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.     

Auxin-Responsive DR5 Promoter Coupled with Transport Assays Suggest Separate but Linked Routes of Auxin Transport during Woody Stem Development in Populus

Polar auxin transport (PAT) is a major determinant of plant morphology and internal anatomy with important roles in vascular patterning, tropic growth responses, apical dominance and phyllotactic arrangement. Woody plants present a highly complex system of vascular development in which isolated bundles of xylem and phloem gradually unite to form concentric rings of conductive tissue. We generated several transgenic lines of hybrid poplar (Populus tremula x alba) with the auxin-responsive DR5 promoter driving GUS expression in order to visualize an auxin response during the establishment of secondary growth. Distinct GUS expression in the cambial zone and developing xylem-side derivatives supports the current view of this tissue as a major stream of basipetal PAT. However, we also found novel sites of GUS expression in the primary xylem parenchyma lining the outer perimeter of the pith. Strands of primary xylem parenchyma depart the stem as a leaf trace, and showed GUS expression as long as the leaves to which they were connected remained attached (i.e., until just prior to leaf abscission). Tissue composed of primary xylem parenchyma strands contained measurable levels of free indole-3-acetic acid (IAA) and showed basipetal transport of radiolabeled auxin (³H-IAA) that was both significantly faster than diffusion and highly sensitive to the PAT inhibitor NPA. Radiolabeled auxin was also able to move between the primary xylem parenchyma in the interior of the stem and the basipetal stream in the cambial zone, an exchange that was likely mediated by ray parenchyma cells. Our results suggest that (a) channeling of leaf-derived IAA first delineates isolated strands of pre-procambial tissue but then later shifts to include basipetal transport through the rapidly expanding xylem elements, and (b) the transition from primary to secondary vascular development is gradual, with an auxin response preceding the appearance of a unified and radially-organized vascular cambium.     

VARICOSE, a WD-domain protein, is required for leaf blade development

To gain insight into the processes controlling leaf development, we characterized an Arabidopsis mutant, varicose (vcs), with leaf and shoot apical meristem defects. The vcs phenotype is temperature dependent; low temperature growth largely suppressed defects, whereas high growth temperatures resulted in severe leaf and meristem defects. VCS encodes a putative WD-domain containing protein, suggesting a function involving protein-protein interactions. Temperature shift experiments indicated that VCS is required throughout leaf development, but normal secondary vein patterning required low temperature early in leaf development. The low-temperature vcs phenotype is enhanced in axr1-3 vcs double mutants and in vcs mutants grown in the presence of polar auxin transport inhibitors, however, vcs has apparently normal auxin responses. Taken together, these observations suggest a role for VCS in leaf blade formation.     

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.     

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.     

Auxin distribution and transport during embryogenesis and seed germi-nation of Arabidopsis

INTRODUCTIONAuxin plays an important role in regu1ating celldivision, e1ongation and differentiatiou, vascular tis-sue fOrmation[1], pollen deve1opment[2] and 1eafyhead fOrmation[3]. Adrin polar transport is be-1ieved to invohe in a variety of important growthand developmenial processes, including the patternfOrmation of eInbryO, leaf morphogenesis and theroot gravity response[4--8]. Auxin po1ar transportinhibitor has been proved essential illterference ofataln transport leading to patte…     

Integrating cellular and organismic aspects of vascular differentiation

Vascular differentiation can be studied at two levels, and they should complement one another: as an aspect of integrated plant development and as cellular processes. The differentiation of organized strands that connect between organs is induced by polar auxin flow, towards the roots. Anatomy, therefore, can be a complementary method of observing polarity and its changes. As expected for a self-correcting and essential system, vascular patterning mutations are relatively rare and have pleiotropic effects, including modifications of responses to auxin and its transport. Tissue polarity both expresses and depends on auxin transport, a feedback that could account for the determined nature of polarity as well as the gradual canalization of differentiation to vascular strands. This predicts that the molecules responsible for polarity will be localized gradually as differentiation proceeds. Further, a modified location of these molecules can be expected to precede anatomical expressions of a new, regenerated, polarity. Tracheary differentiation is probably the best studied example of cell differentiation. Within the plant, however, this differentiation is coupled to oriented cell growth either along or at right angles to the axis of auxin flow, depending on tissue competence. Differentiation is also coupled to the differentiation of the other components of the vascular system. There are, presumably, early joint stages to these differentiation processes, but what they are remains an intriguing problem.     

An auxin-dependent distal organizer of pattern and polarity in the Arabidopsis root

Root formation in plants involves the continuous interpretation of positional cues. Physiological studies have linked root formation to auxins. An auxin response element displays a maximum in the Arabidopsis root and we investigate its developmental significance. Auxin response mutants reduce the maximum or its perception, and interfere with distal root patterning. Polar auxin transport mutants affect its localization and distal pattern. Polar auxin transport inhibitors cause dramatic relocalization of the maximum, and associated changes in pattern and polarity. Auxin application and laser ablations correlate root pattern with a maximum adjacent to the vascular bundle. Our data indicate that an auxin maximum at a vascular boundary establishes a distal organizer in the root.     

Inhibited polar auxin transport results in aberrant embryo development in Norway spruce

Current hypotheses concerning the role of polar auxin transport in embryo development are entirely based on studies of angiosperms, while little is known about how auxin regulates pattern formation in gymnosperms. In this study, different developmental stages of somatic embryos of Norway spruce (Picea abies) were treated with the polar auxin transport inhibitor 1-N-naphtylphthalamic acid (NPA). Effects of the treatments on auxin content, embryo differentiation and programmed cell death (PCD) were analysed. During early embryo development, NPA-treatment led to increased indole-3-acetic acid (IAA) content, abnormal cell divisions and decreased PCD, resulting in aberrant development of embryonal tube cells and suspensors. Mature embryos that had been treated with NPA showed both apical and basal abnormalities. Typically the embryos had abnormal cotyledon formation and irregular cell divisions in the area of the root meristem. Our results show that polar auxin transport is essential for the correct patterning of both apical and basal parts of conifer embryos throughout the whole developmental process. Furthermore, the aberrant morhologies of NPA-treated spruce embryos are comparable with several auxin response and transport mutants in Arabidopsis. This suggests that the role of polar auxin transport is conserved between angiosperms and gymnosperms.     

Alteration of auxin polar transport in the Arabidopsis ifl1 mutants

The INTERFASCICULAR FIBERLESS/REVOLUTA (IFL1/REV) gene is essential for the normal differentiation of interfascicular fibers and secondary xylem in the inflorescence stems of Arabidopsis. It has been proposed that IFL1/REV influences auxin polar flow or the transduction of auxin signal, which is required for fiber and vascular differentiation. Assay of auxin polar transport showed that the ifl1 mutations dramatically reduced auxin polar flow along the inflorescence stems and in the hypocotyls. The null mutant allele ifl1-2 was accompanied by a significant decrease in the expression level of two putative auxin efflux carriers. The ifl1 mutants remained sensitive to auxin and an auxin transport inhibitor. The ifl1-2 mutant exhibited visible phenotypes associated with defects in auxin polar transport such as pin-like inflorescence, reduced numbers of cauline branches, reduced numbers of secondary rosette inflorescence, and dark green leaves with delayed senescence. The visible phenotypes displayed by the ifl1 mutants could be mimicked by treatment of wild-type plants with an auxin polar transport inhibitor. In addition, the auxin polar transport inhibitor altered the normal differentiation of interfascicular fibers in the inflorescence stems of wild-type Arabidopsis. Taken together, these results suggest a correlation between the reduced auxin polar transport and the alteration of cell differentiation and morphology in the ifl1 mutants.     

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.     

Dynamics of MONOPTEROS and PIN-FORMED1 expression during leaf vein pattern formation in Arabidopsis thaliana

Genetic evidence links the Arabidopsis MONOPTEROS (MP) and PIN-FORMED1 (PIN1) genes to the patterning of leaf veins. To elucidate their potential functions and interactions in this process, we have assessed the dynamics of MP and PIN1 expression during vascular patterning in Arabidopsis leaf primordia. Both genes undergo a dynamic process of gradual refinement of expression into files one to two cells wide before overt vascular differentiation. The subcellular distribution of PIN1 is also gradually refined from a non-polar distribution in isodiametric cells to strongly polarized in elongated procambial cells and provides an indication of overall directions of auxin flow. We found evidence that MP expression can be activated by auxin exposure and that PIN1 as well as DR5::GUS expression is defective in mp mutant leaves. Taken together the results suggest a feedback regulatory loop that involves auxin, MP and PIN1 and provide novel experimental support for the canalization-of-auxin-flow hypothesis.     

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.     

VAN3 ARF-GAP-mediated vesicle transport is involved in leaf vascular network formation

Within the leaf of an angiosperm, the vascular system is constructed in a complex network pattern called venation. The formation of this vein pattern has been widely studied as a paradigm of tissue pattern formation in plants. To elucidate the molecular mechanism controlling the vein patterning process, we previously isolated Arabidopsis mutants van1 to van7, which show a discontinuous vein pattern. Here we report the phenotypic analysis of the van3 mutant in relation to auxin signaling and polar transport, and the molecular characterization of the VAN3 gene and protein. Double mutant analyses with pin1, emb30-7/gn and mp, and physiological analyses using the auxin-inducible marker DR5::GUS and an auxin transport inhibitor indicated that VAN3 may be involved in auxin signal transduction, but not in polar auxin transport. Positional cloning identified VAN3 as a gene that encodes an adenosine diphosphate (ADP)-ribosylation factor-guanosine triphosphatase (GTPase) activating protein (ARF-GAP). It resembles animal ACAPs and contains four domains: a BAR (BIN/amphiphysin/RVS) domain, a pleckstrin homology (PH) domain, an ARF-GAP domain and an ankyrin (ANK)-repeat domain. Recombinant VAN3 protein showed GTPase-activating activity and a specific affinity for phosphatidylinositols. This protein can self-associate through the N-terminal BAR domain in the yeast two-hybrid system. Subcellular localization analysis by double staining for Venus-tagged VAN3 and several green-fluorescent-protein-tagged intracellular markers indicated that VAN3 is located in a subpopulation of the trans-Golgi network (TGN). Our results indicate that the expression of this gene is induced by auxin and positively regulated by VAN3 itself, and that a specific ACAP type of ARF-GAP functions in vein pattern formation by regulating auxin signaling via a TGN-mediated vesicle transport system.