Vascular Tissue Differentiation Research Articles

Evolutionary patterns in auxin action.

This review represents the first effort ever to survey the entire literature on auxin (indole-3-acetic acid, IAA) action in all plants, with special emphasis on the green plant lineage, including charophytes (the green alga group closest to the land plants), bryophytes (the most basal land plants), pteridophytes (vascular non-seed plants), and seed plants. What emerges from this survey is the surprising perspective that the physiological mechanisms for regulating IAA levels and many IAA-mediated responses found in seed plants are also present in charophytes and bryophytes, at least in nascent forms. For example, the available evidence suggests that the apical regions of both charophytes and liverworts synthesize IAA via a tryptophan-independent pathway, with IAA levels being regulated via the balance between the rates of IAA biosynthesis and IAA degradation. The apical regions of all the other land plants utilize the same class of biosynthetic pathway, but they have the potential to utilize IAA conjugation and conjugate hydrolysis reactions to achieve more precise spatial and temporal control of IAA levels. The thallus tips of charophytes exhibit saturable IAA influx and efflux carriers, which are apparently not sensitive to polar IAA transport inhibitors. By contrast, two divisions of bryophyte gametophytes and moss sporophytes are reported to carry out polar IAA transport, but these groups exhibit differing sensitivities to those inhibitors. Although the IAA regulation of charophyte development has received almost no research attention, the bryophytes manifest a wide range of developmental responses, including tropisms, apical dominance, and rhizoid initiation, which are subject to IAA regulation that resembles the hormonal control over corresponding responses in seed plants. In pteridophytes, IAA regulates root initiation and vascular tissue differentiation in a manner also very similar to its effects on those processes in seed plants. Thus, it is concluded that the seed plants did not evolve de novo mechanisms for mediating IAA responses, but have rather modified pre-existing mechanisms already operating in the early land plants. Finally, this paper discusses the encouraging prospects for investigating the molecular evolution of auxin action.

Harlequin (hlq) and short blue root (sbr), two Arabidopsis mutants that ectopically express an abscisic acid- and auxin-inducible transgenic carrot promoter and have pleiotropic effects on morphogenesis.

Plant growth and development is regulated by complex interactions among different hormonal, developmental and environmental signalling pathways. Isolation of mutants in these processes is a powerful approach to dissect unknown mechanisms in regulatory networks. The plant hormones abscisic acid (ABA) and auxin are involved in vegetative, developmental and environmental growth responses, including cell division and elongation, vascular tissue differentiation and stress adaptation. The uidA (beta-glucuronidase; GUS) reporter gene driven by the carrot (Daucus carota) late embryogenesis-abundantDc3 promoter in transgenic Arabidopsis thaliana seedlings is ABA-inducible in the root zone of elongation and vasculature. We show here that the ABA-insensitive2-1 mutation (abi2) reduces ABA-inducible Dc3-GUS expression in these root tissues. Dc3-GUS expression is also induced in root cortex cells by indole-3-acetic acid. We mutagenized, with ethyl methane sulfonate, 5100 M1 abi2/abi2 homozygous plants of a line that carries two independent Dc3-GUS reporter genes and screened M2 clonal lines for ABA-inducible Dc3-GUS expression in roots. We isolated two novel single-gene nuclear mutants, harlequin (hlq) and short blue root (sbr), that ectopically express Dc3-GUS in roots and have pleiotropic effects on morphogenesis. The hlq mutant expresses Dc3-GUS in a checkered pattern in epidermis of roots and hypocotyls, accumulates callose and has deformed and collapsed epidermal cells and abnormal and reduced root hairs and leaf trichomes. It (hlq) is also dwarfed, skotomorphogenic and sterile. The sbr mutant is a seedling-lethal dwarf that over-expresses Dc3-GUS in the root and has radially swollen epidermal cells in the root and hypocotyl, supernumerary cell number in the root cortex and epidermis, abnormal vasculature, and abnormal epidermal cell patterning in cotyledons and leaves. It (sbr) also exhibits a semidominant root phenotype of reduced growth and lateral root initiation. The hlq and sbr mutants are not rescued by exogenous application of plant growth regulators. The hlq and sbr mutants do not require the abi2-1 mutant gene for their phenotypes and map to chromosome III and I, respectively. Further characterization of the hlq and sbr phenotypes and genes may provide insights into the relationship of hormone- and stress-regulated gene expression to morphogenesis and plant growth.

The Cleavable N-terminal Domain of Plant Endopolygalacturonases from Clade B May Be Involved in a Regulated Secretion Mechanism

Polygalacturonases represent the most abundant carbohydrate hydrolase family in the Arabidopsis thaliana genome, and they are thought to be involved in nearly all of the developmental processes requiring cell wall modifications during the life cycle of the plant. By phylogenetic analysis, plant polygalacturonases fall into at least three groups, one of which is distinguished from the others by the presence of an additional N-terminal domain. We have used RDPG1, the polygalacturonase involved in pod dehiscence in oilseed rape (Brassica napus), as a model to investigate the function of this domain. We have confirmed that this domain is absent in the mature protein by determination of the N-terminal sequence of mature RDPG1 purified from oilseed rape pod. We have furthermore investigated the accumulation and subcellular localization of the precursor containing the N-terminal domain and of the mature protein throughout the development and maturation of the pod. Using recombinant expression in Pichia pastoris, we have produced the RDPG1 precursor, and we present evidence that the N-terminal domain of plant polygalacturonases is not involved in folding or inactivation of the precursor but may play a role in the intracellular transport of this protein family via a novel regulated secretion pathway.

Foliar and Axial Aspects of Vascular Differentiation: Hypotheses and Evidence

A comparison is made between foliar and axial vascular differentiation. Current thoughts and new evidence are presented on the role of hormones in controlling the differentiation of vascular tissues in organized and tumorous tissues, focusing on the role of auxin and cytokinin in controlling phloem and xylem relationships, vessel size and density, cambium sensitivity, vascular adaptation and xylem evolution in deciduous hardwood trees. The possible role of wounding is also considered. A new hypothesis, namely, the leaf-venation hypothesis, is proposed to explain the hormonal control of vascular differentiation in leaves of dicotyledonous plants. Experimental evidence in support of the hypothesis is presented showing that hydathodes, the watersecreting glands, are the primary sites of auxin synthesis during leaf morphogenesis. Vessel element patterns similar to those found in hydathodes were experimentally induced by exogenous auxin application.

Two differentially regulated Arabidopsis genes define a new branch of the DFR superfamily

Two tandem genes were identified on Arabidopsis chromosome II ( AtCRL1 and AtCRL2) encoding proteins with homology to members of the dihydroflavonol-4-reductase (DFR) superfamily. The encoded CRL1 and CRL2 proteins share 87% mutual amino acid sequence identity whereas their promoter regions are highly divergent, suggesting differential regulation of the CRL genes. Phylogenetic analysis placed CRL1 and CRL2 in a separate branch of the DFR superfamily. Northern blotting showed strong AtCRL1 induction by abscisic acid (ABA), drought, and heat shock, and high expression level in seeds, thus resembling the expression pattern of late embryogenic abundant ABA-responsive genes. Differential expression of the two genes during plant development was confirmed in plants expressing transcriptional fusions between the two promoters and the Escherichia coli β-glucuronidase reporter gene. This showed that, whereas high expression of AtCRL1 in mature seeds declines during subsequent vegetative growth, transcriptional activity from the AtCRL2 promoter increases during vegetative growth. Expression of both genes is restricted to vascular tissue. Based upon their homology to proteins involved in lignin synthesis, we propose that AtCRL2 is involved in generating conducting tissue late in development, while AtCRL1 is involved in vascular tissue differentiation and/or synthesis in the germinating embryos.

Characterization of a new begomovirus from Egypt infecting hollyhock (Althea rosea)

A viral isolate from Egypt associated with symptoms of enations and leaf curling on hollyhock (Althea rosea) was characterized at the cytopathological and molecular levels. Microscopic observations showed that enations resulted from a reorganization of the vascular tissues, including activation of a cambial activity in the phloem, the development of a palisade parenchyma in place of a spongy one and the differentiation of minor vascular tissues. From this isolate, the full-length DNA-A of a begomovirus (family Geminiviridae) was cloned and sequenced. This genome exhibited a genetic organization similar to that of other old-world begomoviruses like Tomato yellow leaf curl virus from Israel and Ageratum yellow vein virus from Singapore. However, its sequence was significantly distinct (similarity < 69%) from any other geminivirus. This begomovirus was thus considered as representative of a new viral species named Althea roseaenation virus (AREV). AREV was agroinfectious on Nicotiana benthamiana, on which it induced a severe leaf-curling and vein distortion, but could not re-establish infection on A. rosea. To determine if AREV was also associated with a similar disease affecting okra in Upper-Egypt, the partial sequence of the coat protein gene of an isolate was determined. It exhibited 90% nt identity with the hollyhock isolate (97% amino acid), suggesting a genetic heterogeneity in the begomovirus population associated with the enation diseases.

Expression of cysteine proteinase mRNA in chickpea ( Cicer arietinum L.) is localized to provascular cells in the developing root

Summary A full-length cDNA clone encoding a chickpea (Cicer arietinum L.) cysteine proteinase, homologous to rd21gene of Arabidopsis as well as to cysteine proteinase genes from legumes, was isolated. Expression of the mRNA in growing radicles has been located in a very precisely defined longitudinal zone in the stele, corresponding to the area of differentiation of vascular tissues. This result suggests that the encoded proteinase may be involved in programmed cell death events that precede vascular development.

Altered cytokinin distribution in the shootless maize mutant ed*41

The distribution of cytokinins is strongly altered in seedlings of the shootless ed*41 mutant of maize (Zea mays L.), compared with wild-type. Immunolocalisation of zeatin and analysis of cytokinin levels clearly indicate that these hormones are not present in the shoot apex zone of the mutant. Since an anomalous differentiation of vascular tissues has also been observed in the mutant, a major role of vascular connection in hormone translocation affecting development of the shoot apical meristem is proposed. Immunolocalisation of zeatin was confined to the root cap, cortex and vascular tissues of both mutant and wild-type seedlings suggesting a tissue-specific synthesis of this hormone in the root. A time-course of cytokinin distribution in the wild-type developing shoot provided evidence that the tunica and corpus zones become competent to respond to cytokinins in subsequent periods, very probably in conjunction with photomorphogenesis. On the contrary, this pathway is totally disrupted in the mutant. Taken together, the data point to a relationship between the ed*41 mutation, inadequate vascular connection and disrupted hormone translocation.

Surgical Experiments on the Differentiation of Vascular Tissue in the Shoot Apex of Carrot (Daucus carota L.)

Surgical techniques were applied to the shoot apex of carrot (Daucus carota L.) to test the interpretation that provascular tissue is the initial stage of vascular differentiation and to localize the sources of the influences that control its differentiation. If the apex is isolated laterally by vertical incisions leaving it at the summit of a plug of pith tissue, vascular differentiation proceeds normally and an independent vascular system is formed in the pith plug. If all leaf primordia are systematically suppressed, provascular tissue continues to differentiate as an acropetal extension of the pre-existing vascular system but no further differentiation occurs. When the apex is isolated laterally and all leaf primordia are suppressed, provascular tissue continues to be formed acropetally and is extended basipetally into the pith plug by redifferentiation of pith cells, but no further differentiation occurs. This tissue reacts positively to histochemical tests for esterase indicating its vascular nature. If only one leaf primordium is allowed to develop on an isolated shoot apex, its vascular system develops normally and extends basipetally into the pith plug, but there is no extension of provascular tissue into the pith plug. These results support the interpretation that the initial stage of vascular differentiation is controlled by the apical meristem but that further maturation of vascular tissue depends upon influences from developing leaf primordia.

The role of the Arabidopsis ELD1 gene in cell development and photomorphogenesis in darkness.

Because cell growth and differentiation are regulated by complex interactions among different signaling pathways, a growth defect affects subsequent differentiation. We report on a growth-defective mutant of Arabidopsis, called eld1 (elongation defective 1). Cell elongation was impaired in every organ examined. Later characteristics of the eld1 phenotype include defective vascular tissue differentiation, the inability to grow in soil, ectopic deposition of suberin around twisted vascular bundles, the de-etiolation phenotype, and continuation of shoot development and flowering in the dark. The dwarf phenotype of eld1 could not be rescued by treatment with exogenous growth regulators. Because defective cell elongation is the earliest and most universal feature detected in eld1 mutants, control of or activity in cell elongation may be the primary function of the ELD1 gene. The impaired cell growth results in pleiotropic effects on cell proliferation and differentiation, and the retardation in hypocotyl elongation enables growth and development in darkness.

In situ hybridization study of the rice phloem thioredoxin h mRNA accumulation – possible involvement in the differentiation of vascular tissues

Thioredoxin (TRX) h was identified as a major protein in rice (Oryza sativa L. cv. Kantou) phloem sap. Accumulation of TRX h mRNA in fully expanded rice leaves was detected only in the companion cells. To clarify the role of TRX h in phloem development, we performed in situ hybridization of TRX h mRNA in rice plants during the course of vascular development. TRX h mRNA was detected in all cell types at an early stage of development and became gradually confined to the companion cells in later stages. The possible roles of TRX h in vascular development are discussed.

Expression of a polygalacturonase associated with tomato seed germination.

Radicle protrusion from tomato (Lycopersicon esculentum Mill.) seeds to complete germination requires weakening of the endosperm tissue opposite the radicle tip. In common with other cell wall disassembly processes in plants, polygalacturonases (PGs) may be involved. Only calcium-dependent exo-PG activity was detected in tomato seed protein extracts. Chromatographic profiles of a partially acid-hydrolyzed fraction of polygalacturonic acid further digested with seed extract were consistent with the presence of only calcium-dependent exo-PG activity. In addition, a transcript encoding a previously unknown PG was detected prior to the completion of germination. The mRNA, produced from a gene (LeXPG1) estimated by Southern analysis to be represented once in the genome, was also present in flowers (anthers) and in lower amounts in roots and stems. LeXPG1 mRNA abundance was low during seed development, increased during imbibition, and was even greater in seeds that had completed germination. Expression of LeXPG1 during germination predominates in the endosperm cap and radicle tip, and in the radicle appears as a distinct band possibly associated with vascular tissue differentiation. We suggest that PG is involved in cell wall loosening of the endosperm necessary for radicle protrusion from tomato seeds and in subsequent embryo and seedling growth.

Histochemical detection and image analysis of non-specific esterase activity and the amount of polyphenols during annual bud development in Norway spruce

This study focused on Norway spruce trees during their annual development and, specifically, on the histochemical analysis of the activity of the non-specific esterase and the amount of polyphenols in the vegetative buds. Azo-coupling reaction with naphthol-AS-acetate as a substrate and Fast Blue B Salt as a coupling reagent was applied to the longitudinal median cryo-sections of the formalin-fixed buds. A method of image analysis was developed to evaluate the intensity of the histochemical reaction quantitatively. High activity of non-specific esterase was detected in the cells of the collenchymatic plate, the youngest bud scales, the leaf primordia, in the zone of differentiation of vascular tissues and procambium of the dormant bud. The activity of non-specific esterase decreased in all tissues during bud break. During the growth of a new shoot enzyme activity was detected only in the vascular bundles of the shoot. Enzyme activity re-occurred at the beginning of the autumn in the cells of the youngest bud scales and in the cells of the collenchymatic plate of the newly establishing buds. Three different types of polyphenols at different intracellular localizations (as verified by scanning electron microscopy) were observed: granular, vacuolar and drop-like polyphenols. The amount and localization of those polyphenols changed during the bud growing cycle. The positive reaction of all three types of polyphenols with vanillin-HCl proved that these polyphenols are tannin in nature. These findings are discussed from the viewpoint of frost tolerance and tissue differentiation.

Initial Differentiation of Vascular Tissue in the Shoot Apex of Carrot (Daucus carotaL.)

Although an initial stage of vascular differentiation preceding procambium has been demonstrated in ferns, its presence in seed plants has not been accepted generally. In the shoot apex of carrot, a short cylinder of provascular tissue is recognized as the initial stage of vascular differentiation. This first becomes apparent through the enlargement and vacuolation of pith and cortical tissue rather than as a result of specific changes in the provascular tissue itself. Procambium in discrete strands differentiates acropetally in the provascular tissue in relation to developing leaf primordia. Provascular tissue is not recognized above the axil of the youngest leaf primordium but it is distinct at or above the level at which the trace of the youngest primordium diverges. Support for the recognition of provascular tissue is provided by a positive reaction to histochemical tests for carboxylesterases in this tissue as well as in procambium and later stages of vascular differentiation.

Polarity, vascularization and auxin

Polarity, vascularization and auxin

Developmental logic: establishing a relationship between developmental process and phylogenetic pattern in primitive vascular plants

Cladistic methods serve as a powerful example of the current shift in epistemology from a largely inductivist stance toward more `hypothetico-deductive' or `strong inference' approaches. However, in the area of character analysis, especially in addressing the roles of developmental and evolutionary processes influencing our understanding of homology, synapomorphy, and evolutionary pattern, it appears that little has changed. This omission has the very real potential of substantially limiting resolution and reliability of cladistic hypotheses, and points to the need for an expanded methodology that simultaneously addresses evolutionary pattern and underlying causal factors. Due to unique aspects of both plant development and evolutionary history, early vascular plants offer attractive features that make them highly suitable as a test case along these lines. Expanding on the Zimmermann's `telome theory' of a strong inference approach to developmental process is suggested by employing an analogy with logic gates and logic circuits in programming theory. Hypothesized units of developmental dynamic, termed `evolutionary developmental gates' (EDG) define a relationship between external and internal `environmental' factors, and developmental outcome, using a logical conditional. EDGs are then assembled into networks, specifying a relationship between developmental processes and resultant morphological structure, that may be tested. Testing utilizes several lines of evidence including comparing EDGs with specific developmental data, computer modelling, and evolutionary comparisons based on fossil and living taxa. To illustrate this approach, and to initiate analysis of what constitutes a set of developmental processes necessary for describing primitive vascular plant form, four developmental modules comprised of networks of EDGs are proposed. These include: (1) establishment of self-recognition at the shoot apex along with initiation of a hormone (auxin) gradient, (2) normal cell division modeled as an iterative and recursive process, (3) developmental cascades leading to tissue differentiation of epidermis, cortex, and vascular tissues, and (4) establishment of new apices either by bifurcation or de novo formation. Although clearly more detail is required to enable important evolutionary comparisons, it is anticipated that networks of EDGs can comprise a more realistic perspective with which to estimate homology in evolutionary studies.

Adventitious root formation in cuttings of loblolly pine (Pinus taeda L .): developmental sequence and effects of maturation

Adventitious root formation in cuttings from fascicular shoots in loblolly pine (Pinus taeda L.) consists of four more or less discontinuous stages: (1) proliferation of cells at the base of the cutting, (2) differentiation of wound vascular tissue and periderm, (3) dedifferentiation of a zone near the wound cambium and wound phloem to form a root initial, and (4) formation of a root meristem. Anatomical changes during adventitious root initiation are described in cuttings from donors of different types and ages. Cuttings from seedlings and 3- to 7-year-old hedged stock plants rooted better than cuttings from 3-year-old tree form donors. It is concluded that the loss of rooting capacity in loblolly pine can be arrested by shearing loblolly pine stock plants to low hedges. The process of root initiation, however, was similar in cuttings from all sources and is apparently not the cause for the rapid decline of rooting potential with increasing age of the donor plant.

Transforming growth factor-beta dynamically regulates vascular smooth muscle differentiation in vivo.

Variations in the levels of smooth muscle-specific isoforms of contractile proteins have been reported to occur in many different vascular diseases. However, although much work has been done in vitro to investigate the regulation of smooth muscle cell differentiation, the molecular mechanisms which regulate the differentiation of vascular smooth muscle tissue in vivo are unknown. Using quantitative immunofluorescence, we show that in rat arteries levels of smooth muscle differentiation markers correlate with the levels of the cytokine TGF-beta. In young mice with one allele of the TGF-beta1 gene deleted, the levels of both TGF-beta1 and smooth muscle differentiation markers are reduced compared to wild-type controls. This regulation of smooth muscle differentiation by TGF-beta during post-natal development also occurs dynamically in the adult animal. Following various pharmacological or surgical interventions, including treatment of mice with tamoxifen and balloon injury of rat carotid arteries, there is a strong correlation between the changes in the levels of TGF-beta and changes in the levels of smooth muscle differentiation markers (r=0. 9, P<0.0001 for n=26 experiments). We conclude that TGF-beta dynamically regulates smooth muscle differentiation in rodent arteries in vivo.

Transforming growth factor-β dynamically regulates vascular smooth muscle differentiation in vivo

ABSTRACT Variations in the levels of smooth muscle-specific isoforms of contractile proteins have been reported to occur in many different vascular diseases. However, although much work has been done in vitro to investigate the regulation of smooth muscle cell differentiation, the molecular mechanisms which regulate the differentiation of vascular smooth muscle tissue in vivo are unknown. Using quantitative immunofluorescence, we show that in rat arteries levels of smooth muscle differentiation markers correlate with the levels of the cytokine TGF-β. In young mice with one allele of the TGF-β1 gene deleted, the levels of both TGF-β1 and smooth muscle differentiation markers are reduced compared to wild-type controls. This regulation of smooth muscle differentiation by TGF-β?during post-natal development also occurs dynamically in the adult animal. Following various pharmacological or surgical interventions, including treatment of mice with tamoxifen and balloon injury of rat carotid arteries, there is a strong correlation between the changes in the levels of TGF-β and changes in the levels of smooth muscle differentiation markers (r=0.9, P&amp;lt;0.0001 for n=26 experiments). We conclude that TGF-β dynamically regulates smooth muscle differentiation in rodent arteries in vivo.

Imaging of coronary artery microstructure (in vitro) with optical coherence tomography

OCT achieves high-resolution and image differentiation of vascular tissues to a degree that has not been previously possible with any method except excisional biopsy. Thus, OCT represents a promising new diagnostic technology for intracoronary imaging, which could permit the in vivo evaluation of critical vascular pathology.