Stage-specific markers define early steps of procambium development in Arabidopsis leaves and correlate termination of vein formation with mesophyll differentiation

During leaf development, ground meristem cells along continuous lines undergo coordinated oriented cell divisions and differentiate to form procambial cells, the precursors of all vascular cells. The molecular genetic dissection of early procambial development suffers from the lack of easily identifiable markers, especially of cell states preceding procambium formation. In this study, we have identified and characterized three reporter gene expression markers that reflect three distinct preprocambial stages, as well as one marker whose expression seems to be perfectly congruent with the appearance of procambial cells. All four markers are invariably expressed in continuous domains connected to pre-existing vasculature and their expression profiles reveal a common spatiotemporal pattern of early vein formation. We observed progressive extension of vascular strands at the preprocambial stage, suggesting that veins are initiated as freely ending preprocambial domains and that network formation occurs through subsequent fusion of these domains. Consistent with this interpretation, we demonstrate that veins are generally not programmed to become freely ending or interconnected network elements. Instead, we found that the progressive extension of preprocambial domains can be interrupted experimentally and that this leads to less complex vein patterns consisting of fewer vein orders, in which even lower-order veins become freely ending. Mesophyll differentiation turned out to be strictly correlated with the termination of preprocambial domain extension. These findings suggest that Arabidopsis vein pattern is not inherently determinate, but arises through reiterative initiation of new preprocambial branches until this process becomes terminated by the differentiation of mesophyll.     

Modification of cell proliferation patterns alters leaf vein architecture in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Formation of leaf vascular pattern requires regulation of a number of cellular processes, including cell proliferation. To assess the role of cell proliferation during vein order formation, leaf development in genetic lines exhibiting aberrant cell proliferation patterns due to altered expression patterns of ANT and ICK1 genes was analyzed. Modification of cell proliferation patterns alters the number of higher order veins and the number of minor tertiary veins remodeled as intersecondary veins in Arabidopsis rosette leaves. Minor vein complexity, as indicated by branch point and freely ending veinlet number, is highly correlated with a decrease or increase in cell proliferation. Observations of procambial strand formation in modified cell proliferation pattern lines showed that vein pattern is specified early in leaf development and that formation of freely ending veinlets is temporally correlated with the expansion of ground meristem when cell proliferation is terminated prematurely. Taken together, our observations indicate that: (1) genes that modulate cell proliferation play a key role in regulating the meristematic competence of ground meristem cells to form procambium and vein pattern during leaf development, and (2) ANT is a crucial part of this regulation.     

Auxin transport-dependent,stage-specific dynamics of leaf vein formation

For centuries, the formation of vein patterns in the leaf has intrigued biologists, mathematicians and philosophers. In leaf development, files of vein-forming procambial cells emerge from seemingly homogeneous subepidermal tissue through the selection of anatomically inconspicuous preprocambial cells. Although the molecular details underlying the orderly differentiation of veins in the leaf remain elusive, gradually restricted transport paths of the plant hormone auxin have long been implicated in defining sites of vein formation. Several recent advances now appear to converge on a more precise definition of the role of auxin flow at different stages of vascular development. The picture that emerges is that of vein formation as a self-organizing, reiterative, auxin transport-dependent process.Key words: arabidopsis, leaf development, polar auxin transport, procambium, vascular patterningThe vascular system of plants is a branching array of cell files extending through all organs.¹ In dicot leaves, these vascular strands, or ‘veins’, are arranged in a ramified pattern that largely reflects the shape of the leaf (Fig. 1A).^2,3 ‘Lateral veins’ branch from a conspicuous central vein (‘midvein’) that is continuous with the stem vasculature. In many species, lateral veins extend along the leaf edge to form ‘marginal veins’, which connect to adjacent lateral veins to form prominent closed loops. Finally, a series of ‘higher-order veins’ branch from midvein and loops and can either terminate in the lamina (‘free-ending veins’) or join two veins (‘connected veins’).Open in a separate windowFigure 1Conceptual summary of dicot leaf vein formation. (A) Schematics of a simplified mature leaf illustrating midvein (M), first, second and third loops (L1, L2 and L3, respectively)—each derived from corresponding lateral (LV) and marginal (MV) veins—free-ending (FV) and connected (CV) higher-order veins, hydathodes (H) and middle-to-margin positions (decreasing green gradient) as used in the text. (B) State transitions in leaf subepidermal cell differentiation. Available evidence suggests that the vein patterning process is limited to ground meristem cells (white), while subepidermal cells that have begun to acquire mesophyll characteristics are incapable of responding to vein-inducing signals.^11,13,19,38 Expression of preprocambial (blue) and mesophyll emergence markers seem to identify two mutually exclusive and typically irreversible cell states, one leading to procambium (pink) and the other to mature mesophyll (green) formation. The transition from ground meristem to differentiated mesophyll could conceivably occur through a cell state that is formally equivalent to the preprocambial state in vascular differentiation. However, the existence of such a ‘premesophyll’ state (faded gray), the extent of its stability, its mutual exclusivity or competition with the preprocambial state and its responsiveness to vein-inducing signals still remain open questions. (C) Stage-specific dynamics of leaf vein patterning and their dependency on auxin levels and transport as exemplified for loop formation, but in general equally applicable to all veins. Upper series: PIN1-labeled auxin transport paths corresponding to preprocambial cell selection zones (yellow). Note how loops are composed of a lateral PIN1 expression domain (LD) and an initially free-ending marginal PIN1 expression domain (MD). Further, note slightly expanded PIN1 expression domains in a fraction of hydathode-associated third loops during normal development, broad PIN1 domains on the side of local auxin application (arrowhead) and nearly ubiquitous PIN1 expression upon systemic auxin transport inhibition. Middle series: directions of Athb8/J1721-marked preprocambial strand formation (blue arrows). Note middle-to-margin progression of preprocambial strand formation during normal loop development. Further, note margin-to-middle preprocambial strand extension in a fraction of third loops during normal development and in all loops forming on the side of auxin application. Finally, note co-existence of middle-to-margin and margin-to-middle polarities of preprocambial strand extension during the formation of individual loops in response to auxin transport inhibition. Lower series: gradual appearance of procambial cell identity acquisition (pink to magenta). Note simultaneous differentiation of lateral and marginal procambial strands in normal loop development. Further, note successive formation of lateral and marginal procambial strands in a fraction of third loops during normal development and in all loops formed on the side of auxin application and under conditions of reduced auxin transport. Arrows temporally connect successive stages of vein formation. See text for additional details.Vascular cells mature from procambial cells: narrow, cytoplasmdense cells, characteristically arranged in continuous strands.⁴ Leaf procambial strands differentiate from files of isodiametric preprocambial cells, which are selected from the anatomically homogeneous subepidermal tissue of the leaf primordium, the ground meristem (Fig. 1B).^5,6 The mechanism by which ground meristem cells are specified to procambial cell fate is unknown, but an instrumental role for auxin transport and resulting auxin distribution patterns in this process has increasingly gained support.^7–13 This brief essay summarizes a recent group of articles that emphasizes the importance of auxin transport in leaf vein formation.     

The RADICLELESS1 gene is required for vascular pattern formation in rice

The molecular mechanisms through which the complex patterns of plant vascular tissues are established are largely unknown. The highly ordered, yet simple, striate array of veins of rice leaves represents an attractive system to study the dynamics underlying pattern formation. Here we show that mutation in the RADICLELESS1 (RAL1) gene results in distinctive vascular pattern defects. In ral1 embryonic scutella, secondary veins are absent and in the prematurely aborted and discontinuous primary veins, cells are misaligned to each other. In ral1 leaves, longitudinal and commissural (transverse) veins display altered spacing and the commissural veins additionally show atypical branching and interruptions in their continuity. The vascular pattern alterations of ral1 occur in the context of normally shaped leaf primordia. Anatomical inspection and analysis of the expression of the procambium specification marker Oshox1-GUS and of the auxin-inducible reporter DR5-GUS demonstrates that all the vascular patterning aberrations of ral1 originate from defects in the procambium, which represents the earliest identifiable stage of vascular development. Furthermore, the ral1 mutant is unique in that procambium formation in leaf primordium development is delayed. Finally, the ral1 vascular patterning distortions are associated with a defective response to auxin and with an enhanced sensitivity to cytokinin. ral1 is the first mutant impaired in both procambium development and vascular patterning to be isolated in a monocot species.     

Gene trapping in Arabidopsis reveals genes involved in vascular development

The procambium is made up of stem cells that give rise to various vascular cells in plants. To understand the molecular nature of procambium cells, we tried to identify genes that characterize procambium cells using Arabidopsis gene trap lines. Among 26,000 gene trap lines, we found 67 lines in which beta-glucuronidase (GUS) staining occurred along vascular tissues in cotyledons and/or adult leaves. Although four gene trap lines showed procambium-preferential GUS expression, their expression patterns differed from each other during procambium development in root tips and young rosette leaves. Genomic regions flanking the gene trap insertion points in 25 of the 67 lines were determined, including three lines showing preferential GUS staining of the procambium. The three procambium-related genes encoded PINHEAD, katanin and an unknown DUF740 domain-containing protein. We discuss procambium development based on the functions and the differential GUS staining patterns of the procambium-related genes.     

Involvement of the VEP1 gene in vascular strand development in Arabidopsis thaliana

A dominant mutant line characterized by abnormal leaf venation pattern was isolated from a transgenic Arabidopsis plant pool that was generated with Agrobacterium culture harboring an Arabidopsis antisense cDNA library. In the mutant line, the phenotype was due to antisense suppression of a gene we named VEP1 (Vein Patterning). The predicted amino acid sequence of the gene contained a motif related to the mammalian death domain that is found in the apoptotic machinery. Reduced expression of the VEP1 gene resulted in the reduced complexity of the venation pattern of the cotyledons and foliar leaves, which was mainly due to the reduced number of the minor veins and their incomplete connection. The analysis of mutant embryos indicated that the phenotype was originated, at least in part, from a defect in the procambium patterning. In the mutant, the stem and root were thinner than those in wild type. This phenotype was associated with reduced vascular development. The promoter activity of the VEP1 gene was detected preferentially in the vascular regions. We propose that the death domain-containing protein VEP1 functions as a positive element required for vascular strand development in Arabidopsis thaliana.     

Cell lineage analysis of maize bundle sheath and mesophyll cells

Maize leaves are divided into repeated longitudinal units consisting of vascular tissue, bundle sheath (BS), and mesophyll (M) cells. We have carried out a cell lineage analysis of these cell types using six spontaneous striping mutants of maize. We show that certain cell division patterns are preferentially utilized, but not required, to form the characteristic arrangement of cell types. Our data suggest that early in development a central cell layer is formed, most frequently by periclinal divisions in the adaxial subepidermal layer of the leaf primordium. Lateral and intermediate veins are initiated in this central layer, most often by divisions which contribute daughter cells to both the procambium and the ground meristem. These divisions generate "half vein" units which comprise half of the bundle sheath cells around a vein and a single adjacent M cell. We show that intermediate veins are multiclonal both in this transverse direction and along their lengths. BS cells are more closely related to M cells in the middle layer of the leaf than to those in the upper and lower subepidermal layers. An examination of sector boundaries has shown that photosynthetic differentiation in M cells is affected by the phenotype of neighboring BS cells.     

Identification of phloem involved in assimilate loading in leaves by the activity of the galactinol synthase promoter

The definition of "minor" veins in leaves is arbitrary and of uncertain biological significance. Generally, the term refers to the smallest vein classes in the leaf, believed to function in phloem loading. We found that a galactinol synthase promoter, cloned from melon (Cucumis melo), directs expression of the gusA gene to the smallest veins of mature Arabidopsis and cultivated tobacco (Nicotiana tabacum) leaves. This expression pattern is consistent with the role of galactinol synthase in sugar synthesis and phloem loading in cucurbits. The expression pattern in tobacco is especially noteworthy since galactinol is not synthesized in the leaves of this plant. Also, we unexpectedly found that expression in tobacco is limited to two of three companion cells in class-V veins, which are the most extensive in the leaf. Thus, the "minor" vein system is defined and regulated at the genetic level, and there is heterogeneity of response to this system by different companion cells of the same vein.     

Genetic regulation of vascular tissue patterning in Arabidopsis

Plants transport water and nutrients through a complex vascular network comprised of interconnected, specialized cell types organized in discrete bundles. To identify genetic determinants of vascular tissue patterning, we conducted a screen for mutants with altered vascular bundle organization in Arabidopsis cotyledons. Mutations in two genes, CVP1 and CVP2 (for cotyledon vascular pattern), specifically disrupt the normal pattern of vascular bundles in cotyledons, mature leaves, and inflorescence stems. The spatial distribution of the procambium, the precursor to mature vascular tissue, is altered in cvp1 and cvp2 embryos, suggesting that CVP1 and CVP2 act at a very early step in vascular patterning. Similarly, in developing stems of cvp1 and leaves of cvp2, the pattern of vascular differentiation is defective, but the maturation of individual vascular cells appears to be normal. There are no discernible alterations in cell morphology in cvp2 mutants. In contrast, cvp1 mutants are defective in directional orientation of the provascular strand, resulting in a failure to establish uniformly aligned vascular cells, and they also show a reduction in vascular cell elongation. Neither cvp1 nor cvp2 mutants displayed altered auxin perception, biosynthesis, or transport, suggesting that auxin metabolism is not generally affected in these mutants.     

Shifts in leaf vein density through accelerated vein formation in C4 Flaveria (Asteraceae)

Background and Aims

Leaf venation in many C₄ species is characterized by high vein density, essential in facilitating rapid intercellular diffusion of C₄ photosynthetic metabolites between different tissues (mesophyll, bundle sheath). Greater vein density has been hypothesized to be an early step in C₄ photosynthesis evolution. Development of C₄ vein patterning is thought to occur from either accelerated or prolonged procambium formation, relative to ground tissue development.

Methods

Cleared and sectioned tissues of phylogenetically basal C₃ Flaveria robusta and more derived C₄ Flaveria bidentis were compared for vein pattern in mature leaves and vein pattern formation in developing leaves.

Key Results

In mature leaves, major vein density did not differ between C₃ and C₄ Flaveria species, whereas minor veins were denser in C₄ species than in C₃ species. The developmental study showed that both major and minor vein patterning in leaves of C₃ and C₄ species were initiated at comparable stages (based on leaf length). An additional vein order in the C₄ species was observed during initiation of the higher order minor veins compared with the C₃ species. In the two species, expansion of bundle sheath and mesophyll cells occurred after vein pattern was complete and xylem differentiation was continuous in minor veins. In addition, mesophyll cells ceased dividing sooner and enlarged less in C₄ species than in C₃ species.

Conclusions

Leaf vein pattern characteristic to C₄ Flaveria was achieved primarily through accelerated and earlier offset of higher order vein formation, rather than other modifications in the timing of vein pattern formation, as compared with C₃ species. Earlier cessation of mesophyll cell division and reduced expansion also contributed to greater vein density in the C₄ species. The relatively late expansion of bundle sheath and mesophyll cells shows that vein patterning precedes ground tissue development in C₄ species.Key words: Bundle sheath, C₄ photosynthesis evolution, Flaveria, heterochrony, leaf development, mesophyll, vein density, vein pattern formation     

Cell Lineage of Vein Formation in Variegated Leaves of the C4Grass Stenotaphrum secundatum

Clonal analysis of variegated leaves of the C₄grass, Stenotaphrumsecundatum, indicates that invasions among meristematic layersoccur during the organogenetic stage of leaf development, resultingin long, broad white and green stripes. These layer invasionscease prior to the second phase of leaf development when delimitationof leaf regions occurs. Vein precursors mostly arise duringthe second phase, so that procambial strand formation is superimposedon the lineage makeup of earlier-formed tissue. Anatomical evidenceindicates that procambium arises through formative divisionswithin ground tissue of leaf primordia and that each strandis derived from a variable number (one–four) of groundmeristem precursors. If a developing vein straddles the boundarybetween previously-formed green and white sectors, then themature vein is half green and half white, reflecting its mixedcell lineage. In Stenotaphrum, 24.8% of the sectors observedwere bounded by such ‘half veins’. The temporalrelationship of layer invasion and tissue system delimitationin this species supports the view that positional signals aremore important than lineage history in the determination oftissue type. However, analysis of planes of cell division indeveloping veins indicates, that, once formed, procambial strandsare discrete lineage units that extend longitudinally by proliferativedivisions. Thus, lineage restrictions may play an importantrole in the third stage of leaf development, differentiationof tissues and cells, which also includes the maintenance ofcell identity.Copyright 2000 Annals of Botany Company C₄photosynthesis, cell lineage, clonal analysis, leaf development, St. Augustine’s grass,Stenotaphrum secundatum , variegation, vein formation     

Ontogeny of the vascular bundles and contiguous tissues in the maize leaf blade

The patterns of initiation and early development of the minor and major veins in the flat portion of the leaf blade of maize (Zea mays L.) follow similar patterns. The veins and their associated bundle sheath cells commonly arise from cell assemblages derived from a single cell lineage, or longitudinal file of cells, rather than from two “half vein units” derived from different cell lineages. In addition, apparently, none of the vascular cells derived from the procambium is directly related ontogenetically to a bundle sheath cell. In veins derived from larger cell assemblages, the lateral bundle sheath cells are more closely related ontogenetically to the mesophyll cells, which are derived from the ground meristem, than to the vascular cells, which are derived from procambium. The bundle sheath cells, accordingly, are interpreted as being ground meristem in origin.     

Early Development and Ultrastructure of the Major Veins and Their Sheath Tissues in the Maize Leaves

The report described the ultrastructural changes that occurred in the major veins and their associated bundle sheaths (BS) of the maize ( Zea mays L. ) leaf blade in the process of their differentiation from three adjacent cells in the middle layer of the ground meristem, the minimal number of cells involved with the initiation of a procambial strand and the associated BS. The inner cell underwent two successive unequal periclinal divisions: a smaller cell that later differentiated into the adaxial BS cell precursor, and a larger one that divided once again periclinally yielding an abaxial BS cell precursor and a centrally located procambial initial cell. One of the two lateral cells immediately adjacent to either side of the inner cell also divided periclinally; these derivatives, along with another lateral cell of the original three-celled unit formed the precursor cells of the lateral BS. Prior to the initiation of protophlcem differentiation, all of the procambial cells showed ultrastructural characteristics basically similar to the procambial initial. They possessed a prominent nucleus with electron-dense aggregates of heterochromatin, a dense cytoplasm rich in ribosomes, proplastids and mitochondria; also a thin wall containing numerous plasmodesmata. In many cases, only short pieces of rough endoplasmic reticulum cistemae and a few small sized vacuoles were present. In adclifton, evidence of cytoplasmic disintegration leading to new vacuole formation was noted in the process of proeambium development. It was observed that certain endoplasmic reticulum was engaged in the sequestration and lysis of cytoplasm. No apparent uhrastmctuml difference was found between the BS cell precursors and the procambial initials, that was, the distinction between the procambium and the surrounding BS cells occurred gradually after vein initiation, The major ultrastmctural changes which occurred during the differentiation of the meristematic BS cells into the vacuolated cells were (1) a proplastid to chloroplast transformation going through a prolamellar body stage, and (2) the appearance of the multi-concentric membrane complex which might play a role in the degradation of some ribosomes and other cytoplasmic components during the differentiation of BS cells.     

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

For the genetic analysis of molecular mechanisms underlying temporal and spatial regulation of vascular pattern formation, we isolated mutants of Arabidopsis thaliana that are impaired in vascular patterning. Microscopic examination of the cotyledonary venation of 3,400 M(3) lines led to the identification of 12 mutant lines. Genetic analysis of 8 of these mutant lines indicated that vein pattern formation in these lines resulted from monogenic recessive mutations in 7 different genes, designated VAN1 through VAN7. Mutations in VAN1 through VAN6 genes caused fragmentation (disconnection or partial loss) of lateral veins of the cotyledon and tertiary veins of the rosette leaf whereas they were less injurious to the formation of major veins. Detailed characterization of the van3 mutant using pAthb8::GUS and pTED3::GUS, as molecular markers for the early stage of vascular tissue formation showed that the provascular tissue of the cotyledonary lateral veins was differentiated in fragments during late embryogenesis. These phenotypes of the van mutants are discussed in relation to the auxin signal flow canalization hypothesis and the diffusion-reaction prepattern hypothesis, with the fragility of the continuity in the minor vein formation favoring the latter hypothesis.     

DIFFERING ONTOGENETIC ORIGINS OF PCR (“KRANZ”) SHEATHS IN LEAF BLADES OF C4 GRASSES (POACEAE)

The origin and early development of procambium and associated ground meristem of major and minor veins have been examined in the leaf blades of seven C₄ grass species, representing different taxonomic groups and the three recognized biochemical C₄ types (NAD-ME, PCK, and NADP-ME). Comparisons were made with the C₃ species, Festuca arundinacea. In “double sheath” (XyMS+) species (Panicum effusum, Eleusine coracana, and Sporoboìus elongatus), the procambium of major veins gives rise to xylem, phloem, and a mestome sheath; associated ground meristem differentiates into PCA (“C₄ mesophyll”) tissue and the PCR (“Kranz”) sheath. Development in the C₃ species parallels this pattern, except that associated ground meristem differentiates into mesophyll and a parenchymatous bundle sheath. In contrast, major vein procambium of “single sheath” (XyMS–) species (Panicum bulbosum, Digitaria brownii, and Cymbopogon procerus) differentiates into xylem, phloem and a PCR sheath; associated ground meristem gives rise to PCA tissue. These observations of major vein development support W. V. Brown's hypothesis that the PCR sheaths of “double sheath” (XyMS+) C₄ grasses are homologous with the parenchymatous bundle sheaths of C₃ grasses, while in “single sheath” (XyMS–) C₄ species they are homologous with the mestome sheath. Although there are some similarities in the development of the major and minor vascular bundle procambium in the C₄ species examined, the ontogeny of the smaller minor veins is characterized by a precocious delineation of the PCR sheath layer that may even precede the appearance of the distinctive cytological features of ground meristem and procambium. This contracted development in minor veins appears to be related to their close spacing in mature leaves and to their comparatively late appearance during leaf ontogeny.     

The SCARFACE gene is required for cotyledon and leaf vein patterning

Mechanisms controlling vein patterning are poorly understood. We describe a recessive Arabidopsis mutant, scarface (sfc), which maps to chromosome 5. sfc mutants have vein pattern defects in cotyledons, leaves, sepals and petals. In contrast to the wild type, in which these organs all have linear veins that are continuous with at least one other vein, in sfc mutants these organs' secondary and tertiary veins are largely replaced by small segments of discontinuous veins, which we call vascular islands. Patterning defects are manifest in cotyledon provascular tissue, suggesting that the patterning defect occurs early in organogenesis. sfc mutants have exaggerated responses to exogenous auxin. Analysis of monopteros (mp(T370)) sfc-1 double mutants suggested that SFC has partially overlapping functions with MP in patterning of both primary and secondary veins.