Vessel contents of leaves after excision: a test of the Scholander assumption

When petioles of transpiring leaves are cut in the air, according to the 'Scholander assumption', the vessels cut open should fill with air as the water is drained away by tissue rehydration and/or continued transpiration. The distribution of air-filled vessels versus distance from the cut surface should match the distribution of lengths of 'open vessels', i.e. vessels cut open when the leaf is excised. A paint perfusion method was used to estimate the length distribution of open vessels and this was compared with the observed distribution of embolisms by the cryo-SEM method. In the cryo-SEM method, petioles are frozen in liquid nitrogen soon after the petiole is cut. The petioles are then cut at different distances from the original cut surface while frozen and examined in a cryo-SEM facility, where it is easy to distinguish vessels filled with air from those filled with ice. The Scholander assumption was also confirmed by a hydraulic method, which avoided possible freezing artefacts. In petioles of sunflower (Helianthus annuus L) the distribution of embolized vessels agrees with expectations. This is in contrast to a previous study on sunflower where cryo-SEM results did not agree with expectations. The reasons for this disagreement are suggested, but further study is required for a full elucidation.     

Cutting Frozen Sections on a Paraffin Microtome

Into a hole drilled in a block of dry ice, a metal microtome object disk is placed to cool. A drop of water is placed on the disk, and the specimen to be cut is fixed in place. By setting the dry ice in a well-insulated box, the specimen is thoroughly frozen. The disk is then clamped in the microtome, and chips of dry ice are wedged between the metal disk and the object clamp of the microtome. This ensures the continued cooling of the specimen while the tissue is being cut.     

Factors affecting ice nucleation in plant tissues

Factors affecting the ice nucleation temperature of plants and plant tissues were examined. The mass of a sample had a marked effect on ice nucleation temperature. Small tissue samples supercooled to −10°C and were not accurate predictors of the nucleation temperature of intact plants in either laboratory or field experiments. This effect was not unique to plant tissues and was observed in autoclaved and control soil samples. Ice nucleation temperatures of bean, corn, cotton, and soybean seedlings were influenced by the length of subzero exposure, presence of ice nucleation active bacteria, and leaf surface wetness. The number of factors influencing ice nucleation temperature suggested that predicting the freezing behavior of plants in the field will be complex.     

Cold requirement for maximal activity of the bacterial ice nucleation protein INAZ in transgenic plants

The bacterial ice nucleation gene inaZ confers production of ice nuclei when transferred into transgenic plants. Conditioning of the transformed plant tissue at temperatures near 0°C greatly increased the ice nucleation activity in plants, and maximum ice nucleation activity was achieved only after low-temperature conditioning for about 48 h. Although the transgenic plants contain similar amounts of inaZ mRNA at both normal and low temperatures, low temperatures are required for accumulation of INAZ protein. We propose that the stability of the INAZ protein and thus ice nucleation activity in the transgenic plants is enhanced by low-temperature conditioning.     

The water-filled versus air-filled status of vessels cut open in air: the 'Scholander assumption' revisited

When petioles of transpiring leaves are cut in the air, according to the ‘Scholander assumption’, the vessels cut open should fill with air as the water is drained away by continued transpiration. The distribution of air‐filled vessels versus distance from the cut surface should match the distribution of lengths of ‘open vessels’, i.e. vessels cut open when the leaf is excised. Three different methods were used to estimate the length distribution of open vessels and compared it to the observed distribution of embolisms by the cryo‐scanning electron microscope (SEM) method. In the cryo‐SEM method, petioles are frozen in liquid nitrogen soon after the petiole is cut. The petioles are then cut at different distances from the original cut surface while frozen and examined in a cryo‐SEM facility, where it is easy to distinguish vessels filled with air from those filled with ice. In petioles of Acer platanoides and Juglans regia, the distribution of embolized vessels agrees with expectations. This is in contrast to a previous study on sunflower where cryo‐SEM results did not agree with expectations. The reason for this disagreement requires further study for a full elucidation.     

Effect of wounding or infection by Phytophthora infestans on the contents of terpenoids in potato tubers

Inoculation of the potato cut surfaces with an incompatiblerace of Phytophthora infestans induced an accumulation of rishitin,but only a trace occurred when infected by a compatible race.When the tuber was cut, a large amount of steroid-glycoalkaloids(solanine) accumulated in the cut surface tissue, although onlya trace was found in intact tissue. Only a small amount of solanine,if any, was contained in the wound periderm tissue. Most ofthe solanine seemed to be distributed in tissue neighbouringthe newly formed meristematic tissue zone. Distribution of solanineas a function of distance from the cut surface was exponential.Infection of the cut surface by an incompatible race of Phytophthorainfestans reduced the accumulation of solanine. The higher theconcentration of zoospore used, the less the solanine content.It has been reported that the higher the concentration of zoosporesused for inoculation of the cut surface, the less the numberof renewed meristematic cells in the wound tissue. In experimentsusing fresh and aged tubers, a good correlation between thenumber of renewed meristematic cells and solanine content wasfound. The accumulation of solanine in the wound tissue andits reduction due to infection by an incompatible race may berelated to renewed meristematic cells formation and its reductioncaused by the infection. No drastic change in carotenoids or sterol contents was found2 days after cutting or inoculation, when the tubers were cut,or cut and then infected by the incompatible race. ¹ Studies on the phytoalexin (No. 10). (6) in References constitutes"Studies on the phytoalexin No. 9". ² Present adress: Plant Pathology Laboratory, Faculty of Agriculture,Nagoya University, Chikusa-ku, Nagoya, 464, Japan. (Received April 1, 1972; )     

Effect of Plant Species and Environmental Conditions on Ice Nucleation Activity of Pseudomonas syringae on Leaves

Selected plant species and environmental conditions were investigated for their influences on expression of ice nucleation activity by 15 Pseudomonas syringae strains grown on plants in constant-temperature growth chamber studies. Ice nucleation frequencies (INFs), the fraction of cells that expressed ice nucleation at −5 or −9°C, of individual strains varied greatly, both on plants and in culture. This suggests that the probability of frost injury, which is proportional to the number of ice nuclei on leaf surfaces, is strongly determined by the particular bacterial strains that are present on a leaf surface. The INFs of strains were generally higher when they were grown on plants than when they were grown in culture. In addition, INFs in culture did not correlate closely with INFs on plants, suggesting that frost injury prediction should be based on INF measurements of cells grown on plants rather than in culture. The relative INFs of individual strains varied with plant host and environment. However, none of seven plant species tested optimized the INFs of all 15 strains. Similarly, incubation for 48 h at near 100% relative humidity with short photoperiods did not always decrease the INF when compared with a 72 h, 40% relative humidity, long-photoperiod incubation. Pathogenic strains on susceptible hosts were not associated with higher or lower INFs relative to their INFs on nonsusceptible plant species. The ice nucleation activity of individual bacterial strains on plants therefore appears to be controlled by complex and interacting factors such as strain genotype, environment, and host plant species.     

用于研究植物质外体空间三维结构的树脂铸型技术

本文介绍了用于研究植物体质外体空间三维结构的树脂铸型技术。植物包含许多重要的质外体空间,如木质部管状分子的腔隙、与气孔相连的叶肉细胞间的通气系统、分泌腔等等。这种空间的三维结构可借助于扫描电子显微镜(SEM)研究。但问题是,用SEM直接观察不到一个组织或细胞切面内部的图像,因此,不能观察它们的全貌。通过运用树脂铸型技术,可以获得完整的组织或腔隙内部空间的铸型。反映管壁结构的各种形象被印在铸型的表面上,在SEM下可对质外体空间进行详细研究。树脂铸型技术在结构植物学的研究上有重要的意义。     

用于研究植物质外体空间三维结构的树脂铸型技术

本文介绍了用于研究植物体质外体空间三维结构的树脂铸型技术。植物包含许多重要的质外体空间,如木质部管状分子的腔隙、与气孔相连的叶肉细胞间的通气系统、分泌腔等等。这种空间的三维结构可借助于扫描电子显微镜（ＳＥＭ）研究。但问题是,用ＳＥＭ直接观察不到一个组织或细胞切面内部的图像,因此,不能观察它们的全貌。通过运用树脂铸型技术,可以获得完整的组织或腔隙内部空间的铸型。反映管壁结构的各种形象被印在铸型的表面     

Gibberellin produced in the cotyledon is required for cell division during tissue reunion in the cortex of cut cucumber and tomato hypocotyls

Cucumber (Cucumis sativus) hypocotyls were cut to one-half of their diameter transversely, and morphological and histochemical analyses of the process of tissue reunion in the cortex were performed. Cell division in the cortex commenced 3 d after cutting, and the cortex was nearly fully united within 7 d. 4',6-Diamidino-2-phenylindole staining and 5-bromo-2'-deoxyuridine labeling experiments indicate that nDNA synthesis occurred during this process. In addition, specific accumulation of pectic substances was observed in the cell wall of attached cells in the reunion region of the cortex. Cell division during tissue reunion was strongly inhibited when the cotyledon was removed. This inhibition was reversed by applying gibberellin (GA, 10(-4) M GA3) to the apical tip of the cotyledon-less plant. Supporting this observation, cell division in the cortex was inhibited by treatment of the cotyledon with 10(-4) M uniconazole-P (an inhibitor of GA biosynthesis), and this inhibition was also reversed by simultaneous application of GA. In contrast to the essential role of cotyledon, normal tissue reunion in cut hypocotyls was still observed when the shoot apex was removed. The requirement of GA for tissue reunion in cut hypocotyls was also evident in the GA-deficient gib-1 mutant of tomato (Lycopersicon esculentum). Our results suggest that GA, possibly produced in cotyledons, is essential for cell division in reuniting cortex of cut hypocotyls.     

An Application of the Frozen Sectioning Technic for Cutting Serial Sections Thru the Brain

The availability of CO₂ ice makes it practical to cut large blocks of cerebral tissue by the freezing method. If the tissue is first treated with 20-30% ethyl alcohol for sufficient time to secure uniform penetration of the alcohol (about 24 hours), formation of hard ice crystals can be controlled and serial sections 25-100 μ thick can be cut with negligible loss. The alcohol can be added to the fixative used for perfusion, or it can be added at any time later in the firing process, or after fixation is completed. The sections are cemented to the slide and groups of slides are manipulated thru staining processes in glass trays. Ordinary cell and fiber stains give satisfactory results. The method is particularly useful for certain neurophysiological purposes such as defining the location of electrode tracks and lesions and certain types of retrogrades. The Prussian blue test for electrolytically deposited iron can be conveniently applied in conjunction with other stains, to determine the point at which a given action potential response was observed, if steel electrodes are used.     

Cryo-ultramicrotomy after coating of the cut surface of the tissue block with carbon and metals before sectioning.

Working under improvised conditions it is shown that coating of the cut surface of a frozen tissue block with metal and carbon improves, when applied prior to each cutting, the sectioning properties and the handling of the subsequent ultrathin cryosection.     

ELECTRON MICROSCOPY OF STRUCTURAL DETAIL IN FROZEN BIOLOGICAL SPECIMENS

A procedure is described whereby preshadowed replicas can be obtained from frozen biological specimens which have been cut and then etched by sublimation of the ice from their surfaces. Electron micrographs showing details of the internal structure of plant virus crystals are presented to demonstrate the values of the procedure. Crystals of purified tobacco ringspot virus and squash mosaic virus and some portions of turnip yellow mosaic virus crystals have been shown to exhibit hexagonal packing. Sections through in situ crystals of tobacco mosaic virus show the rods to be parallel within each layer and arranged in a square net as viewed end on. Individual rods in each layer of the latter measure 300 mµ in length and are somewhat tilted with respect to the rods of adjacent layers. This results in the formation of a herring-bone appearance when a crystal is cut perpendicular to its hexagonal face. It is suggested that the procedure outlined here might well serve to supplement other procedures for the preparation of many cytological specimens for electron microscopy.     

Expression of antifreeze proteins in transgenic plants

The quality of frozen fruits and vegetables can be compromised by the damaging effects of ice crystal growth within the frozen tissue. Antifreeze proteins in the blood of some polar fishes have been shown to inhibit ice recrystallization at low concentrations. In order to determine whether expression of genes of this type confers improved freezing properties to plant tissue, we have produced transgenic tobacco and tomato plants which express genes encoding antifreeze proteins. Theafa3 antifreeze gene was expressed at high steady-state mRNA levels in leaves from transformed plants, but we did not detect inhibition of ice recrystallization in tissue extracts. However, both mRNA and fusion proteins were detectable in transgenic tomato tissue containing a chimeric gene encoding a fusion protein between truncated staphylococcal protein A and antifreeze protein. Furthermore, ice recrystallization inhibition was detected in this transgenic tissue.     

Cryo-ultramicrotomy after coating of the cut surface of the tissue block with carbon and metals before sectioning

Summary Working under improvised conditions it is shown that coating of the cut surface of a frozen tissue block with metal and carbon improves, when applied prior to each cutting, the sectioning properties and the handling of the subsequent ultrathin cryosection.     

Evaluating intramural virtual electrodes in the myocardial wedge preparation: simulations of experimental conditions

While defibrillation is the only means for prevention of sudden cardiac death, key aspects of the process, such as the intramural virtual electrodes (VEs), remain controversial. Experimental studies had attempted to assess intramural VEs by using wedge preparations and recording activity from the cut surface; however, applicability of this approach remains unclear. These studies found, surprisingly, that for strong shocks, the entire cut surface was negatively polarized, regardless of boundary conditions. The goal of this study is to examine, by means of bidomain simulations, whether VEs on the cut surface represent a good approximation to VEs in depth of the intact wall. Furthermore, we aim to explore mechanisms that could give rise to negative polarization on the cut surface. A model of wedge preparation was used, in which fiber orientation could be changed, and where the cut surface was subjected to permeable and impermeable boundary conditions. Small-scale mechanisms for polarization were also considered. To determine whether any distortions in the recorded VEs arise from averaging during optical mapping, a model of fluorescent recording was employed. The results indicate that, when an applied field is spatially uniform and impermeable boundary conditions are enforced, regardless of the fiber orientation VEs on the cut surface faithfully represent those intramurally, provided tissue properties are not altered by dissection. Results also demonstrate that VEs are sensitive to the conductive layer thickness above the cut surface. Finally, averaging during fluorescent recordings results in large negative VEs on the cut surface, but these do not arise from small-scale heterogeneities.     

Ice adhesions in relation to freeze stress

In freezing, competitive interaction between ice and hydrophilic plant substances causes an energy of adhesion to develop through the interstitial liquid. The thermodynamic basis for the adhesion energy is discussed, with estimates of the energies involved. In this research, effects of adhesion energy were observed microscopically in conjunction with energies of crystallization and frost desiccation. The complex character of ice in intact crown tissue of winter barley (Hordeum vulgare L.) and the problems of sectioning frozen tissue without producing artifacts led to an alternative study of single barley cells in a mesh of ice and cell wall polymers. Adhesions between ice, cell wall polymers, and the plasmalemma form a complexly interacting system in which the pattern of crystallization is a major factor in determination of stress and injury.     

Errors in measuring water potentials of small samples resulting from water adsorption by thermocouple psychrometer chambers

The adsorption of water by thermocouple psychrometer assemblies is known to cause errors in the determination of water potential. Experiments were conducted to evaluate the effect of sample size and psychrometer chamber volume on measured water potentials of leaf discs, leaf segments, and sodium chloride solutions. Reasonable agreement was found between soybean (Glycine max L. Merr.) leaf water potentials measured on 5-millimeter radius leaf discs and large leaf segments. Results indicated that while errors due to adsorption may be significant when using small volumes of tissue, if sufficient tissue is used the errors are negligible. Because of the relationship between water potential and volume in plant tissue, the errors due to adsorption were larger with turgid tissue. Large psychrometers which were sealed into the sample chamber with latex tubing appeared to adsorb more water than those sealed with flexible plastic tubing. Estimates are provided of the amounts of water adsorbed by two different psychrometer assemblies and the amount of tissue sufficient for accurate measurements of leaf water potential with these assemblies. It is also demonstrated that water adsorption problems may have generated low water potential values which in prior studies have been attributed to large cut surface area to volume ratios.     

Freezing of nonwoody plant tissues. III. Videotape micrography and the correlation between individual cellular freezing events and temperature changes in the surrounding tissue

A new technique was developed for the observation and recording on videotape of thermal and microscopic changes that occur simultaneously during the freezing of cucumber tissue. The freezing process occurs in two steps. Nucleation and growth of ice crystals in the continuous extracellular liquid phase is followed by nucleation and growth of ice crystals in individual supercooled cells. The freezing of cells in rapid succession causes the average temperature to remain constant for a short time. This mechanism explains the second freezing plateau found in most plant tissue freezing curves.     

Hydrogen peroxide and lignification

The production of hydrogen peroxide in plant tissue is demonstrated quickly with a simple histochemical test. The test solution, 50 mM potassium iodide in a 4% (w/v) potato starch suspension, is applied to the cut surface of the tissue to be tested. Hydrogen peroxide oxidizes iodide ions to iodine; the iodine is complexed by the starch to form a blue-purple color. This test detects hydrogen peroxide production in cells undergoing lignification, i.e. tracheary elements and phloem fibers, and in some epidermal cells. In addition there is a rapid production of hydrogen peroxide in crushed cells. The test is negative under (i) anaerobic conditions and (ii) in the presence of catalase.