Endogenous gibberellin A1 levels control thermoperiodic stem elongation in Pisum sativum

Gibberellin (GA) is believed to be involved in thermoperiodic stem elongation. With this in mind, we studied the correlation between gibberellin A₁ (GA₁) levels and stem elongation affected by alternating day (DT) and night temperature (NT) in 5 genotypes of Pisum sativum differing in their degree of dwarfism. The endogenous GA content in the tissue of two of the genotypes was determined by combined gas chromatography and mass spectrometry. The wild genotype developed 40 to 50% shorter stems and internodes under a low DT and high NT combination (negative difference [DIF] between DT and NT, DT/NT 15.5/21.5 or 14/24°C) than under the opposite regime of high DT and low NT (positive DIF, DT/NT 22.5/16.5 or 24/14°C). The GA biosynthetic mutants ls and le, and the auxin and brassinosteroid mutant lkb responded in a similar way, but not as strongly as the wild type. The stem length of the GA-insensitive slender mutant (la cry^s) was reduced by only 8% under negative compared to positive DIF. In the wild type endogenous GA levels decreased by 60% from positive to negative DIF in the upper part of the stem. Further, there was a corresponding decrease in the levels of precursors to GA₁, i.e. GA₅₃, GA₄₄, GA₁₉ and GA₂₀, while 2β-hydroxylated GA₂₀ and GA₁, GA₂₉ and GA₈, respectively, were unaffected by DIF. A similar increase in the ratios of GA₂₉ to GA₂₀ and GA₈ to GA₁ from positive to negative DIF was seen in the stem tissue of the le mutant as in the wild type. The temperature regimes affected the levels of GA₁ and its precursors in combined leaf and petiole samples and in the shoot tip in a similar manner as in the stem tissue. However, the different temperature regimes did not affect the ratio of GA₈/GA₁ in the shoot tip. The results indicate that altered stem elongation of the pea plants in response to diurnal temperature alternations may be mediated by changes in endogenous levels of GA₁. The GA₁ levels may be controlled by an effect of DIF on both biosynthetic and inactivation steps.     

Decreased D2-40 immunoreactivity in stored paraffin sections and methods for preserving it

D2-40, a monoclonal antibody against podoplanin, is a selective marker of lymphatic endothelium and is widely used for research on and diagnosis of pathology of lymphatic vessels. We examined the relation between the duration of tissue section storage and changes in immunostaining by D2-40 antibody; we evaluated also the effects of preservation methods on changes in immunostaining during storage. Staining by D2-40 was attenuated by long-term preservation of scalp skin and lymph node sections at room temperature. The attenuation of D2-40 staining in stored sections was improved by preservation at low temperature, i.e., 4° or ? 30° C. We investigated also the immunostaining of preserved tissue sections using NZ-1 and Lyve-1, which are antibodies against lymphatic endothelium markers. Staining by NZ-1 or Lyve-1 antibody was detected clearly in sections that had been stored for 16 weeks. Our study suggests that either long-term storage of D2-40 immunostained tissue sections should be avoided or the section should be preserved at low temperature.     

Three-dimensional architecture of the higher plant nucleolonema disclosed on serial ultrathin sections

Summary— The three-dimensional architecture of the nucleolonema of Vicia faba has been studied by applying a silver impregnation technique to serial ultrathin sections. This technique disclosed lateral and transverse segments of the nucleolonema which were heavily impregnated with silver. The lateral profiles of the nucleolonema segments were classified into three main categories; a segment made up of one to several rod-like filaments (type I); a ladder-like segment consisting of two parallel and of transverse filaments (type II); and a last type constructed from two parallel filaments (type III). Tracing of the lateral segments through serial sections has indicated that type I first appears, then either type II or III and finally type I reappears at the corresponding sites on sections. Types II and III remained constant in width, about 1.0 μm, along their longitudinal axes whereas the width of type I was significantly smaller than that of the two former. The lateral filaments of both types II and III showed heterogeneity in width on account of the presence of knobs intermittently distributed along them. The thickness of these knobs was about 0.35 μm. Combining the observations on serial ultrathin sections and the morphometrical data it is very probable that the elementary structure of the nucleolonema is a 0.35-μm thick filament that tightly coils up into a solenoid structure with a thickness of approximately 1.0 μm. This model can explain the appearance of open- and closed-argyrophilic rings in serial sections since transverse segments of the solenoid are expected to show the argyrophilic rings. The elementary filament of the nucleolonema solenoid was sometimes loosened. Judging from our cytochemical data at the electron microscope level, some argyrophilic proteins appear to reside in the axial space of the solenoid but both DNA and RNA were not detectable in this space.     

Internode length in Lathyrus odoratus. The involvement of gibberellins

Evidence was obtained by gas chromatography-mass spectrometry and gas chromatography-selected ion monitoring for the presence of gibberellin A₂₀), GA₁, GA₂₉, GA₈ and 2-epiGA₂₉ in vegetative shoots of tall sweet pea, Lathyrus odoratus L. Both tall (genotype L –) and dwarf (genotype II ) sweet peas elongated markedly in response to exogenous GA₁ attaining similar internode lengths at the highest dose levels. Likewise internode length in both genotypes was reduced by application of the GA biosynthesis inhibitor, PP333. The ratio of leaflet length to width was reduced by application of PP333 to tall plants and this effect was reversed by GA₁. When applied to plants previously treated with PP333, GA₂₀ promoted internode elongation of L – plants as effectively as GA₁, but GA₂₉ was not as effective as GA₁ when applied to II plants. In contrast, GA₂₀ and GA₁ were equally effective when applied to the semidwarf lb mutant but GA-treated lblb plants did not attain the same internode length as comparable GA-treated Lb – plants. The difference in stature between the tall and dwarf types persisted in dark-grown plants. It is concluded that GA₁ may be important for internode elongation and leaf growth in sweet pea. Mutant l may influence GA₁ synthesis by reducing 3β-hydroxylation of GA₂₀ whereas mutant lb appears to affect GA sensitivity.     

Persistent effects of changes in phytochrome status on internode growth in light-grown mustard: Occurrence,kinetics and locus of perception

Extension growth of the first internode in fully de-etiolated mustard (Sinapis alba L.) seedlings (11–12.5 d old) is under the control of both the current phytochrome photoequilibrium (Pfr/P, ratio of the far-red-absorbing form of phytochrome to total phytochrome) and that established by short (<12 h) pretreatments. Plants were pretreated with either light pulses providing different calculated Pfr/P followed by dark incubations of different durations (a), or with a 12-h period of white light establishing different Pfr/P (b). After the pretreatments, the plants received either light pulses providing different Pfr/P, followed by dark incubations (c), or continuous white light with or without addtional far-red light (d). Thus, four experimental approaches were followed: (a)(c); (a)(d); (b)(c) and (b)(d). Extension growth during the second period (c or d) was not only affected by the current phytochrome status, but also by that established during the pretreatment period (a or b). The results show the existence of a long-term promotion of stem growth which persists after the end of the low Pfr/P pretreatment. This effect is different from the previously reported rapid effect of far-red light added to background white light as follows: (i) the duration of low Pfr/P required to effect a full response is longer (2.5 h); (ii) the duration of the promotion after returning to high Pfr/P is longer (approx. 24 h) and (iii) the locus of perception is mainly in the leaves, rather than the growing internode.Abbreviations FR far-red light - PAR photosynthetically active radiation - Pfr/P ratio between the FR-absorbing form and total phytochrome - R red light - WL white light     

Correlative Microscopy of Vitreous Sections Provides Insights into BAR-Domain Organization In Situ

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Clonal analysis of corn plant development

The formation of the lower nodes and internodes in maize (Zea mays L.) and the progression of their differentiation was investigated by generating clonal sectors from cells of the apical meristem. Marked clones were induced by irradiating dry seeds (kernels) and 2-, 8- and 13-day-old seedlings heterozygous for anthocyanin markers (b, pl) and a chlorophyll factor (wd). The extent and apparent number of cells generating the internodes 2–6, which normally remain condensed, were traced by promoting the elongation of these internodes with gibberellic acid. At the mature seed stage, internodes 2 and 3 are undergoing longitudinal expansion and each is represented by two or three circumferential populations of cells. Internodes 4 and 5 are in the process of radial expansion and each is represented by a single circumferential population of cells. At nodes 2–4, the cells for leaves and internodes have separated but such a separation has not occurred for nodes 5 and 6. The formation and expansion of basal six internodes progressed acropetally, i.e. from the base toward distal nodes. Analysis of sectors induced at the seedling stage shows that the formation of middle and top internodes also progress acropetally. The basal, middle and top internodes were found to develop at different apparent cell numbers in the apical meristem.     

Phenotypic plasticity of internode elongation stimulated by deep-seeding and ethylene in wheat seedlings

Deep-seeding and ethylene were found to stimulate extension growth of the first internode of intact wheat (Triticum aestivum L.) seedlings in darkness. Seedlings of Hon Mang Mai emerged from much deeper in the soil than the seedlings of the other varieties used and their first internodes elongated to a much greater extent in response to ethylene. Carbon dioxide slowed elongation of the first internode and inhibited ethylene action. Elongation of the first internode due to deep-seeding and ethylene treatment showed high heritabilities, suggesting a genetic basis underlying those traits.     

Comparative Study of Antigen Retrieval Heating Methods: Microwave, Microwave and Pressure Cooker, Autoclave, and Steamer

We present a study comparing the most popular beating methods currently used for antigen retrieval (AR) immunostaining: the microwave oven, microwave with pressure cooker, autoclave, and steamer heating. A panel of 21 antibodies was tested on formalin fixed, paraffin embedded sections using these heating methods and Tris-HC1 buffer, pH 9.5, plus 5% urea as the AR solution. Three observers independently evaluated the intensity of AR immunostaining. All heating methods yielded good results for AR immunostaining. There were only minor differences among the heating methods for AR when the optimal concentration of primary antibody for normal immunostaining was used; however, background staining may occasionally be troublesome if antibodies are not retitrated and diluted further for use on tissues after AR. Significant differences were observed only after further dilution of the primary antibodies; the microwave pressure cooker, extended microwave heating (5 min × 4) and autoclave heating then showed a similar intensity of staining that was stronger than results obtained with the steamer (20 min) or regular microwave heating (5 min × 2 J. Extension of the steamer heating time, however, yielded equivalent results. This study indicates that different heating methods can yield similar intensities of AR immunostaining if the heating times are adjusted appropriately. It is noteworthy that, in general, the adjusted conditions for maximal retrieval differ from those most widely cited in the literature, or recommended by manufacturers. That several heating devices may provide similar results permits the use of different AR heating methods according to the equipment available. This study also is an early step in standardizing the AR immunostaining protocol by providing uniform conditions for “maximal retrieval” as a common end point for all laboratories.     

Staining Paraffin Sections Without Prior Removal of the Wax

Paraffin sections are usually rehydrated before staining. It is possible to apply aqueous dye solutions without first removing the wax. Staining then occurs more slowly, and only if the embedding medium has not melted or become unduly soft after catting. To avoid this problem, sections are flattened on water no hotter than 45 C and dried overnight at 40 C. Minor technical modifications to the staining procedures are needed. Mercury deposits are removed by iodine, and a 3% solution of sodium thiosnlfate in 60% ethanol is used to remove the iodine from paraffin sections. At room temperature, progressive staining takes 10-20 tunes longer for sections in paraffin than for hydrated sections; at 45 C, this can be shortened to about three times the regular staining time. After staining, the slides are rinsed in water, air dried, dewaxed with xylene, and coverslipped in the usual way. Nuclear staining in the presence of wax was achieved with toluidine blue, O, alum-hematoxylin and Weigert's iron-hematoxylin. Eosin and van Gieson's picric acid-acid fuchsine were effective anionic counterstains. A one-step trichrome mixture containing 3 anionic dyes and phosphomolybdic acid was unsuitable for sections in wax because it Imparted colors that were nninformative and quite different from those obtained with hydrated sections. Advantages of staining in the presence of wax include economy of solvents, reduced risk of overstaining and strong adhesion of sections to slides.