Resin-casting: a method for investigating apoplastic spaces

A method has been developed in which liquid resin can be injected or infiltrated into spaces within a plant body, for example the lumens of vessels, fibers, and intercellular spaces. After polymerization of the resin, plant tissues are digested away with two solutions used in sequence: first, equal parts concentrated hydrogen peroxide and glacial acetic acid; second, concentrated sulfuric acid. Complete digestion renders three-dimensional casts of the spaces in the original tissue. These can be examined with scanning electron microscopy, light microscopy, or a dissecting microscope. Casts have such high fidelity and high resolution that details of pit canals, pit chambers, and perforation plates can be studied. Vessel casts over 15 cm long and revealing the details of several thousand constituent vessel elements have been obtained easily. Casts of the lumens of all cell types and of narrow intercellular spaces are obtained by prolonged infiltration with a low-viscosity resin solution. Alternatively, rapid, brief injection of the resin by vacuum produces casts predominantly of just those spaces that were open to the resin at the cut ends of the sample, for example the lumens of vessels and secretory ducts or the intercellular space network of an aerenchymatous parenchyma.     

Solute permeation across the apoplastic barrier in the perisperm–endosperm envelope in cucumber seeds

An apoplastic barrier consisting of callose and lipid layers in the perisperm–endosperm (PE) envelope is known to restrict inward and outward transport of solutes in cucurbit seeds. The present work examines permeability properties of the barrier using cucumber seed as a model system. Osmometrically determined osmotic potential of the apoplastic fluid was used as a basis for osmotic studies aimed at examining solute exclusion from the apoplastic barrier in the PE envelope. The assessment of apoplastic permeability involved measuring the amount of anionic and cationic organic dyes diffused into agarose gel discs through the PE envelope. Ionic/non-ionic solutes including polyethylene glycols having Stokes radii ≤ 0.6 nm showed considerable permeation through the apoplastic barrier in the PE envelope as indicated by greater seed thickness/breadth ratios. Permeances of dyes across the PE envelope were in the order: 2,6-dichlorophenolindophenol (DCPIP) > 2,3,5-triphenyltetrazolium chloride (TTC) ~ methyl orange ~ methylene blue > Eosin Y ≫ Janus green ~ crystal violet ~ Evans Blue. Permeation time_0.5 for DCPIP and TTC was 9.71 and 9.96 h, respectively. Dyes having Stokes radii < 0.5 nm showed significant inward as well as outward diffusion across the PE envelope in contrast to restricted diffusion of dyes having Stokes radii > 0.5 nm. Size exclusion limit for apoplastic barrier in cucumber PE envelope was resolved to be about 0.5 nm by dye permeation and around 0.8 nm by osmotic studies. Dye permeances depended primarily on particle size as described by a quadratic polynomial function rather than on charge or log D.     

菰适应湿地环境的解剖和屏障结构特征研究

菰(Zizania latifolia)是一种多年生挺水植物,为了探讨该植物根、茎和叶的解剖结构、组织化学及其质外体屏障的通透性生理。该文利用光学显微镜和荧光显微镜,对菰的根、茎、叶进行了解剖学和组织化学研究。结果表明:(1)菰不定根解剖结构由外而内分别为表皮、外皮层、单层细胞的厚壁机械组织层、皮层、内皮层和维管柱;茎结构由外而内分别为角质层、表皮、周缘厚壁机械组织层、皮层、具维管束的厚壁组织层和髓腔。叶鞘具有表皮和具维管束皮层,叶片具有表皮,叶肉和维管束。(2)不定根具有位于内侧的内皮层及其邻近栓质化细胞和外侧的外皮层组成的屏障结构;茎具内侧厚壁机械组织层,外侧的角质层和周缘厚壁机械组织层组成的屏障结构,屏障结构的细胞壁具凯氏带、木栓质和木质素沉积的组织化学特点,叶表面具有角质层。(3)菰通气组织包括根中通气组织,茎、叶皮层的通气组织和髓腔。(4)菰的屏障结构和解剖结构是其适应湿地环境的重要特征,但其茎周缘厚壁层和厚壁组织层较薄。由此推测,菰适应湿地环境,但在旱生环境中分布有一定的局限性。     

Characterization of a membrane-associated apoplastic lipoxygenase in Phaseolus vulgaris L.

An extracytoplasmic 86.7 kDa protein was isolated from intercellular washing fluids (IWF) of Phaseolus vulgaris etiolated hypocotyls. Micro sequencing of tryptic peptides of the 86.7 kDa protein revealed 100% identity with a bean lipoxygenase (LOX) protein fragment. Purified P87-LOX exhibited LOX activity characterized by an optimal pH of 6.0 and linolenic acid as an optimal substrate, and was classified as a 13-LOX with respect to its positional specificity of linoleic acid oxygenation. A protein identical to P87-LOX, as determined by MALDI-TOF analysis and biochemical characterization, was purified from hypocotyl microsomes. Immunoblot analysis showed that P87-LOX is present in plasma membrane-enriched fractions, from which it was solubilized using high ionic strength buffers. These observations suggest that P87-LOX is a peripheral protein associated to the apoplastic face of the plasma membrane.     

Embryonic cuticle establishment: The great (apoplastic) divide

The plant cuticle, a dynamic interface between plants and their environment, is formed by the secretion of hydrophobic lipids and waxes into the outer wall of aerial epidermal cells. Cuticle formation is such a ubiquitous feature of epidermal cells, and is of such fundamental importance for plant survival, that identifying and understanding specific developmental roles for this structure has been a major challenge for plant scientists. In recent work, we have tried to understand the functional relationships between a signaling feedback loop required for epidermal cell specification in developing plant embryos, and a seed specific signaling cascade, involving components localized both in the embryo and in the embryo surrounding endosperm, and necessary for embryo cuticle function. Analysis of the strongly synergistic genetic relationships between these 2 independent pathways, combined with mathematical simulations of the behavior of the signaling feedback loop, have allowed us to propose an important, and hitherto unsuspected, role for the embryonic cuticle as an apoplastic diffusion barrier, necessary for preventing the excessive diffusion of developmentally important signaling molecules away from developing embryo into surrounding tissues.     

Proton density and apoplastic domains within soybean nodules in relation to the oxygen diffusion barrier

A spin-echo pulse sequence was used to obtain ¹H nuclear magnetic resonance microimages of soybean nodules, with resolution to 40 mm and 3D presentation. At an acquisition time (TR) of c. 270 ms and a short echo time (TE) of 8.3 ms, a high proton intensity was detected in infected tissue and vascular strands, relative to the cortex. A longer TE of 15.5 ms was associated with a relatively low proton intensity in infected tissue. Thus protons in infected tissue were characterized by a short T₂. Following detopping of the plant, or treatment of the root system with Ar:O₂, proton intensity (TE 5.9, TR 120 ms) decreased exponentially in a zone corresponding to the inner cortex-outer infected region. This result is interpreted as being caused by a loss of water mobility (i.e. increase in viscosity) or a filling of intercellular air spaces with fluid (i.e. loss of air-water interfaces). The membrane impermeant tracer lucifer yellow was observed to infiltrate from the rhizosphere through the nodule cortex into the infected region in intact nodules of detopped and Ar:O₂-treated, but not control, plants. This result is consistent with the filling of air spaces with fluid following the imposed treatments, allowing infiltration of aqueous tracers. Variation in intercellular air space volume within the inner cortex has been suggested as a mechanism to allow a variable rate of diffusion of O₂ into soybean nodules.     

Apoplastic transport across young maize roots: effect of the exodermis

The uptake of water and of the fluorescent apoplastic dye PTS (trisodium 3-hydroxy-5,8,10-pyrenetrisulfonate) by root systems of young maize (Zea mays L.) seedlings (age: 11–21 d) has been studied with plants which either developed an exodermis (Casparian band in the hypodermis) or were lacking it. Steady-state techniques were used to measure water uptake across excised roots. Either hydrostatic or osmotic pressure gradients were applied to induce water flows. Roots without an exodermis were obtained from plants grown in hydroponic culture. Roots which developed an exodermis were obtained using an aeroponic (=mist) cultivation method. When the osmotic concentration of the medium was varied, the hydraulic conductivity of the root (Lp _r in m³ · m⁻² · MPa⁻¹ · s⁻¹) depended on the osmotic pressure gradient applied between root xylem and medium. Increasing the gradient (i.e. decreasing the osmotic concentration of the medium; range: zero to 40 mM of mannitol), increased the osmotic Lp _r. In the presence of hydrostatic pressure gradients applied by a pressure chamber, root Lp _r was constant over the entire range of pressures (0–0.4 MPa). The presence of an exodermis reduced root Lp _r in hydrostatic experiments by a factor of 3.6. When the osmotic pressure of the medium was low (i.e. in the presence of a strong osmotic gradient between xylem sap and medium), the presence of an exodermis caused the same reduction of root Lp _r in osmotic experiments as in hydrostatic ones. However, when the osmotic concentration of the medium was increased (i.e. the presence of low gradients of osmotic pressure), no marked effect of growth conditions on osmotic root Lp _r was found. Under these conditions, the absolute value of osmotic root Lp _r was lower by factors of 22 (hydroponic culture) and 9.7 (aeroponic culture) than in the corresponding experiments at low osmotic concentration. Apoplastic flow of PTS was low. In hydrostatic experiments, xylem exudate contained only 0.3% of the PTS concentration of the bathing medium. In the presence of osmotic pressure gradients, the apoplastic flow of PTS was further reduced by one order of magnitude. In both types of experiments, the development of an exodermis did not affect PTS flow. In osmotic experiments, the effect of the absolute value of the driving force cannot be explained in terms of a simple dilution effect (Fiscus model). The results indicate that the radial apoplastic flows of water and PTS across the root were affected differently by apoplastic barriers (Casparian bands) in the exodermis. It is concluded that, unlike water, the apoplastic flow of PTS is rate-limited at the endodermis rather than at the exodermis. The use of PTS as a tracer for apoplastic water should be abandoned. Received: 9 October 1997 / Accepted: 5 February 1998     

The apoplastic oxidative burst in response to biotic stress in plants: a three-component system

The oxidative burst, the generation of reactive oxygen species (ROS) in response to microbial pathogen attack, is a ubiquitous early part of the resistance mechanisms of plant cells. It has also become apparent from the study of a number of plant-pathogen interactions and those modelled by elicitor treatment of cultured cells that there may be more than one mechanism operating. However, one mechanism may be dominant in any given species. NADPH oxidases have been implicated in a number of systems and have been cloned and characterized. However, the enzyme system which is the major source of ROS in French bean (Phaseolus vulgaris) cells treated with a cell wall elicitor from Colletotrichum lindemuthianum, appears to be dependent on an exocellular peroxidase. The second component, the extracellular alkalinization, occurs as a result of the Ca(2+) and proton influxes and the K(+) efflux common to most elicitation systems as one of the earliest responses. The third component, the actual reductant/substrate, has remained elusive. The low molecular weight compound composition of apoplastic fluid was compared before and after elicitation. The substrate only becomes available some min after elicitation and can be extracted, so that by comparing the profiles by LC-MS it has been possible to identify possible substrates. The mechanism has proved to be complex and may involve a number of low molecular weight components. Stimulation of H(2)O(2) production was observed with saturated fatty acids such as palmitate and stearate without concomitant oxylipin production. This biochemical evidence is supported by immunolocalization studies on papillae forming at bacterial infection sites that show the peroxidase isoform present at sites of H(2)O(2) production revealed by cerium chloride staining together with the cross-linked wall proteins and callose and callose synthase. The peroxidase has been cloned and expressed in Pichia pastoris and has been shown to catalyse the oxidation reaction with the same kinetics as the purified enzyme. Furthermore, Arabidopsis plants transformed heterologously using the French bean peroxidase in antisense orientation have proved to be highly susceptible to bacterial and fungal pathogens. Thus it is possible that Arabidopsis is another species with the potential to mount an apoplastic oxidative burst and these transformed plant lines may be useful to identify the peroxidase that is responsible.     

Contribution to the exodermis in the rootlets ofFraxinus excelsior L.

The exodermis of ash roots is initiated early in the apical meristem. When fully differentiated, it is composed of alternating “long” and “short” cells measuring approx. 70×25×25 μm and 25×28×25 μm respectively. At a short distance from the apex, the long cells undergo structural and histochemical changes from a “primary” towards a “secondary” stage: an impermeable suberized lamella is formed, cellulose lamellae become impregnated by lignin, and the protoplast dies off. The short cells show a distinctly thickened outer wall (“cap”) which is composed exclusively of cellulose, and possess abundant cytoplasm with a large nucleus. In the process of ageing the structure of short cells is not markedly affected. Following the early disintegration of rhizodermis, the exodermis works as the external protective layer of tho fragile end-root. Its long cells fulfil the mechanical protection, the short cells serve as passage cells for solutions and gases, during the whole life-span of the rootlet.     

The uroepithelium: not just a passive barrier

The uroepithelium lines the inner surface of the renal pelvis, the ureters, and the urinary bladder, where it forms a tight barrier that allows for retention of urine, while preventing the unregulated movement of ions, solutes, and toxic metabolites across the epithelial barrier. In the case of the bladder, the permeability barrier must be maintained even as the organ undergoes cyclical changes in pressure as it fills and empties. Beyond furthering our understanding of barrier function, new analysis of the uroepithelium is providing information about how detergent-insoluble membrane/protein domains called plaques are formed at the apical plasma membrane of the surface umbrella cells, how mechanical stimuli such as pressure alter exocytic and endocytic traffic in epithelial cells such as umbrella cells, and how changes in pressure are communicated to the underlying nervous system.     

The fortysomething barrier: medicine and age discrimination.

Agism in the medical profession is mainly covert but it is not uncommon. It is widely believed that people become less productive as they get older. However, research has shown that older people have less absenteeism, more job stability, and greater output than younger workers. Job losses, which until recently were unheard of in the NHS, usually affect older people first, resulting in the loss of those with the most skill and experience. With an aging population it is important that the government takes steps to discourage age discrimination in the NHS and Britain as a whole.     

Apoplastic transport of abscisic acid through roots of maize: effect of the exodermis

The exodermal layers that are formed in maize roots during aeroponic culture were investigated with respect to the radial transport of cis-abscisic acid (ABA). The decrease in root hydraulic conductivity (Lp_r) of aeroponically grown roots was stimulated 1.5-fold by ABA (500 nM), reaching Lp_r values of roots lacking an exodermis. Similar to water, the radial flow of ABA through roots (J_ABA) and ABA uptake into root tissue were reduced by a factor of about three as a result of the existence of an exodermis. Thus, due to the cooperation between water and solute transport the development of the ABA signal in the xylem was not affected. This resulted in unchanged reflection coeffcients for roots grown hydroponically and aeroponically. Despite the well-accepted barrier properties of exodermal layers, it is concluded that the endodermis was the more effective filter for ABA. Owing to concentration polarisation effects, ABA may accumulate in front of the endodermal layer, a process which, for both roots possessing and lacking an exodermis, would tend to increase solvent drag and hence ABA movement into the xylem sap at increased water flow (J_Vr). This may account for the higher ABA concentrations found in the xylem at greater pressure difference. Received: 26 January 1999 / Accepted: 26 May 1999     

The assessment of enriched apoplastic extracts using proteomic approaches

In plant tissues the extracellular environment or apoplast, incorporating the cell wall, is a highly dynamic compartment with a role in many important plant processes including defence, development, signalling and assimilate partitioning. Soluble apoplast proteins from Arabidopsis thaliana, Triticum aestivum and Oryza sativa were separated by two‐dimensional electrophoresis. The molecular weights and isoelectric points for the dominant proteins were established prior to excision, sequencing and identification by matrix‐assisted laser‐desorption ionisation time of flight mass spectrometry (MALDI ‐ TOF MS). From the selected spots, 23 proteins from O. sativa and 25 proteins from A. thaliana were sequenced, of which nine identifications were made in O. sativa (39%) and 14 in A. thaliana (56%). This analysis revealed that: (i) patterns of proteins revealed by two‐dimensional electrophoresis were different for each species indicating that speciation could occur at the level of the apoplast, (ii) of the proteins characterised many belonged to diverse families reflecting the multiple functions of the apoplast and (iii), a large number of the apoplast proteins could not be identified indicating that the majority of extracellular proteins are yet to be assigned. The principal proteins identified in the aqueous matrix of the apoplast were involved in defence, i.e. germin‐like proteins or glucanases, and cell expansion, i.e. β‐D‐glucan glucohydrolases. This study has demonstrated that proteomic analysis can be used to resolve the apoplastic protein complement and to identify adaptive changes induced by environmental effectors.     

Studies on the physiological sheaths in roots I. Ultrastructure of the exodermis inHoya carnosa L.

Summary InHoya roots most exodermal cells are elongated and a band of suberin lamellae is formed in all their walls early in development; later on carbohydrate tertiary wall layers are deposited inside the suberin lamellae. Some exodermal cells which are restricted to root hair-bearing areas are short and unsuberized but their outer tangential wall is conspicuously thickened. Combined evidence from light microscopy and transmission and scanning electron microscopy reveals the bulk of this cap-formed thickening as a mosaic structure with two different components forming an extensive labyrinth. Irregular masses of a lignified, amorphous substance are separated by radially oriented, tortuous channels containing a very dense, granular-fibrillar material. The innermost wall layer is fibrillar and shows a texture and density similar to the material in the separating channels. The cap contains prominent pits with plasmodesmatal connections between short cells and the epidermis. In mature and non-functional short cells a band of suberin lamellae and eventually tertiary wall layers are deposited.A hypothesis as to the function of the short cells is based on the assumption that the cap functions through differential shrinkage of two components forming the labyrinthine structure. This would ensure a very effective closing of the translocating pathway upon desiccation and shrinkage and a consequent swelling and re-opening upon rehydration. The regulatory function of such mechanism is discussed.     

The apoplastic oxidative burst peroxidase in Arabidopsis is a major component of pattern-triggered immunity

In plants, reactive oxygen species (ROS) associated with the response to pathogen attack are generated by NADPH oxidases or apoplastic peroxidases. Antisense expression of a heterologous French bean (Phaseolus vulgaris) peroxidase (FBP1) cDNA in Arabidopsis thaliana was previously shown to diminish the expression of two Arabidopsis peroxidases (peroxidase 33 [PRX33] and PRX34), block the oxidative burst in response to a fungal elicitor, and cause enhanced susceptibility to a broad range of fungal and bacterial pathogens. Here we show that mature leaves of T-DNA insertion lines with diminished expression of PRX33 and PRX34 exhibit reduced ROS and callose deposition in response to microbe-associated molecular patterns (MAMPs), including the synthetic peptides Flg22 and Elf26 corresponding to bacterial flagellin and elongation factor Tu, respectively. PRX33 and PRX34 knockdown lines also exhibited diminished activation of Flg22-activated genes after Flg22 treatment. These MAMP-activated genes were also downregulated in unchallenged leaves of the peroxidase knockdown lines, suggesting that a low level of apoplastic ROS production may be required to preprime basal resistance. Finally, the PRX33 knockdown line is more susceptible to Pseudomonas syringae than wild-type plants. In aggregate, these data demonstrate that the peroxidase-dependent oxidative burst plays an important role in Arabidopsis basal resistance mediated by the recognition of MAMPs.     

Wintersweet accumulates apoplastic chitinase with a dual role in petals

Most monocotyledons like cereals accumulate antifreeze proteins in the apoplast during cold acclimation, but it is still uncertain whether dicotyledons do. Here we report the isolation and characterisation of a 33-kD apoplastic chitinase extracted from the corolla of wintersweet (Chinmonanthus praecox communis L.), which was purified using successive column chromatography on Phenyl-Sepharose, DEAE-Sepharose, and CM-Sepharose. Antifreezing activity of chitinase was confirmed in terms of the formation of bipyramidal ice crystals and high thermal-hysteresis values. Interestingly, chitinase was also found to affect germination of fungal spores of four major plant pathogens. From these data, we hypothesize that, under natural conditions, wintersweet as one of the overwintering dicotyledons also accumulates apoplastic antifreeze proteins like monocotyledons. To our knowledge, this is the first report on the isolation of dicotyledon apoplastic chitinase with high-level antifreeze and antifungal activities.     

The dermaseptin precursors: a protein family with a common preproregion and a variable C-terminal antimicrobial domain.

Preprodermaseptins are a group of antimicrobial peptide precursors found in the skin of a variety of frog species. Precursors of this family have very similar N-terminal preprosequences followed by markedly different C-terminal domains that correspond to mature antimicrobial peptides. Some of these peptides are 24-34 amino acids long and form well-behaved amphipathic alpha-helices, others are disulfide-linked peptides of 20-46 residues, still others, highly hydrophobic, are the smallest antimicrobial peptides known so far being only 10-13 residues in length. All these peptides are broad-spectrum microbicides that kill many bacteria, protozoa, yeasts and fungi by destroying or permeating the microbial membrane. In frogs belonging to the genus Phyllomedusinae, preprodermaseptins encoded peptides also include dermorphins and deltorphins, D-amino acid-containing heptapeptides which are very potent and specific agonists of the mu- or delta-opioid receptors. The remarkable similarity between preproregions of precursors that give rise to peptides with very different primary structures, conformations and activities suggests that the corresponding genes originate from a common ancestor. The high conservation of the precursor prepropart indicates that this region must have an important function.     

The expression of a mountain cedar allergen comparing plant-viral apoplastic and yeast expression systems

Jun a 3, a major allergenic protein in mountain cedar pollen, causes seasonal allergic rhinitis in hypersensitive individuals. Recombinant Jun a 3 was expressed in Nicotiana benthamiana interstitial fluid (300 microg/g leaf material) and Pichia pastoris (100 microg/ml media). Polyclonal anti-Jun a 3 and IgE antibodies from the sera of allergic patients both reacted with the recombinant protein. Of the two systems, recombinant protein from the plant apoplast contained fewer contaminating proteins. This method allows for a more convenient and inexpensive expression of the recombinant allergen, which will allow for further structural studies and may prove useful in diagnostic and/or immunotherapeutic strategies for cedar allergy.