Auxin Influx Carriers Control Vascular Patterning and Xylem Differentiation in Arabidopsis thaliana

Auxin is an essential hormone for plant growth and development. Auxin influx carriers AUX1/LAX transport auxin into the cell, while auxin efflux carriers PIN pump it out of the cell. It is well established that efflux carriers play an important role in the shoot vascular patterning, yet the contribution of influx carriers to the shoot vasculature remains unknown. Here, we combined theoretical and experimental approaches to decipher the role of auxin influx carriers in the patterning and differentiation of vascular tissues in the Arabidopsis inflorescence stem. Our theoretical analysis predicts that influx carriers facilitate periodic patterning and modulate the periodicity of auxin maxima. In agreement, we observed fewer and more spaced vascular bundles in quadruple mutants plants of the auxin influx carriers aux1lax1lax2lax3. Furthermore, we show AUX1/LAX carriers promote xylem differentiation in both the shoot and the root tissues. Influx carriers increase cytoplasmic auxin signaling, and thereby differentiation. In addition to this cytoplasmic role of auxin, our computational simulations propose a role for extracellular auxin as an inhibitor of xylem differentiation. Altogether, our study shows that auxin influx carriers AUX1/LAX regulate vascular patterning and differentiation in plants.     

Evaluation of the role of hormonal factors in secondary growth of dicots

The present investigation was carried out with a view to finding out the role of hormonal factors in secondary growth in stems ofCapsicum annuum andSolanum melongena. Seedlings were treated with aqueous solutions of growth substances and observations were made in respect to initiation of cambium, magnitude of secondary growth and differentiation of the secondary vascular tissues. Enhancement of secondary growth by auxins (IBA, IAA and NAA) and its partial or complete inhibition by auxin inhibitors (MH, PMN and DCA) clearly establishes the controlling nature of auxins on the process. Evidence has been presented demonstrating that auxins are more involved with differentiation of xylem, whereas GA is more involved with phloem. The investigation may be of economic value in improving the quality and quantity of wood.     

The Control of Vascular Branching in Coleus 1. The Side Bundle

Xylary branching at the proximal end of a differentiating sidebundle was modified by surgical alteration of the surroundingleaf traces and manipulation of their auxin fluxes. Incisionsthrough one corner trace, with the other pre-existing traceintact, resulting in xylem differentiation in the branch ofthe newly formed side bundle toward only the severed trace.Application of IAA to the cut trace allowed xylem branchingof the new strand in both directions. With sufficient auxinimbalance created by increasing the concentration of the appliedIAA, the new xylem strand branched away from the higher auxinsource. Auxin relations were thus able to regulate the courseof differentiation of vascular strands, but their role in regulatingbranching patterns in intact plants may be questioned. Xylembranched exclusively toward an incised trace only when the auxinflux of the incised trace was virtually eliminated. Phloem andprocambium of the differentiating strand were unaffected bythis treatment. Coleus, vascular differentiation, vascular anatomy, vascular branching, vascular patterns, auxin, auxin balance, node     

Polarity and the Induction of Organized Vascular Tissues

This work deals with those properties of plant tissues whichare responsible for the organization of vascular cells in orderedstrands. It is shown that auxin alone is sufficient to causethe differentiation of strands of xylem cells in the parenchymaof pea roots. An artificially induced strand, once it is formed,attracts towards itself newly induced vascular strands, andthis attraction results in the union of old and new strands.It is also shown that the application of auxin to natural vasculartissues prevents their being joined by newly induced vascularstrands. It is proved that this is dependent on a directionaleffect and not simply on a local accumulation of auxin. To understand these results, it must be assumed that the polarityin terms of auxin transport is increased during the processof vascular tissue induction. The same polarity, once established,is maintained by the presence of auxin, so that the differentiationof strands perpendicular to the axis of this polarity is prevented.These characteristics of plant tissues concerning auxin transportexplain the basic phenomena of the organization of vascularcells in defined and ordered strands.     

Patterned cell determination in a plant tissue: the secondary phloem of trees

The secondary vascular tissues (xylem and phloem) of woody plants originate from a vascular cambium and develop as radially oriented files of cells. The secondary phloem is composed of three or four cell types, which are organised into characteristic recurrent cellular sequences within the radial cell files of this tissue. There is a gradient of auxin (indole acetic acid) across both the cambium and the immediately postmitotic cells within the xylem and phloem domains, and it is believed that this morphogen, probably in concert with other morphogenic factors, is closely associated with the determination and differentiation of the different cells types in each tissue. A hypothesis is developed that, in conjunction with the positional values conferred by the graded radial distribution of morphogen, cell divisions at particular positions within the cambium are sufficient to determine not only each of the phloem cell types but also their recurrent pattern of differentiation within each radial cell file.     

Circular Vessels and the Control of Vascular Differentiation in Plants

The occurrence of vessels in the form of rings is used as a critical example for a hypothesis about the control of the pattern of cells in vascular tissues. These vessels, rare in intact plants, are common in the basal or root side of tissues close to transverse wounds of bean seedlings, radish storage tissues, and other plant material. Their formation is promoted, as are normal vascular tissues, by developing parts of the shoot or by a source of the hormone auxin. They are also found in grafts where cells of opposite polarities are close together, and in cut plants where vascular induction occurs from the direction of the roots and is therefore opposite to the original polarity of the tissue. Circular vessels are found, therefore, where the flux of auxin and possibly other signals controlling vascular differentiation is expected to follow a circular route. They show that differentiation is a response of individual cells to the flux rather than the gradient or concentration of the hormonal signals and suggest a hormonal interpretation of differences between apical and basal callus growth.     

The position of regenerating cambia: auxin/sucrose ratio and the gradient induction hypothesis.

To account for the positions in which vascular cambia regenerate in wound callus, a gradient induction hypothesis was proposed in 1961 in terms of gradients in 'some factor as yet unknown'. It now seems likely that the gradient is based on morphogen diffusion between source and sink on opposite sides of existing cambia, with morphogen diffusing into the adjoining wound callus. It is specifically proposed that there are two morphogens, auxin diffusing centrifugally and sucrose diffusing centripetally. The cambium then regenerates along a path where the ratio of auxin to sucrose concentration is similar to that at the original cambium, and its orientation (as regards xylem and phloem formation) is determined by the direction of the gradient in this ratio. These proposals are supported by published evidence on auxin and sucrose concentration gradients across the cambium, and on their sources, movements, and known effects on vascular differentiation. Simulations of the proposed positional control system predict patterns of cambial regeneration and orientation corresponding to those observed in four different types of wound and graft.     

Transformation of the collateral vascular bundles into amphivasal vascular bundles in an Arabidopsis mutant.

Arabidopsis inflorescence stems develop a vascular pattern similar to that found in most dicots. The arrangement of vascular tissues within the bundle is collateral, and vascular bundles in the stele are arranged in a ring. Although auxin has been shown to be an inducer of vascular differentiation, little is known about the molecular mechanisms controlling vascular pattern formation. By screening ethyl methanesufonate-mutagenized populations of Arabidopsis, we have isolated an avb1 (amphivasal vascular bundle) mutant with a novel vascular pattern. Unlike the collateral vascular bundles seen in the wild-type stems, the vascular bundles in the avb1 stems were similar to amphivasal bundles, i.e. the xylem completely surrounded the phloem. Furthermore, branching vascular bundles in the avb1 stems abnormally penetrated into the pith, which resulted in a disruption in the ring-like arrangement of vascular bundles in the stele. The avb1 mutation did not affect leaf venation pattern and root vascular organization. Auxin polar transport assay indicated that the avb1 mutation did not disrupt the auxin polar transport activity in inflorescence stems. The avb1 mutation also exhibited pleiotropic phenotypes, including curled stems and extra cauline branches. Genetic analysis indicated that the avb1 mutation was monogenic and partially dominant. The avb1 locus was mapped to a region between markers mi69 and ASB2, which is covered by a yeast artificial chromosome clone, CIC9E2, on chromosome 5. Isolation of the avb1 mutant provides a novel means to study the evolutionary mechanisms controlling the arrangement of vascular tissues within the bundle, as well as the mechanisms controlling the arrangement of vascular bundles in the stele.     

The Induction of Differentiation of Organized Vessels in a Storage Organ

The organized differentiation of vascular tissues was studiedin a simple system which allowed vessel members to be followedindividually. Local application of auxin to pieces of turnipstorage root resulted in differentiation of vessel and sieveelements within two days. These are normally organized in alongitudinal fashion. The induction of differentiation is inhibitedby triiodobenzoic acid. The number of differentiated cells dependedon the auxin concentration and also on the length of time thetissue was allowed no differentiate. No vessel members wereobserved in less than 48 h and the minimum effective IAA concentrationwas 8 x 10^–6 M. The results established a simple, quantitativesystem for the study of vessel differentiation. Brassica campestris cv. Rapifera, auxin, differentiation, storage root, vessel, xylem     

The arabidopsis ATHB-8 HD-zip protein acts as a differentiation-promoting transcription factor of the vascular meristems

ATHB-8, -9, -14, -15, and IFL1/REV are members of a small homeodomain-leucine zipper family whose genes are characterized by expression in the vascular tissue. ATHB-8, a gene positively regulated by auxin (Baima et al., 1995), is considered an early marker of the procambial cells and of the cambium during vascular regeneration after wounding. Here, we demonstrate that although the formation of the vascular system is not affected in athb8 mutants, ectopic expression of ATHB-8 in Arabidopsis plants increased the production of xylem tissue. In particular, a careful anatomical analysis of the transgenic plants indicated that the overexpression of ATHB-8 promotes vascular cell differentiation. First, the procambial cells differentiated precociously into primary xylem. In addition, interfascicular cells also differentiated precociously into fibers. Finally, the transition to secondary growth, mainly producing xylem, was anticipated in transgenic inflorescence stems compared with controls. The stimulation of primary and secondary vascular cell differentiation resulted in complex modifications of the growth and development of the ATHB-8 transgenic plants. Taken together, these results are consistent with the hypothesis that ATHB-8 is a positive regulator of proliferation and differentiation, and participates in a positive feedback loop in which auxin signaling induces the expression of ATHB-8, which in turn positively modulates the activity of procambial and cambial cells to differentiate.     

Jasmonic acid modulates xylem development by controlling polar auxin transport in vascular tissues

Development of xylem cells is affected by environmental stresses such as drought and oxidative stress, and recent findings suggested that jasmonic acid (JA) mediates this process through interaction with other phytohormones such as cytokinin. In this study, we showed that polar auxin transport regulated by PIN3 and PIN7 is involved in the JA-mediated xylem development in vascular tissues. The mutant plants that lack the activity of PIN3 and PIN7 responsible for the auxin transport developed extra xylems in vascular tissues such as the JA-treated wild-type plants. Visualization of auxin response and xylem development in the roots treated with NPA, an inhibitor of polar auxin transport, suggested that disruption of polar auxin transport is involved in the xylem phenotype of pin3 pin7 double mutants. We also found that cytokinin increases expressions of PIN3 and PIN7 responsible for the auxin transport while JA decreases only PIN7. These suggested that PIN7-mediated polar auxin transport system modulates xylem development in response to JA. The finding that JA affects auxin distribution in root vascular tissues further supported this. Collectively, these suggest that JA promotes xylem development by disrupting auxin transport in vascular tissues, and the auxin efflux genes, more especially PIN7 whose expression is suppressed by JA mediates this process.     

Polarity,Continuity, and Alignment in Plant Vascular Strands

Plant vascular cells are joined end to end along uninterrupted lines to connect shoot organs with roots; vascular strands are thus polar, continuous, and internally aligned. What controls the formation of vascular strands with these properties? The “auxin canalization hypothesis”—based on positive feedback between auxin flow through a cell and the cell's capacity for auxin transport—predicts the selection of continuous files of cells that transport auxin polarly, thus accounting for the polarity and continuity of vascular strands. By contrast, polar, continuous auxin transport—though required—is insufficient to promote internal alignment of vascular strands, implicating additional factors. The auxin canalization hypothesis was derived from the response of mature tissue to auxin application but is consistent with molecular and cellular events in embryo axis formation and shoot organ development. Objections to the hypothesis have been raised based on vascular organizations in callus tissue and shoot organs but seem unsupported by available evidence. Other objections call instead for further research; yet the inductive and orienting influence of auxin on continuous vascular differentiation remains unique.     

EXPERIMENTAL INDUCTION OF VASCULAR TISSUES IN CALLUS OF ANGIOSPERMS

Wetmore , Ralph H. (Harvard U., Cambridge, Mass.), and John P. Rier . Experimental induction of vascular tissues in callus of angiosperms . Amer. Jour. Bot. 50(5): 418–430. Illus. 1963.—Callus tissues in established maintenance culture lack morphological and physiological organization. Such callus consists of homogeneous parenchyma. Movement of auxin and sugar, therefore, must be along diffusion gradients. The only vascular tissues occurring in callus are induced. Experimental induction of vascular tissues has been successful in callus of 3 sp. of the Oleaceae: a tree, Fraxinus americana, and 2 shrubs, Syringa vulgaris and Ligustrum vulgare; another tree, Salix purpurea, var. lambertiana; a vine, Parthenocissus tricuspidata; and an herb, Helianthus tuberosus. In each of these species, an auxin (IAA or NAA in these studies) and a sugar (sucrose or glucose in these studies) prove necessary for the induction and complete differentiation of xylem and phloem in callus tissues. Varying concentrations of sugar alter the proportions of xylem to phloem: low concentrations, 1.5%–2.5%, favor xylem formation; high, 3%–4%, favor phloem. Middle concentrations, 2.5%–3.5%, favor the presence of xylem and phloem, usually with a cambium between. The almost universal association of xylem and phloem may have its explanation in this middle concentration of sugar. Grafting of apices into callus or direct application of appropriate concentrations of an auxin and a sugar in agar to the surface of callus causes nodules of vascular tissue to be formed, mostly in a circular pattern when seen in section transverse to the axis of orientation of the callus in the medium. The diameter of this circle varies directly with the auxin concentration at the place of application, 0.05 mg/liter giving a narrow, and 1 mg/liter, a wide pith. In individual nodules, xylem is characteristically oriented towards the center of the callus and the phloem towards the outside. Variable cross-sectional views of nodule distribution in calli under different treatments suggest experimental approaches to understanding stelar patterns. The induction and differentiation in callus of xylem and phloem tissues has no relation to conduction. Any use of vascular tissues can occur only after their induction.     

Canalization without flux sensors: a traveling-wave hypothesis

In 1969, Tsvi Sachs published his seminal hypothesis of vascular development in plants: the canalization hypothesis. A positive feedback loop between the flux of the phytohormone auxin and the cells' auxin transport capacity would canalize auxin progressively into discrete channels, which would then differentiate into vascular tissues. Recent experimental studies confirm the central role of polar auxin flux in plant vasculogenesis, but it is unclear if and by which mechanism plant cells could respond to auxin flux. In this Opinion article, we review auxin perception mechanisms and argue that these respond more likely to auxin concentrations than to auxin flux. We propose an alternative mechanism for polar auxin channeling, which is more consistent with recent molecular observations.