Freeze avoidance: a dehydrating moss gathers no ice.

Using cryo-SEM with EDX fundamental structural and mechanical properties of the moss Ceratodon purpureus (Hedw.) Brid. were studied in relation to tolerance of freezing temperatures. In contrast to more complex plants, no ice accumulated within the moss during the freezing event. External ice induced desiccation with the response being a function of cell type; water-filled hydroid cells cavitated and were embolized at -4 °C while parenchyma cells of the inner cortex exhibited cytorrhysis, decreasing to ∼ 20% of their original volume at a nadir temperature of -20 °C. Chlorophyll fluorescence showed that these winter acclimated mosses displayed no evidence of damage after thawing from -20 °C while GCMS showed that sugar concentrations were not sufficient to confer this level of freezing tolerance. In addition, differential scanning calorimetry showed internal ice nucleation occurred in hydrated moss at ∼-12 °C while desiccated moss showed no evidence of freezing with lowering of nadir temperature to -20 °C. Therefore the rapid dehydration of the moss provides an elegantly simple solution to the problem of freezing; remove that which freezes.

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.

Cell-specific localization of Na+ in roots of durum wheat and possible control points for salt exclusion.

Sodium exclusion from leaves is an important mechanism for salt tolerance in durum wheat. To characterize possible control points for Na(+) exclusion, quantitative cryo-analytical scanning electron microscopy was used to determine cell-specific ion profiles across roots of two durum wheat genotypes with contrasting rates of Na(+) transport from root to shoot grown in 50 mm NaCl. The Na(+) concentration in Line 149 (low transport genotype) declined across the cortex, being highest in the epidermal and sub-epidermal cells (48 mm) and lowest in the inner cortical cells (22 mm). Na(+) was high in the pericycle (85 mm) and low in the xylem parenchyma (34 mm). The Na(+) profile in Tamaroi (high transport genotype) had a similar trend but with a high concentration (130 mm) in the xylem parenchyma. The K(+) profiles were generally inverse to those of Na(+). Chloride was only detected in the epidermis. These data suggest that the epidermal and cortical cells removed most of the Na(+) and Cl(-) from the transpiration stream before it reached the endodermis, and that the endodermis is not the control point for salt uptake by the plant. The pericycle as well as the xylem parenchyma may be important in the control of net Na(+) loading of the xylem.

Distribution of glucosinolates and sulphur-rich cells in roots of field-grown canola (Brassica napus).

To investigate the role played by the distribution pattern of glucosinolates (GSLs) in root systems in the release of biocides to the rhizosphere, GSLs have been localized, for the first time, to specific regions and cells in field-grown roots. GSL concentrations in separated tissues of canola (Brassica napus) were determined by chemical analysis, and cell-specific concentrations by extrapolation from sulphur concentrations obtained by quantitative cryo-analytical scanning electron microscopy (SEM). In roots with secondary growth, GSL concentrations in the outer secondary tissues were up to 5x those of the inner core. The highest GSL concentrations (from sulphur measurements) were in two cell layers just under the outermost periderm layer, with up to 100x published concentrations for whole roots. Primary tissues had negligible GSL. Release and renewal of the peripheral GSLs is probably a normal developmental process as secondary thickening continues and surface cells senesce, accounting for published observations that intact roots release GSLs and their biocide hydrolosates to the rhizosphere. Absence of myrosin idioblasts close to the root surface suggests that GSLs released developmentally are hydrolysed by myrosinase in the rhizosphere, ensuring a continuous localized source of biotoxic hydrolysates which can deter soil-borne pests, and influence microbial populations associated with long-lived components of the root system.

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.

Relative amounts of soluble and insoluble forms of phosphorus and other elements in intraradical hyphae and arbuscules of arbuscular mycorrhizas.

Transport of phosphorus (P) into host plants and its release to root cells is an important function of arbuscular mycorrhizal fungi (AMF). However, relatively little is known about the forms and water solubilities of P compounds in specific locations in the intraradical fungal structures. We determined concentrations and solubility of P components in these structures in white clover (Trifolium repens L.). Plants were grown in the field (colonised by indigenous AMF) or in the glasshouse (inoculated with Glomus intraradices). Mycorrhizas were cryo-fixed in liquid nitrogen immediately (control) or after treatments designed to destroy cell membranes and extract solubles. Thirty to 70% of total P in hyphae and 100% in arbuscules was not extracted. The unextracted proportion of P was higher in the inoculated plants suggesting an environmental effect. It is proposed that the large component of non-extractable P in the arbuscules is involved in the tight regulation of inorganic P release to the host cells. In control roots magnesium, potassium and P were present in hyphae in molar ratios 1 : 2 : 4, further evidence that this relationship may be universal for AMF, and that other P-balancing cations are present but undetectable by the analytical technique.

Frozen in time: a new method using cryo-scanning electron microscopy to visualize root-fungal interactions.

A new method of sample preparation for cryo-scanning electron microscopy was used to visualize internal infection of wheat (Triticum aestivum) roots by the pathogenic fungus Rhizoctonia solani AG-8. The new method retained fungal hyphae and root cells in situ in disintegrating root tissues, thus avoiding the distortions that can be introduced by conventional preparation by chemical fixation, dehydration and embedding. Infected roots frozen in liquid nitrogen were cryo-planed and etched (sublimed) at -80 degrees C for a critical length of time (up to 9 min) in the microscope column to reveal plant and fungal structures in three dimensions. Root and fungal structures were well preserved irrespective of infection severity. Root and hyphal cell walls were clearly seen and hyphal architecture within and between root cells was preserved. This rapid method permits three-dimensional in situ visualization of fungal invasion within roots and has broad application for examination of diseases caused by other necrotrophic fungi.

Longitudinal and transverse profiles of K+ and Cl- concentration in 'low-' and 'high-salt' barley roots.

The concentrations of K+ and Cl- in meristematic and differentiated cells of barley (Hordeum Vulgare. L. ev. California Mariout) roots were determined by means of quantitative X-ray microanalysis. In roots of seedlings grown in 0.5 mM CaSO4 ('low-salt' roots), the mean cytoplasmic K+ concentration of meristematic cells was 194 mM, while the average vacuolar concentrations at 10, 50 and 100 mm from the root tip were 62,25 and 22 mM, respectively. Such marked differences in vacuolar K+ concentration of differentiated cells were not found in roots of seedlings grown in full strength Johnson solution ('high-salt' roots). Thus the maintenance of K+ at a relatively high concentration in the cytoplasm of meristematic cells of 'low-salt' roots appears to be a priority requirement. The mean Cl- concentration in the cytoplasm of meristematic cells of 'low-salt' roots was 4.6 mM, and the vacuolar Cl- concentration declined towards the root base. These measured concentrations are compatible with previous data, indicating that X-ray microanalysis of fully-hydrated samples, used together with colloidal graphite mixtures as standards, provides a reliable means of measuring ion concentrations at the subcellular level.