Nuclear magnetic resonance imaging in studies of gravitropism in soil mixtures

Gravitropic responses of oat coleoptiles were measured in different growth media; humid air, natural soil and artificial soil (glass beads). The oat coleoptiles in soil and glass beads were monitored by NMR imaging, while those in humid air were imaged in darkness with an infrared-sensitive charge-coupled device (CCD) camera. The present study shows for the first time that gravitropic experiments can be performed in artificial soil using NMR imaging as a convenient and suitable recording method. Not only was it possible to follow the gravitropic curvatures in natural soil, but the artificial soil allowed plant images of sufficient spatial and temporal resolution to be recorded. The advantages of using artificial soil in magnetic resonance imaging studies are that the iron content of glass beads is very low compared with natural soil, and that the artificial soil matrix can easily be standardized with regard to particle size distribution and nutrient content. Two types of glass beads were used, the diameter of the small and the large beads being 300–400 and 420–840 μm, respectively. The growth rate of the coleoptiles in soil and in big beads was roughly the same and only slightly lower than in humid air, whereas small beads reduced the growth rate by approx. 16%. The bending rate of the coleoptiles during the gravitropic response was reduced by c. 65% in soil and 75% in bead mixtures relative to bending in air. It should be noted, however, that the maximum curvature of the coleoptile tip was of the same order in all cases, about 35°. This value may represent the largest possible curvature of the organ. The potential of NMR imaging to study how plant organs penetrate the soil under the influence of gravitropism, mechanical impedance and thigmotropism is also discussed.     

An exo-β-d-glucanase derived from Zea coleoptile walls with a capacity to elicit cell elongation

Cell wall proteins were extracted from maize coleoptiles, Zea mays L. B37 x MO 17, with high concentrations of LiCl. Ion-exchange, chromatofocusing and gel-filtration chromatography were employed extensively to purify exo-β-glucanase activity from the extract. The purified enzyme functioned as an exo-(1→3)-β-glucanase (E.C. 3.2.1.58) and as a glucosidase (E.C. 3.2.1.21) capable of extensive hydrolysis of the native Zea wall (1→3), (1→4)-β- d -glucan, yielding glucose as the final product. The exoglucanase also enhances elongation of maize coleoptile sections in both the presence and absence of exogenous IAA.     

RIP and RALyase cleave the sarcin/ricin domain, a critical domain for ribosome function, during senescence of wheat coleoptiles

Type-I ribosome-inactivating protein (RIP), which is found in many plants, catalyzes depurination of a specific adenine in the sarcin/ricin domain (SRD) of the large rRNA causing loss of ribosomal activity. Previously, we found a RNA apurinic site-specific lyase (RALyase) that catalytically cleaved the phosphodiester bond at the RIP-dependent depurination site by β-elimination reaction. Here we show that both the RIP activity and RIP-RALyase-mediated cleavage of SRD in the cytoplasmic ribosome were induced at the late stage of senescence of wheat coleoptiles. Following this process, tissue death was observed. Furthermore, transgenic tobacco plants expressing glucocorticoid-induced RIP developed senescence-like phenotype. Our results suggest that ribosome inactivation due to the cleavage of SRD by the inducible RIP and constitutively expressed RALyase may be a unique plant system that mediates programmed cell death at the late senescent stage.     

Cell wall hydroxycinnamate esters as UV-A receptors in phototropic responses of higher plants—a new hypothesis

E/Z Photoisomerism, in UV-A, of cell wall ferulate and diferulate-carbohydrate esters is suggested as a mechanism for transduction of light energy leading to changes in wall structure and hence water flux, turgor pressure and growth. Unilateral light would cause phototropism.     

High-frequency somatic embryogenesis of koala fern (<Emphasis Type="Italic">Baloskion tetraphyllum,restionaceae</Emphasis>)

Summary Baloskion tetraphyllum is a member of the Restionaceae and is an important species for the rehabilitation of disused mine sites and wetland areas, and is also highly prized as a cut flower. Its use for restoration of disturbed land is, however, severely limited, due to very poor propagation success by conventional methods. A study was conducted to evaluate the potential of somatic embryogenesis for the large-scale propagation of this species. A variety of auxins (at different concentrations) were investigated for their efficacy in stimulating somatic embryogenesis. Somatic embryos were successfully induced from excised coleoptiles of B. tetraphyllum on half-strength Murashige and Skoog (1/2MS) medium supplemented with 1 μM 2,4-dichlorophenoxyacetic (2,4-D). To scale up the production, proliferation of secondary somatic embryos was achieved using primary somatic embryos as the tissue source, on 1/2MS+1 μM 2,4-D resulting in a 30-fold increase in somatic embryo, numbers. Almost all the somatic embryos developed into plants and were established ex vitro. The other auxins investigated, including p-chlorophenoxyacetic acid, indole-3-acetic acid, α-naphthaleneacetic acid, and picloram, were not as effective as 2,4-D. The age, of the explant material significantly influenced somatic embryogenesis with white, young coleoptiles (5–7 d) producing 50% more somatic embryos than green, more mature (8–14 d) coleoptiles. The protocol developed for B. tetraphyllum has the potential to be commercially viable, with an estimated 22 000 somatic embryos produced from 1 g of plant material. This research may also have a positive impact on the propagation of other important Restionaceae species.     

Effects of red light on the fluence–response relationship for pulse-induced phototropism of maize coleoptiles

Dark-adapted coleoptiles of maize (Zea mays L.) were treated with red light (3min at 10.5 μmol m^?2S^?1) and were Stimulated, after a dark interval, with a pulse of unilateral blue light to induce phototropism. Phototropic fluence-response curves were obtained in this way for different dark intervals. It was confirmed that the bell-shaped fluence-response curve for the first pulse-induced positive phototropism (FPIPP) shifts to higher fluences following the red-light treatment, the maximal shift being achieved at a dark interval of 2h. We found, however, that the two arms of the Fluence-response curve do not shift synchronously. The shift of the descending arm to higher fluences began at 15 min. The ascending arm showed a slight shift to lower fluences before a greater shift to higher flucnces. the change of the shift direction occurring at 30–40min. Accordingly, the fluence-response curve obtained for a 30 min dark interval was comparatively wide. Although dark-adapted coleoptiles showed only fPIPP, another bell-shaped fluence-response curve, representing the second pulse-induced positive phototropism (sPIPP), appeared gradually after the red-light treatment. These changes of the phototropic fluence– respnse curve following exposure to red light are likely to have adaptive values because they favour phototropism under brighter light.     

Is Cell Elongation Regulated by Extracellular Auxin?

Experiments with isolated epidermal strips of maize coleoptiles, pretreated with auxin and further incubated on sucrose agar containing different concentrations of auxin (indole-3-acetic acid, IAA or naphthalene-1-acetic acid, NAA) and/or naphthylphthalamic acid (NPA), are described. Preincubation for 2h with 2 . 10^?4M IAA or 10^?5M NAA in buffer, followed by 30 min wash in buffer results in measurable cell elongation during a subsequent incubation for 6 h on sucrose agar. Addition of 10^?4M NPA inhibited the response to auxin and this inhibition could be reversed by providing IAA in addition to NPA. Inner tissue fragments (without outer epidermis) did not respond to external IAA. These results lead to the conclusion that auxin secretion at the outer epidermis may be an essential step in auxin-regulated coleoptile growth.     

Flooding resistance of Rumex species strongly depends on their response to ethylene: Rapid shoot elongation or foliar senescence

In excised Zea maus L. coleoptiles incubated in aerated media at high fresh weight per volume ratios, indole-3-acetic acid induces transient drops of extracellular pH. Based on the quantitative dependency of the response on the initial auxin concentration we developed a novel auxin bioassay, which allows reliable estimation of IAA concentrations between 10⁻⁸⁵ and 10⁻⁵M. Using the bioassay and complementary concentration measurements by IAA fluorescence we found the transient IAA-induced pH response paralleled by a decrease of auxin activity and concentration in the medium. This decline is rapid and starts immediately upon auxin addition, and insofar differs from the well known IAA degradation by epiphytic bacteria in long-term auxin tests. We conclude that the transient character of the auxin pH response is due to rapid IAA metabolism. The effect occurs under those experimental conditions that are necessary for reliable estimations of auxin-induced shifts of cell wall pH, which considerably complicates the interpretation of the results.     

Sterol biosynthetic capability of purified membrane fractions from maize coleoptiles

Membrane fractions were isolated from etiolated maize coleoptiles by differential and sucrose density gradient centrifugation. Specific membrane components were identified by using marker enzyme activities. Fractions were also tested for α-naphthylacetic acid (NAA).and N-naphthylphthalamic acid (NPA) binding activities. Evidence is presented for the isolation of a plasma-membrane fraction containing specific binding sites for NPA, a high concentration of sterols, and most of the total UDP-glucose sterol glucosyltransferase activity. A fraction rich in endoplasmic reticulum is shown to contain most of the binding sites for NAA and all of the activity of both S-adeno-Syl-L-methionine-Δ24 cycloartenol methyltransferase and cycloeucalenol-obtusifoliol isomerase.     

Separation of presumptive plasma membranes from mitochondria by partition in an aqueous polymer two-phase system

Light-induced absorbance changes (LIAC), indicating the reversible reduction of a b-type cytochrome, and with a possible connection to blue light photomorphogenesis, have been found in a presumptive plasma membrane rich centrifuge fraction from LIAC could be due to plasma membrane vesicles turned inside out or to cytochromes localized in other organelles. Phase partition proved to be a rapid method (results technique membrane particles are separated according to differences in surface properties rather than size and density. LIAC could be separated into two fractions: one partitioning into the polyethylene glycol rich upper phase and another preferring the dextram rich lower phase. Mitochondria (cytochrome c oxidase) were recovered in the lower phase. A dual distribution of LIAC was found with all materials tested: corn coleoptiles, corn shoots, barley shoots and cauliflower inflorescences. About 80–90% of the cytochromes in the upper phase were related to LIAC, whereas only 10–15% of those in the lower phase were of this kind. The LIAC preferring the upper phase was probably bound to the plasma membrane, since plasma membrane vesicles are known to have a high partition in these phase systems. The lower phase LIAC could be due to plasma membrane vesicles turned inside out or to cytochromes localized in other organelles. Phase partition proved to be a rapid method (results within one hour after the initial pelleting) for purification of presumptive plasma membranes, yielding a preparation which contained five times less mitochondrial contamination than the preparation obtained with sucrose gradient centrifugation (the 33/45% w/w sucrose interface fraction).