Production and characterization of a monoclonal antibody specific for the polyamine spermidine and its application in ELISA.

To enable the immunoassay of spermidine in tissue extracts, a monoclonal antibody, JAC-1, specific for free spermidine was raised, using a spermidine-ovalbumin conjugate as immunogen. This antibody was characterized, and found to possess a high degree of specificity for spermidine, with only 4% molar cross-reactivity with spermine. A competitive ELISA using this antibody was developed. This assay is able to detect as little as 10 pmol spermidine extracted from small, circa 10 mg, tissue samples. The assay is unaffected by the presence of up to a 4-fold molar ratio of spermine, whereas the corresponding spermine competitive ELISA is adversely affected by a 0.5-fold molar ratio of spermidine. The spermidine competitive ELISA using JAC-1 was used to estimate the spermidine content of several plant and animal tissue extracts and the results compared with HPLC data. This novel assay is a useful development in the assay of polyamines since, compared to routinely employed HPLC methods, it offers increased convenience, rapidity, and capacity for large numbers of samples.

The use of soft x rays to study the ultrastructure of living biological material.

Imaging biological specimens with soft x rays offers several potential benefits over electron microscopy, and these are briefly reviewed. The disadvantages, most notably radiation-induced structural changes, have been investigated and images of irradiated algal cells (Chlorella) are presented. In soft x-ray contact microscopy the image is recorded rapidly to avoid both natural and radiation-induced movement and this technique has been used to study the ultrastructural effects of electron microscopy fixatives. In the epidermal hairs of tomato plants there are numerous strands of cytoplasm which, by light microscopy, appear to traverse the vacuole but are rarely seen by electron microscopy. However, by soft x-ray contact microscopy these strands and the organelles within them can be successfully imaged. Moreover, examination by soft x-ray contact microscopy of the cytoplasm in a fixed material shows that these strands are not present in chemically fixed material. This paper also reports the use of soft x-ray contact microscopy to examine the abscission cells found within the protonema of a moss (Bryum tenuisetum) and compares the images to those obtained by light and electron microscopy.

Assay of the polyamine spermine by a monoclonal antibody-based ELISA.

Spermine-specific monoclonal antibodies were prepared by immunising mice with protein-spermine conjugates. A resulting monoclonal antibody, IAG-1, exhibited both high affinity for spermine (binding constant 5.5 x 10(7) M-1) and high specificity, cross-reacting only weakly with spermidine. Using this antibody a competitive ELISA was developed with a detection limit of 1 pmol. The assay has been used to quantify spermine content of plant tissues, without derivatization, and producing within 6 h of collection, values for the spermine content which are similar to published data obtained by HPLC.

Soft X-ray contact microscopy of biological materials.

Light microscopy (LM) enables biological specimens to be examined without fixation or dehydration but the resolution is insufficient for studies of cell ultrastructure. Electron microscopy (EM) improves the resolution, but requires the specimen to be fixed or frozen, which may cause alterations in cell structure. Using soft X-rays to image specimens improves the resolution, relative to LM, and avoids tissue pretreatment. Staining is not required since within the 'water window' (2.3-4.4 nm), carbon absorbs more strongly than oxygen. The lower attenuation of soft X-rays, relative to electrons, by biological material allows specimens several microns thick to be examined. Several sources for generating water-window X-rays are briefly described and examples of images obtained with each are presented. The specimens imaged include both plant and animal material either in the fixed or natural state. Of the different systems currently used to collect images only contact imaging is considered in detail. By placing the specimen against photosensitive resist, which acts as the image recording medium, an absorption map of the specimen is produced. This latent image is then chemically developed and, after coating, the resist is examined by scanning EM, or, if a replica is produced, by transmission EM. Using laser-produced plasmas such images are produced within a very short exposure time, typically 1-10 nsec, thus avoiding any radiation-induced damage to the specimen which other X-ray imaging techniques may cause.

Soft X-Ray Contact Microscopy of Biological Specimens: Aluminum-Coated Silicon Nitride Windows as XUV Filters.

The optimum wavelengths for soft x-ray contact microscopy of biological specimens are between 2.2 and 4.4 nm as, relative to water, this gives maximum contrast between carbon- and oxygen-containing constituents. Irradiation outside of this so-called "water window" interferes with either the resolution and/or the contrast that can be obtained. The previous belief that silicon nitride windows act as an effective filter for the longer wavelength, XUV radiation generated from laser-produced plasmas is shown to be suspect. However, in this study the use of aluminum-coated windows, which effectively exclude these longer wavelengths, still permits successful imaging of biological specimens. The added strength imparted to coated windows also ensures their survival during the imaging process, thereby demonstrating that the tissues can remain hydrated at the time that the image is formed in the photosensitive resist material used in this technique.

Improved resolution of soft x-ray contact images using laser-produced plasmas.

Soft x-ray contact images have been obtained of diatom skeletons using laser-generated plasmas of low energy (2 J IR). These images have a resolution of less than 60 nm, which is superior to that reported previously for biological specimens, using this technique.

Studies on flower longevity in Digitalis : The role of ethylene in corolla abscission.

The flowers of Digitalis purpurea respond to pollination by rapid corolla abscission without any loss of corolla turgor, nor any significant loss of corolla constituents, relative to the corollas of unpollinated flowers of a similar age. The corollas of unpollinated flowers too eventually abscise, 6 d after the stigma opens, however, they do so with only a minimal loss of fresh weight or corolla constituents. Pollination causes an increase in ethylene production detectable within 1 h. Increased ethylene production occurs initially only from the upper portion of the style, later from the lower portion, and lastly, between 23 and 48 h after pollination, from the ovary plus calyx. The pollination response can be induced by exogenous ethylene, the degree of weakening of the corolla abscission zone being dependent upon the concentration and duration of the exposure period and on the stage of flower development. The regulation of ethylene biosynthesis and its involvement in the control of pollination-induced corolla abscission are discussed.

Pollen-stigma interaction in Brassica oleracea: the role of stigmatic proteins in pollen grain adhesion.

The adhesion of pollen grains to the stigmas of Brassica oleracea was assayed after treatment of the stigmas wiuth protease and/or cycloheximide. Treatment with protease alone adversely affected pollen grain adhesion. However, the adhesive properties of the stigma recovered fully if the stigmas were not pollinated until 2 h after treatment. Immersion of the stigmas in cycloheximide after protease treatment prevented any recoveryt of the stigmas' adhesive properties. Cycloheximide treatment alone prevented pollen grain adhesion when pollination occurred later than 1--2 h after treatment but did not affect pollen grain adhesion if pollination occurred immediately after treatment. These results indicated not only that the surface-held proteins of the stigma are involved in pollen grain adhesion, but also that their turnover rate is rapid. Isoelectric focusing of extracts derived from stigmas after protease and cycloheximide treatment showed a marked decrease in staining intensity of 3 protein bands, one of which, a glycoprotein, is known to be present only when the self-incompativility system is fully functional. These observations suggest a specificity of adhesion between higher plant cells in the presence of the cell wall.

Pollen stigma interactions in Brassica oleracea.

Recent studies on the mechanism of self-incompatibility in Brassica indicate the location, nature and mode of action of the molecules involved. Characteristics of the pollen surface and the stigma surface are described in detail, together with new information pertaining to the recognition molecules located therein. A sequence of events is outlined leading from pollination, through adhesion, hydration, germination, and tube growth to acceptance and ultimate compatibility. The characteristics of rejection of incompatible grains are described for each stage of the pollen-stigma interaction. It is proposed that recognition of proteins from the coating of self-pollen by the molecules in the pellicle results in the formation of a biologically-active complex which inhibits water supply to the incompatible grain, and that all other manifestations of incompatibility are a consequence of this initial response.

A glycoprotein associated with the acquisition of the self-incompatibility system by maturing stigmas of Brassica oleracea.

Iso-electric focusing of extracts derived from stigmatic homogenates of Brassica oleracea reveals that the mature stigma possesses large quantities of a glycoprotein not present in earlier stages of development in the bud. Pollen germination experiments carried out in parallel with the biochemical tests suggest that the appearance of this glycoprotein, which has an isoelectric point of pH 5.8, is coincident with the development of the self-incompatibility response. The site of this protein, and the role it may play in pollen-stigma interactions are discussed in terms of current models of the self-incompatibility system in Brassica.

Pollen-pistil interaction in Brassica oleracea : Events prior to pollen germination.

Quantitative studies of the adhesion of pollen grains to the stigma in Brassica oleracea revealed that self-pollen is initially less firmly bound than cross-pollen. The pollen grain tryphine, believed to be important in the adhesion process, has been shown to differ in mobility following self- and cross-pollination when observed using fluorescent probes. The hydration of the pollen grains has been investigated in vitro by measuring the changes in shape, volume and fresh weight of the imbibing grains. Whilst little change in volume could be detected there was a considerable increase in fresh weight together with a change of shape. The significance of these events, which occur prior to pollen germination, is discussed in relation to their effect upon subsequent germination and expression of self-incompatibility.

Studies on flower longevity in Digitalis : Pollination induced corolla abscission in Digitalis flowers.

Flower lifespan was terminated by corolla abscission 5-6 days after stigma opening in the unpollinated flower. Increased pollen loads produced increased seed set and reduced flower longevity progressively to a minimum of one day after pollination with pure pollen. Weakening of the abscission zone was detectable 8 h after pollination, whilst the pollen tubes were still within the stigmatic zone, suggesting that a stimulus, moving at 4 mm h(-1) minimum, was transmitted through the style and ovary. Soon after pollination removal of the stigma prevented the pollination-induced corolla abscission. Later it was necessary to remove the stigma and upper style, and later still the whole style to delay abscission. The progressive induction of the stigma and style took place at a rate of 1.5 mm h(-1), in advance of the pollen tubes which grew at 0.75 mm h(-1). It was not possible to reproduce the pollination effects by application of indoleacetic acid (IAA) to the stigma or the style.