Inhibition of phenylalanine ammonia-lyase activity causes the depression of lignin accumulation of secondary wall thickening in isolatedZinnia mesophyll cells

Summary Isolated mesophyll cells ofZinnia elegans L. cv. Canary Bird differentiate into tracheary elements in differentiation (D) medium. These elements develop lignified secondary wall thickenings. The influence of 2-aminoindan-2-phosphonic acid (AIP), an inhibitor of phenylalanine ammonia-lyase (PAL), on lignification ofZinnia tracheary elements was examined. The mesophyll cells were cultured in D and AIP media. The latter medium, in which 100 M AIP was added to the D medium, inhibited PAL activity, though the differentiation proceeded. Morphological differences of secondary wall thickenings cultured in these two types of media were investigated under an UV microscope and a transmission electron microscope. The secondary wall thickenings at 96 h in the D medium showed strong UV absorption. The fibrillar structure of the thickenings observed clearly at 72 h was covered with electron opaque materials by 96 h. The secondary wall thickenings at 96 h in the AIP medium showed weak UV absorption. The thickenings at 96 h had a cracked appearance. Furthermore, the thickenings showed a little irregular or wavy arrangement of cellulose microfibrils and had many pores and spaces between microfibrils. From these results, the role of lignin accumulation in the formation of secondary wall thickenings was discussed.Abbreviations AIP 2-aminoindan-2-phosphonic acid - PAL phenylalanine ammonia-lyase     

THE FINE STRUCTURE OF DIFFERENTIATING XYLEM ELEMENTS

Differentiating xylem elements of Avena coleoptiles have been examined by light and electron microscopy. Fixation in 2 per cent phosphate-buffered osmium tetroxide and in 6 per cent glutaraldehyde, followed by 2 per cent osmium tetroxide, revealed details of the cell wall and cytoplasmic fine structure. The localized secondary wall thickening identified the xylem elements and indicated their state of differentiation. These differentiating xylem elements have dense cytoplasmic contents in which the dictyosomes and elements of rough endoplasmic reticulum are especially numerous. Vesicles are associated with the dictyosomes and are found throughout the cytoplasm. In many cases, these vesicles have electron-opaque contents. "Microtubules" are abundant in the peripheral cytoplasm and are always associated with the secondary wall thickenings. These microtubules are oriented in a direction parallel to the microfibrillar direction of the thickenings. Other tubules are frequently found between the cell wall and the plasma membrane. Our results support the view that the morphological association of the "microtubules" with developing cell wall thickenings may have a functional significance, especially with respect to the orientation of the microfibrils. Dictyosomes and endoplasmic reticulum may have a function in some way connected with the synthetic mechanism of cell wall deposition.     

Non-lignified helical cell wall thickenings in root cortical cells of Aspleniaceae (Polypodiales): histology and taxonomical significance

Background and Aims

Extraxylary helical cell wall thickenings in vascular plants are not well documented, except for those in orchid velamen tissues which have been studied extensively. Reports on their occurrence in ferns exist, but detailed information is missing. The aim of this study is to focus on the broad patterns of structure and composition and to study the taxonomic occurrence of helical cell wall thickenings in the fern family Aspleniaceae.

Methods

Structural and compositional aspects of roots have been examined by means of light, electron, epifluorescence and laser scanning confocal microscopy. To assess the taxonomical distribution of helical cell wall thickenings a molecular phylogenetic analysis based on rbcL sequences of 64 taxa was performed.

Key Results

The helical cell wall thickenings of all examined species showed considerable uniformity of design. The pattern consists of helical, regularly bifurcating and anastomosing strands. Compositionally, the cell wall thickenings were found to be rich in homogalacturonan, cellulose, mannan and xyloglucan. Thioacidolysis confirmed our negative phloroglucinol staining tests, demonstrating the absence of lignins in the root cortex. All taxa with helical cell wall thickenings formed a monophyletic group supported by a 100 % bootstrap value and composed of mainly epiphytic species.

Conclusions

This is the first report of non-lignified pectin-rich secondary cell walls in ferns. Based on our molecular analysis, we reject the hypothesis of parallel evolution of helical cell wall thickenings in Aspleniaceae. Helical cell wall thickenings can mechanically stabilize the cortex tissue, allowing maximal uptake of water and nutrients during rainfall events. In addition, it can also act as a boundary layer increasing the diffusive pathway towards the atmosphere, preventing desiccation of the stele of epiphytic growing species.     

Pectin distribution pattern in the apertural intine of Euphorbia peplus L. (Euphorbiaceae) pollen

The apertural inner layer (intine) of Euphorbia L. pollen grains has a characteristic but original structure that has paired thickenings, one on either side of the colpus. To determine the nature and role of this intine layer, pollen grains of Euphorbia peplus L. were germinated in vivo and in vitro. The germination process involves wall changes that facilitate formation of the pollen tube and its subsequent growth. In the thickenings of the intine of E. peplus, the unesterified pectin epitopes are more densely localised in the inner part of the middle intine. No such epitopes are located in the intine portion adjacent to the plasma membrane (cellulosic endintine). Unesterified pectin epitopes are also localised in the outer part of the intine but are restricted to the centre of the aperture, around and in the pore. The de-esterification of pectins is very advanced at the time of dehiscence and pollen germination. The stratification of the aperture intine may take the following pathway at the time of germination: the thin outer zone of the intine in the pore region becomes disorganised and undergoes dissolution with liberation of unesterified and esterified pectins; the middle intine thickenings undergo an important elastic modification, but without liberation of unesterified pectins; the cellulosic inner intine is the progenitor of the pollen tube wall. This special intine of E. peplus is an adaptation to the hydration process preceding germination, increasing intine and pollen grain wall elasticity.     

Occurrence of Helical Thickenings on the Vessel Element Walls of Dicotyledonous Woods

One-hundred and forty taxa belonging to 38 families were examinedfor helical thickenings on their vessel element walls usingscanning electron microscopy. Helical thickenings covering theentire vessel element wall occur in seventeen taxa and in ninetaxa they are localized. The thickenings are present in somevessel elements only except in members of the Meliaceae. Thelatter have prominent thickenings on the narrow vessel elementwalls and fine thickenings or rarely, no thickenings on thewider vessel element walls. In the Meliaceae they occur as discontinuoushelices which merge with the vessel element walls at intervals.The systematic and diagnostic value of the thickenings is limitedbecause of their wide, morphological variations. There is noevidence for physiological or ecological significance of helicalthickenings. Their frequent presence in temperate and subtropicalregions requires an analysis of their probable adaptive significance. Scanning electron microscopy, wood anatomy, vessel element wall, helical thickenings     

Water transport in xylem conduits with ring thickenings

Helical or annular wall thickenings are not only present in protoxylem, but may also he a feature of the tracheids or vessel elements of secondary wood. The frequency of their occurrence tends to be a function of climatic factors and conduit diameter. In order to obtain a functional explanation for these structures, the hydrodynamic behaviour of xylem conduits with various patterns of annular wall thickenings was investigated numerically using a commercial CFD (Computational Fluid Dynamics) package. The fluid flow phenomena are presented in detail. The calculations show that the developing pressure gradient of the structures with corrugated walls is in each case lower than that of a smooth pipe with a diameter corresponding to the distance between two opposite thickenings. Furthermore, complex flow patterns with circulation zones between the thickenings develop which are dependent on the geometry of the wall. It may be hypothesized that these circulation zones influence the lateral water flow. The results are discussed with regard to the relationships between the water conduction function of the xylem and ecological factors.     

Characterization and genetic mapping of a mutation (ms35) which prevents anther dehiscence in Arabidopsis thaliana by affecting secondary wall thickening in the endothecium

The male sterile mutant, ms35 , of Arabidopsis thaliana was produced by X-irradiation of seeds. The mutant produces fertile pollen, but is male sterile because the anthers do not dehisce. Anther development in ms35 plants occurs as in wild-type Arabidopsis until shortly after microspores are released from meiotic tetrads. Thereafter, in the wild type, bands of lignified, cellulosic secondary wall thickenings are laid down around the cells of the anther endothecium. In contrast, wall thickenings are not formed in the endothecium of the ms35 mutant. Development of other lignified tissues, for example the vascular tissue of the stamen, occurs normally in ms35 plants. In mutant anthers, as pollen maturation is completed, the stomium is cleaved but the anther wall does not retract to release pollen. The block in anther dehiscence in ms35 plants is specifically correlated with the absence of endothecial wall thickenings. The ms35 mutation represents the first genetic evidence in support of the proposed role of the endothecium in anther dehiscence. The ms35 gene was mapped to the top arm of chromosome 3 ( hy2 -(4.17±2.31 cM)- ms35 -(32.14±5.45 cM)- gl1 ).     

Direct Visualization of Lignifying Secondary Wall Thickenings in Zinnia elegansCells in Culture

The lignifying secondary wall thickenings of tracheary elementsthat were differentiating from Zinnia mesophyll cells in suspensionculture were examined by a freeze-etch replica technique. Cellulosemicrofibrils in primary and secondary wall thickenings differedin terms of both width and arrangement. The primary wall wasobserved as a randomly arranged network of microfibrils. Bycontrast to microfibrils in the secondary wall thickenings werehighly organized, with many pores and spaces between them. Numerousfilamentous and granular cross-links were observed in both primarywalls and secondary wall thickenings. As lignifica-tion proceeded,the cellulose microfibrils in secondary wall thickenings becameobscure as a result of the deposition of large numbers of sphericalbodies around and between the microfibrils. This material hadcompletely covered the fibrous matrix by the end of lignification.It might have been composed of the products of the dehydrogenationof monolignols. We also noted that the microfibrils appearedto be slightly irregular or wavy just after the start of lignificationbut were straighter and appeared to be more rigid when lignificationwas complete. (Received July 25, 1996; Accepted April 24, 1997)     

Das Relief der Blattoberfläche

Summary Studies on the fine structural changes accompanying xylem differentiation in wheat coleoptile have indicated that the microtubules are concerned with the inception of a regular wall thickening pattern, and later with wall deposition at the thickening site. The endoplasmic reticulum is situated characteristically in continuous profiles between the thickenings. Radioautographic studies at the electron microscope level using labelled glucoses have shown that the endoplasmic reticulum, golgi bodies and the cytoplasm near the microtubules were often labelled during deposition into nearby thickenings of radioactive materials derived from the tritiated glucoses. Incorporation into the wall occurred mainly at the top of the thickenings. The plastids of the xylem cells were also often labelled, but only during the earlier stages of differentiation; when massive wall deposition was evident, such an incorporation was never observed. The fine structural and radioautographic results are briefly discussed in terms of the possible functions of the organelles in the plant cell.     

A phi Layer in Roots of Ceratonia siliqua L

The central cylinder of the primary root of the carob tree (Ceratonia siliqua) is encircled by a layer of cells with wall thickenings, known as a phi (φ) cell layer. The development of the φ layer and the chemical composition of the cell wall thickenings have been studied in roots of C. siliqua. The results reveal the presence of condensed tannins in the mature phi thickenings and that the development of the φ layer is asynchronous: at 0–1 cm from the root tip φ thickenings appear before endodermis differentiation at the sites opposite phloem, at 1–4 cm new φ thickenings are developed at the sites opposite xylem, at 4–7 cm the φ layer consists of two layers of cells and it completely encloses the central cylinder.     

Cell wall adaptations to multiple environmental stresses in maize roots

A municipal solid-waste bottom slag was used to grow maize plants under various abiotic stresses (high pH, high salt and high heavy metal content) and to analyse the structural and chemical adaptations of the cell walls of various root tissues. When compared with roots of control plants, more intensive wall thickenings were detected in the inner tangential wall of the endodermis. In addition, phi thickenings in the rhizodermis in the oldest part of the seminal root were induced when plants were grown in the slag. The role of the phi thickenings may not be a barrier for solutes as an apoplastic dye could freely diffuse through them. The chemical composition of cell walls from endodermis and hypodermis was analysed. Slag-grown plants had higher amounts of lignin in endodermal cell walls when compared to control plants and a higher proportion of H-type lignin in the cell walls of the hypodermis. Finally, the amount of aliphatic suberin in both endo- and hypodermal cell walls was not affected by growing the plants on slag. The role of these changes in relation to the increase in mechanical strengthening of the root is discussed.     

Developmental Features of Cell Wall Formation in Sieve Elements of the Grass Aegilops comosa var. thessalica

In differentiating sieve elements of Aegilops comosa var. thessalicadictyosomes are abundant and they produce numerous smooth vesicles.Coated vesicles seem to bud from smooth ones. Since both kindsof vesicles appear both in the cytoplasm and in associationwith the plasmalemma, it is proposed that they move to and fusewith the plasmalemma transferring products for cell wall synthesis.During differentiation sub-plasmalemmal microtubules are initiallyscarce and randomly oriented but soon afterwards they becomenumerous and transversely oriented to the long axis. Cellulosemicrofibrils in the cell wall appear to run parallel to themicrotubules and the latter may regulate microfibril orientation. Root protophloem sieve elements develop wave-like wall thickenings,which are, during development, overlaid by microtubules perpendicularto the long axis. Just after maturation these thickenings progressivelybecome smooth and finally the walls appear uniform in thickness.The wave-like wall thickenings may function as stored wall material,utilized in later stages of development when wall material willbe needed and its synthesis will be impossible because of theabsence of a synthesizing mechanism in the highly degraded protoplastsof mature sieve elements. It is suggested that in this way thethickenings may enable root protophloem sieve elements to growand keep pace with the active clongation of the surroundingcells. Aegilops comosa var. thessalica, sieve elements. cell wall, microtubules, dictyosomes, coated vesicles, wave-like thickenings     

PATTERNS OF ENDOTHECIAL WALL THICKENINGS IN ARACEAE: SUBFAMILIES POTHOIDEAE AND MONSTEROIDEAE

A survey of the patterns of endothecial wall thickenings in 106 representative species from 20 genera in the Pothoideae and Monsteroideae was made using cleared anthers, sections and macerations. The wide variety of wall thickenings that is present is based on an annular-helical pattern. Variations in thickenings are related to differences in cell shape, cell orientation, intergradation between helical and annular patterns, pitch of helices, presence of branched thickenings, and various types of discontinuities in thickenings. Notable exceptions to the annular-helical pattern include Culcasia, which lacks a differentiated endothecial layer with thickenings, and Acorus, which has a peculiar stellate pattern that is unique in the family. No single pattern consistently characterizes either subfamily, although continuous helices are common in the Monsteroideae, and rare in the endothecium of Pothoideae (except Anadendrum). Monsteroideae frequently exhibit a series of slanted separate thickenings on anticlinal walls, which is absent from Pothoideae except in Heteropsis. The slanted pattern is considered a variation on a rectangular helix, involving discontinuities of thickenings on the periclinal walls. Some monsteroid genera show considerably more interspecific variation (Rhaphidophora) than others (Monstera). Endothecial thickenings constitute an anatomical character that is useful in the systematic study of Araceae; present results support other anatomical studies in identifying Culcasia and Acorus as highly divergent genera in the Pothoideae.     

Exotesta morphology of the Gardenieae — Gardeniinae (Rubiaceae)

Persson, C. 1995. Exotesta morphology of the Gardenieae-Gardeniinae (Rubiaceae). -Nord. J. Bot. 15: 285–300. CGpenhagen. ISSN 0107–055X.
Exotesta morphology of 68 species in 59 genera of the Gardeniinae were examined with the aid of scanning and light microscopy. The shape of the exotesta cells in surface is either isodiametrical or elongate and both shapes occur among New World and Old World genera. All genera, except for Monosalpinx, Posoqueria and Macrosphyra , are provided with secondary thickenings in the cell walls of the exotesta. The majority of the genera has a thickened radial wall. In the Asian genera Catunaregam, Deccania, Tamilnadia and the mainly Pacific genera, Atractocarpus and Trukia thickenings of the radial wall are very low or absent. In nearly half the genera the inner tangential wall is provided with thickenings, usually in the shape of a continuous plate. However, in four African genera, Atractogyne, Didymosalpinx, Mitriostigma and Sherbournia , the wall is provided with elongate anastomosing ribs. Atractocarpus differs markedly from the rest of the Gardeniinae by its ring-like thickenings in the inner tangential wall. Thickenings, in the shape of a continuous plate, in the outer tangential wall is restricted to three neotropical genera, Alibertia, Amaioua and Borojoa . Presence of distinct knobs is common in palaeotropical genera, whereas in neotropical genera this feature is only present in Casasia and Kotchubaea . It is concluded that data on exotesta morphology alone does not support any of the previously proposed informal groups, but may still be an important character to deduce phylogenetic relationships, primarily on a level just above the genus.     

The Epidermal Cell Structure of the Secondary Pollen Presenter in Vangueria infausta (Rubiaceae: Vanguerieae) Suggests a Functional Association with Protruding Onci in Pollen Grains

Secondary pollen presentation is a well-known phenomenon in the Rubiaceae with particularly conspicuous pollen presenters occurring in the tribe Vanguerieae. These knob-like structures are formed by a modification of the upper portion of the style and stigma, together known as the stylar head complex. In the flower bud and shortly before anthesis, the anthers surrounding the stylar head complex dehisce and release pollen grains which adhere to the pollen presenter. The epidermal cells of the pollen presenter facing the anthers are radially elongated with a characteristic wall thickening encircling the anticlinal walls of each cell towards the distal end. These cells were studied in the pollen presenter of Vangueria infausta using electron and light microscopy in conjunction with histochemical tests and immunohistochemical methods. Other prominent thickenings of the cell wall were also observed on the distal and proximal walls. All these thickenings were found to be rich in pectin and possibly xyloglucan. The terms “thickenings of Igersheim” and “bands of Igersheim” are proposed to refer, respectively, to these wall structures in general and those encircling the anticlinal walls of each cell near the distal end. The epidermal cells have an intricate ultrastructure with an abundance of organelles, including smooth and rough endoplasmic reticulum, Golgi apparatus, mitochondria and secretory vesicles. This indicates that these cells are likely to have an active physiological role. The pollen grains possess prominent protruding onci and observations were made on their structure and development. Walls of the protruding onci are also rich in pectin. Pectins are hydrophilic and known to be involved in the dehydration and rehydration of pollen grains. We hypothesise that the thickenings of Igersheim, as well as the protruding onci of the pollen grains, are functionally associated and part of the adaptive syndrome of secondary pollen presentation, at least in the Vanguerieae.     

Dispersed lignin in tracheary elements treated with cellulose synthesis inhibitors provides evidence that molecules of the secondary cell wall mediate wall patterning

Mesophyll cells of Zinnia elegans var. Envy that had been induced to differentiate into tracheary elements (TEs) in suspension culture were treated with the cellulose synthesis inhibitor 2,6-dichlorobenzonitrile (DCB). The deposition of cellulose into the patterned secondary cell wall thickenings typical of TEs was inhibited as demonstrated by reduced incorporation of [¹⁴C]glucose into acetic/nitric insoluble material and absence of cellulose detectable by fluorescence after staining with Tinopal LPW, polarization optics, or labeling with a specific cellulase. Respiration as indicated by release of ¹⁴CO₂ was inhibited to a much lesser extent, supporting a selective mechanism of action of DCB on the cellulose biosynthetic pathway. Patterned secondary cell wall thickenings were deposited in DCB-treated TEs, but these were smaller and less regularly shaped than those of control TEs. These cellulose-depleted thickenings lacked another abundant component of normal thickenings, the hemicellulose xylan, as indicated by absence of labeling with a specific xylanase or an antibody to xylan. DCB-treated TEs also showed dispersed lignin after staining with phloroglucinol, whereas control TEs contained lignin specifically localized to the secondary cell wall thickenings. Isoxaben, another recently described inhibitor of synthesis of acetic/nitric insoluble cell wall material (putatively cellulose), caused the same absence of detectable cellulose and xylan in the thickenings and dispersed lignin. These data suggest that: (i) the localization of lignin is ultimately dependent on the localization of cellulose; (ii) normal patterned wall assembly in TEs occurs in a self-perpetuating cascade in which some molecules of the secondary cell wall mediate patterning of others.     

Xylem wall deposition

Summary Tritiated leucine, tyrosine, phenylalanine, methyllabelled methionine, and cinnamic acid were used to study xylem wall deposition and lignin formation with radioautography. Leucine did not specifically label xylem thickenings; tyrosine, phenylalanine and methionine were quite good precursors in this regard. Cinnamic acid was also readily taken up by the tissues and was very markedly concentrated in the xylem thickenings; the labelling of thickenings also occurred in empty tracheids. In developing xylem cells, labelling of the cytoplasm indicated that both the endoplasmic reticulum and Golgi bodies were associated with the wall incorporation. Vesicles probably derived from the Golgi bodies, were generally observed to aggregate in the cytoplasm near the bands of wall microtubules (even if secondary wall thickening had not commenced). Simple biochemical analyses showed that incorporation of cinnamic acid into amino acids and proteins was negligible, but some lignin oxidation products were heavily labelled. The results are related to the biochemistry of lignin synthesis, and confirm that cinnamic acid is a highly specific marker for some forms of wall synthesis.     

Structure and development of cell connections in the phloem ofMetasequoia glyptostroboides needles I. Ultrastructural aspects of modified primary plasmodesmata in Strasburger cells

Summary Symplasmic contacts of Strasburger cells in the mature needle ofMetasequoia glyptostroboides were analysed with special regard to changes of plasmodesmata in fine structure and distribution. In meristematic cells simple primary plasmodesmata are evenly distributed throughout the entire wall, whereas in mature Strasburger cells plasmodesmata are aggregated in defined, dome-shaped wall thickenings. The elongated, often multiple-branched cytoplasmic strands show a distinct neck region besides a considerably dilated sleeve region confluent with cavities, which have formed at branching sites of plasmodesmata in various planes of the wall thickening. Most branches radiating from these cavities connect the protoplasts of the adjacent cells; occasionally some strands are discontinuous. The desmotubules of both, continuous and discontinuous plasmodesmal branches exhibit great variability in structure and number: they may be partially dilated, multiple-stranded and branched within single plasmodesmal branches. Fine structurally, plasmodesmata of Strasburger cells show great resemblance with developing sieve pores of conifers. This characteristic fine structure implicates a special role of the endomembrane system for phloem loading in theMetasequoia leaf.Dedicated to Prof. Dr. Dr. h.c. Eberhard Schnepf on the occasion of his retirement     

Protoxylem: the deposition of a network containing glycine-rich cell wall proteins starts in the cell corners in close association with the pectins of the middle lamella

Antibodies were used to localise polysaccharide and protein networks in the protoxylem of etiolated soybean (Glycine max L.) hypocotyls. The deposition of glycine-rich proteins (GRPs) starts in the cell corners between protoxylem elements and xylem parenchyma cells. Finally, the GRPs form a network between two mature protoxylem elements. The network also interconnects the ring- and spiral-shaped secondary wall thickenings, as well as the thickenings with the middle lamellae of living xylem parenchyma cells. In addition to the GRP network, a polysaccharide network composed mainly of pectins is involved in the attachment of the secondary wall thickenings to the middle lamellae of xylem parenchyma cells.