The postembryonic development of the stem vascular system results in an anatomically complex structure. Procambial strands at the apical meristem form protoxylem and protophloem elements of young bundles, which are gradually displaced by metaxylem and metaphloem as each stem segment matures. The anatomy of a mature bundle is, therefore, different from that of a young bundle (Esau, 1962). Partially occluded tracheids of protoxylem are displaced by the larger open vessels of metaxylem in the main stem, except in the vein supplies to leaves, in which tracheids are maintained (Eames and MacDaniels, 1925). Metaphloem contains companion cells and sieve elements to improve transport efficiency and fibers to provide strength.

The neighboring parenchyma and companion cells of the conducting elements in the shoot also differentiate. These cells frequently develop wall ingrowths, characteristic of transfer cells. Xylem parenchyma of the leaf traces develop extensive wall ingrowths in the upper internode region. Phloem companion cells develop transfer cells in the nodal complex and are mostly associated with vascular connections to the axillary bud (Gunning et al., 1970).

Although the anatomy of the stem vascular system has been meticulously studied, the molecular genetics underpinning its inherent heterogeneity is largely mysterious. In this article, we show how the dynamic nature of vascular development is revealed by the complex expression pattern of the ZeRH2.1 gene.

The ZeRH2.1 gene fragment was isolated during a complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) analysis (Milioni et al., 2002) of the time course of differentiation to tracheary elements in the zinnia (Zinnia elegans cv Envy) mesophyll cell system. In the zinnia system, about 60% of freshly isolated leaf mesophyll cells transdifferentiate into tracheary elements upon induction by exogenous auxin and cytokinin (Fukuda and Komamine, 1980). We obtained a full-length cDNA sequence for ZeRH2.1 using a cDNA library derived from the in vitro system. The ZeRH2.1 gene is a member of the large RING domain-containing family (Kosarev et al., 2002) that in plants comprises nearly 400 genes, very few members of which have been characterized. RING domains are defined by a conserved pattern of Cys and His residues capable of coordinating two atoms of zinc within a characteristic cross-brace structure. Various RING proteins show binding to E2 ubiquitin-conjugating enzyme, are known to mediate protein-protein interactions, and are reported to be involved in a wide range of cellular processes, including development, oncogenesis, apoptosis, and viral replication (Saurin et al., 1996; Borden, 2000). At the molecular level, the domains are involved in a variety of functions, from transcription and RNA processing to organelle transport and peroxisomal biogenesis (Borden, 1998). Only a handful of the plant genes with RING domains have been studied in any depth (Kosarev et al., 2002), COP1 being probably the best known (Torii et al., 1999).

In this article, we have integrated a detailed anatomical analysis of the zinnia vasculature with gene expression analysis to explain the complex in situ pattern of ZeRH2.1. We describe how the development of the stem vascular system results in differential expression patterns of the ZeRH2.1 gene in anatomically distinct vascular bundles. We propose a role for the ZeRH2.1 gene in intercellular transport processes and in transfer cell function.

Figure 1. A, Schematic representation of the translated protein sequence of ZeRH2.1, showing a divergent region (blue), a conserved domain of unknown function (yellow), and the RING domain (red stripes). B, ClustalW alignment of the predicted ZeRH2.1 protein sequence (more ...)

We have estimated previously that 80% of the expression patterns observed in cDNA-AFLP gels can be confirmed by reverse transcription (RT)-PCR or northern gel-blot analyses (Milioni et al., 2002). The cDNA-AFLP analysis indicated that ZeRH2.1 was first expressed 4 h after addition of auxin and cytokinin to the culture medium in the zinnia system. However, the expression pattern of ZeRH2.1 using RT-PCR is not consistent with the cDNA-AFLP pattern and showed that the gene is expressed in freshly isolated mesophyll cells and at all stages of the time course of tracheary element formation in the zinnia system (Fig. 1D) and also in cultures containing either auxin or cytokinin alone (data not shown).

Figure 2. In situ hybridization of ZeRH2.1 antisense riboprobe with transverse sections from the mature zone of zinnia stems. A, Transverse section from the mature zone (below the second node counted from the first fully expanded leaf at the shoot apex) of the (more ...)

The vascular system in the zinnia stem is characteristic of many dicotyledons and forms a hollow cylinder sandwiched between dermal and ground tissue (Sachs, 1875). Long-running vascular strands form individual units of the stem vascular system, which are separated by interfascicular regions. These vascular strands are composed of collateral vascular bundles, with phloem located outside the xylem (Fig. 2, A and C).

In situ hybridization of the ZeRH2.1 antisense riboprobe with transverse sections from the mature zone of the zinnia stem showed that almost all cells contain low levels of transcript (Fig. 2C). Control experiments using the sense probe showed no detectable signal (data not shown). However, certain cells in the vascular bundles show a much higher abundance of transcript. The expression pattern of ZeRH2.1 in vascular bundles 1, 3, 5, 7, 9, and 11, as numbered in the transverse section shown in Figure 2A, is different from that in the other bundles in the stem (Fig. 2C). Clearly, two classes of vascular bundles are represented in a single transverse section taken from the mature zone of the zinnia stem (Fig. 2C).

To understand the underlying differences between these two classes of vascular bundles, we need to describe the morphology of the vasculature. The arrangement of leaves along the zinnia stem is decussate, with alternating pairs of leaves at right angles to the pairs of leaves at the nodes above and below (Fig. 2B). Each leaf, which is simple, entire, and obtuse, is connected directly to the stem without a petiole and is trilacunar, that is, supplied by three vascular traces, a midrib and two flanking sideribs (Fig. 2B) that are directly connected to the central stem vasculature. In a stem transverse section, therefore, six such vascular bundles will be present and are referred to here as leaf trace bundles (Eames and MacDaniels, 1925), while the remaining six or more bundles are referred to as stem bundles. In Figure 2A, the six leaf trace bundles are marked with arrows: of these, midrib bundle 1, together with siderib bundles 3 and 11, supply one leaf, while midrib bundle 7, with siderib bundles 9 and 5, supply the opposite leaf.

The ZeRH2.1 expression pattern provides a molecular marker that distinguishes the leaf trace bundles from the stem bundles. The leaf trace bundles (Fig. 2, C and D) show enhanced expression of ZeRH2.1 in xylem parenchyma cells, between the rows of xylem conducting elements, but basal levels in phloem cells. However, stem bundles have only basal levels of transcript in their xylem parenchyma cells, but instead have abundant transcript in particular phloem cells (Fig. 2E). At higher magnification (Fig. 2F), the transcript is most abundant in small companion cells next to larger sieve elements. Abundant transcript is also present in some cells in the interfascicular region (Fig. 2, C and G). As shown in Figure 2G, these small cells appear comparable to the companion cells of Figure 2F and may represent young phloem of new developing vascular tissues. Vascular bundles of the hypocotyl do not have abundant transcript in xylem or phloem cells (data not shown).

Figure 3. Morphology of zinnia leaf trace and stem bundles. A to C, Leaf trace bundles. A, Leaf trace bundle showing characteristic radial arrangement of the xylem tracheids, surrounded by densely cytoplasmic xylem parenchyma cells (arrows). The phloem is largely (more ...)

By contrast, stem bundles (Fig. 3D) have a smaller proportion of phloem fibers and most of the phloem comprises functional companion cells and sieve tube elements (Fig. 3, H and I). The xylem is arranged in an irregular network of vessels and supporting parenchyma cells, which are not so densely cytoplasmic as in leaf trace bundles (Fig. 3D). Micrographs at higher magnifications (Fig. 3, E and F) show that the xylem parenchyma cell walls are secondarily thickened and do not develop transfer cell-like morphology. However, the phloem companion cells of stem bundles have developed peg-like wall ingrowths (Fig. 3I).

Hypocotyl bundles (Fig. 4B) have a morphology characteristic of mature stem bundles. The xylem vessels are irregularly arranged and surrounding parenchyma cells are secondarily thickened. However, unlike young stem bundles, there are few phloem cells and these do not show clear companion cell or sieve element morphology.

Figure 4. A and B, Stem bundle morphology. C to E, Juvenile bundle morphology. F to K, ZeRH2.1 expression pattern in the young zone of zinnia stem. A, Higher magnification of xylem elements from the internal end of the stem bundle after transition in the long-running (more ...)

In the interfascicular zone, where ZeRH2.1 transcript is detected in small cells, young vascular bundles develop. Recent divisions of a phloem mother cell into a sieve element and a companion cell can easily be identified (Fig. 3G). The companion cells have a characteristic triangular shape and dense cytoplasm. Enlarged cells destined to be xylem vessel elements are also identifiable.

The expression pattern of the ZeRH2.1 gene in juvenile bundles is distinct from that of either stem or leaf trace bundles of the mature stem. Expression of ZeRH2.1 is not enhanced in any of the cell types of the xylem (Fig. 4, F–H). Although xylem tracheids of these bundles are radially arranged in a similar fashion to leaf trace bundles (Fig. 4C), the surrounding parenchyma cells are not densely cytoplasmic nor do they show transfer cell-like morphology (Fig. 4D). However, abundant transcript was detected in the phloem (Fig. 4, F–I). As shown in Figure 4G, the expression of the ZeRH2.1 gene is enhanced in three to four cell layers of phloem. Precursors of phloem fiber cells do not express the gene. Almost all of the phloem cells of a recently divided, very young bundle show accumulation of the transcript (Fig. 4H), as no fiber cells have developed yet. At higher magnification (Fig. 4I), gene expression in the juvenile bundles is confined to some of the larger cells of the phloem. The phloem companion cells of juvenile bundles do not develop wall ingrowths and are roughly the same size as sieve elements (Fig. 4E) and parenchyma cells. It is therefore hard to establish in which phloem cell type the gene is expressed.

The transverse section in Figure 4, F and K, shows the anatomy of a node subtending a leaf pair above node 1. The overlaid boundary in Figure 4K marks the outline of one of the two future leaves that will arise at this node. The leaf traces destined to supply the future leaf have already diverged from the stem (Fig. 4F, black arrowheads), although the leaf remains physically attached to the main stem. Xylem parenchyma cells of the diverged midrib bundle, which are likely to be in the transition state to form transfer cells, strongly express ZeRH2.1 (Fig. 4J). A similar pattern of expression was detected in the siderib bundles but was absent from all of the vascular bundles in the central vasculature. In addition to the leaf trace bundles, cells positioned at the top half of the leaf (Fig. 4F, arrows), the future palisade mesophyll cells, also show enhanced gene expression; however, spongy mesophyll cells or the neighboring stem cortical cells do not show up-regulation of the transcript (Fig. 4F).

The expression pattern of the molecular marker ZeRH2.1 in different types of vascular bundles is summarized in Figure 5.

Figure 5. Schematic overview of the bundle morphology and the expression pattern of ZeRH2.1 gene in the zinnia stem vascular system. A, Zinnia plant with young, mature, and hypocotyl zones demarcated. B, Transverse sections showing the vascular bundle arrangements (more ...)

To address these questions, we resolved the arrangement of the vasculature in the zinnia stem (Fig. 6). The hypocotyl has four vascular strands, each of which divides once to bring the number of vascular strands to eight. Four of these eight strands divide once again to supply the first pair of leaves, bringing the total number of vascular strands to 12. This represents the base number for zinnia, which the plant will maintain by continued divisions of the vascular strands. At the apical meristem, all of the bundles are identical and show juvenile bundle morphology. Vascular strands that have diverged out from the central vasculature as leaf traces develop leaf trace bundle morphology. As the stem elongates and internodes develop, continuing leaf traces within the central vasculature also develop leaf trace bundle morphology (indicated by green lines in Fig. 6) in the upper region of the internode. As the vascular strand increases in distance from the node, which it supplied as a leaf trace, it undergoes a morphological transition from leaf trace bundle to stem bundle, represented by transition of green lines into black lines, in Figure 6. The peripheral part of the xylem develops irregular metaxylem, characteristic of stem bundles, whereas the internal part of the newly formed bundle contains protoxylem elements with leaf trace bundle characteristics (Fig. 3D) and xylem parenchyma cells with wall ingrowths (Fig. 4A). In mature stem bundles, lacunae form in the protoxylem as the stem elongates and the cells are subject to stretching forces (Fig. 4B). Interestingly, protoxylem tracheids are not obliterated (Fig. 4B), perhaps because of the mechanical support provided by the thick walls of neighboring transfer cells. Older parts of the stem with very long internodes show only stem bundle morphology, illustrated in Figure 6 by black lines, between the hypocotyl and node 4.

Figure 6. Zinnia stem vascular network, showing arrangement and interconnection of the leaf and stem bundles. The hypocotyl contains four bundles, which divide to produce 12 vascular bundles. Green lines indicate leaf trace bundles and black lines indicate stem (more ...)

The central stem vasculature only contains six leaf trace bundles at one node, as the vascular bundles supplying the leaf do not continue above that node, shown within the blue rectangle of Figure 6. For example, the vascular supply to leaves of node 2 terminates at that node, and node 1 does not contain any leaf trace bundles that have supplied node 2. Similarly, below the supply node as well, leaf trace bundle morphology remains confined to a single internode. As shown in Figure 6, the siderib bundles branch out just below the supply node. This is not the case for midribs, which originate two nodes below the supply node. The midrib bundle for node 2, for example, originates by the merging of two bundles from node 4. The midrib bundles acquire stem bundle morphology before reaching the node below the supply node; node 2 midrib, for example, will show stem bundle morphology when observed at node 3.

The development of the postembryonic stem vascular system involves three processes: (1) the differentiation of juvenile bundles into leaf trace bundles and stem bundles, (2) the specialization of vascular cells for specific function, and (3) the differentiation of the vascular network as a coherent unit. We have used the expression pattern of a zinnia gene, ZeRH2.1, as a molecular marker to help track the differentiation of the zinnia stem vascular system as the plant matures (Fig. 5).

Understanding the differential expression pattern of ZeRH2.1 in different developmental zones of the zinnia stem required careful analysis of the anatomy. As zinnia is an established model system for studying xylogenesis, detailed characterization of the stem vascular network is important for functional analysis of the genes in vivo. We define two zones in the stem, a young zone, above the first fully expanded leaf, and a mature zone, between the second open leaf and the hypocotyl (Fig. 5). Juvenile bundles in the young zone have a uniform anatomy, consisting of protophloem and radially arranged tracheids of protoxylem. Juvenile bundles then develop into leaf trace bundles in which xylem parenchyma cells have differentiated into transfer cells, supporting radially arranged protoxylem tracheids. The phloem of these bundles is characterized by metaphloem and is largely composed of companion cells and sieve elements. As the plant grows, the internodes elongate and the long-running vascular strands develop irregularly arranged metaxylem vessels, characteristic of stem bundle morphology (Figs. 3, D–F, and 6). The internal cells of newly formed stem bundles are protoxylem cells (Figs. 2E and 3D), which are later stretched and displaced as metaxylem is formed (Fig. 4A).

Transfer cell differentiation is coordinated with organ development and occurs at specific locations across particular developmental windows (Pate and Gunning, 1972; Harrington et al., 1997). Wall ingrowths of transfer cells form just as intensive transport starts, and provide efficient and rapid short-distance transport of solutes. Xylem parenchyma cells are best displayed within leaf trace bundles in the upper region of an internode and in the traces leaving the main body of the stem (Gunning et al., 1970). Recently diverged vascular traces supplying young leaves show up-regulation of ZeRH2.1 transcript in xylem parenchyma cells (Fig. 4, F and J), where wall ingrowths are just beginning to form. In the younger part of the stem, near the apical meristem, internodes are not well developed, and xylem parenchyma cells of juvenile bundles do not develop transfer cells and have basal levels of ZeRH2.1 transcript (Fig. 4, F–H). The expression of the ZeRH2.1 gene is, therefore, linked with newly formed transfer cells in leaf traces of the young zone and with functional transfer cells in leaf trace bundles.

The xylem parenchyma cells of the stem bundles have only basal levels of the ZeRH2.1 transcript (Fig. 2E). As shown in Figure 3, D to F, the peripheral xylem cells of stem bundles that are formed in the long-running internodes have thickened xylem parenchyma without transfer cell-like wall ingrowths. However, the internal xylem cells of these bundles consist of protoxylem with characteristic wall ingrowths in xylem parenchyma cells (Fig. 4A). It is possible that older transfer cells associated with the protoxylem at the internal end of the vascular bundle are no longer functional in stem bundles. As stem bundles age, protoxylem is stretched and then becomes displaced (Fig. 4B). Our results show that only the leaf trace bundle transfer cells, which are in close proximity to the node, contain abundant ZeRH2.1 transcript. In hypocotyl bundles, no up-regulated expression of the ZeRH2.1 gene is observed (data not shown) and no transfer cells remain (Fig. 4B).

In the in vitro zinnia system, at least two different cell fates are represented: cells that form tracheary elements and the cells that do not. Several lines of evidence, together with the fact that dilution of cell number in the culture results in suppression of tracheary element formation, suggest that the population of living mesophyll cells that do not differentiate are required to support those differentiating to become tracheary elements (Matsubayashi et al., 1999; McCann et al., 2001; Motose et al., 2001). Most of the riboprobes that we have derived from differentially expressed sequences identified in our cDNA-AFLP screen hybridize to cells within vascular bundles of stem (Milioni et al., 2002). However, we find a variety of cell types are labeled in addition to young xylem tissue, including cambium, phloem, sieve elements, phloem fibers, and xylem parenchyma (Milioni et al., 2002; P. Dahiya and N. Stacey, unpublished data), and some genes are expressed only in subpopulations in the in vitro system (McCann et al., 2001). This raises the possibility that other vascular cell fates are being explored by isolated cells in the in vitro system and that the cells in culture that do not differentiate as tracheids may be required by those that do and may have some of the properties of other vascular cell types.

ZeRH2.1 transcript, however, is also up-regulated in some cells that do not develop transfer cell morphology. In juvenile bundles, the phloem cells accumulate ZeRH2.1 transcript but do not develop wall ingrowths (Fig. 4, E–I). The leaf palisade parenchyma also accumulates transcript without development of transfer cell morphology (Fig. 4, F and K). This observation is consistent with the constitutive expression pattern of ZeRH2.1 in the in vitro zinnia system derived from leaf mesophyll and palisade cells (Fig. 1D). The enhanced levels of ZeRH2.1 transcript in different cell types may be correlated with their involvement in enhanced transportation. Xylem parenchyma cells of leaf trace bundles, companion cells of stem bundles, and phloem cells of juvenile bundles are all placed at crucial junctions of active transportation. Leaf palisade parenchyma cells actively export photosynthates to the rest of the plant. Therefore, we hypothesize that the ZeRH2.1 gene has a role to play during active transport, with more abundant transcript identifying sites of major intercellular transport.

How the ZeRH2.1 gene product might facilitate transport remains to be determined. The protein sequence contains a RING domain (Fig. 1). RING domain-containing proteins are components of supramolecular assemblies in cells that act in a variety of unrelated biochemical reactions. Kentsis et al. (2002) showed that purified RINGs from functionally unrelated proteins, including promyelocytic leukaemia protein (Borden, 1998), human KAP-1/TIF1^β (Peng et al., 2000), lymphocytic choriomeningitis virus Z (Kentsis et al., 2001), breast cancer susceptibility gene product 1, and breast cancer susceptibility gene product 1-associated RING domain protein (Brzovic et al., 2001), self-assemble into supramolecular structures in vitro that resemble those they form in vivo. RING self-assembly creates complexes that act structurally as polyvalent scaffolds, thermodynamically by amplifying activities of partner proteins, and catalytically by spatiotemporal coupling of enzymatic reactions (Kentsis et al., 2002).

In conclusion, our study integrates the complex expression pattern of a molecular marker with the differentiation of the stem vascular system of zinnia. The developmental progression of juvenile bundles to leaf trace bundles, stem bundles, and hypocotyl bundles is marked by the differential expression patterns of the ZeRH2.1 gene. As all four types of bundles show distinct anatomical characteristics, expression patterns of other vascular genes are likely to follow suit. The most similar sequence to ZeRH2.1 belongs to cluster 2.3 in the classification of RING domain-containing genes in Arabidopsis (Kosarev et al., 2002). Our study of the expression pattern of ZeRH2.1 assigns a potential function to genes in this cluster in facilitating active transportation via protein-protein interactions.

Mesophyll cells were isolated from zinnia leaves and induced to differentiate to tracheary elements in vitro as described previously (Domingo et al., 1998).

The 26S rRNA fragment, isolated by cDNA-AFLP, was used as a control for equal loading of total RNA. Primers for the 26S rRNA (5′-AAAGGATTCTACCAGTCGCTTGATGGGA-3′ and 5′-ACGCCTCTAAGTCAGAATCCGGGCTAGA-3′) were mixed with the above reaction.

Upon request, all novel materials described in this publication will be made available in a timely manner for noncommercial research purposes, subject to the requisite permission from any third-party owners of all or parts of the material. Obtaining any permission will be the responsibility of the requestor.

Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession number AJ867840.

We thank Jaap Nijsse (Wageningen University, Wageningen, The Netherlands) for useful advice to determine vascular arrangement, and JIF-Nuffield fellow Ranu Dhalla (John Innes Centre, Colney, UK) for assistance with RT-PCR. P.D. performed the experiments and drafted the manuscript, B.W. sectioned blocks for histology, N.S. performed plant husbandry and cell cultures, D.M. isolated the cDNA-AFLP fragment corresponding to a partial sequence of ZeRH2.1, and K.R. and M.C.M. cosupervised the work and cowrote the manuscript.