M. J. Canny

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 endophytess 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.

VESSEL CONTENTS DURING TRANSPIRATION : EMBOLISMS AND REFILLING

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 days 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 ~5 _g_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 (~25 _g_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 plants 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. Using the assumptions of that theory it is easy to outline a simple mechanism by which the refilling of vessels might be achieved by reverse osmosis, and the adjustment in (3) might be achieved by osmoregulation in the starch sheath.

Pathways and processes of water and nutrient movement in roots

Recent work in our laboratory provides evidence for a revised view of the functioning of roots of maize, and probably of all the grasses. The development of coherent soil sheaths on the distal 30-cm of these roots, and the loss of the sheaths further back, led us to investigate the differences in surface structure, anatomy, carbon exudation and microflora of the sheathed and bare zones. The significant differences are summarized. But the fact which underlies all these differences is the maturation of the late metaxylem (LMX). In the sheathed zones the LMX elements are still alive and non-conducting; only the early metaxylem (EMX) and protoxylem are open. In the bare zones they are open vessels. This leads directly to the dryness of bare zones and the wetness of sheathed zones, and indirectly to the other differences noted. Branch root junctions are shown to be structures of great significance. Besides connecting the branches to the axile systems, they serve also to connect the EMX and LMX vessels, and contain a tracheid barrier which prevents air embolisms entering the main vessels. These discoveries force us to revise the traditional view of water uptake by the root hair zone, and to suggest that much water must also enter bare roots, possibly via the laterals. There is some published evidence for this. The living LMX elements of the sheathed zone accumulate large concentrations of potassium which must joint the transpiration water at the transition to the bare zone. Calculations suggest that this may be only a tenth of the requirement of a mature plant, and that the balance may enter the bare zones with the transpiration water.

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.

APPLICATIONS OF THE COMPENSATING PRESSURE THEORY OF WATER TRANSPORT

Some predictions of the recently proposed theory of long-distance water transport in plants (the Compensating Pressure Theory) have been verified experimentally in sunflower leaves. The xylem sap cavitates early in the day under quite small water stress, and the compensating pressure P (applied as the tissue pressure of turgid cells) pushes water into embolized vessels, refilling them during active transpiration. The water potential, as measured by the pressure chamber or psychrometer, is not a measure of the pressure in the xylem, but (as predicted by the theory) a measure of the compensating pressure P. As transpiration increases, P is increased to provide more rapid embolism repair. In many leaf petioles this increase in P is achieved by the hydrolysis of starch in the starch sheath to soluble sugars. At night P falls as starch is reformed. A hypothesis is proposed to explain these observations by pressure-driven reverse osmosis of water from the ground parenchyma of the petiole. Similar processes occur in roots and are manifested as root pressure. The theory requires a pump to transfer water from the soil into the root xylem. A mechanism is proposed by which this pump may function, in which the endodermis acts as a one-way valve and a pressure-confining barrier. Rays and xylem parenchyma of wood act like the xylem parenchyma of petioles and roots to repair embolisms in trees. The postulated root pump permits a re-appraisal of the work done by evaporation during transpiration, leading to the proposal that in tall trees there is no hydrostatic gradient to be overcome in lifting water. Some published observations are re-interpreted in terms of the theory: doubt is cast on the validity of measurements of hydraulic conductance of wood; vulnerability curves are found not to measure the cavitation threshold of water in the xylem, but the osmotic pressure of the xylem parenchyma; if measures of xylem pressure and of hydraulic conductance are both suspect, the accepted view of the hydraulic architecture of trees needs drastic revision; observations that xylem feeding insects feed faster as the water potential becomes more negative are in accord with the theory; tyloses, which have been shown to form in vessels especially vulnerable to cavitation, are seen as necessary for the maintenance of P, and to conserve the supplementary refilling water. Far from being a metastable system on the edge of disaster, the water transport system of the xylem is ultrastable: robust and self-sustaining in response to many kinds of stress.

Vessel contents of leaves after excision - a test of Scholander's assumption.

A test was attempted of the assumption that, when a leaf is cut, the xylem still contains water under tension beyond the first vessel cross walls. This assumption enabled Scholander to argue that the balance pressure in his pressure chamber measured the tension in water columns in the vessels before cutting. The numbers of embolized vessels were counted, after rapid freezing of petiole and midrib samples of sunflower leaves, in the cryo-scanning electron microscope. Counts were made on leaves still attached to the plant and at intervals after cutting from the plant (up to 16 min) during a short spring days transpiration. The lengths of vessels in the leaves, measured by latex particle perfusion, showed that 8% of vessels in the mid-petioles and 0% in the midribs should be opened by cutting. The changing percentages of embolized vessels (E) with time showed that: (1) in intact plants E was close to zero until midday when it rose to ~40%, and then fell progressively to near zero by 1600; (2) in excised leaves there was no detectable change in E immediately after cutting, and, in all but two time courses, no change as large as the 8% of opened vessels within 16 min; (3) but briefly, when E was high (midday), it rose further after cutting to a plateau (_E = 30-40%) in 4 min. From this rate of emptying, the estimated maximum pressure difference between vessels and parenchyma was of the order of 0.05-0.2 MPa (0.5 to 2 bar) at this time. (4) All these changes occurred in the petioles 1 h before they were found in the midribs. The test failed because the expected large pressure difference between vessels and parenchyma was not present. Further, the embolized vessels were refilled at the time of peak transpiration, which would be impossible with any substantial tension in the vessels. Because these results contradict the whole basis of the Cohesion Theory, a second experiment was carried out to test them, and is reported in a companion paper.

Structural changes in acclimated and unacclimated leaves during freezing and thawing

Freeze-induced damage to leaf tissues was studied at different states of acclimation to low temperatures in snow gum, Eucalyptus pauciflora Sieber ex Sprengel. Intact, attached leaves of plants grown under glasshouse or field conditions were frozen at natural rates (frost-freezing) and thawed under laboratory conditions. Leaves were cryo-fixed unfrozen, during frost-freezing or after thawing for observation in a cryo-scanning electron microscope. Frost-freezing in unacclimated tissues caused irreversible tissue damage consistent with tissue death. Intracellular ice formed in the cambium and phloem, killing the cells and leaving persistent gaps between xylem and phloem. Many other cells were damaged by frost-freeze-induced dehydration and failed to resorb water from thawed extracellular ice, leaving substantial amounts of liquid water in intercellular spaces. In contrast, acclimated leaves showed reversible tissue displacements consistent with leaf survival. In these leaves during freezing, massive extracellular ice formed in specific expansion zones within the midvein. On thawing, water was resorbed by living cells, restoring the original tissue shapes. Possible evolutionary significance of these expansion zones is discussed. Acclimated leaves showed no evidence of intracellular freezing, nor tissue lesions caused by extracellular ice. While the observations accord with current views of freeze-sensitivity and tolerance, cryo-microscopy revealed diverse responses in different tissue types.

Mucilage production by wounded xylem tissue of maize roots: time course and stimulus

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.

Locating water-soluble vital stains in plant tissues by freeze-substitution and resin-embedding

Vital stains, moving in the transpiration stream in leaf apoplast, may be kept in place through freezing, freeze‐substitution, embedding and sectioning, to reveal their position in the living plant. This technique has been used to study the details of movement of water out of the veins of leaves, and has wide application in histochemistry with water‐labile dyes, and for following dye movements in protoplasm. Patterns of water movement in the leaf of Zea mays L. are presented as an example.

Cryo-scanning electron microscopy (CSEM) in the advancement of functional plant biology. Morphological and anatomical applications

Cryo-scanning electron microscopy (CSEM) is reviewed by exploring how the images obtained have changed paradigms of plant functions and interactions with their environment. Its power to arrest and stabilise plant parts in milliseconds, and to preserve them at full hydration for examination at micrometre resolution has changed many views of plant function. For example, it provides the only feasible way of accurately measuring stomatal aperture during active transpiration, and volume and shape changes in guard cells, or examining the contents of laticifers. It has revealed that many xylem conduits contain gas, not liquid, during the day, and that they can be refilled with sap and resume water transport. It has elucidated the management of ice to prevent cell damage in frost tolerant plants and has revealed for the first time inherent biological and physical features of root/soil interactions in the field. CSEM is increasingly used to reveal complementary structural information in studies of metabolism, fungal infection and symbiosis, molecular and genetic analysis.