Int. J. Plant Sci. 165(2):231–242. 2004.
Ó 2004 by The University of Chicago. All rights reserved.
Julie Kang and Nancy Dengler1
Department of Botany, University of Toronto, 25 Willcocks Street, Toronto, Ontario M5S 3B2, Canada
Vein pattern development in the leaves of higher plants requires that a continuous system with hierarchical
vein size classes and regular spacing be formed de novo from ground meristem precursors. In this study, we use
a molecular marker of procambium identity, AtHB-8::GUS, to investigate the procambial stage of vein pattern
formation in adult rosette leaves of Arabidopsis thaliana and to compare the elaboration of AtHB-8-marked
vein pattern with that expressed by xylem differentiation. While the well-studied juvenile leaves of Arabidopsis
have a relatively simple brochidodromous vein pattern with the simultaneous appearance of the looped
secondary veins, adult leaves have a complex semicraspedodromous vein pattern, and a majority of the straight
secondary veins develop progressively. Late-formed secondary veins either arise in the basal portions of the leaf
or are intercalated between earlier-formed secondary veins. Smaller ‘‘connector’’ veins become enhanced
during development to form the subterminal loops of the semicraspedodromous secondary vein pattern.
Higher-order veins, especially freely ending veinlets, are formed throughout leaf expansion, maintaining
a stable vein density. These unique features of adult leaf vein pattern are strongly correlated with the presence
of marginal serrations and a protracted period of leaf expansion. In early leaf development, AtHB-8::GUS
expression precedes any of the hallmark anatomical features of procambial cells in presumptive procambial
strands, defining a ‘‘preprocambial’’ stage. In contrast, AtHB-8::GUS expression was not detected during the
late formation of higher-order veins, indicating that functionally redundant mechanisms guide the development
of leaf vascular pattern.
Keywords: Arabidopsis thaliana, AtHB-8, leaf development, procambium, vein pattern, xylem.
dopsis (Telfer and Poethig 1994; Nelson and Dengler 1997;
Kinsman and Pyke 1998; Van Lijsebettens and Clarke 1998;
Candela et al. 1999; Mattsson et al. 1999; Sieburth 1999).
The elements of the leaf vein hierarchical system are formed
sequentially. First, a single procambial strand extends from
the stem vascular bundles into the leaf primordium to form
the primary vein (Busse and Evert 1999; Kang et al. 2003).
Second, the secondary veins arise as continuous loops, with
the first loops formed in the apical portion of the leaf and
new ones added basipetally (Mattsson et al. 1999; Sieburth
1999). This pattern in which secondary veins are joined in
a series of prominent arches defines the brochidodromous
subtype of pinnate venation (Hickey 1988; Leaf Architecture
Working Group 1999). Tertiary and higher-order veins also
appear as continuous strands that span intercostal areas and
are added basipetally until the complete reticulate vein pattern is formed.
Formation of an individual procambial strand is either simultaneous (anatomically defined procambium appears concurrently along the length of the strand) or progressive
(procambium appears first at one end of the strand and is
elaborated unidirectionally) (Nelson and Dengler 1997). The
simultaneous appearance of secondary veins, a conspicuous
feature of Arabidopsis juvenile leaf vein development, may
be linked to the brochidodromous vein pattern. In contrast,
in most plants with pinnate leaf venation, the secondary
veins terminate in the leaf margin, the craspedodromous subtype (Hickey 1988; Leaf Architecture Working Group 1999).
In the handful of craspedodromous species for which vein
ontogeny has been described, secondary vein formation is
The leaf venation system is required for (1) the import and
distribution of water and solutes to living leaf tissues and (2)
the loading and export of photoassimilates to other regions
of the plant. During the development of leaf vein pattern, formation of certain architectural features is essential: continuity between leaf veins and the leaf traces of the stem,
a hierarchy of vein size classes within the leaf blade, regular
spacing of minor vein elements in the pattern, and a correct
arrangement of specialized vascular tissues within each vein.
In addition, functioning of the leaf venation system depends
on differentiation of conducting cells in two tissue types: tracheary elements that transport water and solutes in the xylem
and sieve tube elements that transport photoassimilates in
the phloem. These specialized conducting cells must develop
in proportion to other nonconducting cell types such as parenchyma and sclerenchyma cells and must develop the appropriate longitudinal continuity and radial arrangement to
carry out their function as the transport machinery within
the plant body.
Previous studies have identified a number of commonalities
of leaf vein pattern ontogeny in the juvenile leaves of Arabi-
1 Author for correspondence; telephone 416-978-3536, fax 416978-5878; e-mail [email protected]
Manuscript received October 2003; revised manuscript received December
described as progressive (reviewed in Nelson and Dengler
Arabidopsis leaves reflect the heteroblasty expressed during
the course of shoot development. In addition to differences in
shape, leaves within the heteroblastic series exhibit differences
in numbers of hydathodes and marginal serrations, in vein
pattern, and in trichome location and density (Telfer and
Poethig 1994; Candela et al. 1999; Tsukaya et al. 2000). Because of the simplicity of vein pattern and tractability for mutant screens, juvenile leaves have been the focus of almost all
previous studies of vein pattern ontogeny in Arabidopsis.
Such emphasis on juvenile leaves assumes that the patterns
and mechanisms of development will be common to all leaves,
but since we wanted to specifically study the origin of a more
complex, hierarchical vein pattern, our observations have
been made on the eighth rosette leaf, one that expresses characteristics unique to the adult phase of shoot development.
Leaf vein pattern is first expressed as a pattern of procambial strands. Procambium is the primary meristematic tissue
that serves as a precursor to xylem and phloem tissues and
can be distinguished on anatomical grounds by its elongate
cell shape, lack of vacuolation, and formation of continuous
narrow files (Esau 1965b; Nelson and Dengler 1997; Mattsson et al. 1999; Dengler 2001; Scarpella et al. 2003). During
leaf development, procambial strands are formed de novo
from ground meristem precursors and become delimited
through distinctive planes of cell division (Esau 1965a; Nelson and Dengler 1997; Dengler and Kang 2001). How procambial cells acquire their distinctive shape and form
a hierarchical pattern of continuous strands within an initially uniform developmental field is still unknown.
Recent observations of leaf vein development using the
auxin reporter construct, DR5::GUS (Ulmasov et al. 1997),
found expression in files of ground meristem cells that are
not distinguished from surrounding cells by shape, as well as
in files of elongate procambial cells (Mattsson et al. 2003).
These results indicate that an auxin response defines a ‘‘preprocambial’’ stage that precedes the anatomical distinction
between procambial and adjacent ground meristem cells.
While many previous studies of leaf vascular pattern ontogeny have exploited the easily visualized, but relatively late,
stages of xylem differentiation as markers of vein pattern
(Telfer and Poethig 1994; Kinsman and Pyke 1998; Candela
et al. 1999; Sieburth 1999), the use of molecular markers of
procambium (and preprocambium) makes it possible to characterize the earliest stages of vein pattern formation (Baima
et al. 1995; Scarpella et al. 2000, 2003; Kang and Dengler
2002; Mattsson et al. 2003).
Expression of the HD-Zip class III transcription factor,
Arabidopsis thaliana homeobox gene-8 (AtHB-8), is reported
to be restricted to procambial cells and to coincide with the
development of anatomically defined procambium (Baima
et al. 1995, 2001). In this study, we characterize the development of the vein pattern of adult leaves of Arabidopsis thaliana using expression of the AtHB-8::GUS construct as
a marker for the procambial stage (Baima et al. 1995, 2001).
We compare this pattern with that expressed by differentiated xylem, using the appearance of secondary cell walls
as a marker for mature tracheary elements. Our observations
identify fundamental aspects of vein pattern ontogeny that
are not revealed in juvenile leaves and also provide circumstantial evidence for the functional role(s) of AtHB-8 expression in leaf vein pattern development.
Material and Methods
Plant Material and Growth Conditions
Seeds (ecotype Columbia) carrying an AtHB-8::GUS construct were provided by Giorgio Morelli (Instituto Nazionale
di Ricerca per gli Alimenti e la Nutrizione, Italy). The
AtHB-8::GUS reporter construct consists of a 1.7-kb DNA
fragment of the AtHB-8 genomic sequence upstream of the
putative initiation codon and is fused in frame to GUS
(Baima et al. 1995).
Seeds were surface sterilized with 20% (v/v) commercial
bleach, rinsed in distilled water, and stored for 1 wk at 4°C
before being sown directly onto sterilized Promix (SunGro
Horticulture, Bellevue, Wash.). Plants were grown in a Conviron E15 growth chamber (Controlled Environments,
Winnipeg, Manitoba) at 22°C under a 10 h day : 14 h night
cycle (150 mmol m2 s1). Leaves 1 and 8 were selected as
the representative of juvenile and adult rosette leaves, respectively. Leaf 8 was selected for detailed analysis since it is
formed well after the transition from juvenile to adult phases
(Telfer et al. 1997) and has a complex vein pattern with welldefined vein orders. Under these growth conditions, leaf 1 is
initiated ca. 5 d after germination and leaf 8 ca. 14 d after
germination (Donnelly et al. 1999). Length of leaves 1 and
8 were measured every second day until growth of leaf
8 ceased, ca. 34 d after germination. Leaf 8 was sampled at
2-d intervals from day 4 through to day 20 (fig. 1H). Since collecting early stages of vascular development was necessary,
leaves were sampled every 6 (for day 0) or 24 h from day 1
through to day 4 (see fig. 1). Six-hour collections were based
on leaf 5 length measurements since leaf 8 is not externally
Fig. 1 Patterns of AtHB-8::GUS expression in cleared whole mounts of developing Arabidopsis leaves at day 0. A, B, High magnification of
the preprocambial stage as indicated by AtHB-8::GUS expression. AtHB-8::GUS expression is restricted to a narrow strand of cells that are not
distinguished from neighboring cells by shape (arrows). Some strands end freely, indicating progressive development. A, ‘‘Connector’’ vein at the
preprocambial stage at ca. 24 h. B, Tertiary veins at ca. 48 h. C, Whole leaf at ca. 12 h. AtHB-8::GUS-expressing cells at the base of the primary
vein are elongate, characteristic of anatomically defined procambial cells (arrow). D–G, Whole leaves collected at 6-h intervals showing pattern of
AtHB-8::GUS expression. D, Primary vein is present at 6 h. E, The first secondary veins are formed by 12 h and appear either as continuous loops
or as short spurs on the primary vein (arrow). Leaf serrations begin to form (asterisk). F, Leaf serrations are more pronounced (asterisks) and are
associated with new secondary veins extending toward the leaf margin. G, Smaller-diameter veins (‘‘connectors’’) diverge just proximal to the
secondary vein termini and connect either acropetally (shown here) or basipetally (fig. 1A) with an earlier-formed secondary vein (arrow). H, Time
course of leaf expansion for leaves 1 and 8. Lengths are mean values for blade plus petiole 6 SE. A, B, Bars ¼ 25 mm; C–G, bars ¼ 50 mm.
visible at initiation. Ca. 15 to 20 replicates were collected for
analysis at each stage.
Histochemical Localization of GUS Activity
and Tissue Preparation
Histochemical localization of GUS activity was performed
using 5-bromo-4-chloro-3-indolyl-b-D-glucuronide (X-gluc)
as a substrate. Leaf tissue samples were placed in 90% acetone on ice for 20 min and then in X-gluc buffer solution
(750 mg mL1 X-Gluc, 100 mM NaPO4 [pH 7], 3 mM K3
F3[CN]6, 10 mM EDTA, 0.1% [w/v] Nonidet-P40) under
vacuum for 4 h. After GUS detection, leaf tissue was postfixed in ethanol : acetic acid (3 : 1, v/v) for 1 h, rinsed in
70% (v/v) ethanol, cleared overnight in saturated chloral hydrate, and mounted in the same solution.
Qualitative and Quantitative Observations
Slides were examined under bright-field (AtHB-8::GUS
expression) or dark-field (xylem tracheary element differentiation) optics on a Reichert-Jung Polyvar microscope (ReichertJung, Vienna). Images were photographed using a Nikon
DXM1200 digital camera and ACT-1 Software (Nikon,
Tokyo). Images were digitized using a GTCO digitizing tablet
(GTCO Calcomp, Scottsdale, Ariz.) and Image-Pro Plus Software (Media Cybernetics, Silver Spring, Md.). For all quantitative analyses, each leaf was divided into four equal sectors
(along the proximal-distal axis), and data were collected
from each sector. Sector data were pooled to give a total distribution for the leaf half. Data were analyzed using SigmaPlot and SigmaStat Software (SPSS, Chicago).
AtHB-8::GUS Expression and Development
of Procambial Strands
The domain of AtHB-8::GUS expression is initially restricted to cells that lack the distinctive shape of procambial
cells but that are positioned where presumptive procambial
strands are predicted to form, indicating that AtHB-8::GUS
expression defines a preprocambial stage of development (fig.
1). Although AtHB-8::GUS expression is comparatively weak
at the preprocambial stage, narrow cell files can be seen to
extend part of the way across intercostal regions, indicating
that the procambial strands are elaborated by the progressive
recruitment of individual ground meristem cells (arrows in
fig. 1A, 1B). Stronger expression of AtHB-8::GUS coincides
with the development of the distinct elongate appearance of
individual procambial cells (arrow, fig. 1C), indicating that
its expression marks both preprocambial and procambial
stages. Although we did not specifically examine the conversion of AtHB-8::GUS-expressing preprocambial cells to those
anatomically defined as procambial cells (that also express
AtHB-8::GUS), we make the assumption that procambial
strand formation mirrors the progressive development of the
preprocambial phase in the following descriptions.
At early stages of leaf development (day 0), the primary
vein develops acropetally into the young primordium as the
leaf emerges from the shoot apical meristem (fig. 1D). The
first secondary vein appears either as a continuous loop from
the apex of the primary vein (the pattern observed for juvenile leaves; Mattsson et al. 1999, 2003) or as a short spur
projecting from the primary vein (arrow, fig. 1E). Subsequently formed secondary veins arise as short spurs that then
develop progressively from the primary vein toward the margin (fig. 1F). Coincident with the formation of these progressive secondary veins, small bulges appear at the leaf margin
opposite the secondary veins (asterisks, fig. 1E, 1F). These
areas of localized growth will develop into the prominent
leaf serrations characteristic of adult leaves. The secondary
veins terminate within these serrations and subsequently become connected to adjacent secondary veins by new smallerdiameter procambial strands that arise proximal to the
secondary vein terminus (arrow, fig. 1G). These ‘‘connector’’
strands become more conspicuous with developmental time
(black arrow, fig. 2A; arrows, fig. 3G) and form the basis for
the semicraspedodromous designation of vein pattern in
which secondary veins branch near the margin, with one
branch terminating at the margin and the other joining the
superadjacent secondary (Hickey 1988; Leaf Architecture
Working Group 1999).
By the end of day 1, two major secondary loops are present in each lamina half (fig. 2A). As the leaf grows in length,
the intercostal regions demarcated by these veins increase in
area, and new major secondary veins are intercalated between these first-formed secondary veins (black arrowhead,
fig. 2A). The intercalated secondaries are narrow in diameter
at first and express GUS weakly; however, these veins rapidly
increase in diameter and express GUS more strongly, so that
they reach a secondary vein level of prominence by days 2–4
(black arrowheads, fig. 2B, 2C, 2G). Subsequently formed
secondary veins are added basipetally along the primary vein
(red arrowheads, fig. 2A–2C). By the end of day 1, the first
tertiary veins form parallel to the primary vein, while additional tertiary veins with various orientations appear within
all intercostal areas by day 2 (red arrows, fig. 2A, 2B). Development of tertiary and higher-order veins is also progressive,
as indicated by AtHB-8::GUS expression at the preprocambial stage (fig. 1B). Expression of AtHB-8::GUS appears
Fig. 2 Patterns of AtHB-8::GUS expression and of differentiated xylem seen in cleared whole mounts of developing Arabidopsis leaves. A–C, G–I,
AtHB-8::GUS expression pattern shown through bright-field optics at days 1–8. Inset in G, High magnification of a leaf serration. AtHB::GUS
expression extends further into the serration than differentiated xylem (arrow in J). Inset in I, High magnification showing that AtHB-8::GUS expression
is absent in all vein orders at the leaf apex. D–F, J–L, Differentiated xylem pattern as shown through dark-field optics. Leaves are the same as those shown
in A–C and G–I at days 1–8. 1 ¼ first-formed secondary vein loop; 2 ¼ second-formed secondary vein; black arrowhead ¼ intercalated secondary vein;
red arrowhead ¼ basal secondary vein; black arrow ¼ smaller-diameter ‘‘connector’’ that joins adjacent secondary vein; red arrow ¼ tertiary vein;
asterisks ¼ leaf serration. A–F, Bars ¼ 200 mm; G–L, bars ¼ 1 mm; inset in G, bar ¼ 50 mm; inset in I, bar ¼ 250 mm.
strongest in the older procambial strands of the primary and
secondary veins, while weaker expression is seen in the new
procambial strands of the tertiary veins (red arrow, fig. 2A).
By days 2–4, higher-order minor veins (quaternary, quinternary, and freely ending veinlets) appear, forming the characteristic reticulate vein pattern of Arabidopsis leaves (fig.
2B, 2C, 2G). Leaf serrations become more prominent, both
in association with basipetally formed secondary veins and
with those intercalated between the apicalmost secondary
veins (asterisks, fig. 2C; inset, fig. 2G, 2J). Expression of
AtHB-8::GUS is strong in all vein categories at this stage of
leaf development (fig. 2G). At about day 6, coinciding with
the beginning of rapid leaf expansion (fig. 1H), AtHB-8::GUS
expression begins to disappear from procambial strands,
beginning at the leaf apex (inset, fig. 2I) and extending basipetally, so that expression is present only in the basal half of the
leaf at day 8 (fig. 2I).
At day 12, AtHB-8::GUS expression is present in all vein
categories in the basal quarter of the leaf (fig. 3A, 3B). AtHB-
8::GUS expression disappears from tertiary veins after day
16, however, and is expressed only in the primary vein and in
proximal regions of the secondary veins by day 20 (arrows,
fig. 3C, 3E). AtHB-8::GUS is not detectable after day 24.
Development of Vein Pattern as Indicated by
Differentiated Xylem
Differentiation of the primary vein xylem begins during
day 1 of leaf 8 development. The first tracheary elements appear near the base of the leaf, and differentiation extends
acropetally toward the leaf apex (fig. 2D, 2E) and basipetally
within the leaf trace (data not shown). Differentiation within
the secondary veins first occurs near the leaf apex and proceeds basipetally (fig. 2F). Xylem differentiation within laterformed secondary veins may extend either basipetally from
previously formed secondary veins, or form islands that later
connect both acropetally and basipetally (arrow, fig. 2F).
This is in contrast to procambial strands where islands are
Fig. 3 Patterns of AtHB-8::GUS expression and differentiated xylem in cleared whole mounts of developing Arabidopsis leaves. A, C, E, Low
magnification of the basal quarter of the leaves under bright-field optics. A, AtHB-8::GUS expression is present in all vein categories at day 12.
C, E, At day 16 and 20, AtHB-8::GUS expression is restricted to the primary vein and proximal regions of the basal secondary veins (arrows).
B, D, F, Same regions as A, C, E under dark-field optics to show differentiated xylem pattern. G, Low magnification of the basal half of a leaf at
day 18 showing ‘‘connector’’ veins (light blue lines with arrows) and termini of secondary veins (blue lines with arrowheads). H, All vein orders are
present by day 18. Bars ¼ 500 mm.
never seen. Xylem of tertiary veins begins to differentiate at
about day 4 (fig. 2J–2L), but it is not until day 18 that veins
of all orders are expressed as differentiated xylem (fig. 3G,
At late stages of leaf development, AtHB-8::GUS is not detectable in most higher-order vascular strands (fig. 3C–3F),
yet vein density continues to increase, suggesting that new
veins are formed without AtHB-8::GUS expression. To test
whether veins are formed at later stages of leaf expansion,
we undertook a quantitative analysis of vein pattern as indicated by AtHB-8::GUS expression and xylem differentiation.
Quantitative Comparison of AtHB-8::GUS
and Xylem Patterns
The most rapid rate of leaf expansion occurs between day 6
and day 12, with a slower rate of increase until day 20 (fig.
4A); this parallels the increase in leaf length (fig. 1H). Vein
density (the ratio between the sum of all vein lengths to lamina
area; Candela et al. 1999) for both AtHB-8::GUS-expressing
strands and strands with differentiated xylem is highest at
day 6, the beginning of rapid lamina expansion, reaching values of 2.1 mm/mm2 (fig. 4B). Vein density for strands with differentiated xylem decreases rapidly to values of ca. 0.5 mm/
mm2 after this stage and is then maintained at this density as
the leaf continues to expand between days 10 and 20 (fig. 4B).
Vein branch point number is also used as an indicator of
vein pattern complexity (Candela et al. 1999). In leaf 8,
branch point numbers for AtHB-8::GUS-expressing strands
increases to day 6 when the total number per lamina half is
180 6 2:9 (inset, fig. 4C). Xylem differentiation within procambial strands lags behind those procambial strands defined
by AtHB-8::GUS expression; the number of branch points
for strands with differentiated xylem is 100 6 1:5 at day 6
and increases to 250 6 2:2 by day 8 (fig. 4C). Since we predicted that branch point number for AtHB-8::GUS-expressing strands and for strands with differentiated xylem should
correspond, we gathered data from an intermediate stage
(day 7) and found similar values at day 7 (260:2 6 3:9) for
AtHB-8::GUS-expressing strands and at day 8 (250:1 6 2:2)
for strands with differentiated xylem. Most strikingly, the
number of xylem branch points continues to increase after
this stage of leaf development until values of 693 6 6:3
branch points per lamina half are seen in day 18 leaves (fig.
4C). The increase in branch point numbers for both AtHB8::GUS-expressing strands and differentiated xylem occurs in
all four sectors and thus is distributed along the entire leaf
length (fig. 4E). A similar pattern is seen for the number of
freely ending veinlets per lamina half, which increases dramatically through to day 18 (fig. 4D), indicating that these
veins are most likely those that are formed at late stages of
leaf development.
As a comparison, a similar quantitative analysis of vein pattern was made for a juvenile leaf. In contrast to adult leaf 8,
the number of branch points detected for AtHB-8::GUSexpressing strands corresponds exactly to the number of
branch points for differentiated xylem by the end of development in juvenile leaf 1 (table 1). Since our quantitative results
indicate that new veins are continually formed during leaf development, we tested these results by looking for anatomically
defined procambial strands that did not express AtHB-8::GUS
during late leaf expansion stages (days 12–18). Freely ending
veinlets were found that lack AtHB-8::GUS expression (fig.
5A, 5C). Dark-field microscopy shows that differentiated
xylem is not present in these veins (fig. 5B). Aniline blue
stain and bright-field microscopy was used to test for the presence of callose, a compound that is abundant only in phloem
sieve tube elements. Callose was identified in the phloem of the
parent strand but not in the freely ending veinlets (inset, fig.
5F). Since all freely ending veinlets were observed to have differentiated xylem in mature leaves, we conclude that these
strands represent an earlier procambial state of development.
AtHB-8::GUS Is Expressed at a Preprocambium Stage
In young leaf primordia, AtHB-8::GUS is expressed in
narrow domains of ground meristem cells that, based on
position, are likely to become the procambial strands.
Since the state of commitment of these cells to a vascular
developmental pathway is unknown, we refer to these
domains as ‘‘preprocambium’’ (Mattsson et al. 2003). These
AtHB-8::GUS-expressing cells do not differ from neighboring
ground meristem cells in shape, indicating that this gene
could play a role in the cellular processes that convert ground
meristem precursors to cells with a distinctive elongate cell
shape, a defining feature of procambium. Recently, similar observations have been made for the auxin reporter DR5::GUS,
which is expressed in narrow strands of preprocambial cells
in juvenile leaves of Arabidopsis (Mattsson et al. 2003). Since
AtHB-8 expression is upregulated in response to auxin
(Baima et al. 1995, 2000, 2001; Mattsson et al. 2003), it is possible that AtHB-8 responds to a signal provided by canalized
auxin flow from a source to a sink within young leaf tissue.
AtHB-8::GUS is also expressed strongly in procambial
strands that consist of anatomically defined procambial cells
and in developing vascular bundles where xylem and phloem
cells are differentiating (Baima et al. 1995; Kang and Dengler
2002; Kang et al. 2003), suggesting that it may play multiple
roles in vascular development (see below).
Development of Preprocambial Strands Is Progressive
Previous descriptions of juvenile leaf procambial pattern
formation based either on anatomical appearance (Mattsson
et al. 1999) or on expression of the DR5::GUS marker
(Mattsson et al. 2003) have shown that, while formation of
the primary vein procambial strand is progressive and acropetal, secondary veins first appear as continuous loops, which
is the simultaneous pattern of vein formation. In contrast,
during the development of the vein pattern of adult leaves reported here, the formation of secondary and tertiary veins, as
indicated by expression of the AtHB-8::GUS construct, is
clearly progressive. Examination of new presumptive procambial strands at high magnification reveals that strands
consist of narrow files of isodiametric cells weakly expressing
GUS with termini that end freely in ground tissue. The shape
similarity between GUS-expressing cells and adjacent cells
that lack GUS expression suggests that additional ground
Fig. 4 Quantitative analysis of vein pattern as indicated by AtHB-8::GUS expression and by differentiated xylem in developing Arabidopsis
leaves. Data represent total values for one half of the lamina (A–D) or for each of the four sectors (E) of developing leaves at the days indicated.
Insets represent data for vein pattern as indicated by AtHB-8::GUS expression, while large boxed graphs represent data for veins with
differentiated xylem. Data for AtHB-8::GUS are presented only through to day 6 since expression diminishes basipetally after this stage. A, Time
course for increase in lamina area. B–D, Time course for increase in total vein density, branch point number, and number of freely ending veinlets.
E, Time course for increase in branch point number per sector. Box ¼ twenty-fifth and seventy-fifth percentiles; whisker ¼ smallest and largest
values; solid line ¼ mean; black bars ¼ sector A; light gray bars ¼ sector B; dark gray bars ¼ sector C; white bars ¼ sector D. N ¼ 1520.
meristem cells are recruited to the termini of presumptive
procambial strands. This progressive pattern might be a peculiarity of adult-phase leaves but might also represent a fleeting
stage in the development of all leaf veins.
The progressive pattern of preprocambial strand formation
is particularly conspicuous during the development of sec-
ondary veins. Although the apicalmost one or two secondary
veins were often detected as continuous loops, all laterformed secondary veins in adult leaves first appear as short
spurs on the midvein that then extend progressively toward
the leaf margin. Conversion of a preprocambial stage of
development to an anatomically defined procambial stage
Table 1
Juvenile Leaf 1 Vein Pattern Complexity Expressed as Total Branch Point Number
Days after germination
ATHB-8::GUS expressing pattern
Differentiated xylem pattern
32.3 6 2.0
12.7 6 4.3
38.9 6 3.9
27 6 2.6
54.8 6 5.6
35.8 6 2.9
53.5 6 1.6
48.8 6 1.8
55.5 6 2.7
56.8 6 2.5
Note. Total branch points for AtHB-8::GUS-expressing strands and for strands with differentiated xylem
pattern correspond closely by day 24. No significant change in branch point number was observed after day 24.
N ¼ 10.
appears to follow the same progressive pattern but was not
examined in detail. The conspicuous straight course of these
secondary order AtHB-8::GUS-expressing strands is consistently associated with the formation of serrations on the leaf
margin, a feature that is absent in juvenile leaves of Arabi-
dopsis (Tsukaya et al. 2000). Expression of the auxin reporter DR5::GUS indicates that the marginal serrations of
adult rosette leaves are sites of auxin accumulation during
development (Aloni et al. 2003). Such localized sources of
auxin might function both to guide the progressive course of
Fig. 5 Anatomical evidence for presence of procambial strands that lack AtHB-8::GUS expression. A, B, Low magnification at approximately
the midpoint of the lamina of a day 16 leaf. Arrows indicate location of putative procambial strand. A, Image under bright-field optics. B, Same
area as in A under dark-field optics showing absence of differentiated xylem. C, High magnification of the same area of the leaf seen in A and B
using differential contrast imaging. The anatomically defined procambial strand lacks AtHB-8::GUS expression. D, High magnification of the
same strand in C stained with aniline blue. No indication of differentiated phloem. Arrow shows aniline blue–positive white fluorescence of
a lateral sieve area callose in phloem of the parent strand. Inset shows high magnification of the lateral sieve area callose. A, B, Bars ¼ 1 mm; C, D,
bars ¼ 100 mm; inset in D, bar ¼ 25 mm.
secondary vein formation and to stimulate the localized
growth required for serration formation (Tsukaya and Uchimiya 1997; Dengler and Kang 2001).
Initial Vein Pattern Is Remodeled to Form the
Hierarchy of Size Classes
Leaf venation in Arabidopsis, and in flowering plants in
general, is characterized by a hierarchy of discrete vein size
classes. Each vein order is defined by its width at the point of
branching from its parent vein; for instance, tertiary veins
are defined by their narrower width where they branch from
the secondary veins and so on (Hickey 1988; Nelson and
Dengler 1997; Leaf Architecture Working Group 1999). In
mature leaves, primary and secondary veins are distinguished
as major because they are associated with ribs of ground tissue, while minor veins are typically embedded in mesophyll
tissue (Esau 1965a). These structural differences reflect functional categories, as only minor veins are involved in phloem
loading (Haritatos et al. 2000). Differences in mature vein
width are closely correlated with the duration of cell proliferation within vein procambium during leaf development, as
indicated by the expression of a cyclin1At::GUS construct
(Kang and Dengler 2002). The major primary and secondary
veins are initiated first during leaf development, and cell cycling continues longest in these veins, giving the relatively
broad diameters that are highlighted in cleared leaves. In
contrast, tertiary and higher-order minor veins are initiated
later and cease cycling earlier, resulting in narrow-diameter
veins (Kang and Dengler 2002). While vein size classes are
defined by their width at the point of branching, the width of
most veins attenuates toward their distal ends (Nelson and
Dengler 1997). This gradient reflects the duration of cell proliferation, since the proximal ends of Arabidopsis secondary
veins continue to express the cyclin1At::GUS reporter for 3–
4 d longer than distal regions of the same vein (Kang and
Dengler 2002). These observations indicate that the somewhat arbitrary designations given to vein orders reflects at
least two different processes: the sequence of vein formation
(secondaries are formed before tertiaries and thus are larger)
and the duration of cell proliferation (veins that are small at
initiation may become larger through prolongation of cell
Our observations of vein pattern formation in developing
leaves of Arabidopsis indicate that the second process, essentially a ‘‘remodeling’’ of smaller procambial strands into
larger ones is an important aspect of venation development.
Later-formed secondary veins first appear as conspicuous
strands extending from the midvein toward the leaf margin.
Smaller-diameter procambial strands lying parallel to the leaf
margin extend from newly formed secondary veins to connect with earlier-formed secondaries. The connector branch
arises in a subdistal position so that the original vein becomes the branch that ends blindly in a hydathode. These
features define the semicraspedodromous vein pattern that
characterizes adult leaves of Arabidopsis. The connectors become more conspicuous, presumably through prolonged cell
proliferation but still represent a less noticeable component
of the secondary vein pattern in mature leaves. This remodeling of vein pattern is likely more important for the adult
leaves with their protracted period of leaf expansion and
more complex vein pattern than for the juvenile leaves in
the heteroblastic shoots of Arabidopsis. In Eucalyptus, the
brochidodromous pattern of the juvenile foliage leaves has
been observed to arise by a similar remodeling (Carr et al.
1986), indicating that this might be a common developmental feature of vein pattern formation in flowering plants.
Another previously undescribed aspect of vein pattern formation in leaves of Arabidopsis is the intercalation of lateformed secondary veins between earlier-formed secondaries.
This feature has not been reported in cotyledons and juvenile
leaves where vein formation is strictly basipetal. The developmental feature may reflect the protracted period of leaf expansion and the functional requirement for an optimal ratio
between major and minor veins in mature leaves (RothNebelsick et al. 2001). During leaf expansion in Arabidopsis,
vein density remains constant, despite the rapid increase in
lamina area, due to the continual addition of new higherorder veins and freely ending veinlets. In adult leaves, the intercostal area within the first secondary loop increases ca. 25
times in size, and intercalation of new secondary veins into
this region might provide a conduit that functions to maintain hydraulic flow between new minor veins and the midvein (Roth-Nebelsick et al. 2001).
Higher-Order Veins Are Formed Independently
of AtHB-8 during Late Leaf Expansion
AtHB-8::GUS expression reveals that a complex pattern of
leaf vein procambial strands is formed several days before
the same pattern is revealed by the presence of differentiated
xylem. Under our growth conditions, maximum complexity
of AtHB-8::GUS-expressing strands (as indicated by the total
number of branch points per lamina half) was reached between the seventh and eighth day of leaf 8 expansion. After
this stage, AtHB-8::GUS expression disappeared along a basipetal gradient, where expression was limited only to the
primary vein and the proximal portions of the secondary
veins in the basal quarter of the leaf by day 20 (Kang and
Dengler 2002; this study). In contrast to the time course of
AtHB-8::GUS-expression pattern, development of vein pattern as indicated by differentiated xylem is delayed and does
not reach the same level of complexity (number of branch
points) until 2–4 d later. Surprisingly, vein pattern as indicated by differentiated xylem pattern continues to increase in
complexity throughout leaf expansion, and the total number
of branch points per half lamina approximately doubles during this period. Similarly, the number of veins of the smallest
order, the freely ending veinlets, continue to increase
throughout leaf expansion, suggesting that the observed increase in overall vein complexity reflects formation of this
vein size class. We tested our methods for assessing vein complexity by making a comparable set of measurements for leaf
1 and found that the vein pattern revealed by AtHB-8::GUS
expression was mirrored exactly by that of strands with differentiated xylem strands by the end of lamina expansion,
suggesting that our methods were reliable and that the discrepancy between procambial and xylem patterns was
a unique feature of adult leaf development. The apparent formation of veins without AtHB-8::GUS expression might
indicate that expression of AtHB-8::GUS is so transient that
these elements of vein pattern were missed during the first
8 d of leaf expansion. Careful observation of leaf tissues
revealed that all anatomically defined procambial strands
expressed AtHB-8::GUS during these early developmental
stages, however. An alternative explanation is that these
higher-order, last-formed veins develop from procambial
strands without the participation of AtHB-8.
Although the downstream targets of this homeodomain
transcription factor are unknown, the restriction of AtHB8::GUS expression to preprocambial and procambial tissue
provides circumstantial evidence that this gene functions in
the early stages of converting an uncommitted precursor tissue to procambium. Several, not mutually exclusive, hypotheses of the specific function(s) of AtHB-8 exist, and the
upregulation of AtHB-8 in response to auxin may play a role
in any or all of them (Baima et al. 1995; Mattsson et al.
2003). The coincidence in spatial and temporal pattern of
AtHB-8::GUS and cyclin1At::GUS expression suggests that
this homeobox gene could regulate the distinctive patterns of
cell division that distinguish procambium from its precursor
ground meristem (Kang and Dengler 2002). Both the highresolution pattern of AtHB-8::GUS expression (Kang and
Dengler 2002; Kang et al. 2003) and the loss-of-function
phenotypes of other members of the HD-ZIP class III homeobox genes (McConnell et al. 2001) indicate that AtHB-8
might function in defining a xylem subdomain within the
procambial strand and therefore participate in establishing
leaf dorsiventral polarity along with other members of its
Regardless of the specific function(s) of AtHB-8 in leaf
vein development, the formation of more than half of the
higher-order venation of adult leaves without its apparent
participation indicates that functionally redundant mechanisms operate in the formation of leaf vein pattern. While
canalized auxin flow clearly plays a key role in vascular pattern formation and other fundamental aspects of plant devel-
opment (Berleth 2000; Berleth and Mattsson 2000; Berleth
et al. 2000), this mechanism most likely interacts with other,
as yet unidentified, factors during the development of a complex biological pattern such as the venation of an adult leaf
(Berleth 2000; Deyholos et al. 2000; Koizumi et al. 2000;
Carland et al. 2002; Clay and Nelson 2002). The greater sensitivity of higher-order veins to auxin transport inhibition
experiments indicates that additional pathways might
contribute to establishing the architecture of the major veins
(Mattsson et al. 1999, 2003; Sieburth 1999). Similarly, when
auxin responses are impaired through mutation, phenotypes
typically display perturbed higher-order venation and disrupted vein continuity (Przemeck et al. 1996; Hamann et al.
1999; Deyholos et al. 2000; Hobbie et al. 2000; Steynen and
Schultz 2003). In contrast, while mutations in the Cotyledon
Vascular Pattern 1 (CVP1) gene also result in vein discontinuity, auxin accumulation and transport are not different
from wild type (Carland et al. 1999, 2002). The finding that
CVP1 encodes a sterol methyltransferase enzyme (Carland
et al. 2002) indicates that sterols may play a role in processes
such as establishing procambial cell continuity since sterol
biosynthesis mutants also exhibit alteration in vasculature
(Jang et al. 2000). Our finding that an established marker of
procambium, AtHB-8::GUS, is not expressed during late formation of higher-order veins may reflect the presence of multiple, functionally redundant mechanisms that guide leaf vein
pattern formation.
This work was supported by the Natural Science and Engineering Research Council of Canada (research grant to N.
Dengler and postgraduate fellowship to J. Kang). We thank
Giorgio Morelli for generously providing AtHB-8::GUS
seeds; Petra Donnelly, Emily Fung, and Ada Wong for technical support and Enrico Scarpella and Thomas Berleth for
helpful suggestions on the manuscript.
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