Ethylene as a natural agent inducing plumular hook formation in pea seedlings

Summary Removing endogenous ethylene by hypobaric treatment, or displacing it with carbon dioxide inhibits hook development in etiolated pea seedlings. When seedlings are returned to a normal atmosphere, hook formation occurs in darkness. Addition of ethylene accelerates this process. When ethylene induces hook formation, cell division in the hook tissue is rather inhibited by the gas. These data suggest that endogenous ethylene causes formation of the hook by inducing expansion of certain cells.     

Ethylene-mediated enhancement of apical hook formation in etiolated Arabidopsis thaliana seedlings is gibberellin dependent

Dark-grown Arabidopsis seedlings develop an apical hook by differential elongation and division of hypocotyl cells. This allows the curved hypocotyl to gently drag the apex, which is protected by the cotyledons, upwards through the soil. Several plant hormones are known to be involved in hook development, including ethylene, which causes exaggeration of the hook. We show that gibberellins (GAs) are also involved in this process. Inhibition of GA biosynthesis with paclobutrazol (PAC) prevented hook formation in wild-type (WT) seedlings and in constitutive ethylene response (ctr)1-1, a mutant that exhibits a constitutive ethylene response. In addition, a GA-deficient mutant (ga1-3) did not form an apical hook in the presence of the ethylene precursor 1-aminocyclopropane-1-carboxylate (ACC). Analysis of transgenic Arabidopsis seedlings expressing a green fluorescent protein (GFP)-repressor of ga1-3 (RGA) fusion protein suggested that ACC inhibits cell elongation in the apical hook by inhibition of GA signaling. A decreased feedback of GA possibly causes an induction of GA biosynthesis based upon the expression of genes encoding copalyl diphosphate synthase (CPS; GA1) and GA 2-oxidase (AtGA2ox1). Furthermore, expression of GASA1, a GA-response gene, suggests that differential cell elongation in the apical hook might be a result of differential GA-sensitivity.     

Lectins in castor bean seedlings

The amounts of the two lectins (ricin and Ricinus communis agglutinin) in tissues of castor bean seedlings were followed during germination and early growth. For measurement, lectins in extracts were separately eluted from Sepharose columns; an antibody to the agglutinin was also used to detect the lectins by immunodiffusion. The endosperm of the dry seed contains 3.5 mg total lectin (5.6% of the total seed protein), which declines by 50% by day 4 and more rapidly thereafter as the tissue is completely consumed. The cotyledons of the dry seed also contain lectins but the amounts are less than 1% of those in the endosperm, and, as in the endosperm, they are constituents of the albumin fraction of the isolated protein bodies. No lectins were detected in the green cotyledons of 10-day seedlings that had been exposed to light from day 5. The embryonic axes of 2-day seedlings contained very small amounts of lectins but they were not detectable in the aerial parts of seedlings grown for 3 weeks or in cells from endosperm grown in tissue culture.

The ability of proteinases and glycosidases (isolated from endosperm of 4-day seedlings) to hydrolyze the lectins was examined. No hydrolysis of the two lectins was observed, but the subunits, separated by reduction with 2-mercaptoethanol, were hydrolyzed slowly by a proteinase and some release of mannose was observed in the presence of the glycosidases. Ricin was converted to its subunits by cysteine and an enzyme in an endosperm extract accelerated chain separation by glutathione.

     

Particulate cytochromes of mung bean seedlings

Efforts have been made to solubilize cytochrome components from particulate fractions of etiolated mung bean seedlings. Low temperature spectrophotometry reveals that the cytochrome composition of mitochondria isolated from whole seedlings is the same as that reported by Bonner for mung bean hypocotyls. On the basis of the identity in position of the α-bands in low temperature difference spectra for mitochondria, for a partially purified haemoprotein from mitochondria, and for purified cytochrome b-555, it is suggested that cytochrome b-555 is an intrinsic component of mung bean mitochondria. Difference spectra show that both the mitochondrial and microsomal fractions contain at least 2 b-type cytochromes. Cytochrome b-555 is almost certainly present in the microsomes, since the low temperature difference spectrum for the cytochrome is identical with the spectrum for this particulate fraction.

By freezing and thawing mung bean mitochondria in 4% cholate and centrifuging, cytochrome oxidase activity can be concentrated in the supernatant fraction, although it is not completely solubilized. The oxidase is inhibited by high concentrations of cytochrome c. A particle-bound cytochrome c can be obtained from mitochondria by digestion with snake venom. However, the autoxidizability of the preparation indicates that the cytochrome has been solubilized in a modified form. A CO-binding pigment can be obtained from mung bean microsomes by digestion with snake venom.

     

The sites of excretion of ninhydrin-positive substances by broad bean seedlings

Summary A simple method is described for determining the sites of excretion of ninhydrin-positive substances from the undamaged roots of seedlings of broad bean. Results are given on the sites of excretion of such substances from the germinating seed and from the young root. It is suggested that in such roots the centres of maximum excretion may be correlated with centres of high proteolytic activity within the root.     

3-Isoxazolidone from jack bean seedlings

3-Isoxazolidone has been isolated from jack bean seedlings.     

Characteristics of galacturonic Acid oligomers as elicitors of casbene synthetase activity in castor bean seedlings

The metabolism of [¹⁴CH₃]2-(2,4-dichlorophenoxy)isobutyric acid (DIB) was studied in plants and cell suspension cultures of Lycopersicon esculentum Mill. sp. `Lukullus'. Both plants and cells in suspension culture showed a rapid uptake of DIB from nutrient media. The metabolites, isolated by extraction with methanol and separated by chromatographic methods, were identified by enzymic, chemical, and spectrometric methods. Two conjugates of the carboxyl with 2 and 3 moles glucose per mole DIB and, to a smaller extent, its β-d-glucopyranosyl ester, were formed in both intact plants and cell suspension cultures, but there were quantitative differences.     

Opening of the hypocotyl hook in seedlings as influenced by light and adjacent tissues

The influence of the cotyledons and apical bud and the root system on the light-induced opening of the hypocotyl hook of etiolated seedlings of Gossypium hirsutum L., Phaseolus vulgaris L., Helianthus annuus L., Ipomoea alla L., Ipomoea sp., Cucumis sativus L., Linum usitatissimum L., Hibiscus esculentus L., and Raphanus sativus L. was studied. Light stimulated the opening of hypocotyl hook in all cases, but the cotyledons and roots had different effects in different plants. Generally, the presence of cotyledons and the remainder of the shoot apical to the hook inhibited light-mediated opening, but in Gossypium the organs stimulated light-mediated opening. Presence of roots either promoted opening, had no effect, or had an effect only when the cotyledons were present. In the dark the adjacent organs had a reduced effect over that shown in the light, but one cultivar of cotton, Acala SJ1, opened the hook in the dark without cotyledons as much as under any condition in the light. The variation between species in hook opening may related to the need of that process for a proper hormonal balance, as affected by light, which must be obtained from adjacent tissues.     

Hydrogen peroxide acts as a signal molecule in the adventitious root formation of mung bean seedlings

Hydrogen peroxide (H₂O₂), an active oxygen species, is widely generated in many biological systems and mediates various physiological and biochemical processes in plants. In this study we demonstrated that the exogenous H₂O₂ was able to promote the formation and development of adventitious roots in mung bean seedlings. Treatments with 1–100 mM H₂O₂ for 8–18 h significantly induced the formation and development of adventitious roots. Catalase (CAT) and ascorbic acid, which are H₂O₂ scavengers or inhibitors, eliminated the adventitious root-promoting effects of exogenous H₂O₂. H₂O₂ may have a downstream signaling function in the auxin signaling pathway and be involved in auxin-induced adventitious root formation. 2,3,5-Triiodobenzoic acid (TIBA), an inhibitor of auxin polar transport, strongly inhibited adventitious rooting of mung bean seedlings; however, the inhibiting effects of TIBA on adventitious rooting can be partially reversed by the exogenous IBA or H₂O₂. Diphenylene iodonium (DPI) strongly inhibits the activity of NADPH oxidase, which is one of the main sources of H₂O₂ formation in plant cells. DPI treatment strongly inhibited the formation of adventitious roots in mung bean, but the inhibitory effects of DPI on rooting can be partially reversed by the exogenous H₂O₂ or IBA. This indicates that the formation of adventitious roots was blocked once the generation of H₂O₂ through NADPH oxidase was inhibited, and H₂O₂ mediated the IBA-induced adventitious root formation. Furthermore, a rapid increase in the endogenous level of H₂O₂ was detected during incubation with water 12–36 h after the primary root removal in mung bean seedlings. Three hours after the primary root removal, the generation of endogenous H₂O₂ was markedly induced in IBA-treated seedlings in comparison with water-treated seedlings. This implies that IBA induced overproduction of H₂O₂ in mung bean seedlings, and that IBA promoted adventitious root formation via a pathway involving H₂O₂. Results obtained suggest that H₂O₂ may function as a signaling molecule involved in the formation and development of adventitious roots in mung bean seedlings.     

Role of growth regulators in the bean hypocotyl hook opening response

Summary The opening of the hypocotyl hook in bean seedlings is due to a rapid elongation of cells on the inner side of the hook elbow. Red light promotes hook opening by inducing this cell elongation.Opening is inhibited by low concentrations of indoleacetic acid (IAA) and 2,4-dichlorophenoxyacetic acid (2,4-D), and higher concentrations of these auxins cause a closure of the hook. In darkness, opening is induced slightly by p-chlorophenoxyisobutyric acid (PCIB), whereas in red light this auxin antagonist promotes opening only when IAA is added simultaneously to inhibit opening.The amount of diffusible auxin released by the hook tissue is not affected by red illumination that is sufficient to induce maximal hook opening.Gibberellic acid (GA) promotes the hook opening. The magnitude of its effect is, however, rather small, especially in darkness. (2-Chloroethyl)-trimethylammonium chloride (CCC) and 2-isopropyl-4-(trimethylammonium-chloride)-5-methylphenyl piperidine-1-carboxylate (Amo-1618) inhibit hook opening in red light, and this inhibition is completely overcome by addition of GA.Cytokinins and abscisic acid at rather high concentrations inhibit hook opening in light but produce no significant effect in darkness.Hook opening is promoted by Ca⁺⁺ and K⁺, and notably by Co⁺⁺ and Ni⁺⁺.It is concluded that 1. endogenous gibberellin assists in hook opening, but light does not act by changing the gibberellin level; 2. light does not act by decreasing the endogenous auxin level; and 3. cytokinins or abscisic acid do not seem to have a special role in the response.     

Distribution of nitrate reductase in ageing bean seedlings

The in vivo activity of nitrate reductase (NR, E.C. 1.6.6.1 [EC] )in the roots, stem and leaves of bean (Phaseolus vulgaris L.)was measured at different ages of seedlings. The leaves alwayshad higher levels of the enzyme than the roots or stem. Thelevel of the enzyme in the very young leaves were low, increasingto a maximum by day 10 to 11 of seedling growth at 26°C,after which it start to decline. The level of the enzyme in7 dayold decotyledonized leaves was about 2.5 times higher thanthat in leaves from intact seedlings. A supply of exogenousnitrate caused a considerable increase in the total organicnitrogen in the leaf only after day 9, when the nitrogen supplyfrom the cotyledons presumably is low. (Received March 22, 1975; )     

Phytochrome-controlled ethylene biosynthesis of intact etiolated bean seedlings

Intact etiolated bean (Phaseolus vulgaris L. cv. Limburgse vroege) seedlings were illuminated with red light (10.5 W·m^-2) for 10 min. After different time intervals ethylene production, and contents of 1-aminocyclopropane-1-carboxylic acid (ACC) and 1-(malonylamino)cyclopropane-1-carboxylic acid were measured. The red-light-induced decrease of ethylene production in 8-d-old intact etiolated bean seedlings was fast, strong and long-lasting ad was mediated through the phytochrome system. This effect appeared to be strictly age-dependent, as it could not be detected in plants younger than 6 d or older than 11 d.The capacity for the conversion of ACC to ethylene was not affected by red light. The inhibitory effect of the light treatment on ethylene production could be related to a reduced free-ACC content. This reduction was a consequence of a temporary non-reversible increase of ACC malonylation and a long-lasting, for a certain time reversible, inhibition of ACC synthesis. The effect of a brief irradiation with red light on the decrease of ethylene production and free-ACC content was completed after about 2 h. Reversibility by far-red, however, persisted for at least 3 h, and was lost between 3 and 6 h.Abbrevation ACC 1-aminocyclopropane-1-carboxylic acid - M-ACC 1-(malonylamino)cyclopropane-1-carboxylic acid     

Involvement of ethylene in responses of etiolated bean hypocotyl hook to coumarin

Coumarin, at concentrations between 1.0 and 0.1 mm, inhibited red light-induced opening of the etiolated bean hypocotyl hook. In addition, anthocyanin synthesis and geotropic bending were inhibited. Coumarin stimulated ethylene synthesis, and ethylene was shown to mediate the inhibitory actions of coumarin. This conclusion was supported by: (a) the parallel concentration dependence and time sequence of hook closing and ethylene synthesis, (b) the restriction of the bulk of coumarin-induced ethylene production to the curved portion of the hook where opening is expressed, (c) the ability of both coumarin and ethylene to reclose partially opened hooks, and (d) the ability of exogenous ethylene, in the amounts produced by coumarintreated hooks, to duplicate the inhibitory effects of coumarin. There was an increasing stimulation of growth of the straight portion of the hypocotyl hook section as coumarin concentrations were increased from 0.1 to 1.0 mm. This action of coumarin was not duplicated by ethylene and occurred regardless of the presence or absence of added ethylene. The results of this study suggest that many actions of coumarin in growth systems are mediated by ethylene produced in response to the coumarin.     

Amino Acid transport in germinating castor bean seedlings

During germination and early growth of the castor bean (Ricinus communis) nitrogenous constituents from the endosperm are transferred via the cotyledons to the growing embryo. Exudate collected from the cut hypocotyl of 4-day seedlings contained 120 millimolar soluble amino nitrogen and glutamine was the predominant amino acid present, comprising 35 to 40% of the total amino nitrogen. To determine the nature of nitrogen transfer, the endosperm and hypocotyl were removed and glutamine uptake by the excised cotyledons was investigated. Uptake was linear for at least 2 hours and the cotyledons actively accumulated glutamine against a concentration gradient. The uptake was sensitive to respiratory inhibitors and uncouplers and efflux of glutamine from the excised cotyledons was negligible. Transport was specific for the l-isomer. Other neutral amino acids were transported at similar rates to glutamine. Except for histidine, the acidic and basic amino acids were transported at lower rates than the neutral amino acids. For glutamine transport, the K(m) was 11 to 12 millimolar and the V(max) was 60 to 70 micromoles per gram fresh weight per hour. Glutamine uptake was diminished in the presence of other amino acids and the extent of inhibition was greatest for those amino acids which were themselves rapidly transported into the cotyledons. The transport of amino acids, on a per seedling basis, was greatest for cotyledons from 4-to 6-day seedlings, when transfer of nitrogen from the endosperm is also maximal. It is concluded that the castor bean cotyledons are highly active absorptive organs transporting both sucrose and amino acids from the surrounding endosperm at high rates.     

Effects of acifluorfen on auxin translocation in bean seedlings

The effect of 5-(2-chloro-4-(trifluoromethyl)phenoxy)-2-nitrobenzoic acid (acifluorfen) on the translocation of the¹⁴C-labeled auxins 2,4,5-trichlorophenoxyacetic acid (2,4,5-T-1-¹⁴C) and indole-3-acetic acid (IAA-1-¹⁴C) was determined. The auxins and acifluorfen were injected into the stem at the cotyledonary node of 9-day-old bean (Phaseolus vulgaris L. cv Tenderpod) seedlings. The plants were harvested 4 h after treatment and analyses of¹⁴C were made of various plant parts. Acifluorfen increased 2,4,5-T,-1-¹⁴C translocation out of the treated area and especially into the large primary leaves. This translocation pattern is indicative of apoplastic translocation and suggests that acifluorfen inhibited vein loading of the auxins. Acifluorfen affected auxin translocation in the dark as effectively as in the light even though the herbicidal effects of acifluorfen are known to be expressed only after light treatment.Journal article no. 4403 of the Agric. Exp. Stn., Oklahoma State Univ.     

Changing cell-wall compositions in hypocotyls of dark-grown bean seedlings

Hypocotyls of dark-grown 6-day-old seedlings of Phaseolus vulgaris L. proved to be sufficiently homogeneous to permit studies relating the rate of cell elongation to the composition of the primary cell walls. Whereas the levels of cellulose and uronic acids remained practically constant during and after cell extension, all other components showed major or minor changes. Cell-wall protein, as such, decreased by more than 50%, but indications are that hydroxyproline-rich glycoprotein increased with a decreasing rate of cell elongation, concomitant with a rise in the degree of arabinosylation of wall-bound hydroxyproline. As cell elongation slowed down, non-cellulosic glucose accumulated, presumably in the form of a -(1–4)glucan closely associated with cellulose. These findings confirm the notion that the primary cell wall is a highly dynamic structure.     

Light exaggerates apical hook curvature through phytochrome actions in tomato seedlings

Contrary to the established notion that the apical hook of dark-grown dicotyledonous seedlings opens in response to light, we found in tomato (Solanum lycopersicum L.) that the apical hook curvature is exaggerated by light. Experiments with several tomato cultivars and phytochrome mutants, irradiated with red and far-red light either as a brief pulse (Rp, FRp) or continuously (Rc, FRc), revealed: the hook-exaggeration response is maximal at the emergence of the hypocotyl from the seed; the effect of Rp is FRp-reversible; fluence–response curves to a single Rp or FRp show an involvement of low and very low fluence responses (LFR, VLFR); the effect of Rc is fluence-rate dependent, but that of FRc is not; the phyA mutant (phyA hp-1) failed to respond to an Rp of less than 10⁻² μmol m⁻² and to an FRp of all fluences tested as well as to FRc, thus indicating that the hook-exaggeration response involves phyA-mediated VLFR. The Rp fluence–response curve with the same mutant also confirmed the presence of an LFR mediated by phytochrome(s) other than phyA, although the phyB1 mutant (phyB1 hp-1) still showed full response probably due to other redundant phytochrome species (e.g., phyB2). Simulation experiments led to the possible significance of hook exaggeration in the field that the photoresponse may facilitate the release of seed coat when seeds germinate at some range of depth in soil. It was also observed that seed coat and/or endosperm are essential to the hook exaggeration.