Paterson’s curse roots were studied with respect to their ability to produce coloured secondary products, napthoquinones, in living roots. Young roots produced large quantities of red coloured anthro- or napthoquinones in outer layers of root periderm. In contrast, mature or aged roots exhibited blackened periderm containing accumulations of dark coloured secondary products, possibly due to oxidation or polymerization of these compounds over time. Ethanolic extracts of young root periderm tissues were bright red or pink in colour and contained several unusual napthoquinones, including acetylshikonin, and 1,3 dihydroxy -3- methylanthraquinone, as detected by LC/MS and GC/MS analyses. Mature or aged root extracts were colourless in appearance, and contained 1,3 dihydroxy-3-methylanthraquinone and other likely related constituents. Production of napthoquinones and colour of root extracts was clearly influenced by location of harvest of Paterson’s curse, time of harvest and age of root tissue. Both young and aged root extracts exhibited strong inhibition of root growth of annual ryegrass, with young root extracts showing greatest phytotoxicity. Shikonin at 1 mg/ml was also phytotoxic to annual ryegrass growth, with increasing phytotoxicity noted with increasing shikonin concentration. The role of napthoquinones in plant invasion and their interactions in the plant rhizosphere require further elucidation, as novel napthoquinones exhibit potent antimicrobial, fungitoxic, and phytotoxic activity due to their impact on electron transport and cellular respiration processes.

Branch root development on the primary root of maize (Zea mays L.) seedlings was followed for 9 d after planting. This period includes the shift from seedling heterotrophy to autotrophy. Linear density of branches in the basal region ranged from ~38 cm-1 at the base to ~10 cm-1 beyond 10 cm. Branch roots in the first ~8 cm were produced before assimilate was available. Branch length decreased from ~26 mm at 1 cm along the primary root to ~8 mm at 10 cm from the base. Without the cotyledon, branch root density in the basal region was ~10 cm-1 and roots were short (~5 mm). Beyond 8-10 cm both measurements matched those of intact seedlings. Dark-grown seedlings had basal branch root densities higher than those without cotyledons but none beyond 10 cm. There were more and smaller diameter sieve tubes in the basal region of the primary root. These decreased distally in number but had larger diameters where branches formed after assimilate was available. Proliferation of basal branch roots in very young seedlings can have major advantages for successful seedling establishment in the field and could be screened for without difficulty.

BACKGROUND AND AIMSSome 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.METHODSPetioles of intact plants were cryo-fixed, planed to smooth transverse faces, and examined in a cryo-SEM.KEY RESULTSWith 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.CONCLUSIONSThe 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.

Storage of phosphorus (P) in stem tissue is important in Mediterranean Proteaceae, because proteoid root growth and P uptake is greatest during winter, whereas shoot growth occurs mostly in summer. This has prompted the present investigation of the P distribution amongst roots, stems, and leaves of Hakea prostrata R.Br. (Proteaceae) when grown in nutrient solutions at ten P-supply rates. Glasshouse experiments were carried out during both winter and summer months. For plants grown in the low-P range (0, 0.3, 1.2, 3.0, or 6.0 micromol d(-1)) the root [P] was > stem and leaf [P]. In contrast, leaf [P] > stem and root [P] for plants grown in the high-P range (6.0, 30, 60, 150, or 300 micromol P d(-1)). At the highest P-supply rates, the capacity for P storage in stems and roots appears to have been exceeded, and leaf [P] thereafter increased dramatically to approximately 10 mg P g(-1) dry mass. This high leaf [P] was coincident with foliar symptoms of P toxicity which were similar to those described for many other species, including non-Proteaceae. The published values (tissue [P]) at which P toxicity occurs in a range of species are summarized. X-ray microanalysis of frozen, full-hydrated leaves revealed that the [P] in vacuoles of epidermal, palisade and bundle-sheath cells were in the mM range when plants were grown at low P-supply, even though very low leaf [P] was measured in bulk leaf samples. At higher P-supply rates, P accumulated in vacuoles of palisade cells which were associated with decreased photosynthetic rates.

Results from a controlled environment system and the field showed that slow root elongation rate was associated with accumulation of Pseudomonas spp. in the rhizosphere; fast root elongation avoided accumulation. In the controlled environment system, total bacteria and bacteria belonging to the genus Pseudomonas were quantified along wheat (Triticum aestivum L. cv. Janz) seminal roots elongating at rates of 2.4 or 0.8 cm d-1 in loose and compacted field soil, respectively. Although total numbers of bacteria were similar for both rates of elongation, more Pseudomonas spp. accumulated on the slow-growing roots and their numbers were greatest 0.5-1 cm from the root tips. A reduced rate of root elongation in compacted soil accelerated the differentiation of root hairs, branch roots and adhesion of rhizosheath soil. Elongation rate and distance between the root tip and the zone of root hair development were positively correlated (r=0.9), providing a morphological indicator of root elongation rate in the field. Slow-growing roots from the field had 20 times more Pseudomonas spp. per unit root length than fast-growing field roots, while total bacteria were 8-fold higher; differences were greatest 0-1 cm from the tips. These results may explain how soil structure and Pseudomonas spp. interact in conservation farming. Rapid root elongation is identified as a desirable trait for avoiding accumulations of bacteria.

As a reaction to invasion by pathogens, plants block their xylem conduits with mucilage, restricting pathogen advance. Wounding soil-grown roots of maize revealed that pectinaceous mucilage could be found in the vessels after 6 h, and abundantly filled most vessels up to 3 cm proximal to the wound after 1 d. Phenolics increased in the mucilage at later times. The same reactions occurred in vessels following mechanical wounding of axenically-grown roots, showing that the presence of microbes is not necessary for the response. The xylem mucilage is similar to root-cap mucilage in mode of extrusion from the periplasmic space of living cells through primary wall, apparent phase transition, and staining indicative of acidic polysaccharides. Whether other known properties of root-cap mucilage which might alter vessel functioning, such as reduction of surface tension and increased viscosity produced by dissolved solutes, are also common to xylem mucilage requires further investigation. However, our results indicate that possible influence of wounding-induced mucilage in xylem vessels should be considered in all experimental investigations of xylem function.

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 (pO2). 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 pO2 values. Colonies of G. diazotrophicus were grown on solid medium at atmospheric pO2 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 O2 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 O2 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 O2 that maintains aerobic respiration while not inhibiting nitrogenase activity.

This paper originates from an address at the 8th International Symposium on Nitrogen Fixation with Non-Legumes, Sydney, NSW, December 2000 Currently proposed means of entry of bacterial endophytes into roots of field-grown crop plants, as well as niches available for their successful colonization of living tissues, are evaluated from a plant biology perspective and the following conclusions drawn. (1) Opportunities for passive ‘crack entry’ into healthy, undisturbed roots in the field may not be as available as studies with laboratory-grown plants have suggested. Consistent entry of endophytes into living root tissues in the field probably requires bacterial capability to hydrolyse the hydrophobic incrustations of the walls of epidermal, hypodermal, endodermal and other cortical cells. (2) Xylem lumen apoplast is an unsuitable niche for endophytes, especially in the grasses, because of the reduced fitness such colonization imposes on plants subjected to field stresses. (3) The intercellular space apoplast is the most suitable niche for endophytes. More data about the environment of this apoplast are urgently needed so that plant varieties can be tailored to provide an optimal environment in these spaces for particular endophytes. It is suggested that many bacterial ‘endophytes’ may not have colonized living tissues, but are living in protective niches in dead surface tissues or closely adhering soil of rhizosheaths. Selection of strains of beneficial bacteria adapted for colonizing these external niches may be desirable.

The reliability of the cryo-SEM technique for stabilizing and quantifying embolisms in vessels of transpiring plants has recently been criticized, on the grounds that the embolisms observed are artefacts of the freezing. One of the tests used was a comparison of the embolisms found in rachises of transpiring walnut leaves (Juglans regia L.) frozen intact on the tree, with both cryo-SEM images of vessel contents and the measured hydraulic conductivity of similar samples whose xylem pressure had been returned to atmospheric pressure by cutting the leaves off under water. Embolisms found in intact frozen rachises were not present in cut rachises, and the high rachis hydraulic conductivities indicated a similar absence of embolisms. We show that the authors’ conclusions are wrong because their test produces a different artefact. When the petiole is cut under dye solution it is obvious that the immersing solution is drawn into the embolized vessels and fills them. Thus, the cryo-SEM images and the high hydraulic conductivities of specimens prepared by this technique do not indicate the contents of the xylem in the intact plant. In fact, this artefact may perhaps be used (with the dye) to measure the embolisms in the intact plant. The embolisms seen in the cryo-SEM are most unlikely to be artefacts. The published work that also shows embolisms and their refilling in transpiring plants by techniques involving no freezing is reviewed.

The time taken to refill embolized xylem vessels in field-grown roots of maize (Zea mays L.) was determined after transpiration was stopped by detopping the plants, or after individual roots were isolated from the plant. At various times after transpiration-driven how was stopped, root segments were frozen in situ and the percentage of large, late metaxylem vessels that were embolized was determined by examination in a cryo-SEM. Control roots frozen before interruption of the transpiration stream had a variable percentage of the vessels embolized, but no embolized vessels were found by about 20 min after isolation. Individual roots and vessels showed large variances in time to refill. In detopped root systems, the mean percentage of embolized vessels showed an exponential decay with a half-time of similar to2 min. The variance in refilling time is ascribed to two causes: the various proportions of gas acid liquid present in an individual vessel at the time of initial measurement, and the balance in composition between water vapour and air in the gas phase. Time to produce exudate from the cut surface of the isolated roots was variable and unrelated to the percentage of vessel embolism. It is proposed that the refilling of the vessels in these isolated roots is by water pushed radially into the vessels by living cells, and that embolized vessels throughout intact transpiring plants are similarly refilled. (C) 2001 Editions scientifiques et medicales Elsevier SAS.

Solute osmotic potentials (Ψx) in the vessels of hydroponically grown maize roots were measured to assess the osmotic-xylem-sap mechanism for generating root pressure (indicated by guttation). Solutes in vessels were measured in situ by X-ray microanalysis of plants frozen intact while guttating. Osmotic potentials outside the roots (Ψo) were changed by adding polyethylene glycol to the nutrient solution. Guttation rate fell when Ψo was decreased, but recovered towards the control value during 3–5 days when Ψo was greater than or equal to −0.3 MPa, but not when Ψo was equal to −0.4 MPa. In roots stressed to Ψo = −0.3 MPa, Ψx, was always more positive than Ψo, and Ψx changed only slightly (ca. 0.05 MPa). Thus the adjustment in the roots which increased root pressure cannot be ascribed to Ψx, contradicting the osmotic-xylem-sap mechanism. An alternative driving force was sought in the osmotic potentials of the vacuoles of the living cells (Ψv), which were analysed by microanalysis and estimated by plasmolysis. Ψv showed larger responses to osmotic stress (0.1 MPa). Some plants were pretreated with abundant KNO3 in the nutrient solution. These plants showed very large adjustments in Ψv (0.4 MPa) but little change in Ψx (0.08 MPa). They guttated by 4 h after Ψo was lowered to −0.4 MPa. It is argued that turgor pressure of the living cells is a likely alternative source of root pressure. Published evidence for high solute concentrations in the xylem sap is critically assessed.

The exudate that wells out of the cut surface of detached rents is commonly thought to flow from the xylem and to be forced up the vessels by root pressure. Direct monitoring of the cut surface of maize (Zea mays L.) roots by optical microscopy and cryomicroscopy of fast-frozen material showed that the liquid originated not only from cut vessels but was also exuded from living tissues and intercellular spaces over the entire cut surface. Indeed, exuded liquid usually appeared last over the vessels. This pattern of exudation was seen also from partially submerged segments: of mature roots in which the lower end had been sealed. Exudation also occurred from similarly prepared root sleeves from which the stele had been removed. Measurements of rates of exudation often showed a periodicity of about 1 h. These observations add to earlier published descriptions of the phenomenon, particularly extensive 19th century studies, which remains unexplained by current understanding of root water relations. (C) 2000 Editions scientifiques et medicales Elsevier SAS.

The plexus of vascular bundles in the nodes of grasses is notoriously complex, where long axial bundles pass through a network of transverse bundles. The xylem pathways for water in maize stems have been investigated anatomically and with dye and particulate tracers, revealing some of the details of this complexity. Only approx. 3 % of axial Vessels pass through nodes without being interrupted by end wails. Axial bundles at nodes differ from those in internodes in having the metaxylem and protoxylem vessels connected by small tracheary elements. So it is only at nodes that exchange of sap occurs between the lame vessels within a bundle. End walls, acting as tilters for particles and gas bubbles, always separate axial vessels from vessels in transverse bundles. The high redundancy of bundle connections in the nodal plexus is interpreted as providing alternative water pathways to bypass embolisms and damaged or diseased sections of the xylem. The pores in the filters at the base of nodes and between axial and transverse vessels within nodes are <20 nm in diameter. Where axial Vessels connect to transverse vessels, a variety of unusual shapes of vessel elements mediate two- and three-way connections within the plexus. (C) 2000 Annals of Botany Company.

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.

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 +/- 0.7 nm, 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. 100 MPa. (C) 2000 Annals of Botany Company.

Embolism and refilling of vessels was monitored directly by cryomicroscopy of field-grown corn (Zea mays L.) roots. To test the reliability of an earlier study showing embolism refilling in roots at negative leaf water potentials, embolisms were counted, and root water potentials (Psiroot) and osmotic potentials of exuded xylem sap from the same roots were measured by isopiestic psychrometry. All vessels were full at dawn (Psiroot -0.1 MPa). Embolisms were first seen in late metaxylem vessels at 8 AM. Embolized late metaxylem vessels peaked at 50% at 10 AM (Psiroot -0.1 MPa), fell to 44% by 12 PM (Psiroot -0.23 MPa), then dropped steadily to zero by early evening (Psiroot -0.28 MPa). Transpiration was highest (8.5 μg cm-2 s-1) between 12 and 2 PM when the percentage of vessels embolized was falling. Embolized vessels were refilled by liquid moving through their lateral walls. Xylem sap was very low in solutes. The mechanism of vessel refilling, when Psiroot is negative, requires further investigation. Daily embolism and refilling in roots of well-watered plants is a normal occurrence and may be a component of an important hydraulic signaling mechanism between roots and shoots.

The recent measurements of high percentage embolized vessels in a sample of maize axile roots during the day raised the question whether such interruptions to water flow could occur in plants that had only a single water-supplying root. Zea mays L. plants were grown in the field on a root system developed entirely from the single primary root. The formation of vessel embolisms in this root was monitored by cryo-scanning electron microscopy. Vessels were sap-filled at dawn, but 60% of late metaxylem (LMX) vessels were embolized by 0830 h. This high percentage persisted until early afternoon, then declined to reach 30% by 1800 h. Leaf balance pressures rose from about 0.3 MPa at dawn to a peak mean of 1.2 MPa at 1300 h, then declined to 0.5 MPa by 1800 h. Peak embolism in LMX vessels was reached in the early morning at balance pressures of 0.7–0.8 MPa. Some refilled in the afternoon while balance pressures were 0.5–0.7 MPa. The six to eight LMX vessels at the root base would deliver most of the transpiration stream to the shoot. Our finding that up to 60% were embolized during the day, while the plants continue to transpire, suggests that the mean flow velocity in the vessels is higher than the value of 270 m h–1 estimated recently by others for completely full vessels in the mesocotyl of single-rooted corn plants.

Rapid wilting and subsequent rapid recovery of the shoots of plants whose roots are cooled and rewarmed (first described by Sachs, 1860([23])), has been investigated by cryoscanning electron microscopy. Squash plants began to wilt within 5 min and were completely wilted 1 h after their roots were placed in nutrient solution at 4 degrees C. Recovery began in 5 min and was complete by 45 min when the roots were returned to solution at 22 degrees C. Some stomata on the abaxial leaf surface remained fully or partially open in the wilted plants and transpiration continued at a low level. Both control and wilted plants had the same proportion (60 %) of targe root vessels partially or totally gas-filled, showing that the supply of water was not limited by the reduction of axial hydraulic conductance due to vessel embolism. However, only 10% of these embolized vessels in the wilted plants contained any liquid, compared to similar to 80 % of similar vessels in control and recovered plants. This is visual evidence of reduced radial hydraulic conductance into the vessels in the cold roots, and that this reduced conductance, together with still open-stomata, produces wilting. These effects were reversed by rewarming.