A22 disrupts the bacterial actin cytoskeleton by directly binding and inducing a low-affinity state in MreB.

S-(3,4-Dichlorobenzyl)isothiourea (A22) disrupts the actin cytoskeleton of bacteria, causing defects of morphology and chromosome segregation. Previous studies have suggested that the actin homologue MreB itself is the target of A22, but there has been no direct observation of A22 binding to MreB and no mechanistic explanation of its mode of action. We show that A22 binds MreB with at least micromolar affinity in its nucleotide-binding pocket in a manner that is sterically incompatible with simultaneous ATP binding. A22 negatively affects both the time course and extent of MreB polymerization in vitro in the presence of ATP. A22 prevents assembly of MreB into long, rigid polymers, as determined by both fluorescence microscopy and sedimentation assays. A22 increases the critical concentration of ATP-bound MreB assembly from 500 nM to approximately 2000 nM. We therefore conclude that A22 is a competitive inhibitor of ATP binding to MreB. A22-bound MreB is capable of polymerization, but with assembly properties that more closely resemble those of the ADP-bound state. Because the cellular concentration of MreB is in the low micromolar range, this mechanism explains the ability of A22 to largely disassemble the actin cytoskeleton in bacterial cells. It also represents a novel mode of action for a cytoskeletal drug and the first biochemical characterization of the interaction between a small molecule inhibitor of the bacterial cytoskeleton and its target.

The management of extracellular ice by petioles of frost-resistant herbaceous plants.

BACKGROUND AND AIMS: Some frost-tolerant herbaceous plants droop and wilt during frost events and recover turgor and posture on thawing. It has long been known that when plant tissues freeze, extracellular ice forms. Distributions of ice and water in frost-frozen and recovered petioles of Trifolium repens and Escholschzia californica were visualized. METHODS: Petioles of intact plants were cryo-fixed, planed to smooth transverse faces, and examined in a cryo-SEM. KEY RESULTS: With frost-freezing, parenchyma tissues shrank to approx. one-third of their natural volume with marked cytorrhysis of the cells, and massive blocks of extracellular icicles grew under the epidermis (poppy) or epidermis and subepidermis (clover), leaving these layers intact but widely separated from the parenchyma except at specially structured anchorages overlying vascular bundles. On thawing, the extracellular ice was reabsorbed by the expanding parenchyma, and surface tissues again contacted the internal tissues at weak junctions (termed faults). These movements of water into and from the fault zones occurred repeatedly at each frost/thaw event, and are interpreted to explain the turgor changes that led to wilting and recovery. Ice accumulations at tri-cellular junctions with intercellular spaces distended these spaces into large cylinders, especially large in clover. Xylem vessels of frozen petioles were nearly all free of gas; in thawed petioles up to 20 % of vessels were gas-filled. CONCLUSIONS: The occurrence of faults and anchorages may be expected to be widespread in frost-tolerant herbaceous plants, as a strategy accommodating extracellular ice deposits which prevent intracellular freezing and consequent membrane disruption, as well as preventing gross structural damage to the organs. The developmental processes that lead to this differentiation of separation of sheets of cells firmly cemented at determined regions at their edges, and their physiological consequences, will repay detailed investigation.

Location and quantification of phosphorus and other elements in fully hydrated, soil-grown arbuscular mycorrhizas: a cryo-analytical scanning electron microscopy study.

• Concentrations of phosphorus (P), potassium (K), magnesium (Mg) and calcium (Ca) were determined in situ in fully hydrated arbuscular mycorrhizas by cryo-analytical scanning electron microscopy. The field- and glasshouse-grown plants (subterranean and white clovers, field pea and leek) were colonized by indigenous mycorrhizal fungi. • The [P] in intraradical hyphae was generally 60-170 mM, although up to 600 mM was recorded, and formed strong linear relationships with [K], up to 350 mM, and [Mg], up to 175 mM. Little Ca was detected. The turgid branches of young arbuscules contained 30-50 mM P, up to 100 mM K and little Mg. Collapsing arbuscule branches and clumped arbuscules had greatly elevated Ca (30-250 mM), but otherwise differed little from young arbuscule branches in elemental concentration. • The [P] was low or undetectable in 86% of uncolonized cortical cell vacuoles, but was generally elevated in vacuoles surrounding an arbuscule and in the liquid surrounding hyphae in intercellular spaces. • Our results suggest that both young arbuscules and intercellular hyphae are sites for P-transfer, that Mg2+ and K+ are probably balancing cations for P anions in hyphae, and that host cells may limit arbuscule lifespan through deposition of material rich in Ca.

Evidence for protection of nitrogenase from O(2) by colony structure in the aerobic diazotroph Gluconacetobacter diazotrophicus.

Gluconacetobacter diazotrophicus is an endophytic diazotroph of sugarcane which exhibits nitrogenase activity when growing in colonies on solid media. Nitrogenase activity of G. diazotrophicus colonies can adapt to changes in atmospheric partial pressure of oxygen (pO(2)). This paper investigates whether colony structure and the position of G. diazotrophicus cells in the colonies are components of the bacterium's ability to maintain nitrogenase activity at a variety of atmospheric pO(2) values. Colonies of G. diazotrophicus were grown on solid medium at atmospheric pO(2) of 2 and 20 kPa. Imaging of live, intact colonies by confocal laser scanning microscopy and of fixed, sectioned colonies by light microscopy revealed that at 2 kPa O(2) the uppermost bacteria in the colony were very near the upper surface of the colony, while the uppermost bacteria of colonies cultured at 20 kPa O(2) were positioned deeper in the mucilaginous matrix of the colony. Disruption of colony structure by physical manipulation or due to 'slumping' associated with colony development resulted in significant declines in nitrogenase activity. These results support the hypothesis that G. diazotrophicus utilizes the path-length of colony mucilage between the atmosphere and the bacteria to achieve a flux of O(2) that maintains aerobic respiration while not inhibiting nitrogenase activity.

The reliability of cryoSEM for the observation and quantification of xylem embolisms and quantitative analysis of xylem sap in situ.

The reliability of cryoSEM for visualizing gas embolisms in xylem vessels of intact, functioning roots is examined and discussed. The possibility that these embolisms form as a result of freezing water columns under tension is discounted by a double-freeze experiment. Two regions of the same root, one frozen under tension, the other isolated from the tension by the first freeze, had the same percentage of embolisms, as did also long pieces of root frozen simultaneously along their length. The reliability of energy-dispersive X-ray analysis to measure xylem sap concentration in situ in frozen tissue was established by measurement of KCl standard solution frozen on stubs, and within xylem vessels. Solute heterogeneity within the vessels varied with freezing procedure; deep-freeze > LN2 > cryopliers > liquid ethane, but only the deep-freeze method gave unsatisfactory estimates of concentration for the standard solution. It is concluded that cryoanalytical SEM is useful for direct observation of gas and liquid-filled compartments, and for solute analyses at depth within intact plant organs.

Daily embolism and refilling of xylem vessels in the roots of field-grown maize.

Embolisms in the vessels of maize axile roots of different types were observed directly after rapid freezing of intact, functioning roots in the field, by cryo-scanning electron microscopy. Quantification of the degree of embolization in each root was made by counting empty and full vessels of both the late and early metaxylem (LMX & EMX), and expressed as percent embolized vessels of the LMX, and %EMX poles containing embolized vessels. Contents of the connecting xylem (CX) at branch root junctions, and of xylem in branch roots were observed also, but not systematically quantified. Records of % embolized vessels were made from dawn to dusk on summer days in Ottawa under moderate irradiance, and in Canberra under high irradiance. Measurements in Canberra were supported by estimates of irradiance, of stomatal conductance, and of chamber balance pressure of bagged and unbagged leaves. Soon after sunrise embolisms appeared in all types of vessel, at balance pressures c. 300-400 kPa, and increased rapidly with increasing irradiance. During the middle of the day % embolized vessels reached a maximum (LMX ≈70% in Ottawa, and ≈80% in Canberra). At all times the EMX vessels were less embolized. The midday maximum was brief in Ottawa, and % embolized vessels fell to a low value during the afternoon. In Canberra the maximum was prolonged into late afternoon. By dusk nearly all vessels were once again filled with sap. The balance pressures measured during vessel refilling in Canberra ranged from 500 kPa to 1200 kPa. At all times of the day sap was seen entering some embolized vessels. Almost all were refilling by mid- to late-afternoon. Such refilling was especially frequent at junctions of branch roots with the axile roots. X-ray microanalysis of the sap entering the vessels, and of the liquid filling or partly filling vessels, showed the concentration of mineral solutes present in the sap was below the threshold of detection (≈12 mM). These results are discussed in relation to current opinions about embolisms and vessel refilling.

Retention in situ and spectral analysis of fluorescent vacuole components in sections of plant tissues.

The contents of plant vacuoles vary in different organs and with the health of the plant, but little is known of the cell-to-cell distribution of soluble organic compounds within plant tissues. Soluble fluorescent phenolic compounds can be immobilized in plant tissues using an anhydrous freeze-substitution and resin embedment process. The vacuolar fluorescence can be characterized in fluorescence photomicrographs for variations in color and intensity, or more quantitatively with spectra obtained using a microspectrofluorometer. This is demonstrated here in freeze-substituted roots and leaves of soybean. Excitation and emission spectra of individual vacuoles can be compared with spectra of pure compounds to form profiles of the varied phenolic contents of plant vacuoles. Such analyses will add an important anatomical dimension to the study of plant defense and stress responses.

Further Evidence that the N(inf2)-Fixing Endophytic Bacterium from the Intercellular Spaces of Sugarcane Stems Is Acetobacter diazotrophicus.

Nitrogen-fixing bacteria, isolated from the sugar solution in intercellular spaces of sugarcane stems, were compared with the type strain of Acetobacter diazotrophicus (PAL-5) and found to be congruent with it in all characters studied. These characters were 37 morphological and biochemical tests, cellular fatty acid composition, and nitrogenase activity. The nitrogenase activity was measured by acetylene reduction and H(inf2) evolution and found to be unusual in that the H(inf2) evolution was suppressed much less than expected by high concentrations of acetylene.

Formation and Stabilization of Rhizosheaths of Zea mays L. (Effect of Soil Water Content).

Field observations have shown that rhizosheaths of grasses formed under dry conditions are larger, more coherent, and more strongly bound to the roots than those formed in wet soils. We have quantified these effects in a model system in which corn (Zea mays L.) primary roots were grown through a 30-cm-deep prepared soil profile that consisted of a central, horizontal, "dry" (9% water content) or "wet" (20% water content) layer (4 cm thick) sandwiched between damp soil (15-17% water content). Rhizosheaths formed in dry layers were 5 times the volume of the subtending root. In wet layers, rhizosheaths were only 1.5 times the root volume. Fractions of the rhizosheath soil were removed from individual roots by three successive treatments; sonication, hot water, and abrasion. Sonication removed 50 and 90% of the soil from rhizosheaths formed in dry and wet soils, respectively. After the heat treatment, 35% of the soil still adhered to those root portions where rhizosheaths had developed in dry soil, compared with 2% where sheaths had formed in wet soil. Root hairs were 4.5 times more abundant and were more distorted on portions of roots from dry layers than from wet layers. Drier soil enhanced adhesiveness of rhizosheath mucilages and stimulated the formation of root hairs; both effects stabilize the rhizosheath. Extensive and stable rhizosheaths may function in nutrient acquisition in dry soils.

A Nitrogen-Fixing Endophyte of Sugarcane Stems (A New Role for the Apoplast).

The intercellular spaces of sugarcane (Saccharum officinarum L.) stem parenchyma are filled with solution (determined by cryoscanning microscopy), which can be removed aseptically by centrifugation. It contained 12% sucrose (Suc; pH 5.5.) and yielded pure cultures of an acid-producing bacterium (approximately 104 bacteria/mL extracted fluid) on N-poor medium containing 10% Suc (pH 5.5). This bacterium was identical with the type culture of Acetobacter diazotrophicus, a recently discovered N2-fixing bacterium specific to sugarcane, with respect to nine biochemical and morphological characteristics, including acetylene reduction in air. Similar bacteria were observed in situ in the intercellular spaces. This demonstrates the presence of an N2-fixing endophyte living in apoplastic fluid of plant tissue and also that the fluid approximates the composition of the endophytes's optimal culture medium. The apoplastic fluid occupied 3% of the stem volume; this approximates 3 tons of fluid/ha of the crop. This endogenous culture broth consisting of substrate and N2-fixing bacteria may be enough volume to account for earlier reports that some cultivars of sugarcane are independent of N fertilizers. It is suggested that genetic manipulation of apoplastic fluid composition may facilitate the establishment of similar symbioses with endophytic bacteria in other crop plants.

Planing frozen hydrated plant specimens for SEM observation and EDX microanalysis.

A procedure is described for forming a flat face on a frozen piece of plant tissue, which may then be observed fully-hydrated or lightly etched, and coated or uncoated with a metal film, in scanning electron microscopy (SEM). The frozen sample was planed with a glass knife at -80 degrees C in a cryo-ultramicrotome. The sections were discarded, and the planed block face placed on the cold stage in the microscope column, either for observation uncoated at low kV, or for light etching (-90 degrees C) to reveal the cell outlines. If a higher accelerating voltage was needed, the face was given an evaporative coating of Al in the cryo-preparation chamber and returned to the column. The advantages of the planed face over the usual fracture face are illustrated: imaging at a chosen rather than a chance position; clearer cellular and subcellular detail; preservation of hydrated gels like mucilage and swollen cell walls; the possibility of making serial parallel sections through the same piece of tissue; opportunities for accurate morphometric analyses on the planed face; capacity to produce longitudinal sections; preservation of very delicate structures that are destroyed by fixation and drying. A major advantage of the Al-coated planed face is the increased accuracy of energy-dispersive X-ray (EDX) microanalyses on a smooth rather than a rough surface. Tests are included which show that neither the light etching employed, nor successive planing, interferes with the analyses of elements in the frozen face.

The epidermal surface of the maize root tip: III. Isolation of the surface and characterization of some of its structural and mechanical properties.

The surface of the young epidermal cells of maize roots is composed o) three layers; the inner layer (LI), which is the outer epidermal Wall, overlaid by a pellicle consisting of a thick, coherent inner layer (L2) and a very thin, loosely organized outer layer (L3). The entire surface can be removed intact to produce either narrow, circumferential strips or apical halftones, by gently prying it loose in the circumferential direction by hand with insect pins. Usually only short remnants of anticlinical walls of the epidermal cells remain attached. These isolated surface pieces always curl outward at the free circumferential edges in the longitudinal direction of the original intact root. When the strips are deliberated stretched alone their long axis (i.e., the original circumferential direction) they elongate irreversibly by us much us two thirds of their length, before showing some elastic deformation and breaking. Some plastic deformation may occur in the original longitudinal direction of the root during removal of the strips. The plastic deformation opens the helicoidal array of microfibrils in the L1 layer. Deformation also produces structural changes over the original radial walls and those transverse anticlinal walls that form boundaries of cell packets derived from single cells. In these positions the L1 layers over adjacent cells separate in the direction of the applied stress. This occurs by the separation of the L I layers of adjacent cells and the stretching of the inward projection of the amorphous L2 layer of the pellicle which lies; over these original anticlinal walls. There is much less or no separation of the L1 layers over anticlinal walls Of adjoining daughter cells in the epidermis. The pellicle always remains firmly attached to the outer epidermal wall during removal of the surface strips. On removal, these strips shorten in their original longitudinal direction in situ, indicating a release of tension imposed by underlying cells. Their outward curling suggests stress between the wall and pellicle of the outer epidermal surface in the intact root. These findings focus attention on structural differences between sites where anticlinal walls of different origin join the outer surface, and on possible differences in surface extensibility at each sites.

The epidermal surface of the maize root tip: II. Abnormalities in a mutant which grows crookedly through soil.

The mutation Ageotropic (Agt) results in defective development of the surface pellicle which overlies the young epidermal cells of mesocotyl and nodal roots of maize. In roots of plants of the parent, cv. Kys, pellicle development is normal. This structure forms a coherent, smooth covering, up to ∼ 13/µm thick, over the epidermal cells distal to the elongation zone. This pellicle is external to the helicoidal outer wall of the epidermal cells and is composed of a thick inner layer with close-packed, longitudinally oriented fibrils, and a thin outer layer with less regularly oriented fibrils. Both layers of the normal pellicle and the underlying epidermal wall are strongly stained by the periodic acid-Schiff's (PAS) reaction. In Agt plants, the pellicle is irregular and diffuse, lacks definition of the two layers and is largely amorphous with occasional wisps of fibrils. Neither this mutant pellicle nor the underlying epidermal wall are PAS-positive. Mesocotyl and nodal roots of Agt are sensitive to gravity but grow crookedly through soil. It is proposed that a normal pellicle maintains the smooth outer contour of young roots and provides stiffness in the region distal to the zone of elongation. When the pellicle is defective, the root tip is more compliant and bends result when the tip encounters small air spaces and barriers in the soil.

Sites of entry of water into the symplast of maize roots.

Following our publication of a new method of calculating rates of water uptake by roots from measurements of the rate of accumulation on the roots of a marker solute, this paper describes the sites of accumulation of the solute, which indicate the sites where the water entered the symplast. Sulphorhodamine G (SR) was supplied in aeroponic mist culture to large maize plants with fully developed root systems. Root samples were collected after 4 to 8 h of transpiration in the dye-mist from both axes and branches of the main roots, and from non-transpiring (detopped) controls, frozen rapidly, freeze-substituted, and embedded and sectioned by an anhydrous procedure that preserves the SR in place. Whole mounts and sections were examined by bright-field, polarizing and epifluorescence microscopy. Major accumulations of SR were all at the outer surface of the roots, on Epidermal or root hair cell walls, or, in older roots where the epidermal cells were separating or dead, on the outer wall of the hypodermis. On some branch roots, though not on any main axes, the accumulations of SR were conspicuously aligned in the grooves over anticlinal cell walls of the epidermis. Non-transpiring plants showed very slight accumulations. Diffusion of SR into the cell wall apoplast was limited by the suberized lamellae and Casparian bands of the hypodermis, except in some branch roots, where SR diffused throughout the cortical cell walls. In parts of roots where the epidermis and hypodermis had been damaged, SR diffused through cell walls of the cortex from the wound site. These patterns of accumulation show that water enters the symplast of roots at the outermost cell membranes of the root, whether they are epidermal or hypodermal cells. Water enters roots with fully developed hypodermises at high rates. The rote of the hypodermal suberization is to limit solute movement in the wall apoplast. A symplastic path for water throughout the cortex, endodermis and living cells of the stele is suggested.

The epidermal surface of the maize root tip: I. Development in normal roots.

The surface of the meristematic epidermis of maize roots is tri-partite. A helicoidal primary wall follows the contours of the tops of the columnar epidermal cells and is continuous with their buttressed anticlinal walls. Two overlying layers form a smooth covering over the root which obscures the cell outlines. This compound surface is similar architecturally to outer epidermal surfaces of shoots. The two outer layers are distinct structurally and in their staining properties from the wall and are together here referred to as the pellicle. Both pellicle layers are fibrillar but not helicoidal. Their development begins in the boundary between the cap and the root proper and they reach maximum thickness over the meristematic region. The outer layer then disintegrates and is absent from the elongation zone. The inner layer thins irregularly as the columnar cells elongate to their final tabular form and usually persists only over the groove above anticlinal walls and at the base of root hairs. The cell wall thins to about half its maximum thickness during this elongation. Emerging root hairs broach the pellicle and the original primary wall. Remnants of both these layers form a short, tight collar at the base of each hair; this collar adheres to the primary wall of the hair which is continuous with a new, thin wall which is formed interior to the original outer wall of the parent cell. Failure to recognize the complex structure and transitory nature of the epidermal pellicle has led to confusion in the literature regarding the nature of root-surface and rhizosphere mucilaginous components and their origin. These interpretations are compared with those arising from this study.

Development of water conducting capacity in the root systems of young plants of corn and some other c4 grasses.

Development of the primary and early nodal roots was studied in Zea mays L., Zea mexicana (Schrad.) Reeves & Mangelsd., Sorghum bicolor (L.) Moench., and Sorghum sudanese (Piper) Stapf. in relation to shoot development. In all the types studied all roots reached lengths of about 30 centimeters before the late metaxylem (LMX) was open, and young plants with total root lengths of around 100 centimeters had almost no open LMX. On average, corn seedlings with up to 36 square centimeters of leaf had no open LMX. The name "immature apices" is suggested for such long but not fully functional roots. In plants up to 50 days old a fairly constant proportion of less than half the total root length had open LMX. A pilot study of stomatal resistance on days of high evaporative demand suggested that young seedlings may show higher resistance than older plants in the afternoon. Estimates of longitudinal permeability of corn roots with only early metaxylem vessels open indicate very steep gradients of water potential would develop under such conditions.

The early development of the sheep trophoblast and the involvement of cell death.

During the period of rapid elongation prior to the initiation of placental attachment (days 12-16 of gestation), the ovine blastocyst consists of a single layer (primarily) of roughly cuboidal trophoblastic cells with an inner lining of flattened endodermal cells. Well-developed spot desmosomes link the adjacent cell borders in both the trophoblastic and endodermal layers. The trophoblastic cells contain acid phosphatase-positive, lysosomelike organelles, the mean diameter of which increases greatly between days 12 and 16 and whose contents vary during development. Also during the developmental period studied, trophoblast cells accumulate lipid; and periodic acid-Schiff-positive binucleate cells appear within the trophoblast layer. A consistent observation throughout the 5 days of rapid growth and differentiation of the blastocyst was the death and disintegration of some trophoblast cells. These disintegrating cells are usually singly dispersed within the trophoblast, although occasionally groups of four or five are observed. The cell death may indicate overall remodelling of the blastocyst, or the cells may represent genetically deficient cells which are unable to respond to the appropriate signal to differentiate.

Evaluation of the specificity of lectin binding to sections of plant tissue.

Hand sections of young corn root tips have been used in a study of problems encountered in the binding of fluorescently-labelled lectins to plant tissues. It was found, surprisingly, that with lectins specific for a sugar known to be present (Lotus and Ulex lectins for L-fucose), with a lectin specific for a sugar thought not to be present (wheat-germ agglutinin for N-acetylglucosamine), with non-lectin glycoprotein and protein (gamma-globulin and bovine serum albumin) and with basophilic dyes (alcian blue and toluidine blue), a coincidental binding pattern similar to the pattern of autofluorescence in the same tissue was obtained. Corn root tissues include cell walls composed of complex polysaccharides esterified with ferulic acid residues, as well as mucilages which are highly hydrated and expanded. In such material, neither standard inhibition controls with haptens nor the use of a wide range of lectin concentrations are adequate to distinguish clearly specific and non-specific binding of fluorescently-labelled lectin. Therefore, lectins are not the simple test probes they have been supposed. Before interpreting results obtained in using fluorescently-labelled lectins on any tissue sections, all available information (biochemical as well as histochemical) about the tissue must be considered.

The cellular localization of chorionic somatomammotrophin in ovine chorion.

Sections of Days 12 to 55 sheep chorion, embedded at low temperature in glycol methacrylate, were exposed to rabbit anti-ovine chorionic somatomammotrophin (oCS) to ascertain the distribution of oCS. This hormone was first detectable on Day 14, when the cytoplasm of each chorionic cell displayed a low level of fluorescence surrounding droplets which were shown to be lipid containing. At this time, all chorionic cells were uninucleate. By Day 28, binucleate chorionic cells had appeared but showed no binding of the oCS antiserum which was confined to a significant proportion of the uninucleate chorionic cells surrounding lipid droplets, as at Day 14. The same pattern of hormone distribution, although with reduced fluorescence intensity, was observed on Day 55; fluorescence indicative of antibody binding was seen only in some of the uninucleate chorionic cells. Hence, oCS was detected in chorionic tissue before the differentiation of binucleate cells (Day 14) and, at all stages, it was confined to the cytoplasm of specific uninucleate chorionic cells in close association with lipid droplets.

The rhizosphere in Zea: new insight into its structure and development.

Some of the nodal roots of field-grown Zea mays L. bear a persistent soil sheath along their entire length underground except for a glistening white soil-free zone which extends approximately 25 mm behind the root cap. These roots are generally unbranched. The histology of the surface and the rhizosphere of the sheathed roots has been examined by correlated light and electron microscopy. All mature peripheral tissues including root hairs, are largely intact and apparently alive where enclosed by the soil sheath. The sheath is permeated by extracellular mucilage which is histochemically distinct from the mucilage at the epidermal surface, but similar to that produced by the root cap. Isolated cells resembling those sloughed from the sides of the root cap persist in the soil sheath along the length of these roots. Fresh whole mounts of the sheath show that these detached cells may be alive and streaming vigorously even at some distance from the root cap. Rhizosphere mucilage is associated with the isolated cells.