Wall ingrowths in epidermal transfer cells of Vicia faba cotyledons are modified primary walls marked by localized accumulations of arabinogalactan proteins

Despite the importance of transfer cells in enhancing nutrient transport in plants, little is known about how deposition of the complex morphology of their wall ingrowths is regulated. We probed thin sections of mature cotyledon epidermal transfer cells of Vicia faba with affinity probes and antibodies specific to polysaccharides and glycoproteins, to determine the distribution of these components in their walls. Walls of these transfer cells consist of the pre-existing primary wall, a uniformly deposited wall layer and wall ingrowths which are comprised of two regions; an electron-opaque inner region and an electron-translucent outer region. The primary wall reacted strongly with antibodies against esterified pectin, xyloglucan, the side chains of rhamnogalaturonan-1 and a cellulase-gold affinity probe. The electron-opaque inner region of wall ingrowths displayed a similar labeling pattern to that of the primary wall, showing strong cross-reactivity with all antibodies tested, except those reacting against highly de-esterified pectins. The electron-opaque outer layer of developmentally more mature wall ingrowths reacted strongly with anti-callose monoclonal and polyclonal antibodies, but showed no reaction for pectin or xyloglucan antibodies or the cellulase-gold affinity probe. The plasma membrane-wall interface was labeled strongly with anti-arabinogalactan protein (AGP) antibodies, with some AGP-reactive antibodies also labeling the electron-translucent zone. Nascent wall ingrowths were labeled specifically with AGPs but not anti-callose. A reduction in wall ingrowth density was observed when developing transfer cells were exposed to beta-d-glucosyl Yariv reagent compared with controls. Our results indicate that wall ingrowths of transfer cells are primary wall-like in composition and probably require AGPs for localized deposition.     

Wall ingrowth formation in transfer cells: novel examples of localized wall deposition in plant cells

The formation of wall ingrowths increases plasma membrane surface areas of transfer cells involved in membrane transport of nutrients in plants. Construction of these ingrowths provides intriguing and diverse examples of localized wall deposition. Flange wall ingrowths resemble secondary wall thickenings of tracheary elements in morphology and probable mechanisms of deposition. By contrast, reticulate wall ingrowths, deposited as discrete papillate projections, branch and fuse to create a fenestrated wall labyrinth representing a novel form of localized wall deposition. Papillate wall ingrowths are initiated as patches of disorganized cellulosic material and are compositionally similar to primary walls, except for a surrounding layer of callose and enhanced levels of arabinogalactan proteins at the ingrowth/membrane interface. How this unusual form of localized wall deposition is constructed is unknown but may involve constraining cellulose-synthesizing rosette complexes at their growing tips.     

Wall ingrowth architecture in epidermal transfer cells ofVicia faba cotyledons

Summary We describe the use of scanning electron microscopy to provide novel views of the three-dimensional morphology of the ingrowth wall in epidermal transfer cells of cotyledons of developingVicia faba seed. Wall ingrowth deposition in these cells amplifies the surface area of plasma membrane available for transport of solutes during cotyledon development. Despite the physiological importance of such amplification, little is known about wall ingrowth morphology and deposition in transfer cells. A detailed morphological analysis of wall deposition in this study clearly established for the first time that wall ingrowths are deposited at scattered, discrete loci as papillate ingrowth projections. The new views of the ingrowth wall revealed that these projections branch and fuse laterally, and fusion occurs by fine connections to form a fenestrated sheet or layer. This sheet of wall material then provides a base for further deposition of ingrowth projections to progressively build many interconnected, fenestrated layers. Consolidation, or filling-in, of the fenestrae in these layers appears to occur from small fingerlike protrusions of wall material which extend laterally from the most recently deposited surface of the fenestrae. We propose that deposition of fenestrated layers may provide a mechanism for maintaining continuous amplification of plasma membrane surface area in the face of turnover of the plasma membrane and transporter proteins associated with it. The techniques reported in this paper will provide new opportunities to investigate wall ingrowth deposition and its regulation in transfer cells.Abbreviations SEM scanning electron microscopy - TEM transmission electron microscopy Dedicated to Professor Brian E. S. Gunning on the occasion of his 65th birthday     

Transfer cell wall architecture: a contribution towards understanding localized wall deposition

Summary. A survey is presented of the architecture of secondary wall ingrowths in transfer cells from various taxa based on scanning electron microscopy. Wall ingrowths are a distinguishing feature of transfer cells and serve to amplify the plasma membrane surface area available for solute transport. Morphologically, two categories of ingrowths are recognized: reticulate and flange. Reticulate-type wall ingrowths are characterized by the deposition of small papillae that emerge from the underlying wall at discrete but apparently random loci, then branch and interconnect to form a complex labyrinth of variable morphology. In comparison, flange-type ingrowths are deposited as curvilinear ribs of wall material that remain in contact with the underlying wall along their length and become variously elaborate in different transfer cell types. This paper discusses the morphology of different types of wall ingrowths in relation to existing models for deposition of other secondary cell walls. Received July 20, 2001 Accepted November 29, 2001     

Nucellar projection transfer cells in the developing wheat grain

Summary Transfer cells in the nucellar projection of wheat grains at 25 ±3 days after anthesis have been examined using light and electron microscopy. Within the nucellar tissue, a sequential increase in non-polarized wall ingrowth differentiation and cytoplasmic density was evident. Cells located near the pigment strand were the least differentiated. The degree of differentiation increased progressively in cells further removed from the pigment strand and the cells bordering the endosperm cavity had degenerated. Four stages of transfer cell development were identified at the light microscope level. Wall ingrowth differentiation followed a sequence from a papillate form through increased branching (antler-shaped ingrowths) which ultimately anastomosed to form a complex labyrinth. The final stage of wall ingrowth differentiation was compression which resulted in massive ingrowths. In parallel with wall ingrowth deposition cytoplasmic density increased. During wall deposition, paramural and multivesicular bodies were prominent and were in close association with the wall ingrowths. The degeneration phase involved infilling of cytoplasmic islets within the wall ingrowths. This was accompanied by complete loss of the protoplast. The significance of this transfer cell development for sucrose efflux to the endosperm cavity was assessed by computing potential sucrose fluxes across the plasma membrane surface areas of the nucellar projection cells. Transfer cell development amplified the total plasma membrane surface area by 22 fold. The potential sucrose flux, when compared with maximal rates of facilitated membrane transport of sugars, indicated spare capacity for sucrose efflux to the endosperm cavity. Indeed, when the total flux was partitioned between the nucellar projection cells at the three stages of transfer cell development, the fully differentiated stage III cells located proximally to the endosperm cavity alone exhibited spare transport capacity. Stage II cells could accommodate the total rate of sucrose transfer, but stage I cells could not. It is concluded that the nucellar projection tissue of wheat provides a unique opportunity to study transfer cell development and the functional role of these cells in supporting sucrose transport.Abbreviations CSPMSA cross sectional plasma membrane surface area - LPMSA longitudinal plasma membrane surface area - PTS tri-sodium 3-hydroxy-5,8,10-pyrenetrisulfonate     

Induction of wall ingrowths of transfer cells occurs rapidly and depends upon gene expression in cotyledons of developing Vicia faba seeds

Summary. Abaxial epidermal cells of developing faba bean (Vicia faba) cotyledons are modified to a transfer cell morphology and function. In contrast, the adaxial epidermal cells do not form transfer cells but can be induced to do so when excised cotyledons are cultured on an agar medium. The first fenestrated layer of wall ingrowths is apparent within 24 h of cotyledon exposure to culture medium. The time course of wall ingrowth formation was examined further. By 2 h following cotyledon excision, a 350 nm thick wall was deposited evenly over the outer periclinal walls of adaxial epidermal cells and densities of cytoplasmic vesicles increased. After 3 h in culture, 10% of epidermal cells contained small projections of wall material on their outer periclinal walls. Thereafter, this percentage rose sharply and reached a maximum of 90% by 15 h. Continuous culture of cotyledons on a medium containing 6-methyl purine (an inhibitor of RNA synthesis) completely blocked wall ingrowth formation. In contrast, if exposure to 6-methyl purine was delayed for 1 h at the start of the culture period, the adaxial epidermal cells were found to contain small wall ingrowths. Treating cotyledons for 1 h with 6-methyl purine at 15 h following cotyledon excision halted further wall ingrowth development. We conclude that transfer cell induction is rapid and that signalling and early events leading to wall ingrowth formation depend upon gene expression. In addition, these gene products have a high turnover rate. Correspondence and reprints: School of Environmental and Life Sciences, Biology Building, University of Newcastle, Callaghan, NSW 2308, Australia.     

Polarized microtubule deposition coincides with wall ingrowth formation in transfer cells ofVicia faba L. cotyledons

Summary The epidermal transfer cells in developingVicia faba L. cotyledons are highly polarized. Extensive wall ingrowths occur on their outer periclinal walls and extend part way down both anticlinal walls. This ingrowth development serves to increase the surface area of the plasma membrane and thus maximize porter-dependent uptake of sugars from the seed apoplasm. In contrast, the inner periclinal walls of these transfer cells do not form wall ingrowths. We have commenced a study of the mechanisms responsible for establishing this polarity by first analysing the microtubule (MT) cytoskeleton in developing transfer cells. Thin sections of fixed cotyledons embedded in methacrylate resin were processed for immunofluorescence microscopy using monoclonal anti--tubulin and counterstained with Calcofluor White to visualize wall ingrowths. In epidermal cells of young cotyledons where wall ingrowths were yet to develop, MT labelling was detected around all cortical regions of the cell. However, in cells where wall ingrowths were clearly established, MT labelling was detected almost exclusively in cortical regions adjacent to the wall ingrowths. Little, if any, MT labelling was detected on the anticlinal or inner periclinal walls of these cells. This distribution of MTs was most prominent in cells with well developed wall ingrowths. In these cells, a subpopulation of MTs were also detected emanating from the subcortex and extending towards the wall ingrowth region. The possible role of MT distribution in establishing transfer cell polarity and wall ingrowth formation is discussed.Abbreviations MT microtubule     

Transfer cells and nematode induced giant cells inHelianthemum

Summary The morphology of wall ingrowths in xylem and phloem transfer cells inHelianthemum is different. It is possible to use nematode infection to induce the formation of giant cells which abut both xylem and phloem elements to test whether ingrowth morphology is controlled by the solutes presumed to be transported across the plasmalemma of the cells. This experiment has been done and it is found that although wall ingrowths develop against both xylem and phloem, the giant cells exhibit only the ingrowth structure characteristic of xylem transfer cells.     

GIGANTEA is a component of a regulatory pathway determining wall ingrowth deposition in phloem parenchyma transfer cells of Arabidopsis thaliana

Transfer cells are specialised transport cells containing invaginated wall ingrowths that generate an amplified plasma membrane surface area with high densities of transporter proteins. They trans‐differentiate from differentiated cells at sites at which enhanced rates of nutrient transport occur across apo/symplasmic boundaries. Despite their physiological importance, little is known of the molecular mechanisms regulating construction of their intricate wall ingrowths. We investigated the genetic control of wall ingrowth formation in phloem parenchyma transfer cells of leaf minor veins in Arabidopsis thaliana. Wall ingrowth development in these cells is substantially enhanced upon exposing plants to high‐light or cold treatments. A hierarchical bioinformatic analysis of public microarray datasets derived from the leaves of plants subjected to these treatments identified GIGANTEA (GI) as one of 46 genes that are commonly up‐regulated twofold or more under both high‐light and cold conditions. Histological analysis of the GI mutants gi‐2 and gi‐3 showed that the amount of phloem parenchyma containing wall ingrowths was reduced 15‐fold compared with wild‐type. Discrete papillate wall ingrowths were formed in gi‐2 plants but failed to develop into branched networks. Wall ingrowth development in gi‐2 was not rescued by exposing these plants to high‐light or cold conditions. In contrast, over‐expression of GI in the gi‐2 background restored wall ingrowth deposition to wild‐type levels. These results indicate that GI regulates the ongoing development of wall ingrowth networks at a point downstream of inputs from environmental signals.     

Calcification and Decalcification During Epithallial Cell Turnover in Coralline Red Algae

Epithallial cells of the coralline red algae are characterized by unusual structural specialization, which include deep invaginations of the distal cell surface, and by unique development, which culminates in senescence, shedding, and replacement of the cells. Electron microscopic study of epithelial cell differentiation in morphologically and taxonomically disparate species suggests that the unusual features of epithelial cell structure and development stem from the fact that these dynamics occur within a calcified matrix. Distal wall ingrowths begin to form on the initial cells, cells whose cleavage eventually gives rise distally to new epithelial cells. After the distal wall ingrowths form, the overlying crosswall becomes rich in organic material. For this organic wall material to be deposited into the existing crosswall, the wall must first be decalcified; therefore, the presence of abundant organic material in the crosswall provides a marker of localized decalcification. We propose that the location and time of origin of distal wall ingrowths indicate a connection between the ingrowths and two coordinated processes: localized secretion of wall material, and decalcification of the overlying cell wall in preparation for the movement of the young epithelial cell into a new location relative to the surrounding calcified matrix. The large plasmalemmal surface area associated with the distal wall ingrowths allows for a greater abundance of membrane‐associated components, such as proton pumps, that could drive localized cell wall decalcification.     

Role of light and jasmonic acid signaling in regulating foliar phloem cell wall ingrowth development

Phloem cells adjacent to sieve elements can possess wall invaginations. The role of light and jasmonic acid signaling in wall ingrowth development was examined in pea companion cells (CCs), Arabidopsis thaliana phloem parenchyma cells (PCs), and in Senecio vulgaris (with ingrowths in both cell types). Features characterized included wall ingrowths (from electron microscopic images), foliar vein density and photosynthetic capacity. In Arabidopsis, wall ingrowths were bulky compared with finger-like invaginations in pea and S. vulgaris. Relative to low light (LL), wall invagination in both CCs and PCs was greater in high light (HL). Treatment with methyl jasmonate in LL had no effect on CCs, but increased PC wall ingrowths. LL-to-HL transfer resulted in significantly less wall ingrowth in the fad7-1 fad8-1 (jasmonate-deficient) Arabidopsis mutant relative to the wild type. These results suggest that chloroplast oxidative status, via chloroplast-derived jasmonates, may modulate phloem structure and function. While CC wall ingrowths facilitate phloem loading by expanding the membrane area available for active uptake, one can speculate that phloem PC ingrowths may have two potential roles: to increase the efflux of sugars and/or protons into the apoplast to augment phloem loading; and/or to protect the phloem against pathogens and/or insects.     

Reactive oxygen species form part of a regulatory pathway initiating trans-differentiation of epidermal transfer cells in Vicia faba cotyledons

Various cell types can trans-differentiate to a transfer cell (TC) morphology characterized by deposition of polarized ingrowth walls comprised of a uniform layer on which wall ingrowths (WIs) develop. WIs form scaffolds supporting amplified plasma membrane areas enriched in transporters conferring a cellular capacity for high rates of nutrient exchange across apo- and symplasmic interfaces. The hypothesis that reactive oxygen species (ROS) are a component of the regulatory pathway inducing ingrowth wall formation was tested using Vicia faba cotyledons. Vicia faba cotyledons offer a robust experimental model to examine TC induction as, on being placed into culture, their adaxial epidermal cells rapidly (hours) form ingrowth walls on their outer periclinal walls. These are readily visualized by electron microscopy, and epidermal peels of their trans-differentiating cells allow measures of cell-specific gene expression. Ingrowth wall formation responded inversely to pharmacological manipulation of ROS levels, indicating that a flavin-containing enzyme (NADPH oxidase) and superoxide dismutase cooperatively generate a regulatory H(2)O(2) signature. Extracellular H(2)O(2) fluxes peaked prior to the appearance of WIs and were followed by a slower rise in H(2)O(2) flux that occurred concomitantly, and co-localized, with ingrowth wall formation. De-localizing the H(2)O(2) signature caused a corresponding de-localization of cell wall deposition. Temporal and epidermal cell-specific expression profiles of VfrbohA and VfrbohC coincided with those of extracellular H(2)O(2) production and were regulated by cross-talk with ethylene. It is concluded that H(2)O(2) functions, downstream of ethylene, to activate cell wall biosynthesis and direct polarized deposition of a uniform wall on which WIs form.     

Extracellular hydrogen peroxide,produced through a respiratory burst oxidase/superoxide dismutase pathway,directs ingrowth wall formation in epidermal transfer cells of Vicia faba cotyledons

The intricate, and often polarized, ingrowth walls of transfer cells (TCs) amplify their plasma membrane surface areas to confer a transport function of supporting high rates of nutrient exchange across apo-/symplasmic interfaces. The TC ingrowth wall comprises a uniform wall layer on which wall ingrowths are deposited. Signals and signal cascades inducing trans-differentiation events leading to formation of TC ingrowth walls are poorly understood. Vicia faba cotyledons offer a robust experimental model to examine TC induction as, when placed into culture, their adaxial epidermal cells rapidly (h) and synchronously form polarized ingrowth walls accessible for experimental observations. Using this model, we recently reported findings consistent with extracellular hydrogen peroxide, produced through a respiratory burst oxidase homolog/superoxide dismutase pathway, initiating cell wall biosynthetic activity and providing directional information guiding deposition of the polarized uniform wall. Our conclusions rested on observations derived from pharmacological manipulations of hydrogen peroxide production and correlative gene expression data sets. A series of additional studies were undertaken, the results of which verify that extracellular hydrogen peroxide contributes to regulating ingrowth wall formation and is generated by a respiratory burst oxidase homolog/superoxide dismutase pathway.     

Role of sugars in regulating transfer cell development in cotyledons of developing Vicia faba seeds

Summary. Transfer cell formation in cotyledons of developing faba bean (Vicia faba L.) seeds coincides with an abrupt change in seed apoplasm composition from one dominated by hexoses to one in which sucrose is the principal sugar. On the basis of these observations, we tested the hypothesis that sugars induce and/or sustain transfer cell development. To avoid confounding effects of in planta developmental programs, we exploited the finding that adaxial epidermal cells of cotyledons, which do not become transfer cells in planta, can be induced to form functional transfer cells when cotyledons are cultured on an agar medium. Growth rates of cotyledons cultured on hexose or sucrose media were used to inform choice of sugar concentrations. The same proportion of adaxial epidermal cells of excised cotyledons were induced to form wall ingrowths independent of sugar species and concentration supplied. In all cases, induction of wall ingrowths coincided with a marked increase in the intracellular sucrose-to-hexose ratio. In contrast, further progression of wall ingrowth deposition was correlated positively with intracellular sucrose concentrations that varied depending upon external sugar species and supply. Sucrose symporter induction and subsequent maintenance behaved identically to wall ingrowth formation in response to an external supply of hexoses or sucrose. However, in contrast to wall ingrowth formation, induction of sucrose symporter activity was delayed. We discuss the possibility of intracellular sugars functioning both as signals and substrates that induce and control subsequent development of transfer cells. Correspondence and reprints: School of Environmental and Life Sciences, Biology Building, University of Newcastle, Callaghan, NSW 2308, Australia.     

Ultrastructure of transfer cells in spontaneous nodules of alfalfa (Medicago sativa)

Summary Spontaneous nodules were formed on the primary roots of alfalfa plants in the absence ofRhizobium. Histologically, these white single-to-multilobed structures showed nodule meristems, cortex, endodermis, central zone, and vascular strands. Nodules were devoid of bacteria and infection threads. Instead, the larger cells were completely filled with many starch grains while smaller cells had very few or none. Xylem parenchyma and phloem companion cells exhibited long, filiform and branched wall ingrowths. The characteristic features of both types of transfer cells were polarity of wall ingrowths, high cytoplasmic density, numerous mitochondria, abundant ribosomes, well-developed nucleus and nucleolus, and vesicles originated from rough endoplasmic reticulum. These results were compared with normal nodules induced byRhizobium. Our results suggest that xylem parenchyma and phloem companion transfer cells are active and probably involved in the short distance transport of solutes in and out of spontaneous nodules. Since younger nodules showed short, papillate, and unbranched wall ingrowths, and older tissue showed elongated, filiform and branched wall ingrowths, the development of wall ingrowths seemed to be gradual rather then abrupt. The occurrence of both type-A and -B wall ingrowths suggests that phloem companion transfer cells may be active in loading and unloading of sieve elements. Since there were no symbiotic bacteria and thus no fixed nitrogen, it is tempting to speculate that xylem parenchyma transfer cells may be re-transporting accumulated carbon from starch grains to the rest of the plant body by loading xylem vessels. Fusion of ER-originated vesicles with wall ingrowth membrane indicated the involvement of ER in the membrane formation for elongating wall ingrowths. Since transfer cells were a characteristic feature of both spontaneous andRhizobium-induced nodules, their occurrence and development is controlled by the genetic make-up of alfalfa plant and not by a physiological source or sink emanating from symbiotic bacteria.Abbreviations ATP adenosine triphosphate - ATPase adenosine triphosphatase - EH emergent root hair - EM electron microscope - Nar nodulation in the absence of Rhizobium - RT root tip - RER rough endoplasmic reticulum - YEMG yeast extract mannitol-gluconate     

Transfer cell induction in cotyledons ofVicia faba L.

Summary Immediately prior to seed fill, a dermal transfer cell complex, comprised of epidermal and subepidermal cells, differentiates on the abaxial surface of the cotyledons in seed ofVicia faba. Over the period of differentiation of this complex in vivo, the principal sugars of the seed apoplasmic sap change from hexoses, glucose and fructose, to sucrose. Cotyledons were removed from seeds before differentiation of the transfer cell complex and cultured for 6 days on an agar-based medium in the dark with their abaxial surface in contact with a medium containing either 100 mM hexoses (glucose and fructose in equimolar concentrations) or 100 mM sucrose. On both media, cotyledon growth rate was maintained throughout the culture period at, or above, that of in vivo grown cotyledons of equivalent developmental age. When cotyledons were cultured on a medium containing glucose and fructose, epidermal cells of both the ab- and adaxial surfaces developed wall ingrowths on their outer periclinal walls and their cytoplasm became dense, vesicular, and rich in mitochondria. Extensive ingrowth deposition also occurred on walls of the subepidermal cells and several rows of underlying storage cells where they abutted intercellular spaces. This latter ingrowth development was apparent on both cotyledon surfaces, but extended into more of the underlying cell layers on the abaxial surface at the funicular end of the cotyledon. In in vivo grown cotyledons, such ingrowth development is restricted to the subepidermal cells of the abaxial surface. Ingrowth morphology was commensurate with that of transfer cells of in vivo grown cotyledons. In contrast to the observed induction on a medium containing glucose and fructose, cotyledons cultured with sucrose as the sole sugar source exhibited no ingrowth deposition or small wall ingrowths in some abaxial epidermal cells. While the potential sugar signalling mechanism is unknown, this culture system offers an exciting opportunity to explore the molecular biology of transfer cell development.Abbreviations DAA days after anthesis - GC-MS gas chromatography and mass spectrometry - PAR photosynthetically active radiation - RGR relative growth rate - SCM standard culture medium     

Immunogold localization of callose and other cell wall components in pea nodule transfer cells

Summary Transfer cells are located adjacent to xylem and phloem elements in pea nodule vascular tissues. The composition of the labyrinthine wall intrusions was investigated by immunogold labeling using specific antibody probes. Callose antigen was found at the base of newly formed cell wall intrusions and also in adjacent plasmodesmata. Sections through developed labyrinthine intrusions revealed that wall ingrowths had an internal structure with small domains of callose suggesting the presence of channels or vents. Xyloglucan and pectin antigens were uniformly distributed within the wall, but the distribution of extensin antigens was variable, with different antigens being detected in different regions of the wall ingrowth. A lectinlike glycoprotein, PsNLEC-1, was localized in intercellular spaces associated with nodule transfer cells. Previously, expression of this component was observed in other types of cells showing complex involution of the plasma membrane, namely root cortical cells harboring arbuscular mycorrhizae and nodule cells harboring nitrogen-fixing rhizobia.     

The Initiation, Development and Removal of Embryo Sac Wall Ingrowths in the Developing Seeds of Solanum nigrum L. An Ultrastructural Study

In developing seeds of Solanum nigrum L., wall ingrowths developedat the extreme micropylar and chalazal ends of the embryo sac.In the micropylar region, the wall ingrowths were initiatedat the three-celled endosperm stage starting at the base ofthe zygote then progressing for a short distance chalazalwards.They developed quickly with the most elaborate around the baseof the suspensor. The chalazal wall ingrowths developed alongthe surfaces of the chalazal cup, the antipodal cup and thehypostase. Those along the hypostase were initiated at the four-celled,those in the chalazal and antipodal cups at the 20-celled endospermstages. The most elaborate developed along the base of the antipodalcup; the most simple were along the base of the chalazal cup.Small electron-lucent invaginations of the plasmalemma whichlater became filled with fibrillar material, were the earliestindication of wall ingrowth formation. Removal of the wall ingrowthscommenced at the mid-globular stage of embryo development andwas completed by the mid-heart-shaped stage. In the micropylarregion, wall ingrowth removal was rapid, starting with the lossof the fibrillar component followed by the thinning of the cellwall. However, along the hypostase and antipodal cup, a heterogeneouslayer of varying electron densities and a thinner, more electrondense layer was laid down over the ingrowths. This was followedby the removal of the fibrillar component. The initiation, removaland location of the embryo sac wall ingrowths is discussed inconnection with understanding the nutritional relationshipsbetween maternal tissue, endosperm and embryo.Copyright 1995,1999 Academic Press Wall ingrowths, Solanum nigrum, transfer cells, zone of separation and secretion, hypostase     

An ultrastructural and cytochemical study of the wall-membrane apparatus of transfer cells using freeze-substitution

Summary A freeze-substitution technique is described which enables the ultrastructure of certain types of plant transfer cells to be preserved with minimal ice crystal damage. The ultrastructure of transfer cells fromFunaria, Lonicera, andSenecio after freeze-substitution has been compared with that of glutaraldehyde-osmium fixed material. The irregular clear zone between wall and plasma membrane, present in conventional preparations, is absent in freeze-substituted tissue. It is proposed that this interfacial zone is an artefact caused by expansion of wall ingrowth material during conventional fixation procedures. In transfer cells with a complex wall labyrinth the swelling of wall material severely disrupts the true structure of the wall-membrane apparatus and results in a large decrease in the surface to volume ratio of the protoplast. These findings are supported in the case ofFunaria by a freezefracture study. The reactivity of the plasma-membrane to the PTA/chromic acid stain is enhanced in freeze-substituted material. Use of theThiéry silver proteinate reagent in conjunction with freeze-substitution has revealed marked differences between the wall ingrowths ofFunaria sporophyte haustorium transfer cells and those ofLonicera nectary trichomes.