The role of elastic stresses on leaf venation morphogenesis

We explore the possible role of elastic mismatch between epidermis and mesophyll as a driving force for the development of leaf venation. The current prevalent ‘canalization’ hypothesis for the formation of veins claims that the transport of the hormone auxin out of the leaves triggers cell differentiation to form veins. Although there is evidence that auxin plays a fundamental role in vein formation, the simple canalization mechanism may not be enough to explain some features observed in the vascular system of leaves, in particular, the abundance of vein loops. We present a model based on the existence of mechanical instabilities that leads very naturally to hierarchical patterns with a large number of closed loops. When applied to the structure of high-order veins, the numerical results show the same qualitative features as actual venation patterns and, furthermore, have the same statistical properties. We argue that the agreement between actual and simulated patterns provides strong evidence for the role of mechanical effects on venation development.     

The FORKED genes are essential for distal vein meeting in Arabidopsis

As in most dicotyledonous plants, the leaves and cotyledons of Arabidopsis have a closed, reticulate venation pattern. This pattern is proposed to be generated through canalization of the hormone auxin. We have identified two genes, FORKED 1 (FKD1) and FORKED 2 (FKD2), that are necessary for the closed venation pattern: mutations in either gene result in an open venation pattern that lacks distal meeting. In fkd1 leaves and cotyledons, the defect is first evident in the provascular tissue, such that the distal end of the newly forming vein does not connect to the previously formed, more distal vein. Plants doubly mutant for both genes have widespread defects in leaf venation, suggesting that the genes function in an overlapping manner at the distal junctions, but act redundantly throughout leaf veins. Expression of an auxin responsive reporter gene is reduced in fkd1 leaves, suggesting that FKD1 is necessary for the auxin response that directs vascular tissue development. The reduction in reporter gene expression and the fkd1 phenotype are relieved in the presence of auxin transport inhibition. The restoration of vein junctions in situations where auxin concentrations are increased indicates that distal vein junctions are sites of low auxin concentration and are particularly sensitive to reduced FKD1 and FKD2 activity.     

Self-organization of the vascular system in plant leaves: inter-dependent dynamics of auxin flux and carrier proteins

The vegetative hormone Auxin is involved in vascular tissues formation throughout the plant. Trans-membrane carrier proteins transporting auxin from cell to cell and distributed asymmetrically around each cell give to auxin a polarized movement in tissues, creating streams of auxin that presume future vascular bundles. According to the canalization hypothesis, auxin transport ability of cells is thought to increase with auxin flux, resulting in the self-enhancement of this flux along auxin paths. In this study we evaluate a series of models based on canalization hypothesis using carrier proteins, under different assumptions concerning auxin flux formation and carrier protein dynamics. Simulations are run on a hexagonal lattice with uniform auxin production. A single cell located in the margin of the lattice indicates the petiole, and acts as an auxin sink. The main results are: (1) We obtain branching auxin distribution patterns. (2) The type of self-enhancement described by the functional form of the carrier proteins regulation responding to the auxin flux intensity in different parts of a cell, has a strong effect on the possibility of generating the branching patterns. For response functions with acceleration in the increase of carrier protein numbers compared to the auxin flux, branching patterns are likely to be generated. For linear or decelerating response functions, no branching patterns are formed. (3) When branching patterns are formed, auxin distribution greatly differs between the case in which the number of carrier proteins in different parts of a cell are regulated independently, and the case in which different parts of a cell compete for a limited number of carrier proteins. In the former case, the auxin level is lower in veins than in the surrounding tissue, while in the latter, the auxin is present in greater abundance in veins. These results suggest that canalization is a good candidate for describing plant vein pattern formation.     

Arabidopsis thickvein mutation affects vein thickness and organ vascularization, and resides in a provascular cell-specific spermine synthase involved in vein definition and in polar auxin transport

Polar auxin transport has been implicated in the induction of vascular tissue and in the definition of vein positions. Leaves treated with chemical inhibitors of polar auxin transport exhibited vascular phenotypes that include increased vein thickness and vascularization. We describe a recessive mutant, thickvein (tkv), which develops thicker veins in leaves and in inflorescence stems. The increased vein thickness is attributable to an increased number of vascular cells. Mutant plants have smaller leaves and shorter inflorescence stems, and this reduction in organ size and height is accompanied by an increase in organ vascularization, which appears to be attributable to an increase in the recruitment of cells into veins. Furthermore, although floral development is normal, auxin transport in the inflorescence stem is significantly reduced in the mutant, suggesting that the defect in auxin transport is responsible for the vascular phenotypes. In the primary root, the veins appear morphologically normal, but root growth in the tkv mutant is hypersensitive to exogenous cytokinin. The tkv mutation was found to reside in the ACL5 gene, which encodes a spermine synthase and whose expression is specific to provascular cells. We propose that ACL5/TKV is involved in vein definition (defining the boundaries between veins and nonvein regions) and in polar auxin transport, and that polyamines are involved in this process.     

Reviewing models of auxin canalization in the context of leaf vein pattern formation in Arabidopsis

In both plants and animals vein networks play an essential role in transporting nutrients. In plants veins may also provide mechanical support. The mechanism by which vein patterns are formed in a developing leaf remains largely unresolved. According to the canalization hypothesis, a signal inducing vein differentiation is transported in a polar manner and is channeled into narrow strands. Since inhibition of auxin transport affects venation patterns, auxin is likely to be part of the signal involved. However, it is not clear whether the canalization hypothesis, initially formulated over 25 years ago, is compatible with recent experimental data. In this paper we focus on three aspects of this question, and show that: (i) canalization models can account for an acropetal development of the midvein if vein formation is sink-driven; (ii) canalization models are in agreement with venation patterns resulting from inhibited auxin transport and (iii) loops and discontinuous venation patterns can be obtained assuming proper spacing of discrete auxin sources.     

Evolution and Function of Leaf Venation Architecture: A Review

The leaves of extant terrestrial plants show highly diverseand elaborate patterns of leaf venation. One fundamental featureof many leaf venation patterns, especially in the case of angiospermleaves, is the presence of anastomoses. Anastomosing veins distinguisha network topologically from a simple dendritic (tree-like)pattern which represents the primitive venation architecture.The high degree of interspecific variation of entire venationpatterns as well as phenotypic plasticity of some venation properties,such as venation density, indicate the high selective pressureacting on this branching system. Few investigations deal withfunctional properties of the leaf venation system. The interrelationshipsbetween topological or geometric properties of the various leafvenation patterns and functional aspects are far from beingwell understood. In this review we summarize current knowledgeof interrelationships between the form and function of leafvenation and the evolution of leaf venation patterns. Sincethe functional aspects of architectural features of differentleaf venation patterns are considered, the review also refersto the topic of individual and intraspecific variation. Onebasic function of leaf venation is represented by its contributionto the mechanical behaviour of a leaf. Venation geometry anddensity influences mechanical stability and may affect, forexample, susceptibility to herbivory. Transport of water andcarbohydrates is the other basic function of this system andthe transport properties are also influenced by the venationarchitecture. These various functional aspects can be interpretedin an ecophysiological context. Copyright 2001 Annals of BotanyCompany Review, leaves, leaf venation, evolution, network, transport, flow, mechanical stabilization     

In silico leaf venation networks: Growth and reorganization driven by mechanical forces

Development commonly involves an interplay between signaling, genetic expression and biophysical forces. However, the relative importance of these mechanisms during the different stages of development is unclear. Leaf venation networks provide a fitting context for the examination of these questions. In mature leaves, venation patterns are extremely diverse, yet their local structure satisfies a universal property: at junctions between veins, angles and diameters are related by a vectorial equation analogous to a force balance. Using a cell proliferation model, we reproduce in silico the salient features of venation patterns. Provided that vein cells are given different mechanical properties, tensile forces develop along the veins during growth, causing the network to deform progressively. Our results suggest that the local structure of venation networks results from a reorganization driven by mechanical forces, independently of how veins form. This conclusion is supported by recent observations of vein development in young leaves and by the good quantitative agreement between our simulations and data from mature leaves.     

Responses of plant vascular systems to auxin transport inhibition.

To assess the role of auxin flows in plant vascular patterning, the development of vascular systems under conditions of inhibited auxin transport was analyzed. In Arabidopsis, nearly identical responses evoked by three auxin transport inhibitor substances revealed an enormous plasticity of the vascular pattern and suggest an involvement of auxin flows in determining the sites of vascular differentiation and in promoting vascular tissue continuity. Organs formed under conditions of reduced auxin transport contained increased numbers of vascular strands and cells within those strands were improperly aligned. In leaves, vascular tissues became progressively confined towards the leaf margin as the concentration of auxin transport inhibitor was increased, suggesting that the leaf vascular system depends on inductive signals from the margin of the leaf. Staged application of auxin transport inhibitor demonstrated that primary, secondary and tertiary veins became unresponsive to further modulations of auxin transport at successive stages of early leaf development. Correlation of these stages to anatomical features in early leaf primordia indicated that the pattern of primary and secondary strands becomes fixed at the onset of lamina expansion. Similar alterations in the leaf vascular responses of alyssum, snapdragon and tobacco plants suggest common functions of auxin flows in vascular patterning in dicots, while two types of vascular pattern alterations in Arabidopsis auxin transport mutants suggest that at least two distinct primary defects can result in impaired auxin flow. We discuss these observations with regard to the relative contributions of auxin transport, auxin sensitivity and the cellular organisation of the developing organ on the vascular pattern.     

Auxin is required for leaf vein pattern in Arabidopsis

To investigate possible roles of polar auxin transport in vein patterning, cotyledon and leaf vein patterns were compared for plants grown in medium containing polar auxin transport inhibitors (N-1-naphthylphthalamic acid, 9-hydroxyfluorene-9-carboxylic acid, and 2,3,5-triiodobenzoic acid) and in medium containing a less well-characterized inhibitor of auxin-mediated processes, 2-(p-chlorophynoxy)-2-methylpropionic acid. Cotyledon vein pattern was not affected by any inhibitor treatments, although vein morphology was altered. In contrast, leaf vein pattern was affected by inhibitor treatments. Growth in polar auxin transport inhibitors resulted in leaves that lacked vascular continuity through the petiole and had broad, loosely organized midveins, an increased number of secondary veins, and a dense band of misshapen tracheary elements adjacent to the leaf margin. Analysis of leaf vein pattern developmental time courses suggested that the primary vein did not develop in polar auxin transport inhibitor-grown plants, and that the broad midvein observed in these seedlings resulted from the coalescence of proximal regions of secondary veins. Possible models for leaf vein patterning that could account for these observations are discussed.     

The procambium specification gene Oshox1 promotes polar auxin transport capacity and reduces its sensitivity toward inhibition

The auxin-inducible homeobox gene Oshox1 of rice (Oryza sativa) is a positive regulator of procambial cell fate commitment, and its overexpression reduces the sensitivity of polar auxin transport (PAT) to the PAT inhibitor 1-N-naphthylphthalamic acid (NPA). Here, we show that wild-type rice leaves formed under conditions of PAT inhibition display vein hypertrophy, reduced distance between longitudinal veins, and increased distance between transverse veins, providing experimental evidence for a role of PAT in vascular patterning in a monocot species. Furthermore, we show that Oshox1 overexpression confers insensitivity to these PAT inhibitor-induced vascular-patterning defects. Finally, we show that in the absence of any overt phenotypical change, Oshox1 overexpression specifically reduces the affinity of the NPA-binding protein toward NPA and enhances PAT and its sensitivity toward auxin. These results are consistent with the hypothesis that Oshox1 promotes fate commitment of procambial cells by increasing their auxin conductivity properties and stabilizing this state against modulations of PAT by an endogenous NPA-like molecule.     

Root meristems in Medicago truncatula tissue culture arise from vascular-derived procambial-like cells in a process regulated by ethylene

Leaf explants of Medicago truncatula were used to investigate the origins of auxin-induced root formation. On the application of auxin there is some callus formation (not the massive amount that occurs in response to auxin plus cytokinin) and roots appear shortly after the first visible callus. Histological examination reveals morphologically distinctive sheets of callus cells that emanate from the veins of the leaf explants and, within this cell type, root primordia are produced as well as some vascular tissue cells. What is suggested is that the vein-derived cells (VDCs) are procambial-like and function as pluripotent stem cells with a propensity to form root meristems or vascular tissues in response to added auxin. The development of root primordia from these pluripotent cells was clearly up-regulated by the use of the sickle (skl) mutant, which is a mutant impaired in ethylene signal transduction while the wild type and the sunn mutant, defective in auxin polar transport, produced similar numbers of roots. The skl mutant in generating many more roots concomitantly formed fewer vascular tissues. The root meristems differentiate similarly to normal roots producing a central cylinder of vascular tissue, which connects with the leaf explant veins. The VDCs appear to be derived from the cells of or near the phloem. The leaf observations suggest that a pool of stem cells exist in vascular tissue that, in combination with auxin and perhaps other factors, drive a diversity of plant development outcomes that is species specific. The way auxin interacts with other hormones is a key factor in determining the stem cell fate. The histological data in this study also assist in the interpretation of the molecular analysis of auxin-induced root formation in cultured leaves of M. truncatula.     

Hydraulic analysis of water flow through leaves of sugar maple and red oak

Leaves constitute a substantial fraction of the total resistance to water flow through plants. A key question is how hydraulic resistance within the leaf is distributed among petiole, major veins, minor veins, and the pathways downstream of the veins. We partitioned the leaf hydraulic resistance (R(leaf)) for sugar maple (Acer saccharum) and red oak (Quercus rubra) by measuring the resistance to water flow through leaves before and after cutting specific vein orders. Simulations using an electronic circuit analog with resistors arranged in a hierarchical reticulate network justified the partitioning of total R(leaf) into component additive resistances. On average 64% and 74% of the R(leaf) was situated within the leaf xylem for sugar maple and red oak, respectively. Substantial resistance-32% and 49%- was in the minor venation, 18% and 21% in the major venation, and 14% and 4% in the petiole. The large number of parallel paths (i.e. a large transfer surface) for water leaving the minor veins through the bundle sheath and out of the leaf resulted in the pathways outside the venation comprising only 36% and 26% of R(leaf). Changing leaf temperature during measurement of R(leaf) for intact leaves resulted in a temperature response beyond that expected from changes in viscosity. The extra response was not found for leaves with veins cut, indicating that water crosses cell membranes after it leaves the xylem. The large proportion of resistance in the venation can explain why stomata respond to leaf xylem damage and cavitation. The hydraulic importance of the leaf vein system suggests that the diversity of vein system architectures observed in angiosperms may reflect variation in whole-leaf hydraulic capacity.     

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.     

Leaf anatomy and ultrastructure of the Crassulacean-acid-metabolism plant Kalanchoe daigremontiana

Light-microscopic analysis of leaf clearings of the obligate Crassulacean-acid-metabolism (CAM) species Kalanchoe daigremontiana Hamet et Perr. has shown the existence of unusual and highly irregular venation patterns. Fifth-order veins exhibit a three-dimensional random orientation with respect to the mesophyll. Minor veins were often observed crossing over or under each other and over and under major veins in the mesophyll. Paraffin sections of mature leaves show tannin cells scattered throughout the mesophyll rather evenly spaced, and a distinct layer of tannin cells below the abaxial epidermis. Scanning electron microscopy showed that bundle-sheath cells are distinct from the surrounding mesophyll in veins of all orders. Transmission electron microscopy demonstrated developing sieve-tube elements in expanded leaves. Cytosolic vesicles produced by dictyosomes undergo a diurnal variation in number and were often observed in association with the chloroplasts. These vesicles are an interesting feature of cell ultrastructure of CAM cells and may serve a regulatory role in the diurnal malic-acid fluctuations in this species.Abbreviations CAM Crassulacean acid metabolism - SEM scanning-electron microscopy - TEM transmission-electron microscopy     

Asymmetric auxin response precedes asymmetric growth and differentiation of asymmetric leaf1 and asymmetric leaf2 Arabidopsis leaves

We have analyzed the development of leaf shape and vascular pattern in leaves mutant for ASYMMETRIC LEAVES1 (AS1) or AS2 and compared the timing of developmental landmarks to cellular response to auxin, as measured by expression of the DR5:beta-glucuronidase (GUS) transgene and to cell division, as measured by expression of the cycB1:GUS transgene. We found that the earliest visible defect in both as1 and as2 first leaves is the asymmetric placement of auxin response at the distal leaf tip. This precedes visible changes in leaf morphology, asymmetric placement of the distal margin gap, formation of margin gaps along the leaf border, asymmetric distribution of marginal auxin, and asymmetry in cell division patterns. Moreover, treatment of developing leaves with either exogenous auxin or an auxin transport inhibitor eliminates asymmetric auxin response and subsequent asymmetric leaf development. We propose that the initial asymmetric placement of auxin at the leaf tip gives rise to later asymmetries in the internal auxin sources, which subsequently result in asymmetrical cell differentiation and division patterns.     

How canalization can make loops: a new model of reticulated leaf vascular pattern formation

Formation of the vascular system in plant leaves can be explained by the canalization hypothesis which states that veins are formed in an initially homogeneous field by a self-organizing process between the plant hormone auxin and auxin carrier proteins. Previous models of canalization can generate vein patterns with branching but fail to generate vein patterns with closed loops. However, closed vein loops are commonly observed in plant leaves and are important in making them robust to herbivore attacks and physical damage. Here we propose a new model which generates a vein system with closed loops. We postulate that the "flux bifurcator" level is enhanced in cells with a high auxin flux and that it causes reallocation of auxin carriers toward neighbouring cells also having a high bifurcator level. This causes the auxin flux to bifurcate, allowing vein tips to attach to other veins creating vein loops. We explore several alternative functional forms for the flux bifurcator affecting the reallocation of efflux carriers and examine parameter dependence of the resulting vein pattern.     

ONTOGENY OF FOLIAR VENATION IN EUPHORBIA FORBESII

Six species of Euphorbia endemic to the Hawaiian Islands have disjunct veins as a normal component of their foliar anatomy. An ontogenic study of the foliar venation of one of these species, E. forbesii, showed a normal development of the foliar procambium as determined by previous studies of dicotyledonous leaves. The disjunct veinlets are isolated early in the histogenesis of the intersecondary veins when certain procambial cells fail to differentiate into vascular tissue. It appears that these cells develop into normal parenchymatous cells of the ground tissue. It is suggested that these cells are physiologically distinct from the rest of the procambial cells. In no instance was a tracheary element seen which appeared to have arisen independently of the normal procambial reticulum.