Induction of myrosinase gene expression and myrosinase activity in radish hypocotyls by phototropic stimulation

The role of myrosinase (beta-thioglucoside glucohydrolase, EC 3.2.3.1) in the phototropic response in radish hypocotyls was investigated. Unilateral illumination with blue light abruptly up-regulated the activity of myrosinase, which releases bioactive 4-methylthio-3-butenyl isothiocyanate (MTBI) from inactive 4-methylthio-3-butenyl glucosinolate (MTBG), in the illuminated halves of radish hypocotyls 10 min after onset of phototropic stimulation, peaking after 30 min and decreasing thereafter. The myrosinase activity in the shaded halves also increased, but was significantly lower than that in the illuminated halves. Furthermore, whether blue light illumination induces myrosinase gene expression was studied. Northern blotting analysis indicated that myrosinase mRNA levels were increased markedly in unilaterally illuminated hypocotyls, reaching maximum signal intensity within 10 min after onset of blue illumination, declining nearly to the control level thereafter. These results suggested that phototropic stimulation promotes myrosinase gene expression and myrosinase activity in the illuminated side, resulting in the conversion of inactive MTBG to active MTBI and simultaneously producing more active raphanusanins, causing a phototropic response.     

Growth patterns and gravitropic curvature of radish hypocotyls

The rapid growth rate of radish cells makes the hypocotyl particularly valuable in research on growth phenomena and gravitropism. An examination of mean data regarding the growth of aetiolated radish seedlings not subjected to gravitropic stimulation showed that there was an increase in the growth rate from day 3 to day 5. When the hypocotyl was placed horizontally all zones showed an increase in growth of the lower surface of the hypocotyl and generally a decrease in the growth rate in the upper surface. The upper apical part of the gravistimulated hypocotyl grew almost the same as controls. In some cases, in both 4- and 5-day-old seedlings, in the upper median part there was a lower growth rate than in controls. The zones of growth during the development of the hypocotyl were different. Analysis of curvature showed that the growth rate of the different zones was a function of their location in the hypocotyl and that the rate of curvature was different in various parts of the hypocotyl.     

Action spectra for the inhibition of growth in radish hypocotyls

In etiolated seedlings of Raphanus sativus L. the inhibition of hypocotyl elongation by continuous light showed a major bimodal peak of action in the red and far-red, and two minor peaks in the blue regions of the spectrum. It is argued that, under conditions of prolonged irradiation, phytochrome is the pigment controlling the inhibition of hypocotyl elongation by red and far-red light, but that its mode of action in far-red is different from that in red. A distinct pigment is postulated for blue light.Abbreviations B blue - FR far red - G green - R red - HIR high irradiance reaction - P_r and P_fr red and far red absorbing forms of phytochrome - R red     

A major factor in gravitropism in radish hypocotyls is the suppression of growth on the upper side of hypocotyls

The changes in length on the two opposite sides of etiolated radish (Raphanus sativus) hypocotyls prior to, and following gravitropic stimulation, were measured using an infrared-imaging system. It was observed that the growth suppression on the upper side began first at least 10 min after the onset of gravitropic stimulation, and after 30 min the acceleration in growth on the lower side started. The gravitropic curvature was steadily induced from 10 min. When radish hypocotyls were switched from a vertical to horizontal position for different durations and then replaced to the vertical position, the growth suppression on the gravity-stimulated (upper) side was observed in all cases, but the acceleration in growth on the opposite (lower) side appeared only in continuously gravity-stimulated seedlings, although it occurred later than the growth suppression on the upper side. These results suggest that the suppression in growth on the upper side of the hypocotyls is a direct effect of gravitropic stimulation, but not the acceleration on the lower side. When 4-methylthio-3-butenyl isothiocyanate (4-MTBI), which has an inhibitory activity against radish hypocotyl growth, was applied on the one side of radish hypocotyls and then the 4-MTBI-applied side or opposite side was placed in a horizontal position, the former showed greater bending than the control, suggesting that the growth suppression on the upper side is enhanced and maintained with MTBI application there. In the latter case, the seedlings showed less bending than the control, suggesting a decrease in growth on the lower side with MTBI application. All the results suggest that gravitropism of radish hypocotyls may be caused by an increase in growth-inhibiting substance(s) induced with gravitropic stimulation in the upper side, inducing growth inhibition there.     

Effects of hypergravity on the elongation growth in radish and cucumber hypocotyls

The elongation growth of the hypocotyls of radish and cucumber seedlings was examined under hypergravity in a newly developed centrifuge (Kasaharaet al. 1995). The effects of hypergravity on elongation growth differed between the two species. The rate of elongation of radish hypocotyls was reduced under basipetal hypergravity (H+20g) but not under acropetal hypergravity (H-13g), as compared to growth under the control conditions (C+1g and C-1g). In cucumber hypocotyls, elongation growth was inhibited not only by basipetal but also by acropetal hypergravity. Under these conditions, the reduction in the elongation growth of both radish and cucumber hypocotyls was accompanied by an increase in their thickness. Although no distinct differences in relative composition of neutral sugars were found, the amounts of cell-wall components (pectic substances, hemicelluloses and cellulose) per unit length of hypocotyls were increased by exposure to hypergravity.     

Effects of hypergravity on the elongation growth in radish and cucumber hypocotyls

The elongation growth of the hypocotyls of radish and cucumber seedlings was examined under hypergravity in a newly developed centrifuge (Kasahara et al. 1995). The effects of hypergravity on elongation growth differed between the two species. The rate of elongation of radish hypocotyls was reduced under basipetal hypergravity (H+2O g) but not under acropetal hypergravity (H-13 g), as compared to growth under the control conditions (C+1 g and C-1 g). In cucumber hypocotyls, elongation growth was inhibited not only by basipetal but also by acropetal hypergravity. Under these conditions, the reduction in the elongation growth of both radish and cucumber hypocotyls was accompanied by an increase in their thickness. Although no distinct differences in relative composition of neutral sugars were found, the amounts of cell-wall components (pectic substances, hemicelluloses and cellulose) per unit length of hypocotyls were increased by exposure to hypergravity.     

Starch content of etiolated radish (Raphanus sativus L.) hypocotyls as affected by chloramphenicol and external sucrose

Abstract

Starch was enzymatically assayed in the hypocotyls of radish (Raphanus sativus L.) seedlings grown in water or chloramphenicol (CAP) 1 × 10^4. CAP inhibits starch formation and its effect is related to the concentration. Both CAP- and water-grown hypocotyls are able to accumulate starch when sucrose is supplied in the medium, thus suggesting that the damage caused by CAP to the amyloplast is not irreversible. Apical segments of both water- and CAP- grown hypocotyls accumulate starch upon incubation in sucrose solutions while basal segments are unable to accumulate starch even in the presence of sucrose. The authors suggest that the basal segments are unable to perform sucrose uptake or that the amyloplast is incapable to starch synthesis. In any case the inability of the basal segment to perform sucrose uptake is independent of CAP. These findings confirm that the radish hypocotyls is not homogeneous along its axes.     

Metal binding induces conversion of B- to the hybrid B-Z-form in natural DNA

Highly polymerized herring testis DNA of the random nucleotide sequence has been studied in solution by circular dichroism and ultra-violet absorption spectrometry under various experimental conditions. At low temperature upon addition of 0.05M NaCl or 1.15M MgSO(4) the DNA formed a helix that belonged to the B-family. As the temperature was increased a transition from the pure B- to the hybrid B-Z-form occurred in the presence of 1.15M MgSO(4). This transition occurred over a large range of temperatures and corresponded to a non-cooperative conformational change. A similar DNA transition was induced with 0.098mM Co(NH(3))(6)Cl(3). However, in the presence of 5.3M NaCl the DNA conformation was not similar to that observed in 1.15M MgSO(4) or 0.098mM Co(NH(3))(6)Cl(3) independently on the environmental temperature. In 5.3M NaCl the DNA is thought to undergo a transition from one to another right-handed conformation that could be intermediate partially dehydrated conformer arising on the first step in the sequential transition to the dehydration of the polynucleotide. Our results show that a realistic model of native DNA, bearing Z-tracts embedded in B-helixes, can be obtained upon binding of alkaline earth or transition metals.     

Phototropic stimulation does not induce unequal distribution of indole-3-acetic acid in maize coleoptiles

Distribution of endogenous diffusible auxin into agar blocks from phototropically stimulated maize coleoptile tips was studied using a bioassay and a physicochemical assay, to clarify whether phototropism in maize coleoptiles involves a lateral gradient in the amount of auxin. At 50 min after the onset of phototropic stimulation, when the phototropic response was still developing, direct assay of the blocks with the Avena curvature test showed that the auxin activity in the blocks from the shaded half-tips was twice that of the lighted side, at both the first and second positive phototropic curvatures. However, physicochemical determination following purification showed that the amount of indole-3-acetic acid (IAA) was evenly distributed in the blocks from lighted and shaded coleoptile half-tips at both the first and second positive phototropic curvatures. The even distribution of the IAA was also confirmed with the Avena curvature test following purification by HPLC. These results indicate that phototropism in maize coleoptiles is not caused by a lateral gradient of IAA itself and thus cannot be described by the Cholodny-Went theory. Furthermore, the lower auxin activity in the blocks from the lighted half-tips suggests the presence of inhibitor(s) interfering with the action of auxin and their significant diffusion from unilaterally illuminated coleoptile tips.     

Phototropic Responses of Vaucheria geminata to Intermittent Blue Light Stimuli

Phototropic responses of a tip-growing coenocytic alga Vaucheria geminata to intermittent blue light pulses were analyzed. Curvatures caused by repeated light pulses separated by dark intervals of various lengths were as large as those obtained by continuous light of the same incident energy, unless the length of the dark interval exceeded a critical value of about 30 to 40 seconds. If the dark interval was longer than the critical length, bending no longer took place. Another response to intermittent irradiation was found. In a narrow range of light pulse and dark interval lengths, i.e. light pulses longer than 10 milliseconds and dark intervals shorter than a value ranging from 15 to 150 milliseconds, intermittent irradiation was more effective than continuous irradiation of the same total energy. In this region, the maximum response to repeated pulses was equal to the curvature caused by a continuous irradiation of the same total elapsed time. It is evident that in this condition the dark interval is not perceived and the alga responds as if it were in light. The results suggest that two different dark reactions may be involved in the phototropic response of this algal cell.     

Gibberellin metabolism in excised lettuce hypocotyls: Response to GA9 and the conversion of [3H]GA9

Elongation growth and gibberellin (GA₉) metabolism in excised hypocotyls of lettuce (Lactuca sativa L. cv. Arctic) were investigated. Exogenously supplied GA₉ stimulates elongation of hypocotyl sections and this response is intermediate between that elicited by GA₁ or GA₂₀ and GA_4/7 mixture. Although uptake of radioactivity from [³H]GA₉ increases with time, this gibberellin does not accumulate in the tissue but is rapidly converted to a compound with HPLC properties resembling those of [³H]GA₂₀. After 2 h incubation in [³H]GA₉, the presumptive GA₂₀ represents 90% of the acidic ethyl acetate-soluble radioactivity in the tissue. Radioactivity is also associated with an acidic butanol-soluble fraction containing two components resolvable by HVE. The major component is similar in electrophoretic properties to a GA-glucosyl ether while the other compares to a GA-glucosyl ester. Conversion of [³H]GA₉ to its [³H]GA₂₀-like metabolite is reduced by addition of carrier GA₉ or GA_4/7 at concentrations as low as 1 M, while GA₁, GA₃ and L-proline are without effect. Formation of the GA₂₀-like compound can be blocked by the addition of 2,2-dipyridyl, and this inhibitory effect of dipyridyl can be reversed by addition of Fe²⁺. At 200 M dipyridyl, elongation growth as well as [³H]GA₉ metabolism are reduced by 80%. The relationship of the metabolism of GA₉ to the growth response is discussed.Abbreviations AB butanol-soluble - AE ethyl-acetate-soluble - GA gibberellin - GA₁, GA₄ gibberellin A₁, gibberellin A₄, etc. - TLC thin layer chromatography - HPLC high performance liquid chromatography - HVE high voltage electrophoresis     

Studies on plant growth-regulating substances LIII. The conversion of certain substituted ethylamines to active growth substances in plants

A number of substituted phenylethylamines have been synthesised and their plant growth-regulating activities, in standard wheat and pea tests, have been determined and compared with those of related substituted phenoxyethylamines, phenylacetic acids and phenoxyacetic acids. The effects of applying the most active amines to various plant species are described and the results are considered in relation to the possible ability of the plant to convert the amine into the physiological active acetic acid derivative.     

Arabidopsis ROOT PHOTOTROPISM2 Contributes to the Adaptation to High-Intensity Light in Phototropic Responses

Living organisms adapt to changing light environments via mechanisms that enhance photosensitivity under darkness and attenuate photosensitivity under bright light conditions. In hypocotyl phototropism, phototropin1 (phot1) blue light photoreceptors mediate both the pulse light-induced, first positive phototropism and the continuous light-induced, second positive phototropism, suggesting the existence of a mechanism that alters their photosensitivity. Here, we show that light induction of ROOT PHOTOTROPISM2 (RPT2) underlies photosensory adaptation in hypocotyl phototropism of Arabidopsisthaliana. rpt2 loss-of-function mutants exhibited increased photosensitivity to very low fluence blue light but were insensitive to low fluence blue light. Expression of RPT2 prior to phototropic stimulation in etiolated seedlings reduced photosensitivity during first positive phototropism and accelerated second positive phototropism. Our microscopy and biochemical analyses indicated that blue light irradiation causes dephosphorylation of NONPHOTOTROPIC HYPOCOTYL3 (NPH3) proteins and mediates their release from the plasma membrane. These phenomena correlate closely with the desensitization of phot1 signaling during the transition period from first positive phototropism to second positive phototropism. RPT2 modulated the phosphorylation of NPH3 and promoted reconstruction of the phot1-NPH3 complex on the plasma membrane. We conclude that photosensitivity is increased in the absence of RPT2 and that this results in the desensitization of phot1. Light-mediated induction of RPT2 then reduces the photosensitivity of phot1, which is required for second positive phototropism under bright light conditions.     

Semaphorin 3A induces CaV2.3 channel-dependent conversion of axons to dendrites

Polarized neurites (axons and dendrites) form the functional circuitry of the nervous system. Secreted guidance cues often control the polarity of neuron migration and neurite outgrowth by regulating ion channels. Here, we show that secreted semaphorin 3A (Sema3A) induces the neurite identity of Xenopus spinal commissural interneurons (xSCINs) by activating Ca(V)2.3 channels (Ca(V)2.3). Sema3A treatment converted the identity of axons of cultured xSCINs to that of dendrites by recruiting functional Ca(V)2.3. Inhibition of Sema3A signalling prevented both the expression of Ca(V)2.3 and acquisition of the dendrite identity, and inhibition of Ca(V)2.3 function resulted in multiple axon-like neurites of xSCINs in the spinal cord. Furthermore, Sema3A-triggered cGMP production and PKG activity induced, respectively, the expression of functional Ca(V)2.3 and the dendrite identity. These results reveal a mechanism by which a guidance cue controls the identity of neurites during nervous system development.     

Proximity of Agrobacterium to living plant tissues induces conversion to a filamentous bacterial form

Changes in Agrobacterium colony and cell morphology were observed following co-culture of the bacterium with a variety of different plant tissues. Bacterial colonies grown in the presence of plant tissue became opaque and appeared to grow as a thick mat of cells. A single bacterial colony would often grow to fill an entire 100-mm-diameter petri dish. Ultrastructural observations of the bacteria in these colonies revealed the formation of a predominantly filamentous form of the bacterium. The bacteria ranged from 5 to 100 µm in length as compared to 2 µm in the non-filamentous form. The filamentous form was observed 2-3 days after co-culture and only if the bacteria were either in direct contact with or in close proximity (<5 mm) to living plant tissues. The filamentous form was observed with both wild-type and engineered Agrobacterium strains. As proximity of the bacteria to living tissue was necessary for induction, plant tissues apparently produce inductive compounds that are either very labile or have their effect only at high concentrations.     

Influence of ions and chemical substances on the response of the frog's tongue to mechanical stimulation

• 1.1. The fungiform papillae responded to the mechanical stimulation of either an air puff or a Frey's hair. but the filiform papillae did not.
• 2.2. The integrated neural response to an air puff was greatest along the margin and at the apex of the dorsum linguae.
• 3.3. More than three quarters of the mechanosensitive fibers responded to CaCl₂ application and one third responded to NaCl application. Mono- and divalent cations had various influences on the response to mechanical stimulation.
• 4.4. Glucose and sucrose dissolved in distilled water depressed the response, but when dissolved in Ringer's solution had no effect. HCl and quinine-HCl depressed the mechanical response.