Electron-translucent angular areas in developing tapetum cell walls and pollen grains ofTilia platyphyllos

Summary InTilia platyphyllos, the anther tapetal cell walls undergo significant modifications from the tetrad stage onwards. During the tetrad stage the inner tangential and radial parts of the tapetal walls begin to dissolve, while the distal parts swell. After the tetrad stage, the distal and outer radial tapetal cell walls become covered by a thick, irregular, highly electron-dense, polysaccharide layer. Striking features of the maturing tapetal walls (microspore stage and later) are electron-translucent, structureless, unstainable angular areas of variable dimensions. Similar electron-translucent areas occur in the exine arcades and apertures, but also isolated in the locular fluid ofT. platyphyllos. Electron-translucent areas, that are also found in the exine arcades and tapetal cells of other angiosperms, can be interpreted as the products of poorly understood metabolic processes.     

Studies onArtemisia afra Jacq.: The phloem in stem and leaf

Summary The structure of the phloem was studied in stem and leaf ofArtemisia afra Jacq., with particular attention being given to the sieve element walls. Both primary and secondary sieve elements of stem and midvein have nacreous walls, which persist in mature cells. Histochemical tests indicated that the sieve element wall layers contained some pectin. Sieve element wall layers lack lignin. Sieve elements of the minor veins (secondary and tertiary veins) lack nacreous thickening, although their walls may be relatively thick. These walls and those of contiguous transfer cells are rich in pectic substances. Transfer cell wall ingrowths are more highly developed in tertiary than in secondary veins.     

Anatomical and biochemical changes in the composition of developing seed coats of annatto (Bixa orellana L.)

Seeds of Bixa orellana (L.) have a sclerified palisade cell layer, which constitutes a natural barrier to water uptake. In fact, newly fully developed B. orellana seeds are highly impermeable to water and thereby dormant. The purpose of this work is to investigate, from a developmental point of view, the histochemical and physical changes in the cell walls of the seed coat that are associated with the water impermeability. Seed coat samples were analyzed by histochemical and polarization microscopy techniques, as well as by fractionation/HPAEC-PAD. For histochemical analysis the tissue samples were fixed, dehydrated, embedded in paraffin and the slides were dewaxed and tested with appropriate stains for different cell wall components. Throughout the development of B. orellana seeds, there was a gradual thickening of the seed coat at the palisade region. This thickening was due to the deposition of cellulose and hemicelluloses in the palisade layer cell walls, which resulted in a highly water impermeable seed coat. The carbohydrate composition of the cell walls changed dramatically at the late developmental stages due to the intense deposition of hemicelluloses. Hemicelluloses were mainly deposited in the outer region of the palisade layer cell walls and altered the birefringent pattern of the walls. Xylans were by far the most abundant hemicellulosic component of the cell walls. Deposition of cellulose and hemicelluloses, especially xylans, could be responsible for the impermeability to water observed in fully developed B. orellana seeds.     

Ultrastructure of infection-thread development during the infection of soybean by Rhizobium japonicum

The location and topography of infection sites in soybean (Glycine max (L.) Merr.) root hairs spot-inoculated with Rhizobium japonicum have been studied at the ultrastructural level. Infections commonly developed at sites created when the induced deformation of an emerging root hair caused a portion of the root-hair cell wall to press against an adjacent epidermal cell, entrapping rhizobia within the pocket between the two host cells. Infections were initiated by bacteria which became embedded in the mucigel in the enclosed groove. Infection-thread formation in soybean appears to involve degradation of mucigel material and localized disruption of the outer layer of the folded hair cell wall by one or more entrapped rhizobia. Rhizobia at the site of penetration are separated from the host cytoplasm by the host plasmalemma and by a layer of wall material that appears similar or identical to the normal inner layer of the hair cell wall. Proliferation of the bacteria results in an irregular, wall-bound sac near the site of penetration. Tubular infection threads, bounded by wall material of the same appearance as that surrounding the sac, emerge from the sac to carry rhizobia roughly single-file into the hair cell. Growing regions of the infection sac or thread are surrounded by host cytoplasm with high concentrations of organelles associated with synthesis and deposition of membrane and cell-wall material. The threads follow a highly irregular path toward the base of the hair cell. Threads commonly run along the base of the hair cell for some distance, and may branch and penetrate into subjacent cortical cells at several points in a manner analagous to the initial penetration of the root hair.     

Deposition of glucuronoxylans on the secondary cell wall of Japanese beech as observed by immuno-scanning electron microscopy

Summary Glucuronoxylans (GXs), the main hemicellulosic component of hardwoods, are localized exclusively in the secondary wall of Japanese beech and gradually increase during the course of fiber differentiation. To reveal where GXs deposit within secondary wall and how they affect cell wall ultrastructure, immuno-scanning electron microscopy using anti-GXs antiserum was applied in this study. In fibers forming the outer layer of the secondary wall (S₁), cellulose fibrils were small in diameter and deposited sparsely on the inner surface of the cell wall. Fine fibrils with approximately 5 nm width aggregated and formed thick fibrils with 12 nm width. Some of these thick fibrils further aggregated to form bundles which labelled positively for GXs. In fibers forming the middle layer of the secondary wall (S₂), fibrils were thicker than those found in S₁ forming fibers and were densely deposited. The S₂ layer labelled intensely for GXs with no preferential distribution recognized. Compared with newly formed secondary walls, previously formed secondary walls were composed of thick and highly packed microfibrils. Labels against GXs were much more prevalent on mature secondary walls than on newly deposited secondary walls. This result implies that the deposition of GXs into the cell wall may occur continuously after cellulose microfibril deposition and may be responsible for the increase in diameter of the microfibrils.Abbreviations GXs glucuronoxylans - PBS phosphate-buffered saline - RFDE rapid-freeze and deep-etching technique - FE-SEM field emission scanning electron microscope - TEM transmission electron microscope     

Carotenoids in the outer cell-wall layer of Scenedesmus (Chlorophyceae)

Scenedesmus obliquus, strain 633, which synthesizes ketocarotenoids and sporopollenin, also forms pink-red-colored cell walls. Both the cell walls left over after autospore liberation and those from homogenates of disrupted green cells have similar carotenoid pigmentation. Canthaxanthin, astaxanthin, an unidentified ketocarotenoid, and lutein were found as integral cell wall components. They are bound to the outer (trilaminar) layer of the complete cell wall which also contains sporopollenin.Abbreviations CWH complete cell walls isolated from the homogenates - CWM maternal cell walls accumulated in the medium - KC ketocarotenoid - SC secondary carotenoids - SP sporopollenin     

Immunocytochemical localization of polygalacturonase during tracheary element differentiation in<Emphasis Type="Italic"> Zinnia elegans</Emphasis>

Polygalacturonase (PG) is a cell wall-associated protein that degrades pectin. A ZePG1 cDNA encoding a putative PG was isolated from Zinnia elegens L. and a rabbit antibody specific to the ZePG1 protein was generated. The level of the ZePG1 protein was up-regulated when tracheary element differentiation was initiated. Using gold-labeled secondary antibodies for light and electron microscopy, ZePG1 protein was localized in cultured Zinnia cells. This protein was preferentially distributed on tracheary elements (TEs). At the subcellular level, the protein was localized on secondary wall thickenings, primary walls, Golgi bodies and vesicles. Thus, the putative role of the ZePG1 protein might be the degradation of pectic substances before lignification. Some non-TE cells also accumulated ZePG1 protein on primary walls, Golgi bodies and vesicles. The accumulation of ZePG1 protein on primary walls seems to be at the elongating tips of non-TE cells. In plants, ZePG1 protein was localized on the secondary wall thickenings of differentiating TEs and phloem regions. These results suggest that the expression of the ZePG1 protein is highly regulated both spatially and temporally during in vitro and in situ TE differentiation.Abbreviations GST Glutathione-S-transferase - PATAg Periodic acid–thiocarbohydrazide–silver proteinate - PG Polygalacturonase - TE Tracheary element     

Preinfection cell wall formation in roots and developing nodules ofAlnus rubra Bong

Summary The establishment of actinorhizal root nodules involves penetration of host cell walls and intracellular colonization by the nitrogen-fixing endosymbiont,Frankia (Actinomycetales). In the early stages of the infection process inAlnus, unusual cell walls with undulate profiles were observed in root tip meristematic derivatives, and in early (preinfection) derivatives of the nodule lobe meristem, inFrankia-inoculated plants. The irregular cell walls attached obliquely to preexisting walls, but were not discontinuous. Serial sections revealed that the unusual walls divided two daughter cells. Microtubules in bundled arrays were abundant near the undulate walls, and radiated in several planes. In the root tips, the anomalous cell walls were observed within one day of inoculation withFrankia.     

A mutant of Arabidopsis thaliana displaying altered patterns of cellulose deposition

A homozygous recessive mutant of Arabidopsis thaliana has been selected which displays altered patterns of cellulose deposition. The mutant was selected because leaf and stem trichomes lacked the strong birefringence under polarized light which is characteristic of plant cells which contain highly ordered cellulose in their secondary cell walls. Compared with wild-type A. thaliana, this mutant (designated tbr for trichome birefringence) also displays reduced birefringence in the xylem of the leaf. Direct chemical analyses of root, stem, and leaf tissues, including isolated leaf trichomes, support the conclusion that tbr is impaired in its ability to deposit secondary wall cellulose in specific cell types, most notably in trichomes where the secondary wall appears to be totally absent. Altered patterns of wound-induced callose deposition in trichomes and surrounding cells is another trait which also co-segregates with the tbr mutation.     

Alfalfa Stem Tissues: Cell-wall Development and Lignification

Alfalfa stems contain a variety of tissues with different patternsof cell-wall development. Development of alfalfa cell wallswas investigated after histochemical staining and with polarizedlight using light microscopy and scanning electron microscopy.Samples of the seventh internode, from the base of stems grownon cut stems, were harvested at five defined stages of developmentfrom early internode elongation through to late maturity. Internodeseven was elongating up to the third sample harvest and internodediameter increased throughout the entire sampling period. Chlorenchyma,cambium, secondary phloem, primary xylem parenchyma and pithparenchyma stem tissues all had thin primary cell walls. Pithparenchyma underwent a small amount of cell-wall thickeningand lignification during maturation. Collenchyma and primaryphloem tissues developed partially thickened primary walls.In contrast to a recent report, the formation of a ring shaped,lignified portion of the primary wall in a number of cells inthe exterior part of the primary phloem was found to precedethe deposition of a thick, non-lignified secondary wall whichwas degradable by rumen microbes. In numerous xylem fibres fromthe fourth harvest date onwards, an additional highly degradablesecondary wall layer was deposited against a previously depositedlignified and undegradable secondary wall. The pattern of lignificationobserved in alfalfa stem tissues suggests that polymerizationof monolignols by peroxidases at the luminal border of the primarycell wall creates an impermeable zone which restricts lignificationof the middle lamella region of tissues with thick primary walls.Copyright1998 Annals of Botany Company Alfalfa,Medicago sativaL., stem tissue, cell wall, development, lignification, degradation.     

Localisation and characterisation of cell wall mannan polysaccharides in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

Polysaccharides containing -1,4-mannosyl residues (mannans) are abundant in the lignified secondary cell walls of gymnosperms, and are also found as major seed storage polysaccharides in some plants, such as legume species. Although they have been found in a variety of angiosperm tissues, little is known about their presence and tissue localisation in the model angiosperm, Arabidopsis thaliana (L.) Heynh. In this study, antibodies that specifically recognised mannans in competitive ELISA experiments were raised in rabbits. Using these antibodies, we showed that Golgi-rich vesicles derived from Arabidopsis callus were able to synthesise mannan polysaccharides in vitro. Immunofluorescence light microscopy and immunogold electron microscopy of Arabidopsis inflorescence stem sections revealed that the mannan polysaccharide epitopes were localised in the thickened secondary cell walls of xylem elements, xylem parenchyma and interfascicular fibres. Similarly, mannan epitopes were present in the xylem of the leaf vascular bundles. Surprisingly, the thickened epidermal cell walls of both leaves and stems also contained abundant mannan epitopes. Low levels were observed in most other cell types examined. Thus, mannans are widespread in Arabidopsis tissues, and may be of particular significance in both lignified and non-lignified thickened cell walls. Polysaccharide analysis using carbohydrate gel electrophoresis (PACE) of cell wall preparations digested with a specific mannanase showed that there is glucomannan in inflorescence stems. The findings show that Arabidopsis can be used as a model plant in studies of the synthesis and functions of mannans.Abbreviations BSA bovine serum albumin - ELISA enzyme-linked immunosorbent assay - PACE polysaccharide analysis by carbohydrate gel electrophoresis     

Effects of hypergravity conditions on elongation growth and lignin formation in the inflorescence stem of Arabidopsis thaliana

The effects of hypergravity on elongation growth and lignin deposition in secondary cell walls of the Arabidopsis thaliana (L.) Heynh. inflorescence stem were examined in plants grown for 3 days after exposure to hypergravity in the direction from shoot to root at 300 g for 24 h. The content of acetylbromide-extractable lignins in a secondary cell wall fraction prepared by enzyme digestion of inflorescence stem segments removing primary cell wall components was significantly increased by the hypergravity stimulus. Xylem vessels, particularly in a region closer to the base of the inflorescence stem, increased in number. Gadolinium chloride at 0.1 mM, a blocker of mechanoreceptors, partially suppressed the effect of hypergravity on lignin deposition in the secondary cell wall fraction. These results suggest that mechanoreceptors are responsible for hypergravity-induced lignin deposition in secondary cell walls in A. thaliana inflorescence stems.     

Distribution of cell wall components in<Emphasis Type="Italic"> Sphagnum</Emphasis> hyaline cells and in liverwort and hornwort elaters

Spiral secondary walls are found in hyaline cells of Sphagnum, in the elaters of most liverworts, and in elaters of the hornwort Megaceros. Recent studies on these cells suggest that cytoskeletal and ultrastructural processes involved in cell differentiation and secondary wall formation are similar in bryophytes and vascular plant tracheary elements. To examine differences in wall structure, primary and secondary wall constituents of the hyaline cells of Sphagnum novo-zelandicum and elaters of the liverwort Radula buccinifera and the hornwort Megaceros gracilis were analyzed by immunohistochemical and chemical methods. Anti-arabinogalactan–protein antibodies, JIM8 and JIM13, labeled the central fibrillar secondary wall layer of Megaceros elaters and the walls of Sphagnum leaf cells, but did not label the walls of Radula elaters. The CCRC-M7 antibody, which detects an arabinosylated (16)-linked -galactan epitope, exclusively labeled hyaline cells in Sphagnum leaves and the secondary walls of Radula elaters. Anti-pectin antibodies, LM5 and JIM5, labeled the primary wall in Megaceros elaters. LM5 also labeled the central layer of the secondary wall but only during formation. In Radula elaters, JIM5 and another anti-pectin antibody, JIM7, labeled the primary wall. The distribution of arabinogalactan–proteins and pectic polysaccharides restricted to specific wall types and stages of development provides evidence for the developmental and functional regulation of cell wall composition in bryophytes. Monosaccharide-linkage analysis of Sphagnum leaf cell walls suggests they contain polysaccharides similar to those of higher plants. The most abundant linkage was 4-Glc, typical of cellulose, but there was also evidence for xyloglucans, 4-linked mannans, 4-linked xylans and rhamnogalacturonan-type polysaccharides.Abbreviations AGP Arabinogalactan–protein - Araf Arabinofuranose - Fucp Fucopyranose - GalAp Galacturonopyranose - Galp Galactopyranose - GlcAp Glucuronopyranose - HGA Homogalacturonan - Manp Mannopyranose - RG Rhamnogalacturonan - Rhap Rhamnopyranose - XG Xyloglucan - Xylp Xylopyranose     

Cellular re-distribution of flavin-containing polyamine oxidase in differentiating root and mesocotyl of <Emphasis Type="Italic">Zea mays</Emphasis> L. seedlings

Plant polyamine oxidases (PAOs; EC 1.5.3.11) are hydrogen peroxide-producing enzymes supposedly involved in cell-wall differentiation processes and defence responses. Maize (Zea mays L.) PAO (MPAO) is a 53 kDa secretory glycoprotein, abundant in primary and secondary cell walls of several tissues. Using biochemical, histochemical, ultrastructural and immunocytochemical techniques, the distribution and sub-cellular compartmentalisation of MPAO in the primary root and mesocotyl of seedlings at different maturation stages or after growth under varying light conditions were analysed. In apical root tissues, MPAO immunoreactivity was mainly detected in the cytoplasmic compartment, while a lower immunoreactivity was observed in the cell walls. In the more mature, basal part of the root, intense immunogold labelling was found in the primary and secondary walls of protoxylem precursors and vessels, while endodermal cells and living metaxylem precursors were immunopositive both in their walls and in their thin cytoplasmic compartments. A re-distribution of MPAO protein from the cytoplasm toward the primary and secondary walls was also recognised when immunoreactivity of basal root tissues from 3-day-old seedlings was compared with that detected in 11-day-old tissues. Accordingly, biochemical analyses revealed MPAO entrapment in the extracellular matrix of mature tissues. In the mesocotyl, an enrichment of MPAO immunolabelling in the cell wall of protoxylem, metaxylem and epidermal tissues, as a function of light exposure, was observed. Taken together, these data support the hypothesised role of PAOs in cell-wall maturation. Moreover, the relevant intraprotoplasmic MPAO localisation observed mainly in differentiating root tissues suggests an additional role in intracellular production of hydrogen peroxide.Alessandra Cona and Sandra Moreno have contributed equally to this paper.     

Changes in the arrangement of cellulose microfibrils associated with the cessation of cell expansion in tracheids

 The relationship between the cessation of cell expansion and formation of the secondary wall was investigated in the early-wood tracheids of Abies sachalinensis Masters by image analysis and field emission scanning electron microscopy. The area of the lumen and the length of the perimeter of the lumen of differentiating tracheids increased from the cambium towards the xylem. These increases had just ceased in the case of tracheids closest to the cambium in which birefringence was first detected by observations with a polarizing light microscope. Cellulose microfibrils (MFs) deposited on the innermost surfaces of radial walls were not well ordered during the expansion of cells, but well ordered MFs were deposited at the subsequent stage of cell wall formation. The first well ordered MFs were oriented in an S-helix. The well ordered MFs had already been deposited at the tracheids where birefringence was first detected under the polarizing light microscope. These results indicate that the deposition of the well ordered MFs, namely, the formation of the secondary wall, begins before the cessation of cell expansion of tracheids. Therefore, it seems that the expansion of tracheids is restricted by the deposition of the secondary wall because the cell walls become rigid simultaneously with the development of the secondary wall and, therefore, the yield point of cell walls exceeds the turgor pressure of the cell. Received: 3 July 1996 / Accepted: 24 September 1996     

Sucrose biosynthesis in Dunaliella

Four independent kinds of observations indicate that the cell wall regenerated by oat (Avena sativa L.) and corn (Zea mays L.) protoplasts in culture is less well developed than that regenerated by tobacco (Nicotiana tabacum L.) protoplasts. Following wall regeneration the cereal protoplasts remained susceptible to osmotic shock upon transfer to water, showed great enlargement, stained poorly with calcofluor white, and maintained a positive internal electrical potential. The development of a negative membrane potential by tobacco protoplasts in culture often occurred simultaneously with the onset of cell division. Since division was observed only in protoplasts which had regenerated good cell walls and had re-established negative membrane potentials it is suggested that culture conditions which favor these two processes should improve protoplast viability.     

The suberin lamella,a possible barrier to water movement from the veins to the mesophyll ofThemeda triandra forsk

Summary Precipitation of ferrous ions by ferricyanide in transpiring leaves ofThemeda triandra Forsk. produced crystalline deposits, which were visible with the light and electron microscope. Prussian blue crystals were formed within the lumina of the tracheary elements and the apoplast, or cell wall continuum of the vascular tissues and bundle-sheath cells. Little if any deposition was noted within the lignified secondary thickenings of the tracheary elements. The localization pattern suggests that the ferrous ions moved from the lumina of the tracheary elements via the exposed primary walls. Prussian blue crystals were abundant in the outer tangential and radial walls of the bundle-sheath cells. By contrast, crystals were lacking in the walls of neighbouring mesophyll cells, suggesting that the suberin lamella in the bundle-sheath walls effectively inhibited the apoplastic movement of ferrous ions and possibly may impede, or restrict the movement of water across the bundle-sheath/mesophyll interface.     

Two separate zones of helicoidally orientated microfibrils are present in the walls ofNitella internodes during growth

Summary The dynamic changes in microfibril architecture in the internode cell walls of the giant unicellular algaNitella translucens were studied during cell expansion. Thin section electron microscopy in conjunction with mild matrix polysaccharide extraction techniques revealed three distinct architectural zones in the walls of fully grown cells. These zones were related to distinct phases of growth by monitoring changes in cell wall architecture of internodes during active cell expansion. The initial microfibril deposition before the onset of active cell growth is helicoidal. A helicoid is a structurally complex but ordered arrangement of microfibrils that has been detected increasingly often in higher plant cell walls. During active cell elongation microfibrils are deposited transversely to the direction of cell elongation as shown in earlier studies by birefringence measurements in the polarizing microscope. The gradual decline in cell elongation corresponds with a final helicoidal deposition which continues after cell expansion ceases entirely.The continual presence of the initial helicoidal zone in the outer wall region during the whole growth process suggests that these microfibrils do not experience strain reorientation and are continually reorganized, or maintained, in a well ordered helicoidal arrangement.     

Localization of glucuronoxylans in Japanese beech visualized by immunogold labelling

Summary An antiserum against glucuronoxylans (GXs) has been raised from a mouse. The dot-blot immunoassay and competitive inhibition test indicated that the antibodies could bind specifically to GXs. Therefore, the antiserum was used for immunogold labelling to investigate the localization of GXs in Japanese beech. Labelling of GXs was seen only in the secondary walls of xylem cells, but not in the primary walls or the middle lamella. GXs were evenly distributed in the secondary walls except for the outer part of the outer secondary-wall layer in which they were less abundant. The labelling density in each secondary-wall layer (S₁, S₂, and S₃) increased during cell wall formation. This result strongly suggests that the deposition of GXs occurs in a penetrative way.     

Cytochemical evidence of a fungal cell wall alteration during infection of Eucalyptus roots by the ectomycorrhizal fungus Cenococcum geophilum

When the ectomycorrhizal fungus Cenococcum geophilum changes from a saprophytic to a symbiotic stage, its cell wall structure becomes simplified. The external hyphal wall layer which, in the saprophytic stage, is highly reactive to the Gomori-Swift test becomes poorly reactive and can no longer be distinguished from the internal wall layer in the Hartig net hyphae. The intensely stained external wall layer was also absent from pure cultures of Cenococcum geophilum grown on a medium with a low sugar content. This cell wall alteration could be due to a decrease in the amount of melanin or of melanin plus cystine-containing proteins. This change may be necessary for increased nutrient exchange between symbionts through hyphal walls.