Explore the vast range of titles available.

• The palisade cell sizes in leaves of Eucalyptus pauciflora were estimated in paradermal sections of cryo-fixed leaves imaged in the cryo-scanning electron microscope, as a quantity called the cell area fraction (CAF). • Cell sizes were measured in detached leaves as a function of leaf water content, in intact leaves in the field during a day's transpiration as a function of balance pressure of adjacent leaves, and on leaf disks equilibrated with air of relative humidities from 100 to 58%. • Values of CAF ranged from 0.82 at saturation to approx. 0.3 in leaves dried to a relative water content (RWC) of 0.5, and in the field to approx. 0.58 at 15 bar (1.5 MPa) balance pressure. At a CAF of 0.58, the moisture content of the cell walls is in equilibrium with air at 90% relative humidity, which is the estimated relative humidity in the intercellular spaces. • It is shown that at this moisture content, the cell walls could be exerting a pressure of approx. 50 bar on the cell contents.

• 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.

Flow of the transpiration stream in the lumen apoplast of the xylem appears hydrodynamically orthodox in being approximately described by the Hagen-Poiseuille Law, and by Murray's Law for branching pipes. Flow may be followed in the major (supply) veins by labelling the stream with dye solutions. Progress of the dye in the stream into the minor (distribution) veins is obscured by surrounding tissues. Observations of the spread of fluorescent tracers from these veins in living leaves gave results that have been seriously misinterpreted to present a false view of the cell wall apoplast. Microscopy of the stabilized water-soluble fluorescent tracers moving out of the minor veins has revealed that: (i) the dye is separated from the water by filtration through cell membranes, and the water moves through the symplast; and (ii) the dye diffuses in the cell wall apoplast at rates 1/100 to 1/10 000 the rate of diffusion in water. As a consequence of (i), high concentrations of dye build up at sites called sumps. In grasses these sumps may be in the intercellular spaces outside the xylem. In dicotyledons these sumps are within the small tracheary elements. In fact, flow in the lumen apoplast is flow through leaky tubes, and is inadequately described by the Hagen-Poiseuille Law. Leaky tubes have a critical radius, below which (for a given pressure gradient) flow cannot occur. As a consequence of this, a wedge of xylem made up of vessels of different radii acts as a unit to concentrate dye tracers in a sump at its apex. Sumps may also be formed by evaporation of the water in the stream, especially at leaf margins. Investigations with the cryo-analytical scanning electron microscope of the natural ions of the transpiration stream reveal high concentrations of K, Cl, P and Ca in the stream in all the sizes of vessel and vein of sunflower leaves. These high concentrations appear to be produced, not by the mechanisms responsible for the formation of sumps of dyes, but by some other processes, probably occurring in the stem. The absence of sump formation by ions at the places where dyes form sumps is probably due to the more rapid penetration of the ions through the cell membranes. Reasons for the discrepancy between these measurements of salt concentrations in the stream and those obtained from sap expressed from leaves by pressure vessels are discussed. Implications of these facts for the design and interpretation of experiments with leaves are presented.

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 10(4 ) 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

The architecture of the connecting xylem network in the vascular plexus linking branch and main root vessels has been examined using cryoSEM, and the limiting porosity of the network determined with tracers (dye, and particles of known size: latex, polystyrene and gold sols). Dye and water move freely throughout the xylem network, while particles are constrained to follow tortuous vessel-like conduits of irregularly-shaped elements linked by large-diameter perforations. These conduits end at special pit membranes (boundary pit membranes) at the periphery of main root vessels. Particles accumulate on the outer side of these filters, often filling the terminal elements of these conduits adjacent to the main root vessels. Some vessel elements within the plexus are isolated from the convoluted conduits by normal pit membranes, and often also from each other, by pit membranes and still-intact end walls in otherwise mature elements. These extra-conduit elements may be an auxiliary filtering system. The boundary pit membranes filtered out particles with mean diameters as small as 4.9±0.7nm, indicating a pore size one or two orders of magnitude smaller than most previous measurements for pit membranes, but close to pore sizes determined for hydrated primary cell walls. It is concluded that boundary pit membranes at branch-root junctions are efficient filters for microbes and particulates entering damaged branch roots. They would also restrict entry of air/water interfaces when main root xylem tension was less than approx. 100MPa.

When freshly cut segments of naturally decorticated steles of horizontal roots of the Australian grass tree (Xanthorrhea preissii) are subjected to gentle suction, bubbles of gas sometimes appear, as well as liquid, in capillaries attached to the aspirated ends of the steles. We tested the hypothesis that this gas comes from vessels embolized in the intact xylem stream, and that the gas volume extruded is therefore an effective measure of the extent of this embolism. To do this, twin samples were taken from individual roots of X. preissii in the field, one was fast-frozen intact for subsequent estimation of vessel embolisms in the cryo-scanning electron microscope (CSEM), the other 15-cm segment rapidly assessed for volume of gas aspirated into a standard micropipette tube. The two measures mutually confirmed one another by showing a strong positive correlation between numbers of embolized vessels and extracted gas volume. Similar gas volumes were obtained from replicate root segments excised directly from a root when the ends of the segment were frozen before excision, and aspiration conducted after subsequent thawing of the ends under water. The pattern of changes in embolisms during unstressed conditions in early summer, shown by both CSEM and aspiration, indicated almost no embolisms before dawn, followed by a rapid rise to a peak in mid morning, than a progressive loss of embolisms in late afternoon. It was also shown that the amount of embolism did not change with time after excision of the roots up to at least 30 min. A comparison of changes in leaf transpiration with gas volumes in steles during a 24-h cycle at peak transpiration stress in mid summer showed rapid rates of transpiration in early morning and late afternoon, with an intervening period of low water loss during the rest of the day. Numbers of embolisms rose to an early morning peak, followed by apparent repair of these before noon. There was a second spate of embolisms in late afternoon, followed by complete refilling of all xylem with liquid by an hour or so after dusk. All vessels then remained fully recharged until the following dawn. We believe that aspiration is a direct and reliable technique, which offers a simple, inexpensive means of assessing the relative extent of embolism of vessels in xylem, and a means to test earlier findings by the other direct method of the CSEM. In a broad context, the technique should provide new opportunities for evaluating water relations of the xylem of whole plants.

A new method of measuring water uptake by roots is applied to whole root systems of large maize plants growing in aeroponic (mist) culture. The method depends on the build-up of concentration of dye (sulphorhodamine G) on and in the root surface, when it is fed in the mist. Water enters the roots rapidly, but the dye is separated from the water by osmotic filtration and penetrates the apoplast only by very slow diffusion. The dye accumulates progressively with time on roots on transpiring plants, but not on roots of non-transpiring controls. The rate of accumulation of dye at a place on the root is translated to a flux of water into the root at that place, using the concentration of dye in the mist. Water fluxes were measured into first-order branches and axes over the root system and expressed both as fluxes per unit length and per unit surface of root. Values are consistent with those found by potometric methods for limited samples of young plants. The variance of the measurements is quite large, possibly reflecting real heterogeneities in water uptake throughout the root system. The maximum water uptake achieved by a few branches was 40 μl h-1 cm-2 root. On average, flux into axes and branches was the same throughout the root system, at about 5 μl h-1 cm-2. The region of the axis at which the late metaxylem vessels mature and become conducting coincides with the region where the branches become active in water uptake (20-30 cm from the tip). Proximal to this, the branches collect about eight times as much water as the axis, having about eight times the surface area. The zone of maximum water collection by branches and axis is 30-60 cm from the tip (6-8 μl h-1 cm-1 axis). In the older, more proximal regions, water collection drops to about a quarter of this.

Changes of view on the course of the transpiration strcam beyond the veins in leaves are followed from the imbibition theory of Sachs, through the (symplastic) endosmotic theory of Pfeffer (which prevailed almost unquestioned until the late 1930s), to Strugger's experiments with fluorescent dye tracers and the epifluorescence microscope. This latter work persuaded many to return to the apoplastic-(wall)-path viewpoint, which, despite early and late criticisms that were never rebutted, is still widely held. Tracer experiments of the same kind are still frequently published without consideration of the evidence that they do not reveal the paths of water movement. Experiments on rehydration kinetics of leaves have not produced unequivocal evidence for either path. The detailed destinies of the solutes that reach the leaf in the transpiration stream have received little attention. Consideration of physical principles governing flow and evaporation in a transpiring leaf emphasizes that: (1) Diffusion over interveinal distances at the rates in water will account for substantial solute movement in a few minutes, even in the absence of flow. (2) Diffusion can occur also against opposing flow. (3) Volume fluxes in veins are determined by the diameter of the largest vessels, and all the leaves examined contain high conductance supply veins which are tapped into by low-conductance distributing veins. (4) Edges and teeth of leaves will be places of especially rapid evaporation, and they often have high-conductance veins leading to them. (5) Solutes in the stream will tend to accumulate at leaf margins. On the basis of recent work, the view is maintained that the water of the stream enters the symplast through cell membranes very close to tracheary elements. Also, that this occurs locally over a small area of membrane. Many solutes in the stream are left outside in the apoplast. This produces regions of high solute concentration in the apoplast and an enrichment of solutes in the stream as it perfuses the leaf. Solutes that enter the symplast are not so easily tracked. Suggestions about where some of them may go can be gained from a fluorescent probe that identifies particular cells (scavenging cells) as having H+-ATPase porter systems to scrub selected solutes from the stream. Unpublished case-histories are presented which illustrate many aspects of these processes and principles. These are: (1) Maize leaf veins, where the symplastic water path starts at the parenchyma sheah; (2) Lupin veins, where the symplastic path starts at the bundle sheath and where solutes are concentrated in blind terminations; (3) The edges of maize leaves where flow is enhanced by a large vein (open to the apoplast), and solutes are deposited in the apoplast by evaporation; (4) Poplar leaf teeth, which receive strong flows, and where the epithem cells are scavenging cells; (5) Mimosa leaf marginal hairs, which have scavenging cells at their base; (6) Active hydathodes, whose epithem cells are scavenging cells; (7) Pine needle transfusion, which is a site of both solute enrichment (in the tracheids), and scavenging (in the parenchyma); (8) Estimates are made of diffusion coefficients of a solute both along and at right angles to the major diffusive pathway in wheat leaves. The first is 1000 times the second, but is 1/100 of free diffusion in water. Five general themes of the behaviour and organization of the transpiration stream are induced form the facts reviewed. These are: (1) The stream is channelled into courses of graded intensities by the interplay of the physical forces with the anatomical features, each course with a distinct contribution to the processing of the stream (2) Water enters the symplast at precise locations as close as possible to the traceary elements. (3) As the stream moves through the leaf its solute concentration is enriched many-fold at predictable sites. (4) Solutes excluded from the symplast diffuse from these sources of high concentration in specially formed wall paths, in precise patterns, at rates which can be measured, and which are low compared with diffusion in water. (5) Other solutes permeate the symplast, often over the surfaces of groups of cells which are organized into recognized structural features.

The fluorescent tracers sulphorhodamine G (SR) and pyrene trisulphonate (PTS) were used to explore the functions of cells and tissues within the pine needle, following their progress after feeding through the transpiration stream. The distributions of tracer in the needles were stabilized for fluorescence microscopy by rapid freezing and freeze-substitution, and anhydrous embedding and sectioning. After short pulse + chase times (up to 2 h), SR and PTS accumulated at higher-than-xylem concentrations in the transfusion tracheids on the flanks of the vascular strand, but did not pass out through the endodermis. The accumulations occurred locally where the transpiration water was separated from the tracers and passed into the symplast of the transfusion parenchyma and endodermis. After a 24 h water chase, SR had entered the symplast through the transfusion parenchyma, and spread through the endodermis and palisade. It is argued that this is evidence of active H+-ATPase systems lowering the external pH of the transfusion parenchyma, and characterizes these cells as scavenging cells similar to those found in the bundle sheath systems of legume leaves. The transport of SR through the endodermis and palisade is the first clear evidence that this tracer can also function as a symplastic tracer. The hypothesis that the transfusion parenchyma acts to scavenge solutes from the transpiration stream was tested by loading the stream with [14C]aspartate and examining the subsequent distribution of 14C by dry autoradiography. After a pulse + chase of (0.75 + 3) h, 14C was found concentrated in the transfusion parenchyma, and at even higher levels in the Strasburger cells. It is proposed that major functions of the transfusion tissue of gymnosperms are (a) the concentrating of solutes from the transpiration stream and (b) the retrieval from the stream of selected solutes that are returned to the phloem through the Strasburger cells, or forwarded through the endodermis to the palisade.

A test was made of the previous unexpected observation that embolized vessels were refilled during active transpiration. The contents of individual vessels in petioles of sunflower plants were examined, after snap-freezing at 2-h intervals during a day's transpiration, in the cryo-scanning electron microscope, and assessed for the presence of liquid or gas (embolism) contents. Concurrent measurements were made of irradiance, leaf temperature, transpiration rate, and leaf water potential (by pressure chamber). Up to 40% of the vessels were already embolized by 0900 (transpiration rate approx 5 micrograms cm-2 s-1, water potential about -300 J/kg), and the proportion declined to a minimum (as low as 4%) at 1500. This was the time of highest transpiration rate (approx 25 micrograms cm-2 s-1) and most negative water potential (-600 to -700 J/kg). Images of vessels with mixed gas and liquid contents showed water being extruded through pits in the walls of the vessels to refill them. The data indicate that: (1) the water columns are weak and break under quite small tensions; (2) embolisms are repaired by refilling the vessels with water on a short time scale (minutes) throughout the day; (3) the vigor of this refilling process is adjusted by the plant on a longer time scale (hours) to the intensity of the water stress; (4) the pressure chamber balance pressure (P) does not measure tension in the vessels; (5) P is also not a measure of water stress (as measured by vessel embolization); and (6) P is a measure of the plant's response to water stress, i.e., a measure of the vigor of the refilling process. The test confirms the previous observations and negates all the assumptions and evidences of the Cohesion Theory. The data are fully consistent with the Compensating Pressure Theory, which predicted the relations demonstrated in this experiment