Cell wall polymers in the Phaeoceros placenta reflect developmental and functional differences across generations.

The placenta of hornworts is unique among bryophytes in the restriction of transfer cells that are characterized by elaborate wall labyrinths to the gametophyte generation. During development, cells around the periphery of the sporophyte foot elongate, forming smooth-walled haustorial cells that interdigitate with gametophyte cells. Using immunogold labeling with 22 antibodies to diverse cell wall polymers, we examined compositional differences in the developmentally and morphologically distinct cell walls of gametophyte transfer cells and sporophyte haustorial cells in the placenta of Phaeoceros. As detected by Calcofluor White fluorescence, cellulose forms the cell wall scaffolding in cells on both sides of the placenta. Homogalacturonan (HG) and rhamnogalacturonan I (RG-I) pectins are abundant in both cell types, and haustrorial cells are further enriched in methyl-esterified HGs. The abundance of pectins in placental cell walls is consistent with the postulated roles of these polymers in cell wall porosity and in maintaining an acidic apoplastic pH favorable to solute transport. Xyloglucan hemicellulose, but not mannans or glucuronoxylans, are present in cell walls at the interface between the two generations with a lower density in gametophytic wall ingrowths. Arabinogalactan proteins (AGPs) are diverse along the plasmalemma of placental cells and are absent in surrounding cells in both generations. AGPs in placental cell walls may play a role in calcium binding and release associated with signal transduction as has been speculated for these glycoproteins in other plants. Callose is restricted to thin areas in cell walls of gametophyte transfer cells. In contrast to studies of transfer cells in other systems, no reaction to the JIM12 antibody against extensin was observed in Phaeoceros.

Transmission Electron Microscopy of Centrioles, Basal Bodies and Flagella in Motile Male Gametes of Land Plants.

Motile male gametes (spermatozoids) of land plants are coiled and contain a modified and precisely organized complement of organelles that includes a locomotory apparatus with two to thousands of flagella. Each flagellum is generated from a basal body that originates de novo as a centriole in spermatogenous cell lineages. Much of what is known about the diversity of plant male gametes was derived from detailed transmission electron microscopic studies. Because the process of spermatogenesis results in complete transformation of the shape and organization of these cells, TEM studies have yielded a wealth of information on cellular differentiation. Because green algal progenitor groups contain centrioles and a variety of motile cells, land plant spermatozoids also provide a plethora of opportunities to examine the evolution and eventual loss of centrioles and locomotory apparatus during land colonization. Here we provide a brief overview of the studies and methodologies we have conducted over the past 20 years that have elucidated not only the structural diversity of these cells but also the development of microtubule organizing centers, the de novo origin of centrioles and the ontogeny of structurally complex motile cells.

The mechanism for explosive seed dispersal in Cardamine hirsuta (Brassicaceae).

PREMISE: Although many highly successful weed species use a ballistic seed dispersal mechanism, little is known about the mechanics of this process. Bittercress (Cardamine hirsuta) siliques are morphologically similar to Arabidopsis siliques, but they can project their seeds up to 5 m, while Arabidopsis seeds are dispersed by gravity. Comparison of these species should enable us to determine which structures might be responsible for ballistic seed dispersal. METHODS: Sections of Arabidopsis and bittercress siliques were immunolabeled with antibodies raised against a variety of polysaccharide epitopes. RESULTS: In bittercress, the second endocarp layer (enB) of the valve had strongly asymmetrical cell wall thickenings, whereas the analogous cells in Arabidopsis were reinforced symmetrically and to a lesser extent. Additionally, an accumulation of mucilaginous pectins was found between the first and second endocarp (enA and enB) layers in the bittercress valve that was not present in Arabidopsis. However, in both species, highly de-esterified homogalacturonan was lost in the dehiscence zone (at the carpel/replum interface) as the siliques matured, thus allowing for separation of the valve at maturity. CONCLUSIONS: Ballistic seed dispersal in bittercress may involve the contraction of the outer pericarp tissue against the highly asymmetrically thickened enB cells, which are hypothesized to bend in one direction preferentially. The stress generated by the differential drying of the inner and outer layers of the valve is released suddenly as the adhesion between the cells of the dehiscence zone is lost, leading to a rapid coiling of the valve and dispersal of the seeds.

Leaf abscission in Impatiens (Balsaminaceae) is due to loss of highly de-esterified homogalacturonans in the middle lamellae.

PREMISE OF STUDY: Abscission zones (AZ) are sites where leaves and other organs are shed. Investigating the AZ by classical biochemical techniques is difficult due to its small size and because the surrounding tissue is not involved in abscission. The goals of this study were to determine whether AZ cell walls are chemically unique from the other cells of the petiole, perhaps making them more susceptible to enzymatic degradation during abscission and to identify which cell wall polysaccharides are degraded during abscission. METHODS: A battery of antibodies that recognize a large number of cell wall polysaccharide and glycoprotein epitopes was used to probe sections of the Impatiens leaf AZ at several time points in the abscission process. KEY RESULTS: Prior to abscission, the walls of the AZ cells were found to be similar in composition to the walls of the cells both proximal and distal to the AZ. Of all the epitopes monitored, only the highly de-esterified homogalacturonans (HG) of the middle lamellae were found to be reduced post-abscission and only at the plane of separation. More highly esterified homogalacturonans, as well as other pectin and xyloglucan epitopes were not affected. Furthermore, cellulose, as detected by an endoglucanase-gold probe and cellulose-binding module staining, was unaffected, even on the walls of the cells facing the separation site. CONCLUSIONS: In the leaf abscission zone of Impatiens, wall alterations during abscission are strictly limited to the plane of separation and involve only the loss of highly de-esterified pectins from the middle lamellae.

A cytochemical and immunocytochemical analysis of the wall labyrinth apparatus in leaf transfer cells in Elodea canadensis.

BACKGROUND AND AIMS: Transfer cells are plant cells specialized in apoplast/symplast transport and characterized by a distinctive wall labyrinth apparatus. The molecular architecture and biochemistry of the labyrinth apparatus are poorly known. The leaf lamina in the aquatic angiosperm Elodea canadensis consists of only two cell layers, with the abaxial cells developing as transfer cells. The present study investigated biochemical properties of wall ingrowths and associated plasmalemma in these cells. METHODS: Leaves of Elodea were examined by light and electron microscopy and ATPase activity was localized cytochemically. Immunogold electron microscopy was employed to localize carbohydrate epitopes associated with major cell wall polysaccharides and glycoproteins. KEY RESULTS: The plasmalemma associated with the wall labyrinth is strongly enriched in light-dependent ATPase activity. The wall ingrowths and an underlying wall layer share an LM11 epitope probably associated with glucuronoarabinoxylan and a CCRC-M7 epitope typically associated with rhamnogalacturonan I. No labelling was observed with LM10, an antibody that recognizes low-substituted and unsubstituted xylan, a polysaccharide consistently associated with secondary cell walls. The JIM5 and JIM7 epitopes, associated with homogalacturonan with different degrees of methylation, appear to be absent in the wall labyrinth but present in the rest of cell walls. CONCLUSIONS: The wall labyrinth apparatus of leaf transfer cells in Elodea is a specialized structure with distinctive biochemical properties. The high level of light-dependent ATPase activity in the plasmalemma lining the wall labyrinth is consistent with a formerly suggested role of leaf transfer cells in enhancing inorganic carbon inflow. The wall labyrinth is a part of the primary cell wall. The discovery that the wall ingrowths in Elodea have an antibody-binding pattern divergent, in part, from that of the rest of cell wall suggests that their carbohydrate composition is modulated in relation to transfer cell functioning.

Immunohistochemical investigation of the necrotrophic phase of the fungus Colletotrichum gloeosporioides in the biocontrol of hemp sesbania (Sesbania exaltata; Papilionaceae).

PREMISE OF THE STUDY: Fungal plant pathogens exert much of their effect on plant cells through alterations in the host cell walls. However, obtaining biochemical proof for this change is difficult because of the relatively small number of cells that are affected by the pathogen relative to the bulk of host tissue. In this study, we examined the differences in host wall composition between infected and uninfected areas of seedlings of the weed hemp sesbania (Sesbania exaltata) that were treated with the biocontrol agent Colletotrichum gloeosporioides. • METHODS: To determine the changes in cell wall composition, we used semi-thin sections and a battery of antibody probes that recognize components of the cell wall and immunogold-silver cytochemistry to visualize the probes. • KEY RESULTS: A loss of specific plant cell wall polysaccharides in the region surrounding the primary fungal infection and the creation of a defensive layer by the plant to limit the fungal invasion were the two most obvious changes noted in this study. At the invasion site, there was significant loss of rhamnogalacturon-1 (RGI) and esterified and de-esterified homogalacturonan (HG)-reactive epitopes from the cell walls. In contrast, boundary tissue between the vascular tissue and the fungal lesion reacted more strongly with antibodies that recognize arabinogalactan proteins (AGPs) and xyloglucans than in unaffected areas. • CONCLUSIONS: These data strongly indicate a role of pectinases in the invasion of the biocontrol agent and the importance of extensins, AGPs, and xyloglucans as defense by the host.

Gelatinous fibers are widespread in coiling tendrils and twining vines.

Although the coiling of tendrils and the twining of vines has been investigated since Darwin's time, a full understanding of the mechanism(s) of this coiling and twining ability has not yet been obtained. In a previous study (Planta 225: 485-498), gelatinous (G) fibers in tendrils of redvine occurred concomitantly with the ability to coil, strongly indicating their role in the coiling process. In this study, tendrils and twining vines of a number of species were examined using microscopic and immunocytochemical techniques to determine if a similar presence and distribution of these fibers exists in other plant species. Tendrils that coiled in many different directions had a cylinder of cortical G fibers, similar to redvine. However, tendrils that coiled only in a single direction had gelatinous fibers only along the inner surface of the coil. In tendrils with adhesive tips, the gelatinous fibers occurred in the central/core region of the tendril. Coiling occurred later in development in these tendrils, after the adhesive pad had attached. In twining stems, G fibers were not observed during the rapid circumnutation stage, but were found at later stages when the vine's position was fixed, generally one or two nodes below the node still circumnutating. The number and extent of fiber development correlated roughly with the amount of torsion required for the vine to ascend a support. In contrast, species that use adventitious roots for climbing or were trailing/scrambling-type vines did not have G fibers. These data strongly support the concept that coiling and twining in vines is caused by the presence of G fibers.

Recovery of microtubules on the blepharoplast of Ceratopteris spermatogenous cells after oryzalin treatment.

Most land plants have ill-defined microtubule-organizing centers (MTOCs), consisting of sites on the nuclear envelope or even along microtubules (MTs). In contrast, the spermatogenous cells of the pteridophyte Ceratopteris richardii have a well-defined MTOC, the blepharoplast, which organizes MTs through the last two division cycles. This allows a rare opportunity to study the organization and workings of a structurally well-defined plant MTOC. In this study, antheridial plants were treated with levels of oryzalin that cause complete MT loss from the cells containing blepharoplasts. The oryzalin was then washed out and plants were allowed to recover for varying amounts of time. If the spermatogenous cells were fixed prior to washing out, the blepharoplasts had an unusual appearance. In the matrix (pericentriolar) material where MT ends are normally found, clear areas of about the diameter of MTs were seen embedded in a much deeper matrix, made more obvious in stereo pairs. Occasionally, the matrix material was highly distended, although the basal body template cylinder morphology appeared to be unaltered. The blepharoplasts often occurred as clusters of 2 or 4, indicating that blepharoplast reproduction is not affected by the lack of MTs, but that their movement to the poles is. Gamma (gamma) tubulin antibodies labeled the edge of the blepharoplast in areas where the pits are located, indicating that these might be sites for MT nucleation. After wash out, the new MTs always re-appeared on the blepharoplast and the recovery occurred within an hour of washout. MT lengths increased with increasing washout time and were indistinguishable from untreated blepharoplasts after 24 h of recovery. After washout, arrays formed in new sperm cells such as the spline and basal bodies were often malformed or present in multiple copies, as were the blepharoplasts in these cells prior to wash out. These data indicate that the blepharoplast serves as the site of MT nucleation and organization even after complete MT de-polymerization.

Unusual trichome structure and composition in mericarps of catchweed bedstraw (Galium aparine).

Catchweed bedstraw is famous for its ability to adhere to other objects due to the presence of numerous trichomes surrounding the stem and mericarps and on the surfaces of the leaves. These trichomes serve as an efficient vector for the movement of the propagules via animals. In this study, we examined the structure and composition of the mericarp trichomes by microscopic and immunocytochemical techniques to determine the distribution of polysaccharides. Trichomes present around the mericarps are distinguished by a pronounced hooked tip, resembling in many ways those on Velcro. In semi-thin sections, the hooked area of the trichome contains little or no lumen but rather appears to be solidly composed of cell wall material. This solid hook appears to be divided into a plug-like zone of material and a highly thickened primary wall. These trichomes are also compositionally unique. They contain very little xyloglucan, even though other tissues in the plant reacted strongly with antibodies that recognize these polysaccharides. The distribution of pectin epitopes on these hooked trichomes was extremely distinctive, with each of the antibodies recognizing domains along the surface of the primary wall and/or in the plug area. Despite the heavily thickened nature of the walls of these trichomes, xylans were not present. Thus, the unique plugged, thickened, and hooked tip of these trichomes appears to be the result of a specific combination and distribution of various pectic polysaccharide molecules. This unusual wall composition may facilitate the formation of highly curved structures that might be difficult to form with the more rigid xyloglucans and xylans.

Immunocytochemical characterization of tension wood: Gelatinous fibers contain more than just cellulose.

Gelatinous fibers (G-fibers) are the active component of tension wood. G-fibers are unlike traditional fiber cells in that they possess a thick, nonlignified gelatinous layer (G-layer) internal to the normal secondary cell wall layers. For the past several decades, the G-layer has generally been presumed to be composed nearly entirely of crystalline cellulose, although several reports have appeared that disagreed with this hypothesis. In this report, immunocytochemical techniques were used to investigate the polysaccharide composition of G-fibers in sweetgum (Liquidambar styraciflua; Hamamelidaceae) and hackberry (Celtis occidentalis; Ulmaceae) tension wood. Surprisingly, a number of antibodies that recognize arabinogalactan proteins and RG I-type pectin molecules bound to the G-layer. Because AGPs and pectic mucilages are found in other plant tissues where swelling reactions occur, we propose that these polymers may be the source of the contractile forces that act on the cellulose microfibrils to provide the tension force necessary to bend the tree trunk.

A cortical band of gelatinous fibers causes the coiling of redvine tendrils: a model based upon cytochemical and immunocytochemical studies.

A cortical band of fiber cells originate de novo in tendrils of redvine [Brunnichia ovata (Walt.) Shiners] when these convert from straight, supple young filaments to stiffened coiled structures in response to touch stimulation. We have analyzed the cell walls of these fibers by in situ localization techniques to determine their composition and possible role(s) in the coiling process. The fiber cell wall consists of a primary cell wall and two lignified secondary wall layers (S(1) and S(2)) and a less lignified gelatinous (G) layer proximal to the plasmalemma. Compositionally, the fibers are sharply distinct from surrounding parenchyma as determined by antibody and affinity probes. The fiber cell walls are highly enriched in cellulose, callose and xylan but contain no homogalacturonan, either esterified or de-esterified. Rhamnogalacturonan-I (RG-I) epitopes are not detected in the S layers, although they are in both the gelatinous layer and primary wall, indicating a further restriction of RG-I in the fiber cells. Lignin is concentrated in the secondary wall layers of the fiber and the compound middle lamellae/primary cell wall but is absent from the gelatinous layer. Our observations indicate that these fibers play a central role in tendril function, not only in stabilizing its final shape after coiling but also generating the tensile strength responsible for the coiling. This theory is further substantiated by the absence of gelatinous layers in the fibers of the rare tendrils that fail to coil. These data indicate that gelatinous-type fibers are responsible for the coiling of redvine tendrils and a number of other tendrils and vines.

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.

The association of peroxisomes with the developing cell plate in dividing onion root cells depends on actin microfilaments and myosin.

We have investigated changes in the distribution of peroxisomes through the cell cycle in onion ( Allium cepa L.) root meristem cells with immunofluorescence and electron microscopy, and in leek ( Allium porrum L.) epidermal cells with immunofluorescence and peroxisomal-targeted green fluorescent protein. During interphase and mitosis, peroxisomes distribute randomly throughout the cytoplasm, but beginning late in anaphase, they accumulate at the division plane. Initially, peroxisomes occur within the microtubule phragmoplast in two zones on either side of the developing cell plate. However, as the phragmoplast expands outwards to form an annulus, peroxisomes redistribute into a ring immediately inside the location of the microtubules. Peroxisome aggregation depends on actin microfilaments and myosin. Peroxisomes first accumulate in the division plane prior to the formation of the microtubule phragmoplast, and throughout cytokinesis, always co-localise with microfilaments. Microfilament-disrupting drugs (cytochalasin and latrunculin), and a putative inhibitor of myosin (2,3-butanedione monoxime), inhibit aggregation. We propose that aggregated peroxisomes function in the formation of the cell plate, either by regulating hydrogen peroxide production within the developing cell plate, or by their involvement in recycling of excess membranes from secretory vesicles via the beta-oxidation pathway. Differences in aggregation, a phenomenon which occurs in onion, some other monocots and to a lesser extent in tobacco BY-2 suspension cells, but which is not obvious in the roots of Arabidopsis thaliana (L.) Heynh., may reflect differences within the primary cell walls of these plants.

Herbicide resistance work in the United States Department of Agriculture-Agricultural Research Service.

Herbicide-resistant weed biotypes are an increasing problem in agriculture, with reports of resistance to almost every herbicide class at some place in the world, and the total number of resistant biotypes at over 250. Agricultural Research Service (ARS) scientists have been key players in this area since the first substantiated occurrence of these resistant biotypes in the 1970s. The most significant of their contributions is the complete unraveling of the mechanism of triazine resistance by Arntzen and colleagues, then with ARS at the University of Illinois. These studies established a high benchmark for research in this area and are a model for all studies in this area. Other ARS scientists have investigated a large number of weed biotypes with resistance to a wide range of herbicide classes and mechanisms of resistance. Collectively, these studies have been used to generate herbicide resistance-management schemes for growers, based upon the herbicide site and the potential for resistance development.

Tissue-specific expression and localization of safener-induced glutathione S-transferase proteins in Triticum tauschii.

Glutathione S-transferase (GST; EC 2.5.1.18) gene expression was examined in the coleoptile and new leaf tissue of etiolated shoots of the diploid wheat species Triticum tauschii (Coss.) Schmal., which is considered to be a progenitor and the D-genome donor to cultivated, hexaploid bread wheat Triticum aestivum L. GST expression (mRNA, protein, and enzyme activity with a herbicide substrate) in these shoot tissues was examined in response to herbicide safener treatment. Two different antibody probes, raised against the same safener-inducible GST protein (TtGSTU1) but differing in their specificity, were utilized to determine tissue distribution and subcellular localization of GST proteins in etiolated shoots. GST transcripts, immunoreactive GST proteins, and herbicide-metabolizing activity were all highest in the coleoptile of etiolated, safener-treated T. tauschii shoots. Anti-GST immunolabeling was strongest in the outer epidermal and adjoining sub-epidermal cells in both coleoptiles and new leaves following safener treatment. Our data indicate that safeners protect grass crops from herbicide injury by dramatically inducing the expression of GST proteins in the outer cell layers of the coleoptile, which prevents the herbicide from reaching the sensitive new leaves of etiolated shoots as they emerge from the soil.

Diversity in the distribution of polysaccharide and glycoprotein epitopes in the cell walls of bryophytes: new evidence for the multiple evolution of water-conducting cells.

• Although histologically much simpler than higher plants, bryophytes display a considerable degree of tissue differentiation, notably in those groups that possess an internal system of specialized water-conducting cells (WCCs). Here, using a battery of monoclonal antibodies, we examined the distribution of cell wall polysaccharide and glycoprotein carbohydrate epitopes in the gametophyte of four hepatics and eight mosses, with special reference to water-conducting cells. • CCRC-M7, an antibody against an arabinogalactan epitope, gave a highly consistent and generally specific labelling of WCCs; more variable results were obtained with other antibodies. The labelling patterns indicate that bryophytes exhibit cell and tissue complexity with respect to cell wall components on a par with higher plants. • A remarkable diversity in the immunocytochemical characteristics of WCCs was observed not only when comparing major bryophyte groups but also within the relatively small and well-circumscribed moss order Polytrichales, indicating that the cell wall biochemistry of WCCs may have been finely tuned in response to specific evolutionary pressures. The immunocytochemical data strengthen the notion that the WCCs in Takakia are not homologous with the hydroids of other mosses nor with the WCCs in Haplomitrium and metzgerialean liverworts. • The presence of several carbohydrate epitopes in hydroid walls runs strongly counter to the notion that their maturation involves hydrolysis of noncellulosic polysaccharides.