Hydrogen ion-induced curvature in cucumber hypocotyls

A strip of filter paper saturated with a pH 3.0 buffer solutionand placed on the upper side of a light-grown cucumber hypocotylseedling can eliminate the negative geotropic curvature of horizontallyplaced seedlings, by promoting elongation of the upper side.These filter paper strips also caused strong curvature of verticallyplaced seedlings and induced cell wall loosening of the epidermis. (Received February 16, 1973; )     

Asparate transcarbamylase activity in etiolated cowpea hypocotyls treated with 2,4-dichlorophenoxyacetic Acid

Treating etiolated cowpea (Vigna unguiculata) seedlings with 2,4-dichlorophenoxyacetic acid resulted in 2.5-, 3.9-, and 6.5-fold increases in DNA, soluble protein, and RNA, respectively, over untreated controls 84 hours after treatment. Aspartate transcarbamylase activity increased within 12 hours after treatment, and by 84 hours it was almost 12-fold greater than that in the untreated controls. During that time, activity in untreated controls dropped 60%. The assay used ¹⁴C-aspartate, which was then separated from the ¹⁴C-ureidosuccinate product by Dowex 50 (H⁺ form) column chromatography. Thin layer chromatography of the reaction product indicated that most of the carbamyl-phosphate-dependent radioactivity co-chromatographed with ureidosuccinate. The reaction has a pH optimum near 10.0 and is inhibited by uridine 5′-phosphate and succinate. The data suggest that aspartate transcarbamylase is important in pyrimidine biosynthesis in 2,4-dichlorophenoxyacetic acid-treated seedlings.     

Polymer mobility in cell walls of cucumber hypocotyls

Cell walls were prepared from the growing region of cucumber (Cucumis sativus) hypocotyls and examined by solid-state 13C NMR spectroscopy, in both enzymically active and inactivated states. The rigidity of individual polymer segments within the hydrated cell walls was assessed from the proton magnetic relaxation parameter, T2, and from the kinetics of cross-polarisation from 1H to 13C. The microfibrils, including most of the xyloglucan in the cell wall, as well as cellulose, behaved as very rigid solids. A minor xyloglucan fraction, which may correspond to cross-links between microfibrils, shared a lower level of rigidity with some of the pectic galacturonan. Other pectins, including most of the galactan side-chain residues of rhamnogalacturonan I, were much more mobile and behaved in a manner intermediate between the solid and liquid states. The only difference observed between the enzymically active and inactive cell walls, was the loss of a highly mobile, methyl-esterified galacturonan fraction, as the result of pectinesterase activity.     

Induction of competence for elicitation of defense responses in cucumber hypocotyls requires proteasome activity

The epidermal cells of hypocotyls from etiolated cucumber seedlings are not constitutively competent for elicitation of the rapid H2O2 defense response. However, elicitor competence developed while conditioning the surface-abraded seedlings by rotating them in buffer for 4 h. Competence development was greatly potentiated by inducers of systemic acquired resistance and suppressed by specific inhibitors of proteasome activity, clastolactacystin beta-lactone (LAC) and carboxybenzoyl-L-leucyl-L-leucyl-L-leucinal (LLL). In the freshly abraded seedlings, chitinase gene activation became evident approximately 4 h after elicitor addition. Accumulation of chitinase mRNA was enhanced upon conditioning prior to elicitation and was inhibited by LAC and LLL, indicating that the process which leads to H2O2 elicitation competence is also superimposed on the elicitation of chitinase mRNA. LAC and LLL caused an accumulation of ubiquitin-conjugated proteins and enhanced the expression of a proteasome alpha-subunit, suggesting that proteasome activity was specifically inhibited and that the effect observed on gene expression was not due to impaired gene induction in general. Together, our results suggest that the ubiquitin-proteasome system may play a crucial role in a process which switches the signaling pathway for diverse plant defense responses into a functional state, as is known for many basic cellular processes in both animals and yeast.     

Effect of white light on meristematic activity in developing sunflower hypocotyls

Summary To determine whether hypocotyl elongation in sunflower seedlings (Helianthus annuus L.) is dependent on cell divisions (meristematic activity), we used a specific inhibitor of DNA synthesis (fluorodeoxyuridine). The seedlings were either grown for 6 days in darkness or continuous white light (WL). Under both conditions hypocotyl growth was retarded by 30–70% in the presence of the inhibitor. Because the nuclei do not become endopolyploid we conclude that hypocotyl growth is dependent on cell reproduction. In the next step an immunocytochemical method was used to detect the percentage of nuclei in S-phase (meristematic activity) in different regions and tissues of the hypocotyls. In the peripheral cell layers (epidermis, cortex) meristematic activity was much greater than in the pith of the organ. In rapidly growing (etiolated) hypocotyls meristematic activity is largely restricted to the closed apical hook of the stem. After transfer to WL the hook opens and hypocotyl elongation is inhibited. In the epidermis and cortex of the apical hook a large WL-induced enhancement in the percentage of nuclei in S-phase occurred, which was followed by a light-mediated retardation of meristematic activity. Our data show that WL exerts a transient stimulatory effect on meristematic activity during photomorphogenesis of the sunflower seedling.Abbreviations BrdUrd 5-bromo-2-deoxyuridine - D darkness - FdUrd 5-fluoro-2-deoxyuridine - TRITC tetramethyl-rhodamine-isothiocyanate - WL white light     

Mechanism of blue-light-induced plasma-membrane depolarization in etiolated cucumber hypocotyls

A large, transient depolarization of the plasma membrane precedes the rapid blue-light (BL)-induced growth suppression in etiolated seedlings of Cucumis sativus L. The mechanism of this voltage transient was investigated by applying inhibitors of ion channels and the plasma-membrane H⁺-ATPase, by manipulating extracellular ion concentrations, and by measuring cell input resistance and ATP levels. The depolarizing phase was not affected by Ca²⁺-channel blockers (verapamil, La³⁺) or by reducing extracellular free Ca²⁺ by treatment with ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA). However, these treatments did reduce the rate of repolarization, indicating an inward movement of Ca²⁺ is involved. No effects of the K⁺-channel blocker tetraethylammonium (TEA⁺) were detected. Vanadate and KCN, used to inhibit the H⁺-ATPase, reduced or completely inhibited the BL-induced depolarization. Levels of ATP increased by 11–26% after 1–2 min of BL. Input resistance of trichome cells, measured with double-barreled microelectrodes, remained constant during the onset of the depolarization but decreased as the membrane voltage became more positive than -90 mV. The results indicate that the depolarization mechanism initially involves inactivation of the H⁺-ATPase with subsequent transient activation of one or more types of ion channels.Abbreviations and Symbols BL blue light - CI current injection - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - TEA⁺ tetraethylammonium - V_m membrane voltage We wish to thank Drs. Adam Bertl and Clifford L. Slayman, Yale School of Medicine, New Haven, Conn., USA, for helpful discussions. This work was supported by a Natural Sciences and Engineering Research Council of Canada Scholarship (E.P.S.) and National Science Foundation Grant DMB-8351030 (D.J.C.).     

Chromatin RNA polymerase activity from soybean hypocotyls treated with gibberellic acid and AMO-1618

Chromatin isolated from control, AMO-1618 [2′-isopropyl-4′-(trimethylammonium chloride)-5′-methylphenyl piperidine carboxylate] and gibberellic acid (GA) treated soybean hypocotyl tissue incorporates labeled nucleoside triphosphates into acid-insoluble RNA. Gibberellic acid, sprayed on intact soybean hypocotyls, is shown to have enhanced the level of chromatin RNA polymerase activity while chromatin isolated from hypocotyls pretreated with AMO-1618 exhibits a lower polymerase activity relative to the control. Chromatin extracted from the treated or untreated seedlings are all sensitive to the inhibition (in varying degrees) by the presence of actinomycin D, pyrophosphate, or ribonuclease. Thus enhanced (or decreased) RNA-synthesizing capacity of chromatin in response to chemical treatments may be due to enhanced (or decreased) synthesis of RNA polymerase.     

Enhanced deoxyribonucleic Acid polymerase activity of chromatin from soybean hypocotyls treated with 2,4-dichlorophenoxyacetic Acid

Chromatin isolated from soybean (Glycine max L., var. Wayne) hypocotyls was capable of catalyzing the polymerization of labeled deoxyribonucleoside triphosphate in the presence of the three other deoxyribonucleoside triphosphates into a trichloroacetic acid-insoluble product. This product was insensitive to base hydrolysis and ribonuclease, but was sensitive to acid hydrolysis and deoxyribonuclease. Chromatin-DNA polymerase required Mg²⁺ and all four deoxyribonucleoside triphosphates for maximal activity. Inorganic pyrophosphate and actinomycin D inhibited the polymerase activity, but 2, 4-dichlorophenoxyacetic acid had no effect in vitro. Chromatin from plants previously treated with 2, 4-dichlorophenoxyacetic acid supported a greater level of DNA synthesis than did chromatin from untreated plants.     

Induction of peroxidases in cucumber hypocotyls by wounding and fungal infection

The induction of peroxidases (EC 1.11.1.7) during elicitation of lignification by α-1,4-linked oligogalacturonides in cucumber hypocotyl segments ( Cucumis sativus L. cv, Wisconsin SMR 58) was investigated. The wounding associated with the preparation of hypocotyl segments induced a 19-fold increase in peroxidase activity during the following 72 h. The increase was partially due to an increase in activity of a constitutive peroxidase with a pI of 8.9 and partially due to the expression of new peroxidase isozymes with pIs of 3.8, 5.4, 6.2, 9.1 and 9.4. The oligogalacturonides did not induce any peroxidase activity in addition to the wound-induced activity. These results suggest that either the constitutive peroxidase isozyme (pI 8.9) of intact hypocotyls or some of the wound-induced peroxidases are involved in the oligogalacturonide-induced lignification.
Induction of the peroxidases by wounding was inhibited by cycloheximide. This indicates that they accumulate as a result of de novo protein synthesis. Actinomycin D caused only a modest inhibition of the wound-induction peroxidases, indicating that the process is regulated at the level of translation.
Peroxidase activity increased more rapidly in resistant than in susceptible cucumber hypocotyls after inoculation with the pathogen Cladosporium cucumerinum Ellis & Arthur. The pattern of isozymes which was induced by fungal infection of resistant hypocotyls was similar to the pattern of isozymes induced by wounding. This suggests that similar induction mechanisms may be involved in the two processes.     

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.     

Mechanism of rapid suppression of cell expansion in cucumber hypocotyls after blue-light irradiation

Rapid suppression of hypocotyl elongation by blue light in cucumber (Cucumis sativus L.) was studied to examine possible hydraulic and wall changes responsible for diminished growth. Cell-sap osmotic pressure, measured by vaporpressure osmometry, was not decreased by blue light; turgor pressure, measured by the pressureprobe technique, remained constant during the growth inhibition; and stem hydraulic conductance, measured by dynamic and static methods, was likewise unaffected by blue light. Wall yielding properties were assessed by the pressure-block technique for in-vivo stress relaxation. Blue light reduced the initial rate of relaxation by 77%, but had little effect on the final amount of relaxation. The results demonstrate that blue irradiation acts to decrease the wall yielding coefficient, but not the yield threshold. Stress-strain (Instron) analysis showed that irradiation of the seedlings had little effect on the mechanical extensibilities of the isolated wall. The results indicate that blue light can reduce cell-wall loosening without affecting bulk viscoelastic properties, and indicate a chemorheological mechanism of cell-wall expansion.Abbreviations and symbols BL blue light - wall yield coefficient - Y wall yield threshold - P turgor pressure - L hydraulic conductance - g radial water-potential gradient supporting cell expansion - osmotic pressure - P_i initial chamber pressure needed to stop growth - P_f final chamber pressure needed to stop growth     

Thigmotropism and the modulation of tropistic curvature by mechanical perturbation in cucumber hypocotyls

Mechanical perturbation (MP) applied unilaterally to cucumber ( Cucumis sativus L.) hypocotyls induced thigmotropic curvature toward the stimulus. Gravitropic or phototropic curvature of the hypocotyl was inhibited by symmetrical application of MP to both sides of the hypocotyl. When both MP and IAA were unilaterally applied simultaneously to the same side, the hypocotyls always bent toward the MP stimulus, as in thigmotropism alone. Thus, the exogenous IAA did not control the direction of curvature. Aminoethoxyvinyl glycine (AVG) blocked thigmotropism as well as gravitropism and phototropism, but promoted IAA-induced curvature. MP-stimulated ethylene evolution peaked about 4 h after MP, followed by a peak of thigmotropic curvature. For all tropisms more ethylene evolved from the stimulated side than from the other side of the hypocotyls. MP-induced ethylene acting as a growth inhibitor, auxin-transport inhibitor, and/or modulator of tissue sensitivity to auxin, may be involved in thigmotropism and MP-induced inhibition of various tropisms. Ethylene produced as a result of MP was not affected by the removal of cotyledons. This MP-induced ethylene was additive to that of phototropically or gravitropically stimulated ethylene.     

A reduced xylem pressure altered the electric and growth responses in cucumber hypocotyls

This study measured the electric and growth responses in excised cucumber hypocotyls and compared them with those in intact seedlings. Root excision (first severing cut) eliminated most of the positive xylem pressure (P_x) in the hypocotyl, caused a rapid, transient drop in the hypocotyl growth rate (GR) and some small, local depolarization near the cut site. Although accompanied by a smaller decrease in P_x, a second, severing cut in the basal hypocotyl caused a decrease in GR which was no longer transient and a depolarization which was increased in both size and extent. These changes were not wound effects because they could be simulated by root incubation in mannitol. The reduced GR recovery occurred also in the absence of electric changes after a second increase in the mannitol concentration incubating the root of intact seedlings. Increased electric sensitivity and altered growth response therefore appear to be two independent examples of physiological changes resulting from a lowered P_x.     

The relationship between xyloglucan endotransglycosylase and in-vitro cell wall extension in cucumber hypocotyls

It has been proposed that cell wall loosening during plant cell growth may be mediated by the endotransglycosylation of load-bearing polymers, specifically of xyloglucans, within the cell wall. A xyloglucan endotransglycosylase (XET) with such activity has recently been identified in several plant species. Two cell wall proteins capable of inducing the extension of plant cell walls have also recently been identified in cucumber hypocotyls. In this report we examine three questions: (1) Does XET induce the extension of isolated cell walls? (2) Do the extension-inducing proteins possess XET activity? (3) Is the activity of the extension-inducing proteins modulated by a xyloglucan nonasaccharide (Glc₄-Xyl₃-Gal₂)? We found that the soluble proteins from growing cucumber (cucumis sativum L.) hypocotyls contained high XET activity but did not induce wall extension. Highly purified wall-protein fractions from the same tissue had high extension-inducing activity but little or no XET activity. The XET activity was higher at pH 5.5 than at pH 4.5, while extension activity showed the opposite sensitivity to pH. Reconstituted wall extension was unaffected by the presence of a xyloglucan nonasaccharide (Glc₄-Xyl₃-Gal₂), an oligosaccharide previously shown to accelerate growth in pea stems and hypothesized to facilitate growth through an effect on XET-induced cell wall loosening. We conclude that XET activity alone is neither sufficient nor necessary for extension of isolated walls from cucumber hypocotyls.     

Appearance of a reversible hydrogenase activity in Anabaena cylindrica grown in high light

In vivo H₂ evolution by Anabaena cylindrica Lemm. strain PCC 7122 grown in the presence of ammonia at low and high light intensities was studied. We found that after 2 h of anaerobic incubation, H₂ evolution [at a rate of 0.5 μmol (mg dry weight)¹ h⁻¹] via reversible hydrogenase occurred in high light grown cells, while this kind of activity was not found in low light grown cells. H₂ evolution was inhibited by 3-(3'. 4'-dichlorophenyl-1, 1-dimethylurea (DCMU). Illuminating the cells in the phycocyanin absorption region resulted in a higher rate of H₂ evolution than illuminating the cells in the chlorophyll absorption region. The results indicate that reversible hydrogenase receives reducing equivalents from photosynthetic water photolysis and that both photosystem II and photosystem I participate in the H₂ production. Hydrogenase activity was found in the soluble fraction after mild sonication in the case of low light grown cells. After this treatment high light grown cells retained 70% of their hydrogenase activity in the particulate fraction, but released it into the soluble fraction in the presence of 2% deoxycholic acid.
In vitro H₂ evolution did not differ significantly in the low and high light grown cells. Hence, the differences in the in vivo H₂ evolution reflect the different availability of endogenous reductants for hydrogenase in the two kinds of cells. On the basis of our results it is suggested that high light grown Anabaena cells eliminate part of the photosynthetically produced excess electrons via an induced reversible hydrogenase activity. This is the first report of H₂ evolution associated with water photolysis and catalyzed by hydrogenase in cyanobacteria.     

Oxidative DNA damage in cucumber cotyledons irradiated with ultraviolet light

DNA was isolated from the cotyledons of cucumber seedlings irradiated with ultraviolet (UV)-C (254 nm) or UV-B+UV-A (280–360 nm; maximum energy at 312 nm) at various fluence rates and durations. Following enzymatic hydrolysis of DNA, the content of 8-hydroxy-2-deoxyguanosine [(8-OHdG), 8-oxo-7,8-dihydro-2-deoxyguanosine], a well-established biomarker closely identified with carcinogenesis and aging in animal cells, was determined using a high-performance liquid chromatograph equipped with an electrochemical detector. The levels of 8-OHdG increased with UV-C and UV-B irradiation in a fluence-dependent manner. This increase was also observed in etiolated cotyledons that had been excised from dark-grown cucumber seedlings and then cultured in vitro under UV light: monochromatic UV light at 270 nm or 290 nm increased the 8-OHdG level considerably, while UV at wavelengths above 310 nm had only small effects. In situ detection of H₂O₂ and quantification of H₂O₂ in plant extracts revealed that H₂O₂ accumulated in cotyledons irradiated with UV light. These results suggest that UV irradiation induces oxidative DNA damage in plant cells.     

Interaction of light and gravitropism with nutation of hypocotyls of Arabidopsis thaliana seedlings

Etiolated seedlings of Arabidopsis thaliana nutated under conditions of physiological darkness while about ten percent of monitored individuals exhibited regular elliptical nutation, circumnutation. Pre-irradiation with red light prevented occurrence of circumnutation without having an effect on the average rate of the nutational movement. Phototropic response of seedlings to unilateral blue light appeared to be superimposed over nutation. Throughout gravitropism, some seedlings continued to exhibit nutation suggesting that these two processes are independently controlled. Based on these results, we suggest that nutation in Arabidopsis probably is not controlled by the mechanism predicted by the theory of gravitropic overshoots.     

Effect of light on peroxidase and lignin synthesis in mungbean hypocotyls

Two days of light irradiance reduced the growth of mungbean hypocotyls as well as the levels of endogenous indole-3-acetic acid (IAA). In hypocotyls, both peroxidase and laccase activities were enhanced by light. The lignin content in mungbean hypocotyls was enhanced twofold by light. The inhibition of mungbean hypocotyl growth caused by light might be due to the decline of endogenous IAA, which could be degraded by a cationic peroxidase. The higher levels of lignin were correlated with the increased anionic peroxidase activity in light-treated tissues.