Humidity gradients in the air spaces of leaves.

Stomata are orifices that connect the drier atmosphere with the interconnected network of more humid air spaces that surround the cells within a leaf. Accurate values of the humidities inside the substomatal cavity, wi, and in the air, wa, are needed to estimate stomatal conductance and the CO2 concentration in the internal air spaces of leaves. Both are vital factors in the understanding of plant physiology and climate, ecological and crop systems. However, there is no easy way to measure wi directly. Out of necessity, wi has been taken as the saturation water vapour concentration at leaf temperature, wsat, and applied to the whole leaf intercellular air spaces. We explored the occurrence of unsaturation by examining gas exchange of leaves exposed to various magnitudes of wsat - wa, or Δw, using a double-sided, clamp-on chamber, and estimated degrees of unsaturation from the gradient of CO2 across the leaf that was required to sustain the rate of CO2 assimilation through the upper surface. The relative humidity in the substomatal cavities dropped to about 97% under mild Δw and as dry as around 80% when Δw was large. Measurements of the diffusion of noble gases across the leaf indicated that there were still regions of near 100% humidity distal from the stomatal pores. We suggest that as Δw increases, the saturation edge retreats into the intercellular air spaces, accompanied by the progressive closure of mesophyll aquaporins to maintain the cytosolic water potential.

The amino acid distribution in rachis xylem sap and phloem exudate of Vitis vinifera 'Cabernet Sauvignon' bunches.

Amino acids are essential to grape berry and seed development and they are transferred to the reproductive structures through the phloem and xylem from various locations within the plant. The diurnal and seasonal dynamics of xylem and phloem amino acid composition in the leaf petiole and bunch rachis of field-grown Cabernet Sauvignon are described to better understand the critical periods for amino acid import into the berry. Xylem sap was extracted by the centrifugation of excised leaf petioles and rachises, while phloem exudate was collected by immersing these structures in an ethylenediaminetetraacetic acid (EDTA) buffer. Glutamine and glutamic acid were the predominant amino acids in the xylem sap of both grapevine rachises and petioles, while arginine and glycine were the principal amino acids of the phloem exudate. The amino acid concentrations within the xylem sap and phloem exudate derived from these structures were greatest during anthesis and fruit set, and a second peak occurred within the rachis phloem at the onset of ripening. The concentrations of the amino acids within the phloem and xylem sap of the rachis were highest just prior to or after midnight while the flow of sugar through the rachis phloem was greatest during the early afternoon. Sugar exudation rates from the rachis was greater than that of the petiole phloem between anthesis and berry maturity. In summary, amino acid and sugar delivery through the vasculature to grape berries fluctuates over the course of the day as well as through the season and is not necessarily related to levels near the source.

Development and persistence of sandsheaths of Lyginia barbata (Restionaceae): relation to root structural development and longevity.

BACKGROUND AND AIMS: Strongly coherent sandsheaths that envelop perennial roots of many monocotyledonous species of arid environments have been described for over a century. This study, for the first time, details the roles played by the structural development of the subtending roots in the formation and persistence of the sheaths. METHODS: The structural development of root tissues associated with persistent sandsheaths was studied in Lyginia barbata, native to the Western Australian sand plains. Cryo-scanning electron microscopy CSEM, optical microscopy and specific staining methods were applied to fresh, field material. The role of root hairs was clarified by monitoring sheath development in roots separated from the sand profile by fine mesh. KEY RESULTS AND CONCLUSIONS: The formation of the sheaths depends entirely on the numerous living root hairs which extend into the sand and track closely around individual grains enmeshing, by approx. 12 cm from the root tip, a volume of sand more than 14 times that of the subtending root. The longevity of the perennial sheaths depends on the subsequent development of the root hairs and of the epidermis and cortex. Before dying, the root hairs develop cellulosic walls approx. 3 µm thick, incrusted with ferulic acid and lignin, which persist for the life of the sheath. The dead hairs remain in place fused to a persistent platform of sclerified epidermis and outer cortex. The mature cortex comprises this platform, a wide, sclerified inner rim and a lysigenous central region - all dead tissue. We propose that the sandsheath/root hair/epidermis/cortex complex is a structural unit facilitating water and nutrient uptake while the tissues are alive, recycling scarce phosphorus during senescence, and forming, when dead, a persistent essential structure for maintenance of a functional stele in the perennial Lyginia roots.

Seasonal water relations of Lyginia barbata (Southern rush) in relation to root xylem development and summer dormancy of root apices.

*Periods of dormancy in shallow roots allow perennial monocotyledons to establish deep root systems, but we know little about patterns of xylem maturation, water-transport capacities and associated economies in water use of growing and dormant roots. *Xylem development, anatomy, conductance and in situ cellular [K] and [Cl] were investigated in roots of field-grown Lyginia barbata (Restionaceae) in Mediterranean southwestern Australia. Parallel studies of gas exchange, culm relative water loss and soil water content were conducted. *Stomatal conductance and photosynthesis decreased during summer drought as soil profiles dried, but rates recovered when dormant roots became active with the onset of wetter conditions. Anatomical studies identified sites of close juxtaposition of phloem and xylem in dormant and growing roots. Ion data and dye tracing showed mature late metaxylem of growing roots was located >or= 100 mm from the tip, but at only

Summer dormancy and winter growth: root survival strategy in a perennial monocotyledon.

Here, we tested the alternation of root summer dormancy and winter growth as a critical survival strategy for a long-lived monocotyledon (Restionaceae) adapted to harsh seasonal extremes of Mediterranean southwest Western Australia. Measurements of growth and the results of comparative studies of the physiology, water content, metabolites, osmotic adjustments, and proteomics of the dormant and growing perennial roots of Lyginia barbata (Restionaceae) were assessed in field-grown plants. Formation of dormant roots occurred before the onset of summer extremes. They resumed growth (average 2.3 mm d(-1)) the following winter to eventually reach depths of 2-4 m. Compared with winter-growing roots, summer dormant roots had decreased respiration and protein concentration and c. 70% water content, sustained by sand-sheaths, osmotic adjustment and presumably hydraulic redistribution. Concentrations of compatible solutes (e.g. sucrose and proline) were significantly greater during dormancy, presumably mitigating the effects of heat and drought. Fifteen root proteins showed differential abundance and were correlated with either winter growth or summer dormancy. None matched currently available libraries. The specific features of the root dormancy strategy of L. barbata revealed in this study are likely to be important to understanding similar behaviour in roots of many long-lived monocotyledons, including overwintering and oversummering crop species.

Invited Review: 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.

Frost tolerance and ice formation in Pinus radiata needles: ice management by the endodermis and transfusion tissues.

Conifers are among the most frost tolerant tree species. Cryo-scanning electron microscopy (cryo-SEM) was used to visualise ice formation in pine needles to better understand how conifer leaves manage extracellular ice. Acclimated and unacclimated needles of Pinus radiata (D.Don) were subjected to freezing treatments (at a rate of 2°C h-1), tested for electrolyte leakage and sampled for cryo-SEM analysis. Half maximal electrolyte leakage occurred at -4 and -12°C for unacclimated and acclimated needles, respectively. Ice nucleation occurred at similar temperatures (-3°C) in both acclimated and unacclimated pine needles, indicating that frost tolerance did not increase supercooling. During freezing and thawing, the tissues outside and inside the endodermis shrank and swelled independently, with little or no transfer of water between the two regions. During freezing, mesophyll cells shrank, exhibiting cytorrhysis, and extracellular ice accumulated in gas spaces of the mesophyll tissue. Mesophyll cells from acclimated needles recovered their structure after thawing, and unacclimated mesophyll showed significant damage. In the vascular cylinder, ice accumulated in transfusion tracheids which expanded to occupy areas made vacant by shrinkage of transfusion parenchyma, Strasburger cells and the endodermis. This behaviour was reversible in acclimated tissue, and may play an important role in the management of ice during freeze/thaw events.

Electrolyte distribution and yolk sac morphology in frozen hydrated equine conceptuses during the second week of pregnancy.

To investigate how equine conceptuses expand rapidly despite the hypo-osmolality of their yolk sac fluid, 18 conceptuses, aged 8-12 days and 0.8-10.0 mm in diameter, were examined by cryoscanning electron microscopy and energy dispersive X-ray microanalysis to determine the distribution of Na, Cl and K in their fluids. No osmotic gradient was found between central and peripheral yolk sac fluid. In conceptuses > or = 6 mm in diameter, the concentrations of both Na and K in the subtrophectodermal compartments were higher than those determined previously in uterine fluid, supporting the concept of osmotic intake of fluid from the uterine environment as far as the compartments. However, electrolyte concentrations in the compartments consistently exceeded those found in the yolk sac, making it likely that 'uphill' water transport, rather than a purely osmotic uptake, is involved in yolk sac fluid accumulation. We also speculate that capsule formation could actively contribute to conceptus expansion and thereby to fluid intake.

Hypothesis. Air embolisms exsolving in the transpiration water - the effect of constrictions in the xylem pipes.

When water flows through a constriction, air can come out of solution (i.e. it can exsolve). This phenomenon is manifested in the transpiration stream of plants. Observations of gas in functioning xylem prompted a hypothesis predicting the daily balance between air and water in wood: a sudden fall in water content at sunrise, followed by an increase in water content during the day. An extended record by time domain reflectometry of volumetric water content (VWC) every 2 h throughout a summer shows the detailed pattern of change of VWC during 25 individual days, giving good agreement with the hypothesis. This hypothesis has wide-ranging consequences for experiments using cut plant parts. Perfusing aqueous solutions through excised xylem also can exsolve air from the water, causing declines in flow. The location of such air was investigated in cryo-fixed perfused vine stems by cryo-scanning electron microscopy. Bubbles formed at residual walls of perforation plates in small vessels, and filled many large vessels. The input surface is revealed as a major source of exsolved air. Precautions to reduce this effect are outlined and discussed.

Branch roots of young maize seedlings, their production, growth, and phloem supply from the primary root.

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.

A second pathway for gas out of the pressure chamber--what is being squeezed?

We report a qualitative description of the flows of gas that occur through a leaf when its balance pressure is measured in the pressure chamber. There are two distinct pathways: (a) a bulk flow of gas through the intercellular air spaces, and (b) a diffusion-driven pathway where gas is dissolved into solution under high pressure and comes out of solution at the liquid/atmosphere surface of the cut end where the pressure is atmospheric. The intercellular space flow is well known. It is argued that this flow shows to a reasonable approximation, that the externally supplied gas is squeezing the non-gaseous part of the leaf, and the outer boundary of the non-gaseous material is the boundary of the system that is being manipulated. The second pathway, the diffusion-driven flow, has not (we believe) been described before, and is analogous to a diver getting the bends. The diffusion-based flow demonstrates that gas spaces can and do form inside the outer boundary of the non-gaseous part of the leaf when a balance pressure is measured. These interior gas spaces alter the value recorded for the balance pressure, and complicate any interpretation of what this measurement tells us about the water status of the plant. A hypothesis is proposed that the diffusion-based flow from the xylem comes from vessels that are embolized, and that percentage embolisms might be measured by the proportion of vessels showing the diffusion-driven flow.

A mechanical interpretation of pressure chamber measurements--what does the strength of the squeeze tell us?

Argument still continues about what properties of a plant organ the pressure chamber measures. A mechanical (as opposed to a thermodynamic) analysis is made of the system squeezed by the pressurized gas, the non-gaseous part of the leaf. The boundary of the system is defined so that it remains at constant mass, and constant density is assumed, during the squeeze. This is equivalent to assuming constant volume. On those assumptions, it is shown that the liquid is brought to the cut surface by a change of shape of the system. Generic mechanical principles are then used to deduce a priori, a quantitative interpretation of the balance pressure. The formal mechanical interpretation involves two variables, the interfacial tension and the change in surface area, which cannot currently be measured. Instead of these, we used two related variables which can be measured, the mass fraction of water in the leaf (Q) and the maximum mass fraction of water at full saturation (Qx) to deduce an approximate mechanical interpretation. When Q is close to Qx, we deduced that the balance pressure (Pb) required for the shape change should be approximately proportional to the reduction in mass in changing from Qx to Q, a variable called the relative water loss (RWL). The constant of proportionality (kappa) is a basic characteristic of the type of leaf used, and the final relation, Pb=kappa (RWL) is called Relation A. We then deduce that the constant kappa should be an approximately linear function of Qx. The linear function is defined by limiting values, so that when Qx is 1, kappa is predicted to be 0 bar, and at the other extreme, when Qx is 0, kappa is predicted to be in the range 500-1000 bar. This is called Relation B. Experiments with 32 leaves from 10 species are used to test the mechanical interpretation. The results showed that Relation A was a reasonable approximation for most of the tested leaves. The data for 10 species, were used to estimate Relation B, confirming that as Qx approached 1, kappa did approach 0 bar as predicted, and that as Qx approached 0, kappa approached approximately 750 bar, consistent with the a priori prediction of 500-1000 bar. The relations were also successfully tested using independent published data. An estimate of Qx is shown to be of considerable practical value in (a) converting Pb to water status and vice versa; (b) characterizing leaf morphology and composition; and (c) rationalizing quantitatively the functional classes of xerophytes, mesophytes and hygrophytes. The assumption of constant density inside the outer boundary of the non-gaseous material cannot be guaranteed, and when this is violated, our (or any other) interpretation of Pb is unreliable. Investigation of the conditions under which this assumption is invalid should be a high priority.

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.