Int. J. Plant Sci. 165(2):231–242. 2004. Ó 2004 by The University of Chicago. All rights reserved. 1058-5893/2004/16502-0001$15.00 VEIN PATTERN DEVELOPMENT IN ADULT LEAVES OF ARABIDOPSIS THALIANA 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. Introduction 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 2003. 231 KANG & DENGLER—LEAF VEIN DEVELOPMENT described as progressive (reviewed in Nelson and Dengler 1997). 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). 233 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. KANG & DENGLER—LEAF VEIN DEVELOPMENT 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). Results 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 235 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. 236 INTERNATIONAL JOURNAL OF PLANT SCIENCES 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. KANG & DENGLER—LEAF VEIN DEVELOPMENT 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, 3H). 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 237 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. Discussion 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 238 INTERNATIONAL JOURNAL OF PLANT SCIENCES 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 KANG & DENGLER—LEAF VEIN DEVELOPMENT 239 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 14 16 18 20 24 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. 240 INTERNATIONAL JOURNAL OF PLANT SCIENCES 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 division). 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 KANG & DENGLER—LEAF VEIN DEVELOPMENT 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 class. 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- 241 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. Acknowledgments 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. Literature Cited Aloni R, S Katja, M Langhans, CI Ullrich 2003 Gradual shifts in sites of free-auxin production during leaf-primordium development and their role in vascular differentiation and leaf morphogenesis in Arabidopsis. Planta 216:841–853. Baima S, F Nobili, G Sessa, S Lucchetti, I Ruberti, G Morelli 1995 The expression of the AtHB-8 homeobox gene is restricted to provascular cells in Arabidopsis thaliana. Development 121: 4171–4182. Baima S, M Possenti, A Matteucci, E Wisman, MM Altamura, I Ruberti, G Morelli 2001 The Arabidopsis ATHB-8 HD-Zip protein acts as a differentiation-promoting transcription factor of the vascular meristem. Plant Physiol 126:643–655. Baima S, M Tomassi, A Matteucci, MM Altamura, I Ruberti, G Morelli 2000 Role of the ATHB-8 gene in xylem formation. Pages 445–455 in R Savidge, R Napiers, eds. Cell and molecular biology of wood formation. BIOS Scientific, Oxford. Berleth T 2000 Plant development: hidden networks. Curr Biol 10: R658–R661. Berleth T, J Mattsson 2000 Vascular development: tracing signals along veins. Curr Opin Plant Biol 3:406–411. Berleth T, J Mattsson, C Hardtke 2000 Vascular continuity, cell axialisation and auxin. Plant Growth Reg 32:173–185. Busse JS, RF Evert 1999 Pattern of differentiation of the first vascular elements in the embryo and seedling of Arabidopsis thaliana. Int J Plant Sci 160:1–13. Candela H, A Martinez-Laborda, JL Micol 1999 Venation pattern formation in Arabidopsis thaliana vegetative leaves. Dev Biol 205: 205–216. Carland FM, BL Berg, JN Fitzgerald, S Jinamornphongs, T Nelson, B Keith 1999 Genetic regulation of vascular tissue patterning in Arabidopsis. Plant Cell 11:2123–2137. Carland FM, S Fujioka, S Takatsuto, S Yoshida, T Nelson 2002 The identification of CVP1 reveals a role for sterols in vascular patterning. Plant Cell 14:2045–2058. Carr DJ, SGM Carr, JR Lenz 1986 Leaf venation in Eucalyptus and other genera of Myrtaceae: implications for systems of classification of venation. Aust J Bot 34:53–62. Clay NK, T Nelson 2002 VH1, a provascular cell-specific receptor kinase that influences leaf cell patterns in Arabidopsis. Plant Cell 14:2707–2722. 242 INTERNATIONAL JOURNAL OF PLANT SCIENCES Dengler NG 2001 Regulation of vascular development. J Plant Growth Regul 20:1–13. Dengler NG, J Kang 2001 Vascular patterning and leaf shape. Curr Opin Plant Biol 4:50–56. Deyholos MK, G Cordner, S Beebe, LE Sieburth 2000 The SCARFACE gene is required for cotyledon and leaf vein patterning. Development 127:3205–3213. Donnelly PM, D Bonetta, H Tsukaya, RE Dengler, NG Dengler 1999 Cell cycling and cell enlargement in developing leaves of Arabidopsis. Dev Biol 215:407–419. Esau K 1965a Plant anatomy. Wiley, New York. ——— 1965b Vascular differentiation in plants. Holt, Rinehart & Winston, New York. Hamann T, U Mayer, G Jürgens 1999 The auxin-insensitive bodenlos mutation affects primary root formation and apical-basal patterning in the Arabidopsis embryo. Development 126:1387–1395. Haritatos E, R Medville, R Turgeon 2000 Minor vein structure and sugar transport in Arabidopsis thaliana. Planta 211:105–111. Hickey LJ 1988 A revised classification of the architecture of dicotyledonous leaves. Pages 25–39 in CR Metcalfe, L Chalke, eds. Anatomy of dicotyledons. Vol 1. Oxford University Press, New York. Hobbie L, M McGovern, LR Hurwitz, A Pierro, N Liu, A Bandyopadhyay, M Estelle 2000 The axr6 mutants of Arabidopsis thaliana define a gene involved in auxin response and early development. Development 127:23–32. Jang JC, S Fujioka, M Tasaka, H Seto, S Takatsuto, A Ishii, M Aida, S Yoshida, J Sheen 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 J, N Dengler 2002 Cell cycling frequency and expression of the homeobox gene AtHB-8 during leaf vein development in Arabidopsis. Planta 216:212–219. Kang J, J Tang, P Donnelly, N Dengler 2003 Primary vascular pattern and expression of AtHB-8 in shoots of Arabidopsis. New Phytol 158:443–454. Kinsman EA, KA Pyke 1998 Bundle sheath cells and cell-specific plastid development in Arabidopsis leaves. Development 125: 1815–1822. Koizumi K, M Sugiyama, H Fukuda 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. Leaf Architecture Working Group (LAWG) 1999 Manual of leaf architecture—morphological description and categorization of di- cotyledonous and net-veined monocotyledonous angiosperms. Smithsonian Institution, Washington, D.C. Mattsson J, W Ckurshumova, T Berleth 2003 Auxin signalling in Arabidopsis leaf vascular development. Plant Physiol 131: 1327–1339. Mattsson J, RZ Sung, T Berleth 1999 Responses of plant vascular systems to auxin transport inhibition. Development 126:2979–2991. McConnell JR, J Emery, Y Eshed, N Bao, J Bowman, MK Barton 2001 Role of PHABULOSA and PHAVOLUTA in determining radial pattern in shoots. Nature 411:709–713. Nelson T, N Dengler 1997 Leaf vascular pattern formation. Plant Cell 9:1121–1135. Przemeck GK, J Mattsson, CS Hardtke, RZ Sung, T Berleth 1996 Studies on the role of the Arabidopsis gene MONOPTEROS in vascular development and plant cell axialization. Planta 200: 229–237. Roth-Nebelsick A, D Uhl, V Mosbrugger, H Kerp 2001 Evolution and function of leaf venation architecture: a review. Ann Bot 87:553–566. Scarpella E, S Rueb, KJM Boot, JHC Hoge, AH Meijer 2000 A role for the rice homeobox gene Oshox1 in provascular cell fate commitment. Development 127:3655–3669. Scarpella E, S Rueb, AH Meijer 2003 The RADICLELESS1 gene is required for vascular pattern formation in rice. Development 130: 645–658. Sieburth L 1999 Auxin is required for leaf vein pattern in Arabidopsis. Plant Physiol 121:1179–1190. Steynen QJ, EA Schultz 2003 The FORKED genes are essential for distal vein meeting in Arabidopsis. Development 130:4695–4708. Telfer A, KM Bollman, RS Poethig 1997 Phase change and the regulation of trichome distribution in Arabidopsis thaliana. Development 124:645–654. Telfer A, RS Poethig 1994 Leaf development in Arabidopsis. Pages 379–401 in EM Meyerowitz, CR Somerville, eds. Arabidopsis. Cold Spring Harbor Laboratory Press, Plainsview, N.Y. Tsukaya H, K Shoda, GT Kim, H Uchimiya 2000 Heteroblasty in Arabidopsis thaliana (L.) Heynh. Planta 210:536–542. Tsukaya H, H Uchimiya 1997 Genetic analysis of the formation of the serrated margin of leaf blades in Arabidopsis: combination of a mutational analysis of leaf morphogenesis with the characterization of a specific marker gene expressed in hydathodes and stipules. Mol Gen Genet 256:231–238. Ulmasov T, J Murfett, G Hagen, TJ Guilfoyle 1997 Aux/IAA proteins repress expression of reporter genes containing natural and highly active synthetic auxin response elements. Plant Cell 9:1963–1971. Van Lijsebettens M, J Clarke 1998 Leaf development in Arabidopsis. Plant Physiol Biochem 36:47–60.
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