodromous Arabidopsis venation pattern. Each 2nd order vein connects precisely defined pre-existing veins with each other and this regularity lends strong support to a genetic specification of network patterns. We therefore explored the formation of the first loop of 2nd order veins in leaf primordia grown under various conditions. Remarkably, when leaf primordia were grown at 30°C and under continuous light (condition C), a significant portion of 2nd order vein loops were not connected at the distal end and fewer higher-order veins were produced (Table 1). Except for the complexity of their venation patterns, leaves grown under all three conditions were morphologically and developmentally similar (Table S2, http://dev.biologists.org/supplemental/).
We conclude that even in vein orders that normally comprise only one vein type (freely ending or connecting), vein types can be changed if the duration of the reiterative branching process is altered. This plasticity of vascular patterning is consistent with the initiation of all veins as freely ending veins, but inconsistent with a rigid specification of network properties.
Mesophyll differentiation and freely ending vein formation
We considered that if freely ending veins become connected through the
fusion of elongating preprocambial domains, it might be possible to identify
cellular events at the preprocambial stage that promote or prevent the
formation of connecting veins. We began our analysis with higher-order
preprocambial domains that predictably give rise to freely ending veins. For
example, 5th order veins in the second IA grown under condition A will
invariably be freely ending (Table
2). We analyzed the anatomy of the respective IA prior to
procambium formation (6 DAI) and noticed that all Athb8-GUS expression domains
were surrounded by mesophyll cells (Table
2), which were readily recognizable by their round shape, their
separation by intercellular spaces and by the appearance of chloroplasts along
the cell surface (Fig. 5G-I).
The simultaneous appearance of all three features characteristic of mesophyll
cells was confirmed in confocal images of GM and mesophyll cells in a line
carrying the plasma membrane marker LTI6b::YFP (line 29-1)
(Cutler et al., 2000). Round
cells separated by intercellular spaces invariably contained large numbers of
strongly autofluorescent plastids along the plasma membrane
(Fig. 5Q), while polygonal
cells were tightly connected and had no or few very weakly autofluorescent
plastids, undetectable in whole-mount preparations
(Fig. 5P). Under condition B,
4th order veins in the first IA are also invariably freely ending
(Table 2), and again we
observed that all corresponding preprocambial Athb8-GUS expression domains
were surrounded by mesophyll cells (Table
2; Fig. 5A-C). The
4th order veins in the first IA grown under condition A and those in the
second IA grown under condition B are not always freely ending
(Table 2). We found that the
corresponding preprocambial Athb8-GUS expression domains were not always
embedded in mesophyll but could instead be surrounded by polygonal GM cells
(Fig. 5D-F). Most strikingly,
the percentage of Athb8-GUS domains surrounded by mesophyll cells nearly
perfectly matched the percentage of freely ending 4th order veins in mature
leaves under the same conditions (Table
2), strongly suggesting that initiation of mesophyll
differentiation interferes with extension of preprocambial strands.
|
In summary, extremely high correlation between observations at the preprocambial and the mature state of vein formation indicates that preprocambial strand formation becomes terminated by the differentiation of the surrounding GM into mesophyll and that the prevalence of connecting versus freely ending veins in a given vein order depends on the relative timing of preprocambium specification (marked by Athb8-GUS expression) versus mesophyll differentiation. It should be emphasized that it is possible that mesophyll and procambium development influence each other mutually, but the design of our experiments can only trace an influence of mesophyll differentiation on the progression of preprocambial domains: in young leaves (3-6 DAI) the initiation of these domains is invariant and their progression becomes arrested only when they are surrounded by mesophyll cells.
Continuity of vascular expression domains
The above observations indicate that preprocambial strands, which are
initially connected to pre-existing vasculature only at one end, can
nevertheless give rise to veins that are eventually connected to vasculature
at both ends. If fusions of vascular strands occur frequently, procambium
might also be initiated as an island, entirely disconnected from pre-existing
vasculature, to become fused at one or both ends at a later stage. In our
extremely large test sample (more than 700 primordia were investigated for
each of the four gene expression markers), the expression domains of all four
markers were always in continuity with pre-existing vasculature. We conclude
that the propensity of GM cells to adopt preprocambial and procambial cell
fate is very strongly promoted by, if not strictly dependent upon, a direct
connection to pre-existing vasculature (see Discussion).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Early markers of vascular strand formation
Given the cell type complexity of mature vascular tissues and their high
degree of functional specialization, it is not surprising that vascular
tissue-specific gene expression profiles are common and readily observed in
enhancer-trap and gene-trap expression pattern surveys (Sundaresan et al.,
1995; Geisler et al., 2002).
However, many of these profiles do not include the earliest stages of vascular
cell fate specification, and even the few markers that are expressed at early
stages, among them Athb8-GUS (Baima et al., 1995;
Kang and Dengler, 2002
) and
553-643 (van den Berg et al., 1995), have not been characterized relative to
each other or relative to anatomical features of early leaf development. The
four markers presented here were selected from a larger collection (Table S1,
http://dev.biologists.org/supplemental/)
as it became apparent that they could identify consecutive stages preceding or
accompanying procambium formation. In the development of Arabidopsis
leaves, expression of these markers is invariably initiated in the temporal
sequence: Athb8-GUS, 553-643/GT5211 (vein-associated weak), GT5211
(strong)/ET1335 (Fig. 6A), the
latter two markers being expressed simultaneously with procambial cell
features. In large numbers of leaf primordia, all aspects of marker gene
expression, including onset, intensity and decline proved to be highly
reproducible. We therefore suggest that the expression of these markers can be
used to visualize distinct stages of procambial cell fate specification that
cannot be identified by anatomical features.
Each of the four markers has an additional expression domain outside the incipient procambial domain. Three markers, Athb8-GUS, 553-643 and ET1335, are visible in a broad region near the tip of the leaf, where not all marker-expressing cells will become procambial. Marker GT5211 is initially weakly expressed in all GM cells, in addition to its strong procambial expression. Although these features show that the expression of none of these markers is absolutely restricted to vascular cells, this does not diminish their usefulness as indicators of specific vascular cell states. While it will be interesting to understand the molecular basis of the additional expression domains, the markers can already assist in the spatial and temporal dissection of early vascular development.
Polarities in Arabidopsis leaf vein ontogeny
Polarity in vein differentiation has been deduced from the directional
differentiation of xylem (Telfer and
Poethig, 1994; Kinsman and
Pyke, 1998
; Candela et al.,
1999
; Sieburth,
1999
). In this study, markers of preprocambial and procambial cell
states revealed that that the progression of vein differentiation at earlier
stages can be strikingly different. For example, xylem differentiation in 2nd
order vein loops is initiated at the leaf tip, while procambium formation
occurs simultaneously along the entire vein and the still earlier cell fate,
marked by Athb8-GUS, progresses from proximal to distal. This result is
important because the polarity of xylem differentiation has fueled discussions
about the position of signaling sources
(Nelson and Dengler, 1997
).
However, if the polar progression of xylem differentiation is not correlated
with that of earlier markers, it is unlikely that this polarity can provide
clues as to the position of signaling sources defining the routes of vascular
strands.
Expression of Athb8-GUS is the earliest marker in our study and, most interestingly, when judged by this marker, veins of all orders seem to have a common ontogeny. In all vein orders, expression of Athb8-GUS is initiated next to pre-existing vasculature and then progresses polarly away from this point of origin. More importantly, the expression of this marker identifies a critical stage in preprocambial cell specification. Expression of Athb8-GUS and the emergence of mesophyll cells seem to mark two mutually exclusive and typically irreversible stages, one leading to vascular and the other to mature mesophyll formation. Mutual exclusivity is evidenced by the fact that Athb8-GUS was never found expressed in cells of mesophyll appearance. Irreversibility of mesophyll differentiation is indicated by the observation that Athb8-GUS domains enclosed by mesophyll cells would not become elongated to form connecting veins. Finally, irreversibility of Athb8-GUS expression is indicated by the fact that islands of Athb8-GUS expression are not observed at any stage. If mesophyll cells could differentiate within Athb8-GUS expression domains, one would expect multiple interruptions of those domains.
A common ontogeny of veins of different orders is not only suggested by
Athb8-GUS expression, but also by procambium formation (or ET1335-GUS
expression). Continued cell division is a defining characteristic of
procambium. Cell cycling frequencies are initially similar in veins of
different orders and differences between vein orders have been attributed to
differential durations of cell cycling in vein development
(Donnelly et al., 1999;
Kang and Dengler, 2002
).
Furthermore, our data suggest that in all veins, procambium becomes
recognizable along the entire length of a strand very rapidly, once the
Athb8-GUS expression domain has reached its maximum extension, and is then
extended by intercalary growth. Our half-day observation intervals do not
exclude minor deviations from simultaneity, but the dynamics of procambium
formation is in striking contrast to the clearly progressive expansion of the
Athb8-GUS expression domain. The polar progression in the acquisition of a
preprocambial cell identity is the first vascular patterning event we can
monitor and it suggests that the vein network is formed through the fusion of
initially free branches.
Formation of closed vascular networks
Vascular strands have existed since the Silurian period (some 400 million
years ago), but were initially arranged in rather simple parallel patterns
along the main axis of the plant (Stein,
1993). With the evolution of leaves in the Devonian and early
Carboniferous, reiterative bifurcation leading to `open' venation patterns was
present in almost all plants with fern-like (macrophylls) leaves (ferns,
progymnosperms and gymnosperms)
(Roth-Nebelsick et al., 2001
).
According to the fossil record, simple network patterns, involving connected
veins (`closed' networks) (Roth-Nebelsick
et al., 2001
) appeared during the Upper Carboniferous
(Kull, 1999
). These network
patterns evolved repeatedly from open venation patterns in different lineages
(e.g. Wagner, 1979
) and were
initially less complex than angiosperm venation patterns
(Wagner, 1979
;
Kull, 1999
). In angiosperms,
these networks further evolved to produce veins of distinct hierarchical
orders. Evolutionary history therefore suggests that closed venation patterns
involving connecting veins evolved repeatedly from open venation patterns.
How vascular networks can be generated has been subject to some discussion. Given the high degree of complexity and reproducibility of vein patterns in many plant species, the existence of detailed genetic programs specifying the precise organization of individual vein orders seems plausible. However, at least for the brochidodromous venation pattern of the Arabidopsis leaf, several of our observations provide support for a reiterative, largely self-organizing mechanism underlying the formation of veins of all orders. First, in all vein orders, vein formation is initiated by the polar progression of a preprocambial cell state. Second, veins in all orders are initially freely ending and (except for the 1st order vein) will go on towards becoming connecting veins if network elaboration in higher vein orders is allowed to continue. Third, the number of vein orders in the Arabidopsis leaf is not genetically defined. Fourth, the developmental event terminating reiterative vein initiation and preprocambial vein elongation, the onset of mesophyll differentiation, occurs outside of the vascular system and is apparently not an inherent part of a vascular patterning program (Table 2; Fig. 6B). We therefore postulate that veins of all orders in the Arabidopsis leaf are initiated through the reiteration of a common branching program rather than through a genetically specified network plan.
Two alternative self-organizing mechanisms are often discussed (see
Nelson and Dengler, 1997). One
involves an apical-basal signal flow that becomes gradually restricted to
vascular differentiation domains (Sachs,
1991
; Sachs,
1981
). The other postulates that vascular strand patterns are
specified locally, for example through autocatalytic,
local-activation-long-range-inhibition mechanisms
(Meinhardt, 1982
; Meinhardt
and Gierer,
http://www.eb.tuebingen.mpg.de/dept4/meinhardt/theory.html).
Both concepts postulate a single self-organizing principle underlying the
formation of all vein orders and are therefore compatible with a reiterative
mode of vein initiation.
One crucial formal distinction between the two self-organizing models is
that apical-basal signal flow involves signaling throughout the entire plant
and thereby postulates that all vascular strands are connected from early on,
while other types of self-organizing processes could generate transiently or
permanently isolated vascular strands. Mutants in support of either of these
models have been identified (see Turner
and Sieburth, 2002). One group of mutants establishes the
connection to apical-basal auxin signaling both phenotypically and
biochemically (Hardtke and Berleth,
1998
; Hamann et al.,
2002
; Hellmann et al.,
2003
). Another group highlights the formation of isolated
procambial islands, thereby questioning the role of continuous apical-basal
signaling in procambium formation (Carland
et al., 1999
; Deyholos et al.,
2000
; Koizumi et al.,
2000
). While the above mutant phenotypes help to explore the
question of whether signaling through a procambial strand is critical in
vascular patterning, our results suggest a common mechanism underlying the
formation of all vein orders without specifically supporting one of the
alternative models. Invariant continuity of expression domains of all four
markers in extremely large numbers of leaf primordia can be considered as
circumstantial support for signaling through emerging veins, but also this
observation does not exclude the existence of isolated procambial strands in
mutant backgrounds, which have been described in Arabidopsis
(Carland et al., 1999
;
Deyholos et al., 2000
;
Koizumi et al., 2000
).
The observed interference between mesophyll and vascular cell specification
introduces an additional patterning influence and therefore has important
implications for the interpretation of venation patterns in accessions or
mutants of Arabidopsis or in other species. For example a surprising
portion of mutant venation patterns in Arabidopsis is characterized
by failure of secondary veins to form closed loops at the leaf tip
(Przemeck et al., 1996;
Carland et al., 1999
;
Koizumi et al., 2000
;
Steynen and Schultz, 2003
).
Our results suggest that because of early mesophyll differentiation in this
position, any delay of vascular differentiation and/or premature mesophyll
differentiation is likely to result in this phenotype, which may explain its
frequent occurrence.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Berleth, T., Mattsson, J. and Hardtke, C. S. (2000). Vascular continuity and auxin signals. Trends Plant Sci. 5,387 -393.[CrossRef][Medline]
Candela, H., Martinez-Laborda, A. and Micol, J. L. (1999). Venation pattern formation in Arabidopsis thaliana vegetative leaves. Dev. Biol. 205,205 -216.[CrossRef][Medline]
Carland, F. M., Fujioka, S., Takatsuto, S., Yoshida, S. and
Nelson, T. (2002). The identification of CVP1
reveals a role for sterols in vascular patterning. Plant
Cell 14,2045
-2058.
Carland, F. M., Berg, B. L., FitzGerald, J. N., Jianamornphongs,
S., Nelson, T. and Keith, B. (1999). Genetic
regulation of vascular tissue patterning in Arabidopsis. Plant
Cell 11,2123
-2137.
Casson, S. A., Chilley, P. M., Topping, J. F., Evans, I. M.,
Souter, M. A. and Lindsey, K. (2002). The POLARIS
gene of Arabidopsis encodes a predicted peptide required for correct
root growth and leaf vascular patterning. Plant Cell
14,1705
-1721.
Choe, S., Noguchi, T., Fujioka, S., Takatsuto, S., Tissier, C.
P., Gregory, B. D., Ross, A. S., Tanaka, A., Yoshida, S., Tax, F. E.
and Feldmann, K. A. (1999). The Arabidopsis
dwf7/ste1 mutant is defective in the delta7 sterol C-5
desaturation step leading to brassinosteroid biosynthesis. Plant
Cell 11,207
-221.
Clay, N. K. and Nelson, T. (2002). VH1, a
provascular cell-specific receptor kinase that influences leaf cell patterns
in Arabidopsis. Plant Cell
14,2707
-2722.
Cutler, S. R., Ehrhardt, D. W., Griffitts, J. S. and Somerville,
C. R. (2000). Random GFP::cDNA fusions enable visualization
of subcellular structures in cells of Arabidopsis at a high
frequency. Proc. Natl. Acad. Sci. USA
97,3718
-3723.
Deyholos, M. K., Cordner, G., Beebe, D. and Sieburth, L. E.
(2000). The SCARFACE gene is required for cotyledon and
leaf vein patterning. Development
127,3205
-3213.
Donnelly, P. M., Bonetta, D., Tsukaya, H., Dengler, R. E. and Dengler, N. G. (1999). Cell cycling and cell enlargement in developing leaves of Arabidopsis. Dev. Biol. 215,407 -419.[CrossRef][Medline]
Esau, K. (1965). Plant Anatomy. New York: John Wiley and Sons.
Foster, A. S. (1952). Foliar venation in angiosperms from an ontogenetic standpoint. Am. J. Bot. 39,752 -766.
Geisler, M., Jablonska, B. and Springer P. S.
(2002). Enhancer trap expression patterns provide a novel
teaching resource. Plant Physiol.
130,1747
-1753.
Gray, W. M., Ostin A., Sandberg, G., Romano, C. P. and Estelle,
M. (1998). High temperature promotes auxin-mediated hypocotyl
elongation in Arabidopsis. Proc. Natl. Acad. Sci. USA
95,7197
-7202.
Hamann, T., Benkova, E., Bäurle, I., Kientz, M. and
Jürgens, G. (2002). The Arabidopsis BODENLOS
gene encodes an auxin response protein inhibiting MONOPTEROS-mediated embryo
patterning. Genes Dev.
16,1610
-1615.
Hardtke, C. S. and Berleth, T. (1998). The
Arabidopsis gene MONOPTEROS encodes a transcription factor
mediating embryo axis formation and vascular development. EMBO
J. 17,1405
-1411.
Hellmann, H., Hobbie, L., Chapman, A., Dharmasiri, S.,
Dharmasiri, N., del Pozo, C., Reinhardt, D. and Estelle M.
(2003). Arabidopsis AXR6 encodes CUL1 implicating SCF E3 ligases
in auxin regulation of embryogenesis. EMBO J.
22,3314
-3325.
Hemerly, A. S., Ferreira, P., de Almeida Engler, J., Van
Montagu, M., Engler, G. and Inze, D. (1993). cdc2a
expression in Arabidopsis is linked with competence for cell
division. Plant Cell 5,1711
-1723.
Holding, D. R. and Springer, P. S. (2002). The vascular prepattern enhancer trap marks early vascular development in Arabidopsis. Genesis 33,155 -159.[CrossRef][Medline]
Inoue, T., Higuchi, M., Hashimoto, Y., Seki, M., Kobayashi, M., Kato, T., Tabata, S., Shinozaki, K. and Kakimoto, T. (2001). Identification of CRE1 as a cytokinin receptor from Arabidopsis. Nature 409,1060 -1063.[CrossRef][Medline]
Jang, J. C., Fujioka, S., Tasaka, M., Seto, H., Takatsuto, S.,
Ishii, A., Aida, M., Yoshida, S. and Sheen, J. (2000).
A critical role of sterols in embryonic patterning and meristem programming
revealed by the fackel mutants of Arabidopsis thaliana.Genes Dev. 14,1485
-1497.
Kang, K. and Dengler, N. (2002). Cell cycling frequency and expression of the homeobox gene ATHB-8 during leaf vein development in Arabidopsis. Planta 216,212 -219.[CrossRef][Medline]
Kinsman, E. A. and Pyke, K. A. (1998). Bundle
sheath cells and cell-specific plastid development in Arabidopsis
leaves. Development 125,1815
-1822.
Koizumi, K., Sugiyama, M. and Fukuda, H.
(2000). A series of novel mutants of Arabidopsis
thaliana that are defective in the formation of continuous vascular
network: calling the auxin signal flow canalization hypothesis into question.
Development 127,3197
-3204.
Kull, U. (1999). Zur Evolution der Adernetze von Blättern, insbesondere der Angiospermen. Profil 16,35 -48.
Lojda, C. (1970). Indigogenic methods for glycosidases II. An improved method for ß-D-galactosidase and its application to localization studies of the enzymes in the intestine and in other tissues. Histochemie 23,266 -288.[Medline]
Mähönen, A. P., Bonke, M., Kauppinen, L., Riikonen,
M., Benfey, P. N. and Helariutta, Y. (2000). A novel
two-component hybrid molecule regulates vascular morphogenesis of the
Arabidopsis root. Genes Dev.
14,2938
-2943.
Mattsson, J., Ckurshumova, W. and Berleth, T.
(2003). Auxin signaling in Arabidopsis leaf vascular
development. Plant Physiol.
131,1327
-1339.
Meinhardt, H. (1982). Models of Biological Pattern Formation. London: Academic Press Inc. Ltd.
Meinhardt, H. and Gierer, A. http://www.eb.tuebingen.mpg.de/dept4/meinhardt/theory.html
Nelson, T. and Dengler, N. (1997). Leaf
vascular pattern formation. Plant Cell
9,1121
-1135.
Petrov, A. P. and Vizir, I. Y. (1982). On seed germination synchronization of Arabidopsis thaliana (L.) Heynh.Arabidopsis Information Service 19 .
Przemeck, G. K. H., Mattsson, J., Hardtke, C. S., Sung, Z. R. and Berleth, T. (1996). Studies on the role of the Arabidopsis gene MONOPTEROS in vascular development and plant cell axialization. Planta 200,229 -237.[Medline]
Roth-Nebelsick, A., Uhl, D., Mosbrugger, V. and Kerp, H.
(2001). Evolution and function of leaf venation architecture: a
review. Ann. Bot. 87,553
-566.
Sachs, T. (1981). The control of the patterned differentiation of vascular tissues. Adv. Bot. Res. 9, 151-262.
Sachs, T. (1991). Cell polarity and tissue patterning in plants. Development Suppl. 1,83 -93.
Sieburth, L. E. (1999). Auxin is required for
leaf vein pattern in Arabidopsis. Plant
Physiol. 121,1179
-1190.
Souter, M., Topping, J., Pullen, M., Friml, J., Palme, K.,
Hackett, R., Grierson, D. and Lindsey, K. (2002).
hydra mutants of Arabidopsis are defective in sterol
profiles and auxin and ethylene signaling. Plant Cell
14,1017
-1031.
Steeves, T. A. and Sussex, I. M. (1989). Patterns in Plant Development. Cambridge University Press: Cambridge, UK.
Stein, W. (1993). Modeling the evolution of stelar architecture in vascular plants. Int. J. Plant Sci. 154,229 -263.[CrossRef]
Steynen, Q. J. and Schultz E. A. (2003). The
FORKED genes are essential for distal vein meeting in
Arabidopsis. Development.
130,4695
-4708.
Szekeres, M., Nemeth, K., Koncz-Kalman, Z., Mathur, J., Kauschmann, A., Altmann, T., Redei, G. P., Nagy, F., Schell, J. and Koncz, C. (1996). Brassinosteroids rescue the deficiency of CYP90, a cytochrome P450, controlling cell elongation and de-etiolation in Arabidopsis. Cell 85,171 -182.[Medline]
Telfer, A. and Poethig, R. S. (1994). Leaf development in Arabidopsis. In Arabidopsis (ed. E. M. Meyerowitz and C. R. Somerville), pp.379 -401. New York: Cold Spring Harbor Press.
Turner, S. and Sieburth, L. (2002). Vascular patterning. In The Arabidopsis Book (ed. C.R. Somerville and E.M. Meyerowitz) American Society of Plant Biologists, Rockville, MD, doi/10.1199/tab.0073, http://www.aspb.org/publications/arabidopsis/
Wagner, W. H. (1979). Reticulate veins in the systematics of modern ferns. Taxon 28, 87-95.
Willemsen, V., Friml, J., Grebe, M., van den Toorn, A., Palme,
K. and Scheres, B. (2003). Cell polarity and PIN
protein positioning in Arabidopsis require STEROL METHYLTRANSFERASE1
function. Plant Cell 15,612
-625.
Ye, Z.-H. (2002). Vascular tissue differentiation and pattern formation in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 53,183 -202.[CrossRef][Medline]