A quantitative analysis of the interspecific plasmodesmata in the non-division walls of the plant chimera Laburnocytisus adamii (Poit.) Schneid.

Non-division walls in petals of the chimera Laburnocytisus adamii (Poit.) Schneid, were screened for the occurrence and distribution of symplasmic connections. The secondary plasmodesmata (PD) between epidermal cells of Cytisus purpureus Scop, and subepidermal cells of Laburnum anagyroides Medik. were compared with the PD of corresponding cell walls in petals of the two parental species. The non-division walls in the petals of L. adamii were traversed mainly by continuous PD and a few half-PD, both being grouped in pit fields. The secondary PD were characterized by a high percentage of branching (82%), with more than 40% consisting of a single strand at the Cytisus cell side interconnected by a median cavity with two strands of the Laburnum subepidermal cell. In addition, more than 30% of all PD showed secondary branching in the subepidermal wall portion. As a consequence, the cross-sectional areas of plasmodesmatal strands on each side of the central cavity differed remarkably in size, representing a “bottleneck” in the epidermal wall portion. In contrast, PD in the petals of the parental species were symmetrically branched. The comparison of cross-sectional areas of PD in the cell wall between the epidermis and subepidermis of petals of L. anagyroides showed a well-tuned system. The occurrence of half-PD in the intraspecific wall indicates a secondary origin. We conclude that, in the chimera, both genotypically different cells take part in the formation of the interspecific PD.     

Changes in the Distribution of Plasmodesmata in Developing Fibre-tracheid Pit Membranes of Sorbus aucuparia L.

The distribution of plasmodesmata in cambial pit fields anddeveloping fibre-tracheid pit membranes in Sorbus aucupariaL. has been studied using conventional and high voltage transmissionelectron microscopy. In cambial pit fields, plasmodesmata arewidely separated while, during cell enlargement and the stageof secondary wall formation, they occur exclusively in a denselypacked cluster, eccentrically located on the pit membrane ina thickening of primary wall material. It is not clear at themoment whether the cluster arises by aggregation of the cambialpit field plasmodesmata, or by de novo formation in a localizedregion coupled with loss of plasmodesmata outside this region.The significance of these changes is discussed. Sorbus aucuparia L., plasmodesmata, pit membranes, xylem differentiation     

Replication and trafficking of a plant virus are coupled at the entrances of plasmodesmata

Plant viruses use movement proteins (MPs) to modify intercellular pores called plasmodesmata (PD) to cross the plant cell wall. Many viruses encode a conserved set of three MPs, known as the triple gene block (TGB), typified by Potato virus X (PVX). In this paper, using live-cell imaging of viral RNA (vRNA) and virus-encoded proteins, we show that the TGB proteins have distinct functions during movement. TGB2 and TGB3 established endoplasmic reticulum–derived membranous caps at PD orifices. These caps harbored the PVX replicase and nonencapsidated vRNA and represented PD-anchored viral replication sites. TGB1 mediated insertion of the viral coat protein into PD, probably by its interaction with the 5′ end of nascent virions, and was recruited to PD by the TGB2/3 complex. We propose a new model of plant virus movement, which we term coreplicational insertion, in which MPs function to compartmentalize replication complexes at PD for localized RNA synthesis and directional trafficking of the virus between cells.     

CELL DIVISION AND FILAMENT FORMATION IN THE DESMID BAMBUSINA BREBISSONII (CHLOROPHYTA)1

Cell division and semicell expansion in the filamentous desmid Bambusina brebissonii Kütz. were investigated using transmission and scanning electron microscopy. Interphase cells are typical of desmids, containing a full complement of organelles and a cell wall penetrated by complex pores, but the cells lack a well-defined median constriction. Cell division involves an open spindle and the centripetal growth of a primary septum formed by the fusion of small, dark-staining vesicles probably derived from dictyosomes. Telophase nuclei are separated by a system of interzonal microtubules and numerous large, lighter-staining vesicles also derived from the dictyosomes. Following cell division, an elaborate replicate cross wall is formed which consists of both primary and secondary wall layers. During semicell expansion, a portion of the primary wall splits apart as the new semicells evaginate and expand to their full size. The primary wall stops splitting at a thick ring of secondary wall material leaving the cells united by the remaining common layer of primary wall. When semicell expansion is completed, the primary wall is not shed from the lateral walls of the new semicells, and pores through both primary and secondary wall layers begin to produce sheath material. However, pores in the end walls of cells do not function unless the filament is broken. The intact primary wall between cells and the absence of sheath production between cells comprise the mechanism serving to hold the cells of Bambusina brebissonii together in long filaments.     

Sieve-element differentiation and fluoresceine translocation in wound-phloem of pea roots after complete severance of the stele

Experimental interruption of the root stele of Pisum sativum L. induces in the cortex tissue the development of wound-sieve tubes which bridge the wound and reconnect the vascular stumps. Outside the stele, sieve plates arise from primary pit fields. This origin is confirmed by the distribution of future sieve pores over the original parenchyma cell wall and by remnants of the pitfield cavity in developing sieve plates. Differentiation of wound-sieve elements is similar to that of bundle-sieve elements and includes the chromatolytic disintegration of nuclei as well as the development of typical sieve pores arising from pit-field plasmodesmata. The completion of first woundsieve tubes (indicated by a continuous chain of anilin-blue-positive sieve plates by-passing the wound) was observed 55–62 h after wounding. However, effective translocation, visualized with fluoresceine as a phloem-mobile marker, was not found until 10 h (on average) later. It is suggested that this time delay corresponds to the maturing of the last link within a chain of wound-sieve-tube members. Presumably, enucleate sieve elements with widened pores are a prerequisite for effective phloem translocation.Abbreviations DAPI 4,6-diamidino-2-phenylindole·2 H₂O - ER endoplasmic reticulum Preliminary results of this investigation have been presented at the International Phloem Transport Conference in Asilomar, Cal., USA 1985 (cf. Schulz 1986c)     

Identification of novel genes in Arabidopsis involved in secondary cell wall formation using expression profiling and reverse genetics

Forward genetic screens have led to the isolation of several genes involved in secondary cell wall formation. A variety of evidence, however, suggests that the list of genes identified is not exhaustive. To address this problem, microarray data have been generated from tissue undergoing secondary cell wall formation and used to identify genes that exhibit a similar expression pattern to the secondary cell wall-specific cellulose synthase genes IRREGULAR XYLEM1 (IRX1) and IRX3. Cross-referencing this analysis with publicly available microarray data resulted in the selection of 16 genes for reverse genetic analysis. Lines containing an insertion in seven of these genes exhibited a clear irx phenotype characteristic of a secondary cell wall defect. Only one line, containing an insertion in a member of the COBRA gene family, exhibited a large decrease in cellulose content. Five of the genes identified as being essential for secondary cell wall biosynthesis have not been previously characterized. These genes are likely to define entirely novel processes in secondary cell wall formation and illustrate the success of combining expression data with reverse genetics to address gene function.     

Ultrastructural study of plasmodesmata in the brown alga Dictyota dichotoma (Dictyotales, Phaeophyceae)

Plasmodesmata are intercellular bridges that directly connect the cytoplasm of neighboring cells and play a crucial role in cell-to-cell communication and cell development in multicellular plants. Although brown algae (Phaeophyceae, Heterokontophyta) are phylogenetically distant to land plants, they nevertheless possess a complex multicellular organization that includes plasmodesmata. In this study, the ultrastructure and formation of plasmodesmata in the brown alga Dictyota dichotoma were studied using transmission electron microscopy and electron tomography with rapid freezing and freeze substitution. D. dichotoma possesses plasma membrane-lined, simple plasmodesmata without internal endoplasmic reticulum (desmotubule). This structure differs from those in land plants. Plasmodesmata were clustered in regions with thin cell walls and formed pit fields. Fine proteinaceous "internal bridges" were observed in the cavity. Ultrastructural observations of cytokinesis in D. dichotoma showed that plasmodesmata formation began at an early stage of cell division with the formation of tubular pre-plasmodesmata within membranous sacs of the cytokinetic diaphragm. Clusters of pre-plasmodesmata formed the future pit field. As cytokinesis proceeded, electron-dense material extended from the outer surface of the mid region of the pre-plasmodesmata and finally formed the nascent cell wall. From these results, we suggest that pre-plasmodesmata are associated with cell wall development during cytokinesis in D. dichotoma.     

A Cytochemical Study of Cell Wall Hydrolysis in the Secondary Xylem of Poplar (Populus italica Moench)

Certain developmental features of cell wall hydrolysis werestudied in the secondary xylem of poplar (Populus italica Moench).At the intervascular pit membrane hydrolysis starts prematurelybefore differentiation of the secondary wall is complete andincreases progressively. Eventually the whole of the middlelamella is hydrolysed, and the primary wall undergoes lyticmodification. The modified polysaccharides are dispersed, presumablyby the transpiration stream. During differentiation the vessel-parenchymapit membrane remains unaltered and undergoes thickening. Thepresent investigation suggests that the plasalemma plays animportant role in the ordered hydrolysis of certain regionsof the primary walls. Populus italicaMoench, poplar, secondaryxylem, xylem, cell wall hydrolysis, plasmalemma, pit membram     

Ontogeny of external glands in the bladderwortUtricularia monanthos

Summary The development of external glands on traps and stolons ofU. monanthos has been studied using transmission electron microscopy. During early differentiation of the epidermis some cells remain narrow and develop a protuberance which subsequently divides into a terminal and a pedestal cell, with the remainder of the original cell forming the basal epidermal cell of the gland. The lateral wall of the pedestal cell soon becomes densely impregnated throughout its thickness, and this is followed by the formation of discontinuous cuticular deposits within the primary wall of the terminal cell. The outer wall of the terminal cell then usually undergoes extensive secondary wall thickening beginning with the formation of ingrowths which for a period characterize the cell as a transfer cell. Later, at the stage when traps begin capturing prey, these ingrowths are overlain by further layers of secondary wall material. Concomitantly, in the pedestal cell, wall ingrowths become fully differentiated on the outer transverse wall and persist throughout the remaining life of the gland.The function of external glands during early ontogeny is discussed. At the stage when the terminal cell is differentiated as a transfer cell it is suggested that the gland is mainly responsible for absorbing solutes from the external medium. Once traps commence capturing prey the gland may become modified for a rôle in water secretion, facilitated by the differentiation of the pedestal cell as a transfer cell, and by the formation of a thick outer wall in the terminal cell.     

Scanning electron microscopy in nematode-induced giant transfer cells.

A study of giant cells induced by the root-knot nematode, Meloidogyne incognita, in roots of Impatiens balsamina was made by scanning electron microscopy. The cytoplasmic contents of giant cells were removed by a procedure based on KOH digestion, to reveal inner wall structure. Wall ingrowths typical of transfer cells are present in giant cells from six days onwards after induction. They develop on walls adjacent to vascular tissues, and their distribution and development was examined. Pit fields contianing plasmodesmata become elaborated in walls between giant cells, but pit fields are lost between giant cells and cells outside them. The distribution of plasmodesmata in pit fields suggests that de novo formation of plasmodesmata occurs in walls between giant cells. Various aspects of giant cell formation and function are discussed and wall ingrowth development is compared in giant cells and normal transfer cells.     

Seed coat development, anatomy and scanning electron microscopy of Harpagophytum procumbens (Devil's Claw), Pedaliaceae

Seed coat development of Harpagophytum procumbens (Devil's Claw) and the possible role of the mature seed coat in seed dormancy were studied by light microscopy (LM), transmission electron microscopy (TEM) and environmental scanning electron microscopy (ESEM). Very young ovules of H. procumbens have a single thick integument consisting of densely packed thin-walled parenchyma cells that are uniform in shape and size. During later developmental stages the parenchyma cells differentiate into 4 different zones. Zone 1 is the multi-layered inner epidermis of the single integument that eventually develops into a tough impenetrable covering that tightly encloses the embryo. The inner epidermis is delineated on the inside by a few layers of collapsed remnant endosperm cell wall layers and on the outside by remnant cell wall layers of zone 2, also called the middle layer. Together with the inner epidermis these remnant cell wall layers from collapsed cells may contribute towards seed coat impermeability. Zone 2 underneath the inner epidermis consists of large thin-walled parenchyma cells. Zone 3 is the sub-epidermal layers underneath the outer epidermis referred to as a hypodermis and zone 4 is the single outer seed coat epidermal layer. Both zones 3 and 4 develop unusual secondary wall thickenings. The primary cell walls of the outer epidermis and hypodermis disintegrated during the final stages of seed maturation, leaving only a scaffold of these secondary cell wall thickenings. In the mature seed coat the outer fibrillar seed coat consists of the outer epidermis and hypodermis and separates easily to reveal the dense, smooth inner epidermis of the seed coat. Outer epidermal and hypodermal wall thickenings develop over primary pit fields and arise from the deposition of secondary cell wall material in the form of alternative electron dense and electron lucent layers. ESEM studies showed that the outer epidermal and hypodermal seed coat layers are exceptionally hygroscopic. At 100% relative humidity within the ESEM chamber, drops of water readily condense on the seed surface and react in various ways with the seed coat components, resulting in the swelling and expansion of the wall thickenings. The flexible fibrous outer seed coat epidermis and hypodermis may enhance soil seed contact and retention of water, while the inner seed coat epidermis maintains structural and perhaps chemical seed dormancy due to the possible presence of inhibitors.     

Spatial regulation of pectic polysaccharides in relation to pit fields in cell walls of tomato fruit pericarp

Scanning electron microscopic examination of intact tomato (Lycopersicon esculentum) pericarp and isolated pericarp cell walls revealed pit fields and associated radiating ridges on the inner face of cell walls. In regions of the cell wall away from pit fields, equivalent ridges occurred in parallel arrays. Treatment of isolated cell walls with a calcium chelator resulted in the loss of these ridges, indicating that they contain homogalacturonan-rich pectic polysaccharides. Immunolabeling procedures confirmed that pit fields and associated radiating ridges contained homogalacturonan. Epitopes of the side chains of pectic polysaccharides were not located in the same regions as homogalacturonan and were spatially regulated in relation to pit fields. A (1-->4)-beta-galactan epitope was absent from cell walls in regions of pit fields. A (1-->5)-alpha-arabinan epitope occurred most abundantly at the inner face of cell walls in regions surrounding the pit fields.     

Spatial and temporal variability of xylan distribution in differentiating secondary xylem of hybrid aspen

Xylans occupy approximately one-third of the cell wall components in hardwoods and their chemical structures are well understood. However, the microdistribution of xylans (O-acetyl-4-O-methylglucuronoxylans, AcGXs) in the cell wall and their correlation with functional properties of cells in hardwood xylem is poorly understood. We demonstrate here the spatial and temporal distribution of xylans in secondary xylem cells of hybrid aspen using immunolocalization with LM10 and LM11 antibodies. Xylan labeling was detected earliest in fibers at the cell corner of the S? layer, and then later in vessels and ray cells respectively. Fibers showed a heterogeneous labeling pattern in the mature cell wall with stronger labeling of low substituted xylans (lsAcGXs) in the outer than inner cell wall. In contrast, vessels showed uniform labeling in the mature cell wall with stronger labeling of lsAcGXs than fibers. Xylan labeling in ray cells was detected much later than that in fibers and vessels, but was also detected at the beginning of secondary cell wall formation as in fibers and vessels with uniform labeling in the cell wall regardless of developmental stage. Interestingly, pit membranes including fiber-, vessel- and ray-vessel pits showed strong labeling of highly substituted xylans (hsAcGXs) during differentiation, although this labeling gradually disappeared during pit maturation. Together our observations indicate that there are temporal and spatial variations of xylan deposition and chemical structure of xylans between cells in aspen xylem. Differences in xylan localization between aspen (hardwood) and cedar (softwood) are also discussed.     

DEVELOPMENT OF THE AMORPHOUS LAYER (PROTECTIVE LAYER) IN XYLEM PARENCHYMA OF CV. GOLDEN DELICIOUS APPLE,CV. LORING PEACH,AND WILLOW

The amorphous layer (AL) in xylem parenchyma may play a prominent role in defining the freezing response of a tissue. A comparative study was undertaken to examine the development of the AL in xylem parenchyma of cv. Golden Delicious apple and cv. Loring peach, which exhibit deep supercooling, and willow, which exhibits only extracellular freezing. AL formation did not begin until secondary wall formation was entirely completed. Deposition of the loose textured AL began at the edges of a pit cavity and spread to the adjacent vessel-parenchyma pit membrane. Upon completion, the AL ensheathed the protoplast of the xylem parenchyma cell and was much thicker in the area of the pit cavity. During AL development, numerous cortical microtubules, RER, and coated vesicles were present in the cytoplasm. Observations indicate that AL development was similar in all 3 species, but differences were found in the time of maturation within an annual ring and AL coloration after staining with toluidine blue O. The latter indicates that compositional differences may exist which may account for the variation in freezing response exhibited by these species.     

SEM studies on vessels in ferns. 11. Ophioglossum

With scanning electron microscopy (SEM), the nature of metaxylem vessel elements and tracheids was examined in Ophioglossum crotalophomides, 0. pendulum subsp. falcatum , and 0. vulgatum roots and rhizomes. Vessels were identified in all species. End walls of vessel elements, which bear perforations, are like lateral wall pitting of those elements in the secondary wall framework and differ only in absence of pit membranes or presence of pit membrane remnants. Some of the perforations contain pit membrane remnants that have large pores, small porosities, or are threadlike or weblike in structure. Dimorphic perforations were found in some vessel elements of rhizomes of 0. pendulum subsp. falcatum. Tracheids are very likely present in addition to vessels in all three species. The secondary wall framework of both tracheids and vessels is basically scalariform, although deviations in pattern are present. Vessel elements of Ophiglossum are entirely comparable to those of leptosporangiate ferns.     

Arrangement of cortical microtubules during formation of bordered pit in the tracheids ofTaxus

Summary The arrangement of cortical microtubules during the development of the secondary wall and bordered pits in the tracheids ofTaxus was examined by immunofluorescence and electron microscopy. The cambial region of radial longitudinal sections of developing young shoots (2–3 years old) contains cells at various stages of differentiation from cambial cells to tracheids. At the early stage of formation of bordered pits, circular bands of microtubules were seen to be associated with the inner edge of the border of the developing pit. In other regions than the pit secondary wall of uniform thickness was laid down, and obliquely oriented cortical microtubules ran parallel to one another. These cortical microtubules also covered the surface of the border of the developing pit on the side facing the center of the cell. As the border of the pit developed, a circular band of MTs remained associated with the inner edge of border, suggesting that the MTs were involved in the formation of the rim of the bordered pit, extending the initial border thickening, which consisted of concentrically oriented cellulose microfibrils. After completion of the formation of the bordered pit, helical thickenings became apparent. The obliquely oriented microtubules were organized in bands parallel to one another, being superimposed on the helical thickenings. The involvement of MTs in the formation of bordered pits and helical thickening is discussed.     

The roles of the cytoskeleton during cellulose deposition at the secondary cell wall

During secondary cell wall formation, developing xylem vessels deposit cellulose at specific sites on the plasma membrane. Bands of cortical microtubules mark these sites and are believed to somehow orientate the cellulose synthase complexes. We have used live cell imaging on intact roots of Arabidopsis to explore the relationship between the microtubules, actin and the cellulose synthase complex during secondary cell wall formation. The cellulose synthase complexes are seen to form bands beneath sites of secondary wall synthesis. We find that their maintenance at these sites is dependent upon underlying bundles of microtubules which localize the cellulose synthase complex (CSC) to the edges of developing cell wall thickenings. Thick actin cables run along the long axis of the cells. These cables are essential for the rapid trafficking of complex-containing organelles around the cell. The CSCs appear to be delivered directly to sites of secondary cell wall synthesis and it is likely that transverse actin may mark these sites.     

On the Robustness of the Geometrical Model for Cell Wall Deposition

All plant cells are provided with the necessary rigidity to withstand the turgor by an exterior cell wall. This wall is composed of long crystalline cellulose microfibrils embedded in a matrix of other polysaccharides. The cellulose microfibrils are deposited by mobile membrane bound protein complexes in remarkably ordered lamellar textures. The mechanism by which these ordered textures arise, however, is still under debate. The geometrical model for cell wall deposition proposed by Emons and Mulder (Proc. Natl. Acad. Sci. 95, 7215–7219, 1998) provides a detailed approach to the case of cell wall deposition in non-growing cells, where there is no evidence for the direct influence of other cellular components such as microtubules. The model successfully reproduces even the so-called helicoidal wall; the most intricate texture observed. However, a number of simplifying assumptions were made in the original calculations. The present work addresses the issue of the robustness of the model to relaxation of these assumptions, by considering whether the helicoidal solutions survive when three aspects of the model are varied. These are: (i) the shape of the insertion domain, (ii) the distribution of lifetimes of individual CSCs, and (iii) fluctuations and overcrowding. Although details of the solutions do change, we find that in all cases the overall character of the helicoidal solutions is preserved.     

Cell wall enzymatic lysis of the yeast form of Pullularia pullulans and wall regeneration by protoplasts

During the process of degradation of the cell wall of the yeast form of Pullularia pullulans by the lytic system of Micromonospora chalcea samples were withdrawn at different times and observed under phase contrast and electron microscope. The progressive lysis of the walls reveals a fibrillar component inside the apparently amorphous wall. Freeze etched preparations of cells during the formation and regeneration of protoplasts show that the cellular membrane is split and this method allows the smooth external face of the membrane and other internal face covered by particles to be seen. The fact that the smooth face of the membrane is only visible during the preparation or the regeneration of protoplasts and very rarely when intact cells are fractured, suggests a strong adherence between cell wall and this external layer of the membrane. During the regeneration which takes place as in most of the yeasts and moulds, a special study of the extension of the cell wall is made and a possible mechanism for this extension of the regenerated cell wall is proposed.     

Cotton fibre differentiation: Occurrence and distribution of coated and smooth vesicles during primary and secondary wall formation

Summary Coated vesicles occur in differentiating cotton fibres during primary and secondary wall formation. The coated vesicles are often associated with the plasmalemma, or with membranes at the secreting face of dictyosomes, corresponding positionally to GERL. During secondary wall formation the number of dictyosome-associated coated vesicles seems to be smaller than during primary wall formation. When sections are stained for periodateoxidizable polysaccharides (Thiéry reaction) the membrane of plasmalemma-associated coated vesicles is intensely stained. The membrane of dictyosome-associated coated vesicles is only weakly stained. On the basis of the present evidence it is not possible to clearly decide, whether the staining in plasmalemma-associated coated vesicles is due to obliquely cut membrane or to vesicle contents. The vesicle coat material is not stained. Possible functions of coated vesicles in differentiating cotton fibres are discussed.Vesicles with contents positively stained with the Thiéry reaction are observed only during primary wall formation. The membrane of these vesicles is smooth and seems to bud from the same cisternae, probably GERL, as do the coated vesicles. During secondary wall formation no vesicles containing periodate-oxidizable polysaccharides could be detected, even under conditions that result in a strong, specific reaction in the cellulosic secondary wall. In some instances polysaccharidic material, resembling secondary wall material, has been seen to adhere to the outside of the plasmalemma. These results are consistent with the hypothesis that, in higher plants, at least part of primary wall material may already be synthesized in dictyosome vesicles, whereas cellulose biosynthesis occurs at the cell surface.